HK1215180B - Lipid formulated compositions and methods for inhibiting expression of eg5 and vegf genes - Google Patents

Description

Lipid-formulated compositions and methods for inhibiting Eg5 and VEGF gene expression

The present application is a divisional application of the invention patent application No. 201080020483.1, filed on March 12, 2010, and entitled “Lipid-formulated composition and method for inhibiting Eg5 and VEGF gene expression”.

Technical Field

The present invention relates to lipid-formulated compositions containing double-stranded ribonucleic acid (dsRNA) and their use in mediating RNA interference to inhibit the expression of gene combinations, such as Eg5 and vascular endothelial growth factor (VEGF) genes. The dsRNA is formulated into a lipid formulation and may include a lipoprotein, such as apolipoprotein E. The invention also includes the use of the compositions in treating pathological processes mediated by Eg5 and VEGF expression, such as cancer.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application Serial No. 61/159,788, filed March 12, 2009, U.S. Provisional Application Serial No. 61/231,579, filed August 5, 2009, and U.S. Provisional Application Serial No. 61/285,947, filed December 11, 2009, all of which are incorporated herein by reference in their entirety for all purposes.

Sequence Listing Reference

This application includes a Sequence Listing created on XX/XX/2010 and submitted electronically as a text file named 16564US_sequencelisting.txt, which is XXX,XXX bytes in size. The Sequence Listing is incorporated by reference.

Background Art

The maintenance of cell populations in an organism is determined by the cellular processes of cell division and programmed cell death. In normal cells, the cellular events related to the start and completion of each process are highly regulated. In proliferative diseases such as cancer, one or both of these processes may be disturbed. For example, cancer cells may lose the regulation of their cell division cycle (checkpoint control) through mutation, overexpression of positive regulators or loss of negative regulators.

Alternatively, cancer cells may lose the ability to undergo programmed cell death through overexpression of negative regulators. Therefore, there is a need to develop new chemotherapeutic agents that can restore checkpoint control and programmed cell death processes in cancer cells.

One approach to treating human cancers is to target proteins essential for cell cycle progression. In order for the cell cycle to progress from one phase to the next, certain prerequisite events must complete. Checkpoints exist within the cell cycle that enforce the proper sequence of events and phases. One such checkpoint is the spindle checkpoint, which occurs during the metaphase stage of mitosis. Small molecules targeting proteins with essential functions in mitosis can trigger the spindle checkpoint to arrest cells in mitosis. Among the small molecules that arrest mitosis, those that clinically demonstrate anti-tumor activity also induce apoptosis—morphological changes associated with programmed cell death. Therefore, an effective chemotherapy for cancer treatment could be one that induces checkpoint control and programmed cell death. Unfortunately, few compounds are known to effectively control these processes within cells. Most compounds known to cause mitotic arrest and apoptosis act as tubulin-binding agents. These compounds alter the dynamic instability of microtubules and indirectly alter the function and structure of the mitotic spindle, thereby causing mitotic arrest. Because most of these compounds specifically target tubulin, a component of all microtubules, they may also affect one or more of the many normal cellular processes in which microtubules play a role. Thus, there remains a need for agents that more specifically target proteins associated with proliferating cells.

Eg5 is one of several kinesin-like motor proteins that are concentrated in the mitotic spindle and is known to be required for the formation and/or function of the bipolar mitotic spindle. Small molecules that interfere with the bipolarity of the mitotic spindle have recently been reported (Mayer, T.U. et al. 1999. Science 286(5441)971-4, incorporated herein by reference). More specifically, the small molecules induce the formation of an abnormal mitotic spindle in which a monostellate array of microtubules emanates from a central pair of centrosomes, with chromosomes bound to the distal ends of the microtubules. Due to the monostellate array, the small molecule has been termed "monastrol". This monostellate array phenotype has previously been observed in mitotic cells immunodepleted of Eg5 dynein. This distinctive monostellate array phenotype has led to the identification of monastrol as a potential inhibitor of Eg5. In fact, monastrol has also been shown in in vitro experiments to inhibit the Eg5 dynein-driven motility of microtubules. The Eg inhibitor monastrol has no significant effect on the related kinesin motors or on the motors responsible for intracellular Golgi movement. Cells that display the monastrol phenotype by immunodepletion of Eg5 or inhibition of Eg5 are arrested in the M-phase of the cell cycle. However, the mitotic arrest induced by immunodepletion or inhibition of Eg5 is transient (Kapoor, T.M., 2000. J Cell Biol 150(5)975-80). Both the monastrol phenotype and the cell cycle arrest in mitosis induced by monastrol are reversible. The cells recover to form a normal bipolar mitotic spindle to complete mitosis and continue the cell cycle and normal cell proliferation. These data suggest that Eg5 inhibitors that induce transient mitotic arrest may not be effective in treating cancer cell proliferation. Nevertheless, the finding that monastrol causes mitotic arrest is intriguing, and further research is needed to identify compounds that can be used to modulate the Eg5 motor protein in a manner that is effective in treating human cancers. There is also a need to study the combination of these compounds with other antitumor agents.

VEGF (vascular endothelial growth factor, also known as vascular permeability factor, VPF) is a multifunctional cytokine that stimulates angiogenesis, epithelial cell proliferation, and endothelial cell survival. VEGF is produced by a variety of tissues, and its overexpression or aberrant expression can lead to a variety of disorders, including cancer and retinal diseases such as age-related macular degeneration and other angiogenic disorders.

Recently, double-stranded RNA molecules (dsRNA) have been shown to block gene expression using a highly conserved regulatory mechanism known as RNA interference (RNAi). WO 99/32619 (Fire et al.) discloses the use of dsRNA of at least 25 nucleotides in length to inhibit gene expression in nematodes. dsRNA has also been shown to degrade target RNA in other organisms, including plants (see, e.g., WO 99/53050, Waterhouse et al.; and WO 99/61631, Heifetz et al.), fruit flies (see, e.g., Yang, D., et al., Curr. Biol. (2000) 10: 1191-1200), and mammals (see, e.g., WO 00/44895, Limmer; and DE 101 00 586.5, Kreutzer et al.). This natural mechanism is currently the focus of a new class of pharmaceutical agents for the treatment of diseases caused by abnormal or deleterious regulation of genes.

Summary of the Invention

The present invention provides compositions and methods for inhibiting the expression of human Eg5/KSP and VEGF genes in cells using lipid-formulated compositions comprising dsRNA.

The present invention comprises a nucleic acid lipid particle comprising a first double-stranded ribonucleic acid (dsRNA) for inhibiting expression of the human kinesin family member 11 (Eg5/KSP) gene in a cell and a second dsRNA for inhibiting expression of human VEGF in a cell. The nucleic acid lipid particle comprises a lipid formulation comprising 45-65 mol% of a cationic lipid, 5 mol% to about 10 mol% of a non-cationic lipid, 25-40 mol% of a sterol, and 0.5-5 mol% of PEG or a PEG-modified lipid. The first dsRNA targeting Eg5/KSP comprises a first sense strand and a first antisense strand, wherein the first sense strand has a first sequence, and the first antisense strand has a second sequence complementary to at least 15 consecutive nucleotides of SEQ ID NO: 1311 (5'-UCGAGAAUCUAAACUAACU-3'), wherein the first sequence and the second sequence are complementary, and wherein the first dsRNA is 15 to 30 base pairs in length. The second dsRNA comprises a second sense strand and a second antisense strand, the second sense strand having a third sequence, and the second antisense strand having a fourth sequence that is complementary to at least 15 consecutive nucleotides of SEQ ID NO: 1538 (5'-GCACAUAGGAGAGAUGAGCUU-3'), wherein the third sequence and the fourth sequence are complementary, and wherein the second dsRNA is 15 to 30 base pairs in length.

In one embodiment, the cationic lipid of the composition has Formula A, wherein Formula A is:

wherein R1 and R2 are independently alkyl, alkenyl or alkynyl, each of which may be optionally substituted, R3 and R4 are independently lower alkyl or R3 and R4 may be taken together to form an optionally substituted heterocycle.

In other embodiments, the cationic lipid is XTC (2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane). In a related embodiment, the cationic lipid is XTC, the non-cationic lipid is DSPC, the sterol is cholesterol, and the PEG lipid comprises PEG-DMG. In another related embodiment, the cationic lipid is XTC and the formulation is selected from the group consisting of:

In another embodiment, the cationic lipid of the composition is ALNY-100 ((3aR,5s,6aS)-N,N-dimethyl-2,2-di((9Z,12Z)-octadeca-9,12-dienyl)tetrahydro-3aH-cyclopenta[d][1,3]dioxol-5-amine). In other embodiments, the cationic lipid is ALNY-100 and the formulation comprises:

In other embodiments, the cationic lipid is MC3 (4-(dimethylamino)butanoic acid (6Z,9Z,28Z,31Z)-heptatriacontac-6,9,28,31-tetraen-19-yl ester). In a related embodiment, the cationic lipid is MC3 and the lipid formulation is selected from the group consisting of:

In another embodiment, the first dsRNA comprises a sense strand consisting of SEQ ID NO: 1534 (5′-UCGAGAAUCUAAACUAACUTT-3′) and an antisense strand consisting of SEQ ID NO: 1535 (5′-AGUUAGUUUAGAUUCCUGATT-3′), and the second dsRNA comprises a sense strand consisting of SEQ ID NO: 1536 (5′-GCACAUAGGAGAGAUGAGCUU-3′) and an antisense strand consisting of SEQ ID NO: 1537 (5′-AAGCUCAUCUCUCCUAUGUGCUG-3′). In yet another embodiment, each strand is modified as follows to include 2'-O-methyl ribonucleotides (represented by lowercase "c" or "u") and phosphorothioates (represented by lowercase "s"): the first dsRNA includes a sense strand consisting of SEQ ID NO: 1240 (5'-ucGAGAAucuAAAcuAAcuTsT-3') and an antisense strand consisting of SEQ ID NO: 1241 (5'-AGUuAGUUUuAGAUUCUCGATsT); the second dsRNA includes a sense strand consisting of SEQ ID NO: 1242 (5'-GcAcAuAGGAGAGAuGAGCUsU-3') and an antisense strand consisting of SEQ ID NO: 1243 (5'-AAGCUcAUCUCUCCuAuGuGCusG-3').

In other embodiments, the first and second dsRNAs include at least one modified nucleotide. In some embodiments, the modified nucleotide is selected from the group consisting of a 2'-O-methyl modified nucleotide, a nucleotide having a 5'-phosphorothioate group, and a terminal nucleotide connected to a cholesterol derivative or a dodecanoic acid didecylamide group. In another embodiment, the modified nucleotide is selected from the group consisting of a 2'-deoxy-2'-fluoro modified nucleotide, a 2'-deoxy-modified nucleotide, a locked nucleotide, an abasic nucleotide, a 2'-amino modified nucleotide, a 2'-alkyl-modified nucleotide, a morpholino nucleotide, a phosphoramidate, and a nucleotide containing a non-natural base. In yet another embodiment, the first and second dsRNAs each comprise at least one 2'-O-methyl modified nucleotide and at least one nucleotide having a 5'-phosphorothioate group.

In some embodiments, the length of each dsRNA is 19-23 bases. In another embodiment, the length of each chain of each dsRNA is 21-23 bases. In yet another embodiment, the length of each chain of the first dsRNA is 21 bases, the sense strand length of the second dsRNA is 21 bases and the antisense strand length of the second dsRNA is 23 bases. In other embodiments, the first and second dsRNA are present in an equimolar ratio. In one embodiment, the composition also contains Sorafenib. In another embodiment, the composition also contains lipoprotein. In another embodiment, the composition also contains apolipoprotein E (ApoE).

In another embodiment, when contacted with a cell expressing Eg5, the composition inhibits Eg5 expression by at least 40%. In yet another embodiment, when contacted with a cell expressing VEGF, the composition inhibits VEGF expression by at least 40%. In other embodiments, administering the composition to a cell reduces the expression of Eg5 and VEGF in the cell. In a related embodiment, the composition is administered at a nM concentration. In another embodiment, administering the composition to a cell increases monoastrocytic formation in the cell.

In other embodiments, administration of the composition to a mammal results in at least one effect selected from the group consisting of: preventing tumor growth, reducing tumor growth, or prolonging survival of the mammal. In some embodiments, the effect is measured using at least one test selected from the group consisting of: body weight measurement, organ weight measurement, visual inspection, mRNA analysis, serum AFP analysis, and survival monitoring.

The present invention also provides methods for inhibiting the expression of Eg5/KSP and VEGF in cells. The methods comprise the step of administering a composition of the present invention to a cell. The present invention also provides methods for inhibiting tumor growth, reducing tumor growth, or prolonging the survival of a mammal in need of cancer treatment. The methods comprise the step of administering a composition of the present invention to a mammal. In one embodiment, the mammal has liver cancer. In another embodiment, the mammal is a human with liver cancer. In some embodiments, a dose containing 0.25 mg/kg to 4 mg/kg of dsRNA is administered to the mammal. In other embodiments, the dsRNA is administered to a human at a dose of about 0.01, 0.1, 0.5, 1.0, 2.5, or 5.0 mg/kg.

In yet another embodiment, the present invention provides a method for reducing tumor growth in a mammal in need of treatment for cancer, the method comprising administering a composition of the present invention to the mammal, the method reducing tumor growth by at least 20%. In another embodiment, the method reduces KSP expression by at least 60%.

Specifically, the present invention relates to the following:

1. A composition comprising a nucleic acid lipid particle comprising a first double-stranded ribonucleic acid (dsRNA) for inhibiting expression of a human kinesin family member 11 (Eg5/KSP) gene in a cell and a second dsRNA for inhibiting expression of human VEGF in a cell, wherein:

The nucleic acid lipid particle contains a lipid formulation comprising 45-65 mol% of a cationic lipid, 5 mol% to about 10 mol% of a non-cationic lipid, 25-40 mol% of a sterol, and 0.5-5 mol% of a PEG or PEG-modified lipid.

The first dsRNA consists of a first sense strand and a first antisense strand, wherein the first sense strand contains a first sequence, and the first antisense strand contains a second sequence complementary to at least 15 consecutive nucleotides of SEQ ID NO: 1311 (5'-UCGAGAAUCUAAACUAACU-3'),

wherein the first sequence and the second sequence are complementary, and wherein the first dsRNA is 15 to 30 base pairs in length; and

The second dsRNA consists of a second sense strand and a second antisense strand, the second sense strand contains a third sequence, and the second antisense strand contains a fourth sequence that is complementary to at least 15 consecutive nucleotides of SEQ ID NO: 1538 (5'-GCACAUAGGAGAGAUGAGCUU-3'),

wherein the third sequence is complementary to the fourth sequence, and wherein the second dsRNA is 15 to 30 base pairs in length.

2. The composition of item 1, wherein the cationic lipid has formula A, wherein formula A is:

wherein R1 and R2 are independently alkyl, alkenyl or alkynyl, each of which may be optionally substituted, R3 and R4 are independently lower alkyl or R3 and R4 may be taken together to form an optionally substituted heterocycle.

3. The composition of item 2, wherein the cationic lipid contains XTC (2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane).

4. The composition of item 2, wherein the cationic lipid comprises XTC, the non-cationic lipid comprises DSPC, the sterol comprises cholesterol, and the PEG lipid comprises PEG-DMG.

5. The composition of item 2, wherein the cationic lipid contains XTC and the formulation is selected from the group consisting of:

6. The composition of item 1, wherein the cationic lipid comprises ALNY-100 ((3aR, 5s, 6aS)-N,N-dimethyl-2,2-di((9Z, 12Z)-octadec-9,12-dienyl)tetrahydro-3aH-cyclopenta[d][1,3]dioxol-5-amine)).

7. The composition of item 6, wherein the cationic lipid comprises ALNY-100 and the formulation is selected from the group consisting of:

8. The composition of item 1, wherein the cationic lipid comprises MC3 (4-(dimethylamino)butyric acid (6Z,9Z,28Z,31Z)-triacontac-6,9,28,31-tetraen-19-yl ester).

9. The composition of item 8, wherein the cationic lipid contains MC3 and the lipid formulation is selected from the group consisting of:

10. The composition of item 1, wherein the first dsRNA consists of a sense strand and an antisense strand, the sense strand consists of SEQ ID NO: 1534 (5’-UCGAGAAUCUAAACUAACUTT-3’), the antisense strand consists of SEQ ID NO: 1535 (5’-AGUUAGUUUAGAUUCCUGATT-3’), and the second dsRNA consists of a sense strand and an antisense strand, the sense strand consists of SEQ ID NO: 1536 (5’-GCACAUAGGAGAGAUGAGCUU-3’), the antisense strand consists of SEQ ID NO: 1537 (5’-AAGCUCAUCUCUCCUAUGUGCUG-3’).

11. The composition of item 10, wherein each chain is modified as follows to include a 2'-O-methyl ribonucleotide represented by a lowercase "c" or "u" and a phosphorothioate represented by a lowercase "s":

The first dsRNA consists of a sense strand and an antisense strand, the sense strand consists of SEQ ID NO: 1240 (5'-ucGAGAAucuAAAcuAAcuTsT-3'), and the antisense strand consists of SEQ ID NO: 1241 (5'-AGUuAGUUuAGAUUCUCGATsT);

The second dsRNA consists of a sense strand consisting of SEQ ID NO: 1242 (5'-GcAcAuAGGAGAGAuGAGCUsU-3') and an antisense strand consisting of SEQ ID NO: 1243 (5'-AAGCUcAUCUCUCCuAuGuGCusG-3').

12. The composition of item 1, wherein the first and second dsRNAs comprise at least one modified nucleotide.

13. The composition of item 12, wherein the modified nucleotide is selected from the group consisting of a 2′-O-methyl modified nucleotide, a nucleotide having a 5′-phosphorothioate group, and a terminal nucleotide linked to a cholesterol derivative or a dodecylamide group.

14. The composition of item 12, wherein the modified nucleotide is selected from the group consisting of 2′-deoxy-2′-fluoro modified nucleotides, 2′-deoxy-modified nucleotides, locked nucleotides, abasic nucleotides, 2′-amino modified nucleotides, 2′-alkyl-modified nucleotides, morpholino nucleotides, aminophosphoesters and nucleotides containing non-natural bases.

15. The composition of item 1, wherein the first and second dsRNAs each comprise at least one 2'-O-methyl modified ribonucleotide and at least one nucleotide having a 5'-phosphorothioate group.

16. The composition of item 1, wherein each strand of each dsRNA is 19-23 bases in length.

17. The composition of item 1, wherein each strand of each dsRNA is 21-23 bases in length.

18. The composition of item 1, wherein each strand of the first dsRNA is 21 bases in length, the sense strand of the second dsRNA is 21 bases in length and the antisense strand of the second dsRNA is 23 bases in length.

19. The composition of item 1, wherein the first and second dsRNAs are present in an equimolar ratio.

20. The composition of item 1 further contains sorafenib.

21. The composition according to item 1 further contains lipoprotein.

22. The composition according to item 1, further comprising apolipoprotein E (ApoE).

23. The composition according to item 1, wherein when contacted with a cell expressing Eg5, the composition inhibits Eg5 expression by at least 40%.

24. The composition of item 1, wherein when contacted with a cell expressing VEGF, the composition inhibits VEGF expression by at least 40%.

25. The composition according to item 1, wherein administering the composition to cells reduces the expression of Eg5 and VEGF in the cells.

26. The composition of item 25, wherein the composition is administered at a nM concentration.

27. The composition of item 1, wherein administering the composition to a cell increases the formation of monoasters in the cell.

28. The composition according to item 1, wherein administration of the composition to a mammal results in at least one effect selected from the group consisting of preventing tumor growth, reducing tumor growth, or prolonging the survival of the mammal.

29. The composition according to item 28, wherein the effect is determined by at least one test selected from the group consisting of body weight measurement, organ weight measurement, macroscopic examination, mRNA analysis, serum AFP analysis, and survival rate monitoring.

30. A method for inhibiting the expression of Eg5/KSP and VEGF in cells, comprising administering the composition of item 1 to the cells.

31. A method for preventing tumor growth, reducing tumor growth, or prolonging the survival of a mammal in need of treatment for cancer, comprising administering the composition of item 1 to the mammal.

32. The method according to claim 31, wherein the mammal has liver cancer.

33. The method according to item 31, wherein the mammal is a human suffering from liver cancer.

34. The method of claim 31, wherein a dose containing 0.25 mg/kg to 4 mg/kg of dsRNA is administered to the mammal.

35. The method of claim 31, wherein the dsRNA is administered to the human at about 0.01, 0.1, 0.5, 1.0, 2.5, or 5.0 mg/kg.

36. A method for reducing tumor growth in a mammal in need of treatment for cancer, said method comprising administering to said mammal the composition of claim 1, said method reducing tumor growth by at least 20%.

37. The method according to item 36, wherein the method reduces KSP expression by at least 60%.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is a graph showing the percentage of liver weight to body weight after administration of SNALP-siRNA to a Hep3B mouse model.

FIG2A is a graph showing the effect of PBS on body weight in a Hep3B mouse model.

FIG2B is a graph showing the effect of SNALP-siRNA (VEGF/KSP) on body weight in a Hep3B mouse model.

FIG2C is a graph showing the effect of SNALP-siRNA (KSP/luciferase) on body weight in the Hep3B mouse model.

FIG2D is a graph showing the effect of SNALP-siRNA (VEGF/luciferase) on body weight in the Hep3B mouse model.

FIG3 is a graph showing the effect of SNALP-siRNA on body weight in a Hep3B mouse model.

FIG4 is a graph showing body weights of untreated control animals.

FIG5 is a graph showing the effect of control luciferase-SNALP siRNA on body weight in a Hep3B mouse model.

FIG6 is a graph showing the effect of VSP-SNALP siRNA on body weight in a Hep3B mouse model.

FIG7A is a graph showing the effect of SNALP-siRNA on human GAPDH levels normalized to mouse GAPDH levels in the Hep3B mouse model.

FIG7B is a graph showing the effect of SNALP-siRNA on serum AFP levels measured by serum ELISA in the Hep3B mouse model.

FIG8 is a graph showing the effect of SNALP-siRNA on human GAPDH levels normalized to mouse GAPDH levels in the Hep3B mouse model.

FIG9 is a graph showing the effect of SNALP-siRNA on human KSP levels normalized to human GAPDH levels in the Hep3B mouse model.

FIG10 is a graph showing the effect of SNALP-siRNA on human VEGF levels normalized to human GAPDH levels in the Hep3B mouse model.

FIG. 11A is a graph showing the effect of SNALP-siRNA on mouse VEGF levels normalized to human GAPDH levels in the Hep3B mouse model.

FIG11B is a set of graphs showing the effects of SNALP-siRNA on human GAPDH levels and serum AFP levels in the Hep3B mouse model.

FIG12A is a graph showing the effects of PBS, luciferase, and ALN-VSP on tumor KSP in the Hep3B mouse model, where tumor KSP was determined as a percentage relative to hKSP mRNA.

Figure 12B is a graph showing the effects of PBS, luciferase, and SNALP-VSP on tumor VEGF in the Hep3B mouse model, where tumor VEGF was measured as a percentage relative to hVEGF mRNA.

Figure 12C is a graph showing the effects of PBS, luciferase, and SNALP-VSP on GAPDH levels in the Hep3B mouse model, where GAPDH levels were determined as a percentage relative to hGAPDH mRNA.

Figure 13A is a graph showing the effect of SNALP si-RNA on the survival rate of mice bearing liver tumors. Treatment was started 18 days after tumor cell inoculation.

Figure 13B is a graph showing the effect of SNALP siRNA on the survival rate of mice bearing liver tumors. Treatment was started 26 days after tumor cell inoculation.

FIG14 is a graph showing the effect of SNALP-siRNA on serum alpha-fetoprotein (AFP) levels.

Figure 15A is an image of an H&E-stained section from a tumor-bearing animal (three weeks after Hep3B cell implantation) administered 2 mg/kg of SNALP-VSP. Twenty-four hours later, the tumor-bearing liver lobe was processed for histological analysis. Arrows indicate single astrocytes.

Figure 15B is an image of an H&E-stained section of a tumor-bearing animal (three weeks after Hep3B cell implantation) administered 2 mg/kg SNALP-Luc. Twenty-four hours later, the tumor-bearing liver lobe was processed for histological analysis.

FIG16 is a graph showing the effect of administration of SNALP-formulated siRNA and sorafenib on survival rate.

FIG17 is a flow chart of the in-line mixing method.

FIG18 is a graph showing the effect of treatment with LNP-08 formulated VSP on the expression of KSP and VEGF in mouse intrahepatic Hep3B tumors.

Figure 19 illustrates the chemical structures of PEG-DSG and PEG-C-DSA.

Figure 20 illustrates the structures of cationic lipids ALNY-100, MC3, and XTC.

FIG21 is a graph showing the effects of VSP formulated with SNALP-1955(Luc), ALN-VSP02, SNALP-T-VSP LNP11, and LNP-12 on the expression of KSP and VEGF in mouse intrahepatic Hep3B tumors.

Figure 22 is a set of graphs comparing the effects of treatment with LNP08-Luc, ALN-VSP02, and VSP formulated with LNP-08 and LNP08-C18 on the expression of KSP and VEGF in mouse intrahepatic Hep3B tumors.

Detailed Description of the Invention

The present invention provides compositions and methods for inhibiting the expression of Eg5 and VEGF genes in cells or mammals using dsRNA. The dsRNA is encapsulated in lipid nucleic acid particles. The present invention also provides compositions and methods for treating pathological conditions and diseases in mammals caused by the expression of Eg5 and VEGF genes, such as liver cancer. The dsRNA controls the sequence-specific degradation of mRNA through a process known as RNA interference (RNAi).

The following detailed description discloses how to prepare and use compositions comprising dsRNA to inhibit the expression of the Eg5 gene and the VEGF gene, respectively, as well as compositions and methods for treating diseases and disorders (e.g., cancer) caused by the expression of these genes. The pharmaceutical compositions characterized by the present invention include dsRNA and a pharmaceutically acceptable carrier, wherein the dsRNA contains an antisense strand comprising a complementary region, wherein the complementary region is less than 30 nucleotides in length, typically 19-24 nucleotides in length, and the complementary region is substantially complementary to at least a portion of the RNA transcript of the Eg5 gene. The compositions characterized by the present invention also include dsRNA, wherein the dsRNA contains an antisense strand comprising a complementary region, wherein the complementary region is less than 30 nucleotides in length, typically 19-24 nucleotides in length, and the complementary region is substantially complementary to at least a portion of the RNA transcript of the VEGF gene.

Therefore, certain aspects of the present invention provide a pharmaceutical composition comprising Eg5 and VEGF dsRNA and a pharmaceutically acceptable carrier, a method of using the composition to inhibit the expression of the Eg5 gene and the VEGF gene, respectively, and a method of using the pharmaceutical composition to treat diseases caused by the expression of the Eg5 and VEGF genes.

I. Definition

For convenience, the meanings of certain terms and phrases used in the specification, examples, and appended claims are provided below. If there is an apparent discrepancy between the usage of a term in other parts of this specification and its definition provided in this section, the definitions in this section shall prevail.

"G", "C", "A" and "U" each generally represent a nucleotide comprising guanine, cytosine, adenine and uracil as bases, respectively. "T" and "dT" are used interchangeably herein to refer to a deoxyribonucleotide in which the nucleobase is thymine, e.g., deoxyribothymine. However, it will be understood that the term "ribonucleotide" or "nucleotide" may also refer to a modified nucleotide, or a surrogate alternative group, as described in detail below. Those skilled in the art will appreciate that guanine, cytosine, adenine and uracil may be replaced by other groups without substantially altering the base pairing properties of an oligonucleotide comprising a nucleotide bearing such an alternative group. For example, and without limitation, a nucleotide comprising inosine as its base may form base pairs with a nucleotide comprising adenine, cytosine or uracil. Thus, a nucleotide comprising uracil, guanine or adenine in a nucleotide sequence of the present invention may be replaced by, for example, a nucleotide comprising inosine. In another example, adenine and cytosine anywhere in the oligonucleotide can be replaced by guanine and uracil, respectively, thereby forming G-U wobble base pairing with the target mRNA. Sequences containing such replacement groups are embodiments of the present invention.

As used herein, "Eg5" refers to human kinesin family member 11, also known as KIF11, Eg5, HKSP, KSP, KNSL1, or TRIP5. The Eg5 sequence can be found as NCBI Gene ID: 3832, HGNC ID: HGNC: 6388, and Ref Seq ID number: NM_004523. The terms "Eg5," "KSP," and "Eg5/KSP" are used interchangeably.

As used herein, "VEGF", also known as vascular permeability factor, is a vascular growth factor. VEGF is a human dimeric 45kDa glycoprotein that exists in at least three different isoforms. VEGF isoforms are expressed in endothelial cells. The VEGF gene comprises 8 exons that express a 189 amino acid protein isoform. The 165 amino acid isoform lacks the residues encoded by exon 6, while the 121 amino acid isoform lacks the residues encoded by exons 6 and 7. VEGF145 is an isoform that is expected to contain 145 amino acids and lack exon 7. VEGF can act on endothelial cells by binding to endothelial tyrosine kinase receptors such as Flt-1 (VEGFR-1) or KDR/flk-1 (VEGFR-2). VEGFR-2 is expressed in endothelial cells and is associated with endothelial cell differentiation and angiogenesis. A third receptor, VEGFR-3, is involved in lymphogenesis.

Various isoforms have different biological activities and clinical significance. For example, VEGF145 induces angiogenesis, and like VEGF189 (but different from VEGF165), VEGF145 effectively binds to the extracellular matrix by a mechanism that is independent of the heparin sulfate associated with the extracellular matrix. VEGF shows activity as an endothelial cell mitogen and chemoattractant in vitro, and induces vascular permeability and angiogenesis in vivo. VEGF is secreted by a variety of cancer cell types and promotes tumor growth by inducing the growth of the vasculature associated with tumors. It has been shown that inhibiting VEGF function can limit the growth of primary experimental tumors and the incidence of metastasis in mice with impaired immune capacity. Relating to a variety of dsRNAs for VEGF is described in co-pending U.S. serial numbers 11/078,073 and 11/340,080, which are incorporated herein by reference in their entirety.

As used herein, "target sequence" means a contiguous portion of the nucleotide sequence of an mRNA molecule formed during transcription of the Eg5/KSP and/or VEGF gene, including mRNAs that are products of RNA processing of the primary transcript.

As used herein, the term "strand comprising a sequence" means an oligonucleotide containing a stretch of nucleotides described by the sequence referred to using standard nucleotide nomenclature.

As used herein, unless otherwise indicated, the term "complementary" when used to describe the relationship between a first nucleotide sequence and a second nucleotide sequence means the ability of an oligonucleotide or polynucleotide comprising the first nucleotide sequence to hybridize and form a duplex structure with an oligonucleotide or polynucleotide comprising the second nucleotide sequence under certain conditions, as understood by those skilled in the art. For example, such conditions can be stringent conditions, wherein stringent conditions can include: 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, hybridization at 50°C or 70°C for 12-16 hours, followed by washing. Other conditions can be used, such as physiologically relevant conditions that may be encountered in an organism. Depending on the ultimate application of the hybridizing nucleotides, those skilled in the art will be able to determine the set of conditions that are most suitable for testing the complementarity of two sequences.

The term "complementary" includes base pairing of an oligonucleotide or polynucleotide comprising a first nucleotide sequence with an oligonucleotide or polynucleotide comprising a second nucleotide sequence over the entire length of the first and second nucleotide sequences. Such sequences may be referred to as "fully complementary" relative to one another in the present invention. However, when a first sequence is referred to as "substantially complementary" relative to a second sequence of the present invention, the two sequences may be fully complementary, or they may form one or more, but generally no more than 4, 3, or 2, mismatched base pairs upon hybridization while maintaining the ability to hybridize under conditions most relevant to their ultimate application. However, when two oligonucleotides are designed to form one or more single-stranded overhangs upon hybridization, such overhangs will not be considered mismatches for purposes of determining complementarity. For example, a dsRNA comprising one oligonucleotide of 21 nucleotides in length and another oligonucleotide of 23 nucleotides in length may still be referred to as "fully complementary" for purposes of the present invention when the longer oligonucleotide comprises a 21-nucleotide sequence that is fully complementary to the shorter oligonucleotide.

As used herein, the term "complementary" sequence may also include non-Watson-Crick base pairs and/or base pairs formed by non-natural and modified nucleotides, or may be formed entirely of non-Watson-Crick base pairs and/or base pairs formed by non-natural and modified nucleotides, as long as the above requirements regarding their hybridization ability are met. Such non-Watson-Crick base pairs include, but are not limited to, G:U Wobble or Hoogstein base pairs.

The terms "complementary," "fully complementary," and "substantially complementary" herein may be used with respect to base pairing between the sense and antisense strands of a dsRNA or between the antisense strand of a dsRNA and a target sequence, as will be understood from the context of their use.

As used herein, a polynucleotide that is "substantially complementary to at least a portion of a messenger RNA (mRNA)" refers to a polynucleotide that is substantially complementary to a contiguous portion of a messenger RNA of interest (e.g., encoding Eg5/KSP and/or VEGF), including the 5' untranslated region (UTR), the open reading frame (ORF), or the 3' UTR. For example, a polynucleotide is complementary to at least a portion of an Eg5 mRNA if the sequence is substantially complementary to a non-interrupted portion of an mRNA encoding Eg5.

As used herein, the term "double-stranded RNA" or "dsRNA" refers to a duplex structure containing two antiparallel and substantially complementary nucleic acid chains as defined above. Typically, the majority of the nucleotides in each chain are ribonucleotides, but as described in detail herein, each chain or both chains may also include at least one non-ribonucleotide, such as a deoxyribonucleotide and/or a modified nucleotide. In addition, as used herein, "dsRNA" may include chemical modifications of ribonucleotides, including substantial modifications at multiple nucleotides and including all types of modifications disclosed herein or known in the art. For the purposes of this specification and claims, any such modification (as used in siRNA-type molecules) is covered by "dsRNA."

The two chains forming the duplex structure can be different parts of a larger RNA molecule, or they can be separate RNA molecules. When the two chains are part of a larger molecule, and therefore connected by a series of continuous nucleotides between the 3' end of a chain and the 5' end of the corresponding other chain forming the duplex structure, the chain connecting the RNA is called a "hairpin loop". When the two chains are covalently linked by a series of continuous nucleotides between the 3' end of a chain and the 5' end of the corresponding other chain forming the duplex structure, the connection structure is called a "connector". The RNA chain can have the same or different numbers of nucleotides. The maximum number of base pairs is the number of nucleotides of the shortest chain of the dsRNA minus any overhangs present in the duplex. In addition to the duplex structure, the dsRNA can include one or more nucleotide overhangs. Generally, the majority of the nucleotides of each chain are ribonucleotides, but as described in detail in the present invention, each chain or two chains can also include at least one non-ribonucleotide, such as deoxyribonucleotides and/or modified nucleotides. In addition, as used in this specification, "dsRNA" can include chemical modifications of ribonucleotides, including substantial modifications at multiple nucleotides and including all types of modifications disclosed herein or known in the art. For the purposes of this specification and claims, any such modification (such as for siRNA-like molecules) is covered by "dsRNA."

As used herein, "nucleotide overhang" refers to one or more unpaired nucleotides that protrude from the duplex structure of a dsRNA when the 3' end of one strand of the dsRNA extends beyond the 5' end of the other strand (or vice versa). "Blunt" or "blunt-ended" means that there are no unpaired nucleotides at that end of the dsRNA, i.e., no nucleotide overhang. A "blunt-ended" dsRNA is a dsRNA that is double-stranded throughout its entire length, i.e., there are no nucleotide overhangs at either end of the molecule. In some embodiments, the dsRNA may have a nucleotide overhang at one end of the duplex and a blunt end at the other end.

The term "antisense strand" means a dsRNA strand that includes a region that is substantially complementary to a target sequence. As used herein, the term "complementary region" means a region on the antisense strand that is substantially complementary to a sequence (e.g., a target sequence as defined herein). When the complementary region and the target sequence are not completely complementary, mismatches may occur in the interior or terminal regions of the molecule. Typically, most tolerated mismatches are located in the terminal regions, e.g., within 6, 5, 4, 3, or 2 nucleotides of the 5' and/or 3' ends.

As used herein, the term "sense strand" means the dsRNA strand that includes a region that is substantially complementary to a region of the antisense strand.

When referring to dsRNA, "introducing into a cell" means facilitating uptake or absorption into the cell, as understood by those skilled in the art. The absorption or uptake of dsRNA can occur by unassisted diffusion or active cellular processes or by adjuvants or devices. The meaning of this term is not limited to cells in vitro. dsRNA can also be "introduced into a cell," wherein the cell is part of a living organism. In this case, introducing into a cell will include delivery to the organism. For example, for in vivo delivery, the dsRNA can be injected into a tissue site or administered systemically. In vitro introduction into cells includes methods known in the art, such as electroporation and lipofection.

The terms "silencing" and "inhibiting expression," "downregulating expression," "preventing expression," and the like, as they relate to the Eg5 and/or VEGF genes, refer to at least partial inhibition of expression of the Eg5 gene, as manifested by a decrease in the amount of Eg5 mRNA and/or VEGF mRNA, which can be isolated from a first cell or group of cells in which the Eg5 and/or VEGF gene is transcribed, and which has been treated such that expression of the Eg5 and/or VEGF gene is inhibited compared to a second cell or group of cells that is substantially identical to the first cell or group of cells but has not been so treated (control cells). The degree of inhibition is generally represented by the following formula:

Alternatively, the degree of inhibition can be determined based on a reduction in a parameter functionally related to Eg5 and/or VEGF gene expression, for example, the amount of protein encoded by the Eg5 and/or VEGF genes produced by cells, or the number of cells displaying a certain phenotype, such as apoptosis. In principle, target gene silencing can be determined in any cell expressing the target (constitutively or by genetic engineering) and by any appropriate assay. However, where a reference is needed, the assays provided in the following examples will serve as such a reference in order to determine whether a given dsRNA inhibits Eg5 gene expression to a certain extent and is therefore included in the present invention.

For example, in some cases, the expression of the Eg5 gene (or VEGF gene) is inhibited by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% or 50% by administering a double-stranded oligonucleotide of the present invention. In some embodiments, the expression of the Eg5 and/or VEGF gene is inhibited by at least about 60%, 70% or 80% by administering a double-stranded oligonucleotide of the present invention. In other embodiments, the expression of the Eg5 and/or VEGF gene is inhibited by at least about 85%, 90% or 95% by administering a double-stranded oligonucleotide of the present invention. The following tables and examples provide expression inhibition values for various Eg5 and/or VEGF dsRNA molecules using various concentrations.

As used herein, in the context of Eg5 expression (or VEGF expression), the terms "treat (verb)", "treating (noun)" and the like refer to alleviating or slowing down a pathological process mediated by Eg5 and/or VEGF expression. In the context of the present invention, to the extent it relates to any of the other conditions described below (other than a pathological process mediated by Eg5 and/or VEGF expression), the terms "treat (verb)", "treating (noun)" and the like refer to alleviating or slowing down at least one symptom associated with such a condition, or delaying or reversing the progression of such a condition, for example, delaying the progression of liver cancer.

As used herein, the phrases "therapeutically effective amount" and "prophylactically effective amount" mean an amount that provides a therapeutic benefit in treating, preventing, or controlling a pathological process mediated by Eg5 and/or VEGF expression, or a significant symptom mediated by Eg5 and/or VEGF expression. The specific therapeutically effective amount can be readily determined by an ordinary practitioner and may vary depending on factors known in the art, such as the type of pathological process mediated by Eg5 and/or VEGF expression, the patient's medical history and age, the stage of the pathological process mediated by Eg5 and/or VEGF expression, and the administration of other agents that counteract the pathological process mediated by Eg5 and/or VEGF expression.

As used herein, a "pharmaceutical composition" comprises a pharmacologically effective amount of a dsRNA and a pharmaceutically acceptable carrier. As used herein, a "pharmacologically effective amount," "therapeutically effective amount," or simply "effective amount" refers to an amount of RNA effective to produce a desired pharmacological, therapeutic, or preventative result. For example, if a given clinical treatment is considered effective when a measurable parameter associated with a disease or condition is reduced by at least 25%, then the therapeutically effective amount of the drug for treating that disease or condition is the amount necessary to cause a reduction in that parameter by at least 25%.

The term "pharmaceutically acceptable carrier" refers to a carrier for administering a therapeutic agent. As described in more detail below, such carriers include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. The term specifically excludes cell culture media. For orally administered drugs, pharmaceutically acceptable carriers include, but are not limited to, pharmaceutically acceptable excipients such as inert diluents, disintegrants, binders, lubricants, sweeteners, flavorings, colorants, and preservatives. Suitable inert diluents include sodium carbonate and calcium carbonate, sodium phosphate and calcium phosphate, and lactose, while corn starch and alginic acid are suitable disintegrants. Binders can include starch and gelatin, while lubricants (if present) are typically magnesium stearate, stearic acid, or talc. If desired, the tablet can be coated with materials such as glyceryl monostearate or glyceryl distearate to delay absorption in the gastrointestinal tract.

As used herein, a "transformed cell" is a cell into which a vector has been introduced, and the cell is capable of expressing a dsRNA molecule from the vector.

II. Double-stranded RNA (dsRNA)

As described in more detail herein, the present invention provides double-stranded ribonucleic acid (dsRNA) molecules for inhibiting the expression of Eg5 and/or VEGF genes in cells or mammals, wherein the dsRNA comprises an antisense strand comprising a complementary region that is complementary to at least a portion of an mRNA formed during the expression of the Eg5 and/or VEGF gene, wherein the complementary region is less than 30 nucleotides in length, typically 19-24 nucleotides in length, and wherein upon contact with a cell expressing the Eg5 and/or VEGF gene, the dsRNA inhibits the expression of the Eg5 and/or VEGF gene. The dsRNA of the present invention may also comprise one or more single-stranded nucleotide overhangs.

The dsRNA can be synthesized by standard methods known in the art as described below, for example, by utilizing, for example, an automated DNA synthesizer commercially available from Biosearch, Applied Biosystems, Inc. The dsRNA comprises two fully complementary chains to hybridize to form a duplex structure. One chain (the antisense strand) of the dsRNA comprises a complementary region substantially complementary to the target sequence of the mRNA sequence formed during the expression of the Eg5 and/or VEGF gene, typically fully complementary, and the other chain (the sense strand) comprises a region complementary to the antisense strand such that, when combined under suitable conditions, the two chains hybridize to form a duplex structure. Typically, the length of the duplex is 15 to 30, or 25 to 30, or 18 to 25, or 19 to 24, or 19 to 21, or 19, 20, or 21 base pairs. In one embodiment, the length of the duplex is 19 base pairs. In another embodiment, the length of the duplex is 21 base pairs. When two different siRNAs are used in combination, the duplex lengths can be the same or different.

The length of each strand of the dsRNA of the present invention is typically 15 to 30, or 18 to 25, or 18, 19, 20, 21, 22, 23 or 24 nucleotides. In other embodiments, each strand is 25-30 base pairs in length. The lengths of the strands of the duplex can be the same or different. When two different siRNAs are used in combination, the lengths of the strands of each siRNA can be the same or different. For example, a composition can contain a dsRNA targeting Eg5 having a sense strand of 21 nucleotides and an antisense strand of 21 nucleotides, and a second dsRNA targeting VEGF having a sense strand of 21 nucleotides and an antisense strand of 23 nucleotides.

The dsRNA of the present invention may comprise one or more single-stranded overhangs of one or more nucleotides. In one embodiment, at least one end of the dsRNA has a single-stranded nucleotide overhang of 1 to 4, typically 1 or 2 nucleotides. In another embodiment, the antisense strand of the dsRNA has an overhang of 1-10 nucleotides, each located at the 3' end and the 5' end of the sense strand. In other embodiments, the sense strand of the dsRNA has an overhang of 1-10 nucleotides, each located at the 3' end and the 5' end of the antisense strand.

Surprisingly, the inhibitory properties of dsRNAs with at least one nucleotide overhang may be superior to those of their blunt-ended counterparts. In some embodiments, the presence of only one nucleotide overhang strengthens the interference activity of the dsRNA without affecting its overall stability. It has been confirmed that dsRNAs with only one overhang are particularly stable and effective in vivo and in various cells, cell culture media, blood, and serum. Typically, the single-stranded overhang is located at the 3' end of the antisense strand, or, at the 3' end of the sense strand. The dsRNA may also have a blunt end that is typically located at the 5' end of the antisense strand. This dsRNA can have improved stability and inhibitory activity, and therefore allows for administration at low doses, i.e., less than 5mg/kg recipient body weight/day. Typically, the antisense strand of the dsRNA has a nucleotide overhang located at the 3' end, and the 5' end is a blunt end. In another embodiment, one or more nucleotides in the overhang are replaced by nucleoside thiophosphates.

As described in more detail herein, the compositions of the present invention comprise a first dsRNA targeting Eg5 and a second dsRNA targeting VEGF. The first and second dsRNAs can have identical overhang configurations, e.g., the number of nucleotide overhangs on each strand, or the dsRNAs can have different specific configurations. In one embodiment, the first dsRNA targeting Eg5 comprises a 2-nucleotide overhang at the 3' end of each strand, and the second dsRNA targeting VEGF comprises a 2-nucleotide overhang at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand (e.g., the 3' end of the sense strand).

In one embodiment, the Eg5 gene targeted by the dsRNA of the present invention is the human Eg5 gene. In one embodiment, the antisense strand of the dsRNA targeting Eg5 comprises at least 15 contiguous nucleotides of one of the antisense sequences in Tables 1-3. In a specific embodiment, the first sequence of the dsRNA is selected from one of the sense strands in Tables 1-3, and the second sequence is selected from the antisense sequences in Tables 1-3. Optional antisense agents targeting other parts of the target sequence provided in Tables 1-3 can be easily determined using the target sequence and flanking Eg5 sequences. In some embodiments, the dsRNA targeting Eg5 will comprise at least two nucleotide sequences selected from the sequences provided in Tables 1-3. One of the two sequences is complementary to the other of the two sequences, and one of the sequences is substantially complementary to the mRNA sequence produced upon expression of the Eg5 gene. Similarly, the dsRNA will comprise two oligonucleotides, one of which is described as the sense strand in Tables 1-3, and the second oligonucleotide is described as the antisense strand in Tables 1-3.

In embodiments where a second dsRNA targeting VEGF is used, such agents are described in the Examples, Tables 4a and 4b, and in co-pending U.S. Patent Serial Nos. 11/078,073 and 11/340,080, incorporated herein by reference. In one embodiment, the dsRNA targeting VEGF has an antisense strand that is complementary to at least 15 contiguous nucleotides of a VEGF target sequence described in Table 4a. In other embodiments, the dsRNA targeting VEGF comprises one of the antisense sequences of Table 4b, one of the sense sequences of Table 4b, or one of the duplexes (sense and antisense strands) of Table 4b.

It is well known to those skilled in the art that dsRNAs containing duplex structures of 20 to 23, and particularly 21, base pairs have been shown to be particularly effective in inducing RNA interference (Elbashir et al., EMBO 2001, 20: 6877-6888). However, it has also been found that shorter or longer dsRNAs may also be effective. In the embodiments described above, by virtue of the properties of the oligonucleotide sequences provided in Tables 1-3, the dsRNAs of the present invention may comprise at least one strand of at least 21 nt in length. It is reasonable to expect that shorter dsRNAs containing one of the sequences of Tables 1-3 minus only a few nucleotides at one or both ends may be similarly effective compared to the above dsRNAs. Thus, the present invention relates to dsRNAs containing a partial sequence of at least 15, 16, 17, 18, 19, 20, or more contiguous nucleotides from one of the sequences of Tables 1-3, and having an ability to inhibit Eg5 gene expression in a FACS assay as described below that differs by no more than 5, 10, 15, 20, 25, or 30% compared to a dsRNA containing the full sequence. In addition, dsRNAs that cleave the target sequences provided in Tables 1 to 3 can be readily prepared using the Eg5 sequence and the provided target sequences. Other dsRNAs targeting VEGF can be designed in a similar manner using the sequences disclosed in Tables 4a and 4b, the Examples, and co-pending U.S. Serial Nos. 11/078,073 and 11/340,080 (incorporated herein by reference).

Furthermore, the RNAi agents provided in Tables 1-3 identify sites within the Eg5 mRNA susceptible to RNAi-based cleavage. Thus, the present invention also encompasses RNAi agents, e.g., dsRNAs, that target within a sequence targeted by one of the agents of the present invention. As used herein, a second RNAi agent is said to target within the sequence of a first RNAi agent if it cleaves the message anywhere within the mRNA that is complementary to the antisense strand of the first RNAi agent. Such a second agent typically consists of at least 15 contiguous nucleotides from one of the sequences provided in Tables 1-3, linked to additional nucleotide sequences from a region adjacent to a selected sequence in the Eg5 gene. For example, the last 15 nucleotides of SEQ ID NO: 1 linked to the next 6 nucleotides from the target Eg5 gene form a 21-nucleotide single-stranded agent based on one of the sequences provided in Tables 1-3. Other RNAi agents, for example, dsRNA targeting VEGF can be designed in a similar manner using the sequences disclosed in Tables 4a and 4b, the Examples, and co-pending U.S. Serial Nos. 11/078,073 and 11/340,080 (incorporated herein by reference).

The dsRNA of the present invention may contain one or more mismatches with the target sequence. In a preferred embodiment, the dsRNA of the present invention contains no more than 3 mismatches. If the antisense strand of the dsRNA contains a mismatch with the target sequence, it is preferred that the mismatch region is not located in the center of the complementary region. If the antisense strand of the dsRNA contains a mismatch with the target sequence, it is preferred that the mismatch is limited to 5 nucleotides from either end, for example, 5, 4, 3, 2 or 1 nucleotides from the 5' or 3' end of the complementary region. For example, for a 23-nucleotide dsRNA chain complementary to the Eg5 gene region, the dsRNA generally does not contain any mismatch within the central 13 nucleotides. The methods described in the present invention can be used to determine whether a dsRNA containing a mismatch with the target sequence can effectively inhibit the expression of the Eg5 gene. It is important to consider the effectiveness of dsRNAs with mismatches in inhibiting Eg5 gene expression, particularly if it is known that a specific complementary region in the Eg5 gene within a population has polymorphic sequence variations.

Modification

In another embodiment, the dsRNA is chemically modified to improve stability. Can be synthesized and/or modified nucleic acid of the present invention by the method that has been used for a long time in this area, for example " Current protocols in nucleic acid chemistry, " Beaucage, S.L. etc. (Edrs.), John Wiley & Sons, Inc., New York, NY, the method described in USA, this document is incorporated herein by reference. The specific example of the preferred dsRNA compound for the present invention includes containing modified backbone or not containing dsRNA of natural internucleoside bond. As defined in this specification, the dsRNA containing modified backbone includes those that retain phosphorus atom in the backbone and those that do not contain phosphorus atom in the backbone. For the purpose of this specification, and once cited in this area, the modified dsRNA that does not contain phosphorus atom in its internucleoside backbone can also be considered as oligonucleoside.

Preferred modified dsRNA backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters; methyl and other alkyl phosphates, including 3'-alkylene phosphates and chiral phosphates, phosphinates; phosphoramidates, including 3'-aminophosphoramidates and aminoalkylphosphoramidates with normal 3'-5' linkages, thionylphosphoramidates, thionylalkylphosphates, thionylalkylphosphotriesters and boranophosphates, 2'-5' linked analogs of these substances, and those with reversed polarity, wherein adjacent nucleoside unit pairs are 3'-5' to 5'-3' or 2'-5' to 5'-2' linked. Various salts, mixed salts and free acid forms are also included.

Typical U.S. patents that teach the preparation of the above-mentioned phosphorus-containing linkages include, but are not limited to, U.S. Patent Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,195; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399, 676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,316; 5,550,111; 5,563,253; 5,571,799; 5,587,361; and 5,625,050, each of which is incorporated herein by reference.

Preferred modified dsRNA backbones that do not include phosphorus atoms have backbones formed from short alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatom or heterocyclic internucleoside linkages. These backbones include those with morpholino linkages (formed in part by the sugar portion of the nucleoside); siloxane backbones; sulfide, sulfoxide, and sulfone backbones; formyl and thioformyl backbones; methyleneformyl and thioformyl backbones; olefin-containing backbones; sulfamate backbones; methyleneimino and methylenehydrazine backbones; sulfonate and sulfamoyl backbones; amide backbones; and other backbones with mixed N, O, S, and CH2 components.

Typical U.S. patents that teach the preparation of the above oligonucleosides include, but are not limited to, U.S. Patent Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,64,562; and 5,677,439, each of which is hereby incorporated by reference.

In other preferred dsRNA mimics, both the sugar and the internucleoside linkage of the nucleotide unit, i.e., the backbone, are replaced by new groups. The base unit is retained for hybridization with an appropriate nucleic acid target compound. One such oligomeric compound, a dsRNA mimic that has been shown to have excellent hybridization properties, is called peptide nucleic acid (PNA). In PNA compounds, the sugar backbone of dsRNA is replaced by an amide containing backbone, particularly an aminoethylglycine backbone. The nucleobases are retained and are bound directly or indirectly to the nitrogen atoms of the amide portion of the backbone. Typical U.S. patents that teach the preparation of PNA compounds include, but are not limited to, U.S. Patent Nos. 5,539,082; 5,714,331; and 5,719,262, each of which is incorporated herein by reference. Further teachings of PNA compounds can be found in Nielsen et al., Science, 1991, 254, 1497-1500.

The most preferred embodiments of the present invention are dsRNAs having phosphorothioate backbones and oligonucleosides having heteroatom backbones, particularly _--CH2 -NH-- _CH2-- , --CH2--N(CH3)--O-- _CH2-- [referred to as a methylene(methylimino) or MMI backbone], _--CH2 --O-- _N ( _CH3 )--CH2--, --CH2--N( _CH3 )--N( _CH3 )-- _CH2-- , and --N( _CH3 )-- _CH2 -- _CH2-- [wherein the natural phosphodiester backbone is represented as --O--P--O-- _CH2-- ] _{of the aforementioned} U.S. Patent No. _5,489,677 , and the amide backbone of the aforementioned U.S. Patent No. 5,602,240. dsRNAs having a morpholino backbone structure of the aforementioned U.S. Patent No. 5,034,506 are also preferred.

Modified dsRNAs can also include one or more substituted sugar moieties. Preferred dsRNAs include one of the following at the 2' position: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S-, or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl, and alkynyl groups can be substituted or unsubstituted C ₁ to C ₁₀ alkyl or C ₂ to C ₁₀ alkenyl and alkynyl groups. Particularly preferred are O[(CH ₂ ) _n O] _m CH ₃ , O(CH ₂ ) _n OCH ₃ , O(CH ₂ ) _n NH ₂ , O(CH ₂ ) _n CH ₃ , O(CH ₂ ) _n ONH ₂ , and O(CH ₂ ) _n ON[(CH ₂ ) _n CH ₃ )] ₂ , wherein n and m are from 1 to about 10. Other preferred dsRNAs include one of the following at the 2' position: _C1 to _C10 lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl _, SH, _SCH3 , OCN, Cl, Br, CN, CF3, _OCF3 , _SOCH3 , _SO2CH3 , _ONO2 , _NO2 , _N3 , _NH2 , heterocycloalkyl, heterocycloalkaryl _, aminoalkylamino, polyalkylamino, substituted silyl, RNA cleavage group, reporter group, intercalating group, group for improving the pharmacokinetic properties of dsRNA or group for improving the pharmacodynamic properties of dsRNA, and other substituents with similar properties. Preferred modifications include 2'-methoxyethoxy (2'-O-- _CH2CH2OCH3 , also known _as 2'-O-(2-methoxyethyl) or 2'-MOE) (Martin et al., Helv. Chim _. Acta, 1995, 78, 486-504), i.e., an alkoxy-alkoxy group. Other preferred modifications include 2'-dimethylaminooxyethoxy, i.e., an O( _CH2 ) _2ON ( _CH3 ) ₂ group, also known as 2'-DMAOE, as described in the Examples below, and 2'-dimethylaminoethoxyethoxy (also known in the art as 2'-O-dimethylaminoethoxyethyl or 2'-DMAEOE), i.e., 2'-O-- _CH2 --O-- _CH2 --N( _CH2 ) ₂ , also described in the Examples below.

Other preferred modifications include 2'-methoxy (2'-OCH ₃ ), 2'-aminopropoxy (2'-OCH ₂ CH ₂ CH ₂ NH ₂ ), and 2'-fluoro (2'-F). Similar modifications can also be made at other positions in the dsRNA, particularly the 3' position of the sugar on the 3' terminal nucleotide or the 5' position of the 5' terminal nucleotide in 2'-5' linked dsRNAs. dsRNAs can also use sugar mimetics, such as cyclobutyl moieties, in place of the pentofuranosyl sugar. Representative U.S. patents that teach such modified sugar structures include, but are not limited to, U.S. Patent Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633; and 5,700,920, some of which are commonly owned by the present application and each of which is incorporated herein by reference in its entirety.

dsRNA can also include modifications or substitutions of nucleobases (commonly referred to in the art as "bases"). As used herein, "unmodified" or "natural" nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C), and uracil (U). Modified nucleobases include other synthetic and natural nucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propyne uracil and cytosine, 6-azouracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-sulfanyl, 8-hydroxy and other 8-substituted adenine and guanine, 5-halouracil and cytosine, 5-propyne uracil and cytosine, 6-azouracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-sulfanyl, 8-hydroxy and other 8-substituted adenine and guanine, 5-halouracil and cytosine, 5- substituted, in particular 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and 3-deazaadenine. Other nucleobases include those disclosed in U.S. Patent No. 3,687,808, those disclosed in The Concise Encyclopedia Of Polymer Science And Engineering, pp. 858-859, Kroschwitz, J.L, ed. John Wiley & Sons, 1990, those disclosed in Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613, and those disclosed in Sanghvi, Y S., Chapter 15, DsRNA Research and Applications, pp. 289-302, Crooke, S.T. and Lebleu, B., Ed., CRC Press, 1993. Certain of these nucleobases are particularly useful for increasing the binding affinity of the oligomeric compounds of the invention. These nucleobases include 5-substituted pyrimidines, 6-azapyrimidines, and N-2, N-6, and O-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil, and 5-propynylcytosine. 5-Methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2 degrees Celsius (Sanghvi, Y.S., Crooke, S.T. and Lebleu, B., Eds., DsRNA Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278), and are currently the preferred base substitutions, especially when combined with a 2'-O-methoxyethyl sugar modification.

Representative U.S. patents that teach the preparation of some of the above-referenced modified nucleobases, as well as other modified nucleobases, include, but are not limited to, the above-referenced U.S. Patent No. 3,687,808 and U.S. Patent Nos. 4,845,205; 5,130,30; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121, 5,596,091; 5,614,617; and 5,681,941, each of which is incorporated herein by reference, and U.S. Patent No. 5,750,692, which is also incorporated herein by reference.

Conjugate

Another modification of the dsRNA of the invention includes chemically attaching to the dsRNA one or more moieties or conjugates that enhance the activity, cellular distribution, or cellular uptake of the dsRNA. Such moieties include, but are not limited to, lipid moieties, such as a cholesterol moiety (Letsinger et al., Proc. Natl. Acid. Sci. USA, 199, 86, 6553-6556), cholic acid (Manoharan et al., Biorg. Med. Chem. Let., 1994 4 1053-1060); thioethers, such as beryl-S-triphenylmethanethiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660, 306-309; Manoharan et al., Biorg. Med. Chem. Let., 1993, 3, 2765-2770), thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20, 533-538); aliphatic chains, such as dodecanediol or undecyl residues (Saison-Behmoaras et al., EMBO J, 1991, 10, 1111-1118; Kabanov et al., FEBS Lett., 1990, 259, 327-330; Svinarchuk et al., Biochimie, 1993, 75, 49-54); phospholipids, such as di-hexadecyl-rac-glycerol or triethyl-ammonium 1,2-di-O-hexadecyl-rac-glycerotrioxy-3-H phosphate (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al., Nucl. Acids Res., 1990, 18, 3777-3783); polyamine or polyethylene glycol chains (Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969-973), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264, 229-237), or an octadecylamine or hexylamine-carbonyl oxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277, 923-937).

Representative U.S. patents that teach the preparation of these dsRNA conjugates include, but are not limited to, U.S. Patent Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717, 5,580,731; 5,591,584; 5,109,124; 5,118,802; ,138,045;5,414,077;5,486,603;5,512,439;5,578,718;5,608,046;4,587,044;4,605,735;4,667,025;4,762,779;4,789,737;4,824,941;4,835,263;4,876,335;4,904,582;4,958,01 3; 5,082,830; 5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098; 5,371,241, 5,391,723; 5,416 ,203, 5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941, each of which is incorporated herein by reference.

It is not necessary to uniformly modify all positions of a given compound; in fact, more than one of the above modifications can be combined in a single compound, or even a single nucleoside within a dsRNA. The present invention also includes dsRNA compounds that are chimeric compounds. In the context of the present invention, a "chimeric" dsRNA compound or "chimera" is a dsRNA compound, particularly a dsRNA, comprising two or more chemically distinct regions, each composed of at least one monomeric unit (i.e., a nucleotide in the case of a dsRNA compound). These dsRNAs typically include at least one region in which the dsRNA is modified to confer increased resistance to nuclease degradation, increased cellular uptake, and/or increased binding affinity to the target nucleic acid. Other regions of the dsRNA can serve as substrates for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. For example, RNase H is a cellular endonuclease that cleaves the RNA strand of an RNA:DNA duplex. Therefore, activation of RNase H results in cleavage of the RNA target, thereby greatly enhancing the effectiveness of the dsRNA in inhibiting gene expression. Thus, when chimeric dsRNAs are used, similar results can generally be achieved with shorter dsRNAs compared to phosphorothioate deoxy dsRNAs that hybridize to the same target region. Cleavage of the RNA target can be routinely monitored by gel electrophoresis and, if necessary, related nucleic acid hybridization techniques known in the art.

In some cases, the dsRNA can be modified with non-ligand groups. Many non-ligand molecules have been conjugated to dsRNA to enhance the activity, cellular distribution or cellular uptake of the dsRNA, and procedures for performing such conjugations are available in the scientific literature. Such non-ligand moieties include lipid moieties, such as cholesterol (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86:6553), cholic acid (Manoharan et al., Bioorg. Med. Chem. Lett., 1994, 4:1053); thioethers, such as hexyl-S-triphenylmethanethiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660:306; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3:2765), thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20:533); aliphatic chains, such as dodecanediol or undecyl residues (Saison-Behmoaras et al., EMBO J., 1991, 10:111; Kabanov et al., FEBS Lett., 1990, 259:327; Svinarchuk et al., Biochimie, 1993, 75:49); phospholipids, such as di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycerotrioxy-3-H-phosphate (Manoharan et al., Tetrahedron Lett., 1995, 36:3651; Shea et al., Nucl. Acids Res., 1990, 18:3777); polyamine or polyethylene glycol chains (Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969), or adamantane acetic acid (Manoharan et al., Tetrahedron The conjugation reaction can be carried out with a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264: 229), or an octadecylamine or hexylamine-carbonyl hydroxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277: 923). Representative U.S. patents teaching the preparation of such dsRNA conjugates are listed above. A typical conjugation protocol involves synthesizing a dsRNA with an amino linker at one or more positions in the sequence. The amino group is then reacted with a molecule conjugated with an appropriate coupling agent or activator. The conjugation reaction can be performed with the dsRNA still bound to a solid support or after cleavage of the dsRNA in solution. Purification of the dsRNA conjugate by HPLC typically yields the pure conjugate.

In some cases, the ligand can be multifunctional and/or the dsRNA can be conjugated to more than one ligand. For example, a dsRNA can be conjugated to one ligand to enhance uptake and to a second ligand to improve release.

Vector-encoded siRNA reagents

In another aspect of the invention, Eg5 and VEGF-specific dsRNA molecules expressed from transcriptional units are inserted into DNA or RNA vectors (see, for example, Couture, A., et al., TIG. (1996), 12: 5-10; Skillern, A., et al., International PCT Publication No. WO 00/22113, Conrad, International PCT Publication No. WO 00/22114, and Conrad, U.S. Pat. No. 6,054,299). These transgenes can be introduced as linear constructs, circular plasmids, or viral vectors that can be incorporated and inherited as transgenes integrated into the host genome. Transgenes can also be constructed so that they are inherited as extrachromosomal plasmids (Gassmann, et al., Proc. Natl. Acad. Sci. USA (1995) 92: 1292).

The individual strands of the dsRNA can be transcribed by promoters located on two separate expression vectors and co-transfected into target cells. Alternatively, the individual strands of the dsRNA can be transcribed by promoters located on the same expression plasmid. In a preferred embodiment, the dsRNA can be expressed as inverted repeats connected by a linker polynucleotide sequence so that the dsRNA has a stem and loop structure.

Recombinant dsRNA expression vectors are typically DNA plasmids or viral vectors. Viral vectors expressing dsRNA can be constructed based on, but not limited to, the following viruses: adeno-associated virus (for review, see Muzyczka, et al., Curr. Topics Micro. Immunol. (1992) 158: 97-129); adenovirus (for example, see Berkner, et al., BioTechniques (1998) 6: 616), Rosenfeld et al. (1991, Science 252: 431-434, and Rosenfeld et al. (1992), Cell 68: 143-155); or alphavirus and other viruses known in the art. Retroviruses have been used to introduce a variety of genes into many different cell types in vitro and/or in vivo, including epithelial cells (see, e.g., Eglitis, et al., Science (1985) 230:1395-1398; Danos and Mulligan, Proc. Natl. Acad. Sci. USA (1998) 85:6460-6464; Wilson et al., 1988, Proc. Natl. Acad. Sci. USA 85:3014-3018; Armentano et al., 1990, Proc. Natl. Acad. Sci. USA 87:6141-6145; Huber et al., 1991, Proc. Natl. Acad. Sci. USA 88:8039-8043; Ferry et al., 1991, Proc. Natl. Acad. Sci. USA 88:8377-8381; Chowdhury et al., 1991, Science 254:1802-1805; van Beusechem. et al., 1992, Proc. Natl. Acad. Sci. USA 89:7640-19; Kay et al., 1992, Human Gene Therapy 3:641-647; Dai et al., 1992, Proc. Natl. Acad. Sci. USA 89:10892-10895; Hwu et al., 1993, J. Immunol. 150:4104-4115; U.S. Patent No. 4,868,116; U.S. Patent No. 4,980,286; PCT Application WO 89/07136; PCT Application WO 89/02468; PCT Application WO 89/05345; and PCT application WO 92/07573). Recombinant retroviral vectors capable of transducing and expressing genes inserted into the cellular genome can be prepared by transfecting the recombinant retroviral genome into appropriate packaging cell lines, such as PA317 and Psi-CRIP (Comette et al., 1991, Human Gene Therapy 2:5-10; Cone et al., 1984, Proc. Natl. Acad. Sci. USA 81:6349). Recombinant adenoviral vectors can be used to infect a variety of cells and tissues in susceptible hosts (e.g., rats, hamsters, dogs, and chimpanzees) (Hsu et al., 1992, J. Infectious Disease, 166:769) and have the advantage of not requiring mitotically active cells for infection.

Any viral vector capable of accepting the coding sequence of the dsRNA molecule to be expressed can be used, for example, vectors derived from adenovirus (AV); adeno-associated virus (AAV); retroviruses (e.g., lentivirus (LV), rhabdovirus, murine leukemia virus); herpes virus, etc. The tropism of the viral vector can be modified by pseudotyping the vector with envelope proteins or other surface antigens from other viruses or, as appropriate, by substituting different viral capsid proteins.

For example, the lentiviral vectors of the present invention can be pseudotyped with surface proteins from vesicular stomatitis virus (VSV), rabies, Ebola, Mokola, and the like. By engineering the vectors to express different capsid protein serotypes, the AAV vectors of the present invention can be targeted to different cells. For example, an AAV vector expressing a serotype 2 capsid on a serotype 2 genome is referred to as AAV2/2. The serotype 2 capsid gene in the AAV2/2 vector can be replaced with a serotype 5 capsid gene to generate an AAV2/5 vector. Techniques for constructing AAV vectors expressing different capsid protein serotypes are within the skill of the art; for example, see Rabinowitz J E et al. (2002), J Virol 76: 791-801, which is incorporated herein by reference in its entirety.

The selection of recombinant viral vectors suitable for use in the present invention, methods for inserting nucleic acid sequences for expressing dsRNA into the vectors, and methods for delivering the viral vectors into desired cells are within the scope of those skilled in the art. See, for example, Dornburg R (1995), Gene Therap. 2: 301-310; Eglitis MA (1988), Biotechniques 6: 608-614; Miller AD (1990), Hum Gene Therap. 1: 5-14; Anderson WF (1998), Nature 392: 25-30; and Rubinson DA et al., Nat. Genet. 33: 401-406, all of which are incorporated herein by reference.

Preferred viral vectors are viruses derived from AV and AAV. In a particularly preferred embodiment, the dsRNA of the present invention is expressed as two separate, complementary single-stranded RNA molecules from a recombinant AAV vector, such as one containing a U6 or H1 RNA promoter, or a cytomegalovirus (CMV) promoter.

Suitable AV vectors for expressing the dsRNA of the present invention, methods for constructing recombinant AV vectors, and methods for delivering the vectors into target cells are described in Xia H et al. (2002), Nat. Biotech. 20: 1006-1010.

Suitable AAV vectors for expressing the dsRNA of the present invention, methods for constructing recombinant AV vectors, and methods for delivering the vectors into target cells are described in Samulski R et al. (1987), J. Virol. 61: 3096-3101; Fisher K J et al. (1996), J. Virol, 70: 520-532; Samulski R et al. (1989), J. Virol. 63: 3822-3826; U.S. Pat. No. 5,252,479; U.S. Pat. No. 5,139,941; International Patent Application No. WO 94/13788; and International Patent Application No. WO 93/24641, all of which are incorporated herein by reference.

The promoter driving expression of the dsRNA of the invention in a DNA plasmid or viral vector can be a eukaryotic RNA polymerase I (e.g., a ribosomal RNA promoter), RNA polymerase II (e.g., a CMV early promoter or an actin promoter or a U1 snRNA promoter), or typically an RNA polymerase III promoter (e.g., a U6 snRNA or a 7SK RNA promoter), or a prokaryotic promoter, such as a T7 promoter, provided that the expression plasmid also encodes the T7 RNA polymerase required for transcription from the T7 promoter. Such promoters can also direct transgene expression to the pancreas (e.g., see the insulin regulatory sequence for pancreas (Bucchini et al., 1986, Proc. Natl. Acad. Sci. USA 83: 2511-2515)).

In addition, transgene expression can be precisely regulated, for example, by using inducible regulatory sequences and expression systems, such as those that are sensitive to certain physiological regulators, such as circulating glucose levels or hormones (Docherty et al., 1994, FASEB J. 8:20-24). Such inducible expression systems suitable for controlling transgene expression in cells or mammals include regulation by ecdysone, estrogen, progesterone, tetracycline, chemical inducers of dimerization, and isopropyl-β-D1-thiogalactopyranoside (EPTG). One skilled in the art will be able to select appropriate regulatory/promoter sequences based on the intended use of the dsRNA transgene.

Typically, the recombinant vector that can express dsRNA molecule is delivered as described below, and remains in the target cell. Alternatively, a viral vector that provides transient expression of the dsRNA molecule can be used. This vector can be repeatedly administered as required. Once expressed, dsRNA and target RNA combine and regulate its function or expression. The delivery of the dsRNA expression vector can be systemic, for example, via intravenous or intramuscular administration, by being administered to the target cell from the patient's explantation, and then reintroducing the patient, or by any other means that can introduce required target cell.

DsRNA expression DNA plasmids are typically transfected into target cells as a complex with a cationic lipid carrier (e.g., Oligofectamine) or a non-cationic lipid-based carrier (e.g., Transit-TKO ^™ ). The present invention also relates to multiple lipid transfections for dsRNA-mediated inhibition targeting different regions of a single EG5 gene (or VEGF gene) or multiple EG5 genes (or VEGF genes) over a period of one week or longer. The successful introduction of the vector of the present invention into the host cell can be monitored using various known methods. For example, a reporter gene, such as a fluorescent marker, such as green fluorescent protein (GFP), can be used to signal transient transfection. Markers that provide resistance to specific environmental factors (e.g., antibiotics and drugs) for transfected cells, such as hygromycin B resistance, can be used to ensure stable transfection of isolated cells.

Eg5-specific dsRNA molecules and VEGF-specific dsRNA molecules can also be inserted into vectors and used as gene therapy vectors for human patients. For example, the gene therapy vector can be delivered to the patient by intravenous injection, local administration (see U.S. Patent No. 5,328,470) or stereotactic injection (see, for example, Chen et al. (1994) Proc. Natl. Acad. Sci. USA 91: 3054-3057). Pharmaceutical preparations of gene therapy vectors can include the gene therapy vector in an acceptable diluent, or can include a slow-release matrix that encapsulates the gene delivery medium. Alternatively, if a complete gene delivery vector, such as a retroviral vector, can be prepared intact from recombinant cells, the pharmaceutical preparation can include one or more cells that produce the gene delivery system.

Pharmaceutical compositions comprising dsRNA

In one embodiment, the present invention provides pharmaceutical compositions comprising the dsRNA described herein and a pharmaceutically acceptable carrier, as well as methods for administering the pharmaceutical compositions. The pharmaceutical compositions comprising the dsRNA are used to treat diseases or conditions associated with the expression or activity of Eg5/KSP and/or VEGF genes, such as pathological processes mediated by Eg5/KSP and/or VEGF expression, such as liver cancer. Such pharmaceutical compositions are formulated based on the delivery method.

dose

The pharmaceutical composition of the present invention is administered in a dosage sufficient to inhibit the expression of the Eg5/KSP and/or VEGF genes. Typically, a suitable dosage of dsRNA is 0.01 to 200.0 milligrams (mg) per kilogram (kg) of recipient body weight per day, typically 1 to 50 mg per kilogram of body weight per day. For example, dsRNA can be administered in a single dose of 0.01 mg/kg, 0.05 mg/kg, 0.5 mg/kg, 1 mg/kg, 1.5 mg/kg, 2 mg/kg, 3 mg/kg, 5.0 mg/kg, 10 mg/kg, 20 mg/kg, 30 mg/kg, 40 mg/kg or 50 mg/kg.

The pharmaceutical composition can be administered once a day, or the dsRNA can be administered as two, three, or more sub-doses at appropriate intervals throughout the day. The effect of a single dose on Eg5/KSP and/or VEGF levels is long-lasting, allowing subsequent doses to be administered at intervals of no more than 7 days or at intervals of no more than 1, 2, 3, or 4 weeks.

In some embodiments, the dsRNA is administered by continuous infusion or delivered by a controlled release formulation. In that case, the dsRNA contained in each sub-dose must be reduced accordingly to reach a total daily dose. The dosage unit can also be mixed and used to deliver within a number of days, for example, using a conventional sustained release formulation that provides a sustained release of the dsRNA within a number of days. Sustained release formulations are well known in the art and are particularly useful for delivering reagents to specific locations, for example, can be used together with reagents of the present invention. In this embodiment, the dosage unit comprises corresponding multiple daily doses.

Those skilled in the art will appreciate that certain factors can influence the dosage and timing required to effectively treat a subject, including, but not limited to, the severity of the disease or condition, previous treatment, the subject's overall health and/or age, and other existing conditions. Furthermore, treatment of a subject with a therapeutically effective amount of a composition can include a single treatment or a series of treatments. Effective dosages and in vivo half-lives of individual dsRNAs contemplated by the present invention can be estimated based on in vivo testing using conventional methods or using appropriate animal models as described elsewhere herein.

Advances in mouse genetics have yielded numerous mouse models for studying various human diseases, such as pathological processes mediated by Eg5/KSP and/or VEGF expression. Such models are useful for in vivo testing of dsRNA and for determining therapeutically effective doses. Suitable mouse models include, for example, mice harboring plasmids expressing human Eg5/KSP and/or VEGF. Another suitable mouse model is a transgenic mouse carrying a transgene expressing human Eg5/KSP and/or VEGF.

The toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, for example, to determine the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio of toxic to therapeutic effects is the therapeutic index, which can be expressed as the ratio LD50/ED50. Compounds with high therapeutic indices are preferred.

The data obtained from cell culture assays and animal studies can be used to formulate dosage ranges for human use. The dosage of the composition characterized by the present invention is generally within a circulating concentration range that includes the ED50 but has little or no toxicity. Depending on the dosage form used and the route of administration used, the dosage may vary within this range. For any compound used in the method characterized by the present invention, a therapeutically effective dose can be initially estimated from cell culture assays. Dosages can be formulated in animal models to obtain a circulating plasma concentration range for the compound, and if appropriate, a circulating plasma concentration range for the polypeptide product of the target sequence (e.g., to obtain a reduced polypeptide concentration), the concentration range including the IC50 (i.e., the concentration at which the test compound achieves half-maximal inhibition of symptoms) determined in cell culture. This information can be used to more accurately determine a useful dosage for humans. For example, plasma levels can be determined by high performance liquid chromatography.

In addition to their administration as discussed above, the dsRNA features of the present invention can be co-administered with other known agents that are effective in treating pathological processes mediated by target gene expression. In any case, a practicing physician can adjust the dosage and timing of dsRNA administration based on the results observed using efficacy criteria known in the art or described herein.

Drug administration

The pharmaceutical compositions of the present invention can be administered in a variety of ways, depending on whether local or systemic treatment is desired and on the area to be treated. Administration can be topical, pulmonary (e.g., by inhalation or insufflation of a powder or aerosol, including by nebulizer), intratracheal, intranasal, epidermal, and transdermal, as well as subcutaneous, oral, or parenteral, e.g., subcutaneous.

Typically, when treating mammals suffering from hyperlipidemia, dsRNA molecules are administered systemically via parenteral administration. Parenteral administration methods include intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; or intracranial, for example, intraparenchymal, intrathecal or intraventricular administration. For example, dsRNA, bound or unbound or formulated with or without liposomes, can be administered intravenously to the patient. To this end, dsRNA molecules can be formulated into compositions, such as sterile and non-sterile aqueous solutions, non-aqueous solutions in common solvents such as alcohols, or solutions in liquid or solid oily matrices. Such solutions can also contain buffers, diluents and other suitable additives. For parenteral, intrathecal or intraventricular administration, dsRNA molecules can be formulated into compositions, such as sterile aqueous solutions, which can also contain buffers, diluents and other suitable additives (e.g., penetration enhancers, carrier compounds and other pharmaceutically acceptable carriers). The present invention describes the formulations in more detail.

The dsRNA can be delivered in a manner that targets a specific tissue, such as the liver (eg, hepatocytes of the liver).

preparation

The pharmaceutical formulations of the present invention, which can be conveniently presented in unit dosage form, can be prepared according to conventional methods well known in the pharmaceutical industry. Such techniques include the step of mixing the active ingredient with a pharmaceutical carrier or excipient. Typically, the formulations are prepared by uniformly and intimately mixing the active ingredient with a liquid carrier or a finely divided solid carrier, or both, and then, if necessary, shaping the product.

The present composition can be formulated into any of many possible dosage forms, such as, but not limited to, tablets, capsules, gel capsules, liquid syrups, soft gels, suppositories, and enemas. The present composition can also be formulated into suspensions in water, non-aqueous, or mixed media. Aqueous suspensions can also contain substances that increase the viscosity of the suspension, including, for example, sodium carboxymethylcellulose, sorbitol, and/or dextran. The suspension can also contain stabilizers.

Pharmaceutical compositions of the present invention include, but are not limited to, solutions, emulsions, and liposome-containing formulations. These compositions can be produced from a variety of components, including but not limited to preformed liquids, self-emulsifying solids, and self-emulsifying semisolids. In one aspect, when treating liver diseases such as hyperlipidemia, the formulation is a formulation targeting the liver.

In addition, dsRNAs targeting EG5/KSP and/or VEGF genes can be formulated into compositions containing dsRNAs mixed, encapsulated, conjugated, or otherwise linked to other molecules, molecular structures, or nucleic acid mixtures. For example, a composition containing one or more dsRNA agents targeting EG5/KSP and/or VEGF genes can include other therapeutic agents, such as other cancer therapeutics or one or more dsRNA compounds targeting non-EG5/KSP and/or VEGF genes.

Oral, parenteral, topical, and biologic agents

Compositions and preparations for oral administration include powders or granules, microparticles, nanoparticles, suspensions or aqueous solutions or non-aqueous media, capsules, gel capsules, bags, tablets or mini tablets. Thickeners, flavoring agents, diluents, emulsifiers, dispersion aids or adhesives may be needed. In some embodiments, oral formulations are formulations in which the dsRNA of the present invention is administered together with one or more penetration enhancers, surfactants and chelating agents. Suitable surfactants include fatty acids and/or their esters or salts, bile acids and/or their salts. Suitable bile acids/salts include chenodeoxycholic acid (CDCA) and ursodeoxycholic acid (UDCA), cholic acid, dehydrocholic acid, deoxycholic acid, glycocholic acid, glycolic acid, glycerodeoxycholic acid, taurocholic acid, taurodeoxycholic acid, tauro-24,25-dihydro-sodium fusidate and glycerol dihydrofusidate sodium. Suitable fatty acids include arachidonic acid, undecanoic acid, oleic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, glyceryl monooleate, glyceryl dilaurate, glyceryl 1-monodecanoate, 1-dodecylazacycloheptane-2-one, acylcarnitines, acylcholines, monoglycerides, diglycerides or their pharmaceutically acceptable salts (e.g., sodium salt). In some embodiments, a penetration enhancer combination is used, e.g., a fatty acid/salt and bile acid/salt combination. A typical combination is the sodium salt of lauric acid, capric acid and UDCA. Other penetration enhancers include polyoxyethylene-9-lauryl esters, polyoxyethylene-20-hexadecyl esters. The dsRNA of the present invention feature can be delivered orally in a granular form comprising spray-dried particles or a granular form comprising complexed micrometers or nanoparticles. dsRNA complexing agents include polyamino acids; polyimines; polyacrylates; polyalkyl acrylates, polyoxethanes, polyalkyl cyanoacrylates; cationized gelatin, albumin, starch, acrylates, polyethylene glycol (PEG) and starch; polyalkyl cyanoacrylates; DEAE-derivatized polyimines, pullulan, cellulose and starch. Suitable complexing agents include chitosan, N-trimethylchitosan, poly-L-lysine, polyhistidine, polyornithine, polyspermine, protamine, polyvinylpyridine, polythiols Diethylaminomethylethylene P(TDAE), polyaminostyrene (e.g., p-amino), poly(methylcyanoacrylate), poly(ethylcyanoacrylate), poly(butylcyanoacrylate), poly(isobutylcyanoacrylate), poly(hexylcyanoacrylate), DEAE-methacrylate, DEAE-hexylacrylate, DEAE-acrylamide, DEAE-albumin and DEAE-dextran, polymethacrylate, polyhexylacrylate, poly(D,L-lactic acid), poly(DL-lactic-co-glycolic acid (PLGA), alginate and polyethylene glycol (PEG). Oral formulations of dsRNA and their preparation are described in detail in U.S. Patent 6,887,906, U.S. Patent Publication No. 20030027780 and U.S. Patent No. 6,747,014, each of which is incorporated herein by reference.

Compositions and formulations for parenteral, intraparenchymal (into the brain), intrathecal, intraventricular, or intrahepatic administration may include sterile aqueous solutions which may also contain buffers, diluents, and other suitable additives such as, but not limited to, penetration enhancers, carrier compounds, and other pharmaceutically acceptable carriers or excipients.

Pharmaceutical compositions and preparations for topical administration can include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous solutions, powders or oily bases, thickeners, etc. may be necessary or desirable. Suitable topical preparations include preparations in which the characteristic compounds of the present invention and local delivery agents such as lipids, liposomes, fatty acids, fatty acid esters, steroids, chelating agents and surfactants are mixed. Suitable lipids and liposomes include neutral (e.g., dioleoylphosphatidyl DOPE ethanolamine, dimyristoylphosphatidylcholine DMPC, distearoylphosphatidylcholine), negative (e.g., dimyristoylphosphatidylglycerol DMPG) and cationic (e.g., dioleoyltetramethylaminopropyl DOTAP and dioleoylphosphatidylethanolamine DOTMA). The dsRNA characterized by the present invention can also be encapsulated in liposomes or can form complexes therewith, particularly with cationic liposomes. Alternatively, the dsRNA can be complexed with lipids, particularly with cationic lipids. Suitable fatty acids and esters include, but are not limited to, arachidonic acid, oleic acid, arachidic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate, 1-dodecylazacycloheptan-2-one, acylcarnitines, acylcholines, or C1-10 alkyl esters (e.g., isopropyl myristate), monoglycerides, diglycerides, or pharmaceutically acceptable salts thereof. Topical formulations are described in detail in U.S. Patent No. 6,747,014, which is incorporated herein by reference. Additionally, dsRNA molecules can be administered to mammals by biological or non-biological means, such as those described in U.S. Patent No. 6,271,359. Non-biological delivery can be accomplished by a variety of methods, including, but not limited to: (1) loading liposomes with dsRNA acid molecules provided herein and (2) complexing dsRNA molecules with lipids or liposomes to form nucleic acid-lipid or nucleic acid-liposome complexes. The liposomes can be composed of cations and neutral lipids commonly used for in vitro transfection of cells. Cationic lipids can be complexed with negatively charged nucleic acids (e.g., charge-related) to form liposomes. Examples of cationic liposomes include, but are not limited to, lipofectin, lipofectamine, lipofectace, and DOTAP. Methods for forming liposomes are known in the art. For example, liposome compositions can be formed from lecithin, dimyristoyl phosphatidylcholine, dipalmitoyl phosphatidylglycerol, or dioleoylphosphatidylethanolamine. Many lipophilic agents are commercially available, including Lipofectin ^™ (Invitrogen/Life Technologies, Carlsbad, Calif.) and Effectene ^™ (Qiagen, Valencia, Calif.). In addition, commercially available cationic lipids such as DDAB or DOTAP can be used to optimize systemic delivery methods, and each cationic lipid can be mixed with a neutral lipid such as DOPE or cholesterol. In some cases, liposomes such as those described by Templeton et al. (Nature Biotechnology, 15: 647-652 (1997)) can be used. In other embodiments, polycations such as polyethyleneimine can be used to achieve in vivo and in vitro delivery (Boletta et al., J. Am Soc. Nephrol. 7: 1728 (1996)). Additional information on the use of liposomes to deliver nucleic acids can be found in U.S. Patent No. 6,271,359, PCT Publication No. WO 96/40964, and Morrissey, D. et al. 2005. Nat Biotechnol. 23(8): 1002-7.

Biological delivery can be realized by several methods, including but not limited to using viral vectors.For example, viral vectors (for example, adenovirus and herpes virus vectors) can be used for dsRNA molecule delivery to hepatocyte.Standard molecular biology techniques can be used for one or more dsRNA provided by the invention are introduced into one of the many different viral vectors developed before, so that nucleic acid is delivered to cell.These viral vectors of gained can be used for, for example, infecting by one or more dsRNA being delivered to cell.

Liposomal preparations

In addition to microemulsions, many organized surface-active structures have been studied and used in pharmaceutical formulations. These include monolayers, micelles, bilayers, and vesicles. Vesicles (e.g., liposomes) have attracted considerable attention due to the specificity and duration of action they provide in drug delivery. As used herein, the term "liposome" refers to a vesicle composed of amphiphilic lipids arranged in a spherical bilayer or multiple spherical bilayers.

Liposomes are unilamellar or multilamellar vesicles with a membrane formed from a lipophilic material and an aqueous interior. The aqueous portion contains the composition to be delivered. Cationic liposomes have the advantage of being able to fuse with cell walls. Non-cationic liposomes, although they cannot fuse effectively with cell walls, are taken up by macrophages in the body.

In order to penetrate intact mammalian skin, lipid vesicles must penetrate a series of pores with a diameter of less than 50 nm under the influence of an appropriate transdermal gradient. Therefore, it is necessary to use liposomes that are highly deformable and able to penetrate such pores.

Other advantages of liposomes include that liposomes derived from natural phospholipids are biocompatible and biodegradable; that liposomes can bind many water- and lipid-soluble drugs; and that liposomes can protect drugs encapsulated in their internal compartments from metabolism and degradation (Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, page 245). Important factors to consider in preparing liposome formulations are the lipid surface charge, vesicle size, and the aqueous volume of the liposomes.

Liposomes can transfer and deliver active ingredients to the site of action. Because the liposome membrane is structurally similar to biological membranes, when liposomes are applied to tissues, they begin to fuse with the cell membrane. Due to liposome fusion and cell progression, the liposome contents flow into cells where the active agent can act.

Liposomal formulations have been the focus of extensive research as a delivery method for many drugs. There is growing evidence that liposomes offer several advantages over other formulations for topical drug delivery. These advantages include reduced side effects associated with high systemic absorption of the administered drug, increased accumulation of the administered drug at the desired target, and the ability to deliver a wide variety of hydrophilic and hydrophobic drugs into the skin.

Several reports have detailed the ability of liposomes to deliver agents containing high molecular weight DNA into the skin. Compounds including analgesics, antibodies, hormones, and high molecular weight DNA have been administered to the skin. Most applications result in targeting the upper epidermis.

Liposomes are divided into two major categories. Cationic liposomes are positively charged liposomes that interact with negatively charged DNA molecules to form a stable complex. The positively charged DNA/liposome complex binds to the negatively charged cell surface and is internalized in endosomes. Due to the acidic pH within the endosomes, the liposomes rupture, releasing their contents into the cytoplasm (Wang et al., Biochem. Biophys. Res. Commun., 1987, 147, 980-985).

pH-sensitive or negatively charged liposomes capture DNA rather than complexing with it. Because both DNA and lipids have similar charges, repulsion occurs rather than complex formation. However, some DNA is trapped in the aqueous interior of these liposomes. pH-sensitive liposomes have been used to deliver DNA encoding the thymidine kinase gene to cultured cell monolayers. Expression of the exogenous gene was detected in the target cells (Zhou et al., Journal of Controlled Release, 1992, 19, 269-274).

A major type of liposome composition comprises a phospholipid that is different from naturally derived phosphatidylcholine. For example, a neutral liposome composition can be formed by dimyristoylphosphatidylcholine (DMPC) or dipalmitoylphosphatidylcholine (DPPC). Anionic liposome compositions are usually formed by dimyristoylphosphatidylglycerol, while anionic gene fusion liposomes are mainly formed by phosphatidylethanolamine (DOPE). Another type of liposome composition is formed by phosphatidylcholine (PC), for example, soybean PC and egg PC. Another type is formed by a mixture of phosphatidylcholine and/or phosphatidylcholine and/or cholesterol.

Several studies have evaluated the topical delivery of liposomal drug formulations to the skin. Liposomes containing interferon were applied to guinea pig skin to reduce herpes sores on the skin, while delivery of interferon via other means (e.g., as a solution or as an emulsion) was ineffective (Weiner et al., Journal of Drug Targeting, 1992, 2, 405-410). In addition, other studies tested the effect of administering interferon as part of a liposomal formulation and administering interferon using an aqueous system, concluding that liposomal formulations were superior to aqueous administration (du Plessis et al., Antiviral Research, 1992, 18, 259-265).

Nonionic liposome systems, particularly systems comprising nonionic surfactants and cholesterol, have also been investigated to determine their effectiveness in delivering drugs to the skin. Nonionic liposome formulations containing Novasome ^™ I (glyceryl dilaurate/cholesterol/polyoxyethylene-10-stearyl ether) and Novasome ^™ II (glyceryl distearate/cholesterol/polyoxyethylene-10-stearyl ether) were used to deliver cyclosporin A into the dermis of mouse skin. The results showed that this nonionic liposome system was able to effectively promote the deposition of cyclosporin A into different layers of the skin (Hu et al., STP Pharma. Sci., 1994, 4, 6, 466).

Liposomes also include "sterically stabilized" liposomes, which, as used herein, refers to liposomes containing one or more specialized lipids that, when incorporated into liposomes, result in increased circulation duration compared to liposomes lacking such specialized lipids. Examples of sterically stabilized liposomes are liposomes in which a portion of the vesicle-forming lipid portion (A) comprises one or more glycolipids, such as monosialoganglioside G _M1 , or (B) is derivatized with one or more hydrophilic polymers, such as polyethylene glycol (PEG) moieties. Without wishing to be bound by any particular theory, it is believed in the art that, at least for sterically stabilized liposomes containing gangliosides, sphingomyelins, or PEG-derivatized lipids, the increased circulation half-life of these sterically stabilized liposomes is derived from reduced uptake into cells of the reticuloendothelial system (RES) (Allen et al., FEBS Letters, 1987, 223, 42; Wu et al., Cancer Research, 1993, 53, 3765).

Various liposomes containing one or more glycolipids are known in the art. Papahadjopoulos et al. (Ann. N.Y. Acad. Sci., 1987, 507, 64) reported the ability of monosialoylganglioside GM1, galactocerebroside sulfate, and phosphatidylinositol to improve the blood half-life of liposomes. These findings were detailed by Gabizon et al. (Proc. Natl. Acad. Sci. U.S.A., 1988, 85, 6949). U.S. Patent No. 4,837,028 and WO 88/04924 to Allen et al. disclose liposomes containing (1) sphingomyelin and (2) ganglioside GM1 or galactocerebroside sulfate. U.S. Patent No. 5,543,152 (Webb et al.) discloses liposomes containing sphingomyelin. Liposomes containing 1,2-sn-dimyristoylphosphatidylcholine are disclosed in WO 97/13499 (Lim et al.).

Many liposomes containing lipids derivatized with one or more hydrophilic polymers and methods for their preparation are known in the art. Sunamoto et al. (Bull. Chem. Soc. Jpn., 1980, 53, 2778) described liposomes containing the nonionic detergent 2C _1215G , which contained a PEG moiety. Illum et al. (FEBS Lett., 1984, 167, 79) noted that polystyrene particles containing a hydrophilic coating of polymerized ethylene glycol resulted in a significantly increased blood half-life. Synthetic phospholipids modified by incorporating carboxyl groups of polyglycols (e.g., PEG) were described by Sears (U.S. Patent Nos. 4,426,330 and 4,534,899). Klibanov et al. (FEBS Lett., 1990, 268, 235) described experiments demonstrating that liposomes containing phosphatidylethanolamine (PE) derivatized with PEG or stearate PEG significantly increased blood circulation half-life. Blume et al. (Biochimica et Biophysica Acta, 1990, 1029, 91) extended this research to other PEG-derivatized phospholipids, for example, DSPE-PEG formed by combining distearoylphosphatidylethanolamine (DSPE) and PEG. Liposomes with covalently bound PEG moieties on their outer surface are described in Fisher's European Patent No. EP 0 445 131 B1 and WO90/04384. Liposome compositions containing 1-20 mole percent of PEG-derivatized PE and methods of use thereof are described by Woodle et al. (U.S. Patent Nos. 5,013,556 and 5,356,633) and Martin et al. (U.S. Patent No. 5,213,804 and European Patent No. EP 0 496 813 B1). Liposomes containing a variety of other lipid-polymer conjugates are disclosed in WO 91/05545 and U.S. Patent Nos. 5,225,212 (Martin et al.) and WO 94/20073 (Zalipsky et al.). Liposomes containing PEG-modified ceramide lipids are described in WO 96/10391 (Choi et al.). U.S. Patent No. 5,540,935 (Miyazaki et al.) and U.S. Patent No. 5,556,948 (Tagawa et al.) describe liposomes containing PEG, the surface of which can be further derivatized with functional moieties.

Many liposomes containing nucleic acids are known in the art. WO 96/40062 to Thierry et al. discloses methods for encapsulating high molecular weight nucleic acids in liposomes. U.S. Patent No. 5,264,221 to Tagawa et al. discloses protein-bound liposomes and claims that the contents of such liposomes can include dsRNA. U.S. Patent No. 5,665,710 to Rahman et al. describes certain methods for encapsulating oligodeoxyribonucleotides in liposomes. WO 97/04787 to Love et al. discloses liposomes containing dsRNA targeting the raf gene.

Transfersomes are another type of liposome, and they are highly deformable lipid aggregates that are attractive candidates for drug delivery vehicles. Transfersomes can be described as lipid droplets that are so highly deformable that they can easily penetrate pores smaller than the droplets. Transfersomes are suitable for the environment in which they are used, for example, they are self-optimizing (suitable for the shape of skin pores), self-repairing, often reach their target without breaking and usually self-loading. To prepare transfersomes, surface edge activators, usually surfactants, can be added to standard liposome compositions. Transfersomes are used to deliver serum albumin to the skin. It has been confirmed that transfersome-mediated serum albumin delivery is as effective as subcutaneous injection of a solution containing serum albumin.

Surfactants are widely used in formulations such as emulsions (including microemulsions) and liposomes. The most common method for classifying and dividing the many different types of surfactants (both natural and synthetic) is by the hydrophile/lipophile balance (HLB). The nature of the hydrophilic group (also known as the "head") provides the most useful means for classifying the different surfactants used in formulations (Rieger, in Pharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, N.Y., 1988, p. 285).

If the surfactant molecule is nonionic, it is classified as a nonionic surfactant. Nonionic surfactants are widely used in medicine and cosmetics and can be used over a wide pH range. Depending on their structure, their HLB value is usually 2 to about 18. Nonionic surfactants include nonionic esters, such as ethylene glycol esters, propylene glycol esters, glycerides, polyglycerol esters, sorbitan esters, sucrose esters and ethoxylated esters. Nonionic alkanolamides and ethers such as ethoxylated fatty alcohols, propoxylated alcohols and ethoxy/propoxylated block copolymers are also included in this class. Polyoxyethylene surfactants are the most commonly used members of the nonionic surfactant category.

When the surfactant molecule carries a negative charge when dissolved or dispersed in water, the surfactant is classified as an anionic surfactant. Anionic surfactants include carboxylic acid esters, such as soaps; acyl lactylates; amino acid amides; sulfates, such as alkyl sulfates and ethoxyalkyl sulfates; sulfonates, such as alkylbenzenesulfonates, acyl isethionates, acyl taurates, and sulfosuccinates; and phosphates. The most important members of the anionic surfactant class are alkyl sulfates and soaps.

When a surfactant molecule carries a positive charge when dissolved or dispersed in water, the surfactant is classified as a cationic surfactant. Cationic surfactants include quaternary ammonium salts and ethoxylated amines. Quaternary ammonium salts are the most commonly used members of this class of surfactants.

If the surfactant molecule can carry either a positive or negative charge, the surfactant is classified as an amphoteric surfactant. Amphoteric surfactants include acrylic acid derivatives, substituted alkylamides, N-alkylbetaines, and phospholipids.

The use of surfactants in pharmaceuticals, formulations, and emulsions is reviewed (Rieger, in Pharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, N.Y., 1988, p. 285).

Nucleic acid lipid particles

In one embodiment, the dsRNA of the present invention is completely encapsulated in a lipid formulation, for example, to form a nucleic acid-lipid particle. Typically, nucleic acid-lipid particles include cationic lipids, non-cationic lipids, sterols, and lipids (e.g., PEG-lipid conjugates) that prevent particle aggregation. Nucleic acid-lipid particles are very useful for systemic applications because they show prolonged circulation duration after intravenous (i.v.) injection and accumulate in distal sites (e.g., physically separated sites from the administration site). In addition, when present in nucleic acid-lipid particles of the present invention, nucleic acid resists nuclease degradation in aqueous solution. Nucleic acid-lipid particles and methods for their preparation are disclosed in, for example, U.S. Patent Nos. 5,976,567; 5,981,501; 6,534,484; 6,586,410; 6,815,432; and PCT Publication No. WO 96/40964.

The nucleic acid-lipid particle can also include one or more other lipids and/or components such as cholesterol. Other lipids can be included in the liposome composition for a variety of purposes, such as to prevent lipid oxidation or to bind ligands to the liposome surface. Any of a variety of lipids can be present, including amphiphilic, neutral, cationic and anionic lipids. Such lipids can be used alone or in combination. The present invention describes specific examples of other lipid components that can be present.

Other components that may be present in the nucleic acid-lipid particles include bilayer-stabilizing components such as polyamide oligomers (see, e.g., U.S. Pat. No. 6,320,017), peptides, proteins, detergents, lipid-derivatives such as PEG coupled to phosphatidylethanolamine and PEG bound to ceramide (see, U.S. Pat. No. 5,885,613).

Nucleic acid-lipid particles can include one or more secondary amino lipids or cationic lipids, neutral lipids, sterols, and lipids selected to reduce aggregation of the lipid particles during formation, which can result from steric stabilization of the particles that prevents charge-induced aggregation during formation.

For example, nucleic acid-lipid particles include SPLP, pSPLP and SNALP. The term "SNALP" refers to a stable nucleic acid-lipid particle containing SPLP. The term "SPLP" refers to a nucleic acid-lipid particle encapsulated with plasmid DNA in a lipid vesicle. SPLP includes "pSPLP," which includes the encapsulated condensing agent-nucleic acid complex listed in PCT Publication No. WO 00/03683.

The particles of the present invention typically have an average diameter of about 50 nm to about 150 nm, more typically about 60 nm to about 130 nm, even more typically about 70 nm to about 110 nm, and most typically about 70 nm to about 90 nm, and are substantially non-toxic.

In one embodiment, the ratio of lipid to drug (mass/mass ratio) is about 1:1 to about 50:1, about 1:1 to about 25:1, about 3:1 to about 15:1, about 4:1 to about 10:1, about 5:1 to about 9:1, or about 6:1 to about 9:1, or about 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1 or 33:1.

Cationic lipids

Nucleic acid-lipid particles of the present invention generally include cationic lipids. For example, the cationic lipids can be N, N-dioleyl-N, N-dimethylammonium chloride (DODAC), N, N-distearyl-N, N-dimethylammonium bromide (DDAB), N-(1-(2,3-dioleoyloxy)propyl)-N, N, N-trimethylammonium chloride (DOTAP), N-(1-(2,3-dioleyloxy)propyl)-N, N, N-trimethylammonium chloride (DOTMA), N, N-dimethyl-2,3-dioleyloxy)propylamine (DODMA), 1,2-dilinoleyloxy-N, N-dimethylaminopropane (DLinDMA), 1,2-dilinoyloxy-N, N-dimethylaminopropane (DLenDMA), 1,2-dilinoleylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP), 1,2-dilinoleyloxy-3-(dimethylamino)acetoxypropane (DLin-DAC), 1,2-dilinoleyloxy-3-morpholinylpropane (DLin-MA), 1,2-dilinoleoyloxy-3-dimethylaminopropane (DLinDAP), 1,2-dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA), 1-linoleoyl-2-linoleyloxy-3 -dimethylaminopropane (DLin-2-DMAP), 1,2-dilinoleyloxy-3-trimethylaminopropane chloride (DLin-TMA.Cl), 1,2-dilinoleoyloxy-3-trimethylaminopropane chloride (DLin-TAP.Cl), 1,2-dilinoleyloxy-3-(N-methylpiperazin-1-yl)propane (DLin-MPZ) or 3-(N,N-dilinoleylamino)-1,2-propanediol (DLinAP), 3-(N,N-dioleylamino)-1,2-propanediol (DOAP), 1,2-dilinoleyloxy-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA) or Its analogs, (3aR, 5s, 6aS) -N, N-dimethyl-2, 2-di ((9Z, 12Z) - octadeca-9, 12-dienyl) tetrahydro -3aH- cyclopenta [d] [1, 3] dioxol-5-amine (ALNY-100), 4- (dimethylamino) butyric acid (6Z, 9Z, 28Z, 31Z) - triacontria-6, 9, 28, 31-tetraen-19-yl ester (MC3), or a mixture thereof.

In addition to the above specific description, other cationic lipids carrying a net positive charge at approximately physiological pH can also be included in the lipid particles of the present invention. Such cationic lipids include, but are not limited to, N, N-dioleyl-N, N-dimethylammonium chloride ("DODAC"); N-(2,3-dioleyloxy)propyl-N, N-N-triethylammonium chloride ("DOTMA"); N, N-distearyl-N, N-dimethylammonium bromide ("DDAB"); N-(2,3-dioleyloxy)propyl)-N, N, N-trimethylammonium chloride ("DOTAP"); 1,2-dioleyloxy-3-trimethylammonium chloride ("DOTMA"); N-(1-(2,3-dioleyloxy)propyl)-N-2-(sperminecarboxamido)ethyl)-N,N-dimethylammonium trifluoroacetate ("DOSPA"); dioctadecylamidoglycylcarboxyspermine ("DOTAP.Cl"); 3β-(N-(N′,N′-dimethylaminoethane)-carbamoyl)cholesterol ("DC-Chol"); N-(1-(2,3-dioleyloxy)propyl)-N-2-(sperminecarboxamido)ethyl)-N,N-dimethylammonium trifluoroacetate ("DOSPA"); dioctadecylamidoglycylcarboxyspermine ("DOTAP.Cl"); In some embodiments, the cationic lipid is an amino lipid. In some embodiments, the cationic lipid is an amino lipid. In some embodiments, the cationic lipid is an amino lipid. In some embodiments, the cationic lipid is an amino lipid. In some embodiments, the cationic lipid is an amino lipid. In some embodiments, the cationic lipid is an amino lipid. In some embodiments, the cationic lipid is an amino lipid. In some embodiments, the cationic lipid is an amino lipid. In some embodiments, the cationic lipid is an amino lipid. In some embodiments, the cationic lipid is an amino lipid. In some embodiments, the cationic lipid is an amino lipid.

As used herein, the term "amino lipid" is meant to include lipids having one or two fatty acid or fatty alkyl chains and an amino head group (including alkylamino or dialkylamino groups), which can be protonated to form cationic lipids at physiological pH.

Other amino lipids comprise the lipid with optional fatty acid group and other dialkylamino groups, comprise the lipid (for example, N-ethyl-N-methylamino-, N-propyl-N-ethylamino-etc.) that wherein alkyl substituent is different.For wherein R ¹¹ and R ¹² all are those embodiments of long-chain alkyl or acyl group, they can be identical or different.Usually, the amino lipid with less saturated acyl chain is easier to control size, particularly for filtration sterilization purpose, the size of described complex must be lower than about 0.3 micron.Preferably comprising carbon chain length is C ₁₄ to C ₂₂ unsaturated fatty acid amino lipid.Other supports also can be used for separating amino and fatty acid or fatty alkyl moiety of amino lipid.Suitable support is well known by persons skilled in the art.

In certain embodiments, the amino or cationic lipids of the present invention have at least one protonatable or deprotonatable group such that the lipid is positively charged at a pH equal to or lower than physiological pH (e.g., pH 7.4) and neutral at a second pH, preferably equal to or greater than physiological pH. Of course, it will be understood that the addition or removal of protons that changes with pH is an equilibrium process, and reference to charged or neutral lipids refers to the properties of the dominant species, without requiring that all lipids exist in a charged or neutral form. However, the present invention does not exclude the use of lipids having more than one protonatable or deprotonatable group, or zwitterionic lipids.

In certain embodiments, the pKa of the protonatable group in the protonatable lipids of the present invention is from about 4 to about 11. The most preferred pKa is from about 4 to about 7 because these lipids will be cationic at the lower pH formulation stage, while the particle surface will be largely (but not completely) neutralized at physiological pH of about pH 7.4. One advantage of this pKa is that at least some nucleic acids associated with the outer surface of the particle lose their electrostatic interactions at physiological pH and can be removed by simple dialysis; thus, the susceptibility of the particle to clearance is greatly reduced.

An example of a cationic lipid is 1,2-dilinolenyloxy-N,N-dimethylaminopropane (DLinDMA). The synthesis and preparation of nucleic acid-lipid particles comprising DlinDMA are described in International Application PCT/CA2009/00496, filed April 15, 2009.

In one embodiment, the cationic lipid XTC (2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane) is used to prepare nucleic acid-lipid particles. The synthesis of XTC is described in U.S. Provisional Patent Application No. 61/107,998, filed October 23, 2008, which is incorporated herein by reference.

In another embodiment, the cationic lipid MC3 (4-(dimethylamino)butanoic acid (6Z, 9Z, 28Z, 31Z)-heptatriacontac-6,9,28,31-tetraen-19-yl ester) (e.g., DLin-M-C3-DMA) is used to prepare nucleic acid-lipid particles. The synthesis of MC3 and preparations containing MC3 is described, for example, in U.S. Provisional Serial No. 61/244,834, filed on September 22, 2009, and U.S. Provisional Serial No. 61/185,800, filed on June 10, 2009, which are incorporated herein by reference.

In another embodiment, the cationic lipid ALNY-100 ((3aR,5s,6aS)-N,N-dimethyl-2,2-di((9Z,12Z)-octadeca-9,12-dienyl)tetrahydro-3aH-cyclopenta[d][1,3]dioxol-5-amine)) is used to prepare nucleic acid-lipid particles. The synthesis of ALNY-100 is described in International Patent Application No. PCT/US09/63933, filed November 10, 2009, which is incorporated herein by reference.

FIG20 illustrates the structures of ALNY-100, MC3, and XTC.

The cationic lipid may comprise from about 20 mol% to about 70 mol% or about 45-65 mol% or about 40 mol% of the total lipid present in the particle.

Non-cationic lipids

The nucleic acid-lipid particles of the present invention may include non-cationic lipids. The non-cationic lipids may be anionic lipids or neutral lipids. Examples include, but are not limited to, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexylamine-1-carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphatidylethanolamine (DMPE), distearoyl-phosphatidyl-ethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE), cholesterol, or mixtures thereof.

Anionic lipids suitable for use in the lipid particles of the present invention include, but are not limited to, phosphatidylglycerol, diphosphatidylglycerol, diacylphosphatidylserine, diacylphosphatidic acid, N-lauroylphosphatidylethanolamine, N-succinylphosphatidylethanolamine, N-glutarylphosphatidylethanolamine, lysylphosphatidylglycerol and other anionic modifying groups attached to neutral lipids.

When present in lipid granules, neutral lipid can be any one of a variety of lipid species, and it exists in the form of uncharged or neutral zwitterionic under physiological pH. Such lipids include, for example, diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, sphingomyelin, dihydrosphingomyelin, cephalin and cerebroside. The selection of the neutral lipid used in the granule described in the present invention is usually guided by, for example, the liposome size and stability of the liposome in the bloodstream. Preferably, the neutral lipid component is a lipid with two acyl groups (that is, diacylphosphatidylethanolamine and diacylphosphatidylethanolamine). The lipid with a variety of acyl chain groups that change chain length and saturation is commercially available, and can be separated or synthesized by familiar technology. In one group of embodiments, preferably containing a carbon chain length is C ₁₄ to C ₂₂ of saturated fatty acids. In another group of embodiments, using a lipid containing a carbon chain length is C ₁₄ to C ₂₂ of mono- or di-unsaturated fatty acids. In addition, the lipid containing a mixture of saturated and unsaturated fatty acid chains can be used. Preferably, the neutral lipid used in the present invention is DOPE, DSPC, POPC or any related phosphatidylcholine. The neutral lipid used in the present invention can also be composed of sphingomyelin, dihydrosphingomyelin or phospholipids with other head groups such as serine and inositol.

In one embodiment, the non-cationic lipid is distearoylphosphatidylcholine (DSPC). In another embodiment, the non-cationic lipid is dipalmitoylphosphatidylcholine (DPPC).

If cholesterol is included, the non-cationic lipid can comprise about 5 mol% to about 90 mol%, about 5 mol% to about 10 mol%, about 10 mol%, or about 58 mol% of the total lipid present in the particle.

Conjugated lipids

Conjugated lipids can be used in nucleic acid-lipid particles to prevent aggregation, including polyethylene glycol (PEG)-modified lipids, monosialoganglioside Gm1, and polyamide oligomers ("PAO"), for example (described in U.S. Patent No. 6,320,017). Other compounds containing uncharged, hydrophilic steric hindering moieties (which prevent aggregation during formulation), such as PEG, Gm1, or ATTA, can also be linked to lipids used in the methods and compositions of the present invention. ATTA-lipids are described, for example, in U.S. Patent No. 6,320,017, and PEG-lipid conjugates are described, for example, in U.S. Patent Nos. 5,820,873, 5,534,499, and 5,885,613. Typically, the concentration of the lipid component selected to reduce aggregation is about 1 to 15% (molar percentage of lipid).

Specific examples of PEG-modified lipids (or lipid-polyoxyethylene conjugates) that can be used in the present invention can have a variety of "anchoring" lipid moieties to fix the PEG moiety to the surface of the lipid vesicle. Examples of suitable PEG-modified lipids include PEG-modified phosphatidylethanolamine and phosphatidic acid, PEG-ceramide conjugates (e.g., PEG-CerC14 or PEG-CerC20) (described in co-pending USSN 08/486,214, incorporated herein by reference), PEG-modified dialkylamines and PEG-modified 1,2-diacyloxypropane-3-amines. Particularly preferred are PEG-modified diglycerides and dialkylglycerols.

In embodiments where a sterically larger moiety, such as PEG or ATTA, is conjugated to a lipid anchor, the choice of lipid anchor depends on the type of form in which the conjugate is conjugated to the lipid particle. It is well known that mePEG(mw2000)-distearoylphosphatidylethanolamine (PEG-DSPE) will remain conjugated to liposomes until the particles are cleared from the circulation, which can be on the order of several days. Other conjugates, such as PEG-CerC20, have similar retention capabilities. However, upon contact with serum, PEG-CerC14 rapidly exchanges out of the formulation, with a T _½ of less than 60 minutes in some experiments. As described in U.S. Patent Application SN 08/486,214, at least three characteristics influence the exchange rate: the length of the acyl chain, the degree of acyl chain saturation, and the size of the sterically hindering head group. Compounds with appropriate variations in these characteristics can be used in the present invention. In some therapeutic applications, it is preferred that the PEG-modified lipid rapidly dissociates from the nucleic acid-lipid particle in vivo, thereby allowing the PEG-modified lipid to have a relatively short lipid anchor. In other therapeutic applications, it is preferred that the nucleic acid-lipid particles have a long plasma circulation time, and thus the PEG-modified lipids will have relatively long lipid anchors. Typical lipid anchors include those with a length of about C ₁₄ to about C ₂₂ , preferably about C ₁₄ to about C _16. In some embodiments, the size of the PEG moiety, e.g., mPEG-NH ₂ , is about 1000, 2000, 5000, 10,000, 15,000, or 20,000 Daltons.

It should be noted that aggregation-preventing compounds do not necessarily require lipid conjugation to function properly. Free PEG or free ATTA in solution may be sufficient to prevent aggregation. If the particles are stable after preparation into a formulation, PEG or ATTA can be dialyzed away before administration to a subject.

The conjugated lipid that inhibits particle aggregation can be, for example, polyethylene glycol (PEG)-lipids, including but not limited to, PEG-diaceryl (DAG), PEG-dialkyloxypropyl (DAA), PEG-phospholipids, PEG-ceramide (Cer) or mixtures thereof. For example, the PEG-DAA conjugate can be PEG-dilauryloxypropyl (Ci ₂ ), PEG-dimyristyloxypropyl (Ci ₄ ), PEG-dipalmityloxypropyl (Ci ₆ ) or PEG-distearyloxypropyl (Ci ₈ ). Other conjugated lipids include polyethylene glycol-dimyristyl glycerol (C14-PEG or PEG-C14, wherein the average molecular weight of PEG is 2000 Da) (PEG-DMG); (R)-2,3-dioctadecyloxypropyl 1-(methoxypoly(ethylene glycol) 2000) propylcarbamate) (PEG-DSG); PEG-carbamoyl-1,2-dimyristyloxypropylamine, wherein the average molecular weight of PEG is 2000 Da (PEG-cDMA); N-acetylgalactosamine-((R)-2,3-dioctadecyloxypropyl 1-(methoxypoly(ethylene glycol) 2000) propylcarbamate)) (GalNAc-PEG-DSG); and polyethylene glycol-dipalmityl glycerol (PEG-DPG).

In one embodiment, the conjugated lipid is PEG-DMG. In another embodiment, the conjugated lipid is PEG-cDMA. In yet another embodiment, the conjugated lipid is PEG-DPG. Alternatively, the conjugated lipid is GalNAc-PEG-DSG.

The conjugated lipid that prevents particle aggregation can be 0 mol% to about 20 mol% or about 0.5 to about 5.0 mol% or about 2 mol% of the total lipid present in the particle.

When present, the sterol component of the lipid mixture may be any sterol commonly used in the art of liposome, lipid vesicle or lipid particle formulation. A preferred sterol is cholesterol.

In some embodiments, the nucleic acid-lipid particle further comprises a sterol, such as cholesterol, comprising, for example, about 10 mol% to about 60 mol% or about 25 to about 40 mol% or about 48% of the total lipids present in the particle.

lipoprotein

In one embodiment, the formulation of the present invention further comprises apolipoprotein. As used herein, the term "apolipoprotein" or "lipoprotein" refers to apolipoprotein and variants and fragments thereof known to those skilled in the art, as well as to apolipoprotein agonists, analogs or fragments thereof as described below.

Suitable apolipoproteins include, but are not limited to, ApoA-I, ApoA-II, ApoA-IV, ApoA-V, and ApoE, and their active polymorphs, isoforms, variants, mutants, and fragments or truncated forms. In certain embodiments, the apolipoprotein is a thiol-containing apolipoprotein. "Thiol-containing apolipoprotein" refers to an apolipoprotein, variant, fragment, or isoform that contains at least one cysteine residue. The most commonly used thiol-containing apolipoproteins are ApoA-IMilano (ApoA- _{IM) and ApoA-I Paris (ApoA-IP )} , which contain one cysteine residue (Jia et al., 2002, Biochem. Biophys. Res. Comm. 297:206-13; Bielicki and Oda, 2002, Biochemistry 41:2089-96). ApoA-II, ApoE2, and ApoE3 are also thiol-containing apolipoproteins. Isolated ApoE and/or active fragments and polypeptide analogs thereof (including recombinantly produced forms thereof) are described in US Patent Nos. 5,672,685; 5,525,472; 5,473,039; 5,182,364; 5,177,189; 5,168,045; 5,116,739, the disclosures of all of which are incorporated herein by reference. ApoE3 is described in Weisgraber, et al., "Human E apoprotein heterogeneity: cysteine-arginine interchanges in the amino acid sequence of the apo-E isoforms," J. Biol. Chem. (1981) 256:9077-9083 and Rall, et al., "Structural basis for receptor binding heterogeneity of apolipoprotein E from type III hyperlipoproteinemic subjects," Proc. Nat. Acad. Sci. (1982) 79:4696-4700 (see also GenBank Accession No. K00396).

In certain embodiments, the apolipoprotein may be its mature form, its preproapolipoprotein form or its proapolipoprotein form. Within the scope of the present invention, pro-ApoA-I and mature ApoA-I (Duverger et al., 1996, Arterioscler. Thromb. Vasc. Biol. 16(12): 1424-29), ApoA-I Milano (Klon et al., 2000, Biophys. J. 79: (3) 1679-87; Franceschini et al., 1985, J. Biol. Chem. 260: 1632-35), ApoA-I Homo- and heterodimers (where applicable) of ApoA-11 (Daum et al., 1999, J. Mol. Med. 77:614-22), ApoA-II (Shelness et al., 1985, J. Biol. Chem. 260(14):8637-46; Shelness et al., 1984, J. Biol. Chem. 259(15):9929-35), ApoA-IV (Duverger et al., 1991, Euro. J. Biochem. 201(2):373-83), and ApoE (McLean et al., 1983, J. Biol. Chem. 258(14):8993-9000).

In certain embodiments, the apolipoprotein can be a fragment, variant or isoform of an apolipoprotein. The term "fragment" refers to any apolipoprotein whose amino acid sequence is shorter than that of a native apolipoprotein, and the fragment retains the activity of the native apolipoprotein, including lipid binding. A "variant" refers to a replacement or change in the amino acid sequence of an apolipoprotein, wherein the replacement or change (e.g., increase or deletion of an amino acid residue) does not eliminate the activity of the native apolipoprotein, including lipid binding. Therefore, a variant can comprise a protein or peptide whose amino acid sequence is substantially identical to that of the native apolipoprotein provided by the present invention, wherein one or more amino acid residues are conservatively substituted by chemically similar amino acids. Examples of conservative substitutions include replacing other residues with at least one hydrophobic residue such as isoleucine, valine, leucine or methionine. Similarly, for example, the present invention relates to substitutions between at least one hydrophilic residue such as arginine and lysine, between glutamine and asparagine, and between glycine and serine (see U.S. Patent Nos. 6,004,925, 6,037,323 and 6,046,166). The term "isoform" refers to proteins that have the same, additional or partial functions and similar, identical or partial sequences and may or may not be products of the same gene and are generally tissue specific (see Weisgraber 1990, J. Lipid Res. 31(8): 1503-11; Hixson and Powers 1991, J. Lipid Res. 32(9): 1529-35; Lackner et al., 1985, J. Biol. Chem. 260(2): 703-6; Hoeg et al., 1986, J. Biol. Chem. 261(9): 3911-4; Gordon et al., 1984, J. Biol. Chem. 259(1): 468-74; Powell et al., 1987, Cell Biol Chem. 261 ... 50(6):831-40; Aviram et al., 1998, Arterioscler. Thromb. Vase. Biol. 18(10):1617-24; Aviram et al., 1998, J. Clin. Invest. 101(8):1581-90; Billecke et al., 2000, Drug Metab. Dispos. 28(11):1335-42; Draganov et al., 2000, J. Biol. Chem. 275(43):33435-42; Steinmetz and Utermann 1985, J. Biol. Chem. 260(4):2258-64; Widler et al., 1980, J. Biol. Chem. 255(21):10464-71; Dyer et al., 1995, J. Lipid Res. 36(1): 80-8; Sacre et al., 2003, FEBS Lett. 540(1-3): 181-7; Weers, et al., 2003, Biophys. Chem. 100(1-3): 481-92; Gong et al., 2002, J. Biol. Chem. 277(33): 29919-26; Ohta et al., 1984, J. Biol. Chem. 259(23): 14888-93 and U.S. Pat. No. 6,372,886).

In certain embodiments, the methods and compositions of the present invention include the use of chimeric structures of apolipoproteins. For example, the chimeric structure of an apolipoprotein can be composed of an apolipoprotein domain with high lipid binding capacity, and the binding capacity is related to an apolipoprotein domain comprising ischemia-reperfusion protective properties. The chimeric structure of an apolipoprotein can be a structure comprising a partition region within an apolipoprotein (i.e., a homologous structure) or a chimeric structure can be a structure comprising a partition region between different apolipoproteins (i.e., a heterogeneous structure). Compositions containing chimeric constructs may also include fragments that are variants of apolipoproteins or fragments designed to have specific properties (e.g., lipid binding, receptor binding, enzyme, enzyme activation, antioxidant, or reduction-oxidation properties) (see Weisgraber 1990, J. Lipid Res. 31(8):1503-11; Hixson and Powers 1991, J. Lipid Res. 32(9):1529-35; Lackner et al., 1985, J. Biol. Chem. 260(2):703-6; Hoeg et al., 1986, J. Biol. Chem. 261(9):3911-4; Gordon et al., 1984, J. Biol. Chem. 259(1):468-74; Powell et al., 1987, Cell 50(6):831-40; Aviram et al., 1998, Arterioscler. Thromb. Vasc. Biol. 18(10):1617-24; Aviram et al., 1998, J. Clin. Invest. 101(8):1581-90; Billecke et al., 2000, Drug Metab. Dispos. 28(11):1335-42; Draganov et al., 2000, J. Biol. Chem. 275(43):33435-42; Steinmetz and Utermann 1985, J. Biol. Chem. 260(4):2258-64; Widler et al., 1980, J. Biol. Chem. 255(21):10464-71; Dyer et al., 1995, J. Lipid Res. 36(1): 80-8; Sorenson et al., 1999, Arterioscler. Thromb. Vasc. Biol. 19(9): 2214-25; Palgunachari 1996, Arterioscler. Throb. Vasc. Biol. 16(2): 328-38: Thurberg et al., J. Biol. Chem. 271(11): 6062-70; Dyer 1991, J. Biol. Chem. 266(23): 150009-15; Hill 1998, J. Biol. Chem. 273(47): 30979-84).

The apolipoprotein used in the present invention also includes recombinant, synthetic, semisynthetic or purified apolipoprotein. The methods for obtaining apolipoprotein or its equivalent used in the present invention are well known in the art. For example, apolipoproteins can be isolated from plasma or natural products, e.g., by density gradient centrifugation or immunoaffinity chromatography, or prepared synthetically or semisynthetically using recombinant DNA techniques known in the art (e.g., see Mulugeta et al., 1998, J. Chromatogr. 798(1-2):83-90; Chung et al., 1980, J. Lipid Res. 21(3):284-91; Cheung et al., 1987, J. Lipid Res. 28(8):913-29; Persson, et al., 1998, J. Chromatogr. 711:97-109; U.S. Pat. Nos. 5,059,528, 5,834,596, 5,876,968 and 5,721,114; and PCT Publications WO 86/04920 and WO 87/02062).

The apolipoproteins used in the present invention also include apolipoprotein agonists, such as peptides and peptide analogs that mimic the activity of ApoA-I, ApoA-IMilano (ApoA- _IM ), ApoA-I Paris (ApoA- _IP ), ApoA-II, ApoA-IV, and ApoE. For example, the apolipoprotein can be any of the apolipoproteins described in U.S. Patent Nos. 6,004,925, 6,037,323, 6,046,166, and 5,840,688, the contents of which are incorporated herein by reference.

Apolipoprotein agonist peptides or peptide analogs can be synthesized or prepared using any peptide synthesis technique known in the art, including, for example, the techniques described in U.S. Patent Nos. 6,004,925, 6,037,323, and 6,046,166. For example, the peptides can be prepared using the solid phase synthesis technique first described by Merrifield (1963, J. Am. Chem. Soc. 85: 2149-2154). Other peptide synthesis techniques can be found in Bodanszky et al., Peptide Synthesis, John Wiley & Sons, 2d Ed., (1976), and other references readily available to those skilled in the art. An overview of peptide synthesis techniques can be found in Stuart and Young, Solid Phase Peptide. Synthesis, Pierce Chemical Company, Rockford, 111., (1984). Peptides can also be synthesized by solution methods such as those described in The Proteins, Vol. II, 3d Ed., Neurath et al., Eds., p. 105-237, Academic Press, New York, N.Y. (1976). Suitable protecting groups for the synthesis of various peptides are described in the above-mentioned references and in McOmie, Protective Groups in Organic Chemistry, Plenum Press, New York, N.Y. (1973). The peptides of the present invention can also be prepared by chemical or enzymatic cleavage of larger portions of, for example, apolipoprotein A-I.

In certain embodiments, the apolipoprotein can be an apolipoprotein mixture. In one embodiment, the apolipoprotein can be a homogeneous mixture, i.e., a single type of apolipoprotein. In another embodiment, the apolipoprotein can be a heterogeneous mixture of apolipoproteins, i.e., a mixture of two or more different apolipoproteins. Embodiments of heterogeneous mixtures of apolipoproteins can, for example, include a mixture of apolipoproteins of animal origin and apolipoproteins of semi-synthetic origin. In certain embodiments, the heterogeneous mixture can, for example, include a mixture of ApoA-I and ApoA-I Milano. In certain embodiments, the heterogeneous mixture can, for example, include a mixture of ApoA-I Milano and ApoA-I Paris. Suitable mixtures for use in the methods and compositions of the present invention will be apparent to those skilled in the art.

If the apolipoprotein is obtained from a natural source, it can be obtained from a plant or animal source. If the apolipoprotein is obtained from an animal source, the apolipoprotein can be from any species. In certain embodiments, the apolipoprotein can be obtained from an animal source. In certain embodiments, the apolipoprotein can be obtained from a human source. In a preferred embodiment of the present invention, the apolipoprotein is derived from the same species as the individual to whom the apolipoprotein is administered.

Other components

In many embodiments, amphiphilic lipids are included in lipid granules of the present invention. " amphiphilic lipids " means any suitable material, wherein the hydrophobic portion of the lipid material points to the hydrophobic phase, and the hydrophilic portion points to the aqueous phase. This compound includes but is not limited to phospholipids, amino lipids and sphingolipids. Typical phospholipids include sphingomyelin, lecithin, phosphatidylethanolamine, phosphatidylserine, phosphatidyl inositol, phosphatidic acid, palmitoyloleoylphosphatidylcholine, lysolecithin, lysolecithin, dipalmitoyloleoylphosphatidylcholine, dioleoylphosphatidylcholine, distearoylphosphatidylcholine or dilinoleylphosphatidylcholine. Other phosphorus-free compounds can also be used, such as sphingolipids, glycosphingolipid family, diglyceride and beta-acyloxy acid. This amphiphilic lipid can easily mix with other lipids such as triglyceride and sterol.

Also suitable for inclusion in the lipid granules of the present invention are designable fusogenic lipids. Such lipid granules have a tendency to fuse with the cell membrane and deliver their payload until a given signal event occurs. This allows the lipid granules to be more evenly distributed after being injected into an organism or lesion, and then they begin to fuse with the cells. The signal event can be, for example, a change in pH, temperature, ionic environment, or time. In the latter case, fusion delays or "covering" components, such as ATTA-lipid conjugates or PEG-lipid conjugates, can simply be exchanged out of the lipid granule membrane over time. Typical lipid anchors include lengths of about C ₁₄ to about C ₂₂ , preferably about C ₁₄ to about C _16. In some embodiments, the size of the PEG moiety (e.g., mPEG-NH ₂ ) is about 1000, 2000, 5000, 10000, 15000, or 20000 daltons.

The lipid particles conjugated to the nucleic acid agent can also include a targeting moiety, for example, a targeting moiety that specifically targets a cell type or tissue. Targeting of lipid particles using a variety of targeting moieties, such as ligands, cell surface receptors, glycoproteins, vitamins (e.g., riboflavin), and monoclonal antibodies has been previously described (e.g., see U.S. Patent Nos. 4,957,773 and 4,603,044). The targeting moiety can include an entire protein or a fragment thereof. The targeting mechanism generally requires that the targeting agent be located on the surface of the lipid particle in such a manner that the targeting moiety is available for interaction with the target (e.g., a cell surface receptor). A variety of different targeting agents and methods are known and available in the art, including those described in Sapra, P. and Allen, TM, Prog. Lipid Res. 42(5): 439-62 (2003); and Abra, RM et al., J. Liposome Res. 12: 1-3, (2002).

Lipid particles, i.e., liposomes, together with a surface coating of hydrophilic polymer chains, such as polyethylene glycol (PEG) chains, have been proposed for targeting (Allen, et al., Biochimica et Biophysica Acta 1237:99-108 (1995); DeFrees, et al., Journal of the American Chemistry Society 118:6101-6104 (1996); Blume, et al., Biochimica et Biophysica Acta 1149:180-184 (1993); Klibanov, et al., Journal of Liposome Research 2:321-334 (1992); U.S. Patent No. 5,013556; Zalipsky, Bioconjugate Chemistry 4:296-299 (1993); Zalipsky, FEBS Letters 353:71-74 (1994); Zalipsky ...321-334 (1992); U.S. Patent No. 5,013556; Zalipsky, Bioconjugate Chemistry 4:321-334 (1992); U.S. Patent No. 5,013556; Zalipsky, Bioconjugate Chemistry 4:321-334 (1993); Za Stealth Liposomes Chapter 9 (Lasic and Martin, Eds) CRC Press, Boca Raton FL (1995). In one approach, a ligand, such as an antibody, for targeting the lipid particle is attached to the polar head group of the lipid that forms the lipid particle. In another approach, the targeting ligand is attached to the distal end of a PEG chain that forms a hydrophilic polymer coating (Klibanov, et al., Journal of Liposome Research 2: 321-334 (1992); Kirpotin et al., FEBS Letters 388: 115-118 (1996)).

Standard methods for coupling targeting agents can be used. For example, phosphatidylethanolamine, which can be activated for attachment of a targeting agent, or derivatized lipophilic compounds, such as lipid-derivatized bleomycin, can be used. Antibody-targeted liposomes can be constructed, for example, using liposomes conjugated to protein A (see, Renneisen, et al., J. Bio. Chem., 265: 16337-16342 (1990) and Leonetti, et al., Proc. Natl. Acad. Sci. (USA), 87: 2448-2451 (1990). Other examples of antibody conjugates are disclosed in U.S. Pat. No. 6,027,726, the teachings of which are incorporated herein by reference. Examples of targeting moieties can also include other proteins specific for cellular components, including antigens associated with tumors or tumors. Proteins used as targeting moieties can be linked to liposomes via covalent bonds (see Heath, Covalent Attachment of Proteins to Liposomes, 149 Methods in Enzymology 111-119 (Academic Press, Inc. 1987)). Other targeting methods include the biotin-avidin system.

Preparation of nucleic acid-lipid particles

In one embodiment, the nucleic acid-lipid particle formulations of the present invention are prepared via extrusion or in-line mixing.

Extrusion method (also referred to as preforming method or batch method) is to first prepare empty liposomes (i.e., without nucleic acid), and then add nucleic acid into the method of empty liposomes. The liposome components are squeezed out through a small-pore polycarbonate membrane or an uneven ceramic membrane to cause a relatively well-defined particle size distribution. Typically, the suspension is circulated through the membrane once or many times until the desired liposome complex particle size distribution is achieved. The liposomes can be continuously extruded through a small-pore membrane to achieve a gradual reduction in liposome size. In some cases, the lipid-nucleic acid composition formed can be used without the need to determine the size. These methods are disclosed in US 5,008,050; US 4,927,637; US 4,737,323; Biochim Biophys Acta. 1979 Oct 19; 557(1): 9-23; Biochim Biophys Acta. 1980 Oct 2; 601(3): 559-7; Biochim Biophys Acta. 1986 Jun 13; 858(1): 161-8; and Biochim. Biophys. Acta 1985 812, 55-65, which are incorporated herein by reference in their entirety.

In-line mixing is a method in which lipids and nucleic acids are added together into a mixing chamber. The mixing chamber can be a simple T-connector or any other mixing chamber known to those skilled in the art. These methods are disclosed in U.S. Patent Nos. 6,534,018 and 6,855,277; US Publication No. 2007/0042031 and Pharmaceuticals Research, Vol. 22, No. 3, Mar. 2005, pp. 362-372, which are incorporated herein by reference in their entirety.

It will also be understood that the formulations of the present invention may be prepared by any method known to those skilled in the art.

Characterization of nucleic acid-lipid particles

Formulations prepared by standard or non-extrusion methods can be characterized in a similar manner. For example, formulations are typically characterized by visual inspection. They should be whitish, transparent solutions free of aggregates or precipitates. The particle size and size distribution of lipid-nanoparticles can be determined by light scattering, for example, using a Malvern Zetasizer Nano ZS (Malvern, USA). The particle size should be approximately 20-300 nm, for example, 40-100 nm. The particle size distribution should be unimodal. A dye exclusion assay can be used to assess the total siRNA concentration and the captured fraction in the formulation. Prepared siRNA samples can be incubated with an RNA-binding dye such as Ribogreen (Molecular Probes) in the presence or absence of a surfactant that disrupts the formulation, such as 0.5% Triton-X100. The total siRNA in the formulation can be determined by comparing the signal emitted in the sample containing the surfactant to a standard curve. The captured fraction is determined by subtracting the "free" siRNA content (determined by the signal in the absence of the surfactant) from the total siRNA content. The percentage of captured siRNA is typically >85%. In one embodiment, the formulation of the invention is at least 75%, at least 80%, or at least 90% entrapped.

For nucleic acid-lipid particle preparations, particle diameter is at least 30nm, at least 40nm, at least 50nm, at least 60nm, at least 70nm, at least 80nm, at least 90nm, at least 100nm, at least 110nm and at least 120nm. Suitable scope is typically about at least 50nm to about at least 110nm, about at least 60nm to about at least 100nm or about at least 80nm to about at least 90nm.

Nucleic acid-lipid particle preparations

LNP01

An example of a synthetic nucleic acid-lipid particle is as follows. Nucleic acid-lipid particles are synthesized using lipidoid ND98·4HCl (MW1487) (Formula 1), cholesterol (Sigma-Aldrich), and PEG-ceramide C16 (Avanti Polar Lipids). The nucleic acid-lipid particles are sometimes referred to as LNP01 particles. Stock solutions of each in ethanol can be prepared as follows: ND98, 133 mg/ml; cholesterol, 25 mg/ml, PEG-ceramide C16, 100 mg/ml. The stock solutions of ND98, cholesterol, and PEG-ceramide C16 can then be combined, for example, in a molar ratio of 42:48:10. The combined lipid solution is mixed with aqueous siRNA (e.g., in sodium acetate at pH 5) such that the final ethanol concentration is about 35-45% and the final sodium acetate concentration is about 100-300 mM. Once mixed, lipid-siRNA nanoparticles typically form spontaneously. In some embodiments, the nanoparticle mixture can be prepared by extruding a mixture of nanoparticles of different sizes using a thermobarrier extruder. The nanoparticles ...

LNP01 formulations are described, for example, in International Application Publication No. WO 2008/042973, which is incorporated herein by reference.

Other typical nucleic acid-lipid particle preparations are described in the table below.Should be understood that the title of the nucleic acid-lipid particle in the table is not restrictive.For example, as used in the present invention, term SNALP means the preparation comprising the cationic lipid DLinDMA.

Formulations containing XTC are described, for example, in U.S. Provisional Serial No. 61/239,686, filed September 3, 2009, which is incorporated herein by reference.

Formulations containing MC3 are described, for example, in U.S. Provisional Serial No. 61/244,834, filed September 22, 2009, and U.S. Provisional Serial No. 61/185,800, filed June 10, 2009, which are incorporated herein by reference.

Formulations containing ALNY-100 are described, for example, in International Patent Application PCT/US09/63933, filed November 10, 2009, which is incorporated herein by reference.

Other typical formulations are described in Tables 25 and 26. Lipid refers to a cationic lipid.

Table 25: Composition (mol %) of typical nucleic acid-lipid particles prepared via extrusion

Table 26: Composition of typical nucleic acid-lipid particles prepared via in-line mixing method

Synthesis of cationic lipids

Any compound used in the nucleic acid-lipid particles of the present invention, such as cationic lipids, can be prepared by known organic synthesis techniques (including the methods described in more detail in the Examples). Unless otherwise indicated, all substituents are defined as follows.

"Alkyl" refers to a straight or branched, acyclic or cyclic, saturated aliphatic hydrocarbon containing 1 to 24 carbon atoms. Typical saturated straight-chain alkyl groups include methyl, ethyl, n-propyl, n-butyl, n-pentyl, and n-hexyl; while saturated branched-chain alkyl groups include isopropyl, sec-butyl, isobutyl, tert-butyl, and isopentyl. Typical saturated cyclic alkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl; while unsaturated cyclic alkyl groups include cyclopentenyl and cyclohexenyl.

"Alkenyl" refers to an alkyl group as defined above that contains at least one double bond between adjacent carbon atoms. Alkenyl groups include cis- and trans-isomers. Typical straight-chain and branched alkenyl groups include ethenyl, propenyl, 1-butenyl, 2-butenyl, isobutenyl, 1-pentenyl, 2-pentenyl, 3-methyl-1-butenyl, 2-methyl-2-butenyl, 2,3-dimethyl-2-butenyl, and the like.

"Alkynyl" refers to any alkyl or alkenyl group as defined above that further comprises at least one triple bond between adjacent carbon atoms. Typical straight and branched chain alkynyl groups include ethynyl, propynyl, 1-butynyl, 2-butynyl, 1-pentynyl, 2-pentynyl, 3-methyl-1-butynyl, and the like.

"Acyl" refers to any alkyl, alkenyl, or alkynyl group substituted with an oxy group at the carbon at the point of attachment, as defined below. For example, -C(=O)alkyl, -C(=O)alkenyl, and -C(=O)alkynyl are acyl groups.

"Heterocycle" refers to a 5- to 7-membered monocyclic or 7- to 10-membered bicyclic or heterocyclic ring, which may be saturated, unsaturated or aromatic, and which contains 1 or 2 heteroatoms independently selected from nitrogen, oxygen and sulfur, and the nitrogen and sulfur heteroatoms may optionally be oxidized, and the nitrogen heteroatom may optionally be quaternized, and the heterocycle also includes a bicyclic ring in which any of the above heterocycles is fused to a benzene ring. The heterocycle may be connected via any heteroatom or carbon atom. Heterocycle includes heteroaryl as defined below. Heterocycle includes morpholinyl, pyrrolidinonyl, pyrrolidinyl, piperidinyl, piperazinyl, hydantoinyl, valerolactamyl, oxirane, oxetanyl, tetrahydrofuranyl, tetrahydropyranyl, tetrahydropyridinyl, tetrahydropyrimidinyl, tetrahydrothiophenyl, tetrahydrothiopyranyl, tetrahydropyrimidinyl, tetrahydrothiophenyl, tetrahydrothiopyranyl, etc.

The terms "optionally substituted alkyl,""optionally substituted alkenyl,""optionally substituted alkynyl,""optionally substituted acyl," and "optionally substituted heterocycle" mean that, when substituted, at least one hydrogen atom is replaced with a substituent. In the case of an oxygen substituent (=O), two hydrogen atoms are replaced. In this regard, substituents include oxygen, halogen, heterocycle, -CN, ^-ORx , ^-NRxRy , ^-NRxC (=O) ^Ry , ^-NRxSO2Ry , ^-C (=O) _Rx , -C(=O) ^ORx , ^-C (=O) ^NRxRy , ^-SONRx _, and ^-SONNRxRy , wherein n is 0, 1, or ² , Rx _and ^Ry are the same or different and are independently hydrogen, ^alkyl , or heterocycle, and each of the alkyl and ^heterocycle substituents may be further substituted with one or more of the ^following : oxygen, halogen, ^-OH , -CN, alkyl, ^-ORx , heterocycle, ^-NRxRy , ^{-NRxC (} =O) ^Ry _, ^-NRxSO2Ry , -C(=O) ^Rx , -C(=O) ^ORx , -C(=O) ^NRxRy , ^-SONRx _, and ^-SONNRxRy _. NR ^x R ^y .

"Halogen" refers to fluorine, chlorine, bromine and iodine.

In some embodiments, the inventive method may need to use a protecting group. Protecting group methods are well known to those skilled in the art (e.g., see Protective Groups in Organic Synthesis, Green, T.W. et al., Wiley-Interscience, New York City, 1999). In short, the protecting group in the context of the present invention is any group that reduces or eliminates the reactivity of unwanted functional groups. Protecting groups can be added to functional groups to shield their reactivity during certain reactions, and then the protecting groups are removed to reveal the original functional groups. In some embodiments, "alcohol protecting groups" are used. "Alcohol protecting groups" are any groups that reduce or eliminate the reactivity of unwanted alcohol functional groups. Technology well known in the art can be used to increase and remove protecting groups.

Synthesis of Formula A

In one embodiment, the nucleic acid-lipid particles of the present invention are formulated using a cationic lipid of formula A:

Wherein R1 and R2 are independently alkyl, alkenyl or alkynyl, each group can be optionally substituted, and R3 and R4 are independently low alkyl or R3 and R4 can be combined to form an optionally substituted heterocycle. In some embodiments, the cationic lipid is XTC (2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane). Generally, the lipid of the above formula A can be prepared by the following reaction schemes 1 or 2, wherein unless otherwise indicated, all substituents are as defined above.

Solution 1

Lipid A can be prepared according to Scheme 1, wherein R ₁ and R ₂ are independently alkyl, alkenyl, or alkynyl, each of which may be optionally substituted, and R ₃ and R ₄ are independently lower alkyl or R ₃ and R ₄ may be combined to form an optionally substituted heterocycle. Ketone 1 and bromide 2 can be purchased or prepared according to methods known to those skilled in the art. The reaction of 1 and 2 produces ketal 3. Ketal 3 is treated with amine 4 to produce a lipid of Formula A. The lipid of Formula A can be converted to the corresponding ammonium salt using an organic salt of Formula 5, wherein X is an anionic counterion selected from halogen, hydroxide, phosphate, sulfate, and the like.

Option 2

Alternatively, the ketone 1 starting material can be prepared according to Scheme 2. Grignard reagent 6 and cyanide 7 can be purchased or prepared according to methods known to those skilled in the art. The reaction of 6 and 7 produces ketone 1. The conversion of ketone 1 to the corresponding lipid of Formula A is described in Scheme 1.

Synthesis of MC3

The preparation of DLin-M-C3-DMA (i.e. 4-(dimethylamino) butyric acid (6Z, 9Z, 28Z, 31Z)-triacontac-6,9,28,31-tetraene-19-base ester)) is as follows.At room temperature stir (6Z, 9Z, 28Z, 31Z)-triacontac-6,9,28,31-tetraene-19-alcohol (0.53g), 4-N, N-dimethylaminobutyric acid hydrochloride (0.51g), 4-N, the methylene chloride (5mL) solution of N-dimethylaminopyridine (0.61g) and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (0.53g) spends the night.With dilute hydrochloric acid washing this solution, then use the sodium bicarbonate aqueous solution washing of dilution.Organic part anhydrous magnesium sulfate drying, filters and removes solvent on rotary evaporator. The residue was passed through a silica gel column (20 g) using a 1-5% methanol/dichloromethane gradient. Fractions containing the purified product were combined and the solvent removed to give a colorless oil (0.54 g).

Synthesis of ALNY-100

The synthesis of ketal 519 [ALNY-100-100] was carried out using the following Scheme 3:

Synthesis of 515:

To a stirred suspension of LiAlH4 (3.74 g, 0.09852 mol) in 200 ml of anhydrous THF in a double-necked RBF (1 L) at 0°C under a nitrogen atmosphere was slowly added 514 (10 g, 0.04926 mol) in 70 ml of THF. After complete addition, the reaction mixture was heated to room temperature and then heated to reflux for 4 h. The progress of the reaction was monitored by TLC. After the reaction was complete (monitored by TLC), the mixture was cooled to 0°C and quenched by carefully adding _a saturated _Na2SO4 solution. The reaction mixture was stirred at room temperature for 4 h and filtered. The residue was appropriately washed with THF. The filtrate and washings were mixed and diluted with 400 ml of dioxane and 26 ml of concentrated HCl and stirred at room temperature for 20 minutes. The volatiles were removed under vacuum to obtain the hydrochloride salt of 515 as a white solid. Yield: 7.12 g, 1H-NMR (DMSO, 400 MHz): δ = 9.34 (broad, 2H), 5.68 (s, 2H), 3.74 (m, 1H), 2.66-2.60 (m, 2H), 2.50-2.45 (m, 5H).

Synthesis of 516:

To a stirred solution of compound 515 in 100 mL of anhydrous DCM in a 250 mL two-necked RBF was added NEt (37.2 mL, 0.2669 mol) and cooled to 0°C under a nitrogen atmosphere. After slowly adding N-(benzyloxy-carbonyloxy)-succinimide (20 g, 0.08007 mol) in 50 mL of anhydrous DCM, the reaction mixture was allowed to warm to room temperature. After completion of the reaction (2-3 h, monitored by TLC), the mixture was washed with 1N HCl solution (1 x 100 mL) and then saturated _NaHCO solution (1 x 50 mL). The organic layer was then dried over _{anhydrous NaSO} and the solvent evaporated to afford the crude material, which was purified by silica gel column chromatography to afford 516 as a sticky mass. Yield: 11 g (89%). 1H-NMR (CDCl3, 400MHz): δ=7.36-7.27 (m, 5H), 5.69 (s, 2H), 5.12 (s, 2H), 4.96 (br., 1H), 2.74 (s, 3H), 2.60 (m, 2H), 2.30-2.25 (m, 2H). LC-MS [M+H] - 232.3 (96.94%).

Synthesis of 517A and 517B:

Cyclopentene 516 (5 g, 0.02164 mol) was dissolved in 220 mL of acetone and water (10:1) in a single-necked 500 mL RBF. N-methylmorpholine-N-oxide (7.6 g, 0.06492 mol) and 4.2 mL of a 7.6% solution of OsO (0.275 g, 0.00108 mol) in tert-butanol were added at room temperature. After the reaction was complete (approximately 3 h), the mixture was quenched by the addition _of solid _Na₂SO₃ and stirred at room temperature for 1.5 h. The reaction mixture was diluted with DCM (300 mL) and washed with water (2 x 100 mL), then saturated _NaHCO₃ (1 x 50 mL), water (1 x 30 mL), and finally brine (1 x 50 mL). The organic phase was dried _over anhydrous _Na₂SO₄ and the solvent was removed in vacuo. The crude material was purified by silica gel column chromatography to give a mixture of diastereomers which were separated by preparative HPLC. Yield: 6 g crude product.

517A-Peak-1 (white solid), 5.13 g (96%). 1H-NMR (DMSO, 400 MHz): δ = 7.39-7.31 (m, 5H), 5.04 (s, 2H), 4.78-4.73 (m, 1H), 4.48-4.47 (d, 2H), 3.94-3.93 (m, 2H), 2.71 (s, 3H), 1.72-1.67 (m, 4H). LC-MS - [M+H] - 266.3, [M+NH4+] - 283.5 present, HPLC - 97.86%. Stereochemistry confirmed by X-ray.

Synthesis of 518:

Compound 518 (1.2 g, 41%) was obtained as a colorless oil using a method similar to that used to synthesize compound 505. 1H-NMR (CDCl 3 , 400 MHz): δ = 7.35-7.33 (m, 4H), 7.30-7.27 (m, 1H), 5.37-5.27 (m, 8H), 5.12 (s, 2H), 4.75 (m, 1H), 4.58-4.57 (m, 2H), 2.78-2.74 (m, 7H), 2.06-2.00 (m, 8H), 1.96-1.91 (m, 2H), 1.62 (m, 4H), 1.48 (m, 2H), 1.37-1.25 (br m, 36H), 0.87 (m, 6H). HPLC-98.65%.

General procedure for the synthesis of compound 519:

A solution of compound 518 (1 eq) in hexane (15 mL) was added dropwise to an ice-cold solution of LAH in THF (1 M, 2 eq). After complete addition, the mixture was heated at 40°C for 0.5 h and then cooled again in an ice bath. The mixture was carefully hydrolyzed with saturated _{aqueous Na2SO4} solution and then filtered through Celite to reduce to an oil. Column chromatography provided purified 519 (1.3 g, 68%) as a colorless oil. 13C NMR＝130.2, 130.1(x2), 127.9(x3), 112.3, 79.3, 64.4, 44.7, 38.3, 35.4, 31.5, 29.9(x2), 29.7, 29.6(x2), 29.5(x3), 29.3(x2), 27.2(x3), 25.6, 24.5, 23.3, 226, 14.1; Electrospray MS (+ve): Molecular weight (M+H)+ calculated for C44H80NO2 654.6, found 654.6.

Therapeutic agent-lipid particle compositions and formulations

The present invention includes compositions containing lipid granules of the present invention and activating agent, wherein said activating agent is combined with said lipid granules.In a specific embodiment, said activating agent is a therapeutic agent.In a specific embodiment, said activating agent is encapsulated in the aqueous interior of lipid granules.In other embodiments, said activating agent is present in one or more lipid layers of lipid granules.In other embodiments, said activating agent is combined with the outside or inner lipid surface of lipid granules.

As used herein, "completely encapsulated" means that the nucleic acid in the particles is not significantly degraded after exposure to serum or nuclease assays that significantly degrade free DNA. In a completely encapsulated system, preferably less than 25% of the particle nucleic acid is degraded in a treatment that normally degrades 100% of the free nucleic acid, more preferably less than 10% and most preferably less than 5% of the particle nucleic acid is degraded. Alternatively, complete encapsulation can be determined experimentally. FLEX(R) is an ultrasensitive fluorescent nucleic acid stain for quantifying oligonucleotides and single-stranded DNA in solution (available from Invitrogen Corporation, Carlsbad, CA). Complete encapsulation also indicates that the particles are serum stable, i.e., they do not rapidly break down into their component parts upon in vivo administration.

Active agents used in the present invention include any molecule or compound capable of exerting a desired effect on a cell, tissue, organ, or subject. This effect can be, for example, biological, physiological, or cosmetic. Active agents can be any type of molecule or compound, including, for example, nucleic acids, peptides, and polypeptides, including antibodies, such as polyclonal antibodies, monoclonal antibodies, and antibody fragments; humanized antibodies, recombinant antibodies, recombinant human antibodies, and Primatized ^™ antibodies; cytokines, growth factors, apoptosis factors, differentiation-inducing factors, cell surface receptors and their ligands; hormones; and small molecules, including small organic molecules or compounds.

In one embodiment, the active agent is a therapeutic agent, or a salt or derivative thereof. Therapeutic agent derivatives can themselves be therapeutically active, or they can be prodrugs that become active after further modification. Thus, in one embodiment, the therapeutic agent derivative retains some or all of the therapeutic activity compared to the unmodified agent, while in another embodiment, the therapeutic agent derivative lacks therapeutic activity.

In various embodiments, therapeutic agents include any therapeutically effective agent or drug, such as anti-inflammatory compounds, antidepressants, stimulants, analgesics, antibiotics, birth control drugs, antipyretics, vasodilators, anti-angiogenic agents, cytovascular agents, signal transduction inhibitors, cardiovascular drugs (e.g., antiarrhythmic drugs), vasoconstrictors, hormones, and steroids.

In certain embodiments, the therapeutic agent is an oncology drug, which may also be referred to as an anti-tumor drug, an anti-cancer drug, a tumor drug, an anti-tumor agent, etc. Examples of oncology drugs that can be used in the present invention include, but are not limited to, adriamycin, levorotatory oncolysin, allopurinol, hexamethylmelamine, Amifostine, anastrozole, araC, arsenic trioxide, azathioprine, bexarotene, biCNU, bleomycin, intravenous busulfan, oral busulfan, capecitabine (Xeloda), carboplatin, carmustine, CCNU, celecoxib, chlorambucil, cisplatin, cladribine, cyclosporine A, fluorouracil, cytarabine, daunorubicin, carboplatin, daunorubicin, dexamethasone, dexrazoxane, docetaxel, adriamycin, adriamycin, DTIC, epirubicin, estramustine, etoposide phosphate, etoposide and VP-16, exemestane, FK506, fludarabine, fluorouracil, 5-FU, gemcitabine (Gemzar), gemtuzumab ozogamicin, goserelin acetate, hydroxyurea, idarubicin, ifosfamide , imatinib mesylate, interferon, irinotecan (Camptostar, CPT-111), letrozole, leucovorin, clalipine, leuprorelin, levamisole, alitretinoin, megestrol acetate, melphalan, L-PAM, sodium mercaptoethanesulfonate, methotrexate, methoxsalen, mithramycin, mitomycin, mitoxantrone, nitrogen mustard, paclitaxel, pamidronate, methoxypolyethylene glycol succinamide adenosine deaminase, pentostatin, porfimer sodium, prednisone, B-cell monoclonal antibody, streptozotocin, STI-571, tamoxifen, taxotere, temozolomide, teniposide, VM-26, topotecan (Hycamtin), toremifene, tretinoin, ATRA, valrubicin, vinblastine, vinblastine, vincristine, VP16, and vinorelbine. Other examples of oncology drugs that can be used in the present invention are ellipticine and ellipticine analogs or derivatives, epothilones, intracellular kinase inhibitors and camptothecins.

Other preparations

emulsion

The compositions of the present invention may be prepared and formulated as emulsions. Emulsions are typically heterogeneous systems in which one liquid is dispersed in another liquid in the form of droplets, typically exceeding 0.1 μm in diameter (Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; Block in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 2, p. 335; Higuchi et al., in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 1985, p. 301). An emulsion is typically a two-phase system comprising two immiscible liquid phases that are intimately mixed and dispersed with one another. Typically, emulsions can be water-in-oil (w/o) or oil-in-water (o/w). When the aqueous phase is finely dispersed and dispersed in fine droplets throughout a bulk oil phase, the resulting composition is referred to as a water-in-oil (w/o) emulsion. Alternatively, when the oil phase is finely dispersed and dispersed in fine droplets throughout a bulk aqueous phase, the resulting composition is referred to as an oil-in-water (o/w) emulsion. In addition to the dispersed phase, an emulsion may contain other components, and the active drug may be present as a solution in the aqueous phase, the oil phase, or itself as a separate phase. If desired, pharmaceutical excipients such as emulsifiers, stabilizers, colorants, and antioxidants may also be present in the emulsion. Pharmaceutical emulsions can also be multiple emulsions consisting of two or more phases, for example, oil-in-water-in-oil (o/w/o) and water-in-oil-in-water (w/o/w) emulsions. Such complex formulations often offer certain advantages over simple two-phase emulsions. In multiple emulsions, single oil droplets surround water droplets in an o/w emulsion to form a w/o/w emulsion. Similarly, a system of water droplets surrounding oil droplets in a stable oil continuous phase provides an o/w/o emulsion.

Emulsions are characterized in that there is almost no or no thermodynamic stability. Usually, the dispersion or discontinuous phase of the emulsion is well dispersed in the external phase or the continuous phase, and the viscosity of the emulsifier or the preparation is maintained in this form. Any phase of the emulsion can be semisolid or solid, and the ointment base and cream of the emulsion type are just like this. Other methods of stabilizing emulsions need to utilize emulsifiers, which can add any phase of the emulsion. Emulsifiers can be roughly divided into four categories: synthetic surfactants, naturally occurring emulsifiers, absorption bases and finely dispersed solids (Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p.199).

Synthetic surfactants, also known as surface active agents, have broad applicability in emulsion formulations and have been reviewed in the literature (Rieger, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 285; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), Marcel Dekker, Inc., New York, N.Y., 1988, volume 1, p. 199). Surfactants are generally amphiphilic molecules and contain hydrophilic and hydrophobic portions. The ratio of the hydrophilicity to the hydrophobicity of a surfactant is called the hydrophilic/lipophilic balance (HLB), which is an important tool for classifying and selecting surfactants in formulation preparation. Based on the nature of the hydrophilic group, surfactants can be divided into different types: nonionic, anionic, cationic and amphoteric (Rieger, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, page 285).

Naturally occurring emulsifiers for emulsion formulations include lanolin, beeswax, phosphatides, lecithin and gum arabic. Absorption bases have hydrophilicity so that they can absorb water to form w/o emulsions while still maintaining their semisolid viscosity, such as anhydrous lanolin and hydrophilic petrolatum. Finely dispersed solids are also used as good emulsifiers, particularly in combination with surfactants and used in viscous formulations. These include polar inorganic solids, such as heavy metal hydroxides, non-swelling clays such as bentonite, palygorskite, hectorite, kaolin, montmorillonite, colloidal aluminum silicate and colloidal magnesium aluminum silicate, pigments and non-polar solids such as carbon or tristearin.

A variety of non-emulsifying materials may also be included in emulsion formulations and contribute to the properties of the emulsion. These include fats, oils, waxes, fatty acids, fatty alcohols, fatty acid esters, humectants, hydrophilic colloids, preservatives, and antioxidants (Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., Vol. 1, p. 335; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., Vol. 1, p. 199).

Hydrophilic colloids or hydrocolloids include naturally occurring gums and synthetic polymers such as polysaccharides (e.g., gum arabic, agar, alginic acid, carrageenan, guar gum, karaya gum, and tragacanth gum), cellulose derivatives (e.g., carboxymethyl cellulose and carboxypropyl cellulose), and synthetic polymers (e.g., carbomers, cellulose ethers, and carboxyvinyl polymers). These substances disperse in water or swell in water to form colloidal solutions that stabilize emulsions by forming a strong interfacial film around the dispersed phase droplets and by increasing the viscosity of the external phase.

Because emulsions typically contain many ingredients that easily support microbial growth, such as carbohydrates, proteins, sterols, and phospholipids, preservatives are often added to these formulations. Common preservatives in emulsion formulations include methylparaben, propylparaben, quaternary ammonium salts, benzyl ammonium chloride, parabens, and boric acid. Antioxidants are also commonly added to emulsion formulations to prevent the formulation from deteriorating. The antioxidants used can be free radical scavengers, such as tocopherols, alkyl gallates, butylated hydroxyanisole, butylated hydroxytoluene; or reducing agents, such as ascorbic acid and sodium metabisulfite; and antioxidant synergists, such as citric acid, tartaric acid, and lecithin.

The use of emulsion formulations via the dermal, oral, and parenteral routes and their methods of preparation have been reviewed in the literature (Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, page 199). Emulsion formulations for oral delivery have been widely used due to their ease of formulation and efficacy in terms of absorption and bioavailability (Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, page 245; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, page 199). Mineral oil-based laxatives, oil-soluble vitamins, and high-fat nutritional preparations are materials commonly administered orally as o/w emulsions.

In one embodiment of the invention, the dsRNA and nucleic acid compositions are formulated as microemulsions. A microemulsion can be defined as a system of water, oil, and amphiphilic molecules that is a single, optically isotropic, and thermodynamically stable liquid solution (Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger, and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., Vol. 1, p. 245). Typically, a microemulsion is prepared by first dispersing an oil in an aqueous surfactant solution and then adding a sufficient fourth component, typically a medium-chain alcohol, to form a transparent system. Therefore, microemulsions are also known as thermodynamically stable, isotropic, transparent dispersions of two immiscible liquids that are stabilized by an interfacial film of surfactant molecules (Leung and Shah, in: Controlled Release of Drugs: Polymers and Aggregate Systems, Rosoff, M., Ed., 1989, VCH Publishers, New York, pp. 185-215). Microemulsions are generally prepared by combining three to five components, including oil, water, surfactant, cosurfactant, and electrolyte. Whether a microemulsion is of the water-in-oil (w/o) or oil-in-water (o/w) type depends on the nature of the oil and surfactant used and the structure and geometrical assembly of the polar head and hydrocarbon tail of the surfactant molecule (Schott, in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 1985, p. 271).

Phenomenological methods using phase diagrams have been extensively studied by those skilled in the art and have yielded comprehensive knowledge of how to formulate microemulsions (Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., Vol. 1, p. 245; Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., Vol. 1, p. 335). Microemulsions have the advantage over traditional emulsions of allowing water-insoluble drugs to be dissolved in thermodynamically stable droplet formulations that form spontaneously.

Surfactants for preparing microemulsions include, but are not limited to, ionic surfactants, nonionic surfactants, Brij96, polyoxyethylene oleyl ethers, fatty acid polyglycerol esters, tetraglyceryl monolaurate (ML310), tetraglyceryl monooleate (MO310), hexaglyceryl monooleate (PO310), hexaglyceryl pentaoleate (PO500), decaglyceryl monocaprate (MCA750), decaglyceryl monooleate (MO750), decaglyceryl sesquioleate (SO750), decaglyceryl decaoleate (DAO750), which are used alone or in combination with cosurfactants. Due to the gaps between surfactant molecules, cosurfactants (typically short-chain alcohols, such as ethanol, 1-propanol, and 1-butanol) are suitable for increasing interfacial fluidity by penetrating into the surfactant film and thus generating an abnormal film. However, microemulsions can be prepared without the use of cosurfactants, and alcohol-free self-emulsifying microemulsion systems are known in the art. Typically, the aqueous phase can be, but is not limited to, water, aqueous pharmaceutical solutions, glycerol, PEG300, PEG400, polyglycerol, propylene glycol, and derivatives of ethylene glycol. The oil phase can include, but is not limited to, materials such as Captex 300, Captex 355, Capmul MCM, fatty acid esters, medium chain (C8-C12) mono-, di-, and triglycerides, polyoxyethylene fatty acid glycerides, fatty alcohols, polyglycolized glycerides, saturated polyglycolized C8-C10 glycerides, vegetable oils, and silicone oils.

Microemulsions are particularly noteworthy for their potential use in drug dissolution and improved drug absorption. Lipid-based microemulsions (o/w and w/o) have been proposed for improving the oral bioavailability of drugs (including peptides) (Constantinides et al., Pharmaceutical Research, 1994, 11, 1385-1390; Ritschel, Meth. Find. Exp. Clin. Pharmacol., 1993, 13, 205). Microemulsions have the following advantages: improved drug dissolution, protection from enzymatic hydrolysis of the drug, potential for improved drug absorption due to surfactant-induced changes in membrane fluidity and permeability, ease of preparation, ease of oral administration compared to solid dosage forms, improved clinical efficacy, and reduced toxicity (Constantinides et al., Pharmaceutical Research, 1994, 11, 1385; Ho et al., J. Pharm. Sci., 1996, 85, 138-143). Microemulsions typically form spontaneously when their components are mixed together at ambient temperature. This may be particularly advantageous when formulating heat-labile pharmaceutical peptides or dsRNA. In cosmetic and pharmaceutical applications, microemulsions are also effective in transdermal delivery of active ingredients. It is expected that the microemulsion compositions and formulations of the present invention will promote increased systemic absorption of dsRNA and nucleic acids from the gastrointestinal tract, as well as improve local cellular uptake of dsRNA and nucleic acids.

The microemulsions of the present invention may also contain other components and additives, such as sorbitan monostearate (Grill3), Labrasol, and penetration enhancers to improve the properties of the formulation and enhance the absorption of the dsRNA and nucleic acids of the present invention. Penetration enhancers used in the microemulsions of the present invention can be classified into one of five major categories: surfactants, fatty acids, bile salts, chelating agents, and non-chelating non-surfactants (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92). Each of these categories has been discussed above.

penetration enhancers

In one embodiment, the present invention uses a variety of penetration enhancers to effectively deliver nucleic acids, particularly dsRNA, to animal skin. Most drugs are present in solution in ionized or non-ionized form. However, usually only lipid-soluble or lipophilic drugs are easy to penetrate the cell membrane. It has been found that if the membrane that needs to be penetrated is treated with a penetration enhancer, even non-lipophilic drugs can also penetrate the cell membrane. Except helping non-lipophilic drugs to diffuse through the cell membrane, penetration enhancers also improve the permeability of lipophilic drugs.

Penetration enhancers can be classified into one of five broad categories, namely surfactants, fatty acids, bile salts, chelating agents, and non-chelating non-surfactants (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92). Each of the above categories of penetration enhancers is described in detail below.

Surfactants: According to the present invention, surfactants are chemical entities that, when dissolved in an aqueous solution, reduce the surface tension of the solution or the interfacial tension between the aqueous solution and another liquid, resulting in increased absorption of dsRNA through the mucosa. In addition to bile salts and fatty acids, these penetration enhancers include, for example, sodium lauryl sulfate, polyoxyethylene-9-lauryl ether, and polyoxyethylene-20-cetyl ether (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92); and perfluorochemical emulsions such as FC-43. Takahashi et al., J. Pharm. Pharmacol., 1988, 40, 252).

Fatty acids: Various fatty acids and their derivatives as penetration enhancers include, for example, oleic acid, lauric acid, capric acid (n-decanoic acid), myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicapric acid, tricapric acid, monoolein (1-monooleoyl-rac-glycerol), dilaurin, caprylic acid, arachidonic acid, 1-monocaprin, 1-dodecylazacycloheptan-2-one, acylcarnitines, acylcholines, their _C1-10 alkyl esters (e.g., methyl, isopropyl, and t-butyl) and their mono- and di-glycerides (i.e., oleate, laurate, caprate, myristate, palmitate, stearate, linoleate, etc.) (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; El Hariri et al., J. Pharm. Pharmacol., 1992, 44, 651-654).

Bile salts: The physiological effects of bile include promoting the dispersion and absorption of lipids and fat-soluble vitamins (Brunton, Chapter 38 in: Goodman & Gilman's The Pharmacological Basis of Therapeutics, 9th Ed., Hardman et al. Eds., McGraw-Hill, New York, 1996, pp. 934-935). Various natural bile salts and their synthetic derivatives act as penetration enhancers. Therefore, the term "bile salts" includes any naturally occurring bile component as well as any synthetic derivative thereof. Suitable bile salts include, for example, cholic acid (or its pharmaceutically acceptable sodium salt, sodium cholate), dehydrocholic acid (sodium dehydrocholate), deoxycholic acid (sodium deoxycholate), glycocholic acid (sodium glycocholate), glycolic acid (sodium glycolate), glycodeoxycholic acid (sodium glycodeoxycholate), taurocholic acid (sodium taurocholate), taurodeoxycholic acid (sodium taurodeoxycholate), chenodeoxycholic acid (sodium chenodeoxycholate), ursodeoxycholic acid (UDCA), sodium tauro-24,25-dihydro-fusidate (STDHF), sodium glyceryl dihydrofusidate, and polyoxyethylene-9-lauryl ether (POE) (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92; Swinyard, Chapter 39 In: Remington's Pharmaceutical Sciences, 18th Ed., Gennaro, ed., Mack Publishing Co., Easton, Pa., 1990, pp. 782-783; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; Yamamoto et al., J. Pharm. Exp. Ther., 1992, 263, 25; Yamashita et al., J. Pharm. Sci., 1990, 79, 579-583).

Chelating agents: As used herein, chelating agents can be defined as compounds that remove metal ions from solution by forming complexes with them, thereby increasing the absorption of dsRNA through mucosal membranes. When used as penetration enhancers in the present invention, chelating agents have the additional advantage of acting as DNase inhibitors, as most characterized DNA nucleases require divalent metal ions for catalysis and are therefore inhibited by chelating agents (Jarrett, J. Chromatogr., 1993, 618, 315-339). Suitable chelating agents include, but are not limited to, disodium ethylenediaminetetraacetate (EDTA), citric acid, salicylates (e.g., sodium salicylate, 5-methoxysalicylate, and homovanillate), N-acyl derivatives of collagen, laureth-9, and N-aminoacyl derivatives of β-diketones (enamines) (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; Buur et al., J. Control Rel., 1990, 14, 43-51).

Non-chelating non-surfactant: As used herein, non-chelating non-surfactant penetration enhancer compounds can be defined as compounds that do not exhibit significant chelating or surfactant activity but still enhance the absorption of dsRNA through nutrient mucosa (Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33). Such penetration enhancers include, for example, unsaturated cyclic urea, 1-alkyl- and 1-alkenylazacyclo-alkanone derivatives (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92); and nonsteroidal anti-inflammatory drugs such as diclofenac, indomethacin, and phenylbutazone (Yamashita et al., J. Pharm. Pharmacol., 1987, 39, 621-626).

Agents that enhance cellular uptake of dsRNA can also be added to the pharmaceutical and other compositions of the present invention. For example, cationic lipids, such as lipofectin (Junichi et al., U.S. Pat. No. 5,705,188); cationic glycerol derivatives; and polycationic molecules, such as polylysine (Lollo et al., PCT Application No. WO 97/30731), are known to enhance cellular uptake of dsRNA.

Other agents that can be used to increase the permeability of administered nucleic acids include glycols, such as ethylene glycol and propylene glycol; pyrroles, such as 2-pyrrole; azones; and terpenes, such as limonene and menthone.

carrier

The dsRNA of the present invention can be formulated in a pharmaceutically acceptable carrier or diluent. A "pharmaceutically acceptable carrier" (also referred to herein as an "excipient") is a pharmaceutically acceptable solvent, suspending agent, or any other pharmacologically inert medium. Pharmaceutically acceptable carriers can be liquid or solid and can be selected based on the intended mode of administration to provide the desired volume, compatibility, and other relevant transport and chemical properties. Typical pharmaceutically acceptable carriers include, but are not limited to: water; saline solutions; binders (e.g., polyvinyl pyrrolidone or hydroxypropyl methylcellulose); fillers (e.g., lactose and other sugars, gels, or calcium sulfate); lubricants (e.g., starch, polyethylene glycol, or sodium acetate); disintegrants (e.g., starch or sodium starch glycolate); and wetting agents (e.g., sodium lauryl sulfate).

Certain compositions of the present invention are also formulated with a carrier compound. As used herein, a "carrier compound" or "carrier" may refer to a nucleic acid or its analog that is inert (i.e., not biologically active itself) but is recognized as a nucleic acid by processes in the body that reduce the bioavailability of the biologically active nucleic acid, for example, by degrading the biologically active nucleic acid or promoting its removal from the circulation. Co-administration of a nucleic acid and a carrier compound (typically in excess of the latter) can result in a significant reduction in the amount of nucleic acid recovered from the liver, kidneys, or other external circulation reservoirs, presumably due to competition between the carrier compound and the nucleic acid for a common receptor. For example, when it is co-administered with polyinosinic acid, dextran sulfate, polycytidylic acid or 4-acetamido-4'-isothiocyanato-stilbene-2,2-disulfonic acid, the recovery rate of some phosphorothioate dsRNAs in liver tissue can be reduced (Miyao et al., DsRNA Res. Dev., 1995, 5, 115-121; Takakura et al., DsRNA & Nucl. Acid Drug Dev., 1996, 6, 177-183).

excipient

In contrast to carrier compounds, a "pharmaceutical carrier" or "excipient" is a pharmaceutically acceptable solvent, suspending agent, or any other pharmacologically inert medium for delivering one or more nucleic acids to an animal. The excipient can be liquid or solid and is selected based on the intended mode of administration so as to provide the desired volume, compatibility, etc. when combined with the nucleic acid and other components of a given pharmaceutical composition. Typical pharmaceutical carriers include, but are not limited to, binders (e.g., pregelatinized corn starch, polyvinyl pyrrolidone, or hydroxypropyl methylcellulose); fillers (e.g., lactose and other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethyl cellulose, polyacrylates, or calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc, silica, colloidal silicon dioxide, stearic acid, metallic stearates, hydrogenated vegetable oils, corn starch, polyethylene glycol, sodium benzoate, sodium acetate, etc.); disintegrants (e.g., starch, sodium starch glycolate, etc.); and wetting agents (e.g., sodium lauryl sulfate, etc.).

The compositions of the present invention can also be formulated using pharmaceutically acceptable organic or inorganic excipients that do not react adversely with nucleic acids and are suitable for non-parenteral administration. Suitable pharmaceutically acceptable carriers include, but are not limited to, water, saline solutions, alcohol, polyethylene glycol, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethyl cellulose, polyvinyl pyrrolidone, and the like.

Preparations for topical administration of nucleic acids may include sterile and non-sterile aqueous solutions, non-aqueous solutions in common solvents such as alcohols, or solutions of nucleic acids in liquid or solid oily matrices. The solutions may also contain buffers, diluents, and other suitable additives. Pharmaceutically acceptable organic or inorganic excipients suitable for non-parenteral administration that do not adversely react with the nucleic acids may also be used.

Suitable pharmaceutically acceptable excipients include, but are not limited to, water, saline solution, alcohol, polyethylene glycol, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinyl pyrrolidone, and the like.

Other components

The present composition can also comprise other auxiliary components that are commonly used for pharmaceutical compositions at the use level determined in this area.Therefore, for example, the composition can comprise other compatible pharmaceutically active substances, for example, antipruritic, astringent, local anesthetic or anti-inflammatory agent, or can comprise other materials for physically preparing the various dosage forms of the present composition, for example, coloring agent, correctives, preservative, antioxidant, sunscreen, thickener and stabilizer. However, when adding such material, it should not unduly interfere with the biological activity of the present composition component. If necessary, the preparation can be sterilized and mixed with auxiliary agents, for example, lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for affecting osmotic pressure, buffer, coloring agents, correctives and/or aromatic substances that do not react adversely with the nucleic acid of the preparation, etc.

Aqueous suspensions may contain substances that increase the viscosity of the suspension, including, for example, sodium carboxymethylcellulose, sorbitol and/or dextran. The suspension may also contain stabilizers.

Combination therapy

On the one hand, the compositions of the present invention can be used for combination therapy. The term "combination therapy" includes combining other biologically active ingredients (such as, but not limited to, a second and different antitumor agent) and non-drug therapy (such as, but not limited to, surgery or radiotherapy) when the compound is administered to the subject. For example, the compounds of the present invention can be used in combination with other pharmaceutically active compounds, preferably compounds that can improve the effect of the compounds of the present invention. The compounds of the present invention can be used simultaneously (as a single preparation or separate preparations) or successively with other drug therapies. Typically, combination therapy is expected to administer two or more drugs during a single cycle or course of treatment.

In one aspect of the invention, the subject compounds can be administered in combination with one or more separate agents to modulate protein kinases associated with a variety of disease states. Examples of such kinases can include, but are not limited to, serine/threonine specific kinases, receptor tyrosine specific kinases, and non-receptor tyrosine specific kinases. Serine/threonine kinases include mitogen-activated protein kinases (MAPKs), meiosis-specific kinases (MEKs), RAFs, and Aurora kinases. Examples of receptor kinase families include epidermal growth factor receptors (EGFRs) (e.g., HER2/neu, HER3, HER4, ErbB, ErbB2, ErbB3, ErbB4, Xmrk, DER, Let23), fibroblast growth factor (FGF) receptors (e.g., FGF-R1, GFF-R2/BEK/CEK3, FGF-R3/CEK2, FGF-R4/TKF, KGF-R), hepatocyte growth/scatter factor receptors (HGFRs) (e.g., MET, RON, SEA, SEX), insulin receptor (e.g., IGF-1 receptor), Eph (e.g., CEK5, CEK8, EBK, ECK, EEK, EHK-1, EHK-2, ELK, EPH, ERK, HEK, MDK2, MDK5, SEK); AxI (e.g., Mer/Nyk, Rse); RET; platelet-derived growth factor receptor (PDGFR) (e.g., PDGFα-R, PDGβ-R, CSF1-R/FMS, SCF-R/C-KIT, VEGF-R/FLT, NEK/FLK1, FLT3/FLK2/STK-1). Non-receptor tyrosine kinase family includes, but is not limited to, BCR-ABL (e.g., p43 ^ab1 , ARG); BTK (e.g., ITK/EMT, TEC); CSK, FAK, FPS, JAK, SRC, BMX, FER, CDK, and SYK.

In another aspect of the invention, the subject compounds can be administered in combination with one or more agents that modulate non-kinase biological targets or processes. Such targets include histone deacetylases (HDACs), DNA methyltransferases (DNMTs), heat shock proteins (e.g., HSP90), and proteosomes.

In one embodiment, the subject compound can be combined with an anti-tumor agent (e.g., small molecules, monoclonal antibodies, antisense RNA, and fusion proteins) that inhibits one or more biological targets, such as Zolinza, Tarceva, Iressa, Lapatinib (Tykerb), Gleevec, Sutent, Dasatinib (Sprycel), Nexavar, Sorafenib, CNF2024, RG108, BMS387032, Affmitak, Avastin, Trastuzumab (Herceptin), Cetuximab (Erbitux), AG24322, PD325901, ZD6474, PD 184322, Obatodax, ABT737, and AEE788. The therapeutic efficacy of this combination is improved relative to the efficacy obtained by using any agent alone, and the emergence of resistant mutant variants can be inhibited or delayed.

In certain preferred embodiments, the compounds of the present invention are administered in combination with a chemotherapeutic agent. Chemotherapeutic agents encompass a wide range of treatments in the field of oncology. These agents are administered at various stages of the disease to shrink tumors, eliminate residual cancer cells left after surgery, induce remission, maintain remission, and/or alleviate symptoms associated with cancer or its treatment. Examples of such agents include, but are not limited to, alkylating agents, such as mustard gas derivatives (mechlorethamine, cyclophosphamide, chlorambucil, melphalan, ifosfamide), ethyleneimines (phosphothioate, hexamethonium), alkyl sulfonates (myleran), hydrazines and triazines (hexamethonium, toluidine, dacarbazine, and temozolomide), nitrosoureas (carmustine, lomustine, and streptozotocin), ifosfamide, and metal salts (carboplatin, cisplatin, oxaliplatin); plant alkaloids, such as podophyllotoxins (etoposide and teniposide), taxanes (paclitaxel, docetaxel), vinca alkaloids (vincristine, vinblastine, vindesine, and vinorelbine), and camptothecin analogs (irinotecan, topotecan); antitumor antibiotics, such as chromomycins (dactinomycin and mithramycin), anthracyclines (adriamycin, daunorubicin), and daunorubicin. oxazolidinone, epirubicin, mitoxantrone, valrubicin, and idarubicin) and other antibiotics, such as mitomycin, dactinomycin, and bleomycin; antimetabolites, such as folate antagonists (methotrexate, pemetrexed, raltitrexed, pteridine), pyrimidine antagonists (5-fluorouracil, floxuridine, fluorouracil, capecitabine, and gemcitabine), purine antagonists (6-mercaptopurine and 6-thioguanine), and adenosine deaminase inhibitors (cladribine, fludarabine, mercaptopurine, clofarabine, thioguanine, nelarabine, and pentostatin); topoisomerase inhibitors, such as topoisomerase I inhibitors (e.g., cyclopenthixone, cyclopenthixone, cyclopenthixone, pyrimidine antagonists, capecitabine, and gemcitabine); In some embodiments, the present invention includes but is not limited to rituximab, rituximab, trastuzumab, ibritumomab tiuxetan, cetuximab, panitumumab, tositumomab, bevacizumab, and other anti-tumor agents, such as ribonucleotide reductase inhibitors (hydroxyurea), corticosteroid inhibitors (mitotane), enzymes (asparaginase and pegaptase), anti-microtubule agents (estramustine), and retinoids (bexarotene, isotretinoin, tretinoin (ATRA). In certain preferred embodiments, the compounds of the present invention are administered in combination with a chemotherapeutic protectant. Chemoprotectants protect the body or minimize the side effects of chemotherapy. Examples of such agents include, but are not limited to, amistin, sodium mercaptoethanesulfonate, and dexrazoxane.

In one aspect of the present invention, the subject compounds are used in conjunction with radiotherapy. Radiation is typically delivered internally (radioactive material is implanted near the cancer site) or externally by a machine using photons (x-rays or gamma rays) or microparticle radiation. When combined therapy also includes radiotherapy, the radiotherapy can be performed at any appropriate time, as long as a beneficial effect can be obtained from the combined effects of the therapeutic agent and the radiotherapy combination. For example, in appropriate circumstances, when radiotherapy is temporarily removed from the administered therapeutic agent, a beneficial effect can still be obtained, perhaps for several days or even weeks.

It will be understood that the compounds of the present invention can be used in combination with immunotherapeutic agents. One form of immunotherapy is to generate an active systemic tumor-specific immune response of host origin by administering a vaccine composition at a location distant from the tumor. Various types of vaccines have been proposed, including isolated tumor-antigen vaccines and anti-idiotypic vaccines. Another approach is to use tumor cells from the subject to be treated, or derivatives of such cells (reviewed by Schirrmacher et al. (1995) J. Cancer Res. Clin. Oncol. 121: 487). U.S. Patent No. 5,484,596 to Hanna Jr. et al. claims a method for treating resectable tumors to prevent recurrence or metastasis, comprising surgically removing the tumor, dispersing the cells with collagenase, irradiating the cells, and vaccinating the patient with at least three consecutive doses of approximately 10 ⁷ cells.

Should be understood that the compounds of this invention can be advantageously used in conjunction with one or more adjunctive therapeutic agents. Examples of suitable agents for adjunctive therapy include steroids, such as corticosteroids (amcinolone acetonide, betamethasone, betamethasone dipropionate, betamethasone valerate, budesonide, clobetasol, clobetasol acetate, clobetasol butyrate, clobetasol 17-propionate, cortisone, deflazacort, desoximetasone, diflumethasone valerate, dexamethasone, dexamethasone sodium phosphate, hydroxyprednisolone, furoate, fluocinonide, fluocinonide, halcitonide, hydrocortisone, hydrocortisone butyrate, hydrocortisone sodium succinate, hydrocortisone valerate, prednicarbate, prednisone, triamcinolone, triamcinolone acetonide, and halobetasol propionate); 5HTi agonists, such as triptans (e.g., sumatriptan or noractin); adenosine Al agonists; EP ligands; NMDA modulators, such as glycine antagonists; sodium channel blockers (e.g., lamotrigine); substance P antagonists (e.g., NKi antagonists); cannabinoids; acetaminophen or phenacetin; 5-lipoxygenase inhibitors; leukotriene receptor antagonists; DMARDs (e.g., methotrexate) amphetamine); gabapentin and related compounds; tricyclic antidepressants (e.g., amitriptyline); neuronal cell stabilizing antiepileptic drugs; monoaminergic uptake inhibitors (e.g., venlafaxine); matrix metalloproteinase inhibitors; nitric oxide synthase (NOS) inhibitors, such as iNOS or nNOS inhibitors; inhibitors of tumor necrosis factor release or action; antibody therapy, such as monoclonal antibody therapy; antiviral agents, such as nucleoside inhibitors (e.g., lamivudine) or immune system modulators (e.g., interferon); opioid analgesics; topical Anesthetics; stimulants, including caffeine; H2-antagonists (such as furazolidone); proton pump inhibitors (such as omeprazole); antacids (such as aluminum or magnesium hydroxide); antiflatulents (such as simethicone); decongestants (such as phenylephrine, phenylpropanolamine, pseudoephedrine, oxymetazoline, epinephrine, naphazoline, bupropion, hexahydroephedrine, or levomethorphan); cough suppressants (such as codeine, hydrocodone, caramiphene, vinpoxetine, or dextromethorphan); diuretics; or sedat or non-sedat antihistamines.

The compounds of this invention can be administered together with siRNA targeting other genes. For example, the compounds of this invention can be administered together with siRNA targeting the c-Myc gene. In one embodiment, AD-12115 can be administered together with c-Myc siRNA. Examples of c-Myc-targeted siRNA are disclosed in U.S. Patent Application No. 12/373,039, which is incorporated herein by reference.

Methods for treating diseases caused by expression of Eg5 and VEGF genes

The present invention particularly relates to the use of compositions containing at least two dsRNAs (one targeting the Eg5 gene and one targeting the VEGF gene) in the treatment of cancer, such as liver cancer, to inhibit tumor growth and tumor metastasis. For example, the compositions, such as pharmaceutical compositions, can be used to treat solid tumors, such as intrahepatic tumors, as may occur in liver cancer. Compositions containing dsRNAs targeting Eg5 and dsRNAs targeting VEGF can also be used to treat other tumors and cancers, such as breast cancer, lung cancer, head and neck cancer, brain cancer, abdominal cancer, colon cancer, colorectal cancer, esophageal cancer, gastrointestinal cancer, glioma, tongue cancer, neuroblastoma, osteosarcoma, ovarian cancer, pancreatic cancer, prostate cancer, retinoblastoma, Wilm's tumor, multiple myeloma, and for the treatment of skin cancer, melanoma, lymphoma, and leukemia. The present invention also relates to the use of compositions containing Eg5 dsRNA and VEGF dsRNA for inhibiting the accumulation of ascites and pleural effusions in various types of cancer, such as liver cancer, breast cancer, lung cancer, head cancer, neck cancer, brain cancer, abdominal cancer, colon cancer, colorectal cancer, esophageal cancer, gastrointestinal cancer, glioma, tongue cancer, neuroblastoma, osteosarcoma, ovarian cancer, pancreatic cancer, prostate cancer, retinoblastoma, Wilm's tumor, multiple myeloma, skin cancer, melanoma, lymphoma, and leukemia. Due to the inhibitory effect on Eg5 and VEGF expression, the compositions of the present invention or pharmaceutical compositions prepared therefrom can improve the quality of life.

In one embodiment, a patient is treated for a tumor associated with AFP expression, or a tumor that secretes AFP, such as liver cancer or a teratoma. In certain embodiments, the patient is treated for a malignant teratoma, an endodermal sinus tumor (yolk sac carcinoma), a neuroblastoma, a hepatoblastoma, a hepatocellular carcinoma, a testicular cancer, or an ovarian cancer.

The present invention also relates to the use of dsRNA or a pharmaceutical composition thereof, for example, for treating cancer or for preventing tumor metastasis, wherein the pharmaceutical composition is combined with other drugs and/or other treatment methods, for example, with known drugs and/or known treatment methods (e.g., methods currently used to treat cancer and/or for preventing tumor metastasis).

The present invention can also be implemented by including specific RNAi agents in combination with other anticancer chemotherapeutic agents, such as any traditional chemotherapeutic agents. The combination of specific binding agents and such other agents can enhance the chemotherapy regimen. Many chemotherapy regimens that can be combined with the methods of the present invention will come to the mind of practitioners in the art. Any chemotherapeutic agent can be used, including alkylating agents, antimetabolites, hormones and antagonists, radioisotopes, and natural products. For example, the compounds of the present invention can be administered together with antibiotics such as adriamycin and other anthracycline analogs, nitrogen mustards such as cyclophosphamide, pyrimidine analogs such as 5-fluorouracil, cisplatin, hydroxyurea, taxol, and their natural and synthetic derivatives. As another example, for mixed tumors, such as adenomas of the breast, where the tumors include gonadotropin-dependent and gonadotropin-independent cells, the compounds can be administered together with leuprorelin or a sex hormone blocker (a synthetic peptide analog of LH-RH). Other anti-tumor approaches include the use of tetracycline compounds and another treatment modality, such as surgery, radiation, etc., which are also referred to as "adjuvant anti-tumor modalities" herein. Therefore, the method of the present invention can be used together with such traditional methods to advantageously reduce side effects and enhance efficacy.

Method for inhibiting the expression of Eg5 gene and VEGF gene

In another aspect, the present invention provides a method for inhibiting the expression of Eg5 and VEGF genes in mammals, comprising administering the composition of the present invention to the mammal, thereby silencing the expression of the target Eg5 and VEGF genes.

In one embodiment, the method for inhibiting Eg5 gene expression and VEGF gene expression comprises administering a composition comprising two different dsRNA molecules to a mammal in need of treatment, wherein the nucleotide sequence of one dsRNA molecule is complementary to at least a portion of the RNA transcript of the Eg5 gene, and the nucleotide sequence of the other dsRNA molecule is complementary to at least a portion of the RNA transcript of the VEGF gene. When the organism in need of treatment is a mammal, such as a human, the composition can be administered by any method known in the art, including but not limited to oral or parenteral routes, including intravenous, intramuscular, subcutaneous, transdermal, airway (aerosol), nasal, rectal, and topical (including buccal and sublingual) administration. In a preferred embodiment, the composition is administered by intravenous infusion or injection.

Method for preparing lipid particles

The methods of the present compositions use certain cationic lipids, the synthesis, preparation and characterization of which are described below and in the accompanying examples. In addition, the present invention provides methods for preparing lipid particles, including those associated with therapeutic agents, such as nucleic acids. In the methods described herein, a lipid mixture is mixed with an aqueous nucleic acid buffer solution to prepare an intermediate mixture containing nucleic acids encapsulated in lipid particles, wherein the encapsulated nucleic acids are present at a nucleic acid/lipid ratio of about 3 wt% to about 25 wt%, preferably 5 to 15 wt%. Optionally, the size of the intermediate mixture can be adjusted to obtain lipid-encapsulated nucleic acid particles, wherein the lipid portion is a unilamellar vesicle, preferably with a diameter of 30 to 150 nm, more preferably about 40 to 90 nm. The pH is then raised to neutralize at least a portion of the surface charge on the lipid-nucleic acid particles, thereby providing a lipid-encapsulated nucleic acid composition that is at least partially surface-neutralized.

As mentioned above, some of these cationic lipids are amino lipids, which are charged at a pH lower than the amino pKa, and are substantially neutral at a pH higher than the pKa. These cationic lipids are called titratable cationic lipids and can be used in preparations of the present invention by using a two-step method. First, at a relatively low pH, lipid vesicles are formed with titratable cationic lipids and other vesicle components in the presence of nucleic acid. In this way, the vesicle will encapsulate and capture nucleic acid. Second, by increasing the pH of the medium to a level higher than the pKa of the titratable cationic lipid present, i.e., physiological pH or higher, the surface charge of the newly formed vesicle is neutralized. The particularly advantageous aspect of this method comprises the nucleic acid that is easy to remove any surface adsorption, and the nucleic acid delivery medium generated has a neutral surface. Liposomes or lipid particles with a neutral surface are expected to avoid being rapidly removed from circulation, and to avoid certain toxicity relevant to the cationic liposome preparation. Additional details regarding these uses of such titratable cationic lipids in nucleic acid-lipid particle formulations are provided in US Patent 6,287,591 and US Patent 6,858,225, which are incorporated herein by reference.

It is also noted that the vesicles formed in this manner provide preparations having uniform vesicle size and high nucleic acid content. Additionally, the size of the vesicles ranges from about 30 to about 150 nm, preferably from about 30 to about 90 nm.

Without being bound by any particular theory, it is believed that the very high efficiency of nucleic acid encapsulation is the result of electrostatic interactions at low pH. At acidic pH (e.g., pH 4.0), the vesicle surface is charged and binds to a portion of the nucleic acid through electrostatic interactions. When an external acidic buffer replaces a neutral buffer (e.g., pH 7.5), the surface of the lipid particles or liposomes is neutralized, thereby removing any external nucleic acid. More detailed information on the formulation method is provided in various publications (e.g., U.S. Patent No. 6,287,591 and U.S. Patent No. 6,858,225).

In view of the above, the present invention provides a method for preparing a lipid/nucleic acid formulation. In the method described herein, a lipid mixture is mixed with a buffered aqueous solution of nucleic acids to prepare an intermediate mixture containing nucleic acids encapsulated in lipid particles, for example, wherein the encapsulated nucleic acids are present at a nucleic acid/lipid ratio of about 10 wt% to about 20 wt%. Optionally, the size of the intermediate mixture can be adjusted to obtain lipid-encapsulated nucleic acid particles, wherein the lipid portion is a unilamellar vesicle, preferably with a diameter of 30 to 150 nm, more preferably about 40 to 90 nm. The pH is then raised to neutralize at least a portion of the surface charge on the lipid-nucleic acid particles, thereby providing a lipid-encapsulated nucleic acid composition that is at least partially surface-neutralized.

In certain embodiments, the lipid mixture comprises at least two lipid components: a first amino lipid component of the present invention selected from lipids having a pKa such that at a pH below the pKa, the lipid is cationic and at a pH above the pKa, the lipid is neutral, and a second lipid component selected from lipids that prevent particle aggregation during lipid-nucleic acid particle formation. In specific embodiments, the amino lipid is a novel cationic lipid of the present invention.

In the preparation of the nucleic acid-lipid particles of the present invention, the lipid mixture is generally a solution of lipids in an organic solvent. The lipid mixture can then be dried to form a film or lyophilized to form a powder, which is then hydrated with an aqueous buffer to form liposomes. Alternatively, in a preferred method, the lipid mixture can be dissolved in a water-soluble alcohol such as ethanol, and the ethanolic solution is added to the aqueous buffer, resulting in spontaneous formation of liposomes. In a most preferred embodiment, alcohol is used in a commercially available form. For example, anhydrous ethanol (100%) or 95% ethanol is used, with the remainder being water. This method is described in more detail in U.S. Patent No. 5,976,567.

According to the present invention, the lipid mixture is mixed with a buffered aqueous solution that may contain nucleic acids. The buffered aqueous solution is typically a solution having a pH less than the pKa of the protonatable lipids in the lipid mixture. Examples of suitable buffers include citrate, phosphate, acetate, and MES. A particularly preferred buffer is citrate buffer. The anion content of the preferred buffer is in the range of 1-1000 mM, depending on the chemical properties of the encapsulated nucleic acid, and optimizing the buffer concentration may be important for achieving high loading levels (e.g., see U.S. Patents 6,287,591 and 6,858,225). Alternatively, pure water acidified to a pH of 5-6 with chloride, sulfate, etc. may be used. In this case, it is suitable to add 5% glucose, or another non-ionic solute, which can balance the osmotic pressure across the particle membrane when the particles are dialyzed to remove ethanol, raise the pH, or mixed with a pharmaceutically acceptable carrier such as saline. The nucleic acid content in the buffer can vary, but is typically about 0.01 mg/mL to about 200 mg/mL, more preferably about 0.5 mg/mL to about 50 mg/mL.

A mixture of lipids and a buffered aqueous solution of a therapeutic nucleic acid is mixed to provide an intermediate mixture. The intermediate mixture is typically a mixture of lipid particles with encapsulated nucleic acids. In addition, the intermediate mixture may also include a portion of nucleic acids, which are attached to the surface of the lipid particles (liposomes or lipid vesicles) due to ionic attraction between the negatively charged nucleic acids and the positively charged lipids on the surface of the lipid particles (the amino lipids or other lipids that make up the protonatable first lipid component are positively charged in a buffer having a pH less than the pKa of the protonatable groups on the lipids). In one preferred embodiment, the lipid mixture is an alcoholic solution of lipids, and the volumes of the solutions are adjusted so that once combined, the resulting alcohol content is from about 20% to about 45% by volume. The method of combining the mixture can include any type of processing, generally depending on the scale of the preparation being prepared. For example, when the total volume is about 10-20 mL or less, the solutions can be mixed in a test tube and stirred together using a vortex mixer. Large-scale processing can be carried out in glassware of appropriate production scale.

Optionally, the size of the lipid-encapsulated therapeutic agent (e.g., nucleic acid) complex prepared by mixing a lipid mixture and a buffered aqueous solution of the therapeutic agent (nucleic acid) can be adjusted to obtain a desired size range and a relatively narrow lipid particle size distribution. Preferably, the mean diameter of the composition provided by the present invention is from about 70 to about 200 nm, more preferably from about 90 to about 130 nm. Several techniques can be used to adjust the liposomes to the desired size. A method for adjusting the size is described in U.S. Patent No. 4,737,323, which is incorporated herein by reference. Ultrasonic treatment of a liposome suspension by bath or probe sonication can gradually reduce the size, thereby obtaining small unilamellar vesicles (SUVs) with a size of less than about 0.05 microns. Homogenization is another method that relies on shear energy to crush larger liposomes into smaller liposomes. In a typical homogenization process, multilamellar vesicles are recirculated through a standard emulsion homogenizer until the selected liposome size is observed, typically from about 0.1 to 0.5 microns. In both methods, the particle size distribution can be monitored by a commonly used laser beam particle sizer. In certain methods of the invention, extrusion is used to obtain uniform vesicle size.

Extrusion of the liposome composition through a small-pore polycarbonate membrane or an asymmetric ceramic membrane results in a relatively well-defined particle size distribution. Typically, the suspension is circulated through the membrane one or more times until the desired liposome complex size distribution is achieved. The liposomes can be continuously extruded through the small-pore membrane to achieve a gradual reduction in liposome size. In some cases, the resulting lipid-nucleic acid composition can be used without the need for size adjustment.

In a specific embodiment, the inventive method also comprises the step of at least some surface charges on the lipid part of neutralization lipid-nucleic acid composition.By neutralizing surface charge at least in part, unencapsulated nucleic acid is discharged from the lipid granule surface, and can use ordinary method to remove from composition.Preferably, the nucleic acid of unencapsulated and surface adsorption is removed from the composition of gained by replacing buffer solution.For example, with HEPES-buffered saline (HBS, pH about 7.5) solution replacement citrate buffer (pH about 4.0, for forming composition) causes the neutralization of liposome surface and the release of nucleic acid from surface.Can remove the nucleic acid of discharge with standard method by chromatography then, then convert pH into the buffer solution that is greater than the pKa of used lipid.

Optionally, lipid vesicles (i.e., lipid particles) can be formed by hydration in an aqueous buffer and sized using any of the methods described above, followed by the addition of nucleic acids. As described above, the pH of the aqueous buffer should be lower than the pKa of the amino lipid. The nucleic acid solution is then added to these size-adjusted, preformed vesicles. To encapsulate the nucleic acids in such "preformed" vesicles, the mixture should contain an alcohol, such as ethanol. Where ethanol is used, it should be present in a concentration of about 20% (w/w) to about 45% (w/w). In addition, depending on the composition of the lipid vesicles and the properties of the nucleic acids, it may be necessary to heat the mixture of the preformed vesicles and the nucleic acids in the aqueous buffer-ethanol mixture to a temperature of about 25° C. to about 50° C. It will be clear to those skilled in the art that optimizing the encapsulation process to obtain the desired nucleic acid level in the lipid vesicles will require controlling variables, such as ethanol concentration and temperature. Examples of suitable conditions for encapsulating nucleic acids are provided in the Examples. Once the nucleic acids are encapsulated in the preformed vesicles, the external pH can be raised to at least partially neutralize the surface charge. Unencapsulated and surface-adsorbed nucleic acids can then be removed as described above.

How to use

Lipid granule of the present invention can be used for in vitro or in vivo therapeutic agent is delivered to cell.In a specific embodiment, the therapeutic agent is nucleic acid, and it uses nucleic acid-lipid granule of the present invention to be delivered to cell.The following description of the various methods using lipid granule of the present invention and related pharmaceutical composition is illustrated by the description relevant to nucleic acid-lipid granule, and it should be understood that these methods and compositions can be easily used for delivering any therapeutic agent that is used to treat any disease that benefits from this treatment or illness.

In certain embodiments, the present invention provides methods for introducing nucleic acids into cells. Preferred nucleic acids for introduction into cells are siRNA, immunostimulatory oligonucleotides, plasmids, antisense and ribozymes. These methods can be performed by contacting the particles or compositions of the present invention with cells for a period of time sufficient for intracellular delivery to occur.

The compositions of the present invention can be adsorbed onto virtually any cell type. Once adsorbed, the nucleic acid-lipid particles can be internalized by a portion of the cell, exchange lipids with the cell membrane, or fuse with the cell. Transfer or binding of the nucleic acid portion of the complex can occur via any of these pathways. Without limiting the scope of the present invention, it is believed that in cases where the particles are taken up by the cell via endocytosis, the particles then interact with the endosomal membrane, causing endosomal membrane destabilization, perhaps through the formation of a non-bilayer phase, resulting in the encapsulated nucleic acid entering the cytoplasm. Similarly, in cases where the particle and the cytoplasmic membrane fuse directly, when fusion occurs, the liposome membrane integrates into the cell membrane, and the liposome contents bind to the intracellular fluid. Contact between the cell and the lipid-nucleic acid composition (when performed in vitro) will occur in a biologically compatible medium. The concentration of the composition can vary depending on the specific application, but is typically between about 1 μmol and about 10 mmol. In certain embodiments, treatment of the cells with the lipid-nucleic acid composition is typically performed at physiological temperature (approximately 37°C) for about 1 to 24 hours, preferably about 2 to 8 hours. For in vitro applications, nucleic acids can be delivered to any cell, whether of plant or animal origin, vertebrate or invertebrate, and of any tissue or type, grown in culture. In a preferred embodiment, the cell is an animal cell, more preferably a mammalian cell, most preferably a human cell.

In one set of embodiments, the lipid-nucleic acid particle suspension is added to 60-80% confluent cells at a cell density of about 10 ³ to about 10 ⁵ cells/mL, preferably about 2×10 ⁴ cells/mL. The concentration of the suspension added to the cells is preferably about 0.01 to 20 μg/mL, more preferably about 1 μg/mL.

Typical applications include providing intracellular delivery of siRNA using well-known methods to inhibit or silence specific cellular targets. In addition, applications include delivering DNA or mRNA sequences encoding therapeutically useful polypeptides. In this way, treatment is provided for genetic diseases by providing insufficient or missing gene products (i.e., for Duchenne muscular dystrophy, see Kunkel, et al., Brit. Med. Bull. 45(3): 630-643 (1989), for cystic fibrosis, see Goodfellow, Nature 341: 102-103 (1989)). Other uses of the compositions of the present invention include introducing antisense oligonucleotides into cells (see Bennett, et al., Mol. Pharm. 41: 1023-1033 (1992)).

Alternatively, the compositions of the present invention can also be used to deliver nucleic acids to cells in vivo by methods known to those skilled in the art. Regarding the use of the present invention for delivering DNA or mRNA sequences, Zhu et al., Science 261:209-211 (1993), incorporated herein by reference, describes the intravenous delivery of a cytomegalovirus (CMV)-chloramphenicol acetyltransferase (CAT) expression plasmid using a DOTMA-DOPE complex. Hyde et al., Nature 362:250-256 (1993), incorporated herein by reference, describes the use of liposomes to deliver the cystic fibrosis transmembrane conductance regulator (CFTR) gene to the airway epithelium and alveoli of the lungs of mice. Brigham et al., Am. J. Med. Sci. 298:278-281 (1989), incorporated herein by reference, describes the in vivo transfection of mouse lungs with a functional prokaryotic gene encoding the intracellular enzyme, chloramphenicol acetyltransferase (CAT). Thus, the compositions of the present invention can be used to treat infectious diseases.

For in vivo administration, the pharmaceutical composition is preferably administered parenterally, i.e., intraarticularly, intravenously, intraperitoneally, subcutaneously, or intramuscularly. In a specific embodiment, the pharmaceutical composition is administered intravenously or intraperitoneally by bolus injection. As an example, see U.S. Patent No. 5,286,634 to Stadler et al., which is incorporated herein by reference. Intracellular nucleic acid delivery is also discussed in Straubringer et al., Methods in Enzymology, Academic Press, New York. 101: 512-527 (1983); Mannino, et al., Biotechniques 6: 682-690 (1988); Nicolau, et al., Crit. Rev. Ther. Drug Carrier Syst. 6: 239-271 (1989), and Behr, Acc. Chem. Res. 26: 274-278 (1993). Other methods of administering lipid-based therapeutics are described, for example, in Rahman et al., U.S. Patent No. 3,993,754; Sears, U.S. Patent No. 4,145,410; Papahadjopoulos et al., U.S. Patent No. 4,235,871; Schneider, U.S. Patent No. 4,224,179; Lenk et al., U.S. Patent No. 4,522,803; and Fountain et al., U.S. Patent No. 4,588,578.

In other methods, the drug formulation can be brought into contact with the target tissue by applying the formulation directly to the tissue. The application can be performed through local, "open," or "closed" procedures. "Local" means applying the drug formulation directly to tissue exposed to the environment, such as the skin, oropharynx, external auditory canal, etc. An "open" procedure involves opening the patient's skin and directly visualizing the underlying tissue to which the drug formulation is applied. This is typically achieved through a surgical procedure, such as a thoracotomy to access the lungs, an abdominal laparotomy to access the abdominal viscera, or other direct surgical procedures close to the target tissue. A "closed" procedure is an invasive procedure in which the target tissue is not directly visualized but is accessed through a small wound in the skin via an inserted instrument. For example, the formulation can be administered to the peritoneum via needle lavage. Similarly, the drug formulation can be administered to the meninges or spinal cord by infusion during a lumbar puncture, and the patient is then positioned as is typically done for spinal anesthesia or spinal mepantocan imaging. Alternatively, the formulation can be administered via an endoscopic device.

Lipid-nucleic acid compositions can also be administered as an aerosol for inhalation into the lungs (see Brigham, et al., Am. J. Sci. 298(4):278-281 (1989)) or by direct injection into the lesion site (Culver, Human Gene Therapy, Mary Ann Liebert, Inc., Publishers, New York. pp. 70-71 (1994)).

The method of the present invention can also be carried out in a variety of hosts. Preferred hosts include mammalian species, such as humans, non-human primates, dogs, cats, cows, horses, sheep, etc.

The dosage of the lipid-therapeutic agent particles of the invention will depend on the ratio of therapeutic agent to lipid and the opinion of the attending physician based on the age, weight, and condition of the patient.

In one embodiment, the present invention provides methods for regulating the expression of a target polynucleotide or polypeptide. These methods generally include contacting a cell with a lipid particle of the present invention, which is bound to a nucleic acid capable of regulating the expression of a target polynucleotide or polypeptide. As used herein, the term "regulate" means changing the expression of a target polynucleotide or polypeptide. In different embodiments, regulation can refer to increasing or enhancing, or it can refer to reducing or lowering. Methods for determining the expression level of a target polynucleotide or polypeptide are known and available in the art, including, for example, methods using reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry techniques. In a specific embodiment, the expression level of a target polynucleotide or polypeptide increases or decreases by at least 10%, 20%, 30%, 40%, 50% or greater than 50% compared to an appropriate control value. For example, if it is necessary to increase polypeptide expression, the nucleic acid can be an expression vector comprising a polynucleotide encoding the desired polypeptide. On the other hand, if it is necessary to reduce the expression of a polynucleotide or polypeptide, the nucleic acid can, for example, be an antisense oligonucleotide, siRNA or microRNA comprising a polynucleotide sequence that specifically hybridizes to the polynucleotide encoding the target polypeptide, thereby interfering with the expression of the target polynucleotide or polypeptide. Alternatively, the nucleic acid may be a plasmid that expresses such antisense oligonucleotide, siRNA or microRNA.

In one embodiment, the invention provides a method for regulating the expression of polypeptides in a cell, comprising providing a lipid particle to the cell, wherein the lipid particle is composed of a cationic lipid of formula A, a neutral lipid, a sterol, PEG or PEG-modified lipid or is substantially composed of a cationic lipid of formula A, a neutral lipid, a sterol, PEG or PEG-modified lipid, such as a molar ratio of about 35-65% of the cationic lipid of formula A, 3-12% of the neutral lipid, 15-45% of the sterol and 0.5-10% of the PEG or PEG-modified lipid, wherein the lipid particle is combined with a nucleic acid capable of regulating the expression of polypeptides. In a specific embodiment, the lipid molar ratio is about 60/7.5/31/1.5 or 57.5/7.5/31.5/3.5 (mol% lipid A/DSPC/cholesterol/PEG-DMG). In another set of embodiments, the neutral lipid in these compositions is replaced by DPPC (dipalmitoyl phosphatidylcholine), POPC, DOPE or SM.

In a specific embodiment, the therapeutic agent is selected from siRNA, microRNA, antisense oligonucleotide and a plasmid capable of expressing siRNA, microRNA or antisense oligonucleotide, and wherein the siRNA, microRNA or antisense RNA comprises a polynucleotide that specifically binds to a polynucleotide encoding the polypeptide or its complement to reduce polypeptide expression.

In other embodiments, the nucleic acid is a plasmid encoding a polypeptide or a functional variant or fragment thereof, such that expression of the polypeptide or a functional variant or fragment thereof is increased.

In a related embodiment, the present invention provides a method for treating a disease or condition characterized by overexpression of a polypeptide in a subject, comprising providing a pharmaceutical composition of the present invention to the subject, wherein the therapeutic agent is selected from siRNA, microRNA, antisense oligonucleotide and a plasmid capable of expressing siRNA, microRNA or antisense oligonucleotide, and wherein the siRNA, microRNA or antisense RNA comprises a polynucleotide that specifically binds to a polynucleotide encoding the polypeptide or its complement.

In one embodiment, the pharmaceutical composition comprises lipid particles, the lipid particles are made up of lipid A, DSPC, cholesterol and PEG-DMG, PEG-C-DOMG or PEG-DMA or are basically made up of lipid A, DSPC, cholesterol and PEG-DMG, PEG-C-DOMG or PEG-DMA, for example, the lipid PEG-DMG, PEG-C-DOMG or PEG-DMA modified by the cationic lipid of the formula A of about 35-65%, the neutral lipid of 3-12%, the sterol of 15-45% and 0.5-10% PEG or PEG-, wherein the lipid particles are combined with therapeutic nucleic acid. In a specific embodiment, the lipid molar ratio is about 60/7.5/31/1.5 or 57.5/7.5/31.5/3.5 (mol% lipid A/DSPC/ cholesterol/PEG-DMG). In another group of embodiments, the neutral lipid in these compositions is replaced by DPPC, POPC, DOPE or SM.

In another related embodiment, the present invention includes a method for treating a disease or condition characterized by underexpression of a polypeptide in a subject, comprising providing to the subject a pharmaceutical composition of the present invention, wherein the therapeutic agent is a plasmid encoding the polypeptide or a functional variant or fragment thereof.

Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as commonly understood by those skilled in the art to which the present invention belongs. Suitable methods and materials are described below, although similar or equivalent methods and materials as described in the present invention can also be used to implement or test the present invention. All publications, patent applications, patents and other references mentioned herein are incorporated herein by reference in their entirety. In the event of conflict, this specification (including definitions) shall prevail. In addition, the materials, methods and examples are merely illustrative and non-restrictive.

Example

Example 1: dsRNA synthesis

Reagent Source

When the source of a reagent is not specifically given in the present invention, such reagent can be obtained from any molecular biology reagent supplier with the quality/purity standards used in molecular biology.

siRNA synthesis

For screening of dsRNA, single-stranded RNA was prepared by solid phase synthesis on a 1 μmol scale using an Expedite 8909 synthesizer (Applied Biosystems, Applera Deutschland GmbH, Darmstadt, Germany) and controlled pore glass (CPG, Proligo Biochemie GmbH, Hamburg, Germany) as a solid support. RNA and RNA containing 2′-O-methyl nucleotides were generated by solid phase synthesis using the corresponding phosphoramidites and 2′-O-methyl phosphoramidites (Proligo Biochemie GmbH, Hamburg, Germany), respectively. These building blocks were incorporated into selected sites within the oligoribonucleotide chain sequence using standard nucleoside phosphoramidite chemistry as described in, for example, Current protocols in nucleic acid chemistry, Beaucage, S.L. et al. (Edrs.), John Wiley & Sons, Inc., New York, NY, USA. Phosphorothioate bonds were introduced by replacing the iodine oxidant solution with a solution of Beaucage reagent (Chruachem Ltd, Glasgow, UK) in acetonitrile (1%). Additional auxiliary reagents were obtained from Mallinckrodt Baker (Griesheim, Germany).

Deprotection and purification of crude oligoribonucleotides were performed by anion exchange HPLC according to the specified procedure. Yield and concentration were determined by UV absorption of the corresponding RNA solution at a wavelength of 260 nm using a spectrophotometer (DU 640B, Beckman Coulter GmbH, Unterschleißheim, Germany). Double-stranded RNA was generated by mixing equimolar complementary strand solutions in annealing buffer (20 mM sodium phosphate, pH 6.8; 100 mM sodium chloride), heating in a water bath at 85-90°C for 3 minutes and cooling to room temperature within 3-4 hours. The annealed RNA solution was stored at -20°C until use.

dsRNA targeting the Eg5 gene

Initial screening group

siRNA design was performed to identify siRNAs targeting Eg5 (also known as KIF11, HSKP, KNSL1, and TRIP5). The human mRNA sequence of Eg5, RefSeq ID: NM_004523, was used.

siRNA duplexes were designed that cross-reacted with both human and mouse Eg5. Twenty-four duplexes were synthesized for screening (Table 1a). A second screening panel was defined using 266 siRNAs targeting human Eg5 and its rhesus monkey ortholog (Table 2a). An expanded screening panel was selected using 328 siRNAs targeting human Eg5, without requiring any hits to Eg5 mRNA from other species (Table 3a).

The sequences of human and partial rhesus monkey Eg5 mRNA were downloaded from the NCBI nucleotide database, and the human sequence was further used as a reference sequence (human Eg5: NM_004523.2, 4908 bp, rhesus monkey Eg5: XM_001087644.1, 878 bp (only the 5′ portion of human Eg5).

For the table: Keywords: A, G, C, U - Ribonucleotide: T - Deoxythymidine: U, C - 2'-O-Methyl nucleotide: S - Phosphorothioate bond.

Table 1a. Sequences of Eg5/KSP dsRNA duplexes

Table 1b. Analysis of Eg5/KSP ds duplex

Table 2a. Sequences of Eg5/KSP dsRNA duplexes

Table 2b. Analysis of Eg5/KSP dsRNA duplexes

Table 3. Sequence and analysis of Eg5/KSP dsRNA duplexes

dsRNA targeting the VEGF gene

Four hundred target sequences were identified within exons 1-5 of the VEGF-A121 mRNA sequence. The reference transcript is: NM_003376.

Table 4a includes the identified target sequences. The corresponding siRNAs targeting these sequences were subjected to bioinformatics screening.

To ensure that the sequence was specific for the VEGF sequence and not for sequences from any other gene, the target sequence was collated against sequences in Genbank using the BLAST search engine provided by NCBI. The use of the BLAST algorithm is described in Altschul et al., J. Mol. Biol. 215:403, 1990; and Altschul and Gish, Meth. Enzymol. 266:460, 1996.

siRNAs were also prioritized based on their ability to cross-react with monkey, rat, and human VEGF sequences.

Of these 400 potential target sequences, 80 were selected for analysis by experimental screening to identify a small number of lead candidates. A total of 114 siRNA molecules were designed for these 80 target sequences (Table 4b).

Table 4a. Target sequences in VEGF-121

Table 4b: Duplexes targeting VEGF

Strand: S = sense, AS = antisense

Example 2: In vitro screening of Eg5 siRNA via cell proliferation

Since silencing of Eg5 has been shown to cause mitotic arrest (Weil, D, et al. [2002] Biotechniques 33:1244-8), a cell viability assay was used for siRNA activity screening. HeLa cells (14,000 per well [screens 1 and 3] or 10,000 per well [screen 2]) were seeded in 96-well plates and transfected simultaneously with ipofectamine 2000 (Invitrogen) at a final siRNA concentration of 30 nM per well, 50 nM for the first screen, and 25 nM for the second screen. A subset of duplexes was tested in the third screen at 25 nM (Table 5).

Seventy-two hours after transfection, WST-1 reagent (Roche) was added to the culture medium, and absorbance at 450 nm was measured to assess cell proliferation. The absorbance value of control (non-transfected) cells was considered 100%, and the absorbance of the siRNA-transfected wells was compared to the control value. Six replicates were performed in each of the three screening runs. Subsets of siRNAs were then tested across a range of siRNA concentrations. The assay was performed in HeLa cells (14,000 cells per well; the method was the same as above, see Table 5).

Table 5: Effects of 25 nM duplexes targeting Eg5 on cell viability

The nine siRNA duplexes that showed the greatest growth inhibition in Table 5 were retested in HeLa cells at a range of siRNA concentrations. The siRNA concentrations tested were 100 nM, 33.3 nM, 11.1 nM, 3.70 nM, 1.23 nM, 0.41 nM, 0.14 nM, and 0.046 nM. The experiment was repeated six times, and the concentration of each siRNA that resulted in 50 percent inhibition of cell proliferation (IC ₅₀ ) was calculated. For each duplex, the dose-response analysis was performed two to four times. The average IC ₅₀ values (nM) are given in Table 6.

Table 6: IC50 of siRNA: Cell proliferation in HeLa cells

duplex AL-DP-6226 15.5 AL-DP-6229 3.4 AL-DP-6231 4.2 AL-DP-6232 17.5 AL-DP-6239 4.4 AL-DP-6242 5.2 AL-DP-6243 2.6 AL-DP-6244 8.3 AL-DP-6248 1.9

Example 3: Eg5 via mRNA suppression In vitro screening of siRNA

Shortly before transfection, HeLa S3 (ATCC-Number: CCL-2.2, LCG Promochem GmbH, Wesel, Germany) cells were seeded at 1.5 x 10 ⁴ cells/well in 75 μl of growth medium (Ham's F12, 10% fetal bovine serum, 100 u penicillin/100 μg/ml streptomycin, all from Bookroom AG, Berlin, Germany) in a 96-well plate (Greiner Bio-One GmbH, Frickenhausen, Germany). Transfection was repeated four times. In each well, 0.5 μl of Lipofectamine 2000 (Invitrogen GmbH, Karlsruhe, Germany) and 12 μl of Opti-MEM (Invitrogen) were mixed and incubated at room temperature for 15 min. For the case where the siRNA concentration is 50 nM in a transfection volume of 100 μl, 1 μl of 5 μM siRNA and 11.5 μl of Opti-MEM were mixed in each well, combined with the Lipofectamine 2000-Opti-MEM mixture and incubated for another 15 minutes at room temperature. The siRNA-Lipofectamine 2000-complex was completely added to the cells, and the cells were incubated for 24 h at 37°C in a humidified incubator (Heroes GmbH, Hanau) with 5% CO _2. Single-dose screening was performed once at 50 nM and 25 nM, respectively.

Cells were harvested by applying 50 μl of a lysis mixture (from the contents of the QuantiGene bDNA-test kit of Genospectra, Fremont, USA) to each well containing 100 μl of growth medium and lysed at 53°C for 30 min. 50 μl was then incubated with probe sets specific for human Eg5 and human GAPDH and operated according to the supplier's protocol for QuantiGene. Finally, chemiluminescence was measured as RLU (relative light units) in a Victor2-Light (Perkin Elmer, Wiesbaden, Germany), and the values obtained with the hEg5 probe set were standardized relative to the corresponding GAPDH values in each well. The values obtained with the siRNA for Eg5 were correlated with the values obtained with the nonspecific siRNA (for HCV) set to 100% (Tables 1b, 2b, and 3b).

The effective siRNA characterization that screening obtains is made further with dose-response curve.The transfection of dose-response curve is carried out with following concentration: 100nM, 16.7nM, 2.8nM, 0.46nM, 77picoM, 12.8picoM, 2.1picoM, 0.35picoM, 59.5fM, 9.9fM and analogies (do not have siRNA), and is diluted to the ultimate density of 12.5 μ l with Opti-MEM according to said scheme.Use Microsoft Excel embedding software XL-fit 4.2 (IDBS, Guildford, Surrey, UK) and application dose-response model 205 to carry out data analysis (table 1b, 2b and 3b).

Lead siRNA AD12115 was additionally analyzed by applying the WST-proliferation assay from Roche (as described above).

A subset of the 34 duplexes from Table 2 that exhibited maximal activity was tested by transfection in HeLa cells at final concentrations ranging from 100 nM to 10 fM. Transfections were performed in quadruplicate. Dose-response experiments were performed twice for each duplex. The concentrations of each duplex that reduced KSP mRNA by 20% (IC20), 50% (IC50), and 80% (IC80) were calculated (Table 7).

Table 7: Dose-response mRNA inhibition of Eg5/KSP duplexes in HeLa cells

Concentration given in pM

IC20s IC50s IC80s Duplex name First screening Second screening First screening Second screening First screening Second screening AD12077 1.19 0.80 6.14 October 16 38.63 76.16 AD12078 25.43 25.43 156.18 156.18 ND ND AD12085 9.08 1.24 40.57 8.52 257.68 81.26 AD12095 1.03 0.97 9.84 4.94 90.31 60.47 AD12113 4.00 5.94 17.18 28.14 490.83 441.30 AD12115 0.60 0.41 3.79 3.39 23.45 23.45 AD12125 31.21 22.02 184.28 166.15 896.85 1008.11 AD12134 2.59 5.51 17.87 22.00 116.36 107.03 AD12149 0.72 0.50 4.51 3.91 30.29 40.89 AD12151 0.53 6.84 4.27 10.72 22.88 43.01 AD12152 155.45 7.56 867.36 66.69 13165.27 ND AD12157 0.30 26.23 14.60 92.08 14399.22 693.31 AD12166 0.20 0.93 3.71 3.86 46.28 20.59 AD12180 28.85 28.85 101.06 101.06 847.21 847.21 AD12185 2.60 0.42 15.55 13.91 109.80 120.63 AD12194 2.08 1.11 5.37 5.09 53.03 30.92 AD12211 5.27 4.52 11.73 18.93 26.74 191.07 AD12257 4.56 5.20 21.68 22.75 124.69 135.82

AD12280 2.37 4.53 6.89 20.23 64.80 104.82 AD12281 8.81 8.65 19.68 42.89 119.01 356.08 AD12282 7.71 456.42 20.09 558.00 ND ND AD12285 ND 1.28 57.30 7.31 261.79 42.53 AD12292 40.23 12.00 929.11 109.10 ND ND AD12252 0.02 18.63 6.35 68.24 138.09 404.91 AD12275 25.76 25.04 123.89 133.10 1054.54 776.25 AD12266 4.85 7.80 10.00 32.94 41.67 162.65 AD12267 1.39 1.21 12.00 4.67 283.03 51.12 AD12264 0.92 2.07 8.56 15.12 56.36 196.78 AD12268 2.29 3.67 22.16 25.64 258.27 150.84 AD12279 1.11 28.54 23.19 96.87 327.28 607.27 AD12256 7.20 33.52 46.49 138.04 775.54 1076.76 AD12259 2.16 8.31 8.96 40.12 50.05 219.42 AD12276 19.49 6.14 89.60 59.60 672.51 736.72 AD12321 4.67 4.91 24.88 19.43 139.50 89.49

(ND - not determined)

Example 4: Silencing of Eg5/KSP in the liver of young rats after a single bolus injection of LNP01-formulated siRNA

Eg5/KSP expression is detectable in the liver of growing rats from birth until approximately 23 days of age. Target silencing of formulated Eg5/KSP siRNA was assessed in juvenile rats using duplex AD-6248.

KSP duplex assay

method

Animal Dosing. Male, juvenile Sprague-Dawley rats (19 days old) were administered a single dose of liposome-formulated siRNA ("LNP01") via tail vein injection. Ten animals in each group received 10 milligrams per kilogram of body weight (mg/kg) of AD6248 or nonspecific siRNA. The dose level refers to the amount of siRNA duplex administered in the formulation. A third group received phosphate-buffered saline. Two days after siRNA administration, the animals were sacrificed. The livers were excised, quickly frozen in liquid nitrogen, and ground into a powder.

mRNA Assay. Eg5/KSP mRNA levels were measured in livers from all treatment groups. Each liver powder sample (approximately 10 mg) was homogenized in tissue lysis buffer containing proteinase K. The levels of Eg5/KSP and GAPDH mRNA were measured in triplicate using the Quantigene branched DNA assay (GenoSpectra). The mean values for Eg5/KSP in each sample were normalized to the mean GAPDH values. The group mean values for each experiment were determined and normalized to the PBS group.

Statistical Analysis: Significance was determined by ANOVA followed by Tukey's post hoc test.

result

Data Summary

The mean values (± standard deviation) of Eg5/KSP mRNA are shown. Statistical significance (p value) compared with the PBS group is shown (ns, not significant [p>0.05]).

Table 8. Experiment 1

A statistically significant reduction in hepatic Eg5/KSP mRNA was obtained following administration of formulation AD6248 at a dose of 10 mg/kg.

Example 5: Silencing of rat liver VEGF after intravenous infusion of LNP01-formulated VSP

A "lipidoid" formulation containing an equimolar mixture of two siRNAs was administered to rats. As used herein, VSP refers to a composition containing two siRNAs, one directed against Eg5/KSP and the other against VEGF. In this experiment, the duplexes AD3133, targeting VEGF, and AD12115, targeting Eg5/KSP, were used. Because Eg5/KSP expression is nearly undetectable in adult rat liver, only VEGF levels were measured after siRNA treatment.

Administered siRNA duplex (VSP)

Keywords: A, G, C, U-ribonucleotides; C, U-2′-O-Me nucleotides; S-phosphorothioate. The unmodified form of each strand and the target of each siRNA are as follows:

method

Animal Dosing. Adult, female Sprague-Dawley rats were administered siRNA formulated with lipidoid ("LNP01") via infusion into the femoral vein over a two-hour period. Groups of four animals received doses of 5, 10, and 15 milligrams per kilogram of body weight (mg/kg) of formulated siRNA. The dose level refers to the total amount of siRNA duplex administered in the formulation. A fourth group received phosphate-buffered saline. Animals were sacrificed 72 hours after the completion of the siRNA infusion. The livers were excised, flash-frozen in liquid nitrogen, and ground into a powder.

Preparation method

Lipidoid ND98·4HCl (MW 1487) (Formula 1 above), cholesterol (Sigma-Aldrich), and PEG-ceramide C16 (Avanti Polar Lipids) were used to prepare lipid-siRNA nanoparticles. Stock solutions of each in ethanol were prepared as follows: ND98, 133 mg/ml; cholesterol, 25 mg/ml; and PEG-ceramide C16, 100 mg/ml. The ND98, cholesterol, and PEG-ceramide C16 stock solutions were then mixed at a molar ratio of 42:48:10. The combined lipid solution was mixed with aqueous siRNA (in sodium acetate at pH 5) to a final ethanol concentration of approximately 35-45% and a final sodium acetate concentration of approximately 100-300 mM. Upon mixing, lipid-siRNA nanoparticles spontaneously formed. Depending on the desired particle size distribution, the resulting nanoparticle mixture is extruded through a polycarbonate membrane (100 nm cutoff) using a hot barrier extruder (Lipex Extruder, Northern Lipids, Inc) in some cases. In other cases, the extrusion step is omitted. Ethanol removal and simultaneous buffer exchange can be accomplished by dialysis or tangential flow filtration. The buffer is exchanged with phosphate buffered saline (PBS) at pH 7.2.

Characterization of formulations

Formulations prepared by standard methods or extrusion-free methods can be characterized in a similar manner. The formulations are first characterized by visual inspection. They should be whitish, translucent solutions free of aggregates or precipitates. The particle size and size distribution of the lipid-nanoparticles are determined by dynamic light scattering using a Malvern Zetasizer Nano ZS (Malvern, USA). The particle size should be approximately 20-300 nm, ideally 40-100 nm. The particle size distribution should be unimodal. A dye exclusion assay is used to assess the total siRNA concentration and the captured fraction in the formulation. Formulated siRNA samples are incubated with the RNA-binding dye Ribogreen (Molecular Probes) in the presence or absence of the formulation-disrupting surfactant 0.5% Triton-X100. The total siRNA in the formulation can be determined by comparing the signal from the surfactant-containing sample with the standard curve. The captured fraction is determined by subtracting the "free" siRNA content (determined by the signal in the absence of the surfactant) from the total siRNA content. The percentage of captured siRNA is typically >85%. For SNALP formulations, the particle size is at least 30 nm, at least 40 nm, at least 50 nm, at least 60 nm, at least 70 nm, at least 80 nm, at least 90 nm, at least 100 nm, at least 110 nm, and at least 120 nm. A preferred range is from about at least 50 nm to about at least 110 nm, preferably from about at least 60 nm to about at least 100 nm, and most preferably from about at least 80 nm to about at least 90 nm. In one example, each particle size comprises at least about a 1:1 ratio of Eg5 dsRNA and VEGF dsRNA.

mRNA Assay. Each liver powder sample (approximately 10 mg) was homogenized in tissue lysis buffer containing proteinase K. VEGF and GAPDH mRNA levels were determined for each sample using the Quantigene Branched DNA Assay (GenoSpectra) in triplicate. The mean VEGF value for each sample was normalized to the mean GAPDH value. The group mean for each experiment was determined and normalized to the PBS group.

Protein determination

Each liver powder sample (approximately 60 mg) was homogenized in 1 ml of RIPA buffer. Total protein concentration was determined using a Micro BCA protein assay kit (Pierce). VEGF protein levels were determined using a VEGF ELISA assay (R&D Systems) using total protein samples from each animal. Group means for each experiment were determined and normalized to the PBS group.

Statistical Analysis: Significance was determined by ANOVA followed by Tukey's post hoc test.

result

Data Summary

The mean values (± standard deviation) of mRNA (VEGF/GAPDH) and protein (rel.VEGF) for each treatment group are shown. The statistical significance (p value) of each experiment compared to the PBS group is shown.

Table 9

Statistically significant reductions in hepatic VEGF mRNA and protein were measured at all three siRNA dose levels.

Example 6: Evaluation of VSP SNALP in a mouse model of human liver tumors

These studies utilized a VSP siRNA mixture containing a dsRNA targeting KSP/Eg5 and a dsRNA targeting VEGF. As used herein, VSP refers to a composition containing two siRNAs, one directed against Eg5/KSP and the other against VEGF. In this experiment, duplexes AD3133 (directed against VEGF) and AD12115 (directed against Eg5/KSP) were used. The siRNA mixture was formulated into SNALP as described below.

The maximum study size was 20-25 mice. To test the efficacy of the siRNA-SNALP mixture in treating liver cancer, 1 x 10^6 tumor cells were injected directly into the left lateral lobe of the test mice. The incision was closed with sutures, and the mice were allowed to recover for 2-5 hours. Mice fully recovered within 48-72 hours. SNALP siRNA treatment was initiated 8-11 days after tumor inoculation.

The SNALP formulations used were (i) VSP (KSP + VEGF siRNA mixture (1:1 molar ratio)); (ii) KSP (KSP + Luc siRNA mixture); and (iii) VEGF (VEGF + Luc siRNA mixture). All formulations contained equal amounts (mg) of each active siRNA. All mice received a total siRNA/lipid dose, and each mixture was formulated using the original citrate buffer conditions to a 1:57 cDMA SNALP (1.4% PEG-cDMA; 57.1% DLinDMA; 7.1% DPPC and 34.3% cholesterol), 6:1 lipid:drug ratio.

Human Hep3B Study A: Antitumor Activity of VSP-SNALP

Human hepatoma Hep3B tumors were established in scid/beige mice by intrahepatic inoculation. Group A (n=6) animals were administered PBS; Group B (n=6) animals were administered VSP SNALP; Group C (n=5) animals were administered KSP/Luc SNALP; Group D (n=5) animals were administered VEGF/Luc SNALP.

SNALP treatment was initiated eight days after tumor inoculation. SNALP was administered at 3 mg/kg total siRNA twice weekly (Monday and Thursday) for a total of six doses (cumulative 18 mg/kg siRNA). The final dose was administered on day 25, with the endpoint on day 27.

Tumor burden was determined by (a) body weight; (b) liver weight; (c) visual inspection + photography on day 27; (d) human-specific mRNA analysis; and (e) blood alpha-fetoprotein levels on day 27.

Table 10 below shows the results of visual scoring of tumor burden measured in the inoculated (left) liver lobe. Scores: "-" = no visible tumor; "+" = obvious tumor tissue at the injection site; "++" = discrete tumor nodules protruding from the liver lobe; "+++" = large tumor protruding from both lobes; "++++" = large tumor, multiple nodules, spread throughout the liver lobe.

Table 10

The percentage of liver weight relative to body weight is shown in Figure 1. Figures 2A, 2B, 2C and 2D show the effects of PBS, VSP, KSP and VEGF on body weight of mice bearing human hepatoma Hep3B tumors.

The following conclusions were drawn from this study: (1) VSP SNALP showed potential antitumor effects in the Hep3B 1H model; (2) the antitumor activity of the VSP mixture appeared to be closely related to the KSP component; (3) anti-KSP activity was confirmed by single-dose histological analysis; and (4) VEGF siRNA showed no detectable effect on the inhibition of tumor growth in this model.

Human Hep3B Study B: Prolonged Survival Following Treatment with VSP

In the second Hep3B study, human hepatoma Hep3B tumors were established by intrahepatic inoculation into scid/beige mice. These mice were deficient in lymphocytes and natural killer cells, which minimizes the extent of immune-mediated anti-tumor effects. Group A (n=6) mice were untreated; Group B (n=6) mice were given luciferase (luc) 1955 SNALP (Lot No. AP10-02); and Group C (n=7) mice were given VSP SNALP (Lot No. AP10-01). SNALP was a 1:57 cDMA SNALP with a 6:1 lipid:drug ratio.

SNALP treatment began eight days after tumor inoculation. SNALP was administered at 3 mg/kg siRNA twice weekly (Monday and Thursday) for a total of six doses (cumulative 18 mg/kg siRNA). The final dose was administered on day 25, and the study endpoint was on day 27.

Tumor burden was determined by (1) body weight; (2) visual inspection + photography on day 27; (3) human-specific mRNA analysis; and (4) blood alpha-fetoprotein levels on day 27.

FIG3 shows the body weights measured on each day of administration (Days 8, 11, 14, 18, 21, and 25) and on the day of sacrifice.

Table 11

Scoring: “-” = no visible tumor; “+” = obvious tumor tissue at the injection site; “++” = discrete tumor nodules protruding from one lobe; “+++” = large tumor protruding from both lobes; “++++” = large tumor with multiple nodules throughout the lobes.

The correlation between body weight and tumor burden is shown in Figures 4, 5, and 6. Figure 4 shows the percentage of body weight of untreated mice over 27 days. Figure 5 shows the percentage of body weight of 1955Luc SNALP-treated mice over 27 days. Figure 6 shows the percentage of body weight of VSPSNALP-treated mice over 27 days.

A single dose of VSP SNALP (2 mg/kg) administered to Hep3B mice resulted in the formation of mitotic spindles in liver tissue samples as detected by histological staining.

Tumor burden was quantified by quantitative RT-PCR (pRT-PCR) (Taqman). Human GAPDH was normalized to mouse GAPDH by species-specific Taqman assay. FIG7A shows the tumor scores in the above table as determined by visual inspection relative to GAPDH levels.

Serum ELISA was performed to measure alpha-fetoprotein (AFP) secreted by the tumor. As described below, if AFP levels decreased after treatment, the tumor did not grow. Figure 7B shows that treatment with VSP reduced AFP levels in some animals compared to those treated with the control.

Human HepB3 Study C :

In a third study, human HCC cells (HepB3) were injected directly into the livers of SCID/beige mice, and treatment began 20 days later. Group A animals received PBS; Group B animals received 4 mg/kg Luc-1955 SNALP; Group C animals received 4 mg/kg SNALP-VSP; Group D animals received 2 mg/kg SNALP-VSP; and Group E animals received 1 mg/kg SNALP-VSP. A single dose of intravenous (iv) treatment was administered, and mice were sacrificed 24 hours later.

Tumor burden and target silencing were determined by qRT-PCR (Taqman). Tumor scores were also determined visually as described above, and the results are shown in the table below. The hGAPDH levels shown in Figure 8 correlated with the visually observed tumor scores shown in the table below.

Table 12

Scoring: "-" = visible tumor/some small tumors; "++" = discrete tumor nodules protruding from one lobe; "+++" = large tumors protruding from both lobes.

Human (tumor-derived) KSP silencing was determined by Taqman analysis, and the results are shown in Figure 9. hKSP expression was normalized to hGAPDH. Approximately 80% tumor KSP silencing was observed at 4 mg/kg SNALP-VSP, with significant efficacy at 1 mg/kg. The clear bar graph in Figure 9 shows the results from small (low GAPDH) tumors.

Human (tumor-derived) VEGF silencing was determined by Taqman analysis, and the results are shown in Figure 10. hVEGF expression was normalized to hGAPDH. Approximately 60% tumor VEGF silencing was observed at 4 mg/kg SNALP-VSP, with efficacy evident at 1 mg/kg. The clear bar graph in Figure 10 shows the results from small (low GAPDH) tumors.

Mouse (liver-derived) VEGF silencing was determined by Taqman analysis, and the results are shown in Figure 11A. mVEGF expression was normalized to hGAPDH. Approximately 50% liver VEGF silencing was observed at 4 mg/kg SNALP-VSP, and efficacy was significant at 1 mg/kg.

Human HepB3 Study D: Contribution of Each dsRNA to Tumor Growth

In the fourth study, human HCC cells (HepB3) were injected directly into the livers of SCID/beige mice, with treatment starting 8 days later. Treatment was by intravenous (iv) bolus injection twice weekly for a total of six doses. The final dose was administered on day 25, with the endpoint on day 27.

Tumor burden was determined by gross histology, human-specific mRNA analysis (hGAPDH qPCR), and blood alpha-fetoprotein levels (serum AFP via ELISA).

In Study 1, Group A was treated with PBS, Group B was treated with SNALP-KSP+Luc (3 mg/kg), Group C was treated with SNALP-VEGF+Luc (3 mg/kg), and Group D was treated with SNALP-VSP (3 mg/kg).

In Study 2, Group A was treated with PBS, Group B was treated with SNALP-KSP+Luc (1 mg/kg), and Group C was treated with ALN-VSP02 (1 mg/kg).

Both GAPDH mRNA levels and serum AFP levels were decreased after treatment with SNALP-VSP (as shown in FIG11B ).

Histological studies :

Human hepatoma Hep3B tumors were established by intrahepatic inoculation in mice. SNALP treatment was initiated 20 days after tumor inoculation. Tumor-bearing mice (three per group) were treated intravenously (IV) with a single dose of (i) VSP SNALP or (ii) control (Luc) SNALP (2 mg/kg total siRNA).

Liver/tumor samples were collected 24 hours after a single SNALP administration for routine H&E histology.

Large tumor nodules (5-10 mm) were evident macroscopically at autopsy.

Effects of SNALP-VSP in Hep3B mice :

Treatment with SNALP-VSP (a mixture of KSP dsRNA and VEGF dsRNA) reduced tumor burden and tumor-derived KSP and VEGF expression. A decrease in GAPDH mRNA levels (a measure of tumor burden) was also observed following administration of SNALP-VSP dsRNA (shown in Figures 12A, 12B, and 12C). A reduction in macroscopic tumor burden was also evident following administration of SNALP-VSP.

A single IV bolus injection of SNALP-VSP also resulted in mitotic spindle formation, which was clearly detected in Hep3B mouse liver tissue samples. This result indicates cell cycle arrest.

Example 7: Survival of SNALP-VSP animals versus SNALP-Luc treated animals

To determine the effect of siRNA SNALP on survival in cancer subjects, mice were treated with SNALP-siRNA to establish tumors via intrahepatic inoculation. These studies used a VSP siRNA cocktail containing dsRNAs targeting KSP/Eg5 and VEGF. A control was a dsRNA targeting Luc. The siRNA cocktail was formulated with SNALP.

Tumor cells (human hepatoma Hep3B, 1x10^6) were injected directly into the left lateral lobe of scid/beige mice. These mice are deficient in lymphocytes and natural killer (NK) cells, which is the minimum range for immune-mediated anti-tumor effects. The incision was closed by suture, and the mice were allowed to recover for 2-5 hours. The mice fully recovered within 48-72 hours.

All mice received a total siRNA/lipid intravenous (iv) dose, and each mixture was formulated as 1:57 cDMA SNALP (1.4% PEG-cDMA; 57.1% DLinDMA; 7.1% DPPC and 34.3% cholesterol), 6:1 lipid:drug using native citrate buffer conditions.

siRNA-SNALP treatment was started on the days shown below (18 or 26 days) after tumor inoculation. siRNA-SNALP was administered twice weekly at a dose of 4 mg/kg after 18 or 26 days for three weeks. Survival was monitored and animals were sacrificed based on human surrogate endpoints (e.g., animal weight, abdominal distension/discoloration, and overall health).

Survival data for treatment starting 18 days after tumor inoculation are summarized in Tables 13, 14, and Figure 13A.

Table 13. Kaplan-Meier (survival) data (% survival)

Table 14. Survival days of each animal

Figure 13A shows the mean survival of SNALP-VSP animals and SNALP-Luc treated animals relative to the number of days after tumor inoculation. The mean survival of SNALP-VSP animals was approximately 15 days longer than that of SNALP-Luc treated animals.

Table 15. Serum alpha-fetoprotein (AFP) concentrations of each animal before and at the end of treatment (concentrations expressed in μg/ml)

Tumor burden was monitored during the experiment using serum AFP levels. Alpha-fetoprotein (AFP) is the major plasma protein produced by the yolk sac and liver during fetal life. This protein is considered the fetal counterpart of serum albumin, and the human AFP and albumin genes are located in tandem on chromosome 4 with the same transcriptional orientation. AFP is found in monomeric, dimer, and trimer forms and binds copper, nickel, fatty acids, and bilirubin. AFP levels gradually decrease after birth, reaching adult levels between 8 and 12 months of age. AFP levels in normal adults are low but detectable. AFP has no known function in normal adults; however, AFP expression in adults is often associated with a subset of tumors, such as hepatoma and teratoma. AFP is a tumor marker used to monitor testicular cancer, ovarian cancer, and malignant teratomas. The main tumors that secrete AFP include endodermal sinus tumors (yolk sac carcinoma), neuroblastoma, hepatoblastoma, and hepatocellular carcinoma. In patients with AFP-secreting tumors, serum AFP levels often correlate with tumor size. Serum levels can be used to assess response to treatment. Typically, if AFP levels decrease after treatment, the tumor is not growing. A temporary increase in AFP immediately after chemotherapy may not indicate tumor growth, but rather that it is shrinking (and releasing AFP due to tumor cell death). Resection is usually accompanied by a decrease in serum levels. As shown in Figure 14, tumor burden was significantly reduced in animals treated with SNALP-VSP.

The experiment was repeated with SNALP-siRNA treatment on days 26, 29, 32, 35, 39, and 42 post-implantation. The data are shown in Figure 13B. The average survival of SNALP-VSP animals was extended by approximately 15 days, and that of SNALP-Luc-treated animals was extended by approximately 19 days, or 38%.

Example 8: Induction of Monoastrocytes in Established Tumors

Inhibition of KSP in dividing cells leads to the formation of monoasters, which can be readily observed in tissue sections. To determine whether monoaster formation occurs in SNALP-VSP-treated tumors, 2 mg/kg of SNALP-VSP was administered via tail vein injection to tumor-bearing animals (three weeks after Hep3B cell implantation). Control animals received 2 mg/kg of SNALP-Luc. Each mixture was formulated using the original citrate buffer conditions at a ratio of 1:57 cDMA SNALP (1.4% PEG-cDMA; 57.1% DLinDMA; 7.1% DPPC and 34.3% cholesterol), 6:1 lipid:drug.

Twenty-four hours later, the animals were sacrificed and the tumor-bearing liver lobes were processed for histological analysis. Representative images of H&E-stained tissue sections are shown in Figure 15. Extensive monoastrocytic formation was evident in tumors treated with SNALP-VSP (A) but not SNALP-Luc (B). In the latter, normal mitotic figures were evident. Monoastrocytic formation is a hallmark of KSP inhibition and provides evidence that SNALP-VSP has significant activity in established liver tumors.

Example 9: Preparation method and product specifications of ALN-VSP02 (SNALP-VSP)

The ALN-VSP02 product contains 2 mg/mL of the drug substance, ALN-VSPDS01, formulated in a sterile lipid particle formulation (referred to as SNALP) for IV administration via infusion. The drug substance, ALN-VSPDS01, consists of two siRNAs (ALN-12115, which targets KSP, and ALN-3133, which targets VEGF) in equimolar ratios. The drug product is packaged in 10 mL glass vials with a 5 mL fill volume.

The drug substance can be formulated with the cationic lipids XTC, ALNY-100 and MC3 to form other nucleic acid-lipid particle preparations described in the present invention.

This invention uses the following terms:

*Optional name = AD-12115, AD12115; **Optional name = AD-3133, AD3133

9.1. Preparation of Drug Substance ALN-VSPDS01

The two siRNA components of the drug substance ALN-VSPDS01, ALN-12115 and ALN-3133, were synthesized using commercially available synthesis equipment and raw materials. The preparation method involves synthesizing two single-stranded oligonucleotides of each duplex (19562 sense and 19563 antisense for ALN 12115 and 3981 sense and 3982 antisense for ALN 3133) via conventional solid-phase oligonucleotide synthesis using phosphoramidite chemistry and a 5'-dimethoxytriphenylmethyl (DMT) protecting group with either tert-butyldimethylsilyl (TBDMS) protection or replacement of the 2' hydroxyl group with a 2' methoxy ('OMe) group. The oligonucleotide chains are assembled on a solid support such as controlled pore glass or polystyrene using the phosphoramidite method. The cycle consists of 5' deprotection, coupling, oxidation, and capping reactions. Each coupling reaction is performed by activating an appropriately protected ribose, 2'OMe, or deoxyribonucleoside amide using a 5(ethylmercapto)1H tetrazolium reagent, followed by coupling to the free 5' hydroxyl group of the support-immobilized protected nucleoside or oligonucleotide. After an appropriate number of cycles, the final 5' protecting group is removed by acid treatment. The crude oligonucleotide is cleaved from the solid support by treatment with aqueous methylamine, accompanied by removal of the cyanoethyl protecting group and the nucleobase protecting group. The 2'OTBDMS group is then cleaved using a reagent containing hydrogen fluoride to produce a crude oligoribonucleotide, which is purified using strong anion exchange high-performance liquid chromatography (HPLC) followed by ultrafiltration desalting. The purified single strands are analyzed to confirm the correct molecular weight, molecular sequence, impurity profile, and oligonucleotide content, and then annealed to form a duplex. The annealed duplex intermediates, ALN 12115 and ALN 3133, are either lyophilized and stored at 20°C or mixed in a 1:1 molar ratio and lyophilized to produce the drug substance, ALN VSPDS01. If the duplex intermediates were stored as dry powders, they were redissolved in water before mixing. The mixing process was monitored by HPLC to achieve an equimolar ratio.

Instance specifications are shown in Table 16a.

Table 16a. Instance specifications for ALN-VSPDS01

The results of the stability study of the ALN-VSPDS01 drug substance up to 12 months are shown in Table 16b. Assays were selected to assess physical properties (appearance, pH, water content), purity (by SEC and denaturing anion exchange chromatography), and potency (by denaturing anion exchange chromatography) [AX-HPLC].

Table 16b: Stability of Drug Substance

9.2 Preparation of Drug Product ALN-VSP02

ALN VSP02 is a sterilized formulation of two siRNAs (in a 1:1 molar ratio) and a lipid excipient in an isotonic buffer. The lipid excipient binds to the two siRNAs, protecting them from degradation in the circulatory system and facilitating their delivery to target tissues. The specific lipid excipients and their respective quantitative ratios (shown in Table 17) were selected through a repeated series of experiments comparing the physicochemical properties, stability, pharmacodynamics, pharmacokinetics, toxicity, and product manufacturability of a large number of different formulations. The excipient DLinDMA is a titratable amino lipid that is positively charged at low pH, such as those found in mammalian cell endosomes, but is relatively uncharged at the more neutral pH of whole blood. This feature promotes efficient encapsulation of negatively charged siRNAs at low pH, preventing the formation of empty particles, but allows the formulation buffer to be replaced with a more neutral storage buffer before use to adjust (reduce) the particle charge. Cholesterol and the neutral lipid DPPC were introduced to provide physicochemical stability to the particles. The polyethylene glycol lipid conjugate PEG2000C DMA contributes to drug product stability and provides optimized circulation time for the proposed use. The ALN VSP02 lipid particles have an average diameter of approximately 80-90 nm and low polydispersity. At neutral pH, the particles are essentially uncharged, with a zeta potential of less than 6 mV. Based on this preparation method, there is no evidence of empty (unloaded) particles.

Table 17: Quantitative composition of ALN-VSP02

*The 1:1 molar ratio of the two siRNAs in the drug product was maintained throughout the particle size distribution of the drug product particles.

Solutions of lipids (in ethanol) and the ALN VSPDS01 drug substance (in aqueous buffer) were mixed and diluted to form a colloidal dispersion of siRNA lipid particles with an average particle size of approximately 80-90 nm. The dispersion was then filtered through a 0.45/0.2 μm filter, concentrated, and diafiltered by tangential flow filtration. After in-process testing and adjustment to a concentration of 2.0 mg/mL, the product was filter-sterilized, aseptically filled into vials, stoppered, capped, and stored at 5 ± 3°C. Ethanol and all aqueous buffer components were USP grade; all water used was USP Sterile Water for Injection grade. ALN-VSP02.

Similar methods were used to formulate other lipid formulations of ALN-VSPDS01, for example, those containing the cationic lipids XTC, ALNY-100, and MC3.

Example 10: In vitro efficacy of ALN-VSP02 on human cancer cell lines

The efficacy of ALN-VSP02 treatment on human cancer cell lines was determined by measuring KSP mRNA, VEGF mRNA and cell viability after treatment. The IC50 (nM) values of KSP and VEGF were determined in each cell line.

Table 19: Cell lines

On day one, cells were seeded in complete medium in 96-well plates to reach 70% confluence by day two. The following day, the medium was replaced with Opti-MEM Reduced Serum Medium (Invitrogen Cat N: 11058-021) and the cells were transfected with ALN-VSP02 or the control SNALP-Luc at concentrations ranging from 1.8 μM to 10 pM. Six hours later, the medium was replaced with complete medium. Each cell line was seeded in triplicate for each experiment.

ALN-VSP02 was formulated as described in Table 17.

Cells were harvested 24 hours after transfection. KSP levels were determined using bDNA and VEGF mRNA levels were determined using human TaqMan assay.

Viability was determined at 48 and/or 72 hours using Cell Titer Blue reagent (Promega Cat N: G8080) according to the manufacturer's recommendations.

As shown in Table 20, nM concentrations of VSP02 can effectively reduce the expression of KSP and VEGF in various human cell lines.

Table 20: Results

Example 11: Antitumor efficacy of VSP SNALP versus sorafenib in established Hep3B intrahepatic tumors

The antitumor effects of multiple doses of VSP SNALP were investigated in scid/beige mice bearing established Hep3B intrahepatic tumors compared with sorafenib, a small molecule inhibitor of protein kinases approved for the treatment of hepatocellular carcinoma (HCC).

As described herein, tumors were formed by intrahepatic inoculation of scid/beige mice. Treatment was initiated 11 days after inoculation. Mice were treated with sorafenib and control siRNA-SNALP, sorafenib and VSP siRNA-SNALP, or only VSP siRNA-SNALP. Control mice were treated with buffer only (DMSO replaced sorafenib, and PBS replaced siRNA-SNALP). Sorafenib was administered parenterally from Monday to Friday for three weeks at a dose of 15 mg/kg body weight for a total of 15 injections. Sorafenib was administered at least 1 hour after the injection of SNALP. siRNA-SNALPS was administered intravenously via the lateral tail vein at 3 mg/kg based on the most recently recorded body weight (10 ml/kg) on days 1, 4, 7, 10, 14, and 17 for three weeks (6 doses in total).

Each siRNA-SNALP was formulated as a 1:57 cDMA SNALP (1.4% PEG-cDMA; 57.1% DLinDMA; 7.1% DPPC and 34.3% cholesterol), 6:1 lipid:drug using native citrate buffer conditions.

Mice were sacrificed based on assessment of tumor burden, including gradual weight loss and clinical signs including sickness, abdominal distension/discoloration, and mobility.

The survival percentage data are shown in Figure 16. Co-administration of VSP siRNA-SNALP and sorafenib increased the survival rate compared to administration of sorafenib or VSP siRNA-SNALP alone. VSP siRNA-SNALP increased the survival rate compared to sorafenib.

Example 12: In vitro efficacy of VSP using variants of AD-12115 and AD-3133

Two sets of duplexes targeting Eg5/KSP and VEGF were designed and synthesized. Each set consisted of duplexes tiling 10 nucleotides in each direction of the target site of either AD-12115 or AD-3133.

The target, sense, and antisense strand sequences for each duplex are shown in the table below.

The inhibition of expression by each duplex is determined using the assays described herein. The duplexes are administered alone and/or in combination, for example, Eg5/KSP dsRNA and VEGF dsRNA combination. In some embodiments, the dsRNA is administered in the form of a nucleic acid lipid particle (e.g., a SNALP formulation described herein).

Table 21: dsRNA sequences targeting VEGF and Eg5/KSP (extended)

Example 13: dsRNA targeting VEGF with a single blunt end

A panel of dsRNA duplexes targeting VEGF was designed and synthesized. Each panel included duplexes extending 10 nucleotides in each direction of the AD-3133 target site. Each duplex included a 2-base overhang at the end corresponding to the 3' end of the antisense strand and no overhang, e.g., a blunt end, at the end corresponding to the 5' end of the antisense strand.

The sequences of each strand of these duplexes are shown in the table below.

The inhibition of expression by each duplex was determined using the assays described herein. The VEGF duplexes were administered alone and/or in combination with Eg5/KSP dsRNA (e.g., AD-12115). In some embodiments, the dsRNA is administered in the form of nucleic acid lipid particles (e.g., SNALP formulations described herein).

Table 22: Target sequences of blunt-ended dsRNAs targeting VEGF

Table 23: Strand sequences of blunt-ended dsRNAs targeting VEGF

Example 14: dsRNA oligonucleotide synthesis

synthesis

All oligonucleotides were synthesized on an AKTA oligopilot synthesizer using a commercially available controlled pore glass solid support (dT-CPG, Prime Synthesis) and RNA phosphoramidites with standard protecting groups, 5'-O-dimethoxytrityl N6-benzoyl-2'-tert-butyldithiocyanate, and 1'-dimethoxytrityl N6-benzoyl-2'-tert-butyldithiocyanate. Oligonucleotides were synthesized using methylsilyl-adenosine-3′-O-N,N′-diisopropyl-2-nitrileethyl phosphoramidite, 5′-O-dimethoxytrityl-N4-acetyl-2′-tert-butyldimethylsilyl-cytidine-3′-O-N,N′-diisopropyl-2-nitrileethyl phosphoramidite, 5′-O-dimethoxytrityl-N2-isobutyl-2′-tert-butyldimethylsilyl-guanosine-3′-O-N,N′-diisopropyl-2-nitrileethyl phosphoramidite, and 5′-O-dimethoxytrityl-2′-tert-butyldimethylsilyl-uridine-3′-O-N,N′-diisopropyl-2-nitrileethyl phosphoramidite (Pierce Nucleic Acids Technologies). 2'-F phosphoramidite, 5'-O-dimethoxytrityl-N4-acetyl-2'-fluoro-cytidine-3'-O-N,N'-diisopropyl-2-cyanoethyl-phosphoramidite, and 5'-O-dimethoxytrityl-2'-fluoro-uridine-3'-O-N,N'-diisopropyl-2-cyanoethyl-phosphoramidite were purchased from Promega. All phosphoramidites were used at a 0.2 M concentration in acetonitrile (CH3CN) except guanosine, which was used at a 0.2 M concentration in 10% THF/ANC (v/v). A coupling/recycle time of 16 minutes was used. The activator was 5-ethylmercaptotetrazole (0.75 M, American International Chemicals); iodine/water/pyridine was used for PO oxidation, and PADS (2%) in 2,6-lutidine/ACN (1:1 v/v) was used for PS oxidation.

The 3'-ligand conjugated chain is synthesized with a solid support containing the corresponding ligand. For example, the cholesterol unit is introduced into the sequence by hydroxyprolinol-cholesterol phosphoramidite. Cholesterol is bound to trans-4-hydroxyprolinol via a 6-aminocaproate bond to obtain the hydroxyprolinol-cholesterol portion. 5'-end Cy-3 and Cy-5.5 (fluorophore) labeled siRNAs are synthesized by the corresponding Quasar-570 (Cy-3) phosphoramidite purchased from Biosearch Technologies. Ligand conjugates that are bound to the 5'-end and or internal positions are obtained by using appropriately protected ligand-phosphoramidite structural units. In the presence of a 5-(ethylmercapto)-1H-tetrazole activator, a 0.1M solution of phosphoramidite in anhydrous CH3CN is extended for 15min with the oligonucleotide bound to the solid support. Internucleotide phosphite oxidation to phosphate was performed using standard iodine-water as reported in (1) or by treatment with tert-butyl hydroperoxide/acetonitrile/water (10:87:3) (10 min oxidation wait time for conjugated oligonucleotides). Phosphothioates were introduced by oxidation of phosphites to phosphorothioates using sulfur transfer agents such as DDTT (available from AM Chemicals), PADS, and/or Beaucage reagent. Cholesterol phosphoramidites were synthesized in-house and used at a concentration of 0.1 M in dichloromethane. The coupling time for cholesterol phosphoramidites was 16 min.

Deprotection I (Nucleobase Deprotection)

After the synthesis is complete, the support is transferred to a 100 mL glass bottle (VWR). The oligonucleotide is cut from the support and the base and phosphate groups are deprotected at 55°C for 6.5 h with 80 mL of an ethanolamine [ammonia: ethanol (3: 1)] mixture. The bottle is briefly cooled on ice and the ethanolamine mixture is then filtered into a new 250 mL bottle. The CPG is washed with 2 x 40 mL portions of ethanol/water (1: 1 v/v). The mixture volume is then reduced to approximately 30 mL by a rotary evaporator (roto-vap). The mixture is then frozen on dry ice and dried in a vacuum using a speed vac.

Deprotection II (removal of 2'-TBDMS group)

The dried residue was resuspended in 26 mL of triethylamine, triethylamine trihydrofluoride (TEA·3HF) or pyridine-HF and DMSO (3:4:6) and heated at 60°C for 90 minutes to remove the tert-butyldimethylsilyl group (TBDMS) at the 2' position. The reaction was then quenched with 50 mL of 20 mM sodium acetate and the pH was adjusted to 6.5. The oligonucleotides were stored in a refrigerator until purification.

analyze

Oligonucleotides are analyzed by high performance liquid chromatography (HPLC) and then purified, with the choice of buffer and column depending on the sequence and/or the nature of the conjugated ligand.

HPLC purification

The ligand-conjugated oligonucleotides were purified by reverse phase preparative HPLC. The unconjugated oligonucleotides were purified by anion exchange HPLC on an internally packed TSK gel column. The buffer was 20 mM sodium phosphate (pH 8.5) in 10% _CH3CN (buffer A) and 20 mM sodium phosphate (pH 8.5) in 10% _CH3CN , 1 M NaBr (buffer B). The fractions containing the full-length oligonucleotides were combined. Desalted and lyophilized. Approximately 0.15 OD of the desalted oligonucleotide was diluted to 150 μL in water and then pipetted into a special vial for CGE and LC/MS analysis. The compound was then analyzed by LC-ESMS and CGE.

siRNA preparation

To prepare siRNA, equimolar amounts of the sense and antisense strands were heated in 1xPBS at 95°C for 5 min and slowly cooled to room temperature. The integrity of the duplex was confirmed by HPLC analysis. AD-3133 and AD-AD-12115 were synthesized as described herein.

Example 15: Synthesis of conjugated lipids

Synthesize PEG-lipids, such as mPEG2000-1,2-di-O-alkyl-sn3-carbamoylglycerol (PEG-DMG), using the following method:

mPEG2000-1,2-di-O-alkyl-sn3-carbamoylglycerol

Preparation of compound 4a: by 1,2-bis-O-tetradecyl-sn-glyceride 1a (30g, 61.80mmol) and N, N '-succinimide carbonate (DSC, 23.76g, 1.5eq) add in dichloromethane (DCM, 500mL) and stir in ice-water mixture.Triethylamine (25.30mL, 3eq) is added in the solution stirred, and the reaction mixture is stirred at ambient temperature and spends the night.Reaction process is monitored by TLC.With DCM (400mL) diluted reaction mixture, water (2X500mL), NaHCO _The aqueous solution (500mL) washs organic layer, is then standard operation.The residue obtained is dried and spends the night in high vacuum at ambient temperature.After drying, the crude carbonate 2a thus obtained is dissolved in dichloromethane (500mL) and stirs in ice bath. To the stirred solution was added mPEG ₂₀₀₀ -NH ₂ (3, 103.00 g, 47.20 mmol, purchased from NOF Corporation, Japan) and anhydrous pyridine (80 mL, excess) under argon. In some embodiments, x in methoxy-(PEG) x-amine is 45-49, preferably 47-49, more preferably 49. The reaction mixture was then stirred at ambient temperature overnight. The solvent and volatiles were removed under vacuum, and the residue was dissolved in DCM (200 mL) and loaded onto a silica gel column packed with ethyl acetate. The column was eluted first with ethyl acetate and then with a gradient of 5-10% methanol in dichloromethane to afford the desired PEG-lipid 4a as a white solid (105.30 g, 83%). ¹ H NMR (CDCl ₃ , 400 MHz) δ=5.20-5.12 (m, 1H), 4.18-4.01 (m, 2H), 3.80-3.70 (m, 2H), 3.70-3.20 (m, -O-CH ₂ -CH ₂ -O-, PEG-CH ₂ ), 2.10-2.01 (m, 2H), 1.70-1.60 (m, 2H), 1.56-1.45 (m, 4H), 1.31-1.15 (m, 48H), 0.84 (t, J=6.5 Hz, 6H). MS range found 2660-2836.

Preparation of 4b: 1,2-Di-O-hexadecyl-sn-glyceride 1b (1.00 g, 1.848 mmol) and DSC (0.710 g, 1.5 eq) were added to dichloromethane (20 mL) and cooled to 0°C in an ice-water mixture. Triethylamine (1.00 mL, 3 eq) was added and stirred overnight. The reaction was followed by TLC, diluted with DCM, washed with water (twice), _NaHCO₃ solution, and dried over sodium sulfate. The solvent was removed under reduced pressure, and the residue 2b was kept under high vacuum overnight. This compound was used in the next reaction without additional purification. MPEG ₂₀₀₀ - _NH₂₃ (1.50 g, 0.687 mmol, purchased from NOF Corporation, Japan) and compound 2b from the previous step (0.702 g, 1.5 eq) were dissolved in dichloromethane (20 mL) under argon. The reaction was cooled to 0°C. To this was added pyridine (1 mL, in excess) and stirred overnight. The reaction was monitored by TLC. The solvent and volatiles were removed under vacuum and the residue was purified by chromatography (first with ethyl acetate and then with 5-10% MeOH/DCM gradient elution) to obtain the required compound 4b as a white solid (1.46 g, 76%). ¹ H NMR (CDCl ₃ , 400 MHz) δ=5.17 (t, J=5.5 Hz, 1H), 4.13 (dd, J=4.00 Hz, 11.00 Hz, 1H), 4.05 (dd, J=5.00 Hz, 11.00 Hz, 1H), 3.82-3.75 (m, 2H), 3.70-3.20 (m, -O-CH ₂ -CH ₂ -O-, PEG-CH ₂ ), 2.05-1.90 (m, 2H), 1.80-1.70 (m, 2H), 1.61-1.45 (m, 6H), 1.35-1.17 (m, 56H), 0.85 (t, J=6.5 Hz, 6H). MS range found: 2716-2892.

Preparation of 4c: 1,2-Di-O-octadecyl-sn-glyceride 1c (4.00 g, 6.70 mmol) and DSC (2.58 g, 1.5 eq) were added to dichloromethane (60 mL) and cooled to 0°C in an ice-water mixture. Triethylamine (2.75 mL, 3 eq) was added and stirred overnight. The reaction was followed by TLC, diluted with DCM, washed with water (twice), _NaHCO₃ solution, and dried over sodium sulfate. The solvent was removed under reduced pressure, and the residue 2b was kept under high vacuum overnight. This compound was used in the next reaction without additional purification. MPEG ₂₀₀₀ - _NH₂₃ (1.50 g, 0.687 mmol, purchased from NOF Corporation, Japan) and compound 2c from the previous step (0.760 g, 1.5 eq) were dissolved in dichloromethane (20 mL) under argon. The reaction was cooled to 0°C. To this was added pyridine (1 mL, in excess) and stirred overnight. The reaction was monitored by TLC. The solvent and volatiles were removed under vacuum and the residue was purified by chromatography (first with ethyl acetate and then with 5-10% MeOH/DCM gradient elution) to give the desired compound 4c as a white solid (0.92 g, 48%). ¹ H NMR (CDCl ₃ , 400 MHz) δ=5.22-5.15 (m, 1H), 4.16 (dd, J=4.00 Hz, 11.00 Hz, 1H), 4.06 (dd, J=5.00 Hz, 11.00 Hz, 1H), 3.81-3.75 (m, 2H), 3.70-3.20 (m, -O-CH ₂ -CH ₂ -O-, PEG-CH ₂ ), 1.80-1.70 (m, 2H), 1.60-1.48 (m, 4H), 1.31-1.15 (m, 64H), 0.85 (t, J=6.5 Hz, 6H). MS range found: 2774-2948.

Example 16: General protocol for extrusion

Lipids (e.g., lipid A, DSPC, cholesterol, DMG-PEG) are dissolved and mixed in ethanol according to the desired molar ratio. Liposomes are formed by ethanol injection, where the mixed lipids are added to a sodium acetate buffer at a pH of 5.2. This results in spontaneous formation of liposomes in 35% ethanol. The liposomes are extruded through a 0.08 μm polycarbonate membrane at least twice. siRNA stock solutions are prepared in sodium acetate, and 35% ethanol is added to the liposomes for loading. The siRNA-liposome solution is incubated at 37°C for 30 min and subsequently diluted. The ethanol is removed and replaced with PBS buffer by dialysis or tangential flow filtration.

Example 17: General Scheme of In-Line Mixing Method

Prepare separate and distinct stock solutions: one containing lipids and the other containing siRNA. Prepare a lipid stock solution containing, for example, lipid A, DSPC, cholesterol, and PEG lipids by dissolving them in 90% ethanol. The remaining 10% is low pH citrate buffer. The concentration of the lipid stock solution is 4 mg/mL. Depending on the type of fusogenic lipid used, the pH of this citrate buffer can range from 3 to 5. siRNA is also dissolved in citrate buffer at a concentration of 4 mg/mL. For small-scale production, prepare 5 mL of each stock solution.

The stock solution is completely transparent and the lipid must be completely dissolved before mixing with the siRNA. Therefore, the stock solution can be heated to completely dissolve the lipid. The siRNA used in this method can be an unmodified oligonucleotide or a modified oligonucleotide and can be conjugated to a lipophilic moiety such as cholesterol.

The individual stock solutions were mixed by pumping each solution into a T-junction. A double-ended Watson-Marlow pump was used to simultaneously control the start and stop of the two streams. The 1.6 mm polypropylene tubing was further reduced to a 0.8 mm tube to increase the linear flow rate. Polypropylene tubing (ID = 0.8 mm) was connected to either side of the T-junction. The linear edge of the polypropylene T was 1.6 mm, resulting in a volume of 4.1 mm ³ . Each large end (1.6 mm) of the polypropylene tubing was placed in a test tube containing the dissolved lipid stock solution or dissolved siRNA. A single pipe was set after the T-junction, where the combined flow would flow out. The pipe then extended into a container containing 2 volumes of PBS. The PBS was stirred rapidly. The flow rate of the pump was set at 300 rpm or 110 mL/min. The ethanol was removed and replaced with PBS by dialysis. The lipid preparation was then concentrated to the appropriate working concentration by centrifugation or diafiltration.

FIG17 shows a schematic diagram of the in-line mixing method.

Example 18: Silencing of siRNA in Hep3B tumors in mice by LNP-08 formulated VSP

Silencing of VSPs (VEGF and KSP) was performed in orthotopic (intrahepatic) Hep3B tumors following intravenous administration of siRNA formulated in nucleic acid-lipid particles containing XTC, such as LNP-08.

Tumors were established by transplanting 1×10 ⁶ Hep3B cells into the right flank of 8-week-old female Fox scid/beige mice. The cells were genetically engineered to stably express firefly luciferase. Tumor burden was monitored weekly by in vivo biophotonic imaging using the IVIS system (Caliper, Inc.). Approximately 4 weeks after tumor implantation, groups of tumor-bearing animals received intravenous (tail vein) injections of the following test articles:

LNP08-1955 is a lipid nanoparticle formulated with siRNA AD-1955 (targeting firefly luciferase) containing XTC (60 mol%), DSPC (7.5 mol%), cholesterol (31 mol%), and PEG-cDMG (1.5 mol%) with an N:P ratio of approximately 3.0.

LNP08-VSP is a lipid nanoparticle formulated with siRNAs AD-12115 (targeting KSP) and AD-3133 (targeting VEGF) in a 1:1 molar ratio. It contains XTC (60 mol%), DSPC (7.5 mol%), cholesterol (31 mol%), and PEG-cDMG (1.5 mol%) with an N:P ratio of approximately 3.0.

One day after treatment, animals were sacrificed and tumor-bearing liver lobes were harvested for analysis. Total RNA was extracted and cDNA was synthesized using random primers. Human KSP and human VEGF levels were determined using human-specific custom assays (Applied Biosystems, Inc.) normalized to human GAPDH. Group means were calculated and normalized to the LNP08-1955 treated group.

As shown in Figure 18, treatment with LNP08-VSP (Group 2) resulted in a greater than 60%, eg, 68% (p < 0.001) reduction in tumor KSP mRNA and at least a 40% reduction in VEGF mRNA (p < 0.05) compared to LNP08-1955 treatment (Group 1).

Example 19: Evaluation of LNP-011 and LNP-012 lipid formulations in the mouse Hep3b tumor model

The effects of various VSP formulations on KSP and VEGF expression in mouse intrahepatic Hep3B tumors were compared. Thirty-five female Fox Scid beige mice were injected with 1×10^6 Hep3B-Luc cells suspended in 0.025cc PBS via direct intrahepatic surgery. Tumor growth was monitored by Xenogen via Luc readings.

Mice received a single bolus injection (4 mg/kg) of one of the following formulations: SNALP-1955 (luciferase control); ALN-VSP02; SNALP-T-VSP (containing C-18 PEG)-VSP; LNP-11-VSP; and LNP-12VSP. Twenty-four hours after dosing, animals were sacrificed, and TaqMan assays were used to determine tumor-specific KSP and VEGF inhibition.

The results are shown in Figure 21. SNAPL-T-VSP, LNP-11-VSP and LNP-12VSP showed increased inhibition of KSP expression compared to ALN-VSP02.

Example 20: Evaluation of LNP-08+/-C18 lipid formulation in mouse Hep3b tumor model

The following VSP formulations were tested in the HEP3B tumor model. One of the following formulations, prepared according to the above method, was injected (intrahepatically) into tumor-bearing mice as a single bolus IV dose:

ALN-VSP02 was prepared as described in Example 9.

LNP08-Luc is a lipid nanoparticle formulated with siRNA AD-1955 (targeting firefly luciferase) containing XTC (60 mol%), DSPC (7.5 mol%), cholesterol (31 mol%), and PEG-cDMG (1.5 mol%) with an N:P ratio of approximately 3.0.

LNP08-VSP is a lipid nanoparticle formulated with siRNA AD-12115 (targeting KSP) and AD-3133 (targeting VEGF) in a 1:1 molar ratio. It contains XTC (60 mol%), DSPC (7.5 mol%), cholesterol (31 mol%), and PEG-cDMG (1.5 mol%) with an N:P ratio of approximately 3.0.

LNP08-C18-VSP is a lipid nanoparticle formulated with siRNA AD-12115 (targeting KSP) and AD-3133 (targeting VEGF) in a 1:1 molar ratio. It contains XTC (60 mol%), DSPC (7.5 mol%), cholesterol (31 mol%), and PEG-cDSG (1.5 mol%) with an N:P ratio of approximately 3.0.

The chemical structures of PEG-DSG and PEG-C-DSA are illustrated in Figure 19. PEG-DSG is polyethylene glycol distyrylglycerol, where PEG is C18-PEG or PEG-C18, and the average molecular weight of PEG is 2000 Da.

Twenty-four hours after treatment, animals were sacrificed and tumors were harvested for analysis. Total RNA was extracted from the tumors, and cDNA was synthesized using random primers. Human KSP and human VEGF levels were determined normalized to human GAPDH using human-specific custom assays (Applied Biosystems, Inc.).

The results are shown graphically in Figure 22, which demonstrates silencing of KSP and VEGF comparable to that caused by ALN-VSP02.

Example 21: Role of ApoE in the Cellular Uptake of Liposomes in HeLa Cells

LNP-formulated dsRNA was prepared by adding recombinant human ApoE. The resulting LNP-ApoE-formulated dsRNA was tested in HeLa cells for its effect on cellular uptake of the dsRNA. Compositions and methods using ApoE and ionizable lipids are described in International Patent Application No. PCT/US10/22614, which is incorporated herein by reference in its entirety.

Experimental methods

HeLa cells were seeded at 6,000 cells per well in 96-well plates (Grenier) overnight. Three different liposome formulations of Alexa-fluor 647-labeled GFP siRNA: 1) LNP01, 2) SNALP, and 3) LNP05 were diluted to a final concentration of 50 nM in one of three media conditions. The media tested were OptiMem, DMEM with 10% FBS, or DMEM with 10% FBS plus 10 μg/mL human recombinant ApoE (Fitzgerald Industries). The designated liposomes were added to the cells in culture medium or in culture medium pre-complexed with ApoE for 10 minutes for 4, 6, or 24 hours. Each experimental condition was performed in triplicate. After addition to the HeLa cells at the designated time points, the cells were fixed in 4% paraformaldehyde for 15 minutes, and the nuclei and cytoplasm were then stained with DAPI and Syto dye. Images were acquired using an Opera spinning disk automated confocal system from Perkin Elmer. Alexa Fluor 647 siRNA uptake was quantified using Acapella software. Four different parameters were quantified: 1) cell number, 2) number of siRNA-positive spots per area, 3) number of siRNA-positive spots per cell, and 4) integrated spot signal, or the average number of siRNA spots per cell multiplied by the average spot intensity. The average spot signal thus provides a rough estimate of the total siRNA content per cell.

Additionally, four different LNP-ApoE formulated dsRNAs (SNALP (DLinDMa), XTC, MC3, ALNY-100) were tested in the following cell lines and the effects on cellular dsRNA uptake were determined:

A375 (melanoma), B16F10 (melanoma), BT-474 (breast cancer), GTL-16 (gastric cancer), Hct116 (colon cancer), Hep3b (liver cancer), HepG2 (liver cancer), HeLa (cervical cancer), HUH 7 (liver cancer), MCF7 (breast cancer), Mel-285 (uveal melanoma), NCI-H1975 (lung cancer), OMM-1.3 (uveal melanoma), PC3 (prostate cancer), SKOV-3 (ovarian cancer), U87 (glioblastoma).

Example 22: _Kd of KSP siRNA in the presence of ApoE

The effect of ApoE on the Kd (affinity) of LNP-08 formulated siRNA targeting KSP was evaluated in various cell lines. siRNAs formulated with LNP08 and LNP08 and C18PEG were used. The siRNA duplex targeting KSP was AL-DP-6248.

The following cell lines were used.

cell lines Cell type species HeLa Cervical adenocarcinoma people HCT116 colon cancer people A375 melanoma people MCF7 Breast cancer people

B16F10 melanoma mice Hep3b Liver cancer people HUH 7 Liver cancer people HepG2 Liver cancer people Skov 3 Ovarian cancer people U87 Glioblastoma people PC3 Prostate cancer people

On day one, cells were seeded at 20,000 cells/well in a 96-well plate. On day two, the formulated siRNA was incubated in serum-containing medium +/- ApoE at 37°C for 15-30 minutes. The medium was removed from the cells, and 100 μL/well of the pre-warmed complex was plated onto the cells at a 20 nM siRNA concentration. ApoE concentration was titrated at 1.0, 3.0, 9.0, and 20.0 μg/ml. Cells were incubated with the formulated duplex for 24 hours. On day three, cells were lysed and prepared for bDNA analysis and kD calculation.

The presence of Apo E improved kD in a number of cell lines including HCT-116, HeLa, A375, and B16F10 (data not shown).

Example 23: _IC50 of KSP siRNA in the presence of ApoE

The effect of ApoE on the _IC50 (efficacy) of LNP-08 formulated siRNA targeting KSP was evaluated in various cell lines. siRNAs formulated with LNP08 and LNP08 and C18PEG were used. The siRNA duplex targeting KSP was AL-DP-6248.

On day 0, cells were seeded at 15,000-20,000 cells/well in 96-well plates. On day one, serum-containing culture medium, formulated duplex, and +/- 3 ug/ml ApoE were incubated at 37°C for 15-30 minutes. Serial dilutions of siRNA were used in the range of 0.01 nM to 1.0 μM. Culture medium was removed from the cells, and 100 uL/well of the pre-warmed complex was plated onto the cells. The cells were incubated with siRNA for 24 hours. On day two, the cells were lysed and prepared for bDNA analysis as described herein. KSP mRNA levels were measured using Quantigene 1.0 to determine KSP levels and compared to GAPDH. The negative control was siRNA targeting luciferase, AD-1955.

The results are shown in the table below. LNP-08 formulated siRNA was active in all cell lines. In some cell lines, the addition of ApoE improved the efficacy of siRNA treatment, as evidenced by lower _IC50 .

Example 24: Inhibition of Eg5/KSP and VEGF expression in humans

A pharmaceutical composition, such as a nucleic acid-lipid particle containing a dsRNA targeting the Eg5/KSP gene and a dsRNA targeting the VEGF gene, is used to treat a human subject to inhibit the expression of the Eg5/KSP and VEGF genes in the nucleic acid-lipid particle. For example, the nucleic acid-lipid particle comprises XTC, MC3, or ALNY-100.

A subject in need of treatment is selected or identified. The subject may be in need of treatment for cancer, such as liver cancer.

At time zero, an appropriate first dose of the composition is administered subcutaneously to the subject. The composition is formulated as described herein. After a period of time, the subject's condition is assessed, for example, by measuring tumor growth, serum AFP levels, etc. This assessment can be accompanied by measuring Eg5/KSP and/or VEGF expression in the subject, and/or determining successful siRNA-targeted products of Eg5/KSP and/or VEGF mRNA. Other relevant criteria can also be measured. The number and intensity of doses are adjusted based on the subject's needs.

After treatment, the subject's condition is compared to the condition that existed before treatment, or to the condition of untreated subjects with similar conditions.

Those skilled in the art will be aware of methods and compositions other than those specifically set forth herein which will allow them to practice the invention within the full scope of the appended claims.

Claims (5)

1. A composition comprising nucleic acid lipid particles, said nucleic acid lipid particles containing a first double-stranded RNA (dsRNA) for inhibiting the expression of the human kinin family member 11 (Eg5/KSP) gene in cells and a second dsRNA for inhibiting the expression of human VEGF in cells, wherein: The nucleic acid lipid particles contain a lipid formulation comprising 45-65 mol% cationic lipids, 5-10 mol% non-cationic lipids, 25-40 mol% sterols, and 0.5-5 mol% PEG or PEG-modified lipids, wherein the cationic lipids contain MC3 (4-(dimethylamino)butyric acid (6Z,9Z,28Z,31Z)-heptadec-6,9,28,31-tetraen-19-yl ester), and the sterols contain cholesterol. The first dsRNA consists of a sense strand and an antisense strand. The sense strand is composed of SEQ ID NO:1240 (5’-ucGAGAAucuAAAcuAAcuTsT-3’) and the antisense strand is composed of SEQ ID NO:1241 (5’-AGUuAGUUuAGAUUCUCGATsT). The second dsRNA consists of a sense strand and an antisense strand, the sense strand being composed of SEQ ID NO:1242 (5’-GcAcAuAGGAGAGAuGAGCUsU-3’) and the antisense strand being composed of SEQ ID NO:1243 (5’-AAGCUcAUCUCUCCuAuGuGCusG-3’). Each chain is modified to include 2'-O-methylribonucleotides, as indicated by lowercase "c" or "u", and phosphate thioesters, as indicated by lowercase "s". The first and second dsRNAs are present in an equimolar ratio. 2. The composition of claim 1, wherein the cationic lipid contains MC3 and the lipid formulation is selected from the group consisting of: 3. The composition of claim 1 or 2 further comprises sorafenib. 4. The composition of claim 1 or 2 further contains lipoproteins. 5. The composition of claim 1 or 2 further comprises apolipoprotein E (ApoE).