TW201717970A - Therapeutic compositions and methods for malignant tumors with RNAi molecules targeted to Hsp47 and p21 - Google Patents

Abstract

This invention provides compositions and methods for preventing or treating a malignant tumor in a mammal in need thereof, by administering to the mammal a therapeutically effective amount of a composition comprising RNAi molecules, which RNAi molecules can be active in reducing expression of Hsp47, or a combination of RNAi molecules active for Hsp47 and p21.

Description

Therapeutic compositions and methods for malignant tumors using RNAi molecules targeting Hsp47 and p21

The present invention relates to the field of biological agents and therapeutic agents composed of nucleic acid-based molecules. More particularly, the present invention relates to methods and compositions for delivering RNA interfering agents to prevent, treat or ameliorate the effects of diseases and diseases involving malignancies.

Sequence table

The present application includes a sequence listing of an ASCII file via electronic submission, created on December 20, 2015, entitled ND5123767 WO_SL.txt, having a size of 100,000 bytes and the entire contents of which is incorporated herein by reference.

The growth of malignant tumors is associated with extracellular matrix (ECM). The molecular chaperone "heat shock" protein Hsp47 is involved in the regulation of the ECM gene transcription network.

Hsp47 expression may be activated in breast cancer and other cancers. Reducing Hsp47 can reduce the growth of breast cancer cells and limit tumor growth.

Increased performance of Hsp47 may be a factor in poor survival outcomes in breast cancer patients. Hsp47 expression may contribute to cancer progression, in part due to increased ECM protein. See, for example, Cancer Res, 2015, 75(8); 1580-91.

Hsp47 is highly expressed in pancreatic cancer. Hsp47 can be a useful specific marker for detecting oral cancer.

Hsp47 or a homologous gene sequence thereof is disclosed, for example, as GenBank No. AB010273 (human), X60676 (mouse) or M69246 (rat, gp46).

Agents for suppressing Hsp47 have been disclosed for use in inhibiting fibrosis. See, for example, US 8,173,170 B2 and US 8,710,209 B2. However, there is limited information on the effects of inhibiting the development, progression and growth of malignant tumors.

P21 is a cell cycle regulatory protein encoded by the CDKN1A gene and belonging to the CIP/KIP family. This protein has a function of inhibiting the action of the complex by binding to a cyclin-CDK complex to inhibit cell cycle progression in the G1 phase and the G2/M phase. Specifically, the p21 gene undergoes activation by the p53 gene (one of the tumor suppressor genes). It has been reported that when p53 is activated by DNA damage, etc., p53 activates p21, thereby preventing the cell cycle from being arrested in the G1 phase and the G2/M phase.

P21 is overexpressed in a variety of human cancers, including prostate cancer, cervical cancer, breast cancer, and squamous cell carcinoma, and in many cases, upregulation of p21 is positively associated with tumor grade, invasion, and aggressiveness. See, for example Chang et al., Proc. Natl. Acad. Sci. USA, 2000, Vol. 97, No. 8, pp. 4291-96. In addition, up-regulation of p21 has been reported to be associated with poor prognosis of tumorigenic and various forms of cancer, including brain cancer, prostate cancer, ovarian cancer, breast cancer, and esophageal cancer. See, for example, Winters et al., Breast Cancer Research, 2003, Vol. 5, No. 6, pp. R242-R249. In addition, the disease may be age-related diseases including atherosclerosis, Alzheimer's disease, amyloidosis and arthritis. See, for example, Chang et al., Proc. Natl. Acad. Sci. USA, 2000, Vol. 97, No. 8, pp. 4291-96.

There is an urgent need for methods and compositions for the development of therapies for patients with malignant tumors, such as siRNA sequences, compounds and structures for inhibiting the expression of Hsp47 and p21.

What is needed is a method and composition for preventing or treating a malignant tumor. There is a continuing need for RNAi molecules that target Hsp47, p21, and other structures, as well as compositions for preventing, treating, or reducing malignancies.

The present invention relates to the surprising discovery that the size of a malignant tumor in vivo can be reduced by treatment with a siRNA inhibitor of Hsp47 and a combination of an inhibitor of Hsp47 and an inhibitor of p21.

The present invention relates to methods and compositions for the incorporation of nucleic acid-based therapeutic compounds for delivery to various organs for the prevention, treatment or amelioration of conditions and diseases of malignancies. In some implementations, the invention provides for A composition of an RNA interference molecule (RNAi molecule) that does not express a gene of various targets associated with malignant tumors.

The present invention can provide compositions for delivery of therapeutic molecules and methods of their use. The various RNA-based and pharmaceutical compositions of the present invention are useful in methods of preventing or treating malignancies.

The present invention relates to a method for a malignant tumor and a composition of a nucleic acid-based therapeutic compound. In some embodiments, the invention provides RNAi molecules, structures and compositions that render Hsp47 and Hsp47 and p21 non-expressing. The structures and compositions of the present disclosure can be used to prevent, treat or reduce the size of a malignant tumor.

In certain embodiments, the invention provides a double-stranded nucleic acid molecule that is an RNAi molecule, such as siRNA or shRNA, for use in suppressing Hsp47. Embodiments of the invention may also provide RNAi molecules for use in the suppression of p21.

In certain embodiments, the inhibitory nucleic acid molecule can be an antisense nucleic acid molecule, a small interfering RNA (siRNA), or a double stranded RNA (dsRNA).

The RNAi molecule of the present invention has an activity capable of not expressing a gene, for example, a dsRNA having an activity which renders a gene non-expressing, an siRNA having a gene which is incapable of expressing a gene, a microRNA or a shRNA, and a DNA-directed RNA (ddRNA) Piwi interaction RNA (piRNA) and repeat-related siRNA (rasiRNA).

In still other embodiments, the methods of the invention reduce transcription or translation of Hsp47 and/or p21 in a malignant tumor.

In a particular embodiment, the invention includes methods for reducing the expression of Hsp47 in a malignant cell or for reducing the expression of Hsp47 and p21 in a malignant cell, wherein the cell can be a human cell, a neoplastic cell, alive Cells in cells or tubes in vivo.

Embodiments of the invention may also provide methods for treating an individual having a tumor, wherein the tumor cancer cells exhibit an abnormal level of Hsp47 expression. The method can involve administering to the individual an effective amount of an inhibitory nucleic acid molecule, wherein the inhibitory nucleic acid molecule reduces Hsp47 expression, or wherein the combination of RNAi molecules reduces the performance of Hsp47 and p21, thereby treating the tumor. In some embodiments, the method of the invention reduces the size of the tumor relative to the size of the tumor prior to or without treatment.

In various embodiments, the inhibitory nucleic acid molecule can be delivered in a liposome, polymer, microsphere, nanoparticle, gene therapy vector, or naked DNA vector.

In a further aspect, the invention features a method for treating an individual, such as a human patient, having a tumor wherein the tumor cancer cell exhibits Hsp47. In certain embodiments, the method can comprise administering to the individual an effective amount of an inhibitory nucleic acid molecule (wherein the inhibitory nucleic acid molecule is an antisense nucleic acid molecule) or an RNAi molecule or a combination thereof, which inhibits the Hsp47 polypeptide Performance, or inhibition of both Hsp47 polypeptides and p21 polypeptides.

In a particular embodiment, the tumor cells overexpress Hsp47.

In certain embodiments, the tumor can be a malignant tumor, Or lung cancer or pancreatic cancer.

Embodiments of the present invention can provide a pharmaceutical composition for treating a malignant tumor, the composition comprising a nanoparticle encapsulating an RNAi molecule, wherein the RNAi molecule targets Hsp47. In some embodiments, the invention includes a method for dispensing an active agent to an individual to treat a malignancy, the method comprising administering to the individual a pharmaceutical composition as described above.

In a further embodiment, the invention includes a pharmaceutical composition for treating a malignant tumor, the composition comprising a nanoparticle encapsulating an RNAi molecule, wherein one of the RNAi molecules partially targets Hsp47 and a portion of the RNAi molecule is targeted To p21.

The invention further contemplates a method for preventing, treating or ameliorating the symptoms of one or more malignancies in a mammal in need thereof, the method comprising administering to the mammal a therapeutically effective amount of an RNAi molecule comprising an activity having reduced Hsp47 expression. Composition.

In a further aspect, the invention includes a method for preventing, treating or ameliorating the symptoms of one or more malignancies in a mammal in need thereof, the method comprising administering to the mammal a therapeutically effective amount of an RNAi-containing molecule A composition wherein a portion of the RNAi molecule has an activity capable of reducing the expression of Hsp47 and a portion of the RNAi molecule has an activity capable of reducing p21 expression.

In certain aspects, the invention includes a method for reducing the growth rate or proliferation of cancer stem cells in a mammal in need thereof, the method comprising administering to the mammal a therapeutically effective amount of a composition comprising an RNAi molecule, wherein One part of the RNAi molecule has the ability to reduce Hsp47 expression It is active and one part of the RNAi molecule has an activity that reduces p21 expression.

A further embodiment of the invention may provide a composition for dispensing an active agent to treat a malignant tumor in an individual, wherein the composition comprises liposomal nanoparticle. The composition can be used in a method of dispensing an active agent into an organ of an individual to treat a malignant tumor.

Detailed description of the invention

The invention provides methods of treating a tumor in an individual using a therapeutic composition that reduces the expression of the Hsp47 nucleic acid molecule or polypeptide. In certain embodiments, the invention provides methods of treating a tumor in an individual using a therapeutic composition that reduces the expression of the Hsp47 nucleic acid molecule or polypeptide, and the p21 nucleic acid molecule or polypeptide.

In still other aspects, embodiments of the invention can provide methods and compositions for reducing the rate of growth or proliferation of cancer stem cells. The present invention relates to a surprising effect of inhibiting the growth or proliferation of cancer stem cells in vivo by treatment with a combination of an siRNA inhibitor of Hsp47 and an inhibitor of Hsp47 and an inhibitor of p21.

Therapeutic compositions of the invention can include inhibitory nucleic acid molecules, including RNAi molecules such as siRNA, shRNA, and antisense RNA.

The invention encompasses RNAi molecules, ribozymes, antisense nucleic acids, DNA/RNA chimeric polynucleotides, and vectors for expressing them and Hsp47 dominant negative variants for use in suppressing DNA encoding Hsp47.

In general, individuals are diagnosed with tumors (eg lung cancer) After treatment with pancreatic cancer, a treatment involving the suppression of Hsp47 or the suppression of Hsp47 and p21 is selected.

Examples of the agent for suppressing Hsp47 as used herein include a drug which inhibits the production and/or activity of Hsp47 and a drug which promotes degradation and/or inactivation of Hsp47. Examples of drugs for suppressing the production of Hsp47 include RNAi molecules, ribozymes, antisense nucleic acids, DNA/RNA chimeric polynucleotides encoding DNA of Hsp47 or vectors expressing them.

Examples of the medicament for inhibiting p21 as used herein include a medicament for suppressing p21 production and/or activity and a medicament for promoting p21 degradation and/or inactivation. Examples of drugs for suppressing p21 production include RNAi molecules, ribozymes, antisense nucleic acids, DNA/RNA chimeric polynucleotides encoding DNA of p21, or vectors expressing the same.

RNAi molecule

One of ordinary skill in the art will appreciate that the reported sequence can be altered over time and correspondingly incorporate any changes required for the nucleic acid molecules of the invention.

Embodiments of the present invention can provide compositions and methods that use small nucleic acid molecules to render the Hsp47 gene unexpressed. Additional embodiments of the invention may provide compositions and methods for using small nucleic acid molecules to render the Hsp47 and p21 genes unexpressed.

The RNAi molecule of the present invention has an activity capable of not expressing a gene, for example, a dsRNA having an activity capable of not expressing a gene, an siRNA having a activity capable of expressing a gene, a microRNA or a shRNA, and DNA-directed RNA (ddRNA), Piwi-interacting RNA (piRNA), and repeat-related siRNA (rasiRNA). These molecules are capable of mediating RNA interference.

The compositions and methods disclosed herein can also be used to treat a variety of malignancies in an individual.

The nucleic acid molecules and methods of the present invention may be used in combination or in combination with the expression of the gene encoding Hsp47 and down-regulation of the genes encoding Hsp47 and p21.

The composition and method of the present invention may comprise a nucleic acid molecule which modulates or modulates the expression of the Hsp47 protein and/or the gene encoding the protein, or which modulates or modulates the Hsp47 protein and/or the gene encoding the protein and the p21 protein and / or the performance of a gene encoding the protein, and the expression of a protein associated with the maintenance and/or development of a disease or disorder or disorder (such as a malignant tumor) associated with Hsp47 and/or a gene encoding the protein.

The compositions and methods of the present invention are described with reference to exemplary sequences of Hsp47 and p21. Those of ordinary skill in the art will appreciate that the various aspects and embodiments of the present invention are directed to any related Hsp47 or p21 gene, sequence or variant, such as homologous genes and transcript variants and polymorphisms, including to any Hsp47 and p21 gene-related single nucleotide polymorphisms (SNPs).

The RNAi molecules of the invention can target Hsp47 or p21 and any homologous sequences, for example using complementary sequences or by incorporating non-standard base pairs that provide additional target sequences (eg, mismatched and/or wobble base pairs (wobble Base pair)).

In cases where a mismatch is identified, non-standard base pairs (e.g., mismatches and/or wobble bases) can be used to generate nucleic acid molecules that target more than one gene sequence.

For example, non-standard base pairs (such as UU and CC base pairs) can be used to generate nucleic acid molecules capable of targeting sequences to distinguish targets that share sequence homology. Thus, RNAi molecules can target nucleotide sequences that are retained between homologous genes, while a single RNAi molecule can be used to inhibit the expression of more than one gene.

In some aspects, the methods and compositions of the invention include RNAi molecules having activity against any portion of Hsp47 mRNA. The RNAi molecule can include a sequence that is complementary to any mRNA encoding a Hsp47 sequence.

In a further aspect, the methods and compositions of the invention comprise an RNAi molecule having activity against any portion of the p21 mRNA. The RNAi molecule can include a sequence that is complementary to any mRNA encoding the p21 sequence.

In some embodiments, an RNAi molecule of the disclosure can have an activity against Hsp47 RNA, wherein the RNAi molecule comprises a sequence that is complementary to an RNA having a variant Hsp47 coding sequence, such as is known in the art to be associated with a malignancy. Mutation of the Hsp47 gene.

In a further embodiment, the RNAi molecule of the invention may comprise a nucleotide sequence that mediates the non-expression of the Hsp47 or p21 gene.

An RNAi molecule as used herein refers to any molecule that causes RNA interference, including duplex RNA, such as siRNA (small dry Disturbed RNA), miRNA (microRNA), shRNA (short hairpin RNA), ddRNA (DNA-directed RNA), piRNA (Piwi interaction RNA) or rasiRNA (repeated related siRNA) and their modified forms. These RNAi molecules are commercially available or can be designed and prepared based on known sequence information, and the like. Antisense nucleic acids include RNA, DNA, PNA or a complex thereof. A DNA/RNA chimeric polynucleotide as used herein includes a double-stranded polynucleotide consisting of DNA and RNA which inhibit expression of a target gene.

In one embodiment, the agent of the invention contains siRNA as a therapeutic agent. The siRNA molecule can be from about 10 to 50 or more nucleotides in length. The siRNA molecule can be from about 15 to 45 nucleotides in length. The siRNA molecule can be from about 19 to 40 nucleotides in length. The siRNA molecule can be from 19 to 23 nucleotides in length. The siRNA molecules of the invention can mediate RNAi against the mRNA of the target. Commercially available design tools and kits, such as those available from Ambion (Austin, TX) and the Whitehead Biomedical Research Institute of Massachusetts Institute of Technology (Cambridge, MA), allow for the design and manufacture of siRNA.

Method for treating malignant tumors

Embodiments of the invention may provide RNAi molecules useful for down-regulating or inhibiting the expression of Hsp47 and/or Hsp47 proteins, and down-regulating or inhibiting the expression of p21 and/or p21 proteins.

In some embodiments, the RNAi molecules of the invention are useful for down-regulating or inhibiting Hsp47 and/or Hsp47 proteins caused by Hsp47 haplotype polymorphisms, which may be associated with diseases or conditions such as malignancies. which performed.

Monitoring Hsp47 protein or mRNA levels, and/or p21 protein or mRNA levels can be used to characterize gene silencing and determine the potency of the compounds and compositions of the invention.

The RNAi molecules of the present disclosure may be used alone or in combination with other siRNAs for regulating the expression of one or more genes.

The RNAi molecules of the present disclosure may be used alone or in combination with other drugs known to prevent or treat diseases, or to ameliorate the symptoms of Hsp47-related disorders or disorders, including malignancies.

The RNAi molecules of the invention can modulate or inhibit the expression of Hsp47 or p21 in a sequence-specific manner.

The RNAi molecules of the present disclosure may comprise a guide strand, a series of contiguous nucleotides of the guide strand being at least partially complementary to Hsp47 mRNA or p21 mRNA.

In certain aspects, a malignant tumor can be treated by RNA interference using one or more RNAi molecules of the invention.

The treatment of malignant tumors can be characterized in a suitable cell-based model and in an in vitro or in vivo animal model.

The treatment of malignant tumors can be determined by measuring the level of Hsp47 mRNA or the level of Hsp47 protein in the cells of the affected tissue, and/or by measuring the level of p21 mRNA or the level of p21 protein in the cells of the affected tissue. Characterization.

Treatment of malignant tumors can be characterized by non-invasive medical scans of affected organs or tissues.

Embodiments of the invention may include methods for preventing, treating or ameliorating the symptoms of a disease or condition associated with Hsp47 in an individual in need thereof.

In certain embodiments, a combination of Hsp47 siRNA and p21 siRNA can unexpectedly increase cancer cell death. In a further embodiment, the combination of Hsp47 siRNA and p21 siRNA can unexpectedly advantageously reduce cancer cell proliferation.

In some embodiments, a method for preventing, treating or ameliorating a symptom of a malignant tumor in an individual can comprise administering to the individual an RNAi molecule of the invention to modulate Hsp47 gene and/or p21 gene expression in the individual or organism. .

In some embodiments, the present invention contemplates methods for downregulating the expression of a Hsp47 gene in a cell or organism by contacting the cell or organism with an RNAi molecule of the invention. In certain embodiments, the invention contemplates methods of downregulating the expression of the Hsp47 gene and the p21 gene in a cell or organism by contacting the cell or organism with two or more RNAi molecules of the invention.

The inhibitory nucleic acid molecule can be a nucleotide oligomer that can be employed in the form of a single or double stranded nucleic acid molecule to reduce gene expression. In one method, the inhibitory nucleic acid molecule is a double-stranded RNA for expression of a knockdown gene mediated by RNA interference (RNAi). In one embodiment, the preparation comprises 8 to 25 (eg, 8, 10, 12, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25) nucleotide oligos of the invention. A double-stranded RNA (dsRNA) molecule of a contiguous nucleotide of a polymer. The dsRNA can be double helix Two complementary strands of RNA, or a single strand of RNA (small hairpin (sh) RNA) with its own double helix.

In some embodiments, the dsRNA is about 21 or 22 base pairs, but can be shorter or longer, up to about 29 nucleotides. Double stranded RNA can be produced using standard techniques, such as chemical synthesis or intravitreal transcription. Kits are available, for example, from Ambion (Austin, TX) and Epicentre (Madison, Wisconsin).

Methods for expressing dsRNA in mammalian cells are described in Brummekamp et al. Science 296: 550-553, 2002; Paddison et al. Genes & Devel. 16: 948-958, 2002; Paul et al. Biotechnol. 20: 505-508, 2002; Sui et al., Proc. Natl. Acad. Sci. USA 99: 5515-5520, 2002; Yu et al. Proc. Natl. Acad. Sci. USA 99: 6047-6052 , 2002; Miyagishi et al., Nature Biotechnol. 20: 497-500, 2002; and Lee et al., Nature Biotechnol. 20: 500-5052002, each of which is incorporated herein by reference.

An inhibitory nucleic acid molecule "corresponding" to the Hsp47 gene comprising at least one double-stranded gene fragment such that each strand of the double-stranded inhibitory nucleic acid molecule is capable of binding to a complementary strand of the target Hsp47 gene. The inhibitory nucleic acid molecule does not have to correspond exactly to the reference Hsp47 sequence.

In one embodiment, the siRNA has at least about 85%, 90%, 95%, 96%, 97%, 98%, or even 99% sequence identity to the nucleic acid of the target. For example, a 19 base pair duplex having a 1 to 2 base pair mismatch is considered useful in the methods of the invention. For other implementations In the aspect, the nucleotide sequence of the inhibitory nucleic acid molecule exhibits 1, 2, 3, 4, 5 or more mismatches.

The inhibitory nucleic acid molecules provided herein are not limited to siRNA, but include any nucleic acid molecule sufficient to reduce the expression of a Hsp47 or p21 nucleic acid molecule or polypeptide. The DNA sequences provided herein can be used, for example, to discover and develop therapeutic antisense nucleic acid molecules to reduce the performance of the encoded protein. The invention further provides catalytic RNA molecules or ribozymes. These catalytic RNA molecules can be used to inhibit the performance of nucleic acid molecules of a target in vivo. The inclusion of a ribozyme sequence within the antisense RNA confers cleavage activity on the molecule, thereby increasing the activity of the construct. The design and use of the target RNA-specific ribozymes is described in Haseloff et al., Nature 334: 585-591. 1988 and US 2003/0003 469 A1, each of which is incorporated herein by reference.

In various embodiments of the invention, the catalytic nucleic acid molecule is formed in a hammerhead or hairpin motif. Examples of such hammerhead motifs are described in Rossi et al., Aids Research and Human Retroviruses, 8: 183, 1992. Examples of hairpin motifs are described in Hampel et al., Biochemistry, 28: 4929, 1989 and Hampel et al., Nucleic Acids Research, 18: 299, 1990. Those skilled in the art will be aware of the specific substrate binding sites required for enzymatic nucleic acid molecules that are complementary to the RNA region of one or more targets, and which have a substrate for conferring RNA cleavage activity on the molecule. A nucleotide sequence within or surrounding the binding site.

Compared with cells in which the inhibitor is not used, the target The containment condition can be determined by the performance or activity of the corresponding protein in the depressed cell. The performance of the protein can be assessed by any known technique; examples include immunoprecipitation using antibodies, EIA, ELISA, IRA, IRMA, Western blotting, immunohistochemistry, immunocytochemistry, flow cytometry A method of hybridizing a nucleic acid which specifically hybridizes with a nucleic acid encoding the protein or a unique fragment thereof, or a transcription product (for example, mRNA) or a splicing product of the nucleic acid, a northern blotting method, a Southern blotting method, and various PCR methods.

The activity of the protein can be assessed by any known method, such as, for example, immunoprecipitation, Western blotting, accumulation analysis, pull-down or surface plasmon resonance (SPR), to assess the known activity of the protein, including Proteins such as, for example, Raf-1 (especially phosphorylated Raf-1) or EGFR (especially phosphorylated EGFR) bind.

In one aspect, the invention features features of a vector encoding an inhibitory nucleic acid molecule of any of the above aspects. In a particular embodiment, the vector is a retrovirus, adenovirus, adeno-associated virus or lentiviral vector. In another embodiment, the vector contains a promoter suitable for expression in mammalian cells.

The amount of the active RNA interference-inducing component formulated in the composition of the present invention may be an amount which does not cause an adverse effect exceeding the administration benefit. The same amount can be determined using cultured cells, by in-tube test, or in an animal or mammalian (such as mouse, rat, dog or pig, etc.) model, and such test methods are techniques of the art. It is known to those skilled in the art.

The amount of the active ingredient to be formulated may be based on the agent or composition The way things are changed. For example, when a plurality of constituent units are used in one administration, the amount of the active ingredient to be formulated in one composition unit can be obtained by dividing the amount of the active ingredient required for one administration by the plurality of units. Decide.

The invention also relates to the use of a medicament for the manufacture of a medicament or composition for the inhibition of Hsp47 or p21, and for inhibiting Hsp47 or inhibiting the composition of Hsp47 and p21 for reducing or reducing malignancy.

RNA interference

RNA interference (RNAi) refers to sequence-specific post-transcriptional gene silencing mediated by short interfering RNA (siRNA) in animals. See, for example, Zamore et al., Cell, 2000, Vol. 101, pp. 25-33; Fire et al., Nature, 1998, Vol. 391, pp. 806811; Sharp, Genes & Development, 1999, Vol. , pp. 139-141.

RNAi responses in cells can be triggered by double-stranded RNA (dsRNA), although this mechanism is not fully understood. Certain dsRNAs in cells can undergo the action of Dicer, a ribonuclease III. See, for example, Zamore et al., Cell, 2000, Vol. 101, pp. 25-33; Hammond et al., Nature, 2000, Vol. 404, pp. 293-296. Dicer can process dsRNA into short dsRNA fragments, which are siRNAs.

In general, siRNA can be from about 21 to about 23 nucleotides in length and includes a base pair double helix region of about 19 nucleotides in length.

RNAi is involved in an endonuclease complex called RNA-induced silencing complex (RISC). The siRNA has an antisense strand or guide strand that enters the RISC complex and mediates cleavage of a single strand of RNA target having a sequence complementary to the antisense strand of the siRNA duplex. The other strand of siRNA is a follower. The target RNA is cleaved in the middle of the region complementary to the antisense strand of the siRNA duplex. See, for example, Elbashir et al., Genes & Development, 2001, Vol. 15, pp. 188-200.

The term "sense strand" as used herein refers to the nucleotide sequence of a siRNA molecule that is partially or fully complementary to at least a portion of the corresponding antisense strand of the siRNA molecule. The sense strand of the siRNA molecule can include a nucleic acid sequence that has homology to the nucleic acid sequence of the target.

The term "antisense strand" as used herein refers to the nucleotide sequence of an siRNA molecule that is partially or fully complementary to at least a portion of the target nucleic acid sequence. The antisense strand of the siRNA molecule can comprise a nucleic acid sequence that is complementary to a corresponding sense strand of at least a portion of the siRNA molecule.

RNAi molecules can down-regulate or knock down gene expression by mediating RNA interference in a sequence-specific manner. See, for example, Zamore et al., Cell, 2000, Vol. 101, pp. 25-33; Elbashir et al., Nature, 2001, Vol. 411, pp. 494-498; Kreutzer et al., WO 2000/044895; Zernicka-Goetz et al., WO 2001/36646; Fire et al., WO 1999/032619; Plaetinck et al., WO 2000/01846; Mello et al., WO 2001/029058.

The term "inhibiting," "down-regulating," or "reducing" as used herein with respect to gene expression refers to the expression or encoding of one or more of the genes. The level of the mRNA molecule of the protein, or the activity of one or more encoded proteins, is reduced to less than that observed in the absence of the RNAi molecule or siRNA of the invention. For example, the level of performance, the level of mRNA, or the level of encoded protein activity can be reduced to at least 1%, or at least 10%, or at least 20% lower than the level observed in the absence of the RNAi molecule or siRNA of the invention. Or at least 50% or at least 90%.

RNAi molecules can also be used to knock down viral gene expression, thereby affecting viral replication.

RNAi molecules can be made from individual polynucleotide strands (sense stocks or followers, and antisense stocks or guide strands). The Guide Unit and the Followers are at least partially complementary. The guide strand and the follower strand can form a double helix region having from about 15 to about 49 base pairs.

In some embodiments, the double helix region of the siRNA can have 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 , 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48 or 49 base pairs.

In certain embodiments, the RNAi molecule is active in the RISC complex and the active length in the RISC is a double helix region.

In still other embodiments, the RNAi molecule can have activity as a Dicer substrate to be converted into an RNAi molecule that is active in the RISC complex.

In some aspects, the RNAi molecule can have a complementary guide and a follower sequence at the opposite end of the long molecule such that the molecule can form a duplex region with the complementary sequence portion, and the strand is by nucleotide or non-nuclear A glycoside linker connects one end of the double helix region. For example, hairpin arrangement or stem and ring arrangement. The interaction of the linker with the strand can be a covalent bond or a non-covalent interaction.

An RNAi molecule of the disclosure can include a nucleotide, non-nucleotide or mixed nucleotide/non-nucleotide linker joining a sense region of a nucleic acid to an antisense region of a nucleic acid. The nucleotide linker can be a linker of ≧2 nucleotides in length (e.g., about 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides in length). The nucleotide linker can be an aptamer. As used herein, "aptamer" or "nucleic acid aptamer" refers to a nucleic acid molecule that specifically binds to a molecule of a target, wherein the nucleic acid molecule has a sequence comprising a sequence that is recognized by the molecule of the target in its natural environment. Alternatively, the aptamer can be a nucleic acid molecule that binds to a molecule of the target, wherein the molecule of the target does not naturally bind to the nucleic acid. For example, the aptamer can be used to bind to a ligand binding domain of a protein, thereby preventing the naturally occurring ligand from interacting with the protein. See, for example, Gold et al., Annu Rev Biochem, 1995, Vol. 64, pp. 763-797; Brody et al., J. Biotechnol., 2000, Vol. 74, pp. 5-13; Hermann et al. , Science, 2000, Vol. 287, pp. 820-825.

Examples of non-nucleotide linkers include abasic nucleotides, polyethers, polyamines, polyamines, peptides, carbohydrates, lipids, polyhydrocarbons, or other polymeric compounds (eg, polyethylene glycols, such as those having 2 Up to 100 ethylene glycol units). Some examples are described in the following literature: Seela et al., Nucleic Acids Research, 1987, Vol. 15, pp. 3113-3129; Cload et al., J. Am. Chem. Soc., 1991, Vol. 113, pp .6324-6326; Jaeschke et al., Tetrahedron Lett., 1993, Vol. 34, pp. 301; Arnold et al., WO 1989/002439; Usman et al., WO 1995/006731; Dudycz et al., WO 1995/011910, and Ferentz et al., J. Am. Chem. Soc., 1991, Vol. 113, pp. 4000-4002.

The RNAi molecule can have one or more overhangs from the double helix region. The overhangs, which are non-base paired, single-stranded regions, can be from 1 to 8 nucleotides in length or longer. The overhang may be a 3' overhang, wherein the 3' end of one strand has a single strand of 1 to 8 nucleotides in length. The overhang may be a 5' end, wherein the 5' end of one strand has a single strand of 1 to 8 nucleotides in length.

The overhangs of the RNAi molecules can be of the same length or can be of different lengths.

The RNAi molecule can have one or more blunt ends, wherein the double helix region ends do not have overhangs and the strand is base paired with the ends of the double helix region.

The RNAi molecules of the present disclosure may have one or more blunt ends, or may have one or more overhangs or may have a combination of blunt ends and overhangs.

The 5' end of one of the RNAi molecules can be at the blunt end or can be at the overhang. The 3' end of one of the RNAi molecules may be at the blunt end or may be at the overhang.

The 5' end of one of the RNAi molecules can be at the blunt end and the 3' end is at the overhang. The 3' end of one of the RNAi molecules can be at the blunt end and the 5' end is at the overhang.

In some embodiments, the RNAi molecule is blunt at both ends end.

In another embodiment, the RNAi molecule has an overhang at both ends.

The protruding ends at the 5' and 3' ends may have different lengths.

In certain embodiments, the RNAi molecule can have a blunt end, wherein the 5' end of the antisense strand and the 3' end of the sense strand do not have any overhanging nucleotides.

In a further embodiment, the RNAi molecule can have a blunt end, wherein the 3' end of the antisense strand and the 5' end of the sense strand do not have any overhanging nucleotides.

RNAi molecules may have mismatches in base pairing in the double helix region.

Any nucleotide in the overhang of the RNAi molecule can be a deoxyribonucleotide or a ribonucleotide.

One or more deoxyribonucleotides may be located at the 5' end, wherein the 3' end of the other strand of the RNAi molecule may not have an overhang or may not have a deoxyribonucleotide overhang.

One or more deoxyribonucleotides may be located at the 3' end, wherein the 5' end of the other strand of the RNAi molecule may not have an overhang or may not have a deoxyribonucleotide overhang.

In some embodiments, one or more or all of the overhang nucleotides of the RNAi molecule can be 2'-deoxyribonucleotides.

Dicer-derived RNAi molecule

In some aspects, the RNAi molecule can have a length suitable as a Dicer substrate that can be processed to produce a RISC active RNAi molecule. See, for example, Rossi et al., US 2005/0244858.

The dicer-derived dsRNA can be of sufficient length to allow it to be treated by Dicer to produce an active RNAi molecule, and can further comprise one or more of the following properties: (i) Dicer-derived dsRNA can be asymmetric For example, a 3' overhang on the antisense strand, and (ii) a modified strand of the dicer-derived dsRNA may have a modified 3' end to direct the dicer directed binding and to process the dsRNA into an active RNAi molecule.

RNAi molecule for p21

Examples of RNAi molecules of the target p21 mRNA of the present invention are shown in Table 1.

Key to Table 1: Capital letters A, G, C, and U refer to ribose-A, ribose-G, ribose-C, and ribose-U, respectively. Lowercase letters a, g, c, t represents 2'-deoxy-A, 2'-deoxy-G, 2'-deoxy-C and thymidine, respectively. mU is 2'-methoxy-U.

Examples of RNAi molecules of the target p21 mRNA of the present invention are shown in Table 2.

Key to Table 2: Capital letters A, G, C, and U refer to ribose-A, ribose-G, ribose-C, and ribose-U, respectively. The lowercase letters a, u, g, c, t are 2'-deoxy-A, 2'-deoxy-U, 2'-deoxy-G, 2'-deoxy-C and deoxythymidine, respectively (dT=T= t). Underlined refers to 2'-OMe-substituted, such as U. N is A, C, G, U, U, a, c, g, u, t or a modified, reversed or chemically modified nucleotide.

An RNAi molecule as used herein refers to any molecule that causes RNA interference, including bihelical RNA, such as siRNA (small interfering RNA), miRNA (microRNA), shRNA (short hairpin RNA), ddRNA (DNA-directed RNA). , piRNA (Piwi interaction RNA) or rasiRNA (repeated related siRNA) and their modified forms. These RNAi molecules are commercially available or can be designed and prepared based on known sequence information, and the like. Antisense nucleic acids include RNA, DNA, PNA or a complex thereof. A DNA/RNA chimeric polynucleotide as used herein includes a double-stranded polynucleotide consisting of DNA and RNA which inhibits the expression of a target gene.

In one embodiment, the agent of the invention contains siRNA as a therapeutic agent. The siRNA molecule can be from about 10 to 50 or more nucleotides in length. The siRNA molecule can be from about 15 to 45 nucleotides in length. The siRNA molecule can be from about 19 to 40 nucleotides in length. The siRNA molecule can be from 19 to 23 nucleotides in length. The siRNA molecules of the invention can mediate RNAi against the target mRNA. Commercially available design tools and kits (such as those available from Ambion (Austin, TX) and the Massachusetts Institute of Technology's Whitehead Biomedical Research Institute (Cambridge, MA) allow for the design and manufacture of siRNA.

P21 is present in a variety of animals, including humans. The sequence information of human CDKN1A (p21) can be: NM_000389.4, NM_078467.2, NM_001291549.1, NM_001220778.1, NM_001220777.1 (NP_001207707.1, NP_001278478.1, NP_001207706.1, NP_510867.1, NP_000380.1 Found in ).

An exemplary target nucleic acid sequence of p21 mRNA is disclosed GenBank accession number NM_000389.4 (CDKN1A) is 2175 nucleotides in length.

Target RNAi molecule of Hsp47

In some embodiments, the invention provides a series of RNAi molecules and compositions for regulating the expression of heat shock protein 47 (Hsp47), a collagen-specific molecular chaperone for intracellular trafficking and maturation.

Some examples of siRNAs for Hsp47 are listed in US Pat. No. 8,710,209, the disclosure of which is incorporated herein by reference in its entirety herein.

Hsp47 or a homologous gene sequence thereof is disclosed, for example, as GenBank No. AB010273 (human), X60676 (mouse) or M69246 (rat, gp46).

Agents for suppressing Hsp47 have been disclosed for inhibiting fibrosis. See, for example, US Pat. No. 8,173,170 B2, the entire disclosure of which is incorporated herein by reference in its entirety. However, there is limited information on the effects of inhibiting the development, progression and growth of Hsp47 in malignant tumors.

In some embodiments, each strand of the siRNA molecule of the invention can be 15 to 60 nucleotides in length or 15 to 40 nucleotides in length or 19 to 25 nucleotides in length.

In certain embodiments, the present invention provides a pharmaceutical composition for treating a malignant tumor comprising an RNAi molecule, the pharmaceutical composition being a standard RNAi molecule of target Hsp47.

Examples of RNAi molecules that target the Hsp47 mRNA of the present disclosure are shown in Table 3.

Key to Table 3: Capital letters A, G, C, and U refer to ribose-A, ribose-G, ribose-C, and ribose-U, respectively. The lowercase letter d stands for "deoxygenation".

Further examples of RNAi molecules that target Hsp47 mRNA of the present disclosure are shown in Table 4.

Key to Table 4: Name: r X stands for ribonucleotide, m X stands for 2'-O-methylribonucleotide, 25r X stands for ribonucleotide with 2'-5' linkage, C3 stands for 1 , 3-propanediol spacer, idAB represents reverse 1,2-dideoxy-D-ribose, and P represents a phosphate group at the 3'-end.

How to use RNAi molecules

The nucleic acid molecules and RNAi molecules of the invention can be delivered to cells or tissues by direct administration of the molecule or by combining the molecule with a carrier or diluent.

The nucleic acid molecules and RNAi molecules of the invention can be administered by direct administration of the molecule to a carrier, or diluent or any other delivery vehicle (eg, viral sequence, viral material, or lipid or lipid) for assisting, facilitating, or accelerating the entry of the molecule into the cell. The body modulator) is delivered or administered to a cell, tissue, organ or individual.

The nucleic acid molecules and RNAi molecules of the invention can be complexed with cationic lipids, packaged within liposomes, or otherwise delivered to cells or tissues of the target. The nucleic acid or nucleic acid complex can be administered topically to the relevant tissue in vitro or in vivo by direct administration to the skin, transdermal administration or injection.

Delivery systems can include, for example, aqueous and non-aqueous gels, creams, emulsions, microemulsions, liposomes, ointments, aqueous and non-aqueous solutions, lotions, aerosols, hydrocarbon bases, and powders, and can contain Shape agents such as solubilizers and penetration enhancers.

The inhibitory nucleic acid molecules or compositions of the invention can be administered in unit dosage form in pharmaceutically acceptable diluents, carriers or excipients. Conventional pharmaceutical practice can be used to provide a suitable modulator or composition to administer the compound to a patient suffering from a disease caused by excessive cell proliferation. Administration can begin before the patient develops symptoms. Any suitable route of administration may be used, for example, administration via the parenteral, intravenous, intraarterial, subcutaneous, neoplastic Internal, intramuscular, intracranial, intraorbital, intraocular, intraventricular, intrahepatic, intracapsular, intrathecal, intracisternal, intraperitoneal, intranasal routes, aerosols, suppositories, or oral administration. For example, the therapeutic preparation may be in the form of a liquid solution or suspension; in the case of oral administration, the preparation may be in the form of a tablet or a capsule; and in the case of an intranasal preparation, a powder, a nasal drop or an aerosol Form.

The compositions and methods of the present disclosure can include a performance vector comprising a nucleic acid sequence encoding at least one RNAi molecule of the invention in a manner that permits expression of the nucleic acid molecule.

The nucleic acid molecules and RNAi molecules of the invention can be expressed from the transcription unit inserted into the DNA or RNA vector. The recombinant vector can be a DNA plasmid or a viral vector. Viral vectors can be used to provide transient expression of nucleic acid molecules.

For example, the vector may contain a sequence encoding a binary or self-complementary single nucleic acid molecule of a duplex RNAi molecule to form an RNAi molecule. A performance vector can include a nucleic acid sequence encoding two or more nucleic acid molecules.

Nucleic acid molecules can be expressed within the cell from eukaryotic promoters. Those skilled in the art will appreciate that any nucleic acid from a suitable DNA/RNA vector can be expressed in eukaryotic cells.

In some aspects, a viral construct can be used to introduce a expression construct into a cell to transcribe a dsRNA construct encoded by the expression construct.

The lipid modulator can be administered to the animal by intravenous, intramuscular, or intraperitoneal injection, or by oral administration, or by inhalation or other methods known in the art.

Pharmaceutically acceptable modulators for administration of oligonucleotides are known and can be used.

In one embodiment of the above method, the dosage of the inhibitory nucleic acid molecule is from about 5 to 500 mg/m ² /day, such as 5, 25, 50, 100, 125, 150, 175, 200, 225, 250. , 275 or 300 mg/m ² /day.

In some embodiments, the inhibitory nucleic acid molecules of the invention are administered systemically at a dose of from about 1 to 100 mg/kg, such as 1, 5, 10, 20, 25, 50, 75 or 100 mg/kg.

In further embodiments, the dosage may range from about 25 to 500 mg/m ² /day.

Methods for preparing modulators known in the art can be found, for example, in "Remington: The Science and Practice of Pharmacy" Ed. A. R. Gennaro, Lippincourt Williams & Wilkins, Philadelphia, Pa., 2000.

Formulations for parenteral administration may, for example, contain excipients, sterile water or saline, polyalkylene glycols (such as polyethylene glycol), vegetable derived oils or hydrogenated naphthalenes. Biocompatible, biodegradable lactide polymers, glycolide/lactide copolymers or polyoxyethylene-polyoxypropylene copolymers can be used to control compound release. Other potentially useful parenteral delivery systems for inhibitory nucleic acid molecules include ethylene-vinyl acetate copolymer particles, osmotic pumps, implantable infusion systems, and liposomes. The preparation for inhalation may contain an excipient such as lactose, or may be an aqueous solution containing, for example, polyoxyethylene-9-lauryl ether, glycocholate, and deoxycholate, or may be used for An oily solution administered as a nasal drop or in the form of a gel.

The modulator can be administered to a human patient in a therapeutically effective amount (e.g., in an amount to prevent, eliminate, or ameliorate pathological conditions) to provide a therapy for a neoplastic disease or condition. Preferred dosages of the nucleotide oligomers of the invention may depend, for example, on the type and extent of the condition, the overall health of the particular patient, the formulation of the compound excipient, and the route of administration.

The pharmaceutical composition of the present invention is effective for treating Hsp47-related diseases. Examples of the disease include diseases caused by abnormal cell proliferation, and diseases exhibiting excessive expression of Hsp47.

All of the above methods for reducing malignant tumors can be in-tube or in vivo methods. Dosage measurements can be made using cultured cells, etc., as determined by art in the glass tube assay. An effective amount can be to reduce tumor size by at least 10%, at least 20%, or at least 30%, or at least 40%, or at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90. %, up to 100% of the amount. An effective amount can reduce proliferation of cancer cells by at least 10%, at least 20%, or at least 30%, or at least 40%, or at least 50%, or at least 60%, or at least 70%, compared to a control group, Or at least 80%, or at least 90% or up to 100%.

Examples of diseases caused by abnormal cell proliferation include malignant tumors, hyperplasia, keloid, Cushing's syndrome, aldosteronism, erythroplakia, and true erythrocytosis ( Polycythemia vera), leukoplakia, hypertrophic scar, lichen planus, and lentiginosis.

Examples of diseases caused by excessive expression of Hsp47 include malignant tumors.

Examples of cancer include sarcomas such as fibrosarcoma, fibrous histiocytoma, liposarcoma, rhabdomyosarcoma, leiomyosarcoma, angiosarcoma, kaposi Kaposi's sarcoma, lymphangiosarcoma (lymphangiosarcoma), synovial sarcoma, chondrosarcoma and osteosarcoma, carcinoma, such as brain tumor, head and neck cancer, breast cancer, lung cancer, esophageal cancer , gastric cancer, duodenal cancer, appendix cancer, colon cancer, rectal cancer, liver cancer, pancreatic cancer, gallbladder cancer, cholangiocarcinoma, anal cancer, kidney cancer, ureteral cancer, bladder cancer, prostate cancer, testicular cancer, uterine cancer, ovarian cancer , skin cancer, leukemia and malignant lymphoma.

Cancer includes epithelial malignancies and non-epithelial malignancies. Cancer can occur in any part of the body, such as the brain, head and neck, chest, limbs, lungs, heart, thymus, esophagus, stomach, small intestine (duodenum, jejunum, ileum), large intestine (colon, cecum, appendix, rectum), liver , pancreas, gallbladder, kidney, urinary tract, bladder, prostate, testis, uterus, ovary, skin, striated muscle, smooth muscle, synovium, cartilage, bone, thyroid, adrenal gland, peritoneum, mesentery, bone marrow, blood, vascular system, lymph Systems such as lymph nodes, lymph, etc.

In another embodiment, the cancer comprises cancer cells that exhibit hormone- or growth factor-independent proliferation. Further implementation Among them, cancer includes cancer cells that exhibit excessive expression of Hsp47.

Nanoparticle

Embodiments of the invention provide liposomal nanoparticle compositions. The ionizable molecules of the present invention can be used to form liposome compositions which can have bilayered lipid-like molecules.

The nanoparticle composition can have one or more ionizable molecules of the invention in a liposome structure, a bilayer structure, a micelle, a layered structure, or a mixture thereof.

In some embodiments, the composition can include one or more liquid carrier components. Liquid carriers suitable for delivery of the active agents of the present invention can be pharmaceutically acceptable liquid carriers. The liquid carrier can include an organic solvent or a combination of water and an organic solvent.

Embodiments of the invention provide lipid nanoparticles having a size of from 10 to 1000 nm. In some embodiments, the liposome nanoparticles are from 10 to 150 nm in size.

In certain embodiments, the liposomal nanoparticle of the invention encapsulates the RNAi molecule and retains at least 80% of the encapsulated RNAi molecule 1 hour after exposure to human serum.

Pharmaceutical composition

The present invention further contemplates a method of treating a malignant tumor by administering an individual of the present invention to the organ of the individual by administering the composition of the present invention. Treatable organs include lung, liver, pancreas, colon, heart, bone Bone, skin, small intestine and joints.

In some embodiments, the invention provides methods of treating a lung malignancy disorder by administering to a subject a composition of the invention.

In a further aspect, the invention provides a range of pharmaceutical modulators.

The pharmaceutical modulators herein may include the active agents and pharmaceutical carriers, or the lipids of the present invention, together with a pharmaceutically acceptable carrier or diluent. In general, the active agents in this specification include siRNA (the active agent for malignant tumors) and any small molecule drug.

The drug carrier allows the composition to reach the stellate cell target. The pharmaceutical carrier may contain the drug inside or be attached to the outside of the drug-containing substance, or may be mixed with the drug as long as the drug carrier includes a retinoid derivative and/or a vitamin A analog, and the retinoid is derived. The vitamin A and/or vitamin A analog is at least partially exposed to the exterior of the formulation. The composition or preparation may be covered with a suitable material such as, for example, an enteric coating or a substance that disintegrates over time, or a material that can be incorporated into a suitable drug delivery system.

The pharmaceutical preparation of the present invention may contain one or more of the following agents: a surfactant, a diluent, an excipient, a preservative, a stabilizer, a dye, and a suspending agent.

Some pharmaceutical carriers, diluents and components for use in pharmaceutical preparations, and methods for formulating and administering the compounds and compositions of the present invention are described in Remington's Pharmaceutical Sciences, 18th Ed., Mack Publishing Co., Easton, Penn. (1990).

Examples of preservatives include sodium benzoate, ascorbic acid and esters of p-hydroxybenzoic acid.

Examples of the surfactant include alcohols, esters, sulfated aliphatic alcohols.

Examples of excipients include sucrose, glucose, lactose, starch, crystallized cellulose, mannitol, light anhydrous citrate, magnesium aluminate, aluminum magnesium citrate, synthetic aluminum citrate, calcium carbonate, acid Sodium carbonate, calcium hydrogen phosphate and calcium carboxymethyl cellulose.

Examples of suspending agents include coconut oil, olive oil, sesame oil, peanut oil, soybean, cellulose acetate phthalate, methyl acetate-methacrylate copolymer, and phthalate.

The therapeutic modulator of the present invention for delivering one or more molecules having an activity which renders the gene non-expressible can be administered to a mammal in need thereof. A therapeutically effective amount of a modulator and an active agent (which may be encapsulated in a liposome) can be administered to a mammal for the prevention or treatment of a malignancy.

The route of administration can be local or systemic.

The therapeutically effective modulators of the present invention can be administered by a variety of routes including intravenous, intraperitoneal, intramuscular, subcutaneous and oral.

Routes of administration can include, for example, parenteral delivery, including intramuscular, subcutaneous, intravenous, intramedullary injection, as well as intrathecal, direct intraventricular, intraperitoneal, intranasal or intraocular injection.

The modulator may also be administered in a sustained release or controlled release dosage form (including long-acting injections, osmotic pumps, and the like) for long-term administration and/or timing, pulsed administration at a predetermined rate.

The compositions of the present invention may be administered by a variety of routes, including both oral and parenteral routes, examples of which include, but are not limited to, oral, intravenous, intramuscular, subcutaneous, topical, intrapulmonary, intragastric, intratracheal, intrabronchial, Transnasal, rectal, intra-arterial, intra-cavity, intraventricular, intramedullary, intralymphatic, intralymphatic, intracerebral, intrathecal, intraventricular, transmucosal, transdermal, intranasal, intraperitoneal, and intrauterine routes, and The composition can be formulated into a dosage form suitable for each route of administration. Such dosage forms and methods of preparation may be selected from any known dosage form and method, as appropriate. See, for example, Hyojun Yakuzaigaku, Standard Pharmaceutics, edited by Yoshiteru Watanabe, et al., Nankodo, 2003.

Examples of dosage forms suitable for oral administration include, but are not limited to, powders, granules, tablets, capsules, liquids, suspensions, emulsions, gels, and syrups, and examples of suitable dosage forms for parenteral administration include injections. Such as injectable solutions, injectable suspensions, injectable emulsions, and ready-to-use injections. Modulators for parenteral administration may be in the form of, for example, aqueous or nonaqueous isotonic sterile solutions, or suspensions.

Pharmaceutical formulations which are administered parenterally, for example by bolus injection or continuous infusion, comprise an aqueous solution of the active modulator in water soluble form. Suspensions of the active compounds can be prepared in a suitable oily injection suspension. Aqueous injection suspensions may contain materials which increase the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol or dextran. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the compound to allow for the preparation of highly concentrated solutions.

Modulators for injection can be presented in unit dosage form. For example in ampoules or multi-dose containers with added preservatives. The preparation may be in the form of a suspension, solution or emulsion such as in an oily or aqueous vehicle, and may contain formulating agents such as suspending, stabilizing and/or dispersing agents. Alternatively, the active ingredient can be in powder form for constitution with a suitable carrier, such as sterile pyrogen-free water, before use.

In addition to the formulations described above, the modulator can also be formulated as a depot preparation. These long acting modulators can be administered by intramuscular injection. Thus, for example, the preparation may be formulated as a suitable polymeric or hydrophobic material, such as an emulsion in an acceptable oil or ion exchange resin or a sparingly soluble derivative such as a sparingly soluble salt.

The compositions and modulators of the present invention may also be formulated for topical delivery and may be applied to the skin of an individual using any suitable method for administering a topical delivery vehicle. For example, the modulator can be applied by hand, using an applicator, or by a method involving both. After administration, the modulator can be applied to the skin of the individual by, for example, rubbing. It can be administered several times a day or on a daily basis. For example, the modulator can be applied to the skin of the individual once a day, twice a day or multiple times a day, or once every two days, once every three days, or once a week, once every two weeks, or once every few weeks.

The modulators or pharmaceutical compositions described herein can be administered to the subject by any suitable means. Examples of administration methods also include (a) administration via subcutaneous, intraperitoneal, intravenous, intramuscular, intradermal, intraocular, intracapsular, intraspinal, intrasternal injection, etc., including infusion pump delivery; (b) Topical administration, such as by direct injection into the kidney or heart area, for example by implantation Into the reservoir; and a method of contacting the active compound with the living tissue as deemed appropriate by those skilled in the art.

The precise modulator, route of administration and dosage of the pharmaceutical composition can be selected by the individual physician in view of the condition of the patient. See, for example Goodman &Gilman's The Pharmacological Basis of Therapeutics, 12 th Ed., Sec.1,2011. Generally, the dosage of the composition administered to the patient can range from about 0.5 to about 1000 mg/kg (patient weight). The dose can be administered as a single dose or as a series of two or more doses during the course of one or more days. In the case where a human compound dose has been established for at least some conditions, the dose will be about the same as the established human dose, or from about 0.1% to about 500% of the established human dose, more preferably about 25% to about 250%. When no human dose is established, as in the case of newly discovered pharmaceutical compositions, a suitable human dose can be inferred from the ED 50 or ID 50 value, or other suitable value derived from a glass tube or in vivo study ( As evidenced by the Institute of Toxicity Research and Efficacy in Animals).

Method for preventing or treating malignant tumors

The invention further relates to a method for controlling the activity or growth of a malignant tumor, the method comprising administering to the individual in need thereof an effective amount of the composition. An effective amount as referred to herein, in the method for treating a malignant tumor, refers to an amount that relieves its symptoms, or delays or halts its progression, and preferably prevents or cures the onset or recurrence of a malignant tumor. The preferred amount is also such that the adverse effects caused by it do not exceed the benefits derived from the administration of the quantity. Such An effective amount can be determined by using a cultured cell as appropriate, by a glass tube test, or by an experiment in an animal or mammal (e.g., mouse, rat, dog or pig) model, and the test methods are Those skilled in the art are well known. Further, the dosage of the active agent in the carrier and the dosage of the active agent used in the methods of the present invention are known to those skilled in the art, or may be determined by the above-described tests as appropriate.

The frequency of administration depends on the nature of the composition used and the above conditions of the individual, and may be administered multiple times per day (ie 2, 3, 4, 5 or more per day), once a day, every few times. Days (ie every 2, 3, 4, 5, 6 or 7 days, etc.) are administered once, weekly, every week or every few weeks (ie every 2, 3, 4 weeks, etc.) several times (eg weekly 2, 3, 4 times, etc.).

In some embodiments, the invention also relates to a method of delivering a drug to a malignant tumor cell using the above vector. The method comprises the step of administering or adding a carrier having a substance to be delivered to a living body or a medium (e.g., a medium) containing extracellular matrix-producing cells in the lung. These steps can be suitably carried out according to any of the known methods or the methods described in the present invention. Furthermore, the above method includes a mode performed in a glass tube and a mode in which the malignant tumor cells in the lungs in the body are targeted.

The therapeutically effective modulator of the present invention can be administered by systemic delivery which provides for broad biodistribution of the active agent.

Embodiments of the invention may provide a therapeutic modulator comprising a therapeutic molecule of the invention and a pharmaceutically acceptable carrier.

The effective dose of the modulator of the present invention can be administered from 1 to 12 times per day, or once a week. The duration of the investment can be 1, 2, 3, 4, 5, 6 or 7 days, or may be 1, 2, 3, 4, 5, 6, 8, 10 or 12 weeks.

Figure 1: Figure 1 shows the results of an in vivo study of a pancreatic cancer model using Hsp47 as a single target to test tumor growth inhibition. As shown in Figure 1, tumors, if any, grew slowly during the course of the study in the group treated with the Hsp47 siRNA modulator (2M). No weight loss was detected. Conversely, the tumor volume in the vehicle control group was doubled in only 22 days. In conclusion, Hsp47 siRNA at a dose of 0.75 mpk was observed to completely inhibit tumor growth, indicating that a modulator containing Hsp47 siRNA is an effective anticancer therapeutic.

Figure 2: Figure 2 shows the gene expression of Hsp47, type I collagen and type IV collagen in human cancer cell lines.

Figure 3: Figure 3 shows an untreated sample used in a method of suppressing proliferation of SW480 colon cancer cells with siRNA targeting Hsp47.

Figure 4: Figure 4 shows the effect of 10 nM negative siRNA in a method of suppressing proliferation of SW480 colon cancer cells with siRNA targeting Hsp47.

Figure 5: Figure 5 shows the effect of 50 nM negative siRNA in a method of suppressing proliferation of SW480 colon cancer cells with siRNA targeting Hsp47.

Figure 6: Figure 6 shows the target Hsp47 The effect of siRNA against 100 nM negative siRNA in the method of SW480 colon cancer cell proliferation.

Figure 7: Figure 7 shows the effect of 10 nM of active siRNA in a method of suppressing proliferation of SW480 colon cancer cells with siRNA targeting Hsp47.

Figure 8: Figure 8 shows the effect of 50 nM of active siRNA in a method of suppressing proliferation of SW480 colon cancer cells with siRNA targeting Hsp47.

Figure 9: Figure 9 shows the effect of 100 nM of active siRNA in a method of suppressing proliferation of SW480 colon cancer cells with siRNA targeting Hsp47.

Figure 10: Figure 10 shows the results of growth analysis in a method of suppressing proliferation of SW480 colon cancer cells with siRNA targeting Hsp47. The vertical axis is the number of cells × 10 ⁴ . The solid circle is an untreated sample. Marker X is a negative siRNA. The triangular markers represent samples treated with active siRNA targeting Hsp47.

Figure 11: Figure 11 shows the results of a dye exclusion assay in a method of inhibiting proliferation of SW480 colon cancer cells with siRNA targeting Hsp47. The vertical axis is the percentage of dead cells. The open circle is a sample treated with active siRNA targeting Hsp47. Marker X is a negative siRNA. The solid circle mark represents the unprocessed sample.

Figure 12: Figure 12 shows an untreated sample in a method of suppressing proliferation of HCT116 colon cancer cells with siRNA targeting Hsp47.

Figure 13: Figure 13 shows the effect of 10 nM negative siRNA in a method of suppressing proliferation of HCT116 colon cancer cells by siRNA targeting Hsp47.

Figure 14: Figure 14 shows the effect of 10 nM of active siRNA in a method of suppressing proliferation of HCT116 colon cancer cells with siRNA targeting Hsp47.

Figure 15: Figure 15 shows the results of growth analysis in a method of suppressing proliferation of HCT116 colon cancer cells with siRNA targeting Hsp47. The vertical axis is the number of cells × 10 ⁴ . The solid circle is an untreated sample. The open circles in the ascending curve are labeled as negative siRNA. The open circle mark in the flat curve represents a sample treated with the active siRNA of the target Hsp47.

Figure 16: Figure 16 shows the results of dye exclusion analysis in a method of inhibiting proliferation of HCT116 colon cancer cells by siRNA targeting Hsp47. The vertical axis is the percentage of dead cells. The open circle in the ascending curve is a sample treated with active siRNA targeting Hsp47. The open circles in the flat curve are labeled as negative siRNA. The solid circle mark represents the unprocessed sample.

Figure 17: Figure 17 shows the untreated samples used in the method of suppressing proliferation of A549 lung cancer cells by siRNA targeting Hsp47.

Figure 18: Figure 18 shows the effect of 10 nM negative siRNA in a method of suppressing proliferation of A549 lung cancer cells by siRNA targeting Hsp47.

Figure 19: Figure 19 shows the target Hsp47 The effect of 50 nM negative siRNA in the method of siRNA to suppress proliferation of A549 lung cancer cells.

Figure 20: Figure 20 shows the effect of 100 nM negative siRNA in a method of suppressing proliferation of A549 lung cancer cells by siRNA targeting Hsp47.

Figure 21: Figure 21 shows the effect of 10 nM of active siRNA in a method of suppressing proliferation of A549 lung cancer cells by siRNA targeting Hsp47.

Figure 22: Figure 22 shows the effect of 50 nM of active siRNA in a method of suppressing proliferation of A549 lung cancer cells by siRNA targeting Hsp47.

Figure 23: Figure 23 shows the effect of 100 nM of active siRNA in a method of inhibiting proliferation of SW480 colon cancer cells with siRNA targeting Hsp47.

Figure 24: Figure 24 shows the results of a growth assay in a method of suppressing proliferation of A549 lung cancer cells by siRNA targeting Hsp47. The vertical axis is the number of cells × 10 ⁴ . The solid circle is an untreated sample. The open circles are labeled as negative siRNA. The triangular markers represent samples treated with active siRNA targeting Hsp47.

Figure 25: Figure 25 shows the results of the dye exclusion assay in a method of suppressing proliferation of A549 lung cancer cells by siRNA targeting Hsp47. The vertical axis is the percentage of dead cells. The open circle is a sample treated with active siRNA targeting Hsp47. Marker X is a negative siRNA. The solid circle mark represents the unprocessed sample.

Figure 26: Figure 26 shows the untreated samples used in the method of suppressing proliferation of HepG2 hepatoma cells by siRNA targeting Hsp47.

Figure 27: Figure 27 shows the effect of 10 nM negative siRNA in a method of suppressing proliferation of HepG2 liver cancer cells by siRNA targeting Hsp47.

Figure 28: Figure 28 shows the effect of 50 nM negative siRNA in a method of suppressing proliferation of HepG2 liver cancer cells by siRNA targeting Hsp47.

Figure 29: Figure 29 shows the effect of 100 nM negative siRNA in a method of suppressing proliferation of HepG2 liver cancer cells by siRNA targeting Hsp47.

Figure 30: Figure 30 shows the effect of 10 nM of active siRNA in a method of suppressing proliferation of HepG2 hepatoma cells by siRNA targeting Hsp47.

Figure 31: Figure 31 shows the effect of 50 nM of active siRNA in a method of suppressing proliferation of HepG2 hepatoma cells by siRNA targeting Hsp47.

Figure 32: Figure 32 shows the effect of 100 nM of active siRNA in a method of suppressing proliferation of HepG2 hepatoma cells by siRNA targeting Hsp47.

Figure 33: Figure 33 shows the results of growth analysis in a method of suppressing proliferation of HepG2 hepatoma cells by siRNA targeting Hsp47. The vertical axis is the number of cells × 10 ⁴ . The solid circle is an untreated sample. The open circles are labeled as negative siRNA. Marker X represents a sample treated with active siRNA targeting Hsp47.

Figure 34: Figure 34 shows the results of dye exclusion analysis in a method of suppressing proliferation of HepG2 liver cancer cells by siRNA targeting Hsp47. The vertical axis is the percentage of dead cells. The open circle is a sample treated with active siRNA targeting Hsp47. Marker X is a negative siRNA. The solid square mark represents the unprocessed sample.

Figure 35: Figure 35 shows the results of the method for detecting Annexin V and PI in colon cancer cells SW480 transfected with active Hsp47 siRNA on day 2 after siRNA transfection.

Figure 36: Figure 36 shows the results of the method for detecting Annexin V and PI in colon cancer cell HCT116 transfected with active Hsp47 siRNA on day 2 after siRNA transfection.

Figure 37: Figure 37 shows the results of a method for detecting the expression of caspase-3 and Hsp47 proteins in SW480 colon cancer cells transfected with active Hsp47 siRNA.

Figure 38: Figure 38 shows the results of a method for detecting the expression of caspase-3 and Hsp47 proteins in HepG2 liver cancer cells transfected with active Hsp47 siRNA.

Figure 39: Figure 39 shows the results of a method for detecting the expression of caspase-3 and Hsp47 proteins in A549 lung cancer cells transfected with active Hsp47 siRNA.

Figure 40: Figure 40 shows the detection of caspase in colon cancer cell SW480 transfected with Hsp47 siRNA and p21 siRNA The result of the method of enzyme-3/7 activity. These data show that the combined use of Hsp47 siRNA and p21 siRNA in colon cancer cells can result in an unexpectedly unexpected increase in the level of caspase, which surprisingly increases apoptosis.

Example 1: In vivo studies were performed to test Hsp47 as a single target to inhibit tumor growth. As shown in Table 5, a pancreatic cancer model derived from PANC-1 was selected because it is enriched in collagen.

Research methods:

Inoculation of PANC-1 cells into females via the subcutaneous route The right abdomen of the thymus nude mouse.

Tumor size was measured and calculated using the formula: tumor volume = length x width ² /2. When the established tumor reached approximately 200 mm ³ , the mice were randomly assigned for test article injection.

The animals were injected via an intravenous route with 0.75 mg/kg, q1w of a modulator (2M) containing Hsp47-siRNA for a total of 4 doses.

Animal weight and tumor volume were monitored twice a week.

RESULTS AND CONCLUSION: Tumor growth was slow in this PANC-1 model and the doubling time of the vehicle group was 22 days. No weight loss was detected. A modulator (2M) based on Compound 81 and comprising Hsp47-siRNA was observed to completely inhibit tumor growth at a dose of 0.75 mpk. In summary, inhibition of Hsp47 inhibited tumor growth, indicating that the modulator is an effective anti-cancer therapeutic.

Results and Conclusions: Figure 1 shows in vivo results of a pancreatic cancer model using Hsp47 as a single target to test tumor growth inhibition. As shown in Figure 1, tumors, if any, grew slowly during the course of the study in the group treated with the Hsp47 siRNA modulator (2M). No weight loss was detected. Conversely, the tumor volume in the vehicle control group was doubled in only 22 days. In conclusion, Hsp47 siRNA at a dose of 0.75 mpk was observed to completely inhibit tumor growth, indicating that a modulator containing Hsp47 siRNA is an effective anticancer therapeutic.

Example 2: Figure 2 shows Hsp47, type I collagen and type IV collagen in human cancer cell lines A549, MCF7, MDA-MB-231, HCT116, M7609, COLO230HSR, Gene expression in SW480, PANC-1, SW1990, MIA-PaCa-2, HepG2, HT1080 and HaLa. The genes in SW480 and HepG2 performed significantly.

Example 3: Figures 3 to 11 show the results of a method for suppressing proliferation of SW480 colon cancer cells with siRNA targeting Hsp47.

Figure 3 shows untreated samples used in a method of suppressing proliferation of SW480 colon cancer cells with siRNA targeting Hsp47. Figure 4 shows the effect of 10 nM negative siRNA in a method of suppressing proliferation of SW480 colon cancer cells with siRNA targeting Hsp47. Figure 5 shows the effect of 50 nM negative siRNA in a method of suppressing proliferation of SW480 colon cancer cells with siRNA targeting Hsp47. Figure 6 shows the effect of 100 nM negative siRNA in a method of suppressing proliferation of SW480 colon cancer cells with siRNA targeting Hsp47. Figure 7 shows the effect of 10 nM of active siRNA in a method of suppressing proliferation of SW480 colon cancer cells with siRNA targeting Hsp47. Figure 8 shows the effect of 50 nM of active siRNA in a method of suppressing proliferation of SW480 colon cancer cells by siRNA targeting Hsp47. Figure 9 shows the effect of 100 nM of active siRNA in a method of suppressing proliferation of SW480 colon cancer cells with siRNA targeting Hsp47.

A comparison of Figures 4 and 7, 5 and 8 and 6 and 9 shows that active siRNA targeting Hsp47 would suppress SW480 colon cancer cells.

Figure 10 shows the results of growth analysis in a method of suppressing proliferation of SW480 colon cancer cells with siRNA targeting Hsp47. The vertical axis is the number of cells × 10 ⁴ . The solid circle is an untreated sample. Marker X is a negative siRNA. The triangular markers represent samples treated with active siRNA targeting Hsp47.

Figure 11 shows the results of dye exclusion analysis in a method of suppressing proliferation of SW480 colon cancer cells with siRNA targeting Hsp47. The vertical axis is the percentage of dead cells. The open circle is a sample treated with active siRNA targeting Hsp47. Marker X is a negative siRNA. The solid circle mark represents the unprocessed sample.

Example 4: The results of the method of suppressing proliferation of HCT116 colon cancer cells by siRNA targeting Hsp47 are shown in Figures 12 to 16.

Figure 12 shows an untreated sample in a method of suppressing proliferation of HCT116 colon cancer cells with siRNA targeting Hsp47. Figure 13 shows the effect of 10 nM negative siRNA in a method of suppressing proliferation of HCT116 colon cancer cells by siRNA targeting Hsp47. Figure 14 shows the effect of 10 nM of active siRNA in a method of suppressing proliferation of HCT116 colon cancer cells by siRNA targeting Hsp47. A comparison of Figures 12, 13 and 14 shows that active siRNA targeting Hsp47 can suppress HCT116 colon cancer cells.

Figure 15 shows the results of growth analysis in a method of suppressing proliferation of HCT116 colon cancer cells by siRNA targeting Hsp47. The vertical axis is the number of cells × 10 ⁴ . The solid circle is an untreated sample. The open circles in the ascending curve are labeled as negative siRNA. The open circle mark in the flat curve represents a sample treated with the active siRNA of the target Hsp47.

Figure 16 shows the siRNA containment of the target Hsp47 The results of the dye exclusion assay in the method of HCT116 colon cancer cell proliferation. The vertical axis is the percentage of dead cells. The open circle in the ascending curve is a sample treated with active siRNA targeting Hsp47. The open circles in the flat curve are labeled as negative siRNA. The solid circle mark represents the unprocessed sample.

Example 5: The results of the method of suppressing proliferation of A549 lung cancer cells by siRNA targeting Hsp47 are shown in Figures 17 to 25.

Figure 17 shows an untreated sample in a method of suppressing proliferation of A549 lung cancer cells with siRNA targeting Hsp47. Figure 18 shows the effect of 10 nM negative siRNA in a method of suppressing proliferation of A549 lung cancer cells by siRNA targeting Hsp47. Figure 19 shows the effect of 50 nM negative siRNA in a method of suppressing proliferation of A549 lung cancer cells by siRNA targeting Hsp47. Figure 20 shows the effect of 100 nM negative siRNA in a method of suppressing proliferation of A549 lung cancer cells by siRNA targeting Hsp47. Figure 21 shows the effect of 10 nM of active siRNA in a method of suppressing proliferation of A549 lung cancer cells by siRNA targeting Hsp47. Figure 22 shows the effect of 50 nM of active siRNA in a method of suppressing proliferation of A549 lung cancer cells by siRNA targeting Hsp47. Figure 23 shows the effect of 100 nM of active siRNA in a method of suppressing proliferation of SW480 colon cancer cells with siRNA targeting Hsp47.

Comparison of Figures 18 and 21, 19 and 22 and 20 and 23 shows that active siRNA targeting Hsp47 can suppress A549 lung cancer cells.

Figure 24 shows the results of growth analysis in a method of suppressing proliferation of A549 lung cancer cells by siRNA targeting Hsp47. The vertical axis is the number of cells × 10 ⁴ . The solid circle is an untreated sample. The open circles are labeled as negative siRNA. The triangular markers represent samples treated with active siRNA targeting Hsp47.

Figure 25 shows the results of dye exclusion analysis in a method of suppressing proliferation of A549 lung cancer cells by siRNA targeting Hsp47. The vertical axis is the percentage of dead cells. The open circle is a sample treated with active siRNA targeting Hsp47. Marker X is a negative siRNA. The solid circle mark represents the unprocessed sample.

Example 6: Results of a method for suppressing proliferation of A549 lung cancer cells by siRNA targeting Hsp47 are shown in Figures 26 to 34.

Figure 26 shows an untreated sample in a method of suppressing proliferation of HepG2 liver cancer cells by siRNA targeting Hsp47. Figure 27 shows the effect of 10 nM negative siRNA in a method of suppressing proliferation of HepG2 hepatoma cells by siRNA targeting Hsp47. Figure 28 shows the effect of 50 nM negative siRNA in a method of suppressing proliferation of HepG2 liver cancer cells by siRNA targeting Hsp47. Figure 29 shows the effect of 100 nM negative siRNA in a method of suppressing proliferation of HepG2 hepatoma cells by siRNA targeting Hsp47. Figure 30 shows the effect of 10 nM of active siRNA in a method of suppressing proliferation of HepG2 hepatoma cells by siRNA targeting Hsp47. Figure 31 shows the effect of 50 nM of active siRNA in a method of suppressing proliferation of HepG2 hepatoma cells by siRNA targeting Hsp47. Figure 32 shows the effect of 100 nM of active siRNA in a method of suppressing proliferation of HepG2 hepatoma cells by siRNA targeting Hsp47.

Comparison of Figures 27 and 30, 28 and 31 and 29 and 32 shows that active siRNA targeting Hsp47 can suppress HepG2 liver cancer cells.

Figure 33 shows the results of growth analysis in a method of suppressing proliferation of HepG2 liver cancer cells by siRNA targeting Hsp47. The vertical axis is the number of cells × 10 ⁴ . The solid circle is an untreated sample. The open circles are labeled as negative siRNA. Marker X represents a sample treated with active siRNA targeting Hsp47.

Figure 34 shows the results of dye exclusion analysis in a method of suppressing proliferation of HepG2 liver cancer cells by siRNA targeting Hsp47. The vertical axis is the percentage of dead cells. The open circle is a sample treated with active siRNA targeting Hsp47. Marker X is a negative siRNA. The open square mark represents the untreated sample.

Example 7: The results of the method for detecting annexin V and PI in colon cancer cells (SW480, HCT116) transfected with Hsp47 siRNA are shown in Figures 35 to 36.

Figure 35 shows the results of a method for detecting annexin V and PI in colon cancer cells SW480 on day 2 after transfection with active Hsp47 siRNA. Figure 36 shows the results of a method for detecting annexin V and PI on day 2 after transfection of siRNA with Hsp116 transfected with active Hsp47 siRNA.

Example 8: The results of the method for detecting the expression of caspase-3 and Hsp47 proteins in cancer cells transfected with Hsp47 siRNA are shown in Figures 37 to 39.

Figure 37 shows the detection of active Hsp47 siRNA Results of methods for expression of caspase-3 and Hsp47 proteins in transfected SW480 colon cancer cells. Figure 38 shows the results of a method for detecting the expression of caspase-3 and Hsp47 proteins in HepG2 hepatoma cells transfected with active Hsp47 siRNA. Figure 39 shows the results of a method for detecting the expression of caspase-3 and Hsp47 proteins in A549 lung cancer cells transfected with active Hsp47 siRNA.

Example 9: Figure 40 shows the results of a method for detecting caspase-3/7 activity in colon cancer cells (SW480) transfected with Hsp47 siRNA and p21 siRNA. These data indicate that the combination of Hsp47 siRNA and p21 siRNA in colon cancer cells can result in an unexpectedly unexpected increase in the level of caspase. The surprising increase in the level of this caspase in cancer cells has shown an alarming increase in apoptosis of this cell.

Example 10: Intravitreal transfection was performed in A549 cell lines to determine siRNA knockdown potency. The modulator of the present invention containing the RNAi molecule of the target Hsp47 was used to observe dose-dependent knockdown of Hsp47 mRNA.

Protocol for knockdown in glass tubes: One day before transfection, cells were seeded at a density of 2 x 10 ³ cells in 96-well plates containing 100 μl of DMEM containing 10% FBS (HyClone, Inc., Catalog No. SH30243.01) and the 96-well plate was incubated at 37 ° C in a humidified atmosphere incubator with 5% CO ₂ in air. The medium was replaced with 90 μl of Opti-MEM I low serum medium (Life Technologies, Inc., catalog number 31985-070) containing 2% FBS prior to transfection. 0.2 μl of Lipofectamine RNAiMax (Life Technologies, catalog number 13778-100) was mixed with 4.8 μl of Opti-MEM I for 5 minutes at room temperature. 1 μl of siRNA was mixed with 4 μl of Opti-MEM I, combined with LF2000 solution, and then gently mixed without mixing by shaking. Wait at room temperature for 5 minutes. The mixture was incubated for 10 minutes at room temperature to allow for the formation of an RNA-RNAiMax complex. 10 μl of RNA-RNAiMax complex was added to each well and the plate was gently shaken by hand. The cells were incubated for 2 hours at 37 ° C in a humidified atmosphere incubator containing 5% CO ₂ in air. The medium was changed to fresh MEM I low serum medium (Life Technologies, Cat. No. 31985-070) containing 2% FBS. 24 hours after transfection, the cells were washed once with ice-cold PBS. The cells were lysed in 50 μl of Cell-to-Ct Cell Lysis Buffer (Life Technologies, Cat. No. 4391851 C) for 5 to 30 minutes at room temperature. 5 μl of the stop solution was added and incubated for 2 minutes at room temperature. The mRNA level was immediately measured by TAQMAN by RT-qPCR. Alternatively, the sample can be frozen at -80 ° C and analyzed later.

Example 11: Efficacy of Hsp47 siRNA to inhibit tumors. Using a pancreatic cancer xenograft model, a relatively low dose of the target Hsp47 siRNA of 0.75 mg/kg was performed. The combined siRNA showed significant and unexpectedly favorable tumor inhibition potency on day 28.

In this experiment, A549 and PANC-1 cell lines were obtained from ATCC. The cell suspension was mixed with ice-cold thawed BD matrigel in a ratio of 1:1 for injection. 0.1 ml of 2×10 ⁶ (A549) cells or 2.5 were inoculated into the right ventral side of each mouse (6 to 8 weeks old Charles River laboratory athymic female nude mice) via a subcutaneous route using a 25G needle and syringe. × 10 ⁶ cells (PANC-1) was inoculated with cells (1 per mouse was inoculated twice). The mice were anesthetized when inoculated On the day the established tumor reached approximately 250 to 350 mm ³ (A549) or 150 to 250 mm ³ (PANC-1), the animals were injected with a bolus through the tail vein. The animals were sacrificed with excess carbon dioxide and the tumors were dissected at various time points after dosing. The wet weight of the tumor was first measured and then divided into three sections to measure Hsp47 knockdown, biodistribution of siRNA, and biomarker analysis. Samples were snap frozen in liquid nitrogen and stored at -80 °C until ready for processing for bioanalysis.

Example 12: Efficacy of siRNA encapsulated in liposome modulators was assessed in a mouse model of in situ A549 lung cancer.

Experimental animals: A total of 60 male NCr nu/nu mice, 5 to 6 weeks old, were used in the study. Experimental animals were grown and grown at AntiCancer. They were kept in the HEPA filtered environment during the experiment. The cage, food and bedding are all autoclaved. The animal diet was obtained from Harlan Teklad Corporation (Madison, Wisconsin).

Preparation of liposome modulator: The modulator was prepared and stored at 4 °C. The modulator was warmed to room temperature 10 minutes before injection into the mice.

A549 Human Lung Cancer In Situ Model (SOI): On the day of SOI, tumors were harvested from the subcutaneous site of animals bearing A549 tumor xenografts and placed in RPMI-1640 medium. Necrotic tissue was removed and the viable tissue was cut into 1.5 to 2 mm ³ pieces. Animals were inhaled with isoflurane to anesthetize and disinfect the surgical area with iodine and alcohol. A transverse incision about 1.5 cm in length was made on the left chest wall of the mouse using surgical scissors. An intercostal incision is made between the third and fourth ribs and the left lung is exposed. An A549 tumor fragment was transplanted to the surface of the lung with 8-0 surgical suture (nylon). The chest wall was closed with a 6-0 surgical suture (wire). Using a 3 cc syringe with a 25G X 11⁄2 needle, the remaining air in the chest cavity was withdrawn by intrathoracic puncture to reinflate the lungs. The chest wall was closed with a 6-0 surgical silk suture. All of the above surgical procedures were performed under a HEPA filter layer flow hood using a 7x magnification microscope (Olympus).

Three days after tumor implantation, tumor-bearing mice were randomized into groups of ten mice each. Treatment of each group of mice was started three days after implantation of the tumor.

End point: Experimental mice were killed 42 days after the start of treatment. Primary tumors were excised and weighed on an electronic scale for subsequent analysis.

The anti-tumor efficacy of the modulator against human lung cancer A549 was assessed by comparing the final primary tumor weight measured at autopsy between each treatment group and the vehicle control group. The average tumor weight of each group was measured.

Estimation of Compound Toxicity: The average body weight of mice in the treatment and control groups was maintained within the normal range throughout the experiment. Roughly observed mice also did not detect other signs of toxicity.

Conclusion: By comparing the final tumor weight obtained at the end of the experiment, it can be concluded that compared with the control group, the treatment group can significantly and surprisingly reduce human lung cancer when treated with a modulator containing 2 mg/kg of Hsp47 siRNA. Tumor growth and tumor volume of A549. No toxicity was observed.

Example 13: Effect of small interfering RNA (siRNA) targeting Hsp47 on growth and angiogenesis of A549 cells in nude mice in chorioallantoic membrane (CAM) assay. Construction of three pairs of Hsp47 siRNA plasmids And non-silencing plasmids, and transfected into A549 cells via LIPOFECTAMINE 2000, respectively. The most efficient Hsp47 siRNA plasmid pair was selected by ELISA and real-time RT-PCR. A549 cells transfected with the selected Hsp47 siRNA-plasmid, A549 cells transfected with non-silencing plasmid, and untransfected A549 cells were inoculated into nude mice, respectively. Chicken embryos were randomly divided into four groups and CAM was treated with different solutions for 48 hours: medium DMEM as a negative control group, untransfected A549 cell culture supernatant as a positive control group, and Hsp47 siRNA A549 as a Hsp47 siRNA group. Cell culture supernatants and non-silencing siRNA A549 cell culture supernatants as non-silencing siRNA groups. CAM was harvested on day 12 for microscopic analysis.

Compared with the control group, Hsp47 siRNA plasmid induced A549 cells to reduce the secretion of Hsp47, accompanied by a decrease in Hsp47 mRNA. The mean tumor volume of the mouse xenografts in the Hsp47 siRNA group was reduced compared to the non-silent siRNA group; the xenografts were grown to a time delay of 50 ^mm3 . The Hsp47 content in the xenografts is reduced. In the CAM analysis, the Hsp47 content in the negative group was zero, whereas the Hsp47 content in the Hsp47 siRNA group was reduced by 20-70% compared to the non-silencing siRNA group or the positive group; compared with the negative group, the Hsp47 siRNA group or The vascular branching points of CAM in the non-silencing siRNA group or the positive group increased; the total vascular length of CAM in the Hsp47 siRNA group was increased compared to the negative group, and it was increased in the non-silencing siRNA group or the positive group. Compared with the negative control group, when Hsp47 was added to the cell culture supernatant of the Hsp47 siRNA group, the proliferation of microvessels was significantly increased, and significant proliferation of blood vessels was observed in the non-silencing siRNA group or the positive control group.

Example 14: Cell culture. The human non-small cell lung cancer cell line A549 was cultured in F-12K medium (ATCC) supplemented with 10% FBS (FBS, Invitrogen) at 37 ° C in a humidified atmosphere with 5% CO ₂ . A549TR cells were transduced with individual lentiviral transduction granules according to the manufacturer's instructions (Sigma-Aldrich) to generate cells stably expressing the control or Hsp47 siRNA. Resistant plants were selected for 12 days in 2.5 μg/mL puromycin (Invivogen), isolated using a colonization cylinder, and subsequently expanded and maintained in a medium containing puromycin.

Example 15: siRNA targeting Hsp47 resulted in a substantial regression of tumor volume in vivo.

A lipid modulator was used to encapsulate the siRNA and deliver it in the nanoparticles to xenografts of human A549 lung cancer cells in scid mice. The xenografts were tested to identify the presence or absence of KRAS mutations or abnormal performance levels compared to normal cells. When tumors were established (>100 mm ³ ), mice were treated once every 2 days with siRNA targeting Hsp47 or control (non-specific) siRNA for 2 weeks. The test was stopped when the control group had to be euthanized.

RESULTS: Treatment with siRNA targeting Hsp47 prevented tumor expansion and resulted in a significant reduction in tumor volume.

The recovered tumors were sectioned and visually examined by TUNEL staining. Tumors treated with siRNA targeting Hsp47 showed significantly higher levels of apoptosis. RNA was extracted from the tumor and subjected to real-time PCR to examine specific knockdown of Hsp47.

RESULTS: Treatment with siRNA targeting Hsp47 significantly reduced Hsp47 expression in vivo.

Example 16: The siRNA of the target p21 of the present invention was found to have an activity capable of rendering a gene in a glass tube. The dose-dependent gene knockdown activity of p21 siRNA showed an IC50 of less than about 3 picomoles (pM) and can be as low as 1 pM.

Intracellular transfection was performed in A549 cells to determine the siRNA knockdown potency. The dose-dependent knockdown of p21 mRNA was observed with the siRNA of Table 1, as shown in Table 6.

As shown in Table 6, the activity of the p21 siRNA of Table 1 was in the range of 0.3 to 10 pM, which is suitable for various uses, including as an agent for use in vivo.

Example 17: The p21 siRNA construct of the invention having deoxynucleotides located in the seed region of the antisense strand of siRNA can provide an unexpected and advantageous increase in gene knockdown activity.

Intra-glass transfection was performed in the A549 cell line to determine the knockdown potency of p21 siRNA based on structure 1735' (SEQ ID NO: 57 and 71). As shown in Table 7, p21 siRNA based on structure 1735' was observed to knock down p21 mRNA in a dose-dependent manner.

As shown in Table 7, the activity of p21 siRNA with three deoxynucleotides in the antisense strand seed region was surprising based on the structure 1735' compared to the p21 siRNA without the deoxynucleotide in the double-stranded region. And unexpectedly increased by up to 300 times.

These data indicate that p21 siRNA with deoxynucleotides in the antisense strand seed region in its structure provides surprisingly increased gene knockdown activity compared to p21 siRNA without deoxynucleotides in the double-stranded region.

The activity of p21 siRNA having three deoxynucleotides in the antisense strand seed region shown in Table 7 is in the range of 0.001 to 0.1 pM, which range is particularly suitable for many uses, including as an agent for in vivo use.

The embodiments described herein are not limiting and those skilled in the art can readily appreciate the specific groups of modifications described herein. It is possible to test for identification of nucleic acid molecules with improved RNAi activity without undue experimentation.

All publications, patents, and literatures specifically referred to herein are hereby incorporated by reference in their entirety for all purposes.

It is to be understood that the invention is not limited to the specific methods, protocols, materials and reagents described, as these may vary. It is also understood that the terminology used herein is for the purpose of describing the particular embodiments It will be readily understood by those skilled in the art that the present invention may be substituted and modified without departing from the scope and spirit of the present disclosure, and those embodiments are within the scope of the specification and the appended claims. Inside.

It must be noted that the singular forms "a", "the" and "the" Similarly, the terms "a" or "an", "an" or "an" It should also be noted that the terms "comprises, "comprising", "containing", "including", and "having" are used interchangeably and are intended to be interpreted in a broad and unrestricted manner.

Recitation of ranges of values herein are merely intended to serve as a short-term method of individually referring to the various values falling within the range, and each individual value is included in this patent specification as if it were individually recited herein. In the case of the Markush group, those skilled in the art will recognize that the description includes individual members of the Markusi group and subgroups of members.

Without further elaboration, the skilled artisan skilled in the art can utilize the invention to the fullest extents thereof. Therefore, the following specific embodiments are to be construed as illustrative only and not limiting of the remainder of the disclosure.

All of the features disclosed in this patent specification can be combined in any combination. The various features disclosed in this patent specification can be replaced by alternative features for the same, equivalent or similar purpose.

<110> NITTO DENKO CORPORATION

<120> Therapeutic compositions and methods for malignant tumors using RNAi molecules targeting Hsp47 and p21

<130> ND5123767WO

<140> PCT/US15/67558

<141> 2015-12-28

<150> JP2014_266198

<151> 2014-12-26

<150> 62/266,670

<151> 2015-12-13

<150> 62/184,195

<151> 2015-06-24

<160> 134

<170> PatentIn version 3.5

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<220>

<223> Description of Artificial Sequence: Synthetic Oligonucleotides

<220>

<221> Modified _ base

<222> (20)..(21)

<223> 2'-methoxy-nucleotide

<400> 34

<210> 35

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Description of Artificial Sequence: Synthetic Oligonucleotides

<220>

<221> Modified _ base

<222> (20)..(21)

<223> 2'-methoxy-nucleotide

<400> 35

<210> 36

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Description of Artificial Sequence: Synthetic Oligonucleotides

<220>

<221> Modified _ base

<222> (20)..(21)

<223> 2'-methoxy-nucleotide

<400> 36

<210> 37

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Description of Artificial Sequence: Synthetic Oligonucleotides

<220>

<221> Modified _ base

<222> (20)..(21)

<223> 2'-methoxy-nucleotide

<400> 37

<210> 38

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Description of Artificial Sequence: Synthetic Oligonucleotides

<220>

<221> Modified _ base

<222> (20)..(21)

<223> 2'-methoxy-nucleotide

<400> 38

<210> 39

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Description of Artificial Sequence: Synthetic Oligonucleotides

<220>

<221> Modified _ base

<222> (20)..(21)

<223> 2'-methoxy-nucleotide

<400> 39

<210> 40

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Description of Artificial Sequence: Synthetic Oligonucleotides

<220>

<221> Modified _ base

<222> (20)..(21)

<223> 2'-methoxy-nucleotide

<400> 40

<210> 41

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Description of Artificial Sequence: Synthetic Oligonucleotides

<220>

<221> Modified _ base

<222> (20)..(21)

<223> 2'-methoxy-nucleotide

<400> 41

<210> 42

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Description of Artificial Sequence: Synthetic Oligonucleotides

<220>

<221> Modified _ base

<222> (20)..(21)

<223> 2'-methoxy-nucleotide

<400> 42

<210> 43

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Description of Artificial Sequence: Synthetic Oligonucleotides

<220>

<221> Modified _ base

<222> (20)..(21)

<223> 2'-methoxy-nucleotide

<400> 43

<210> 44

<211> 21

<212> RNA

<213> Human master sequence

<220>

<223> Description of Artificial Sequence: Synthetic Oligonucleotides

<220>

<221> Modified _ base

<222> (20)..(21)

<223> 2'-methoxy-nucleotide

<400> 44

<210> 45

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Description of Artificial Sequence: Synthetic Oligonucleotides

<220>

<221> Modified _ base

<222> (20)..(21)

<223> 2'-methoxy-nucleotide

<400> 45

<210> 46

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Description of Artificial Sequence: Synthetic Oligonucleotides

<220>

<221> Modified _ base

<222> (20)..(21)

<223> 2'-methoxy-nucleotide

<400> 46

<210> 47

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Description of Artificial Sequence: Synthetic Oligonucleotides

<220>

<221> Modified _ base

<222> (20)..(21)

<223> 2'-methoxy-nucleotide

<400> 47

<210> 48

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Description of Artificial Sequence: Synthetic Oligonucleotides

<220>

<221> Modified _ base

<222> (20)..(21)

<223> 2'-methoxy-nucleotide

<400> 48

<210> 49

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Description of Artificial Sequence: Synthetic Oligonucleotides

<220>

<221> Modified _ base

<222> (20)..(21)

<223> 2'-methoxy-nucleotide

<400> 49

<210> 50

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Description of Artificial Sequence: Synthetic Oligonucleotides

<220>

<221> Modified _ base

<222> (20)..(21)

<223> 2'-methoxy-nucleotide

<400> 50

<210> 51

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Description of Artificial Sequence: Synthetic Oligonucleotides

<220>

<221> Modified _ base

<222> (20)..(21)

<223> 2'-methoxy-nucleotide

<400> 51

<210> 52

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Description of Artificial Sequence: Synthetic Oligonucleotides

<220>

<221> Modified _ base

<222> (20)..(21)

<223> 2'-methoxy-nucleotide

<400> 52

<210> 53

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Description of Artificial Sequence: Synthetic Oligonucleotides

<220>

<221> Modified _ base

<222> (20)..(21)

<223> 2'-methoxy-nucleotide

<400> 53

<210> 54

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Description of Artificial Sequence: Synthetic Oligonucleotides

<220>

<221> Modified _ base

<222> (20)..(21)

<223> 2'-methoxy-nucleotide

<400> 54

<210> 55

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Description of Artificial Sequence: Synthetic Oligonucleotides

<220>

<221> Modified _ base

<222> (20)..(21)

<223> 2'-methoxy-nucleotide

<400> 55

<210> 56

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Description of Artificial Sequence: Synthetic Oligonucleotides

<220>

<221> Modified _ base

<222> (20)..(21)

<223> 2'-methoxy-nucleotide

<400> 56

<210> 57

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of Artificial Sequence: Synthetic Oligonucleotides

<220>

<223> Description of combined DNA/RNA molecules: synthetic oligonucleotides

<220>

<221> Modified _ base

<222> (20)..(21)

<223> a, c, t, g, u, unknown or other

<400> 57

<210> 58

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Description of Artificial Sequence: Synthetic Oligonucleotides

<220>

<221> Modified _ base

<222> (1)..(3)

<223> 2'-OMe-nucleotide

<220>

<221> Modified _ base

<222> (8)..(8)

<223> 2'-OMe-nucleotide

<220>

<221> Modified _ base

<222> (12)..(12)

<223> 2'-OMe-nucleotide

<220>

<221> Modified _ base

<222> (17)..(17)

<223> 2'-OMe-nucleotide

<220>

<221> Modified _ base

<222> (20)..(21)

<223> 2'-OMe-nucleotide

<400> 58

<210> 59

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Description of Artificial Sequence: Synthetic Oligonucleotides

<220>

<221> Modified _ base

<222> (1)..(3)

<223> 2'-OMe-nucleotide

<220>

<221> Modified _ base

<222> (8)..(8)

<223> 2'-OMe-nucleotide

<220>

<221> Modified _ base

<222> (12)..(12)

<223> 2'-OMe-nucleotide

<220>

<221> Modified _ base

<222> (14)..(15)

<223> 2'-OMe-nucleotide

<220>

<221> Modified _ base

<222> (17)..(17)

<223> 2'-OMe-nucleotide

<220>

<221> Modified _ base

<222> (20)..(21)

<223> 2'-OMe-nucleotide

<400> 59

<210> 60

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Description of Artificial Sequence: Synthetic Oligonucleotides

<220>

<221> Modified _ base

<222> (1)..(3)

<223> 2'-OMe-nucleotide

<220>

<221> Modified _ base

<222> (8)..(8)

<223> 2'-OMe-nucleotide

<220>

<221> Modified _ base

<222> (12)..(12)

<223> 2'-OMe-nucleotide

<220>

<221> Modified _ base

<222> (14)..(15)

<223> 2'-OMe-nucleotide

<220>

<221> Modified _ base

<222> (17)..(17)

<223> 2'-OMe-nucleotide

<220>

<221> Modified _ base

<222> (20)..(21)

<223> 2'-OMe-nucleotide

<400> 60

<210> 61

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Description of Artificial Sequence: Synthetic Oligonucleotides

<220>

<221> Modified _ base

<222> (1)..(3)

<223> 2'-OMe-nucleotide

<220>

<221> Modified _ base

<222> (8)..(8)

<223> 2'-OMe-nucleotide

<220>

<221> Modified _ base

<222> (12)..(12)

<223> 2'-OMe-nucleotide

<220>

<221> Modified _ base

<222> (17)..(17)

<223> 2'-OMe-nucleotide

<220>

<221> Modified _ base

<222> (20)..(21)

<223> 2'-OMe-nucleotide

<400> 61

<210> 62

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Description of Artificial Sequence: Synthetic Oligonucleotides

<220>

<221> Modified _ base

<222> (20)..(21)

<223> 2'-OMe-nucleotide

<400> 62

<210> 63

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Description of Artificial Sequence: Synthetic Oligonucleotides

<220>

<221> Modified _ base

<222> (20)..(21)

<223> 2'-OMe-nucleotide

<400> 63

<210> 64

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Description of Artificial Sequence: Synthetic Oligonucleotides

<220>

<221> Modified _ base

<222> (20)..(21)

<223> 2'-OMe-nucleotide

<400> 64

<210> 65

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Description of Artificial Sequence: Synthetic Oligonucleotides

<220>

<221> Modified _ base

<222> (20)..(21)

<223> 2'-OMe-nucleotide

<400> 65

<210> 66

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Description of Artificial Sequence: Synthetic Oligonucleotides

<220>

<221> Modified _ base

<222> (20)..(21)

<223> 2'-OMe-nucleotide

<400> 66

<210> 67

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Description of Artificial Sequence: Synthetic Oligonucleotides

<220>

<221> Modified _ base

<222> (20)..(21)

<223> 2'-OMe-nucleotide

<400> 67

<210> 68

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Description of Artificial Sequence: Synthetic Oligonucleotides

<220>

<221> Modified _ base

<222> (8)..(8)

<223> 2'-OMe-nucleotide

<220>

<221> Modified _ base

<222> (12)..(12)

<223> 2'-OMe-nucleotide

<220>

<221> Modified _ base

<222> (17)..(17)

<223> 2'-OMe-nucleotide

<220>

<221> Modified _ base

<222> (20)..(21)

<223> 2'-OMe-nucleotide

<400> 68

<210> 69

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Description of Artificial Sequence: Synthetic Oligonucleotides

<220>

<221> Modified _ base

<222> (1)..(3)

<223> 2'-OMe-nucleotide

<220>

<221> Modified _ base

<222> (20)..(21)

<223> 2'-OMe-nucleotide

<400> 69

<210> 70

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Description of Artificial Sequence: Synthetic Oligonucleotides

<220>

<221> Modified _ base

<222> (1)..(3)

<223> 2'-OMe-nucleotide

<220>

<221> Modified _ base

<222> (8)..(8)

<223> 2'-OMe-nucleotide

<220>

<221> Modified _ base

<222> (12)..(12)

<223> 2'-OMe-nucleotide

<220>

<221> Modified _ base

<222> (17)..(17)

<223> 2'-OMe-nucleotide

<220>

<221> Modified _ base

<222> (20)..(21)

<223> 2'-OMe-nucleotide

<400> 70

<210> 71

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of Artificial Sequence: Synthetic Oligonucleotides

<220>

<223> Description of combined DNA/RNA molecules: synthetic oligonucleotides

<220>

<221> Modified _ base

<222> (20)..(21)

<223> a, c, t, g, u, unknown or other

<400> 71

<210> 72

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of Artificial Sequence: Synthetic Oligonucleotides

<220>

<223> Description of combined DNA/RNA molecules: synthetic oligonucleotides

<220>

<221> Modified _ base

<222> (2)..(2)

<223> 2'-OMe-nucleotide

<220>

<221> Modified _ base

<222> (7)..(7)

<223> 2'-OMe-nucleotide

<220>

<221> Modified _ base

<222> (11)..(11)

<223> 2'-OMe-nucleotide

<220>

<221> Modified _ base

<222> (20)..(21)

<223> 2'-OMe-nucleotide

<400> 72

<210> 73

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of Artificial Sequence: Synthetic Oligonucleotides

<220>

<223> Description of combined DNA/RNA molecules: synthetic oligonucleotides

<220>

<221> Modified _ base

<222> (2)..(2)

<223> 2'-OMe-nucleotide

<220>

<221> Modified _ base

<222> (7)..(7)

<223> 2'-OMe-nucleotide

<220>

<221> Modified _ base

<222> (11)..(11)

<223> 2'-OMe-nucleotide

<220>

<221> Modified _ base

<222> (20)..(21)

<223> 2'-OMe-nucleotide

<400> 73

<210> 74

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of Artificial Sequence: Synthetic Oligonucleotides

<220>

<223> Description of combined DNA/RNA molecules: synthetic oligonucleotides

<220>

<221> Modified _ base

<222> (2)..(2)

<223> 2'-OMe-nucleotide

<220>

<221> Modified _ base

<222> (20)..(21)

<223> 2'-OMe-nucleotide

<400> 74

<210> 75

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of Artificial Sequence: Synthetic Oligonucleotides

<220>

<223> Description of combined DNA/RNA molecules: synthetic oligonucleotides

<220>

<221> Modified _ base

<222> (2)..(2)

<223> 2'-OMe-nucleotide

<220>

<221> Modified _ base

<222> (20)..(21)

<223> 2'-OMe-nucleotide

<400> 75

<210> 76

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of Artificial Sequence: Synthetic Oligonucleotides

<220>

<223> Description of combined DNA/RNA molecules: synthetic oligonucleotides

<220>

<221> Modified _ base

<222> (7)..(7)

<223> 2'-deoxy-nucleotide

<220>

<221> Modified _ base

<222> (20)..(21)

<223> 2'-OMe-nucleotide

<400> 76

<210> 77

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of Artificial Sequence: Synthetic Oligonucleotides

<220>

<223> Description of combined DNA/RNA molecules: synthetic oligonucleotides

<220>

<221> Modified _ base

<222> (7)..(7)

<223> 2'-deoxy-nucleotide

<220>

<221> Modified _ base

<222> (20)..(21)

<223> 2'-OMe-nucleotide

<400> 77

<210> 78

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of Artificial Sequence: Synthetic Oligonucleotides

<220>

<223> Description of combined DNA/RNA molecules: synthetic oligonucleotides

<220>

<221> Modified _ base

<222> (1)..(1)

<223> 2'-deoxy-nucleotide

<220>

<221> Modified _ base

<222> (7)..(7)

<223> 2'-deoxy-nucleotide

<220>

<221> Modified _ base

<222> (20)..(21)

<223> 2'-OMe-nucleotide

<400> 78

<210> 79

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of Artificial Sequence: Synthetic Oligonucleotides

<220>

<223> Description of combined DNA/RNA molecules: synthetic oligonucleotides

<220>

<221> Modified _ base

<222> (7)..(7)

<223> 2'-deoxy-nucleotide

<220>

<221> Modified _ base

<222> (20)..(21)

<223> 2'-OMe-nucleotide

<400> 79

<210> 80

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of Artificial Sequence: Synthetic Oligonucleotides

<220>

<223> Description of combined DNA/RNA molecules: synthetic oligonucleotides

<220>

<221> Modified _ base

<222> (20)..(21)

<223> 2'-OMe-nucleotide

<400> 80

<210> 81

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Description of Artificial Sequence: Synthetic Oligonucleotides

<220>

<221> Modified _ base

<222> (2)..(2)

<223> 2'-OMe-nucleotide

<220>

<221> Modified _ base

<222> (7)..(7)

<223> 2'-OMe-nucleotide

<220>

<221> Modified _ base

<222> (11)..(11)

<223> 2'-OMe-nucleotide

<220>

<221> Modified _ base

<222> (20)..(21)

<223> 2'-OMe-nucleotide

<400> 81

<210> 82

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Description of Artificial Sequence: Synthetic Oligonucleotides

<220>

<221> Modified _ base

<222> (20)..(21)

<223> 2'-OMe-nucleotide

<400> 82

<210> 83

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Description of Artificial Sequence: Synthetic Oligonucleotides

<220>

<221> Modified _ base

<222> (20)..(21)

<223> 2'-OMe-nucleotide

<400> 83

<210> 84

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Description of Artificial Sequence: Synthetic Oligonucleotides

<220>

<221> Modified _ base

<222> (20)..(21)

<223> 2'-OMe-nucleotide

<400> 84

<210> 85

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Description of Artificial Sequence: Synthetic Oligonucleotides

<400> 85

<210> 86

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of Artificial Sequence: Synthetic Oligonucleotides

<220>

<223> Description of combined DNA/RNA molecules: synthetic oligonucleotides

<400> 86

<210> 87

<211> 25

<212> RNA

<213> Artificial sequence

<220>

<223> Description of Artificial Sequence: Synthetic Oligonucleotides

<400> 87

<210> 88

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of Artificial Sequence: Synthetic Oligonucleotides

<220>

<223> Description of combined DNA/RNA molecules: synthetic oligonucleotides

<400> 88

<210> 89

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of Artificial Sequence: Synthetic Oligonucleotides

<220>

<223> Description of combined DNA/RNA molecules: synthetic oligonucleotides

<400> 89

<210> 90

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of Artificial Sequence: Synthetic Oligonucleotides

<220>

<223> Description of combined DNA/RNA molecules: synthetic oligonucleotides

<400> 90

<210> 91

<211> 25

<212> RNA

<213> Artificial sequence

<220>

<223> Description of Artificial Sequence: Synthetic Oligonucleotides

<400> 91

<210> 92

<211> 25

<212> RNA

<213> Artificial sequence

<220>

<223> Description of Artificial Sequence: Synthetic Oligonucleotides

<400> 92

<210> 93

<211> 25

<212> RNA

<213> Artificial sequence

<220>

<223> Description of Artificial Sequence: Synthetic Oligonucleotides

<400> 93

<210> 94

<211> 26

<212> DNA

<213> Artificial sequence

<220>

<223> Description of Artificial Sequence: Synthetic Oligonucleotides

<220>

<223> Description of combined DNA/RNA molecules: synthetic oligonucleotides

<400> 94

<210> 95

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> Description of Artificial Sequence: Synthetic Oligonucleotides

<400> 95

<210> 96

<211> 33

<212> DNA

<213> Artificial sequence

<220>

<223> Description of Artificial Sequence: Synthetic Oligonucleotides

<220>

<223> Description of combined DNA/RNA molecules: synthetic oligonucleotides

<400> 96

<210> 97

<211> 26

<212> DNA

<213> Artificial sequence

<220>

<223> Description of Artificial Sequence: Synthetic Oligonucleotides

<220>

<223> Description of combined DNA/RNA molecules: synthetic oligonucleotides

<400> 97

<210> 98

<211> 29

<212> DNA

<213> Artificial sequence

<220>

<223> Description of Artificial Sequence: Synthetic Oligonucleotides

<220>

<223> Description of combined DNA/RNA molecules: synthetic oligonucleotides

<400> 98

<210> 99

<211> 29

<212> DNA

<213> Artificial sequence

<220>

<223> Description of Artificial Sequence: Synthetic Oligonucleotides

<220>

<223> Description of combined DNA/RNA molecules: synthetic oligonucleotides

<400> 99

<210> 100

<211> 33

<212> DNA

<213> Artificial sequence

<220>

<223> Description of Artificial Sequence: Synthetic Oligonucleotides

<220>

<223> Description of combined DNA/RNA molecules: synthetic oligonucleotides

<400> 100

<210> 101

<211> 37

<212> DNA

<213> Artificial sequence

<220>

<223> Description of Artificial Sequence: Synthetic Oligonucleotides

<220>

<223> Description of combined DNA/RNA molecules: synthetic oligonucleotides

<400> 101

<210> 102

<211> 37

<212> DNA

<213> Artificial sequence

<220>

<223> Description of Artificial Sequence: Synthetic Oligonucleotides

<220>

<223> Description of combined DNA/RNA molecules: synthetic oligonucleotides

<400> 102

<210> 103

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of Artificial Sequence: Synthetic Oligonucleotides

<220>

<223> Description of combined DNA/RNA molecules: synthetic oligonucleotides

<400> 103

<210> 104

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of Artificial Sequence: Synthetic Oligonucleotides

<220>

<223> Description of combined DNA/RNA molecules: synthetic oligonucleotides

<400> 104

<210> 105

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of Artificial Sequence: Synthetic Oligonucleotides

<220>

<223> Description of combined DNA/RNA molecules: synthetic oligonucleotides

<400> 105

<210> 106

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Description of Artificial Sequence: Synthetic Oligonucleotides

<400> 106

<210> 107

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of Artificial Sequence: Synthetic Oligonucleotides

<220>

<223> Description of combined DNA/RNA molecules: synthetic oligonucleotides

<400> 107

<210> 108

<211> 27

<212> RNA

<213> Artificial sequence

<220>

<223> Description of Artificial Sequence: Synthetic Oligonucleotides

<400> 108

<210> 109

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of Artificial Sequence: Synthetic Oligonucleotides

<220>

<223> Description of combined DNA/RNA molecules: synthetic oligonucleotides

<400> 109

<210> 110

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of Artificial Sequence: Synthetic Oligonucleotides

<220>

<223> Description of combined DNA/RNA molecules: synthetic oligonucleotides

<400> 110

<210> 111

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of Artificial Sequence: Synthetic Oligonucleotides

<220>

<223> Description of combined DNA/RNA molecules: synthetic oligonucleotides

<400> 111

<210> 112

<211> 27

<212> RNA

<213> Artificial sequence

<220>

<223> Description of Artificial Sequence: Synthetic Oligonucleotides

<400> 112

<210> 113

<211> 27

<212> RNA

<213> Artificial sequence

<220>

<223> Description of Artificial Sequence: Synthetic Oligonucleotides

<400> 113

<210> 114

<211> 27

<212> RNA

<213> Artificial sequence

<220>

<223> Description of Artificial Sequence: Synthetic Oligonucleotides

<400> 114

<210> 115

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of Artificial Sequence: Synthetic Oligonucleotides

<220>

<223> Description of combined DNA/RNA molecules: synthetic oligonucleotides

<400> 115

<210> 116

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of Artificial Sequence: Synthetic Oligonucleotides

<220>

<223> Description of combined DNA/RNA molecules: synthetic oligonucleotides

<400> 116

<210> 117

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of Artificial Sequence: Synthetic Oligonucleotides

<220>

<223> Description of combined DNA/RNA molecules: synthetic oligonucleotides

<400> 117

<210> 118

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of Artificial Sequence: Synthetic Oligonucleotides

<220>

<223> Description of combined DNA/RNA molecules: synthetic oligonucleotides

<400> 118

<210> 119

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of Artificial Sequence: Synthetic Oligonucleotides

<220>

<223> Description of combined DNA/RNA molecules: synthetic oligonucleotides

<400> 119

<210> 120

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of Artificial Sequence: Synthetic Oligonucleotides

<220>

<223> Description of combined DNA/RNA molecules: synthetic oligonucleotides

<400> 120

<210> 121

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of Artificial Sequence: Synthetic Oligonucleotides

<220>

<223> Description of combined DNA/RNA molecules: synthetic oligonucleotides

<400> 121

<210> 122

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of Artificial Sequence: Synthetic Oligonucleotides

<220>

<223> Description of combined DNA/RNA molecules: synthetic oligonucleotides

<400> 122

<210> 123

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of Artificial Sequence: Synthetic Oligonucleotides

<220>

<223> Description of combined DNA/RNA molecules: synthetic oligonucleotides

<400> 123

<210> 124

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of Artificial Sequence: Synthetic Oligonucleotides

<220>

<223> Description of combined DNA/RNA molecules: synthetic oligonucleotides

<400> 124

<210> 125

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of Artificial Sequence: Synthetic Oligonucleotides

<220>

<223> Description of combined DNA/RNA molecules: synthetic oligonucleotides

<400> 125

<210> 126

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Description of Artificial Sequence: Synthetic Oligonucleotides

<220>

<223> Description of combined DNA/RNA molecules: synthetic oligonucleotides

<400> 126

<210> 127

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> Description of Artificial Sequence: Synthetic Oligonucleotides

<220>

<223> 5'-direction 1,2-dideoxy-D-ribose

<220>

<221> misc_ characteristics

<222> (15)..(19)

<223> 2'-5' linkage between nucleotides

<220>

<223> 3'-C3 spacer

<400> 127

<210> 128

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> Description of Artificial Sequence: Synthetic Oligonucleotides

<220>

<221> Modified _ base

<222> (2)..(2)

<223> 2'-O-methyl ribonucleotides

<220>

<221> Modified _ base

<222> (4)..(4)

<223> 2'-O-methyl ribonucleotides

<220>

<221> Modified _ base

<222> (6)..(6)

<223> 2'-O-methyl ribonucleotides

<220>

<221> misc_ characteristics

<222> (7)..(8)

<223> 2'-5' linkage between nucleotides

<220>

<221> Modified _ base

<222> (8)..(8)

<223> 2'-O-methyl ribonucleotides

<220>

<221> Modified _ base

<222> (11)..(11)

<223> 2'-O-methyl ribonucleotides

<220>

<221> Modified _ base

<222> (13)..(13)

<223> 2'-O-methyl ribonucleotides

<220>

<221> Modified _ base

<222> (15)..(15)

<223> 2'-O-methyl ribonucleotides

<220>

<221> Modified _ base

<222> (17)..(17)

<223> 2'-O-methyl ribonucleotides

<220>

<221> Modified _ base

<222> (19)..(19)

<223> 2'-O-methyl ribonucleotides

<220>

<223> 3'-C3-C3 spacer

<400> 128

<210> 129

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> Description of Artificial Sequence: Synthetic Oligonucleotides

<220>

<223> 5'-direction 1,2-dideoxy-D-ribose

<220>

<221> Modified _ base

<222> (4)..(4)

<223> 2'-O-methyl ribonucleotides

<220>

<221> misc_ characteristics

<222> (9)..(10)

<223> 2'-5' linkage between nucleotides

<220>

<221> Modified _ base

<222> (11)..(11)

<223> 2'-O-methyl ribonucleotides

<220>

<221> Modified _ base

<222> (13)..(13)

<223> 2'-O-methyl ribonucleotides

<220>

<221> Modified _ base

<222> (17)..(17)

<223> 2'-O-methyl ribonucleotides

<220>

<223> 3'-C3 spacer

<400> 129

<210> 130

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> Description of Artificial Sequence: Synthetic Oligonucleotides

<220>

<221> Modified _ base

<222> (1)..(1)

<223> 2'-O-methyl ribonucleotides

<220>

<221> Modified _ base

<222> (4)..(4)

<223> 2'-O-methyl ribonucleotides

<220>

<221> misc_ characteristics

<222> (6)..(7)

<223> 2'-5' linkage between nucleotides

<220>

<221> Modified _ base

<222> (8)..(8)

<223> 2'-O-methyl ribonucleotides

<220>

<221> Modified _ base

<222> (11)..(11)

<223> 2'-O-methyl ribonucleotides

<220>

<221> Modified _ base

<222> (15)..(15)

<223> 2'-O-methyl ribonucleotides

<220>

<223> 3'-C3-C3 spacer

<400> 130

<210> 131

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> Description of Artificial Sequence: Synthetic Oligonucleotides

<220>

<223> 5'-direction 1,2-dideoxy-D-ribose

<220>

<221> misc_ characteristics

<222> (15)..(19)

<223> 2'-5' linkage between nucleotides

<220>

<223> 3'-C3-spacer-phosphoride

<400> 131

<210> 132

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> Description of Artificial Sequence: Synthetic Oligonucleotides

<220>

<221> Modified _ base

<222> (1)..(1)

<223> 2'-O-methyl ribonucleotides

<220>

<221> Modified _ base

<222> (3)..(3)

<223> 2'-O-methyl ribonucleotides

<220>

<221> Modified _ base

<222> (5)..(5)

<223> 2'-O-methyl ribonucleotides

<220>

<221> misc_ characteristics

<222> (7)..(8)

<223> 2'-5' linkage between nucleotides

<220>

<221> Modified _ base

<222> (9)..(9)

<223> 2'-O-methyl ribonucleotides

<220>

<221> Modified _ base

<222> (11)..(11)

<223> 2'-O-methyl ribonucleotides

<220>

<221> Modified _ base

<222> (13)..(13)

<223> 2'-O-methyl ribonucleotides

<220>

<221> Modified _ base

<222> (15)..(15)

<223> 2'-O-methyl ribonucleotides

<220>

<221> Modified _ base

<222> (17)..(17)

<223> 2'-O-methyl ribonucleotides

<220>

<221> Modified _ base

<222> (19)..(19)

<223> 2'-O-methyl ribonucleotides

<220>

<223> 3'-C3-C3- spacer

<400> 132

<210> 133

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> Description of Artificial Sequence: Synthetic Oligonucleotides

<220>

<223> 5'-direction 1,2-dideoxy-D-ribose

<220>

<221> Modified _ base

<222> (2)..(3)

<223> 2'-O-methyl ribonucleotides

<220>

<221> Modified _ base

<222> (5)..(5)

<223> 2'-O-methyl ribonucleotides

<220>

<221> Modified _ base

<222> (7)..(8)

<223> 2'-O-methyl ribonucleotides

<220>

<221> misc_ characteristics

<222> (9)..(10)

<223> 2'-5' linkage between nucleotides

<220>

<221> Modified _ base

<222> (12)..(12)

<223> 2'-O-methyl ribonucleotides

<220>

<221> Modified _ base

<222> (14)..(15)

<223> 2'-O-methyl ribonucleotides

<220>

<221> Modified _ base

<222> (17)..(17)

<223> 2'-O-methyl ribonucleotides

<220>

<223> 3'-C3-spacer

<400> 133

<210> 134

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> Description of Artificial Sequence: Synthetic Oligonucleotides

<220>

<221> Modified _ base

<222> (2)..(2)

<223> 2'-O-methyl ribonucleotides

<220>

<221> misc_ characteristics

<222> (6)..(7)

<223> 2'-5' linkage between nucleotides

<220>

<221> Modified _ base

<222> (7)..(7)

<223> 2'-O-methyl ribonucleotides

<220>

<221> Modified _ base

<222> (11)..(11)

<223> 2'-O-methyl ribonucleotides

<220>

<221> Modified _ base

<222> (16)..(16)

<223> 2'-O-methyl ribonucleotides

<220>

<223> 3'-C3-C3- spacer

<400> 134

Claims (77)

A pharmaceutical composition for treating a malignant tumor, the composition comprising a nanoparticle encapsulating an RNAi molecule, wherein the RNAi molecule targets Hsp47. The pharmaceutical composition of claim 1, wherein the composition retains at least 80% of the activity of the encapsulated RNAi molecule after exposure to human serum for 1 hour. The pharmaceutical composition of claim 1, wherein the RNAi molecule for treating the malignant tumor is siRNA or shRNA. The pharmaceutical composition of claim 1, wherein each RNAi molecule comprises a double-stranded region, wherein the double-stranded region comprises a nucleotide sequence corresponding to a target sequence of Hsp47 mRNA. A method for dispensing an active agent to an individual for treating a malignant tumor, the method comprising administering to the individual a pharmaceutical composition as claimed in claim 1. A pharmaceutical composition for treating a malignant tumor, the composition comprising a nanoparticle encapsulating an RNAi molecule, wherein one of the RNAi molecules partially targets Hsp47 and one of the RNAi molecules is partially targeted to p21. The pharmaceutical composition of claim 6, wherein the composition retains at least 80% of the activity of the encapsulated RNAi molecule after exposure to human serum for 1 hour. The pharmaceutical composition of claim 6, wherein the RNAi molecule for treating the malignant tumor is siRNA or shRNA. For example, the pharmaceutical composition of claim 6 of the patent scope, wherein There is a portion of an RNAi molecule that is capable of reducing the activity of Hsp47, each RNAi molecule comprising a double-stranded region, wherein the double-stranded region comprises a nucleotide sequence corresponding to a target sequence of Hsp47 mRNA. The pharmaceutical composition of claim 6, wherein each of the RNAi molecules comprises a double-stranded region, wherein the double-stranded region comprises a nucleoside corresponding to a target sequence of p21 mRNA, in a portion of the RNAi molecule capable of reducing p21 expression Acid sequence. A method for dispensing an active agent to an individual for the treatment of a malignant tumor, the method comprising administering to the individual a pharmaceutical composition as claimed in claim 6 of the patent. The method of claim 11, wherein the malignant tumor is located in the lung, colon, pancreas, liver, heart, bone, skin or small intestine. The method of claim 11, wherein the RNAi molecule is administered at a dose of 0.01 to 2 mg/kg, administered at least once a day for up to 12 weeks. The method of claim 11, wherein the administration provides an average AUC (0-final) of the Hsp47 RNAi molecule of from 1 to 1000 ug*min/mL and an average _{Cmax of} from 0.1 to 50 μg/mL. A method for preventing, treating or ameliorating one or more symptoms of a malignant tumor in a mammal in need thereof, the method comprising administering to the mammal a therapeutically effective amount of a composition comprising an RNAi molecule having activity to reduce Hsp47 expression Things. The method of claim 15, wherein the mammal For human and the Hsp47 is human Hsp47. The method of claim 15, wherein the malignant tumor overexpresses Hsp47. The method of claim 15, wherein the RNAi molecule reduces the expression of Hsp47 in the mammal. The method of claim 15, wherein the administering reduces the performance of Hsp47 in the mammal by at least 5% for at least 5 days. The method of claim 15, wherein the administering reduces the volume of the malignant tumor of the mammal by at least 5%, or at least 10%, or at least 20%, or at least 30%, or at least 40% or at least 50. %. The method of claim 15, wherein the method reduces one or more symptoms of the malignant tumor, or delays or terminates the progression of the malignant tumor. The method of claim 15, wherein the administering reduces the growth of malignant cells of the individual. The method of claim 15, wherein the administering reduces the growth of the malignant cells of the individual by at least 2%, or at least 5%, or at least 10%, or at least 15% or at least 20%. The method of claim 15, wherein the tumor cell overexpresses wild-type Hsp47 RNA or protein. The method of claim 15, wherein the tumor is a sarcoma selected from the group consisting of lung adenocarcinoma, mucinous adenoma, ductal carcinoma of the pancreas, and colorectal cancer. The method of claim 15, wherein the malignant tumor is a sarcoma selected from the group consisting of lung adenocarcinoma, mucinous adenoma, ductal carcinoma of the pancreas, colorectal cancer, breast cancer, and fibrosarcoma. The method of claim 15, wherein the malignant tumor is located in an anatomical region selected from the group consisting of lung, liver, pancreas, colon, kidney, heart, bone, skin, small intestine and joint, and the like Any combination. The method of claim 15, wherein the administration is performed 1 to 12 times a day. The method of claim 15, wherein the administration is continued for 1, 2, 3, 4, 5, 6 or 7 days. The method of claim 15, wherein the administration is continued for 1, 2, 3, 4, 5, 6, 8, 10 or 12 weeks. The method of claim 15, wherein the RNAi molecule is administered at a dose of 0.01 to 2 mg/kg, administered at least once a day for up to 12 weeks. The method of claim 15, wherein the administration provides an average AUC (0-final) of the Hsp47 RNAi molecule of from 1 to 1000 ug*min/mL and an average _{Cmax of} from 0.1 to 50 μg/mL. The method of claim 15, wherein the administration is intravenous injection, intradermal injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, oral, topical, drip or inhalation. A method for preventing, treating or ameliorating one or more symptoms of a malignant tumor in a mammal in need thereof, the method comprising administering the feeding A therapeutically effective amount of a composition comprising an RNAi molecule, wherein one portion of the RNAi molecule has activity to reduce Hsp47 expression and one portion of the RNAi molecule has activity to reduce p21 expression. The method of claim 34, wherein the mammal is human and the Hsp47 is human Hsp47. The method of claim 34, wherein the malignant tumor overexpresses Hsp47. The method of claim 34, wherein the RNAi molecule reduces the expression of Hsp47 and p21 in the mammal. The method of claim 34, wherein the administering reduces the performance of Hsp47 and p21 of the mammal by at least 5% for at least 5 days. The method of claim 34, wherein the administering reduces the volume of the malignant tumor of the mammal by at least 5%, or at least 10%, or at least 20%, or at least 30%, or at least 40% or at least 50. %. The method of claim 34, wherein the method reduces one or more symptoms of the malignant tumor, or delays or terminates the progression of the malignant tumor. The method of claim 34, wherein the administering reduces the growth of malignant cells of the individual. The method of claim 34, wherein the administering reduces the growth of the malignant cells of the individual by at least 2%, or at least 5%, or at least 10%, or at least 15% or at least 20%. The method of claim 34, wherein the tumor cell Excessive expression of wild-type Hsp47 RNA or protein. The method of claim 34, wherein the tumor is a sarcoma selected from the group consisting of lung adenocarcinoma, mucinous adenoma, ductal carcinoma of the pancreas, and colorectal cancer. The method of claim 34, wherein the malignant tumor is a sarcoma selected from the group consisting of lung adenocarcinoma, mucinous adenoma, ductal carcinoma of the pancreas, colorectal cancer, breast cancer, and fibrosarcoma. The method of claim 34, wherein the malignant tumor is located in an anatomical region selected from the group consisting of lung, liver, pancreas, colon, kidney, heart, bone, skin, small intestine and joint, and the like Any combination. The method of claim 34, wherein the administration is performed 1 to 12 times a day. The method of claim 34, wherein the administration is continued for 1, 2, 3, 4, 5, 6 or 7 days. The method of claim 34, wherein the administration is continued for 1, 2, 3, 4, 5, 6, 8, 10 or 12 weeks. The method of claim 34, wherein the RNAi molecule is administered at a dose of 0.01 to 2 mg/kg, administered at least once a day for up to 12 weeks. The method of claim 34, wherein the administration of the Hsp47 RNAi molecule provides an average AUC (0-final) of from 1 to 1000 ug*min/mL and an average _{Cmax of} from 0.1 to 50 [mu]g/mL. For example, the method of claim 34, wherein the administration is static Intrapulmonary injection, intradermal injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, oral, topical, instillation or inhalation. A method for reducing the growth rate or proliferation of a cancer stem cell of a mammal in need thereof, the method comprising administering to the mammal a therapeutically effective amount of a composition comprising an RNAi molecule, wherein a portion of the RNAi molecule is capable of reducing Hsp47 The activity is expressed and a portion of the RNAi molecule has an activity that reduces p21 expression. The method of claim 53, wherein the mammal is human and the Hsp47 is human Hsp47. The method of claim 53, wherein the RNAi molecule reduces the expression of Hsp47 and p21 in the mammal. The method of claim 53, wherein the administering reduces the Hsp47 and p21 performance of the mammal by at least 5% for at least 5 days. The method of claim 53, wherein the administering reduces the volume of the malignant tumor of the mammal by at least 5%, or at least 10%, or at least 20%, or at least 30%, or at least 40% or at least 50. %. The method of claim 53, wherein the administering reduces the growth of the cancer stem cells of the individual by at least 2%, or at least 5%, or at least 10%, or at least 15% or at least 20%. The method of claim 53, wherein the cancer cell overexpresses wild-type Hsp47 RNA or protein. The method of claim 53, wherein the cancer cell is in a sarcoma selected from the group consisting of lung adenocarcinoma, mucinous gland Tumor, pancreatic ductal cancer and colorectal cancer. The method of claim 53, wherein the cancer cell is in a sarcoma selected from the group consisting of lung adenocarcinoma, mucinous adenoma, ductal carcinoma of the pancreas, colorectal cancer, breast cancer, and fibrosarcoma. The method of claim 53, wherein the cancer cell line is located in an anatomical region selected from the group consisting of lung, liver, pancreas, colon, kidney, heart, bone marrow, skin, small intestine, eyes, and any of them. combination. The method of claim 53, wherein the administration is performed 1 to 12 times a day. The method of claim 53, wherein the administration is continued for 1, 2, 3, 4, 5, 6 or 7 days. The method of claim 53, wherein the administration is continued for 1, 2, 3, 4, 5, 6, 8, 10 or 12 weeks. The method of claim 53, wherein the administering is at least one 0.01 to 2 mg/kg of the RNAi molecular dose per day for up to 12 weeks. The method of claim 53, wherein the administration provides an average AUC (0-final) of the Hsp47 RNAi molecule of from 1 to 1000 ug*min/mL and an average _{Cmax of} from 0.1 to 50 [mu]g/mL. The method of claim 53, wherein the administration is intravenous, intradermal, subcutaneous, intramuscular, intraperitoneal, oral, topical, instillation or inhalation. A composition for treating a malignant tumor of an individual, wherein the The composition comprises liposomal nanoparticle, which encapsulates an RNAi molecule that targets Hsp47. The composition of claim 69, wherein the malignant tumor is located in the lung, colon or pancreas. The composition of claim 69, wherein the malignant tumor is located in the liver, heart, bone, skin or small intestine. The composition of claim 69, wherein the composition comprises liposome nanoparticles having a size of 10 to 1000 nm. The composition of claim 69, wherein the composition comprises liposome nanoparticles having a size of 10 to 150 nm. A composition according to claim 69, wherein one of the RNAi molecules is partially targeted to Hsp47 and one of the RNAi molecules is partially targeted to p21. The composition of claim 74, wherein the liposomal nanoparticle retains at least 80% of the encapsulated RNAi molecule after exposure to human serum for 1 hour. A method for dispensing an active agent to an organ of an individual for treating a malignant tumor, the method comprising administering to the individual a composition as claimed in claim 69. The method of claim 76, wherein the malignant tumor is located in the lung, colon, kidney, pancreas, liver, bone marrow, skin, eyes or small intestine.