TW201023898A - Use of a truncated eIF-5A1 polynucleotide to induce apoptosis in cancer cells - Google Patents

Abstract

The present invention relates to the combinatorial use of an siRNA targeted against an endogenous gene to knock out or knock down expression of the endogenous gene in a host and a delivery of a polynucleotide encoding the gene in a delivery vehicle/expression vector to the host to provide expression in the host of the protein encoded by the polynucleotide. The present invention also relates to a truncated form of eIF-5A1 that is useful for inducing apoptosis and killing of cells, especially cancer cells in a mammal.

Description

201023898 VI. INSTRUCTIONS OF THE INVENTION: FIELD OF THE INVENTION The present invention provides an isolated polynucleotide encoding a truncated form of eIF-5Al and a truncated eIF-5Al multipeptide. The truncated eIF-5Al polynucleotide is useful for inducing apoptosis and killing cancer cells in one body. This application claims the priority of U.S. Provisional Application No. 61/093,749, filed on Sep. 3, 2008, and the priority of U.S. Application Serial No. 12/400,742, filed on March 19, 2009. The content is incorporated by reference. [Prior Art] Quite recently, researchers have observed that double-stranded RNA ("dsRNA") can be used to inhibit protein expression. This ability to inhibit a gene has broad potential to treat human diseases, and many researchers and commercial entities are investing considerable resources in developing treatments based on this technology. Gene suppression induced by double-stranded RNA can occur in at least three levels: # (1) transcription deactivation, which means RNA-directed DNA or histone methylation; (ii) siRNA-induced mRNA degradation; and (iii) mRNA-induced transcriptional weakening. The main mechanism by which RNA-induced inhibition (RNA interference, or RNAi) is generally thought to be in mammalian cells is the degradation of mRNA. In mammalian cells, the initial use of RNAi focused on the use of long-chain dsRNA. However, these attempts to induce RNAi have been limited in success, in part because of the induction of interferon responses, which have led to a general inhibition of protein synthesis that targets the target. Therefore, the use of long-chain dsRNA for RNAi is not a viable option in mammalian systems.

More recently, it has been shown that when a short (18-30 bp) RNA duplex is introduced into a mammalian cell in culture, sequence-specific inhibition of the target mRNA can be achieved without inducing an interferon reaction. Certain dsRNAs in these short dsRNAs, such as small fragments of inhibitory RNA ("siRNAs"), can act catalytically at sub-molecular concentrations to cleave greater than 95% of the target mRNA in the cell. The mechanism of siRNA activity and some applications thereof are described in Provost et al. (2002) Ribonuclease Activity and RNA Binding of Recombinant Human Dicer, EMBO J. 21(21): 5864-5874; Tabara et al. (2002) ) The dsRNA

Binding protein RDE-4 Interacts with RDE-1, DCR-1 and a DexH-box Helicase to Direct RNAi in C. elegans, Cell 109(7):861-71 ; Ketting et al. (2002) Dicer Functions in RNA Interference And in Synthesis of Small RNA Involved in Developmental Timing in C. elegans ; Martinez et al.,

Single-Stranded Antisense siRNAs Guide Target RNA Cleavage in RNAi, Cell 110(5):563; Hutvagner & Zamore (2002) A microRNA in a multiple-turnover RNAi enzyme complex,Science 297:2056 ° From a mechanical point of view, Long double-stranded RNA is introduced into plants and invertebrate cells and is fragmented into siRNA by the action of a type III endonuclease. The type 3 endonuclease is known as Dicer (Sharp, RNA interference--2001, Genes Dev. 2001, 15:485) ° 142866.doc -4 - 201023898

Dicer, a type 3 ribonuclease-like enzyme, processes dsRNA into short-segment interfering RNAs of 19-23 base pairs, characterized by a 2 base 3· overhang. Bernstein, Caudy, Hammond, & Hannon (2001) Role for a bidentate ribonuclease in the initiation step of RNA interference, Nature 409:363. The siRNAs then bind to an RNA-induced inhibitory complex (RISC) in which one or more helicases unwind the siRNA duplex, allowing the complementary antisense strand to direct the identification of the stem. Nykanen, Haley, & Zamore (2001), ATP ❹ requirements and small interfering RNA structure in the RNA interference pathway, Cell 107:309. Upon binding to the appropriate target mRNA, one or more endonucleases in the RISC cleave the target, thereby inducing inhibition. Elbashir, Lendeckel, & Tuschl (2001) RNA interference is mediated by 21 - and 22-nucleotide RNAs, Genes Dev. 15:188, Figure 1. This interference potency is long-lasting and can be detected after many cell divisions. In addition, RNAi exhibits sequence specificity. Kisielow, M. et al. w (2002), Isoform-specific knockdown and expression of adaptor protein ShcA using small interfering RNA, J. Biochem. 363:1-5 o Therefore, the RNAi mechanism can specifically reduce one type of transcription. And not ψ> affects closely related related mRNA. These properties make siRNA a potential and useful tool for suppressing gene expression and studying gene function and drug target confirmation. In addition, siRNAs have the potential to be used in medical assays to combat (1) diseases caused by excessive or misrepresentation of genes; and (2) diseases caused by the expression of genes comprising mutations. 142866.doc 201023898 The successful gene suppression of siRNA relies on many factors. One of the most controversial issues in RNAi is the question of the necessity of siRNA design, which is to consider the siRNA sequences used. In early nematode and plant studies, long-chain dsRNA was introduced to circumvent this problem (see, for example, Fire, a. et al. (1998) Nature 391: 806-811). In this simple organism, Dicer cleaves long-chain dsRNA molecules into siRNAs, thus creating a multi-variant dimeric population that strongly covers all transcription. When some fragments of these molecules are not functional (i.e., induce little or no inhibition), one or more will have a high degree of functional potential, thereby inhibiting the gene of interest and reducing the need for siRNA design. Unfortunately, this same method of interferon response is not feasible for mammalian systems. When this effect can be circumvented by bypassing the Dicer's cleavage step and introducing siRNA directly, this approach may carry a non-functional or semi-functional risk to the selected siRNA sequence. Many studies have published the idea that siRNA design is not a decisive element of RNAi. On the other hand, others in the field have begun to explore the possibility of creating more efficient RNAi by focusing on the design of siRNA. In order to treat different diseases or disorders, the upward regulation of certain proteins is desirable, but this may not be all that is needed. For example, a combination of siRNAs may be required to reduce or eliminate the expression of an endogenous protein or a different protein. The present invention meets this need and provides a method of treating cancer, particularly multiple bone tumors. Cancer, including multiple myeloma, is a disease that benefits the ability to induce apoptosis. Traditional medical treatment for multiple myeloma includes chemotherapy, stem cell migration, high-dose chemotherapy for stem cell transplantation, and salvage surgery. Chemotherapy includes thalidomide® (thalidomide), bortezomib, ardidia® (pamidronate), steroids And Zometa® (zoledronic acid). However, many chemotherapeutic drugs are toxic to active dividing non-cancerous cells, such as the bone marrow, stomach wall, intestinal wall, and hair follicles. Therefore, chemotherapy can lead to a decrease in the number of blood cells, nausea, vomiting, diarrhea and hair loss. Traditional chemotherapy, or standard dose chemotherapy, is a typical primary or initial treatment for patients with multiple myeloma. Patients may also receive chemotherapy for high-dose chemotherapy or stem cell transplantation. Introduction therapy (traditional chemotherapy prior to stem cell transplantation) can be used prior to transplantation to reduce tumor weight. Some chemotherapy drugs are more suitable for introduction therapy than others because they are less toxic to the bone marrow and can cause higher amounts of stem cells from the bone marrow. Examples of chemotherapeutic drugs suitable for introduction therapy include dexamethasone, salivir/dexamethasone, VAD [vincristine, adrimycin® (doxorubicin) (Adriamycin® (Adriamycin® ( Doxorubicin)) and dexamethasone combination] and DVd [pegylated liposomal doxil® (Calylyx®), Changchun new test and reduction The combination of dexamethasone]. The standard treatment for multiple myeloma is a combination of melphala and prednisone (a corticosteroid) that achieves a 50% response rate. Unfortunately, mold flanges are a burning agent and are less suitable for introduction therapy. Corticosteroids (especially dexamethasone) are sometimes used alone in multiple myeloma treatments, especially in older patients or those who cannot tolerate chemotherapy 142866.doc 201023898. Dexamethasone is also used alone or in combination with other agents for introduction therapy. VAD is most commonly used for induction therapy, but recently it has been shown that DVd is effective in induction therapy. Recently approved for the treatment of multiple myeloma, but its toxicity is very high. However, none of the existing therapies offer a clear potential for cure. Therefore, there is still a need to find a suitable treatment for cancer and multiple osteosarcoma. The present invention meets this need. SUMMARY OF THE INVENTION The present invention provides an isolated polynucleotide encoding a truncated form of eIF-5Al and a truncated polypeptide. This truncated eIF 5A1 polynucleotide is useful in inducing apoptosis and killing cancer cells in one body. The truncated polynuclear acid can be used in a performance vector which is then administered to a body. This truncated form of eIF_5A is expressed in the mammal and kills cancer cells. The truncated eIF-5Al protein is about 16 kDA relative to about 17 full length eIF5Ai protein. A polynucleotide encoding a truncated eIF5Al protein can be used to produce an agent for inducing apoptosis in a cancer cell or a tumor of a body. The polynucleus (IV) encoding a truncated eIF5A1 protein can be used with other specific embodiments described herein (eg, in combination with a polynucleotide encoding a full length eIF5A) and a multicore encoding a full length mutation eIF5A The combination of glycosides and/or in combination with a siRNA targeting the 3, UTR of eIF5 A). The invention also relates to the use of a combination of a target for an endogenous gene for siRNA to eliminate or reduce one of the endogenous genes; see and to encode a gene with a delivery tool/expression vector Nucleotide delivery to the host' to provide for expression of the protein encoded by the multinuclear (tetra) in the host 142866.doc 201023898 to the host encoding a normal (non-defective) protein (or the protein itself) __Multinuclear #acid and exhibits the polynuclear (four) (in the case of the polynucleotide) 'making the normal protein perform its necessary function. Preferably, the siRNA is designed to target a region of the gene such that it reduces or eliminates the endogenous expression of the defective protein, but at the same time does not affect the endogenous expression of the polynucleotide to which the normal protein is administered. The present invention provides a composition comprising an eIF5Al siHNA complex targeting the eIF5A123i terminus, and a expression vector comprising a polynucleotide encoding a mutant eIF5A1, wherein the mutant eIF5A1 is incapable of being lysine by carboxyresinamine (hypusinated), and wherein the siRNA and the expression carrier are complexed to polyethyleneimine to form a complex. The present invention provides a composition comprising an siRNA targeting a target gene to inhibit endogenous expression of the target gene in a body, and a polynucleotide encoding a target protein, the target Protein can be expressed in this individual. In certain embodiments, the polynucleotide is in the plastid of an anti-RNAi (not inhibited by the siRNA). The siRNA and the plastid are preferably complexed to polyethyleneimine to form a complex. In certain embodiments, the siRNA has a sequence ' as shown in Figure 25 and wherein the polynucleoside encoding the mutant eIF5Al is eIF5A1K5〇R. The expression vector comprises a polynucleotide encoding a mutant eIF5 A1 and a promoter operably linked to provide expression of the polynucleotide in a single body. The promoter is preferably tissue specific or systemic. For example, if the composition is used to treat cancer, it is preferred that the promoter is tissue-specific for the tissue at which the cancer is located. For example, for the treatment of multiple bone marrow 142866.doc 201023898 tumors, preferably a B cell specific promoter such as B29. In certain embodiments, the expression vector comprises a pCpG plastid. In certain embodiments, the eIF5Al siRNA and the expression vector comprising the mutant eIF5Al polynucleotide are complexed by themselves to a polyethyleneimine, such as in vivo JetPEITM. In other specific embodiments, the eiF5Al siRNA and the expression vector comprising the mutant eiF5Al polynucleotide are combined together to a polyethyleneimine. The present invention further provides a composition comprising an eIF5Al siRNA targeting the 3' terminus of eIF5Al and a expression vector comprising a polynucleotide encoding a mutant eIF5Al, wherein the mutant eIF5Al is incapable of being carboxypyramine Acidified, and wherein the siRNA and the expression vector are delivered to a body to treat cancer. The cancer can be any cancer that contains multiple bone tumors. The invention further provides a method of treating cancer comprising administering a composition of the invention (including but not limited to mammals and humans); the composition can be administered via any acceptable route, for example, but not Limited to intravenous (IV), intraperitoneal (IP), subcutaneous or intratumoral (IT). The siRNA and the expression vector can be administered in different numbers or via different routes, or can be administered simultaneously via the same route. For example, but not limited to, the siRNA can be delivered naked or complexed to a vehicle such as in viv〇jetPEI via IV, and the expression vector can be administered in vivo, or the siRNA and the expression vector can be administered via IV or Tumors are administered in vivo, and so on. The present invention provides a method of inhibiting the growth of cancer cells and/or killing cancer cells. The invention also provides methods of inhibiting or slowing the ability of a cancer cell to metastasize. Inhibition of cancer growth includes a reduction in the size of the tumor, a decrease in the growth of the tumor 142866.doc •10-201023898 and may also include a complete remission of the tumor. The cancer can be any cancer or tumor including, but not limited to, colon cancer, colorectal adenocarcinoma, bladder cancer, cervical adenocarcinoma, and lung cancer. Preferably, the cancer is a multiple bone tumor. In a preferred embodiment, the eIF-5A is a mutation that is not lysylated by carboxyresin. Exemplary mutations are described herein. In addition to providing eIF-5A or a polynucleotide encoding eIF-5 A to a single body (to provide performance of the eIF-5A), the siRNA system provides endogenous performance to eliminate or reduce 611?-5A. The invention also provides for the use of eIF5A, a polynucleotide encoding eIF5Al, and an siRNA against eIF5Al to produce a single myeloma cell in an individual with one agent for treating cancer and killing one of multiple myeloma. Preferably, the polynucleotide encoding a mutant eIF_5A is not lysylated by carboxyresulin. The invention also provides a method of treating sickle-type anemia. A polynucleotide encoding a healthy heme gene (e.g., HBB) is administered to a patient having a sickle type poor gold. In cooperation, the patient is also administered an siRNA encoding the defective heme gene (e.g., a gene encoding the mutant HbS) to reduce or eliminate the performance of the defective protein. The treatment may further comprise administering other known drugs or treatments commonly used to treat sickle-type anemia. [Embodiment] The present invention provides an isolated polynucleic acid' that encodes a truncated form eIF_5A1 and a truncated eIF-ΑΙ multipeptide. The truncated eIF-5Al polynucleus 142866.doc -11 - 201023898 Glyceric acid is useful for inducing apoptosis and killing cancer cells. The truncated polypeptide can be used in a performance vector which is then administered to a mammal. This truncated form of eIF-5 A is expressed in the mammal and kills cancer cells. The truncated eIF-5Al protein is approximately 16 kDA relative to the full length, approximately 17 kDa of eIF-5Al protein. The formation of a smaller molecular weight form of eIF5 A (~16 kDa) has been observed in cells undergoing apoptosis. This has also been observed in beta cell island cells treated with a cytokine cocktail and human myeloma cells (KAS cells) treated with a cytotoxic drug (actinomycin D). See Figure 35. This smaller molecular weight form of elf5A has also been found to accumulate in apoptotic HeLa cervical cancer cells following infection with Ad-eIF5Al, suggesting that this smaller molecular weight form of eIF5A is derived from cleavage by protease rather than alternative splicing ( The result of alternative splicing). The accumulation of eIF5A in this smaller molecular form was observed to be accompanied by a dramatic decrease in the amount of full-length carboxypyramine lysine eiF5A, which more supports the smaller molecular weight form of eIF5A because of cleavage rather than selective splicing. See Figure 36. The present invention provides a composition comprising an eIF5Al polynucleotide encoding a truncated eIF5Al protein. This composition is useful for the manufacture of an agent for inducing apoptosis in a cancer cell or a tumor in one body. The eIF5Al polynucleotide encodes a truncated eiF5Al protein which preferably comprises the amino acid sequence as set forth in SEQ ID NO: 37 shown in Figure 38. In certain embodiments, the eIF5Al polynucleotide encodes a truncated eIF5Al protein of about 16 kDA. The composition and/or the agent can be administered to a mammal, including human 142866.doc 12 201023898. As used herein, "individual" includes mammals and humans. Induction of apoptosis can have the following potencies in cancer cells or tumors: slowing down cancer cell or tumor growth, preventing cancer cell or tumor cell growth or killing the cancer cell or reducing the tumor size' and any combination of the above. Any cancer can be treated, and in certain embodiments, the cancer is multiple bone tumors. In certain embodiments, the eIF5 A1 polynuclear acid is included in the sequence referred to in seq ID NO: 38 (as shown in Figure 41). In certain embodiments, the eIF5 A1 polynucleotide is contained in a plastid or expression vector. The plastid and expression vectors are described in more detail below. In certain embodiments, the expression vector is an adenovirus expression vector or pHM6. In certain embodiments, the expression vector comprises a tissue-specific promoter, such as when the composition or agent is used to treat multiple myeloma, the promoter is a B cell-specific promoter ( That is, B29). The expression vector can comprise a pCpG plastid. The performance carrier can be complexed to polyethyleneimine as discussed in more detail below. Preferably, the composition or agent is administered intratumorally, intravenously or subcutaneously. The invention also provides a truncated eIF5Al protein-isolated polynucleotide, wherein the polynucleotide is contained in SEq ID NO : 38 proposed sequence (as shown in Figure 41). The invention is also long: a single truncated eIF5 A1 protein-isolated polynucleotide is provided, wherein the truncated protein comprises the amino acid sequence set forth in SEq ID NO:37. As discussed below, the eIF5Al is formed by cleavage of thiocysteine protease 142866.doc -13 - 201023898. The invention therefore further provides an isolated truncated eIF5Al multipeptide which is formed by cleavage of a cysteine protease-mediated eIF5Al. The invention further provides for the use of a composition or composition for the manufacture of an eIF5Al comprising An agent that combines a polynucleotide with a full length eiF5Al polynucleotide that encodes a truncated eIF5Al protein. The composition is useful for making a medicament to induce apoptosis of a cancer cell or tumor in one of the bodies. As described above, the eIF5Al polynucleotide encodes a truncated eIF5A protein. The full length elF5Al polynucleotide encodes a protein comprising the amino acid sequence set forth in SEQ ID NO:35. The full length eIF5Al polynucleotide is described in more detail below. "In certain embodiments, the eIF5Al polynucleotide comprises the sequence set forth in SEQ ID NO: 38" and the full length eIF5Al polynucleotide comprises The sequence set forth in SEQ ID NO: 43 (shown in Figure 53). The full length and truncated polynucleotides can be present in the expression vector as described herein. Furthermore, the vector may comprise the promoter described herein as well as any other useful promoter. In certain embodiments, the full length eIF5A polynucleotide encodes a mutated eIF5Al, wherein the mutation prevents or inhibits carboxyresin lysine by deoxycarboxyresinamine lysine synthetase, and/ Or wherein the mutation is present at the ubiquitination site and/or the acetylation site. This mutation is described in more detail below. In certain embodiments, the mutation is selected from the group consisting of K50A, K50R, K67A, K47R, K67R, K50A/K67A, K50A/K47R, K50A/K67R, K50R/K67A, K50R/K47R, K50R/K67R, and K47A/K67A The group formed. 142866.doc -14· 201023898 The invention also provides a composition comprising a polynucleotide encoding a truncated eIF5A, a nucleotide encoding a mutant eIF5A, and a target for the 3'UTR of eIF5A An siRNA. The combination/dual use of a nucleotide encoding a mutant eIF5A with an siRNA targeting the 3' UTR of eIF5A is more fully described below. The composition is useful for producing a medicament for administration to a body to induce apoptosis in cancer cells or tumors. In certain preferred embodiments, the siRNA targets the sequence in eIF5Al: 5'-GCT GGA CTC CTC CTA CAC A-3'. The siRNA is φ dsRNA, and one of the strands of the dsRNA comprises the sequence: 5'-GCU GGA CUC CUC CUA CAC A-3'. In certain embodiments, the siRNA is stabilized to prevent degradation in serum. The full length eIF5Al polynucleotide and/or the eIF5Al polynucleotide encoding the truncated eIF5A protein may be present in a expression vector. In certain embodiments, the full length eIF5Al polynucleotide and/or the eIF5Al polynucleotide encoding the truncated eIF5A protein and/or the siRNA is complexed to polyethyleneimine. The full-length eIF5Al polynucleotide and/or the eIF5Al polynucleotide encoding the truncated eIF5A protein and/or the siRNA are independently complexed to polyethyleneimine. The invention also provides a method of inducing apoptosis in a cancer cell of a mammal or in a tumor of the mammal by providing a composition or agent described herein, for example, the composition or agent, for example a nucleotide comprising a truncated eIF5Al, optionally comprising a nucleotide encoding a full length eIF5A or a nucleotide encoding a full length mutation eIF5A, and optionally comprising a target against eIF5A 3 'UTR' an siRNA. In some embodiments 142866.doc 15 201023898 the cancer is multiple myeloma, and the composition/agent is administered intravenously, intraperitoneally or intratumorally. The invention also relates to the use of a combination of an siRNA target for an endogenous gene to knock out or reduce the expression of the endogenous gene in a body, and the polynucleotide encoding the gene is located Provided to a delivery vehicle/expression carrier of the individual to provide expression of the protein encoded by the polynucleotide in the host. This combination is useful in treating an individual having a disease or condition caused by the presence of a defective or mutated protein, i.e., the protein produced in the individual is unable to perform its necessary function, or the protein The interaction of a metabolic pathway or biomolecule is disrupted by its defective structure. The siRNA is designed to target the gene encoding the defective protein and to reduce or eliminate the performance of the defective protein. A polynucleotide encoding a normal (non-defective) protein is administered to the sputum and expressed in the individual such that the normal protein can perform its necessary function. In another specific embodiment, the protein is administered to the individual without the polynucleotide encoding the protein. The terms, protein, peptide and polypeptide are used interchangeably herein. Preferably, the siRNA is designed to target a region of the gene such that it reduces or eliminates the endogenous expression of the defective protein without affecting the exogenous expression of the polynucleotide encoding the normal protein administered. . For example, the siRNA can target the 3' UTR so it does not affect the performance of the sense construct (the polynucleic acid encoding the protein). By reducing or eliminating the endogenous expression of the defective gene by 142866.doc _ 16 - 201023898, there will be fewer or no such defective protein competing with the normal protein expressed from the exogenous polynucleotide. An example of a disease state useful for this application is about sickle-type anemia. Sickle anemia is a blood disease that affects hemoglobin, a protein found in red blood cells (RBCs) that helps carry oxygen throughout the body. Recording anemia occurs when a person inherits two abnormal genes (from one parent each), which results in the performance of a mutant hemoglobin (Hbs). This mutant heme causes the RBCS to change shape. Red blood cells with normal hemoglobin (heme A, or HbA) can easily move through the bloodstream, transporting oxygen to cells throughout the body. They can even "squeeze" very small blood vessels. Sickle anemia occurs because abnormal forms of hemoglobin (Hbs) tend to clump together, making red blood cells sticky, hard, and more fragile, and causing them to form a curved knife-like shape. Although hundreds of HBB gene variants are known, sickle-type anemia is most commonly caused by heme variant HbS. In this variation, the sixth amino acid position in the HBB multi-peptide chain 'the hydrophobic amino acid, valine acid, replaces the hydrophilic glutamic acid. This substitution creates a hydrophobic point on the outside of the protein structure that binds to a hydrophobic region adjacent to the beta bond of the heme molecule. This HbS molecule aggregates (polymerizes) into hard fibers, causing a "sickle-shaped" red blood cell. Polymerization only occurs when red blood cells have carried oxygen molecules to different tissues throughout the body and released them. Once the red blood cells return to the lungs where heme can bind oxygen, 142866.doc -17- 201023898, the long fiber HbS molecules are depolymerized or broken into a single molecule. The cycle between the polypeptidation and the depolymerization causes the red blood cell membrane to harden. When these red blood cells do not carry oxygen, their hard and tortuous shape can cause blockage of small blood vessels. This blockage can cause pain and can damage the organ. Sickle anemia is a disease of a somatic chromosome recessive gene. One must inherit two HbS variants or one HbS and one other variant to show the disease. The so-called original person's have a normal Hbb gene (HbA) and an HbS, which are characteristic of sickle-shaped cells and do not exhibit symptoms of the disease. Thus, a specific embodiment of the invention provides a method of treating an individual with sickle-type anemia. The siRNA labeled to the HBB gene was administered to the patient. The siRNA is designed to reduce and preferably degrade the performance of the Hb of heme. A polynucleotide encoding a normal heme is supplied to the individual, so that the individual exhibits a normal heme. The siRNA is also designed such that it does not interfere with the expression of the exogenous polynucleotide encoding the normal erythropoietin. Thus, the individual no longer produces mutated hemoglobin (or greatly reduces manufacturing)' but creates normal healthy hemoglobin, resulting in more normal normal red blood cells. It is also useful in the context of the present invention that a post-transcriptional modification occurs in the protein' which causes or causes a disease state. Using siRNA to reduce the expression of the endogenous protein, no or less protein can be used for this post-transcriptional modification. A polynucleic acid encoding a protein is then provided to the patient for exogenous expression. The protein was modified such that it was post-translationally modified without the 142866.doc -18-201023898 method. This protein can then be used in the body for its proper use, but because it cannot be post-translationally modified, it does not cause the disease state. Those skilled in the art will be aware of different post-translational modifications. For example, by translating other biochemical functional groups, such as acetates, phosphates, different lipids and carbohydrates, by changing the chemical nature of an amino acid (eg, citrulline), or by causing a structure Changes, such as disulfide bridging, post-translational modification of the amino acid broadens the functional range of the protein. Also, the enzyme removes the amino acid from the ammonia end of the protein or cleaves the peptide chain from the middle. For example, the peptide hormone insulin is cleaved twice after the formation of the disulfide bond, and a proeptid is removed from the middle of the chain, and the formed protein consists of two disulfide bonds. Constructed by linked multi-peptide chains. Also, most of the initially produced multi-peptides start with an amino acid, methionine, because the "starting" code on the mRNA also encodes the amino acid. This amino acid is usually removed during the post-translational modification. Other modifications, such as phosphorylation, are part of a common mechanism for controlling the behavior of a protein, such as activating or deactivating an enzyme. Another post-translational modification includes carboxyresinamine lysineization of eukaryotic initiation factor 5A (eIF5A) with deoxycarboxylate lysine synthetase (dhs). Accordingly, the present invention provides a method of altering the expression of a gene in a body, wherein a polynucleotide encoding a protein is provided to a patient and expressed in the patient. The protein can be a normal/wild type protein or a mutant protein. The expression of the corresponding endogenous gene is inhibited by the siRNA administered to the individual. The method further comprises providing a construct comprising a polynuclear phytic acid 142866.doc -19-201023898 encoding the target protein' wherein the polynucleotide is expressed in the individual to produce the target dry protein. In certain embodiments, when the endogenous gene exhibits a defective protein, the polynucleotide is designed to encode a normal, healthy protein. The siRNA is administered to inhibit the expression of the defective endogenous protein. In certain embodiments, 'when the endogenous gene exhibits a normally healthy protein' the polynucleotide is designed to encode a mutant protein that cannot be post-translationally modified, and this post-translational modification is in a normal/healthy It can occur in non-mutated proteins. The siRNA is administered to inhibit the expression of the endogenous protein, so that less of this protein can be used for post-translational modification. In certain embodiments, the siRNA is selected or designed to target the region of the gene such that the expression of the exogenous polynucleotide is not affected. For example, the siRNA can target the 3, UTR or 3, terminus. »Hai siRNA can be delivered to the patient as naked s1rnA or naked siRNA that is stable to serum. The siRNA can be injected systemically, i.e., IP or iv. Alternatively, the siRNA can be injected or delivered locally to the desired body part. In certain embodiments, the siRNA can be administered using a delivery device such as, but not limited to, a dendrimer, a liposome or a polymer. The polynucleotide encoding the desired protein can be administered via any delivery method that provides or permits expression of the nucleotide. The terms polynucleotide and nucleotide are used interchangeably herein to be delivered via any viral or non-viral mechanism, such as, but not limited to, plastids, expression vectors, viral constructs, adenoviral constructs, dendrimers. A substance, a liposome or a polymer. 142866.doc • 20· 201023898 In some embodiments, a plastid having a reduced CpG dinucleotide is used to express the polynucleotide. Any promoter that promotes the expression of the polynucleotide can be used, which can be selected based on the desired application for the treatment. For example, in order to kill multiple myeloma, a promoter specific for B cells may be desirable, such as the human B29 promoter/enhancer. In other specific embodiments, the promoter may be a promoter specific for another tissue, or a system promoter. The polynucleotide encoding the marker protein can be delivered via IV or subcutaneous injection, or any other biocompatible delivery mechanism. Alternatively, the polynucleotide may be delivered in a liposome or any other suitable "carrier" or "tool" that provides for delivery of the DNA (or plastid or expression vector) to the target tumor or cancer cell. See, for example, Luo, Dan, et al., Nature Biotechnology, Vol. 18, January 2000, ρρ·33-37 for a review of synthetic DNA delivery systems. Thus, it is preferred to deliver the nucleotide/plastid/expression carrier' such as a liposome, a dendrimer, or a similar non-toxic nanoparticle via a nanometer sized tool. The tool preferably protects the nucleotide/plastid/expression vector from premature clearance or when delivering an effective amount of the nucleotide/plastid/expression vector to the individual, tumor or cancer cell Free from causing an immune response. Exemplary tools can range from a simple nanoparticle that binds to the nucleotide/plastid/expression carrier to a more complex polyethylene glycol modified (pegylated) tool such as a ligand with a ligand. Ethylene glycol modified liposome attached to its surface to target a specific cellular receptor. Liposomes and polyethylene glycol modified microliposomes are known in the art. In the conventional liposome, the molecule to be delivered (i.e., small molecule drug, protein, nucleotide or plastid) is contained in the central chamber of the liposome. Those skilled in the art will recognize that there are also "stealth", standard and cationic liposomes that can be used for molecular delivery. See, for example, Hortobagyi, Gabriel N., et al., J. Clinical Oncology, Vol. 19, Issue 14 (July) 2001: 3422-3433 and Yu, Wei, et al., Nucleic Acids Research. 2004, 32(5 ); e48. The liposomes are injected intravenously and can be modified to make their surfaces more hydrophilic [by the addition of pegylated to the bilayer], which increases their circulation time in the bloodstream. These are known as "sneaky" liposomes, and are particularly useful as carriers for hydrophilic (water-soluble) anticancer drugs, such as doxorubicin and mitoxantrone. In order to promote the specific binding characteristics of a drug-carrying liposome to a target cell, such as a tumor cell, a specific molecule such as an antibody, a protein, a peptide, or the like may be attached to the surface of the liposome. For example, an antibody to a receptor present on a cancer cell can be used to target the liposome to the cancer cell. In the case of a target multiple myeloma, for example, folate, sputum-6 or transferrin can be used to target the liposome to multiple myeloma cells. Dendrimers are known in the art and provide a preferred delivery tool. See, for example, Marjoros, Istvan, J., et al, "PAMAM dendrimer-Based Multifunctional Conjugate for Cancer Therapy: Synthesis, Characterization, and Functionality",

Biomacromolecules, Vol. 7, No. 2, 2006; 572-579, and

Majoros, Istvan J., et al., J. Med. Chem, 2005. 48, 5892-5899 for a discussion of dendrimers o 142866.doc -22- 201023898

In a preferred embodiment, the delivery means comprises a polyethyleneimine nanoparticle " an exemplary polyethyleneimine nanoparticle is in vivo_jetPEITM, currently manufactured by Polyplus Transfection, Inc. In vivo-jetPEITM is a cationic polymer transfection agent used as a DNA and siRNA delivery agent. Polyplus Transfection's In vivo-jetPEITM is a linear polyethyleneimine reagent that provides reliable nucleic acid delivery in animals. It is used for gene therapy (Ohana et al., 2004. Gene Ther Mol Bio 8:181-192; Vernejoul et al., 2002. Cancer Research 62:6124-31), RNA interference (Urbain-Klein et al., 2004) Gene therapy 23: 1-6; Grezelinski et al., 2006. Human Gene Therapy 17: 751-66) and gene inoculation (Garzon et al., 2005. Vaccine 23: 1384-92). In vivo JET-PEI is currently used as a delivery vehicle in cancer gene therapy in human clinical trials. (Lemkine et al., 2002. Mol. Cell. Neurosci. 19: 165-174).

In vivo-jetPEITM concentrates the nucleic acid into nanoparticles of about 50 nm, which are stable for several hours. Due to this unique protection mechanism, blood cell aggregation after injection is reduced compared to other agents, thereby preventing restricted diffusion, red blood cell aggregation, and microembolism in a tissue. These nanoparticles are small enough to diffuse into the tissue and enter the cell by pinocytosis. In vivo-jetPEITM helps release nucleic acids from the endosome and transfer through the nuclear membrane. In a preferred embodiment, the siRNA and a vector/plast containing the polynucleotide are administered to the individual via an in vivo-jet PEITM complex. The siRNA and the vector/plastid comprising the polynucleotide may be combined together via a 142866.doc •23·201023898 polymeric complex, such as a polyethyleneimine or the in vivo jetPEITM, or may be separately Composite to a polymer. For example, when the siRNA and the vector/plastid comprising the polynucleotide are separately administered to the individual (separately means different times and/or different delivery sites), preferably the siRNA is used. And the polynucleotide is complexed to a different sturdy band. When the action of the administration is simultaneous and at the same site, it may be preferable to complex the siRNA and the doxorubicin. In another specific embodiment, the protein itself is delivered to the individual rather than a plastid or carrier to deliver a polynucleotide that will behave in the individual. The protein can be isolated or synthetic. One embodiment of the invention provides a method of treating cancer in a body'. The individual comprises a mammal and a human. Treating cancer includes, but is not limited to, inducing > death in cancer cells, killing cancer cells, reducing the number of cancer cells, and reducing the volume/weight of the tumor. The method comprises administering a composition comprising a eIF5Al siRNA and a polynucleotide encoding a mutant elF5Al. The composition and the eIF5Al siRNA and the polynucleotide encoding a mutant eIF5Al are discussed below. @All cells make eukaryotic initiation factor 5A ("eIF-5A") (also referred to as "factor 5A"). Mammalian cells produce two isomers (eIF- 5Al and eIF-5A2^ eIF-5Al are called apoptosis-specific eIF_. 5A, which is regulated upwards as the cells undergo apoptosis. Human eIF_5Ai has an accession number NM 001970, and It is shown in j. It is generally believed that eIF-5Al is responsible for transporting mRNAs of some proteins required for apoptosis out of the nucleus. eIF_5A2 is called proliferation elF 5A, 142866.doc -24 - 201023898 because it is generally believed that it is responsible for some coding The mRNA of the protein required for cell division is transported out of the nucleus. See Liu & Tartakoff (1997) Supplement to Molecular Biology of the Cell, 8, 426a. Abstract No. 2476, 37th American Society for Cell Biology Annual Meeting, and Rosorius et Al· (1999) J. Cell Science, 112, 2369-2380. Both factors, 5A, were modified by deoxycarboxylate lysine synthetase (“DHS”). DHS carboxypyramine lysine The eIF-5A. Carboxyurin lysine (Hypusi) Ne), a unique amino acid found in all eukaryotic cells and archaea tested, but not in eubacteria, and eiF-5A is the only known carboxy-containing putrescine Acidic protein. Park (1988) J. Biol. Chem., 263, 7447-7449; Schumann & Klink (1989) System. Appl. Microbiol., 11, 103-107; Bartig et al. (1990) System. Appl. Microbiol., 13, 112-116; Gordon et al. (1987a) J. Biol. Chem., 262, 16585-16589. Carboxyuramine lysineized eIF-5A is a step after two post-translational modifications Formed in: the first step is to form a deoxygenated putrescine by transposing the 4-aminobutyl moiety of the semitriamine to one of the eIF-5 A precursors specific to the οι-amine group of the amine acid. The lysine residue, the step of formation is catalyzed by deoxycarboxylate lysine synthetase. The second step involves hydroxylating the 4-aminobutyl moiety by deoxycarboxylate lysine hydroxylase to form carboxyresinamine lysine. The amino acid sequence of eIF-5 A is sufficiently retained between species, and the amino acid sequence surrounding the carboxypyramine lysine residue in eIF-5 A is completely retained, suggesting this modification. It may be important for survival. Park et al. (1993) Biofactors, 4, 95-104. This hypothesis is further supported by the phenomenon observed in the following 142S66.doc -25-201023898: Deactivation of two eIF-5A isomers starting from yeast or catalyzing the first step of the DHS in its activation Deactivation of genes blocks cell division. Schnier et al. (1991) Mol. Cell.

Biol, 11, 3105-3114; Sasaki et al. (1996) FEBS Lett., 384, 151-154; Park et al. (1998) J. Biol. Chem., 273, 1677-1683. However, depletion of the eIF-5 A protein in yeast only results in a small reduction in total protein, suggesting that eIF-5A may be required for translation of a particular portion of the mRNA, but not for total protein synthesis. . Kang et al. (1993), "Effect of initiation factor elF-Spring 5A depletion on cell proliferation and protein synthesis", in Tuite, M. (ed.), Protein Synthesis and Targeting in Yeast, NATO Series H. The discovery that ligands that bind eIF-5A have a highly retained motif has recently been shown to support the importance of eIF-5A. Xu & Chen (2001) J. Biol. Chem., 276, 2555-2561. Furthermore, it was found that the carboxycisyramine lysine residue of the modified eIF-5A is essential for sequence-specific binding to RNA, and the binding does not provide protection against ribonuclease. Θ The inventors have shown that when a polynucleotide encoding eIF-5A is administered to a cell, apoptosis of those cells is increased. They have shown that they can promote apoptosis of the cancer cells by administering an eIF-5Al polynucleotide that is subsequently expressed in cancer cells. See, for example, the application Serial Nos. 10/200, 148, 11/287, 460, 11/293, 391, and 11/637, 835, the entire contents of each of which is incorporated herein by reference. The inventors have additionally confirmed that when a cell has an increased carboxyresosamine lysine factor 5A, the cell does not undergo apoptosis as usual, and 142866.doc -26· 201023898 will enter a survival mode and Does not undergo apoptosis. It is noteworthy that in cancer cells, there is a significant amount of carboxypyramine lysine factor 5A, and therefore, the cells do not enter apoptosis (and do not die). Thus, in order to treat cancer by killing the cancer cell (promoting the cancer cell to enter the apoptotic pathway), a polynucleotide encoding eIF-5A1 is administered to the individual or to the cancer cell or tumor to provide an increase. In eIF-5Al, the eIF-5Al in turn leads to apoptosis of the cancer cells and ultimately to cell death and tumor shrinkage. However, if only the polynucleotide encoding the eIF-5Al protein is provided to upregulate the φ gene expression of eiF-5Al' and siRNA is not used to reduce the endogenous performance of eIF-5Al, there is a competitive confrontation: The eIF-5Al expression of the cells towards the apoptotic pathway competes with the presence of the vasopressin-dependent factor 5A that directs the cells towards the cell's survival pathway. The present invention eliminates the competition for this confrontation 'and presents an improvement' which only increases the performance of eIF-5Al. The polynucleotide administered to the individual or cell is mutated such that the resulting protein is not lysylated by carboxyresosamine. Furthermore, the endogenous performance of Factor 5 a was knocked out or reduced by targeting siRNA against eIF-5A, so no/less endogenous eIF-5Al would be lysine by carboxyresosamine. Therefore, since there is no (or greatly reduced) carboxypyramine lysine eiF-5A in the cell 'they will not be pushed into the survival mode. Preferably, the polynucleotide encoding a mutant eIF-5 A1 is mutated so that it cannot be lysineized by carboxyresosamine' and thus cannot be used to drive the cell into a mode of survival. For example, in a particular embodiment, the polynucleotide encoding eIF_5A is mutated such that the lysine (κ) at position 50 (which is typically lysine by DHS carboxypyramine) is changed to monoalanine (A ) (it cannot be acidified by carboxy putrescine lysine 142866.doc • 27- 201023898). This mutation is called K50A. In another embodiment, the amine acid at position 67 becomes a arginine (R). This mutation is called (K67R). In another embodiment, the lysine (K) at position 67 becomes alanine (A) and is referred to as (K67A). In another embodiment, the lysine (κ) at position 50 becomes - arginine (K50R), and another embodiment provides a mutation wherein the lysine at position 47 ( K) becomes a arginine (K47R). A double pair of mutations is used in other embodiments. A double mutation is at position 50 that the lysine (K) becomes a arginine (R), and at position 67 the amide acid (K) becomes a arginine (R). This double mutation is called K50R/K67R. This double mutation was similarly incapable of being lysine by carboxyresosamine, but the change in the amino acid was not as much altered for the 3-D structure of eIF-5Al as the single mutation (K50A). This double mutation thus provides a protein structure that is very similar to the wild protein type in the 3-d structure and folding, and is therefore more stable than this single mutation. Because it is more stable, it exists longer in the body to provide longer medical benefits. Therefore, the body will have the factor 5A' required for normal cell function but it will not be lysine by carboxyresosamine, so the cell will not be stuck in the cell survival mode and escape from apoptosis. Another double mutation is that the lysine at position 47 becomes a arginine (R)' and the lysine at position 50 becomes a arginine (R). This mutation is called (K47R/K50R). The present invention provides another double mutation in which the lysine (K) at position 50 becomes an alanine (a), and the lysine at position 67 becomes an alanine (A). This mutation is called (K5〇A/K67A). Since the body requires factor 5A for normal cell survival and proliferation of healthy cells 142866.doc -28-201023898, if the siRNA is delivered systemically, it is preferred not to completely shut down the performance in the individual with the siRNA. . Control of eIF-5A performance can be achieved by using siRNA that is not good at turning off performance (ie, suspending or reducing performance but not completely turning off performance), or using a dose and/or treatment regimen to balance performance. Allows normal growth and function of healthy cells and promotes cancer cells to undergo apoptosis. Alternatively, local delivery of siRNA can be used. If the siRNA is delivered locally to cancer cells or tumors, the performance is preferably rejected. By rejecting the expression, there is no factor 5 A around which carboxyresosamine can be lysine, so there is no carboxyresosamine lysine-containing eIF-5 A to lock the cell into a survival mode. Since the siRNA is delivered locally to the cancer or tumor, it is not necessary to have eIF-5A which can be used to regulate cell growth. In certain embodiments, the endogenous gene is eIF5A1. Labeling (siRNA against eIF5Al is administered to the individual to inhibit the expression of the endogenous eiF_5Al. In certain embodiments, the siRNA comprises SEQ ID ΝΟ: 1 or SEQ ID NO: 2 or eIF5Al, which inhibits the expression of endogenous eIF-5Al. In some embodiments, the eIF5A1 is human eIF-5Al (as shown in Figure 1), and the individual is a human. Other stems are directed against human eIF-5Al The siRNAs are known and are disclosed in the applications 11/134,445, 11/287,460, ii/184,982, 11/293,391, 11/725,520, 11/725,470, 11/637,835.

In other specific embodiments, the individual is a mammal and the eIF5 A1 is specific for the mammal. For example, the individual is a dog and the eIF5Al is a dog's eIF5Al. In certain embodiments, the siRNA 142866.doc -29-201023898 is necessarily composed of the siRNA construct shown in Figure 25. For example, the siRNA comprises a nucleic acid that targets the eIF5Al, but also includes a prominent portion, such as a u or tau nucleic acid, or also a tag, such as a set of his acid tags (often referred to as HA tags, which are commonly used in trials). Research within the tube). As long as the siRNA construct can reduce the expression of the stem gene, it can comprise a molecule attached to the 5 or 3 terminus (or even included in the string of nucleic acids as shown in Figure 25) or additional Nucleic acid, and the molecule or additional nucleic acid falls within the scope of the "essential composition" of the stomach. Preferably, the siRNA targets the region of the eIF5Al gene such that it does not affect the performance of the exogenous polynucleotide. For example, the eIF5A1 siRNA targets the 3, UTR or the 3, terminus. The siRNA shown in Figure 25 is an exemplary eIF5A1 siRNA. The polynucleotide encodes eIF5Al, wherein the polynucleotide is mutated to encode an eIF5A1 variant. The mutated eIF5Al was designed such that the mutated eIF5Al was not post-translationally modified (cannot be lysine by carboxyresosamine). Exemplary mutations are discussed above. In cases involving solid tumors, the S1 RNA may be desired and delivered directly to the tumor. With respect to time and the delivery site, the sputum polynucleotide can be delivered separately, or can be delivered simultaneously and/or at the same delivery site. Those skilled in the art will appreciate that the timing of administration of the siRNA may have to be made when the endogenous protein is being translated and not after it has been completed. However, the inventors have shown that eIF5A1 is not toxic to normal tissues (see in the middle of the request, in the case of the fine 5 years U, please refer to the serial number 11/293,391 ', which is hereby fully incorporated by reference. ), it may be preferred to 142866.doc -30- 201023898 a delivery complex (compared to direct administration of the eIF5A polynucleotide/plastid/expression vector). A preferred delivery system provides an effective amount of eIF5Al to the individual, tumor or cancer cell population, and preferably provides delivery of a target to the tumor or cancer cell population. Thus, in certain embodiments, the eIF5Al nucleotide/plasto/expression vector, such as a liposome, dendrimer, or a similar non-toxic nanoparticle, is preferably delivered via a nano-sized tool. Such as a polyethyleneimine polymer (such as an in vivo JetPEITM complex). φ The eIF5Al protein can also be delivered directly to the site of the tumor. Those skilled in the art will be able to determine the dosage and length of the eIF5Al protein delivery treatment regimen. The molecular basis of apoptosis induced by eIF5Al is discussed below. Death receptor signaling by treatment of cancer cells with Ad-eIF5Al (adenovirus with a wild-type eIF5Al) or Ad-eIF5Al (K50A) induced thiocysteine 8 and thiocysteine 3, thiocysteine Lysinase 8 is initiated by the binding of a death receptor and a ligand, and thiocyl protease 3 is a thiosyl protease. These are likely to be direct effects of eIF5Al, and the fact that thiocysteine protease 8 and thiocysteine protease 3 are also activated after treatment with eIF5Al (K50A) which cannot be lysine by carboxyresosamine represents this effect. Can be attributed to 50 eIF5Al due to lysine. Treatment with Ad-eIF5Al also appeared to result in upregulation of the death receptor, as indicated by the upward regulation of TNFR as previously shown. The indirect involvement of the granulocyte pathway in the afferent glandular gland pathway from the direct or 142866.doc -31 - 201023898 of the amino acid 5〇eIF5Al is supported by many observations including the discovery of thiocyl protease 9 It is activated by treatment of cancer cells with eIF5Al or eIF5Al (K50A). Similarly, p53' plays a role in the activation of the apoptotic pathway of the granulocyte and appears to be regulated by eIF5Al. For example, treatment of cancer cells with actinomycin D upregulates P53, and this up-regulation of p53 is inhibited by eIF5Al siRNA. Consistent with this, treatment of cancer cells with Ad-eIF5Al upregulated P53 mRNA. Treatment of cancer cells with eIF5Al also induces the migration of Bax from the cytosol to the granulocytes, followed by loss of granulosa membrane potential and release of cytochrome C from the intergranular space of the granulosa to the cytosol. In addition, this treatment resulted in the up-regulation of Bcl2, Bim after cleavage and Bim after splicing, which are all pro-apoptotic. MAPK message delivery Furthermore, the inventors have obtained evidence that eIF5Al is involved in the transmission of apoptosis-related MAPK messages. For example, treatment of cancer cells with Ad-eIF5Al up-regulates P-JNK, which in turn inhibits anti-apoptotic Bcl2. In addition, both Ad_eIF5Al and Ad-eIF5Al (K50A) induce the formation of P-p38, which affects a wide range of pro-apoptotic agents, including TNFR1 and TNF, FAS and FASL, and thiocysteine 8 , Bid, cytochrome C and thiocysteine protease 3, followed by initiation of apoptosis. NF-κΒ message delivery There is evidence that NF-κΒ message delivery supports myeloma growth. For example, adhesion of myeloma cells to bone marrow stromal cells induces up-regulation of NF-κΒ-dependent IL-6, a growth and anti-apoptotic factor in multiple myeloma [Chauhan et al. (1996) S7, 1104.]. In addition, TNF-α secreted by the cells of the bone chain tumor activates NF-κΒ in bone marrow stromal cells, thereby up-regulating the translation and secretion of IL-6. TNF-α also activates NF-κΒ in myeloma cells, resulting in intercellular adhesion molecule-1 (ICAM-1; CD54) and vascular cell adhesion molecule-1 (VCAM-) on myeloma cells and bone marrow stromal cells. 1; Upward adjustment of CD106). [Hideshima et al_ (2001) Oncogene 20, 4519]. This in turn increases the association of osteoblastoma cells with bone marrow stromal cells. [Hideshima et al. (2001) Owcogewe 20, 4519]. Conversely, these effects are inhibited by blocking TNF-α-induced NF-κΒ activation [Hideshima et al. (2001) 20, 4519]. Marriage, like NF-κΒ mediates anti-TNF-α-induced apoptosis in myeloma cells. [Hideshima et al. (2002) JBC 277, 16639]. These and other observations have led to the notion that NF-κΒ signaling may be a compelling target in multiple myeloma therapy. The inventors have shown that eIF5 A1 siRNA inhibits the activation of NF-κΒ and the formation of ICAM-1 in human myeloma cells. These observations represent that eIF5Al plays a role in the activation of NF-κΒ, and since the subsequent effect of NF-κΒ activation is essentially pro-survival, we predict that this activation is ED95 by carboxyresosamine lysine Direct or indirect media. A feature of IL-1 multiple myeloma is that myeloma cells overproduce the pro-inflammatory cytokine, IL-1, which over-manufacture leads to deterioration of bone tissue. It has been shown that eIF5Al siRNA in mice dramatically reduces IL-1 overproduction induced by an LPS stimulation. A specific embodiment of the invention provides a method of treating multiple myeloma 142866.doc-33-201023898. Multiple myeloma (ΓΜΜ) is a progressive and fatal disease characterized by the proliferation of malignant plasma cells and the presence of osteopathy in the bone marrow. More than 14 months of Zhao tumor, an incurable but treatable plasma cell cancer. Plasma cells are an important part of the immune system, and are important immunoglobulins (antibodies) that help fight infections and diseases. Multiple myeloma is characterized by a very large number of abnormal plasma cells in the bone marrow as well as intact individual immunoglobulins (IgG, IgA, IgD or IgE; "Μ-protein") or this week's protein (free individual plants) Over-manufacturing of the chain). Hypocalcemia, anemia, kidney damage, increased sensitivity to bacterial infections, and reduced production of normal immunoglobulins are common clinical manifestations of multiple myeloma. Multiple myeloma is often characterized by diffuse osteoporosis, usually in the pelvis, spine, ribs, and skull. The present invention appears to be well suited for the treatment of multiple myeloma due to the feedback loop found in multiple myeloma. For example, multiple myeloma produces low concentrations of IL-1 in the epiphysis. This IL-1 then stimulates stromal cells to produce il-6' which then stimulates the growth of multiple myeloma. The inventors have previously shown (see application 11/725, 539 and 11/184, 982) that siRNA targeting 61卩-5Α1 can inhibit the expression of pro-inflammatory interleukins such as IL-1, TNF-α and IL. -8. Therefore, the siRNA does not only reduce the performance of eiF-5A and can be used for carboxyresosamine lysineization, which also interrupts or reduces the feedback loop of IL_1/il-6. One siRNA of the standard human eIF5 A was used to inhibit the amount of endogenous serotonin lysine eIF5A in the tumor, while the nucleoside of the primary antibody expressing an eIF5A (eIF5AK50R) mutation was used to provide unmodified eiF5 A in vivo. The amount of the eIF5A (eIF5AK5GR) mutation cannot be lysine by carboxyresosamine. Intratumoral injection of PEI nanocomplex containing eiF5A siRNA was inhibited by more than 80% compared to 142866.doc •34·201023898 intratumoral injection of a control group siRNA complex (***ρ=〇 · 〇〇〇 3) MM tumor growth, which represents an inhibitory amount of carboxypyramine lysine eIF5A has an anti-tumor effect. A PEI complex comprising an elF5AK50R-expressing plastid has a similar effect and inhibits tumor growth by more than 70% (** = p 0.001) relative to a complex comprising a control set of plastids. Thus, the growth of MM tumors can be inhibited by the growth promoting defibrin lysine eIF5A or by the increased amount of eIF5A in the acidified form of the pro-apoptotic antiseptic amine lysine φ. In vivo delivery of a complex comprising eIF5A siRNA and an anti-RNAi eIF5AK5GR plastid has a synergistic effect on tumor growth and results in significant tumor shrinkage, inhibiting tumor growth by 94% (***p=0.0002). Intravenous delivery of the eIF5A siRNA/eIF5AK50R PEI complex also effectively reduced tumors by up to 95% (**p=0.002), which represents a therapeutic systemic delivery that is feasible. Local or systemic delivery of the eIF5A siRNA/eIF5AK50R pDNA PEI complex results in a significant anti-tumor effect in multiple myeloma. ® The present invention further provides a composition for the treatment of cancer comprising multiple myeloma. In a preferred embodiment, the composition is a complex of a plastid DNA and eIF5Al siRNA encoding a point mutation eIF5Al which is not lysine by carboxyresosamine, the eIF5Al siRNA selective, selective Endogenous human eIF5A is inhibited, but the point mutation eIF5Al encoded by the plastid has no utility. The eIF5Al siRNAs and the chimeric mutant eIF5Al polynucleotide are discussed above. Both the plastid DNA and the siRNA are preferably complexed to PEI (polyethyleneimine) nanoparticles. They can be compounded separately and administered separately or together, or they can be compounded together. When the nanoparticles are ingested into the cells, the DNA and the RNA bind to the positively charged amine groups on the PEI and are released. Cells in both dividing and non-dividing have been shown to efficiently ingest PEI-nucleic acid complexes. This plastid DNA preferably encodes eIF5Al (K50R), which is eIF5Al (K50A) and is not lysine-derivatized by carboxyresin, and thus has a strong atrophic property. This performance of eIF5Al (K50R) is preferably regulated by a B cell-specific promoter. The eIF5Al siRNA is preferably specific for endogenous human eIF5Al and has no effect on the transgenic eIF5Al (K50R) expression. An exemplary preferred eIF5Al siRNA comprises, consisting of, or consisting of the siRNA shown in Figure 25. The basic rationale for the inclusion of the eIF5Al siRNA is: (1) in order to deplete endogenous eIF5Al, which is almost carboxyresphate lysine, thus promoting the survival form; (2) in order to inhibit the activation of NF-κΒ, and thereby Reduction of production of IL-6 and formation of intercellular adhesion molecules; and (3) inhibition of IL-1 formation. eIF5Al siRNA acts synergistically with eIF5Al (K50R) to induce apoptosis in myeloma cells. Since (2) and (3) above are conditions for survival, they may be mediated by carboxyresinamine lysine-derived eIF5Al and thus are not affected by eIF5Al (K50R) which cannot be lysine by carboxyresylamine. This method results in a large amount of uncarboxylated putrescine-induced eIF5A, which contains multiple myeloma cells with minimal effect on healthy cells. A preferred composition is referred to herein as SNS01. SNS0I is a complex comprising an anti-RNAi plastid encoding eIF5AK5C)R and a siRNA targeting human eIF5A, which is driven by a promoter for increased safety 142866.doc -36- 201023898 The promoter is restricted to the expression of cells derived from B cells (including bone tumor cells) having a prominent dTdT 3' to enhance resistance to nucleases, and the siRNA and the plastid are complexed to in vivo JetPEI TM. EXAMPLES Example 1: Transfection of HeLaS3 cells with wild-type and variant eIF-5Al. Lipofectamine 2000 was used to transfect plastids with HA-tagged eIF5Al variants into HeLa S3 cells, including wild-type eIF5Al (WT). ), eIF5AlK50R (K50R), eIF5AlK67R (K67R), φ eIF5AlK67A (K67A), eIF5 A1K47R/K50R (K4750R), eIF5AlK50R/K67R (K5067R) or eIF5AlK50A/K67A (K5067A). A plastid representing LacZ was used as a control group. At 24 and 48 hours (A) or 28 and 52 hours (B) after transfection, cell lysates were harvested and fragments were separated by SDS-PAGE. An antibody against HA was used to detect the amount of transfected eIF5Al. Results: Compared to wild type (A), the eIF5Al mutation (K67R) of an lysine at the putative ubiquitination site increased the accumulation of the eIF5Al transgene. Compared to wild-type eIF5Al(B), the eIF5Al mutation (K50R) of lysine required for carboxyresphate lysine ® increased the accumulation of eIF5Al transgene. A double mutant form of eIF5Al (K50A/K67A) performed particularly well compared to the unmutated wild-type eIF5Al transgene (A+B). See Figure 2. Example 2: Transfection of KAS cells with wild-type and variant eIF-5Al

PAMAM dendrimer (FMD44) was used to transfect plastids expressing HA-tagged eIF5Al variants into KAS cells, including wild-type eIF5Al (5Al), eIF5AlK67A (K67A), eIF5AlK50A/K67A 142866.doc - 37-201023898 (K50A K67A), eIF5AlK50R (K50R), eIF5AlK47R (K47R), eIF5AlK67R (K67R), eIF5A1K47R/K50R (K47R K50R) or eIF5AlK50R/K67R (K50R K67R). A plastid representing LacZ was used as a control group. Forty-eight hours after transfection, cell lysates were harvested and the fragments were separated by SDS-PAGE. An antibody against HA was used to detect the amount of transfection of eIF5Al. An antibody against actin was used to confirm the administration of the same amount (Equal loading). Results: Compared to the wild type, the eIF5Al mutation (K67A or K67R) of an lysine at the putative ubiquitination site increased the accumulation of the eIF5Al transgene. Compared to wild-type eIF5A, the eIF5Al mutation (K50R) of the lysine or the mutation at the acetylation site (K47R) required for carboxysulphate Q lysine increased the accumulation of the eIF5Al transgene. A double mutant form of eIF5Al (K50A/K67A) also showed a higher amount than the unmutated wild-type eIF5Al transgene. See Figure 3. Example 3: Transfection of KAS cells using PAMAM dendrimers PAMAM dendrimer (FMD45-2) was used to transfect plastids with HA-tagged eIF5Al variants into KAS cells, including eIF5AlK50R ( K50R), eIF5Α1Κ50Α/Κ67Α (Κ50Α/Κ67Α) Ο or eIF5AlK50R/K67R (K50R K67R). A plastid representing LacZ was used as a control group. 72 hours after transfection, cells were stained with Annexin/PI and analyzed by FACS. Cells that were positive for Annexin V staining and negative for PI (propidium iodide) were in the early stage of death (Ann+/PI-), and cells thought to be positive for both Annexin V and PI staining were in apoptosis. Late (Ann+/PI+). RESULTS: Compared to the amount of the LacZ control group, the eIF5Al mutation in the carboxyresinamine lysine site (K50R) or the putative ubiquitination moiety 142866.doc -38 - 201023898 (K67R) This double mutation (K50A/K67A) caused significant apoptosis in KAS cells. See Figure 4. Example 4: Transfection of plastid KAS cells expressing eIF5Al and eIF5Al variants Lipofectamine 2000 was used to transfect plastids expressing HA-tagged eIF5Al variants into KAS cells, including eIF5AlK50A (K50A) and eIF5AlK50R (K50R) ), eIF5AlK67R (K67R), eIF5AlK50A/K67A (K50A/K67A) or eIF5AlK50R/K67R (K50R K67R). A plastid representing LacZ was used as a control group. 72 hours after the transfection, the cells were stained with Annexin/PI and analyzed by FACS. Cells that were positive for Annexin V staining and negative for PI (propidium iodide) (Ann+/PI-) were in the early stage of apoptosis (Ann+/PI-), and cells thought to be positive for both Annexin V and PI staining were Late stage of apoptosis (Ann+/PI+). RESULTS: Compared to the amount of the LacZ control group, the eIF5Al mutation at the carboxyresosamine lysine site (K50R) or at the putative ubiquitination site (K67R) and the double mutation (K5〇A/) K67A) caused significant apoptosis in KAS cells. See Figure 5. ® Example 5: Treatment of KAS cells with mutated eIF5Al leads to apoptosis Using Lipofectamine 2000 to transfect plastids with HA-tagged eIF5Al variants into KAS cells, the eIF5Al variant is eIF5AlK50R (K50R) or eIF5AlK50A/K67A (K50A/ K67A). A plastid representing LacZ was used as a control group. 72 hours after transfection, cells were stained with Annexin/PI and analyzed by FACS. Cells that were positive for Annexin V staining and negative for PI (propidium iodide) (Αηη+/ΡΙ·) were in the early stage of apoptosis (Αηη+/ΡΙ-), and both Annexin V and sputum staining were positive 142866. The cells of doc-39-201023898 are in the late stage of apoptosis (Ann+/PI+). RESULTS: Compared to the amount of the LacZ control group, the eIF5Al mutation (K50R) of an lysine at the carboxyperain lysine site or the mutation in the carboxysulphonated lysine site and the putative ubiquitination site eIF5Al caused significant apoptosis in KAS cells. See Figure 6 〇Example 6A: Killing multiple myeloma cells KAS (human multiple myeloma) cells cultured in siRNA medium with siRNA/adenovirus vector [RPMI 1640, 10% fetal with 4 ng/ml IL-6] Bovine serum (FBS) and penicillin/streptomycin (P/S)]. Lipofectamine 2000 (Invitrogen) was used to transfect 58.7 pmole of siRNA into KAS cells. Pretending transfected cells were treated with Lipofectamine 2000 in the absence of siRNA. Transfection was carried out in antibiotic-free medium S10. a) Targeting human siRNAs of eIF5Al:

eIF5Al siRNA target #1 (this siRNA targets this region of human eIF5Al): 5, -AAGCTGGACTCCTCCTACACA-3' (SEQ ID NO: _)). This siRNA sequence is shown in Figure 25 and is often referred to herein as h5Al. 10 eIF5Al siRNA target #2 eIF5Al (This siRNA targets this region of human eIF5Al: 5,-AAAGGAATGACTTCCAGCTGA-3· (SEQ ID NO: 1)). (The siRNA sequence is often referred to herein as h5Al-ALT) b) Control group siRNA: The control group siRNA has the following sequence: sense strand, 5'-ACACAUCCUCCUCAGGUCGdTdT-3'; and antisense strand, 3,-dTdTUGUGUAGGAGGAGUCCAGC_5M. Other control groups that have been used include non-standard shoes from Dharmacon* 142866.doc •40· 201023898 Effective siRNAs, as they have been tested with microarrays to limit unwanted off-targeting effects» In the in vitro work of studying NF-κΒ, the control group used was Dharmacon's non-standard siRNA's (sequence D-001700-01), and worked in vivo, using the control group, Dharmacon's (sequence D- 001810-01). Four hours after transfection, the cells were pelleted and resuspended in 1 ml of S10 medium. Transfected KAS cells were counted 72 hours after initial siRNA transfection and cultured in 24-well plates at a density of 300,000 cells per well (300,000 φ cells/well) and transfected a second time with the same siRNA. . Four hours after transfection, the cells were pelleted and resuspended in 1 ml of S10 medium (without IL-6) containing 3000 ifu of Ad-LacZ (adenovirus expressing beta-galactosidase) or Ad -5A1M (an adenovirus expressing human eIF5AlK5GA). After 72 hours, the cells were harvested and stained with Annexin V-FITC and PI (BD Bioscience) and analyzed by FACS analysis. ® a) Early apoptosis was defined as cells positive for Annexin-FITC staining and negative for PI staining (Ann+/PI-). b) Total apoptosis is defined as the total number of cells in early apoptosis (Ann+/PI-) or late apoptosis/bad death (Ann+/PI+). The 5A1 siRNA Target #1 targets this 3'UTR of human eIF5Al and therefore does not affect the performance of eIF5Al from adenovirus. The 5A1 siRNA target #2 is dried within the open reading frame of human eIF5Al, so it can potentially interfere with the performance of eIF5Al from adenovirus. 142866.doc •41 - 201023898 Results: Cells infected with siRNA and infected with adenovirus expressing eIF-5Al K50A variant had a greater amount of apoptosis compared to untreated cells and cells treated with siRNA only. See Figure 7. Example 6B: Pretreatment with eIF5Al siRNA against eIF5Al Target #1 (shown in Figure 25) reduced the expression of endogenous eIF5Al, but accumulates the anti-RNAi eIF5Alk5GA exhibited by adenovirus. Lipofectamine 2000 was used to transfect one of the control group siRNA (C) or two siRNA targets eIF5Al (#1 and #2) into KAS cells. The eIF5Al siRNA#l targets the 3'UTR of eIF5Al and therefore does not interfere with eIF5Al expression from adenovirus because it contains only the open reading region of eIF5Al. The sequence of this siRNA is shown in Figure 25. The eIF5Al #2 siRNA targets this open reading region of eIF5Al and therefore will affect the performance of endogenous and proliferative eIF5Al. The cells were transfected a second time with the same siRNA 72 hours after the initial transfection hours. After 4 hours, the transfection complex was removed from the cells and replaced with growth medium (-) IL6 containing Ad-LacZ (L) or Ad-eIF5AlK50A (5M). After 72 hours, the cell lysate was harvested and analyzed by Western blotting using antibodies against eIF5A and actin. See Figure 7B. The accumulation of eIF5Al (lane 1 to lane 2) of the virus was observed, and it was clearly seen that the expression of eIF5A of #1 and #2 by the eIF5Al siRNA target was decreased (lanes 5 and 7 versus lane 3) ). As expected, the eIF5Al siRNA #1 did not affect the accumulation of eIF5AlK5GA expressed by the virus (lane 6 to lane 4), whereas the eIF5Al siRNA #2 only slightly affected the performance of the virus expressing the transgene (8th lane) For the fourth track). 142866.doc •42- 201023898 Example 6C: Pretreatment with eIF5Al siRNA against eIF5Al Target #1 reduced the performance of phosphorylated NF-κΒ in human multiple bone tumor cells prior to adenovirus infection. Lipofectamine 2000 was used to transfect a control panel siRNA (hC) or a siRNA target eIF5Al (#l) into KAS cells. The eIF5Al siRNA #1 targets the 3'UTR of eIF5Al and therefore does not interfere with eIF5Al expression from adenoviruses as it only contains the open reading region of eIF5Al. After 72 hours of initial transfection, cells were transfected with the second φ with the same siRNA. After 4 hours, the transfection complex was removed from the cells and replaced with growth medium (+) IL6 containing Ad-LacZ (L) or Ad-eIF5AlK5GA (5M). After 24 hours, the cell lysate was harvested and analyzed by Western blotting using antibodies against phosphorylated-NF-kB p65 (Ser 536) and eIF5A. As expected, this eIF5Al siRNA #1 did not affect the performance of the virus by the 61?5 in/5 accumulation. The phosphorylation of NF-kB p65 in serine 536 regulates activation, nuclear localization, protein-protein interaction. Role and transcriptional activity. See Figure 7C. ® Example 6D: Pretreatment with eIF5Al siRNA #1 reduced the expression of phosphorylated NF-κΒ and ICAM-1 in human multiple myeloma cells prior to adenovirus infection. Using Lipofectamine 2000 One of the control group siRNA (C) or two 'siRNA target eIF5Al (#1 and #2) was transfected into KAS cells. After 72 hours of initial transfection, the cells were incubated with the same siRNA. A second transfection was performed. After 4 hours, the transfection complex was removed from the cells and replaced with growth medium (+) IL6. 24 hours after the second transfection 142866.doc -43- 201023898 At this time, the cells were stimulated with 40 ng/ml TNF-α, and cell lysates were harvested at 0, 4 or 24 hours, and anti-phospho-NF-kB p65 (Ser 536), ICAM-1 and myoblast were used. Protein antibodies were analyzed by Western blotting. After transfection with two eIF5Al-specific siRNAs, TNF- was observed. Induction of NF-kB p65 phosphorylation (ser 536) and decreased expression of ICAM-1. Phosphorylation of NF-kB p65 in serine 536 regulates activation, nuclear localization, protein-protein interaction, and transcriptional activity. -1 is an intercellular attachment surface glycoprotein that is believed to be involved in the pathogenesis of multiple myeloma. See Figure 7D. Example 6E: Pretreatment of KAS cells with siRNA increases delivery of eIF5Alk5GR* in the presence of IL-6 Apoptosis induced. Lipofectamine 2000 was used to transfect control group siRNA (hcon) or human eIF5Al siRNA (h5Al) into KAS cells. The cells were re-transfected with siRNA after 72 hours. 4 hours after removal of siRNA transfection medium The empty vector (mcs) or the eIF5Alk5GR (K50R) plastid PEI complex was added to the cells. The growth medium used throughout the study contained IL-6. After 72 hours, the cells were stained with Annexin/PI and FACS analysis to measure > weekly death. See Figure 7F. Example 7: Co-administration of eIF-5Al plastid and eIF-5Al siRNA delayed the growth of multiple subcutaneous bone tumors (Figure 8-10). Subcutaneous injection of KAS cells To SCID mice. When observed The tumor was touched and the 10 million KAS-6/1 myeloma cells in 200 pL PBS were injected into the right abdomen of six 3-5 week old SCID/CB 17 mice. Treatment begins when the tumor reaches a minimum size of 4 mm3. 142866.doc •44- 201023898 The pEI complex containing pCpG-mcs (empty vector) and control group siRNA was injected intratumorally twice a week into control group mice (control group consisting of 3 mice: Cl, C-2 and C-3). A PEI complex comprising the anti-RNAi plastids pCpG_eIF5Alk5〇R and eIF5Al siRNA was injected intratumorally twice a week into treated mice. (The treatment group consisted of 3 mice: 5A-1, 5A-2 and 5A-3). Inject into multiple sites in the tumor to prevent backflow and use a slow injection rate to increase intake. The data in Figure 8 shows the tumor volume of all mice in this group. The data shown in Figure 9 is the mean tumor volume of each group of +/_ standard errors. The asterisk represents statistical significance (*=p<〇.〇25; n=3). Figure 10 shows that co-administration of eIF5Al plastids and eIF5A1 siRNA reduced the weight of multiple bone marrow subcutaneous tumors. The data shown is the mean tumor weight for each group of +/- standard errors. The asterisk represents statistical significance (*=p<0.05; n=3). 2x0.1 ml of JET-PEI (P〇lyPlus) was used in this in vivo test. The N/P ratio is 8. The PEI/DNA/siRNA complex was formed in a total volume of 0.1 ml of 5% glucose. The procedure for forming the composite is as follows. 1. Reheat the ingredients to room temperature. Keep sterility.

2. Dilute 20 pg of plastid DNA (2 mg/ml approximately 10 μΐ) and 1 μL siRNA (1 mg/ml '10 μΐ) to a total volume of 25 μl. Use sterile water to fill in the insufficient amount. 3. Adjust the volume of the sputum solution to 50 μΐ of 5% glucose by adding 25 μM of 1% glucose. Gently mix and centrifuge briefly. 4. Dilute 4·8 μΐ of in vivo JETPEI to a total volume of 25 μΐ of 10% glucose. Adjust the volume to 50 μl with sterile water to a final concentration of 5〇/〇 142866.doc •45· 201023898 Glucose. Gently mix and centrifuge briefly. 5. Immediately add 50 μΐ of diluted PEI to the dilution of the 5 μ μ1 to avoid reverse order. Gently mix and centrifuge immediately (spind〇wn). 6. Incubate for 15 minutes before injection. The complex is stable for 6 hours. CpG-free selection vectors and pCpG plastids were obtained from InviV〇Gen. These plastids were completely free of CpG binuclear acid and were named pCpG. These plastids produce high levels of transgenic genes in both in vitro and in vivo, providing sustained in vivo performance relative to the cmv-based plastids. The pCpG plastid contains an element such as a gene encoding a selectable marker or reporter, which is a naturally deficient CpG dinucleotide, modified to remove all CpG or completely synthesized. of. The fact that CG is the only non-essential and replaceable dinucleotide between the 1/ dinucleotides forming the gene code makes the synthesis of these new dual genes possible. The eight codons contain a cg encoding five different amino acids. All 8 codons can be replaced by at least one of the two codons encoding the same amino acid to create a new dual gene encoding a protein having the same amino acid sequence as the wild type, thus The wild type counterpart is active. These new dual genes can be individually removed from a plastid that is pM〇D and can be easily removed from the plastid. The pCpG plastid allows for long-lasting sustained expression in vivo and represents a useful tool to study the utility of CpG on gene expression in expressing TLR9 in vivo and in vitro, and their utility to the natural and acquired immune system . The empty vector, pCpG-mcs (Invivogen), is a vector that does not exhibit the gene product 142866.doc -46 - 201023898, has only a multiple cloning site' and serves as a control group vector. A HA-tagged eIF5Alk5()R cDNA was sub-selected into the Ncol and Nhel site of a pCpG-LacZ vector (Invivogen), which has been removed from the vector to generate the processing vector pCpG-eIF5Al (K50R) ). The DNA was prepared using an Endo-Free Qiagen kit. The measured endotoxic amount was <0.03 EU/ug; DNA should be dissolved in water to 2 mg/ml. The control panel siRNA used in this experiment was a non-target control group siRNA (D-001810-01) from Dharmacon confirmed by a microarray. The siRNA was obtained by a modification (siSTABLE) to prevent degradation in serum. The eIF5Al siRNA used in this experiment was designed to target the 3'UTR of human eIF5Al. There is no similarity between this eIF5Al siRNA and mouse eIF5Al, so this siRNA should only inhibit human (but not mouse) eIF5Al. This siRNA also has no similarity to eIF5A2 (human or mouse). The siRNA was obtained by a modification method (siSTABLE) to prevent degradation in serum. The eIF5Al siRNA has the following target sequences: 5' GCU GGA CUC CUC CUA CAC A (UU) 3 Dissolve the siSTABLE siRNA in water to 1 mg/ml (packed at -20 °C). Tumor length (1) and width (w) dimensions were measured 2-3 times per week using a digital caliper. Tumor volume was calculated according to the following equation: 1 = length; minimum size w = width; maximum size 142866.doc -47- 201023898 Tumor volume (mm3) = l2 * w * 0, 5 Statistical analysis using Student's t test (Student's /- Test) for statistical analysis. A letter of reliance on 95% (p<〇.〇5) is considered significant. Example 8: Co-administration of eIF5Al plastids and eIF5Al siRNA delayed the growth of multiple subcutaneous bone tumors and led to tumor shrinkage. In another study, again, KAS cells were injected subcutaneously into SCID mice. Treatment begins when a touchable tumor is observed. PEI complexes containing pCpG-mcs (empty vector) and control group siRNA were injected intratumorally twice a week into control group mice (control groups G-1, G-2 and G-3). A PEI complex containing anti-RNAi plastid PCpG-eIF5Alk50R (20 pg of plastid DNA) and eIF5Al siRNA (10 pg of siRNA) was injected intratumorally twice a week into treated mice (treatment group G_4, G) -5 and G-6). The data shown in Figure 11 is the mean tumor volume for each group of +/- standard errors. The asterisk represents statistical significance (*=p<0.025; n=3). Six injections (red arrow) were administered over a 21-day period. Example 9: Intravenous administration (i.v.) eiF5Al siRNA and intratumoral administration (l.t.) PEI/eIF5AlK50R plastid complex resulted in a reduction in multiple bone marrow subcutaneous tumors (Group 2B). KAS cells were injected subcutaneously into SCID mice. When a audible tumor was observed, treatment was initiated with an initial tail injection of 50 μg of control group siRNA (control group) or human eiF5 A1 siRNA (treatment group). Control group mice were treated twice a week with intratumoral injection of PEI complex containing pCpG-mcs (empty vector; control group; g-1, G-2 and G-3). Treated mice 142866.doc •48· 201023898 followed by a PEI complex comprising the anti-RNAi plastid pCpG-eIF5Alk50R (20 pg plastid DNA) (treatment group; G-4, G-5 and G-6) Intratumoral injections were processed twice a week. Control group mice continued to receive i.v. injection control group siRNA once a week (control groups R-1, R-2 and R-3). Treatment group mice were followed by one week of i.v. injection of human eIF5Al siRNA (20 μβ) (treatment groups R-4, R-5 and R-6). The nurturing in Figure 12 is the tumor volume of all mice in each group. Six intra-injection of PEI/DNA (red arrow) and four times of siRNA (blue arrow) i.v. injection were administered over a period of 21 days. ❿ Figure 13 provides an overview of the results from Examples 8 and 9. The data shown in Figure 13 is the mean tumor volume of mice in each group of +/- standard errors. The asterisk represents the statistical significance between the treatment and control groups (**=ρ<〇.〇1;***=p<0.001; n=3) ° The process of forming the PEI complex: 1. Warming the ingredients back To room temperature. Keep sterility. 2. Dilute plastid DNA or plastid DNA + siRNA to a total volume of 25 μM. Use sterile water to adjust the volume. ® a) For complexes with only plastid DNA: Dilute 20 pg of plastid DNA (10 μM at 2 mg/ml) to a total volume of 25 μM. Fill the difference volume with sterile water. b) For plastid DNA + siRNA complex: 20 pg of plastid DNA (~10 μΐ at 2 mg/ml) and 10 pg of siRNA (10 μΐ at 1 mg/ml) were diluted to a total volume of 25 μΐ. Fill the difference volume with sterile water.

3. Adjust the volume of the DNA 142866.doc •49·201023898 solution to 50 μΐ of 5% glucose by adding 25 μM of 10% glucose (provided by the ΡΕΙ kit). Gently mix and centrifuge briefly. 4. Dilute JETPEI in vivo to a total volume of 25 μΐ of 10% glucose. a) For complexes with only plastid DNA: 3.2 μL of in vivo JETPEI was diluted to a total volume of 25 μL of 10% glucose. Adjust the volume to 50 μΐ with sterile water to give a final concentration of 5% glucose. Gently mix and centrifuge briefly. b) For plastid DNA + siRNA complex: 4.8 μΐ of in vivo JETPEI was diluted to a total volume of 25 μΐ of 10% glucose. Adjust the volume to 50 μΐ with sterile water to give a final concentration of 5% glucose. Gently mix and centrifuge briefly. 5. Immediately add 50 μΐ of diluted sputum to the 50 μ ΐ diluted DNA (do not reverse the order!). Gently mix and centrifuge immediately. 6. Incubate for 15 minutes before injection. The complex is stable for 6 hours. Regarding the tail vein injection of siRNA, the initial siRNA injection was 50 micrograms. The siRNA was diluted to 0.4 mg/ml in PBS. Each mouse was injected with 125 μΐ (50 pg) into the tail vein. Subsequent injections of stable siRNA in 20 pg of serum per mouse were administered twice a week. The siRNA was diluted to 0.4 mg/ml in PBS. Each mouse was injected with 50 μΐ (20 μδ) into the tail vein. Figure 13A shows that co-administration of eIF5Al plastids and eIF5Al siRNA results in tumor shrinkage. KAS cells were injected subcutaneously into SCID mice. Treatment begins when a touchable tumor is observed. The PEI complex containing the anti-RNAi plastids pCpG-eIF5Alk50R and eIF5Al siRNA was injected into mice per week 2 142866.doc • 50- 201023898 tumors (treatment group; G-4, G-5 and G-) 6). Six injections were administered over a period of 21 days. At 42 days after the start of treatment, the mice were sacrificed, the skin under the tumor was opened, and evidence of tumor growth was examined. Tumor growth was not observed in any of the 2A treated mice. Figure 13C shows in vivo (i.v.) administration of eIF5Al siRNA and intratumoral (i.t.) administration of PEI/eIF5AlK50R plastid complex to cause tumor shrinkage in multiple subcutaneous tumors of bone marrow. KAS cells were injected subcutaneously into SCID mice. When an audible tumor was observed, treatment was started with an initial injection of 50 micrograms of human eIF5Al siRNA (treatment group). Next, the PEI complex containing the anti-RNAi plastid pCpG-eIF5Alk50R was injected intratumorally twice a week to treat the mice (treatment group, R-4, R·5 and R-6). Mice continued Human eIF5 A1 siRNA was received by i.v. injection once a week. The processing is ended 21 days after the initial processing. At 42 days after the start of treatment, the mice were sacrificed, the skin under the tumor was opened, and evidence of tumor growth was examined. No tumor growth was observed in one of the mice in this treatment group (Group 2B). Example 10: Intravenous co-administration of eIF5Al plastids and eIF5Al siRNA delayed the growth of multiple bone marrow subcutaneous tumors. KAS cells were injected subcutaneously into SCID mice. When a audible tumor was observed, treatment was initiated with an initial injection of 50 micrograms of control group siRNA (control group) or human eIF5A1 siRNA (treatment group). The mice were then treated intravenously (red arrow) or intraperitoneally (green arrow) to 2 times per week to include PEI complexes of pCpG_mcs (empty vector; control group; Al, A2 and A3) or contain anti-RNAi PEI complex of the bulk pCpG-eIF5Alk50R (treatment group; A4, 142866.doc 51 201023898 A5 and A6). Mice continued to receive control group siRNA (; control group Ai, 2 and A3) or human eIF5Al siRNA (treatment group; A4, A5 and A6), once a week by intravenous injection (blue arrow) ^ Tumor volume of all mice in each group. The data shown in Figure 14 is the tumor volume of all mice in each group. Example 11: Intravenous (iv) administration of elF5Al siRNA and intravenous (i.vj or intraperitoneal (ip.) administration of PEI/eIF5AlK50R plastid complex resulted in delayed growth of multiple bone marrow subcutaneous tumors. SCID mice were KAS cells were injected subcutaneously. When the audible tumor was observed, the initial injection initiation treatment with 50 μg of control siRNA (control group) or human eIF5A1 siRNA (treatment group) was started. The control group was sequentially treated with vein or abdominal cavity. The PEI complex containing pCpG_mcs (empty vector) was treated by injection (once a week) (three mice in the control group, Bl, B2 and B3). The mice in the treatment group were injected intravenously or intraperitoneally (once a week). Treatment of PEI complex containing anti-interference RNA (RNAi-resistant) plastid pCpG-eIF5AlK50R (treatment group; B4, B5 and B6). The mice continued to receive control siRNA intravenously every week (control group, Bi, B2 and B3) Or human eIF5Ai siRNA (three mice in the treatment group, B4, B5 and B6). This test started with an initial 5 μg microgram of siRNA injection (day 2 of Figure 15). The subsequent injections used 20 micrograms per week. siRNA. siRNA is naked when administered. That is, there is no delivery vehicle. The PEI complex contains 2 〇pg of plastid DNA. The initial PEI injection is administered by intraperitoneal injection, and the subsequent injection is administered intravenously. The data shown in Figure 5 is in each group. Tumor volume of all mice. Figure 1612 is for the contours of Figure 1 and Figure 11. §ciD mice were injected subcutaneously with 142866.doc -52- 201023898 cells. This treatment was started when the audible tumor was already visible. The first group of mice received an intravenous injection of control siRNA (control group; group A) or eIF5Al siRNA (treatment group; group A) and intravenous or intraperitoneal injection of PEI complex containing pCpG-mcs (control group; Group A) or anti-RNAi plastid complex (treatment group; group A). The second group of mice were injected intravenously or intraperitoneally twice a week with PEI complex containing pCpG-mcs (empty vector) and control siRNA ( Control group; Group B) or PEI complex containing anti-RNAi plastids pCpG-eIF5AlK50R and eIF5Al siRNA φ (treatment group; sputum group). Data are expressed as the positive and negative standard deviation of the mean tumor volume of mice in each group. Group and control group There are significant differences on the tenth (*=ρ <0·05; ***=p <0.001; n=3) ° The experimental steps for preparing the PEI complex and siRNA have been described in detail in the previous examples. : Co-administration of eIF5Al plastids and eIF5Al siRNA can delay the growth of multiple myeloma and lead to tumor shrinkage. SCID mice were first injected subcutaneously with KAS cells and treatment was initiated after observation of the sensible swollen tumor formation. The control group mice were first injected intraperitoneally with PEI complex containing pCpG-mcs (empty vector) and control siRNA twice (control group consisted of three mice, control group 1, control group 2 and control group). 3). In the treatment group, PEI complex containing anti-RNAi plastid pCpG-eIF5AlK50R and eIF5Al siRNA was injected intratumorally twice a week (four mice in the treatment group were: 5A-1, 5A-2, respectively). 5A-3 and 5A-4). This tumor was injected with a PEI complex containing 20 micrograms of plastid DNA and 10 micrograms of siRNA. The results of the product of 142866.doc -53- 201023898 for all mice in each group are shown in Figure 17. Example 13: Subcutaneous multiple myeloma was reduced by intravenous administration of eIF5Al siRNA and intratumoral injection and PEI/eIF5AlK5GR plastids. SCID mice were injected subcutaneously with KAS cells. When a measurable tumor was observed, 50 μg of control group siRNA was injected (three mice in control group, control group 1, control group 2 and control group 3) or It is human eIF5Al siRNA (there are three mice in the treatment group, respectively: 5A-1, 5A-2, 5A-3). Intratumoral injection of PEI complex containing pCpG-mcs (20 μg) was administered twice a week after control mice (control group 1-3). The mice in the treatment group received intratumoral injections of anti-RNAi plastid pCpG-eIF5AlK5()R (20 μg) (5A-1, 5 A-2 and 5 A-3) twice a week. The control group mice were injected twice a week through the tail vein and continued to receive control siRNA (20 μg). The mice in the treatment group were administered twice a week through the tail vein and continued to receive human eIF5Al siRNA (20 μg). These injections were completed 48 hours prior to intratumoral injection. Small fragment siRNA is naked when administered (i.e., not constructed on a vector). The experimental results of tumor volume of all mice in each group are shown in Fig. 18. Example 14: Co-administration of the eIF5AlK5GR plastid and eIF5Al siRNA against the EF 1 or B29 promoter can delay the growth of subcutaneous multiple myeloma and lead to tumor shrinkage. SCID mice were injected subcutaneously with KAS cells and treatment was initiated when a palpable tumor was observed. The mice were intratumorally injected with PEI complex twice a week containing control vectors (G1 and G2) or eIF5Al plastids driven by the B29 promoter (G3 and G4) or the EF1 promoter, and control siRNA (Gl, 142866.doc -54- 201023898 G3, G5) or h5Al siRNA (G2, G4, G6). The results for each group are expressed as the positive and negative standard deviations of the mean tumor volume. Note: The B29 promoter represents a B-cell-specific promoter, however, the B29 promoter/mCMV enhancer used in this experiment drives high-performance HA-eIF5AlK5〇R, but non-B-cells, in KAS cells in vitro. Specific performance (may be partly caused by CMV enhancers). See Figure 19. Example 15: Co-administration of eIF5Al siRNA and EF1 or B29 promoter-driven eIF5AlK5GR plastids can increase the anti-myeloma growth of 61卩5 into 11^()11 φ bodies and reduce tumors The load caused. SCID mice were injected subcutaneously with KAS cells and treatment was initiated when a palpable tumor was observed. Mice were injected intratumorally twice weekly with control vectors (G1 and G2) or eIF5Al plastids driven by the B29 promoter (G3 and G4) or the EF1 promoter, as well as control siRNA (Gl, G3, G5) or h5Al siRNA. (G2, G4, G6). After 24 hours from the start of treatment, multiple myeloma was taken and the weight of the tumor was measured. The results for each group are expressed as the mean negative standard deviation of the tumor weight mean. See Figure 20. ® Example 16: eIF5Al siRNA synergistically increases apoptosis induction driven by Ad-eIF5A-infected lung adenocarcinoma cells.

A549 cells were first infected with Ad-LacZ or Ad-eIF5A. The cells were then transfected with siRNA and virus infection for 4 hours with the addition of control siRNA or siRNA targeting human eIF5Al (h5Al), and the cells were recultured in fresh medium for 72 hours. The apoptosis was then detected by Annexin/PI labeling. Note: Over-expression of eIF5A in this cell line 'will be due to DHS 142866.doc •55· 201023898 and the amount of DOHH, resulting in the accumulation of unhypusinated eIF5A' and thus the overexpression of eIF5 AK5GR caused by early holes Death, produces a similar effect. These results indicate that the synergistic effect of eIF5A, which simultaneously inhibits hypusinated and eIF5A, which is excessively unhypusinated, can also be observed in non-myeloid tumor cells. See Figure 21. Example 17: Constructing the pExp5A plastid. pExp5A is a characterization of a cytoplasm with a reduced CpG dinucleotide that drives human eIF5AlK5GR to express its significance in B cell lines. The vector is derived from pCpG-LacZ, which is completely deficient in CpG dinucleotide (Invitrogen). None of the factors used to replicate and screen E. coli contained CpG dinucleotides. All of the original CMV enhancer/promoter and LacZ genes from the CpG LacZ vector were replaced with the human minimal B cell specific promoter (B29/CD79b; Invitrogen) and human eIF5AlK50V; t to drive B cells. Dedicated to eIF5AlK5()R. The B29 DHS4.4 3' enhancer was also added to this vector downstream of the eIF5Al expression, to enhance the role of the B29 promoter to reduce non-specific expression in non-B cells (Malone alpha human 2006. B29 gene silencing In pituitary cells are regulated by its 3' enhancer· J. Mol. Biol. 362: 173-183). When the B29 minimal promoter is added to the vector, the eIF5AlK5GR*B29 DHS4.4 3' enhancer will carry 32 CpG dinucleotides into the vector. A factor used for E. coli expression. The starting point of replication: five.co/z· R6K gamma ori. *Because the R6K gamma origin of replication is on the plastid, the pCpG plastid is only 142866.doc -56· 201023898 can be amplified in E. coli strains with the alpha mutant gene, which cannot be found in standard E. coli Replication ^ Therefore, the pCpG plastid is simultaneously provided (Invivogen) together with the α/? mutated gene and the Dcm methylated (Invivogen) Escherichia coli strain GT115. Bacterial promoter: EM2K, a CpG-free bacterial EM7 promoter. Screening markers: anti-ZeocinTM gene; synthetic genes that are synthetic without CpGs. Factor for expression in mammalian cells Φ Mammalian promoter: Human-167 bp minimal B29 (CD79b) promoter for tissue-specific expression in B cells. A synthetic intron (I 140) appears in the 5' untranslated region. Polyadenylation signal: A polyadenylation signal of a Late SV40 without a CpG dinucleotide. 3% enhancer: human B29 DHS4.4 3 · end enhancer. MAR: Two matrix-free regions (MAR) without CpG are constructed between bacterial and mammalian translation units. One of the W-hanging regions is derived from the human IFN-β gene, and the other is derived from the 5, end region of the β-erythroglobulin gene. The predicted ρΕχρ5Α gene sequence (3371 bp is provided in Figure 23). Amino acid sequence of eIF5AlK5GR * K50R mutation is indicated by the bottom line

MADDLDFETGDAGASATFPMQCSALRKNGFVVLKGRPCK

IVEMSTSKTGRHGHAKVHLVGIDIFTGKKYEDICPSTHNM

DVPNIKRNDFQLIGIQDGYLSLLQDSGEVREDLRLPEGDLG 142866.doc -57· 201023898 KEIEQKYDCGEEILITVLSAMTEEAAVAIKAMAK Constructing pExp5A-Building a large network Step 1: Selecting B29 DHS4.4 3' enhancer and re-colonization into pGEM T easy vector (Promega) - producing pGEM-4.4enh #8. Step 2: Re-selection of the least B29 promoter region into pCpG-LacZ (Invivogen) - production B29-5 #3 〇 Step 3: Re-selection of HA-eIF5AlK5QR into B29-5 #3 vector-generating B29-5 #3-eIF5AlK50R. Step 4: Create a new multi-enzyme cleavage site region on pCpG-mcs (Invivogen) to generate pCpG-Linker4. Step 5: Re-selection of the B29 DS4.4 3' enhancer into the pCpG-Linker4 region - production of pCpG-DHS4.4. Step 6: Re-selection of the B29 promoter and HA-eIF5AlK5GR plus SV40 pA expression 匣 Enter pCpG-DHS4.4 to generate pExp-5. Step 7: Replace the HA-eIF5AlK5()R into a non-HAeIF5AlK5()R at pExp-5 to generate the final vector pExp5A. Detailed steps for construction Step 1: Selection of B29 DHS4.4 3' enhancer and re-colonization into pGEM T easy vector (Promega) - yield pGEM-4.4enh #8. The B29 DHS4.4 3' enhancer was subjected to a PCR reaction using the following primers to extract the genomic DNA extracted from KAS cells (human multiple myeloma cell line). The positive-strand primer is 5'-CAGCAAGGGAGCACCTATG-3' and the anti-strand primer is 5'-GTTGCAGTGAGCGGAGATG-3'. The primer sequence was designed using the sequence of the human CD79B/GH-1 intergenic region (Accession AB062674) 142866.doc -58 - 201023898. This 608 bp PCR reaction fragment was re-sorted into the pGEM® T easy selection vector (Promega) and sequenced. Komatsu et al. 2002. Novel regulatory regions found downstream of the rat B29/Ig-b gene. Eur. J. Biochem. 269: 1227-1236. The sequence of the B29 DHS4.4 3' enhancer PCR fragment (297 bp) in pGEM-4.4enh #8 is expressed as follows

ACCACCCTGGGCCAGGCTGGGCCAAGCCAGGCGGCCCC φ TGTGTTTTCCCCAGTCTCTGGGCTGCTGGAGGGAACCAG GTTGTTTTGGCATCAGCCTCTACTGAGrcGGAGrrrTTr CTTTCCTGCTGCTTTGCATAGTGGCACTAATTCCGTCCT CCTACCTCCACCAGGGACCTAGGCAGCCGGGTAGATGGT GGGAGGAGGCTTCACTTCTCCCCCAAGCAGGGTCTCCAC CTGCTTG ^ GGCTGCCCTGGGTTGGGGGAGGCCTTGGCTT TACCTAAAGACTTTTTAACACCTCT

The 〇+4.4 region includes locations where many transcription factors can be combined • SRY GTTGTTT

_ GATA CATCAGC _ OCT-X GCTGCTTTGCATAG NF-KB GAGGCTGCCC Alignment of B29 DHS4.4 3' enhancer PCR fragment (297 bp) and human CD79B/GH-1 intergenic region in pGEM-4.4enh #8 (Accession AB062674 Sequence 142866.doc -59- 201023898 1 10 2Φ 30 40 50 60 70 80 90 100 110 120 190 I Circle-circle -4--------♦---------♦- --------— -----♦-------♦ One by one | C6aWOICCTSCq>CCT«aaMCaW6reaWTCW>CCreamT6CIMMCCTCCCTCCaW6CCB66CTCTSCTCCIICTTCCT6nfiRCCCT6Sft6S6flflTCCTTCGftG6CCCCTCT6CTBTTCCT8

131 140 190 160 170 180 190 2Φ0 21Φ 220 230 240 250 260 I·····—»«——-4——............♦---------♦- ------ a ♦ a _ a, a cTCTawTTca »e« > awiBcwrraT «: T6mM6CT« xHHiiMcmh «WTawBKce6aciiGC6awcTB6Ta > gwcwmaxHimTcac £ asftTTCccTCT66iWTTCCB66TG6 261 270 280 290 300 310 | II» - | c «4» fiCCftfi6T68CTW > S6CCCCaiC6CCraaCTrroiMIWMTnCTCCIiaaCCCT66eCdi66CT066CCmBCai66C66CCCCTfiTCTmCCCa >STCTCTSC6CTCCTa»66CRiiCa>66r PCR IICCI>CtCTCCeCCftCGCTC6CCCIWCCa>6CCC6aXCTGT6TTTTCCCCIi6TCTCT6G6CTSl «ASUS ..................................... ...........anmCTG6GCCRGGC7G&GCC»KCCflGGCGGCCCCmGnnCCCCfl6TCTCT6GGCTGl

Fenb PCR

391400 410 420 430 440450460 I .........•丨.......·-------------- rrT6GCHTCft6CCTCTWCTSWGCCeBB6CCL I ItLII lIXThtlltl HMJIIII6T6SC«CTB<ITTCC6TO

________STUGRTO m8GCftTCflGCCTCTRCTSHSC «ai« CCCTTCrmCCTIiCTamGCftTI «T6SC ^ TRBTltXSTaTCCTiCCimCCfle86 ^ raG6CBSCCCGeT« »TeeT6eSB6Gft6eCTTCHCTTC Tn6GCaTCB6CCTCTHCreagXfifiBBCCCTTCXmiXT6CTBCm6DITft6TS6C« CTiWnCCeTmCCTfgCTCCHCC «8fi6HCCTWgCB6CC666TI« aTeeTB (i6fleC «GGCTTCICTTC cenb PCR Consensus I-1-1-1 --- · ---------- ------4-»..... I--------♦-------------------1 TccecawBCBaccTCTCC«XTGCTTai6arreca:T6B6TT666Gaic6CCTTSGcmRCCTWwa« :TTTTraBcaccTCTawc«cflCft6mcccTeiw>CTTTCTMCTM ιβιιιιιιιιη TCCCCCM«CRG6GTCTCCflCCTGCTTGfl6eCTBCCCre6GTTGGGGGMGCCTTG6CmilCCTW«GfCmTTinCflCCTCT TCCGCCineCRGGSTCTCCINXTGCTTGRGBCTGCCCTGGGTTG6SBBIGGa:TT8GCTTTRCCTMW6RCTTnTfnaCCTCT................................... ..........

Step 2: Re-selection of the minimal B29 promoter into pCpG-LacZ φ (Invivogen)-producing Β29-5 #3 ° Minimal-167 human Β29 promoter is a commercial substance with a full-length human Β29 promoter In the body (pDrive-hB29; Invivogen), the following primers were used: the positive-strand primer S'-CCAACTAGTGCGACCGCCAAACCTTAGC-S': and the anti-strand primer 5'-CAAAAGCTTGACAACGTCCGAGGCTCCTTGG-3, and the PCR reaction was amplified. The PCR fragment was then digested with Spel and Hindlll and ligated into pCpG- with Spel and Hindlll cleavage sites.

In the LacZ vector (Invivogen) to produce B29-5 #3. 〇 Determine the sequence of the least B29 promoter PCR fragment (188 bp) in B29-5 #3

GCGACCGCCAAACCTTAGCGGCCCAGCTGACAAAAGCC

TGCCCTCCCCCAGGGTCCCCGGAGAGCTGGTGCCTCCCC

TGGGTCCCAATTTGCATGGCAGGAAGGGGCCTGGTGAG

GAAGAGGCGGGGAGGGGACAGGCTGCAGCCGGTGCAGT

TACACGTTTTCCTCCAAGGAGCCTCGGACGTTGTC Alignment of the least B29 promoter PCR fragment (B29_min) in B29-5 #3 -60- 142866.doc

I 201023898 column and full-length human B29 promoter sequence from pDrive-hB29 Ι- ΙΟ

Ctre〇IB6aXCflCTilCTMBCfieil6BCTTSTa«a»ICTMaiBBTCqKftMWWCflCqacanTTflBatTCTCTTCCTCTCCTS6GGGTC6B6GBTGftGttflCB>«HRHCTB6CTSCai6GMftC

CTBIBaCTBTB6CTCaiTCC«C«CTCT6HBaiCftSeCIWOTl

I

•I

820 830 840 850 inCBTflttHCBSCTSCCTBTTIIJBLTaiaiBCCCMeiWTl mGRCTTGCCSeGt

TCCCCCCTTCCM«l6TT6CCIIGIC06aiGe6C6M66CT66CTC6CCai6C6BOTOI

1100 1110 1120 1130 1140 1150 USO 1170 CCTGCrgg8flBfleCCT6CCCTCCCCCB6fiSTCCCC66 »6afiCT66TgXTCCCCT6fieTCCaWITTT6afffi6ai6aMSeB6CCTB6TCT6aW6ftSfiCCCGCnBCCaiai6gnrSCfl6CC6CTfiCH6 CCflSCTfiBaMBRBCCTOXCTCCCCCaS6eTaXCSSft6MCTC6TBXTCCCCTCfi6TCCPWm6aire6CflttBBBB66CCTfi6TCn〇CWiCnCCCC6Bg > aagiOIC8CmB6CCecreC« 6 CCfl6CTgCaWB6CCT6CCCTCCCCCfl6B6TCCCCMa6R6CT66T6CCTCCCCT6S6TCCawm6COTC6CB6 «WBSCeCCT6eTqi0« WGHC6CCCSGncaaiRCftSCCTgimCS6T6CB6 1190: CRfl6GnGi rrGGT6CCTCCCCT68GTa 1220 1230 1171 U80 eleven B29_pron TTnCflCeTTTTCCTCC Ming GGRGCCTC Ming RCCTT6TCfK666m666GTC66G6fKMMC66rm: B29 ^) in T ftCIC6TmcCTCCfn6GRGCCTCGGAC6mTC - C6GRCG1 &quot?;

Comensus TTnCKGTnTaTCCf»6GAGCCTI iTTSTC* Step 3: Re-implantation of HA-eIF5AlK5GR into the B29-5#3 vector - production of pB29-eIF5AlK50R_7.

HA-eIF5AlK5()R uses pHM6_eIF5AlK5()R as a DNA template, using the following bow I: Orthoptera I: 5'-CGCCATGGACATGTACCCTT ACGACGTCCCAGACTACGCTGCAGATGATTTGGACTTCG AG-3, and anti-strand bow | 5'-CGCGCTAGCCAGTTATTTTGCCATCGC C-3 'Amplification was performed, and the resulting PCR fragment was subjected to Ncol and Nhel digestion, and then cloned into the B29-5 #3 vector carrying the Ncol and Nhel restriction sites to replace the LacZ gene. Sequence of the HA-eIF5AlKS0R PCR fragment in pB29-eIF5AlK50RJ7 -61 142866.doc 201023898 Column (497 bp)

ACATGTACX: CTTACGACGTCCCAGACTACGCTGCAGATGATTTGGACTTCGAG

ACAGGAGATGCAGGGGCCTCAGCCACCTTCCCAATGCAGTGCTCAGCATTAC

GTAAGAATGGTnTGTGGTGCTCAAGGGCCGGCCATGTAAGATCGTCGAGAT

GTCTACTTCGAAGACTGGCAGGCATGGCCATGCCAAGGTCCATCTGGTTGGC

ATTGATATTTTTACTGGGAAGAAATATGAAGATATCTGCCCGTCGACTCATAA

CATGGATGTCCCCAACATCAAAAGGAATGATTTCCAGCTGATTGGCATCCAG

GATGGGTACCTATCCCTGCTCCAGGACAGTGGGGAGGTACGAGAGGACCTTC

GTCTGCCTGAGGGAGACCTTGGCAAGGAGATTGAGCAGAAGTATGACTGTGG

AGAAGAGATCCTGATCACAGTGCTGTCCGCCATGACAGAGGAGGCAGCTGTT

GCAATCAAGGCGATGGCAAAATAACTG pB29-eIF5AlK50R_7 HA sequence after translation of HA, eIF5AlKS0R PCR fragment

HA epitope determinant eIF5AlK50R

K50R

MDMYPYDVPDYAADDLDFETGDAGASATFPMOCSALRKN

GFVVLKGRPCKIVEMSTSKTGigHGHAKVHLVGIDIFTGKK

YEDICPSTHNMDVPNIKRNDFOLIGIODGYLSLLODSGEVR

EDLRLPEGDLGKEIEOKYDCGEEILITVLSAMTEEAAVAIK

AMAK alignment in pB29-eIF5AlK50R_7 HA-eIF5AlK50R PCR fragment and human eIF5Al (Accession # NP_001961) sequence 10 20 30 40 50 60 70 eIF5ft

eIF5fl m>5R_K50R Consensus

wmjSFEr TRRODLDFE' ♦aflDDUSTTGDRQISITFPI

TPtlQCSnLRKHGFWI FPMQCSIIRKMGFVVI FPHQCSflLRKNGFVVI

80 90 100 110 120 130 一-♦一一一· I GKKYEDlCPSTHtMDVPfaKR»ffiFQLI6IQOGYLSLL〇DS6EVREDLfU.PEGDL

lKGRPCiaVEnSTS) < TGKH »mt (VHLy61DlFT6 VLKGRPaaVBtSTSKTGIttmiKVHLVeZDIFTGKKYEOKPSTHIteVPItIKRNDFQLmQniYLSLLQOSGEVREOLRLPEGOL VU (QRPCICIVEHSTSKTGrHGHflKVHLV6IDIFTGKKYEDICPSTHiM) VPNIKRNDFQLIGIQ06YLSLL«) SeEVRE0LRLPEG0L 131 140 150 160 165 I ------------_ ♦ --1

8KEIEQKYDCGEEILnVtSmTE£l«V{aKmRK

GKaEQKYDCGEEILimsmTEEfWVflIKflnflK 6KEZEQKyDCGEE3LrrVLSHHTEErwVflI»mRIC 142866.doc -62- 201023898 Step 4: Create a new multi-enzyme cleavage site region on pCpG-mcs (Invivogen) - produce pCpG-Linker 4. The pCpG selection vector is a pCpG-mcs G2 (Invivogen), which is first digested with EcoRI to remove the mCMV enhancer contained in the mammalian performance, hEF 1 promoter, synthetic intron, and more. The cleavage site, as well as the SV40 polyadenylation signal. This EcoRI-treated pCpG-mcs G2 vector was ligated to a synthetic linker with EcoRI binding to create a new vector without a promoter but with multiple cleavage sites (pCpG- Linker4). Synthetic joint, Linker4 with 2 EcoRI sticks:

EcoRI Adhesive AATTCTCGAGTCATCGATAAGCGGCCGCAOACGCGI? I "gAGCTCAGTAGC^ATTCGCCGGCSTOTGCGCATTAA Xhol Clal NoU Mlul

EcoRI sticky sequence near the new Doceu site on pCpG-Linker4

51 101 151 201 251 301 351 401 451 501 551 601 651

GGCATGTGAACTGGCTGTCTTGGTTTTCATCTGTACTTCATCTGCTACCT CTGTGACCTGAAACATATTTATAATTCCATTAAGCTGTGCATATGATAGA TTTATCATATGTATTTTCCTTAAAGGATTTTTGTAAGAACTAATTGAATT GATACCTGTAAAGTCTTTATCACACTACCCAATAAATAATAAATCTCTTT GTTCAGCTCTCTGTTTCTATAAATAT6TACAAGTTTTATTGI I Μ IAGTG GTAGTGATTTTATTCTCTTTCTATATATATACACACACATGTGTGCATTC VVTATATACAATTTTTATGAATAAAAAATTATTAGCAATCAATATTG CCACTGAIΠ I1GTTTATGTGAGCAAACAGCAGATTAAAAGGAATT CTCGAGTCATCGATAAGCGGCCGCAGACGCGTAATTCAGTCAATATGTTC ACCCCAAAAAAGCTGTTTGTTAACTTGCCAACCTCATTCTAAAATGTATA TAGAAGCCCAAAAGACAATAACAAAAATA7TCTTGTAGAACAAAATGGGA AAGAATGTTCCACTAAATATCAAGATTTAGAGCAAAGCATGAGATGTGTG GGGATAGACAGTGAGGCTGATAAAATAGAGTAGAGCTCAGAAACAGACCC ATTGATATATGTAAGTGACCTATGAAAAAAATATGGCATTTTACAATGGG ATAAA1 AAAACt 50 100 150 200 250 300 350 400 450 500 550 600 650 700 142866.doc -63- 201023898 701 751 801 851 901 9511001

\TATTTATAT ZAAAAAAAAT \AATcmrrA \GAAAAATTT rAACCACAAAGAAAAGATTGTTAATTAGATTGC mTTAAAATTAAAAAACCATTAAGAAAAGTCA

! AAAATGATGATCTTTTTCTTmTAGAAAAACAGGGAAATAT / GTAAAAAATAAAAGGGAACCCATATGTCATACCATACACAC / TCCAGTGAATTATAAGTCTAAATGGAGAAGGCAAAACTTTAAATCrrTTA GAAAATAATATAGAAGCATGCCATCAAGACTTCAGTGTAGAGAAAAATTT CTTATGACTCAAAGTCCT / ATGAATATTAAGACTTAT GGCCATAGAATGACAGAAAATATTTGCAAC 750 8008S0 900 9501000 1030 numbers in the sequence above is for ease of explanation the following characteristics: flGlo MAR (nucleotides 1-380); EcoRI recognition sequence (nucleotides 396- 401); 11 〇 1 recognition sequence (nucleotides 401-406); (: 131 recognition sequence (nucleotides 409-414); Notl recognition sequence (nucleotides 417-424); Mlul recognition sequence (nucleosides Acid 427-432); IFNfl S/MAR (nucleotide 438-1, 030). ▲

W Step 5: Re-selection of the B29 DS4.4 3'-end enhancer into the pCpG-Linker4 region - production of pCpG-DHS4.4. The B29 DHS4.4 3'-end enhancer was PCR-based with pGEM-4.4enh #8 as a template for the following primers. The positive-strand primer Jb 5I-GAAGCGGCCGCAC CACCCTGGGCCAGGCTGG-3' and the anti-strand primer are 5.-CCACGCGTA GAGGTGTTAAAAAGTCTTTAGGTAAAG-3. The resulting PCR fragment was digested with Notl and Mlul, and then ligated into the vector pCpG-Linker4 Q with a new polymorphic site of Notl and Mlul restriction sites to generate pCpG-DHS4.4. ≫ pCpG-DHS4.4 full-length sequence as bad Shu J (2,282 bp) 1 TTAATTAAAATTATCTCTAAGGCATGTGAACTGGCTGTCTTGGTTTTCAT 50 51 CTGTACTTCATCTGCTACCTCTGTGACCTGAAACATATTTATAATTCCAT 100 101 TAAGCTGTGCATATGATAGATTTATCATATGTATTTTCCTTAAAGGATTr 150 151 TTGTAAGAACTAATTGAATTGATACCTGTAAAGTCTTTATCACACTACCC 200 201 AATAAATAATAAATCTCTTTGTTCAGCTCTCTGTTTCTATAAATATGTAC 250 251 AAGTTTTATTGT1 II IAGTGGTAGTGATTTTATTCTCTTTCTATATATAT 300 301 ACACACACATGTGTGCATTCATAAATATATACAATTTTTATGAATAAAAA 350 351 ATTATTAGCAATCAATATTGAAAACCACTGATI ITTGTTTATGTGAGCAA 400 -64 - 142866.doc 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 950 1000 1050 1100 1150 1200 1250 1300 1350 1400 1450 1500 1550 1600 1650 1700 1750 1800 1850 1900 1950 2000 2050 2100 2150 2200 2250 2282

ACAGCAGATTAAAAGGAATTCTCGAGTCATCGATAAGCGGCCGCACCACC CTGGGCCAGGCTGGGCCAAGCCAGGCGGCCCCTGTGTTTTCCCCAGTCTC TGGGCTGCTGGAGGGAACCAGGTTGTTTTGGCATCAGCCTCTACTGAGCC GGAGCCCTTCCTTTCCTGCTGCTTTGCATAGTGGCACTAATTCCGTCCTC CTACCTCCACCAGGGACCTAGGCAGCCGG6TAGATGGTGGGAGC CACTTCTCCCCCAAGCAGGGTCTCCACCTGCTTGAGGCTG < GGGGAGGCCTTGGCTTTACCTAAAGACT f I I TAACACCTCT / i TCAGTCAATATGTTCACCCCAAAAAAGCTGTTTGTTAACTTGCC ATTCTAAAATGTATATAGAAGCCCAAAAGACAATAACAAAAATATTCTTG TAGAACAAAATGGGAAAGAATGTTCCACTAAATATCAAGATTTAGAGCAA AGCATGAGATGTGTGGGGATAGACAGTGAGGCTGATAAAATAGA6TAGAG CTCAGAAACAGACCCATTGATATATGTAAGTGACCTATGAAAAAAATATG GCATTTTACAATGGGAAAATGATGATCT

3GAGGAGGCTT

3CCCTGGGTTG

TACGCGTAAT

TGCCAACCTC

TCTTT 7 TTAGAAAAACAGG GAAATATATTTATATGTAAAAAATAAAAGGGAACCCATATGTCATACCAT ACACACAAAAAAATTCCAGTGAATTATAAGTCTAAATGGAGAAGGCAAAA CTTTAAATCTTTTAGAAAATAATATAGAAGCAT6CCATCAAGACTTCAGT GTAGAGAAAAATTTCTTATGACTCAAAGTCCTAACCACAAAGAAAAGATT GTTAATTAGATTGCATGAATATTAAGACTTA'I I I I IAAAATTAAAAAACC ATTAAGAAAAGTCAGGCCATAGAATGACAGAAAATATTTGCAACACCCCA GTAAAGAGAATTGTAATATGCAGATTATAAAAAGAAGTCTTACAAATCAG TAAAAAATAAAACTAGACAAAAATTTGAACAGATGAAAGAGAAACTCTAA ATAATCATTACACATGAGAAACTCAATCTCAGAAATCAGAGAACTATCAT TGCATATACACTAAATTAGAGAAATATTAAAAGGCTAAGTAACATCTGTG GCTTAATTAAAACAGGTAGTTGACAATTAAACATTGGCATAGTATATCTG CATAGTATAATACAACTCACTATAGGAGGGCCATCATGGCCAAGTTGACC AGTGCTGTCCCAGTGCTCACAGCCAGGGATGTGGCTGGAGCTGTTGAGTT ctggactgacaggttggggttctccagagattttgtggaggatgactttg

CAGGTGTGGTCAGAGATGATGTCACCCTGTTCATCTCAGCAGTCCAGCAC CAGGTGGTGCCTGACAACACCCTGGCTTGGGTGTGGGTGAGAGGACTGGA TGAGCTGTATGCTGAGTGGAGTGAGGTGGTCTCCACCAACTTCAGGGATG CCAGTGGCCCTGCCATGACAGAGATTGGAGAGCAGCCCTGGGGGAGAGAG TTTGCCCTGAGAGACCCAGCAGGCAACTGTGTGCACTTTGTGGCAGAGGA GCAGGACTGAGGATAACCTAGGAAACCTTAAAACCTTTAAAAGCCTTATA TATTCII Π I I IICTTATAAAACTTAAAACCTTAGAGGCTATTTAAGTTG

CTGATTTATATTAATTTTATTGTTCAAACATGAGAGCTTAGTACATGAAA

CATGAGAGCTTAGTACATTAGCCATGAGAGCTTAGTACATTAGCCATGAG

GGTTTAGTTCATTAAACATGAGAGCTTAGTACATTAAACATGAGAGCTTA

The number in the sequence above GTACATACTATCAACAGGTTGAACTGCTGATC is used to facilitate the description of the following characteristics: flGlo MAR (nucleotide 1-400); EcoRI identification sequence (nucleotides 416-421); and 11〇1 recognition sequence (nucleotide 421- 426); (: 1 & 1 recognition sequence (nucleotides 429-434); Notl recognition sequence (nucleotides 437-444); DHS4.4 (nucleotides 142866.doc-65-201023898 445-741); Mlul recognition sequence (nucleotides 742-747); FNB S/MAR (nucleotides 753-1, 569). Step 6: Re-selection of B29 promoter and HA-eIF5AlK5QR plus SV40 pA showed 匣 entry into pCpG-DHS4. 4-Generating pExp-5 B29-eIF5Al Representation 匣 Contains minimal B29 promoter, synthetic intron, HA-eIF5AlK5°R and SV40 pA, which was generated from the pB29-eIF5AlK50R_7 vector using the following primers Amplification (step 3). The positive-strand primer is 5'-GTTATCGATACTAGTGCGACCGC CAAACC-3'; and the anti-strand primer is 5,-CAAGCGGCCGCCATACCA 10 CATTTGTAGAGGTTTTAC-3'. The resulting PCR fragment is digested with Clal and Notl. The reaction was ligated into the pCpG-DHS4_4 vector harboring the Clal and Notl cleavage sites to generate pExp-5. : Replacement of HA-eIF5AlK5()R into non-HA eIF5AlK5()R in pExp-5 yields the final vector pExp5A. The pExp-5 plastid first removes HA_eIF5AlK50R with two restriction enzymes, Ncol and Nhel. The eIF5AlK50R PCR fragment was amplified by PCR in pHM6-eIF5AlK5GR. The following is the primer sequence used for the primer, the 5'-CACCATGGCAGATGATTTG GACTTC-3' and the anti-strand bow | -CGCGCTAGCCAGTTATTTTGC CATCGCC-3'. The product of the PCR reaction was digested by Ncol and Nhel, and then ligated into B29-5 #3 carrying a Ncol and Nhel cleavage site to generate a new vector, B29 -K50R. B29-K50R was digested with Ncol and Nhel again, and a 470 bp eIF5AlK5QR fragment was obtained by gel purification. This fragment was ligated to Ncol 142866.doc-66·201023898 and Nhel digestion. The site of the pEXp-5 vector produces the final expression vector pExp5A °

Example 18: Testing of pExp5A A plastids were transferred to different cell lines using the Lipofectamine 2000 method. The expression level of HA-eIF5AlK50R was detected by Western blotting using an anti-HA antibody (Roche) 24 hours after transfection. The cell lines used in this experiment were P3X63Ag8.653 (mouse B lymphoblastoid-bone marrow tumor), KAS (human bone tumor), HepG2 (human hepatocellular carcinoma), T24 (human bladder tumor), HT-29 (human colorectal tumor), HEK-293 (human embryonic kidney cell), PC3 (human prostate tumor), HeLa (human cervical adenocarcinoma) and A549 (lung tumor). See Figure 24. The expression of HA-eIF5AlK5()R in the pExp_5 plastids in human and mouse osteophyte tumor cell lines was comparable to that of the plastids using the continuously expressed EF1 promoter (CpG-eIF5AlK5()R). However, if HA-eIF5AlK5GR expressed in pExp-5 plastid is used, the region of expression is limited to non-B-cell lines, but the region expressed by HA-eIF5AlK5GR ® is not limited to non-B when using the persistently expressed EF1 promoter. - in the cell line. With one exception, in HEK-293 (human embryonic kidney cells), a human embryonic kidney cell line was observed to have a high expression of HA-eIF5AlK5QR after transfection with pExp-5. This phenomenon may result from the embryonic properties of this cell line. But at the same time we still don't know if ρΕχρ-5 will also behave in mature kidney cells. Finally, this pExp-5 plastid is used for toxicological analysis studies, and in clinical trials, the non-ΗΑ-e-labeled eIF5AlK50R replaces HA-eIF5AlK50R (ρΕχρ5Α). Εχρ-5 contains the HA marker 142866.doc-67-201023898 eIF5AlK5GR, eIF5A1K5〇R expression and the sustained expression of the full-length B29 promoter-driven plastid expression phase driven by the human Β29 promoter/enhancer Comparison. Example 19: Formation of in vivo JETPEITM nanoparticles This example illustrates how to form a living JETPEFM nanoparticle complex for injection of mice weighing approximately 20 grams. The dose used was 1.5 mg/kg (0.1 ml) '1.5 mg/kg equal to ΐ·〇 mg pExp5A/kg plus 0.5 mg h5Al/kg, and the ratio of DNA to siRNA was 2:1. The plastid DNA and siRNA were diluted to a total volume of 25 ml. Use sterile water to adjust the volume. * Dilute 20 mg of plastid DNA (10 ml, concentration 2 mg/ml) and 10 mg of siRNA (l〇 ml, concentration 1 mg/ml) in a total volume of 25 ml. Use sterile water to adjust the volume. 25 ml of 10% glucose (provided by the PEI kit) was added to adjust the volume of the DNA solution to 50 ml of a 5% glucose solution. Slightly shake and centrifuge. Dilute the living JETPEITM into a total volume of 25 ml of water ^ Dilute 36 ml of live JETPEITM into a total volume of 25 ml of water, adjust the final volume to 5 〇 ml (containing 10% glucose solution), the final concentration is 5% glucose Solution. Slightly shake the plate and centrifuge. Immediately add 50 ml of diluted pEI to 5 mL of diluted DNA (do not reverse the order of manipulation), shake gently and centrifuge immediately. When the PEI complex is formed, it is stable for 8 to 1/2 hours. Its N/P ratio should be 6〇N/P ratio representing the number of positively charged nitrogen residues in the living 拎(3)耵 compared to the number of negatively charged phosphate residues of DNA and siRNA. Each gram of DNA and siRNA contains the same number of phosphate residues, so the N/p ratio is used to measure the ion balance in the complex. Increasing the ratio of N/p 142866.doc -68- 201023898 of the complex increases the toxicity of the complex. The live JET-PEI is provided as a 150 mM solution (expressed as a nitrogen residue), in which 1 mg of DNA contains 3 nmole of anionic linalic acid. The final concentration of DNA should not exceed 0.5 mg/ml in the total volume. DNA must be of high quality and prepared in water. Before use, the live-jetPEI and 10% glucose solution should be warmed to room temperature. Example 20: Find the optimal dose and intravascular injection of SNS01 and SNS-EF1/UU in mice for repeated dose experiments. ❿ SNS01 is an embodiment of the present invention and is a tumor biotarget treatment for the treatment of multiple myeloma. SNS01 is composed of three parts: DNA vector, which is used to express the early apoptotic mutant gene of eIF5A (see Figure 22); siRNA can attack the native eIF5A which promotes tumor cell growth/anti-apoptosis (see Figure A sequence of 25); and a synthetic polymer, polyethylenimine (7>? Wvo-jetPEI; Polyplus Transfection Inc.), which serves as a carrier for transportation. The purpose of this experiment was to determine the highest dose that can be tolerated in mice and the therapeutic dose of long-term ® administered intravascular SNS01. Two separate experiments were conducted to investigate this purpose. The highest tolerable dose (Study ID: MTD) was an 8-day study. In this study, mice received two intravenous injections of SNS01 (from 2.2 mg/kg to 3.7 mg/kg). Toxicity is assessed by observing clinical signs, body weight, organ weight, and liver enzymes. In addition, the 9-week repeat dose study (Study ID: EX6) was used to assess the toxicity of SNS-EF1/UU after two long-term doses (1.5 mg/kg), as well as other factors. SNS-EF1/UU is a 142866.doc -69-201023898 SNS-01 preclinical version, which differs from SNS01 in that in the SNS01 complex, eIF5AK5GR is expressed by a sustained promoter. Driven (it can be performed at any time and in any tissue), rather than being driven by a B-cell-specific promoter. Use of the B cell-specific promoter B29 in SNS01 by limiting early apoptosis 61?5 VIII & 5 () 11 mutations can only be used in B cells, including myeloma cells, to enhance the safety of treatment . The EX6 study also included the administration of mouse-specific eIF5A siRNA to a population of mice. This experiment was used to determine if cytotoxicity is caused by inhibition of eIF5A in mouse tissues. In repeated dose studies, toxicity was assessed in terms of clinical signs, body weight, histopathology, liver enzymes, and liver histopathology.

Experiment ID Animal model Injection schedule treatment time Test substance MTD CD-1 (female) twice a week (intravenous) 8 days (2 injections) SNS01 EX6 Balb/c (female) twice a week (intravenous) 9 weeks - SNS-EF1/UU-SNS-EF1/UU These two items were tested separately - Oxygen eIF5A siRNA test substance plastid DNA siRNA Loaded material grade SNS013 pExp5Ac (eIF5AK5()R expression driven by B29 B cell specific promoter) eIF5A siRNA (dTdT highlights d) human-specific eIF5 A siRNA in vivo-jetPEITM GLP-grade material SNS-EFl/UUb pCpG-HA-eEF5AK50R (HA-eIF5AK50R is driven by the broad EF1 promoter) h5Al (UU prominent d Human-specific eIF5A siRNA in vivo-jetPEITM Research grade material 142866.doc •70- 201023898 a- SNS01 contains GLP-grade material and is clinically produced b-SNS-EF1/UU is a preclinical trial of SNS01 development The substance c-pExp5A plastid is an anti-RNAi plastid because it has only one eiF5A translational region, whereas eIF5A siRNA (h5Al) is the 31-terminal region that attacks eIF5A' d-SNS01 eIF5A siRNA and h5Al siRNA sequences are identical, except At 31.48, the end of the end is containing dTdT protrusions instead of UU protrusions. The dTdT protrusion does not affect the selectivity or effectiveness of SiNA on the target, but has been proposed to increase its stability. Preclinical trials indicated that the therapeutic concentration of SNS01 ranged from 0.75 mg/kg to 1.5 mg/kg (Study EX9). In the 8-day dose range analysis study (Study ID: MTD), a higher dose than the original treatment concentration was used to test to determine the upper limit of the highest Han. Twice a week intravascular injection of test substances showed that the tolerable low doses were 2.2 mg/kg and 2.9 mg/kg, although one mouse developed symptoms after receiving 2.9 mg/g and was subsequently euthanized. When the dose is up to or greater than 3.3 mg/kg, the survival rate is about 20-25%, so the maximum tolerable dose is between 2.2 mg/kg and 2.9 mg/kg, while the dose range with good treatment is 0.75. Mg/kg to 1.5 mg/kg. In a nine-week repeat dose experiment (Study ID: EX6), mice received SNS-EF1/UU (1.5 mg/kg) in the tail vein twice a week. No toxicity associated with the test was observed during the experiment. DNA and siRNA were tested separately in this experiment and mice were tolerant of both tests. Since eIF5A siRNA administered to humans has no effect in mice, eIF5A siRNA administered to mice is also included in this test. During the nine-week test period, no toxicity was observed in long-term administration of mice. These results indicate that the therapeutic doses of SNS-01 and SNS-EF1/UU are not toxic to mice even after long-term administration. 142866.doc •71 201023898 Test substance plastid DNA composition manufacturer siRNA composition manufacturer PEI composition of manufacturer test substance formation SNS01 VGXI Lot# pExp5A.08L007 Avecia Polyplus Transfection Inc. GLP-grade ingredient contains 10% glucose, Forming a nanocomposite; this complex is used for injection of SNS-EF1/UU Qiagen EndoFree plastid Mega Kit <0.1 EU>g DNA Thermo Scientific Dharmacon RNAi Technologies Polyplus Transfection Inc. Research-grade component contains 10 in 2> % glucose, forming a nanocomplex; this complex is used in injection storage conditions (stability) -20 °C for 2*4 hours) -20° pan 1 year) 4° (21 years) room temperature 6 hours) Test System and Experimental Design All views of this experiment were guided by guidelines established by the University of Waterloo Animal Care Committee (Waterloo, Ontario, Canada), established by the Canadian Council on Animal Care and the Province of Ontario. . Experimental Animals CD-I and BALB/c mice were obtained from Charles River Laboratories (Quebec, Canada). Both mice received a tail vein injection of the test substance twice a week. A slow injection (about 2-3 minutes) is used when injecting a volume greater than 0.2 ml. The mice used for the 8-day experiment were about 6-9 weeks old at the beginning of the experiment. Mice used for 9-week repeated dose experiments were about 5-6 weeks old at the start of the experiment. 142866.doc -72- 201023898 Experiment ID: ]S group Animals only (female) Test substance N/P ratio 3 dose (mg/kg) Injection volume injection number MTD-C 5 5% glucose - 0.33 mL 2 MTD -PA 5 «NS01 6 2.2 mg/kg 0.20 mL 2 MTD-PB 5 SNS01 6 2.9 mg/kg 0.27 mL 2 MTD-PC 4 SNS01 (5 3.3 mg/kg 0.30 mL T* MTD-PD 5 ^§01 Γ6 3.7 Me/ke 0.33 mL a- N/P ratio = in P] b - positively charged nitrogen due to toxicity in bi and negatively charged phosphate in nucleic acid t. should produce 'survived mice not injected 2nd : Value. Experiment ID: Ε : X6 group Animals only (female) Test substance N/P Specific dose (mg/kg) Injection volume Injection number Ex6-Gl 4 5% Glucose - 0.10 mL 20 Ex6-G2 5 Placebo Control a Control3 s 1.5 mg/kg 0.10 mL 20 Ex6-G3 5 siRNAb 8 1.5 mg/kg 0.10 mL 20 Ex6-G4 6 8 1.5 mg/kg 0.10 mL 20 Ex6-G5 4 SNS- EF1/UU 8 1.5 mg /kg 0.10 mL 20 Ex6-G6 6 Mouse siRNAd 8 1.5 mg/kg 0.10 mL 20 Complex contains a non-expressing plastid (having the same vector as ρΕχρ5 A) and a non-aggressive b-PEI complex containing a non-expression quality (ΡΕχρ5Α the same vector) and a h5Al siR] STA. C- PEI complexes containing pCpG-HA-eIF5AK5GR a plastid and not a siRNA〇 offensive

The d-PEI complex contains a non-expressing plastid (having the same vector as pExp5A) and an eIF5A siRNA against e.g. (human eIF5A siRNA does not respond in mice). Wang's Eight-Day Maximum Tolerance Dose Experiment (MTD) Two-dose eight-day trials were dose-range experiments used to determine the maximum tolerated dose of SNS01. No clinically toxic symptoms were observed in the clinical dose range of SNS01 at 2.2 mg/kg-3.7 mg/kg and previous 〇75 142866.doc •73·201023898 mg/kg-1.5 mg/kg. There were no clinical signs of toxicity at the lowest dose of SNS01 (2.2 mg/kg), except for one mouse. The fur of this mouse was slightly wrinkled and the reaction was lowered (this phenomenon was resolved within an hour). No clinical signs of toxicity were found after the second dose of 2.2 mg/kg SNS01 was injected. In this experiment, all mice maintained their body weight. The organ did not change under the microscope. In the control group, the ratio of body weight to body weight did not change except for a moderate increase in the ratio of liver weight to body weight. However, since the increase in the ratio of liver weight to body weight was not observed in any of the high dose experimental groups, the increase in the ratio was independent of the test substance. At the dose of SNS01 at 2.9 mg/kg, more than 4 of the 5 mice had no clinical signs of toxicity. However, within 1 hour after the injection of the test agent, one mouse developed convulsions and difficulty breathing, so it must be euthanized in a humane manner. In surviving mice, following the second dose of SNS01, the clinical signs of no toxicity appeared. During the course of the experiment, the mice maintained their body weight. Under the microscope observation, the organs did not change, and the ratio of body weight to body weight did not change. In the second dose of 2.9 mg/kg of SNS〇H^, the serum level of ALT increased slightly. Unexpectedly, the SNS01 dose of 3_3 mg/kg or more was not tolerated by all mice in both experimental groups and must be euthanized in a humane manner. In all cases, the clinical condition caused by one hour of injection was shown to be consistent with other reported high-dose PEI-injected mice 142866.doc •74·201023898. Mice that survived the injection of 3.3 mg/kg and 3.7 mg/kg of SNS01, after four hours of injection, were fully recovered and their body weight was maintained during the experiment, although they were no longer injected with the second dose. Therefore, the maximum tolerated dose of SNS01 is between 2.2 mg/kg and 2.9 mg/kg. Nine-week Repeated Dosing Experiment (EX6) The purpose of the nine-week repeat dose trial was to evaluate the clinical safety of the therapeutic dose of SNS-EF1/UU (1.5 mg/kg). During the development of SNS01, SNS-EF1/UU was a complex used in preclinical experiments. There is no significant difference between SNS-φ EF1/UU and SNS01. The most obvious difference is that SNS-EF1/UU is an experimental grade agent, and the performance of eIF5AK5QR is initiated by continuous expression of the human EF1 promoter (human EF1 promoter is in different cell types). There are performances in it). However, SNS01 uses a B cell-specific promoter to drive the expression of eIF5AK5GR. In a safety assay, the persistence of a promoter can be used to assess the toxicity of the mutant eIF5AK5GR protein accumulation in non-B cell tissue. On the other hand, the nine-week re-dosing experiment included testing the safety of the individual components of SNS-EF1/UU. The DNA group reagent (EX6-G3) contains - a complex of eIF5 A plastid and a non-aggressive control siRNA, whereas the siRNA group reagent (Ex6-G4) contains human eIF5A (h5Al) siRNA and a non-expressing plastid Complex. Since the test substance SNS-EF1/UU contains a human eIF5A siRNA (which does not affect the performance of endogenous mouse eIF5A), another feature of this experiment is that the Ex6-G6 experimental reagent (containing a non-expressing plastid and an effective attack) A PEI complex composed of siRNA of mouse eIF5 A). The Ex6-G6 experimental reagent can assess the safety of clinical administration of active eIF5A siRNA. 142866.doc -75- 201023898 All mice survive to the planned killing day. During the nine-week experiment, no clinical signs of toxicity appeared in any of the experimental groups. All mice continued to increase their body weight. In the third and sixth weeks after the start of the injection, the number of red gold balls and white blood cells in all the mice in the experimental group was normal. After all mice were slaughtered, the liver enzyme content in Qiqing was within the normal range. Under the microscope, the appearance of the organs of all mice was normal. Histopathological analysis of the major organs was performed by an independent pathologist, showing that the test substance did not cause toxicity. Mice can tolerate clinically administered therapeutic doses of SNS-EF1/UU and no adverse reactions were observed. In addition, no toxicity was found in the clinical management of mouse-specific eIF5A siRNA, suggesting that the administration of PEI complex containing human eIF5A siRNA is safe for humans. Example 21: Efficacy of intravenous injection of SNS-B29/UU and SNS01 in mice with multiple bone tumors. SNS01 is introduced as before. The test substance SNS-B29/UU is a clinical product of SNS01. There is only a slight difference between SNS-B29/UU and SNS01. The main difference is that the components in SNS-B29/UU are experimental grades instead of GLP grades. The primary objective of this experiment was to evaluate the minimum effective dose of SNS-B29/UU and to demonstrate the GLP grade component consisting of SNS01 with the same efficacy as the experimental grade components used in clinical studies. The repeated dose tumor test (experiment ID: EX9) lasted for five weeks. In order to evaluate the clinical dose suitable for SNS01, the evaluation was for growth in mice during the weekly dose of SNS-B29/UU that was gradually less than twice a week. Inhibition of subcutaneous tumors. By monitoring clinical symptoms, weight and organ weight, 142866.doc -76-201023898 mode' also assesses the toxicity of the injected mice. Experimental ID animal model injection schedule duration test substance EX9 C.B17 (SCID) (Female) SNS-B29/UU mice (KAS-6/1) administered intravenously for 25 days twice a week with subcutaneous human multiple myeloma. SNS01 test substance plastid DNA siRNA method Material grade SNS01a pExp5Ac (eIF5AK5<)R expression, driven by a promoter expressed only in B29B cells) eIF5A siRNAd (dTdT overhang e) to human-specific eIF5A siRNA intracellular _ jetPEITM GLF grade material SNS-B29/UU0 pExp5Ac h5Al (UU highlights e) on human-specific eIF5A siRNA intracellular-jetPEITM experimental grade material a-SNS01 contains GLP grade material, this reagent b-SNS-B29/UU is used in preclinical experiments c-pExp5A There is RNAi resistance, due to frame), when eIF5A siRNA (h5Al>ic^(d-eIF5A siRNA and h5AlsiRNAs are two; ^ prominent and h5AlsiRNAs are UU prominence or potency at the 3' end, and enhance siRNA stability The test substance that is being developed for clinical application, this experiment led to the development of SNS01. • Its plastid contains only the translatable region of eIF5A (open reading; untranslatable region of IF5A3' (3'UTR) » complete Similarly, except for the eIF5A siRNA at the 3' end of dTdT, this dTdT overhang does not affect the siRNA targeting of the selective Lu SNS-B29/UU in the human subcutaneous multiple myeloma mouse (SCID) treatment range. SNS-B29 The /UU dose is between 0.15 mg/kg and 1.5 mg/kg. The antitumor efficacy of the test substance is determined by measuring the tumor volume twice a week and by weighing the tumor tissue after killing the mouse. The dose of SNS-B29/UU is between 0.75 mg/kg and 1.5 mg/kg, resulting in a markedly smaller tumor, showing an effective dose of SNS-B29/UU between 0.75 mg/kg and 1.5 mg/kg. When the dose of the test substance SNS-B29/UU was 0.38 mg/kg, it was also observed that 142866.doc -77-201023898 inhibited the growth of subcutaneous tumors, although no tumor was observed at this dose. To. At low doses (o.l5 mg/kg SNS-B29/UU) at the low dose (o.l5 mg/kg SNS-B29/UU), there was some inhibition of tumor growth, indicating a large effective dose range for SNS-B29/UU. See Figure 26 and Figure 27. SNS01 consisting of GLP grade components has similar inhibitory effects on tumor growth as SNS-B29/UU. Tumor-bearing mice had a good tolerance for injections of SNS01 and SNS-B29/UU, and these mice continued to gain weight during the course of the experiment. Test substance and tool test substance plastid DNA composition of manufacturer siRNA consisting of manufacturer PEI consisting of manufacturer test substance formation SNS01 VGXI Lot# pExp5A.08L007 Avecia Polyplus Transfection Inc. GLP_ grade substance combined with 10% glucose, formed Nanocomposite, this complex was injected in 2 to 4 hours. SNS-B29/UU Qiagen EndoFree plastid Mega Kit <0.1 EU/pg DNA ThermoScientific Dharmacon RNAi Technologies Polyplus Transfection Inc. The experimental grade component combines with 10% glucose to form a nanocomposite, which is in 2 to 4 hours. Being injected. Storage conditions (stability) -20 ° C years) -20. Pan 1 year) 4° fel year) Room temperature (2 6 hours) Test system and experimental design C.B.17/IcrHsd-Prkdc (SCID) Although mice were obtained from Harlan (Indianapolis, IN, USA). Six cells which can develop into KAS-6/l (human multiple myeloma) were injected into the right side of the mouse 5 to 6 weeks old by injection to cause subcutaneous tumor formation. The SNS-B29/UU injection begins at a tumor size of 20 to 40 cubic centimeters (approximately four weeks after tumor cell injection). SNS01 injection begins at a tumor size of 130 cm3 (approximately six weeks after tumor cell injection 142866.doc -78 - 201023898). The mice receive the test substance via a tail vein injection. Experiment ID: Number of animals in the EX9 group (female) Test substance N/P Specific dose mg/kg Injection volume Injection number Ex9-Gl 3 Control group a 6 1.5 mg 0.1 ml~~Τϊ Ex9-G2 4 SNS- B29/UU 6 1-5 mg 0.1 ml TI Ex9-G3 4 SNS- B29/UU 6 0.75 mg / kg 0.05 ml π Ex9-G4 4 SNS- B29/UU 6 0.38 mg / kg 0.025 ml 11. Ex9-G5 3 SNS- B29 /UU 6 0.15 mg / · 0.01 ml 11 Ex9-G9 3 SNS01 6 1.5 mg / kg 0.1 ml 7 a- PEI compound! Do not contain non-expressing plastids (with pExp5A; 1 question vector), no aggressive siRNA. Repeated dose tumor trials Repeated dose tumor experiments were designed to determine the minimum effective dose of SNS-B29/UU and to demonstrate that GLP-grade SNS01 test substances can contain tumor suppressor activity as demonstrated by the experimental grade test substance SNS-B29/UU . A secondary objective was to assess the toxicity of the treatment by monitoring the clinical response of the experimental mice's body weight and organ weight. The tumor suppressing activity of the test substance was determined by two tumor volume measurements per one-lane (using a digital caliper). After killing the mouse, the tumor was cut and weighed. All mice survived until the scheduled sacrifice period. The control group mice treated with the PEI nanocomplex had an average tumor size of 284 mm3 at sacrifice, and the nanocomplex contained a non-expressing plastid and a non-target siRNA at 1.5 mg/kg SNS-B29 The mice treated with /UU only had an average tumor size of 13 mm3, which reduced tumor growth by 95% (*Ρ=〇·〇26). However, attempts were made to excise tumors from mice treated with 1.5 mg/kg SNS-B29/UU 142866.doc -79-201023898' No evidence of tumors found in any of the mice. Reducing half the dose of SNS-B29/UU to 0.75 mg/kg still resulted in a reduction of 91% tumor volume (*ρ=0·03) and 87% body weight (*p=〇.〇4), one of the mice. The tumor has completely disappeared. Therefore, the ideal therapeutic dose of SNS-B29/UU twice a week appears to be between 0.75 mg/kg and 1.5 mg/kg. The twice-week dose of SNS-B29/UU dose is as low as 〇. 1 5 mg/kg, which still results in a 60% decrease in the final tumor volume, indicating that SNS-B29/UU has potential resistance over a wide range of doses. Tumor activity. In addition to inhibiting tumor growth, treatment with 0.75 mg/kg SNS-B29/UU and 1.5 mg/kg SNS01 resulted in a significant decrease in tumor hoarding, suggesting that this treatment may induce tumor regression by triggering tumor apoptosis. The percentage change in tumor size in tumor-bearing mice treated with SNS-B29/UU was -244% and -245% at doses of 0.75 mg/kg and 1.5 mg/kg, respectively. At the same time, the size of the control group mice increased by more than 2000%. Injecting SNS01 once every two weeks also significantly reduced multiple bone tumors. The percentage change in tumor volume of mice treated with 1.5 mg/kg SNS01 was ·349%, indicating that SNS01 was effective as SNS-B29/UU. Because the tumor volume treated with SNS01 was reduced by 349% after only 25 days of treatment, and the tumor volume treated by SNS-B29/UU reached a 245% reduction after 35 days of treatment, the fact of using GLP-grade materials Biological activity may have increased. In addition, the tumor treated by SNS01 was quite large (about 130 mm3), indicating that SNS01 was treated to combat established tumors. All mice were well tolerated and no clinical signs of toxicity were observed. All groups of mice continued to gain weight during the course of the study. At 142866.doc -80 · 201023898, no visible changes in the appearance of the organ were observed during the autopsy, and no significant changes in the ratio of organ weight to body weight occurred. Therefore, SNS01 (and its preclinical version of SNS-B29/UU) is highly effective for SCID mice, and it is extremely effective in treating human multiple myeloma under the skin when it is delivered twice a week via intravenous injection. All doses of SNS-B29/UU tested were effective for inhibiting tumor growth, but the highest dose of 1.5 mg/kg successfully eliminated tumors in all treated mice. Example 22: Biodistribution of plastid DNA and siRNA polyethyleneimine (JetPEI) complex. The green fluorescent protein (GFP)-expression construct is used to determine the location of the plastid DNA delivered by the PEI complex. Two promoters were used to drive GFP expression: EF1: ubiquitous promoter (EF1::GFP) or B29: B cell-specific promoter (B29 "GFP") containing 20 micrograms of GFP plastid DNA and 10 micrograms The PEI complex of h5Al siRNA of fluorescein (DY547) was prepared at an N/P ratio of 6. BalB/C mice were injected intravenously with 5% glucose or PEI complex for two consecutive days. After 72 hours of the first injection The mouse was killed and the organs were removed, and the GFP expression was analyzed with DY547-siRNA by conjugated focal microscope. Bone marrow: Most mice showed DY547-siRNA but no GFP. The time of organ removal may not match GFP. Spikes in performance, and GFP may be 'shadowed' or GFP may not be expressed. However, in some cases it was observed that GFP and DY547 are co-located in the same bone marrow cells. Therefore, this provides PEI nanoparticles that can be injected intravenously. Evidence of transfection of bone marrow cells in living animals. 142866.doc -81 · 201023898 Lung: In some cases only DY547-siRNA is present without GFP. The time to remove the organ may not match the peak of GFP expression, or GFP may Shading or GFP may not be expressed. Spleen: Evidence of GFP expression (when driven by the EF1 promoter) co-located in cells that also have DY547-siRNA can be seen. When driven by the B29 promoter, GFP is expressed in spleen cells. It is lower. This shows that PEI nanoparticles seem to transfect spleen cells. Kidney: No GFP or DY547 is observed, indicating that nanoparticles may not enter the kidney. © Liver: In most cases, there is DY547-siRNA There is no evidence of GFP expression. This provides evidence that PEI nanoparticle is being transfected into liver cells. Heart: EF1::GFP is found in heart tissue and DY547-siRNA coexist, indicating that PEI nanoparticles may be transfected This organ. No GFP was observed driven by the B29 promoter. Example 23: Effect of DNAisiRNA ratio on HA-eIF5AK5GR expression KAS cells contain B29-HA-eIF5AK5()R (B cell-specific promoter-driven plasmon-driven plasmon) Transfection of nanoparticles with h5Al siRNA. JET PEITM nanoparticles containing different ratios of pExp5A and h5Al siRNA were prepared and placed at room temperature before addition to KAS cells. After 4 hours, 4 hours after transfection, the medium containing the nanoparticles was replaced with fresh medium. After 24 hours, the cell lysate was collected and the antibody against HA was used for Western blot analysis. The ratio of DNA: siRNA was different. The standard ratio is 2:1, and the ratio of 1:8, 3:1, and 2:1 accumulates 11-8-61卩5八1^()11 to the peak. See Figure 30. 142866.doc -82 - 201023898 Example 24: DNA: siRNA ratio for the effect of apoptosis induced by nanoparticle transfection. Nanoparticles containing different ratios of pExp5A and h5Al siRNA were prepared and allowed to stand at room temperature for 4 hours before adding KAS cells. " 4 hours after transfection, the medium containing nanoparticles was replaced with fresh medium. . Cells were harvested after 48 hours, calibrated with Annexin V/PI and analyzed by FACS. The initiation of apoptosis was highest in cells transfected with nanoparticles with a standard DNA:siRNA ratio of 2:1. See Figure 31. φ Example 25: Administration of a PEI complex (N/P = 6 or 8) containing eIF5AlK50R plastid and eIF5Al siRNA (siSTABLE or non-siSTABLE) inhibited the growth of multiple myeloma subcutaneous tumors and caused tumor shrinkage. SCID mice were injected subcutaneously into KAS cells. Treatment begins when a touchable tumor is observed. Mice were injected intravenously twice a week: (G1) PEI complex containing 20 mg pCpG-mcs (empty sputum) and 10 mg N/P=8 control group siRNA (medium dose); (G5) containing 20 Mg anti-RNAi plastid pCpG· eIF5Alk50R and 10 mg N/P=8 of siSTABLE h5Al siRNA (medium® dose, siSTABLE) PEI complex; (G8) contains 20 mg anti-RNAi plastid pCpG-eIF5Alk50R and 10 mg A PEI complex of h5Al siRNA (medium dose, N/P = 6) with N/P = 6. The data shown is the individual tumor volume of each group of mice. The final injection was administered on the 40th day after the start of the treatment. See Figure 32. Example 26: JET PEITM nanoparticles are effectively absorbed by tumor tissue and the nanoparticles transport plastids and siRNA to the same cells. Tumor sections were taken 48 hours after injection of nanoparticles containing pExp-GFP (control of GFP-specific B promoter specificity 142866.doc • 83 - 201023898) and DY547-siRNA (fluorescence-targeted siRNA). The common expression of GFP and DY547 was observed in tumor sections by conjugated focus microscopy, indicating that the nanoparticles were being effectively absorbed by the tumor tissue, and the nanoparticles were transporting the plastid and siRNA to the same cells. See Figure 33. Example 27: Creating an adenovirus construct for a truncation study. Adenovirus (serotype 5, E3-delete) 1) Ad-eIF5AlA(2-6) [Δ(2-6)]_ This is a human eIF5Al lacking amino acids 2 to 6. _ 2) Ad-eIF5AlK50RA(2-6)[A(2-6)/K50R] This is a human eIF5Al lacking amino acids 2 to 6. Further, "K50R" means that the amino acid (K) at position 50 has been mutated to arginine (R), and therefore it is believed that the carboxyresin amine lysine modification cannot be performed by DHS. 3) Ad-eIF5AlD6E[D6E] - This is a predicted human eIF5Al (D6 to E) whose mutation position has been mutated. 4) Ad-eIF5AlD6E/K50R[D6E/K50R] - This is the predicted human eIF5AlK50R (D6 to E) in which the cleavage position has been mutated. Further, G "K50R" means that the amide acid (K) at position 50 has been mutated to arginine (R), and therefore it is believed that the carboxyresinamine lysine modification cannot be performed by DHS. Example 28: eIF5 A cleavage of the caspase vector. To identify the position of the cut, protein lysates isolated from Actinomycin D-treated KAS cells were isolated by two-dimensional gel electrophoresis (Figure 37), and the point corresponding to the smaller molecular weight eIF5A cleavage product was removed from the gel. 142866.doc • 84- 201023898 Cut and sequence with mass spectrometer (Figure 38B). Although the full length sequence of the cleavage product has been obtained, it is determined that the cleavage occurs after the sixth amino acid from the fN end of the protein (Fig. 38A). It is hypothesized that the leading sequence of the cleavage position is DDLD", and 疋 has been identified as the sequence of the cleavage site of thiocyl protease (Chay et al" 2002). In order to determine whether the production of the cleavage fragment is related to thiocysteine protease, Test for the ability of a thiocysteine protease inhibitor to block the production of cleavage fragments produced during activin D-induced apoptosis in human myeloma cells (Fig. 4 - thiosporin Protease inhibitors and specific inhibitors of thiocysteine proteases 3, 8 and 9 strongly prevent KAS cells from finally cleavage of eIF5A during AccD-induced apoptosis. Indole inhibitors are also reduced, but do not completely prevent the formation of cleavage products. Immobilization with thiocysteine protease inhibitors, preventing the accumulation of cleavage products' also inhibits the reduction of eIF5a's slow-tolerant amine lysine form (Figure 41) , indicating that the decrease in carboxypyramine lysine eIF5A during activin D-induced apoptosis is the result of cleavage. • The thiosinase cleavage of the protein during the death of the bacterium may have numerous OBJECTIVE: 1) Deactivation of pro-survival or anti-apoptotic proteins. For example, the translation of eukaryotic cells, the initiation factor 4G1, is cleaved by a thiocysteine protease to deactivate it, thereby inhibiting translation. The initiation factor binds to the 5-terminal cap structure of the mRNA and causes capped mRNA to bind to the 4〇S ribosomal subunit; 2) creates a dominant inhibitory pattern of the protein (for example, s, NF-kappa B p65 is cleaved by thiocysteine protease during apoptosis to create a dominant suppressor fragment that binds to DNA I42866.doc -85- 201023898 but does not have anti-theta activity, thus acting as NF.KB The dominant inhibitor of inhibition) 3) The cleavage of the gain function (gain〇f_-n) leads to the activation of the protein by removing the will or regulatory region, thus causing the formation of a fragment with a new activity or increased activity. (For example, BRCA-mediated cell cycle arrest and activation of apoptotic breast cancer inhibitor protein protein f is released - a red-cleaved fragment screams ^ eIF4G family member - activated after thiocysteine cleavage The cutting product Translation of apoptotic-associated proteins); 4) Conversion of anti-apoptotic proteins to pro-cavitation proteins (for example, 'expansion inhibitors such as cl xL and c IAP1, which can be transmitted through thiocysteine Protease cleavage to transform into pro-apoptotic protein; and 5) intracellular redistribution (for example, when cleaved by thiocysteine protease 8, Bid promotes _ apoptotic pl5 fragment is cleaved by exposure Acid residues are citrated (myrist〇yUti〇n), followed by transfer of Bid to granulocytes that enhance cytochrome c release.) In order to determine eIF5A of thiocysteine-mediated mediators during activin D-induced apoptosis Cleavage was a common phenomenon or the appearance of excised ejF5A in HeLa cells treated with actinomycin D was specifically examined for myeloma cells (Fig. 42). No accumulation of eIF5A cleavage fragments in HeLa cells treated with actinomycin D was observed. In an attempt to determine the post-translational modification (eg, phosphorylation or ethosylation) of eIF5A, it may be necessary to test the deacetylated inhibitor, nicotinamide, for the formation of cleavage fragments. effect. eIF5A acetylation has been observed by mass spectrometry of eIF5A in our and other laboratories (feet ~ w 2006). Furthermore, yeast eIF5A6 was identified as a receptor for Sir2-related deacetylase Hst2 (Canb (1)· ei 142S66.doc -86- 201023898 2008), and the authors observed that eIF5A is deacetylated only in yeast lacking Hst2. Or observed in yeast overexpressing eIF5A, indicating that eIF5A generally appears in unacetified form. The HeLa cells treated with actinomycin D were allowed to stand with the Sir2 deacetylase inhibitor nicotinic guanamine, resulting in accumulation of eIF5A cleavage products, indicating that eIF5 A acetylation was required for the cleavage of thiocysteine protease. These data suggest that eIF5 A is normally protected from thiocysteine activity until the apoptotic signal triggers eIF5 A acetylation and allows cleavage of the thiocysteine protease medium. φ References

Chay KO, Park SS, Mushinski JF (2002). Linkage of caspase-mediated degradation of paxillin to apoptosis in Ba/F3 murine pro-B lymphocytes. J Biol Chem. 277, 14521-14529.

Fischer U, Janicke RU, Schulze-Osthoff K (2003). Many cuts to ruin: a comprehensive 10 update of caspase substrates. Cell Death and Differentiation 10, 76-100.

Kim, SC, Sprung, R., Chen, Y., Xu, Y, Ball, H., Pei, J., Cheng, T., Kho, Y, Xiao, H., Xiao, L., Grishin, NV , White, M., Yang, XJ, and Zhao, Y. (2006) Substrate and Functional Diversity of Lysine Acetylation Revealed by Climbing Pro Proomomics Survey. Mol Cell 23, 607-618.

Shirai et al. (2008). Global analysis of gel mobility of proteins and its use in target identification J. Biol. Chem. 283, 10745-10752. [Simplified illustration] Figure 1 provides the amine group of human eIF-5Al The acid sequence shows 25 different important parts. Figure 2 shows that mutations in e50-5Al at K50 and K67 increase the accumulation of transfected proteins. See example 1. 142866.doc •87- 201023898 Figure 3 shows that mutations in e475Al at K47, K50 and K67 increase the accumulation of transfected proteins. See example 2. Figure 4 shows that mutation of eIF5Al at K50 and K67 leads to induction of apoptosis when transfected into KAS cells. See example 3. Figure 5 shows that mutation of eIF5Al at K50 and K67 leads to induction of apoptosis when transfected into KAS cells. See example 4. Figure 6 shows that mutation of eIF5Al at K50 and K67 leads to induction of apoptosis when transfected into KAS cells. See example 5. Figure 7A shows that treatment with siRNA and modification of an adenovirus expressing eIF-5Al results in apoptosis of KAS cells. See example 6A. Figure 7B shows that pretreatment with eIF5Al siRNA [for Target #1 (SEQ ID ΝΟ: 1)] (the siRNA construct sequence shown in Figure 25) reduced the expression of endogenous eIF5Al, but let the adenovirus The accumulation of anti-RNAi eIF5Alk5QA was demonstrated. See example 6B. Figure 7C shows that pretreatment with eIF5Al siRNA against Target #1 reduced the performance of acidified NF-κΒ in human multiple bone tumor cells prior to infection with adenovirus. See example 6C. Figure 7D shows that pretreatment with eiF5Al siRNA against Target #1 reduced the performance of acidified NF-kB and ICAM-1 in human multiple bone tumor cells prior to infection with adenovirus. See example 6D.

Figure 7E shows that inhibition of NFkB DNA binding activity by LPS mediation by siRNA-mediated eIF5A inhibition in human multiple bone tumor cells. Since NF-kB regulates many pro-survival and anti-apoptotic pathways, when combined with overexpressed eIF5 AK5GR, this NFkB 142866.doc -88 * 201023898 activity, which is inhibited by eIF5 A siRNA, may explain its increased ability to induce apoptosis. . Figure 7F shows that KAS cells pretreated with siRNA increased apoptosis by delivery of the eIF5Alk50R gene in the presence of IL-6. See example 6E. Figure 8 shows that co-administration of eiF5Al plastids and eiF5Al siRNA delays the growth of multiple bone marrow subcutaneous tumors. The data shown is the tumor volume of all mice in each group. See example 7. Figure 9 shows that co-administration of eIF5Al plastids and eIF5Al siRNA delays the growth of multiple bone marrow subcutaneous tumors. The data shown is the mean tumor volume for positive and negative standard errors in each group. See example 7. Figure 10 shows that co-administration of eIF5Al plastids and elF5Al siRNA reduced the weight of multiple bone marrow subcutaneous tumors. See example 7. Figure 11 shows that co-administration of eIF5Al plastids and eIF5Al siRNA delays the growth of multiple bone marrow subcutaneous tumors and leads to tumor shrinkage. See example 8. Figure 12 shows that intravenous administration of (i.v.) eIF5Ai siRNA and intratumoral administration of (i_t.) PEI/eIF5AlK50R plastid complex resulted in a reduction in multiple bone marrow subcutaneous tumors. See example 9. Figure 13A shows that treatment with eiF5Al plastids and eIF5Al siRNA delayed the growth of multiple bone marrow subcutaneous tumors and resulted in tumor shrinkage. Figure 13B shows that co-administration of eiF5Al plastids and eIF5Al siRNA results in tumor shrinkage. Figure 13C shows that intratumoral (i.v.) administration of eiF5Al siRNA and intratumoral administration of PEI/eIF5AlK5GR plastid complex resulted in tumor shrinkage of multiple bone marrow subcutaneous tumors. 142866.doc • 89- 201023898 Figure Η shows that intravenous co-administration of eiF5Al plastids and elF5Al siRNA delays the growth of multiple bone marrow subcutaneous tumors. See example 1〇. Figure 15 shows that intravenous (iv) or intraperitoneal (i p ) administration of intravenous (i.v.) and PEI/eIF5AlK50R plastid complexes of eIF5Al siRNA delayed the growth of multiple bone marrow subcutaneous tumors. See example n. Figure 16 shows that treatment with eF5Al plastids and elF5Al siRNA delayed the growth of multiple bone marrow subcutaneous tumors. Figure 17 shows that co-administration of elF5Al plastids and eIF5Al siRNA delays the growth of multiple bone marrow subcutaneous tumors and leads to tumor shrinkage. See Example 12 ° Figure 18 shows that administration of eiF5Al siRNA intravenously (i.v.) and administration of PEI/eIF5AlK5QR plastid complex tumors in vivo resulted in tumor shrinkage in multiple bone marrow subcutaneous tumors. See example 13. Figure 19 shows that co-administration of eiF5AlK50R plastid and elF5Al siRNA delays the growth of multiple bone marrow subcutaneous tumors and leads to tumor shrinkage (KAS_SQ-5), which is driven by the EF1 or B29 promoter. See example 14. Figure 20 shows that co-administration of eiF5Al siRNA increases the anti-tumor efficacy of eIF5AlK50R plastids against multiple bone marrow subcutaneous tumors and results in a reduced tumor weight (KAS-SQ-5), which is derived from EF1 or Driven by the B29 promoter. See example 丨 5. Figure 21 shows that eIF5Al siRNA synergistically increases apoptosis induced by Ad-eIF5A infection in lung adenocarcinoma cells. See example 16. Figure 22 shows a map of ρΕχρ5 A, which is described in Example 17 as 142866.doc -90- 201023898. Figure 23 shows the predicted sequence of pExp5A (3371 bp). See example 17. Figure 24 shows the performance of eIF5AlK5GR in different cell lines. See Example 18 ° . Figure 25 shows the sequence of the target and a preferred eIF5Al siRNA sequence. Figure 26 shows the efficacy study in multiple bone marrow tumors. See example 21. Figure 27 provides the results of an efficacy study in multiple bone marrow tumors. See example 21. φ Figure 28 provides the sequence of the eIF-5Alk50R cDNA. Figure 29 provides a calibration of human eIF5Al for human 61?5 eight Shanghai 5()11. Figure 30 shows the effect of DNA:siRNA ratio on 11 816 卩 5 8 10 () 11 expression. See example 23. Figure 31 shows the effect of DNA:siRNA ratio on apoptosis induced by nanoparticle transfection. See example 24. Figure 32 shows that administration of a PEI complex (N/P = 6 or 8) containing elF5AlK5 (DR plastid and eIF5Al siRNA (si stable or non-si stable) inhibits the growth of multiple subcutaneous bone tumors and leads to tumors Zoom out. See Example 25. Figure 33 shows that JET PEITM nanoparticles are efficiently taken up by tumor tissue, and nanoparticles deliver plastids and siRNA to the same cells. See Example 26 ° Figure 34 shows actinomycetes from KAS cells Apoptosis was observed in response to D. Untreated or treated KAS cells at 0.5 μg/ml actinomycin D. At 24 hours after the start of treatment, the cells were harvested, washed, and the apoptotic cells were Annexin/PI (BD Bioscience). Marked and analyzed by flow cytometry. 142866.doc •91· 201023898 Figure 35 shows the reaction of actinomycin treatment D to produce a truncated form of elF-SAl accumulation. Actinomycin d at 0.5 μg/ml KAS cells were treated for a period of time (0 hours to 30 hours). Cell lysates were harvested and Western blot analysis was performed using antibodies against eIF5A1. Smaller molecular weights were observed starting from the activin 〇 treatment for nearly 8 hours (cutting) Post) form eIF5 ai accumulates and is thus present during the remainder of the treatment. Using the anti-actin antibody, the same amount is shown on the same membrane by Western blotting. Figure 36 shows the truncated form of eIF-5Al ratio The full-length eIF-5Al has a horse-like isoelectric point. KAS cells were untreated or treated with activin d at 0.5 μg/ml. The lysate was harvested 1 - 7 hours after treatment, and 2-D gel electrophoresis was performed. Separation of 'subsequent to Western blot analysis using antibodies against eIF5A1. A large amount of full-length eIF5Al' was observed in untreated samples and was observed in actinomycin D-treated samples. The IF cut form of eIF5Al accumulates. Figure 37 provides a photograph of a truncated 2-D gel of eiF-5Al. KAS cells were treated with 55 μg/ml actinomycin D. 17 hours after the start of treatment, harvest The cell lysate was separated by 2-D gel electrophoresis' followed by staining with Coomassie Blue. The position of the cleaved form of eIF5Al was indicated and collected for mass spectrometry. Figure 38 provides The truncated eIF_5A1 is qualitative The results of the analyzer sequencing. (A) The full length amino acid sequence of eIF5Al is shown in black. The bottomed line (A) shows the sequenced peptide identified by mass spectrometer (B), which is full length. Calibration of the eIF5Al sequence. Note that the first 6 amino acids of eiF5Al are depleted from the sequenced peptide. 142866.doc -92· 201023898 Figure 39 shows inhibition of caspase 3, 8 and 9 The agent inhibits the formation of the truncated form eIF5Al. Human multiple myeloma KAS cells were incubated with different thiocyl protease inhibitors for 16 hours prior to treatment with activin D (ActD) at 0.5 μg/ml for 16 hours. Collect cell lysates. Proteins were separated by 2D-PAGE and analyzed by Western blotting using antibodies against eIF5A. The cysteines protease inhibitors used are Z-VAD-FMK (general thiopurine protease inhibitor), Z-LEHD-FMK (thiosyl protease 9 inhibitor), Z-DEVD-FMK (Sulfur φ cystine protease 3/7 inhibitor), Z-IETD-FMK (thiocysteine protease 6/8 inhibitor), and Z-YVAD-FMK (thiocysteine protease 1 inhibitor). The data indicate that both the general thiocysteine protease inhibitor and the inhibitors of thiocysteine proteases 3, 8 and 9 strongly prevent the formation of this cleavage form of eIF5 A during ActD-induced apoptosis in KAS cells. accumulation. The thiosinase 1 inhibitor was also reduced, but did not completely block the formation of the cleavage product. These results indicate that eIF5Al is cleaved by a thiocysteine protease (induced by actinomycin D) after genotoxic stress; cleavage of eIF5Al can alter its activity, ie, increase pro-apoptotic activity. Figure 40 shows that a deacetylase inhibitor (nicotine decylamine) promotes the formation of truncated eIF-5Al. eIF5A, which can be acetylated on amino acid 47, is a Sir2-related deacetylase Hst2 (Shirai et al., 2008). Human cervical cancer Hela S3 cells were treated with • 0.5 μg/ml actinomycin D (ActD) and/or 20 μM nicotine guanamine for a specified length of time. Nicotine guanamine was a potent inhibitor of Sir2. Cell lysates were collected. Proteins were separated by 2D-PAGE and analyzed by Western blotting using an antibody against eIF5A 142866.doc -93- 201023898. Treatment of He la S3 cells with ActD alone or with pro-amine did not induce accumulation of eIF5A in the cleavage form. However, in HeLa cells treated with both actinomycin D and nicotinamide, the cut form of eIF5 A was up-regulated after exposure to ActD for at least 4 hours, suggesting that acetamylation promotes EIF5 A cleavage during apoptosis. Figure 41 provides the truncated DNA sequence of human eIF5Al. Figure 42 provides full length eIF5Al, truncated eIF5As and the protein sequence of the cleavage site. Figure 43: Infection of adenoviral constructs causes accumulation of eIF5A transgene in Hela cells. Hela S3 cells were infected with different adenoviral constructs at 500 infectious units per cell. Cell lysates were collected at 48 and 72 hours post infection and subjected to SDS-PAGE and Western blot analysis using antibodies against eIF5A and actin. The accumulation of truncated eIF5 A was observed. Figure 44: Accumulation of a truncated form of eIF5Al (eIF5AIAl-6) in human myeloma cells via apoptosis induced by actinomycin D, sodium nitroferric chloride or removal of IL-6. KAS-6/1 cells were treated as indicated, and then subjected to 2D-PAGE and Western blot analysis using antibodies against eIF5A. Accumulation of truncated eIF5Al was induced in KAS-6/1 cells via apoptosis induced by actinomycin D, NO donor, sodium nitroferrocyanide or IL-6 deficiency. Excessive expression of truncated eIF5Al caused by accumulation of eIF5Al protein induced by Ad-AlA(2-6) or Ad-A1A(2-6)/K50R is shorter than the shortened 61?5 produced during apoptosis Eight 101?5 eight one eight 1-6) have smaller? 1, which represents other post-translational 142866.doc -94- 201023898 modifications present on eIF5AlAl-6. Figure 45: eIF5AlD6E and eIF5AlD6E/K50R are resistant to cleavage induced by actinomycin D during apoptosis. Human multiple myeloma KAS-6/1 cells were treated as indicated, and then subjected to 2D-PAGE and Western blot analysis using anti-eIF5A antibodies. Excessive expression of eIF5Al (Ad-Al) was cleaved during treatment with actinomycin D. However, eIF5AlD6E or eIF5AlD6E/K50R is resistant to cleavage after actinomycin D treatment. These results are consistent with the hypothesis that the amino acid 3 to 6 of eIF5Al constitutes a thiocysteine φ protease cleavage site. Figure 46: EIF5A1 was cleaved in vitro using recombinant thiocysteine protease. Human eIF5Al (Al) and mutant eIF5AlD6E (D6E) and eIF5AlD6E/K50R (D6E/K50R) were subcloned into the pHM6 vector (Roche), which resulted in a HA-tagged protein. Transcribe and translate ρΗΜ6-ΗΑ-Α1, pHM6-HA-Al(D6E) and pHM6-HA-Al(D6E/K50R) (TNT T7 Quick Coupled Transcription/Translation Systems; Promega) in vitro and follow the manufacturer's instructions. Labeled with Transcend Non-Radioactive Translation ® Detection Systems (Promega). 3 μl of the translation product in each tube with 1 μl of recombinant thiocysteine protease (thiosporin 1, 2, 3, 6, 7, 8, 9 or 10; 〇also 11^〇 (;1^111) Incubate for 2 hours at 37 ° C in thiocysteine protease reaction buffer (50 mM Hepes, 0.1% CHAPS, 10% sugarcane* sugar, 100 mM NaCl, 10 mM DTT, 1 mM EDTA) The final product was subjected to SDS-PAGE and biotinylated protein was visualized by incubation with avidin-HRP (Streptavidin-HRP) followed by chemical fluorescence detection. 142866.doc -95- 201023898 3,6,7,8,9 and 10 cut wild-type eIF5Al, but not cleavage with thiocysteine 1. In vitro, HA-tagged eIF5AlD6E and eIF5AlD6E/K50R were thiosporin Acid proteases 3, 7 and 8 cut efficiently, but were not efficiently cleaved by thio-deacetylases 9 and 10. However, this cleavage may be due to the extremely sufficient thiocylase activity in the in vitro system and glutamate. High structural similarity with aspartic acid, thereby allowing the cleavage of eIF5AlD6E, albeit at a reduced efficiency The following (especially because eIF5AlD6E and eIF5AlD6E/K50R are resistant to cleavage in in vivo assays) (Figure 45) Figure 47: Ad-eIF5AlA compared to infection with wild-type Ad-eIF5Al or Ad-eIF5AlK50R Infection of Hela cells by (2-6) or Ad-eIF5AlK50RA (2-6) resulted in increased apoptosis. Human adenoviral constructs were infected with human cervical cancer Hela S3 cells at 500 cells per cell. At 48 or 72 hours after infection, cells were harvested and subjected to Annexin V/PI staining. Cells were then sorted by flow cytometry, showing early in apoptosis (Annexin V+/PI-) and in late death (Annexin) Percentage of cells of V+/PI+). The truncation of amino acids 2 to 6 increases the apoptotic activity of eIF5Al or eIF5AlK50R. Conversely, mutations at the cut are predicted to be reduced, but the apoptotic activity of eIF5Al or eIF5AlK50R is not eliminated. Results The cleavage of eIF5Al, which represents a thiocysteine protease vector, resulted in a truncated form of eIF5Al with increased apoptotic activity.Figure 48 provides a model of eIF-5Al truncation of thiocysteine protease mediators in apoptosis. In the KAS human bone marrow After the cells to actinomycin D treatment 142866.doc • 96 · 201023898, eIF5A after cutting accumulate in the nucleus. Figure 50 shows that cleaved eIF5A accumulates in the nucleus after treatment with actinomycin K in KAS human myeloma cells. Key: un = treatment; actD = treatment with actinomycin D; C = cytoplasmic fragment; N = nucleus. Fragments, α-tubulin (α-tubilin) and α-PCNA are control groups. This figure also shows that this truncated form accumulates significantly in the nucleus, while the full length eIF5A is evenly distributed between the cytoplasm and the nucleus. Figure 51 shows that the truncated form of eIF5 A is accumulated in a variety of cell lines and cell types in response to 9 different apoptotic stimuli, and eIF5A cleavage by thiocyl protease 14 is common during apoptosis. The phenomenon. This figure also shows that this truncated form accumulates significantly in the nucleus, while the full length eIF5 A is evenly distributed between the cytoplasm and the nucleus. Figure 52 provides the results of a study showing that truncated eiF5 A is produced in a variety of cell lines in response to different apoptotic stimuli to produce an IL-6 deficiency or IL in KAS human multiple myeloma cells. -6 and FBS were deficient in treatment and treated with actinomycin zero D in UACC 1598 (human ovarian cancer cell line). Figure 53 provides the nucleotide sequences of human elF5Al and eIF5A2. 142866.doc • 97· 201023898 Sequence Listing <110> US-based Nuosko Technology Inc. <12 0> Truncated EIF-5A1 Polynucleotide for Inducing Apoptosis of Cancer Cells <140> 098129755 < 141> 2009-09-03 <150> 12/400,742 <151> 2009-03-09 <150> 61/093,749 <151> 2008-09-03 <160> 51

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Leu Lys Gly Arg Pro Cys Lys Le Val Glu Met Ser Thr Ser Lys Thr 35 40 45

Gly Arg His Gly His Ala Lys Val His Leu Val Gly lie Asp lie Phe 50 55 60

Thr Gly Lys Lys Tyr Glu Asp lie Cys Pro Ser Thr His As Met Asp 65 70 75 80

Val Pro Asn lie Lys Arg Asn Asp Phe Gin Leu lie Gly lie Gin Asp 85 90 95

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142866 - Sequence Listing. DOC 201023898 115 120 125

He Leu He Thr Val Leu Ser Ala Met Thr Glu Glu 135 140

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<223> Description of artificial sequence: synthetic primer <400> 7 cagcaaggga gcacctatg <210> 8 <211> 19 <212> DNA <213> artificial sequence <220><223> Description: Synthetic primer <400〉 8 gttgcagtga gcggagatg

Cys Gly Glu Glu 130 <210> 9 <211> 297 <212> DNA <213> Artificial Sequence <220><223> Description of Artificial Sequence: Synthetic Polynucleotide <400> 9 accaccctgg ctgctggagg cctgctgctt gccgggtaga aggctgccct gccaggctgg gaaccaggtt tgcatagtgg tggtgggagg gggttggggg gccaagccag gttttggcat cactaattcc aggcttcact aggccttggc gcggcccctg cagcctctac gtcctcctac tctcccccaa tttacctaaa tgttttcccc tgagccggag ctccaccagg gcagggtctc gactttttaa agtctctggg cccttccttt gacctaggca cacctgcttg cacctct < 210 > 10

142,866 * SEQUENCE LISTING .DOC 201023898 < 211> 800 < 212 > DNA < 213> Homo sapiens < 400> 10 gggaacagct gccagctggg agaccaagtg caatcaacct gcacgtgcaa agcctccctc 60 ccaagccagg ctgtgctcca cttcctgttg accctggagg gaatccttcg aggcccctct 120 gctattcctg ctctgaattc cagcaaggga gcacctatgc tgtgggagct gccagtttaa 180 ctggggaatc aagaccagca caggggaact agtgagaaca gtgccaattt tcaccagatt 240 ccctctggaa ttccaggtgg ggcaggtggg taaggccccc acgcctgcag tttcaggtaa 300 atctctccac caccctgggc caggctgggc caagccaggc ggcccctgtg ttttccccag 360 tctctgggct gctggaggga accaggttgt tttggcatca gcctctactg agccggagcc 420 cttcctttcc tgctgctttg catagtggca ctaattccgt cctcctacct ccaccaggga 480 cctaggcagc cgggtagatg gtgggaggag gcttcacttc tcccccaagc agggtctcca 540 cctgcttgag gctgccctgg gttgggggag gccttggctt tacctaaaga ctttttaaca 600 cctctgaaca acacagtttc Cctgagactt tgaagctctt gttttattta tttatttatt 660 tatttattta cttatttatt tatttgcaga cagagtctca ctctgttgcc cagactggag 720 tgcagtggca ccatctccgc tcactgcaac ctccgtctcc tgagttcaag caattctcct 7 80 gcctcagcct ccaaagtacc 800 <210> 11 <211> 28 <212> DNA <213>Artificial sequence <220><223> Description of artificial sequence: synthetic primer <400> 11 ccaactagtg cgaccgccaa accttagc 28 <210> 12 <211> 31 <212> DNA <213>ΛΧ sequence<220><223> Description of artificial sequence: synthetic primer <400> 12 caaaagcttg acaacgtccg aggctccttg g 31 • 4.

142866· Sequence Listing. DOC 201023898 <210> 13 <211> 188 <212> DNA <213> Artificial Sequence <220><223> Description of Artificial Sequence: Synthetic Polynucleotide <400> 13 gcgaccgcca agagctggtg ggcggggagg acgttgtc aaccttagcg cctcccctgg ggacaggctg gcccagctga gtcccaattt cagccggtgc caaaagcctg gcatggcagg agttacacgt ccctccccca aaggggcctg tttcctccaa gggtccccgg gtgaggaaga ggagcctcgg 60 120 180 188

≪ 210 > 14 < 211 > 1237 < 212 > DNA < 213> Homo sapiens < 400 > 14 cctgcagggc ccgacgattt gccaggaaac gtacacttgt aggggcaggg actctgagga ttccctgccc gttaagaact ccacctacat ggcctctcac ttgctccaga gcactccagc tgccctgtcc agctaagttc gacttgccgg ccactagtaa agcatctctt ataaaattca gcatgttgtg gcgcaaaggt caggcaggat aagaccccct ccctgaagaa ccaccctgtt caagacctgg acctctgtgg caagaggctc ttcttcagtg aggttcattc gctcggccct acggagggtt cctctcctgg gagggctcag ccctgcacaa gagtttatat acagctggag ggacctgcag gcccccagtg tggcagcccc actgaatctt ctcccatcct tgctctgggc ctcctcttcc ataggacagg tcggggagtt gtgaggagag gggtcgagga ctgcagggct gggcatctct cagttcctga gacctgaggg acaacaattc gctgcgtggt tacatactct ctcccagtgg ccacagggtc ccctccagat cgctgggtga tgcctatttc ggcagacggc tgagaggtgg tgagagacaa gaggtctgca gaaggggctg gcactgtggc ctcccccaca aacgcactca ggattttcgc ttcacagcat ctgccacacc aacttccaac gcctgacctg ggaatagttc gctcacggcc Agaggggagg acagagggca aaaagaagct agcatgctgt cactggaccc tccatccagc ccagctcctg gagtcccaca aaagctgtct gaggaag Gga tgacctgctc atggctgcct ggtctgtggc aggacagagg caggaataga ctggctggcc 60 120 180 240 300 360 420 480 540 600 660 720 780 840 900

142866- Sequence Listing .DOC 201023898 caggggatga ccaccggtgg gactgagagg ccccccagag gaccgccaaa ccttagcggc agctggtgcc tcccctgggt cggggagggg acaggctgca gttgtcacgg gtttggggtc ggtaagcaca gacagagggg gcatccacag aggaccccag ccagctgaca aaagcctgcc cccaatttgc atggcaggaa gccggtgcag ttacacgttt ggggacagag cggtgac agcacaggct tcccccagaa ctgtgctgcc caagctgggc ctcccccagg gtccccggag ggggcctggt gaggaagagg tcctccaagg agcctcggac 960 1020 1080 1140 1200 1237 < 210 & gt 15 <211> 61 <212> DNA <213> artificial sequence <220><223> Description of artificial sequence: synthetic primer <400> 15 cgccatggac atgtaccctt acgacgtccc agactacgct gcagatgatt tggacttcga g 60 61 <210> 16 <211> 28 <212> DNA <213> Artificial sequence <220><223> Description of artificial sequence: synthetic primer <400> 16 cgcgctagcc agttattttg ccatcgcc 28 <210> 17 <;211> 497 <212> DNA <213> Artificial Sequence <220><223> Description of Artificial Sequence: Synthetic Polynucleotide <400> 17 acatgtaccc Ttacgacgtc ccagactacg ctgcagatga tttggacttc gagacaggag atgcaggggc ctcagccacc ttcccaatgc agtgctcagc attacgtaag aatggttttg tggtgctcaa gggccggcca tgtaagatcg tcgagatgtc tacttcgaag actggcaggc atggccatgc caaggtccat ctggttggca ttgatatttt tactgggaag aaatatgaag 60 120 180

142,866 * Sequence Listing .DOC 240 201023898 atatctgccc gtcgactcat aacatggatg tccccaacat caaaaggaat gatttccagc tgattggcat ccaggatggg tacctatccc tgctccagga cagtggggag gtacgagagg accttcgtct gcctgaggga gaccttggca aggagattga gcagaagtat gactgtggag aagagatcct gatcacagtg ctgtccgcca tgacagagga ggcagctgtt gcaatcaagg cgatggcaaa ataactg < 210 > 18 < 211 > 165 < 212 > PRT <213>Artificial sequence <220><223> Description of artificial sequence: synthetic peptide

Met Asp Met Tyr Pro Tyr Asp Val Pro Asp Tyr Ala Ala Asp Asp Leu 15 10 15

Asp Phe Glu Thr Gly Asp Ala Gly Ala Ser Ala Thr Phe Pro Met Gin 20 25 30

Cys Ser Ala Leu Arg Lys Asn Gly Phe Val Val Leu Lys Gly Arg Pro 35 40 45

Cys Lys lie Val Glu Met Ser Thr Ser Lys Thr Gly Arg His Gly His 50 55 60

Ala Lys Val His Leu Val Gly lie Asp lie Phe Thr Gly Lys Lys Tyr 65 70 75 80

Glu Asp lie Cys Pro Ser Thr His As Met Asp Val Pro Asn lie Lys 85 90 95

Arg Asn Asp Phe Gin Leu lie Gly lie Gin Asp Gly Tyr Leu Ser Leu 100 105 110

Leu Gin Asp Ser Gly Glu Val Arg Glu Asp Leu Arg Leu Pro Glu Gly 115 120 125

Asp Leu Gly Lys Glu lie Glu Gin Lys Tyr Asp Cys Gly Glu Glu lie 130 135 140

142866· Sequence Listing. DOC 201023898

Leu lie Thr Val Leu Ser Ala Met Thr Glu Glu Ala Ala Val Ala lie 145 150 155 160

Lys Ala Met Ala Lys 165 <210> 19 <211> 154 <212> PRT <213> Homo sapiens <400> 19

Met Ala Asp Asp Leu Asp Phe Glu Thr Gly Asp Ala Gly Ala Ser Ala 15 10 15

Thr Phe Pro Met Gin Cys Ser Ala Leu Arg Lys Asn Gly Phe Val Val 20 25 30

Leu Lys Gly Arg Pro Cys Lys lie Val Glu Met Ser Thr Ser Lys Thr 35 40 45

Gly Lys His Gly His Ala Lys Val His Leu Val Gly lie Asp lie Phe 50 55 60

Thr Gly Lys Lys Tyr Glu Asp Labor Le Cys Pro Ser Thr His As Met Asp 65 70 75 80

Val Pro Asn work le Lys Arg Asn Asp Phe Gin Leu lie Gly lie Gin Asp 85 90 95

Gly Tyr Leu Ser Leu Leu Gin Asp Ser Gly Glu Val Arg Glu Asp Leu 100 105 110

Arg Leu Pro Glu Gly Asp Leu Gly Lys Glu lie Glu Gin Lys Tyr Asp 115 120 125

Cys Gly Glu Glu lie Leu lie Thr Val Leu Ser Ala Met Thr Glu Glu 130 135 140

Ala Ala Val Ala lie Lys Ala Met Ala Lys 145 150

<210> 20 <211> 36 <212> DNA

142866· Sequence Listing. DOC 36 201023898 <213> ΛΧ Sequence <220> <223> Description of Artificial Sequence: Synthetic Oligonucleotide <400> 20 aattctcgag tcatcgataa gcggccgcag acgcgt <210> 21 <211&gt ; 36 <212> DNA <213>Artificial Sequence<220>

<223> Description of artificial sequence: synthetic oligonucleotide <400> 21 36 aattacgcgt ctgcggccgc ttatcgatga ctcgag

<210> 22 <211> 1030 <212> DNA <213> Artificial Sequence <220><223> Description of Artificial Sequence: Synthetic Polynucleotide <400> 22 ggcatgtgaa ctggctgtct tggttttcat aaacatattt ataattccat taagctgtgc taaaggattt ttgtaagaac taattgaatt aataaataat aaatctcttt gttcagctct gtttttagtg gtagtgattt tattctcttt ataaatatat acaattttta tgaataaaaa atttttgttt atgtgagcaa acagcagatt ccgcagacgc gtaattcagt caatatgttc acctcattct aaaatgtata tagaagccca caaaatggga aagaatgttc cactaaatat gggatagaca gtgaggctga taaaatagag gtaagtgacc tatgaaaaaa atatggcatt ttttagaaaa acagggaaat atatttatat ctgtacttca tctgctacct ctgtgacctg atatgataga tttatcatat gtattttcct gatacctgta aagtctttat cacactaccc ctgtttctat aaatatgtac aagttttatt ctatatatat acacacacat gtgtgcattc attattagca atcaatattg Aaaaccactg aaaaggaatt ctcgagtcat cgataagcgg accccaaaaa agctgtttgt taacttgcca aaagacaata acaaaaatat tcttgtagaa caagatttag agcaaagcat gagatgtgtg tagagctcag aaacagaccc attgatatat ttacaatggg aaaatgatga tctttttctt gtaaaaaata aaagggaac c catatgtcat 60 120 180 240 300 360 420 480 540 600 660 720 •9-

142,866 * Sequence Listing .DOC 780 201023898 accatacaca caaaaaaaat tccagtgaat tataagtcta aatggagaag gcaaaacttt 840 aaatctttta gaaaataata tagaagcatg ccatcaagac ttcagtgtag agaaaaattt 900 cttatgactc aaagtcctaa ccacaaagaa aagattgtta attagattgc atgaatatta 960 agacttattt ttaaaattaa aaaaccatta agaaaagtca ggccatagaa tgacagaaaa 1020 tatttgcaac 1030 < 210 > 23 < 211 > 31 <212> DNA <213>Artifical sequence <220><223> Description of artificial sequence: synthetic primer <400> 23 gaagcggccg caccaccctg ggccaggctg g 31 <210> 24 <211> 36 <212> DNA <213>Artificial sequence <220><223> Description of artificial sequence: synthetic primer <400> 24 ccacgcgtag aggtgttaaa aagtctttag gtaaag 36 <210> 25 <211> 2282 <212> DNA <213 〉Artifical sequence <220><223> Description of artificial sequence: synthetic polynucleotide <400> 25 ttaattaaaa ttatctctaa ggcatgtgaa ctggctgtct tggttttcat ctgtacttca 60 tctgctacct ctgtgacctg aaacatattt ataattccat taagctgtgc atatgataga 120 Tttatcatat gtattttcct taaaggattt ttgtaagaac taattgaatt gatacctgta 180 aagtctttat cacactaccc aataaataat aaatctcttt gttcagctct ctgtttctat 240 aaatatgtac aagttttatt gtttttagtg gtagtgattt tattctcttt ctatatatat 300 acacacacat gtgtgcattc ataaatatat acaattttta tgaataaaaa attattagca 360 -10·

142866 - Sequence Listing. DOC 201023898

atcaatattg aaaaccactg atttttgttt ctcgagtcat cgataagcgg ccgcaccacc cctgtgtttt ccccagtctc tgggctgctg ctactgagcc ggagcccttc ctttcctgct ctacctccac cagggaccta ggcagccggg ccaagcaggg tctccacctg cttgaggctg taaagacttt ttaacacctc tacgcgtaat tgccaacctc attctaaaat aatattcttg tagaacaaaa tgggaaagaa agcatgagat gtgtggggat agacagtgag gacccattga tatatgtaag tgacctatga gatgatcttt ttctttttta gaaaaacagg gaacccatat gtcataccat acacacaaaa gaaggcaaaa ctttaaatct tttagaaaat gtagagaaaa atttcttatg actcaaagtc ttgcatgaat attaagactt atttttaaaa agaatgacag aaaatatttg tttgttaact caacacccca aaagaagtct tacaaatcag taaaaaataa gaaactctaa ataatcatta cacatgagaa tgcatataca ctaaattaga gaaatattaa aacaggtagt tgacaattaa acattggcat tataggaggg ccatcatggc caagttgacc gtggctggag ctgttgagtt ctggactgac gatgactttg caggtgtggt cagagatgat caggtggtgc ctgacaacac cctggcttgg gctgagtgga gtgaggtggt ctccaccaac gagattggag agcagccctg ggggagagag gtgcactttg tggcagagga gcaggactga aagccttata tattcttttt tttcttataa atgtgagcaa acagcagatt aaaaggaatt ctgggccagg ctgggccaag ccaggcggcc gagggaacca ggttgttttg gcatcagcct gctttgcata gtggcactaa ttccgtcctc tagatggtgg gaggaggctt cacttctccc ccctgggttg ggggaggcct tggctttacc tcagtcaata tgttcacccc caataacaaa aaaaaagctg gtatatagaa gcccaaaaga ttgtaatatg cagattataa aactagacaa tgttccacta aatatcaaga tttagagcaa gctgataaaa tagagtagag ctcagaaaca aaaaaatatg gcattttaca atgggaaaat gaaatatatt tatatgtaaa aaataaaagg aaattccagt gaattataag tctaaatgga aatatagaag catgccatca agacttcagt ctaaccacaa agaaaagatt gttaattaga ttaaaaaacc attaagaaaa gtcaggccat gtaaagagaa aaatttgaac agatgaaaga actcaatctc agaaatcaga gaactatcat aaggctaagt aacatctgtg gcttaattaa agtatatctg catagtataa tacaactcac agtgctgtcc cagtgctcac agccagggat aggttggggt tctccagaga ttttgtggag gtcaccctgt tcatctcagc agtccaggac gtgtgggtga gaggactgga tgagctgtat ttcagggatg ccagtggccc tgccatgaca tttgccctga gagacccagc aggcaactgt ggataaccta ggaaacctta aaacctttaa aacttaaaac cttagaggct atttaagttg -11 -

142866- Sequence Listing .DOC 201023898 ctgatttata ttaattttat tgttcaaaca tgagagctta gtacatgaaa catgagagct 2160 tagtacatta gccatgagag cttagtacat tagccatgag ggtttagttc attaaacatg 2220 agagcttagt acattaaaca tgagagctta gtacatacta tcaacaggtt gaactgctga 2280 tc 2282 < 210 > 26 < 211 > 29 < 212 > DMA < 213> Artificial Sequence <220><223> Description of Artificial Sequence: Synthetic Primer <400> 26 gttatcgata ctagtgcgac cgccaaacc 29 <210> 27 <211> 36 <212> DNA <213>Artificial Sequence <220>;<223> Description of artificial sequence: synthetic primer <400> 27 caagcggccg ccataccaca tttgtagagg ttttac 36 <210> 28 <211> 25 <212> DNA <213>artificial sequence <220>< 223 > Description of Artificial Sequence: Synthetic Primer <400> 28 caccatggca gatgatttgg acttc 25 <210> 29 <211> 154 <212> PRT < 213 > 213 > Artificial Sequence <220><2 2 3> Description of Artificial Sequence: Synthetic Peptide <400> 29

Met Ala Asp Asp Leu Asp Phe Glu Thr Gly Asp Ala Gly Ala Ser Ala •12-

142866·List of contents DOC 201023898 5 10 15

Thr Phe Pro Met Gin Cys Ser Ala Leu Arg Lys 20 25

Asn Gly Phe Val Val 30

Leu Lys Gly Arg Pro Cys Lys lie Val Glu Met 35 40

Ser Thr Ser Lys Thr 45

Gly Lys His Gly His Ala Lys Val His Leu Val 50 55

Gly lie Asp lie Phe 60

Thr Gly Lys Lys Tyr Glu Asp lie Cys Pro Ser 65 70 75

Thr His Asn Met Asp 80

Val Pro Asn lie Lys Arg Asn Asp Phe Gin Leu 85 90 lie Gly lie Gin Asp 95

Gly Tyr Leu Ser Leu Leu Gin Asp Ser Gly Glu 100 105

Val Arg Glu Asp Leu 110

Arg Leu Pro Glu Gly Asp Leu Gly Lys Glu lie 115 120

Glu Gin Lys Tyr Asp 125

Cys Gly Glu Glu lie Leu lie Thr Val Leu Ser 130 135

Ala Met Thr Glu Glu 140

Ala Ala Val Ala lie Lys Ala Met Ala Lys 145 150

<210> 30 <211> 3370 <212> DNA <213> Artificial sequence<220><223> Description of artificial sequence: synthetic polynucleotide <400> 30 ttaattaaaa tctgctacct tttatcatat aagtctttat ttatctctaa ctgtgacctg Ggttttcct cacactaccc ggcatgtgaa aaacatattt taaaggattt aataaataat ctggctgtct ataattccat ttgtaagaac aaatctcttt tggttttcat ctgtacttca taagctgtgc atatgataga taattgaatt gatacctgta gttcagctct ctgtttctat -13-

142,866 * Sequence Listing .DOC 201023898 aaatatgtac aagttttatt gtttttagtg gtagtgattt tattctcttt ctatatatat 300 acacacacat gtgtgcattc ataaatatat acaattttta tgaataaaaa attattagca 360 atcaatattg aaaaccactg atttttgttt atgtgagcaa acagcagatt aaaaggaatt 420 ctcgagtcat cgatactagt gcgaccgcca aaccttagcg gcccagctga caaaagcctg 480 ccctccccca gggtccccgg agagctggtg cctcccctgg gtcccaattt gcatggcagg 540 aaggggcctg gtgaggaaga ggcggggagg ggacaggctg cagccggtgc agttacacgt 600 tttcctccaa ggagcctcgg acgttgtcaa gcttctgcct tctccctcct gtgagtttgg 660 taagtcactg actgtctatg cctgggaaag ggtgggcagg agatggggca gtgcaggaaa 720 agtggcacta tgaaccctgc agccctagga atgcatctag acaattgtac taaccttctt 780 ctctttcctc tcctgacagg ttggtgtaca gtagcttcca ccatggcaga tgatttggac 840 ttcgagacag gagatgcagg ggcctcagcc accttcccaa tgcagtgctc agcattacgt 900 aagaatggtt ttgtggtgct caagggccgg ccatgtaaga tcgtcgagat gtctacttcg 960 aagactggca ggcatggcca tgccaaggtc catctggttg gtattgatat ttttactggg 1020 aagaaatatg aagatatctg Cccgtcgact cataacatgg atgtccccaa catcaaaagg 1080 aatgatttcc agctgattgg catccaggat gggtacctat ccctgctcca ggacagtggg 1140 gaggtacgag aggaccttcg tctgcctgag ggagaccttg gcaaggagat tgagcagaag 1200 tatgactgtg gagaagagat cctgatcaca gtgctgtccg ccatgacaga ggaggcagct 1260 gttgcaatca aggcgatggc aaaataactg gctagctggc cagacatgat aagatacatt 1320 gatgagtttg gacaaaccac aactagaatg cagtgaaaaa aatgctttat ttgtgaaatt 1380 tgtgatgcta ttgctttatt tgtaaccatt ataagctgca ataaacaagt taacaacaac 1440 aattgcattc attttatgtt tcaggttcag ggggaggtgt gggaggtttt ttaaagcaag 1500 taaaacctct acaaatgtgg tatggcggcc gcaccaccct gggccaggct gggccaagcc 1560 aggcggcccc tgtgttttcc ccagtctctg ggctgctgga gggaaccagg ttgttttggc 1620 atcagcctct actgagccgg agcccttcct ttcctgctgc tttgcatagt ggcactaatt 1680 ccgtcctcct acctccacca gggacctagg cagccgggta gatggtggga ggaggcttca 1740 cttctccccc aagcagggtc tccacctgct tgaggctgcc ctgggttggg ggaggccttg 1800 gctttaccta aagacttttt aacacctcta cgcgtaattc agtcaatatg ttcaccccaa 1860 aaaagctgtt tgttaacttg ccaacctcat tct Aaaatgt atatagaagc ccaaaagaca 1920 ataacaaaaa tattcttgta gaacaaaatg ggaaagaatg ttccactaaa tatcaagatt 1980 ·] 4·

142866 - Sequence Listing. DOC 201023898

tagagcaaag cagaaacaga gggaaaatga ataaaaggga taaatggaga acttcagtgt taattagatt caggccatag gattataaaa atgaaagaga actatcattg ttaattaaaa caactcacta ccagggatgt ttgtggagga tccaggacca agctgtatgc ccatgacaga gcaactgtgt acctttaaaa ttaagttgct tgagagctta taaacatgag actgctgatc catgagatgt cccattgata tgatcttttt acccatatgt aggcaaaact agagaaaaat gcatgaatat aatgacagaa agaagtctta aactctaaat catatacact caggtagttg taggagggcc ggctggagct tgactttgca ggtggtgcct tgagtggagt gattggagag gcactttgtg gccttatata gatttatatt gtacattagc agcttagtac gtggggatag tatgtaagtg cttttttaga cataccatac ttaaatcttt ttcttatgac taagacttat aatatttgca caaatcagta aatcattaca aaattagaga acaattaaac atcatggcca gttgagttct ggtgtggtca gacaacaccc gaggtggtct cagccctggg gcagaggagc ttcttttttt aattttattg catgagagct attaaacatg acagtgaggc acctatgaaa aaaacaggga acacaaaaaa tagaaaataa tcaaagtcct ttttaaaatt acaccccagt aaaaataaaa catgagaaac aatattaaaa attggcatag agttgaccag ggactgacag gagatgatgt tggcttgggt ccaccaactt ggagagagtt aggactgagg tcttataaaa ttcaaacatg tagtacatta agagcttagt tgataaaata aaaatatggc aatatattta attccagtga tatagaagca aaccacaaag aaaaaaccat aaagagaatt ctagacaaaa tcaatctcag ggctaagtaa tatatctgca tgctgtccca gttggggttc caccctgttc gtgggtgaga cagggatgcc tgccctgaga ataacctagg cttaaaacct agagcttagt gccatgaggg acatactatc gagtagagct attttacaat tatgtaaaaa attataagtc tgccatcaag aaaagattgt taagaaaagt gtaatatgca atttgaacag aaatcagaga catctgtggc tagtataata gtgctcacag tccagagatt atctcagcag ggactggatg agtggccctg gacccagcag aaaccttaaa tagaggctat acatgaaaca tttagttcat aacaggttga 2040 2100 2160 2220 2280 2340 2400 2460 2520 2580 2640 2700 27 60 2820 2880 2940 3000 3060 3120 3180 3240 3300 3360 3370 <210> 31 <211> 21 <212> DNA <213> Homo sapiens <220> 15-

142866· Sequence Listing. DOC 201023898 <223> Description of the combined DNA/RNA molecule: Homo sapiens <400> 31 gcuggacucc uccuacacat t 21 <210> 32 <211> 21 <212> DNA <213> Description of artificial sequence <220><223> combination of DNA/RNA molecules: Synthetic oligonucleotide <220><223> Description of artificial sequence: synthetic oligonucleotide <400> 32 uguguaggag Gaguccagct t 21 <210> 33 <211> 537 <212> DNA <213>Artificial sequence <220><2 2 3> Description of artificial sequence: synthetic polynucleotide <400> 33 atagggagac ccaagctgga gaccatgtac ccatacgacg tcccagacta cgctggaagc 60 ttaatggcag atgatttgga cttcgagaca ggagatgcag gggcctcagc caccttccca 120 atgcagtgct cagcattacg taagaatggt tttgtggtgc tcaagggccg gccatgtaag 180 atcgtcgaga tgtctacttc gaagactggc aggcatggcc atgccaaggt ccatctggtt 240 ggtattgata tttttactgg gaagaaatat gaagatatct gcccgtcgac tcataacatg 300 gatgtcccca acatcaaaag gaatgatttc cagctgattg gcatccagga tgggtaccta 360 tccctgctcc aggacagtgg ggaggtacga gaggacctt c gtctgcctga gggagacctt 420 ggcaaggaga ttgagcagaa gtatgactgt ggagaagaga tcctgatcac agtgctgtcc 480 gccatgacag aggaggcagc tgttgcaatc aaggccatgg caaaataact ggaattc 537 <210> 34 <211> 167 <212> PRT <213>Artificial sequence <220><223> Artificial sequence Description: Synthetic poly--16-

142866· Sequence Listing. DOC 201023898 <400> 34

Met Tyr Pro Tyr Asp Val Pro Asp Tyr Ala Gly Ser Leu Met Ala Asp 15 10 15

Asp Leu Asp Phe Glu Thr Gly Asp Ala Gly Ala Ser Ala Thr Phe Pro 20 25 30

Met Gin Cys Ser Ala Leu Arg Lys Asn Gly Phe Val Val Leu Lys Gly 35 40 45

Arg Pro Cys Lys lie Val Glu Met Ser Thr Ser Lys Thr Gly Arg His 50 55 60

Gly His Ala Lys Val His Leu Val Gly lie Asp lie Phe Thr Gly Lys 65 70 75 80

Lys Tyr Glu Asp lie Cys Pro Ser Thr His Asm Met Asp Val Pro Asn 85 90 95 lie Lys Arg Asn Asp Phe Gin Leu lie Gly lie Gin Asp Gly Tyr Leu 100 105 110

Ser Leu Leu Gin Asp Ser Gly Glu Val Arg Glu Asp Leu Arg Leu Pro 115 120 125

Glu Gly Asp Leu Gly Lys Glu lie Glu Gin Lys Tyr Asp Cys Gly Glu 130 135 140

Glu lie Leu lie Thr Val Leu Ser Ala Met Thr Glu Glu Ala Ala Val 145 150 155 160 Ala lie Lys Ala Met Ala Lys 165 <210> 35 <211> 154 <212> PRT <213> Homo sapiens <;400> 35

Met Ala Asp Asp Leu Asp Phe Glu Thr Gly Asp Ala Gly Ala Ser Ala 15 10 15

Thr Phe Pro Met Gin Cys Ser Ala Leu Arg Lys Asn Gly Phe Val Val 17·

142866 · Sequence Listing. DOC 201023898 20 25 30

Leu Lys Gly 35

Arg Pro Cys Lys lie Val Glu Met Ser Thr Ser Lys Thr 40 45

Gly Lys His 50

Gly His Ala Lys Val His Leu Val Gly lie Asp lie Phe 55 60

Thr Gly Lys 65

Lys Tyr Glu Asp lie Cys Pro Ser Thr His As Met Asp 70 75 80

Val Pro Asn lie Lys Arg Asn Asp Phe Gin Leu lie Gly lie Gin Asp 85 90 95

Gly Tyr Leu

Ser Leu Leu Gin Asp Ser Gly Glu Val Arg Glu Asp Leu 100 105 110

Arg Leu Pro 115

Glu Gly Asp Leu Gly Lys Glu lie Glu Gin Lys Tyr Asp 120 125

Cys Gly Glu 130

Glu lie Leu lie Thr Val Leu Ser Ala Met Thr Glu Glu 135 140

Ala Ala Val 145

Ala lie Lys Ala Met Ala Lys 150 <210> 36 <211> 154 <212> PRT <213> Homo sapiens <400> 36 Met Ala Asp 1

Asp Leu Asp Phe Glu Thr Gly Asp Ala Gly Ala Ser Ala 5 10 15

Thr Phe Pro

Met Gin Cys Ser Ala Leu Arg Lys Asn Gly Phe Val Val 20 25 30

Leu Lys Gly 35

Arg Pro Cys Lys lie Val Glu Met Ser Thr Ser Lys Thr 40 45

Gly Lys His 50

Gly His Ala Lys Val His Leu Val Gly lie Asp lie Phe 55 60

142866 - Sequence Listing. DOC 18 _ 201023898

Thr Gly Lys Lys Tyr Glu Asp lie Cys Pro Ser Thr His As Met Asp 65 70 75 80

Val Pro Asn lie Lys Arg Asn Asp Phe Gin Leu lie Gly lie Gin Asp 85 90 95

Gly Tyr Leu Ser Leu Leu Gin Asp Ser Gly Glu Val Arg Glu Asp Leu 100 105 110

Arg Leu Pro Glu Gly Asp Leu Gly Lys Glu lie Glu Gin Lys Tyr Asp 115 120 125

Cys Gly Glu Glu lie Leu lie Thr Val Leu Ser Ala Met Thr Glu Glu 130 135 140

Ala Ala Val Ala lie Lys Ala Met Ala Lys 145 150 <210> 37 <211> 148 <212> PRT <213> Homo sapiens <400> 37

Phe Glu Thr Gly Asp Ala Gly Ala Ser Ala Thr Phe Pro Met Gin Cys 15 10 15

Ser Ala Leu Arg Lys Asn Gly Phe Val Val Leu Lys Gly Arg Pro Cys 20 25 30

Lys lie Val Glu Met Ser Thr Ser Lys Thr Gly Lys His Gly His Ala 35 40 45

Lys Val His Leu Val Gly lie Asp lie Phe Thr Gly Lys Lys Tyr Glu 50 55 60

Asp lie Cys Pro Ser Thr His Asm Met Asp Val Pro Asn lie Lys Arg 65 70 75 80

Asn Asp Phe Gin Leu lie Gly lie Gin Asp Gly Tyr Leu Ser Leu Leu 85 90 95

Gin Asp Ser Gly Glu Val Arg Glu Asp Leu Arg Leu Pro Glu Gly Asp 100 105 110 •19·

142866 - Sequence Listing. DOC 201023898

Leu Gly Lys Glu lie Glu Gin Lys Tyr Asp Cys Gly Glu Glu lie Leu 115 120 125 lie Thr Val Leu Ser Ala Met Thr Glu Glu Ala Ala Val Ala lie Lys 130 135 140

Ala Met Ala Lys 145 < 210 > 38 < 211 > 447 < 212 > DNA < 213> Homo sapiens < 400 > 38 ttcgagacag gagatgcagg ggcctcagcc accttcccaa tgcagtgctc agcattacgt aagaatggct ttgtggtgct caaaggccgg ccatgtaaga tcgtcgagat gtctacttcg aagactggca agcacggcca cgccaaggtc catctggttg gtattgacat ctttactggg aagaaatatg aagatatctg cccgtcaact cataatatgg atgtccccaa catcaaaagg aatgacttcc agctgattgg catccaggat gggtacctat cactgctcca ggacagcggg gaggtacgag aggaccttcg tctccctgag ggagaccttg gcaaggagat tgagcagaag tacgactgtg gagaagagat cctgatcacg gtgctgtctg ccatgacaga ggaggcagct gttgcaatca aggccatggc aaaataa < 210 > 39 < 211 > 154 < 212 > PRT < 213> Homo sapiens < 400 > 39

Met Ala Asp Asp Leu Asp Phe Glu Thr Gly Asp Ala Gly Ala Ser Ala 15 10 15

Thr Phe Pro Met Gin Cys Ser Ala Leu Arg Lys Asn Gly Phe Val Val 20 25 30

Leu Lys Gly Arg Pro Cys Lys lie Val Glu Met Ser Thr Ser Lys Thr 35 40 45

Gly Lys His Gly His Ala Lys Val His Leu Val Gly lie Asp lie Phe 50 55 60 •20- β O 12 0 18 0 2 4 0 30 0 3 6 0 4 2 CD 4 4 7

142866* Sequence Listing. DOC 201023898

Thr Gly Lys Lys Tyr Glu Asp lie Cys Pro Ser Thr His As Met Asp 65 70 75 80

Val Pro Asn lie Lys Arg Asn Asp Phe Gin Leu lie Gly lie Gin Asp 85 90 95

Gly Tyr Leu Ser Leu Leu Gin Asp Ser Gly Glu Val Arg Glu Asp Leu 100 105 110

Arg Leu Pro Glu Gly Asp Leu Gly Lys Glu lie Glu Gin Lys Tyr Asp 115 120 125

Cys Gly Glu Glu lie Leu lie Thr Val Leu Ser Ala Met Thr Glu Glu 130 135 140

Ala Ala Val Ala lie Lys Ala Met Ala Lys 145 150 <210> 40 <211> 148 <212> PRT <213> Homo sapiens <400> 40

Phe Glu Thr Gly Asp Ala Gly Ala Ser Ala Thr Phe Pro Met Gin Cys 15 10 15

Ser Ala Leu Arg Lys Asn Gly Phe Val Val Leu Lys Gly Arg Pro Cys 20 25 30

Lys lie Val Glu Met Ser Thr Ser Lys Thr Gly Lys His Gly His Ala 35 40 45

Lys Val His Leu Val Gly lie Asp lie Phe Thr Gly Ly.s Lys Tyr Glu 50 55 60

Asp lie Cys Pro Ser Thr His Asm Met Asp Val Pro Asn lie Lys Arg 65 70 75 80

Asn Asp Phe Gin Leu lie Gly lie Gin Asp Gly Tyr Leu Ser Leu Leu 85 90 95

Gin Asp Ser Gly Glu Val Arg Glu Asp Leu Arg Leu Pro Glu Gly Asp •21·

142866 · Sequence Listing. DOC 201023898 100 110

Leu Gly Lys Glu lie Glu Gin Lys 115 120

Asp Cys Gly Glu Glu lie Leu 125 lie Thr Val Leu Ser Ala Met Thr 130 135

Glu Ala Ala Val Ala lie Lys 140

Ala Met Ala Lys 145 <210> 41 <211> 154 <212> PRT <213> Homo sapiens <400> 41 Met Ala Asp Asp Leu Asp Phe Glu 1 5 Thr Phe Pro Met Gin Cys Ser Ala 20 Leu Lys Gly Arg Pro Cys Lys lie 35 40 Gly Lys His Gly His Ala Lys Val 50 55 Thr Gly Lys Lys Tyr Glu Asp lie 65 70 Val Pro Asn lie Lys Arg Asn Asp 85 Gly Tyr Leu Ser Leu Leu Gin Asp 100 Arg Leu Pro Glu Gly Asp Leu Gly 115 120 Cys Gly Glu Glu lie Leu lie Thr 130 135

Gly Asp Ala Gly Ala Ser Ala 10 15

Arg Lys Asn Gly Phe Val Val 30

Glu Met Ser Thr Ser Lys Thr 45

Leu Val Gly lie Asp lie Phe 60

Pro Ser Thr His Asn Met Asp 75 80

Gin Leu lie Gly lie Gin Asp 90 95

Gly Glu Val Arg Glu Asp Leu 110

Glu lie Glu Gin Lys Tyr Asp 125

Leu Ser Ala Met Thr Glu Glu 140 -22·

142866 - Sequence Listing. DOC 201023898

Ala Ala Val Ala lie Lys Ala Met Ala Lys 145 150 <210> 42 <211> 6 <212> PRT <213> Homo sapiens <400>

Met Ala Asp Asp Leu Asp 1 5

aggcagctgt tgcaatcaag 43 atggcagatg acttggactt cgagacagga cagtgctcag cattacgtaa gaatggcttt gtcgagatgt ctacttcgaa gactggcaag attgacatct ttactgggaa gaaatatgaa gtccccaaca tcaaaaggaa tgacttccag ctgctccagg acagcgggga ggtacgagag aaggagattg agcagaagta cgactgtgga atgacagagg; < 210 > 43 < 211 > 465 < 212 > DNA < 213 > Homo sapiens < 400 & gt Gatgcagggg cctcagccac cttcccaatg gtggtgctca aaggccggcc atgtaagatc cacggccacg ccaaggtcca tctggttggt gatatctgcc cgtcaactca taatatggat ctgattggca tccaggatgg gtacctatca gaccttcgtc tccctgaggg agaccttggc gaagagatcc tgatcacggt gctgtctgcc gccatggcaa aataa 60 120 180 240 300 360 420 465

gtggtgctca catggtcatg 44 atggcagacg cagtgctcgg gtggagatgt attgatattt gttccaaata ctgctgacag aaagaaatag aaattgattt ccttgcgcaa caacttccaa tcacgggcaa ttaagagaaa aaactggtga agggaaaata cactactgga aaacggcttc aactggaaag aaaatatgaa tgattatcaa agttcgtgag caatgcaggt gatgccgggg; < 210 > 44 < 211 > 462 < 212 > DNA < 213> Homo sapiens < 400 & gt Gatatttgtc ctgatatgca gatcttaaac gaagatgtac cttccagcac aaggacgacc ccaaggttca cttctactca ttcaagatgg tgccagaagg aggtgtctgt ttaccctatg atgcaaaata ccttgttgga caacatggat ttacctttcc tgaactaggc catgtgtgca 60 120 180 240 300 360 420 •23-

142866· Sequence Listing DOC 201023898 atgagtgaag aatatgctgt agccataaaa ccctgcaaat aa 462 <210> 45 <211> 19 <212> DNA <213> Homo sapiens <400> 45 gctggactcc tcctacaca 19 <210> 46 <211> 19 <212> RNA <213> Homo sapiens <400> 46 gcuggacucc uccuacaca 19 <210> 47 <211> 4 <212> PRT <213> Homo sapiens <400> 47 Asp Asp Leu Asp 1 <210> 48 <211> 4 <212> PRT < 213 > 213 > artificial sequence<220><223> Description of artificial sequence: synthetic peptide <400> 48 Leu Glu His Asp 1 <210> 49 <211> 4 <212> PRT <213>Artificial sequence <220><223> Description of artificial sequence: synthetic peptide <400> 49 Asp Glu Val Asp 1

142866 - Sequence Listing. DOC • 24-201023898 <210> 50 <211> 4 <212> PRT <213> Artificial Sequence <220><223> Description of Artificial Sequence: Synthetic Peptide <400>; 50 lie Glu Thr Asp 1

<210> 51 <211> 4 <212> PRT < 213 > artificial sequence <220><223> Description of artificial sequence: synthetic peptide <400> 51 Tyr Val Ala Asp 1

142866 - Sequence Listing. DOC -25-

Claims (1)

201023898 VII. Patent Application Range: L—The use of the eIF5Al polynucleotide of the short eIF5Al protein of the bat code is used to prepare a drug that induces apoptosis of cancer cells or tumors in an individual. 2. The use of claim 1, wherein the eIF5Al polynucleotide encodes a truncated eIF5Al protein comprising the amino acid sequence of SEQ ID NO: 37 as shown in Figure 38. 3. 4. 5. 6. 7. 8. The use of claim 1, wherein the eIF5Al polynucleotide encodes a short eIF5A1 protein of about 16 kDA. The use of claim 1 wherein the individual is a mammal. The use of claim 4, wherein the mammal is a human. For example, the use of cells, which are caused by cancer cells or tumors, can slow down the growth of cancer cells or tumors, prevent cancer cells or tumor cells from growing or poisoning cancer cells or reduce tumor volume. The use of claim 1, wherein the cancer is multiple myeloma. The use of claim 1, wherein the eIF5A1 polynucleotide comprises the sequence set forth in SEQ ID NO: 38 (shown in Figure 41). 9. The use of claim 1, wherein the eIF5A1 polynucleotide is contained within a plastid or expression vector. 10. The use of claim 1, wherein the expression vector is a gland expression vector or pHM6. 11. The use of claim 1 wherein the expression vector comprises a tissue-specific promoter. 12. The use of claim 11, wherein the tissue-specific promoter is a B 142866.doc 201023898 cell-specific promoter. 13. For the purposes of claim 12, B29. 14 15 wherein the B cell specific promoter is </w~, wherein the expression carrier and the polyethyleneimine are used as the target, wherein the expression vector has a reduced [pc dinucleotide. 17. The use of claim 1, wherein the agent is administered intratumorally, intravenously or subcutaneously. 18. An isolated polynucleotide encoding a truncated eIF5A1 protein wherein the polynucleotide comprises the sequence set forth in SEQ ID NO: 38 (shown in Figure 41). 19. An isolated polynucleotide encoding a truncated elF5Al protein' wherein the truncated protein comprises the amino acid sequence set forth in SEQ ID NO: 37 (shown in Figure 38). 20. An isolated truncated eIF5Al polypeptide formed by a cleavage-mediated cysteine protease of eIF5Al. 21. Use of an eIF5Al polynucleic acid encoding a truncated eIF5Al protein and a full length eIF5Al polynucleotide for use in the preparation of a medicament for eliciting apoptosis in cancer cells or tumors in an individual. 22. The use of claim 21, wherein the eIF5Al polynucleotide encodes a truncated eIF5Al protein comprising the amino acid sequence of SEQ ID NO: 37, and wherein the full length eIF5Al polynucleotide encoding comprises SEQ ID NO: 35 Amine 142866.doc 201023898 Protein of the base acid sequence. 23. The use of claim 21, wherein the eIF5Al polynucleotide encodes a truncated eIF5Al protein of about 16 kDA. 24. The use of claim 21, wherein the individual is a mammal. 25. The use of claim 24, wherein the mammal is a human. 26. The use of claim 21, wherein the cancer cells or tumor-induced cells &gt; peripheral effects can slow the growth of cancer cells or tumors, prevent cancer cells or tumor cells from growing or poisoning cancer cells or reduce tumor volume. ❹ 27. The use of claim 21, wherein the cancer is multiple myeloma. 28. The use of claim 21, wherein the eIF5Al polynucleotide comprises the sequence of SEQ ID ΝΟ.38, and wherein the full length eIF5Al polynucleotide comprises the sequence of SEQ ID NO: 43 (shown in Figure 53). 29. The use of claim 23, wherein the full-length eIF5Al polynucleotide encodes a mutated eIF5Al, wherein the mutation prevents or inhibits hypusination by deoxy-helper synthase, and/or Wherein the mutation is present in the ubiquitination site and/or the acetylation site. ® 30. The composition of claim 23, wherein the mutant is from K50A, K50R, K67A, K47R, K67R, K50A/K67A, K50A/K47R, K50A/K67R, K50R/K67A, K50R/K47R, *K50R/ Selected from the group consisting of K67R and K47A/K67A. 31. The use of claim 29, further comprising targeting a 3'UTR siRNA against eIF5A. 32. The use of claim 31, wherein the siRNA target is aligned with the sequence 5 of eIF5A, -GCT GGA CTC CTC CTA CAC A-3'. 142866.doc 201023898 33. The use of claim 32, wherein the siRNA is a dsRNA and a strand of the dsRNA comprises a sequence of 5'-GCU GGA CUC CUC CUA CAC A-3'. 34. The use of claim 32, wherein the siRNA is stable to prevent degradation of serum. 35. The use of claim 31, wherein the full length eiF5Al polynucleotide and/or the elF5Al polynucleotide encoding the truncated eIF5Al protein is present in a performance vector. 36. The use of claim 35, wherein the full length eiF5Al polynucleotide and/or the elF5Al polynucleotide encoding the truncated eIF5Al protein and/or the siRNA are mismatched with a polyethyleneimine. 37. The use of claim 36, wherein the full length eIF5A1 polynucleotide and/or the elF5Al polynucleotide encoding the truncated eIF5Al protein and/or the siRNA are independently mismatched with a polyethyleneimine. A method of inducing tumor apoptosis in a mammalian cancer cell or a mammal by providing the mammal a composition according to any one of claims 1 to 38. 39. The method of claim 38, wherein the composition is administered intravenously, intraperitoneally, or intratumorally. 40. The method of claim 38, wherein the cancer is multiple myeloma. 41. A composition comprising a complex formed by an eIF5Ai siRNA targeting an eIF5Ai 3, a terminal ORF and a expression vector comprising a polynucleotide encoding a mutant eIF5A1, wherein the mutant eIF5Al is incapable of being lysine by carboxyresinamine, Wherein the siRNA and the expression vector are mismatched with polyethyleneimine to form a mismatch 142866.doc 201023898. 42. A composition comprising a siRNA that targets a target gene to inhibit endogenous expression of the target gene in an individual, and a polynucleotide encoding a target protein that is expressed in an individual against an RNAI plastid, Wherein the siRNA. is mismatched with the plastid system and polyethyleneimine to form a complex. 43. The composition of claim 41, wherein the siRNA has the sequence shown in Figure 25, and wherein the polynucleotide encoding the mutant eIF5Al is eIF5AlK5GR. 44. The composition of claim 43, which comprises a tissue-specific promoter. Φ 45. The composition of claim 44, which comprises a B cell specific promoter. 46. The composition of claim 45, wherein the B cell specific promoter is B29. 47. The composition of claim 43, wherein the expression vector comprises a pCpG plastid. 48. The composition of claim 41, wherein the eIF5Al siRNA and the expression vector comprising the mutant eIF5Al polynucleotide are independently mismatched with polyethyleneimine. 49. The composition of claim 41, wherein the eIF5Al siRNA and the expression vector comprising the mutant eIF5Al polynucleotide are together with the polyethyleneimine. ® 50. A composition comprising an eIF5Al siRNA targeting the 3' end of eIF5Al and a expression vector comprising a polynucleotide encoding the mutant eIF5Al, wherein the mutant eIF5Al is not lysylated by carboxyresosamine, and wherein the siRNA is A sputum expression vector is administered to an individual to treat cancer. 5. The composition of claim 50, wherein the cancer is multiple myeloma. 52. A method of treating cancer comprising administering to a subject a composition of claim 50. 53. A method of treating cancer comprising administering to a subject a combination of claim 41 142866.doc 201023898. 54. The method of claim 52, wherein the composition is administered intravenously, intraperitoneally, or intratumorally. 55. The method of claim 53, wherein the siRNA targeting the 3' end of eIF5Al and the expression vector comprising the polynucleotide encoding the mutant eIF5Al are administered by different routes. 56. The method of claim 52, wherein the composition is administered at a dose of from about 0.15 mg/kg to about 1.5 mg/kg twice a week. 57. The method of claim 52, wherein the composition is administered at a dose of from about 0.75 mg/kg to about 1.5 mg/kg twice a week. 142866.doc