CN102202693A - Use of a truncated elf-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-5Al that is useful for inducing apoptosis and killing of cells, especially cancer cells in a mammal.

Description

The purposes of truncated-type EIF-5A1 polynucleotide cancer cell specific induction of apoptosis

Related application

The application requires the priority of the U. S. application number 12/400,742 of the U.S. Provisional Application number application on March 19th, 61/093,749 and 2009 that JIUYUE in 2008 submitted on the 3rd, they all by reference integral body be attached to herein.

Background of invention

Recently, research worker observes double-stranded RNA (" dsRNA ") and can be used for the expression of Profilin matter, the ability of this silent gene of double-stranded RNA has the extensive potentiality of treatment human diseases, and now, many research worker and commercial entity's great amount of investment exploitation are based on the therapy of this technology.

At least 3 kinds of different levels can appear in double-stranded RNA induced gene silence: (i) transcribe inactivation, i.e. the DNA of RNA guiding or histone methylated; The (ii) inductive mRNA degraded of siRNA; The (iii) inductive transcription attenuation of mRNA.

It is generally acknowledged that to induce the main mechanism of silence (RNA disturb or RNAi) be the mRNA degraded to RNA in mammalian cell.In mammalian cell, attempt at first using RNAi mainly to be to use long-chain dsRNA.Yet these induce the obtained effect of the trial of RNAi limited, and partly cause is to have induced ifn response, and ifn response is opposite with target-specific, and it causes protein synthesis generally to suppress.Therefore, long-chain dsRNA is not a feasible selection for the RNAi of mammlian system.

Recently, evidence suggests, in the time will lacking the mammalian cell of (18-30bp) RNA duplex introducing cultivation, can realize that the sequence-specific of said target mrna suppresses and not inducing interferon reaction.Some short dsRNA is called as little inhibition type RNA (" siRNA "), can carry out the catalytic effect in inferior molar concentration, with the said target mrna more than 95% in the incising cell.The description of siRNA mechanism of action and some are used referring to Provost et al. (2002) Ribonuclease Activity and RNA Binding of Recombinant Human Dicer, and EMBO is (21) J.21: 5864-5874; Tabara et al. (2002) The dsRNA Binding Protein RDE-4 Interacts with RDE-I, DCR-I 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

From mechanistic approach, the III type Cobra venom endonuclease that the long dsrna of introduced plant and no spinal animals cell is called as Dicer is decomposed into siRNA.Sharp，RNA?interference-2001，Genes?Dev.2001，15：485。Dicer is a kind of ribonuclease-III-sample enzyme, and it is processed as the short interfering rna s of 19-23 base pair, 3 ' jag with distinctive 2 bases with dsRNA.Bernstein，Caudy，Hammond，&?Hannon(2001)Role?for?a?bidentate?ribonuclease?in?the?initiation?step?of?RNA?interference，Nature?409：363。SiRNAs mixes in the inductive reticent complex of RNA-(RISC) then, and wherein one or more unwindases siRNA two strands of untwisting makes complementary antisense strand instruct target identification.Nykanen，Haley，&?Zamore(2001)，ATP?requirements?and?small?interfering?RNA?structure?in?the?RNA?interference?pathway，Cell?107：309。When being attached to suitable said target mrna, one or more Cobra venom endonuclease cutting said target mrnas among the RISC are to induce silence.Elbashir, Lendeckel ， ﹠amp; Tuschl (2001) RNA interference is mediated by 21-and 22-nucleotide RNAs, Genes Dev.15:188, Fig. 1.

Described interference effect can continue for a long time, and many times still can detect behind the cell division.And RNAi has sequence-specific.Kisielow，M.et?al.(2002)，Isoform-specific?knockdown?and?expression?of?adaptor?protein?ShcA?using?small?interfering?RNA，J.Biochem.363：1-5。Therefore, but RNAi mechanism specificity suppresses one type transcript, can not influence closely-related with it mRNA simultaneously.The useful tool that these characteristics make siRNA may become inhibition of gene expression, research gene function and confirm the medicine target.And siRNA may be used as following treatment of diseases agent: (1) is by the disease that mistake is expressed or false demonstration causes of gene; (2) disease that causes by the gene expression that comprises sudden change.

Successful siRNA-dependent gene silence depends on many factors.About RNAi one of the most controversial problem be the problem of siRNA design necessity, promptly consider the sequence of used siRNA.The early stage work of carrying out with Caenorhabditis elegans (C.elegans) and plant walked around because of the long dsRNA of introducing this siRNA sequential design problem (referring to, for example, Fire, A.et al. (1998) Nature 391:806-811).In this primitive organism, long dsRNA molecule is cut into siRNA by Dicer, thereby produces various duplexs, and it may cover the complete transcriptional thing.Although some part right and wrong in these molecules functional (that is, induce seldom or not reticent), one or more molecules may have high functionality, therefore can reticent target gene, and not too need to design siRNA.Unfortunately, since ifn response, the inapplicable mammlian system of same method.Yet, avoid the Dicer cutting step, directly to introduce siRNA and can avoid this influence, the risk of implementing this strategy is that the siRNA sequence possibility right and wrong of choosing are functional or partly functional.

Many researchs have shown that the siRNA design is not the viewpoint of the key factor of RNAi.On the other hand, other research worker in this field has been set about research emphasis siRNA design and has been made more effectively probability of RNAi.

In order to treat various diseases or obstacle, need to raise some protein, but this may not need whole.For example, may need to be used in combination the expression that siRNA suppresses or knock out endogenous protein or different proteins.The present invention addresses that need and provides the method for treatment cancer, especially multiple myeloma.

Cancer comprises multiple myeloma, is the disease that can benefit from cell death inducing.The traditional remedies of multiple myeloma comprises chemotherapy, stem cell transplantation, high dose chemotherapy associating stem cell transplantation and complementary therapy.Chemotherapy comprises uses following Drug therapy: Thalomid

(Thalidomide), bortezomib, Aredia (pamidronic acid), steroid and Zometa

(zoledronic acid).For example medullary cell, gastrointestinal mucosa and hair follicle all are toxic yet many chemotherapeutics are to mitotically active non-cancerous cell.So chemotherapy may cause blood cell count minimizing, nausea,vomiting,diarrhea and alopecia.

Conventional chemotherapy or standard dose chemotherapy are typical elementary treatment of multiple myeloma patients or initial therapy.The patient also can accept the chemotherapy and the stem cell transplantation of high dose chemotherapy preparation.Before can be used for transplanting, antilepsis (conventional chemotherapy before the stem cell transplantation) reduces tumor load.Compare with other therapies, some chemotherapeutics is more suitable for antilepsis, because they are less and make bone marrow produce more stem cell to medullary cell toxicity.The chemotherapeutics example that is suitable for antilepsis comprises dexamethasone, Thalidomide/dexamethasone, VAD (vincristine, Adriamycin

(doxorubicin) and dexamethasone make up) and DVd (pegylated liposomal doxorubicin (Doxil

Caelyx

), the dexamethasone combination of vincristine and the shortening course of treatment (reduced schedule)).

The standard care of multiple myeloma be melphalan in conjunction with prednisone (a kind of corticosteroids), response rate reaches 50%.Unfortunately, thus melphalan is an alkylating agent is not suitable for antilepsis very much.Cortical steroid (especially dexamethasone) is used for multiple myeloma therapy, especially gerontal patient sometimes separately and those are impatient at the patient of chemotherapy.The same associating separately or with other medicines of dexamethasone is used for antilepsis.VAD is the most frequently used antilepsis, and proves that recently DVd is effective in antilepsis.Recently, bortezomib is approved for the treatment multiple myeloma, but its toxicity is very big.Yet, do not have a kind of obvious probability that shows healing in the existing therapy.Therefore, still be necessary the to seek treatment suitable therapy of cancer and multiple myeloma.The present invention addresses that need.

Brief summary of the invention

The invention provides separation polynucleotide and the truncated-type eIF-A1 polypeptide of coding truncated-type eIF-5A1.Truncated-type eIF-5A1 polynucleotide can be used for inducing patient's cancer cell-apoptosis and kill cancer cell.Can in expression vector, use the truncated-type polynucleotide, give the patient with it then.Truncated-type eIF-5A form can be at mammal expression in vivo and kill cancer cell.The about 16kDA of truncated-type eIF-5A1 protein, and the about 17kDa of total length eIf-5A1 protein.The proteinic polynucleotide of coding truncated-type eIF5A1 can be used to prepare the medicine that is used to induce patient's cancerous cell or tumor death.The proteinic polynucleotide of coding truncated-type eIF5A1 can be united other embodiment described herein and be used (for example the siRNA of the 3 ' UTR of the polynucleotide of the polynucleotide of combined coding total length eIF5A1, combined coding total length sudden change eIF5A1 and/or associating targeting eIF5A uses).

The invention still further relates to unite uses the targeting endogenous gene to express in the host with the protein that described polynucleotide encoding is provided to the host with the siRNA that knocks out or suppress endogenous gene and express in the host and the polynucleotide of sending the described gene of coding with delivery media (a delivery vechicle)/expression vector.Normal (non-defective) the proteinic polynucleotide (or protein itself) of will encoding give the host and express (under the polynucleotide situation), make normal protein matter can fulfil its necessary function.Preferably siRNA is designed to the described gene region of targeting, it can not influence the heterogenous expression of the polynucleotide of the coding normal protein matter that is given when suppressing or knocking out the endogenous expression of deficient protein matter like this.

The invention provides a kind of compositions, its comprise targeting eIF5A1 3 ' terminal eIF5A1siRNA, comprise the complex of expression vector of the polynucleotide of encoding mutant eIF5A1, wherein said sudden change eIF5A1 can not be by hydroxyl fourth lysineization, and wherein said siRNA and expression vector and polyaziridine (polyethylenimine) are compounded to form complex.

The invention provides a kind of compositions, it comprises the polynucleotide of the target protein that the targeting target gene can express with the siRNA that suppresses the endogenous expression of this target gene of patient and coding in the patient.In certain embodiments, described polynucleotide are present in the RNAi r plasmid and (can not be suppressed by siRNA).Described siRNA and plasmid preferably are compounded to form complex with polyaziridine.

In certain embodiments, siRNA has sequence shown in Figure 25, and wherein the polynucleotide of encoding mutant eIF5A1 are eIF5A1K ^50RExpression vector comprises the polynucleotide of encoding mutant eIF5A1 expresses in the patient so that polynucleotide to be provided with the promoter that effectively is connected.Promoter is preferably tissue-specific or systematic.For example, if compositions is used for the treatment of cancer, preferred promoter is tissue-specific to the residing tissue of this cancer so.For example, for the treatment multiple myeloma, preferably use the B cell specificity promotor, for example B29.In certain embodiments, expression vector comprises the pCpG plasmid.

In certain embodiments, eIF5A1 siRNA is independent compound with polyaziridine with the expression vector that comprises sudden change eIF5A1 polynucleotide, for example In vivo JetPEI ^TMIn other embodiments, eIF5A1 siRNA is combined with each other with the expression vector and the polyaziridine that comprise sudden change eIF5A1 polynucleotide.

The present invention also provides a kind of compositions, it comprises the eIF5A1 siRNA and the expression vector that comprises the polynucleotide of encoding mutant eIF5A1 of the 3 ' end of targeting eIF5A1, wherein said sudden change eIF5A1 can not be by hydroxyl fourth lysineization, and wherein said siRNA and expression vector are delivered to the patient with the treatment cancer.Described cancer can be any cancer, comprises multiple myeloma.

The present invention also provides a kind of treatment method for cancer, and it comprises and gives patient's (including but not limited to mammal and people) compositions of the present invention.

The present composition gives by any acceptable approach, in intravenous, intraperitoneal, subcutaneous or tumor.SiRNA and expression vector can give through different approaches in different time, perhaps can give together through same approach in the same time.Such as but not limited to, siRNA directly or with carrier compound (for example In vivo jetPEI) can be sent through IV, but and give in the described expression vector tumor, perhaps can the two gives in IV or tumor etc. with described siRNA and expression vector.

The invention provides the method for a kind of anticancer growth and/or kill cancer cell.The present invention also provides a kind of method that suppresses or slow down the cancer cell metastasis ability.Suppress growth of cancers and comprise and reduce the tumor size, delay tumor growth, also can comprise thorough elimination tumor.Described cancer can be any cancer or tumor, includes but not limited to colon cancer, knot rectal adenocarcinoma, bladder cancer, adenocarcinoma of the uterine cervix and pulmonary carcinoma.Preferred cancer is a multiple myeloma.

In preferred embodiments, described eIF-5A be a kind of can not be by the mutant of hydroxyl fourth lysineization.This paper has described typical mutant.

Except polynucleotide that eIF-5A or coding eIF-5A are provided to patient's (expressing eIF-5A) to provide, also provide siRNA to knock out or to suppress the endogenous expression of eIF-5A.

The purposes of medicine that the present invention also provides the polynucleotide of eIF5A, coding eIF5A1 and is used to prepare treatment cancer, kill multiple myeloma patient's multiple myeloma cells at the siRNA of eIF5A1.The polynucleotide of optimized encoding sudden change eIF-5A are not by hydroxyl fourth lysineization.

The present invention also provides a kind of method for the treatment of sicklemia.The patient that the polynucleotide of healthy hemoglobin gene (for example HBB) of will encoding are suffered from sicklemia.Also unite and give the patient siRNA, described siRNA targeting coding defective hemoglobin gene (for example gene of encoding mutant HbS) is expressed to suppress or to knock out described deficient protein matter.Described treatment also can comprise give other the treatment sicklemia in normally used known drug or treatment.

Brief description

Fig. 1 provides the aminoacid sequence of people eIF-5A1 and illustrates different important sites.

Fig. 2 illustrates eIF-5A1 increases the proteic accumulation of transfection in the sudden change of K50 and K67.Referring to embodiment 1.

Fig. 3 illustrates eIF5A1 increases the proteic accumulation of transfection in the sudden change of K47, K50 and K67.Referring to embodiment 2.

The eIF5A1 transfection that Fig. 4 illustrates K50 and K67 sudden change cell death inducing in the KAS cell time.Referring to embodiment 3.

The eIF5A1 transfection that Fig. 5 illustrates K50 and K67 sudden change cell death inducing in the KAS cell time.Referring to embodiment 4.

The eIF5A1 transfection that Fig. 6 illustrates K50 and K67 sudden change cell death inducing in the KAS cell time.Referring to embodiment 5.

Fig. 7 A diagram is with the siRNA transfection with causing the KAS apoptosis through modifying the adenovirus processing of expressing eIF-5A1.Referring to embodiment 6A.

The pretreatment (at target #1 (SEQ ID NO:1)) (siRNA construct sequence shown in Figure 25) of Fig. 7 B diagram eIF5A1 siRNA has reduced endogenous eIF5A1 to be expressed, but makes the RNAi-resistance eIF5A1 of gland virus expression ^K50AAccumulation.Referring to embodiment 6B.

Fig. 7 C diagram is used at the eIF5A1 siRNA pretreatment of target #1 and is carried out the phosphorylation NF-κ B expression that adenovirus infection reduces people's multiple myeloma cells then.Referring to embodiment 6C.

Fig. 7 D diagram is used at the eIF5A1 siRNA pretreatment of target #1 and is carried out phosphorylation NF-kB and the ICAM-1 expression that adenovirus infection reduces people's multiple myeloma cells then.Referring to embodiment 6D.

The NFkB DNA-that Fig. 7 E is shown in the eIF5A inhibition LPS-mediation of siRNA-mediation in people's multiple myeloma cells induces in conjunction with active.EIF5A siRNA to NFkB active inhibition can explain and cross expression eIF5A ^K50RIn conjunction with the time because NF-kB regulates numerous short survivals and approach anti-apoptotic, so it strengthens the ability of apoptosis-inducing.

Fig. 7 F is shown in IL-6 and exists down, and siRNA pretreatment KAS cell increases eIF5A1 ^K50RThe apoptosis that gene delivery causes.Referring to embodiment 6E.

Fig. 8 diagram gives the growth that eIF5A1 plasmid and eIF5A1 siRNA delay the multiple myeloma Subcutaneous tumor simultaneously.Shown in data are gross tumor volumes of every group of all mices.Referring to embodiment 7.

Fig. 9 diagram gives the growth that eIF5A1 plasmid and eIF5A1 siRNA delay the multiple myeloma Subcutaneous tumor simultaneously.Shown in data be every group of mean tumour volume+/-standard error.Referring to embodiment 7.

Figure 10 diagram gives the eIF5A1 plasmid simultaneously and eIF5A1 siRNA alleviates multiple myeloma Subcutaneous tumor weight.Referring to embodiment 7.

Figure 11 diagram gives the eIF5A1 plasmid simultaneously and eIF5A1 siRNA delays multiple myeloma Subcutaneous tumor growth and causes tumor shrinkage (reducing/dwindle) (shringkage).Referring to embodiment 8.

Figure 12 illustrates intravenous and gives that (i.t.) gives PEI/eIF5A1 in (i.v.) eIF5A1 siRNA and the tumor ^K50RPlasmid composite causes multiple myeloma Subcutaneous tumor shrinkage.Referring to embodiment 9.

Figure 13 A diagram delays multiple myeloma Subcutaneous tumor growth and causes tumor to reduce with eIF5A1 plasmid and eIF5A1 siRNA treatment.Figure 13 B diagram gives the eIF5A1 plasmid simultaneously and eIF5A1 siRNA causes tumor shrinkage.Figure 13 C diagram intravenous gives (i.v.) eIF5A1siRNA and tumor interior (i.t.) gives PEI/eIF5A1 ^K50RPlasmid composite causes multiple myeloma Subcutaneous tumor shrinkage.

Figure 14 illustrates intravenous and gives eIF5A1 plasmid and eIF5A1 siRNA simultaneously and delay multiple myeloma Subcutaneous tumor growth.Referring to embodiment 10.

Figure 15 illustrates intravenous and gives (i.v.) eIF5A1 siRNA and intravenous (i.v.) or intraperitoneal (i.p.) and give PEI/eIF5A1 ^K50RPlasmid composite delays multiple myeloma Subcutaneous tumor growth.Referring to embodiment 11.

Figure 16 illustrates with eIF5A1 plasmid and eIF5A1 siRNA treatment and delays multiple myeloma Subcutaneous tumor growth.

Figure 17 diagram gives the eIF5A1 plasmid simultaneously and eIF5A1 siRNA delays multiple myeloma Subcutaneous tumor growth and cause tumor to reduce.Referring to embodiment 12.

Figure 18 illustrates intravenous and gives that (i.t.) gives PEI/eIF5A1 in (i.v.) eIF5A1 siRNA and the tumor ^K50RPlasmid composite causes multiple myeloma Subcutaneous tumor shrinkage.Referring to embodiment 13.

Figure 19 diagram gives the eIF5A1 of EF1 or B29 promoters driven simultaneously ^K50RPlasmid and eIF5A1 siRNA delay multiple myeloma Subcutaneous tumor growth and cause tumor to reduce (KAS-SQ-5).Referring to embodiment 14.

Figure 20 diagram gives eIF5A1 siRNA simultaneously and strengthens eIF5A1 ^K50RPlasmid (EF1 or B29 promoters driven) is to the antitumor action of multiple myeloma Subcutaneous tumor, and causes tumor load descend (KAS-SQ-5).Referring to embodiment 15.

Figure 21 illustrates eIF5A1 siRNA cooperation and strengthens the apoptosis that Ad-eIF5A infection lung adenocarcinoma cell causes.Referring to embodiment 16.

Figure 22 illustrates the collection of illustrative plates of pExp5A, and its structrual description is referring to embodiment 17.

Figure 23 illustrates the expected sequence (3371bp) of pExp5A.Referring to embodiment 17.

Figure 24 illustrates eIF5A1 ^K50RExpression in different cell lines.Referring to embodiment 18.

Figure 25 illustrates target sequence and preferred eIF5A1 siRNA sequence.

Figure 26 provides the efficacy study result of multiple myeloma.Referring to embodiment 21.

Figure 27 provides the efficacy study result of multiple myeloma.Referring to embodiment 21.

Figure 28 provides eIF-5A1 ^K50RThe sequence of cDNA.

Figure 29 provides people eIF-5A1 and people eIF5A1 ^K50RBetween comparison.

Figure 30 illustrates DNA: the comparison HA-eIF5A of siRNA ^K50RThe influence of expressing.Referring to embodiment 23.

Figure 31 illustrates DNA: the inductive apoptotic influence of comparison nanoparticle transfection of siRNA.Referring to embodiment 24.

Figure 32 diagram comprises eIF5A1 ^K50RThe PEI complex (N/P=6 or 8) of plasmid and eIF5A1 siRNA (siSTABLE or non-siSTABLE) suppresses multiple myeloma Subcutaneous tumor growth and causes tumor to reduce.Referring to embodiment 25.

Figure 33 illustrates JET PEI ^TMNanoparticle is effectively absorbed by tumor tissues, and nanoparticle is sent plasmid and siRNA to identical cell.Referring to embodiment 26.

Figure 34 illustrates the apoptosis that the KAS cell is caused by actinomycin D.The KAS cell is untreated or with 0.5 μ g/ml actinomycin D treatment.After initial treatment 24 hours, harvesting, washing with Annexin/PI (BD Bioscience) labelling apoptotic cell, were passed through flow cytometry then.

Figure 35 illustrates the truncated-type eIF-5A1 accumulation that actinomycin D treatment causes.With the KAS cell with 0.5 μ g/ml actinomycin D treatment 0-30 hour.The harvesting lysate uses the antibody at eIF5A1 to pass through western blot analysis then.After actinomycin D treatment about 8 hours, begin to observe the accumulation of the eIF5A1 of small-molecular weight form (cutting type/cracking type), and this accumulation runs through remaining processing.Western blotting proof loading by same film equates at actin antibody in use.

Figure 36 illustrates the height of the isoelectric point, IP of truncated-type eIF-5A1 than total length eIf-5A1.The KAS cell is untreated or with 0.5 μ g/ml actinomycin D treatment.Beginning to handle back 17 hours, the harvesting lysate separates the back by the 2-D gel electrophoresis and uses the antibody at eIF5A1 to carry out western blot analysis.In untreated samples, observe a large amount of total length eIF5A1, in the actinomycin D treatment sample, observe the accumulation of the cutting type eIF5A1 of higher PI value.

Figure 37 provides truncated-type eIF-5A1 2-D the photo of gel.The KAS cell is through 0.5 μ g/ml actinomycin D treatment.Beginning to handle back 17 hours, the harvesting lysate carries out carrying out Coomassie blue stain after the 2-D gel electrophoresis separates.Collect cutting type eIF5A1, its position is indicated in mass spectral analysis.

Figure 38 provides the result of truncated-type eIF-5A1 mass spectrum order-checking.(A) full length amino acid sequence of eIF5A1 is with black display.With eIF5A1 full length sequence comparison, the peptide (B) of the order-checking that mass spectrum is identified shows (A) with underscore.Be noted that preceding 6 aminoacid that in the order-checking peptide, do not have eIF5A1.

Figure 39 illustrates caspase (caspase) 3,8 and 9 inhibitor suppress truncated-type eIF-5A1 formation.With people's multiple myeloma KAS cell and different caspase inhibitor incubation 8 hours, and then with 0.5 μ g/ml actinomycin D (ActD) coprocessing 16 hours.The collecting cell lysate.Protein separates by 2D-PAGE, uses the antibody at eIF5A to carry out western blot analysis.The caspase inhibitor that uses is Z-VAD-FMK (common caspase inhibitor), Z-LEHD-FMK (caspase 9 inhibitor), Z-DEVD-FMK (caspase 3/7 inhibitor), Z-IETD-FMK (caspase 6/8 inhibitor) and Z-YVAD-FMK (caspase 1 inhibitor).Data show that common caspase inhibitor and caspase 3,8 and 9 inhibitor all can stop cutting type eIF5A to form effectively, and cutting type eIF5A accumulates in the inductive apoptosis of the ActD of KAS cell.Caspase 1 inhibitor also reduces but not exclusively blocks the formation of cleaved products.These results show that eIF5A1 is by the caspase cracking behind genotoxicity pressure (inducing by actinomycin D); The cutting of eIF5A1 can change its activity, promptly increases pro-apoptosis bioactivity.

Figure 40 illustrates deacetylase inhibitors (nicotiamide) and promotes truncated-type eIF-5A1 to form.Can on lysine 47, acetylizad eIF5A be the substrate (Shiraiet al., 2008) of the relevant deacetylase Hst2 of Sir2.Human cervical carcinoma HelaS3 cell continues to handle the fixed time through 0.5 μ g/ml actinomycin D (ActD) and/or 20 μ M nicotiamide (effective inhibitor of Sir2).The collecting cell lysate.Protein separates the back by 2D-PAGE and uses the antibody at eIF5A to carry out western blot analysis.The accumulation of not inducing cutting type eIF5A with ActD or nicotiamide individual processing HelaS3 cell.Yet, handle the HeLa cell with actinomycin D and nicotiamide, after being exposed to ActD at least 4 hours, cutting type eIF5A raised, and the expression acetylation can promote the eIF5A cutting during the apoptosis.

Figure 41 provides the DNA sequence of truncated-type human eIF5A1.

Figure 42 provides protein sequence, truncated-type eIF5A and the cleavage site of total length eIF5A1.

Figure 43: infect the Hela cell with adenovirus construct and cause the accumulation of eIF5A transgenic.With different adenovirus constructs with 500 infectious units/cell infection HelaS3 cell.In infecting the 48 and 72 hours collecting cell lysates in back, carry out SDS-PAGE, use and carry out western blot analysis at eIF5A and actin antibody.Can be observed the accumulation of truncated-type eIF5A.

Figure 44: eliminate cell death inducing with actinomycin D, sodium nitroprusside processing or IL-6 and cause truncated-type eIF5A1 (eIF5A1 Δ 1-6) in human myeloma cell, to accumulate.The KAS-6/1 cell is handled as shown, carries out 2D-PAGE then and uses the antibody at eIF5A to carry out western blot analysis.In the KAS-6/1 cell, all induce truncated-type eIF5A1 accumulation through the apoptosis-inducing of actinomycin D, NO donor sodium nitroprusside or IL-6 hunger.It is low to use Ad-A1 Δ (2-6) or Ad-A1 Δ (2-6)/K50R to cross truncated-type eIF5A1 (eIF5A1 Δ 1-6) the pI value that produces when expression truncated-type eIF5A1 induces cumulative eIF5A1 protein pI value than apoptosis, shows to have other post translational modification on eIF5A1 Δ 1-6.

Actinomycin D during Figure 45: eIF5A1D6E and the eIF5A1D6E/K50R pair cell apoptosis-inductive resistance that is cut with.People's multiple myeloma KAS-6/1 cell is handled as shown, carries out 2D-PAGE then and uses the antibody at eIF5A to carry out western blot analysis.Cross the eIF5A1 (Ad-A1) that expresses and during actinomycin D treatment, experience cutting.Yet eIF5A1D6E or eIF5A1D6E/K50R are to the resistance that is cut with after the actinomycin D treatment.These results meet the hypothesis of the aminoacid 3-6 formation caspase cleavage site of eIF5A1.

Figure 46: the external cutting that the recombinant type caspase carries out eIF5A1.People eIF5A1 (A1) and mutant eIF5A1D6E (D6E) and eIF5A1D6E/K50R (D6E/K50R) sub-clone are gone into pHM6 carrier (Roche), and it causes the HA labelled protein to be expressed.PHM6-HA-A1, pHM6-HA-A1 (D6E) and pHM6-HA-A1 (D6E/K50R) are in vitro transcription and translation (TNT T7 Quick Coupled Transcription/Translation Systems; Promega), and according to manufacturers instruction with Transcend Non-Radioactive Translation Detection Systems (Promega) labelling.With every kind of external translation product of 3 microlitres and 1 microlitre recombinant type caspase (caspase 1,2,3,6,7,8,9 or 10; Calbiochem) in caspase reaction buffer (50mM Hepes, 0.1%CHAPS, 10% sucrose, 100mM NaCl, 10mM DTT, 1mM EDTA) in 37 ℃ of incubations 2 hours.End product manifests biotinylated protein matter through SDS-PAGE with Succ-PEG-DSPE-HRP incubation, carries out chemiluminescence detection then.Wild type eIF5A1 is cut and is not cut by caspase 1 by caspase 2,3,6,7,8,9 and 10.The eIF5A1D6E of HA-labelling and eIF5A1D6E/K50R be by caspase 3,7 and 8 external effective cuttings, but can not effectively be cut by caspase 9 and 10.Yet, this cutting may be owing to the structure height similarity between high abundance caspase activity in the vitro system and glutamic acid and the aspartic acid, therefore allow cutting eIF5A1D6E, but cutting efficiency lower (especially because eIF5A1D6E and eIF5A1D6E/K50R measure in vivo to being cut with resistance (Figure 45)).

Figure 47: with Ad-eIF5A1 Δ (2-6) or Ad-eIF5A1 ^K50RΔ (2-6) infects the Hela cell and causes apoptosis than using wild type Ad-eIF5A1 or Ad-eIF5A1 ^K50RThe apoptosis that infects increases.With human cervical carcinoma Hela S3 cell with different adenovirus constructs with 500 infectious unit/cell infections.In infecting the 48 or 72 hours collecting cells in back, carry out Annexin V/PI dyeing.Sort by the flow cytometry pair cell then.The apoptosis early stage (Annexin V+/PI-) and the cell percentage in late period (Annexin V+/PI+) have been shown.Amputation aminoacid 2-6 strengthens eIF5A1 or eIF5A1 ^K50RThe apoptosis activity.On the contrary, the sudden change of the cleavage site of expection reduces but does not eliminate eIF5A1 or eIF5A1 ^K50RThe apoptosis activity.These results show that the eIF5A1 cutting of caspase mediation causes having the active truncated-type eIF5A1 that increases of apoptosis.

Figure 48 provides the action model of truncate eIF-5A1 in apoptosis of caspase mediation.

Figure 49 illustrates the accumulation of cutting type eIF5A in nucleus behind the actinomycin D treatment KAS human myeloma cell.

Figure 50 illustrates the accumulation of cutting type eIF5A in nuclear behind the actinomycin D treatment KAS human myeloma cell.Key word: UN=is untreated; The actD=actinomycin D treatment; C=kytoplasm part; N=examines part, and alpha-tubulin (tubilin) and α-PCNA are contrasts.This figure shows that also truncated-type eIF5A mainly accumulates in nucleus, and total length eIF5A is evenly distributed between kytoplasm and the nuclear.

Figure 51 illustrates truncated-type eIF5A and reply the accumulation that different apoptosis stimulates in various kinds of cell system and cell type, and it is common phenomenon in apoptosis that the eIF5A that shows caspase-mediation cuts.This figure shows that also clipped form mainly accumulates in nuclear, and total length eIF5A is evenly distributed between kytoplasm and the nuclear.

The result of study that Figure 52 provides shows that truncate eIF5A replys generation--KAS people's multiple myeloma cells that different apoptosis stimulates in different cell lines: with IL-6 hunger or IL-6 and the hungry processing of FBS; UACC1598 (Proliferation of Human Ovarian Cell system): use actinomycin D treatment.

Figure 53 provides the nucleotide sequence of people eIF5A1 and eIF5A2.

Detailed Description Of The Invention

The invention provides separation polynucleotide and the truncated-type eIF-A1 polypeptide of coding truncated-type eIF-5A1.Truncated-type eIF-5A1 polynucleotide are used for cell death inducing and kill cancer cell.Described truncated-type polynucleotide can be used for giving mammal with expression vector then in the expression vector.Truncated-type eIF-5A form is expressed in mammal and kill cancer cell.The about 16kDA of truncated-type eIF-5A1 albumen, and the about 17kDa of total length eIf-5A1 albumen.

Formation than the eIF5A (about 16kDa) of small-molecular weight form is observed in the experience apoptotic cells.This phenomenon is also observed in β islet cells of handling with cytokine mixture and the human myeloma cell (KAS cell) with the cytotoxic drug actinomycin D treatment.Referring to Figure 35.Also find after the HeLa cervical cancer cell infects Ad-eIF5A1, to accumulate in the experience apoptotic cells, show that eIF5A than the small-molecular weight form is because protease cracking produces rather than the result of alternative splicing than the eIF5A of small-molecular weight form.The quantity that is accompanied by total length hydroxyl fourth lysineization (hypusinated) eIF5A than the eIF5A of small-molecular weight form accumulation sharply reduces, and this observed result supports that further the eIF5A than the small-molecular weight form is because the viewpoint that cracking rather than alternative splicing form.Referring to Figure 36.

The invention provides a kind of compositions, it comprises the proteinic eIF5A1 polynucleotide of coding truncated-type eIF5A1.This compositions can be used for preparing the medicine of inducing patient's cancerous cell or apoptosis of tumor cells.The truncated-type eIF5A1 protein of described eIF5A1 polynucleotide encoding preferably comprises the aminoacid sequence of SEQ ID NO:37 shown in Figure 38.In certain embodiments, the about 16kDA of truncated-type eIF5A1 protein of described eIF5A1 polynucleotide encoding.

Can give mammal with compositions and/or medicine, comprise the people." patient " used herein comprises mammal and people.

Cell death inducing can produce following effect in cancerous cell or tumor: delay cancerous cell or tumor growth, stop cancerous cell or growth of tumour cell, perhaps kill cancer cell or reduction tumor size and above-mentioned any effect combination.Can treat any cancer, in certain embodiments, cancer is a multiple myeloma.

In certain embodiments, the eIF5A1 polynucleotide comprise the sequence (as shown in figure 41) shown in the SEQ ID NO:38.In certain embodiments, the eIF5A1 polynucleotide are included in plasmid or the expression vector.Plasmid and expression vector are described in further detail hereinafter.In certain embodiments, expression vector is adenovirus expression carrier or pHM6.In certain embodiments, when compositions or medicine were used for the treatment of multiple myeloma, expression vector comprised tissue-specific promoter, for example B cell specificity promotor (being B29).Expression vector can comprise the pCpG plasmid.As hereinafter being described in further detail, expression vector can be compound with polyaziridine.

In the preferred tumor of described compositions or medicine, intravenous or subcutaneous giving.

The present invention also provides coding truncated-type eIF5A1 proteinic separation polynucleotide, and wherein said polynucleotide comprise the sequence (as shown in figure 41) shown in the SEQ ID NO:38.

The present invention also provides a kind of coding truncated-type eIF5A1 proteinic separation polynucleotide, and wherein said truncated-type protein comprises the aminoacid sequence shown in the SEQ ID NO:37.

As mentioned below, described eIF5A1 forms by the cracking/cutting of caspase mediation.Therefore, the present invention also provides the isolating truncated-type eIF5A1 polypeptide by the eIF5A1 cracking formation of caspase mediation.

The present invention further provides the purposes that a kind of compositions or said composition prepare medicine, described compositions comprises coding proteinic eIF5A1 polynucleotide of truncated-type eIF5A1 and total length eIF5A1 polynucleotide.This compositions is used to prepare the medicine of inducing patient's cancerous cell or tumor death.The proteinic eIF5A1 polynucleotide of coding truncated-type eIF5A as mentioned above.Total length eIF5A1 polynucleotide encoding comprises the protein of the aminoacid sequence shown in the SEQ ID NO:35.Total length eIF5A1 polynucleotide are described in further detail hereinafter.In certain embodiments, described eIF5A1 polynucleotide comprise the sequence shown in the SEQ ID NO:38, and described total length eIF5A1 polynucleotide comprise the sequence (shown in Figure 53) shown in the SEQ ID NO:43.

Described total length and truncated-type polynucleotide can be present in the expression vector as herein described.In addition, carrier can comprise promoter as herein described and any other useful promoter.

In certain embodiments; total length eIF5A polynucleotide encoding sudden change eIF5A1, wherein said sudden change prevents or the hydroxyl fourth lysine that suppresses deoxidation hydroxyl fourth lysine synthase (deoxyhypusine synthase) turns usefulness (hypusination) into and/or wherein said sudden change is present on ubiquitination site and/or the acetylation site.Mutant/sudden change is described in further detail hereinafter.In certain embodiments, mutant is selected from K50A, K50R, K67A, K47R, K67R, K50A/K67A, K50A/K47R, K50A/K67R, K50R/K67A, K50R/K47R, K50R/K67R and K47A/K67A.

The present invention also provides a kind of compositions, and it comprises the siRNA of the 3 ' UTR of the nucleotide of polynucleotide, encoding mutant eIF5A of coding truncated-type eIF5A and targeting eIF5A.The siRNA of the 3 ' UTR of the nucleotide of combination/binary use encoding mutant eIF5A and targeting eIF5A hereinafter is described in detail in detail.Described compositions can be used for preparing medicine, gives the patient with inducing cancer cell or tumor death with it.

In certain preferred aspects, 5 '-GCT GGA CTC CTC CTA CAC A-3 ' sequence of siRNA targeting eIF5A1.SiRNA is that the chain of dsRNA and dsRNA comprises sequence 5 '-GCU GGA CUC CUC CUA CAC A-3 '.In certain embodiments, siRNA is stabilized preventing and degrades in serum.

Total length eIF5A1 polynucleotide and/or the proteinic eIF5A polynucleotide of coding truncated-type eIF5A1 may reside in the expression vector.In certain embodiments, total length eIF5A1 polynucleotide and/or the coding proteinic eIF5A polynucleotide of truncated-type eIF5A1 and/or siRNA and polyaziridine are compound.Total length eIF5A1 polynucleotide and/or the coding proteinic eIF5A polynucleotide of truncated-type eIF5A1 and/or described siRNA are independent compound with polyaziridine.

The present invention also provides a kind of method of inducing mammalian cancer cells or mammal tumor apoptosis, it provides compositions as herein described or medicine to mammal, for example comprises the nucleotide of coding truncated-type eIF5A1, the optional nucleotide of coding total length eIF5A or nucleotide and the optional siRNA that comprises the 3 ' UTR of targeting eIF5A of coding total length sudden change eIF5A of comprising.In certain embodiments, cancer is a multiple myeloma, will give in compositions/medicine intravenous, intraperitoneal or the tumor.

The invention still further relates to unite and use the targeting endogenous gene to offer the polynucleotide of patient's the described gene of coding, in the host, express with the protein that described polynucleotide encoding is provided with the siRNA that knocks out or suppress patient's endogenous gene and express with delivery media/expression vector.

This combination can be used for treating the patient because disease or the disease that exists deficient protein or mutain to cause, promptly, wherein the protein that the patient produced can not implement it must function, thereby perhaps because its fault of construction upsets (fowls up) metabolic pathway or bio-molecular interaction.SiRNA is designed to the gene of targeting coding deficient protein matter, and suppresses or knock out the expression of deficient protein matter.Normal (non-defective) the proteinic polynucleotide of will encoding give the patient and express in the patient, so as normal protein matter can implement it must function.

In yet another embodiment, give the patient with protein, rather than the polynucleotide of the desired protein of encoding.Term protein, peptide and polypeptide are used interchangeably in this article.

Preferably SiRNA is designed to a certain zone of the described gene of targeting, makes its inhibition or knock out the endogenous expression of deficient protein matter, but can not influence the heterogenous expression of the polynucleotide of the coding normal protein matter that gives simultaneously.For example, but siRNA targeting 3 ' UTR make its can not influence give adopted construct (these proteinic polynucleotide of encoding) heterogenous expression arranged.By suppressing or knock out the endogenous expression of dcc gene, normal protein matter less or that do not have deficient protein matter and exogenous polynucleotide to express is competed.

A kind of disease example that advantageous application of the present invention relates to is a sicklemia.Sicklemia is a kind of hematologic effects that influences hemoglobin, and the hemoglobin in the erythrocyte (RBC) helps transportation oxygen to whole body everywhere.

When an individual inheritance to two aberrant gene (from one of each heredity of father and mother), it causes expressing sudden change hemoglobin (HbS), and sicklemia will take place.The hemoglobin of sudden change causes the RBC alteration of form.Erythrocyte with Hb A hemoglobin adult (hemoglobin A or HbA) moves by blood flow easily, and oxygen is transported to the whole body all cells.They can easily " squeeze " and cross very tiny blood vessel.What meniscocyte's property was poor is because hemoglobin variant (HbS) often flocks together, and makes erythrocyte be viscosity, stiff and easier fragmentation, makes them form crooked sickle shaped.

Although known hundreds of kind HBB genetic mutation, sicklemia is the most common to be caused by haemoglobin variant HbS.In this variant, the hydrophobic amino acid valine is substituted in the hydrophilic glutamic acid of the 6th amino acid position of HBB polypeptide chain.This outside that is substituted in protein structure produces hydrophobic speckle, and it clings the hydrophobic region of adjacent haemoglobin molecule β chain.This makes and produces " reaping hook " shape erythrocyte by the HbS molecular aggregates stiff fiber of (polymerization) formation together.

Polymerization only takes place after erythrocyte discharges oxygen molecule, and oxygen molecule is sent to the various tissues of whole body.Wherein hemoglobin can be in conjunction with the pulmonary of oxygen in case erythrocyte is got back to, and the long fibre of HbS molecule will depolymerization or is cracked into single molecule.Polymerization and depolymerize between circulation erythrocyte membrane is ossify.These erythrocytic rigid shape distortions that reach can cause little blood vessel blockage when not carrying oxygen.This obstruction can cause the pain outbreak and can damage organ.

Sicklemia is autosomal recessive disease.The people who manifests this disease must be genetic to the HbS variant of two copies or be genetic to a copy HbS and another variant that copies.Carrier with a copy normal HBB gene (HbA) and a copy HbS has sickle cell trait and can not show disease symptoms.

Therefore, one embodiment of the invention provide a kind of method of the patient's of treatment sicklemia.Give patient's targeting HBB the siRNA of gene.Design SiRNA is to suppress, preferably to knock out the expression of Hb H bs variant.The polynucleotide of coding Hb A hemoglobin adult are offered the patient and make described patient express normal hemoglobin.It also designs SiRNA so that can not disturb the expression of the exogenous polynucleotide of coding Hb A hemoglobin adult.Therefore, described patient no longer produces variant hemoglobin (perhaps seldom producing), replaces the hemoglobin that produces normal health, to produce the normal normocyte of greater functionality.

The present invention can be used for also taking place that protein post-translational modification causes or the situation of the disease that causes.SiRNA is used to suppress endogenous protein to express, and making does not have or seldom can be used for post translational modification.Then, provide the polynucleotide of coded protein to be used for heterogenous expression to the patient.Modifying protein is modified so that it can not be translated the back.This then protein can suitably be used by body, but because it can not be translated the back modification, so can not cause disease.It will be appreciated by those skilled in the art that different post translational modifications.For example, after translation, proteinic envelop of function is expanded in amino acid whose post translational modification by the following method: connect other biochemical function and roll into a ball for example acetate, phosphate, various lipid and carbohydrate; Change amino acid whose chemical property (for example citrullineization (citrullination)) or carry out structural change, as forming disulfide bond.In addition, enzyme can be removed aminoacid from proteinic amino terminal, perhaps from centre cutting peptide chain.For example, the peptide hormone insulin is formed back cutting twice at disulfide bond, propetide is removed from middle-of-chain; The protein of gained is made up of two polypeptide chains that disulfide bond connects.Equally, most of newly-generated polypeptide begin with amino acids methionine, this seed amino acid because " opening the beginning " codon on mRNA is also encoded.When post translational modification, remove this aminoacid usually.Other is modified as phosphorylation is the part of the common mechanism of control protein acts, for example activates or inactivator.Other post translational modification comprises by deoxidation hydroxyl fourth lysine synthase (DHS) the hydroxyl fourth lysine of eukaryotic cell translation initiation factor 5A (eIF5A) is turned into usefulness.

Therefore, the invention provides a kind of method of the patient's of change gene expression, wherein the polynucleotide of coded protein are offered the patient and in the patient, express.Protein can be normal/wild-type protein or mutein.The siRNA that the expression of corresponding endogenous gene is given the patient suppresses.

This method also comprises the construct that the polynucleotide that comprise the target protein of encoding are provided, and wherein said polynucleotide are expressed in the patient to produce target protein.Endogenous gene is expressed in some embodiment of deficient protein matter therein, and the design polynucleotide are with the normal/health-care eggs white matter of encoding.Give SiRNA and suppress the expression of defective endogenous protein.Endogenous gene is expressed in normal/healthy proteinic some embodiment therein, and the design polynucleotide are with encoding mutant protein, and it is modified with the same back that can not be translated that normal/health-care eggs white matter or not mutated protein take place.Give SiRNA and suppress the endogenous protein expression, make this protein seldom can be used for post translational modification.

In certain embodiments, choose or design the zone of siRNA, do not express so that do not influence exogenous polynucleotide with target gene.For example, but siRNA targeting 3 ' UTR or 3 ' end.

SiRNA can be delivered to the patient as naked siRNA or for the stable naked siRNA of serum.SiRNA can inject by system, i.e. IP or IV.Perhaps, but siRNA local injection or be delivered to the body desired area.In certain embodiments, siRNA can delivery media give, such as but not limited to dendrimer, liposome or polymer.

The polynucleotide of coding desired protein can give by any mode of sending that provides or allow nucleotide to express.Term polynucleotide and nucleotide this paper are used interchangeably.Sending can be by any virus or non-virus mechanism, such as but not limited to plasmid, expression vector, virus formulation body, adenovirus construct, dendrimer, liposome or polymer.

In certain embodiments, the expression plasmid that will have a CpG dinucleotide of minimizing is used to express polynucleotide.Can use anyly can promote the polynucleotide expression promoter, the needs that this therapy is used are depended in the selection of promoter.For example, kill multiple myeloma may need the promoter to the B cell-specific, for example people B29 promoter/enhancer.In other embodiments, promoter can be other tissue-specific promoter, perhaps can be system start-up.The polynucleotide of coding target protein can be sent by IV or subcutaneous injection or any other biologically acceptable delivery mechanism.Perhaps, polynucleotide can be sent in suitable " carrier " or " medium " of target tumor or cancerous cell with liposome or other any DNA delivery (or plasmid or expression vector) that provides.Referring to for example, Luo, Danet al., Nature Biotechnology, Vol.18, January 2000, pp.33-37, synthetic DNA delivery system summary.Therefore, preferably send nucleotide/plasmid/expression vector through nanometer size medium (for example liposome, dendrimer or similar nontoxic nanoparticle).Preferable medium protection nucleotide/plasmid/expression vector is not removed too early, and the nucleotide/plasmid/expression vector that perhaps ought send effective dose does not produce immunne response to patient tumors or cancerous cell.Exemplary media comprises extremely more complicated Pegylation medium of simple and the bonded nanoparticle of nucleotide/plasmid/expression vector, for example has to be connected the pegylated liposomal of its lip-deep part with the targeting specific cell receptor.

Liposome known in the art and pegylated liposomal.In traditional liposomal, the molecule that send (being little medicine, protein, nucleotide or plasmid) is included in the central chamber of liposome.It will be appreciated by those skilled in the art that also have to can be used for " stealth ", targeting, the cationic-liposome that molecule is sent.Referring to for example, Hortobagyi, Gabriel N.et al., J.Clinical Oncology, Vol.19, Issue 14 (July) 2001:3422-3433; Yu, Weiet al., Nucleic Acids Research.2004,32 (5); E48.But the intravenous injection liposome, but modified liposome makes their surface have more hydrophilic (by adding Polyethylene Glycol (" Pegylation ")) to bilayer, increases their circulation times in blood.These are called as " stealth " liposome, especially can be used as for example carrier of doxorubicin and mitoxantrone of hydrophilic (water solublity) cancer therapy drug.For the liposome of strengthening carrying medicine and the target cell specificity binding characteristic of tumor cell for example, specific molecular (for example antibody, protein, peptide etc.) can be connected on the surface of liposome.For example, the antibody of the receptor on the anticancer cell can be used for the liposome target cancer cell.Under the situation of targeting multiple myeloma, for example folic acid, II-6 or transferrins can be used for liposome targeting multiple myeloma cells.

The same known dendrimer in this area and preferred delivery media is provided.Referring to following document for example about the argumentation of dendrimer: 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; Majoros, Istvan J.et al., J.Med.Chem, 2005.48,5892-5899.

In preferred embodiments, delivery media comprises the polyaziridine nanoparticle.Exemplary polyaziridine nanoparticle is In vivo-jetPEI ^TM, at present by Polyplus Transfection, Inc. produces.In vivo-jetPEI ^TMBe the cationic polymer transfection agents, can be used as DNA and siRNA delivery agents.The In vivo-jetPEI that Polyplus Transfection produces ^TMBe linear polyaziridine reagent, it provides reliable delivery of nucleic acids in animal.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 disturbs (Urbain-Kleinet al., 2004.Gene therapy 23:1-6; Grezelinskiet al., 2006.Human Gene Therapy 17:751-66) and genetic vaccine (Garzon et al., 2005.Vaccine 23:1384-92).In vivo jet-PEI in people's clinical trial, be used as at present the oncogene therapy delivery vector (Lemkineet al., 2002.Mol.Cell.Neurosci.19:165-174).

In vivo-jetPEI ^TMNucleic acid is condensed into about 50nm nanoparticle, can stablizes some hrs.Because the protection mechanism of this uniqueness, injection bleeding from anus cell aggregation is low than other reagent, therefore stops and organizes diffusion limited, erythrocyte aggregation and micro-embolization.These nanoparticles are very little, can diffuse in the tissue and by endocytosis and enter cell.In vivo-jetPEI ^TMHelp nucleic acid to pass nuclear membrane from endosome release and transhipment.

In preferred embodiments, with siRNA and comprise polynucleotide carrier/plasmid these two through In vivo-jetPEI ^TMComplex gives the patient.Can be with SiRNA and the carrier/plasmid that comprises polynucleotide through polymer composites for example polyaziridine or In vivo jetPEI ^TMComplex is combined with each other, perhaps can be separately compound with polymer.For example, when siRNA and the carrier/plasmid that comprises polynucleotide separately need be given the patient (the time and/or send on the site separately) time, preferably that siRNA and polynucleotide and different carriers are compound.Give to carry out simultaneously with to give the site identical when described, preferred siRNA and polynucleotide are combined with each other.

In yet another embodiment, protein itself is delivered to the patient, rather than gives plasmid or carrier to send the polynucleotide of in the patient, expressing.Described protein can be isolated protein or synthetic protein.

One embodiment of the invention provide a kind of treatment patient method for cancer, and the patient comprises mammal and people.The treatment cancer includes but not limited to cancer cell specific induction of apoptosis, kill cancer cell, reduction cancerous cell quantity and reduces gross tumor volume/weight.This method comprises and gives a kind of compositions that it comprises the polynucleotide of eIF5A1 siRNA and encoding mutant eIF5A1.The polynucleotide of compositions, eIF5A1siRNA and encoding mutant eIF5A1 are discussed hereinafter in detail.

All cells all produces eukaryotic initiation factor 5A (" eIF-5A ") (this paper is also referred to as " factor 5A ").Mammalian cell produces two kinds of isotype eIF-5A (eIF-5A1 and eIF-5A2).EIF-5A1 is called as apoptosis specificity eIF-5A, because it raises in meeting with apoptotic cells.The accession number of people eIF-5A1 is NM001970, is shown in Fig. 1.It is believed that eIF-5A1 is responsible for the proteinic mRNA group that the Codocyte apoptosis is essential and transports nucleus.EIF-5A2 is called as propagation eIF-5A, is responsible for that Codocyte is bred essential proteinic mRNA group and transports nucleus because it is believed that it.Referring to Liu ﹠amp; 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.

Two kinds of factor 5A carry out post translational modification by deoxidation hydroxyl fourth lysine synthase (" DHS ").DHS makes eIF-5A hydroxyl fourth lysineization.Hydroxyl fourth lysine (Hypusine) is a kind of aminoacid of uniqueness, see eukaryotic cell and the archeobacteria checked to some extent, but do not find that in eubacteria eIF-5A is unique known protein that comprises hydroxyl fourth lysine.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。Hydroxyl fourth lysine eIF-5A forms in the step after two translations: the first step is the formation of deoxidation hydroxyl fourth lysine residue, and the catalysis of deoxidation hydroxyl fourth lysine synthase partly is transferred to the amino butyl of the 4-of spermidine on the alpha-amido of specific lysine of precursor eIF-5A; Second step related to by the amino butyl of deoxidation hydroxyl fourth lysyl hydroxylase hydroxylating 4-and partly forms hydroxyl fourth lysine.

The aminoacid sequence of eIF-5A is very conservative in species, and strict conservative aminoacid sequence is round the hydroxyl fourth lysine residue of eIF-5A, and it shows that this modification may be important for survival.Parket?al.(1993)Biofactors，4，95-104。This imagination also obtains the further support of following observed result: only in yeast, find the inactivation of two kinds of isotype eIF-5A or the inactivation of DHS gene up to now, and the first step of DHS catalytic activation, thus stop cell division.Schnier?et?al.(1991)Mol?Cell.Biol，11，3105-3114；Sasakiet?al.(1996)FEBS?Lett.，384，151-154；Parket?al.(1998)J.Biol.Chem.，273，1677-1683。Yet the proteinic shortage of the eIF-5A in the yeast only makes that gross protein is synthetic to be reduced slightly, show that eIF-5A is that the translation of specific part mRNA is essential, rather than protein is always synthetic necessary.Kanget?al.(1993)，″Effect?of?initiation?factor?eIF-5A?depletion?on?cell?proliferation?and?protein?synthesis，″in?Tuite，M.(ed.)，Protein?Synthesis?and?Targeting?in?Yeast，NATO?Series?H.。Recent findings, the bonded eIF-5A of part has the motif of high conservative, also proves the importance of eIF-5A.Xu?&?Chen(2001)J.Biol.Chem.，276，2555-2561。In addition, find that the hydroxyl fourth lysine residue of the eIF-5A of modification is essential for sequence-specificity in conjunction with RNA, and in conjunction with the protection that does not provide ribonuclease.

The inventor shows, when giving cell with the polynucleotide of coding eIF-5A, can increase the apoptosis of those cells.They show that also they can promote cancer cell-apoptosis thereby it is expressed by giving eIF-5A1 polynucleotide in cancerous cell.Referring to common pending application 10/200,148; 11/287,460; 11/293,391 and 11/637,835, they all by reference integral body be attached to herein.

The inventor determines in addition: when cell had the hydroxyl of enhancing fourth lysine form factor 5A, cell entered existential mode, and can not experience the apoptosis that they take place along with the time usually.It should be noted that has the hydroxyl of significant quantity fourth lysine factor 5A in the cancerous cell, therefore, cell does not carry out apoptosis (with can not be dead).Therefore, by kill cancer cell treatment cancer (promote cancerous cell and enter apoptotic pathways), give patient or cancerous cell or tumor to increase expression eIF-5A1 with the polynucleotide of coding eIF-5A1, it causes cancer cell-apoptosis again, final cell death and tumor shrinkage.Yet, if only provide the proteinic polynucleotide of coding eIF-5A1 to raise eIF-5A1 gene expression and do not use siRNA to suppress the endogenous expression of eIF-5A1 in addition, competition will occur: the eIF-5A1 that cell guiding enters apoptotic pathways expresses the hydroxyl fourth lysine factor 5A competition that enters the cell survival approach with the cell guiding that exists.The present invention has eliminated this competition, and the improvement of performance has not just increased the eIF-5A1 expression.Make the polynucleotide sudden change that gives patient or cell, make that the expressing protein that produces can not be by hydroxyl fourth lysineization.In addition, the endogenous expression of factor 5A knocks out/suppresses with the siRNA of targeting eIF-5A, do not have around making/less endogenous eIF-5A1 is by hydroxyl fourth lysineization.Therefore, owing to do not have (or not having substantially) hydroxyl fourth lysine eIF-5A in the cell, so they can not promote survivability mode.

The polynucleotide of encoding mutant eIF-5A1 are preferably suddenlyd change, so that it can not be by hydroxyl fourth lysineization, therefore can not drive cell enters survivability mode.For example, in one embodiment, the polynucleotide sudden change of coding eIF-5A makes 50 lysine (K) (it is usually by DHS hydroxyl fourth lysineization) be changed to alanine (A) (its can not by hydroxyl fourth lysineization).This sudden change is expressed as K50A.

In another embodiment, 67 lysines are changed to arginine (R).This mutant is expressed as (K67R).In yet another embodiment, 67 lysines (K) are changed to alanine (A), are expressed as (K67A).In yet another embodiment, 50 lysines (K) are changed to arginine (K50R); Another embodiment provides mutant, and wherein 47 lysines (K) are changed to arginine (K47R).

In other embodiments, use double-mutant.A kind of double-mutant is that wherein 50 lysines (K) are changed to arginine (R), and 67 lysines (K) are changed to arginine (R).This double-mutant is called as K50R/K67R.This double-mutant equally can not be by hydroxyl fourth lysineization, and still described amino acid whose variation does not change the 3-D structure of eIF-5A1, and is similar with single sudden change (K50A).The protein that therefore this two sudden change provides very similar 3-D shape and fold as wild type, therefore more stable than single mutant.Because more stable, it exists the long period so that more persistent therapeutical effect to be provided in body.Therefore, the factor 5A that body has can satisfy the normal cell function but it can not hydroxyl fourth lysineization, thereby cell can not enter the cell survival pattern and avoids apoptosis so.

Another kind of double-mutant is that wherein 47 lysines (K) are changed to arginine (R) and the lysine on 50 is changed to arginine (R), and this mutant is expressed as (K47R/K50R).The invention provides another kind of double-mutant, wherein the lysine on 50 (L) is changed to alanine (A), and the lysine on 67 is changed to alanine (A), and this mutant is expressed as (K50A/K67A).

, keeps body normal cell survival and healthy cell propagation because needing factor 5A, so if siRNA is sent by system, then preferred siRNA not exclusively blocks patient's expression.EIF-5A expresses control and can realize like this: use the siRNA that not exclusively closes expression (promptly close or reduce and express but not exclusively close expression) or utilize dosage form and/or therapeutic scheme with the balance expression, thereby growth and the function of both having kept healthy cell are normal, promote cancer cell-apoptosis again.

Perhaps, but local delivery siRNA.If to cancerous cell or tumor, expressing so, local delivery siRNA preferably knocks out.By knocking out expression, just do not change factor 5A on every side, thereby do not have hydroxyl fourth lysine eIF-5A blockade cell to enter survivability mode by hydroxyl fourth lysine.Because local delivery siRNA is to cancer or tumor, so do not need to provide eIF-5A to be used for normal cell growth.

In certain embodiments, endogenous gene is eIF5A1.Give the patient to suppress endogenous eIF-5A1 expression with the siRNA of targeting eIF5A1.In certain embodiments, siRNA comprises the siRNA of SEQ ID NO:1 or SEQ ID NO:2 or any targeting eIF5A1, and it will suppress endogenous eIF-5A1 and express.In certain embodiments, eIF5A1 is that people eIF-5A1 (shown in Figure 1) and described patient are the people.The siRNA of known other targeting people eIF-5A1 and be disclosed in common pending application 11/134,445; 11/287,460; 11/184,982; 11/293,391; 11/725,520; 11/725,470; 11/637,835.In other embodiments, the patient is that mammal and eIF5A1 are that mammal is specific.For example, the patient is that Canis familiaris L. and eIF5A1 are Canis familiaris L. eIF5A1.In certain embodiments, siRNA is made up of siRNA construct shown in Figure 25 basically.For example, siRNA comprises the nucleic acid of targeting eIF5A1 but comprises jag for example U or T nucleic acid equally, perhaps also comprises label, for example his label (be commonly referred to HA label-its be generally used in vitro study).Can be included in 5 ' or 3 ' the terminal molecule that connects or other nucleic acid (for example even be included in the continuous chain of nucleic acid shown in Figure 25), these molecules or nucleic acid belong to the part of " forming basically ", and condition is the expression that the siRNA construct can reduce target gene.The zone of preferred siRNA targeting eIF5A1 gene makes that not influencing exogenous polynucleotide expresses.For example eIF5A1 siRNA targeting 3 ' UTR or 3 ' end.SiRNA shown in Figure 25 is exemplary eIF5A1 siRNA.

Polynucleotide encoding eIF5A1, wherein said polynucleotide sudden change is with coding eIF5A1 variant.Design sudden change eIF5A1 makes mutant eIF5A1 can not be translated the back and modifies (can not by hydroxyl fourth lysineization).Exemplary mutant as mentioned above.

When relating to the cancer of entity tumor, may need directly to send siRNA to tumor.With regard to the time with send with regard to the site, siRNA can separate with polynucleotide and gives, perhaps can in simultaneously and/or the identical site of sending give together.It will be appreciated by those skilled in the art that the appropriate time that gives siRNA may be when endogenous protein is translated rather than after translation.

Although it is nontoxic (referring to the pending application series number 11/293 of application on November 28th, 2005 to normal structure that the inventor shows eIF5A1 for a long time, 391, its by reference integral body be attached to herein), than directly giving eIF5A polynucleotide/plasmid/expression vector, still preferred delivery complexes.The eIF5A1 that preferred delivery system provides effective dose also preferably provides targeted delivery to tumor or cancer cell population to patient tumors or cancer cell population.Therefore, in certain embodiments, preferably send eIF5A1 nucleotide/plasmid/expression vector by nanometer size medium for example liposome, dendrimer or similar nontoxic nanoparticle for example polyaziridine polymer (for example In vivo jetPEI complex) send.

EIF5A1 protein can directly be delivered to tumor sites equally.Those skilled in the art can determine to send the dosage and the time length of the proteinic therapeutic scheme of eIF5A1.

Molecular basis by the eIF5A1 cell death inducing is discussed hereinafter.

The death receptor signal transduction

Handling cancerous cell with Ad-eIF5A1 (adenovirus of wild type eIF5A1) or Ad-eIF5A1 (K50A) induces caspase 8 (it passes through death receptor-part in conjunction with starting) and caspase 3 (executor's caspase) to activate.These may be the remote-effects of eIF5A1, and in fact using can not be by after the eIF5A1 of hydroxyl fourth lysineization (K50A) processing or the treatment, and caspase 8 and caspase 3 are activated equally, show that this effect is by lysine ₅₀EIF5A1 causes.Handle also demonstration with Ad-eIF5A1 and cause death receptor to raise, the same with previous TNFR1 rise.

The mitochondrion path

Apoptotic mitochondrion path directly or indirectly relates to lysine ₅₀EIF5A1, the support that this obtains a large amount of observed results comprises that discovery activates caspase 9 with eIF5A1 or eIF5A1 (K50A) processing cancerous cell.The p53 demonstration of working in activating mitochondrion apoptosis path equally, is regulated by eIF5A1.For example, raise p53 with the actinomycin D treatment cancerous cell, and this rise of p53 is suppressed by eIF5A1 siRNA.Consistent therewith is to handle cancerous cell with Ad-eIF5A1 and raise p53mRNA.Handle cancerous cell with eIF5A1 and induce Bax to migrate to mitochondrion from cytosol equally, then mitochondrial membrane potential loss and cytochrome C space in mitochondrial membrane are released into the cytosol.In addition, this processing causes cutting type Bcl2, Bim and montage type Bim to raise, and they all are that promotion is apoptotic.

The MAPK signal transduction

In addition, the inventor has obtained the evidence that eIF5A1 participates in the MAPK signal transduction of apoptosis dependency.For example, handle cancerous cell with Ad-eIF5A1 and raise P-JNK, it suppresses the Bcl2 of anti-apoptosis again.In addition, Ad-eIF5A1 and Ad-eIF5A1 (K50A) both induces P-p38 to form, and it starts apoptosis by the various short apoptosis agent of influence again, and short apoptosis agent comprises TNFR1 ﹠amp; TNF; FAS ﹠amp; FASL; Caspase 8; Bid; Cytochrome C and caspase 3.

NF-κ B signal transduction

Evidence suggests TNF-κ B signal TransductionThe growth of support myeloma.For example, the myeloma cell adheres to marrow stromal cell and induces NF-κ B-dependent transcription to raise IL-6, and its somatomedin that is multiple myeloma is again anti-apoptosis factor [Chauhanet al. (1996) Blood 87,1104].In addition, the NF-κ B in the excretory TNF-α of the myeloma cell activated bone marrow stromal cell, thus rise IL-6 transcribes and secretes.TNF-α is the NF-κ B in the activated bone marrow oncocyte also, and it causes the intracellular adhesion molecule-1 (ICAM-1 on myeloma cell and the marrow stromal cell; CD54) and vascular cell adhesion molecule-1 (VCAM-1; CD106) raise [Hideshimaet al. (2001) Oncogene 20,4519].This has strengthened the association [Hideshimaet al. (2001) Oncogene 20,4519] of myeloma cell and marrow stromal cell again.On the contrary, these effects are suppressed [Hideshimaet al. (2001) Oncogene 20,4519] by stoping the inductive NF-kB activation of TNF α.In fact, might mediate among the myeloma cell at the inductive apoptotic protection of TNF α [Hideshimaet al. (2002) JBC277,16639] by NF-κ B.These and other observed result shows that the NF-kB signal transduction may be a noticeable target spot in the multiple myeloma therapy.

The inventor shows that eIF5A1 siRNA suppresses NF-κ B activation in human myeloma cell and ICAM-1 forms these two.These observed results show that eIF5A1 works in NF-κ B activates, and because the effect after the NF-κ B activation in fact all is short survival, so we predict that this activation is directly or indirectly mediated by hydroxyl fourth lysine eIF5A1.

IL-1

The excessive generation proinflammatory cytokine of myeloma cell IL-1 is the feature of multiple myeloma, and it causes osseous tissue to be degenerated.EIF5A1 siRNA has shown that LPS attacks the excessive generation of inductive IL-1 in the rapid reduction mice.

One embodiment of the invention provide a kind of method for the treatment of multiple myeloma.Multiple myeloma (" MM ") is a kind of gradual fatal disease, it is characterized in that pernicious plasma cell breeds in bone marrow and have an osteolytic lesion.But multiple myeloma is a kind of can not the healing medicable plasma cell cancer.Plasma cell is immune important component part, and it produces the immunoglobulin (antibody) that helps opposing infection and disease.Multiple myeloma is characterised in that abnormal plasmocyte excessive in the bone marrow and complete monoclonal immunoglobulin (IgG, IgA, IgD or IgE; " M-protein ") or the excessive generation of Bence-Jones protein (no monoclonal light chain).Hypocalcemia, anemia, renal damage, bacterial infection susceptibility increase and normally the immunoglobulin generation is impaired all is the common clinical manifestation of multiple myeloma.Multiple myeloma is characteristics with the dispersivity osteoporosis usually also, sees pelvis, spinal column, rib and skull usually.

It seems that the present invention very be suitable for treating multiple myeloma, because found the stimulation feedback circuit in multiple myeloma.For example, multiple myeloma produces Il-1 with low concentration in bone marrow.Il-1 stimulates stromal cell to produce IL-6 again, and it stimulates the multiple myeloma growth then.The inventor before showed (referring to pending application 11/725,539 and 11/184,982), and directly the siRNA at eIF-5A1 can suppress expression of proinflammatory cytokines, for example Il-1; TNF-α and Il-8).Therefore, siRNA not only suppresses eIF-5A and expresses (less like this generation hydroxyl fourth lysine turns usefulness into), and cuts off or reduction Il-1/Il-6 feedback circuit.

The siRNA of targeting people eIF5A is used for suppressing the level of the endogenous hydroxyl fourth of tumor lysine eIF5A, and expresses eIF5A mutant (eIF5A ^K50R) the RNAi-r plasmid of (its can not by hydroxyl fourth lysineization), be used to improve the eIF5A level of unmodified in the body.The PEI nano-complex that injection comprises eIF5A siRNA in the tumor suppress the MM tumor growth surpass 80% ( ^{* *}P=0.0003 and comprises the complex that contrasts siRNA and compares), show that the level that suppresses hydroxyl fourth lysine eIF5A has antitumous effect.Comprise eIF5A ^K50RThe PEI complex of expression plasmid have similar effects and suppress tumor growth surpass 70% ( ^*P=0.001 compares with the complex that comprises control plasmid).Therefore, by suppressing somatotrophic hydroxyl fourth lysine eIF5A or can suppressing the MM tumor growth by the level that increases the not hydroxyl fourth lysine form eIF5A that urgees apoptosis.Intra-tumor delivery comprises eIF5A siRNA and RNAi-resistance eIF5A ^K50RThe complex of plasmid has the co acting effect and tumor is obviously dwindled tumor growth, suppress tumor growth reach 94% ( ^{* *}P=0.0002).Intravenous is sent eIF5AsiRNA/eIF5A ^K50RThe PEI complex is same effectively reduce tumor growth reach 95% ( ^*P=0.002), show that it is practicable that described therapeutic agent is sent by system.

EIF5A siRNA/eIF5A sends in local delivery and system ^K50RPDNA PEI complex all causes the obvious Graft Versus Tumor of multiple myeloma.

The present invention also provides a kind of cancer compositions of (comprising multiple myeloma) that can be used for treating.In a preferred embodiment, described compositions is the complex of plasmid DNA and eIF5A1siRNA, described plasmid DNA coding can not be by the point mutation eIF5A1 of hydroxyl fourth lysineization, and described eIF5A1 siRNA selectivity suppresses endogenous people eIF5A1 but described plasmid-encoded point mutation eIF5A1 is not influenced.The polynucleotide of eIF5A1 siRNA and encoding mutant eIF5A1 as mentioned above.Plasmid DNA and siRNA are preferably compound with PEI (polyaziridine) nanoparticle.They can separate compound and separately give or give together, and perhaps they can be combined with each other.DNA and RNA combine and discharge when nanoparticle is absorbed into cell with the positive charge on the PEI is amino.Proved that the PEI-nucleic acid complexes effectively is absorbed into somatoblast and not in the somatoblast.

Plasmid DNA optimized encoding eIF5A1 (K50R), it can not be by hydroxyl fourth lysineization as eIF5A1 (K50A), thereby be short forcefully apoptosis.EIF5A1 (K50R) expresses preferred by the adjusting of B cell specificity promotor.

3 ' of the preferably endogenous people eIF5A1 of eIF5A1 siRNA-end is specific and do not influence the expression of trans eIF5A1 (K50R).Exemplary preferred eIF5A1 siRNA comprises that siRNA/ shown in Figure 25 is made up of siRNA shown in Figure 25 basically or is exactly to be made up of siRNA shown in Figure 25.The basic reason of including eIF5A1 siRNA in is: (1) eliminates endogenous eIF5A1, and it is almost all by hydroxyl fourth lysineization thereby for urging the survival form; (2) activation of inhibition NF-κ B, the generation and the intracellular adhesion molecule that reduce IL-6 thus form; (3) suppressing IL-1 forms.Described eIF5A1 siRNA and eIF5A1 (K50R) synergism are induced myeloma cell's apoptosis.Because above (2) and (3) all are short survival incidents,, thereby are not subjected to and are influenced by the eIF5A1 of hydroxyl fourth lysineization (K50R) so they are probably by hydroxyl fourth lysine eIF5A1 mediation.This method produces a large amount of not eIF5A of hydroxyl fourth lysineization, and it causes that cancerous cell comprises the multiple myeloma cells apoptosis, but to almost not influence of healthy cell.

Preferred compositions refers to SNS01 at this paper.SNS01 is the complex of a kind of RNAi-of comprising r plasmid DNA and siRNA, and described RNAi-r plasmid dna encoding is by the eIF5A of promoters driven ^K50R, strengthening safety, and the people eIF5A that the siRNA targeting has dTdT a 3 ' jag to be improving the nuclease resistance in the cell (comprising the myeloma cell) of B-cell source for described promoter limiting expression, and this siRNA and plasmid and In vivo JetPEI ^TMCompound.

Embodiment

Embodiment 1: with wild type eIF-5A1 and variant eIF-5A1 transfection HeLaS3 cell

With the plasmid transfection of HeLaS3 cell with the eIF5A1 variant of Lipofectamine 2000 and expression HA-labelling, described variant comprises wild type eIF5A1 (WT), eIF5A1K50R (K50R), eIF5A1K67R (K67R), eIF5A1K67A (K67A), eIF5A1K47R/K50R (K4750R), eIF5A1K50R/K67R (K5067R) or eIF5A1K50A/K67A (K5067A).The plasmid of expressing LacZ is with comparing.After transfection 24 and 48 hours (A) or 28 and 52 hours (B), the harvesting lysate separates by SDS-PAGE.The expression of transfection eIF5A1 uses the antibody test at HA.The lysine mutation (K67R) of result: eIF5A1 in the ubiquitination site of inferring increases the genetically modified accumulation of eIF5A1 and surpasses wild type (A).EIF5A1 turns into essential lysine mutation (K50R) at hydroxyl fourth lysine and increases the genetically modified accumulation of eIF5A1 equally above wild type eIF5A1 (B).Compare with the wild type eIF5A1 transgenic that do not suddenly change, the eIF5A1 of two mutant forms (K50A/K67A) expresses especially fully (A+B).Referring to Fig. 2.

Embodiment 2: with wild type eIF-5A1 and variant eIF-5A1 transfection KAS cell

With PAMAM dendrimer (FMD44) transfection of KAS cell with the plasmid with the eIF5A1 variant of expressing the HA-labelling, variant comprises wild type eIF5A1 (5A1), eIF5A1K67A (K67A), eIF5A1K50A/K67A (K50A K67A), eIF5A1K50R (K50R), eIF5A1K47R (K47R), eIF5A1K67R (K67R), eIF5A1K47R/K50R (K47R K50R) or eIF5A1K50R/K67R (K50R K67R).The plasmid of expressing LacZ is with comparing.After the transfection 48 hours, the harvesting lysate separated by SDS-PAGE.The expression of transfection eIF5A1 uses the antibody test at HA.Use confirms to equate to load at actin antibody.The sudden change (K67A or K67R) of result: eIF5A1 on the lysine in the ubiquitination site of inferring increases the genetically modified accumulation of eIF5A1 and surpasses wild type.Hydroxyl fourth lysine turns into sudden change of the eIF5A1 on the lysine that needs (K50R) or the sudden change of the eIF5A1 on the acetylation site (K47R) and increases the genetically modified accumulation of eIF5A1 equally above wild type eIF5A.Compare with the wild type eIF5A1 transgenic that do not suddenly change, the eIF5A1 of two mutant forms (K50A/K67A) expresses with higher level equally.Referring to Fig. 3.

Embodiment 3: use PAMAM dendrimer transfection KAS cell

With PAMAM dendrimer (FMD45-2) transfection of KAS cell with the plasmid that is loaded with the eIF5A1 variant of expressing the HA-labelling, variant comprises eIF5A1K50R (K50R), eIF5A1K50A/K67A (K50A/K67A) or eIF5A1K50R/K67R (K50R K67R).The plasmid of expressing LacZ is with comparing.After the transfection 72 hours, cell is carried out facs analysis after with Annexin/PI dyeing.The negative cell of Annexin V stained positive and PI (propidium iodide) dyeing considered to be in apoptosis early stage (Ann+/PI-), and Annexin V and PI uniform dyeing positive cells then are considered to be in apoptosis late period (Ann+/PI+).Result: eIF5A1 on the lysine in hydroxyl fourth lysine site sudden change (K50R) or in the ubiquitination site mutation (K67R) of inferring and two sudden change (K50A/K67A), cause the KAS apoptosis significantly to surpass the LacZ control level.Referring to Fig. 4.

Embodiment 4: with the plasmid transfection KAS cell of expressing eIF-5A1 and eIF-5A1 variant

With the plasmid transfection of KAS cell with the eIF5A1 variant of Lipofectamine 2000 and expression HA-labelling, variant comprises eIF5A1K50A (K50A), eIF5A1K50R (K50R), eIF5A1K67R (K67R), eIF5A1K50A/K67A (K50A/K67A) or eIF5A1K50R/K67R (K50R K67R).The plasmid of expressing LacZ is with comparing.After the transfection 72 hours, with passing through facs analysis behind the Annexin/PI staining cell.The negative cell of Annexin V stained positive and PI (propidium iodide) dyeing considered to be in apoptosis early stage (Ann+/PI-), and Annexin V and PI uniform dyeing positive cells then are considered to be in apoptosis late period (Ann+/PI+).Result: eIF5A1 turns into the lysine mutation on the site (K50R) or the ubiquitination site mutation (K67R) of inferring and double-mutant (K50A/K67A) at hydroxyl fourth lysine and all causes the KAS apoptosis significantly to surpass the LacZ control level.Referring to Fig. 5.

Embodiment 5: use the eIF-5A1 processing KAS cell of sudden change to cause apoptosis

The KAS cell is used Lipofectamine 2000 and expressed the eIF5A1 variant eIF5A1K50R (K50R) of HA-labelling or the plasmid transfection of eIF5A1K50A/K67A (K50A/K67A).The plasmid of expressing LacZ is with comparing.After the transfection 72 hours, with passing through facs analysis behind the Annexin/PI staining cell.The negative cell of Annexin V stained positive and PI (propidium iodide) dyeing considered to be in apoptosis early stage (Ann+/PI-), and Annexin V and PI uniform dyeing positive cells then are considered to be in apoptosis late period (Ann+/PI+).Result: turn into the eIF5A1 on the lysine in site sudden change (K50R) or at hydroxyl fourth lysine at hydroxyl fourth lysine and to turn into, cause the KAS apoptosis significantly above the LacZ control level with the eIF5A1 sudden change (K50A/K67A) on the site and the ubiquitination site of inferring.Referring to Fig. 6.

The adenovirus mediated kill multiple myeloma cells of embodiment 6A:siRNA/

KAS (people's multiple myeloma) cell is remained in the S10 culture medium [RPMI 1640, comprise 4ng/ml IL-6,10% hyclone (FBS) and penicillin/streptomycin (P/S)].Use Lipofectamine 2000 (Invitrogen) with the 58.7pmole siRNA transfection of KAS cell.With Lipofectamine 2000 but there is not siRNA treatment of simulated transfectional cell.Transfection is not carried out in having antibiotic S10 culture medium.

A) siRNA of targeting people eIF5A1:

EIF5A1 siRNA target #1 (siRNA targeting people eIF5A1 zone: 5 '-AAG CTG GAC TCC TCC TAC ACA-3 ' (SEQ ID NO:__).The SiRNA sequence is shown in Figure 25, and this paper is commonly referred to h5A1.

EIF5A1 siRNA target #2 eIF5A1 (siRNA targeting people eIF5A1 zone: 5 '-AAAGGA ATG ACT TCC AGC TGA-3 ' (SEQ ID NO:__).(this siRNA sequence this paper is commonly referred to h5A1-ALT)

B) contrast siRNA: contrast siRNA has following sequence: positive-sense strand, 5 '-ACA CAU CCU CCU CAG GUC GdTdT-3 '; And antisense strand, 3 '-dTd TUG UGU AGG AGG AGU CCA GC-5 ' ".

Other contrast of having used comprises the siRNA of the non-target validation that derives from Dharmacon, because they are checked to limit the unwanted effect of missing the target by microarray.For example, for the external NFkB that studies, the contrast of use is the non-target siRNA (sequence D-001700-01), and the contrast of studying use in the body is the siRNA (sequence D-001810-01) of Dharmacon of Dharmacon.

After the transfection 4 hours, resuspending was in the S10 of 1ml culture medium behind the cell precipitation.After 72 hours, the KAS cell of counting transfection is seeded on 24 orifice plates with 300,000 cells/well, carries out the transfection second time with identical siRNA then in initial siRNA transfection.

After the transfection 4 hours, resuspending was in the S10 of 1ml culture medium (not having IL-6) behind the cell precipitation, and it comprises Ad-LacZ (expressing the adenovirus of tilactase) or Ad-5A1M (the expressing human eIF5A1 of 3000ifu ^K50AAdenovirus).

After 72 hours, harvesting also carries out the apoptosis analysis: with Annexin V-FITC and PI (BD Bioscience) dyeing carrying out subsequently facs analysis.

A) early stage apoptosis is defined as the Annexin-FITC stained positive and cell (Ann+/PI-) that PI dyeing is negative

B) the apoptosis sum be defined as early stage apoptosis (Ann+/PI-) or late period apoptosis/necrosis (Ann+/PI+) total cellular score

3 ' the UTR of 5A1 siRNA target #1 targeting people eIF5A1, thereby can not influence gland virus expression eIF5A1.In the open open reading-frame of 5A1siRNA target #2 targeting people eIF5A1, so it may potential interference gland virus expression eIF5A1.

Result: handle and experience apoptotic number greater than untreated cell and only with the apoptosis number of the cell of siRNA processing with the cell of the adenovirus infection of expressing the eIF-5A1K50A variant with siRNA.Referring to Fig. 7.

Embodiment 6B: use eIF5A1 siRNA pretreatment (shown in Figure 25), reduce endogenous eIF5A1 and express but make the RNAi-resistance eIF5A1 of gland virus expression at eIF5A1 target #1 ^K50AAccumulation.

The KAS cell is used Lipofectamine 2000 and one of the two kinds of siRNA (#1 and #2) that contrast siRNA (C) or targeting eIF5A1 transfection.3 ' the UTR of eIF5A1 siRNA#1 targeting eIF5A1, thereby can not disturb gland virus expression eIF5A1, because it only comprises the open reading frame of eIF5A1.The sequence of siRNA is shown in Figure 25.The open reading frame of eIF5A1#2 siRNA targeting eIF5A1, thereby can influence endogenous and external source and express eIF5A1.After initial transfection 72 hours, cell is carried out the transfection second time with identical siRNA.After 4 hours, remove transfection composite, to comprise Ad-LacZ (L) or Ad-eIF5A1 from cell ^K50AGrowth medium (-) IL6 (5M) replaces.After 72 hours, the harvesting lysate also uses and passes through western blot analysis at eIF5A and actin antibody.Referring to Fig. 7 B.Can be observed the accumulation (1 road/2 roads) of the eIF5A1 of expressing viral, obviously pass through the reduction (5 roads and 7 roads/3 roads) of the eIF5A of eIF5A1 siRNA target #1 and #2 expression.As expection, eIF5A1 siRNA#1 does not influence the eIF5A1 of expressing viral ^K50AThe accumulation in (6 roads/4 roads), the only appropriate genetically modified expression (8 roads/4 roads) that influences expressing viral of eIF5A1 siRNA#2 simultaneously.

Embodiment 6C: use eIF5A1 siRNA pretreatment adenovirus infection then, reduce the expression of phosphorylation NF-κ B in people's multiple myeloma cells at target #1.

The KAS cell is used Lipofectamine 2000 and siRNA (#1) transfection that contrasts siRNA (hC) or targeting eIF5A1.Therefore 3 ' the UTR of eIF5A1 siRNA#1 targeting eIF5A1 can not disturb gland virus expression eIF5A1, because it only comprises the open reading frame of eIF5A1.After 72 hours, cell is carried out the transfection second time with identical siRNA in initial transfection.After 4 hours, transfection composite is removed from cell, to comprise Ad-LacZ (L) or Ad-eIF5A1 ^K50AGrowth medium (+) IL6 (5M) replaces.After 24 hours, the harvesting lysate uses the antibody at phosphoric acid-NF-kB p65 (Ser 536) and eIF5A to carry out western blot analysis.As expection, eIF5A1 siRNA#1 does not influence the eIF5A1 of expressing viral ^K50AAccumulation.Activate, appraise and decide position, protein protein interaction and transcriptional activity in the NF-kB p65 of serine 536 phosphorylations adjusted.Referring to Fig. 7 C.

Embodiment 6D:, reduce phosphorylation NF-kB and the expression of ICAM-1 in people's multiple myeloma cells with eIF5A1 siRNA#1 pretreatment adenovirus infection then.

The KAS cell is used Lipofectamine 2000 and one of the two kinds of siRNA (#1 and #2) that contrast siRNA (C) or targeting eIF5A1 transfection.After initial transfection 72 hours, cell is carried out the transfection second time with identical siRNA.After 4 hours, transfection composite is removed from cell, replaced with growth medium (+) IL6.After the transfection 24 hours for the second time,,, use and carry out western blot analysis at phosphoric acid-NF-kB p65 (Ser 536), ICAM-1 and actin antibody in 0 hour, 4 hours or 24 hours harvesting lysates with 40ng/mlTNF-α irritation cell.Observe with after the transfection of two kinds of eIF5A1-specific siRNAs, express reduction in the inductive NF-kB p65 of TNF-α phosphorylation (ser 536) and ICAM-1.Position, protein protein interaction and transcriptional activity are regulated and activate, appraised and decided to the phosphorylation of NF-kB p65 on serine 536.ICAM-1 is the intercellular adhesion surface glycoprotein, it is believed that it participates in the pathogeny of multiple myeloma.Referring to Fig. 7 D.

Embodiment 6E: in the presence of IL-6, strengthen eIF5A1 with siRNA pretreatment KAS cell ^K50RThe apoptosis of gene delivery.

The KAS cell is used Lipofectamine 2000 and contrast siRNA (hcon) or people eIF5A1 siRNA (h5A1) transfection.After 72 hours, with siRNA transfectional cell again.With empty carrier (mcs) or eIF5A1 ^K50R(K50R) the PEI complex of plasmid adds to cell, after 4 hours, removes the siRNA transfection media.The growth medium that whole research is used comprises IL-6.Detect apoptosis after 72 hours: with carrying out facs analysis behind the Annexin/PI staining cell.Referring to Fig. 7 F.

Embodiment 7: give eIF-5A1 plasmid and eIF-5A1siRNA simultaneously and delay multiple myeloma Subcutaneous tumor growth (Fig. 8-10).

To SCID mouse subcutaneous injection KAS cell.Begin treatment when observing palp tumor.The right rib side of body 6 3-5 SCID/CB17 mices in age in week is injected at 1 * 10 among the 200 μ L PBS ⁷Individual KAS-6/1 myeloma cell is when tumor reaches minimum volume 4mm ³Begin treatment during size.

Control mice is injected the PEI complex that comprises pCpG-mcs (empty carrier) and contrast siRNA (matched group is 3 mice: C-1, C-2 and C-3) in 2 tumors weekly.The treatment mice is injected the PEI complex (the treatment group is 3 mice: 5A-1,5A-2 and 5A-3) that comprises RNAi-r plasmid pCpG-eIF5A1k50R and eIF5A1 siRNA in 2 tumors weekly.Anti-backflow is injected in a plurality of sites in tumor, and the injection speed that slows down absorbs to improve.The gross tumor volume of all mices in Fig. 8 data show group.Data shown in Figure 9 be every group of mean tumour volume+/-standard error.Asterisk represent statistical significance ( ^*=p＜0.025; N=3).

Figure 10 demonstration gives the eIF-5A1 plasmid simultaneously and eIF-5A1siRNA alleviates the weight of multiple myeloma Subcutaneous tumor.Data presented be every group of average tumor weight+/-standard error.Asterisk represent statistical significance ( ^*=p＜0.05; N=3).

JET-PEI (PolyPlus) is used for the body build-in test with 2 * 0.1ml.The ratio of N/P is 8.The PEI/DNA/siRNA complex forms in 5% glucose of cumulative volume 0.1ml.The scheme that forms complex is as follows.

1. make the component temperature to room temperature.Keep aseptic.

2. (about 10 μ l, 2mg/ml) (10 μ l 1mg/ml) are diluted to cumulative volume 25 μ l with 10 μ g siRNA with 20 μ g plasmid DNA.Use the sterilized water part of covering the shortage.

3. 10% glucose that adds 25 μ l is regulated volume to 50 μ l 5% glucose of dna solution.Of short duration centrifugal behind the vortex gently.

4. the In vivo JETPEI dilution with 4.8 μ l is 10% glucose of cumulative volume 25 μ l.Be adjusted to volume 50 μ l with sterilized water, ultimate density is 5% glucose.Of short duration centrifugal behind the vortex gently.

5. the DNA (order can not be put upside down) that adds PEI to the 50 μ l dilution of 50 μ l dilution immediately.Rotation is slowed down immediately behind the of short duration vortex.

6. incubation was injected after 15 minutes.Stable composite 6 hours.

No CpG cloning vehicle and pCpG plasmid derive from InvivoGen.These plasmids do not have the CpG dinucleotide fully, are called pCpG.All produce high-caliber transgene expression in the external and body of these plasmids, and opposite with CMV type plasmid, its make proper in continuous expression.The natural CpG of the not having dinucleotide of the element that the pCpG plasmid comprises; It is modified to remove all CpG; Or all synthetic, the gene of the selected marker of for example encoding or reporter protein.These neomorphs can the synthetic fact be: in 16 dinucleotide that form genetic code, CG is only nonessential alternative dinucleotide.8 kinds of codons comprise the CG of the 5 kinds of different aminoacids of encoding.8 kinds of codons all can be encoded same amino acid whose two kinds of at least a replacements of codon with generation neomorph (protein of its coding and wild type same acid sequence), thereby it has activity equally as wild type counterparts.These neomorphs can independently be present in the plasmid (they can easily therefrom cut off) that is called pMOD.

Can be the in vivo long-term continuous expression of pCpG plasmid, and be useful tool, its can be used in the body and in vitro study CpG to the influence (using the cell line of expressing TLR9) of gene expression and they are to the congenital and influence acquired immunity system.Empty carrier pCpG-mcs (Invivogen) does not have expressing gene product, the carrier of a multiple clone site only, and as control vector.EIF5A1 with the HA-labelling ^K50RThe cDNA sub-clone is gone into NcoI and NheI site (Invivogen) of pCpG-LacZ carrier, and the LacZ gene is removed from it, to produce treatment carrier pCpG-eIF5A1 (K50R).Use Endo-Free Qiagen test kit to prepare DNA.Measure level of endotoxin＜0.03EU/ug; DNA is 2mg/ml in the water.

The contrast siRNA that experiment is used be the microarray checking non-target contrast siRNA (Dharmacon, D-001810-01).The siRNA that acquisition has a modification (siSTABLE) degrades in serum preventing.

The eIF5A1 siRNA that experiment is used is designed to 3 ' UTR at people eIF5A1.Do not have similarity between eIF5A1 siRNA and the mice eIF5A1, therefore, siRNA only suppresses people (non-mice) eIF5A1.SiRNA and eIF5A2 (people or mice) do not have similarity equally.The siRNA that acquisition has a modification (siSTABLE) degrades in serum preventing.EIF5A1siRNA has following target sequence:

5’GCU?GGA?CUC?CUC?CUA?CAC?A(UU)3

With siSTABLE siRNA with 1mg/ml water-soluble (storing) in-branches such as 20C.

Use digital calipers 2-3 time/week measure the size of tumor long (l) and wide (w).Calculate gross tumor volume according to following formula:

L=length; Minimum dimension

The w=width; Full-size

Gross tumor volume (mm ³)=l ²* w*0.5

Statistical analysis

Use t-check the carrying out statistical analysis of Student.Confidence level greater than 95% that be considered as having significance (p＜0.05).

Embodiment 8: give eIF5A1 plasmid and eIF5A1 siRNA simultaneously and delay multiple myeloma Subcutaneous tumor growth and cause tumor to reduce.

In another research, to SCID mouse subcutaneous injection KAS cell.Begin treatment when observing palp tumor.Control mice is injected the PEI complex that comprises pCpG-mcs (empty carrier) and contrast siRNA (matched group G-1, G-2 and G-3) in 2 tumors weekly.The treatment mice is injected the PEI complex (treatment group G-4, G-5 and G-6) that comprises RNAi-r plasmid pCpG-eIF5A1k50R (20 μ g plasmid DNA) and eIF5A1 siRNA (10 μ g siRNA) in 2 tumors weekly.Figure ll shown in data be every group of mean tumour volume+/-standard error.Asterisk represent statistical significance ( ^*=p＜0.025; N=3).In 21 days, carry out 6 injections (red arrow).

Embodiment 9: intravenous gives to give in (i.v.) eIF5A1 siRNA and the tumor (i.t.) PEI/eIF5A1K50R plasmid composite and causes the tumor of multiple myeloma Subcutaneous tumor to reduce (2B group).

To SCID mouse subcutaneous injection KAS cell.When observing palp tumor, initially inject begin treatment with 50 μ g contrast siRNA (matched group) or people eIF5A1 siRNA (treatment group) afterbody.Subsequently control mice is treated (empty carrier by the PEI complex that injection in 2 tumors weekly comprises pCpG-mcs; Matched group; G-1, G-2 and G-3).Subsequently the treatment mice is treated (treatment group by the PEI complex that injection in 2 tumors weekly comprises RNAi-r plasmid pCpG-eIF5A1k50R (20 μ g plasmid DNA); G-4, G-5 and G-6).Control mice continues to accept 1 i.v. injection contrast siRNA (matched group R-1, R-2 and R-3) weekly.The treatment mice continues to accept 1 i.v. injection people eIF5A1siRNA (20 μ g) (treatment group R-4, R-5 and R-6) weekly.Data shown in Figure 12 are gross tumor volumes of every group of all mices.In 21 days, give to inject PEI/DNA (red arrow) and 4 i.v. injection siRNA (blue arrow) in 6 tumors.

Figure 13 provides embodiment 8 and 9 results' stacking chart.To SCID mouse subcutaneous injection KAS cell.Data shown in Figure 13 be every group of mice mean tumour volume+/-standard error.Asterisk represent between treatment group and the matched group statistical significance ( ^*=p＜0.01; ^{* *}=p＜0.001; N=3).

Form the method for PEI complex:

1. make the component temperature to room temperature.Keep aseptic.

2. plasmid DNA or plasmid DNA+siRNA are diluted to cumulative volume 25 μ l.Use the sterilized water adjusted volume.

A) complex of plasmid DNA only:

(10 μ l 2mg/ml) are diluted to cumulative volume 25 μ l with 20 μ g plasmid DNA.Use the sterilized water part of covering the shortage.

B) plasmid DNA+siRNA complex:

(about 10 μ l, 2mg/ml) (10 μ l 1mg/ml) are diluted to cumulative volume 25 μ l with 10 μ g siRNA with 20 μ g plasmid DNA.Use the sterilized water part of covering the shortage.

3. 10% glucose (PEI kit provides) that adds 25 μ l is regulated volume to 50 μ l 5% glucose of dna solution.Of short duration centrifugal behind the vortex gently.

4. In vivo JETPEI dilution is 10% glucose of cumulative volume 25 μ l.

A) complex of plasmid DNA only:

3.2 μ l In vivo JETPEI dilution is 10% glucose of cumulative volume 25 μ l.Be adjusted to volume 50 μ l with sterilized water, ultimate density is 5% glucose.Of short duration centrifugal behind the vortex gently.

B) plasmid DNA+siRNA complex:

4.8 μ l In vivo JETPEI dilution is 10% glucose of cumulative volume 25 μ l.Be adjusted to volume 50 μ l with sterilized water, ultimate density is 5% glucose.Of short duration centrifugal behind the vortex gently.

5. immediately the PEI of 50 μ l dilution being added to 50 μ l dilution DNA (could not transpose! ) in.Slow down immediately behind the of short duration vortex.

6. incubation was injected after 15 minutes.Stable composite 6 hours.

With regard to tail vein injection siRNA, the initial injection of siRNA is 50 μ g.SiRNA is diluted to the PBS of 0.4mg/ml.(50 μ g) is injected into tail vein with 125 μ l/ mices.Inject serum stable type siRNA weekly for 2 times with 20 μ g/ mices subsequently.SiRNA is diluted to the PBS of 0.4mg/ml.(20 μ g) is injected into tail vein with 50 μ l/ mices.

Figure 13 B demonstration gives the eIF5A1 plasmid simultaneously and eIF5A1 siRNA causes tumor to reduce.To SCID mouse subcutaneous injection KAS cell.Begin treatment when observing palp tumor.Mice is injected PEI complex (the treatment group that comprises RNAi-r plasmid pCpG-eIF5A1k50R and eIF5A1 siRNA in 2 tumors weekly; G-4, G-5 and G-6).Injection is 6 times in 21 days.Behind begin treatment 42 days, put to death mice, cut knub position skin examination tumor growth situation.In treatment mice 2A group, do not observe any tumor growth.

Figure 13 C shows that intravenous gives to give in (i.v.) eIF5A1 siRNA and the tumor (i.t.) PEI/eIF5A1K50R plasmid composite and causes the multiple myeloma Subcutaneous tumor to reduce.To SCID mouse subcutaneous injection KAS cell.When observing palp tumor, initially inject begin treatment with 50 μ g people eIF5A1 siRNA (treatment group).Subsequently mice is injected PEI complex (the treatment group that comprises RNAi-r plasmid pCpG-eIF5A1k50R in 2 tumors weekly; R-4, R-5 and R-6).Mice continues to accept i.v. injection people eIF5A1 siRNA weekly for 1 time.21 days finish to treat behind begin treatment.After initial therapy, put to death mice in 42 days, cut knub position skin examination tumor growth situation.In treatment group (2B group), there is a mice not have tumor growth.

Embodiment 10: intravenous gives the eIF5A1 plasmid simultaneously and eIF5A1 siRNA delays multiple myeloma Subcutaneous tumor growth.

To SCID mouse subcutaneous injection KAS cell.When observing palp tumor, initially inject begin treatment with 50 μ g contrast siRNA (matched group) or people eIF5A1 siRNA (treatment group).PEI complex (the empty carrier that subsequently mice is comprised pCpG-mcs by the injection of about 2 intravenouss (red arrow) or intraperitoneal (green arrow) weekly; Control group A 1, A2 and A3) or comprise PEI complex (the treatment group of RNAi-r plasmid pCpG-eIF5A1k50R; A4, A5 and A6).Mice continues to accept 1 i.v. injection (blue arrow) contrast siRNA (control group A 1, A2 and A3) or people eIF5A1 siRNA (treatment group A4, A5 and A6) weekly.Shown in data are gross tumor volumes of every group of all mices.Data shown in Figure 14 are gross tumor volumes of every group of all mices.

Embodiment 11: intravenous gives (i.v.) eIF5A1 siRNA and intravenous (i.v.) or intraperitoneal (i.p.) and gives the PEI/eIF5A1K50R plasmid composite and delay multiple myeloma Subcutaneous tumor growth.

To SCID mouse subcutaneous injection KAS cell.When observing palp tumor, initially inject begin treatment with 50 μ g contrast siRNA (matched group) or people eIF5A1 siRNA (treatment group).About weekly 1 intravenous of control mice or the peritoneal injection PEI complex that comprises pCpG-mcs is treated (empty carrier subsequently; Matched group 3 mice: B1, B2 and B3).Subsequently treatment about weekly 1 intravenous of mice or peritoneal injection are comprised RNAi-r plasmid pCpG-eIF5A1 ^K50RThe PEI complex treat (treatment group; B4, B5 and B6).Mice continues to accept contrast siRNA (matched group B1, B2 and B3) or people eIF5A1 siRNA (3 mice mice: B4 of treatment group, B5 and B6) i.v. injects 1 time weekly.Experiment is initially injected beginning (on Figure 15 curve the 2nd day) with 50 μ g siRNA.Inject 20 μ g siRNA 1 time weekly subsequently.Directly give SiRNA, promptly do not have delivery media.The PEI complex comprises 20 μ g plasmid DNA.Initial p EI injection is the i.v. injection for i.p. injection injection subsequently.Data shown in Figure 15 are gross tumor volumes of every group of all mices.

Figure 16 provides the stacking chart as a result of embodiment 10 and 11.To SCID mouse subcutaneous injection KAS cell.Begin treatment when observing palp tumor.One group of mice is accepted 1 i.v. injection contrast siRNA (contrast weekly; A organizes) or eIF5A1 siRNA (treatment; A group) and i.v. or the i.p. injection PEI complex that comprises pCpG-mcs (contrast; A organizes) or comprise RNAi-r plasmid pCpG-eIF5A1 ^K50RThe PEI complex (treatment; The A group).Second group of mice accepted the PEI complex that about weekly 2 i.v. or i.p. injection comprise pCpG-mcs (empty carrier) and contrast siRNA and (contrasts; Or comprise the PEI complex (treatment of RNAi-r plasmid pCpG-eIF5A1k50R and eIF5A1siRNA B group); The B group).Data presented be every group of mice mean tumour volume+/-standard error.Asterisk represent between treatment group and the matched group statistical significance ( ^*=p＜0.05; ^{* *}=p＜0.001; N=3).

The scheme of preparation PEI complex and siRNA as previously described in the embodiment.

Embodiment 12: give eIF5A1 plasmid and eIF5A1 siRNA simultaneously and delay multiple myeloma Subcutaneous tumor growth and cause tumor to reduce.

To SCID mouse subcutaneous injection KAS cell.Begin treatment when observing palp tumor.Control mice weekly in 2 tumors injection comprise pCpG-mcs (empty carrier) and contrast siRNA the PEI complex (matched group has 3 mices: contrast 1, contrast 2 and contrast 3).Treatment mice 2 interior injections of tumor weekly comprises RNAi-r plasmid pCpG-eIF5A1 ^K50RPEI complex (the treatment group has 4 mice: 5A-1,5A-2,5A-3 and 5A-4) with eIF5A1 siRNA.The PEI complex of injection comprises the siRNA of 20 μ g plasmid DNA and 10 μ g in the tumor.Data shown in Figure 17 are gross tumor volumes of every group of all mices.

Embodiment 13: intravenous gives to give (i.t.) PEI/eIF5A1 in (i.v.) eIF5A1 siRNA and the tumor ^K50RPlasmid composite causes the multiple myeloma Subcutaneous tumor to reduce.

To SCID mouse subcutaneous injection KAS cell.When observing palp tumor, initially inject begin treatment with 50 μ g contrast siRNA (matched group has 3 mices: contrast 1, contrast 2 and contrast 3) or people eIF5A1 siRNA (the treatment group has 3 mice: 5A-1,5A-2 and 5A-3).Inject the PEI complex treatment control mice (matched group 1-3) that comprises pCpG-mcs (20 μ g) in 2 tumors weekly subsequently.2 interior injections of tumor comprise RNAi-r plasmid pCpG-eIF5A1 weekly subsequently ^K50RThe PEI complex treatment treatment mice (5A-1, A-2,5A-3) of (20 μ g).Control mice continues to accept 2 tail vein i.v. injection any contrast siRNA (20 μ g) weekly.The treatment mice continues to accept 2 tail vein i.v. injection people eIF5A1siRNA (20 μ g) weekly.Described injection gives to give after 48 hours injection in the tumor.SiRNA gives as naked SiRNA, does not promptly have delivery media.Data shown in Figure 180 are gross tumor volumes of every group of all mices.

Embodiment 14: give eIF5A1 simultaneously ^K50RPlasmid (by EF1 or B29 promoters driven) and eIF5A1 siRNA delay multiple myeloma Subcutaneous tumor growth and cause tumor to reduce.

To SCID mouse subcutaneous injection KAS cell.Begin treatment when observing palp tumor.Mice is injected the PEI complex in 2 tumors weekly, the eIF5A1 plasmid that described PEI complex comprises control vector (G1 and G2) or drives by B29 promoter (G3 and G4) or EF1 promoter (G5 and G6), and contrast siRNA (G1, G3, G5) or h5A1siRNA (G2, G4, G6).Video data be every group of mean tumour volume+/-standard error.Attention: the B29 promoter is B-cell-specificity promoter.Yet, can drive KAS cells in vitro high expressed HA-eIF5A1 although find B29 promoter/mCMV enhancer that this research is used ^K50R, but as if it be not B-cell-specific (may be because cmv enhancer).Referring to Figure 19.

Embodiment 15: give the eIF5A1 that eIF5A1 siRNA strengthens EF1 or B29 promoters driven simultaneously ^K50RPlasmid is to the antitumor action of multiple myeloma Subcutaneous tumor and cause tumor load to reduce.

To SCID mouse subcutaneous injection KAS cell.Begin treatment when observing palp tumor.Mice is injected the PEI complex in 2 tumors weekly, described PEI complex comprises the eIF5A1 plasmid that control vector (G1 and G2) or B29 promoter (G3 and G4) or EF1 promoter (G5 and G6) drive, and contrast siRNA (G1, G3, G5) or h5A1 siRNA (G2, G4, G6).Behind begin treatment, put to death mice in 24 days, downcut Subcutaneous tumor and weigh.Video data be each the group average tumor weight+/-standard error.Referring to Figure 20.

The collaborative Ad-eIF5A of raising of embodiment 16:eIF5A1 siRNA infects the apoptosis-inducing that lung adenocarcinoma cell produced.

Infect the A549 cell with Ad-LacZ or Ad-eIF5A.SiRNA transfectional cell with contrast siRNA or targeting people eIF5A1 (h5A1): transfection media is added to cell, and then add virus.At with the siRNA transfection and after 4 hours with viral infection, replace with fresh culture, and incubation cell 72 hours, then with the Annexin/PI labelling to detect apoptotic cell.Attention: the eIF5A in this cell line crosses to express and causes the not accumulation of hydroxyl fourth lysine eIF5A, because the amount of DHS and DOHH is limited, thereby and eIF5A ^K50RCross to express equally, cause identical apoptosis-promoting effect.These data show, in the tumorous type of non-myeloma, observe equally suppress simultaneously hydroxyl fourth lysine eIF5A and not hydroxyl fourth lysine eIF5A cross the synergism of expressing the pair cell apoptosis that causes.Referring to Figure 21.

Embodiment 17: make up plasmid pExp5A.

PExp5A is the expression plasmid of the CpG dinucleotide with minimizing of design, mainly drives people eIF5A1 in the B cell line cell ^K50RExpress.Support source is from pCpG-LacZ, a kind of plasmid (Invivogen) that does not at all have the CpG dinucleotide.The whole elements that duplicate in escherichia coli and need to select all do not have the CpG dinucleotide.Former cmv enhancer/promoter in the CpG-LacZ carrier and LacZ gene are respectively by the minimum B cell specificity promotor of people (B29/CD79b; Invivogen) and people eIF5A1 ^K50RDisplacement is so that drive B-cell specific expression eIF5A1 ^K50RB29DHS4.4 3 ' enhancer has been introduced the plasmid downstream of eIF5A1 expression cassette, so that strengthen the B29 promoter activity and reduce non-B cellular expression (Malone et al.2006.B29 gene silencing in pituitary cells is regulated by its 3 ' enhancer.J.Mol.Biol.362:173-183).Mix B29 minimal promoter, eIF5A1 ^K50RAnd B29DHS4.43 ' enhancer has been introduced the 32CpG dinucleotide in the carrier.

Escherichia coli (E.coli) Expression element

Origin of replication: escherichia coli R6K γ starting point (ori.)

^*Owing to there is R6K γ origin of replication, the pCpG plasmid increases in the escherichia coli mutants which had of expressing the pir mutant gene only.They can not duplicate in the standard coli strain.Therefore, the pCpG plasmid offers escherichia coli GT115Bacterial strain, the Dcm also pir mutant (Invivogen) of defective that methylates.

Bacillary promoter: EM2K, a kind of bacillary EM7 promoter of not having the CpG form.

Selected marker: Zeocin ^TMResistant gene, a kind of synthetic allele that does not have CpG.

Expression element in the mammalian cell

Mammalian promoter: for B cell tissue-specific expressed minimum B29 (CD79b) promoter of people-167bp.A kind of synthetic intron (I 140) also is present among the 5 ' UTR.

Polyadenylation signal: the SV40 polyadenylation signal in late period of no CpG dinucleotide form.

3 ' enhancer: people B29 DHS4.43 ' enhancer.

MAR:2 no CpG Matrix bonding pad (MAR) is present between bacillary transcript unit and the mammal transcript unit.A MAR is derived from people IFN-β gene 5 ' district, and another is derived from betaglobulin gene 5 ' district.

The expected sequence of pExp5A (3371bp) is seen Figure 23.

EIF5A1 ^K50RAminoacid sequence

^*K50R sudden change underscore illustrates

MADDLDFETGDAGASATFPMQCSALRKNGFVVLKGRPCKIVEMSTSKTGRH

GHAKVHLVGIDIFTGKKYEDICPSTHNMDVPNIKRNDFQLIGIQDGYLSLLQD

SGEVREDLRLPEGDLGKEIEQKYDCGEEILITVLSAMTEEAAVAIKAMAK

Make up pExp5A-structure main points:

Step 1: clone's B29 DHS4.4 3 ' enhancer and sub-clone are gone into pGEM Teasy (Promega)--set up pGEM-4.4enh#8.

Step 2: minimum B29 promoter sub-clone is gone into pCpG-LacZ (Invivogen)--set up B29-5#3.

Step 3: with HA-eIF5A1 ^K50RSub-clone is gone into the B29-5#3 carrier--set up B29-5#3-eIF5A1 ^K50R

Step 4: in pCpG-mcs (Invivogen), set up novel multiple clone site--set up pCpG-Linker4.

Step 5: B29 DS4.4 3 ' enhancer sub-clone is gone into pCpG-Linker4--set up pCpG-DHS4.4.

Step 6: with B29 promoter+HA-eIF5A1 ^K50R+ SV40pA expression cassette sub-clone is gone into pCpG-DHS4.4--and is set up pExp-5.

Step 7: with the HA-eIF5A1 among the pExp-5 ^K50RWith non--HA eIF5A1 ^K50RDisplacement--set up final carrier pExp5A.

Make up and describe in detail:

Step 1: clone's B29 DHS4.4 3 ' enhancer and sub-clone are gone into pGEM T easy (Promega)--set up pGEM-4.4enh#8.

To use following primer from the isolating genomic DNA of KAS cell (people's multiple myeloma cells system) by PCR clone B29DHS4.4 3 ' enhancer:

Forward primer 5 '-CAG CAA GGG AGC ACC TAT G-3 ' and reverse primer 5 '-GTT GCA GTG AGC GGA GAT G-3 '.The sequential design primer of end user CD79B/GH-1 intergenic region (Accession AB062674).The 608bp PCR fragment sub-clone of gained is gone into pGEM

T easy cloning vehicle (Promega) and order-checking.Komatsuet?al.2002.Novel?regulatory?regions?found?downstream?of?the?rat?B29/Ig-b?gene.Eur.J.Biochem.269：1227-1236。

The sequence of B29DHS4.43 ' the enhancer PCR fragment (297bp) in pGEM-4.4enh#8

ACCACCCTGGGCCAGGCTGGGCCAAGCCAGGCGGCCCCTGTGTTTTCCCC

AGTCTCTGGGCTGCTGGAGGGAACCAG GTTGTTTTGG

CTCTACT

GAGCCGGAGCCCTTCCTTTCCT

TGGCACTAATTCCG

TCCTCCTACCTCCACCAGGGACCTAGGCAGCCGGGTAGATGGTGGGAGGA

GGCTTCACTTCTCCCCCAAGCAGGGTCTCCACCTGCTT

TGG

GTTGGGGGAGGCCTTGGCTTTACCTAAAGACTTTTTAACACCTCT

The o+4.4 zone comprises some transcription factor binding site points

■SRY? GTTGTTT

■GATA

■OCT-X?

■NF-KB?

B29 DHS4.4 3 ' enhancer PCR fragment (297bp) that will be in pGEM-4.4enh#8 with The sequence of people CD79B/GH-1 intergenic region (Accession AB062674) is compared

Step 2: minimum B29 promoter sub-clone is gone into pCpG-LacZ (Invivogen)--set up B29-5#3.

From being purchased the plasmid (pDrive-hB29 that carries total length people B29 promoter; Invivogen) minimum-167 people B29 promoteres of amplification are used following primer: forward primer, 5 '-CCA ACT AGTGCG ACC GCC AAA CCT TAG C-3 '; Reverse primer, 5 '-CAA AAG CTTGAC AAC GTC CGA GGC TCC TTG G-3 '.The PCR fragment of gained with SpeI and HindIII digestion and connect into the SpeI of pCpG-LacZ carrier and HindIII site (Invivogen) to set up B29-5#3.

B29 minimal promoter PCR fragment (188bp) sequence in B29-5#3

GCGACCGCCAAACCTTAGCGGCCCAGCTGACAAAAGCCTGCCCTCCCCCA

GGGTCCCCGGAGAGCTGGTGCCTCCCCTGGGTCCCAATTTGCATGGCAGG

AAGGGGCCTGGTGAGGAAGAGGCGGGGAGGGGACAGGCTGCAGCCGGTG

CAGTTACACGTTTTCCTCCAAGGAGCCTCGGACGTTGTC

B29 minimal promoter PCR fragment (B29min) and pDrive-hB29 that will be in B29-5#3 Total length people B29 promoter compare

Step 3: with HA-eIF5A1 ^K50RSub-clone is gone into the B29-5#3 carrier--set up pB29-eIF5A1K50R_7.

HA-eIF5A1 ^K50RBy pcr amplification, use pHM6-eIF5A1 ^K50RBe dna profiling and the following primer of use: forward primer 5 '-CGC CAT GGA CAT GTA CCC TTA CGA CGT CCC AGA CTA CGC TGC AGA TGA TTT GGA CTT CGA G-3 ', reverse primer 5 '-CGC GCT AGCCAG TTA TTT TGC CAT CGC C-3 '.The PCR fragment of gained is gone into the NcoI of B29-5#3 and NheI site with displacement LacZ with NcoI and NheI digestion and sub-clone.

HA-eIF5A1 in pB29-eIF5A1K50R_7 ^K50R The sequence of PCR fragment (497bp)

ACATGTACCCTTACGACGTCCCAGACTACGCTGCAGATGATTTGGACTTCG

AGACAGGAGATGCAGGGGCCTCAGCCACCTTCCCAATGCAGTGCTCAGCA

TTACGTAAGAATGGTTTTGTGGTGCTCAAGG6CCGGCCATGTAAGATCGT

CGAGATGTCTACTTCGAAGACTGGCAGGCATGGCCATGCCAAGGTCCATC

TGGTTGGCATTGATATTTTTACTGGGAAGAAATATGAAGATATCTGCCCGT

CGACTCATAACATGGATGTCCCCAACATCAAAAGGAATGATTTCCAGCTG

ATTGGCATCCAGGATGGGTACCTATCCCTGCTCCAGGACAGTGGGGAGGT

ACGAGAGGACCTTCGTCTGCCTGAGGGAGACCTTGGCAAGGAGATTGAGC

AGAAGTATGACTGTGGAGAAGAGATCCTGATCACAGTGCTGTCCGCCATG

ACAGAGGAGGCAGCTGTTGCAATCAAGGCGATGGCAAAATAACTG

HA-eIF5A1 in pB29-eIF5A1K50R_7 ^K50R The segmental translation of PCR

eIF5A1 ^K50R

MD

ADDLDFETGDAGASATFPMQCSALRKNGFVVLKGRPCKI

IQDGYLSLLQDSGEVREDLRLPEGDLGKEIEQKYDCGEEILITVLSAMTEEAAV

AIKAMAK

HA-eIF5A1 that will be in pB29-eIF5A1K50R_7 ^K50R PCR fragment and people EIF5A1 (Accession#NP 001961) compares

Step 4: in pCpG-mcs (Invivogen), set up new multiple clone site--set up pCpG-Linker4.

PCpG cloning vehicle pCpG-mcs G2 (Invivogen) removes the mammal expression cassette with EcoRI digestion, and it comprises mCMV enhancer, hEF1 promoter, synthetic intron, multiple clone site and SV40 polyadenylation signal.The pCpG-mcs G2 carrier of EcoRI digestion is connected to synthetic linker with EcoRI sticky end has new multiple clone site with foundation no promoter vector (pCpG-Linker4).

Synthetic linker promptly has the joint 4 of 2 Eco RI sticky ends

The sequence of peripheral region, site falls in Xin Duoke in pCpG-joint 4

Above sequence numbering is following for convenience of description sequence signature: β Glo MAR (nucleotide 1-380); EcoRI recognition sequence (nucleotide 396-401); XhoI recognition sequence (nucleotide 401-406); ClaI recognition sequence (nucleotide 409-414); NotI recognition sequence (nucleotide 417-424); MluI recognition sequence (nucleotide 427-432); IFN β S/MAR (nucleotide 438-1,030).

Step 5: B29 DS4.4 3 ' enhancer sub-clone is gone into pCpG-Linker4--produce pCpGDHS4.4.

By pcr amplification B29 DHS4.4 3 ' enhancer, use pGEM-4.4enh#8 is as template and use following primer: forward primer 5 '-GAA GCG GCC GCA CCA CCCTGG GCC AGG CTG G-3 '; Reverse primer 5 '-CC A CGC GTA GAG GTG TTA AAA AGT CTT TAG GTA AAG-3 '.The PCR fragment of gained is with NotI and MluI digestion, connects in NotI in the new multiple clone site of pCpG-joint 4 and the MluI site to set up pCpG-DHS4.4.

The pCpG-DHS4.4 full length sequence (2,282bp)

Above sequence numbering is following for convenience of description sequence signature: β Glo MAR (nucleotide 1-400); EcoRI recognition sequence (nucleotide 416-421); XhoI recognition sequence (nucleotide 421-426); ClaI recognition sequence (nucleotide 429-434); NotI recognition sequence (nucleotide 437-444); DHS4.4 (nucleotide 445-741); MluI recognition sequence (nucleotide 742-747); IFN β S/MAR (nucleotide 753-1,569).

Step 6: with B29 promoter+HA-eIF5A1 ^K50R+ SV40pA expression cassette sub-clone is gone into pCpG-DHS4.4--and is produced pExp-5.

((it comprises minimum B29 promoter, synthetic intron, HA-eIF5A1 to step 3) amplification B29-eIF5A1 expression cassette from pB29-eIF5A1K50R_7 by PCR ^K50RAnd SV40pA), use following primer: forward primer 5 '-GTT ATC GAT ACT AGT GCG ACC GCCAAA CC-3 '; Reverse primer 5 '-CAA GCG GCC GCC ATA CCA CAT TTGTAG AGG TTT TAC-3 '.The PCR fragment of gained is with ClaI and NotI digestion, and sub-clone is gone in ClaI in the multiple clone site in pCpG-DHS4.4 and the NotI site to produce pExp-5.

Step 7: with the HA-eIF5A1 among the pExp-5 ^K50RWith non--HA eIF5A1 ^K50RDisplacement is to produce final carrier pExp5A.

Digest the pExp-5 plasmid to remove HA-eIF5A1 with NcoI and NheI ^K50RBy PCR from pHM6-eIF5A1 ^K50RThe eIF5A1 of non--HA-labelling increases ^K50RThe PCR fragment is used following primer: forward primer 5 '-CAC CAT GGC AGA TGA TTT GGA CTT C-3 '; Reverse primer 5 '-CGC GCT AGCCAG TTA TTT TGC CAT CGC C-3 '.The PCR product of gained is connected among the NcoI and NheI site of B29-5#3 after digesting with NcoI and NheI, to produce B29-K50R.With NcoI and NheI digestion B29-K50R, gel-purified 470bp eIF5A1 ^K50RBe connected to the pExp-5 of NcoI/NheI-digestion after the fragment, to produce final expression vector pExp5A.

Embodiment 18:pExp5A test

Use the various cell lines of Lipofectamine 2000 usefulness plasmid transfections, after the transfection 24 hours with anti--HA antibody (Roche) by Western blotting mensuration HA-eIF5A1 ^K50RExpress.The different cell lines of test are P3X63Ag8.653 (mice B lymphoblast-myeloma), KAS (human myeloma), HepG2 (human hepatocellular carcinoma), T24 (human bladder cancer); HT-29 (people ties rectal adenocarcinoma), HEK-293 (HEKC), PC3 (human prostata cancer); HeLa (people's adenocarcinoma of the uterine cervix) and A549 (pulmonary carcinoma).Referring to Figure 24.

The pExp-5 plasmid is expressed HA-eIF5A1 people and mouse myeloma cell line ^K50RLevel be comparable to plasmid (CpG-eIF5A1 with composing type EF1 promoter ^K50R) level.Yet compare the HA-eIF5A1 that pExp-5 drives with the expression of constitutive promoter ^K50RExpression is limited to non-B cell line.An exception is HEK-293 cell (human embryonic kidney cell line), observes high-caliber HA-eIF5A1 after this cell line pExp-5 transfection ^K50RExpress-this may be because the embryonal cause of this cell line; At present, we do not know whether pExp-5 is becoming the human kidney cells invading the exterior to reach.The final plasmid that toxicity research and clinical trial are used is a kind of like this pExp-5: HA-eIF5A1 wherein ^K50RReplace with the eIF5A1 of non--HA labelling ^K50R(pExp5A).PExp-5 comprises the eIF5A1 of HA-labelling under the control of person of low position B29 promoter/enhancer ^K50RWith HA-eIF5A1 ^K50RExpression and the expression that drives of constitutive expression plasmid and the expression that comprises the plasmid of total length B29 promoter compare.

Embodiment 19:In vivo JETPEI ^TMNanoparticle forms

Providing this embodiment is in order to form In vivo JETPEI ^TMThe nanoparticle complex, so that with 1.5mg/kg (0.1mL)--1.5mg/kg=1.0mg pExp5A/kg+0.5mgh5A1/kg--DNA: siRNA=2: 1 dosage is injected the mice to 20g.

Plasmid DNA and siRNA are diluted to cumulative volume 25ml.Use sterilized water to adjust volume. ^*(10ml, 2mg/ml) (10ml 1mg/ml) is diluted to cumulative volume 25ml with 10mg siRNA with the 20mg plasmid DNA.Use the sterilized water part of covering the shortage.10% glucose (PEI kit provides) that adds 25ml is regulated the dna solution volume to 50ml 5% glucose.Of short duration centrifugal behind the vortex gently.With In vivo JETPEI ^TMDilution is the aqueous solution of cumulative volume 25ml. ^*With 3.6mlIn vivo JETPEI ^TMDilution is the aqueous solution of cumulative volume 25ml.Regulate final volume with 10% glucose and be adjusted to 50ml, final concentration is 5% glucose.Of short duration centrifugal behind the vortex gently.Immediately the PEI of 50ml dilution being added to the 50ml dilution DNA (could not transpose! ).Rotation is slowed down immediately behind the of short duration vortex.

After complex forms, leave standstill and stablized in 8-10 hour.The ratio of complex N/P is 6.The ratio of N/P is an In vivo-jetPEI positive charge nitrogen residue number and the ratio of DNA and siRNA negative charge phosphoric acid radix.Every gram DNA and siRNA comprise the phosphate of similar number.Therefore, the ratio of N/P reflection complex intermediate ion balance.The ratio that improves complex N/P can improve the toxicity of complex.The 150mM solution (expression nitrogen residue) of In vivo JET-PEI is provided, and 1mgDNA comprises 3nmole anion phosphate simultaneously.The DNA final concentration is no more than 0.5mg/ml in the final volume.DNA should be high-quality and prepare in water.In vivo-jetPEI and 10% glucose to room temperature are used then.

Embodiment 20: carry out dosage range and the repeated doses research that intravenous gives SNS01 and SNS-EF1/UU mice.

SNS01 is one embodiment of the invention, it be a kind of biology target be to treat the treatment of cancer method of multiple myeloma.SNS01 is grouped into by three kinds of one-tenth: the dna vector (referring to Figure 22) of expressing the short apoptosis mutant of eIF5A; Targeting promotes the siRNA (referring to the sequence of Figure 25) of the natural eIF5A of growth of cancer cells/anti-apoptosis; And as synthetic polymer (the In vivo-jetPEI of the polyaziridine by name of delivery media; Polyplus Transfection Inc.).

This research purpose is to determine that maximum tolerated dose and long-term intravenous give the feasibility of the SNS01 of mice therapeutic dose.Carry out two independently researchs.Maximum tolerated dose (research ID:MTD) research is research in 8 days, wherein accepts the SNS01 (from 2.2mg/kg-3.7mg/kg) that two agent doses increase progressively in the mouse vein, by monitoring clinical symptom, body weight, organ weight's regulating liver-QI enzyme evaluate toxicity.Repeated dose study of 9-week (research ID:EX6) is intended to estimate the toxicity that gives for a long time for 2 times weekly behind therapeutic dose (1.5mg/kg) SNS-EF1/UU and the toxicity of heterogeneity thereof.SNS-EF1/UU be a kind of clinical before type SNS01, difference mainly is by constitutive promoter (always institute the promoter that reaches of invading the exterior) in a organized way non-B-cell-specificity promoter driving eIF5A in the SNS01 complex ^K50RExpression.In SNS01, use B cell-specific b 29 promoteres, be intended in the cell of the B-cell source that comprises the myeloma cell, express by limiting short apoptosis eIF5A mutant, thus the safety of enhancing therapeutic agent.EX6 research also comprises one group of mice: wherein with the eIF5A that determines the inhibition mouse tissue whether any toxic action is arranged with mice-specificity eIF5A siRNA administration.By monitoring the toxicity in clinical symptom, body weight, hematology, liver enzyme and the research of histopathological evaluation repeated doses.

Experiment shows before clinical, and the therapeutic dose of SNS01 is 0.75mg/kg-1.5mg/kg (research EX9).Seek (research ID:MTD) in the research at 8 days dosage range, carried out dosage test, to measure the upper limit of dosage range apparently higher than the therapeutic dose scope.Be subjected to test product weekly the dosage that gives of 2 intravenouss when the low dosage level of 2.2mg/kg and 2.9mg/kg, be fully tolerance, but have a mice morbid state to occur and implemented euthanasia in 2.9mg/kg.Survival rate is approximately 20-25% when 3.3mg/kg or the above dosage of 3.3mg/kg.Therefore, maximum tolerated dose is between 2.2mg/kg and the 2.9mg/kg, and it is higher than therapeutic domain 0.75mg/kg-1.5mg/kg far away.

In 9 all repeated doses researchs (research ID:EX6), mice is accepted the SNS-EF1/UU of tail vein injection therapeutic dose (1.5mg/kg) weekly for 2 times, does not observe the toxic reaction relevant with being subjected to test product during studying.In this research, also separately tested DNA and siRNA, the two all is fully to tolerate for mice.Because people eIF5A siRNA does not have activity to mice, therefore, this research also comprises mice eIF5A-specific siRNA.In 9 weeks, do not observe and give mice eIF5A siRNA relevant toxic reaction for a long time.

These results show, even give the SNS01 and the SNS-EF1/UU of mice therapeutic dose for a long time, also all be do not have toxic.

Be subjected to test product and medium

Test macro and research design

The University of Waterloo Animal Care Committee (Waterloo that the various aspects of this research are all set up according to Canadian Council on Animal Care and Province of On tarioAnimals for Research Act, Ontario, Canada) guilding principle of Zhi Dinging is carried out.

CD-1 and BALB/c mouse derive from Charles River Laboratories (Quebec, Canada).The mice of two groups of researchs is all accepted weekly, and 2 tail vein injections are subjected to test product.Use slowly injection (about 2-3 minute) so as delivery volume greater than 0.2ml.

The all ages of mice about 6-9 when research begins that are used for research in 8 days.The all ages of mice about 5-6 when research begins that are used for 9 all repeated doses researchs.

Maximum tolerated dose research (MTD) in 8-days

Two doses of researchs in 8 days are the research of seeking dosage range, are intended to determine the maximum tolerated dose of SNS01.Dosage range is 2.2mg/kg-3.7mg/kg, and it is far longer than the therapeutic dose scope of 0.75mg/kg-1.5mg/kg.During the lowest dose level of SNS01 (2.2mg/kg), do not observe the clinical toxicity symptom, exception be to have the slight wrinkling and activity of mice performance fur to reduce (these symptoms disappeared in 1 hour) a little.Behind the SNS01 of the 2nd injection 2.2mg/kg, do not observe the clinical toxicity symptom.All mice body weight remain unchanged in whole research.Organ does not have macroscopic variation.With respect to matched group, except that liver weighs: the ratio moderate of body weight increased, the organ weight did not change with the ratio of body weight.Yet, increase owing in any higher dose levels group, observe this ratio, thus unlikely be subjected to test product relevant.

The SNS01 dosage of 4/5 mice tolerance 2.9mg/kg, no clinical toxicity symptom.Yet in back 1 hour of injection, a mice is had a convulsion and slight respiratory distress, is sentenced euthanasia by hommization then.All the other mices are carried out not observing the clinical toxicity symptom after the 2nd the SNS01 injection.The weight of mice remains unchanged in whole research, does not have macroscopic variation in the organ, and perhaps the organ weight does not change with the ratio of body weight.Just Serum ALT levels has slight increase behind the SNS01 of two doses of 2.9mg/kg dosage.

As expection, SNS01 toleration when 3.3mg/kg or the above dosage of 3.3mg/kg is not good, and all mices (except 1) have no alternative but be sentenced euthanasia by hommization in two groups.Mice ill clinical symptoms occurs and sentenced by hommization under all situations of euthanasia, and clinical symptoms all occurs in back 1 hour of injection, and is consistent with other report research of using high dose PEI.Though 3.3mg/kg and 3.7mg/kg survival mice are not accepted dosage the 2nd time, in back 4 hours of injection, recover fully and in whole research, remain its body weight.Therefore the maximum tolerated dose of SNS01 is between 2.2mg/kg and the 2.9mg/kg.

9-week repeated doses research (EX6)

The purpose of 9-week repeated doses research is to assess to give therapeutic dose (1.5mg/kg) SNS-EF1/UU the safety of (at SNS01 a kind of complex that is used for preclinical study in development period) for a long time.SNS-EF1/UU obviously is different from SNS01, and the main distinction is that the SNS-EF1/UU material is research grade and eIF5A ^K50RExpression is by all having active composing type people EF1 promoters driven for all cells type.Although SNS01 uses B-cell-specificity promoter to drive eIF5A ^K50RExpress, but in this safety research, use constitutive promoter can assess because sudden change eIF5A ^K50RProtein is accumulated the toxicity that produces in non--B-cell tissue.On the other hand, 9-week repeated doses research comprises the experimental group of the various component safeties of testing SNS-EF1/UU.Give plasmid that DNA group (Ex6-G3) comprises eIF5A and the complex of non-targeting contrast siRNA, give the complex that siRNA group (Ex6-G4) comprises people eIF5A (h5A1) siRNA and non-expression plasmid simultaneously.Do not influence the people eIF5A siRNA that endogenous mouse eIF5A expresses because comprised by test product SNS-EF1/UU, so another feature of this research is the group (Ex6-G6) that comprises the PEI complex of the siRNA that comprises non-expression plasmid and efficient targeting mice eIF5A.This group can be assessed the safety that gives active eIF5AsiRNA for a long time.

All animals survived are to the predetermined date of putting to death.In 9 all research process, all do not observe the clinical toxicity symptom in any group, and the mice body weight of all groups continues to increase in research process.3 week and 6 weeks after initial therapy, monitoring erythrocyte and leukocyte count, the erythrocyte of all dosage groups and leukocyte count all are normal.Measure the serum liver enzyme level after putting to death mice, the serum liver enzyme level of all mices is all in normal range.All do not observe macroscopic organ cosmetic variation in any group.Independent pathology expert has carried out histopathological analysis to major organs, shows to be subjected to the test product can toxigenicity.

The SNS-EF1/UU toleration that gives the mice therapeutic dose for a long time well and is not observed side effect.In addition, give mice-specificity eIF5A siRNA for a long time and show non-toxic reaction, show that the PEI complex that administration of human comprises people eIF5A siRNA should be safe.

Embodiment 21: to using the therapeutic efficiency research of SNS-B29/UU and SNS01 in the mouse vein that has multiple myeloma.

SNS01 as mentioned above.Being subjected to test product SNS-B29/UU is clinical preceding type SNS01.SNS-B29/UU and SNS01 difference are minimum, and the main distinction is that each component is research grade but not GLP-level.The research purpose of this paper report is to measure the minimum effective dose of SNS-B29/UU, and conclusive evidence comprises the GLP-level material of SNS01 and the same the working of research grade material of preclinical study use.Repeated doses tumor research (research ID:EX9) is 5-week research: wherein assessment reduces the ability that SNS-B29/UU dosage suppresses mice Subcutaneous tumor growth weekly for 2 times gradually, is in order to measure the optimal therapeutic dosage of SNS01.Equally, assess the signs of toxicity of treatment animal by monitoring clinical symptoms, body weight and organ weight.

In the SCID of subcutaneous carrier's multiple myeloma mice, measure the therapeutic dose scope of SNS-B29/UU.The dosage of test SNS-B29/UU is between 0.15mg/kg and 1.5mg/kg.Determined by measuring gross tumor volume weekly for 2 times by the antitumor efficacy of test product, and the tumor resection tissue is weighed after putting to death mice.0.75mg/kg cause obvious tumor to reduce with the SNS-B29/UU dosage of 1.5mg/kg, the therapeutic dose scope that shows SNS-B29/UU is between 0.75mg/kg and 1.5mg/kg.Also observe effective inhibition Subcutaneous tumor growth, but not observing tumor reduces in the SNS-B29/UU of 0.38mg/kg.The dosage that is low to moderate the SNS-B29/UU of 0.15mg/kg is even observed certain tumor growth inhibition, shows the therapeutic dose wide ranges.Referring to Figure 26 and 27.

To use the SNS01 of GLP-level composition preparation and the effect contrast of SNS-B29/UU to find, both effects aspect the inhibition tumor growth are suitable.The toleration for the treatment of the SCID mice of carrying tumor with SNS01 and SNS-B29/UU is good, and mice body weight in whole research continues to increase.

Be subjected to test product and medium

Test macro and research design

(Indianapolis, IN USA) obtain female C.B.17/IcrHsd-Prkdc (SCID) mice from Harlan.Form Subcutaneous tumor: with 10x10 ⁶Individual survival KAS-6/1 (people's multiple myeloma) injection cell is gone into the right rib side of body of 5-6 mice in age in week.When tumor reaches about 20-40mm ³When size (about 4 weeks behind the injection tumor cell) used the SNS-B29/UU begin treatment.When tumor reaches about 130mm ³(about 6 weeks behind tumor cell injection) use the SNS01 begin treatment.Mice is accepted weekly, and 2 interior injections of tail vein are subjected to test product.

The repeated doses tumor research

The repeated doses tumor research is intended to determine the minimum dose therapeutically effective of SNS-B29/UU, and proved by test product SNS-B29/UU by research grade to confirm that GLP-level SNS01 is subjected to test product to keep tumors inhibition activity.Next is any toxic reaction that clinical symptoms, body weight and the organ weight of monitor therapy mice assesses treatment.Thereby use digital calipers to measure the anti-tumor activity that the monitoring of gross tumor volume value is subjected to the test product treatment weekly for 2 times.Putting to death the back tumor resection weighs.

All mice survivals are put to death day to predetermined.With the PEI nano-complex treatment control mice that comprises non-expression plasmid and non-targeting siRNA, the mean tumour volume during execution is 284mm ³, and only be 13mm with the mean tumour volume of the mice of 1.5mg/kg SNS-B29/UU treatment ³, tumor growth reduction by 95% ( ^*P=0.026).Yet when attempting to excise the tumor of the mice for the treatment of with 1.5mg/kgSNS-B29/UU, all mices are not all found tumor.Reduce SNS-B29/UU dosage half during to 0.75mg/kg, also cause respectively gross tumor volume reduce 91% ( ^*P=0.03) and tumor weight alleviate 87% ( ^*And the tumor complete obiteration of a mice arranged p=0.04).Therefore, the optimal therapeutic dosage of 2 injection SNS-B29/UU be it seems between 0.75mg/kg and 1.5mg/kg weekly.Give SNS-B29/UU dosage weekly for 2 times and be low to moderate 0.15mg/kg and still cause last gross tumor volume to reduce by 60%, show that SNS-B29/UU has the effective antitumor activity in the broad dosage range.

Except that suppressing tumor growth, also cause obviously reducing of gross tumor volume with SNS-B29/UU and SNS01 with 0.75mg/kg and 1.5mg/kg treatment, show that this treatment can induced tumor decline, inducing apoptosis of tumour cell probably.The mice of carrying tumor changed with the percentage ratio of the gross tumor volume of dosage level 0.75mg/kg and 1.5mg/kg treatment with SNS-B29/UU be respectively-244% and-245%.In the identical time, the tumor of control mice size increases above 2000%.2 injection SNS01 equally obviously dwindle multiple myeloma weekly.Percentage ratio with the gross tumor volume of the mice of 1.5mg/kgSNS01 treatment is changed to-349%, shows that SNS01 and SNS-B29/UU effect are same.In fact use GLP-level material can increase biological activity, because with SNS01 treatment only after 25 days, gross tumor volume just reduces by 349%, and the SNS-B29/UU treatment is after 35 days, and gross tumor volume has reduced by 245%.In addition, the tumor with the SNS01 treatment is sizable (about 130mm ³), show that the tumor that fully forms with the SNS01 treatment is effective.

All mice is good to the toleration of treatment, does not observe the clinical toxicity symptom.In whole research, all group mices continue to increase body weight.Do not have macroscopic organ cosmetic variation during autopsy, and significant change does not take place in the ratio of organ weight and body weight.

Therefore, when 2 intravenous injections were sent weekly, the SCID mice was good to the toleration of SNS01 (with its clinical preceding type SNS-B29/UU), and extremely effectively treats the Subcutaneous tumor of people's multiple myeloma.All of SNS-B29/UU are subjected to amount of reagent all effectively to suppress tumor growth, but maximum dose level 1.5mg/kg successfully eliminates the tumor of all mices of receiving treatment.

Embodiment 22: distribute in the body of plasmid DNA and siRNA polyaziridine (JetPEI) complex

Green fluorescent protein (" GFP ") GFP-expression construct is used to measure the location of the plasmid DNA that the PEI complex sends.Use 2 kinds of promoters driven GFP to express: EF1, omnipresence promoter (EF1::GFP); Or B29, B-cell specificity promotor (B29::GFP).The PEI complex that comprises 20 μ g GFP plasmid DNA and 10 μ g fluorescently-labeled (DY547) h5A1 siRNA with N/P than=6 preparations.Continuous 2 days to injection 5% glucose or PEI complex in the BalB/C mouse vein.Mice is put to death in back 72 hours of the 1st injection, gathers in the crops its organ, analyzes GFP with confocal microscopy and expresses and DY547-siRNA.

Bone marrow: under most of situation, have DY547-siRNA still not have the evidence that GFP expresses.The time and the GFP expression peak that may gather in the crops organ are inconsistent; The GFP signal might quencher or not expression of GFP.Yet, observe and navigate to myelocytic GFP of same bone and DY547 in some cases altogether.Therefore, but this proof intravenous injection gives the medullary cell of PEI nanoparticle transfection living animal.

Lung: under most of situation, have DY547-siRNA still not have the evidence that GFP expresses.It is inconsistent that the time of results organ and GFP express the peak, and perhaps the GFP signal might quencher or not expression of GFP.

Spleen: visible GFP expresses (when the EF1 promoters driven) and is positioned the evidence that there is cell in the DY547-siRNA positive altogether.When the B29 promoters driven, being expressed in the splenocyte of GFP is very low.This shows that the PEI nanoparticle seems the transfection splenocyte.

Kidney: do not observe GFP or DY547, show that nanoparticle may not enter kidney.

Liver: under most of situation, have DY547-siRNA still not have the evidence that GFP expresses.This provides evidence for PEI nanoparticle transfection hepatocyte.

Heart: visible EF1::GFP and DY547-siRNA are positioned heart tissue altogether, but therefore show this organ of PEI nanoparticle transfection.Do not observe GFP with the B29 promoter.

The comparison HA-eIF5A of embodiment 23:DNA: siRNA ^K50RThe influence of expressing.

With the KAS cell with comprising B29-HA-eIF5A ^K50RThe nanoparticle transfection of (by the plasmid of B-cell-specificity promoter driving) and h5A1siRNA.Preparation comprises the pExp5A of different proportion and the JET PEI of h5A1siRNA ^TMNanoparticle in room temperature incubation 4 hours, adds to the KAS cell then.After the transfection 4 hours, the culture medium that will comprise nanoparticle was replaced by fresh culture.After 24 hours, the harvesting lysate uses the antibody at HA to carry out western blot analysis.The ratio of DNA: siRNA is more different than 2: 1 with standard.HA-eIF5A ^K50RReached the accumulation summit at 1: 0,3: 1 and 2: 1 o'clock in ratio.Referring to Figure 30.

The inductive apoptotic influence of comparison nanoparticle transfection of embodiment 24:DNA: siRNA.

Preparation comprises the nanoparticle of pExp5A and h5A1siRNA of different proportion, in room temperature incubation 4 hours, adds to the KAS cell then.After the transfection 4 hours, the culture medium that will comprise nanoparticle was replaced by fresh culture.After 48 hours, harvesting is analyzed with Annexin V/PI labelling and by FACS.Have DNA in nanoparticle: when siRNA was 2: 1 a standard proportional, inductive apoptosis was the highest in its cells transfected.Referring to Figure 31.

Embodiment 25: comprise eIF5A1 ^K50RThe PEI complex (N/P=6 or 8) of plasmid and eIF5A1 siRNA (siSTABLE or non-siSTABLE) suppresses the growth of multiple myeloma Subcutaneous tumor and causes tumor to reduce.

To SCID mouse subcutaneous injection KAS cell.Begin treatment when observing palp tumor.To mice 2 following complex of intravenous injection weekly: (G1) comprise the pCpG-mcs (empty carrier) of 20mg and the PEI complex of 10mg contrast siRNA, N/P=8 (moderate); (G5) comprise the RNAi-r plasmid pCpG-eIF5A1 of 20mg ^K50RWith the PEI complex of the siSTABLE h5A1siRNA of 10mg, and N/P=8 (moderate, siSTABLE); (G8) comprise the RNAi-r plasmid pCpG-eIF5A1 of 20mg ^K50RWith the PEI complex of the h5A1siRNA of 10mg, and N/P=6 (moderate, N/P=6).Shown in data are single gross tumor volumes of every group of mice.After initial therapy, gave last injection in 40 days.Referring to Figure 32.

Embodiment 26:JET PEI ^TMNanoparticle is effectively absorbed by tumor tissues and nanoparticle is delivered to identical cell with plasmid with siRNA.

Inject back 48 hours making tumor biopsies with the nanoparticle that comprises pExp-GFP (GFP under the control of B cell specificity promotor) and DY547-siRNA (fluorescently-labeled siRNA).According to confocal microscopy, in tumor biopsy, observe localized altogether GFP and DY547 and express, show that nanoparticle is effectively absorbed by tumor tissues and nanoparticle is delivered to identical cell with plasmid with siRNA.Referring to Figure 33.

Embodiment 27: adenovirus construct carries out truncate research.

Adenovirus (serotype 5, E3-disappearance)

1) Ad-eIF5A1 Δ (2-6) [Δ (2-6)]-this is the people eIF5A1 that lacks aminoacid 2-6.

2) Ad-eIF5A1 ^K50RΔ (2-6) [Δ (2-6)/K50R]-this is the people eIF5A1 that lacks aminoacid 2-6.In addition, " K50R " is meant that 50 lysines (K) have sported arginine (R), therefore it is believed that it can not be by DHS hydroxyl fourth lysineization.

3) Ad-eIF5A1D6E[D6E]-this is people eIF5A1, wherein expects cleavage site suddenly change (D6 sports E).

4) Ad-eIF5A 1D6E/K50R[D6E/K50R]-this is people eIF5A1 ^K50R, wherein expect cleavage site suddenly change (D6 sports E).In addition, " K50R " is meant that 50 lysines (K) have sported arginine (R), and therefore it is believed that can not be by DHS hydroxyl fourth lysineization.

Embodiment 28: the eIF5A cracking of caspase mediation

In order to identify cleavage site, to from the KAS cell of actinomycin D treatment, separate (Figure 37) by the 2-D gel electrophoresis by isolating protein cleavage thing, downcut and the corresponding speckle of eIF5A pyrolysis product from gel, by mass spectrum order-checking (Figure 38 B) than small-molecular weight.Although do not obtain the pyrolysis product of full length sequence, determine that cutting occurs in (Figure 38 A) after terminal the 6th amino acids of protein N.The sequence that was right after before the cleavage site of being suspected is ' DDLD ', it is the sequence (Chayet al., 2002) that has been accredited as the caspase cleavage site.

For whether the generation of determining crack fragment is that caspase is dependent, in human myeloma cell during the actinomycin D cell death inducing, the ability (Figure 41) that test caspase inhibitor stops crack fragment to produce.The strong all prevention of general caspase inhibitor and specificity caspase 3,8 and 9 inhibitor forms cutting type eIF5A, and cutting type eIF5A accumulates during the inductive KAS apoptosis of ActD-.Caspase 1 inhibitor is same to be reduced but the non-formation that stops pyrolysis product fully.Not only stop the accumulation of pyrolysis product with caspase inhibitor incubation, and suppress the loss (Figure 41) of the eIF5A of hydroxyl fourth lysine form, show that the hydroxyl fourth lysine eIF5A loss in actinomycin D-cell death inducing is cracked result.

Protein caspase cracking during the apoptosis is (referring to the summary of Fisher etc., 2003) may have following multiple purpose: 1) short survivin matter of inactivation or anti-apoptotic proteins matter-for example: eukaryotic cell translation initiation factor 4G1 (it is in conjunction with 5 ' cap of mRNA and promote the mRNA that adds medicated cap combine with 40S ribosomal subunit) is by caspase cracking inactivation, thereby inhibition is translated; 2) protein of setting up the negative form of dominance (for example, thereby NF-κ B p65 produces dominant negative fragment by the caspase cracking during apoptosis, it does not still have transactivation activity in conjunction with DNA, therefore as the negative inhibitor of the dominance of NF-kB); 3) the acquired cracking of function, cause activating pro apoptotic protein by removing inhibitory removal domain or modulability domain, therefore cause having new activity or increase active fragment and (for example form, BRCA-1, a kind of mediated cell cycle stops and apoptotic breast carcinoma Profilin matter, and it discharges short apoptosis crack fragment by the caspase cracking and is activated.DAP5, it is one of member of eIF4G family, it is activated by caspase cutting back; The translation of pyrolysis product irritation cell apoptosis-related protein matter); 4) transforming anti-apoptotic proteins is that (for example, inhibitors of apoptosis is as BCl-x for pro apoptotic protein _LAnd c-IAP1, become pro apoptotic protein by the caspase cracking); With 5) cell reallocation (for example, after by caspase 8 cracking, the short apoptosis p15 fragment of Bid carry out myristoylization on the glycine residue that cracking exposes, and it makes the Bid transposition to mitochondrion subsequently, and it improves cytochrome c release at mitochondrion).

For the eIF5A cracking of determining the caspase-mediation in actinomycin D-inductive apoptosis is that common phenomenon or myeloma cell are specific, check whether the HeLa cell of handling with lonizing radiation rhzomorph D exists cutting type eIF5A (Figure 42).In the HeLa cell that lonizing radiation rhzomorph D-handles, do not observe the accumulation of eIF5A crack fragment.In order to determine whether eIF5A post translational modification (for example phosphorylation or acetylation) may be the cracked prerequisite of caspase, check the influence that the deacetylase inhibitors nicotiamide forms crack fragment.Our laboratory and other laboratory (Kimet al., 2006) have been observed the acetylation of eIF5A by mass spectral analysis eIF5A.In addition; identified that yeast eIF5A is substrate (the Shiraiet al. of the relevant deacetylase Hst2 of Sir2-; 2008); the author notice just the yeast that does not have Hst2 or wherein eIF5A cross the acetylation of observing eIF5A in the yeast of expression, show that eIF5A exists with non-acetylated form usually.HeLa cell and Sir2 deacetylase inhibitors nicotiamide that incubation lonizing radiation rhzomorph D handles cause the accumulation of eIF5A pyrolysis product, show that the cracking of generation caspase-mediation needs the eIF5A acetylation.These data show that before the cracking that apoptotic signal triggers the acetylation of eIF5A and makes caspase mediate took place, possibility eIF5A was protected and avoids by the caspase effect usually.

List of 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?update?of?caspase?substrates.Cell?Death?and?Differentiation?10，76-100。

Kim，S.C，Sprung，R.，Chen，Y.，Xu，Y.，Ball，H.，Pei，J.，Cheng，T.，Kho，Y.，Xiao，H.，Xiao，L.，Grishin，N.V.，White，M.，Yang，X.J.，and?Zhao，Y.(2006)Substrate?and?Functional?Diversity?of?Lysine?Acetylation?Revealed?by?a?Proteomics?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。

Claims (57)

1. proteinic eIF5A1 polynucleotide of the truncated-type eIF5A1 that encodes are used for inducing the purposes of the apoptotic medicine of patient's cancerous cell or tumor in preparation.

2. the purposes of claim 1, wherein said eIF5A1 polynucleotide encoding truncated-type eIF5A1 protein, this protein comprises the aminoacid sequence shown in the SEQ ID NO:37, as shown in figure 38.

3. the purposes of claim 1, the truncate eIF5A1 protein of the about 16kDA of wherein said eIF5A1 polynucleotide encoding.

4. the purposes of claim 1, wherein said patient is a mammal.

5. the purposes of claim 4, wherein said mammal is the people.

6. the purposes of claim 1, wherein said inducing cancer cell or tumor death delay cancerous cell or tumor growth, prevention cancerous cell or growth of tumour cell, perhaps kill cancer cell or reduce gross tumor volume.

7. the purposes of claim 1, wherein said cancer is a multiple myeloma.

8. the purposes of claim 1, wherein said eIF5A1 polynucleotide comprise the sequence shown in the SEQ ID NO:38, as shown in figure 41.

9. the purposes of claim 1, wherein said eIF5A1 polynucleotide are included in plasmid or the expression vector.

10. the purposes of claim 1, wherein said expression vector is adenovirus expression carrier or pHM6.

11. the purposes of claim 10, wherein said expression vector comprises tissue-specific promoter.

12. the purposes of claim 11, wherein said tissue-specific promoter is the B cell specificity promotor.

13. the purposes of claim 12, wherein said B cell specificity promotor is B29.

14. the purposes of claim 10, wherein said expression vector comprises the pCpG plasmid.

15. the purposes of claim 10, wherein said expression vector and polyaziridine are compound.

16. the purposes of claim 10, wherein said expression vector have the CpG dinucleotide of minimizing.

17. the purposes of claim 1, wherein said medicine in tumor, intravenous or subcutaneous giving.

18. isolating polynucleotide, its coding truncated-type eIF5A1 protein, wherein said polynucleotide comprise the sequence shown in the SEQ ID NO:38, as shown in figure 41.

19. isolating polynucleotide, its coding truncated-type eIF5A1 protein, wherein said truncated-type protein comprises the aminoacid sequence shown in the SEQ ID NO:37, as shown in figure 38.

20. an isolating truncated-type eIF5A1 polypeptide, its eIF5A1 cutting by the caspase mediation forms.

21. coding proteinic eIF5A1 polynucleotide of truncated-type eIF5A1 and the combination of total length eIF5A1 polynucleotide are used for preparing the purposes of the medicine of inducing patient's cancerous cell or apoptosis of tumor cells.

22. the purposes of claim 21, wherein said eIF5A1 polynucleotide encoding comprises the truncate eIF5A protein of the aminoacid sequence shown in the SEQID NO:37, and wherein total length eIF5A1 polynucleotide encoding comprises the protein of the aminoacid sequence shown in the SEQ ID NO:35.

23. the purposes of claim 21, the truncate eIF5A protein of the about 16kDA of wherein said eIF5A1 polynucleotide encoding.

24. the purposes of claim 21, wherein said patient is a mammal.

25. the purposes of claim 24, wherein said mammal is the people.

26. the purposes of claim 21, wherein said apoptosis-inducing delay cancerous cell or tumor growth, prevention cancerous cell or growth of tumour cell or kill cancer cell or reduce gross tumor volume.

27. the purposes of claim 21, wherein said cancer is a multiple myeloma.

28. the purposes of claim 21, wherein said eIF5A1 polynucleotide comprise the sequence shown in the SEQ ID NO:38, and wherein total length eIF5A1 polynucleotide comprise the sequence shown in the SEQ ID NO:43, shown in Figure 53.

29. the purposes of claim 23; wherein said total length eIF5As polynucleotide encoding sudden change eIF5A1, wherein said sudden change prevents or the hydroxyl fourth lysine that suppresses deoxidation hydroxyl fourth lysine synthase turn into and/or wherein said sudden change be present in ubiquitination site and/or acetylation site.

30. the purposes of claim 23, wherein said sudden change is selected from: K50A, K50R, K67A, K47R, K67R, K50A/K67A, K50A/K47R, K50A/K67R, K50R/K67A, K50R/K47R, K50R/K67R and K47A/K67A.

31. the purposes of claim 29, it also comprises the siRNA of the 3 ' UTR of targeting eIF5A.

32. the purposes of claim 31,5 '-GCT GGA CTC CTC CTA CAC A-3 ' sequence of wherein said siRNA targeting eIF5A1.

33. the purposes of claim 32, wherein said siRNA is dsRNA, and the chain of dsRNA comprises sequence 5 '-GCU GGA CUC CUC CUA CAC A-3 '.

34. the purposes of claim 32, wherein said siRNA stabilisation are degraded in serum preventing.

35. the purposes of claim 31, wherein said total length eIF5A1 polynucleotide and/or the proteinic eIF5A polynucleotide of coding truncated-type eIF5A1 are present in the expression vector.

36. the purposes of claim 35, wherein said total length eIF5A1 polynucleotide and/or the coding proteinic eIF5A polynucleotide of truncated-type eIF5A1 and/or siRNA and polyaziridine are compound.

37. the purposes of claim 36, wherein said total length eIF5A1 polynucleotide and/or the coding proteinic eIF5A polynucleotide of truncated-type eIF5A1 and/or siRNA are independent compound with polyaziridine.

Thereby 38. one kind induce the apoptotic method of mammalian cancer cells or tumor by offering the compositions of any one among the mammal claim 1-38.

39. the method for claim 38, wherein said compositions gives in intravenous, intraperitoneal or tumor.

40. the method for claim 38, wherein said cancer is a multiple myeloma.

41. compositions, its comprise targeting eIF5A1 3 ' terminal eIF5A1 siRNA, comprise the complex of expression vector of the polynucleotide of encoding mutant eIF5A1, wherein said sudden change eIF5A1 can not be by hydroxyl fourth lysineization, and wherein said siRNA and expression vector and polyaziridine are compounded to form complex.

42. a compositions, it comprises the siRNA of targeting target gene with the endogenous expression of inhibition patient target gene; With can be with RNAI r plasmid coding at the polynucleotide of the target protein of patient's expression in vivo, wherein said siRNA and plasmid and polyaziridine are compounded to form complex.

43. the compositions of claim 41, wherein said siRNA has sequence shown in Figure 25, and wherein the polynucleotide of encoding mutant eIF5A1 are eIF5A1 ^K50R

44. the compositions of claim 43, it comprises tissue-specific promoter.

45. the compositions of claim 44, it comprises the B cell specificity promotor.

46. the compositions of claim 45, wherein said B cell promoter is B29.

47. the compositions of claim 43, wherein said expression vector comprises the pCpG plasmid.

48. the compositions of claim 41, wherein said eIF5A1 siRNA is independent compound with polyaziridine with the expression vector that comprises sudden change eIF5A1 polynucleotide.

49. the compositions of claim 41, wherein said eIF5A1 siRNA is combined with each other with the expression vector and the polyaziridine that comprise sudden change eIF5A1 polynucleotide.

50. compositions, it comprises the eIF5A1 siRNA and the expression vector that comprises the polynucleotide of encoding mutant eIF5A1 of the 3 ' end of targeting eIF5A1, wherein said sudden change eIF5A1 can not be by hydroxyl fourth lysineization, and wherein said siRNA and expression vector are delivered to the patient with the treatment cancer.

51. the compositions of claim 50, wherein said cancer is a multiple myeloma.

52. a treatment method for cancer, it comprises the compositions that gives patient's claim 50.

53. a treatment method for cancer, it comprises the compositions that gives patient's claim 41.

54. the method for claim 52, wherein said compositions gives in intravenous, intraperitoneal or tumor.

55. the method for claim 53,3 ' the terminal siRNA of wherein said targeting eIF5A1 sends through different approaches with the expression vector that comprises the polynucleotide of encoding mutant eIF5A1.

56. the method for claim 52, wherein said compositions 2 dosage injections with the about 1.5mg/kg of about 0.15mg/kg-weekly give.

57. the method for claim 52, wherein said described compositions 2 dosage injections with the about 1.5mg/kg of about 0.75mg/kg-weekly give.

Images