795 Efficient In Vivo Transduction Using RNA Replicon Vector Derived from Sendai Virus Self Replicating Ribonucleoprotein Complexes such as intcrleukin 12, in several tumor models It was of great inte[.]
such as intcrleukin 12, in several tumor models It was of great interest to study if SPV vectors could escape tumors after intratumoral injection and if they could target tumor nodules by systemic administration We analyzed the biodistribution of an SPV vector expressing luciferase (SFV-Luc) after intravenous, intraperitoneal, or intratumoral administration in immunocompetent mice SFV-Luc systemic inoculation led to high infectivity in heart and lung, and medium infectivity in spleen, kidney, and gonads, without gender influence Tumor specific infection was only achieved by intratumoral inoculation, without vector spreading to other tissues We also investigated the effect of SFV preinoculations on subsequent vector administrations Systemic inoculation with one dose of 1O'vp (viral particles) or two doses of 106vp ofSFV-LacZ were able to strongly inhibit luciferase expression in animals reinoculated systemically with SFV-Luc, which correlated with high sera neutralizing antibodies titers However, intratumoral preinoculation with up to lO'vp of SPV-LacZ only moderately impaired tumor reinfection, indicating that it could be feasible to treat tumors with several doses of SPV vectors 793 Integration-Defective Lentiviral Vectors as a Platform for Sleeping Beauty TransposaseMediated Gene Insertion Nicklas H Staunstrup, Maria Jakobsen, Brian Moldt, Jacob G Mikkelsen 'Human Genetics, University ofAarhus Aarhus, Denmark Sleeping Beauty (SB) is a widely studied Tc I/mariner-like transposon reconstructed from ancient salmonid fish genomes The binary SB-derived vector system is comprised of an integrating part, the transposon, and the ancillary enzyme, the transposase SB transposes efficiently in a wide range of cells and integrates with no observed bias towards any specific region of the genome SB demonstrates long-term transgene expression and thereby circumvents a primary limitation of non-transposon, DNA-mediated gene delivery However, as with other non-viral systems, SB is inferior to the viral vectors in terms of DNA delivery In an attempt to overcome this shortcoming, we have studied the possibility of combining SB with an integration-disabled lentiviral vector aiming at the creation of a hybrid viral vector that facilitates transposasedriven gene insertion We first generated SIN HIV-I lentiviral (LV) vectors containing the left and right inverted repeats (IRs) ofSB as well as a puro" expression cassette In plasmid transfection assays based on co-delivery with either a hyperactive variant ofSB (HSB3) or an inactive mutant (mSB) as a control, the SB/LV vector proved as a highly effective substrate for transposition Furthermore, in transduction assays the titer of vectors containing the SB IRs was reduced only -5-fold compared to the wildtype vector By Southern blot analyses ofHIRT-extracted episomal DNA we detected circular I-LTR and 2-LTR lentiviral DNA episomes, considered dead-end by-products of lentiviral infection Notably, we found an increased level of circles generated during transduction of integrase-deficient vectors compared to integration-proficient vectors This is of major importance, since circular transposition substrates, in contrast to linear substrates, are believed to facilitate efficient SB transposition In initial assays SB/LV-transduced cells were transfected with transposase-expressing plasmid DNA Preliminary findings indicate that HSB3 elevates the Icvel of circle integration above the level obtained with the inactive mSB control However, the transduction efficiency is still reduced compared to integration-proficient lentiviral vectors We are currently studying transposase delivery in the context of integration-defective lentiviral vectors In addition, in an effort to selectively exclude background insertions of integration-defective lentiviral vectors we have produced vectors that express thymidine kinase from viral insertions but not from transposase-mediated insertions Molecular Therapy Volume15 Supplement ~ br 2007 Copyright © T he American Socie ty o r G ene "1l1f:r:lpy 794 Development of Novel Defective Sendai Virus Vectors Capable of Expressing Therapeutic Genes Persistently without Chromosomal Integration Ken Nishimura,' Hiroaki Segawa,' Mahito Nakanishi.' Research Laboratory, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, lbaraki, Japan f Biotherapeuttc Scndai virus (SeV) vectors occupy a unique position among virus vectors, because the replication and transcription occurs throughout in the cytoplasm using single-stranded RNA genome as a template This characteristic is advantageous for evading undesirable genotoxicity by random chromosomal integration Nevertheless, cytopathic nature of SeV has hindered their application requiring long-term gene expression Previous approaches for achieving stable gene expression by altering ScV vector backbone were largely unsueccssful, partly because molecular mechanisms underlying SeV-mediated cytotoxicity remain obscure In this presentation, we report the detail characteristics of a novel defective SeV vector capable of long-term gene expression by minimizing the cytotoxicity This vector has been developed based on a temperature-sensitive noncytopathic mutant strain SeV clone 151 (Nishimura, et al., ASGT 2005) It was established that cellular anti-virus reactions induced by SeV infection , including induction of interferon-beta and apoptosis, was mediated through RIG-IIIRP-3 pathway (e.g., Kato, H., et al., Nature, 441, 10I-I 05,2006) We identified three independent mutant loci in the genome RNA of SeV clone lSI with which this virus could escape from the fatal anti-virus reactions None of these loci is directly involved in active interference with RIG-IIIRF-3 pathway, while one ofthe loci is closely related to regulation ofthe interferon gene , possibly by modulating the expression of RNA molecule(s) triggering RIG-IIIRF-3 pathway By combining this mutant locus with deletion of structural genes (P, HN and M), we succeeded to develop novel defective SeV vectors capable ofexpressing installed genes persistently without detectable cytotoxicity Installing a drug resistant gene (e.g., Blastcydin S deaminase) allow us to select the cells carrying defective SeV vector genome, but the gene expression is maintained stably even in the absence of selective pressure, for at least 50 days in various cultured cells We also introduce the technical improvements enabling us to rescue defective SeV vectors reproducibly and possible applications ofthis vector system to gene therapy and to the production of protein pharmaceutics 795 Efficient In Vivo Transduction Using RNA Replicon Vector Derived from Sendai Virus SelfReplicating Ribonucleoprotein Complexes Takashi Hironaka, Makoto Inoue, Eisaku Suzuki, Hitoshi Iwasaki, Kentaro Washizawa, Satoshi Fujikawa, Akihiro Iida, Mamoru Hasegawa 'Department ofScience & Technology Develpoment, DNAVEC Corporation, Tsukuba, Ibaraki, Japan The cytoplasmic RNA vector would be promising for use in gene therapy and gene vaccines because ofits important genotoxicity-free nature Especially, recombinant Sendai virus (SeV) vector as well as its modified non-transmissible variants can efficiently transfer and express foreign genes for a broad range of mammalian cells and tissues However, the immunogenicity of the SeV might limit its application in some cases Two envelope glycoproteins, HN and P that mediate the virus attachment and penetration into target cells, are major determinants for neutrali zing response in transduced animals As an alternative type ofthe vector that solves this problem, we have developed a novel SeV RNA replicon vector thattakes advantage of SeV self-replicating ribonucleoprotein complexes (RNP) consisting ofthe RNA genome, nucleocapsid protein (NP) , phosphoprotein (P) S305 and large protein (L) The SeV RNA replicon vector (RNP vector) mixed with (or without) lipofection reagents efficientlytransferred foreign genes to several mammalian cell lines in vitro, and dosedependently expressed them The induction of the RNP into the cells seems to be via endocytosis because it was affected by the chloroquineor cytochalasinB treatment.The success in preparinga largeamount of this RNPvector from the cells transduced with SeV vector and establishinga preservingmethodenabled administration ofthis vector in vivo Weexamined the delivery ofthe RNP vector carrying either luciferase or green fluorescent protein (OFP) genes with/without Iipofeetion reagent in several tissue in vivo The apparent expression of the luciferase was detected in tibialis anterior muscle of rats without transfeetion reagent Very importantly, the equal transgene expression was detected even when the second administration ofRNP vectorwas doneafterthe primaryadministration of SeV or RNP vector In addition, about a IDO-fold of luciferase activity and vigorous EOFP expression was observed at the muscle with bupivacain treatment.These results indicatesthat the SeV RNP vector system will be useful for cytoplasmic expression of foreign genes in vitro/in vivo and may be a powerful and safety tool of its application to human gene therapy or vaccination 796 In Vitro Evolution of a Replicating, Chimeric Murine Leukemia Virus-Gibbon Ape Leukemia Virus Vector Christopher R Logg,' Brian T Baranick,' Nathan A Lemp,' Noriyuki Kasahara.':' I Department a/Medicine University a/California Los Angeles Los Angeles CA; lMolecular Biology Institute University 0/ California Los Angeles Los Angeles CA; 'Department a/Molecular & Medical Pharmacology, University ofCalifornia, Los Angeles Los Angeles CA The abilityof retrovirusparticlesto incorporateenvelopeproteins from other retroviral strains and genera and even from other virus families has been widely exploited for the generation of replication-defective rctroviral vectors bearing heterologous envelope proteins Wepreviouslydevelopedreplication-competent retroviral (RCR)vectors,basedon full-length MLVgenomes,that can transmit transgenesto mammaliancells withhigh efficiencyand stability We desired to investigate the possibility of "genetically" pseudotyping RCR vectors by replacing the native env gene with that of another virus.Tothis end, we substitutedthe env gene in one ofthese vectors with that ofOALV Although the OALVEnv protein can be used to make high titer pscudotypcsof defectiveMLVvectors, the env gene substitution rendered the chimeric virus incapable of replication However,passage of abortively infectedcells resulted in outgrowth of mutant viruses exhibiting rapid replicationkinetics,and different variantsarose in different infections.Two had acquiredmutationsat or near the spliceacceptorsite and three had acquireddual mutations within the LTR Analysis of the levels of unspliced and spliced viral RNAproduced by the parental and evolved viruses showed that the mutationsgained by each ofthese variants functioned to reverse an imbalancein splicingcausedby the substitution ofthc env gene Our results demonstrate the presence of previously unknown cis-acting sequences in MLV that modulate splicing of the viral transcript and illustrate an efficient approach to generating and screening for replicating mutantsof replication-impaired recombinant retroviruses using forced evolution 797 Regulated HIV-1 and HIV-2 Lentiviral Vectors Suresh K AI)'aYAdriana Zingone,' Satomi Adaka,'J Oeetanjali SachdevaY 'Center for Cancer Research, National Cancer Institute National Institutes 0/ Health Bethesda MD; lDivision ofCancer Treatment and Diagnosis National Cancer Institute National Institutes a/Health Bethesda MD; "Chtba University Chiba, Japan ; "National Institute/or Research in Reproduction Mumbai, India Conventional lentiviral vectors contain an internal promoter to drive transgene expression This promoter generally is a constitutive strong promoter, such as CMY The vector may instead carry a special promoter, such as a tissue-specificpromoter or one that can be modulated by external signals Weattempted to design lentiviral vectors for gene therapy of cancer - chronic disease requiring long-term expression of the ' therapeutic gene' Since the fate of a cell depends on the balance of pro-life and pro-death signals, we surmised that combinationtargeting of both signals will be additive or synergistic Thus, we set out to create vectors carrying anti-Bcl2 shRNA to down regulate pro-survival signals and Bax transgenc to up regulate pro-death signals Because both genes arc normal cellular genes, the issue arose how to achieve tumor cell selectivity Wedesigned a trans regulatory loop to achieve selectivity The idea was to drive therapeutic gene expression with viral LTR (no internal promoter) which would need to be trans activated by the viral Tat, and drive Tat expression with the TERT promoter for tumor cell scleetivity To our surprise, our HIV-2 vector carrying the marker OFPgene withoutthe internalpromoterfailed to express OFP to any significant level, even when provided with Tat in trans This was not the case for the analogous HIV-I vector The HIV-l vector gave some OFP expression without Tat, which was boosted several foldwhen providedwithTat, as expected This dichotomous behavior was reminiscent of other differences of detail between HIV- I and HIV-2 noted previously, and which could have biological consequences We hypothesized that the genetic determinants of this difference in phenotype lie within the viral gcnomes, Wethus closely re-examined the genetic structures of the HIV-1 and HIV2 vectors, and noted the following: the LTR of HIV-2 was longer than that of HIV-I and contained additional regulatory elements as noted before; the leader ofHIV-2 is also longer than that of HIV-I, again with additional regulatory clements; the leader-based splice donor ofHIV-2 had been mutated to prevent fortuitous splicing out of packaging signal located downstream; and there arc two short open reading frames in the HIV-2 leader located upstream of the OFP marker gene, and which if utilized, will make OFP a classic downstream gene, and thus poorly expressed We are now testing these possibilities by 3'-truncating the HIV-2 LTR, restoring its splice donor site, and introducing an IRES element between the leader and OFP gene It will be interesting to further dissect this phenomenon by creating hybrid or chimeric vectors 798 Expression of Transduced Proteinase Inhibitor Is Upregulated in Human Islets Exposed to Pro-Apoptotic Stimuli; Implications for Protection of Transplanted Islets Sirlene Ccchin,' M Sofia Ochoa,' Ingrid Perez-Alvarez,' Michael A Curran,' Elizabeth S Fcnjves.' I Diabetes Research Institute University ofMiami Miami FL; l l m m l n o l o ~ Memorial Sloan Kettering Cancer Center; Nell' lark Nt: BACKGROUND: Type I diabetes isan autoimmunedisease resulting from the destructionof the insulinsecretingbeta cells within pancreatic islets Islet transplantation can reestablish regulated insulin secretion; however, large numbers of transplanted islets are S306 Molecular Therapy Volum e 15 Supplcrncnr t, ~b )' 2007 Copyright © Oll ie 'uuericm Society o f Gene Therapy ... with SeV vector and establishinga preservingmethodenabled administration ofthis vector in vivo Weexamined the delivery ofthe RNP vector carrying either luciferase or green fluorescent protein (OFP)... illustrate an efficient approach to generating and screening for replicating mutantsof replication-impaired recombinant retroviruses using forced evolution 797 Regulated HIV-1 and HIV-2 Lentiviral Vectors...and large protein (L) The SeV RNA replicon vector (RNP vector) mixed with (or without) lipofection reagents efficientlytransferred foreign genes to several mammalian cell lines in vitro, and dosedependently