- Email: [email protected]
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 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 9 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
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 9 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