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592. Effective Transduction of Nondividing PBL, Monocytes and Monocyte-Derived Macrophages with SV40-Derived Vectors Protects from HIV-1 Challenge
INFECTIOUS DISEASES & VACCINES specifically bond by the p36/HLA-A0201 tetramer. Tranduced cells expressing either of the two functional pairs of anti-JCV CIRs were not stained by unrelated p36 or p100/HLA-A0201 tetramer, indicating the functional paired CIRs remain their antigen specificity. Further functional tests of the CIRs with human T cells are currently undergoing.
592. Effective Transduction of Nondividing PBL, Monocytes and Monocyte-Derived Macrophages with SV40-Derived Vectors Protects from HIV-1 Challenge Elena I. Marusich,1 David S. Strayer.1 Pathology, Jefferson Medical College, Philadelphia, PA.
1
One of the main challenges in application of gene therapy to treatment of HIV-1 infection is the need to protect non-dividing lymphocytes and monocytes, as well as monocyte-derived macrophages (MDM), from HIV-1. Since monocyte-derived macrophages represent a major reservoir HIV-1 in vivo, and because at any given time most T cells, monocytes and macrophages in the body are quiescent, gene transfer to these types of cells is very important, yet very difficult, in considering gene delivery to protect from HIV-1. Recombinant SV40-derived vectors (rSV40s) transduce resting cells, especially lymphocytes, monocytes and macrophages, very efficiently. Therefore we tested the effectiveness of a series of anti-HIV-1 transgenes delivered to human PBL, monocytes and MDM by rSV40-derived vectors. We have engineered a series of constructs carrying: single chain Fv antibodies (SFvs) against the DNA-binding and catalytic regions of HIV-1 integrase (IN), SFvs binding two different regions of HIV1 reverse transcriptase (RT) and the trans-dominant mutant of HIV1 Rev, RevM10. In all cases, transgene expression was driven by CMV-IEP. Normal human PBMC were fractionated into lymphocytes (PBL) and monocytes by differential adherence. Monocytes were differentiated into macrophages using standard cytokine cocktails. Unstimulated PBL were transduced with these vectors by sequential exposure over 3 days at a cumulative MOI=16. Control cultures were mock-transduced or treated with control rSV40s. Transgene expression was measured by FACS. The quiescent state of the PBL at the time of transduction was verified by propidium iodide staining of their DNA, followed by flow cytometry to confirm that cells were in the G0/G1 phase of the cycle. After transduction, cultures were challenged using 80 IU and 400 infectious units (IU) HIV-1NL4-3, or with 400 IU and 4000 IU primary isolate HIV-1 92HT594. PBL were then stimulated (to maintain viability) with IL-2, and maintained in this condition for 1 month of challenge. At 80 IU challenge dose PBL treated with rSV40 vectors carrying RevM10, either of the anti-IN SFvs or either anti-RT SFvs were completely protected, compared with controls. Similarly, at the higher 400 IU challenge dose of HIV-1, virus replication was also completely inhibited for all test transduced infectious cultures. PBL transduced with the anti-HIV-1-integrase SFvs were better protected at highest 4000 IU dose HIV-1 primary isolate 92HT594, in comparison with other transduced cultures. Comparably high protective efficiency was also observed in challenge studies using monocytes and MDM transduced with all of the tested SV40-delivered constructs. HIV-1 replication was inhibited at least 9095%, at all challenge doses of HIV-1, compared with controls. Thus, we verified the potency of an array of anti-HIV-1 transgenes delivered by SV40 based vectors to human non-dividing PBL, monocytes and macrophages. Anti-HIV-1 transgenes so transferred to all these cell types proved protective when tested against multiple strains of HIV-1. Therefore, rSV40 gene delivery to resting HIV-1-susceptible blood cells provides effective protection from even high dose HIV-1 challenge. Molecular Therapy Volume 13, Supplement 1, May 2006 Copyright The American Society of Gene Therapy
593. Combination Anti-HIV-1 Gene Therapy Simultaneously Targeting CCR5 and Rev Protein Using SV40-Derived Vectors Inhibits Replication of HIV Alena A. Chekmasova,1 David S. Strayer.1 Pathology, Jefferson Medical College, Philadelphia, PA.
1
CXCR4 and CCR5 chemokine receptors are respectively the principal coreceptors for T-cell tropic and macrophage (M)- tropic human immunodeficiency virus 1 (HIV-1) isolates. The CCR5 chemokine receptor is important for most clinical strains of HIV to establish infection. Individuals with naturally occurring polymorphisms in the CCR5 gene who have reduced or absent CCR5 are apparently otherwise healthy but are resistant to HIV infection. CCR5 is thus an excellent therapeutic target in HIV therapy. RevM10, a trans-dominant mutant form of HIV-1 Rev, has been shown to be effective in inhibiting HIV replication in T cell lines and primary T cells. In this study our goal was to reduce CCR5, protect CCR5+ cells from R5-tropic HIV and inhibit replication of HIV. We used a Tagdeleted SV40-derived vector, SV(VARNAiR5#5m/h/RevM10), to deliver interfering RNA against CCR5 (siRNA vs. CCR5) driven by the adenovirus VA1 promoter, a pol III promoter, and CMV-IEPdriven anti-Rev (RevM10) transgenes to human cell lines SupT1 and SupT1/CCR5. The effectiveness of transduction was assessed by immunostaining and flow cytometry. The RevM10 transgene carried a C-termial AU1 epitope tag, detected using commercial anti-AU1 antibody. Effective expression of the RNAi was detected as a decrease in cell membrane CCR5. Protection from HIV-1 was measured by p24 antigen levels in culture supernatants. Transduction with SV(VARNAiR5#5m/h/RevM10) provided transgene expression in >95% of cultured cells. Expression of RevM10 was observed, as evidenced by immunodetection of AU1, and cell membrane CCR5 was decreased, compared to control rSV40 vectors. In addition, simultaneous delivery of these transgenes in this combinatorial vector protected from HIV-1 replication: HIV-1 p24 in culture supernatants was decreased for both SupT1 and SupT1/CCR5 cell lines infected respectively with 100 infectious units of either X4-tropic HIV-1NL4-3 or R5-tropic HIV-1Ba-L. It is expected that all cell types will be protected from HIV by this vector, since RevM10 is effective vs. both X4- and R5-tropic strains. However, results to date suggest that the combination of the two transgenes in one vector provides enhanced protection for CCR5-bearing cells. Thus, our data results demonstrate that simultaneous targeting of more then one HIV-1 function provides effective inhibition of HIV-1 replication. Targeting in one vector a cellular protein (CCR5) necessary for HIV-1 entry may decrease the likelihood of escape mutation and provide enhanced protection from HIV-1 infection.
594. Protection Against In Vivo Tumor Growth after Electroporation-Enhanced DNA Vaccination with the Hepatitis C Virus Non-Structural 3/4A Rune Kjeken,1 Gustaf Ahlen,2 Jonas Soderholm,2 Torunn Tjelle,1 Iacob Mathiesen,1 Matti Sallberg.2 1 Research and Development, Inovio Inc, San Diego, CA; 2 Division of Clinical Virology, F68, Karollinska University Hospital Huddinge, Stockholm, Sweden. Hepatitis C virus (HCV) infection is a major global health problem with at least 170 million chronically infected persons worldwide. Although antiviral combination therapy with pegylated interferonalpha and ribavirin has proven efficient for treating some subtypes of HCV, for patients infected with HCV 1a or 1b, the response rates are less then 50%. Therapeutic vaccination therefore remains an attractive alternative to improve therapy for these patients. S229
592. Effective Transduction of Nondividing PBL, Monocytes and Monocyte-Derived Macrophages with SV40-Derived Vectors Protects from HIV-1 Challenge Elena I. Marusich,1 David S. Strayer.1 Pathology, Jefferson Medical College, Philadelphia, PA.
1
One of the main challenges in application of gene therapy to treatment of HIV-1 infection is the need to protect non-dividing lymphocytes and monocytes, as well as monocyte-derived macrophages (MDM), from HIV-1. Since monocyte-derived macrophages represent a major reservoir HIV-1 in vivo, and because at any given time most T cells, monocytes and macrophages in the body are quiescent, gene transfer to these types of cells is very important, yet very difficult, in considering gene delivery to protect from HIV-1. Recombinant SV40-derived vectors (rSV40s) transduce resting cells, especially lymphocytes, monocytes and macrophages, very efficiently. Therefore we tested the effectiveness of a series of anti-HIV-1 transgenes delivered to human PBL, monocytes and MDM by rSV40-derived vectors. We have engineered a series of constructs carrying: single chain Fv antibodies (SFvs) against the DNA-binding and catalytic regions of HIV-1 integrase (IN), SFvs binding two different regions of HIV1 reverse transcriptase (RT) and the trans-dominant mutant of HIV1 Rev, RevM10. In all cases, transgene expression was driven by CMV-IEP. Normal human PBMC were fractionated into lymphocytes (PBL) and monocytes by differential adherence. Monocytes were differentiated into macrophages using standard cytokine cocktails. Unstimulated PBL were transduced with these vectors by sequential exposure over 3 days at a cumulative MOI=16. Control cultures were mock-transduced or treated with control rSV40s. Transgene expression was measured by FACS. The quiescent state of the PBL at the time of transduction was verified by propidium iodide staining of their DNA, followed by flow cytometry to confirm that cells were in the G0/G1 phase of the cycle. After transduction, cultures were challenged using 80 IU and 400 infectious units (IU) HIV-1NL4-3, or with 400 IU and 4000 IU primary isolate HIV-1 92HT594. PBL were then stimulated (to maintain viability) with IL-2, and maintained in this condition for 1 month of challenge. At 80 IU challenge dose PBL treated with rSV40 vectors carrying RevM10, either of the anti-IN SFvs or either anti-RT SFvs were completely protected, compared with controls. Similarly, at the higher 400 IU challenge dose of HIV-1, virus replication was also completely inhibited for all test transduced infectious cultures. PBL transduced with the anti-HIV-1-integrase SFvs were better protected at highest 4000 IU dose HIV-1 primary isolate 92HT594, in comparison with other transduced cultures. Comparably high protective efficiency was also observed in challenge studies using monocytes and MDM transduced with all of the tested SV40-delivered constructs. HIV-1 replication was inhibited at least 9095%, at all challenge doses of HIV-1, compared with controls. Thus, we verified the potency of an array of anti-HIV-1 transgenes delivered by SV40 based vectors to human non-dividing PBL, monocytes and macrophages. Anti-HIV-1 transgenes so transferred to all these cell types proved protective when tested against multiple strains of HIV-1. Therefore, rSV40 gene delivery to resting HIV-1-susceptible blood cells provides effective protection from even high dose HIV-1 challenge. Molecular Therapy Volume 13, Supplement 1, May 2006 Copyright The American Society of Gene Therapy
593. Combination Anti-HIV-1 Gene Therapy Simultaneously Targeting CCR5 and Rev Protein Using SV40-Derived Vectors Inhibits Replication of HIV Alena A. Chekmasova,1 David S. Strayer.1 Pathology, Jefferson Medical College, Philadelphia, PA.
1
CXCR4 and CCR5 chemokine receptors are respectively the principal coreceptors for T-cell tropic and macrophage (M)- tropic human immunodeficiency virus 1 (HIV-1) isolates. The CCR5 chemokine receptor is important for most clinical strains of HIV to establish infection. Individuals with naturally occurring polymorphisms in the CCR5 gene who have reduced or absent CCR5 are apparently otherwise healthy but are resistant to HIV infection. CCR5 is thus an excellent therapeutic target in HIV therapy. RevM10, a trans-dominant mutant form of HIV-1 Rev, has been shown to be effective in inhibiting HIV replication in T cell lines and primary T cells. In this study our goal was to reduce CCR5, protect CCR5+ cells from R5-tropic HIV and inhibit replication of HIV. We used a Tagdeleted SV40-derived vector, SV(VARNAiR5#5m/h/RevM10), to deliver interfering RNA against CCR5 (siRNA vs. CCR5) driven by the adenovirus VA1 promoter, a pol III promoter, and CMV-IEPdriven anti-Rev (RevM10) transgenes to human cell lines SupT1 and SupT1/CCR5. The effectiveness of transduction was assessed by immunostaining and flow cytometry. The RevM10 transgene carried a C-termial AU1 epitope tag, detected using commercial anti-AU1 antibody. Effective expression of the RNAi was detected as a decrease in cell membrane CCR5. Protection from HIV-1 was measured by p24 antigen levels in culture supernatants. Transduction with SV(VARNAiR5#5m/h/RevM10) provided transgene expression in >95% of cultured cells. Expression of RevM10 was observed, as evidenced by immunodetection of AU1, and cell membrane CCR5 was decreased, compared to control rSV40 vectors. In addition, simultaneous delivery of these transgenes in this combinatorial vector protected from HIV-1 replication: HIV-1 p24 in culture supernatants was decreased for both SupT1 and SupT1/CCR5 cell lines infected respectively with 100 infectious units of either X4-tropic HIV-1NL4-3 or R5-tropic HIV-1Ba-L. It is expected that all cell types will be protected from HIV by this vector, since RevM10 is effective vs. both X4- and R5-tropic strains. However, results to date suggest that the combination of the two transgenes in one vector provides enhanced protection for CCR5-bearing cells. Thus, our data results demonstrate that simultaneous targeting of more then one HIV-1 function provides effective inhibition of HIV-1 replication. Targeting in one vector a cellular protein (CCR5) necessary for HIV-1 entry may decrease the likelihood of escape mutation and provide enhanced protection from HIV-1 infection.
594. Protection Against In Vivo Tumor Growth after Electroporation-Enhanced DNA Vaccination with the Hepatitis C Virus Non-Structural 3/4A Rune Kjeken,1 Gustaf Ahlen,2 Jonas Soderholm,2 Torunn Tjelle,1 Iacob Mathiesen,1 Matti Sallberg.2 1 Research and Development, Inovio Inc, San Diego, CA; 2 Division of Clinical Virology, F68, Karollinska University Hospital Huddinge, Stockholm, Sweden. Hepatitis C virus (HCV) infection is a major global health problem with at least 170 million chronically infected persons worldwide. Although antiviral combination therapy with pegylated interferonalpha and ribavirin has proven efficient for treating some subtypes of HCV, for patients infected with HCV 1a or 1b, the response rates are less then 50%. Therapeutic vaccination therefore remains an attractive alternative to improve therapy for these patients. S229