Human anti-HIV-1 tat sFv intrabodies for gene therapy of advanced HIV-1-infection and AIDS

Marasco, Wayne A.; LaVecchio, Joyce; Winkler, Aaron

doi:10.1016/s0022-1759(99)00159-3

Cited by 63 publications

(35 citation statements)

References 86 publications

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“…The use of intrabodies in other cell-based systems have not been reported to alter growth and development. [64][65][66] Although CCR5 intrabody expression is functional and limits the amount and duration of viral spread at the population level in CD4 + T cells isolated from the human thymus (Figure 3), additional studies are required to determine the variability of CAD-R5-mediated protection provided to individual CD4 + T cells derived from transduced CD34 + progenitors.…”

Section: Ccr5 Intrabody-mediated Protection and Enrichment Of T Cellsmentioning

confidence: 99%

T-cell protection and enrichment through lentiviral CCR5 intrabody gene delivery

et al. 2006

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CCR5 is the chemokine co-receptor for R5-tropic human immunodeficiency virus type 1 (HIV-1) isolates most often associated with primary infection. We have developed an HIV-1 self-inactivating vector, CAD-R5, containing a CCR5 single-chain antibody (intrabody) gene, which when expressed in T-cell lines and primary CD4 + T cells disrupts CCR5 cell surface expression and provides protection from R5-tropic isolate exposure. Furthermore, CAD-R5 intrabody expression in primary CD4 + T cells supports significant growth and enrichment over time during HIV-1-pulsed dendritic cell-T-cell interactions. These results indicate that CCR5 intrabody-expressing CD4 + T cells are refractory against this highly efficient primary route of infection. CD34 + cells transduced with the CAD-R5 vector gave rise to CD4 + and CD8 + thymocytes in non-obese diabetic (NOD)/ severely combined-immunodeficient (SCID)-human thymus/ liver (hu thy/liv) mice, suggesting that CCR5 intrabody expression can be maintained throughout differentiation without obvious cellular effects. CD4 + T cells isolated from NOD/SCID-hu thy/liv mice were resistant to R5-tropic HIV-1 challenge demonstrating the maintenance of protection. Our findings demonstrate delivery of anti-HIV-1 activity through CCR5 intrabodies in primary CD4 + T cells and CD34 + cell-derived T-cell progeny. Thus, gene delivery strategies that provide a selective survival and growth advantage for T effector cells may provide a therapeutic benefit for HIV-1-infected individuals who have failed conventional therapies.

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Section: Ccr5 Intrabody-mediated Protection and Enrichment Of T Cellsmentioning

confidence: 99%

T-cell protection and enrichment through lentiviral CCR5 intrabody gene delivery

et al. 2006

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“…Various intrabodies were shown to be effective against human immunodeficiency virus replication and infection (Marasco et al, 1999;Mhashilkar et al;Poznansky et al, 1998;Kitamura et al, 1999;BouHamdan et al, 1999;Goncalves et al, 2002). Other intrabodies were used successfully against various oncogenes (Jean et al, 2000;Deshane et al, 1995;Chen et al, 2002).…”

Section: Discussionmentioning

confidence: 99%

“…Successful transduction has been demonstrated with, for example, retrovirus and lentivirus vectors (BouHamdan et al, 1999;Chen et al, 1994b;Marasco et al, 1999;Richardson et al, 1998). To date the first clinical studies involving patients with ovarian cancer are ongoing using adenovirus vectors to express anti-erbB-2 intrabodies (Alvarez et al, 2000;Deshane et al, 1995).…”

Section: Discussionmentioning

confidence: 99%

Abrogation of hepatitis C virus NS3 helicase enzymatic activity by recombinant human antibodies

Artsaenko

Tessmann

Sack³

et al. 2003

Journal of General Virology

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The hepatitis C virus (HCV) NS3 protein possesses both protease and helicase activities and is essential for virus replication and maturation. Specific inhibition of NS3 enzymatic activity can be achieved by antibody binding. Transduction of hepatocytes with encoding cDNA leading to intracellular expression of antibody fragments is expected to terminate HCV replication in infected cells. The objective of the present study was the generation of human antibody fragments that neutralize the viral NS3 helicase activity for gene therapeutic applications and drug design. A human immunoglobulin phage-display library cloned from bone marrow aspirate of patients infected with HCV was used for affinity selection against HCV NS3 helicase. Antibody fragments with high affinity to HCV helicase were isolated. To evaluate the inhibitory potential of isolated single-chain antibody fragments, a helicase-mediated, DNA-unwinding enzymatic assay was developed in ELISA format. Recombinant protein comprising the full-length HCV NS3 helicase domain was expressed in the baculovirus expression system. Recombinant antibodies that inhibit the HCV helicase at nanomolar concentrations, with efficacies ranging from 20 % to complete abrogation of enzymatic unwinding activity, were identified. These antibody fragments may be useful for novel gene therapeutic strategies that employ intracellular immunization and may provide new insights into the design of small molecule inhibitors of essential HCV proteins.

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“…In previous studies we have demonstrated that an intrabody to HIV-1 Tat could significantly inhibit HIV-1 replication in vitro (12,13) indicating that the Tat transactivation pathway could be targeted by intrabodies specific for the viral component of this pathway. Based on these studies, we further hypothesized that, because the molecular interaction between HIV-1 Tat and the N-terminal region of hCyclinT1 is necessary for HIV-1 replication, the selective disruption of this binding by anti-hCyclinT1 intrabodies could inhibit Tat-mediated transactivation and HIV-1 replication without having a detrimental effect on basal transcription.…”

Section: The Hivmentioning

confidence: 95%

Inhibition of Tat-mediated Transactivation and HIV-1 Replication by Human Anti-hCyclinT1 Intrabodies

Bai¹,

Sui²,

Zhu³

et al. 2003

Journal of Biological Chemistry

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Human immunodeficiency virus, type 1 (HIV-1) replication requires the interaction of Tat protein with the human cyclinT1 (hCyclinT1) subunit of the positive transcription elongation factor (P-TEFb) complex, which then cooperatively binds to transactivation response element (TAR) RNA to transactivate HIV transcription. In this report, a non-immune human singlechain antibody (sFv) phage display library was used to isolate anti-hCyclinT1 sFvs that could disrupt hCyclinT1-Tat interactions. The N-terminal 272 residues of hCyclinT1, including the entire cyclin domains and the Tat⅐TAR recognition motif (TRM), that fully support Tat transactivation was used for panning, and of the five unique anti-hCyclinT1 sFvs that were obtained, three bound to the cyclin box domains and two bound to TRM. All sFvs could be expressed as intrabodies at high levels in transiently transfected 293T and in stable Jurkat and SupT1 transfectants and could specifically co-immunoprecipitate co-expressed hCyclinT1 in 293T cells with varying efficacy without disrupting hCyclinT1-Cdk9 interactions. In addition, two sFv clones (3R6-1 and 2R6-21) that mapped to the cyclin box domains markedly inhibited Tat-mediated transactivation in several transiently transfected cell lines without inhibiting basal transcription or inducing apoptosis. When HIV-1 challenge studies were performed on stable 3R6-1-expressing Jurkat T cells, near complete inhibition of viral replication was obtained at a low challenge dose, and 74 -88% inhibition to HIV-1 replication was achieved at a high infection dose in SupT1 cells. These results provide proof-in-principle that anti-hCyclinT1 intrabodies can be designed to block HIV-1 replication without causing cellular toxicity, and as a result, they may be useful agents for "intracellular immunization"-based gene therapy strategies for HIV-1 infection/AIDS.

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Human anti-HIV-1 tat sFv intrabodies for gene therapy of advanced HIV-1-infection and AIDS

Cited by 63 publications

References 86 publications

T-cell protection and enrichment through lentiviral CCR5 intrabody gene delivery

T-cell protection and enrichment through lentiviral CCR5 intrabody gene delivery

Abrogation of hepatitis C virus NS3 helicase enzymatic activity by recombinant human antibodies

Inhibition of Tat-mediated Transactivation and HIV-1 Replication by Human Anti-hCyclinT1 Intrabodies

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