- Email: [email protected]
208. Inhibition of Human Immunodeficiency Virus (HIV) Type 1 Replication through Expression of RNAi Effectors Targeting HIV-Dependency Factor HTATSF1
INFECTIOUS DISEASES AND VACCINES I 207. Evaluation of the Immune Responses in the Treatment of RPE65 Deficient Dogs by Subretinal Injection of a Recombinant AAV Serotype 4 Nathalie Provost,1 Lise Libleau,1 Alexandra Mendes Madeira,1 Jack-Yves Deschamps,3 Michel Weber,2 Guylène LeMeur,2 Yan Cherel,3 Philippe Moullier,1 Fabienne Rolling,1 Christine Chauveau.1 1 INSERM U649, Nantes, France; 2Service d’Ophtalmologie, CHU Hotel Dieu, Nantes, France; 3Ecole Nationale Veterinaire, Nantes, France.
Leber’s Congenital Amaurosis (LCA) is a group of hereditary retinal degeneration leading to total blindness in adulthood. We previously demonstrated that targeted gene transfer to the RPE cells using a recombinant adeno-associated virus serotype 4 (rAAV2/4) can be used to correct the RPE65 defect in a LCA model of RPE65/- purebred Briard dogs. We aim to evaluate the role of the immune responses in RPE65-/- dogs treated by subretinal rAAV2/4.hrpe65 mediated gene delivery in order to determine (i) whether the subretinal injection of the therapeutic dose of rAAV2/4.hrpe65 leads to development of immune responses to the vector or the RPE65 transgene, (ii) the immune consequences of the subretinal injection of rAAV2/4.hrpe65 in the contralateral eye of dogs with a first eye previously treated, (iii) whether a preexisting immunity to AAV4 could limit the therapeutic effectiveness of the subretinal injection of rAAV2/4.hrpe65. Three RPE65-/- dogs were treated by subretinal injection of rAAV2/4.hrpe65 in one eye. ELISA analysis showed that none of the animals had detectable serum antibodies (Abs) neither against the transgene, nor against the AAV4 vector capsid in the 4 months following the treatment. IFNγ enzyme-linked immunosorbent spot (ELISpot) assays performed on peripheral blood mononuclear cells (PBMCs) did not show any cellular responses to the transgene or to the AAV4 vector capsid at any time. Four months after the treatment of the first eye, the contralateral eye of the dogs was treated by a subretinal injection of rAAV2/4.hrpe65. Analysis of the sera of the animals did not allow to detect Abs to the transgene or the AAV4 capsid. IFNγ ELISpot assays performed on PBMCs did not show any cellular responses to the transgene or,the AAV4 capsid. In addition, IFNγ ELISpot performed with splenocytes 12 months after the first subretinal injection did not allow detection of any cellular responses to the transgene or to the AAV4 vector capsid. Because data indicate that low levels of neutralizing Abs can abrogate transduction with high titers of AAV vectors, we aim to evaluate the impact of a preexisting immunity to AAV4 on the transduction efficacy of subretinal AAV2/4. hrpe65 mediated gene delivery. In order to induce a high titer of antiAAV4 Abs in serum of the animals, two dogs were pre-immunized with AAV4 2 months before subretinal injection of AAV2/4.hrpe65. Bilateral full field electroretinographic recordings performed after gene transfer showed that a strong preexisting immunity to AAV4 did not block AAV2/4.hrpe65-mediated gene delivery and long term function recovery in RPE65-/- dogs. To conclude, our findings suggest (i) that subretinal administration of the therapeutic dose of rAAV2/4.hrpe65 does not induce a detectable immune response to the transgene or the AAV4 vector capsid, (ii) that sequential subretinal readministration of rAAV2/4.hrpe65 is safe and potent, (iii) that preexisting immunity to AAV4 does not block subretinal AAV2/4. hrpe65 mediated gene transfer.
Molecular Therapy Volume 19, Supplement 1, May 2011 Copyright © The American Society of Gene & Cell Therapy
Infectious Diseases and Vaccines I 208. Inhibition of Human Immunodeficiency Virus (HIV) Type 1 Replication through Expression of RNAi Effectors Targeting HIV-Dependency Factor HTATSF1
Victoria Green,1 Patrick Arbuthnot,1 Marco S. Weinberg.1 Antiviral Gene Therapy Research Unit, Department of Molecular Medicine and Haematology, University of the Witwatersrand, Johannesburg, South Africa. 1
Given the high mutability of human immunodeficiency virus type 1 (HIV-1), an appealing antiviral strategy is to minimise the emergence of escape mutants by silencing cellular factors required for viral replication. Despite many putative HIV-1 dependency factors (HDFs) being identified in genome-wide screens, few have subsequently been validated as potential therapeutic targets. Ten RNA Pol III-driven short hairpin (sh) RNA cassettes were constructed targeting expression of the predicted HDF HTATSF1, and confirmed HDF p75 as a control. Three shRNAs targeting htatsf1 decreased endogenous mRNA and protein levels by ∼50%, as determined by qRT-PCR and Western blot, respectively. Processing of guide strands were confirmed by Northern blot, supporting silencing via an RNAi mechanism. No significant cytotoxic effects were observed on shRNA expression. TZM-bl cells, a luciferase reporter cell line, were used to determine the efficacy of shRNAs. HTATSF1-targeting shH1 significantly inhibited luciferase activity in a dose-dependent manner when transiently expressed in TZM-bl cells infected with various HIV-1 strains. shH1 also decreased infectious virion production by 68%. As CD4+ T cells are a physiological substrate of HIV, lentiviruses were used to generate T cell lines with stable shRNA expression. Replication kinetics of the HIV-1 molecular clone p81A-4 were determined from p24 antigen levels up to 18 days post-infection. SupT1 T cells with stable expression of shH1 exhibited decreased p24 production throughout the time course (<1% at day 10), and to a greater extent than the viral genome-targeting positive control. Moreover, HTATSF1 inhibition had no affect on splicing of cellular and viral transcripts but did slightly reduce cellular proliferation. The results demonstrate that post-transcriptional gene silencing of HTATSF1 can lead to decreased HIV-1 replication without cytotoxic effects in a T cell line. Anti-htatsf1 RNAi effectors may, therefore, be a useful addition to existing HIV-1 combinatorial gene therapies.
209. Protection Against Divergent Influenza H1N1 Virus by a Centralized Influenza Hemagglutinin Eric A. Weaver,1 Richard Webby,2 Michael A. Barry.1 Mayo Clinic, Rochester, MN; 2St. Jude Children’s Research Hospital, Memphis, TN.
1
Influenza poses a persistent worldwide threat to the human population. As evidenced by the 2009 H1N1 pandemic, current vaccine technologies are unable to respond rapidly to this constantly diverging pathogen. We tested the utility of adenovirus (Ad) vaccines expressing centralized consensus influenza antigens. Ad vaccines were produced within 2 months and protected against influenza in mice within 3 days of vaccination. Ad vaccines were able to protect at doses as low as 1e7 virus particles/kg indicating that approximately 1,000 human doses could be rapidly generated from standard Ad preparations. To generate broadly cross-reactive immune responses, centralized consensus antigens were constructed against H1 influenza and against H1 through H5 influenza. Twenty full-length H1 HA sequences representing the main branches of the H1 HA phylogenetic tree were used to create a synthetic centralized gene, HA1-con. HA1con minimizes the degree of sequence dissimilarity between the vaccine and existing circulating viruses. HA1-con induced stronger S81
Leber’s Congenital Amaurosis (LCA) is a group of hereditary retinal degeneration leading to total blindness in adulthood. We previously demonstrated that targeted gene transfer to the RPE cells using a recombinant adeno-associated virus serotype 4 (rAAV2/4) can be used to correct the RPE65 defect in a LCA model of RPE65/- purebred Briard dogs. We aim to evaluate the role of the immune responses in RPE65-/- dogs treated by subretinal rAAV2/4.hrpe65 mediated gene delivery in order to determine (i) whether the subretinal injection of the therapeutic dose of rAAV2/4.hrpe65 leads to development of immune responses to the vector or the RPE65 transgene, (ii) the immune consequences of the subretinal injection of rAAV2/4.hrpe65 in the contralateral eye of dogs with a first eye previously treated, (iii) whether a preexisting immunity to AAV4 could limit the therapeutic effectiveness of the subretinal injection of rAAV2/4.hrpe65. Three RPE65-/- dogs were treated by subretinal injection of rAAV2/4.hrpe65 in one eye. ELISA analysis showed that none of the animals had detectable serum antibodies (Abs) neither against the transgene, nor against the AAV4 vector capsid in the 4 months following the treatment. IFNγ enzyme-linked immunosorbent spot (ELISpot) assays performed on peripheral blood mononuclear cells (PBMCs) did not show any cellular responses to the transgene or to the AAV4 vector capsid at any time. Four months after the treatment of the first eye, the contralateral eye of the dogs was treated by a subretinal injection of rAAV2/4.hrpe65. Analysis of the sera of the animals did not allow to detect Abs to the transgene or the AAV4 capsid. IFNγ ELISpot assays performed on PBMCs did not show any cellular responses to the transgene or,the AAV4 capsid. In addition, IFNγ ELISpot performed with splenocytes 12 months after the first subretinal injection did not allow detection of any cellular responses to the transgene or to the AAV4 vector capsid. Because data indicate that low levels of neutralizing Abs can abrogate transduction with high titers of AAV vectors, we aim to evaluate the impact of a preexisting immunity to AAV4 on the transduction efficacy of subretinal AAV2/4. hrpe65 mediated gene delivery. In order to induce a high titer of antiAAV4 Abs in serum of the animals, two dogs were pre-immunized with AAV4 2 months before subretinal injection of AAV2/4.hrpe65. Bilateral full field electroretinographic recordings performed after gene transfer showed that a strong preexisting immunity to AAV4 did not block AAV2/4.hrpe65-mediated gene delivery and long term function recovery in RPE65-/- dogs. To conclude, our findings suggest (i) that subretinal administration of the therapeutic dose of rAAV2/4.hrpe65 does not induce a detectable immune response to the transgene or the AAV4 vector capsid, (ii) that sequential subretinal readministration of rAAV2/4.hrpe65 is safe and potent, (iii) that preexisting immunity to AAV4 does not block subretinal AAV2/4. hrpe65 mediated gene transfer.
Molecular Therapy Volume 19, Supplement 1, May 2011 Copyright © The American Society of Gene & Cell Therapy
Infectious Diseases and Vaccines I 208. Inhibition of Human Immunodeficiency Virus (HIV) Type 1 Replication through Expression of RNAi Effectors Targeting HIV-Dependency Factor HTATSF1
Victoria Green,1 Patrick Arbuthnot,1 Marco S. Weinberg.1 Antiviral Gene Therapy Research Unit, Department of Molecular Medicine and Haematology, University of the Witwatersrand, Johannesburg, South Africa. 1
Given the high mutability of human immunodeficiency virus type 1 (HIV-1), an appealing antiviral strategy is to minimise the emergence of escape mutants by silencing cellular factors required for viral replication. Despite many putative HIV-1 dependency factors (HDFs) being identified in genome-wide screens, few have subsequently been validated as potential therapeutic targets. Ten RNA Pol III-driven short hairpin (sh) RNA cassettes were constructed targeting expression of the predicted HDF HTATSF1, and confirmed HDF p75 as a control. Three shRNAs targeting htatsf1 decreased endogenous mRNA and protein levels by ∼50%, as determined by qRT-PCR and Western blot, respectively. Processing of guide strands were confirmed by Northern blot, supporting silencing via an RNAi mechanism. No significant cytotoxic effects were observed on shRNA expression. TZM-bl cells, a luciferase reporter cell line, were used to determine the efficacy of shRNAs. HTATSF1-targeting shH1 significantly inhibited luciferase activity in a dose-dependent manner when transiently expressed in TZM-bl cells infected with various HIV-1 strains. shH1 also decreased infectious virion production by 68%. As CD4+ T cells are a physiological substrate of HIV, lentiviruses were used to generate T cell lines with stable shRNA expression. Replication kinetics of the HIV-1 molecular clone p81A-4 were determined from p24 antigen levels up to 18 days post-infection. SupT1 T cells with stable expression of shH1 exhibited decreased p24 production throughout the time course (<1% at day 10), and to a greater extent than the viral genome-targeting positive control. Moreover, HTATSF1 inhibition had no affect on splicing of cellular and viral transcripts but did slightly reduce cellular proliferation. The results demonstrate that post-transcriptional gene silencing of HTATSF1 can lead to decreased HIV-1 replication without cytotoxic effects in a T cell line. Anti-htatsf1 RNAi effectors may, therefore, be a useful addition to existing HIV-1 combinatorial gene therapies.
209. Protection Against Divergent Influenza H1N1 Virus by a Centralized Influenza Hemagglutinin Eric A. Weaver,1 Richard Webby,2 Michael A. Barry.1 Mayo Clinic, Rochester, MN; 2St. Jude Children’s Research Hospital, Memphis, TN.
1
Influenza poses a persistent worldwide threat to the human population. As evidenced by the 2009 H1N1 pandemic, current vaccine technologies are unable to respond rapidly to this constantly diverging pathogen. We tested the utility of adenovirus (Ad) vaccines expressing centralized consensus influenza antigens. Ad vaccines were produced within 2 months and protected against influenza in mice within 3 days of vaccination. Ad vaccines were able to protect at doses as low as 1e7 virus particles/kg indicating that approximately 1,000 human doses could be rapidly generated from standard Ad preparations. To generate broadly cross-reactive immune responses, centralized consensus antigens were constructed against H1 influenza and against H1 through H5 influenza. Twenty full-length H1 HA sequences representing the main branches of the H1 HA phylogenetic tree were used to create a synthetic centralized gene, HA1-con. HA1con minimizes the degree of sequence dissimilarity between the vaccine and existing circulating viruses. HA1-con induced stronger S81