- Email: [email protected]
42. Intensive Pharmacological Immunosuppression Allows for Repetitive Liver Gene Transfer with Recombinant Adenovirus in Nonhuman Primates
IMMUNOLOGIC & HOST RESPONSES IN GENE & CELL THERAPY indicate that the development of anti-VSV-G antibodies, even if likely targeted to the infused cells, does not have the capability to affect the potency of the infused product. In conclusion the development of an anti-VSV-G immunity was well tolerated in all subjects and was not found to have any effect on the therapeutic efcacy of Lexgenleucel-T treatment.
42. Intensive Pharmacological Immunosuppression Allows for Repetitive Liver Gene Transfer with Recombinant Adenovirus in Nonhuman Primates
Antonio Fontanellas,1 Sandra Hervás-Stubbs,1 Itsaso Mauleón,1 Juan Dubrot,1 Uxua Mancheño,1 María Collantes,2 Ana Sampedro,1 Carmen Unzu,1 Carlos Alfaro,1 Asis Palazón,1 Rafael Enríquez de Salamanca,3 Cristian Smerdou,1 Alberto Benito,4 Jesús Prieto,1 Iván Peñuelas,5 Ignacio Melero.1 1 Gene Therapy and Hepatology Area, Centre for Applied Medical Research, University of Navarra, Pamplona, Spain; 2MicroPET Research Unit, CIMA-CUN, University of Navarra, Pamplona, Spain; 3Research Center, Hospital Universitario 12 de Octubre, Madrid, Spain; 4Department of Radiology, Clínica Universidad de Navarra, University of Navarra, Pamplona, Spain; 5Department of Nuclear Medicine, Clínica Universidad de Navarra, University of Navarra, Pamplona, Spain. Repeated administration of gene therapies is hampered by host immunity towards vectors and transgenes. Attempts to circumvent anti-vector immunity include pharmacological immunosuppression or alternating different vectors and vector serotypes with the same transgene. The aim of this study was to combine clinically available immunosuppressant drugs permitting repeated liver gene transfer to macaques. The immunosuppression regime included (i) Rituximab (20mg/kg/dose i.v.) at days -9, -6, -3, immediately before adenovirus injections and weekly following the adenoviral administration, (ii) two doses of 3 mg/kg of rabbit antithymocyte immunoglobulin (ATG) at days -2 and -1 before the adenovirus injection. (iii) Methylprednisolone was applied intramuscularly 10 min before the ATG infusion at a dose of 100 mg on day -2 and 50 mg on day -1 to avoid systemic inammation. (iv) Mycophenolate mofetil (MMF) doses were 25-30 mg/kg/day and (v) for FK506 0.25 mg /kg/day. MMF and FK506 were orally given daily from day -2 during three months after vector infusion. Our studies show that B-cell depletion with anti-CD20 monoclonal antibody and concomitant T-cell inhibition with clinically available drugs permit repeated liver gene transfer to a limited number of non-human primates with recombinant adenovirus. Adenoviral vector-mediated transfer of the herpes simplex virus type 1 thymidine kinase (HSV1-tk) reporter gene was visualized in vivo with a semiquantitative transgene-specic positron emission tomography (PET) technique, liver immunohistochemistry and immunoblot for the reporter transgene in needle biopsies. Neutralizing antibody and T cell-mediated responses towards the viral capsids were sequentially monitored and found to be repressed by the drug combinations tested. Repeated liver transfer of the HSV1-tk reporter gene with the same recombinant adenoviral vector was achieved in macaques undergoing a clinically feasible immunosuppressive treatments that ablated humoral and cellular immune responses. This strategy allows measurable gene retransfer to the liver as late as 15 months following the rst adenoviral exposure in a macaque, which has undergone a total of four successful gene-transfer procedures with the same adenoviral vector.
43. Induction of Regulatory T Cells as an Approach To Modulate CTL Responses Against AAV Capsid Epitopes
Daniel J. Hui,1 Gary Pien,1 Etiena Basner-Tschakarjan,1 Federico Mingozzi,1 Jonathan D. Finn,1 Shangzhen Zhou,1 William Martin,2 Anne S. De Groot,2 Katherine A. High.1,3 1 Pediatrics – Hematology, Children’s Hospital of Philadelphia, Philadelphia, PA; 2EpiVax, Inc., Providence, RI; 3Howard Hughes Medical Institute, Philadelphia, PA. Adeno-associated viral (AAV) vectors are promising tools for gene transfer strategies. However, the potential for untoward immune responses to the viral vector remains one of the last barriers to successful gene transfer, as was the case in the rst clinical trial using AAV serotype 2 to deliver Factor IX to Hemophilia B subjects via a liver-directed approach. Results from that trial showed that expression of F.IX was short-lived, followed by transient elevation of liver enzymes, which we hypothesized to result from the reactivation of AAV capsid-specic memory CD8+ T cells generated during a previous exposure to AAV. We have been able to model this hypothesis in vitro using a human hepatocyte cytotoxicity assay, showing that AAV-specic effector CD8+ T cells expanded from human donors were able to lyse target hepatocyte cells transduced with AAV (J Clin Invest. 2009; 119:1688-95). Using this in vitro model, we investigated the engagement of regulatory T cells as a possible alternative to immunosuppression strategies for circumventing unwanted immune responses associated with viral gene transfer. Using a MHC Class II-restricted T cell epitope in the Fc region of IgG, which has been previously shown to induce regulatory T cells (Tregitope, Blood 2008; 112:3303-3311), we have been able to demonstrate the inhibition of AAV capsid-specic CTL killing of target hepatocytes. Regulatory T cell populations also expanded in the presence of the Tregitope, further showing the potential of this approach. Here we report Tregitope activity in an additional MHC Class I system: HLA allele A*0101 which binds to the AAV capsid epitope SADNNNSEY. Co-culture of effector cells with both peptide and Tregitope inhibited CTL-mediated killing by 60% in both peptideloaded and AAV-2 transduced hepatocytes. To date, Tregitope activity has been shown using PBMCs expanded from 8/8 donors with different HLA haplotypes, suggesting a high level of promiscuity of binding among different MHC Class II molecules. Polyfunctional analysis for markers of T cell activation was performed, and cumulative results from multiple donors show that CD8+ T cells incubated with the Tregitope have a 6-fold decrease in the production of IL-2, 4-fold decrease in TNF-α, and 3-fold decrease of IFN-γ. To elucidate the mechanism of Tregitope-mediated inhibition, we used a depletion approach to separate populations of CD8+ T cells and CD4+ Tregs prior to performing the assay and observed that removal of Tregs after expansion still resulted in a loss of CTL activity (60%), suggesting the CD8+ cells are inhibited during expansion. Furthermore, CTL activity of CD8+ T cells expanded against AAV separately was only slightly (30%) inhibited by the CD4+ T cell fraction when added after expansion, suggesting time of incubation could also play a role in the inhibition of CTL responses. We conclude that the use of Tregitopes represents a novel approach for antigen-specic modulation of capsid-specic T cell responses.
44. Long-Term Phenotypic Correction of Hemophilia A after Neonatal Administration of AAV
Chuhong Hu,1 Fides D. Lay,1 Joseph Springer,1 Ronald W. Busuttil,1 Gerald S. Lipshutz.1 1 Surgery, UCLA School of Medicine, Los Angeles, CA.
Hemophilia A is a genetic disorder caused by mutations in the factor VIII gene leading to a coagulation abnormality. Clinical manifestations can lead to serious complications and recombinant S18
Molecular Therapy Volume 18, Supplement 1, May 2010 Copyright © The American Society of Gene & Cell Therapy
42. Intensive Pharmacological Immunosuppression Allows for Repetitive Liver Gene Transfer with Recombinant Adenovirus in Nonhuman Primates
Antonio Fontanellas,1 Sandra Hervás-Stubbs,1 Itsaso Mauleón,1 Juan Dubrot,1 Uxua Mancheño,1 María Collantes,2 Ana Sampedro,1 Carmen Unzu,1 Carlos Alfaro,1 Asis Palazón,1 Rafael Enríquez de Salamanca,3 Cristian Smerdou,1 Alberto Benito,4 Jesús Prieto,1 Iván Peñuelas,5 Ignacio Melero.1 1 Gene Therapy and Hepatology Area, Centre for Applied Medical Research, University of Navarra, Pamplona, Spain; 2MicroPET Research Unit, CIMA-CUN, University of Navarra, Pamplona, Spain; 3Research Center, Hospital Universitario 12 de Octubre, Madrid, Spain; 4Department of Radiology, Clínica Universidad de Navarra, University of Navarra, Pamplona, Spain; 5Department of Nuclear Medicine, Clínica Universidad de Navarra, University of Navarra, Pamplona, Spain. Repeated administration of gene therapies is hampered by host immunity towards vectors and transgenes. Attempts to circumvent anti-vector immunity include pharmacological immunosuppression or alternating different vectors and vector serotypes with the same transgene. The aim of this study was to combine clinically available immunosuppressant drugs permitting repeated liver gene transfer to macaques. The immunosuppression regime included (i) Rituximab (20mg/kg/dose i.v.) at days -9, -6, -3, immediately before adenovirus injections and weekly following the adenoviral administration, (ii) two doses of 3 mg/kg of rabbit antithymocyte immunoglobulin (ATG) at days -2 and -1 before the adenovirus injection. (iii) Methylprednisolone was applied intramuscularly 10 min before the ATG infusion at a dose of 100 mg on day -2 and 50 mg on day -1 to avoid systemic inammation. (iv) Mycophenolate mofetil (MMF) doses were 25-30 mg/kg/day and (v) for FK506 0.25 mg /kg/day. MMF and FK506 were orally given daily from day -2 during three months after vector infusion. Our studies show that B-cell depletion with anti-CD20 monoclonal antibody and concomitant T-cell inhibition with clinically available drugs permit repeated liver gene transfer to a limited number of non-human primates with recombinant adenovirus. Adenoviral vector-mediated transfer of the herpes simplex virus type 1 thymidine kinase (HSV1-tk) reporter gene was visualized in vivo with a semiquantitative transgene-specic positron emission tomography (PET) technique, liver immunohistochemistry and immunoblot for the reporter transgene in needle biopsies. Neutralizing antibody and T cell-mediated responses towards the viral capsids were sequentially monitored and found to be repressed by the drug combinations tested. Repeated liver transfer of the HSV1-tk reporter gene with the same recombinant adenoviral vector was achieved in macaques undergoing a clinically feasible immunosuppressive treatments that ablated humoral and cellular immune responses. This strategy allows measurable gene retransfer to the liver as late as 15 months following the rst adenoviral exposure in a macaque, which has undergone a total of four successful gene-transfer procedures with the same adenoviral vector.
43. Induction of Regulatory T Cells as an Approach To Modulate CTL Responses Against AAV Capsid Epitopes
Daniel J. Hui,1 Gary Pien,1 Etiena Basner-Tschakarjan,1 Federico Mingozzi,1 Jonathan D. Finn,1 Shangzhen Zhou,1 William Martin,2 Anne S. De Groot,2 Katherine A. High.1,3 1 Pediatrics – Hematology, Children’s Hospital of Philadelphia, Philadelphia, PA; 2EpiVax, Inc., Providence, RI; 3Howard Hughes Medical Institute, Philadelphia, PA. Adeno-associated viral (AAV) vectors are promising tools for gene transfer strategies. However, the potential for untoward immune responses to the viral vector remains one of the last barriers to successful gene transfer, as was the case in the rst clinical trial using AAV serotype 2 to deliver Factor IX to Hemophilia B subjects via a liver-directed approach. Results from that trial showed that expression of F.IX was short-lived, followed by transient elevation of liver enzymes, which we hypothesized to result from the reactivation of AAV capsid-specic memory CD8+ T cells generated during a previous exposure to AAV. We have been able to model this hypothesis in vitro using a human hepatocyte cytotoxicity assay, showing that AAV-specic effector CD8+ T cells expanded from human donors were able to lyse target hepatocyte cells transduced with AAV (J Clin Invest. 2009; 119:1688-95). Using this in vitro model, we investigated the engagement of regulatory T cells as a possible alternative to immunosuppression strategies for circumventing unwanted immune responses associated with viral gene transfer. Using a MHC Class II-restricted T cell epitope in the Fc region of IgG, which has been previously shown to induce regulatory T cells (Tregitope, Blood 2008; 112:3303-3311), we have been able to demonstrate the inhibition of AAV capsid-specic CTL killing of target hepatocytes. Regulatory T cell populations also expanded in the presence of the Tregitope, further showing the potential of this approach. Here we report Tregitope activity in an additional MHC Class I system: HLA allele A*0101 which binds to the AAV capsid epitope SADNNNSEY. Co-culture of effector cells with both peptide and Tregitope inhibited CTL-mediated killing by 60% in both peptideloaded and AAV-2 transduced hepatocytes. To date, Tregitope activity has been shown using PBMCs expanded from 8/8 donors with different HLA haplotypes, suggesting a high level of promiscuity of binding among different MHC Class II molecules. Polyfunctional analysis for markers of T cell activation was performed, and cumulative results from multiple donors show that CD8+ T cells incubated with the Tregitope have a 6-fold decrease in the production of IL-2, 4-fold decrease in TNF-α, and 3-fold decrease of IFN-γ. To elucidate the mechanism of Tregitope-mediated inhibition, we used a depletion approach to separate populations of CD8+ T cells and CD4+ Tregs prior to performing the assay and observed that removal of Tregs after expansion still resulted in a loss of CTL activity (60%), suggesting the CD8+ cells are inhibited during expansion. Furthermore, CTL activity of CD8+ T cells expanded against AAV separately was only slightly (30%) inhibited by the CD4+ T cell fraction when added after expansion, suggesting time of incubation could also play a role in the inhibition of CTL responses. We conclude that the use of Tregitopes represents a novel approach for antigen-specic modulation of capsid-specic T cell responses.
44. Long-Term Phenotypic Correction of Hemophilia A after Neonatal Administration of AAV
Chuhong Hu,1 Fides D. Lay,1 Joseph Springer,1 Ronald W. Busuttil,1 Gerald S. Lipshutz.1 1 Surgery, UCLA School of Medicine, Los Angeles, CA.
Hemophilia A is a genetic disorder caused by mutations in the factor VIII gene leading to a coagulation abnormality. Clinical manifestations can lead to serious complications and recombinant S18
Molecular Therapy Volume 18, Supplement 1, May 2010 Copyright © The American Society of Gene & Cell Therapy