CTL response to AAV Vector

Summary

Principal Investigator: Richard J Samulski
Abstract: Adeno-associated virus (AAV) is a very promising gene therapy vector in pre-clinical and clinical trials. However, recent studies have demonstrated that the AAV2 capsid can induce a cytotoxic T lymphocyte (CTL) response via both classical antigen presentation and cross-presentation pathways, thereby raising concerns associated with immune response to AAV vectors. In particular, it has been suggested that capsid specific CTLs eliminated AAV2 transduced liver cells and resulted in therapeutic failure in a hemophilia B clinical trial. The goal of this proposal is to understand the mechanisms of presentation of AAV capsid antigens in vitro and in vivo and more importantly, to devise strategies to evade the immune response. Our long term goals are to enhance the safety and efficacy of AAV vectors through formulation of novel immune evasion strategies. Since data from animal models have contradicted clinical observations outlined above (possibly due to poor immunogenicity of the AAV capsid in mice), we have integrated a strong immune domain OVA epitope SIINFEKL into the VP3 protein of the AAV2 capsid (AAV2-OVA). Decreased transgene expression was seen in mice with memory OVA CTLs following liver transduction with AAV2-OVA vector. In the current proposal, we will use AAV2-OVA vector to investigate the kinetics and mechanisms of AAV capsid cross-presentation in transduced cells in vitro and in vivo (Aim 1 and 2). Through these studies, we expect to thoroughly characterize the CTL response to AAV capsid proteins in mice that more accurately represents data obtained in humans. After AAV vector binds on cell surface, via endosomal uptake, AAV2 capsids must uncoat enroute to the nucleus prior to vector genome transcription. This trafficking route suggests that antigen presentation of capsids after transduction may follow a classical MHC-class I pathways. Many viruses (For example herpes, EB) evade host immune response by synthesizing small peptides called viral proteins interfering with antigen presentation (VIPR), we propose that integration of VIPR into AAV capsid evade host CTL mediated elimination of AAV transduced target cells (Aim 3). By engineering VIPRs into AAV2 capsid proteins, we will ensure that antigen presentation will be attenuated only in AAV2 transduced cells without systemic side effects on the immune system (as would be the case with immunosuppressive drugs or application of regulator T cells). These experiments rely on predetermined domains in AAV capsid proteins for incorporation of VIPR domains. A timely understanding is critical for the continued use of AAV therapy under the current protocols (i.e. without immunosuppression addendums). PUBLIC HEALTH RELEVANCE: Lay summary Adeno-associated virus (AAV) has been used in over 50 clinical trials and proves to be very promising for gene therapy, due to long-term therapeutic gene expression after delivery of AAV vectors. Recently, data from one clinical trial for hemophilia B suggested that AAV2 could induce an immune response and thus eliminate AAV2 infected liver cells. However, the results from a mouse model study did not support these findings. One explanation of these contradictory results could be the weak immunogenic activity of AAV capsids in mice. To more accurately mimic the clinical trial and establish an appropriate mouse model, we propose to insert a strong immunogen into AAV2 capsid and use these mutant AAV capsids to make recombinant virus. After injection of these viruses into mouse, we will investigate whether immunogen specific CTLs eliminate AAV2 transduced target cells in mice. Additionally, to reduce any immune response elicited by the AAV capsid, we will insert the Epstein-Barr virus product EBNA-1 into a non-essential region of the AAV capsid. We will test whether EBNA-1 will mediate inhibition of an immune response and prevent eradication of AAV infected cells. The long-term goals of this proposal are to critically evaluate the immune response to AAV-infected cells and to develop a novel AAV vector that will evade an immune response without compromising function, thus improving AAV as a gene therapy tool and enhancing its therapeutic value.
Funding Period: 2009-12-01 - 2014-11-30
more information: NIH RePORT

Top Publications

  1. pmc Mutagenesis of adeno-associated virus type 2 capsid protein VP1 uncovers new roles for basic amino acids in trafficking and cell-specific transduction
    Jarrod S Johnson
    Department of Pharmacology, Gene Therapy Center, University of North Carolina, Chapel Hill, North Carolina 27599 7352, USA
    J Virol 84:8888-902. 2010
  2. pmc Promyelocytic leukemia protein is a cell-intrinsic factor inhibiting parvovirus DNA replication
    Angela M Mitchell
    Gene Therapy Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA
    J Virol 88:925-36. 2014
  3. pmc K137R mutation on adeno-associated viral capsids had minimal effect on enhancing gene delivery in vivo
    Chunping Qiao
    1 Division of Molecular Pharmaceutics, Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599
    Hum Gene Ther Methods 25:33-9. 2014
  4. pmc Mechanistic insights into the enhancement of adeno-associated virus transduction by proteasome inhibitors
    Angela M Mitchell
    Gene Therapy Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA
    J Virol 87:13035-41. 2013
  5. pmc Oversized AAV transductifon is mediated via a DNA-PKcs-independent, Rad51C-dependent repair pathway
    Matthew L Hirsch
    1 Gene Therapy Center, University of North Carolina, Chapel Hill, North Carolina, USA 2 Department of Ophthalmology, University of North Carolina, Chapel Hill, North Carolina, USA
    Mol Ther 21:2205-16. 2013
  6. pmc β-cell-specific IL-2 therapy increases islet Foxp3+Treg and suppresses type 1 diabetes in NOD mice
    Mark C Johnson
    Department of Microbiology and Immunology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina
    Diabetes 62:3775-84. 2013
  7. pmc Kinetics of adeno-associated virus serotype 2 (AAV2) and AAV8 capsid antigen presentation in vivo are identical
    Yi He
    Institute of Hematology and Blood Diseases Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Tianjin 300020, China
    Hum Gene Ther 24:545-53. 2013
  8. pmc Adeno-associated virus capsid antigen presentation is dependent on endosomal escape
    Chengwen Li
    Gene Therapy Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA
    J Clin Invest 123:1390-401. 2013
  9. pmc Arsenic trioxide stabilizes accumulations of adeno-associated virus virions at the perinuclear region, increasing transduction in vitro and in vivo
    Angela M Mitchell
    Gene Therapy Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA
    J Virol 87:4571-83. 2013
  10. pmc Cytoplasmic trafficking, endosomal escape, and perinuclear accumulation of adeno-associated virus type 2 particles are facilitated by microtubule network
    Ping jie Xiao
    Gene Therapy Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA
    J Virol 86:10462-73. 2012

Detail Information

Publications19

  1. pmc Mutagenesis of adeno-associated virus type 2 capsid protein VP1 uncovers new roles for basic amino acids in trafficking and cell-specific transduction
    Jarrod S Johnson
    Department of Pharmacology, Gene Therapy Center, University of North Carolina, Chapel Hill, North Carolina 27599 7352, USA
    J Virol 84:8888-902. 2010
    ..e., subnuclear mobilization or uncoating) limit successful AAV infection...
  2. pmc Promyelocytic leukemia protein is a cell-intrinsic factor inhibiting parvovirus DNA replication
    Angela M Mitchell
    Gene Therapy Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA
    J Virol 88:925-36. 2014
    ..Additionally, PMLII inhibition of AAV second-strand synthesis and replication, which are processes necessary for all parvoviruses, suggests implications for replication of other parvoviruses. ..
  3. pmc K137R mutation on adeno-associated viral capsids had minimal effect on enhancing gene delivery in vivo
    Chunping Qiao
    1 Division of Molecular Pharmaceutics, Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599
    Hum Gene Ther Methods 25:33-9. 2014
    ..In addition, no difference was found in transgene expression with either AAV7-K137R or AAV9-K137R mutants. Our results indicated that the K137R mutation on AAV7, AAV8, and AAV9 had minimal to no effect on transduction efficiency in vivo...
  4. pmc Mechanistic insights into the enhancement of adeno-associated virus transduction by proteasome inhibitors
    Angela M Mitchell
    Gene Therapy Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA
    J Virol 87:13035-41. 2013
    ..We determined that proteasome inhibitors act on rAAV through proteasome inhibition and not serine or cysteine protease inhibition, likely through positive changes late in transduction. ..
  5. pmc Oversized AAV transductifon is mediated via a DNA-PKcs-independent, Rad51C-dependent repair pathway
    Matthew L Hirsch
    1 Gene Therapy Center, University of North Carolina, Chapel Hill, North Carolina, USA 2 Department of Ophthalmology, University of North Carolina, Chapel Hill, North Carolina, USA
    Mol Ther 21:2205-16. 2013
    ..5 kb expression cassette restored dysferlin levels in a dystrophic model. Collectively, oversized AAV genome transduction requires unique DNA repair pathways and offers an alternative, more efficient strategy for large-gene therapy...
  6. pmc β-cell-specific IL-2 therapy increases islet Foxp3+Treg and suppresses type 1 diabetes in NOD mice
    Mark C Johnson
    Department of Microbiology and Immunology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina
    Diabetes 62:3775-84. 2013
    ..Collectively, these findings demonstrate that β-cell-specific IL-2 expands an islet-resident Foxp3(+)Tregs pool that effectively suppresses ongoing type 1 diabetes long term. ..
  7. pmc Kinetics of adeno-associated virus serotype 2 (AAV2) and AAV8 capsid antigen presentation in vivo are identical
    Yi He
    Institute of Hematology and Blood Diseases Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Tianjin 300020, China
    Hum Gene Ther 24:545-53. 2013
    ....
  8. pmc Adeno-associated virus capsid antigen presentation is dependent on endosomal escape
    Chengwen Li
    Gene Therapy Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA
    J Clin Invest 123:1390-401. 2013
    ..These results should facilitate the design of effective strategies to evade capsid-specific CTL-mediated elimination of AAV-transduced target cells in future clinical trials...
  9. pmc Arsenic trioxide stabilizes accumulations of adeno-associated virus virions at the perinuclear region, increasing transduction in vitro and in vivo
    Angela M Mitchell
    Gene Therapy Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA
    J Virol 87:4571-83. 2013
    ..In summary, our data support a model wherein As(2)O(3) increases rAAV transduction both in vitro and in vivo and maintains perinuclear accumulations of capsids, facilitating productive nuclear trafficking...
  10. pmc Cytoplasmic trafficking, endosomal escape, and perinuclear accumulation of adeno-associated virus type 2 particles are facilitated by microtubule network
    Ping jie Xiao
    Gene Therapy Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA
    J Virol 86:10462-73. 2012
    ....
  11. pmc Single amino acid modification of adeno-associated virus capsid changes transduction and humoral immune profiles
    Chengwen Li
    Gene Therapy Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA
    J Virol 86:7752-9. 2012
    ..Our results suggest that AAV types can be utilized not only as templates to generate mutants with enhanced transduction efficiency but also as substrates for repeat administration...
  12. pmc The AAV vector toolkit: poised at the clinical crossroads
    Aravind Asokan
    Gene Therapy Center, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599 7352, USA
    Mol Ther 20:699-708. 2012
    ..This review will provide an overview of preclinical studies with the ever-expanding AAV vector portfolio in large animal models and an update on new lead AAV vector candidates poised for clinical translation...
  13. pmc Quantitative 3D tracing of gene-delivery viral vectors in human cells and animal tissues
    Ping jie Xiao
    Gene Therapy Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599 7352, USA
    Mol Ther 20:317-28. 2012
    ....
  14. pmc Phase 1 gene therapy for Duchenne muscular dystrophy using a translational optimized AAV vector
    Dawn E Bowles
    Department of Surgery, Division of Surgical Sciences, Duke University Medical Center, Durham, North Carolina, USA
    Mol Ther 20:443-55. 2012
    ..g., limb infusion gene delivery) and should usher in the next generation of viral delivery systems for human gene transfer...
  15. pmc Combination therapy utilizing shRNA knockdown and an optimized resistant transgene for rescue of diseases caused by misfolded proteins
    Chengwen Li
    Gene Therapy Center, Department of Pediatrics, University of North Carolina, Chapel Hill, NC 27599, USA
    Proc Natl Acad Sci U S A 108:14258-63. 2011
    ..Based on these observations, similar gene-therapy strategies could be considered for any diseases caused by accumulation of misfolded proteins...
  16. pmc AAV-6 mediated efficient transduction of mouse lower airways
    Wuping Li
    State Key Laboratory for Molecular Virology and Genetic Engineering, Institute of Pathogen Biology at Beijing and The Institute of Blood Transfusion at Chengdu, Chinese Academy of Medical Sciences, 100730 China
    Virology 417:327-33. 2011
    ..Thus compared with AAV1, AAV6 has a unique ability to escape proteasome-mediated degradation, which is likely responsible for its higher transduction efficiency in mouse airway epithelium...
  17. ncbi Neutralizing antibodies against adeno-associated virus examined prospectively in pediatric patients with hemophilia
    C Li
    Gene Therapy Center, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA
    Gene Ther 19:288-94. 2012
    ..The results may guide rational design of clinical trials using alternative AAV serotypes and suggest that younger patients who are given AAV gene therapy will benefit from the lower prevalence of NAbs...
  18. ncbi Inducible adeno-associated virus-mediated IL-2 gene therapy prevents autoimmune diabetes
    Kevin S Goudy
    Department of Microbiology and Immunology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA
    J Immunol 186:3779-86. 2011
    ..These results demonstrate that inducible IL-2 gene therapy is an effective and safe approach to manipulate Foxp3(+)Treg and suppress T cell-mediated autoimmunity and that under the conditions employed, IL-2 is more potent than TGF-β1...
  19. pmc Parkinson's disease gene therapy: success by design meets failure by efficacy
    Raymond T Bartus
    1 Ceregene, Inc, San Diego, California, USA 2 RTBioconsultants, Inc, San Diego, California, USA
    Mol Ther 22:487-97. 2014
    ....