Immunological Barriers to Gene Therapy

Gene therapy has emerged as an important therapeutic tool for treatment of genetic diseases. The initial emphasis of the field for vectors for delivery of foreign genes has shifted to the immunological barriers that prevent long term transgene expressing and readministration. Extensive studies have shown that transient transgene expression is primarily due to immune responses to vector and also to the transgene product. The Immunology Core focuses on development state-of-the-art assays to assist in the understanding of mechanisms of immune-mediated transgene elimination and readministration of vectors, immune suppression/modulation regimens.

The concepts of gene therapy have opened new avenues to treatment of diseases. The central role of cellular and humoral immune responses against vector and transgene products have been appreciated since the earliest attempts of in vivo gene transfer. Several components of the immune system participate in generation of a co-ordinated cascade of events, which culminate in activation of effector cells, namely cytotoxic T cells and antibody secreting B cells. Several studies have shown that CD4 T helper cells play a central role in induction of both cytotoxic T cells and B cells. The first barrier for infections is the innate immunity, which comprises of neutrophils, tissue macrophages, NK cells, a host of soluble factors including complement, anti-microbial agents and inflammatory cytokines. Presentation of endogenous antigens by MHC class I (above) , and exogenous antigens by MHC class II (below) initiate the immune responses by activation of both CD8 and CD4 T cells. Recent advances in the mechanism of antigen presentation will allow manipulation antigen processing and presentation to interfere with activation or modulation of antigen-specific immune activation. A central role for the dendritic cells (antigen-presenting cells) in regulating immune responses has been established by the demonstration that these cells capture and process antigens, express lymphocyte co-stimulatory molecules, migrate to lymphoid organs and secrete cytokines.

Activation of CD4+ T cells (below) requires two signals; one transduced by recognition of antigen in the context of MHC class II molecules, and the second by co-stimulatory molecules e.g. CD28. Several studies have suggested that interaction of CD40L on T cells with CD40 on antigen presenting cells leads to expression of CD80/CD86 molecules which in turn bind to CD28 on T cells. Interruption of this sequential cascade of events results in T cell unresponsiveness. Delineation of critical events leading to complete T cell effector function has allowed development of novel strategies in regulation of immune responses, which may ultimately lead to long term transgene expression following gene therapy.

Regulated activation of B cells also involves a complex series of events leading to immunoglobulin secretion, which neutralizes soluble transgene expression, and impedes re-administration potential of viral vectors. Antigen is first complexed by components of complement systems and then presented to B cells in complex form on follicular dendritic cells in the lymph nodes. The complexed antigen is taken up by B cells through the BCR and processed and presented by MHC class II to T helper cells. Activated B cells expressing CD80/CD86 trigger activation of CD4 helper T cells. The helper function provided by CD4+ T cells to induce B cells to differentiate in to immunoglobulin secretion includes cell - cell contact, through molecular interactions, e.g. CD40L - CD40, or soluble cytokines, e.g. IL-4. It is in these responses that B cells generate immunological memory, and increase affinity by somatic mutation and finally differentiate into antibody secreting plasma cells.

A majority of the pharmaceutical intervention strategies that have been extensively investigated in clinical trials for transplantation and are being adapted for gene therapy are inhibitors of cell mediated immune function. With the advent of novel vectors, e.g. AAV and lentiviruses which result in stable gene expression over prolonged periods for the first time in treatment of human disease, immuno suppressive therapies need to be targeted to inhibition of antigen-specific B cells.

References

The following references represent some of the work which the GTP Immunology Group has published.

1. McClane, S.J., N. Chirmule, C.V. Burke and S.E. Raper, Characterization of the immune response after local delivery of recombinant adenovirus in murine pancreas and successful strategies for readministration.Hum Gene Ther, 8(18):2207-16, Dec 1997.
2. Chirmule, N., J.V. Hughes, G.P. Gao, S.E. Raper and J.M. Wilson, Role of E4 in eliciting CD4 T-cell and B-cell responses to adenovirus vectors delivered to murine and nonhuman primate lungs.J Virol, 72(7):6138-45, Jul 1998.
3. Bennett, J., A.M. Maguire, A.V. Cideciyan, M. Schnell, E. Glover, V. Anand, T.S. Aleman, N. Chirmule, A.R. Gupta, Y. Huang, G.P. Gao, W.C. Nyberg, J. Tazelaar, J. Hughes, J.M. Wilson and S.G. Jacobson, Stable transgene expression in rod photoreceptors after recombinant adeno-associated virus-mediated gene transfer to monkey retina. Proc Natl Acad Sci U S A, 1999. 96(17): p. 9920-5.
4. Chirmule, N., A. Avots, S.M. LakshmiTamma, S. Pahwa and E. Serfling, CD4-mediated signals induce T cell dysfunction in vivo. J Immunol, 1999. 163(2): p. 644-9.
5. Chirmule, N., J. Moffett, P. Dhagat, J. Tazelaar and J.M. Wilson, Adenoviral vector-mediated gene therapy in the mouse lung: no role of Fas-Fas ligand interactions for elimination of transgene expression in bronchioepithelial cells. Hum Gene Ther, 1999. 10(17): p. 2839-46.
6. Chirmule, N., A.D. Moscioni, Y. Qian, R. Qian, Y. Chen and J.M. Wilson, Fas-Fas ligand interactions play a major role in effector functions of cytotoxic T lymphocytes after adenovirus vector-mediated gene transfer. Hum Gene Ther, 1999. 10(2): p. 259-69.
7. Chirmule, N., K. Propert, S. Magosin, Y. Qian, R. Qian and J. Wilson, Immune responses to adenovirus and adeno-associated virus in humans. Gene Ther, 1999. 6(9): p. 1574-83.
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10. Zuckerman, J.B., C.B. Robinson, K.S. McCoy, R. Shell, T.J. Sferra, N. Chirmule, S.A. Magosin, K.J. Propert, E.C. Brown-Parr, J.V. Hughes, J. Tazelaar, C. Baker, M.J. Goldman and J.M. Wilson, A phase I study of adenovirus-mediated transfer of the human cystic fibrosis transmembrane conductance regulator gene to a lung segment of individuals with cystic fibrosis. Hum Gene Ther, 1999. 10(18): p. 2973-85.
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12. Chirmule, N., J. Tazelaar and J.M. Wilson, Th2-dependent B cell responses in the absence of CD40-CD40 ligand interactions. J Immunol, 2000. 164(1): p. 248-55.
13. Chirmule, N., W. Xiao, A. Truneh, M.A. Schnell, J.V. Hughes, P. Zoltick and J.M. Wilson, Humoral immunity to adeno-associated virus type 2 vectors following administration to murine and nonhuman primate muscle. J Virol, 2000. 74(5): p. 2420-5.
14. Sarkar, R., G.P. Gao, N. Chirmule, J. Tazelaar and H.H. Kazazian, Jr., Partial correction of murine hemophilia A with neo-antigenic murine factor VIII. Hum Gene Ther, 2000. 11(6): p. 881-94.
15. Gao, G.P., M.R. Alvira, L. Wang, R. Calcedo, J. Johnston and J.M. Wilson, Novel adeno-associated viruses from rhesus monkeys as vectors for human gene therapy. Proc Natl Acad Sci U S A, 2002. 99(18): p. 11854-9.
16. Gao, G., M.R. Alvira, S. Somanathan, Y. Lu, L.H. Vandenberghe, J.J. Rux, R. Calcedo, J. Sanmiguel, Z. Abbas and J.M. Wilson, Adeno-associated viruses undergo substantial evolution in primates during natural infections. Proc Natl Acad Sci U S A, 2003. 100(10): p. 6081-6.
17. Gao, G., C. Lebherz, D.J. Weiner, R. Grant, R. Calcedo, B. McCullough, A. Bagg, Y. Zhang and J.M. Wilson, Erythropoietin gene therapy leads to autoimmune anemia in macaques. Blood, 2004. 103(9): p. 3300-2.
18. Gao, G., L.H. Vandenberghe, M.R. Alvira, Y. Lu, R. Calcedo, X. Zhou and J.M. Wilson, Clades of Adeno-associated viruses are widely disseminated in human tissues. J Virol, 2004. 78(12): p. 6381-8.
19. Roy, S., G. Gao, Y. Lu, X. Zhou, M. Lock, R. Calcedo and J.M. Wilson, Characterization of a family of chimpanzee adenoviruses and development of molecular clones for gene transfer vectors. Hum Gene Ther, 2004. 15(5): p. 519-30.
20. Roy, S., D.S. Clawson, R. Calcedo, C. Lebherz, J. Sanmiguel, D. Wu and J.M. Wilson, Use of chimeric adenoviral vectors to assess capsid neutralization determinants. Virology, 2005. 333(2): p. 207-14.
21. Varnavski, A.N., R. Calcedo, M. Bove, G. Gao and J.M. Wilson, Evaluation of toxicity from high-dose systemic administration of recombinant adenovirus vector in vector-naive and pre-immunized mice. Gene Ther, 2005. 12(5): p. 427-36.
22. Wang, L., R. Calcedo, T.C. Nichols, D.A. Bellinger, A. Dillow, I.M. Verma and J.M. Wilson, Sustained correction of disease in naive and AAV2-pretreated hemophilia B dogs: AAV2/8-mediated, liver-directed gene therapy. Blood, 2005. 105(8): p. 3079-86.
23. Roy, S., Y. Zhi, G.P. Kobinger, J. Figueredo, R. Calcedo, J.R. Miller, H. Feldmann and J.M. Wilson, Generation of an adenoviral vaccine vector based on simian adenovirus 21. J Gen Virol, 2006. 87(Pt 9): p. 2477-85.
24. Vandenberghe, L.H., L. Wang, S. Somanathan, Y. Zhi, J. Figueredo, R. Calcedo, J. Sanmiguel, R.A. Desai, C.S. Chen, J. Johnston, R.L. Grant, G. Gao and J.M. Wilson, Heparin binding directs activation of T cells against adeno-associated virus serotype 2 capsid. Nat Med, 2006. 12(8): p. 967-71.
25. Zhi, Y., J. Figueredo, G.P. Kobinger, H. Hagan, R. Calcedo, J.R. Miller, G. Gao and J.M. Wilson, Efficacy of severe acute respiratory syndrome vaccine based on a nonhuman primate adenovirus in the presence of immunity against human adenovirus. Hum Gene Ther, 2006. 17(5): p. 500-6.
26. Limberis, M.P., J. Figueredo, R. Calcedo and J.M. Wilson, Activation of CFTR-specific T Cells in cystic fibrosis mice following gene transfer. Mol Ther, 2007. 15(9): p. 1694-700.
27. Roy, S., G.P. Kobinger, J. Lin, J. Figueredo, R. Calcedo, D. Kobasa and J.M. Wilson, Partial protection against H5N1 influenza in mice with a single dose of a chimpanzee adenovirus vector expressing nucleoprotein. Vaccine, 2007. 25(39-40): p. 6845-51.
28. Wang, L., J. Figueredo, R. Calcedo, J. Lin and J.M. Wilson, Cross-presentation of adeno-associated virus serotype 2 capsids activates cytotoxic T cells but does not render hepatocytes effective cytolytic targets. Hum Gene Ther, 2007. 18(3): p. 185-94.

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