Aim Proposal

SPECIFIC AIMS

Adeno-associated virus (AAV) vector has been successfully applied to target the liver in clinical trials with hemophilia patients1,2. These trials have suggested that the AAV capsid specific cytotoxic T lymphocytes (CTLs) eliminate AAV vector targeted liver cells, following AAV2 or AAV8 transduction, and result in therapeutic failure. Our studies and others have demonstrated that both classical antigen presentation and cross-presentation pathways are involved in mounting an AAV capsid specific CTL response3-7. In clinical trials, ion exchange chromatography has been used to purify AAV vectors. Unlike CsCl purification approach, the chromatographic method cannot currently separate genome-containing AAV capsids (full particles) from empty particles. The contamination of empty virions potentially increases the AAV capsid antigen load in transduced cells and it has been demonstrated that empty virion contamination in vector preparations induces liver damage, which potentially enhances capsid antigen presentation from full virion transduction8,9. Although we have observed a lower capsid antigen presentation from AAV empty virion infection compared to full particles in vitro10, our in vivo preliminary result demonstrated that AAV empty capsids still elicit capsid antigen presentation. In this proposal we will investigate the kinetics of capsid antigen presentation from empty virions and the effect of empty particles on antigen presentation from full particle transduction (Aim 1a and 1b). We have demonstrated that AAV capsid cross-presentation is dependent on virion endosomal escape and proteasome-mediated capsid degradation in AAV transduced cells in vitro10. However, the mechanistic insights of the work were largely elucidated in vitro, and over a limited time period (24 to 48 hrs), and therefore it remains unclear which aspects of our discoveries translate in vivo regarding long-term antigen processing and presentation. The mechanism of capsid antigen presentation from empty virions and full particles in vivo will be performed using mouse models deficient in the genes responsible for classical class-I antigen presentation (TAP -/- mice) or classical class-II antigen presentation (Cat S -/- mice) (Aim 1c). Our data have shown that capsid antigen presentation is dose-dependent and requires capsid ubiquitination for proteasome mediated degradation11,12. To decrease antigen presentation on AAV transduced cells for avoiding capsid specific CTL-mediated elimination, it has been proposed to modify the AAV capsid surface in order to enhance AAV transduction while lowering the effective dose, or to escape capsid ubiquitination13,14. It is unclear whether the enhancement of liver transduction with AAV mutants or a decrease in capsid ubiquitination influences capsid antigen presentation in vivo (Aim 2a). Proteasome inhibitors have been shown to enhance AAV transduction and inhibit antigen presentation12,15. However, our further in vitro study demonstrated varying effects of the proteasome inhibitor on capsid antigen cross-presentation in a dose related manner. A high dose of the proteasome inhibitor bortezomib blocks capsid antigen presentation, while a lower dose of bortezomib increases capsid antigen presentation without enhanced transduction. We hypothesize that proteasome inhibitor treatment will change the profile of AAV antigen presentation in vivo and the combination of AAV mutants and proteasome inhibitors will further increase AAV transduction while inhibiting capsid antigen presentation (Aim 2b and 2c). It is well-known that the transduction of AAV vectors in mouse models does not always translate into the human host. To address this, a mouse model xenografted with human hepatocytes has been used to develop AAV vectors for human liver targeting gene therapy16. In this proposal we will explore the directed evolution approach combined with a rational design strategy to isolate AAV vectors with human hepatocyte specific tropism and the ability to evade a capsid specific CTL response in humanized mice (Aim 3). Elucidation of AAV empty capsid antigen presentation in vivo and the development of an AAV vector with enhanced human liver transduction and CTL immune-evasion, will allow us to design safer and more effective strategies that address the current clinical complications for human liver gene therapy using AAV. To address these issues, we will execute the following specific aims: 1. Study the effect of AAV empty particles on AAV capsid antigen cross-presentation in vivo.

a. The kinetics and dose-response of AAV capsid antigen presentation from AAV empty virions in vivo. b. The effect of empty particles on capsid antigen presentation from full-particle AAV transduction in vivo. c. AAV capsid antigen presentation in TAP-/- and in Cat S-/- mice.

2. Investigate AAV capsid antigen presentation following administration of AAV mutants and/or proteasome inhibitors for enhanced liver transduction in vivo. a. Capsid antigen presentation from AAV mutants with enhanced liver transduction in mice. b. The effect of proteasome inhibitors (high vs low dose) on natural AAV capsid antigen presentation in

vivo. c. The effect of a combination of AAV mutants with proteasome inhibitors on antigen presentation in vivo.

3. Isolate AAV chimeric capsids with human hepatocyte tropism and the capacity for CTL evasion. a. Verify AAV human liver transduction efficiency in xenograft mice. b. Characterization of AAV mutants recovered from human liver xenografted mice. c. Investigation of capsid CTL evasion from humanized AAV mutants.

Contact PD/PI: Li, Chengwen

Specific Aims Page 33

Contact PD/PI: Li, Chengwen

LITERATURE CITED

1 Manno, C. S. et al. Successful transduction of liver in hemophilia by AAV-Factor IX and limitations imposed by the host immune response. Nat Med 12, 342-347, doi:10.1038/nm1358 (2006).

2 Nathwani, A. C. et al. Adenovirus-associated virus vector-mediated gene transfer in hemophilia B. N Engl J Med 365, 2357-2365, doi:10.1056/NEJMoa1108046 (2011).

3 Li, C. et al. Adeno-associated virus type 2 (AAV2) capsid-specific cytotoxic T lymphocytes eliminate only vector-transduced cells coexpressing the AAV2 capsid in vivo. J Virol 81, 7540-7547, doi:10.1128/JVI.00529-07 (2007).

4 Chen, J., Wu, Q., Yang, P., Hsu, H. C. & Mountz, J. D. Determination of specific CD4 and CD8 T cell epitopes after AAV2- and AAV8-hF.IX gene therapy. Molecular therapy : the journal of the American Society of Gene Therapy 13, 260-269, doi:10.1016/j.ymthe.2005.10.006 (2006).

5 Wang, L., Figueredo, J., Calcedo, R., Lin, J. & Wilson, J. M. Cross-presentation of adeno-associated virus serotype 2 capsids activates cytotoxic T cells but does not render hepatocytes effective cytolytic targets. Human gene therapy 18, 185-194, doi:10.1089/hum.2007.001 (2007).

6 Sabatino, D. E. et al. Identification of mouse AAV capsid-specific CD8+ T cell epitopes. Molecular therapy : the journal of the American Society of Gene Therapy 12, 1023-1033, doi:10.1016/j.ymthe.2005.09.009 (2005).

7 Li, H. et al. Pre-existing AAV capsid-specific CD8+ T cells are unable to eliminate AAV-transduced hepatocytes. Molecular therapy : the journal of the American Society of Gene Therapy 15, 792-800, doi:10.1038/sj.mt.6300090 (2007).

8 Wright, J. F. AAV empty capsids: for better or for worse? Molecular therapy : the journal of the American Society of Gene Therapy 22, 1-2, doi:10.1038/mt.2013.268 (2014).

9 Gao, K. et al. Empty virions in AAV8 vector preparations reduce transduction efficiency and may cause total viral particle dose-limiting side effects. Molecular Therapy — Methods & Clinical Development 1, 1-8 (2014).

11 He, Y. et al. Kinetics of adeno-associated virus serotype 2 (AAV2) and AAV8 capsid antigen presentation in vivo are identical. Human gene therapy 24, 545-553, doi:10.1089/hum.2013.065 (2013).

12 Li, C. et al. Adeno-associated virus capsid antigen presentation is dependent on endosomal escape. J Clin Invest 123, 1390-1401, doi:10.1172/JCI66611 (2013).

13 Shen, S. et al. Engraftment of a galactose receptor footprint onto adeno-associated viral capsids improves transduction efficiency. J Biol Chem 288, 28814-28823, doi:10.1074/jbc.M113.482380 (2013).

14 Zhong, L. et al. Next generation of adeno-associated virus 2 vectors: point mutations in tyrosines lead to high-efficiency transduction at lower doses. Proc Natl Acad Sci U S A 105, 7827-7832, doi:10.1073/pnas.0802866105 (2008).

15 Mitchell, A. M. & Samulski, R. J. Mechanistic insights into the enhancement of adeno-associated virus transduction by proteasome inhibitors. J Virol, doi:10.1128/JVI.01826-13 (2013).

16 Lisowski, L. et al. Selection and evaluation of clinically relevant AAV variants in a xenograft liver model. Nature 506, 382-386, doi:10.1038/nature12875 (2014).

17 Inaba, K. & Inaba, M. Antigen recognition and presentation by dendritic cells. Int J Hematol 81, 181- 187, doi:10.1532/IJH97.04200 (2005).

18 Gao, G. et al. Purification of recombinant adeno-associated virus vectors by column chromatography and its performance in vivo. Human gene therapy 11, 2079-2091, doi:10.1089/104303400750001390 (2000).

19 Lock, M., Alvira, M. R. & Wilson, J. M. Analysis of particle content of recombinant adeno-associated virus serotype 8 vectors by ion-exchange chromatography. Hum Gene Ther Methods 23, 56-64 (2012).

20 Qu, G. et al. Separation of adeno-associated virus type 2 empty particles from genome containing vectors by anion-exchange column chromatography. J Virol Methods 140, 183-192, doi:10.1016/j.jviromet.2006.11.019 (2007).

21 Urabe, M. et al. Removal of empty capsids from type 1 adeno-associated virus vector stocks by anion- exchange chromatography potentiates transgene expression. Molecular therapy : the journal of the American Society of Gene Therapy 13, 823-828, doi:10.1016/j.ymthe.2005.11.024 (2006).

References Cited Page 51