Proteasome Inhibitors Decrease AAV2 Capsid derived Peptide Epitope Presentation on MHC Class I Following Transduction

Abstract

Adeno-associated viral (AAV) vectors are an extensively studied and highly used vector platform for gene therapy applications. We hypothesize that in the first clinical trial using AAV to treat hemophilia B, AAV capsid proteins were presented on the surface of transduced hepatocytes, resulting in clearance by antigen-specific CD8+ T cells and consequent loss of therapeutic transgene expression. It has been previously shown that proteasome inhibitors can have a dramatic effect on AAV transduction in vitro and in vivo. Here, we describe using the US Food and Drug Administration-approved proteasome inhibitor, bortezomib, to decrease capsid antigen presentation on hepatocytes in vitro, whereas at the same time, enhancing gene expression in vivo. Using an AAV capsid-specific T-cell reporter (TCR) line to analyze the effect of proteasome inhibitors on antigen presentation, we demonstrate capsid antigen presentation at low multiplicities of infection (MOIs), and inhibition of antigen presentation at pharmacologic levels of bortezomib. We also demonstrate that bortezomib can enhance Factor IX (FIX) expression from an AAV2 vector in mice, although the same effect was not observed for AAV8 vectors. A pharmacological agent that can enhance AAV transduction, decrease T-cell activation/proliferation, and decrease capsid antigen presentation would be a promising solution to obstacles to successful AAV-mediated, liver-directed gene transfer in humans.

Enhanced liver gene transfer and evasion of preexisting humoral immunity with exosome-enveloped AAV vectors

“…Second, the ability to lower the vector dose to achieve full therapeutic efficacy would confer a clear advantage in terms of avoidance of CD8 1 T-cell responses directed to the capsid. 11 This point can be extrapolated from a number of preclinical and clinical studies, 12,[37][38][39][40] suggesting that at low vector doses it is less likely to experience vector-related immunotoxicities. 3 Last, as suggested here by the higher frequency of Tregs found in lymph nodes of exo-AAV-treated animals, higher transgene expression levels are likely to result in more robust induction of immunological tolerance.…”

Engineered AAV vector minimizes in vivo targeting of transduced hepatocytes by capsid-specific CD8+ T cells

“…22,23 Further details are provided in the supplemental data ("Materials"; see the Blood Web site). 38 Alanine transaminase (ALT) and aspartate transaminase (AST) levels in plasma were measured by Small Animal Veterinary diagnostics laboratory at the University of Florida in a blinded fashion.…”

“…21 In vitro studies have been helpful in studying T-cell targeting of transduced human hepatocytes. 22,23 However, development of a preclinical in vivo model of AAV vector immunogenicity has been frustrating and largely unsuccessful. Immunization against capsid by various methods designed to induce capsid-specific CD8 1 T cells failed to eliminate transduced cells in mice, 20,24-28 despite major histocompatibility complex class I (MHC I) presentation of capsid for several weeks following AAV gene transfer.…”

Celastrol enhances AAV1-mediated gene expression in mice adipose tissues

“…It has been reported to promote the transduction of various AAV serotypes in many cell lines, 17 the notable ones include tripeptidyl aldehydes such as MG132 (Z-LLL), anthracyclines 18 and bortezomib (Velcade). 19 Although AAV capsid proteins can be ubiquitinated, the AAV proteasomal degradation does not seem to negatively correlate with the AAV transduction efficiency. 17 Rather, the uncoated AAV genomes translocated in the nuclear are often seen after proteasome inhibitor treatment, suggesting that the intracellular trafficking, which is the ratelimiting process for AAV transduction in many cell types, has been altered.…”