Cellular immune response to cryptic epitopes during therapeutic gene transfer

Abstract

The immune response has been implicated as a critical factor in determining the success or failure of clinical gene therapy trials. Generally, such a response is elicited by the desired transgene product or, in some cases, the delivery system. In the current study, we report the previously uncharacterized finding that a therapeutic cassette currently being used for human investigation displays alternative reading frames (ARFs) that generate unwanted protein products to induce a cytotoxic T lymphocyte (CTL) response. In particular, we tested the hypothesis that antigenic epitopes derived from an ARF in coagulation factor IX (F9) cDNA can induce CTL reactivity, subsequently killing F9-expressing hepatocytes. One peptide (p18) of 3 candidates from an ARF of the F9 transgene induced CD8 ؉ T cell reactivity in mice expressing the human MHC class I molecule B0702. Subsequently, upon systemic administration of adeno-associated virus (AAV) serotype 2 vectors packaged with the F9 transgene (AAV2/F9), a robust CD8 ؉ CTL response was elicited against peptide p18. Of particular importance is that the ARF epitope-specific CTLs eliminated AAV2/F9-transduced hepatocytes but not AAV2/F9 codon-optimized (AAV2/F9-opt)-transduced liver cells in which p18 epitope was deleted. These results demonstrate a previously undiscovered mechanism by which CTL responses can be elicited by cryptic epitopes generated from a therapeutic transgene and have significant implications for all gene therapy modalities. Such unforeseen epitope generation warrants careful analysis of transgene sequences for ARFs to reduce the potential for adverse events arising from immune responses during clinical gene therapy protocols.factor IX ͉ gene therapy ͉ CTL ͉ AAV R ecombinant adeno-associated virus (rAAV) vectors possess several attractive features, including lack of pathogenicity, broad tissue tropism, and the ability to promote sustained transgene expression, that make them particularly well-suited for clinical gene therapy applications (1). Driven by a rapidly expanding understanding of AAV biology and a wide range of preclinical studies in mice, dogs, and primates, more than 50 clinical trials have been approved for diseases ranging from Parkinson's to Duchenne Muscular Dystrophy. Recombinant AAV vectors do not contain any viral genome elements, with the exception of 145-bp terminal repeats required for packaging, and persistent transgene expression lasting several years has been observed in preclinical animal models. Given the aforementioned observations, it has been perceived generally that despite generating a strong humoral immune response, AAV vector administration does not elicit a cellular immune response to the AAV capsid that could potentially result in the clearance of AAV vector-transduced target cells.In the first successful clinical trial with an AAV vector, hemophilia B patients were treated by using an AAV serotype 2 (AAV2) vector to deliver coagulation factor IX (F9) cDNA into the liver. Within 2 weeks, therapeutic levels of F9 were detect...

Immune responses to AAV vectors: overcoming barriers to successful gene therapy

“…87 Screening of subjects for anti-ARF T-cell reactivity did not support the model. Animal studies showed that AAV2 binds more efficiently to APCs than AAV8.…”

“…81 The inability to recapitulate the human findings in an animal model led to ongoing controversy about the pathophysiological basis of the transaminase elevation and loss of F.IX expression, 82 and multiple alternative hypotheses were advanced (Table 2). [83][84][85][86][87][88] However, if the observed toxicity was indeed related to the catabolism of the capsid, then this aspect of drug metabolism needed to be understood in greater detail. Early studies by Engelhardt and colleagues 89,90 showed that AAV vector particles are indeed ubiquitinated once internalized in the cytosol, a step that is fundamental for targeting intracellular proteins for degradation through This approach has been shown to be effective in some instances.…”

Adeno-associated virus capsid antigen presentation is dependent on endosomal escape

“…To address whether the OVA SIINFEKL peptide was processed and presented on the surface of HepG2/H-2Kb with H-2Kb molecules after AAV2-OVA infection, we transduced HepG2/H-2Kb cells by AAV2-OVA/AAT or AAV2/AAT viruses. Additionally, the OVA-derived SIINFEKL peptide and p18 from an alternative ORF of human coagulation factor IX cDNA were used to pulse HepG2/H-2Kb cells (34). Twenty-four hours later, HepG2/H-2Kb cells were fixed and washed thoroughly, and OT-1 spleen cells were added to HepG2/H-2Kb cells overnight.…”

Systemic Elimination ofde novoCapsid Protein Synthesis from Replication-Competent AAV Contamination in the Liver

“…The rcAAV particle contamination could be a key factor not only affecting the safety of rAAV vectors, but also de- termining whether or not transgene expression can persist long-term (Manno et al, 2006;Murphy et al, 2008;Hauck et al, 2009;Li et al, 2009a;Mays and Wilson, 2009;Pien et al, 2009;Vandenberghe et al, 2009a,b). Many strategies, such as altering the helper sequences to minimize homology between the AAV ITR and the rep and cap gene sequences, can significantly reduce rcAAV generation through homologous recombination events.…”

“…The rcAAV particle is an undesirable contaminant that may affect transgene expression or elicit a hazardous immune response (Manno et al, 2006;Murphy et al, 2008;Hauck et al, 2009;Li et al, 2009a;Pien et al, 2009). Various strategies have been proposed to minimize the homologous events that lead to rcAAV formation.…”