A short hepatitis C virus NS5A peptide expression by AAV vector modulates human T cell activation and reduces vector immunogenicity

Abstract

Viral vector-mediated gene therapies have the potential to treat many human diseases; however, host immune responses against the vector and/or the transgene pose a safety risk to the patients and can negatively impact product efficacy. Thus, novel strategies to reduce vector immunogenicity are critical for the advancement of these therapies. T cell activation (TCA) is required for the development of immune responses during gene therapy. We hypothesized that modulation of TCA by incorporating a novel viral immunomodulatory factor into a viral vector may reduce unwanted TCA and immune responses during gene therapy. To test this hypothesis, we identified an immunomodulatory domain of the hepatitis C virus (HCV) NS protein 5A (NS5A) protein and studied the effect of viral vectors expressing NS5A peptide on TCA. Lentiviral vector-mediated expression of a short 20-mer peptide derived from the NS5A protein in human T cells was sufficient to inhibit TCA. Synthetic 20-mer NS5A peptide also inhibited TCA in primary human T cells. Mechanistically, the NS5A protein interacted with Lck and inhibited proximal TCR signaling. Importantly, NS5A peptide expression did not cause global T cell signaling dysfunction as distal T cell signaling was not inhibited. Finally, recombinant adeno-associated virus (AAV) vector expressing the 20-mer NS5A peptide reduced both the recall antigen and the TCR-mediated activation of human T cells and did not cause global T cell signaling dysfunction. Together, these data suggest that expression of a 20-mer NS5A peptide by an AAV vector may reduce unwanted TCA and may contribute to lower vector immunogenicity during gene therapy.

AAV Immunotoxicity: Implications in Anti-HBV Gene Therapy

“…Transgene expression from weaker hepatocyte-specific promoters and RNAi-mediated transgene self-regulation [140][141][142] Avoid TLR 9 signaling activation CpG island reduction or TLR9 inhibition via oligonucleotide incorporation in the genome [143][144][145] Avoid dsRNA sensing Engineering of the expression cassette, such that transcription from 3 ITR is inhibited [146] Repress the activation of primary and memory T-cell responses Co-expressing NS5A with the transgene [147] Capsid Reduce dose, de-target APCs, and induce tolerance Rational designs and directed evolution to generate capsids with high hepatocyte transduction efficiencies [148][149][150][151] Avoid complement activation Rational designs and directed evolution to generate capsids that will evade pre-existing antibodies [135,[152][153][154] Avoid TLR9 signaling activation TLR9 inhibiting peptides insertion in the capsid [155] Reduce antigen presentation Mutation of surface-exposed tyrosine residues [156,157] Enhance Treg proliferation and reduce CD8+ T cell activity Incorporation of the MHC II epitopes derived from IgG in the AAV capsid [158] 11.…”

“…A recent study showed that the AAV-mediated delivery of short immunomodulatory peptides, such as the hepatitis C virus non-structural protein 5A (NS5A), suppressed the unwanted activation of primary T-cells ex vivo. The expression of this proline-rich hydrophilic, 20-mer peptide represses the activation of memory T cell responses in the absence of CD3 (see Table 1) [147]. Designing anti-HBV AAVs that co-express this peptide may provide a strategy to overcome the anti-AAV adaptive immune response.…”