(F-221) Development of Phosphoserine-containing Zwitterionic Peptides for Reduction of Immunogenicity of Adeno-associated Virus Vector-mediated Gene Therapy
Introduction:: Recombinant adeno-associated virus (AAV) is a prominent gene vector used in DNA-based gene therapy and has been approved by the FDA. However, preclinical studies on animals and human patients have shown that a single dose of AAV injection can lead to significant activation of both humoral and cell-mediated immune responses, which makes re-administration of AAVs difficult. Our previous work has demonstrated that modification of AAV with phosphoserine-containing zwitterionic (ZPS) peptides induced AAV-specific immune tolerance and mitigated its immunogenicity. In this work, a new ZPS peptide is developed to maximize its immunosuppressive activities without negatively impacting the transduction efficiency of the vectors.
Materials and Methods:: A monomer for the new ZPS peptide is synthesized in-house via click chemistry, which is then polymerized on a peptide synthesizer (CEM) through 9 coupling cycles to form a polypeptide. A cysteine is added to the N-terminus peptide for the subsequent conjugation. The newly synthesized peptide is then cleaved from the resin, deprotected, and purified. Peptide conjugation of AAVs is performed as shown in Figure 1A. AAVs are first activated by AMAS crosslinker (Thermo) and then reacted with ZPS peptide via one-step conjugation. The ratio of the crosslinker, the peptide, and the AAV is adjusted for an optimized conjugation. Ultracentrifugation is performed to purify the AAVs. The final products are characterized in gel-permeation chromatography and proceeded to the in vitro and in vivo experiments. The transduction efficiency of the modified AAV is analyzed by in vitro Fluc expression experiment in HEK 293T cells as shown in Figure 1C. The modified AAV express fluorescent protein, GFP, is injected into mice to test the in vivo transduction efficacy.
Results, Conclusions, and Discussions:: Development of the new ZPS peptide-modified AAVs follows two principles. First, an optimized peptide sequence is used containing different amino acids to provide maximal flexibility of the ZPS peptide (Figure 1B). The increased flexibility minimizes the steric hindrance from the conjugated peptides that compromise the transduction efficiency of the AAVs. Second, optimization of the conjugation method is performed to increase the ZPS peptide density on the AAVs and to reduce AAV lost during chemical conjugation. A sufficient density of ZPS peptides is critical to provide maximal functional moieties, PS, to enhance the immune protection of AAVs. Different amounts of ZPS could achieve different transduction efficiencies. Subsequently, the in vivo transduction experiment is performed to compare the efficiency of AAVs and the biodistribution changes after modifications. Immunological assays are performed to confirm the induction of AAV-specific immunotolerance, which is proved by the reduction of capsid-specific antibody titers after modification and an increased level of Treg populations. Compared to the previous method, this new chemistry allows the maximal immune tolerance capacity while maintaining the transduction potency.
In this work, our ZPS-peptide-modified AAVs can successfully induce capsid-specific immune tolerance while maintaining substantial yield and transduction rate. The strategy here provides a promising solution for the hardly achievable re-administration of AAV vectors in gene therapy.
