Empty virions in AAV8 vector preparations reduce transduction efficiency and may cause total viral particle dose-limiting side effects

Abstract

Empty virions are inadvertent by-products of recombinant adeno-associated virus (rAAV) packaging process, resulting in vector lots with mixtures of full and empty virions at variable ratios. Impact of empty virions on the efficiency and side-effects of rAAV transduction has not been well characterized. Here, we generated partially and completely empty AAV8 virions, fully packaged rAAV8 lots as well as mixtures of empty and fully packaged virions with variable ratios of empty virions (REVs). The aforementioned dosing formulations of rAAV8 expressing either cellular (EGFP or nuclear-targeted (n) LacZ) or secreted (human α1-antitrypsin, hA1AT) reporter genes were intravenously injected into two different mouse strains, followed by analyses of transgene expressions and serum alanine aminotransferase (ALT) levels at different time points. We found that addition of empty particles to the fixed doses of rAAV8 preparations repressed liver transduction up to 64% (serum hA1AT) and 44% (nLacZ) in C57BL/6 mice, respectively. The similar trend in inhibiting EGFP expression together with concurrent elevations of serum ATL levels were observed in the BALB/c mice, indicating that empty particles may also exacerbate side-effects of rAAV8EGFP transduction. Our results suggest that removal of empty particles from rAAV preparations may improve efficacy and safety of AAV in clinical applications.

Obstacles and future of gene therapy for hemophilia

“…These data tabulated in (Table 1) suggest that total capsid (cp) doses of at least 1 × 10 12 cp/kg may be necessary to surmount even low level of preexisting NAbs. Indeed, Mingozzi et al showed in a mouse model of anti-AAV NAbs that empty capsids can enhance FIX transgene expression in a dose-dependent manner that is also a function of NAb titer; however, excessive empty capsids also lead to decreased transgene expression, likely due to competition with vector at cellular binding sites [24] since the addition of empty capsids significantly decreases transduction in mice without NAbs [25]. Together, the human and mouse data suggest that reintroduction of empty viral capsids is a promising strategy for avoiding NAbs, especially at low titer levels.…”

Syngeneic AAV Pseudo-particles Potentiate Gene Transduction of AAV Vectors

“…Most of the negatively stained pseudo-vectors appeared to be empty because they displayed donut-like shapes of virions, and no significant difference was found among the three types of pseudo-vectors (Figure 3A) 15 . We also did not observe self-assembly intermediates, which should be smaller than the regular particles 20 .…”

“…Recently, Gao et al. demonstrated that pseudo-vectors suppressed transgene expression and contributed to hepatic transaminase elevation in mice 15 . Moreover, the pseudo-vectors cogenerated and separated from the bona fide AAV8 vectors, resulting in higher elevations of liver enzyme than the vector alone or mixed with a completely empty vector.…”

Widespread and targeted gene expression by systemic AAV vectors: Production, purification, and administration

“…Immunogenicity of AAVs may be exacerbated by empty capsid contamination in viral preparations 35,36 . The viral purification protocol (Steps 15-31) provided here reduces, but does not eliminate, empty capsids (Fig.…”