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, and mixtures of empty and fully packaged virions with variable ratios of empty virions. The aforementioned dosing formulations of rAAV8 expressing either cellular (EGFP (enhanced green fluorescent protein) 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 ALT levels were observed in the BALB/c mice, indicating that empty particles may also exacerbate side effects of rAAV8 EGFP transduction. Our results suggest that removal of empty particles from rAAV preparations may improve efficacy and safety of AAV in clinical applications.
Highlights
IntroductionAdeno-associated virus (AAV), a small single-stranded DNAcontaining nonpathogenic human parvovirus, is an efficient gene transfer vehicle for gene transfer to different tissues including liver, without apparent vector-related toxicities.[1,2,3] Recombinant AAV (rAAV) has been clinically evaluated for in vivo gene therapy applications, including treatment of hemophilia.[4,5,6,7,8,9] The rAAV serotype 2–mediated liver gene transfer for treatment of hemophilia in human resulted in a transient increase in hepatic enzymes and loss of transgene expression subsequently indicating activation of CD8+ T-cell responses against AAV2 capsids.[10,11] The magnitude of such adaptive immune responses appeared to be dose and serotype of AAV capsid dependent, suggesting that a high liver-tropic and low immunity AAV vector may be needed for effective liver-directed gene therapy.[10,11,12] Nonhuman primate–derived rAAV serotype 8 outperformed all other AAV serotypes[13] in transducing hepatocytes in vivo and could be an ideal candidate for this purpose
Our results documented that the empty virions as well as other impurity proteins of cellular and transgene origins were efficiently removed from rAAV8 vector preparations by the CsCl gradient sedimentation method described by Ayuso et al.[18]
RAAV8hFIX lots that have currently been administrated to hemophilia patients are tinted with up to 90% of carried-over nontherapeutic but rather potentially immunotoxic empty AAV8 capsids from Recombinant associated virus (AAV) (rAAV) production process.[16,17]
Summary
Adeno-associated virus (AAV), a small single-stranded DNAcontaining nonpathogenic human parvovirus, is an efficient gene transfer vehicle for gene transfer to different tissues including liver, without apparent vector-related toxicities.[1,2,3] Recombinant AAV (rAAV) has been clinically evaluated for in vivo gene therapy applications, including treatment of hemophilia.[4,5,6,7,8,9] The rAAV serotype 2–mediated liver gene transfer for treatment of hemophilia in human resulted in a transient increase in hepatic enzymes and loss of transgene expression subsequently indicating activation of CD8+ T-cell responses against AAV2 capsids.[10,11] The magnitude of such adaptive immune responses appeared to be dose and serotype of AAV capsid dependent, suggesting that a high liver-tropic and low immunity AAV vector may be needed for effective liver-directed gene therapy.[10,11,12] Nonhuman primate–derived rAAV serotype 8 outperformed all other AAV serotypes[13] in transducing hepatocytes in vivo and could be an ideal candidate for this purpose. Several patients with high doses of AAV8 vector delivery had transient increases in transaminases associated with increased AAV capsid–specific T cells and decreased circulating hF.IX levels, such a vector-related immunotoxicity seemed to be resolvable by anti-inflammation steroid regimens of prednisolone.[14,15]
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