Preclinical in vivo evaluation of pseudotyped adeno-associated virus vectors for liver gene therapy

Abstract

We report the generation and use of pseudotyped adeno-associated viral (AAV) vectors for the liver-specific expression of human blood coagulation factor IX (hFIX). Therefore, an AAV-2 genome encoding the hfIX gene was cross-packaged into capsids of AAV types 1 to 6 using efficient, large-scale technology for particle production and purification. In immunocompetent mice, the resultant vector particles expressed high hFIX levels ranging from 36% (AAV-4) to more than 2000% of normal (AAV-1, -2, and -6), which would exceed curative levels in patients with hemophilia. Expression was dose-and time-dependent, with AAV-6 directing the fastest and strongest onset of hFIX expression at all doses. Interestingly, systemic administration of 2 ؋ 10 12 vector particles of AAV-1, -4, or -6 resulted in hFIX levels similar to those achieved by portal vein delivery. For all other serotypes and particle doses, hepatic vector administration yielded up to 84-fold more hFIX protein than tail vein delivery, corroborated by similarly increased vector DNA copy numbers in the liver, and elicited a reduced immune response against the viral capsids. Finally, neutralization assays showed variable immunologic cross-reactions between most of the AAV serotypes. Our technology and findings should facilitate the development of AAV pseudotype-based gene therapies for hemophilia B and other liver-related diseases.

Single Amino Acid Modification of Adeno-Associated Virus Capsid Changes Transduction and Humoral Immune Profiles

“…Although a high prevalence of NAbs against AAV2 is seen in humans, the incidence of NAbs to alternative types appears to be nonexistent or at lower titers (23,27). In vitro NAb assays, including those performed in this study, have demonstrated a lack of cross-reactivity between different types (22,23,26). The utilization of other types of AAV vectors to overcome AAV2 NAb activity has been performed in several animal models (27,55,64).…”

Augmentation of AAV-mediated cardiac gene transfer after systemic administration in adult rats

“…With identification of more AAV serotypes, the natural diversity of AAV capsids was utilized in terms of tissue-tropism and transduction efficiency by generation of pseudotyped vectors. 21 In comparison to AAV-2, AAV-6-pseudotyped vectors lead to higher myocardial transduction after intramyocardial injection and systemic administration. [22][23][24][25] Other AAV serotype vectors such as AAV-8 or -9 resulted in even higher cardiac transduction levels after intravenous injection in adult mice.…”

Intracellular trafficking of adeno-associated viral vectors

“…15,16 These AAV serotypes share a common genome structure, but have varying abilities to infect different cell types and tissue based on their capsid protein recognition by cell surface receptors. The repertoire of rAAV vectors has also been greatly expanded by the development of technologies to pseudo-package rAAV genomes, [17][18][19][20] package AAV genomes with two different ITR serotypes, 21 generate mosaic rAAV particles with more than one capsid serotype, [22][23][24] retarget AAV by generating rAAV capsid modification [25][26][27][28][29] and generate rAAV with chemically modified capsids. 30 These technologies have greatly expanded the ability to tailor rAAV for specific applications in gene therapy.…”