Syngeneic AAV Pseudo-particles Potentiate Gene Transduction of AAV Vectors

Qizhao Wang,Biao Dong,Katie A Pokiniewski,Jenni Firrman,Zhongren Wu,Mario P.S Chin,Xiongwen Chen,Linshu Liu,Ruian Xu,Yong Diao,Weidong Xiao

doi:10.1016/j.omtm.2016.12.004

Abstract

Adeno-associated virus (AAV) vectors have emerged as a safe and efficient gene therapy platform. One complication is that a significant amount of empty particles have always been generated as impurities during AAV vector production. However, the effects of such particles on AAV vector performance remain unclear. Here we systemically evaluated the biological properties of three types of “empty” AAV particles: syngeneic pseudo-vectors with partial AAV genomes derived from DNA of the corresponding full particles, allogeneic pseudo-vectors with partial genomes different from the corresponding full particles, and null pseudo-vectors with no DNA inside the capsids. The syngeneic particles in excess increased the corresponding full AAV vector transgene expression both in vivo and in vitro. However, such effects were not observed with null or allogeneic particles. The observed differences among these pseudo-AAV particles may be ascribed to the syngeneic pseudo-vector DNA facilitating the complementary DNA synthesis of the corresponding full AAV particles. Our study suggests that the DNA content in the pseudo-vectors plays a key role in dictating their effects on AAV transduction. The effects of residual “empty” particles should be adequately assessed when comparing AAV vector performance. The syngeneic AAV pseudo-vectors may be used to enhance the efficacy of gene therapy.

Highlights

Adeno-associated virus (AAV) is a non-pathogenic parvovirus with a 4.7-kb single-stranded DNA viral genome that encodes two large open reading frames (ORFs) flanked by two inverted terminal repeats (ITRs)
Syngeneic AAV8 Pseudo-vectors Enhance Transduction of the Corresponding Full Vectors In Vivo To investigate whether pseudo-vector AAV particles reduce transduction efficiency in vivo, three different pseudo-vectors were generated and intravenously injected into a BALB/c hemophilia A (HA) mouse model together with full AAV8 vectors
The purified full AAV8 vector was AAV8-TTR-hF8 (5.1 kb)[19] (Figure 4A). syngenic to AAV8-TTRhF8 itself (sAAV8) was a by-product purified during the AAV8-TTR-hF8 production process, which carried partial genomes of AAV-TTR-hF8 (Figures 3 and 4). nAAV8 was produced from HEK293 cells transfected with the AAV8 packaging and adenoviral helper plasmids only, which do not carry any genomic DNA of the AAV vector. AAV8-hHC vector or other AAV8 vectors (aAAV8) was a by-product of AAV8-hHC production, whose genome (Figure 4A) carries the human heavy chain of the hF8 gene driven by the AAT promoter in combination with an ApoE enhancer. aAAV8 was named allogeneic AAV pseudovector in reference to sAAV8 because it was derived from AAV8hHC instead of AAV8-TTR-hF8

Summary

INTRODUCTION

Adeno-associated virus (AAV) is a non-pathogenic parvovirus with a 4.7-kb single-stranded DNA (ssDNA) viral genome that encodes two large open reading frames (ORFs) flanked by two inverted terminal repeats (ITRs). It is speculated that the presentation of pseudo-vectors in clinic-grade preparations can substantially increase the amount of AAV capsid proteins and potentially lead to unwanted immunological consequences. One example of such side effects was observed during a clinical trial for the treatment of hemophilia with an AAV8 vector that contained a 10-fold excess of empty capsids over the full vector.[4] Recently, Gao et al demonstrated that pseudo-vectors suppressed transgene expression and contributed to hepatic transaminase elevation in mice.[15] 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. We systemically analyzed the physical properties and effects of AAV pseudo-vectors on AAV vector transduction performance

RESULTS

DISCUSSION

MATERIALS AND METHODS