High AAV vector purity results in serotype- and tissue-independent enhancement of transduction efficiency

Abstract

The purity of adeno-associated virus (AAV) vector preparations has important implications for both safety and efficacy of clinical gene transfer. Early-stage screening of candidates for AAV-based therapeutics ideally requires a purification method that is flexible and also provides vectors comparable in purity and potency to the prospective investigational product manufactured for clinical studies. The use of cesium chloride (CsCl) gradient-based protocols provides the flexibility for purification of different serotypes; however, a commonly used first-generation CsCl-based protocol was found to result in AAV vectors containing large amounts of protein and DNA impurities and low transduction efficiency in vitro and in vivo. Here, we describe and characterize an optimized, secondgeneration CsCl protocol that incorporates differential precipitation of AAV particles by polyethylene glycol, resulting in higher yield and markedly higher vector purity that correlated with better transduction efficiency observed with several AAV serotypes in multiple tissues and species. Vectors purified by the optimized CsCl protocol were found to be comparable in purity and functional activity to those prepared by more scalable, but less flexible serotype-specific purification processes developed for manufacture of clinical vectors, and are therefore ideally suited for pre-clinical studies supporting translational research.

Enhanced liver gene transfer and evasion of preexisting humoral immunity with exosome-enveloped AAV vectors

“…In agreement with several reports showing efficient release of AAV vectors in culture media during production, [19][20][21] yields in production were similar for exo-and standard AAV vector preparations across several serotypes, with the exception of AAV6, which gave much higher yields in the exo-AAV fraction (supplemental Table 1). Analysis of the exosome profile of exo-AAV8 vectors showed an average size of ;115 nm ( Figure 1A).…”

“…The conventional AAV sample presented well-preserved viruses, as expected ( Figure 1D), with no detectable exosomes, reflecting the use of a detergent step during purification. 19 In addition to isolated AAV and empty exosomes, vesicular structures containing several exosomes, which ranged in size from 150 to 250 nm and contained a large number of viruses, were also observed (supplemental Figure 1A). …”

“…17 From the same production, standard AAV vectors were purified from cell lysates using 2 successive ultracentrifugation rounds in cesium chloride density gradient. 19 The AAV genome in both standard AAV and exo-AAV vectors was quantified using quantitative real-time polymerase chain reaction (qPCR).…”

Robust ZFN-mediated genome editing in adult hemophilic mice

“…AAV serotype 8 vectors were produced and titered as previously described. 9,10 Factor IX levels and activity…”

Safety Study of Adeno-Associated Virus Serotype 2-Mediated Human Acid Sphingomyelinase Expression in the Nonhuman Primate Brain

“…AAV serotype 2 vector carrying cDNA sequence encoding human acidic sphingomyelinase (hASM) downstream from a cytomegalovirus (CMV) promoter was packaged at the clinical Vector Core facility at Children's Hospital of Philadelphia (Philadelphia, PA) (Matsushita et al, 1998;Ayuso et al, 2010). Undiluted vector was used for the rats (thalamus only) and for the high-dose NHPs (4.3 · 10 12 vector genomes/ml) and this was diluted 10-fold with vehicle (PBS-0.001% Pluronic [v/v]; Invitrogen, Carlsbad, CA) for lower doses.…”