Evidence for Encapsidation of Prokaryotic Sequences during Recombinant Adeno-Associated Virus Production and Their in Vivo Persistence after Vector Delivery

Abstract

Recombinant adeno-associated virus vectors (rAAV) have been successfully used for long-term gene expression in animal models and in patients. However, while the therapeutic potential of rAAV appears promising, safety issues, including contaminants found in vector stocks, must be further evaluated. We previously reported that a cis-acting replication element present within the AAV-2 p5 promoter was responsible for the encapsidation of rep-cap sequences observed during rAAV production. In that study, we also noticed that plasmid-derived prokaryotic sequences (such as the ampicillin resistance gene) could be found packaged into AAV capsids. In this report, first we confirmed and extended the latter observation by analyzing rAAV stocks produced using different procedures. Second, we demonstrated that these plasmid-derived sequences were transferred and persisted in vivo after rAAV injection into different tissues. Third, our data showed that at least some of these packaged plasmid molecules were linked to the AAV ITRs and were present in vivo in a form that could be rescued through bacterial transformation. This study highlights the need for more stringent characterization of rAAV stocks and provides useful information on the development of rAAV production methods that are able to circumvent or limit the generation of such undesirable particles.

Immune responses to AAV vectors: overcoming barriers to successful gene therapy

“…81 The inability to recapitulate the human findings in an animal model led to ongoing controversy about the pathophysiological basis of the transaminase elevation and loss of F.IX expression, 82 and multiple alternative hypotheses were advanced (Table 2). [83][84][85][86][87][88] However, if the observed toxicity was indeed related to the catabolism of the capsid, then this aspect of drug metabolism needed to be understood in greater detail. Early studies by Engelhardt and colleagues 89,90 showed that AAV vector particles are indeed ubiquitinated once internalized in the cytosol, a step that is fundamental for targeting intracellular proteins for degradation through This approach has been shown to be effective in some instances.…”

Clearance and characterization of residual HSV DNA in recombinant adeno-associated virus produced by an HSV complementation system

“…7,8 To determine whether such undesirable packaging of non-transgene DNA sequences might be reduced during rHSV-based rAAV production, two lots of rAAV1 vector expressing the human a-1 antitrypsin (rAAV1-CB-hAAT) generated and purified as described in Materials and Methods were subjected to an extensive nuclease treatment to remove any nonencapsidated nucleic acid impurities, and assessed for the presence of particles containing HSV DNA by a real-time quantitative PCR (qPCR) assay targeted to the HSV UL36 gene sequence. The HSV UL36 sequence was found to be packaged at 0.0076-0.0094% of the total rAAV vg, accounting for 6.4-7.8 ng of total HSV DNA per 1Â10 12 rAAV vg ( Table 1).…”

“…[7][8][9] Using an rHSV-based method to produce rAAV1-CB-hAAT vectors, we found that residual HSV DNA sequences were randomly packaged, and the average HSV DNA sequence copy numbers detected were approximately 0.012% of the total rAAV vg, corresponding to approximately 10 ng of total HSV DNA per 1Â10 12 vg of rAAV. This is at least 100-fold less than the level of residual plasmid DNA (1.2-6.3% of the total rAAV vg) reported by Chadeuf et al, 8 and, when expressed as ng of residual DNA per 1Â10 12 vg of rAAV, is comparable with the lowest amount of residual plasmid DNA reported by Hauck et al 9 (10 ng vs 8.6 ng). The encapsidated residual HSV DNA sequences are most likely single-stranded DNA based on the observations from Southern blot analysis of DNA transferred under neutral conditions, although we could not completely exclude the possibility that part of the encapsidated HSV DNA are double stranded.…”

“…6 Several recent reports on rAAV preparations produced by either the plasmid transient transfection method or stable producer cells have documented that DNA sequences other than the therapeutic transgene expression cassette are encapsidated into rAAV particles. [7][8][9] Non-transgene DNA sequences, including AAV2 rep and antibiotic resistance gene sequences (Amp r and/or Kan r ), were reported to be present at levels up to 6.3% of the total rAAV vector genomes (vg). 7,8 Transcription of encapsidated rep and cap sequences in rAAV preparations has also been reported, and was suggested to have a potential impact on vector performance in vivo.…”

“…[7][8][9] Non-transgene DNA sequences, including AAV2 rep and antibiotic resistance gene sequences (Amp r and/or Kan r ), were reported to be present at levels up to 6.3% of the total rAAV vector genomes (vg). 7,8 Transcription of encapsidated rep and cap sequences in rAAV preparations has also been reported, and was suggested to have a potential impact on vector performance in vivo. 7 Evaluation of an rAAV2-hFIX vector made by plasmid transient transfection and used in a clinical trial also found that non-transgene DNA sequences were packaged, but no transcription of AAV2 cap or other DNA sequences was detected.…”