Evidence for Packaging of rep-cap Sequences into Adeno-Associated Virus (AAV) Type 2 Capsids in the Absence of Inverted Terminal Repeats: a Model for Generation of rep -Positive AAV Particles

Abstract

We previously reported that a 350-bp region of the adeno-associated virus (AAV) type 2 rep gene contains a cis-acting element responsible for the Rep-dependent replication of a transiently transfected rep-cap plasmid. In this study, we further report that replicated rep-cap sequences can be packaged into AAV capsids in the absence of the inverted terminal repeats.The usual procedure for recombinant adeno-associated virus (rAAV) assembly involves transfection of the vector and the rep-cap plasmid into cells which are either infected with adenovirus or cotransfected with an adenoviral helper plasmid (4,11,15). Despite the lack of homologous sequences between the rep-cap and vector sequences, the precise characterization of rAAV preparations indicated that they were contaminated to various extents with particles containing rep-cap AAV sequences. We designated these contaminating particles rep positive (rep ϩ ), because they were able to transfer a Rep function, as detected by a replication center assay (RCA) (10). Previous studies have indicated that most of these particles were replication competent and that they arose from nonhomologous recombination events between the rep-cap plasmid and the inverted terminal repeats (ITRs) in the rAAV vector (1, 14). Deletion of critical ITR sequences involved in the nonhomologous recombination events prevented the formation of such replication-competent particles. However, rAAV preparations remained contaminated by replication-defective AAV particles containing rep-cap genomes, further suggesting that these sequences had been packaged in the absence of the ITRs (14).Altogether, these observations suggested that some additional cis-acting elements were present in the rep-cap sequences, allowing their replication and encapsidation. We and others have previously reported the presence of a cis-acting replication element (CARE) located in the 5Ј portion of the rep gene (9, 12). The CARE was localized in a 350-bp region that included the p5 promoter and the 5Ј portion of the rep coding sequence (nucleotides 190 to 540 of wild-type AAV), and it was demonstrated that this element behaved in vitro and in vivo as a Rep-dependent origin of replication in the absence of both ITRs (9).In the present study, we investigated whether the presence of CARE could also lead to the packaging of rep-cap sequences into AAV capsids in the absence of the viral ITRs. A critical element in this study was the need to distinguish between DNA truly packaged inside the particles and that simply contaminating the preparations. The conventional procedure to extract viral DNA from a cell lysate or a purified rAAV stock relies on the use of DNase I to digest contaminating DNA before extraction of packaged sequences. The activity of DNase I was checked by mixing purified rAAVLZ particles (rAAV encoding the nucleus-localized ␤-galactosidase) with up to 400 ng of X174 DNA (either double or single stranded). After digestion for 1 h at 37°C with 50 U of DNase I (Roche) in 500 l of Dulbecco modified Eagle medium, pack...

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 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. 9 Furthermore, characterization of rAAV-like particles produced in human embryonic kidney (HEK) 293 cells by an adenovirus-assisted method has revealed that small (100 bp) to moderately sized (700 bp) DNA sequences can be encapsidated in the absence of all AAV transand cis-acting elements (rep and inverted-terminal repeat (ITR)).…”

“…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.…”

Viral vectors for in vivo gene transfer in Parkinson's disease: Properties and clinical grade production

“…Wild-type virus may contaminate vector preparations, and RCV may be formed during production due to recombination between the vector and the helper sequences found in complementary cell lines or plasmids (Escarpe et al, 2003;Murakami et al, 2002;Nony et al, 2003;Sastry et al, 2005) The presence of wild-type virus or RCV may affect vector expression and mobilization.…”