Accurate Identification and Quantification of DNA Species by Next-Generation Sequencing in Adeno-Associated Viral Vectors Produced in Insect Cells

Abstract

Recombinant adeno-associated viral (rAAV) vectors have proven excellent tools for the treatment of many genetic diseases and other complex diseases. However, the illegitimate encapsidation of DNA contaminants within viral particles constitutes a major safety concern for rAAV-based therapies. Moreover, the development of rAAV vectors for early-phase clinical trials has revealed the limited accuracy of the analytical tools used to characterize these new and complex drugs. Although most published data concerning residual DNA in rAAV preparations have been generated by quantitative PCR, we have developed a novel single-strand virus sequencing (SSV-Seq) method for quantification of DNA contaminants in AAV vectors produced in mammalian cells by next-generation sequencing (NGS). Here, we describe the adaptation of SSV-Seq for the accurate identification and quantification of DNA species in rAAV stocks produced in insect cells. We found that baculoviral DNA was the most abundant contaminant, representing less than 2.1% of NGS reads regardless of serotype (2, 8, or rh10). Sf9 producer cell DNA was detected at low frequency (£0.03%) in rAAV lots. Advanced computational analyses revealed that (1) baculoviral sequences close to the inverted terminal repeats preferentially underwent illegitimate encapsidation, and (2) single-nucleotide variants were absent from the rAAV genome. The high-throughput sequencing protocol described here enables effective DNA quality control of rAAV vectors produced in insect cells, and is adapted to conform with regulatory agency safety requirements.

Pharmacology of Recombinant Adeno-associated Virus Production

“…The percentages of DNA contaminants were published in Lecomte et al., 64 Ye et al., 67 and Penaud-Budloo et al. 68 …”

“… 67 Moreover, less than 0.04% of rep and cap sequences contaminated the rAAV1, rAAV2, and rAAV8 vectors produced in the OneBac2.0 cell line by baculovirus infection. 23 Finally, we recently published that less than 0.1% of Illumina reads corresponded to rep-cap genes in rAAV8 batches produced in the dual-baculovirus/Sf9 cells system 68 ( Table 3 ).…”

“…In the dual-baculovirus/Sf9 platform, we demonstrated that the baculoviral DNA can account for up to 2.1% of rAAV8 vector genomes content, with an increased proportion for the sequences adjacent to ITRs to be encapsidated, such as the gentamycin-resistance gene in the Bac-to-Bac backbone. 68 Thus, undesirable packaging of vector plasmid sequences is strongly thought to be ITR dependant.…”

“…However, we have demonstrated that the vast majority of contaminating DNA in purified rAAV stocks is protected by the AAV capsid and is thus resistant to drastic DNase treatment. 64 , 68 …”

Intrinsic Differential Scanning Fluorimetry for Fast and Easy Identification of Adeno-Associated Virus Serotypes

“…rAAV vectors were produced and purified based on previous studies in adherent HEK293 13,14 or Sf9 cells 15 and finally buffered in 1Â PBS (phosphate buffered solution). Empty and full particles from some AAV vectors were purified by 2 rounds of cesium chloride gradients as described previously in Ayuso et al 16 When indicated, AAV vectors produced in HEK293 cells were purified by tangential flow filtration (TFF) using a 300 kDa modified polyester membrane from Repligen (Waltham, MA) and buffered in 1Â PBS (10 mM Na 2 PO 4 , 1.8 mM KH 2 PO 4 , 4.27 mM KCl, and 137 mM NaCl, pH 7.4).…”