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

Abstract

Encapsidation of cellular-or plasmid-derived DNA sequences during recombinant adeno-associated virus (rAAV) production has been well documented. However, most of the published data were generated from rAAV vectors manufactured by the plasmid transient transfection method. We previously reported a novel, scalable method for rAAV manufacturing based on a recombinant herpes simplex virus (rHSV) complementation system. In this report, we evaluated clearance of DNA impurities during rAAV purification, by determining the quantity of residual herpes simplex virus and cellular DNA at each process step. A single Benzonase treatment during the upstream process effectively reduced unprotected HSV and cellular DNA to o300 bp fragments, and subsequent chromatography steps completely removed these small DNA fragments. Further analysis showed that trace amounts of residual, DNase-resistant HSV and cellular DNA were present at static concentrations during subsequent purification steps, and the residual HSV DNA sequences were single stranded, ranging from 0.8 to 4.2 kb. After transduction of human embryonic kidney 293 cells with purified rAAV, the residual HSV DNA fragments were neither transcribed nor translated into HSV proteins. In summary, this manufacturing process for rAAV production was effective in removing DNA and protein contaminants and achieving a highly purified product, suitable for human clinical application.

Direct Head-to-Head Evaluation of Recombinant Adeno-associated Viral Vectors Manufactured in Human versus Insect Cells

“…With many clinical trials under way, assessing the genetic identity of rAAV stocks manufactured by different protocols becomes a pressing regulatory issue. Many groups have reported a collateral encapsidation of sequences derived from packaging host cells, 25,30,31 bacterial helper plasmid backbones, 25,32 helper viruses, 8,31 and wild-type (WT) AAV rep/cap sequences. 25,33 To evaluate the genetic identity of the packaged rAAV cassette, we set to conduct NGS analysis of encapsidated single-stranded viral DNA.…”

A Novel Next-Generation Sequencing and Analysis Platform to Assess the Identity of Recombinant Adeno-Associated Viral Preparations from Viral DNA Extracts

“…Several studies have reported a range of common DNA contaminants in rAAV preparations, including plasmid backbone sequences such as antibiotic resistance markers or elements from the capsid and helper packaging plasmids, and genomic DNA from the packaging cells. [2][3][4][5][6][7][8][9][10][11] Although the effect of contaminating DNA on downstream applications remains unclear, it has been demonstrated that it can persist in mammalian systems for months after the vector has been administered. 3 Previous work using single molecule real-time sequencing (SMRT), ligation of thymine and adenine overhangs (TA-based ligation), and tagmentation-based library preparation methods have proved that NGS is an effective tool to confirm rAAV genome sequence and identify vector preparation anomalies such as truncated genomes, reverse packaging, and the presence of contaminating DNA.…”

Ocular Inflammatory Response to Intravitreal Injection of Adeno-Associated Virus Vector: Relative Contribution of Genome and Capsid

“…Separation of full and empty capsids was performed as previously described. 19 Briefly, an aliquot of purified Master rAAV2tYF-CMV-hGFP (4.2 • 10 13 vg) made by rHSV helper viruses was supplemented with CsCl to a final density of 1.40 gcm -3 , loaded into a Beckman ultracentrifuge tube and subjected to ultracentrifugation at 60,000 r.p.m. for 20 h in a 70Ti rotor.…”