Abstract Details

A6 - AAV Vectors - Product Development Manufacturing and Approval Considerations

61: Producing High-Purity rAAV Vectors by Recombination-Dependent Minicircle Dual Transfection

Type: Oral Abstract Session

Presentation Details

Session Title: AAV Manufacturing II






Triple transfection of HEK293 cells is the most widely used method for producing recombinant adeno-associated virus (rAAV), a leading vector for human gene therapy. It involves three plasmids co-transfected to cells at roughly equal molar or mass ratio: a helper plasmid that delivers adenoviral helper genes (pHelper), a trans plasmid that expresses AAV rep and cap genes (pTrans), and a cis plasmid that harbors a therapeutic transgene cassette flanked by AAV inverted terminal repeats (pCis). Depside its tremendous success, this method still faces many challenges. One important concern is vector impurity, including plasmid backbone encapsidation that can not be removed by downstream processing and empty capsid that compromises vector potency. Here we developed a recombination-dependent minicircle dual transfection method that reduces the backbone DNA encapsidation by 20-40 folds and improves full capsid ratio by 2-3 folds compared with triple transfection, while generating comparable rAAV yield with reduced plasmid demand in the upstream rAAV production stage. This methodology consists of two plasmids, pHelper-Bxb1 that provides adenoviral helper genes and the recombinase Bxb1, and pTrans/Cis with the attP- and attB-flanked cis element inserted into the C-terminus of Rep in pTrans plasmid (See Figure). When pHelper-Bxb1 and pTrans/Cis are co-transfected into HEK293 cells, Bxb1 catalyzes the recombination between attP and attB sites in pTrans/Cis, generating a functional pTrans-attR that expresses Rep and Cap proteins, and a minicircle pCis that contains only ITR-flanked transgene without any plasmid backbone sequences, which serves as the template for further vector genome amplification. In this way, the packaged AAV vectors are devoid of plasmid backbone DNA. When HEK293 cells receive only pTrans/Cis, the ITR-flanked transgene cassette embedded in the Rep gene serves as a disrupting insertion that abolishes Cap expression, thus avoiding empty capsid formation. Using EGFP as a model transgene, we demonstrated that this newly developed dual transfection method dramatically improved vector purity compared with triple transfection: (1) it reduced bacterial backbone encapsidation from 5.2% to 0.14% for rAAV2, and from 3.3% to 0.16% for rAAV9; (2) the full capsid ratios in crude lysate, as determined by ddPCR of genome titer normalized to ELISA capsid titer, increased from 11% to 23% for rAAV2, and from 14% to 46% for rAAV9. Mechanistically, we found that the asynchronous presence of pTrans and pCis contributes to high empty capsid levels in triple transfection. Intracellular genetic coupling of pTrans and pCis in triple transfection improves the full capsid ratio to similar levels of recombination-dependent minicircle dual transfection. Altogether, these results demonstrate that recombination-dependent minicircle dual transfection serves as a cost-saving and easy-to-implement manufacturing method that produces rAAV with significantly improved vector purity. In addition, it highlights the importance of spatial synchronization of pTrans and pCis plasmids in mitigating empty capsid formation. G.G. and D.W. are corresponding authors.

Plain Language Summary

Recombinant adeno-associated virus (rAAV) serves as a leading vector for human gene therapy. Manufacturing high-purity rAAV vectors holds the key for clinical success. Here we developed a new plasmid transfection-based AAV manufacturing method, recombination-dependent minicircle dual transfection, that was able to reduce the packaged plasmid backbone contaminants by 20-40 folds and the empty capsid generation by 2-3 folds while saving roughly 20% plasmid consumption in rAAV production phase. This novel AAV manufacturing method is expected to advance human gene therapy by producing high-purity rAAV vectors.

Hao Liu¹, Lingzhi Ren¹, Nan Liu¹, Chen Zhou¹, Guangping Gao^1,2, Dan Wang<sup>1,3

1</sup>Horae Gene Therapy Center, University of Massachusetts Chan Medical School, Worcester, MA,²Department of Microbiology and Physiological Systems, University of Massachusetts Chan Medical School, Worcester, MA,³RNA Therapeutics Institute, University of Massachusetts Chan Medical School, Worcester, MA"