Efficient production of adeno-associated virus vectors using split-type helper plasmids.

Abstract

Adeno‐associated virus (AAV) vectors are potentially useful vehicles for the delivery of therapeutic genes into human cells. To determine the optimal expression pattern of AAV proteins (Rep78, Rep68, Rep52, Rep40, and Cap proteins) for packaging the recombinant AAV genome, helper plasmids were split into two portions. In this study, two sets of split‐type helper plasmids were prepared; i.e., 1) a Rep expression plasmid (pRep) and Cap expression plasmid (pCap), and 2) a large Rep expression plasmid (pR78/68) and small Rep plus Cap expression plasmid (pR52/40Cap). When AAV vectors were produced using these sets of split‐type helper plasmids at various ratios, the optimal ratio of (large) Rep expression plasmid and Cap expression plasmid was 1 to 9 for both sets. More importantly, the titers were comparable to or even higher than that of a conventional helper plasmid (pIM45) (4.9±2.1×1011 vector particles/10 cm dish for pRep and pCap; 2.9±1.6×1011 vector particles/10 cm dish for pR78/68 and pR52/40Cap; and 1.8±0.16×1011 particles/10 cm dish for pIM45). Western analysis of AAV proteins suggests that the expression of a relatively small amount of large Rep and a large amount of Cap is important for optimal vector production. The present study shows that the AAV helper plasmid can be split without losing the ability to package the recombinant AAV genome, and provides us with valuable basic information for the development of efficient AAV packaging cell lines.