Improved system for helper-dependent adenoviral vector production

Abstract

Helper-dependent adenoviral vectors (HDAds) are devoid of all viral coding sequences and have demonstrated tremendous potential for gene therapy by providing increased cloning capacity (up to 37 kb) and long-term, high-level transgene expression in vivo with negligible toxicity. Currently, the most widely used method of producing HDAds is the Cre/loxP system developed by Graham and co-workers. However, two major obstacles currently hinder progress of this promising technology: (1) the difficulty of large-scale vector production and (2) helper virus (HV) contamination. We have developed an improved producer cell line, HV, and protocols that have successfully addressed these problems. With this system, >1 x 10(13) viral particles (vp) can be easily produced from 3 liters of cells within 2 weeks of vector rescue, with specific yields of >10,000 vp/cell and with exceedingly low HV contamination of 0.4-0.1% without relying on density-based vector purification and 0.02-0.01% following CsCl purification. This new system represents a major improvement over the original method in terms of simplicity, speed, vector yield, and purity, and it will significantly improve our ability to assess this promising gene therapy technology, especially in large animal models and, ultimately, for clinical applications.

Chromatographic purification of recombinant adenoviral and adeno-associated viral vectors: methods and implications

“…29,77,78 Sandig et al 77 addressed the problem of helper Ad contamination by minimizing the homology between the packaging signal (c) of helper Ad and HDAd, thus reducing the probability of homologous recombination. Palmer et al 78 reversed the orientation of c with respect to HDAd, making the genome of recombinants too large to be packaged and also selected a producer cell line with higher intracellular Cre levels for efficient c excision.…”

Gutless adenovirus: last-generation adenovirus for gene therapy

“…However, since both helper and gutless vectors have the same viral capsid, separation must be addressed before purification. Thus far, strategies have been based on reducing the packaging efficiency of the helper genome compared to the gutless genome, either by mutating its packaging signal, [18][19][20] by the different 13 (genomes bigger or smaller than the optimal do not package efficiently), or by specific elimination of its packaging signal during viral production. 21 Initial strategies consisted in the use of helperdependent adenoviruses carrying a defective packaging signal.…”

“…A recent and elegant improvement in the Cre-loxP system has been the reversion of the packaging signal of the helper virus in order to avoid generation of a replication competent adenovirus (RCA) by recombination with the gutless vector, which has reduced the helper contamination to levels down to 0.02-0.1%. 18 However, Cre and FLP are bidirectional recombinases that permit excision of the packaging signal and also its re-entrance, favoring contamination by the helper adenovirus. Improving…”

“…However, to achieve large quantities of high quality helper-free gutless vectors needed for clinical assays, it is fundamental to develop and improve the methodology in the amplification and purification steps as well as in vector quality. Thus, development of several cell lines able to grow in suspension, like human 293S, 31 PER.C6, 32 and 293Cre suspension cell lines 18 facilitates large-scale amplification in bioreactors, although purification methods like non-ultracentrifuge-based methods and elimination of helper contamination still need to be improved.…”

Self-inactivating helper virus for the production of high-capacity adenoviral vectors

“…Comparing different packaging cells, the intensity of Cre expression correlated with their ability to cleave C, and this contributed to the improvement of HC-Ad production. 12 However, it has been described that the expression of Cre recombinase in the packaging cells decreases in late stages of vector co-amplification as a result of the shut-off of cellular protein synthesis produced by the HV. 13 This is the moment when the function of the Cre recombinase should be most intense in order to cleave C in an increasing number of HV genome copies.…”