Abstract
A number of packaging systems are available for production of recombinant adeno-associated virus vectors (rAAVs). Among these, the use of a two-plasmid cotransfection system, in which Rep and Cap genes and Ad helper genes are on the same plasmid, has not been frequently employed for good manufacturing practices (GMP) production, even though it presents some practical advantages over the common three-plasmid (triple) transfection method. To confirm and expand the utility of the two-plasmid system, we generated GMP-compatible versions of this system and used those package reporter genes in multiple capsid variants in direct comparison with triple transfection. Vector yields, purity, and empty-to-full ratios were comparable between double and triple transfection methods for all capsid variants tested. We performed an in vivo side-by-side comparison of double and triple transfection vectors following both intravenous injection and intramuscular injection in mice. Expression and transduction were evaluated in muscle and liver 4 weeks after injection. Additional studies of bioactivity were conducted in vivo using packaged vectors carrying a variety of cargos, including the therapeutic transgene, microRNA, and single- or double-stranded vector. Results showed that cargos packaged using double transfection were equivalently bioactive to those packaged using a triple transfection system. In conclusion, these data suggest the utility of midrange (1E12-1E16) GMP-compatible packaging of adeno-associated virus (AAV) vectors for several AAV capsids.
Highlights
The production of recombinant adeno-associated virus (AAV) vectors in the relative absence of wild-type AAV was first accomplished by Samulski, Chang, and Shenk using cotransfection of adenovirus 5-infected HeLa cells with an AAV2 inverted terminal repeat (ITR)-flanked vector cassette and AAV2 ITR-deleted construct to provide rep and cap functions in trans.[1]
We report that the use of a two-plasmid system instead of a three-plasmid system simplifies the transfection step and decreases by one-third the cost of manufacturing the good manufacturing practices (GMP)-compatible plasmid, resulting in significant cost savings in the current good manufacturing practices (cGMP) packaging of recombinant adeno-associated virus vectors (rAAVs).[26]
To package rAAVs using this system, the pQT plasmid is transfected with the transgene plasmid flanked by AAV ITRs (Fig. 1A)
Summary
The production of recombinant adeno-associated virus (AAV) vectors in the relative absence of wild-type AAV was first accomplished by Samulski, Chang, and Shenk using cotransfection of adenovirus 5-infected HeLa cells with an AAV2 inverted terminal repeat (ITR)-flanked vector cassette and AAV2 ITR-deleted construct to provide rep and cap functions in trans.[1].
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