Efficient Production of Dual Recombinant Adeno-Associated Viral Vectors for Factor VIII Delivery

Abstract

Recombinant adeno-associated viral (rAAV) vectors have gained attention for human gene therapy because of their high safety and clinical efficacy profile. For factor VIII gene delivery, splitting the coding region between two AAV vectors remains a viable strategy to avoid the packaging capacity limitation (*5.0 kb). However, it is time-consuming and labor-intensive to produce two rAAV vectors in separate batches. Here we demonstrated successful production of dual rAAV vectors for hemophilia A gene therapy in a single preparation. When the AAV vector plasmids carrying the human factor VIII heavy chain (hHC) and the light chain (hLC) expression cassettes were cotransfected into 293 cells along with the AAV rep&cap and mini-adenovirus helper plasmids, both rAAV-hHC and rAAV-hLC were produced at the desired ratio and in high titer. Interestingly, the rAAVhHC vectors always yielded higher titers than rAAV-hLC vectors as a result of more efficient replication of rAAV-hHC genomes. The resulting vectors were effective in transducing the tissue culture cells in vitro. When these vectors were administered to hemophilia A mice, factor VIII was detected in the mouse plasma by both the activated partial thromboplastin time assay and enzyme-linked immunosorbent assay. The functional activity as well as the antigen levels of secreted factor VIII were similar to those of vectors produced by the traditional method. The dual-vector production method has been successfully extended to both AAV2 and AAV8 serotypes. In conclusion, cotransfection of vector plasmids presents an efficient method for producing dual or multiple AAV vectors at significantly reduced cost and labor.

High-Density Recombinant Adeno-Associated Viral Particles are Competent Vectors forIn VivoTransduction

“…17,23,25,26,29,35 The AAV particles purified from these bands were reported to have significantly different infectious properties in vitro. De la Maza and Carter found a 16-fold difference in infectivity between the regular and heavy AAV2 particles, 17 whereas Roy et al reported a 300-fold difference in infectivity.…”

“…35 Briefly, pAAV-Rep&Cap (serotype 8), pAd helper, and the transgene plasmids were cotransfected into HEK 293 cells cultured in roller bottles at a ratio of 1:1:1. Vectors from both the transfected cells and medium were harvested 72 h post transfection, and purified by two rounds of CsCl gradient ultracentrifuge.…”

“…To elucidate the genome copy number in the liver of injected mice, the total DNA was extracted from the liver cells using a GeneJET genomic DNA purification Kit (K0721; Thermo Scientific). All qPCR analysis was performed using the Fast SYBR Green Master Mix, which has been previously described, 35,37 using the following primers: hHC forward primer, 5¢-CTGAAATGGATGTGGTCAGG-3¢; hHC reverse primer, 5¢-AGTCCCAGTCCTCCTCT TCA-3¢; Gluc forward primer, 5¢-CCACCGAGAAC AACGAAGA-3¢; Gluc reverse primer, 5¢-CATCTC TTTGAGCACCTCCA-3¢; EGFP forward primer, 5¢-TGACCCTGAAGTTCATCTGC-3¢; EGFP reverse primer, 5¢-GAAGTCGTGCTGCTTCATGT-3¢. The genome copy number of vector found in each sample was determined by comparison with a standard curve made from serial dilutions of plasmid containing the corresponding AAV genomes.…”

“…Multiple doses of scAAV8-Gluc vector were injected into mice via the tail vein as previously described. 35 For these injections, the high dose was 6 · 10 11 vg/mouse and the low dose was 2 · 10 11 vg/mouse. Mouse plasma was harvested post vector administration by retro orbital eye bleeding at regular intervals as described.…”

Development of Optimized AAV Serotype Vectors for High-Efficiency Transduction at Further Reduced Doses

“…21 Briefly, human Huh7 cells were transduced with 1 · 10 3 vg/cell with QM-ssAAV2-WT-Fluc, QM-ssAAV2-LC1-Fluc, QM-ssAAV2-LC2-Fluc, DM-ssAAV3-WT-Fluc, DMssAAV3-LC1-Fluc, or DM-ssAAV3-LC2-Fluc vectors and incubated for 72 hr. Transgene expression was determined by Luciferase Assay System (mean -SD) as described previously.…”