A Fragmented Adeno-Associated Viral Dual Vector Strategy for Treatment of Diseases Caused by Mutations in Large Genes Leads to Expression of Hybrid Transcripts

“Given that the fAAV approach will generate a heterogeneous transgene population [3-5], we performed further in vitro transduction experiments to discover what these truncated transgenes would initiate once inside cells.”

“Adeno-associated virus (AAV) vectors are known to have an optimal packaging capacity of ~4.7 kb (the size of their native genome) but can be encouraged to package slightly larger transgenes [1].”

“In 2008, Allocca et al. [2] attempted to package much larger genomes of up to 8.9 kb and given the positive outcomes of their strategy, assumed the packaging to have been successful.”

“is now acknowledged to be due to AAV capsids truncating the oversized transgenes during the packaging process [5].”

“Within the host nucleus these have been predicted to regenerate the original oversized transgene through homologous recombination [7] or annealing of plus and minus strand complementary regions prior to second-strand synthesis [8], Trapani et al. have shown success with the fAAV approach in HEK293…”

“…synthesis [8], Trapani et al. have shown success with the fAAV approach in HEK293 cells [9] and Hirsch et al. suggested fAAV vectors are a better gene therapy approach for delivering large genes to the retina and skeletal muscle than an alternative trans-splicing dual vector approach [10].”

“have shown success with the fAAV approach in HEK293 cells [9] and Hirsch et al.”

“This method provides no designated limit of packaging at the 5’ end of either the plus or minus strands of the transgene and results in a collection of AAV particles carrying transgenes of different length containing only a single ITR, referred to herein as fragmented AAV (fAAV) [5,6].”

“The mechanisms by which transgenes recombine are still being elucidated but both non-homologous end joining (NHEJ) [11] and homologous recombination [10] has been implicated.”

“Packaging fragmented single-ITR AAV genomes from over-sized transgenes has been shown by several research groups to successfully lead to expression of the target protein in transduced cells in vitro and in vivo with proof of principle demonstrated in muscle, lung and retina [2,7,9,10,12].”

“transduction efficiencies as well as the aberrant production of truncated proteins from single AAV vectors have been two major limitations [103,104].”

“In addition, these sorts of fragmented transgenes present the clear risk of imperfect reconstitution of the normal coding sequence upon exposure to the cellular DNA doublestrand break repair machinery.(33) To overcome these drawbacks for cDNAs exceeding the size limit of AAV vectors, dual vector strategies for oversiz…”

“Further assessments showed that the fragmented AAV-ABCA4 vectors could recombine to create the full ABCA4 gene transcript but generated hybrid transcripts with unintentional AAV genome integrations [44].”

“Investigators have explored dual-AAV systems to reconstitute large genes in situ or have turned to alternative vectors, but efficient and consistent reassembly remains challenging [69–71].”