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Characteristics of Minimally Oversized Adeno-Associated Virus Vectors Encoding Human Factor VIII Generated Using Producer Cell Lines and Triple Transfection.

ABSTRACT: Several ongoing clinical studies are evaluating recombinant adeno-associated virus (rAAV) vectors as gene delivery vehicles for a variety of diseases. However, the production of vectors with genomes >4.7 kb is challenging, with vector preparations frequently containing truncated genomes. To determine whether the generation of oversized rAAVs can be improved using a producer cell-line (PCL) process, HeLaS3-cell lines harboring either a 5.1 or 5.4 kb rAAV vector genome encoding codon-optimized cDNA for human B-domain deleted Factor VIII (FVIII) were isolated. High-producing "masterwells" (MWs), defined as producing >50,000 vg/cell, were identified for each oversized vector. These MWs provided stable vector production for >20 passages. The quality and potency of the AAVrh8R/FVIII-5.1 and AAVrh8R/FVIII-5.4 vectors generated by the PCL method were then compared to those prepared via transient transfection (TXN). Southern and dot blot analyses demonstrated that both production methods resulted in packaging of heterogeneously sized genomes. However, the PCL-derived rAAV vector preparations contained some genomes >4.7 kb, whereas the majority of genomes generated by the TXN method were ≤4.7 kb. The PCL process reduced packaging of non-vector DNA for both the AAVrh8R/FVIII-5.1 and the AAVrh8R/FVIII-5.4 kb vector preparations. Furthermore, more DNA-containing viral particles were obtained for the AAVrh8R/FVIII-5.1 vector. In a mouse model of hemophilia A, animals administered a PCL-derived rAAV vector exhibited twofold higher plasma FVIII activity and increased levels of vector genomes in the liver than mice treated with vector produced via TXN did. Hence, the quality of oversized vectors prepared using the PCL method is greater than that of vectors generated using the TXN process, and importantly this improvement translates to enhanced performance in vivo.

SUBMITTER: Nambiar B

PROVIDER: S-EPMC5314997 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Usher syndrome 1B (USH1B) is a severe, autosomal recessive, deaf-blind disorder caused by mutations in myosin 7A (MYO7A). Patients are born profoundly deaf and exhibit progressive loss of vision starting in their first decade. MYO7A is expressed in human photoreceptors and retinal pigment epithelium, but disease pathology begins in photoreceptors, highlighting the need to develop a gene replacement strategy that effectively targets this cell type. For its safety and efficacy in clinical trials and ability to transduce postmitotic photoreceptors, we have focused on developing a clinically applicable adeno-associated virus (AAV) platform for delivering full-length MYO7A cDNA (∼6.7 kb). Packaging of full-length MYO7A cDNA in AAV produces vectors with heterogeneous, fragmented genomes ("fAAV") capable of reconstituting full-length cDNA postinfection. We previously showed that fAAV vectors effectively delivered full-length MYO7A in vitro and in vivo. However, fAAV vectors are relatively inefficient and their heterogeneous genomes preclude definitive characterization, a drawback for clinical translatability. The aim of this study was to overcome these limitations by creating dual-AAV-vector platforms for USH1B with defined genomes. Human MYO7A was cloned in AAV vector pairs, each containing genomes <5 kb and intact inverted terminal repeats. One vector contained a promoter and 5' portion of the cDNA and the partner vector contained a 3' portion and polyadenylation signal. "Simple overlap" vectors share a central part of the MYO7A cDNA sequence. "Trans-splicing" and "hybrid" vectors utilize splice donor and acceptor sites with and without an additional central recombinogenic sequence, respectively. Vector pairs expressed full-length MYO7A in vitro and in vivo with equal or higher efficiency than fAAV, with a hybrid platform being most efficient. Importantly, analysis of MYO7A mRNA derived from each dual-vector platform revealed 100% fidelity to the predicted sequence. Our results suggest that dual AAV vectors with defined genetic payloads are a potential treatment option for USH1B.

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Characteristics of Minimally Oversized Adeno-Associated Virus Vectors Encoding Human Factor VIII Generated Using Producer Cell Lines and Triple Transfection.

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