Dual adeno-associated virus vectors result in efficient in vitro and in vivo expression of an oversized gene, MYO7A

Abstract

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.

FIG. 1.

Schematic representation of the dual-AAV-vector pairs created for this study. (A) Fragmented vector (fAAV). (B) Simple overlap: the 1365 bp shared between the two vectors is shaded gray. (C) Trans-splicing vector. (D) AP hybrid vector: the 270 bp element shared between the two vectors is marked with diagonal gradient shading (1/3 head as described by Ghosh et al., 2011). (E) The native intron hybrid vectors utilizing the natural intron 23 of MYO7A sharing 250 bp overlapping sequence. 3′MYO7A, 3′ portion of MYO7A; 5′MYO7A, 5′ portion of MYO7A; AAV, adeno-associated virus; AP, alkaline phosphatase; intron, intron 23 of MYO7A; pA, polyadenylation signal; SA, splice acceptor site; SD, splice donor site; smCBA, cytomegalovirus immediate early/chicken β-actin chimeric promoter.

FIG. 2.

Human embryonic kidney (HEK293) cells express human MYO7A after infection with simple overlap vectors (MOI of 10,000 for both vectors) packaged in AAV2(tripleY-F). Equal amounts of protein were separated on 7.5% sodium dodecyl sulfate polyacrylamide gel electrophoresis and stained for MYO7A (A). HEK293 cells infected with AAV2(tripleY-F) at MOIs of 10,000, 2000, and 400 (B). Time course of MYO7A expressed in HEK293 cells. Cells were harvested 3–7 days after infection (C). I, HEK293 cells infected with AAV2 dual vectors; MOI, multiplicity of infection; T, HEK293 cells transfected with full-length MYO7A plasmid; U, untreated HEK293 cells.

FIG. 3.

Comparison of AAV2 and AAV2(tripleY-F)-based vectors in HEK293 cells. Cells were infected with single fAAV, AP hybrid, and simple overlap MYO7A dual-vector platforms packaged in AAV2 or AAV2(tripleY-F) at an MOI of 10,000. HEK293 cells transfected with MYO7A plasmid were used as positive control.

FIG. 4.

Human MYO7A expressed in HEK293 cells. Cells were infected with AAV2-based vector platforms. For each of the dual-vector systems, the corresponding 5′ and 3′ vectors or only the 5′ vector were used for infection. HEK293 cells transfected with MYO7A plasmid were used as positive control. Cells were infected with the MYO7A dual-vector pairs at an MOI of 10,000 for each vector. Protein samples were analyzed on Western blot with an antibody against MYO7A (A). Each platform's relative ability to promote reconstitution was compared by quantifying the amount of 5′ vector-mediated truncated protein product in the presence or absence of the respective 3′ vector (B). Full-length MYO7A expression mediated by dual vectors was quantified relative to transfection control (C).

FIG. 5.

Characterization of MYO7A dual vectors' restoration of coding sequence. Experimental plan is shown in (A). HEK293 cells were infected with AAV2-based dual-vector platforms and RNA was extracted and PCR-amplified with gene-specific primers. Control digests with BglII (B) and PpuMI (P) revealed the predicted banding pattern shown in (B). Undigested (U) PCR product is shown as control and a DNA size marker for reference (M). Separately, products were digested with KpnI and AgeI and cloned into pUC57 for sequencing of the entire overlap region (C). Ten clones per vector platform were analyzed. M13 forward and reverse primers specific for the subclone vector were used to obtain sense and antisense reads (each ∼1000 bp) resulting in ∼140 bp for which sense and antisense reads overlapped. PCR, polymerase chain reaction. Color images available online at www.liebertpub.com/hgtb

FIG. 6.

Dual-vector-mediated MYO7A (HA) expression in vivo. C57BL/6J mice were injected subretinally with AAV2-based dual vectors containing a C′ terminal HA tag. Retinal protein expression was analyzed 4 weeks later by immunohistochemistry and Western blot. Ten-micrometer frozen retinal cross sections were imaged at 10×and 60×. Equal amounts of protein were separated on a 4–15% polyacrylamide gel and stained with an HA antibody. For comparison, endogenous MYO7A from C57BL/6J retina was probed with an antibody against MYO7A to confirm that HA-tagged MYO7A migrated at the appropriate size. GCL, ganglion cell layer; INL, inner nuclear layer; IS, inner segments; ONL, outer nuclear layer; OS, outer segments; PR, photoreceptors; RPE, retinal pigment epithelium.