Person:

Vandenberghe, Luk

The use of viral vectors for inner ear gene therapy is receiving increased attention for treatment of genetic hearing disorders. Most animal studies to date have injected viral suspensions into neonatal ears, via the round window membrane. Achieving transduction of hair cells, or sensory neurons, throughout the cochlea has proven difficult, and no studies have been able to efficiently transduce sensory cells in adult ears while maintaining normal cochlear function. Here, we show, for the first time, successful transduction of all inner hair cells and the majority of outer hair cells in an adult cochlea via virus injection into the posterior semicircular canal. We used a “designer” AAV, AAV2/Anc80L65, in which the main capsid proteins approximate the ancestral sequence state of AAV1, 2, 8, and 9. Our injections also transduced ~10% of spiral ganglion cells and a much larger fraction of their satellite cells. In the vestibular sensory epithelia, the virus transduced large numbers of hair cells and virtually all the supporting cells, along with close to half of the vestibular ganglion cells. We conclude that this viral vector and this delivery route hold great promise for gene therapy applications in both cochlear and vestibular sense organs.

In this report, we describe the development of a modified adeno-associated virus (AAV) capsid and promoter for transduction of retinal ON-bipolar cells. The bipolar cells, which are post-synaptic to the photoreceptors, are important retinal targets for both basic and preclinical research. In particular, a therapeutic strategy under investigation for advanced forms of blindness involves using optogenetic molecules to render ON-bipolar cells light-sensitive. Currently, delivery of adequate levels of gene expression is a limiting step for this approach. The synthetic AAV capsid and promoter described here achieves high level of optogenetic transgene expression in ON-bipolar cells. This evokes high-frequency (∼100 Hz) spiking responses in ganglion cells of previously blind, rd1, mice. Our vector is a promising vehicle for further development toward potential clinical use.

Widespread gene transfer to the retina is challenging as it requires vector systems to overcome physical and biochemical barriers to enter and diffuse throughout retinal tissue. We investigated whether exosome-associated adeno-associated virus, (exo-AAV) enabled broad retinal targeting following intravitreal (IVT) injection, as exosomes have been shown to traverse biological barriers and mediate widespread distribution upon systemic injection. We packaged an AAV genome encoding green fluorescent protein (GFP) into conventional AAV2 and exo-AAV2 vectors. Vectors were IVT injected into the eyes of adult mice. GFP expression was noninvasively monitored by fundus imaging and retinal expression was analyzed 4 weeks post-injection by qRT-PCR and histology. Exo-AAV2 outperformed conventional AAV2 in GFP expression based on fundus image analysis and qRT-PCR. Exo-AAV2 demonstrated deeper penetration in the retina, efficiently reaching the inner nuclear and outer plexiform, and to a lesser extent the outer nuclear layer. Cell targets were ganglion cells, bipolar cells, Müller cells, and photoreceptors. Exo-AAV2 serves as a robust gene delivery tool for murine retina, and the simplicity of production and isolation should make it widely applicable to basic research of the eye.

Efforts to develop gene therapies for hearing loss have been hampered by the lack of safe, efficient, and clinically relevant delivery modalities1, 2. Here we demonstrate the safety and efficiency of Anc80L65, a rationally designed synthetic vector3, for transgene delivery to the mouse cochlea. Cochlear explants incubated with Anc80L65 encoding eGFP demonstrated high level transduction of inner and outer hair cells (60–100%). Injection of Anc80L65 through the round window membrane resulted in highly efficient transduction of inner and outer hair cells, a substantial improvement over conventional adeno-associated virus (AAV) vectors. Anc80L65 round window injection was well tolerated, as indicated by sensory cell function, hearing and vestibular function, and immunologic parameters. The ability of Anc80L65 to target outer hair cells at high rates, a requirement for restoration of complex auditory function, may enable future gene therapies for hearing and balance disorders.

Adeno-associated viruses (AAVs) are used extensively as a gene delivery vehicle for retinal gene therapy, yet its ability to target the anterior segment of the eye, critical to unlocking therapeutic opportunities, is less characterized. Previously, self-complimentary (sc) AAV was shown to be necessary for transduction of the cornea and trabecular meshwork (TM), limiting the size of the gene transfer cassette, likely due to a block in second strand synthesis thought to be required for functional transduction. Here, we evaluated several AAV capsids in a single stranded (ss) genome conformation for their ability to overcome the need for scAAV for targeting corneal endothelium and TM. AAV2, 8, and a recently synthetically developed AAV called Anc80L65 were evaluated in vitro and in vivo by intracameral injection in mice. Results show that although scAAV2 demonstrated superior infectivity in vitro including Human Trabecular meshwork (HTM) immortalized cell lines; Anc80L65 transduced following a single intracameral injection efficiently all components of the mouse anterior segment, including the TM, corneal stroma, and endothelial cells. These results suggest that Anc80L65 is able to overcome the requirement for scAAV genomes to enable TM and corneal targeting, expanding the potential experimental and therapeutic use of AAV gene transfer in the anterior segment of the eye.

Retinal gene therapy has come a long way in the last few decades and the development and improvement of new gene delivery technologies has been exponential. The recent promising results from the first clinical trials for inherited retinal degeneration due to mutations in RPE65 have provided a major breakthrough in the field and have helped cement the use of recombinant adeno-associated viruses (AAV) as the major tool for retinal gene supplementation. One of the key problems of AAV however, is its limited capacity for packaging genomic information to a maximum of around 4.8 kb. Previous studies have demonstrated that homologous recombination and/or inverted terminal repeat (ITR) mediated concatemerization of two overlapping AAV vectors can partially overcome the size limitation and help deliver larger transgenes. The aim of this study was to investigate and compare the use of different AAV dual-vector approaches in the mouse retina using a systematic approach comparing efficiencies in vitro and in vivo using a unique oversized reporter construct. We show that the hybrid approach relying on vector genome concatemerization by highly recombinogenic sequences and ITRs sequence overlap offers the best levels of reconstitution both in vitro and in vivo compared to trans-splicing and overlap strategies. Our data also demonstrate that dose and vector serotype do not affect reconstitution efficiency but a discrepancy between mRNA and protein expression data suggests a bottleneck affecting translation.

Frame-disrupting mutations in the DMD gene, encoding dystrophin, compromise myofiber integrity and drive muscle deterioration in Duchenne muscular dystrophy (DMD). Removing one or more exons from the mutated transcript can produce an in-frame mRNA and a truncated but still functional protein. In this study, we develop and test a direct gene editing approach to induce exon deletion and recover dystrophin expression in the mdx mouse model of DMD. Delivery by adeno-associated virus (AAV) of clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 endonucleases coupled with paired guide RNAs flanking the mutated Dmd exon23 resulted in excision of intervening DNA and restored Dystrophin reading frame in myofibers, cardiomyocytes and muscle stem cells following local or systemic delivery. AAV-Dmd CRISPR-treatment partially recovered muscle functional deficiencies and generated a pool of endogenously corrected myogenic precursors in mdx mouse muscle.