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Single AAV-mediated mutation replacement genome editing in limited number of photoreceptors restores vision in mice.

ABSTRACT: Supplementing wildtype copies of functionally defective genes with adeno-associated virus (AAV) is a strategy being explored clinically for various retinal dystrophies. However, the low cargo limit of this vector allows its use in only a fraction of patients with mutations in relatively small pathogenic genes. To overcome this issue, we developed a single AAV platform that allows local replacement of a mutated sequence with its wildtype counterpart, based on combined CRISPR-Cas9 and micro-homology-mediated end-joining (MMEJ). In blind mice, the mutation replacement rescued approximately 10% of photoreceptors, resulting in an improvement in light sensitivity and an increase in visual acuity. These effects were comparable to restoration mediated by gene supplementation, which targets a greater number of photoreceptors. This strategy may be applied for the treatment of inherited disorders caused by mutations in larger genes, for which conventional gene supplementation therapy is not currently feasible.

SUBMITTER: Nishiguchi KM

PROVIDER: S-EPMC6981188 | biostudies-literature | 2020 Jan

REPOSITORIES: biostudies-literature

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Single AAV-mediated mutation replacement genome editing in limited number of photoreceptors restores vision in mice.

Nishiguchi Koji M KM Fujita Kosuke K Miya Fuyuki F Katayama Shota S Nakazawa Toru T

Nature communications 20200124 1

Supplementing wildtype copies of functionally defective genes with adeno-associated virus (AAV) is a strategy being explored clinically for various retinal dystrophies. However, the low cargo limit of this vector allows its use in only a fraction of patients with mutations in relatively small pathogenic genes. To overcome this issue, we developed a single AAV platform that allows local replacement of a mutated sequence with its wildtype counterpart, based on combined CRISPR-Cas9 and micro-homolo ...[more]

PMID: 31980606

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Project description:Development of viral vectors capable of transducing photoreceptors by less invasive methods than subretinal injection would provide a major advancement in retinal gene therapy. We sought to develop novel AAV vectors optimized for photoreceptor transduction following intravitreal delivery and to develop methodology for quantifying this transduction in vivo. Surface exposed tyrosine (Y) and threonine (T) residues on the capsids of AAV2, AAV5 and AAV8 were changed to phenylalanine (F) and valine (V), respectively. Transduction efficiencies of self-complimentary, capsid-mutant and unmodified AAV vectors containing the smCBA promoter and mCherry cDNA were initially scored in vitro using a cone photoreceptor cell line. Capsid mutants exhibiting the highest transduction efficiencies relative to unmodified vectors were then injected intravitreally into transgenic mice constitutively expressing a Rhodopsin-GFP fusion protein in rod photoreceptors (Rho-GFP mice). Photoreceptor transduction was quantified by fluorescent activated cell sorting (FACS) by counting cells positive for both GFP and mCherry. To explore the utility of the capsid mutants, standard, (non-self-complementary) AAV vectors containing the human rhodopsin kinase promoter (hGRK1) were made. Vectors were intravitreally injected in wildtype mice to assess whether efficient expression exclusive to photoreceptors was achievable. To restrict off-target expression in cells of the inner and middle retina, subsequent vectors incorporated multiple target sequences for miR181, an miRNA endogenously expressed in the inner and middle retina. Results showed that AAV2 containing four Y to F mutations combined with a single T to V mutation (quadY-F+T-V) transduced photoreceptors most efficiently. Robust photoreceptor expression was mediated by AAV2(quadY-F+T-V) -hGRK1-GFP. Observed off-target expression was reduced by incorporating target sequence for a miRNA highly expressed in inner/middle retina, miR181c. Thus we have identified a novel AAV vector capable of transducing photoreceptors following intravitreal delivery to mouse. Furthermore, we describe a robust methodology for quantifying photoreceptor transduction from intravitreally delivered AAV vectors.

Project description:Visual speech facilitates auditory speech perception, but the visual cues responsible for these benefits and the information they provide remain unclear. Low-level models emphasize basic temporal cues provided by mouth movements, but these impoverished signals may not fully account for the richness of auditory information provided by visual speech. High-level models posit interactions among abstract categorical (i.e., phonemes/visemes) or amodal (e.g., articulatory) speech representations, but require lossy remapping of speech signals onto abstracted representations. Because visible articulators shape the spectral content of speech, we hypothesized that the perceptual system might exploit natural correlations between midlevel visual (oral deformations) and auditory speech features (frequency modulations) to extract detailed spectrotemporal information from visual speech without employing high-level abstractions. Consistent with this hypothesis, we found that the time-frequency dynamics of oral resonances (formants) could be predicted with unexpectedly high precision from the changing shape of the mouth during speech. When isolated from other speech cues, speech-based shape deformations improved perceptual sensitivity for corresponding frequency modulations, suggesting that listeners could exploit this cross-modal correspondence to facilitate perception. To test whether this type of correspondence could improve speech comprehension, we selectively degraded the spectral or temporal dimensions of auditory sentence spectrograms to assess how well visual speech facilitated comprehension under each degradation condition. Visual speech produced drastically larger enhancements during spectral degradation, suggesting a condition-specific facilitation effect driven by cross-modal recovery of auditory speech spectra. The perceptual system may therefore use audiovisual correlations rooted in oral acoustics to extract detailed spectrotemporal information from visual speech.

Dataset Information

Single AAV-mediated mutation replacement genome editing in limited number of photoreceptors restores vision in mice.

Publications

Single AAV-mediated mutation replacement genome editing in limited number of photoreceptors restores vision in mice.

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