The accessible and immune-privileged environment of the eye makes it ideal for gene therapy. It has been 8 years since successful gene therapy was first reported for patients with an inherited form of progressive retinal degeneration (RD), Leber Congenital Amaurosis (LCA2), following treatment in one eye with AAV2-RPE65.1, 2, 3 Now, Bennett et al.4 have reported improved vision without adverse effects, such as immunogenicity, following treatment of the second eye with AAV2-RPE65, in children with LCA2. These results strongly support the use of in situ gene therapy in patients who can be treated prior to the onset of RD.
Paramount to this approach is the early identification of patients, before the retina has undergone any irreversible changes. While most forms of RD are detected only after significant retinal pathogenesis, Usher syndrome types 1 and 2 present congenital deafness that facilitates genetic identification of the disease in infancy; Usher 1 patients are born profoundly deaf and Usher 2 patients are insensitive to high frequencies.5 Nowadays, the deafness can be treated with cochlear implants, and genetic testing of deaf infants is used to identify Usher syndrome and thus predict ensuing RD.
The first retinal gene therapy studies with Usher genes were carried out using lentiviral (LV) delivery of MYO7A, the gene responsible for Usher syndrome 1B (USH1B). MYO7A is present in both the photoreceptor and retinal pigment epithelial (RPE) cells6 (Figure 1a). Injection of LV-MYO7A into the subretinal space of Myo7a-deficient mice was found to correct mutant phenotypes in both these cell types.7 A phase I/II clinical trial, using LV-MYO7A to treat RD in USH1B, has been under way since 2012 (https://clinicaltrials.gov/ct2/show/NCT01505062).
Because the MYO7A coding sequence is 6.7 kb, it was thought that a viral vector such as LV was needed for delivery, since it features a larger carrying capacity than the reported maximum of 5 kb for adeno-associated virus (AAV). However, more recently, it was found that use of AAV, including AAV2, as used in the LCA2 treatments, mentioned above, resulted in WT levels of MYO7A and correction of retinal phenotypes in mutant mice.8, 9, 10
A significant body of research has now demonstrated that oversized AAV genomes can be packaged into high titer AAV as 5′ truncated sense and anti-sense genomes, termed fragmented vectors (fAAV). The truncated genomes are efficiently reassembled, with high fidelity, into the full transgene product following transduction of target cells, as a result of recombination that is biased towards homologous recombination (HR) rather than non-homologous end joining (NHEJ)11, 12, 13 (Figure 1b). Oversized gene replacement therapy, using fAAV expressing MYO7A cDNA, not only demonstrated reconstitution of the intact transgene product in vivo but also reported more reliable phenotypic correction of the underlying mutation than a dual AAV vector expression system.9 Similar success was also reported for fAAV expressing a 7.5 kb dysferlin transgene.14 Recently, an optimized dual vector system was shown to be comparable to fAAV vectors in a mouse model of Stargardt’s RD.15 There is an ongoing debate over which AAV system is the best for delivery of large transgenes.10, 14, 16 However, it is likely that many factors, including the transgene sequence and the epigenetic and transcriptional state of the target tissue will influence the success of each approach.
Despite the promise of gene augmentation therapy for genetic RDs, this approach is not amenable for a gene whose functional cDNA is very large, or which expresses multiple essential isoforms. All the Usher genes have been reported to express multiple isoforms, although their relative importance in the human retina is unknown (Table 1). The expression of two major isoforms of MYO7A in the human retina17 is a concern for the current USH1B clinical trial, which is using the single cDNA that was generated in the original mouse studies.7 This isoform corrects mouse retinal phenotypes,7, 9 but the relative isoform expression may differ between mouse and human retinas.
Direct targeting of genetic mutations can overcome these limitations. Recently, antisense oligonucleotides (ASOs) were used to correct a splice-site mutation in CEP290, a large gene defective in another form of LCA, and whose protein, like most of the Usher proteins, functions in the photoreceptor cilium.18 Similarly, ASOs were used to target a cryptic splice site in the orthologue of USH1C, thereby rescuing hearing and vestibular functions in a mouse model for USH1C.19 ASOs are limited, however, to diseases amenable to repair by blocking translation or a specific splice site.
Other mutations can potentially be repaired by gene editing strategies, including the clustered, regularly interspaced, palindromic repeats (CRISPR)-associated (Cas) system, which has revolutionized the field of genomic engineering since its introduction.20 Adapted from the microbial immune system, this technology uses a short guide RNA to target the Cas endonuclease to a specific locus in the genome. The Cas endonuclease can then generate double-stranded breaks in the DNA, which can be repaired by one of the two mechanisms: (1) NHEJ, an error-prone process that often results in insertions or deletions, or (2) homology-directed repair (HDR), which requires a repair template to introduce modifications to the targeted genetic locus.21 NHEJ occurs at a higher frequency, while HDR is more suitable for repairing mutations. Recent RD studies demonstrated correction of rd1 via CRISPR-mediated HDR of mutant mouse zygotes,22 and allele-specific ablation of a dominant mutant allele of Rho by CRISPR-mediated NHEJ with neonatal mice.23
Viral delivery of CRISPR-Cas components offers high transduction efficiency, limited only by the size of the donor HDR template with respect to viral capacity (see above). HDR is currently also limited by its low efficiency, especially in post-mitotic cells.24 Nonetheless, the observation that an oversized AAV genome is regenerated in vivo by photoreceptor and RPE cells9, 10 indicates the presence of HDR in these cell types,11, 13, 25 and corroborates previous work, demonstrating HDR in developed adult photoreceptors.26
One strategy to help overcome limitations of HDR is to take advantage of different classes of CRISPR-Cas systems. For example, unlike the commonly used Cas9, the endonuclease Cpf1 generates staggered cuts with 5′ overhangs.27 The resulting cleavage could mediate the insertion of a DNA fragment to correct a mutation by NHEJ, the more dominant repair mechanism. Another strategy is to use homology-independent targeted integration (HITI), whereby the nuclease cuts both donor and genomic DNA, resulting in ligation of the donor fragment into a genomic locus using NHEJ. This approach has been demonstrated very recently in a study that included in vivo gene editing of the Mertk gene, which is expressed in the RPE and is essential for ingestion of the photoreceptor outer segment disks by the RPE. The HITI-edited gene resulted in morphological and physiological repair that was more significant than that obtained using an HDR-based approach.28
Novel innovations in the field of gene editing will provide opportunities to optimize gene repair for RDs, however, a genetic model with well-characterized cellular phenotypes would be useful to test and optimize the efficiency of gene editing. Among RDs, the cellular phenotypes resulting from loss of MYO7A in mutant mouse retinas have been particularly well characterized. These phenotypes, such as melanosome localization in the RPE29 and opsin concentration in the proximal photoreceptor cilium,30 can be scored on a cell-by-cell basis,9, 31 thus giving a direct readout of efficiency, making them potentially useful in optimization studies.
In conclusion, because patients with Usher syndrome are typically identified before RD begins, they are particularly suitable for gene therapy approaches. Preclinical tests are needed to determine if only one isoform is essential to prevent RD; in this case, all but 2 or 3 of the largest genes would appear suitable for augmentation by AAV, fAAV or dual AAV vector delivery of a single cDNA. Subtypes that are associated with a very large gene (USH2A and 2C) or more than one essential retinal isoform represent appropriate candidates for testing AAV vectors in the context of new gene-editing strategies (Table 1; Figure 1c).
References
Bainbridge JW, Smith AJ, Barker SS, Robbie S, Henderson R, Balaggan K et al. Effect of gene therapy on visual function in Leber's congenital amaurosis. N Engl J Med 2008; 358: 2231–2239.
Maguire AM, Simonelli F, Pierce EA, Pugh EN Jr, Mingozzi F, Bennicelli J et al. Safety and efficacy of gene transfer for Leber's congenital amaurosis. N Engl J Med 2008; 358: 2240–2248.
Hauswirth WW, Aleman TS, Kaushal S, Cideciyan AV, Schwartz SB, Wang L et al. Treatment of leber congenital amaurosis due to RPE65 mutations by ocular subretinal injection of adeno-associated virus gene vector: short-term results of a phase I trial. Hum Gene Ther 2008; 19: 979–990.
Bennett J, Wellman J, Marshall KA, McCague S, Ashtari M, DiStefano-Pappas J et al. Safety and durability of effect of contralateral-eye administration of AAV2 gene therapy in patients with childhood-onset blindness caused by RPE65 mutations: a follow-on phase 1 trial. Lancet 2016; 388: 661–672.
Kimberling WJ, Moller C . Clinical and molecular genetics of Usher syndrome. J Am Acad Audiol 1995; 6: 63–72.
Liu X, Vansant G, Udovichenko IP, Wolfrum U, Williams DS . Myosin VIIa, the product of the Usher 1B syndrome gene, is concentrated in the connecting cilia of photoreceptor cells. Cell Motil Cytoskeleton 1997; 37: 240–252.
Hashimoto T, Gibbs D, Lillo C, Azarian SM, Legacki E, Zhang XM et al. Lentiviral gene replacement therapy of retinas in a mouse model for Usher syndrome type 1B. Gene Ther 2007; 14: 584–594.
Allocca M, Doria M, Petrillo M, Colella P, Garcia-Hoyos M, Gibbs D et al. Serotype-dependent packaging of large genes in adeno-associated viral vectors results in effective gene delivery in mice. J Clin Invest 2008; 118: 1955–1964.
Lopes VS, Boye SE, Louie CM, Boye S, Dyka F, Chiodo V et al. Retinal gene therapy with a large MYO7A cDNA using adeno-associated virus. Gene Ther 2013; 20: 824–833.
Trapani I, Colella P, Sommella A, Iodice C, Cesi G, de Simone S et al. Effective delivery of large genes to the retina by dual AAV vectors. EMBO Mol Med 2014; 6: 194–211.
Dong B, Nakai H, Xiao W . Characterization of genome integrity for oversized recombinant AAV vector. Mol Ther 2010; 18: 87–92.
Hirsch ML, Green L, Porteus MH, Samulski RJ . Self-complementary AAV mediates gene targeting and enhances endonuclease delivery for double-strand break repair. Gene Ther 2010; 17: 1175–1180.
Wu Z, Yang H, Colosi P . Effect of genome size on AAV vector packaging. Mol Ther 2010; 18: 80–86.
Hirsch ML, Li C, Bellon I, Yin C, Chavala S, Pryadkina M et al. Oversized AAV transductifon is mediated via a DNA-PKcs-independent, Rad51C-dependent repair pathway. Mol Ther 2013; 21: 2205–2216.
Trapani I, Toriello E, de Simone S, Colella P, Iodice C, Polishchuk EV et al. Improved dual AAV vectors with reduced expression of truncated proteins are safe and effective in the retina of a mouse model of Stargardt disease. Hum Mol Genet 2015; 24: 6811–6825.
Ghosh A, Yue Y, Duan D . Efficient transgene reconstitution with hybrid dual AAV vectors carrying the minimized bridging sequences. Hum Gene Ther 2011; 22: 77–83.
Weil D, Levy G, Sahly I, Levi-Acobas F, Blanchard S, El-Amraoui A et al. Human myosin VIIA responsible for the Usher 1B syndrome: a predicted membrane-associated motor protein expressed in developing sensory epithelia. Proc Natl Acad Sci USA 1996; 93: 3232–3237.
Garanto A, Chung DC, Duijkers L, Corral-Serrano JC, Messchaert M, Xiao R et al. In vitro and in vivo rescue of aberrant splicing in CEP290-associated LCA by antisense oligonucleotide delivery. Hum Mol Genet 2016; 25: 2552–2563.
Lentz JJ, Jodelka FM, Hinrich AJ, McCaffrey KE, Farris HE, Spalitta MJ et al. Rescue of hearing and vestibular function by antisense oligonucleotides in a mouse model of human deafness. Nat Med 2013; 19: 345–350.
Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N et al. Multiplex genome engineering using CRISPR/Cas systems. Science 2013; 339: 819–823.
Yang L, Mali P, Kim-Kiselak C, Church G . CRISPR-Cas-mediated targeted genome editing in human cells. Methods Mol Biol 2014; 1114: 245–267.
Wu WH, Tsai YT, Justus S, Lee TT, Zhang L, Lin CS et al. CRISPR repair reveals causative mutation in a preclinical model of retinitis pigmentosa. Mol Ther 2016; 24: 1388–1394.
Bakondi B, Lv W, Lu B, Jones MK, Tsai Y, Kim KJ et al. In vivo CRISPR/Cas9 gene editing corrects retinal dystrophy in the S334ter-3 rat model of autosomal dominant retinitis pigmentosa. Mol Ther 2016; 24: 556–563.
Ran FA, Hsu PD, Wright J, Agarwala V, Scott DA, Zhang F . Genome engineering using the CRISPR-Cas9 system. Nat Protoc 2013; 8: 2281–2308.
Lai Y, Yue Y, Duan D . Evidence for the failure of adeno-associated virus serotype 5 to package a viral genome ⩾8.2 kb. Mol Ther 2010; 18: 75–79.
Chan F, Hauswirth WW, Wensel TG, Wilson JH . Efficient mutagenesis of the rhodopsin gene in rod photoreceptor neurons in mice. Nucleic Acids Res 2011; 39: 5955–5966.
Zetsche B, Gootenberg JS, Abudayyeh OO, Slaymaker IM, Makarova KS, Essletzbichler P et al. Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system. Cell 2015; 163: 759–771.
Suzuki K, Tsunekawa Y, Hernandez-Benitez R, Wu J, Zhu J, Kim EJ et al. In vivo genome editing via CRISPR/Cas9 mediated homology-independent targeted integration. Nature 2016; 540: 144–149.
Liu X, Ondek B, Williams DS . Mutant myosin VIIa causes defective melanosome distribution in the RPE of shaker-1 mice. Nat Genet 1998; 19: 117–118.
Liu X, Udovichenko IP, Brown SDM, Steel KP, Williams DS . Myosin VIIa participates in opsin transport through the photoreceptor cilium. J Neurosci 1999; 19: 6267–6274.
Jacobson SG, Cideciyan AV, Aleman TS, Sumaroka A, Roman AJ, Gardner LM et al. Usher syndromes due to MYO7A, PCDH15, USH2A or GPR98 mutations share retinal disease mechanism. Hum Mol Genet 2008; 17: 2405–2415.
Nagel-Wolfrum K, Becker M, Goldmann T, Müller C, Vetter J, Wolfrum U . USH1C transcripts and harmonin proteinexpression in human retina. Invest Ophthalmol Vis Sci 2011; 52: 45–45.
Lagziel A, Overlack N, Bernstein SL, Morell RJ, Wolfrum U, Friedman TB . Expression of cadherin 23 isoforms is not conserved: implications for a mouse model of Usher syndrome type 1D. Mol Vis 2009; 15: 1843–1857.
Ahmed ZM, Riazuddin S, Aye S, Ali RA, Venselaar H, Anwar S et al. Gene structure and mutant alleles of PCDH15: nonsyndromic deafness DFNB23 and type 1 Usher syndrome. Hum Genet 2008; 124: 215–223.
Riazuddin S, Belyantseva IA, Giese A, Lee K, Indzhykulian AA, Nandamuri SP et al. Mutations in CIB2, a calcium and integrin binding protein, cause Usher syndrome type 1J and nonsyndromic deafness DFNB48. Nat Genet 2012; 44: 1265–1271.
van Wijk E, Pennings RJ, te Brinke H, Claassen A, Yntema HG, Hoefsloot LH et al. Identification of 51 novel exons of the Usher syndrome type 2A (USH2A) gene that encode multiple conserved functional domains and that are mutated in patients with Usher syndrome type II. Am J Hum Genet 2004; 74: 738–744.
Weston MD, Luijendijk MW, Humphrey KD, Moller C, Kimberling WJ . Mutations in the VLGR1 gene implicate G-protein signaling in the pathogenesis of Usher syndrome type II. Am J Hum Genet 2004; 74: 357–366.
Ebermann I, Scholl HP, Charbel Issa P, Becirovic E, Lamprecht J, Jurklies B et al. A novel gene for Usher syndrome type 2: mutations in the long isoform of whirlin are associated with retinitis pigmentosa and sensorineural hearing loss. Hum Genet 2007; 121: 203–211.
Vastinsalo H, Jalkanen R, Dinculescu A, Isosomppi J, Geller S, Flannery JG et al. Alternative splice variants of the USH3A gene Clarin 1 (CLRN1). Eur J Hum Genet 2011; 19: 30–35.
Acknowledgements
The authors are currently supported by NIH grants R01EY013408, R01EY026526, P30EY00331 (DSW), F32EY026318 (AC), T32EY007026 (RH) and R01MH111534 (DG).
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Williams, D., Chadha, A., Hazim, R. et al. Gene therapy approaches for prevention of retinal degeneration in Usher syndrome. Gene Ther 24, 68–71 (2017). https://doi.org/10.1038/gt.2016.81
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DOI: https://doi.org/10.1038/gt.2016.81