Gene therapy rescues cone function in congenital achromatopsia.

The successful restoration of visual function with recombinant adeno-associated virus (rAAV)-mediated gene replacement therapy in animals and humans with an inherited disease of the retinal pigment epithelium has ushered in a new era of retinal therapeutics. For many retinal disorders, however, targeting of therapeutic vectors to mutant rods and/or cones will be required. In this study, the primary cone photoreceptor disorder achromatopsia served as the ideal translational model to develop gene therapy directed to cone photoreceptors. We demonstrate that rAAV-mediated gene replacement therapy with different forms of the human red cone opsin promoter led to the restoration of cone function and day vision in two canine models of CNGB3 achromatopsia, a neuronal channelopathy that is the most common form of achromatopsia in man. The robustness and stability of the observed treatment effect was mutation independent, but promoter and age dependent. Subretinal administration of rAAV5-hCNGB3 with a long version of the red cone opsin promoter in younger animals led to a stable therapeutic effect for at least 33 months. Our results hold promise for future clinical trials of cone-directed gene therapy in achromatopsia and other cone-specific disorders.

[1]  P. Sieving,et al.  Mutations in the CNGB3 gene encoding the beta-subunit of the cone photoreceptor cGMP-gated channel are responsible for achromatopsia (ACHM3) linked to chromosome 8q21. , 2000, Human molecular genetics.

[2]  J. Milunsky,et al.  A locus for autosomal recessive achromatopsia on human chromosome 8q , 1999, Clinical genetics.

[3]  P. Lennie,et al.  Packing arrangement of the three cone classes in primate retina , 2001, Vision Research.

[4]  G H Jacobs,et al.  Color vision in the dog , 1989, Visual Neuroscience.

[5]  G. Acland,et al.  Application of a New Subretinal Injection Device in the Dog , 2004, Cell transplantation.

[6]  E. Zrenner,et al.  Variant phenotypes of incomplete achromatopsia in two cousins with GNAT2 gene mutations. , 2004, Investigative ophthalmology & visual science.

[7]  Jean Bennett,et al.  Gene therapy restores vision in a canine model of childhood blindness , 2001, Nature Genetics.

[8]  L. Rubin,et al.  The electroretinogram in dogs with inherited cone degeneration. , 1975, Investigative ophthalmology.

[9]  D. Hunt,et al.  Progressive cone and cone-rod dystrophies: phenotypes and underlying molecular genetic basis. , 2006, Survey of ophthalmology.

[10]  W. Hauswirth,et al.  Human blue-opsin promoter preferentially targets reporter gene expression to rat s-cone photoreceptors. , 2006, Investigative ophthalmology & visual science.

[11]  W. Hauswirth,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. , 2008, Human gene therapy.

[12]  G. Acland,et al.  Canine cone transducin-gamma gene and cone degeneration in the cd dog. , 1998, Investigative ophthalmology & visual science.

[13]  P. Luthert,et al.  Topographical characterization of cone photoreceptors and the area centralis of the canine retina , 2008, Molecular vision.

[14]  Herbert Jägle,et al.  Reorganization of human cortical maps caused by inherited photoreceptor abnormalities , 2002, Nature Neuroscience.

[15]  Rappold,et al.  Human Molecular Genetics , 1996, Nature Medicine.

[16]  A. J. Roman,et al.  Human RPE65 gene therapy for Leber congenital amaurosis: persistence of early visual improvements and safety at 1 year. , 2009, Human gene therapy.

[17]  K. Koeppen,et al.  Clinical and genetic features of Hungarian achromatopsia patients. , 2005, Molecular vision.

[18]  E. Ostrander,et al.  Canine CNGB3 mutations establish cone degeneration as orthologous to the human achromatopsia locus ACHM3. , 2002, Human molecular genetics.

[19]  C. Klaver,et al.  Genetic etiology and clinical consequences of complete and incomplete achromatopsia. , 2009, Ophthalmology.

[20]  I. Maumenee,et al.  Genetic basis of total colourblindness among the Pingelapese islanders , 2000, Nature Genetics.

[21]  L. Rubin,et al.  Pathology of hemeralopia in the Alaskan malamute dog. , 1974, Investigative ophthalmology.

[22]  L. Rubin.  Clinical features of hemeralopia in the adult Alaskan malamute. , 1971, Journal of the American Veterinary Medical Association.

[23]  C. Klaver,et al.  Homozygosity mapping reveals PDE6C mutations in patients with early-onset cone photoreceptor disorders. , 2009, American journal of human genetics.

[24]  P. Sieving,et al.  Remapping of the RP15 locus for X-linked cone-rod degeneration to Xp11.4-p21.1, and identification of a de novo insertion in the RPGR exon ORF15. , 2000, American journal of human genetics.

[25]  R. Barlow,et al.  Impaired cone function and cone degeneration resulting from CNGB3 deficiency: down-regulation of CNGA3 biosynthesis as a potential mechanism. , 2009, Human molecular genetics.

[26]  R. Quatrano.  Genomics , 1998, Plant Cell.

[27]  Rubin Lf.  Hemeralopia in Alaskan Malamute pups. , 1971 .

[28]  A. J. Roman,et al.  Safety of recombinant adeno-associated virus type 2-RPE65 vector delivered by ocular subretinal injection. , 2006, Molecular therapy : the journal of the American Society of Gene Therapy.

[29]  G. H. Jacobs,et al.  Response to Comment on "Emergence of Novel Color Vision in Mice Engineered to Express a Human Cone Photopigment" , 2007, Science.

[30]  G. Acland,et al.  Selective absence of cone outer segment beta 3-transducin immunoreactivity in hereditary cone degeneration (cd). , 1996, Experimental eye research.

[31]  T. L. McGee,et al.  A Null mutation in the rhodopsin gene causes rod photoreceptor dysfunction and autosomal recessive retinitis pigmentosa , 1992, Nature Genetics.

[32]  W. Hauswirth,et al.  Restoration of cone vision in a mouse model of achromatopsia , 2007, Nature Medicine.

[33]  W. Hauswirth,et al.  Longitudinal evaluation of expression of virally delivered transgenes in gerbil cone photoreceptors , 2008, Visual Neuroscience.

[34]  Y. Péréon,et al.  Postsurgical assessment and long-term safety of recombinant adeno-associated virus-mediated gene transfer into the retinas of dogs and primates. , 2005, Archives of ophthalmology.

[35]  Nick Tyler,et al.  Effect of gene therapy on visual function in Leber's congenital amaurosis. , 2008, The New England journal of medicine.

[36]  Y. Chérel,et al.  Recombinant adeno-associated virus serotype 4 mediates unique and exclusive long-term transduction of retinal pigmented epithelium in rat, dog, and nonhuman primate after subretinal delivery. , 2003, Molecular therapy : the journal of the American Society of Gene Therapy.

[37]  T. Dryja,et al.  Digenic retinitis pigmentosa due to mutations at the unlinked peripherin/RDS and ROM1 loci. , 1994, Science.

[38]  Donald J. Zack,et al.  A locus control region adjacent to the human red and green visual pigment genes , 1992, Neuron.

[39]  Jean Bennett,et al.  Gene Therapy for Leber's Congenital Amaurosis is Safe and Effective Through 1.5 Years After Vector Administration , 2009, Molecular therapy : the journal of the American Society of Gene Therapy.

[40]  R. Hess,et al.  Night vision : basic, clinical, and applied aspects , 1990 .

[41]  G. H. Jacobs,et al.  Photopigments of dogs and foxes and their implications for canid vision , 1993, Visual Neuroscience.

[42]  S. Jacobson,et al.  Human rod monochromacy: linkage analysis and mapping of a cone photoreceptor expressed candidate gene on chromosome 2q11. , 1998, Genomics.

[43]  Frank Müller,et al.  Rod and cone photoreceptor cells express distinct genes for cGMP-gated channels , 1993, Neuron.

[44]  S. Leal,et al.  Homozygosity mapping of the Achromatopsia locus in the Pingelapese. , 1999, American journal of human genetics.

[45]  W. Hauswirth,et al.  Production and purification of serotype 1, 2, and 5 recombinant adeno-associated viral vectors. , 2002, Methods.

[46]  G. Ying,et al.  Evaluation of a behavioral method for objective vision testing and identification of achromatopsia in dogs. , 2010, American journal of veterinary research.

[47]  Marc Korczykowski,et al.  Canine and Human Visual Cortex Intact and Responsive Despite Early Retinal Blindness from RPE65 Mutation , 2007, PLoS medicine.

[48]  W. Hauswirth,et al.  Long-term restoration of rod and cone vision by single dose rAAV-mediated gene transfer to the retina in a canine model of childhood blindness. , 2005, Molecular therapy : the journal of the American Society of Gene Therapy.

[49]  A. Moore,et al.  The cone dystrophies , 1998, Eye.

[50]  Jay Neitz,et al.  Gene therapy for red-green colour blindness in adult primates , 2009, Nature.

[51]  D. Hunt,et al.  Mapping of a novel locus for achromatopsia (ACHM4) to 1p and identification of a germline mutation in the α subunit of cone transducin (GNAT2) , 2002, Journal of medical genetics.

[52]  M. Varnum,et al.  Subunit Configuration of Heteromeric Cone Cyclic Nucleotide-Gated Channels , 2004, Neuron.

[53]  J. Rine,et al.  A novel retinal degeneration locus identified by linkage and comparative mapping of canine early retinal degeneration. , 1999, Genomics.

[54]  Edwin M Stone,et al.  Human gene therapy for RPE65 isomerase deficiency activates the retinoid cycle of vision but with slow rod kinetics , 2008, Proceedings of the National Academy of Sciences.

[55]  Kathleen A. Marshall,et al.  Safety and efficacy of gene transfer for Leber's congenital amaurosis. , 2008, The New England journal of medicine.

[56]  Tiansen Li,et al.  AAV-mediated expression targeting of rod and cone photoreceptors with a human rhodopsin kinase promoter. , 2007, Investigative ophthalmology & visual science.

[57]  L. D. Del Priore,et al.  Drusen in age-related macular degeneration: pathogenesis, natural course, and laser photocoagulation-induced regression. , 1999, Survey of ophthalmology.

[58]  S. Jacobson,et al.  Mutations in the cone photoreceptor G-protein alpha-subunit gene GNAT2 in patients with achromatopsia. , 2002, American journal of human genetics.

[59]  Rubin Lf.  Clinical features of hemeralopia in the adult Alaskan malamute. , 1971 .

[60]  S. Bolz,et al.  A homologous genetic basis of the murine cpfl1 mutant and human achromatopsia linked to mutations in the PDE6C gene , 2009, Proceedings of the National Academy of Sciences.

[61]  L. Sharpe,et al.  Total colour blindness: an introduction , 1990 .

[62]  A. Hendrickson,et al.  Human photoreceptor topography , 1990, The Journal of comparative neurology.

[63]  M. Sandberg,et al.  Recessive mutations in the gene encoding the β–subunit of rod phosphodiesterase in patients with retinitis pigmentosa , 1993, Nature Genetics.

[64]  A. Milam,et al.  Distribution and morphology of human cone photoreceptors stained with anti‐blue opsin , 1991, The Journal of comparative neurology.

[65]  G. Acland,et al.  Retinal degenerations in the dog: IV. Early retinal degeneration (erd) in Norwegian elkhounds. , 1987, Experimental eye research.

[66]  M. Biel,et al.  Molecular Cloning and Functional Characterization of a New Modulatory Cyclic Nucleotide-Gated Channel Subunit from Mouse Retina , 2000, The Journal of Neuroscience.

[67]  D. Hunt,et al.  The cone dysfunction syndromes , 2004, British Journal of Ophthalmology.

[68]  Algis J Vingrys,et al.  Loss of cone function in age-related maculopathy. , 2003, Investigative ophthalmology & visual science.

[69]  S. Zolotukhin,et al.  Production of recombinant adeno-associated virus vectors. , 2005, Human gene therapy.

[70]  H. Spekreijse,et al.  The “silent substitution” method in visual research , 1982, Vision Research.

[71]  S. Jacobson,et al.  CNGA3 mutations in hereditary cone photoreceptor disorders. , 2001, American journal of human genetics.

[72]  S. Haverkamp,et al.  Impaired opsin targeting and cone photoreceptor migration in the retina of mice lacking the cyclic nucleotide-gated channel CNGA3. , 2005, Investigative ophthalmology & visual science.

[73]  W. Hauswirth,et al.  Gene therapy restores vision-dependent behavior as well as retinal structure and function in a mouse model of RPE65 Leber congenital amaurosis. , 2006, Molecular therapy : the journal of the American Society of Gene Therapy.

[74]  Alexander Sumaroka,et al.  Vision 1 year after gene therapy for Leber's congenital amaurosis. , 2009, The New England journal of medicine.

[75]  C. Curcio,et al.  Photoreceptor loss in age-related macular degeneration. , 1996, Investigative ophthalmology & visual science.

[76]  K. Bartoli,et al.  Transmembrane S1 mutations in CNGA3 from achromatopsia 2 patients cause loss of function and impaired cellular trafficking of the cone CNG channel. , 2005, Investigative ophthalmology & visual science.

[77]  G. Fishman,et al.  ABCA4 gene sequence variations in patients with autosomal recessive cone-rod dystrophy. , 2003, Archives of ophthalmology.

[78]  S. Jacobson,et al.  Total colourblindness is caused by mutations in the gene encoding the α-subunit of the cone photoreceptor cGMP-gated cation channel , 1998, Nature Genetics.

[79]  W. Hauswirth,et al.  Targeting gene expression to cones with human cone opsin promoters in recombinant AAV , 2008, Gene Therapy.

[80]  J. Lupski,et al.  Achromatopsia: the CNGB3 p.T383fsX mutation results from a founder effect and is responsible for the visual phenotype in the original report of uniparental disomy 14 , 2007, Human Genetics.

[81]  G. Acland,et al.  A frameshift mutation in RPGR exon ORF15 causes photoreceptor degeneration and inner retina remodeling in a model of X-linked retinitis pigmentosa. , 2006, Investigative ophthalmology & visual science.

[82]  P. Sieving,et al.  CNGB3 mutations account for 50% of all cases with autosomal recessive achromatopsia , 2005, European Journal of Human Genetics.

[83]  E. Traboulsi,et al.  CNGA3 mutations in two United Arab Emirates families with achromatopsia , 2008, Molecular vision.

[84]  Kathleen A. Marshall,et al.  Age-dependent effects of RPE65 gene therapy for Leber's congenital amaurosis: a phase 1 dose-escalation trial , 2009, The Lancet.