Retinal gene therapy

Retinal gene therapy Abstract Introduction Inherited retinal diseases are the leading cause of sight impairment in people of working age in England and Wales, and the second commonest in childhood. Gene therapy offers the potential for benefit. Sources of data Pubmed and clinicaltrials.gov. Areas of agreement Gene therapy can improve vision in RPE65-associated Leber Congenital Amaurosis (RPE65-LCA). Potential benefit depends on efficient gene transfer and is limited by the extent of retinal degeneration. Areas of controversy The magnitude of vision improvement from RPE65-LCA gene therapy is suboptimal, and its durability may be limited by progressive retinal degeneration. Growing points The safety and potential benefit of gene therapy for inherited and acquired retinal diseases is being explored in a rapidly expanding number of trials. Areas timely for developing research Developments in vector design and delivery will enable greater efficiency and safety of gene transfer. Optimization of trial design will accelerate reliable assessment of outcomes. gene therapy, retina, retinal diseases Introduction The retina is a highly specialized, multi-layered tissue that includes a layer of light-sensitive cone- and rod-photoreceptor cells, which initiate neuronal signalling in response to light stimulation by means of a highly sophisticated cascade of enzymatic reactions (phototransduction). The photoreceptor cells are supported by a monolayer of pigmented cells, the retinal pigment epithelium (RPE), which performs many key processes including the regeneration of visual pigment that is bleached following light exposure (the visual cycle). Diseases of the retina, including age-related macular degeneration (AMD), inherited retinal diseases (IRDs), diabetic retinopathy and vascular occlusion, represent the commonest cause of severe sight impairment in the developed world. Inherited retinal diseases are the leading cause of blindness in people of working age in England and Wales, and the second commonest cause in children.1 Defects in genes encoding proteins involved in the phototransduction cascade or the visual cycle account for a large proportion of IRDs. Gene therapy offers an opportunity to improve outcomes of inherited monogenic disorders of the retina. In its simplest and commonest form, gene ‘supplementation’ therapy compensates for a genetic deficiency, resulting from loss-of-function mutations in the endogenous gene, by provision of the normal gene to the cells in which it is required.2 The therapeutic gene, typically delivered using a viral vector, is utilized by the transcriptional machinery of the target cell to generate the normal gene product that is otherwise lacking. Alternative gene therapy techniques aim to suppress the undesirable expression of a harmful protein product resulting from gain-of-function mutations, with or without simultaneous provision of the normal gene.3 Also being investigated are gene editing strategies to correct harmful mutations in endogenous genes, and anti-sense oligonucleotide mediated exon skipping to mitigate their impact. Clinical trials of gene therapies have included applications for primary immune deficiencies, haemoglobinopathies, haemophilia B, neurological diseases and cancer.4 The first gene therapy product approved by the European Medicines Agency was alipogene tiparvovec (Glybera), developed for the treatment of lipoprotein lipase deficiency.4 Here we describe the key strategies of ocular gene therapy and focus on its application to disease of the retina, with emphasis on experimental therapies in clinical trials. Figure 1 is a schematic diagram of the location of genes in cells of the outer retina targeted in current gene therapy trials. Fig. 1 View largeDownload slide Schematic diagram showing the outer retina and the cellular location of poducts of the genes targeted by current gene therapy trials (red) and the genes explored for future trials (black). Fig. 1 View largeDownload slide Schematic diagram showing the outer retina and the cellular location of poducts of the genes targeted by current gene therapy trials (red) and the genes explored for future trials (black). Gene therapy and the eye The retina has specific advantages as a target organ for gene therapy. The transparency of the ocular media provides accessibility for microsurgical delivery of vector suspension to the retina under direct visualization, and for cellular level imaging to target intervention and assess its impact. Vector suspension can be targeted to the retina with minimal systemic dissemination owing to the contained nature and compartmentalization of the intraocular tissues. The intraocular environment provides the retina with a degree of immune privilege, which limits immune responses that could adversely affect retinal function and limit expression of the therapeutic gene. Since inherited retinal disease typically causes bilateral disease with significant symmetry, the untreated contralateral eye offers a valuable control for natural history, learning effects and intra-individual variability in performance. For gene transfer to retinal cells, most clinical applications currently employ recombinant adeno-associated virus (AAV) or lentivirus vectors. AAV is a small, non-pathogenic single stranded DNA virus widely used for gene delivery in IRDs. AAV vectors can mediate efficient and sustained transduction of photoreceptor cells, RPE, and ganglion cells. First generation AAV2 vectors are limited by relatively slow onset of expression and small capacity (4.7 kB).5 However, the isolation of alternative serotypes and the development of self-complementary vectors and novel variants, by rational design and/or directed evolution, have provided a broad range of alternatives with more rapid expression and cell tropisms.6,7 Measures to address the limited capacity include dual AAV vector strategies in which a large gene delivered in component parts by AAV is reconstituted by splicing.8 Since lentiviral vectors have substantially greater capacity (approximately 8 kB) than AAV, they can naturally accommodate larger genes. Lentiviral vectors mediate efficient transduction that is typically limited to RPE cells but one type of lentiviral vector, derived from the equine infectious anaemia virus (EIAV), mediates variable transgene expression in photoreceptor cells.9 Vector capacity is a fundamentally important issue given that the most common genes causing IRD are large, including ABCA4 (Stargardt Disease) and USH2A (syndromic and non-syndromic Retinitis Pigmentosa). Intraocular administration Since defects in genes involved in phototransduction or the visual cycle account for many IRDs, photoreceptors and RPE cells are important target populations for gene therapy. Viral vectors deliver genes to these cells efficiently when the vector suspension is placed in direct contact with the cells in the outer retina. This is typically achieved by injecting the vector suspension between the RPE and the overlying photoreceptor cell layer. Injection into this potential subretinal space is typically performed using a fine cannula that is advanced through the sclera anteriorly, across the vitreous cavity and through the inner retina (Fig. 2A). This generates a bleb of vector suspension that temporarily separates the neurosensory retina from the underlying RPE, before it is absorbed over a period of hours or days. Injection of vector suspension into the vitreous cavity (Fig. 2B) can result in gene delivery to cells of the inner retina, including ganglion cells. Although intravitreal vector is technically simpler than subretinal injection, anatomical barriers prevent efficient gene delivery to the outer retina and this route of administration may be more immunogenic than subretinal injection.10 Compromise of inner retinal integrity, in conditions such as X-linked Retinoschisis, may enable enhanced access of intravitreal vector suspensions to the inner retina.11 Delivery of vector suspensions into the suprachoroidal potential space (between the sclera and choroid) may enable targeting of the choroid, for conditions such as AMD and idiopathic polypoidal choroidal vasculopathy.12 Fig. 2 View largeDownload slide Schematic diagram identifying ocular structures and location of (A) subretinal injection and (B) intravitreal injection. Fig. 2 View largeDownload slide Schematic diagram identifying ocular structures and location of (A) subretinal injection and (B) intravitreal injection. Current gene therapy clinical trials RPE65 Leber Congenital Amaurosis (LCA), first described by Theodore Leber in 1869, is a group of recessively inherited infantile-onset rod-cone dystrophies. The prevalence ranges from 1 in 33 000 to 1 in 81 000.13 LCA accounts globally for 5% of IRDs and 20% of children attending specialist schools for students with sight impairment.14 Mutation of one of several genes, including RPE65, causes impaired vision from birth/early infancy and typically progresses to severe sight impairment. RPE65 is expressed in the RPE and encodes a 65-kD protein that is a key component of the visual cycle, a biochemical pathway that regenerates the visual pigment after exposure to light.15,16 A lack of functional RPE65 results in deficiency of 11-cis retinal such that rod-photoreceptor cells are unable to respond to light.17 Cone photoreceptor cells have access to 11-cis retinaldehyde chromophore through an alternative pathway that does not depend on RPE-derived RPE65 thus allowing cone-mediated vision in children.18 However, progressive degeneration of cone photoreceptor cells ultimately results in the loss of cone-mediated vision. The appearance of the retina on clinical examination is typically normal at an early age, but retinal degeneration becomes in advanced disease (Fig. 3). Gene-replacement therapy can improve visual function in rodent models of RPE65-LCA, and in the Swedish Briard dog, which has a naturally occurring mutation in RPE65.19 The treated eyes of dogs showed improved responses on electroretinography, pupillometry and flash-evoked cortical potentials in the dark-adapted state, with improvements sustained for as long as 10 years.19 Fig. 3 View largeDownload slide Colour fundus photographs of (A) a patient with RPE65-associated Leber congenital amaurosis and (B) an unaffected individual for comparison. (A) shows peripheral RPE atrophy with a tessellated appearance to the fundus owing to the choroidal vasculature, with central preservation of retinal structure. Fig. 3 View largeDownload slide Colour fundus photographs of (A) a patient with RPE65-associated Leber congenital amaurosis and (B) an unaffected individual for comparison. (A) shows peripheral RPE atrophy with a tessellated appearance to the fundus owing to the choroidal vasculature, with central preservation of retinal structure. The first trial of gene therapy in humans with RPE65-LCA was reported in 2008.20 Several phase I/II trials have provided evidence that subretinal injection of a recombinant AAV 2/2 vector containing the RPE65 cDNA can improve retinal function and vision.20–23 The findings of an open-label randomized controlled trial confirm benefit at 1 year to night vision, as indicated by improved performance in a test of vision-guided mobility in low luminance.24 In 2017 the US FDA approved voretingene neparvovec (Luxturna, Spark Therapeutics Inc.) for the treatment of RPE65-associated LCA. Long-term findings indicate that, despite improved function of surviving retina, the benefits of gene therapy can be limited by progressive retinal degeneration.23,25–27 To investigate the hypothesis that greater provision of RPE65 will provide more durable, robust benefit, the effect of a more efficient optimized AAV 2/5 vector is being investigated.28 Figure 4 illustrates one example of improved retinal sensitivity in RPE65-associated LCA following intervention gene therapy, as demonstrated by advanced hill of vision modelling.29 Fig. 4 View largeDownload slide An oblique topographical view of the central hill of vision, produced by Visual Field Modelling and Analysis (VFMA, Office of Technology Transfer and Business Development [OHSU], Portland, Oregon, USA)29 in a subject (A) prior to and (B) following gene therapy intervention (A) shows residual central visual field prior to intervention, in comparison to (B) which demonstrates increased retinal sensitivity and therefore a larger and taller hill of vision at 1 year post-intervention. Fig. 4 View largeDownload slide An oblique topographical view of the central hill of vision, produced by Visual Field Modelling and Analysis (VFMA, Office of Technology Transfer and Business Development [OHSU], Portland, Oregon, USA)29 in a subject (A) prior to and (B) following gene therapy intervention (A) shows residual central visual field prior to intervention, in comparison to (B) which demonstrates increased retinal sensitivity and therefore a larger and taller hill of vision at 1 year post-intervention. CNGA3, CNGB3 Achromatopsia (ACHM) is a recessively inherited disorder of cone photoreceptor function affecting approximately 1 in 30 000 people. The condition causes severe impairment of sight from birth, with poor visual acuity, absent or markedly reduced colour vision, disabling photophobia and pendular nystagmus.30 ACHM can be caused by mutations in any one of at least six genes including CNGA3 and CNGB3, which encode the alpha and beta subunits, respectively, of the cone photoreceptor cyclic nucleotide gated channels, and account for the majority of people affected.30 Structural preservation of individual cone photoreceptors can be determined in affected humans by adaptive optics scanning laser ophthalmoscopy (AO-SLO),30 to evaluate their potential to benefit from gene therapy. AAV-mediated gene-replacement therapy can improve cone function in rodent models of both CNGA3-ACHM31 and CNGB3-ACHM,32 and in canine models of CNGB3-ACHM.30 Early phase clinical trials of gene therapy for both CNGA3-ACHM and CNGB3-ACHM will determine the extent to which this approach might benefit visual acuity, colour discrimination, photophobia and nystagmus in affected humans (NCT02599922, NCT03001310, NCT02610582, NCT02935517) (Table 1). Table 1 Clinical trials of retinal gene therapy (LCA, Leber Congenital Amaurosis; RP, Retinitis Pigmentosa) Disease Gene Gene function Vector Mode Phase Current status Clinical trials ID Sponsor Location LCA-2 RPE65 Retinoid cycle AAV2/2 Subretinal III Ongoing NCT00999609 Spark Therapeutics UK NCT01208389 LCA-2 RPE65 Retinoid cycle AAV2/2 Subretinal I Ongoing NCT00516477 Spark Therapeutics USA LCA-2 RPE65 Retinoid cycle AAV2/5 Subretinal I/II Ongoing NCT02781480 MeiraGTx UK II Ltd UK NCT02946879 LCA-2 RPE65 Retinoid cycle AAV2 Subretinal I Unknown NCT00821340 Hadassah Medical Organization Israel LCA-2 RPE65 Retinoid cycle AAV2/2 Subretinal I Ongoing NCT00481546 University of Pennsylvania and National Eye Institute USA LCA-2 RPE65 Retinoid cycle AAV2 Subretinal I/II Ongoing NCT00749957 Applied Genetic Technologies Corp USA LCA-2 RPE65 Retinoid cycle AAV2/2 Subretinal I/II Completed NCT00643747 University College London UK LCA-2 RPE65 Retinoid cycle AAV2/4 Subretinal I/II Completed NCT01496040 Nantes University Hospital France LCA-10 CEP290 Ciliary transport N/A* Intravitreal I/II Ongoing NCT03140969 ProQR Therapeutics USA, Belgium Stargardt Disease ABCA4 Retinoid transfer EIAV Subretinal I/II Ongoing NCT01367444 Sanofi/Oxford BioMedica USA, France RP assoc. with Usher’s Syndrome Type 1B MYO7A Intracellular transport EIAV Subretinal I/II Ongoing NCT01505062 Sanofi/Oxford BioMedica USA, France Choroideremia REP1 Intracellular trafficking AAV2 Subretinal II Ongoing NCT02553135 University of Miami USA Choroideremia REP1 Intracellular trafficking AAV2 Subretinal II Ongoing NCT02671539 STZ Eyetrial Germany Choroideremia REP1 Intracellular trafficking AAV2 Subretinal I/II Ongoing NCT01461213 University of Oxford/ NightstaRx UK Choroideremia REP1 Intracellular trafficking AAV2 Subretinal I/II Ongoing NCT02077361 University of Alberta/ NightstaRx Canada Choroideremia REP1 Intracellular trafficking AAV2 Subretinal II Ongoing NCT02407678 University of Oxford/ NightstaRx UK Choroideremia CHM Intracellular trafficking AAV2 Subretinal I/II Ongoing NCT02341807 Spark Therapeutics USA MERTK-RP MERTK Cellular signalling AAV2 Subretinal I Ongoing NCT01482195 King Khaled Eye Specialist Hospital Saudi Arabia X-Linked RP RPGR Intracellular transport AAV8 Subretinal I/II Ongoing NCT03116113 NightstaRx UK X-Linked RP RPGR Intracellular transport AAV2/5 Subretinal I/II Ongoing NCT03252847 MeiraGTx UK II Ltd UK Achromatopsia CNGB3 Phototransduction cascade AAV2 Subretinal I/II Ongoing NCT02599922 Applied Genetic Technologies Corp USA Achromatopsia CNGB3 Phototransduction cascade AAV2/8 Subretinal I/II Ongoing NCT03001310 MeiraGTx UK II Ltd UK Achromatopsia CNGA3 Phototransduction cascade AAV8 Subretinal I/II Ongoing NCT02610582 STZ Eyetrial/University Hospital Tuebingen Germany Achromatopsia CNGA3 Phototransduction cascade AAV2 Subretinal I/II Ongoing NCT02935517 Applied Genetic Technologies Corp USA, Israel X-Linked Retinoschisis RS1 Intercellular adhesion AAV8 Intravitreal I/II Ongoing NCT02317887 National Eye Institute USA X-Linked Retinoschisis RS1 Intercellular adhesion AAV2 Intravitreal I/II Ongoing NCT02416622 Applied Genetic Technologies Corp USA Neovascular-AMD sFLT01 Anti-angiogenesis AAV2 Intravitreal I Ongoing NCT01024998 Sanofi USA Neovascular-AMD AntiVEGFfab Anti-angiogenesis AAV8 Subretinal I Ongoing NCT03066258 Regenxbio USA Neovascular-AMD sFLT01 Anti-angiogenesis AAV2 Subretinal I/II Completed NCT01494805 Lions Eye Institute Australia Neovascular-AMD Endostatin/Angiostatin Anti-angiogenesis EIAV Subretinal I Completed NCT01301443 Oxford BioMedica USA Dry-AMD sCD59 MAC inhibitory protein AAV2 Intravitreal I Ongoing NCT03144999 Hemera Biosciences USA Retinitis Pigmentosa ChR2 Light-gated ion channels AAV2 Intravitreal I/II Ongoing NCT02556736 Allergan USA Disease Gene Gene function Vector Mode Phase Current status Clinical trials ID Sponsor Location LCA-2 RPE65 Retinoid cycle AAV2/2 Subretinal III Ongoing NCT00999609 Spark Therapeutics UK NCT01208389 LCA-2 RPE65 Retinoid cycle AAV2/2 Subretinal I Ongoing NCT00516477 Spark Therapeutics USA LCA-2 RPE65 Retinoid cycle AAV2/5 Subretinal I/II Ongoing NCT02781480 MeiraGTx UK II Ltd UK NCT02946879 LCA-2 RPE65 Retinoid cycle AAV2 Subretinal I Unknown NCT00821340 Hadassah Medical Organization Israel LCA-2 RPE65 Retinoid cycle AAV2/2 Subretinal I Ongoing NCT00481546 University of Pennsylvania and National Eye Institute USA LCA-2 RPE65 Retinoid cycle AAV2 Subretinal I/II Ongoing NCT00749957 Applied Genetic Technologies Corp USA LCA-2 RPE65 Retinoid cycle AAV2/2 Subretinal I/II Completed NCT00643747 University College London UK LCA-2 RPE65 Retinoid cycle AAV2/4 Subretinal I/II Completed NCT01496040 Nantes University Hospital France LCA-10 CEP290 Ciliary transport N/A* Intravitreal I/II Ongoing NCT03140969 ProQR Therapeutics USA, Belgium Stargardt Disease ABCA4 Retinoid transfer EIAV Subretinal I/II Ongoing NCT01367444 Sanofi/Oxford BioMedica USA, France RP assoc. with Usher’s Syndrome Type 1B MYO7A Intracellular transport EIAV Subretinal I/II Ongoing NCT01505062 Sanofi/Oxford BioMedica USA, France Choroideremia REP1 Intracellular trafficking AAV2 Subretinal II Ongoing NCT02553135 University of Miami USA Choroideremia REP1 Intracellular trafficking AAV2 Subretinal II Ongoing NCT02671539 STZ Eyetrial Germany Choroideremia REP1 Intracellular trafficking AAV2 Subretinal I/II Ongoing NCT01461213 University of Oxford/ NightstaRx UK Choroideremia REP1 Intracellular trafficking AAV2 Subretinal I/II Ongoing NCT02077361 University of Alberta/ NightstaRx Canada Choroideremia REP1 Intracellular trafficking AAV2 Subretinal II Ongoing NCT02407678 University of Oxford/ NightstaRx UK Choroideremia CHM Intracellular trafficking AAV2 Subretinal I/II Ongoing NCT02341807 Spark Therapeutics USA MERTK-RP MERTK Cellular signalling AAV2 Subretinal I Ongoing NCT01482195 King Khaled Eye Specialist Hospital Saudi Arabia X-Linked RP RPGR Intracellular transport AAV8 Subretinal I/II Ongoing NCT03116113 NightstaRx UK X-Linked RP RPGR Intracellular transport AAV2/5 Subretinal I/II Ongoing NCT03252847 MeiraGTx UK II Ltd UK Achromatopsia CNGB3 Phototransduction cascade AAV2 Subretinal I/II Ongoing NCT02599922 Applied Genetic Technologies Corp USA Achromatopsia CNGB3 Phototransduction cascade AAV2/8 Subretinal I/II Ongoing NCT03001310 MeiraGTx UK II Ltd UK Achromatopsia CNGA3 Phototransduction cascade AAV8 Subretinal I/II Ongoing NCT02610582 STZ Eyetrial/University Hospital Tuebingen Germany Achromatopsia CNGA3 Phototransduction cascade AAV2 Subretinal I/II Ongoing NCT02935517 Applied Genetic Technologies Corp USA, Israel X-Linked Retinoschisis RS1 Intercellular adhesion AAV8 Intravitreal I/II Ongoing NCT02317887 National Eye Institute USA X-Linked Retinoschisis RS1 Intercellular adhesion AAV2 Intravitreal I/II Ongoing NCT02416622 Applied Genetic Technologies Corp USA Neovascular-AMD sFLT01 Anti-angiogenesis AAV2 Intravitreal I Ongoing NCT01024998 Sanofi USA Neovascular-AMD AntiVEGFfab Anti-angiogenesis AAV8 Subretinal I Ongoing NCT03066258 Regenxbio USA Neovascular-AMD sFLT01 Anti-angiogenesis AAV2 Subretinal I/II Completed NCT01494805 Lions Eye Institute Australia Neovascular-AMD Endostatin/Angiostatin Anti-angiogenesis EIAV Subretinal I Completed NCT01301443 Oxford BioMedica USA Dry-AMD sCD59 MAC inhibitory protein AAV2 Intravitreal I Ongoing NCT03144999 Hemera Biosciences USA Retinitis Pigmentosa ChR2 Light-gated ion channels AAV2 Intravitreal I/II Ongoing NCT02556736 Allergan USA *RNA anti-sense oligonucleotides are administered without a vector. Table 1 Clinical trials of retinal gene therapy (LCA, Leber Congenital Amaurosis; RP, Retinitis Pigmentosa) Disease Gene Gene function Vector Mode Phase Current status Clinical trials ID Sponsor Location LCA-2 RPE65 Retinoid cycle AAV2/2 Subretinal III Ongoing NCT00999609 Spark Therapeutics UK NCT01208389 LCA-2 RPE65 Retinoid cycle AAV2/2 Subretinal I Ongoing NCT00516477 Spark Therapeutics USA LCA-2 RPE65 Retinoid cycle AAV2/5 Subretinal I/II Ongoing NCT02781480 MeiraGTx UK II Ltd UK NCT02946879 LCA-2 RPE65 Retinoid cycle AAV2 Subretinal I Unknown NCT00821340 Hadassah Medical Organization Israel LCA-2 RPE65 Retinoid cycle AAV2/2 Subretinal I Ongoing NCT00481546 University of Pennsylvania and National Eye Institute USA LCA-2 RPE65 Retinoid cycle AAV2 Subretinal I/II Ongoing NCT00749957 Applied Genetic Technologies Corp USA LCA-2 RPE65 Retinoid cycle AAV2/2 Subretinal I/II Completed NCT00643747 University College London UK LCA-2 RPE65 Retinoid cycle AAV2/4 Subretinal I/II Completed NCT01496040 Nantes University Hospital France LCA-10 CEP290 Ciliary transport N/A* Intravitreal I/II Ongoing NCT03140969 ProQR Therapeutics USA, Belgium Stargardt Disease ABCA4 Retinoid transfer EIAV Subretinal I/II Ongoing NCT01367444 Sanofi/Oxford BioMedica USA, France RP assoc. with Usher’s Syndrome Type 1B MYO7A Intracellular transport EIAV Subretinal I/II Ongoing NCT01505062 Sanofi/Oxford BioMedica USA, France Choroideremia REP1 Intracellular trafficking AAV2 Subretinal II Ongoing NCT02553135 University of Miami USA Choroideremia REP1 Intracellular trafficking AAV2 Subretinal II Ongoing NCT02671539 STZ Eyetrial Germany Choroideremia REP1 Intracellular trafficking AAV2 Subretinal I/II Ongoing NCT01461213 University of Oxford/ NightstaRx UK Choroideremia REP1 Intracellular trafficking AAV2 Subretinal I/II Ongoing NCT02077361 University of Alberta/ NightstaRx Canada Choroideremia REP1 Intracellular trafficking AAV2 Subretinal II Ongoing NCT02407678 University of Oxford/ NightstaRx UK Choroideremia CHM Intracellular trafficking AAV2 Subretinal I/II Ongoing NCT02341807 Spark Therapeutics USA MERTK-RP MERTK Cellular signalling AAV2 Subretinal I Ongoing NCT01482195 King Khaled Eye Specialist Hospital Saudi Arabia X-Linked RP RPGR Intracellular transport AAV8 Subretinal I/II Ongoing NCT03116113 NightstaRx UK X-Linked RP RPGR Intracellular transport AAV2/5 Subretinal I/II Ongoing NCT03252847 MeiraGTx UK II Ltd UK Achromatopsia CNGB3 Phototransduction cascade AAV2 Subretinal I/II Ongoing NCT02599922 Applied Genetic Technologies Corp USA Achromatopsia CNGB3 Phototransduction cascade AAV2/8 Subretinal I/II Ongoing NCT03001310 MeiraGTx UK II Ltd UK Achromatopsia CNGA3 Phototransduction cascade AAV8 Subretinal I/II Ongoing NCT02610582 STZ Eyetrial/University Hospital Tuebingen Germany Achromatopsia CNGA3 Phototransduction cascade AAV2 Subretinal I/II Ongoing NCT02935517 Applied Genetic Technologies Corp USA, Israel X-Linked Retinoschisis RS1 Intercellular adhesion AAV8 Intravitreal I/II Ongoing NCT02317887 National Eye Institute USA X-Linked Retinoschisis RS1 Intercellular adhesion AAV2 Intravitreal I/II Ongoing NCT02416622 Applied Genetic Technologies Corp USA Neovascular-AMD sFLT01 Anti-angiogenesis AAV2 Intravitreal I Ongoing NCT01024998 Sanofi USA Neovascular-AMD AntiVEGFfab Anti-angiogenesis AAV8 Subretinal I Ongoing NCT03066258 Regenxbio USA Neovascular-AMD sFLT01 Anti-angiogenesis AAV2 Subretinal I/II Completed NCT01494805 Lions Eye Institute Australia Neovascular-AMD Endostatin/Angiostatin Anti-angiogenesis EIAV Subretinal I Completed NCT01301443 Oxford BioMedica USA Dry-AMD sCD59 MAC inhibitory protein AAV2 Intravitreal I Ongoing NCT03144999 Hemera Biosciences USA Retinitis Pigmentosa ChR2 Light-gated ion channels AAV2 Intravitreal I/II Ongoing NCT02556736 Allergan USA Disease Gene Gene function Vector Mode Phase Current status Clinical trials ID Sponsor Location LCA-2 RPE65 Retinoid cycle AAV2/2 Subretinal III Ongoing NCT00999609 Spark Therapeutics UK NCT01208389 LCA-2 RPE65 Retinoid cycle AAV2/2 Subretinal I Ongoing NCT00516477 Spark Therapeutics USA LCA-2 RPE65 Retinoid cycle AAV2/5 Subretinal I/II Ongoing NCT02781480 MeiraGTx UK II Ltd UK NCT02946879 LCA-2 RPE65 Retinoid cycle AAV2 Subretinal I Unknown NCT00821340 Hadassah Medical Organization Israel LCA-2 RPE65 Retinoid cycle AAV2/2 Subretinal I Ongoing NCT00481546 University of Pennsylvania and National Eye Institute USA LCA-2 RPE65 Retinoid cycle AAV2 Subretinal I/II Ongoing NCT00749957 Applied Genetic Technologies Corp USA LCA-2 RPE65 Retinoid cycle AAV2/2 Subretinal I/II Completed NCT00643747 University College London UK LCA-2 RPE65 Retinoid cycle AAV2/4 Subretinal I/II Completed NCT01496040 Nantes University Hospital France LCA-10 CEP290 Ciliary transport N/A* Intravitreal I/II Ongoing NCT03140969 ProQR Therapeutics USA, Belgium Stargardt Disease ABCA4 Retinoid transfer EIAV Subretinal I/II Ongoing NCT01367444 Sanofi/Oxford BioMedica USA, France RP assoc. with Usher’s Syndrome Type 1B MYO7A Intracellular transport EIAV Subretinal I/II Ongoing NCT01505062 Sanofi/Oxford BioMedica USA, France Choroideremia REP1 Intracellular trafficking AAV2 Subretinal II Ongoing NCT02553135 University of Miami USA Choroideremia REP1 Intracellular trafficking AAV2 Subretinal II Ongoing NCT02671539 STZ Eyetrial Germany Choroideremia REP1 Intracellular trafficking AAV2 Subretinal I/II Ongoing NCT01461213 University of Oxford/ NightstaRx UK Choroideremia REP1 Intracellular trafficking AAV2 Subretinal I/II Ongoing NCT02077361 University of Alberta/ NightstaRx Canada Choroideremia REP1 Intracellular trafficking AAV2 Subretinal II Ongoing NCT02407678 University of Oxford/ NightstaRx UK Choroideremia CHM Intracellular trafficking AAV2 Subretinal I/II Ongoing NCT02341807 Spark Therapeutics USA MERTK-RP MERTK Cellular signalling AAV2 Subretinal I Ongoing NCT01482195 King Khaled Eye Specialist Hospital Saudi Arabia X-Linked RP RPGR Intracellular transport AAV8 Subretinal I/II Ongoing NCT03116113 NightstaRx UK X-Linked RP RPGR Intracellular transport AAV2/5 Subretinal I/II Ongoing NCT03252847 MeiraGTx UK II Ltd UK Achromatopsia CNGB3 Phototransduction cascade AAV2 Subretinal I/II Ongoing NCT02599922 Applied Genetic Technologies Corp USA Achromatopsia CNGB3 Phototransduction cascade AAV2/8 Subretinal I/II Ongoing NCT03001310 MeiraGTx UK II Ltd UK Achromatopsia CNGA3 Phototransduction cascade AAV8 Subretinal I/II Ongoing NCT02610582 STZ Eyetrial/University Hospital Tuebingen Germany Achromatopsia CNGA3 Phototransduction cascade AAV2 Subretinal I/II Ongoing NCT02935517 Applied Genetic Technologies Corp USA, Israel X-Linked Retinoschisis RS1 Intercellular adhesion AAV8 Intravitreal I/II Ongoing NCT02317887 National Eye Institute USA X-Linked Retinoschisis RS1 Intercellular adhesion AAV2 Intravitreal I/II Ongoing NCT02416622 Applied Genetic Technologies Corp USA Neovascular-AMD sFLT01 Anti-angiogenesis AAV2 Intravitreal I Ongoing NCT01024998 Sanofi USA Neovascular-AMD AntiVEGFfab Anti-angiogenesis AAV8 Subretinal I Ongoing NCT03066258 Regenxbio USA Neovascular-AMD sFLT01 Anti-angiogenesis AAV2 Subretinal I/II Completed NCT01494805 Lions Eye Institute Australia Neovascular-AMD Endostatin/Angiostatin Anti-angiogenesis EIAV Subretinal I Completed NCT01301443 Oxford BioMedica USA Dry-AMD sCD59 MAC inhibitory protein AAV2 Intravitreal I Ongoing NCT03144999 Hemera Biosciences USA Retinitis Pigmentosa ChR2 Light-gated ion channels AAV2 Intravitreal I/II Ongoing NCT02556736 Allergan USA *RNA anti-sense oligonucleotides are administered without a vector. RPGR Retinitis Pigmentosa (RP) is a progressive of rod-cone retinal degeneration, caused by mutations in more than 60 genes and affects about 1 in 3000 people. X-linked RP (XLRP), a particularly severe form, accounts for 15–25% of RP with onset in childhood and rapid progression. Seventy to eighty percent of XLRP is caused by sequence variants in the RPGR gene, which encodes the retinitis pigmentosa GTPase regulator.33 In both rodent and canine models of the condition, AAV-mediated gene supplementation using truncated or codon-optimized constructs to improve transgene stability have slowed retinal degeneration.34,35 Phase I/II trials of AAV-mediated gene therapy in affected humans are ongoing (NCT03116113, NCT03252847). CHM Choroideremia is an X-linked recessive disorder, characterized by night-blindness in childhood, with gradually progressive constriction of peripheral vision leading to impairment of central vision in later life. Choroideremia is caused by sequence variants in the 1.9 kb CHM gene that encodes Rab escort protein 1 (REP1), which is required for intracellular vesicular transport.36 In a phase I/II clinical trial, subretinal injection of AAV2 vector expressing REP1 is reported to be well tolerated and associated with improved function in some instances.5,37 Long-term follow-up will determine whether gene therapy can protect against progressive constriction of visual fields owing to retinal degeneration (NCT01461213, NCT02553135, NCT02671539, NCT02077361). MERTK Defects in the gene encoding MERTK, a receptor tyrosine kinase essential for removal of waste material from photoreceptors, result in accumulation of debris and progressive rod-cone retinal degeneration (retinitis pigmentosa).38,39 In a rodent model of the disease, gene replacement therapy by subretinal administration of AAV2 vectors encoding Mertk can improve the outcome.40,41 In a phase I/II clinical trial, an AAV2-MERTK vector was well tolerated in affected humans with a suggestion of temporary benefit in some participants.42 RS1 X-Linked Retinoschisis (XLRS) causes impairment of sight in childhood, with variable progression in later adulthood. The prevalence is estimated to be 1 in 10 000 men. Associated features include strabismus, refractive error and anisometropia. Gene defects in RS1, which encodes retinoschisin, impair adhesion and connectivity between photoreceptor and bipolar cells, leading to schisis of the macula and retinal dysfunction. About 50% of those affected also have peripheral retinoschisis which presents a risk of retinal detachment and vitreous haemorrhage. Intravitreal delivery of AAV2 or AAV8 vectors encoding RS1 improves the outcome in Rs1-deficient mice, and appears safe in rabbits.11,43,44 Phase I/II dose-escalation clinical trials of intravitreal gene replacement of RS1 using AAV2 and AAV8 vectors are ongoing (NCT02317887, NCT02416622). Candidate genes for future clinical trials AIPL1 Aryl hydrocarbon receptor-interacting protein-like 1 (AIPL1) is a molecular chaperone of phosphodiesterase 6, which mediates rod-photoreceptor-specific phototransduction. Mutations in AIPL1 cause a particularly severe, rapidly progressive form of LCA (AIPL1-LCA). Affected individuals present with severe sight impairment from birth and rapid retinal degeneration. Although the natural history is very poor, some preservation of retinal structure during infancy in humans45 indicates a window of opportunity for intervention by gene replacement therapy. The potential for benefit has been demonstrated experimentally in a rodent model.46,47 GUCY2D Retinal guanylate cyclase-1 is essential in photoreceptor cells for timely recovery from photoexcitation. Mutations in the GUCY2D gene account for 10–20% of LCA and photoreceptor architecture is relatively well preserved.48 Demonstration of improved retinal function following gene replacement therapy using AAV5 and AAV2/8 and AAV8 vectors indicate the potential for benefit in affected humans.48,49 CEP290 CEP290 encodes a centrosomal protein involved in trafficking through the connecting cilia of photoreceptor cells. Mutations in CEP290 account for 30% of LCA. The size of the full-length gene exceeds the capacity of AAV vectors for a gene replacement strategy. However, the most common disease-causing variant, the intronic variant c.2991 + 1655 A > G, creates a cryptic splice donor site and premature stop codon that may be addressed by alternative strategies such as anti-sense oligonucleotides or CRISPR/Cas9-mediated gene editing.50 ABCA4 Stargardt disease (STGD1) results from mutations in the photoreceptor-specific flippase ABCA4, leading to intracellular accumulation of a toxic retinoid, and degeneration of the outer retina. STGD1 is the most common inherited macular degeneration, with an incidence of 1 in 10 000,51 causing progressively severe impairment of sight from early childhood or adulthood. The full-length ABCA4 gene (6.8 kb) exceeds the carrying capacity of AAV vectors. Alternative strategies for gene replacement include the use of oversized AAV5 vectors,52 trans-splicing and hybrid AAV2 dual vector systems.8 The use of a lentiviral vector based on Equine Infectious Anaemia Virus (EIAV) is reported to improve the outcome in a rodent model of the condition53 and to be well-tolerated in non-human primates.54 A phase I/II dose-escalation clinical trial of EIAV-ABCA4 vector is ongoing (NCT01367444). MYO7A Usher Syndrome is a recessively inherited condition characterized by impairment of sensorineural hearing and progressive impairment of sight owing to rod-cone degeneration. It is clinically and genetically heterogeneous, and is associated with defects in as many as 10 genes.55 Usher syndrome type 1B is caused by defects in the myosin VIIa (MYO7A) gene, resulting in abnormal accumulation of opsin in the cilia of photoreceptor cells. Like ABCA4, the large size of the MYO7A (8.1 kb) gene52 exceeds the carrying capacity of conventional AAV2/2 vectors. Alternative strategies include the use of dual vectors,8,56 to deliver gene fragments that are reassembled by homologous recombination following transduction, oversized AAV5 vectors,52 and lentiviral vectors.57,58 Subretinal injection of EIAV-MYO7A is well tolerated in non-human primates, and the potential for benefit to vision in humans is being explored in an ongoing phase I/II clinical trial (NCT01505062).58 RHO Many disease-causing sequence variants in RHO, which encodes the rod-photoreceptor pigment rhodopsin, have dominant negative effects in which the protein interferes with essential cell functions leading to variably severe rod-cone degeneration.59 More than 150 such mutations in RHO have been identified, accounting for 20–30% of autosomal dominant retinitis pigmentosa.60 Strategies to suppress expression of the disease allele specifically are currently difficult owing to the substantial mutational heterogeneity. One alternative approach is to suppress both the mutant and normal allele non-specifically, and simultaneously to provide a replacement functional gene that has been modified to resist suppression.61 This approach can improve the outcome in a rodent model but is yet to be explored in affected humans. Age-related macular degeneration AMD is the leading cause of vision loss in individuals over the age of 60. AMD can be classified as neovascular (wet) or atrophic (dry). Impairment of sight from neovascular-AMD and proliferative diabetic retinopathy is a direct consequence of pathological neovascularization which is driven strongly by the up-regulation of vascular endothelial growth factor (VEGF). The use of therapeutic anti-VEGF antibodies has resulted in substantially improved outcomes, but sustained benefit requires repeated intraocular injections. Vector-mediated gene expression of angiostatic proteins offers the opportunity to achieve sustained intraocular delivery following a single injection. Subretinal injection of a rAAV 2/2 vector expressing the soluble VEGF receptor sFlt-1 appears to be well tolerated in humans with neovascular-AMD, though a larger study will be needed to determine benefit (NCT0149805).62,63 Intravitreal injection of an AAV2 vector expressing sFlt-1 in advanced neovascular-AMD was followed by improved structure in a minority.64 Intraocular expression of sFlt-1 was sustained but variable and possibly limited by antibody-mediated immune responses to AAV2, with clinical signs of inflammation evident in some instances (NCT01024998). Subretinal delivery of Retinostat™, a non-replicating EIAV vector containing genes encoding the angiostatic proteins Endostatin and Angiostatin, appears safe in rodent65 and non-human primate models.66 In humans with neovascular-AMD, subretinal injection of Retinostat™ results in sustained intraocular transgene expression, and appears well tolerated (NCT01301443).67 Dry-AMD is characterized by dysfunction and degeneration of RPE cells, associated with inappropriate activation of the complement cascade.68 The protein CD59 can protect against cellular injury by blocking the membrane attack complex (MAC).69 A gene therapy product to increase expression of a soluble form of CD59 (sCD59) is currently being investigated (NCT03144999). The future The benefits of gene therapy for LCA-RPE65 have led to approval by the FDA of the first gene therapy for ocular disease, and to a rapid expansion in clinical trials exploring the safety and potential benefit of gene therapy for other inherited and acquired retinal diseases. The results will help define for each condition the potential window of opportunity for effective intervention. Further developments in vector design and delivery will enable greater efficiency and safety of gene transfer. Rapid reliable assessment of outcomes will be accelerated by optimization of clinical trial design. Conflict of interest statement MM, AJS, RRA and JWBB declare financial interests in MeiraGTx. References 1 Liew G , Michaelides M , Bunce C . A comparison of the causes of blindness certifications in England and Wales in working age adults (16–64 years), 1999-2000 with 2009-2010 . BMJ Open 2014 ; 4 : e004015 . doi:10.1136/bmjopen-2013-004015 . 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Invest Ophthalmol Vis Sci 2017 ; 58 : 3073 – 85 . doi:10.1167/iovs.16-20083 . Google Scholar CrossRef Search ADS PubMed 69 Lachmann PJ . The control of homologous lysis . Immunol Today 1991 ; 12 : 312 – 5 . doi:10.1016/0167-5699(91)90005-E . Google Scholar CrossRef Search ADS PubMed © The Author(s) 2018. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png British Medical Bulletin Oxford University Press

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Abstract

Abstract Introduction Inherited retinal diseases are the leading cause of sight impairment in people of working age in England and Wales, and the second commonest in childhood. Gene therapy offers the potential for benefit. Sources of data Pubmed and clinicaltrials.gov. Areas of agreement Gene therapy can improve vision in RPE65-associated Leber Congenital Amaurosis (RPE65-LCA). Potential benefit depends on efficient gene transfer and is limited by the extent of retinal degeneration. Areas of controversy The magnitude of vision improvement from RPE65-LCA gene therapy is suboptimal, and its durability may be limited by progressive retinal degeneration. Growing points The safety and potential benefit of gene therapy for inherited and acquired retinal diseases is being explored in a rapidly expanding number of trials. Areas timely for developing research Developments in vector design and delivery will enable greater efficiency and safety of gene transfer. Optimization of trial design will accelerate reliable assessment of outcomes. gene therapy, retina, retinal diseases Introduction The retina is a highly specialized, multi-layered tissue that includes a layer of light-sensitive cone- and rod-photoreceptor cells, which initiate neuronal signalling in response to light stimulation by means of a highly sophisticated cascade of enzymatic reactions (phototransduction). The photoreceptor cells are supported by a monolayer of pigmented cells, the retinal pigment epithelium (RPE), which performs many key processes including the regeneration of visual pigment that is bleached following light exposure (the visual cycle). Diseases of the retina, including age-related macular degeneration (AMD), inherited retinal diseases (IRDs), diabetic retinopathy and vascular occlusion, represent the commonest cause of severe sight impairment in the developed world. Inherited retinal diseases are the leading cause of blindness in people of working age in England and Wales, and the second commonest cause in children.1 Defects in genes encoding proteins involved in the phototransduction cascade or the visual cycle account for a large proportion of IRDs. Gene therapy offers an opportunity to improve outcomes of inherited monogenic disorders of the retina. In its simplest and commonest form, gene ‘supplementation’ therapy compensates for a genetic deficiency, resulting from loss-of-function mutations in the endogenous gene, by provision of the normal gene to the cells in which it is required.2 The therapeutic gene, typically delivered using a viral vector, is utilized by the transcriptional machinery of the target cell to generate the normal gene product that is otherwise lacking. Alternative gene therapy techniques aim to suppress the undesirable expression of a harmful protein product resulting from gain-of-function mutations, with or without simultaneous provision of the normal gene.3 Also being investigated are gene editing strategies to correct harmful mutations in endogenous genes, and anti-sense oligonucleotide mediated exon skipping to mitigate their impact. Clinical trials of gene therapies have included applications for primary immune deficiencies, haemoglobinopathies, haemophilia B, neurological diseases and cancer.4 The first gene therapy product approved by the European Medicines Agency was alipogene tiparvovec (Glybera), developed for the treatment of lipoprotein lipase deficiency.4 Here we describe the key strategies of ocular gene therapy and focus on its application to disease of the retina, with emphasis on experimental therapies in clinical trials. Figure 1 is a schematic diagram of the location of genes in cells of the outer retina targeted in current gene therapy trials. Fig. 1 View largeDownload slide Schematic diagram showing the outer retina and the cellular location of poducts of the genes targeted by current gene therapy trials (red) and the genes explored for future trials (black). Fig. 1 View largeDownload slide Schematic diagram showing the outer retina and the cellular location of poducts of the genes targeted by current gene therapy trials (red) and the genes explored for future trials (black). Gene therapy and the eye The retina has specific advantages as a target organ for gene therapy. The transparency of the ocular media provides accessibility for microsurgical delivery of vector suspension to the retina under direct visualization, and for cellular level imaging to target intervention and assess its impact. Vector suspension can be targeted to the retina with minimal systemic dissemination owing to the contained nature and compartmentalization of the intraocular tissues. The intraocular environment provides the retina with a degree of immune privilege, which limits immune responses that could adversely affect retinal function and limit expression of the therapeutic gene. Since inherited retinal disease typically causes bilateral disease with significant symmetry, the untreated contralateral eye offers a valuable control for natural history, learning effects and intra-individual variability in performance. For gene transfer to retinal cells, most clinical applications currently employ recombinant adeno-associated virus (AAV) or lentivirus vectors. AAV is a small, non-pathogenic single stranded DNA virus widely used for gene delivery in IRDs. AAV vectors can mediate efficient and sustained transduction of photoreceptor cells, RPE, and ganglion cells. First generation AAV2 vectors are limited by relatively slow onset of expression and small capacity (4.7 kB).5 However, the isolation of alternative serotypes and the development of self-complementary vectors and novel variants, by rational design and/or directed evolution, have provided a broad range of alternatives with more rapid expression and cell tropisms.6,7 Measures to address the limited capacity include dual AAV vector strategies in which a large gene delivered in component parts by AAV is reconstituted by splicing.8 Since lentiviral vectors have substantially greater capacity (approximately 8 kB) than AAV, they can naturally accommodate larger genes. Lentiviral vectors mediate efficient transduction that is typically limited to RPE cells but one type of lentiviral vector, derived from the equine infectious anaemia virus (EIAV), mediates variable transgene expression in photoreceptor cells.9 Vector capacity is a fundamentally important issue given that the most common genes causing IRD are large, including ABCA4 (Stargardt Disease) and USH2A (syndromic and non-syndromic Retinitis Pigmentosa). Intraocular administration Since defects in genes involved in phototransduction or the visual cycle account for many IRDs, photoreceptors and RPE cells are important target populations for gene therapy. Viral vectors deliver genes to these cells efficiently when the vector suspension is placed in direct contact with the cells in the outer retina. This is typically achieved by injecting the vector suspension between the RPE and the overlying photoreceptor cell layer. Injection into this potential subretinal space is typically performed using a fine cannula that is advanced through the sclera anteriorly, across the vitreous cavity and through the inner retina (Fig. 2A). This generates a bleb of vector suspension that temporarily separates the neurosensory retina from the underlying RPE, before it is absorbed over a period of hours or days. Injection of vector suspension into the vitreous cavity (Fig. 2B) can result in gene delivery to cells of the inner retina, including ganglion cells. Although intravitreal vector is technically simpler than subretinal injection, anatomical barriers prevent efficient gene delivery to the outer retina and this route of administration may be more immunogenic than subretinal injection.10 Compromise of inner retinal integrity, in conditions such as X-linked Retinoschisis, may enable enhanced access of intravitreal vector suspensions to the inner retina.11 Delivery of vector suspensions into the suprachoroidal potential space (between the sclera and choroid) may enable targeting of the choroid, for conditions such as AMD and idiopathic polypoidal choroidal vasculopathy.12 Fig. 2 View largeDownload slide Schematic diagram identifying ocular structures and location of (A) subretinal injection and (B) intravitreal injection. Fig. 2 View largeDownload slide Schematic diagram identifying ocular structures and location of (A) subretinal injection and (B) intravitreal injection. Current gene therapy clinical trials RPE65 Leber Congenital Amaurosis (LCA), first described by Theodore Leber in 1869, is a group of recessively inherited infantile-onset rod-cone dystrophies. The prevalence ranges from 1 in 33 000 to 1 in 81 000.13 LCA accounts globally for 5% of IRDs and 20% of children attending specialist schools for students with sight impairment.14 Mutation of one of several genes, including RPE65, causes impaired vision from birth/early infancy and typically progresses to severe sight impairment. RPE65 is expressed in the RPE and encodes a 65-kD protein that is a key component of the visual cycle, a biochemical pathway that regenerates the visual pigment after exposure to light.15,16 A lack of functional RPE65 results in deficiency of 11-cis retinal such that rod-photoreceptor cells are unable to respond to light.17 Cone photoreceptor cells have access to 11-cis retinaldehyde chromophore through an alternative pathway that does not depend on RPE-derived RPE65 thus allowing cone-mediated vision in children.18 However, progressive degeneration of cone photoreceptor cells ultimately results in the loss of cone-mediated vision. The appearance of the retina on clinical examination is typically normal at an early age, but retinal degeneration becomes in advanced disease (Fig. 3). Gene-replacement therapy can improve visual function in rodent models of RPE65-LCA, and in the Swedish Briard dog, which has a naturally occurring mutation in RPE65.19 The treated eyes of dogs showed improved responses on electroretinography, pupillometry and flash-evoked cortical potentials in the dark-adapted state, with improvements sustained for as long as 10 years.19 Fig. 3 View largeDownload slide Colour fundus photographs of (A) a patient with RPE65-associated Leber congenital amaurosis and (B) an unaffected individual for comparison. (A) shows peripheral RPE atrophy with a tessellated appearance to the fundus owing to the choroidal vasculature, with central preservation of retinal structure. Fig. 3 View largeDownload slide Colour fundus photographs of (A) a patient with RPE65-associated Leber congenital amaurosis and (B) an unaffected individual for comparison. (A) shows peripheral RPE atrophy with a tessellated appearance to the fundus owing to the choroidal vasculature, with central preservation of retinal structure. The first trial of gene therapy in humans with RPE65-LCA was reported in 2008.20 Several phase I/II trials have provided evidence that subretinal injection of a recombinant AAV 2/2 vector containing the RPE65 cDNA can improve retinal function and vision.20–23 The findings of an open-label randomized controlled trial confirm benefit at 1 year to night vision, as indicated by improved performance in a test of vision-guided mobility in low luminance.24 In 2017 the US FDA approved voretingene neparvovec (Luxturna, Spark Therapeutics Inc.) for the treatment of RPE65-associated LCA. Long-term findings indicate that, despite improved function of surviving retina, the benefits of gene therapy can be limited by progressive retinal degeneration.23,25–27 To investigate the hypothesis that greater provision of RPE65 will provide more durable, robust benefit, the effect of a more efficient optimized AAV 2/5 vector is being investigated.28 Figure 4 illustrates one example of improved retinal sensitivity in RPE65-associated LCA following intervention gene therapy, as demonstrated by advanced hill of vision modelling.29 Fig. 4 View largeDownload slide An oblique topographical view of the central hill of vision, produced by Visual Field Modelling and Analysis (VFMA, Office of Technology Transfer and Business Development [OHSU], Portland, Oregon, USA)29 in a subject (A) prior to and (B) following gene therapy intervention (A) shows residual central visual field prior to intervention, in comparison to (B) which demonstrates increased retinal sensitivity and therefore a larger and taller hill of vision at 1 year post-intervention. Fig. 4 View largeDownload slide An oblique topographical view of the central hill of vision, produced by Visual Field Modelling and Analysis (VFMA, Office of Technology Transfer and Business Development [OHSU], Portland, Oregon, USA)29 in a subject (A) prior to and (B) following gene therapy intervention (A) shows residual central visual field prior to intervention, in comparison to (B) which demonstrates increased retinal sensitivity and therefore a larger and taller hill of vision at 1 year post-intervention. CNGA3, CNGB3 Achromatopsia (ACHM) is a recessively inherited disorder of cone photoreceptor function affecting approximately 1 in 30 000 people. The condition causes severe impairment of sight from birth, with poor visual acuity, absent or markedly reduced colour vision, disabling photophobia and pendular nystagmus.30 ACHM can be caused by mutations in any one of at least six genes including CNGA3 and CNGB3, which encode the alpha and beta subunits, respectively, of the cone photoreceptor cyclic nucleotide gated channels, and account for the majority of people affected.30 Structural preservation of individual cone photoreceptors can be determined in affected humans by adaptive optics scanning laser ophthalmoscopy (AO-SLO),30 to evaluate their potential to benefit from gene therapy. AAV-mediated gene-replacement therapy can improve cone function in rodent models of both CNGA3-ACHM31 and CNGB3-ACHM,32 and in canine models of CNGB3-ACHM.30 Early phase clinical trials of gene therapy for both CNGA3-ACHM and CNGB3-ACHM will determine the extent to which this approach might benefit visual acuity, colour discrimination, photophobia and nystagmus in affected humans (NCT02599922, NCT03001310, NCT02610582, NCT02935517) (Table 1). Table 1 Clinical trials of retinal gene therapy (LCA, Leber Congenital Amaurosis; RP, Retinitis Pigmentosa) Disease Gene Gene function Vector Mode Phase Current status Clinical trials ID Sponsor Location LCA-2 RPE65 Retinoid cycle AAV2/2 Subretinal III Ongoing NCT00999609 Spark Therapeutics UK NCT01208389 LCA-2 RPE65 Retinoid cycle AAV2/2 Subretinal I Ongoing NCT00516477 Spark Therapeutics USA LCA-2 RPE65 Retinoid cycle AAV2/5 Subretinal I/II Ongoing NCT02781480 MeiraGTx UK II Ltd UK NCT02946879 LCA-2 RPE65 Retinoid cycle AAV2 Subretinal I Unknown NCT00821340 Hadassah Medical Organization Israel LCA-2 RPE65 Retinoid cycle AAV2/2 Subretinal I Ongoing NCT00481546 University of Pennsylvania and National Eye Institute USA LCA-2 RPE65 Retinoid cycle AAV2 Subretinal I/II Ongoing NCT00749957 Applied Genetic Technologies Corp USA LCA-2 RPE65 Retinoid cycle AAV2/2 Subretinal I/II Completed NCT00643747 University College London UK LCA-2 RPE65 Retinoid cycle AAV2/4 Subretinal I/II Completed NCT01496040 Nantes University Hospital France LCA-10 CEP290 Ciliary transport N/A* Intravitreal I/II Ongoing NCT03140969 ProQR Therapeutics USA, Belgium Stargardt Disease ABCA4 Retinoid transfer EIAV Subretinal I/II Ongoing NCT01367444 Sanofi/Oxford BioMedica USA, France RP assoc. with Usher’s Syndrome Type 1B MYO7A Intracellular transport EIAV Subretinal I/II Ongoing NCT01505062 Sanofi/Oxford BioMedica USA, France Choroideremia REP1 Intracellular trafficking AAV2 Subretinal II Ongoing NCT02553135 University of Miami USA Choroideremia REP1 Intracellular trafficking AAV2 Subretinal II Ongoing NCT02671539 STZ Eyetrial Germany Choroideremia REP1 Intracellular trafficking AAV2 Subretinal I/II Ongoing NCT01461213 University of Oxford/ NightstaRx UK Choroideremia REP1 Intracellular trafficking AAV2 Subretinal I/II Ongoing NCT02077361 University of Alberta/ NightstaRx Canada Choroideremia REP1 Intracellular trafficking AAV2 Subretinal II Ongoing NCT02407678 University of Oxford/ NightstaRx UK Choroideremia CHM Intracellular trafficking AAV2 Subretinal I/II Ongoing NCT02341807 Spark Therapeutics USA MERTK-RP MERTK Cellular signalling AAV2 Subretinal I Ongoing NCT01482195 King Khaled Eye Specialist Hospital Saudi Arabia X-Linked RP RPGR Intracellular transport AAV8 Subretinal I/II Ongoing NCT03116113 NightstaRx UK X-Linked RP RPGR Intracellular transport AAV2/5 Subretinal I/II Ongoing NCT03252847 MeiraGTx UK II Ltd UK Achromatopsia CNGB3 Phototransduction cascade AAV2 Subretinal I/II Ongoing NCT02599922 Applied Genetic Technologies Corp USA Achromatopsia CNGB3 Phototransduction cascade AAV2/8 Subretinal I/II Ongoing NCT03001310 MeiraGTx UK II Ltd UK Achromatopsia CNGA3 Phototransduction cascade AAV8 Subretinal I/II Ongoing NCT02610582 STZ Eyetrial/University Hospital Tuebingen Germany Achromatopsia CNGA3 Phototransduction cascade AAV2 Subretinal I/II Ongoing NCT02935517 Applied Genetic Technologies Corp USA, Israel X-Linked Retinoschisis RS1 Intercellular adhesion AAV8 Intravitreal I/II Ongoing NCT02317887 National Eye Institute USA X-Linked Retinoschisis RS1 Intercellular adhesion AAV2 Intravitreal I/II Ongoing NCT02416622 Applied Genetic Technologies Corp USA Neovascular-AMD sFLT01 Anti-angiogenesis AAV2 Intravitreal I Ongoing NCT01024998 Sanofi USA Neovascular-AMD AntiVEGFfab Anti-angiogenesis AAV8 Subretinal I Ongoing NCT03066258 Regenxbio USA Neovascular-AMD sFLT01 Anti-angiogenesis AAV2 Subretinal I/II Completed NCT01494805 Lions Eye Institute Australia Neovascular-AMD Endostatin/Angiostatin Anti-angiogenesis EIAV Subretinal I Completed NCT01301443 Oxford BioMedica USA Dry-AMD sCD59 MAC inhibitory protein AAV2 Intravitreal I Ongoing NCT03144999 Hemera Biosciences USA Retinitis Pigmentosa ChR2 Light-gated ion channels AAV2 Intravitreal I/II Ongoing NCT02556736 Allergan USA Disease Gene Gene function Vector Mode Phase Current status Clinical trials ID Sponsor Location LCA-2 RPE65 Retinoid cycle AAV2/2 Subretinal III Ongoing NCT00999609 Spark Therapeutics UK NCT01208389 LCA-2 RPE65 Retinoid cycle AAV2/2 Subretinal I Ongoing NCT00516477 Spark Therapeutics USA LCA-2 RPE65 Retinoid cycle AAV2/5 Subretinal I/II Ongoing NCT02781480 MeiraGTx UK II Ltd UK NCT02946879 LCA-2 RPE65 Retinoid cycle AAV2 Subretinal I Unknown NCT00821340 Hadassah Medical Organization Israel LCA-2 RPE65 Retinoid cycle AAV2/2 Subretinal I Ongoing NCT00481546 University of Pennsylvania and National Eye Institute USA LCA-2 RPE65 Retinoid cycle AAV2 Subretinal I/II Ongoing NCT00749957 Applied Genetic Technologies Corp USA LCA-2 RPE65 Retinoid cycle AAV2/2 Subretinal I/II Completed NCT00643747 University College London UK LCA-2 RPE65 Retinoid cycle AAV2/4 Subretinal I/II Completed NCT01496040 Nantes University Hospital France LCA-10 CEP290 Ciliary transport N/A* Intravitreal I/II Ongoing NCT03140969 ProQR Therapeutics USA, Belgium Stargardt Disease ABCA4 Retinoid transfer EIAV Subretinal I/II Ongoing NCT01367444 Sanofi/Oxford BioMedica USA, France RP assoc. with Usher’s Syndrome Type 1B MYO7A Intracellular transport EIAV Subretinal I/II Ongoing NCT01505062 Sanofi/Oxford BioMedica USA, France Choroideremia REP1 Intracellular trafficking AAV2 Subretinal II Ongoing NCT02553135 University of Miami USA Choroideremia REP1 Intracellular trafficking AAV2 Subretinal II Ongoing NCT02671539 STZ Eyetrial Germany Choroideremia REP1 Intracellular trafficking AAV2 Subretinal I/II Ongoing NCT01461213 University of Oxford/ NightstaRx UK Choroideremia REP1 Intracellular trafficking AAV2 Subretinal I/II Ongoing NCT02077361 University of Alberta/ NightstaRx Canada Choroideremia REP1 Intracellular trafficking AAV2 Subretinal II Ongoing NCT02407678 University of Oxford/ NightstaRx UK Choroideremia CHM Intracellular trafficking AAV2 Subretinal I/II Ongoing NCT02341807 Spark Therapeutics USA MERTK-RP MERTK Cellular signalling AAV2 Subretinal I Ongoing NCT01482195 King Khaled Eye Specialist Hospital Saudi Arabia X-Linked RP RPGR Intracellular transport AAV8 Subretinal I/II Ongoing NCT03116113 NightstaRx UK X-Linked RP RPGR Intracellular transport AAV2/5 Subretinal I/II Ongoing NCT03252847 MeiraGTx UK II Ltd UK Achromatopsia CNGB3 Phototransduction cascade AAV2 Subretinal I/II Ongoing NCT02599922 Applied Genetic Technologies Corp USA Achromatopsia CNGB3 Phototransduction cascade AAV2/8 Subretinal I/II Ongoing NCT03001310 MeiraGTx UK II Ltd UK Achromatopsia CNGA3 Phototransduction cascade AAV8 Subretinal I/II Ongoing NCT02610582 STZ Eyetrial/University Hospital Tuebingen Germany Achromatopsia CNGA3 Phototransduction cascade AAV2 Subretinal I/II Ongoing NCT02935517 Applied Genetic Technologies Corp USA, Israel X-Linked Retinoschisis RS1 Intercellular adhesion AAV8 Intravitreal I/II Ongoing NCT02317887 National Eye Institute USA X-Linked Retinoschisis RS1 Intercellular adhesion AAV2 Intravitreal I/II Ongoing NCT02416622 Applied Genetic Technologies Corp USA Neovascular-AMD sFLT01 Anti-angiogenesis AAV2 Intravitreal I Ongoing NCT01024998 Sanofi USA Neovascular-AMD AntiVEGFfab Anti-angiogenesis AAV8 Subretinal I Ongoing NCT03066258 Regenxbio USA Neovascular-AMD sFLT01 Anti-angiogenesis AAV2 Subretinal I/II Completed NCT01494805 Lions Eye Institute Australia Neovascular-AMD Endostatin/Angiostatin Anti-angiogenesis EIAV Subretinal I Completed NCT01301443 Oxford BioMedica USA Dry-AMD sCD59 MAC inhibitory protein AAV2 Intravitreal I Ongoing NCT03144999 Hemera Biosciences USA Retinitis Pigmentosa ChR2 Light-gated ion channels AAV2 Intravitreal I/II Ongoing NCT02556736 Allergan USA *RNA anti-sense oligonucleotides are administered without a vector. Table 1 Clinical trials of retinal gene therapy (LCA, Leber Congenital Amaurosis; RP, Retinitis Pigmentosa) Disease Gene Gene function Vector Mode Phase Current status Clinical trials ID Sponsor Location LCA-2 RPE65 Retinoid cycle AAV2/2 Subretinal III Ongoing NCT00999609 Spark Therapeutics UK NCT01208389 LCA-2 RPE65 Retinoid cycle AAV2/2 Subretinal I Ongoing NCT00516477 Spark Therapeutics USA LCA-2 RPE65 Retinoid cycle AAV2/5 Subretinal I/II Ongoing NCT02781480 MeiraGTx UK II Ltd UK NCT02946879 LCA-2 RPE65 Retinoid cycle AAV2 Subretinal I Unknown NCT00821340 Hadassah Medical Organization Israel LCA-2 RPE65 Retinoid cycle AAV2/2 Subretinal I Ongoing NCT00481546 University of Pennsylvania and National Eye Institute USA LCA-2 RPE65 Retinoid cycle AAV2 Subretinal I/II Ongoing NCT00749957 Applied Genetic Technologies Corp USA LCA-2 RPE65 Retinoid cycle AAV2/2 Subretinal I/II Completed NCT00643747 University College London UK LCA-2 RPE65 Retinoid cycle AAV2/4 Subretinal I/II Completed NCT01496040 Nantes University Hospital France LCA-10 CEP290 Ciliary transport N/A* Intravitreal I/II Ongoing NCT03140969 ProQR Therapeutics USA, Belgium Stargardt Disease ABCA4 Retinoid transfer EIAV Subretinal I/II Ongoing NCT01367444 Sanofi/Oxford BioMedica USA, France RP assoc. with Usher’s Syndrome Type 1B MYO7A Intracellular transport EIAV Subretinal I/II Ongoing NCT01505062 Sanofi/Oxford BioMedica USA, France Choroideremia REP1 Intracellular trafficking AAV2 Subretinal II Ongoing NCT02553135 University of Miami USA Choroideremia REP1 Intracellular trafficking AAV2 Subretinal II Ongoing NCT02671539 STZ Eyetrial Germany Choroideremia REP1 Intracellular trafficking AAV2 Subretinal I/II Ongoing NCT01461213 University of Oxford/ NightstaRx UK Choroideremia REP1 Intracellular trafficking AAV2 Subretinal I/II Ongoing NCT02077361 University of Alberta/ NightstaRx Canada Choroideremia REP1 Intracellular trafficking AAV2 Subretinal II Ongoing NCT02407678 University of Oxford/ NightstaRx UK Choroideremia CHM Intracellular trafficking AAV2 Subretinal I/II Ongoing NCT02341807 Spark Therapeutics USA MERTK-RP MERTK Cellular signalling AAV2 Subretinal I Ongoing NCT01482195 King Khaled Eye Specialist Hospital Saudi Arabia X-Linked RP RPGR Intracellular transport AAV8 Subretinal I/II Ongoing NCT03116113 NightstaRx UK X-Linked RP RPGR Intracellular transport AAV2/5 Subretinal I/II Ongoing NCT03252847 MeiraGTx UK II Ltd UK Achromatopsia CNGB3 Phototransduction cascade AAV2 Subretinal I/II Ongoing NCT02599922 Applied Genetic Technologies Corp USA Achromatopsia CNGB3 Phototransduction cascade AAV2/8 Subretinal I/II Ongoing NCT03001310 MeiraGTx UK II Ltd UK Achromatopsia CNGA3 Phototransduction cascade AAV8 Subretinal I/II Ongoing NCT02610582 STZ Eyetrial/University Hospital Tuebingen Germany Achromatopsia CNGA3 Phototransduction cascade AAV2 Subretinal I/II Ongoing NCT02935517 Applied Genetic Technologies Corp USA, Israel X-Linked Retinoschisis RS1 Intercellular adhesion AAV8 Intravitreal I/II Ongoing NCT02317887 National Eye Institute USA X-Linked Retinoschisis RS1 Intercellular adhesion AAV2 Intravitreal I/II Ongoing NCT02416622 Applied Genetic Technologies Corp USA Neovascular-AMD sFLT01 Anti-angiogenesis AAV2 Intravitreal I Ongoing NCT01024998 Sanofi USA Neovascular-AMD AntiVEGFfab Anti-angiogenesis AAV8 Subretinal I Ongoing NCT03066258 Regenxbio USA Neovascular-AMD sFLT01 Anti-angiogenesis AAV2 Subretinal I/II Completed NCT01494805 Lions Eye Institute Australia Neovascular-AMD Endostatin/Angiostatin Anti-angiogenesis EIAV Subretinal I Completed NCT01301443 Oxford BioMedica USA Dry-AMD sCD59 MAC inhibitory protein AAV2 Intravitreal I Ongoing NCT03144999 Hemera Biosciences USA Retinitis Pigmentosa ChR2 Light-gated ion channels AAV2 Intravitreal I/II Ongoing NCT02556736 Allergan USA Disease Gene Gene function Vector Mode Phase Current status Clinical trials ID Sponsor Location LCA-2 RPE65 Retinoid cycle AAV2/2 Subretinal III Ongoing NCT00999609 Spark Therapeutics UK NCT01208389 LCA-2 RPE65 Retinoid cycle AAV2/2 Subretinal I Ongoing NCT00516477 Spark Therapeutics USA LCA-2 RPE65 Retinoid cycle AAV2/5 Subretinal I/II Ongoing NCT02781480 MeiraGTx UK II Ltd UK NCT02946879 LCA-2 RPE65 Retinoid cycle AAV2 Subretinal I Unknown NCT00821340 Hadassah Medical Organization Israel LCA-2 RPE65 Retinoid cycle AAV2/2 Subretinal I Ongoing NCT00481546 University of Pennsylvania and National Eye Institute USA LCA-2 RPE65 Retinoid cycle AAV2 Subretinal I/II Ongoing NCT00749957 Applied Genetic Technologies Corp USA LCA-2 RPE65 Retinoid cycle AAV2/2 Subretinal I/II Completed NCT00643747 University College London UK LCA-2 RPE65 Retinoid cycle AAV2/4 Subretinal I/II Completed NCT01496040 Nantes University Hospital France LCA-10 CEP290 Ciliary transport N/A* Intravitreal I/II Ongoing NCT03140969 ProQR Therapeutics USA, Belgium Stargardt Disease ABCA4 Retinoid transfer EIAV Subretinal I/II Ongoing NCT01367444 Sanofi/Oxford BioMedica USA, France RP assoc. with Usher’s Syndrome Type 1B MYO7A Intracellular transport EIAV Subretinal I/II Ongoing NCT01505062 Sanofi/Oxford BioMedica USA, France Choroideremia REP1 Intracellular trafficking AAV2 Subretinal II Ongoing NCT02553135 University of Miami USA Choroideremia REP1 Intracellular trafficking AAV2 Subretinal II Ongoing NCT02671539 STZ Eyetrial Germany Choroideremia REP1 Intracellular trafficking AAV2 Subretinal I/II Ongoing NCT01461213 University of Oxford/ NightstaRx UK Choroideremia REP1 Intracellular trafficking AAV2 Subretinal I/II Ongoing NCT02077361 University of Alberta/ NightstaRx Canada Choroideremia REP1 Intracellular trafficking AAV2 Subretinal II Ongoing NCT02407678 University of Oxford/ NightstaRx UK Choroideremia CHM Intracellular trafficking AAV2 Subretinal I/II Ongoing NCT02341807 Spark Therapeutics USA MERTK-RP MERTK Cellular signalling AAV2 Subretinal I Ongoing NCT01482195 King Khaled Eye Specialist Hospital Saudi Arabia X-Linked RP RPGR Intracellular transport AAV8 Subretinal I/II Ongoing NCT03116113 NightstaRx UK X-Linked RP RPGR Intracellular transport AAV2/5 Subretinal I/II Ongoing NCT03252847 MeiraGTx UK II Ltd UK Achromatopsia CNGB3 Phototransduction cascade AAV2 Subretinal I/II Ongoing NCT02599922 Applied Genetic Technologies Corp USA Achromatopsia CNGB3 Phototransduction cascade AAV2/8 Subretinal I/II Ongoing NCT03001310 MeiraGTx UK II Ltd UK Achromatopsia CNGA3 Phototransduction cascade AAV8 Subretinal I/II Ongoing NCT02610582 STZ Eyetrial/University Hospital Tuebingen Germany Achromatopsia CNGA3 Phototransduction cascade AAV2 Subretinal I/II Ongoing NCT02935517 Applied Genetic Technologies Corp USA, Israel X-Linked Retinoschisis RS1 Intercellular adhesion AAV8 Intravitreal I/II Ongoing NCT02317887 National Eye Institute USA X-Linked Retinoschisis RS1 Intercellular adhesion AAV2 Intravitreal I/II Ongoing NCT02416622 Applied Genetic Technologies Corp USA Neovascular-AMD sFLT01 Anti-angiogenesis AAV2 Intravitreal I Ongoing NCT01024998 Sanofi USA Neovascular-AMD AntiVEGFfab Anti-angiogenesis AAV8 Subretinal I Ongoing NCT03066258 Regenxbio USA Neovascular-AMD sFLT01 Anti-angiogenesis AAV2 Subretinal I/II Completed NCT01494805 Lions Eye Institute Australia Neovascular-AMD Endostatin/Angiostatin Anti-angiogenesis EIAV Subretinal I Completed NCT01301443 Oxford BioMedica USA Dry-AMD sCD59 MAC inhibitory protein AAV2 Intravitreal I Ongoing NCT03144999 Hemera Biosciences USA Retinitis Pigmentosa ChR2 Light-gated ion channels AAV2 Intravitreal I/II Ongoing NCT02556736 Allergan USA *RNA anti-sense oligonucleotides are administered without a vector. RPGR Retinitis Pigmentosa (RP) is a progressive of rod-cone retinal degeneration, caused by mutations in more than 60 genes and affects about 1 in 3000 people. X-linked RP (XLRP), a particularly severe form, accounts for 15–25% of RP with onset in childhood and rapid progression. Seventy to eighty percent of XLRP is caused by sequence variants in the RPGR gene, which encodes the retinitis pigmentosa GTPase regulator.33 In both rodent and canine models of the condition, AAV-mediated gene supplementation using truncated or codon-optimized constructs to improve transgene stability have slowed retinal degeneration.34,35 Phase I/II trials of AAV-mediated gene therapy in affected humans are ongoing (NCT03116113, NCT03252847). CHM Choroideremia is an X-linked recessive disorder, characterized by night-blindness in childhood, with gradually progressive constriction of peripheral vision leading to impairment of central vision in later life. Choroideremia is caused by sequence variants in the 1.9 kb CHM gene that encodes Rab escort protein 1 (REP1), which is required for intracellular vesicular transport.36 In a phase I/II clinical trial, subretinal injection of AAV2 vector expressing REP1 is reported to be well tolerated and associated with improved function in some instances.5,37 Long-term follow-up will determine whether gene therapy can protect against progressive constriction of visual fields owing to retinal degeneration (NCT01461213, NCT02553135, NCT02671539, NCT02077361). MERTK Defects in the gene encoding MERTK, a receptor tyrosine kinase essential for removal of waste material from photoreceptors, result in accumulation of debris and progressive rod-cone retinal degeneration (retinitis pigmentosa).38,39 In a rodent model of the disease, gene replacement therapy by subretinal administration of AAV2 vectors encoding Mertk can improve the outcome.40,41 In a phase I/II clinical trial, an AAV2-MERTK vector was well tolerated in affected humans with a suggestion of temporary benefit in some participants.42 RS1 X-Linked Retinoschisis (XLRS) causes impairment of sight in childhood, with variable progression in later adulthood. The prevalence is estimated to be 1 in 10 000 men. Associated features include strabismus, refractive error and anisometropia. Gene defects in RS1, which encodes retinoschisin, impair adhesion and connectivity between photoreceptor and bipolar cells, leading to schisis of the macula and retinal dysfunction. About 50% of those affected also have peripheral retinoschisis which presents a risk of retinal detachment and vitreous haemorrhage. Intravitreal delivery of AAV2 or AAV8 vectors encoding RS1 improves the outcome in Rs1-deficient mice, and appears safe in rabbits.11,43,44 Phase I/II dose-escalation clinical trials of intravitreal gene replacement of RS1 using AAV2 and AAV8 vectors are ongoing (NCT02317887, NCT02416622). Candidate genes for future clinical trials AIPL1 Aryl hydrocarbon receptor-interacting protein-like 1 (AIPL1) is a molecular chaperone of phosphodiesterase 6, which mediates rod-photoreceptor-specific phototransduction. Mutations in AIPL1 cause a particularly severe, rapidly progressive form of LCA (AIPL1-LCA). Affected individuals present with severe sight impairment from birth and rapid retinal degeneration. Although the natural history is very poor, some preservation of retinal structure during infancy in humans45 indicates a window of opportunity for intervention by gene replacement therapy. The potential for benefit has been demonstrated experimentally in a rodent model.46,47 GUCY2D Retinal guanylate cyclase-1 is essential in photoreceptor cells for timely recovery from photoexcitation. Mutations in the GUCY2D gene account for 10–20% of LCA and photoreceptor architecture is relatively well preserved.48 Demonstration of improved retinal function following gene replacement therapy using AAV5 and AAV2/8 and AAV8 vectors indicate the potential for benefit in affected humans.48,49 CEP290 CEP290 encodes a centrosomal protein involved in trafficking through the connecting cilia of photoreceptor cells. Mutations in CEP290 account for 30% of LCA. The size of the full-length gene exceeds the capacity of AAV vectors for a gene replacement strategy. However, the most common disease-causing variant, the intronic variant c.2991 + 1655 A > G, creates a cryptic splice donor site and premature stop codon that may be addressed by alternative strategies such as anti-sense oligonucleotides or CRISPR/Cas9-mediated gene editing.50 ABCA4 Stargardt disease (STGD1) results from mutations in the photoreceptor-specific flippase ABCA4, leading to intracellular accumulation of a toxic retinoid, and degeneration of the outer retina. STGD1 is the most common inherited macular degeneration, with an incidence of 1 in 10 000,51 causing progressively severe impairment of sight from early childhood or adulthood. The full-length ABCA4 gene (6.8 kb) exceeds the carrying capacity of AAV vectors. Alternative strategies for gene replacement include the use of oversized AAV5 vectors,52 trans-splicing and hybrid AAV2 dual vector systems.8 The use of a lentiviral vector based on Equine Infectious Anaemia Virus (EIAV) is reported to improve the outcome in a rodent model of the condition53 and to be well-tolerated in non-human primates.54 A phase I/II dose-escalation clinical trial of EIAV-ABCA4 vector is ongoing (NCT01367444). MYO7A Usher Syndrome is a recessively inherited condition characterized by impairment of sensorineural hearing and progressive impairment of sight owing to rod-cone degeneration. It is clinically and genetically heterogeneous, and is associated with defects in as many as 10 genes.55 Usher syndrome type 1B is caused by defects in the myosin VIIa (MYO7A) gene, resulting in abnormal accumulation of opsin in the cilia of photoreceptor cells. Like ABCA4, the large size of the MYO7A (8.1 kb) gene52 exceeds the carrying capacity of conventional AAV2/2 vectors. Alternative strategies include the use of dual vectors,8,56 to deliver gene fragments that are reassembled by homologous recombination following transduction, oversized AAV5 vectors,52 and lentiviral vectors.57,58 Subretinal injection of EIAV-MYO7A is well tolerated in non-human primates, and the potential for benefit to vision in humans is being explored in an ongoing phase I/II clinical trial (NCT01505062).58 RHO Many disease-causing sequence variants in RHO, which encodes the rod-photoreceptor pigment rhodopsin, have dominant negative effects in which the protein interferes with essential cell functions leading to variably severe rod-cone degeneration.59 More than 150 such mutations in RHO have been identified, accounting for 20–30% of autosomal dominant retinitis pigmentosa.60 Strategies to suppress expression of the disease allele specifically are currently difficult owing to the substantial mutational heterogeneity. One alternative approach is to suppress both the mutant and normal allele non-specifically, and simultaneously to provide a replacement functional gene that has been modified to resist suppression.61 This approach can improve the outcome in a rodent model but is yet to be explored in affected humans. Age-related macular degeneration AMD is the leading cause of vision loss in individuals over the age of 60. AMD can be classified as neovascular (wet) or atrophic (dry). Impairment of sight from neovascular-AMD and proliferative diabetic retinopathy is a direct consequence of pathological neovascularization which is driven strongly by the up-regulation of vascular endothelial growth factor (VEGF). The use of therapeutic anti-VEGF antibodies has resulted in substantially improved outcomes, but sustained benefit requires repeated intraocular injections. Vector-mediated gene expression of angiostatic proteins offers the opportunity to achieve sustained intraocular delivery following a single injection. Subretinal injection of a rAAV 2/2 vector expressing the soluble VEGF receptor sFlt-1 appears to be well tolerated in humans with neovascular-AMD, though a larger study will be needed to determine benefit (NCT0149805).62,63 Intravitreal injection of an AAV2 vector expressing sFlt-1 in advanced neovascular-AMD was followed by improved structure in a minority.64 Intraocular expression of sFlt-1 was sustained but variable and possibly limited by antibody-mediated immune responses to AAV2, with clinical signs of inflammation evident in some instances (NCT01024998). Subretinal delivery of Retinostat™, a non-replicating EIAV vector containing genes encoding the angiostatic proteins Endostatin and Angiostatin, appears safe in rodent65 and non-human primate models.66 In humans with neovascular-AMD, subretinal injection of Retinostat™ results in sustained intraocular transgene expression, and appears well tolerated (NCT01301443).67 Dry-AMD is characterized by dysfunction and degeneration of RPE cells, associated with inappropriate activation of the complement cascade.68 The protein CD59 can protect against cellular injury by blocking the membrane attack complex (MAC).69 A gene therapy product to increase expression of a soluble form of CD59 (sCD59) is currently being investigated (NCT03144999). The future The benefits of gene therapy for LCA-RPE65 have led to approval by the FDA of the first gene therapy for ocular disease, and to a rapid expansion in clinical trials exploring the safety and potential benefit of gene therapy for other inherited and acquired retinal diseases. The results will help define for each condition the potential window of opportunity for effective intervention. Further developments in vector design and delivery will enable greater efficiency and safety of gene transfer. Rapid reliable assessment of outcomes will be accelerated by optimization of clinical trial design. Conflict of interest statement MM, AJS, RRA and JWBB declare financial interests in MeiraGTx. References 1 Liew G , Michaelides M , Bunce C . A comparison of the causes of blindness certifications in England and Wales in working age adults (16–64 years), 1999-2000 with 2009-2010 . BMJ Open 2014 ; 4 : e004015 . doi:10.1136/bmjopen-2013-004015 . 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British Medical BulletinOxford University Press

Published: Mar 1, 2018

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