Gene Therapy in Retinitis Pigmentosa (RP) Henry J Kaplan, MD Evans Professor of Ophthalmology Chair, Department of Ophthalmology & Visual Sciences Director, Kentucky Lions Eye Center University of Louisville Iran 2015

Overview - Gene Therapy in RP Gene therapy in Leber’s Congenital Amaruosis, type 2 (LCA 2) – Childhood form of RP – Gene mutation for LCA 2 in RPE65 gene Gene therapy in RP – Most common form of ADRP in North America – Pro23His mutation in Rhodopsin

Objective of Gene Therapy in Photoreceptor Degeneration Basic objective is delivery of genes to cells induce protein expression Where the disease is the result of an absent gene (i.e. a null gene mutation) the objective is to replace the missing protein Where the disease is the result of an abnormal protein (mutant gene mutation) the objective is to overwhelm the function of the abnormal protein with the normal or wild-type (WT) protein

Objective of Gene Therapy in Photoreceptor Degeneration  Viruses are most frequently used to deliver genes, e.g. AAV (adeno-associated virus), but nanoparticles, liposomes and other approaches can be used.  Viral induced expression of a normal protein in null mutations (i.e.where no protein is expressed) and is straightforward, e.g. AAV-RPE65 Leber’s Congenital Amaurosis: Bennett, Wilson & colleagues

Gene Therapy Protocols for delivering genes to retinal tissues most frequently use adenoviral (AAV) vectors AAV vectors efficient in delivering the gene to the targeted cell but are epigenetic and don’t integrate into the host cell genome in contrast to retroviral vectors Expression is not permanent and repeated delivery may be required Host immune repsonse may develop with repeated injections even though the eye is immunologically priviledged

Leber’s Congenital Amaurosis (LCA) LCA is a group of diseases with early-onset childhood retinal dystrophy characterized by vision loss, nystagmus, and severe retinal dysfunction. Usually present at birth with profound or modest vision loss, pendular nystagmus, nonrecordable ERG and other clinical findings - hypermetropia, photophobia, oculodigital sign, keratoconus, cataracts, and a variable appearance to the fundus.

LCA At least 19 different types which result from mutations in at least 14 genes Mutations in CEP290, CRB1, GUCY2D, and RPE65 genes are most common Occurs in 2 to 3 per 100,000 newborns and is responsible for ~ 5% retinal dystrophies The loss of cone function at birth or shortly thereafter clinically distinguishes LCA from RP

LCA 2 Inherited most frequently as autosomal recessive disease LCA2 is distinguished by moderate visual impairment at infancy that progresses to total blindness by mid to late adulthood. One of the unique qualities of LCA2 is that, even with profound early visual impairment, retinal cells are relatively preserved.

LCA2 Fundus photo with yellow dots and mottling of RPE Yellow dots, mottling RPEPosterior staphyloma

Gene Therapy for Leber’s Congenital Amaurosis (LCA) 3 independent initial clinical trials were performed to evaluate safety and efficacy in LCA2 caused by the gene mutation in RPE65. A deficiency of 11-cis-retinal causes rod photoreceptors unable to respond to light. RPE OS Photoreceptors

Initial Clinical Trials Patients were treated with a single unilateral subretinal injection of adenoassociated virus 2 (AAV2) carrying the RPE65 gene in the eye with the worst vision No serious adverse effects from AAV2 Short-term follow-up showed an improvement in selected measures of vision - including BCVA, kinetic VF, nystagmus, pupillary light reflex, microperimetry, DA perimetry, and DA full-field ERG

3 Year F/U in CHOP LCA2 Trial Showed that in 5 patients the improvements in visual and retinal function that had been achieved a few months after treatment remained stable. The maximum improvement was achieved within 6 months after treatment. Improved BCVA only achieved post- treatment in patients without nystagmus. No serious adverse effects. F Testa, et al. Ophthalmology 2013;120:1283–1291

Clinical Data from Trial BCVAFAF

Jacobson SG. et al. Improvement and Decline in Vision with Gene Therapy in Childhood Blindness N Engl J Med May 14; 372(20): 1920–1926. Three years after therapy in 5 patients, improvement in vision was maintained in 3, but the rate of loss of photoreceptors in the treated retina was the same as that in the untreated retina. Long-term follow-up data from three treated patients showed topographic maps of visual sensitivity in treated regions, nearly 6 years after therapy for two of the patients and 4.5 years after therapy for the third patient, indicate progressive diminution of the areas of improved vision.

J.W.B. Bainbridge et al. Long-Term Effect of Gene Therapy on Leber’s Congenital Amaurosis. N Engl J Med May 14; 372(20): 1887–1897 Improvements in retinal sensitivity, to varying extents, in 6 of 12 participants for up to 3 years, peaking at 6 to 12 months after treatment and then declining. No associated improvement in retinal function was detected by means of ERG. Three participants had intraocular inflammation, and two had clinically significant deterioration of visual acuity. The reduction in central retinal thickness varied among participants. CONCLUSIONS—Gene therapy with rAAV2/2 RPE65 vector improved retinal sensitivity, albeit modestly and temporarily. In dogs, RPE65 gene therapy with the same vector at lower doses improved vision-guided behavior, but only higher doses resulted in improvements in retinal function that were detectable with the use of ERG.

Retinitis Pigmentosa Classic fundus presentation Attenuated retinal vessels Mottling/granularity of RPE Bone-spicule intraretinal pigmentation Optic nerve head pallor Clinical symptoms – night blindness, constricted peripheral visual field Diagnostic testing - abnormal ERG recordings

Why do patients ultimately go blind in RP? Although rod photoreceptor loss results in nyctalopia and a constricted visual field, blindness and major visual disability results from cone photoreceptor dysfunction and ultimately death in late stages of the disease

Objective of Gene Therapy in Pro23His Retinopathy – Most Common Form AD RP But when a mutant protein is expressed, can expression of a virally induced native protein offset the mutant protein? – AAV- cDNA Rho  expression of Rho – AAV-siRNA Rho  suppression of Rho

Preliminary conclusion – gene therapy with cDNA Rho causes no harm and appears to delay cone loss in P23H retinopathy

Gene Therapy for Pro23His Mutation in ADRP WW Hauswirth and colleagues. Long-Term Rescue of Retinal Structure and Function by Rhodopsin RNA Replacement with a Single Adeno-Associated Viral Vector in P23H RHO Transgenic Mice. HUMAN GENE THERAPY 23:356–366 (April 2012) They delivered both a modified cDNA and an siRNA by a single AAV vector and observed long-term rescue of ADRP in this mouse model. Because the siRNA targets human as well as mouse rhodopsin mRNAs, the combination vector may be useful for the treatment of human disease.

LONG-TERM RESCUE OF MOUSE PHOTORECEPTORS BY mRNA REPLACEMENT

Conclusion The molecular ability to shutdown the mutant protein produced by the diseased cell with siRNA and then produce normal protein with simultaneously introduced “hardened” cDNA is an exciting innovation that we are exploring with Hauswirth, Lewin and colleagues in our model of P23H retinopathy in the mini-swine. Gene therapy for LCA 2 and P23H retinopathy in RP are being intensively explored and hold great promise.