Rpe65 mutant dogs Pde6A mutant dogs Cngb1 mutant dogs rAAV RPE65 Mutant Dog/ Leber Congenital Amaurosis Null mutation in Rpe65 retinal function (ERG & dim light vision) Failure of 11-cis retinal supply to photoreceptors (visual cycle) Retina only slow degeneration (S-cones and area centralis degeneration – variable) RPE lipid inclusions 8 Mo 3.5 yr The Visual (Retinoid) Cycle retinal pigment All-trans-retinol epithelium (Vitamin A) RPE65 11-cis-retinal Visual pigments All-trans-retinal rod and cone outer segments All-trans-retinol Gene supplementation therapy for RPE65 Leber Congenital Amaurosis Initial trials in dogs – very successful Outcome in humans Some improvement in visual function Appears to not preserve photoreceptors in longer term Questions Is there preservation of photoreceptors? Why is outcome in humans not so successful? Does RPE65 Gene Therapy Preserve Photoreceptors? Rpe65-/- dogs: Early loss of S-cones Slow LM cone loss Very slow rod loss Exception – region of high density of photoreceptors – rapid loss Gene therapy preservation of photoreceptors Limitations to Human Functional Rescue and Photoreceptor Preservation Hypothesis The dose of gene therapy delivered is a limiting factor for the efficacy of treatment Specific aim To compare the clinical efficacy and the levels of expression of RPE65 protein and the end product of RPE65 function (11-cis retinal) of various doses of RPE65 gene therapy in Rpe65 -/- dogs Methods Tested total dose of 8x108 to 1x1011 vg/eye ERG Scotopic b wave Vision testing % correct choice RPE65 protein expression Dose of gene therapy +/+ 8x108 4x109 2x1010 1x1011 RPE65 GAPDH RPE65 protein expression RPE65/DAPI/ autofluorescence Chromophore levels 11-cis retinal levels undetectable In Rpe65 -/- All-trans retinal Chromophore vs clinical outcomes Scotopic b wave r2 = 0.91 p < 0.0001 Vision testing % correct choice r2 = 0.58 p = 0.02 RPE65 gene expression Human vs. canine 4 samples/species Human 2.4x higher Conclusions In dogs, the clinical effect of RPE65 gene therapy is dose-dependent Robust ERG and vision rescue at highest doses Vision improvement without ERG rescue at lower doses Only the highest dose delivered resulted in detectable RPE65 protein expression 11-cis retinal levels correlate with clinical outcomes Retinal RPE65 expression in human > dog Human gene supplementation therapy may be improved by higher expression of RPE65? Pde6A Mutant Cardigan Welsh Corgi Adapted from Baehr & Frederick Vis Res 2009 1-base pair deletion codon 616 Premature stop codon Cell Rod Phototransduction Membrane Discs in rod outer segment c Dark: Channel c open Tβ Tα γ α β γ R Tα Tα Tγ Light: c Channel closed cGMPcGMP Light GMP Pde6A Mutant Cardigan Welsh Corgi Rod ERG fails to develop (cone a-wave) Rods do not mature Rapid loss of rods Peak photoreceptor apoptosis 28 days Slower cone degeneration 9 wks Normal Affected Tuntivanich et al IOVS 2009 Pde6a-/-. Lack total PDE6, Rhodopsin is mislocalized Pde6a+/+ Pde6a-/- Pde6a+/+ Pde6a-/- Pde6a Gene Therapy AAV2/8(733)-CBA-PDE6a Subretinal injection Pde6A gene supplementation therapy 43 days post inj Pde6a Vision Testing – 5 Mo Vision testing 4 weeks after injection Optical Coherence Treated eye Control injected eye Tomography Photorecepto layer Histology Treated eye Untreated eye hCAR Photoreceptor layer Treated eye Untreated eye Phosphodiesterase (PDE) Expression localized No to OS expression Rhodopsin Mislocalized Localized to rod cell to OS body Pde6a Gene Supplementation Conclusions Treated dogs gain rod-mediated function ERG – evidence of rod function Vision testing – convincing PDE6 expressed in outer segments Preservation of retinal structure Reduced rhodopsin mislocalization Outer nuclear layer preservation Subretinal AAV2/5-GRK1-Cngb1 Background: 6 Mo Control Affected 10 Mo 14 Mo 50 μVolt 50 mSec Background: Canine Cngb1 PRA Exon 26 Wild-Type Mutant Background: CNGB1 Expression in Rods • CNGB1 produces multiple transcripts in retina encoding CNGB1a, GARP1 & GARP2 CNGB1a GARP2 Cell Rod Phototransduction Membrane Discs in rod outer segment c Dark: Channel c open Light: Tβ Tα γ α β γ R Channel Tγ closed c cGMPcGMP Light GMP Background: CNGB1 Expression in Rods • 1 CNGB1a + 3CNGA1 forms the CNG channel • GARP2 important structurally and has several interactions Pearring et al 2013 Background: CNGB1 Mutations – Other Species • Retinitis pigmentosa – humans. Two mouse models 1. GARP region mutations 2. Downstream mutations • Mouse model – structural rod • Mouse model – slow rod disc abnormalities, faster degeneration degeneration • RP patients • RP patients – legally blind in • Night blind from childhood teens • RP diagnosed in 30’s Cngb1-X1 mouse Cngb1-X26 mouse CNGB1a GARP2 Background: Canine Cngb1-/- Phenotype Wild-type Cngb1-/- • Lack full-length Cngb1a • Garps present • Very reduced Cnga1 • Rod function ↓ ↓ reduced • ↓ ↓ reduced Rod ERG • Blind in dim light • Relatively slow photoreceptor loss Hypothesis: Slow structural degeneration provides opportunity for rAAV gene augmentation Methods • Dogs: 5 Cngb1-/- dogs, 14.5 to 28 weeks of age • Baseline Measures: • ERG, Vision Testing, OCT • Subretinal Injections • 1 eye - AAV2/5-GRK1-cCngb1: 1.75x1011 vp (low dose) • 7 eyes - AAV2/5-GRK1-cCngb1: 1.25x1012 – 1.5x1012 vp (high dose) • Outcome measures: • ERG, Vision Testing, OCT • Histology • One dog (2 eyes) followed up to 18 months. Results – Cngb1 and Cnga1 present in rod OS Pearring et al 2013 Results: Rod ERG Rescued Scotopic ERG series Scotopic (rod) flicker 50 µV 50 mSec A-wave modeling Results: Rod ERG Rescued & Maintained Results: Treatment Results in Rod Vision • Outcome measures: 1. Tunnel exit choice 2. Time to exit Conclusions • Prior to treatment and untreated Cngb1-/- dogs: • Lack Cngb1 expression in rod outer segments • Very reduced Cnga1 expression in rod outer segments • Markedly reduced rod function (ERG and Vision Testing) • AAV2/5-GRK1-cCngb1 delivered subretinally: • Formation of rod CNG channel subunits in the treated area • Rescues rod ERG function • Scotopic response threshold improved by 2+ log units • Significant increase in scotopic ERG a- and b-wave amplitudes • Results in vision at low light levels • Rescue is maintained to at least 18 months post treatment Future Investigations 18 Mo post • Duration of rod rescue AAV5-CNGB1 Untreated • Structural preservation • Maintenance of cone function • Success when rod loss more advanced Translational Potential • Potential for treating CNGB1 RP Challenges for Retinal Gene Therapy Delivery Vectors that transduce photoreceptors from vitreous Vector optimization Transduction levels Preservation of residual photoreceptors Can they recover from disease compromise? Can cones be preserved? Therapy for more challenging disease Large genes Dominant-negative mutation Transcription factor mutation.