Two-step Reactivation of Dormant Cones in Retinitis Pigmentosa Henry J Kaplan, MD Evans Professor of Ophthalmology Chair, Ophthalmology & Visual Sciences Director, KY Lions Eye Center University of Louisville 25th Iranian Congress of Ophthalmology2015

Collaborators UofL Ophthalmology NSRRC Iowa State University Faculty Doug Dean Maureen McCall Wei Wang Patrick Scott Paul DeMarco Kaplan Research team Sang Joon Lee Xiaoqin Lu Douglas Emery Yongqin Liu Eric Vukmanic Juan Fernandez de Castro NSRRC Randall Prather Eric Walters Iowa State University Jason Ross Missouri U Toshiko Ezashi Michael R Roberts U Florida William Hauswirth Al Lewin UC Berkeley John Flannery

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 full-field ERG recordings

Functional Vision Loss Occurs with Cone Dysfunction and Death Most RP mutations occur in rod-specific photoreceptor genes leading to progressive rod destruction, and the loss of peripheral vision and dark adaptation However, it is the subsequent loss of cone function, marked by the sequential loss of the visual pigment-rich cone OS and mitochondrial-rich cone IS that results in severe functional visual impairment

Overview Transplantation of stem cells to replace degenerated rods in RP and restore rod vision may not be possible Primary reason the number of cells required for rod vision may not be achievable since < 5% of transplanted cells integrate into the ONL However, functional blindness in RP begins with loss of cone function We asked the question whether rod stem cell transplantation could prevent cone loss, and even rescue degenerated cones

Concept of Cone Dormancy As rods lose function and die, cones rapidly lose synthesis of their cone OS, and thus, central (i.e. macula) visual function Subsequently, cone IS that are critical for aerobic metabolism, disassemble Yet despite this loss of cone IS there is long-term persistence of cone cell bodies in RP retinas in a non-functional state (i.e. dormant cones).

Novel Hypothesis – Loss of cone function is a 2-step process and is amenable to treatment These dormant cones can regenerate IS and OS following stem cell transplantation Although NP agents, such as RdCVF, may delay the onset of cone dormancy, they can’t reactivate dormant cones – i.e. they can’t reassemble cone IS and regenerate cone OS However, the persistence of dormant cones in RP raises the possibility of regeneration of cone IS and synthesis of cone OS synthesis to maintain or restore lost central vision

Generation of a model of Pro23His (P23H) Retinopathy in the Pig Autosomal dominant RP is the most common inheritance pattern (~30% of cases) P23H mutation in Rhodopsin (RHO) most common form of autosomal dominant RP in North America Somatic cell nuclear transfer - transgenic model Ross et al. Invest Ophthalmol Vis Sci (IOVS) 2012; 53(1):501

Why use the pig as a model of cone dormancy in RP? The study of dormant cones needs to occur in a species where there is an area of high cone density in the retina The visual streak of the pig, analogous to the macula of man is such an area located ~ 2.5 mm superior to the optic disc Somatic cell nuclear transfer - transgenic model Ross et al. Invest Ophthalmol Vis Sci (IOVS) 2012; 53(1):501

Clinical phenotype of P23H retinopathy in pig P Scott et al. IOVS 2014 4;55:2452–2459 Morphology Wt P23H P23H swine at P3 lack ROS; CIS appear enlarged; COS (black arrows) abut the RPE; the ONL is thinner Rapid loss of rod PR in ONL by P30, with loss of RHO expression in the few remaining rod nuclei By P60, there are only cone PR nuclei in ONL

Clinical phenotype of P23H retinopathy in pig ff ERG JP Fernandez de Castro et al. IOVS 2014 55:2460–2468 At P0, there is no scotopic (i.e. rod) ERG in P23H retinopathy, in contrast to Wt However, the photopic (i.e. cone) ERG in P23H retinopathy matures normally until P30, and then responses decline P30 P90

Retinal Stem Cells Used for Transplantation Three types of stem cells being studied ESC (embryonic stem cells) Fetal retinal progenitor cells (E65) Swine retinal progenitor cells (NSRRC – Univ Missouri) iPSC (induced pluripotent stem cells) Swine iPSC (M Roberts ) Gestational cycle ~ 115 days for pigs; we have used fetal cells at various time points from E45-E105 for transplantation iPSC were differentiated in vitro to express rod photoreceptor cell markers and then transplanted

Technique of stem cell transplantation beneath retina in pig Zhou et al. Stem Cells 2011; 29:972

Method of counting of cone nuclei and OS The number of cone nuclei and OS were counted along the visual streak as a function of distance from the site of transplantation (injection bleb) in retinal sections in 200 µm lengths from the margin of the bleb to 2000 µm distal.

Three Stages of Cone Dormancy in Our Pig of P23H Retinopathy P14 – analogous to onset of central vision loss in RP in man Rods are lost, cone OS are malformed P60 – analogous to poor central vision in man Rods are lost, cone OS are lost, but cone IS are maintained P120 – analogous to end-stage of central vision in man Rods are lost, cone OS are lost and cone IS collapse

Transplanted stem cells preserved cone OS at P14 When we transplanted E65 or rod-differentiated iPSCs into the SRS at P14 (rods lost, cone OS malformed) there was regeneration of cone OS and an enhanced mfERG response ≤ 1000μ from the transplant at P60 (rods lost, cone OS lost)

P23H Retinopathy Transplanted at P14 and Assessed at P60

Transplanted stem cells regenerated cone IS and OS at P120 When E65 or rod-differentiated iPSCs were transplanted at P120 (after rods and cone OS lost, cone IS collapsed) there was regeneration of both cone IS and OS, as well as an increased mfERG response function ≤ 1400μ

P23H Retinopathy Transplanted at P120 and Assessed at P155

Additionally, cone IS assembly and OS synthesis are separate functions Transplanted rods triggered both IS reassembly and reactivation of OS in dormant cones But IS reassembly extended farther beyond reactivation of OS synthesis Implying these are separable functions of the transplanted rods

Cone Dormancy can be Reversed and Central Vision Recovered Step 1: Cone IS reassembly Rods initially transmit a signal for reassembly of the ER and mitochondria into a polarized mitochondrial-rich IS Step 2: Cone OS synthesis Mitochondrial-based aerobic metabolism is returns for fatty acid synthesis and cone OS formation Since cones lose OS synthesis well before IS disassembly, therapies aimed at reversing decreased aerobic metabolism might preserve cone function prior to end-stage IS loss. But ultimately reversal of IS loss, e.g. by rod-differentiated iPSC transplants, may be possible

Conclusions Swine model of P23H retinopathy suggests a 2-step process for reactivation of dormant cones Step 1, reassembly of cone IS, is dependent on rod progenitor cells and/or rod-differentiated iPSCs Step 2, cone OS synthesis, is dependent on induced aerobic metabolism Recognition of this 2-step process suggests multiple potential translational interventions

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Sahel Hypothesis Ait-Slu N et al. Cell 2015 Circulating GL in the choroid is transported to the subretinal space by the RPE, and then is taken up by a Bsg/Glut1 complex on cone IS Recent mouse studies show that RdCVF (a thioredoxin) binds Bsg1/Glut1 on cone IS to promote GL uptake in vitro

RdCVF doesn’t reverse cone dormancy Although, overexpression of RdCVF in the SRS in murine RP delays cone OS loss and function The NP effect of RdCVF doesn’t reverse cone dormancy Bryne LC et al. JCI 2015 RdCVF1 ko (Nxnl1) mice display normal cone EP function until 12 mos These results suggest that RdCVF is not essential for cone development or function until perhaps challenged by oxidative stress

Transplanted stem cells Multifocal ERG Response Density Maps After Transplantation in P23H Retinopathy 4 weeks post-surgery OD Transplanted stem cells OS BSS Control Therefore, not only is cone morphology rescued/regenerated but electrical function as well