1. 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

2. 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

3. 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

4. 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

5. 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

6. 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).

7. 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

8. 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

9. 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

10. Clinical phenotype of P23H retinopathy in pig
P Scott et al. IOVS ;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

11. 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

12. 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

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

14. 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.

15. 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

16. 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)

17. P23H Retinopathy Transplanted at P14 and Assessed at P60

18. 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

19. P23H Retinopathy Transplanted at P120 and Assessed at P155

20. 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

21. 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

22. 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

23. Thank you!

24. 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

25. 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

26. 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