“WHAT’S THE GENE?” COVER FOCUS An update on gene therapy from a choroideremia perspective.

BY THOMAS L. EDWARDS, PhD, FRANZCO

“What’s the gene?” is unvaryingly the first WHY CHOROIDEREMIA? question posed to trainees and vitreoretinal Although choroideremia is relatively uncommon, affect- fellows after an anonymous color fundus ing approximately 1 in 50,000 people, it is well suited to a photograph is presented for their scrutiny study of gene therapy for three main reasons. First, because at the weekly professorial teaching session a patient’s central retina is usually preserved until late in the at the Oxford Eye Hospital. The case, which disease course, visual acuity and macular sensitivity remain shows strikingly pale fundi with exten- reliable outcome measures. Second, relative preservation sive chorioretinal atrophy and an island of central retinal thickness may safeguard the target region of relative sparing at the posterior pole, concerns that of during subretinal injection. Third, the choroideremia gene a 28-year-old man with 6/6 vision. A trainee mumbles, CHM has a relatively short coding sequence (1962 base “Choroideremia?” prompting the follow up question, “So, pairs), which is less than the maximum capacity for recom- how big is the gene?” binant adeno-associated virus serotype 2 (AAV2); hence the That genetics (and choroideremia in particular) is the follow-up question in the above tutorial concerning gene size. topic of these vitreoretinal tutorials should come as no sur- This virus is able to transduce all layers of the neurosensory prise; a number of clinical trials of gene therapy for inher- retina,7 making it ideally suited for targeting the affected cells ited retinal degenerations (IRDs) are under way around the in choroideremia. world, including one for choroideremia being conducted here in Oxford.1 Gene therapy for RPE652-4 and MERTK5 PHASE 1/2 TRIAL (both for retinitis pigmentosa) are in clinical trials at other Given that the majority of CHM pathologic variants are centers, and additional genes—such as RS1 (X-linked predicted to result in loss of gene function, gene replace- juvenile retinoschisis), CNGB3 (a cause of achromatopsia) ment is a sound strategy for this X-linked IRD. An ongoing and RPGR (the most common cause of X-linked retinitis phase 1/2 trial demonstrated sustained improvement of pigmentosa)—will likely be targeted in clinical trials in the central visual acuity 3.5 years after administration of AAV2 near future. A summary of some of these trials was pro- containing the nonmutated CHM transgene.1 This was an vided by Boye et al in their comprehensive review of retinal encouraging finding, given that two recent reports of AAV gene therapy.6 usage for Leber congenital amaurosis found that patients’ early gains in visual function subsequently declined.4,8 Moreover, the study dose (1 x 1010 viral genome particles) in our choroideremia trial was expected to be at the lower end of the therapeutic range because the primary outcome was safety. A subsequent clinical trial using a higher gene dose is under way to further resolve the optimal treatment dose.

CURRENT AND FUTURE DETERMINING EFFICACY STUDY GENES Determining the best outcome measures for assessing gene therapy efficacy in slowly progressive IRDs is a signifi- RPE65 — retinitis pig- RS1 — X-linked juvenile cant challenge. In choroideremia, central visual acuity is mentosa, Leber congenital retinoschisis often spared until middle age. Consider this article’s opening amaurosis CNGB3 — achromatopsia tutorial case as an example: If this patient received gene ther- MERTK — retinitis RPGR — X-linked retinitis apy, researchers might need to wait years before observing a pigmentosa pigmentosa rescue effect relative to the untreated fellow eye. An array of tests—including visual acuity, contrast

2016/ISSUE 3 | NEW RETINA MD 23 sensitivity, color vision, autofluorescence, and micrope- rimetry—are deployed throughout the follow-up period to detect changes in visual function. These tests are also performed in untreated individuals to help build a better WATCH IT NOW understanding of the disease’s natural history. For example, our laboratory recently developed an index that predicts the Gene Therapy for Choroideremia rate at which clinicians should expect to observe contraction Dr. Edwards sits down with Eyetube to discuss gene of the residual area of autofluorescence in choroideremia.9 therapy for choroideremia during Euretina in Nice. One can see how this could become a useful clinical tool for counseling patients and monitoring treatment responses in trial participants. Furthermore, it could be used to identify female carriers with a progressive male-pattern phenotype in

COVER FOCUS COVER whom gene therapy would likely be beneficial.10

DELIVERING A VIRAL VECTOR While the phase 1/2 study established the safety of our AAV2 vector in the eye, the emerging challenge for vitreoret- inal surgeons will be how to consistently administer a precise bit.ly/edwards316 volume of viral vector safely into the subretinal space, partic- ularly in eyes with fragile degenerate retinas. The technically simpler alternative—intravitreal administration—requires a overcome with the aid of technological innovations, such larger dose of virus to produce an equivalent multiplicity of as robot-assisted surgery, that facilitate micron-level preci- infection, increasing the risks both of retinal toxicity and of sion and steadiness of intraocular instruments during gene an adverse immune reaction to the larger viral load. therapy procedures. n In the ongoing Oxford trial, the vector is delivered 1. Edwards TL, Jolly JK, Groppe M, et al. Visual acuity after retinal gene therapy for choroideremia. N Engl J Med. to the subretinal space using the viscous fluid injection 2016;374(20):1996-1998. mode on the vitrectomy machine and a custom 41-gauge 2. Jacobson SG, Cideciyan AV, Ratnakaram R, et al. Gene therapy for Leber congenital amaurosis caused by RPE65 muta- tions: safety and efficacy in 15 children and adults followed up to 3 years. Arch Ophthalmol. 2012;130:9-24. Teflon-tipped cannula held by the surgeon in the subretinal 3. Testa F, Maguire AM, Rossi S, et al. Three-year follow-up after unilateral subretinal delivery of adeno-associated virus in space. Inevitably, improvements in safety and reliability will patients with Leber congenital amaurosis type 2. Ophthalmology. 2013;120:1283-1291. 4. Bainbridge JWB, Mehat MS, Sundaram V, et al. Long-term effect of gene therapy on Leber’s congenital amaurosis. be driven by technological innovation. Already, operating N Engl J Med. 2015;372:1887-1897. microscopes equipped with optical coherence tomography 5. Ghazi NG, Abboud EB, Nowilaty SR, et al. Treatment of retinitis pigmentosa due to MERTK mutations by ocular subreti- nal injection of adeno-associated virus gene vector: results of a phase I trial. Hum Genet. 2016;135(3):327-343. devices are being used by some centers to observe in real 6. Boye SE, Boye SL, Lewin AS, Hauswirth WW. A comprehensive review of retinal gene therapy. Mol Ther. 2013;21:509-519. time the extent and location of the subretinal bleb during 7. Ali RR, Reichel MB, Thrasher AJ, et al. Gene transfer into the mouse retina mediated by an adeno-associated viral vector. Hum Mol Genet. 1996;5(5):591-594. gene therapy procedures. In the near future, robot-assisted 8. Jacobson SG, Cideciyan AV, Roman AJ, et al. Improvement and decline in vision with gene therapy in childhood blind- instrument manipulation will enable precision placement of ness. N Engl J Med. 2015;372:1920-1926. 11,12 9. Jolly JK, Edwards TL, Moules J, et al. A qualitative and quantitative assessment of fundus autofluorescence patterns in a cannula under the retina. The cannula tip could be held patients with choroideremia. Invest Ophthalmol Vis Sci. 2016. [In press]. perfectly still in the subretinal space by the robot, enabling 10. Edwards TL, Groppe M, Jolly JK, et al. Correlation of retinal structure and function in choroideremia carriers. Ophthal- mology. 2015;122:1274-1276. an injection to take place over several minutes or even lon- 11. Meenink T, Naus G, de Smet M, et al. Robot assistance for micrometer precision in vitreoretinal surgery. Invest ger, reducing the risk to the retina from damaging stretch Ophthalmol Vis Sci. 2013;54:5808-5808. 12. Meenink HCM, Hendrix R, Naus GJL, et al. Robot-assisted vitreoretinal surgery. In: Medical Robotics. Philadelphia: forces or surgeon tremor. Elsevier; 2012:185-209. THE FUTURE OF GENE THERAPY Thomas L. Edwards, PhD, FRANZCO A number of IRDs may soon become amenable to n clinical academic vitreoretinal fellow, Nuffield Laboratory of subretinal injection of a viral vector capable of slowing or Ophthalmology, University of Oxford; and Oxford Eye Hospital, halting retinal degeneration. Identifying the causative gene Oxford University Hospital NHS Foundation Trust, both in will become increasingly important for genetic counseling Oxford, United Kingdom and predicting the expected rate of progression, based on n financial disclosure: none a growing body of gene-specific natural history data. The n [email protected] surgical challenges associated with subretinal delivery will be

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