Release Date: July 1, 2018 July 2018 Expiration Date: July 31, 2019 Last Review: May 25, 2018

A CME-Accredited Activity Brave New World: THERAPY FOR INHERITED DISEASE

Distinguished Faculty This CME activity is supported by an unrestricted educational grant from Bart P. Leroy, MD, PhD (Chair) . Mark E. Pennesi, MD, PhD Christina M. Ohnsman, MD A CME activity sponsored by

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IRD_Supp_EyeNet.indd 1 5/25/18 3:12 PM Bart P. Leroy, MD, PhD (Course Chair) Intended Audience Head of Department & Chairman This activity has been designed to meet the educational Department of needs of comprehensive ophthalmologists, pediatric Staff Member, Center for Medical Genetics Ghent Associate Professor, Ophthalmic Genetics ophthalmologists, and specialists. & Visual Electrophysiology Ghent University & Ghent University Hospital Disclaimer Ghent, Belgium The opinions expressed in this educational activity are those Division of Ophthalmology and of the faculty, and do not represent those of the Institute. Center for Cellular & Molecular Therapeutics Learners should appraise the information presented critically Children’s Hospital of Philadelphia Philadelphia, PA, USA and draw conclusions only after careful consideration of all available scientific information. Mark E. Pennesi, MD, PhD Conflicts of Interest/Disclosures Division Chief, Ophthalmic Genetics Associate Professor of Ophthalmology The Institute takes great pride in developing education that School of Medicine is fair-balanced and unbiased. As such, and in accordance Oregon Health & Science University with the ACCME Standards for Commercial Support, balance, Portland, Oregon, USA independence, objectivity, and scientific rigor are ensured in all CME activities. This requires everyone who is in a position to control the content of a CME activity to disclose all relevant Christina M. Ohnsman, MD financial relationships with any commercial interest. In addition, Former Clinical Instructor the content of this material has been approved by an Department of Pediatric Ophthalmology independent peer reviewer. Wills Eye Hospital Philadelphia, PA, USA Disclosures are as follows: Bart P. Leroy, MD, PhD has affiliations with Spark Therapeutics (Scientific Advisory Board, Grant Recipient); ProQR Therapeutics and GenSight Biologics (Principal Investigator) Mark E. Pennesi MD, PhD has affiliations with Nacuity Pharmaceuticals, Horama (Scientific Advisory Board); Ionis, Learning Objectives After participating in this activity, participants will be better Editas, Biogen, RegenexBio, Astellas, Opthotech, Spark able to demonstrate the ability to: Therapeutics (Clinical Trial Consultant); Nacuity (Shareholder); • Recognize the urgency of early and accurate genetic AGTC, Sanofi, Nightstar, Foundation Fighting Blindness diagnosis in IRD (Grant Recipient) • Discuss the challenges of living with an IRD, including Christina M. Ohnsman, MD has affiliations with Spark impact on functional vision and quality of life Therapeutics (Consultant). • Describe the variety of approaches to , (Peer Reviewer) has disclosed that including current and emerging treatment options for IRD Rudolph S. Wagner, MD he has no relevant financial relationships specific to the subject • Summarize the safety and efficacy of investigational IRD matter within the past 12 months. gene therapies • Design a patient-centered approach to the diagnosis and Cathy Pagano (Institute for Continuing Healthcare Education) management of IRD has disclosed that she has no relevant financial relationships specific to the subject matter within the past 12 months. Accreditation Statement This activity has been planned and implemented in accordance Disclosure of Unlabeled Use with the accreditation requirements and policies of the This activity will review off-label or investigational Accreditation Council for Continuing Medical Education by information. the Institute for Continuing Healthcare Education (Institute). This activity has been supported by an unrestricted educational The Institute is accredited by the ACCME to provide continuing grant from Spark Therapeutics. medical education for physicians. For any questions, please email us at [email protected] Designation Statement The Institute designates this enduring material for a maximum ©2018 The Institute for Continuing Healthcare Education. of 2.0 AMA PRA Category 1 CreditsTM. Physicians should claim only the credit commensurate with the extent of their participation in the activity.

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IRD_Supp_EyeNet.indd 2 5/25/18 3:12 PM Introduction nherited retinal diseases (IRDs) are a class of rare, single- ticipation in social activities.7 Such patients also have high Igene disorders that represent the major cause of fa- levels of depression, anxiety, and isolation.8 Additionally, milial blindness in the Western world and, until recently, 60-70% of blind Americans are unemployed, while al- have been untreatable.1 The recent approval of the fi rst most 30% live below the poverty line.9,10 Only 15% earn a gene therapy for IRD, for retinal bachelor’s degree or higher.9 Therefore, career limitations dystrophies caused by biallelic RPE65 mutations, and and unemployment are major psychological stressors for the growing number of active clinical gene therapy trials IRD patients.7 It is critical for physicians to understand the for other IRDs signal the dawn of a new era in caring for functional impact that gene therapy can have on a pa- patients with IRDs. For the fi rst time, ophthalmologists tient’s life. Although perfect vision may not be restored can offer hope. with treatment, any visual acuity, visual fi eld, and/or light Few natural history studies have been completed for sensitivity that is retained or improved is signifi cant and IRDs, resulting in a lack of information regarding disease may ameliorate some of the psychosocial impacts of pro- course and outcomes.2-6 This limited information com- gressive vision loss. bined with a lack of direct experience with IRD patients In this supplement, we present updated guidelines results in limited understanding of the extent to which dis- for diagnosis, referral patterns, and treatment—all of ease progression affects a patient’s daily activities, or how which have changed dramatically in the past year. While even small improvements in functional vision can translate it is impossible to provide a comprehensive text on this to signifi cant gains in quality of life. One in-depth study huge and rapidly evolving subject in such limited space, showed that patients with pigmentosa (RP) have our goal is to present a common-sense approach to IRD signifi cant diffi culties adjusting to blindness across mul- management that can be used in every eye care set- tiple psychosocial domains, including distressed relation- ting to optimize outcomes for patients. For all of us— ships with healthcare professionals and family members, clinicians and patients alike—it is, indeed, a brave new poor ability to adjust at work or school, and impaired par- world. – Bart P. Leroy, MD, PhD

Inherited Retinal Disease Overview FIGURE 1 Collectively, IRDs are blinding conditions affecting ap- proximately 200,000 people in the US, often presenting in The Spectrum of Inherited Retinal Disease childhood and persisting for life.11,12 In addition to severe : Inheritance: ABCA4 AD, AR, XL, Mitochondrial vision loss, an IRD may be part of a systemic syndrome, CEP290 Age of Onset: CHM possibly as the fi rst presenting feature, requiring timely < 1 year – LCA CNGA3 1-5 years – SECORD CNGB3 diagnosis and appropriate multidisciplinary management 5-10 years – XLRP MYO7A 12 20-40 years – AR or AD RP ND4 for the patient’s optimal health and development. > 55 years - Autoimmune RPGR RPE65 Syndromic: Layer: Classifi cation of Inherited Retinal Disease Usher Syndrome Vitreoretinal The IRD spectrum may be described in a variety of Bardet-Beidl Retinal RPE overlapping ways, including the type of functional loss, Senior-Loken Chorioretinal Refsum region or layer of the retina affected, inheritance, pro- Alstrom gressive nature, etc., as shown in Figure 1. As we will Organelles: Region: see, these classifi cations are useful in reaching a clinical Lysosomes Macula diagnosis, but ultimately, all clinical descriptions must Peroxisomes Pericentral Mitochondria Posterior Pole lead to genetic testing for defi nitive diagnosis. Mid-periphery Centriole Functional Loss: Concentric To date, more than 260 genes causing IRDs have been Rod- Pan Retina Cone-Rod Dystrophy identifi ed and another 37 have been mapped to a chro- Cone Dystrophy mosomal location with gene identifi cation in progress.13 The pace of discovery has been rapid, with approximate- testing is incomplete. ly 50 genes identifi ed every four years over the last two While a genetic diagnosis is now recommended for all decades. 13 However, mutations in the same gene can IRDs, the clinical description remains an important fi rst step cause a range of clinical presentations, or phenotypes, in focusing genetic testing. As current gene screening tech- whereas similar phenotypes can result from abnormali- niques often identify multiple potentially causative chang- ties in one of many different genes.13,14 Therefore, a clear es, these always need to be interpreted in a clinical con- one-to-one genotype-phenotype correlation is rare in text. Consequently, it is essential that clinicians recognize this group of diseases, which is currently considered the the various categories of IRDs. However, the complexity of most genetically heterogeneous in humans.11,13,14 For clinical diagnoses (Table 1) may be overwhelming to most this reason, a clinical diagnosis in the absence of genetic physicians, especially considering that IRDs are rare. The

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IRD_Supp_EyeNet.indd 3 5/25/18 3:12 PM FIGURE 2 parison, mutations in most oth- GeneticGenetic and and Clinical Clinical DiversityDiversity of of Mutations Mutations er IRD-associated genes cause AssociatedAssociated with with Retinitis Pigmentosa fewer than 100 new cases per year.15 The majority of patients

Congenital Stationary Leber Congenital experiencing Blindness Amaurosis due to an undiagnosed IRD are CLUAP1, DTHD1, GDF6, GNB3, GPR179, GRK1, IQCB1, LCA5, NMNAT1, initially seen by a pediatrician, GRM6, LRIT3, NYX, OTX2, RD3 SLC24A1, TRPM1 optometrist, or general oph- 16,17 CRB1, IFT140, thalmologist. Most of these PDE6B, RHO, IMPDH1, LRAT, MERTK, SAG RDH12, RPE65, AIPL1, CABP4 physicians have little or no di- SPATA7, TULP1 GUCY2D, Cone/Cone-Rod RPGRIP1 Dystrophies rect experience with the diag- CRX, PRPH2 ADAM9, ATF6, C21orf2, AGBL5, ARL2BP, ARL3, BEST1, C2orf71, CA4, CACNA2D4, CDHR1, nosis or management of IRDs, CNGA1, CNGB1, CYP4V2, DHDDS, DHX38, EMC1, CNGA3, CNGB3, CNNM4, Retinitis EYS, FAM161A, FSCN2, GPR125, GUCA1B, HGSNAT, COD2,* CORD4,* CORD8,* which leads to multiple rounds HK1, IDH3B, IMPG2, KIAA1549, KIZ, KLHL7, MAK, ABCA4, CERKL, CORD17,* GNAT2, GUCA1A, Pigmentosa MVK, NEK2, NEUROD1, NR2E3, NRL, OFD1, PDE6A, PROM1, RPGR, KCNV2, PDE6C, PDE6H, of referrals and variable patient PDE6G, POMGNT1, PRCD, PRPF3, PRPF31, PRPF4, SEMA4A PITPNM3, POC1B, RAB28, 16 PRPF6, PRPF8, RBP3, RGR, RLBP1, ROM1, RP1L1, RAX2, RCD1,* RIMS1, access to specialist services. RP1, RP2, RP6,* RP9, RP22,* RP24,* SLC7A14, TTLL5, UNC119 SNRNP200, SPP2, TOPORS, TRNT1, As a result, rare-disease patients ZNF408, ZNF513 C8orf37 CACNA1F, GNAT1, CSNB such as these see up to 8 physi- CLRN1, ARL6, BBS1, RDH5 USH2A BBS2, IFT172, TTC8 cians and receive 2 to 3 misdi- ABHD12, ADGRV1, agnoses over the course of 5-7 CDH23, CEP250, CIB2, HARS, MYO7A, ADIPOR1, BBIP1, BBS10, CEP290, PCDH15, USH1C, USH1E,* years before receiving a correct BBS12, BBS4, BBS5, BBS7, KCNJ13 LCA USH1G, USH1H,* 8 BBS9, IFT27, INPP5E, USH1K,* WHRN diagnosis. Eliminating or reduc- Usher LZTFL1, MKKS, MKS1, NPHP1, SDCCAG8, TRIM32 Syndrome ing this delay in reaching an ac- Bardet-Biedel curate and complete diagnosis Syndrome may prevent irreparable vision loss, lost opportunities to re- gene most commonly mutated is the ABCA4 gene. Bial- ceive current and emerging treatments and access to early lelic mutations in this gene cause , with intervention services, and harm to a patient’s general health an estimated 400 new cases per year in the US.15 In com- and development if the IRD is associated with a systemic

TABLE 1 Clinical Classifi cation of Inherited Retinal Disease Retinal Degeneration Abnormal Full Field ERG Normal Full Field ERG Progressive Stationary Progressive Stationary Nonsyndromic RP Cone Dysfunction Syndromes ABCA4 Spectrum North Carolina Macular Dystrophy • Rod-cone Dystrophies • Stargardt/ Flavimaculatus • Cone-rod Dystrophies • Oligocone Trichromacy - Macular Dysfunction Isolated Foveal Hypoplasia • Cone Dystrophies • Blue Cone - Peripheral Cone • Enhanced S-cone Syndrome - Peripheral Rod/Cone Albinism Leber Congenital Amaurosis • Bradyopsia • Oculocutaneous Albinism SECORD/Jevenile RP Bestrophinopathies (BEST1) • Ocular Albinism X-Linked RP Rod System Function Disorders • AD Best Vitelliform Macular AR RP • Congenital Stationary Night Blindness Dystrophy AD RP ERG Patterns • AR Best Vitelliform Macular Schubert-Bornschein, Riggs, etc. Dystrophy Syndromic RP Genes • AR Bestrophinopathy AD: GNAT1, RHO, PDE6B • AD VitreoRetinoChoroidopathy • Usher Syndrome XL: NYX , CACNA1F • Bardet-Biedl Syndromes AR: GRM6, TRPM1, LRIT1, etc. PRPH2 Spectrum • Joubert Syndrome • Fundus albipunctata (RDHS) • Pattern Dystrophy • Senior Loken Syndrome • Oguchi Disease (ARR, GRK1) • Central Areolar Choroidal Dystrophy • Alström Syndrome Mitochondrial Disorders X-Linked EFEMP1 Spectrum • Kearns-Sayre Syndrome, etc. Dominant Drusen Peroxisomal Disorders Fleck Dystrophies Doyne Honeycomb Dystrophy • Zellweger Spectrum, etc. • Benign Familial Flecked Retina Malattia Levintenese Lysosomal Storage Disorders • Retinitis Punctata Albescens • Hurler, Hurler-Scheie, Scheie • Alport Sorsby Macular Dystrophy Disease, etc. Spinocerebellar Ataxia 7 Occult Macular Dystrophy Neuronal Ceroid Lipofuscinoses • CLN3-Related, etc.

Choroidal Degenerations Gyrate Atrophy Bietti Crystalline Dystrophy Late Onset Retinal Dystrophy

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IRD_Supp_EyeNet.indd 4 5/25/18 3:12 PM syndrome. In addition, significant reduction in anxiety is the result of an early diagnosis. For this reason, we present a LACK OF GENOTYPE-PHENOTYPE simplified approach to evaluation of these diseases. CORRELATIONS IN IRD • Clinical findings of most IRDs are rarely Photoreceptor Diseases pathognomonic of a particular genetic mutation. Nearly two-thirds of IRDs are photoreceptor diseases.15 • A given genetic mutation may result in a variety of Classically, these were described as rod-mediated and clinical IRD presentations. cone-mediated diseases, but this sharp distinction has • One phenotype may be caused by mutations in a been discarded. Some conditions are progressive and variety of genes. are called dystrophies, while others are mostly stationary. In most, if not all, photoreceptor dystrophies, the func- tion of both rod and cone systems is compromised, some- FIGURE 3 times in the early stages of the disease. Photoreceptor dys- trophies comprise a spectrum of diseases, ranging from Differentiation of Retinal and Macular IRD predominantly rod dystrophies to predominantly cone dystrophies, with disorders intermediate between the two having varying involvement of both systems.18 Therefore, these are more properly called generalized photoreceptor IRDs of the rod-cone, cone-rod, or cone types.18 The most common clinical subgroup among all general- ized, progressive photoreceptor IRDs is RP, a diverse group of peripheral retinal dystrophies affecting photoreceptors and the retinal pigment epithelium (RPE). In general, RP is characterized by reduced sensitivity to light resulting in A. Bone-spicule fundus B. Fundus changes due to night blindness—causing difficulty navigating in moderate- appearance in RP, a ABCA4-related Stargardt ly low light—and delayed dark , followed by a generalized photoreceptor macular dystrophy progressive loss of peripheral visual fields (VFs), and, even- IRD tually, reduction of best-corrected visual acuity (BCVA) in many cases. Patients with RP lose 5%-10% of their remain- and variable degrees of and . ing VF every year once the disease process has begun,19-21 Family history is also very useful to learn, whether approximately 50% every 5 years. RP is also the most ge- other family members have had similar symptoms, at netically diverse subgroup of IRD, with disease-causing what age, and whether these were progressive. mutations in more than 100 genes despite similar clinical Fundus appearance may be helpful, such as the presentations (Figure 2).1,13 Likewise, mutations in one of bone-spicule appearance classically associated with reti- these genes may cause a range of clinical presentations, nal IRD such as R P, or the pisciform flecks associated with as demonstrated in a recent natural history study reporting the most frequent macular IRD, ABCA4-related Stargardt over 20 distinct clinical diagnoses for autosomal recessive disease (Figure 3). However, it is important to know that RPE65 mutations.2 In fact, the clinical findings of most IRDs many IRD cases do not have these classic textbook find- are rarely pathognomonic of a single genetic mutation, ings, so one must be aware of more subtle fundus ab- with few exceptions. This lack of genotype-phenotype cor- normalities. In fact, some cases of IRD have no visible relations is one of the most critical factors driving change in fundus abnormalities at all in the early stages. For this the IRD diagnostic pathway. Clinical descriptions, such as reason, any patient suspected of having an IRD must un- R P, are no longer considered final diagnoses; they are now dergo further workup, including additional imaging and merely the first step toward a definitive genetic diagnosis functional testing. via molecular testing. Useful forms of imaging for the evaluation of IRD in- clude color fundus photos, spectral domain ocular coher- Retinal vs. Macular Classification ence tomography (SD-OCT), fundus autofluorescence, It is helpful to use practical, broad categories based on and reflectance imaging with blue and near-infrared light. clinical presentation. For example, does the IRD primar- In addition, formal VF testing—including kinetic fields, mi- ily affect the entire retina or only the macula? One study croperimetry, and, in some cases, static VF evaluation—is at a tertiary care IRD subspecialty practice reported that essential whenever possible. (ERG) 65% of IRDs are generalized photoreceptor diseases and is particularly important in differentiating forms of IRD. another 28% are localized, macular dystrophies.15 The The full-field flash ERG will be abnormal in generalized ocular history is useful for differentiation here, as the vast retinal disease, but not in disease limited to the macula. majority of retinal IRD is of the rod-cone type,15 typical- Many forms of IRD have a distinct pattern on ERG, making ly presenting with night blindness () and con- this test indispensable in the evaluation of these patients. stricted VFs. On the other hand, macular IRDs present However, an ERG must be performed according to inter- with reduced central vision, color vision abnormalities, nationally accepted International Society for Clinical Elec-

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IRD_Supp_EyeNet.indd 5 5/25/18 3:13 PM trophysiology of Vision (ISCEV) standards and interpreted properly for accurate results.22 While detailed knowledge ENSURING OPTIMAL OUTCOMES FOR of each subcategory of IRD and the associated test results PATIENTS WITH IRD is commendable, it is not necessary for most eye care pro- • Awareness of IRD clinical spectrum fessionals. Instead, the best way to ensure optimal out- • Order workup comes for these patients is to consider the possibility of – Commensurate with local physician’s expertise an IRD, order a workup commensurate with your exper- – Based on local availability tise and availability of tests, and, most importantly, make a • Prompt referral to IRD specialist for further prompt referral to an IRD specialist for further evaluation, evaluation, genetic testing, and potential genetic testing, and potential treatment. treatment

Progressive vs. Non-Progressive IRDs Indeed, mutations in one of several genes, including As mentioned above, it is important to distinguish RPE65, LRAT, MERTK, SPATA7, and TULP1 may cause whether an IRD is progressive or stable. Some IRDs, rod-cone dystrophy with onset from birth to early adult- such as congenital stationary night blindness (CSNB) and hood (Figure 2), limiting the usefulness of age of on- achromatopsia, are either non-progressive or only mini- set for diagnosis.23-28 While a clinical classification is not mally progressive, and therefore have a better prognosis completely out of date, a more modern one is based on than IRDs that inexorably progress to complete blindness. genotypes, accommodating the diversity of phenotypes However, their clinical presentations often mimic those related to mutations in genes implicated in IRD. For of progressive IRDs, making it challenging to distinguish example, in a retrospective natural history study of 70 progressive from non-progressive diseases.12 For exam- patients with biallelic RPE65 mutations, over 20 distinct ple, CSNB is characterized by night blindness similar to a clinical diagnoses were used at the patients’ first visits, rod-cone IRD, and achromatopsia is often mistaken for a which occurred from 1 to 43 years of age.2 Nine patients progressive cone-rod dystrophy. In addition, progression had clinical diagnoses of both Leber congenital amauro- of high in some IRDs may be mistaken for pro- sis (LCA) and RP over the course of their follow-up. Clear- gression of the disease, adding to the confusion. Misdi- ly, then, clinical diagnoses alone may be misleading, with agnosis is particularly common in cases where an ERG has genetic testing required for a more accurate diagnosis. not been performed or has been misinterpreted, result- Furthermore, genetic testing can distinguish LCA from ing in unnecessary anguish for patients and their parents achromatopsia, albinism, CSNB and syndromic disease, who have been told to prepare for eventual blindness. all of which may be clinically similar at their onset but This is another crucial reason for consulting an IRD spe- have markedly different prognoses for visual function.39 cialist as soon as an IRD is suspected, so that progressive IRDs may be differentiated from non-progressive condi- Syndromic IRDs tions with rod and/or cone system dysfunction. The importance of recognizing systemic disease asso- ciated with a rod-cone IRD cannot be overstated. Again, Chorioretinopathies it is the genetic analysis that will help in making an ear- Another category of IRD involves the choroid in ad- ly, specific diagnosis. In general, genetic mutations that dition to the retina. Chorioretinopathies often have dis- result in abnormalities of cellular organelles common to tinct appearances with loss of choroidal vessels and RPE, retinal and non-ocular cells cause syndromic IRDs. For although this is not always the case, especially early in example, Usher syndrome, familiar to all eye care profes- the disease course. For example, choroideremia is often sionals, describes a group of autosomal recessive con- mistaken for RP. However, choroideremia is an X-linked ditions characterized by rod-cone dystrophy and partial disease while RP may be autosomal recessive, autosomal or complete congenital sensorineural hearing loss.40 In dominant, or X-linked. Misdiagnosis may therefore have some subtypes, vestibular function is also abnormal. The implications in terms of family planning. affected organelles are the specialized cilia in the photo- receptors and in the hair cells of the inner ear. Age of Onset Usher syndrome is now recognized early because Historically, age of onset has been a major feature many, if not all, children with congenital sensorineural in the clinical classification of IRDs. For example, in the hearing loss are screened for mutations in genes involved spectrum of rod-cone dystrophies, infants presenting in genetically determined deafness. At a minimum, they with nystagmus, failure to fix and follow, and non-record- are screened for retinal involvement annually. However, able ERGs were diagnosed with Leber congenital am- an IRD may be the first presenting feature of other cil- aurosis (LCA) while adults with nyctalopia, peripheral VF iopathies, including Joubert syndrome and Senior-Lo- loss, peripheral retinal abnormalities, and abnormal sco- ken syndrome, requiring magnetic resonance imaging topic ERGs were diagnosed with RP. Children with similar (MRI) of the brain and kidney function testing. Likewise, signs and symptoms received a diagnosis of early onset Bardet-Biedl syndrome is another that causes R P. However, the clinical classifications have progressive- renal insufficiency. Therefore, it is imperative to reach a ly lost importance with the advent of the molecular era. conclusive genetic diagnosis as quickly as possible to

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IRD_Supp_EyeNet.indd 6 5/25/18 3:13 PM guide systemic evaluation and treatment. Peroxisomal disorders are another category of syn- GENETIC TESTING OPTIONS dromic IRD, with severe deficiency of one or more of • Single gene nearly 50 biochemical reactions that take place in per- • Gene panel, grouped by clinical diagnosis oxisomes. Peroxisomal syndromes range from the severe • Whole Exome Sequencing (WES) systemic involvement and infant mortality of Zellweger • Whole Genome Sequencing (WGS) syndrome to single or abnormalities such as adult-type Refsum disease, associated with serious example, a genetic diagnosis may sometimes reduce the neurologic and cardiac dysfunction in adulthood.41 Some need for additional electrophysiologic testing, clarify the aspects of peroxisomal disorders, such as the accumu- need for evaluation for syndromic disease, and determine lation of phytanic acid in Refsum disease, are managed when to change medications and supplements, such as with dietary modifications, while others, like the adrenal the avoidance of supplemental vitamin A in ABCA4-mac- insufficiency associated with X-linked adrenoleukodys- ular dystrophy.49 Furthermore, a genetic IRD diagnosis is ir- trophy, are treatable with medications. refutable evidence to a school district that a child needs ed- While some IRD-associated syndromes present with ucational accommodations that may otherwise be denied. dysmorphic features that signal a systemic disorder, many In addition, better insight into inheritance risk and family present only as an IRD. For example, CLN3-related neuro- planning, including planning of prenatal or pre-implantation nal ceroid lipofuscinosis, previously known as Batten dis- diagnosis, are directly dependent on an accurate genetic ease, is often only recognized after an initial diagnosis of diagnosis. a retinal dystrophy. In this syndrome, vision loss is often the first presenting symptom, and patients are frequently Genetic Testing Options: Selecting the Appropriate misdiagnosed with Stargardt disease, although ERG may Genetic Test differentiate between the two. Subsequent progressive After providing accurate clinical input to guide ge- mental and neuromotor degeneration follow, very often netic testing, a clinician’s diagnostic work is not finished. ending in early death.42 However, isolated retinal dystro- Selecting the appropriate test is the next step. However, phies due to specific mutations in CLN genes have recent- genetic testing, particularly in a genetically diverse set of ly been described, further illustrating the need for accurate diseases such as IRD, is not as straightforward as ordering genotyping. Therefore, the best method of detecting or a complete blood count (CBC). In recent years, IRD ge- ruling out systemic involvement is through early, appropri- netic testing options have expanded to include numerous ate genetic testing, as the majority of genetic mutations choices, including single-gene testing, gene-panel tests mediating syndromes have been identified. that include multiple IRD genes grouped by clinical diag- nosis, whole-exome sequencing (WES, all coding portions Genetic Testing Is Required for Accurate, Complete of DNA sequenced), and whole-genome sequencing IRD Diagnosis (WGS, all coding and non-coding DNA sequenced).1,44 As discussed, a variety of clinical diagnoses may be All of these options except single-gene testing employ used to describe a single genetic mutation (e.g., ABCA4 next-generation sequencing (NGS), the use of parallel se- and RPE65), and numerous mutations may cause a “sin- quencing of millions of short segments of DNA that are gle” clinical diagnosis (e.g., RP) due to a lack of strict then matched with a human reference genome using bio- genotype-phenotype correlations.1,12,43 For this reason, informatics. Unexpected DNA variations are then report- genetic testing is the only accurate way to make a spe- ed. NGS has revolutionized genetic testing, reducing cost cific diagnosis. American Academy of Ophthalmology and time, so that it is now possible to sequence an entire (AAO) guidelines now state that genetic testing is re- within a day.50 However, NGS testing is not quired to pinpoint the causative mutation of IRDs, there- sensitive at picking up large deletions or duplications, so by directing disease management, defining prognosis, some alleles can be missed. informing genetic counselling and family planning, and Experts recommend ordering the most specific test helping identify those for whom gene-based or other available given the patient’s clinical findings to avoid the therapies might be appropriate.44-46 However, the out- financial and emotional cost of discovering unrelated dated concept that IRDs are untreatable has presented IRD-causing mutations and so-called variants of unknown a huge obstacle for patient access to genetic testing. significance (VUS).46,51 To avoid this and simultaneously With the availability of numerous clinical trials and the control costs, some researchers have developed algo- recent approval of voretigene neparvovec,47 there is an rithms for tiered testing guided by clinical diagnosis, al- urgent need to reduce the time from onset of symptoms though this can prolong the wait for results.12,15,51 On the to genotyping in order to identify patients who can ben- other hand, in patients with clinically hard-to-distinguish efit from gene therapy treatments.48 cases, sequencing a larger set of retinal disorder-related In IRDs without current options for gene-based treat- genes can increase the chance of identifying the genetic ments, genetic testing may still direct medical manage- cause.52 This should be reserved for times when an IRD ment in meaningful ways and help to better understand expert cannot pinpoint the phenotype well enough to or- disease mechanisms in preparation for future therapy. For der a narrower panel, for an important reason: WES may

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IRD_Supp_EyeNet.indd 7 5/25/18 3:13 PM FIGURE 4 Vector: What is Gene Therapy? Carries therapeutic gene Subretinal Injection Gene Augmentation: Free RNA Nucleotides Mutation Replace No Protein Replace missing or Antisense Strand of DNA Missing Protein disabled protein 3’ 3’ Gene Editing: Correct a mutant protein by correcting a mutant gene Intravitreal Injection 5’ 5’ RNA Transcript Editing Direction of Transcription Sense Strand of DNA Decrease (Antisense oligonucleotide therapy): mRNA Bad Protein Stop production of mutant protein or correct mature mRNA

Mutant Protein

reveal several potential inherited -causing be carefully evaluated in the context of the clinical phe- mutations per patient, challenging even the most experi- notype to avoid errors in diagnosis.15,16 Results may be enced IRD specialist to make a diagnosis when interpret- inconclusive, often revealing sequence VUS that may or ing these results. Furthermore, whole-exome results may may not be pathogenic. Terminology describing these reveal other mutations with serious health implications, variants has been confusing, prompting recommenda- such as predisposition to cancers or neurodegenerative tions for classifying and labeling them.54 In these incon- disease.48 Therefore, although WES is certainly gaining clusive cases, interpretation requires a level of expertise traction as the go-to technology, interpretation of its re- that is lacking in most ophthalmologists because only a sults often requires input from an IRD expert. For the same few US board-certifi ed ophthalmologists are also board reasons, genetic counseling may be extremely helpful for certifi ed as medical geneticists by the American Board of these families. Additionally, testing may be determined Medical Genetics.16,55 While a brief CME program could by the patient’s insurance and by what the family can af- never substitute for a one- or two-year fellowship in med- ford. When comprehensive testing is fi nancially prohib- ical genetics, it is critical to recognize the sometimes-in- itive for some families in the US, initial research testing conclusive nature of genetic test results and the related may be an affordable option to help identify a gene of need to identify appropriate ocular geneticists and ge- interest, since only confi rmation testing of mutation(s) in netic counselors for consultation. Otherwise, ophthal- a single gene is required. mologists risk misdiagnosis, which can cause irreparable The vast majority of ophthalmologists do not have harm to patients. A causative mutation can be identifi ed training in choosing among these genetic testing options. in up to 60%-80% of patients with IRDs.15,44 A medical geneticist, a genetic counselor with expertise in ocular disease, and/or an IRD specialist can help deter- Genetic Counseling in IRD mine the best genetic test to use, based on current test Genetic counseling should be provided before IRD methodology, the number of genes to be tested, detection genetic testing is ordered and after results are ob- rate, price of the test, and potential insurance reimburse- tained, according to the AAO Recommendations on ment. IRD specialists are best positioned to provide this Clinical Assessment of Patients with Inherited Retinal service in concert with genetic counselors, but there are Degenerations 2016, AAO Recommendations for Ge- only 68 members of the International Society of Genetic netic Testing of Inherited Eye Diseases 2014, and oth- Eye Disease and Retinoblastoma worldwide.53 Therefore, ers.44,46,55 This can take place in the physician’s offi ce if it becomes incumbent upon every ophthalmologist and the physician employs a genetic counselor or is quali- optometrist to familiarize themselves with genetic testing fi ed to provide this service (although few are). Other- for IRDs, if only to expedite referral to one of these ex- wise, a referral to a clinical geneticist or an in-person perts. It is also important to identify genetic counselors or telephone-based genetic counselor with expertise in with the appropriate expertise to assist in managing these genetically determined eye disorders is required. Prior patients, and for genetic counselors themselves to ex- to genetic testing, genetic counselors collect detailed pand their knowledge of IRD genetic testing options. The family histories, draw pedigrees, provide genetic and Genetic Testing Registry website (https://www.ncbi.nlm. prognostic counseling, and review molecular testing nih.gov/gtr/) is a helpful resource for identifying available options, implications, and limitations with patients. Ge- genetic tests and associated laboratories for IRDs as well netic counselors review genetic variants in test reports as other genetic conditions. and research their potential contribution to disease. Following genetic testing, genetic counselors may co- Interpretation of Genetic Test Results ordinate additional testing of family members, enroll Unlike the straightforward interpretation of most lab the patient in research or patient registries and discuss tests, genetic test results—the genotype—must always clinical trials.44 They may also provide support for pa-

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IRD_Supp_EyeNet.indd 8 5/25/18 3:13 PM Vector: Carries therapeutic gene Subretinal Injection Enhancer Promoter Gene Augmentation: Free RNA Nucleotides Mutation Replace ITR No Protein Replace missing or Antisense Strand of DNA Missing Protein disabled protein Promoter Therapeutic Gene Enhancer Exon/ ITR 3’ 3’ Therapeutic Gene ITR ITR Terminator Gene Editing: Exon/Intron Terminator Enhancer Correct a mutant protein byPromoter correcting a mutantITR gene Intravitreal Injection 5’ 5’

RNA Transcript Editing Therapeutic Gene Direction of Transcription Sense Strand of DNA Decrease (Antisense oligonucleotide therapy): Exon/Intron mRNA Bad Protein Stop production of mutant protein or correct mature mRNA ITR Terminator Mutant Protein

tients who are disappointed to learn that the test results tion, antisense oligonucleotides (AON), gene silencing, are negative, meaning that treatment is not available to and genome editing. Each type of therapy has its own them currently.49 distinct mechanism, and the availability of clinical safety ITR As we have seen, most ophthalmologists are not quali- and effi cacy data is highly variable. With the wide array Promoter Therapeutic Gene Terminator fi ed to offer these complex and time-consuming services, Enhancer of gene therapy technologies being developed and the

Exon/Intron and therefore need to collaborateITR with a genetic counsel- rapid pace at which genomic medicine is advancing, it or trained in ocular genetics and/or an ocular geneticist. A is critical to understand that each genetic therapeutic, recent study showed that 92% of patients with RP desire even within the same category (e.g., gene augmenta- genetic counseling, demonstrating its value to patients.56 tion therapy), is unique. However, lack of access to a qualifi ed genetic counselor In order to understand gene therapy, a brief review due to availability or insurance coverage is a signifi cant of basic genetics is in order. DNA contains the coding barrier to proper diagnosis and management of IRDs. sequences, or genes, for every protein needed in the body, arranged in 23 pairs of found in Gene Therapy for IRD every human cell. When a protein is needed, DNA un- The ultimate purpose of genetic testing is not only accu- winds and is transcribed into messenger RNA (mRNA), rate diagnosis, prognosis, family planning, research, etc., which subsequently undergoes maturation through but also treatment. Prior to the possibility of gene thera- splicing (the cellular mechanism of cutting out non-cod- py, physicians were taught to avoid the potential harm of ing sequences called and keeping the needed ordering genetic testing for untreatable diseases. For this coding sequences, called exons). Mature mRNA then reason, the growing availability of treatment for IRDs will carries the “sense” genetic instructions to ribosomes, be the most powerful motivating factor for the widespread where the protein is produced (Figure 4). A mutation adaptation of genetic testing of these diseases. is an error in a gene, usually resulting in production of As gene mapping for IRDs has exploded, the possibility an abnormal protein that has one of two consequences: of gene therapy has become a reality. Many patients with loss of function or gain of function. Most biallelic muta- these monogenic diseases are excellent candidates for tions, in autosomal recessive conditions, result in loss of gene therapy. The most advanced gene therapy, voreti- function, either through decreased protein production gene neparvovec, has been approved by the US Food and or production of a malfunctioning protein. On the other Drug Administration (FDA) as the fi rst gene therapy for hand, heterozygous mutations in autosomal dominant an inherited disease (RPE65 mutation-associated IRD).47 disease may cause gain of function, or may interfere Numerous other gene therapies are also under develop- with the function of the normal protein encoded by the ment for IRDs such as Stargardt disease, Usher syndrome normal allele (a “dominant negative” mutation). Type 1B, RPGR-related X-linked RP, LCA, X-linked reti- Gene therapy may be used to address mutations in noschisis, choroideremia, Leber hereditary optic neurop- several ways, as shown in Figure 4: to replace the missing athy (LHON), and achromatopsia.28 As these treatments or disabled protein by supplying a normal copy of the become available in clinical trials and commercially, there gene that remains separate from the target cell’s DNA is an urgent need to reduce the time from onset of symp- (gene augmentation), to correct the genetic code for the toms to accurate diagnosis with genotyping in order to protein within the target cell’s native DNA (gene editing), identify patients who would benefi t from gene therapies. to stop production of a harmful mutant protein by pre- Before discussing gene therapy programs for individ- venting the translation of mRNA (RNA transcript editing ual IRDs, it is useful to understand the concepts of gene via AON therapy), or to correct abnormal intron splicing therapy itself. This is not a one-size-fi ts-all approach to in mature mRNA via AON. The use of AON therapy is treatment, but rather, a number of highly diverse tech- described in more detail below. nologies that address strategies for gene augmenta- While gene augmentation therapy may be com-

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IRD_Supp_EyeNet.indd 9 5/25/18 3:13 PM TABLE 2 Comparison of Recombinant Viral Vectors Used in Gene Therapy57-60

Adenovirus Adeno-Associated Virus (AAV) Lentivirus (retrovirus)

Pathogenic Low No No

Integration Into Target No No* Yes Cell Genome

Immunogenicity High Very low with subretinal delivery; Low Higher with intravitreal delivery

Infects Dividing Cells Yes Yes Yes

Infects Non-dividing Cells Yes Yes Yes (with less efficiency)

Transgene Expression Transient Prolonged Prolonged (Transient or stable) (Transient or stable)

Relative Viral Titer Very High High High

Carrying Capacity 7.5 kb 4.5-4.9 kb 8 kb

*Native AAV will integrate, but recombinant AAV rarely does

pared with administering a medication, gene editing ogy is only designed to block one single, specific mutation has been described as genome surgery.61 In this tech- at a time, so that a different mutation in the same gene nique, the defective gene is corrected directly, using causing the same IRD will not be addressed. AON therapy molecular scissors called endonucleases to cut certain is currently under study for CEP290-mediated IRD.63 sequences of DNA. The mutated gene may be cut and revised, cut and removed, or cut and replaced. Earlier An Overview of Vectors for Gene Therapy versions of gene editing have been largely replaced Regardless of the gene therapy modality used, the ther- by Clustered Regularly Interspaced Short Palindromic apeutic DNA or RNA must be delivered into the target cell. Repeats (CRISPR) technology, an adaptation of a Strep- Only AONs do not require vectors. The vehicle used to tococcus pyogenes endonuclease guided to the mu- deliver the therapeutic gene is called a vector and may be tated gene by RNA. Researchers hope to begin the first designed to target specific cells. Several viruses have been Phase 1 trial of a potential CRISPR gene-editing treat- used as vectors including adenovirus, adeno-associated ment for CEP290-mediated IRD this year.61,62 virus (AAV2, AAV8), and lentivirus. Differences among the Another potential approach to treating inherited dis- types of viral vectors include packaging capacity, stability, eases is the use of gene editing to correct a mutation in level of immune response, infectivity of dividing or non-di- a patient’s own stem cells, followed by transplantation viding cells, and DNA integration into chromosomes (in- of those cells into the affected tissue. However, this ap- tegrating) vs. remaining extrachromosomal (episomal), as proach to IRD is in very early preclinical stages.61 shown in Table 2.64 Since gene therapy is delivered to spe- The last gene therapy strategy, AON, stops the trans- cific target cells and not reproductive cells, the resulting lation of harmful, abnormal by blocking a seg- changes cannot be passed to offspring. ment or correcting intron splicing of mRNA. Recall that A vector carries the transgene (therapeutic gene) along one strand of DNA is the “sense” strand, containing the with helper plasmids. In addition, vectors may contain an actual genetic instructions for proteins, and the other is enhancer, responsible for cell-specific gene regulation, the “antisense” strand (Figure 4). During transcription, and a promoter, a coding sequence that initiates transcrip- so-called pre-messenger RNA, containing exons and in- tion of the therapeutic gene. trons and that is complementary to the antisense strand, AAV is a nonpathogenic virus incapable of causing in- is produced, so that the mRNA contains the correct sense fection in the host. In addition, it is an episomal vector, sequence. Before the latter is translated into a protein, meaning that its DNA remains separate from the host cell’s splicing ensures that introns are correctly spliced out and DNA and therefore does not disturb the normal expression all exons are combined. However, if the DNA contains a of host genes under typical conditions. For these reasons, mutation, the mRNA carries an incorrect sequence and an it is considered a safer approach than other viral vectors.64 abnormal protein will result. A small antisense RNA seg- Furthermore, AAV has a low potential to cause an immune ment, an “antisense oligonucleotide,” can be sent to bind response in the subretinal space and is durable. with the mutated sense mRNA message and either correct AAV2, the best characterized and most commonly used splicing or block protein synthesis altogether. This technol- AAV, targets the RPE cells in the eye, as well as the central

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IRD_Supp_EyeNet.indd 10 5/25/18 3:13 PM nervous system and kidneys. AAVs have a limited carrying capacity of 4.5-4.9 kb, thereby limiting the size of the gene MEANINGFUL CLINICAL TRIAL ENDPOINTS carried. While some genes, including RPE65, are appropri- MUST: ately sized for this vector, others like ABCA4 and MYO7A • Reflect the real-life effect of an IRD on functional are too large. For these larger genes, the carrying capacity vision and visual function of lentivirus (such as equine infectious anemia virus [EIAV]) • Consider the expected rate of disease progression may be more appropriate, although lentivirus has proven relative to the endpoint less efficient at transducing photoreceptor cells than RPE cells. Furthermore, lentiviruses are retroviruses, inserting themselves into host DNA randomly, with a consequent such as fundus photography, OCT, fluorescein angiogra- small risk of cancer or other serious mutations. phy, and autofluorescence, further facilitate assessment Packaging parts of large genes such as MYO7A, of efficacy and safety.64,75 In addition, the eye’s immune ABCA4, and ALMS1 into dual or even triple AAV, with privilege makes systemic circulation of the vector less inserts recombining after delivery into the target cell, is likely, reducing the risk of a harmful immune response.64 an alternative strategy that is currently being studied.65-67 Gene therapy may be delivered to the retina via intra- In addition to viral vectors, liposomal nanoparti- vitreal (IVT) injection or subretinal injection (Figure 4). IVT cle vectors are under development, with the intention injection is very familiar to retina specialists due to wide- of compacting DNA or RNA and packaging it to travel spread, frequent use of vascular endothelial growth factor across cell membranes.68 Liposomal nanoparticle vectors inhibitors in retinal vascular diseases. In adults, this is an may have the capacity to deliver genes that are too large in-office procedure, with relatively low risk of complica- to be carried by lentiviruses. tions. However, IVT injection with the most common viral Once the building blocks of the vector have been de- vectors has been shown to achieve only limited transduc- signed, gene therapy manufacturing can take place in tion efficiency, confined to cells in the inner retinal layers. cells acting as biological factories. For example, voreti- Furthermore, extra-ocular distribution and shedding of gene neparvovec is an AAV2 vector manufactured using vector after IVT injection is measurable, resulting in a sys- human-derived HEK293 (human embryonic kidney) cells. temic humoral immune response.75,76 Nevertheless, the The manufacturing process includes multiple purification ease of administration with IVT may outweigh these con- steps to reduce the number of empty capsids, maximiz- cerns, certainly if sufficient immunosuppression is applied. ing the potential for full capsids containing RPE65 to be Subretinal injection, on the other hand, delivers vector delivered to the RPE target cells.69 A surfactant is added to the outer retinal layers, including photoreceptors and at the end of the manufacturing process to help prevent RPE cells, over a localized area. Unlike IVT injection, this adherence of the product to the inside of vials or syringes. technique requires pars plana vitrectomy in an operating It is critical to note that gene therapies using the same room under retrobulbar or general anesthesia. While the gene and targeting the same tissue, even with the same or subretinal injection technique is not as ubiquitous as IVT similar vector capsid, can vary in multiple important ways, injection, it is nevertheless within the skill set of retina including helper sequences, purification of empty cap- surgeons. Risks of this technique are those of vitrectomy, sids, final formulation, dose optimization, surgical deliv- including infection, , as well as macular holes or ery procedure, and adjuvant immunomodulatory therapy. .27,77 In addition, thinning of the outer This may explain why various RPE65 gene therapy trials, nuclear layer has been reported, although this has been with different administration procedures, gene constructs, determined to be clinically nonsignificant.78 vector formulations, and/or surgical approaches, showed Subretinal injection is preferred when the outer retinal improvements in retinal function but variable durability of layers or RPE are the target, causing the minimal immune effect.64,70-74 response associated with localized vector. On the other hand, IVT injection is useful when inner retinal layers and Vector Administration wide areas of the retina are to be treated, especially when Even the best-designed, most efficient vector must retinal structural damage from underlying disease would ultimately be delivered to the appropriate target cell. In prevent successful subretinal injection, and where no systemic diseases, this may be accomplished through an major concerns over systemic shedding and off-target intravenous infusion. As with all systemic medications, the transduction are present.75 concentration of vector that ultimately reaches the target cell is limited as the treatment travels through the body’s IRD Gene Therapy Clinical Trials circulation. In contrast, the eye is an ideal site for gene Ideally, a clinical trial for any IRD treatment is designed therapy for several reasons. First, its small size and en- with endpoints measuring aspects of visual function and closed compartment require only relatively small amounts functional vision affected by the pathophysiology of of vector to achieve the necessary concentration, limiting that particular IRD. Measures of visual function, such as local toxic reactions. The eye’s transparent media allow BCVA and VF, are familiar clinical trial endpoints (Table direct visualization during examination and during surgi- 3). On the other hand, measures of functional vision, of- cal procedures. The ability to perform non-invasive tests, ten described as the ability to integrate multiple aspects

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IRD_Supp_EyeNet.indd 11 5/25/18 3:13 PM TABLE 3 Photoceptor-Associated Impairments and Visual Function Assessments

Associated Impairment Visual Function Assessment

Rods Decreased light sensitivity/night blindness • Full-field light sensitivity threshold (FST) • ERG--rod response

Poor adaptation to changing light levels Dark adaptometry

Decreased peripheral visual field Dark-adapted perimetry

Cones Poor central vision Visual acuity

Impaired color vision Specialized color vision testing

Overall cone function • Full-field light sensitivity threshold (FST) with chromatic stimuli • ERG—cone response

Decreased peripheral visual field Peripheral visual field testing

Photophobia Light discomfort test

Central Central visual field testing, including microperimetry

of visual function to conduct visually dependent activi- has been found to be too small to be a sensitive out- ties of daily living, are less often included in clinical trial come measure for ABCA4 treatment trials of 1 year’s du- design despite their importance to overall quality of life. ration.79 This may be due to a ceiling or floor effect in Examples of functional vision are reading and mobility the data. On the other hand, microperimetry has been or navigation. When evaluating clinical trial results, one identified as an important outcome measure for future must consider whether the endpoints are meaningful to ABCA4 clinical trials,80,81 and incidence of definitely de- the disease and treatment being studied. For example, creased fundus autofluorescence (DDAF) may serve asa BCVA is of interest in any ocular clinical trial. However, monitoring tool for interventional clinical trials that aim this measure of cone-mediated, foveal visual function is to slow disease progression.82 A similar natural history much more relevant to a cone, cone-rod, or macular dys- study called RUSH2A is underway for patients with Usher trophy than to a predominantly rod-mediated rod-cone syndrome type 2 caused by USH2A mutations.83 dystrophy that often leaves the macula intact until later Without an available appropriate functional vision out- stages of disease. Likewise, VF testing is typically done come measure for a rod-cone dystrophy like RPE65 muta- under photopic conditions, thereby measuring only cone tion-associated IRD, researchers in the voretigene nepar- function. As a result, the extent of rod-mediated VF con- vovec gene therapy trials developed a novel endpoint to striction affecting navigational ability at night is not mea- measure change in rod- and cone-mediated functional sured with standard VF testing. Furthermore, the clinical vision. The Multi-Luminance Mobility Test (MLMT) was manifestations of an IRD, such as very limited retinal and developed to measure functional, ambulatory vision at macular function with photophobia or nystagmus, often light levels encountered during activities of daily living.84 of congenital or early onset, may limit the ability to per- This mobility course with 12 standardized configurations form certain evaluations. One of the unique challenges in was designed to be performed by children as young as IRD clinical trial design, then, is to select measurable end- age 3. Subjects were evaluated for accuracy and speed on points that reflect the real-life visual impact of the disease. the MLMT at 7 standardized light levels, ranging from 1 Knowledge of disease progression relative to an end- to 400 lux, where 1 lux is comparable to the light level of point is also essential when selecting meaningful out- a moonless summer night or a nightlight and 400 lux is come measures. This is the founding principal behind equivalent to a brightly lit office. A normally sighted am- ProgSTAR, the largest natural history study of Stargardt bulatory person would be able to complete the course at disease, which is caused by autosomal recessive ABCA4 1 lux with no or minimal errors, while someone with RPE65 mutations. The ProgSTAR study group is dedicated to mutation-associated IRD or other rod-cone dystrophy understanding disease progression and determining would have significant challenges at the 1 to 10 lux range. the best outcome measures to accelerate evaluation of In this way, changes in MLMT performance were designed emerging treatments.5 For example, BCVA, as a measure to correlate with changes in real-world, clinically meaning- of foveal, cone-mediated function, would appear to be ful functional vision. Subjects passed at a given light level an ideal endpoint for a macular dystrophy like Stargardt if they succeeded in both accuracy and time assessments. disease, but annual change in BCVA in these patients The corresponding lux score was recorded for the lowest

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IRD_Supp_EyeNet.indd 12 5/25/18 3:13 PM FIGURE 5 Results from Phase 3 Voretigene Neparvovec Trial

A. Mean Bilateral MLMT Change Scores Over Time B. Mean Change in FST White Light Over Time

4 -4

-3 3

-2 2

-1 Bilateral change score change Bilateral 1 Change from BL for both eyes BL for from Change

OI Population 0 OI Population 0 DI Population DI Population

1 -1

BL D90 Y1 XD90 XY1/Y2 BL D90 Y1 XD90 XY1/Y2 D30 D180 XD30 XD180 XY2/Y3 D30 D180 XD30 XD180 XY2/Y3 Study visit 2 Study visit 1

Data presented as mean ± SE. For the DI group, change is Data presented as mean ± SE. log10(cd.s/m2), log 10 (candela second relative to injection baseline after year 1. per meter squared).

light level at which the subject had passed.84 In addition most 2 light levels for the intervention group reflects a gain to change in MLMT score, full-field light sensitivity (FST) of functional vision allowing independent navigation over a threshold (a test of rod photoreceptor function measuring wider range of illuminance levels encountered in daily life. the lowest illumination detectable over the entire retina) On average, the intervention group moved from passing and visual acuity (VA) were other endpoints used in vore- the MLMT at the level of 50 lux, comparable to light found tigene neparvovec clinical trials.70 Variations of MLMT are in an indoor stairwell or train station at night, to passing at currently used in several gene therapy trials. the level of 4 lux, or light associated with holiday lights or an outdoor parking lot at night. Thirteen (65%) of the 20 mITT Gene Therapy Phase 3 Clinical Trials intervention subjects passed MLMT at the lowest luminance Voretigene Neparvovec for RPE65 Mutation- level tested (1 lux) at 1 year, demonstrating the maximum Associated IRD MLMT improvement possible. By contrast, no control sub- The phase 3 open-label, randomized, controlled study jects passed MLMT at 1 lux at 1 year. A change of 1 light lev- of gene augmentation by sequential, bilateral, subretinal el in passing the MLMT was considered clinically significant, administration of voretigene neparvovec (VN) for RPE65 especially against the backdrop of the progressive nature mutation-associated IRD was the first phase 3 trial of a gene of this condition. The design of the MLMT with 7 steps of therapy for IRD.70 Biallelic RPE65 mutation is responsible predetermined light levels precluded evaluation of subjects for 8% to 16% of LCA cases, presenting with profoundly at illuminance lower than 1 lux, creating a ceiling effect that impaired vision and nystagmus in infancy, and about 1% to limited the ability to measure the true scale of improvement 3% of RP cases, presenting with nyctalopia and peripheral in some subjects. In addition, the MLMT change score does VF loss in childhood or young adulthood.23,85-89 As previ- not reflect the full extent of improvement, including the ously discussed, due to its variable phenotype, patients speed with which subjects complete the course. with RPE65 mutation-associated IRD have been given a Increased light sensitivity, as expressed by MLMT number of other clinical diagnostic labels.2 scores, was further corroborated by mean FST (white light The primary efficacy endpoint of the phase 3 VN trial [reported as log10(cd.s/m2)] averaged over both eyes) in was the change in bilateral MLMT performance (change the intervention group, which showed a rapid 1.89 log unit in lux score for the lowest passing light level) at 1 year rel- improvement by day 30 in light sensitivity that increased to ative to baseline. A positive change score indicates pass- over 2 log units by 1 year (Figure 5B).70 The control group ing the MLMT at a lower light level. Thirty-one subjects showed no meaningful change in this measure over 1 year. with biallelic RPE65 mutations comprising the intent-to- The difference of −2.11 between the ITT intervention and treat (ITT) group were randomized 2:1 to the intervention control groups was significant (P=0.0004). group and the control group, respectively. One subject Mean Goldmann VF III4e nearly doubled compared from each group withdrew prior to receiving VN, leaving with baseline after treatment with VN (Figure 6), increas- 20 intervention and 9 control subjects in the modified ing by 92% in the intervention group and decreasing by ITT (mITT) population and safety analysis populations. 16% in the control group. Mean BCVA averaged over At 1 year, the mean bilateral MLMT change score for the both eyes improved by 8.1 letters from baseline for the ITT population was 1.8 in the original intervention group intervention group and 1.6 letters for the control group, and 0.2 in the control group, a statistically significant differ- which was not statistically significant.70 ence (P=0.0013, Figure 5A).70 This average change of al- No harmful immune responses associated with VN

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IRD_Supp_EyeNet.indd 13 5/25/18 3:13 PM FIGURE 6

Representative Goldmann Visual Field III4e Before and After Treatment With VN

were observed. The most frequently reported ocular treat- ly recognized mitochondrial disease, presenting most fre- ment-emergent adverse events among phase 3 subjects quently in young men as painless, acute, profound vision were consistent with vitrectomy and the subretinal injection loss in one eye, followed within weeks or months and procedure, including transient mild ocular inflammation, sometimes years by similar manifestations in the other eye. transient elevated intraocular pressure, cataract, and intra- LHON is caused by mutations in mitochondrial genes, with operative retinal tears treated with laserpexy. One subject the most severe and common mutation occurring in the experienced loss of VA.70 ND4 gene (11778G>A). These mutations cause death of After one year, phase 3 control subjects crossed over the retinal ganglion cells, leading to optic atrophy.92 LN is a to receive treatment with VN. These delayed intervention recombinant AAV2 containing DNA encoding human mito- subjects had similar results to those of the original inter- chondrial ND4 protein that includes a mitochondrial target- vention group.90 Improvements in MLMT and FST have ing sequence. The protein is assembled in the ganglion cell remained durable for 3 years in phase 3 subjects (Figure 6), and then imported into the mitochondria. as have improvement in VF and stabilization of VA.91 Topline results from the REVERSE randomized, dou- ble-masked, sham-controlled trial, in which one eye Lenadogene Nolparvovec for ND4 Mutation- received IVT injection of LN and the contralateral eye Associated Leber Hereditary received a sham injection, were recently released.93 The Three ongoing phase 3 studies of lenadogene nolpar- primary efficacy endpoint was change in BCVA at48 vovec (LN) are investigating its efficacy and safety in sub- weeks post-injection. Treated eyes (n=37) had a mean jects with vision loss duration of ≤6 months (RESCUE trial), improvement of 11 letters as compared with baseline. >6 months to 1 year (REVERSE), and <1 year (REFLECT) Unexpectedly, untreated contralateral eyes (treated with in Leber Hereditary Optic Neuropathy (LHON) due to the a sham injection) also showed a similar improvement of 11778G>A mutation in ND4. LHON is the most common- 10 letters (Figure 7). Due to this improvement in un- treated eyes, the trial did not meet its primary endpoint. FIGURE 7 Mean change in retinal ganglion cell macular volume Mean Change from Baseline in the measured on SD-OCT from baseline demonstrated a statistically significant difference (P=0.0189) between REVERSE Lenadogene Nolparvovec Trial LN-treated eyes and sham-treated eyes, with untreated 3 Visual Acuity in All Eyes eyes losing 0.038 mm of macular ganglion cell volume 15 while the ganglion cell volume of treated eyes was es- 11 3 93 10 8 sentially unchanged (-0.003 mm ). 10 6 Mean change in thickness of the temporal quadrant and 5 4 3 2 5 papillomacular bundle of the retinal nerve fiber layer from 3 1 2 1 baseline demonstrated a statistically significant difference 0 (P=0.0359) between LN-treated eyes and sham-treated -3 -3 LN (N=37) -5 eyes, with untreated eyes showing a loss of 3.4 μm com- SHAM (N=37) 93 Data presented as mean ± SE. pared with a loss of 0.6 μm in treated eyes.

Mean Change From Baseline in ETDRS Leters Mean Change From -10 0 5 10 15 20 25 30 35 40 50 Ocular treatment-emergent adverse events among

Weeks After Administration phase 3 subjects were related to the injection procedure and to LN itself. Specifically, intraocular inflammation,

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IRD_Supp_EyeNet.indd 14 5/25/18 3:13 PM References FIGURE 8 1. Lee K, Garg S. Navigating the current landscape of clinical genetic testing for inherited 94 retinal dystrophies. Genet Med 2015;17:245-252. Gene Augmentation Clinical Trials 2. Reape KZ, Schaefer G, et al. Natural history of individuals with retinal degeneration due to biallelic mutations in the gene [poster]. Presented at the Annual Meeting of the Phase 1/2 Phase 3 Post-Approval RPE65 Association for Research in Vision and Ophthalmology, Baltimore, MD, May 7-11, 2017. RPE65 3. Valkenburg D LS, Bax N, Groenewoud J, et al. Differential progression of Stargardt disease between young infancy and late adulthood [poster]. Presented at the Annual Meeting of the ND4 Association for Research in Vision and Ophthalmology, Baltimore, MD, May 7-11, 2017. MERTK 4. Foundation Fighting Blindness. QLT Inc. completes enrolment in natural history study for RP and LCA. https://www.fightingblindness.ie/newsx/qlt-inc-completes-enrolment-in-natu- REP1 ral-history- study-for-rp-and-lca/. Accessed March 21, 2018. 5. ProgSTAR.org website. http://progstar.org/. Accessed March 21, 2018. ABCA4 6. ClinicalTrials.gov. NCT03146078. Rate of Progression in USH2A Related Retinal Degener- MYO7A ation (RUSH2A). https://clinicaltrials.gov/ct2/show/NCT03146078?term=rush2a&rank=1. Accessed March 21, 2018. CNGA3 7. Jangra D, Ganesh A, Thackray R, et al. Psychosocial adjustment to visual loss in patients with retinitis pigmentosa. Ophthalmic Genet 2007;28:25-30. CNGB3 8. Shire. Rare disease impact report: Insights from patients and the medical community 2013. https:// RPGR globalgenes.org/wp-content/uploads/2013/04/ShireReport-1.pdf. Accessed March 21, 2018. 9. National Federation of the Blind. Statistical facts about blindness in the United States. CHM https://nfb.org/blindness-statistics. 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Summaries of genes and loci causing retinal diseases. https://sph.uth.edu/retnet/ sum-dis.htm. Updated April 16, 2018. Accessed May 22, 2018. 14. Veleri S, Lazar CH, Chang B, et al. Biology and therapy of inherited retinal degenerative disease: Insights from mouse models. Dis Model Mech 2015;8:109-129. Emerging Gene Therapy Treatments 15. Stone EM, Andorf JL, Whitmore SS, et al. Clinically focused molecular investigation of 1000 Other emerging gene therapy trials are summarized consecutive families with inherited retinal disease. Ophthalmology 2017;124:1314-1331. in Figure 8. Clearly, gene therapy is on the horizon for 16. Moore T, Burton H. A needs assessment & review of specialist services for genetic eye disor- ders: Report for the United Kingdom Genetic Testing Network (PHG Foundation) 2008 http:// many IRDs, from the breakthrough of VN for RPE65 www.phgfoundation.org/documents/167_1210674534.pdf. Accessed March 21, 2018. mutation-associated IRD to the promise of LN for ND4 17. 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In most cases, optimal care will be given by an in a consanguineous family with a splice sitemutation in the MERTK gene. Br J Ophthalmol IRD specialist, aided by genetic counselors and low 2009;93(7):920-925. 33. Ksantini M, Lafont E, Bocquet B, et al. Homozygous mutation in MERTK causes severe auto- vision specialists. In this brave new world of gene somal recessive retinitis pigmentosa. Eur J Ophthalmol 2012;22(4):647-653. therapy, we can finally offer hope of improvement in 34. Online Mendelian Inheritance in Man. Leber congenital amaurosis 3; LCA3. https://www. omim.org/entry/604232. Updated May 17, 2016. Accessed May 22, 2018. visual function and functional vision to patients who 35. Iqbal M, Naeem MA, Riazuddin SA, et al. Association of pathogenic mutations in TULP1 would have otherwise become blind. with retinitis pigmentosa in consanguineous Pakistani families. Arch Ophthalmol 2011;129(10):1351–1357.

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