Table A1.

Summary Trial Inclusion, Exclusion Eligibility and Endpoints.

Study Author		Bainbridge et al., 2015 (NEJM). NCT00643747		Jacobson et al., 2012 (Arch Ophthalmol). NCT00481546		Le Meur et al., 2018 (Mol Ther). NCT01496040		Russell et al., 2017 (Lancet). NCT00999609		Testa et al., 2013 (Ophthalmology). NCT00516477		Weleber et al., 2016 (Ophthalmology). NCT00749957
Principal Investigator		Study Director: Robin R Ali, PhD University College, London		Samuel G. Jacobson, MD, PhD; University of Pennsylvania		Michel WEBER, Professor; CHU Nantes		Albert M Maguire, MD (Children’s Hospital of Philadelphia) and Stephen R Russell, MD (University of Iowa).		Study Director: Clinical Director, Spark Therapeutics		J Timothy Stout, MD, PhD, MBA; Casey Eye Institute, Oregon Health & Science University
Sponsor (Academic/Industry)		University College, London; (Moorfields Eye Hospital NHS Foundation Trust; Targeted Genetics Corporation)		University of Pennsylvania		Nantes University Hospital		Spark Therapeutics		Spark Therapeutics		Applied Genetic Technologies Corporation; (Oregon Health and Science University; University of Massachusetts, Worcester.
Official title		An Open-Label Dose Escalation Study of an Adeno-associated Virus Vector (AAV2/2-hRPE65p-hRPE65) for Gene Therapy of Severe Early-Onset Retinal Degeneration		Phase I Trial of Ocular Subretinal Injection of a Recombinant Adeno-Associated Virus (rAAV2-CBSB-hRPE65) Gene Vector to Patients With Retinal Disease Due to RPE65 Mutations (Clinical Trials of Gene Therapy for Leber Congenital Amaurosis)		Prospective Monocentric Open Label Non Randomized Uncontrolled Phase I/II Clinical Gene Therapy Protocol for the Treatment of Retinal Dystrophy Caused by Defects in RPE65		A Safety and Efficacy Study in Subjects With Leber Congenital Amaurosis (LCA) Using Adeno-Associated Viral Vector to Deliver the Gene for Human RPE65 to the Retinal Pigment Epithelium (RPE) [AAV2-hRPE65v2-301]		A Phase 1 Safety Study in Subjects With Leber Congenital Amaurosis (LCA) Using Adeno-Associated Viral Vector to Deliver the Gene for Human RPE65 Into the Retinal Pigment Epithelium (RPE) [AAV2-hRPE65v2-101]		A Multiple-Site, Phase 1/2, Safety and Efficacy Trial of a Recombinant Adeno-associated Virus Vector Expressing RPE65 (rAAV2-CB-hRPE65) in Patients With Leber Congenital Amaurosis Type 2
Study design		Phase 1–2, open-label, non-randomized;		Phase 1, open-label, non-randomized;		Phase 1/2, open, non-randomized;		Phase 3, open-labelled, randomised (RCT);		Phase 1, open-label, non-randomized (3-year study);		Phase 1–2, open-label, non-randomized;
Treatment		rAAV 2/2. hRPE65p.hRPE65		rAAV2-RPE65		AAV2/4.-RPE65-RPE65		AAV2-hRPE65v2		AAV2-hRPE65v2		rAAV2-CB-hRPE65
Inclusion Criteria:	1	Clinical diagnosis of severe early-onset retinal dystrophy confirmed missense mutation(s) in RPE65	1	RPE65-associated retinal disease (two disease-causing RPE65 mutations);	1	Mutations that code for abnormal RPE65 protein	1	Willingness to adhere to protocol and long-term follow-up as evidenced by written informed consent or parental permission and subject assent (where applicable).	1	Male and female subjects of any ethnic group are eligible for participation in this study, providing they meet the following criteria:	1	Retinal disease consistent with a diagnosis of Leber congenital amaurosis and documented mutations in the RPE65 gene (including null mutations and mutations that code for abnormal RPE65 protein);
			2	Clinical diagnosis of Leber congenital amaurosis (LCA)/early-onset retinal degeneration (EORD) and of severely impaired visual and retinal function, and best corrected visual acuity of 20/40 or worse in the study eye;	2	Presence of characteristic abnormalities in fundus	2	Diagnosis of LCA due to RPE65 mutations; molecular diagnosis is to be performed, or confirmed, by a CLIA-approved laboratory.	2	Must be willing to adhere to protocol and companion protocol for long-term follow-up as evidenced by written informed consent or parental permission and subject assent.	2	At least 6 years of age;
			3	Ability to perform tests of visual and retinal function;	3	Dramatic reduction of both rods ans cones ERG responses	3	Age three years old or older.	3	Adults and children diagnosed with LCA.	3	Good general health without significant physical examination findings or clinically significant abnormal laboratory results;
			4	Visible photoreceptor layer on a standard OCT scan;	4	Low visual acuity <0.32	4	Visual acuity worse than 20/60 (both eyes) and/or visual field less than 20 degrees in any meridian as measured by a III4e isopter or equivalent (both eyes).	4	Molecular diagnosis of LCA due to RPE65 mutations (homozygotes or compound heterozygotes) by a CLIA-approved laboratory.	4	Able to perform tests of visual and retinal function;
			5	Good general health;	5	inform consent signed	5	Sufficient viable retinal cells as determined by non-invasive means, such as optical coherence tomography (OCT) and/or ophthalmoscopy. Must have either: 1) an area of retina within the posterior pole of >100 µm thickness shown on OCT; 2) ≥ 3 disc areas of retina without atrophy or pigmentary degeneration within the posterior pole; or 3) remaining visual field within 30 degrees of fixation as measured by a III4e isopter or equivalent.	5	Age eight years old or older at the time of administration.	5	Visual acuity not better than 20/60 and not worse than hand motion in both the treated eye and the fellow eye;
			6	Ability to comply with research procedures;			6	Subjects must be evaluable on mobility testing (the primary efficacy endpoint) to be eligible for the study. Evaluable is defined as: 1) The ability to perform mobility testing within the luminance range evaluated in the study. Individuals must receive an accuracy score of ≤ 1 during screening mobility testing at 400 lux or less to be eligible; individuals with an accuracy score of > 1 on all screening mobility test runs at 400 lux, or those who refuse to perform mobility testing at screening, will be excluded. 2) The inability to pass mobility testing at 1 lux. Individuals must fail screening mobility testing at 1 lux to be eligible; individuals that pass one or more screening mobility test runs at 1 lux will be excluded.	6	Visual acuity ≤ 20/160 or visual field less than 20 degrees in the eye to be injected.	6	Visible photoreceptor (outer nuclear) layer on a standard optical coherence tomography (OCT) scan;
			7	Specific for Cohorts 1, 2 and 4: 18 years of age and older;							7	Acceptable hematology, clinical chemistry and urine laboratory parameters;
			8	Specific for Cohorts 3 and 5: Between 8 and 17 years of age, inclusive.							8	For females of childbearing potential, a negative pregnancy test at screening and at baseline, and agreement to use effective contraception for 12 months after administration of rAAV2-CB-hRPE65, for sexual activity that could lead to pregnancy;
											9	For males of reproductive potential, agreement to use effective contraception for 12 months after administration of rAAV2-CB-hRPE65, for sexual activity that could lead to pregnancy
Exclusion Criteria:	1	Visual acuity in the study eye better than 6/36 Snellen	1	AAV antibody titers greater than two standard deviations above normal at baseline;	1	Patients with chronic conditions such a haematological, cardiac, renal diseases	1	Unable or unwilling to meet requirements of the study, including receiving bilateral subretinal vector administrations.	1	SUBJECTS WILL NOT BE EXCLUDED BASED ON THEIR GENDER, RACE OR ETHNICITY. Subjects who meet any of the following conditions are excluded from the clinical study: Subjects who meet any of the following conditions are excluded from the clinical study:	1	Pre-existing eye conditions that would preclude the planned surgery or interfere with interpretation of study endpoints or complications of surgery (e.g., glaucoma, corneal or lenticular opacities, or history or retinal detachment);
	2	Hypertension	2	Humoral immune deficiency as evidenced by low tetanus toxoid IgG antibody titers;	2	Patients with, within the past 6 months, a clinically significant cardiac disease or known congestive heart failure, cardiac rhytm and conduction abnormalities	2	Any prior participation in a study in which a gene therapy vector was administered.	2	Unable or unwilling to meet requirements of the study.	2	Presence of epiretinal membrane on OCT;
	3	Diabetes mellitus	3	Pre-existing eye conditions that would preclude the planned surgery or interfere with the interpretation of study endpoints or surgical complications;	3	Patients with pulmonaty dysfunction	3	Participation in a clinical study with an investigational drug in the past six months.	3		3	History of immunodeficiency or other medical conditions that might increase the risk of rAAV2-CB-hRPE65 administration;
	4	Tuberculosis	4	Complicating systemic diseases;	4	Patients with suspected rheumatoid arthritis	4	Use of retinoid compounds or precursors that could potentially interact with the biochemical activity of the RPE65 enzyme; individuals who discontinue use of these compounds for 18 months may become eligible.	4	Participation in a clinical study with an investigational drug in the past six months.	4	Use of anticoagulants or anti-platelet agents within 7 days prior to study agent administration;
	5	Renal impairment	5	Use of anti-platelet agents that may alter coagulation within 7 days prior to study agent administration;	5	Patients with current systemic infection……..	5	Prior intraocular surgery within six months.	5	Pre-existing eye conditions that would preclude the planned surgery or interfere with the interpretation of study endpoints (for example, glaucoma, corneal or lenticular opacities).	5	History of allergy or sensitivity to medications planned for use in the peri-operative period;
	6	Immunocompromise	6	Use of immunosuppressive medications;			6	Known sensitivity to medications planned for use in the peri-operative period.	6	Lack of sufficient viable retinal cells as determined by non-invasive means, such as optical coherence tomography (OCT) and/or ophthalmoscopy. Specifically, if indirect ophthalmoscopy reveals less than 1 disc area of retina which is not involved by complete retinal degeneration (indicated by geographic atrophy, thinning with tapetal sheen, or confluent intraretinal pigment migration), these eyes will be excluded. In addition, in eyes where optical coherence tomography (OCT) scans of sufficient quality can be obtained, areas of retina with thickness measurements less than 100 um, or absence of neural retina, will not be targeted for delivery of AAV2-hRPE65v2.	6	For females of childbearing potential, a positive pregnancy test at screening or baseline (within 2 days before rAAV2-CB-hRPE65 administration);
	7	Osteoporosis	7	Pregnancy or breastfeeding;			7	Pre-existing eye conditions or complicating systemic diseases that would preclude the planned surgery or interfere with the interpretation of study. Complicating systemic diseases would include those in which the disease itself, or the treatment for the disease, can alter ocular function. Examples are malignancies whose treatment could affect central nervous system function (for example: radiation treatment of the orbit; leukemia with CNS/optic nerve involvement). Subjects with diabetes or sickle cell disease would be excluded if they had any manifestation of advanced retinopathy (e.g., macular edema or proliferative changes). Also excluded would be subjects with immunodeficiency (acquired or congenital) as there could be susceptibility to opportunistic infection (such as CMV retinitis).	7	Complicating systemic diseases or clinically significant abnormal baseline laboratory values. Complicating systemic diseases would include those in which the disease itself, or the treatment for the disease, can alter ocular function. Examples are malignancies whose treatment could affect central nervous system function (for example, radiation treatment of the orbit; leukemia with CNS/optic nerve involvement). Also excluded would be subjects with immuno-compromising diseases, as there could be susceptibility to opportunistic infection (such as CMV retinitis). Subjects with diabetes or sickle cell disease would be excluded if they had any manifestation of advanced retinopathy (e.g., macular edema or proliferative changes). Subjects with juvenile rheumatoid arthritis could be excluded due to increased infection risk after surgery due to poor wound healing. Subjects who are positive for hepatitis B, C, and HIV will be excluded.	7	Females who are breast feeding;
	8	Gastric ulceration	8	Individuals (males and females) of childbearing potential who are unwilling to use effective contraception;			8	Individuals of childbearing potential who are pregnant or unwilling to use effective contraception for four months following vector administration.	8	Prior ocular surgery within six months.	8	Use of any investigational agent, or systemic corticosteroids or other immunosuppressive drug(s), within 3 months prior to enrollment;
	9	Severe affective disorder)	9	Any condition that would prevent a subject from completing follow-up examinations during the course of the study;			9	Individuals incapable of performing mobility testing (the primary efficacy endpoint) for reason other than poor vision, including physical or attentional limitations.	9	Known sensitivity to medications planned for use in the peri-operative period.	9	Prior receipt of any AAV gene therapy product;
	10	Pregnancy or lactation	10	Any condition that makes the subject unsuitable for the study;			10	Any other condition that would not allow the potential subject to complete follow-up examinations during the course of the study or, in the opinion of the investigator, makes the potential subject unsuitable for the study.	10	Individuals of childbearing potential who are pregnant or unwilling to use effective contraception for the duration of the study.	10	Any condition which leads the investigator to believe that the participant cannot comply with the protocol requirements or that may place the participant at an unacceptable risk for participation.
			11	Current, or recent participation, in any other research protocol involving investigational agents or therapies;			11	Subjects will not be excluded based on their gender, race, or ethnicity.	11	Any other condition that would not allow the potential subject to complete follow-up examinations during the course of the study and, in the opinion of the investigator, makes the potential subject unsuitable for the study.
			12	Recent receipt of an investigational biologic therapeutic agent.					12	Subjects will be excluded if immunological studies show presence of neutralizing antibodies to AAV2 above 1:1000.
Primary Outcome Measures:	1	intraocular inflammation [Time Frame: at intervals up to 12 months]	1	The primary safety endpoint in this trial is the standard ocular examination. Toxicity will also be assessed by measurement of vision, hematology and serum chemistries, assays for vector genomes, reported subject history of symptoms and adverse events. [Time Frame: 15 years]	1	The drug safety evaluation after administration [Time Frame: After administration of the gene therapy product.The patient will be folloed for the duration of the hospital stay, an average of 7 days]	1	Multi-luminance Mobility Testing (MLMT), Bilateral [Time Frame: One year (change from baseline)]; The MLMT measures changes in functional vision, as assessed by the ability to navigate a course accurately and at a reasonable pace at different levels of environmental illumination. MLMT was assessed using both eyes at 1 or more of 7 levels of illumination, ranging from 400 lux (a brightly lit office) to 1 lux (a moonless summer night). Each light level was assigned a score code ranging from 0 to 6. A higher score indicated that a subject was able to pass the MLMT at a lower light level. A score of −1 was assigned to those who could not pass MLMT at 400 lux. The MLMT of each subject was videotaped and assessed by independent graders. The MLMT score was determined by the lowest light level at which the subject was able to pass the MLMT. The MLMT score change was defined as the difference between the score at Baseline and the score at Year 1. A positive MLMT score change from Baseline to Year 1 visit indicated that the subject was able to complete the MLMT at a lower light level.	1	The primary outcome measures are safety and tolerability. Secondary outcome measure(s) include changes in visual function as measured by subjective, psychophysical tests and by objective, physiologic tests. [Time Frame: Visual function will be measured at designated intervals from baseline visits through 5 years as stated in the protocol.]	1	Number of Participants Experiencing Ocular or Non-ocular Adverse Events [Time Frame: 2 years]
Secondary Outcome Measures:	1	visual function [Time Frame: intervals up to 12 months]	1	Visual function will be quantified prior to and after vector administration in order to determine whether vector administration affects visual function. [Time Frame: 15 years]	2	Biodistribution: Urine sampling and nasal secretion will be collected at several time points after administration of the gene therapy product during all the duration of hospital stay, an average of 7 days.	1	Full-field Light Sensitivity Threshold (FST) Testing: White Light [Time Frame: One year (change from baseline)]; Measures the light sensitivity of the entire visual field by recording the luminance at which a subject reliably reports seeing the dimmest flash.			2	Participants With Changes in Visual Fields [Time Frame: 2 years]; Improvement in the central 30 degree visual field, measured by static perimetry, at one or more time points after treatment, that was greater than the limit of agreement for baseline values.
					3	Different efficacy parameters and immune parameters have to be measured to conclude on the overall amelioration of quality of life of enrolled patients [Time Frame: Between Day −120 and Day −7, Day 5, Day 14, Day 30 Day 60, Day 90, Day 120, Day 180, Day 360]	2	Multi-luminance Mobility Testing (Monocular) [Time Frame: One year (change from baseline)]; The MLMT measures changes in functional vision, as assessed by the ability to navigate a course accurately and at a reasonable pace at different levels of environmental illumination. MLMT was assessed using the first eye at 1 or more of 7 levels of illumination, ranging from 400 lux (a brightly lit office) to 1 lux (a moonless summer night). Each light level was assigned a score code ranging from 0 to 6. A higher score indicated that a subject was able to pass the MLMT at a lower light level. A score of -1 was assigned to those who could not pass MLMT at 400 lux. The MLMT of each subject was videotaped and assessed by independent graders. The MLMT score was determined by the lowest light level at which the subject was able to pass the MLMT. The MLMT score change was defined as the difference between the score at Baseline and the score at Year 1. A positive MLMT score change from Baseline to Year 1 visit indicated that the subject was able to complete the MLMT at a lower light level.			3	Participants With Changes in Best Corrected Visual Acuity [Time Frame: 2 years]; Increase in BCVA of 7 or more letters at Year 2 visit compared to average baseline value
					4	Recording global ERG (electroretinogram)	3	Visual Acuity [Time Frame: One year (change from baseline)]; Measurement of the sharpness of vision, determined by the ability to read letters on a standardized chart from a specified distance.
					5	Patient efficacy questionnaire
					6	Testing of far and near visual acuity, color vision, pupillometry, microperimetry and dark adaptation.