Materials and Methods s14

MATERIALS AND METHODS

GMP Vector Production

Crude Cell Pellets: 10 cell factories of GMP qualified HEK293 cells at 80% confluency were transfected with GLP plasmid DNA containing the AAV2-VMD2-hMERTK vector cassette and

GLP plasmid DNA containing the AAV2 capsids genes needed for packaging the hMERTK vector genome. At 72 hours post-DNA transfection the cells were harvested in individual cell factory lots, pelleted by centrifugation and frozen at -20C until all ten cell factories had been collected.

Purified Bulk Vector: Three transfected cell pellets were thawed at 37 C. Pellets were then resuspended in 20mM Tris, 150mM NaCl with 0.5% OPE Buffer. The resuspended pellets were incubated for one hour at 37°C in the presence of Benzonase (30 units/mL) and MgCl2. The cell suspension was then transferred to a bioprocess container and microfluidized. The microfluidized cell lysate was centrifuged and the supernatant was collected and transferred to a bioprocess container (HE Load). The HE Load was introduced onto a 150mL Heparin 6

Sepharose FPLC Column, washed with 20mM Tris, 150mM NaCl with 0.5% OPE Buffer, then

PBS and eluted in PBS + 200mM NaCl (HE Intermediate) and collected into a bioprocess container. The HE Intermediate was stored overnight at 2 to 8°C. The HE Intermediate was then warmed to room temperature and adjusted to 1.1M NaCl by the addition of 4M NaCl. This was loaded onto a 20mL Phenyl Sepharose FPLC Column and the flow through collected into a bioprocess container (PS Intermediate). The PS Intermediate was diluted seven-fold with WFI and loaded onto a 20mL SP FPLC Column. The SP column was washed with PBS then eluted in

PBS + 200mM NaCl (Purified Bulk). Samples were collected for bioburden testing, viral identity and purity testing and viral genome titer testing. The Purified Bulk sterile filtered then stored at -70 to -90°C. Two Purified Bulk lots were manufactured.

Vector Filtration and Concentration: Two Purified Bulk Lots were thawed overnight at 2 to 8°C.

The 300,000 MW Tangential Flow Filtration Cartridge was washed with 70% IPA, rinsed with

WFI then equilibrated in PBS + 200mM NaCl. Both Purified Bulk Lots were loaded into the filtration system then washed with 10X volume of PBS + 200mM NaCl. The final volume was reduced to 12mL. The filtered concentrated bulk was sterile filtered then stored overnight at 2 to

AAV2-VMD2-hMERTK Final Fill: The filtered concentrated bulk was warmed to room temperature then sterile filtered. An automatic pipette set to 150µL was used to fill a total of 72 vials. Vials have been stored at -70 to -90°C. Based on preliminary viral titer assays using real time PCR, each vial contains sufficient vector to treat one patient eye. It is expected that 40-45 vials will be available for this application after full GMP-compliant testing is completed.

Vector Testing: To be a GMP-compliant product suitable for human clinical testing according to the US FDA, the vector must pass a series of post-production tests. Random vials of vector were selected for:

• Sterility testing

• Endotoxin testing

• Benzonase contamination testing

• HEK293 DNA contamination testing

• Viral genome titer testing • Viral identity and purity testing

• Infectious titer testing

• Replication competent AAV testing

• DNA sequencing

• Appearance, pH and conductivity testing

• AAV2 capsid testing.

An AAV2 capsid protein gel to test for vector purity has been run and shows good purity, easily passing vector release criteria (Figure S2).

Human Subjects

This is an open-label, dose escalation, Phase I clinical trial of subretinal administration of rAAV2-VMD2-hMERTK. The goal of this trial is to assess the safety of gene transfer in subjects with MERTK-associated retinal disease. The study has been approved by the IRB committee at the King Khaled Eye Specialist Hospital (KKESH) and is registered at www.clinicaltrials.gov

NCT01482195.

The study population includes subjects who fulfilled the following criteria:

 Genetically confirmed MERTK-associated retinitis pigmentosa

 VA: 20/100 or less in the worse eye

 Good general health based on a complete physical examination and hematology and

chemistry studies performed at a pre-treatment evaluation;  Ability to comply with research procedures

The eye with worse visual function was chosen for rAAV2-VMD2-hMERTKvector administration.

Subjects were excluded from the study according to the following criteria:

 Pre-existing eye conditions that would preclude the planned surgery or interfere with

the interpretation of study endpoints or surgical complications

 Complicating systemic diseases (such as medical conditions causing

immunosuppression) that would preclude the gene transfer, ocular surgery or known

sensitivity or allergy to medications planned for use in the peri-operative period

 Use of anti-platelet agents that may alter coagulation within 7 days prior to study

agent administration

 Use of immunosuppressive medications

 Pregnancy or breastfeeding

 Individuals (males and females) of childbearing potential who are unwilling to use

effective contraception for 1 year following agent administration and barrier

contraception for 3 months following agent administration

 Any other condition that would prevent a subject from completing follow-up

examinations during the course of the study and that, in the opinion of the

investigator, makes the subject unsuitable for the study

 Current, or recent (within the past 30 days, or 10 half lives of the drug) participation,

in any other research protocol involving investigational agents or therapies

 Recent (within past 6 months) receipt of an investigational biologic therapeutic agent. In order to be eligible to participate, all women of childbearing potential must have had two negative pregnancy tests within the month prior to rAAV2-VMD2-hMERTK administration.

Candidates were asked to sign an informed consent in accordance with study protocol and IRB regulations. Children 14 years of age and older were included in this clinical trial according to specific guidelines. Prior to enrolling children, an Assent Form and a Parental Permission Form was submitted to the relevant institutional regulatory agencies for review and approval.

Study Agent

The study agent, rAAV2-VMD2-hMERTK, is a non-replicating, rep/cap-deleted, recombinant adeno-associated viral construct containing the human MERTK gene. The manufacturing site is the Powell Gene Therapy Center/Human Applications Laboratory of the University of Florida,

Gainesville, FL, a GMP level facility. The vector is formulated in BSS and sterile water for injection.

The initial protocol mandated a single subretinal injection of 5.96x1010vg in 150 µl of rAAV2-

VMD2-hMERTK. This dose is equivalent to the 0.3X dose used in preclinical studies which was not associated with acute or chronic toxicities. The 1X dose is considered to be below the “no observable adverse effect level” (NOAEL). Following injecting the initial two subjects with no safety concerns upon follow-up, the protocol was amended and the dose was increased to 450 µl for the subsequent patients (1X dose). Complete instructions for drug preparation and administration are available in the Study Operations Manual.

Surgical procedure: Subjects were asked to discontinue the use of aspirin, aspirin-containing products, and other drugs that may alter coagulation, 10 days prior to receipt of study agent after approval from their primary or ordering physician. Decisions regarding the timing of restarting these medications were made by the retinal surgeon.

The surgery was performed under peribulbar anesthetic block accompanied by monitored intravenous sedation or under general anesthesia, at the discretion of the surgeon and subject.

Standard sterile ophthalmic surgical procedures including the use of 5% povidone-iodine and a sterile lid speculum were utilized. A standard 3-port, 20-gauge pars plana vitrectomy setup was performed starting with the core vitrectomy. This was followed by triamcinolone-assisted peeling of the posterior hyaloid with or without the use of adjuvant maneuvers such as the use of a Tano scraper. Peeling the hyaloid allows unobstructed access to the posterior pole and subretinal space for drug delivery. After drawing the study agent into a 39-gauge injection cannula (Surmodics Inc., O’Fallon, MO) attached to a syringe, it was injected manually into the subretinal space at a predetermined site within the posterior pole. The predetermined injection site was selected after careful assessment of the retina including a preoperative macular optical coherence tomography such that a site adjacent to preserved residual photoreceptors and RPE and away from retinal vessels was chosen. All efforts were exerted to avoid detaching the center of the fovea with the bleb of subretinal fluid in order to avoid the occurrence of an iatrogenic macular hole. Also the injection site was chosen to be away from the center of the fovea because pre-clinical studies have shown localized retinal toxicity secondary to the retinotomy created by the injection cannula 23. The number of injection sites was tailored intraoperatively depending on the volume of study agent injected and the spread of the subretinal fluid bleb. The aim was to cover as much as possible of the macular area without detaching the center of the fovea. No air- fluid exchange or endolaser to the injection site/s was planned. Careful examination of the peripheral retina was performed to ensure the absence of any iatrogenic retinal breaks. The sclerotomy sites were secured with 7.0 vicryl sutures. At the end of the procedure, the conjunctiva was closed with interrupted vicryl sutures, subconjunctival antibiotics and steroids were administered, and the eye was covered with a patch and Fox shield over atropine ointment.

Following study agent administration, patients were admitted to the hospital for close monitoring as per protocol. They were instructed to maintain a supine position for the first two days during which only bedside assessment was performed. Starting the third postoperative day, extensive systemic and ophthalmic evaluations were performed (Please see below for details).

Study Visits and other procedures:

Table S4 outlines the schedule of planned study evaluations at KKESH. Study agent administration occurred on Day 0. Due to the extensive nature of the study procedures, an acceptable timeframe was permitted within which the remainder of the planned study evaluations can occur. These timeframes are outlined in Table S5.

Systemic evaluation:

For each subject, a detailed medical history and physical examination were obtained by an internist that is part of the study team during the first baseline visit and specific post-vector administration evaluations according to protocol. These evaluations were mainly targeted to assess for the potential occurrence of systemic adverse events. Studies obtained included a 12- lead electrocardiogram, chest X-ray, complete blood count with differential, prothrombin time with INR, partial thromboplastin time, serum electrolytes, full serum chemistries including calcium and measures of hepatocellular integrity and of renal function, as well as a urinalysis. In addition, measurements of antibody titers to AAV2 capsid components and antigen-specific reactivity (ASR, data not shown) assays were performed at baseline and during post-vector administration evaluations as markers of a systemic immune response to vector administration, as previously described23. Moreover, peripheral blood analysis by DNA PCR was performed at baseline and up to one year following vector administration to detect vector spread. Genomic

DNA (gDNA) was extracted from patient blood samples according to manufacturer’s instructions using a DNeasy blood and tissue kit (Qiagen) and quantified using a biophotometer

(Eppendorf). Real time PCR was performed on the blood gDNA using an ABI 7900HT sequence detection system (Life Technologies), as previously described, using primers and probe specific to the SV40 poly-A of the VMD2-hMERTK-pA vector cassette19.

Prescribed and over-the-counter medications used two weeks prior to baseline visits were recorded. Any changes in these medications were also recorded during each subsequent evaluation.

Ophthalmic evaluation:

A detailed ophthalmic evaluation including ophthalmic history recording (full history at baseline and abbreviated history subsequent to intervention) as well as a detailed examination including slit lamp examination, applanation tonometry, indirect ophthalmoscopy, biomicroscopy and spectral domain optical coherence tomography (SD-OCT) (Spectralis, Heidelberg, Germany) were performed at baseline and specific post-vector administration visits according to protocol.

Although candidates may have very severe loss of function, an attempt was made to measure a best-corrected visual acuity using early treatment diabetic retinopathy (ETDRS) charts, and measurements were recorded as the number of letters read on each line of the chart 24. If a patient was unable to read at least three letters of the first line correctly, the chart distance was progressively halved from the standard 4 m until either the first line was correctly read or the shortest distance of 0.5 m was reached. Regardless of test distance, visual acuity was expressed as a Snellen equivalent ranging from 20/20 or better to 20/1600. Patients who were unable to read any letters on the chart were tested for light perception and if they perceived light they were assigned the acuity score equivalent of 20/3200. Baseline lens opacities, if any, were graded using the LOCs classification system 25.

Following administration of rAAV2-VMD2-hMERTK, ophthalmic examinations primarily focused to detect any differences from baseline such as corneal abnormalities, afferent pupillary defect, signs of intraocular inflammation, cataract, and intraocular pressure changes. Fundus photographs to document the retinal findings were obtained at baseline and at post-vector administration according to protocol.

SD-OCT imaging was performed using a macular cube acquisition protocol with a scan pattern of 25 × 20° consisting of a raster of 19 horizontal line-scans with a scanning density of 512 A- scans per line and an averaging parameter of 9 times (RTA= 9) while correcting for eye movements using the proprietary TruTrack function. All line scans of each macular study were reviewed for errors in automated retinal boundary detection and those with errors where corrected manually. Following that, the center of the fovea was localized and the retinal thickness map was corrected accordingly in case of off-center imaging to yield the correct central macular (CMT) and central foveal thickness (CFT) measurements.

Additional acquisition protocols such as radial scans and higher density scans (61 line scans) were also performed in an effort to collect as much information as possible regarding the distribution and integrity of the macular photoreceptors and RPE at baseline and following therapy.

Retinal Function Measurement:

Retinal function was measured using full-field dark-adapted psychophysics following pupil dilation using tropicamide 1% and phenylephrine 2.5%. Candidates for this gene transfer safety trial were expected to have severe retina-wide dysfunction. Traditional tests of function, such as perimetry, was not utilized as a standard procedure expected since most subjects had unstable fixation often accompanied by nystagmus and was not utilized as a standard procedure. Even for patients with stable fixation, the dynamic range of traditional instruments is not sufficient for testing the severe loss of function 26. In such cases, full-field stimulus threshold testing (FST1) based on a commercially available automated perimeter (Humphrey Field Analyzer, model 750i,

Zeiss-Humphrey, Dublin, CA) can be utilized. The full-field nature of the stimulus makes the test mostly independent of fixation and it has been reported that achromatic stimuli could be detected by all patients under dark-adapted conditions including those with severe retinopathies

27. More recently this technique has been extended (FST2) so that stimuli are delivered with an unmodified colordome desktop ganzfeld and Espion console (Diagnosys LLC, Littleton, MA).

In this trial, subjects were dilated and dark-adapted (>45 min), then were positioned in front of the FST2 stimulator. Test sessions were performed in each eye while the contralateral eye was

“triple” patched with a combination of adhesive eye patch (Opticlude, Mexcare, 3M Health Care,

St. Paul, MN), black opaque photographic tape (Scotch, 3M, St. Paul, MN), and black eye patch with elastic band (Wilson Ophthalmic, Palatine, IL). Pre-testing with the white stimulus was used for explanation of the test and practice. All lights were turned off and infrared video monitoring was used throughout the session to confirm open eyelids and attentive subjects. Each uniocular test session consisted of repeated measurements of psychophysical threshold to a full- field stimulus using flashes of 200 ms duration. A short pause was given between threshold determinations to avoid fatigue. Repeat testing was performed at similar times of the day. Details on this method of evaluating visual function in individuals with severe retinal degenerations have been published 28,29. Statistical analysis of the FST data was performed as follows. Descriptive statistics for the continuous variables were reported as mean ± standard deviation. Two-way repeated measures ANOVA, followed by post-hoc tests were performed. The statistical level of significance was set at p < 0.05. The statistical analysis was performed by using SAS 9.3

(Statistical Analysis System, SAS Institute Inc., Cary, NC, USA) and Partek Genomics Suite

(Partek Inc., St. Louis, MO, USA).

Primary and secondary endpoints:

The primary endpoint in this trial is safety at 2-year follow-up that was assessed at both the ophthalmological and systemic levels. The standard ocular evaluation as well as the ophthalmological tests detailed above were utilized to assess for any ocular side effects.

Systemic toxicity was also assessed through detailed evaluations by the medical practitioner at regularly scheduled time points as well as measurement of hematology and serum chemistry parameters, assays for vector genomes, reported subject history of any symptoms and adverse events. Specific details regarding the grading of ocular and systemic side effects were developed and are available in the operating manual of the trial. Adverse events, when present, were classified as serious or not, graded, recorded, reported, treated and followed according to the detailed criteria provided in the manual of operations of the trial. Dose-limiting toxicity (DLT) was defined as any adverse event of grade 3 or higher that was possibly, probably or definitely related to the study agent. The investigators conferred with the data and safety monitoring board (DSMB) as well as the Institutional Review Board (IRB) and the Saudi Food and Drug administration (SFDA) on all grade 3 or higher adverse events, when present, that were possibly, probably, or definitely related to the study agent according to regulatory requirements.

Visual function as a secondary endpoint was quantified as detailed above utilizing ETDRS visual acuity measurements as well as FST for retinal function. Central macular and foveal thickness measurements by SD-OCT were also included in the trial as a specific secondary endpoint measure.

To ensure safety of human subjects and integrity of data in this trial, a data and safety- monitoring plan was established. A DSMB met at specific intervals according to protocol and the frequency of DSMB meetings were increased at the discretion of the investigators or at the request of Saudi FDA and other regulatory agencies, when needed.

A long-term follow-up will occur for up to 15 years after study agent administration. Only the two-year data are reported herein.

For the primary endpoint, the dose was defined to have exceeded the maximally tolerated dose

(MTD) if either of the following occurred: (a) At least two of three subjects in a cohort have

Dose Limiting Toxicity (DLT) or (b) If exactly one of the first three subjects has DLT, and DLT occurred in one of a possible two additional subjects accrued to this dose. If the dose did not exceed the MTD in this initial group of subjects, the study proceeded to dosing of other subjects using the same dose pending DSMB approval. If the first dose level exceeded the MTD, the investigators conferred with the DSMB and Saudi FDA about the possibility of studying one lower level of drug dosing in the same way.