An Open-label Dose Escalation Study of an Adeno-associated Virus Vector (scAAV2-P1ND4v2) for Gene Therapy of Leber's Hereditary Optic Neuropathy (LHON) Caused by the G11778A Mutation in Mitochondrial DNA
Phase: PHASE1
Conditions: Leber's Hereditary Optic Neuropathy
Sponsor: Byron Lam

 
 
 
An Open-label Dose Escalation Study of an Adeno-associated Virus 
Vector (scAAV2-P1ND4v2) for Gene Therapy of Leber's Hereditary 
Optic Neuropathy (LHON) caused by the G11778 mutation in 
mitochondrial DNA. 
NCT02161380 
Date: February 9, 2019. IRB 20140248. 

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1) 
Protocol Title 
An Open-label Dose Escalation Study of an Adeno-associated Virus Vector (scAAV2-
P1ND4v2) for Gene Therapy of Leber's Hereditary Optic Neuropathy (LHON) caused 
by the G11778 mutation in mitochondrial DNA 
SHORT TITLE: Leber’s hereditary optic neuropathy (LHON): Gene Therapy 
Clinical Trial  
IND NO.: 15941 
IND SPONSOR:  John Guy, M.D. 
   - LHON: Gene Therapy Clinical Trial is registered on the Clinical      Trials.gov website 
(http://clinicaltrials.gov). Clinicaltrials.gov number: NCT02161380 
 
2) 
IRB Review History* 
N/A 
3) 
Objectives* 
Our goal is to test safety. 
The purpose of this dose-escalation study is to assess the safety and 
tolerability of scAAV2-P1ND4v2 (abbreviated as AAV-ND4) gene 
replacement therapy in subjects confirmed with the G11778A mutation in 
mtDNA responsible for Leber’s Hereditary Optic Neuropathy. Ocular and 
systemic toxicity will be assessed following vector administration to 
determine if there are adverse changes that may be associated with vector 
administration. 
This first-in-man (FIM) clinical trial will assess safety, tolerability, and 
potentially efficacy of a single intravitreal injection in patient groups 
reflecting the acute, presymptomatic and chronic stages and 
manifestation of the LHON disease.  
 
          Aim 1. To Evaluate the Safety of AAV-ND4 in subjects with long-
standing bilateral acuity loss (Group 1). Phase I will consist of an open-
label, unilateral single-dose, intravitreal injection of AAV-ND4 per subject 
in a dose-escalation study investigating the safety of four vector doses 
(Low: 1.18 x109 vg*, Medium: 5.81.x 109vg*(*based on CHOP titers as 
recommended by FDA), High: 2.40x1010 vg and Higher: 1x1011 vg) in 
subjects with molecularly confirmed G11778A-mutated mitochondrial 
DNA.  The bilateral chronic group will include patients with ≥12 months 
since onset in one eye and at least 6 months onset in the more recently 
affected eye. Both eyes must have acuity reduced to ≤ 35 letters.  If both 
eyes have ≥12 months since onset, the eye with worse visual acuity will be 

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injected. If both eyes have the same acuity the eye with longest onset will 
be injected. If one eye has <12 months and >6month since onset and the 
difference in acuity between the eyes is ≤10 letters, the eye with ≥12 
months will be injected.  If eyes have an acuity difference >10 letters, the 
eye with worse acuity will be injected. 
 
Aim 2. To Evaluate the Safety of AAV-ND4 in subjects with acute 
(<12 months) bilateral loss of visual acuity to < 35 ETDRS letters (Snellen = 
20/200) (Group 2). The eye with worse acuity will be injected. 
           Aim 3. To Evaluate the Safety of AAV-ND4 in subjects with unilateral 
loss of acuity to <35 ETDRS letters, but who have “good acuity” ≥ 70 letters 
(20/40 Snellen) in the contralateral eye (Group 3). The eye with better 
acuity will be selected for injection. 
For each group, administration of study drug will follow an adaptive plan 
to identify the maximum tolerated dose. If none of the three subjects at a 
given dose level develops a major toxicity, the next cohort will receive the 
next higher dose. We will wait a minimum of 6 weeks between injections 
of the same dose in successive subjects, and will wait until 3 months after 
the last subject is injected with this dose before moving to the cohort 
receiving the next higher dose. We will test the lowest dose first in 3 
subjects of Aim I, before moving on to testing this low dose in Aim 2, then 
lastly in Aim 3. Safety will be monitored for loss of 15 letters in Aims 1 and 
2, but a difference of 15 letters worse in the treated eyes relative to 
untreated eyes for Aim 3. If acuity, and visual field testing demonstrate no 
toxicity in Aims 1 and 2, we will proceed with Aim 3. 
The objective of this proposed clinical trial is to evaluate the safety of 
mitochondrially targeted ND4 gene therapy with the adenoassociated viral 
vector in appropriate LHON patients in a Phase I clinical trial.  If warranted 
by the results of the Phase I trial, another aim will be to obtain a 
preliminary estimate of the therapy’s efficacy. We provide a summary of 
the extensive preclinical testing in relevant rodent animal models to 
support the efficacy of the approach proposed in this clinical trial. 
The primary hypothesis being tested is there will be no systemic toxicity or loss of 
vision to No Light Perception in the injected eye.  
 
4) 
Background* 
Background and Significance 
Leber’s Hereditary Optic Neuropathy (LHON) is a maternally inherited form 
of bilateral optic neuropathy causing severe and permanent visual loss in 
young adults, usually between the second and fourth decades of life. LHON 

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usually presents as a loss of central vision that typically progresses over 
weeks without pain, until bilateral central scotomas and visual loss remain. 
Visual loss may occur in both eyes simultaneously, or in one eye with the 
fellow eye affected weeks to months later. The deterioration of vision may 
progress over several months to a maximum loss of function which 
stabilizes at visual acuities worse than 20/200. 
Characterization of the hereditary nature of LHON led early researchers to 
postulate that it was a disorder of mitochondrial inheritance. Molecular 
analysis has confirmed that the basic etiology results from a point 
mutation in mitochondrial DNA (mtDNA). Three primary mutations at 
nucleotide pair 3460, 11778, and 14484 are generally agreed to be the 
primary causes of LHON, accounting for 98% of LHON cases. All primary 
LHON mutations alter mtDNA-encoded protein subunits, which comprise 
NADH-ubiquinone oxidoreductase (complex I) of the mitochondrial 
respiratory chain. Therefore, it is generally hypothesized that a 
dysfunctional respiratory chain impairs ganglion cell function leading to 
ganglion cell layer and optic nerve damage resulting in vision loss. While 
the 
relationship 
between 
genotype 
and 
phenotype 
appears 
straightforward, the selective clinical presentation observed in LHON 
patients indicates a complex pathogenesis. Although a primary LHON 
mutation is necessary for the expression of the disease, it is unclear how 
the mutations interact with other genetic and/or epigenetic factors to 
cause tissue specific disease in only select individuals.  
To date an effective treatment for LHON has not been identified. 
Anecdotal reports of treatment efficacy have been difficult to interpret 
given the potential for spontaneous recovery in certain genotypes. 
Treatments including systemic steroids, hydroxycobalamin, cyanide 
antagonists, and naturally occurring cofactors (e.g., coenzyme Q10, 
succinate and idebenone) have not produced definitive evidence of 
treatment or prevention of visual loss. 
We have taken an alternative approach for correcting point mutations in 
mitochondrial protein-coding genes—by expressing these genes in the 
nucleus in conjunction with an N-terminal targeting sequence for 
mitochondrial import. Most mitochondrial proteins are in fact made this 
way and are transported into the organelles after their translation on 
cytoplasmic polyribosomes.  The mechanism of their import and assembly 
has been a popular research topic for over 20 years. There are two major 
obstacles to correcting mitochondrial mutations in this way.  First, 
mitochondrial translation employs a partially different genetic code than 
the nuclear-cytoplasmic system, and second, some mitochondrial proteins 
may be too hydrophobic to be imported and properly assembled outside 

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the mitochondria.  The first obstacle can be addressed by completely 
recoding mitochondrial genes so that they can be expressed on 
cytoplasmic ribosomes, an approach coined “allotopic expression”. This 
process involves using overlapping oligonucleotides to rebuild the genes in 
the universal genetic code, choosing codons that permit high-level 
translation ~ or using in situ mutagenesis to change codons that differ 
between the mitochondria and nucleus.  The second problem is more 
difficult.  
Our investigative team headed by John Guy was the first group to 
overcome the deficiency in oxidative phosphorylation of LHON cells by 
constructing a ''nuclear version" of the mitochondrial gene (ND4) then 
targeted the cytoplasmically synthesized protein to the mitochondria by 
using a targeting sequence appended to the reading frame (allotopic 
expression). When delivered to cultured cells containing a mutant ND4 
respiratory function was restored.  Recently our group showed in vivo 
allotopic ND4 expression is safe in rodents and primates suggesting it may 
be useful in LHON patients.  
In previous work, we have made major strides towards determining the 
pathogenesis and testing a treatment for LHON. First, we discovered that 
G11778A ND4 mutant cells have a severe reduction in ATP synthesis, even 
though mild reductions in complex I activity appear insufficient to induce 
disease. Since no technology existed to introduce DNA directly into 
mitochondria, we overcame this deficiency in oxidative phosphorylation 
by constructing a "nuclear version" of the mitochondrial gene then 
targeted the cytoplasmically synthesized protein to the mitochondria by 
using a targeting sequence appended to the reading frame (allotopic 
expression). When delivered to cultured cells containing a mutant ND4, 
respiratory function was restored. Next, we created a bona fide animal 
model for LHON. Using site directed mutagenesis of the nuclear version of 
ND4 we replaced the codon for arginine to that for histidine at amino acid 
340. Injection of this construct into the mouse visual system disrupted 
mitochondrial cytoarchitecture, elevated reactive oxygen species, induced 
swelling of the optic nerve head and induced apoptosis, with a progressive 
demise of ganglion cells in the retina and their axons comprising the optic 
nerve. In contrast, ocular expression of the wild-type human ND4 subunit 
appeared safe, suggesting that it may be useful for treatment of patients 
with LHON. Here by using the scientific and clinical knowledge acquired to 
date, we will begin the journey towards genetic therapy for human optic 
neuropathy and mitochondrial disease. 
In summary, LHON is a worthy target for mitochondrial gene therapy that 
we are proposing here by allotopic expression of a normal ND4 gene. While 

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other experimental approaches such as importing genes from other 
species, changing the ratio of heteroplasmy with specific restriction 
endonucleases, selecting for respiratory function or regeneration (in 
muscle), none of these techniques is directly applicable to the treatment 
of LHON caused by 100% mutated mtDNA. We propose to apply gene 
therapy to a human disease caused by mutated mtDNA. Success here may 
have implications well beyond these initially targeted patients and organ 
systems besides the eye. 
 
rAAV Human Clinical Trials: General and Specific Experience 
The NIH Genetic Modification Clinical Research Information System 
(GeMCRIS®) (http://www.gemcris.od.nih.gov/) currently lists 9 active 
clinical trial protocols involving rAAV gene transfer in a number of different 
diseases, including ophthalmic disorders. Administration of the vector is 
by a wide variety of different routes, such as aerosolized inhalation, 
intramuscular, intrahepatic, intracerebral and intraocular injections. 
Specific background for this proposed protocol includes the human rAAV 
studies performed at the University of Florida. The University of Florida 
(UF) Powell Gene Therapy Center’s core facilities and investigators have 
substantial experience in current Good Manufacturing Practice (cGMP) 
production of rAAV vectors and in the performance of Phase I clinical trials 
of rAAV vectors in humans. Among the first human trials with rAAV for any 
disease were those performed by T.R. Flotte, which began in November, 
1995, with Dr. Flotte as PI at Johns Hopkins University and continued by 
the Hopkins group and the Flotte group (later at the University of Florida), 
beginning in July,1996. The several Phase I and II clinical studies (using 
rAAV2 instilled in nose or maxillary sinus or by aerosol administration), 
completed or in progress, in cystic fibrosis (CF) show no significant vector-
related toxicity. These studies have included individuals from ages 12 to 43 
years and they have ranged in severity of their underlying CF lung disease. 
Overall, vector administration to the respiratory tract has been very well-
tolerated in these human subjects and there is clear evidence of the 
feasibility of gene transfer. There is a trend to indicate that the vector is 
biologically active for the correction of chloride secretion.  Further relevant 
background to the current proposal is the human study at the University 
of Florida using a rAAV2-CB-hRPE65 vector with intraocular administration 
to clinical subjects [56]. The production and purification protocol of the 
cGMP vector for the proposed LHON clinical trial will be essentially 
identical to that used for the currently active Phase I trial of rAAV2-CB-
hRPE65 vector with 2 differences. Here we used a self complementary AAV 
containing both positive and negative strands with 3 tyrosine to 

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phenylalanine modifications in the VP3 AAV capsid to increase the speed 
and efficiency of transgene expression. Unlike RPE 65 mutations causing 
Leber’s congenital amaurosis that have slowly progressive visual loss since 
childhood, LHON presents with acute visual loss. Therefore, speed and 
efficiency of transgene delivery are critical in LHON. 
A summary of findings from preclinical studies 
Preclinical studies of scAAV2-P1ND4v2 by intraocular delivery have 
included proof-of-principle studies in a mouse model, and safety studies 
to evaluate the potential for toxicity and the biodistribution of intravitreal 
rAAV-ND4 in normal rats and normal non-human primates. 
Proof-of-Principle Studies:  
To demonstrate the safety and efficacy of the gene therapy vector to be 
used in the Leber's hereditary optic neuropathy gene therapy (LHON) 
clinical trial, we modified the ND4 subunit of complex I in the nuclear 
genetic code for import into mitochondria.  The protein was targeted into 
the organelle by agency of a targeting sequence. The gene was packaged 
into adenoassociated viral (AAV) vectors, then vitreally injected into 
rodents, primates and ex vivo human eyes that were tested for expression 
and integration by immunohistochemistry and blue native polyacrylamide 
gel electrophoresis. Animals were followed by serial fundus photography, 
OCT, multifocal or pattern ERGs. Rescue of visual loss was tested in rodent 
eyes also injected with a mutant G11778A ND4 homologue responsible for 
most cases of LHON. FLAG-tagged human ND4 expressed in almost all 
mouse retinal ganglion cells a week after injection and it integrated into 
the rodent complex I. Furthermore, in mouse eyes also injected with a 
mutant ND4, wild-type ND4 prevented defective ATP synthesis, 
suppressed visual loss, reduced apoptosis of retinal ganglion cells and 
prevented demise of axons in the optic nerve.  ND4 injected into the ex 
vivo human eye expressed in most retinal ganglion cells. Primate(s) 
vitreally injected with untagged wild-type ND4 and followed for 3 months 
had no serious adverse reactions.  
Thus, expression of our tagged ND4 vector in the ex vivo human eye, safety 
of the test article, rescue of the LHON mouse model and the severe 
irreversible loss of retinal ganglion cells in LHON support clinical testing in 
our patients with mutated G11778A mitochondrial DNA. 
Safety Studies (Rodent): 
A GLP compliant 1-month and 3-month dose based toxicity and 
biodistribution study of scAAV2-P1ND4v2 vector delivered by intravitreal 
injection into the eyes of normal rodents was performed at the University 
of Florida, Powell Gene Therapy Center Toxicology Core Facility.  

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The test article (vector) used in this study is the same as that planned for 
clinical investigation. The study consisted of 4 experimental groups. The 
animals were injected intravitreally with either Balanced Salt Solution 
(vehicle control group) or scAAV2-P1ND4v2 at a total dose of 6.56 x 106, 
6.56 x 107 and 1.97 x 108 vector genomes. Each arm consisted of 20 
animals, which included 10 males and 10 females, half were sacrificed 30 
days post injection and the remaining half were sacrificed 90 days post 
injection (+/-3 days). The primary safety endpoint of this study was the 
histopathology examination and biodistribution determination of the 
Skeletal muscle (quadriceps), Diaphragm, Heart, Cerebrum, Lung, Spleen, 
Liver, Kidney, Pancreas, Gonads, Jejunum, Parotid Gland, Mesenteric 
Lymph node, Un-injected eye with optic nerve (PCR only) and Injected eye 
with optic nerve (histopathology only). Clinical assessments were also 
measured by CBC and serum chemistry analysis. Clinical observations, 
including body weights, were also performed. 
Intravitreal administration of vector genomes was well tolerated. There 
were no gross or microscopic findings that were related to administration 
of the test article. Daily clinical observations and weekly body weight 
measurements showed no test article-related trends.  
Minimal test-article related clinical chemistry changes were observed in 
male rats in all vector dose groups, which was characterized by transient 
increases in alkaline phosphatase activity on Day 30. No vector or 
treatment related trends were seen in the complete blood count analyses. 
Vector genomes were not present in any non-target tissues and blood. 
Safety Studies (Primate) 
A 3-month dose based toxicity and biodistribution study of scAAV2-
P1ND4v2 vector delivered by intravitreal injection into the eyes of normal 
non-human primates was performed at the Oregon National Primate 
Research Center at Oregon Health & Science University. 
The test article (vector) used in this study is the same as that planned for 
clinical investigation. Primates were injected intravitreally with either 
Balanced Salt Solution (vehicle control group) or scAAV2-P1ND4v2 at a 
total dose of 2.46 x 1010 vector genomes in one eye (group 1) or both eyes 
(group 2). Each group consisted of 3 animals. Primates were sacrificed 90-
117 days post injection. The primary safety endpoint of this study was the 
histopathology examination and biodistribution determination of brain, 
heart, lung, liver, pancreas, kidney, spleen, jejunum, gonads, skeletal 
muscle (quadriceps), and lymph nodes (mandibular, mesenteric and 
tracheobronchial).  Also evaluated by histopathology were eyes, colon, 
sternum and bone marrow.  Also evaluated for biodistribution were ocular 

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anterior chamber fluid and vitreous; specific brain structures evaluated for 
biodistribution included optic chiasm, optic tract, lateral geniculate 
nucleus, superior colliculus, visual cortex (occipital lobe), thalamus and 
cerebellum. 
All major organs and were negative (≤ 100 copies/ug genomic DNA) at the 
time of sacrifice, with the exceptions of low levels in the spleen of the 2 
vector-injected animals in group 2 and in the mandibular lymph nodes of 
one animal in each group. At sacrifice, there were no gross or microscopic 
findings that were related to administration of the test article. 
In vivo, ocular status was evaluated by retinal color fundus photography, 
retinal ocular coherence tomography (OCT) imaging and intraocular 
pressure measurements. Retinal function was also assessed by multifocal 
electroretinography. Clinical assessments included differential CBC and 
serum chemistry analysis a multiple time points.  Clinical observations, 
including body weights, were also performed. 
Intravitreal administration of vector genomes was well tolerated. Retinal 
evaluations by retinal photography and OCT showed no retinal 
abnormalities and only mild and transient post-injection vitreal changes in 
some cases. Multifocal electroretinography showed no treatment-related 
changes in retinal function.  
No test-article related clinical chemistry changes were observed in 
monkeys in both groups, and no vector or treatment related trends were 
seen in the complete blood count analyses. Daily clinical observations 
detected no test article-related findings, and body weight measurements 
were stable or gradually increasing in all animals. Assessments of ocular 
health by imaging and by clinical electrophysiology showed no test article-
related abnormalities.  
Conclusions from Safety Studies 
Studies in rats and non-human primates described above suggest that 
intravitreal administration of scAAV2-P1ND4v2 at doses of up to 2.46 x 
1010 vector genomes: 
(a) Do not lead to any acute or long term ocular pathology, 
(b) Do not lead to any systemic toxicity by histopathological analysis of all 
major organs groups, and 
(c) Do not lead to significant vector spread (migration) beyond the 
eye/optic nerve (target site) in the vector-injected eye (treatment).  
All the safety data at hand shows no observed adverse effect level of up to 
2.46 x 1010 vector genomes in primates. This suggests that translation of 

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scAAV2-P1ND4v2 to human trials should be safe. However, we plan to take 
a conservative approach to dosing in man by starting the clinical 
investigation with a vector dose equivalent to 1 log unit below this level – 
at 5.0 x 109 vector genomes. 
 
Summary of the known and potential risks and benefits to human 
subjects 
This clinical trial is the first human study of scAAV2-P1ND4v2 so the risks 
associated with vector administration are not precisely known. Based on 
our preclinical studies of scAAV2-P1ND4v2 in normal rats and normal non-
human primates there is a high likelihood that intravitreal administration 
of scAAV2-P1ND4v2 will be safe in humans. Safety of rAAV2 vectors in 
humans is also supported by previous and on-going clinical trials. Most 
relevant are studies performed at the University of Florida’s Powell Gene 
Therapy Center. These trials include several Phase I and II clinical studies 
in individuals with cystic fibrosis   with alpha-1-antitrypsin deficiency, with 
RPE65-associated retinal disease. Overall, to date, results indicate no 
significant vector-related toxicity and demonstrate the feasibility of gene 
transfer. Potential risks associated with scAAV2-P1ND4v2 and the study 
procedures are discussed below. 
Specifically associated with intravitreal-injections of scAAV2-P1ND4v2, 
there is a risk of degeneration of lens and cataract formation in the injected 
eye. Detachment of the retina is also possible. 
There is a potential risk of vector spread to other organs. In our safety 
studies, all major organs of rats and primates were essentially negative (≤ 
100 copies/ug genomic DNA) for vector. Our biodistribution results after 
scAAV2-P1ND4v2 (intravitreal) administration to rats and non-human 
primates indicate only limited vector spread outside the injected eye and 
no evidence of a vector dose-related effect. The risks of vector spread and 
germline transmission (all gonad samples were negative) are therefore 
presumed to be low in the planned human trial. 
There is a risk that subjects may develop an immune response to the 
vector. Our neutralizing antibody assay data from scAAV2-P1ND4v2 
administration in large animals suggests that the risk of adverse immune 
response in humans is minimal. It is possible, but unlikely, that scAAV2-
P1ND4v2 could interact with other viruses with which the subject comes 
in contact, forming a new virus that could produce new side effects.  
In preclinical studies, there was minimal ocular inflammation which 
subsided with time after administration; in one animal of the non-human 
primate studies. Administration of corticosteroids and antibiotics 

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following intravitreal injection of the study agent is planned for this clinical 
trial to minimize the risk of local inflammation. There is potential that 
vision of subjects could be further reduced. Based on preclinical studies in 
animal models and in normal animals, the risk of further visual impairment 
due to scAAV2-P1ND4v2 is believed to be low. 
There is a chance that the study agent could damage the DNA in the cells 
of a subject’s retina. In the unlikely event that this occurred, it could put 
the subject at risk for developing retinal tumors in the future. In animal 
and human studies to date, cancer has not developed and therefore, the 
risk is considered to be low. In our preclinical work with the AAV vector, 
the majority of vector DNA appears to persist as episomal rather than 
integrated into genomic DNA, which makes it very unlikely that 
mutagenesis and tumor development will occur. 
Intravitreal administration of scAAV2-P1ND4v2, as mentioned above, 
carries a risk of retinal cell loss from administration trauma. There is also a 
risk of cataract development that could require additional surgery. There 
is a risk of retinal tears or retinal detachment and there is the possibility 
that these could lead to worse vision. Exogenous intraocular infection 
(endophthalmitis) is rare but can occur. The most common pathogens can 
be treated but poor vision (in patients with better vision than those 
entering this study) can result from infection despite successful antibiotic 
treatment. Intraocular hemorrhage is rare in patients without bleeding 
diathesis. Vitreous hemorrhage is benign and usually resolves 
spontaneously. Choroidal or subretinal hemorrhage is usually self-limited 
and could affect vision (or potential vision) significantly. Temporary 
increases in intraocular pressure can occur following ocular/retinal 
injections and are treatable with topical or systemic medications to lower 
eye pressure. Risk of anesthesia, which is local (retrobulbar) is low. There 
is a small risk of penetration of the eye or optic nerve from retrobulbar 
injection and also the possibility of hemorrhage, which can be 
decompressed if necessary Patients have complained of feeling a burning 
or painful sensation in the injected eye after the anesthesia wears off. In 
addition to these risks, there may be other longer-term risks which are not 
known. 
In addition to these risks, there may be other longer-term risks which are 
not known. 
Benefits: Due to the initial, safety establishing nature of this investigation, 
there is no anticipated benefit to the subjects in this trial. 
Description of and Justification for the Route of Administration, Dosage, 
Dosage Regimen, and Treatment Period(s): 

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The route of administration, dosage, dosage regimen, and treatment 
period(s) proposed in this protocol is based on several considerations.  The 
preclinical studies have been performed with the clinical vector.  Only 
single injections have been made across the rodent and primate studies 
with a few exceptions.  The dose delivered has varied as different 
parameters such as transfection efficiency and toxicity were explored. 
Using naïve rodents we found that injections into uninjured ocular tissue 
are well tolerated. Doses of up to 1.97 x 108 vector genomes showed no 
observable effects of toxicity. There were no gross or microscopic findings 
that were related to administration of the vector. Daily clinical 
observations and weekly body weight measurements showed no vector-
related trends. Minimal test-article related clinical chemistry changes were 
observed in male rats in all vector dose groups, which was characterized 
by transient increases in alkaline phosphatase activity on Day 30. No vector 
or treatment related trends were seen in the complete blood count 
analyses. Vector genomes were not present in any non-target tissues and 
blood. 
In normal non-human primates, observed for up to 3-4 months post 
treatment, intravitreal administration of vector genomes (of up to 2.46 x 
1010 vector genomes) in one or both eyes was well tolerated. No ocular or 
retinal abnormalities were observed and only mild and transient post-
injection vitreal changes were observed. No vector related clinical 
chemistry changes were observed, and no vector or treatment related 
trends were seen in the complete blood count analyses. Daily clinical 
observations detected no vector-related findings, and body weight 
measurements were stable or gradually increasing in all animals.  
Assessments of ocular health by imaging and by clinical electrophysiology 
showed no vector-related abnormalities. At sacrifice, there were no gross 
or microscopic findings that were related to administration of the vector. 
Vector genomes were not present in any non-target tissues, with the 
exceptions of low levels in spleen of the 2 vector-injected animals and in 
the mandibular lymph nodes of two other animals. 
The frequent study visits and safety assessments in year’s 1-3 post-
injection and the long-term follow-up will assure substantial opportunity 
for observation for adverse events and efficacy signals. 
 
 
Use of Adeno-associated Virus (AAV) and Recombinant AAV 
AAV is a parvovirus with a 4.7 kb single-stranded DNA genome. It was 
discovered as a laboratory contaminant of adenovirus cultures and was 

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subsequently found to require adenovirus or another helper virus to 
replicate under most circumstances. At least 9 AAV serotypes (1-9) have 
been isolated and cloned currently. None of the AAV serotypes has been 
associated with any human disease. AAV serotype 2 binds to cells via a 
heparin sulfate proteoglycan receptor. Once attached, AAV entry is 
dependent upon the presence of a co-receptor, which may consist of 
either the fibroblast growth factor receptor (FGF-R) or the v-5 integrin 
molecule.  
The AAV life cycle is quite unusual. Cells infected with AAV and a helper 
virus (or another adjunctive agent, such as UV irradiation or treatment 
with genotoxic agents) will undergo productive replication of AAV prior to 
cell lysis, which is induced by the helper rather than by AAV. Human cells 
infected with AAV alone, however, will become persistently infected. This 
latency pathway results in colinear integration of AAV sequences within 
the host cell genome, within a specific site on human chromosome 19, the 
AAVS1 site. While this site is not strictly homologous to AAV, it contains 
consensus elements required for binding and nicking by the AAV Rep 
protein, a non-structural protein that is also involved in productive 
replication and in transcriptional regulation of the virus. Once AAV is 
integrated, it will remain stable within infected cells for prolonged periods 
of time, up to 100 passages.  Episomal forms of the virus may also be 
present for extended periods in some circumstances. If latently infected 
cells are subsequently infected with a helper virus, the genome will be 
excised and productive AAV replication and packaging will ensue.   
 Recombinant AAV (rAAV) vectors have been developed by replacement of 
the viral coding sequences with a transgene of interest. The ITR sequences 
must be retained in rAAV since these serve as origins for viral DNA 
replication and contain the packaging signals. The maximum packaging 
capacity of rAAV is approximately 5kb, including the ITRs, the transgene, 
its promoter, and polyadenylation signal. The full exploitation of rAAV for 
gene transfer has been limited in the past primarily by the packaging and 
purification process. In particular, contamination of rAAV vector 
preparations with wild-type AAV has been found to alter the biological 
behavior of the vector, and limitations on the titers and infectivity of the 
vectors have limited their widespread use in the past. Advances in the 
packaging and purification technology have resulted in a dramatic 
improvement in the expression levels that have been achievable in vivo.  
rAAV vectors are uniquely suitable for in vivo gene transfer for genetic and 
metabolic disorders, since they are non-toxic, highly efficient when used 
at high titers, relatively non-immunogenic, and very stable in their pattern 
of expression. The utility of rAAV vectors for in vitro and in vivo gene 

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transfer has now been well established. There appear to be important 
tissue specific differences in rAAV effects. However, rAAV vectors have 
been found to be particularly efficient for gene transfer into terminally 
differentiated cells such as neurons, myofibers, and photoreceptor cells. 
Gene transfer to the bronchial epithelium has also been demonstrated as 
has rAAV transduction of hepatocytes. Very high dosages of rAAV-CB-hAAT 
(alpha 1 antitrypsin) vectors given via the portal vein to adult mice or via 
superficial temporal vein to neonatal mice will result in long-term 
expression of high levels of hAAT. In fact, a single dose of 3x1011i.u. of 
rAAV2-CB-AAT vector given intraportal to young adult mice resulted in 
expression levels in excess of 2 mg/ml (well above the threshold for 
therapeutic effect of 0.5 to 0.8mg/ml) that were sustained for over 1 year 
without any detectable pathology. This same study showed that rAAV 
persisted primarily as high MW episomes and was not associated with 
tumorigenesis. 
We have shown that intravitreal injections of AAV serotype 2 efficiently 
transduces the cell type, retinal ganglion cells affected in LHON (Koilkonda 
et al 2014), that wild-type ND4 does not lead to ocular toxicity. In fact it 
prevents visual, RGC and optic nerve axonal loss due to the effects of an 
AAV carrying the mutant ND4 allele} 
Our group has performed specific preclinical toxicology and biodistribution 
studies with intravitreal scAAV2-P1ND4V2.  
 
5) 
Inclusion and Exclusion Criteria* 
Subjects with LHON with the G11778A mutation confirmed by genetic 
testing during screening evaluation will be classified into three (3) groups 
based on long-standing (chronic), recent onset (acute bilateral) or acute 
unilateral (presymptomatic eye) disease manifestation. 
The study population will include , males and females 15 years old or older 
diagnosed with G11778A-associated Leber’s Hereditary Optic Neuropathy. 
Inclusion Criteria 
 
1. Age15 or older  
2. Patients with LHON and the G11778A mitochondrial DNA mutation.  A previous 
CLIA certified genetic lab result showing the LHON G11778A mutation will be 
accepted for inclusion; 
3. Ability to perform tests of visual and retinal function; 
4. Ability to comply with research procedures; 
5. Able and willing to provide informed consent before undergoing any study related 
procedures. 

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6. Good general health as based on the investigator's assessment of the history, 
physical examination and laboratory testing performed at the baseline 
examination. 
Exclusion Criteria 
1. 
Unwilling or unable to give consent,  
2. 
 Unable or unlikely to return for scheduled protocol visits 
3. 
Pregnant or nursing women or unwillingness for subject with 
childbearing potential to use contraception during the first year of the 
study. 
4. 
Optic disc drusen on exam or in previous history. 
5. 
Ocular diseases or visual dysfunction conditions other than refractive 
error (e.g. amblyopia, glaucoma, etc.) in the eye selected for the 
injection. 
6. 
Previous eye surgery in the eye selected for injection. 
7. 
  Aspartate transaminase (AST)/alanine transaminase (ALT) >5.0 x upper limit 
of normal (ULN); Total bilirubin >3  x ULN; Hemoglobin < 8 g/dL; neutrophil 
count <1.0 x 109/L; or platelet count < 50 x 109/L 
a) 
   Any laboratory screening test that meets the abnormality criteria stated 
above can be repeated once between Baseline one to Baseline 2.   
8. 
Type I diabetes or the presence of diabetic retinopathy 
9. 
History of neurodegenerative conditions (e.g. multiple sclerosis, 
neuromyelitis optica, Parkinson disease) 
10. 
History of autoimmune conditions (e.g. systemic lupus 
erythematosus) 
11. 
 Systemic diseases having ocular manifestations likely to confound 
assessment of study resultsHistory of cancer within five years other 
than localized basal or squamous cell carcinoma not near the orbital 
area. Patients with a prior history of cancer will need documentation 
from their cancer specialist that the cancer was cured at least 5 years 
before study entry.  
12. 
Allergy to pupil dilating drops or narrow angles precluding safe 
dilation. 
13. 
        No Light Perception (NLP) vision in either eye. 
 
, “No special populations are to be included in this research, such as 
children 14 years old or younger, prisoners, or pregnant women.  
 
 Exclude: All subjects who are unable to consent 
 Exclude: Children 14 years old or younger 
 Exclude: Pregnant women  

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 Exclude: Prisoners   
 
6) 
Number of Subjects*  
Number of Study Centers: One (1), Bascom Palmer Eye Institute, 
University of Miami, Miami, FL. 
For each group patient recruitment will consist of 3 cohorts, until the last cohort which 
will consist of both Group I and III individuals.  
Table 1 Proposed Sample Size 
 
Low-dose 
(1.18x109 vg)*
Medium dose 
(5.81x109 vg)* 
High dose (2.40x1010 
vg) 
Higher dose 
(1x1011vg) 
  Group I 
3 
3 
3 
 
  Group II 
3 
3 
 
3 
  Group III 
3 
3 
 
 
  Group I 
    And** 
  Group III 
 
 
 
3 
 
3 
       *=based on CHOP titers as recommended by FDA 
 
As summarized above, dose escalation will follow the adaptive Phase 1 plan of 
Rubinstein and Simon (http://linus.nci.nih.gov/techreport/phaseIctd.pdf). At each 
dose, escalation will occur until 2 of 3 (or 6) injected eyes manifest the safety 
endpoint. (See reference for detailed description of dose escalation strategy) Table 2 
of the reference contains operating characteristics of this approach. Following the 
algorithm of Rubinstein and Simon with three doses, the sample size could range 
between 3 and 18 for each group.  As an example, if the true probabilities of 
noteworthy safety events in the proposed three doses were 0.05, 0.25, 0.5, 
respectively, the most likely sample sizes for each group would range between N=9 
with probability 20%, N=12 with probability 54%, or N=15, with probability 22%. These 
sample sizes could potentially be sufficient to observe a treatment effect when 
compared in a matched fashion to the uninjected eyes in groups 1 and 3. 
 
**Escalation from high dose group I to higher dose group II was previously approved. 
Since chronic bilateral group I individuals are considered at lower risk than group II, 
there are no additional safety concerns to injecting group I intermixed with group III 
individuals, after the 3rd group II injection and 3 month safety period. In this combined 
cohort, each of the 6 injections will be spaced at least 6 weeks apart. Ordering of 
participants will be determined by their presentation to the study and PI discretion, 
with the aim of ensuring injection of the more difficult to recruit group III individuals. 

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7) 
Study-Wide Recruitment Methods* N/A for Multi-center trial 
  
Number of Study Centers: One (1), Bascom Palmer Eye Institute, University of 
Miami, Miami, FL. 
 
Phase I.  A review of the LHON patients currently being seen by Dr. John Guy and 
Dr. Byron Lam with long-standing severe vision loss of 20/200 or worse, shows 
that we have 23 patients who would be eligible for the Phase 1 study in Groups 1 
and 2. We expect that the availability of a potential treatment will draw more 
patients in groups 2 and 3. Thus, we should have a sizable pool of patients for the 
Phase 1 safety study of the AAV-ND4 vector. 
Individuals who have been diagnosed with G11778A mutation associated Leber’s 
hereditary Optic Neuropathy and who are eligible based on inclusion and 
exclusion criteria will be invited to participate in this clinical trial. A complete 
physical examination and clinical chemistries will be performed at a pre-
treatment, screening evaluation to further determine eligibility. Subjects will be 
considered enrolled when they sign the Informed Consent. 
Enrollment Procedures:  
 
Eligibility will be assessed at a study team meeting prior to the baseline 2 exam. 
This meeting must include at least one MD coinvestigator in addition to the PI. Other 
participants may include clinical staff, biostatistics center staff, and the CRORS 
office.  Minutes will be recorded during these meetings and a sign in sheet will be 
kept. If the injecting retina specialist is unable to attend the assessment meeting, 
they will be asked to review and sign off on the eligibility criteria checklist prior to 
the injection. If the patient meets all inclusion and exclusion criteria a request will 
be forwarded to the research pharmacist for Study Drug dilution. 
 
 
8) 
Study Timelines* 
 The duration of an individual subject’s participation in the study:10 years 
 The duration anticipated to enroll all study subjects: 3 years 
 The estimated date for the investigators to complete this study 
(complete  primary analyses): 5 years after first patient has received 
administration of study agent. 
9)    Study Endpoints* 

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         Assessment of Primary Endpoint – Toxicity 
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 any symptoms and adverse events. 
Ocular. Routine eye examinations will be performed before and after 
vector administration, and surgeons administering the injections will be 
alert to injection administration related complications. The clinical ocular 
examinations for toxicity will include slit lamp examination, measurements 
of intraocular pressure (applanation tonometry or Tono-pen) and 
gonioscopy (as needed in cases of increased intraocular pressure to 
examine for anterior chamber abnormalities). Some potential ocular 
toxicities are included in Table 6-6 below. These are modifications of the 
Ocular/Visual Adverse Events tables of the Common Terminology Criteria 
for Adverse Events v3.0 (CTCAE), considering the LHON patient population 
and the circumstances of the intervention. Direct and indirect 
ophthalmoscopy and biomicroscopy will be the mainstay of the retinal 
examinations. Fundus photography (with infrared illumination) will also be 
taken to document fundus appearance.   
Systemic. Physical examination at regularly-scheduled time points will be 
performed by a medical practitioner to allow detection of and response to 
any acute symptoms. Complete blood count with differential, prothrombin 
time (with INR), partial thromboplastin time, serum chemistries, including 
measures of hepatocellular integrity and of renal function (including BUN 
and creatinine), and a urinalysis will be performed at baseline visit (serving 
also as a pre-injection evaluation) and on post-treatment evaluations. This 
testing will help detect toxicity in a number of organ systems. For research 
purposes measurements of neutralizing antibodies to human ND4 and AAV 
capsid components and cellular assays will be performed prior to and after 
vector administration as markers of the immune response. At each contact 
with the subject, information will be obtained on adverse events by 
specific questioning. Systemic toxicity will be graded based on the scale 
included in Table 6-6 and 6-7. 
 

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Table 6-6: Ocular toxicity scale  
Toxicity 
Grade 1 
Grade 2 
Grade 3 
Grade 4 
Keratitis  
(corneal 
inflammatio
n / corneal 
ulceration) 
Abnormal 
ophthalmologic 
changes only; 
intervention 
not indicated 
Symptomatic
; medical 
intervention 
indicated  
Symptomati
c; surgical 
intervention 
indicated 
Perforation or 
blindness 
(worse than 
baseline 
visual 
function)* 
Glaucoma 
Elevated 
intraocular 
pressure (EIOP) 
warranting 
topical agent 
for 
intervention 
EIOP 
requiring 
topical 
agents and 
oral agents 
for 
intervention
EIOP 
warranting 
operative 
intervention
EIOP 
uncontrolled, 
resulting in 
blindness 
(worse than 
baseline 
visual 
function)*  
Cataract 
Mild change 
from baseline, 
detected on 
ophthalmologic 
exam; not 
warranting 
intervention 
Moderate 
change from 
baseline, 
detected on 
exam; not 
warranting 
intervention
Severe 
change from 
baseline, 
detected on 
exam; not 
warranting 
intervention
Severe 
change 
warranting 
operative 
intervention*
Uveitis 
Mild anterior 
uveitis not 
warranting 
intervention 
Moderate 
anterior 
uveitis 
warranting 
medical 
intervention
Severe 
posterior or 
pan-uveitis 
warranting 
medical 
intervention
Severe uveitis 
warranting 
operative 
intervention 
and 
threatening 
ocular 
integrity* 
Vitreous 
hemorrhage
Mild 
hemorrhage 
detected on 
ophthalmologic 
exam; not 
warranting 
intervention 
Moderate 
hemorrhage 
not 
warranting 
intervention
Severe 
hemorrhage  
warranting 
vitrectomy 
Uncontrolled 
vitreous 
hemorrhage, 
threatening 
ocular 
integrity 

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Table 6-6: Ocular toxicity scale  
Toxicity 
Grade 1 
Grade 2 
Grade 3 
Grade 4 
Retinal 
detachment
Localized and 
intervention 
not indicated 
Not resolving 
but 
intervention 
not yet 
indicated 
Operative 
intervention 
indicated 
Operative 
failure 
threatening 
ocular 
integrity* 
*  
  For group III, due to the natural history of LHON where visual loss is expected in the 
asymptomatic eye, a vision-related SAE will be considered a decrease in acuity in the 
injected eye to 0.3 logMAR units (15-letter equivalent) worse than the worst acuity 
measured in the uninjected fellow eye at the current or any prior study visit. 
  
 
 
 
 
 
 
 
Table 6-7: Systemic toxicity scale 
Grade
Definition 
1 
Mild toxicity, usually transient, requiring no special treatment and 
generally not interfering with usual daily activities. 
2 
Moderate toxicity which may be ameliorated by simple 
therapeutic maneuvers, and impairs usual activities. 
3 
Severe toxicity which requires therapeutic intervention and 
interrupts usual activities.  Hospitalization may or may not be 
required. 
4 
Life-threatening toxicity which requires hospitalization. 
 
Evaluation of Secondary Endpoint – Visual Function Changes 
Visual function will be quantified prior to and after vector administration 
in order to determine whether vector administration affects visual 

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function. The main measure of visual function will include best corrected 
ETDRS visual acuity. 
 
10)    Procedures Involved* 
Method of Assigning Subjects to Treatment Groups: 
Subjects enrolled in the study will be assigned to disease groups based on 
disease manifestation and screening evaluation. All subjects in the study 
will receive active treatment (one injection of scAAV2-P1ND4v2) at the 
Low, Medium or High dose as explained in section 6, Number of Subjects. 
Study Visits:  
Table 6-4 outlines the schedule of planned study evaluations. Subjects will 
be enrolled and undergo baseline assessments prior to Day 0.  Study agent 
administration which will occur on Day 0 and details of the procedure are 
included below. 
Table 6-4 Study Visit Summary Procedures 
Time point 
Baseline 
Day  
Year 
B1 
B2 
0 
▼
1 
7 
3 
0 
6 
0 
9 
0 
1 
8 
0 
2 
7 
0 
3 
6 
5 
1.5 
2 
3 
4 -10 
Informed consent/assent 
■  
 
 
 
 
■ 
 
 
 
 
■ 
 
 
 
 
■ 
 
 
 
 
 
■
 
Blood test for G11778A 
mutation 
■  
History and physical exam
■ 
Electrocardiogram, chest 
X-ray 
■  
 
 
 
 
 
 
 
 
 
Pregnancy test
■ 
■   
 
 
 
 
 
 
 
 
Vital signs monitoring 
 
■ 
■ 
■  
 
 
 
 
 
 
Hematology, chemistry, 
urinalysis 
■  
■ 
■ 
■ 
■  
■  
 
■
■
■ 
Coagulation Panel
 
■ 
 
 
 
 
 
 
 
 
AAV antibody and
 
 
 
 
 
 
 
 
 
 
 
specific reactivity (ASR)
■
■
■
■
measurement
Peripheral blood PCR 
■+  
 
■+ ■+  
 
 
 
 
 
Visual Acuity 
■ 
■ 
■ 
■ 
■ 
■ ■ ■ ■ ■ ■
■ 
■
■ 
Perimetry
■ 
■ 
 
■ 
■ 
■ ■ ■ ■ ■ ■
■ 
■
■ 
PERG
■ 
■ 
 
■ 
■ 
■ ■ ■ ■ ■ ■
■ 
■
■ 
Neuro-ophthalmic Exam
■ 
■ 
■ 
■ 
■ 
■ ■ ■ ■ ■ ■
■ 
■
■ 
OCT Imaging
■ 
■  
 
■  
■  
 
 
■ 
■
■ 

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Fundus photography
■  
 
■ 
■ 
■ ■  
 
 
■ 
■
■ 
Quality of life questions 
(NEI VFQ-25) 
■  
 
 
 
 
■  
■  
■ 
■ 
 
■
 
■ 
 
Adverse event recording
■ 
■ 
■ 
■ 
■ 
■ ■ ■ ■ ■ ■
Long-term follow-up
 
 
■ 
■ 
 
        Baseline visits are no more than 1 month before day 0, date of vector 
administration;  
           + testing will continue until two consecutive samples test negative 
 
Detailed Study Procedures 
Informed Consent 
The use of the informed consent process will ensure that subjects are 
adequately informed about the study procedures and associated risks and 
benefits. An investigator will also provide complete explanation of the 
study to potential participants and will ensure that all questions are 
answered prior to decisions about participation. Participation in this 
clinical trial is completely voluntary and great care will be taken to ensure 
that individuals do not feel coerced to participate. Subjects will be able to 
terminate their participation at any time without prejudice. In order to 
assess the voluntary nature of participation in the study, a decision 
monitor will speak with all participants prior to beginning the study 
procedures.  
Complete History 
A detailed medical history will be obtained from each subject during the 
first baseline visit to ensure that exclusion from participation is not 
indicated.  
Physical Examination 
During baseline and post-vector administration evaluations, physical 
examinations will be performed. These exams will assess occurrence of 
systemic adverse events.  
Electrocardiogram, Chest X-ray  
12-lead electrocardiogram and chest X-ray will be performed at a baseline 
visit as part of the pre-vector administration evaluation. 
Hematology, Chemistries, Urinalysis, Coagulation Panel  
Complete blood count, with platelet count, serum chemistries including 
BUN, creatinine, electrolytes, liver function and coagulation panel tests 

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will be obtained. Blood samples will be collected at baseline visit (serving 
also as a pre-vector administration evaluation) and during post-vector 
administration evaluations. This testing will help detect toxicity in a 
number of organ systems. In order to be eligible to participate, all women 
of childbearing potential must have two negative pregnancy tests within 
the month prior to study agent administration. 
Vital Signs Monitoring 
Following vector administration, subjects will be closely observed and vital 
signs (e.g. blood pressure, pulse, respiration, and temperature) will be 
monitored. Subjects will be discharged after IOP is confirmed to be less 
than 30 mm. 
AAV Antibody and Antigen-specific Reactivity (ASR) Measurement (For 
Research Only) 
Measurements of neutralizing antibody titers to AAV2 capsid components 
and ASR assays will be performed at a baseline visit and during post-vector 
administration evaluations as markers of any systemic immune response 
to vector administration. 
Human ND4 Antibody (For Information Only) 
Currently, there is no human ND4 antibody available for detection of 
transgene expression. In preclinical studies, we used the FLAG tagged ND4 
for immunodetection. However, FLAG is not included in the clinical vector. 
Therefore, on a For Information Only basis, detection of antibodies against 
the human ND4 transgene will be attempted at a baseline visit and during 
post-vector administration evaluations as markers of a systemic immune 
response to the transgene ND4. Serum will be reacted against Western 
blots of human mitochondrial proteins before and after administration of 
scAAV2-P1ND4v2. Bands within this size range of P1ND4v2 (approximately 
50 kilodaltons) that develop post treatment will be excised and submitted 
for identification by mass spectroscopy to determine if they are against 
human ND4.  
Peripheral Blood Q-PCR (For Research Only) 
To detect vector spread, peripheral blood analysis for vector DNA by Q-
PCR will be performed at a baseline visit and following vector 
administration. If positive (>100 vg) testing will continue until two 
consecutive samples test negative. 
 Visual Acuity 
Visual acuity is an important outcome variable.  Visual acuity is measured 
before pupil dilation or any other technique that could affect vision. 

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Refraction is performed prior to formal measurement of ETDRS visual 
acuity testing at all visits. 
Subjective Refraction: 
The use of a phoropter or trial frame is permitted to determine best-
corrected visual acuity.  The left eye is occluded first.  An approximate 
beginning refraction may be determined by retinoscopy, automated 
refraction, or a subjective refraction from a prior visit.  The sphere is 
refined first.  The cylinder is then refined, first the axis followed by the 
power.  Finally, the sphere is rechecked.  The right eye is then occluded, 
and the procedure is repeated for the left eye. 
If the patient wears contact lenses and has glasses also, he or she is 
instructed not to wear the contact lenses on the day of the examination.  
In the event that the patient either has no glasses or has forgotten the 
instructions and reported for the assessment wearing contact lenses, the 
contact lenses are removed and at least thirty minutes allowed to elapse 
before subjective refraction and visual acuity testing is performed. 
ETDRS Visual Acuity: 
The logmar visual acuity testing has been adapted from the Early 
Treatment of Diabetic Retinopathy (ETDRS).  The logmar visual acuity scale 
facilitates statistical analysis and simplifies quantification of acuity at 
various distances.  The right eye is tested with ETDRS logmar chart 1, and 
then the left eye is tested with ETDRS logmar chart 2.  Each chart is hidden 
from view until the eye being examined is ready for testing.   
The room illumination should be at a level of 50 to 100 foot candles, and 
between 50 and 125 foot candles should illuminate the ETDRS visual acuity 
chart.  The distance from the patient’s eye to the visual acuity chart is 
exactly 4.0 meters.  The patient may sit or stand, but he or she is not 
allowed to lean forward or backward so a constant testing distance is 
maintained.  After proper instruction, refraction, and placement of the 
appropriate lenses in a trial frame, the left eye is occluded and testing is 
begun with the right eye.  The patient is instructed that the chart has 
letters only and no numbers.  If the patient forgets this information and 
reads a number, he or she is reminded that the chart contains only letters 
and the examiner requests a letter in lieu of the number.  Each letter that 
is identified correctly is circled on the ETDRS Visual Acuity Worksheet.  The 
patient is advised to read slowly, so as to achieve the best identification of 
each letter.  When the patient says he or she cannot read a letter, he or 
she is encouraged to guess.  The patient should be encouraged to fix 
eccentrically if this improves the visual acuity, but care must be taken to 
ensure that the fellow eye remains covered. 

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Eyes reading fewer than 20 letters correctly at a test distance of 4.0 meters 
are tested at 1.0 meter.  Before testing at 1.0 meter, +0.75 sphere is added 
to the 4.0 meter correction already in the trial frame to compensate for 
the closer testing distance.  The patient is asked to read only the first six 
lines at 1.0 meter, so the maximum score attainable at that distance is 30.  
Correctly identified letters are circled on the ETDRS Visual Acuity 
Worksheet.  If the patient’s visual acuity is so poor that he or she cannot 
read the largest letter at 1.0 meter, assess his or her ability to count 
fingers.  After testing of the right eye is completed, chart 1 is replaced by 
chart 2 and the procedure is repeated for the left eye. 
Each letter read correctly and circled on the ETDRS Visual Acuity 
Worksheet is scored as one point.  The score for each line (which is zero if 
no letters were read correctly) and the total score is recorded after testing 
is completed.  If testing at 1.0 meter is not required (i.e. 20 or more letters 
were seen with testing at 4.0 meters), 30 points are automatically scored 
for the 1.0 meter test.  The total score, equaling the sum of the 4.0 meter 
and 1.0 meter scores, is recorded on the data form.   
Testing for Finger Counting: 
After proper instruction and refraction, the examiner’s hand is viewed at a 
distance of two feet from the patient’s eye.  The fellow eye is closed and 
completely occluded by the palm of the patient’s or assistant’s hand.  The 
examiner presents a random number of fingers to the patient.  The patient 
is asked to indicate the number of fingers seen.  If the number of fingers 
shown is correctly identified on four or more of five presentations, vision 
is recorded as count fingers. If the number of fingers presented cannot be 
identified on four or more of five presentations, test for hand motions. 
Testing for Hand Motions: 
In testing for hand motion, the examiner’s hand is viewed with all fingers 
extended and separated at a distance of two feet from the patient’s eye.  
The fellow eye is closed and completely occluded by the palm of the 
patient’s or assistant’s hand.  The patient’s glasses are not to be worn.  The 
examiner’s hand is presented in a random order under three conditions:  
stationary, moving back and forth horizontally, and moving up and down 
vertically.  The speed of movement is approximately one complete cycle of 
movement (up and down or back and forth) per second.  The patient is 
instructed that the examiner’s hand will be presented in one of these 
conditions.  He or she is asked to respond to the question, “what is my 
hand doing now?” with either, “still”, “back and forth”, or “up and down”.  
The process is repeated five times.  It is considered a correct response if 
the patient states the hand is still or he or she cannot see it while it is 

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stationary, and he or she is able to recognize movement and identify its 
direction.  If hand motions are correctly identified on four or more of five 
presentations, vision is recorded as hand motions.  If hand motions cannot 
be identified on four or more of five presentations, test for light 
perception. 
Testing for Light Perception: 
Light perception is tested using the same complete occlusion of the fellow 
eye with no other bright lights visible from the patient’s position.  The 
patient’s glasses are not worn.  The light of an indirect ophthalmoscope is 
directed into the eye from a distance of 2 feet for one or two seconds, and 
then turned away.  The patient is asked to report “on” when he or she sees 
the light, and “off” when it disappears.  The process is repeated five times 
in a nonrhythmic fashion.  The visual acuity is recorded as light perception 
if the patient responds correctly four or more out of five times. 
Perimetry 
Visual field assessment is an important outcome measure.  Quantitative 
automated perimetry is performed using the Humphrey Field Analyzer.  
Visual field testing is performed before any technique that could affect 
vision.  A visual field should be attempted in any eye that has sufficient 
vision to permit finger counting at two feet.  Eyes with poor central vision 
may have an intact, off-center island of vision which may be measured with 
perimetry. 
A 30-2 threshold test is performed in all patients using a size III white 
stimulus.  Visual field testing is performed with the standard Swedish 
Interactive Thresholding Algorithm (SITA) threshold strategy; the same 
testing strategy must be used throughout the duration of the study.  The 
pupil diameter should be 3 mm or greater before visual field testing is 
undertaken, and this may require pharmacologic dilation.  Standardized 
refraction is performed to determine the patient’s distance refraction and 
best-corrected visual acuity prior to visual field testing.  The age 
appropriate plus lens is added to the distance refraction.  Patient 
education is provided, and the instrument is set up for the test.  The 
technician should monitor fixation and patient responses during testing. 
Two HVF 30-2 SITA STANDARD will be done at baseline with a minimum of 
30 minutes to 4 days between tests. 
 Pattern Electroretinography (PERG) 
The pattern electroretinogram (PERG) is a special kind of ERG that is 
generated when a high-contrast patterned stimulus (alternating stripes or 
checkerboards) is viewed on a TV monitor. The primary generators of the 
PERG are retinal ganglion cell (RGC), whereas the traditional flash ERG is 

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generated in the outer retina. The PERG ─ but not the ERG ─ is altered in 
conditions that impair RGC function, and is abolished when RGC are 
degenerated. Therefore, the PERG has been extensively employed for 
detection and monitoring of RGC function in glaucoma and optic nerve 
diseases. Recently, a user-friendly protocol  for PERG recording (PERGLA) 
has been developed at the Bascom Palmer Eye Institute,   and it has been 
implemented in a commercially available instrument (Lace Elettronica, 
Italy). As this protocol uses skin electrodes, is fast and reproducible, it has 
been rapidly adopted by many investigators for longitudinal studies ─ 
including LHON  ─ and for testing the effect of treatments. In this proposal 
we will use the PERGLA protocol. 
A small patch (approximately 1 cm2) of the skin of the central forehead, of 
the temples, and lower eyelids is gently cleansed with electrode skin 
preoperative preparation pads (containing 70% isopropyl alcohol and 
pumice) to reduce skin impedance at less than 5000 Ohm. Five skin 
electrode cups (9 mm diameter) are filled with conductive jelly and taped 
(surgical tape, 1.5 × 1.5 cm) over the cleansed skin. The central forehead 
electrode is placed 3 cm above the bridge of the nose. The temporal 
electrodes are placed 3 cm lateral to the lateral canthus. For lower-eyelid 
electrodes, the upper margin of the electrode is kept 5 mm below 
eyelashes. The subject leans his or her chin and forehead over a chin rest 
placed in front of a television display, placed at 30 cm distance from the 
subject’s eyes. To record the PERG, the subject fixates with both eyes on a 
target at the center of the pattern display. The television display is 
contained within a Ganzfeld bowl to provide a fixed amount of background 
adaptation (4 cd/m2). The pupils are undilated.  
Neuro-ophthalmic Examination 
Ocular Motility: 
The range of duction of each eye is assessment in the 9 cardinal gazes. 
Alternate cover test is used to detect ocular misalignment in the primary 
gaze if no duction defects are detected. If duction is impaired in one or 
both eyes, alternate cover test is also performed in the cardinal gazes. 
Measurements of Intraocular Pressure: 
Measurement of intraocular pressure will be done by either the Tono-pen 
instrument or by Goldmann applanation. The Tono-pen has a self-
calibration program. If it fails to calibrate another instrument that does will 
be used. The calibration of the Goldmann applanation tonometer is 
checked every 3 months, as described in the Haag-Streit Goldmann 
Applanation Tonometer Operator’s Manual.    The intraocular pressure of 
the right eye is always tested first.  Following instillation of a drop of 0.5% 

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proparacaine, a fluorescein strip is placed near the lateral canthus in the 
lower conjunctival sac.  Once the lacrimal fluid has been sufficiently 
colored, the fluorescein strip is removed.  Alternatively, one drop of 
premixed fluorescein and anesthetic may be instilled.  The patient’s head 
is properly positioned in the chin rest and against the forehead rest 
without leaning forward or straining.  Any tight-fitting neckwear is 
loosened.  The patient is asked to look straight ahead at a distant object or 
fixation target.  If it is necessary to hold the eyelids open, the investigator 
holds the eyelids open against the orbital rim taking care not to apply any 
pressure on the globe.  The patient is instructed not to hold his or her 
breath.  If corneal astigmatism is greater than 3.0 diopters, the prism is 
rotated so that the axis of the minus cylinder on the prism graduation 
corresponds to the red mark on the prism holder.  The investigator looks 
through the slit lamp and gently brings the tip of the prism in contact with 
the center of the cornea.  The mires should be well focused, centered 
horizontally, and positioned vertically so that they are of equal 
circumference above and below the horizontal dividing line.  If the mires 
are narrower than approximately one tenth their diameter, the 
investigator instills additional fluorescein.  The investigator adjusts the 
measuring drum until the inner borders of the two mires just touch each 
other.  If pulsation is present, the measuring drum is adjusted until the 
mires separate a given distance during systole and overlap the same 
distance during diastole.  The investigator removes the prism from the 
cornea and records the measurement.  After testing of the right eye is 
complete, testing of the left eye follows the same technique. 
Biomicroscopy 
Examination of the anterior segment using slit lamp biomicroscopy is 
performed at the Qualifying Assessment to document the status of the eye 
before therapy, and at all follow-up examinations to detect any changes in 
ocular status during the course of the study which may be attributable to 
the disease or treatment.  Slit lamp biomicroscopy may be performed with 
any commercially available instrument, and it is used in a standard fashion 
starting anteriorly and working posteriorly.  Standardizing subjective 
grading of lenticular opacities is difficult.  However, it is expected that 
subjective grading by each investigator is relatively reproducible.   
Lids: 
The lids are examined for signs of blepharitis, including scales and crusting 
of the lashes, inspissated Meibomian glands, and erythema of the lid 
margin.   
Conjunctiva: 

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The conjunctiva is examined for scarring from prior trauma or cicatrizing 
disease (e.g. Stevens Johnson syndrome, ocular pemphigoid).  The 
presence and extent of conjunctival scarring may be evaluated using a 
cotton tipped applicator to test the mobility of the conjunctiva.   
Cornea: 
The cornea is examined  to evaluate the epithelium, stroma, and 
endothelium.  The techniques of diffuse illumination, scleral scatter, and 
retroillumination may be used.  The presence of corneal epithelial or 
stromal edema is noted.   
Anterior Chamber: 
Before fluorescein instillation or pupillary dilation, the degree of anterior 
chamber cell and flare is determined.  Eyes with chronic or recurrent 
uveitis are excluded from the study.  Careful assessment of the anterior 
chamber depth is made after therapy.  If the anterior chamber is shallow, 
the central anterior chamber depth is measured relative to the corneal 
thickness.  The appropriate gradation of > 3 CT, > 2 CT, > 1 CT, < 1 CT, or 
lens-cornea touch is documented.   
Iris: 
Before pupillary dilation, the pupillary iris is examined at high 
magnification for the presence of neovascularization.  If rubeosis iridis is 
present, the eye is ineligible for the study. 
Lens: 
After pupillary dilation, the investigator assesses the lens and grades any 
cataract present as mild, moderate, or severe. 
Fundoscopic Examination: 
After pupil dilation with appropriate mydriatics, the vitreous will be 
assessed for determination of cell and flare and posterior vitreous 
detachment. The optic nerve and posterior pole are examined at the slit 
lamp using a Volk 90 diopter, 78 diopter, or 60 diopter lens.  A head-
mounted indirect ophthalmoscope and hand held condensing lens (20 
diopter or 28 diopter Nikon aspheric lens) is used to evaluate the retinal 
periphery. 
   Ocular Coherence Tomography (OCT) 
Optical Coherence Tomography (OCT) is a noninvasive imaging technique 
that can be used to measure the thickness of retinal nerve fiber layer 
(RNFL). The pupils should be dilated to help insure optimal quality scans. 
OCT should be performed after all other visual function testing. A spectral-
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used to obtain circular peripapillary scans (Fast RNFL protocol). This will 
include three 3.4 mm diameter retinal scans that will be averaged to 
provide the RNFL thickness at 256 points spaced along the circumference 
of the circular scan in each eye after dilation. The software provided with 
the instrument calculates average NFL thickness and compares it to a 
database of age-matched control subjects, for patients 15 years of age or 
older giving a percentile score relative to this control database. The 
average and sector (divided into nasal, temporal, inferior, and superior 
quadrants) measurements obtained for each patient’s affected and 
unaffected eye will be considered abnormal if ranked below the 5th 
percentile. OCT testing  will be performed by a trained technician. Macular 
OCT will be performed to assess for the presence of any cystoid macular 
edema or macular alteration.  Macular OCT also provides RNFL data in the 
macular region. 
The patient will be seated at the instrument and his/her chin is positioned 
on the chinrest. The following scans will be acquired on each eye: at least 
one acceptable 200x200 macular cube and one acceptable 200x200 optic 
disc cube. Each scan will be repeated until 2 similar images are obtained 
and are deemed acceptable. 
The right eye will be imaged first. 
The operator will choose the type of scan to be performed from the 
available menu at the top of the screen. The patient will be asked to fixate 
on the internal fixation target. In cases of poor central vision, the patient 
may be unable to fixate on the internal fixation target. In these cases, the 
external fixation target should be presented to the fellow eye, and used to 
position the fovea centrally (macular cube) and nerve centrally (Optic Disc 
cube). The pupil is aligned in the center of the video image and the focus 
is adjusted by using the arrows to move the chinrest. 
The LSO fundus image is centered and focused. 
The OCT scan image is enhanced.  
The image is acquired clicking on the capture button. 
The operator will review all Cirrus HD-OCT scans as soon as they are 
acquired to ensure acceptable image quality, according to the criteria 
described above. If the image is deemed acceptable it will be saved, if not 
the operator will try again. 
The steps above are repeated to complete all the scans required.  
When the process is completed the operator will repeat the procedure to 
image the left eye.  

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If more than one dataset is saved for a given eye, scan type, and session, 
the dataset with the best image quality will be used for analysis. Scans may 
be exported from the instrument to perform additional analysis using 
custom software. 
   Fundus Photography 
Using an infrared camera to avoid excess visible light exposure, fundus 
photographs of two standard fields of the fundus will be taken at a baseline 
visit and at post-vector administration evaluations to document fundus 
appearance. 
The following descriptions of the standard fields assume that there are two 
cross hairs in the camera ocular, one vertical and the other horizontal 
intersecting in the center of the ocular. 
Field 1: Stereoscopic pairs of photos of the disc (2X): Center the temporal 
edge of the optic disc at the intersection of the cross hairs in the ocular. 
Field 2: Stereoscopic pairs of photos of the posterior pole (45 degrees): 
Center the macula near the intersection of the cross hairs in the ocular. 
The intersection of cross hairs should be placed about 1/8 - 1/4 disc 
diameter (DD) above the center of Macula. 
   Quality of Life Questionnaire (NEI VFQ-25) 
The Clinical Coordinator for the study will administer the National Eye 
Institute’s Visual Function Questionnaire (NEI VFQ-25) to all patients 
enrolled in the study at pre-injection, and Month 2, 6 and 12.   A special 
note will be made of patients’ responses to the questions concerning 
ocular pain.  The Biostatistics Center at Bascom Palmer Eye Institute has 
extensive experience in analyzing data from the NEI VFQ-25 in patients 
with several diagnoses, including glaucoma, low vision, and age-related 
macular degeneration.  
 
Detailed Study Agent Administration Procedures 
One-time intravitreal injection in a single eye will be administered. 
Procedure for Intravitreal Injection  
Patients in this trial will undergo unilateral intravitreal injection. All intravitreal 
injections will be performed by Dr. Janet Davis, Dr. Thomas Albini or Dr. Harry 
Flynn who have extensive experience in this procedure. The injection protocol is 
as follows: 

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 All intravitreal procedures shall be performed using aseptic technique and 
standard precautions protocol. 
 Preparations for the injection should not begin until the ophthalmologist is 
present within the unit. 
 A“time out” immediately prior to the procedure will be conducted by two 
persons as a final verification of the correct patient, procedure and site. The 
following equipment will be in the treatment room used for the injections. 
1. 1.0 cc syringe containing Study Drug Solution (Low, Medium, High or 
Higher dose) 
2. Dilating Drops 
3. Sterile eye pad 
4. Alcohol swap 
5. Sterile caliper 
6. Sterile eye lid speculum 
7. Optional-intravitreal kit with disposable calipers and lid speculum 
8. Tonopen with cover 
9. Lidocaine 4% vial 
10. Betadine 5% vial 
11. Betadine swab 
12. Medication to be injected 
13. 30 and 32 gauge needles 
14. Antibiotic drops 
15. Proparacaine drops 
16. Sterile cotton tip applicators 
17. Gloves 
18. Needle “box” holder 
19. Intravitreal Injection Form 

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20. Patient Discharge Instructions Form (Patient Education Assessment) 
21. Vital signs monitor 
Adequate documentation of the procedure will be done. 
1. The procedure used to prepare the eye for intravitreal injection will be 
documented on the Intravitreal Injection Form. 
2. The person who performs the eye prep will write or check mark the exact 
Procedure used to prepare the eye. 
3. The physician performing the intravitreal injection will document the Name of 
the drug given, the dose and amount of the drug given, and the Location of 
the injection on the Intravitreal Injection Form. 
The following procedure will be carefully followed: 
1. The physician writes an order for the intravitreal injection.  
2. The physician obtains an informed consent from the patient. 
3. The patient’s identity is confirmed using two patient identifiers. (Name and 
Date of Birth) 
4. The Nurse/Injection Specialist will initiate documentation by utilizing the 
Intravitreal Injection form. 
5. The Nurse/Injection specialist will assess the patient’s desire to learn barriers 
of learning, any physical or cognitive limitations and complete the Patient 
Education Assessment. 
6. The Nurse/Injection Specialist will explain the procedure to the patient. 
7. The patient’s blood pressure and pulse are monitored by the nurse/injection 
specialist. Abnormal findings are immediately reported to the patient’s 
physician. 
8. The Nurse/Injection specialist will verify the correct procedure and site (right 
or left eye) with the patient, the order and the consent form. 
9. The Nurse/Injection Specialist will verify and mark the site with skin marker. 
10. The Nurse/Injection Specialist will dilate the eye to be injected if not already 
dilated. 
11. The Nurse/Injection Specialist will recline patient and prepare the patient for 
injection. 

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12. The Nurse/Injection Specialist will wash his/her hands and immediately put on 
gloves. 
13. The Physician removes the 1 cc syringes prepared by the Sylvester 
Investigation Drug Service, containing Study drug from the transport 
container. 
14. The Nurse/Injection Specialist will instill one drop of Proparacaine on the eye 
designated to receive an intravitreal injection. 
15. The Nurse/Injection Specialist will instill one drop of Lidocaine 4% on the 
designated to receive an intravitreal injection. 
16. The Nurse/Injection Specialist will instill one drop of Betadine 5% on the eye 
designated to receive an intravitreal injection. 
17. The Nurse/Injection Specialist will instruct the patient to close his/her eye for 
a moment. 
18. The Nurse/Injection Specialist will repeat steps #15-17, two more times. 
19. The Nurse/Injection Specialist will use a Betadine swab to clean the eye lashes 
and lids, of the eye designated to receive an intravitreal injection. The eye will 
be swabbed from inner canthus to outer canthus. 
20. The Nurse/Injection Specialist will wipe the designated eye with sterile eye 
pad. 
21. The Nurse/Injection Specialist will place the eye lid speculum on the eye 
designated to receive an intravitreal injection. 
22. The Nurse/Injection Specialist will saturate a sterile cotton tip applicator with 
drops of  Lidocaine 4% without touching the applicator. 
23. The Nurse/Injection Specialist will utilize the cotton tip applicator, saturated 
with lidocaine 4% drops, to apply pressure on the area to be injected. (i.e. 6:00, 
7:00, 5:00 O’clock etc.) 
24. The Nurse/ Injection Specialist will repeat steps 22-23 two more times. Apply 
A drop of Lidocaine 4% to the cornea between each cotton tip applicator 
pressure application. 
25. The Nurse/Injection Specialist will call the physician and continue steps 22- 23 
until the physician enters the room. 

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26. As the physician enters the room, the nurse/injection specialist will instill one 
drop of Betadine 5% to the area of the eye designated for injection and where 
pressure is being applied. 
27. TIME OUT- The Nurse/Injection Specialist and Physician will conduct the final 
verification of the patient information, physician order, procedure and/or drug 
and eye designated to receive an intravitreal injection. 
28. OPTIONAL- The Physician will use a sterile caliper to measure the area to be 
injected. 
29. The Physician performs an anterior chamber paracentesis to remove 0.15 to 
0.2 ml in order to reduce risk of reflux after injection using a 3 cc syringe 
mounted with a 32-gauge needle. The anterior chamber fluid will be stored 
for future analysis. The syringe is immediately discarded in the biohazards 
sharps container. 
 
30. The Physician will check the drug dosage and inject the drug into the eye. 
31. As the physician withdraws the needle, the nurse/injection specialist will instill 
two drops of antibiotic solution. 
32. The Nurse/Injection Specialist or physician will remove the eye lid speculum. 
33. The Nurse/Injection Specialist will check the patient for light perception in the 
Treated eye and notify the physician immediately of no light perception (NLP). 
Procedure for Monitoring subjects after injection  
1. The Nurse/Injection Specialist will monitor the intraocular pressure (IOP) with 
a Tonopen every 15 minutes post-injection until the IOP is under 30. If, the IOP 
is under 30, the patient can be discharged. 
2. If the IOP is over 30, then the patient can be discharged if approved by the 
physician OR the IOP and vision will be monitored every 15 minutes and the 
patient discharged when the IOP is Under 30. 
3. The IOP at discharge is recorded. 
4. The Nurse/Injection Specialist will review discharge instructions and have the 
Patient sign the Patient Discharge Instruction form. 
 
Risks Associated with Study Procedures 
Risks associated with visual function tests include risks from eye drops. 
Rarely, a patient may experience redness, discomfort or an allergic reaction 

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to the eye drops used to dilate the pupil or anesthetize the cornea. Angle 
closure glaucoma may be precipitated by the dilating drops in a susceptible 
individual. If a patient has hypertension or cardiac disease, dilating drops can, 
in rare circumstances, temporarily exacerbate these conditions.  
 
Phlebotomy has the risks of discomfort, psychological stress leading to 
syncopal reactions and hematomas at the site of venipuncture.  
 
The chest X-ray performed as part of this study is not necessary for a 
subject’s medical care and will occur only as a result of participation in this 
study. Risks associated with radiation exposure of the magnitude in a chest 
X-ray are considered to be extremely low. 
 
Adequacy of Protection against Risks 
Recruitment and Informed Consent 
Many individuals and families with LHON have expressed interest and 
willingness to participate in a clinical trial. Additional volunteer participants 
will be recruited from patients of Dr. Byron Lam at Bascom Palmer Eye 
Institute, University of Miami. We expect continued inquiries from newly 
diagnosed LHON patients and families who are interested in the research and 
clinical studies due to the protocol listing on GeMCRIS and widely-available 
genetic testing for retinal degeneration-associated genes.  
 
Before subjects are enrolled in this trial, an Informed Consent document will 
be approved by the IRB of University of Miami. An investigator will 
completely explain the study to potential participants. At the discretion of 
the potential participants, friends and/or family members will be included in 
these discussions. Subjects will be encouraged to ask questions prior to 
providing written informed consent. Participation in this clinical trial is 
completely voluntary and great care will be taken to ensure that individuals 
do not feel coerced to participate. Subjects will be able to terminate their 
participation at any time without prejudice. 
 
Protection against Risk  
The following procedures are likely to minimize the potential risks associated 
with study procedures.  
 
The medical history and physical examination performed prior to study agent 
administration will identify patients with medical or ophthalmic conditions 
that would increase the risks associated with study procedures. Those 
subjects will be excluded from participation. Surgical administration of the 
study agent will be performed in standard fashion by experienced retinal 

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surgeons and with strong attention paid to the risks listed above. 
Administration of topical corticosteroids and antibiotics following intravitreal 
injection of the study agent is planned for this clinical trial to minimize the 
risk of local inflammation or infection. To minimize the risks of potential 
adverse events, following intravitreal injection, study subjects will be 
monitored and discharged only after their intraocular pressure (IOP) is less 
than 30 mm. Subjects will also be examined one day after  injection and 
again on day 7. If ocular inflammation is noted at day 1, an additional visit 
will be scheduled before the day 7 visit. Subjects will be provided with 
contact numbers for any questions or concerns arising regarding the possible 
effects of the administration of scAAV2-P1ND4v2. Results of all laboratory 
and safety examinations will be reviewed by an investigator throughout the 
study. Since the risks of rAAV vector administration to pregnancies and 
developing fetuses are unknown, female subjects of childbearing potential 
will have two pregnancy tests prior to administration and pregnant and 
breastfeeding females will be excluded from participation. Subjects (men and 
women) of reproductive ability will be required to use effective 
contraception for 1 year following gene transfer. Subjects must also be 
willing to use barrier contraception following gene transfer. If subjects do not 
agree to use contraception, they will be excluded from the study.  
 
A slit lamp exam will be performed to be certain no abrasions were 
sustained. 
 
Phlebotomy will be performed by experienced phlebotomists, thus 
decreasing the likelihood of discomfort, stress and the occurrence of 
bruising.  
Subjects will be provided with emergency contact information for Dr. Guy 
and will be encouraged to call if they feel that an adverse event is occurring. 
The Bascom Palmer Eye Institute maintains a 24-hour fully staffed emergency 
room. In the event of an adverse event, immediate medical care will be 
provided without cost to the subject or the subject’s family.   
 
Confidentiality of research materials obtained through this study will be 
maintained by coding data collection forms. Patients included as subjects in 
reports or publications will be referenced with numbers. 
 
 
This is the first time study agent, scAAV2(Y444+500+730)-smCBA-P1ND4v2, 
abbreviated as scAAV2-P1ND4v2, will be used in Humans.  
 

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The purpose of this dose-escalation study is to assess the safety and 
tolerability of scAAV2-P1ND4v2 gene replacement therapy in subjects 
confirmed with the G11778A mutation in mtDNA responsible for Leber’s 
Hereditary Optic Neuropathy. Ocular and systemic toxicity will be assessed 
following vector administration to determine if there are adverse changes that 
may be associated with vector administration. 
 
This first-in-man (FIM) clinical trial will assess safety, tolerability, and 
potentially efficacy of a single intravitreal injection in patient groups reflecting 
the acute, presymptomatic and chronic stages and manifestation of the LHON 
disease.  
 
The Food and Drug Administration (FDA), IND#15941 and National Institutes 
of Health/National Eye Institute (NIH/NEI) grant number: 1U10EY023558-
01A1, have approved this clinical trial. 
Sources of Materials 
Research materials obtained from human subjects related to this trial will 
include history and physical assessments, results of blood, urine, and LHON 
specific analyses, results of retinal and ophthalmic evaluations, AE reports, 
chest x-rays, electrocardiogram. Copies of CRFs, original test results, subject 
medical records, signed informed consent forms, correspondence, and all 
other trial-related documents will be kept at the clinical sites in locked 
cabinets (McKnight Building, Room 408, 1638 NW 10TH AVE, MIAMI, FL 
33136). All material collected as part of the studies will be obtained 
specifically for research purposes and only trained personnel directly 
involved in the study will be able to access study materials. Subjects will be 
identified by code number on all data collection forms and in any reports or 
publications. All information about study subjects will be kept confidential 
and managed according to the requirements of the Health Insurance 
Portability and Accountability Act of 1996 (HIPAA). Data management will be 
21 CFR compliant.  
 
Long-term follow-up of trial participants will be performed according to 
regulatory guidelines by Dr. Guy or his designees. 
 Long-term follow-up will occur for 10 years after study agent 
administration. All subjects will be provided with written contact 
information for Dr. Guy should they experience any SAE that they consider 
possibly related to study treatment or study participation. Subjects will be 
instructed to notify Dr. Guy of changes in their contact information. 

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Annually, subjects will be contacted by mail or by telephone and 
information concerning any cancer, neurological, autoimmune or 
hematological disorder that developed or worsened since the previous 
contact will be obtained. The subjects will also be asked for information 
concerning hospitalizations and use of medications.   
At the time of death, no matter what the cause, permission for an autopsy 
will be requested of subjects' families. Subjects will be asked to advise their 
families of this request and of its scientific and medical importance. 
11)  Data and Specimen Banking (for research purposes). Specimens of blood  
and anterior chamber fluid will be stored for analysis of vector DNA and anti-AAV 
antibodies. 
 
 
 

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12)  Data Management* 
 
Study data will be managed by the Biostatistics Center as part of a separate 
National Eye Institute grant U10EY024247 which has already been I.R.B. 
approved (protocol 20140294, approval date 9-June-2014). The following is 
abstracted from that protocol. 
 
Assignment of Patient Identification (ID) Number 
The Baseline Qualifying Assessment establishes whether the patient satisfies the 
eligibility criteria.  If the patient appears to be eligible for the study, the Clinical 
Center (CC) completes the Qualifying Assessment Form and Pre-injection Form.  
Both forms are faxed to the Biostatistics Center (BC), along with a copy of the 
consent form.  The BC reviews each of the inclusion and exclusion criteria to 
ensure that the patient is eligible.  
Any patient who is confirmed by the BC to meet the eligibility criteria and is 
enrolled in the Phase 1 Safety Study is assigned a patient identification (ID) 
number. The patient identification number is a 3 digit, 3 letter code which is 
unique for each patient.   The first digit refers to the Group (1-3). The second 
digit refers to the dose (1=low, 2=medium, 3=high, or  4=higher).  The third digit 
is a sequential number for each patient.  The 3 letters are the subjects initials. A 
line through will be used in the center box for those subjects with only two 
initials.  .  For example, ID number 2-3-1-AZE refers to the first patient enrolled 
with the high dose in group 2. 
 
  Data Management 
 
A master log is kept of each patient in the Study.  An appointment schedule is 
made for each patient and sent to the Clinical Center.  When a data form is 
received at the Biostatistics Center, it is processed for filing and data entry.  Each 
form is data entered by a data entry clerk and then verified by double entry by 
the BC Research Coordinator.  Edit checks, such as missing data and out-of-range 
values, will be clarified within the Clinical Center. 
BC procedures provide for  
(i) 
CRF tracking and annotation  
(ii) 
Audit from source document to database 

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(iii) 
Database design  
(iv) 
Data entry 
(v) 
Data validation 
(vi) 
Discrepancy management 
(vii) 
Database lock  
 
(i) 
The BC maintains a stand-alone patient log recording dates of enrollment 
and study visits as they occur, as well as documenting receipt of CRFs from 
the clinical center study coordinator. The BC will accept photocopies of the 
original CRFs or will copy them for the study coordinator if necessary. 
Originals will be returned to the coordinator and the copies retained at the 
BC. The data entry operator inspects the forms visually for missing fields and 
reports these to the clinical coordinator who either supplies the missing data 
or provides an explanation for why the data were not collected. CRFs 
awaiting data entry are filed in the data entry operator’s office – access to 
this office is by magnetic card key.  
 
 
(ii) 
A separate dataset and entry screen is provided by the data manager for 
each of the study forms: Inclusion-Exclusion/Group Assignment, Adverse 
Event, AntiAAV Antibodies, Baseline / Follow Up Exam, Clinical Chemistry, 
Coagulation, Chest Xray Exam, Demographic Data, Electrocardiogram, 
Hematology, Injection/Treatment Day 0, Concomitant Medications, Vital 
Signs/Physical Exam, Serum Pregnancy, Urinalysis, NEI VFQ 25 Quality of Life 
Questionnaire. The family tree CRF is not data entered.  
 
(iii) 
Computer data entry will be performed in two stages. 
a. Initial data entry will be performed by the BC data entry operator into the 
21 CRF part 11 compliant Velos eResearch system which is maintained by 
the University of Miami’s Office of Research Information Management, 
which supports audit trails and controls access to the datasets.  
b. These data will be exported to IBM/SPSS data collection version 7, which 
supports two pass verification to foster data quality. Any errors found in 
the verification pass will be corrected in the Velos datasets and these 
corrections recorded with audit trails.  
As a further monthly check on database integrity, the current dataset will be 
compared with the previous month’s backup and differences will be 
reconciled. The CRFs are data entered and a subsequent verification data 
entry pass is performed, typically, days or weeks later, after which the 
CRF copies are stored in locked file cabinets within the BC.  
 

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(iv) 
Following entry, data will be subjected to range and consistency checks. 
Data entered from the CRFs are merged by patient ID to create a single 
record for each patient and these records are subjected to range and 
consistency checks by the study statistician. Data entry errors uncovered 
in this way are corrected by the data entry operator in the Velos system 
and documented with its audit trail feature.  
 
(v) 
Similarly, data fields containing values demonstrated to be unlikely or 
impossible by consistency checks are reported to the clinical coordinator 
for resolution. Necessary corrections are made and documented as with 
data entry errors. Interim versions of these data files are maintained for 
reference by the BC. Any updates to the paper forms (i), based on this 
procedure are dated and made to the paper forms in a different color ink 
from that originally used to fill out the form.  
 
(vi) 
After a proper quality check and assurance, the final data validation is 
run. If there are no discrepancies, the datasets are finalized by the study 
statistician and data manager. BC hardware and software will be 
identified and available for inspection by the FDA or the representatives 
of the NEI or DSMC.  
The BC ensures that safety events, systemic, ocular, and/or acuity loss (described 
below), are captured on study CRFs (Adverse Event and Follow Up Exam) and reported 
to the FDA, NEI, and DSMC. 
 
Data Security 
 
The paper data forms for the Study are kept in file cabinets in the Biostatistics facility in 
the  Dominion Tower.  The building is locked and access is by card key entry.  A security 
guard is present during working hours.  The computer files for the study are kept on 
computers in the same location.  These rooms are kept locked when not in use, and the 
study computer files are password-protected as described in Resources.  The data files 
are backed up daily and are stored at a remote facility.  Computer data files used for 
publication are saved and stored as separate files. 
Data Forms 
The data forms will be designed to be self-explanatory.  Their completion should not 
require reference to separate information manuals.  The data forms contain information 
to be collected at a given point in time during the study.  Information collected at 
another date is incorporated into a separate form.  Data forms are faxed to the 

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Biostatistics Center for data entry.  Forms will be reviewed periodically and revised as 
dictated by protocol changes.   
Data Analysis 
The analysis of data will be performed by the Biostatistics Center under the supervision 
of WJ Feuer.  He is the principal investigator biostatistics Center for the Phase 1 Safety 
trial, descriptive statistics with 95% confidence intervals around the success rates and 
the adverse event rates will be calculated.  To reiterate, the results of the ongoing 
monitoring of each patient’s course by the BC and the Data and Safety Monitoring 
Committee (DSMC) will be evaluated to decide about continuing recruitment in this 
phase of the trial; statistical considerations will only provide guidelines. 
 
Assessments of change in visual acuity will be calculated from the Baseline 2 
measurement to guard against regression to the mean from the screening acuity, 
Baseline 1. 
 
These patients will also be monitored for ocular and systemic adverse events which will 
be reviewed by the DSMC on an ongoing basis as deemed necessary by the DSMC and 
the NEI. 
 
 
Ancillary data analysis will include assessments of the changes in acuity, visual fields, 
and OCT RNFL thickness as well as correlations between these measurements to the 
limited extent that this can be studied with such a small sample size.  Assuming that the 
earlier the stage, the more likely the benefit of treatment, we will examine and develop 
outcomes that can be used in later  phases and other trials that could include a wide 
range of these patients before the onset of permanent visual deficits. 
 
Evaluations of Efficacy in a phase I trial.  
Patients with molecularly confirmed G11778A mitochondrial DNA will be recruited into 
one of 3 groups. 
 
 
Low-dose 
(1.18x109 vg) 
Medium dose 
(5.81x109 vg) 
High  dose 
(2.40x1010 vg) 
Higher dose 
(1x1011vg) 
  Group I 
3 
3 
3 
 
  Group II 
3 
3 
 
3 
  Group III 
3 
3 
 
3 
    
The certified technician performing the visual acuity assessments will be masked to 
treatment. The elimination of improvement to ≥70 letters from the efficacy endpoint 
increases the likelihood of a success by natural history and correspondingly decreases 
power. To obtain the requisite patient numbers needed to complete the efficacy 

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assessments, we will submit for another U10 for phase II, if phase I safety assessment 
supports further investigation.  Our previous power estimates were based on 
improvement by 15 letters and acuity of 20/40 and included both best and worst eyes of 
patients (Aim 1 and 2). The new endpoint, which drops the latter requirement, is more 
common in natural history but is assessed only on the 31 worst eyes eligible for Aim 1 and 
Aim 2. We include the upper 95% confidence intervals along with the point estimates as 
recommended. 
  
 
Power for detecting 15 letter improvement with N=9 in group I  
 
 
 
 
 
 
 
Treatment Efficacy 
 
Natural History Estimate 
 
 
50% 
 
75% 
 
95% 
 
Point:  
 
  9% 
 
Power 35% 
 
83% 
 
>95% 
 
Upper 95% CI:               28% 
 
Power 13% 
 
41% 
 
92% 
 
 
Power for detecting 15 letter improvement with N=9 in group 3  
 
 
 
 
 
 
 
Treatment Efficacy 
 
Natural History Estimate 
 
 
50% 
 
75% 
 
95% 
 
Point:  
 
33% 
 
Power   9% 
 
30% 
 
88% 
 
Upper 95% CI:              70% 
 
Power  NA 
 
NA 
 
17% 
 
Sample size for 80% (90%) power to detect a 15 letter improvement in group I  
 
 
 
 
 
 
 
Treatment Efficacy 
 
Natural History Estimate 
 
 
50% 
 
75% 
 
95% 
 
Point:  
 
  9% 
 
N 
18 (22)  
  9 (10)  
  6 (6) 
 
Upper 95% CI:               28% 
 
N 
76 (100) 
18 (21)  
  9 (9) 
 
Sample size for 80% (90%) power to detect a 15 letter improvement in group 3  
 
 
 
 
 
 
 
Treatment Efficacy 
 
Natural History Estimate 
 
 
50% 
 
75% 
 
95% 
 
Point:  
 
33% 
 
N 
130 (175) 
22 (27)  
  9 (11) 
 
Upper 95% CI:              70% 
 
N 
NA 
 
NA 
 
34 (41) 
 
 
 
13)   Provisions to Monitor the Data to Ensure the Safety of Subjects* 
 
The DSMC is responsible for the ethical conduct of the study.  This 
committee oversees the informed consent process and major changes in the 

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protocol.  The DSMC reviews the accumulating data for evidence of adverse 
and beneficial treatment effects.  This committee will meet as often as they 
deem appropriate, but at least twice each year for the duration of the study.  
Telephone conferences will occur as needed.  The responsibilities of the 
DSMC are as follows: 
 To review the study design and study documents before the start of the 
study to identify any problems that may affect patient safety 
 To monitor adherence to the study protocol  
 To review treatment reports prepared by the BC for evidence of adverse and 
beneficial treatment effects 
 To terminate the study if treatment risk is so high that continuation of the 
trial is deemed unethical 
 To collaborate with the investigators on interpretation of study data. 
 To recommend changes to the study protocol based on periodic data analysis 
 To review and approve all publications and presentations 
 To determine when data collected in the study should be released to study 
investigators, study patients, the medical community, and the public 
 
14)   Withdrawal of Subjects* 
             
Subjects may withdraw or be withdrawn from the study for any of the following 
reasons: 
 Occurrence of intolerable adverse events, including clinically significant abnormal 
laboratory findings which, in the opinion of the investigator, preclude further 
participation in the study; 
 Voluntary withdrawal by participant; 
 Subject non-compliance with study procedures; 
 Investigator decision that withdrawal from further participation would be in the 
subject’s best interest; 
 Instruction to terminate study by regulatory, safety, institutional authorities or the 
DSMB. 
The reason for withdrawal will be documented for each subject. Subjects who 
withdraw due to an adverse event will be followed until the event has stabilized 

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or resolved. Subjects will be considered withdrawn from the study at the time that 
any of the above criteria for withdrawal are met. If subjects withdraw prematurely 
from the study, data collection will occur throughout the protocol-defined follow-
up period. If a subject withdraws consent for participation, attempts will be made 
to record at least survival data through the end of the follow-up period. If a subject 
withdraws from the study and contact is lost, three attempts will be made to 
contact the subject by phone after which next-of-kin will be contacted in order to 
obtain survival data.  Any subject who withdraws prior to administration of study 
agent will be replaced. 
15)  Risks to Subjects* 
     Summary of the known and potential risks and benefits to human subjects 
Subjects may experience burning or pain in the eye after the anesthesia given for 
the injection wears off.  This clinical trial is the first human study of scAAV2-
P1ND4v2 so the risks associated with vector administration are not precisely 
known. Based on our preclinical studies of scAAV2-P1ND4v2 in normal rats and 
normal non-human primates  there is a high likelihood that intravitreal 
administration of scAAV2-P1ND4v2 will be safe in humans. Safety of rAAV2 
vectors in humans is also supported by previous and on-going clinical trials. Most 
relevant are studies performed at the University of Florida’s Powell Gene Therapy 
Center. These trials include several Phase I and II clinical studies in individuals with 
cystic fibrosis   with alpha-1-antitrypsin deficiency, with RPE65-associated retinal 
disease. Overall, to date, results indicate no significant vector-related toxicity and 
demonstrate the feasibility of gene transfer. Potential risks associated with 
scAAV2-P1ND4v2 and the study procedures are discussed below. 
Specifically associated with intravitreal-injections of scAAV2-P1ND4v2, there is a 
risk of degeneration of lens and cataract formation in the injected eye. 
Detachment of the retina is also possible. 
There is a potential risk of vector spread to other organs. In our safety studies, all 
major organs of rats and primates were essentially negative (≤ 100 copies/ug 
genomic DNA) for vector. Our biodistribution results after scAAV2-P1ND4v2 
(intravitreal) administration to rats and non-human primates indicate only limited 
vector spread outside the injected eye and no evidence of a vector dose-related 
effect. The risks of vector spread and germline transmission (all gonad samples 
were negative) are therefore presumed to be low in the planned human trial. 
There is a risk that subjects may develop an immune response to the vector. Our 
neutralizing antibody assay data from scAAV2-P1ND4v2 administration in large 
animals suggests that the risk of adverse immune response in humans is minimal. 
It is possible, but unlikely, that scAAV2-P1ND4v2 could interact with other viruses 

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with which the subject comes in contact, forming a new virus that could produce 
new side effects.  
In preclinical studies, there was minimal ocular inflammation which subsided with 
time after administration; in one animal of the non-human primate studies. 
Administration of corticosteroids and antibiotics following intravitreal injection of 
the study agent is planned for this clinical trial to minimize the risk of local 
inflammation. There is potential that vision of subjects could be further reduced. 
Based on preclinical studies in animal models and in normal animals, the risk of 
further visual impairment due to scAAV2-P1ND4v2 is believed to be low. 
There is a chance that the study agent could damage the DNA in the cells of a 
subject’s retina. In the unlikely event that this occurred, it could put the subject at 
risk for developing retinal tumors in the future. In animal and human studies to 
date, cancer has not developed and therefore, the risk is considered to be low. In 
our preclinical work with the AAV vector, the majority of vector DNA appears to 
persist as episomal rather than integrated into genomic DNA, which makes it very 
unlikely that mutagenesis and tumor development will occur. 
Intravitreal administration of scAAV2-P1ND4v2, as mentioned above, carries a risk 
of retinal cell loss from administration trauma. There is also a risk of cataract 
development that could require additional surgery. There is a risk of retinal tears 
or retinal detachment and there is the possibility that these could lead to worse 
vision. Exogenous intraocular infection (endophthalmitis) is rare but can occur. 
The most common pathogens can be treated but poor vision (in patients with 
better vision than those entering this study) can result from infection despite 
successful antibiotic treatment. Intraocular hemorrhage is rare in patients without 
bleeding diathesis. Vitreous hemorrhage is benign and usually resolves 
spontaneously. Choroidal or subretinal hemorrhage is usually self-limited and 
could affect vision (or potential vision) significantly. Temporary increases in 
intraocular pressure can occur following ocular/retinal injections and are 
treatable with topical or systemic medications to lower eye pressure. Risk of 
anesthesia, which is local (retrobulbar) is low. There is a small risk of penetration 
of the eye or optic nerve from retrobulbar injection and also the possibility of 
hemorrhage, which can be decompressed if necessary. In addition to these risks, 
there may be other longer-term risks which are not known. 
 
16)     Potential Benefits to Subjects* 
 
Benefits: Due to the initial, safety establishing nature of this investigation, there 
is no anticipated benefit to the subjects in this trial. 
 

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This Phase I clinical trial will define the safety of the proposed procedures and 
study agent. Since this is the first study of scAAV2-P1ND4v2, the potential risks 
and benefits are unknown and there are no expected direct benefits to the 
individual subjects. The results of this trial will provide information on the 
biological response to intravitreal gene delivery in this human blinding disease 
and may be extended to other degenerative diseases caused by mutated 
mitochondrial genes.  
 
 
17)    Vulnerable Populations* 
       
No special populations are to be included in this research, such as children 14 
years old or younger, prisoners, or pregnant women.  We wish to exclude 
pregnant women.  There is no reason to unnecessarily endanger the unborn.  The 
data obtained from this study may provide additional data to warrant extension 
of similar, future studies to additional populations. 
 
 18)    Multi-Site Research*N/A 
 
 19)    Community-Based Participatory Research* N/A 
 
  20)    Sharing of Results with Subjects* 
 
DSMC will  determine when data collected in the study should be released to 
study investigators, study patients, the medical community, and the public 
 
21)    Setting  
Describe the sites or locations where your research team will conduct the 
research. 
Bascom Palmer Eye Institute 
Clinics of Drs. Guy, Lam, Davis, Albini, Pasol  and Flynn. 
See Manual Of Procedures for the methods that will be used in the 
examinations and testing as well as the injection procedure. 
22)    Resources Available 
 Phase I.  A review of the LHON patients currently being seen by Dr. John Guy and 
Dr. Byron Lam with long-standing severe vision loss of 20/200 or worse, shows 
that we have 44 patients who would be eligible for the Phase 1 study in Groups 1 
and 2. We expect that the availability of a potential treatment will draw more 
patients in groups 2 and 3. We have also followed 45 asymptomatic carriers. One 

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of them has converted to affected. Others may also. Thus, we should have a 
sizable pool of patients for the Phase 1 safety study of the AAV-ND4 vector. 
 
 The Principal Investigator is listed as 40% effort for this study.  The co 
investigator’s including doctors Lam is listed at 5%. Dr. Davis is listed at 
10% for year 2 of this NIH funded grant.  
Bascom Palmer Eye Institute: Founded in 1962, Bascom Palmer Eye 
Institute serves as the Department of Ophthalmology for the University of 
Miami Miller School of Medicine. The Institute comprises clinical care, 
teaching, and research activities. In the past two years, Bascom Palmer Eye 
Institute was rated as the number one eye hospital in the nation by board-
certified ophthalmologists from across the United States. It has been either 
first or second in the rankings since the survey began. Ophthalmology 
Times, an industry periodical for and by ophthalmologists consistently 
rates Bascom Palmer’s patient care and residency programs as among the 
best in the nation. 
 
 Currently there are 42 board-certified ophthalmologists on the full-time clinical 
faculty, almost all fellowship-trained. In addition, 10 adjunct faculty members take 
part in various training activities for 22 board-certified fellows and 20 residents. 
All ophthalmic sub-specialties are represented within this group. More than half 
of the Institute’s clinical faculty members are listed in Castle Connolly “Top 
Doctors in America” including the PI.  
 
 In 2001, Bascom Palmer began an intensive recruitment effort to build its research 
program. There are currently 21 research faculty members in the department. 
Hiring continues, including scientists and clinician-scientists for the research 
program, as well as clinicians to staff the expanding clinical facilities.  
 
 The Anne Bates Leach Eye Hospital is Bascom Palmer’s main facility for 
ophthalmic care. Operating within a six story building located on the medical 
campus (opened in 1976), the hospital serves individuals of all ages.  More than 
200,000 patients are seen each year and at least 10,000 surgical procedures are 
performed. Additionally, 24-hour emergency care and community-based 
ophthalmic care for indigent and low-income patients of Miami is provided. The 
hospital employs numerous optometrists as well as other professional staff.  
 
 The neuro-ophthalmology clinic is run 5 days a week.  3 of these days are staffed 
by Dr. Lam and one of these days staffed by Dr. Guy.  Therefore we have 
coverage 80% of the time of every week for our LHON patients.  Bascom Palmer 

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mint maintains a fully staffed eye emergency room 24/7.  Therefore we have 
coverage for any conceivable problem that could arise.   
 
 
 
 
 
 
The manual of procedures delineates the protocol to be followed by all 
personnel in the study.  Drs. Guy and Lam have just completed a five-year 
study of patients with LHON that included all evaluations proposed here 
with the exception of the intravitreal injections of study drug.   
23)  Prior Approvals 
University of Miami Institutional Review Board, University of Miami 
Biosafety Committee and University of Miami Environmental  Health and 
Safety Department. 
 Approvals from the Federal Drug Administration and National Institutes of 
Health/National Eye Institute have been obtained by Sponsor-Investigator, 
Dr. John Guy.  
  24)  Recruitment Methods 
Patients in this single center study will be recruited from the existing 
LHON patient pool in Dr John Guy and Dr Byron Lam’s clinics, where 
access to records is already given outside of research, and from referrals 
of outside ophthalmologists. This is the same strategy used to recruit 
patients for the completed natural history study of Leber’s Hereditary 
Optic Neuropathy (references below). Sufficient participants (N>40) were 
recruited in this way, even without the possibility of offering participants 
the possibility of an active treatment. We do not anticipate the need for 
advertisements or other recruitment materials. 
Patients will be reimbursed for travel expenses as needed and in 
accordance with the budget in the grant application approved by the 
national eye Institute 
  25)  Local Number of Subjects: N/A 
 
Number of Study Centers: There is only a single study center at, Bascom Palmer Eye 
Institute, University of Miami, Miami, FL. 
 
Many individuals and families with LHON have expressed interest and willingness 
to participate in a clinical trial.  Phase I.  A review of the LHON patients currently 

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being seen by Dr. John Guy and Dr. Byron Lam with long-standing severe vision 
loss of 20/200 or worse, shows that we have 23 patients who would be eligible for 
the Phase 1 study in Groups 1 and 2. We expect continued inquiries from newly 
diagnosed LHON patients and families who are interested in the research and 
clinical studies due to the protocol listing on GeMCRIS and widely-available 
genetic testing for retinal degeneration-associated genes. We expect that the 
availability of a potential treatment will draw more patients in groups 2 and 3. 
Thus, we should have a sizable pool of patients for the Phase 1 safety study of the 
AAV-ND4 vector. 
 Individuals who have been diagnosed with G11778A mutation associated Leber’s 
hereditary Optic Neuropathy and who are eligible based on inclusion and 
exclusion criteria will be invited to participate in this clinical trial. A complete 
physical examination and clinical chemistries will be performed at a pre-
treatment, screening evaluation to further determine eligibility. Subjects will be 
considered enrolled when they sign the Informed Consent. 
26)  Confidentiality: N/A 
This is not a multicenter study. 
27)  Provisions to Protect the Privacy Interests of Subjects 
Patients enrolled in the study are assigned a patient identification (ID) number. 
The patient identification number is a 3 digit, 3 letter code  used  are the subjects 
initials. A line through will be used for subjects without a middle initial .   This ID 
number is used on CRFs instead of the patient name. 
 
Access to datasets, including Personal Health Information, is restricted to 
Biostatistics Center (BC) personnel associated with the study and identified as 
such in Eprost.  For a complete description of data management procedures see 
section  12.  
 
Section 12 also describes the physical facilities of the BC and measures taken to 
ensure data security.  
 
Direct Access To Source Data/Documents  
Qualified representatives of regulatory agencies, internal auditors or 
monitors will have the right, both during and after this study, to conduct 
inspections and to audit and review medical records and/or study 
documents (CRFs, source data/documents, etc.) pertinent to the clinical 
study as permitted by the regulations (21 CFR 312.58; 312.68, ICH-GCP 
E6; 6.10).  

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Quality Control & Quality Assurance 
Monitoring of the study will be provided through the University of Miami’s Office 
of Clinical Research Operations and Regulatory Support (CRORS) or an 
experienced external monitoring service. 
 
28)  Compensation for Research-Related Injury 
            Provisions for Injury  
In event of injury resulting from procedures associated with the clinical trial, the 
cost of medical treatment in excess of that covered by a third party payer will be 
provided to the subject but financial compensation is not available for injury. 
29)  Economic Burden to Subjects (From ICF form) 
If you choose to take part in this study, will it cost you anything? 
Subject nor their insurance company will be charged for any tests or 
procedures that are done for the purposes of the research study. Study 
agent will be provided without charge. Subject or Subject’s  insurance 
company will be responsible for costs of tests or procedures that are 
part of your routine medical care. 
 
Will you receive payment for your participation in this study? 
Study Subject will not receive financial compensation for participation in 
this study. Travel will be provided and arranged by our research staff. In 
the event that we are unable to provide this service, with documentation 
of expenses, Study Subject  will be compensated for travel expenses. This 
reimbursement for costs will be done in accordance with the travel 
policies and procedures of Bascom Palmer Eye Institute and the 
University of Miami School of Medicine. Study Subject's name and social 
security number will be reported to University administrative personnel 
for purposes of making and recording the payment.  The research staff 
will arrange for payment or reimbursement of procedures associated 
with the study and performed at non-University locations. 
 
 30)  Consent Process Consent process will take place in the Bascom 
Palmer Eye Institute.  
 The informed consent form will be read to all subjects through a laptop 
using the Dragon Software.  A hardcopy of the consent form will be 
provided to the patients to follow while the ICF is being read to them.  If 

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the subject is a minor the hardcopy will be provided to the parent.   A 
study member is present during the consent process and he/she can 
pause, or make adjustments to volume and reading speed to subjects 
satisfaction.  After the consent form has been read the subject will have 
the opportunity to ask questions.   
 The waiting period will be as long as the prospective Subject requires.  
 Patient will be asked at the start of every visit whether they wish to 
continue participating in LHON: Gene Therapy Clinical Trial. 
 An investigator will also provide complete explanation of the study to 
potential participants and will ensure that all questions are answered 
prior to decisions about participation.  
 Time that will be devoted to the consent discussion will as much time as 
potential participant requires.  
 At the discretion of the potential participants, friends and/or family 
members will be included in these discussions. Subjects will be encouraged 
to ask questions prior to providing written informed consent. Participation 
in this clinical trial is completely voluntary and great care will be taken to 
ensure that individuals do not feel coerced to participate. Subjects will be 
able to terminate their participation at any time without prejudice. 
 To ensure Subjects understand in participating in the clinical trial, subject 
will be asked questions regarding ICF.  
 As the PI and co investigators speak only English no non-English speaking 
subjects will be enrolled to ensure communications between studies 
subjects and investigators. 
  Patients who do not speak English will not be enrolled.  
 Signatures will be obtained on an IRB approved assent form for all subjects 
ages 15-17 years old.   
Waiver or Alteration of Consent Process (consent will not be obtained, 
required information will not be disclosed, or the research involves 
deception) – N/A 
Subjects who are not 15 years old or older- Excluded 
Cognitively Impaired Adults - Excluded 
Adults Unable to Consent - Excluded 
Adults Unable to Consent - Excluded 
.            
31)   Process to Document Consent in Writing 
After the patient is given verbal informed consent, he or she will be asked 
to sign the written informed consent form. 

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32)   Drugs  
Handling of Investigational Agent 
The study agent will be stored according to regulatory guidelines (CFR 21, Part 
312.69). The investigators will take adequate precautions, including storage of the 
investigational agent in a securely locked (McKnight Building) , substantially 
constructed cabinet or other securely locked (Locked freezer) to prevent theft or 
diversion of the substance into illegal channels of distribution. Only the PI has the 
key to this freezer. 
Disposition of Study Agent 
The investigators, in conjunction with the Research Pharmacy Services will 
maintain adequate records of the disposition of the drug, including dates, 
quantity, and use by subjects. If the trial is terminated, suspended, discontinued, 
or completed, the investigators will provide for disposition of the unused supplies, 
in accordance with regulatory guidelines (CFR 21, Part 312.62). 
 
 
All requirements completed per FDA regulations 
 
Applicable to: 
FDA Regulation 
IND Studies 
21 CFR 11 
X 
21 CFR 54 
X 
21 CFR 210 
X 
21 CFR 211 
X 
21 CFR 312 
X 
21 CFR 812 
 
21 CFR 820 
 
 
The FDA has given us approval for the low and medium dose injections.  
We will be supplying them pre-clinical data for the high dose injections 
before administering that dose to our patients with LHON.  They have 
approved the injection procedure and our manual of procedures which we 
will follow according to all FDA regulations.  In addition the Data Safety 
and Monitoring Committee constituted by the NIH will be reviewing our 
forms and data every 6 months. 
 

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33)  Record Keeping 
Accurate and complete study records will be maintained and kept in the 
McKnight Bldg  
 
34)  Publication and Presentation Policy 
A Study publication is one which contains details of the design, methods, or results of 
the Study, and is written by investigators.  Any paper classified as a Study publication 
must be approved by the Data and Safety Monitoring Committee prior to submission for 
publication.  Similarly, any presentation made on behalf of the Study must be approved 
by the Data Safety and Monitoring Committee.