CRISPR/Cas9 mRNA Instantaneous Gene Editing Therapy Assisted Corneal Transplantation in the Treatment of Refractory Viral Keratitis
Phase: NA
Conditions: Viral Keratitis, Blindness Eye, Herpes Simplex Virus Infection, Cornea
Sponsor: Shanghai BDgene Co., Ltd.

 
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Identifier: NCT04560790 
 
 
Official Title: 
CRISPR/Cas9 mRNA Instantaneous Gene Editing Therapy 
Assisted Corneal Transplantation in the Treatment of 
Refractory Viral Keratitis 
 
 
 
 
 
Date: April 12, 2020 
 
 
 

 
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Protocol 
Unique Protocol ID: JYMS-CXL#02 
This trial protocol has been provided by the authors to give readers additional information about their 
work.  
 
 

 
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DESCRIPTION 
This supplement contains the following items:  
1. Original protocol, final protocol, summary of changes.  
2. Original and final statistical analysis plan. 
 
 
 

 
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Table of Contents 
ORIGINAL STUDY PROTOCOL .......................................................................................................5 
CURRENT STUDY PROTOCOL.......................................................................................................24 
SUMMARY OF PROTOCOL CHANGES .........................................................................................43 
ORIGINAL AND CURRENT STATISTICAL ANALYSIS PLAN....................................................45 
 
 

 
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Original Study Protocol 
 
 

 
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Protocol Approval ID: 2020023-2-v1 
Safety and Efficacy of CRISPR/Cas9 mRNA Instantaneous Gene 
Editing Therapy to Treat Refractory Viral Keratitis 
Investigator:       Jiaxu Hong, M.D., Ph.D, M.PA. 
                 Deputy Chief Physician 
                 Department of Ophthalmology and Visual Science 
Research Institute:  Eye, and ENT Hospital, 
Shanghai Medical College, Fudan University, Shanghai, China 
Founding:         National Natural Science Foundation of China,  
81970766, 8217040684 and 31971364 
the Program for Professor of Special Appointment 
(Eastern Scholar) at Shanghai Institutions of Higher Learning, 
TP2019046 
The Shanghai Innovation Development Program, 
2020-RGZN-02033 
the Shanghai Key Clinical Research Program,  
SHDC2020CR3052B 
Startup funding from Shanghai Center for Systems Biomedicine 
Shanghai Jiao Tong University, WF220441504 
Collaborators:      Key Laboratory of Systems Biomedicine, 
Shanghai Jiao Tong University 
 
Version 1.0 
Version Date: December 28, 2019 
 

 
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Brief Summary: 
The purpose of this study is to evaluate the safety, tolerability and efficacy of a single dose of 
CRISPR/Cas9 mRNA Instantaneous Gene Editing Therapy administered via corneal injection in 
participants with refractory herpetic viral keratitis. 
 
Condition or disease 
Intervention/treatment 
Herpes Simplex Virus Infection 
Drug: HELP (BD111) Adult single group Dose 
Refractory Viral Keratitis 
(Administered by corneal injection surgery. Dosage form: 
injection solution. Dose: 250μL. Frequency of 
administration: one time injection.) 
 
Description: 
 
This is an open-label, single dose study of HELP in adult (ages 18 to 70) participants with refractory 
herpetic viral keratitis. Six participants will be enrolled. HELP is a novel gene editing product designed 
to clear Herpes simplex virus type I (HSV-1) that results in herpetic stromal keratitis in both acute and 
recurrent infection models which is the leading factor for infectious blindness. 
The follow-up period was 360 days, and the patients will be followed up 3±1 days, 7±2 days, 30±7 
days, 90±14 days, 180±21 days, and 360+31 days after treatment. 
 
Outcomes Measures: 
 Primary Outcome Measure 
1. Concentration of dose limiting toxicities 
Observe and record AE, SAE incidence of dose limiting toxicities related with HELP administration. 
2. Concentration of maximum tolerated dose 
Observe and record AE, SAE at maximum tolerated dose when occurs dose limiting toxicities. 
3. Clearance effective of HSV-1 genome 

 
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Judge HSV-1 genome clearance effective according to DNA sequencing results by methods of Plaque 
assay, Elisa, PCR etc. 
4. Grafted corneal survival time 
Observe the survival time of grafted corneal in subjects, whether the grafted corneal is transparent or 
opacity. 
5. Visual improvement compared with baseline 
Judge the visual recovery progress according to visual examination results on day 3±1，7±2，30±7，
90±14，180±21，360±31. 
 
 Secondary Outcome Measure 
1. Changes of herpes virus load compared with baseline 
Herpes virus content before and after treatment were determined by methods of plaque assay, ELISA, 
PCR etc. Compare the viral content changes with baseline. 
 
 

 
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Table of Contents 
1. Introduction…………………………………………………………………………………….11 
1.1 Study Objects……….………………………………………………………………….11 
1.2 Backgrounds of HELP………………….…….…………………………………………11 
2. Eligibility Criteria………………………………………………………………………………11 
2.1 Inclusion Criteria……………………………………………………………………….11 
2.2 Exclusion Criteria……………………………………………………….………………12 
3. Study Implementation…………………………………………………….…………………….13 
3.1 Study Design……………………………………………………………………………13 
3.2 Preparation of HELP Formulation……………………………………………………....14 
3.3 Corneal Transplant and HELP Administration………………………….………………15 
3.4 Post-operation Management………………………………………………….…………16 
4. CRISPR/Cas9 Therapy Interruption…………………………….………………….…………17 
5. Quality Control……………………………………………………………………….…………17 
5.1 Quality Control and Quality Assurance…………………………………….…………...16 
5.2 Monitoring…………………………………………………………………………. ….16 
6. Data Collection and Evaluation……………………………………………….……………….18 
7. Statistical Considerations……………………………………………….……………………...19 
8. Adverse Event Reporting……………………………………………………………………….19 
8.1 Adverse Event……………………………………………….………………………….19 
8.2 Serious Adverse Event……………………………………………….………….……...19 
8.3 Recording and Reporting……………………………………………….……………….20 
9. Record Form & Retention……………………………………………….………….………….21 
10. Ethics……………………………………………….……….………….………….…………….21 

 
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10.1 
Ethical Conduct of the Study……………………………………………….……...21 
10.2 
Patient Information and Consent……………………………………………….….21 
11. Reference……………………………………………….…………………….………….………23 
 
 
 

 
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1. Introduction 
1.1 Study Objectives 
The primary objective of this study is to determine the safety of CRISPR/Cas9 mRNA Instantaneous 
Gene Editing Therapy in the form of intracorneal injected HELP. The secondary objective is to 
evaluate the HSV-1 erasing efficiency of HELP with the assistance of corneal transplantation for the 
treatment of refractory HSK patients. In addition, potential long-term off-target effect of HELP 
injection should also be determined during the 360 days of follow-ups. 
 
1.2 Backgrounds 
HELP, a mRNA-carrying lentiviral particles that include SpCas9 mRNA and HSV-1-gene-targeting 
guide RNAs (designated HSV-1-erasing lentiviral particles, termed HELP). The guide RNA 
expression cassette simultaneously were targeted two genes of HSV-1, UL8 and UL29, and co-
packaged it with SpCas9 mRNA in an mRNA-carrying lentiviral particle. The gRNA expression 
cassette is reverse transcribed and maintained as circular episomal DNA, corresponding to an 
integration-defective lentiviral vector. Lentiviral transporters have been reported increasingly in terms 
of gene editing. As reported in other studies,1-5 only transient, mild ocular inflammation following 
surgery was noted and the presence of vector was limited to the injected eye after treatment. 
 
 
2. Eligibility Criteria 
2.1 Inclusion Criteria 
Patients with refractory keratitis caused by herpes virus type I who has acute corneal perforation or 
had at least one time failed corneal transplant due to the virus relapse. 
 Age between 18 to 75 years 
 No systemic immune eye disease 
 Good eyelid structure and blink function 
 Exists the potential of visual recovery by evaluation of ocular structure and function 

 
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 Identify patients who are repeatedly infected with HSV-1 virus(only under the condition that the 
patients are obviously suffering relapse infections, corneal perforation and need corneal 
transplantation) 
 No retinal detachment and normal visual function 
 No history of corneal trauma 
 Subjects or their legal guardians voluntarily participate in this study, sign informed consent, good 
compliance and cooperation with follow-up visits 
 
2.2 Exclusion Criteria 
 Lacrimal coating and blink function loss 
 Schirmer's test result is less than 2mm for severe dry eye disease 
 Pregnant and lactating women (pregnancy defined in this study as positive urine pregnancy test) 
 Currently is involved in clinical trials of other drugs or medical devices 
 Active eye infection (including but not limited to: blepharitis, infectious conjunctivitis, keratitis, 
sclerotitis, endophthalmitis) in target eye or contralateral eye within 30 days prior to enrollment 
 Ocular surface malignant tumor 
 A history of allergic reaction or allergy to sodium luciferin, allergy to protein products used for 
treatment or diagnosis, allergy to 2 drugs or non-drug factors, or current allergic disease 
 current in an infectious disease requiring oral, intramuscular or intravenous administration 
 Patients with systemic immune diseases 
 Any uncontrolled clinical problems (such as severe mental, neurological, cardiovascular, 
respiratory and other systemic diseases and malignant neoplasms) 
 Not effective contraception 
 In uncontrolled hypertension, systolic is no less than 160 mmHg, diastolic is no less than 100 
mmHg 
 In uncontrolled diabetes, fasting glucose is no less than 10.0umol/L 

 
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 Renal insufficiency, serum creatinine is more than 133umol/L 
 Arrhythmia, myocardial ischemia, myocardial infarction (diagnosed by electrocardiogram) 
 Liver dysfunction, al ANINE aminotransferase and aspartate aminotransferase levels are higher 
than 80 IU/L 
 Platelet level is below 100,000 /uL or above 450,000 /uL 
 Hemoglobin level is below 10.0g/dL (male) or 9.0g/dL (female) 
 No anticoagulant was used, prothrombin time is higher than 16s, and thrombin time of activated 
part is higher than 50s 
 HIV infection (HIV-positive) 
 Subjects lack compliance with the study or the ability to sign informed consent 
 There are currently signs of systemic infection, including fever and ongoing antibiotic treatment 
(in this study, systemic infection was defined as deviation from normal values of white blood cells, 
lymphocytes, and neutrophils on routine blood tests) 
 Administration of Glucocorticoids and other systemic immunosuppressive drugs 
 The investigator judges other conditions unsuitable for the trial 
 
 
3. Study Implementation 
3.1 Study Design 
The enrollment of participants initiates relevant processes immediately. After the completion of 
preoperative examinations, participants with corneal perforation will be arranged for transplantation 
as soon as possible. During the usual procedure of the surgery, CRISPR/Cas9 mRNA formulation in 
the form of HELP will be administrated for the treatment of refractory HSK. The hospital observations 
of participants will persist for 7 days. The postoperative recovery condition, HSV-1 testing outcome, 
adverse effects of the participants will be examined at every follow-up time. After their hospital 
discharge on 7th day, follow-up will be conducted on 1 month, 2 months, 3 months, 6 months, and 12 
months after the surgery. The basic information is shown in the Table 1. below. 

 
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Table 1. Basic Information of Our Clinical Study 
Item 
Content 
Study Type 
Interventional (Clinical Trial) 
Estimated Enrollment 
6 participants 
Allocation 
N/A 
Intervention Model 
Single Group Assignment 
Intervention Model Description 
Single group target value: Since there is no 
similar products approved on the market or 
similar comparative treatment methods, the 
single group target value method is adopted 
Masking 
None (Open Label) 
Primary Purpose 
Treatment 
Actual Study Start Date 
November 4, 2020 
Estimated Primary Completion Date 
November 2021 
Estimated Study Completion Date 
May 2022 
 
In this study, each participant diagnosed with terminal refractory HSK will undergo HELP injection 
during the normal procedure of penetrating keratoplasty (PK). Before the surgery, they will receive 
conventional topical or systemic antiviral treatment. During the operation, single dose CRISPR/Cas9 
mRNA formulation in the form of HELP will be injected into the recipient graft bed. Then the 
participants will no longer receive any forms of antiviral treatment unless occurs an uncontrollable 
virus recurrence, when the conventional antiviral therapy continues. At follow-up visits, participants 
will undergo a series of examinations, including best-corrected visual acuity, intraocular pressure, 
photograph of anterior segment, postoperative B-scan ultrasonography, time-domain optical coherence 
tomography (OCT), spectral-domain OCT, electroretinography (ERG), ELISA of blood samples, deep 
sequencing of corneal epithelial cells, HSV-1 assessment and potential adverse events. 
 
3.2 Preparation of HELP Formulation 
HELP (BD111) was produced following Good Manufacturing Practice (GMP) guidelines. Briefly, 
293T cells were seeded in 10-tray stacks cell factory (Thermo Fisher Scientific) and transfected with 
6 packaging plasmids using PEI. Cell culture medium was refreshed 6 h post-transfection. 

 
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Supernatants were harvested 48 h later and pooled before filtration through 0.45 μm. The downstream 
purification process included a benzonase treatment overnight at 4°C, followed by ultrafiltration and 
purification using ion-exchange chromatography. The resulting BD111 was dissolved in PBS, passed 
through 0.22-μm filters, and stored at -80℃. 
 
3.3 Corneal Transplant and HELP Administration 
3.3.1 Preoperative Medications 
The preoperative medications for participants having a PK are listed below: 
 Topical antiviral drugs: Acyclovir eye drops 6 times daily and oral acyclovir tablets 3 times daily 
till the day before surgery 
 Miotics: 1% Pilocarpine drops for phakic patients undergoing PK, instilled every 10 minutes for 
30 minutes before the operation 
 Intravenous Mannitol 20%: Dosage of 1 g/kg given over 20–30 minutes before the operation to 
lower the intraocular pressure 
3.3.2 Anesthesia 
PK will be performed under retrobulbar block for patients with good cooperation or those who are at 
risk for undergoing general endotracheal anesthesia in our hospital. The procedure is performed under 
monitored anesthesia care, including the control of blood pressure, heart rate, oxygen saturation and 
anxiety. Patients should supine and look straight forward, with their heads maintained in a neutral 
position. After sterilization of the surgical area, a needle (27 Gauge, 3 cm long) is inserted at the 
inferolateral border of the bony orbit and directed straight back until its tip has passed the equator of 
the globe. The needle is then directed medially and cephalad toward the apex of the orbit. Upon a 
negative aspiration for blood, 3-4mls of local anesthetic solution is injected and the needle is then 
withdrawn. 2% Lidocaine (Xylocaine) is used as anesthetic agent. Patients are then draped in sterile 
fashion using a surgical film, which isolates eyelashes from the sterile field.  
3.3.3 Procedural Technique 
After the placement of a lid speculum, an appropriately sized trephine (usually 6.5-7.5mm) marks the 
epithelium on the host cornea prior to trephination. Once centered, trephination to approximately 90% 
depth is performed. Through the base of the trephination groove enters the anterior chamber using a 

 
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75 blade. The cornea is then carefully removed using corneal scissors, avoiding any inadvertently 
cutting or straying outside of the groove. After removing the cornea, viscoelastic is instilled to maintain 
the anterior chamber and provide protection to the intraocular tissues. The removed host tissue will be 
sent to pathology for pathogen detection. 
The donor corneal button is cut endothelial side up using a trephine-loaded punch instrument, which 
should be completed prior to trephining the host. In globe perforation participants, donor grafts should 
be oversized by 0.5-0.75 mm compared with the host bed. Once the donor button is cut, the tissue is 
carefully protected for later use. Before transferring to the recipient bed, the donor graft is irrigated to 
remove storage solution. 10-0 nylon suture and interrupted sutures are preferred for securing the donor 
tissue to the host bed. Initially 4 interrupted cardinal sutures in sequence at 12, 6, 3, 9 o’clock position 
are performed, and 8-12 more sutures may be necessary before the graft is secure. Loose ends of the 
sutures should be cut short and the knots be buried in the host tissue. Finally, additional viscoelastic is 
applied in the angle and to the anterior chamber. 
3.3.4 HELP Administration 
The HELP formulation stored in -80℃ will be transferred in 4 ℃ from the lab to the surgery room 30 
min earlier. Then it will be recovered to the room temperature 10 min before its usage, where in total 
of 0.25 ml formulation is drawn into a 27g ophthalmic syringe for the later injection. Penetrating 
keratoplasty will be performed following the routine procedures described above. After sewing up the 
donor cornea, the HELP formulation in the syringe is injected in evenly divided 6-8 locations of the 
recipient graft bed. The indication for its successful entrance into the stroma will be the visible edema 
and whitening of the corneal bed in the injection sites, which should cover the entire recipient bed area 
in the end. 
 
3.4 Post-operation Management 
 Antimicrobials: for an infectious cause, topical antimicrobials should be selected based on cultures 
after PK. Initially, the antibiotic should be broad spectrum with low toxicity (e.g. 
fluoroquinolones), typically four times daily during the first week 
 Immune Modulators: topical corticosteroid drops (such as difluprednate 0.05% ophthalmic 
suspension or prednisolone acetate 1.0% ophthalmic suspension four times daily (average). 
Steroids are tapered depending on the patient 

 
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 Intraocular Pressure Lowering Agents: intraocular Pressure should be monitored at each 
postoperative visit, agents for lowering pressure should be used if significantly elevated 
 Ocular Lubricants: non-preserved artificial tears can be started early in the postoperative period 
to maintain and preserve the corneal epithelium 
 
4. CRISPR/Cas9 Therapy Interruption 
A CRISPR/Cas9 therapy interruption is a restart of discontinued conventional anti-viral therapy, since 
the CRISPR gene editing therapy is single dose applied. The CRISPR/Cas9 therapy interruption will 
be performed under certain conditions. As HSV-1 testing result is the main outcome of this study, the 
CRISPR/Cas9 therapy interruption will only be performed when HSV-1 testing result is at a certain 
range. 
4.1 Criteria for CRISPR/Cas9 Therapy Interruption 
 A written informed consent was provided by the patient 
 HSV-1 testing results rises to excessive range (Ct value <25) 
 Noticeable corneal or systemic adverse events related to CRISPR/Cas9 therapy 
 
 
5. Quality Control 
5.1 Quality Control and Quality Assurance 
During this study, the Emergency Response Group will conduct regular visits to ensure the protocol 
and Gond Clinical Practices (GCPs) being followed. The monitors may inspect the accuracy of the 
recorded data through source documents. The investigators and their relevant personnel are available 
and cooperate during the monitoring visits and possible audits or inspections. 
5.2 Monitoring 
The potential audits and inspections, including source data verification, must be accepted by 
representatives designated by the Protocol Chair. Through these audits and inspections, monitors will 
examine whether the medical activities and documents related to this study were following the protocol, 

 
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institutional regulation, and any applicable policy requirements. Monitoring will start at the first time 
of participant registration and will continue during the entire protocol performance. 
 
 
6. Data Collection and Evaluation 
Unless noted, the procedures for all patients must be identical. Any AE/SAE will be documented and 
reported throughout the study. The period of data collection will be divided into three sections, 
including pre-study screening, study procedure and post-study follow-up. Screening procedures for 
baseline data will be taken 3~14 days prior to CRISPR/Cas9 gene editing treatment. An, and 
institutional Review Board (IRB) approved informed consent document (ICD), must be signed and 
dated before any specific procedures are done. Results from standard clinical assessments or laboratory 
tests, prior to the date of informed consent but within the allowed time restraint for screening 
procedures, can be used for determining the patient's eligibility. The patient's source documents must 
clearly document the use of pre-consent results. A screening will contain the following items: 
 Sign form of informed consent 
 Disease history and prior treatment 
 Complete ophthalmic examinations 
 Vital signs 
 Laboratory evaluation 
 Other necessary items depending on specific situations 
During the study period, each patient will receive corresponding treatment according to the protocol, 
which was elaborated in the “Study Implementation” section. After the procedure, all the patients will 
be scheduled a follow-up for 360 days or more, the patients will be followed up 7±2 days, 30±7 days, 
60±10 days 90±14 days, 180±21 days and 360±31 days after treatment, where their post-treatment 
status will be carefully evaluated. 
 
 
 

 
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7. Statistical Considerations 
This is an open-label, single-arm clinical study. The investigators conducted this research to evaluate 
the safety and efficacy of HSV-1-targeting CRISPR formulation HELP (BD111) for in vivo treatment 
of refractory HSK patients. In this study, participants will receive a single dose of HELP formulation 
by intrastromal injection during the penetrating keratoplasty for acute corneal perforation. At each 
follow-up point, a series of safety and effectiveness evaluations will be performed. 
The primary endpoint, HSV-1 in tear swabs will be evaluated at various test points according to the 
data collection plan, as summarized by simple descriptive summary. For safety evaluations, clinical 
examinations (slit-lamp examination, measurement of best-corrected visual acuity, intraocular 
pressure, B-scan ultrasonography, the time-domain OCT, the spectral-domain OCT, and the ERG) will 
be presented as simple descriptive statistics in the form of pictures, line charts or summarized tables. 
Deep sequencing of the corneal epithelial cells (acquired by corneal wipes) will also be reported as 
descriptively summarized tables. Blood ELISA will be reported graphically with the aid of the 
Student’s test. Other laboratory and imaging correlative studies will also be reported descriptively and 
graphically. 
 
 
8. Adverse Event Reporting 
8.1 Adverse Event 
AE is defined as an undesirable experience or medical condition that occurs after the administration 
of the procedures or operations specified in the protocol. AE includes all events that may or may not 
be related to the study. All observed or volunteered AEs regardless of suspected causal relationship to 
the CRISPR/Cas9 gene editing treatment will be reported as described in the following sections. For 
all AEs, the investigator must obtain adequate information both to evaluate the outcome of AEs and 
to assess whether it fits the criteria as a SAE, which requires immediate notification to designated 
representatives. Follow-ups by the investigator are also required until the event stabilizes at an 
acceptable level to the investigator. 
 
8.2 Serious Adverse Event Reporting 

 
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SAE is an AE with a potential outcome of death, prolonged hospitalization, disability or permanent 
damage, congenital anomaly/birth defect, or other serious important medical events. Specifically, SAE 
is any untoward medical occurrence that:  
 Results in death 
 Life-threatening 
 Requires extra hospitalization or prolongation of existing hospitalization 
 Results in persistent or significant disability/incapacity 
 Results in congenital anomaly/birth defect 
All suspected SAEs related to the study should be followed up according to the institution's standard 
of care, preferably in consultation with leading specialists. 
 
8.3  Recording and Reporting 
An emergency contact will be assigned to each participant in case of any unexpected circumstances. 
In occurrence of an AE, it will be graded according to the DAIDS severity grading system ICH 6: 
 Mild: participant was able to perform all normal activities 
 Moderate: the participant had to discontinue some activities due to the AE 
 Severe: the participant was incapacitated by the AE and unable to perform normal activities 
 Life-threatening: participant experienced extreme limitation in activity, significant assistance 
required; significant medical intervention/therapy required, hospitalization probable 
Investigators will assess the occurrence of AEs and SAEs at all follow up time points during the study. 
All AE/SAE will be recorded and reported in detail, with further investigation into the relationship 
between each AE and the clinical procedures. 
 
 
 
 

 
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9. Record Form & Retention 
All data related to this study in both paper and electronic form will be available only to the research 
team and the legal supervisors. The original data are the property of the investigation team and are 
inaccessible to any other third parties. The researchers from the team are ultimately responsible for the 
collection and analyze of the data. Any alterations to the original data must be documented. All the 
original data will be retained securely by the investigators both during and after the closure of the study 
in order to ensure evaluations or audits from regulatory authorities. If the investigator is unable to 
continue retaining the study records for the required period for any reason, the Emergency Response 
Group should be prospectively notified. 
 
 
10. Ethics 
The conduct of this study will abide by the laws and principles proclaimed in the Declaration of 
Helsinki. All managements and operations will be carried out under the supervision of The Institutional 
Review Board (IRB) & Hospital Ethics Committee (HEC).   
10.1 Ethical Conduct of the Study 
The study will be conducted in accordance with the protocol and the requirements according to the 
concerned laws. The project will be submitted to the supervision parties for censorship. The study will 
be initiated only with the approval of the IRB & HEC. All the data and information collected from the 
study will be presented to the supervision parties, especially the information related to AE & SAE. 
Alteration of the project plan will not be made without the consideration and authorization of the 
supervision parties. A full report will be provided after the closure of the study. 
 
10.2 Patient Information and Consent 
All participants and/or their legal representatives will be fully informed of the objectives and details 
of this study. All possible outcomes and risks will be acknowledged. A consent of written form will 
be obtained from each patient or their representatives. The consent process will be witnessed by an 
impartial supervisor. A copy of the consent will be provided if requested by the participants. The 
consent documents will be stored by the investigators along with other data collected during the study 

 
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for a period required by the protocol. All the personal information and data from the patients will only 
be available to the investigators and supervision parties to guarantee privacy. The data will not be used 
for purposes other than this study. 
 
 
 

 
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11. Reference 
1. 
Maude SL, Frey N, Shaw PA, et al. Chimeric antigen receptor T cells for sustained remissions in 
leukemia. N Engl J Med 2014;371(16):1507-17. (In eng). DOI: 10.1056/NEJMoa1407222. 
2. 
Thompson AA, Walters MC, Kwiatkowski J, et al. Gene Therapy in Patients with Transfusion-
Dependent 
β-Thalassemia. 
N 
Engl 
J 
Med 
2018;378(16):1479-1493. 
(In 
eng). 
DOI: 
10.1056/NEJMoa1705342. 
3. 
Mamcarz E, Zhou S, Lockey T, et al. Lentiviral Gene Therapy Combined with Low-Dose Busulfan in 
Infants 
with 
SCID-X1. 
N 
Engl 
J 
Med 
2019;380(16):1525-1534. 
(In 
eng). 
DOI: 
10.1056/NEJMoa1815408. 
4. 
Kohn DB, Booth C, Kang EM, et al. Lentiviral gene therapy for X-linked chronic granulomatous 
disease. Nat Med 2020;26(2):200-206. (In eng). DOI: 10.1038/s41591-019-0735-5. 
5. 
Khan A, Barber DL, Huang J, et al. Lentivirus-mediated gene therapy for Fabry disease. Nat Commun 
2021;12(1):1178. (In eng). DOI: 10.1038/s41467-021-21371-5. 
 
 
 
 
 

 
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Current Study Protocol 
 
 

 
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Protocol Approval ID: 2020023-2-v2 
In Vivo CRISPR Gene Editing in Patients with Herpes Stromal 
Keratitis 
Investigator:       Jiaxu Hong, M.D., Ph.D, M.PA. 
                 Deputy Chief Physician 
                 Department of Ophthalmology and Visual Science 
Research Institute:  Eye, and ENT Hospital, 
Shanghai Medical College, Fudan University, Shanghai, China 
Founding:         National Natural Science Foundation of China,  
81970766, 8217040684 and 31971364 
the Program for Professor of Special Appointment 
(Eastern Scholar) at Shanghai Institutions of Higher Learning, 
TP2019046 
The Shanghai Innovation Development Program, 
2020-RGZN-02033 
the Shanghai Key Clinical Research Program,  
SHDC2020CR3052B 
Startup funding from Shanghai Center for Systems Biomedicine 
Shanghai Jiao Tong University, WF220441504 
Collaborators:      Key Laboratory of Systems Biomedicine, 
Shanghai Jiao Tong University 
 
Version 2.0 
Version Date: April 12, 2020 

 
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Brief Summary: 
The purpose of this study is to evaluate the safety, tolerability and efficacy of a single dose of 
CRISPR/Cas9 mRNA Instantaneous Gene Editing Therapy administered via corneal injection in 
participants with refractory herpetic viral keratitis. 
 
Condition or disease 
Intervention/treatment 
Herpes Simplex Virus Infection 
Drug: HELP (BD111) Adult single group Dose 
Refractory Viral Keratitis 
(Administered by corneal injection surgery. Dosage 
form: injection solution. Dose: 200μL. Frequency of 
administration: one time injection.) 
 
Description: 
This is a single-arm, single dose study of mRNA-carrying HELP (HSV-1-erasing lentiviral particles) 
in adult (ages 18 to 70) participants with refractory herpetic viral keratitis. 3-6 participants will be 
enrolled and receive HELP injection during the usual procedure of corneal transplantation. HELP is a 
novel gene editing formulation designed to clear Herpes simplex virus type I (HSV-1) that results in 
herpetic stromal keratitis in both acute and recurrent infection models which is the leading factor for 
infectious blindness. The main outcomes of this study include HSV-1 testing outcome, graft survival, 
visual acuity and adverse effects of the participants. 
 
 
Outcomes Measures: 
1. Primary Outcome Measure 
HSV-1 testing results of the intervention eye. Tear swab of 1 week, 1 month, 2 months, 3 months, 6 
months and 12 months post-operation will be collected and quantified for HSV-1 genome by real-time 
polymerase chain reaction (RT-PCR). The virus testing outcome was indicated as positive (+) or 
negative (-) by Ct values. Ct value of above 40 will be considered negative, 36-40 be considered 
weakly positive, and below 35 will be considered a strong positive. 

 
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2. Secondary Outcome Measure 
 Patient graft survival. The corneal grafts of participants will be monitored under a complete 
ophthalmic examination, including slit-lamp examination and anterior segment optical coherence 
tomography (OCT), at every follow up visit. 
 Best-corrected visual acuity. Changes of visual acuity of participants along the clinical trial. 
 Adverse effects, including possible alteration of ocular structure and function, Cas9-specific 
immunoreaction or off-target editing of CRISPR in the bloodstream. Postoperative B-scan 
ultrasonography, time-domain OCT, spectral-domain OCT, and electroretinography (ERG) will 
be used to evaluate ocular status. ELISA and Whole-genome sequencing (WGS) will be conducted 
on the blood samples collected at 3-month and 6-month visit, respectively. 
 
 
 
 

 
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Table of Contents 
1. Introduction………………………………………………………………………………..........30 
1.1 Study Objects……….……………………………………………………………..........30 
1.2 Backgrounds of HELP………………….…….……………………………………........30 
2. Eligibility Criteria………………………………………………………………………………30 
2.1 Inclusion Criteria…………………………………………………………………..........30 
2.2 Exclusion Criteria……………………………………………………….………………31 
3. Study Implementation…………………………………………………….…………………….32 
3.1 Study Design……………………………………………………………………………32 
3.2 Preparation of HELP Formulation………………………………………………………33 
3.3 Corneal Transplant and HELP Administration………………………….………………34 
3.4 Post-operation Management………………………………………………….…………35 
4. CRISPR/Cas9 Therapy Interruption…………………………….………………….…………36 
5. Quality Control……………………………………………………………………….…………36 
5.1 Quality Control and Quality Assurance…………………………………….…………...36 
5.2 Monitoring…………………………………………………………………………. ….36 
6. Data Collection and Evaluation……………………………………………….……………….37 
7. Statistical Considerations……………………………………………….……………………...38 
8. Adverse Event Reporting……………………………………………………………………….38 
8.1 Adverse Event……………………………………………….………………………….38 
8.2 Serious Adverse Event……………………………………………….………….……...38 
8.3 Recording and Reporting……………………………………………….……………….39 
9. Record Form & Retention……………………………………………….………….………….40 
10. Ethics……………………………………………….……….………….………….…………….40 

 
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10.1 
Ethical Conduct of the Study……………………………………………….……...40 
10.2 
Patient Information and Consent……………………………………………….….40 
11. Reference……………………………………………….…………………….………….………42 
 
 
 

 
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1. Introduction 
1.1 Study Objectives 
The primary objective of this study is to evaluate the HSV-1 erasing efficiency of HELP with the 
assistance of corneal transplantation for the treatment of refractory HSK patients. The secondary 
objective is to determine the safety of CRISPR/Cas9 mRNA Instantaneous Gene Editing Therapy in 
the form of intracorneal injected HELP. In addition, potential long-term off-target effect of HELP 
injection should also be determined during the 360 days of follow-ups. 
 
1.2 Backgrounds 
HELP, mRNA-carrying lentiviral particle that delivers SpCas9 mRNA and a guide RNA-expressing 
cassette targeting HSV-1 genome (designated HSV-1-erasing lentiviral particle, termed HELP). The 
viral genome encodes two guide RNAs simultaneously targeting HSV-1 UL8 and UL29 gene, 
respectively, and is co-packaged with SpCas9 mRNA in the lentiviral particle. The gRNA expression 
cassette is reverse transcribed in the integration-defective lentiviral vector and maintained as circular 
episomal DNA in the nucleus. Lentiviral delivery has been reported increasingly in gene therapy and 
is tolerated in the clinical trials as reported in other studies.1-5 In the preclinical study, HELP efficiently 
blocks HSV-1 replication and the occurrence of herpetic stromal keratitis (HSK) in different corneal 
infection models without finding notable adverse effects.6  
 
 
2. Eligibility Criteria 
2.1 Inclusion Criteria 
 Age between 18 to 70 years old; 
 No systemic immune eye disease; 
 Good eyelid structure and blink function; 
 Exists the potential of visual recovery by evaluation of ocular structure and function; 

 
Page|31 
 Patients with refractory keratitis who are repeatedly infected with HSV-1 virus (more than three 
times per year) and resistant to topical or systemic anti-viral agents, with no response to regular 
immunosuppressive agents; 
 Patients who are obviously suffering from relapse HSV infections with corneal perforation, 
requiring corneal transplantation; 
 No history of corneal trauma; 
 Subjects or their legal guardians voluntarily participate in this study, sign informed consent, good 
compliance and cooperation with follow-up visits. 
 
2.2 Exclusion Criteria 
 Lacrimal coating and blink function loss; 
 Schirmer's test result is less than 2mm for severe dry eye disease; 
 Pregnant and lactating women (pregnancy defined in this study as positive urine pregnancy test); 
 Currently is involved in clinical trials of other drugs or medical devices; 
 Active eye infection (including but not limited to: blepharitis, infectious conjunctivitis, keratitis, 
sclerotitis, endophthalmitis) in target eye or contralateral eye within 30 days prior to enrollment; 
 Ocular surface malignant tumor; 
 A history of allergic reaction or allergy to sodium luciferin, allergy to protein products used for 
treatment or diagnosis, allergy to ≥ 2 drugs or non-drug factors, or current allergic disease; 
 current in an infectious disease requiring oral, intramuscular or intravenous administration; 
 Patients with systemic immune diseases; 
 Any uncontrolled clinical problems (such as severe mental, neurological, cardiovascular, 
respiratory and other systemic diseases and malignant neoplasms); 
 Not effective contraception; 
 In uncontrolled hypertension, systolic is no less than 160 mmHg, diastolic is no less than 100 
mmHg; 
 In uncontrolled diabetes, fasting glucose is no less than 10.0umol/L; 

 
Page|32 
 Renal insufficiency, serum creatinine is more than 133umol/L; 
 Arrhythmia, myocardial ischemia, myocardial infarction (diagnosed by electrocardiogram); 
 Liver dysfunction, al ANINE aminotransferase and aspartate aminotransferase levels are higher 
than 80 IU/L; 
 Platelet level is below 100,000 /μL or above 450,000 /μL; 
 Hemoglobin level is below 10.0g/dL (male) or 9.0g/dL (female); 
 No anticoagulant was used, prothrombin time is higher than 16s, and thrombin time of activated 
part is higher than 50s; 
 HIV infection (HIV-positive); 
 Subjects lack compliance with the study or the ability to sign informed consent; 
 There are currently signs of systemic infection, including fever and ongoing antibiotic treatment 
(in this study, systemic infection was defined as deviation from normal values of white blood cells, 
lymphocytes, and neutrophils on routine blood tests); 
 Administration of Glucocorticoids and other systemic immunosuppressive drugs; 
 The investigator judges other conditions unsuitable for the trial 
 
 
3. Study Implementation 
3.1 Study Design 
The enrollment of participants initiates relevant processes immediately. After the completion of 
preoperative examinations, participants with corneal perforation will be arranged for transplantation 
as soon as possible. During the usual procedure of the surgery, CRISPR/Cas9 mRNA formulation in 
the form of HELP will be administrated for the treatment of refractory HSK. The hospital observations 
of participants will persist for 7 days. The postoperative recovery condition, HSV-1 testing outcome, 
adverse effects of the participants will be examined at every follow-up time. After their hospital 
discharge on 7th day, follow-up will be conducted on 1 month, 2 months, 3 months, 6 months and 12 
months after the surgery. The basic information is shown in the Table 1. below. 

 
Page|33 
 
 
Table 1. Basic Information of Our Clinical Study 
Item 
Content 
Study Type 
Interventional (Clinical Trial) 
Estimated Enrollment 
3-6 participants 
Allocation 
N/A 
Intervention Model 
Single Group Assignment 
Intervention Model Description 
Single group target value: Since there is no 
similar products approved on the market or 
similar comparative treatment methods, the 
single group target value method is adopted 
Masking 
None (Open Label) 
Primary Purpose 
Treatment 
Actual Study Start Date 
November 4, 2020 
Estimated Primary Completion Date 
November 2021 
Estimated Study Completion Date 
May 2022 
 
In this study, each participant diagnosed with terminal refractory HSK will undergo HELP injection 
during the normal procedure of penetrating keratoplasty (PK). Before the surgery, they will receive 
conventional topical or systemic antiviral treatment. During the operation, single dose CRISPR/Cas9 
mRNA formulation in the form of HELP will be injected into the recipient graft bed. Then the 
participants will no longer receive any forms of antiviral treatment unless occurs an uncontrollable 
virus recurrence, when the conventional antiviral therapy continues. At follow-up visits, participants 
will undergo a series of examinations, including best-corrected visual acuity, intraocular pressure, 
photograph of anterior segment, postoperative B-scan ultrasonography, time-domain optical coherence 
tomography (OCT), spectral-domain OCT, electroretinography (ERG), ELISA of blood samples, deep 
sequencing of corneal epithelial cells, HSV-1 testing and potential adverse events. 
 
3.2 Preparation of HELP Formulation 

 
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HELP (BD111) was produced following Good Manufacturing Practice (GMP) guidelines. Briefly, 
293T cells were seeded in 10-tray stacks cell factory (Thermo Fisher Scientific) and transfected with 
6 packaging plasmids using PEI. Cell culture medium was refreshed 6 h post-transfection. 
Supernatants were harvested 48 h later and pooled before filtration through 0.45 μm. The downstream 
purification process included a benzonase treatment overnight at 4°C, followed by ultrafiltration and 
purification using ion-exchange chromatography. The resulting BD111 was dissolved in PBS, passed 
through 0.22-μm filters, and stored at -80℃. 
 
3.3 Corneal Transplant and HELP Administration 
3.3.1 Preoperative Medications 
The preoperative medications for participants having a PK are listed below: 
 Topical antiviral drugs: Acyclovir eye drops 6 times daily and oral acyclovir tablets 3 times daily 
till the day before surgery 
 Miotics: 1% Pilocarpine drops for phakic patients undergoing PK, instilled every 10 minutes for 
30 minutes before the operation 
 Intravenous Mannitol 20%: Dosage of 1 g/kg given over 20–30 minutes before the operation to 
lower the intraocular pressure 
3.3.2 Anesthesia 
PK will be performed under retrobulbar block for patients with good cooperation or those who are at 
risk for undergoing general endotracheal anesthesia in our hospital. The procedure is performed under 
monitored anesthesia care, including the control of blood pressure, heart rate, oxygen saturation and 
anxiety. Patients should supine and look straight forward, with their heads maintained in a neutral 
position. After sterilization of the surgical area, a needle (27 Gauge, 3 cm long) is inserted at the 
inferolateral border of the bony orbit and directed straight back until its tip has passed the equator of 
the globe. The needle is then directed medially and cephalad toward the apex of the orbit. Upon a 
negative aspiration for blood, 3-4mls of local anesthetic solution is injected and the needle is then 
withdrawn. 2% Lidocaine (Xylocaine) is used as anesthetic agent. Patients are then draped in sterile 
fashion using a surgical film, which isolates eyelashes from the sterile field.  
3.3.3 Procedural Technique 

 
Page|35 
After the placement of a lid speculum, an appropriately sized trephine (usually 6.5-7.5mm) marks the 
epithelium on the host cornea prior to trephination. Once centered, trephination to approximately 90% 
depth is performed. Through the base of the trephination groove enters the anterior chamber using a 
75 blade. The cornea is then carefully removed using corneal scissors, avoiding any inadvertently 
cutting or straying outside of the groove. After removing the cornea, viscoelastic is instilled to maintain 
the anterior chamber and provide protection to the intraocular tissues. The removed host tissue will be 
sent to pathology for pathogen detection. 
The donor corneal button is cut endothelial side up using a trephine-loaded punch instrument, which 
should be completed prior to trephining the host. In globe perforation participants, donor grafts should 
be oversized by 0.5-0.75 mm compared with the host bed. Once the donor button is cut, the tissue is 
carefully protected for later use. Before transferring to the recipient bed, the donor graft is irrigated to 
remove storage solution. 10-0 nylon suture and interrupted sutures are preferred for securing the donor 
tissue to the host bed. Initially 4 interrupted cardinal sutures in sequence at 12, 6, 3, 9 o’clock position 
are performed, and 8-12 more sutures may be necessary before the graft is secure. Loose ends of the 
sutures should be cut short and the knots be buried in the host tissue. Finally, additional viscoelastic is 
applied in the angle and to the anterior chamber. 
3.3.4 HELP Administration 
The HELP formulation stored in -80℃ will be transferred in 4 ℃ from the lab to the surgery room 30 
min earlier. Then it will be recovered to the room temperature 10 min before its usage, where in total 
of 0.25 ml formulation is drawn into a 27g ophthalmic syringe for the later injection. Penetrating 
keratoplasty will be performed following the routine procedures described above. After sewing up the 
donor cornea, the HELP formulation in the syringe is injected in evenly divided 6-8 locations of the 
recipient graft bed. The indication for its successful entrance into the stroma will be the visible edema 
and whitening of the corneal bed in the injection sites, which should cover the entire recipient bed area 
in the end. 
 
3.4 Post-operation Management 
 Antimicrobials: for an infectious cause, topical antimicrobials should be selected based on cultures 
after PK. Initially, the antibiotic should be broad spectrum with low toxicity (e.g. 
fluoroquinolones), typically four times daily during the first week 

 
Page|36 
 Immune Modulators: topical corticosteroid drops (such as difluprednate 0.05% ophthalmic 
suspension or prednisolone acetate 1.0% ophthalmic suspension four times daily (average). 
Steroids are tapered depending on the patient 
 Intraocular Pressure Lowering Agents: intraocular Pressure should be monitored at each 
postoperative visit, agents for lowering pressure should be used if significantly elevated 
 Ocular Lubricants: non-preserved artificial tears can be started early in the postoperative period 
to maintain and preserve the corneal epithelium 
 
 
4. CRISPR/Cas9 Therapy Interruption 
A CRISPR/Cas9 therapy interruption is a restart of discontinued conventional anti-viral therapy, since 
the CRISPR gene editing therapy is single dose applied. The CRISPR/Cas9 therapy interruption will 
be performed under certain conditions. As HSV-1 testing results is the main outcome of this study, the 
CRISPR/Cas9 therapy interruption will only be performed when virus testing outcome is at a certain 
range. 
4.1 Criteria for CRISPR/Cas9 Therapy Interruption 
 A written informed consent was provided by the patient 
 HSV-1 testing outcome rises to excessive range (Ct value <25) 
 Noticeable corneal or systemic adverse events related to CRISPR/Cas9 therapy 
 
 
5. Quality Control 
5.1 Quality Control and Quality Assurance 
During this study, the Emergency Response Group will conduct regular visits to ensure the protocol 
and Gond Clinical Practices (GCPs) being followed. The monitors may inspect the accuracy of the 
recorded data through source documents. The investigators and their relevant personnel are available 
and cooperate during the monitoring visits and possible audits or inspections. 

 
Page|37 
5.2 Monitoring 
The potential audits and inspections, including source data verification, must be accepted by 
representatives designated by the Protocol Chair. Through these audits and inspections, monitors will 
examine whether the medical activities and documents related to this study were following the protocol, 
institutional regulation, and any applicable policy requirements. Monitoring will start at the first time 
of participant registration and will continue during the entire protocol performance. 
 
 
6. Data Collection and Evaluation 
Unless noted, the procedures for all patients must be identical. Any AE/SAE will be documented and 
reported throughout the study. The period of data collection will be divided into three sections, 
including pre-study screening, study procedure and post-study follow-up. Screening procedures for 
baseline data will be taken 3~14 days prior to CRISPR/Cas9 gene editing treatment. An, and 
institutional Review Board (IRB) approved informed consent document (ICD), must be signed and 
dated before any specific procedures are done. Results from standard clinical assessments or laboratory 
tests, prior to the date of informed consent but within the allowed time restraint for screening 
procedures, can be used for determining the patient's eligibility. The patient's source documents must 
clearly document the use of pre-consent results. A screening will contain the following items: 
 Sign form of informed consent 
 Disease history and prior treatment 
 Complete ophthalmic examinations 
 Vital signs 
 Laboratory evaluation 
 Other necessary items depending on specific situations 
During the study period, each patient will receive corresponding treatment according to the protocol, 
which was elaborated in the “Study Implementation” section. After the procedure, all the patients will 
be scheduled a follow-up for 360 days or more, the patients will be followed up 7±2 days, 30±7 days, 
60±10 days 90±14 days, 180±21 and 360±31 days after treatment, where their post-treatment status 
will be carefully evaluated. 

 
Page|38 
 
 
7. Statistical Considerations 
This is an open-label, single-arm clinical study. The investigators conducted this research to evaluate 
the safety and efficacy of HSV-1-targeting CRISPR formulation HELP for in vivo treatment of 
refractory HSK patients. In this study, participants will receive a single dose of HELP formulation by 
intrastromal injection during the penetrating keratoplasty for acute corneal perforation. At each follow-
up point, a series of safety and effectiveness evaluations will be performed. 
The primary endpoint, HSV-1 in tear swabs will be evaluated at various test points according to the 
data collection plan, as summarized by simple descriptive summary. For safety evaluations, clinical 
examinations (slit-lamp examination, measurement of best-corrected visual acuity, intraocular 
pressure, B-scan ultrasonography, the time-domain OCT, the spectral-domain OCT, and the ERG) will 
be presented as simple descriptive statistics in the form of pictures, line charts or summarized tables. 
Deep sequencing of the corneal epithelial cells (acquired by corneal wipes) will also be reported as 
descriptively summarized tables. Blood ELISA will be reported graphically with the aid of the 
Student’s test. Other laboratory and imaging correlative studies will also be reported descriptively and 
graphically. 
 
 
8. Adverse Event Reporting 
8.1 Adverse Event 
AE is defined as an undesirable experience or medical condition that occurs after the administration 
of the procedures or operations specified in the protocol. AE includes all events that may or may not 
be related to the study. All observed or volunteered AEs regardless of suspected causal relationship to 
the CRISPR/Cas9 gene editing treatment will be reported as described in the following sections. For 
all AEs, the investigator must obtain adequate information both to evaluate the outcome of AEs and 
to assess whether it fits the criteria as a SAE, which requires immediate notification to designated 
representatives. Follow-ups by the investigator are also required until the event stabilizes at an 
acceptable level to the investigator. 

 
Page|39 
 
8.2 Serious Adverse Event Reporting 
SAE is an AE with a potential outcome of death, prolonged hospitalization, disability or permanent 
damage, congenital anomaly/birth defect, or other serious important medical events. Specifically, SAE 
is any untoward medical occurrence that:  
 Results in death 
 Life-threatening 
 Requires extra hospitalization or prolongation of existing hospitalization 
 Results in persistent or significant disability/incapacity 
 Results in congenital anomaly/birth defect 
All suspected SAEs related to the study should be followed up according to the institution's standard 
of care, preferably in consultation with leading specialists. 
 
8.3 Recording and Reporting 
An emergency contact will be assigned to each participant in case of any unexpected circumstances. 
In occurrence of an AE, it will be graded according to the DAIDS severity grading system ICH 6: 
 Mild: participant was able to perform all normal activities 
 Moderate: the participant had to discontinue some activities due to the AE 
 Severe: the participant was incapacitated by the AE and unable to perform normal activities 
 Life-threatening: participant experienced extreme limitation in activity, significant assistance 
required; significant medical intervention/therapy required, hospitalization probable 
Investigators will assess the occurrence of AEs and SAEs at all follow up time points during the study. 
All AE/SAE will be recorded and reported in detail, with further investigation into the relationship 
between each AE and the clinical procedures. 
 
 

 
Page|40 
 
 
9. Record Form & Retention 
All data related to this study in both paper and electronic form will be available only to the research 
team and the legal supervisors. The original data are the property of the investigation team and are 
inaccessible to any other third parties. The researchers from the team are ultimately responsible for the 
collection and analyze of the data. Any alterations to the original data must be documented. All the 
original data will be retained securely by the investigators both during and after the closure of the study 
in order to ensure evaluations or audits from regulatory authorities. If the investigator is unable to 
continue retaining the study records for the required period for any reason, the Emergency Response 
Group should be prospectively notified. 
 
 
10. Ethics 
The conduct of this study will abide by the laws and principles proclaimed in the Declaration of 
Helsinki. All managements and operations will be carried out under the supervision of The Institutional 
Review Board (IRB) & Hospital Ethics Committee (HEC).   
10.1 Ethical Conduct of the Study 
The study will be conducted in accordance with the protocol and the requirements according to the 
concerned laws. The project will be submitted to the supervision parties for censorship. The study will 
be initiated only with the approval of the IRB & HEC. All the data and information collected from the 
study will be presented to the supervision parties, especially the information related to AE & SAE. 
Alteration of the project plan will not be made without the consideration and authorization of the 
supervision parties. A full report will be provided after the closure of the study. 
 
10.2 Patient Information and Consent 
All participants and/or their legal representatives will be fully informed of the objectives and details 
of this study. All possible outcomes and risks will be acknowledged. A consent of written form will 
be obtained from each patient or their representatives. The consent process will be witnessed by an 

 
Page|41 
impartial supervisor. A copy of the consent will be provided if requested by the participants. The 
consent documents will be stored by the investigators along with other data collected during the study 
for a period required by the protocol. All the personal information and data from the patients will only 
be available to the investigators and supervision parties to guarantee privacy. The data will not be used 
for purposes other than this study. 
 
 
 
 

 
Page|42 
11  Reference 
1. 
Maude SL, Frey N, Shaw PA, et al. Chimeric antigen receptor T cells for sustained remissions in 
leukemia. N Engl J Med 2014;371(16):1507-17. (In eng). DOI: 10.1056/NEJMoa1407222. 
2. 
Thompson AA, Walters MC, Kwiatkowski J, et al. Gene Therapy in Patients with Transfusion-
Dependent 
β-Thalassemia. 
N 
Engl 
J 
Med 
2018;378(16):1479-1493. 
(In 
eng). 
DOI: 
10.1056/NEJMoa1705342. 
3. 
Mamcarz E, Zhou S, Lockey T, et al. Lentiviral Gene Therapy Combined with Low-Dose Busulfan in 
Infants 
with 
SCID-X1. 
N 
Engl 
J 
Med 
2019;380(16):1525-1534. 
(In 
eng). 
DOI: 
10.1056/NEJMoa1815408. 
4. 
Kohn DB, Booth C, Kang EM, et al. Lentiviral gene therapy for X-linked chronic granulomatous 
disease. Nat Med 2020;26(2):200-206. (In eng). DOI: 10.1038/s41591-019-0735-5. 
5. 
Khan A, Barber DL, Huang J, et al. Lentivirus-mediated gene therapy for Fabry disease. Nat Commun 
2021;12(1):1178. (In eng). DOI: 10.1038/s41467-021-21371-5. 
6.     Yin D, Ling SK, Wang DW, et al. Targeting herpes simplex virus with CRISPR-Cas9 cures herpetic 
stromal keratitis in mice. Nat Biotechnol 2021;39(5):567-577. DOI: 10.1038/s41587-020-00781-8. 
 
 
 

 
Page|43 
Summary of Protocol Changes 
 
 

 
Page|44 
Section of Current 
protocol 
Rationale 
Current Version 
Basic Information 
Adjust the number of 
participants enrolled 
3-6 participants will be enrolled and 
receive HELP injection during the usual 
procedure of corneal transplantation. 
Outcomes Measures 
Adjust the contents and 
priorities of outcomes 
Primary outcome: HSV-1 testing outcome 
of the intervention eye;  
Secondary outcome: patient graft survival; 
best-corrected visual acuity; adverse 
effects 
2.1 
Adjust the inclusion criteria 
Patients with refractory keratitis caused by 
herpes virus type I who has acute corneal 
perforation or had at least one time failed 
corneal transplant due to the virus relapse. 
 
 
 

 
Page|45 
Original and Current Statistical Analysis Plan 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
Page|46 
Statistical Analysis Plan 
This is an open-label, single-arm, non-randomized interventional trial. The investigators conducted 
this research to evaluate the safety and efficacy of HSV-1-targeting CRISPR formulation HELP 
(BD111) for in vivo treatment of refractory HSK patients. In this study, participants will receive a 
single dose of HELP formulation by intrastromal injection during the penetrating keratoplasty for acute 
corneal perforation. At each follow-up point, a series of safety and effectiveness evaluations will be 
performed. 
The primary endpoint, HSV-1 in tear swabs will be evaluated at various test points according to the 
data collection plan, as summarized by simple descriptive summary. For safety evaluations, clinical 
examinations (slit-lamp examination, measurement of best-corrected visual acuity, intraocular 
pressure, B-scan ultrasonography, the time-domain OCT, the spectral-domain OCT, and the ERG) will 
be presented as simple descriptive statistics in the form of pictures, line charts or summarized tables. 
Deep sequencing of the corneal epithelial cells (acquired by corneal wipes) will also be reported as 
descriptively summarized tables. Blood ELISA will be reported graphically with the aid of the 
Student’s test. Other laboratory and imaging correlative studies will also be reported descriptively and 
graphically. Data were analysed using GraphPad Prism 8 and presented as mean ± SEM in all 
experiments (n=3). Unpaired two-tailed Student’s t-tests were performed to determine the P values 
(95% confidence interval). n.s., non-significant.