Human Umbilical Cord Mesenchymal Stem Cells to Treat Neuromyelitis Optica Spectrum Disorder (hUC-MSC-NMOSD): A Study Protocol for a Prospective, Multicenter, Randomized, Placebo-Controlled Clinical Trial


 Background：Neuromyelitis optica spectrum disorder (NMOSD) is a severe relapsing and disabling inflammatory autoimmune disease of the central nervous system. Despite the progress made in understanding the pathogenesis of the disease, the optimal first line treatment to reduce relapse rate and ameliorate neurological disability remains unclear. Mesenchymal stem cells (MSC) are known for having anti-inflammatory and regenerative properties and there are a number of studies on the use of human umbilical cord MSCs (hUC-MSCs) in NMOSD patients. Therefore, we will conduct a prospective, multicenter, randomized, placebo-controlled clinical trial to study the safety and effectiveness of hUC-MSCs in the treatment of NMOSD.Methods：The trial is planned to recruit 430 AQP4-IgG seropositive NMOSD patients. The multicenter study will be conducted in six clinical centers. The whole clinical trial consists of three consecutive stages. The first stage will be carried out in the leading center (Ren Ji Hospital) only and it will last 24 months. In the first stage the primary objective is to evaluate the safety of hUC-MSCs. Patients (n=30) will be treated with three different doses of hUC-MSC: the low dose group (n=10, 1×106 MSC / kg·weight), the medium dose group (n=10, 2×106 MSC / kg·weight) and the high dose group (n=10, 5×106 MSC / kg·weight). The second stage, which aims to find the optimal dosage, will last 24 months and will be carried out in six centers (one leading center and five branch centers). Patients (n=160) will be randomized into four groups by 1:1:1:1 allocation ratio: the low, medium, high dose groups and the controlled group (n=40). The third stage, which aims to evaluate the effectiveness, will lasts for 24 months and will be developed in six centers. Patients (n=320) will be 1:1 randomized into two groups: the optimal dose group (n=160) and the controlled group (n=160). HUC-MSC infusion will be given four times to the intervention groups and stem cell solution will be given four times to the control group every 3 months; besides the primary drugs will be provided to both groups. Primary endpoint is the first recurrent time and secondary endpoints are the recurrent times, EDSS scores, MRI lesion numbers, OSIS scores, Hauser walking index and SF-36 (quality of life short Form-36) scores. Exploratory endpoints will include: serum lymphocyte subsets, cytokines, complements, serum anti-AQP4 antibody titers etc. Endpoint events and side-effects will be evaluated at baseline and every 3 months for a total of 24 months follow-up.Discussion：Although hUC-MSC has shown promising treatment effect of NMOSD in preclinical study, there is still a lack of well-designed clinical trials to evaluate the safety and effectiveness of hUC-MSC among NMOSD patients. As far as we know, this trial will be the first one to systematically demonstrate the clinical safety and efficacy of hUC-MSC in treating NMOSD and might be able to determine the optimal dose of hUC-MSC for NMOSD patients.Trial registration：The study was registered with Chinese Clinical Trial Registry (CHICTR.org.cn) on Mar 2, 2016 (registration No. ChiCTR-INR-16008037), and the revised trial protocol (Protocol version 1.2.1) was released on March 16, 2020.


Abstract
Background Neuromyelitis optica spectrum disorder (NMOSD) is a severe relapsing and disabling in ammatory autoimmune disease of the central nervous system. Despite the progress made in understanding the pathogenesis of the disease, the optimal rst line treatment to reduce relapse rate and ameliorate neurological disability remains unclear. Mesenchymal stem cells (MSC) are known for having anti-in ammatory and regenerative properties and there are a number of studies on the use of human umbilical cord MSCs (hUC-MSCs) in NMOSD patients. Therefore, we will conduct a prospective, multicenter, randomized, placebo-controlled clinical trial to study the safety and effectiveness of hUC-MSCs in the treatment of NMOSD.

Methods
The trial is planned to recruit 430 AQP4-IgG seropositive NMOSD patients. The multicenter study will be conducted in six clinical centers. The whole clinical trial consists of three consecutive stages. The rst stage will be carried out in the leading center (Ren Ji Hospital) only and it will last 24 months. In the rst stage the primary objective is to evaluate the safety of hUC-MSCs. Patients (n=30) will be treated with three different doses of hUC-MSC: the low dose group (n=10, 1×10 6 MSC / kg·weight), the medium dose group (n=10, 2×10 6 MSC / kg·weight) and the high dose group (n=10, 5×10 6 MSC / kg·weight). The second stage, which aims to nd the optimal dosage, will last 24 months and will be carried out in six centers (one leading center and ve branch centers). Patients (n=160) will be randomized into four groups by 1:1:1:1 allocation ratio: the low, medium, high dose groups and the controlled group (n=40). The third stage, which aims to evaluate the effectiveness, will lasts for 24 months and will be developed in six centers. Patients (n=320) will be 1:1 randomized into two groups: the optimal dose group (n=160) and the controlled group (n=160). HUC-MSC infusion will be given four times to the intervention groups and stem cell solution will be given four times to the control group every 3 months; besides the primary drugs will be provided to both groups. Primary endpoint is the rst recurrent time and secondary endpoints are the recurrent times, EDSS scores, MRI lesion numbers, OSIS scores, Hauser walking index and SF-36 (quality of life short Form-36) scores. Exploratory endpoints will include: serum lymphocyte subsets, cytokines, complements, serum anti-AQP4 antibody titers etc. Endpoint events and sideeffects will be evaluated at baseline and every 3 months for a total of 24 months follow-up.

Discussion
Although hUC-MSC has shown promising treatment effect of NMOSD in preclinical study, there is still a lack of well-designed clinical trials to evaluate the safety and effectiveness of hUC-MSC among NMOSD patients. As far as we know, this trial will be the rst one to systematically demonstrate the clinical safety and e cacy of hUC-MSC in treating NMOSD and might be able to determine the optimal dose of hUC-MSC for NMOSD patients.

Trial registration
The study was registered with Chinese Clinical Trial Registry (CHICTR.org.cn) on Mar  Neuromyelitis optica spectrum disorder (NMOSD) is a severe disabling in ammatory autoimmune disease of the central nervous system (CNS) featured with recurrent relapses of optic neuritis and longitudinally extensive transverse myelitis [1]. Autoantibodies against the water channel protein aquaporin-4 (AQP4) are the diagnostic markers of the disorder and more than two-thirds of patients meeting clinical criteria for NMOSD are AQP4-IgG seropositive [2,3]. Frequent relapses are associated with a stepwise accumulation of neurological disability. Therefore, relapse prevention is particularly important to reduce the risk of a systemic disability over time [4].
Azathioprine, mycophenolate mofetil, and rituximab are the most commonly used therapies for patients with NMOSD. However, a substantial number of patients who have had these therapies have relapsed [5,6]. So far, the optimal rst line treatment to reduce relapse rate remains unclear. Furthermore, no drug has been proved to improve neurological disability in NMOSD patients.
Mesenchymal stem cells (MSCs) are stromal precursor cells residing in many tissues, including bone marrow, umbilical cord (UC), fetal liver and adipose tissue [7][8][9]. MSCs are considered to be multipotent, and have antiin ammatory as well as regenerative properties [10,11]. MSCs derived from the human umbilical cord (hUC-MSCs) are more primitive, and possess multiple advantages including ethical agreeableness, a less invasive procedure for isolation, low immunogenicity, high proliferation capacity, and multi-lineage differentiation capability [12,13].
Treatment with MSC improves the course of the preclinical model of multiple sclerosis, experimental autoimmune encephalomyelitis (EAE), when administered at early stages. In EAE, MSC have a profound antiin ammatory and immune-modulating effect, but they also exhibit neuroprotective features and foster remyelination endogenous neurogenesis with scarce evidence of differentiation in neural cells [14][15][16]. A recent pilot clinical trial showed that infusion of autologous MSC derived from bone marrow is safe, can reduce the relapse frequency, and mitigates neurological disability with the recovery of neural structures in the optic nerve and spinal cord in NMOSD patients [17].
However, as far as we know, there are very few reports on the use of hUC-MSCs in NMOSD patients. Here we present the protocol of human Umbilical Cord Mesenchymal Stem Cells to Treat Neuromyelitis Optica Spectrum Disorder (hUC-MSC-NMOSD), a prospective, multicenter, randomized, placebo-controlled clinical trial.

Setting and participants
The study will be conducted in six clinical centers in Shanghai, China. The leading center is Shanghai Ren Ji Hospital, which is one of the main teaching hospitals of the Shanghai Jiao Tong University School of medicine. Ren Ji Hospital is also one of the biggest clinical centers of neuroimmunological disorders in China. The ve branch centers are: Shanghai Rui Jin Hospital, Shanghai Xin Hua Hospital, Shanghai General Hospital, Shanghai Sixth People's Hospital and Shanghai Ninth People's Hospital. In this study protocol, only the patients who ful lled the diagnostic criteria of NMOSD established by Wingerchuket al. in 2015 [18] will be included.
The study protocol has been approved by the Ethics Committees of Ren Ji Hospital (2016-071K).
The clinical study will be conducted in accordance with all national regulations. A signed written informed consent form will be obtained from all the participants before enrollment. This clinical trial complies with the principles of the World Medical Association Declaration of Helsinki-Ethical Principles for Medical Research Involving Human Subjects, with all its amendments, as well as with the principles of Good Clinical Practice.

Study design
This is a prospective study for an open-labeled, prospective, multicenter, randomized, placebo-controlled clinical trial. The estimated study time period is from Jan 2017 to Dec 2022. NMOSD patients in the remission phase will be randomized into two groups: the treatment group (hUC-MSC infusion plus regular treatment) and the controlled group (stem cell base solution infusion plus regular treatment). All the patients will be treated and followed up for two years.
The whole clinical trial includes three consecutive stages.
The rst stage of the trial, which will last 24 months, is an open-labeled study and will be carried out in the leading center only. The aim of the rst stage is to evaluate the safety of the infusion of hUC-MSC in NMOSD patients. Patients (n = 30) will be treated with three different doses of hUC-MSC. According to the process of enrollment, the rst to the tenth participant are in the low dose group (n = 10, 1 × 10 6 MSC / kg·weight); the eleventh to the twentieth participant are in the medium dose group (n = 10, 2 × 10 6 MSC / kg·weight); the twentyrst to thirtieth participant are in the high dose group (n = 10, 5 × 10 6 MSC / kg·weight). The study starts from the low dose. Only when the lower dose group ends the observation period of 3 months, the study will proceed to a higher dose group. In case of unacceptable toxic reaction occurs in the dose escalation, the study will be ended immediately. In relation to safety consideration, before enrolling the patients in the low dose group, we will give a single medium dose of hUC-MSC to one patient and follow up the therapy for one year. The outcome of this dosage will be used only for safety evaluation, not to evaluate the therapy effectiveness.
The second and third stages of the study protocol are part of the multi-center double blinded randomized clinical trial. The second stage, which aims to screen out the optimal dosage, will last 24 months and will be carried out in six centers (one leading center and ve branch centers). Patients (n = 160) will be randomized into four groups: the low, medium, high dose groups and the controlled group (n = 40 respectively). The aim of the second stage is to screen out the optimal dosage of hUC-MSC for NMOSD patients. The third stage, which aims to evaluate the effectiveness, will lasts for 24 months and will be developed in six centers. Patients (n = 320) will be randomized into two groups: the optimal dose group (n = 160) and the controlled group (n = 160). The aim of the third stage is to evaluate the effectiveness and safety of hUC-MSC for NMOSD patients. The study ow chart is shown in Fig. 1 For any subject withdrawal, the reasons for withdrawal will be documented.

The transportation and storage of hUC-MSC
The hUC-MSC will be immersed in liquid nitrogen, carried in a portable liquid nitrogen container and will be transported to the research centers by a cold chain logistic company. There will be a real-time recording of the temperature inside the liquid nitrogen container. The hUC-MSC will be stored in liquid nitrogen until resuscitation and infusion.

Resuscitation of hUC-MSC
A thermostat water bath will be preheated to 37 ℃. A metal clip will be precooled in liquid nitrogen and then the freezing bags of MSC will be extracted. The freezing bags will be quickly dropped into the water bath and swayed back and forth gently until totally melting (time duration: 2 minutes).

Inspections of hUC-MSC
The subjects can be infused hUC-MSC only after the sample tests meets the following requirements: (1) Total cell amount 5*10 7 ±10% per package; cell viability over 85%; (2) Bacterial endotoxin test negative; Gram staining negative.

Transfusion of hUC-MSC
Subjects in the intervention groups will receive four hUC-MSC infusions every 3 months besides the primary drugs. The dosages are 1, 2 or 5 × 10 6 MSC per kilogram of body weight. The hUC-MSC will be infused via the subjects' peripheral veins. The drip rate is 30 drops per minute in the rst 15 minutes of infusion and 45 drops per minute in the next 15 minutes. If there are no adverse effects, the drip rate can be gradually increased, but never exceeding 70 drops per minutes. The time of infusion should last no more than one hour. After the infusion of MSC, subjects will be infused 500 ml normal saline.
Subjects in the control group will receive four stem cell base solution infusions (the same packaging bag as hUC-MSC) every 3 months besides the primary drugs.

Participant safety protection measures
(1) Subjects will be admitted to the hospital when receiving the infusion; (2) Intravenous infusion of dexamethasone of 5 mg will be given to subjects 15 minutes before the infusion of hUC-MSC; (3) Subjects will have electrocardiogram monitoring during the whole process of infusion; (4) Subjects will be discharged after 2-4 days observation period after the infusion.

Group allocation, randomization and blinding
The rst phase of the trial is an open-label, dose-escalation study that will be performed in leading center, the Shanghai Ren Ji Hospital. For stage 2 and 3, permuted blocks randomization (block sizes of four) will be performed centrally using an integrated web response system (IWRS). In the stage 2, there are four arms, which are the low dose group (1 × 10 6 MSC / kg·weight), the medium dose group (2 × 10 6 MSC / kg·weight), the high dose group (5 × 10 6 MSC / kg·weight) and the control group (stem cell base solution). Patients will be assigned randomly in a 1:1:1:1 ratio to hUC-MSC group and control group, respectively. In the stage 3, there are two treatment arms. Patients will be assigned randomly in a 1:1 ratio to hUC-MSC group and control group, respectively.
In stage 2 and 3, all participants, investigators and the statistician are blinded to group allocation until the end of the study and data analysis. Only pharmacists who in charge of preparing study drug are not blinded. Doubleblinding will be performed via a centralized randomization system.

Sample size estimation
Based on previous epidemiology studies on NMOSD among Asian, the replasing rate for the patients in the rst year of the onset event is estimated at 55% [19]. In this study, we expected that the relapsing rate can be reduced to 40% in the MSC group, according to our preliminary data. In the rst stage of exploratory study, each dosage cohort will include 10 patients to meet the safety evaluation goal, which is 30 patients in total. In the adaptive design stage 2 and 3, a total sample size of 400 patient would have 80% power to detect 15% relapsing rate reduction at a two-sided 5% signi cant level, with 20% expected loss to follow-up rate. Therefore, we expected to enroll a total number of 430 subjects in this study.

Discontinuation of Study Intervention
Unexpected discontinuation of study intervention criteria are: (1)

Adverse Events
Adverse events (AEs) refer to any unwanted symptoms or signs in a clinical investigation subject, and do not necessarily have a causal relationship with the applied intervention [20]. All AEs will be coded according to the World Health Organization's Adverse Reactions Terminology. At the end of the clinical trial, the intensity and relationship of any AEs with the study intervention will be identi ed.
If any SAE is encountered, even with extremely low incidence, it will be reported to the trial's the principal investigator, the stem cell research ethics committee, and the state supervision agency within 24 hours of its occurrence.
There are two types of side effects that need intensive monitoring, side effects during infusion and long-term side effects.
Side effects during infusion include allergic reactions (such as fever, tachycardia, dyspnea) and systematic complications (infection, embolism, multiple organ failure); While monitoring long-term side effects, the following items will be tested every 3 months: tumor markers (AFP, CEA, CA199, CA125, CA153, CA242); complete blood count and urinalysis, liver and kidney function, blood glucose and electrolyte, coagulation tests, tests for infective diseases (HIV, RPR, hepatitis B and C; tested at baseline, 9 months and 2 years of follow-up), urine HCG; electrocardiogram, chest X-ray or CT, and abdominal ultrasound.

Follow-up
Primary, secondary, exploratory endpoint events and side-effects will be evaluated in all participating subjects at baseline and every 3 months. Magnetic resonance scans and optical coherence tomography (OCT) will be evaluated at baseline, year 1 and year 2 of follow-up.
For safety consideration, during stage one of the trial, all subjects will receive a telephone interview within one week after hUC-MSC infusion and then every two weeks until the second infusion. The telephone interviewing will include information on symptoms' change and side effects.
The study schedule is shown in Table 1.

Liver and kidney function
Tests for infective diseases

Data management
All study documents from all the centers will be considered highly con dential and will be stored in locked ling cabinets in a room with restricted access. All data will be translated into electronic format and stored in a database. Access to the database will be strongly restricted. The principal investigator and the biostatistician will be able to log into the data set and get full access to the information only upon permission from the head of this study. Data backups will be performed regularly by trial coordinators. If required, data transfer between centers will be encrypted, and any information capable of identifying individuals will be removed.
Trial quality assurance This study will be implemented according to high quality standards and will be delivered in accordance with the present trial protocol, which will not be amended unless any SAE occurs during its implementation. All research staff, including investigators, research assistants, and outcome evaluators, will be trained in advance to be able to competently administer all the items as per the protocol. Once the clinical trial begins, an independent trial inspector will visit each study site monthly and will be responsible for reviewing the following: overall research progress and integrity, adherence to the selection criteria for all the included subjects, compliance with the scheduled intervention for each participant, compliance with national regulations, and the handling of practical problems. Moreover, this trial inspector may occasionally provide suggestions to the principal investigator, who will make any nal decisions about trial modi cations, continuation, or termination.

Statistical analysis
The Kolmogrov-Smirnov Z test will be used for checking data normality. Categorical variables will be summarized as counts (percentage) and continuous variables as the means (standard error, SE) or medians (interquartile ranges, IQR), if not normally distributed. Statistical comparisons between the groups will be performed using Student's t-test or the Mann-Whitney U test for continuous variables, as well as the χ2 and Fisher's exact tests for categorical variables, as deemed appropriate. Correlations between continuous variables will be assessed by Pearson's correlation coe cient or Spearman's correlation coe cient.
In this study, two analysis sets will be required. For the safety set (SS) analysis, all safety indicators will be obtained from those who have undergone at least one dose of hUC-MSC infusions. For the effectiveness analysis set, intention to treat (ITT) analysis set will be used.
Lan-DeMets alpha-spending function with an O'Brien-Fleming boundary will be used to control the family-wise of type I error. Inverse normal combination test will be used to combined data from stage 2 and 3 in the nal analysis. An independent Data and Safety Monitoring Boards (DSMB) will review and evaluate the accumulated study data for participant safety, study conduct and progress at each stage of study. Only members of DSMB could have access to the data and results of analysis. DSMB can make recommendations to terminate the study on the basis of one of the following conditions: a signi cant difference in terms of effectiveness between groups; a signi cant risk-bene t ratio in one group; low degree of success in a reasonable period (such as poor compliance, low incidence of endpoint events). The advice of the DSMB meeting will be shared with the steering committee of the trial.

Discussion
To the best of our knowledge, this study will be the rst one to systematically demonstrate the clinical safety and e cacy of hUC-MSC in treating NMOSD, which leads to severe neurological disability in young population. Several studies have reported preliminary results on the e cacy of hUC-MSC in treating different types of diseases, such as stroke, spinal cord injury, hereditary spinocerebellar ataxia, multiple system atrophy, multiple sclerosis and other systemic diseases such as hematological diseases, immunological diseases, and etc [21][22][23][24][25][26][27]. Toxicity is rarely reported in these studies. In addition, these studies have a small number of subjects, lack of control, and they are not well designed. Therefore, there is a need for high quality evidence on the e cacy of hUC-MSC transfusion.
Some studies with small sample sizes have reported the e cacy of MSC in treating NMOSD, but they use invasive methodologies in performing stem cell transplantation [17,[28][29][30]. As far as we know, there was only one study using hUC-MSC to treat NMO till the present day. This study was published in 2012. In this study, hUC-MSCs transplantation was used to treat ve NMO patients with a follow-up period of 18 months. Among the ve cases, four showed therapeutic improvement after hUC-MSCs treatment. Both symptoms and signs improved and relapse frequencies were reduced [31].
The majority of previous studies only tested one dose of hUC-MSC [27]. Therefore, the optimal transfusion dose of hUC-MSC has not been determined till now. We use three different doses (1, 2 or 5 × 10 6 MSC per kilogram of body weigh) in stage 1 and 2 of the trial in order to determine the optimal dose of hUC-MSC in NMOSD patients, balancing both e cacy and safety.
The current study is a well-designed protocol for a clinical trial, and it includes a variety of safety indicators, functional assessments, imaging evaluations, and humoral indicators. We believe that this study protocol will allow to elucidate the therapeutic e cacy and safety concerns of hUC-MSC in treating AQP4-positive NMOSD and nally to determine the best dosage to be used.
In summary, hUC-MSC holds great promise for an effective treatment of NMOSD.
The results of this study may represent a milestone and may be used for future revisions of NMOSD guidelines.

Trial Status
The study was registered with CHICTR.