AN OPEN LABEL PHASE I STUDY OF hCT-MSC, AN UMBILICAL CORD-DERIVED MESENCHYMAL STROMAL CELL PRODUCT IN YOUNG CHILDREN WITH AUTISM SPECTRUM DISORDER
Phase: PHASE1
Conditions: Autism Spectrum Disorder
Sponsor: Duke University

Open Label Phase I HCT-MSC in Toddlers with ASD (TACT)
May 17, 2021 
Page 1 of 55 
AN OPEN LABEL PHASE I STUDY OF hCT-MSC, AN UMBILICAL CORD-DERIVED 
MESENCHYMAL STROMAL CELL PRODUCT IN YOUNG CHILDREN WITH AUTISM 
SPECTRUM DISORDER 
IND 17313 
Principal Investigator: 
Joanne Kurtzberg, MD 
Robertson Clinical and Translational Cell Therapy Program 
Duke Translational Research Institute 
Duke University Medical Center 
Box 3850 Durham, NC 27710 
Phone:  919-668-1100 
Co-Principal Investigator: 
Geraldine Dawson, PhD, FAPA, FAPS 
Duke Center for Autism and Brain Development 
Duke University Medical Center 
2608 Erwin Road 
Durham, NC 27710 
Phone: 919-668-0070 
Co-Principal Investigator: 
Jessica Sun, MD 
Robertson Clinical and Translational Cell Therapy Program 
Duke Translational Research Institute 
Duke University Medical Center 
Box 3850 Durham, NC 27710 
Phone:  919-668-1100 
NCT04294290

 
 
Open Label Phase I HCT-MSC in Toddlers with ASD (TACT) 
 
May 17, 2021 
 
Page 2 of 55 
 
Additional Investigators: 
 
Scott Compton, PhD 
Duke Center for Autism and Brain 
Development 
Duke University Medical Center 
2608 Erwin Road Durham, NC 
27710 
919-681-1100 
 
Dr Lauren Franz, M.B., CH.B.  
Duke Center for Autism and Brain 
Development 
Duke University Medical Center 
2608 Erwin Road Durham, NC 
27710 
919-681-0023 
 
Nicole Heilbron, PhD 
Duke Center for Autism and Brain 
Development 
Duke University Medical Center 
2608 Erwin Road Durham, NC 
27710 
919-668-0072 
 
Jill Howard, PhD 
Duke Center for Autism and Brain 
Development 
Duke University Medical Center 
2608 Erwin Road Durham, NC 
27710 
919-681-0033 
 
Lynn Cheatham, PhD 
GMP Lab Manager 
Duke University Medical Center 
Box 3350 Durham, NC 27710 
919-668-8700 
 
Barbara Waters-Pick, BS, MT (ASCP) 
STCL Lab Supervisor 
Duke University Medical Center 
2400 Pratt Street, Suite 1300, Box 3350 
Durham, NC 27705 
919-668-1178 
 
 
 
 
 
Guillermo Sapiro, PhD 
Department of Electrical and Computer 
Engineering 
Duke Univrsity 
Durham, NC 27708 
919-660-5386 
 
Elena Tenenbaum, PhD 
Duke Center for Autism and Brain 
Development 
Duke University Medical 
Center 2609 Erwin Road 
Durham, NC 27710 
919-668-2943 
 
Jesse Troy, PhD, MPH 
Statistician 
Duke University Medical 
Center Box 3850 Durham, NC 
27710 919-668-2932 
 
Kimberly Carpenter, PhD 
Duke Center for Autism and Brain 
Development 
Duke University Medical 
Center 2609 Erwin Road 
Durham, NC 27710 
919-668-2904 
 

Open Label Phase I HCT-MSC in Toddlers with ASD (TACT) 
May 17, 2021 
Page 3 of 55 
TABLE OF CONTENTS 
INVESTIGATOR SIGNATURE PAGE ..................................................................................................................................... 5 
PROTOCOL SUMMARY ...................................................................................................................................................... 6 
ABBREVIATIONS ............................................................................................................................................................... 7 
1.0 
PURPOSE ............................................................................................................................................................... 8 
2.0 
BACKGROUND AND HYPOTHESIS .......................................................................................................................... 8 
2.1 
AUTISM SPECTRUM DISORDER ..................................................................................................................................8 
2.2 
IMMUNE DYSREGULATION IN ASD .............................................................................................................................8 
2.3  
SYNAPTIC DYSFUNCTION IN ASD ................................................................................................................................9 
2.4 
MESENCHYMAL STROMAL CELLS (MSCS) ...................................................................................................................9 
2.5 
PRECLINICAL STUDIES OF CELL THERAPIES IN ASD ...................................................................................................17 
2.6 
OUTCOMES OF CELL THERAPIES IN CHILDREN WITH ASD ........................................................................................18 
2.7 
STUDY RATIONALE AND HYPOTHESIS ......................................................................................................................24 
2.8 
STUDY DESIGN .........................................................................................................................................................24 
2.9 
RISKS AND BENEFITS ................................................................................................................................................25 
3.0 
STUDY OBJECTIVES .............................................................................................................................................. 25 
4.0 
STUDY DESIGN ..................................................................................................................................................... 25 
4.1 
GENERAL DESIGN .....................................................................................................................................................25 
4.2 
STUDY FLOW CHART ................................................................................................................................................26 
4.3 
STUDY ENDPOINTS ...................................................................................................................................................26 
5.0 
RESEARCH PARTICIPANT SELECTION AND WITHDRAWAL .................................................................................... 27 
5.1 
STUDY POPULATION ................................................................................................................................................27 
5.2 
INCLUSION CRITERIA ................................................................................................................................................27 
5.3 
EXCLUSION CRITERIA ...............................................................................................................................................27 
5.4 
RESEARCH PARTICIPANT RECRUITMENT AND SCREENING ......................................................................................28 
5.5 
EARLY WITHDRAWAL OF RESEARCH PARTICIPANTS ................................................................................................29 
6.0      STUDY PRODUCT .................................................................................................................................................. 29 
6.1 
HUMAN UMBILICAL CORD TISSUE-DERIVED MESENCHYMAL STROMAL CELLS (HCT-MSC)......................................29 
6.2 
DONOR SCREENING AND TESTING ...........................................................................................................................30 
6.3 
PROCESS AND FINAL FORMULATION .......................................................................................................................30 
6.4 
PACKAGING OF STUDY PRODUCT .............................................................................................................................31 
6.5 
ADMINISTRATION OF PRODUCT ..............................................................................................................................31 
6.6 
SAFETY FOLLOW-UP .................................................................................................................................................31 
7.0 
STUDY PLAN ........................................................................................................................................................ 32 
7.1 
OVERVIEW ...............................................................................................................................................................32 
7.2 
PARTICIPANT SCREENING ........................................................................................................................................32 
7.3 
STUDY INFUSIONS ....................................................................................................................................................32 
7.4 
CARE DURING UNEXPECTED EVENTS .......................................................................................................................33 
7.5 
ASSESSMENTS ..........................................................................................................................................................33 
7.5.1 Assessments for Diagnosis of Autism Spectrum Disorder: ......................................................................................33 
7.5.2 Other Clinician Assessments: ..................................................................................................................................34 
7.5.3 Parent Caregiver Questionnaires: ...........................................................................................................................35 
7.6 
REQUIRED EVALUATIONS .........................................................................................................................................36 
7.6.1 Medical and Safety Assessments: ...........................................................................................................................36 

Open Label Phase I HCT-MSC in Toddlers with ASD (TACT) 
May 17, 2021 
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Safety and return assessments should be performed within a month of the indicated time point. ................................37 
7.6.2  Diagnostic, Behavioral, Cognitive/Language, and Attention evaluations. ............................................................37 
8.0 
STATISTICAL CONSIDERATIONS ........................................................................................................................... 38 
8.1 
STUDY DESIGN .........................................................................................................................................................38 
8.2 
STUDY DURATION ....................................................................................................................................................38 
8.3 
DEMOGRAPHICS AND BASELINE CHARACTERISTICS ................................................................................................38 
8.4 
SAMPLE SIZE AND POWER CALCULATIONS ..............................................................................................................38 
8.5 
PRIMARY ENDPOINTS ..............................................................................................................................................38 
8.6 
EFFICACY ENDPOINTS ..............................................................................................................................................38 
9.0 
SAFETY AND ADVERSE EVENT REPORTING .......................................................................................................... 39 
9.1 
DEFINITIONS ............................................................................................................................................................39 
9.2 
ADVERSE EVENT REPORTING ...................................................................................................................................39 
9.3 
SERIOUS ADVERSE EVENT REPORTING .....................................................................................................................40 
9.4 
ELICITING ADVERSE EVENT INFORMATION ..............................................................................................................40 
9.5 
STOPPING GUIDELINES ............................................................................................................................................40 
9.6 
PARTICIPANT REPLACEMENT ...................................................................................................................................41 
10.0 
DATA SAFETY MONITORING BOARD (DSMB) ....................................................................................................... 41 
11.0 
DATA HANDLING AND QUALITY ASSURANCE ...................................................................................................... 41 
11.1 
CASE REPORT FORMS ...............................................................................................................................................41 
11.2 
VIDEO AND AUDIO RECORDINGS .............................................................................................................................41 
11.3 
INSPECTION OF RECORDINGS ..................................................................................................................................41 
11.4 
STUDY RECORD RETENTION .....................................................................................................................................42 
12.0 
ADMINISTRATIVE ASPECTS .................................................................................................................................. 43 
12.1 
CONFIDENTIALITY ....................................................................................................................................................43 
12.2 
INSTITUTIONAL REVIEW BOARD APPROVAL ............................................................................................................43 
12.3 
MODIFICATION OF THE PROTOCOL..........................................................................................................................43 
12.4 
INFORMED CONSENT ...............................................................................................................................................43 
12.5 
PROTOCOL VIOLATIONS AND DEVIATIONS ..............................................................................................................44 
12.6 
STUDY REPORTING REQUIREMENTS ........................................................................................................................44 
12.7 
FINANCIAL OBLIGATIONS .........................................................................................................................................44 
12.8 
STUDY CONDUCT .....................................................................................................................................................45 
12.9 
PUBLICATIONS .........................................................................................................................................................45 
13.0 
REFERENCES ........................................................................................................................................................ 45 
14.0 APPENDIX 1: SAFETY QUESTIONNAIRE .................................................................................................................... 51 

 
 
Open Label Phase I HCT-MSC in Toddlers with ASD (TACT) 
 
May 17, 2021 
 
Page 5 of 55 
 
 
INVESTIGATOR SIGNATURE PAGE 
 
 
 
 
AN OPEN LABEL PHASE I STUDY OF hCT-MSC, AN UMBILICAL CORD-DERIVED 
MESENCHYMAL STROMAL CELL PRODUCT IN YOUNG CHILDREN WITH AUTISM 
SPECTRUM DISORDER 
 
 
 
I agree to conduct and supervise this clinical study in accordance with the design and 
specific provisions of this protocol; modifications to the study or protocol are acceptable 
only with a mutually agreed upon protocol amendment except when necessary to protect 
the safety of participants.  I agree to await IRB approval for the protocol and informed 
consent before initiating the study, to obtain informed consent from participants prior to 
their enrollment in the study.  I agree to report to responsible regulatory agencies and the 
IRB (when necessary) adverse events that occur in the course of this investigation.  I 
agree to maintain accurate and adequate records in the case report forms as required by 
this protocol and maintain those records for the period of time required.  I will make the 
study documentation available for safety oversight committee review and/or for other 
inspections as required.  I agree to maintain study documentation for the period of time 
required.  I agree to comply with all other requirements regarding the obligations of 
clinical investigators according to FDA regulations and guidance.  I agree to ensure that 
all people assisting in the conduct of this study are informed in meeting the above 
commitments. 
 
 
 
 
(Investigator’s printed name) 
 
 
 
(Investigator’s signed name) 
 
 
 
 
 
 
 

 
 
Open Label Phase I HCT-MSC in Toddlers with ASD (TACT) 
 
May 17, 2021 
 
Page 6 of 55 
 
PROTOCOL SUMMARY  
 
Protocol Number: 
 
 
Title: 
 
AN OPEN LABEL PHASE I STUDY OF hCT-MSC, AN 
UMBILICAL CORD TISSUE-DERIVED MESENCHYMAL 
STROMAL CELL PRODUCT IN YOUNG CHILDREN 
WITH AUTISM SPECTRUM DISORDER 
 
Study Phase: 
I 
 
Study Site: 
Single site; Duke University, Durham NC 
 
Study Therapy, Dosage, and 
Route of Administration: 
 
Umbilical Cord Tissue Mesenchymal Stromal Cells, 
isolated and expanded from umbilical cord donors.  One 
dose of 2x106/kg administered intravenously. 
 
Objectives: 
To determine the safety of a single intravenous infusion of 
hCT-MSC in very young children with ASD. 
 
Research Participant Population: 
Twelve toddlers, ages 18 months to <4 years (3 years 
and 364 days), with an established diagnosis of autism 
spectrum disorder (ASD) 
 
Study Design: 
 
Prospective 
Safety Assessments/Endpoints: 
1. Incidence of infusion reactions 
2. Incidence of infections 
3. Incidence of clinically significant anti-HLA antibody 
formation 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
 
Open Label Phase I HCT-MSC in Toddlers with ASD (TACT) 
 
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ABBREVIATIONS 
ADOS 
 
Autism Diagnostic Observation Schedule 
AE 
 
Adverse Event 
ASD 
 
Autism Spectrum Disorder 
BOSA 
 
Brief Observation of Symptoms of Autism 
CB 
 
Human Umbilical Cord Blood 
CDI 
 
Communication Development Inventories  
CFR 
 
Code of Federal Regulations 
CGI 
 
Clinical Global Impression 
CMP 
 
Complete Metabolic Panel 
CMV 
 
Cytomegalovirus 
CNS 
 
Central Nervous System 
CNV 
 
Copy Number Variation 
CRF 
 
Case Report Form 
CVA 
 
Computer Vision Analysis  
CTCAE 
 
Common Terminology Criteria for Adverse Events 
DAYC-2 
 
Developmental Assessment of Young Children-Second Edition 
DSMB 
 
Data Safety Monitoring Board 
DMSO 
 
Dimethyl Sulfoxide 
DSM-5 
 
Diagnostic and Statistical Manual of Mental Disorders, Fifth Edition 
EDC 
 
Electronic Data Capture  
EEG 
 
Electroencephalography 
EOWPVT 
 
Expressive One Word Picture Vocabulary Test 
FACT 
 
Foundation for the Accreditation of Cellular Therapy  
FDA 
 
Federal Drug Administration 
GCP 
 
Good Clinical Practice 
GMP 
 
Good Manufacturing Practice 
GvHD 
 
Graft versus Host Disease 
HBV 
 
Hepatitis B Virus 
hCT-MSC 
 
Human Umbilical Cord Tissue-derived Mesenchymal Stromal Cells 
HCV 
 
Hepatitis C Virus 
HIV 
 
Human Immunodeficiency Virus 
HLA 
 
Human Leukocyte Antigen 
HRPP 
 
Human Research Protections Program 
HSCT 
 
Hematopoietic Stem Cell Transplantation 
HTLV 
 
Human T-lymphotropic Virus 
ICH 
 
International Council for Harmonisation 
IRB 
 
Institutional Review Board 
ISBT 
 
International Society for Blood Transfusion 
IV 
 
Intravenous 
IVIG 
 
Intravenous Immunoglobulin 
JERI 
 
Joint Engagement Rating Inventory  
MBHQ 
 
Medical/Behavioral History Questionnaire  
mITT 
 
Modified Intention to Treat 
MSC 
 
Mesenchymal Stromal Cell 
NMDP 
 
National Marrow Donor Program 
NSAID 
 
Non-steroidal Anti-inflammatory Drug 
PCIT 
 
Parent-Child Interaction Task 
PDDBI 
 
Pervasive Developmental Disorder Behavior Inventory 
PET 
 
Positron Emission Test 
PRA 
 
Panel Reactive Antibody 
RBS-EC 
 
Repetitive Behavior Scale for Early Childhood 
S2K 
 
Sense to Know 
SAE 
 
Serious Adverse Event 
SNP 
 
Single-nucleotide Polymorphism 
TNC 
 
Total Nucleated Cells 
VABS 
 
Vineland Adaptive Behavior Scales 
WBC 
 
White Blood Cell 

 
 
Open Label Phase I HCT-MSC in Toddlers with ASD (TACT) 
 
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Page 8 of 55 
 
1.0 
PURPOSE 
The purpose of this Open Label Phase I study is to determine the feasibility and safety of 
treatment via a single intravenous infusion of human umbilical cord tissue-derived 
mesenchymal stromal cells (hCT-MSC) in very young children with autism spectrum disorder 
(ASD). 
2.0 
BACKGROUND AND HYPOTHESIS 
2.1 
AUTISM SPECTRUM DISORDER 
Autism Spectrum Disorder (ASD) is a heterogeneous neurodevelopmental disorder with onset 
early in life. It is characterized by impairments in social communication and the presence of 
repetitive behaviors and a restricted range of activities.1 ASD is 4 to 5 times more prevalent in 
boys, with approximately 1 in 59 children in the United States diagnosed with ASD.2 ASD is 
often accompanied by intellectual disability and is usually a chronic, disabling disorder that 
compromises the full potential of the affected individual. The majority of individuals with ASD 
are not able to live independently and require lifelong support or accommodations. 
Accordingly, the lifetime cost of supporting an individual with ASD is estimated to be $1.4 
million. The cost is $2.4 million for those who also have an intellectual disability.3 
 
Early behavioral intervention leads to improved outcomes in individuals with ASD.4 Other 
treatments include occupational and speech therapy, and specialized educational and 
vocational support. Despite receiving high quality early behavioral intervention and related 
therapies, many individuals with ASD continue to have significant impairments, with a 
substantial percentage not acquiring speech.  All of the currently available medical treatments, 
such as psychotropic medications, are intended to ameliorate associated co-morbid 
symptoms, such as irritability, but do not address core symptoms, such as the ability to socially 
interact and communicate. In light of this, there is a large unmet need for effective medical 
treatments for ASD that address core autism symptoms. 
 
The etiology of ASD is complex and includes both genetic and environmental factors. In 
recent years, genetic sequencing and analysis have identified several de novo mutations, 
copy number variants (CNV), and single-nucleotide polymorphisms (SNP) that are associated 
with an increased risk of ASD. However, it is estimated the specific genetic cause can only be 
identified in less than 20% of cases, with the remaining causes likely being caused by an 
interaction between multiple genetic and environmental risk factors.5 Several environmental 
factors have also been associated with an increased risk of ASD, including prematurity, birth 
complications, maternal teratogen exposure, environmental toxins, and advanced paternal 
age. Finally, inflammation, particularly neuroinflammation, and immune dysregulation have 
been implicated in the etiology of ASD. Therefore, immunomodulatory therapies may have a 
role in the treatment of children with ASD. 
2.2 
IMMUNE DYSREGULATION IN ASD 
A role of immune activation and/or dysregulation in the etiology of ASD is supported by 
several different observations. Multiple epidemiologic studies have demonstrated an 
increased rate of ASD in children born to mothers who had an infection during pregnancy. 
This association has been consistent across countries and time periods, having been 
reported as early as the 1970s after a rubella pandemic in the United States6 and throughout 
the decades in Denmark,7 Sweden, and other countries. Maternal fever, antibiotic treatment, 
elevated levels of inflammatory markers, and infection with several different microbes have 
been associated with increased risk for the development of ASD in the child, indicating that 

 
 
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Page 9 of 55 
 
the immune response to infection is likely to be more causative than the infectious agent 
itself. Accordingly, animal models have yielded offspring with ASD phenotypes by inducing 
immune activation in the pregnant mothers.8,9 
 
Potential pathophysiologic mechanisms include alterations in maternal-fetal immune tolerance 
(via maternal antibodies and/or cellular immunity) and fetal brain inflammation, which lead to 
changes in brain cytokine profiles and microglial activation that may be detrimental to 
neurodevelopment. Evidence of increased numbers of microglia and increased microglial 
activation in ASD has been obtained via autopsy studies, positron emission tomography (PET) 
brain imaging, and animal models. In addition, abnormal functioning in aspects of the immune 
system in the brain (such as microglia that are tasked with providing support to neuronal 
synapses) has been described.10,11 Increased plasma cytokine levels, upregulated genes 
associated with microglial activation, localized inflammation and pathological astrocyte 
activation have also been associated with ASD.12 Of note, many cytokines and molecules 
classically associated with immune regulation are now recognized as also playing a role in 
normal neurodevelopment. This dual functionality may prove to be an important link in the 
association of immune-related changes and abnormal neurodevelopment in ASD. 
2.3  
SYNAPTIC DYSFUNCTION IN ASD 
Synapses are points of communication between neurons, allowing the organized passage of 
information via electrical and chemical signaling. While there is a period of intensified 
synaptogenesis early in development, synapses retain plasticity throughout life, enabling, for 
example, learning and memory. Normal synaptic development and maintenance are essential 
to proper neuronal function, and abnormalities in either process have been associated with 
multiple neurodevelopmental conditions, including ASD. Mutations in genes encoding synaptic 
proteins have been implicated in ASD. Additionally, human and animal studies have 
demonstrated a reduction in the size, number, and morphology of dendritic spines and an 
increase in immature spine morphology in ASD and autism related disorders.13 It is also likely 
that environmental factors may also influence synaptic changes. These alterations may lead 
to problematic neuronal connectivity, such as large-range under-connectivity and short-range 
over-connectivity.14,15 
 
Altogether, these observations suggest that both environmental and genetic risk 
factors may contribute to the development of ASD by causing immune dysregulation 
and/or abnormal neuronal connectivity that adversely affect normal brain development 
and cause core symptomatology observed in individuals with ASD. 
2.4 
MESENCHYMAL STROMAL CELLS (MSCs) 
hCT-MSC is a third party, allogeneic, human mesenchymal stromal cell (MSC) product 
manufactured from donated umbilical cord tissue, after written informed consent for donation 
and commercialization, harvested from discarded birthing tissues, that is digested, expanded 
for two passages in culture in xeno free media, harvested, cryopreserved, and stored in the 
vapor phase of liquid nitrogen until use. The umbilical cord tissue is donated by healthy 
mothers delivering full term babies via Cesarean Section after written informed consent. The 
cells are cryopreserved and stored in the Robertson CT2 GMP laboratory (2400 Pratt Street, 
Duke University, Durham, NC). 
 
Mesenchymal stromal cells (MSCs): 
MSCs are a heterogeneous group of undifferentiated, multipotent cells that can be isolated 
from several different tissues including bone marrow, adipose tissue, and birth tissues 
(umbilical cord blood, umbilical cord tissue, amniotic fluid, placenta). While MSCs can give rise 

 
 
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to mesodermal tissue types including bone, cartilage, and fat, their primary mechanism of 
action is thought to result from paracrine effects on immunomodulation, antiapoptosis, 
angiogenesis, support of the grown and differentiation of local stem and progenitor cells, 
antifibrosis, and chemoattraction (figure 1).16 MSCs have demonstrated a multitude of 
immunomodulatory effects on both humoral and cell-mediated immune responses. These 
include, but are not limited to, inhibiting B-, T-, NK, dendritic-cell, and microglial proliferation, 
decreasing pro-inflammatory cytokine production, and blocking neutrophil recruitment. 
 
Figure 1: Paracrine effects of MSCs, including a subset of factors secreted by cultured MSCs. (Figure adapted 
from Singer, Annu. Rev. Pathol. Mech. Dis., 2011.16) 
 
 
Despite their ability to modulate the immune response, MSCs themselves have low 
immunogenicity. MSCs express low levels of MHC class I molecules on their surface and lack 
expression of MHC class II and several costimulatory molecules. This allows MSCs to be 
used in the allogeneic setting, without the need for donor-recipient HLA matching typical of 
other cellular therapies or transplantation. In fact, in a review of 13 human studies of 
intravenous allogeneic MSC administration, there were no reports of infusional toxicity17 or 
longer term SAEs, supporting the notion that MSCs are “immune-privileged” and can avoid 
immunological allorecognition. When utilized as a therapeutic cell, it is widely accepted that 
MSCs exert effects via paracrine and trophic signaling. They do not engraft in the recipient. 
 
Safety of MSCs: 
MSCs manufactured from bone marrow and adipose tissue have been studied in hundreds of 
clinical trials worldwide involving thousands of individuals and a wide variety of human 
conditions. Their safety has been repeatedly demonstrated and is summarized in a 2012 
systematic review and meta-analysis17 of 36 clinical trials in 14 countries using MSCs in over 
1,000 recipients with cardiovascular, neurological, oncologic, metabolic, gastrointestinal, and 
post-transplant conditions. Based on a combination of randomized controlled trials and 
uncontrolled studies with follow-up periods of weeks to up to 5 years, there was no association 
Chemoattraction 
(MSC Migration: CXCR4, 
CXCR3) 
Immune 
modulation 
(IDO, PGE2, IL-10, TGF-
Beta, iNOS, HO,T Reg’s, 
Inhibition of Monocyte 
Maturation) 
Anti-
Inflammatory/ 
Antiproliferative 
 (TSG-6) 
 
Anti-apoptosis 
(IGF-1, bFGF, VGEF, 
HGF, 
TGF-Beta) 
Pro-angiogeneic 
effects 
(IGF-1, VEGF, bFGF 
MCP-1) 
Antifibrotic 
effects 
HGF, bFGF 

 
 
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between MSC treatment and acute infusional toxicity, organ system complications, infection, 
death, or malignancy. The only side effect associated with MSC treatment was transient fever, 
which did not cause any long-term sequelae. Importantly, no malignancies were reported in 
patients without a prior history of cancer. 
 
After intravenous administration, approximately 70% of infused MSCs are engulfed by lung 
macrophages. While there is a theoretical risk of respiratory complications, the only 
randomized trial in which acute pulmonary reactions have been observed was conducted in 
patients with chronic ischemic heart failure who received intracoronary MSCs.18 Thus, this risk 
seems to be limited to patients whose underlying condition makes them susceptible to 
developing pulmonary edema. Although a few cardiovascular studies have reported 
arrhythmias, the incidence was not statistically different from control groups in meta-analysis.17 
 
Umbilical cord tissue is an attractive source of MSCs as it is readily available and easily 
obtained without risk to the maternal or newborn baby donor, is non-controversial, and has a 
higher proliferative potential than MSCs from other postnatal sources.19 Numerous preclinical 
studies have not demonstrated any evidence of tumorigenicity or toxicity of cord tissue-derived 
MSCs.20 A summary of early phase clinical trials published in English that utilized cord tissue-
derived MSCs is shown in Table 1. Among these 37 studies including 710 patients and at least 
1,431 doses of cord tissue-derived MSCs with follow-up ranging from three months up to six 
years, no severe adverse events were reported. Several more clinical trials of cord-tissue 
derived MSCs in various disease conditions are underway (clinicaltrials.gov). 

 
 
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Table 1: Umbilical Cord Tissue-Derived MSCs Studied in Humans 
 
Indication 
Report 
Ages, yrs 
(range or 
mean) 
Route of 
administration 
Dose 
# of 
doses 
(interval) 
Duration 
of f/u 
(months) 
AEs due to CT-
MSCs 
Cao, 201821 
Intrauterine Adhesions 
26 
36 
Intrauterine 
1x107 
1 
30 
none 
Xaio, 201822 
Spinal Cord Injury 
2 
29 
Spinal cord 
4x107 
1 
12 
none 
Riordan, 201823 
Multiple Sclerosis 
20 
41 
IV 
2x107 
7 (daily) 
12 
HA, fatigue 
Sun, 201824 
Kidney Transplant 
21 
41 
IV, intra-arterial 
2-5x106 
2 
12 
none 
Xu, 201825 
Severe Aplastic Anemia 
24 
18 
IV 
5x105 
1 
13 
none 
Rahyussalim, 
201726 
Spinal Cord 
Entrapment, Kidney 
Failue 
1 
62 
IV, IT 
1.6x107 
36 
8 
none 
Park, 201727 
Osteoarthritis 
6 
59 
Intra-articular 
1.15-2x107 
1 
84 
arthralgia, back pain, 
bladder distension,  
antithyroglobulin 
antibody 
Bartolucci, 
201728 
Heart Failure 
15 
57 
IV 
1x106/kg 
1 
12 
none 
Kim, 201729 
Atopic Dermatitis 
34 
29 
Subcutaneous 
2.5-5x107 
1 
3 
none 
Wang, 201730,31 
Lupus 
40 
17-54 
IV 
1x106/kg 
2 
(weekly) 
12-72 
 
none 
Chen, 201632 
Osteonecrosis 
9 
>18 
Intra-arterial 
5-10x107 
1 
24 
none 
Li, 201633,34 
Autism 
20 
3-14 
IT 
1x106/kg 
2 
(5-7 days) 
6 
transient fever (n=4) 
Xie, 201635 
Hypoxic Ischemic 
Encephalopathy 
12 
45-68 
IV 
1x108 
1 
6 
none 
Wang, 201636 
Juvenile Idiopathic 
Arthritis 
10 
2-15 
IV 
4x107 
2 
(3 months) 
12 
none 
Chen, 201637 
Psoriasis 
2 
26-35 
IV 
1x106/kg 
1, 5 
48, 60 
none 
Cai, 201638 
Type I Diabetes 
21 
5-28 
Intra-arterial 
(pancreatic artery) 
1.1x106/kg 
1 
12 
none 
Hu, 201639 
Type 2 Diabetes 
31 
42-63 
IV 
1x106/kg 
2 
(monthly) 
36 
none 
Hua, 201640 
Spinal Cord Injury 
1 
25 
IT 
1x107 
4 
(3 days) 
12 
none 
Qin, 201641 
Diabetic Foot 
28 
N/A 
Endovascularly 
N/A 
N/A 
3 
none 
Zhao, 201542 
Heart Failure 
30 
53.2 
Intracoronary 
NR 
1 
6 
none 
Liang, 201543 
Radiation Myelitis 
1 
37 
IV, IT 
5.2x107 IV, 
1.1x107 IT 
1 
18 
none 
Wang, 201544 
Cerebral Palsy 
16 
4-12 
IT 
1-1.5x107 
4 
6 
NR 
Li, 201545 
Coronary Artery 
Occlusion 
15 
N/A 
Intracoronary 
3-5x106 
1 
24 
none 

 
 
Open Label Phase I HCT-MSC in Toddlers with ASD (TACT) 
 
May 17, 2021 
 
Page 13 of 55 
 
 
Indication 
Report 
Ages, yrs 
(range or 
mean) 
Route of 
administration 
Dose 
# of 
doses 
(interval) 
Duration 
of f/u 
(months) 
AEs due to CT-
MSCs 
Rajput, 201546 
Duchenne Muscular 
Dystrophy 
11 
5-18 
IV & IM 
1x106/kg 
4 
(monthly) 
36 
none 
Gu, 201547 
Lupus Nephritis 
58 
12-55 
IV 
1x106/kg 
1 
12 
none 
Pan, 201548 
Bone Marrow Necrosis 
1 
11 
Intrabone 
2x107 
1 
38 
none 
Wu, 201549 
GvHD 
24 
14-44 
IV 
0.5-1x106/kg 
1 
1-24 
none 
Zhu, 201550 
Leukemia, undergoing 
haplo HSCT 
25 
4-17 
IV 
1-1.4x106/kg 
1 
3-25 
none 
Miao, 201551 
Neurological disorders 
88 
2-68 
IT 
NR 
4-6 
(5-7 days) 
NR 
transient HA, fever, 
back/leg pain 
Li, 201552 
Becker Muscular 
Dystrophy 
3 
6-46 
IV 
3-5x107 
1 
3 
none 
Wang, 201553 
Hemorrhagic Cystitis 
7 
11-38 
IV 
0.8-1.6x106/kg 
1-3 
NR 
none 
Cheng, 201454 
Spinal Cord Injury 
10 
35.3 
Spinal Cord 
Injection 
2x107 
2 
(10 days) 
6 
none 
Chang, 201455 
Bronchopulmonary 
Dysplasia 
9 
10 days 
Intratracheal 
1-3x107 
1 
3 
none 
Li, 201456 
Multiple Sclerosis 
13 
42 
IV 
4x106/kg 
3 
(2 weeks) 
12 
none 
Kong, 201457 
Type 2 Diabetes 
18 
 
IV 
N/A 
3 
6 
slight fever 
Wang, 201358 
Traumatic Brain Injury 
20 
5-48 
IT 
1x107 
4 
(5-7 days) 
6 
HA, dizziness 
Wang, 201359 
Primary Biliary Cirrhosis 
7 
33-58 
IV 
0.5x106/kg 
3 
(monthly) 
12 
none 
Zhang, 201360 
HIV 
7 
26-49 
IV 
0.5x106/kg 
3 
(monthly) 
12 
none 
Jin, 201361 
Spinocerebellar Ataxia 
16 
 
IV & IT 
N/A 
N/A 
12 
none 
Wu, 201362 
Leukemia, undergoing 
CBT 
8 
3-12 
IV 
2-10x106/kg 
1 
8-27 
none 
Jiang, 201363 
Stroke 
4 
40-59 
Intra-arterial (MCA) 
2x107 
1 
6 
none 
Hu, 201364 
Type I Diabetes 
15 
17.6 
IV 
1.5-3.2x107 
1 
24 
none 
Shi, 201265 
Liver Failure 
24 
24-59 
IV 
0.5x106/kg 
3 
(monthly) 
18 
none 
Zhang, 201266 
Liver Cirrhosis 
30 
25-64 
IV 
0.5x106/kg 
3 
(monthly) 
12 
none 
Qu, 200967 
Bone Nonunion 
36 
36 
Intrabone 
1x106 - 1x107 
1 
13 
none 
Totals:   
    45 studies 
 
819 
0-68 
 
 
 >1,700  
3-84 
6 studies 
(3 IT route) 
Abbreviations:  HA-headache, IM-intramuscular, IT-intrathecal, IV-intravenous, N/A-not available, NR- not recorded 
 

 
 
Open Label Phase I HCT-MSC in Toddlers with ASD (TACT) 
 
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Experience with MSCs in Children: 
The most well-studied MSC product given to children to date is a bone marrow-derived product 
originally manufactured by Osiris as Prochymal® and subsequently acquired by Mesoblast as 
Remestemcel-L used to treat patients with GvHD after a hematopoietic stem cell transplant. 
Prochymal® received regulatory approval in Canada in 2012 and in Japan in 2018 as TEMCELL® 
for use in children with acute steroid refractory GvHD. A recent expanded access study of 
Remestemcel-L enrolled 241 children (median age 9.6 years) with refractory GvHD from 2007-
2014.68 Eight MSC doses (2x106 cells/kg/dose) were given intravenously over a four week time 
period, with four additional weekly infusions in patients who demonstrated a partial or mixed 
response. There were no incidences of ectopic tissue formation in 2434 total doses, with follow-up 
of 2-9 years. The most frequent severe adverse events were infections (24%) and respiratory 
disorders (16%), common issues in post-transplant patients. There were only 11 severe adverse 
events and one infusion reaction related to MSC treatment. Patients with primary steroid-refractory 
GvHD who had only been treated with steroids demonstrated an 81% response rate to MSCs. 
Overall, 65% of patients demonstrated an improvement in their GvHD, with higher response rates 
in patients with less severe disease. This demonstrates that repeated doses of MSCs are safe and 
well-tolerated in children, and suggests that they provide anti-inflammatory and 
immunomodulatory effects in the setting of GvHD. A more recent phase III open-label, single arm 
study of Remestemcel-L in 55 children with primary steroid refractory GVHD replicated these 
results.  
 
In a recent study, a German group pooled bone marrow cells from 8 healthy donors to create an 
MSC cell bank.69 They treated 26 children, ages 1-19 years old with steroid-refractory GvHD, 
with a total of 81 doses of MSCs from this bank (median 3 doses per patient, median 2.2x106/kg 
MSCs IV per dose). Treatment was well-tolerated, with only one reported headache and one 
episode of nausea across all doses. Six patients died from causes unrelated to MSCs 
(thromboembolism, disease progression, GvHD, sepsis). Response rate, defined as complete or 
partial remission of GvHD, was 77%. 
 
Others are investigating the use of MSCs in patients with other conditions. There have been case 
reports of intracoronary administration of autologous bone marrow-derived MSCs in children, ages 
4 months to 14 years, with severe dilated cardiomyopathy and/or heart failure with subsequent 
short- and intermediate-term improvement in clinical condition and B-type natriuretic peptide 
values.70-72 Studies have also been conducted in patients with type 1 diabetes.38 One such study 
administered umbilical cord-derived MSCs intravenously in 29 patients, including several children, 
with newly diagnosed type 1 diabetes.64 Treatments were well-tolerated, and moderate 
improvements in some metabolic measures were observed. Early phase studies of MSCs are also 
underway in preterm infants with bronchopulmonary dysplasia. In one published study, cells were 
delivered intratracheally and no significant side effects were reported in these young babies.55 
None of these early phase studies have reported toxicity related to the MSC therapy. Per Table 1, 
over 105 children with various conditions have been treated with human umbilical cord tissue-
derived MSCs without apparent adverse events. 
 
MSCs in Neurologic Conditions: 
Numerous preclinical studies using MSCs for diseases of the central nervous system suggest 
that MSCs can act through release of different neurotrophic, anti-inflammatory, and anti-
apoptotic factors to promote recovery the injured area and prevent further damage. The majority 
of clinical experience has focused on adults with stroke, with a few studies in patients with 
neurodegenerative conditions or multiple sclerosis (MS). 
 

 
 
Open Label Phase I HCT-MSC in Toddlers with ASD (TACT) 
 
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Several small studies of autologous bone marrow-derived MSCs have been conducted in adult 
patients with acute or chronic stroke, with no significant side effects.73 In a phase II study of an 
allogeneic MSC product (MultiStem) conducted in 126 adult patients with stroke (65 treated, 61 
placebo), MSC therapy was well-tolerated.74 While there was no difference between placebo and 
treated patients in measures of stroke recovery, the treatment group had a lower rate of mortality 
and infections, associated with down regulation of inflammatory biomarkers including IL-6. 
Patients who received MSCs earlier (24-36 hours post-stroke vs. 36-48 hours post-stroke) 
demonstrated more favorable recovery than patients who received later treatment or placebo. 
 
A few clinical trials of autologous MSC therapy have been conducted in patients with multiple 
sclerosis (MS). Of 25 patients with progressive MS treated with a single intrathecal injection of 
autologous bone marrow-derived MSCs, the disease stabilized in half of patients over a one-year 
time period.75 Side effects, all transient and self-limited including low-grade fever, 
nausea/vomiting, lower limb weakness, and headache, were likely related to the intrathecal route 
of administration and were consistent with those reported from other small studies of intrathecal 
MSC treatment in patients with MS.76,77 Another study that treated 10 patients with progressive 
visual deficits due to MS with a single intravenous dose of autologous bone marrow-derived 
MSCs demonstrated improvements in visual acuity, visual evoked response latency, and optic 
nerve area.78 
 
One clinical trial has been conducted in seven patients with Parkinson’s disease who received 
a single dose of autologous bone marrow-derived MSCs transplanted to the subventricular 
zone using stereotaxic surgery. With a follow-up of 10 to 36 months, three patients 
demonstrated an improvement in disease symptoms. Two patients also reported subjective 
improvement of symptoms and reduction in drug dosage.79 
 
Potential Mechanism of MSCs in ASD: 
The exact mechanism of action of MSCs in ASD is the subject of ongoing investigations, but there 
are several potential means through which MSCs may exert therapeutic effects, including cell-
mediated immunomodulation, molecular-mediated neuroprotection, and restoration of functional 
neurologic circuitry. 
 
MSCs have been shown to exert immunomodulatory effects on cell-mediated immune 
responses by inhibiting T-cell proliferation and decreasing production of pro-inflammatory 
cytokines, including TNF-α and interferon gamma.80 As ongoing immune dysregulation may 
contribute to the pathophysiology of ASD, suppressing the cell-mediated immune response with 
MSCs could have potential therapeutic benefit. hCT-MSCs have demonstrated the ability to 
suppress T-cell response in vitro, as measured by thymidine uptake four days after incubation of 
mononuclear cells with increasing numbers of hCT-MSCs and CD3/CD28 beads to stimulate T-
cell proliferation (figure 2).  
 
MSCs may also provide neuroprotection through anti-inflammatory mechanisms by inhibiting 
neural apoptosis, microglial activation, astrocyte proliferation, and oxidative stress molecules.81 
Koh, et al.82 demonstrated that cord tissue-derived MSCs promote neuron survival via secretion 
of neurotrophic factors. In several other models, MSCs have demonstrated the ability to 
decrease both the number and activation of microglial cells, which are thought to play a critical 
role in the development of ASD.83,84 It is unclear if this phenomenon is caused by a direct effect 
of the MSCs or if it is mediated through activation of cytokines (ie. TCP, IL-6). 
 

 
 
Open Label Phase I HCT-MSC in Toddlers with ASD (TACT) 
 
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Page 16 of 55 
 
 
 
 Figure 3: hCT-MSCs decrease microglial activation.  Panel A: Peripheral blood mononuclear cells (PBMCs) 
were obtained from three donors, stimulated with anti-CD3 and anti-CD28 antibodies on beads.  hCT-MSCs were 
irradiated before addition to the PBMC cultures. T-cell suppression is measured by suppression of 3H-thymidine 
uptake in PBMCs. Panel B: Murine cerebellar slice culture model demonstrating decreased microglial activation in 
hCT-MSC treated cultures after demyelination and microglial activation via LPC exposure.  
 
 
Potency: Inhibition of T-cell Proliferation 
No Interferon (IFN) 
With Interferon (IFN) and/or MT 
Figure 2. hCT-MSCs suppress T cell proliferation 
MSC were thawed and plated in fibronectin-coated wells of a 96 well 
plate. After allowing the MSC to adhere overnight, T cell proliferation 
assays were established either in the presence or absence of the 
MSC. T cell proliferation assays were performed in the presence of a 
diminishing number of MSC, with the highest being 33,000 MSC per 
reaction. In each assay, 100,000 MNC were incubated with anti-
CD3/CD28 beads to stimulate T cell proliferation. After 4 days, the 
level of cell proliferation was measured, using 3H-thymidine 
incorporation. The data represent the responses of two different blood 
donors when cultured with one of the cell lines produced during 
validation runs. 
A 
B 

 
 
Open Label Phase I HCT-MSC in Toddlers with ASD (TACT) 
 
May 17, 2021 
 
Page 17 of 55 
 
 
Finally, MSCs may also aid in synaptogenesis and restoration/regeneration of functional 
neurological pathways by supplying bioactive agents that stimulate the action of intrinsic neural 
progenitor cells. Various molecular targets have been implicated, including tissue plasminogen 
activator (tPA), synaptophysin, brain-derived neurotrophic factor (BDNF), and neurotransmitter 
receptors. Through a series of in vitro experiments via coculture, patch-clamp, inhibitory, and 
biochemical techniques, Koh, et al.82 demonstrated that (1) human umbilical cord-tissue derived 
MSCs can induce synapse formation and enhance synaptic function and (2) thrombospondin 
proteins are both produced by cord tissue-derived MSCs and necessary for their synaptic effects. 
In addition to chemokines associated with the capacity to repair endothelium, hCT-MSCs also 
produce thrombospondin-1, indicating that they may also have the capacity to restore effective 
synapses. Research at Duke University demonstrated that hCT-MSCs produce and secrete 
multiple cytokine and chemokines using cytokine arrays by RayBiotech (Norcross, GA). Follow up 
studies using Bioplex assays and ELISA measured levels of selected cytokines/chemokines as 
shown in Table 2. 
 
Table 2.  Cytokine and chemokine production by hCT-MSCs. 
Cytokine/chemokine 
P2 supernatant 
range, pg/mL 
BioPlex 
 
IL-6 
178 - 1134 
CCL2 /MCP-1 
270 - 453 
CXCL1/GRO  
250 - 1280 
CXCL5 
880 - 3025 
CXCL8/IL-8 
250 - 837 
ELISA 
 
Thrombospondin-1 
150 - 415 
A Bioplex has validated many of the findings from the cytokine arrays.  
ELISA assays have been used to quantify the production of Thrombospondin-1 and -2.  
 
2.5 
PRECLINICAL STUDIES OF CELL THERAPIES IN ASD 
Mouse models of single gene disorders that are associated with the ASD phenotype, such as 
Fragile X syndrome, Rett syndrome, and Tuberous Sclerosis Complex syndrome, have been 
used to study the effects of cellular therapy, such as MSCs, on both brain and behavior. 
Reported data from such animal studies may reflect differences related to the specific genetic 
subtype of ASD and may not generalize to all cases (e.g. idiopathic ASD). 
 
Derecki et al85 report recovery of function in a mouse model of Rett syndrome, an X-linked 
condition associated with ASD typically caused by a mutation of the MECP2 gene. This gene 
encodes a methyl-CpG-binding protein, and the mutation leads to deficient phagocytic function in 
glial cells. Transplantation of cells from wild type bone marrow via intravenous infusion arrested 
disease development in the mouse model of Rett syndrome (Mecp2-null C57BL/6 mice). Following 
engraftment, survival was improved, breathing patterns normalized, apneas were reduced, body 
weight increased, and locomotor activity was improved. 
 
The BTBR T+ Itpr3tfI (BTBR) mouse strain, derived from the inbred Black and Tan BRachyury 
strain, is another mouse model of ASD. In addition to impaired social behavior, aberrant 
communication, increased repetitive behaviors, and increased cognitive rigidity, BTBR mice also 
exhibit increased levels of peripheral CD4+ T-cells, peripheral B-cells, and serum and brain 

 
 
Open Label Phase I HCT-MSC in Toddlers with ASD (TACT) 
 
May 17, 2021 
 
Page 18 of 55 
 
immunoglobulin levels, among other immune abnormalities.86 Segal-Gavish, et al87 delivered 
human MSCs to BTBR mice via intraventricular injection into the central nervous system. Mice 
were immunosuppressed with cyclosporine before and after treatment. In this model, 
improvements in all three domains – social behavior, stereotyped behaviors, and cognitive rigidity 
– were observed in MSC-treated mice compared to controls. Differences in anxiety-related 
behaviors and locomotion were not observed. 
 
These mouse models demonstrate the potential for benefit from cellular therapies in at least 
certain subtypes of ASD. 
2.6 
OUTCOMES OF CELL THERAPIES IN CHILDREN WITH ASD 
The autism and cellular therapies teams at Duke have partnered to test whether intravenous 
infusions of autologous or allogeneic cord blood or allogeneic cord tissue derived MSCs would be 
safe, feasible and improve core symptoms of autism in affected individuals. Results of these 
studies are summarized below. 
 
DukeABCs Clinical Trial: Duke investigators recently completed and published the results of an 
open-label Phase 1 safety and tolerability study in 25 children (ages 2-5 mean age 4.5 years), 
diagnosed with ASD who were treated with a single intravenous infusion of autologous CB 
and followed for a year (ClinicalTrials.gov ID: NCT02176317).88 CB was administered as a 
single infusion (median infused dose: 2.6x107/kg, range: 1.0x107/kg to 8.1x107/kg). No 
immunosuppression was administered prior to infusion. 
 
The safety and tolerability profile of autologous CB infusion in ASD was excellent. No serious 
adverse events were reported, and adverse events, in general, were sparse. Three children 
had mild allergic reactions associated temporally with the infusions, consisting of cough and 
hives during infusion for 1 child and cough post-infusion for 2 children. Also, one child was 
noted to be more irritable for 2 days post-infusion. No participants discontinued prematurely 
from the study due to adverse events. 
 
With regard to preliminary assessments of efficacy, improvements in social communication 
abilities were noted on the caregiver-completed Vineland Adaptive Behavior Scales-Second 
Edition (VABS-II) and on the Pervasive Developmental Disorder Behavior Inventory (PDDBI) 
(figure 4). The Clinical Global Impression-Improvement scale, completed by clinicians, reflected 
beneficial changes during the 6-month period post-infusion in core ASD symptoms in 
approximately 60% of the participants, as manifested by the participants’ increased social 
communication skills, receptive/expressive language, decreased repetitive behavior, and 
decreased sensory sensitivities. In computerized eye-tracking assessments, the participants 
manifested improvements in social attention. Participants, on average, showed a 20% increase 
in odds of gazing at the actress’ eyes over time (OR=1.20, 95% CI: 1.00, 1.43, p5.048). A 7-
point change in VABS-II socialization standard score was associated with a 14% increase in 
odds of gazing at the actress (OR=1.14, 95% CI: 1.07,1.21; p<.001).88 
 
Electrophysiological recordings were taken during viewing of dynamic social and nonsocial 
stimuli at 6 and 12 months post-treatment. Significant changes in EEG spectral characteristics 
were found by 12 months post-infusion, which were characterized by normalization of the EEG 
spectrum, reflected in increased alpha and beta power and decreased EEG theta power.89 
 
 
 
 

 
 
Open Label Phase I HCT-MSC in Toddlers with ASD (TACT) 
 
May 17, 2021 
 
Page 19 of 55 
 
Figure 4: Changes in ASD Symptoms after Autologous CB Infusion. Panel A: Vineland-II socialization domain 
standard scores at baseline and six months after infusion (p=0.02 baseline to six months). Panel B: Distribution of 
Vineland-II socialization domain standard scores stratified by nonverbal IQ. Panel C: Vineland-II communication 
domain standard scores at baseline and six months after infusion (p<0.01 baseline to six months). Panel D: 
Distribution of CGI-I scores at six (blue) and 12 (red) months post-infusion. Sample sizes are N=25 for baseline and 
6-month time points and N=22 at 12-months.  
 
 
 
 

 
 
Open Label Phase I HCT-MSC in Toddlers with ASD (TACT) 
 
May 17, 2021 
 
Page 20 of 55 
 
There was also some evidence that improvements in social behavior were found to be associated 
with increases in connectivity across brain regions when assessed via diffusion tensor imaging 
(obtained via MRI scans). In an open label trial evaluating the efficacy of autologous cord blood 
for treating autism symptoms, improvement on 3 behavioral outcome measures was correlated 
with increased connectivity between the left temporal pole and the left hippocampus (VABS II 
p<0.001; Expressive One Word Picture Vocabulary Test-Fourth Edition (EOWPVT-4) p<0.05; 
CGI-I p<0.0001). Improvement on both the VABS-II and the EOWPVT-4 was correlated with 
increased connectivity between the fusiform and putamen (VABS II p<0.05; EOWPVT-4 p=0.01) 
as well as between the inferior temporal gyrus and the hippocampus (both p<0.05), both in the left 
hemisphere. Finally, there was a significant correlation between improvement on both the VABS-II 
and the CGI-I and increased connectivity between the inferior temporal gyrus and superior 
temporal gyrus (VABS-II p<0.05, CGI-I p<0.01) and between the temporal pole and globus 
pallidus (VABS II p<0.01; CGI-I p<0.05) in the left hemisphere. Improvement on the VABS II and 
CGI-I was also significantly correlated with increased connectivity between the frontal pole and 
globus pallidus and the insula and putamen in the right hemisphere (all p<0.05). Based on these 
data, a Phase 2 randomized study was conducted to evaluate the efficacy of CB therapy versus 
placebo in children with ASD. Data analysis of this trial is currently in progress. 
 
MSCs in ASD: One Chinese study enrolled 37 children with ASD and treated them with 4 doses 
of either umbilical cord blood mononuclear cells (CBMNC, given both intravenously and 
intrathecally) (n=14), CBMNC + intrathecal umbilical cord-derived mesenchymal stem cells 
(UCMSC) (n=9), or standard therapy (n=14).34 The only treatment-related side effect was 
transient fever in five patients. At six months post-treatment, both treated groups demonstrated 
greater improvement in multiple ASD measures (Childhood Autism Rating Scale, Clinical Global 
Impression scale, Aberrant Behavior Checklist) than the placebo group, indicating a potential 
therapeutic response. 
 
Results of a Phase I Trial of human cord tissue MSCs in children with ASD: At Duke, we have 
treated 12 children, ages 2-11 years, with ASD with hCT-MSC on a phase I open label trial to 
determine the safety of single and repeated IV doses of hCT-MSC. In this study, the first cohort of 
three patients received a single dose, the second cohort of three patients received two doses 
given two months apart, and the third cohort of six patients received three hCT-MSC infusions 
with a two-month interval between doses. Each dose contained 2x106/kg hCT-MSC (see study 
schema in Figure 5). 
 
Figure 5: Study Design for Phase I Study of hCT-MSC in Children with ASD 
     
 
Infusion1
Infusion2
Infusion3
Follow-up
Cohort1
N=3
MSCs
F/U
F/U
Cohort2
N=3
MSCs
MSCs
F/U
F/U
Cohort3
N=6
MSCs
MSCs
MSCs
F/U
F/U
Baseline
2months
4months
6months
6&12months
fromfinaldose

 
 
Open Label Phase I HCT-MSC in Toddlers with ASD (TACT) 
 
May 17, 2021 
 
Page 21 of 55 
 
Twelve children, three girls, nine boys with a mean age of 6.4 years (range 4-9 years), were 
enrolled between June and October 2017. Eleven participants were white, one was Asian; two 
were Hispanic or Latino. A total of 27 hCT-MSC doses were administered from 3 different 
manufacturing lots. Target dose at each administration was 2.0x106 TNC/kg based on the post 
thaw count. The mean of the actual dose infused was 2.0x106 TNC/kg with a standard deviation of 
0.4x10 6 TNC/kg. One patient received a lower dose of 1.79x106 TNC/kg (Patient 12 at Infusion 3) 
due to a laboratory complication. All children were premedicated with an intravenous dose of 
diphenhydramine 0.5mg/kg and solumedrol 0.5mg/kg approximately 30 minutes before the MSC 
dose. The MSCs were administered over 30 minutes. IV hydration was administered for 15-60 
minutes post MSC infusion. 
 
Three reactions were reported in 2 patients within 24 hours of infusion. One patient in Cohort 2 
experienced Allergy and Moderate Hypotension during infusion 2 associated with a deviation in 
the premedication schedule for the infusion. Due to an adverse reaction of extreme agitation to IV 
Benadryl during the initial infusion, this patient received oral Atarax as an alternative 
premedication for the second infusion. Shortly after initiation of the second hCT-MSC infusion, 
the patient developed diffuse erythroderma and a cough followed by mild hypotension and 
hypoxia. The infusion was immediately stopped, and the patient recovered completely with a fluid 
bolus and an extra dose of IV Solumedrol. The remainder of the hCT-MSC infusion was 
successfully completed after a dose of IV Benadryl. One patient in Cohort 3 experienced 
Moderate Hypotension after infusion 3 and received additional IV fluids. 
 
A total of 66 non-serious Adverse Events (AEs) that were not specifically attributed to the study 
product were reported among 11 of the 12 enrolled participants (Figure 6).  All of these reported 
events were Mild. The most frequently occurring event, agitation during the infusion procedure, 
was associated with the requirements of placing and maintaining an IV and being confined to a 
hospital room for the infusion, and all of these events resolved on the same day.  A total of 22 
other psychiatric or behavioral symptoms were reported in 7 different participants: 2 participants/2 
events in Cohort 1, 1 participant/5 events in Cohort 2, and 4 participants/15 events in Cohort 3. 
These symptoms included aggression (n=2), agitation (n=5), anxiety (n=3), defiant behavior (n=2), 
depression (n=1), emotional labiality (n=1), insomnia (n=3), intentional self-injury (n=1), and 
stereotopies (n=4).  Of note, 3 participants accounted for 17/22 of the psychiatric or behavioral 
adverse events.  Aside from agitation during the infusion procedure, there was a trend of 
increasing frequency of non-serious adverse events with increasing number of doses 
administered, but this was not statistically significant. There were no differences observed in the 
frequency of AEs according to lot number (GMP-075: median=9 events/participant, range: 1-12; 
GMP-087: median=4.5, range: 0-13; GMP-088: median=3.5, range: 3-50) (PKruskal-Wallis=0.58).  
There were no concerning changes in blood counts, chemistries, basic inflammatory markers 
(CRP, ESR), or humoral and cellular immune profiles throughout the study.  All participants 
remained Coombs negative.  
 
Six months after the initial hCT-MSC dose, development of anti-HLA class I antibodies was 
observed in 5 of 9 participants who did not have detectable HLA antibodies pre-treatment. HLA 
antibody data is available on all 12 participants at baseline and 6 months, and 11/12 participants 
at ≥12 months after their final hCT-MSC dose. In those participants who developed new HLA 
antibodies by six months, the HLA antibodies persisted at ≥12 months (figure 7). The HLA 
antibodies were directed against HLA alleles/antigens expressed on the MSCs and not by the 
patient. There were no associated clinical events in patients who developed anti-HLA antibodies. 
 
 
 

 
 
Open Label Phase I HCT-MSC in Toddlers with ASD (TACT) 
 
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Figure 6: Adverse Events Observed during Phase I hCT-MSC Study. A total of 2 events were related 
to study product—Hypotension and Infusion Related Reaction (discussed above). 
 
 
 
Class I HLA antibodies by number of doses, lot of hCT-MSC, and degree of HLA match are 
shown in Figure 5. New class I anti-HLA antibodies developed in 1/3 participants who 
received one dose of hCT-MSC, 1/3 participants who received 2 doses, and 3/6 participants 
who received 3 doses. All three participants who developed broad-spectrum class I HLA 
antibodies were treated with the same lot of hCT-MSC. In addition, 4/4 participants who 
were at least haploidentical to their hCT-MSC donor developed new-onset HLA antibodies 
(broad spectrum or donor-specific), versus 1/8 participants who were 0/8, 1/8, 2/8, or 3/8 
HLA-matched at HLA-A, B, C, and DRB1. No detected anti- HLA antibodies have been 
clinically significant. With the small numbers of patients enrolled on this study, there was no 
ability to assess effect of numbers of doses administered on the development of these 
antibodies. 
 
Results of behavioral outcome assessments are shown in Table 3. Measures reported below 
include assessments of social communications skills (VABS-3) with increases of 3 points and 
above indicating improvement, severity of autism symptoms (PDDBI) with decreases of at least 5 
points indicating improvement, and expert clinical judgment (CGI) ranging from no improvement to 
much improvement. Fifty percent (6/12) of patients showed an improvement on at least 2/3 
measures, 33% (4/12) showed an improvement on all three measures, and 16% (2/12) showed an 
improvement on 2/3 measures. Of the children who improved on 2/3 of the outcome measures, 
two had one dose, two had two doses, and two had three doses. 
 
 
 

 
 
Open Label Phase I HCT-MSC in Toddlers with ASD (TACT) 
 
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Figure 7:  Class I anti-HLA Antibodies.  Panel A: Presence of Class I HLA antibodies at baseline, 6 
months, and >12 months by participant (≥12 month data not available for participants 3, 4, 11, 12). Panel B: 
Class I HLA antibodies and baseline and 6 months by number of hCT-MSC doses. Panel C: Class I HLA 
antibodies at baseline and 6 months by lot of hCT-MSC. Panel D: Class I HLA antibodies by HLA match (at 
HLA-A, B, C, DRB1) between hCT-MSC donor and recipient. 
 
 
 
Table 3:  Behavioral Evaluations, Phase I Study of hCT-MSC in Children with ASD 
 
 
 
 
 
 
DQ=Developmental Quotient, VABS=Vineland Adaptive Behavior Scales-Third Edition (VABS-3) Socialization 
Standard Score, PDDBI=Pervasive Developmental Disorder Behavior Inventory Autism Composite, CGI=Clinical 
Global Impression-Improvement scale 
*Clinically significant improvement = 3 points. 
0
20
40
60
80
100
1 2 3 4 5 6 7 8 9 101112
%RelativeClassIHLAAntibody

Participants
Baseline
6months
~12months
0
20
40
60
80
100
1
1
1
2
2
2
3
3
3
3
3
3
%RelativeClassIHLAAntibody
#ofDoses
6months
Baseline
0
20
40
60
80
100
A A A A
B B B B C C C C
%RelativeClassIHLAAntibody
MSClot
6months
Baseline
0
20
40
60
80
100
0
0
0
0
0
1
3
3
4
4
4
6
%RelativeClassIHLAAntibody
TotalHLAmatch
6months
Baseline
ID 
Dose 
Sex 
Nonverbal 
DQ 
VABS* 
PDDBI 
CGI 
# of 
assessments 
indicating 
Improvement 
1 
1 
M 
62 
-2 
- 
Min 
1 
2 
1 
M 
68 
4 
6 
Min 
2 
3 
1 
M 
45 
22 
-22 
Min 
3 
4 
2 
F 
59 
0 
-6 
Much 
2 
5 
2 
M 
40 
-10 
-1 
No 
0 
6 
2 
M 
36 
8 
-22 
Min 
3 
7 
3 
M 
42 
-2 
0 
Min 
1 
8 
3 
M 
54 
-8 
-4 
No 
0 
9 
3 
M 
71 
-3 
6 
Min 
1 
10 
3 
M 
82 
19 
-20 
Min 
3 
11 
3 
F 
59 
4 
-7 
Min 
3 
12 
3 
F 
95 
7 
-2 
No 
1 

 
 
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2.7 
STUDY RATIONALE AND HYPOTHESIS 
The mechanistic rationale and overarching theory of this line of investigation is that hCT-MSC can 
act through paracrine and trophic mechanisms to favorably modulate ongoing inflammation and/or 
immune pathology in the brain, suppress microglial activation, and possibly protect neurons from 
further damage. The primary hypothesis of this phase I clinical trial is that administration of hCT-
MSCs will be safe and tolerable and result in improvement in the social communication abilities, 
as measured via objective ratings of social communication abilities during caregiver-child 
interaction.  
 
In many contexts, MSCs suppress, rather than augment, immunological and inflammatory 
responses. Documented mechanisms include shifts in effector T cells such as generation of 
regulatory T cell populations and changes in monocyte/dendritic cell cytokine generation leading 
to anti-inflammatory cytokines. Therefore, it is plausible to consider a population of MSCs as an 
immunological and/or anti-inflammatory agent. Both postmortem brain tissue studies and PET 
imaging data from living individuals with ASD have revealed evidence of increased microglial 
activation, suggesting that immune and/or inflammatory mediated brain damage plays a role in 
the etiology of ASD as discussed above.90 Thus, hCT-MSC may be a candidate therapy for ASD 
because of the immunomodulatory activities of MSCs. 
 
2.8 
STUDY DESIGN 
This is a single site, prospective study of one intravenous infusion of human umbilical cord tissue-
derived mesenchymal stromal cells (hCT-MSC) in toddlers with autism spectrum disorder (ASD).  
Toddlers 18 months to <4 years (3 years 364 days) of age with a confirmed diagnosis of ASD will 
be eligible to participate. Diagnosis will be confirmed at the time of the eligibility visit at the Duke 
Center for Autism and Brain Development.  All participants will receive a single intravenous dose 
of 2x106/kg hCT-MSC per kilogram at baseline.  Assessments will be conducted at baseline and 6 
months, with remote follow-up assessments at 12 months.  
The primary purpose of this study is to evaluate safety and feasibility.  Safety assessments include 
monitoring of acute infusion reactions, adverse events, incidence of infections, and markers of 
alloimmunization.  Clinical outcome measures will also be described.  A key clinical outcome 
measure is the change in social communication abilities from baseline to 6 months based on the 
Joint Engagement Rating Inventory (JERI), a commonly-used and well-validated coding system 
for rating the quality and quantity of social communication skills in toddlers with and without 
ASD.91  JERI coding rates social communication abilities on a 1 to 7 scale and factors in both the 
quantity and quality of skills.  The total joint engagement score as well as ratings on all JERI 
subscales that comprise the total score will be described.  
 
Other clinical endpoints will include the PDD Behavior Inventory (PDDBI) autism composite score, 
the mean of the Socialization Subscale Standard Score and Communication Subscale Standard 
Score on the Vineland Adaptive Behavior Scales (VABS-3), the Clinical Global Impression Scale 
(CGI) – Severity and Improvement Scales, the Communicative Development Inventories (CDI-2): 
Words & Sentences subscales, and the Pediatric Quality of Life Scale (PedsQL Generic Core 
Scale).   
 
Exploratory clinical endpoints will include autism symptoms measured by an app that elicits and 
records autism symptoms on an iPad (SenseToKnow), PDD Behavior Inventory (PDDBI) 
Subscales, and VABS-3 Standard Score and age equivalent for the following  subscales: 
Socialization, Communication, and Daily Living and the Standard Score and age equivalent for the 
VABS-3 Adaptive Behavior Composite.  

 
 
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Safety and VABS-3 assessments will also be conducted remotely at three and 12 months. 
Duration of study participation will be 12 months from the time of the baseline infusion. 
2.9 
RISKS AND BENEFITS 
Potential risks associated with infusion of MSCs include a reaction to the product (rash, shortness 
of breath, coughing, wheezing, difficulty breathing, hypotension, hypertension, hypoxia, swelling 
around the mouth, throat or eyes, tachycardia, bradycardia, diaphoresis), transmission of infection, 
and HLA sensitization.  Theoretical risks that must be considered but have not been associated 
with MSC administration in humans include the possibility of immune suppression and ectopic 
tissue formation.  Cord blood collected with the cord tissue used to manufacture hCT-MSC is 
screened for infection, and the product must meet release criteria prior to infusion, as described 
below.  Participants will not receive immunosuppressive therapy prior to or after infusion of hCT-
MSC cells.  Additionally, an inherent risk in completing study procedures in person is possible 
exposure to COVID-19; however, social distancing, screening, masking, cleaning and other safety 
procedures in line with the most recent guidelines from Duke Health and the Duke University 
School of Medicine have been implemented to minimize this risk as much as is feasible.    
 
Potential benefits of this intervention include the possibility that hCT-MSC may, via direct or 
indirect mechanisms, induce changes that result in the reduction of the participant’s core ASD 
symptomatology and improvement in abilities affected by ASD symptoms. 
3.0 
STUDY OBJECTIVES 
1. To determine the safety and tolerability of a single intravenous infusion of hCT-MSC in very 
young children with ASD. 
2. To determine whether there are improvements in social communication abilities during 
caregiver-child interaction from baseline to six months following an infusion at baseline.   
4.0 
STUDY DESIGN 
4.1 
GENERAL DESIGN 
This is a single site, phase I, open-label, prospective study of one intravenous infusion of human 
umbilical cord tissue-derived mesenchymal stromal cells (hCT-MSC) in 12 toddlers ≥18 months to 
<4 years (3 years 364 days) of age with autism spectrum disorder (ASD).  All participants will 
receive a single intravenous dose of 2x106 hCT-MSC per kilogram at baseline.  The primary 
outcome is safety, as measured by the incidences of infusion reactions, infections, and new-onset 
clinically significant anti-HLA antibodies.  The main clinical outcome measure is the change in 
social communication abilities from baseline to 6 months based on the Joint Engagement Rating 
Inventory (JERI).91  Duration of study will be 12 months from the time of the initial infusion.   

 
 
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4.2 
STUDY FLOW CHART 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
4.3 
STUDY ENDPOINTS 
 
Safety Endpoints:   
Safety of hCT-MSC infusion in children with ASD will be assessed by: 
1. Incidence and severity of infusion reactions  
2. Incidence and severity of product-related infections  
3. Evidence of alloimmunization via anti-HLA antibodies  
4. Incidence and severity of graft vs. host disease  
5. Incidence and severity of unexpected adverse events, by relation to study product  
 
Efficacy Endpoints:  
The primary clinical outcome measure is the change in social communication abilities from 
baseline to 6 months based on total score (1-7) on the Joint Engagement Rating Inventory (JERI), 
a commonly-used and well-validated coding system for rating the quality and quantity of social 
communication skills in toddlers with and without ASD.91     
 
Change measured on the following endpoints from baseline to six months will be compared: 
1. PDD Behavior Inventory Autism Composite Score (PDDBI) 
2. Mean of the Socialization Subscale and Communication Subscale standard scores on the 
Vineland Adaptive Behavior Scales (VABS-3) 
3. Clinical Global Impression Scale (CGI) – Severity and Improvement (Improvement at 6 
months only 
12 Month Remote 
Evaluations
6 Month Follow 
Up Visit
3 Month Remote 
Visit
Baseline Visit
Patient Screening
Review Records, Labs, Videos, Photos, Remote 
Cognitive Evaluations
Confirm Eligibility, Consent, clinical and 
behavioral assessments, Parent Questionnaires & 
Interviews, Safety Assessments, Single Dose 
Infusion of hCT-MSC
Parent Questionnaires & Interview; Safety Assessment
Clinical and behavioral assessments, Parent 
Questionnaires & Interviews, Safety 
Assessments
Parent Questionnaires & Interviews, Safety 
Assessments, Laboratory Tests & Follow Up

 
 
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4. Communicative Development Inventories (CDI-2): Words & Sentences subscales  
 
Exploratory Endpoints: 
Change measured from baseline to six months will be evaluated on the following outcome 
measures.  
1. Changes in auditory orienting, facial expressions, look duration, attention to social and 
nonsocial stimuli, postural sway, and vocalizations as measured via computer vision 
analysis by an app that elicits and records autism symptoms on an iPad (SenseToKnow) 
 
2. PDD Behavior Inventory (PDDBI) Subscales  
3. VABS-3 Socialization Subscale Standard Score and age equivalent 
4. VABS-3 Communication Subscale Standard Score and age equivalent 
5. VABS-3 Daily Living Subscale Standard Score and age equivalent 
6. VABS-3 Adaptive Behavior Composite Standard Score and age equivalent 
7. Pediatric Quality of Life Scale – Generic Core 
5.0 
RESEARCH PARTICIPANT SELECTION AND WITHDRAWAL  
 
5.1 
STUDY POPULATION 
Twelve toddlers ages 18 months to <4 years (3 years 364 days) with a confirmed diagnosis of 
ASD. 
5.2 
INCLUSION CRITERIA 
1. Age 18 months to <4 years (3 years 364 days) at the time of consent 
2. Confirmed clinical DSM-5 diagnosis of Autism Spectrum Disorder using the DSM-5  
    Checklist as informed by the Brief Observation of Symptoms of Autism (BOSA) Assessment. 
3. Fragile X testing performed and negative; constitutional microarray (CMA) and/or whole exome 
sequencing performed and results not linked to autism diagnosis 
4. Stable on current psychoactive medication regimen (dose and dosing schedule) for at least 2 
months prior to infusion of study product 
5. Normal absolute lymphocyte count (≥1500/uL) 
6. Participant and parent/guardian are English speaking 
7. Able to travel to Duke University for two multi-day visits (baseline and six months) and 
parent/guardian is able to participate in interim surveys and interviews  
8. Parental consent from at least one parent or guardian. 
 
5.3 
EXCLUSION CRITERIA 
1. General: 
a. Review of medical records indicates ASD diagnosis not likely  
b. Screening data suggests that participant would not be able to comply with the 
requirements of the study procedures as assessed by the study team 
c. Family is unwilling or unable to commit to participation in all study-related 
assessments, including protocol follow up  
d. Sibling is enrolled in this (Duke TACT) study 
2. Genetic:  
a. Records indicate that child has a known genetic syndrome such as (but not limited to) 
Fragile X syndrome, neurofibromatosis, Rett syndrome, tuberous sclerosis, PTEN 
mutation, cystic fibrosis, muscular dystrophy or a genetic mutation known to be 
associated with ASD 

 
 
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b. Known pathogenic mutation or copy number variation (CNV) associated with ASD 
(e.g., 16p11.2, 15q13.2, 2q13.3) 
3. Infectious: 
a. Known active CNS infection  
b. Evidence of uncontrolled infection based on records or clinical assessment 
c. Known HIV positivity 
d. Exposure to COVID-19 in the preceding 14 days or positive COVID-19 test in the 
previous 28 days. Subjects with a past history of infection with COVID-19 must be 
symptom-free for 14 days prior to the initial visit 
 
4. Medical: 
a. Known metabolic disorder  
b. Known mitochondrial dysfunction 
c. History of unstable epilepsy or uncontrolled seizure disorder, infantile spasms, Lennox 
Gastaut syndrome, Dravet syndrome, or other similar chronic seizure disorder 
d. Active malignancy or prior malignancy that was treated with chemotherapy 
e. History of a primary immunodeficiency disorder 
f. History of autoimmune cytopenias (i.e., ITP, AIHA) 
g. Coexisting medical condition that would place the child at increased risk for 
complications of study procedures   
h. Concurrent genetic or acquired disease or comorbidity(ies) that could require a future 
stem cell transplant 
i. 
Significant sensory (e.g., blindness, deafness, uncorrected hearing impairment) or 
motor (e.g., cerebral palsy) impairment  
j. 
Impaired renal or liver function as determined by serum creatinine >1.5mg/dL or total 
bilirubin >1.3mg/dL, except in patients with known Gilbert’s disease  
k. Significant hematologic abnormalities defined as: Hemoglobin <10.0 g/dL, WBC < 
3,000 cells/mL, ALC <1000/uL, Platelets <150 x 10e9/uL  
l. 
Known clinically relevant physical dysmorphology associated with neurodevelopmental 
conditions. 
5. Current/Prior Therapy:  
a. History of prior cell therapy 
b. Current or prior use of IVIG or other anti-inflammatory medications with the exception 
of NSAIDs 
c. Current or prior immunosuppressive therapy  
i. No systemic steroid therapy that has lasted >5 days within 4 weeks prior to 
enrollment.  Topical and inhaled steroids are permitted. 
 
5.4 
RESEARCH PARTICIPANT RECRUITMENT AND SCREENING 
Patients may be recruited through IRB-approved advertising for the study on the websites of 
private CB banks, parent sponsored websites, the NMDP website, selected autism societies, local 
medical providers, or the Duke Center for Autism and Brain Development, through a record of 
inquiries for previous studies (brain injury database), and through the Duke Center for Autism and 
Brain Development research registry.  Separate IRB approval will be obtained for any 
advertisements.   
 
If a participant has consented to the Duke Center for Autism Research Repository (Pro00054178), 
study team members may access these data for purposes of screening, eligibility, or as source 
data in this study. Participants screened for or enrolled in the TACT study may also be 
approached per repository protocol to determine their interest in storing data from this study and 

 
 
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any future studies into the repository for future use.  The consent form and participant-facing 
materials related to the Duke Center for Autism Research Repository are submitted and approved 
separately, through Pro00054178.   
 
Screening for the study is conducted under a separate, IRB-approved screening protocol 
(Pro00063563).  Under this protocol, after written informed consent is obtained from a 
parent/guardian, the patient’s medical and psychological records, therapy records, photographs, 
behavioral videos, and results of genetic testing are obtained and reviewed by two teams.  The 
medical review is conducted by a team of pediatric nurses, nurse practitioners, and physicians to 
identify the presence of any metabolic, immunologic, neurologic, sensory, genetic, or laboratory 
exclusion criteria.  If no exclusion criteria are identified, the psychiatric review is then conducted by 
a combination of psychologists, psychiatrists, and other clinically trained staff with expertise in 
diagnosing and treating children with ASD.  They perform an extensive review of the patient’s 
psychological records as well as any therapy records available. A patient must be approved by 
both medical and psychiatric screening teams to proceed with further laboratory, phone, 
teleconference, or in-person screening and study enrollment.  Should a concern for a previously 
undiagnosed condition or genetic finding arise during the screening process, this will be discussed 
with the patient’s parent(s)/guardian(s) and a referral will be made to an appropriate medical or 
psychiatric provider for evaluation and treatment, if indicated. 
 
5.5 
EARLY WITHDRAWAL OF RESEARCH PARTICIPANTS 
 
Criteria for Removal from Protocol Therapy: 
1. Diagnosis of a genetic disease while under evaluation or on study. 
2. Change in medical condition that precludes study participation.  
 
Patients who are off protocol therapy are to be followed until they meet any one of the off-study 
criteria (see below).  Follow-up data will be obtained on off-protocol participants unless consent is 
withdrawn.  Participants that are taken off study prior to infusion of hCT-MSC will be considered 
not evaluable and can be replaced with another participant. 
 
Off-Study Criteria: 
1. Death. 
2. Lost to follow-up. 
3. Withdrawal of consent for any further data collection. 
4. Completion of the final study visit. 
6.0      STUDY PRODUCT 
6.1 
HUMAN UMBILICAL CORD TISSUE-DERIVED MESENCHYMAL STROMAL CELLS (hCT-
MSC) 
hCT-MSCs are a product of allogeneic cells manufactured from digested umbilical cord tissue that 
is expanded in culture, cryopreserved and banked.  hCT-MSCs are manufactured from umbilical 
cord tissue donated to the Carolinas Cord Blood Bank, an FDA-licensed, FACT-accredited, public 
cord blood bank at Duke University Medical Center, after written informed consent from the baby’s 
mother.  Cord tissue is harvested from the placentas of male babies delivered by elective C-
section after a normal, full-term pregnancy.   Donor screening questionnaires are completed by 
the maternal donor, and maternal blood is tested for communicable diseases by the CLIA-certified 
donor screening laboratory.  Donors must be eligible for donation to a public cord blood bank for 
allogeneic use.  After delivery of the placenta and cord, the cord blood is aseptically drained from 
the placenta.  Then the cord is dried and cleaned with chloropreps, separated from the base of the 

 
 
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placenta, placed in a sterile bottle containing Plasmalyte A, and transported to the Robertson CT2 
GMP cell processing laboratory at room temperature in a validated container.  
 
In the clean room manufacturing suite, in a biosafety cabinet, the cord tissue is removed from the 
media, placed in sterile dishes, cut into small pieces and then minced and digested in the Miltenyi 
Biotec GentleMacs Octo Dissociator with GMP-grade enzymes:  hyaluronidase, DNase, 
collagenase, papain.  The resultant cell suspension is placed in culture in Prime XV MSC 
Expansion XSFM (Irvine Scientific) media with 1% platelet lysate and grown to confluence (~7-14 
days) to establish the P0 culture.  To establish the master cell bank, P0 is harvested and 
cryopreserved in cryovials with Cryostor 10 media (BioLife), and stored in the vapor phase of 
liquid nitrogen.  P1 and P2 cultures are grown under similar conditions, in hyperflasks or 
hyperstacks without platelet lysate, as needed to create the working cell bank and product for 
administration, respectively. Cells from P1 and P2 are removed from plastic cultureware using 
TrypLE (Gibco). The final product is derived from the P2 cultures which are harvested into 
plasmalyte with 5% human serum albumin, washed and cryopreserved in 5 compartment 
cryobags (Syngen) in 5 mL containing 50-100 million cells in a final concentration of 10% DMSO 
with dextran (Akron Scientific).  On the day of administration, one compartment is thawed, diluted 
in 10-40 mls of plasmalyte IV solution, placed in a syringe or bag and transported to the bedside 
for administration over 30-60 minutes.    
 
At each passage, the cell product is characterized by assessing cell surface phenotype by flow 
cytometry and functional assays via T-cell proliferation and organotypic models of microglial 
activation.  Each lot, prior to cryopreservation of P2, will also be tested for sterility, endotoxin and 
mycoplasma and these tests must meet specifications.  For dosing, release testing after thaw and 
dilution will include TNCC and viability via cellometer.  Sterility cultures (14 day) are also initiated 
but results are not available at the time of infusion.  See section 6.4 for management plan if 
sterility cultures subsequently turn positive post infusion. Patients will be dosed with 2x106 hCT-
MSCs/kg based on the post thaw count. 
 
6.2 
DONOR SCREENING AND TESTING 
Donor screening and testing is performed per Carolinas Cord Blood Bank standard operating 
procedures to meet all requirements in 21CFR Part 1271.  The screening and testing is current 
with recommendations and is approved by the FDA under biological license number 1870. 
Maternal donors of umbilical cord blood are screened and tested for HIV-1, HIV-2, hepatitis B virus 
(HBV, surface and core antigen), hepatitis C virus (HCV), Treponema pallidum (syphilis), CJD 
(screening only), Chagas, human T-lymphotropic virus types 1 and 2 (HTLV-1, HTLV-2) and CMV. 
Nucleic acid testing for HIV-1/2/O, HBV, WNV and HCV are also performed on maternal blood. 
Screening for Zika virus may also performed. 
 
 
Because the cord tissue used for this study will be obtained from donors consented for cord blood 
donation to the Carolinas Cord Blood Bank, they will undergo donor screening and infectious 
disease testing per Carolinas Cord Blood Bank standard operating procedures.  The cord blood-
associated maternal samples and cord tissue MSC samples will be retained as reference samples 
for future testing as part of this study. 
 
6.3 
PROCESS AND FINAL FORMULATION 
hCT-MSC is manufactured from a single umbilical cord tissue in a series of three steps that 
generate a master cell bank, a working cell bank, and the study product.  The product for each 
step is frozen and stored in vapor phase in liquid nitrogen freezer. At P2, a representative cryobag 

 
 
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will be thawed and qualified prior to the treatment of any patients with that lot of product. Testing 
will include cell count, viability, phenotype, functional assays, endotoxin, mycoplasm, gram stain 
and sterility.    
 
On the day of treatment, cells are thawed per SOP CT2-MSC-006, diluted in 10-40 mLs of 
plasmalyte-A + 5% HSA, and an aliquot removed for cell count, viability, and sterility culture.  If the 
cells meet release criteria, the final product volume is adjusted to deliver the appropriate dose 
(2x106 cells/kg) to the study participant.  The cells are delivered to the bedside in a bag or syringe 
containing plasmalyte-A, 5% HSA, and residual DMSO.  Any removed cell suspension is 
inoculated into aerobic and anaerobic culture bottles for sterility testing. The cells have a four-hour 
expiry post thaw. 
 
The hCT-MSC final product will be released conditionally for administration to the patient after 
testing a post thaw cell count and viability.  Final release will occur after the 14-day sterility culture 
period for the study product.  In the event that a sterility culture turns positive after administration 
of the product, the organism will be identified and antibiotic sensitivities performed.  The patient’s 
family will be contacted to determine if they are symptomatic (ie fever).  Asymptomatic patients will 
not be treated with antibiotics.  Symptomatic patients will be evaluated and treated accordingly, 
with blood cultures and antibiotics as appropriate.  All patients receiving a product with 
subsequent positive sterility test will be followed with close, regular contact by a study nurse for 14 
days after the positive sterility test is noted. 
 
6.4 
PACKAGING OF STUDY PRODUCT 
All umbilical cord tissues will be assigned an ISBT Demand 128 bar code label or unique identifier, 
which is carried through to all in-process and final hCT-MSC products.  In addition, the CT2 GMP 
facility will provide a final product label for each hCT-MSC product.  The product label will include 
a space to affix the bar code label as well as space for the participant number, date and time of 
product expiry, and any other pertinent information.  As a participant is enrolled, a participant 
number will be assigned which will link to the 12 digit ISBT number bar code number assigned to 
the umbilical cord blood tissue.  The final product will be assigned a lot number (manufacturing 
operation number) and expiry date and time that will be denoted on the Certificate of Analysis and 
product label. The participant number and ISBT bar code number of the product will be also listed 
on the Certificate of Analysis.  All products will be transported from the CT2 laboratory to the 
infusion center in a validated cooler by courier. 
 
6.5 
ADMINISTRATION OF PRODUCT 
Patients will be admitted to the infusion center on the day of their scheduled infusion.  Patients 
may require some sedation prior to the IV placement if they are unable to remain still or cooperate. 
A peripheral IV will be placed by clinical or study staff.  Patients will be premedicated with 
Benadryl 0.5mg/kg/dose IV and Solumedrol 0.5-1mg/kg IV.  The hCT-MSCs or placebo product 
will be administered intravenously over 30-60 minutes.  Vital signs (heart rate, blood pressure, 
temperature, respiratory rate) will be attempted upon arrival to the clinic and monitored as 
clinically indicated.  Pulse oximetry will be monitored continuously throughout the infusion and for 
at least 5 minutes post infusion.  Patients should be observed a minimum of 15-30 minutes post 
infusion.   
6.6 
SAFETY FOLLOW-UP 
On Day 1 following each infusion, the participant will be evaluated by study staff to assess for any 
infusion related adverse reactions or complications.  This assessment can be done in person, over 

 
 
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the phone, by email or a teleconference.  At 7-10 days post each infusion, a member of the study 
team will contact the parent or guardian via phone, teleconference, or email to assess patient 
status and any adverse events.  A questionnaire (see Appendix 1) will be administered at each 
subsequent visit and at 3, 9 and 12 months to assess for serious adverse events.  PRA will be 
obtained pre-treatment and at 6 and 12 months after the initial infusion.  
7.0 
STUDY PLAN 
7.1 
OVERVIEW 
Parents/Guardians who have previously contacted our program and have a child who may meet 
eligibility criteria for this study will be notified that this study is available.  After initial contact, 
parents/guardians of potential research participants will have an initial phone or teleconference 
interview with study personnel to describe the study, verify basic eligibility criteria, and confirm 
their interest in participation.  The participant’s eligibility will then be screened through review of 
medical and psychological records, therapy records, video, photos, cognitive and diagnostic 
testing, and laboratory testing under a separate screening protocol and consent.  
 
Once all screening is complete and the participant is likely to meet study criteria, the participant 
will travel to Duke for their first visit.  During their baseline visit at the Duke Center for Autism and 
Brain Development, the informed consent will be obtained and participant eligibility will be 
determined by physical observation and verification of ASD diagnosis per BOSA and DSM-5 
criteria. During his/her first visit, he/she will also undergo additional clinical and observational 
evaluations, and SenseToKnow app assessments.  Participants will be evaluated the day after 
infusion either in person, via teleconference, or by phone call and parents will be contacted 7-10 
days after infusion for follow up safety evaluation.  Participants will return to Duke six months later 
for repeated clinical and observational evaluations, and SenseToKnow app assessments, and 
safety follow-up.  At 12 months post-infusion, remote safety assessment and parent-completed 
behavioral questionnaires will be completed.  
 
7.2 
PARTICIPANT SCREENING 
Screening for the study is conducted under a separate, IRB-approved screening protocol 
(Pro00063563).  Under this protocol, after written informed consent is obtained from a 
parent/guardian, the participant’s medical and psychological records, therapy records, 
photographs, behavioral videos, and results of all genetic testing are obtained and reviewed by 
two teams.  The medical review is conducted by a team of pediatric nurses, nurse practitioners, 
and physicians to identify the presence of any metabolic, immunologic, neurologic, sensory, 
genetic, or laboratory exclusion criteria.  If no such exclusion criteria are identified, the psychiatric 
review is then conducted by a combination of psychologists, psychiatrists, and other clinically 
trained staff with expertise in diagnosing and treating children with ASD.  They perform an 
extensive review of the participant’s psychological records as well as any therapy records 
available.  A participant must be approved by both medical and psychiatric screening teams to 
proceed with further laboratory or phone, teleconference, or in-person screening and study 
enrollment.  Should a concern for a previously undiagnosed condition or genetic finding arise 
during the screening process, this will be discussed with the participant’s parent(s)/guardian(s) 
and a referral will be made to an appropriate medical or psychiatric provider for evaluation and 
treatment, if indicated. 
7.3 
STUDY INFUSIONS 
All participants will receive an infusion of allogeneic hCT-MSC cells.  On the day of infusion, hCT-
MSC cells will be thawed and prepared by the CT2 GMP laboratory and/or Duke Stem Cell Lab 

 
 
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per standard operating procedure and provided for infusion of the participant in the clinic under the 
supervision of the study team and Pediatric Blood and Marrow Transplant Program staff.  Baseline 
vital signs (heart rate, blood pressure, temperature, respiratory rate) will be obtained.  A peripheral 
IV will be placed by clinical staff, anesthesia or a member of the study team.  Prior to the infusion, 
premedications (Benadryl, Solumedrol) will be administered.  The study product will be infused 
over 30-60 minutes.  The child will be observed in the clinic for a minimum of 30 minutes after the 
infusion.  IV fluids (D5 ½ NS) at 1.5 maintenance will be attempted. Participants will be discharged 
from clinic after at least 1 hour providing all vital signs are at their baseline and they are 
asymptomatic with no evidence of toxicity.  Participants will be evaluated by study staff the day 
after the infusion to assess for any infusion-related adverse reactions or complications.  A phone 
call to parents/guardians by study staff to assess safety of the infusion will be conducted 7-10 
days after the infusion. 
 
If a participant has evidence of illness on the day of planned infusion, including but not limited to 
fever >38.5 C, vomiting, diarrhea, or respiratory distress, the infusion will be postponed. 
7.4 
CARE DURING UNEXPECTED EVENTS 
In the event that a participant develops signs or symptoms of anaphylaxis including urticaria, 
difficulty breathing, cough, wheezing, or vomiting during his/her study infusion, the infusion will be 
interrupted and appropriate medical therapy initiated.  
7.5 
ASSESSMENTS 
The following child assessments will be conducted at the Duke Center for Autism and Brain 
Development according to the schedule outlined in section 7.6: 
 
Respondent 
Measure 
Domain 
Length of 
Administration 
Clinical Assessments  
Brief Observation of Symptoms of Autism 
(BOSA)* 
ASD Diagnosis 
45-60 minutes 
Developmental Assessment of Young 
Children, Second Addition (DAYC-2) * 
Cognitive/Language 
45-60 minutes 
Clinical Global Impressions Scale – S and I 
Autism symptoms 
10 minutes 
JERI rating based on Parent-Child 
Interaction Task (PCIT) 
Social communication 
12 minutes 
Biomarker Assessments  
 
 
 
 
 
 
SenseToKnow (S2K) Application with 
Computer Vision Analysis (CVA) 
Autism symptoms 
10 minutes 
*The DAYC-2 and BOSA are only conducted during screening 
 
7.5.1 Assessments for Diagnosis of Autism Spectrum Disorder: 
Diagnosis of ASD will be confirmed by the DSM-5 Checklist, which will be informed by the Brief 
Observation of Symptoms of Autism (BOSA) Assessment.  Clinical research staff that have been 
certified as research reliable in the administration of the ADOS-2 will complete diagnostic 
evaluations.  
Brief Observation of Symptoms of Autism (BOSA): This assessment will be used to help inform a 
DSM-5 diagnosis of ASD.  The BOSA is a semi-structured parent-child interaction that lasts 
between 12 to 16 minutes.  Age and verbal ability are used to determine the appropriate module of 
the BOSA. A clinician who is research-reliable on the ADOS-2 observes the BOSA and scores a 
corresponding ADOS-2 protocol.  Following completion of the BOSA, an algorithm is used to map 
these binary codes onto a DSM checklist.  The BOSA is only completed at the baseline visit. 
 
 

 
 
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Diagnostic Statistical Manual-5 Checklist (DSM-5): This is a clinician checklist based off of 
diagnostic criteria for ASD within the Diagnostic and Statistical Manual of Mental Disorders, Fifth 
Edition.  Diagnostic criteria are taken from the DSM-5, and are utilized in order to confirm 
appropriate ASD diagnosis for inclusion in the study.  This checklist is completed by trained 
clinicians using the clinician’s best judgment and is informed by information gathered during the 
administration of the BOSA. The DSM-5 Checklist takes about 3-5 minutes to complete.  This 
checklist is completed at baseline. 
 
 
7.5.2 Other Clinician Assessments:  
Developmental Assessment of Young Children- Second Addition (DAYC-2): 92 The Developmental 
Assessment of Young Children (DAYC-2) is a cognitive based assessment designed to identify 
delays in very young children and preschoolers from birth through 5 years. Only the Cognitive 
scale will be administered. Test administration time varies from 10-20 minutes per domain. This 
assessment will be conducted during the baseline visit only. 
 
SenseToKnow (S2K) Application (app): The application consists of a series of brief dynamic 
stimuli (including short, <1 minute movies and games) which are designed to elicit early risk 
behaviors related to autism. The application records the child's behavior via the front-facing 
camera as he or she interacts with the stimuli. Algorithms based on computer vision and machine 
learning are employed to quantify a multitude of autism symptom behaviors, including attentional 
responses, preferential gaze, facial affect, and motor responses. Dawson and Sapiro have 
conducted studies in in-clinic and at-home environments demonstrating that CVA can objectively 
and reliably quantify autism symptoms.93-97 The application takes about 10 minutes will be 
conducted at the baseline and 6 month visits. 
 
JERI Rating based on Parent-Child Interaction Task (PCIT): During a 12-minute Parent-Child 
Interaction Task (PCIT), the participant will be observed during two contiguous sessions 
conducted in the same room comprised of (1) a six-minute interactive play session in which the 
parent joins the child in play and (2) a six-minute independent play session with toys available 
during which the caregiver will be sitting in the corner of the room completing a questionnaire. The 
Parent-Child Interaction Task will be completed at the baseline and 6 month visits.  Behavior will 
be video recorded with audio recording. Behavior will then be characterized through two methods: 
1) automated video tracking with computer vision analysis, and 2) behavioral coding of child social 
communication abilities during interactive play using the Joint Engagement Rating Inventory 
(JERI).91  Videotapes will be coded using JERI by trained and reliable raters.  Computer vision 
analysis software will be used to automatically track the child’s movements and the parent’s 
movements. The purpose of the video tracking is to automatically measure social approach or 
avoidance and exploration. The primary dependent variable is the time spent in each core region 
(center of room, periphery of room, near parent, activity table). Exploratory dependent variables 
will be related to latency, frequency, velocity and movement.  
 
The JERI will be used to code for child engagement states during the first 6-minute parent-child 
interactive play session. Behaviors that will be rated include: unengaged, object engaged, 
supported joint engagement, coordinated joint engagement, symbol-infused joint engagement, 
and total joint engagement. All items are rated on a 1 to 7 scale and factor in both the quantity and 
quality of each behavioral state of engagement.  Dependent variables will include total joint 
engagement (primary endpoint) and ratings on all items (secondary endpoints).  
 
Vineland Adaptive Behavior Scales, Third Edition (VABS-3) Survey Interview Form:98 This will be 
used to assess social, communication, motor, and adaptive behavior. The assessment is 
administered to the parent/caregiver using a semi-structured interview format. This assessment 

 
 
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takes about 60-120 minutes to administer.  The VABS-3 is a well-standardized measure of several 
domains of adaptive functioning including socialization, communication, daily living, and motor 
skills. Norms are available from birth to 90 years.  The Socialization subdomain assesses play, 
interpersonal relationships and coping skills. The Communication subdomain assesses receptive, 
expressive and written language skills. The Daily Living Skills subdomain assesses personal, 
domestic and community living skills. The Motor Skills subdomain assesses gross and fine motor 
skills. The VABS-3 will be completed at the baseline, 6, and 12 months.  
 
Clinical Global Impression (CGI): The CGI is a commonly used rating scale with two components 
that measures symptom severity and treatment response.  
 
The Clinical Global Impression – Severity Scale (CGI-S) is a 7-point scale that requires the 
clinician to rate the severity of the participant’s symptoms based on parent interview and child 
observation at the baseline and 6 month visits.  The CGI-S requires the clinician to rate the 
severity of the participant’s symptoms of ASD at the time of the assessment, relative to the 
clinician’s past experience with participants who have the same diagnosis. There will be three 
separate CGI-S ratings; these include social communicative functioning, restricted/repetitive 
interests and behaviors, and overall. The CGI-S will be completed at the baseline and 6 months.  
 
The Clinical Global Impression – Improvement (CGI-I) is a 7-point scale that requires the clinician 
to assess how much the participant has improved or worsened relative to baseline based on 
parent interview and child observation. There will be three separate CGI-I ratings; social 
communicative functioning, restricted/repetitive interests and behaviors, and overall improvement. 
The CGI-I will be completed at the 6-month visit.  
 
7.5.3 Parent Caregiver Questionnaires: 
All caregiver questionnaires will be completed online through a Duke approved EDC system or by 
paper. The EDC survey tool is available for Duke users through a university-wide site license. The 
EDC is integrated with Duke’s NetID authentication system but allows sharing of surveys with non-
Duke users.   
 
Pervasive Developmental Disorder Behavior Inventory (PDDBI):99 The PDDBI was developed to 
assess responsiveness to intervention in children with ASD. The PDDBI is an informant-based 
rating scale that is designed for children 1 year, 6 months to 12 years, 5 months.  It assesses 
problem behaviors as well as appropriate social, language, and learning/memory skills. The 
PDDBI assesses both social impairments typically associated with the active but odd subtype of 
ASD and development of pro-social skills that are integral to improved reciprocal social behavior. 
The PDDBI renders raw scores as well as t-scores based on comparisons to a standardized ASD 
population.  The PDDBI has been validated in a PDDBI development sample of 311 children 
between the ages of 1 and 17 years old. This is a parent questionnaire with 188 items that takes 
approximately 30-45 minutes to complete.  The PDDBI will be administered every three months.  
 
Intervention History Questionnaire: This questionnaire is completed by a primary caregiver to 
obtain detailed information on behavioral health interventions that the child/family has been 
involved in over the past 3 months or since the questionnaire was last administered.  Information 
is collected about the type and quantity of interventions, services, and treatments the child is 
receiving. This questionnaire will be administered at baseline, 3 months and 6 months. 
 
MacArthur-Bates Communicative Development Inventories, Second Edition (CDIs):100 
The CDIs are standardized caregiver-completed report measures which capture information about 
children’s developing abilities in early language, including gestures, production, vocabulary, 

 
 
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comprehension, and grammar.  It was developed for use in infants and toddlers ages 8 to 30 
months but it may also be used with older children with developmental delays.  Because language 
changes so dramatically during that time span, there are three separate forms for different age 
ranges and language abilities.  Each form generally takes 20-40 minutes to complete.   
 
CDI: Words and Sentences form is designed for children ages 16 to 30 months.  This form yields 
scores for vocabulary production and a number of aspects of grammatical development, including 
sentence complexity and the mean length of the child's longest utterances. The CDI words and 
sentences version will be used for all participants. This form will be collected at baseline, 3 
months, 6 and 12 months. 
 
 
Pediatric Quality of Life (PedsQL) 4.0 Generic Core Scales: The PedsQL 4.0 Generic Core Scales 
is a 5-minute parent questionnaire that measures the child’s functioning in the dimensions of: 
physical, emotion, social and school. The Toddler Parent Report version is composed of 21 items 
and will be completed at the baseline and 6 month visits and remotely at 12 months.   
 
 
Center for Autism and Brain Development (CABD) Demographics Form: The CABD demographics 
form collects race and ethnicity according to the NIH reporting standards. Additional questions 
collect data to help characterize the population. This parent questionnaire takes less than 10 
minutes to complete and will only be completed at the baseline visit. 
 
Medical and Behavioral History Questionnaire (MBHQ): The MBHQ is a broad assessment of 
medical and behavioral issues. It is completed at baseline, three, six, nine and 12 months. 
 
COVID-19 Exposure and Family Impact Survey (CEFIS): 101 The CEFIS is an open-source parent 
report questionnaire that conceptualizes exposure to potentially traumatic aspects of COVID-19 
and assesses the impact of the pandemic on the family.  The CEFIS will be completed every three 
months.  Data collected with the CEFIS measure will be de-identified and shared with the tool 
developers through a CSV file sent securely using DukeBOX.  
 
7.6 
REQUIRED EVALUATIONS 
       
7.6.1 Medical and Safety Assessments: 
 

 
 
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*CBCD, CMP, PRA may be obtained at initial visit or within 6 months prior to consent; HLA, fragile X, CMA/WES any time prior to enrollment.  
Safety and return assessments should be performed within a month of the indicated time point.  
7.6.2  Diagnostic, Behavioral, Cognitive/Language, and Attention evaluations.  
Time Points# 
Screening 
Baseline 
(Visit 1) 
7-10 days 
post-infusion 
6  
months 
 
12 months 
CBCD*, CMP*, participant HLA, fragile X, 
CMA/WES 
X 
 
 
 
 
Review of prior records ± videos 
X 
 
 
 
 
History & Physical 
 
X 
 
X 
 
Samples for storage of DNA & viable 
mononuclear cells, DNA extract and hold 
 
X 
 
 
 
Donor Referral Panel 
 
X 
 
 
 
CBCD, CMP, HLA Antibody Screen (PRA), 
Immune Reconstitution Panel, Humoral Immune 
Profile 
 
X* 
 
X 
X 
Neuropsychological evaluation 
 
X 
 
X 
X 
Safety Assessment –in person  
      (Day post-infusion) 
 
X            
 
 
 
Safety Assessment – phone call/survey 
 
 
X 
 
 
 
 
 
Time Points 
 
Measure 
Time (min) 
Baseline 
(visit) 
3 mo. 
(remote) 
6 mo. 
(visit) 
9 mo. 
(remote) 
 12 
mo.(remo
te) 
Clinician 
Assessme
nt w/ Child 
Brief Observation of Symptoms of 
Autism (BOSA) 
45-60 
X 
 
 
 
 
 
 Developmental Assessment of Young 
Children – Second Edition (DAYC-2) 
45-60 
X 
 
 
 
 
 
Other 
Assessme
nts w/ 
Child 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
SenseToKnow (S2K) app 
10 
X 
 
X 
 
 
Observatio
n of child 
Parent-Child Interaction (PCI) for JERI 
rating 
 
12 
X 
 
X 
 
 
Caregiver 
Interviews  
Vineland Adaptive Behavior Scales, 
Third Edition Survey Interview Form 
60-120 
X 
 
X 
 
X 
Clinical Global Impression Parent 
Interview 
30-45 
X 
 
X 
 
 
Medical/Behavioral History 
Questionnaire (MBHQ)   
15-30 
X 
X 
X 
X 
X 
Caregiver 
Questionn
aires 
(Conducte
d with the 
Caregiver 
Only) 
Pervasive Developmental Disorder 
Behavior Inventory (PDDBI) 
30-45 
X 
X 
X 
X 
X 
Intervention History  
15 
X 
X 
X 
X 
X 
MacArthur-Bates Communicative 
Development Inventories, Second 
Edition (CDIs) 
20-40 
X 
X 
X 
 
X 
COVID-19 Exposure and Family 
Impact Survey (CEFIS) 
15 
X 
X 
X 
X 
X 
 
Pediatric Quality of Life (PedsQL) 
5-10 
X 
 
X 
 
X 
Demographic Form  
10 
X 
 
 
 
 
 
Clinical Global Impression – Severity 
(CGI-S)  
Clinical Global Impression – 
Improvement (CGI-I)  
15 
X 
 
X 
 
 
Other 
Clinician 
Completed  
Diagnostic Statistical Manual-5 (DSM-
5) Checklist 
3-5 
X 
 
 
 
 
 
Clinical Global Impressions - Severity 
3-5 
X 
 
X 
 
 

 
 
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8.0 
STATISTICAL CONSIDERATIONS  
8.1 
STUDY DESIGN 
This is a single site, Phase I, prospective study of intravenous hCT-MSC infusion in 12 children 18 
months to <4 years (3 years 364 days) of age with ASD.  All participants will be treated with hCT-
MSC.  Participants will receive a single hCT-MSC infusion at baseline and evaluated at 6 (in 
person) and 12 (remotely) months post-infusion. Duration of study participation will be 12 months 
from the time of the first infusion.  
8.2 
STUDY DURATION 
Research participants will be enrolled in the study for 12 months after the administration of the 
hCT-MSC infusion. 
8.3 
DEMOGRAPHICS AND BASELINE CHARACTERISTICS 
Demographics and baseline characteristics will be summarized for all research participants.  
Characteristics to be examined include age, sex, race/ethnicity, IQ, and baseline behavioral 
status. 
8.4 
SAMPLE SIZE AND POWER CALCULATIONS 
The proposed trial is an open label Phase I trial of one intravenous infusion of hCT-MSC in 
toddlers 18 months to <4 years (3 years 364 days) of age with a diagnosis of ASD.  The primary 
outcome is safety, as measured by the incidences of infusion reactions, infections, and new-onset 
clinically significant anti-HLA antibodies.  Efficacy measures will also be assessed.  The primary 
efficacy measure is coded time spent in sustained engagement with the caregiver in a brief semi-
structured parent child interaction task (PCIT).  The change on this outcome measure from 
Baseline to Month 6 will be analyzed.  
8.5 
PRIMARY ENDPOINTS 
The primary outcome is safety.  Safety endpoints will include the incidence of infusion reactions, 
infections, and new-onset clinically significant anti-HLA antibodies.  Presence of infusion reactions 
and infections will be assessed in person at the time of infusion, 24 hours after infusion, and at six 
months and will be assessed remotely at 7-10 days post-infusion and 12 months and will be 
reported descriptively.  Anti-HLA antibodies will be assessed at six and 12 months post-infusion.  
8.6 
EFFICACY ENDPOINTS 
Primary Endpoint:  
The primary clinical outcome measure is the change in social communication abilities from 
baseline to 6 months based on total score (1-7) on the Joint Engagement Rating Inventory (JERI), 
a commonly-used and well-validated coding system for rating the quality and quantity of social 
communication skills in toddlers with and without ASD.91     
 
Secondary Endpoints: 
Change measured on the following endpoints from baseline to six months will be compared: 
1. PDD Behavior Inventory Autism Composite Score (PDDBI) 
2. Mean of the Socialization Subscale and Communication Subscale standard scores on the 
Vineland Adaptive Behavior Scales (VABS-3) 
3. Clinical Global Impression Scale (CGI) – Severity and Improvement (Improvement at 6 
months only 
 
Clinical Global Impressions - 
Improvement 
3-5 
 
 
X 
 
 

 
 
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4. Communicative Development Inventories (CDI-2): Words & Sentences subscales  
 
Exploratory Endpoints: 
Change measured from baseline to six months will be evaluated on the following outcome 
measures.  
1. As measured by an app that elicits and records autism symptoms on an iPad 
(SenseToKnow), changes in auditory orienting, facial expressions, look duration, attention 
to social and nonsocial stimuli, postural sway, and vocalizations as measured via computer 
vision analysis 
 
2. PDD Behavior Inventory (PDDBI) Subscales  
3. VABS-3 Socialization Subscale Standard Score and age equivalent 
4. VABS-3 Communication Subscale Standard Score and age equivalent 
5. VABS-3 Daily Living Subscale Standard Score and age equivalent 
6. VABS-3 Adaptive Behavior Composite Standard Score and age equivalent 
9.0 
SAFETY AND ADVERSE EVENT REPORTING 
9.1 
DEFINITIONS 
Adverse Event (AE):  An adverse event is any untoward medical occurrence associated with the 
use of the investigational product regardless of whether it is considered related to the 
investigational product.   
 
Serious Adverse Event (SAE):  An adverse event or suspected adverse reaction is considered 
“serious” if, in the view of either the investigator or sponsor, it results in any of the following 
outcomes:  death, a life threatening adverse event, inpatient hospitalization or prolongation of 
existing hospitalization, a persistent or significant incapacity or substantial disruption of the ability 
to conduct normal life functions, or a congenital anomaly/birth defect.  Important medical events 
that may not result in death, be life-threatening, or require hospitalization may be considered 
serious when, based upon appropriate medical judgment, they may jeopardize the participant and 
may require medical or surgical intervention to prevent one of the outcomes listed in this definition. 
 
Grade/Severity:  Grade/severity will be assessed according to CTCAE v5.0 guidelines. 
 
Suspected Adverse Reaction:  A suspected adverse reaction is any adverse event for which there 
is a reasonable possibility that the investigational product caused the adverse event.  “Reasonable 
possibility” means there is evidence to suggest a causal relationship between the investigational 
product and the adverse event. 
 
Causality:  The investigator will use the following question when assessing causality of an adverse 
event to the investigational product: “Is there a reasonable possibility that the investigational 
product caused the event?”  An affirmative answer designates the event as a suspected adverse 
reaction. 
9.2 
ADVERSE EVENT REPORTING 
All adverse events reported or observed during the study beginning at the time of the initial 
infusion must be recorded.  AEs occurring in 5% of enrolled participants will be reported in 
clinicaltrials.gov.  Information to be reported includes date when the site became aware of the 
event, investigator-specified assessment of severity and relationship to study therapy, whether 
there is an alternative etiology, seriousness, as well as any required treatment or evaluations, and 

 
 
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outcome.  In general, investigators should report adverse events as diseases or syndromes 
whenever possible, instead of reporting individual component symptoms, signs, laboratory 
abnormalities, and sequelae. 
Severe adverse infusion reactions (fatal, life-threatening or requiring hospitalization) will be 
reported within seven calendar days of receipt of the information.  All fatal or life threatening SAEs 
will be reported by the investigator or its representatives to the FDA by telephone or fax within 
seven calendar days after receipt of the information, following FDA guidelines.  All serious and 
unexpected AEs will be reported to the FDA via a written report within 15 days of receipt of the 
information (21 CFR 312.32).  If the principal investigator assesses an event to be unrelated to the 
study, then the event will not require expedited reporting but will be included in the annual 
summary report. 
The following events within 24 hours of hCT-MSC infusion will also be recorded in the e-CRF: 
infusion reaction, sinus bradycardia, sinus tachycardia, hypertension, hypotension, fever, 
rigors/chills, nausea, vomiting, infection, dyspnea, hypoxia, and hemoglobinuria. 
9.3 
SERIOUS ADVERSE EVENT REPORTING 
The Principal Investigator or its representative will be responsible for telephone or fax reporting of 
any unexpected SAEs to the FDA.  The Principal Investigator or its representative will notify the 
FDA by telephone or fax of any fatal or life threatening experience (expedited report) associated 
with the use of the study therapy as soon as possible but no later than seven calendar days after 
receipt of the information.  Initial notification will be followed by a written report within 15 calendar 
days.  For SAEs associated with the use of the study therapy, the Principal Investigator will notify 
the FDA as soon as possible, but no later than 15 days, of the initial receipt of the information.  
The Principal Investigator or Sub-Investigator is responsible for informing the Institutional Review 
Board (IRB) and DSMB of any study related and unexpected SAEs.   
9.4 
ELICITING ADVERSE EVENT INFORMATION 
In addition to research participant observations, AEs will be documented from any data collected 
throughout the study including clinically significant laboratory values or physical exam findings. 
9.5 
STOPPING GUIDELINES 
The following stopping guidelines will be monitored during the duration of the study.  The stopping 
guidelines will be monitored by the study team and are to be used to indicate boundaries requiring 
discussion by the investigators and DSMB.  The study will be stopped for a safety review if: 
 
Any participant experiences a grade 4-5 infusion reactions within 48 hours of infusion; OR 
 
Two or more grade 4-5 adverse events determined to be temporally related to the study 
product by the medical safety monitor and/or the DSMB occur; 
OR 
 
Any participant experiences a blood stream infection within 6 months of infusion; 
OR 
 
Any participant develops grade II-IV GvHD; 
OR 
 
Any death. 
 
A consensus decision to stop the study will be made by the investigators and the DSMB.  Such a 
decision with its supporting documentation and possible future plans for the study will be 
submitted to, and discussed with, the FDA. 

 
 
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9.6 
PARTICIPANT REPLACEMENT 
In the event that any enrolled participants do not receive treatment, additional participants may be 
randomized in order to reach the target number of evaluable, treated participants.   
10.0 DATA SAFETY MONITORING BOARD (DSMB) 
A DSMB will be formed and a charter established.  Members of the DSMB will be independent of 
Duke University and comprised of a minimum of three members, including a clinician with 
experience in the treatment of ASD and a physician with experience in cell therapy.  The DSMB 
will be notified immediately for all SAEs directly related to the study product throughout the study.  
A total safety assessment will be prepared on an annual basis and forwarded to the DSMB for 
review as well.  The DSMB and its members will have the right to make decisions and act 
independently.  Policies of the DSMB will be described in the DSMB charter and signed by all 
members. 
 
All study related and unexpected SAEs reported or observed during the study beginning at the 
time of the study infusion must be recorded and maintained in the study participant’s paper files.  
Severe adverse infusion reactions (fatal, life threatening or requiring hospitalization) will be 
reported to the IRB and FDA in accordance with HRPP policies. 
11.0 DATA HANDLING AND QUALITY ASSURANCE 
11.1 
CASE REPORT FORMS 
As part of the responsibilities assumed by participating in the study, the Principal Investigator or 
Sub-Investigators agree to maintain adequate case histories of the research participants treated 
as part of the research under this protocol.  The Principal Investigator or Sub-Investigator agrees 
to maintain accurate CRFs and source documentation as part of the case histories.  Duke 
University will supply the CRF electronically (eCFR) through secured electronic data entry 
systems. 
11.2 
VIDEO AND AUDIO RECORDINGS 
Video recordings of potential participants from parents and guardians may be submitted and used 
for determining study eligibility.  Audio and video recordings may also be obtained of portions of 
the evaluations and interviews if indicated with parental consent, and may include full facial 
features and audio.  The recordings will be used solely for analysis by the research team or for 
educational purposes if consent is obtained from the parent/guardian.  They will be stored 
electronically on a password-protected server and identified by the participants’ study ID.  
Software used to conduct and record visit assessments completed remotely will be done on Duke 
approved platforms and participants will receive instructions to enhance privacy and security 
measures.  
11.3 
INSPECTION OF RECORDINGS 
The Principal Investigator or Sub-Investigators and institutions involved in the study will permit 
study-related monitoring, audits, IRB review, and regulatory inspection(s) by providing direct 
access to all study records.  In the event of an audit, the Principal Investigator or Sub-Investigator 
agrees to allow the Food and Drug Administration (FDA) or other regulatory agency access to all 
study records.  The Principal Investigator or Sub-Investigators should promptly notify all relevant 
parties of any audits scheduled by any regulatory authorities and promptly forward copies of any 
audit reports received to the both. 

 
 
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11.4 
STUDY RECORD RETENTION 
Study results will be retained in the participant’s research record for six years after the study is 
completed or until the participant reaches the age of 21, whichever is longer.  Essential 
documents should be retained until at least two years after the last approval of a marketing 
application in an International council for Harmonisation (ICH) region and until there are no 
pending or contemplated marketing applications in an ICH region or at least two years have 
elapsed since the formal discontinuation of clinical development of the investigational product.  
These documents should be retained for a longer period, however, if required by the applicable 
regulatory requirements.  
11.5 
Process for adding the Study Diagnostic Report to Patient’s Duke Medical Record 
As part of this study, it the participant (or their LAR, as applicable) also receives clinical services at 
Duke outside of the study, they may request have their clinical evaluation report(s) added to their Duke 
Maestro chart. Doing this will allow researchers and clinicians at the Duke Center for Autism and Brain 
Development to better integrate our research and clinical programs. If the patient is getting care outside of 
the study at Duke, they may benefit from this by making clinical evaluations and care in the non-research 
context more seamless, allowing providers to review reports from clinical assessments conducted as part of 
the study in order to inform clinical care, and avoiding unnecessary duplication of efforts on the part of both 
research participant and study team. Most participants of the study are not patients receiving regular 
medical care at Duke outside of the study. However, in the event that a participant would like to have the 
report added to the medical record, they may make a request to do so.  
 
When receiving a request, the study team will let the participant know, or remind them, that the diagnostic 
report will summarize diagnoses, developmental function, and cognitive skills and other symptoms and 
abilities that were assessed as part of the study; that we will not “list” or change diagnoses in the medical 
record, but will make the report available for other Duke Health providers to review. We will also note that 
the report will indicate that the individual received the evaluation as part of a study. Documentation of 
requests will be stored with the participant’s research record. 
 
11.6 
Participant Engagement 
Participants of this study may be contacted after their study participation to share their perspective 
and opinions on their study experience, including any parts that they enjoyed and/or any 
challenges. They will be reassured that this is completely optional. Questions include: their 
experience in the study generally, their opinion on research study assessments, the quality of print 
materials and interactions with our staff, anything about the study they found helpful and any 
challenges encountered, and what changes might be helpful to consider in the future. Interviews 
may be recorded on Duke-approved platforms if the family agrees. These open-ended questions 
and responses will not be asked as a part of this study, or used for data analysis, or to make 
conclusions about the study, but may be used in efforts to support the Duke Center for Autism and 
Brain Development’s efforts in connecting with the broader community via social media, 
conferences, newsletters, seminars and other events/media about our research efforts and impact 
at the Duke Center for Autism. This activity will be key to the Center’s efforts in promoting active 
engagement and bidirectional exchange with the families we serve and the broader community. 
These interviews may also help inform designing protocols and/or making changes as needed to 
enhance inclusivity, cultural appropriateness, and feasibility. If staff perform post-study interviews, 
participants will be asked whether they want to publish their identity in any publicly published 
information or would prefer to not be named. The relevant information will be added to a HIPAA 
waiver provided by the News office and authorization will be obtained through signing of the 
HIPAA waiver. Although these interviews and dissemination and outreach core (DOC) activities 
are not part of the study, we wish to inform the IRB that these interactions may take place as it 
may entail additional interactions with research participants.  
  

 
 
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Additionally, participants who are willing to have their image(s) or video(s) (including voice) used 
for purposes of media, news, trainings, articles, conferences and other dissemination activities, 
will also be asked to sign the appropriate HIPAA waiver provided by the DUHS News office with 
their selections and permissions noted in the form.   
12.0 ADMINISTRATIVE ASPECTS 
The following administrative items are meant to guide the Principal Investigator or Sub-
Investigator in the conduct of the study but may be participant to change based on industry and 
government Standard Operating Procedures or Working Practice Documents or Guidelines.   
12.1 
CONFIDENTIALITY  
All laboratory specimens, evaluation forms, reports, and other records will be identified in a 
manner designed to maintain research participant confidentiality.  All records will be kept in a 
secure storage area with limited access.  Clinical information will not be released without the 
written permission of the research participant’s guardian except as necessary for monitoring and 
auditing. 
 
The Principal Investigator or Sub-Investigator and all employees and coworkers involved with this 
study may not disclose or use for any purpose other than performance of the study, any data, 
record, or other unpublished, confidential information disclosed to those individuals for the 
purpose of the study.   
12.2 
INSTITUTIONAL REVIEW BOARD APPROVAL 
Federal regulations and the ICH guidelines require that approval be obtained from an IRB prior to 
participation of human research participants in research studies.  Prior to the study onset, the 
protocol, informed consent, any advertisement used to recruit study participants, and any other 
written information regarding this study to be provided to the research participant or the research 
participant’s legal guardian must be approved by the IRB.   
 
All IRB approvals should be signed by the IRB Chairman or designee and must identify the IRB 
name and address, the clinical protocol by title and/or protocol number, and the date the approval 
and/or favorable opinion was granted. 
 
The Principal Investigator or Sub-Investigator is responsible for obtaining continued review of the 
clinical research at intervals not exceeding one year or otherwise specified by the IRB.  The 
Principal Investigator or Sub-Investigator must supply the Sponsor or its designee with written 
documentation of continued review of the clinical research. 
12.3 
MODIFICATION OF THE PROTOCOL 
Any changes in this research activity, except those necessary to remove an apparent, immediate 
hazard to the research participant, must be reviewed and approved by the IRB.   
12.4 
INFORMED CONSENT 
A written informed consent in compliance with Part 50 of Title 21 of the Code of Federal 
Regulations (CFR) and Institutional IRB shall be obtained from each research participant prior to 
entering the study or performing any unusual or non-routine procedure that involves risk to the 
research participant.   
Before enrollment, each prospective research participant and/or at least one legal guardian will be 
given a full explanation of the study and be allowed to read the approved informed consent form.  

 
 
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Once the Principal Investigator or Sub-Investigator is assured that the research participant/legal 
guardian understands the implications of participating in the study, the research participant/legal 
guardian will be asked to give consent to participate in the study by signing the informed consent 
form. 
The Principal Investigator or Sub-Investigator shall provide a signed/dated copy of the signed 
informed consent to the research participant and/or legal guardian.   
12.5 
PROTOCOL VIOLATIONS AND DEVIATIONS 
The Principal Investigator or Sub-Investigator or designee must document and explain in the 
research participant’s source documentation any deviation from the approved protocol.  The 
Principal Investigator or Sub-Investigator may implement a deviation from, or a change of, the 
protocol to eliminate an immediate hazard to study research participants without prior IRB 
approval.  As soon as possible after such an occurrence, the implemented deviation or change, 
the reasons for it, and any proposed protocol amendment(s) should be submitted to the IRB for 
review and approval, to the Sponsor for agreement, and to the regulatory authorities, if required. 
 
A deviation from the protocol is an unintended and/or unanticipated departure from the procedures 
and/or processes approved by the Sponsor and the IRB and agreed to by the Principal 
Investigator or Sub-Investigator.  Deviations usually have an impact on individual research 
participants or a small group of research participants and do not involve inclusion/exclusion or 
primary endpoint criteria.  A protocol violation occurs when there is non-adherence to the protocol 
that results in a significant, additional risk to the research participant, when the research 
participant or Principal Investigator or Sub-Investigator has failed to adhere to significant protocol 
requirements (inclusion/exclusion criteria) and the research participant was enrolled without prior 
Sponsor approval, or when there is non-adherence to FDA regulations and/or ICH GCP 
guidelines. 
 
Protocol violations and deviations will be documented by the study coordinator and the clinical 
monitor will notify study staff and update protocol deviation documentation throughout the course 
of monitoring visits. Principal Investigators or Sub-Investigators will be notified of violations and/or 
deviations in writing by the study coordinator and/or the study monitor. The IRB should be notified 
of all protocol violations and deviations meeting criteria in a timely manner as required by the site’s 
IRB. 
12.6 
STUDY REPORTING REQUIREMENTS 
By participating in the study, the Principal Investigator or Sub-Investigator agrees to submit reports 
of serious adverse events according to the timeline and method outlined in the protocol.  In 
addition, the Principal Investigator or Sub-Investigator agrees to submit annual reports to his/her 
IRB as appropriate.  The Principal Investigator or Sub-Investigator also agrees to provide the 
Sponsor with an adequate report shortly after completion of the Principal Investigator’s or Sub-
Investigator’s participation in the study. 
12.7 
FINANCIAL OBLIGATIONS 
Duke University is not financially responsible for further testing/treatment of any medical condition 
that may be detected during the screening progress. In addition, in the absence of specific 
arrangements, Duke University is not financially responsible for further treatment of the research 
participant’s disease. 

 
 
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12.8 
STUDY CONDUCT 
The Principal Investigator agrees that the study will be conducted according to the principles of the 
ICH E6 Guideline on GCP and the principles of the World Medical Association Declaration of 
Helsinki.  The Principal Investigator will conduct all aspects of this study in accordance with all 
national, state, and local laws or regulations. 
12.9 
PUBLICATIONS 
Following completion of the study, the data may be considered for reporting at a scientific meeting 
or for publication in a scientific journal.  In these cases, Duke University will be responsible to 
determine how the manuscript is written and edited, the number and order of authors, the 
publication to which it will be submitted, and other related issues. 
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Fenson L, Marchman V, Thal D, Dale P, Reznick J, Bates E. MacArthur-Bates Communicative 
Development Inventories:  User’s Guide and Technical Manual 2nd ed. Baltimore, MD: Brookes; 
2006. 
101. 
Kazak, A., Canter, K., Phan-Vo, T., McDonnell, G., Hildenbrand, A., Alderfer, M., ... & Deatrick, J. 
(2020). COVID-19 exposure and family impact survey. Philadelphia, PA: The Center for Pediatric 
Traumatic Stress. 

 
 
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14.0 APPENDIX 1: SAFETY QUESTIONNAIRE  
 
BASELINE MEDICAL & BEHAVIORAL HISTORY QUESTIONNAIRE 
 
Please tell us if your child has ever experienced any significant problems with any 
of the following conditions using the column marked EVER. If so, tell us whether they 
have continued to experience significant problems with that condition during the past 2 
months in the next column. 
 
EVER 
Past 2 
months 
No 
Yes 
No 
Yes 
1 Has your child ever had any fevers with no clear cause? 
2 Has your child ever had any serious or recurrent infections? 
3 Has your child ever had any allergic reactions? 
4 Has your child ever had significant problems with autoimmune disorders? 
5 Has your child ever had any significant problems with rashes or abnormal skin 
lesions? 
6 Has your child ever had jaundice (yellowing of the skin or eyes), except as a 
newborn? 
7 Has your child ever received a blood transfusion? 
8 Has your child ever had any significant problems with bloody noses, easy bruising or 
prolonged bleeding? 
9 Has your child ever had any abnormal blood tests? 
10 Has your child ever had any problems with breathing, asthma or coughing? 
11 Has your child ever had any problems that make you concerned about his/her heart? 
12 Has your child ever had any significant problems with his/her mouth or teeth? 
13 Has your child ever had any bowel problems, including any bowel accidents? 
14 Does your child have ongoing problems with diarrhea? 
15 Has your child ever had any ongoing episodes of vomiting? 
16 Has your child ever had any significant problems with appetite? 
17 Has your child ever had any significant problems with weight? 
18 Has your child ever had any significant problems with his/her muscles or joints? 
19 Has your child ever had any problems with swelling in any part of his or her body? 
20 Has your child ever had any problems with his/her urinary tract or problems when 
he/she urinates? 
21 Has your child ever had any problems with his/her breasts/nipples or private parts? 
22 Has your child ever had any problems with how thirsty he or she is? 
23 Has your child ever had any problems with his/her ears or hearing? 
24 Has your child ever had any eye or vision problems? 
25 Has your child ever had problems with severe or frequent headaches? 
 
 
 
 
 
 

 
 
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EVER 
Past 2 
months 
No 
Yes 
No 
Yes 
26  Has your child ever had any significant problems with dizziness? 
27 Has your child ever had any seizures? 
   28 Has your child ever had any problems with involuntary movements? 
29 Is your child chronically tired or fatigued? 
30 Has your child ever had cancer? 
31 Has your child ever had a tumor? 
32 Has your child ever had any unexplained masses or growths? 
33 
Have you ever had any other concerns about your child’s medical health? If so, 
please explain below. 
  
34 Has your child ever had any significant problems sleeping at night? 
35 Has your child ever had any significant problems being too sleepy during the day? 
  36 
Has your child ever had differences in sensory processing such as sensory seeking 
behaviors or sensory aversion behaviors (including food aversion)? 
37 
Has your child ever had any problems with rituals, being flexible, repetitive behaviors 
or repetitive language? 
38 Has your child ever had any problems with hyperactivity or impulsivity? 
39 
Has your child ever had any significant problems with refusing to follow directions that 
he/she understands? 
40 
Has your child ever had any significant problems with aggression, irritability or getting 
frustrated easily? 
41 
Has your child ever had significant problems (more frequent/severe than other kids) 
with meltdowns or agitation? 
42 Has your child ever had any problems with staying motivated? 
43 Has your child ever had ever any problems with worries or sadness? 
44 
Has your child ever had any problems with hurting him/herself on purpose or 
wanting to die? 
45 Has your child ever had any significant problems with mood swings? 
46 
Has your child ever had any problems with believing things that aren’t true or 
seeing/hearing things that aren’t there? 
47 
Has your child ever had any problems with seeming not to know where he/she was 
or what was really happening? 
48 
Have you ever had any other concerns about your child’s behavior, which is not 
addressed in questions 33-45? If so, please explain below. 
 
 
Please use the space below to explain any “yes” answers. 
 
 
 
 
 
 
 
 
 

 
 
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FOLLOW-UP MEDICAL & BEHAVIORAL HISTORY QUESTIONNAIRE 
Please tell us if your child has ever experienced any significant problems with any of the 
following conditions SINCE YOU LAST COMPLETED THIS QUESTIONNAIRE. 
Past 3 
months 
No 
Yes 
1 Has your child had any fevers? 
2 Has your child had infections? 
3 Has your child had any allergic reactions? 
4 Has your child had any new or worse problems with autoimmune disorders? 
5 Has your child had any new rashes or abnormal skin lesions? 
6 Has your child developed jaundice (yellowing of the skin or eyes)? 
7 Has your child received a blood transfusion? 
8 Has your child had any new problems with bloody noses, easy bruising or prolonged bleeding? 
9 Has your child had any abnormal blood tests since their last visit? 
10 Has your child had any new or worsening problems with breathing, asthma or coughing? 
11 Has your child had any new or worsening problems with their heart? 
12 Has your child had any new problems with his/her mouth or teeth? (Do not include losing baby 
teeth.) 
13 Has your child had any new or worsening bowel problems, including bowel accidents? 
14 Has your child had any new or worsening diarrhea? 
15 Has your child had any new or worsening episodes of vomiting? 
16 Has your child had any new or worsening problems with appetite? 
17 Has your child had any new or worsening problems with weight? 
18 Has your child had any new or worsening problems with his/her muscles or joints? 
19 Has your child had any new or worsening swelling in any part of his or her body? 
20 Has your child had any new or worsening problems when he/she urinates? 
21 Has your child had any new or worsening problems with his/her breasts/nipples or private parts? 
22 Has your child had any changes in thirstiness? 
23 Has your child had any new or worsening problems with his/her ears or hearing? 
24 Has your child had any new or worsening eye or vision problems? 
25 Has your child had any new or worsening headaches? 
26 Has your child had any new or worsening problems with dizziness? 
27 Has your child had any new or worsening seizures? 
28 Has your child had any new or worsening problems with involuntary movements? 
29 Has your child had any new or worsening chronic tiredness or fatigue? 
 
 
 
 
 
 
 

 
 
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Past 3 
months 
No 
Yes 
30 Has your child ever had cancer? 
31 Has your child ever had a tumor? 
32 Has your child ever had any unexplained masses or growths? 
33 Has your child been hospitalized?  If so, please explain below. 
   34 Has your child had any new significant injuries? 
35 
Have you ever had any other concerns about your child’s medical health? If so, please explain 
below. 
 
36 Has your child had any new or worsening problems sleeping at night? 
37 Has your child had any new or worsening problems being too sleepy during the day? 
38 
Has your child had any new or worsening problems with differences in sensory processing such 
as sensory seeking behaviors or sensory aversion behaviors (including food aversion)? 
39 
Has your child had any new or worsening problems with rituals, being flexible, repetitive behaviors 
or repetitive language? 
40 Has your child had any new problems with hyperactivity or impulsivity? 
41 
Has your child had any new or worsening problems with refusing to follow directions that he/she 
understands? 
42 
Has your child had any new or worsening problems with aggression, irritability or getting frustrated 
easily? 
43 Has your child had any new or worsening with meltdowns or agitation? 
44 Has your child had any new or worsening problems with staying motivated? 
45 Has your child had any new or worsening problems with worries or sadness? 
46 Has your child had any new or worsening problems with hurting him/herself or wanting to die? 
47 Has your child had any new or worsening problems with mood swings? 
48 
Has your child had any new or worsening problems with believing things that aren’t true or 
seeing/hearing things that aren’t there? 
49 
Has your child had any new or worsening problems with knowing where he/she is or who is with 
him/her? 
50 Has your child had any new or worsening problems with their thinking? 
51 
Have there been any other new or worsening problems with your child’s behavior, which is not 
addressed in questions 35-50? If so, please explain below. 
 
Please use the space below to explain any “yes” answers.

 
 
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