Efficacy of Intravenous Umbilical Cord Blood Infusion as Cell Therapy for Children With Autism Spectrum Disorder (ASD): Duke ACT
Phase: PHASE2
Conditions: Autism Spectrum Disorder, ASD, Autism, PDD
Sponsor: Joanne Kurtzberg, MD

March 15, 2018 
A Phase II Study of the Efficacy of Intravenous Umbilical Cord Blood Infusion 
as Cell Therapy for Children with Autism Spectrum Disorder (ASD):  
DukeACT 
IND 15949 
NCT02847182
Principal Investigator 
Joanne Kurtzberg, MD 
Robertson Clinical and Translational Cell 
Therapy Program 
Duke Translational Research Institute 
Duke University Medical Center  
Co- Principal Investigator 
Geraldine Dawson, PhD, FAPA, FAPS 
Duke Center for Autism and Brain 
Development 
Duke University Medical Center 
Co- Principal Investigator 
Jessica Sun, MD 
Robertson Clinical and Translational Cell 
Therapy Program 
Duke Translational Research Institute 
Duke University Medical Center 

 
March 15, 2018 
2
Additional Investigators: 
Jennifer H. Baker, RN 
Clinical Trials Manager 
Duke Translational Medicine Institute 
Robertson Clinical and Translational Cell Therapy 
Program 
Duke University Medical Center  
 
 
 
Jayne Cash, RN, BSN 
Nurse Clinician 
Duke Translational Research Institute 
Duke University Medical Center  
 
   
 
Nicole Heilbron, PhD 
Assistant Professor 
Duke Center for Autism and Brain Development 
Department of Psychiatry and Behavioral 
Sciences  
Duke University Medical Center 
 
 
 
Jill Lorenzi, PhD 
Medical Instructor 
Department of Psychiatry and Behavioral 
Sciences 
Duke Center for Autism and Brain Development 
 
 
 
Anne Fitzgerald, RN 
Nurse Clinician 
Duke Translational Research Institute 
Duke University Medical Center 
 
   
 
Jesse Troy, PhD, MPH 
Statistician 
Duke Translational Research Institute 
Duke University Medical Center 
 
 
 
 
 
 
Lauren Franz, MBChB, MPH 
Assistant Professor 
Duke Center for Autism and Brain Development 
Department of Psychiatry and Behavioral 
Sciences  
Duke University Medical Center 
 
 
 
Joseph Horrigan, MD 
Associate Consulting Professor 
Psychiatry and Behavioral Sciences 
Duke University Medical Center 
 
 
 
Colleen McLaughlin, DNP, RN, CPNP 
Duke Translational Research Institute 
Duke University Medical Center  
 
 
 
Michael Murias, PhD 
Assistant Professor 
Duke Center for Autism and Brain Development 
Duke Institute for Brain Sciences 
Duke University Medical Center  
 
 
 
Linmarie Sikich, MD 
Associate Professor 
Department of Psychiatry and Behavioral 
Sciences 
Duke Center for Autism and Brain Development 
Duke University Medical Center 
 
 
 
Allen W. Song, PhD 
Director, Brain Imaging and Analysis Center 
Professor of Radiology, Psychiatry, 
Neurobiology, and BME 
Duke University Medical Center  
 
 
 
 
 
 

 
March 15, 2018 
3
Scott Compton, PhD 
Associate Professor 
Department of Psychiatry and Behavioral Science 
and Department of Psychology and Neuroscience 
Duke University Medical Center 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
March 15, 2018 
4
TABLE OF CONTENTS 
INVESTIGATOR SIGNATURE PAGE ...................................................................................................... 6 
PROTOCOL SUMMARY ............................................................................................................................ 6 
ABBREVIATIONS ........................................................................................................................................ 7 
1.0 
PURPOSE ............................................................................................................................................. 9 
2.0 
BACKGROUND AND HYPOTHESIS .............................................................................................. 9 
2.1 
AUTISM SPECTRUM DISORDER .......................................................................................................... 9 
2.2 
RATIONALE FOR CELLULAR THERAPY ............................................................................................. 10 
2.3 
PRECLINICAL STUDIES OF CELL THERAPIES IN ASD ....................................................................... 10 
2.4 
PRECLINICAL STUDIES DEMONSTRATING MECHANISM OF ACTION OF CB CELLS TO 
RESCUE/REPAIR BRAIN CELLS .................................................................................................................. 11 
2.5 
TREATMENT OF PEDIATRIC PATIENTS WITH NEUROLOGICAL CONDITIONS WITH AUTOLOGOUS 
CB 
14 
2.6 
ALLOGENEIC UMBILICAL CORD BLOOD .......................................................................................... 19 
2.7 
PRELIMINARY EXPERIENCE USING ALLOGENEIC CORD BLOOD IN PATIENTS WITH NEUROLOGICAL 
CONDITIONS ............................................................................................................................................... 20 
2.8 
SOURCE OF UNRELATED CORD BLOOD UNITS FOR THIS TRIAL ...................................................... 22 
2.9 
SPECIFICATIONS FOR QUALIFICATION OF PRIVATE OR PUBLIC CB UNITS FOR CELL THERAPY 
STUDIES ..................................................................................................................................................... 22 
2.10 
CLINICAL SAFETY SUMMARY ........................................................................................................ 23 
2.11 
STUDY RATIONALE AND HYPOTHESES .......................................................................................... 23 
2.12 
STUDY DESIGN ............................................................................................................................... 24 
2.13 
RISKS AND BENEFITS ..................................................................................................................... 24 
3.0 
STUDY OBJECTIVES ...................................................................................................................... 25 
4.0 
STUDY DESIGN ................................................................................................................................ 26 
4.1 
GENERAL DESIGN ............................................................................................................................ 26 
4.2 
STUDY FLOW CHART ....................................................................................................................... 27 
4.3 
STUDY ENDPOINTS ........................................................................................................................... 27 
5.0 
RESEARCH PARTICIPANT SELECTION AND WITHDRAWL ............................................. 28 
5.1 
STUDY POPULATION ......................................................................................................................... 28 
5.2 
INCLUSION CRITERIA ....................................................................................................................... 28 
5.3 
EXCLUSION CRITERIA ...................................................................................................................... 29 
5.4 
RESEARCH PARTICIPANT RECRUITMENT AND SCREENING .............................................................. 30 
6.0 
STUDY PRODUCT ........................................................................................................................... 31 
6.1 
UMBILICAL CORD BLOOD ................................................................................................................ 31 
6.2 
HLA MATCHING .............................................................................................................................. 31 
6.3 
DONOR SCREENING FOR CB UNITS .................................................................................................. 31 
6.4 
CB UNIT CHARACTERIZATION AND SHIPPING ................................................................................. 32 
6.5 
PLACEBO PRODUCT .......................................................................................................................... 32 
6.6 
PACKAGING OF STUDY PRODUCT .................................................................................................... 33 
6.7 
ADMINISTRATION OF STUDY PRODUCT ........................................................................................... 33 
6.8 
SAFETY FOLLOW-UP ........................................................................................................................ 33 
7.0 
STUDY PLAN .................................................................................................................................... 33 
7.1 
OVERVIEW ........................................................................................................................................ 33 
7.2  
PATIENT SCREENING ....................................................................................................................... 34 
7.3 
CB UNIT SELECTION ........................................................................................................................ 34 

 
March 15, 2018 
5
7.4  
PSYCHOLOGICAL ASSESSMENTS ..................................................................................................... 35 
7.5 
NEUROPHYSIOLOGY AND NEUROIMAGING ...................................................................................... 41 
7.6 
BIOLOGICAL SAMPLES ..................................................................................................................... 42 
7.7 
STUDY PRODUCT INFUSION.............................................................................................................. 42 
7.8 
CARE DURING UNEXPECTED EVENTS .............................................................................................. 43 
7.9 
REQUIRED EVALUATIONS ................................................................................................................ 44 
8.0 
STATISTICAL CONSIDERATIONS ............................................................................................. 45 
8.1 
STUDY DESIGN ................................................................................................................................. 46 
8.2 
ACCRUAL ......................................................................................................................................... 46 
8.3 
STUDY DURATION ............................................................................................................................ 46 
8.4 
DEMOGRAPHICS AND BASELINE CHARACTERISTICS ....................................................................... 46 
8.5 
DESCRIPTION OF THE PRIMARY OUTCOME MEASURE ..................................................................... 46 
8.6 
DESCRIPTION OF THE VABS-3 SOCIALIZATION DOMAIN STANDARD SCORE ................................. 47 
8.7 
SAMPLE SIZE AND POWER CALCULATIONS ..................................................................................... 47 
8.8 
POWER FOR SUBGROUP ANALYSES .................................................................................................. 48 
8.9 
CORRECTION FOR MULTIPLE TESTING ............................................................................................. 49 
8.10 
RANDOMIZATION PLAN ................................................................................................................. 49 
8.11 
ANALYSIS PLAN ............................................................................................................................. 50 
8.12 
LOSS TO FOLLOW-UP AND DISCONTINUATION .............................................................................. 50 
8.13 INTERIM ANALYSIS ............................................................................................................................ 50 
9.0 
SAFETY AND ADVERSE EVENT REPORTING ........................................................................ 50 
9.1 
DEFINITIONS ..................................................................................................................................... 50 
9.2 
ADVERSE EVENT REPORTING .......................................................................................................... 51 
9.3 
STOPPING GUIDELINES ..................................................................................................................... 51 
9.4 
PARTICIPANT REPLACEMENT ........................................................................................................... 52 
9.5 
EMERGENCY UNBLINDING ............................................................................................................... 52 
10.0 
DATA SAFETY MONITORING BOARD (DSMB) .................................................................... 52 
11.0 
DATA HANDLING AND QUALITY ASSURANCE .................................................................. 52 
11.1 
CASE REPORT FORMS .................................................................................................................... 52 
11.2 
VIDEO AND AUDIO RECORDINGS ................................................................................................... 53 
11.3 
INSPECTION OF RECORDS ............................................................................................................... 53 
11.4 
STUDY RECORD RETENTION .......................................................................................................... 53 
12.0 
ADMINISTRATIVE ASPECTS .................................................................................................... 53 
12.1 
CONFIDENTIALITY ......................................................................................................................... 53 
12.2 
INSTITUTIONAL REVIEW BOARD APPROVAL ................................................................................. 54 
12.3 
MODIFICATION OF THE PROTOCOL ................................................................................................ 54 
12.4 
INFORMED CONSENT ...................................................................................................................... 54 
12.5 
PROTOCOL VIOLATIONS AND DEVIATIONS .................................................................................... 54 
12.6 
STUDY REPORTING REQUIREMENTS .............................................................................................. 55 
12.7 
FINANCIAL OBLIGATIONS .............................................................................................................. 55 
12.8 
STUDY CONDUCT ........................................................................................................................... 55 
12.9 
PUBLICATIONS ............................................................................................................................... 55 
13.0 
REFERENCES ................................................................................................................................ 56 
14.0 
APPENDIX 1:  SAFETY QUESTIONNAIRE .............................................................................. 62 
15.0 
APPENDIX 2:  BIOLOGICAL SAMPLES .................................................................................. 66 
16.0 
APPENDIX 3:  SEDATION GUIDELINES ................................................................................. 67 

 
March 15, 2018 
6
 
 
INVESTIGATOR SIGNATURE PAGE 
 
 
 
A Phase II Study of the Efficacy of Intravenous Umbilical Cord Blood 
Infusion as Cell Therapy for Children with Autism Spectrum Disorder (ASD):  
DukeACT 
 
 
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) 
 
 
 
 
 
 
 

 
March 15, 2018 
6
PROTOCOL SUMMARY 
Protocol Number: 
 
DukeACT 
Title: 
 
A Phase II Study of the Efficacy of Intravenous Umbilical Cord Blood 
Infusion as Cell Therapy for Children with Autism Spectrum Disorder 
(ASD):  DukeACT 
 
Study Phase: 
II 
 
Study Site: 
Single site; Duke University, Durham NC 
 
Study Therapy, Dosage, 
and Route of 
Administration: 
 
Autologous or allogeneic unrelated umbilical cord blood (CB), minimum 
pre-cryopreservation cell dose of 2.5 x 107 cells/kilogram, administered as a 
single intravenous infusion 
 
Objectives: 
1. To determine, in a randomized, placebo controlled, best available 
donor source trial, the efficacy of a single intravenous infusion of 
umbilical CB in improving the core symptoms of autism in young 
children with ASD. 
a. To determine whether there is a difference in mean response at 6 
months between all cell sources and placebo. 
b. To determine whether there is a difference in mean response at 6 
months between autologous CB and placebo. 
c. To determine whether there is a difference in mean response at 6 
months between allogeneic CB and placebo. 
d. To determine whether there is a difference in mean response at 6 
months between autologous CB and allogeneic CB. 
2. To describe the safety and tolerability of a single intravenous infusion 
of unrelated donor CB in children with ASD.  
3. To explore whether patient age or IQ correlate with response. 
4. To explore whether changes in MRI, EEG, and eye tracking are 
observed after CB treatment. 
5. To identify biomarkers that positively or negatively correlate with 
response to treatment. 
 
Research Participant 
Population: 
Up to 190 children ages two to seven years with ASD 
 
Study Design: 
 
This is a Phase II, single site, prospective, randomized, double-blind study 
of a single intravenous autologous or allogeneic, unrelated CB infusion.  
Subjects will be randomly assigned to Sequence A, consisting of a single 
infusion of CB cells at baseline followed 6 months later by a single infusion 
of placebo, or Sequence B, consisting of an infusion of placebo at baseline 
followed 6 months later by an infusion of CB cells. The primary endpoint 
will be evaluated 6 months after the first infusion.   
 
Safety Assessments: 
1. Incidence and severity of infusion reactions 
2. Incidence and severity of product-related infections 
3. Evidence of alloimmunization assessed by the presence of anti-HLA 
and anti-RBC antibodies and nonspecific markers of systemic 
inflammation (ESR, CRP) 
4. Incidence and severity of any graft vs. host disease 

 
March 15, 2018 
7
5. Incidence and severity of adverse events, by relation to the study 
product 
ABBREVIATIONS 
 
 
 
AE 
 
Adverse Event 
ASD 
 
Autism Spectrum Disorder 
CB 
 
Umbilical Cord Blood 
CBC 
 
Complete Blood Count 
CFR 
 
Code of Federal Regulations 
CFU 
 
Colony Forming Unit 
CMV 
 
Cytomegalovirus 
CRF 
 
Case Report Form 
CNS 
 
Central Nervous System 
CP 
 
Cerebral Palsy 
CT 
 
Computed Tomography 
DMSO 
 
Dimethyl Sulfoxide 
DTI 
 
Diffusion Tensor Imaging 
FDA 
 
Federal Drug Administration 
GCP 
 
Good Clinical Practice 
CGI-I 
 
Clinical Global Impression-Improvement  
CGI-S 
 
Clinical Global Impression-Severity 
GMFM-66 
 
Gross Motor Function Measure - 66 
GvHD 
 
Graft versus Host Disease 
HIE 
 
Hypoxic Ischemic Encephalopathy 
HIV 
 
Human Immunodeficiency Virus 
HLA 
 
Human Leukocyte Antigen 
HSCT 
 
Hematopoietic Stem Cell Transplantation 
ICH 
 
International Conference on Harmonisation 
IRB 
 
Internal Review Board 
IV 
 
Intravenous 
MRI 
 
Magnetic Resonance Imaging 
MSCs 
 
Mesenchymal Stromal Cells 
PCR 
 
Polymerase Chain Reaction 
PDD-BI 
 
Pervasive Developmental Disorder-Behavior Inventory 
SAE 
 
Serious Adverse Event 
STCL 
 
Stem Cell Laboratory 
TNC 
 
Total Nucleated Cells 
VABS-3 
 
Vineland Adaptive Behavior Scales, ThirdEdition 

 
March 15, 2018 
8
WBC 
 
White Blood Cell 
 
 
 

 
March 15, 2018 
9
1.0 
PURPOSE 
The purpose of this study is to determine whether a single intravenous infusion of human 
umbilical cord blood (CB) can improve the core symptoms of autism in children with 
autistic spectrum disorder (ASD) through a randomized, placebo controlled, best 
available donor source phase II clinical trial. 
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 repetitive behaviors, a restricted range of 
activities, and impairments in social communication.1  This disorder is 4 to 5 times more 
prevalent in boys and affects over 2 million individuals in the US, with approximately 1 
in 68 American children identified as falling on the autism spectrum.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   
 
The treatment of ASD is generally supportive and is often multimodal.  Approaches 
include medication, behavioral therapy, occupational and speech therapies, and 
specialized educational and vocational support. All of the currently available medical 
treatments, such as psychotropic medications, are intended to ameliorate associated co-
morbid symptoms, such as irritability, but they are not disease-modifying. In light of this, 
there is a large unmet need for better, more effective and disease-modifying medical 
treatments for ASD.  
 
The etiology of ASD is unknown, but recent studies indicate that genetic and 
environmental factors contribute to the disease. A common pathophysiological finding is 
abnormal synaptic functioning in areas of the brain, including the cerebral cortex. White 
matter abnormalities have also been reported, potentially implicating irregularities in 
myelination or axonal development in the pathophysiology of ASD.4  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.5,6  
Increased plasma cytokine levels, upregulated genes associated with microglial 
activation, localized inflammation and pathological astrocyte activation have been 
associated with ASD.7  In this study, we hypothesize that infusion of CB will facilitate 
neural cell protection/repair and reduce inflammation resulting in improvement in 
the core autistic symptoms of children with ASD.   
 
Although the etiology of ASD is likely to be multifactorial, and the phenotypes can be 
variable, symptoms may be mediated by a final common pathway involving microglial 
activation.  Nonetheless, it is possible that risk-based analyses performed at the 
conclusion of this study may uncover differential treatment responses in subsets of 

 
March 15, 2018 
10
subjects.  Future studies utilizing induced pluripotent stem cells (IPSC) may be useful in 
further investigating any such findings.    
2.2 
Rationale for Cellular Therapy   
Human umbilical cord blood (CB) is rich in highly proliferative stem and progenitor cells 
mobilized by placental signals promoting homing to developing organs and is an 
established source of stem cells for hematopoietic transplantation.  We have previously 
shown that allogeneic unrelated donor umbilical cord blood transplantation, after 
preparation with high dose, myeloablative chemotherapy, can halt progression of disease 
in children with inherited leukodystrophies.8-10  In these patients, donor cells cross the 
blood brain barrier and engraft as microglial cells in the brain.  We have also 
demonstrated the safety of intravenous autologous cord blood infusion in young children 
with brain injuries11 and ASD.  In several animal models of brain injury and cerebral 
palsy, it has been shown that administration of xenogeneic human CB leads to improved 
motor function.12  Improvements in autistic symptoms have also been reported in two 
different mouse models of ASD after administration of bone marrow cells or 
mesenchymal stromal cells (MSCs). We hypothesize that cell therapy utilizing CB may 
improve the symptoms of ASD by modifying the immune response and inflammation in 
the brain and/or initiating repair of damaged brain circuitry. 
2.3 
Preclinical Studies of Cell Therapies in ASD  
Mouse models of ASD have been developed to provide a method to identify candidate 
genes and chromosomal regions associated with limited subpopulations of ASD such as 
Fragile X, Rett, and Angelman syndromes.  Reported data from animal studies is 
confounded by the many differences in the phenotype of the ASD disorder.   
 
Derecki, et al13 report recovery of function in a model of Rett syndrome, an X-linked 
autism spectrum disorder usually 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.  BTBR mice exhibit impaired social 
behavior, aberrant communication, increased repetitive behaviors, and increased 
cognitive rigidity.  Segal-Gavish, et al14 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.     

 
March 15, 2018 
11
2.4 
Preclinical Studies Demonstrating Mechanism of Action of CB Cells to 
Rescue/Repair Brain Cells 
A common pathophysiological finding in ASD is abnormal synaptic functioning in the 
brain. Development and maintenance of neural synapses is a complex and continual 
process that begins early in development and continues throughout life.  While synapses 
were once thought simply to be connections between neurons, it is now recognized that 
many different cell types, including astrocytes and microglia, play an integral role in 
synaptic pruning and maintenance.  Microglia, in particular, play critical but incompletely 
understood roles in propagation and resolution of central nervous system injuries. These 
cells modulate neuroinflammation, produce factors that regulate activities of astrocytes, 
oligodendrocytes, and neurons, aid in maintenance of synapses, and clear debris to 
provide an environment for oligodendrocytes to begin to remyelinate neurons.15,16  
Preclinical data indicate that stem cell treatments have the ability to affect numerous cell 
types in the central nervous system via trophic or paracrine effects, thereby potentially 
altering the course of neurologic and neuropsychiatric diseases.   Selected examples are 
detailed below. 
Neurogenesis and Microglial Modulation:   
Microglia are thought to regulate changes in hippocampal neurogenesis through the 
production of inflammatory mediators such as tumor necrosis factor alpha (TNF-α) and 
interleukin-1 beta and growth factors such as insulin-like growth factor-1 and brain-
derived neurotrophic factor (BDNF).17-19 Neuroinflammation inhibits neurogenesis in the 
adult hippocampus, and inflammatory blockade restores neurogenesis.20   
 
Studies with human CB cells have shown benefit in xenograft models.  In an aging rat 
model, human CB mononuclear cells have been shown to increase the proliferation of 
neural stem cells and decrease the number of activated microglia in the dentate gyrus of 
the hippocampus and the subgranular zone.21 
 
More recently, Shahaduzzaman, et al22 demonstrated that CB-derived T cells increase 
survival and proliferation of adult neural stem cells in vitro. In a mouse model, they 
showed that CB CD4+ T cells promoted proliferation of neural stem cells in the dentate 
gyrus of aging rats and restored dendritic spine density of hippocampal pyramidal cells 
one week post-injection. Longer term, they demonstrated that a single IV injection of CB 
CD4+ T cells increased proliferation in the dentate gyrus and decreased the amount of 
activated microglia in the aged rat brain. These results suggest that CB T cells may 
provide trophic support to aging neurons, thereby enhancing proliferation and 
maintaining dendritic and axonal connections in the aging brain. 
 
Microglial Inflammation: 
In a mouse model of Alzheimer’s disease, Lee et al. demonstrated that infusion of CB-
MSCs resulted in reversal of disease-associated microglial neuroinflammation, as 
evidenced by decreased microglia-induced proinflammatory cytokines, elevated 
alternatively activated microglia, and increased anti-inflammatory cytokines.  This was 
associated with reductions in amyloid-β peptide deposition, β-secretase 1 levels, and tau 
hyperphosphorylation, known biomarkers of Alzheimer’s disease.  The mice also 
demonstrated improved spatial learning and memory decline.  The authors suggest that 

 
March 15, 2018 
12
CB-MSCs produced their sustained neuroprotective effect by inducing a feed-forward 
loop involving alternative activation of microglial neuroinflammation, thereby 
ameliorating disease pathophysiology and reversing the cognitive decline associated with 
Aβ deposition in Alzheimer’s disease mice.23 
 
Oligodendrocytes & Myelination: 
Our group has developed DUOC-01, a cell therapy product cultured from banked human 
CB mononuclear cells.24  Immunodepletion and selection studies demonstrate that 
DUOC-01 cells are derived from CB CD14+ monocytes. In a NOD/SCID/IL2R
null 
mouse model of demyelination via cuprizone feeding, the corpus callosum of mice 
treated with DUOC-01 showed enhanced myelination (figure 1), a higher proportion of 
fully myelinated axons, decreased gliosis and cellular infiltration (figure 2), and more 
proliferating oligodendrocyte lineage cells than controls.25  DUOC-01 cells also express 
higher levels of transcripts for several factors that can dampen inflammation, promote 
oligodendrocyte restoration, and promote myelination and tissue remodeling after injury.  
In addition, CD14 monocytes in thawed CB demonstrate anti-inflammatory and 
immunomodulatory activity, suggesting that without manipulation thawed CB cells have 
the capacity to exert anti-inflammatory and pro-neurogenic activity. 

 
March 15, 2018 
13
 
 
 
 
 
 
 
  
 
 
Ringer’s 
DUOC-01 
CD14 
20x 
400x 
0
0.5
1
1.5
2
2.5
3
Myelination score 
** 
* 
Control
CPZ
Ringer's
DUOC-01
CD14+
Ringers 
DUOC-01 
A
C
B
Figure 1:  Remyelination Effects of DUOC-01 Cells.  Panels A&B: LFB-PAS staining analysis of effect of DUOC-01 
treatment on remyelination following cessation of CPZ treatment. (A) LFB-PAS staining one week after IC injection of 
CD14+ monocytes (lower panels), DUOC-1 cells (middle panels), or Ringer’s solution (upper panels) in CPZ fed NSG 
mice. Midline CC area is shown by dotted green line. (B) Myelination score based on LFB-PAS staining of mice fed 
normal chow (control) or CPZ for 5 weeks one week after treatment of CPZ-treated mice with CD14+ monocytes, DUOC-
01 cells, or Ringer’s. DUOC-01 treatment for 1 week significantly increased the myelination in the CC area compared to 
Ringer’s injected controls. ** indicate significant p-value < 1.2x10-15 for this study. CD14+- cell treated sample showed 
increased amount of remyelination compared to the Ringer’s treated group, but it was significantly less than the DUOC-
01 treated group (p< 2.4x10-5). Panel C: Electron microscopic analysis of remyelination status upon DUOC-01 treatment. 
Representative 8800x electron micrographs of corpus callosum region of CPZ-fed mice 1 week after injection of Ringer’s 
solution (upper panel) or DUOC-01 cells (lower panel). Blue arrows indicate un-myelinated axons. Red arrow-heads 
indicate mitochondria; enlarged mitochondria are clearly visible in the Ringer’s-treated group. Scale bar=1.0μm 
% area covered by Iba1 
0
10
20
30
40
50
** 
0
10
20
30
40
50
% area covered by GFAP 
** 
Control
CPZ
Ringer's
DUOC-01
0
0.5
1
1.5
2
2.5
3
Cellularity 
** 
Figure 2:  DUOC-01 cell treatment reduces severe astrogliosis and microglial infiltration. Panel A:  A quantitative 
cellularity scoring (by counting the nucleus in CC region of LFB stained brain slices) on a scale of 0 to 3, by a 3 blinded 
readers. ** indicates significance (n≥5, p≤1.8x10-13). ‘Control’ is no cuprizone fed, ‘CPZ’ is Cuprizone fed, ‘Ringer’s’ is 
1week after Ringer’s injection, ‘DUOC-01’ is one week after DUOC-01 injection. Panel B: Cellularity score from LFB 
stained slides were further supported by immunohistochemical staining using astrocyte specific (GFAP, pink, Right 
panels) and microglia specific (Iba1, blue left panels) markers. Midline CC areas are shown in each picture. Scale 
bar=100μm. Panel C: Quantitative analysis of area covered by Iba1 (upper panel) and GFAP (lower panel) stained cells, 
indicative of their numbers, along the CC. Both the numbers of Iba1-positive (microglia) and GFAP- positive (astrocytes) 
cells were significantly lower (p-value<0.002 and <0.01 respectively; n=3 mice per group) in the DUOC-01 treated mice. 
Areas covered by the each channel (either GFAP or Iba1) per microscopic field were quantified by ImageJ software. 
A
C
B

 
March 15, 2018 
14
These and other preclinical studies indicate that CB cells may effectively modulate 
multiple types of cells in the central nervous system, including decreasing microglial 
activation and inflammation.  Given the increasing evidence that synaptic and microglial 
dysfunction play a role in ASD, CB may be a candidate therapy for patients with ASD 
due to its ability to modulate microglial and other cells in the brain. 
2.5 
Treatment of Pediatric Patients with Neurological Conditions with Autologous CB 
At Duke, we have been studying autologous CB infusion for the past 11 years in over 
600 children, ages one day to 15 years, with cerebral palsy (CP), hypoxic ischemic 
encephalopathy (HIE), hypoplastic left heart syndrome post ECMO, congenital 
hydrocephalus, ASD, and other brain injuries.  No safety concerns were raised in this 
experience, which is detailed below. 
 
 
Safety:  
The first trial, conducted from 2004-2009, confirmed the safety of administration of 
autologous CB as an intravenous infusion in the outpatient setting.11  In this study, 184 
infants and children with HIE, cerebral palsy, congenital hydrocephalus and other brain 
injuries received a total of 198 autologous CB infusions (14 patients received two 
infusions based on availability of larger cell doses).  The majority of parents had elected 
to store their child’s cord blood privately when they were born.  Autologous CB units 
were obtained from 24 different cord blood banks:  149 (81%) CB units came from 11 
private U.S. banks (113 from two of the larger private U.S. banks), 13 (7%) CBUs from 
11 international banks, and 22 (12%) CB units from 2 public banks. The median volume 
of cord blood collected was 60 ml (range 5-180 ml) and median total nucleated cell count 
(TNCC) contained in the cord blood, as reported by the cord blood bank at the time of 
cryopreservation, was 4.7x108 (range 0.3-33.8x108) total nucleated cells and 1.8 x106 
(range 0-19.1 x106) CD34 cells.  All infusions were administered through a peripheral IV 
after premedication with oral Tylenol 15mg/kg, IV Benadryl 0.5mg/kg and IV 
Solumedrol 0.5mg/kg.  Median post thaw recovery of TNCC was 82% (range 13-200%), 
and patients were dosed with a median of 2.0x107 TNC/kg (range 0.1-13.3x107), 0.7x105 
CD34+ cells/kg (range 0.04-6.4x105), and 6.5x104 CFU/kg (range 0-315x104).   
 
Three patients (1.5%) experienced anaphylactic reactions during their CB infusion 
characterized by wheezing with or without urticaria 2-10 minutes after the IV infusion 
was initiated.  The reactions resolved after discontinuation of the infusion and treatment 
with additional IV Benadryl and bronchodilators.  The remainders of the CB cells were 
discarded for two of the infusions stopped prior to completion; one patient was able to 
restart and complete the CB infusion.  One patient’s mother experienced an allergic 
reaction consisting of urticaria, presumably due to contact with DMSO exhaled onto her 
face and neck by her child receiving a CB infusion.  The reaction resolved with oral 
Benadryl.  With up to 11 years of follow-up, no infections, autoimmune diseases, tumors, 
or other adverse events were observed.11,26-28  This series demonstrated safety and 
feasibility of autologous CB infusion, and anecdotal reports of improved function were 
common. 
 
Hypoxic Ischemic Encephalopathy (HIE):   

 
March 15, 2018 
15
In phase I trial of newborns with HIE at birth conducted at Duke, fresh, non-
cryopreserved autologous CB processed to reduce volume and RBC content on a Sepax 1 
bioprocessor (Biosafe, Geneva) was infused in 1, 2, or 4 doses within the first 72 hours of 
life in babies with moderate-to-severe encephalopathy qualifying for therapeutic 
hypothermia.29 These babies (n=39) were compared to a concomitant group of babies 
who were also cooled at Duke but did not receive CB cells (n=146).  CB infusions were 
found to be safe in these critically ill babies, and babies receiving cells had increased 
survival rates to discharge (100% vs. 89%, p=0.03).  Of the 25 CB recipients with known 
one-year outcomes, 16 (64%) survived with Bayley III scores ≥ 85 in all three domains, 
and of the 63 cooled-only infants with known one year outcomes, 25 (40%) survived with 
Bayley III scores ≥ 85 in all three domains (p = 0.04).  A phase II randomized, placebo 
controlled, multicenter trial has recently been activated to formally test the efficacy of 
autologous CB infusion in these babies.   
Cerebral Palsy:   
The CP-AC study was a phase II, prospective, randomized, double blind, placebo 
controlled crossover study testing the efficacy of a single IV infusion of autologous CB 
in children ages 1-6 years with cerebral palsy (IND #14360).  A total of 63 children with 
a median baseline age of 2 years (range 1-6) were enrolled and treated from 2010-2015 
(32 CB, 31 placebo). Children were eligible if they were (1) Gross Motor Function 
Classification System (GMFCS) level 2-4 or (2) GMFCS level 1 with hemiplegia if they 
used their affected hand as an assist only. Children with known genetic conditions, 
intractable seizures, or severe microcephaly were ineligible.  Autologous CB units were 
eligible if they had a precyropreservation TNCC of ≥1x107/kg, negative sterility tests, 
and negative maternal infectious disease tests. Subjects were evaluated at baseline, one 
year, and two years with functional evaluations (GMFM-66, Peabody, Assisting Hand 
Assessment, Bayley) and brain MRI with DTI tractography and connectivity analyses. 
They were randomized to the order of CB and placebo infusions (given one year apart). 
The placebo consisted of TC-199 tissue culture medium with 1% DMSO. The primary 
endpoint was change in GMFM-66 score at one year after the initial infusion (CB dosed 
at 1-5x107 cells/kg or placebo). Cells or placebo were administered IV over 5-10 minutes 
in the outpatient setting after premedication with Tylenol, Benadryl, and Solumedrol. 
Subjects received IV fluids and were monitored for 2-4 hours post-infusion. 
 
The median TNCC of CB infused in this study was 2x107/kg (range 0.8–4.8) and median 
infused CD34 dose 0.7x105/kg (range 0.1–4). Despite negative pre-cryopreservation 
cultures, one CB unit grew β-hemolytic strep upon thaw. There were no clinical 
infections, and no patients were treated with antibiotics. One participant had a transient 
infusion reaction with urticaria and fever, which resolved.  
 
Analysis of the 63 patients at one year showed no difference in GMFM-66 change scores 
between placebo and treated groups (6.9 vs. 7.5, p=0.72). However, treated subjects with 
cell doses above or below the median precryopreservation or infused doses of 3x107/kg 
and 1.98x107/kg, respectively, demonstrated improvement in GMFM-66 change scores 
compared to subjects who received lower cell doses (p<0.01 for precryopreservation 
dose, p=0.05 for infused dose, Figure 2). Infused cell dose was not correlated with age 
(p=0.43) or type (p=0.32) or severity (p=0.46) of CP. Although the primary endpoint of 

 
March 15, 2018 
16
the study was change in the GMFM score in treated versus placebo patients 1 year post 
infusion, the observation that children receiving higher cell doses had a change in motor 
function, is important and influenced the minimum cell dose for this study (see section 
6.4). The choice of this cell dose threshold is further supported by the fact that this same 
cell dose has been identified as the minimum therapeutic cell dose facilitating 
engraftment after allogeneic hematopoietic stem cell transplant.  
 
Using a subject’s baseline GMFM-66 score, GMFCS level, age, and published 
percentiles,30 we calculated the expected one-year GMFM-66 raw score for each patient. 
Since such percentile values are only available for children ≥2 years old, one-year old 
subjects (n=25) were excluded from this analysis. In the evaluable patients (n=38), the 
difference between the actual one-year GMFM-66 score and the expected one-year 
GMFM-66 score was calculated. In the entire autologous CB group, the median actual-
expected difference in GMFM-66 scores was 1.7 (range -6.1-14.5), versus 2.2 (range -
6.2-12.6) in the placebo group. When the CB group was analyzed by infused cell dose, 
subjects who received above the median infused dose of 1.98x107/kg (n=9) improved a 
median 4.3 (range -1.7 to 14.5) points greater than expected (p=0.05 vs placebo), whereas 
subjects who received below the median infused dose improved a median 1.9 points less 
than expected (range -6.1 to 12.9; p=0.02 vs high dose, p=0.07 vs placebo).   
Figure 3:  GMFM-66 Scores by Randomized Treatment Assignment and Cell Dose Panel A: Distribution of GMFM-66 
score at baseline and one year in patients randomized to placebo and autologous CB. Lines connect the group means (circles) 
over time. Panel B: Distribution of raw GMFM-66 change score from baseline to 1 year in subjects assigned to placebo and in 
treated subjects who received above or below the median infused cell dose (Low: <1.98x107/kg, N=16 vs. High: ≥1.98x107/kg, 
N=16). Panel C: Raw GMFM-66 change scores based on median cell doses (Precryopreservation doses: Low, <3x107/kg, N=16 
vs. High, ≥3x107/kg, N=16; Infused doses: <1.98x107/kg, N=16 vs. High: ≥1.98x107/kg, N=16). Panel D: Actual-Expected 
GMFM-66 scores in patients ≥2 years of age (N=38) at one year based on infused dose. 

 
March 15, 2018 
17
 
Forty-eight patients were also eligible for analysis of the Peabody Developmental Motor 
Scales-2 (PDMS-2), which assesses motor skills in children from birth through age 5. 
The median one-year change from baseline in the Gross Motor Quotient was 0.0 in the 
autologous CB group and -1.0 in the placebo group (p=0.38). When the subjects treated 
with autologous CB were analyzed by infused cell dose (>/< 1.98x107/kg), a significant 
change was detected in the Gross Motor Quotient (3.0 vs. -2.0, p= 0.02).  
 
MRI data was also analyzed to explore relationships between change in GMFM-66 
scores, total brain connectivity, and cell dose. Accurate anatomical image parcellation 
could not be obtained in approximately one-third of subjects due to substantial 
morphologic brain abnormalities, leaving 23 treated patients and 15 placebo patients with 
usable connectivity data. However, the images from 2 of 4 patients ≥3 years who 
received high cell doses were unusable. There were no statistically significant differences 
in CP type, GMFCS level, or age between patients with and without analyzable images. 
As previously described,31 there was a moderate correlation between change in GMFM-
66 score and total connectivity at one-year in all analyzable subjects (n=38, Spearman 
r=0.53; 95% CI: 0.25, 0.73; p<0.001). Total connectivity change was not related to 
baseline GMFCS level, typography of CP, or gender, but was inversely correlated with 
age (Spearman r=-0.52; 95% CI: -0.72, -0.23; p=0.001). Though not statistically 
significant, there was a trend of increasing normalized connectivity at higher doses in the 
high-dose group, but no such relationship in the low-dose group.   
 
The CP-AC was limited by small sample size of the dosing groups, as cell dose was not a 
pre-specified analysis.  In addition to confirming safety of autologous CB infusion, the 
A
B 
C 
D: Actual-Expected Scores  
Figure 4: Brain Connectivity via MRI/DTI  Panel A: Correlation between change in GMFM-66 raw score and total connectivity 
in all analyzable subjects (n=38). Panel B: Infused cell dose in treated patients (n=23) versus change in normalized connectivity 1 
year post-infusion. Patients age ≤3 years are in green, >3 years in red. Panel C: Representative MRI/DTI images of 2 subjects. The 
top patient received a high cell dose and had a large increase in GMFM score and number of trackable nerve fibers (likely due to 
increased anisotropy or conductivity) and thus increased connectivity. The patient in the bottom panel, who received a placebo 
infusion, had a slightly negative change in GMFM score and had little to no increase in trackable nerve fibers and connectivity. 
A 
B 
C 
           Baseline 
 
  One-Year 

 
March 15, 2018 
18
CP-AC study demonstrated that when appropriately dosed, autologous CB infusion may 
be beneficial in children with CP.  
 
 
Autism Spectrum Disorder (ASD):  
Duke investigators recently completed 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). CB was administered as a single infusion 
(median infused dose: 2.6x107/kg, range: 1.0x107/kg to 8.1x107/kg). No 
immunosuppression was implemented 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 (PDD-BI) (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, while their 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). This data suggests that a larger Phase 
2 randomized study to evaluate the efficacy of CB therapy versus placebo is justified.  

 
March 15, 2018 
19
 
 
2.6 
Allogeneic 
Umbilical Cord Blood 
Many children who 
might benefit from CB therapies will not have their autologous CB banked.  In order to 
extend these therapies to all patients, use of an allogeneic product will be necessary.  
Allogeneic CB has been used extensively in the field of hematopoietic transplantation, 
and we have begun investigating its use in children and adults with brain injury.  
Background information is provided below.  
 
Allogeneic CB for Hematopoietic Reconstitution:   
Allogeneic human CB as a source of cells for hematopoietic reconstitution after 
myeloablative therapy has a proven track record of over 25 years of use in the clinic, 
with over 35,000 transplants performed.32  Allogeneic CB transplantation has been shown 
to be safe and has not been shown to cause tumors or cellular dysregulation.  Compared 
to other cell sources, CB has the following advantages: 
1. CB is an abundantly available source of stem cells that can be harvested at no risk to 
the mother or infant.  It is routinely collected, cryopreserved, and banked.  
2. Public CB donors are screened for risks of transmitting infectious agents through 
blood per CFR1271, subpart C donor screening regulations.  Important infectious 
agents, particularly cytomegalovirus (CMV), are much less common in the newborn 
than adults, and are less likely to contaminate CB units. 
3. CB units, cryopreserved and banked, are available on demand, and can be easily 
shipped and thawed for use when needed, eliminating delays and uncertainties that 
complicate bone marrow collection from unrelated donors. 
Time Point 
Median 
Mean 
SD 
 
Time Point 
Median 
Mean 
SD 
Baseline 
75.00 
74.40 
11.41 
 
Baseline 
76.00 
75.48 
20.32 
6 Months 
76.50 
80.17 
17.88 
 
6 Months 
86.00 
81.38 
22.75 
12 Months 
81.00 
79.13 
16.68 
 
12 Months 
86.00 
80.25 
23.34 
Figure 5:  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:  Vineland-II 
communication domain standard scores at baseline and six months after infusion (p<0.01 baseline to six months). Panel 
C: 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. 
 
A 
B 
C 
Vineland Socialization Domain 
CGI-I 
Vineland Communication Domain 

 
March 15, 2018 
20
4.  CB lymphocytes are more immunotolerant of a new host.  Thus, the intensity of 
graft-versus-host reactivity of fetal lymphocytes is less than that of adult cells, so 
transplantation of CB after myeloablabtion and immunosuppression where 
engraftment of donor cells is needed to treat the underlying disease results in less 
graft versus host disease (GvHD) than transplantation of bone marrow or other adult 
hematopoietic stem cell sources. 
5. CB contains pluripotent stem cells that have demonstrated the ability to differentiate 
into numerous types of cells throughout the body, including in the brain.  Thus CB 
may provide a source of cells for non-hematopoietic tissue repair or regeneration. 
6.   A minimum pre cryopreserved cell dose of 25 million cells per kg has been 
established as the threshold for successful hematopoietic engraftment after marrow 
and immune ablation.  
 
In this study, we propose to use allogeneic CB donor without the use of chemotherapy or 
other immunosuppressive therapies.  We are fully aware of the fact that engraftment of 
donor cells will not occur. Rather the mechanisms of action is paracrine signaling of 
endogenous cells to repair.  Since there is no myelo or marrow ablation and only 
immunocompetent children will be treated, and engraftment is not expected, the risk of 
GVHD is virtually non-existent.   
2.7 
Preliminary Experience Using Allogeneic Cord Blood in Patients with Neurological 
Conditions 
More recently, based on substantial preclinical data, CB has been investigated as a 
potential therapy for patients with brain injuries and neurological conditions.  As all 
adults and most children do not have access to autologous CB, allogeneic cells are 
frequently utilized in clinical trials of cellular therapies for neurologic conditions.  
Examples of allogeneic CB studies in human patients are described below.  
 
Stroke:  
The CoBIS study is an IRB approved, FDA IND sponsored, prospective, open-label, 
multi-center, Phase 1 safety study of a single intravenous infusion of non-HLA matched, 
ABO matched, allogeneic CB in 10 adults ages 18-80 years old.  CB units are selected by 
ethnicity, blood type, and ability to supply a dose of 0.5 – 1.5 x 107 TNCC/kg.  Eligible 
patients include those experiencing a recent, acute cortical, hemispheric, ischemic stroke 
in the middle cerebral artery (MCA) distribution as detected by MRI as a diffusion 
weighted abnormality and are enrolled if their National Institutes of Health Stroke Scale 
(NIHSS) is 8-15 (right hemisphere) or 8-18 (left hemisphere).  Participants who receive 
tPA or undergo mechanical perfusion are eligible for inclusion. Participants are not pre-
treated with immunosuppressive drugs. The primary endpoint is safety as assessed by the 
frequency and severity of adverse events within 24 hours of CB infusion and during 
the12 month period post CB infusion.  Secondary outcome measures include Modified 
Rankin Scale (mRS), NIHSS, the Barthel Index (BI), and European Quality of Life (EQ-
5D-3L), Patient Health Questionnaire Scale (PHQ8), Telephone Interview for Cognitive 
Status (TICS), and a self-reporting survey of rehabilitation therapy.  MRI will be used to 
evaluate changes in the brain 3 months post infusion.  
  

 
March 15, 2018 
21
To date (March 2016), all 10 participants have been enrolled and treated with allogeneic 
unrelated donor non-HLA matched, ABO matched CB within 10 days of their stroke. 
There have been no serious adverse events with a follow-up of 1-8 months.  Thus, early 
safety data suggests intravenous infusion of unmatched, allogeneic, CB cells is well 
tolerated.  If the safety profile remains favorable, we will move to a randomized, placebo 
controlled Phase 2 study with the goal of using unrelated non-HLA matched CB to down-
regulate inflammation and promote neuroprotection and neurorepair in patients with 
ischemic stroke.    
 
Cerebral Palsy at Duke:   
Given the benefit in motor improvement observed in the CP-AC study utilizing 
autologous CB, and recognizing that most children do not have a suitably qualified 
autologous CB unit available, we recently initiated a phase I clinical trial to evaluate the 
safety of fully HLA-matched or haploidentical allogeneic sibling CB infusion in children 
with CP (ClinicalTrials.gov ID NCT02599207).  As of April 2016, 13 children have been 
treated.  There have been no acute infusion-related toxicities, and no significant adverse 
events with short follow-up. 
 
Cerebral Palsy in Other Countries:   
Studies of allogeneic CB infusion in children with CP have also been conducted in Korea 
(n=105) and Russia (n=80).  The Korean study had three groups:  allogeneic CB + 
cyclosporine + erythropoietin; erythropoietin alone; and a control group.33  One severely 
affected patient died in her sleep 14 weeks after CB administration, and this was 
determined to be unrelated to the CB infusion.  Eight other patients experienced serious 
adverse events requiring hospitalization (pneumonia–4, seizure–1, influenza–2, urinary 
tract infection–1), but the distribution did not differ between groups.  Non-serious 
adverse events that were more common in the CB group were pneumonia and irritability.  
At one year of follow-up, there were no reported prolonged or delayed serious adverse 
events.  The authors reported greater improvements in cognitive and select motor 
functions in children who received CB and erythropoietin versus controls.  There was no 
CB-only group for comparison.  In the Russian study, children ages 1-12 years with 
cerebral palsy received 1-6 intravenous infusions of allogeneic CB with an average dose 
of 2.5x108 viable cells per infusion.34  Most patients who received four or more infusions 
showed improvement in tone, motor, and/or cognitive function, but there was no control 
group for comparison.  In their series, factors that impacted treatment response included 
age, severity of brain damage, and number of CB infusions, with more responses 
observed in younger, less severely impaired children and those who received more than 
two infusions. 
 
Autism Spectrum Disorder (ASD) in Other Countries: 
In China, 37 autistic children, ages 3-12 years, were enrolled in a study utilizing 
allogeneic CB mononuclear cells (CBMNC) and/or umbilical cord-derived mesenchymal 
stem cells (UCMSC).35  The children were divided into three groups:  CBMNC (n=14), 
CBMNC+UCMSC (n=9), and control (n=14).  All children received standard 
rehabilitation therapy.  Cells were given in four doses, 5-7 days apart, via intravenous 
and/or intrathecal administration.  In the cell recipients, transient fever (5/23) was the 

 
March 15, 2018 
22
only reported adverse event related to the therapy.  Compared to control patients at six 
months, they observed greater improvements in the Childhood Autism Rating Scale 
(CARS) in the CBMNC+UCMSC group and in the Clinical Global Impression scale 
(CGI) in both cell groups.  This was a small, non-randomized study designed primarily to 
assess safety.  Nonetheless, the functional data suggests the potential for benefit.  
2.8 
Source of Unrelated Cord Blood Units for this Trial 
The Carolinas Cord Blood Bank (CCBB) is one of the largest public cord blood banks in 
the nation. Established in 1998 with support from the National Heart and Blood Institute 
of the NIH, the CCBB has over 30,000 CB units in inventory and has distributed over 
2,500 CB units for transplant to date. In 2012 the CCBB received approval from the FDA 
for its BLA application to market DUCORD, a stem cell product derived from umbilical 
cord blood, for use in transplants between unrelated donors and recipients. DUCORD is 
approved for use in hematopoietic stem cell reconstitution for patients with disorders 
affecting the hematopoietic system that are inherited, acquired, or result from 
myeloablative treatment. The CCBB currently collects from 10 hospital sites (8 in North 
Carolina, 1 in Atlanta, GA and 1 in Boston, MA). It also accepts CB donations from 
mothers delivering in any hospital in North Carolina and Atlanta through a kit donation 
program. 
2.9 
Specifications for Qualification of Private or Public CB Units for Cell Therapy 
Studies 
Most hematopoietic stem cell transplants utilizing CB are carried out with allogeneic 
unrelated donor units obtained from public CB banks after myeloablative chemotherapy 
with or without total body irradiation. There are over 200 family CB banks worldwide 
that store autologous CB units after parental request and reimbursement.  Most CB units 
are cryopreserved as red cell and plasma depleted, mononuclear cell enriched cell 
populations.  Reference samples are also stored as attached segments or in vials for 
additional testing and qualification.  High quality public and private banks screen 
maternal donors for the risk of transmitting blood-borne infectious diseases, as surrogates 
for the infant donor, with FDA-cleared donor screening tests in CLIA approved donor 
screening laboratories. 
 
Based on established criteria utilizing allogeneic CB for hematopoietic stem cell 
transplantation and our experience in treating more than 600 children with autologous CB 
for neurological conditions, we have established the following criteria to qualify public or 
privately banked CB units for cell therapy studies.  The CB unit must have: 
 
1. Pre-cryopreservation total nucleated cell count (TNCC) documented and at least 
2.5 x 107/kg 
2. Pre-cryopreservation viability ≥70% (total or CD34+ cell population) 
3. Pre-cryopreservation sterility culture performed and negative 
4. Maternal infectious disease screening as follows:  Testing must include negative 
results for Hepatitis B, Hepatitis C, HIV, HTLV, and syphilis. These tests are 
superseded by the testing requirements of the country in which the cord blood was 
banked (ie. HTLV testing is not required or routinely performed in certain parts of 
the world where the infection is not endemic). Additional screening, which is 

 
March 15, 2018 
23
dependent on the timing of the CB collection, may be performed based on local 
and national regulations.  If a screening test is positive, confirmatory testing must 
be negative, except for CMV.  Units from mothers who have a positive CMV 
antibody screen may be used.  Autologous units from mothers who test positive 
for Hep B core antibody but who test negative for Hep B Surface Antigen and 
Hep B NAT may be used. 
5. Test sample available for identity confirmation and potency testing 
6. HLA typing performed and meets study-specific parameters 
7. CD45+ viability ≥40% or CD34+ viability ≥70% on thawed test sample 
 
These same criteria will be utilized for this clinical trial and, along with procedures for 
CB administration, are detailed again in section 6.0. 
2.10 
Clinical Safety Summary 
In summary, in this study, we will administer a single intravenous infusion of either 
autologous or partially HLA-matched, allogeneic unrelated donor cord blood in young 
pediatric patients with autism. All children will have documentation of normal immune 
function prior to study enrollment.  No chemotherapy or immunosuppressive 
pretreatment will be utilized. The infused cells are not expected to engraft. Rather they 
will exert a therapeutic effect via paracrine signaling which will stimulate endogenous 
cells to facilitate neural cell repair and connectivity. 
 
Our experience in over 600 pediatric patients with brain injuries including Cerebral 
Palsy, HIE, ASD, and other injuries infused intravenously with autologous CB 
demonstrates that the procedure is safe and well-tolerated in children with neurological 
conditions and supports further testing in the pediatric ASD population.   In addition, 
infusions in immunocompetent patients with brain injuries (adults with stroke and 
children with cerebral palsy), although smaller in number, also demonstrate a favorable 
safety profile and support further studies of allogeneic CB to treat children with ASD.  
Furthermore, infusions of allogeneic CB after myeloablative therapy in >35,000 patients 
have been demonstrated to be safe for over 25 years, although in this setting because 
engraftment of donor cells must occur, a risk of graft versus host disease is present. This, 
however, is not the case in the current study because infused children are 
immunocompetent, will reject the donor cells and, accordingly, will not be at risk for 
GvHD. 
2.11 
Study Rationale and Hypotheses 
The major objective of this study is to determine whether a single intravenous infusion of 
CB can improve the core symptoms of autism in children with ASD.  The rationale for 
the study and for the potential benefit of CB is based upon the following hypotheses: 
 The developing brain exhibits remarkable plasticity, making young children ideal 
candidates for deriving maximal benefit from the pluripotential properties of CB.   
 Cellular therapy, acting through paracrine or trophic mechanisms, may repair or 
facilitate improvement in areas of the brain with aberrant functioning by repairing 
or enhancing neural connectivity.  
 Cellular therapy may modify immune response and inflammation that may be 
linked to improvements in the core symptomatology of ASD, including in areas 
such as social communication. 

 
March 15, 2018 
24
 Intravenous administration of allogeneic cord blood cells to immunocompetent 
hosts should be safe and effective.  It is expected that the cells will be 
immunologically rejected within days to weeks of administration, eliminating a 
risk of Graft versus Host Disease (GvHD) or aberrant cell proliferation post-
infusion. 
 
The mechanistic rationale for this clinical study hypothesizes that CB can act through 
paracrine and allocrine mechanisms to modulate on-going inflammation and/or immune 
pathology in the brain and possibly protect neurons from further damage.  In many 
contexts, CB cells dampen, 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 CB cells 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.36  Thus, CB may be a candidate therapy for ASD because of the CB’s 
immunomodulatory activities. 
 
The projected rationale for this therapy focuses on the ability of CB to modify 
inflammation through downstream effects on glial cells and cytokines.  CB cells may not 
be required to proliferate or persist in the recipient.  Given that the recipients will not be 
conditioned in any way and have a full complement of stem and progenitor cells prior to 
treatment, we feel it is unlikely that allogeneic CB cells will engraft in the recipient or 
that engrafted stem and progenitor cells from the CB treatment will contribute 
significantly to any therapeutic effect.  
2.12 
Study Design 
This is a single site, prospective, randomized, double-blind study of a single intravenous 
autologous or allogeneic, unrelated CB infusion in children ages 2-7 years with ASD.  
Participants will be randomly assigned to Sequence A, consisting of a single infusion of 
CB cells at baseline followed 6 months later by a single infusion of placebo, or Sequence 
B, consisting of an infusion of placebo at baseline followed 6 months later by an infusion 
of CB cells.  All participants will ultimately be treated with CB cells at some point during 
the study.  Participants with an available qualified autologous CB unit will receive 
autologous cells, and those without a suitable autologous CB unit available will receive 
cells from a ≥4/6 HLA-matched, allogeneic, unrelated donor CB unit from the Carolinas 
Cord Blood Bank.  All infusions will be double-blinded.  The primary outcomes will be 
assessed 6 months after the initial infusion in the sequence.  Additional testing for 
secondary exploratory analyses will be performed at 12 months.  Duration of study 
participation will be 12 months from the time of baseline infusion. 
2.13 
Risks and Benefits 
CB cells will be prepared and infused using standard operating procedures that have been 
used in over 35,000 individuals worldwide.  The potential risks associated with infusion 
of autologous CB cells or the placebo product include an allergic reaction to the product 

 
March 15, 2018 
25
(rash, shortness of breath, wheezing, difficulty breathing, hypotension, swelling around 
the mouth, throat or eyes, tachycardia, diaphoresis) to the product or transmission of 
infection.  Additional risks associated with infusion of allogeneic CB cells include 
hypertension, bradycardia, anaphylaxis, hematuria, acute hemolytic reaction, rejection of 
cells, immune dysregulation (develop of HLA directed antibodies), and development of 
graft-versus-host disease.  All CB units are screened for infection and must meet release 
criteria prior to infusion, as described below.  Participants will not receive 
immunosuppressive therapy prior to or after infusion of CB cells.   
 
Participants will likely require sedation in order to complete a brain MRI and infusion.  
Risks associated with sedation include nausea, vomiting, blood vessel injury, nerve 
injury, lung injury, heart attack, allergy to medications, brain damage, respiratory 
insufficiency, hypoxia, hypotension, and anaphylaxis, and death.  Medications used for 
sedation may include midazolam, dexmedetomidine, and/or propofol.  Potential risks 
associated with the use of midazolam include excessive sleepiness or sedation, headache, 
hiccoughs, cardiac arrest, involuntary movements, agitation, changes in breathing 
(decreased, slowing or stopping of breathing), and decreased oxygen in the blood.  Risks 
associated with the use of dexmedetoidine include increased or decreased blood pressure, 
nausea, vomiting, dry mouth, decreased red blood cells, fever, changes in heart rhythms 
(abnormal rhythms or a fast or slow heart rate), cardiac arrest, changes in oxygen levels 
or the amount of acid in the blood, fluid on the lungs, slowing of breathing.  Risks 
associated with the use of propofol include pain at the injection site, nausea, vomiting, 
involuntary movements, changes in heart rhythm, heart failure, high blood pressure, 
inflammation of the pancreas (pancreatitis), anaphylaxis or severe allergic reaction, 
seizure, kidney failure, priapism (male erection that doesn’t resolve without medical 
care), changes in breathing (including apnea or the stopping of breathing), increased acid 
in the blood, or infection in the blood.  These associated risks are described in detail in 
the consent form. Risks associated with blood draws and IVs include momentary 
discomfort or pain, bruising, infection, bleeding, clotting, and fainting.  Risks associated 
with genetic testing include medical, psychosocial, and economic risks, effects on 
insurability and employability, limits on educational options, and social stigma.      
 
Potential benefits of this intervention include the possibility that the CB cells 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, such as social communication.  
3.0 
STUDY OBJECTIVES 
1. To determine, in a randomized, placebo controlled, best available donor source trial, 
the efficacy of a single intravenous infusion of umbilical CB in improving the core 
symptoms of children with ASD. 
a. To determine whether there is a difference in mean response at six months 
between all cell sources and placebo. 
b. To determine whether there is a difference in mean response at six months 
between autologous CB and placebo. 

 
March 15, 2018 
26
c. To determine whether there is a difference in mean response at six months 
between allogeneic CB and placebo. 
d. To determine whether there is a difference in mean response at six months 
between autologous CB and allogeneic CB. 
2. To describe the safety and tolerability of a single intravenous infusion of unrelated 
donor CB in children with ASD.  
3. To explore whether patient age or IQ correlate with response. 
4. To explore whether changes in MRI, EEG, and eye tracking are observed after CB 
treatment. 
5. To identify biomarkers that positively or negatively correlate with response to 
treatment. 
4.0 
STUDY DESIGN 
4.1 
General Design 
This is a single site, prospective, randomized, double-blind study of a single intravenous 
autologous or allogeneic, unrelated CB infusion in children ages 2-7 years with ASD.  
Participants will be randomly assigned to Sequence A, consisting of a single infusion of 
CB cells at baseline followed 6 months later by a single infusion of placebo, or Sequence 
B, consisting of an infusion of placebo at baseline followed 6 months later by an infusion 
of CB cells.  All participants will ultimately be treated with CB cells at some point during 
the study.  Participants with an available qualified autologous CB unit will receive 
autologous cells, and those without a suitable autologous CB unit available will receive 
cells from a ≥4/6 HLA-matched allogeneic, unrelated donor CB unit from the Carolinas 
Cord Blood Bank.  All infusions will be double-blinded.  The primary outcomes will be 
assessed 6 months after the initial infusion in the sequence.  Additional testing for 
secondary exploratory analyses will be performed at 12 months.  Duration of study 
participation will be 12 months from the time of baseline infusion. 

 
March 15, 2018 
27
4.2 
Study Flow Chart 
 
 
4.3 
Study Endpoints 
 
Primary Endpoint:   
The primary endpoint of this study is the change in social communication skills (a core 
symptom of autism) from baseline to six months after the initial study infusion, as 
measured by the Vineland Adaptive Behavior Scale (VABS)-3 Survey Interview Form, 
Socializations Subscale Standard Score.  Control (placebo) and treated patients will be 
compared.   
 
Secondary Endpoints:   
Additional data regarding baseline to six month changes in autism symptom severity, 
problem behaviors, social, language skills, and treatment response will be assessed via 
both parent-reported and clinician-assessed measures, using the following measures: 
1. Vineland Socialization domain raw score 
2. Vineland Socialization domain age equivalent 
3. Pervasive Developmental Disorder Behavior Inventory (PDD-BI) composite 
standard score (parent questionnaire) 
12 Month Evaluations
6 Month Evaluations 
(Primary Endpoint) & 
Infusion
Baseline Evaluations & 
Infusion
Enroll & Randomize
Qualifying Visit
CBU Screening
Patient Screening
Review Records, 
Labs, Videos, 
Photos
Review CBU 
Report, HLA & 
Potency testing
Final Eligibility 
Determination
Sequence A
CB
Placebo
Sequence B
Placebo
CB
Remote 
Assessments

 
March 15, 2018 
28
4. CGI-S and CGI-I (clinician assessment) 
5. Expressive One-Word Picture Vocabulary Test raw score (clinician assessment)  
6. Vineland Adaptive Behavior Communication subscale standard score and age 
equivalent, Daily Living subscale standard score and age equivalent, and the 
Adaptive Behavior Composite standard score and age equivalent 
7. Individual subscales of the PDD-BI t scores. 
 
Safety and tolerability of donor CB 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 and anti-RBC antibodies and 
nonspecific markers of systemic inflammation (ESR, CRP) 
4. Incidence and severity of graft vs. host disease 
5. Incidence and severity of unexpected adverse events, by relation to study product 
 
Exploratory Analyses:   
 
The following data will be collected for exploratory analyses: 
1. Neurophysiological response to social and nonsocial stimuli via EEG and eye 
gaze tracking to social and nonsocial stimuli 
2. Gastrointestinal symptoms via the Pediatric Quality of Life Inventory (PedsQL) 
Gastrointestinal Symptoms Scales 
3. Brain connectivity via MRI with DTI 
5.0 
RESEARCH PARTICIPANT SELECTION AND WITHDRAWL 
 
5.1 
Study Population 
Up to 190 children ages 2-7 years with a confirmed diagnosis of ASD and an available 
qualified autologous or unrelated donor cord blood unit. 
 
5.2 
Inclusion Criteria 
1. Age ≥ 2 years to ≤ 7 years (7 years, 364 days) at the time of visit 1 
2. Confirmed clinical DSM-5 diagnosis of Autism Spectrum Disorder using the DSM-5 
Checklist 
3. Fragile X testing performed and negative 
4. Available and qualified umbilical cord blood unit with a minimum banked total nucleated 
cell dose of ≥ 2.5 x 107 cells/kg that meets criteria outlined in Section 6.0, either: 
a. Autologous umbilical cord blood unit OR 
b. ≥4/6 HLA-matched allogeneic unrelated umbilical cord blood unit from the 
Carolinas Cord Blood Bank  
5. Stable on current psychiatric medication regimen (dose and dosing schedule) for at least 
2 months prior to infusion of study product 
6. Normal absolute lymphocyte count (≥1500/uL) 
7. Participant and parent/guardian are English speaking 
8. Able to travel to Duke University two times (baseline and 6 months post-baseline), and 
parent/guardian is able to participate in interim surveys and interviews monthly 
9. Parental consent 

 
March 15, 2018 
29
5.3 Exclusion Criteria 
1. General: 
a. Review of medical records indicates ASD diagnosis not likely  
b. Known diagnosis of any of the following coexisting psychiatric 
conditions:  depression, bipolar disorder, schizophrenia, obsessive compulsive 
disorder, Tourette syndrome 
c. Screening data suggests that participant would not be able to comply with the 
requirements of the study procedures, including study outcome measures, as 
assessed by the study team 
d. Family is unwilling or unable to commit to participation in all study-related 
assessments, including follow up for approximately 12 months 
e. Sibling is enrolled in this (DukeACT) 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 
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. HIV positivity 
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 sedation or other 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. Evidence of clinically relevant physical dysmorphology indicative of a genetic 
syndrome as assessed by the PIs or other investigators, including a medical 
geneticist and psychiatrists trained in identifying dysmporphic features associated 
with neurodevelopmental conditions. 
 

 
March 15, 2018 
30
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 >2 weeks, and no systemic 
steroids within 3 months 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 cerebral 
palsy societies, local medical providers, 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.   
 
Screening for the DukeACT 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 cord blood report (if applicable), medical 
records, school 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 and psychiatrists with expertise in diagnosing and treating 
children with ASD.  They perform an extensive review of the patient’s psychological 
records as well as any school and therapy records available.  Both teams review the 
patient’s photographs and records to evaluate for dysmorphic features.  Any patients with 
questionable facial features or findings on genetic testing are then reviewed by a medical 
geneticist with expertise in genetic conditions associated with ASD.  A patient must be 
approved by both medical and psychiatric screening teams to proceed with further 
laboratory 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 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 the CB will be 
considered not evaluable and can be replaced with another participant. 
 

 
March 15, 2018 
31
 
 
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 
Umbilical Cord Blood 
Patient enrollment is dependent on the availability of a banked unit of autologous CB at a 
private or public cord blood bank or an allogeneic unrelated donor CB from the Carolinas 
Cord Blood Bank.  The unit must have appropriate degree of HLA matching, donor 
screening, and product characterization as detailed below.   
6.2 
HLA Matching 
All potential study participants will undergo HLA typing at HLA-A, B, and HLA-DRB1 
via blood or buccal swab.  HLA testing will be performed at low resolution for patients 
with autologous CB units and high resolution for patients utilizing allogeneic CB units.  
In addition to the HLA matching criteria, each CB unit must meet the infectious disease 
and dosing requirements detailed in the sections below.  Matching requirements are as 
follows: 
 
Autologous CB units:  Identity confirmation via low resolution 6/6 HLA match. 
Allogeneic CB units:  Patients’ HLA typing will be performed on two separate samples for 
confirmation.  Allogeneic units that are potential matches will initially be identified from a 
search of the Carolinas Cord Blood Bank.  The best available HLA-matched (≥4/6) CB unit 
with a pre-cryopreservation nucleated cell dose ≥2.5 x 107 cells/kg will be selected.  Once a 
unit is selected, HLA typing will be used to confirm the original HLA typing and to select 
the best matching unit.  When possible, at least 1 match at each HLA loci will be prioritized.   
 A 6/6 matched donor will be selected before a 5/6, which will be selected before a 4/6 
antigen matched donor.  An UCB unit must be at least 4/6 HLA-matched with the 
patient.  
 When a unit is partially mismatched, compatibility at the DR locus will be ranked as 
most important, followed by compatibility at the B locus and lastly the A locus.   
6.3 
Donor Screening for CB Units 
Screening of maternal blood collected within 30 days before or after delivery is used for 
CB donor infectious disease screening.  Maternal testing must have been performed in a 
CLIA (or equivalent for CB units from international banks) certified donor testing 
laboratory.  Testing must include negative results for:  Hepatitis B, Hepatitis C, HIV, 
HTLV, and syphilis.  These tests are superseded by the testing requirements of the 
country in which the cord blood was banked (ie. HTLV testing is not required or 
routinely performed in certain parts of the world where the infection is not endemic). 
Additional screening (i.e. West Nile, Chagas), which is dependent on the timing of the 

 
March 15, 2018 
32
cord blood collection, may be performed based on local and national regulations.  If a 
screening test is positive, confirmatory testing must be negative, except for CMV.  Units 
from mothers who have a positive CMV antibody screen may be used.  Autologous units 
from mothers who test positive for Hep B core antibody but who test negative for Hep B 
Surface Antigen and Hep B NAT may be used. 
6.4 
CB Unit Characterization and Shipping 
Results of initial testing at the cord blood bank must include a pre-cryopreservation TNC, 
viability and sterility culture.  Pre-cryopreservation TNC must be ≥2.5x107/kg, sterility 
cultures must have been negative and total viability or CD34+ cell viability, if performed, 
must have been >70%.  
 
A test vial or segment must be available from each CB unit for potency testing and 
confirmatory HLA typing for identity confirmation.  The test sample will be 
sent/transferred to the Duke University Stem Cell Laboratory (STCL) in the frozen state.  
At the laboratory, the sample will be thawed and tested for viability, CFU, and CD34.  
HLA typing will be performed on the same or a related sample either at Duke or another 
certified laboratory.  Units will be deemed acceptable for the trial if the CD45 cell 
population is >40% viable and/or the CD34+ population is >70% viable.  CFU growth 
and CD34 will be described but will not be a specification for study enrollment.   
 
After testing, CB units from CB banks outside of Duke will be shipped in a dry shipper 
containing a data logger to monitor temperature to the STCL at Duke University in the 
frozen state.  Upon receipt, the CB unit will be examined for appropriate labeling and 
integrity of the cryopreservation bag and ports and the confirmation of the frozen state.  
The CB will be transferred from the dry shipper to a liquid nitrogen freezer and stored 
until the day of infusion. 
 
On the infusion day, all or part of the CB will be thawed and washed with dextran 40 + 
5% human serum albumin in the standard fashion per Standard Operating Procedure.  At 
the time of thawing, standard studies including total nucleated cell count, viability, viable 
CD34+ cell count, CFU, and bacterial culture will be performed on the CB unit in the 
Duke STCL.  A research sample of the CB will be retained for future analyses, and a 
sample of allogeneic CB units will be retained for later chimerism testing.  A goal dose of 
2.5-5x107 TNCC/kg of participant body weight based on the pre-cryopreservation TNCC 
will be prepared for infusion in a volume of approximately 30 mL or no more than 5cc/kg 
as determined by the ordering physician.  
6.5 
Placebo Product 
Cryopreserved UCB that has been thawed and washed in the standard fashion has a pink 
color due to the cellular and or/media content and a distinctive scent due to the DMSO 
used in cryopreservation.  In order for patients and families to be truly blinded to the type 
of infusion they are receiving, the placebo product must be similar in both appearance 
and odor.  Therefore, the placebo product will be acellular and will consist of TC-199 
(pink) with 1% DMSO which are standard ingredients in cellular products.  The volume 
of placebo product will be ~30 ml, in the range of a typical UCB unit that has been 

 
March 15, 2018 
33
washed and thawed after cryopreservation.  The placebo solution will be placed in the 
same final container so the clinical staff remains blinded to study product or placebo.    
6.6 
Packaging of Study Product 
All UCB products receive a unique identification number (ISBT Demand 128 bar code) 
to ensure integrity of the product and maintain chain of custody.  The clinical site or cord 
blood bank will assign an ISBT Demand 128 bar code label to the CB unit, which is 
placed on the thawed CB unit bag or via tie tag.  A similar label will be generated for the 
placebo products, which will be packaged in identical bags or syringes to ensure blinding 
of the patient and family.  All products will be transported from the STCL to the infusion 
site in a validated cooler by courier. 
6.7 
Administration of Study Product 
Patients will arrive in clinic on the morning of their scheduled infusion.  A peripheral IV 
will be placed either by an anesthesiologist, clinical staff or study staff.  After the CB is 
thawed and transported to the clinic, premedication with Benadryl 0.5mg/kg/dose IV, 
Solumedrol 0.5-1mg/kg IV will be administered.   The infusion product (CB or placebo) 
will be administered intravenously over 2 to 25 minutes under direct physician 
supervision.  Vital signs (heart rate, blood pressure, temperature, respiratory rate) will be 
checked upon arrival to the clinic and as clinically indicated.  Pulse oximetry will be 
monitored continuously throughout the infusion and for 15-30 minutes post infusion.  
Patients will be observed and hydrated with standard IV hydration for approximately one 
hour post infusion.  Patients will be discharged from clinic after a minimum of 1 hour 
providing all vital signs are at their baseline and they are awake, taking oral fluids, and 
asymptomatic with no evidence of toxicity. 
6.8 
Safety Follow-up 
On Day 1 following the infusion, the participant will be seen by study staff to assess for 
any infusion related adverse reactions or complications.  At ~2 weeks post administration 
of the CB, a member of the study team will contact the parent or guardian via phone or 
email to assess patient status and any adverse events.  A questionnaire (see Appendix 1) 
will be administered at 3, 6, 9, and 12 months post-infusion to assess for serious adverse 
events.  Included in the safety assessment, concomitant medications will also be collected 
at a minimum of at the baseline, 3, 6, 9, and 12-month time points.  
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 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 records, video, photos, and laboratory testing 
under a separate screening protocol.  Information on any autologous CB units will also be 
obtained and reviewed.  If the child appears eligible, and does not have an autologous CB 
unit available, a suitable unrelated donor CB unit will be identified at the Carolinas Cord 

 
March 15, 2018 
34
Blood Bank.  The CB unit (autologous or allogeneic) will be screened as described in 
section 6.   
 
Once all screening is complete and the patient is likely to meet study criteria, the patient 
will travel to Duke for their first visit.  On day 1, the informed consent will be obtained 
and patient eligibility will be determined by a physical observation and verification of 
ASD diagnosis per DMS-5 criteria.  If the child is deemed eligible, they will be enrolled 
on study and randomized to the order in which they receive CB and placebo infusions 
(Sequence A or Sequence B).  During their first visit, they will also have a brain MRI, 
neuropsychological evaluations, EEG, and their initial infusion (CB or placebo).  
Participants will be evaluated the day after the infusion and parents will be contacted 7-
10 days after the infusion.  Participants will return to Duke six months later, when they 
will undergo the same assessments and receive whichever infusion they did not receive at 
the first visit.  Additional safety assessments will be performed via phone or email at 3, 9, 
and 12 months post-infusion. 
7.2  
Patient Screening 
Initial patient screening will be conducted with informed consent under a separate 
protocol and will include a review of medical records, videos, photos, initial laboratory 
testing, screening of autologous CB units as per section 5.4.  If no exclusion criteria are 
identified, patient questionnaires and the Autism Diagnostic Interview- Revised (ADI-R), 
Shortened Version are administered.  If a qualified autologous CB unit is not available, 
unrelated donor CB unit will be identified at the Carolinas Cord Blood Bank.  
 
After study eligibility appears likely based on patient and CB criteria, potential 
participants will travel to Duke for initial evaluation.  Evaluations and treatments will be 
conducted in the outpatient setting.  After informed consent is obtained, a brief physical 
exam and baseline psychological testing will be conducted to confirm eligibility.  If no 
exclusion criteria are realized, the patient will be enrolled, randomized, and undergo the 
remainder of the study evaluations.   
7.3 
CB Unit Selection 
If the child has an available, banked autologous CB unit, the CB report for that unit will 
be obtained from the bank at which it is stored, if not already available.  If the autologous 
CB unit meets the donor screening and characterization requirements outlined in section 
6, a sample of the patient’s blood/buccal mucosa, and CB unit will be obtained for HLA 
typing confirm that the CB unit belongs to the child.   
   
If a participant does not have a suitable autologous CB unit available, an allogeneic 
unrelated donor CB unit will be identified at the Carolinas Cord Blood Bank.  HLA 
typing will be obtained on the patient, and the best available HLA-matched CB unit with 
a pre-cryopreservation nucleated cell dose ≥2.5x107 cells/kg will be chosen according to 
the principles below. 
 When possible, at least 1 match at each HLA loci will be prioritized.   
 When a unit is partially mismatched, compatibility at the DR locus will be ranked 
as most important, followed by compatibility at the B locus and A locus. 
 

 
March 15, 2018 
35
Once a suitable autologous or allogeneic CB unit has been deemed an acceptable match, 
a sample of the CB unit will be tested for potency in the Duke STCL.  If results of these 
tests are satisfactory and the CB unit is stored outside of Duke, the CB unit will be 
delivered to the Duke STCL in the frozen state. 
7.4  
Psychological Assessments 
The following measurements will be conducted with the child participant at Duke during 
each clinic visit: 
Respondent 
Measure 
Domain  
Length of 
Administration 
Clinician Assessments 
with the Child 
(completed at Duke) 
Autism Diagnostic Observation Schedule, 
Second Edition (ADOS-2) 
ASD-Related 
Symptoms 
45-50 minutes 
Mullen Scales of Early Learning, AGS 
Edition or the Differential Ability Scales, 
Second Edition* 
Cognitive/ 
Language 
45-60 minutes 
Expressive One-Word Picture Vocabulary 
Test, Fourth Edition 
Cognitive/ 
Language 
10-30 minutes 
Physiological and 
Functional Assessments 
of the Child (completed 
at Duke) 
Electrophysiological Response to Social and 
Nonsocial Stimuli 
EEG 
20 minutes 
Eye Gaze Tracking of Social Stimuli 
ASD-Related 
Symptoms 
10-15 minutes 
Clinician Observation 
of Parent/Child 
Parent-Child Interaction with Noldus 
EthoVision 
Parent-Child 
Interaction 
12 minutes 
 
 
 
 
* The Mullen Scales of Early Learning or Differential Ability Scales is only conducted during the baseline visit.   
 
7.4.1 Assessments for Diagnosis of Autism Spectrum Disorder: 
Diagnosis of ASD will be confirmed by the DSM-5 Checklist, which will be informed by 
the Autism Diagnostic Observation Schedule, Second Edition (ADOS-2) and the Autism 
Diagnostic Interview-Revised (ADI-R)-Shortened Version.  Diagnostic evaluations will 
be completed by clinical research staff that have been certified as research reliable in the 
administration of the ADOS-2 and ADI-R. 
 
Autism Diagnostic Interview-Revised (ADI-R), Shortened Version:37 This is completed 
during the screening process of DukeACT, prior to the participant’s arrival at Duke 
University and will be used to help inform a DSM-5 diagnosis of ASD.  The ADI-R is a 
comprehensive parent interview that assesses early functioning in three domains: 
language/communication, reciprocal social interactions, and restricted, repetitive, and 
stereotyped behaviors and interests.  The research reliable interviewer follows 
standardized procedures for obtaining information and recording responses.  Interview 
questions include various content areas, including the participant’s background, 
behaviors, early development and milestones, language acquisition, current 
communicative functioning, social development and play, interests and behaviors, and 
any other clinically relevant behaviors. The ADI-R shortened version follows the same 
protocols as the standard ADI-R, but some of the questions not required for making a 
diagnosis are omitted. The ADI-R shortened version takes between 90-150 minutes 
depending on the age of the child and the complexity of behaviors.    
 

 
March 15, 2018 
36
Autism Diagnostic Observation Schedule, Second Edition (ADOS-2):38 This assessment 
will be used to help inform a DSM-5 diagnosis of ASD. The ADOS-2 is a standardized 
observational assessment of core ASD symptomatology. Age- and language-dependent 
modules are composed of a series of activities led by a trained, research-reliable clinician 
to observe the child’s communication, social interactions, play, restricted, and repetitive 
behaviors. ADOS-2 protocols are designed to elicit behaviors that directly map onto the 
ASD DSM-5 criteria.  The ADOS-2 can be administered to toddlers as young as 12 
months of age. Age and verbal ability are used to determine the appropriate module 
(toddler, 1, 2, 3 or 4). For each ADOS-2 Toddler Module assessment, a score will be 
generated using the Toddler Module Calibrated Severity Scores developed by Esler et al. 
(2015) to facilitate comparison to other modules. Severity Scores will also be generated 
for Modules 1-4. The ADOS-2 takes around 45-60 minutes to administer. This 
assessment will be completed at baseline and 6 month visits. 
 
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 edition 5.  
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 clinician’s best judgment and is informed by information 
gathered during the administration of the ADI-R, Shortened Version and the ADOS-2. 
The DSM-5 takes less than 5 minutes to complete and will be completed at baseline and 6 
month visits.      
 
7.4.2 Other Clinician Assessments:  
Clinical Global Impression (CGI): The CGI is a commonly used rating scale with two 
components that measures symptom severity and treatment response or change in clinical 
presentation between time points. It takes 5 minutes to complete. 
 
The Clinical Global Impression – Severity Scale (CGI-S): The CGI-S is a 7 point scale 
completed at the baseline and 6-month visits that requires the clinician to rate the severity 
of the participant’s symptoms of ASD at the time of assessment, relative to the clinician’s 
past experience with participants who have the same diagnosis.  Based on the clinician’s 
lifetime clinical experience, a participant is assessed on severity of ASD symptoms at the 
time of rating - 1, normal, no symptoms; 2, borderline level of symptoms; 3, mild 
symptoms; 4, moderate symptoms; 5, marked symptoms; 6, severe symptoms; or 7, 
extremely severe symptoms. The 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 baseline and 6 month visits. 
 
The Clinical Global Impression – Improvement (CGI-I): The CGI-I is a 7 point scale that 
requires the clinician to assess how much the participant’s ASD symptoms have 
improved or worsened relative to a baseline assessment. The symptoms are rated as: 1, 
very much improved; 2, much improved; 3, minimally improved; 4, no change; 5, 
minimally worse; 6, much worse; or 7, very much worse. 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. 

 
March 15, 2018 
37
 
Continuous Visual Scale of Change (Visual Change): The Visual Change is completed at 
the 6-month visit as a measure of improvement for the key domains of impairment 
associated with ASD. There will be three separate scales of change completed:  social 
communicative functioning, restricted/repetitive interests and behaviors, and overall 
improvement. The rater places a vertical line on the scale indicating how much he/she 
feels the participant has improved or worsened compared to the baseline visit, with the 
center of the scale indicating “No Meaningful Changes.”  When data is entered, the 
distance to the line and total length of the bar will be measured.  In contrast to the CGI-
S and I, it is not specifically anchored to pre-specified levels of abnormal behaviors, 
functional abilities or level of supports, or pre-specified "amounts of change." It is a 
continuous measure based on the individual clinician's judgment.  The rater should base 
his or her judgment on the amount of change observed during various assessments and on 
the CGI and Visual Scales Parent Interview. Completion of the Visual Scale takes less 
than 5 minutes and is completed at the 6-month visit.  
 
CGI and Visual Scales Parent Interview: The CGI and Visual Scales Parent Interview is 
completed by a clinician and conducted with the primary caregiver. The interview focuses 
on the participant’s social communication abilities and challenges, restricted interests, 
repetitive behaviors, and overall functioning. The clinician will obtain details about 
frequency and quality of behaviors in different contexts, such as at home, in school, and in 
the community, as well as details about the level of support the child requires to function 
in each setting. The interview will be completed at the baseline visit and updated at the 6-
month visit. The interview will take around 30 minutes to complete at the baseline visit 
and will take around 15 minutes to complete at the 6-month visit.  
 
Differential Ability Scales, Second Edition (DAS-II):39 The DAS-II is an assessment 
administered by a trained clinician to observe behavior and compute a score to assess 
cognitive abilities. This test is appropriate for ages 2 years, 6 months through 17 years, 11 
months and will be used for participants aged 4 years, 0 months and older at baseline. 
This assessment takes about 45 minutes and will be conducted at the baseline visit only.  
 
Mullen Scales of Early Learning, AGS Edition:40 The Mullen Scales of Early Learning is 
a cognitive functioning assessment specifically designed for very young children and 
preschoolers, from birth to 68 months.  It measures five scales of cognitive functioning: 
Gross Motor, Visual Reception, Fine Motor, Expressive Language, and Receptive 
Language (note: for DukeACT, the Gross Motor subscale will not be 
administered).  Each scale is tested individually, and the exam is not timed.  Some of the 
questions may require parental input in order to assess the child’s ability.  The exam is 
interactive and includes toys and manipulatives for the child to engage with during the 
exam. Test administration time varies from approximately 15 minutes for a one-year-old 
to an hour for 5-year-olds. The Mullen will be used with participants under 4 years, 0 
months at the baseline visit. This assessment takes about 60 minutes and will be 
conducted at the baseline visit only.  
 

 
March 15, 2018 
38
Parent-Child Interaction with Noldus EthoVision: During the Parent-Child Interaction 
Task, Noldus EthoVision will be used. The purpose of the video tracking is to determine 
whether automated tracking of children’s movements related to social approach or 
avoidance can be reliability administered and provide a valid measure of social 
communication in children with ASD. This paradigm is designed to automatically track 
movements related to social approach and avoidance behavior of children with ASD with 
a familiar adult. The primary dependent variable is the time spent in the periphery of a 
room versus near the adult.  Children will be observed during two contiguous sessions 
conducted in the same room comprised of (1) a six-minute free-play session with toys 
available during which the caregiver will be silently reading a magazine in the corner on 
the room and (2) a six-minute parent-child interaction, where the parent joins the child 
for interactive play to see how the child plays with the adult and vice versa.  Behavior 
will be recorded from a ceiling-mounted camera and software will be used to 
automatically track the child’s movements.  Dependent variables include percentage of 
session spent in the caregiver region of interest (ROI), latency to approach the caregiver 
ROI, and percentage of time spent in the periphery ROI. The Parent-Child Interaction 
Task will be conducted at baseline and 6 month visits.   
 
Expressive One-Word Picture Vocabulary Test, Fourth Edition:41 This is a child 
observation by a trained clinician that tests an individual’s ability to match a spoken word 
with an image of an object, action, or concept. It tests an individual’s ability to name, 
with one word, objects, actions, and concepts when presented with color illustrations. 
This test is appropriate for ages 2-80 years and takes about 10-30 minutes to complete; 
completion time is determined by an individual’s verbal ability. This assessment will be 
conducted at baseline and 6 month visits. 
 
Vineland Adaptive Behavior Scales (VABS) - 3 Survey Interview Form:42 This will be 
used to assess adaptive behavior. The assessment is administered to the parent/caregiver 
using a semi-structured interview format. This assessment can be used with ages 0-90 
years of age and takes about 60 minutes to administer.  The VABS-3 is a well-
standardized measure of adaptive functioning, assessing adaptive behavior in 
communication, daily living, socialization, motor, and maladaptive behaviors. Norms are 
available from birth to 90 years.  The Socialization subdomain assesses play and 
interpersonal relationships.  This assessment will be conducted at baseline and 6 month 
visits and will also be conducted remotely at 12 months post-baseline. 
 
7.4.3 Parent Caregiver Questionnaires: 
These questionnaires will be completed during baseline and 6 month visits at Duke and 
will be sent to the parent/guardian to complete remotely throughout the study. All parent 
questionnaires will be completed online through the survey tool Qualtrics. The Qualtrics 
survey tool is available for Duke users through a university-wide site license. Qualtrics is 
integrated with Duke’s NetID authentication system but allows sharing of surveys with 
non-Duke users.  Parents/guardians will access the Qualtrics links through a secure, 
personalized parent portal built by RTI International.  The parent portal will be password 
protected. 
 

 
March 15, 2018 
39
Pervasive Developmental Disorder Behavior Inventory (PDD-BI):43 The PDD-BI was 
developed to assess responsiveness to intervention in children with ASD. The PDD-BI is 
an informant-based rating scale that is designed for children 1 year, 6 mos. to 12 years, 5 
mos. It assesses problem behaviors as well as appropriate social, language, and 
learning/memory skills. The PDD-BI 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 PDD-BI renders raw scores 
as well as t-scores based on comparisons to a standardized ASD population.  The PDD-
BI has been validated in a PDD-BI 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 PDD-BI will be completed at baseline and 
6-month visits and will also be completed remotely at 3, 9, and 12 months post-baseline. 
 
Aberrant Behavior Checklist-Community (ABC-C):44 This parent-completed rating scale 
will be used to measure aberrant behaviors associated with ASD, with an emphasis on 
social withdrawal. The ABC-C is a validated scale that can assess drug and other 
treatment effects in studies with developmentally-disabled individuals. Separate factor 
analyses of data from samples of institutionalized participants (n = 418 [mean age 29.5 
yrs.] in Stage 1 and n = 509 [mean age 25.9 yrs.] in Stage 2) resulted in a 5-factor scale 
comprising 58 items. The factors were labeled as (1) Irritability, Agitation, Crying; (2) 
Social Withdrawal; (3) Stereotypic Behavior; (4) Hyperactivity, Noncompliance; and (5) 
Inappropriate Speech. The ABC-SQW has been used in other clinical trials focusing on 
the core social and communication symptoms of autism.  The ABC-C has 58 items, and 
each item is rated as 0= not at all a problem, 1= the behavior is a problem, but slight in 
degree, 2= the problem is moderately serious, or 3= the problem is severe in degree. This 
parent-completed rating scale takes approximately 10 to 15 minutes to complete. The 
ABC-C will be completed at baseline and 6 month visits and will be completed remotely 
at 3, 9, and 12 months post-baseline. 
 
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 
on a monthly basis. 
 
Behavior Rating Inventory of Executive Function-Preschool Version (BRIEF-P):45 This 
assessment is a questionnaire for parents of preschool-aged children that enables 
professionals to assess executive function behaviors in the home and preschool 
environments. It is designed for a board range of preschool children including those with 
emergent learning disabilities and attentional disorders, language disorders, traumatic 
brain injuries, lead exposure, pervasive developmental disorders, and other 
developmental neurological, psychiatric, and medical conditions. The BRIEF-P contains 
63 items and takes about 15 minutes to complete. The BRIEF-P will be used with 
participants under the age of 5 years at baseline.  This questionnaire will be completed at 
baseline and 6 month visits and remotely 12 months post-baseline. 

 
March 15, 2018 
40
 
Behavior Rating Inventory of Executive Function (BRIEF):46 This assessment is a 
questionnaire for parents of school age children that enables professionals to assess 
executive function behaviors in the home and school environments. The parent form of 
the BRIEF contains 86 items within eight theoretically and empirically derived clinical 
scales that measure different aspects of executive functioning: Inhibit, Shift, Emotional 
Control, Initiate, Working Memory, Plan/Organize, Organization of Materials, and 
Monitor. The BRIEF takes about 15 minutes to complete and will be used with 
participants aged 5 years and older at baseline. This questionnaire will be completed at 
baseline and 6 month visits and remotely 12 months post-baseline.  
 
Sensory Experiences Questionnaire, Version 2.1 (SEQ):47,48 The SEQ 2.1 asks parents to 
respond, on a 5-point Likert scale, to 45 questions about the frequency of their children’s 
responses to sensory stimuli in the context of daily activities and routines. This study will 
use a modified version that only asks parents to complete the Likert scale questions and 
omits all open-ended questions. The SEQ 2.1 has been validated for children with autism 
ages 2 -12 years, been shown to discriminate children with ASD from developmental 
delay and typically developing controls, and has a high internal consistency (α = 0.80).49 
Summary scores will be derived for hyper-responsiveness (SOR). The SEQ 2.1 takes 
around 10 minutes to complete and will only be completed at the baseline visit.  
 
Early Life Exposures Assessment Tool (ELEAT): This is a parent questionnaire that 
assesses for a multitude of environmental exposures that the parents/child may have been 
exposed to in the child’s early life and development. The questionnaire asks a variety of 
questions about life before pregnancy, during pregnancy, and during the child’s first year 
of life, and gives a very detailed, quantitative look at any possible food or chemical 
exposures that may have been harmful. A modified and shortened version of the ELEAT 
which only includes relevant variables will be used for the study.  This assessment takes 
about 10 minutes to complete and will only be completed at the baseline visit.  
 
Pediatric Quality of Life Inventory (PedsQL) Gastrointestinal Symptoms Scales:50 The 
PedsQL Gastrointestinal Symptoms Scales is a 10 minute parent questionnaire that 
measures gastrointestinal symptoms in children as over 50% of children with ASD suffer 
from gastrointestinal discomfort. This assessment will be completed at baseline and 6 
month visits and remotely 12 months post-baseline.  
 
Demographic information will be obtained at the baseline visit for each participant using 
the Duke Center for Autism and Brain Development Demographics Form.  Interval 
medical and social histories will be obtained at the six-month visit in person and with the 
aid of interval history questionnaires. 
 
7.4.4 Other Assessments:  
The Visit Preparation Inventory is a brief questionnaire that is completed with the parent 
prior to the child’s visit. Information about the child’s preferences and dislikes is assessed 
on this form that is then utilized to prepare reinforcement techniques for the child’s visit. 
In addition, sensory sensitivities related to the completion of EEG or eye tracking are 

 
March 15, 2018 
41
assessed in order to best prepare for the child’s EEG sessions.  This questionnaire is 
completed by the clinical research coordinator during a phone call with the caregivers 
one to two weeks before baseline and 6 month visits. 
 
  
7.5 
Neurophysiology and Neuroimaging 
7.5.1 Neurophysiology:   
Electroencephalography (EEG):  EEG is a non-invasive measure of brain activity. A 
flexible dense array of electrodes (EGI System 400, Electrical Geodesics Inc., Eugene, 
Oregon) is placed over the head and secured with a chin strap. EEG signals are amplified 
and sent to recording computer for on-line viewing.  EEG is used here to investigate 
patterns of brain activity elicited by social and non-social stimuli. The EEG takes around 
20 minutes to complete and will be completed at baseline and 6 month visits.  
 
EEG will be used to assess changes in cortical activation (as reflected by changes in 
alpha, theta, and beta rhythms) and functional connectivity (as reflected by changes in 
EEG coherence) during a baseline condition and while viewing social and non-social 
stimuli.  Alpha oscillations have been shown to emerge from activations of the thalamo-
cortical network and have also been demonstrated to be present in subcortical areas, 
including the hippocampal region.  Alpha frequencies result from a reciprocal interplay 
between excitatory and inhibitory neurons and are influenced by cholinergic, 
serotonergic, and glutamaterigic mechanisms.  Alpha activity increases during a relaxed 
state and decreases during active stimulus processing.  Theta oscillations are especially 
prominent in the hippocampal region, are influenced by the interaction between 
glutamatergic and gamma-animobutyric acidergic (GABAergic) neurons, and may 
correlate with synaptic plasticity.    
 
EEG will be recorded during a resting baseline state and while viewing standardized 
videotaped segments of social (female singing a nursery rhyme) and nonsocial (activated 
toys) stimuli.  In a study of preschool aged children with ASD, alpha and theta EEG 
power during viewing of social and nonsocial stimuli have been shown to change as a 
function of behavioral treatment and are specifically correlated with improvements in 
social behavior.51   EEG data will be recorded from 128-channel dense electrode array 
Geodesic sensor nets (recorded online with reference to the vertex) at 500 Hz, high-pass 
filtered at 0.1 Hz and low-pass filtered at 200 Hz. EEG will be edited both through 
automatic artifact-detection software (Net Station 4.4) and hand-editing without 
knowledge of group membership.  
 
Spectral analyses. EEG data will be Fourier-transformed using Welch's method 
(implemented in Matlab, R2012b).  Power estimates will be averaged across electrode 
groups (right posterior and anterior, left posterior and anterior, midline occipital 
electrodes) and natural log-transformed to reduce skew. Planned analyses contrasted 
power in the theta (5-7 Hz) and alpha (9-11 Hz) bands during the presentation of face 
versus object stimuli, allowing the analyses to account for individual differences in 
absolute power.  Alpha and theta power will be analyzed using repeated measures 
ANOVAs, with stimulus (face, object) and region as within-participant variables.  

 
March 15, 2018 
42
 
Eye Tracking of Social Stimuli (EGT): The EGT is a 15 minute technical assessment 
using specialized equipment in a controlled setting to track visual attention via eye 
movements of children with autism.  Children with ASD glean useful social information 
from different parts of the observed face as compared to a control group of children. This 
assessment is appropriate for children of 1 year to adult. The children will be shown 
videotapes of scenes involving social and nonsocial stimuli.  The depending variables 
include how much time the child spends looking at the social versus non-social stimuli in 
the videotapes, including the specific aspects of the social stimuli that attract and hold the 
child’s attention. The EGT will be conducted at baseline and 6 month visits.     
 
7.5.2  Neuroimaging:  
MRI will be used to assess potential structural/anatomic modifications, and functional 
changes, in the brain before and after cell therapy. MRI images will also be used to 
correlate brain iron with ASD symptoms.  Changes in MRI images may reflect alterations 
in astrocyte activity, blood perfusion, fiber tract integrity, brain network integrity, myelin 
microstructural integrity and brain functional connectivity.  The following imaging will 
be utilized: 
 
 High-Resolution T1 Imaging, 3D IR-prepped FSPGR, 1x1x1 mm resolution, 
estimated imaging time 3 min. 
 High-resolution perfusion imaging, 3D spiral with arterial spin labeling, target 
resolution 1.5x1.5x1.5 mm (will evaluate 1x1x1 provided SNR is sufficient), 
estimated imaging time 3 min. 
 High-resolution diffusion tensor imaging, 2D multi-shot DW EPI base 
sequence, 25 diffusion encoding directions, b factor 800 s/mm2, target 
resolution 1.5.x1.5x1.5 mm  (will evaluate 1x1x1 provided SNR is sufficient), 
estimated imaging time 10 min. 
 High-resolution fcMRI, 2D single- or two-shot EPI base sequence, target 
resolution 1.5x1.5x1.5 mm (will evaluate 1x1x1 provided SNR is sufficient), 
estimated imaging time 10 min. (5 min. per run, two runs). 
 High-resolution quantitative susceptibility imaging, multi-echo 3D FSPGR 
based acquisition, target resolution 1x1x1 mm, estimated imaging time 5 min. 
7.6 
Biological Samples 
The following samples will be obtained for future biomarker studies (see Appendix 2 for 
additional details): 
 Blood for immunological, DNA, and RNA studies 
 Blood for generation of induced pluripotent stem cells (IPSCs) 
 Urine 
 Saliva 
 Parental blood samples 
7.7 
Study Product Infusion 
On the day of infusion, CB cells or placebo product will be prepared by the STCL and 
provided for infusion of the patient in the outpatient clinic under the supervision of the 

 
March 15, 2018 
43
study team and Pediatric Blood and Marrow Transplant Program staff.  A peripheral IV 
will be placed by clinical staff, anesthesia or a member of the study team.  Prior to the 
study infusion, premedications (Benadryl and Solumedrol) will be administered.  The 
infusion will be given over approximately 2-30 minutes using standard practices.  The 
child will receive 1-1.5x maintenance IV fluids as described below and be observed in 
the clinic for a minimum of 1 hour after the infusion.  Patients will be discharged from 
clinic after at least 1 hour providing all vital signs are at their baseline and they are awake 
and asymptomatic with no evidence of toxicity.  Patients 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 10 days to 2 weeks after the infusion. 
 
Maintenance IV Fluid Rate (Holliday-Segar Method from Harriet Lane Handbook) 
Body weight:  
mL/kg per day 
1st 10 kg 
 
100 
 
divided by 24hr/day 
2nd 10 kg 
 
 50  
 
divided by 24 hr/day 
each add'l kg  
 20 
 
divided by 24 hr/day 
 
If a patient 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. 
 
7.8 
Care During Unexpected Events 
In the event that a patient develops signs or symptoms of anaphylaxis including urticaria, 
difficulty breathing, cough, wheezing, or vomiting during their CB infusion, the infusion 
will be terminated and appropriate medical therapy initiated.  
 
 

 
March 15, 2018 
44
7.9 
Required Evaluations 
 
7.9.1  Medical and Safety Assessments: 
 
Time Points# 
Screening 
Baseline 
(Visit 1) 
2 weeks 
post-
infusion 
3 mo. 
6 mo. 
(visit 2) 
2 weeks 
post-
infusion 
9 
mo. 
12 
mo.& 
CBCD*, CMP*, Type & Screen*, patient HLA 
X 
 
 
 
 
 
 
 
CBU potency; HLA confirmatory typing 
X 
 
 
 
 
 
 
 
Review of prior records ± videos 
X 
 
 
 
 
 
 
 
History & Physical 
 
X 
 
 
X 
 
 
 
Donor Referral Panel 
 
X 
 
 
X 
 
 
 
Patient DNA sample for chimerism 
 
X 
 
 
 
 
 
 
Donor DNA sample for chimerism+ 
 
X+ 
 
 
X+ 
 
 
 
CBCD*, CMP*, Type & Screen*, Direct Coombs, 
HLA Antibody Screen (PRA), ESR, CRP, 
Ferritin, Immune Reconstitution Panel, Humoral 
Immune Profile 
 
X 
 
 
X 
 
 
X 
In-person neuropsychological evaluation 
 
X 
 
 
X 
 
 
 
Remote psychological evaluation 
 
 
 
X 
 
 
X 
X 
Brain MRI 
 
X 
 
 
X 
 
 
 
Peripheral blood chimerism 
(donor CB recipients only) 
 
 
 
 
X 
 
 
X 
CB infusion (TNCC, viability, CD34+ cells, CFU, 
sterility cultures) 
 
X 
 
 
X 
 
 
 
Safety Assessment – in-person evaluation 
 
X 
(Day 
after 
infusion) 
 
 
X 
(Day 
after 
infusion) 
 
 
 
Safety Assessment – phone call/survey 
 
 
X 
X 
X 
X 
X 
X 
* CBCD, CMP, and Type & Screen may be obtained at initial visit or within 6 months prior to enrollment 
&12 month laboratory evaluations may be obtained remotely or mailed to Duke 
+Donor DNA will be obtained at the time of CB thaw 
#Safety and return assessments should be performed within a month of the indicated time point. 
 
 
 
 

 
March 15, 2018 
45
7.9.2  Diagnostic, Behavioral, Neurocognitive, and Neurophysiological Evaluations: 
 
 
Measure 
Length  
Time Points# 
Baseline 
(visit) 
3 mo. 
(remote) 
6 mo. 
(visit) 
9  mo. 
(remote) 
12 mo. 
(remote) 
Pre-Visit 
Clinician 
Assessment 
w/Parent 
Autism Diagnostic Interview, Revised, 
Shortened Version  (ADI-R) 
90-150 minutes 
X  
(pre-
visit) 
 
 
 
 
Clinician 
Assessment 
w/Child  
Autism Diagnostic Observation, Second 
Edition (ADOS-2) 
 
45-60 minutes 
X 
 
X 
 
 
 
Mullen Scales of Early Learning, AGS 
Edition  
60 minutes 
X 
 
 
 
 
 
Differential Ability Scales, Second Edition 
(DAS-II) 
45 minutes 
X 
 
 
 
 
 
Parent-Child Interaction (proximity seeking) 
 
12 minutes 
X 
 
X 
 
 
 
Expressive One-Word Picture Vocabulary 
Test, Fourth Edition (EOWPVT) 
10-30 minutes 
X 
 
X 
 
 
Physiological and 
Functional 
Assessments 
w/Child  
Electrophysiological Response to Social 
Stimuli (EEG) 
20 minutes 
X 
 
X 
 
 
 
Eye Gaze Tracking of Social Stimuli (EGT) 
10-15 minutes 
X 
 
X 
 
 
Clinician 
Assessments 
w/Parent 
Vineland Adaptive Behavior Scales, Third 
Edition, Survey Interview Form 
60 minutes 
X 
 
X 
 
X 
Parent 
Questionnaires 
Pervasive Developmental Disorder-Behavior 
Inventory (PDD-BI) 
30 minutes 
X 
X 
X 
X 
X 
 
Intervention History* 
15 minutes 
X 
X 
X 
X 
X 
 
The Early Life Exposures Assessment Tool 
(ELEAT) 
10 minutes 
X 
 
 
 
 
 
Aberrant Behavior Checklist-Community 
10 minutes 
X 
X 
X 
X 
X 
 
Sensory Experiences Questionnaire 2.1 
10 minutes 
X 
 
 
 
 
 
Brief Rating Inventory of Executive 
Functioning or Brief Rating Inventory of 
Executive Function-Preschool 
15 minutes 
X 
 
X 
 
X 
 
PedsQL-Gastrointestinal Symptoms 
Inventory 
10 minutes 
X 
 
X 
 
X 
Other Clinician 
Assessments 
Diagnostic Statistical Manual 5-Checklist 
(DSM-5) 
3-5 minutes 
X 
 
X 
 
 
 
Clinical Global Impression-Severity and 
Improvement and Visual Scales 
15 minutes 
X 
 
X 
 
 
*Intervention history will be collected on a monthly basis. 
#Assessments should be performed within a month of the indicated time point. 
 
8.0 
STATISTICAL CONSIDERATIONS 
This study will enroll autistic children age 2 to 7 years of age. Participants will be 
randomized in a 2:1 allocation to cord blood or placebo with stratification by important 
prognostic factors (see below for randomization plan). The primary objective of the study 
will be fulfilled by comparing the mean of the 6-month change in VABS-3 socialization 
domain standard score between cord blood and placebo arms. The study also includes 
pre-planned subgroup analyses comparing the same outcome between treated patients 
receiving autologous or allogeneic cord blood vs. placebo, and between patients receiving 

 
March 15, 2018 
46
autologous and allogeneic cord blood. The approach to sample size estimation was based 
on the primary comparison of cord blood to placebo. Power for the pre-planned subgroup 
analyses is described using the sample size planned for the primary comparison. 
Note: All children in the placebo arm will receive cord blood after reaching their six-
month evaluation. This serves simultaneously as a recruitment tool and to inform 
exploratory analyses. However, sample size and power calculations are based only on the 
planned comparisons between patients assigned cord blood vs. placebo at six months.  
8.1 
Study Design 
This is a single site, prospective, randomized, double-blind, parallel group study of a 
single intravenous autologous or allogeneic, unrelated CB infusion in children ages 2-7 
years with ASD.  Participants will be randomly assigned to Sequence A, consisting of a 
single infusion of CB cells at baseline followed 6 months later by a single infusion of 
placebo, or Sequence B, consisting of an infusion of placebo at baseline followed 6 
months later by an infusion of CB cells.  All participants will be treated with CB cells; 
participants with an available qualified autologous CB unit will receive autologous cells, 
and those without suitable autologous CB available will receive ≥4/6 HLA-matched, 
allogeneic, unrelated donor CB cells from the Carolinas Cord Blood Bank.  All infusions 
will be double-blinded.  The primary outcomes will be assessed 6 months after the initial 
infusion in the sequence.  Duration of study participation will be 12 months from the time 
of baseline infusion. 
8.2 
Accrual 
It is estimated that up to 4 research participants will be enrolled each week and that 
approximately 15 months of accrual will be necessary to enroll 190 participants.   
8.3 
Study Duration 
Research participants will be followed for safety for 6 months after the second study 
infusion. 
8.4 
Demographics and Baseline Characteristics 
Demographics and baseline characteristics will be summarized for all research 
participants.  Characteristics to be examined include age, sex, race/ethnicity, and baseline 
behavioral status. 
8.5 
Description of the Primary Outcome Measure 
The primary objective of the study is to determine, in a randomized, placebo controlled, 
best available donor source trial, the efficacy of a single intravenous infusion of umbilical 
CB in improving the core symptoms of children with ASD. Because there is no single 
best outcome measure recommended for use in interventional clinical trials of autism,52 a 
pilot study (the DukeABC trial) was conducted that enrolled 25 children with ASD and 
evaluated the utility of several measures for documenting change in ASD symptoms over 
a short time period post-treatment with cord blood. In the DukeABC trial, clinically and 
statistically significant change was observed over a 6-month period on several outcome 
measures that quantify ASD core symptoms, including the VABS-II, PDDBI, and CGI.  
The VABS-3 socialization domain standard score was selected as the primary outcome 
measure in this trial as it assesses a core autism symptom, social communication that is 

 
March 15, 2018 
47
hypothesized to be affected by the therapy under study. The PDD-BI and CGI are 
included in this study as secondary outcomes. 
8.6 
Description of the VABS-3 Socialization Domain Standard Score 
The VABS-3 will be administered to the participant’s parents by interview in this study. 
The VABS-3 measures adaptive behavior in several domains including socialization, 
which is the domain used as the primary outcome measure in this trial. The socialization 
domain is a summary of measures in three subdomains: interpersonal relationships, play 
and leisure time, and coping skills. Raw scores within these subdomains are standardized 
to V-scale scores for each domain (mean=15, SD=3). The sum of the subdomain V-scale 
scores are then standardized to a normal distribution with mean 100 and standard 
deviation 15, thus resulting in the socialization standard score. Our study will use the 
difference between this standard score measured at baseline and 6-months post-treatment. 
Positive change over a six-month period indicates gains in socialization behaviors. The 
median change in socialization standard score over a six-month period (n=24) in the 
DukeABC trial was 2 points (range: -8 to 30) and this was significantly different from 
zero (P=0.02, Wilcoxon signed rank test). Children with non-verbal IQ less than 55 (n=9) 
had a median change of -1 point (range: -8 to 14) whereas children with non-verbal IQ 
>=55 (n=15) had a median change of 6 points (range: -6 to 30) (P=0.02, Wilcoxon rank 
sum test). No association was observed between the change score and sex (P=.07, 
Wilcoxon rank sum test, n=4 females) or baseline age (Spearman r = -0.18, P=0.41).  
8.7 
Sample Size and Power Calculations 
We estimated the sample size required for 80% power to detect various standardized 
effect sizes comparing patients randomized to cord blood vs. placebo. The Type I error 
rate was fixed at 2.5% for this comparison to account for the additional pre-planned 
comparisons described in later sections.  
 
The null and alternative hypotheses for the primary comparison are:  
 
𝐻0: 𝜇𝐶𝐵−𝜇𝑃= 0 
𝐻𝐴: 𝜇𝐶𝐵−𝜇𝑃≠0 
 
Where 𝜇𝐶𝐵 is the population mean 6-month change in VABS II socialization standard 
score in autistic children treated with cord blood and 𝜇𝑃is the analogous mean in autistic 
children treated with placebo. 
 
Sample size was estimated assuming a two-sample, equal variance t-test. Calculations 
were done using PASS 12.0 using non-central t-distributions. The table below shows the 
sample sizes required for 80% to detect various standardized effect sizes assuming 
alpha=0.025 and 2:1 allocation to cord blood or placebo. 
 
 
 
 
 
 

 
March 15, 2018 
48
Actual Power 
NPlacebo 
NCord Blood 
NTotal 
Cohen’s d 
0.80055 
357 
714 
1,071 
0.20 
0.80145 
229 
458 
687 
0.25 
0.80201 
160 
320 
480 
0.30 
0.80284 
118 
236 
354 
0.35 
0.80023 
90 
180 
270 
0.40 
0.80454 
72 
144 
216 
0.45 
0.80094 
58 
116 
174 
0.50 
0.80018 
48 
96 
144 
0.55 
0.80588 
41 
82 
123 
0.60 
0.80510 
35 
70 
105 
0.65 
0.80073 
30 
60 
90 
0.70 
0.81320 
27 
54 
81 
0.75 
0.81617 
24 
48 
72 
0.80 
0.80871 
21 
42 
63 
0.85 
0.81275 
19 
38 
57 
0.90 
0.80900 
17 
34 
51 
0.95 
0.82502 
16 
32 
48 
1.00 
 
 
A total sample size of 144 (96 on cord blood and 48 on placebo) will provide 80% power 
to detect a moderate (d=0.55) sized treatment effect given the stated design parameters. 
Expansion of the sample size by ~15% to allow for dropout, and to facilitate  piloting the 
study procedures with the first 2-3 enrolled subjects yields an approximate sample size of 
165 participants (110 on cord blood and 55 on placebo). Therefore, with a sample size 
of 165 participants allocated 2:1 to cord blood: placebo, the trial will have greater 
than 80% power to detect a moderate sized treatment effect with strict control of 
Type I error. However, in recognition of the potential need to modify the sample size to 
maintain power for the planned alternative hypothesis (see Section 8.13, Interim 
Analysis) we set an anticipated maximum of 190 participants. 
 
8.8 
Power for Subgroup Analyses 
In this study it is of interest to compare treatment effects by best available cell source. 
However, the actual power available for the requisite subgroup analyses is dependent 
upon the size of the groups, which cannot be known a priori. Therefore, we adopted a 
descriptive approach to power analysis for these comparisons under what we believe to 
be likely accrual eventualities, and using a simplified analytical model.  
 
To explore power and sample size we selected a 2-way ANOVA framework where the 
main effects were treatment (cord blood or placebo) and cell source (autologous or 
allogeneic). Within this model the investigation of cell source is formulated as the F-test 
of interaction in the 2-way ANOVA, which compares the treatment effect by cell source. 
This test uses the Bonferroni-corrected alpha specified in the primary analysis (0.025). A 
primary assumption of this model is that group sizes are equal. Given the design of our 
trial this is unlikely to occur. Nonetheless, illustration of detectable effect sizes with 
balanced deigns can be instructive as they provide the “best case scenario.” Given a fixed 

 
March 15, 2018 
49
power, sample size, and Type I error the actual detectable effect size will be larger in 
unbalanced designs than balanced designs. Therefore, we estimated the detectable effect 
size under different sized balanced designs to describe a possible range of detectable 
effect sizes in our trial. 
 
We began by calculating power assuming a balanced design with an average of 41.25 
patients per group (the planned accrual of 165 patients divided among four groups 
defined by cord blood/placebo and autologous/allogeneic) and a common main effect and 
interaction effect size. Using PASS version 12.0 we estimated that with this sample size 
and the aforementioned design parameters our study would have approximately 80% 
power to detect a standardized effect size of 0.25 (considered a medium ANOVA effect 
size by Cohen) for the interaction test. Using an average sample size that is equal to the 
smallest expected group sample size in our trial (n=27.5, assuming the placebo group is 
evenly divided by cell source) the detectable effect size is 0.33, which is closer to the 
large (0.4) effect size defined by Cohen.  
 
Therefore, we can be confident that our trial will be able to detect moderate to large 
differences in the treatment effect by cell source with strict control of Type I error using 
an unbalanced 2-way ANOVA. 
8.9 
Correction for Multiple Testing 
Our sample size plan incorporates the Bonferroni adjustment for control of Type I error 
across the pre-planned statistical tests involving the primary endpoint; i.e., the test of cord 
blood vs. placebo and the test of interaction that evaluates treatment effect by cell source.  
 
Secondary endpoints will be tested assuming a per-comparison alpha of 0.05. The False 
Discovery Rate (FDR) procedure will then be applied to determine which results are 
significant after consideration for multiple testing. The primary endpoint comparison of 
cord blood vs. placebo will be included in the FDR procedure. 
 
Further details are available in the Statistical Analysis Plan (SAP). 
8.10 
Randomization Plan 
The study will randomize participants to cord blood or placebo with stratification by age 
(< 5 vs. >=5 years), non-verbal IQ (<55 vs. >=55), and best available cell source 
(autologous vs. allogeneic) and will utilize a randomly varying block size with 2:1 
allocation to cord blood and placebo. The DukeABC trial showed that IQ was a likely 
confounder of the change in Vineland socialization standard score over a 6-month period. 
Age is also a likely confounder as children’s developmental rate varies with age. The 
availability of autologous or allogeneic cord blood prior to randomization is a potential 
confounder as it might correlate with socioeconomic status, and this may influence the 
use of therapy outside the trial. Although there is a dearth of efficacious therapies for 
improvement of the core symptoms of autism, the use of therapy outside the trial may 
result in a tendency of parents to over-report positive outcomes on the Vineland-3 or the 
PDD-BI. Thus, by stratifying our randomization on available cell source we will preserve 
the validity of our comparison of the treated and cord blood groups.  

 
March 15, 2018 
50
8.11 
Analysis Plan 
Full details of the planned analysis are available in the accompanying Statistical Analysis 
Plan (SAP). Briefly, safety analyses will be primarily descriptive and will rely on 
graphical displays and tabulated descriptive statistics. The primary efficacy analysis 
comparing cord blood to placebo is based on a two-sample t-test of the mean difference 
(from baseline to 6 months) in the VABS 3 socialization domain standard score 
comparing the cord blood and placebo arms. Study procedures will be piloted in the first 
2-3 enrolled subjects and therefore these subjects may not be included in the primary 
analysis. Additional analyses are described in the SAP. 
 
Analysis Populations: 
Full Analysis Population 
This population constitutes all enrolled participants and is used for descriptive purposes; 
e.g. to identify subjects who are not evaluable for the primary efficacy analysis. 
  
Modified Intention to Treat (mITT) Population 
This analysis set will include only subjects who complete all of the baseline and follow-
up VABS-3 socialization domain standard score.  Subjects who are missing one or both 
of these evaluations cannot contribute to the primary efficacy analysis because the change 
score on the VABS-3 socialization domain is not observable.  Subjects will be analyzed 
according to the treatment they were assigned (regardless of what they actually receive).  
  
Safety Population 
The safety population will include all subjects who received at least 1 infusion. Analyses 
of the Safety Population will be conducted using an as-treated approach, which considers 
each patient according the treatment actually received rather than the treatment they were 
assigned. 
  
Each subject’s status with regard to each analysis population will be determined prior to 
breaking the blind.  
8.12 
Loss to Follow-up and Discontinuation 
Patients who are lost to follow-up or are discontinued from the study will not be replaced, 
and additional participants will not be randomized. 
8.13 Interim Analysis 
A blinded re-estimation of sample size will be conducted when the primary endpoint is 
evaluable on approximately 75% of the participants. 
 
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.   
 

 
March 15, 2018 
51
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 patient or 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 v4.0 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 AEs reported or observed during the study beginning at the time of the study infusion 
must be recorded.  Information to be reported includes 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, any treatment or evaluations, and 
outcome.  In general, investigators should report AEs as diseases or syndromes whenever 
possible, instead of reporting individual component symptoms, signs, laboratory 
abnormalities, and sequelae. 
SAEs (fatal, life-threatening or requiring hospitalization) will be reported within 7 
calendar days of receipt of the information.  All fatal or life threatening SAEs will be 
reported by the investigator/representative to the FDA by phone or fax within 7 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. 
9.3 
Stopping Guidelines 
Stopping guidelines based on safety will be monitored by the CRO and will 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 reaction within 48 hours of 
infusion;  

 
March 15, 2018 
52
OR 
 Two or more grade 4-5 adverse events determined to be temporally related 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. 
9.4 
Participant Replacement 
 
The sponsor may replace any participant who has not been dosed.   
9.5 
Emergency Unblinding 
All participants will be treated with CB infusion, but the time course will vary between 
groups and participants will be blinded to the order in which they receive CB and placebo 
infusions.  If an SAE occurs and is related to either the study product, the participant and 
family will be unblinded to the infusion that they received.  Documentation of unblinding 
will be indicated on the CRF and on the SAE report if one is required.  
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 unexpected 
SAEs directly related to the study product throughout the study.  Interim reports of safety 
and tolerability information will be prepared and will be forwarded to the DSMB for 
review three months after 50 participants have received their initial infusion.  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 

 
March 15, 2018 
53
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.  The recordings will be used solely for analysis by 
the research team or for educational purposes if written consent is obtained from the 
parent/guardian.  They will be stored electronically on a password-protected server and 
identified by the participants’ study ID.   
11.3 
Inspection of Records 
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. 
11.4 
Study Record Retention 
Study results will be retained in the patient’s research record for six years after the study 
is completed or until the patient 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 Conference on 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.  
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. 

 
March 15, 2018 
54
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 
patients, 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 his/her legal guardian 
will be given a full explanation of the study and be allowed to read the approved 
informed consent form.  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, 

 
March 15, 2018 
55
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 nonadherence 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 nonadherence to FDA regulations and/or ICH GCP guidelines. 
Protocol violations and deviations will be documented by the clinical monitor throughout 
the course of monitoring visits.  Principal Investigators or Sub-Investigators will be 
notified of violations and/or deviations in writing by the monitor.  The IRB should be 
notified of all protocol violations and deviations 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. 
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 and/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. 

 
March 15, 2018 
56
13.0 
REFERENCES 
1. 
KING BH, NAVOT N, BERNIER R, WEBB SJ. UPDATE ON DIAGNOSTIC 
CLASSIFICATION IN AUTISM. CURR OPIN PSYCHIATRY. MAR 
2014;27(2):105-109. 
2. 
DEVELOPMENTAL DISABILITIES MONITORING NETWORK 
SURVEILLANCE YEAR PRINCIPAL I, CENTERS FOR DISEASE C, 
PREVENTION. PREVALENCE OF AUTISM SPECTRUM DISORDER AMONG 
CHILDREN AGED 8 YEARS - AUTISM AND DEVELOPMENTAL 
DISABILITIES MONITORING NETWORK, 11 SITES, UNITED STATES, 2010. 
MMWR SURVEILL SUMM. MAR 28 2014;63(2):1-21. 
3. 
BUESCHER AV, CIDAV Z, KNAPP M, MANDELL DS. COSTS OF AUTISM 
SPECTRUM DISORDERS IN THE UNITED KINGDOM AND THE UNITED 
STATES. JAMA PEDIATR. AUG 2014;168(8):721-728. 
4. 
HARDAN AY, FUNG LK, FRAZIER T, ET AL. A PROTON SPECTROSCOPY 
STUDY OF WHITE MATTER IN CHILDREN WITH AUTISM. PROG 
NEUROPSYCHOPHARMACOL BIOL PSYCHIATRY. APR 3 2016;66:48-53. 
5. 
TAKANO T. ROLE OF MICROGLIA IN AUTISM: RECENT ADVANCES. DEV 
NEUROSCI. 2015;37(3):195-202. 
6. 
ZANTOMIO D, CHANA G, LASKARIS L, ET AL. CONVERGENT EVIDENCE 
FOR MGLUR5 IN SYNAPTIC AND NEUROINFLAMMATORY PATHWAYS 
IMPLICATED IN ASD. NEUROSCI BIOBEHAV REV. MAY 2015;52:172-177. 
7. 
GOINES PE, ASHWOOD P. CYTOKINE DYSREGULATION IN AUTISM 
SPECTRUM DISORDERS (ASD): POSSIBLE ROLE OF THE ENVIRONMENT. 
NEUROTOXICOL TERATOL. MAR-APR 2013;36:67-81. 
8. 
BEAM D, POE MD, PROVENZALE JM, ET AL. OUTCOMES OF UNRELATED 
UMBILICAL CORD BLOOD TRANSPLANTATION FOR X-LINKED 
ADRENOLEUKODYSTROPHY. BIOL BLOOD MARROW TRANSPLANT. JUN 
2007;13(6):665-674. 
9. 
ESCOLAR ML, POE MD, PROVENZALE JM, ET AL. TRANSPLANTATION OF 
UMBILICAL-CORD BLOOD IN BABIES WITH INFANTILE KRABBE'S 
DISEASE. N ENGL J MED. MAY 19 2005;352(20):2069-2081. 

 
March 15, 2018 
57
10. 
PRASAD VK, MENDIZABAL A, PARIKH SH, ET AL. UNRELATED DONOR 
UMBILICAL CORD BLOOD TRANSPLANTATION FOR INHERITED 
METABOLIC DISORDERS IN 159 PEDIATRIC PATIENTS FROM A SINGLE 
CENTER: INFLUENCE OF CELLULAR COMPOSITION OF THE GRAFT ON 
TRANSPLANTATION OUTCOMES. BLOOD. OCT 1 2008;112(7):2979-2989. 
11. 
SUN J, ALLISON J, MCLAUGHLIN C, ET AL. DIFFERENCES IN QUALITY 
BETWEEN PRIVATELY AND PUBLICLY BANKED UMBILICAL CORD 
BLOOD UNITS: A PILOT STUDY OF AUTOLOGOUS CORD BLOOD 
INFUSION IN CHILDREN WITH ACQUIRED NEUROLOGIC DISORDERS. 
TRANSFUSION. SEP 2010;50(9):1980-1987. 
12. 
DROBYSHEVSKY A, COTTEN CM, SHI Z, ET AL. HUMAN UMBILICAL 
CORD BLOOD CELLS AMELIORATE MOTOR DEFICITS IN RABBITS IN A 
CEREBRAL PALSY MODEL. DEVELOPMENTAL NEUROSCIENCE. MAR 17 
2015. 
13. 
DERECKI NC, CRONK JC, LU Z, ET AL. WILD-TYPE MICROGLIA ARREST 
PATHOLOGY IN A MOUSE MODEL OF RETT SYNDROME. NATURE. APR 5 
2012;484(7392):105-109. 
14. 
SEGAL-GAVISH H, KARVAT G, BARAK N, ET AL. MESENCHYMAL STEM 
CELL TRANSPLANTATION PROMOTES NEUROGENESIS AND 
AMELIORATES AUTISM RELATED BEHAVIORS IN BTBR MICE. AUTISM 
RES. JAN 2016;9(1):17-32. 
15. 
CHUNG WS, WELSH CA, BARRES BA, STEVENS B. DO GLIA DRIVE 
SYNAPTIC AND COGNITIVE IMPAIRMENT IN DISEASE? NAT NEUROSCI. 
NOV 2015;18(11):1539-1545. 
16. 
NAYAK D, ROTH TL, MCGAVERN DB. MICROGLIA DEVELOPMENT AND 
FUNCTION. ANNU REV IMMUNOL. 2014;32:367-402. 
17. 
GEMMA C, BACHSTETTER AD, BICKFORD PC. NEURON-MICROGLIA 
DIALOGUE AND HIPPOCAMPAL NEUROGENESIS IN THE AGED BRAIN. 
AGING DIS. DEC 1 2010;1(3):232-244. 
18. 
SHETTY AK, HATTIANGADY B, SHETTY GA. STEM/PROGENITOR CELL 
PROLIFERATION FACTORS FGF-2, IGF-1, AND VEGF EXHIBIT EARLY 
DECLINE DURING THE COURSE OF AGING IN THE HIPPOCAMPUS: ROLE 
OF ASTROCYTES. GLIA. AUG 15 2005;51(3):173-186. 

 
March 15, 2018 
58
19. 
EKDAHL CT, CLAASEN JH, BONDE S, KOKAIA Z, LINDVALL O. 
INFLAMMATION IS DETRIMENTAL FOR NEUROGENESIS IN ADULT 
BRAIN. PROC NATL ACAD SCI U S A. NOV 11 2003;100(23):13632-13637. 
20. 
ASAHARA T, KAWAMOTO A. ENDOTHELIAL PROGENITOR CELLS FOR 
POSTNATAL VASCULOGENESIS. AM J PHYSIOL CELL PHYSIOL. SEP 
2004;287(3):C572-579. 
21. 
BACHSTETTER AD, PABON MM, COLE MJ, ET AL. PERIPHERAL 
INJECTION OF HUMAN UMBILICAL CORD BLOOD STIMULATES 
NEUROGENESIS IN THE AGED RAT BRAIN. BMC NEUROSCI. 2008;9:22. 
22. 
SHAHADUZZAMAN M, GOLDEN JE, GREEN S, ET AL. A SINGLE 
ADMINISTRATION OF HUMAN UMBILICAL CORD BLOOD T CELLS 
PRODUCES LONG-LASTING EFFECTS IN THE AGING HIPPOCAMPUS. AGE 
(DORDR). DEC 2013;35(6):2071-2087. 
23. 
LEE HJ, LEE JK, LEE H, ET AL. HUMAN UMBILICAL CORD BLOOD-
DERIVED MESENCHYMAL STEM CELLS IMPROVE NEUROPATHOLOGY 
AND COGNITIVE IMPAIRMENT IN AN ALZHEIMER'S DISEASE MOUSE 
MODEL THROUGH MODULATION OF NEUROINFLAMMATION. 
NEUROBIOL AGING. MAR 2012;33(3):588-602. 
24. 
KURTZBERG J, BUNTZ S, GENTRY T, ET AL. PRECLINICAL 
CHARACTERIZATION OF DUOC-01, A CELL THERAPY PRODUCT 
DERIVED FROM BANKED UMBILICAL CORD BLOOD FOR USE AS AN 
ADJUVANT TO UMBILICAL CORD BLOOD TRANSPLANTATION FOR 
TREATMENT OF INHERITED METABOLIC DISEASES. CYTOTHERAPY. JUN 
2015;17(6):803-815. 
25. 
SAHA A, BUNTZ S, SCOTLAND P, ET AL. A CORD BLOOD MONOCYTE-
DERIVED CELL THERAPY PRODUCT ACCELLERATES BRAIN 
REMYELINATION. JOURNAL OF CLINICAL INVESTIGATION. 2016. 
26. 
KURTZBERG J. IND 15949 MAY 20, 2016 2016. 
27. 
KURTZBERG J. IND 14360 JUNE 9, 2015 2015. 
28. 
COTTEN CM. IND 14753 SEPTEMBER 30, 2015 2015. 

 
March 15, 2018 
59
29. 
COTTEN CM, MURTHA AP, GOLDBERG RN, ET AL. FEASIBILITY OF 
AUTOLOGOUS CORD BLOOD CELLS FOR INFANTS WITH HYPOXIC-
ISCHEMIC ENCEPHALOPATHY. J PEDIATR. MAY 2014;164(5):973-979 E971. 
30. 
HANNA SE, BARTLETT DJ, RIVARD LM, RUSSELL DJ. REFERENCE 
CURVES FOR THE GROSS MOTOR FUNCTION MEASURE: PERCENTILES 
FOR CLINICAL DESCRIPTION AND TRACKING OVER TIME AMONG 
CHILDREN WITH CEREBRAL PALSY. PHYS THER. MAY 2008;88(5):596-607. 
31. 
ENGLANDER ZA, SUN J, LAURA C, MIKATI MA, KURTZBERG J, SONG AW. 
BRAIN STRUCTURAL CONNECTIVITY INCREASES CONCURRENT WITH 
FUNCTIONAL IMPROVEMENT: EVIDENCE FROM DIFFUSION TENSOR 
MRI IN CHILDREN WITH CEREBRAL PALSY DURING THERAPY. 
NEUROIMAGE CLIN. 2015;7:315-324. 
32. 
BALLEN KK, GLUCKMAN E, BROXMEYER HE. UMBILICAL CORD BLOOD 
TRANSPLANTATION: THE FIRST 25 YEARS AND BEYOND. BLOOD. JUL 25 
2013;122(4):491-498. 
33. 
MIN K, SONG J, KANG JY, ET AL. UMBILICAL CORD BLOOD THERAPY 
POTENTIATED WITH ERYTHROPOIETIN FOR CHILDREN WITH 
CEREBRAL PALSY: A DOUBLE-BLIND, RANDOMIZED, PLACEBO-
CONTROLLED TRIAL. STEM CELLS. MAR 2013;31(3):581-591. 
34. 
ROMANOV YA, TARAKANOV OP, RADAEV SM, ET AL. HUMAN 
ALLOGENEIC AB0/RH-IDENTICAL UMBILICAL CORD BLOOD CELLS IN 
THE TREATMENT OF JUVENILE PATIENTS WITH CEREBRAL PALSY. 
CYTOTHERAPY. JUL 2015;17(7):969-978. 
35. 
LV YT, ZHANG Y, LIU M, ET AL. TRANSPLANTATION OF HUMAN CORD 
BLOOD MONONUCLEAR CELLS AND UMBILICAL CORD-DERIVED 
MESENCHYMAL STEM CELLS IN AUTISM. J TRANSL MED. 2013;11:196. 
36. 
ESTES ML, MCALLISTER AK. IMMUNE MEDIATORS IN THE BRAIN AND 
PERIPHERAL TISSUES IN AUTISM SPECTRUM DISORDER. NAT REV 
NEUROSCI. AUG 2015;16(8):469-486. 
37. 
RUTTER M, LECOUTEUR A, LORD C. AUTISM DIAGNOSTIC INTERVIEW-
REVISED. TORRANCE, CA: WESTERN PSYCHOLOGICAL SERVICES; 2008. 

 
March 15, 2018 
60
38. 
LORD C, RUTER M, DILAVORE PC, RISI S. AUTISM DIAGNOSTIC 
OBSERVATION SYSTEM. LOS ANGELES, CA: WESTERN PSYCHOLOGICAL 
SERVICES; 1999. 
39. 
ELLIOTT CD. DIFFERENTIAL ABILITY SCALES, 2ND EDITION. SAN 
ANTONIO, TX: HARCOURT ASSESSMENT; 2007. 
40. 
MULLEN EM. MULLEN SCALES OF EARLY LEARNING: WESTERN 
PSYCHOLOGICAL SERVICES; 1995. 
41. 
MARTIN N, BROWNELL R. EXPRESSIVE ONE-WORD PICTURE 
VOCABULARY TEST 4. NOVATO, CA: ACADEMIC THERAPY 
PUBLICATIONS; 2011. 
42. 
SPARROW SS, BALLA DA, CICCHETTI DV. VINELAND ADAPTIVE 
BEHAVIOR SCALES:  INTERVIEW EDITION. CIRCLE PINES, MN: 
AMERICAN GUIDANCE SERVICE; 1984. 
43. 
COHEN IL, SCHMIDT-LACKNER S, ROMANCZYK R, SUDHALTER V. THE 
PDD BEHAVIOR INVENTORY: A RATING SCALE FOR ASSESSING 
RESPONSE TO INTERVENTION IN CHILDREN WITH PERVASIVE 
DEVELOPMENTAL DISORDER. J AUTISM DEV DISORD. FEB 2003;33(1):31-
45. 
44. 
AMAN MG, SINGH NN, STEWART AW, FIELD CJ. THE ABERRANT 
BEHAVIOR CHECKLIST: A BEHAVIOR RATING SCALE FOR THE 
ASSESSMENT OF TREATMENT EFFECTS. AM J MENT DEFIC. MAR 
1985;89(5):485-491. 
45. 
GIOIA GA, ESPY KA, ISQUITH PK. BEHAVIOR RATING INVENTORY OF 
EXECUTIVE FUNCTION-PRESCHOOL VERSION (BRIEF-P): 
PSYCHOLOGICAL ASSESSMENT RESOURCES; 2003. 
46. 
GIOIA GA. BRIEF:  BEHAVIOR RATING IVENTORY OF EXECUTIVE 
FUNCTION. ODESSA, FL: PSYCHOLOCIAL ASSESSMENT RESOURCES; 
2000. 
47. 
LITTLE KG, WILKS SE. PSYCHOMETRIC EVALUATION OF A PROPOSED 
ALZHEIMER'S AGGRESSION SCALE. AM J ALZHEIMERS DIS OTHER 
DEMEN. AUG 2011;26(5):373-380. 

 
March 15, 2018 
61
48. 
BARANEK GT. SENSORY EXPERIENCES QUESTIONNAIRE - VERSON 3.0 
(SEQ): UNIVERSITY OF NORTH CAROLINA AT CHAPEL HILL; 2009. 
49. 
BARANEK GT, DAVID FJ, POE MD, STONE WL, WATSON LR. SENSORY 
EXPERIENCES QUESTIONNAIRE: DISCRIMINATING SENSORY FEATURES 
IN YOUNG CHILDREN WITH AUTISM, DEVELOPMENTAL DELAYS, AND 
TYPICAL DEVELOPMENT. J CHILD PSYCHOL PSYCHIATRY. JUN 
2006;47(6):591-601. 
50. 
VARNI JW, KAY MT, LIMBERS CA, FRANCIOSI JP, POHL JF. PEDSQL 
GASTROINTESTINAL SYMPTOMS MODULE ITEM DEVELOPMENT: 
QUALITATIVE METHODS. J PEDIATR GASTROENTEROL NUTR. MAY 
2012;54(5):664-671. 
51. 
DAWSON G, JONES EJ, MERKLE K, ET AL. EARLY BEHAVIORAL 
INTERVENTION IS ASSOCIATED WITH NORMALIZED BRAIN ACTIVITY 
IN YOUNG CHILDREN WITH AUTISM. J AM ACAD CHILD ADOLESC 
PSYCHIATRY. NOV 2012;51(11):1150-1159. 
52. 
AMAN MG, NOVOTNY S, SAMANGO-SPROUSE C, ET AL. OUTCOME 
MEASURES FOR CLINICAL DRUG TRIALS IN AUTISM. CNS SPECTR. JAN 
2004;9(1):36-47. 
 
 
 
 

 
March 15, 2018 
62
14.0 
APPENDIX 1:  SAFETY QUESTIONNAIRE 
 
 
BASELINE MEDICAL & BEHAVIORAL HISTORY QUESTIONNAIRE 
 
(To be completed on Monday of the baseline visit) 
 
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? 
  
  
  
  
  

 
March 15, 2018 
63
  
  
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. 
  
  
  
  
  
 
 
 
 
 
 
 
33 
Has your child ever had any significant problems sleeping at night? 
  
  
  
  
34 
Has your child ever had any significant problems being too sleepy during the day? 
  
  
  
  
35 
Has your child ever had problems where he/she had to move around a lot, or had a hard time 
sleeping, or was upset because it seemed like he or she wasn’t comfortable in his/her body? 
  
  
  
  
  
36 
Has your child ever had any problems with rituals, being flexible, repetitive behaviors or language? 
  
  
  
  
37 
Has your child ever had any problems with hyperactivity or impulsivity?  
  
  
  
  
38 
Has your child ever had any significant problems with refusing to follow directions that he/she 
understands? 
  
  
  
  
  
39 
Has your child ever had any significant problems with aggression, irritability or getting frustrated 
easily?  
  
  
  
  
  
40 
Has your child ever had significant problems (more frequent/severe than other kids) with meltdowns 
or agitation? 
  
  
  
  
41 
Has your child ever had any problems with staying motivated? 
  
  
  
  
42 
Has your child ever had ever any problems with worries or sadness? 
  
  
  
  
  
43 
Has your child ever had any problems with hurting him/herself on purpose or wanting to die?  
  
  
  
  
  
43 
Has your child ever had any significant problems with mood swings?  
  
  
  
  
44 
Has your child ever had any problems with believing things that aren’t true or seeing/hearing things 
that aren’t there? 
45 
Has your child ever had any problems with seeming not to know where he/she was or what was 
really happening?  
46 
Have you ever had any other concerns about your child’s behavior?  If so, please explain below. 
 
 
 
 
 
 
 
 
 
 
 
 
Please use the space below to explain any “yes” answers. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
March 15, 2018 
64
 
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? 
 
 
 

 
March 15, 2018 
65
  
  
Past 3 
months 
  
  
No 
Yes   
30 
Has your child been diagnosed with cancer?  
 
 
 
31 
Has your child developed a new tumor? 
 
 
 
32 
Has your child developed a new mass or growth? 
 
 
 
33 
Has your child been hospitalized?  If so, please explain below. 
 
 
 
34 
Has your child had any new significant injuries? 
 
 
 
35 
Is there anything else that concerns you about your child’s medical health?  If so, please explain below. 
  
  
  
 
 
 
 
 
35 
Has your child had any new or worsening problems sleeping at night? 
  
  
36 
Has your child had any new or worsening problems being too sleepy during the day? 
  
  
37 
Has your child had any new or worsening problems finding a comfortable body position or feeling like something 
isn’t right in his/her body? 
  
  
  
38 
Has your child had any new or worsening problems with rituals, being flexible, repetitive behaviors or language? 
  
  
39 
Has your child had any new or worsening problems with hyperactivity or impulsivity?  
  
  
40 
Has your child had any new or worsening problems with refusing to follow directions that he/she understands? 
  
  
  
41 
Has your child had any new or worsening problems with aggression, irritability or getting frustrated easily?  
  
  
  
42 
Has your child had any new or worsening with meltdowns or agitation? 
  
  
43 
Has your child had any new or worsening problems with staying motivated? 
  
  
44 
Has your child had any new or worsening problems with worries or sadness? 
  
  
  
45 
Has your child had any new or worsening problems with hurting him/herself or wanting to die?  
  
  
  
46 
Has your child had any new or worsening problems with mood swings?  
  
  
47 
Has your child had any new or worsening problems with believing things that aren’t true or seeing/hearing things 
that aren’t there? 
48 
Has your child had any new or worsening problems with knowing where he/she is or who is with him/her?  
49 
Has your child had any new or worsening problems with their thinking? 
 
 
 
50 
Is there anything else that concerns you about your child’s behavior?  If so, please explain below. 
 
 
 
 
 
 
 
 
Please use the space below to explain any “yes” answers. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
March 15, 2018 
66
 
15.0 
APPENDIX 2:  BIOLOGICAL SAMPLES 
 
The following biological samples will be obtained and stored for future studies: 
 
 
Subject 
Sample Type 
Collection Vessel 
Amount 
Patient 
Blood sample for immune panel and DNA 
Green top tube 
2 x 3 mL 
Blood sample for IPSCs 
Purple top tube 
2 mL 
Blood sample for RNA 
PAX gene tube 
2.5 mL 
Urine sample 
Cotton balls 
2 mL 
(1 mL per tube) 
Saliva 
Sponge 
2 mL 
Buccal Swab 
Kit 
back up if unable 
to obtain blood 
Parents 
of 
Patient 
Blood samples from parents 
Purple top tube 
4 mL 
 
 
 
 
 
 
 
 

 
March 15, 2018 
67
16.0 
APPENDIX 3:  SEDATION GUIDELINES 
 
DukeACT Sedation Guidelines 
 
Procedural sedation is intended to result in a depressed level of consciousness that allows the 
patient to maintain independent and continuous airway control.  All sedation will be performed 
by a pediatric anesthesiologist per their practice guidelines and based on each child’s clinical 
status.  The guidelines for the sedation procedure are detailed below: 
 
Equipment: 
MRI compatible oxygen delivery system 
Oxygen saturation monitor and probe 
Appropriate sized ambu bag and mask 
Suction 
Blood pressure monitoring capability 
Continuous heart monitoring 
End tidal CO2 monitoring device 
Supplies to obtain IV access 
 
 
Premedication: (for IV placement/sedation) 
Midazolam 0.5mg/kg orally or 0.2mg/kg intranasally, administered 15-30 minutes prior to the 
procedure.  A healthcare professional will remain with the patient to monitor level of 
consciousness through direct observation. 
 
 
Sedation options:  
Dexmedetomidine (load of up to 2mcg/kg and an infusion of 0.2-2.0mcg/kg/hr) IV 
 
Or 
 
Propofol (load 0.5-1.0mg/kg) IV and continue as a continuous infusion until procedure is 
completed. 
 
 
Intra-Procedure: 
a) Patients will be continually monitored by MD/RN for signs of hypoventilation, hypotension, 
apnea and bradycardia. 
b) Continuous monitoring of heart rate, oxygen saturation using pulse oximetry, and end tidal 
CO2 using the MRI safe devices will be performed during the procedure.  Blood pressure will be 
obtained at regular intervals. 
c) Patients will receive supplemental oxygen throughout the procedure. 
d) At no time will a patient be left unattended. 
 
 
Post-Procedure Monitoring: 

 
March 15, 2018 
68
Continuous monitoring of heart rate and oxygen saturation using pulse oximetry will be 
performed until the patient is awake. 
 
 
Discharge: 
A. Patients may be discharged when the following criteria are met: 
a) A RASS (RICHMOND AGITATION SEDATION SCALE) Score of -1 or 0. 
b) Responsiveness and orientation reflects pre-procedure status 
c) Patient is able to ambulate with minimal assistance or mobility returned to baseline. 
B. Patients will be discharged in the care of their caregiver with post-procedure instructions, 
including a contact phone number in case of emergency.