Hematopoietic Stem Cell Transplantation for Patients With Severe Sickle Cell Disease Using Myeloablative Conditioning and αβ+ T-cell Depleted Hematopoietic Stem Cells From Partially Matched Familial Donors
Phase: NA
Conditions: Sickle Cell Disease
Sponsor: University of Chicago

 
 
UCPEDS 
SCDHAP 
 
Version: June 22, 2021 
 
HEMATOPOIETIC STEM CELL TRANSPLANTATION FOR PATIENTS WITH 
SEVERE SICKLE CELL DISEASE USING MYELOABLATIVE 
CONDITIONING AND αβT-CELL-DEPLETED HEMATOPOIETIC STEM 
CELLS FROM PARTIALLY MATCHED FAMILIAL DONORS 
 
 
Principal Investigator  
John M. Cunningham, MD1,3 
 
Co-Investigators 
 
Sandeep Gurbuxani, MD5 
Michael Bishop, MD1,4 
Scott Hunter, PhD7 
James LaBelle, MD, PhD1,3 
 
Gabrielle Lapping-Carr, MD1,2,3  
Theodore Karrison, PhD6 
Radhika Peddinti, MD2,3 
 
1 Stem Cell Transplantation Program 
2 Sickle Cell Disease Program  
3 Department of Pediatrics 
4Department of Medicine 
5Section of Hematopathology, Department of Pathology  
6 Department of Public Health Sciences 
7Department of Psychiatry and Behavioral Neurosciences  
 
Biological Sciences Division &  
University of Chicago Medicine Comer Children’s Hospital 
University of Chicago  
5721 S. Maryland Avenue,  
 Chicago, IL 60637 
(773) 702-6808 
 
Contents of this document may not be extracted without permission of the Principal 
Investigator. 

                                                                                                                            
 
  
 
 
TABLE OF CONTENTS 
 
 
1.0 
EXECUTIVE SUMMARY & OBJECTIVES ...........................................................1 
2.0 
BACKGROUND AND RATIONALE ......................................................................3 
3.0 
ELIGIBILITY CRITERIA.........................................................................................13 
4.0  
DESIGN .....................................................................................................................15 
5.0 
TREATMENT OR INTERVENTION ......................................................................18 
6.0 
DRUG INFORMATION ...........................................................................................27 
 
7.0 
EVALUATION CRITERIA ......................................................................................32 
8.0 
OFF-STUDY CRITERIA ..........................................................................................33 
9.0  
REPORTING CRITERIA ..........................................................................................34 
 
10.0 
STATISTICAL CONSIDERATIONS.......................................................................36 
11.0 
HUMAN SUBJECTS ................................................................................................38 
12.0 
OBTAINING INFORMED CONSENT ....................................................................39 
13.0 
DATA ACQUISITION AND QUALITY ASSURANCE MONITORING … .........39 
14.0 
REFERENCES ..........................................................................................................40 
 
APPENDICES 
 
Appendix I:    Schedule of Events: Clinical and Laboratory Evaluations ........................................ 45  
Appendix II:   Neuropsychological Testing ..................................................................................... 47 
Appendix III:  Criteria for Adverse Event Evaluation and Reporting ............................................. 48 
 
 

                                                                                                                            
 
 
SCDHAP 1 
 
 
1.0 
EXECUTIVE SUMMARY, HYPOTHESIS & OBJECTIVES  
1.1 
Executive Summary 
Sickle cell disease (SCD) is an autosomal recessive disorder with pathognomonic mutations in 
the β-globin gene. The resultant ‘sickling’ of the hemoglobin tetramer in red blood cells leads to 
vasoocclusive and hemolytic crises, pain, and end-organ damage. [1] SCD has a multi-system 
pathophysiology that includes life-threatening infection, splenic dysfunction, stroke, 
neurocognitive deficits and a shortened life span. [2] Despite improvements in medical 
management of severe SCD, the only known cure for this disease is allogeneic hematopoietic 
stem cell transplantation (HSCT) and the most experience and success has been demonstrated 
using matched sibling donors (MSD). Successful outcomes result in retardation of end organ 
damage, outstanding long-term survival and low rates of chronic transplant-related complications. 
However, fewer than 15% of patients with severe disease have a matched sibling donor, and an 
even smaller fraction have matched unrelated donors in the international donor registries. [3, 4] 
Strategies to expand the donor pool with unrelated cord blood donors have been unsuccessful due 
to high rates of graft failure, a direct result of the inherent low stem cell doses in these graft 
products. [5-8] In contrast, early transplantation studies using grafts with large numbers or 
‘megadoses’ of haploidentical parental hematopoietic stem cells depleted of alloreactive T-cells 
in β-thalassemia, a related hemoglobinopathy, have shown significant promise. [9, 10] In this 
study, we propose a pilot approach to evaluate a comparable megadose strategy to transplantation 
of patients with SCD. Specifically, our approach takes advantage of a novel technique that results 
in extensive depletion of alloreactive T-cell receptor (TCR) αβ+ T-cells (<1x105  cells/kg), and 
enrichment of stem cells (CD34, >10x106 cells/kg). Furthermore, unlike previous graft 
engineering strategies, we will infuse large numbers of TCRγδ+ T-cells, Natural Killer (NK) cells 
and other cells that facilitate engraftment and effective immune reconstitution, with the potential 
of minimizing severe acute graft-versus-host disease (aGvHD) and post- transplantation viral 
disease. 
 
1.2 
Hypothesis 
We hypothesize that research subjects with severe SCD who receive a haploidentical CD34+ 
megadose hematopoietic stem cell graft depleted of αβ T-cells will experience similar outcomes 
to patients in historical studies of MSD hematopoietic stem cell transplants (HSCT), including 
similar rates of graft rejection and peri-transplant toxicity.  
 
1.3 
Primary Objective 
 
1.3.1 
To assess the safety of αβ T-cell depleted haploidentical hematopoietic 
stem cell transplantation (Hap-HSCT) for children, adolescents and 
young adults with severe SCD. The treatment plan will be considered 
safe if it lacks excessive toxicity. Toxicity is defined as failure to engraft 
(graft failure), grade III/IV irreversible end organ toxicity, grade III/IV 
aGvHD or death within 100 days post- Hap-HSCT.  
 
Our approach will also provide preliminary observational data on the 
efficacy of Hap-HSCT for children, adolescents and young adults with 
severe SCD. Efficacy is defined as stable donor engraftment (>5% total 

                                                                                                                            
 
 
SCDHAP 2 
 
 
nucleated cell DNA) and donor erythropoiesis that corrects the SCD 
hematologic phenotype (<50% HbS in the peripheral blood). 
 
 
1.4 
Secondary Objectives 
 
1.4.1 
To estimate 1-year overall and event-free survival after Hap-HSCT.  
1.4.2 
To observe the incidence and severity of acute and chronic GvHD after 
Hap-HSCT.   
 
1.5 
Exploratory Objectives 
1.5.1 
To obtain preliminary information regarding relative donor engraftment 
(chimerism) 
among 
different 
cell 
subsets, 
including 
unsorted 
mononuclear cell, bone marrow erythroid precursors, and T- and B-
lymphoid subsets. 
1.5.2 
To assess the proportion of research participants requiring stem cell 
boosts or donor lymphocyte infusions after Hap-HSCT. 
1.5.3 
To evaluate systemic end organ function and/or complications post Hap-
HSCT in a longitudinal manner especially growth and development, 
neurologic, neurovascular and neurocognitive status, and systemic organ 
function.  
1.5.4 
To investigate immune reconstitution after Hap-HSCT.  

                                                                                                                            
 
 
SCDHAP 3 
 
 
2.0 
BACKGROUND AND RATIONALE 
 
2.1 
Introduction 
 
With advances in screening and supportive care, 85-95% of children with SCD survive to their 
twentieth birthday. However, compared to unaffected African-Americans, overall life expectancy 
is reduced by 25 or more years.[2, 11] Furthermore, among the approximately 1,000 children with 
SCD born each year, greater than 20% will develop a severe clinical phenotype with recurrent 
vaso-occlusive crises (VOC), manifesting as frequent episodes of severe pain, acute chest 
syndrome (ACS) and/or stroke.[3] Indeed, these individuals have higher rates of mortality than 
the SCD population as a whole. Scheduled red blood cell transfusions reduce the incidence of 
recurrent stroke following an initial cerebrovascular event, but there is a significant risk of stroke 
recurrence (approximately 45%) even with adequate compliance.[12]  
 
Significant morbidity and mortality related to chronic transfusion therapy warrants the 
investigation of alternate strategies. Agents that induce fetal hemoglobin such as daily 
hydroxyurea (HU) administration, as well as allogeneic HSCT, have received significant 
investigatory focus. While the use of HU therapy has increased and demonstrated improvement in 
many clinical symptoms of disease in some patients with SCD, this is not a curative strategy. [13] 
Indeed, long term efficacy has only been observed in a subset of patients. Furthermore, effective 
use of HU has not been associated with retardation in chronic SCD-mediated end organ damage, 
or prolongation of life. Furthermore, it is frequently associated with poor compliance. [13, 14]  
 
As the SCD patient population reaches young adulthood, the disease phenotype is characterized 
by complications such as avascular necrosis, priapism, physical growth and sexual development 
delay, pulmonary hypertension, congestive heart failure and renal dysfunction which are less 
responsive to current non-transplant modalities. [15] Recent data show that children and young 
adults have high rates of cognitive dysfunction with lower IQ scoring compared to age matched 
sibling and peer controls, suggesting an urgent need for novel strategies that correct the disease 
phenotype effectively. [16] 
 
In contrast to these outcomes, long-term studies of hematopoietic stem cell transplantation 
(HSCT) from a matched sibling donor (MSD) have demonstrated curative potential with excellent 
overall and disease-free survival (Table 1).[17-21] Impressively, all data to date show a 
significant amelioration of progressive chronic organ damage after allogeneic stem cell 
transplantation.   
 
2.2 
HSCT for Sickle Cell Disease 
 
The ability of HSCT to cure SCD was first demonstrated in a patient with coincident acute 
myeloid leukemia in 1984. This patient remains in complete remission of both diseases over 30 
years later.[22] Subsequently, over 400 MSD allografts for patients with SCD have been 
performed. In fact, 327 patients with sickle cell disease received MSD transplantation between 
2010-2014. [23] Selection of patients has been agreed by physicians and patient advocates, and is 
based on the severity of the presenting phenotype including previous strokes, recurrent acute 
chest syndrome, and/or recurrent vaso-occlusive crises. [24] Summarizing these results, overall 
survival and disease-free survival range from 91-97% and 73-86%, respectively. [17-21] Peri-
transplant toxicity in patients who receive MSD transplantation is acceptable with grade II-IV 
acute GvHD (aGvHD) occurring in fewer than 20% of transplanted individuals, chronic GvHD 
(cGvHD) in fewer than 29%, and death in 6% of patients. (See Tables 1 and 2) In the most 

                                                                                                                            
 
 
SCDHAP 4 
 
 
recent studies, graft failure occurred in 8.6% of patients. Long-term follow-up data demonstrates 
stabilization of chronic organ damage with no further SCD-related events in the majority of 
patients. [25-28] Adverse events include transplant-related end organ damage, sterility, and a 
slightly increased risk of a secondary malignancy.[29] 
 
 
 
Our unpublished experience at the University of Chicago, and previously at St. Jude Children's 
Research Hospital (SJCRH), is consistent with these outcomes. Of 32 patients transplanted, all 
have full donor engraftment, with 29 (91%) patients demonstrating disease-free survival without 
long-term SCD-related sequelae (JMC, unpublished). Three patients died of transplant-related 
complications. Chronic GvHD post transplantation was the major complication precipitating 
death and occurred in all three of these patients greater than one-year post-HSCT.  
 
 
 
To reduce the incidence of pre- and post-transplant toxicities, MSD HSCT with reduced intensity 
conditioning regimens has been evaluated. Overall survival in these cohorts approaches 100%, 
with event-free survival between 86-100% (Table 2). [30-32] The University of Chicago Sickle 
Cell Disease program is currently performing either matched sibling donor or fully matched 
unrelated donor transplantation for patients with severe phenotype sickle cell disease or using 
ameliorative medical approaches including chronic hydroxyurea therapy and/or red blood cell 
transfusions. Individuals who fulfill disease eligibility criteria and have a MSD donor or fully 
matched unrelated donor would be excluded from this study.   
 
2.3 
Reduction in SCD-related Organ Damage in MSD Transplant Survivors  
Investigative groups have evaluated not only engraftment and peri-transplant toxicities as 
outcomes, but also the acute and long-term effects of corrected hematopoiesis on SCD-mediated 

                                                                                                                            
 
 
SCDHAP 5 
 
 
end-organ damage. For example, detailed central nervous system (CNS) evaluation by the SJCRH 
group 
demonstrated 
stable 
neurologic 
examinations, 
MRI/MRA 
scans, 
and 
IQ/reading/mathematics scores over a 3-5 year follow-up period when evaluated with 
longitudinal neuroimaging and neurocognitive testing. [25, 26] Interestingly, most children had 
clinically non-significant subtle parenchymal changes on MRI. Changes included rare incidences 
of presumed transplant-related leukoencephalopathy or new vascular lacunae. Other investigators 
have demonstrated that with successful donor engraftment, patients who had stroke prior to SCT 
had no recurrent CNS infarction, which is a considerable improvement from the 45% re-
infarction rate seen with patients who are cared for with chronic transfusion therapy alone. [12, 
27] 
 
In a similar manner, resolution of recurrent vaso-occlusive crises (VOCs), hyposplenism, growth 
and development delays, chronic pulmonary injury, endocrinopathy and other SCD-related end-
organ damage have been assessed longitudinally in post-transplant patients.[27, 28] These studies 
have demonstrated normalization of hematopoiesis, universal resolution of VOCs in all 
individuals, reversal of splenic defects, delayed puberty and improvement in other SCD-related 
damage in many instances. We propose to evaluate organ-specific outcomes longitudinally in a 
descriptive manner in this study.   
 
2.4 
Approaches to Expand the Therapeutic Use of HSCT in SCD  
 
Although results of HSCT for SCD using MSD are encouraging, it is estimated that fewer than 
15% of patients will have a non-affected HLA-identical sibling.[3, 4] For example, an 
unpublished review by our principal investigator of patients treated with chronic transfusions at 
SJCRH demonstrated that of the 47 patients eligible for HSCT based on clinical features, only 
four patients had appropriate MSDs. Thus, the lack of availability of MSDs has been a major 
barrier to transplantation for patients with severe SCD.  The goal of our program is to develop a 
successful transplant strategy for patients who lack a MSD.  This strategy must reduce the 
procedure-related morbidity and mortality to levels that are comparable to those observed in 
patients receiving MSD grafts.  
 
To date, investigators have considered three potential alternate sources of donor hematopoietic 
cells which have been evaluated with varying success: matched unrelated donors (MUD), 
unrelated umbilical cord blood (UCB) grafts, haploidentical unmanipulated bone marrow and 
peripheral blood stem cell selected grafts with post infusion cyclophosphamide and haploidentical 
CD34+ selected stem cell grafts.  A brief summary of our current understanding of these 
approaches is provided below: 
 
MUD Donors African-Americans are underrepresented on MUD registries creating substantial 
difficulties in identifying unrelated donors. [4, 33] Fewer than 60% of African-Americans have a 
potentially suitable donor compared to 75-80% of Caucasians. As a result, there is minimal data 
on the outcomes of patients receiving matched unrelated donor transplantation. [34] Although we 
have had two survivors in our pediatric cohort at Comer Children’s Hospital (CCH), we have 
evaluated innumerable other patients without observing a 10/10 Human Leukocyte Antigen 
(HLA) match, the currently accepted standard for the use of MUD grafts in non-malignant 
disease. Thus, this approach is not feasible as an accessible, safe approach to alternate 
transplantation in children with SCD. We propose that in the unlikely event that a potential 
research subject has a 10/10 HLA MUD, he/she will be ineligible for this study.  
 
Cord Blood Donors The national and international registries have large banks of unrelated UCB 

                                                                                                                            
 
 
SCDHAP 6 
 
 
grafts. Indeed, this graft source has been used successfully to treat children and adults with both 
malignant and non-malignant diseases. However, the results in patients with hemoglobinopathies 
have been disappointing. Multiple groups have reported rates of graft rejection ranging from 44-
62.5%, with a cGvHD rate of 12-14% (Table 3). [5, 6, 8, 35] A recent large Blood and Marrow 
Transplant Clinical Trials Network trial was closed early to accrual due to graft rejection and 
related morbidity and mortality. A direct relationship between toxicity to low stem cell dose was 
observed in these studies. [6] Given the poor outcomes with this approach, we propose that 
individuals with severe SCD as defined by the eligibility criteria, and who lack a suitable 10/10 
MSD or MUD donor, will be eligible for the study regardless of the availability of appropriate 
cord blood units.  
 
 
 
Haploidentical Donors: The effective and safe use of partially matched familial donors for 
children and adults with hematologic disease has been a strong aspiration since the feasibility of 
transplantation was first demonstrated. Family members who are heterozygotes for the HbS 
genotype are highly attractive donors given their accessibility and willingness to donate stem 
cells. Indeed, haploidentical donors are being used increasingly for children and adults with 
malignant diseases, a successful expansion of the donor pool. [36, 37] 
 
 
 
However, major concerns with this approach include the potential for severe life-threatening 
GvHD and graft rejection/failure. The haploidentical state is associated with major HLA class I or 
II mismatches. Although these mismatches may be beneficial for malignant disease control due to 
the graft versus tumor (GvT) benefit of an allograft, they are unnecessary and potentially 
detrimental in patients with non-malignant diseases due to their GvHD potential. Approaches to 
reduce this key toxicity include the use of post-transplant cyclophosphamide as GvHD 
prophylaxis or allograft engineering with CD34 hematopoietic stem cell selection and associated 
rigorous and broad T-cell depletion. [38] Both approaches have been associated with very poor 
engraftment and immune reconstitution rates when used for patients with SCD, the latter being 
associated with a high incidence of viral reactivation. Interestingly, rates of severe acute and 

                                                                                                                            
 
 
SCDHAP 7 
 
 
chronic GvHD were relatively low (<10%) in these studies (Table 4). [39, 40] 
 
In summary, these studies show that a) new haploidentical stem cell transplant approaches are 
warranted, and b) key components necessary for a successful outcome of alternate donor HSCT 
for SCD must include: 1) a reduction in the rate of GvHD and graft failure after haploidentical 
transplantation; 2) the ability to achieve rapid myeloid and lymphoid engraftment to reduce the 
incidence of opportunistic infection and viral reactivation; 3) the need to use less toxic 
conditioning regimens to prevent treatment related morbidity.   
 
 
 
 
2.5 
Rationale for a Novel Haploidentical Transplantation Approach: an αβ+ T-cell 
depleted stem cell infusion with a fixed γδ+ T-cell dose and a megadose of CD34+ stem 
cells in children, adolescents, and young adults with SCD.  
 
Extensive animal and human transplant studies have demonstrated that two graft characteristics 
are critical for successful haploidentical hematopoietic stem cell transplantation 1) a high CD34 
dose and 2) a low T-cell dose. Murine studies have shown the value of supplementing T-cell 
depleted bone marrow with megadoses of blood stem cells.[9] In vitro studies have demonstrated 
the presence of tolerizing “veto cells” in mobilized human CD34+ hematopoietic progenitor cells. 
These cells are capable of inducing immunologic tolerance against donor cells while preserving 
anti-third party reactivity.[41] In human studies, ‘megadoses’ of CD34+ cells have been 
associated with more rapid engraftment and lower probability of graft failure.[9, 41] Megadoses 
are defined as > 10 × 106/kg. Other studies have reported more rapid hematopoietic recovery, a 
reduced incidence of fungal infections, reduced number of febrile days, lower transplantation-
related mortality, and higher 180 day survival with higher CD34+ cell doses. [42] In the context 
of unmanipulated, reduced-intensity, allogeneic matched-sibling donor transplants, higher CD34+ 
cell doses were associated with more rapid complete donor chimerism in donor T-lymphocytes 
and a lower relapse rate in high-risk malignancies, but a higher rate of chronic GvHD. [43] 
However, unlike unmanipulated grafts, CD34-selected grafts have high rates of graft failure, and 
are associated with viral reactivation and poor immune reconstitution. [44] 
 
To address this deficit, we propose to evaluate the efficacy and toxicity associated with 
transplantation of αβ+-depleted haploidentical grafts into eligible individuals with severe SCD. 
These grafts will be composed of megadoses of CD34+ cells with measurable numbers of γδ+ T-
cells, NK cells, and other cells that facilitate engraftment. The latter cellular populations, which 
are depleted in CD34+ selected grafts, are associated with higher rates of myeloid and immune 
reconstitution in pre-clinical and recently published studies. [45-48]  
  
Donor T-cell identification of host as non-self is the primary insult in the development of GvHD. 
αβ+ T-cells are the primary culprit in this phenomenon. They constitute the vast majority of T-
cells and when biopsied, tissues affected by GvH demonstrate the enrichment of αβ+ T-cells. 
[49-51] Furthermore, lethally irradiated mice transplanted with bone marrow-depleted of αβ+ T-
cells and enriched for γδ+ T-cells were protected from GvHD and mortality when compared to 
mice transplanted with either unmanipulated or pan T-cell depleted grafts respectively. 
 
These studies have resulted in clinical studies of αβ+-depleted haploidentical stem cell 
transplantation using the Miltenyi CliniMACS system in patients with hematologic malignancies 
and non-malignant, non-SCD hematologic disorders. These studies demonstrate favorable 
morbidity and mortality profiles. Specifically, Bertaina et al. described their experience with 23 

                                                                                                                            
 
 
SCDHAP 8 
 
 
children with non-malignant hematologic diseases. In their cohort, only three (13%) developed 
grade I-II GvHD, with no grade III-IV GvHD, visceral GvHD or chronic GvHD.[40, 48] This 
strategy therefore is likely to significantly limit the GvHD seen in our study population.  
 
The use of αβ+ T-cell-depleted stem cell haploidentical grafts allows for infusion of megadoses 
of CD34+ stem cells by removal of T-cells speculated to cause GvHD but retention of supportive 
cells that help facilitate engraftment. We hypothesize that this strategy will overcome the barriers 
limiting engraftment for patients with SCD receiving haploidentical grafts. Finally, the rates of 
viral reactivation were lower (approximately 38%) in γδ+ T-cell enriched grafts when compared 
to other haploidentical graft strategies that reported rates of viral reactivation as high as 77%. [48, 
52] Again, we contend that this apparent advantage should be evaluated in the SCD population at 
high risk of adverse disease-related morbidity and mortality. 
 
2.6 
Justification for the Use of Pediatric Patients  
 
Despite advances in the medical management of patients with sickle cell disease, patients 
continue to suffer from significant morbidity secondary to disease related end organ damage and 
early mortality. [1, 2, 53, 54] Hematopoietic stem cell transplantation is the only known cure. The 
progressive end organ damage patients experience as a result of their sickle cell disease puts them 
at higher risk of transplant related complications as they age. A recent study evaluating the 
outcomes of 1000 patients transplanted for severe sickle cell disease demonstrated not only that 
patients aged <16 years have superior overall and event free survival but that even within these 
groups increasing age is inversely associated with survival rates suggesting that an older age is a 
risk for poor outcome and transplantation of pediatric patients yields optimal outcomes. As result, 
pediatric patients aged 10 years or older will be included in Stage I of this study. Should results of 
Stage I of this study demonstrate that the strategy is safe in the initial patients, we would extend 
the lower age limit to two year olds. Additional stopping rules will be put in place for this subset 
of patients to ensure safety in younger children during the Stage II phase. Please see section 10.0 
for details.   
 
In addition to being optimal for individual subject outcomes, the inclusion of pediatric patients in 
clinical trials of transplantation in sickle cell disease is standard as evidenced by the trials 
outlined in tables 1-4. In fact, including only adult patients in this study would very likely create a 
negative selection bias as the trials in the historic literature have been composed largely of 
pediatric subjects.  
 
2.7 
Chemotherapy Considerations 
 
Busulfan pharmacokinetic monitoring: Ablation of host hematopoiesis is critical to successful 
transplantation for many diseases and particularly when using mismatched donor grafts. 
[55] To achieve this goal, we will use a busulfan-containing conditioning regimen given highly 
successful engraftment rates reported with this approach. 
 
Specifically, busulfan levels will be checked per institutional standard. The goal for the area 
under the curve (AUC) value will be 1000-1200 μmol/min/L. Previous studies have demonstrated 
that in patients with sickle cell disease who received transplantation with busulfan, full chimerism 
was seen more commonly when AUC was higher (median 1018 +/- 122 μmol/min/L). Patients 
who had a lower AUC (median 863 +/- 72 μmol/min/L) had mixed chimerism or graft loss. In 
addition, there was no significant difference in rates of toxicity, specifically VOD, between the 
two groups.[56]  

                                                                                                                            
 
 
SCDHAP 9 
 
 
 
Use of Fludarabine in Sickle Cell Disease: Fludarabine will also be used in this regimen. This 
agent has previously been described in haploidentical transplantation regimens, including αβ+T-
cell and CD19+ B-cell depleted graft transplantation in non-malignant hematologic diseases 
excluding SCD.[48] Its use in SCD needs to be considered carefully due to its association with 
neurotoxicity, and the likelihood for SCD patients to have overt or covert neurologic injury prior 
to, and during, the stem cell transplant procedure. However, the cumulative fludarabine doses of 
160 mg/m2 used in this haploidentical transplant protocol is far below the threshold of 480 mg/m2 
associated with neurotoxicity in adults. Indeed, fludarabine has been utilized recently in the 
conditioning regimens for patients with SCD. Neurotoxicity was observed in less than 5% of 
children, with the predominant insult being posterior reversible encephalopathy. It is likely that 
concurrent calcineurin inhibitor therapy was a factor in the development of neurotoxicity in these 
patients. Importantly, all events were reversible and did not result in any long-term sequelae. 
Research subjects on this study will not receive calcineurin inhibitor prophylaxis, reducing the 
potential risk. [32, 57] 
 
2.7 
Prevention and Management of Graft Failure in SCD Patients Undergoing Allogeneic 
Stem Cell Transplantation 
 
Studies of alternative donor transplant for hemoglobinopathies, including SCD and β–
thalassemia, have shown high rates of primary and secondary graft failure. Sodani reported on 33 
pediatric patients with thalassemia who received MSD BMTs, all were first transplants. All 
patients had poor prognosis with Lucarelli Class III classifications (severe iron overload, hepatic 
fibrosis, poor iron chelation) and all received hydroxyurea 30 mg/kg/day, azathioprine 3 mg/kg, 
G-CSF twice a week, and hypertransfusion with packed red blood cells prior to transplantation.  
Conditioning consisted of busulfan (14 mg/kg) and cyclophosphamide (120-160 mg/kg).  GvHD 
prophylaxis consisted of cyclophosphamide 7.5 mg/kg IV on day 1, methotrexate 10 mg/m2 on 
days 3 and 6, methylprednisolone 0.5 mg/kg/day from day -1 to day +25, and cyclosporine for 1 
year.  Using this regimen, overall and event-free survival was 96% and 90%, respectively. The 
rate of rejection was 8% and the death rate was 4%. Previous historical controls at the time 
demonstrated rejection of 30% when hydroxyurea and azathioprine were not used. [58] Taking a 
similar approach, others have demonstrated similar improvements in non-engraftment rates in 
adults with SCD using three months of hydroxyurea and azathioprine prior to transplant. [18] 
 
As a preparative regimen, hydroxyurea and azathioprine will be maintained for 3 months, if 
tolerated, until the day before the conditioning regimen. In addition, red blood cell transfusions or 
exchange will be performed to target hemoglobin S levels of less than 30% just prior to initiation 
of conditioning regimen.  
 
The etiology of graft rejection in haploidentical transplantation is multifactorial, and attributed to 
a combination of cellular-mediated graft rejection and allo-immunization Strategies proven to 
improve engraftment secondary to host-versus-graft immune system mediated graft failure 
include myeloablative chemotherapy and T-cell depletion of the host prior to transplantation. [59] 
Our preparative regimen will include myeloablative chemotherapy as well as anti-thymocyte 
globin (ATG) to address both major mechanisms.  
 
Graft failure will be considered donor chimerism less than 5% whole blood DNA or symptomatic 
cytopenias. We will consider the graft a success if the patient does not have graft failure and 
achieved a HbS <50%. These criteria were selected specifically given previous data on the 
kinetics of graft failure in patients with SCD. [60, 61]  

                                                                                                                            
 
 
SCDHAP 10 
 
 
 
Other investigators have demonstrated that in patients transplanted for SCD who develop mixed 
myeloid and lymphoid chimerism following transplantation, there is enrichment of donor 
peripheral erythrocytes (predominant donor erythroid chimerism). [61, 62] This state is associated 
with disease amelioration, even in instances where whole blood DNA chimerism evaluation 
demonstrates host myelo-lymphopoiesis.  [62] This data suggests that non-HbSS donor erythroid 
precursors outcompete host erythroid precursors. [61]  
 
Poor graft function and graft failure following stem cell transplant are associated with increased 
morbidity and mortality secondary to infection and hemorrhagic complications. [63] In previous 
haploidentical studies, patients with graft failure/poor graft function who receive an infusion of a 
CD34+ enriched stem cell boosts have demonstrated both improved trilineage hematopoiesis as 
well as decreased GvHD and treatment related mortality when compared to patients who received 
neither a stem cell infusion or unmanipulated bone marrow boost. [63] Should graft failure occur 
despite preventative strategies described above, a CD34+ enriched stem cell boost will be infused 
at the discretion of the principal investigator.  Our stem cell boost strategy in the context of graft 
failure/poor graft function mirrors this approach.  
 
 
2.8 
Special Considerations in SCD Patients Undergoing Allogeneic Stem Cell 
Transplantation  
 
In the peri-transplant period, several SCD-specific thresholds for transfusion support will be 
adopted to reduce previously observed cytopenia-related toxicities. Thus, platelet counts will be 
maintained at > 50 × 109/L, and hemoglobin will be kept between 9 and 11 g/dL to reduce the risk 
of stroke. [17, 64] Lastly, penicillin V-potassium will be given twice daily from transplant day 
+21 or when tolerating PO medications, consistent with institutional standard of care. Treatment 
will continue until patients have completed pneumococcal vaccinations and developed positive 
titers, due to the hyposplenic state of all individuals with SCD.  
 
 
2.9 
Justification for monitoring of immune reconstitution and viral reactivation after γδ+ 
T-cells allografts 
 
Haploidentical hematopoietic stem cell transplantation has expanded the donor pool for patients 
who lack HLA matched donors. Haploidentical donor transplantation requires T-cell depletion or 
intensive immune suppression to avoid life-limiting GvHD. Compared to more traditional HLA 
matched donor transplantation approaches, patients who receive haploidentical transplantation 
often experience delayed T-cell mediated immune reconstitution when compared to those 
receiving MSD grafts. [65] This defect can be associated with disease relapse (particularly in 
malignant diseases) and viral infections. [44] However early engraftment of various lymphocyte 
subsets predicts both improved survival and decreased treatment-related mortality, including viral 
infection. [66-71] We anticipate the infusion of large numbers of γδ+ T-cells in our haploidentical 
stem cell transplantation approach will improve immune reconstitution post-transplant. Support 
for this conclusion includes a randomized control trial of  haploidentical stem cell grafts that 
compared either a) ex vivo CD3+T-cell depletion and in vivo CD19+ B cells depletion (negative 
selection), or  b) CD34+ positive selection grafts. More rapid reconstitution of cytolytic NK cells 
and enhanced resistance to viral reactivation were demonstrated in patients receiving CD3+ 
CD19+ depleted grafts. [72] 
 

                                                                                                                            
 
 
SCDHAP 11 
 
 
Viral reactivation following allogeneic stem cell transplantation is a common complication that 
can contribute to significant morbidity and mortality.[73, 74] Patients are at particularly high risk 
if they experience delayed immune reconstitution. Major risk factors for viral reactivation, and 
consequent viral disease include the use of mismatched donors, myeloablative conditioning, T-
cell depletion and increased GvHD prophylaxis. The result is not only a slowing in the tempo of 
T- and B-cell reconstitution, but a reduction in the quality of the response.[73, 75] Previous 
studies of αβ+T-cell depleted haploidentical transplantation in non-malignant hematologic 
disease other than SCD have demonstrated low rates of viral reactivation when compared to other 
alternative donor strategies. This outcome has been attributed to improved immune reconstitution 
related to the partial T-cell depleted nature of the graft as compared to complete T-cell depletion. 
[48]  
 
Understanding both the rate and quality of immune reconstitution as well as the rates of viral 
reactivation following αβ+T-cell-depleted haploidentical transplantation is therefore of critical 
importance to improving the safety and expected non-relapse related morbidity and mortality 
risks in these patients. As a secondary goal, we propose to examine defined lymphoid/NK 
peripheral blood subsets at regular intervals post-transplantation, allowing a comprehensive 
picture of lymphoid engraftment to be generated. Coupled with these studies, we will examine 
cytomegalovirus (CMV), Epstein-Barr virus (EBV), Adenovirus and BK DNA titers in the serum 
to correlate in a descriptive manner the tempo of functional immune reconstitution.   
 
Monitoring of viral reactivation and preemptive treatment has proven to be a cornerstone of 
successful treatment of viral reactivation events, decreasing morbidity and mortality associated 
with specifically CMV and EBV related illnesses. [76, 77] The more recent availability of agents 
active against adenovirus and systemic BK viremia suggests that similar monitoring for these 
organisms should be contemplated. These approaches are part of the standard of care in the first 
365 days following transplantation at the University of Chicago and will be utilized throughout 
the study.  
 
2.9 
The Central Nervous System in SCD  
 
Silent Infarction, Stroke, and Neuropsychological Testing in Children with SCD: Silent infarcts 
were present in 22% of children with sickle cell anemia who were studied in the Cooperative 
Study of Sickle Cell Disease (CSSCD). [78] Silent infarcts were predictive of subsequent strokes 
and new or more extensive silent infarcts in the CSSCD cohort. Importantly, there is a well-
documented relationship between decreased performance on neuropsychological testing and the 
presence of silent infarcts. [79]  
 
Overt stroke occurs in 5-10% of children with SCD who are less than 15 years of age. [80, 81] 
Chronic transfusion therapy is inadequate to prevent recurrent stroke after transfusions were 
discontinued. [82] Furthermore, although effective, transfusion therapy is associated with morbidity 
and does not prevent all recurrent strokes. Indeed, roughly 25% of children receiving chronic 
transfusion therapy for stroke experienced recurrent overt stroke and 45% suffer either over stroke or 
additional silent stroke. [12] 
 
Neuropsychological testing is frequently compromised in children with SCD even in patients who 
have not had an overt stroke or a demonstrable silent infarct on brain MRI. [16] This protocol will 
include detailed pre- and post-HSCT brain MRI and neuropsychological studies. 
 

                                                                                                                            
 
 
SCDHAP 12 
 
 
Treatment of CNS effects of SCD: Relatively little information is available regarding interventions 
which prevent, limit, or restore compromised CNS function.  Evidence-based conclusions are limited 
to those of the Stroke Prevention Trial in Sickle Cell Anemia (STOP) trial, in which chronic 
transfusion was superior to observation in the prevention of primary stroke in high-risk children 
identified by transcranial Doppler (TCD) studies. [83] The consensus from single institution trials is 
that chronic transfusion is effective in prevention of secondary stroke in patients who have 
experienced an initial stroke. [84, 85] Recently, the efficacy of stem cell transplantation in secondary 
stroke prevention has been demonstrated. [27] We propose to study the CNS outcomes of the 
patients enrolled in this study in a descriptive manner.   
 
2.10 
Community Consensus on use of Allogeneic HSCT for Severe Sickle Cell Disease 
 
To address the issues involved in expanding HSCT to all eligible patients with SCD, the first 
consensus conference to discuss the medical, psychosocial, safety, and ethical issues associated 
with alternate donor transplantation was convened at SJCRH in June 2000.[86] This conference 
was modeled after a similar meeting held prior to the initiation of a multi-institutional study of 
MSD BMT in SCD. Data was presented on risk factors for severe SCD, the state of allogeneic 
HSCT, the ethics of experimental therapy in SCD, and the views of patient advocates. Based on 
the information presented, the participants agreed that pilot trials of haploidentical HSCT for 
severe SCD were worthy of exploration. All agreed that the criteria for success of these studies 
with respect to safety, toxicity and long-term donor engraftment should closely match those 
achieved in the MSD setting. Similar meetings of experts, patient advocates, and ethicists have 
endorsed and extended these conclusions including the recent Sickle Cell Annual Meeting at the 
National Institute of Health (NIH) in August 2016. [87] 
 
We have received many inquiries from parents and physicians regarding haploidentical 
transplantation. We propose to use an approach similar to that used for other allogeneic donor 
transplants with respect to supportive care. We plan to involve our social workers and Child Life 
specialists in all patient and family evaluations/discussions, and offer hospital patient advocates 
and chaplains for families who consider this investigational approach. These efforts will be 
documented in the clinical record. 
 
2.11 
Summary 
 
Although haploidentical HSCT has been historically associated with high rates of morbidity and 
mortality, the technology and supportive care necessary to achieve a successful outcome has 
improved significantly over the past several years.  The mortality of MSD HSCT for SCD has 
been approximately 6%.  It is our goal to perform haploidentical HSCT with no more than 10% 
mortality.  In studies to date this has been feasible, but the rates of graft failure, GvHD and 
effective immune reconstitution suggest that significant additional improvements are required. To 
achieve this goal, we propose that research participants will be treated in a closely monitored two 
stage design with plans to continue enrollment into the second stage design if strict safety 
parameters are met in the first phase portion of the study. (See section 10.0) 
 
For this trial, we propose to use: 
a) A conditioning regimen that consists of busulfan, fludarabine, low-dose thiotepa, 
antithymocyte globulin (ATG) and rituximab, to limit toxicity.  Busulfan, fludarabine and 
ATG have been used safely and effectively in children with SCD with matched sibling 
donors.  Low-dose thiotepa and rituximab have been used in haploidentical transplant 
protocols with modest toxicity. Specifically, the role of the latter agent is to reduce EBV-

                                                                                                                            
 
 
SCDHAP 13 
 
 
related post-transplant lymphoproliferative disease. This is a standard of care approach in 
the context of haploidentical HSCT. 
b) Megadoses of CD34+ stem cells (Target: >10x106/kg) for children and young adults with 
severe SCD. These cells will be purified using the Miltenyi CliniMACS system designed 
for αβ+ T-cell receptor selection using immunomagnetic beads. A fixed T-cell dose of 
approximately <1.0 x 105 αβCD3+ cells/kg will be infused. No greater than 1.0 x 108 
γδ+CD3+-cells/kg will be infused.  
c) No post-transplant GvHD prophylaxis will be used in an attempt to accelerate immune 
reconstitution. We will add the use of GvHD prophylaxis should we find we are 
approaching unacceptable rates of acute GvHD as defined by our stopping rules. See 
Section 10.0. 
d) Close monitoring for toxicity and safety of the protocol.  
e) Stem cell boosts to improve engraftment in the context of declining donor chimerism. In 
general, stem cell boosts will be given for cytopenias or aplasia with or without declining 
donor chimerism. See Section 5.9 for more details 
 
We will also study: 
a) The kinetics of hematopoietic engraftment and immune reconstitution following stem cell 
infusion. 
b) The effects of haploidentical HSCT on SCD-related organ damage with a particular focus 
on the central nervous system.  
 
 
3.0 
ELIGIBILITY 
 
3.1 
Transplant Research Recipient Inclusion Criteria 
3.1.1. Hemoglobin SS, SC, S-β0 Thalassemia, or SO-Arab Sickle Cell Disease. 
3.1.2. Age 10-25 years, inclusive (Stage I); 2-25 years, inclusive (Stage II). 
3.1.3. Lack of fully matched (10/10) family donor or fully matched (10/10) unrelated 
donor registered in the National Marrow Donor Program (NMDP).  
3.1.4. Partially-matched family member with hemoglobin AA (normal) or hemoglobin 
AS (sickle trait) phenotype. 
3.1.5. SCD with Severe Phenotype, defined by the following criteria: 
a) Neurologic manifestations of sickle disease including cerebral 
vascular accident (CVA), transient ischemic event (TIA) or 
abnormal MRI findings suggestive of silent infarct.  
b) Two or more episodes of acute chest syndrome (ACS) requiring 
admission for transfusional or respiratory support including 
supplemental oxygen within [two years] of enrollment in study 
despite hydroxyurea therapy. Patients who cannot tolerate 
hydroxyurea and who experience multiple episodes of ACS will 
also be eligible.  
c) History of severe vaso-occlusive (VOC) disease requiring 
hospitalization and intravenous narcotics on 3 or more occasions 

                                                                                                                            
 
 
SCDHAP 14 
 
 
per year over the two years prior to enrollment despite 
hydroxyurea therapy. Patients who cannot tolerate hydroxyurea 
and who experience multiple episodes of VOC will also be 
eligible.  
d) Other severe phenotype as evidenced by end organ dysfunction 
related to sickle cell disease.  
 
3.2 
Transplant Research Recipient Exclusion Criteria 
3.2.1  Karnofsky or Lansky score < 60%. 
3.2.2  Acute hepatitis or evidence of moderate or severe portal fibrosis on biopsy.  
(Biopsy will be obtained if patient has been on chronic transfusion therapy > 6 
months or has a ferritin > 1000 ng/ml) or AST or ALT >5 times the upper limit 
of normal. 
3.2.3  Severe renal impairment (as evidenced by creatinine clearance of <50ml/minute 
glomerular filtration rate (GFR) < 50% predicted normal).  
3.2.4  Cardiac function that demonstrates shortening fraction less than 26% by cardiac 
echocardiogram or pulmonary hypertension. 
3.2.5  Pregnant Female. 
3.2.6   Lactating female. 
3.2.7  HIV Seropositivity. 
3.2.8  Pulmonary function with baseline O2 saturation <85% or Diffusing Capacity for 
Carbon Monoxide (DLCO) on pulmonary function testing (PFT) with a DLCO 
<40%. 
 
3.3 
Donor Eligibility (Inclusion/Exclusion) Criteria 
3.3.1 
Is a partially matched family member. 
3.3.2 
Donor does not have sickle cell disease. 
3.3.3 
Donor cannot be HIV seropositive. 
3.3.4  Donor cannot be positive for anti-Hepatitis C antibodies or anti-Hepatitis B core 
antibody.  
3.3.5 
Donor cannot have invasive fungal infection. 
3.3.6 
Lactating or pregnant females are excluded. 
3.3.7 
Medical conditions that preclude apheresis. 

                                                                                                                            
 
 
SCDHAP 15 
 
 
3.3.8 
Donor must be evaluated by their physician and deemed medically safe for 
apheresis.  
3.3.9 
If the selected donor is <18 years old, he/she will be meet with the donor 
advocate to provide additional support to the donor and his/her family as well as 
to minimize emotional and psychosocial risk to the donor as per institutional 
guidelines and transplant standard operating procedures. 
 
 
3.4 
Graft Eligibillity (Graft Release) Criteria 
3.4.1 
Graft must have >10x106 CD34+ cells. 
3.4.2 
Graft must have <1.0x105 αβ+ T-cells. 
3.4.3 
Graft must have >1x108 γδ+  T-cells. 
3.4.4 
Graft must have a negative gram stain and culture. 
3.4.5 
Graft must have an endotoxin test demonstrating less than 5 EU/kg body 
weight/hour 
3.4.6 
Graft must have >90% viability testing based on trypan blue test 
Note: All graft release criteria will be performed on the final device output product prior to 
cryopreservation. Graft must meet these criteria prior to initiation of conditioning chemotherapy. 
Once met graft with by cryopreserved and conditioning chemotherapy will begin.  
Gram stain and culture performed on the final device output will be available prior to 
cryopreservation and be included in the graft release criteria as indicated above. Additional gram 
stain and culture will be performed after thawing will not be available prior to infusion as the 
products are thawed at the patient’s bedside and infused immediately to ensure the best possible 
viability. This will be monitored closely and if a positive test is obtained, the subject will initiate 
appropriate antibiotics immediately in conjunction with guidance from our immunocompromised 
infectious disease team. 
 
4.0 
DESIGN  
4.1 
Aims of Current Study 
4.1.1 
Safety 
 
The primary aim of this study is to determine the safety of αβ T-cell depleted 
haploidentical HSCT in children, adolescents and young adults with severe SCD.   
The treatment plan will be considered safe if there is no excessive toxicity. 
Excessive toxicity is defined as failure to engraft/graft rejection or overall grade 
III/IV aGvHD or death within 100 days post transplantation. Graft failure will 

                                                                                                                            
 
 
SCDHAP 16 
 
 
include both primary and secondary graft failure. Primary graft failure is defined 
as failure to achieve an absolute neutrophil count (ANC) of >500/uL at 28 days 
post stem cell transplant. Secondary graft failure is defined as loss of neutrophil 
engraftment (ANC <500/uL for three consecutive days) and <5% total nucleated 
cell DNA of donor origin despite G-CSF administration and/or platelet   
<30X10^3/uL with <5% total nucleated cell DNA of donor origin despite initial 
stem cell infusion.  [88, 89] We will also consider graft failure if the HgB S 
percentage is greater than sixty. These criteria are based on standard criteria to 
define graft failure and an understanding of graft failure specifically in the setting 
of transplantation for sickle cell disease. [60, 61, 90, 91] 
 
We will evaluate the primary objective of safety of HSCT within the framework 
of a two-stage design. In the first stage, we will enroll up to eleven research 
participants to receive HSCT.  The treatment will provisionally be considered 
safe if no more than two of eleven research participants have overall grade III or 
IV acute GvHD, no more than four of eleven participants have grade II or higher 
acute GvHD, no more than four of eleven participants have graft failure and no 
more than two of eleven participants die of transplant toxicity. Once it is 
determined that the treatment plan is provisionally safe, we will begin enrollment 
of eligible and consented research subjects into a subsequent second phase (up to 
twenty-seven) to assess safety as well as the efficacy of the transplant procedure. 
Further details are given in Section 10.0 (Statistics).  
 
Contingency plans for the study: Should any of the above defined stopping rules 
be met during enrollment of the first eleven research participants, study 
enrollment will be halted and the protocol will require amendment of the 
conditioning regimen with resubmission of a revised protocol for review by the 
IRB and the appropriate regulatory agencies.   
 
We will follow the above algorithm with one additional modification.  If the rate 
of aGHVD in the first 11 patients is too high (as defined), we will modify the 
treatment protocol to include immune suppression prophylaxis and begin anew, 
enrolling 11 patients in the first stage and up to 26 in the second stage if no 
further stopping boundary is met.   
 
 
4.1.2 
Mixed Chimerism and Immune Reconstitution 
 
A goal of our study is to monitor hematopoietic chimerism in children and young 
adults with SCD who receive a haploidentical HSCT.  Based on previous 
experience, we would anticipate at least 80% of patients will have durable (100 
days) donor cell engraftment sufficient to correct the erythroid defect of SCD.  
Our approach will be to determine the donor contribution to hematopoiesis at 
multiple time points after transplantation. This will include measurements of 
initial bone marrow chimerism after engraftment, followed by sequential 
measurement of unsorted peripheral blood chimerism.  It is standard of care to 
measure chimerism following stem cell transplant. This is not experimental.  
Donor chimerism analysis will be evaluated from the bone marrow and 
peripheral blood at the time of full engraftment (roughly between day 21-28 post 
stem cell Hap-HSCT) and then approximately every two weeks until 100 days 

                                                                                                                            
 
 
SCDHAP 17 
 
 
post Hap-HSCT. Chimerism will then be followed monthly until day 180 post 
Hap-HSCT and at least every three months until one (1) year post Hap-HSCT. 
Chimerism is measured by a small tandem repeats (STR) PCR technique using 
total nucleated cell DNA from peripheral blood or bone marrow leukocytes. [92, 
93] 
 
Erythroid chimerism will be assessed based on the ratio of βA to βS chains by 
hemoglobin electrophoresis every two weeks post-transplantation until day +100 
approximately monthly until day 180, then approximately every three months 
until one year.  
 
Functional and quantitative studies of T-cells will be performed as part of the 
standard of care for post stem cell transplant patients. Investigations of immune 
reconstitution will include characterization of T-cell subsets (CD3, CD4, CD8) at 
approximately day 28 and day 100 post HSCT, then every three months for two 
years. Both TCRαβ and γδ+ presentation will be assessed. Additional testing on 
T-cell number and function including testing T-cell receptor diversity by 
spectratyping, TCR-rearrangement excision circles (TRECs), and assays of T-cell 
function including T-cell responses to mitogens may be assessed if clinically 
indicated.  
 
The tempo of B-cell reconstitution will also be a focus, including enumeration of 
B-cells and determination of functional activity through measurement of 
immunoglobin levels. Finally, we will assess NK number. These tests will be 
performed at approximately day 28 and day 100 post HSCT, then every three 
months for two years as part of the standard of care for post stem cell transplant 
patients.  
 
 
4.1.3   Assessing Effects of HapSCT on End Organ Damage 
 
Previous studies have demonstrated that patients with SCD who undergo 
successful stem cell transplantation have cessation and even in some instances 
improvement in their previously documented end organ dysfunction secondary to 
their SCD that occurred prior to transplantation. We will measure outcomes 
related to end organ dysfunction including cardiac, pulmonary, liver, renal and 
CNS both prior to transplantation and at 6 monthly intervals after transplantation. 
Evaluation of end organ dysfunction as described below is standard of care in the 
post-transplant setting. These evaluations are not experimental or related to 
research.  
 
Growth and development: Each subject will have height and weight measured 
prior to initiation of transplantation. When available, growth curves from the 
years preceding transplantation will also be evaluated. Height and weight will be 
measured at every post- transplantation visit to evaluate growth in a longitudinal 
manner following the intervention.  
 
Cardiac: Echocardiography will be performed at 12 months post-transplantation 
and then yearly for at least five years following transplantation to evaluate for 
cardiac dysfunction and evidence of pulmonary hypertension as part of our 

                                                                                                                            
 
 
SCDHAP 18 
 
 
standard of care for post-transplantation patients. A cardiac T2* will also be 
performed to assess for iron overload. 
 
Pulmonary: Pulmonary function testing will occur at approximately day 180 
post-transplantation and then at approximately yearly intervals post-
transplantation for two years in asymptomatic individuals with normal O2 
saturations at routine visits. Those individuals with aberrant O2 saturations will 
be followed more regularly as determined by our pulmonology colleagues. 
 
Renal: Creatinine and BUN evaluation will be performed daily starting from day 
+1 until discharge from the hospital. These values will be assessed at each clinic 
visit following discharge, and/or at least every other week through the first 180 
days post-transplant. They will be performed at approximately monthly intervals 
through one year. Creatinine clearance will be assessed at day +180 and 1 year 
post-transplantation.  
 
Liver: Liver function testing (including AST, ALT, GGT and bilirubin level) will 
be performed daily starting from day +1 until discharge from the hospital. These 
values will be assessed at each clinic visit following discharge, and 
approximately weekly through the first 180 days post-transplant. They will be 
performed at approximately monthly intervals through one year.  Additionally, 
liver MRI with coronal and axial T2 star sequences will be performed at 12 
months post-transplant if clinically indicated.  
 
CNS: Research participants who undergo Hap-HSCT will undergo detailed 
neurological, neuroimaging and neurocognitive evaluation.  Assessment will be 
at BASELINE, and at approximately 12, 24, 36, and 48 months post-
transplantation as determined by our neuropsychology colleagues. We will 
evaluate sickle vasculopathy and brain parenchyma in SCD research participants 
by conventional MRI/MRA, as well as arterial spin labeling (ASL) perfusion and 
diffusion tensor imaging (DTI) of white matter.   
 
Neurocognitive development will be measured by Dr. Scott Hunter and his 
clinical team. (See appendix II) Full assessments will include evaluation of 
neurocognitive function, mood/behavior and executive functioning. This testing 
will occur at baseline and then at one year post-transplantation. Testing at 6 and 
18 months post transplantation with focus on behavioral testing. Focused 
assessment based on individual patient will be assessed yearly starting at two 
years post transplantation and continue to five years.  Many of these testing 
strategies have been previously used to evaluate neurocognitive development in 
patients with sickle cell disease post–transplantation, and as part of routine 
care.[26]  
 
5.0 
TREATMENT OR INTERVENTION 
 
The treatment protocol is outlined in the schema below. The treatment of patients with 
myeloablative chemotherapy followed by stem cell infusion and subsequent clinical monitoring is 
considered standard of care therapy for patients undergoing stem cell transplantation. The 
mobilization of peripheral blood stem cells using GCSF and apheresis is standard of care for stem 

                                                                                                                            
 
 
SCDHAP 19 
 
 
cell transplant donors. The only experiemental components of treatment is the use of the Miltenyi 
CliniMACS device for αβ T-cell depletion.  
 
Figure 1. Study Schema  
GCSF indicates granulocyte colony-stimulating factor. D indicates days. Yr 
indicates years.  
 
5.1 
Research Participant Selection 
 
Research participants will be recruited from the UCM sickle cell disease joint pediatric and adult 
cohort.  Children, adolescents and young adults followed at other institutions are also eligible.  It 
is anticipated that accrual for Stage I of this study will be achieved within approximately 18-24 
months.  We currently have eight children/adolescents in the process of evaluation for 
haploidentical HSCT for SCD.  This protocol will treat an underserved and vulnerable 
(pediatric/adolescent/young adult) population using an investigational approach to HSCT. 
Eligibility criteria for this protocol are stricter than those for MSD transplants, and delineate those 
children/adolescents with the poorest prognosis for whom therapeutic options are limited. [24]     
 
5.2 
Donor Selection: 
 
Donor selection prior to stem cell transplant is standard of care and includes HLA typing of any 
potential donors and medical evaluation to determine suitability of donor. This study will 
consider parental donors in addition to sibling donors but each potential donor will be evaluated 
per standard of care procedures. This is not experiemental or for the purposes of research.  
 
5.2.1 
All family members who are at least a 5/10 HLA match will be considered. 
 
5.2.2 
Potential donors will be screened for hemoglobinopathy by electrophoresis. 
Patients with sickle cell disease trait will be considered eligible. Any patient with 
sickle cell disease will not be eligible to donate.  
 
5.2.3  Donors will be excluded from consideration if any anti-donor HLA antibodies 
are detected during pre-transplant evaluation. 
 

                                                                                                                            
 
 
SCDHAP 20 
 
 
5.2.4 
Donors must be medically fit to undergo therapy with G-CSF and peripheral stem 
cell mobilization and cleared by their physician to undergo procedure.   
 
5.2.6  If multiple donors are eligible, the transplant physician team will choose the best 
donor. Priority will first be given to the donor with the most HLA allele matches. 
Following that preference will be given to maternal donors given improved 
survival noted in maternal versus paternal haploidentical donors attributed to 
bidirectional immunological tolerance in utero.[94, 95]  
 
5.3 
Stem Cell Mobilization/Graft Collection: 
 
Donors will undergo stem cell mobilization per our institutional standard following evaluation by 
their physician and resultant medical clearance to donate. Donors will receive GCSF 10mcg/kg 
with close observation of peripheral CD34+ cell count. When the peripheral CD34+ cell count is 
adequate (20 cells/uL), the donor will have a peripheral intravenous line (when possible) or an 
apheresis catheter placed in conjunction with the blood bank and processing lab, and cells will be 
collected. Donors may undergo pheresis for two consecutive days if a single day of pheresis does 
not allow for a sufficient collection. This is standard of care for stem cell mobilization and graft 
collection. This process is not experiemental or for research.  
 
 
 
5.4 
Collection of Autologous Back-up Bone Marrow, Pre-Conditioning and Conditioning 
Therapy   
 
5.4.1 
Collection of Autologous Bone marrow:       
 
  
Each recipient will undergo bone marrow harvest 1-2 months prior to stem cell 
infusion. This stem cell source will serve as a stem cell rescue if a patient 
develops primary or secondary engraftment failure after a subsequent stem cell 
boost. Stem cells will be stored for future use of the patient only. Patient will be 
admitted the evening prior to their bone marrow harvest. The patient will receive 
intravenous fluid and transfusion of donor pRBC to reach a goal hemoglobin 
level of 10 g/dL prior to general anesthesia with the goal of preventing vaso-
occlusive manifestations of SCD following anesthesia. This is standard of care. 
This process is not experiemental or for research. 
 
5.4.2  Pre-conditioning therapy: 
 
  
Hydroxyurea will be given for approximately 3 months prior to the conditioning 
therapy, (approximately day -102 to day -8).  Hydroxyurea will begin with a 
starting dose 20 mg/kg/day and titrated up to a goal of 30mg/kg/day.  The dosage 
may be adjusted based on the absolute neutrophil count.  Doses may be held for 
an ANC < 500/mm3. Doses may also be held prior to collection of autologous 
bone marrow. Dose modifications or changes in doses or days of administration 
for HU, which are done for safety reasons or as clinically indicated, will be done 
without causing protocol variations or violations. This is standard of care for 
transplant in sickle cell disease. This process is not experiemental or for research. 
 
 
 
 

                                                                                                                            
 
 
SCDHAP 21 
 
 
5.4.3  Conditioning Regimen 
 
1. Thiotepa 
Dosage and Route of Administration 
• 
The dose is 5 mg/kg/dose intravenously every 12 hours (2 doses) on Day -8  
 
2. Busulfan 
Dosage and Route of Administration 
• 
The dose is 0.8 mg/kg/dose intravenously every 6 hours (16 doses) on days -7, -6, 
-5, -4 and -3 
 
3. Fludarabine 
Dosage and Route of Administration 
• 
The dose is 40 mg/m2/dose intravenously every 24 hours (4 doses) on days -6, -5, 
-4, and -3 
 
4. ATG 
Dosage and Route of Administration 
• 
The dose is 4 mg/kg/dose intravenously every 24 hours (3 doses) on days -5, -4, 
and -3 
 
5. Rituximab 
Dosage and Route of Administration 
• 
The dose is 200mg/m2/dose intravenously every 24 hours (3 doses) on days -5, -4, 
and -3 
 
This conditioning regimen is considered standard therapy in the context of haploidentical stem 
cell transplant. These agents have also been used safely in patients with sickle cell disease and 
pediatric patients. This regimen is not experiemental or being used for the purpose of research.  
 
 
 
Table 5. Schema of Conditioning Regimen 
Day 
Treatment 
-8 
Thiotepa 
-7 
Busulfan 
-6 
Busulfan, Fludarabine 
-5 
Busulfan, Fludarabine, ATG 
-4 
Busulfan, Fludarabine, ATG  
-3 
Busulfan, Fludarabine, ATG  
-2 
Rest 
-1 
Rituximab 
 0 
Stem Cell Infusion 
 
For details of source, pharmacology, formulation, supplier, toxicity and route of 
administration, see Section 6.0. The term “every” used in this table is an 
approximate term meaning that these medications noted will be administered 
approximately “every” 12 hours. The drug administration timing in the case of 
“every 12 hours” may be modified +/- 4 hours as clinically indicated such as in 

                                                                                                                            
 
 
SCDHAP 22 
 
 
the case of surgical procedures or to accommodate other necessary medication 
delivery or procedures (such as an emergency CT scan) without resulting in 
protocol variation or violation.  
 
All medication dosing may be modified for research recipients based upon actual 
body weight or adjusted ideal body weight when clinically indicated. 
Specifically, busulfan dosing may be modified to target ideal area under the 
curve based on patient’s pharmacokinetic evaluations per institutional standard. 
See Section 2.6 for additional details. Medication doses may be rounded to the 
nearest integer or to the nearest appropriate quantity when clinically or 
pharmaceutically indicated as per the MD and PharmD.   
    5.5    Research Subject Pre-Transplant Evaluation  (see Appendix I for Roadmap)   
5.5.1 
Time Period  
 
Consent must be obtained any time within 6 months prior to initiation of 
chemotherapy, specifically day-10. Pre-transplantation evaluations will follow 
institutional standards for transplantation of patients with SCD as outlined below.  
5.5.2  General Assessment obtained for all transplantation candidates 
 
  
As per our institutional standards, we will evaluate for signs and symptoms of 
pre- transplant infection with standard pre-transplant infectious disease markers, 
PPD, CT chest/abdomen/pelvis and sinuses and Panorex in combination with 
dental evaluation. Respiratory viral panel from nasal secretions will be tested. 
Blood, nasal, urine and stool cultures will also be sent. Organ function will be 
assessed with ECHO, PFTs, GFR assessment, liver function studies, audiogram. 
PTH, TSH, free T4, IGFBP3, IGF1, and 25 hydroxy-vitamin D level and if the 
subject is greater than 8 years of age testosterone, estrogen, FSH and LH will 
also be assessed. Lipid panel and coagulation profile will be sent. Consultation 
with psychology, social work, nutrition, dentistry, child life, and ophthalmology 
will occur prior to transplantation. This is standard of care. This process is not 
experiemental or for research. 
 
 
5.5.3  Organ specific evaluations 
 
  
These evaluations are standard of care. This process is not experiemental or for 
research. 
 
Central nervous system: Patients will have MRI/MRA brain as well as arterial 
spin labeling (ASL) perfusion and diffusion tensor imaging (DTI) of white matter 
prior to transplantation.  
Psychological Assessment: Neurocognitive functioning will be assessed using a 
comprehensive neuropsychological test battery including measures of general 
intelligence, academic achievement, attention and concentration, memory, visual-
motor and visual-perceptual function, and planning/organization. A listing of the 
specific neuropsychological measures includes Wechsler Intelligence Scales and 
Wide Ranging Achievement testing can be seen in Appendix II.  These tests will 

                                                                                                                            
 
 
SCDHAP 23 
 
 
be performed only if the research participants speak and comprehend English 
adequately as equivalent test instruments are unavailable in other languages. 
Liver: Non-infused Liver MRI will be performed with coronal and axial T2 star 
sequences to measure iron overload. Optional (to be done at discretion of PI) 
Liver biopsy with iron quantitation if clinically indicated as judged by the PI (ie. 
on chronic transfusion therapy for > 6 months or ferritin > 1000 ng/ml). 
Cardiac: Cardiac MRI with T2 star sequences will be performed to evaluate for 
cardiac iron overload. Baseline echocardiogram and electrocardiogram (EKG) 
will be evaluated to assess for function.  
Hematologic and Immunologic: Within four weeks of initiation of conditioning 
chemotherapy, we will evaluate hemoglobin electrophoresis, ferritin, iron, 
transferrin, and percent saturation. CBC will be evaluated within one week of 
conditioning and every two weeks prior to that to allow for titration of 
hydroxyurea. Lymphocyte subset analysis and quantitative immunoglobins will 
also be assessed within four weeks of initiation of chemotherapy.  
Renal: Kidney function will be assessed through either nuclear medicine 
Glomerular Filtration Rate (GFR) evaluation or 24 hour urine creatinine 
clearance. BUN and serum creatinine will also be measured prior to initiation of 
chemotherapy.  
Pulmonary: Pulmonary function testing will be assessed prior to transplantation 
including DLCO and oxygen saturation testing.  
Growth and Development: At a final pre-transplantation visit, height and weight 
will be measured. When possible, growth curves will be obtained from years 
prior to transplant presentation from the research subject’s primary care provider 
or hematologist.  
 
 
5.6 
Pre- transplant donor evaluations 
 
If the donor meets criteria as outlined in section 3.3 and is selected as outlined in section 
5.2, he/she will undergo the following work up: complete history and physical, donor 
questionnaire, chest X-ray, EKG and infectious disease evaluation, CBC with differential, 
reticulocyte count, coagulation studies, LDH, comprehensive metabolic panel with 
phosphorous, magnesium and uric acid. Urinalysis and urine or serum pregnancy testing 
(for female participants great than 12 years old) within 7 days of mobilization. This is 
standard of care. This process is not experiemental or for research. 
 
The infection disease evaluation, in compliance with FDA regulations for all cellular 
product donors includes screening for HIV-1, HIV-2, Hepatitis B (surface and core 
antigen), hepatitis C virus (HCV), Treponema pallidium, HTLV-1, HTLV-2 and CMV. 
The donors will be screened for exposure to Creutzfeldt-Jakob disease and Zika Virus. 
Additionally, as per institutional standards, we will also test for west nile virus and 
Chagas disease. Testing is all done at the American Red Cross, National Testing 
Laboratory which is a CLIA and Centers for Medicare and Medicaid Services (CMS) 

                                                                                                                            
 
 
SCDHAP 24 
 
 
certified site. If a donor is found to be “ineligible” based on this testing the subject and/or 
the guardians will be made aware of testing and eligibility. Should the medical team and 
the family decide to proceed despite ineligibility the device output will have the 
following label: “WARNING: Advise patient of communicable disease risk,” and when 
applicable, “WARNING: Reactive test results for (name of disease agent or disease).” 
This testing will all be completed within 30 days prior to recovery of cellular product.  
 
 
 
 
5.7 
Research Participant Peri- and Post-Hap-HSCT Evaluation (see Appendix I for 
Roadmap) 
 
5.7.1  General and Organ Specific Safety Evaluations for Research Participants per 
Institutional Standard for peri- and post-transplant follow up. 
 
 
 
General Physical Evaluations daily while inpatient (typically admitted at 
initiation of primary conditioning, i.e. day -10) then at least once per week until 
Day 100 and then at least once per month to one year and then at least annually 
to five years post-transplantation.   
 
 
 
CBC with differential at least daily while inpatient until first hospital discharge 
post stem cell transplantation then at least weekly until Day 100 then at least 
once per month till one year and then at least annually till five years.   
 
 
 
Comprehensive metabolic panel with magnesium and phosphate at least daily 
while inpatient until first hospital discharge then at least weekly until Day 100 
then at least once per month till one year and then at least annually till five years.   
 
 
At day 28, day 100 and day 365 evaluations, we will also assess with Urinalysis 
and CXR. On day 100 and day 365 25-hydroxy vitamin D and PTH will be 
evaluated. Ferritin, iron at approximately 3, 6, 12, 24 and 36 months post-
transplantation. 
 
 
MRI/MRA using protocol for SCD with perfusion, diffusion, and DTI at 
approximately 6, 12, 24, 36 and 48 months post-transplantation. TCD study at 
approximately 6 months, 12 months, and 24 months post SCT at the discretion of 
the investigator and if technically feasible. 
 
 
On day 365 dental evaluation, CXR, PFTs, EKG and Echocardiogram, bone 
density will also be obtained. 
 
  
This is standard of care. This process is not experiemental or for research. 
 
5.7.2 
Infectious disease evaluation and monitoring for viral reactivation 
 
CMV, BK and Adenoviral PCRs will be evaluated weekly starting on day +15 
through day 100 and then monthly for one year. Antiviral preemptive or 
therapeutic interventions will be adjusted accordingly to institutional guidelines. 
Additionally, should the patient develop fevers, cultures will be drawn and 
broad-spectrum antibiotics will be initiated. Additional evaluations will be 
obtained at the discretion of the clinical team and principal investigator. 

                                                                                                                            
 
 
SCDHAP 25 
 
 
 
Research participants will have EBV DNA levels monitored in an effort to detect 
early viral reactivation as a harbinger of lymphoproliferative disorder (EBV-
LPD), allowing for preemptive therapy. Samples of peripheral blood will be 
drawn weekly from approximately day +15 to day +100 post-transplant and 
assayed for quantitative EBV DNA levels.  This time period may be extended for 
research participants with delayed immune reconstitution.  Research participants 
with EBV DNA levels > 1,000 copies/mL on two consecutive measurements or 
those with EBV-LPD may be treated with rituximab (monoclonal anti-CD20) 375 
mg/m2 IV and then as clinically indicated. [96] 
  
This is standard of care. This process is not experiemental or for research. 
 
5.7.3 
Measurement of Chimerism 
 
Molecular chimerism: Peripheral blood engraftment analysis will be obtained 
weekly starting on day +21 and weekly thereafter until day 100. After day 100 
these studies will be checked approximately monthly until 1 year post 
transplantation and then approximately every 3-6 months in the subsequent 5 
years. Bone marrow evaluation at day 28, day 100, and day 365 will be obtained 
to evaluate for marrow engraftment as per current institutional standard.  
 
Erythroid chimerism: Hemoglobin electrophoresis of with quantitation of F, A2, 
S at approximately two week intervals post transplantation until day 100 and then 
monthly until one year. It will subsequently be checked yearly through five years 
post-transplant. If a bone marrow aspirate is clinically indicated in the post-
transplant period, it will be tested for erythroid precursor chimerism.  
  
This is standard of care. This process is not experiemental or for research.  
 
5.7.4 
Immune reconstitution 
Immune reconstitution will be evaluated with a focus on lymphocyte recovery 
and function. Functional and quantitative studies of T-cells will be performed as 
part of the standard of care for post stem cell transplant patients. Investigations of 
immune reconstitution will include characterization of T-cell subsets (CD3, CD4, 
CD8) at approximately day 28 and day 100 post HSCT, then every three months 
for two years. Both TCRαβ and γδ+ presentation will be assessed. Additional 
testing on T-cell number and function including testing T-cell receptor diversity 
by spectratyping, TCR-rearrangement excision circles (TRECs), and assays of T-
cell function including T-cell responses to mitogens may be assessed if clinically 
indicated.  
 
 
The tempo of B-cell reconstitution will also be a focus, including enumeration of 
B-cells and determination of functional activity through measurement of 
immunoglobin levels. Finally we will assess NK number. These tests will be 
performed at approximately day 28 and day 100 post HSCT, then every three 
months for two years as part of the standard of care for post stem cell transplant 
patients. 

                                                                                                                            
 
 
SCDHAP 26 
 
 
 
This is standard of care. This process is not experiemental or for research 
5.7.5 
Neuropsychologic Assessment 
Neuropsychologic: Evaluations will be performed at approximately 12 months 
post transplantation and yearly post-transplant where feasible. These tests will be 
performed only if the research participants speak and comprehend English 
adequately as equivalent test instruments are unavailable in other languages. See 
appendix II for additional details.  
This is standard of care. This process is not experiemental or for research. 
 
 
5.8 
Supportive Care Guidelines Specific for Patients with SCD Post-Transplant 
 
Anticonvulsants will be prescribed to all research recipients starting on day -10 and to continue 
for three months post transplant. This approach is standard of care for patients with SCD who 
have a known risk of neurotoxicity in the peri-transplant period. [17, 64] For safety reasons, 
research participants’ hemoglobin should be kept at approximately 9-11 mg/dl.  Platelets should 
be maintained at approximately >50,000mm3.  Maintenance of these levels has been proven to 
prevent CNS events in patients previously transplanted for SCD. [17, 64] This is standard of care. 
This process is not experiemental or for research. 
 
 
5.9 
Stem Cell Boosts 
 
Donor chimerism studies will be performed by small tandem repeat (STR) analysis on 
either bone marrow or peripheral blood beginning on or about day 21 after engraftment, 
approximately weekly thereafter until day 100, and then as indicated in section 5.7.3 
above or as clinically indicated.  
 
These studies will determine the need for stem cell boosts.  Criteria will generally be the 
level of donor chimerism (<5%) in whole blood DNA or hemoglobin electrophoresis 
demonstrating >60% hemoglobin S:  
 
5.9.2 
A second peripheral blood stem cell graft will be obtained from the transplant 
donor and processed using a Miltenyi CD34+ selection column in the Cell 
Processing Laboratory according to applicable cGMP and cGTP guidelines. 
 
5.9.3 
Research participants with low-level donor whole blood or bone marrow 
chimerism, elevated HbS percentage (>50% on two occasions at least 2 days 
apart), and/or low peripheral blood counts may receive an infusion of αβ+ T-cell 
depleted stem cells from the original donor when possible. 
 
5.9.4 
Should graft rejection occur, research participants will be reconditioned with 
anti-T Cell antibody, steroids, cyclosporine and receive a CD34+-selected stem 
cell boost from the original donor at the discretion of the treating physician and 
in agreement with the patient/family.  A separate informed consent document is 
required to be signed by the legal guardian for this procedure. 

                                                                                                                            
 
 
SCDHAP 27 
 
 
 
5.9.5 
Should graft failure occur following a CD34+-selected stem cell boost, re-
infusion of host bone marrow cells will be recommended and the research subject 
will be taken off study.  
 
This is standard of care. This process is not experiemental or for research. 
 
5.10 
Compliance with post-transplantation evaluations 
Compliance with post-transplantation evaluations is highly contingent upon the 
participant’s/legal guardian’s compliance with returning to the clinic for follow-up 
appointments.  Therefore, once the research participant is four months post-transplant, the 
required monthly follow-up evaluation schedule may be adjusted by +/-7 days without 
causing protocol violations/variations; this applies only in the case when the research 
participant fails to return to the clinic for regularly scheduled clinic visits.  If the 
scheduled weekly or monthly evaluation falls on a holiday or during a holiday time 
period, a reasonable adjustment in the follow-up clinic visit due to the holiday schedule is 
expected and would not be noted as a protocol variation or violation.  Every effort will be 
made to encourage and have the research participant/legal guardian return for the 
scheduled clinic visits. 
 
  
6.0 
DRUG & DEVICE INFORMATION 
 
6.1 
THIOTEPA (Thioplex by Immunex) (TESPA, TSPA) 
 
Source and Pharmacology: Thiotepa is a cell-cycle nonspecific polyfunctional alkylating 
agent.  It reacts with DNA phosphate groups to produce cross-linking of DNA strands 
leading to inhibition of DNA, RNA and protein synthesis.  Thiotepa is extensively 
metabolized 
in 
the 
liver 
to 
metabolites 
that 
retain 
activity, 
primarily 
triethylenephosphoramide (TEPA).  The main route of elimination is via the urine, 
mainly as metabolites; the elimination half-life of the thiotepa is 2.5 hours, and that of 
TEPA is 17.6 hours. 
 
Formulation and Stability: Thiotepa is supplied in single-use vials containing 15 mg of 
lyophilized thiotepa, 80 mg NaCl and 50 mg NaHCO3.  The intact vials should be stored 
under refrigeration and protected from light.  Each vial should be reconstituted with 1.5 
ml of sterile water for injection to yield a concentration of 10 mg/ml.  Further dilution 
with sterile water for injection to a concentration of 1 mg/mL yields an isotonic solution; 
if larger volumes are desired for intracavitary, IV infusion, or perfusion therapy, this 
solution may then be diluted with 5% dextrose or 0.9% NaCl containing solutions.  The 
10 mg/ml reconstituted solution is chemically stable when stored in the refrigerator for up 
to 5 days, however, it is recommended that solutions be prepared just prior to 
administration since they do not contain a preservative.  Reconstituted solutions should 
be clear to slightly opaque: the solutions may be filtered through a 0.22 micron filter to 
eliminate haze. 
 
Supplier:  Commercially available; manufactured by Immunex. 
 

                                                                                                                            
 
 
SCDHAP 28 
 
 
Toxicity:  Dose limiting toxicity is myelosuppression.  The leukocyte nadir may occur at 
any time from 10 to >30 days.  Other toxicities include pain at the injection site, nausea 
and vomiting, anorexia, mucositis, dizziness, headache, amenorrhea, interference with 
spermatogenesis, and depigmentation with topical use.  Allergic reactions, including skin 
rash and hives, have been reported rarely.  Rare cases of apnea, hemorrhagic cystitis, and 
renal failure have occurred.  Thiotepa is mutagenic, carcinogenic, and teratogenic in 
animals.  Pregnancy category D.  
 
Route of Administration: Thiotepa will be administered as an intravenous infusion. 
  
 6.2 
METHYLPREDNISOLONE (Medrol, Solu-Medrol) 
 
Source and Pharmacology: This is an adrenal corticosteroid, anti-inflammatory, immuno-
suppressant.  Decreases inflammation by suppression of migration of polymorphonuclear 
leukocytes and reversal of increased capillary permeability. 
 
Formulation and Stability: Tablet (4mg), Sodium Succinate power for Inj. 125, 500mg. 
 
Supplier: Commercially available. 
 
Toxicity: Toxicities include edema, hypertension, vertigo, seizures, psychoses, headache, 
pseudotumor cerebri, acne, skin atrophy, impaired wound healing, Cushing’s syndrome, 
pituitary adrenal axis suppression, growth suppression, glucose intolerance, hypokalemia, 
alkalosis, peptic ulcer, nausea, vomiting, transient leukocytosis, muscle weakness, 
osteoporosis, fractures, cataracts, glaucoma, and increased risk of infection. 
 
Route of Administration: oral, intravenous infusion. 
 
6.3 
BUSULFAN (Myeleran) 
 
Pharmacology:  Busulfan is a polyfunctional alkylating agent. It interferes with the 
normal function of DNA by alkylating intracellular nucleophiles and cross-linking DNA 
strands.  Busulfan is cell cycle phase non-specific. It is well absorbed orally and is 
metabolized by the liver.  Drugs that induce hepatic metabolism (e.g., phenytoin) increase 
clearance and those that inhibit hepatic metabolism (e.g., itraconazole) may decrease 
clearance.  The plasma half-life is ≈ 2.5 hours in adults, but children may have higher 
clearances.  
Formulation and Stability: Busulfan is available as a 2mg tablet and as a solution or 
suspension for intravenous administration. 
 
Supplier: Parenteral solutions are commercially available.  Parental suspension is 
investigational. 
 
  
Toxicity:  Acute dose limiting toxicity is myelosuppression including leukopenia, 
thrombocytopenia and anemia.  This effect is delayed with a nadir of 14-21 days.  Some 
patients may develop bone marrow fibrosis.  Nausea and vomiting are generally mild.  
Other GI symptoms include diarrhea, anorexia and associated weight loss.  Seizures are 
associated with high doses.  Other side effects include liver dysfunction, skin 
hyperpigmentation, skin rash, gynecomastia, sterility, cataracts and alopecia.  Secondary 
cancers have occurred.  “Busulfan lung”, manifested by diffuse interstitial pulmonary 

                                                                                                                            
 
 
SCDHAP 29 
 
 
fibrosis, persistent cough, fever, rales and dyspnea may occur, most commonly after high 
doses or prolonged therapy. 
 
 
Route of administration: intravenous infusion. 
 
 
6.4 
G-CSF (Filgrastim) (Neupogen) 
Source and Pharmacology:  G-CSF (granulocytic colony stimulating factor), is a 
biosynthetic hematopoietic agent that is made using recombinant DNA technology in 
cultures of Escherichia coli.  G-CSF stimulates production, maturation and activation of 
neutrophils.  In addition, endogenous G-CSF enhances certain functions of mature 
neutrophils, including phagocytosis, chemotaxis and antibody--dependent cellular 
cytotoxicity. 
 
Formulation and Stability:  G-CSF is supplied in vials containing 300 mcg and 480 mcg 
of G-CSF at a concentration of 300 mcg/ml.  The intact vials should be stored under 
refrigeration.   The vials can be left out of refrigeration for 24 hours, but should be 
discarded if left at room temperature for longer periods of time.  G-CSF can be drawn up 
into tuberculin syringes for administration and stored under refrigeration for up to 7 days 
prior to usage.  G-CSF can be further diluted for IV infusion in 5% dextrose.  Do not 
dilute in saline---precipitate may form.  If the final concentration of this product is < 15 
mcg/ml, it is recommended that albumin be added to a final concentration of 2mg/ml 
(0.2%) to minimize adsorption of the drug to infusion containers and equipment.   
 
Supplier:  Commercially available 
 
Toxicity:  G-CSF causes marked leukocytosis.  Adverse reactions reported commonly 
include bone pain, thrombocytopenia, diarrhea, nausea, rash, alopecia, fever, anorexia 
and pain or bruising at the injection site.  Allergic reactions, MI, atrial fibrillation, and 
splenomegaly have been reported rarely.  G-CSF is contraindicated in patients with 
allergy to E. coli derived products. 
Dosage and Route of Administration:  Subcutaneous or intravenous. 
6.5 
HYDROXYUREA (Hydrea®) 
Source and Pharmacology: Hydroxyurea is an antimetabolite. As an antineoplastic, 
hydroxyurea is considered a cell-cycle specific agent effective in the S-phase of mitosis. 
Hydroxyurea inhibits the conversion of DNA bases by blocking ribonucleotide reductase. 
Since the drug causes cells to arrest at the G1-S interface, and irradiation sensitivity is 
maximum during this time, concomitant hydroxyurea and irradiation result in synergistic 
toxicity (Gilman et al, 1990).  
 
The known pharmacological effects of hydroxyurea which may be beneficial in sickle 
cell anemia include increasing hemoglobin F levels in red blood cells (RBCs), decreasing 
neutrophils, increasing water content of RBCs, increasing deformability of sickled cells, 
and altering the adhesion of RBCs to endothelium (Prod Info Droxia(R), 2001).  
 

                                                                                                                            
 
 
SCDHAP 30 
 
 
Formulation and Stability: 500 mg capsules.  Store in a tightly closed container at room 
temperature (25 degrees Celsius, 77 degrees Fahrenheit); avoid excessive heat.  
 
Supplier:  Commercially available 
Toxicity: Toxicities include myelosuppression, anorexia N/V/D, constipation, stomatitis, 
drowsiness, dizziness,  headaches, skin and facial erythema, skin rash, nail changes, 
alopecia, skin changes, dysuria, abnormal LFTs, jaundice, abnormal BUN and creatinine, 
seizures and auditory hallucinations.  
Dosage and administration: Oral administration; Hydroxyurea will begin with a starting 
dose 15 mg/kg/day as a single dose.  The dosage may increase by 5mg/kg/day weekly if 
the absolute neutrophil count (ANC) remains > 1000/mm3 to a maximum of 30 
mg/kg/day.  Dose may be held for an ANC < 1000/mm3. 
 
6.6 
RITUXIMAB (Rituxan ™) 
 
 
Source and Pharmacology: Rituximab is a murine /human chimeric monoclonal antibody. 
It is specific for the CD20 antigen located on B-cells. Rituximab has been shown to 
mediate complement-dependent tumor cell lysis and antibody-dependent cellular 
cytotoxicity. Direct binding to the CD20 antigen is thought to play a role in inhibition of 
cell growth. Rituximab is administered intravenously. The mean serum half-life after a 
single IV dose of 375 mg/m2 is 59.8 hours (range 11.1-104.6 hours).  
 
Formulation and Stability: Rituximab is available as 100 mg/10 ml single-use and 500 
mg/50 ml single-use vials. Each vial also contains sodium chloride 9 mg/ml, sodium 
citrate 7.35 mg/ml, polysorbate 80 0.7 mg/ml and water for injection. Rituximab for 
injection concentration must be diluted with 5% Dextrose or 0.9% NaCl prior to 
administration. After dilution, unused drug is stable for 24 hours when refrigerated (2-8 
degrees Celsius) and 12 hours at room temperature. Vials should be protected from direct 
sunlight.  
 
Supplier:  Commercially available. 
 
Toxicity:  Hypersensitivity reactions may occur; therefore, premedication with 
acetaminophen and diphenhydramine should be considered before each infusion. The 
most common toxicities are infusion related and may include chills, fever, headache, 
nausea, vomiting, angioedema (13%), hypotension (10%), bronchospasm (8%), and 
arrhythmias. Other possible adverse reactions include thrombocytopenia, myalgias, 
arthralgias, asthenia, and throat irritation. 
 
Note: Intravenous; Do not administer as an intravenous push or bolus.   
 
6.7 
ATG (anti-thymocyte globin)  
 
Source and Pharmacology: Immunosuppressant involved in the elimination of antigen-
reactive T lymphocytes (killer cells) in peripheral blood or alteration in the function of T-
lymphocytes, which are involved in humoral immunity and partly in cell-mediated 
immunity. 

                                                                                                                            
 
 
SCDHAP 31 
 
 
 
Formulation and Stability: For IV use only. Infuse over at least 4 hours through a 0.2 to 1 
micron inline filter. Allow solution to reach room temperature prior to infusion. Infusion 
must be completed within 24 hours of preparation. May cause vein irritation (chemical 
phlebitis) if administered peripherally; high flow veins are preferred to reduce phlebitis 
(infuse into vascular shunt, arterial venous fistula, or high flow central vein). 
 
Monitor closely throughout infusion for allergic reaction. Appropriate resuscitative 
equipment should be nearby during administration. May require premedication with an 
antipyretic, antihistamine, and/or corticosteroid to prevent reactions. Discontinue infusion 
for anaphylaxis or respiratory distress. Administer epinephrine, corticosteroids, 
antihistimines, and/or antipyretics as indicated to manage reactions.  
 
Due to possible infusion-related reactions, it may be preferable to avoid initiation 
treatment late in the day or on weekends; consider withholding beta-blockers prior to 
administration to avoid suppressing compensatory responses to anaphylaxis.  
 
Toxicity: Chills, headaches, dermatologic reaction, pruritus, skin rash, urticarial, 
leukopenia, thrombocytopenia, arthralgia, fever, bradycardia, cardiac disease/failure, 
chest pain, edema, hypertension, hypotension, myocarditis, phlebitis, thrombophlebitis, 
agitation, burning sensation, dizziness, encephalitis, generalized ache, lethargy, seizure, 
diaphoresis, night sweats, diarrhea, nausea, stomatitis, vomiting, proteinuria, 
lymphadenopathy, hepatosplenomegaly, hepatic function tests, anaphylaxis, serum 
sickness, viral infection, back pain, joint stiffness, myalgia, periorbital edema, renal 
function abnormality, dyspnea, pleural effusion, respiratory distress 
 
6.8 
FLUDARABINE (fludara) 
 
Source and pharmacology: Inhibits DNA synthesis by inhibition of DNA polymerase and 
ribonucleotide reductase; also inhibits DNA primase and DNA ligase. 
  
Formulation and Stability: Intravenous. The manufacturer recommends administering 
over ~30minutes. Continuous infusion and IV bolus over 15 minutes have been used for 
some off label protocols. Store intact vials under refrigeration or at room temperature, as 
specified according to each manufacturer’s labeling. Reconstituted solution or vials of the 
solution for injection that have been punctured (in use) should be used within 8 hours.  
 
Supplier: commercially available 
 
Toxicity: Edema, fever, fatigue, pain, chills, rash, nausea/vomiting, anorexia, diarrhea, 
gastrointestinal bleeding, urinary tract infections, myelosuppression, anemia, neutropenia, 
thrombocytopenia, weakness, myalgia, paresthesia, visual disturbance, cough, 
pneumonia, dyspnea, upper respiratory infection, diaphoresis, angina, arrhythmia, 
cerebrovascular accident, heart failure, MI, supraclavicular tachycardia, deep vein 
thrombosis, phlebitis, aneurysm, transient ischemic attack, malaise, headache, sleep 
disorder, cerebellar syndrome, depression, mentation impairment, alopecia, pruritis, 
seborrhea, hyperglycemia, dehydration, stomatitis, esophagitis, constipation, mucositis, 
dysphagia, dysuria, hesitancy, hemorrhage, cholelithiasis, liver function abnormality, 
liver failure, osteoporosis, arthralgia, hearing loss, hematuria, renal failure, renal function 
test abnormality, proteinuria, pharyngitis, allergic pneumonitis, hemoptysis, sinusitis, 
bronchitis, epistaxis, hypoxia, anaphylaxis. 

                                                                                                                            
 
 
SCDHAP 32 
 
 
 
6.9 
General description of the CliniMACS System 
 
This system is described in depth in the Investigators Brochure.  Below is a brief 
description of the operation of the system: 
 
The mechanism of action of the CliniMACS Cell Selection System is based on magnetic-
activated cell sorting.  The CliniMACS device is a powerful tool for the isolation of many 
cell types.  Heterogeneous cell mixtures, e.g. apheresis products, can be separated in a 
magnetic field using an immunomagnetic label specific for the cell type of interest, such 
as CD34+ human hematopoietic cell selection, TCRαβ+ CD3+ T-cell depletion, or 
CD56+ NK cell enrichment. The cells to be isolated or depleted are specifically labeled 
with superparamagnetic particles by an antibody directed toward a cell surface antigen.  
After magnetic labeling, the cells are separated using a high-gradient magnetic separation 
column. The magnetically labeled cells are retained in the magnetic column while the 
unlabeled cells flow through.  The retained cells are eluted by removing the magnetic 
field from the column, the washing the desired cells out and collecting them. 
 
The superparamagnetic particles are small in size (about 50 nm in diameter) and are 
composed of iron oxide/hydroxide and dextran conjugated to monoclonal antibodies.  
These magnetic particles form a stable colloidal solution and do not precipitate or 
aggregate in magnetic fields.  The CliniMACS device is comprised of a computer-
controlled instrument incorporating a strong permanent magnet and a separation column 
with a ferromagnetic matrix to separate the cells labeled with the magnetic particles.  The 
high-gradient system allows the application of strong magnetic forces and a rapid 
demagnetization.  Small ferromagnetic structures, such as the column matrix, placed in a 
magnetic field, concentrate this homogenous field and thereby produce high magnetic 
gradients. In their immediate proximity, the ferromagnetic structures generate magnetic 
forces 10,000-fold greater than in the absence of those structures enabling the retention of 
magnetically-labeled cells. After removing the column from the magnet, the rapid 
demagnetization of the column matrix allows the release of retained cells. The 
CliniMACS device is comprised of a closed-system sterile tubing set containing columns 
with a coated ferromagnetic matrix and a paramagnetic, cell specific, labeling reagent.  
The instrument will separate the cells in a fully automated process.  
 
7.0 
EVALUATION CRITERIA  
 
7.1 
The primary goal of this study is:  
 
 
To determine the safety of αβ T-cell depleted haploidentical HSCT in research 
participants with severe SCD. To assess the safety of haploidentical hematopoietic stem 
cell transplantation (HSCT) for children and young adults with sickle cell disease (SCD) 
and stroke (persistent neurologic deficit lasting >24 hours and present on MRI) or 
recurrent ACS (>2 in one year) or recurrent VOC requiring hospital admission >3 times in 
one year. The treatment plan will be considered safe if there is not excessive toxicity.  
Toxicity for this protocol is defined as failure to engraft/graft rejection, or grade III/IV 
aGvHD or death within 100 days post transplantation. 
Our approach will also provide preliminary observational data on the efficacy of Hap-
HSCT for children, adolescents and young adults with severe SCD. Efficacy is defined as 

                                                                                                                            
 
 
SCDHAP 33 
 
 
stable donor engraftment (>5% total nucleated cell DNA) and donor erythropoiesis that 
corrects the SCD hematologic phenotype (<50% HbS in the peripheral blood). 
 
7.2 
The secondary objectives are as follows: 
7.2.1 
To estimate 1-year overall and event-free survival after Hap-HSCT. 
7.2.2 
To observe the incidence and severity of acute and chronic GvHD after Hap-
HSCT. 
7.3 
The exploratory objectives are as follows: 
7.3.1 
To observe the degree of hematopoietic chimerism among different cells subsets 
after haploidentical stem cell transplantation. 
7.3.2  To obtain preliminary information on the degree of engraftment necessary to 
ameliorate the features of SCD and the proportion of participants requiring stem 
cell boosts.  
7.3.3 
To evaluate systemic end organ function and/or complications post Hap-HSCT in 
a longitudinal manner. 
7.3.4 
To monitor immune reconstitution after Hap-HSCT. 
 
8.0 
OFF-STUDY or OFF-THERPAY CRITERIA 
    8.1 The following criteria will be utilized to censor stem cell transplant research 
participants: 
8.1.1 
Withdrawal of research participation - research participants or guardians may 
withdraw their consent and/or permission to participate in this study at any time.  
Research participant and/or legal guardian who withdraw consent and/or 
permission after initiating conditioning will be counted as failures. 
8.1.2 
Failure to obtain >5 X 106 CD34+ cells/kg from two successive donors (after a 
maximum of attempted 2 cycles of G-CSF/apheresis per donor).  Cells from only 
one donor will be infused into the research participant. 
8.1.3 
Documented graft rejection or graft failure after stem cell infusion (boost). 
8.1.4 
Research participant death prior to starting conditioning. 
8.1.5 
Research participant/legal guardian lost to follow-up. 
8.1.6 
Discretion of the Principal Investigator.   
  

                                                                                                                            
 
 
SCDHAP 34 
 
 
     8.2   The following criteria will be used  for stem cell donors to be exluded from ongoing 
medical care:  
 
Stem Cell donors are consented as a standard of care. It is also standard of care for those 
patients to be followed by an apheresis physician and to have the opportunity to withdraw 
from care at any time.  
8.2.1  Donor participant withdrawal. Donor participants may withdraw their consent to 
participate as medical stem cell donors at any time.   
8.3.2  Donor participant lost to follow-up during their donation of stem cells. 
8.3.3 Discretion of the Principal Investigator.   
 
9.0 
REPORTING CRITERIA 
9.1 
Adverse Events1 
During the first year post initiation of primary stem cell transplant, the Principal 
Investigator and the clinical research staff assigned to the study will monitor for adverse 
events and score them according to the criteria specified by the National Cancer 
Institute’s (NCI) Common Toxicity Criteria (CTC) Version 3.0.   
For this Phase I study, NCI grade I–IV AEs will be collected for on-study research 
participants from the initiation of HU/azathioprine until the first 100 days post-transplant.  
After the day 100 time point, AE data collected will be limited to grade III and IV NCI 
AEs and only any clinically significant grade I and II AEs (significant as per the 
judgment of the PI) up to the one-year post-transplant time point.  
Post-haploidentical transplant off-study research participants who are off-study due to 
graft failure/graft rejection after boosts or as per Principal Investigator’s discretion will 
continue to be followed for safety data for 5 years post-initial protocol transplant infusion 
despite off-study status through the late effects data collection methodology detailed 
below.  
 
Research participants who are enrolled but go off-study due to inadequate cell collection 
from 2 successive donors (i.e. research participant enrolled but goes off study and does 
not receive a transplant on this protocol) will be followed for 30 days post last 
HU/azathioprine dose only.  
 
Post-one year late effects monitoring: After the first year post treatment, adverse event 
(AE) data collecting and reporting for on-study participants will be done concurrently 
with the research participant’s annual follow-up visit in the transplant clinic.  After 
conducting a full physical exam, the physician will complete a systems-based late events 
checklist noting any (significant) new or previously diagnosed medical conditions or 
 
1 See also Appendix III: Criteria for Adverse Event Evaluation and Reporting 
 

                                                                                                                            
 
 
SCDHAP 35 
 
 
diagnoses that were present during the time frame since the last annual evaluation.  Any 
clinically significant adverse events noted in these annual (approximately annual) follow-
up visits that in the opinion of the Principal Investigator warrant expeditious reporting 
will be reported to the IRB and FDA accordingly.  All other late effects noted in this 
assessment will be summarized in the IRB and FDA annual review reports.  Annual 
reporting of late effects is mandated until five years post-transplant.   
 
 
Stem cell donor research participants will be followed for grade III and IV NCI adverse 
event from the time period of the initiation of mobilization treatment to 30 days after the 
last day of apheresis only.   If a complete repeat pheresis procedure is required, the 
adverse event collection will restart with the time period of the initiation of the second 
mobilization treatment and will stop 30 days after the last day of the second pheresis 
procedure.  Adverse event reporting for the donor research recipients will be conducted 
according to the same guidelines set forth for research participants in terms of reporting 
to FDA and IRB.  Adverse event information for research participant donors will be 
included in the continuing review reports under a separate table. 
9.2 
Reporting Requirements to the IRB 
 
 
In addition to the continuing review reports to the IRB, the Principal Investigator is 
responsible for reporting serious and unexpected adverse events that impact the safety of 
or risk to research participants.  Non-serious adverse events and expected SAEs will be 
reported to the IRB in the continuing review reports. Serious, unexpected-fatal or serious, 
unexpected-life threatening events are to be reported to the IRB within 48 hours of 
notification of the event to the principal investigator or designees.  All other unexpected 
(non-fatal or non-life threatening) SAEs will be reported within 10 working days of 
notification of the event.  The term “life threatening” refers to an event in which the 
research participant was at risk for death at the time of the event, not an event that 
hypothetically might have caused death if it were more severe.   
 
9.3 
Reporting Requirements to the FDA 
Any unexpected fatal or unexpected life-threatening event that occurs during the time 
period of the initiation of conditioning through the first year post-transplant that is judged 
by the principal investigator to possibly be due to the investigational treatment, will be 
reported to the FDA by telephone or fax as soon as possible but no later than 7 calendar 
days after notification of the event and followed by a complete a written safety report as 
possible within 8 additional calendar days (i.e. full report 15 calendar days total after 
notification of event).   
Unexpected, non-life-threatening SAEs which occur from the time of the initiation of 
conditioning through the first year post transplant that are considered to be due to the 
investigational treatment will be reported to the FDA by written safety report as soon as 
possible but no later than 15 calendar days of the notification of the occurrence of the 
event. Expected SAEs including expected death, unexpected SAEs even unexpected fatal 
SAEs considered by the PI to be not related to the study treatment and non-serious AEs 
will be reported to the FDA in the Annual Review Report.  
All FDA correspondence and reporting will be conducted through the BSD Office of 
Clinical Research (BSD OCR).  The Principal Investigator will submit a copy of the 
Adverse Event Report (AER) form that was submitted to the IRB to the BSD OCR.   

                                                                                                                            
 
 
SCDHAP 36 
 
 
9.4 
Continuing Review Reports 
Reports of progress and summaries of adverse events will be filed with the IRB and the 
Food and Drug Administration at least annually.  
9.6 
Data Submission to Miltenyi Biotec 
Clinical and safety related data will be provided to Miltenyi Biotec, the manufacturer of 
the CliniMACSTM system.  Data will include but is not limited to variables such as the 
transplant research participant’s age and diagnosis, the relationship of the donor to the 
transplant recipient (i.e. mother, father, sister, brother) and donor product(s) related 
information including the stem cell mobilization, processing and infusion procedure. 
Outcome data including lymphohematopoietic reconstitution, immunological response, 
disease response and transplant related complications will be shared with Miltenyi 
Biotec.  Representatives from Miltenyi Biotec will be able to review the research 
participants (donor and transplant research recipient) laboratory and medical record for 
data verification purposes. Copies of all reports to the governing regulatory bodies will 
also be accessible to Miltenyi Biotec upon request.   Information is submitted using a 
unique research participant number.  
9.7 
Reporting to Bone Marrow Transplant Registries 
The Transplant Program reports transplant information to the Center for International 
Bone Marrow Transplant Registry/ Autologous Bone Marrow Transplant Registries 
(CIBMTR).  The CIBMTR is a voluntary research organization of scientists and doctors 
who study various issues in transplantation.  The CIBMTR collects information from 
more than 450 transplant centers from 48 countries.  Information is submitted using a 
unique research participant number.  
9.8 
Study Stopping Rules 
This study will suspend accrual if a stopping criteria is met as described in section 10.0 
below.   Should these events occur, this would prompt discussion amongst investigators. 
Safety analyses will include all subjects receiving any components of the investigational 
therapy.   
 
10.0 
STATISTICAL CONSIDERATIONS 
For the primary analysis, we will test the null hypothesis that the GF rate is 35% against 
the alternative that it is 15%.  We will employ a two-stage design to enable stopping for 
futility if the GF rate is high. [97] Eleven patients will be enrolled in the Stage I and if 4 
or more have GF the trial will be terminated.  (Accrual will be suspended for 100 days 
after the 11th patient has been enrolled to await his/her outcome if exactly 3 GFs have 
been observed up to that point in time.)  If 3 or fewer patients have GF an additional 26 
patients will be entered for a total of 37.  Then if 10 or more GFs have been observed the 
treatment will be rejected, whereas if 9 or fewer have GF, i.e., < 24%, the regimen will be 
considered sufficiently promising to warrant further trials.  This design has an alpha level 

                                                                                                                            
 
 
SCDHAP 37 
 
 
(type I error rate) of 10% and 90% power if the true GF rate is 15%.  The probability of 
terminating after the first 11 patients is 0.57 if the true GF rate is 35%.   
 
Three additional stopping rules will be employed in the event of an unacceptable number 
of deaths, occurrences of aGvHD, or unexpected grade III or higher toxicities deemed 
related to the therapy.  If, at any time, more than two deaths occur, we will suspend the 
trial and consider early termination pending review of the causes of death. We will also 
consider early termination if there is evidence that the rate of grade II or higher aGvHD 
exceeds 25% or the rate of grade III or higher aGvHD exceeds 10%.  Specifically, if 5 of 
the first 11 patients or 13 of 37 develop grade II or higher aGvHD we will consider early 
termination of the trial. (In both instances, the lower, one-sided 90% confidence bound 
exceeds 25%.)  Similarly, if 3 of the first 11 or 7 of 37 exhibit grade III or higher aGvHD, 
termination of the study will be considered (in both cases, the lower confidence bound 
exceeds 10%).  Finally, we will consider early termination if there is evidence that the 
rate of grade III or higher treatment-related toxicities exceeds 25%.  Thus if 5 of the first 
11 or 13 of 37 patients experience irreversible grade III or higher treatment-related 
toxicity, the study will be suspended.  
 
We will follow the above algorithm with three additional modifications:   
i) As an additional safeguard, the first three patients in Stage I of the study will be 
enrolled at a rate of one (1) patient per month. Accrual will be suspended for 100 
days after the 3rd  patient has been enrolled to assess excessive acute and 
subacute toxicities from the  treatment regimen. If excessive toxicity or a 
stopping rule, is not reached in that time frame, we will proceed to complete 
enrollment of the additional 8 research subjects to Stage I of the study. 
ii) If the rate of aGvHD in the first 11 patients is too high (as defined), we will 
modify the treatment protocol to include immune suppression prophylaxis and 
begin anew, enrolling 11 patients in Stage I and up to 26 in the Stage II if no 
further stopping boundary is met.   
iii) If the Stage I data does not meet criteria for stopping rules, we will proceed to 
Stage II. As Stage I of this study will indicate preliminary safety, we will extend 
the study to include patients from 2 years to 25 years. The patients who were not 
previously included (ages 2 to 9 years) will be analyzed both within the entire 
cohort and as a subset population. Should there be two or more patients age 2-9 
who experience death, grade III/IV GvHD, grade III/IV treatment-related toxicity 
or graft failure, the study will be suspended and a meeting will be convened 
among the internal Safety Monitoring committee. In addition to stopping rules 
for this specific cohort, we will also apply a staggering of the enrollment within 
this subset of subjects. Similar to Stage I, we will stagger the enrollment of 
patients ages 2-9 for the first three patients in this age range. We will enroll 
patients at a rate of one (1) patient per month, and accrual will be suspended for 
100 days after the 3rd  patient has been enrolled to assess excessive acute and 
subacute toxicities from the  treatment regimen.  
 
 

                                                                                                                            
 
 
SCDHAP 38 
 
 
Since we will employ stopping rules in the event of an unacceptable number of deaths, 
occurrences of aGvHD, or high-grade toxicity we will stagger enrollment so that, if a 
signal emerges, we will not have exposed an excessive number of patients. We anticipate 
that we will see the vast majority of aGvHD develop prior to 60 days post Hap-HSCT. 
[98-101] 
 
We will continue enrollment at a rate not to exceed 2 patients per month. If we are 
approaching the boundary of any of the stopping rules (3 deaths; 5 of 11 in Stage I or 13 
of 37 > grade II aGvHD in Stage II; 3 of 11 in Stage I or 7 of 37 > grade III aGvHD in 
Stage II; 5 of 11 in Stage I or 13 of 37 grade III or higher treatment-related toxicity in 
Stage II), we will convene a meeting of the principal investigator, co-investigator, and 
statistician to decide whether curtailment of enrollment or its rate is warranted. Please see 
Section 2.5.6 and Appendix 4 for additional information on the monitoring committee. 
 
Cumulative incidence curves for time to GF will be calculated with treatment-related mortality as 
a competing risk. [102] Overall and event-free survival will be estimated using the Kaplan-Meier 
method. [103] Descriptive statistics (mean and standard deviation for continuous variables, 
frequency summaries for categorical data) will be generated for secondary endpoints (immune 
reconstitution, end organ function), together with 90% confidence intervals for parameters of 
interest. Mixed effects regression models will be fit to analyze changes in QOL measures over 
time. [104]  
 
11.0    HUMAN SUBJECTS 
Potential risks of this study include the following: 
 
1. Toxicity from conditioning therapy, including mucosal damage (mucositis), intestinal damage 
(diarrhea) and liver damage (veno-occlusive disease): Based on the proposed conditioning 
regimen and preliminary experience using similar regimens, we anticipate a low (<10% 
incidence of NCI grade IV toxicity). 
2. Risk of infection due to low blood cell counts and weakened immune system: We anticipate a 
fatal infection rate of <5%. 
3. Risk of GvHD: Based on our preliminary experience using highly purified stem cell 
transplantation, we anticipate a risk of severe GvHD (overall grade III/IV acute GvHD or 
extensive chronic GvHD) of less than 10%. 
4. Risk of graft failure, resulting in return of SCD:  We anticipate a risk of graft rejection of up 
to 33%. 
5. Death.  We anticipate the mortality rate will be <10%. 
6. Late effects such as impaired growth, fertility, or treatment-related malignancy.  We 
anticipate that, due to the absence of irradiation, growth will not be significantly impaired.  
The risk of treatment-related malignancy with this therapy is likely less than 1% given the 
absence of cyclophosphamide and irradiation.  Fertility is likely to be impaired given the 
presence of busulfan.  Fertility will be serially assessed at the appropriate time. Participants 
will be educated on this risk prior to transplantation.  
 
 
 

                                                                                                                            
 
 
SCDHAP 39 
 
 
12.0 
OBTAINING INFORMED CONSENT 
 
Significant safeguards must be put in place prior to proceeding with HSCT in each patient. 
Education of the patient and their family will be mandatory, which will include the following: 
 
Dr. Cunningham will conduct an initial presentation of the protocol.  The transplant procedure as 
well as alternative forms of therapy will be presented as objectively as possible.  The risks and 
hazards of the procedure are explained to the patient's responsible family members/legal 
guardians and to the patient.  It will be pointed out that patients with sickle cell anemia have a 
variable course and that there is no clear agreement about the role or optimal time to perform 
alternate donor hematopoietic stem cell transplantation. There will be ample opportunity for 
questions related to the protocol, consents, or issues related to stem cell transplantation for sickle 
cell disease. Patients will also have the opportunity to meet with the social worker and the patient 
advocate before the signing portion of the informed consent process is initiated unless they 
decline. 
 
Additional discussions and question and answer sessions between the PI and the family 
members/legal guardians will be done as needed.  A final session, which will be of similar 
format, at which time the enrollment consent may be signed. Assent will be obtained in all 
children and adolescents > 8 years of age. The social worker, the patient advocate or a designee 
(in the case where the social worker/patient advocate is not available) must be present as well as a 
member of the nursing staff.  Any person(s) of the patient/parent’s choosing will be encouraged 
to attend. 
 
 
13.0   DATA ACQUISITION AND QUALITY ASSURANCE MONITORING  
 
13.1  
Enrollment on Study 
 
Confirm subject eligibility as defined in Section 3.0 Patient Eligibility using the Case 
Report Forms (CRFs). Form will be signed by a PI. Complete and sign the ‘Patient 
Eligibility Checklist’ and “Donor Eligibility Checklist.” Be sure that all required values 
and dates are filled in.  
   
13.2   Data Submission 
 
The study data manager will review Case Report Forms (CRF) for accuracy and 
completeness. Data collected using these forms will then be entered into a secure 
REDCap database accessible to the study investigators and personnel.  
  
13.3  
Quality Assurance Monitoring 
 
Monitoring of timeliness of adverse and serious adverse event reporting will be done as 
events are reported.  Monitoring of modified adverse event reporting, data required for 
primary objectives, and compliance with the conduct of the protocol will be conducted at 
3-month intervals.  
 
 
 

                                                                                                                            
 
 
SCDHAP 40 
 
 
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Horan J, H.A., Lagerlof Diogaurdi, Brown C, Grizzle A, Shelman C, Kanter J, Hale G, Nieder M, 
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Sodani, P., et al., New approach for bone marrow transplantation in patients with class 3 
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Hsieh, M.M., C.J. Wu, and J.F. Tisdale, In mixed hematopoietic chimerism, the donor red cells 
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Walters, M.C., et al., Stable mixed hematopoietic chimerism after bone marrow transplantation 
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Larocca, A., et al., Boost of CD34+-selected peripheral blood cells without further conditioning in 
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Walters, M., et al., Neurologic complications after allogeneic marrow transplantation for sickle 
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Chang, 
Y.J., 
et 
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J Clin Immunol, 2012. 32(2): p. 268-80. 

                                                                                                                            
 
 
SCDHAP 43 
 
 
66. 
Savani, B.N., et al., Absolute lymphocyte count on day 30 is a surrogate for robust hematopoietic 
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Kim, H.T., et al., Absolute lymphocyte count recovery after allogeneic hematopoietic stem cell 
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Lin, R. and Q. Liu, Diagnosis and treatment of viral diseases in recipients of allogeneic 
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Green, M.L., et al., Efficacy of a viral load-based, risk-adapted, preemptive treatment strategy for 
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Russell, M.O., et al., Effect of transfusion therapy on arteriographic abnormalities and on 
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Wilimas, J., et al., Efficacy of transfusion therapy for one to two years in patients with sickle cell 
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Woodard, P., et al., Summary of symposium: the future of stem cell transplantation for sickle cell 
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88. 
Olsson, R., et al., Graft failure in the modern era of allogeneic hematopoietic SCT. Bone Marrow 
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Nollet, F., et al., Standardisation of multiplex fluorescent short tandem repeat analysis for 
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Kristt, D., et al., Hematopoietic chimerism monitoring based on STRs: quantitative platform 
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Stern, M., et al., Survival after T cell-depleted haploidentical stem cell transplantation is improved 
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Tamaki, S., et al., Superior survival of blood and marrow stem cell recipients given maternal 
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Lee, S.E., et al., Risk and prognostic factors for acute GVHD based on NIH consensus criteria. 
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SCDHAP 45 
 
 
Appendix I: Schedule of Events: Clinical and Laboratory Evaluations 
 
 
Required Evaluations of End Organ Function 
Observation 
Prior to 
Transplantation 
Day 28 
Day 100 
Day 180 
Year 1 
Year 2, 4 
Year 3, 5 
Echocardiogram 
X 
 
 
 
X 
X 
X 
EKG 
X 
 
 
 
X 
 
 
PFTs 
X 
 
 
X 
X 
X 
X 
Chest XR 
X 
X 
X 
 
X 
 
 
Cr Clearance or 
GFR 
X 
 
 
X 
X 
 
 
Urinalysis 
X 
X 
X 
 
X 
 
 
T2 Star MRI (liver 
& heart) 
X 
 
 
 
X 
 
 
Brain MRI/MRA  
X 
 
 
X 
X 
X 
X 
Neurocognitive 
Testing 
X 
 
X 
X 
X 
X 
X 
Dental Evaluation 
X 
 
 
 
X 
 
 
Bone Density 
 
 
 
 
X 
 
 
Endocrine 
Evaluation 
X 
 
X 
 
X 
 
 
Lipid Panel 
X 
 
 
 
 
 
 
Bone Marrow 
Evaluation 
X 
X 
X 
 
X 
 
 
Coagulation 
Testing (PTT/INR) 
X 
 
X 
 
 
 
 
 See appendix II for specific neurocognitive and psychological testing to be performed at 
various time points 
 Endocrine evaluation will include PTH, 25 hydroxy vitamin D level, IGFB3, IGF1 and if >8 
years testosterone, estrogen, LH/FSH at initial evaluation. At day 100 post-transplant we will 
obtain TSH/T4, PTH and vitamin D.  
 MRI/MRA will be evaluated at year 3 and year 5 when deemed clinically appropriate. 
 
All of the above evaluations are standard of care. They are not experimental or for research. 
 
 
Required Infectious Disease Evaluation Prior to Transplantation 
CT sinus 
CT chest/abdomen/pelvis 
Infectious Disease Markers per institutional standards 
Panorex 
Nasal Respiratory Viral Panel 
Cultures (blood, urine, stool, nasal) 

                                                                                                                            
 
 
SCDHAP 46 
 
 
Additional studies or repeat studies will be performed during transplantation as deemed clinically 
appropriate by clinical transplant team. CMV, EBV, BK and adenoviral PCRs will be evaluated 
approximately weekly starting at day +15 through day 100, then monthly for one year. 
 
All of the above evaluations are standard of care. The evaluations are not experimental or for research. 
 
 
 
 

                                                                                                                            
 
 
SCDHAP 47 
 
 
 
Appendix II: Neuropsychological Testing 
As described previously, neurocognitive and psychological testing will occur prior to 
transplantation and post transplantation at 12 months and yearly through five years post 
transplantation. Baseline and one year post transplant evaluations will be performed that will 
include neurocognitive, mood/behavior and executive functioning assessments. At six and 
eighteen months post transplantation limited behavioral assessments will be performed.  Testing 
that occurs yearly starting at two years post transplantation will be focused and address patient 
specific needs as determined by the Dr. Scott Hunter and his clinical team. Below are the 
evaluations that will be incorporated into these evaluations.   
 
Neurocognitive Functioning 
Wechsler Abbreviated Scale of Intelligence, Second Edition (WASI-2) 
Wide Range Achievement Test, Fourth Edition (WRAT-4) 
California Verbal Learning Test for Children (CVLT-C) 
Rey Complex Figure Test (RCFT) 
Beery-Buktenica Developmental Test of Visual-Motor Integration 
Connors Continuous Performance Test 
Delis-Kaplan Executive Function Test: Trail Making Tests, Sorting Test, Color-Word 
Identification Test 
 
Assessment of Mood/Behavior and Executive Functioning 
Behavioral Assessment System for Children, Second Edition (Parent, Teacher and Self-Report 
forms) 
Behavior Rating Inventory of Executive Function (Parent, Teacher, and Self-Report forms) 
 
All of the above evaluations are standard of care. The evaluations are not experimental or for research. 
 
 
 
 
 

                                                                                                                            
 
 
SCDHAP 48 
 
 
 
 
Appendix III: 
Criteria for Adverse Event Evaluation and Reporting 
Specialized criteria are required for the reporting of adverse events in transplant clinical trials as 
some medical events, even severe medical and adverse events/side effects are not only anticipated 
in these research participants but are intended (iatrogenic) events.  For example, abnormal 
hematological values including (moderate to life threatening) levels of neutropenia, leukopenia, 
anemia and thrombocytopenia are deliberately induced by conditioning regimens in an effort to 
provide space in the bone marrow compartment for the donor stem cells.    
 
In order to be able to provide the highest quality of accurate and pertinent protocol safety data it 
is necessary to clearly specify the criteria for capturing and reporting what constitutes an adverse 
event for this protocol.  However, these guidelines are not considered all-inclusive and/or 
unconditional.  Medical and scientific judgment should be exercised in deciding whether 
particular medical/adverse events may require reporting despite the criteria put forth in this 
document.     
 
Section 1: General Definitions 
Adverse Event (AE)– An adverse event is any unfavorable or unintended symptom, sign, or 
disease (including an abnormal laboratory finding), temporally associated with the use of a 
medical device, treatment or procedure that may or may not be considered related to the medical 
device, treatment or procedure. 
Serious Adverse Event (SAE):  An SAE is any untoward medical occurrence that: 
• 
Results in death or 
• 
Is life-threatening or 
• 
Requires inpatient hospitalization of greater than 24 hours or causes a significant 
prolongation of existing hospitalization or 
• 
Results in persistent or clinically significant disability/incapacity, second cancer or 
• 
Is a congenital anomaly/birth defect (in an offspring) 
The term “life threatening” refers to an event in which the research participant was at risk for 
death at the time of the event, not an event that hypothetically might have caused death if it were 
more severe. 
 
Section 2: Guidelines for Transcription and Reporting of Adverse Events 
 
The following guidelines pertain to adverse event reporting in this transplant protocol: 
 
1. Time period for adverse event reporting will begin at the time of initiation of the conditioning 
regimen pre-transplantation. The timeframe for the completion of AE reporting and required 
NCI grades to be reported is specified in the reporting section of this protocol.  
2. Information collected concerning AEs will include the name of the event; onset date/ 
resolution date (if ongoing at time off study, it will be noted only as “ongoing at time off 

                                                                                                                            
 
 
SCDHAP 49 
 
 
study” in a specific field in the database with no resolution date; NCI CTC Version 3.0 grade 
where applicable (example, GvHD will only be graded by Seattle Criteria); SAE 
determination; Expected/ unexpected; A relationship to research therapy.  
3. Any medical diagnoses, diseases, signs and symptoms that are present prior to the initiation 
of conditioning should not be recorded as adverse events unless they worsen in severity or 
frequency with causality appropriately identified.  
4. AEs will be reported as diseases or syndromes whenever possible (as long as clearly related) 
instead of reporting as individual component symptoms, signs or laboratory abnormalities.  
For example in the case of medically diagnosed veno-occlusive disease, the sub-components 
of this disease such as weight gain, increased bilirubin, abdominal distension, abdominal 
cramping/discomfort/pain requiring medication, and/or ascites would not be reported 
individually as AEs as they are known symptoms and manifestations of the primary disease.  
5. Disease progression and/or disease relapse will not be considered an adverse event, as this is 
part of the potential natural course of the underlying disease. Any adverse event that is 
determined to be clearly related to the research recipients underlying disease/disease 
progression will not be reported as an adverse event.  Please note that disease progression 
and/or disease relapse are reported in other contexts within transplants studies. 
6. The following are considered provisions for best clinical management for this patient 
population and will not be documented and reported as adverse events as specified:  
• 
All blood product infusions including exchange transfusions. 
• 
Loss of appetite requiring initiation of total parenteral nutrition (TPN) or initiation of 
TPN for nutritional prophylaxis pre-transplant and up to day +100 post-transplant.  TPN 
administration is a standard therapy administered to the majority of transplant recipients 
regardless of the degree of loss of appetite. If TPN is required to be initiated after day 
+100 post-transplant (i.e., the AE or causative event leading to initiation of TPN occurs 
after day 100), then the etiology for the initiation of the TPN (e.g. anorexia/loss of 
appetite) may be deemed an AE as determined by the PI.   
• 
If the research participant requires an additional infusion of donor stem cell (boost), the 
guideline for AE reporting of initiation of TPN will resume (restart) from date of boost to 
day 100 post boost.   
7. Due to the deliberate myeloablation/myelosupression required for stem cell transplantation 
the following criteria will be applicable to abnormal hematological laboratory values in 
relation to adverse event reporting in this stem cell transplant patient population: 
Platelets/Thrombocytopenia: Thrombocytopenia will be reported as an adverse event under 
the following conditions regardless of study phase/grade of AEs noted to be collected in 
Reporting section:  
If the platelet counts drop to < 20,000/mm3 requiring platelet transfusions after the following 
criteria have been met:  
1. If the patient has exhibited post-transplant platelet engraftment as defined as a platelet 
count of >20,000/mm3 with no platelet transfusions within 7 days  

                                                                                                                            
 
 
SCDHAP 50 
 
 
AND (both criteria must be met) 
2. The research recipient continues to maintain a platelet transfusion independent state* for 
a minimum of 1 month after this initial (above specified) engraftment time period.  
(*Exceptions – prophylaxis platelet transfusions for surgical procedures, BMA, BX, LP, 
endoscopy, BAL, etc, hemorrhage will not be factored into this time period) 
     If thrombocytopenia occurs after this time point, an adverse event will be captured for this  
     disorder.  
Hemoglobin/Anemia will be transcribed and reported as an adverse event if the patient has 
exhibited RBC engraftment (Hgb >8 without transfusion for at least 30 days) and then would 
require red blood cell infusion for anemia of <8.0 (Exceptions – prophylaxis RBC infusions 
for surgical procedures, Bone marrow aspirates/biopsies, lumbar punctures, etc. would not be 
factored into this criteria)    
Neutrophils/Neutropenia – neutropenia will be reported as an AE if the patient has exhibited 
by an absolute neutrophil count (ANC) of > 1,000/mm3 without granulocyte colony 
stimulating factors (G-CSF) for at least 30 days and would require further G-CSF treatment 
for an ANC of < 500/mm3 
Leukocytes/Leukopenia: Leukocyte values will not be evaluated and graded as adverse events 
in the context of this study as engraftment/related events are based on the corresponding 
neutrophil count and treat according to ANC (see neutrophil section). 
8. Metabolic laboratory and vital sign abnormalities should not be reported as adverse events 
unless criterion for a SAE is fulfilled or the investigator feels the abnormality should be 
reported as an AE. 
9. Reporting of SAE:  Any hospitalizations regardless of duration which meet the following 
criteria will not be reported as SAEs as they are part of the protocol therapy and/or conditions 
of best clinical management for this patient population: 
• 
Apheresis, line placement, conditioning /stem cell infusions/boosts/DLI administration 
• 
Disease evaluation procedures such as bone marrow aspirates/ biopsies, liver biopsies, 
staging/re-staging procedures, or scans.  
• 
Planned hospitalization for administration of medication/ chemotherapy and related 
disease or protocol required treatment. 
• 
Planned /scheduled treatment (including surgical intervention) for underlying primary 
disease related interventions.  
• 
Concomitant radiotherapy (if applicable) 
• 
Transfusion support, exchange transfusions, chelation therapy, etc. 
 

                                                                                                                            
 
 
SCDHAP 51 
 
 
10. Every effort will be made to obtain records as soon as possible from outside facilities in the 
case where a patient may require planned (if applicable) or unplanned medical or surgical 
intervention at an outside institution.   These documents will be reviewed upon receipt to 
UCM for adverse events and will be reported according to the reporting guidelines specified 
in this document. 
11. Only the primary causative diagnoses/clinical indication(s) for a research participant being 
admitted to the hospital for > 24 hours will be identified as the SAE in the database and 
reported to the IRB in the SAE continuing review reports.  Other concurrent diagnoses 
occurring at the time of this hospitalization that - in and of themselves - would not have 
resulted in hospitalization and therefore were not the reason for the hospitalization (do not 
meet criteria of SAE), will not be reported as SAEs but will be reported accordingly as 
adverse events.   Although these concurrent events may be reported within the context of the 
describing the research participant’s overall clinical situation in an SAE report long form to 
the IRB/FDA, they will not be reported separately as SAE since they do not meet the criteria 
of SAEs.  
 
For example, if a research participant is evaluated in the clinic and is noted to have an NCI 
CTC grade III headache, grade I left foot pain and grade II fever with neutropenia.  The 
Principal Investigator feels the headache and foot pain, although significant, do not warrant 
hospitalization.  However, the Principal Investigator deems that the fever does require 
hospitalization of greater than 24 hours.  In this case, the SAE is the fever with neutropenia.  
The headache and foot pain would be noted where appropriate as AEs rather than SAEs.