Nonmyeloablative Allogeneic Peripheral Blood Mobilized Hematopoietic Precursor Cell Transplantation for Severe Congenital Anemias Including Sickle Cell Disease (SCD) and B-Thalassemia
Phase: PHASE1
Conditions: Congenital Hemolytic Anemia, Sickle Cell Disease
Sponsor: National Heart, Lung, and Blood Institute (NHLBI)

 
 Protocol with Statistical Analysis Plan Cover Page:   
 
Official Title: 
Nonmyeloablative allogeneic peripheral blood mobilized hematopoietic 
precursor cell transplantation for severe congenital anemias including sickle cell 
disease (SCD) and β-thalassemia 
NCT number: 
 
NCT00061568 
Document Type:  
Protocol with Statistical Analysis Plan (SAP)  
Document Date: 
October 03, 2023  
 

03-H-0170 
John Tisdale, M.D. 
October 03, 2023 (Amendment KKK) 
1
CLINICAL RESEARCH PROTOCOL 
 
 
 
 
 Project #03-H-0170 
 
 
 
 
 
 
 
 
 
Drug: Sirolimus/Alemtuzumab 
 
 
 
 
 
 
 
 
 
IND: Exempt 
Date: 03 OCT 2023 
 
 
Title: Nonmyeloablative allogeneic peripheral blood mobilized hematopoietic precursor cell 
transplantation for severe congenital anemias including sickle cell disease (SCD) and β-thalassemia 
 
Other underlying words: Peripheral blood stem cells, host-donor chimerism, graft-versus-host disease, 
graft-versus-marrow, sirolimus (Rapamune®), low dose irradiation, alemtuzumab (Campath®), donor 
apheresis 
 
Principal Investigator:  John F. Tisdale MD, CMTB, NHLBI 
 
 
 
Subjects of study: 
 
Number 
 
Sex 
 
Age range 
 
Patients: 
 
  60-75  
 
either  
>4  
 
 
Donors  
 
  60-75  
 
either  
Age range 2 to 80 years 
 
(accrual will stop after at least 50 successfully transplanted and 15 donors returning for 1 yr. post-
transplant testing) 
 
 
Project involves ionizing radiation? 
 
Yes  
Off site project? 
 
 
 
No 
Multi-Institutional project? 
 
 
No 
DSMB  
 
 
 
 
Yes 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

03-H-0170 
John Tisdale, M.D. 
October 03, 2023 (Amendment KKK) 
2
PRECIS 
Nonmyeloablative allogeneic peripheral blood stem cell (PBSC) transplants are currently being 
investigated in phase I/II trials assessing engraftment, efficacy, and toxicity at a number of transplant 
centers.  Preliminary data have shown a high rate of complete donor engraftment with a relatively low 
toxicity profile. The decreased risk of transplant-related complications associated with 
nonmyeloablative transplants expands eligibility to patients with nonmalignant hematological 
disorders curable by allogeneic transplantation; however, significant toxicity with current regimens 
persists including severe graft versus host disease (GVHD) leading to significant morbidity and 
mortality. Moreover, mixed chimerism has been shown to be sufficient to induce clinical remissions 
in children with nonmalignant hematologic disorders undergoing conventional allogeneic 
transplantation.  Therefore, newer regimens need to be developed that are more applicable to patients 
with non-malignant disorders in whom no graft vs. leukemia effect is needed, and where mixed 
chimerism is sufficient for disease amelioration.  
 
In this protocol, we propose transplantation in patients with severe beta-globin disorders including 
SCD and β- thalassemia, considered at high risk for complications from or ineligible for standard 
BMT, with allogeneic PBSCs from an HLA identical sibling using a novel immunosuppressive 
regimen without myeloablation in an attempt to further decrease the transplant related 
morbidity/mortality. The low intensity nonmyeloablative conditioning regimen will consist of low 
dose radiation, Alemtuzumab (Campath®) and Sirolimus (Rapamune®) as a strategy to provide 
adequate immunosuppression to allow sufficient engraftment for clinical remission with a lower risk 
of GVHD development.  T-cell replete, donor-derived, granulocyte colony-stimulating factor 
(filgrastim, G-CSF) mobilized PBSCs will be used to establish hematopoietic and lymphoid 
reconstitution.  
 
The primary endpoint of this study is treatment success at one year, defined as full donor type 
hemoglobin on hemoglobin electrophoresis for patients with SCD and transfusion-independence for 
patients with β-thalassemia.  Other end points include degree of donor-host chimerism necessary for 
long-term graft survival and disease amelioration, incidence of acute and chronic GVHD, incidence 
of graft rejection, transplant-related morbidity, as well as disease-free and overall survival. 
 
 
 

03-H-0170 
John Tisdale, M.D. 
October 03, 2023 (Amendment KKK) 
3
 
TABLE OF CONTENTS 
 
1.0 
Objectives 
2.0 
Background 
3.0 
Scientific and Clinical Justification 
4.0 
Study Design 
5.0 
Eligibility Assessment 
6.0 
Clinical Evaluation of the Patient 
7.0 
Treatment Plan 
8.0 
Donor Evaluation and Plan 
9.0 
Management of Patient Complications 
10.0 
Research Studies 
11.0 
Biostatistical Considerations 
12.0 
Data and Safety Monitoring  
13.0 
Human Subject Protections 
14.0 
Conflict of Interest 
15.0 
Pharmaceuticals 
16.0 
Compensation 
17.0 
References 
Appendices: 
A 
ECOG, Lansky, and Karnofsky Performance Status Scales  
B 
Transfusions 
C 
Exchange transfusion and donor stem cell apheresis procedure 
D  
Preparative regimen  
E 
Busulfan administration 
 
Addendum A:  Neuropsychological functioning and quality of life in patients with severe 
congenital anemias  
Addendum B:  Reproductive Health Assessment 
 
SEE ALSO  
BONE MARROW TRANSPLANT CONSORTIUM GUIDELINES 
 
Marrow stem cell transplant section 
 
Hematology branch, NHLBI 
 
 
http://intranet.cc.nih.gov/bmt/   
 
 

03-H-0170 
John Tisdale, M.D. 
October 03, 2023 (Amendment KKK) 
4
1.0 
OBJECTIVES 
 
1.1 
To evaluate the safety, efficacy, and toxicity of a low intensity nonmyeloablative preparative 
regimen followed by an allogeneic granulocyte colony-stimulating factor (filgrastim, G-CSF) 
mobilized peripheral blood stem cell transplant in a population of patients with severe congenital 
anemias including SCD and β-thalassemia, at increased risk for complications with or ineligible 
for standard myeloablative allo-transplantation.  
 
1.2 
To examine the level of chimerism required to maintain both graft survival as well as 
hematologic normalcy.  
 
1.3 
To determine the incidence and severity of acute and chronic graft versus host disease (GVHD) 
using a novel nonmyeloablative conditioning regimen 
 
1.4 
To determine the rate of graft rejection using a novel nonmyeloablative conditioning regimen 
 
1.5 
To determine disease-free survival, overall survival, relapse, transplant-related mortality, and 
death from all causes using a novel nonmyeloablative conditioning regimen. 
 
1.6 
To evaluate the longitudinal effects of a novel nonmyeloablative conditioning regimen on 
neuropsychological functioning and quality of life (see Addendum B) 
 
 
2.0  
BACKGROUND 
 
2.1  
Introduction 
Allogeneic bone marrow (and peripheral blood) transplantation (BMT) is the only available cure for 
SCD and β- thalassemia, but has been infrequently pursued due to its associated complications. The 
unacceptable risk of death from conventional BMT renders many patients, especially those with 
nonmalignant disorders, ineligible for what may otherwise be curative therapy.   Recently however, in 
both malignant and non-malignant disorders, it has been shown that these high intensity regimens are 
not necessary for engraftment and survival, and many centers are currently exploring 
nonmyeloablative conditioning regimens in order to reduce the toxicity associated with this treatment 
modality.  While successful engraftment has been reported in the majority of patients conditioned 
with reduced intensity regimens, these regimens still carry significant toxicity and have not 
significantly reduced the risk of graft versus host disease (GVHD).  For patients with congenital 
severe anemias, only the replacement of abnormal or absent erythroid cells with normal donor derived 
erythroid cells is required for disease amelioration.  These disorders constitute an ideal situation for a 
nonmyeloablative conditioning regimen as only a proportion of normal cells will need to engraft 
given the survival and proliferative advantage of the donor derived erythroid cells as compared to the 
host cells. Further, as a nonmyeloablative regimen will allow autologous recovery with a low risk of 
adverse consequences to the recipient if the graft should fail, graft failure is preferable to the 
development of severe GVHD. As such, we propose the development of an immunosuppressive but 
nonmyeloablative transplant regimen consisting of Alemtuzumab, low dose radiation, and sirolimus 
in patients with SCD or β- thalassemia.    
 
2.2 Sickle Cell Disease 
 
2.2.1  Pathophysiology 

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John Tisdale, M.D. 
October 03, 2023 (Amendment KKK) 
5
Sickle cell disease (SCD) is a well described genetic disorder associated with significant morbidity 
and mortality.  It affects one of every 600 African Americans in the United States alone.[1] The 
disease is characterized by recurrent vaso-occlusive crises as a consequence of abnormal hemoglobin 
polymerization in areas of low oxygen tension. As a result, patients develop functional asplenism 
leading to a high risk of infections from encapsulated organisms, recurrent pain crises, acute chest 
syndrome, and neurologic events, as well as sudden death as the most serious consequences of this 
disease.[2]  More recently, sickle hepatopathy and iron overload have been discovered to increase 
mortality in patients with SCD, as patients with ferritin >1000 ug/L or direct bilirubin >0.4mg/dL led 
to significantly decreased survival as compared to patients with ferritin <1000ug/L and direct 
bilirubin <0.4 mg/dL.[3]   
 
In addition, patients with platelet counts in the lowest quartile of the cohort (<267,000/ul) had a 2.7-
fold increase in direct bilirubin after controlling for WBC, to exclude the effect of generalized bone 
marrow suppression as a cause for thrombocytopenia (OR 2.70 95% CI 1.11-6.56, p=0.029). 
 
The medical costs of this disease are enormous, with estimates of $40,000 per patient per year (year 
2000 figures) for chronic transfusion and chelation alone, but do not include the impact on quality of 
life of those with the disease.[2,4] An overlapping symptom complex also occurs in patients with the 
double heterozygous forms of sickle disease, sickle-SC, and sickle β-thal0 disease, and in fact these 
patients cannot be differentiated clinically but only by means of a laboratory test.   
 
2.2.2 Treatment options 
Chronic transfusion therapy decreases the incidence of stroke in pediatric patients with abnormal 
trans-cranial Doppler velocities (internal carotid or middle cerebral artery velocity >200 cm/s).[5]  
While transfusions can prevent further neurologic events in patients at risk, iron overload is common, 
resulting in significant end-organ toxicity. Currently, stroke treatment in SCD has remained mostly 
with exchange transfusions, and less with  hydroxyurea.[6,7,8]   
 
Hydroxyurea results in a significant reduction in the number of painful crises per year and a 
decreased frequency of acute chest syndrome,[9] and has become the treatment of choice for the 
majority of individuals with SCD who lack a suitable matched sibling donor.  Unfortunately, neither 
of these approaches is curative, nor do they appear to reverse established end-organ damage.  
 
Several important interventions have led to an improvement in the overall life expectancy of patients 
with SCD, most notable among these are the use of pneumococcal vaccines and the prophylactic use 
of penicillin during childhood.  Hydroxyurea has also been suggested to improve survival in patients 
with SCD.  However, life expectancy remains significantly shortened compared to the national 
average with that of an affected male being 47 years versus the national average of 72.[10,11]  There 
are no factors to predict better survival among patients, further complicating the decision to proceed 
with higher risk treatments, especially during childhood when such treatments may be better 
tolerated. In one study of 3764 patients, 18 percent of the patients died with overt organ failure, and 
early mortality was highest among patients with symptomatic disease, but another 33 percent who 
appeared to be clinically free of organ failure died during an acute sickle crisis.[2] 
 
The only established cure for patients with SCD is allogeneic bone marrow transplantation; however, 
the procedure has only been applied in children.[12,13,14,15] In adults, the higher burden of 
accumulated end-organ damage would be expected to result in higher transplant associated mortality 
and morbidity, beyond that reported in children, including seizures and intracranial 
hemorrhages.[16,17] Several recent publications continue to show that adults with SCD have ongoing 
organ injury and have premature death.[18,19]  As a result, this method has only been offered to those 

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John Tisdale, M.D. 
October 03, 2023 (Amendment KKK) 
6
patients less than the age of 16 with either end-organ damage or symptomatic disease due to their 
demonstrated higher early mortality rate. 
 
2.3 
β-Thalassemia 
 
2.3.1 Pathophysiology 
Thalassemia is the most common genetic disorder worldwide[20] and is a result of either a defective 
or absent synthesis of one or more of the globin chain subunits of the hemoglobin tetramer.  
Inadequate accumulation of the globin subunit results in hypochromia and microcytosis while the 
unbalanced accumulation of globin subunits results in ineffective erythropoiesis.  There is marked 
heterogeneity ranging from profound anemia resulting in death in utero to a relatively benign anemia.  
Thalassemias are designated by both the globin chain, α or β, whose synthesis is affected, and by 
major or minor, denoting homozygosity or heterozygosity. β-thalassemia major, or Cooley’s anemia, 
is a clinically severe anemia caused by the inheritance of two β-thalassemia alleles.  As a result, 
circulating red blood cells are small and distorted, containing very small amounts of hemoglobin.  
Further, the accumulation of free α-globin molecules in erythrocytes results in ineffective 
erythropoiesis.   
 
2.3.2 Treatment options 
In patients with β-thalassemia major, the resulting anemia is so severe that most patients require 
lifelong red blood cell transfusions. This chronic transfusion therapy necessitates the use of iron 
chelation to prevent the long-term consequences of iron overload. Other treatments such as butyrate, 
and hydroxyurea have been explored, but these have had only limited success, (less so than with 
SCD), are not well tolerated (especially butyrate), and most importantly, are not curative. 
 
Prior to the regular use of deferoxamine for iron chelation, only 25% of patients survived to the age of 
25 years. For patients born after 1967, for whom such chelation has been available, the estimated 
probability of being alive at 20 years of age remains low at 67%[21] and only 40% among 20-year-
old patients will be free of complications. The most common cause of death related to iron overload is 
heart disease, followed by infection and liver disease.[22]  Even with chelation, red cell transfusion 
incurs other risks including transmission of blood borne pathogens such as HIV and hepatitis C, as 
well as the risk of transfusion reactions.    
 
Allo-immunization makes access to blood products more difficult, and even more expensive to 
continue.  Deferoxamine although necessary to prevent iron overload, is itself not benign.  Side 
effects from iron chelation include visual and auditory neurotoxicity, and allergic reactions, including 
anaphylaxis.[23,24,25] There are newer oral chelators, deferasirox (Exjade and Jadenu),[26,27] and 
deferiprone [28,29,30] with each having their own side effect profile that requires periodic 
monitoring. Moreover, the therapy is expensive and not well tolerated, impacting on quality of life 
and often results in poor compliance, especially in adolescents.   
 
The only available cure for β-thalassemia major is bone marrow transplantation; however as in other 
disorders, BMT is not risk free. Risk groups have been identified based upon three variables, 
hepatomegaly > 2 cm, portal fibrosis of any degree, and inadequate compliance with chelation 
therapy, with Class I, II and III having none or 1, 2 or all three of the factors, respectively.  Results 
are best in Class I children who undergo transplantation early, with a DFS of 90-93% and TRM of 3-
4%.[31] For higher risk patients, the survival and DFS decrease to 86 and 82% and drops further to 
62 and 51% in the highest risk category.  Results in adults (>16) are similar at 65 and 62% disease 
free and overall survival respectively.[32] Adults have a higher rejection rate and also a higher 
incidence of GVHD making it necessary to devise better conditioning regimens.  For high risk 

03-H-0170 
John Tisdale, M.D. 
October 03, 2023 (Amendment KKK) 
7
children (Class II or III), improvements have been seen when using lower dose conditioning, resulting 
in less morbidity and mortality, suggesting the rational to apply nonmyeloablative methods to 
adults.[33,34] Although there are newer iron chelators available, cost/access to medication and 
compliance continue to be the main issues in suboptimal chelation. In the present day, the morbidity 
associated with iron overload remains, and extends into transplant related outcomes. The Pesaro 
classification is still being used currently.[35,36,37] 
 
3.0 
SCIENTIFIC AND CLINICAL JUSTIFICATION 
 
In disorders such as hematologic malignancies, the curative effect of bone marrow transplantation has 
been ascribed to the use of myeloablative chemo-radiotherapy and the antileukemic effect of the 
transplant (the graft-versus-leukemia (GVL) effect).[38]  The assumption that the intensive 
myeloablative preparative regimen is essential for the cure of the malignancy went unchallenged until 
the demonstration by Kolb et al, (subsequently confirmed by numerous investigators), that donor 
lymphocytes alone exert a powerful antileukemic effect in the context of patients relapsing with 
myeloid leukemias after BMT.[39,40,41]  This observation has important implications.  First, it may 
be possible to cure some hematologic malignancies with preparative regimens of lower intensity, 
designed to immunosuppress the recipient to allow lymphocyte and stem cell engraftment without 
major cytoreduction of the malignancy by myeloablation.  Second, such low-intensity preparative 
regimens appear to have decreased toxicity and may make transplantation appropriate in patients 
where procedural mortality is usually prohibitive, including patients with more indolent hematologic 
diseases such as those with severe congenital anemias, as well as patients with co-morbid diseases 
and older patients. Unlike patients who undergo allogeneic peripheral blood stem cell (PBSC) 
transplantation for malignant indications, patients with non-malignant disorders such as SCD or β-
thalassemia, do not require full and/or rapid donor engraftment for cure of the disease.[42,43,44] 
While it is generally accepted that graft versus host disease (GVHD) is less severe in patients 
conditioned with low intensity preparative regimens,[45] graft rejection is preferable to the 
development of lethal GVHD in the setting of severe congenital anemias.  
 
Several groups, including our own, have begun to investigate this approach to improve the 
applicability and outcome following allogeneic BMT, and preliminary results were 
encouraging.[46,47,48] The first so called “mini-transplants” were performed by simply lowering the 
doses of standard agents; however, these doses were still sufficiently toxic to incur prolonged 
cytopenias and patients continued to experience significant regimen-related toxicities. Our own 
experience at the NIH using a nonmyeloablative combination of fludarabine and cyclophosphamide 
has included over 100 patients with engraftment seen in the majority,[49,50] and extension to patients 
previously excluded from allogeneic transplant trials such as those infected with the human 
immunodeficiency virus has been proven feasible.[51]  However, GVHD remains a significant 
problem, occurs at a rate not different from that observed with conventional allogeneic PBSC 
transplantation, and continues to be the principle cause of death. 
 
In attempts to decrease these toxicities, and to develop immunosuppressive as opposed to 
myeloablative regimens, many centers have now been exploring different combinations of regimens 
including the use of low dose radiation. Low dose radiation alone has been shown to have anti-
leukemic properties as demonstrated by Shulman et al. where 200 cGy was adequate for inducing 
remission, albeit short lived, in refractory patients ineligible for other standard treatments.[52] Doses 
of 100 to 500 cGy have been shown to have very little toxicity in both murine and rhesus transplant 
models.[53,54,55,56] Even patients with Fanconi anemia who would be considered high risk for 
radiation induced toxicities, tolerate moderate dose (500 cGy) radiation based regimens.[57]  While 
regarded as a method designed for the creation of “space” within the marrow in bone marrow 
transplantation (BMT) regimens, radiation has long been the basis of the establishment of 

03-H-0170 
John Tisdale, M.D. 
October 03, 2023 (Amendment KKK) 
8
immunosuppressive protocols for both BMT and solid organ transplantation in animal models.[58,59] 
There are also data in canines demonstrating stable mixed chimerism with the use of 200cGy total 
body irradiation alone, followed by GVHD prophylaxis with cyclosporine (CsA) and mycophenolate 
mofetil (MMF).[60]  Initial human trials using this regimen of 200 cGy with MMF and CsA were 
encouraging with approximately 20 percent graft failure; and more recent data using fludarabine, an 
immunosuppressive but nonmyeloablative agent, in addition to 200cGy have shown similar results in 
44 patients transplanted to date.[61]  
 
In order to develop a regimen with potential application to individuals with varying degrees of organ 
dysfunction such as those with SCD or other congenital anemias, we have sought conditioning which 
could be applied in such a context, avoiding renally excreted drugs, and relying on the 
immunosuppressive effects of total body irradiation as the basis for such an approach. Patients with 
SCD and β-thalassemia, who have been frequently transfused may be at an increased risk for graft 
rejection as compared to patients with hematologic malignancies because frequent exposure to blood 
products may lead to donor HLA sensitization.  A modest increase in the dosage of TBI from 200 
cGy used in prior studies to 300 cGy may increase both the degree of myelosuppression and 
immunosuppression without significantly altering the side effect profile.  Moreover, drugs such as 
anti-thymocyte globulin which may increase the risk of inciting a sickle cell crisis, or by their side 
effect profile, mimic the symptoms of a pain crisis need to be avoided specifically in the chosen 
patient population of this trial.   
 
Use of alemtuzumab  
Further immunosuppression with the lymphocyte depleting agent Alemtuzumab will also be 
employed. Alemtuzumab is a humanized monoclonal antibody directed against CD52 (which is 
abundantly expressed on all human lymphocytes), and causes T cell activation in vitro as well as 
complement mediated lysis and antibody dependent cellular toxicity. As a result, it depletes both T 
and B cells efficiently in vivo. It is currently being used in clinical trials as monotherapy for certain 
autoimmune disorders including rheumatoid arthritis and multiple sclerosis,[62,63,64,65] T and B cell 
malignancies[66] treatment of solid organ rejection,[67,68,69] and has been approved for use in 
chronic lymphocytic leukemia, a B cell malignancy, as a result of its profound immunosuppressive 
properties.[70,71] 
 
More recently, alemtuzumab has been used prospectively to prevent graft rejection in human 
allotransplantation.[72,73,74,75] 31 patients have been transplanted using 20 mg of Alemtuzumab on 
Day 0 and 1 of transplantation in combination with half dose cyclosporine, which has been shown to 
be ineffective when used alone.  At the 21 months follow-up, only two grafts were lost to 
rejection.[69] Further, data suggest that the use of alemtuzumab, as compared to fludarabine, reduces 
the risk of GVHD, even in the unrelated donor setting.[76]  In one study of 44 patients, including 8 
patients receiving unmanipulated marrow from matched unrelated donors who would therefore be at 
very high risk for developing GVHD, only 2 patients had acute GVHD, both of which were grade 2. 
Only 1 patient developed chronic GVHD.[72] Follow up to this study has included a further 39 
patients undergoing unrelated bone marrow transplantation (including patients having failed a prior 
transplant and/or having a mismatch in either HLA class I or II alleles) for a total of 47 patients, with 
only three patients developing Grade III GVHD and none developing Grade IV.[76]  In another study 
of 12 high risk patients undergoing haplo-identical BMT, one patient experienced Grade II and one 
patient developed Grade III GVHD; none developed Grade IV GVHD.[75] This reduced risk of 
GVHD, as well as its immunosuppressive properties, appears to be due to an in vivo T cell depleting 
effect on the incoming graft. Unlike ATG, which is a nonspecific antibody directed against 
lymphocytes and is also used in conditioning regimens, Alemtuzumab is also better tolerated and has 
no risk of causing serum sickness.  
 

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John Tisdale, M.D. 
October 03, 2023 (Amendment KKK) 
9
Use of sirolimus 
Finally, to improve the odds for graft acceptance without GVHD, we will employ sirolimus, a novel 
immunosuppressive agent, instead of the conventional agent, CysA, based on sirolimus’ distinct 
properties as a tolerogenic agent.[77,78]  Sirolimus is an immunophilin drug similar to CysA; 
however, unlike CysA which inhibits the phosphatase calcineurin and therefore prevents the 
production of interleukin 2, sirolimus prevents translation of mRNAs encoding cell-cycle regulators.  
As a result, sirolimus only inhibits the ability of lymphocytes to proliferate in response to interleukin-
2.  We demonstrated that cells cultured and stimulated in the presence of sirolimus became anergic, 
while cells cultured in the presence of CysA did not.[77]     
 
To confirm these results in an in vivo model, we sought to compare the use of sirolimus to the 
standard post transplant immunosuppressant, CysA, in a nonmyeloablative setting.  We therefore 
transplanted F1 hybrid mice into their parental strain – a model designed to promote graft rejection- 
using only low dose radiation and sirolimus. C57Bl6 recipient mice were injected with either 
sirolimus at 5 mg/kg or CysA, beginning one day before cell infusion.[78] On day 0, they were given 
300 cGy TBI followed by 100 x 106 splenocytes obtained from G-CSF mobilized BalbC/C57Bl6 
hybrid donors.   
 
The immunosuppressants were continued for four weeks beyond cell infusion, and chimerism was 
monitored using flow cytometric analysis.  The mice transplanted with sirolimus showed moderate 
donor engraftment by week 2 which subsequently increased over time even after the discontinuation 
of the sirolimus, reaching levels of greater than 75%.  In contrast, those treated with CysA lost their 
engraftment even while receiving the CysA. Application of this regimen to transgenic mice 
expressing human sickle hemoglobin exclusively produced correction of anemia and red cell sickling 
even at very low levels of donor chimerism.[78]  
 
In a series of experiments performed by Hale et al., sirolimus also proved superior to CysA at 
prolonging skin graft survival in Class I and Class II disparate, fully mismatched, and xenogeneic 
recipients, and the use of sirolimus was superior to CysA when added to ALG and BM in a murine 
model.  Further, mice receiving sirolimus accepted a second same donor skin graft, but rejected third 
party grafts, demonstrating the development of tolerance.[79,80,81]  Sirolimus has also been 
employed in the bone marrow transplant setting, with matched and mismatched sibling and unrelated 
donors.[82]  One study involving 41 patients with hematologic malignancies used sirolimus, 
tacrolimus, and methotrexate as GVHD prophylaxis.  All evaluable patients engrafted, and grades 0-I, 
II, III, and IV acute GVHD occurred in 75%, 13%, 8%, and 5% of patients, respectively.[82]  
Another study included 14 evaluable patients with hematologic malignancies, and used sirolimus and 
mycophenolate mofetil as GVHD prophylaxis.  All patients engrafted, and grades II-IV acute GVHD 
occurred in 21%, and chronic GVHD in 30% of patients.[83] 
  
Sirolimus also has less renal toxicity as compared to CysA.  A randomized trial comparing the 
addition of sirolimus at either 2 or 5mg vs. azathioprine to CysA and prednisone for prophylaxis of 
renal allograft rejection showed a significantly lower rate of acute rejection episodes at both doses of 
the sirolimus as compared to azathioprine (16.9% and 12.0% vs. 29.8%).[84]  In a similar study, 
comparing sirolimus vs. CysA as adjuncts to azathioprine and prednisone, there were similar rates of 
graft survival and incidence of biopsy confirmed graft rejection (98% vs. 90% and 41% vs. 38% 
respectively), but significantly lower serum creatinine in the sirolimus group.[85]  Moreover, in renal 
transplant studies, sirolimus has been shown to be equally effective in preventing graft rejection and 
has been approved as an alternative to CysA. 
 
Our group is currently using a combination of 300 cGy TBI, alemtuzumab, and sirolimus to transplant 
high risk patients with SCD and, β-thalassemia who are 16 years of age and older.  Furthermore, we 

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John Tisdale, M.D. 
October 03, 2023 (Amendment KKK) 
10
have established the safety of PBSC mobilization in individuals with sickle cell trait (SCT), likely to 
represent a sizable fraction of sibling donors for patients with SCD.[86]  Additionally, for patients 
with SCD, higher platelet counts are maintained.[87,88,89]  Patients with SCD who are not routinely 
transfused for their therapy undergo exchange transfusion prior to transplant to lower their 
hemoglobin S to less than 30%.[12]  
 
Given the lower toxicities seen with nonmyeloablative regimens, the possibility of cure with mixed 
chimerisms, and the potential improvement of SCD after transplant with even autologous recovery, 
the extension of low-intensity allogeneic peripheral stem cell transplants to those with homozygous 
SCD, sickle SC, sickle β−thal0, and β-thalassemia, who would normally be offered a standard 
transplant but are considered at a higher risk due to their age (i.e. 18 or older) or other co-morbidities 
is justified. We will use a combination of low dose irradiation, Alemtuzumab, and sirolimus as our 
conditioning regimen. This combination is designed for slow engraftment and tolerance induction 
resulting in partial to full chimerism with reduced rates of graft versus host disease. To optimize our 
approach, we will use peripheral blood stem cells from sibling related donors, as preliminary studies, 
including our own experience with PBPC (peripheral blood progenitor cell) transplants and low 
intensity preparative regimens, indicate that transplant-related mortality and severe acute GVHD is 
uncommon when matched family donors are used.[45]  
 
If there is the same or even less toxicity but successful engraftment in these higher risk patients as we 
anticipate, this technique could be offered to all those who have histocompatible donors regardless of 
risk factors as the risk benefit ratio would clearly be in favor of this potential cure.  As a secondary 
endpoint, we will also examine the levels of donor myeloid chimerism sufficient to provide full 
erythroid chimerism, i.e. the level of overall engraftment needed to achieve disease amelioration for 
SCD, and β-thalassemia.  This information is also necessary for future applications of gene 
replacement therapy which is the ultimate goal for the cure of SCD and β-thalassemia. 
 
Continuation of this protocol 
Unfortunately, the treatments for these beta globin disorders have not changed substantially in the last 
decade and allo-HSCT remains the most accessible curative option.  While gene transfer and gene 
editing approaches are beginning to show promise, the availability to these clinical trials are limited.   
 
Most of the efforts in allo-HSCT in SCD and β-thalassemia continue to be in children, thus this is the 
major reason to continue accrual. We have had anecdotal reports from Israel, Saudi Arabia, London, 
Buffalo, and Alberta, who used this same approach and demonstrated the efficacy of this approach 
(although unpublished).  Recently the transplant group at the University of Illinois in Chicago 
published their results using our approach,[90] and confirmed safety and efficacy in 13 patients.  
Interestingly, ABO incompatibility was not an exclusion in their protocol, and two patients with 
major ABO incompatibility were among the 12 successfully engrafted. Thus, we hope to continue 
enrollment, maximizing safety while gaining knowledge, particularly for the secondary endpoints. 
 
 
4.0 
 STUDY DESIGN  
The current overall protocol success rate is 85-90% in individuals ≥16 years old.  While the accrual in 
younger children is expected to be low, it is still of research interest to test in pediatric population to 
see if fertility preservation can be achieved.  This protocol also serves to capture children who are 
ineligible for standard myeloablative transplant because they met exclusion criteria with poor lung, 
kidney, or liver function.  Similarly, with β-thalassemia, some with Pesaro Class 2 and all with Class 
3 are excluded from myeloablative transplant.  Thus this study will continue to be open for children 
with SCD or β-thalassemia.   

03-H-0170 
John Tisdale, M.D. 
October 03, 2023 (Amendment KKK) 
11
 
We also plan to continue accruing different genotype/phenotypes of SCD, as both are β-globin 
disorders and continue to be reported together in the current literature. Having separate studies for 
each of the sickle subtypes is not standard practice in transplant centers: similarly, in all AML are 
included in one treatment or transplant study, regardless of 7 subtypes; or treating aplastic anemia, 
regardless if it is hepatitis associated, drug induced, or from telomere disease.  
 
A human lymphocyte antigen (HLA)-matched sibling donor will receive filgrastim (G-CSF) 10 to16 
µg/kg/d subcutaneously or intravenously for up to 6 days with apheresis collections of PBPC on day 5 
(and day 6 if required) as a standard procedure under a separate NHLBI protocol (94H0010 or 
20H0099).  The product will be collected and frozen at the NIH Department of Transfusion Medicine 
(DTM) at least two weeks prior to the recipient beginning his/her conditioning.    
 
The patient will receive a preparative regimen of Alemtuzumab (Campath®) to be infused on days –7 
to –3, followed by 300 cGy TBI given as a single dose on day –2. Sirolimus (Rapamune®) at a dose 
of 5mg/day in patients >16 years of age or 1mg/m2/dose to a maximum daily dose of 5mg in patients 
<16 years of age to maintain trough levels between 5-15 ng/ml will be started on day –1. The PBPC 
graft targeted to deliver ≥5 x 106 CD34+ cells/kg will be infused on day 0.   
   
The design of the study incorporates the following features: 
 
1)  This is a phase I/II pilot study to determine the safety and therapeutic potential of a new 
transplant approach (disease-free survival, GVM effect) and to evaluate its toxicity profile (immediate 
toxicity, graft-versus-host disease, graft rejection, mortality) in a patient population with severe 
congenital anemias. 
 
2)  The patient cohort to be studied: Those patients with severe SCD and β-thalassemia, who have 
risk factors for high mortality and morbidity related to their disease (see inclusion criteria in section 
5.1.1 and 5.1.2).   
 
3)  Transplant conditioning regimen - immunosuppression without myeloablation: Patients will 
receive conditioning sufficient to allow donor lympho-hematopoietic engraftment without complete 
marrow ablation.  If the graft is rejected, the patient will reconstitute autologous marrow function.   
We will use a combination of low dose irradiation, Alemtuzumab (Campath®), and sirolimus. 
 
4)  Peripheral blood hematopoietic progenitor cell (PBPC) transplant: An unmanipulated peripheral 
blood stem cell collection from a filgrastim (G-CSF) stimulated HLA-matched donor should improve 
the chance of engraftment because of the high stem cell dose (≥5 x 106/kg CD34+ cells) and the 
presence of donor lymphocytes.   To reduce the risk of GVHD, patients will receive sirolimus before 
and after the transplant. The sirolimus will be tapered as necessary to minimize any graft versus host 
disease while still maintaining adequate chimerism.   
 
 
5.0 
ELIGIBILITY ASSESSMENT 
 
5.1 
Inclusion criteria – recipients (must fulfill one disease category in 5.1.1 and all of 5.1.2) 
 
5.1.1 
Disease specific: 
 

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John Tisdale, M.D. 
October 03, 2023 (Amendment KKK) 
12
5.1.1.1 
Patients with sickle cell disease at high risk for disease related morbidity or mortality, 
defined by having irreversible end-organ damage (A, B, C, D, or E) or potentially reversible 
complication(s) not ameliorated by hydroxyurea (F): 
 
A. Stroke defined as a clinically significant neurologic event that is accompanied by an infarct 
on cerebral MRI OR an abnormal trans-cranial Doppler examination (≥200 m/s); OR 
B. Sickle cell related renal insufficiency defined by a creatinine level ≥1.5 times the upper limit 
of normal (see table below) and kidney biopsy consistent with sickle cell nephropathy OR 
nephrotic syndrome OR creatinine clearance <60mL/min/1.73m2 for patients < 16 years of 
age or < 50mL/min for patients >16 years of age OR requiring peritoneal or hemodialysis. 
.[91,92,93] OR 
Age (Years) 
Upper limit of normal serum creatinine (mg/dl) 
< 5 
0.8 
5 < age < 10 
1.0 
10 < age < 15 
1.2 
> 15 
1.3 
C. Tricuspid regurgitant jet velocity (TRV) of ≥2.5 m/s in patients ≥18 years of age at least 3 
weeks after a vaso-occlusive crisis;[94,95] OR 
D. Recurrent tricorporal priapism defined as at least two episodes of an erection lasting ≥4 hours 
involving the corpora cavernosa and corpus spongiosa;[96] OR 
E.  Sickle hepatopathy defined as EITHER ferritin >1000mcg/L OR direct bilirubin >0.4 mg/dL 
at baseline in patients > 18 years of age; OR 
F. Any one of the below complications 
 
Complication 
Eligible for hydroxyurea* 
Eligible for HSCT 
Vaso-occlusive crises 
At least 3 hospital admissions in 
the last  year[97]  
More than 1 hospital admission 
per year while on maximal 
tolerated dose  of  
hydroxyurea*[2] 
Acute chest syndrome (ACS) 
2 prior ACS while > 3 years of 
age and adequately treated for 
asthma 
any ACS while on 
hydroxyurea*[98]    
Osteonecrosis of 2 or more 
joints 
And significantly affecting their 
quality of life by Karnofsky score 
50-60 (see Appendix A) 
And on hydroxyurea* where total 
hemoglobin increase less than 1 
g/dL or fetal hemoglobin 
increases < 2.5 times the baseline 
level 
Red cell alloimmunization 
Transfusion-dependent 
Total hemoglobin increase < 1 
g/dL while on hydroxyurea* 
*hydroxyurea at maximum tolerated dose  
 
5.1.1.2 
Patients with β-thalassemia who have grade 2 or 3 iron overload, determined by the 
presence of 2 or more of the following: 
 
portal fibrosis by liver biopsy  
 
inadequate chelation history  (defined as failure to maintain adequate compliance with chelation 
with deferoxamine initiated within 18 months of the first transfusion and administered 
subcutaneously for 8-10 hours at least 5 days each week[31]) 
 
hepatomegaly of greater than 2 cm below the costochondral margin 

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John Tisdale, M.D. 
October 03, 2023 (Amendment KKK) 
13
 
5.1.2 
Non-disease specific: 
 
5.1.2.1 
Ages >4 years 
5.1.2.2 
6/6 HLA matched family donor available 
5.1.2.3 
Ability to comprehend and willing to sign an informed consent, assent obtained from 
minors 
5.1.2.4 
Negative serum β-HCG, when applicable 
5.1.2.5 
Pediatric patients < 16 years of age must decline myeloablative bone marrow 
transplantation 
 
5.2 
Exclusion criteria –recipient (any of the following would exclude the subject from participating) 
5.2.1 
ECOG performance status of 3 or more, or Lanksy performance status of <40 (See 
Appendix A).  
5.2.2 
Diffusion capacity of carbon monoxide (DLCO) <35% predicted (corrected for 
hemoglobin and alveolar volume). 
5.2.3 
Baseline oxygen saturation of <85% or PaO2 <70 
5.2.4 
Left ventricular ejection fraction: <35% estimated by ECHO.  
5.2.5 
Transaminases > 5x upper limit of normal for age 
5.2.6 
Evidence of uncontrolled bacterial, viral, or fungal infections (currently taking 
medication and progression of clinical symptoms) within one month prior to starting the 
conditioning regimen 
5.2.7 
Major anticipated illness or organ failure incompatible with survival from PBSC 
transplant. 
5.2.8 
Pregnant or lactating 
5.2.9 
Major ABO mismatch (See Appendix B and C)  
 
5.3 
Inclusion criteria - Donor 
Donor deemed suitable and eligible, and willing to donate per clinical evaluations, who are 
additionally willing to donate blood for research and undergo a neuropsychological test. Donors 
will be evaluated in accordance with existing Standard NIH Policies and Procedures for 
determination of eligibility and suitability for clinical donation under a separate NHLBI protocol. 
Note that participation in this study is offered to all donors, but is not required for a donor to 
make a stem cell donation, so it is possible that not all donors will enroll onto this study. 
 
5.4 
Exclusion criteria- donor:  
None 
 
6.0  
CLINICAL EVALUATION OF THE RECIPIENT  
 
Samples will be ordered and tracked through the CRIS Screens.  Should a CRIS screen not be 
available, the NIH form 2803-1 will be completed and will accompany the specimen and be filed in 
the medical record. 
 
6.1  
Pre-study evaluation 
6.1.1 
History, physical examination to establish baseline symptoms, height and weight 
6.1.2 
ECOG or Lanksy performance status 
6.1.3 
Low resolution molecular HLA- A, B, DR typing of patient and as many family members 
as possible to confirm complete matching of the donor, HLA confirmatory testing of recipient 
once donor is identified 

03-H-0170 
John Tisdale, M.D. 
October 03, 2023 (Amendment KKK) 
14
6.1.4 
Hemoglobin electrophoresis and/or flow cytometric analysis of hemoglobin A, F and SS, 
SC or Sβthal0 as appropriate (for both patient and donor). 
6.1.5 
Antibody screen for HBV, HCV, HIV, HTLV-I/II, CMV, EBV, toxoplasma, syphilis.  
Consider PPD test for patients from areas where tuberculosis is prevalent. 
6.1.6 
Coagulation screen, CBC with differential 
6.1.7 
comprehensive metabolic panel 
6.1.8 
Extended red cell phenotyping, lymphocyte phenotyping TBNK, d-dimer 
6.1.9 
HLA antibody screen if indicated 
6.1.10 
β-HCG pregnancy test for females of childbearing potential 
6.1.11  
CXR, pulmonary function testing (in patients > 5 years of age): vital capacity FEV-1, 
DLCO 
6.1.12 
Sinus, chest, abdomen, and pelvis CT scans as clinically indicated 
6.1.13 
Brain MRI for appropriate patients  
6.1.14   
CNS Doppler flow analysis (to be performed at Children’s National Medical Center 
Department of Radiology) for appropriate patients  
6.1.15 
Cardiac function: EKG, trans-thoracic ECHO, 24 hour holter   
6.1.16 
Troponin T 
6.1.17 
Nutritional assessment. 
6.1.18 
Dental review 
6.1.19 
Social worker interview 
6.1.20 
Ophthalmology consultation 
6.1.21 
Endocrine consultation and testing which will include bone density scan, thyroid panel, 
insulin-like growth factor 1, morning cortisol, ACTH stimulation test, fasting glucose, fasting 
insulin, oral glucose tolerance test, hemoglobin A1C (or serum fructosamine level in patients with 
SCD),  25 hydroxy vitamin D, and in male patients at least 12 years of age, testosterone, 
luteinizing hormone level (LH), and follicle stimulating hormone (FSH), and in female patients at 
least 10 years of age LH, FSH, anti-mullerian hormone, and estradiol 
6.1.22 
Interview with members of primary care team and visit to unit. 
6.1.23 
Consent/Assent documents signed. 
6.1.24 
Complete lipid profile with triglycerides 
6.1.25 
Pulmonary hypertension evaluation by tricuspid regurgitant velocity (TRV) analysis using 
standard trans-thoracic echocardiography 
6.1.26 
Bone marrow aspirate and/or biopsy to rule out other hematologic disorders  
6.1.27 
Quality of life assessment (see Addendum A) 
6.1.28 
Neuropsychologic testing (see Addendum A) 
6.1.29 
Reproductive health assessment (Addendum B) 
6.1.30 
To evaluate sickle cell nephropathy in patients with SCD: cystatin C, c reactive protein 
(CRP), and the following urine studies: urinalysis, phosphate, uric acid, protein to creatinine ratio, 
albumin to creatinine ratio, and 24 hour urine for creatinine, protein, microalbumin, phosphate, uric 
acid, creatinine clearance, and urine protein electrophoresis (UPEP)  
6.1.30    
Evaluation of splenic function via abdominal ultrasound and/or quantitative Howell-Jolly 
body analysis in appropriate patients 
6.1.31  
Hepatology, Transfusion Medicine, Pulmonary, and other consult as indicated 
6.1.32  
STR profile 
 
6.2 
Inpatient monitoring 
All patients with a sickling disorder undergoing transplant will be listed as having a contraindication 
to the use of filgrastim (G-CSF).  
 
Once daily: CBC with differential, comprehensive metabolic panel, temperature, pulse, blood 
pressure, respiratory rate, weight 

03-H-0170 
John Tisdale, M.D. 
October 03, 2023 (Amendment KKK) 
15
 
Twice weekly: reticulocytes, pre-albumin, sirolimus level, and coagulation screen 
 
Weekly: CMV/EBV surveillance  
 
Every two weeks: serum cholesterol, triglycerides 
 
After the last dose Campath: Troponin T, ECHO, 24 hour holter as clinically indicated 
 
 
6.3 
Follow up to day 100: outpatient 
Once a week up to 30 days, blood samples may be collected to test for alemtuzumab levels. 
 
At least weekly up to 30 days then on return follow-up visits and when clinically indicated: CBC, 
hemoglobin electrophoresis to assess HbS, and Hb F levels in appropriate patients, coagulation 
screen, comprehensive metabolic panel, sirolimus level, temperature, pulse, blood pressure, 
respiratory rate, weight; CMV/EBV.  A complete physical exam will be repeated at each visit.  
 
Monthly (+/- 7 days):  Serum cholesterol, triglycerides, pregnancy test  
Peripheral blood will be drawn on days, +30, +60 and +100 to assess for donor-host chimerism in the 
lymphoid, myeloid, and erythroid cell lines including the use of Hb S and Hb F levels.   
 
100 Days (+/- 1 week) after the last dose Campath:  ECHO (if clinically indicated), Troponin (if 
clinically indicated), d-dimer, and in patients with SCD Cystatin C, CRP, and the following urine 
studies: urinalysis, phosphate, uric acid, protein to creatinine ratio, and albumin to creatinine ratio 
 
Days 100 (+/- 7 days): Neuropsychologic testing, Quality of life testing (See Addendum B) 
 
 
 
6.4 
Beyond day 100 
At 6, 12, (optional) 18, 24 (+/- 1 month), 36, 48, and 60 months (+/- 6 month):  CBC, Hb S and Hb F 
levels, comprehensive metabolic panel. Serum cholesterol, triglycerides, echo, and pulmonary 
function tests if clinically indicated.   
 
At 6, 12, and (optional) 18 months (+/- 1 month):  Blood will be drawn for preparation of plasma and 
lymphocytes for in vitro studies.  Based on the results, studies may be repeated every 6 to 12 months 
thereafter 
 
At 6 and 12 months (+/-1 month): Lymphocyte subpopulations and immunoglobulin levels will be 
checked, and the tests will be performed yearly until normalization. 
 
At 6 months in patients with SCD (when possible): Cystatin C, CRP, and the following urine studies: 
urinalysis, phosphate, uric acid, protein to creatinine ratio, and albumin to creatinine ratio 
 
At 12 months in patients with SCD (when possible): Cystatin C, CRP, and the following urine 
studies: urinalysis, phosphate, uric acid, protein to creatinine ratio, albumin to creatinine ratio, Bone 
marrow aspirate and/or biopsy samples will be obtained at day 100 post-transplant and/or when full 
donor erythroid chimerism (patient may refuse) is attained or if clinically indicated.  
 

03-H-0170 
John Tisdale, M.D. 
October 03, 2023 (Amendment KKK) 
16
At 12, 24 (+/- 1 month), 36, 48, and 60 months (+/- 6 months): bone density scan (if indicated), 
thyroid stimulating hormone, free thyroxine, insulin-like growth factor 1, morning cortisol level, 
fasting glucose, fasting insulin, hemoglobin A1C (or serum fructosamine level in patients whose 
donors have sickle cell trait), 25 hydroxy vitamin D, and in male patients at least 12 years of age, 
testosterone level, luteinizing hormone level (LH), and follicle stimulating hormone level (FSH), and 
in female patients at least 10 years of age LH, FSH, anti-mullerian hormone level, and estradiol level. 
All these endocrine labs may be repeated as clinically indicated.    
 
At 12, 24 (+/- 1 month), 36, 48, and 60 months (+/- 6 month): CNS Doppler flow for appropriate 
patients (to be performed at Children’s National Medical Center Department of Radiology) 
 
At 12, and 24 months (+/-1 month): Quality of life testing, neuropsychologic testing, brain MRI (see 
Addendum B) 
 
At 12 and 24 months (+/-1 month):  Reproductive health assessment (see Addendum C) 
 
Splenic function will be measured at 2 years post-transplant in appropriate patients. 
 
At 36, 48, and 60 months’ post-transplant, there is a window of +/- 6 months for their annual visit. 
After 5 years, follow-up visits are not mandatory, but yearly communication with the patient and the 
referring physician may continue.  
 
After 60 months (5 years post-transplant), follow-up visits are not mandatory, but yearly 
communications with the patient and the referring physician will continue to monitor adverse events 
that may be related to the transplant procedure.  
 
7.0  
TREATMENT PLAN FOR THE RECIPENT 
Patients with fever or suspected minor infection should await resolution of symptoms before starting 
the conditioning regimen.  Iron chelation must be discontinued > 48 hours before initiating the 
conditioning regimen.  Hydroxyurea must be discontinued one day prior to initiating the conditioning 
regimen 
 
7.1 
Central venous line placement 
A central venous catheter will be placed by an interventional radiologist, vascular access device 
specialist, or a surgeon prior to transplantation. 
 
7.2 
Infection prophylaxis   
Penicillin VK 250 mg PO BID from day 0 until day 100 until pneumococcal vaccination complete 
post-transplant. Prophylaxis and treatment of infections will otherwise be administered according to 
BMT consortium guidelines. 
 
 
If patients develop significant transaminitis with ALT and/or AST > 2x their baseline value, nystatin 
may be substituted for fluconazole as anti-fungal prophylaxis. 
 
Donors will be offered the influenza vaccination at the NIH Clinical Center as seasonally indicated 
pre-transplant (see section 10.5).  Patients will be offered influenza vaccination, as seasonally 
indicated, when they are at least 6 months post-transplant per CDC HSCT guidelines. 
7.3 
Fever regimen (See BMT Consortium Guidelines)  
 

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John Tisdale, M.D. 
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17
7.4 
Exchange transfusion (See Appendix C) 
Prior to the transplant, those patients with SCD who are not routinely (exchange) transfused will 
undergo an exchange transfusion per DTM procedure for a goal HbS near 30%, just prior to receiving 
the preparative regimen in order to decrease the likelihood of neurologic and other sickling events 
that may be precipitated by the transplant procedure.  
 
7.5 
Bleeding prophylaxis 
When possible, platelet counts will be maintained at or higher than 50,000/ul for patients with SCD 
(which is higher than usually maintained for non-sickle cell patient transplants) throughout the 
transplant to diminish the risk of intracranial bleeding.  Peri-transplant target hemoglobin will be kept 
above 9 g/dL in patients with SCD.  Otherwise, packed red blood cell and platelet transfusions will be 
given according to BMT consortium guidelines.   
 
7.6 
Preparative regimen (see Appendix D)  
 
7.7 
Peripheral blood progenitor cell transplant (see Appendix C)  
 
The target collection number for progenitor cells is ≥10 x 106 CD34+ cells/kg.   This product will be 
collected in advance as a standard procedure under a separate NHLBI protocol (94H0010 or 
20H0099) and cryo-preserved at DTM until the day of transplant.  
 
7.8  
GVHD prophylaxis  
Sirolimus (Rapamune®) will be started on day -1 with a loading dose of 5mg PO q4h for three doses 
in patients >16 years of age and 1mg/m2/dose PO q4h for three doses in patients <16 years of age 
(maximum 5mg per dose), then continued at 5 mg/day in patients > 16 years of age and 1mg/m2/dose 
daily in patients <16 years of age (maximum 5mg per dose) with goal trough levels being 5-15 ng/ml. 
The sirolimus will be given for a minimum of one year however the total duration of administration 
will be determined by the presence or absence of GVHD and the level of donor /recipient chimerism.  
 
If at least one year post-transplant the recipient displays >50% lymphoid donor chimerism, the 
sirolimus dose can be tapered.  Donor/recipient chimerism levels will be checked periodically during 
and after the taper.  If lymphoid and/or myeloid donor chimerism levels decrease by > 20%, the 
sirolimus and/or other immunosuppression may be restarted clinically indicated. 
 
Subjects will be advised not to take medication with grapefruit juice and not to take St. John’s wort 
while on sirolimus.  Subjects must also be advised about limiting exposure to sunlight and UV light 
due to an increased risk of skin cancer. Women of childbearing potential will be informed of the 
potential risks during pregnancy and that they should use effective contraception prior to initiation of 
drug.  
 
7.9 
Transfusion support (See Appendix B) 
Filtered and irradiated blood products will be used in all patients, regardless of CMV status.    
 
7.10 
Nutrition  
Parenteral nutrition will be instituted as clinically indicated.  
 
7.11 
Hospital discharge 
Patient will be discharged when the following criteria are fulfilled:   
 
Patient afebrile, positive weight balance; no parenteral feeding required 
 
Platelet transfusion requirement absent or manageable as an outpatient 
 
Patient or family able to care for central line 
 

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John Tisdale, M.D. 
October 03, 2023 (Amendment KKK) 
18
7.12 
Contraindication listings 
Filgrastim (G-CSF) will be listed as a contraindication for all patients with sickling disorders and will 
only be given with the consent of either the principal investigator, lead associate investigator or the 
attending protocol investigator. 
 
8.0 
DONOR CELLS FOR RESEARCH 
 
Evaluation of related donor suitability and eligibility will be done in accordance with existing 
Standard Policies and Procedures under a separate NHLBI protocol as follows: 
 
8.1 
Pre-study consult and evaluation 
 
1. HLA-A, -B, -DR typing of as many  family members as necessary 
2. Confirm HLA identity of donor with patient 
3. History and physical examination 
4. Hemoglobin electrophoresis 
5. Hepatitis B, C, HIV, HTLV-I/II, CMV antibodies, RPR, West Nile virus, T. cruzi 
6.  CBC with differential, coagulation screen, comprehensive metabolic panel 
7. Extended red cell phenotyping,  
8. HLA antibody screening if indicated 
9. Fit to donate:  Orientation - visit to Department of Transfusion Medicine - inspection of veins to 
determine the need for a central line for apheresis. 
10. Donors less than 18 years of age will be evaluated by a mental health specialist with pediatric 
expertise or a social worker about their participation as a donor 
11. Consent/assent to undergo filgrastim (G-CSF) mobilization.   
12. Donors with sickle cell trait will be evaluated with assessment of oxygen saturation by pulse 
oximetry on days 3, 4, and 5 of the mobilization and have a follow-up visit with physician within 2 
weeks after PBPC collection. 
13. If a central venous line is required, blood counts and coagulation studies will be performed before 
line placement  
 
8.2 
Blood donation for research 
 
Up to 50 mL of donor blood may be collected prior to cell donation (and prior to GCSF mobilization) 
for research.  The amount of blood that may be drawn from adult donors (i.e., those persons 18 years 
of age or older) for research purposes shall not exceed 10.5 mL/kg or 550 mL, whichever is smaller, 
over any eight-week period. For pediatric donors, no more than 5 mL/kg may be drawn for research 
purposes in a single day, and no more than 9.5 mL/kg may be drawn over any eight-week period. 
 
Donors will undergo standard cell collections under a separate NHLBI protocol (94H0010 or 
20H0099). 
 
8.3 
Neuropsychological and quality of life testing 
Donors will undergo neuropsychologic testing (please see Addendum A) 
 
 
9.0 
MANAGEMENT OF RECIPIENT COMPLICATIONS 
 
The major complications are cytomegalovirus reactivation, acute and chronic GVHD, and relapse of 
the original disease.  Patients with these complications will be treated along the following lines: 
 

03-H-0170 
John Tisdale, M.D. 
October 03, 2023 (Amendment KKK) 
19
9.1 
CMV reactivation: See BMT Consortium Guidelines 
  
9.2 
Acute GVHD:  See BMT Consortium Guidelines 
 
9.3 
Chronic GVHD:  
Sirolimus will be continued with the duration dependent on the presence or absence of GVHD. 
Prednisone will be dosed according to severity.  Change to alternate day steroid along with sirolimus 
therapy when response is established. 
 
Non-responding patients may be treated with other standard of care therapies (such as cyclosporine, 
psoralen + ultraviolet-A (PUVA), photopheresis, azathioprine, thalidomide, daclizumab, infliximab, 
mycophenolate or tacrolimus) at the discretion of the attending physician. 
 
9.4 
Graft rejection and falling chimerism (poor engraftment): 
This transplant protocol uses a nonmyeloablative preparative regimen. Therefore, autologous 
recovery is anticipated in patients who fail to engraft. For falling chimerism in the myeloid and/or 
CD3 compartment, unselected stem cell product (‘boost’) from the original donor may be used after 
preconditioning, with Busulfan IV 3.2 mg/kg on days -5, -4, and -3 and Campath IV 0.03mg/kg on 
day -7, 0.1mg/kg on day -6, and 0.2mg/kg on days -5 and -4 followed by sirolimus or equivalent 
immunosuppressant as in protocols 14-H-0111 and 15-H-0098. Busulfan dosing may be reduced or 
substituted with melphalan 100-140mg/m2 in those with renal or liver dysfunction to minimize organ 
injury. These medications are widely used at the Clinical Center and other transplant centers, and 
their side effects are well known. Recipient bone marrow harvest or plerixafor mobilization before 
stem cell boost is likely not needed, but may be considered if one or more risk factors for 
pancytopenia post infusion are present (e.g. age >35, low stem cell number of the boost, or low blood 
counts before the start of the pre-conditioning regimen). Marrow harvest procedure is a common 
practice among transplant community to ensure a back-up source of cells is available to avoid 
prolonged period of pancytopenia.  
 
For patients who had initial donor engraftment and subsequent graft rejection (donor myeloid and 
CD3 chimerism of 0%), there is currently no standard approach; watch and wait until resurgence of 
sickle related complications (pain crisis, acute chest syndrome, etc), restart hydroxyurea, or another 
stem cell transplant are all options one can consider. We will discuss these options, and the patient 
can be offered to participate in other applicable clinical trials, or be referred back to their primary 
physician depending on what is considered to be in the best interest of the patient.    
 
 
 
9.5 
Relapse of disease 
Patients with relapse may be treated with standard of care treatment options under a separate NHLBI 
protocol (94H0010 or 20H0099), with or without stem cell rescue (2nd transplant) or referred back to 
their primary physician depending on what is considered to be in the best interest of the patient (also 
see section 11.5.2).   
 
10.0 
RESEARCH STUDIES:  
Human specimen use, disposition, tracking and storage of samples and data 
During the course of participating on this study, blood, tissue and data will be collected for correlative 
laboratory research studies.  Specimens collected strictly for research purposes will not be read by a 
pathologist.   
 
 
 
 

03-H-0170 
John Tisdale, M.D. 
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20
Specimen management 
 
Storage:  Research specimens will be stored in the principal investigator’s laboratory. Samples will 
never be labeled with the patient’s name. Samples will be assigned a unique code known only to the 
principal investigator, which will serve as a link to the patient’s clinical information collected as part 
of this research protocol.  No samples will be provided to investigators outside the branch without 
IRB notification and an executed MTA. Therefore, confidentiality is protected. 
Tracking:  Samples will be ordered and tracked through the CRIS Research Screens.  Should a CRIS 
screen not be available, the NIH form 2803-1 will be completed and will accompany the specimen 
and be filed in the medical record.  Samples will not be sent outside NIH without IRB notification and 
an executed MTA.  
End of study procedures:  Samples from consenting subjects will be stored until they are no longer 
of scientific value or if a subject withdraws consent for their continued use, at which time they will be 
destroyed.  
Loss or destruction of samples:  Should we become aware that a major breech in our plan for 
tracking and storage of samples has occurred, the IRB will be notified. 
 
Sharing:  Samples and/or medical data may be provided to other investigators at the NIH or at other 
institutions for studies related to sickle cell disease and immune system function, without any 
personal identifying information.  These samples cannot be traced back to individual subjects by 
collaborating investigators. These de-identified samples and/or data may be shared with other 
investigators involved with CIBMTR in collaborative studies to compare HSCT outcomes, such as 
mixed chimerism, GVHD, infection, changes in organ function, and mortality.  
 
Data management 
The principal investigator will be responsible for overseeing entry of data into an in-house password 
protected electronic system or locked research file system and ensuring data accuracy, consistency 
and timeliness. The principal investigator, associate investigators/research nurses and/or a contract 
data manager will assist with the data management efforts.  
 
All human subjects personally identifiable information (PII), eligibility and consent verification will 
be recorded.  Primary data obtained during the conduct of the protocol will be kept in secure network 
drives or in approved alternative sites that comply with NIH security standards.  Primary and final 
analyzed data will have identifiers so that research data can be attributed to an individual human 
subject participant, e.g., study-specific identifying number (SSPIN) or other unique code, or 
minimum PII required for subject identification.   
  
 
Data will be abstracted from Clinical Center progress notes as well as from progress notes forwarded 
from home physicians. Laboratory data from NIH will be imported electronically from CRIS into an 
in-house database. Laboratory values from referring home physicians will be entered into the system. 
 
End of study procedures:  Data will be stored in locked cabinets and in a password protected 
database until it is no longer of scientific value.  
 
Loss or destruction of data:  Should we become aware that a major breech in the plan to protect 
patient confidentiality and trail data has occurred, the IRB will be notified.  Data will not be sent 
outside NIH without IRB notification and an executed MTA or CTA. 

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October 03, 2023 (Amendment KKK) 
21
 
 
10.1 
Chimerism studies 
We will use PCR analysis of microsatellites to identify the contribution of the donor marrow to post-
transplant hematopoiesis and to detect donor lymphocytes in the circulation at 6, 12, 24 months. We 
will also use gel electrophoresis and/or flow cytometry to assess the amount of normal hemoglobin 
within the erythroid cells. 
 
10.2 
Bone marrow samples  
A volume (up to 25 ml) of bone marrow aspirate will be collected for research studies at the pre-
transplant evaluation, day 100 post-transplant and/or when full donor erythroid chimerism is attained 
(patient may refuse) or if clinically indicated.  These will be used to help elucidate the contribution of 
the progenitor cells to the circulating component. 
 
10.3 
Transthoracic echocardiography  
Transthoracic echocardiography will be performed to assess TR jet velocity at study onset and yearly 
post transplantation, or more frequently as clinically indicated. 
 
10.4  
Immune reconstitution  
Lymphocyte subpopulations (absolute number of CD3, CD4, CD8, CD16, CD56, and CD19 cells) 
and immunoglobulin levels (IgG, IgA, and IgM) will be quantified at 6 and 12 months post-transplant 
(and yearly thereafter until results have normalized).  Splenic function will be measured in 
appropriate patients by Tc99m-labeled radiocolloid scan of the liver and spleen 2 years’ post-
transplant. This will be compared to the scan from pre-transplant.  Liver and spleen scans have only 
been shown to be reversible in the children with SCD after transplant, thus it is not performed in those 
16 and older.  The spleen of older adolescents and adults with SCD are atrophic, and likely not to 
regain function after transplant, thus the liver and spleen scans are not performed in those age 16 and 
older. 
 
10.5 
Assessment of cytokines and lymphocyte functions 
Donor serum and lymphocytes will be collected pre-transplant.  Patient serum and lymphocytes will 
be collected pre-transplant and at 6, 12, and 18 months post-transplant.  Serum tumor necrosis factor, 
interferon-gamma, and interleukin-17 may be quantified.  Levels may continue to be followed every 6 
to 12 months thereafter based on the results during the first 18 months. 
 
Patient effector T cell subsets may be measured at each time point by intracellular cytokine staining 
for interferon gamma, interleukin-4, interleukin-17, and FoxP3.  Biochemical analysis of mTOR 
signaling complexes in patient T cells will be measured by Western blot for phospho p70 S6 kinase 
and phospho AKT in response to anti CD3 plus anti CD28 stimulation in vitro. 
 
CMV pp65 specific T cell responses may be measured in cases where either donor or recipient is 
CMV positive.  These will be assessed by intracellular cytokine staining for interferon-gamma and 
FoxP3 in response to pooled overlapping pp65 peptides. 
 
In cases where patient/donor pairs have received influenza vaccine due to the seasonal timing of the 
transplant, we may measure influenza specific T cell responses by intracellular cytokine staining for 
interferon-gamma, interleukin-4, interleukin-17, and FoxP3 in response to influenza peptides. 
 
In cases where GVHD or graft rejection is observed, we may assess donor/host allo-reactivity by 
CFSE dilution assay in the presence and absence of sirolimus in vitro.  We will also measure mTOR 
signaling in patient T cells by Western blot for phospho p70 S6 kinase and phospho AKT in response 
to anti CD3 plus anti CD28 stimulation in the presence or absence of sirolimus in vitro. 

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All of these research tests may be run at the laboratory of Dr. Jonathan Powell at Johns Hopkins 
Hospital.   Samples will be assigned a unique code known only to the principal and associate 
investigators.  
 
Transplant recipient blood samples may be sent to the Immune Tolerance Network (ITN) run by 
NIAID for the study of immune function and tolerance in the setting of stem cell transplantation.  
Samples will be assigned a unique code know only to the principal and associate investigators.   
 
10.6    Dual X-ray Absorptiometry (DEXA) Scan  
 
For adult subjects (age 18 and older), up to 2 DEXA scans (to allow for repeat measurement if there 
are areas of the bone that need clarification) will be performed at baseline and every 1-2 years post-
transplant to evaluate patients for osteopenia and osteoporosis.  The frequency will be determined by 
the severity of their bone disease. 
 
10.7 
Neuropsychologic testing:  see Addendum B  
 
10.8 
Quality of life testing: see Addendum B  
 
 
11.0 
BIOSTATISTICAL CONSIDERATIONS 
 
11.1 
Sample size 
This study will accrue a range of 60-75 recipients and 60-75 donors, to allow for at least 50 
successfully transplant patients and at least 15 donors who complete the 1 year post-transplant 
neuropsychological testing. 
 
11.2 
The parameters to be monitored are:  
1) CD34+ cell dose, CD3+ cell dose 
2) Degrees of donor-recipient lymphoid, myeloid and erythroid chimerism by microsatellite PCR 
analysis and normal hemoglobin quantitation-either by gel electrophoresis or if necessary by flow 
analysis – using peripheral blood as appropriate 
3) Neutrophil recovery (days to neutrophil count of 0.5 x 109/l and 1.0 x 109/l). 
4) Platelet recovery (days to platelet count of 50 x 109, days to transfusion independence) 
5) Red cell recovery (days to transfusion independence) 
6) Incidence and severity of acute GVHD 
7) Incidence and severity of chronic GVHD 
8) Incidence of Graft Rejection 
9) Non-hematologic effects attributable to the preparative regimen 
10)  Transplant related mortality by day 200  
11)  Disease-free survival and overall survival 
12)  Hemoglobin F and S, levels in appropriate patients 
13)  Development of further neurologic disease and assessment of neurocognitive status in 
appropriate patients 
14)  Gonadal organ function as reflected by hormone levels and normal menstrual cycles  
15)  Assessment of immune reconstitution 
16)  Assessment of cytokines and lymphocyte function 
17)  Quality of life 
18)  Neuropsychologic function 
 

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11.3 
Study endpoints 
 
Primary endpoint 
The primary endpoint is treatment success at one year, defined as full donor type hemoglobin on 
hemoglobin electrophoresis for patients with SCD and transfusion-independence for patients with β-
thalassemia. 
 
This trial is designed to estimate treatment success, which is anticipated to be about 80%.  The study 
started with a sample size of 25 and this will allow us to estimate the success of engraftment.  For 
example, if the estimated rate is .80, the 95% confidence interval would be approximately (.64, .96).  
This would allow us to rule out rate of treatment success of less than .64.  If the estimated rate is .70, 
the 95% confidence interval would be approximately (.52, .88) and we could rule out rate of treatment 
success below .50.  If the lower bound of the 95% confidence interval is raised to 0.7, the number of 
subjects needed to accrue with respect to success rate is listed below.  
 
Success rate 
0.90 
0.89 
0.88 
0.87 
0.86 
0.85 
0.84 
0.83 
0.82 
0.81 
0.80 
0.79 
Number of 
subjects 
needed 
9 
11 
13 
16 
19 
22 
27 
33 
40 
49 
62 
79 
 
Secondary endpoints 
1) The level of chimerism required to maintain both graft survival as well as hematologic normalcy.  
The chimeric status of patients will be measured on days, +30, , +60, and +100 by microsatellite 
analysis of the peripheral blood.  More frequent monitoring may be required.   
2)  Incidence of acute and chronic GVHD or relapse rate.  GvHD or relapse together count together 
toward the combined endpoint for regimen failure (see section 11.4 below). 
3) Disease-free survival and overall survival 
4) Transplant-related mortality  
 
 
Exploratory endpoints:  
1) Immunologic function post-transplant 
2) Quality of life and neuropsychologic function post-transplant (both donors and recipients) (see 
Addendum B) 
3) Effect of transplant on end-organ function (e.g. renal function, see Addendum A) 
 
The accrual ceiling range would allow for about 50 successfully transplanted recipients, assuming a 
graft rejection rate of about 10-15%. Among the transplanted patients, about 10% of the patients have 
β-thalassemia, sickle β-thalassemia (+), or sickle β-thalassemia (0) – collectively sickle/thal group, 
and another 10% have sickle SC disease. These expected accrual figures in the subgroups are small 
and statistical power for detecting differences in engraftment rates are low for moderate differences, 
e.g. power is less than 20% for engraftment rates of 90% and 70% in the larger and smaller 
subgroups, respectively. Thus, when the subgroups’ engraftment rates are reported the discussion will 
explicitly acknowledge the small sample size supporting the observations so interpretations are 
appropriately cautious.   
 
Additionally, the accrual ceiling range would also allow for at least 15 donors who return for their 1 
year post-transplant testing to repeat neuropsychologic testing. 
 
 
11.4 
Stopping rules 
For regimen failures: Because this regimen is designed to decrease the risk of severe acute and 
chronic GVHD in patients with non-life threatening conditions, the study will be stopped and the 

03-H-0170 
John Tisdale, M.D. 
October 03, 2023 (Amendment KKK) 
24
design re-evaluated after any death. We will also monitor the study for excessive graft rejection and 
excessive severe (acute Grade III-IV and chronic extensive) GVHD which we would consider a 
failure of the regimen. So regimen failure is a composite endpoint, which means graft rejection, acute 
Grade III-IV GVHD, or chronic extensive GVHD.   As adult patients have already been enrolled on 
our protocol, regimen failures will be monitored separately for pediatric patients. 
 
Regimen failure will be evaluated for each individual during the 12 month period following the start 
of transplant.   We will stop the study if posterior probability that the regimen failure rate is greater 
than 30% exceeds 0.90. Stopping means that serious consideration will be given to modifying or 
terminating the protocol.    Stopping boundaries are given in the table below.  Beta prior distributions 
are used for these calculations with parameters alpha, beta = 2.1, 4.9 for regimen failure.  
 
Note that one need not evaluate the number of patients in the first column in order to stop.  For 
example, if 4 of the first 4 patients fail to engraft, we will have crossed the boundary given in the first 
row.  For 50 patients, stopping rule would be met if 29 patients failed. 
Group Sequential Monitoring Plan  
# of patients with 12 month transplant anniversary  
Regimen Failures 
5 
4 
10 
6 
15 
8 
20 
10 
25 
12 
30 
13 
35 
15 
40 
17 
45 
18 
50 
20 
55 
22 
60 
24 
   
The monitoring plan was evaluated by simulation.  If the true regimen failure rate is .3, we will stop 
about 10% of the time.  True regimen failure rates of .2, .4, and .6 entail stopping about 2%, 34% and 
90% of the time respectively.  This is acceptable performance for a stopping rule.   
 
 
For neuropsychologic testing: We propose to stop the study if accrual is such that we cannot meet the 
target of 15 donor-recipient paired neuropsychology responses within our sample size limit of 75 
transplants. For instance, if after the 70th transplant, we have only 9 donor-recipient pairs, we will be 
unable to obtain data on 15 donor-recipient pairs and further enrollment will be stopped. Our stopping 
rule is to halt enrollment if: 
 
we reach 56 transplants but data from only 7 donor-recipient pairs are available, or 
 
we reach 66 transplants but data from only 8 donor-recipient pairs are available, or 
 
we reach 70 transplants but data from only 9 donor-recipient pairs are available, or 
 
we reach 71 transplants but data from only 10 donor-recipient pairs are available, or 
 
we reach 72 transplants but data from only 11 donor-recipient pairs are available, or 
 
we reach 73 transplants but data from only 12 donor-recipient pairs are available, or 
 
we reach 74 transplants but data from only 13 donor-recipient pairs are available.     
 
11.5 
Off study criteria 
 
11.5.1 
Withdrawal by the patient from the transplant procedure 

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John Tisdale, M.D. 
October 03, 2023 (Amendment KKK) 
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Patients and their donors will be given ample opportunity to withdraw from the study prior to 
admission for transplant.  Thereafter, the nature of the procedure does not permit safe withdrawal 
from the protocol. 
 
The patient and donor have the right at any time to elect not to participate in the research aspects of 
the protocol (donation of blood and bone marrow for non-routine tests). 
 
11.5.2 
Withdrawal by the physician from experimental protocol 
Patients with disease relapse may be taken off protocol but will continue to be monitored by our 
institution for a minimum of 6 months post-transplant for possible infectious complications related to 
the conditioning regimen.  The patient will then be consented to the Hematology Branch evaluation 
and treatment protocol (94-H-0010) or to the Sickle Cell Branch natural history protocol (04-H-0161) 
for alternative treatments or referred back to his/her referring physician depending on what is 
considered to be in the best interest of the patient.  
 
12.0 
DATA AND SAFETY MONITORING  
 
12.1 
Safety monitoring 
 
Principal Investigator: The safety of interventions and treatments associated with this protocol will 
be under continuous review by the investigative team.  Accrual, efficacy and safety data will be 
monitored by the PI.   
 
 
NIH Intramural IRB. Prior to implementation of this study, the protocol and the proposed patient 
consent forms will be reviewed and approved by the properly constituted Institutional Review Board 
(IRB) operating according to the 45 CFR 46 code of federal regulations.  This committee will also 
approve all amendments to the protocol or informed consent, and conduct continuing annual review 
so long as the protocol is open to accrual or follow up of subjects.  
 
DSMB: The NHLBI Data safety and Monitoring Board will review the protocol at 6-12 month 
intervals.  A progress report will be forwarded to the DSMB at these times and their 
recommendations will be expeditiously implemented. The DSMB may recommend early termination 
of the study for considerations of safety and efficacy. 
 
12.2 
Grading of adverse events:  
 Definitions: Definitions will be in accordance to HRPP Policy 801 “Reporting Research Events” 
The AEs will be attributed (unrelated, unlikely, possibly, probably or definitely) to study medication 
and/or disease and graded by severity utilizing CTCAE version 4.0.  A copy of the criteria can be 
down-loaded from the CTEP home page at   http://ctep.cancer.gov/reporting/ctc.html.  
 
Grading of adverse events: 
 
Grade 
Category  
 
Description 
1 
Mild  
 
Mild; asymptomatic; clinical or diagnostic observations only; 
intervention not indicated  
2 
Moderate  
 
Moderate; minimal, local or noninvasive intervention indicated; 
limiting age-appropriate instrumental ADL  

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John Tisdale, M.D. 
October 03, 2023 (Amendment KKK) 
26
3 
Severe  
 
Severe or medically significant but not immediately life-threatening; 
hospitalization or prolongation of hospitalization indicated; 
disabling; limiting self-care ADL  
4 
Life 
threatening  
 
Life-threatening consequences; urgent intervention indicated 
5 
Death  
 
Death related to AE  
 
Attribution of Adverse Events: 
Relationship  
Attribution  
Description  
Unrelated to investigational 
agent/intervention1 
Unrelated  
The AE is clearly NOT 
related to the intervention  
 
Unlikely 
The AE is doubtfully related 
to the intervention 
Related to investigational 
agent/intervention1 
Possibly 
The AE may be related to the 
intervention 
Probably 
The AE is likely related to 
the intervention  
Definitely 
The AE is clearly related to 
the intervention  
1NOTE: AEs listed as ‘possibly, probably, or definitely’ related to the investigational agent/intervention are considered 
to have a suspected ‘reasonable causal relationship’ to the investigational agent/intervention (ICH E2A). 
 
 
12.3  NIH IRB and CD reporting 
Expedited Reporting 
Events requiring expedited reporting will be submitted to the IRB per HRPP Policy 801 “Reporting 
Research Events”. 
 
Reports to the IRB at the time of Continuing Review: 
The PI or designee will refer to HRPP Policy 801 “Reporting Research Events” to determine IRB 
reporting requirements. 
 
Reports to the CD: 
The PI or designee will refer to NHLBI DIR guidelines to determine CD reporting requirements and 
timelines. 
 
DSMB:  
Serious unanticipated problem reports will be forwarded immediately to the Data and Safety Monitoring 
Board (DSMB).  All SAEs and a summary of the adverse events that are determined to be possibly, 
probably or definitely related to this investigational treatment will be included for review by the DSMB. 
 
 
 
13.0 
HUMAN SUBJECT PROTECTIONS 
 
13.1 
Rationale for subject selection 
 

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John Tisdale, M.D. 
October 03, 2023 (Amendment KKK) 
27
All patients with confirmed β-thalassemia and/or SCD as defined in section 5.0 will be considered for 
the protocol.  Gender, ethnic background, and/or race will not be taken into consideration. 
 
Strategies for patient recruitment: Hematologists and internists throughout the country will be 
informed of the protocol by letter.  Information about the protocol will be posted on 
Clinicaltrials.gov, Clinical Center studies, and the NHLBI Patient recruitment websites. The protocol 
will also be listed in the physician’s data query (PDQ).    
 
13.2 
Participation of children 
 
13.2.1  As stem cell transplant recipients  
To date, all of the patients are living, none of the patients experienced GVHD, and most patients are 
free from their SCD.  Therefore, we feel that this nonmyeloablative and potentially curative approach 
is reasonable in children.  Four years of age will be the minimum age allowed for recipients on the 
protocol due to safety limits at the NIH.  
 
13.2.3  As participants in laboratory research studies.   
 
Pediatric participants may participate in those laboratory studies that the IRB finds involves no 
greater than minimal risk to children provided that adequate provisions are made for soliciting the 
assent of the children and the permission of their parents or guardians (see section 14.6). 
 
13.3 
Hazards and discomforts - recipient 
 
13.3.1 
Related to the transplant 
The mortality from conventional BMT may be as high as 40%.  Although our data as well as that of 
others suggest a significant reduction in transplant related mortality with nonmyeloablative “mini” PBSC 
transplantation, the procedure nevertheless carries significant risk.  It is therefore only appropriate to carry 
out this experimental procedure in the context of debilitating or life-threatening conditions and with full 
informed consent from the patient, donor and immediate family. We have sought to develop a 
conditioning regimen, which avoids the use of renally excreted drugs, and relies on the 
immunosuppressive effects of total body irradiation as the basis for such an approach.  A modest increase 
in the dosage of TBI from the 200 cGy used in prior studies to 300 cGy may increase both the degree of 
myelosuppression and immunosuppression without significantly altering the side effect profile. The 
specific hazards of this study using a nonmyeloablative preparative regimen and high PBPC content graft 
are graft rejection, graft-versus-host disease, and disease relapse.  The major discomforts are those of 
nausea, anorexia, diarrhea, fever and malaise, and intolerance of the isolation period.  The initial ten 
patients with SCD that were treated according to this protocol at the NIH did not experience significant 
toxicity related to the transplant regimen or GVHD.  In less than 10% of our transplanted patients, there 
have been episodes of bleeding from development of an inhibitor to coagulation factor(s).  The amount of 
bleeding can range from minor to severe, and patients typically need hospitalization for treatment and 
monitoring. Other groups have also reported this complication [99,100,101,102]. Severe hemolytic 
anemia from antibodies detected post-transplant. Treating this form of hemolysis include corticosteroids, 
IVIg, and/or other adjunct therapy. There may be an increased risk for hematologic malignancy, which 
may be related to severe sickle cell disease, inflammatory marrow from frequent vaso-occlusive crises, 
iron overload with increased reactive oxygen species, prior hydroxyurea, radiation, immunosuppression, 
infection, and other unknown factors. Lastly, organ injury and/or dysfunction in the brain, heart, kidney, 
thyroid, muscles, liver, and lung can result from one or more portions of the transplant process 
(conditioning regimen, medications, and infections). 
 

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October 03, 2023 (Amendment KKK) 
28
13.3.2 
Related to alemtuzumab (Campath®) 
 
The safety and efficacy of alemtuzumab were evaluated in a multicentre, open-label, non-comparative 
study in 93 patients with refractory B-cell chronic lymphocytic leukemia (B-CLL) who had been 
previously treated with alkylating agents and had failed treatment with fludarabine and side effects 
are detailed below.  Previous treatment with alkylating agents and fludarabine may have contributed 
to both the range and severity of the side effects observed. 
 
Infusion-related: adverse events resulted in discontinuation of alemtuzumab therapy in 6% of the 
patients. The most commonly reported infusion-related adverse events include rigors in 89% of 
patients, drug-related fever in 83%, nausea in 47%, vomiting in 33%, and hypotension in 15%. Other 
frequently reported infusion-related events include rash in 30% of patients, fatigue in 22%, urticaria 
in 22%, dyspnea in 17%, pruritus in 14%, headache in 13%, and diarrhea in 13%. Acute infusion-
related events were most common during the first week of therapy. Antihistamines, acetaminophen, 
antiemetics, meperidine, and corticosteroids as well as incremental dose escalation were used to 
prevent or ameliorate infusion-related events. 
 
Infections:   In the earlier studies all patients were required to receive anti-herpes and anti-PCP 
prophylaxis.  Forty (43%) of 93 patients experienced 59 infections (one or more infections per 
patient) during treatment or within 6 months of the last dose. Of these, 34 (37%) patients experienced 
42 infections that were of Grade 3 or 4 severity; 11 (18%) were fatal. Fifty-five percent of the Grade 
3 or 4 infections occurred during treatment or within 30 days of last dose. In addition, one or more 
episodes of febrile neutropenia (ANC 500 cells/µL were reported in 10% of patients). The following 
types of infections were reported: Grade 3 or 4 sepsis in 12% of patients with one fatality, Grade 3 or 
4 pneumonia in 15% with five fatalities, and opportunistic infections in 17% with four fatalities. 
Candida infections were reported in 5% of patients; CMV infections in 8% (4% of Grade 3 or 4 
severity); Aspergillosis in 2% with fatal Aspergillosis in 1%; fatal Mucormycosis in 2%; fatal 
Cryptococcal pneumonia in 1%; Listeria monocytogenes meningitis in 1%; disseminated Herpes 
zoster in 1%; Grade 3 Herpes simplex in 2%; and Torulopsis pneumonia in 1%. PCP pneumonia 
occurred in one (1%) patient who discontinued PCP prophylaxis.  In one of the earlier studies where 
anti-herpes and anti-PCP prophylaxis was optional, 37 (66%) patients had 47 infections while or after 
receiving Campath therapy. 
 
Boxed Warning 
Hematologic Toxicity: Serious and, in rare instances fatal, pancytopenia/marrow hypoplasia, 
autoimmune idiopathic thrombocytopenia, and autoimmune hemolytic anemia have occurred in 
patients receiving Campath therapy. Single doses of Campath greater than 30 mg or cumulative 
doses greater than 90 mg per week should not be administered because these doses are 
associated with a higher incidence of pancytopenia.  
Infusion Reactions: Campath can result in serious, and in some instances fatal, infusion reactions. 
Patients should be carefully monitored during infusions and Campath discontinued if indicated.  
Gradual escalation to the recommended maintenance dose is required at the initiation of 
therapy and after interruption of therapy for 7 or more days.  
Infections, Opportunistic Infections: Serious, sometimes fatal bacterial, viral, fungal, and 
protozoan infections have been reported in patients receiving Campath therapy. Prophylaxis directed 
against Pneumocystis carinii pneumonia (PCP) and herpes virus infections has been shown to 
decrease, but not eliminate, the occurrence of these infections.                                 

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John Tisdale, M.D. 
October 03, 2023 (Amendment KKK) 
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Immunosuppression/Opportunistic Infections:  Alemtuzumab induces profound lymphopenia. Anti-
infective prophylaxis is recommended upon initiation of therapy and for a minimum of 2 months 
following the last dose of Alemtuzumab or until CD4+ counts are 200 cells/µL. The median time to 
recovery of CD4+ counts to 200/µL was 2 months, however, full recovery (to baseline) of CD4+ and 
CD8+ counts may take more than 12 months. Because of the potential for Graft versus Host Disease 
(GVHD) in severely lymphopenic patients, irradiation of any blood products administered prior to 
recovery from lymphopenia is recommended. 
 
Hematologic: 
Pancytopenia/Marrow Hypoplasia: Alemtuzumab therapy was permanently discontinued in six (6%) 
patients due to pancytopenia/marrow hypoplasia. Two (2%) cases of pancytopenia/ marrow 
hypoplasia were fatal. 
Anemia: Forty-four (47%) patients had one or more episodes of new onset NCI-CTC Grade 3 or 4 
anemia. Sixty-two (67%) patients required RBC transfusions. In addition, erythropoietin use was 
reported in nineteen (20%) patients. Autoimmune hemolytic anemia secondary to Alemtuzumab 
therapy was reported in 1% of patients. Positive Coombs test without hemolysis was reported in 2%. 
Neutropenia: Sixty-five (70%) patients had one or more episodes of NCI-CTC Grade 3 or 4 
neutropenia. Median duration of Grade 3 or 4 neutropenia was 28 days (range: 2 – 165 days). 
Thrombocytopenia: Forty-eight (52%) patients had one or more episodes of new onset Grade 3 or 4 
thrombocytopenia. Median duration of thrombocytopenia was 21 days (range: 2 – 165 days). Thirty-
five (38%) patients required platelet transfusions for management of thrombocytopenia. Autoimmune 
thrombocytopenia was reported in 2% of patients with one fatal case of Alemtuzumab -related 
autoimmune thrombocytopenia. 
Lymphopenia: The median CD4+ count at 4 weeks after initiation of Alemtuzumab therapy was 2 
(two)/µL, at 2 months after discontinuation of Alemtuzumab therapy, 207/µL, and 6 months after 
discontinuation, 470/µL. The pattern of change in median CD8+ lymphocyte counts was similar to 
that of CD4+ cells. In some patients treated with Alemtuzumab, CD4+ and CD8+ lymphocyte counts 
had not returned to baseline levels at longer than 1-year post therapy. 
 
Cardiac: The following were reported in at least one patient treated on studies where Campath-1H 
was used as a single agent: cardiac failure, cyanosis, atrial fibrillation, cardiac arrest, ventricular 
arrhythmia, ventricular tachycardia, angina pectoris, coronary artery disorder, myocardial infarction, 
and pericarditis. Some of these cardiac abnormalities may be irreversible.  For this reason, we will 
monitor subjects with an echocardiogram, a 24 hour holter monitor and serum troponin levels before 
treatment begins; monitoring after the last dose of Campath-1H and at the 3 month follow up visit 
will be done as clinically indicated. We will also closely monitor subjects for cardiac symptoms and 
ask them to immediately report any cardiac symptoms (palpitations, irregular pulse, difficulty in 
breathing, dizziness, swelling in the ankles, chest discomfort or pain).  
 
13.3.3 
Related to sirolimus: 
The anticipated toxicities of sirolimus in this trial are those related to its immunosuppressive 
properties, such as an increased likelihood of infection, and mucous ulcers.  Other possible toxicities 
are listed here and include those reported with >/=3% and <20% incidence in patients in any 
Sirolimus treatment group in the two controlled clinical trials for the prevention of acute organ graft 
rejection,  
 
Body as a whole: abdomen enlarged, abscess, ascites, cellulitis, chills, face edema, flu syndrome, 
generalized edema, hernia, Herpes zoster infection, lymphocele, delayed/impaired wound healing, 
malaise, pelvic pain, peritonitis, sepsis; 

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John Tisdale, M.D. 
October 03, 2023 (Amendment KKK) 
30
Cardiovascular System: atrial fibrillation, congestive heart failure, hemorrhage, hypervolemia, 
hypotension, palpitation, peripheral vascular disorder, postural hypotension, syncope, tachycardia, 
thrombophlebitis, thrombosis, vasodilatation, pericarditis;  
Digestive System: anorexia, dysphagia, eructation, esophagitis, flatulence, gastritis, gastroenteritis, 
gingivitis, gum hyperplasia, ileus, liver function tests abnormal, mouth ulceration, oral moniliasis, 
stomatitis;  
Endocrine System: Cushing's syndrome, diabetes mellitus, glycosuria, hypercholesterolemia, 
hyperlipidemia;  
Hematologic and Lymphatic System: ecchymosis, leukocytosis, lymphadenopathy, polycythemia, 
thrombotic thrombocytopenic purpura (hemolytic-uremic syndrome); 
Metabolic and Nutritional: acidosis, alkaline phosphatase increased, BUN increased, creatine 
phosphokinase increased, dehydration, healing abnormal, hypercalcemia, hyperglycemia, 
hyperphosphatemia, hypocalcemia, hypoglycemia, hypomagnesemia, hyponatremia, lactic 
dehydrogenase increased, SGOT increased, SGPT increased, weight loss;  
Musculoskeletal System: arthrosis, bone necrosis, leg cramps, myalgia, osteoporosis, tetany:  
Nervous System: anxiety, confusion, depression, dizziness, emotional lability, hypertonia, 
hyperesthesia, hypotonia, insomnia, neuropathy, paresthesia, somnolence;  
Respiratory System: asthma, atelectasis, bronchitis, cough increased, epistaxis, hypoxia, lung edema, 
pleural effusion, pneumonia, rhinitis, sinusitis;  
Skin and Appendages: fungal dermatitis, hirsutism, pruritus, skin hypertrophy, skin ulcer, sweating;  
Special Senses: abnormal vision, cataract, conjunctivitis, deafness, ear pain, otitis media, tinnitus;  
Urogenital System: albuminuria, or proteinuria, bladder pain, dysuria, hematuria, hydronephrosis, 
impotence, kidney pain, kidney tubular necrosis, kidney injury, nocturia, oliguria, pyelonephritis, 
pyuria, scrotal edema, testis disorder, toxic nephropathy, urinary frequency, urinary incontinence, 
urinary retention.  
 
Less frequently occurring adverse events included: mycobacterial infections, Epstein-Barr virus 
infections, and pancreatitis.  
 
13.3.4 Related to radiation 
The side effects of radiation have been well described.101 The most common include nausea and 
mucositis.  There also exists a risk of hypothyroidism, cataracts, interstitial pneumonitis, nephropathy, 
and an unspecified long term risk of developing secondary malignancies.[103]  Importantly, the 
majority of the non-neoplastic effects  were sub clinical and/or reversible.[104]   Studies attempting 
to evaluate the risk induced by radiation alone suggest that there is a higher rate of solid tumors after 
radiation based regimens.  Curtis et al. reported on 19,229 patients and  found a cumulative incidence 
rate of 2.2% at 10years, and 6.7% at 15 years, with higher doses of TBI associated with a higher risk 
of solid cancers.[105]   However, the more important risk factor appears to be related to the level of 
immunosuppression as GVHD was also strongly linked to an increased risk of solid tumor 
development. In fact, some studies have shown no increased risk with radiation therapy,[106,107] but 
the highest risk factor was felt to be the presence of chronic graft versus host disease, and long term 
treatment with cyclosporine.[108,109]  Therefore the actual risk cannot be quantified for the low dose 
of 300cGy to be used in this trial; however is presumed to be minimal.  
 
The additional radiation exposure from DEXA or liver/spleen scans is small and is not expected to 
significantly increase the risk presented by TBI. 
 
13.3.5 Related to antimicrobials in general: Allergic reactions, renal impairment (gentamicin, 
vancomycin, amphotericin, acyclovir), "red man" syndrome - vancomycin, hepatic damage (acyclovir, 
rifampicin) 
 

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31
13.3.6 Related to bone marrow aspirate and biopsy:   No major risks are involved with bone marrow 
aspirate and biopsy. However, a small risk of infections, pain, bleeding, and hematoma formation at 
the site of the aspiration exists with the procedure. 
 
13.3.7 Related to blood draws:   No major risks are involved with blood draws. Minor complications 
including bleeding, pain, and hematoma formation at the site of blood draws, vasovagal reactions or 
infections may rarely occur. 
 
13.3.8 Related to cardiac monitoring 
 
EKG:  An electrocardiogram (EKG) is a test that measures the electrical activity of the 
heartbeat.  With each beat, an electrical impulse (or “wave”) travels through the heart.  This wave 
causes the muscle to squeeze and pump blood from the heart.  A technician will put patches 
(electrodes) on the chest, arms and legs.  The electrodes are soft and don’t cause any discomfort when 
they’re put on or taken off by the technician.  The machine only records the EKG.  It doesn’t send 
electricity into the body.  There’s no pain or risk associated with having an electrocardiogram 
 
Transthoracic ECHO:   The ECHO uses sound waves to visualize and evaluate the function of the 
heart. There are no associated risks 
 
Holter monitor: The Holter involves wearing a monitor for 24 hours during which time the electrical 
activity of the heart is recorded. There are no associated risks other than the inconvenience of wearing 
the apparatus.  
 
13.3.9 Related to central line placement:   
 
A catheter may be placed in a large vein of the neck, chest, or arm using local anesthetic. Patients will 
sign a separate consent for the placement procedure. Only trained experienced staff will place the line 
in order to minimize these procedure related risks 
 
The risks from the procedure are low; they include bleeding, bruising, or infection at the site of 
insertion. Very rarely (less than 1% of the time), the line placement may nick a vein causing one lung 
to collapse during line insertion. If the lung collapses, a tube may have to be inserted into the chest 
and remain in place until the lung re-expands. Because of this risk, patients will have a chest x-ray 
following the procedure to make sure the line is in the correct place and that the lung is not collapsed. 
Once placed, the line will remain in place until drug administration is complete.   
 
13.3.10 Related to brain magnetic resonance imaging 
There is a very small risk that the patient will experience an allergic reaction, and these are usually 
easily controlled with medications.  Anaphylactic reactions can rarely occur.  Patients with severe 
kidney disease requiring dialysis who undergo MRI with intravenous contrast can develop a rare but 
severe disease called nephrogenic systemic fibrosis that leads to hardening of skin, joints, internal 
organs, and eyes.  Patients with severe kidney disease will not receive intravenous contrast.  The 
majority of patients will not require sedation.  However, patients that do require sedation will be 
offered monitored anesthesia care through the Department of Anesthesia and Surgical Services 
(DASS), Clinical Center, NIH, and a separate consent will be completed as needed.    
 
13.3.11 Related to neuropsychiatric testing 
Depending on the patient’s age, neuropsychiatric testing will take up to 1.5 to 2 hours to perform.  
During this time, the patient may experience frustration, fatigue, or distress.  If these symptoms occur, 
the patient will be allowed to take a break.  He or she will not have to answer every question, and 

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32
testing will be discontinued if the patient asks.  The clinical social worker and/or study investigators 
will be available if the patient would like to discuss his/her concerns. 
 
13.3.12 Related to busulfan (Busulfex®) 
The most common adverse events (>10%) expected with busulfan are as follows: 
Cardiovascular: Fast heart rate, swelling in your legs, chest pain, high blood pressure, low blood 
pressure 
Central nervous system: Insomnia, headache, fever, chills, dizziness, anxiety, depression, confusion 
Dermatologic: Rash, hair loss, itching 
Endocrine and Metabolic: Electrolyte imbalance, high blood sugar 
Gastrointestinal: Nausea, vomiting, diarrhea, inflammation of the digestive tract, poor appetite, 
abdominal pain 
Hematologic: decrease in blood cell production 
Hepatic: changes in liver enzymes 
Local: Site injection reaction 
Musculoskeletal: Back pain, weakness, joint pain, muscle pain 
Renal: Kidney damage 
Respiratory: Cough, runny nose, shortness of breath, pneumonia, sore throat 
 
Less common (1-10%) include abnormal heart rhythm, heart failure, hallucination, delirium, seizures, 
brain hemorrhage, severe rash, blockage of the intestine, inflammation of the pancreas, blood in the 
urine or lungs, asthma and liver failure. 
 
13.3.13 Related to Plerixafor, if required prior to stem cell boost 
Side effects reported in healthy volunteers who received subcutaneous injections of plerixafor with 
doses ranging from 40 to 480 μg/kg included abdominal distension, abdominal pain, diarrhea, 
flatulence, nausea, vomiting, and decreased appetite. Injection reactions also occurred including 
erythema, burning, bruising, pain, pruritus, and swelling. Other adverse events included dizziness, 
headache, disorientation, paresthesia, chest tightness, palpitations, tinnitus, vertigo, and ear 
congestion. None of the adverse events were severe. 
 
13.4 
Hazards and discomforts - donor 
 
 
13.4.1 Related to neuropsychiatric testing 
Neuropsychiatric testing will take approximately 1.5 to 2 hours to perform.  During this time, the 
donor may experience frustration, fatigue, or distress.  If these symptoms occur, the donor will be 
allowed to take a break.  He or she will not have to answer every question, and testing will be 
discontinued if the donor asks.  The clinical social worker and/or study investigators will be available 
if the donor would like to discuss his/her concerns. 
 
13.4.1 Related to blood draws 
No major risks are involved with blood draws. Minor complications including bleeding, pain, and 
hematoma formation at the site of blood draws, vasovagal reactions or infections may rarely occur. 
 
13.4.5 Consenting to pregnancy testing in minors of childbearing age 
We will inform the minor during the assent process that for safety, we need to do a pregnancy test. 
She will also be told that if it is positive, we will counsel her and help her tell her parents. If the minor 
does not want to proceed, she will be advised not to sign the assent, and her enrollment on this 
screening protocol will end. 
 
13.5 
Risks in relation to benefit 

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33
 
13.5.1 For adult transplant subjects 
Clinically the approach is ethically acceptable because we are targeting a patient group with a 
debilitating and often lethal hematological disease, incurable with conventional treatments other than 
allogeneic BMT.  The protocol aims to decrease the risk of transplant related mortality, thus making 
more patients candidates for potentially curative therapy. 
 
13.5.2 
For pediatric transplant subjects (ages 4-18) 
The inclusion of children satisfies the criteria set forth in 45 Code of Federal Regulations 46, Subpart 
D as follows: 
 
 (a) the risk is justified by the anticipated benefit to the subjects: We are offering pediatric subjects 
with a probably lethal hematological disease, incurable with conventional treatments other than 
allogeneic BMT, an alternative to symptomatic therapy. This regimen, with its nonmyeloablative 
intent, has substantially lower risk for chemotherapy related toxicities, less injury to organs, and 
based on data in our current patients, lower risk for transplant related mortality and GvHD. On the 
other hand, there is a higher risk of graft rejection, but the magnitude of increase is unknown. 
 
(b) the relation of the anticipated benefit to the risk is at least as favorable to the subjects as that 
presented by available alternative approaches. The protocol aims to decrease the risk of transplant-
related mortality, thus making more patients candidates for potentially curative therapy. Recently a 
reduced intensity study in pediatric patients with SCD showed that there is a chance for fertility 
preservation.[15]  Thus the use of low dose TBI and alemtuzumab would be expected to have higher 
potential for fertility preservation; and 
  
 (c) adequate provisions are made for soliciting the assent of the children and permission of their 
parents or guardians, as set forth in 46.408. 
 
13.5.3   
For adults and pediatric donors participating in blood collection for research and 
neuropsychological testing  
These tests are considered to be no greater than minimal risk to the subject. 
 
13.6       Informed consent 
Informed consent will be conducted following OHSRP Policy 301- Informed Consent. 
An IRB-approved consent form will be provided to the participant electronically or by hard copy for 
review prior to consenting. The initial consent process as well as re-consent, when required, may take 
place in person or remotely (e.g., via telephone or other NIH approved platforms). The investigational 
nature and objectives of this trial, the procedures, and their attendant risks and discomforts and 
potential benefits will be carefully explained to the participant in a private setting. The participant 
will be given as much time as they need to review the document and to consult with their family, 
friends, and personal health care providers. In addition, a study team member will be available to 
answer any questions. 
A signed and dated informed consent document will be obtained by any investigator authorized to 
consent (See Key Study Personnel Page) prior to entry onto the study. Consent may be obtained with 
required signatures on the hard copy of the consent  or on the electronic document.   
When a document that is in electronic format is used for obtaining  consent, this study may use the 
iMed platform which is 21 CFR, Part 11 compliant, to obtain the required signatures. 

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34
During the consent process, participants and investigators may view the same approved consent 
document simultaneously when participant is being consented in person at the Clinical Center or both 
may view individual copies of the approved consent document on screens in their respective locations 
remotely. Signatures may be obtained either by both directly signing on the device that the consenting 
investigator is using (when in person) or through iMed Mobile Signature Capture  (remotely) which 
allows texting or emailing a link to the participant. That link allows the participant to review the 
consent, then proceed to sign on the device they are using. 
Whether hard copy or electronic, both the investigator and the participant will sign the document with 
a hand signature using a pen (if using hard copy), finger, stylus, or mouse (if electronic). 
When done remotely, if the participant prefers to sign a hard copy, they may be instructed to sign and 
date the consent document during the discussion and mail, secure email or fax the signed document to 
the consenting investigator. 
Whether in person or remotely, the privacy of the participant will be maintained. 
Finally, the fully signed informed consent document will be stored in the electronic medical record, 
and the participant will receive a copy of the signed informed consent document.  
 
Assent Procedures: 
Participants 14 years of age or older will review and discuss the adult consent with the parents and 
research team, and sign the standard adult consent in the assent line. If a minor is between the ages of 
7 and 13 years of age, then the minor will sign the minor assent form (donor or recipient). Minors 
under 7 years of age will provide verbal assent for participation in this study. 
Adult consent for the minor participation will be obtained from one parent or guardian given that the 
study involves no more than minimal risk to individual minor subjects who are donors, and greater 
than minimal risk for recipients, but with the prospect of direct benefits . In cases where parents share 
joint legal custody in making medical decisions of their child (e.g. by a custody agreement or court 
order) both parents must give their parental permissions regardless of level of risk of the research. 
Exceptions may be made if one parent is deceased, becomes incompetent or is not reasonably 
available (e.g. in prison). The parent or guardian will sign on the designated line on the informed 
consent attesting to the fact that the child had given assent. 
At any time during participation in the protocol that new information becomes available relating to 
risks, adverse events, or toxicities, this information will be provided orally or in writing to all enrolled 
or prospective patient participants.  Documentation will be provided to the IRB and if necessary the 
informed consent amended to reflect relevant information. 
Consent for Minors when they reach the age of majority:  
When a pediatric subject reaches age 18, continued participation will require consenting of the now 
adult with the standard protocol consent document to ensure legally effective informed consent has 
been obtained. Should sample or data analysis continue following completion of active participation 
and the subject has reached 18 years of age, we will attempt to contact the subject using the last 
known contact information to obtain consent for continued use of data or samples collected during 
their prior visit. Given the length of time that may have transpired for some of the subjects since their 
last visit for this study, we request waiver of informed consent for those individuals who after good 
faith efforts to contact them, we are unable to contact and for subjects who are off study.  
Requirements for Waiver of Consent consistent with 45 CFR 46.116 (d), each of which must be 
addressed in relation to the protocol:  
(1) The research involves no more than minimal risk to the subjects.  

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35
a. Analysis of samples and data from this study involves no additional risks to subjects. 
(2) The waiver or alteration will not adversely affect the rights and welfare of the subjects.  
a. This is an FDA-regulated study and as such, we are mandated to retain all samples, once 
collected, regardless of the age of the subject at the time of collection. Retention of these samples 
or data does not affect the welfare of subjects.  
(3) The research could not practicably be carried out without the waiver or alteration.  
a. Considering the length of time between a minor’s enrollment and their age of majority, it is 
possible that more than a few subjects may be lost to follow up. A significant reduction in the 
number of samples analyzed could impact the quality of the research.  
(4) Whenever appropriate, the subjects will be provided with additional pertinent information after 
participation.  
a. We only plan to request a waiver of re-consent for those subjects who have been lost to follow-
up. 
 
14.0 
CONFLICT OF INTEREST 
 
The Principal Investigator will assure that each associate investigator listed on the protocol title page 
receives a copy of the NIH Guide to preventing conflict of interest.  Investigators added subsequent to 
the initial circulation will be provided a copy of the document when they are added and a copy of the 
Conflict of Interest Statement will be forwarded to the Clinical Director.  Any potential conflicts will 
be reported to the IRB and the resolution of the conflict summarized.    
 
15.0 
PHARMACEUTICALS 
 
15.1  
Alemtuzumab (Campath-1H, Campath®) 
Supply:  Available through the Campath Distribution Program (The Sanofi Foundation for North 
America 1-877-422-6728). Vials are provided through this program upon completion of a patient 
specific request form. Prior to submission of a drug request the patient must provide authorization for 
the release of medical information (NIH-527). Refer to the Pharmacy Department or Clinical 
Pharmacy Specialist for additional details on drug procurement. 
Product description: Alemtuzumab is available in single use vials. Each single use, clear glass vial of 
Campath contains 30 mg alemtuzumab in 1 mL of solution (8.0 mg sodium chloride, 1.44 mg dibasic 
sodium phosphate, 0.2 mg potassium chloride, 0.2 mg monobasic potassium phosphate, 0.1 mg 
polysorbate 80, and .0187 mg disodium edetate dihydrate). No preservatives are added. Each carton 
contains three Campath vials (NDC 50419-357-03) or one Campath vial (NDC 50419-357-01). 
Storage and stability: Vials of alemtuzumab should be stored at 2-8°C (36-46°F) and protected from 
sunlight. The vial should not be frozen; if the vial has been frozen it should be discarded. 
Alemtuzumab contains no antimicrobial preservative. Alemtuzumab should be used within 8 hours 
after dilution. Alemtuzumab solutions may be stored at room temperature (15-30°C) or refrigerated. 
Alemtuzumab solutions should be protected from light. 
Preparation: The vial should be inspected for visible particulate matter and discoloration prior to 
administration. If particulate matter is present or the solution is discolored, the vial should not be 
used. The vial should not be shaken prior to use. The necessary amount of alemtuzumab should be 
withdrawn from the vial into a syringe. The vial contains no preservatives and is intended for single 
use only; the vial should be discarded with any unused portion after withdrawal of desired dose. The 
desired dose is then injected into 100 mL sterile 0.9% Sodium Chloride USP or 5% Dextrose in 
Water USP. The bag should be gently inverted to mix the solution. The syringe is discarded. 

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John Tisdale, M.D. 
October 03, 2023 (Amendment KKK) 
36
Administration: Alemtuzumab should be administered intravenously only. The infusion should be 
administered over a 2-hour period. 
 
15.2 
Filgrastim (G-CSF, Neupogen) 
Supply: Commercially available. 
Product description: Filgrastim injection is available in a concentration of 300mcg/ml in 1ml 
(300mcg) and 1.6ml (480mcg) vials. 
Preparation: For subcutaneous administration, the appropriate prescribed dose is drawn up from the 
vial with no further dilution prior to administration. For intravenous administration, the commercial 
solution for injection should be diluted prior to administration. It is recommended that the prescribed 
dose be diluted with dextrose 5% in water (DO NOT DILUTE WITH NORMAL SALINE) to a 
concentration greater than 5mcg/ml. Filgrastim diluted to concentrations between 5 and 15mcg/ml 
should be protected from absorption to plastic materials by the addition of Albumin (Human) to a 
final concentration of 2mg/ml. When diluted in 5% dextrose or 5% dextrose plus Albumin (Human), 
filgrastim is compatible with glass bottles, PVC and polyolefin IV bags, and polypropylene syringes. 
Storage and Stability: Filgrastim for injection should be stored in the refrigerator at 2o to 8oC (36o to 
46oF). Avoid shaking. 
Route of administration: Subcutaneous injection or intravenous infusion over 15-30 minutes. 
 
15.3 
Sirolimus (Rapamune®) 
Supply: Commercially available. 
Product description: Sirolimus is available in 1 mg and 2 mg tablets and an oral solution (1 mg/mL 
concentration) in a 60 mL amber glass bottle. 
Preparation: For administration of the oral liquid, the measured dose may be diluted in water or 
orange juice prior to administration. Empty the correct amount of sirolimus oral liquid into only a 
glass or plastic container holding at least two ounces of water or orange juice. No other liquids, 
including grapefruit juice, should be used for dilution. Stir vigorously and drink at once. Refill the 
container with an additional volume (minimum 4 ounces or 120 mL) of water or orange juice, stir 
vigorously, and drink at once. 
Storage and Stability: Oral tablets should be stored at room temperature: 20-25 °C (68-77 °F). Oral 
solution should be refrigerated (2-8 °C or 36-46 °F). Once the oral solution bottle is opened, the 
contents should be used within one month. If necessary, the patient may store the bottles at room 
temperatures up to 250 °C for a short period of time (e.g. not more than 15 days for the bottles). 
Route of administration: Oral. 
 
15.4  
Busulfan 
Busulfan is a bifunctional alkylating agent approved for use as a conditioning agent prior to 
allogeneic hematopoietic stem cell transplantation. The IV formulation is commercially available as 
busulfan (Otsuka America Pharmaceutical, Inc.) 
Generic: busulfan 
Classification: alkylating agent 
Action: Alkylates and crosslinks DNA. It has a more marked effect on myeloid cells than on 
lymphoid cells. 
Source: For patient administration, IV busulfan is purchased by the NIH Clinical Center Pharmacy 
Department from commercial sources. The drug is supplied as a clear, colorless sterile solution in 
10mL single use vials. Each vial of busulfan contains 60mg (6mg/mL) of busulfan. 
Stability and Storage: Unopened vials of parenteral busulfan are stable until the date indicated on the 
package when stored under refrigeration at 2-6 degrees C (36-46 degrees F). 
Product description: Injection 
Preparation: Parenteral busulfan must be diluted prior to use with either 0.9% sodium chloride or 5% 
dextrose injection. The diluent quantity should be 10 times the volume of busulfan so that the final 

03-H-0170 
John Tisdale, M.D. 
October 03, 2023 (Amendment KKK) 
37
concentration of busulfan is approximately 0.5mg/mL. Busulfan should be administered 
intravenously via a central venous catheter as a 3-hour infusion every 24 hours for a total of 3 doses 
over 3 days. 
Route: Intravenous 
 
15.5  
Plerixafor (AMD3100, Mozobil®)  
Generic: plerixafor  
Classification: hematopoietic stem cell mobilizer  
Action: Reversibly inhibits binding of stromal cell-derived factor-1-alpha (SDF-1α), expressed on 
bone marrow stromal cells, to the CXC chemokine receptor 4 (CXCR4), resulting in mobilization of 
hematopoietic stem and progenitor cells from bone marrow into peripheral blood.  
Availability: Commercial  
Storage and stability: Store at 25oC (77oF); excursions permitted to 15o-30oC (59o-86oF). [see USP 
Controlled Room temperature] Each vial of plerixafor injection is intended for single use only. Any 
unused drug remaining after injection must be discarded.  
Production description: Plerixafor injection is a sterile, preservative-free, clear, colorless to pale 
yellow, isotonic solution for subcutaneous injection. Each mL of the sterile solution contains 20mg of 
plerixafor. Each single-use vial is filled to deliver 1.2mL of the sterile solution that contains 240μg of 
plerixafor and 5.9mg of sodium chloride in Water for Injection adjusted to a pH of 6.0 to 7.5 with 
hydrochloric acid and with sodium hydroxide, if required.  
Route: subcutaneous injection. 
 
15.6 
Regulatory Considerations for Off Label Usage 
Sirolimus, alemtuzumab, and plerixafor will be used in this study beyond what is indicated in the package 
inserts. Sirolimus is approved by the FDA as an immunosuppressive agent for the prophylaxis of organ 
rejection in patients aged ≥ 13 years receiving renal transplants, in renal patients at high immunologic 
risk, and in lymphangioleiomyomatosis. Alemtuzumab is approved for the treatment of relapsing forms of 
multiple sclerosis. Plerixafor is indicated in combination with granulocyte-colony stimulating factor (G-
CSF) to mobilize hematopoietic stem cells (HSCs) to the peripheral blood for collection and subsequent 
autologous transplantation in patients with non-Hodgkin's lymphoma and multiple myeloma. The use of 
these drugs meet the requirements for an exemption from the IND regulations, 21 CFR 312, specifically: 
 
1. The investigational drug is lawfully marketed in the United States 
2. The investigation is not intended to be reported to the FDA as a well-controlled study in support of a 
new indication for use of the drug product 
3. The investigation is not intended to support a significant change in advertising to an existing lawfully 
marketed prescription drug product 
4. The investigation does not involve a route of administration or dosage level or use in a patient 
population or other factor that significantly increases the risks (or decreases the acceptability of the 
risks) associated with the use of the drug product. 
5. The investigation will be conducted in compliance with the requirements for institutional review set 
forth in FDA regulations 21 CFR 56, and requirements for informed consent as set forth in FDA 
regulations 21 CFR 50 
6. The investigation will be conducted in compliance with FDA regulations 21 CFR 312.7: Promotion and 
charging for investigational drugs. 
 
16.0 
Compensation 
Compensation will be provided to donors for their time and inconvenience. They will receive 
$150 for testing at their 1 year post- transplant visit. 1 night of lodging will be paid for returning 
for testing.  For patient under 18 years old an additional $20 will be paid for escort fee. 
 

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Procedure 
Inconvenience Unit 
(IU) or Time 
Payment 
Neuropsychological Exam 
4 hours 
$50.00 
Neuropsychological Exam (memory testing, 
processing, verbal and nonverbal reasoning 
and the quality of life questionnaires) 
10 IU 
$100.00 
Total Compensation 
$150.00 
 
 
APPENDIX A: PERFORMANCE STATUS GRADING 
 
ECOG PERFORMANCE STATUS SCALE 
GRADE 
DESCRIPTION 
0 
Fully active, able to carry on all pre-disease activities without restriction. 
1 
Restricted in physically strenuous activities and able to carry out work of a light or sedentary 
nature, e.g. light housework, office work. 
2 
Ambulatory and capable of all self-care but unable to carry out any work activities.  Up and 
about more than 50% of waking hours. 
3 
Capable of only limited self-care; confined to bed or chair more than 50% of waking hours. 
4 
Completely disabled. Cannot carry on any self-care. Totally confined to bed or chair. 
5 
Dead 
 
 LANSKY PERFORMANCE STATUS SCALE 
100  
Fully active, normal 
90  
Minor restrictions in strenuous physical activity 
80  
Active, but tired more quickly 
70  
Greater restriction of play and less time spent in play activity 
60  
Up and around, but active play minimal; keeps busy by being involved in quieter activities 
50  
Lying around much of the day, but gets dressed; no active playing participates in all quiet 
play and activities 
40  
Mainly in bed; participates in quiet activities 
30  
Bedbound; needing assistance even for quiet play 
20  
Sleeping often; play entirely limited to very passive activities 
10  
Doesn't play; does not get out of bed 
0  
Unresponsive 
KARNOFSKY PERFORMANCE STATUS SCALE 
100 
Normal; no complaints; no evidence of disease 
90 
Able to carry on normal activity; minor signs or symptoms of disease 
80 
Normal activity with effort; some sign or symptoms of disease 
70 
Cares for self; unable to carry on normal activity or do active work 
60 
Requires occasional assistance, but is able to care for most personal needs 
50 
Requires considerable assistance and frequent medical care 
40 
Disabled; requires special care and assistance 
30 
Severely disabled; hospitalization is indicated, though death not imminent 
20 
Very sick; hospitalization necessary; active support treatment is necessary 
10 
Moribund; fatal processes progressing rapidly 

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October 03, 2023 (Amendment KKK) 
39
0 
Dead 
 
 
APPENDIX B: TRANSFUSION OF RED CELLS TO RECIPIENTS OF ABO INCOMPATIBLE 
MARROW 
 
Major ABO incompatible recipient - donor 
 
Patient  
 
 
Donor  
 
 
Transfused RBC = patients group 
O 
 
 
 
A, B or AB 
 
 
 
O 
A 
 
 
 
B or AB 
 
 
 
A or O 
B 
 
 
 
A or AB 
 
 
 
B or O 
 
Minor ABO incompatibility recipient - donor 
 
Patient  
 
 
Donor  
 
Transfused RBC = donor group 
A, B or AB 
 
 
O 
 
 
 
O 
AB 
 
 
 
B 
 
 
 
B or O 
AB  
 
 
 
A 
 
 
 
A or O 
 
TRANSFUSION OF PLATELETS TO RECIPIENTS OF ABO INCOMPATIBLE MARROW 
Give donor group or volume reduce or provide product as per transfusion medicine standard practice. 
Additional pre- and post-transplant monitoring will be performed to monitor donor erythropoiesis and 
immune hemolysis as described in Appendix D. 
 
APPENDIX C:  TRANSFUSION MEDICINE GUIDELINES FOR APHERESIS PROCEDURES 
IN SICKLE CELL PATIENTS RECEIVING NONMYELOABLATIVE ALLOGENEIC PBSC 
TRANSPLANTS 
 
 
Introduction 
 
Sickle cell disease (SCD) patients are at risk for vaso-occlusive crises such as cerebrovascular accidents 
and the acute chest syndrome which occur due to the viscosity and sickling properties of sickle 
hemoglobin S.  Pre- and peri-transplant maneuvers, such as transfusion or red cell exchanges with 
allogeneic red cells, may be performed to reduce the risk of ischemic events by reducing the levels of S 
hemoglobin.  Pre-transplant coordination with the Department of Transfusion Medicine is critical because 
these patients may have had prior transfusions and have developed alloantibodies.  In addition, the 
distribution of red blood cell phenotypes in patients with SCD will reflect their ethnic heritage and may 
differ from that in the NIH donor pool.  Recruitment of adequate numbers of compatible units thus 
requires careful advance planning and knowledge of the patient’s phenotype and antibody screen.  
 
Allogeneic transplant patients who receive a lymphocyte-replete PBSC graft will also be at risk for 
increased red cell requirements if there is an ABO incompatibility with the donor.  Minor ABO 
incompatibility, such as O donors into A, B or AB recipients, is associated with hemolysis due to 
production of anti-recipient isohemagglutinins by passenger lymphocytes.  Major ABO incompatibility, 
such as A or B donors into O patients, may be associated with a delayed onset of effective donor 
erythropoiesis resulting in pure red cell aplasia after conversion to full donor hematopoiesis. This event 
appears to be most common after non-myeloablative conditioning regimens that are permissive for 
persistent production of anti-donor, host-type isohemagglutinins.  These patients may have a further 

03-H-0170 
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increased red cell requirement and need for advance planning and recruitment. Regarding patients with 
minor ABO incompatibility, immediate complications at the time of infusion due to ABO compatibility 
can be managed by standard DTM policies for manipulation of the graft to remove plasma.  The 
appropriate transfusion policy in the peri-transplant period for the ABO group of red cell, plasma and 
platelet transfusions in patients with ABO incompatible donors is also managed according to standard 
DTM transplant policies.  In addition, the DTM will use red cell serologic testing to carefully monitor 
those patients who have minor ABO incompatibility with the donor for evidence of hemolysis in the peri-
transplant period using a standard operating procedure.  As major ABO incompatibility may lead to pure 
red cell aplasia and therefore absence of erythroid cells which are necessary to cure patients with 
congenital anemias, those with major ABO incompatibility will be ineligible for the protocol.  
 
A potential adverse event in hematopoietic transplantation for congenital anemias is rejection of the 
PBSC graft, especially in patients who have been heavily transfused.  Obtaining higher numbers of donor 
stem cells may reduce the risk of graft rejection.  To achieve this goal, the DTM will collect stem cells 
using a single very large volume apheresis (∼4-5 donor blood volumes) on day 5 after filgrastim (G-CSF) 
administration, which reduces the incidence of thrombocytopenia in the donor associated with apheresis, 
reduces apheresis time and the time with which central venous catheters remain in place, and produces the 
same yields as two smaller donor blood volume procedures performed consecutively on days 5 and 6.    
 
All sickle cell transplant candidates and their identified donors will need to have a full red cell phenotype, 
antibody screen, and quantitative hemoglobin electrophoresis obtained during initial evaluation, well in 
advance of any apheresis procedures. The DTM will enter appropriate restrictions for blood product 
transfusion based on this information.   
 
Procedures for red cell exchanges 
 
Sickle cell patients who are not receiving long term transfusion therapy will be evaluated by the DTM 
fellow/senior staff and considered for a prophylactic red cell exchange prior to transplant to bring the 
target fraction of hemoglobin S to less than 30% to reduce the incidence of post-transplant stroke and 
other events that may be associated with high hemoglobin S levels. ADSOL leuko-reduced packed red 
cells will be used for the exchange.  
 
These patients will have a hemoglobin electrophoresis performed to determine their initial fraction of S 
hemoglobin (%S).  They must also have a full type and screen performed to identify alloantibodies and 
allow for recruitment of donors prior to the exchange.  The patient total blood volume will be computed 
from an algorithm using the COBE computer, and the volume of replacement PRBC required for the 
exchange estimated by utilizing this computer in conjunction with the initial hemoglobin S content and 
the desired end hematocrit and hemoglobin S concentration.   For this protocol, the calculation of the 
COBE computer may be verified using the following calculations for determination of the volume of 
replacement PRBCs needed for the red cell exchange.  
 
Blood volume x patient hematocrit (hct) = Patient's Total Packed Red Cell Volume (PRCV) 
 
(PRCV) x %S = Patient Total Packed RBC Volume of S Hemoglobin (PRCV-S) 
 
The volume of ADSOL PRBC needed to bring the residual fraction of red cells to 30% is 1.25 
exchange volumes. (=  1.25 x (PRCV-S)) 
 
Since increasing the hematocrit in patients with high levels of hemoglobin S may precipitate vaso-
occlusive crises, the red cell exchange will replace the red cells that are removed with an equal volume of 
infused red cells.  Patients who are significantly anemic may have further transfusions given after the 

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John Tisdale, M.D. 
October 03, 2023 (Amendment KKK) 
41
exchange to bring their final hematocrit up to 35%.  In these cases, the target %S should be 30% after the 
final transfusions bring the hematocrit up to 35%.   
 
The target for the %S after the exchange (before additional transfusions) is 35/Hct x 30%, where Hct is 
the patient hematocrit before the exchange.  After the exchange the patient will then receive a volume of 
red cells equal to approximately (0.35-Hct)(wt)(70 ml/kg). 
 
Patients undergoing red cell exchange may experience citrate toxicity from the anticoagulant used in the 
apheresis procedure and contained in the ADSOL red cells.  A citrate infusion rate will be calculated by 
the DTM fellow based on the flow rate of returned red cells plus 2/3 of the citrate infusion rate.  Patients 
who receive more than 1.2 mg of citrate per kilogram per minute will receive intravenous calcium 
through the return line at a rate of 0.5 mg of calcium ion per 21 mg of citrate.   
 
Donor apheresis procedures 
 
I.  
Donor stem cell mobilization with filgrastim (G-CSF) 
 
After medical evaluation and clearance for suitability as an allogeneic donor, each donor will undergo 
mobilization with G-CSF, usually as an outpatient.  The G-CSF will be administered in a dose of 10 to 16 
ug/kg/day for 6-7 days, subcutaneously.  The doses for days 1-4 may be given at any time of day, but the 
doses for day 5 and if necessary, day 6 must be given early in the morning, at least one hour prior to 
starting apheresis. Predictable side effects of G-CSF, including headache, bone pain, and myalgia, will be 
treated with acetaminophen or ibuprofen.  Prophylactic treatment of these side effects with the same 
medications may be elected.  Other side effects will be evaluated and treated accordingly. 
 
II. 
Donor Stem cell collection 
 
 
 
The target CD34 dose is 10 x 106/kg, and the minimum is 5 x 106/kg.   Donors will receive calcium 
chloride prophylaxis to prevent citrate toxicity in accordance with standard DTM policies.  
 
The volume processed per apheresis procedure will be determined by DTM medical staff on the day of 
apheresis, based on peak CD34 cell mobilization response to filgrastim (G-CSF) and the CD34 cell dose 
needed, based on kilogram weight of recipient.  This will range from 15 to 35 liters processed per day for 
1 to 3 days, not to exceed a total of 75 liters over 3 days.  In pediatric subjects, defined as less than 40 kg, 
a maximum of 8 blood volumes will be processed per day, for up to 1-3 days. 
 
The goal is to provide a sufficient number of CD34 cells to ensure engraftment and test the efficacy of 
this modality against disease relapse. 
 
III. 
Filgrastim (G-CSF) administration.  
 
G-CSF will be administered according to a vial based algorithm to reduce wastage, improve patient 
compliance, and increase the total G-CSF dose to lighter weight donors in order to improve CD34 yields. 
 
Donor Weight 
 
Total G-CSF Dose (range) 
 
20-30kg 
 
 
300 mcg (10.0 to 15 mcg/kg) 
31-37kg 
 
 
480 mcg (13.0 to 15.5 mcg/kg) 
38-48 kg                      
600 mcg (12.5 to 15.8 mcg/kg) 
49-56 kg                     
780 mcg (13.9 to 15.9 mcg/kg) 
57-60 kg                       
900 mcg (15.0 to 15.8 mcg/kg) 

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John Tisdale, M.D. 
October 03, 2023 (Amendment KKK) 
42
61-67 kg                       
960 mcg (14.3 to 15.7 mcg/kg) 
68-108 kg                     
1080 mcg (10.0 to 15.9 mcg/kg) 
> 109 kg                       
1200 mcg (11.0 or less) 
 
IV. 
 Plerixafor administration 
 
Plerixafor may be used alone or as described in the prescribing information 
(http://products.sanofi.us/Mozobil/mozobil.html). On day 5 of G-CSF and the donor is undergoing 
apheresis, stem cell mobilization is considered suboptimal (CD34+ count <30), and total projected stem 
cell number collected after the planned 2 days of apheresis is unlikely to meet the minimum of 5 x 10e6 
CD34+ cells/kg of recipient weight, plerixafor may be given at 240 ug/kg subcutaneously in the evening 
of day 5, with another dose of G-CSF, and followed by apheresis the next day. If the minimum CD34 
count is not reached after 2 apheresis, the patient can return about 1 month later to be treated again with 
G-CSF with or without plerixafor, based on the patient’s previous response. The GCSF and plerixafor 
dosing will be discussed between the PI and DTM. G-CSF will be given subcutaneously for five 
consecutive days. On the evening of the day 4, plerixafor at 240 µg/kg subcutaneously will be given 
followed by apheresis on day 5. If the goal CD34 yield is not met, the patient can receive a 6th dose of G-
CSF on the evening of day 5 and a second dose of plerixafor, followed by second apheresis the next day. 
The patient will return within one week of apheresis (+/- 2 days) for history and physical, complete blood 
count, and chemistry/liver panel if needed. Alternative dosing regimen may be used based on published 
literature (Micallef IN, Sinha S et al, BBMT 2013) and discussion with DTM. 
 
V. 
Ex vivo processing of PBPC and lymphocytes 
 
The target cell doses for the PBPC graft are outlined in the section above. 
 
For this protocol, there will be no T cell depletion of the PBPC or bone marrow. The PBPC, and 
lymphocyte products will be cryo-preserved in 5% DMSO/pentastarch for later thawing and infusion.  In 
cases of RBC incompatibility, product manipulations will be done prior to cryopreservation.  For minor 
ABO or other red cell incompatibility, PBPC, bone marrow products, and lymphocyte products will 
undergo plasma removal, with resuspension in an infusible isotonic solution, according to SOPs in the 
DTM Cell Processing Laboratory.  Donors with major ABO mismatch will be ineligible for the protocol. 
 
All products will be prepared for infusion by SOPs of the DTM Cell Processing Laboratory. 
 
 
APPENDIX  D: 
 PREPARATIVE REGIMEN 
 
Patients will receive the preparative regimen as described below.  All drugs will be given intravenously if 
possible based on the dosing formulation. All other concomitant medications or special procedures noted 
in the protocol shall follow standard transplant protocols/procedures per current institutional practice. 
  
Admission, exchange transfusion if necessary 
 
Days –7 to –3  Alemtuzumab (Campath®) IV given in a dose escalation  schedule over a total of 5 
days as follows:  
 
Day –7 
Diphenhydramine 1mg/kg (maximum 50mg) I.V., Acetaminophen 10-15mg/kg 
(maximum 650mg) PO then followed 30 minutes later by Alemtuzumab 0.03mg/kg in 100mL normal 
saline infused over 2 hours 
 

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John Tisdale, M.D. 
October 03, 2023 (Amendment KKK) 
43
Day –6 
Diphenhydramine 1mg/kg (maximum 50mg) IV, Acetaminophen 10-15mg/kg (maximum 
650mg) PO, then followed 30 minutes later by Alemtuzumab 0.1 mg/kg in 100mL normal saline 
infused over 2 hours 
 
Day  -5 to Day -3  Diphenhydramine  1mg/kg (maximum 50mg) IV, Acetaminophen 10-15mg/kg 
(maximum 650mg) PO, then followed 30  minutes later by Alemtuzumab 0.3 mg/kg in 100mL 
normal saline infused over 2 hours 
 
Day –2  
Total Body Irradiation (TBI), 300 cGy radiation given as per the Department of 
Radiology standard of practice 
 
Day –1 
Begin sirolimus (Rapamune®) 5 mg PO q4h x three doses in patients > 16 years of age 
and 1mg/m2/dose PO q4h x three doses in patients less than 16 years of age with a maximum of 5mg 
per dose. 
 
Day 0 
Continue sirolimus (Rapamune®) 5mg PO daily in patients > 16 years of age and 
1mg/m2/dose PO daily in patients <16 years of age with a maximum of 5mg per dose.  Trough levels 
will be maintained between 5-15 ng/ml. Infusion of unmanipulated Granulocyte colony-stimulating 
factor (Neupogen®) - mobilized peripheral blood stem cells 
 
 
APPENDIX E: Administration Scheme for Busulfan 
Busulfan will be administered at a dose based on the calculated AUC of a busulfan test dose of 0.8 mg/kg 
that will be given 5-14 days prior to the preparative regimen. The busulfan conditioning dose will then be 
calculated to obtain a targeted AUC of 4800 µM.min. (The PI will have discretion to use a target range of 
3600 to 6000 µM.min.)  If the test dose cannot be completed or the pharmacokinetic data from the test 
dose cannot be accurately interpreted, the conditioning busulfan dose will default to a dose of 3.2 
mg/kg/dose once daily for 3 consecutive days via IV infusion.  The total cumulative dose of the regimen 
is about 75% of the myeloablative dose.  Keppra (levetiracetam) and clonazepam will be initiated at least 
12 hours before the first dose of the conditioning dose of busulfan for seizure prophylaxis and both drugs 
will be discontinued following the morning dose after the last dose of busulfan.  To further minimize the 
risk of seizures and other neurologic complications, magnesium deficiency will be corrected, arterial 
hypertension will be strictly controlled, and the goal hemoglobin will be 9g/dL and goal platelet count 
50K/uL peri-transplant. 
 
Busulfan will be administered at a dose based on the calculated AUC of a busulfan test dose of 0.8 mg/kg 
that will be given 5-14 days prior to the preparative regimen. The busulfan conditioning dose will then be 
calculated to obtain a targeted AUC of 4800 µM.min. (The PI will have discretion to use a target range of 
3600 to 6000 µM.min.)  If the test dose cannot be completed or the pharmacokinetic data from the test 
dose cannot be accurately interpreted, the conditioning busulfan dose will default to a dose of 3.2 
mg/kg/dose once daily for 3 consecutive days via IV infusion.  The total cumulative dose of the regimen 
is about 75% of the myeloablative dose.  Keppra (levetiracetam) and clonazepam will be initiated at least 
12 hours before the first dose of the conditioning dose of busulfan for seizure prophylaxis and both drugs 
will be discontinued following the morning dose after the last dose of busulfan.  To further minimize the 
risk of seizures and other neurologic complications, magnesium deficiency will be corrected, arterial 
hypertension will be strictly controlled, and the goal hemoglobin will be 9g/dL and goal platelet count 
50K/uL peri-transplant. 
 
Busulfan dosing will be based on ideal body weight or actual body weight, whichever is lower. For 
recipients who are greater than 120% of ideal body weight, busulfan will be dosed on an adjusted ideal 
body weight (ideal body weight plus 25% of the difference between ideal and actual weight). Four 

03-H-0170 
John Tisdale, M.D. 
October 03, 2023 (Amendment KKK) 
44
busulfan blood samples will be drawn following the administration of the test dose in a green top (sodium 
heparin) collection tube (1 mL specimen volume). Optimally, samples should be obtained from a 
peripheral vein in the arm opposite to the central line where busulfan is infused. If a peripheral vein 
sample is not feasible, the samples should be obtained from a different central catheter lumen than that 
used for the busulfan infusion. The first specimen should be drawn immediately after termination of the 
2-hour intravenous infusion of 0.8 mg/kg busulfan. Additional specimens should also be drawn at 1 hour, 
2 hours and 4 hours after termination of infusion. Each sample should be placed on wet ice immediately 
after collection. All samples will be sent to the NIH Clinical Center laboratory for send out to the NIH 
contract laboratory (Mayo Medical Laboratories). 
 
 
 
 

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45
17.0 
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transplantation using a straightforward radiotherapy technique. Clin Oncol 10: 379-383. 

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