Nonmyeloablative Peripheral Blood Mobilized Hematopoietic Precursor Cell Transplantation for Sickle Cell Disease and Beta-thalassemia in People With Higher Risk of Transplant Failure

Our ongoing nonmyeloablative allogeneic peripheral blood stem cell (PBSC) transplant protocol
(03-H-0170) for patients with severe sickle cell disease (SCD) and B-thalassemia from
HLA-matched family donors has excellent results thus far. Our long term leukocyte engraftment
rate is 85-90% with the same disease-free survival. None of the engrafted patients had acute
sickle-related events, significant toxicity associated with the conditioning regimen, or any
evidence of graft versus host disease (GVHD).

While these results rival the transplant outcomes from low risk transplant patients with
B-thalassemia, there are areas for improvement. The first is the 10-15% graft rejection rate,
where a majority of these individuals were male donor and female recipient pairs. Another
limitation is the significant delay in donor red cell engraftment in one recipient who had
pre-existing allo-antibody to donor red cells from previous transfusions. Also we have
excluded another group of individuals with preformed antibodies, recipients having major ABO
incompatibility to the donors.

To overcome these limitations (and reduce the transplant failure rate) in this new protocol,
we will continue our nonmyeloablative approach in the patients with SCD and B-thalassemia
with HLA-matched family donors, but using an increased intensity regimen in a subset
considered at high risk for transplant failure. This modified regimen consists of pentostatin
and oral cyclophosphamide, which we hypothesize will reduce both the T cells that mediate
leukocyte rejection and the B/plasma cells that produce anti-donor erythrocyte antibodies.
The main transplant backbone will remain as alemtuzumab, low dose total body irradiation of
300 cGy, and sirolimus; the transplant graft will remain as unmanipulated G-CSF mobilized,
T-cell replete, PBSC product for hematopoietic and lymphoid reconstitution.

The primary endpoint of this study is the percentage/number of patients who have sustained
donor type hemoglobin at 1 year post transplant for male donors female recipients. The
primary endpoint for those with pre-existing antibodies is the presence of donor red cells
with reticulocytes greater than or equal to30 k/uL at 2 years post-transplant. Other
endpoints include the toxicity of the pentostatin-cyclophosphamide regimen, the 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. Since SCD and B-thalassemia are
non-malignant disorders of red cells, severe GVHD, lack of donor erythrocyte (prolonged donor
red cell aplasia), or graft rejection is collectively considered transplant failure.

- INCLUSION CRITERIA- recipients (must fulfill one disease category in 1.0 and all of
2.0)

1.0 Disease specific

1.1 Patients with sickle cell disease (Hb SS, SC, or S beta-thal) at high risk for
disease-related morbidity or mortality, defined by having severe end-organ damage (A, B, C,
D, or E) or potentially modifiable 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 cerebral arteriopathy requiring chronic transfusion
therapy; OR

- B. Sickle cell-related renal insufficiency defined by a creatinine level greater than
or equal to 1.5 times the upper limit of normal and kidney biopsy consistent with
sickle cell nephropathy OR nephrotic syndrome OR creatinine clearance less than <
50mL/min OR requiring peritoneal or hemodialysis; OR

- C. Tricuspid regurgitant jet velocity (TRV) of greater than or equal to 2.5 m/s 40, 41
at baseline; OR

- D. Recurrent priapism defined as at least 2 episodes of an erection lasting >4 hours
involving the corpora cavernosa and corpus spongiosa; OR

- E. Sickle hepatopathy defined as EITHER ferritin >1000mcg/L OR direct bilirubin >0.4
mg/dL at baseline

- 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/ More
than one hospital admission in the last year while on maximal tolerated dose
of hydroxyurea (at MTD for at least 6 months)

- Acute chest syndrome/ 2 prior ACS/ any ACS while on hydroxyurea (at MTD for
at least 6 months)

- Osetonecrosis of 2 or more joints/ And significantly affecting their quality
of life by Karnofsky score 50-60/ And on hydroxyurea where total hemoglobuin
increases less than 1 g/dL or fetal hemoglobin increases less than 2.5 times
the baseline level

- Red cell alloimmunization/ Transfusion dependent/ Total hemoglobin increases
less than 1g/dL while on hydroxurea (hydroxurea for at least 6 months)

1.2 Patients with 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)

- hepatomegaly of greater than 2cm below the costochondral margin

2.0 Non-disease specific:

2.1 Age greater than or equal to 4 years

2.2 6/6 HLA matched family donor available

2.3 Ability to comprehend and willing to sign an informed consent

2.4 Negative beta-HCG, when applicable

EXCLUSION CRITERIA - recipient (any of the following would exclude the subject from
participating)

1. ECOG performance status of 3 or more

2. 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. Patients with fever or suspected minor infection should
await resolution of symptoms before starting the conditioning regimen.

3. Major anticipated illness or organ failure incompatible with survival from PBSC
transplant

4. Pregnant or lactating

5. We will measure anti-A, anti-B, and/or other red cell antibody titers from the first
cohort to determine the feasibility of transplanting patients with pre-existing
antibodies (major ABO mismatch or other anti-donor red cell antibody). The information
gathered from this cohort of patients will help to determine if the titer or antibody
to a specific antigen needs to be incorporated in the exclusion criteria.

INCLUSION CRITERIA - donor

1. 6/6 HLA matched family donor

2. Weight greater than or equal to 20 kg (insofar that the weight difference between
recipient and donor does not exceed a reasonable likelihood of being able to obtain an
adequate cell dose from the donor within two aphereses)

3. Fit to receive filgrastim (G-CSF) and to give peripheral blood stem cells (blood
counts and blood pressure within DTM standards no history of congestive heart failure
or unstable angina, and no history of stroke)

4. Ability to comprehend and willing to sign an informed consent; assent obtained from
minors

EXCLUSION CRITERIA- donor: (any of the following would exclude the donor from
participating)

1. Pregnant or breastfeeding

2. HIV positive

3. Hemoglobin S greater than or equal to 50%, or B-thalassemia intermedia