Allogeneic Hematopoietic Stem Cell Transplantation for Severe Aplastic Anemia and Other Bone Marrow Failure Syndromes Using G-CSF Mobilized CD34+ Selected Hematopoietic Precursor Cells Co-Infused With a Reduced Dose of Non-Mobilized Donor T-cells

Allogeneic hematopoietic stem cell transplantation (aHSCT) can cure patients with a variety
of bone marrow failure syndromes (BMFS) including severe aplastic anemia (SAA), paroxysmal
nocturnal hemoglobinuria (PNH) or myelodysplastic syndrome (MDS) associated with cytopenias.
Patients with BMFS have traditionally been transplanted with bone marrow (BM) as a stem cell
source. Although chronic graft versus host disease (cGVHD) occurs less commonly with BM
compared to filgrastim (G-CSF) mobilized peripheral blood stem cell (PBSC) transplants, BM
allografts have lower CD34+ progenitor cell numbers, which increases the risk of graft
rejection in heavily transfused BMFS patients to 15-20 percent. To overcome this risk, our
group developed a novel transplant approach for patients at high risk for graft rejection
that utilized cyclophosphamide, fludarabine and anti-thymocyte globulin (ATG) conditioning
followed by infusion of a CD34+ cell rich, T-cell replete G-CSF mobilized PBSC allograft.
Remarkably, in 56 consecutive BMFS patients who had multiple risk factors for graft rejection
who underwent this transplant approach graft rejection did not occur, with all patients
achieving complete donor lymphohematopoietic chimerism. Unfortunately, recipients of G-CSF
mobilized PBSC had a higher incidence of chronic GVHD than has historically been observed
with BM transplantation (72 percent vs. 50 percent cumulative incidence of cGVHD at 1 year
respectively). G-CSF mobilized PBSC transplants contained approximately a 20 fold higher dose
of T-cells that had undergone a TH- 2 type cytokine polarization, a factor which likely
contributed to this high incidence of cGVHD. In this protocol, we attempt to prevent graft
failure and to reduce the incidence of cGVHD by transplanting high numbers of CD34+ selected
PBSC co-infused with a reduced dose of non-mobilized donor T-cells that have not undergone a
TH-2 cytokine polarization.

Subjects with BMFS at high risk for graft rejection will undergo allogeneic stem cell
transplantation from an HLA identical sibling or match unrelated donor using the identical
conditioning regimen utilized in protocol 99-H-0050. Using the Miltenyi CliniMACs system,
recipients will receive an allograft on day 0 containing donor CD34+ cells that have been
positively selected and T-cell depleted following G-CSF mobilization (goal CD34+ cell dose of
5 times 10(6) CD34+ cells /kg recipient) combined with 2 times 10(7) cells/kg of
non-mobilized CD3+ T-cells previously collected and cryopreserved from the same donor by
apheresis prior to G-CSF mobilization.

Primary objective: To evaluate whether administering a CD34+ selected, T-cell depleted
peripheral blood stem cell graft with a concomitant infusion of non-mobilized donor T-cells
at a dose that matches the T-cell dose that is infused in historical bone marrow transplant
cohorts will reduce the incidence of cGVHD at 1 year to that observed with a conventional
bone marrow transplant (50 percent) without increasing the risk of graft failure. This trial
design will allow the trial to stop early if it is unlikely that we have reduced the
proportion of one year cGVHD to 50 percent or if the combined event rate for failed donor
engraftment or treatment related mortality (TRM) at day 100 exceeds 20 percent.

The primary endpoint of this study will be cGVHD at day 365.

Secondary end points include transplant related mortality, engraftment, degree of donor-host
chimerism, incidence of acute and chronic graft versus host disease (GVHD), transplant
related morbidity and overall survival. Health related quality of life will also be assessed
as a secondary outcome measure pre-transplant, 30 and 100 days post transplant and every 6
months until 5 years post transplant.

- INCLUSION CRITERIA:

- Recipient:

- Patients diagnosed with one of the following hematologic diseases which are
associated with reasonable longevity, shown to be curable by allogeneic BMT but
where concern for a high procedural mortality with conventional BMT may delay or
prevent such treatment:

- 1) Paroxysmal nocturnal hemoglobinuria (PNH) associated with
life-threatening thrombosis, and/or cytopenia, and/or transfusion dependence
and/or recurrent and debilitating hemolytic crisis

- 2) Severe aplastic anemia (SAA) or pure red cell aplasia (PRCA [acquired or
congenital]) associated with transfusion dependence and/or neutropenia in
patients who are not candidates for, or who have failed immunosuppressive
therapy

- 3) Refractory anemia (RA) or RARS MDS patients who have associated
transfusion dependence and/or neutropenia.

- Ages 4 to 80 (both inclusive), and weight >18kg

- Availability of HLA identical or single HLA locus mismatched family donor or
10/10 matched unrelated donor at the allelic level (HLA alleles A, B, C, DR, and
DQ).

- 9/10 donors where all the HLA sequences have the same antigen/peptide binding
domains in key exons to the patient. This can result in identical protein
sequences between patient and donor. Allele mismatches in p and g groups can be
considered acceptable due to the exact matching which exists in the binding
domains.

- Telomere Length Testing

- Germline/Inherited gene panel in patients where a suspicion for a familial bone
marrow failure syndrome (BMFS) exists, hTERC and hTERT, GATA2 mutation testing
will be performed on protocol 04-H-0012 or performed elsewhere prior to enrolling
on 04-H-0012.

EXCLUSION CRITERIA:

-Recipient: any of the following

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

- Diffusion capacity of carbon monoxide (DLCO) <40% predicted (patients under the age of
10 may be excluded from this criterion if they have difficulty performing the test
correctly and thus are unable to have their DLCO assessed) using DL Adj and DL/VA/Adj.

- Left ventricular ejection fraction <40% (evaluated by ECHO) or <30% (evaluated by
MUGA)

- Serum creatinine greater than 2.5mg/dl or creatinine clearance less than 50 ml/min by
24 hr urine collection

- Serum bilirubin greater than 4 mg/dl, transaminases greater than 5 times the upper
limit of normal

- Pregnant or lactating

- Fanconi s anemia

- ECOG performance status of 3 or more (See Bone & Marrow Transplant Consortium
Supportive Care Guidelines for HSCT Recipients)

- Other malignant diseases liable to relapse or progress within 5 years, with the
exception of a separate hematologic malignancy where allogeneic stem cell transplant
has been shown to be potentially curative.

- Presence of an active infection not adequately responding to appropriate therapy.

- Inability to comprehend the investigational nature of the study and provide informed
consent. The procedure will be explained to subjects age 8 -17 years with formal
consent being obtained from parents or legal guardian.

INCLUSION CRITERIA:

-Related Donor:

- HLA identical or single HLA mismatched family donor

- Age greater than or equal to 4 and less than or equal to 80 years old

- Weight > 18 kg

- If there is a suspicion of familial BMFS in the recipient, then the donor must have
underong genetic testing for genes associated with BMFS -performed at a CLIA-certified
laboratory, prior to enrolling in this protocol.

EXCLUSION CRITERIA:

-Related Donor: any of the following

- Pregnant or lactating

- Unfit to receive filgrastim (G-CSF) or undergo apheresis (history of stroke, MI,
unstable angina, uncontrolled hypertension, severe heart disease or palpable spleen)

- HIV positive (donors who are positive for HBV, HCV or HTLV-I/II, T.cruzi [Chagas] may
be used at the discretion of the investigator following counseling and approval from
the recipient)

- Sickling hemoglobinopathies including HbSS or HbSC. Donors with HbAS are acceptable

- Inability of donor or guardian of donor to comprehend the investigational nature of
the study and provide informed consent.

- Screening test positive for Chagas disease (Trypanosoma cruzi /T.
cruzi/trypanosomiasis) confirmed by the Center for Disease Control (CDC).

INCLUSION CRITERIA & EXCLUSION CRITERIA: Unrelated Donor

-The NMDP unrelated donor inclusion criteria will be used as outlined in document (link).
Donor eligibility will be completed per NMDP standards and in accordance with most recent
and stringent FDA guidelines.