Gene Transfer for Sickle Cell Disease

This is an open-label, non-randomized, single center, pilot and feasibility, single arm
cohort study involving a single infusion of autologous bone marrow derived CD34+ HSC cells
transduced with the lentiviral vector containing a short-hairpin RNA targeting BCL11a.
Accrual will be a maximum of 7 evaluable patients with SCD. The study will have three strata:

1. Stratum 1: ages ≥18-40

2. Stratum 2: ages ≥12-<18

3. Stratum 3: ages ≥3-<12

To determine the feasibility and safety of administering a lentiviral gene transfer vector
encoding a small hairpin (sh) RNA targeting the γ-globin gene repressor, BCL11A, in patients
with severe SCD.

Patients will undergo standard work-up for autologous bone marrow transplantation according
to institutional guidelines and then undergo two bone marrow harvests at a minimum of 4 weeks
apart that will be used for a back-up marrow (minimum of 2 x 106 CD34+ cells/kg) and for a
harvest of autologous bone marrow for gene transfer.

Patients will receive blood transfusions for a period of 3 months prior to the planned date
of product infusion, with a goal of achieving a HbS level ≤ 30% by the time of gene transfer.
If the subject is already on a chronic transfusion regimen as part of baseline disease
management, the regimen may be continued. The timing of transfusions will be coordinated to
occur within 7 days prior to any procedures requiring anesthesia such as bone marrow harvest.

Hematopoietic cells will be collected from the patient in advance of the treatment, to serve
as a salvage procedure ("back-up graft"), should there be no hematopoietic recovery observed
following the injection of genetically-manipulated cells, or should manipulated cells fail to
meet release criteria. Bone marrow (up to 20 mL/kg) will be harvested from the patient under
general anesthesia from the posterior iliac crests on both sides by multiple punctures at a
minimum of 4 weeks prior to gene therapy. A portion of the bone marrow containing at least 2
x 106 CD34+ cells/kg will be frozen and stored unmanipulated according to standard clinical
procedures for autologous bone marrow collection to constitute the back-up graft. If the
number of CD34+ cells that exceeds 2 x 106 cells/kg is greater than or equal to 1 x 106
cells/kg, these excess cells would be processed for transduction, transduced, and then
frozen. Transduction will be carried out on the selected CD34+ cells and transduced cells
will be cryopreserved. If cells from the first harvest were transduced and frozen, these
gene-modified cells will be thawed and prepared for infusion in parallel with the product
from the subsequent harvest(s). In this case two separate products would be infused. All cell
manipulation procedures and release testing will be performed in the transduction facility in
accordance with Good Manufacturing Practice (GMP) following process specific standard
operating procedures. Final Drug Product will be accompanied by a Certificate of Analysis,
documenting that all release testing is complete and within specification. Subjects will
receive myeloablative conditioning with Busulfan administered on days -5 to -2, prior to
infusion of transduced cells. Cells will be infused intravenously over 30-45 minutes after
standard prehydration and premedication according to Boston Children's Hospital Hematopoietic
Stem Cell Transplantation Unit standard guidelines. This standard requires that the patient
be on continuous cardiac, respiratory and oxygen saturation monitor throughout the infusion
and for 30 minutes afterwards. Vital signs will be measured and recorded pre-transfusion, 15
minutes into transfusion, every hour for duration of infusion, and end of transfusion. The RN
will stay with the patient for the first 5 minutes of the transfusion. If two transduction
products are administered, the second transduced product will be administered without delay
after the first.

Inclusion Criteria:

- Diagnosis of SCD with genotype HbSS, HbS/0 thalassemia, HbSD, or HbSO

- Severe symptomatic SCD, defined by the presence of one or more of the following
clinical complications:

1. Minimum of two episodes of acute chest syndrome (ACS) in the 2 years before study
entry.

2. History of three or more episodes of severe pain events requiring a visit to a
medical facility and treatment with parenteral opioids in the 2 years before
study entry.

3. Recurrent priapism (> 2 episodes) in the 2 years before study entry.

4. Red-cell alloimmunization (>2 antibodies) during long-term transfusion therapy.

5. Receiving, or indicated to receive (based on prior stroke or elevated
transcranial Doppler (TCD) results), chronic transfusions for primary or
secondary stroke prophylaxis.

- Age 3 years to 40 years .

- Failure of hydroxyurea therapy due to lack of clinical improvement or inability to
tolerate due to side effects (e.g., myelosuppression, gastrointestinal symptoms, or
hepatic enzyme elevations). Clinical criteria must be met despite taking hydroxyurea
for greater than or equal to 6 months, unless contraindicated or not tolerated.
Patients taking hydroxyurea who still meet all inclusion criteria are eligible for the
trial.

- No HLA-genotypically identical related bone marrow donor available

- Parental/guardian/patient signed informed consent

- Willingness to return for follow-up for 15 years

- White blood cell (WBC) count within the range of 3.0 - 20.0 x 109 /L Hemoglobin within
the range of 5 - 11 g/dL Platelet count within the range of 100 - 600 x 109 /L PT and
PTT within normal limits, unless prolonged due to anticoagulation requirement.

- Adequate organ function and performance status:

1. Performance status ≥70% (Lansky play for age <16 years, Karnofsky for age ≥16
years)

2. Left ventricular ejection fraction >40% or shortening fraction >25%

3. Direct bilirubin ≤ 2.0 mg/dL

4. Serum creatinine clearance or GFR >/= 70 mL/min/1.73 m2.

5. For ages > 7 years, DLCO (corrected for hemoglobin), FEV1, FVC >50% of predicted;
if age < 7 years, then oxygen saturation >92% on room air.

Exclusion Criteria:

- Contraindication to bone marrow harvest, or to administration of conditioning
medication (busulfan).

- Subjects who have undergone allogeneic transplant previously.

- Known positive HIV serology or HIV nucleic acid testing, or positive serology for HCV,
HBV, or HTLV.

- Uncontrolled infection.

- Active malignancy.

- Known myelodysplasia of the bone marrow or abnormal bone marrow cytogenetics.

- Receipt of an investigational study drug or procedure within 90 days of study
enrollment.

- Pregnancy, or breastfeeding in a postpartum female, or absence of adequate
contraception for fertile subjects. Females of child-bearing potential must agree to
use a medically acceptable method of birth control such as oral contraceptive,
intrauterine device, barrier and spermicide, or contraceptive implant/injection from
Screening through at least 6 months after drug product infusion. Male subjects must
agree to use effective contraception (including condoms) from Screening through at
least 6 months after drug product infusion.

- Acute hepatitis or evidence of moderate or severe portal fibrosis or cirrhosis on
prior biopsy.

- An assessment by the Investigators that the subject will not comply with the study
procedures outlined in the study protocol