Use of G-CSF to Obtain Blood Cell Precursors

The goal of this study is to obtain CD34+ hematopoietic stem cells (HSC) from peripheral
blood and/or bone marrow, and Mononuclear Cells (lymphocytes and monocytes), and granulocytes
(grans) from peripheral blood. These collections maybe used for clinical treatment or
laboratory research.

Participants include: 1. Patients with any primary immune deficiency (PID) where collection
is for clinical use to benefit the patient; 2. Patients with any primary immune deficiency
(PID) where collection is for laboratory research use only. 3. Healthy sibling or other
related donor of those patients with PID in need of an allogeneic stem cell transplant direct
donation for a designated patient; 4. Healthy adult volunteers.

The majority of subjects will have HSC collected from peripheral blood by apheresis. Daily
subcutaneous injections of G-CSF (granulocyte colony stimulating factor/filgrastim) for 5 to
6 days is a standard method used to mobilize HSC to the peripheral blood prior to apheresis,
and will be used for most subjects. Plerixafor (Mozobil) is approved for use in combination
with GCSF to mobilize HSC and will be used in patients, or sibling/related donors undergoing
collection of HSC by apheresis.

Some patients and healthy sibling/related donors will have a clinical scale aspiration
collection of bone marrow to obtain HSC for clinical use. Some participants may have a small
sample needle aspiration collection of bone marrow obtained for research purposes.

Mononuclear cells and/or Granulocytes (gran) will be collected from peripheral blood by
apheresis following no stimulation, G-CSF alone, or a combined single dose of G-CSF (480mcg)
and Dexamethasone (8mg) prior to collection as is used in the Department of Transfusion
Medicine.

HSC, mononuclear cells, and gran collection from patients with PID may be used for laboratory
research or may be designated for future clinical treatment of the patient under separate
treatment protocol. HSC collections from healthy sibling/related donor of those patients with
PID in need of an allogeneic stem cell transplant will be designated for clinical treatment
of the related patient. HSC, mononuclear cells, and gran collection from healthy volunteers
will be designated entirely for laboratory research.

HSC will be used for the following three clinical purposes, where clinical treatments would
occur under separate IRB approved protocols: 1. Autologous HSC from patients may be
genetically modified and infused into the patient for treatment of an infection or the
underlying disease (Gene therapy); 2. Autologous HSC from patients may serve as back up
(rescue product) for patients undergoing matched unrelated donor transplantation or
haploidential transplantation; 3. HSC from a sibling/related donor may be used as a directed
donation for allogeneic transplant of the related patient. Mononuclear Cells (lymphocytes and
monocytes) and granulocytes will be used for the following clinical purposes, 1. Autologous
lymphocytes may be genetically modified and infused into the patient for treatment of an
infection or the underlying disease (Gene therapy); 2. Autologous lymphocytes, monocytes and
granulocytes (neutrophils) may be transfected with mRNAs to transiently express a therapeutic
protein for treatment of an infection or the underlying disease (Gene therapy).

HSC, lymphocytes, monocytes and granulocytes will be used for laboratory research studies
that include: Delineating the pathophysiology of inherited immune deficiencies; Delineating
the physiology of and improving engraftment of hematopoietic stem cells; Determining how
hematopoietic stem cells may be maintained in ex vivo culture without losing pluripotent
potential; Delineating the molecular mechanisms responsible for lineage specific
differentiation; Developing efficient methods for gene transfer into hematopoietic stem cells
for corrective gene therapy; Developing methods for restoration of function in defective
peripheral blood monocytes and/or granulocytes; Further characterization of peripheral blood
monocytes and/or granulocytes from patients with PID.

- ELIGIBILITY CRITERIA:

Patients (Patients with a genetically defined PID or clinical history consistent with PID)

1. Patients will have a genetically defined PID or have a clinical history of recurrent
infections or other problems suggestive of PID, must be 2-70 years of age,

2. Is at least 10 kilograms body weight.

3. Some patients may have active bacterial or fungal infection at the time of study
entry.

4. Preserved renal function (creatinine less than or equal to 2.5 mg/dL; less than or
equal to 3+ proteinuria); preserved hepatic function (bilirubin less than or equal to
2.0 mg/dl); preserved hematologic function (WBC greater than or equal
to1000/mm^3;granulocytes greater than or equal to 500/mm^3; platelets greater than or
equal to 100,000; hematocrit greater than or equal to 25). Of note, patients with PID
often have associated chronic thrombocytopenia. Patients with stable chronic
thrombocytopenia will be eligible for collection, at the investigator s discretion,
with the caveat that patients with platelet count <40,000 the day prior to collection
will be transfused with platelets on the morning of collection. Platelets may also be
given to these patients following the collection if medically indicated..

5. Patients of childbearing potential may be entered if using effective contraception and
having a negative serum or urine pregnancy test within one week of beginning G-CSF
administration.

6. Patients may remain on their regimen of prophylactic treatments as deemed necessary by
the investigator.

7. Willingness to allow blood cell samples to be stored

8. Willingness to allow blood and/or bone marrow samples to be modified to iPS cells

Healthy Donors (HLA matched sibling or other related donor of a patient with PID in need of
an allogeneic hematopoietic stem cell transplant)

1. Healthy sibling or other relative of a patient with PID in need of an allogeneic
transplant,

between 2-70 years of age,

2. Is fully or closely HLA tissue matched to the patient with PID that the hematopoietic
stem cell PBSC or bone marrow collection is designated to clinically benefit,

3. Is at least 10 kilograms body weight.

4. Normal renal function (creatinine less than or equal to 1.5 mg/dL; less than or equal
to 1+ proteinuria); normal hepatic function (bilirubin less than or equal to 1.5
mg/dL); normal hematologic function (WBC greater than or equal to 2500/mm^3;
granulocytes greater than or equal to 1200/mm^3; platelets greater than or equal to
120,000; hematocrit greater than or equal to 38).

5. Normal female donors of childbearing potential may be entered if using effective
contraception and having a negative serum or urine pregnancy test within one week of
beginning G-CSF administration.

6. Willingness to allow blood cell samples to be stored

7. Willingness to allow blood and/or bone marrow samples to be modified to iPS cells

Healthy Volunteers

1. Healthy adults aged 18-65 without active current infection or history of recurrent
infection,

2. Weighs at least 50kg.

3. Normal renal function (creatinine less than or equal to 1.5 mg/dL; less than or equal
to 1+ proteinuria); normal hepatic function (bilirubin less than or equal to 1.5
mg/dL); normal hematologic function (WBC greater than or equal to 2500/mm^3;
granulocytes greater than or equal to 1200/mm^3; platelets greater than or equal to
120,000; hematocrit greater than or equal to 38).

4. Normal female volunteers of childbearing potential may be entered if using effective
contraception and having a negative serum or urine pregnancy test within one week of
beginning GCSF administration.

5. Willingness to allow blood cell samples to be stored

6. Willingness to allow blood and/or bone marrow samples to be modified to iPS cells

For PBMCs and grans collections, adult subjects with known genetic mutations may
participate as healthy volunteers for research purposes as long as the other criteria
listed above are fulfilled.

EXCLUSIONS:

Patients

1. Patients who are hemodynamically unstable (systolic or diastolic blood pressure fall
of 20 mm Hg from the stable patient s baseline measurement) or requiring mechanical
respiratory assistance are excluded.

2. Female patients who are pregnant or lactating as determined by history and/or positive
pregnancy test are excluded.

3. Must be negative by routine blood donor eligibility testing criteria including tests
for syphilis (RPR) and TTV Donor Transplant Panel testing (list is modified
periodically, but may include hepatitis B and C, HIV and HTLV, T. cruzi). This does
not apply to leukapheresis patients, as these tests are not required by DTM.

1. XSCID patients do not make antibodies and false positives may occur because they
receive periodic infusions of pooled donations of IVIG. We have observed positive
anti-HBc testing in these patients. If this occurs, more specific DNA or antigen
testing will be done and must be negative.

2. Autologous HSC Transplant patients - may be positive for Hepatitis B and C if the
investigator deems it necessary to be collected and used as a safety back-up

Healthy Volunteers and Healthy Donors

1. Active bacterial, fungal or viral infection as evidenced by history, physical exam
(temperature >38 degress C), or WBC >9000 are excluded.

2. Females who are pregnant or lactating as determined by history and/or pregnancy test
are excluded.

3. Must be negative by routine blood donor eligibility testing criteria, including tests
for syphilis (RPR) and TTV Recipient Transplant Panel (list is modified periodically,
but may include hepatitis B and C, HIV and HTLV, T. cruzi) This does not apply to
leukaphersis patients, as these tests are not required by DTM policy.

4. Someone without peripheral venous access in arm veins adequate for apheresis (healthy
volunteers only).

5. If in the opinion of the investigator participation in this study places the healthy
volunteer or donor at undue risk.

Patients or Healthy Donors being considered for clinical scale bone marrow harvesting

1. Who are unable to lie prone during the bone marrow harvesting procedure.

2. Who are unable to tolerate general anesthesia during the bone marrow harvesting
procedure.