Lymphocyte Gene Therapy for Mild Hunter Syndrome 
4.4 Nature Of Procedures Or Therapeutic Agents 
Peripheral blood lymphocytes (PBL) will be harvested from the patient by apheresis, 
stimulated to initiate the growth of T-lymphocytes, transduced with IDS-containing retrovirus 
I L2SN, and then re-infused into the patient. An outline of the procedure is diagrammed below 
(Fig- 1). 
4.4.1 Isolation And Culture Of Peripheral Blood Lymphocytes 
The patient will be admitted to the Clinical Research Center at the University of Minnesota 
Hospital Clinic (UMHC). Apheresis will be performed by the UMHC Blood Blank Donor Center 
according to an established procedure which obtains 5 - 6 x 10^ lymphocytes from an adult during 
a 3-hour pheresis run (or a proportionately lower amount from a child). 
Fresh peripheral mononuclear cells (MNC) will be separated from the red cells and 
neutrophils by Ficoll-Hypaque density gradient centrifugation. The MNC will then be washed, 
counted, and cultured. At the initiation of each new culture, 10 ng/ml anti-CD3 antibody (Ortho) 
monoclonal antibody will be added to stimulate the outgrowth of T-lymphocytes. Cultures will 
be grown in plastic Petri plates or in Lifecore bags (Baxter) at a concentration of approximately 0.5 
- 2 x 10 6 cells/ml in media which consists of AJM-V (GIBCO) with 2 mM glutamine, 50 U/ml 
penicillin, 50 ug/ml streptomycin, 2.5 ug/ml Fungizone, and 200 U/ml of FL-2 (Cetus). The cells 
will be cultured at 37°C in a humidified incubator with 5% CO 2 . 
All patient cell manipulations and transductions will be carried out by the University of 
Minnesota Hospital Blood Bank (Dr. David Stoncek, Director) and Cell Processing Center (Dr. 
Jeffrey McCullough, Director). The conditions of culture and lymphocyte stimulation may be 
modified by the PI during the course of this protocol to take advantage of improvements in 
technique or media. 
4.4.2 Growth And Transduction Of IDS-Deficient T-lymphocytes 
Following 3-1/2 days of growth in culture to initiate T-lymphocyte proliferation, cells will 
be transduced by aspirating off the top half of the medium, and then adding L2SN vector- 
containing supernatant (see 6.4 Virus production) in the presence of protamine sulfate (5 - 10 
pg/ml) and IL-2 (200 U/ml). Lymphocytes will be harvested after 6 hours of exposure to virus, 
and placed in fresh medium. This transduction procedure will be repeated on consecutive days 
for a total of 4 days. After the final exposure to retroviral vector, the cells will be fed with fresh 
media and cultured another 18 hours prior to administration to the patient. 
Approximately 80% of the culture will then be infused into the patient. The remaining cells 
will be returned to culture for continued growth and selection and/or analyzed for vector 
integration. Selected cultures will be periodically analyzed for the above features and 
cryopreserved for future patient infusion or additional studies. 
An aliquot of PBL infused into the patient will be assayed by competitive PCR to 
quantitate the frequency of L2SN insertion. Cells will also be tested for IDS enzyme activity and 
for the presence of replication-competent retrovirus by amplification in Mus dunni cells and S+L- 
assay. 
4.4.3 Infusion Of IDS-transduced T-lymphocytes 
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