Lymphocyte Gene Therapy for Mild Hunter Syndrome 
of storage materials accumulated in different tissues accessed by the blood. These results thus 
constitute an in vitro model for gene therapy of Hunter syndrome by IDS gene transfer into 
lymphoid cells, supporting this clinical trial. 
While the studies described above on have provided an initial indication of the 
effectiveness of the constructed IDS viruses in providing high-level expression and metabolic 
correction, transduction of lymphocytes cultured from the peripheral blood of patients with 
Hunter syndrome will more directly test the potential applicability of this approach to human 
therapy. Peripheral blood mononuclear cells were therefore isolated and then cultured in the 
presence of 100 units/ml IL2 and anti-CD3 to engage the T-cell receptor and stimulate growth of 
T-cells. Cells were transduced with LXSN and L2SN retroviral vectors at a multiplicity of 
approximately 2:1 for either 3 or 4 rounds of infection. The enzymatic activity of heterogeneous 
(non-selected) PBL populations is compared to the level of normal, uncultured leukocytes (WBC) 
in Table 4. 
Table 4 - IDS Expression In L2SN-Transduced Peripheral Blood Lymphocytes 
IDS Enzymatic Activity (U/mg/hr) 
Cells 
Mean 
Range 
SD 
n 
WBCfsJormal 
807 
418 - 1,250 
252 
23 
PBLMPS-LXSN 
32 
8.2 - 98 
30 
8 
PBLmps _ L2SN 
470 
124 - 775 
248 
10 
IDS activity was virtually undetectable in extracts from untransduced or LXSN (control virus) 
transduced lymphocytes. All heterogeneous (unselected) PBLmps cultures transduced with 
therapeutic vector L2SN expressed IDS levels comparable with normal leukocytes. 
We have also demonstrated enhanced clearance of 35 SC> 4 -GAG by fibroblasts grown in co- 
culture (64). Thus, ex vivo lymphocyte gene therapy for Hunter syndrome may be feasible 
despite current limitations on efficiency of gene transfer into hematopoietic cells . 
The capability of culturing and transducing large number of has recently been "scaled up" 
at the Section of Transfusion Cellular Therapies Facility at the University of Minnesota Hospital. 
We have demonstrated apheresis of the requisite number of cells 5 - 9 x 10 9 during a 2 - 3 hour 
pheresis run, removal of contaminating erythrocytes (with 82% recovery of mononuclear cells), 
and subsequent culture in 250 ml, 500 ml, and 1,000 ml sterile, gas-permeable culture bags. Initial 
studies have demonstrated the effect of various concentrations of IL-2 (100 -1,000 U/ml rIL-2), and 
have achieved a 17-fold expansion of the cell population over 10 days (Fig. 5). Bacterial and 
fungal cultures showed no contamination. 
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Recombinant DNA Research, Volume 20 
