Lymphocyte Gene Therapy for Mild Hunter Syndrome 
with rnntinnpH ADA pnz.ymp production in PBL. These results lend confidence to the idea that~ 
PBL can be accessed, stimulated to grow in culture, subjected to retroviral-mediated gene transfer 
and then infused into patients for gene expression as therapy for an inherited genetic disease. 
3.0 PRELIMINARY STUDIES 
Infusion of lymphocytes from MPS patients which have been genetically modified to 
express the deficient enzyme could provide a therapeutic alternative for these patients. Exocytosis 
of soluble enzyme and uptake by mannose-6-phosphate receptor-mediated endocytosis has been 
postulated to be the primary mechanism of enzyme transfer. However, other more recent studies 
have also demonstrated enzyme transfer by cell-to-cell contact; in vitro, enzyme may be 
transferred from lymphocytes to fibroblasts (58, 59). 
A limited trial of infusion of normal allogeneic lymphocytes as therapy for MPS types I and 
II (38) resulted in respiratory improvement. Because very small amounts of enzyme appear to be 
capable of normal GAG catabolism (60), and because infusion of allogeneic leukocytes has been 
shown to decrease airway disease in patients with mild Hunter syndrome, the use of transduced 
lymphocytes appears to be a viable approach to treatment in some patients. We anticipate that 
repeated infusions of genetically modified autologous PBL would be effective in providing 
enzyme analogous to the response of prolonged normal marrow engraftment. 
We have recently constructed a series of IDS retroviral vectors including L2SN (Fig. 2) 
exhibiting high-level IDS expression after transduction of cells from patients with Hunter 
syndrome (61-63). The G418-selectable virus L2SN was packaged by transfection into GP+E86 
cells, collecting transient-generated ecotropic virus and using this to infect PA317 cells. Resultant 
G418 resistant PA317 clones were then screened for those producing the highest titer of virus (3 x 
10^/ml). Proviral structure in virus producer cell lines was intact as assessed by overlapping PCR 
reactions extending from 5' of the neo gene to the polypurine tract in the retroviral vector 
downstream of the IDS coding sequence. 
Fig. 2 - IDS Retroviral Vector L2SN 
L2SN 
IDS 
MS3 
As an initial test system for studying the function and effectiveness of the L2SN vector, we 
have used lymphoblastoid cell lines (LCL) established from patients with Hunter syndrome as a 
target cell population (LCLmps)* Extensive initial work has been conducted (61-63) using L2SN to 
infect LCLmps, selecting for positive transductants in medium containing 1.0 mg/ml G418. 
Analysis of two different transductant cell populations indicated that the L2SN-transduced 
LCLmps expressed levels of IDS enzyme increased 10- to nearly 70-fold over the level observed in 
LCL established from normal individuals, i.e., LCLNormal (Table 2). These findings are consistent 
with results recently reported by several different laboratories, indicating a low endogenous level 
of many of the lysosomal enzymes, with subsequent high-level expression after gene transfer 
using strong heterologous regulatory elements. This high-level IDS expression after retroviral- 
mediated gene transfer facilitates studies into the subsequent correction of lysosomal metabolism 
(see below). 
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Recombinant DNA Research, Volume 20 
