Lymphocyte Gene Therapy for Mild Hunter Syndrome 
Table 2 - IDS Expression By L2SNTransduced Lymphoblastoid Cell Lines from Patients with 
Hunter Syndrome (LCLmps) 
Cell Line IDS Enzyme Activity (± S.D.) 
U/mg/hr 
LCLj\j orma i 
829 ± 131 
LCLmps 
<10 
lclmps-lxsn 
<10 
LCLmps-L2SN 
8,766 ±3,468 
LCLmps-L2SN 
55,763 ±18,729 
Amphotropically packaged IDS retroviral vectors have now been used to transduce 
LCLmps, monitoring the exposed cell population for IDS enzyme activity expressed and also for 
integrant frequency. LCLmps were exposed to virus at a multiplicity of infection of approximately 
4 and then cultured for a period of 3 days before preparation of crude cell extracts for IDS enzyme 
assay. Genomic DNA was also extracted to quantitate the frequency of IDS proviral integrants 
using a quantitative PCR assay. IDS proviral integration was assessed in a semiquantitative PCR- 
based assay. This technique indicated the presence of IDS proviral integrants at a level of 0.5% to 
3.0%. IDS enzyme activity was present in all transduced cell populations at a level of 70 to 130 x 
10 3 units per proviral integrant (Table 3). After G4 18-selection for 1 month, IDS activity increased 
to 30-fold of normal levels (18,600 U/mg/h for L2SN). These results verify the function of the IDS 
retroviral vector construct as packaged virus as well as the technical capability to assess IDS 
enzyme expression and the frequency of even low-frequency proviral integration. In summary, 
heterogeneous, unselected LCLmps cultures expressed IDS levels comparable with normal 
although only 0.5-2% of targeted cells were infected in these initial pilot studies. 
Table 3 - Transduction Frequency and IDS Expression Of L2SN In LCLmps 
IDS 
Titer 
LCL Transduction 
IDS Activity 
IDS Activity 
Virus 
cfu/ml 
Frequencyb 
in LCLmps 0 
per Integrantd 
L2SN 
3x 10 6 
2.5% 
1,370 
54,800 
bSemiquantitatiave PCR. cAssayed in IDS-deficient LCL^p^; IDS in normal LCL ~ 700 U/mg/h. 
dlDS activity divided by fraction of cells infected. 
The L2SN-transduced LCLmps (described above) were assayed for accumulation of 
glycosaminoglycans by incubation in the presence of 35 SC> 4 , monitoring incorporation into 
macromolecular material (Fig. 3). Control (LXSN-transduced) LCLmps accumulated 35 SC >4 over 
three days, while normal LCL did not accumulate 35 S 04 beyond the first 24 hrs. However, 
LCLmps transduced with L2SN did not accumulate 35 SC >4 beyond the first day, indicating that 
lysosomal metabolism had been corrected in these transduced cells by high-level expression of 
IDS. 
Recombinant DNA Research, Volume 20 
[287] 
