Recombinant DNA Advisory Committee - 2/10-11/92 
Peripheral blood mononuclear cells from the second patient were plated and a normal 
number of CFU resulted compared to the control. No neomycin resistant colonies were 
observed; therefore, if there was transduction of myeloid progenitor cells, it was not 
detected. T lymphocytes were expanded from this patient for two weeks in culture with 
interleukin-2 (IL-2) and anti-CD3 antibody. The investigators were unable to show any 
CFU-C survival. This result was predicted since there were no growth factors present 
(e.g., IL-3, IL-6, or hematopoietic growth factor) that would be capable of sustaining CFU- 
C cells in culture. Without interleukin-3, there was some evidence that early progenitor 
cells and stem cells underwent apoptosis. 
Dr. Dunbar compared the original LASN retroviral vector with the proposed vector, 
GlNa.SvAd. She explained that the original LASN vector had the ADA gene inserted in 
the first position and the neomycin resistance gene in the second position with an SV40 
promoter. GlNa.SvAd simply switches the position of the ADA and neomycin resistance 
genes. These two vectors are distinguishable by PCR because of the different sizes. 
Preliminary data from experiments using these same vectors (without the ADA gene 
inserted) indicate that CFU-C cells isolated from the CD34 enriched peripheral blood cell 
fraction are transduced by these vectors without G-CSF mobilization. Transduction 
efficiencies were comparable to those of bone marrow stem cell fractions. Transduction 
efficiency was assessed by: (1) expansion of CFU-C from the neomycin resistant 
populations, (2) PCR analysis of neomycin resistant cells, and (3) PCR analysis of the 
CD34 enriched cells to determine gene copy number. 
Dr. Dunbar described a series of experiments that compared transduction of CD34 
enriched normal peripheral blood cells with the GlNa.40 (neomycin resistance gene) and 
GlNa.SvAd (ADA gene) vectors. Results indicated that 16% of the CFU-C cells were 
positive for GlNa.40 and 10% for GlNa.SvAd. Semi-quantitative PCR analysis 
demonstrated transduction efficiencies of approximately 1%. 
Dr. Dunbar stated that it has been very difficult to assess ADA gene expression. 
Expression is often detectable in earlier progenitor cells, but disappears in later myeloid 
progenitors. There have been at least three published series of experiments looking at 
murine bone marrow that has been transduced with different ADA vectors. One 
experiment was very similar in construction to the proposed vector in this amended 
protocol. In addition, expression has been observed in the thymus cells of animals that 
received human ADA transduced bone marrow cells at three and six months following 
treatment. Unpublished data from Dr. Dinko Valerio in the Netherlands shows long-term 
ADA expression in peripheral blood mononuclear fractions isolated from rhesus bone 
marrow. 
Discussion 
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Recombinant DNA Research, Volume 15 
