Recombinant DNA Advisory Committee - 12/3-4/92 
Dr. Nienhuis responded to the RAC's concerns regarding the protective effect of 
transduced ABM cells during taxol administration. Quantitative PCR will be employed 
to determine the proportion of ABM cells that contain the transduced MDR gene. 
Increased levels of MDR expression will indicate amplification of the genetically- 
modified population of cells. 
Dr. Nienhuis discussed the current status of vector development. The vector that was 
used for the animal experiments was a Harvey-based vector that contained extraneous 
sequences, i.e., the entire VL30 coding region. The Moloney-based vectors are being 
proposed for the human experiments because the extraneous sequences have been 
minimized. The amphotropic producer clone G1 MD1-15 made by Dr. McDonagh was 
used for the rhesus animal experiments. The cDNA derived from this clone exhibits a 
functional splice donor and splice acceptor site that are part of the MDR cDNA in 
different exons. Although these sequences probably do not function as splice sites in the 
natural in vivo transcript, this splice occurs in a proportion of the transcripts. The 
original cDNA has a valine substitution in codon 185 that increases colchicine resistance 
and decreases vinblastine resistance. This observation was critical in the development of 
the vector. Dr. McDonagh created the AB-1 vector where splice donor and acceptor 
sites were eliminated and glycine was conservatively substituted for valine. 
AB-1 was the vector that was originally proposed for use in the human studies; however, 
several problems were encountered. First, sequencing of this vector revealed that there 
was a frame shift mutation in the C-terminus of the protein. Second, although the gene 
could be expressed in primary transfected cells in which the DNA had been introduced, 
the retrovirus vector did not provide efficient transfer of the gene due to the corrected 
splice sites. 
The second vector, G1 MD3, contained the corrected frame shift but the splice sites 
remained deleted. Due to the deleted splice sites, this vector was ineffective at providing 
gene transfer. The final vector proposed for this protocol contains the natural cDNA. 
This vector contains the splice donor and acceptor sites and incorporates a glycine 
substitution for valine. Although this amino acid substitution eliminates the C-terminus 
frame shift, vinblastine resistance is enhanced. Amphotropic producer clones are 
currently being characterized that will prove satisfactory for clinical purposes, i.e., 
capable of CD34( + ) cell transduction and producing a titer of approximately 5 x 10 6 . 
Dr. Parkman asked for a comparison between the Harvey-based vector and the new 
vector in an animal model. Dr. Nienhuis asked Dr. McDonagh to respond to Dr. 
Parkman's question. 
Recombinant DNA Research, Volume 16 
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