Recombinant DMA Advisory Committee - 09/9-10/93 
In regard to the issue of compensation for research-related injury, Dr. Walters noted that 
a statement is included in the Informed Consent document explaining that the NIH 
Clinical Center will provide short-term medical care, but not long-term care, for physical 
injuries resulting from a patient's participation in research. This statement is a 
modification over the usual statement that no care or compensation will be provided. 
Investigator Response—Drs. Neinhuis and Sorrentino 
if 7 
In response to Dr. Post's comments about the use of stem cell growth factor, Dr. 
Neinhuis stated that stem cell growth factor will be used in the human clinical trial due 
to the fact that it has recently become available. Responding to the question of MDR-1 
MRNA splicing. Dr. Neinhuis said that the protein encoded by the spliced MRNA is a 
small truncated variant gene product and is presumably nonfunctional. Approximately 
50% of the MDR-1 gene transcript from the DNA construct based on either the Harvey 
or Moloney vector is spliced. Both full length and spliced RNA species are co-packaged 
as virions and are transferred to target cells. mRNA expression of the normal genomic 
MDR-1 gene, however, is not similarly spliced. 
Dr. Sorrentino summarized preclinical studies with the murine model. While earlier 
studies were performed using a Harvey-based vector, recent results were obtained with 
the new Moloney-based construct, G1MD. A high titer amphotropic producer clone of 
the G1MD vector has been isolated from the PA317 packaging cell fine. Transfer of the 
MDR-1 gene to the target cells was assayed by the rhod amine efflux assay. Expression 
of the transduced MDR-1 gene yielded a "dull" phenotype as compared to a "bright" 
phenotype for untransduced cells as observed under a fluorescence microscope. These 
two types of cells were fractionated by a fluorescence activated cell sorter (FACS). 
MDR-1 gene expression by the G1MD construct in mouse hematopoietic cells was 
demonstrated. In the in vivo murine model, Taxol conferred drug resistance to 
hematopoietic cells. Data also demonstrated MDR-1 gene expression after transduction 
with the clinical grade G1MD vector in human CD34( + ) cells. MDR-1 proviral DNA 
was also demonstrated. Quantitative assays for the transduced MDR-1 gene were 
performed in primates. 
Dr. Sorrentino said that similar mRNA splicing of the MDR-1 gene probably occurred in 
constructs used in other previously approved protocols. Mutations made to eliminate the 
splice donor and acceptor sites resulted in a construct that expressed the encoded MDR- 
1 gene, but the vector RNA was unable to be packaged in the producer cell. Presumably 
all packaging cells produced viral particles containing both RNA species. 
Responding to Dr. Miller's question about the replication competent retrovirus (RCR) 
testing of the clinical grade vector, Dr. Neinhuis said that Genetic Therapy, Inc., has 
performed extensive testing to confirm the absence of RCR. 
Committee Motion 
A motion was made by Dr. Parkman and seconded by Dr. Miller to approve the 
Recombinant DNA Research, Volume 18 
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