C. HESDORFFER 4/93 
from patients routinely undergoing ABMT in association with high- 
dose anti-tumor chemotherapy for solid tumors not involving the 
bone marrow; 2) Insertion of the human MDR gene into these cells 
using retroviral vectors; 3) Treatment of the patient with high- 
dose chemotherapy; and 4) Reinfusion of the transduced bone 
marrow cells containing and expressing the transferred genes. On 
subsequent exposure to MDR-responsive drugs, the marrow of these 
patients should be enriched for cells resistant to the 
chemotherapy; the reduced marrow toxicity may permit escalation 
of doses of chemotherapy in the hope of cancer cure in selected 
patients . 
Efficient and safe gene transfer and high-level MDR expression 
are the major goals in this gene therapy-autotransplantation 
scheme. We will use: 1) Retroviral vectors that maximize MDR 
gene transfer and expression into human bone marrow cells; 2) 
Purification of bone marrow hematopoietic precursor cells (BM- 
HPC) using antibodies and cell-sorting to increase the ratio of 
virus: cells; 3) Growth factors to increase cycling necessary for 
retroviral infection and the number of BM-HPC; and 4) In vitro 
assays to assess the number of BM-HPC, and assays for human MDR 
DNA and MDR protein to determine MDR transfer and expression, 
respectively . 
The background for the proposed protocol includes progress made 
recently in the following areas: 
A. Transfer and Expression of Foreign Genes into Mice : 
Retroviral vectors are the most efficient means of 
gene transfer described to date (9) . Defective packaging lines 
having mutations that do not allow the encapsulation of an intact 
Moloney murine leukemia virus genome are available (10-17). 
These packaging lines transfected with retroviral vectors are 
called producer lines. Retroviral vectors have been used to 
transfer the neomycin resistance (neo R ) gene into mouse bone 
marrow stem cells; this results in reconstitution of the host 
mainly with cells that contain the transferred gene (18-24); this 
is especially true if preselection of cells with G418, a neomycin 
analogue, is used ex vivo (21,23,24). This is important because 
if donor stem cells are untransduced they will compete with 
transduced stem cells in reconstituting the host marrow. From 
these experiments, we infer that a selectable gene transferred 
along with a co-transferred non-selectable gene on the same 
retroviral vector would enrich for cells containing both genes. 
G418 cannot be used in live animals because of its toxicity, and, 
thus, the MDR gene has been chosen as a selectable gene. Human 
ADA (25), CD8 (26), B globin (27-29), and glucocerebrosidase (30) 
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Recombinant DNA Research, Volume 18 
