C. HESDORFFER 4/93 
gene transduction is shown to be safe, the results could then be 
used to design a subsequent trial whose goal would be to 
establish the efficacy of this treatment by either: 1) Changing 
the transduction protocol; or 2) Escalating doses of the MDR- 
responsive drugs used, i.e., taxol or adriamycin, following 
marrow transduction, to obtain an improved therapeutic result, 
i.e., prolonged survival of these cancer patients. 
These studies will be carried out initially at CPMC, 
Sloan-Kettering Cancer Center, and Montefiore Hospital. 
Dr. Charles Hesdorffer, a participant in the mouse 
transplantation experiments and Director of the Bone Marrow 
Transplantation Program at Columbia University, and 
Dr. Karen Antman, Director of the Division of Oncology at 
Columbia University beginning July 1, 1993 and an investigator 
with experience in ABMT, will be co-principal investigators. 
Drs. Peter Wiernik (Montefiore) and Subhash Gulati (Sloan- 
Kettering) will also collaborate (letters attached) . There are 
more than adequate numbers of patients available at these centers 
to carry out this protocol. The details of the trial are in the 
protocol at the end of this section. 
Patients will be hospitalized for 24 hours to undergo the bone 
marrow harvest as is standard practice at CPMC. Greater than 
3 X 10® cells/kg will be harvested from each individual who 
agrees to undergo the gene transfer procedure. The marrow will 
first be treated to separate mononuclear cells with a Ficoll- 
Hypaque density gradient. Once this is completed, 30-50% of the 
cells will be sorted for CD34 + cells depending on the number of 
cells harvested. We will use both CellPro columns (Ceprate™SC 
Stem Cell concentrator) containing beads with biotinylated anti- 
CD3 4 antibody, 12.8 (64,65), and AIS CD34 antibody-coated plates 
with anti-CD34 antibody, ICH3 (112) , to purify CD34+ cells prior 
to transduction. An aliquot of the CD34 + population of cells 
will be plated in methylcellulose for CFU-GEMM and BFU-E assays 
directly. The bulk of the CD34+ marrow cells will be pre- 
incubated for 24 hours with growth factors (IL-3, IL-6, and human 
SCF) , and then co-cultured with MDR virus producer cell AM12MDR 
supernatants; an aliquot will be taken for CFU-GEMM and BFU-E 
analyses. The cells will be kept in culture for 3-4 days during 
which time the patients will be given high-dose chemotherapy and 
will be prepared to receive MDR-transduced autologous bone marrow 
cells. At this time, the patients will receive enough nucleated 
cells of their own untreated unfractionated stored marrow to 
permit marrow reconstitution alone, in addition to the transduced 
marrow, representing approximately the same number of total 
marrow cells. In this way, we will ensure marrow engraftment and 
avoid exposing the patient to the risk of delayed engraftment. 
Following the infusion of marrow patients will be treated with 
Recombinant DNA Research, Volume 18 
