III. 
Exposure of the peripheral blood collection which was CD34 
selected to a safety-modified retrovirus which contains a MDR-1 
cDNA will be undertaken following the protocol outlined in 
Appendix D. The peripheral blood or marrow back up collection 
which was not CD34 selected will be frozen in unmodified form. 
PCR assay for the MDR cDNA will be performed following the 
transduction using the assay outlined above. The PCR assay will 
be used to identify if the transduction was successful. A 
transduction frequency of 1% will be considered successful. The 
marrow will be frozen away using programmed freezing, as a backup 
for the peripheral blood transplant. The viability of the frozen 
marrow will be tested using the CFUGM assay. 
IV. Preparative therapy will be delivered a single time before the 
transplantation and will involve cyclophosphamide, BCNU and 
thiotepa, in doses outlined earlier in this protocol. The 
administration of the preparative therapy will not begin until 
the patient has recovered from all of the toxicities of the non 
ablative dose therapy, and until the total white cell count is 
2,000/cu mm, and the ANC is 1,800/cu mm, and until the hemoglobin 
is 8 gm/dcl, and the platelet count is 100,000/cu mm. The 
preparative therapy must also be delayed until all of the quality 
control data for the retroviral modification of the cells, and 
for the determination of the integrity of the hematopoietic cells 
stored is determined. This usually requires 3-4 weeks between ; 
the completion of the collection of the hematopoietic cells and 
the initiation of the preparative therapy. 
V. Infusion of the peripheral blood with collection of a sample for 
RT PCR for MDR-1 of the thawed specimen before infusion. 
VI. PCR for MDR-1 and functional assays for MDR-1 on the 
hematopoietic cells following a recovery of an ANC of 2000/mm^ is 
achieved. 
VII. Delivery of 12 courses of relatively intensive but not ablative , 
levels of Taxol chemotherapy, at a starting dose of 60 mg/m^ | 
every three to four weeks, and the dose will be increased as t 
follows (60, 120, 180, 225 and 275 mg/m^) as is allowed by the | 
rate of hematopoietic recovery. PCR assays will be conducted to | 
assess the percentage of hematopoietic cells which are positive | 
for the MDR-1 cDNA after each course of chemotherapy. 1 
VIII PCR for MDR-1 on colonies of peripheral blood and marrow | 
hematopoietic cells every three to four weeks following each i 
course of therapy, as long as that therapy is administered, or | 
until relapse, at which time the therapy of Taxol maintenance 
will no longer be administered. In situ functional analysis of j 
the number of cells in peripheral blood and marrow by rhodamine j 
efflux will be performed at the same time as RT PCR of MDR-1 to ! 
enumerate the number of cells with increased MDR-1 in the marrow ; 
and peripheral blood. After discontinuation of Taxol therapy, ’ 
the PCR assays will be conducted every three months for two | 
years, and every six months until a 5 year period has elapsed i 
following the initial transplantation of modified marrow. i 
6.0 PRE-TREATMENT EVALUATION J 
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Recombinant DNA Research, Volume 19 
