monoclonal antibody and then passed through a column of avidin-coated beads. Cells 
binding antibody are retained due to the high affinity between avidin and biotin. Bound 
cells are eluted from the column by mechanical agitation of the beads which disrupts the 
CD34 antibody interaction. 
The purity of the selected population ranged from 35-95% with a CFU-C enrichment of 
22-180 fold. Patients received 0.4-3 x 10^ CD34 cell/kg. In the breast cancer study the 
median time to an ANC of 500 was 20 days in patients receiving CD34 selected marrow 
without post transplant growth factors and 1 1 days in patients treated with GCSF post 
transplant. 
3.4 Ex vivo expansion of CD34 selected cells 
Autologous stem cell rescue has two apparently conflicting aims - a short period of 
aplasia with harvesting of as few stem cells as possible. One way to achieve both these 
aims is by expanding committed and uncommitted progenitor cells ex vivo. Several 
investigators have evaluated the ability of growth factors to expand CD34 enriched 
marrow cells ex-vivo.‘^'“* Brugger et al‘^ have shown that the combination of ILl,, IL6, 
SCF, Erythropoietin and IL3 results in a 260 fold expansion in total cell numbers, and 
a 3 fold increase in CD34 cells. We have obtained similar results in our preclinical 
studies (Appendix A). We and others have, however, noted little expansion of the most 
primitive cell populations, so that while treated marrow might produce earlier 
engraftment, its capacity to produce long term reconstitution remains unknown. 
3.5 Use of gene marking to compare reconstitution 
The ideal of ex vivo expansion is to increase both committed and non-committed 
progenitor cells thereby accelerating initial engraftment and ensuring sustained 
reconstitution. An acceptable alternative is simply to expand committed progenitor cells 
to reduce the period of post transplant aplasia provided sufficient pluripotent stem cells 
were retained in an undifferentiated state to permit long term recovery. Gene marking 
of marrow ex vivo allows determination of whether adequate numbers of stem cells 
remain in vivo. In this study we plan to use genetic marking of marrow ex vivo with two 
distinct vectors to compare the subsequent in vivo reconstitution of two aliquots of CD34 
selected marrow - one treated and one unmanipulated. This protocol will therefore 
answer questions about the possibility of accelerating hemopoietic reconstitution. It will 
be a generic processing and transduction protocol for patients receiving autologous BMT 
for pediatric malignancy. 
3.6 Advantages of gene marking 
One of the major difficulties in assessing the impact of growth factors on marrow 
recovery is the wide interpatient variability. This may be further compounded by 
differences in marrow involvement with tumor, pre-transplant therapy and in conditioning 
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Recombinant DNA Research, Volume 19 
