Three aspects of the proposed neo gene marking study are unique 
among the human gene transfer studies conducted to date: 1) The 
use of longer-term (2-6 days) infections of the bone marrow cells 
with the retroviral vector-containing supernatant; 2) The use of 
hematopoietic growth factors in the infection culture; and 3) 
Infection of the CD34+ subpopulation of marrow cells. All three of 
these methods are aimed at increasing the efficiency of gene 
transfer into human stem cells. We will also harvest patients' bone 
marrows during the late part of the nadir recovery period following 
the last cycle of induction chemotherapy in order to capture as many 
proliferating stem cells as possible. 
Turhan and Eaves, et al. have achieved marrow engraftment in three 
patients with CML transplanted with autologous hematopoietic cells 
grown in culture for 10 days to enrich for normal Phi -negative stem 
cells (48). It appears, therefore, that human stem cells and 
progenitors cultured for 2-6 days prior to reinfusion are capable of 
contributing to successful marrow engraftment. 
Naparstek, et al. have shown that pretransplant in vitro activation of 
bone marrow cells with IL-3 and GM-CSF may prove an efficient and 
safe method for acceleration of hematopoietic recovery after BMT. 
Patients undergoing allogeneic BMT were found to have significantly 
faster neutrophil and platelet recovery when one-third of their 
marrow was incubated with GM-CSF for three days and IL-3 on Day 4 
compared to historical controls from the same institution (49). Our 
plan to culture the CD34+ cells with hematopoietic growth factors 
prior to reinfusion appears safe based on this study as well as data 
obtained using this approach in primates (36). Lastly, Berenson has 
demonstrated that CD34+ bone marrow cells are capable of 
reconstituting hematopoiesis in breast cancer patients after high 
dose chemotherapy, and in some cases, total body irradiation (15). 
Berenson et al. separated the CD34+ suboopulation from the bone 
marrow harvests of 7 patients with breast cancer and 2 with 
neuroblastoma and demonstrated that the CD34+ ce lls were capable 
of reconstituting the patients' marrows after high dose 
chemotherapy and, in 3 patients, total body irradiation. The median 
Recombinant DNA Research, Volume 16 
