not interfere with the growth of normal progenitor cells when the two are co- 
cultured (see Data Appendix A). 
Therefore while the primary purpose of transducing autologous marrow with 
a marker gene is to study the source of relapse (3.2) we will also attempt to 
detect the gene after ABMT in cells derived from different lineages. We will 
look for evidence of multipotent progenitor transfection by seeking a single 
insertion site in multiple lineages (see below). We will also look for long term 
expression (3 months to 5 years). 
If expression of transduced genes is detected in vivo we will be able to analyze 
those exogenous and endogenous stimuli which promote cycling of the 
transduced stem cells, including infection and further chemotherapy. 
Ultimately this information would allow protocols to be devised which would 
induce optimum stem cell cycling and cytokine responsiveness of marrow pre 
ABMT for optimum recovery post ABMT. The information would also 
facilitate techniques aimed at growing the marrow ex vivo from small aliquots, 
potentially allowing multiple ABMT from a single marrow harvest (see 1.2 
above). 
TRANSDUCTION STUDY OUTLINE AND SAFETY 
This study proposes to transduce a neomycin resistance marker gene into aliquots of 
marrow obtained for ABMT in treatment of relapsed neuroblastoma. 
The vector to be used in the transduction studies is the helper free LNL-6 retrovirus, 
which has been extensively studied in rodent models and in non-human-primates and 
which was approved for use in man in 1989. The results of the first in vivo human 
study on transduced marrow derived cells have been reported at The American 
Society for Clinical Oncology and in the New England Journal of Medicine. The safety 
data required for the granting of the IND (#3042) under which this first study took 
place are contained in the four volume directories made available to the Chairman 
of the IRB. The current proposal will cross reference these data. The major safety 
concerns and their resolution are also summarized in an article described in Appendbc 
B, and are abstracted below (Cometta et al., 1990). 
4.1 The LNL-6 Vector 
The LNL-6 vector (Figure 1) was developed in the laboratory of A Dusty 
Miller and is a safety-modified version of the retroviral vector N2. The vector 
was constructed by modifying the Moloney murine leukemia virus (MoMLV) 
by removing the viral genes and replacing them with the bacterial neo^ gene. 
The vector has a number of modifications to decrease the likelihood of 
Recombinant DNA Research, Volume 14 
