Early studies demonstrated that retroviral vectors transduce cells at high 
efficiency in tissue culture. In addition, the transduced genes were shown to 
function in established cell lines by measurement of RNA transcripts, enzyme 
activity in vitro , and metabolic resistance to toxic agents. 
Much of the relevant i_n vivo experience with these vectors has been gained 
using murine hematopoietic cells. To develop a bone marrow transplantation 
system more applicable to man, we used retroviral -mediated gene transfer to 
introduce retroviral genes into a number of larger species, including non-human 
primates, dogs, and fetal sheep. The data obtained from these systems will be 
summarized below and in the accompanying supplementary material in Appendix A. 
For gene therapy to be feasible, the proposed system must meet a number of 
requirements. First, the system should be efficient: the vector should be 
capable of transferring the gene into a significant proportion of the target 
cells. For ADA in particular, the target cells are primarily multi potential 
hematopoietic progenitors (stem cells) which can give rise to cells of the 
lymphoid lineage, particularly T cells. Once Integrated Into the chromosome of 
the host cell, the provirus should remain intact and unaltered In daughter 
cells which may undergo a process of differentiation and expansion In myeloid 
and/or lymphoid lineages. Second, continued expression of the ADA gene during 
the various stages of the hematopoietic differentiation pathway is probably 
necessary, in addition to expression in the mature T cell (see Section 
I.B.2.b). And third, some measure of safety of the proposed system must be 
assured (see Section I.B.2.c). 
Methods of Analysis : A variety of techniques have been used in order to 
demonstrate both the presence and stability of retroviral vectors in cells. 
1. DNA analysis. Either the method of Southern or DNA dot blots can 
demonstrate the presence of proviral DNA in transduced cells. Both methods 
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Recombinant DNA Research, Volume 12 
