bleeding. Every effort will be used to maintain sterility and 
prevent bleeding. 
The development of replication-competent retrovirus. As 
discussed (Section IV. E.). 
Survival of Vector Producer Cells in the Host. The PA317 
cells can survive only 7-14 days in a subcutaneous site in 
syngeneic (H-2b) mice and only 7-14 days in xenogeneic rat 
brain. Likewise in immunosuppressed monkeys, the producer 
cells could not be identified > 14 days after injection. 
Taken together, these findings suggest that if any PA137 cells 
are not killed by GCV will not have the ability to survive 
long term in the human brain. 
Dissemination of GlTkSvNa.29 Our MoMLV amphotropic retroviral 
vectors are directly inactivated by human complement without 
antibody. Therefore, escape of free vector into the cerebral 
spinal fluid or the vascular space should result in immediate 
inactivation. The gene transfer in this direct injection 
system is most likely due to the intimate contact of the tumor 
and VPC . Any vector particles that are released in the area 
of injection will be quickly bound by the thousands of 
amphotropic vector receptors on each tumor cell and other host 
cells. Vectors binding to non-dividing cells in the brain 
will be lost. Even if all of the vector particles produced 
were able to escape direct transfer into adjacent tumor cells 
of binding to non-dividing cells and cross the blood-barrier, 
the number of vector particles relative to the number of 
receptors in any organ would still be very small suggesting 
a minimal risk of injury to proliferating cells in any non- 
CNS organ. Any direct transfer into neurons by cell-to-cell 
contact will not result in HS-tk gene integration and 
therefore, should not pose a risk for their destruction with 
GCV treatment. 
Transduction of surrounding brain tissue. There is no 
evidence in our animal model that transduction of surrounding 
normal brain tissue is likely to occur (Section 3. I. A. 2.). 
However, if the HS-tk gene is introduced into a large number 
of normal dividing cells within the CNS (such as endothelial 
cells and astroglial cells) , vasculitis like symptoms 
(headaches, convulsions, bleeding) may develop. Such changes 
however will be localized to the immediate vicinity of the 
tumor as had been shown in our experiments (3.I.C.2). 
Insertional Mutagenesis. Retroviral vector DNA is inserted 
randomly into the genome of proliferating cells, The random 
nature of v this integration has the potential of an untoward 
insertional event. If the insertion disrupts a gene essential 
for maintaining cell function, that particular cell will die. 
Since the gene transfer will occur most predominantly in tumor 
cells, the vector insertion site will result in the death of 
a few tumor cells without GCV, which should not pose a 
problem. The risk of oncogenic transformation with these 
replication-incompetent retroviral vectors cannot be 
accurately estimated since that has never been a documented 
occurrence in animals or man. While this is a real risk, this 
risk must be very low, especially in this protocol, where all 
vector containing cells will be killed by GCV within 2-3 weeks 
Recombinant DNA Research, Volume 18 
