engraftment of autologous cells removed from a patient and treated ex vivo with 
retroviral vectors even if the patient’s marrow is not ablated. 
Safety issues of retrovirus-mediated gene transduction 
The retrovirus vector LgGC and the PG13 packaging cell line (28) will be used for 
transduction of the CD34+ PBRC. The LgGC vector is derived from the LN vector. 
The vector LN is an improved version of the vector LNL6 (29), which has been studied 
extensively in rodent models and in non-human primates. It was approved for use in 
man in 1989. The results of the first in vivo human study using the vector LNL6 to 
transduce tumor-infiltrating lymphocytes have been reported (30). No side effects have 
been seen and no infectious virus has been detected in the patients. 
1. The LgGC vector 
The basis for the LgGC vector is the N2 vector (31), a derivative of Moloney 
murine leukemia virus in which the viral gag, pol, and env genes have been 
removed or truncated and the bacterial neo gene inserted. The retroviral 
packaging signal in N2, however, also contains the start codons for the gag 
protein of the parental helper virus, Pr65 gag and a larger glycosylated protein, 
g Pr85 gag, that initiates at a CTG codon upstream of and in frame with the 
ATG codon of Pr65. To prevent synthesis of viral proteins, the N2 vector was 
modified to LNL6 by inserting a stop codon in place of the Pr65 gag start codon 
and by replacing the upstream region of the vector with the homologous region 
from Moloney murine sarcoma virus, which is very similar to Moloney murine 
leukemia virus but does not make the glycosylated gag protein. However, 
undesirable generation of helper virus could still occur in these packaging cells by 
homologous recombination between viral sequences endogenous in the packaging 
cells and sequences in the constructed vectors, although this has not been 
observed so far. To prevent this possibility the vector LNL6 was modified to LN 
by removing all env sequences. The LgGC vector is derived from the LN vector 
by replacing the neo gene with human GC gene. The defective helper virus used 
to make PG13 packaging cells was truncated immediately after env; thus, the lack 
of homologous overlap between the vector LgGC and viral DNA in the PG13 
packaging cells excludes the possibility of helper virus generation by homologous 
recombination and further increases the safety of the vector LgGC for use in 
humans. 
2. Safety of retrovirus-mediated gene transduction into canine marrow or peripheral 
blood-derived repopulating cells 
Nine of a total of 13 dogs engrafted after transplantation of transduced marrow 
or peripheral blood cells as described above. Eight of the nine dogs have been 
observed so far for more than 1 year after transplantation. Two dogs are alive 
more than 4 years after transplant, three more than 2 years, and two more than 1 
year. One dog (C874) died 3 years after transplantation due to metastatic 
prostate carcinoma, and one dog (D425) died on day 84 from chronic canine 
distemper sclerosing encephalitis. Peripheral blood of the nine engrafted dogs 
was tested three times for replication -competent virus using the S + L' helper virus 
assay as described (32) (last test on 7-93). The S + L-assay is a sensitive assay for 
detection of replication-competent virus. It measures the ability of a virus sample 
to mobilize a retroviral vector from cells that contain but do not produce a 
vector. None of these tests showed the presence of replication-competent virus. 
Recombinant DNA Research, Volume 18 
