These data support the view that human CD8 + CTL exist in HIV- 1 -infected individuals 
and can be stimulated by HIV-IT (V)-transduced autologous cells. It will be important 
to ascertain whether a similar triggering phenomenon can occur in vivo. 
2.4.5 The quantity of HIV-IT (V) injected into mice, Rhesus monkeys, and baboons, as well 
as the projected dose for human clinical studies is very small relative to body cell mass. 
Due to the non-replicating nature of HIV-IT (V), widespread distribution of the vector 
after IM injection would not be expected, particularly in the absence of RCR. The 
majority of HIV-IT (V) should, therefore, remain at the injection site. Although a 
complete accounting of all HIV-IT (V) material within an injected animal is not feasible 
with current assays, vector DNA, if present in tissue, should be detectable using 
sensitive molecular techniques. Two approaches have been used: (1) administration of 
35S methionine/cysteine-labeled vector to mice followed by measurement of radioactive 
counts in various tissues to track vector particles; and (2) polymerase chain reaction 
(PCR) monitoring of injection sites and testes in mice to track the vector integration of 
HIV-1 env DNA in the tissues. 
The data indicate that the majority of IM administered, S-met/cys-labeled vector 
appears to remain at the site of injection and does not traffic throughout the animal. 
Further results demonstrate that vector HIV-1 env DNA is detectable by PCR at the site 
of injection after one or two injections in all mice tested. Results have also indicated 
that genomic DNA obtained from testes of HIV-IT (V)-injected mice are PCR negative 
for HIV- 1 env DNA. These data suggest that HIV-IT (V) injected IM remains 
primarily at the injection site until cleared by the immune system and that there is no 
evidence of vector associated with germline tissue. 
2.4.6 In order to determine the acute safety and biological activity of multiple intramuscular 
injections of HIV-IT (V), a four-week IM toxicity study in male and female BALB/c 
mice was conducted by Hazelton Laboratories (Vienna, Virginia) in accordance with the 
Good Laboratory Practice Regulations. 
Animals were observed for clinical indications of morbidity and mortality daily, and 
body weights were recorded weekly. Potential clinical pathology was assessed by 
hematologic evaluation pre- and post-injection. Gross necropsy and histopathologic 
evaluation of the injection site, gonads, thymus, mesenteric lymph nodes, marrow, 
liver, kidneys, brain and any gross lesion in all animals, was performed. The Findings 
of clinical observations, body weights, food consumption, clinical hematology, and 
necropsy data indicate no evidence of toxicity in mice injected with HIV-IT (V). 
2.4.7 In summary, preclinical data support the view that HIV-IT (V) can be safely 
administered to both mice and non-human primates, that CD8 + CTL and antibody 
responses can be induced in animal models and that cross-reactive cytotoxic immune 
responses directed against cells infected with divergent HIV- 1 -strains can be 
demonstrated by murine CTL induced by HIV-IT (V). In addition, mouse toxicology 
studies have found no HIV-IT (V)-related abnormalities. These data provide the 
impetus to initiate studies of HIV-IT (V) in humans. Based on the successful 
production of CTL responses achieved in non-human primates that received three doses 
of HIV-IT (V) and on a careful analysis of these data. Phase I clinical trial subjects will 
receive three doses of HIV-IT (V) at monthly intervals. 
2.5 Clinical Studies 
Three successive biweekly injections of autologous fibroblasts transduced with HIV-IT (V) 
have been administered to each of four asymptomatic HIV-1 infected subjects in a Phase I 
study conducted at the Shared Medical Research Foundation in Tarzana, California. These 
subjects have been followed for intervals of from two weeks to two months following their 
third injection. No product-attributable adverse events have occurred. It is still too early to 
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Recombinant DNA Research, Volume 18 
