Abstract
The first large randomized phase III trial in gene therapy demonstrated
no improvement in the survival of patients injected with packaging cells that
produced conventional replication-defective retroviral vectors carrying the
herpes simplex virus thymidine kinase gene, a disappointing result that was
attributed to extremely poor levels of transduction efficiency. To circumvent
this problem, we have developed a modified replication-competent retrovirus
(RCR) that is capable of transducing human glioma cell lines A-172, U-87, T-98G,
U-373, and U-138 and rat glioma cell lines C6 and 9L, over multiple infection
cycles
in vitro
, resulting in a tremendous enhancement in
transduction efficiency over conventional replication-defective retroviral
vectors at the same dose. Whereas the transduction efficiency of conventional
retroviral vectors injected into pre-established subcutaneous U-87 tumors at a
dose of 1.0 × 10
5
transducing units (TU) was only 0.2% at 6
weeks postinjection, the same dose of RCR vector resulted in up to 97.2%
transduction. When RCR vectors at a dose of 1.0 × 10
4
TU were
injected into pre-established intracranial U-87 tumors, transduction efficiency
at 2 and 3 weeks was 74 and 98.1%, respectively. Notably, however, intracranial
injection of RCR vectors did not result in detectable infection of normal brain
cells. Furthermore, using a sensitive polymerase chain reaction assay, no
detectable RCR signal could be observed in any extracerebral tissues, including
lung, liver, kidney, upper gastrointestinal tract (esophagus and stomach), lower
gastrointestinal tract (colon and small intestine), skin, spleen, and bone
marrow. Treatment of U-87 intracranial gliomas with RCR vectors carrying the
yeast cytosine deaminase suicide gene followed by 5-fluorocytosine prodrug
administration resulted in 100% survival over a 60-day follow-up period,
compared with 0% survival of control groups receiving vector alone or prodrug
alone. Our results demonstrate that RCR vectors can achieve therapeutically
significant levels of transduction in malignant human gliomas, and that RCR
vector spread after intratumoral injection is restricted to the tumor
itself.
