Creating a Gene Therapy For Chronic Pain and Spinal Cord Disorders

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We began with a straightforward comparison in rats of viral transduction after infusions into the intrathecal space (the CSF space around the spinal cord) or infusions directly into the cord tissue itself (intraparenchymal) (1). Adenovirus was a vast improvement over plasmid: We achieved nearly 60-fold increases over baseline in β-galactosidase activity upon intraparenchymal injections. We injected directly into the ventral horn to provide a good seal around the cannula tract"and were nearly instantly gratified by transduction of the motor neurons, which turned blue in a matter of minutes. The motor neurons filled up, from the dendritic tree all the way out to the axons in the ventral roots. I thought we had solved the problem of neuronal gene transfer! At the very least, we had one vector we could use for intraparenchymal injection.

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In the meantime, the viral stocks of the βÆendorphin-secreting virus were delivered. We sent some to Mannes in Philadelphia. He infected cells and reported back that the media contained very high concentrations of β-endorphin! Finegold began investigating this in vivo. First, we injected the virus into the lateral ventricle in the brain. This was convenient to examine because CSF could be withdrawn readily from the cisterna magna, which is spatially remote from the ventricular injection site. Andrea Mastrangli's group in the NHLBI Pulmonary Branch had shown that β1-antitrypsin could be secreted by an adenovirus injected intraventricularly and that the virus entered the ependymal cells lining the ventricle but did not enter the brain tissue proper (2)2. This is exactly what we observed as well with co-injection of the β-endorphinÆsecreting virus and a β-galactosidaseÆexpressing virus. Significant β-endorphin secretion could be measured within 24 hours and reached concentrations more than 10-fold greater than the basal peptide content.

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In the summer of 1998, we were joined by two students, Jamie Bourque, from the University of Virginia in Charlottesville, and Brian Schulman, an HHMI summer student from the University of Pennsylvania. These two individuals pushed the behavioral aspects of the project to conclusion. They, too, demonstrated the basic antihyperalgesic action of the β-endorphinÆexpressing virus. They also demonstrated the reversal of this effect by the broad-spectrum opioid antagonist naloxone, indicating that the effect was opioid mediated. These results are in press (3).

We are now using different viruses to increase the longevity of expression and designing cassettes for regulated control. We hope to be testing the adenovirus in chronic pain patients in the near future. Exactly when will depend on how the toxicology results turn out.

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