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The primary cultures worked up to a point: The lacZ test gene expressed ?β-galactosidase only in the "feeder layer" of flattened glial cells at the bottom of the plate. The neurons, which in these cultures are like groups of round balls sitting atop the flat glia, never seemed to pick up and express the plasmid. In vivo, plasmid transfer was weak, and the amount of measurable transgene expression was dismal. We could assay a small increase in ?β-galactosidase activity biochemically but could not see the cells with histochemical methods. We tried direct injections of plasmid into tissue and even prolonged infusions (for a week, using an osmotic mini-pump) of about 10 billion plasmid molecules into the cerebrospinal fluid (CSF) space surrounding the spinal cord. We played around with ligand-derivatized polylysines and the timing of plasmid incubations, among other tacks.

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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.

Viral infusion into the CSF, however, was not so happy"there was almost no expression in the spinal cord tissue. We found that the pia mater, one of the meningeal layers surrounding the spinal cord, was a very effective barrier to viral entry from the CSF space into the cord tissue proper. We could literally strip off the pia and stain it histochemically for ?β-galactosidase activity. The pial covering turned blue, but the spinal cord just underneath was devoid of reaction product.

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As luck would have it, we found an ideal gene cassette in the literature. Earlier studies had explored cell transplantation therapy as a potential means of treating pain"using either human cadaver or bovine xenografts of adrenal chromaffin cells (a rich source of enkephalin opioid peptides) or cells that had been engineered to secrete enkephalin. In the latter case, Rusty Gage's group at the Salk Institute in La Jolla, California, had constructed a cassette that allowed fibroblasts to secrete the powerful endogenous opioid ?β-endorphin. They had fused human ?β-endorphin at the COOH-terminus of the leader sequence of nerve growth factor (NGF) to direct the secretion of ?β-endorphin to the nonvesicular secretory pathway.

The idea was to stably incorporate the NGFÆ ?β-endorphin cassette into fibroblasts through a retroviral transduction, isolate secreting fibroblast clones, expand the cells, and transplant them into the intrathecal space"a somatic cell gene therapy approach. Gage's group had already characterized the ability of the construct to secrete authentic ?β-endorphin from cultured fibroblasts but had not used the system in vivo before they dropped this line of research. While the somatic cellÆfibroblast approach seemed cumbersome, the cassette itself seemed tailor-made for the connective tissue cells of the pia.

Thus, we were able to simplify the procedure considerably by using direct gene transfer. Fortunately, Gage was able to dig the plasmid out of the freezer and send it to us for subcloning into adenovirus. At this time, Mannes' NIH fellowship ended, and a new postdoctoral fellow, Alan Finegold, joined the group and began making several adenovirus shuttle vectors containing the NGFÆ ?β-endorphin cassette and several other sense and antisense constructs.

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In the meantime, the viral stocks of the ?ÆendorphinβÆendorphin-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 ?1β1-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.

In the spring of 1998, Finegold began rat studies involving intrathecal injections of the virus in conjunction with the application of hot radiant thermal stimuli to the hindpaw. In this test, the rat is unrestrained and can terminate the trial at any time by twitching the paw away from the heat source"a radiant heat lamp with an attached timer. Interestingly, Sprague Dawley rats never cue to the light coming on or the warming phase of the stimulus. However, once the temperature becomes hot, the rat flicks its paw away, which automatically stops the clock and terminates the power to the lamp. Thus, we can obtain an objective measure of nociceptive sensitivity in an unrestrained rat by recording the latency for paw withdrawal. In addition, we can perturb the system by making it hyper-responsive, using an inflammation in one hind paw. Because the inputs to the cord are lateralized, one paw can be used to assess hyperalgesic responses and the other paw of the same animal can be used to assess normal nociceptive responses. Recently, we have used this test to discriminate between different types of pain-reducing drugs. Rob Caudle in our lab has shown that some drugs such as a ?β-opiateÆselective ligand are analgesic and increase the withdrawal latency in both the inflamed and noninflamed paw. Rob has shown that other compounds have a "pain stateÆdependent effect," increasing the latency of the inflamed paw only. Certain types of K-opioid agonists (K2 agonists) and blockers of the N-methyl D-aspartate glutamate receptor exhibit this property, which we term antihyperalgesic. Several days after the intrathecal injection of the ?β-endorphinÆsecreting adenovirus, we produced a unilateral inflammation and tested the rat's thermal nociceptive responses.

The virus produced an antihyperalgesic effect when the inflamed paw was tested but no effect when the noninflamed paw was examined. Injections of a ?β-galactosidaseÆexpressing adenovirus did not influence the inflammation-induced hyperalgesia.

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).

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This approach represents a new way to deliver peptides to the nervous system. One can imagine a host of new avenues to peptide pharmacology when a "genetic generator" for peptide production is deposited in or near the target tissue. One of the strengths of this approach, therefore, is its versatility"all 20 amino acids are at one's commands command. It also bypasses one of the major stumbling blocks to using peptides as drugs"delivery. Working with the spinal cord makes the paracrine approach easy. The spinal subarachnoid CSF space is readily accessible by lumbar puncture, a common medical procedure, and injections by lumbar puncture may eventually suffice to place the viral vector into the pia.

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