Converging Pathways of Pain Research at NIDCR

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Pain: Study It or Treat It?

This work began in the summer of 1993 with a small program in therapeutics"small because I was able to carve out only limited time over two summers with an HHMI high school student, Susan Lee (who has since gone on to Harvard Medical School in Boston). I had always been a basic bench scientist, and my lab had been studying synaptic-induced gene regulation in the spinal cord in models of persistent peripheral inflammation. I had discovered that persistent pain up-regulates the opioid peptide dynorphin in the dorsal spinal cord, the first synaptic processing station for pain"first observing this with a radioimmunoassay for dynorphin peptide and later measuring the corresponding mRNA increases, performing studies to localize the spinal neurons involved, and eventually examining seven base pairs of enhancer sequence in the promoter.

The transition to translational research was sparked through our weekly laboratory meetings. What was then the Neurobiology and Anesthesiology Branch contained both basic and clinical research groups, and the clinical group sometimes presented patients with chronic pain problems. This was my first exposure to patients with chronic neuropathic pain disorders, and it was a real eye-opener. Chronic neuropathic pain is notoriously difficult to control with currently available drugs and procedures, and the subjects we were seeing exemplified this clinical state of the art. Often, what had begun as relatively minor nerve damage after a traumatic injury progressed to a severe chronic pain disorder. Patients experienced high levels of spontaneous pain and mechanical allodynia (pain from a normally nonpainful stimulus). Just brushing the skin in the neuropathic zone was enough to cause them excruciating pain. This exposure stimulated us to begin exploring new treatments for pain, in addition to studying the molecular neurobiology of pain.

First Steps

In choosing among treatment approaches, we wanted to do something new and to use some of the molecular methods that we had expertise in and control over within our own lab. At the time, we were performing transient transfections to investigate those seven base pairs in the dynorphin promoter. Moreover, there was real excitement over the beginnings of gene therapy, much of which was occurring here on the NIH campus. So the idea of adapting techniques of gene transfer to pain treatment seemed like a natural extension of our current program. Still uncertain as to the exact gene to use in pain treatment, we nonetheless needed to assess the basic process of in vivo gene transfer.

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Nothing worked very well either in vivo or in the primary culture test system, where the glial cells acted as incredible sponges for DNA, no matter what form it was in or what coating was around it. We needed to look elsewhere.

Virus to the Rescue

We turned our attention to virus. Fortunately, my institute had already established a program in gene therapy run by Brian O'Connell, who helped us get started by providing virus reagents and guidance in filing the necessary paperwork with the Biosafety Committee. We were also lucky to have anesthesiologist Drew Mannes join the group, through a joint agreement with the University of Pennsylvania Anesthesiology Department in Philadelphia. For Mannes, the highest priority for research was that it have clinical relevance.

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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.

Evolution of the Paracrine Paradigm

At first we reasoned that we would have to break through the pia for virus to gain access to the spinal cord. There ensued a series of increasingly invasive manipulations, starting with hyperosmotic mannitol shock and proceeding to partial enzymatic digestion with intrathecally applied proteases.

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This proved to be a difficult job for adenovirus, and we slowly came to the conclusion that through no fault in technique, only motor neurons were appropriate targets for adenovirus; the others were apparently impervious to it. Furthermore, we observed that the spread of the virus in the cord was nonuniform. It is an underappreciated fact that the nervous system contains many barriers to free diffusion or dispersal of large viral particles (~90 nM for adenovirus). When the virus encounters axon bundles, it tends to track along the bundle rather than diffuse through it. Tightly packed cells are another barrier. Aside from these physical issues, the cord appears to unevenly express the receptors for adenovirus binding or attachment (CAR) and internalization (integrins). (We are now investigating receptor sites and targeting strategies.)

Preclinical Testing in Vivo

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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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.

Implications and Further Steps

Our studies have delineated a direct in vivo approach for treating pain using gene therapy techniques. Finegold coined the term "paracrine paradigm," because the therapeutic gene product is secreted by cells in the vicinity of the relevant neurons.

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