More than two thirds of patients with diabetes develop neuropathy, which generally affects the sensory neurons within the peripheral nervous system and is characterized by such symptoms as numbness, tingling, pins and needles or loss of sensation, most often to the legs and feet. As a result, patients may be unaware of the presence of ulcers or infections or that they've experienced injury. No treatments exist that can stop progression of neuropathy, let alone reverse any damage to nerves.
Five weeks after a one-time inoculation, diabetic animals receiving gene therapy had complete reversal of established peripheral neuropathy and restoration of lost nerve endings to their feet, reported James R. Goss, Ph.D., research assistant professor of molecular genetics and biochemistry at the University of Pittsburgh School of Medicine. He and colleagues had previously found the same gene therapy approach could prevent the development of neuropathy in diabetic animals.
While the exact cause of diabetic neuropathy is unknown, there is evidence to suggest it is associated with a deficiency or dysfunction of certain neurotrophic factors, which are proteins essential for the survival and proper function of neurons. Therefore, the Pitt researchers sought to develop a therapy that would shuttle the genes responsible for their production directly into affected neurons. To gain entry into the cells, the team designed vectors of inactivated herpes simplex virus (HSV), which normally infects sensory neurons. The HSV vectors were encoded with genes for one of two neurotrophin factors: nerve growth factor (NFG) and neurotrophin-3 (NT-3).
"Herpes simplex virus appears to be an ideally suited gene therapy vector for diabetic neuropathy, in part because very little of the virus is needed to get inside the cell. But in addition, an important advantage with this approach is that we can deliver the gene directly and solely to affected neurons, without bombarding the entire the nervous system," explained Dr. Goss.
In recent years, research conducted by other groups had suggested that large doses of NGF protein given systemically by injection could reverse neuropathy in diabetic rodents, but the approach failed to meet expectations in human trials. By giving NGF protein systemically, high doses are needed to reach the target sensory neurons. But given at low doses, there was no benefit, and at the site of injection, it caused pain that was not well tolerated by the patients in the study.
Sensory neurons throughout the body convey information about touch, pain, temperature and joint position to the spinal cord, which in turn transmits the information to the brain in order to register a particular sensation. As such, to deliver the HSV vectors encoded with the neurotrophic factor genes for NGF or NT-3, the researchers inoculated an area just under the skin, directly to sensory neurons. Once delivered, the HSV vectors travel up the nerve to the sensory neuron cell body located within a cluster of other neuron cell bodies called the dorsal root ganglia. Inside the neuron cell body, the encoded genes are used to make natural NGF or NT-3 proteins, thereby protecting the neurons within the dorsal root ganglia from damage.
In their study, the researchers induced diabetes in mice, which resulted in the development of peripheral neuropathy within six weeks. Once neuropathy was established, they treated two groups with gene therapy -- one received the vector encoded for NGF and a second group received the vector encoded with NT-3. To compare outcomes, another group of diabetic animals was treated with the HSV vector encoded for a gene with no therapeutic value, and a fourth group received no treatment. Five weeks after inoculation, the neuropathy was reevaluated using measurements of heat sensitivity and the ability of nerves in the feet to conduct electrical impulses. In diabetic animals treated with either the NGF or NT-3 vectors, signs of neuropathy had disappeared, whereas in the untreated diabetic animals and the diabetic animals treated with the "empty" vector, neuropathy was still present.
Results of another Pitt study from the same group, also presented today, found that genes introduced by HSV vectors were still expressed six months after single inoculation and could still protect against the development of neuropathy. That study looked at gene expression for NT-3 and two other neurotrophic factors.
"We are encouraged by these outcomes, but in order to bring this approach to a clinical trial we must first carry out toxicology studies with the HSV vector to support its highly safe design. The vector itself is highly defective and incapable of reproducing itself," noted Dr. Goss. "Most of us have been exposed to herpes simplex virus and we need to be concerned that in a patient with wild type virus there is no possibility of our vector recombining with wild type and creating a new infectious virus."
The University of Pittsburgh is unique in that it has the capability to create and manufacture clinical-grade virus vectors and is one of the only institutions developing and researching HSV-based vectors. Within the next few years, Pitt researchers, in collaboration with a team at the University of Michigan, will conduct a human gene therapy trial for the treatment of cancer pain that will employ an HSV vector. According to Dr. Goss, once an additional series of studies are conducted, he and his team may propose a clinical trial for the treatment of diabetic neuropathy.
In addition to Dr. Goss, other authors included on the abstract presented today are William F. Goins, Ph.D.; Darren Wolfe, Ph.D., and Joseph Glorioso III, Ph.D., all from the department of molecular genetics and biochemistry at the University of Pittsburgh School of Medicine; and Marina Mata, M.D., and David Fink, M.D., formerly of the University of Pittsburgh and now with the University of Michigan.
Their research was supported by the National Institutes of Health, the Veterans Affairs Administration and the Juvenile Diabetes Research Foundation International.