Bohn Laboratory :: Bohn Laboratory

The Bohn lab is generally interested in the application of developmental mechanisms to neurodegenerative diseases, particularly Parkinson's disease, amyotrophic lateral sclerosis and brain tumors. The present focus is on delivery of genes to the nervous system that provide increased expression of neurotrophic factors and/or gene constructs that interfere with or promote cell death in the nervous system. Studies are also ongoing to determine the effects in neurons of increased expression of a foreign gene and to optimize methods for gene delivery to specific neurons.

GDNF (glial cell line-derived neurotrophic factor)
Several years ago we discovered that glial cells in the embryonic brain stem secrete factors that promote the survival and differentiation of dopamine neurons. One of these factors turned out to be a novel neurotrophic factor, glial cell line-derived neurotrophic factor (GDNF) and led to the identification of a new family of neurotrophic factors. GDNF a potent factor not only for dopamine neurons, but also motor neurons, cholinergic neurons and several other types of neurons, suggesting that GDNF has therapeutic potential for several neurodegenerative diseases, such as Parkinson's, Lou Gehrig's and Alzheimer' diseases. In animals, numerous laboratories around the world have shown that injection of recombinant GDNF protein into the nervous system improves the survival of specific types of neurons that are normally killed by neurotoxins or physical lesions. While supporting the potential use of GDNF in the clinic, there is at present no method for delivering GDNF, or any neurotrophic factor protein for that matter, to the central nervous system (CNS) in a manner that is safe, non-invasive and which results in chronically increased levels of biologically active protein. To address this issue, we have been investigating ex vivo and in vivo gene transfer methods for delivering neurotrophic factor genes of the CNS in rats, mice, and more recently, non-human primates.

Gene therapy using viral vectors.
Gene therapy is a field that is its infancy and the development of viral vectors that are able to deliver genes to specific tissues and result in long-term transgene expression is an active area of research. Gene transfer to the CNS presents unique challenges due to the relative inaccessibility of the brain and spinal cord, as well as the incredible cellular complexity of the CNS. On the other hand, the goal of being able to target the expression of a therapeutic gene to a specific group or type of cell type in the CNS is one that has the potential of shaping a medical frontier in the treatment of diseases and injuries to the CNS, as well as genetic diseases and birth defects of the CNS. Consequently, our lab is making viral vectors designed to express transgenes in specific cell types in the nervous system. In addition, we have developed quantitative methods for assessing the efficacy of viral vectors in the CNS so that as new generation vectors become available, they can be systematically evaluated. These methods involve anatomical morphometry, ELISA and quantitative polymerase chain reaction (PCR).

Gene therapy using viral vectors in rat models of Parkinson's disease.
Our initial gene therapy studies focused on one class of viral vectors, adenoviral vectors, to deliver a therapeutic gene, GDNF, and control genes, ß-galactosidase and an inactive mutant GDNF, to the rat brain. Following injection of these vectors into the rat brain, sustained levels of transgene expression were observed over a period of two months, the longest time studied. The levels of GDNF that could be delivered in this manner were found to be in the nanogram range, a level that is biologically active and about an order magnitude higher than the affinity of the GDNF receptors in the CNS. Effects of these vectors have been studied in a rat model of Parkinson's disease in which the dopamine neurons were chemically lesioned with the neurotoxin 6-hydroxydopamine (6-OHDA). This rat model also permits quantitative assessment of those dopamine neurons (in the substantia nigra) that specifically projected their axons to the lesion site (in the striatum). Injection of the adenoviral vector harboring the GDNF gene, but not those vectors harboring the control genes, into the vicinity of the dopamine neuron cell bodies was found to protect these from 6-OHDA induced cell death. Only 10-20% of the lesioned dopamine neurons died in the GDNF treated rats, whereas an average of 70% of the dopamine neurons died in control groups. Recent studies have also shown that dopamine neurons can be rescued by GDNF gene delivery even after 6-OHDA induced damage has commenced. Other studies have shown that the effects of GDNF gene delivery on dopamine-dependent behaviors in this rat model of Parkinson's disease are brain region and time dependent. Recent experiments using in vivo microdialysis to study the effects of GDNF on extracellular dopamine levels showed that GDNF gene therapy also increases the availability of dopamine in the region of the brain important to control of movement, i.e. the striatum.

Goals for bringing GDNF gene therapy to the clinic.
The incredible degree of protection elicited by the GDNF adenoviral vector suggested that GDNF gene therapy in humans with Parkinson's disease could inhibit or at least delay the progression of the disease process. Bringing this concept to the clinic, however, will rely on several crucial advances. First, it needed to be demonstrated that vector delivery of GDNF to the terminals of the dopamine neurons would also be effective. We have recently completed studies in the rat demonstrating that injection of a GDNF adenoviral vector into the striatum near the dopamine terminals also protects neurons from 6-OHDA induced cell death. This was important since this area of the brain is surgically accessible for vector injection, whereas the injection of vectors into the brainstem where the dopamine neurons are located would be less safe in humans. Secondly, although we had demonstrated that the dopamine neurons had been protected from cell death, it also had to be shown that these neurons were functional. We have now shown in two behavioral tests of dopamine motor function that the rats that were treated with the GDNF adenoviral vector did not develop deficits in dopamine-dependent behavior, whereas the control rats developed asymmetry in dopamine-dependent motor tasks. Thirdly, we needed to study the neurochemical effects in the brain resulting from experimentally increasing GDNF, a potent factor that may affect a variety of neurotransmitter system and have shown that GDNF gene delivery can upregulate the dopamine system. Finally, we studied gene therapy approaches in the aged rat since Parkinson's disease is a disease of aging and the aged brain may respond differently to GDNF and viral vectors. These studies clearly showed that the effects of GDNF gene delivery are dependent on the brain region treated.
The GDNF gene therapy approach is now in the process of being translated to clinical trials. We have now switched to a different viral vector platform, AAV (adeno-associate virus). AAV is excellent for delivering genes to neurons and is a safe vector that is non-pathogenic in human. AAV is presently is use in several clinical trials in the human brain. Our lab has been working on putting regulatable promoters in AAV so that the expression of GDNF and other therapeutic genes can be turned off should high, prolonged expression of GDNF in the human brain result in unexpected untoward side effects. We recently reported on a tetracycline-regulated vector that can be turned off in rat brain by putting an analog of tetracycline in the rat's drinking water. We are now in the process of doing the final studies in rat and monkey that will be necessary to use these regulated vectors in clinical trials.

Gene therapy in monkey CNS.
The success of these gene therapy studies in rat have led us to gene therapy studies in the non-human primate brain. There is an excellent monkey model of Parkinson's disease that closely mimics clinical symptoms and is more likely to predict outcome of novel therapeutic approaches in human than testing in rat models of Parkinson's disease. In addition, testing gene transfer methods in the non-human primate brain represents the most complex arena for testing prior to undertaking clinical trials. Consequently, we have embarked on a collaboration with Dr. Krys Bankiewicz at UCSF to test our regulated AAV vectors in a chronic hemi-parkinsonian monkey model of Parkinson's disease. Studies are in planned to test AAV vectors with a number of therapeutic candidate genes for Parkinson's disease.

Small Interference RNA vectors and cell death genes.
A small percentage of cases of Parkinson's disease are linked to mutations in the gene alpha-synuclein. In addition, alpha-synuclein is known to aggregate in cells forming Lewy bodies. Lewy bodies are characteristic of both Parkinson's disease and other neurodegenerative diseases called synucleinopathies. Our lab has recently generated a viral vector that expresses a small inhibitory double stranded RNA in cells that completely turns off expression of alpha-synuclein. This vector is now being testing in experimental models of Parkinson's disease. In addition, we are exploring other genes that may be upregulated in endoplasmic reticulum (ER) stress induced neuronal cell death with the idea of using viral vectors to interfere directly with cell death pathways. Conversely, we are exploring the potential of gene therapy vectors to promote cell death in glioblastoma and/or to render the tumor cells more vulnerable to chemotherapy.

Selected References:

Choi-Lundberg, D.L., Lin, Q. , Chang, Y.-N., Chiang, Y.L., Hay, C.M., Mohajeri, H., Davidson, B. and Bohn, M.C. Dopaminergic neurons protected from degeneration by GDNF gene therapy. Science 275, 838-841 (1997).

Choi-Lundberg, D.L. and Bohn, M.C Applications of gene therapy for neurological diseases and injuries. In: Stem Cell Biology and Gene Therapy, Quesenberry, P.J., Stein, G.S., Forget, B. and Weissman, S. (Eds), J. Wiley & Sons, New York, pp. 503-553 (1998).

Bohn, M.C. A commentary on GDNF: From a glial secreted molecule to gene therapy. Biochem. Pharm. 57: 135-142 (1999).

Mohajeri, M.H., Figlewicz, D.A., and Bohn, M.C. Intramuscular grafts of myoblasts genetically modified to secrete glial cell line-derived neurotrophic factor (GDNF) prevent motoneuron loss and disease progression in a mouse model of familial amyotrophic lateral sclerosis. Human Gene Therapy 10: 1853-1866 (1999).

Connor, B., Kozlowski, D.A. , Schallert, T., Tillerson, J.L. , Davidson, B.L. and M.C. Bohn Differential effects of glial cell line-derived neurotrophic factor (GDNF) in the striatum and substantia nigra of the aged Parkinsonian rat. Gene Therapy 6: 1936-1951 (1999).

Kozlowski, D.A., Connor, B., Tillerson, J.L., Schallert, T., Bohn, M.C. Delivery of a GDNF gene into the substantia nigra after a progressive 6-OHDA lesion maintains functional nigrostriatal connections. Exper. Neurol. 166:1-15(2000).

Jiang, L., Rampalli, S., George, D., Press, C., Bremer, E.G., O’Gorman, M. R. G., and Bohn, M.C. Tight regulation of a single tet-off rAAV vector as demonstrated by flow cytometry and quantitative, real-time PCR. Gene Therapy 11: 1057-1067 (2004).

Kozlowski, D.A., Miljan, E. A., Bremer, E.G., Harrod, C.G., Gerin, C., Connor, B. George, D., Larson, B., and Bohn, M.C. “Quantitative analyses of GFR_-1 and GFR_-2 mRNAs and tyrosine hydroxylase (TH) protein in the nigrostriatal system reveals bilateral compensatory changes following unilateral 6-OHDA lesions”. Brain Research, 1016: 170-181 (2004).

Ebert, A.D. Chen, F., He, X., Cryns, V. and Bohn, M.C. A tetracycline-regulated adenovirus encoding dominant-negative caspase-9 is regulated in rat brain and protects against neurotoxin induced cell death in vitro, but not in vivo. Experimental Neurology (2004, in press).

Bohn, M.C. Motoneurons Crave Glial Cell Line-Derived Neurotrophic Factor (GDNF): A commentary. Experimental Neurology (2004, in press).

Yates, JW, Sapru, MK, and Bohn, MC. Silencing of CHOP Decreases the Sensitivity of Dopaminergic Neuronal cells to Parkinsonian Toxins. (2007, submitted)

Research Support:
NINDS, NIMH, the US Army Department of Defense, the Walden W. and Jean Y. Shaw Foundation, the Medical Research Institute Council and the Parkinson's Disease Foundation.