For Researchers > Basic/Pre-Clinical Research > Movement Disorders > Molecular Chaperone Gene Therapy for Polyglutamine Diseases
Molecular Chaperone Gene Therapy for Polyglutamine Diseases
Diane E. Merry, Ph.D., Lead Investigator
Our aim in this program is to develop gene therapeutic approaches to deliver the molecular chaperones Hsp70 and Hsp40 to the nervous system (primarily spinal motor neurons) of mice. Our finding that the molecular chaperones Hsp70 and Hsp40 promote the degradation of the mutant expanded polyglutamine androgen receptor suggests that increasing expression of these proteins through pharmacologic or gene therapy approaches would be of benefit to patients with SBMA and other polyglutamine diseases. Indeed, delivering these genes to the appropriate brain region may be beneficial for any disease marked by abnormal protein accumulation. For our first experiments, we are developing a lentiviral delivery system to deliver and direct the expression of Hsp70 and Hsp40 to motor neurons.
We had previously created a bicistronic construct with Hsp40 and Hsp70 driven by the cytomegalovirus (CMV) promoter. We are creating lentiviral constructs containing this bicistronic cassette driven by the CMV promoter, the PGK promoter, and the PrP promoter. Expression levels will be measured in our motor neuron cultures to determine the optimal promoter prior to treatment of animals.
Lentiviral vectors based on equine infectious anemia virus (EIAV) have been shown to provide efficient and long-term expression (greater than 5 months) in the nervous system. Recent developments suggest that intramuscular delivery of gene therapeutics would provide efficient delivery and expression of molecular chaperones to the spinal cord. For example, researchers have found that including rabies virus glycoprotein pseudotyping in lentiviral vectors allows the efficient retrograde transport of viral particles to spinal motor neurons and beyond. Retrograde transport of adenoassociated virus delivery IGF-1 to motor neurons has also recently been demonstrated in a SOD1 mouse model of ALS, providing proof of principle (albeit using a different viral vector) for this approach. Lentiviral vectors and packaging cell lines are commercially available, and we are collaborating with Dr. Nicholas Mazarakis at Oxford Biomedica Ltd. (UK), on the rabies pseudotyping of our packaged lentivirus.
Our collaborator's recent paper, entitled “VEGF delivery with retrogradely transported lentivector prolongs survival in a mouse ALS model” Nature 429:413, May 27, 2004, suggests that this approach is feasible.
Optimizing the Vector
We confirmed expression of Hsp40 in Cos-7 cells, and while we observed low levels of Hsp70 expression, the levels of Hsp70 were far lower than those we previously showed to be effective in promoting mutant AR turnover. Through a
number of experiments, we have concluded that the low level of Hsp70 expression is likely due to an inadequate Kozak consensus translation start site present in the Hsp70 sequence. While this suboptimal consensus sequence does not pose a problem when the gene is expressed by itself, it dooms the expression of Hsp70 downstream of the IRES (internal ribosome entry sequence). Thus, we are in the process of mutagenizing this sequence to an ideal Kozak consensus, using Stratagene QuikChange mutagenesis kit. We are creating several mutants to test the effects of both distance from the IRES as well as Kozak consensus in the expression of Hsp70.
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