STEREOTAXIC GENE THERAPY TO THE RHESUS CNS

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The central nervous system (CNS) is a primary target for gene therapy because the broad spectrum of inherited and acquired disorders that affect the brain. Neurodegenerative diseases such as Parkinson's disease, Alzheimer's disease, and amyotrophic lateral sclerosis are prime targets for gene therapy. Although rare, inherited lysosomal storage disorders and leukodystrophies, such as mucopolysaccharidosis and Krabbe's disease, are also key targets for gene therapy. Treatment options for most CNS disorders are limited to supportive care, thus providing few therapeutic options. Direct CNS gene transfer may be the most effective option for eliminating the disease and dysfunction. The goal of these studies was the direct comparison of the transduction of adeno-associated virus (AAV) vector systems with regard to levels of and pattern of gene transfer in the CNS of rhesus macaques. The transduction patterns of the various vectors were mapped by determination of the levels of gene transfer to selected regions of the brain and delineating the cell types transduced by immunohistochemistry and microscopy. We compared the ability of six AAV serotypes of recombinant AAV vectors (AAV2/1, AAV2 genome and AAV1 capsid, AAV2/2 and AAV2/7, AAV2/8, AAV2/9 and AAV2/rh10) in the CNS of adult rhesus monkeys (3-6 years old) by sterotaxic-guided injection. Rhesus macaques underwent injections of PBS, or an AAV serotype into individual hemispheres of the brain. All animals were necropsied four weeks after surgery. The injection of all of the AAV serotypes resulted in detectable EGFP activity by direct fluorescent confocal microscopic analysis. A high frequency of EGFP positive cells were detected immediately surrounding the injection tract and in both the white and gray matter. EGFP positive cells were readily detected in the following structures: caudate nucleus, hippocampus, putamen, substantia nigra, globus pallidus, amygdala, crus cerebri, and spinocerebellar tract for all of the vectors tested. Once EGFP positive cells were detected, immunohistochemistry was performed using several neuronal and glial cell antibodies, including GFAP, S100, IBA-1, NeuN, Map2, and NSE. EGFP-positive cells were found to colocalize with all of the tested antigens in various structures throughout the CNS. All of the vector serotypes, except AAV2/8, produced significant levels of EGFP positive cells in the white matter. Cells expressing EGFP were detected as far as 4-6 mm from the injection tract. The transfer of vector to the contralateral hemisphere revealed EGFP protein positive axonal bodies, but no detectable vector transduced cells. There was no evidence of inflammation or degeneration related to the surgical procedures or injection of any of the vectors in the brain or spinal cord. The results of the proposed translational studies will serve as invaluable information on the design and execution of CNS-directed gene therapy protocols in humans, especially children.