660. Quantitative Comparison of AAV Serotypes AAV2, AAV5, and AAVrh.10 Efficiency for CNS Gene Therapy Following Intracranial Gene Delivery
Authors: Dolan Sondhi, Daniel A. Peterson, Elizabeth Vassallo, Christine T. Sanders, Jamie A. Stratton, Neil R. Hackett, Guangping Gao, James M. Wilson, Ronald G. Crystal
Journal: Molecular Therapy (2005)

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Late infantile neuronal ceroid lipofuscinosis (Batten disease) is a fatal pediatric neurodegenerative disease resulting from mutation of the CLN2 gene encoding for a tripeptidyl peptidase (TPP-I). Deficiency of TPP-I results in aberrant degradation of membrane proteins in the lysosome with development of inclusion bodies which lead to neuronal cell death throughout the brain. We have previously shown that AAV2 based vectors expressing human CLN2 cDNA driven by the CMV-enhanced chicken-b-actin hybrid promoter can mediate long term expression of TPP-I in rat brain. In the present study, we have compared the ability of an AAV2-based vector to mediate distribution of TPP-I production in the rat CNS to that of recombinant AAV2-based vectors pseudotyped with capsids derived from human AAV5 and rh.10, a rhesus macaque derived AAV. Vectors (2.5|[times]|109 genome copies) were injected into the striatum of 8 wk old male F344 rats. Fluorometric enzyme assay for TPP-I in the injected region after 4 wk revealed that while AAV2- and AAV5-mediated gene delivery produced TPP-I enzyme levels that were 1.2 and 1.8 fold of endogenous (p<0.01 compared to naive) respectively, AAVrh.10-mediated delivery provided TPP-I levels that approached 17-fold above endogenous levels (p<0.01 compared to either naive or AAV5-treated animals). Immunohistochemical staining showed vector-derived TPP-I in the striatum of rats injected with each of the three vectors, with AAV5 and AAVrh.10 producing the greatest striatal filling. Volumetric analysis showed that while infection by AAV2-derived vector lead to TPP-I detection in 5.1|[plusmn]|0.4% of the striatum, AAV5 and AAVrh.10 resulted in TPP-I expression in 46.9|[plusmn]|2.9% and 51.2|[plusmn]|6.1% of the striatum respectively. Co-immunofluorescence studies demonstrated that virtually all neurons in the region of AAVrh.10 injection were TPP-I positive but no glia stained TPP-I positive. When the whole brain was examined, AAVrh.10 mediated more extensive TPP-I expression, with TPP-I being detected in the globus pallidus and basal forebrain. There was also prominent substantia nigra staining at 4 wk with AAVrh.10 delivery, suggesting retrograde distribution. In addition TPP-I positive cells were readily detected in the frontal cortex and the thalamus, both of which project to the location of the injection in the striatum. When non-striatal staining was quantitated, AAV2 administration resulted in TPP-I positive cells in 0.4|[plusmn]|0.1% of the hemisphere compared to 4.8|[plusmn]|0.6% for AAV5 and 8.6|[plusmn]|1.4% for AAVrh.10. The data show that capsids from other AAV serotypes give more widespread and higher levels of gene transfer than AAV2 vectors and suggests that high transgene expression levels may correlate with a wider distribution of transgene product. In particular, AAV vectors pseudotyped with the capsid from AAVrh.10 may be applicable to gene therapy for diffuse neurological diseases.