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Our data demonstrate that vectors derived from recombinant feline immunodeficiency virus (rFIV) and adeno-associated virus type 5 (rAAV5) transduce cerebellar cells following direct injection into the cerebellar lobules of mice. Both recombinant viruses mediated gene transfer predominantly to neurons, with up to 2500 and 1500 Purkinje cells transduced for rAAV5 or rFIV-based vectors, respectively. The vectors also transduced stellate, basket and Golgi neurons, with occasional transduction of granule cells and deep cerebellar nuclei. rAAV5 also spread outside the cerebellum to the inferior colliculus and ventricular epithelium, while rFIV demonstrated the ability to undergo retrograde transport to the physically close lateral vestibular nuclei. Thus, AAV5 and FIV-based vectors show promise for targeting neurons affected in the hereditary spinocerebellar ataxias. These vectors could be important tools for unraveling the pathophysiology of these disorders, or in testing factors which may promote neuronal survival.

Orthograde labeling and immunocytochemical techniques were used to study the postnatal spatial and temporal development of corticospinal projections in cats. Findings from the orthograde labeling studies indicate that there are three major phases in the spatial development of corticospinal projections: an early period (1-10 postnatal days) when cortical axons grow into the spinal gray from the white matter; an intermediate period (2-5 postnatal weeks) where corticospinal axons develop terminal arborizations in a rostral to caudal, medial to lateral and intermediate gray to dorsal and ventral horn sequence; and, a late period (6-7 postnatal weeks) during which some corticospinal projections are eliminated. The time period over which cortical axons grow into the spinal cord was determined immunocytochemically using a monoclonal antibody against a microtubule associated protein (MAP 1B) present in growing axons. The corticospinal tracts were strongly immunoreactive for MAP 1B during the first three postnatal weeks. MAP 1B immunostaining of these tracts started to decline in the fourth postnatal week and was completely absent by five weeks of age. These findings indicate that the postnatal development of corticospinal projections is spatially and temporally protracted in cats.

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Classical late-infantile neuronal ceroid lipofuscinosis (LINCL) is caused by mutations in tripeptidyl peptidase I (TPP-I), a pepstatin-insensitive lysosomal protease, resulting in neurodegeneration, acute seizures, visual and motor dysfunction. In vitro studies suggest that TPP-I is secreted from cells and subsequently taken up by neighboring cells, similar to other lysosomal enzymes. As such, TPP-I is an attractive candidate for enzyme replacement or gene therapy. In the present studies, we examined the feasibility of gene transfer into mouse brain using recombinant adenovirus (Ad), feline immunodeficiency virus (FIV) and adeno-associated virus (AAV) vectors expressing TPP-I, after single injections into the striatum or cerebellum. A dual TPP-I-and b-galactosidase-expressing adenovirus vector (AdTTP-I/nlsbgal) was used to distinguish transduced (b-galactosidase positive) cells from cells that endocytosed secreted TTP-I. Ten days after striatal injection of AdTTP-I/nlsbgal, b-galactosidasepositive cells were concentrated around the injection site, corpus callosum, ependyma and choroid plexus. In cerebellar injections, b-galactosidase expression was confined to the region of injection and in isolated neurons of the brainstem. Immunohistochemistry for TPP-I expression showed that TPP-I extended beyond areas of b-galactosidase activity. Immunohistochemistry for TTP-I after FIVTTP-I and AAV5TTP-I injections demonstrated TPP-I in neurons of the striatum, hippocampus and Purkinje cells. For all three vectors, TPP-I activity in brain homogenates was 3-7-fold higher than endogenous levels in the injected hemispheres. Our results indicate the feasibility of vector-mediated gene transfer of TPP-I to the CNS as a potential therapy for LINCL.