| Human disorders of cerebellar development often present as motor abnormalities affecting gait, posture and dexterity with concomitant cerebellar hypoplasia or degeneration. Recent evidence suggests that the cerebellum is an important component of cognitive and executive pathways as well. Disruption of these cerebellar functions can result in aberrations of learning, behavior and complex social skills, best illustrated by autism, fetal alcoholism and dyslexia. Unfortunately, the majority of these disorders are refractory to current treatments. However, the advent of gene therapy strategies offers great promise for the amelioration of many of these diseases. Recombinant feline immunodeficiency virus (FIV) was evaluated for its capacity to transduce Purkinje cells (PCs) during early mouse postnatal development. Efficient transduction of PCs was observed both in vivo and in primary cultures with a GFP-expressing, reporter FIV pseudotyped with the G protein of vesicular stomatitis virus. Significant granule cell transduction was also observed. To narrow this viral tropism, two alternative envelopes, p56 glycoprotein of Borna disease virus and a membrane/envelope complex from West Nile virus, were utilized to pseudotype recombinant FIV. Although positive transduction events were observed with both in several cell lines, additional modifications are required to achieve a useful efficiency. In a murine model of abrogated cerebellar development, the staggerer (sg) mouse, recombinant FIV encoding wild-type retinoic acid-related orphan receptor alpha (RORα), the underlying gene defect in sg mice, was generated to test whether the morphological and gene expression abnormalities of sg PCs could be rescued in vivo and in vitro. However, expression of RORα by a strong viral promoter in the packaging line inhibited virus production. Re-engineering the viral transfer vector to limit RORα expression with a PC-restricted, calbindin promoter restored virus production, but RORα expression could not be demonstrated in surrogate cell lines or functional assays. Substitution of RORα with the red fluor, DsRed, to track expression from the calbindin cassette revealed a host cell-dependent pattern of both leaky expression and transcriptional inhibition, poorly correlated with GFP-marked transduction in the complex enhancer environment of the recombinant genome. Additional vector modifications to eliminate extraneous enhancer elements or insulate the calbindin cassette must be tested. |
