AAV-mediated Gene Correction in a Mutant Transgenic LacZ Mouse

Abstract

Abstract: : Purpose: AAV may be used to introduce targeted insertions, deletions and substitutions into homologous chromosomal sequences in vitro. The present studies aim to correct a single point mutation in the lacZ gene to restore the enzymatic activity of beta-galactosidase in vivo, using an AAV-mediated gene-targeting approach and a transgenic mouse containing a single copy of the mutant lacZ gene. Methods: AAV2/5-lacZ-3082 (2.0X10^12p/ml) was prepared. This virus encodes 3082 bp of the lacZ gene and is devoid of a promoter and polyA tail. Subretinal (2X10^9 particles) or intramuscular (5X10^10 particles) injections were performed to administer virus unilaterally to mutant lacZ transgenic mice, age-matched non-transgenic littermates and C57BL/6 mice. The contralateral eye or leg of each animal received a saline injection and served as a control. Animals were sacrificed 21 days (subretinal injection) or 15 days (intramuscular injection) post-treatment. Control and experimental tissues were harvested, fixed in 4% paraformaldehyde, stained with X-gal, cryoprotected and embedded in tissue freezing medium. Sections (10 micron) were obtained and examined microscopically for the presence of blue staining. Results: There was no evidence of blue cell staining in control tissue. Notably we readily identified targeted cells in both muscle and retina. Blue muscle fibers were clearly visible on the surface of the treated muscle specimen even prior to tissue sectioning and light microscopy analysis. Within the retina, we were able to detect blue retinal pigment epithelial (RPE) cells at the site of injection, as well as small numbers of stained photoreceptor cells. Conclusions:Our preliminary data demonstrate that this method may be used to modify single-base pair mutations in muscle fibers, photoreceptors and RPE cells in vivo. These exciting results suggest that AAV-mediated gene correction may prove to be useful in permanently reversing genetic diseases that affect these tissues. Inherited diseases of the retina may be especially amenable to this treatment, as the retina is a small, isolated tissue with immune-privileged properties. Furthermore, many of the mutations responsible for inherited retinal disease have been identified, and a number of well-characterized animal models exist.

Keywords: gene transfer/gene therapy • retina