Functional correction of established central nervous system deficits in an animal model of lysosomal storage disease with feline immunodeficiency virus-based vectors

Abstract

Gene transfer vectors based on lentiviruses can transduce terminally differentiated cells in the brain; however, their ability to reverse established behavioral deficits in animal models of neurodegeneration has not previously been tested. When recombinant feline immunodeficiency virus (FIV)-based vectors expressing beta-glucuronidase were unilaterally injected into the striatum of adult beta-glucuronidase deficient [mucopolysaccharidosis type VII (MPS VII)] mice, an animal model of lysosomal storage disease, there was bihemispheric correction of the characteristic cellular pathology. Moreover, after the injection of FIV-based vectors expressing beta-glucuronidase into brains of beta-glucuronidase-deficient mice with established impairments in spatial learning and memory, there was dramatic recovery of behavioral function. Cognitive improvement resulting from expression of beta-glucuronidase was associated with alteration in expression of genes associated with neuronal plasticity. These data suggest that enzyme replacement to the MPS VII central nervous system goes beyond restoration of beta-glucuronidase activity in the lysosome, and imparts improvements in plasticity and spatial learning.

Figure 1

FIV accessory proteins are not required for transduction of rodent CNS. FIV vectors encoding β-galactosidase containing both or neither of the Vif and Orf2 accessory proteins were generated as described (Materials and Methods) and injected into mice striata. (A) FIV packaging, vector, and envelope constructs. (B) Photomicrograph of a representative section stained for β-galactosidase activity. Mice were injected with FIVβgalΔvifΔorf2 18 weeks earlier. (C) The volume of β-galactosidase expression in FIV-injected hemispheres. (D) A representative confocal photomicrograph of the injected striatum after immunohistochemical staining for β-galactosidase (green) and NeuN (red) antigens. Cell soma colabeled for β-galactosidase and NeuN appear yellow in this merged image. (E) Occasional transduced glia could be identified in sections stained for glial fibrillary acid protein (GFAP, red) and β-galactosidase (green; arrowhead).

Figure 2

β-Glucuronidase expression after FIV-mediated gene transfer. (A) Transgene-positive cells near the region of the injection as revealed by in situ RNA analyses. (B) β-Glucuronidase activity in the brain of a MPS VII mouse injected with FIVβgluc and stained for β-glucuronidase activity (dark red reaction product). (C, E, and G) Representative examples of the lysosomal storage in the striatum (C), cortex (E) and hippocampus (G) of 8- to 12-week-old MPS VII mice. (D, F, and H) Correction of the storage defect in the contralateral striatum (D), cortex (F) and hippocampus (H) 6 weeks after injection of FIVβgluc into an 8-week-old MPS VII mouse.

Figure 3

Behavioral analyses in untreated and FIV-transduced mice. RAPC analysis was used to investigate baseline and posttreatment differences between MPS VII mice and age-matched heterozygous controls. (A) Behavioral sessions before and after gene transfer were performed according to the time line (Materials and Methods). MPS VII mice demonstrated significant baseline impairment in learning (solid red squares) relative to control mice (solid gray circles) as seen by both measures (latency in B and errors in C). There were no significant differences in the performance component of the assay between MPS VII (open red squares) and control (open gray circles) mice. After baseline testing, both groups were segregated randomly before bilateral striatal injection with FIVβgal or FIVβgluc. Behavioral tests on FIVβgal-treated (D and E) and FIVβgluc-treated (F and G) mice resumed 4 weeks after injection. FIVβgal-injected MPS VII mice continued to demonstrate a severe learning impairment in both latency and error (D and E). MPS VII mice injected with FIVβgluc exhibited no significant differences in learning compared with control mice in error or latency measures (F and G; P ≥ 0.05), and reflect a recovery of the learning impairment seen in the baseline measurements (B and C). Solid symbols, learning component. Open symbols, performance component.

Figure 4

Microarray analysis of murine hippocampus after treatment with FIV vectors. Hippocampi from FIVMCS- and FIVβgluc-treated animals were extracted 8 weeks after injection, and gene expression was analyzed on Affymetrix high-density oligonucleotide arrays. (A) A representation of the effects of FIVβgluc gene transfer on gene expression in the hippocampus. (B) A scatter plot depicts the average difference distribution of all genes examined, comparing FIVMCS with FIVβgluc samples. Red points depict genes that are called present, whereas blue points represent genes changing from absent to present or vice versa. Green lines indicate the magnitude of change with intervals of 2, 10, and 30-fold relative to baseline. One gene of interest, Pitpn (U96726), is circled in orange (arrow) and exhibits a significant change in gene expression as depicted by differences in the raw data images (C). (C) The increase in selective binding to the perfect match (pm) vs. the mismatch (mm) probes. (D) The increase or decrease in fold expression of RNA specific to selected genes from FIVβgluc relative to FIVMCS-treated mice. Data normalization and analyses were completed by using algorithms in the Affymetrix Microrarray Analysis suite and Data Mining Tools and significance analysis of microarrays (27).