Preferred transduction with AAV8 and AAV9 via thalamic administration in the MPS IIIB model: A comparison of four rAAV serotypes

Abstract

Sanfilippo syndrome type B (MPS IIIB) is a lysosomal storage disease caused by a deficiency of N-acetyl-glucosaminidase (NAGLU) activity. Since early therapeutic intervention is likely to yield the most efficacious results, we sought to determine the possible therapeutic utility of rAAV in early gene therapy based interventions. Currently, the application of recombinant adeno-associated virus (AAV) vectors is one of the most widely used gene transfer systems, and represents a promising approach in the treatment of MPS IIIB. From a translational standpoint, a minimally invasive, yet highly efficient method of vector administration is ideal. The thalamus is thought to be the switchboard for signal relay in the central nervous system (CNS) and therefore represents an attractive target. To identify an optimal AAV vector for early therapeutic intervention, and establish whether thalamic administration represents a feasible therapeutic approach, we performed a comprehensive assessment of transduction and biodistribution profiles of four green fluorescent protein (GFP) bearing rAAV serotypes, -5, -8, -9 and -rh10, administered bilaterally into the thalamus. Of the four serotypes compared, AAV8 and -9 proved superior to AAV5 and -rh10 both in biodistribution and transduction efficiency profiles. Genotype differences in transduction efficiency and biodistribution patterns were also observed. Importantly, we conclude that AAV8 and to a lesser extent, AAV9 represent preferable candidates for early gene therapy based intervention in the treatment of MPS IIIB. We also highlight the feasibility of thalamic rAAV administration, and conclude that this method results in moderate rAAV biodistribution with limited treatment capacity, thus suggesting a need for alternate methods of vector delivery.

Keywords: AAV; ANOVA, analysis of variance; Adeno-associated virus; BBB, blood brain barrier; CNS, central nervous system; GFAP, glial fibrillary acidic protein; Gene therapy; Lysosomal storage disease; MPS IIIB; MPS IIIB, mucopolysaccharidosis type III B; Mucopolysaccharidosis type III B; NeuN, neuronal nuclear protein; hGFP, humanized green fluorescent protein; rAAV, recombinant adeno-associated virus.

Fig. 1

Regional differences in rAAV biodistribution are observed. (A) Representative images showing thalamic connections within the brain (A). Sites of rAAV vector administration into thalamus (white arrows), and predicted rAAV spread within the CNS using 2% Evans Blue dye are shown (B). Representative tissue sections of interest (C) were analyzed for GFP expression in the cortex, hippocampus, thalamus and cerebellum of three month old MPS IIIB animals after rAAV administration. Scale bar: hippocampus (8 ×), 300 μm; and cortex, thalamus and hippocampus (20 ×), 100 μm.

Fig. 2

rAAVs exhibit differential biodistributions which may be genotype specific. Mid-sagittal brain sections of Control and MPS IIIB 3 month old littermates were assessed for the presence of AAV–GFP (A, dark areas of grayscale images, scale bar = 2 mm). Inset images depict robust staining of ependymal cells by AAV5. The percentage of regional GFP positive area was then quantitated in the cortex, hippocampus, thalamus and cerebellum (B). All sagittal sections were scanned at 20 × magnification and GFP expression was quantified in indicated regions using Aperio ImageScope algorithm. Two-way ANOVA was used to assess differences within region based on serotype, n = 3–4/cohort, *p < 0.05; **p < 0.01, and ^#p < 0.001. Data represented as mean ± SEM.

Fig. 3

AAV serotype dependent general CNS biodistribution. Three months after rAAV administration, brains are sectioned sagittally (A) into four relatively equidistant sections (B) and collected to assess tissue biodistribution following thalamic infusion. Each tissue section in the Control (C) and MPS IIIB (D) treated groups was analyzed for the presence and percentage of GFP positive area. Two-way ANOVA was used to assess differences within tissue section based on serotype and one-way ANOVA was used to test individual differences between serotype, n = 3–4/cohort, *p < 0.05; **p < 0.01, ^#p < 0.001, ^$p < 0.0001. Data represented as mean ± SEM.

Fig. 4

Quantitative PCR for vector genomes in the brain. The right sagittal half of brains from 3 month old control and MPS IIIB mice were dissected into three parts: front, middle and back as shown. The total copies of vector DNA/μg gDNA detected in the CNS of AAV5, -8, -9 and -rh10 treated animals are shown. The positive cutoff limit, ≥ 100 vg/μg gDNA, is presented by the horizontal trend line. gDNA, genomic DNA. n = 3/cohort, *p < 0.05; **p < 0.01, ^#p < 0.001, ^$p < 0.0001. Data represented as mean ± SEM.