Preclinical biodistribution, tropism, and efficacy of oligotropic AAV/Olig001 in a mouse model of congenital white matter disease

Jeremy S Francis, Vladimir Markov, Irenuez D Wojtas, Steve Gray, Thomas McCown, R Jude Samulski, Marciano Figueroa, Paola Leone, Jeremy S Francis, Vladimir Markov, Irenuez D Wojtas, Steve Gray, Thomas McCown, R Jude Samulski, Marciano Figueroa, Paola Leone

Abstract

Recent advances in adeno-associated viral (AAV) capsid variants with novel oligotropism require validation in models of disease in order to be viable candidates for white matter disease gene therapy. We present here an assessment of the biodistribution, tropism, and efficacy of a novel AAV capsid variant (AAV/ Olig001) in a model of Canavan disease. We first define a combination of dose and route of administration of an AAV/Olig001-GFP reporter conducive to widespread CNS oligodendrocyte transduction in acutely symptomatic animals that model the Canavan brain at time of diagnosis. Administration of AAV/Olig001-GFP resulted in >70% oligotropism in all regions of interest except the cerebellum without the need for lineage-specific expression elements. Intracerebroventricular infusion into the cerebrospinal fluid (CSF) was identified as the most appropriate route of administration and employed for delivery of an AAV/Olig001 vector to reconstitute oligodendroglial aspartoacylase (ASPA) in adult Canavan mice, which resulted in a dose-dependent rescue of ASPA activity, motor function, and a near-total reduction in vacuolation. A head-to-head efficacy comparison with astrogliotropic AAV9 highlighted a significant advantage conferred by oligotropic AAV/Olig001 that was independent of overall transduction efficiency. These results support the continued development of AAV/Olig001 for advancement to clinical application to white matter disease.

Conflict of interest statement

S.G. is an inventor on a patent for the Olig001 capsid (US patent #9636370) and has received royalty income from Asklepios BioPharmaceutical related to this invention. All other authors declare no competing interests.

© 2021 The Author(s).

Figures

Graphical abstract
Graphical abstract
Figure 1
Figure 1
Stereological estimates of AAV/Olig001-GFP transduction via the four indicated ROAs (A) Representative image of a GFP-positive cell at 100× magnification with an optical dissector counting frame. Positive soma (arrow) were counted throughout each region of interest (ROI). (B) Cortical, subcortical white matter (SCWM), striatal, and cerebellar ROIs were sampled (respective color codes highlight sampled regions) for GFP-positive soma in a total of 60 serial sections per brain, with a sampling interval (k) of 4. (C–F) Stereological estimates of GFP-positive cells in the cortex (C), SCWM (D), striatum (E), and cerebellum (F) of animals transduced by the indicated ROAs and at the indicated doses. Significant dose-dependent differences within each ROA cohort are denoted by red asterisks (∗p ≤ 0.05; ∗∗p ≤ 0.01; ∗∗∗p ≤ 0.001). For each dose cohort, n = 5 animals, with mean number ± SEM presented.
Figure 2
Figure 2
Comparison of effect of ROA on transduction by AAV/Olig001-GFP at 1 × 1011 dose in each of the ROIs (A–D) Cortex (A), SCWM (B), striatum (C), and cerebellum (D). Mean ± SEM of n = 5 shown. Significant differences between ROA in each ROI are indicated by red asterisks. (E) Native GFP fluorescence in sagittal section brains transduced with 1 × 1011 AAV/Olig001-GFP vector genomes (vgs) via intraparenchymal (IP), intrathecal (IT), intracerebroventricular (i.c.v.), and intracisternamagna (ICM) routes. The IP and i.c.v. routes gave comparably high numbers of GFP-positive cells, but the i.c.v. ROA resulted in much greater spread from injection sites. (F–H) Schematic showing anatomy of sections distal to injection sites and examples of GFP transduction in distal sections (F) from IP (G) and i.c.v. (H) brains. (I) High magnification image of a single cortical GFP-positive cell from the early i.c.v. section shown in (H) highlighting characteristic oligodendrocyte multiprocess-bearing morphology.
Figure 3
Figure 3
Analysis of vg copy number in indicated regions for each ROA cohort receiving 1 × 1011 total vgs Mean vg copy number per group presented as vg per milligram wet tissue weight (n = 4). Tissue was dissected from each ROA in 22-week-old brains after transduction at 6 weeks of age with AAV/Olig001-GFP. DNA was isolated and real-time PCR performed using a custom-designed TaqMan probe/primer set specific for the bovine growth hormone (BGH) polyadenylation sequence in the transgene expression cassette. (A–D) vg number per milligram of wet tissue weight is presented for the cortex (A), SCWM (B), striatum (C), and cerebellum (D). The mean ± SEM is presented for each indicated ROA.
Figure 4
Figure 4
Effect of ROA on tropism of AAV/Olig001-GFP delivered at a 1 × 1011 dose via the indicated ROA Dual channel stacks of GFP/Olig2-positive and GFP/NeuN-positive cells were collected by systematic sampling and colabeled entities scored using the optical fractionator in the cortex, SCWM, striatum, and cerebellum. (A and B) Example of differing degree of Olig2 and NeuN colabeling with native GFP in the cortex (A) and SCWM (B). (C and M) Striatal GFP colabeling was predominantly Olig2 in the striatum (C), but Olig2 colabeling in the cerebellum (D) was relatively rare . (E–L) Stereological estimates of GFP-Olig2 colabeling in the cortex (E), SCWM (G), striatum (I), and cerebellum (K) alongside GFP-NeuN colabeling in these regions in adjacent sections (F, H, J, and L, respectively). Significant differences in GFP colabeling are denoted by red asterisks. For each dose cohort, n = 5, with mean number ± SEM presented. (N) Stereological estimates of BrDU-positive cells within white matter tracts of wild type and nur7 mice at indicated ages. Mean + SEM presented, n=5 per genotype at each age. (O) representative co-labeling of GFP and BrdU (red) in SCWM of the nur7 brain 2 weeks after transduction with AAV/Olig001-GFP at 6 weeks of age.
Figure 5
Figure 5
Striatal injection of 5 × 1010 vg of either AAV/Olig001-GFP or AAV9-GFP results in contrasting glial tropism (A) Mean percent colabeling with either Olig2 (blue) or GFAP (orange), ± SEM presented (n = 4). (B) Representative images of GFP-GFAP colabeling in AAV/Olig001-GFP-transduced brains (upper 3 panels) and AAV9-GFP-transduced brains (lower 3 panels), showing higher levels of GFAP colabeling in AAV9 brains. (C) Animals treated at the indicated doses with AAV/Olig001-ASPA (blue) or AAV9-ASPA (red) and assessed for rotarod latency to fall at 10, 14, 18, and 22 weeks of age. Sham nur7 (purple) and naive wild-type controls (black) are included. Mean ± SEM for each group presented (n = 12). Red asterisk indicates significant improvement in AAV/Olig001-ASPA-treated animals, as determined by repeated-measures ANOVA.
Figure 6
Figure 6
AAV/Olig001 and AAV9 Vector genome biodistribution and function (A) vg copy number in whole brains of 22-week-old animals treated with the indicated dose of either AAV/Olig001-ASPA (blue) or AAV9-ASPA (red). vg expressed as copy number per milligram of wet tissue weight (vg/mg), with group mean ± SEM presented (n = 6). Asterisks denote significant differences in vg copy number at the indicated dose. (B) vg copies per mg of tissue from the spinal cord, liver, and kidney of AAV/Olig001-ASPA and AAV9-ASPA 22-week-old animals administered via the ROA at the 2.5 × 1011 dose. Group means for each tissue are indicated in blue and red script (n = 6). (C) Reconstitution of ASPA protein in both AAV/Olig001-ASPA- and AAV9-ASPA-treated 22-week-old nur7 brains. Sham nur7 negative controls show the absence of detectable protein. (D) Whole-brain NAA in indicated treated and control groups at 22 weeks of age. The decrease of the dose of both AAV/Olig001-ASPA (blue) and AAV9-ASPA (red) results in a corresponding increase in NAA. Age-matched wild-type naive brains (orange) and sham nur7 brains (green) are provided to highlight chronically elevated NAA in nur7 brains. Mean ± SEM for each group provided (n = 6).
Figure 7
Figure 7
Comparative rescue of vacuolation in AAV/Olig001- and AAV9-treated brains (A) Representative H&E-stained sagittal sections of a 22-week-old nur7 brain (upper) and age-matched wild-type brain (lower) highlighting severe vacuolation in the thalamus, pons, and cerebellar white matter. (B) Vacuole volume fraction in brains was scored using an object volume fraction probe that scored the percentage of ROIs occupied by vacuoles in H&E-stained sections. (C) Relative vacuole volume fraction in AAV/Olig001-ASPA (blue)- and AAV9-ASPA (red)-treated 22-week-old brains with data for both the thalamus and pons/cerebral white matter presented. AAV/Olig001 rescued a greater proportion of each region (i.e., greater reduction in vacuole volume) than did AAV9 at all of the three indicated doses. Mean volume fraction (percent of total ROI sampled) shown ± SEM (n = 6). (D) Representative H&E-stained sections from AAV/Olig001-ASPA-treated (left 3 panels) and AAV9-ASPA-treated (right 3 panels) 22-week-old nur7 mice at the indicated doses, showing progressively more vacuolation in AAV9 brains with decreasing dose.

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