An in vitro and in vivo efficacy evaluation of gene therapy candidate SBT101 in mouse models of adrenomyeloneuropathy and in NHPs

Abstract

Adrenomyeloneuropathy is a progressive neurodegenerative disease caused by pathogenic variants in the ABCD1 gene, resulting in very-long-chain fatty acid (VLCFA) accumulation that leads to dying-back axonopathy. Our candidate gene therapy, SBT101 (AAV9-human ABCD1 [hABCD1]), aims to ameliorate pathology by delivering functional copies of hABCD1 to the spinal cord. Transduced cells produce functional ABCD1 protein, thereby repairing the underlying biochemical defect. In vitro and in vivo mouse studies were conducted to assess the biochemical and functional efficacy of SBT101 and show effective delivery to target tissues involved in the disease pathology: spinal cord and dorsal root ganglia. Administration of SBT101 to mixed glial cell cultures from Abcd1-Null mice, and to male Abcd1 knockout (Abcd1 ^-/y ) and double-knockout (Abcd1 ^-/y /Abcd2 ^-/- ) mice led to increased hABCD1 production and reduced VLCFA. Double-knockout mice also exhibited improved grip strength. Furthermore, we conducted biodistribution and safety assessments in nonhuman primates. Six-hour intrathecal lumbar infusions demonstrated effective transduction throughout target tissues, supporting the clinical feasibility of the procedure. SBT101 was well tolerated, with no observed SBT101-related mortality or clinical signs. These findings not only provide preclinical efficacy data for SBT101 but also inform clinically relevant SBT101 dose selection for patients with adrenomyeloneuropathy.

Keywords: SBT101; adeno-associated virus; adrenomyeloneuropathy; biodistribution; gene therapy; human ABCD1; in vivo mouse model; neurodegenerative disease; nonhuman primates; very-long-chain fatty acids.

Graphical abstract

Figure 1

Increased hABCD1 expression and VLCFA reduction in mixed glial cell cultures following SBT101 administration Human ABCD1 expression (A) and VLCFA levels (B) in mixed glial cell cultures (from Abcd1-Null and WT mice) exposed to SBT101. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.0001. Data represent mean ±95% confidence interval.

Figure 2

Increased levels of hABCD1 and mtDNA, and reduced VLCFA levels, in Abcd1^−/y mice 8 weeks after SBT101 administration Viral genomes copy number (A), hABCD1 expression (B), VLCFA levels (C), and mtDNA levels (D) within the spinal cord of Abcd1^−/y mice assessed at 8 weeks after SBT101 administration. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.0001. Data represent mean ± 95% confidence interval.

Figure 3

Increased levels of hABCD1 and mtDNA, and reduced VLCFA levels in Abcd1^−/y mice at 13 and 24 weeks after SBT101 administration Viral genomes copy number (A and E), hABCD1 expression (B and F), VLCFA levels (C and G), and mtDNA levels (D and H) within the spinal cord of Abcd1^−/y mice assessed at 13 weeks (A–D) and 24 weeks (E–H) after SBT101 administration. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.0001. Data represent mean ± 95% confidence interval.

Figure 4

Biochemical improvements following SBT101 administration in Abcd1^−/y/Abcd2^−/− DKO mice Viral genome distribution (A) and hABCD1 mRNA expression (B) in the spinal cord of Abcd1^−/y/Abcd2^−/− DKO mice at 18 months of age (approximately 11 months after SBT101 administration). VLCFA levels (C) and inflammatory cytokine TNF-α levels (D) in the lumbar spinal cord of Abcd1^−/y/Abcd2^−/− DKO mice at 18 months of age. ∗p < 0.05, ∗∗p < 0.01. Data represent mean ± 95% confidence interval.

Figure 5

Improved grip strength in Abcd1^−/y/Abcd2^−/− DKO mice after SBT101 administration Four-paw grip strength in Abcd1^−/y/Abcd2^−/− DKO mice at 15 months of age. ∗p < 0.05. Data represent mean ± 95% confidence interval.

Figure 6

Vector genome biodistribution in the spinal cord and DRG according to delivery method in NHP study 1 Vector genome biodistribution in the spinal cord and DRG was assessed via ddPCR quantification of GFP copies (A) and via immunohistochemical quantification of neuron positivity (B). Plotted values represent mean ± 95% confidence interval. n = 3 NHPs per treatment group, and A1–A3 represent individual NHPs. Percentage neuron positivity is presented as grades 1–5: grade 1, <1%; grade 2, 1%–25%; grade 3, 26%–50%; grade 4, 51%–75%; grade 5, 76%–100%; blanks, unremarkable (no observable GFP immunoreactivity). C2, cervical section 2; ddPCR, droplet digital PCR; DRG, dorsal root ganglia; GFP, green fluorescent protein; HPRT1, hypoxanthine-guanine phosphoribosyltransferase 1; IT-C, intrathecal cervical; IT-L, intrathecal lumbar; L5, lumbar section 5; T5, thoracic section 5; vg/an, vector genomes/animal.

Figure 7

Vector genome biodistribution and hABCD1 mRNA expression Vector genome biodistribution (A, C, and E) and hABCD1 mRNA expression (B, D, and F) in NHP target tissues at 1 month (A and B), 3 months (C and D), and 6 months (E and F) post SBT101 administration in NHP study 4. Data represent mean ± 95% confidence interval. Vector genome copies and hABCD1 mRNA expression were below the limit of detection (LOD) for almost all controls (n = 3; data not plotted). Control NHPs received vehicle control (poloxamer [Kolliphor P188], potassium chloride, potassium dihydrogen phosphate dibasic in sterile water). ^aOne NHP did not have a calculated measurement. ^bOne NHP had a measurement below the limit of quantification. ^cOne NHP had a measurement below the LOD. Vector genome biodistribution and hABCD1 mRNA expression in NHP study 3 and NHP study 5 are shown in Figures S9 and S10, respectively. Vector genome biodistribution and hABCD1 mRNA expression in other tissues are shown in Figures S11–S13. DRG, dorsal root ganglia; hABCD1 human adenosine triphosphate-binding cassette sub-family D member 1; mRNA, messenger RNA; vg/an, vector genomes/animal.

Figure 8

ALT and AST levels post SBT101 administration in NHP study 4 ALT and AST activity were assessed in samples collected before and after a 6-h lumbar infusion of SBT101. (A and B) show samples collected before SBT101 administration on days −14 and −6, and after administration on days 7, 15, 21, 29, 56, 93, and 183. Data represent mean ± 95% confidence interval. Blue shaded areas indicate normal ranges for ALT and AST levels. Control NHPs received vehicle control (poloxamer [Kolliphor P188], potassium chloride, potassium dihydrogen phosphate dibasic in sterile water). (A and B) Data at day 56 and day 93 were based on n = 6 for controls and both low- and medium-dose SBT101 and n = 8 for high-dose SBT101; data at day 183 were based on n = 3 for controls and both low- and medium-dose SBT101 and n = 4 for high-dose SBT101. ALT and AST levels in NHP study 3 and NHP study 5 are shown in Figures S14 and S15, respectively. ALT, alanine aminotransferase; AST, aspartate aminotransferase; vg/an, vector genomes/animal.