Systemic gene therapy corrects the neurological phenotype in a mouse model of NGLY1 deficiency
Ailing Du, Kun Yang, Xuntao Zhou, Lingzhi Ren, Nan Liu, Chen Zhou, Jialing Liang, Nan Yan, Guangping Gao, Dan Wang
Ailing Du, Kun Yang, Xuntao Zhou, Lingzhi Ren, Nan Liu, Chen Zhou, Jialing Liang, Nan Yan, Guangping Gao, Dan Wang
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Research Article Neuroscience Therapeutics

Systemic gene therapy corrects the neurological phenotype in a mouse model of NGLY1 deficiency

Abstract

The cytoplasmic peptide:N-glycanase (NGLY1) is ubiquitously expressed and functions as a de–N-glycosylating enzyme that degrades misfolded N-glycosylated proteins. NGLY1 deficiency due to biallelic loss-of-function NGLY1 variants is an ultrarare autosomal recessive deglycosylation disorder with multisystemic involvement; the neurological manifestations represent the main disease burden. Currently, there is no treatment for this disease. To develop a gene therapy, we first characterized a tamoxifen-inducible Ngly1-knockout (iNgly1) C57BL/6J mouse model, which exhibited symptoms recapitulating human disease, including elevation of the biomarker GlcNAc-Asn, motor deficits, kyphosis, Purkinje cell loss, and gait abnormalities. We packaged a codon-optimized human NGLY1 transgene cassette into 2 adeno-associated virus (AAV) capsids, AAV9 and AAV.PHPeB. Systemic administration of the AAV.PHPeB vector to symptomatic iNgly1 mice corrected multiple disease features at 8 weeks after treatment. Furthermore, another cohort of AAV.PHPeB-treated iNgly1 mice were monitored over a year and showed near-complete normalization of the neurological aspects of the disease phenotype, demonstrating the durability of gene therapy. Our data suggested that brain-directed NGLY1 gene replacement via systemic delivery is a promising therapeutic strategy for NGLY1 deficiency. Although the superior CNS tropism of AAV.PHPeB vector does not translate to primates, emerging AAV capsids with enhanced primate CNS tropism will enable future translational studies.

Authors

Ailing Du, Kun Yang, Xuntao Zhou, Lingzhi Ren, Nan Liu, Chen Zhou, Jialing Liang, Nan Yan, Guangping Gao, Dan Wang

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Figure 8

rAAV.PHPeB treatment lowers GNA in the CNS of iNgly1 mice.

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rAAV.PHPeB treatment lowers GNA in the CNS of iNgly1 mice. (A) Quantific...
(A) Quantification of mouse endogenous Ngly1 mRNA expression (cDNA) in the brain, spinal cord, liver, heart, and TA muscle. N = 5 to 6 mice per group. (B) GNA levels in various tissues of Ngly1fl/fl mice and Ngly1fl/fl iCre mice with or without rAAV.PHPeB treatment. N = 5 mice per group. (C) Representative Western blotting images of NGLY1 protein expression in the brain, spinal cord, liver, heart, and TA muscle of the mice. Quantification of NGLY1 signal (normalized to GAPDH expression) is shown below the images. N = 5 to 6 mice per group. (D) Correlation between GNA level (y axis) and NGLY1 protein expression (x axis) in the brain of Ngly1fl/fl iCre mice with or without rAAV.PHPeB treatment. Each dot represents 1 animal. In A–C, data are mean ± SD of biological repeats; each white square represents an individual male mouse. In A and B, statistical analysis was performed by 1-way ANOVA followed by Tukey’s multiple comparisons test. In C, statistical analysis was performed by 2-tailed Student’s t test. In D, statistical analysis was performed by simple linear regression.