Highly conserved brain vascular receptor ALPL mediates transport of engineered viral vectors across the blood-brain barrier

Moyer, Tyler C.; Hoffman, Brett A.; Chen, Weitong; Shah, Ishan; Ren, Xiao-Qin; Knox, Tatiana; Liu, Jiachen; Wang, Wei; Li, Jiangyu; Khalid, Hamza; Kulkarni, Anupriya S.; Egbuchulam, Munachiso; Clement, Joseph; Bloedel, Alexis; Child, Matthew; Kaur, Rupinderjit; Rouse, Emily; Graham, Kristin; Maura, Damien; Thorpe, Zachary; Sayed-Zahid, Ambreen; Chung, Charlotte Hiu-Yan; Kutchin, Alexander; Johnson, Amy; Yao, Johnny; Thompson, Jeffrey; Pande, Nilesh; Nonnenmacher, Mathieu E.

doi:10.1101/2024.03.12.584703

Cited by 4 publications

References 57 publications

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Characterization of brain transduction capability of a BBB-penetrant AAV vector in mice, rats and macaques reveals differences in expression profiles

Bunuales,

Garduno,

Chillon

et al. 2024

Gene Ther

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Different screening methods are being developed to generate adeno-associated viral vectors (AAV) with the ability to bypass the blood-brain barrier (BBB) upon intravenous administration. Recently, the AAV9P31 stood out as the most efficient version among a library of peptide-displaying capsids selected in C57BL/6 mice using RNA-driven biopanning. In this work we have characterized in detail its biodistribution in different mouse strains (C57BL/6 and Balb/c), as well as in Sprague Dawley rats and non-human primates (Macaca fascicularis). Using GFP and NanoLuc reporter genes, we confirmed homogeneous infection and transgene expression across the CNS of mice injected intravenously with AAV9P31. A more restricted pattern was observed upon either intracerebroventricular or intraparenchymal injection. Following intravenous delivery, region- and cell-specific differential patterns of transduction were observed in the mouse brain, including a preferential transduction of astrocytes and neurons in the cerebral cortex and striatum, whereas neurons were the only transduced cell type in subcortical locations across the hippocampus, thalamus, hypothalamus, mesencephalon, brainstem and cerebellum. Furthermore, transduced microglial cells were never found in any CNS location. Peripheral organs transduced upon intravenous administration included lung, liver, peritoneum, heart and skeletal muscle. However, a comparable performance of AAV9P31 to bypass the BBB in rats and macaques was not observed, although a more limited neuronal transduction was found in the brainstem of rats upon intravenous delivery. Finally, intracerebroventricular delivery in macaques resulted in neuronal transduction in cortical, subcortical structures and cerebellum following a patchy pattern. In conclusion, the widespread CNS transduction obtained in mice upon intravenous delivery of AAV9P31 represents a powerful tool for modeling a wide variety of neurological disorders as well as an appealing choice for the evaluation of gene therapy-based therapeutics.

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Characterization of brain transduction capability of a BBB-penetrant AAV vector in mice, rats and macaques reveals differences in expression profiles

Bunuales,

Garduno,

Chillon

et al. 2024

Gene Ther

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A Novel Engineered U7 Small Nuclear RNA Scaffold Greatly Increasesin vitroandin vivoADAR-Mediated Programmable RNA Base Editing

Byrne,

Burleigh,

Fragoza

et al. 2024

Preprint

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Custom RNA base editing using the endogenous human Adenosine Deaminase Acting on RNA (ADAR) enzyme presents a promising approach for precision therapeutics, alleviating concerns of permanent DNA damage or immunogenicity from foreign bacterial proteins such as CRISPR/Cas. ADAR can be directed to act on therapeutic RNA targets by antisense guide RNAs (gRNAs) that create a substrate for ADAR′s adenosine-to-inosine (effectively A-to-G) deamination activity. Delivery of gRNAs via a DNA expression construct provided by Adeno-Associated Virus (AAV) might allow life-long duration of the therapy. However, a major challenge for RNA editing using gene-encoded gRNAs and endogenous levels of ADAR is achieving sufficient gRNA activity inside cells, especially in therapeutic situations where AAV delivery may provide as low as one viral genome per cell. Here we show that embedding antisense gRNAs into a U7 small nuclear RNA (snRNA) framework and adding hnRNP A1 binding domains greatly increases the efficiency of custom RNA editing. This increased editing efficiency allows for detectable RNA editing from a single genomic insertion of gRNA construct per cell, which enabled a pooled library screen of 750+ gRNA variations to further improve the SmOPT U7 hairpin system. The screen revealed critical residues responsible for RNA editing and generated new SmOPT and U7 hairpin variants that further boosted RNA editing. The final design, combined with an improved synthetic U7 promoter, resulted in up to 76% targeted editing with a single integrated copy of construct per cell, representing a 10- to 100-fold increase over existing circular gRNA approaches. Using systemicin vivoAAV delivery, we achieved an unprecedented 75% RNA editing in the total brain of a mouse model of Hurler syndrome. Our novel SmOPT U7 system also improved published antisense oligos for DMD exon skipping, currently in clinical trials, by up to 25-fold in differentiated myoblasts, and therefore represents a universal scaffold for ADAR-based RNA editing as well as other antisense RNA therapies.

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

Du,

Yang,

Zhou

et al. 2024

JCI Insight

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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.

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Highly conserved brain vascular receptor ALPL mediates transport of engineered viral vectors across the blood-brain barrier

Cited by 4 publications

References 57 publications

Characterization of brain transduction capability of a BBB-penetrant AAV vector in mice, rats and macaques reveals differences in expression profiles

Characterization of brain transduction capability of a BBB-penetrant AAV vector in mice, rats and macaques reveals differences in expression profiles

A Novel Engineered U7 Small Nuclear RNA Scaffold Greatly Increasesin vitroandin vivoADAR-Mediated Programmable RNA Base Editing

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

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