Viral Vector Targets Transferrin Receptor to Deliver GBA1 to Brain

Over the last three decades, scientists have devised molecular shuttles that slip enzymes, antibodies, and other molecules into the central nervous system (CNS) by attaching them to the transferrin receptor, which ferries the cargo across the blood-brain barrier. Now, researchers led by Benjamin Deverman at the Broad Institute in Cambridge, Massachusetts, have piggy-backed on that strategy to deliver genes into the brain. In the May 16 Science, they reported proof-of-concept data, showing that a transferrin-binding motif boosted delivery of adeno-associated virus 9 (AAV9) into the brain 30-fold. The virus, carrying the GBA1 gene, produces seven times as much β-glucocerebrosidase in the mouse brain as did a control virus. Glucocerebrosidase deficiency causes Gaucher's disease, a rare childhood neurodegenerative disorder, and mutations in the enzymes increase the risk of Parkinson's disease. Other gene therapies aim to boost the enzyme's activity (Jul 2018 news).

• Transferrin receptor on endothelial cells can shuttle large molecules into the brain.
• A virus targeting the receptor sneaks genes into the brain.
• It boosts viral delivery 30-fold.

"A critically needed improvement for CNS gene therapy is the characterization of vectors able to efficiently cross the blood-brain barrier after administration in the bloodstream," wrote Olivier Danos of Maryland-based Regenxbio (comment below). "This paper is exciting because it shows, through a series of impeccably executed studies, that some of the current limitations of the technology can be solved."

Joy Zuchero of Denali Therapeutics in South San Francisco called the strategy clever. "This elegant paper represents a significant advancement and opens the door for an exciting new approach in the area of CNS gene delivery," she wrote (comment below). Denali targets the transferrin receptor to deliver a TREM2 antibody, the progranulin protein, and iduronate-2-sulfatase, an enzyme defective in Hunter syndrome, into the brain (Jan 2023 news; Apr 2023 news; Jun 2023 news). Roche uses a similar strategy to deliver trontinemab, a version of their anti-amyloid antibody, gantenerumab (Mar 2021 conference news).

Seeing Scarlet. An AAV9 targeting the transferrin receptor delivered the gene for mScarlet fluorescent protein throughout the brain (top), but an AAV9 carrying the same gene could not (bottom). [Courtesy of Huang et al., Science, 2024.]

Widely used for gene therapies, AAVs come in subtypes that selectively infect certain tissues or cells. AAV9, for one, crosses the BBB and is commonly used for CNS therapies, such as the FDA-approved spinal muscular atrophy treatment Zolgensma (Nov 2019 conference news). Deverman and colleagues thought to enhance this BBB penetration by creating a virus that can be shuttled by the transferrin receptor (TfR). Co-first authors Qin Huang and Ken Chan modified AAV9 viruses, randomly mutating a stretch of seven amino acids within the viral capsid sequence, then screening the tens of millions of viral variants for binding to human TfR. One AAV, called BI-hTFR1 after the Broad Institute, bound tightest to the receptor.

Would this Trojan horse work in vivo? The scientists loaded BI-hTFR1 viruses with luciferase genes, then added them to primary and cultured human brain endothelial cells. These cells shunt transferrin from the blood into the brain parenchyma. BI-hTFR1 delivered 35- and 55-fold more luciferase into the primary and cultured cells, than did AAV9.

Could BI-hTFR1 cross the BBB in vivo? Huang and Chan intravenously injected BI-hTFR1 or AAV9 carrying the gene for the mScarlet fluorescent protein into mice that had the human TfR gene knocked in. Three weeks later, cells glowed throughout the brains of animals injected with BI-hTFR1 but not in those given AAV9 (image above). Across the cortex, striatum, and thalamus, about one-quarter of the oligodendrocytes, half the neurons and almost all the astrocytes expressed mScarlet, measured via fluorescence and immunohistochemistry. There was little expression in microglia. "It will be of great value to continue to evolve the capsid for microglia targeting as well, given this cell type's role in a number of genetically linked neurodegenerative diseases," wrote Zuchero.

The authors anticipate that BI-THFR1 could better deliver therapeutic genes into the brain. To test this, Huang and Chan injected AAVs carrying GBA1 into the veins of human TfR knock-in mice. Three weeks later, 30 times as much BI-hTFR1 viral DNA was in the animals' brains as AAV9 DNA. BI-hTFR1-treated mice widely expressed GBA1 in the brain, mainly in neurons and astrocytes (image below). Their brains had 6.7 times more β-glucocerebrosidase activity than did AAV9-treated mice, suggesting that higher gene expression translated into more enzyme.

Transferring GBA1. AAV9-GBA1 at a dose of 1 x 1014 viral genomes/kg (purple, left) barely infected any neurons (green) in TfR knock-in mice. However, BI-hTFR1 given at the same dose (right) infected neurons throughout the brain. [Courtesy of Huang et al., Science, 2024.]

"It is promising to see successful delivery of GBA1 using BI-hTfR1 as a proof of concept," Zuchero wrote. Deverman has made the plasmid for BI-hTFR1 available for other researchers through Addgene. He said his group is creating a gene therapy for an adult neurodegenerative disease using BI-hTFR1. New York-based Apertura Gene Therapy, a company he co-founded, has two programs based on the AAV. Deverman would not say which diseases they are targeting.—Chelsea Weidman Burke

News Citations

1. Gene Therapy Treats Mouse Models of Gaucher's Disease 19 Jul 2018
2. Ferried Into Brain, TREM2 Antibody Stirs Microglia 20 Jan 2023
3. Pumping Up Progranulin: Scientists Show New Efforts to Get It Done 7 Apr 2023
4. Treatment for Lysosomal Storage Disorder Lowers NfL 23 Jun 2023
5. Shuttle Unloads More Gantenerumab Into the Brain 25 Mar 2021
6. Time to Try Again: Gene-Based Therapy for Neurodegeneration 27 Nov 2019

Therapeutics Citations

1. DNL919
2. Trontinemab
3. Gantenerumab

External Citations

1. Addgene

No Available Further Reading