Engineered AAV Vectors Designed to Bypass the Blood-Brain Barrier (BBB)

 

In a recent article published in Molecular Therapy, researchers from Voyage Therapeutics reported the development of a novel adeno-associated virus (AAV) capsid capable of crossing the blood-brain barrier (BBB) with unprecedented efficiency across multiple species while minimizing liver toxicity. Using an RNA-guided platform in cynomolgus macaques, the team engineered this capsid to enable efficient cross-species central nervous system (CNS) delivery and reduced off-target expression in the liver. This innovation addresses a major limitation of natural AAVs, such as AAV9, which require high systemic doses to achieve limited CNS delivery—thereby increasing the risk of off-target effects.

Why Crossing the Blood-Brain Barrier Is Critical for AAV Gene Therapy

The blood-brain barrier (BBB) blocks over 95% of therapeutics, including gene therapies, from reaching the brain, posing a major hurdle in treating neurological diseases. Recombinant AAV gene therapy, exemplified by Zolgensma for spinal muscular atrophy (SMA)—shows promise but is constrained by the poor BBB penetration of natural capsids like AAV9. This necessitates either high systemic doses or invasive brain injections, increasing toxicity risk and restricting applications to young patients.

Engineered AAVs with enhanced BBB permeability could enable safe, non-invasive delivery (e.g., via intravenous injection) for treating conditions such as ALS, Huntington’s disease, and lysosomal storage disorders. New variants developed through directed evolution and machine learning, such as the PAL family, BI-hTFR1, and AAVhu.32-PLUS, offer up to threefold improvements in BBB penetration, neuronal targeting, and reduced liver uptake. These advances overcome major barriers in CNS access and systemic toxicity, paving the way for broader and safer gene therapy applications.

RNA-Guided Directed Evolution Platform for Rapidly Engineering AAV Capsids

Voyage Therapeutics previously introduced a novel RNA-guided directed evolution platform, TRACER (Tropism Redirection of AAV by Cell-type-specific Expression of RNA), designed to rapidly engineer AAV capsids with enhanced BBB crossing and CNS transduction capabilities. Unlike conventional methods relying on transgenic animals or DNA recovery from tissues, TRACER selects functional capsids based on recovery of mRNA transcribed from CNS-specific promoters, such as SYN (neuronal) or GFAP (astrocytic). This enables direct identification of transcriptionally active, CNS-tropic capsids in non-transgenic animals after systemic delivery.

After just two rounds of in vivo selection in mice, TRACER yielded hundreds of AAV9-derived variants demonstrating up to 400-fold higher CNS transduction efficiency compared to AAV9. These capsids also exhibited significantly reduced off-target expression in peripheral organs like the liver and heart—crucial for clinical safety. Importantly, because TRACER relies on RNA expression rather than DNA recovery, it is applicable to clinically relevant species like non-human primates. Its adaptability to any tissue-specific promoter further expands its utility for developing targeted vectors for gene delivery.

Engineering the AAV9 Capsid via RNA-Guided Directed Evolution

To enhance BBB penetration, researchers created an AAV9 peptide display library by inserting random six-amino-acid peptides into the VR-IV surface loop near the capsid’s threefold axis. This library (~40 million variants) was intravenously injected into macaques, followed by two rounds of in vivo selection. Sequencing viral RNA from brain tissue revealed enriched variants, culminating in the discovery of VCAP-102. Structural analysis via cryo-EM confirmed the presence of a flexible serine-proline-histidine (SPH) motif critical for BBB transport, inserted without compromising capsid integrity.

Enhanced CNS Delivery in Rodents and Primates

In BALB/c mice, intravenous administration of VCAP-102 at 1×10¹³ viral genomes (VG)/kg resulted in 20- to 40-fold higher levels of viral DNA and transgene RNA in the brain and spinal cord compared to AAV9, along with a 14-fold reduction in liver expression. Dose-response studies showed transduction of up to 40% of cortical cells, with a balanced distribution between neurons and astrocytes.

In non-human primates, VCAP-102 achieved 4- to 24-fold higher brain genome delivery and 16- to 186-fold greater transgene RNA expression relative to AAV9, while maintaining low liver accumulation. In African green monkeys, VCAP-102 transduced approximately 50% of brain cells, predominantly astrocytes. In marmosets, the vector delivered 280-fold more genomes and 500-fold higher RNA expression than AAV9.

ALPL Identified as a Key Receptor for BBB Transport

Screening studies identified tissue-nonspecific alkaline phosphatase (ALPL), a conserved, GPI-anchored receptor, as the mediator of VCAP-102’s BBB translocation. In vitro, ALPL overexpression enhanced transduction, while siRNA knockdown or pharmacological inhibition (e.g., with SBI-425) reduced it. Surface plasmon resonance demonstrated direct, nanomolar-affinity binding of VCAP-102 to ALPL at neutral pH, with dissociation under acidic endosomal conditions. Transwell assays confirmed ALPL-dependent transcytosis, mimicking natural receptor-mediated transport mechanisms.

Age-Dependent Efficacy and Therapeutic Implications

Aged mice with elevated ALPL expression showed 1.8-fold greater VCAP-102 transduction than younger mice, underscoring ALPL’s role in vector efficacy. Despite ALPL expression in the liver, VCAP-102 maintained low liver tropism, likely due to differences in endothelial context.

Conclusion and Broader Applications

VCAP-102’s cross-species efficiency, reduced off-target expression, and dependence on ALPL position it as a highly translatable vector for CNS gene therapies. Its success also highlights ALPL as a promising molecular shuttle for other therapeutics, including antibodies and nanobodies. This study provides a compelling blueprint for using conserved receptor biology to enhance delivery precision and safety in gene therapy. The TRACER platform and VCAP-102 collectively represent significant steps toward overcoming translational barriers in CNS-targeted treatments.

References

  1. Highly conserved brain vascular receptor ALPL mediates transport of engineered AAV vectors across the blood-brain barrier.
  2. Single-Dose Gene-Replacement Therapy for Spinal Muscular Atrophy.
  3. Systemic administration of novel engineered AAV capsids facilitates enhanced transgene expression in the macaque CNS. 
  4. Blood-Brain Barrier and Delivery of Protein and Gene Therapeutics to Brain.
  5. An engineered adeno-associated virus mediates efficient blood-brain barrier penetration with enhanced neurotropism and reduced hepatotropism.
  6. An AAV capsid reprogrammed to bind human transferrin receptor mediates brain-wide gene delivery.
  7. A Multifaceted Approach to Optimizing AAV Delivery to the Brain for the Treatment of Neurodegenerative Diseases. 
  8. Rapid evolution of blood-brain-barrier706 penetrating AAV capsids by RNA-driven biopanning.

 

About Marin Biologic Laboratories

Our Recent Publication/Meeting Presentation on Gene Therapy

1.  Development of a Pharmacokinetic (PK) Mouse Serum GLP ELISA for an Anti–CD19–AntiCD3 Diabody
bioRxiv 2025.03.19.644217; doi: https://doi.org/10.1101/2025.03.19.644217
2. Cell-Based Potency Assay for Anti-CD3-Anti-CD19 Diabody. bioRxiv 2025.04.15.648836v1 https://www.biorxiv.org/content/10.1101/2025.04.15.648836v1
3.  American Society of Hematology (ASH) Annual Meeting 2024.
Abstract link: Using Gene Therapy to Solve Challenges with CAR-T Cell Immunotherapy: Lead Selection and Preclinical Development of an Adeno-Associated Virus with Reduced Immunogenicity  Exhibiting Efficient and Long-Term Expression of an Anti-CD19 T-Cell Engager.

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