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Multiplexed Cre-dependent selection yields systemic AAVs for targeting distinct brain cell types.

Sripriya Ravindra Kumar1, Timothy F Miles1, Xinhong Chen1

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Nature Methods
|April 22, 2020
PubMed
Summary

Researchers developed Multiplexed-CREATE (M-CREATE) to evolve adeno-associated virus (AAV) capsids for targeted gene delivery in the brain. This method accelerates the discovery of novel AAV variants for neuroscience and gene therapy.

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Area of Science:

  • Molecular Biology
  • Neuroscience
  • Gene Therapy

Background:

  • Recombinant adeno-associated viruses (rAAVs) are effective gene delivery vectors but natural serotypes have limited tropisms.
  • Targeting specific cell types in the adult mouse brain requires evolved viral capsids.

Purpose of the Study:

  • To expand the utility of AAV vectors by evolving capsid variants for efficient transduction of specific cell types in the adult mouse brain.
  • To develop an advanced platform, Multiplexed-CREATE (M-CREATE), for identifying AAV variants with desired tropisms.

Main Methods:

  • Utilized the Cre-recombination-based AAV targeted evolution (CREATE) platform, enhanced into Multiplexed-CREATE (M-CREATE).
  • M-CREATE employs next-generation sequencing, synthetic library generation, and a dedicated analysis pipeline with multiple selection criteria.
  • Evolved AAV capsids through iterative positive and negative selections.

Main Results:

  • Identified novel AAV capsid variants with enhanced capabilities for central nervous system transduction.
  • Discovered variants with specific tropisms, including bias toward vascular cells and astrocytes, and enhanced neuronal targeting.
  • Found variants capable of crossing the blood-brain barrier effectively in diverse mouse strains.

Conclusions:

  • The M-CREATE methodology significantly accelerates the discovery of engineered AAV capsids.
  • These findings provide valuable tools for neuroscience research and gene therapy applications requiring precise AAV-mediated gene delivery.
  • Evolved AAV capsids offer expanded tropism and improved blood-brain barrier penetration for therapeutic development.