Multiplex viral tropism assay in complex cell populations with single-cell resolution

Area of Science:

• Biotechnology
• Genetics
• Molecular Biology

Background:

• Gene therapy holds immense promise for treating diseases.
• Key challenges include ensuring efficient and specific delivery of therapeutic agents to target cells.
• Current methods for evaluating delivery vectors are often limited in scope and throughput.

Purpose of the Study:

• To develop an integrated bioinformatics and experimental pipeline for multiplex measurement of gene therapy vector transduction efficiency and specificity.
• To enable high-throughput assessment of how diverse delivery vectors interact with various cell types.
• To provide a robust platform for optimizing the safety and efficacy of therapeutic delivery systems.

Main Methods:

• Developed a library-on-library approach to simultaneously test multiple vector variants against multiple cell types.
• Integrated high-throughput sequencing for AAV (adeno-associated virus) variant identification with single-cell RNA transcriptomic sequencing.
• Utilized human cerebral and ocular organoids to model in vivo conditions and validate findings in non-human primates.

Main Results:

• Successfully mapped the transduction patterns of natural and engineered AAV variants at a single-cell resolution within organoids.
• Demonstrated that efficient AAV transduction observed in organoid models is accurately recapitulated in vivo in non-human primates.
• Validated the pipeline's ability to measure both transduction efficiency and specificity across diverse cell populations.

Conclusions:

• The developed library-on-library technology offers a powerful tool for comprehensively evaluating gene therapy delivery vectors.
• This approach significantly advances the ability to determine the safety and efficacy of vectors before clinical application.
• The findings pave the way for accelerated development and improved design of gene therapies.