Identification a Compact Promoter using a New Promoter Selection Strategy and Engineering Hybrid Pol II/III Enable...

Area of Science:

• Gene Therapy
• Molecular Biology
• Bioengineering

Background:

• Adeno-associated virus (AAV) vectors are key for in vivo gene delivery but face packaging limitations with large gene editing tools like Streptococcus pyogenes Cas9 (SpCas9).
• Current strategies using smaller nucleases or dual-vector systems have drawbacks, impacting therapeutic efficiency.
• Developing compact promoters is crucial for efficient gene expression within AAV packaging constraints.

Purpose of the Study:

• To identify and engineer compact RNA polymerase II (Pol II) and hybrid Pol II/III promoters for single-AAV CRISPR-Cas9 gene therapy.
• To enable efficient co-expression of SpCas9 and guide RNA (gRNA) within the AAV packaging limit.
• To improve genome editing efficiency in target cells, including human retinal ganglion cells (RGCs).

Main Methods:

• Analysis of ~300 compact Pol II promoters to identify cell-preferred promoters based on exogenous expression.
• Development of a compact Pol II promoter (Pro2) for robust transgene expression in RGCs.
• Engineering of hybrid Pol II/III promoters by combining Pro2 with minimal Pol III promoters (H1, 7SK, U6) for SpCas9 and gRNA co-expression.
• CRISPR/Cas9 reporter assays to determine minimal functional promoter lengths and assess genome editing efficiency.

Main Results:

• Identification of Pro2 (133 bp), a compact Pol II promoter driving strong transgene expression in RGCs.
• Engineering of three compact hybrid Pol II/III promoters (276, 294, 323 bp) enabling single-AAV co-delivery of SpCas9 and gRNA.
• Achieved high genome editing efficiency in HEK293 cells (approaching 100%) and significant efficiency in human RGCs (up to 55.9%).

Conclusions:

• Established a framework for developing single-AAV CRISPR-based gene therapy strategies by engineering compact promoters.
• Demonstrated the feasibility of single-AAV delivery for SpCas9 and gRNA, overcoming AAV packaging limitations.
• The developed promoters enhance the potential for efficient in vivo genome editing in therapeutic applications.