Enhanced Genome Editing Technologies for Long Polynucleotide Integration

Background

Prime editing represents a promising frontier in genome editing, primarily enabling precise single-point mutations and small insertions or deletions. However, these methods traditionally support modifications up to about 100 base pairs, leaving a gap in the capability to efficiently integrate longer sequences vital for complex genetic therapies. Emerging needs in medical and research fields call for advanced systems that extend these capabilities to integrate substantially longer polynucleotides with high precision and efficiency.

Technology Overview

This invention significantly advances prime editing by introducing systems and methods for paired prime editing and prime assembly to achieve targeted integration of donor polynucleotides ranging from 250 to over 3,000 nucleotides. This innovation leverages a pair of prime editing guide RNAs (pegRNAs) and a unique genome editing complex that includes a programmable DNA binding protein with DNA nickase activity fused to a reverse transcriptase. The pegRNAs feature codon-optimized templates that, while not identical to the target gene, facilitate sequence replacement or integration effectively.

Parallel Solutions:

• Extended-Length Integration: Techniques for integrating longer polynucleotide sequences (up to 10,000 nucleotides) via designed 3’ overhangs.
• VPX and dNTP Supplementation: Utilizing viral particle X (VPX) alongside exogenous dNTPs to enhance editing efficiency.
• Engineered pegRNAs (epegRNAs): These advanced guides include stabilizing sequences that improve durability and binding efficiency.

Applications

• In Vivo and In Vitro Therapeutics: Implementing precise edits in TINF2, ATP1A1, IL2RG, TRAC, and AAVS1 loci for gene therapies targeting conditions like dyskeratosis congenita (DC).
• Advanced Research Capabilities: Facilitating complex genomic study by enabling extensive modifications in cellular models.

Advantages

• Scalability and Precision: Enables configurations for diverse lengths and types of genomic editing, using sophisticated sequence binding strategies.
• Broad Applicability: Suitable for both standard and complex genetic treatments, including regulatory sequence integration and heterologous polypeptide encoding.
• Corrections and Modifications: Provides reliable tools for gene correction, sequence replacement, and incorporating entire open reading frames or regulatory elements seamlessly.