Modular Prime Editing System for AAV Delivery

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Solution Overview

Problem

Conventional prime editing systems, such as those involving Cas9 nickase fused with reverse transcriptase, are hindered by large size, making them incompatible with adeno-associated virus (AAV) and RNA-based delivery platforms, limiting their clinical application due to size constraints and stability issues.

Innovation Solution

A modular prime editing system comprising independent Cas9 nickase RNA and reverse transcriptase template RNA molecules, allowing for precise and efficient genome editing without the need for fusion constructs, enabling delivery through AAV and RNA-based platforms by separating the RNA components to reduce overall size and improve stability.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Manufacturing precision

If conventional prime editing systems use Cas9 nickase fused with reverse transcriptase, then genome editing precision is improved, but system size increases making AAV delivery incompatible

Engineering Contradiction:

Improvegenome editing precisionVSAvoidsystem size

Core Design Contradiction:

Manufacturing precisionVSLength of moving object

Solution Approach 1:

The prime editing system is divided into separate functional modules: Cas9 nickase, reverse transcriptase, and pegRNA operate as independent components rather than a fused construct. This segmentation reduces the size of individual delivery vectors while maintaining the complete editing functionality when components are delivered together via multiple AAV vectors.

Inventive Principle:

Principle #1Segmentation

Solution Approach 2:

The Cas9 nickase and reverse transcriptase components can be delivered as separate AAV vectors, allowing each vector to be optimized for its specific function and size constraints. This multi-vector approach enables AAV delivery compatibility while preserving the precision of prime editing through the coordinated action of all components.

Inventive Principle:

Principle #6Universality (Multi-functionality)

2Adaptability or versatility

If prime editor components are delivered as separate RNA molecules, then delivery compatibility with AAV is improved, but system complexity increases

Engineering Contradiction:

Improvedelivery compatibilityVSAvoidsystem complexity

Core Design Contradiction:

Adaptability or versatilityVSDevice complexity

Solution Approach 1:

The system separates the prime editor into distinct RNA components (Cas9 RNA and RT RNA) that can be independently packaged into AAV vectors. This segmentation enables compatibility with AAV size constraints while the modular nature simplifies vector design and delivery optimization for each component.

Inventive Principle:

Principle #1Segmentation

Solution Approach 2:

The pegRNA serves as an intermediary molecule that coordinates the separate Cas9 nickase and reverse transcriptase components, guiding them to the target site and facilitating their coordinated action. This intermediary approach manages system complexity by providing a central organizing element that integrates the separate components.

Inventive Principle:

Principle #24Intermediary (Mediator)

3Productivity

If fusion constructs are used for prime editing, then delivery efficiency is reduced, but component coordination is simplified

Engineering Contradiction:

Improvedelivery efficiencyVSAvoidcomponent coordination

Core Design Contradiction:

ProductivityVSDevice complexity

Solution Approach 1:

By delivering Cas9 nickase and reverse transcriptase as separate RNA molecules in distinct AAV vectors, the system avoids the delivery bottlenecks associated with large fusion constructs. Each vector can be optimized for efficient transduction, improving overall delivery efficiency while the pegRNA ensures proper coordination of the separate components at the target site.

Inventive Principle:

Principle #1Segmentation

Applied Scientific Principles

This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.

Function Achieved in This Case

This modular approach facilitates precise genome editing in cells and adult mouse liver, achieving comparable efficiency to conventional systems while allowing for AAV integration and improved delivery, enhancing the potential for clinical use by reducing molecular size and enhancing stability.

Implementation Method 1

a modular prime editing system comprising independent Cas9 nickase RNA and reverse transcriptase template RNA molecules

Methodology Applied

Scientific EffectComplementary base pairing:

Implementation Method 2

reverse transcriptase template (RTT) RNA

Methodology Applied

Scientific EffectReverse transcription:

Data Source

PatentUS20240376499A1Modular prime editing systems for genome engineering

Publication Date: 2024.11.14 UNIV OF MASSACHUSETTS

AI summary

A modular prime editing (sPE) system is disclosed with separate and independent Cas9 nickase and reverse transcriptase template. This sPE system results in precise and efficient genome editing in cells and in adult mouse liver which is advantageous over conventional fusion proteins. Additionally, a pegRNA can be separated into an sgRNA and a prime editor template RNA, designated as a petRNA. This flexible, and modular system, is an improvement in the art to obtain precise genome editing.