Split Intein Base Editor Delivery for AAV Packaging Limits

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Solution Overview

Problem

The challenge of delivering CRISPR/Cas9 components, such as SpCas9 and sgRNA, for genome editing is hindered by the small packaging capacity of adeno-associated viruses (AAVs), which limits the inclusion of additional elements like polypeptide domains, promoters, reporters, and DNA templates.

Innovation Solution

A composition comprising split intein-fused fragments of Cas9, including a deaminase, is used to form a base editor system, which is delivered via polynucleotides in vectors like adeno-associated viral vectors, allowing for efficient delivery and editing.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Reliability

If AAV vectors are used to deliver CRISPR/Cas9 components, then delivery to cells is achieved, but packaging capacity is limited

Engineering Contradiction:

Improvedelivery efficiencyVSAvoidpackaging capacity

Core Design Contradiction:

ReliabilityVSQuantity of substance

Solution Approach 1:

The Cas9 protein is divided into two separate fragments (N-terminal fragment and C-terminal fragment) that are delivered separately via AAV vectors. These fragments are then reconstituted in the cell to form the functional Cas9 protein, allowing the packaging capacity limitation to be overcome while maintaining delivery efficiency.

Inventive Principle:

Principle #1Segmentation

Solution Approach 2:

A reconstitution mechanism serves as an intermediary process that occurs within the cell. This mechanism allows the separate Cas9 fragments to be joined together to form the functional protein, enabling the system to work around the packaging capacity constraint of AAV vectors.

Inventive Principle:

Principle #24Intermediary (Mediator)

2Adaptability or versatility

If additional elements are included to meet gene editing objectives, then editing capability is improved, but packaging capacity is exceeded

Engineering Contradiction:

Improveediting capabilityVSAvoidpackaging capacity

Core Design Contradiction:

Adaptability or versatilityVSQuantity of substance

Solution Approach 1:

The base editor system is segmented into separate functional components (Cas9 fragment, deaminase, guide RNA) that can be delivered independently. This allows each component to be optimized and packaged separately, overcoming the total payload limit while maintaining complete editing functionality.

Inventive Principle:

Principle #1Segmentation

Solution Approach 2:

The system uses a modular approach where the same delivery mechanism (AAV vectors) can deliver different combinations of components for various editing objectives. The reconstitution mechanism provides universal functionality, allowing the system to adapt to different editing needs without increasing packaging requirements.

Inventive Principle:

Principle #6Universality (Multi-functionality)

Data Source

PatentUS12529041B2Compositions and methods for delivering a nucleobase editing system

Publication Date: 2026.01.20 BEAM THERAPEUTICS INC

AI summary

The invention provides compositions and methods for delivering first and second polynucleotides each encoding a fragment of an A-to-G Base Editor fusion protein comprising one or more deaminases (e.g., adenosine deaminases) and nCas9, wherein the first polynucleotide encodes an N-terminal fragment of nCas9 fused to an intein-N of a split intein pair and the second polynucleotide encodes a C-terminal fragment of nCas9 fused to an intein-C of a split intein pair, and methods for delivering these fragments together with an sgRNA to a cell (e.g., AAV delivery), where the fragments are spliced together by a split intein system, thereby reconstituting a functional base editing system in the cell.