AAV Vector Splitting for Large Transgene Integration

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Solution Overview

Problem

The limited packaging capacity of adeno-associated virus (AAV) vectors restricts the size of donor DNA templates that can be used for CRISPR/Cas9-mediated genome editing, preventing the efficient integration of large transgenes into specific genetic loci in cells.

Innovation Solution

A method utilizing two AAV vectors with non-overlapping homology arms to split a large transgene, allowing sequential homologous recombination and CRISPR/Cas9-mediated integration into specific genetic loci, enabling the insertion of transgenes exceeding the single AAV vector capacity.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Quantity of substance

If a single AAV vector is used to deliver donor DNA template for CRISPR/Cas9-mediated genome editing, then the delivery system is simple and efficient, but the size of the donor DNA template is limited to about 4.5 kb packaging capacity

Engineering Contradiction:

Improvesize of donor DNA templateVSAvoidnumber of AAV vectors

Core Design Contradiction:

Quantity of substanceVSDevice complexity

Solution Approach 1:

The donor DNA template is divided into multiple segments, each fitting within the 4.5 kb packaging capacity of individual AAV vectors. The patent describes splitting a large donor template (e.g., 7.2 kb CFTR correction template) into at least two separate AAV vectors, each carrying a portion of the homologous arms and donor sequence. This segmentation allows delivery of total donor DNA exceeding single-vector capacity while maintaining AAV's efficient delivery mechanism.

Inventive Principle:

Principle #1Segmentation

2Quantity of substance

If the donor DNA template size exceeds 4.0 kb, then larger transgenes can be integrated, but a single AAV vector cannot accommodate the donor template

Engineering Contradiction:

Improveinsert size of transgeneVSAvoidcompatibility with single AAV vector system

Core Design Contradiction:

Quantity of substanceVSAdaptability or versatility

Solution Approach 1:

Large transgenes exceeding 4.0 kb are segmented across multiple AAV vectors. The patent demonstrates this by dividing the CFTR donor template into segments that fit within individual AAV vectors, enabling integration of the complete large transgene through sequential homologous recombination of the segmented donors.

Inventive Principle:

Principle #1Segmentation

Solution Approach 2:

The solution transitions from a single-dimension (single vector) to multi-dimension (multiple vectors) approach. By utilizing multiple AAV vectors simultaneously, the system overcomes the size limitation of individual vectors while maintaining the benefits of AAV-mediated delivery, effectively adding a dimensional aspect to the delivery system.

Inventive Principle:

Principle #17Another dimension (Dimensionality change)

3Quantity of substance

If multiple AAV vectors are used to deliver split donor templates, then large transgenes can be integrated, but the system complexity increases

Engineering Contradiction:

Improvesize of integrated transgeneVSAvoidmulti-vector system

Core Design Contradiction:

Quantity of substanceVSDevice complexity

Solution Approach 1:

The donor template is segmented into multiple AAV vectors with non-overlapping homology arms. Each vector contains specific segments that, when combined through sequential homologous recombination, reconstruct the complete large transgene integration. This segmentation strategy enables large transgene integration while organizing the complexity into manageable, functionally-defined segments.

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

Enables the precise and efficient integration of large transgenes into cells, overcoming the size limitations of single AAV vectors and facilitating targeted genome editing in various cell types, including primary and stem cells.

Implementation Method 1

designer nucleases (e.g., ZFNs and TALENs) or RNA-guided nucleases (e.g., CRISPR/Cas9), which create site-specific double-strand breaks (DSBs) that stimulate homologous recombination (HR)

Methodology Applied

Scientific EffectCRISPR/Cas9-mediated double-strand break:

Implementation Method 2

stimulate homologous recombination (HR) when supplied with a homologous donor DNA template

Methodology Applied

Scientific EffectHomologous recombination:

Implementation Method 3

adeno associated viral (AAV) vectors to serve as donor template DNA during homologous recombination

Methodology Applied

Scientific EffectViral vector delivery:

Data Source

PatentUS11773409B2CRISPR/Cas 9-mediated integration of polynucleotides by sequential homologous recombination of AAV donor vectors

Publication Date: 2023.10.03 THE BOARD OF TRUSTEES OF THE LELAND STANFORD JUNIOR UNIV

AI summary

The present invention relates to a system and method for efficiently modifying the genome of cells to treat diseases via sequential homologous recombination using CRISPR/Cas-mediated genome editing with donor DNA delivered by two or more adeno-associated virus (AAV) vectors.