Single AAV Vector Delivery of Smaller Cas9 Orthologs for DMD Gene Editing

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Solution Overview

Problem

Current gene editing strategies for Duchenne muscular dystrophy (DMD) using CRISPR systems face challenges due to the large size of Streptococcus pyogenes Cas9, requiring two AAV vectors and limited design flexibility, which complicates effective delivery and administration.

Innovation Solution

Employing smaller Cas9 orthologs from Staphylococcus aureus (SaCas9) and Staphylococcus lugdunensis (SluCas9) to enable a single AAV vector delivery of these enzymes along with multiple guide RNAs, enhancing design flexibility and reducing manufacturing and administrative complexities.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Reliability

If Streptococcus pyogenes Cas9 is used in AAV-based CRISPR/Cas systems, then effective gene editing can be achieved, but two AAV vectors are required increasing delivery complexity

Engineering Contradiction:

Improvegene editing effectivenessVSAvoiddelivery vector complexity

Core Design Contradiction:

ReliabilityVSDevice complexity

Solution Approach 1:

The patent combines the Cas9 enzyme and guide RNA into a single AAV vector system using smaller Cas9 orthologs (SaCas9 or SluCas9), eliminating the need for separate vectors. This merging approach maintains gene editing effectiveness while significantly reducing delivery complexity and manufacturing costs.

Inventive Principle:

Principle #5Merging (Combining)

Solution Approach 2:

The patent changes the parameter of Cas9 enzyme size by using orthologs from Staphylococcus aureus or Staphylococcus lugdunensis instead of Streptococcus pyogenes. These smaller Cas9 variants enable single-vector delivery while maintaining the essential gene editing function, thus resolving the contradiction between reliability and complexity.

Inventive Principle:

Principle #35Parameter changes

2Adaptability or versatility

If two AAV vectors are used to deliver Cas9 and guide RNA separately, then complete CRISPR system components are provided, but manufacturing and administration complexity increases

Engineering Contradiction:

ImproveCRISPR system completenessVSAvoidmanufacturing complexity

Core Design Contradiction:

Adaptability or versatilityVSEase of manufacture

Solution Approach 1:

The invention merges the delivery of Cas9 enzyme and guide RNA into a single AAV vector, simplifying the manufacturing process and administration protocol. This single-vector approach maintains system completeness by including both essential components in one deliverable unit, thereby improving ease of manufacture without sacrificing versatility.

Inventive Principle:

Principle #5Merging (Combining)

3Device complexity

If smaller Cas9 orthologs are used, then single AAV vector delivery is enabled reducing complexity, but design flexibility may be limited

Engineering Contradiction:

Improvevector delivery complexityVSAvoidguide RNA design flexibility

Core Design Contradiction:

Device complexityVSAdaptability or versatility

Solution Approach 1:

The patent achieves multi-functionality by incorporating multiple guide RNAs targeting different genomic locations within the same DMD gene into a single AAV vector along with the smaller Cas9 ortholog. This universal system allows simultaneous or sequential editing at multiple sites, maintaining design flexibility while enjoying the simplicity of single-vector delivery.

Inventive Principle:

Principle #6Universality (Multi-functionality)

4Productivity

If multiple guide RNAs are incorporated on a single vector with smaller Cas9, then design flexibility and efficiency improve, but vector capacity requirements increase

Engineering Contradiction:

Improvegene editing efficiencyVSAvoidvector nucleic acid content

Core Design Contradiction:

ProductivityVSQuantity of substance

Solution Approach 1:

The patent utilizes parameter changes in vector design, specifically selecting AAV serotypes and vector configurations that optimize capacity for accommodating multiple guide RNAs and the Cas9 ortholog. By carefully managing the nucleic acid content and using smaller Cas9 variants, the system achieves high editing efficiency with multiple guides while staying within viable vector capacity constraints.

Inventive Principle:

Principle #35Parameter changes

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 approach improves the efficiency of gene editing by allowing for the use of multiple guide RNAs on a single vector, simplifying delivery, and reducing costs, thereby providing a more effective treatment for DMD.

Implementation Method 1

The approximately 20 nucleotides at the 5′ end of the guide RNA serves as the guide or spacer sequence that can be any sequence complementary to one strand of a genomic target location

Methodology Applied

Scientific EffectComplementary base pairing:

Implementation Method 2

CRISPR-based genome editing can provide sequence-specific cleavage of genomic DNA using a Cas9 and a guide RNA

Methodology Applied

Scientific EffectNuclease cleavage:

Implementation Method 3

To repair these breaks, cells typically use an error prone mechanism of non-homologous end joining (NHEJ) which can lead to disruption of function in the target gene through insertions or deletion of codons

Methodology Applied

Scientific EffectNon-homologous end joining:

Data Source

PatentUS20220096606A1Compositions and Methods for Treatment of Duchenne Muscular Dystrophy

Publication Date: 2022.03.31 VERTEX PHARMACEUTICALS INC

AI summary

Compositions and methods for treating Duchenne Muscular Dystrophy (DMD) are encompassed.