HITI Gene Editing for DMD Exon Replacement

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Solution Overview

Problem

Current treatments for muscular dystrophy, particularly Duchenne Muscular Dystrophy (DMD), are limited in effectively addressing mutations within the DMD gene, leading to progressive muscle degeneration and lack of functional dystrophin protein.

Innovation Solution

The use of Homology-Independent Targeted Integration (HITI) technology, involving Cas9 to generate DNA double-stranded breaks, guide RNAs to target specific sites, and a donor DNA sequence for knock-in, facilitates the replacement of large segments of the DMD gene, including exons 1-19 and 41-55, to restore functional dystrophin expression.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Reliability

If current treatments for muscular dystrophy are used, then existing therapeutic options are available, but they are limited in effectively addressing mutations within the DMD gene

Engineering Contradiction:

Improveeffectiveness of treatmentVSAvoidability to address diverse DMD mutations

Core Design Contradiction:

ReliabilityVSAdaptability or versatility

Solution Approach 1:

The CRISPR-Cas9 gene editing system provides a universal platform that can address multiple types of DMD mutations (deletions, duplications, point mutations) through a single therapeutic approach. The system uses programmable guide RNAs to target different genomic locations, making it adaptable to diverse mutation profiles while maintaining a consistent mechanism of action.

Inventive Principle:

Principle #6Universality (Multi-functionality)

Solution Approach 2:

The invention changes the fundamental parameter of genetic correction from symptomatic management to precise genomic editing. By introducing CRISPR-Cas9 technology, the treatment transitions from addressing downstream effects to directly modifying the underlying genetic defect, thereby improving reliability across various mutation types.

Inventive Principle:

Principle #35Parameter changes

2Reliability

If large segments of the DMD gene are replaced using HITI technology, then functional dystrophin expression is restored, but the complexity of the gene editing process increases

Engineering Contradiction:

Improverestoration of dystrophin expressionVSAvoidcomplexity of gene editing process

Core Design Contradiction:

ReliabilityVSDevice complexity

Solution Approach 1:

The invention uses an intermediary AAV vector to deliver the CRISPR-Cas9 components and donor DNA into muscle cells. This mediator simplifies the delivery of complex gene editing machinery and enables in vivo editing without requiring complex surgical procedures or cell culture manipulations.

Inventive Principle:

Principle #24Intermediary (Mediator)

Solution Approach 2:

The gene editing process is segmented into distinct functional modules: guide RNA for target recognition, Cas9 enzyme for DNA cleavage, and donor DNA for homology-independent integration. This segmentation allows each component to be optimized independently and facilitates modular design of different editing strategies.

Inventive Principle:

Principle #1Segmentation

3Manufacturing precision

If multiple exons are replaced to correct DMD mutations, then the reading frame is restored and functional protein is produced, but the treatment requires precise targeting of multiple genomic sites

Engineering Contradiction:

Improveprecision of mutation correctionVSAvoidnumber of target sites to be edited

Core Design Contradiction:

Manufacturing precisionVSDevice complexity

Solution Approach 1:

The invention performs preliminary design of guide RNAs that target specific intronic regions flanking multiple exons. By pre-calculating the optimal gRNA sequences and their expected cutting patterns, the system simplifies the complexity of coordinating multiple editing events, as the donor DNA is designed to accommodate the anticipated recombination outcomes.

Inventive Principle:

Principle #10Preliminary action

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 enables the restoration of full-length dystrophin protein, potentially offering a one-time genomic correction for a wide range of DMD mutations, thereby delaying disease progression and improving muscle function.

Implementation Method 1

involving Cas9 to generate DNA double-stranded breaks, guide RNAs to target specific sites

Methodology Applied

Scientific EffectCRISPR-Cas9 DNA cleavage:

Implementation Method 2

a donor DNA sequence for knock-in, facilitates the replacement of large segments of the DMD gene

Methodology Applied

Scientific EffectHomology-independent targeted integration:

Data Source

PatentUS20230357795A1AAV-mediated homology-independent targeted integration gene editing for correction of diverse DMD mutations in patients with muscular dystrophy

Publication Date: 2023.11.09 RES INST AT NATIONWIDE CHILDRENS HOSPITAL

AI summary

Disclosed herein are products, methods, and uses for a new gene therapy for treating, ameliorating, delaying the progression of, and/or preventing a muscular dystrophy involving a mutation amenable to DNA repair including, but not limited to, any mutation involving, surrounding, or affecting various regions of the DMD gene. Specifically, the disclosure provides products and methods for fixing diverse DMD mutations by replacement of large segments of the DMD gene comprising multiple exons, using CRISPR/Cas9 and Homology-Independent Targeted-Integration (HITI) to accomplish high efficiency knock-in or make large replacements using the non-homologous end-joining (NHEJ) DNA repair pathway, previously not achievable. In particular, the disclosure provides products, methods and uses for the replacement of DMD exons 1-19, 2-19, or 41-55.