Muscle-Specific Regulatory Cassettes for Skeletal-Selective AAV Expression

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Solution Overview

Problem

AAV-mediated gene therapies for muscle disorders face challenges due to cardiotoxicity from transgene expression in the heart and the need for smaller regulatory cassettes that provide high activity in skeletal muscles with minimal or no activity in cardiac muscle.

Innovation Solution

Development of muscle-specific regulatory cassettes comprising modified enhancer sequences, including Trex, AT-rich, and MEF2 sequences, linked to a −80 to +50 promoter, which provide high expression in skeletal muscle and low expression in cardiac muscle.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Productivity

If conventional regulatory cassettes are used to drive high-level transgene expression in skeletal muscle, then expression activity is improved, but cardiotoxicity increases due to transgene expression in the heart

Engineering Contradiction:

Improvetransgene expression activityVSAvoidcardiotoxicity

Core Design Contradiction:

ProductivityVSObject-affected harmful factors

Solution Approach 1:

The regulatory cassette is engineered with tissue-specific enhancer elements (MEF2, E-box, AT-rich sequences) that create different expression patterns in different muscle types. The cassette provides high expression in skeletal muscle while maintaining low expression in cardiac muscle, thereby achieving high productivity in the target tissue without the harmful cardiotoxicity effect

Inventive Principle:

Principle #3Local quality

2Device complexity

If regulatory cassettes are minimized to reduce size for AAV packaging, then device complexity is reduced, but expression activity may be compromised

Engineering Contradiction:

Improveregulatory cassette sizeVSAvoidexpression activity

Core Design Contradiction:

Device complexityVSProductivity

Solution Approach 1:

The invention extracts and retains only the essential regulatory elements (three modified enhancer sequences and core promoter) needed for high skeletal muscle expression, removing non-essential sequences. This minimization reduces cassette size for AAV packaging while preserving high expression activity through the optimized core elements

Inventive Principle:

Principle #2Taking out (Extraction)

Solution Approach 2:

The enhancer sequences are modified with specific parameter changes including altered E-box orientations, adjusted spacing between elements, and optimized AT-rich regions. These parameter optimizations maintain high expression activity in the minimized cassette structure

Inventive Principle:

Principle #35Parameter changes

3Productivity

If existing muscle-specific cassettes are used, then skeletal muscle expression is achieved, but the cassette size is larger than required for optimal AAV packaging

Engineering Contradiction:

Improveskeletal muscle expressionVSAvoidcassette size

Core Design Contradiction:

ProductivityVSWeight of moving object

Solution Approach 1:

The regulatory cassette is segmented into three modified enhancer sequences positioned upstream of the promoter, with each sequence containing specific elements (Trex, AT-rich, E-box, MEF2). This segmentation allows for compact arrangement and minimization of total cassette size while maintaining functional integrity and high skeletal muscle expression

Inventive Principle:

Principle #1Segmentation

Data Source

PatentUS20260021200A1Muscle-specific regulatory cassettes

Publication Date: 2026.01.22 NEVADA RESEARCH & INNOVATION CORP

AI summary

The present disclosure relates to nucleic acid regulatory cassettes useful for the expression of transgene payloads in specific muscle tissues. Also included in the present disclosure are vectors, including adeno-associated virus (AAV) vectors, comprising the regulatory cassettes of the invention, as well as compositions and methods of treating genetic diseases or disorders also comprising said regulatory cassettes.