Intron Cassette Design for Cell-Type Specific Gene Expression

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Solution Overview

Problem

Current gene therapy vectors face challenges in ensuring precise delivery to specific cell types, leading to off-target effects and toxicity due to non-specific gene expression, which existing methods like viral coat proteins and custom minimal promoters cannot adequately address.

Innovation Solution

The development of nucleic acid constructs incorporating a start codon and an intron cassette with cell-specific exon sequences, splice donor and acceptor sites, and branch sites, allowing for alternative splicing to restrict gene expression to specific cell types by utilizing evolutionarily conserved alternative exons, enabling precise targeting through splicing-linked expression design (SLED).

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Manufacturing precision

If viral coat proteins or custom minimal promoters are used to target specific cell types, then cell type specificity is improved, but these methods have significant limitations and cannot adequately address off-target delivery

Engineering Contradiction:

Improvecell type specificityVSAvoidoff-target delivery control

Core Design Contradiction:

Manufacturing precisionVSReliability

Solution Approach 1:

The invention changes the parameter of gene expression control from transcriptional level (promoters) to post-transcriptional level (splicing). By modifying splicing patterns through intron cassette design, the system achieves more precise cell type specificity that overcomes the limitations of traditional promoter-based approaches.

Inventive Principle:

Principle #35Parameter changes

Solution Approach 2:

The invention introduces an intermediary mechanism (intron cassette with cell-specific exon sequences) between gene delivery and gene expression. This intermediary controls whether the therapeutic gene is expressed in the target cell type, allowing viral vectors to deliver genes to specific cell types while preventing off-target expression through splicing regulation.

Inventive Principle:

Principle #24Intermediary (Mediator)

2Adaptability or versatility

If genes are delivered to non-specific cell types, then gene therapy coverage is improved, but off-target delivery leads to side effects and toxicity

Engineering Contradiction:

Improvegene therapy coverageVSAvoidside effects and toxicity

Core Design Contradiction:

Adaptability or versatilityVSObject-affected harmful factors

Solution Approach 1:

The invention segments the gene expression control into two independent functions: (1) viral vector-mediated gene delivery to target cells, and (2) splicing-regulated gene expression within those cells. This segmentation allows broad delivery coverage while restricting harmful expression to only the intended cell type through cell-specific splicing patterns.

Inventive Principle:

Principle #1Segmentation

Solution Approach 2:

The invention converts the previously harmful phenomenon of non-specific gene expression into a beneficial feature by using the cell type-specific splicing patterns that naturally exist in different cells. The splicing machinery's cell type-specificity, which was not previously exploited, becomes the mechanism for ensuring safe and selective gene expression.

Inventive Principle:

Principle #22Blessing in disguise (Convert harm into benefit)

3Manufacturing precision

If alternative splicing is used to control gene expression specificity, then cell type specificity is improved, but the mechanism complexity increases

Engineering Contradiction:

Improvegene expression specificityVSAvoidnucleic acid construct complexity

Core Design Contradiction:

Manufacturing precisionVSDevice complexity

Solution Approach 1:

The invention uses universal splicing mechanisms (splice donor sites, branch sites, acceptor sites) that are naturally present in all eukaryotic cells. By designing intron cassettes that exploit these universal splicing components, the system achieves cell type-specific gene expression without requiring cell type-specific machinery, thereby managing complexity while maintaining high specificity.

Inventive Principle:

Principle #6Universality (Multi-functionality)

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 ensures precise and cell-type specific gene expression, reducing off-target effects and enhancing the safety and efficacy of gene therapy by using alternative splicing to couple translational reading frames with cell-type specificity, compatible across species and applicable in various delivery methods including AAV and lentivirus.

Implementation Method 1

Strategies to overcome this issue include using different viral coat proteins or custom minimal promoters. However, these methods have significant limitations.

Methodology Applied

Scientific EffectAlternative splicing:

Data Source

PatentUS20240108756A1Compositions and methods for using alternative splicing to control specificity of gene therapy

Publication Date: 2024.04.04 JOHNS HOPKINS UNIVERSITY

AI summary

Disclosed herein are compositions and methods that can be used to express a nucleotide sequence in a specific cell type. The compositions can comprise nucleic acid constructs comprising a start codon; and an intron cassette. The intron cassette can comprise a cell specific exon sequence, a splice donor site, a branch site, and an acceptor site. The cell specific exon sequence is out of frame with the start codon and comprises one or more frameshift mutations. The compositions can be used to treat human diseases.