Hybrid AAV-Transposon Vector for Stable Gene Integration

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Solution Overview

Problem

Current AAV-based vectors face challenges in maintaining stable transgene expression in proliferating cells, such as those in the juvenile liver and bone marrow, limiting their effectiveness in treating genetic diseases like pediatric liver diseases and cancer.

Innovation Solution

A method involving recombinant AAV vectors flanked by transposon-derived inverted terminal repeat sequences, combined with a transposase that recognizes these sequences, to facilitate genomic integration and stable expression of transgenes in proliferating cells.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Productivity

If recombinant AAV vectors are used for transduction of proliferating cells, then transduction efficiency is improved, but stable transgene expression is lost

Engineering Contradiction:

Improvetransduction efficiencyVSAvoidstable transgene expression

Core Design Contradiction:

ProductivityVSReliability

Solution Approach 1:

The patent combines AAV vector system with transposon elements (such as piggyBac, Sleeping Beauty, or Tc1) to create a hybrid vector system. The AAV vector delivers the transgene flanked by transposon-derived inverted terminal repeats, and a transposase enzyme mediates the integration of the transgene into the host genome. This merging of AAV transduction capability with transposon integration capability resolves the contradiction by maintaining high transduction efficiency while achieving stable long-term expression.

Inventive Principle:

Principle #5Merging (Combining)

Solution Approach 2:

The patent introduces transposase as an intermediary enzyme that facilitates the integration of the transgene into the host genome. The transposase recognizes the transposon-derived inverted terminal repeat sequences and mediates the actual genomic integration process. This intermediary mechanism enables stable integration without requiring the transgene to integrate directly through AAV, thus resolving the stability issue while maintaining transduction efficiency.

Inventive Principle:

Principle #24Intermediary (Mediator)

2Duration of action of moving object

If episomal AAV vectors are used in proliferating cells, then initial transgene expression is achieved, but vector loss and elimination occur rapidly

Engineering Contradiction:

Improveinitial transgene expression durationVSAvoidvector loss time

Core Design Contradiction:

Duration of action of moving objectVSLoss of time

Solution Approach 1:

The patent performs preliminary action by pre-flanking the transgene with transposon-derived inverted terminal repeat sequences before using the AAV vector for delivery. This preliminary preparation ensures that when the transposase is expressed, the transgene is ready for immediate and stable integration into the host genome. This prevents the vector loss issue by ensuring the transgene is permanently integrated rather than maintained as a transient episome.

Inventive Principle:

Principle #10Preliminary action

3Ease of manufacture

If AAV vectors are used to treat genetic diseases in proliferating cells, then transgene delivery is achieved, but therapeutically effective expression levels are not maintained

Engineering Contradiction:

Improvetransgene deliveryVSAvoidtherapeutically effective expression

Core Design Contradiction:

Ease of manufactureVSReliability

Solution Approach 1:

The patent changes the key parameter of gene integration mechanism from episomal maintenance to genomic integration. By using transposon-derived inverted terminal repeats and transposase, the system transforms the transgene delivery outcome from transient episomal expression to permanent genomic integration. This parameter change ensures that therapeutically effective expression levels are maintained long-term in proliferating cells, resolving the reliability issue while maintaining ease of delivery.

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 significantly increases stable transgene expression in proliferating cells, enabling effective treatment of diseases like OTC deficiency, ASS deficiency, and progressive familial intrahepatic cholestasis, and shows promise in cancer and bone marrow diseases by ensuring long-term transgene expression.

Implementation Method 1

a source of a transposase that recognises said transposon-derived inverted terminal repeat sequences and directs the genomic integration of the transgene into the genome of the proliferating cell

Methodology Applied

Scientific EffectTransposase-mediated genomic integration:

Data Source

PatentUS11351272B2Stable gene transfer to proliferating cells

Publication Date: 2022.06.07 MOUNT SINAI HOSPITAL

AI summary

Provided herein are methods for facilitating or inducing stable transgene integration and expression in a proliferating cell, comprising administering to the cell (i) a recombinant AAV (rAAV) vector comprising the transgene flanked by transposon-derived inverted terminal repeat sequences, which sequences are in turn flanked by AAV-derived inverted terminal repeat regions, and (ii) a source of a transposase that recognises said transposon-derived inverted terminal repeat sequences and directs the genomic integration of the transgene into the genome of the proliferating cell. Also provide are methods and transgene delivery systems for the treatment or prevention of diseases affecting, associated with or characterised by proliferating cells.