Dual-AAV Vector System for Full-Length PCDH15 Expression

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Solution Overview

Problem

Current treatments for Usher Syndrome type 1F, caused by PCDH15 mutations, are limited to cochlear implants, and there is no effective treatment for the associated blindness, as the PCDH15 coding sequence is too large to fit into a single AAV capsid, preventing wild-type PCDH15 expression in inner ear and eye cells.

Innovation Solution

A dual-AAV vector system is employed, where two isolated nucleic acids, each encoding a portion of the PCDH15 protein, are used to form a full-length PCDH15 coding sequence in target cells through trans splicing or homologous recombination, enabling expression of the PCDH15 protein in the inner ear and eye.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Quantity of substance

If a single AAV capsid is used for gene delivery, then the delivery system is simple and well-characterized, but the PCDH15 coding sequence cannot be accommodated due to size limitations

Engineering Contradiction:

Improvetransgene capacityVSAvoidvector system complexity

Core Design Contradiction:

Quantity of substanceVSDevice complexity

Solution Approach 1:

The PCDH15 coding sequence is divided into two separate fragments, each fitting within one AAV capsid. The first fragment contains the 5' portion of the gene and the second fragment contains the 3' portion. When both AAV vectors infect the same cell, the fragments are reconstituted to form the complete PCDH15 coding sequence, enabling expression of the full-length protein while using standard AAV delivery systems.

Inventive Principle:

Principle #1Segmentation

2Reliability

If the PCDH15 coding sequence is truncated to fit in a single AAV capsid, then the gene can be delivered, but the full-length wild-type PCDH15 protein cannot be expressed

Engineering Contradiction:

Improvefunctional protein expressionVSAvoidcoding sequence length

Core Design Contradiction:

ReliabilityVSLength of moving object

Solution Approach 1:

The complete PCDH15 coding sequence is segmented into two portions that can each be packaged into separate AAV vectors. The first AAV vector contains the 5' portion including the promoter and early coding regions, while the second AAV vector contains the 3' portion including the remaining coding sequence and polyadenylation signal. Together, these segments reconstruct the full-length functional gene.

Inventive Principle:

Principle #1Segmentation

3Quantity of substance

If dual-AAV vectors are used to deliver the complete PCDH15 coding sequence, then full-length protein expression is achieved, but the vector system complexity increases

Engineering Contradiction:

Improvecomplete PCDH15 coding sequenceVSAvoiddual-vector system

Core Design Contradiction:

Quantity of substanceVSDevice complexity

Solution Approach 1:

The PCDH15 gene is split into two manageable segments that can be independently packaged into separate AAV vectors. This segmentation allows each vector to maintain optimal size for efficient transduction while collectively delivering the complete coding sequence. The segments are designed with compatible boundaries that enable proper reconstitution of the open reading frame.

Inventive Principle:

Principle #1Segmentation

Solution Approach 2:

The invention uses cellular machinery as an intermediary to reconstitute the full-length PCDH15 coding sequence from the two separate AAV-delivered fragments. The cell's transcription and translation systems process both vectors simultaneously, and the fragments are assembled into a complete functional gene through natural cellular mechanisms, eliminating the need for complex in vitro reconstitution procedures.

Inventive Principle:

Principle #24Intermediary (Mediator)

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 allows for the expression of full-length PCDH15 protein in target cells, potentially rescuing hearing and vision loss associated with Usher Syndrome type 1F, as demonstrated by improved auditory function and hair bundle morphology in animal models.

Implementation Method 1

two isolated nucleic acids, each encoding a portion of the PCDH15 protein, are used to form a full-length PCDH15 coding sequence in target cells through trans splicing or homologous recombination

Methodology Applied

Scientific EffectTrans splicing:

Implementation Method 2

two isolated nucleic acids, each encoding a portion of the PCDH15 protein, are used to form a full-length PCDH15 coding sequence in target cells through trans splicing or homologous recombination

Methodology Applied

Scientific EffectHomologous recombination:

Data Source

PatentUS20240016955A1Dual-AAV vector delivery of PCDH15 and uses thereof

Publication Date: 2024.01.18 PRESIDENT & FELLOWS OF HARVARD COLLEGE

AI summary

The present disclosure, at least in part, provides a dual-AAV vector system and compositions thereof for expression full-length PCDH15 in target cells. The present disclosure also provides the method of using the dual rAAV system for delivering full-length PCDH15 to a target cell (e.g., inner cells or cells in the eye) for treating deafness and/or blindness (e.g., Usher syndrome 1F).