Modified Parvovirus ITRs Limit Viral Vector Mobilization

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Solution Overview

Problem

Current AAV vectors using ITR2s for therapeutic purposes pose a safety risk due to the ubiquity of AAV2 in the human population, leading to potential vector mobilization out of target tissues or into other individuals.

Innovation Solution

Development of modified parvovirus inverted terminal repeats (ITRs) that do not functionally interact with wild-type large Rep proteins, along with synthetic Rep proteins that interact specifically with these modified ITRs, to create novel Rep-ITR interactions that prevent vector mobilization.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Reliability

If modified ITRs are used to prevent vector mobilization, then safety is improved, but compatibility with wild-type Rep proteins deteriorates

Engineering Contradiction:

ImprovesafetyVSAvoidcompatibility

Core Design Contradiction:

ReliabilityVSAdaptability or versatility

Solution Approach 1:

The ITR is divided into functional domains (nicking stem, RBE, spacer) where specific segments are modified to create Rep-ITR specificity. The nicking stem sequence is altered to prevent recognition by wild-type Rep proteins while maintaining interaction with synthetic Rep proteins, thereby segmenting the ITR function to achieve both safety and controlled compatibility.

Inventive Principle:

Principle #1Segmentation

Solution Approach 2:

Specific local modifications are introduced at critical interaction sites within the ITR, particularly at the nicking stem and RBE regions. These localized changes create selective recognition patterns that distinguish modified ITRs from wild-type ITRs, enabling safety through specificity while preserving necessary functional interactions with designed Rep proteins.

Inventive Principle:

Principle #3Local quality

2Reliability

If synthetic Rep proteins are designed to interact specifically with modified ITRs, then vector mobilization is limited, but system complexity increases

Engineering Contradiction:

ImprovesafetyVSAvoidsystem complexity

Core Design Contradiction:

ReliabilityVSDevice complexity

Solution Approach 1:

The amino acid sequence parameters of Rep proteins are specifically modified to recognize altered ITR sequences. By changing key residues in the Rep protein that contact the ITR, synthetic Rep proteins achieve selective binding to modified ITRs while avoiding wild-type ITRs, thereby limiting vector mobilization through parameter optimization rather than system redesign.

Inventive Principle:

Principle #35Parameter changes

3Reliability

If ITR modifications are introduced to prevent interaction with wild-type Rep proteins, then vector spread is limited, but manufacturing flexibility is reduced

Engineering Contradiction:

ImprovesafetyVSAvoidmanufacturing flexibility

Core Design Contradiction:

ReliabilityVSEase of manufacture

Solution Approach 1:

The ITR modifications are pre-designed and incorporated into the vector construct before production. The modified ITR sequences are established as fixed elements in the plasmid design, allowing standard manufacturing processes to be used while the pre-built specificity prevents unwanted Rep-ITR interactions during production and after delivery.

Inventive Principle:

Principle #10Preliminary action

Data Source

PatentUS11939597B2Restrictive inverted terminal repeats for viral vectors

Publication Date: 2024.03.26 THE UNIV OF NORTH CAROLINA AT CHAPEL HILL

AI summary

This invention relates to modified parvovirus inverted terminal repeats (ITRs) that do not functionally interact with wild-type large Rep proteins, synthetic Rep proteins that functionally interact with the modified ITRs, and methods of using the same for delivery of nucleic acids to a cell or a subject. The modifications provide a novel Rep-ITR interaction that limits vector mobilization, increasing the safety of viral vectors.