Modified ITR Sequences Prevent Host Protein Binding in Gene Therapy

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Solution Overview

Problem

Adeno-associated virus (AAV) gene therapy vectors face limitations due to the inhibitory effects of host cell proteins binding to the T-shaped hairpin structures of inverted terminal repeats (ITRs), which hinder persistent and efficient transgene expression.

Innovation Solution

The use of modified first and second inverted terminal repeats (ITRs) that are shorter or alternative to wild-type ITRs, derived from parvoviruses like B19 or goose parvovirus (GPV), which flank a genetic cassette containing a heterologous polynucleotide sequence, to enhance expression while avoiding T-shaped hairpin formation and subsequent protein inhibition.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Reliability

If wild-type AAV ITRs are used, then viral replication and packaging are maintained, but host cell proteins bind to T-shaped hairpin structures and inhibit transgene expression

Engineering Contradiction:

Improveviral replication and packagingVSAvoidhost cell protein binding to T-shaped hairpin structures

Core Design Contradiction:

ReliabilityVSObject-generated harmful factors

Solution Approach 1:

The patent extracts and removes the problematic T-shaped hairpin structure from the ITR sequence while retaining the essential functional elements for viral replication and packaging. This is achieved by deleting specific nucleotide sequences that form the hairpin structure, thereby eliminating the binding site for host cell proteins that cause inhibition.

Inventive Principle:

Principle #2Taking out (Extraction)

Solution Approach 2:

The patent changes the nucleotide sequence parameters of the ITR to alter its structural properties. By modifying the sequence to prevent T-shaped hairpin formation while maintaining other functional characteristics, the patent resolves the contradiction between viral replication and avoiding host protein binding.

Inventive Principle:

Principle #35Parameter changes

2Productivity

If modified ITRs are used to prevent host protein binding, then transgene expression is improved, but viral replication and packaging efficiency may be compromised

Engineering Contradiction:

Improvetransgene expression efficiencyVSAvoidviral replication and packaging

Core Design Contradiction:

ProductivityVSReliability

Solution Approach 1:

The patent applies local quality changes by modifying only specific regions of the ITR sequence that are responsible for hairpin formation, while leaving other essential functional regions intact. This localized modification allows prevention of host protein binding without compromising overall viral replication and packaging functions.

Inventive Principle:

Principle #3Local quality

Solution Approach 2:

The patent creates a composite ITR structure that combines elements from different viral sources or uses chimeric sequences that integrate functional domains for viral replication with modified regions that prevent host protein binding, achieving both improved expression and maintained viral functions.

Inventive Principle:

Principle #40Composite materials

Data Source

PatentUS20220243201A1Engineered ITR sequences and methods of use

Publication Date: 2022.08.04 BIOVERATIV THERAPEUTICS INC

AI summary

The present disclosure provides nucleic acid molecules comprising a first inverted terminal repeat (ITR), a second ITR, and a genetic cassette encoding a target sequence. In some embodiments, the first ITR and/or the second ITR is an ITR of a non-adeno-associated virus (AAV). Also disclosed are methods of using the nucleic acid molecules in gene therapy applications.