Rep Plasmid Accessory Protein Layout for High-Potency rAAV Production

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Solution Overview

Problem

Current recombinant adeno-associated virus (rAAV) vector production methods face challenges in achieving high titers with high potency, flexibility for various gene therapies, improved economics, and control over full versus empty rAAV particles, leading to reduced transduction efficiency and effective treatment of genetic disorders.

Innovation Solution

A rep plasmid comprising adeno-associated virus replication protein coding sequences and accessory protein coding sequences without encoding a functional cap protein, combined with a cap plasmid encoding capsid proteins, to enhance rAAV vector production and transduction efficiency.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Adaptability or versatility

If a rep/cap split plasmid system is used for rAAV production, then flexibility for various gene therapies is improved, but transduction efficiency deteriorates

Engineering Contradiction:

Improveflexibility for various gene therapiesVSAvoidtransduction efficiency

Core Design Contradiction:

Adaptability or versatilityVSReliability

Solution Approach 1:

The patent divides the helper plasmid into two separate plasmids: a rep plasmid containing Rep proteins and accessory proteins (AAP, MAAP, X), and a cap plasmid containing capsid proteins. This segmentation maintains the flexibility of the split system while restoring transduction efficiency by ensuring all necessary viral components are properly provided.

Inventive Principle:

Principle #1Segmentation

Solution Approach 2:

The accessory proteins (particularly AAP and MAAP) act as intermediaries that mediate between the Rep proteins and the capsid proteins, facilitating proper viral assembly and function. This intermediary role restores the coordination lost in traditional split systems.

Inventive Principle:

Principle #24Intermediary (Mediator)

2Reliability

If accessory proteins are added to the rep plasmid, then transduction efficiency is improved, but plasmid complexity increases

Engineering Contradiction:

Improvetransduction efficiencyVSAvoidplasmid structure

Core Design Contradiction:

ReliabilityVSDevice complexity

Solution Approach 1:

The patent merges the accessory protein coding sequences with the rep protein coding sequences into a single rep plasmid. This combining approach improves transduction efficiency by ensuring proper stoichiometry and coordination of viral proteins, while the modular design keeps the plasmid manageable.

Inventive Principle:

Principle #5Merging (Combining)

3Productivity

If high-titer rAAV particles are produced, then productivity is improved, but control over full versus empty particles deteriorates

Engineering Contradiction:

ImproverAAV titerVSAvoidcontrol over full versus empty particles

Core Design Contradiction:

ProductivityVSManufacturing precision

Solution Approach 1:

The accessory proteins provide feedback mechanisms that monitor and regulate the viral assembly process. AAP and MAAP proteins help ensure proper genome packaging by monitoring capsid formation and preventing assembly of empty particles, thus maintaining precision even at high titers.

Inventive Principle:

Principle #23Feedback

Data Source

PatentEP4671264A1AAV plasmids comprising an accessory protein coding sequence

Publication Date: 2025.12.31 SARTORIUS STEDIM CELLCA GMBH

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AI summary

The invention relates to a rep plasmid comprising at least one adeno-associated virus replication protein coding sequence encoding at least one functional rep protein, and at least one accessory protein coding sequence, wherein the rep plasmid is not capable of encoding a functional cap protein.