AAV Vector Stuffer Sequences for Packaging Efficiency
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Solution Overview
Problem
Adeno-associated virus (AAV) vector genomes face challenges with packaging efficiency due to size constraints, leading to reduced production of functional vectors when larger than native size, and increased unintended 'reverse packaged' sequences when smaller, necessitating the use of 'stuffer sequences' to maintain optimal packaging and minimize toxicity.
Innovation Solution
Incorporation of stuffer sequences within the plasmid backbone that are inert and do not confer unfavorable properties, specifically designed to be around 1300-2300 nucleotides in length, with high identity to specific sequences, to maintain vector genome size near the natural packaging limit and reduce toxicity, while minimizing truncations and adverse effects.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If the AAV vector genome size is increased beyond the native size, then the payload capacity is improved, but the production of functional AAV vectors is reduced
Solution Approach 1:
The vector genome is segmented into functional payload regions and inert stuffer regions. The stuffer sequences serve as inert filler material that occupies space within the capsid without contributing harmful biological activity, allowing the vector to reach optimal packaging size while maintaining production efficiency
Solution Approach 2:
The patent optimizes the length and composition of stuffer sequences to achieve the desired genome size parameter. By carefully controlling the stuffer sequence length (typically 100-500 nucleotides), the vector genome size is adjusted to maximize packaging efficiency while maintaining functional vector production
2Productivity
If the AAV vector genome size is reduced below the packaging limit, then the production efficiency is improved, but the packaging becomes suboptimal
Solution Approach 1:
Stuffer sequences are pre-designed and incorporated into the vector construct before packaging. These sequences are specifically engineered to be inert and non-toxic, providing the necessary bulk to reach optimal packaging size without requiring post-packaging adjustments
3Ease of manufacture
If the plasmid backbone contains antibiotic resistance genes close in size to the intended AAV vector genome, then the plasmid can be propagated in bacteria, but the amount of unintended reverse packaged sequence increases
Solution Approach 1:
The patent extracts or removes potentially harmful sequences from the plasmid backbone, such as antibiotic resistance genes and origin of replication sequences, that could be inadvertently packaged into the AAV vector. This is achieved by designing the plasmid backbone to minimize the presence of such sequences or by using alternative selection markers
Solution Approach 2:
The patent converts the potential harm of plasmid backbone sequences by using them as a source for designing inert stuffer sequences. The stuffer sequences are derived from plasmid regions but are modified to remove harmful elements while retaining the structural properties needed for optimal packaging
4Productivity
If inert stuffer sequences are used to maintain optimal packaging size, then the packaging efficiency is improved, but the risk of toxicity from payload-free AAV increases
Solution Approach 1:
The patent applies local quality by ensuring that only specific regions of the vector genome (the stuffer sequences) are inert, while the payload regions retain their functional properties. The stuffer sequences are specifically designed to lack open reading frames and regulatory elements that could cause toxicity, while the payload regions maintain their therapeutic function
Data Source
AI summary
The present disclosure provides vector stuffer polynucleotides and compositions thereof, including expression constructs and vectors, such as viral vectors and methods of delivering a therapeutic agent (e.g., inhibitory nucleic acid) to a mammal or treating a disease.