Synthetic Polypeptides for AAV Delivery of Split Gene-Editing Payloads
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Solution Overview
Problem
The challenge of delivering large polynucleotide payloads, such as those encoding polypeptides, using adeno-associated viruses (AAVs) or other vectors with limited packaging capacities is a significant hurdle in genome editing applications.
Innovation Solution
The use of synthetic polypeptides, including trans-splicing inteins and their functional fragments, along with encoding polynucleotides, to facilitate delivery of polynucleotides to cells using vectors like AAVs, enabling the assembly of split polypeptides within cells to achieve efficient gene editing.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If large polynucleotide payloads are delivered using AAV vectors, then gene editing objectives can be met, but the limited packaging capacity of AAVs prevents successful delivery
Solution Approach 1:
The polynucleotide payload is divided into multiple smaller segments that can be individually packaged within AAV vectors. Each segment contains a portion of the genetic material, and multiple AAV vectors work together to deliver the complete set of segments to the target cell, overcoming the packaging capacity limitation while achieving delivery of large polynucleotide payloads.
Solution Approach 2:
Multiple polynucleotide segments are organized in a hierarchical structure where smaller functional units are nested within larger organizational frameworks. This allows efficient packing of substantial genetic material into the constrained AAV vector capacity by arranging segments in nested configurations that maximize space utilization.
2Productivity
If all elements required for gene editing are packaged into a single AAV vector, then delivery efficiency improves, but the packaging capacity is exceeded
Solution Approach 1:
Multiple AAV vectors, each carrying different polynucleotide segments, are combined to deliver the complete gene editing toolkit. The vectors work synergistically, with each contributing a necessary component, thereby achieving high delivery efficiency for the full set of editing elements without requiring any single vector to exceed its packaging capacity.
Solution Approach 2:
The system uses multiple AAV vectors that collectively perform the function of delivering a complete gene editing payload. Each vector is designed to carry specific functional modules (e.g., Cas9, gRNA, payload), and together they provide a universal solution for delivering large genetic constructs that would otherwise not fit in a single vector.
3Device complexity
If the polynucleotide payload is reduced to fit AAV packaging capacity, then delivery becomes feasible, but the functional completeness of the gene editing system is compromised
Solution Approach 1:
The complete gene editing system is segmented into multiple functional modules, each fitting within AAV packaging capacity. Critical elements such as Cas9, guide RNA, and payload sequences are divided into separate deliverable units that maintain their individual functionality while collectively providing the complete gene editing capability when assembled in the target cell.
Solution Approach 2:
The polynucleotide segments are pre-assembled into organized structures within each AAV vector before delivery. This preliminary organization ensures that when the vectors infect the target cell, the segments are already positioned and prepared for efficient assembly into the complete functional gene editing system, thereby maintaining reliability without requiring full payload in a single vector.
Data Source
AI summary
Provided herein are synthetic polypeptides including trans-splicing inteins, functional fragments thereof, and polynucleotides encoding the same for use in systems, compositions, kits, and methods for delivering one or more polynucleotides (e.g., polynucleotides encoding a split polypeptide) to a cell using a vector (e.g., a viral vector, such as an adeno-associated virus vector) having limited packaging capacity.