Split-Intein Nucleobase Editors for AAV Packaging Limits
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Solution Overview
Problem
The large size of Cas9 and Cas9-based genome-editing agents restricts their efficient delivery via recombinant adeno-associated virus (rAAV) due to packaging size limits, hindering the application of base editors in gene therapy, particularly for point mutations in living animals.
Innovation Solution
A split-base editor dual AAV strategy is employed, where the base editor is divided into N-terminal and C-terminal halves fused with fast-splicing split-inteins, allowing co-infection and protein splicing to form a full-length, functional base editor within cells, bypassing the packaging size limit of AAV.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If base editors are packaged in AAV for in vivo delivery, then gene therapy application is enabled, but the packaging size limit of AAV (≤5 kb) prevents inclusion of full-length base editor genes (5.2 kb for SpCas9-based editors)
Solution Approach 1:
The base editor is divided into two separate polypeptide fragments (N-terminal fragment and C-terminal fragment), each encoded by a separate AAV genome. This segmentation allows each fragment to be packaged within the AAV size limit while collectively delivering the complete base editor functionality when both fragments are expressed in the same cell.
Solution Approach 2:
The invention uses a self-splicing intein mechanism where the N-terminal fragment contains an N-terminal intein and the C-terminal fragment contains a C-terminal intein. When both fragments are expressed, the inteins mediate spontaneous protein splicing to reconstitute the full-length base editor, effectively nesting the complete functional protein within the combined output of two smaller AAV-delivered fragments.
2Productivity
If the base editor gene size is reduced to fit AAV packaging, then delivery efficiency improves, but the editing functionality and robustness may be compromised
Solution Approach 1:
By segmenting the base editor into two fragments that are delivered separately by AAV, the invention achieves efficient packaging within size limits while maintaining complete editing functionality through in vivo reconstitution of the full-length protein.
Solution Approach 2:
The intein-mediated splicing system is self-contained and autonomously reconstitutes the full-length base editor within the target cell without requiring external assistance, cell line-specific factors, or additional delivery steps, ensuring reliable functionality across diverse cell types.
3Adaptability or versatility
If split-base editor fragments are delivered via dual AAV co-infection, then packaging size limit is bypassed, but the complexity of delivery system increases
Solution Approach 1:
The intein sequences serve as molecular intermediaries that facilitate the joining of the two separately delivered base editor fragments. The inteins mediate the splicing process, acting as a bridge between the N-terminal and C-terminal fragments to reconstitute the functional base editor.
Solution Approach 2:
The dual AAV system leverages the self-splicing capability of inteins to automatically reconstitute the full-length base editor upon co-expression, eliminating the need for complex external assembly procedures, cell line engineering, or additional delivery vehicles.
4Manufacturing precision
If intein splicing is used to reconstitute full-length base editor, then a native peptide bond is regenerated without exogenous sequences, but the delivery requires coordinated expression of multiple components
Solution Approach 1:
The intein sequences are strategically inserted at the N- and C-termini of the base editor fragments, allowing them to be 'taken out' or removed through self-splicing. This extraction of the intein sequences during the splicing process leaves behind a clean, native peptide bond joining the base editor fragments without any exogenous amino acid residues.
Solution Approach 2:
The inteins function as temporary mediators that facilitate the joining of base editor fragments and are subsequently removed, ensuring precise reconstitution of the native protein sequence without permanent alteration or inclusion of foreign sequences.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
This approach enables efficient base editing in somatic tissues, achieving therapeutically relevant editing thresholds in tissues like liver, heart, muscle, retina, and brain, and has shown efficacy in treating diseases such as Niemann-Pick disease type C and congenital deafness by correcting causal mutations in vivo.
Implementation Method 1
protein splicing in trans reconstitutes full-length nucleobase editor
Implementation Method 2
intein splicing removes all exogenous sequences and regenerates a native peptide bond at the split site
Data Source
AI summary
Provided herein are methods of delivering “split” Cas9 protein or nucleobase editors into a cell, e.g., via a recombinant adeno-associated vims (rAAV), to form a complete and functional Cas9 protein or nucleobase editor. The Cas9 protein or the nucleobase editor is split into two sections, each fused with one part of an intein system (e.g., intein-N and intein-C encoded by the dnaE-n and dnaE-c genes, respectively). Upon co-expression, the two sections of the Cas9 protein or nucleobase editor are ligated together via intein-mediated protein splicing. Nucleic acid molecules encoding the N-terminal portion of a Cas9 protein or a nucleobase editor fused to an intein, and nucleic acid molecules encoding the C-terminal portion of a Cas9 protein or nucleobase editor, are provided. Recombinant AAV vectors (e.g, vectors comprising one or more of these nucleic acid molecules each comprising an intein) and particles for the delivery of the split Cas9 protein or nucleobase editor, compositions comprising such AAV vectors and particles, and methods of using such rAAV vectors and particles are also provided. Methods of administering such compositions and AAV particles to a subject are further provided. Cells and compositions comprising these nucleic acid molecules rAAV vectors, and rAAV particles are also provided.