Sequential Stacking of Nucleic Acid Sequences into Genomic Locus
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Solution Overview
Problem
Current viral vectors, such as adeno-associated virus (AAV), have limited DNA cargo capacity, restricting the size of genetic material that can be effectively incorporated into a genome for gene editing, particularly for applications requiring larger transgenes.
Innovation Solution
The use of two recombinant viruses, each carrying a portion of a long transgene, allows for the sequential introduction of nucleic acid sequences into a cell's genome, exceeding the packaging capacity of a single virus by utilizing engineered nucleases and specific nucleic acid sequences for targeted insertion.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If a single AAV vector is used to deliver transgene DNA, then the vector can efficiently deliver genetic material into host cells, but the size of the transgene is limited by the 4.7 kb packaging capacity of the virus
Solution Approach 1:
The patent divides the transgene into multiple segments that can be accommodated within the 4.7 kb packaging capacity of individual AAV vectors. Multiple AAV vectors, each carrying a different segment, are co-delivered to the same host cell. The segments are then assembled into a complete transgene through homologous recombination, enabling delivery of transgenes larger than the capacity of a single vector while maintaining the simplicity of using standard AAV delivery systems.
2Quantity of substance
If the DNA cargo size is increased to exceed 4.7 kb, then larger transgenes can be delivered, but the efficiency of viral packaging and transduction decreases
Solution Approach 1:
By segmenting the large transgene into smaller pieces that each fit within the 4.7 kb capacity, the patent ensures that each individual AAV vector maintains high packaging efficiency and transduction capability. The segmented approach allows the system to deliver large overall DNA cargo sizes while each vector component retains optimal transduction efficiency.
Solution Approach 2:
The patent employs a nested strategy where multiple AAV vectors, each containing a specific segment of the transgene flanked by homology arms, are delivered simultaneously to the same cell population. The vectors nest their functions within the same cellular target, and the homology arms enable the segments to be assembled into the complete transgene structure through homologous recombination, ensuring reliable integration of the full-length transgene.
3Quantity of substance
If multiple AAV vectors are used to deliver transgene segments, then larger DNA sequences can be incorporated into the genome, but the complexity of the delivery system increases
Solution Approach 1:
The patent designs each AAV vector to be multifunctional: they serve as both delivery vehicles for transgene segments and as templates for homologous recombination. The homology arms on each vector serve dual purposes: facilitating efficient recombination with the target genome and enabling assembly of adjacent segments. This universality reduces the need for additional complex components while achieving large DNA incorporation.
Solution Approach 2:
The patent prepares all necessary transgene segments with appropriate homology arms in advance, packaged in separate AAV vectors. This preliminary segmentation and packaging allows for controlled delivery to the same cell population, where the segments are then assembled in a predictable manner through homologous recombination. The pre-organized structure of segments simplifies the overall process compared to attempting to deliver and assemble large DNA molecules de novo.
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 method enables the successful incorporation of larger DNA sequences into targeted genomic locations, overcoming the size limitations of single-virus vectors and facilitating more extensive genetic modifications.
Implementation Method 1
Transduction using AAV vectors can be used to supply cargo DNA for insertion by homologous directed recombination (HDR) into double-stranded breaks generated by site-specific engineered nucleases.
Data Source
AI summary
The present invention encompasses compositions and methods for the sequential stacking of donor nucleic acids into a single genomic locus within a cell to allow for the introduction of relatively long nucleic sequences. This allows for insertion into the genome of a donor nucleic acid sequence that exceeds the packaging capacity of a single adeno-associated viral vector.