CRISPR-Cas9 Self-Targeting Vector for Therapeutic DNA Removal
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Solution Overview
Problem
Current methods for removing therapeutic DNA from host cells are inefficient, as they either leave behind recombination sequences or require cells to be lost after treatment, which is not feasible in gene therapy.
Innovation Solution
The use of CRISPR RNA (gRNA) specifically hybridizing to therapeutic polynucleotides, combined with a CRISPR-associated endonuclease, to inactivate therapeutic DNA by introducing insertions or deletions, thereby allowing for the removal of therapeutic sequences without leaving behind residual DNA.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Loss of substance
If recombinase systems like Cre/lox are used for excision of transgene, then transgene can be removed, but recombination sequences are left behind and must be included in the transgene before application
Solution Approach 1:
The patent extracts and removes the therapeutic transgene sequence from the host cell genome using CRISPR-Cas9 endonuclease system. The gRNA specifically targets the therapeutic sequence for cleavage, and the cell's repair mechanisms (NHEJ or HDR) remove the sequence without leaving behind recombination sequences, thus achieving complete extraction of the unwanted genetic material
Solution Approach 2:
The patent replaces the mechanical/recombinase-based excision system (Cre/lox) with a CRISPR-Cas9 molecular scissor system. Instead of using recombinase enzymes that require specific recombination sequences, the invention uses guide RNA-directed endonuclease activity to precisely cut and remove the therapeutic sequence, substituting one molecular mechanism for another more precise one
2Loss of substance
If inducible suicide genes are used to remove cells carrying transgene, then cells can be eliminated, but this approach is only feasible if cells may be lost after treatment
Solution Approach 1:
Instead of killing cells to remove therapeutic DNA, the patent extracts and removes the therapeutic sequence directly from the host cell genome using CRISPR-Cas9. This allows the cell to remain viable and functional while the unwanted genetic material is precisely excised, eliminating the need for cell loss as a mechanism for removing the transgene
3Adaptability or versatility
If therapeutic vectors are introduced into cells, then gene therapy can be performed, but the vectors are difficult or impossible to remove once introduced
Solution Approach 1:
The patent applies preliminary anti-action by designing the therapeutic vector to include self-targeting gRNA sequences that will direct CRISPR-Cas9 to cleave the therapeutic DNA itself. This built-in suicide mechanism allows the vector to be removed on demand, counteracting its own persistent presence in the cell genome
Solution Approach 2:
The therapeutic vector is designed to encode its own destruction mechanism by including gRNA sequences that target the vector's own genetic material. When CRISPR-Cas9 is introduced, the vector's encoded gRNA guides the endonuclease to cleave the therapeutic sequence, enabling the vector to service its own removal without external intervention
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 effectively inactivates therapeutic polynucleotides by introducing targeted insertions or deletions, allowing for the complete removal of therapeutic DNA from host cells, preventing adverse effects and ensuring the sequences are not integrated into the genome.
Implementation Method 1
contacting said host cell with a clustered regularly interspaced short palindromic repeats (CRISPR) RNA (gRNA) specifically hybridizing to said therapeutic polynucleotide
Implementation Method 2
inactivating said therapeutic polynucleotide by introducing insertions or deletions into said therapeutic polynucleotide
Data Source
AI summary
The present invention relates to a method for inactivating a therapeuticpolynucleotide in a host cell, comprising (a) contacting said host cell with a clustered regularly interspaced short palindromic repeats (CRISPR) RNA (gRNA) specifically hybridizing to said therapeutic polynucleotide and with a CRISPR-associated endonuclease, and, thereby, (b) inactivating said therapeuticpolynucleotide. Moreover, the present invention relates to a targeting polynucleotide comprising expressible polynucleotide sequences encoding (i) a gRNA comprising a first targeting sequence specifically hybridizing to a first target sequence of interest, and, (ii) optionally, a CRISPR-associated endonuclease; wherein said targeting polynucleotide further comprises at least one inactivation sequence positioned such that such said targeting polynucleotide is inactivated by a CRISPR-associated endonuclease activity, wherein said inactivation sequence is identical to said first target sequence or is a second target sequence being non-identical to said first target sequence, preferably wherein said target sequence is identical to said first target sequence.The present invention further relates to kits, vectors, and host cells comprising said targeting polynucleotides and to the medical use of said targeting polynucleotides.