HITI Donor Constructs for Mutation-Independent Gene Replacement
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Solution Overview
Problem
Current gene therapy methods for dominant Mendelian diseases are limited by their inability to avoid degenerative effects of gain-of-function mutations and are restricted to specific mutations, lacking efficacy in differentiated cells and requiring stable, mutation-independent gene correction.
Innovation Solution
The use of Homology-Independent Targeted Integration (HITI) for integrating exogenous DNA sequences into target genes, utilizing a donor nucleic acid flanked by inverted targeting sequences and a nuclease to achieve high integration rates in non-dividing cells, allowing for the replacement of both mutant and wildtype alleles with a correct copy of the gene, and stable expression under endogenous promoters.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If Homology-Directed Repair (HDR) is used for gene correction, then precise correction of the mutant allele is achieved, but efficiency is limited by low homologous recombination activity in differentiated cells
Solution Approach 1:
The invention changes the repair mechanism parameter from HDR to NHEJ, and modifies the donor DNA structure by flanking it with inverted targeting sequences instead of homology arms. This parameter change enables efficient gene correction in differentiated cells by utilizing the constitutively active NHEJ pathway rather than the cell-cycle-dependent HDR pathway.
Solution Approach 2:
The invention inverts the traditional HDR approach by using inverted targeting sequences flanking the donor DNA. This inversion allows the donor DNA to be integrated via NHEJ after Cas9 cleavage, reversing the dependency from homology-directed repair to end-joining repair while achieving targeted integration.
2Reliability
If allele-specific knockout is performed to silence the mutant allele, then the wildtype allele remains unaffected, but the approach is restricted to specific mutations requiring separate therapeutic vectors for each mutation
Solution Approach 1:
The invention creates a universal therapeutic platform where a single donor DNA construct with inverted targeting sequences can replace both mutant and wildtype alleles regardless of the specific mutation. This multi-functional approach eliminates the need for mutation-specific vector design while maintaining therapeutic efficacy through complete gene replacement.
Solution Approach 2:
The invention extracts the disease-causing element by completely removing both mutant and wildtype alleles and replacing them with a corrected gene copy. This extraction approach eliminates the need for allele-specific targeting and works universally across different mutations in the same gene.
3Reliability
If conventional gene therapy is used to replace gene function, then loss-of-function mutations are corrected, but gain-of-function mutations cannot be avoided and continue to cause degenerative effects
Solution Approach 1:
The invention converts the harmful effect of gain-of-function mutations into a benefit by using the same NHEJ mechanism that causes random insertions/deletions to achieve precise targeted replacement. By utilizing NHEJ with inverted targeting sequences, the therapy replaces both mutant and wildtype alleles with a corrected copy, eliminating the harmful GOF mutation while maintaining the simplicity of NHEJ repair.
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
HITI enables efficient gene correction in differentiated cells and tissues, providing stable, therapeutic levels of protein expression, suitable for diseases like retinitis pigmentosa and mucopolysaccharidosis VI, overcoming limitations of conventional gene therapies.
Implementation Method 1
CRISPR-Cas9 is a ribonucleoprotein that binds a sequence called guide RNA (gRNA) and uses it to recognize the target DNA sequence by Watson-Crick base complementarity.
Implementation Method 2
After Cas9 is targeted to a particular location in the genome, it generates a double-strand brake (DSB)
Implementation Method 3
Non homologous end joining (NHEJ) is the most dominant mechanism in most cell types, since it is active in all phases of the cell cycle, and consists of the insertion or deletion of random bases in the site of the DSB in order to repair it.
Implementation Method 4
Homology-Directed Repair (HDR) is a process that occurs mainly in the G and S2 phases of the cell cycle, and uses a homologous template, which can be provided by an external donor DNA or by the other allele, for precise correction of the DSB.
Implementation Method 5
HITI uses a donor DNA that is flanked by the same gRNA target sequences within the gene of interest. After Cas9 cleaves both the gene and the donor DNA, the NHEJ machinery of the cell can include the donor DNA in the repairing of the cleavage, with a surprisingly high (60-80%) rate of integration in the absence of INDELS.
Data Source
AI summary
The present invention relates to a method of integrating an exogenous DNA sequence into a genome of a cell comprising contacting the cell with a) a donor nucleic acid comprising: —at least one STOP codon and a translation initiation sequence (TIS) or —a ribosomal skipping sequence, and —said exogenous DNA sequence wherein said donor nucleic acid is flanked at 5′ and 3′ by inverted targeting sequences; b) a complementary strand oligonucleotide homologous to the targeting sequence and c) a nuclease that recognizes the targeting sequence.