Allele-Specific CRISPR Therapy for ADRP Without HDR
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current treatments for autosomal dominant retinitis pigmentosa (ADRP) are limited, and CRISPR-based therapies face challenges in targeting single nucleotide changes and require homology-directed repair, which is difficult in post-mitotic cells like retinal photoreceptor cells.
Innovation Solution
A CRISPR-based agent comprising a Cas endonuclease and a guide RNA specifically targeting the RHOP23H or NR2E3G56R mutant alleles, using non-homologous end-joining repair mechanisms to inactivate the mutant alleles without relying on HDR, forming a CRISPR-Cas complex to prevent expression of toxic proteins.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If CRISPR methodologies are used to target single nucleotide changes in dominantly-inherited diseases, then the ability to specifically target mutant alleles is improved, but the requirement for homology-directed repair (HDR) makes the therapy difficult to implement in post-mitotic cells like retinal photoreceptor cells
Solution Approach 1:
The patent extracts the requirement for HDR from the CRISPR therapy process by using base editing technology that directly chemically converts the mutant nucleotide without requiring cellular repair mechanisms. This removes the dependency on cell division and HDR pathways that are unavailable in post-mitotic retinal photoreceptor cells
Solution Approach 2:
The patent replaces the mechanical/cellular repair process (HDR requiring cell division) with a direct chemical conversion process. The base editor uses a catalytically impaired Cas9 fused to a base mismatch repair enzyme to directly chemically convert C•G to T•A or A•T to G•C pairs, substituting a biochemical mechanism for a cellular repair mechanism
2Manufacturing precision
If HDR process is used for gene editing, then the correction of mutant alleles can be achieved, but the process requires cell division which is not possible in post-mitotic retinal photoreceptor cells
Solution Approach 1:
The patent extracts the cell division requirement from the gene editing process by employing base editing technology that performs direct nucleotide conversion within the existing cellular context, eliminating the need for HDR and cell division cycles
Solution Approach 2:
The patent changes the operational parameters of gene editing from a division-dependent process (HDR) to a division-independent process (base editing). The base editor maintains high editing accuracy while operating under different biochemical parameters that do not require S/G2 phase cellular conditions
3Productivity
If conventional CRISPR-Cas9 is used for dominantly-inherited diseases, then the general gene editing capability is achieved, but the ability to specifically target single nucleotide changes without affecting wild-type alleles is reduced
Solution Approach 1:
The patent applies local quality by designing the base editor system with allele-specific targeting capability. The guide RNA is designed to match the mutant allele sequence, and the base editor only activates when bound to the mutant sequence, creating a localized editing effect at the specific mutant site without affecting wild-type alleles
Solution Approach 2:
The patent introduces an intermediary mechanism (the base mismatch repair enzyme fused to Cas9) that acts as a mediator between the guide RNA binding and the actual nucleotide conversion. This intermediary ensures that editing only occurs when the correct mutant sequence is recognized, enhancing allele-specific precision while maintaining editing efficiency
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
Potentially halts or reverses retinal degeneration by inactivating the mutant alleles, offering a viable therapy for ADRP without the need for HDR, and can be administered early to prevent or delay disease onset.
Implementation Method 1
a CRISPR-Cas complex (a ribonucleoprotein complex; RNP) formed from expressed Cas endonuclease and the gRNA is capable of specifically targeting and cleaving the target mutant allele
Implementation Method 2
using non-homologous end-joining repair mechanisms to inactivate the mutant alleles without relying on HDR
Data Source
AI summary
An agent for treating a common form of autosomal dominant retinitis pigmentosa (ADRP) is disclosed, wherein the agent comprises a first nucleotide sequence encoding a CRISPR-associated (Cas) endonuclease which binds to an NG or NNGRRT PAM (protospacer adjacent motif) sequence, and a second nucleotide sequence encoding or comprising a guide RNA (gRNA) capable of forming a CRISPR-Cas complex with said Cas endonuclease, wherein the gRNA is specifically targeted to a target mutant allele selected from RHOP23H and NR2E3G56R.