Non-Integrating Episomal dCas9 Activation for Haploinsufficiency
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Solution Overview
Problem
Existing methods for treating diseases caused by reduced gene transcription or activity, such as haploinsufficiency, are inadequate, as they often require genome integration and may not provide sufficient transcriptional activation to treat the condition effectively.
Innovation Solution
The use of a non-integrating episomal vector, such as an adeno-associated viral vector, containing a guide RNA and a catalytically inactive CRISPR nuclease to target and activate transcription of a haploinsufficient gene through a promoter or enhancer region, without modifying the genome, using a dCAS9/gRNA complex.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If a wild-type copy of the gene is delivered into the genome, then the amount of gene product is increased, but the genome is permanently modified through integration
Solution Approach 1:
The patent uses an episomal vector as an intermediary carrier to deliver the wild-type gene copy without integrating it into the host genome. The vector remains separate from the genomic DNA, allowing gene product production while avoiding permanent genomic modification and its associated risks
Solution Approach 2:
The patent extracts the gene therapy approach from the context of genomic integration and places it in an episomal context. By separating the therapeutic gene delivery from genome integration, the solution achieves gene product augmentation without the harmful effects of genomic modification
2Productivity
If CRISPR-based targeted introduction is used, then transcription is increased, but the genome is modified through endonuclease cleavage and repair
Solution Approach 1:
The patent employs an episomal vector as an intermediary system that enables transcriptional activation without requiring endonuclease-mediated genome editing. The CRISPR components operate on the episomal DNA rather than the genomic DNA, achieving productivity enhancement without harmful genome modification
Solution Approach 2:
The patent segments the CRISPR system into two separate functions: one acting on the episomal vector (for transcriptional activation) and the other acting on the genomic DNA (which remains unmodified). This segmentation allows increased transcription while avoiding genome modification by directing the transcriptional activation mechanism to the episomal rather than genomic compartment
3Productivity
If genome integration is used to treat haploinsufficiency, then sufficient transcriptional activation is achieved, but the complexity of the treatment increases
Solution Approach 1:
The episomal vector serves as a simplified intermediary that delivers therapeutic genes without requiring the complex mechanisms of genomic integration. This approach achieves sufficient transcriptional activation while reducing treatment complexity by eliminating the need for precise genomic targeting and integration site selection
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 achieves sufficient transcriptional activation of target genes to treat diseases like haploinsufficiency, providing therapeutic effects without genome modification, and can be applied to various haploinsufficient genes in mammalian cells, including those in the brain.
Implementation Method 1
a targeting region that, under conditions present in a nucleus of the cell, specifically hybridizes to a promoter region or an enhancer region operably linked to a wild-type copy of a haploinsufficient gene
Implementation Method 2
a CRISPR nuclease-binding region that specifically binds a CRISPR nuclease under conditions present in a nucleus of the cell
Implementation Method 3
the complex activates transcription of the wild-type copy of the haploinsufficient gene in an amount and for a duration sufficient to treat the haploinsufficiency disease in the subject
Data Source
AI summary
Methods and compositions are provided for activating transcription in a mammalian cell.