Source 1primary paper2026MED
Toolkit/rs1800378-targeted allele-selective CRISPR/Cas9 VWF disruption strategy
rs1800378-targeted allele-selective CRISPR/Cas9 VWF disruption strategy
Also known as: common SNP-targeted VWF allele-selective gene editing strategy, CRISPR/Cas9 allele-selective disruption of pathogenic VWF variants
Taxonomy: Mechanism Branch / Architecture. Workflows sit above the mechanism and technique branches rather than replacing them.
Summary
Our strategy permanently inactivates VWF variants by selectively disrupting the pathogenic allele's open reading frame via the introduction of indels by Cas9. To circumvent the challenge of designing variant-specific strategies, we targeted the common single nucleotide polymorphism (SNP) rs1800378 in VWF.
Usefulness & Problems
Why this is useful
This strategy uses Cas9 to introduce indels that disrupt the open reading frame of the pathogenic VWF allele in an allele-selective manner. The selectivity is achieved by targeting the common VWF SNP rs1800378 rather than the disease-causing variant itself.; allele-selective inactivation of pathogenic VWF alleles; designing VWD treatments without mutation-specific guide redesign
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This strategy uses Cas9 to introduce indels that disrupt the open reading frame of the pathogenic VWF allele in an allele-selective manner. The selectivity is achieved by targeting the common VWF SNP rs1800378 rather than the disease-causing variant itself.
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allele-selective inactivation of pathogenic VWF alleles
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designing VWD treatments without mutation-specific guide redesign
Problem solved
It is intended to overcome the need for variant-specific therapeutic designs in a genetically heterogeneous disease. It also aims to suppress dominant-negative pathogenic VWF while preserving the nontargeted allele.; addresses heterogeneous mutational landscape by targeting a common SNP instead of each disease-causing variant; aims to preserve expression from the nontargeted VWF allele while disrupting the pathogenic allele
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It is intended to overcome the need for variant-specific therapeutic designs in a genetically heterogeneous disease. It also aims to suppress dominant-negative pathogenic VWF while preserving the nontargeted allele.
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addresses heterogeneous mutational landscape by targeting a common SNP instead of each disease-causing variant
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aims to preserve expression from the nontargeted VWF allele while disrupting the pathogenic allele
Problem links
addresses heterogeneous mutational landscape by targeting a common SNP instead of each disease-causing variant
LiteratureIt is intended to overcome the need for variant-specific therapeutic designs in a genetically heterogeneous disease. It also aims to suppress dominant-negative pathogenic VWF while preserving the nontargeted allele.
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It is intended to overcome the need for variant-specific therapeutic designs in a genetically heterogeneous disease. It also aims to suppress dominant-negative pathogenic VWF while preserving the nontargeted allele.
aims to preserve expression from the nontargeted VWF allele while disrupting the pathogenic allele
LiteratureIt is intended to overcome the need for variant-specific therapeutic designs in a genetically heterogeneous disease. It also aims to suppress dominant-negative pathogenic VWF while preserving the nontargeted allele.
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It is intended to overcome the need for variant-specific therapeutic designs in a genetically heterogeneous disease. It also aims to suppress dominant-negative pathogenic VWF while preserving the nontargeted allele.
Published Workflows
Objective: Develop a broadly applicable allele-selective gene editing strategy for heterozygous dominant-negative VWF variants in VWD by permanently inactivating the pathogenic allele while preserving the nontargeted allele.
Why it works: The workflow is based on using a common SNP to achieve allele selectivity without redesigning a strategy for each pathogenic variant, then confirming both DNA-level editing and proteoform-level reduction while checking for reversal of cellular disease phenotypes.
Cas9-induced indels disrupting the pathogenic allele open reading framecommon SNP-guided allele discriminationCRISPR/Cas9 editingnext-generation sequencing analysisvariant mapping mass spectrometryex vivo patient-derived ECFC testing
Stages
- 1.Common SNP-targeted strategy design(library_design)
This stage exists to circumvent the challenge of designing variant-specific strategies in a disease with a heterogeneous mutational landscape.
Selection: Target the common VWF SNP rs1800378 instead of designing variant-specific strategies.
- 2.Ex vivo proof-of-principle testing in patient-derived ECFCs(functional_characterization)
This stage exists to demonstrate ex vivo proof of principle in patient-derived endothelial cells relevant to VWF production.
Selection: Test the strategy in ECFCs from patients with VWD2A and VWD2B carrying heterozygous pathogenic VWF variants.
- 3.Sequencing-based editing assessment(secondary_characterization)
This stage exists to verify that the editing outcome is allele selective and preserves the nontargeted allele.
Selection: Assess efficient allele-selective VWF knockout while maintaining expression of the nontargeted allele.
- 4.Proteoform confirmation and phenotype readout(confirmatory_validation)
This stage exists to confirm that DNA-level editing translates into selective reduction of variant VWF proteoforms and improved cellular phenotype.
Selection: Confirm selective reduction of variant allele expression at the proteoform level and assess associated reversal of cellular disease phenotypes.
Steps
- 1.Choose rs1800378 as the allele-discriminating targetengineered editing strategy
Enable allele-selective disruption of pathogenic VWF alleles without requiring variant-specific strategy design.
The abstract states this choice was made to circumvent the challenge of designing variant-specific strategies in a heterogeneous disease.
- 2.Test the editing strategy in patient-derived ECFCs from VWD2A and VWD2B casesengineered editing strategy under test
Demonstrate ex vivo proof of principle in disease-relevant endothelial cells carrying heterozygous pathogenic VWF variants.
After strategy design, the authors moved to patient-derived ECFCs to establish proof of principle in a relevant cellular context.
- 3.Measure allele-selective knockout by next-generation sequencing
Determine whether editing efficiently knocks out VWF in an allele-selective manner while maintaining expression of the nontargeted allele.
This analysis follows ex vivo testing to establish the primary editing outcome before protein-level confirmation.
- 4.Confirm selective reduction of variant proteoforms and assess phenotype reversal by variant mapping mass spectrometryconfirmatory assay
Confirm that allele-selective editing reduces variant VWF proteoforms and is accompanied by reversal of cellular disease phenotypes.
This confirmatory step follows sequencing to verify that DNA-level editing produces the intended protein-level and cellular consequences.
Taxonomy & Function
Primary hierarchy
Mechanism Branch
Architecture: A composed arrangement of multiple parts that instantiates one or more mechanisms.
Target processes
editingImplementation Constraints
cofactor dependency: cofactor requirement unknownencoding mode: genetically encodedimplementation constraint: context specific validationimplementation constraint: multi component delivery burdenoperating role: sensorswitch architecture: multi component
The abstract supports a need for CRISPR/Cas9 editing and a targetable rs1800378-containing VWF allele in endothelial cells. The proof-of-principle experiments used patient-derived ECFCs plus next-generation sequencing and variant-mapping mass spectrometry readouts.; requires presence of the targeted common SNP rs1800378 in VWF; requires CRISPR/Cas9 editing in endothelial cells
The abstract does not show in vivo delivery, whole-organism efficacy, or clinical treatment. It also does not establish applicability to patients lacking the targeted SNP.; shown as ex vivo proof of principle in patient-derived ECFCs; abstract does not describe in vivo delivery or clinical validation
Validation
Cell-freeBacteriaMammalianMouseHumanTherapeuticIndep. Replication
Supporting Sources
Ranked Claims
Variant mapping mass spectrometry confirmed selective reduction of variant VWF allele expression and this was accompanied by reversal of cellular disease phenotypes in ECFCs.
Variant mapping mass spectrometry that discriminates between wild-type and variant VWF proteoforms confirmed selective reduction of variant allele expression, which was accompanied by reversal of cellular disease phenotypes in ECFCs.
The allele-selective editing strategy targets the common VWF SNP rs1800378 to avoid designing variant-specific strategies.
To circumvent the challenge of designing variant-specific strategies, we targeted the common single nucleotide polymorphism (SNP) rs1800378 in VWF.
In patient-derived ECFCs from VWD2A and VWD2B cases, next-generation sequencing showed efficient allele-selective VWF knockout while maintaining expression of the nontargeted allele.
We used endothelial colony-forming cells (ECFCs) from patients with VWD2A and VWD2B with heterozygous p.C1190R and p.R1306W variants, respectively, to demonstrate ex vivo proof of principle. Using next-generation sequencing analysis, we show efficient and allele-selective knockout of VWF, while maintaining VWF expression of the nontargeted allele.
The study supports feasibility of a broadly applicable VWD gene editing strategy based on targeting a common SNP rather than being constrained by disease-causing variant, pathogenic mechanism, or VWD subtype.
This study shows the feasibility of a novel gene editing strategy for VWD that, by virtue of its targeting of a common SNP, can be broadly applicable and can be used to design treatments for VWD without being constrained by the disease-causing variant, pathogenic mechanism, or VWD subtype.
The study developed a gene therapy strategy for heterozygous dominant-negative VWF variants that selectively disrupts the pathogenic allele open reading frame using Cas9-induced indels.
In this study, we developed a novel gene therapy strategy for patients with VWD caused by heterozygous dominant-negative VWF variants. Our strategy permanently inactivates VWF variants by selectively disrupting the pathogenic allele's open reading frame via the introduction of indels by Cas9.
Approval Evidence
1 source5 linked approval claimsfirst-pass slug rs1800378-targeted-allele-selective-crispr-cas9-vwf-disruption-strategy
Our strategy permanently inactivates VWF variants by selectively disrupting the pathogenic allele's open reading frame via the introduction of indels by Cas9. To circumvent the challenge of designing variant-specific strategies, we targeted the common single nucleotide polymorphism (SNP) rs1800378 in VWF.
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confirmatory assay resultsupports
Variant mapping mass spectrometry confirmed selective reduction of variant VWF allele expression and this was accompanied by reversal of cellular disease phenotypes in ECFCs.
Variant mapping mass spectrometry that discriminates between wild-type and variant VWF proteoforms confirmed selective reduction of variant allele expression, which was accompanied by reversal of cellular disease phenotypes in ECFCs.
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design rationalesupports
The allele-selective editing strategy targets the common VWF SNP rs1800378 to avoid designing variant-specific strategies.
To circumvent the challenge of designing variant-specific strategies, we targeted the common single nucleotide polymorphism (SNP) rs1800378 in VWF.
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ex vivo resultsupports
In patient-derived ECFCs from VWD2A and VWD2B cases, next-generation sequencing showed efficient allele-selective VWF knockout while maintaining expression of the nontargeted allele.
We used endothelial colony-forming cells (ECFCs) from patients with VWD2A and VWD2B with heterozygous p.C1190R and p.R1306W variants, respectively, to demonstrate ex vivo proof of principle. Using next-generation sequencing analysis, we show efficient and allele-selective knockout of VWF, while maintaining VWF expression of the nontargeted allele.
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feasibilitysupports
The study supports feasibility of a broadly applicable VWD gene editing strategy based on targeting a common SNP rather than being constrained by disease-causing variant, pathogenic mechanism, or VWD subtype.
This study shows the feasibility of a novel gene editing strategy for VWD that, by virtue of its targeting of a common SNP, can be broadly applicable and can be used to design treatments for VWD without being constrained by the disease-causing variant, pathogenic mechanism, or VWD subtype.
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strategy descriptionsupports
The study developed a gene therapy strategy for heterozygous dominant-negative VWF variants that selectively disrupts the pathogenic allele open reading frame using Cas9-induced indels.
In this study, we developed a novel gene therapy strategy for patients with VWD caused by heterozygous dominant-negative VWF variants. Our strategy permanently inactivates VWF variants by selectively disrupting the pathogenic allele's open reading frame via the introduction of indels by Cas9.
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Comparisons
Source-stated alternatives
The abstract contrasts this approach with variant-specific strategies, which it seeks to avoid. No other direct therapeutic alternative is described in the abstract beyond existing VWF concentrate infusion as current treatment.
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The abstract contrasts this approach with variant-specific strategies, which it seeks to avoid. No other direct therapeutic alternative is described in the abstract beyond existing VWF concentrate infusion as current treatment.
Source-backed strengths
permanent inactivation via Cas9-induced indels; broad applicability claimed through targeting of a common SNP; maintains expression of the nontargeted allele in ex vivo ECFC proof-of-principle
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permanent inactivation via Cas9-induced indels
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broad applicability claimed through targeting of a common SNP
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maintains expression of the nontargeted allele in ex vivo ECFC proof-of-principle
Compared with CRISPR/Cas9
rs1800378-targeted allele-selective CRISPR/Cas9 VWF disruption strategy and CRISPR/Cas9 address a similar problem space because they share editing.
Shared frame: same top-level item type; shared target processes: editing
Strengths here: may avoid an exogenous cofactor requirement.
Relative tradeoffs: appears more independently replicated.
Compared with CRISPR/Cas9 system
rs1800378-targeted allele-selective CRISPR/Cas9 VWF disruption strategy and CRISPR/Cas9 system address a similar problem space because they share editing.
Shared frame: same top-level item type; shared target processes: editing
Relative tradeoffs: appears more independently replicated; looks easier to implement in practice.
Compared with Cry/Vip pyramiding
rs1800378-targeted allele-selective CRISPR/Cas9 VWF disruption strategy and Cry/Vip pyramiding address a similar problem space because they share editing.
Shared frame: same top-level item type; shared target processes: editing
Ranked Citations
- 1.