Hereditary Angioedema (HAE) Cell Models for Research

Hereditary Angioedema (HAE) is a rare autosomal dominant disorder characterized by recurrent episodes of severe swelling in the subcutaneous and submucosal tissues. The global prevalence is estimated at 1 in 50,000, with no significant gender or ethnic predilection. The disease is caused by mutations in the SERPING1 gene (type I) or F12 gene (type III), leading to deficiency or dysfunction of C1 inhibitor (C1-INH). Attacks can be life-threatening when involving the larynx, with a mortality rate of up to 30% if untreated. The clinical impact includes significant morbidity, reduced quality of life, and economic burden due to emergency care and lost productivity. Early diagnosis and prophylactic therapy are crucial, but many patients remain undiagnosed or misdiagnosed. Research into the molecular mechanisms and novel therapeutic targets is essential.

HAE serves as an excellent model for studying the complement system, contact activation pathway, and vascular biology. The disease is monogenic, making it amenable to gene editing and functional studies. Public datasets, such as those from ClinVar and the Human Gene Mutation Database, provide extensive mutation data. Open questions include the variability in clinical presentation, the role of modifier genes, and the mechanisms of bradykinin-mediated vascular permeability. Gene-edited cell models allow for precise dissection of these pathways and are invaluable for drug discovery and development.

The primary defect in HAE is a deficiency or dysfunction of C1 inhibitor (C1-INH), a serine protease inhibitor that regulates the complement, contact, and fibrinolytic systems. The contact activation pathway is particularly critical:

1. Activation of factor XII (FXII) on negatively charged surfaces.

2. FXIIa converts prekallikrein to kallikrein.

3. Kallikrein cleaves high-molecular-weight kininogen (HK) to release bradykinin.

4. Bradykinin binds to bradykinin B2 receptor (BDKRB2) on endothelial cells.

5. This triggers nitric oxide and prostaglandin release, leading to vasodilation and increased vascular permeability.

In HAE, C1-INH deficiency leads to uncontrolled activation of this pathway, resulting in excessive bradykinin production and angioedema. Other pathways, such as the complement system, may also contribute to the inflammatory response.

Data from ClinVar and the Human Gene Mutation Database. The majority of SERPING1 mutations are private, with over 700 distinct variants reported.

The key signaling networks in HAE involve:

• • Contact activation pathway: FXII, kallikrein, HK, bradykinin, BDKRB2.
• • Complement pathway: C1 complex, C4, C2, and C1-INH regulation.
• • Fibrinolytic pathway: plasminogen, tissue plasminogen activator, and C1-INH.
• • Endothelial signaling: nitric oxide (NO), prostaglandins, and vascular endothelial growth factor (VEGF).

These networks are interconnected. For example, plasmin can activate FXII, and bradykinin can stimulate NO production. Gene-edited cell models targeting these nodes can help dissect the pathways and identify therapeutic targets.

These cell lines are commonly used for transfection and gene editing. However, they do not endogenously express high levels of SERPING1 or the contact activation components. Therefore, researchers often use primary cells or differentiated cells. Organoids, such as endothelial organoids, can better recapitulate vascular permeability and are being developed for HAE modeling. Gene-edited organoids with SERPING1 knockout or F12 mutations are valuable for studying the disease in a more physiologically relevant context.

Animal models for HAE include:

• • SERPING1 knockout mice: These mice exhibit increased vascular permeability and are used to study the pathophysiology and test therapeutics.
• • Kininogen-deficient mice: Used to study the role of bradykinin.
• • Rat models: Induced by C1-INH depletion using antibodies.
• • Zebrafish models: Transgenic zebrafish with SERPING1 knockdown have been used for drug screening.

These models are useful for in vivo studies, but they have limitations in recapitulating the exact human phenotype. Gene-edited cell models offer a more controlled and human-specific platform.

CRISPR-based gene editing has enabled the creation of isogenic cell lines with precise mutations in SERPING1 or F12. For example:

• • SERPING1 knockout cell lines: Generated by introducing frameshift mutations via CRISPR-Cas9, resulting in loss of C1-INH expression. These lines are useful for studying the complement and contact pathways.
• • SERPING1 point mutation knock-in lines: For type II HAE, specific missense mutations (e.g., Ala436Thr) can be introduced to study the dysfunctional protein.
• • F12 knock-in lines: For type III HAE, the Thr328Lys mutation can be introduced to enhance FXII activity.

These gene-edited cell models are commercially available and sequence-verified, ensuring reproducibility. They are essential for functional genomics, drug screening, and target validation.

Gene-edited cell lines are used to validate the function of genes involved in HAE. For example:

• • SERPING1 knockout lines can be used to study the downstream effects of C1-INH loss on complement activation and bradykinin production.
• • F12 knock-in lines can be used to assess the impact of specific mutations on FXII activation and kallikrein activity.

These models allow researchers to perform loss-of-function and gain-of-function studies in a controlled environment.

Isogenic cell lines are ideal for high-throughput screening of potential therapeutics. For example:

• • SERPING1 knockout cells can be used to test the efficacy of C1-INH replacement therapies or small molecule inhibitors of kallikrein.
• • F12 knock-in cells can be used to screen for inhibitors of FXIIa.

These models can also be used to study resistance mechanisms, such as mutations that confer resistance to specific drugs.

CRISPR-based synthetic lethality screens can identify genes that are essential for cell survival in the context of SERPING1 deficiency. This can lead to the discovery of novel biomarkers and therapeutic targets. For example, a screen in SERPING1 knockout cells might identify genes involved in the contact activation pathway that, when inhibited, cause cell death. These genes could serve as biomarkers for disease severity or as targets for new drugs.