Gene therapy for C1 esterase inhibitor deficiency in a Murine Model of Hereditary angioedema

Abstract

Background: Hereditary angioedema (HAE) is a life-threatening, autosomal dominant disorder characterized by unpredictable, episodic swelling of the face, upper airway, oropharynx, extremities, genitalia, and gastrointestinal tract. Almost all cases of HAE are caused by mutations in the SERPING1 gene resulting in a deficiency in functional plasma C1 esterase inhibitor (C1EI), a serine protease inhibitor that normally inhibits proteases in the contact, complement, and fibrinolytic systems. Current treatment of HAE includes long-term prophylaxis with attenuated androgens or human plasma-derived C1EI and management of acute attacks with human plasma-derived or recombinant C1EI, bradykinin, and kallikrein inhibitors, each of which requires repeated administration. As an approach to effectively treat HAE with a single treatment, we hypothesized that a one-time intravenous administration of an adeno-associated virus (AAV) gene transfer vector expressing the genetic sequence of the normal human C1 esterase inhibitor (AAVrh.10hC1EI) would provide sustained circulating C1EI levels sufficient to prevent angioedema episodes.

Methods: To study the efficacy of AAVrh.10hC1EI, we used CRISPR/Cas9 technology to create a heterozygote C1EI-deficient mouse model (S63±) that shares characteristics associated with HAE in humans including decreased plasma C1EI and C4 levels. Phenotypically, these mice have increased vascular permeability of skin and internal organs.

Results: Systemic administration of AAVrh.10hC1EI to the S63± mice resulted in sustained human C1EI activity levels above the predicted therapeutic levels and correction of the vascular leak in skin and internal organs.

Conclusion: A single treatment with AAVrh.10hC1EI has the potential to provide long-term protection from angioedema attacks in affected individuals.

Keywords: C1 esterase inhibitor; complement; gene therapy; hereditary angioedema; vascular permeability.

Figure 1.

Characterization of the S63+/− mouse. A. CRISPR/Cas9 deleted sequences in exon 3 of the murine SERPING1 gene. B. PCR assessment of exon 3 of wild-type and S63 mice. C. Heterozygote S63+/− and wild-type serum C1EI levels as measured by ELISA. D. Heterozygote S63+/− and wild-type serum C4 levels as measured by ELISA. E. Increased vascular permeability in S63+/− mutant mice 30 min after Evans blue dye injection. Shown are photographs of wild-type (WT) and S63+/− males and female rear paws and snouts.

Figure 2.

AAVrh.10hC1EI-mediated persistent expression of human C1EI levels over time, following a single intravenous administration (10¹¹ gc) to S63+/− mice. AAVrh.10hα1AT (10¹¹ gc) and PBS were controls. Values are presented as means ± SEM. Numbers of mice in each treatment cohort are shown in the labels. The clinical threshold for C1EI is shown (0.7 U/ml) by the upper black dashed line. Limit of detection for the assay (0.15 U/ml) is shown by the lower black dashed line.

Figure 3.

AAVrh.10hC1EI correction of vascular permeability in S63+/− mice. Shown is assessment of S63+/− mice 2, 6 and 24 wk after treatment with AAVrh.10hC1EI (10¹¹ gc) or PBS. Extravasation of dye in rear paws and snouts of S63+/− untreated and S63+/− treated mice 30 min after Evans blue dye administration. Age-matched C57BL/6J mice were used as wild-type controls. Shown are data for males and females.

Figure 4.

Spectrophotometric analysis of vascular permeability of various organs of S63+/− mice following AAVrh.10hC1EI therapy. Shown is data over time for S63+/− mice and wild-type controls. Evans blue dye was administered intravenously and tissues sampled following necropsy at 30 min. Whole tissue was incubated with 1 ml formamide to extract the extravasated Evans blue dye. Optical density was measured at 600 nm and the measurements converted into ng dye extravasated per mg tissue. A. Rear paws; B. kidneys; C. small intestines; D. lung; E. spleen; and F. heart. p values between treated and untreated S63+/− mice are presented in the table for all time-points. n=3–5 mice/group at 2, 6, and 24 wk time-points.