Restriction of C1-inhibitor activity in hereditary angioedema by dominant-negative effects of disease-associated SERPING1 gene variants.

BACKGROUND: Patients with hereditary angioedema experience recurrent, sometimes life-threatening, attacks of edema. It is a rare genetic disorder characterized by genetic and clinical heterogenicity. Most cases are caused by genetic variants in the SERPING1 gene leading to plasma deficiency of the encoded protein C1 inhibitor (C1INH). More than 500 different hereditary angioedema-causing variants have been identified in the SERPING1 gene, but the disease mechanisms by which they result in pathologically low C1INH plasma levels remain largely unknown. OBJECTIVES: The aim was to describe trans-inhibitory effects of full-length or near full-length C1INH encoded by 28 disease-associated SERPING1 variants. METHODS: HeLa cells were transfected with expression constructs encoding the studied SERPING1 variants. Extensive and comparative studies of C1INH expression, secretion, functionality, and intracellular localization were carried out. RESULTS: Our findings characterized functional properties of a subset of SERPING1 variants allowing the examined variants to be subdivided into 5 different clusters, each containing variants sharing specific molecular characteristics. For all variants except 2, we found that coexpression of mutant and normal C1INH negatively affected the overall capacity to target proteases. Strikingly, for a subset of variants, intracellular formation of C1INH foci was detectable only in heterozygous configurations enabling simultaneous expression of normal and mutant C1INH. CONCLUSIONS: We provide a functional classification of SERPING1 gene variants suggesting that different SERPING1 variants drive the pathogenicity through different and in some cases overlapping molecular disease mechanisms. For a subset of gene variants, our data define some types of hereditary angioedema with C1INH deficiency as serpinopathies driven by dominant-negative disease mechanisms.

Production and Validation of Lentiviral Vectors for CRISPR/Cas9 Delivery.

Genetic information transferred by HIV-1-based lentiviral vectors as single-stranded RNA is converted to double-stranded DNA by reverse transcription and subsequently inserted into the genome of recipient cells. Integration into the genome allows stable, long-term expression of genes-of-interest driven by promoter sequences contained within the vector. This technology can be used as a standard method for production of cells stably expressing Cas9 protein and single guide RNA (sgRNA), the key components of the CRISPR genome editing system. Here, we provide a protocol for production and validation of VSV-G-pseudotyped lentiviral vectors for delivery of the CRISPR system and generation of knockout cell lines.

Dominant-negative SERPING1 variants cause intracellular retention of C1 inhibitor in hereditary angioedema.

Hereditary angioedema (HAE) is an autosomal dominant disease characterized by recurrent edema attacks associated with morbidity and mortality. HAE results from variations in the SERPING1 gene that encodes the C1 inhibitor (C1INH), a serine protease inhibitor (serpin). Reduced plasma levels of C1INH lead to enhanced activation of the contact system, triggering high levels of bradykinin and increased vascular permeability, but the cellular mechanisms leading to low C1INH levels (20%-30% of normal) in heterozygous HAE type I patients remain obscure. Here, we showed that C1INH encoded by a subset of HAE-causing SERPING1 alleles affected secretion of normal C1INH protein in a dominant-negative fashion by triggering formation of protein-protein interactions between normal and mutant C1INH, leading to the creation of larger intracellular C1INH aggregates that were trapped in the endoplasmic reticulum (ER). Notably, intracellular aggregation of C1INH and ER abnormality were observed in fibroblasts from a heterozygous carrier of a dominant-negative SERPING1 gene variant, but the condition was ameliorated by viral delivery of the SERPING1 gene. Collectively, our data link abnormal accumulation of serpins, a hallmark of serpinopathies, with dominant-negative disease mechanisms affecting C1INH plasma levels in HAE type I patients, and may pave the way for new treatments of HAE.

Time-Restricted PiggyBac DNA Transposition by Transposase Protein Delivery Using Lentivirus-Derived Nanoparticles.

Continuous innovation of revolutionizing genome engineering technologies calls for an intensified focus on new delivery technologies that not only match the inventiveness of genome editors but also enable the combination of potent delivery and time-restricted action of genome-modifying bits and tools. We have previously demonstrated the use of lentivirus-derived nanoparticles (LNPs) as a protein delivery vehicle, incorporating and transferring DNA transposases, designer nucleases, or RNA-guided endonucleases fused to the N terminus of the Gag/GagPol polypeptide. Here, we establish LNP-directed transfer of the piggyBac DNA transposase protein by fusing the transposase to the integrase protein in the C-terminal end of GagPol. We show protein incorporation and proteolytic release of the DNA transposase within matured LNPs, resulting in high levels of DNA transposition activity in LNP-treated cells. Importantly, as opposed to conventional delivery methods based on transfection of plasmid DNA or in-vitro-transcribed mRNA, protein delivery by LNPs effectively results in time-restricted action of the protein (<24 hr) without compromising overall potency. Our findings refine LNP-directed piggyBac transposase delivery, at present the only available direct delivery strategy for this particular protein, and demonstrate a novel strategy for restricting and fine-tuning the exposure of the genome to DNA-modifying enzymes.