Epigenome Editing Durability Varies Widely Across Cardiovascular Disease Target Genes.

Background: Hepatic knockdown of the proprotein convertase subtilisin/kexin type 9 ( PCSK9 ) gene or the angiopoietin-like 3 ( ANGPTL3 ) gene has been demonstrated to reduce blood low-density lipoprotein cholesterol (LDL-C) levels, and hepatic knockdown of the angiotensinogen ( AGT ) gene has been demonstrated to reduce blood pressure. Genome editing can productively target each of these three genes in hepatocytes in the liver, offering the possibility of durable "one-and-done" therapies for hypercholesterolemia and hypertension. However, concerns around making permanent gene sequence changes via DNA strand breaks might hinder acceptance of these therapies. Epigenome editing offers an alternative approach to gene inactivation, via silencing of gene expression by methylation of the promoter region, but the long-term durability of epigenome editing remains to be established. Methods: We assessed the ability of epigenome editing to durably reduce the expression of the human PCSK9, ANGPTL3 , and AGT genes in HuH-7 hepatoma cells. Using the CRISPRoff epigenome editor, we identified guide RNAs that produced efficient gene knockdown immediately after transfection. We assessed the durability of gene expression and methylation changes through serial cell passages. Results: Cells treated with CRISPRoff and PCSK9 guide RNAs were maintained for up to 124 cell doublings and demonstrated durable knockdown of gene expression and increased CpG dinucleotide methylation in the promoter, exon 1, and intron 1 regions. In contrast, cells treated with CRISPRoff and ANGPTL3 guide RNAs experienced only transient knockdown of gene expression. Cells treated with CRISPRoff and AGT guide RNAs also experienced transient knockdown of gene expression; although initially there was increased CpG methylation throughout the early part of the gene, this methylation was geographically heterogeneous-transient in the promoter, and stable in intron 1. Conclusions: This work demonstrates precise and durable gene regulation via methylation, supporting a new therapeutic approach for protection against cardiovascular disease via knockdown of genes such as PCSK9 . However, the durability of knockdown with methylation changes is not generalizable across target genes, likely limiting the therapeutic potential of epigenome editing compared to other modalities.

Base Editing and Prime Editing: Potential Therapeutic Options for Rare and Common Diseases.

Collectively, genetic disorders affect approximately 350 million individuals worldwide and are a major global health burden. Despite substantial progress in identification of new disease-causing genes, variants, and molecular etiologies, nearly all rare diseases have no targeted therapeutics that can address their underlying molecular causes. Base editing (BE) and prime editing (PE), two newly described iterations of CRISPR-Cas9 genome editing, represent potential therapeutic strategies that could be used to precisely, efficiently, permanently, and safely correct patients' pathogenic variants and ameliorate disease sequelae. Unlike "standard" CRISPR-Cas9 genome editing, these technologies do not rely on double-strand break (DSB) formation, thus improving safety by decreasing the likelihood of undesired insertions and deletions (indels) at the target site. Here, we provide an overview of BE and PE, including their structures, mechanisms, and differences from standard CRISPR-Cas9 genome editing. We describe several examples of the use of BE and PE to improve rare and common disease phenotypes in preclinical models and human patients, with an emphasis on in vivo editing efficacy, safety, and delivery method. We also discuss recently developed delivery methods for these technologies that may be used in future clinical settings.

Dysregulated myosin in Hermansky-Pudlak syndrome lung fibroblasts is associated with increased cell motility.

Hermansky-Pudlak syndrome (HPS) is an autosomal recessive disorder characterized by improper biogenesis of lysosome-related organelles (LROs). Lung fibrosis is the leading cause of death among adults with HPS-1 and HPS-4 genetic types, which are associated with defects in the biogenesis of lysosome-related organelles complex-3 (BLOC-3), a guanine exchange factor (GEF) for a small GTPase, Rab32. LROs are not ubiquitously present in all cell types, and specific cells utilize LROs to accomplish dedicated functions. Fibroblasts are not known to contain LROs, and the function of BLOC-3 in fibroblasts is unclear. Here, we report that lung fibroblasts isolated from patients with HPS-1 have increased migration capacity. Silencing HPS-1 in normal lung fibroblasts similarly leads to increased migration. We also show that the increased migration is driven by elevated levels of Myosin IIB. Silencing HPS1 or RAB32 in normal lung fibroblasts leads to increased MYOSIN IIB levels. MYOSIN IIB is downstream of p38-MAPK, which is a known target of angiotensin receptor signaling. Treatment with losartan, an angiotensin receptor inhibitor, decreases MYOSIN IIB levels and impedes HPS lung fibroblast migration in vitro. Furthermore, pharmacologic inhibition of angiotensin receptor with losartan seemed to decrease migration of HPS lung fibroblasts in vivo in a zebrafish xenotransplantation model. Taken together, we demonstrate that BLOC-3 plays an important role in MYOSIN IIB regulation within lung fibroblasts and contributes to fibroblast migration.

Progressive pulmonary fibrosis in a murine model of Hermansky-Pudlak syndrome.

BACKGROUND: HPS-1 is a genetic type of Hermansky-Pudlak syndrome (HPS) with highly penetrant pulmonary fibrosis (HPSPF), a restrictive lung disease that is similar to idiopathic pulmonary fibrosis (IPF). Hps1ep/ep (pale ear) is a naturally occurring HPS-1 mouse model that exhibits high sensitivity to bleomycin-induced pulmonary fibrosis (PF). Traditional methods of administering bleomycin as an intratracheal (IT) route to induce PF in this model often lead to severe acute lung injury and high mortality rates, complicating studies focusing on pathobiological mechanisms or exploration of therapeutic options for HPSPF. METHODS: To develop a murine model of HPSPF that closely mimics the progression of human pulmonary fibrosis, we investigated the pulmonary effects of systemic delivery of bleomycin in Hps1ep/ep mice using a subcutaneous minipump and compared results to oropharyngeal delivery of bleomycin. RESULTS: Our study revealed that systemic delivery of bleomycin induced limited, acute inflammation that resolved. The distinct inflammatory phase preceded a slow, gradually progressive fibrogenesis that was shown to be both time-dependent and dose-dependent. The fibrosis phase exhibited characteristics that better resembles human disease with focal regions of fibrosis that were predominantly found in peribronchovascular areas and in subpleural regions; central lung areas contained relatively less fibrosis. CONCLUSION: This model provides a preclinical tool that will allow researchers to study the mechanism of pulmonary fibrosis in HPS and provide a platform for the development of therapeutics to treat HPSPF. This method can be applied on studies of IPF or other monogenic disorders that lead to pulmonary fibrosis.

Automated Digital Quantification of Pulmonary Fibrosis in Human Histopathology Specimens.

Pulmonary fibrosis is characterized by abnormal interstitial extracellular matrix and cellular accumulations. Methods quantifying fibrosis severity in lung histopathology samples are semi-quantitative, subjective, and analyze only portions of sections. We sought to determine whether automated computerized imaging analysis shown to continuously measure fibrosis in mice could also be applied in human samples. A pilot study was conducted to analyze a small number of specimens from patients with Hermansky-Pudlak syndrome pulmonary fibrosis (HPSPF) or idiopathic pulmonary fibrosis (IPF). Digital images of entire lung histological serial sections stained with picrosirius red and alcian blue or anti-CD68 antibody were analyzed using dedicated software to automatically quantify fibrosis, collagen, and macrophage content. Automated fibrosis quantification based on parenchymal tissue density and fibrosis score measurements was compared to pulmonary function values or Ashcroft score. Automated fibrosis quantification of HPSPF lung explants was significantly higher than that of IPF lung explants or biopsies and was also significantly higher in IPF lung explants than in IPF biopsies. A high correlation coefficient was found between some automated quantification measurements and lung function values for the three sample groups. Automated quantification of collagen content in lung sections used for digital image analyses was similar in the three groups. CD68 immunolabeled cell measurements were significantly higher in HPSPF explants than in IPF biopsies. In conclusion, computerized image analysis provides access to accurate, reader-independent pulmonary fibrosis quantification in human histopathology samples. Fibrosis, collagen content, and immunostained cells can be automatically and individually quantified from serial sections. Robust automated digital image analysis of human lung samples enhances the available tools to quantify and study fibrotic lung disease.