PCSK9 activation promotes early atherosclerosis in a vascular microphysiological system.

Atherosclerosis is a primary precursor of cardiovascular disease (CVD), the leading cause of death worldwide. While proprotein convertase subtilisin/kexin 9 (PCSK9) contributes to CVD by degrading low-density lipoprotein receptors (LDLR) and altering lipid metabolism, PCSK9 also influences vascular inflammation, further promoting atherosclerosis. Here, we utilized a vascular microphysiological system to test the effect of PCSK9 activation or repression on the initiation of atherosclerosis and to screen the efficacy of a small molecule PCSK9 inhibitor. We have generated PCSK9 over-expressed (P+) or repressed (P-) human induced pluripotent stem cells (iPSCs) and further differentiated them to smooth muscle cells (viSMCs) or endothelial cells (viECs). Tissue-engineered blood vessels (TEBVs) made from P+ viSMCs and viECs resulted in increased monocyte adhesion compared to the wild type (WT) or P- equivalents when treated with enzyme-modified LDL (eLDL) and TNF-α. We also found significant viEC dysfunction, such as increased secretion of VCAM-1, TNF-α, and IL-6, in P+ viECs treated with eLDL and TNF-α. A small molecule compound, NYX-1492, that was originally designed to block PCSK9 binding with the LDLR was tested in TEBVs to determine its effect on lowering PCSK9-induced inflammation. The compound reduced monocyte adhesion in P+ TEBVs with evidence of lowering secretion of VCAM-1 and TNF-α. These results suggest that PCSK9 inhibition may decrease vascular inflammation in addition to lowering plasma LDL levels, enhancing its anti-atherosclerotic effects, particularly in patients with elevated chronic inflammation.

In Vivo Gene Editing of Muscle Stem Cells with Adeno-Associated Viral Vectors in a Mouse Model of Duchenne Muscular Dystrophy.

Delivery of therapeutic transgenes with adeno-associated viral (AAV) vectors for treatment of myopathies has yielded encouraging results in animal models and early clinical studies. Although certain AAV serotypes efficiently target muscle fibers, transduction of the muscle stem cells, also known as satellite cells, is less studied. Here, we used a Pax7nGFP;Ai9 dual reporter mouse to quantify AAV transduction events in satellite cells. We assessed a panel of AAV serotypes for satellite cell tropism in the mdx mouse model of Duchenne muscular dystrophy and observed the highest satellite cell labeling with AAV9 following local or systemic administration. Subsequently, we used AAV9 to interrogate CRISPR/Cas9-mediated gene editing of satellite cells in the Pax7nGFP;mdx mouse. We quantified the level of gene editing using a Tn5 transposon-based method for unbiased sequencing of editing outcomes at the Dmd locus. We also found that muscle-specific promoters can drive transgene expression and gene editing in satellite cells. Lastly, to demonstrate the functionality of satellite cells edited at the Dmd locus by CRISPR in vivo, we performed a transplantation experiment and observed increased dystrophin-positive fibers in the recipient mouse. Collectively, our results confirm that satellite cells are transduced by AAV and can undergo gene editing to restore the dystrophin reading frame in the mdx mouse.

Myogenic Progenitor Cell Lineage Specification by CRISPR/Cas9-Based Transcriptional Activators.

Engineered CRISPR/Cas9-based transcriptional activators can potently and specifically activate endogenous fate-determining genes to direct differentiation of pluripotent stem cells. Here, we demonstrate that endogenous activation of the PAX7 transcription factor results in stable epigenetic remodeling and differentiates human pluripotent stem cells into skeletal myogenic progenitor cells. Compared with exogenous overexpression of PAX7 cDNA, we find that endogenous activation results in the generation of more proliferative myogenic progenitors that can maintain PAX7 expression over multiple passages in serum-free conditions while preserving the capacity for terminal myogenic differentiation. Transplantation of human myogenic progenitors derived from endogenous activation of PAX7 into immunodeficient mice resulted in a greater number of human dystrophin+ myofibers compared with exogenous PAX7 overexpression. RNA-sequencing analysis also revealed transcriptome-wide differences between myogenic progenitors generated via CRISPR-based endogenous activation of PAX7 and exogenous PAX7 cDNA overexpression. These studies demonstrate the utility of CRISPR/Cas9-based transcriptional activators for controlling cell-fate decisions.

Increasing the specificity of CRISPR systems with engineered RNA secondary structures.

CRISPR (clustered regularly interspaced short palindromic repeat) systems have been broadly adopted for basic science, biotechnology, and gene and cell therapy. In some cases, these bacterial nucleases have demonstrated off-target activity. This creates a potential hazard for therapeutic applications and could confound results in biological research. Therefore, improving the precision of these nucleases is of broad interest. Here we show that engineering a hairpin secondary structure onto the spacer region of single guide RNAs (hp-sgRNAs) can increase specificity by several orders of magnitude when combined with various CRISPR effectors. We first demonstrate that designed hp-sgRNAs can tune the activity of a transactivator based on Cas9 from Streptococcus pyogenes (SpCas9). We then show that hp-sgRNAs increase the specificity of gene editing using five different Cas9 or Cas12a variants. Our results demonstrate that RNA secondary structure is a fundamental parameter that can tune the activity of diverse CRISPR systems.

RNA-guided transcriptional silencing in vivo with S. aureus CRISPR-Cas9 repressors.

CRISPR-Cas9 transcriptional repressors have emerged as robust tools for disrupting gene regulation in vitro but have not yet been adapted for systemic delivery in adult animal models. Here we describe a Staphylococcus aureus Cas9-based repressor (dSaCas9KRAB) compatible with adeno-associated viral (AAV) delivery. To evaluate dSaCas9KRAB efficacy for gene silencing in vivo, we silenced transcription of Pcsk9, a regulator of cholesterol levels, in the liver of adult mice. Systemic administration of a dual-vector AAV8 system expressing dSaCas9KRAB and a Pcsk9-targeting guide RNA (gRNA) results in significant reductions of serum Pcsk9 and cholesterol levels. Despite a moderate host response to dSaCas9KRAB expression, Pcsk9 repression is maintained for 24 weeks after a single treatment, demonstrating the potential for long-term gene silencing in post-mitotic tissues with dSaCas9KRAB. In vivo programmable gene silencing enables studies that link gene regulation to complex phenotypes and expands the CRISPR-Cas9 perturbation toolbox for basic research and gene therapy applications.

Exhaustion of Airway Basal Progenitor Cells in Early and Established Chronic Obstructive Pulmonary Disease.

RATIONALE: Up to 40% of smokers develop chronic obstructive pulmonary disease (COPD) over a period that spans decades. Despite the importance of COPD, much remains to be learned about susceptibility and pathogenesis, especially during early, prediagnostic stages of disease. Airway basal progenitor cells are crucial for lung health and resilience because of their ability to repair injured airways. In COPD, the normal airway epithelium is replaced with increased basal and secretory (mucous) cells and decreased ciliated cells, suggesting that progenitors are impaired. OBJECTIVES: To examine airway basal progenitor cells and lung function in smokers with and without COPD. METHODS: Bronchial biopsies taken from smokers at risk for COPD and lung cancer were used to acquire airway basal progenitor cells. They were evaluated for count, self-renewal, and multipotentiality (ability to differentiate to basal, mucous, and ciliated cells), and progenitor count was examined for its relationship with lung function. MEASUREMENTS AND MAIN RESULTS: Basal progenitor count, self-renewal, and multipotentiality were all reduced in COPD versus non-COPD. COPD progenitors produced an epithelium with increased basal and mucous cells and decreased ciliated cells, replicating the COPD phenotype. Progenitor depletion correlated with lung function and identified a subset of subjects without COPD with lung function that was midway between non-COPD with high progenitor counts and those with COPD. CONCLUSIONS: Basal progenitor dysfunction relates to the histologic and physiologic manifestations of COPD and identifies a subset that may represent an early, prediagnostic stage of COPD, indicating that progenitor exhaustion is involved in COPD pathogenesis.

Incomplete MyoD-induced transdifferentiation is associated with chromatin remodeling deficiencies.

Our current understanding of cellular transdifferentiation systems is limited. It is oftentimes unknown, at a genome-wide scale, how much transdifferentiated cells differ quantitatively from both the starting cells and the target cells. Focusing on transdifferentiation of primary human skin fibroblasts by forced expression of myogenic transcription factor MyoD, we performed quantitative analyses of gene expression and chromatin accessibility profiles of transdifferentiated cells compared to fibroblasts and myoblasts. In this system, we find that while many of the early muscle marker genes are reprogrammed, global gene expression and accessibility changes are still incomplete when compared to myoblasts. In addition, we find evidence of epigenetic memory in the transdifferentiated cells, with reminiscent features of fibroblasts being visible both in chromatin accessibility and gene expression. Quantitative analyses revealed a continuum of changes in chromatin accessibility induced by MyoD, and a strong correlation between chromatin-remodeling deficiencies and incomplete gene expression reprogramming. Classification analyses identified genetic and epigenetic features that distinguish reprogrammed from non-reprogrammed sites, and suggested ways to potentially improve transdifferentiation efficiency. Our approach for combining gene expression, DNA accessibility, and protein-DNA binding data to quantify and characterize the efficiency of cellular transdifferentiation on a genome-wide scale can be applied to any transdifferentiation system.

Tracheal dysplasia precedes bronchial dysplasia in mouse model of N-nitroso trischloroethylurea induced squamous cell lung cancer.

Squamous cell lung cancer (SCC) is the second leading cause of lung cancer death in the US and has a 5-year survival rate of only 16%. Histological changes in the bronchial epithelium termed dysplasia are precursors to invasive SCC. However, the cellular mechanisms that cause dysplasia are unknown. To fill this knowledge gap, we used topical application of N-nitroso-tris chloroethylurea (NTCU) for 32 weeks to induce squamous dysplasia and SCC in mice. At 32 weeks the predominant cell type in the dysplastic airways was Keratin (K) 5 and K14 expressing basal cells. Notably, basal cells are extremely rare in the normal mouse bronchial epithelium but are abundant in the trachea. We therefore evaluated time-dependent changes in tracheal and bronchial histopathology after NTCU exposure (4, 8, 12, 16, 25 and 32 weeks). We show that tracheal dysplasia occurs significantly earlier than that of the bronchial epithelium (12 weeks vs. 25 weeks). This was associated with increased numbers of K5+/K14+ tracheal basal cells and a complete loss of secretory (Club cell secretory protein expressing CCSP+) and ciliated cells. TUNEL staining of NTCU treated tissues confirmed that the loss of CCSP+ and ciliated cells was not due to apoptosis. However, mitotic index (measured by bromodeoxyuridine incorporation) showed that NTCU treatment increased proliferation of K5+ basal cells in the trachea, and altered bronchial mitotic population from CCSP+ to K5+ basal cells. Thus, we demonstrate that NTCU-induced lung epithelial dysplasia starts in the tracheal epithelium, and is followed by basal cell metaplasia of the bronchial epithelium. This analysis extends our knowledge of the NTCU-SCC model by defining the early changes in epithelial cell phenotypes in distinct airway locations, and this may assist in identifying new targets for future chemoprevention studies.