Campbell, Jarryd M.

Mitochondria are a dynamic eukaryotic innovation that play diverse roles in biology and disease. The mitochondrial genome is remarkably conserved in all vertebrates, encoding the same 37-gene set and overall genomic structure, ranging from 16,596 base pairs (bp) in the teleost zebrafish (Danio rerio) to 16,569 bp in humans. Mitochondrial disorders are amongst the most prevalent inherited diseases, affecting roughly 1 in every 5000 individuals. Currently, few effective treatments exist for those with mitochondrial ailments, representing a major unmet patient need. Mitochondrial dysfunction is also a common component of a wide variety of other human illnesses, ranging from neurodegenerative disorders such as Huntington’s disease and Parkinson’s disease to autoimmune illnesses such as multiple sclerosis and rheumatoid arthritis. The electron transport chain (ETC) component of mitochondria is critical for mitochondrial biology and defects can lead to many mitochondrial disease symptoms. Here, we present a publicly available collection of genetic mutants created in highly conserved, nuclear-encoded mitochondrial genes in Danio rerio. The zebrafish system represents a potentially powerful new opportunity for the study of mitochondrial biology and disease due to the large number of orthologous genes shared with humans and the many advanced features of this model system, from genetics to imaging. This collection includes 15 mutant lines in 13 different genes created through locus-specific gene editing to induce frameshift or splice acceptor mutations, leading to predicted protein truncation during translation. Additionally, included are 11 lines created by the random insertion of the gene-breaking transposon (GBT) protein trap cassette. All these targeted mutant alleles truncate conserved domains of genes critical to the proper function of the ETC or genes that have been implicated in human mitochondrial disease. This collection is designed to accelerate the use of zebrafish to study many different aspects of mitochondrial function to widen our understanding of their role in biology and human disease.

Genome engineering has gone mainstream because of breakthroughs in defining and harnessing naturally occurring, customizable DNA recognition cursors (protein or RNA-guided). At present, most gene editing relies on these cursors to direct custom DNA endonucleases to a specific genomic sequence to induce a double-strand break. New tools for genome engineering are continuously being explored, and another advance in DNA targeting has recently been described. Argonaute isolated from Natronobacterium gregoryi (NgAgo) is an ssDNA-based cursor that thus far has no known limitations in sequence recognition, shows promise for high specificity, and for many applications may represent a potentially more accessible genome-editing system over prior tools as it requires only a single, 24-base, 5′ phosphorylated ssDNA for DNA targeting. Genome engineering is in a remarkable moment of unprecedented growth with exponential reduction in costs reminiscent of Moore's law in electronics. Many questions remain with regard to Argonaute utility in specific systems, but there is no doubt that genome engineering is expanding into new and exciting areas from synthetic biology to gene therapy.

Transcription activator-like effectors (TALEs) are extremely effective, single-molecule DNA-targeting molecular cursors used for locus-specific genome science applications, including high-precision molecular medicine and other genome engineering applications. TALEs are used in genome engineering for locus-specific DNA editing and imaging, as artificial transcriptional activators and repressors, and for targeted epigenetic modification. TALEs as nucleases (TALENs) are effective editing tools and offer high binding specificity and fewer sequence constraints toward the targeted genome than other custom nuclease systems. One bottleneck of broader TALE use is reagent accessibility. For example, one commonly deployed method uses a multitube, 5-day assembly protocol. Here we describe FusX, a streamlined Golden Gate TALE assembly system that (1) is backward compatible with popular TALE backbones, (2) is functionalized as a single-tube 3-day TALE assembly process, (3) requires only commonly used basic molecular biology reagents, and (4) is cost-effective. More than 100 TALEN pairs have been successfully assembled using FusX, and 27 pairs were quantitatively tested in zebrafish, with each showing high somatic and germline activity. Furthermore, this assembly system is flexible and is compatible with standard molecular biology laboratory tools, but can be scaled with automated laboratory support. To demonstrate, we use a highly accessible and commercially available liquid-handling robot to rapidly and accurately assemble TALEs using the FusX TALE toolkit. Together, the FusX system accelerates TALE-based genomic science applications from basic science screening work for functional genomics testing and molecular medicine applications.

Recent advances in the targeted modification of complex eukaryotic genomes have unlocked a new era of genome engineering. From the pioneering work using zinc-finger nucleases (ZFNs), to the advent of the versatile and specific TALEN systems, and most recently the highly accessible CRISPR/Cas9 systems, we now possess an unprecedented ability to analyze developmental processes using sophisticated designer genetic tools. In this Review, we summarize the common approaches and applications of these still-evolving tools as they are being used in the most popular model developmental systems. Excitingly, these robust and simple genomic engineering tools also promise to revolutionize developmental studies using less well established experimental organisms.

Recent advances in the burgeoning field of genome engineering are accelerating the realization of personalized therapeutics for cardiovascular disease. In the postgenomic era, sequence-specific gene-editing tools enable the functional analysis of genetic alterations implicated in disease. In partnership with high-throughput model systems, efficient gene manipulation provides an increasingly powerful toolkit to study phenotypes associated with patient-specific genetic defects. Herein, this review emphasizes the latest developments in genome engineering and how applications within the field are transforming our understanding of personalized medicine with an emphasis on cardiovascular diseases.

BACKGROUND B-type natriuretic peptide (BNP), a key cardiac hormone in cardiorenal homeostasis, is produced as a 108 amino acid prohormone, proBNP1-108, which is converted to a biologically active peptide BNP1-32 and an inactive N-terminal (NT)-proBNP1-76. The widely accepted model is that the normal heart releases a proteolytically processed BNP1-32 and NT-proBNP, whereas the diseased heart secretes high amounts of unprocessed/glycosylated proBNP1-108 or inappropriately processed BNPs. In contrast, circulating proBNP1-108 has recently been identified in healthy individuals, indicating that the normal heart also secretes unprocessed proBNP1-108. However, the mechanism of proBNP1-108 secretion from the normal heart remains elusive. Our goal was to determine the molecular mechanisms underlying proBNP1-108 intracellular trafficking and secretion from the normal heart. METHODS We expressed preproBNP in cardiomyocytes, and determined the subcellular localization and dominant intracellular and extracellular forms of BNP. RESULTS Intracellular immunoreactive BNPs were first accumulated in the Golgi apparatus, and then distributed throughout the cytoplasm as secretory vesicles. The predominant intracellular form of BNP was nonglycosylated proBNP1-108, rather than BNP1-32. Glycosylated proBNP1-108, but not nonglycosylated proBNP1-108, was detected as the major extracellular form in the culture supernatants of preproBNP-expressing cell lines and primary human cardiomyocytes. Ablation of O-glycosylation of proBNP1-108 at T71 residue, near the convertase recognition site, reduced the extracellular proBNP1-108 and increased extracellular BNP1-32. CONCLUSIONS Intracellular proBNP trafficking occurs through a conventional Golgi–endoplasmic reticulum pathway. Glycosylation of proBNP1-108 controls the stability and processing of extracellular proBNP1-108. Our data establish a new BNP secretion model in which the normal cardiac cells secrete glycosylated proBNP1-108.

Background— Diastolic dysfunction associated with high blood pressure (BP) leads to cardiac remodeling and fibrosis and progression to congestive heart failure. B-type natriuretic peptide (BNP) has BP-lowering, antifibrotic, and antihypertrophic properties, which makes BNP an attractive agent for attenuating the adverse cardiac remodeling associated with hypertension. In the current study, we tested the effects of sustained cardiac proBNP gene delivery on BP, cardiac function, and remodeling in spontaneously hypertensive rats (SHR). Methods and Results— We used the myocardium-tropic adeno-associated virus serotype 9 (AAV9) vector to achieve continuously enhanced cardiac rat proBNP expression. In SHR, a single systemic administration of AAV9 vector allowed long-term cardiac BNP overexpression, resulting in reductions in systolic and diastolic BP for 9 months after injection. Left ventricular (LV) thickness, LV end-systolic dimensions, and LV mass were reduced, whereas ejection fraction was significantly increased, in BNP-treated compared with untreated SHR. Circumferential systolic strain and strain rate of the early phase of diastole were improved in BNP-treated compared with untreated SHR. Noncardiac overexpression of BNP via AAV2 vector was not associated with changes in BP and plasma BNP in SHR. Furthermore, normal Wistar rats injected with AAV9 proBNP vector showed significantly reduced heart weights 4 weeks after injection without BP reduction. Conclusions— AAV9 vector facilitates sustained cardiac proBNP overexpression and improves LV function in hypertensive heart disease. Long-term proBNP delivery improved both systolic and diastolic function. The effects on cardiac structure and function occurred independently of BP-lowering effects in normal Wistar rats.