Engineered PsCas9 enables therapeutic genome editing in mouse liver with lipid nanoparticles.

Clinical implementation of therapeutic genome editing relies on efficient in vivo delivery and the safety of CRISPR-Cas tools. Previously, we identified PsCas9 as a Type II-B family enzyme capable of editing mouse liver genome upon adenoviral delivery without detectable off-targets and reduced chromosomal translocations. Yet, its efficacy remains insufficient with non-viral delivery, a common challenge for many Cas9 orthologues.

Universal toxin-based selection for precise genome engineering in human cells.

Prokaryotic restriction enzymes, recombinases and Cas proteins are powerful DNA engineering and genome editing tools. However, in many primary cell types, the efficiency of genome editing remains low, impeding the development of gene- and cell-based therapeutic applications. A safe strategy for robust and efficient enrichment of precisely genetically engineered cells is urgently required.

Development of an ObLiGaRe Doxycycline Inducible Cas9 system for pre-clinical cancer drug discovery.

The CRISPR-Cas9 system has increased the speed and precision of genetic editing in cells and animals. However, model generation for drug development is still expensive and time-consuming, demanding more target flexibility and faster turnaround times with high reproducibility. The generation of a tightly controlled ObLiGaRe doxycycline inducible SpCas9 (ODInCas9) transgene and its use in targeted ObLiGaRe results in functional integration into both human and mouse cells culminating in the generation of the ODInCas9 mouse.

Cranial irradiation at early postnatal age impairs stroke-induced neural stem/progenitor cell response in the adult brain.

Cranial irradiation (IR) is commonly used to treat primary brain tumors and metastatic diseases. However, cranial IR-treated patients often develop vascular abnormalities later in life that increase their risk for cerebral ischemia. Studies in rodents have demonstrated that IR impairs maintenance of the neural stem/precursor cell (NSPC) pool and depletes neurogenesis.

IDOL regulates systemic energy balance through control of neuronal VLDLR expression.

Liver X receptors limit cellular lipid uptake by stimulating the transcription of Inducible Degrader of the LDL Receptor (IDOL), an E3 ubiquitin ligase that targets lipoprotein receptors for degradation. The function of IDOL in systemic metabolism is incompletely understood. Here we show that loss of IDOL in mice protects against the development of diet-induced obesity and metabolic dysfunction by altering food intake and thermogenesis.

In vivo genome and base editing of a human PCSK9 knock-in hypercholesterolemic mouse model.

Background: Plasma concentration of low-density lipoprotein (LDL) cholesterol is a well-established risk factor for cardiovascular disease. Inhibition of proprotein convertase subtilisin/kexin type 9 (PCSK9), which regulates cholesterol homeostasis, has recently emerged as an approach to reduce cholesterol levels. The development of humanized animal models is an important step to validate and study human drug targets, and use of genome and base editing has been proposed as a mean to target disease alleles.

PA28αβ overexpression enhances learning and memory of female mice without inducing 20S proteasome activity.

Background: The proteasome system plays an important role in synaptic plasticity. Induction and maintenance of long term potentiation is directly dependent on selective targeting of proteins for proteasomal degradation. The 20S proteasome activator PA28αβ activates hydrolysis of small nonubiquitinated peptides and possesses protective functions upon oxidative stress and proteinopathy.

In vivo CRISPR editing with no detectable genome-wide off-target mutations.

CRISPR-Cas genome-editing nucleases hold substantial promise for developing human therapeutic applications but identifying unwanted off-target mutations is important for clinical translation. A well-validated method that can reliably identify off-targets in vivo has not been described to date, which means it is currently unclear whether and how frequently these mutations occur. Here we describe 'verification of in vivo off-targets' (VIVO), a highly sensitive strategy that can robustly identify the genome-wide off-target effects of CRISPR-Cas nucleases in vivo.

Therapeutic Genome Editing With CRISPR/Cas9 in a Humanized Mouse Model Ameliorates α1-antitrypsin Deficiency Phenotype.

α1-antitrypsin (AAT) is a circulating serine protease inhibitor secreted from the liver and important in preventing proteolytic neutrophil elastase associated tissue damage, primarily in lungs. In humans, AAT is encoded by the SERPINA1 (hSERPINA1) gene in which a point mutation (commonly referred to as PiZ) causes aggregation of the miss-folded protein in hepatocytes resulting in subsequent liver damage. In an attempt to rescue the pathologic liver phenotype of a mouse model of human AAT deficiency (AATD), we used adenovirus to deliver Cas9 and a guide-RNA (gRNA) molecule targeting hSERPINA1.

Photothrombosis-induced infarction of the mouse cerebral cortex is not affected by the Nrf2-activator sulforaphane.

Sulforaphane-induced activation of the transcription factor NF-E2 related factor 2 (Nrf2 or the gene Nfe2l2) and subsequent induction of the phase II antioxidant system has previously been shown to exert neuroprotective action in a transient model of focal cerebral ischemia. However, its ability to attenuate functional and cellular deficits after permanent focal cerebral ischemia is not clear. We assessed the neuroprotective effects of sulforaphane in the photothrombotic model of permanent focal cerebral ischemia.

Long-term stimulation of neural progenitor cell migration after cortical ischemia in mice.

Background And Purpose: Cortical ischemia induces neural progenitor cell migration toward the injury site; however, whether these cells are capable of maintaining the migratory response for a longer period after injury remains uncertain.

Methods: We analyzed progenitor migration up to 1 year after induction of photothrombotic stroke to the mouse neocortex. Migrating progenitors identified as doublecortin positive cells (DCX+) were assessed using the immunohistochemistry and immunofluorescence.

Trauma-induced reactive gliosis is reduced after treatment with octanol and carbenoxolone.

Background: Reactive gliosis and scar formation after brain injury can inhibit the recovery process. As many glial cells utilize gap junctions for intercellular signaling, this study investigated whether two commonly used gap junction blockers, octanol and carbenoxolone, could attenuate reactive gliosis following a minor traumatic brain injury.

Methods: Octanol (710 mg/kg) or carbenoxolone (90 mg/kg) was administered 30 minutes before or after a needle track injury in adult male Sprague-Dawley rats.

Changes in the solubility and phosphorylation of α-synuclein over the course of Parkinson's disease.

Lewy bodies are made from insoluble, phosphorylated α-synuclein, but the earliest changes that precipitate such pathology still remain conjecture. In this study, we quantify and identify relationships between the levels of the main pathologic form of phosphorylated α-synuclein over the course of Parkinson's disease in regions affected early through to end-stage disease. Brain tissue samples from 33 cases at different disease stages and 13 controls were collected through the Australian Network of Brain Banks.

Different strokes for different folks: the rich diversity of animal models of focal cerebral ischemia.

No single animal model is able to encompass all of the variables known to affect human ischemic stroke. This review highlights the major strengths and weaknesses of the most commonly used animal models of acute ischemic stroke in the context of matching model and experimental aim. Particular emphasis is placed on the relationships between outcome and underlying vascular variability, physiologic control, and use of models of comorbidity.

ACE inhibition reduces infarction in normotensive but not hypertensive rats: correlation with cortical ACE activity.

Angiotensin-converting enzyme (ACE) inhibition can reduce stroke risk by up to 43% in humans and reduce the associated disability, and hence understanding the mechanism of improvement is important. In animals and humans, these effects may be independent of the blood pressure-lowering effects of ACE inhibition. Normotensive (Wistar-Kyoto (WKY)) and hypertensive (spontaneously hypertensive rat (SHR)) animals were treated with the ACE inhibitors ramipril or lisinopril for 7 or 42 days before 2 hours of transient middle cerebral artery occlusion (MCAo).

Can animal models of disease reliably inform human studies?

H. Bart van der Worp and colleagues discuss the controversies and possibilities of translating the results of animal experiments into human clinical trials.

Comparison of inflammation in the brain and spinal cord following mechanical injury.

Inflammation in the CNS predominantly involves microglia and macrophages, and is believed to be a significant cause of secondary injury following trauma. This study compares the microglial and macrophage response in the rat brain and spinal cord following discrete mechanical injury to better appreciate the degree to which these cells could contribute to secondary damage in these areas. We find that, 1 week after injury, the microglial and macrophage response is significantly greater in the spinal cord compared to the brain.

Stimulation of axonal sprouting by trophic factors immobilized within the wound core.

Traumatic injury to the CNS results in peri-wound sprouting without significant axonal growth beyond the lesion edge. We have previously demonstrated that dopaminergic sprouting in the injured striatum follows an increasing gradient of BDNF and GDNF expression, with sprouting ceasing at the point of maximal factor expression. Progressively more complicated associations of sprouting fibers with increasingly activated microglia and macrophages suggest these factors are localized to the cell surface.

Striatal dopaminergic neurons are lost with Parkinson's disease progression.

Increased numbers of dopaminergic neurons are described in the striatum of patients with Parkinson's disease. In postmortem striatal tissue from Parkinson's disease patients with short disease duration (< or =8 years), the number of dopaminergic neurons is approximately four times that in patients with long duration (> or =16 years). The data suggest the possibility that the presence of large numbers of these striatal dopaminergic neurons may be harmful and may accelerate the disease process.

Dopaminergic innervation of the human striatum in Parkinson's disease.

In Parkinson's disease (PD), dopaminergic input to the caudate nucleus and a band of putaminal tissue abutting the external globus pallidus seems well preserved on immunohistochemical staining for the dopamine transporter. Counting of dopaminergic terminals showed that terminal density in these regions in PD was the same as that in controls, which indicates that input is truly preserved and not a consequence of a compensatory upregulation of metabolism in a reduced pool of surviving terminals. When the branching pattern of dopaminergic axons coursing through the globus pallidus was examined, we found no evidence for increased axonal sprouting in PD that might have contributed to preservation of dopaminergic input to the putamen or caudate nucleus.

Imaging the ischemic penumbra with 18F-fluoromisonidazole in a rat model of ischemic stroke.

Background And Purpose: The ischemic penumbra is a major focus of stroke research. 18F-fluoromisonidazole (18F-FMISO), a positron emission tomography (PET) marker of hypoxic cells, has shown promise as a technique to image the penumbra in humans. Our aim was to delineate the pattern of 18F-FMISO binding in a rat middle cerebral artery transient thread-occlusion model, and correlate this with tissue outcome at 24 hours.