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.
Purpose of Review
Low back pain encompasses three distinct sources: axial lumbosacral, radicular, and referred pain. Annually, the prevalence of low back pain in the general US adult population is ...10–30%, and the lifetime prevalence of US adults is as high as 65–80%.
Recent Findings
Patient history, physical exam, and diagnostic testing are important components to accurate diagnosis and identification of patient pathophysiology. Etiologies of low back pain include myofascial pain, facet joint pain, sacroiliac joint pain, discogenic pain, spinal stenosis, and failed back surgery. In chronic back pain patients, a multidisciplinary, logical approach to treatment is most effective and can include multimodal medical, psychological, physical, and interventional approaches.
Summary
Low back pain is a difficult condition to effectively treat and continues to affect millions of Americans every year. In the current investigation, we present a comprehensive review of low back pain and discuss associated pathophysiology, diagnosis, and treatment.
Early in development, GABA, an inhibitory neurotransmitter in adults, is excitatory. NKCC1 (SLC12A2) encodes one of two cation chloride cotransporters mediating the conversion of GABA from excitatory ...to inhibitory. Using 3' and 5' RACE and PCR, we verified previously characterized alternative transcripts of NKCC1a (1-27) and NKCC1b (1-27(Δ21)), identified new NKCC1 transcripts, and explored their expression patterns during human prefrontal cortical development. A novel ultra-short transcript (1-2a) was expressed preferentially in the fetus. Expression of NKCC1b and 1-2a were decreased in schizophrenia compared with controls (NKCC1b: 0.8-fold decrease, p = 0.013; 1-2a: 0.8-fold decrease, p = 0.006). Furthermore, the expression of NKCC1b was associated with NKCC1 polymorphism rs3087889. The minor allele at rs3087889, associated with reduced NKCC1b expression (homozygous for major allele: N = 37; homozygous for minor allele: N = 15; 1.5-fold decrease; p < 0.01), was also associated with a modest increase in schizophrenia risk in a case-control sample (controls: N = 435; cases: N = 397, OR = 1.5). This same allele was then found associated with cognitive (n = 369) and fMRI (n = 313) intermediate phenotypes associated with schizophrenia-working memory (Cohen's d = 0.35), global cognition or g (d = 0.18), and prefrontal inefficiency (d = 0.36) as measured by BOLD fMRI during a working memory task. Together, these preclinical and clinical results suggest that variation in NKCC1 may increase risk for schizophrenia via alterations of mRNA expression at the molecular level and impairment of optimal prefrontal function at the macro or systems level.
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). Hps1
...(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.
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 Hps1
mice using a subcutaneous minipump and compared results to oropharyngeal delivery of bleomycin.
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.
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.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Abstract only
Overactivation of the renin-angiotensin system contributes to many common and rare cardiovascular diseases such as hypertension, atherosclerosis, and genetic thoracic aortopathies. ...Current pharmacologic therapies that target this pathway have been proven safe and effective, yet they require individuals with these conditions to adhere to daily medications. CRISPR-Cas9-mediated hepatic inactivation of the angiotensinogen (
AGT
) gene, the precursor of all angiotensin peptides, could be a promising “one and done” therapeutic approach for these disorders. However, disruption of
AGT
by Cas9 nuclease would result in irreversible changes to the genome and would likely have negative consequences if targeted to organs outside of the liver. CRISPRoff is a CRISPR-based epigenome editing tool consisting of catalytically inactive Cas9 fused to a DNA methyltransferase, as well as a guide RNA (gRNA) designed to target a genomic promoter of interest. Targeting to a gene promoter can result in DNA methylation and subsequent silencing of gene expression, a phenomenon that has the potential to be reversed by CRISPRon, a catalytically inactive Cas9 fused to a demethylase that could be targeted to the same site using the same gRNA. We hypothesized that CRISPRoff can be used to efficiently and durably silence
AGT
expression by methylation of the
AGT
promoter. To test this, we screened HuH-7 hepatoma cells with CRISPRoff and either individual gRNAs or dual combinations of gRNAs targeting the
AGT
promoter. We measured
AGT
mRNA expression by RT-qPCR and selected the two combinations of gRNAs that resulted in the greatest decrease in
AGT
expression for methylation analysis. We found that
AGT
expression was decreased by ≈50% in treated cells, and this correlated with substantially increased methylation throughout the
AGT
promoter. A long-term in vitro study to assess the durability of this methylation is underway. We are also generating a humanized
AGT
mouse model to test whether these CRISPRoff gRNAs can silence the human
AGT
gene in vivo. These results provide a proof of concept of CRISPRoff for silencing of
AGT
expression as a therapeutic approach for common and rare cardiovascular disorders.
Abstract only Pseudoxanthoma elasticum (PXE) is a rare, autosomal recessive disorder caused by pathogenic variants in ABCC6 , resulting in systemic depletion of the anti-mineralization compound ...pyrophosphate. Pyrophosphate depletion drives ectopic calcification of elastic fibers within the skin, retina, and vasculature. Arterial calcification can lead to intermittent claudication of peripheral arteries and coronary artery disease. There are currently no targeted therapies for PXE. Genome editing may be a promising therapeutic option because ABCC6 is predominantly expressed in the liver, a site that is amenable to current genome editing delivery modalities. We sought to assess whether adenine base editing could efficiently correct one of the most frequent ABCC6 variants in PXE patients, c.3490C>T (p.R1164X) in human hepatocytes and a humanized mouse model. We used prime editing to generate a homozygous R1164X human hepatoma cell line. Treatment of R1164X cells with mRNA encoding the adenine base editor ABE8.8 and a synthetic guide RNA (gRNA) resulted in efficient correction of the variant. OligoNucleotide Enrichment and sequencing (ONE-seq) was used to nominate candidate off-target sites, which were evaluated by targeted amplicon sequencing of edited R1164X cells. To mitigate off-target editing, we generated 21 hybrid gRNAs in which various RNA nucleotides in the protospacer were replaced by DNA nucleotides. All hybrid gRNAs maintained on-target editing, and interestingly, several hybrid gRNAs improved on-target editing compared to the standard gRNA. All hybrid gRNAs improved off-target editing, with multiple hybrid gRNAs eliminating off-target editing at multiple off-target sites. To assess in vivo correction of the R1164X variant, we generated a homozygous R1164X humanized mouse model which displays depletion of plasma pyrophosphate. Studies to correct the variant in the humanized mouse model by lipid nanoparticle delivery of the lead candidate hybrid gRNA are underway. Efficient correction of the R1164X variant in the liver by adenine base editing and restoration of pyrophosphate would represent one of the first examples of using genome editing to improve the multisystemic manifestations of a heritable connective tissue disorder.
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.
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