In Vitro Models for the Evaluation of Antisense Oligonucleotides in Skin

The genodermatosis dystrophic epidermolysis bullosa (DEB) is caused by mutations in the COL7A1 gene which encodes type VII collagen (C7). In the cutaneous basement membrane zone, C7 secures attachment of the epidermal basal keratinocyte to the papillary dermis by means of anchoring fibril formation. The complete absence of these anchoring fibrils leads to severe blistering of skin and mucosa upon the slightest friction and early mortality. To date, although preclinical advances toward therapy are promising, treatment for the disease is merely symptomatic. Therefore, research into novel therapeutics is warranted.Antisense oligonucleotide (ASO)-mediated exon skipping is such a therapy . Clinical examination of naturally occurring exon skipping suggested that this mechanism could most likely benefit the most severely affected patients. The severe form of DEB is caused by biallelic null mutations. Exon skipping aims to bind an ASO to the mutated exon of the pre-mRNA in the cell nucleus. Thereby, the ASO inhibits the recognition of the mutated exon by the splicing machinery, and as a result, the mutated exon is spliced out from the mRNA with its surrounding introns, i.e., it is skipped. Here, we describe in vitro methods to evaluate ASO-mediated exon skipping in a preclinical setting

In Vivo Models for the Evaluation of Antisense Oligonucleotides in Skin

Here, we describe an in vivo model in which antisense oligonucleotides were preclinically evaluated in reconstituted patient and healthy control skin. The aim was to investigate the effect of antisense oligonucleotides upon local or systemic administration. This allows for clinically relevant evaluation of antisense oligonucleotides in an in vivo setting. In this model, primary human keratinocytes and fibroblasts were placed into silicone grafting chambers, implanted onto the back of athymic nude mice. After sufficient cells were expanded, within a few weeks, human skin grafts were generated with a high success rate. These mice bearing grafts were subsequently treated with antisense oligonucleotides targeting exon 105 of the COL7A1 gene which encodes type VII collagen. Patients completely lacking expression of type VII collagen develop severe blistering of skin and mucosa, i.e., recessive dystrophic epidermolysis bullosa. In this chapter, we describe the in vivo model used for the preclinical evaluation of antisense oligonucleotides as therapeutic approach for recessive dystrophic epidermolysis bullosa

Multi-gene panel testing for hereditary cancer predisposition in unsolved high-risk breast and ovarian cancer patients.

PurposeMany women with an elevated risk of hereditary breast and ovarian cancer have previously tested negative for pathogenic mutations in BRCA1 and BRCA2. Among them, a subset has hereditary susceptibility to cancer and requires further testing. We sought to identify specific groups who remain at high risk and evaluate whether they should be offered multi-gene panel testing.MethodsWe tested 300 women on a multi-gene panel who were previously enrolled in a long-term study at UCSF. As part of their long-term care, all previously tested negative for mutations in BRCA1 and BRCA2 either by limited or comprehensive sequencing. Additionally, they met one of the following criteria: (i) personal history of bilateral breast cancer, (ii) personal history of breast cancer and a first or second degree relative with ovarian cancer, and (iii) personal history of ovarian, fallopian tube, or peritoneal carcinoma.ResultsAcross the three groups, 26 women (9%) had a total of 28 pathogenic mutations associated with hereditary cancer susceptibility, and 23 women (8%) had mutations in genes other than BRCA1 and BRCA2. Ashkenazi Jewish and Hispanic women had elevated pathogenic mutation rates. In addition, two women harbored pathogenic mutations in more than one hereditary predisposition gene.ConclusionsAmong women at high risk of breast and ovarian cancer who have previously tested negative for pathogenic BRCA1 and BRCA2 mutations, we identified three groups of women who should be considered for subsequent multi-gene panel testing. The identification of women with multiple pathogenic mutations has important implications for family testing

Analysis of the functional consequences of targeted exon deletion in COL7A1 reveals prospects for dystrophic epidermolysis bullosa therapy

Genetically evoked deficiency of collagen VII causes dystrophic epidermolysis bullosa (DEB)-a debilitating disease characterized by chronic skin fragility and progressive fibrosis. Removal of exons carrying frame-disrupting mutations can reinstate protein expression in genetic diseases. The therapeutic potential of this approach is critically dependent on gene, protein, and disease intrinsic factors. Naturally occurring exon skipping in COL7A1, translating collagen VII, suggests that skipping of exons containing disease-causing mutations may be feasible for the treatment of DEB. However, despite a primarily in-frame arrangement of exons in the COL7A1 gene, no general conclusion of the aptitude of exon skipping for DEB can be drawn, since regulation of collagen VII functionality is complex involving folding, intra-and intermolecular interactions. To directly address this, we deleted two conceptually important exons located at both ends of COL7A1, exon 13, containing recurrent mutations, and exon 105, predicted to impact folding. The resulting recombinantly expressed proteins showed conserved functionality in biochemical and in vitro assays. Injected into DEB mice, the proteins promoted skin stability. By demonstrating functionality of internally deleted collagen VII variants, our study provides support of targeted exon deletion or skipping as a potential therapy to treat a large number of individuals with DEB

A physically active lifestyle is related to a lower level of skin autofluorescence in a large population with chronic-disease (LifeLines cohort)

Background: Physical activity (PA) has substantial health benefits and is important in combatting chronic diseases, which have been associated with elevated levels of advanced glycation endproducts (AGEs). AGEs play a role in the aging process, and an association between PA and AGEs has been reported. We aimed to investigate the relationship between PA and AGE accumulation in a general population and in a population with chronic diseases. Methods: This large cross-sectional population study used data from adult participants in the LifeLines project, with participant information drawn from the LifeLines database as well data from patients with diabetes mellitus or renal and/or cardiovascular diseases. Tissue AGE accumulation was assessed non-invasively by skin-autofluorescence (SAF) using an AGE reader (DiagnOptics Technologies BV, Groningen, the Netherlands). PA was assessed using the short questionnaire to assess health-enhancing physical activity (SQUASH). Multivariate linear regression analyses were adjusted for age, body mass index, sex, and smoking status. Results: Data from 63,452 participants (general population n = 59,177, chronic disease n = 4275) were analyzed. The general population was significantly younger (43.58 +/- 11.77 years, mean +/- SD) and had significantly lower SAF (1.90 +/- 0.42 arbitrary units (AU)) compared to the population with chronic disease (age: 55.51 +/- 12.07 years; SAF: 2.27 +/- 0.51 AU). In the group with chronic disease, more hours of moderate to vigorous physical activities per week were associated with lower SAF (beta= -0.002, 95% confidence interval (95%CI): -0.002 to -0.001). For the general population, there was no association between hours of moderate to vigorous activity and SAF (beta= 3.2 x 10(-5), 95%CI: 0.000-0.001, p = 0.742). However, there was an association in the general population between total hours of PA per week and SAF (beta= 4.2 x 10(-4), 95%CI: 0.000-0.001, p < 0.001), but this association was not found in the chronic disease population (beta = -3.2 x 10(-4), 95%CI: -0.001 to 0.000, p = 0.347). Conclusion: Our study demonstrates that an inverse relationship exists between PA and AGE accumulation in the population with chronic disease. More hours of moderate to vigorous activity is associated with a significantly decreased SAF. More PA is associated with a lower SAF, even after adjusting for the established predictors (age, body mass index, smoking status, and sex). Our findings could help to promote health and prolong longevity

The utility of a portable muscle ultrasound in the assessment of muscle alterations in children with acute lymphoblastic leukaemia

Background:During treatment for acute lymphoblastic leukaemia (ALL), children are prone to musculoskeletal deterioration. However, non-invasive tools to measure muscle mass and intramuscular alterations are limited. In this study we explored the feasibility of muscle ultrasound in children with ALL. Additionally, we analysed whether automated ultrasound outcomes of muscle size and intramuscular fat infiltration (IMAT) were associated with appendicular skeletal muscle mass (ASMM), muscle strength and physical performance. Methods: Children with ALL, aged 3–18 years were included during maintenance therapy. Bilateral images of the rectus femoris muscle were captured using a portable linear array transducer connected to a tablet. Subsequently, an automated image annotation software (MuscleSound) was used to estimate cross-sectional area, muscle thickness and IMAT. Feasibility was assessed using acceptance (percentage of children approached who were enrolled), practicality (percentage of children that completed the ultrasound measurement after enrolment) and implementation (percentage of children that had sufficient imaging to be processed and analysed by the software). Assessments of ASMM by bioimpedance analysis, muscle strength using handheld dynamometry and timed physical performance tests were administered at the same visit. Multivariable linear models were estimated to study the associations between muscle ultrasound outcomes and ASMM, strength and physical performance, adjusted for sex, age, body mass index and ALL treatment week. Results: Muscle ultrasound was performed in 60 out of 73 invited patients (76.9%), of which 37 were boys (61.7%), and median age was 6.1 years (range: 3–18.8 years). The acceptance was 98.7%, practicality 77.9% and implementation was 100%. Patients who refused the examination (n = 13) were younger (median: 3.6, range: 3–11.2 years) compared with the 60 examined children (P = 0.0009). In multivariable models, cross-sectional area was associated with ASMM (β = 0.49 Z-score, 95% confidence interval [CI]:0.3,2.4), knee-extension strength (β = 16.9 Newton [N], 95% CI: 4.8, 28.9), walking performance (β = −0.46 s, 95% CI: −0.75, −0.18) and rising from the floor (β = −1.07 s, 95% CI: −1.71, −0.42). Muscle thickness was associated with ASMM (β = 0.14 Z-score, 95% CI: 0.04, 0.24), knee-extension strength (β = 4.73 N, 95% CI: 0.99, 8.47), walking performance (β = −0.13 s, 95% CI: −0.22, −0.04) and rising from the floor (β = −0.28 s, 95% CI: −0.48, −0.08). IMAT was associated with knee-extension strength (β = −6.84 N, 95% CI: −12.26, −1.41), walking performance (β = 0.2 s, 95% CI: 0.08, 0.32) and rising from the floor (β = 0.54 s, 95% CI: 0.27, 0.8). None of the muscle ultrasound outcomes was associated with handgrip strength. Conclusions: Portable muscle ultrasound appears a feasible and useful tool to measure muscle size and intramuscular alterations in children with ALL. Validation studies using magnetic resonance imaging (gold standard) are necessary to confirm accuracy in paediatric populations.</p

The clinical and prognostic value of late Gadolinium enhancement imaging in heart failure with mid-range and preserved ejection fraction

Heart failure (HF) with mid-range or preserved ejection fraction (HFmrEF; HFpEF) is a heterogeneous disorder that could benefit from strategies to identify subpopulations at increased risk. We tested the hypothesis that HFmrEF and HFpEF patients with myocardial scars detected with late gadolinium enhancement (LGE) are at increased risk for all-cause mortality. Symptomatic HF patients with left ventricular ejection fraction (LVEF) > 40%, who underwent cardiac magnetic resonance (CMR) imaging were included. The presence of myocardial LGE lesions was visually assessed. T1 mapping was performed to calculate extracellular volume (ECV). Multivariable logistic regression analyses were used to determine associations between clinical characteristics and LGE. Cox regression analyses were used to assess the association between LGE and all-cause mortality. A total of 110 consecutive patients were included (mean age 71 +/- 10 years, 49% women, median N-terminal brain natriuretic peptide (NT-proBNP) 1259 pg/ml). LGE lesions were detected in 37 (34%) patients. Previous myocardial infarction and increased LV mass index were strong and independent predictors for the presence of LGE (odds ratio 6.32, 95% confidence interval (CI) 2.07-19.31, p = 0.001 and 1.68 (1.03-2.73), p = 0.04, respectively). ECV was increased in patients with LGE lesions compared to those without (28.6 vs. 26.6%, p = 0.04). The presence of LGE lesions was associated with a fivefold increase in the incidence of all-cause mortality (hazards ratio 5.3, CI 1.5-18.1, p = 0.009), independent of age, sex, New York Heart Association (NYHA) functional class, NT-proBNP, LGE mass and LVEF. Myocardial scarring on CMR is associated with increased mortality in HF patients with LVEF > 40% and may aid in selecting a subpopulation at increased risk

Natural Exon Skipping Sets the Stage for Exon Skipping as Therapy for Dystrophic Epidermolysis Bullosa

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