Peptide-based capture-and-release purification of extracellular vesicles and statistical algorithm enabled quality assessment.

Circulating extracellular vesicles (EVs) have gained significant attention for discovering tumor biomarkers. However, isolating EVs with well-defined homogeneous populations from complex biological samples is challenging. Different isolation methods have been found to derive different EV populations carrying different molecular contents, which confounds current investigations and hinders subsequent clinical translation. Therefore, standardizing and building a rigorous assessment of isolated EV quality associated with downstream molecular analysis is essential. To address this need, we introduce a statistical algorithm (ExoQuality Index, EQI) by integrating multiple EV characterizations (size, particle concentration, zeta potential, total protein, and RNA), enabling direct EV quality assessment and comparisons between different isolation methods. We also introduced a novel capture-release isolation approach using a pH-responsive peptide conjugated with NanoPom magnetic beads (ExCy) for simple, fast, and homogeneous EV isolation from various biological fluids. Bioinformatic analysis of next-generation sequencing (NGS) data of EV total RNAs from pancreatic cancer patient plasma samples using our novel EV isolation approach and quality index strategy illuminates how this approach improves the identification of tumor associated molecular markers. Results showed higher human mRNA coverage compared to existing isolation approaches in terms of both pancreatic cancer pathways and EV cellular component pathways using gProfiler pathway analysis. This study provides a valuable resource for researchers, establishing a workflow to prepare and analyze EV samples carefully and contributing to the advancement of reliable and rigorous EV quality assessment and clinical translation.

CRISPR-Cas9 Engineered Extracellular Vesicles for the Treatment of Dominant Progressive Hearing Loss.

Clinical translation of gene therapy has been challenging, due to limitations in current delivery vehicles such as traditional viral vectors. Herein, we report the use of gRNA:Cas9 ribonucleoprotein (RNP) complexes engineered extracellular vesicles (EVs) for in vivo gene therapy. By leveraging a novel high-throughput microfluidic droplet-based electroporation system (µDES), we achieved 10-fold enhancement of loading efficiency and more than 1000-fold increase in processing throughput on loading RNP complexes into EVs (RNP-EVs), compared with conventional bulk electroporation. The flow-through droplets serve as enormous bioreactors for offering millisecond pulsed, low-voltage electroporation in a continuous-flow and scalable manner, which minimizes the Joule heating influence and surface alteration to retain natural EV stability and integrity. In the Shaker-1 mouse model of dominant progressive hearing loss, we demonstrated the effective delivery of RNP-EVs into inner ear hair cells, with a clear reduction of Myo7ash1 mRNA expression compared to RNP-loaded lipid-like nanoparticles (RNP-LNPs), leading to significant hearing recovery measured by auditory brainstem responses (ABR).

A minimal clinically important difference measured by the Cambridge Pulmonary Hypertension Outcome Review for patients with idiopathic pulmonary arterial hypertension.

Several patient-reported outcome measures have been developed to assess health status in pulmonary arterial hypertension. The required change in instrument scores needed, to be seen as meaningful to the individual, however remain unknown. We sought to identify minimal clinically important differences in the Cambridge Pulmonary Hypertension Outcome Review (CAMPHOR) and to validate these against objective markers of functional capacity. Minimal clinically important differences were established from a discovery cohort (n = 129) of consecutive incident cases of idiopathic pulmonary arterial hypertension with CAMPHOR scores recorded at treatment-naïve baseline and 4-12 months following pulmonary arterial hypertension therapy. An independent validation cohort (n = 87) was used to verify minimal clinically important differences. Concurrent measures of functional capacity relative to CAMPHOR scores were collected. Minimal clinically important differences were derived using anchor- and distributional-based approaches. In the discovery cohort, mean (SD) was 54.4 (16.4) years and 64% were female. Most patients (63%) were treated with sequential pulmonary arterial hypertension therapy. Baseline CAMPHOR scores were: Symptoms, 12 (7); Activity, 12 (7) and quality of life, 10 (7). Pulmonary arterial hypertension treatment resulted in significant improvements in CAMPHOR scores (p < 0.05). CAMPHOR minimal clinically important differences averaged across methods for health status improvement were: Symptoms, -4 points; Activity, -4 points and quality of life -3 points. CAMPHOR Activity score change ≥minimal clinically important difference was associated with significantly greater improvement in six-minute walk distance, in both discovery and validation populations. In conclusion, CAMPHOR scores are responsive to pulmonary arterial hypertension treatment. Minimal clinically important differences in pulmonary hypertension-specific scales may provide useful insights into treatment response in future clinical trials.

CAMPHOR score: patient-reported outcomes are improved by pulmonary endarterectomy in chronic thromboembolic pulmonary hypertension.

BACKGROUND: Pulmonary endarterectomy (PEA) is the recommended treatment for eligible patients with chronic thromboembolic pulmonary hypertension (CTEPH). The Cambridge Pulmonary Hypertension Outcome Review (CAMPHOR) score is an internationally validated patient-reported outcome (PRO) measure for CTEPH. It assesses three domains: activity, quality of life (QoL) and symptoms. We assessed PROs in patients with CTEPH undergoing PEA. METHODS: This retrospective observational study of consecutive CTEPH patients undergoing PEA at the UK national PEA centre between 2006 and 2017 assessed change in CAMPHOR score from baseline (pre-PEA) until up to 5 years post-PEA. CAMPHOR scores were compared between 1) those with and without clinically significant residual pulmonary hypertension and 2) those undergoing PEA and propensity-matched CTEPH patients who were not operated on. The minimally clinically important difference (MCID) was calculated using an anchor-based method. RESULTS: Out of 1324 CTEPH patients who underwent PEA, 1053 (80%) had a CAMPHOR score recorded pre-PEA, 934 (71%) had a score recorded within a year of PEA and 784 (60%) had both. There were significant improvements between pre- and post-PEA in all three CAMPHOR domains (median±interquartile range activity -5±7, QoL -4±8, symptoms -7±8; all p<0.0001). Improvements in CAMPHOR score were greater and more sustained in those without clinically significant residual pulmonary hypertension. CTEPH patients undergoing PEA had better CAMPHOR scores than those not operated on. The MCID in CAMPHOR score was -3±5 for activity, -4±7 for QoL and -6±7 for symptoms. CONCLUSIONS: PROs are markedly improved by PEA in patients with CTEPH, more so in those without clinically significant residual pulmonary hypertension.

Bone tissue engineering by way of allograft revitalization: mechanistic and mechanical investigations using a porcine model.

PURPOSE: "Allograft revitalization" is a process in which cadaveric bone is used to generate well-vascularized living bone. We had previously found that porcine allograft hemimandibles filled with autologous adipose-derived stem cells (ASCs) and recombinant human bone morphogenetic protein-2-soaked absorbable collagen sponge (rhBMP-2/ACS) were completely replaced by vascularized bone, provided the construct had been incubated within a periosteal envelope. The present study sought to deepen our understanding of allograft revitalization by investigating the individual contributions of ASCs and rhBMP-2 in the process and the mechanical properties of the revitalized allograft. MATERIALS AND METHODS: Porcine allograft hemimandible constructs were implanted bilaterally into rib periosteal envelopes in 8 pigs. To examine the contributions of ASCs and rhBMP-2, the following groups were assessed: group 1, periosteum alone; group 2, periosteum+ASCs; group 3, periosteum+rhBMP-2/ACS; and group 4, periosteum+ASCs+rhBMP-2/ACS. After 8 weeks, the allograft constructs were harvested for micro-computed tomography (CT) and histologic analyses and 3-point bending to assess the strength. RESULTS: On harvesting, the constructs receiving rhBMP-2/ACS had significantly greater bone shown by micro-CT than those receiving periosteum only (51,463 vs. 34,310 mm3; P = .031). The constructs receiving ASCs had increased bone compared to group 1 (periosteum only), although not significantly (P = .087). The combination of rhBMP-2/ACS with ASCs produced bone (50,399 mm3) equivalent to that of the constructs containing rhBMP-2/ACS only. The 3-point bending tests showed no differences between the 4 groups and a nonimplanted allograft or native mandible (P = .586), suggesting the absence of decreased strength of the allograft bone when revitalized. CONCLUSIONS: These data have shown that rhBMP-2/ACS significantly stimulates new bone formation by way of allograft revitalization and that the revitalized allograft has equivalent mechanical strength to native bone.