68 research outputs found
Detection of Argonaute (Ago) protein associated MiRNA by combining anti-ago antibody recognition with real-time PCR
MicroRNAs (miRNAs) are small molecules of noncoding RNA that range between ~19-22 nucleotides in length. In recent years, scientists have observed that these small RNA molecules exist in the extracellular environment within eukaryotic organisms. Furthermore, these microRNA molecules are known to associate with a family of proteins named Argonaute proteins. These microRNA/Argonaute protein complexes are the core of a larger assembly of proteins that compose the RNA Induced Silencing Complex (RISC). The RISC has exhibited the ability to inhibit the translation of, or cleave, its target messenger RNA (mRNA), the latter of which being exclusive only to Argonaute 2 protein (Ago2). It has also been observed in the literature that these Argonaute/miRNA complexes often target genomic regions associated with various cancers in humans and, currently, more than 2000 miRNAs have been discovered and published in the literature. Current methods for analyzing miRNA expression involve total RNA extraction using methods such as ethanol precipitation. However, total RNA extraction does not take into consideration that the major functional component of post-transcriptional inhibition is indeed that Argonaute protein/miRNA complex and not the miRNA alone. In this study, we investigate a novel method to capture and detect the active Ago2/miRNA (miRNP) complex and quantitate associated miRNAs by utilizing an antibody against Ago2 and subsequent application of real-time PCR to successfully capture the active miRNP complex and quantitate the associated miRNAs
A translational approach for limb vascular delivery of the micro-dystrophin gene without high volume or high pressure for treatment of Duchenne muscular dystrophy
Background: Duchenne muscular dystrophy (DMD) is an X-linked recessive disorder with monogenic mutations
setting the stage for successful gene therapy treatment. We have completed a study that directly deals with the following
key issues that can be directly adapted to a gene therapy clinical trial using rAAV considering the following criteria: 1) A
regional vascular delivery approach that will protect the patient from widespread dissemination of virus; 2) an approach
to potentially facilitate safe passage of the virus for efficient skeletal muscle transduction; 3) the use of viral doses to
accommodate current limitations imposed by vector production methods; 4) and at the same time, achieve a clinically
meaningful outcome by transducing multiple muscles in the lower limb to prolong ambulation.
Methods: The capacity of AAV1, AAV6 or AAV8 to cross the vascular endothelial barrier carrying a micro-dystrophin
cDNA was compared under identical conditions with delivery through a catheter placed in the femoral artery of the mdx
mouse. Transduction efficiency was assessed by immuno-staining using an antibody (Manex1a) that recognizes the Nterminus
of micro-dystrophin. The degree of physiologic correction was assessed by measuring tetanic force and
protection from eccentric contraction in the extensor digitorum longus muscle (EDL). The vascular delivery paradigm
found successful in the mouse was carried to the non-human primate to test its potential translation to boys with DMD.
Results: Regional vascular delivery resulted in transduction by rAAV8.micro-dystrophin reaching 94.5 ± 0.9 (1 month),
91.3 ± 3.1 (2 months), and 89.6 ± 1.6% (3 months). rAAV6.micro-dystrophin treated animals demonstrated 87.7 ± 6.8
(1 month), 78.9 ± 7.4 (2 months), and 81.2 ± 6.2% (3 months) transduction. In striking contrast, rAAV1 demonstrated
very low transduction efficiency [0.9 ± 0.3 (1 month), 2.1 ± 0.8 (2 months), and 2.1 ± 0.7% (3 months)] by vascular
delivery. Micro-dystrophin delivered by rAAV8 and rAAV6 through the femoral artery significantly improved tetanic
force and protected against eccentric contraction. Mouse studies translated to the hindlimb of cynamologous macaques
using a similar vascular delivery paradigm. rAAV8 carrying eGFP in doses proportional to the mouse (5 Ă— 1012 vg/kg in
mouse vs 2 × 1012 vg/kg in monkey) demonstrated widespread gene expression [medial gastrocnemius – 63.8 ± 4.9%,
lateral gastrocnemius – 66.0 ± 4.5%, EDL – 80.2 ± 3.1%, soleus – 86.4 ± 1.9%, TA – 72.2 ± 4.0%.
Conclusion: These studies demonstrate regional vascular gene delivery with AAV serotype(s) in mouse and non-human
primate at doses, pressures and volumes applicable for clinical trials in children with DMD
Multi-Ancestry Genome-Wide Association Analyses Improve Resolution of Genes and Pathways Influencing Lung Function and Chronic Obstructive Pulmonary Disease Risk
Lung-function impairment underlies chronic obstructive pulmonary disease (COPD) and predicts mortality. In the largest multi-ancestry genome-wide association meta-analysis of lung function to date, comprising 580,869 participants, we identified 1,020 independent association signals implicating 559 genes supported by ≥2 criteria from a systematic variant-to-gene mapping framework. These genes were enriched in 29 pathways. Individual variants showed heterogeneity across ancestries, age and smoking groups, and collectively as a genetic risk score showed strong association with COPD across ancestry groups. We undertook phenome-wide association studies for selected associated variants as well as trait and pathway-specific genetic risk scores to infer possible consequences of intervening in pathways underlying lung function. We highlight new putative causal variants, genes, proteins and pathways, including those targeted by existing drugs. These findings bring us closer to understanding the mechanisms underlying lung function and COPD, and should inform functional genomics experiments and potentially future COPD therapies
Worldwide Argus II implantation: recommendations to optimize patient outcomes
Abstract
Background
A position paper based on the collective experiences of Argus II Retinal Prosthesis System investigators to review strategies to optimize outcomes in patients with retinitis pigmentosa undergoing retinal prosthesis implantation.
Methods
Retinal surgeons, device programmers, and rehabilitation specialists from Europe, Canada, Middle East, and the United States were convened to the first international Argus II Investigator Meeting held in Ann Arbor, MI in March 2015. The recommendations from the collective experiences were collected. Factors associated with successful outcomes were determined.
Results
Factors leading to successful outcomes begin with appropriate patient selection, expectation counseling, and preoperative retinal assessment. Challenges to surgical implantation include presence of staphyloma and inadequate Tenon’s capsule or conjunctiva. Modified surgical technique may reduce risks of complications such as hypotony and conjunctival erosion. Rehabilitation efforts and correlation with validated outcome measures following implantation are critical.
Conclusions
Bringing together Argus II investigators allowed the identification of strategies to optimize patient outcomes. Establishing an on-line collaborative network will foster coordinated research efforts to advance outcome assessment and rehabilitation strategies.http://deepblue.lib.umich.edu/bitstream/2027.42/134581/1/12886_2016_Article_225.pd
Systemic Gene Delivery in Large Species for Targeting Spinal Cord, Brain, and Peripheral Tissues for Pediatric Disorders
Adeno-associated virus type 9 (AAV9) is a powerful tool for delivering genes throughout the central nervous system (CNS) following intravenous injection. Preclinical results in pediatric models of spinal muscular atrophy (SMA) and lysosomal storage disorders provide a compelling case for advancing AAV9 to the clinic. An important translational step is to demonstrate efficient CNS targeting in large animals at various ages. In the present study, we tested systemically injected AAV9 in cynomolgus macaques, administered at birth through 3 years of age for targeting CNS and peripheral tissues. We show that AAV9 was efficient at crossing the blood–brain barrier (BBB) at all time points investigated. Transgene expression was detected primarily in glial cells throughout the brain, dorsal root ganglia neurons and motor neurons within the spinal cord, providing confidence for translation to SMA patients. Systemic injection also efficiently targeted skeletal muscle and peripheral organs. To specifically target the CNS, we explored AAV9 delivery to cerebrospinal fluid (CSF). CSF injection efficiently targeted motor neurons, and restricted gene expression to the CNS, providing an alternate delivery route and potentially lower manufacturing requirements for older, larger patients. Our findings support the use of AAV9 for gene transfer to the CNS for disorders in pediatric populations
The Banff 2022 Kidney Meeting Work Plan:Data-driven refinement of the Banff Classification for renal allografts
The XVIth Banff Meeting for Allograft Pathology was held in Banff, Alberta, Canada, from September 19 to 23, 2022, as a joint meeting with the Canadian Society of Transplantation. In addition to a key focus on the impact of microvascular inflammation and biopsy-based transcript analysis on the Banff Classification, further sessions were devoted to other aspects of kidney transplant pathology, in particular T cell–mediated rejection, activity and chronicity indices, digital pathology, xenotransplantation, clinical trials, and surrogate endpoints. Although the output of these sessions has not led to any changes in the classification, the key role of Banff Working Groups in phrasing unanswered questions, and coordinating and disseminating results of investigations addressing these unanswered questions was emphasized. This paper summarizes the key Banff Meeting 2022 sessions not covered in the Banff Kidney Meeting 2022 Report paper and also provides an update on other Banff Working Group activities relevant to kidney allografts.</p
The Banff 2022 Kidney Meeting Work Plan:Data-driven refinement of the Banff Classification for renal allografts
The XVIth Banff Meeting for Allograft Pathology was held in Banff, Alberta, Canada, from September 19 to 23, 2022, as a joint meeting with the Canadian Society of Transplantation. In addition to a key focus on the impact of microvascular inflammation and biopsy-based transcript analysis on the Banff Classification, further sessions were devoted to other aspects of kidney transplant pathology, in particular T cell–mediated rejection, activity and chronicity indices, digital pathology, xenotransplantation, clinical trials, and surrogate endpoints. Although the output of these sessions has not led to any changes in the classification, the key role of Banff Working Groups in phrasing unanswered questions, and coordinating and disseminating results of investigations addressing these unanswered questions was emphasized. This paper summarizes the key Banff Meeting 2022 sessions not covered in the Banff Kidney Meeting 2022 Report paper and also provides an update on other Banff Working Group activities relevant to kidney allografts.</p
Prediction of overall survival for patients with metastatic castration-resistant prostate cancer : development of a prognostic model through a crowdsourced challenge with open clinical trial data
Background Improvements to prognostic models in metastatic castration-resistant prostate cancer have the potential to augment clinical trial design and guide treatment strategies. In partnership with Project Data Sphere, a not-for-profit initiative allowing data from cancer clinical trials to be shared broadly with researchers, we designed an open-data, crowdsourced, DREAM (Dialogue for Reverse Engineering Assessments and Methods) challenge to not only identify a better prognostic model for prediction of survival in patients with metastatic castration-resistant prostate cancer but also engage a community of international data scientists to study this disease. Methods Data from the comparator arms of four phase 3 clinical trials in first-line metastatic castration-resistant prostate cancer were obtained from Project Data Sphere, comprising 476 patients treated with docetaxel and prednisone from the ASCENT2 trial, 526 patients treated with docetaxel, prednisone, and placebo in the MAINSAIL trial, 598 patients treated with docetaxel, prednisone or prednisolone, and placebo in the VENICE trial, and 470 patients treated with docetaxel and placebo in the ENTHUSE 33 trial. Datasets consisting of more than 150 clinical variables were curated centrally, including demographics, laboratory values, medical history, lesion sites, and previous treatments. Data from ASCENT2, MAINSAIL, and VENICE were released publicly to be used as training data to predict the outcome of interest-namely, overall survival. Clinical data were also released for ENTHUSE 33, but data for outcome variables (overall survival and event status) were hidden from the challenge participants so that ENTHUSE 33 could be used for independent validation. Methods were evaluated using the integrated time-dependent area under the curve (iAUC). The reference model, based on eight clinical variables and a penalised Cox proportional-hazards model, was used to compare method performance. Further validation was done using data from a fifth trial-ENTHUSE M1-in which 266 patients with metastatic castration-resistant prostate cancer were treated with placebo alone. Findings 50 independent methods were developed to predict overall survival and were evaluated through the DREAM challenge. The top performer was based on an ensemble of penalised Cox regression models (ePCR), which uniquely identified predictive interaction effects with immune biomarkers and markers of hepatic and renal function. Overall, ePCR outperformed all other methods (iAUC 0.791; Bayes factor >5) and surpassed the reference model (iAUC 0.743; Bayes factor >20). Both the ePCR model and reference models stratified patients in the ENTHUSE 33 trial into high-risk and low-risk groups with significantly different overall survival (ePCR: hazard ratio 3.32, 95% CI 2.39-4.62, p Interpretation Novel prognostic factors were delineated, and the assessment of 50 methods developed by independent international teams establishes a benchmark for development of methods in the future. The results of this effort show that data-sharing, when combined with a crowdsourced challenge, is a robust and powerful framework to develop new prognostic models in advanced prostate cancer.Peer reviewe
Author Correction: Multi-ancestry genome-wide association analyses improve resolution of genes and pathways influencing lung function and chronic obstructive pulmonary disease risk
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