Gene editing restores dystrophin expression in a canine model of Duchenne muscular dystrophy

Mutations in the gene encoding dystrophin, a protein that maintains muscle integrity and function, cause Duchenne muscular dystrophy (DMD). The deltaE50-MD dog model of DMD harbors a mutation corresponding to a mutational “hotspot” in the human DMD gene. We used adeno-associated viruses to deliver CRISPR gene editing components to four dogs and examined dystrophin protein expression 6 weeks after intramuscular delivery (n = 2) or 8 weeks after systemic delivery (n = 2). After systemic delivery in skeletal muscle, dystrophin was restored to levels ranging from 3 to 90% of normal, depending on muscle type. In cardiac muscle, dystrophin levels in the dog receiving the highest dose reached 92% of normal. The treated dogs also showed improved muscle histology. These large-animal data support the concept that, with further development, gene editing approaches may prove clinically useful for the treatment of DMD

A mouse model for adult cardiac-specific gene deletion with CRISPR/Cas9

Clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) 9 genomic editing has revolutionized the generation of mutant animals by simplifying the creation of null alleles in virtually any organism. However, most current approaches with this method require zygote injection, making it difficult to assess the adult, tissue-specific functions of genes that are widely expressed or which cause embryonic lethality when mutated. Here, we describe the generation of cardiac-specific Cas9 transgenic mice, which express high levels of Cas9 in the heart, but display no overt defects. In proof-of-concept experiments, we used Adeno-Associated Virus 9 (AAV9) to deliver single-guide RNA (sgRNA) that targets the Myh6 locus exclusively in cardiomyocytes. Intraperitoneal injection of postnatal cardiac-Cas9 transgenic mice with AAV9 encoding sgRNA against Myh6 resulted in robust editing of the Myh6 locus. These mice displayed severe cardiomyopathy and loss of cardiac function, with elevation of several markers of heart failure, confirming the effectiveness of this method of adult cardiac gene deletion. Mice with cardiac-specific expression of Cas9 provide a tool that will allow rapid and accurate deletion of genes following a single injection of AAV9-sgRNAs, thereby circumventing embryonic lethality. This method will be useful for disease modeling and provides a means of rapidly editing genes of interest in the heart

Ablation of CaMKIId oxidation by CRISPR-Cas9 base editing as a therapy for cardiac disease

CRISPR-Cas9 gene editing is emerging as a prospective therapy for genomic mutations. However, current editing approaches are directed primarily toward relatively small cohorts of patients with specific mutations. Here, we describe a cardioprotective strategy potentially applicable to a broad range of patients with heart disease. We used base editing to ablate the oxidative activation sites of CaMKIId, a primary driver of cardiac disease. We show in cardiomyocytes derived from human induced pluripotent stem cells that editing the CaMKIId gene to eliminate oxidation-sensitive methionine residues confers protection from ischemia/reperfusion (IR) injury. Moreover, CaMKIId editing in mice at the time of IR enables the heart to recover function from otherwise severe damage. CaMKIId gene editing may thus represent a permanent and advanced strategy for heart disease therapy

Experiences of Kenyan Healthcare Workers Providing Services to Men Who Have Sex With Men: Qualitative Findings from a Sensitivity Training Programme

Introduction Men who have sex with men (MSM) in Kenya are at high risk for HIV and may experience prejudiced treatment in health settings due to stigma. An on-line computer-facilitated MSM sensitivity programme was conducted to educate healthcare workers (HCWs) about the health issues and needs of MSM patients. Methods Seventy-four HCWs from 49 ART-providing health facilities in the Kenyan Coast were recruited through purposive sampling to undergo a two-day MSM sensitivity training. We conducted eight focus group discussions (FGDs) with programme participants prior to and three months after completing the training programme. Discussions aimed to characterize HCWs’ challenges in serving MSM patients and impacts of programme participation on HCWs’ personal attitudes and professional capacities. Results Before participating in the training programme, HCWs described secondary stigma, lack of professional education about MSM, and personal and social prejudices as barriers to serving MSM clients. After completing the programme, HCWs expressed greater acknowledgement of MSM patients in their clinics, endorsed the need to treat MSM patients with high professional standards and demonstrated sophisticated awareness of the social and behavioural risks for HIV among MSM. Conclusions Findings provide support for this approach to improving health services for MSM patients. Further efforts are needed to broaden the reach of this training in other areas, address identified barriers to HCW participation and evaluate programme effects on patient and HCW outcomes using rigorous methodology

Effects of superovulated heifer diet type and quantity on relative mRNA abundances and pyruvate metabolism in recovered embryos.

This study investigated the effects of quantity and type of diet fed to superovulated donor heifers on molecular and metabolic indices of embryonic development. These effects included the relative abundances of mRNAs for the alpha 1 subunit of Na/K-ATPase and the antioxidant enzyme Cu/Zn-SOD, as well as pyruvate utilization in bovine morulae and blastocysts developed in vivo. Heifers were fed a daily ration of either grass silage and a citrus-beet pulp-based concentrate or grass silage and a barley-based concentrate for 116 days, both at 3 kg per day or ad libitum. In embryos derived from heifers fed the pulp-based diets, the relative abundances of the transcripts were not affected by either day of collection or quantity of diet. In embryos derived from heifers fed the barley-based diets, the relative abundances of the Na/K-ATPase transcripts were also not changed by these main effects, while the relative abundances of the Cu/Zn-SOD transcripts were affected by day of collection and by the quantity of diet. Pyruvate metabolism was affected by day of collection, and was significantly increased in day 8 embryos compared with day 7 and day 6 embryos. Diet quantity did not affect pyruvate utilization, whereas diet type did increase pyruvate metabolism in the barley group when compared with the pulp group. The results of this study show for the first time that molecular and metabolic variations may exist in embryos derived in vivo and developed in donor heifers on nutritional regimens differing in type and quantity. Differences in embryos collected on different developmental days may be attributed to varying cell numbers. Alterations in the relative abundances of the Cu/Zn-SOD transcripts and pyruvate metabolism caused by the quantity of diet fed to the donor animal were likely to have been due to alterations in metabolic end products that accumulate in reproductive tract fluids, whereas differences in embryonic metabolism caused by type of diet are related to the composition of the diet. These findings characterize embryos produced in vivo at the molecular level, indicating that the molecular markers used in the present study can differentiate between populations of embryos produced under different nutritional regimens and determine conditions conductive to the production of good quality embryos

A Cardiac MicroRNA Governs Systemic Energy Homeostasis by Regulation of MED13

SummaryObesity, type 2 diabetes, and heart failure are associated with aberrant cardiac metabolism. We show that the heart regulates systemic energy homeostasis via MED13, a subunit of the Mediator complex, which controls transcription by thyroid hormone and other nuclear hormone receptors. MED13, in turn, is negatively regulated by a heart-specific microRNA, miR-208a. Cardiac-specific overexpression of MED13 or pharmacologic inhibition of miR-208a in mice confers resistance to high-fat diet-induced obesity and improves systemic insulin sensitivity and glucose tolerance. Conversely, genetic deletion of MED13 specifically in cardiomyocytes enhances obesity in response to high-fat diet and exacerbates metabolic syndrome. The metabolic actions of MED13 result from increased energy expenditure and regulation of numerous genes involved in energy balance in the heart. These findings reveal a role of the heart in systemic metabolic control and point to MED13 and miR-208a as potential therapeutic targets for metabolic disorders.PaperCli

The psychology of “cure” - unique challenges to consent processes in HIV cure research in South Africa

BACKGROUND : Consent processes for clinical trials involving HIV prevention research have generated considerable debate globally over the past three decades. HIV cure/eradication research is scientifically more complex and consequently, consent processes for clinical trials in this field are likely to pose a significant challenge. Given that research efforts are now moving toward HIV eradication, stakeholder engagement to inform appropriate ethics oversight of such research is timely. This study sought to establish the perspectives of a wide range of stakeholders in HIV treatment and research to inform consent processes for cure research. METHODS : In total, 68 South African stakeholders participated in two qualitative research modalities. In-depth interviews (IDIs) were conducted with a purposive sample of 42 individuals - audiotaped with consent. Twenty-six stakeholders participated in three focus group discussions (FGDs). Thematic analysis of transcribed IDIs and FGDs was conducted. RESULTS : The majority of respondents indicated that there could be unique challenges in HIV cure research requiring special attention. In particular, given the complexity of cure science, translation of concepts into lay language would be critical for potential participants to adequately appreciate risks and benefits in early phase research with experimental interventions. Furthermore, to aid understanding of risks and benefits against a background of desperation for a cure, specially trained facilitators would be required to assist with a psychological assessment prior to consent to avoid curative misconceptions. Long-term participant engagement to assess durability of a cure would mean that the consent process would be prolonged, necessitating annual re-consent. Building trust to maintain such long-term relationships would be critical to retain study participants. CONCLUSION : Unique consent requirements for cure research in South Africa would include significant efforts to maximise understanding of trial procedures, risks and the need for long-term follow-up. However, the psychological dimension of cure must not be underestimated. Beyond an understanding of cure science, the emotional impact of HIV cure advances the discourse from cure to healing. Consequently, the consent process for cure research would need to be enhanced to include psychological support and counselling. This has several important implications for research ethics review requirements for consent in HIV cure research.Fogarty International Center and Office of AIDS Research, OD and National Institute of Allergy and Infectious Diseases and the National Institute of Mental Health of the National Institutes of Health.http://www.biomedcentral.com/bmcmedethicsam2020Immunolog

Dynamic Modulation of Thymic MicroRNAs in Response to Stress

thymocyte subsets. Several of the differentially regulated murine thymic miRs are also stress responsive in the heart, kidney, liver, brain, and/or spleen. The most dramatic thymic microRNA down modulated is miR-181d, exhibiting a 15-fold reduction following stress. This miR has both similar and distinct gene targets as miR-181a, another member of miR-181 family. Many of the differentially regulated microRNAs have known functions in thymopoiesis, indicating that their dysregulation will alter T cell repertoire selection and the formation of naïve T cells. This data has implications for clinical treatments involving anti-inflammatory steroids, ablation therapies, and provides mechanistic insights into the consequences of infections

A renewable energy-centred research agenda for planning and financing Nexus development objectives in rural sub-Saharan Africa

In rural sub-Saharan Africa – the global poverty hotspot – the vast majority of cropland is rainfed only, resulting in reduced and unstable yields. Smallholder farmers account for 80% of agricultural production but they have limited access to relevant services to support both commercial operations and their livelihoods: more than two-thirds of rural dwellers have no access to electricity (crucial for crop irrigation, processing, and storage) and about 40% have no access to clean water. Previous research has analysed integrated technological and resource management approaches to tackle these overlapping development gaps. To finance and implement such transformations in resource-constrained settings, it is now crucial to understand the business and investment implications, also considering the strong regional population growth and the increasing frequency and intensity of climate extremes. Here, we lay out a research agenda that promotes the integration of multi-scale modelling excellence along the climate-water-renewable energy-agriculture-development Nexus and the creation of robust business models for private companies that can sustainably support private smallholder farmers of SSAin their effort to eradicate poverty and inequality. The proposed agenda is a cornerstone of the EC-H2020 project LEAP-RE RE4AFAGRI (“Renewable Energy for African Agriculture: Integrating Modelling Excellence and Robust Business Models”). In proposing the agenda, we highlight the importance of integrating energy access into the Nexus framework from both research and investment perspectives