A Novel CRISPR-Cas9 Strategy to Target DYSTROPHIN Mutations Downstream of Exon 44 in Patient-Specific DMD iPSCs.

Mutations in the DMD gene cause fatal Duchenne Muscular Dystrophy (DMD). An attractive therapeutic approach is autologous cell transplantation utilizing myogenic progenitors derived from induced pluripotent stem cells (iPSCs). Given that a significant number of DMD mutations occur between exons 45 and 55, we developed a gene knock-in approach to correct any mutations downstream of exon 44. We applied this approach to two DMD patient-specific iPSC lines carrying mutations in exons 45 and 51 and confirmed mini-DYSTROPHIN (mini-DYS) protein expression in corrected myotubes by western blot and immunofluorescence staining. Transplantation of gene-edited DMD iPSC-derived myogenic progenitors into NSG/mdx4Cv mice produced donor-derived myofibers, as shown by the dual expression of human DYSTROPHIN and LAMIN A/C. These findings further provide proof-of-concept for the use of programmable nucleases for the development of autologous iPSC-based therapy for muscular dystrophies.

The adult environment promotes the transcriptional maturation of human iPSC-derived muscle grafts.

Pluripotent stem cell (PSC)-based cell therapy is an attractive option for the treatment of multiple human disorders, including muscular dystrophies. While in vitro differentiating PSCs can generate large numbers of human lineage-specific tissue, multiple studies evidenced that these cell populations mostly display embryonic/fetal features. We previously demonstrated that transplantation of PSC-derived myogenic progenitors provides long-term engraftment and functional improvement in several dystrophic mouse models, but it remained unknown whether donor-derived myofibers mature to match adult tissue. Here, we transplanted iPAX7 myogenic progenitors into muscles of non-dystrophic and dystrophic mice and compared the transcriptional landscape of human grafts with respective in vitro-differentiated iPAX7 myotubes as well as human skeletal muscle biospecimens. Pairing bulk RNA sequencing with computational deconvolution of human reads, we were able to pinpoint key myogenic changes that occur during the in vitro-to-in vivo transition, confirm developmental maturity, and consequently evaluate their applicability for cell-based therapies.

Establishment of Skeletal Myogenic Progenitors from Non-Human Primate Induced Pluripotent Stem Cells.

Pluripotent stem (PS) cells enable the scalable production of tissue-specific derivatives with therapeutic potential for various clinical applications, including muscular dystrophies. Given the similarity to human counterparts, the non-human primate (NHP) is an ideal preclinical model to evaluate several questions, including delivery, biodistribution, and immune response. While the generation of human-induced PS (iPS)-cell-derived myogenic progenitors is well established, there have been no data for NHP counterparts, probably due to the lack of an efficient system to differentiate NHP iPS cells towards the skeletal muscle lineage. Here, we report the generation of three independent Macaca fascicularis iPS cell lines and their myogenic differentiation using PAX7 conditional expression. The whole-transcriptome analysis confirmed the successful sequential induction of mesoderm, paraxial mesoderm, and myogenic lineages. NHP myogenic progenitors efficiently gave rise to myotubes under appropriate in vitro differentiation conditions and engrafted in vivo into the TA muscles of NSG and FKRP-NSG mice. Lastly, we explored the preclinical potential of these NHP myogenic progenitors in a single wild-type NHP recipient, demonstrating engraftment and characterizing the interaction with the host immune response. These studies establish an NHP model system through which iPS-cell-derived myogenic progenitors can be studied.

Impact of the National Heart, Lung, and Blood Institute's Loan Repayment Program Funding on Retention of the National Institutes of Health Biomedical Workforce.

Background: The National Institutes of Health (NIH) Loan Repayment Programs (LRPs) were established by Congress in 2000 to help attract and retain highly qualified health professionals in biomedical careers by relieving financial pressure incurred from educational loans obtained during medical school and other advanced-degree clinical training programs. In 2019, the NIH LRP Program increased the maximum repayment from $35,000 per year to $50,000 per year for an individual's educational debt in return for two years of research performed in an NIH mission-relevant area (https://www.lrp.nih.gov/eligibility-programs). In addition, in 2020, the National Heart, Lung, and Blood Institute (NHLBI) increased its participation in the LRP by adding the Health Disparities Research Program to Clinical Research and Pediatric Research Programs. Objective: Before these substantive changes took effect, we sought to determine the impact of the NHLBI's participation in the LRP program on retention of scientists in the biomedical research workforce over the past 20 years. Methods: NHLBI LRP applicant cohorts from 2003 and 2008 were carefully examined with a 10-year follow-up period to measure the impact of applying for and obtaining NIH LRP funding on subsequent K- and R-level application and award rates, publication number, and average relative citation ratio as metrics to assess recruitment and retention of scientists in the biomedical research workforce. Results: Obtaining the LRP award was strongly associated with increased submission of and success in obtaining K- and RPG-grant funding and publications for both the 2003 and 2008 NHLBI LRP cohorts. An analysis of subgroups in the 2008 LRP cohort without prior F, K, or RPG funding revealed a consistently strong association between obtaining an LRP award and subsequent K- or RPG-award submission and success as well as potential synergy between obtaining an LRP award and participation on a T grant toward subsequent K- or RPG-award success rates. Conclusion: The LRP award appears to enhance retention in the biomedical research workforce when measured using metrics of grant application and award rates as well as research publications over a 10-year period.

A universal gene correction approach for FKRP-associated dystroglycanopathies to enable autologous cell therapy.

Mutations in the fukutin-related protein (FKRP) gene result in a broad spectrum of muscular dystrophy (MD) phenotypes, including the severe Walker-Warburg syndrome (WWS). Here, we develop a gene-editing approach that replaces the entire mutant open reading frame with the wild-type sequence to universally correct all FKRP mutations. We apply this approach to correct FKRP mutations in induced pluripotent stem (iPS) cells derived from patients displaying broad clinical severity. Our findings show rescue of functional α-dystroglycan (α-DG) glycosylation in gene-edited WWS iPS cell-derived myotubes. Transplantation of gene-corrected myogenic progenitors in the FKRPP448L-NSG mouse model gives rise to myofiber and satellite cell engraftment and, importantly, restoration of α-DG functional glycosylation in vivo. These findings suggest the potential feasibility of using CRISPR-Cas9 technology in combination with patient-specific iPS cells for the future development of autologous cell transplantation for FKRP-associated MDs.

Genomic Safe Harbor Expression of PAX7 for the Generation of Engraftable Myogenic Progenitors.

Inducible expression of PAX7 in differentiating pluripotent stem cells (PSCs) allows massively scalable generation of human myogenic progenitors, which upon transplantation into dystrophic muscles give rise to donor-derived myofibers and satellite cells. Therefore, PSC-derived PAX7+ myogenic progenitors represent an attractive therapeutic approach to promote muscle regeneration. Work to date has used lentiviral vectors (LVs) that randomly integrate inducible PAX7 transgenes. Here, we investigated whether equivalent induction of the myogenic program could be achieved by targeting the PAX7 transgene into genomic safe harbor (GSH) sites. Across multiple PSC lines, we find that this approach consistently generates expandable myogenic progenitors in vitro, although scalability of expansion is moderately reduced compared with the LV approach. Importantly, transplantation of GSH-targeted myogenic progenitors produces robust engraftment, comparable with LV counterparts. These findings provide proof of concept for the use of GSH targeting as a potential alternative approach to generate therapeutic PSC-derived myogenic progenitors for clinical applications.

Abnormal Endothelial Gene Expression Associated With Early Coronary Atherosclerosis.

Background We examined feasibility of a unique approach towards gaining insight into heritable risk for early atherosclerosis: surveying gene expression by endothelial cells from living subjects. Methods and Results Subjects aged <50 years (mean age, 37; range, 22-49) without obstructive coronary artery disease underwent coronary reactivity testing that identified them as having normal or abnormal coronary endothelial function. Cultures of Blood Outgrowth Endothelial Cells (BOEC) from 6 normal and 13 abnormal subjects passed rigorous quality control and were used for microarray assessment of gene expression. Of 9 genes differentially expressed at false discovery rate <0.1%, we here focus upon abnormal subjects having elevated expression of HMGB1 (high mobility group box 1) which we unexpectedly found to be linked to low LAMC1 (laminin gamma 1) expression. This linkage was corroborated by 3 of our past studies and confirmed bio-functionally. Compared with normal BOEC, abnormal BOEC released 13±3-fold more HMGB1 in response to lipopolysaccharide; and they deposited one tenth as much LAMC1 into collagen subendothelial matrix during culture. Clinical follow-up data are provided for 4 normal subjects (followed 13.4±0.1 year) and for 12 abnormal subjects (followed 9.1±4.5 years). Conclusions The known pathogenic effects of high-HMGB1 and low-LAMC1 predict that the combination would biologically converge upon the focal adhesion complex, to the detriment of endothelial shear responsiveness. This gene expression pattern may comprise a heritable risk state that promotes early coronary atherosclerosis. If so, the testing could be applied even in childhood, enabling early intervention. This approach offers a way to bridge the information gap between genetics and clinical phenotype.

Screening identifies small molecules that enhance the maturation of human pluripotent stem cell-derived myotubes.

Targeted differentiation of pluripotent stem (PS) cells into myotubes enables in vitro disease modeling of skeletal muscle diseases. Although various protocols achieve myogenic differentiation in vitro, resulting myotubes typically display an embryonic identity. This is a major hurdle for accurately recapitulating disease phenotypes in vitro, as disease commonly manifests at later stages of development. To address this problem, we identified four factors from a small molecule screen whose combinatorial treatment resulted in myotubes with enhanced maturation, as shown by the expression profile of myosin heavy chain isoforms, as well as the upregulation of genes related with muscle contractile function. These molecular changes were confirmed by global chromatin accessibility and transcriptome studies. Importantly, we also observed this maturation in three-dimensional muscle constructs, which displayed improved in vitro contractile force generation in response to electrical stimulus. Thus, we established a model for in vitro muscle maturation from PS cells.

Gene Correction of LGMD2A Patient-Specific iPSCs for the Development of Targeted Autologous Cell Therapy.

Limb girdle muscular dystrophy type 2A (LGMD2A), caused by mutations in the Calpain 3 (CAPN3) gene, is an incurable autosomal recessive disorder that results in muscle wasting and loss of ambulation. To test the feasibility of an autologous induced pluripotent stem cell (iPSC)-based therapy for LGMD2A, here we applied CRISPR-Cas9-mediated genome editing to iPSCs from three LGMD2A patients to enable correction of mutations in the CAPN3 gene. Using a gene knockin approach, we genome edited iPSCs carrying three different CAPN3 mutations, and we demonstrated the rescue of CAPN3 protein in myotube derivatives in vitro. Transplantation of gene-corrected LGMD2A myogenic progenitors in a novel mouse model combining immunodeficiency and a lack of CAPN3 resulted in muscle engraftment and rescue of the CAPN3 mRNA. Thus, we provide here proof of concept for the integration of genome editing and iPSC technologies to develop a novel autologous cell therapy for LGMD2A.

The National Heart, Lung, and Blood Institute Strategic Vision Implementation for Health Equity Research.

The National Heart, Lung, and Blood Institute (NHLBI) provides global leadership for a research, training, and education program to promote the prevention and treatment of heart, lung, and blood diseases and enhance the health of all individuals so that they can live longer and more fulfilling lives. Inherent in this mission is the commitment to advance health equity research as an avenue for enhancing the health of all individuals. Additionally, the four goals and eight research objectives of the NHLBI Strategic Vision directly support the commitment to health equity. In this article, we present selected examples of the NHLBI Strategic Vision implementation approaches for advancing health equity research in our mission areas of heart, lung, and blood diseases. Examples of diseases for which the burden of health inequities and our strategic vision implementation approaches are discussed include hypertension, heart failure, vascular dementia, asthma, and sickle cell disease. Examples are provided of new avenues of Institute-solicited research to stimulate and address compelling scientific questions and critical challenges to advance health equity. We also highlight the emerging fields of implementation science and predictive analytics as important opportunities to accelerate the translation of discovery science into health impact for all and to advance health equity.

Generating evidence to inform an update of asthma clinical practice guidelines: Perspectives from the National Heart, Lung, and Blood Institute.

Asthma is the most prevalent chronic respiratory disease worldwide. Its increasing prevalence and evidence of suboptimal control require renewed efforts in the development and widespread implementation of clinical practice guidelines for prevention, treatment, and control. Given the rapidly changing landscape and evolving best practices for guideline development, the National Heart, Lung, and Blood Institute made a commitment to support rigorous systematic evidence reviews that frontline health care providers and stakeholders could use to create new or update existing guidelines. This article describes the protocols, key questions, methodology, and analytic framework to support the update of the 2007 National Asthma Education and Prevention Program Expert Panel Report 3 (EPR-3) on the diagnosis and management of asthma in adults and children. It also describes the expert panel's practical experience in managing asthmatic patients across the age and severity spectrum. The article explains the process for ensuring that the expert panel's deliberations are conducted in accordance with the Institute of Medicine's standards and recommendations for guideline development. The outcome of this ambitious effort will be an update of the EPR-3 asthma guidelines and publication of the key recommendations in the Journal of Allergy and Clinical Immunology. Importantly, several novel approaches will be explored and incorporated as appropriate to accelerate adoption and sustained implementation of the guidelines.