AAV capsid engineering identified two novel variants with improved in vivo tropism for cardiomyocytes.

AAV vectors are promising delivery tools for human gene therapy. However, broad tissue tropism and pre-existing immunity against natural serotypes limit their clinical use. We identified two AAV capsid variants, AAV2-THGTPAD and AAV2-NLPGSGD, by in vivo AAV2 peptide display library screening in a murine model of pressure overload-induced cardiac hypertrophy. Both variants showed significantly improved efficacy in in vivo cardiomyocyte transduction compared with the parental serotype AAV2 as indicated by a higher number of AAV vector episomes in the nucleus and significant improved transduction efficiency. Both variants also outcompeted the reference serotype AAV9 regarding cardiomyocyte tropism, reaching comparable cardiac transduction efficiencies accompanied with liver de-targeting and decreased transduction efficiency of non-cardiac cells. Capsid modification influenced immunogenicity as sera of mice treated with AAV2-THGTPAD and AAV2-NLPGSGD demonstrated a poor neutralization capacity for the parental serotype and the novel variants. In a therapeutic setting, using the long non-coding RNA H19 in low vector dose conditions, novel AAV variants mediated superior anti-hypertrophic effects and revealed a further improved target-to-noise ratio, i.e., cardiomyocyte tropism. In conclusion, AAV2-THGTPAD and AAV2-NLPGSGD are promising novel tools for cardiac-directed gene therapy outperforming AAV9 regarding the specificity and therapeutic efficiency of in vivo cardiomyocyte transduction.

Generation of human induced pluripotent stem cell line MHHi029-A from a male Fabry disease patient carrying c.959A > T mutation.

Fabry disease (FD) is a rare and inherited monogenetic disease caused by mutations in the X-chromosomal alpha-galactosidase A gene GLA concomitant with accumulation of its substrate globotriaosylceramide (Gb3) and multi-organ symptoms. We derived an induced pluripotent stem cell line, MHHi029-A, from a male FD patient carrying a c.959A > T missense mutation in the GLA gene. The hiPSCs show a normal karyotype, expression of pluripotency markers and trilineage differentiation capacity. Importantly, they present the patient-specific mutation in the GLA gene and are therefore a valuable resource for investigating the FD mechanism and identifying novel therapies.

Circulating cardiovascular microRNAs in critically ill COVID-19 patients.

AIMS: Coronavirus disease 2019 (COVID-19) is a still growing pandemic, causing many deaths and socio-economic damage. Elevated expression of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) entry receptor angiotensin-converting enzyme 2 on cardiac cells of patients with heart diseases may be related to cardiovascular burden. We have thus analysed cardiovascular and inflammatory microRNAs (miRs), sensitive markers of cardiovascular damage, in critically ill, ventilated patients with COVID-19 or influenza-associated acute respiratory distress syndrome (Influenza-ARDS) admitted to the intensive care unit and healthy controls. METHODS AND RESULTS: Circulating miRs (miR-21, miR-126, miR-155, miR-208a, and miR-499) were analysed in a discovery cohort consisting of patients with mechanically-ventilated COVID-19 (n = 18) and healthy controls (n = 15). A validation study was performed in an independent cohort of mechanically-ventilated COVID-19 patients (n = 20), Influenza-ARDS patients (n = 13) and healthy controls (n = 32). In both cohorts, RNA was isolated from serum and cardiovascular disease/inflammatory-relevant miR concentrations were measured by miR-specific TaqMan PCR analyses. In both the discovery and the validation cohort, serum concentration of miR-21, miR-155, miR-208a and miR-499 were significantly increased in COVID-19 patients compared to healthy controls. Calculating the area under the curve using receiver operating characteristic analysis miR-155, miR-208a and miR-499 showed a clear distinction between COVID-19 and Influenza-ARDS patients. CONCLUSION: In this exploratory study, inflammation and cardiac myocyte-specific miRs were upregulated in critically ill COVID-19 patients. Importantly, miR profiles were able to differentiate between severely ill, mechanically-ventilated Influenza-ARDS and COVID-19 patients, indicating a rather specific response and cardiac involvement of COVID-19.