Nevin Witman

MRNA technology, which has enabled the rapid development of vaccines for infectious diseases, also holds great promise for a new generation of therapies for a host of rare and common diseases. A decade of clinical trials are beginning to clarify the key barriers to unlock the transformative potential of mRNA drugs, which are being addressed with novel interdisciplinary technical advances that, in some cases, are integrating the fields of gene, cell and mRNA therapies. Here, we review the scientific insights from a select group of clinical studies on mRNA-based drugs, including enzyme replacement therapies for rare diseases, cancer immunotherapies, genome-modifying therapies, and immune cell reprogramming therapies for cancer and autoimmune diseases. Several innovative approaches such as clinically tractable in vivo delivery systems, the development of completely 'immune-silent' mRNA-vehicle formulations that allow repeated administration and the development of approaches for preferential delivery to organs other than the liver would expedite the development of mRNA therapeutics 2.0.

The dysregulated physical interaction between two intracellular membrane proteins, the sarco/endoplasmic reticulum Ca²⁺ ATPase and its reversible inhibitor phospholamban, induces heart failure by inhibiting calcium cycling. While phospholamban is a bona-fide therapeutic target, approaches to selectively inhibit this protein remain elusive. Here, we report the in vivo application of intracellular acting antibodies (intrabodies), derived from the variable domain of camelid heavy-chain antibodies, to modulate the function of phospholamban. Using a synthetic VHH phage-display library, we identify intrabodies with high affinity and specificity for different conformational states of phospholamban. Rapid phenotypic screening, via modified mRNA transfection of primary cells and tissue, efficiently identifies the intrabody with most desirable features. Adeno-associated virus mediated delivery of this intrabody results in improvement of cardiac performance in a murine heart failure model. Our strategy for generating intrabodies to investigate cardiac disease combined with modified mRNA and adeno-associated virus screening could reveal unique future therapeutic opportunities.

The contribution of the epicardium, the outermost layer of the heart, to cardiac regeneration has remained controversial due to a lack of suitable analytical tools. By combining genetic marker-independent lineage-tracing strategies with transcriptional profiling and loss-of-function methods, we report here that the epicardium of the highly regenerative salamander species Pleurodeles waltl has an intrinsic capacity to differentiate into cardiomyocytes. Following cryoinjury, CLDN6⁺ epicardium-derived cells appear at the lesion site, organize into honeycomb-like structures connected via focal tight junctions and undergo transcriptional reprogramming that results in concomitant differentiation into de novo cardiomyocytes. Ablation of CLDN6⁺ differentiation intermediates as well as disruption of their tight junctions impairs cardiac regeneration. Salamanders constitute the evolutionarily closest species to mammals with an extensive ability to regenerate heart muscle and our results highlight the epicardium and tight junctions as key targets in efforts to promote cardiac regeneration.

Yohimbine is a highly selective and potent α₂-adrenoceptor antagonist, which is usually treated as an adjunction for impotence, as well for weight loss and natural bodybuilding aids. However, it was recently reported that Yohimbine causes myocardial injury and controversial results were reported in the setting of cardiac diseases. Here, we used human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) as a model system to explore electrophysiologic characterization after exposure to Yohimbine. HiPSC-CMs were differentiated by employment of inhibitory Wnt compounds. For analysis of electrophysiological properties, conventional whole-cell patch-clamp recording was used. Specifically, spontaneous action potentials, pacemaker currents (I_f), sodium (Na⁺) channel (I_Na), and calcium (Ca⁺⁺) channel currents (I_Ca) were assessed in hiPSC-CMs after exposure to Yohimbine. HiPSC-CMs expressed sarcomeric-α-actinin and MLC2V proteins, as well as exhibited ventricular-like spontaneous action potential waveform. Yohimbine inhibited frequency of hiPSC-CMs spontaneous action potentials and significantly prolonged action potential duration in a dose-dependent manner. In addition, rest potential, threshold potential, amplitude, and maximal diastolic potential were decreased, whereas APD₅₀/APD₉₀ was prolonged. Yohimbine inhibited the amplitude of I_Na in low doses (IC₅₀ = 14.2 μM, n = 5) and inhibited I_Ca in high doses (IC₅₀ = 139.7 μM, n = 5). Whereas Yohimbine did not affect the activation curves, treatment resulted in left shifts in inactivation curves of both Na⁺ and Ca⁺⁺ channels. Here, we show that Yohimbine induces direct cardiotoxic effects on spontaneous action potentials of I_Na and I_Ca in hiPSC-CMs. Importantly, these effects were not mediated by α₂-adrenoceptor signaling. Our results strongly suggest that Yohimbine directly and negatively affects electrophysiological properties of human cardiomyocytes. These findings are highly relevant for potential application of Yohimbine in patients with atrioventricular conduction disorder.