Engineering Prokaryotic Sodium Channels for Excitable Tissue Therapies
Voltage-gated sodium channels (VGSCs) enable generation and spread of action potentials in electrically excitable cells and tissues of all metazoans, from jellyfish to humans. The functional, pore-forming α-subunit of eukaryotic VGSCs is formed from a large polypeptide chain of ~2000 amino acids (~260 kDa), comprising four homologous domains. In humans, VGSC loss-of-function mutations are associated with various neuronal, cardiac, and skeletal muscle disorders characterized by a decrease or complete loss of tissue excitability. Similarly, permanent excitability loss due to acute tissue injuries (e.g. stroke, spinal cord injury, heart attack) could lead to long-term disability and death. Whilst an increase in sodium current through stable gene transfer could improve such conditions, eukaryotic VGSC genes are too large (>6 kbp) to be efficiently delivered to cells by existing viral vectors. In contrast, prokaryotic voltage-gated sodium channels (BacNav) consist of four identical subunits, individually transcribed and translated from single genes of only ~800 bp in size. Therefore, it is plausible that small BacNav genes can be efficiently packaged into viral vectors, either alone or with other ion channel genes, and used to stably introduce or modify electrical excitability of primary human cells. The objective of this thesis is thus to develop the methodology to screen, optimize, and assess BacNav channels as potential substitutes for eukaryotic VGSCs. Specifically, we sought to utilize engineered BacNav to create de novo excitable human tissues and to rescue impaired action potential conduction in vitro.
First, by using a monoclonal HEK293 line stably expressing the potassium channel Kir2.1 and gap junction channel Cx43, we were able to select, among various BacNav orthologs and variants, the channel NavRosD G217A that yielded action potential propagation with highest maximum capture rate. Lentiviral transduction of each of the three channels (NavRosD G217A, Kir2.1, and Cx43) into human fibroblasts yielded robust expression and expected electrical properties as confirmed by patch clamp recordings. By co-expressing all three channels, we were able for the first time to stably convert human fibroblasts into electrically excitable and actively conducting cells. However, the conduction velocity of engineered fibroblast tissue was low, largely due to the slow activation kinetics of NavRosD channel.
In order to improve the conduction properties of engineered fibroblasts, we shifted our focus to NavSheP channel, currently the fastest known BacNav ortholog. Due to the overly hyperpolarized voltage dependency of the wild-type NavSheP channel, we generated a library of NavSheP mutants exhibiting a wide range of shifts in voltage-dependent activation and inactivation and, with the guidance from computational modeling, identified three mutants that yielded ~2.5-fold increases in conduction velocity compared to NavRosD G217A. Importantly, we demonstrated that engineered fibroblasts retained stable functional properties despite extensive expansion or differentiation into myofibroblasts and exhibited strong viability while supporting AP propagation in 3D settings. Furthermore, in an in vitro model of interstitial fibrosis, engineered excitable and actively-conducting fibroblasts rescued impaired cardiac conduction to healthy level. These results strongly suggested that engineered fibroblasts could be used as a robust source for potential cell-based therapies for cardiac diseases.
In addition to the generation of excitable fibroblasts, BacNav channels could also serve as potential substitutes for impaired VGSC in various excitable tissue disorders. The channel NavSheP D60A (ShePA) was chosen for direct expression in mammalian excitable tissues as it yielded fastest conduction in previous studies. By performing codon optimization and adding appropriate endoplasmic-reticulum export signal, we were able to significantly improve membrane expression of ShePA channels. Expression of ShePA in excitable HEK293 tissue (Ex293) rescued impaired conduction upon membrane depolarization and decoupling. Furthermore, cultures of neonatal rat ventricular myocytes (NRVMs) transduced with ShePA virus exhibited enhanced conduction properties and increased resistance to conduction failure in an in vitro model of regional ischemia. Lastly, ShePA expression in highly-arrhythmogenic cardiomyocyte-fibroblast co-cultures led to significant reduction in incidence of reentry. Taken together, these results demonstrated the potential applications of engineered BacNav channels for cardiac gene therapies.
In summary, this dissertation presents the first experimental evidences supporting the use of prokaryotic sodium channels for the induction, control, and rescue of mammalian tissue excitability. The encouraging in vitro results shown in these studies will stimulate the development of BacNav-based therapies for the treatment of cardiac diseases. Furthermore, the experimental methodology developed in this work will serve as a useful framework for the screening, optimization, and assessment of engineered BacNav for specific therapeutic applications.
cardiac cell therapies
cardiac gene therapies
prokaryotic sodium channel
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