Browsing by Subject "genetic engineering"
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Item Open Access Engineering Prokaryotic Sodium Channels for Excitable Tissue Therapies(2017) Nguyen, HungVoltage-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.
Item Open Access Reading the Book of Life: Contingency and Convergence in Macroevolution(2008-01-01) Powell, RussellThis dissertation explores philosophical problems in biology, particularly those relating to macroevolutionary theory. It is comprised of a series of three papers drawn from work that is currently at the publication, re-submission, and review stage of the journal refereeing process, respectively. The first two chapters concern the overarching contours of complex life, while the third zeroes in on the short and long-term prospects of human evolution.
The rhetorical journey begins with a thought experiment proposed by the late paleontologist Stephen Jay Gould. Gould hypothesized that replaying the "tape of life" would result in radically different evolutionary outcomes, both with respect to animal life in general and the human species in particular. Increasingly, however, biologists and philosophers are pointing to convergent evolution as evidence for replicability and predictability in macroevolution. Chapters 1 and 2 are dedicated to fleshing out the Gouldian view of life and its antithesis, clarifying core concepts of the debate (including contingency, convergence, constraint and causation), and interpreting the empirical data in light of these conceptual clarifications. Chapter 3 examines the evolutionary biological future of the human species, and the ways in which powerful new biotechnologies can shape it, for better and for worse. More detailed chapter summaries are provided below.
In Chapter 1, I critique a book-length excoriation of Gould's contingency theory written by the paleobiologist Simon Conway Morris, in which he amasses and marshals a good bulk of the homoplasy literature in the service of promoting a more robust, counter-factually stable account of macroevolution. I show that there are serious conceptual and empirical difficulties that arise in broadly appealing to the frequency of homoplasy as evidence for robustness in the history of life. Most important is Conway Morris's failure to distinguish between convergent (`externally' constrained) and parallel (`internally' constrained) evolution, and to consider the respective implications of these significantly different sources of homoplasy for a strong adaptationist view of life.
In so doing, I propose a new definition of parallel evolution, one intended to rebut the common charge that parallelism differs from convergence merely in degree and not in kind. I argue that although organisms sharing a homoplastic trait will also share varying degrees of homology (given common decent), it is the underlying developmental homology with respect to the generators directly causally responsible for the homoplastic event that defines parallel evolution and non-arbitrarily distinguishes it from convergence. I make use of the philosophical concept of `screening-off' in order to distinguish the proximate generators of a homoplastic trait from its more distal genetic causes (such as conserved master control genes).
In Chapter 2, I critically examine a recent assessment of the contingency debate by the philosopher John Beatty, in which he offers an interpretation of Gould's thesis and argues that it is undermined by iterative ecomorphological evolution. I develop and defend alternative concepts of contingency and convergence, and show how much of the evidence generally held to negate the contingency thesis not only fails to do so, but in fact militates in favor of the Gouldian view of life. My argument once again rests heavily on the distinction between parallelism and convergence, which I elaborate on and defend against a recent assault by developmental biologists, in part by recourse to philosophical work on the ontological prioritization of biological causes.
In Chapter 3, I explore the probable (and improbable) evolutionary biological consequences of intentional germ-line modification, particularly in relation to human beings. A common worry about genetic engineering is that it will reduce the pool of genetic diversity, creating a biological monoculture that could not only increase our susceptibility to disease, but even hasten the extinction of our species. Thus far, however, the evolutionary implications of human genetic modification have remained largely unexplored. In this Chapter, I consider whether the widespread use of genetic engineering technology is likely to narrow the present range of genetic variation, and if so, whether this would in fact lead to the evolutionary harms that some authors envision. By examining the nature of biological variation and its relation to population immunity and evolvability, I show that not only will genetic engineering have a negligible impact on human genetic diversity, but that it will be more likely to ensure rather than undermine the health and longevity of the human species. To this end, I analyze the relationship between genotypic and phenotypic variation, consider process asymmetries between micro and macroevolution, and investigate the relevance of evolvability to clade-level persistence and extinction.