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After an MI, the death of cardiomyocytes impairs the pump capacity of the heart, which, if left untreated, can (and often does) lead to heart failure. Current treatments are limited, with transplantation being the only treatment which addresses the loss of cardiac tissue directly. The amount of transplants carried out is severely limited by lack of healthy donor tissue available, and an alternative source of tissue would go some way to address this issue. HESC have been proposed and investigated as such a potential therapy, with their properties of indefinite self renewal and capacity to differentiate to any cell type found in the human proving extremely attractive. Several laboratories have studied cardiac differentiation in hESC, although no standardised protocol to generate functional cardiomyocytes exists (Laflamme et al., 2007, Ivey et al., 2008a, Xu et al., 2002, Graichen et al., 2008, Yang et al., 2008).

MicroRNAs have been proposed to regulate several physiological and pathological processes, including cardiac differentiation and development (Ivey et al., 2008a, Elia et al., 2009), angiogenesis, arrhythmias, hypertrophy and remodelling (van Rooij et al., 2006, van Rooij et al., 2007, Zhao et al., 2007b, Care et al., 2007). As part of the same transcriptional unit, miR-1 and miR-133 have been shown to have specialised roles in cardiac development, disease and differentiation. MiR- 1, which is widely conserved between species, is muscle specific and has been previously studied in cardiac development (Kwon et al., 2005b, Zhao et al., 2007b, Zhao et al., 2005). The relationship between miR-1 expression and cardiac differentiation has been further understood by studies which show its regulation of several cardiac markers such as Hand2, MyoD, Mef2, Nkx2.5, cyclin- dependant kinase-9, α-actin, myogenin and myosin heavy chain (Chen et al., 2006, Takaya et al., 2009a, Zhao et al., 2005).

MiR-133 has also been reported to be associated with muscle and cardiac function and disease. Double deletion of miR-133a-1 and miR-133a-2 in mice resulted in cardiac morphogenetic defects and a proliferative phenotype of cardiomyocytes, however single deletion resulted in a normal phenotype being observed, suggesting that these miRNAs function in concert and that if 25% of the normal levels of miR-133 are present, then the organism has no ill effects (Liu et al., 2008).

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In a previous publication, Ivey et al. reported that miR-1 and miR-133 were enriched in hESC-derived cardiomyocytes when differentiated using an embryoid body formation protocol (Ivey et al., 2008a). Interestingly, over-expression of these miRNAs elicited opposing effects, with miR-1 over-expression resulting in promotion of cardiac lineage commitment, and miR-133 over-expression appearing to block specification to cardiac muscle, suggesting that miR-1 and miR-133 are critically important miRNA in mesoderm and cardiac differentiation (Ivey et al., 2008a).

HESC are refractory to several transfection methods, and due to their potent self-renewal capacity long lasting and stable expression would be useful qualities in any miRNA modulation approach. Useful due to their ability to integrate into the host genome providing long term gene expression (Naldini et al., 1996), lentiviruses provide opportunities for long lived and efficient microRNA manipulation. Lentiviruses are a member of the retrovirus family retroviridae, and include human immunodeficiency virus 1 (HIV-1), feline immunodeficiency virus (FIV) and simian immunodeficiency virus (SIV). Other advantages to using lentiviral technology include their ability to transduce both dividing and non dividing cells, the ease at which the transgene or promoter can be modified and their relatively simple production (Naldini et al., 1996). Lentiviral technologies used in this study were based on HIV-1 Self Inactivating vectors, that is, they were unable to replicate and form new infectious virus particles. Lentiviral vectors have been produced by deletion of viral genes gag, pol and env and replacement with a transgene of interest under the control of a promoter of choice. Most of these self inactivating vectors are produced by transient triple transfection of a packaging plasmid, an expression plasmid containing the promoter driving the transgene of interest and a vesicular stomatitis virus glycoprotein (VSV-g) envelope plasmid in the presence of polyethylenimine (PEI) (Sinn et al., 2005, Cockrell and Kafri, 2007). VSV-g remains a common choice for a lentiviral vector envelope protein as it has broad cell tropism (Seganti et al., 1986). The lentiviral vectors used in this study are known as “second generation” vectors; that is they had vif, vpr, vpu and nef deleted from the packaging plasmid thereby ensuring if mutation or recombination events had occurred, the viral particles would not have contained virulence factors. These modifications have also been shown to have no significant effect on efficiency or vector titres

(Zufferey et al., 1997). The expression plasmid had further modifications for improved biosafety profile, namely the deletion of key transcriptional regulatory sequences including the homogenous viral enhancer-promoter unit. This has been achieved by a deletion within the U3 region of the 3’ Long Terminal Repeat (LTR) of the DNA transcript. This was reverse transcribed conferring the deletion to the 5’ LTR of the proviral DNA. Transcriptional activity was therefore sufficiently hampered so that no full length viral RNA could be produced (Zufferey et al., 1998). Modifications created to increase the overall functionality of the lentiviral vectors included the introduction of the rev- responsive regulatory element (RRE), the central polypurine tract (cPPT) element and woodchuck hepatitis virus post-transcriptional regulatory element (WPRE). RRE is important as it allowed the Rev-mediated shuttle of mRNA from the nucleus to the cytoplasm for translation to occur and as such is also present on the packaging plasmid (Cochrane et al., 1990, Dull et al., 1998). The cPPT element enhanced the nuclear translocation, degree of vector genome integration into the host genome and had also been shown to increase the achievable titre (Demaison et al., 2002a, Logan et al., 2004, VandenDriessche et al., 2002). The WPRE improved viral genome transcript packaging, regulated the stability of transgene mRNA by polyadenylation, increased RNA export from the nucleus and enhanced translation of transgene mRNA (Zufferey et al., 1999, Demaison et al., 2002a).

It was previously assumed that integrating lentiviruses were not suitable for clinical therapies due to their propensity to integrate semi-randomly but with some common integration sites (CIS) within oncogenes. However, upon further study it was discovered that for the most part the CIS lie in benign genes (Modlich et al., 2009, Cavazzana-Calvo et al., 2010, Biffi et al., 2011) and there are current studies using lentivirus in patients with Wiscott-Aldrich syndrome (Scaramuzza et al., 2012). Lentiviral vectors remain very useful tools in preclinical studies, allowing interrogation of the miRNA involved in hESC lineage commitment.

The aim of this chapter was to design and produce suitable lentiviral vectors for miRNA overexpression, to allow interrogation of miR-1 and miR-133b in cardiac lineage commitment in a feeder-free monolayer hESc culture system (Figure 3.1).

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Figure 3.1: Schematic of cardiac differentiation and lentivirus infection of hESC.

Lentiviruses were generated by triple transfection of a packaging plasmid (p8.74) an envelope plasmid (pMGD) and a microRNA expression plasmid (LNT-SFFV-miR) in the presence of PEI in HEK293T cells. Lentiviruses were collected in the cell media at 48 and 72 hours before being sterile filtered, concentrated by ultracentrifugation and subjected to titration to determine functional virus titre. Lentiviral transduction in the presence of polybrene was then carried out in pluripotent hESC, after which cells were maintained pluripotent as shown by Oct 4 staining, or differentiated towards cardiac lineage as shown by positive staining of TNNT2 and TPM1.