Developing new genomic integration-free gene and cell therapy strategies for muscular dystrophy

Duchenne muscular dystrophy (DMD) is caused by mutations on the Xlinked dystrophin gene and primarily affects skeletal muscles, resulting in disability and premature death. This thesis looks at different strategies to circumvent substantial obstacles in the development of therapies for this incurable disease. Here I hypothesise that the limited availability of large number of cells and the large size of the dystrophin gene (2.4Mb) can be tackled by combining human artificial chromosome (HAC)-based gene correction and induced pluripotent stem cell (iPSC)-mediated production of transplantable myogenic cells. However, another significant hurdle is posed by cell delivery, as skeletal muscle is the most abundant human tissue; therefore I also focused on developing a novel strategy to make the aforementioned DMD iPSC-derived myogenic population systemically deliverable. I hypothesised that cell fate modulators of native skeletal myoblasts could enhanced migratory properties also to human iPSCderived myogenic progenitors. I show that exposure to the Notch ligand DLL4 and PDGF-BB can induce the acquisition of some key properties such as a perivascular marker expression profile and an improved migration in vitro. Taken together these results lay the foundation for a small molecule-based strategy to allow systemic delivery of geneticallycorrected, genomic-integration-free, iPSC-derived myogenic cells for the autologous gene and cell therapy of DMD

A transcriptomic-based drug repositioning approach for the identification of novel muscle-specific therapies for spinal muscular atrophy

Spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disorder (NMD) caused by depleted survival of motor neuron (SMN) levels and characterised by neuronal degeneration and progressive muscle atrophy. Although three approved SMN-dependent treatments have significantly halted disease progression, they are unfortunately not cures. Thus, additional musclespecific therapies are most likely also required to synergistically ameliorate symptoms in SMA patients. One useful strategy for the discovery of novel SMA muscle-specific therapies is drug repositioning (or repurposing), as using existing approved pharmacological compounds allows for the development of more costeffective treatments compared to traditional drug discovery. We have previously investigated drug repositioning in SMA and demonstrated that prednisolone, a synthetic glucocorticoid (GC), improved muscle health and survival in SMA mice. However, the adverse effects associated with chronic GC use limit prednisolone’s long-term therapeutic potential in SMA. We thus wanted to discover prednisolone-targeted genes and pathways in SMA skeletal muscle and identify commercially available drugs that similarly modulate these effectors. We initially performed an RNA sequencing, bioinformatics and drug repositioning database pipeline on muscle from symptomatic post-natal day (P)7 prednisolonetreated and untreated Smn-/-;SMN2 SMA mice. These revealed that genes associated with atrophy, metabolism and muscle function pathways were targeted and normalised by prednisolone in SMA skeletal muscle. Furthermore, a total of 223 commercially approved compounds were predicted to similarly target these genes and pathways. We thus selected metformin, a generic antihyperglycaemic biguanide and oxandrolone, an anabolic steroid, for further investigation in SMA, based on their oral bioavailability, safety in infants and previously reported benefits in related conditions. Metformin was predicted to emulate prednisolone’s activity by upregulating Prkag3 and downregulating Forkhead box O (FoxO) expression. We indeed confirmed that Prkag3 was significantly downregulated in muscle from Smn-/- ;SMN2 and Smn2B/- SMA mice. Furthermore, in vitro experiments in C2C12 myoblast-like cells suggest that the dysregulation of metformin’s molecular targets are SMN-independent and linked to atrophy. However, metformin treatment in both C2C12 cells and Smn2B/- SMA mice (200 mg/kg/day) did not improve disease progression. Furthermore, a higher dose of metformin (400 mg/kg/day) significantly exacerbated disease progression in Smn2B/- SMA mice, which were most likely due to mitochondrial marker dysfunction in the spinal cord. On the other hand, oxandrolone was predicted to upregulate the expression of the androgen receptor (Ar) and its downstream components. However, analyses in both C2C12 cells and muscle from Smn-/-;SMN2 and Smn2B/- SMA mice revealed that most of the predicted oxandrolone targets were in fact not dysregulated. Still, oxandrolone treatment rescued canonical atrophy in C2C12 myotubes and slightly improved survival in Smn2B/- SMA mice (4 mg/kg/day). Taken together, our in vitro and in vivo experiments revealed that metformin and oxandrolone did not successfully emulate prednisolone’s activity in SMA, suggesting that our in silico approach requires refinement for a better prediction of valid drug candidates. Nevertheless, our discovery of prednisolone-targeted pathways and extensive list of drug candidates supports the usefulness of a transcriptomic-based drug repositioning strategy, and that with alterations, it can be quite beneficial for future therapeutic endeavours in SMA

Modern Tools for Genetic Engineering

Site-specific endonucleases create double-strand breaks within the genome and can be targeted to literally any genetic mutation. Together with a repair template, a correction of the defective locus becomes possible. This book offers insight into the modern tools of genome editing, their hurdles and their huge potential. A new era of in vivo genetic engineering has begun

Identification of novel synaptic components by transcriptome profiling of the murine neuromuscular junction

The neuromuscular junction (NMJ) has been studied for over a century, yet we still do not have a complete picture of all its structural and functional components, knowledge of which is paramount in devising treatment strategies for neuromuscular diseases. Previous microarray-based approaches aimed at elucidating novel NMJ components were hindered by technological limitations. Recent technological advancements propelled next-generation RNA-sequencing with its wider dynamic range to the forefront of transcriptome-level gene expression profiling. We utilized laser-capture microdissection to isolate myonuclei underlying the NMJ combined with RNA-sequencing and successfully generated NMJ gene expression profiles of fast-twitch extensor digitorum longus (EDL) and slow-twitch soleus (SOL) muscles and identified a large number of potential novel NMJ genes. The expression levels of canonical NMJ genes were nearly identical between the EDL and SOL, which suggests that the core NMJ gene program might be well conserved between different skeletal muscle types. We used in vivo muscle electroporation to overexpress one of our candidate genes, the transcription factor T-box 21 (TBX21), in the tibialis anterior (TA) muscle and observed an increased density of postsynaptic acetylcholine receptors. TBX21 may thus represent a novel transcription factor contributing to the regulation of the NMJ gene program, with a role in postsynaptic sensitivity. We also generated NMJ gene expression profiles of the TA muscle of 10-month-old (“young”) and 30-month-old (“old”) mice to investigate the effect of aging on the NMJ gene program. Strikingly, the NMJ gene program was remarkably stable, with nearly identical expression levels of canonical NMJ genes between young and old mice. This implies that age-related perturbations of the NMJ are likely caused by external factors, such as accumulated myofiber damage and changes in nerve input, rather than by gradual dysregulation of the NMJ gene program with increasing age. Our findings argue against the hypothesis that aging leads to a broad deterioration of the NMJ gene program that would contribute to perturbations of NMJ structure and function. Furthermore, functional annotation analysis of our different NMJ gene expression datasets strongly indicates the importance of an extensive number of hitherto unknown glycoproteins, as well as of posttranslational modifications, especially glycosylations, at the synaptic basal lamina. We highlight a set of candidate genes that encode for enzymes putatively involved in these processes at the NMJ, and which are potentially involved in the pathophysiology of neuromuscular diseases such as congenital myasthenic syndromes. This thesis expands our understanding of the complexity of the NMJ and lays the foundation for further research that will functionally characterize novel synaptic components and provide the basis for novel therapeutic treatment strategies

Complement-mediated cooperation between immunocytes in the compound ascidian Botryllus schlosseri

Two main kinds of innate immune responses are present in ascidians: phagocytosis and cytotoxicity. They are mediated by two different types of circulating immunocytes: phagocytes and cytotoxic morula cells (MCs). MCs, once activated by non-self-recognition, can stimulate phagocytosis by the release of soluble factors able to act as opsonins. BsC3, the complement C3 homologue, like mammalian C3, contains the thioester bond required to split the molecule into BsC3a and BsC3b. BsC3b likely represents the MC opsonin as it can enhances phagocytosis. The tenet is supported by the observed reduction in phagocytosing cells after exposure of hemocytes to compstatin, a drug preventing C3 activation, or after the bsc3 knockdown by iRNA injection. In addition, the transcript for BsCR1, homologous to mammalian CR1, is present in Botryllus phagocytes and the transcription is modulated during the blastogenetic cycle. MCs also release cytokines (chemokines) able to recruit immunocytes to the infection site. The activity is inhibited by antibodies raised against human TNFa. Since no genes for TNFa are present in the Botryllus genome, the observed activity is probably related to a TNF-domain containing protein, member of the Botryllus complement system. Conversely, activated phagocytes release a rhamnose-binding lectin able to interact with microbial surfaces and act as opsonin. It can also activate MCs by inducing the release of the reported cytokine and stimulate their degranulation. Overall, the results obtained so far indicate the presence of a well-defined cross-talk between the two types of immunocytes during the immune responses of B. schlosseri