Manu Tewari PhD
Research Fellow: Discher lab, Dept of Chemical and Molecular Eng. UPENN
Signaling mechanisms in muscular dystrophies: Contractility, adhesion and differentiation:
Normal development and function of tissue cells depends on the ability of cells to sense and set an optimal balance between adhesion and cellular contractility. Normal myocytes possess both integrin-based adhesions and dystroglycan-complex adhesions, but the latter are missing or perturbed in muscular dystrophies due to dystrophin or gamma-sarcoglycan deficiency both associated with the DGC complex of sarcolemma. Integrin upregulation seems to occur naturally as part of cellular mechanisms of compensation, and focal adhesion and cytoskeletal phospho-protein/s like paxillin, MAPKs and others are highly regulated in dystrophic muscle. The mechano-sensing ability of these downstream molecules is studied in context to muscle differentiation, contractility and adhesion.
Muscle differentiation and Adipogenesis:
The switching of a differentiated cell type from one lineage to another is called trans-differentiation. The mdx- dystrophic muscle, exhibit regeneration and degeneration of muscle cells between 2-12 weeks of age and the pathogenic mechanism in muscular dystropy is more complex than previously understood. In this project we are studying the mechanism of de-differentiation of myotubes into myocytes and trans-differentiation to adipocytes.
Molecular and structural extensibility of BMD phenotypes by manipulative exon splicing in dystrophin gene:
Antisense oligo nucleotide (AON)-induced skipping of dystrophic pre-mRNA based gene therapy, allows the synthesis of a largely functional but truncated dystrophin, associated with a milder phenotype. Core fragments for two such BMD phenotypes, dystrophin nano-constructs, created by in frame exon skipping of exon 49 (corrected for a premature stop codon in exon 49) and exon 45 (corrected for exon 46-51 deletion), R18~linker~R21 and R16~linker~R21 respectively will be examined with force (AFM) and structural (solution studies) probes to study the biophysical properties of the nano-constructs.
Efficient Nuclear Delivery of Antisense Oligonucleotides in vitro and in vivo by Neutral, Nano-Transforming Polymersomes
Delivery of antisense oligonucleotides, AON in muscle , presents some of the same challenges as delivery of many nucleic acids eg control over stability, uptake into cells, endolysosomal escape, and entry into the nucleus. We have demonstrated efficient and functional nuclear delivery of AONs in muscle after loading into nano-transforming, neutral 'polymersomes'
In vitro delivery of antisense oligonucleotide using biodegradable polymer vesicles. (A) Bright field image showing a single patterned myotube post-AON delivery with no signs of toxicity(left). Fluorescent image of delivered AON in nuclei of the top-layer of the myotube (right). Scale bar is 10 microns (B) Delivery of AON in nuclei of myotubes grown in petri dish. Overlay shows the FAM-AON and blue nuclei(Hoechst dye). (C) AON encapsulated in non-degradable PEO-PBD vesicles do not show release , and remain in the cytoplasm.