Generation of Human iPSC-Derived Myotubes to Investigate RNA-Based Therapies In Vitro.

Alternative pre-mRNA splicing can be cell-type specific and results in the generation of different protein isoforms from a single gene. Deregulation of canonical pre-mRNA splicing by disease-associated variants can result in genetic disorders. Antisense oligonucleotides (AONs) offer an attractive solution to modulate endogenous gene expression through alteration of pre-mRNA splicing events.

Ready for Repair? Gene Editing Enters the Clinic for the Treatment of Human Disease.

We present an overview of clinical trials involving gene editing using clustered interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9), transcription activator-like effector nucleases (TALENs), or zinc finger nucleases (ZFNs) and discuss the underlying mechanisms. In cancer immunotherapy, gene editing is applied in T cells, transgenic T cell receptor (tTCR)-T cells, or chimeric antigen receptor (CAR)-T cells to improve adoptive cell therapy for multiple cancer types. This involves knockouts of immune checkpoint regulators such as PD-1, components of the endogenous TCR and histocompatibility leukocyte antigen (HLA) complex to generate universal allogeneic CAR-T cells, and CD7 to prevent self-destruction in adoptive cell therapy.

Sharpening the Molecular Scissors: Advances in Gene-Editing Technology.

The ability to precisely modify human genes has been made possible by the development of tools such as meganucleases, zinc finger nucleases, TALENs, and CRISPR/Cas. These now make it possible to generate targeted deletions, insertions, gene knock outs, and point variants; to modulate gene expression by targeting transcription factors or epigenetic machineries to DNA; or to target and modify RNA. Endogenous repair mechanisms are used to make the modifications required in DNA; they include non-homologous end joining, homology-directed repair, homology-independent targeted integration, microhomology-mediated end joining, base-excision repair, and mismatch repair.

Large-Scale Expansion of Human iPSC-Derived Skeletal Muscle Cells for Disease Modeling and Cell-Based Therapeutic Strategies.

Although skeletal muscle cells can be generated from human induced pluripotent stem cells (iPSCs), transgene-free protocols include only limited options for their purification and expansion. In this study, we found that fluorescence-activated cell sorting-purified myogenic progenitors generated from healthy controls and Pompe disease iPSCs can be robustly expanded as much as 5 × 10-fold. At all steps during expansion, cells could be cryopreserved or differentiated into myotubes with a high fusion index.