Mutant mitochondrial elongation factor G1 and combined oxidative phosphorylation deficiency

Although most components of the mitochondrial translation apparatus are encoded by nuclear genes, all known molecular defects associated with impaired mitochondrial translation are due to mutations in mitochondrial DNA. We investigated two siblings with a severe defect in mitochondrial translation, reduced levels of oxidative phosphorylation complexes containing mitochondrial DNA (mtDNA)–encoded subunits, and progressive hepatoencephalopathy. We mapped the defective gene to a region on chromosome 3q containing elongation factor G1 (EFG1), which encodes a mitochondrial translation factor. Sequencing of EFG1 revealed a mutation affecting a conserved residue of the guanosine triphosphate (GTP)–binding domain. These results define a new class of gene defects underlying disorders of oxidative phosphorylation

Disease-specific, neurosphere-derived cells as models for brain disorders

There is a pressing need for patient-derived cell models of brain diseases that are relevant and robust enough to produce the large quantities of cells required for molecular and functional analyses. We describe here a new cell model based on patient-derived cells from the human olfactory mucosa, the organ of smell, which regenerates throughout life from neural stem cells. Olfactory mucosa biopsies were obtained from healthy controls and patients with either schizophrenia, a neurodevelopmental psychiatric disorder, or Parkinson's disease, a neurodegenerative disease. Biopsies were dissociated and grown as neurospheres in defined medium. Neurosphere-derived cell lines were grown in serum-containing medium as adherent monolayers and stored frozen. By comparing 42 patient and control cell lines we demonstrated significant disease-specific alterations in gene expression, protein expression and cell function, including dysregulated neurodevelopmental pathways in schizophrenia and dysregulated mitochondrial function, oxidative stress and xenobiotic metabolism in Parkinson's disease. The study has identified new candidate genes and cell pathways for future investigation. Fibroblasts from schizophrenia patients did not show these differences. Olfactory neurosphere-derived cells have many advantages over embryonic stem cells and induced pluripotent stem cells as models for brain diseases. They do not require genetic reprogramming and they can be obtained from adults with complex genetic diseases. They will be useful for understanding disease aetiology, for diagnostics and for drug discovery

Reprogramming glioblastoma multiforme cells into neurons by protein kinase inhibitors

Abstract Background Reprogramming of cancers into normal-like tissues is an innovative strategy for cancer treatment. Recent reports demonstrate that defined factors can reprogram cancer cells into pluripotent stem cells. Glioblastoma multiforme (GBM) is the most common and aggressive malignant brain tumor in humans. Despite multimodal therapy, the outcome for patients with GBM is still poor. Therefore, developing novel therapeutic strategy is a critical requirement. Methods We have developed a novel reprogramming method that uses a conceptually unique strategy for GBM treatment. We screened a kinase inhibitor library to find which candidate inhibitors under reprogramming condition can reprogram GBM cells into neurons. The induced neurons are identified whether functional and loss of tumorigenicity. Results We have found that mTOR and ROCK kinase inhibitors are sufficient to reprogram GBM cells into neural-like cells and “normal” neurons. The induced neurons expressed neuron-specific proteins, generated action potentials and neurotransmitter receptor-mediated currents. Genome-wide transcriptional analysis showed that the induced neurons had a profile different from GBM cells and were similar to that of control neurons induced by established methods. In vitro and in vivo tumorigenesis assays showed that induced neurons lost their proliferation ability and tumorigenicity. Moreover, reprogramming treatment with ROCK-mTOR inhibitors prevented GBM local recurrence in mice. Conclusion This study indicates that ROCK and mTOR inhibitors-based reprogramming treatment prevents GBM local recurrence. Currently ROCK-mTOR inhibitors are used as anti-tumor drugs in patients, so this reprogramming strategy has significant potential to move rapidly toward clinical trials

Generation of helper-dependent adenoviral vector for skin disorder

Gene therapy is one of the most compromising treatment of a vast number of genetic disorders, that could not be treated with proper methods decades ago. With the technological advance and extensive basic researches, many of these diseases have been reported to be cured with gene therapy. Severe combined immune deficiency, hemophilia and leukemia are now target of clinical trials of this therapy. Furthermore, patient suffering metabolic disorders, neural diseases, and even cancer are now being treated with different kind of vectors. The gene modification is a such complex process that involves the entrance of vectors into the host cells, the delivery and expression of the genetic information that serves as a therapeutic purpose. The strategy strongly depends on the target cells and the type of vector used, such that every approach is specific and unique. For epidermolysis bullosa, such as RDEB and JEB, long-term skin regeneration based on ex vivo gene addition therapy has been achieved with great results in the research center CIEMAT, but some problems were stated in this kind of therapy. The viral gene delivery efficiency is still low; the expression of the gene of interest cannot be accurately regulated to occur in the desired place and time; and the risk of insertional mutagenesis due to the usage of integrative viral vectors. Hence, gene correction was considered as a more precise alternative, in which the engineered nucleases perform site-specific cleavage in the mutated gene. In a previous study (Cristina Chamorro, 2016), first-generation adenoviral vectors were used for correction of RDEB based on HDR and NHEJ. In Spain, the insertion of Cytosine base at positon 6527, within exon 80, (mutation c.6527insC) causes a recurrent frameshift mutation in 46% of recessive DEB alleles. Due to this high prevalence, the gene editing was focused on the exon 80. It showed that the deletion of exon 80 by using TALENS, delivered by adenoviral vectors, could restore the correct reading frame and subsequently, the collagen VII normal function in the adhesion between epidermis and dermis of skin. The design and production of TALENS is tricky and time-consuming because the nucleases need to be modified according to the sequence of interest. For a generalized gene editing to treat many other mutations causing DEB, CRISPR-Cas9 system is considered a better tool. For this purpose, several dual sgRNA were tested to choose the sgRNA pair that results in the highest exon 80 deletion percentage. The ribonucleoprotein complex (sgRNA-Cas9) was delivered into the primary keratinocytes through nucleofection and was found to restore the reading frame of COL7A1 in a large proportion of cells, which consequently synthesize functional collagen VII variant (Jose Bonafont, 2019). Back to the delivery system for sgRNA-Cas9 complex, adenoviral vectors have been chosen because of their ability to infect quiescent cells like stem cells. But the first-generation is limited for in vivo application due to the relatively high toxicity due to the viral genome remaining in the host cells.Therefore, this study aims at generating a less toxic version of adenoviral vector carrying a gene encoding sgRNA-Cas9 that can delete the specific exon and, in this manner, correct the collagen VII expression, as illustrated in Figure 14. For this purpose, simple 2D cultures are assessed in term of infection while more complex 3D cultures mimicking in vivo gene therapy are evaluated in DNA level to confirm the gene editing through NHEJ, following the workflow indicated in Figure 15...Ingeniería Biomédica (Plan 2010

One-step in vitro generation of ETV2-null pig embryos

Each year, tens of thousands of people worldwide die of end-stage organ failure due to the limited availability of organs for use in transplantation. To meet this clinical demand, one of the last frontiers of regenerative medicine is the generation of humanized organs in pigs from pluripotent stem cells (PSCs) via blastocyst complementation. For this, organ-disabled pig models are needed. As endothelial cells (ECs) play a critical role in xenotransplantation rejection in every organ, we aimed to produce hematoendothelial-disabled pig embryos targeting the master transcription factor ETV2 via CRISPR-Cas9-mediated genome modification. In this study, we designed five different guide RNAs (gRNAs) against the DNA-binding domain of the porcine ETV2 gene, which were tested on porcine fibroblasts in vitro. Four out of five guides showed cleavage capacity and, subsequently, these four guides were microinjected individually as ribonucleoprotein complexes (RNPs) into one-cell-stage porcine embryos. Next, we combined the two gRNAs that showed the highest targeting efficiency and microinjected them at higher concentrations. Under these conditions, we significantly improved the rate of biallelic mutation. Hence, here, we describe an efficient one-step method for the generation of hematoendothelial-disabled pig embryos via CRISPR-Cas9 microinjection in zygotes. This model could be used in experimentation related to the in vivo generation of humanized organs

Mesenchymal Stem Cells for Treatment of CNS Injury

Brain and spinal cord injuries present significant therapeutic challenges. The treatments available for these conditions are largely ineffective, partly due to limitations in directly targeting the therapeutic agents to sites of pathology within the central nervous system (CNS). The use of stem cells to treat these conditions presents a novel therapeutic strategy. A variety of stem cell treatments have been examined in animal models of CNS trauma. Many of these studies have used stem cells as a cell-replacement strategy. These investigations have also highlighted the significant limitations of this approach. Another potential strategy for stem cell therapy utilises stem cells as a delivery mechanism for therapeutic molecules. This review surveys the literature relevant to the potential of mesenchymal stem cells for delivery of therapeutic agents in CNS trauma in humans

Investigation of therapeutic effects in the wound healing of chitosan/pGM-CSF complexes

Granulocyte macrophage colony-stimulating factor (GM-CSF) has been shown to promote the growth, proliferation, and migration of endothelial and keratinocyte cells. Chitosan has been widely used as a biopolymer in wound-healing studies. The aim of this study was to investigate the in vitro proliferative effects of chitosan/pGM-CSF complexes as well as the therapeutic role of the complexes in an in vivo rat wound model. The effect of complexes on cell proliferation and migration was examined. Wounds were made in Wistar-albino rats, and examined histopathologically. The cell proliferation and migration were increased weight ratio- and time-dependently in HaCaT and NIH-3T3 cell lines. Wound healing was significantly accelerated in rats treated with the complexes. These results showed that the delivery of pGM-CSF using chitosan complexes could play an accelerating role in the cell proliferation, migration, and wound-healing process

Following fibroblast lineages in dermal development and scars

Fibroblast heterogeneity studies have shown that it vastly influences the outcome of wound repair (Griffin et al., 2020; Rinkevich et al., 2015). We aimed to characterize the origins of these heterogeneous populations in the publication I. Here we have shown that during dermal development there is an inherent inversion of fibroblast populations from abundant regenerative fibroblasts (ENFs) at early fetal stages of development, to abundant scar-producing fibroblasts (EPFs) during perinatal and adult life. Using novel imaging and analysis approaches, we have charted the dermal maturation dynamics of EPFs during the transition from scar-less (E12) to scarring stages (E16.5) of development. We then followed up on the role of scar-forming fibroblasts in postnatal and adult stages. In publication III, we identified the subcutaneous fascia as the main anatomical contributor of scars upon deep skin injury. Next, we followed the role of fascial scar producing cells, EPFs, and its contribution to scar formation in publication II. Here, we developed a relevant ex-vivo model called “scar-like tissue in a dish”- termed SCAD. We show that scars on SCADs emulate the bona fide in-vivo scar phenotype. Using this model, we visualize and chart live migration dynamics of EPFs at all stages of scar development. Further, using antibody screening and CRISPR-Cas9 based genetic approaches, we identified that N-Cadherin is the adhesion molecular that orchestrates EPF and fascial response to scarring. Finally, to check the clinical relevance, we validated our N-cadherin mechanistic findings in human skin biopsies from various anatomical locations. These findings provide a range of therapeutic avenues in modulating subcutaneous fascial response and prevention of pathological scars