Hematopoietic stem and progenitors cells gene editing: Beyond blood disorders.

Lessons learned from decades-long practice in the transplantation of hematopoietic stem and progenitor cells (HSPCs) to treat severe inherited disorders or cancer, have set the stage for the current gene therapies using autologous gene-modified hematopoietic stem and progenitor cells that have treated so far, hundreds of patients with monogenic disorders. With increased knowledge of hematopoietic stem and progenitor cell biology, improved modalities for patient conditioning and with the emergence of new gene editing technologies, a new era of hematopoietic stem and progenitor cell-based gene therapies is poised to emerge. Gene editing has the potential to restore physiological expression of a mutated gene, or to insert a functional gene in a precise locus with reduced off-target activity and toxicity.

Lentiviral standards to determine the sensitivity of assays that quantify lentiviral vector copy numbers and genomic insertion sites in cells.

With an increasing number of gene therapy clinical trials and drugs reaching the market, it becomes important to standardize the methods that evaluate the efficacy and safety of gene therapy. We herein report the generation of lentiviral standards which are stable, cloned human cells prepared from the diploid HCT116 cell line and which carry a known number of lentiviral vector copies in their genome. These clones can be used as reference cellular materials for the calibration or qualification of analytical methods that quantify vector copy numbers in cells (VCN) or lentiviral vector genomic integration sites (IS).

CRISPR-Cas9 Gene Editing of Hematopoietic Stem Cells from Patients with Friedreich's Ataxia.

Friedreich's ataxia (FRDA) is an autosomal recessive neurodegenerative disorder caused by expansion of GAA repeats in intron 1 of the frataxin () gene, leading to significant decreased expression of frataxin, a mitochondrial iron-binding protein. We previously reported that syngeneic hematopoietic stem and progenitor cell (HSPC) transplantation prevented neurodegeneration in the FRDA mouse model YG8R. We showed that the mechanism of rescue was mediated by the transfer of the functional frataxin from HSPC-derived microglia/macrophage cells to neurons/myocytes.

Interaction between galectin-3 and cystinosin uncovers a pathogenic role of inflammation in kidney involvement of cystinosis.

Inflammation is involved in the pathogenesis of many disorders. However, the underlying mechanisms are often unknown. Here, we test whether cystinosin, the protein involved in cystinosis, is a critical regulator of galectin-3, a member of the β-galactosidase binding protein family, during inflammation.

Potential use of stem cells as a therapy for cystinosis.

Cystinosis is an autosomal recessive metabolic disease that belongs to the family of lysosomal storage disorders (LSDs). Initial symptoms of cystinosis correspond to the renal Fanconi syndrome. Patients then develop chronic kidney disease and multi-organ failure due to accumulation of cystine in all tissue compartments.

Transplantation of wild-type mouse hematopoietic stem and progenitor cells ameliorates deficits in a mouse model of Friedreich's ataxia.

Friedreich's ataxia (FRDA) is an incurable autosomal recessive neurodegenerative disease caused by reduced expression of the mitochondrial protein frataxin due to an intronic GAA-repeat expansion in the gene. We report the therapeutic efficacy of transplanting wild-type mouse hematopoietic stem and progenitor cells (HSPCs) into the YG8R mouse model of FRDA. In the HSPC-transplanted YG8R mice, development of muscle weakness and locomotor deficits was abrogated as was degeneration of large sensory neurons in the dorsal root ganglia (DRGs) and mitochondrial capacity was improved in brain, skeletal muscle, and heart.

Treatment of Inherited Eye Defects by Systemic Hematopoietic Stem Cell Transplantation.

Purpose: Cystinosis is caused by a deficiency in the lysosomal cystine transporter, cystinosin (CTNS gene), resulting in cystine crystal accumulation in tissues. In eyes, crystals accumulate in the cornea causing photophobia and eventually blindness. Hematopoietic stem progenitor cells (HSPCs) rescue the kidney in a mouse model of cystinosis.

BRCA2 is needed for both repair and cell cycle arrest in mammalian cells exposed to S23906, an anticancer monofunctional DNA binder.

Repair of DNA-targeted anticancer agents is an active area of investigation of both fundamental and clinical interest. However, most studies have focused on a small number of compounds limiting our understanding of both DNA repair and the DNA damage response. S23906 is an acronycine derivative that shows strong activity toward solid tumors in experimental models.

Impairment of chaperone-mediated autophagy leads to selective lysosomal degradation defects in the lysosomal storage disease cystinosis.

Metabolite accumulation in lysosomal storage disorders (LSDs) results in impaired cell function and multi-systemic disease. Although substrate reduction and lysosomal overload-decreasing therapies can ameliorate disease progression, the significance of lysosomal overload-independent mechanisms in the development of cellular dysfunction is unknown for most LSDs. Here, we identify a mechanism of impaired chaperone-mediated autophagy (CMA) in cystinosis, a LSD caused by defects in the cystine transporter cystinosin (CTNS) and characterized by cystine lysosomal accumulation.

Brief reports: Lysosomal cross-correction by hematopoietic stem cell-derived macrophages via tunneling nanotubes.

Despite controversies on the potential of hematopoietic stem cells (HSCs) to promote tissue repair, we previously showed that HSC transplantation could correct cystinosis, a multisystemic lysosomal storage disease, caused by a defective lysosomal membrane cystine transporter, cystinosin (CTNS gene). Addressing the cellular mechanisms, we here report vesicular cross-correction after HSC differentiation into macrophages. Upon coculture with cystinotic fibroblasts, macrophages produced tunneling nanotubes (TNTs) allowing transfer of cystinosin-bearing lysosomes into Ctns-deficient cells, which exploited the same route to retrogradely transfer cystine-loaded lysosomes to macrophages, providing a bidirectional correction mechanism.

A southern blot protocol to detect chimeric nuclease-mediated gene repair.

Gene targeting by homologous recombination at chromosomal endogenous loci has traditionally been considered a low-efficiency process. However, the effectiveness of such so-called genome surgery or genome editing has recently been drastically improved through technical developments, chiefly the use of designer nucleases like zinc-finger nucleases (ZFNs), meganucleases, transcription activator-like effector nucleases (TALENs) and CRISPR/Cas nucleases. These enzymes are custom designed to recognize long target sites and introduce double-strand breaks (DSBs) at specific target loci in the genome, which in turn mediate significant improvements in the frequency of homologous recombination.

Upregulation of the Rab27a-dependent trafficking and secretory mechanisms improves lysosomal transport, alleviates endoplasmic reticulum stress, and reduces lysosome overload in cystinosis.

Cystinosis is a lysosomal storage disorder caused by the accumulation of the amino acid cystine due to genetic defects in the CTNS gene, which encodes cystinosin, the lysosomal cystine transporter. Although many cellular dysfunctions have been described in cystinosis, the mechanisms leading to these defects are not well understood. Here, we show that increased lysosomal overload induced by accumulated cystine leads to cellular abnormalities, including vesicular transport defects and increased endoplasmic reticulum (ER) stress, and that correction of lysosomal transport improves cellular function in cystinosis.

Hematopoietic stem cell gene therapy for the multisystemic lysosomal storage disorder cystinosis.

Cystinosis is an autosomal recessive metabolic disease that belongs to the family of lysosomal storage disorders (LSDs). The defective gene is CTNS encoding the lysosomal cystine transporter, cystinosin. Cystine accumulates in all tissues and leads to organ damage including end-stage renal disease.

Trabectedin and its C subunit modified analogue PM01183 attenuate nucleotide excision repair and show activity toward platinum-resistant cells.

PM01183 is a novel marine-derived covalent DNA binder in clinical development. PM01183 is structurally similar to trabectedin (yondelis, ecteinascidin-743) except for the C subunit, and this modification is accompanied by different pharmacokinetics in cancer patients. We here characterize the interaction of PM01183 with the nucleotide excision repair (NER) pathway in comparison with trabectedin.

Ataxia telangiectasia mutated- and Rad3-related kinase drives both the early and the late DNA-damage response to the monofunctional antitumour alkylator S23906.

Numerous anticancer agents and environmental mutagens target DNA. Although all such compounds interfere with the progression of the replication fork and inhibit DNA synthesis, there are marked differences in the DNA-damage response pathways they trigger, and the relative impact of the proximal or the distal signal transducers on cell survival is mainly lesion-specific. Accordingly, checkpoint kinase inhibitors in current clinical development show synergistic activity with some DNA-targeting agents, but not with others.

The NER proteins XPC and CSB, but not ERCC1, regulate the sensitivity to the novel DNA binder S23906: implications for recognition and repair of antitumor alkylators.

S23906 belongs to a novel class of alkylating anticancer agents forming bulky monofunctional DNA adducts. A unique feature of S23906 is its "helicase-like" activity leading to the destabilization of the surrounding duplex DNA. We here characterize the recognition and repair of S23906 adducts by the nucleotide excision repair (NER) machinery.