A p38 MAPK-ROS axis fuels proliferation stress and DNA damage during CRISPR-Cas9 gene editing in hematopoietic stem and progenitor cells.

Ex vivo activation is a prerequisite to reaching adequate levels of gene editing by homology-directed repair (HDR) for hematopoietic stem and progenitor cell (HSPC)-based clinical applications. Here, we show that shortening culture time mitigates the p53-mediated DNA damage response to CRISPR-Cas9-induced DNA double-strand breaks, enhancing the reconstitution capacity of edited HSPCs. However, this results in lower HDR efficiency, rendering ex vivo culture necessary yet detrimental.

Genetic engineering meets hematopoietic stem cell biology for next-generation gene therapy.

The growing clinical success of hematopoietic stem/progenitor cell (HSPC) gene therapy (GT) relies on the development of viral vectors as portable "Trojan horses" for safe and efficient gene transfer. The recent advent of novel technologies enabling site-specific gene editing is broadening the scope and means of GT, paving the way to more precise genetic engineering and expanding the spectrum of diseases amenable to HSPC-GT. Here, we provide an overview of state-of-the-art and prospective developments of the HSPC-GT field, highlighting how advances in biological characterization and manipulation of HSPCs will enable the design of the next generation of these transforming therapeutics.

Mesenchymal stromal cells improve the transplantation outcome of CRISPR-Cas9 gene-edited human HSPCs.

Mesenchymal stromal cells (MSCs) have been employed in vitro to support hematopoietic stem and progenitor cell (HSPC) expansion and in vivo to promote HSPC engraftment. Based on these studies, we developed an MSC-based co-culture system to optimize the transplantation outcome of clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 gene-edited (GE) human HSPCs. We show that bone marrow (BM)-MSCs produce several hematopoietic supportive and anti-inflammatory factors capable of alleviating the proliferation arrest and mitigating the apoptotic and inflammatory programs activated in GE-HSPCs, improving their expansion and clonogenic potential in vitro.

Follicular helper T cell signature of replicative exhaustion, apoptosis, and senescence in common variable immunodeficiency.

Common variable immunodeficiency (CVID) is the most frequent primary antibody deficiency whereby follicular helper T (Tfh) cells fail to establish productive responses with B cells in germinal centers. Here, we analyzed the frequency, phenotype, transcriptome, and function of circulating Tfh (cTfh) cells in CVID patients displaying autoimmunity as an additional phenotype. A group of patients showed a high frequency of cTfh1 cells and a prominent expression of PD-1 and ICOS as well as a cTfh mRNA signature consistent with highly activated, but exhausted, senescent, and apoptotic cells.

Oncogene-induced senescence in hematopoietic progenitors features myeloid restricted hematopoiesis, chronic inflammation and histiocytosis.

Activating mutations in the BRAF-MAPK pathway have been reported in histiocytoses, hematological inflammatory neoplasms characterized by multi-organ dissemination of pro-inflammatory myeloid cells. Here, we generate a humanized mouse model of transplantation of human hematopoietic stem and progenitor cells (HSPCs) expressing the activated form of BRAF (BRAF). All mice transplanted with BRAF-expressing HSPCs succumb to bone marrow failure, displaying myeloid-restricted hematopoiesis and multi-organ dissemination of aberrant mononuclear phagocytes.

Premature Senescence and Increased Oxidative Stress in the Thymus of Down Syndrome Patients.

Down syndrome (DS) patients prematurely show clinical manifestations usually associated with aging. Their immune system declines earlier than healthy individuals, leading to increased susceptibility to infections and higher incidence of autoimmune phenomena. Clinical features of accelerated aging indicate that trisomy 21 increases the biological age of tissues.

RNA Modulates the Expression of Cell Cycle Genes in Human Fibroblasts.

retroelements, whose retrotransposition requires prior transcription by RNA polymerase III to generate RNAs, represent the most numerous non-coding RNA (ncRNA) gene family in the human genome. transcription is generally kept to extremely low levels by tight epigenetic silencing, but it has been reported to increase under different types of cell perturbation, such as viral infection and cancer. RNAs, being able to act as gene expression modulators, may be directly involved in the mechanisms determining cellular behavior in such perturbed states.

Precise Gene Editing Preserves Hematopoietic Stem Cell Function following Transient p53-Mediated DNA Damage Response.

Precise gene editing in hematopoietic stem and progenitor cells (HSPCs) holds promise for treating genetic diseases. However, responses triggered by programmable nucleases in HSPCs are poorly characterized and may negatively impact HSPC engraftment and long-term repopulation capacity. Here, we induced either one or several DNA double-stranded breaks (DSBs) with optimized zinc-finger and CRISPR/Cas9 nucleases and monitored DNA damage response (DDR) foci induction, cell-cycle progression, and transcriptional responses in HSPC subpopulations, with up to single-cell resolution.

An early-senescence state in aged mesenchymal stromal cells contributes to hematopoietic stem and progenitor cell clonogenic impairment through the activation of a pro-inflammatory program.

Hematopoietic stem and progenitor cells (HSPC) reside in the bone marrow (BM) niche and serve as a reservoir for mature blood cells throughout life. Aging in the BM is characterized by low-grade chronic inflammation that could contribute to the reduced functionality of aged HSPC. Mesenchymal stromal cells (MSC) in the BM support HSPC self-renewal.

Whole-genome expression analysis of mammalian-wide interspersed repeat elements in human cell lines.

With more than 500,000 copies, mammalian-wide interspersed repeats (MIRs), a sub-group of SINEs, represent ∼2.5% of the human genome and one of the most numerous family of potential targets for the RNA polymerase (Pol) III transcription machinery. Since MIR elements ceased to amplify ∼130 myr ago, previous studies primarily focused on their genomic impact, while the issue of their expression has not been extensively addressed.

Identification of RNA polymerase III-transcribed Alu loci by computational screening of RNA-Seq data.

Of the ∼ 1.3 million Alu elements in the human genome, only a tiny number are estimated to be active in transcription by RNA polymerase (Pol) III. Tracing the individual loci from which Alu transcripts originate is complicated by their highly repetitive nature.

The Murine PSE/TATA-dependent transcriptome: evidence of functional homologies with its human counterpart.

A series of recent studies demonstrated an unexpectedly high frequency of intronic RNA polymerase (pol) III transcription units spread throughout the human genome. The investigation of a subset of these transcripts revealed their tissue/cell-specific transcription together with the involvement in relevant physiopathological pathways. Despite this evidence, these transcripts did not seem to have murine orthologs, based on their nucleotide sequence, resulting in a limitation of the experimental approaches aimed to study their function.

Identification of RNA polymerase III-transcribed genes in eukaryotic genomes.

The RNA polymerase (Pol) III transcription system is devoted to the production of short, generally abundant noncoding (nc) RNAs in all eukaryotic cells. Previously thought to be restricted to a few housekeeping genes easily detectable in genome sequences, the set of known Pol III-transcribed genes (class III genes) has been expanding in the last ten years, and the issue of their detection, annotation and actual expression has been stimulated and revived by the results of recent high-resolution genome-wide location analyses of the mammalian Pol III machinery, together with those of Pol III-centered computational studies and of ncRNA-focused transcriptomic approaches. In this article, we provide an outline of distinctive features of Pol III-transcribed genes that have allowed and currently allow for their detection in genome sequences, we critically review the currently practiced strategies for the identification of novel class III genes and transcripts, and we discuss emerging themes in Pol III transcription regulation which might orient future transcriptomic studies.