Expansion of human hematopoietic stem cells by inhibiting translation (original) (raw)
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Gene Editing of Hematopoietic Stem Cells: Hopes and Hurdles Toward Clinical Translation
Frontiers in Genome Editing, 2021
In the field of hematology, gene therapies based on integrating vectors have reached outstanding results for a number of human diseases. With the advent of novel programmable nucleases, such as CRISPR/Cas9, it has been possible to expand the applications of gene therapy beyond semi-random gene addition to site-specific modification of the genome, holding the promise for safer genetic manipulation. Here we review the state of the art of ex vivo gene editing with programmable nucleases in human hematopoietic stem and progenitor cells (HSPCs). We highlight the potential advantages and the current challenges toward safe and effective clinical translation of gene editing for the treatment of hematological diseases.
Therapeutic Base Editing of Human Hematopoietic Stem Cells
Blood, 2019
Base editing with fusions of RNA-guided DNA-binding proteins and nucleotide deaminases represents a promising approach to permanently remedy genetic blood disorders without obligatory double strand breaks, however its application in engrafting hematopoietic stem cells (HSCs) remains unexplored. Here we purified A3A(N57Q)-BE3 protein for RNP electroporation of human peripheral blood (PB) mobilized CD34+ hematopoietic stem and progenitor cells (HSPCs). We found that sgRNAs targeting for cytidine base editing the core GATA1 binding motif of the BCL11A +58 erythroid enhancer resulted in efficient on-target base edits (81% allele frequency) with low indels. There was similar HbF induction in erythroid progeny as compared to Cas9:sgRNA RNP nuclease mediated modification of the same target sequence (36% median HbF in base edited cells with 81% allele modifications, 39% HbF in 3xNLS-SpCas9:sgRNA#1617 nuclease edited cells with 99% indels, and 5%HbF in unedited cells). A single therapeutic b...
Genomic Engineering in Human Hematopoietic Stem Cells: Hype or Hope?
Frontiers in Genome Editing, 2021
Many gene editing techniques are developed and tested, yet, most of these are optimized for transformed cell lines, which differ from their primary cell counterparts in terms of transfectability, cell death propensity, differentiation capability, and chromatin accessibility to gene editing tools. Researchers are working to overcome the challenges associated with gene editing of primary cells, namely, at the level of improving the gene editing tool components, e.g., the use of modified single guide RNAs, more efficient delivery of Cas9 and RNA in the ribonucleoprotein of these cells. Despite these efforts, the low efficiency of proper gene editing in true primary cells is an obstacle that needs to be overcome in order to generate sufficiently high numbers of corrected cells for therapeutic use. In addition, many of the therapeutic candidate genes for gene editing are expressed in more mature blood cell lineages but not in the hematopoietic stem cells (HSCs), where they are tightly pa...
Ex vivo expansion of hematopoietic stem cells: mission accomplished?
Swiss Medical Weekly, 2011
A small number of hematopoietic stem cells (HSCs) with self-renewal and multi-lineage repopulation capacity maintain hematopoiesis during the lifetime of an individual. Moreover, HSCs and their potential exist in excess as one individual can share its HSCs with another leading to creation of a genetically identical hematopoietic system. For over half a century this property of HSCs has been utilised by successful allogeneic clinical HSC transplantation for treatment of patients with inherited or acquired genetic and neoplastic diseases of the hematopoietic and immune system. There are now more than twenty thousand allogeneic HSC transplants per year worldwide [1]. However, although more than 17.5 million potential HSC donors are registered and additional 500,000 cord bloods are stored for potential allogeneic HSC transplantation [2], timely availability of appropriately human leukocyte antigen (HLA)compatible HSCs with sufficient quality for patients still poses a problem in the field. Even if a donor is available, toxicity of the procedure could be reduced by increasing HSC numbers in transplants. One way to solve these issues would be by generation of quality-controlled, off the shelf HSC products via in vitro HSC expansion, a "holy grail" procedure many have been hunting for. Here, we discuss accumulating knowledge on signalling pathways involved in HSC maintenance as well as recent achievements to apply the findings to ex vivo HSC expansion for clinical use. Although the specific issue concerns only highly specialised medicine today, newly generated knowledge will be critical for the whole field of stem cell transplantation and regenerative medicine in the future.
Improving Gene Therapy Efficiency through the Enrichment of Human Hematopoietic Stem Cells
Molecular Therapy, 2017
Lentiviral vector (LV)-based hematopoietic stem cell (HSC) gene therapy is becoming a promising clinical strategy for the treatment of genetic blood diseases. However, the current approach of modifying 1 Â 10 8 to 1 Â 10 9 CD34 + cells per patient requires large amounts of LV, which is expensive and technically challenging to produce at clinical scale. Modification of bulk CD34 + cells uses LV inefficiently, because the majority of CD34 + cells are short-term progenitors with a limited posttransplant lifespan. Here, we utilized a clinically relevant, immunomagnetic bead (IB)-based method to purify CD34 + CD38 À cells from human bone marrow (BM) and mobilized peripheral blood (mPB). IB purification of CD34 + CD38 À cells enriched severe combined immune deficiency (SCID) repopulating cell (SRC) frequency an additional 12-fold beyond standard CD34 + purification and did not affect gene marking of long-term HSCs. Transplant of purified CD34 + CD38 À cells led to delayed myeloid reconstitution, which could be rescued by the addition of non-transduced CD38 + cells. Importantly, LV modification and transplantation of IB-purified CD34 + CD38 À cells/non-modified CD38 + cells into immune-deficient mice achieved long-term gene-marked engraftment comparable with modification of bulk CD34 + cells, while utilizing $7-fold less LV. Thus, we demonstrate a translatable method to improve the clinical and commercial viability of gene therapy for genetic blood cell diseases.
2020
Hematopoietic stem and progenitor cells (HSPCs) have great therapeutic important in the medical science due to its ability to both self-renew and differentiate. A genetically modified HSPCs could result in correction of the entire hematopoietic system in patients with various hematological disorders, due to this unique properties. One of the evolutionary discovery in the field of biology is the ability to altered and modify the functional output of genome. Zincfinger nuclease (ZFNs) and transcriptional activator-like effector nucleases (TALENs) are the genome editing technologies used at both the basic and clinical level from the past decades. But after knowing many obstacles are associated with this technologies, scientists leads to a new discovery of a advance technology in the field of genetic engineering and this tool was known as clustered regularly interspaced short palindromic repeats (CRISPR)associated protein 9 (Cas9) system. In this review, we describe the mechanism of CRI...
STEM CELLS, 2018
Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated system (Cas9)-mediated gene editing of human hematopoietic stem cells (hHSCs) is a promising strategy for the treatment of genetic blood diseases through site-specific correction of identified causal mutations. However, clinical translation is hindered by low ratio of precise gene modification using the corrective donor template (homology-directed repair, HDR) to gene disruption (nonhomologous end joining, NHEJ) in hHSCs. By using a modified version of Cas9 with reduced nuclease activity in G1 phase of cell cycle when HDR cannot occur, and transiently increasing the proportion of cells in HDR-preferred phases (S/G2), we achieved a four-fold improvement in HDR/NHEJ ratio over the control condition in vitro, and a significant improvement after xenotransplantation of edited hHSCs into immunodeficient mice. This strategy for improving gene editing outcomes in hHSCs has important implications for the fie...
Functional Profiling of Single CRISPR/Cas9-Edited Human Long-Term Hematopoietic Stem Cells
In the human hematopoietic system, rare self-renewing multi-potent long-term hematopoietic stem cells (LT-HSCs) are responsible for the lifelong production of mature blood cells and are the rational target for clinical regenerative therapies. However, the heterogeneity in the hematopoietic stem cell compartment and variable outcomes of CRISPR/Cas9 editing make functional interrogation of rare LT-HSCs challenging. Here, we report high efficiency LT-HSC editing at single cell resolution using electroporation of modified synthetic gRNAs and Cas9 protein. Targeted short isoform expression of the GATA1 transcription factor elicited distinct differentiation and proliferation effects in single LT-HSC when analyzed with functional in vitro differentiation and long-term repopulation xenotransplantation assays. Our method represents a blueprint for systematic genetic analysis of complex tissue hierarchies at single cell level.
Gene Editing in Adult Hematopoietic Stem Cells
Modern Tools for Genetic Engineering, 2016
Over the last years, an important development has allowed the scientific community to address a precise and accurate modification of the genome. The first probe of concept appeared with the design and use of engineered zinc-finger nucleases (ZFNs), which was expanded later on with the discovery and engineering of meganucleases and transcription activator-like effector nucleases (TALENs) and finally democratized and made easily available to the whole scientific community with the discovery of the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 nuclease combination technology. The availability of these tools has allowed a precise gene editing, such as knockout of a specific gene or the correction of a defective gene by means of homologous recombination (HR), taking advantage of the endogenous cell repair machinery. This process was already known and used but was inefficient-efficiency that has been increased more than 100-fold with the addition of the mentioned specific nucleases to the process. Apart from the proper design of the nucleases to recognize and cut the selected site in the cell genome, two main goals need to be adequately addressed to optimize its function: the delivery of the tools into the desired cells and the selection of those where the gene editing process has occurred correctly. Both steps can be easily solved when the source of cells is extensive or can be expanded and manipulated in vitro extensively, such as immortalized cell lines or pluripotent stem cells (embryonic stem cells and induced pluripotent stem cells). However, both steps are critical in the case of primary cells, such as the hematopoietic stem cells (HSCs). HSCs are a rare cell population present in the bone marrow (BM) of higher mammals, and it is the responsible for the maintenance and