title={Near-perfect precise on-target editing of human hematopoietic stem and progenitor cells},
author={Cloarec-Ung, Fanny-Mei and Beaulieu, Jamie and Suthananthan, Arunan and Lehnertz, Bernhard and Sauvageau, Guy and Sheppard, Hilary M. and Knapp, David J. H. F.},
title={
Near-perfect precise on-target editing of human hematopoietic stem and
progenitor cells
},
author={
Cloarec-Ung, Fanny-Mei and Beaulieu, Jamie and Suthananthan, Arunan and
Lehnertz, Bernhard and Sauvageau, Guy and Sheppard, Hilary M. and Knapp,
David J. H. F.
},
year=2024,
month=jun,
journal={eLife},
...
...
@@ -8,192 +15,26 @@
pages={RP91288},
doi={10.7554/eLife.91288},
issn={2050-084X},
abstractnote={Precision gene editing in primary hematopoietic stem and progenitor cells (HSPCs) would facilitate both curative treatments for monogenic disorders as well as disease modelling. Precise efficiencies even with the CRISPR/Cas system, however, remain limited. Through an optimization of guide RNA delivery, donor design, and additives, we have now obtained mean precise editing efficiencies >90% on primary cord blood HSCPs with minimal toxicity and without observed off-target editing. The main protocol modifications needed to achieve such high efficiencies were the addition of the DNA-PK inhibitor AZD7648, and the inclusion of spacer-breaking silent mutations in the donor in addition to mutations disrupting the PAM sequence. Critically, editing was even across the progenitor hierarchy, did not substantially distort the hierarchy or affect lineage outputs in colony-forming cell assays or the frequency of high self-renewal potential long-term culture initiating cells. As modelling of many diseases requires heterozygosity, we also demonstrated that the overall editing and zygosity can be tuned by adding in defined mixtures of mutant and wild-type donors. With these optimizations, editing at near-perfect efficiency can now be accomplished directly in human HSPCs. This will open new avenues in both therapeutic strategies and disease modelling.},
title={Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9},
author={Doench, John G. and Fusi, Nicolo and Sullender, Meagan and Hegde, Mudra and Vaimberg, Emma W. and Donovan, Katherine F. and Smith, Ian and Tothova, Zuzana and Wilen, Craig and Orchard, Robert and Virgin, Herbert W. and Listgarten, Jennifer and Root, David E.},
year=2016,
month=feb,
journal={Nature Biotechnology},
publisher={Nature Publishing Group},
volume=34,
number=2,
pages={184–191},
doi={10.1038/nbt.3437},
issn={1546-1696},
rights={2015 Springer Nature America, Inc.},
abstractnote={Genome-wide sgRNA libraries based on rules for on-target activity improve results of Cas9-based screens and facilitate a further refinement of on- and off-target prediction algorithms.},
title={Na+ riboswitches regulate genes for diverse physiological processes in bacteria},
author={White, Neil and Sadeeshkumar, Harini and Sun, Anna and Sudarsan, Narasimhan and Breaker, Ronald R.},
year=2022,
month=aug,
journal={Nature Chemical Biology},
publisher={Nature Publishing Group},
volume=18,
number=88,
pages={878–885},
doi={10.1038/s41589-022-01086-4},
issn={1552-4469},
rights={2022 The Author(s)},
abstractnote={Only one protein factor is known that senses Na+ and controls gene expression. The Breaker Laboratory describes a bacterial riboswitch class selective for Na+ that regulates genes important for Na+ homeostasis, pH maintenance, osmotic stress response and ATP synthesis.},
language={en}
}
@article{Iwawaki_Akai_2006,
title={Analysis of the XBP1 splicing mechanism using endoplasmic reticulum stress-indicators},
author={Iwawaki, Takao and Akai, Ryoko},
year=2006,
month=nov,
journal={Biochemical and Biophysical Research Communications},
volume=350,
number=3,
pages={709–715},
doi={10.1016/j.bbrc.2006.09.100},
issn={0006-291X},
abstractnote={Under endoplasmic reticulum (ER) stress conditions, XBP1 mRNA is processed by unconventional splicing and translated into a functional transcription factor. ER stress-specific XBP1 splicing is also known to be activated by IRE1. However, many aspects of the molecular mechanism of XBP1 splicing remain to be elucidated. We previously developed an indicator system that enabled detection of XBP1 splicing using fluorescent proteins as the reporter signals. Here, we use a modification of this method that employs modified ER stress-indicators and mutant IRE1 in vivo and in vitro to analyze XBP1 splicing mechanisms. Our analyses suggest that the 506–579nt region of the XBP1 mRNA is necessary and sufficient for XBP1 splicing, that XBP1 splicing can occur in the cytoplasm, and that cleavage and ligation of XBP1 mRNA during splicing may occur as a coupled reaction.}
title={XBP1 splicing contributes to endoplasmic reticulum stress-induced human islet amyloid polypeptide up-regulation},
author={Zhang, Yun and Lin, Susan and Yao, Jing and Cai, Wantong and Chen, Huaqiu and Aierken, Ailikemu and Wang, Zhe and Song, Weihong},
year=2023,
month=oct,
journal={Genes & Diseases},
volume=11,
number=5,
pages=101148,
doi={10.1016/j.gendis.2023.101148},
issn={2352-4820},
abstractnote={As a pathological hallmark of type 2 diabetes mellitus (T2DM), islet amyloid is formed by the aggregation of islet amyloid polypeptide (IAPP). Endoplasmic reticulum (ER) stress interacts with IAPP aggregates and has been implicated in the pathogenesis of T2DM. To examine the role of ER stress in T2DM, we cloned the hIAPP promoter and analyzed its promoter activity in human β-cells. We found that ER stress significantly enhanced hIAPP promoter activity and expression in human β-cells via triggering X-box binding protein 1 (XBP1) splicing. We identified a binding site of XBP1 in the hIAPP promoter. Disruption of this binding site by substitution or deletion mutagenesis significantly diminished the effects of ER stress on hIAPP promoter activity. Blockade of XBP splicing by MKC3946 treatment inhibited ER stress-induced hIAPP up-regulation and improved human β-cell survival and function. Our study uncovers a link between ER stress and IAPP at the transcriptional level and may provide novel insights into the role of ER stress in IAPP cytotoxicity and the pathogenesis of T2DM.}
author={Wei, Tuo and Sun, Yehui and Cheng, Qiang and Chatterjee, Sumanta and Traylor, Zachary and Johnson, Lindsay T. and Coquelin, Melissa L. and Wang, Jialu and Torres, Michael J. and Lian, Xizhen and Wang, Xu and Xiao, Yufen and Hodges, Craig A. and Siegwart, Daniel J.},
abstract={Approximately 10\% of Cystic Fibrosis (CF) patients, particularly those with CF transmembrane conductance regulator (CFTR) gene nonsense mutations, lack effective treatments. The potential of gene correction therapy through delivery of the CRISPR/Cas system to CF-relevant organs/cells is hindered by the lack of efficient genome editor delivery carriers. Herein, we report improved Lung Selective Organ Targeting Lipid Nanoparticles (SORT LNPs) for efficient delivery of Cas9 mRNA, sgRNA, and donor ssDNA templates, enabling precise homology-directed repair-mediated gene correction in CF models. Optimized Lung SORT LNPs deliver mRNA to lung basal cells in Ai9 reporter mice. SORT LNP treatment successfully corrected the CFTR mutations in homozygous G542X mice and in patient-derived human bronchial epithelial cells with homozygous F508del mutations, leading to the restoration of CFTR protein expression and chloride transport function. This proof-of-concept study will contribute to accelerating the clinical development of mRNA LNPs for CF treatment through CRISPR/Cas gene correction.},
title={Polyethylene glycol (PEG): The nature, immunogenicity, and role in the hypersensitivity of PEGylated products},
author={Mohamed Ibrahim and Eslam Ramadan and Nehal E. Elsadek and Sherif E. Emam and Taro Shimizu and Hidenori Ando and Yu Ishima and Omar Helmy Elgarhy and Hatem A. Sarhan and Amal K. Hussein and Tatsuhiro Ishida},
abstract={Polyethylene glycol (PEG) is a versatile polymer that is widely used as an additive in foods and cosmetics, and as a carrier in PEGylated therapeutics. Even though PEG is thought to be less immunogenic, or perhaps even non-immunogenic, with a variety of physicochemical properties, there is mounting evidence that PEG causes immunogenic responses when conjugated with other materials such as proteins and nanocarriers. Under these conditions, PEG with other materials can result in the production of anti-PEG antibodies after administration. The antibodies that are induced seem to have a deleterious impact on the therapeutic efficacy of subsequently administered PEGylated formulations. In addition, hypersensitivity to PEGylated formulations could be a significant barrier to the utility of PEGylated products. Several reports have linked the presence of anti-PEG antibodies to incidences of complement activation-related pseudoallergy (CARPA) following the administration of PEGylated formulations. The use of COVID-19 mRNA vaccines, which are composed mainly of PEGylated lipid nanoparticles (LNPs), has recently gained wide acceptance, although many cases of post-vaccination hypersensitivity have been documented. Therefore, our review focuses not only on the importance of PEGs and its great role in improving the therapeutic efficacy of various medications, but also on the hypersensitivity reactions attributed to the use of PEGylated products that include PEG-based mRNA COVID-19 vaccines.}
}
@article{jiang_combinatorial_2024,
title={Combinatorial development of nebulized {mRNA} delivery formulations for the lungs},
author={Jiang, Allen Y. and Witten, Jacob and Raji, Idris O. and Eweje, Feyisayo and MacIsaac, Corina and Meng, Sabrina and Oladimeji, Favour A. and Hu, Yizong and Manan, Rajith S. and Langer, Robert and Anderson, Daniel G.},
title={In vitro platform to model the function of ionocytes in the human airway epithelium},
author={Vilà-González, Marta and Pinte, Laetitia and Fradique, Ricardo and Causa, Erika and Kool, Heleen and Rodrat, Mayuree and Morell, Carola Maria and Al-Thani, Maha and Porter, Linsey and Guo, Wenrui and Maeshima, Ruhina and Hart, Stephen L. and McCaughan, Frank and Granata, Alessandra and Sheppard, David N. and Floto, R. Andres and Rawlins, Emma L. and Cicuta, Pietro and Vallier, Ludovic},
Background Pulmonary ionocytes have been identified in the airway epithelium as a small population of ion transporting cells expressing high levels of CFTR (cystic fibrosis transmembrane conductance regulator), the gene mutated in cystic fibrosis. By providing an infinite source of airway epithelial cells (AECs), the use of human induced pluripotent stem cells (hiPSCs) could overcome some challenges of studying ionocytes. However, the production of AEC epithelia containing ionocytes from hiPSCs has proven difficult. Here, we present a platform to produce hiPSCderived AECs (hiPSC-AECs) including ionocytes and investigate their role in the airway epithelium.
Methods hiPSCs were differentiated into lung progenitors, which were expanded as 3D organoids and matured by air-liquid interface culture as polarised hiPSC-AEC epithelia. Using CRISPR/Cas9 technology, we generated a hiPSCs knockout (KO) for FOXI1, a transcription factor that is essential for ionocyte specification. Differences between FOXI1 KO hiPSC-AECs and their wild-type (WT) isogenic controls were investigated by assessing gene and protein expression, epithelial composition, cilia coverage and motility, pH and transepithelial barrier properties.
Results Mature hiPSC-AEC epithelia contained basal cells, secretory cells, ciliated cells with motile cilia, pulmonary neuroendocrine cells (PNECs) and ionocytes. There was no difference between FOXI1 WT and KO hiPSCs in terms of their capacity to differentiate into airway progenitors. However, FOXI1 KO led to mature hiPSC-AEC epithelia without ionocytes with reduced capacity to produce ciliated cells.
Conclusion Our results suggest that ionocytes could have role beyond transepithelial ion transport by regulating epithelial properties and homeostasis in the airway epithelium.
abstractnote={
Precision gene editing in primary hematopoietic stem and progenitor cells
(HSPCs) would facilitate both curative treatments for monogenic disorders as
well as disease modelling. Precise efficiencies even with the CRISPR/Cas
system, however, remain limited. Through an optimization of guide RNA
delivery, donor design, and additives, we have now obtained mean precise
editing efficiencies >90% on primary cord blood HSCPs with minimal toxicity
and without observed off-target editing. The main protocol modifications
needed to achieve such high efficiencies were the addition of the DNA-PK
inhibitor AZD7648, and the inclusion of spacer-breaking silent mutations in
the donor in addition to mutations disrupting the PAM sequence. Critically,
editing was even across the progenitor hierarchy, did not substantially
distort the hierarchy or affect lineage outputs in colony-forming cell assays
or the frequency of high self-renewal potential long-term culture initiating
cells. As modelling of many diseases requires heterozygosity, we also
demonstrated that the overall editing and zygosity can be tuned by adding in
defined mixtures of mutant and wild-type donors. With these optimizations,
editing at near-perfect efficiency can now be accomplished directly in human
HSPCs. This will open new avenues in both therapeutic strategies and disease
modelling.
},
language={en}
}
@book{book,
title={Genome Editing and Biological Weapons: Assessing the Risk of Misuse},
author={Paris, Katherine},
year=2023,
month={01},
pages={},
doi={10.1007/978-3-031-21820-0},
isbn={978-3-031-21819-4}
}
@article{wickiser2020biosecurity,
title={The democratization of biology: how CRISPR and synthetic biology usher in new biosecurity threats},
