Endogenous Promoter Research Articles

Natural Transformation, Protein Expression, and Cryoconservation of the Filamentous Cyanobacterium Phormidium lacuna.

Cyanobacteria are the focus of basic research and biotechnological projects in which solar energy is utilized for biomass production. Phormidium lacuna is a newly isolated filamentous cyanobacterium. This paper describes how new filamentous cyanobacteria can be isolated from marine rockpools. It also describes how DNA can be extracted from filaments and how the genomes can be sequenced. Although transformation is established for many single-celled species, it is less frequently reported for filamentous cyanobacteria. A simplified method for the natural transformation of P. lacuna is described here. P. lacuna is the only member of the order Oscillatoriales for which natural transformation is established. This paper also shows how natural transformation is used to express superfolder green fluorescent protein (sfGFP). An endogenous cpcB promoter induced approximately 5 times stronger expression than cpc560, A2813, or psbA2 promoters from Synechocystis sp. PCC6803. Further, a method for the cryopreservation of P. lacuna and Synechocystis sp. CPP 6803 was established, and methods for assessing motility in a liquid medium and on agar and plastic surfaces are described.

Inducible caspase-9 suicide gene under control of endogenous oct4 to safeguard mouse and human pluripotent stem cell therapy

Pluripotent stem cells (PSCs) are promising in regenerative medicine. A major challenge of PSC therapy is the risk of teratoma formation because of the contamination of undifferentiated stem cells. Constitutive promoters or endogenous SOX2 promoters have been used to drive inducible caspase-9 (iCasp9) gene expression but cannot specifically eradicate undifferentiated PSCs. Here, we inserted iCasp9 gene into the endogenous OCT4 locus of human and mouse PSCs without affecting their pluripotency. A chemical inducer of dimerization (CID), AP1903, induced iCasp9 activation, which led to the apoptosis of specific undifferentiated PSCs in vitro and in vivo. Differentiated cell lineages survived because of the silence of the endogenous OCT4 gene. Human and mouse PSCs were controllable when CID was administrated within 2 weeks after PSC injection in immunodeficient mice. However, an interval longer than 2 weeks caused teratoma formation and mouse death because a mass of somatic cells already differentiated from the PSCs. In conclusion, we have developed a specific and efficient PSC suicide system that will be of value in the clinical applications of PSC-based therapy.

A novel and accurate full-length HTT mouse model for Huntington's disease.

Here, we report the generation and characterization of a novel Huntington's disease (HD) mouse model BAC226Q by using a bacterial artificial chromosome (BAC) system, expressing full-length human HTT with ~226 CAG-CAA repeats and containing endogenous human HTT promoter and regulatory elements. BAC226Q recapitulated a full-spectrum of age-dependent and progressive HD-like phenotypes without unwanted and erroneous phenotypes. BAC226Q mice developed normally, and gradually exhibited HD-like psychiatric and cognitive phenotypes at 2 months. From 3 to 4 months, BAC226Q mice showed robust progressive motor deficits. At 11 months, BAC226Q mice showed significant reduced life span, gradual weight loss and exhibited neuropathology including significant brain atrophy specific to striatum and cortex, striatal neuronal death, widespread huntingtin inclusions, and reactive pathology. Therefore, the novel BAC226Q mouse accurately recapitulating robust, age-dependent, progressive HD-like phenotypes will be a valuable tool for studying disease mechanisms, identifying biomarkers, and testing gene-targeting therapeutic approaches for HD.

Episome-Based Gene Expression Modulation Platform in the Model Diatom Phaeodactylum tricornutum.

Chemically inducible gene expression systems have been an integral part of the advanced synthetic genetic circuit design and are employed for precise dynamic control over genetically engineered traits. However, the current systems for controlling transgene expression in most algae are limited to endogenous promoters that respond to different environmental factors. We developed a highly efficient, tunable, and reversible episome-based transcriptional control system in the model diatom alga, Phaeodactylum tricornutum. We assessed the time- and dose-response dynamics of each expression system using a reporter protein (eYFP) as a readout. Using our circuit configuration, we found two inducible expression systems with a high dynamic range and confirmed the suitability of an episome expression platform for synthetic biological applications in diatoms. These systems are controlled by the presence of β-estradiol and digoxin. Addition of either chemical to transgenic strains activates transcription with a dynamic range of up to ∼180-fold and ∼90-fold, respectively. We demonstrated that our episome-based transcriptional control systems are tunable and reversible in a dose- and time-dependent manner. Using droplet digital polymerase chain reaction (PCR), we also confirmed that inducer-dependent transcriptional activation starts within minutes of inducer application without any detectable transcript in the uninduced controls. The system described here expands the molecular and synthetic biology toolkits in algae and will facilitate future gene discovery and metabolic engineering efforts.

Increased mutation efficiency of CRISPR/Cas9 genome editing in banana by optimized construct.

The CRISPR/Cas9-mediated genome editing system has been used extensively to engineer targeted mutations in a wide variety of species. Its application in banana, however, has been hindered because of the species’ triploid nature and low genome editing efficiency. This has delayed the development of a DNA-free genome editing approach. In this study, we reported that the endogenous U6 promoter and banana codon-optimized Cas9 apparently increased mutation frequency in banana, and we generated a method to validate the mutation efficiency of the CRISPR/Cas9-mediated genome editing system based on transient expression in protoplasts. The activity of the MaU6c promoter was approximately four times higher than that of the OsU6a promoter in banana protoplasts. The application of this promoter and banana codon-optimized Cas9 in CRISPR/Cas9 cassette resulted in a fourfold increase in mutation efficiency compared with the previous CRISPR/Cas9 cassette for banana. Our results indicated that the optimized CRISPR/Cas9 system was effective for mutating targeted genes in banana and thus will improve the applications for basic functional genomics. These findings are relevant to future germplasm improvement and provide a foundation for developing DNA-free genome editing technology in banana.

Heterologous expression of human C-reactive protein in the green alga Chlamydomonas reinhardtii.

C-reactive protein (CRP) participates in human inflammatory responses and is an important indicator in clinical diagnoses. At present, the use of monoclonal antibodies to detect CRP in the human body is high, but they are unstable and expensive. Understanding the CRP expression pathway is of great significance for developing CRP tests and reagents. Chlamydomonas reinhardtii is a model organism that has great potential as a foreign protein expression system. This study is the first attempt to express human CRP in C. reinhardtii. We selected the endogenous constitutive Rbcs2 promoter and terminator and used ble as a selective gene to construct a C. reinhardtii nuclear expression vector containing CRP. After transformation using the glass bead method, six positive transformants were obtained. At the molecular level, full-length CRP was transformed into the genome of C. reinhardtii CC400 cells, and human CRP was expressed. This study provides new insights into obtaining active CRP. PRACTICAL APPLICATIONS: Based on the nuclear transformation system of C. reinhardtii, it can construct an exogenous protein expression system that produces a variety of high value-added products and can be used to produce a variety of high value-added proteins, functional drugs, and industrial raw materials. It has broad market prospects and huge application prospects.

Screening of CHO-K1 endogenous promoters for expressing recombinant proteins in mammalian cell cultures

For the production of recombinant protein therapeutics in mammalian cells, a high rate of gene expression is desired and hence strong viral-derived promoters are commonly used. However, they usually induce cellular stress and can be susceptible to epigenetic silencing. Endogenous promoters, which coordinates their activity with cellular and bioprocess dynamics while at the same time they maintain high expression levels, may help to avoid such drawbacks. In this work, new endogenous promoters were discovered based on high expression levels in RNA-seq data of CHO-K1 cells cultured in high density. The promoters of Actb, Ctsz, Hmox1, Hspa5, Vim and Rps18 genes were selected for generating new expression vectors for the production of recombinant proteins in mammalian cells. The in silico-derived promoter regions were experimentally verified and the majority showed transcriptional activity comparable or higher than CMV. Also, stable expression following a reduction of culture temperature was investigated. The characterized endogenous promoters (excluding Rps18) constitute a promising alternative to CMV promoter due to their high strength, long-term expression stability and integration into the regulatory network of the host cell. These promoters may also comprise an initial panel for designing cell engineering strategies and synthetic promoters, as well as for industrial cell line development.

Characterization of Endogenous Constitutive Promoters from Lactobacillus Salivarius for Finely-Tuning Gene Expression

Characterization of Endogenous Constitutive Promoters from Lactobacillus Salivarius for Finely-Tuning Gene Expression

Ptf1a Promoter-Driven Cre Expression During Spermatogenesis Causes Germline Recombination.

Two independently developed Ptf1a-Cre mouse lines, Ptf1atm1(cre)Hnak and Ptf1atm1(Cre)Cvw, are widely used in pancreatic research. Recently, Ptf1atm1(cre)Hnak line was reported to transmit unwanted paternal recombination. We aimed to investigate whether this exists in the Ptf1atm1(Cre)Cvw line. Ptf1atm1(Cre)Cvw mice were crossed with R26-LSL-LacZ reporter mice. DNA recombination and gene expression were examined by recombination-specific polymerase chain reaction, reverse transcription-polymerase chain reaction, and X-Gal staining. R26 locus recombination was detected in the pancreas as well as the testes and sperm of the double transgenic mice. Positive ptf1a mRNA expression from testes revealed that there was endogenous Ptf1a promoter activity in this extrapancreatic tissue. Of the 15 progenies that inherited LacZ from the male double transgenic mice, 4 (26.7%) were positive for complete whole-body recombination. The presence of recombination in R26 only mice suggested that the recombination occurred before meiosis. Paternal germline recombination exists in the Ptf1atm1(Cre)Cvw mouse line. Ptf1a promoter-driven Cre expression during spermatogenesis before meiosis is the cause of germline recombination. Therefore, when male Ptf1a-Cre mice are used in compound mice breeding, it is necessary to genotype not only floxed alleles but also recombined alleles to examine unwanted recombinations.

Retinal degeneration in humanized mice expressing mutant rhodopsin under the control of the endogenous murine promoter

RHO is one of the most common genetic causes of autosomal dominant retinitis Pigmentosa (adRP) and there is no effective therapy for this disease. While rapidly developed CRISPR/Cas9 gene editing technology presents a promising therapeutic strategy to treat adRP. A large number of studies for treating adRP using CRISPR/Cas9 have been performed based on transgenic mouse models which are affected with adRP caused by mutant mouse rhodopsin allele, the counterpart of human rhodopsin. Recently, some RHO humanized mouse models like T17M, P23H are generated, which permit testing of the therapeutic effect of CRISPR/Cas9 in preclinical in vivo systems, without putting humans at risk. While available humanized mouse models are few compared to the number of known RHO mutations, but it is time-consuming and costly to build humanized mice for each mutation. We wonder whether a humanized mouse model having several mutations simultaneously can be developed, although which rarely occurs in patients, to investigate the therapeutic effect of CRISPR/Cas9 for RHO-mediated adRP in preclinical in vivo systems. Homology directed repair strategy combing with CRISPR/Cas9 was employed to introduce human RHO genomic fragment containing the replacement of mouse exon1(mE1) after the start codon to mE5 before the stop codon and all introns by the human counterparts. The human rhodopsin could express under the control of the endogenous murine promoter both transcriptionally and translationally in vivo. Human rhodopsin in humanized mouse lines (without mutation) could replace murine rhodopsin morphologically and functionally. While human rhodopsin containing T17M, G51D, G114R, R135W and P171R mutations simultaneously in mutant humanized (Mut-Rhowt/hum and Mut-Rhohum/hum) mouse lines caused retinal degeneration. Mut-Rhohum/hum mice suffered from severe retinal degeneration with defective formation of rod outer segment, leaving nonrecordable electroretinogram (ERG) at 3 months. Mut- Rhowt/hum mice had a slower rate of photoreceptors loss. In 7-month-old Mut- Rhowt/hum mice, statistically reduced scotopic ERG responses were visible compared with age-matched WT mice, but the shortened outer segment and thinner outer nuclear layer could be observed from 3 months. From 7 months to 9 months, significantly abnormal scotopic ERG responses were visible and photoreceptors loss were also obvious in 9-month-old Mut-Rhowt/hum mice. In 12-month-old Mut- Rhowt/hum mice, statistically reduced scotopic and photopic ERG responses and retinal degeneration throughout the retina were visible. Because scotopic responses were more affected than photopic responses in mutant humanized mice, demonstrating that rods dysfunction was more severe than cones dysfunction and deteriorated earlier, the pattern of retinal degeneration caused by mutant human rhodopsin was a typical rod-cone decay. Immunocytochemistry in cells indicated human rhodopsin proteins with 5 mutations aggregated in the cytoplasm and were also retained in the endoplasmic reticulum. The mutant human rhodopsin also accumulated in rod inner segments and cellular bodies in vivo. In conclusion, our humanized models provide excellent opportunities to study the human rhodopsin expression patterns. Our mutant humanized heterozygotes can provide opportunities to explore gene editing therapies via CRISPR/Cas9 for these five mutations in preclinical studies, it is time-saving and cost-effective.

Absence of PEXEL-Dependent Protein Export in Plasmodium Liver Stages Cannot Be Restored by Gain of the HSP101 Protein Translocon ATPase.

Host cell remodeling is critical for successful Plasmodium replication inside erythrocytes and achieved by targeted export of parasite-encoded proteins. In contrast, during liver infection the malarial parasite appears to avoid protein export, perhaps to limit exposure of parasite antigens by infected liver cells. HSP101, the force-generating ATPase of the protein translocon of exported proteins (PTEX) is the only component that is switched off during early liver infection. Here, we generated transgenic Plasmodium berghei parasite lines that restore liver stage expression of HSP101. HSP101 expression in infected hepatocytes was achieved by swapping the endogenous promoter with the ptex150 promoter and by inserting an additional copy under the control of the elongation one alpha (ef1α) promoter. Both promoters drive constitutive and, hence, also pre-erythrocytic expression. Transgenic parasites were able to complete the life cycle, but failed to export PEXEL-proteins in early liver stages. Our results suggest that PTEX-dependent early liver stage export cannot be restored by addition of HSP101, indicative of alternative export complexes or other functions of the PTEX core complex during liver infection.

Wnt/β-catenin signaling and p68 conjointly regulate CHIP in colorectal carcinoma

Emerging evidences suggest abundant expression of Carboxy terminus of Hsc70 Interacting Protein or CHIP (alias STIP1 Homology and U–box Containing Protein 1 or STUB1) in colorectal carcinoma, but the mechanistic detail of this augmented expression pattern is unclear. The signature driver of canonical Wnt pathway, β-catenin, and its co-activator RNA helicase p68, are also overexpressed in colorectal carcinoma. In this study, we describe a novel mechanism of Wnt/β-catenin and p68 mediated transcriptional activation of CHIP gene leading to enhanced proliferation of colorectal carcinoma cells. Bioinformatic analyses reconfirmed an elevated CHIP expression level in colorectal carcinoma datasets. Wnt3A treatment and pharmacological activation of canonical Wnt signaling pathway resulted in increased nuclear translocation of β-catenin, augmenting CHIP expression. Likewise, immunoblotting and Real time PCR following overexpression and knockdown of β-catenin and p68 demonstrated upregulated and downregulated CHIP expression, respectively, at both mRNA and protein levels. p68 along with β-catenin were found to occupy Transcription Factor 4 (TCF4) binding sites on endogenous CHIP promoter and regulate its transcription. After cloning CHIP promoter, the increased and decreased promoter activities of CHIP induced by overexpression and knockdown of either β-catenin or p68 further confirmed transcriptional regulation of CHIP gene by Wnt/β-catenin signaling cascade. Finally, enhanced cellular propagation and migration of colorectal carcinoma cells induced by ‘Wnt/β-catenin-p68-CHIP’ axis established the significance of this pathway in oncogenesis. To the best of our knowledge, this is the first report elucidating the mechanistic details of transcriptional regulation of CHIP (STUB1) gene expression.

Endogenous U6 promoters improve CRISPR/Cas9 editing efficiencies in Sorghum bicolor and show potential for applications in other cereals.

Endogenous U6 promoters increase CRISPR/Cas9 editing efficiency in sorghum and may be useful for gene editing applications in other cereals.

Applications of CRISPR/Cas9 in a rice protein expression system via an intron-targeted insertion approach

The sugar starvation-inducible rice αAmy3 promoter and signal peptide are widely used to produce valuable recombinant proteins in rice suspension culture cells. Conventionally, the recombinant gene expression cassette is inserted into the genome at random locations by Agrobacterium- or particle bombardment-mediated transformation. CRISPR/Cas9 gene editing enables gene insertion at a precise target site in the genome. In this study the CRISPR/Cas9 approach was modified for intron-targeted insertion by adding an artificial 3′ splicing site upstream of the recombinant gene. Knock-in transgenic rice cell lines containing the recombinant GFP gene inserted in intron 1 of αAmy3 were generated. The endogenous αAmy3 promoter regulated recombinant gene expression and the αAmy3 signal peptide directed secretion of the recombinant GFP protein into the culture medium. In addition, the recombinant GFP protein was localized in amyloplasts, identical to the subcellular localization of endogenous αAmy3 reported previously. This modified CRISPR/Cas9 knock-in approach is simple and highly efficient, and the recombinant gene insertion frequency attained 12.5%. The approach can be applied in the production of pharmaceutical proteins in rice suspension cell cultures. The high efficiency of the GFP reporter gene knock-in method and the maintenance of target gene behavior also make the strategy applicable to endogenous gene functional studies in rice.

Pb103 Regulates Zygote/Ookinete Development in Plasmodium berghei via Double Zinc Finger Domains.

Sexual reproduction of Plasmodium parasites takes place in anopheline mosquitoes, where male and female gametes fuse to form zygotes and then ookinetes. These processes are orchestrated by stage-specific protein expression, which is mediated in part by translational repression. Accumulating evidence shows that RNA binding proteins (RBPs) play crucial roles in these processes. Here, we report the characterization of P. berghei 103 (Pb103), which encodes a protein possessing double zinc finger domains (ZFs), an RBP. Reporter parasites expressing azami green fluorescent protein (AGFP) under the endogenous Pb103 gene promoter (Pb103-AGFP reporter) showed that the AGFP fluorescent signal was detected from gametes to ookinetes, while AGFP mRNA was translationally repressed in female gametocytes. The Pb103-disrupted parasites (Pb103(−)) grew and produced gametocytes with similar efficiencies to those of wild-type parasites. However, no oocysts were formed in mosquitoes fed Pb103(−). An in vitro fertilization assay showed abortion at the zygote stage in Pb103(−), suggesting that Pb103 plays a critical role in zygote/ookinete development. Cross-fertilization assays with Pb103(−) and male- or female-sterile parasites revealed that Pb103 was essential exclusively for female gametes. To identify the domains critical for zygote/ookinete development, transgenic parasites expressing partially deleted Pb103 were generated and assayed for ookinete maturation. As a result, deleting either of two ZFs but not the C-terminal region abolished zygote/ookinete development, highlighting the indispensable roles of ZFs in parasite sexual development, most likely via translational repression.

Abstract 14153: Novel Tools to Decipher the Molecular Regulation of the Mitochondrial Sodium-Calcium Exchanger, NCLX

The precise control of mitochondrial calcium ( m Ca 2+ ) exchange allows the cardiomyocyte to balance the regulation of mitochondrial metabolism against the risk of m Ca 2+ overload and cell death. The mitochondrial sodium-calcium exchanger (NCLX) is a major route of m Ca 2+ efflux and thus is one of the key proteins that govern m Ca 2+ homeostasis. Recent studies have demonstrated that increasing NCLX-dependent m Ca 2+ efflux is a promising therapeutic objective for pathologies characterized by deleterious m Ca 2+ overload such as ischemia-reperfusion injury, heart failure, and neurodegeneration. A substantial barrier to the development of NCLX-based therapies is that no pharmacologic activators of NCLX are currently available, and indeed, very little is known about the fundamental mechanisms that stimulate or inhibit NCLX activity in vivo . Therefore, we generated a plasmid encoding a biotin ligase domain fused to the C-terminus of human NCLX (hNCLX-BioID2HA) and used this construct to perform an in vitro proximity biotinylation screen for the unbiased detection of proteins that interact with NCLX and/or modulate NCLX function. We identified ~120 candidate NCLX-proximal mitochondrial proteins in AC16 cardiomyocytes and ~80 in HeLa cells, including several of unknown function. Twenty-five proteins were detected in both data sets, suggestive of conserved function between these two cell types. Current studies are defining the specific molecular function of these mitochondrial proteins and their effects on NCLX expression, NCLX activity, and overall m Ca 2+ homeostasis. We are also developing a novel NCLX-3xFLAG knock-in mouse model to enable in vivo expression of C-terminal epitope-tagged NCLX under the control of the endogenous murine promoter. We expect this model to aid in overcoming the significant proteomic challenges of manipulating this 13-transmembrane protein. Together, these approaches will help to uncover the post-translational mechanisms that regulate NCLX-dependent m Ca 2+ efflux and to identify new therapeutic targets for heart failure and other diseases characterized by disrupted m Ca 2+ homeostasis.

Rescue of Murine IL-7 Receptor Deficiency with Human IL-7 Receptor Gene Therapy

▪In humans, interleukin 7 (IL-7) receptor (IL-7R) deficiency causes approximately 10% of cases of severe combined immunodeficiency (SCID). IL-7R deficient SCID is a T-B+NK+ SCID and is caused by autosomal recessive deficiency of the IL-7R alpha chain gene (IL7R). IL-7R is a heterodimeric receptor comprised of the alpha chain and the IL-2 receptor common gamma chain (IL2RG). In both mouse and human, IL-7R is a marker of the common lymphoid progenitor cell, and IL-7 signaling leads to STAT5 phosphorylation and proliferation of developing T and B cells. Mice lacking IL7R, Il7r -/-, lack both T and B cells (Peschon, JJ, et al. J Exp Med. 1994). T cells do not progress to TCR beta chain rearrangement and B cell development is halted at the pre-pro-B cell stage. Similar to the mouse, IL-7 signaling in humans is required for T cell receptor beta gene rearrangement and T cell maintenance, however humans lacking IL-7R can develop B cells.A prior attempt to rescue murine IL-7R deficiency utilized a retroviral vector (mouse stem cell virus, MSCV), the MSCV retroviral promoter, and the murine Il7r gene (Jiang, Q, et al. Gene Therapy. 2005). This strategy did restore T cells and had variable restoration of B cells. However, retroviral-based gene addition of Il7r led to a myeloproliferative condition with significant neutrophilia and splenomegaly. Transduced bone marrow cells formed myeloid progenitors in response to IL-7 in vitro. We evaluated a novel gene therapy for IL-7R deficient SCID that utilizes the human IL7R gene. To prevent lineage skewing, we sought to limit ectopic expression of IL7R in non-lymphoid cells by utilizing the endogenous enhancers and promoters of IL7R. These sequences were identified as sites of high sequence conservation across species and DNA accessibility/hypersensitivity (DHS) in human lymphocytes. We are testing these sequences alone or in combination with the constitutive phosphoglycerate kinase promoter (PGK) in VSV-G pseudotyped lentiviral vectors (LV): vPGK_DHS_hIL7R and vDHS_hIL7R. Here we present the first data evaluating the ability of the human IL-7R protein to functionally replace the murine IL-7R protein and the ability of IL7R gene addition to rescue the murine Il7r -/- immunodeficient phenotype in vivo.Transduction of Il7r -/- bone marrow cells with IL7R encoding LV rescued the formation of lymphocyte precursors from murine bone marrow cells in colony forming unit (CFU) assays (pre-B CFU with human IL-7), with the most robust response seen with vPGK_DHS_hIL7R. Mouse bone marrow from Il7r -/- animals transduced ex vivo engrafted in lethally irradiated (8 Gy) Il7r -/-oppositegender recipients and there were no significant aberrations in absolute neutrophil count, hemoglobin or platelet count. Absolute lymphocyte counts in mice receiving transduced Il7r -/-bone marrow cells was higher (mean 2555/μL) than in mice receiving untransduced bone marrow (mean 1410/μL). The proportion of leukocytes that were T cells was 4.2-fold and 9.8-fold higher at 1 and 2 months post-transplant, respectively. B cells were only seen in mice receiving vPGK_DHS_hIL7R: 7.4% of leukocytes versus 1.5% in controls. A reciprocal decrease in the fraction of Gr1+ cells (neutrophils and monocytes) was seen at two months post-transplant in transduced marrow recipients compared to untransduced controls: 36.5% versus 63% Gr1+, respectively. Lymphocyte subsets are being further analyzed, bone marrow and thymic lymphoid precursors assessed, and T and B cell function in response to immunizations are in progress. Further evaluation in human derived IL7R deficient human cells is warranted.For individuals with IL-7R deficient SCID, but no HLA matched hematopoietic stem cell (HSC) donor, there is a difficult choice between the risks of GVHD with a mismatched HSC donor and supportive care with the hope of identifying a matched HSC donor in the future. In immunodeficiencies however age and serious infection are both associated with increased mortality (Pai, SY, et al. NJEM. 2014). This novel approach to IL7R gene replacement has the potential to be a therapeutic and expedient option for those without a matched donor. Additionally, this would be an ideal disorder for HSC conditioning with less toxic, HSC-targeted strategies given gene corrected lymphocytes and progenitors will preferentially expand post-transplant. DisclosuresRivella: Disc Medicine: Consultancy, Membership on an entity's Board of Directors or advisory committees; Keros Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene Corporation: Consultancy; Ionis Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; MeiraGTx: Consultancy, Membership on an entity's Board of Directors or advisory committees; Forma Theraputics: Consultancy; Incyte: Consultancy.

Modeling the Development of SRSF2 Mutated Myeloid Malignancies By CRISPR/Cas9 Mediated Genome Engineering of Primary Human Hematopoietic Stem and Progenitor Cells

Introduction: Acute Myeloid Leukemia (AML) is a malignant disease of the bone marrow that can arise from a premalignant condition called clonal hematopoiesis of indeterminate potential (CHIP). Mutations in Serine and Arginine-rich Splicing Factor 2 (SRSF2) are detected in CHIP and mediate a high risk for AML development. Here we used CRISPR/Cas9-mediated genome engineering to introduce a heterozygous SRSF2P95H mutation into primary human hematopoietic stem and progenitor cells (HSPCs) and investigated its functional consequences using both in vitro and in vivo assays.Methods: We used CRISPR/Cas9 technology to introduce a heterozygous mutant (mut) SRSF2P95H into the endogenous SRSF2 gene locus of healthy cord blood HSPCs. Our approach is based on homologous recombination using DNA repair templates delivered by adeno-associated virus serotype 6 (AAV6) (Figure A). This allows for targeted in-frame integration of mut and/or wildtype (WT) SRSF2 cDNA under the control of the endogenous SRSF2 promoter. Notably, an integrated fluorescent reporter enables the isolation and tracking of heterozygously mutated HSPCs (Figure B). Methylcellulose colony and long-term competition assays of SRSF2 mut and WT HSPCs were performed in vitro. Cells were analyzed by flow cytometry and characterized cytomorphologically. In addition, bulk RNA-seq analyses were performed to characterize differential gene expression and abnormal splicing events. Xenotransplantation into NSG-SGM3 mice was performed in order to assess stem cell characteristics and the in vivo leukemogenic potential of SRSF2 mut HSPCs. Finally, we investigated the mutation-specific effect of the splicing inhibitor Indisulam to determine if SRSF2 mut cells are particularly vulnerable to splicing inhibition.Results: Colony assays (n=9) revealed impaired erythroid and increased monocytic differentiation of SRSF2 mut HSPCs. Quantification of colonies showed a lower frequency of erythroid BFU-E in SRSF2 mut compared to SRSF2 WT HSPCs (mean ± SD; 33.3 ± 12.5% vs. 17.4 ± 10.8%, p=0.00002). In contrast, the frequency of myeloid CFU-M colonies was higher in SRSF2 mut HSPCs compared to SRSF2 WT HSPCs (38.3 ± 7.3% vs. 22.6 ± 6.8%, p = 0.0003) (Figure C). Long-term in vitro competition assays revealed an outgrowth of SRSF2 mut over WT cells in 2 out of 7 donors. Strikingly, after three months of in vitro culture, in one donor, the SRSF2 mut cells developed a blast-like morphology with strong CD34 expression (Figure D). To assess stem cell characteristics and the leukemogenic potential in vivo, we transplanted SRSF2 mut HSPCs from 4 different donors into immunodeficient NSG-SGM3 mice (n=11). SRSF2 mut cells showed a myeloid-skewed engraftment. Cytomorphologic analysis of long-term engrafted SRSF2 mut myeloid cells revealed dysplastic changes such as nuclear abnormalities and extensive cytoplasmic vacuolization. In 4 out of 11 xenografts, human engraftment substantially increased over time with a parallel outgrowth of the SRSF2 mut clone and the appearance of blast-like cells resembling transformation into myeloid leukemia (Figure E). Comparative RNA-seq analysis identified 138 differentially spliced genes, with exon skipping being the dominant altered splicing type. Gene ontology (GO) analysis on differentially expressed genes revealed “Acute Myeloid Leukemia” among the most enriched terms (p-val = 8.2E-07, min FDR = 1.486E-04). When testing the SRSF2-mutation specific effect of the splicing inhibitor Indisulam, SRSF2 mut HSPCs show a significantly lower IC-50 than WT cells (977nM vs. 3574 nM). Strikingly, in competition- and CFU-assays, Indisulam preferentially eradicates SRSF2 mut hematopoietic cells, while sparing WT cells.Conclusion: Using our CRISPR/Cas9 approach, we can successfully introduce heterozygous SRSF2P95H mutants in primary human HSPCs. Mutant SRSF2P95H leads to increased monocytic differentiation, impaired erythroid differentiation, and phenocopy SRSF2P95H driven diseases in patients. Importantly, we show for the first time that the SRSF2 mutation alone is sufficient to induce dysplastic features and even transform healthy human HSPCs into AML-like blasts. Our model allows the identification and therapeutic investigation of specific cellular vulnerabilities caused by SRSF2 mutations and highlights Indisulam as a potential compound to specifically treat individuals carrying a SRSF2 mutation. [Display omitted] DisclosuresEdiriwickrema: Nanosive SAS: Patents & Royalties. Greinix: Novartis: Consultancy; Celgene: Consultancy; Takeda: Consultancy; Sanofi: Consultancy; Therakos: Consultancy. Sill: Astellas: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; AbbVie: Consultancy, Membership on an entity's Board of Directors or advisory committees. Zebisch: Celgene: Consultancy, Honoraria; AbbVie: Consultancy; Novartis: Consultancy. Majeti: BeyondSpring Inc.: Membership on an entity's Board of Directors or advisory committees; CircBio Inc.: Membership on an entity's Board of Directors or advisory committees; Kodikaz Therapeutic Solutions Inc.: Membership on an entity's Board of Directors or advisory committees; Coherus Biosciences: Membership on an entity's Board of Directors or advisory committees; Acuta Capital Partners: Consultancy; Gilead: Patents & Royalties: inventor on a number of patents related to CD47 cancer immunotherapy licensed to Gilead Sciences, Inc.. Reinisch: Pfizer: Consultancy; Celgene: Research Funding.

Introduction and Genetic Correction of Calreticulin Mutations in Human Hematopoietic Stem and Progenitor Cells Sheds Light on MPN Pathogenesis

Introduction:Recurrent mutations in calreticulin (CALR) are present in 70% to 80% of essential thrombocythemia (ET) and primary myelofibrosis (PMF) patients without a JAK2 or MPL mutation. Despite recent advances in understanding mutant CALR, the detailed mechanisms are not fully elucidated, and current knowledge is mainly based on transgenic mouse models or human cancer cell lines. Thus, to more faithfully model MPN pathogenesis, we first aimed to introduce heterozygous type-1 and type-2 CALR mutations into healthy human hematopoietic stem and progenitor cells (HSPCs) via targeted CRISPR/Cas9-mediated gene knock-in (KI) and investigate its impact on HSPC function in vitro and in vivo. Second, we aimed to correct CALR mutations in patient-derived HSPCs to study their dependence on the initial driver event to exert an MPN phenotype.Methods:We used CRISPR/Cas9 to introduce heterozygous CALR mutations into the endogenous gene locus of healthy cord blood-derived HSPCs. Our approach is based on homologous recombination using DNA repair templates delivered by adeno-associated virus serotype 6 (AAV6). Briefly, Cas9-sgRNA ribonucleoprotein (RNP) was used to cut the DNA. Simultaneously AAV6, carrying either a mutation-bearing or a wildtype control cDNA, was co-delivered to allow for targeted in-frame integration. This way, mutant CALR remains under the control of the endogenous promoter. Concurrent integration of a fluorescent reporter downstream of the mutated exon, enabled purification and tracking of modified cells via flow cytometry. Purified CRISPR-modified HSPCs were used for in vitro collagen-based colony-forming assays, proliferation and differentiation assays in liquid culture, and intrafemoral transplantation into immunodeficient NSG mice to assess their pathogenic potential.Results:Our CRISPR/Cas9 KI strategy enabled us to efficiently generate and enrich for heterozygous CALR mutant human HSPCs. Modified cells harbor the mutation at the endogenous CALR locus with intact gene regulatory regions. Correct integration and transcript expression were confirmed on DNA and RNA level by sanger sequencing. Additionally, CALR mutant protein expression was confirmed via immunohistochemistry using a diagnostically approved mutant-specific antibody.Type-1 and type-2 CALR mutations led to TPO-independent growth of CD34 + HSPC-derived cells and a two-fold (p<0.01) increase of megakaryocyte colonies in collagen-based media compared to wildtype control KI. These findings were corroborated by significantly enhanced CD41 + CD42b + megakaryocyte formation of CALR mutant HSPCs upon liquid culture differentiation.When transplanted into sublethally irradiated immunodeficient NSG mice, CALR mutant HSPCs showed robust engraftment in the bone marrow with a myeloid lineage skewing, outcompetition of wildtype cells and increased formation of CALR mutant CD41 + megakaryocyte progenitors.To investigate, if removal of type-1 and type-2 CALR mutations can ameliorate MPNs, we utilized our KI strategy to correct both CALR mutations in MPN patient-derived HSPCs by replacing them with wildtype sequences. A successful correction was confirmed on DNA and RNA level and by the absence of mutant CALR protein. Opposite to the results from introducing CALR mutations, correcting the mutations led to a two-fold decrease in megakaryocyte colony formation. Interestingly this was only seen in ET and post-ET MF samples, whereas primary MF samples were unaffected, underscoring the importance of other secondary genetic driver events in the pathogenesis of primary MF.Conclusion:Our system allows us to investigate human MPN pathogenesis prospectively and shed light on the transforming mechanisms of mutant CALR in primary HSPCs. We could show that CALR mutations prime HSPCs toward the formation of platelet-producing megakaryocytes. Genetic correction of CALR mutations in MPN patient-derived HSPCs revealed a dependence on the oncogenic mutant CALR driver event in ET and post-ET MF patients, opening the possibility of an ex vivo gene correction approach to remove mutant CALR in patient-derived HSPCs . Lastly, since MPN patient-derived cells have notoriously low engraftment potential in mice, our CRISPR/Cas9-engineered CALR mutant model also provides a powerful new strategy to generate MPN xenotransplants with defined genotypes for the evaluation of novel therapies. DisclosuresGreinix: Celgene: Consultancy; Therakos: Consultancy; Takeda: Consultancy; Sanofi: Consultancy; Novartis: Consultancy. Sill: Astellas: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; AbbVie: Consultancy, Membership on an entity's Board of Directors or advisory committees. Zebisch: Novartis: Consultancy; AbbVie: Consultancy; Celgene: Consultancy, Honoraria. Reinisch: Celgene: Research Funding; Pfizer: Consultancy.

Construction of a CRISPR/Cas9-Mediated Genome Editing System in Lentinula edodes

CRISPR/Cas9 genome editing systems have been established in a broad range of eukaryotic species. Herein, we report the first method for genetic engineering in pyogo (shiitake) mushrooms (Lentinula edodes) using CRISPR/Cas9. For in vivo expression of guide RNAs (gRNAs) targeting the mating-type gene HD1 (LeA1), we identified an endogenous LeU6 promoter in the L. edodes genome. We constructed a plasmid containing the LeU6 and glyceraldehyde-3-phosphate dehydrogenase (LeGPD) promoters to express the Cas9 protein. Among the eight gRNAs we tested, three successfully disrupted the LeA1 locus. Although the CRISPR-Cas9–induced alleles did not affect mating with compatible monokaryotic strains, disruption of the transcription levels of the downstream genes of LeHD1 and LeHD2 was detected. Based on this result, we present the first report of a simple and powerful genetic manipulation tool using the CRISPR/Cas9 toolbox for the scientifically and industrially important edible mushroom, L. edodes.