Gene editing of PKLR gene in human hematopoietic progenitors through 5' and 3' UTR modified TALEN mRNA

Pyruvate Kinase Deficiency (PKD) is a rare erythroid metabolic disease caused by mutations in the PKLR gene, which encodes the erythroid specific Pyruvate Kinase enzyme. Erythrocytes from PKD patients show an energetic imbalance and are susceptible to hemolysis. Gene editing of hematopoietic stem cells (HSCs) would provide a therapeutic benefit and improve safety of gene therapy approaches to treat PKD patients. In previous studies, we established a gene editing protocol that corrected the PKD phenotype of PKD-iPSC lines through a TALEN mediated homologous recombination strategy. With the goal of moving toward more clinically relevant stem cells, we aim at editing the PKLR gene in primary human hematopoietic progenitors and hematopoietic stem cells (HPSCs). After nucleofection of the gene editing tools and selection with puromycin, up to 96% colony forming units showed precise integration. However, a low yield of gene edited HPSCs was associated to the procedure. To reduce toxicity while increasing efficacy, we worked on i) optimizing gene editing tools and ii) defining optimal expansion and selection times. Different versions of specific nucleases (TALEN and CRISPR-Cas9) were compared. TALEN mRNAs with 5' and 3' added motifs to increase RNA stability were the most efficient nucleases to obtain high gene editing frequency and low toxicity. Shortening ex vivo manipulation did not reduce the efficiency of homologous recombination and preserved the hematopoietic progenitor potential of the nucleofected HPSCs. Lastly, a very low level of gene edited HPSCs were detected after engraftment in immunodeficient (NSG) mice. Overall, we showed that gene editing of the PKLR gene in HPSCs is feasible, although further improvements must to be done before the clinical use of the gene editing to correct PKD. Institutode Investigacio ' n Sanitaria de la Fundacio '

Gene editing of PKLR gene in human hematopoietic progenitors through 5' and 3' UTR modified TALEN mRNA

TheauthorswouldliketothankMiguelA.MartinforthecarefulmaintenanceofNSGmice,andRebecaSa ́nchezandOmairaAlberquillafortheirtechnicalassistanceinflowcytometry.TheauthorsalsothankFundacio ́n Botı ́n forpromotingtranslationalresearchattheHemato-poieticInnovativeTherapiesDivisionoftheCIEMATPyruvate Kinase Deficiency (PKD) is a rare erythroid metabolic disease caused by mutations in the PKLR gene, which encodes the erythroid specific Pyruvate Kinase enzyme. Erythrocytes from PKD patients show an energetic imbalance and are susceptible to hemolysis. Gene editing of hematopoietic stem cells (HSCs) would provide a therapeutic benefit and improve safety of gene therapy approaches to treat PKD patients. In previous studies, we established a gene editing protocol that corrected the PKD phenotype of PKD-iPSC lines through a TALEN mediated homologous recombination strategy. With the goal of moving toward more clinically relevant stem cells, we aim at editing the PKLR gene in primary human hematopoietic progenitors and hematopoietic stem cells (HPSCs). After nucleofection of the gene editing tools and selection with puromycin, up to 96% colony forming units showed precise integration. However, a low yield of gene edited HPSCs was associated to the procedure. To reduce toxicity while increasing efficacy, we worked on i) optimizing gene editing tools and ii) defining optimal expansion and selection times. Different versions of specific nucleases (TALEN and CRISPR-Cas9) were compared. TALEN mRNAs with 5' and 3' added motifs to increase RNA stability were the most efficient nucleases to obtain high gene editing frequency and low toxicity. Shortening ex vivo manipulation did not reduce the efficiency of homologous recombination and preserved the hematopoietic progenitor potential of the nucleofected HPSCs. Lastly, a very low level of gene edited HPSCs were detected after engraftment in immunodeficient (NSG) mice. Overall, we showed that gene editing of the PKLR gene in HPSCs is feasible, although further improvements must to be done before the clinical use of the gene editing to correct PKD.S

Generation of a High Number of Healthy Erythroid Cells from Gene-Edited Pyruvate Kinase Deficiency Patient-Specific Induced Pluripotent Stem Cells

Pyruvate kinase deficiency (PKD) is a rare erythroid metabolic disease caused by mutations in the PKLR gene. Erythrocytes from PKD patients show an energetic imbalance causing chronic non-spherocytic hemolytic anemia, as pyruvate kinase defects impair ATP production in erythrocytes. We generated PKD induced pluripotent stem cells (PKDiPSCs) from peripheral blood mononuclear cells (PB-MNCs) of PKD patients by non-integrative Sendai viral vectors. PKDiPSCs were gene edited to integrate a partial codon-optimized R-type pyruvate kinase cDNA in the second intron of the PKLR gene by TALEN-mediated homologous recombination (HR). Notably, we found allele specificity of HR led by the presence of a single-nucleotide polymorphism. High numbers of erythroid cells derived from gene-edited PKDiPSCs showed correction of the energetic imbalance, providing an approach to correct metabolic erythroid diseases and demonstrating the practicality of this approach to generate the large cell numbers required for comprehensive biochemical and metabolic erythroid analyses.info:eu-repo/semantics/publishedVersio

Involvement of KSRP in the post-transcriptional regulation of human iNOS expression–complex interplay of KSRP with TTP and HuR

We purified the KH-type splicing regulatory protein (KSRP) as a protein interacting with the 3′-untranslated region (3′-UTR) of the human inducible nitric oxide (iNOS) mRNA. Immunodepletion of KSRP enhanced iNOS 3′-UTR RNA stability in in vitro-degradation assays. In DLD-1 cells overexpressing KSRP cytokine-induced iNOS expression was markedly reduced. In accordance, downregulation of KSRP expression increases iNOS expression by stabilizing iNOS mRNA. Co-immunoprecipitations showed interaction of KSRP with the exosome and tristetraprolin (TTP). To analyze the role of KSRP binding to the 3′-UTR we studied iNOS expression in DLD-1 cells overexpressing a non-binding mutant of KSRP. In these cells, iNOS expression was increased. Mapping of the binding site revealed KSRP interacting with the most 3′-located AU-rich element (ARE) of the human iNOS mRNA. This sequence is also the target for HuR, an iNOS mRNA stabilizing protein. We were able to demonstrate that KSRP and HuR compete for this binding site, and that intracellular binding to the iNOS mRNA was reduced for KSRP and enhanced for HuR after cytokine treatment. Finally, a complex interplay of KSRP with TTP and HuR seems to be essential for iNOS mRNA stabilization after cytokine stimulation

Impaired Embryonic Development in Mice Overexpressing the RNA-Binding Protein TIAR

TIA-1-related (TIAR) protein is a shuttling RNA-binding protein involved in several steps of RNA metabolism. While in the nucleus TIAR participates to alternative splicing events, in the cytoplasm TIAR acts as a translational repressor on specific transcripts such as those containing AU-Rich Elements (AREs). Due to its ability to assemble abortive pre-initiation complexes coalescing into cytoplasmic granules called stress granules, TIAR is also involved in the general translational arrest observed in cells exposed to environmental stress. However, the in vivo role of this protein has not been studied so far mainly due to severe embryonic lethality upon tiar invalidation.Journal ArticleResearch Support, Non-U.S. Gov'tSCOPUS: ar.jinfo:eu-repo/semantics/publishe

Temporally Regulated Traffic of HuR and Its Associated ARE-Containing mRNAs from the Chromatoid Body to Polysomes during Mouse Spermatogenesis

International audienceBACKGROUND: In mammals, a temporal disconnection between mRNA transcription and protein synthesis occurs during late steps of germ cell differentiation, in contrast to most somatic tissues where transcription and translation are closely linked. Indeed, during late stages of spermatogenesis, protein synthesis relies on the appropriate storage of translationally inactive mRNAs in transcriptionally silent spermatids. The factors and cellular compartments regulating mRNA storage and the timing of their translation are still poorly understood. The chromatoid body (CB), that shares components with the P. bodies found in somatic cells, has recently been proposed to be a site of mRNA processing. Here, we describe a new component of the CB, the RNA binding protein HuR, known in somatic cells to control the stability/translation of AU-rich containing mRNAs (ARE-mRNAs). METHODOLOGY/PRINCIPAL FINDINGS: Using a combination of cell imagery and sucrose gradient fractionation, we show that HuR localization is highly dynamic during spermatid differentiation. First, in early round spermatids, HuR colocalizes with the Mouse Vasa Homolog, MVH, a marker of the CB. As spermatids differentiate, HuR exits the CB and concomitantly associates with polysomes. Using computational analyses, we identified two testis ARE-containing mRNAs, Brd2 and GCNF that are bound by HuR and MVH. We show that these target ARE-mRNAs follow HuR trafficking, accumulating successively in the CB, where they are translationally silent, and in polysomes during spermatid differentiation. CONCLUSIONS/SIGNIFICANCE: Our results reveal a temporal regulation of HuR trafficking together with its target mRNAs from the CB to polysomes as spermatids differentiate. They strongly suggest that through the transport of ARE-mRNAs from the CB to polysomes, HuR controls the appropriate timing of ARE-mRNA translation. HuR might represent a major post-transcriptional regulator, by promoting mRNA storage and then translation, during male germ cell differentiation

Breast Cancer Epigenetics: From DNA Methylation to microRNAs

Both appropriate DNA methylation and histone modifications play a crucial role in the maintenance of normal cell function and cellular identity. In cancerous cells these “epigenetic belts” become massively perturbed, leading to significant changes in expression profiles which confer advantage to the development of a malignant phenotype. DNA (cytosine-5)-methyltransferase 1 (Dnmt1), Dnmt3a and Dnmt3b are the enzymes responsible for setting up and maintaining DNA methylation patterns in eukaryotic cells. Intriguingly, DNMTs were found to be overexpressed in cancerous cells, which is believed to partly explain the hypermethylation phenomenon commonly observed in tumors. However, several lines of evidence indicate that further layers of gene regulation are critical coordinators of DNMT expression, catalytic activity and target specificity. Splice variants of DNMT transcripts have been detected which seem to modulate methyltransferase activity. Also, the DNMT mRNA 3′UTR as well as the coding sequence harbors multiple binding sites for trans-acting factors guiding post-transcriptional regulation and transcript stabilization. Moreover, microRNAs targeting DNMT transcripts have recently been discovered in normal cells, yet expression of these microRNAs was found to be diminished in breast cancer tissues. In this review we summarize the current knowledge on mechanisms which potentially lead to the establishment of a DNA hypermethylome in cancer cells

Post-transcriptional control during chronic inflammation and cancer: a focus on AU-rich elements

A considerable number of genes that code for AU-rich mRNAs including cytokines, growth factors, transcriptional factors, and certain receptors are involved in both chronic inflammation and cancer. Overexpression of these genes is affected by aberrations or by prolonged activation of several signaling pathways. AU-rich elements (ARE) are important cis-acting short sequences in the 3′UTR that mediate recognition of an array of RNA-binding proteins and affect mRNA stability and translation. This review addresses the cellular and molecular mechanisms that are common between inflammation and cancer and that also govern ARE-mediated post-transcriptional control. The first part examines the role of the ARE-genes in inflammation and cancer and sequence characteristics of AU-rich elements. The second part addresses the common signaling pathways in inflammation and cancer that regulate the ARE-mediated pathways and how their deregulations affect ARE-gene regulation and disease outcome