scholarly journals A Functional Analysis of SNP on Anthracycline-Induced Cardiotoxicity in a Uniform Genetic Background Using CRISPR/Cas9 Mediated Single-Base-Edited iPSCs

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 5748-5748
Author(s):  
Takayuki Nakamura ◽  
Shinichi Mizuno ◽  
Hidetoshi Ozawa ◽  
Yoshitaka Yamasaki ◽  
Shuki Oya ◽  
...  
Keyword(s):  
Functional Analysis ◽  
Genome Editing ◽  
Genetic Background ◽  
Conflicts Of Interest ◽  
Lentiviral Vector ◽  
Nicotinamide Adenine Dinucleotide Phosphate ◽  
Target Site ◽  
Expression Vectors ◽  
Single Base

[Introduction] Anthracycline-induced cardiotoxicity (ACT) has been a major problem in leukemia therapy, and reported to be associated with several candidates of single nucleotide polymorphisms (SNPs). C242T polymorphism (rs4673) in the cytochrome b-245 alpha chain (CYBA) gene, which encodes superoxide-generating nicotinamide adenine dinucleotide phosphate (NADPH) oxidase light chain subunit, is focused on as one of ACT related SNPs. Several clinical studies indicated that rs4673 T allele increases the risk of ACT, however, it is unclear how rs4673 affects ACT. Here, we established a series of induced pluripotent stem cells (iPSCs) with a genotype of C/C, T/T or C/T of rs4673 in a uniform genetic background by using CRISPR/ Cas9, and evaluated the effect of each SNP genotype on ACT in vitro. [Methods and Results] To edit the SNP site using CRISPR/Cas9, three guide RNAs (gRNAs) were designed to induce double strand break (DSB) near the target site. Cas9 nuclease and gRNA expression vectors were transfected into HEK293T cells using jetPEI (Polyplus-transfection SAS), and the genome editing activity was assessed by T7 endonucleaseⅠ(T7E1) assay to select more efficient gRNA for cleavage near the target site. The indel frequencies of sgRNAs 1,2 and 3 were 29.4%, 25.6%, and 18.6%, respectively. Furthermore, oligo-DNA (100 nt) to edit single-base in the SNP site was prepared. To generate Cas9/gRNA ribonucleoprotein (RNP) complexes, 1 μg of Cas9 protein and 0.2 μg of gRNA molecules were mixed. RNP complexes and 50 pmol of oligo-DNA were electroporated into human iPSC 201B7cells (Riken BioResource Center) using Neon electroporation device (Invitrogen),single-base-edited iPSCs (C/C and T/T from C/T genotype in rs4673) were established by homology-directed repair (HDR). Successful HDR events were verified by Sanger sequencing. These established iPSCs were differentiated into cardiomyocytes using PSC Cardiomyocyte Differentiation Kit (Thermo Fisher Scientific) in vitro. To facilitate nascent mesoderm induction, TBX6 was expressed transiently by a tetracycline-inducible lentiviral vector. Differentiated cardiomyocytes were revealed to be contracting and be positive for NK2 transcription factor related locus 5 (NKX2-5) and troponin T2 (TNNT2) by immunofluorescence staining.In order to evaluate the ACT depending on the difference of single base in rs4673, doxorubicin (DOX)was administered to each edited iPSC-derived cardiomyocyte. NADP+/NADPH before and after administration of DOX (1μM) in C/C cardiomyocytes was 1±0.18 and 0.93±0.14, respectively. On the other hand, it was 1±0.07 and 1.29±0.03 in T/T cardiomyocytes (p<0.05, Welch test). T/T cardiomyocytes activated NADP+/NADPH cycling after administration of DOX, while C/C cardiomyocytes did not. [Conclusion] SNPs are a factor of genetic diversity among individuals, and have been revealed to be associated with various traits and diseases by Genome-Wide Association Study (GWAS). However, the diverse genetic backgrounds often hamper to analyze the influence of each SNP on the trait. In this study, we analyzed the effects of rs4673 on ACT in a uniform genetic background by using CRISPR/Cas9 edited iPSCs. Our study showed that T/T cardiomyocytes activated NADP+/NADPH cycling after administration of DOX. These results suggest that rs4673T allele may increase the risk of ACT. Genome editing mediated single-base-alteration will provide us a novel system for functional analysis of SNPs. Disclosures No relevant conflicts of interest to declare.

scholarly journals A Novel Type V CRISPR System with Potential for Genome Editing in the Liver

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 1862-1862
Author(s):  
Gregory J. Cost ◽  
Morayma Temoche-Diaz ◽  
Janet Mei ◽  
Cristina N. Butterfield ◽  
Christopher T. Brown ◽  
...  
Keyword(s):  
Amino Acid ◽  
Genome Editing ◽  
Mammalian Cells ◽  
Conflicts Of Interest ◽  
Attractive Alternative ◽  
Vitro Assay ◽  
Crispr System ◽  
Type V

Abstract RNA guided CRISPR genome editing systems can make specific changes to the genomes of mammalian cells and have the potential to treat a range of diseases including those that can be addressed by editing hepatocytes. Attempts to edit the liver in vivo have relied almost exclusively on the Cas9 nucleases derived from the bacteria S treptococcus pyogenes or Staphylococcus aureus to which humans are commonly exposed. Pre-existing immunity to both these proteins has been reported in humans which raises concerns about their in vivo application. In silico analysis of a large metagenomics database followed by testing in mammalian cells in culture identified MG29-1, a novel CRISPR system which is a member of the Type V family but exhibits only 41 % amino acid identity to Francisella tularensis Cas12a/cpf1. MG29-1 is a 1280 amino acid RNA programmable nuclease that utilizes a single guide RNA comprised of a 22 nucleotide (nt) constant region and a 20 to 25 nt spacer, recognizes the PAM KTTN (predicted frequency 1 in 16 bp) and generates staggered cuts. MG29-1 was derived from a sample taken from a hydrothermal vent and it is therefore unlikely that humans will have developed pre-existing immunity to this protein. A screen for sgRNA targeting serum albumin in the mouse liver cell line Hepa1-6 identified 6 guides that generated more than 80% INDELS. The MG29-1 system was optimized for in vivo delivery by screening chemical modifications to the guide that improve stability in mammalian cell lysates while retaining or improving editing activity. Two lead guide chemistries were evaluated in mice using MG29-1 mRNA and sgRNA packaged in lipid nanoparticles (LNP). Three days after a single IV administration on-target editing was evaluated in the liver by Sanger sequencing. The sgRNA that was the most stable in the in vitro assay generated INDELS that ranged from 20 to 25% while a sgRNA with lower in vitro stability failed to generate detectable INDELs. The short sgRNA and small protein size compared to spCas9 makes MG29-1 an attractive alternative to spCas9 for in vivo editing applications. Evaluation of the potential of MG29-1 to perform gene knockouts and gene additions via non-homologous end joining is ongoing. Disclosures No relevant conflicts of interest to declare.

Fli1 Expression Level Affects Megakaryopoiesis and Thrombopoiesis: More Is Not Always a Bad Thing

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 3876-3876
Author(s):  
Karen K Vo ◽  
Danuta Jadwiga Jarocha ◽  
Randolph B Lyde ◽  
Spencer K. Sullivan ◽  
Deborah French ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Genome Editing ◽  
Platelet Activation ◽  
Transgene Expression ◽  
Conflicts Of Interest ◽  
Function Analysis ◽  
Ipsc Line ◽  
Protein Levels ◽  
Platelet Disorder

Abstract Friend leukemia integration 1 (FLI1) is a critical transcription factor responsible for terminal megakaryocyte differentiation. This transcription factor is amongst the genes missing in the inherited disorder Jacobsen syndrome, resulting from a hemizygous deletion on chromosome 11q. The deletion causes dysmegakaryopoiesis and macrothrombocytopenia termed Paris Trousseau syndrome (PTSx). Also, FLI1 mutations in its DNA-binding domain region results in thrombocytopenia in affected patients. We examined induced pluripotent stem cell- (iPSC) derived megakaryocytes (iMegs) to determine if the platelet disorder observed in PTSx could be replicated and if varied levels of Fli1 expression affected megakaryopoiesis and thrombopoiesis. Beginning with a normal control (WT) iPSC line, genome editing was performed to generate three lines with: 1) one copy of FLI1 disrupted (FLI1+/-), 2) hemizygous transgene expression of Fli1 in the adeno-associated virus site 1 (AAVS1) safe harbor locus using a megakaryocyte-specific GP1balpha promoter (WT-overexpressing line, WT-OE), and 3) homozygous transgene expression in AAVS1 (WT-OE2). Additionally, we established an iPSC line from a PTSx patient and edited this line for a similar hemizygous transgene expression of FLI1 (PTSx-OE). Data described here are summarized in the tables below. We confirm our genome editing strategies by examining mRNA and protein levels of iMegs and found WT-OE and WT-OE2 iMegs have ~3X higher mRNA and ~7X higher protein levels than WT iMegs. FLI1+/- and PTSx iMegs both have lower mRNA and ~0.5X the protein levels of WT iMegs, while PTSx-OE iMegs had comparable levels of mRNA and protein as WT-OE iMegs. Megacult colony assays showed WT-OE and WT-OE2 yielded more CFU-Megs than WT HPCs (p²0.01, p²0.05, respectively). PTSx and FLI1+/- had much less CFU-Megs compared to PTSx-OE and WT (p²0.0001). After growth in liquid culture, WT-OE and -OE2 lines had an increase in iMeg numbers (p=0.29, p²0.01) while FLI1+/- and PTSx iMeg numbers were 43% and 10% that of the WT control (p²0.01, p²0.001). PTSx-OE iMeg numbers were comparable to WT. Surface marker CD41 and CD42b levels were increased compared to WT iMegs in the WT-OE and -OE2 iMegs (p=0.25, p²0.05) and less on FLI1+/- and PTSx iMegs (p²0.01, p²0.05). PTSx-OE iMegs were normal compared to WT, but p²0.05 vs. PTSx. Infused CD41+CD42b+ iMegs into NSG mice showed a trend toward same yield of platelets in the OE lines and lower yield in FLI1+/- and PTSx lines compared to WT. However, when calculations were made from HPCs rather than iMegs infused, the FLI1+/- and PTSx iMegs generated significantly less number of released platelets (p²0.05, p²0.01). The half-life of WT-OE and -OE2 released platelets were increased at 4 and 10 hours compared to WT, whereas FLI1+/- and PTSx released platelets have lower half-lives of 2 hours and 0.5 hours, respectively. These decreased platelet half-lives are due to the majority of platelets being defective and cleared at a higher rate. This was corrected to WT in the PTSx-OE-released platelets. Both in vitro iMegs and in vivo released platelets within the murine blood were assessed for function. Analysis of activation by thrombin was performed via FACS for PAC-1 binding and cell surface P-selectin levels, which are both indicators of platelet activation. Initial studies show no difference between WT and the WT-OE and WT-OE2 lines, but FLI1+/- platelet activation was slightly impaired. In summary, we show that studies of iMegs with decreased Fli1 levels replicate many of the clinical features previously described: less iMegs, lower CD41 and CD42b density with less platelets released while having shorter half-lives. On the other hand, increased Fli1 levels resulted in higher number of iMegs with more surface antigen density and released platelets with increased half-lives. Based on overexpression studies of other transcription factors during megakaryopoiesis, such as GATA1, the expectation would have been that the excess of Fli1 in the OE lines would have also resulted in defects in megakaryopoiesis and thrombopoiesis, but instead improvements were seen. The basis for this difference when overexpressing different transcriptional factors needs further analysis. That high Fli1 levels enhance iMeg yield with maintained numbers and function of released platelets may be of value for generating platelets for clinical use from in vitro grown Megs. Figure 1 Figure 1. Figure 2 Figure 2. Disclosures No relevant conflicts of interest to declare.

scholarly journals Robust Genome Editing of Single-Base PAM Targets with Engineered ScCas9 Variants

2019 ◽  
Author(s):  
Pranam Chatterjee ◽  
Noah Jakimo ◽  
Joseph M. Jacobson
Keyword(s):  
Genome Editing ◽  
Streptococcus Pyogenes ◽  
Sequence Space ◽  
Target Site ◽  
Gene Repression ◽  
Single Base ◽  
Base Editing ◽  
Homology Directed Repair ◽  
Protospacer Adjacent Motif

Programmable CRISPR enzymes are powerful and versatile tools for genome editing. They, however, require a specific protospacer adjacent motif (PAM) flanking the target site, which constrains the accessible sequence space for position-specific genome editing applications, such as base editing and homology-directed repair. For example, the standard Cas9 from Streptococcus pyogenes requires a PAM sequence of 5’-NGG-3’ downstream of its RNA-programmed target. Recently, three separate Cas9 enzymes (xCas9-3.7, SpCas9-NG, and ScCas9) have been independently engineered or discovered to reduce the PAM specificity to a single guanine (G) nucleotide, thus greatly expanding the number of targetable sequences. In this study, we have employed motifs from closely-related orthologs to engineer and optimize ScCas9 to exhibit enhanced genome editing and higher fidelity. Our engineered variants demonstrate superior activity within gene repression and nucleolytic contexts and possess effective base editing capabilities.

Hypoxia-Inducible Factor (HIF)-1a Is A Therapeutic Target in Myeloma-Induced Angiogenesis and Bone Destruction in Vivo.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2947-2947
Author(s):  
Marina Bolzoni ◽  
Paola Storti ◽  
Gaetano Donofrio ◽  
Irma Airoldi ◽  
Daniela Guasco ◽  
...  
Keyword(s):  
Mouse Models ◽  
Therapeutic Target ◽  
Conflicts Of Interest ◽  
Lentiviral Vector ◽  
Hypoxia Inducible Factor ◽  
Bone Destruction ◽  
Tumor Burden ◽  
Control Vector

Abstract Abstract 2947 It has been previously reported that bone marrow (BM) microenvironment is hypoxic in multiple myeloma (MM) patients and that hypoxia inducible factor (HIF)-1α is overexpressed by MM cells. However, the potential role of HIF-1α as a therapeutic target in MM is not known and is currently under investigation. In this study we explored the effect of persistent HIF-1α inhibition by expression of a lentivirus shRNA pool in MM cells on proliferation, survival and transcriptional and pro-angiogenic profiles of MM cells either in vitro or in vivo in mouse models. A HIF-1α Lentivirus shRNA pool was used for HIF-1α stable knock-down in human myeloma cell lines (HMCL)s and the pKLO.1 lentiviral vector was used as the empty control vector. HMCLs were infected and then selected with puromycin. Selected clones were screened for HIF-1α, HIF-1β, HIF-2α and HIF-3α. The transcriptional profiles were evaluated in the HMCL JJN3 cells transduced with shRNA forHIF-1α (JJN3-anti-HIF-1α) and on those infected with the control vector pKLO.1 (JJN3-pKLO.1) by U133 Plus2.0 Arrays (Affymetrix®) either in hypoxic or normoxic conditions. Microarray data were further validated by quantitative real time PCR and by ELISA assays for protein levels. Finally the effect of HIF-1α inhibition in MM cells was assessed in vivo in NOD/SCID mice both in subcutaneous and intratibial models. Together with tumor volume and weight, microvascular density was evaluated by CD34 immunostaining. Cortical bone thickness was determined by microQcT in the intratibial mouse model. Among the genes significantly modulated by HIF-1α inhibition (327 and 361 genes in hypoxic and normoxic condition, respectively), we found that the pro-angiogenic molecules VEGF, IL8, IL10, CCL2, CCL5, MMP9 were down-regulated by HIF-1α inhibition. Interestingly some pro-osteoclastogenic cytokines were also inhibited including IL-7 and CCL3/MIP-1α. In the in vivo mouse models, we found that mice, injected either subcutaneously or intratibially with JJN3-anti-HIF-1α, showed a dramatic reduction in the weight and volume of the tumor burden compared to mice inoculated with the JJN3-pKLO.1. A significant reduction in the number of vessels X field and VEGF immunostaining were observed in both mouse models. Moreover in the intratibial experiments HIF-1α inhibition significantly blocked MM-induced bone destruction. Overall our data indicate that HIF-1α suppression in myeloma cells significantly blocks MM-induced angiogenesis and reduces the MM tumor burden and bone destruction in vivo suggesting that HIF-1α is a potential therapeutic target in MM. Disclosures: No relevant conflicts of interest to declare.

scholarly journals The VWRPY Domain Is Essential for RUNX1 Function in Hematopoietic Progenitor Cell Maturation and Megakaryocyte Differentiation

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1319-1319
Author(s):  
Halah Alkadi ◽  
David McKellar ◽  
Tao Zhen ◽  
Tatiana Karpova ◽  
Lisa J Garrett ◽  
...  
Keyword(s):  
Genome Editing ◽  
Conflicts Of Interest ◽  
Hematopoietic Progenitor Cell ◽  
In Vivo Model ◽  
Transactivation Domain ◽  
Hematopoietic Stem ◽  
Platelet Disorder ◽  
The Impact ◽  
Runx1 Mutation

Abstract RUNX1 is a transcription factor essential during definitive hematopoiesis. Germline mutations in RUNX1 results in a disorder called Familial Platelet Disorder with Associated Myeloid Malignancy (FPDMM). FPDMM patients have abnormal bleeding due to reduced platelet count and/or function. Importantly, 20-60% of the FPDMM patients develop hematological malignancies, which are mainly myeloid. Reported RUNX1 mutations in FPDMM families are mostly clustered in the N-terminal runt domain and the C-terminal transactivation domain. Recently, three mutations have been reported at or near the end of the C-terminal repression domain, the VWRPY motif. However, the mechanism behind the VWRPY motif involvement in the FPDMM pathogenesis has not been studied. Interestingly, these VWRPY-mutated RUNX1 proteins still have intact runt and transactivation domains, but patients still show FPDMM phenotype. Here, we evaluate the functional defects of a RUNX1 mutation, L472fsX, which was reported in a FPDMM family, using three different experimental models. Our study is aimed to unravel the significance of the VWRPY motif in FPDMM pathogenesis. The RUNX1 L472fsX mutation is caused by a GC insertion upstream of the VWRPY motif. The mutation results in a frameshift and a run on protein for an additional 123 amino acids. The frameshift abolishes the VWRPY motif, which is responsible for the binding between RUNX1 and a co-repressor protein, TLE1. As expected, from both FRET and co-IP assays, the mutated RUNX1 lost binding with TLE1. Interestingly, we observed increased binding between the mutated RUNX1 and its co-factor CBFβ in the FRET assay, as compared to the wildtype RUNX1. Furthermore, in reporter assays we found that TLE1 failed to repress the expression of a RUNX1 target, M-CSFR promoter, when co-transfected with the mutated RUNX1, which is contrary to what has been seen with wildtype RUNX1. Consistent with increased binding between the mutated RUNX1 and CBFβ in the FRET assay, co-transfecting mutant RUNX1 and CBFβ resulted in a significant increase of M-CSFR promoter expression as compared to wildtype RUNX1 with CBFβ. For another RUNX1 target, Hmga2, wildtype RUNX1 and CBFβ decreased Hmga2 expression, which could be restored by adding TLE1. Transfected mutant RUNX1 and CBFβ also decreased Hmga2 expression, but TLE1 could not restore Hmga2 expression when co-transfected with the mutant RUNX1. These findings suggest that the VWRPY-disrupting L472fsX mutation leads to the loss of binding between mutant RUNX1 and TLE1, which in turn resulted in defective repression of the RUNX1 activity by TLE1. To assess for hematopoietic defects in the FPDMM patients with the L472fsX mutation, blood cells from two family members were reprogrammed to induced pluripotent stem cells (iPSCs). Similar to previous studies, iPSCs from these patients gave rise to fewer megakaryocyte progenitors and mature megakaryocytes during in vitro differentiation. In addition, these FPDMM iPSCs showed decrease in hematopoietic stem cell (HSCs) maturation and differentiation to progenitors. The L472fsX mutation in the iPSCs was then corrected by genome editing using zinc finger nuclease. Importantly, the hematopoietic defects of the FPDMM iPSCs mentioned above were rescued after mutation correction. Overall, the findings in these iPSCs differentiation assays showed that the VWRPY motif is essential for RUNX1 activity in megakaryocytes differentiation. In addition, the VWRPY motif is important for HSCs maturation and differentiation to progenitors. To evaluate the impact of this VWRPY-deletion mutation on hematopoiesis in an in vivo model, CRISPR-mediated genome editing was used to generate mice with frameshift mutations that remove the VWRPY domain. Preliminary observations showed that the mutant mice have minor defects in the peripheral blood. More data on this mouse model will be presented at the meeting. In conclusion, we present a novel RUNX1 mutation (L472fsX) with unique hematopoietic defect that has not been reported previously in FPDMM. Our findings imply the significance of the VWRPY motif in megakaryopoiesis, as well as HSCs maturation and differentiation. Disclosures No relevant conflicts of interest to declare.

scholarly journals AddTag, a two-step approach with supporting software package that facilitates CRISPR/Cas-mediated precision genome editing

2021 ◽  
Author(s):  
Thaddeus D Seher ◽  
Namkha Nguyen ◽  
Diana Ramos ◽  
Priyanka Bapat ◽  
Clarissa J Nobile ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Genome Editing ◽  
Binding Sites ◽  
Software Package ◽  
Gene Editing ◽  
Target Site ◽  
Genomic Features ◽  
Single Base ◽  
Gene Complementation ◽  
Single Base Pair

Abstract CRISPR/Cas-induced genome editing is a powerful tool for genetic engineering, however targeting constraints limit which loci are editable with this method. Since the length of a DNA sequence impacts the likelihood it overlaps a unique target site, precision editing of small genomic features with CRISPR/Cas remains an obstacle. We introduce a two-step genome editing strategy that virtually eliminates CRISPR/Cas targeting constraints and facilitates precision genome editing of elements as short as a single base-pair at virtually any locus in any organism that supports CRISPR/Cas-induced genome editing. Our two-step approach first replaces the locus of interest with an “AddTag” sequence, which is subsequently replaced with any engineered sequence, and thus circumvents the need for direct overlap with a unique CRISPR/Cas target site. In this study, we demonstrate the feasibility of our approach by editing transcription factor binding sites within Candida albicans that could not be targeted directly using the traditional gene editing approach. We also demonstrate the utility of the AddTag approach for combinatorial genome editing and gene complementation analysis, and we present a software package that automates the design of AddTag editing.

scholarly journals Functional Dissection of Chromosome 7q Loss and Haploinsufficient Gene Discovery Using iPSC Models of MDS

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 524-524
Author(s):  
Andriana Kotini ◽  
Jeffrey J Delrow ◽  
Timothy A. Graubert ◽  
Stephen Nimer ◽  
Eirini P Papapetrou
Keyword(s):  
Genome Editing ◽  
Conflicts Of Interest ◽  
Gene Discovery ◽  
Patient Specific ◽  
Telomeric Region ◽  
Loss Of Function ◽  
New Paradigm ◽  
Definitive Evidence ◽  
Chromosomal Deletions

Abstract Somatic loss of one copy of the long arm of chromosome 7 [del(7q)] is a characteristic cytogenetic abnormality in MDS and other myeloid malignancies, well-recognized for decades and associated with unfavorable prognosis. Despite compelling clinical evidence that the del(7q) holds a key to the pathogenesis of MDS, the mechanism remains elusive. Gene haploinsufficiency has been proposed as a plausible mechanism, but definitive evidence is lacking. Narrowing down the responsible region and identifying the critical genes has proved challenging with existing approaches. Chr7q deletions are typically very large and modeling in the mouse is problematic, as the genomic regions syntenic to the human chr7q are dispersed into 4 different mouse chromosomes. More than one commonly deleted regions (CDRs) have been proposed by physical mapping studies in patient cells. A handful of genes on chr7q have been implicated through candidate gene approaches and knockout studies in the mouse. However, despite the intense efforts, the contribution of the del(7q) to the disease phenotype and the critical gene or genes on chr7q that mediate it remain unclear. To overcome the limitations of existing tools (primary patient cells, mouse models) to study del(7q)-MDS, we developed a new model harnessing reprogramming and genome editing technologies. First we derived del(7q)-, in parallel with isogenic karyotypically normal induced pluripotent stem cells (iPSCs) from bone marrow hematopoietic cells of two MDS patients. By whole exome sequencing, we were able to identify somatic variants of the MDS clone and show that they are present in the del(7q)-MDS-iPSCs, but not in the karyotypically normal iPSCs, which therefore unambiguously originate from residual normal cells. We used these isogenic and fully genetically characterized patient-derived iPSCs to characterize disease-relevant cellular phenotypes specific to the MDS-iPSCs, which included severely reduced hematopoietic potential and clonogenicity and increased apoptosis. We next found that iPSC clones spontaneously acquiring a second copy of chr7q had an in vitro growth advantage, which enabled us to isolate one clone that completely rescued its hematopoietic differentiation ability upon restoration of a diploid dosage of a ~30Mb chr7q telomeric region. This result provides the first definitive evidence that the del(7q) abnormality confers a profound loss of hematopoietic potential and that this defect is mediated through reduced dosage, consistent with haploinsufficiency of one or more genes. To further narrow down the critical region, we developed genome editing technologies to engineer large chromosomal deletions for the first time in human cells. Combining gene targeting with a modified Cre-loxP approach and the CRISPR/Cas9 endonuclease technology, we were able to generate a panel of 12 iPSC lines harboring hemizygous deletions of various defined segments spanning the entire long arm of chr7. By asking which of them recapitulate the MDS hematopoietic phenotype, we were able to “functionally map” the critical segment in a region spanning cytobands q32.3 - q36.1. To identify critical gene(s) on chr7q, we designed a phenotype-rescue screen. We selected 62 candidate haploinsufficient genes on the basis of significantly reduced expression in del(7q)- compared to isogenic normal iPSCs. We constructed a barcoded lentiviral library of these ORFs and performed a pooled library screen for rescue of hematopoiesis in del(7q)-MDS-iPSCs, i.e. enrichment in CD45+ hematopoietic progenitors. We selected the top 6 genes within our region that were found recurrently enriched in at least 2 independent experiments. Four of them could be individually validated: dosage complementation partially rescued hematopoiesis and knockdown studies mimicking haploinsufficiency (50% knockdown) in normal primary CD34+ hematopoietic progenitor cells had a detrimental effect in hematopoiesis. The four genes include EZH2 and LUC7L2 – two genes found to harbor recurrent heterozygous loss-of-function mutations in MDS – as well as two genes with no previously known role in MDS, located in close genomic proximity to the former two. This approach, constituting a new paradigm of functional human genetics with patient-specific iPSCs, can be more broadly applicable to the study of the phenotypic consequences of segmental chromosomal deletions and to haploinsufficient gene discovery. Disclosures No relevant conflicts of interest to declare.

scholarly journals Introduction of Two Simultaneous Mutations By Genome Editing Greatly Enhances the Accumulation of the Endogenous Fetal Hemoglobin in Human Normal Erythroid Cells

Blood ◽  
2017 ◽  
Vol 130 (Suppl_1) ◽  
pp. 947-947
Author(s):  
Nikoleta Psatha ◽  
Chang Li ◽  
Hongjie Wang ◽  
Demetri Dalas ◽  
George Stamatoyannopoulos ◽  
...  
Keyword(s):  
Genome Editing ◽  
Conflicts Of Interest ◽  
Fetal Hemoglobin ◽  
Erythroid Differentiation ◽  
Nuclease Activity ◽  
Erythroid Cell ◽  
Erythroid Progenitors ◽  
Knock Out ◽  
Gamma Globin

Abstract Thalassemia or sickle cell patients with increased fetal hemoglobin have an ameliorated clinical picture up to transfusion independency. Genome editing with targeted nucleases of the γ-globin silencer BCL11a or of the HBG promoter has been shown to reenact an HPFH-like phenotype by significantly increasing the endogenous fetal globin (HbF) expression. In order to achieve higher efficiency in HbF reactivation we developed an innovative approach, mediated through a highly customizable helper dependent-adenoviral vector (HD-Ad5/35++) with nuclease activity. This vector bares the Cas9 gene and two sequential gRNAs, permitting simultaneous targeting of two different DNA loci, specifically, the erythroid enhancer of BCL11a and the HBG promoter to recreate the -114 to -102 HPFH deletion. This double targeting vector was compared to the respective single gRNA vectors. We observed that disruption of the BCL11a-enhancer increased Gγ globin, whereas disruption of the HBG promoter had a greater impact on the Αγ globin expression. Simultaneous disruption of both loci in human erythroid progenitors increased the overall HbF expression from less than 1% in the control to more than 20%, through a possibly synergistic effect of the two mutations (HbF:2.2% in BCL11a-enhancer only knock-out, 10.8% in HBG-only knock out) by affecting both gamma globin chains. The editing efficiency per locus was similar between the single gRNA and double gRNA vectors. Erythroid cell morphology and phenotypic profile of the double knock-out cells did not differ compared to the single knock-out cells. In addition to our in vitro experiments, we observed that mobilized peripheral blood CD34+ cells transduced and edited by the double gRNA vector, can efficiently engraft in NSG mice. Furthermore, the engrafted edited cells after erythroid differentiation expressed significantly higher levels of gamma globin compared to the control. This strategy has the potential to induce higher levels of HbF reactivation with a clinical benefit in patients with beta globin disorders. Disclosures No relevant conflicts of interest to declare.

scholarly journals Sex-dependent dysregulation of human neutrophil responses by bisphenol A

2021 ◽  
Vol 20 (1) ◽  
Author(s):  
Wioletta Ratajczak-Wrona ◽  
Marzena Garley ◽  
Malgorzata Rusak ◽  
Karolina Nowak ◽  
Jan Czerniecki ◽  
...  
Keyword(s):  
Nadph Oxidase ◽  
Bisphenol A ◽  
Total Concentration ◽  
Nicotinamide Adenine Dinucleotide Phosphate ◽  
Human Neutrophils ◽  
Boyden Chamber ◽  
No Production ◽  
Pathogen Elimination ◽  
Latex Beads

Abstract Background In the present study, we aimed to investigate selected functions of human neutrophils exposed to bisphenol A (BPA) under in vitro conditions. As BPA is classified among xenoestrogens, we compared its action and effects with those of 17β-estradiol (E2). Methods Chemotaxis of neutrophils was examined using the Boyden chamber. Their phagocytosis and nicotinamide adenine dinucleotide phosphate hydrogen (NADPH) oxidase activity were assessed via Park’s method with latex beads and Park’s test with nitroblue tetrazolium. To assess the total concentration of nitric oxide (NO), the Griess reaction was utilized. Flow cytometry was used to assess the expression of cluster of differentiation (CD) antigens. The formation of neutrophil extracellular traps (NETs) was analyzed using a microscope (IN Cell Analyzer 2200 system). Expression of the investigated proteins was determined using Western blot. Results The analysis of results obtained for both sexes demonstrated that after exposure to BPA, the chemotactic capacity of neutrophils was reduced. In the presence of BPA, the phagocytic activity was found to be elevated in the cells obtained from women and reduced in the cells from men. Following exposure to BPA, the percentage of neutrophils with CD14 and CD284 (TLR4) expression, as well as the percentage of cells forming NETs, was increased in the cells from both sexes. The stimulatory role of BPA and E2 in the activation of NADPH oxidase was observed only in female cells. On the other hand, no influence of E2 on the expression of CD14 and CD284, chemotaxis, phagocytosis, and the amount of NET-positive neutrophils was found for both sexes. The study further showed that BPA intensified NO production and iNOS expression in the cells of both sexes. In addition, intensified expression of all tested PI3K-Akt pathway proteins was observed in male neutrophils. Conclusions The study demonstrated the influence of BPA on neutrophil functions associated with locomotion and pathogen elimination, which in turn may disturb the immune response of these cells in both women and men. Analysis of the obtained data showed that the effect of this xenoestrogen on the human neutrophils was more pronounced than E2.

scholarly journals Lentiviral Vectors for Delivery of Gene-Editing Systems Based on CRISPR/Cas: Current State and Perspectives

Viruses ◽  
2021 ◽  
Vol 13 (7) ◽  
pp. 1288
Author(s):  
Wendy Dong ◽  
Boris Kantor
Keyword(s):  
Large Scale ◽  
Lentiviral Vector ◽  
Clinical Applications ◽  
Scale Production ◽  
Base Editing ◽  
Epigenome Editing ◽  
Vector Systems ◽  
Dividing Cells

CRISPR/Cas technology has revolutionized the fields of the genome- and epigenome-editing by supplying unparalleled control over genomic sequences and expression. Lentiviral vector (LV) systems are one of the main delivery vehicles for the CRISPR/Cas systems due to (i) its ability to carry bulky and complex transgenes and (ii) sustain robust and long-term expression in a broad range of dividing and non-dividing cells in vitro and in vivo. It is thus reasonable that substantial effort has been allocated towards the development of the improved and optimized LV systems for effective and accurate gene-to-cell transfer of CRISPR/Cas tools. The main effort on that end has been put towards the improvement and optimization of the vector’s expression, development of integrase-deficient lentiviral vector (IDLV), aiming to minimize the risk of oncogenicity, toxicity, and pathogenicity, and enhancing manufacturing protocols for clinical applications required large-scale production. In this review, we will devote attention to (i) the basic biology of lentiviruses, and (ii) recent advances in the development of safer and more efficient CRISPR/Cas vector systems towards their use in preclinical and clinical applications. In addition, we will discuss in detail the recent progress in the repurposing of CRISPR/Cas systems related to base-editing and prime-editing applications.

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