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Li, Hojun, Jiahai Shi, and Harvey F. Lodish. "Genome Editing in Erythroid Progenitor Cells Mediated By Crispr/Cas9." Blood 124, no. 21 (December 6, 2014): 1345. http://dx.doi.org/10.1182/blood.v124.21.1345.1345.
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Abstract Investigation of the genetic underpinnings of erythrocyte development holds great value not only in the development of potential therapeutics for hematologic disorders, but also for elucidating basic biological principles. A robust model system for studying erythropoiesis is the mouse fetal liver system, as murine fetal liver is predominantly composed of erythroid progenitor cells at 2 weeks gestation. Upon isolation, these cells can be cultured in the presence of erythropoietic cytokines and follow distinct phases of development, from immature erythroid progenitors to terminally differentiated erythrocytes with robust enucleation and hemoglobinization. To date, loss of function genetic studies of erythropoiesis using the mouse fetal liver system have relied on mouse strains deficient in a gene of interest, or RNA interference inhibiting translation of a gene product of interest. Both strategies have limitations in terms of either time-intensive generation of genetically deficient mice, or inability of RNA interference to faithfully model homozygous deficiency, or haploinsufficiency. The development of genome editing technology based on a RNA-guided system for inducing targeted DNA double strand breaks (DSBs) raises the possibility of faithfully modeling homozygous deficiency or haploinsufficiency in a significantly higher throughput manner. This system consists of RNA-based Clustered Regularly Interspersed Short Palindromic Repeat (CRISPR) elements complexed with the Cas9 nuclease. Upon expression of both components in eukaryotic cells, a CRISPR single guide RNA (sgRNA), base pairs with a genomic target, guiding Cas9 to induce a DSB at that site. Genome editing then occurs at the site of the break if repair occurs via the non-homologous end joining DNA repair pathway, which produces mutational insertions, deletions, and substitutions during the process of DSB repair. In this study, we aimed to develop a system for CRISPR/Cas9-mediated genome editing in murine fetal liver cells. We constructed a retroviral vector co-expressing the Cas9 nuclease and an sgRNA. We initially designed sgRNAs targeting 2 genes non-essential for erythroid development, Gata3, which encodes a transcription factor required for T-cell development, and Lcp2, which encodes an adapter protein required for signal transduction during T-cell activation. These genes were chosen in order to assay genome editing efficiency without the occurrence of negative selection against disruption of genes required for erythroid development. Transduction of fetal liver cells isolated on embryonic day 14.5 (E14.5) with a retroviral vector expressing Cas9 and an sgRNA targeting Gata3 resulted in editing of 38% of Gata3 alleles. Transduction of E14.5 fetal liver cells with vector targeting Lcp2 resulted in editing of 15% of Lcp2 alleles. No editing was detected in control cells transduced with a retroviral vector expressing Cas9 and a scrambled sgRNA. Genome editing was detected using the Surveyor nuclease assay, which quantifies allelic frequency of gene mutations resulting from DSB repair by non-homologous end joining. We next designed an sgRNA targeting the Bcl11a gene, which encodes a protein shown to be instrumental in the embryonic to adult globin switch in mice. Transduction of E14.5 fetal liver cells with vector targeting Bcl11a resulted in editing of 49% of Bcl11a alleles. We then assessed if constitutive expression of Cas9 and an sgRNA affects the ability of fetal liver cells to undergo terminal erythroid differentiation. Compared to cells transduced with vector expressing only GFP, fetal liver cells transduced with retroviral vectors expressing Cas9 and scrambled sgRNAs had no significant difference in enucleation rate, a marker of terminal erythroid differentiation. In this study we demonstrate the ability to induce robust levels of genome editing at various genomic sites in mouse fetal liver cells using CRISPR/Cas9. We also demonstrate constitutive expression of Cas9 does not have any detrimental effect on enucleation. These results open the possibility of high-throughput modeling of homozygous genetic deficiency and genetic haploinsufficiency in studies of erythropoiesis. Disclosures No relevant conflicts of interest to declare.
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Diakatou, Michalitsa, Gaël Manes, Beatrice Bocquet, Isabelle Meunier, and Vasiliki Kalatzis. "Genome Editing as a Treatment for the Most Prevalent Causative Genes of Autosomal Dominant Retinitis Pigmentosa." International Journal of Molecular Sciences 20, no. 10 (May 23, 2019): 2542. http://dx.doi.org/10.3390/ijms20102542.
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Inherited retinal dystrophies (IRDs) are a clinically and genetically heterogeneous group of diseases with more than 250 causative genes. The most common form is retinitis pigmentosa. IRDs lead to vision impairment for which there is no universal cure. Encouragingly, a first gene supplementation therapy has been approved for an autosomal recessive IRD. However, for autosomal dominant IRDs, gene supplementation therapy is not always pertinent because haploinsufficiency is not the only cause. Disease-causing mechanisms are often gain-of-function or dominant-negative, which usually require alternative therapeutic approaches. In such cases, genome-editing technology has raised hopes for treatment. Genome editing could be used to (i) invalidate both alleles, followed by supplementation of the wild type gene, (ii) specifically invalidate the mutant allele, with or without gene supplementation, or (iii) to correct the mutant allele. We review here the most prevalent genes causing autosomal dominant retinitis pigmentosa and the most appropriate genome-editing strategy that could be used to target their different causative mutations.
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Bailey, Charles, Cynthia Metierre, Yue Feng, Kinsha Baidya, Galina Filippova, Dmitri Loukinov, Victor Lobanenkov, Crystal Semaan, and John Rasko. "CTCF Expression is Essential for Somatic Cell Viability and Protection Against Cancer." International Journal of Molecular Sciences 19, no. 12 (November 30, 2018): 3832. http://dx.doi.org/10.3390/ijms19123832.
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CCCTC-binding factor (CTCF) is a conserved transcription factor that performs diverse roles in transcriptional regulation and chromatin architecture. Cancer genome sequencing reveals diverse acquired mutations in CTCF, which we have shown functions as a tumour suppressor gene. While CTCF is essential for embryonic development, little is known of its absolute requirement in somatic cells and the consequences of CTCF haploinsufficiency. We examined the consequences of CTCF depletion in immortalised human and mouse cells using shRNA knockdown and CRISPR/Cas9 genome editing as well as examined the growth and development of heterozygous Ctcf (Ctcf+/−) mice. We also analysed the impact of CTCF haploinsufficiency by examining gene expression changes in CTCF-altered endometrial carcinoma. Knockdown and CRISPR/Cas9-mediated editing of CTCF reduced the cellular growth and colony-forming ability of K562 cells. CTCF knockdown also induced cell cycle arrest and a pro-survival response to apoptotic insult. However, in p53 shRNA-immortalised Ctcf+/− MEFs we observed the opposite: increased cellular proliferation, colony formation, cell cycle progression, and decreased survival after apoptotic insult compared to wild-type MEFs. CRISPR/Cas9-mediated targeting in Ctcf+/− MEFs revealed a predominance of in-frame microdeletions in Ctcf in surviving clones, however protein expression could not be ablated. Examination of CTCF mutations in endometrial cancers showed locus-specific alterations in gene expression due to CTCF haploinsufficiency, in concert with downregulation of tumour suppressor genes and upregulation of estrogen-responsive genes. Depletion of CTCF expression imparts a dramatic negative effect on normal cell function. However, CTCF haploinsufficiency can have growth-promoting effects consistent with known cancer hallmarks in the presence of additional genetic hits. Our results confirm the absolute requirement for CTCF expression in somatic cells and provide definitive evidence of CTCF’s role as a haploinsufficient tumour suppressor gene. CTCF genetic alterations in endometrial cancer indicate that gene dysregulation is a likely consequence of CTCF loss, contributing to, but not solely driving cancer growth.
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Shi, Zhong-Dong, Kihyun Lee, Dapeng Yang, Sadaf Amin, Nipun Verma, Qing V. Li, Zengrong Zhu, et al. "Genome Editing in hPSCs Reveals GATA6 Haploinsufficiency and a Genetic Interaction with GATA4 in Human Pancreatic Development." Cell Stem Cell 20, no. 5 (May 2017): 675–88. http://dx.doi.org/10.1016/j.stem.2017.01.001.
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McDermott, David H., Paejonette Jacobs, Qian Liu, Jiliang Gao, and Philip M. Murphy. "CXCR4 Gene Dosage Is Critical for HSC Engraftment." Blood 126, no. 23 (December 3, 2015): 3066. http://dx.doi.org/10.1182/blood.v126.23.3066.3066.
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Abstract Introduction: Warts, Hypogammaglobulinemia, Infections and Myelokathexis Syndrome (WHIMS) is an autosomal dominant immunodeficiency resulting from gain-of-function mutations in the chemokine receptor CXCR4. We recently described a unique WHIMS patient who underwent spontaneous genetic and phenotypic reversion at approximately age 30 after being severely affected as a child. Her reversion was due to a single catastrophic genetic event known as chromothripsis (chromosome shattering) resulting in the deletion of one copy of 163 genes in addition to her mutant copy of CXCR4 on chromosome 2. This event was traced to a hematopoietic stem cell (HSC) that had spontaneously repopulated her bone marrow; however, which of the genes was responsible and the mechanism required further investigation. Methods: Mouse models of CXCR4 haploinsufficiency (Cxcr4+/o) and WHIMS (Cxcr4+/S338X) were used in competitive bone marrow repopulation experiments transplanting whole bone marrow cells or purified HSC. Recipient mice were treated with / without lethal irradiation prior to transplant. Genome editing with TALENs and CRISPR-Cas9 technology was used to target CXCR4 for deletion in human cell lines. Results: Cxcr4 haploinsufficiency markedly enhanced HSC engraftment potential in recipient WHIM mice whether the donor HSC were purified from whole bone marrow cells or not, and whether the recipient was conditioned by lethal irradiation or not. Enhanced engraftment by Cxcr4 haploinsufficient donor HSC also occurred in wild-type mouse recipients, but to a lesser extent, and was also HSC intrinsic. Genome editing experiments have been successful at deleting one or both copies of CXCR4 in human cell lines in up to 40% of treated cells, and in reducing CXCR4 surface expression. Conclusion: While CXCR4 was already understood to be important in HSC biology, this patient and subsequent murine experiments have proven that the gene dosage of CXCR4 is a critical factor affecting HSC engraftment. Genome editing is a promising technology for deleting one copy of CXCR4, ideally the WHIM allele,in autologous HSC as a strategy to cure WHIM syndrome. Disclosures McDermott: US National Institutes of Health: Employment, Patents & Royalties: pending. Jacobs:US National Institutes of Health: Employment, Patents & Royalties: pending. Liu:US National Institutes of Health: Employment, Patents & Royalties: pending. Gao:US National Institutes of Health: Employment, Patents & Royalties: pending. Murphy:US National Institutes of Health: Employment, Patents & Royalties: pending.
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De Ravin, Suk See, and Julie Brault. "CRISPR/Cas9 applications in gene therapy for primary immunodeficiency diseases." Emerging Topics in Life Sciences 3, no. 3 (May 23, 2019): 277–87. http://dx.doi.org/10.1042/etls20180157.
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AbstractPrimary immunodeficiency diseases (PIDs) encompass a range of diseases due to mutations in genes that are critical for immunity. Haploinsufficiency and gain-of-function mutations are more complex than simple loss-of-function mutations; in addition to increased susceptibility to infections, immune dysregulations like autoimmunity and hyperinflammation are common presentations. Hematopoietic stem cell (HSC) gene therapy, using integrating vectors, provides potential cure of disease, but genome-wide transgene insertions and the lack of physiological endogenous gene regulation may yet present problems, and not applicable in PIDs where immune regulation is paramount. Targeted genome editing addresses these concerns; we discuss some approaches of CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas system applicable for gene therapy in PIDs. Preclinical repair of gene mutations and insertion of complementary DNA restore endogenous gene regulation and they have shown very promising data for clinical application. However, ongoing studies to characterize off-target genotoxicity, careful donor designs to ensure physiological expression, and maneuvers to optimize engraftment potential are critical to ensure successful application of this next-gen targeted HSC gene therapy.
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Roessler, Reinhard, Johanna Goldmann, Chikdu Shivalila, and Rudolf Jaenisch. "JIP2 haploinsufficiency contributes to neurodevelopmental abnormalities in human pluripotent stem cell–derived neural progenitors and cortical neurons." Life Science Alliance 1, no. 4 (June 25, 2018): e201800094. http://dx.doi.org/10.26508/lsa.201800094.
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Phelan–McDermid syndrome (also known as 22q13.3 deletion syndrome) is a syndromic form of autism spectrum disorder and currently thought to be caused by heterozygous loss of SHANK3. However, patients most frequently present with large chromosomal deletions affecting several additional genes. We used human pluripotent stem cell technology and genome editing to further dissect molecular and cellular mechanisms. We found that loss of JIP2 (MAPK8IP2) may contribute to a distinct neurodevelopmental phenotype in neural progenitor cells (NPCs) affecting neuronal maturation. This is most likely due to a simultaneous down-regulation of c-Jun N-terminal kinase (JNK) proteins, leading to impaired generation of mature neurons. Furthermore, semaphorin signaling appears to be impaired in patient NPCs and neurons. Pharmacological activation of neuropilin receptor 1 (NRP1) rescued impaired semaphorin pathway activity and JNK expression in patient neurons. Our results suggest a novel disease-specific mechanism involving the JIP/JNK complex and identify NRP1 as a potential new therapeutic target.
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Romano, Robert, Shahnaz Ghahremani, Talia Zimmerman, Nicholas Legere, Ketan Thakar, Feria A. Ladha, Anthony M. Pettinato, and J. Travis Hinson. "Reading Frame Repair of TTN Truncation Variants Restores Titin Quantity and Functions." Circulation 145, no. 3 (January 18, 2022): 194–205. http://dx.doi.org/10.1161/circulationaha.120.049997.
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Background: Titin truncation variants (TTNtvs) are the most common inheritable risk factor for dilated cardiomyopathy (DCM), a disease with high morbidity and mortality. The pathogenicity of TTNtvs has been associated with structural localization as A-band variants overlapping myosin heavy chain–binding domains are more pathogenic than I-band variants by incompletely understood mechanisms. Demonstrating why A-band variants are highly pathogenic for DCM could reveal new insights into DCM pathogenesis, titin (TTN) functions, and therapeutic targets. Methods: We constructed human cardiomyocyte models harboring DCM-associated TTNtvs within A-band and I-band structural domains using induced pluripotent stem cell and CRISPR technologies. We characterized normal TTN isoforms and variant-specific truncation peptides by their expression levels and cardiomyocyte localization using TTN protein gel electrophoresis and immunofluorescence, respectively. Using CRISPR to ablate A-band variant–specific truncation peptides through introduction of a proximal I-band TTNtv, we studied genetic mechanisms in single cardiomyocyte and 3-dimensional, biomimetic cardiac microtissue functional assays. Last, we engineered a full-length TTN protein reporter assay and used next-generation sequencing assays to develop a CRISPR therapeutic for somatic cell genome editing TTNtvs. Results: An A-band TTNtv dose-dependently impaired cardiac microtissue twitch force, reduced full-length TTN levels, and produced abundant TTN truncation peptides. TTN truncation peptides integrated into nascent myofibril-like structures and impaired myofibrillogenesis. CRISPR ablation of TTN truncation peptides using a proximal I-band TTNtv partially restored cardiac microtissue twitch force deficits. Cardiomyocyte genome editing using SpCas9 and a TTNtv-specific guide RNA restored the TTN protein reading frame, which increased full-length TTN protein levels, reduced TTN truncation peptides, and increased sarcomere function in cardiac microtissue assays. Conclusions: An A-band TTNtv diminished sarcomere function greater than an I-band TTNtv in proportion to estimated DCM pathogenicity. Although both TTNtvs resulted in full-length TTN haploinsufficiency, only the A-band TTNtv produced TTN truncation peptides that impaired myofibrillogenesis and sarcomere function. CRISPR-mediated reading frame repair of the A-band TTNtv restored functional deficits, and could be adapted as a one-and-done genome editing strategy to target ≈30% of DCM-associated TTNtvs.
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Tulli, Susanna, Andrea Del Bondio, Valentina Baderna, Davide Mazza, Franca Codazzi, Tyler Mark Pierson, Alessandro Ambrosi, et al. "Pathogenic variants in the AFG3L2 proteolytic domain cause SCA28 through haploinsufficiency and proteostatic stress-driven OMA1 activation." Journal of Medical Genetics 56, no. 8 (March 25, 2019): 499–511. http://dx.doi.org/10.1136/jmedgenet-2018-105766.
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BackgroundSpinocerebellar ataxia type 28 (SCA28) is a dominantly inherited neurodegenerative disease caused by pathogenic variants in AFG3L2. The AFG3L2 protein is a subunit of mitochondrial m-AAA complexes involved in protein quality control. Objective of this study was to determine the molecular mechanisms of SCA28, which has eluded characterisation to date.MethodsWe derived SCA28 patient fibroblasts carrying different pathogenic variants in the AFG3L2 proteolytic domain (missense: the newly identified p.F664S and p.M666T, p.G671R, p.Y689H and a truncating frameshift p.L556fs) and analysed multiple aspects of mitochondrial physiology. As reference of residual m-AAA activity, we included SPAX5 patient fibroblasts with homozygous p.Y616C pathogenic variant, AFG3L2+/− HEK293 T cells by CRISPR/Cas9-genome editing and Afg3l2−/− murine fibroblasts.ResultsWe found that SCA28 cells carrying missense changes have normal levels of assembled m-AAA complexes, while the cells with a truncating pathogenic variant had only half of this amount. We disclosed inefficient mitochondrial fusion in SCA28 cells caused by increased OPA1 processing operated by hyperactivated OMA1. Notably, we found altered mitochondrial proteostasis to be the trigger of OMA1 activation in SCA28 cells, with pharmacological attenuation of mitochondrial protein synthesis resulting in stabilised levels of OMA1 and OPA1 long forms, which rescued mitochondrial fusion efficiency. Secondary to altered mitochondrial morphology, mitochondrial calcium uptake resulted decreased in SCA28 cells.ConclusionOur data identify the earliest events in SCA28 pathogenesis and open new perspectives for therapy. By identifying similar mitochondrial phenotypes between SCA28 cells and AFG3L2+/− cells, our results support haploinsufficiency as the mechanism for the studied pathogenic variants.
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Tan, Siyuan, Kai-Hsin Chang, Sarah Smith, Kai Chen, Timonthy Sullivan, Qianhe Zhou, Andreas Reik, et al. "Genome Editing of the Bcl11A Erythroid Specific Enhancer in Bone Marrow Derived Hematopoietic Stem and Progenitor Cells for the Treatment of Sickle Cell Disease." Blood 126, no. 23 (December 3, 2015): 203. http://dx.doi.org/10.1182/blood.v126.23.203.203.
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Abstract Ablation of Bcl11A could be a viable approach for the treatment of β-hemoglobinopathies such as β-thalassemia and sickle cell disease (SCD), since patients with Bcl11A haploinsufficiency have persistently high levels of fetal hemoglobin (HbF) (up to 30%), which are associated with development of minimal to no disease symptoms. Genome editing by engineered zinc-finger nucleases that target either the exon 2 (exon ZFN) or the GATA motif of the erythroid specific enhancer (enhancer ZFN) of Bcl11A has been shown to increase HbF level in erythroid progeny from mobilized peripheral hematopoietic stem and progenitor cells (PB-CD34+ HSPCs). However, peripheral mobilization of CD34+ cells is associated with high risk and currently is not an option for SCD patients. Therefore, we investigated the efficacy of genome editing of Bcl11A in bone marrow derived CD34+ cells (BM-CD34+ HSPCs). We first established a clinically compatible large-scale process to isolate CD34+ HSPCs from human bone marrow aspirates and to transiently express the ZFN protein by mRNA electroporation. The CD34+ isolation process resulted in ~ 95% pure CD34+ cells with greater than 90% viability. Both the exon and the enhancer ZFN drove 50-60% Bcl11A gene editing, resulting in a robust elevation of HbF in the erythroid progeny. Notably, the BM-CD34+ HSPCs were found to contain a small population (10 to 25%) of CD34+CD19+ pro-B cells that were refractory to ZFN transfection under our current electroporation condition. Since CD34+CD19+ pro-B cells are not expected to contribute to reconstituting the hematopoietic system other than B-cell lineage, the Bcl11A editing efficiency in the multipotent BM-CD34+ HSPC could be even higher. The engraftment abilities of Bcl11A edited BM-CD34+ cells were then investigated in an immunodeficient NOD/scid/gamma (NSG) mouse model. At a dose of 1 million cells per mouse, treatment with either the exon ZFN or the enhancer ZFN did not detectably impact engraftment or multi-lineage reconstitution compared with untreated cells. However, Bcl11A marking in engrafted human cells was found to be markedly higher in the mice treated by the enhancer ZFN than that by the exon ZFN. The exon ZFN resulted in a strong bias towards in-frame mutations across multi-lineages with the strongest effect observed in the B-cell lineage, suggesting that a threshold level of Bcl11A is required for efficient hematopoietic reconstitution and that cells fully lacking it due to disruption of the coding sequence are at a disadvantage. In contrast, the enhancer ZFN resulted in comparable Bcl11A marking across all lineages with no apparent selection for cells with a functional GATA sequence. Collectively, these data indicate that genome editing of the erythroid specific enhancer of Bcl11A in BM-CD34+ promotes HbF reactivation in the erythroid progeny while maintaining the engraftment and multi-lineage repopulating activities of edited BM-CD34+ HSPCs, which supports further clinical development of this approach for the treatment of SCD. Disclosures Tan: Biogen: Employment, Equity Ownership. Chang:Biogen: Employment, Equity Ownership. Smith:Biogen: Employment, Equity Ownership. Chen:Biogen: Employment, Equity Ownership. Sullivan:Biogen: Employment, Equity Ownership. Zhou:Biogen: Employment, Equity Ownership. Reik:Sangamo BioSciences: Employment, Equity Ownership, Patents & Royalties: Patent applications have been filed based on this work. Urnov:Sangamo BioSciences: Employment, Equity Ownership, Patents & Royalties: Patent applications have been filed based on this work. Rebar:Sangamo BioSciences: Employment. Danos:Biogen: Employment, Equity Ownership. Jiang:Biogen: Employment, Equity Ownership.
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Fortschegger, Klaus, Anna-Maria Husa, Dagmar Schinnerl, Karin Nebral, and Sabine Strehl. "Expression of RUNX1-JAK2 in Human Induced Pluripotent Stem Cell-Derived Hematopoietic Cells Activates the JAK-STAT and MYC Pathways." International Journal of Molecular Sciences 22, no. 14 (July 15, 2021): 7576. http://dx.doi.org/10.3390/ijms22147576.
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A heterogeneous genetic subtype of B-cell precursor acute lymphoblastic leukemia is driven by constitutive kinase-activation, including patients with JAK2 fusions. In our study, we model the impact of a novel JAK2 fusion protein on hematopoietic development in human induced pluripotent stem cells (hiPSCs). We insert the RUNX1-JAK2 fusion into one endogenous RUNX1 allele through employing in trans paired nicking genome editing. Tagging of the fusion with a degron facilitates protein depletion using the heterobifunctional compound dTAG-13. Throughout in vitro hematopoietic differentiation, the expression of RUNX1-JAK2 is driven by endogenous RUNX1 regulatory elements at physiological levels. Functional analysis reveals that RUNX1-JAK2 knock-in cell lines yield fewer hematopoietic progenitors, due to RUNX1 haploinsufficiency. Nevertheless, these progenitors further differentiate toward myeloid lineages to a similar extent as wild-type cells. The expression of the RUNX1-JAK2 fusion protein only elicits subtle effects on myeloid differentiation, and is unable to transform early hematopoietic progenitors. However, phosphoprotein and transcriptome analyses reveal that RUNX1-JAK2 constitutively activates JAK-STAT signaling in differentiating hiPSCs and at the same time upregulates MYC targets—confirming the interaction between these pathways. This proof-of-principle study indicates that conditional expression of oncogenic fusion proteins in combination with hematopoietic differentiation of hiPSCs may be applicable to leukemia-relevant disease modeling.
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Kotini, Andriana, Jeffrey J. Delrow, Timothy A. Graubert, Stephen Nimer, and Eirini P. Papapetrou. "Functional Dissection of Chromosome 7q Loss and Haploinsufficient Gene Discovery Using iPSC Models of MDS." Blood 124, no. 21 (December 6, 2014): 524. http://dx.doi.org/10.1182/blood.v124.21.524.524.
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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.
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König, Saskia, Manfred Fliegauf, Manuel Rhiel, Bodo Grimbacher, Tatjana I. Cornu, Toni Cathomen, and Claudio Mussolino. "Allele-Specific Disruption of a Common STAT3 Autosomal Dominant Allele Is Not Sufficient to Restore Downstream Signaling in Patient-Derived T Cells." Genes 13, no. 10 (October 20, 2022): 1912. http://dx.doi.org/10.3390/genes13101912.
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Dominant negative mutations in the STAT3 gene account for autosomal dominant hyper-IgE syndrome (AD-HIES). Patients typically present high IgE serum levels, recurrent infections, and soft tissue abnormalities. While current therapies focus on alleviating the symptoms, hematopoietic stem cell transplantation (HSCT) has recently been proposed as a strategy to treat the immunological defect and stabilize the disease, especially in cases with severe lung infections. However, because of the potentially severe side effects associated with allogeneic HSCT, this has been considered only for a few patients. Autologous HSCT represents a safer alternative but it requires the removal of the dominant negative mutation in the patients’ cells prior to transplantation. Here, we developed allele-specific CRISPR-Cas9 nucleases to selectively disrupt five of the most common STAT3 dominant negative alleles. When tested ex vivo in patient-derived hematopoietic cells, allele-specific disruption frequencies varied in an allele-dependent fashion and reached up to 62% of alleles harboring the V637M mutation without detectable alterations in the healthy STAT3 allele. However, assessment of the gene expression profiles of the STAT3 downstream target genes revealed that, upon activation of those edited patient cells, mono-allelic STAT3 expression (functional haploinsufficiency) is not able to sufficiently restore STAT3-dependent signaling in edited T cells cultured in vitro. Moreover, the stochastic mutagenesis induced by the repair of the nuclease-induced DNA break could further contribute to dominant negative effects. In summary, our results advocate for precise genome editing strategies rather than allele-specific gene disruption to correct the underlying mutations in AD-HIES.
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Chang, Kai-Hsin, Timothy Sullivan, Mei Liu, Xiao Yang, Chao Sun, Benjamin Vieira, Ming Zhang, et al. "Clonal Analysis of Human Bone Marrow CD34+ Cells Edited By BCL11A-Targeting Zinc Finger Nucleases Reveals Clinically Relevant Levels of Fetal Globin Expression in Edited Erythroid Progeny." Blood 126, no. 23 (December 3, 2015): 3234. http://dx.doi.org/10.1182/blood.v126.23.3234.3234.
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Abstract Sickle cell disease (SCD) is one of the most common inherited blood disorders and is caused by a mutation at the adult beta globin gene resulting in substitution of valine for glutamic acid at position 6 in the encoded protein. While SCD can be cured by hematopoietic stem cell transplant (HSCT), complete donor chimerism is not required to achieve clinical benefits. Stable mixed chimerism of 10-15% in bone marrow or peripheral blood nucleated cells with >70% donor-derived RBCs has been reported to achieve transfusion independence and a symptom-free state in a SCD patient. It has also been proposed that SCD can be treated by reactivating developmentally silenced fetal gamma globin to form fetal hemoglobin (alpha2gamma2, HbF), which inhibits polymerization of HbS. The effect of HbF is predicted to be maximal when HbF content per cell exceeds 10 pg (~30% of total Hb). Furthermore, pathology is prevented when protective F cells (>30% HbF per cell) constitute >70% of total RBCs. We hypothesize that in a gene therapy setting, if >15% of SCD patients' autologous HSCs are programmed to produce protective F cells during erythropoiesis, it will translate into >70% protective F cells in circulation and provide significant alleviation of clinical symptoms. Genome wide association studies have identified BCL11A as a major modifier of HbF levels. Subsequent studies have shown that BCL11A plays a critical role in the fetal to adult globin developmental switch and in repressing fetal globin expression in adult erythroid cells. Conditional inactivation of BCL11A in adult erythroid cells leads to high levels of pan-cellular fetal globin expression and correction of hematologic and pathologic defects in a humanized SCD mouse model. Previously, we have reported that zinc finger nucleases (ZFNs) targeting BCL11A either in the coding region or the GATAA motif in the erythroid-specific enhancer efficiently disrupt the BCL11A locus in human primary CD34+ cells following electroporation of ZFN-encoding mRNA. Elevated fetal globin expression in bulk erythroid cultures was observed following disruption. To determine what percentage of HSPCs have been modified and whether the HbF/F cell content has reached the hypothesized therapeutic level, we analyzed erythroid cells clonally derived from ZFN-transfected CD34+ cells. Genotype of each clonal culture was determined by deep sequencing and globin production was analyzed by a highly sensitive UPLC method. We found that up to 80% of the BFU-Es had both BCL11A alleles edited, half of which had KO/KO alleles (either out of frame mutations for coding region or elimination of the GATAA motif in the enhancer). BCL11A coding KO/KO cells expressed on average 79.1% ± 12.2% fetal globin (Mean ± SD) whereas GATAA motif enhancer region KO/KO cells expressed approximately 48.4% ± 14.1% fetal globin, in comparison with 14.5% ± 9.6% in WT/WT cells . These levels of fetal globin should be sufficiently high to confer protection against HbS polymerization in sickle cells. WT/KO cells in both coding and enhancer editing experiments showed an intermediate phenotype with fetal globin averaging 26.9%± 9.9% and 25.79% ± 12.6%, respectively. Interestingly, when background (WT/WT) fetal globin level was subtracted, the fetal globin levels in WT/KO cells are comparable to those observed in patients with BCL11A haploinsufficiency, which average 14.6%± 10.3%. Together, our data demonstrate that genome editing of BCL11A using highly efficient ZFNs can lead to clinically relevant levels of fetal globin expression in KO/KO erythroid cells. If the frequency of KO/KO BFU-Es we observed in vitro reflects the frequency of KO/KO HSCs in bone marrow after autologous transplantation, genome editing of BCL11A has the potential to provide significant clinical benefit for patients with SCD. Disclosures Chang: Biogen: Employment, Equity Ownership. Sullivan:Biogen: Employment, Equity Ownership. Liu:Biogen: Employment, Equity Ownership. Yang:Biogen: Employment, Equity Ownership. Sun:Biogen: Employment, Equity Ownership. Vieira:Biogen: Employment, Equity Ownership. Zhang:Biogen: Employment. Hong:Biogen: Employment, Equity Ownership. Chen:Biogen: Employment, Equity Ownership. Smith:Biogen: Employment, Equity Ownership. Tan:Biogen: Employment, Equity Ownership. Reik:Sangamo BioSciences: Employment, Equity Ownership, Patents & Royalties: Patent applications have been filed based on this work. Urnov:Sangamo BioSciences: Employment, Equity Ownership, Patents & Royalties: Patent applications have been filed based on this work. Rebar:Sangamo BioSciences: Employment. Danos:Biogen: Employment, Equity Ownership. Jiang:Biogen: Employment, Equity Ownership.
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Fink, Emma C., Jan Krönke, Slater N. Hurst, Namrata D. Udeshi, Tanya Svinkina, Rebekka K. Schneider, Marie E. McConkey, et al. "Lenalidomide Induces Ubiquitination and Degradation of CSNK1A1 in MDS with Del(5q)." Blood 124, no. 21 (December 6, 2014): 4. http://dx.doi.org/10.1182/blood.v124.21.4.4.
Full textAbstract:
Abstract The immunomodulatory (IMiD) drug lenalidomide is a highly effective treatment for multiple myeloma and myelodysplastic syndrome (MDS) with deletion of chromosome 5q (del(5q)). Recently, we and others demonstrated that lenalidomide activates the CRBN-CRL4 E3 ubiquitin ligase to ubiquitinate IKZF1 and IKZF3. Degradation of these lymphoid transcription factors explains lenalidomide’s growth inhibition of multiple myeloma cells and increased IL-2 release from T cells. However, it is unlikely that degradation of IKZF1 and IKZF3 accounts for lenalidomide’s activity in MDS with del(5q). Instead, we hypothesized that ubiquitination of a distinct CRBN substrate in myeloid cells explains the efficacy of lenalidomide in del(5q) MDS. Applying quantitative proteomics in the myeloid cell line KG-1, we identified a novel target, casein kinase 1A1 (CSNK1A1), that had increased ubiquitination and decreased protein abundance following lenalidomide treatment. CSNK1A1 is encoded in the del(5q) commonly deleted region and is thus a potential lenalidomide target in del(5q) MDS. Previous studies have demonstrated that Csnk1a1 is a therapeutic target in a murine model of acute myeloid leukemia. We validated that lenalidomide treatment decreased CSNK1A1 protein levels in multiple human cell lines in a dose-dependent manner without altering CSNK1A1 mRNA levels. Moreover, lenalidomide treatment increased ubiquitination of CSNK1A1 in cell lines. The decrease in CSNK1A1 protein levels in response to lenalidomide was abrogated by treatment with the proteasome inhibitor MG132 and by Cullin-RING ubiquitin ligase inhibition with MLN4924. CSNK1A1 co-immunoprecipitated with CRBN in the presence of lenalidomide, demonstrating direct interaction of CSNK1A1 with the substrate adaptor for the ubiquitin ligase. Homozygous genetic inactivation of the CRBN gene by CRISPR/Cas9 genome editing in 293T cells eliminated lenalidomide-induced degradation of CSNK1A1. In aggregate, these experiments demonstrate that CSNK1A1 is a CRBN-CRL4 substrate that is ubiquitinated and degraded in the presence of lenalidomide. We next explored how degradation of CSNK1A1 might explain the specificity of lenalidomide for cells with del(5q). ShRNA-mediated knockdown of CSNK1A1 sensitized primary human CD34+ cells to lenalidomide treatment, indicating that haploinsufficiency for CSNK1A1 might increase lenalidomide sensitivity in del(5q) hematopoietic cells. We sought to further validate this finding in a genetically defined Csnk1a1 conditional knockout mouse model. While murine cells are resistant to the effects of IMiDs, murine Ba/F3 cells overexpressing human CRBN (hCRBN), but not murine CRBN, degraded CSNK1A1 in response to lenalidomide. To examine the effect of Csnk1a1 haploinsufficiency on lenalidomide sensitivity, we isolated hematopoietic stem and progenitor cells from Csnk1a1+/- and Csnk1a1+/+ mice and transduced them with a retroviral vector expressing hCRBN. When treated with lenalidmide, Csnk1a1+/- cells expressing hCRBN were depleted over time relative to wild-type controls. The enhanced sensitivity of Csnk1a1+/- cells to lenalidomide was associated with induction of p21 and was rescued by heterozygous deletion of p53, demonstrating a critical downstream role for p53 consistent with clinical observations that TP53 mutations confer lenalidomide resistance. In aggregate, these studies demonstrate that lenalidomide induces the ubiquitination and consequent degradation of CSNK1A1 by the CRBN-CRL4 E3 ubiquitin ligase. del(5q) cells have only one copy of CSNK1A1, so they are selectively depleted over wild-type cells, explaining lenalidomide’s clinical efficacy in del(5q) MDS. Although the idea that heterozygous deletions could be cancer vulnerabilities was first proposed 20 years ago, lenalidomide provides the first example of an FDA-approved and clinically effective drug that derives its therapeutic window from specifically targeting a haploinsufficient gene. Disclosures Ebert: Celgene: Research Funding; Genoptix: Consultancy.
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Amazit, Larbi, Mattia Barbot, Isabelle Beau, Jérôme Bouligand, Isabelle Bourdeau, Philippe Chanson, Lucie Cloix, et al. "OR04-4 Loss of KDM1A in Bilateral Macronodular Adrenal Hyperplasia With GIP-Dependent Cushing's Syndrome and in Acromegaly With Paradoxical GH Response to Oral Glucose." Journal of the Endocrine Society 6, Supplement_1 (November 1, 2022): A81. http://dx.doi.org/10.1210/jendso/bvac150.168.
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Abstract Context Primary bilateral macronodular adrenal hyperplasia (PBMAH) with glucose-dependent insulinotropic polypeptide (GIP)-dependent Cushing's syndrome is caused by ectopic expression of GIP receptor (GIPR) in the adrenal lesions. Such ectopic expression of GIPR was also reported in other endocrine neoplasm, notably in somatotroph pituitary adenomas from acromegalic patients with paradoxical increase of GH after oral glucose load, suggesting a common molecular pathogenesis. We aimed to identify the driver event responsible for GIP-dependent PBMAH with Cushing's syndrome and ectopic GIPR expression in somatotropinomas. Methods We conducted an international, multicenter, cohort study. We collected blood and adrenal samples from patients who had undergone unilateral or bilateral adrenalectomy for GIP-dependent PBMAH with Cushing's syndrome. Adrenal samples from patients with PBMAH and Cushing's syndrome without food-dependent cortisol production were used as controls. We further collected somatotropinoma specimens from acromegaly patients followed at two expert endocrine centers in France. Results 17 patients with familial or sporadic GIP-dependent PBMAH with Cushing's syndrome were studied. We identified germline heterozygous mutations in the lysine demethylase 1A (KDM1A) gene in all 17 patients. We further identified a recurrent deletion of the short arm of chromosome 1 harboring the KDM1A locus in the adrenal lesions of affected patients. None of the 25 patients in the control group had KDM1A germline or somatic alterations. Concomitant genetic inactivation of both KDM1A alleles resulted in loss of KDM1A expression in the adrenal lesions. RNA-sequencing revealed the global impact of KDM1A loss in adrenal tissue on gene transcription and identified differentially regulated genes including those encoding for GIPR and other G-Protein-Coupled Receptors that may be involved in adrenal tumorigenesis and regulation of steroidogenesis. In vitro pharmacologic inhibition, silencing and knock-out by CRISPR-Cas9 genome editing of KDM1A led to an increase in GIPR transcripts and protein in human adrenocortical H295R cells. Somatotropinoma samples from 78 patients with acromegaly were studied. 24% of these patients presented with a paradoxical rise of GH after oral glucose load and expressed ectopically GIP-receptor in their somatotropinoma. None of the somatotropinomas harbored KDM1A pathogenic variants, but those from patients with paradoxical GH response displayed a recurrent chromosome 1p loss. Discussion We identified germline inactivating KDM1A mutations and loss of heterozygosity as a genetic predisposition to GIP-dependent PBMAH with Cushing's syndrome following a tumor suppressor gene model of tumorigenesis. We currently perform genetic screening in first-degree relatives of patients with GIP-dependent PBMAH with Cushing's syndrome and clinical examination with biochemical testing in asymptomatic KDM1A variant carriers. We did not identify somatic KDM1A mutations in somatotropinomas expressing GIPR ectopically, however their recurrent 1p chromosome loss suggests that KDM1A haploinsufficiency may contribute to GIPR expression in those tumors. Presentation: Saturday, June 11, 2022 12:15 p.m. - 12:30 p.m.
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Singh, Ram Kumar, Richard J. Jones, Samuel Hong, Fazal Shirazi, Hua Wang, Isere Kuiatse, Andreas Pahl, and Robert Z. Orlowski. "HDP101, a Novel B-Cell Maturation Antigen (BCMA)-Targeted Antibody Conjugated to α-Amanitin, Is Active Against Myeloma with Preferential Efficacy Against Pre-Clinical Models of Deletion 17p." Blood 132, Supplement 1 (November 29, 2018): 593. http://dx.doi.org/10.1182/blood-2018-99-118412.
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Abstract Background: Deletion (del) of 17p involving the p53 tumor suppressor (TP53) remains an adverse prognostic factor in multiple myeloma (MM) despite the use of novel agents as well as high-dose chemotherapy with autologous stem cell rescue. Genomic TP53 deletion can cause haploinsufficiency of nearby genes, such as RNA polymerase II subunit A (POLR2A), which ia also located on 17p13.1. We therefore hypothesized that del 17p could reduce POLR2A expression and enhance sensitivity to a-Amanitin, a potent and specific inhibitor of POLR2A and RNA polymerase III. Methods: Pre-clinical studies were performed using HDP101, a monoclonal antibody-drug conjugate (ADC) targeting BCMA linked to a-Amanitin, along with unconjugated BCMA antibody, a-Amanitin, and a non-targeting control ADC in myeloma cell line models. The latter included H929, MM1.S, and MOLP-8 TP53 wild-type (WT) lines and isogenic cells in which TP53 had been knocked out (KO) using CRISPR/Cas9 genome editing techniques. To further model del 17p and POLR2A haploinsufficiency, POLR2A expression was knocked down using shRNAs. Results: Analysis of the Multiple Myeloma Research Foundation CoMMpassSM database revealed that del 17p13 by SeqFISH was associated with a significant reduction in POLR2A expression by RNASeq (26.05431 fragments per kilobase of transcript per million mapped reads (FPKM) with WT vs. 19.2983 FPKM with del 17p13; p<0.0001). Also, patients within the lower quartile of POLR2A expression, which included those with and without del 17p, had an inferior overall survival (p<0.0011) and a trend towards a worse progression-free survival, suggesting that low POLR2A levels by themselves are an adverse feature. The POLR2A inhibiting anti-BCMA/a-Amanitin conjugate HDP101 induced a time- and dose-dependent reduction in myeloma cell viability post 96-hours of drug exposure starting at concentrations as low as in the picomolar range. This reduction with HDP101 was much greater than that seen with controls, which included a-Amanitin alone, the anti-BCMA antibody without a-Amanitin, or an anti-digoxigenin antibody conjugated to a-Amanitin. When myeloma cells were co-cultured with HS-5 human marrow stromal cells, only a-Amanitin and, to a much greater extent, HDP101 induced a loss of cell viability in myeloma cells, while the stromal cells were spared by HDP101. Loss of cell viability due to HDP101 was associated with induction of apoptosis as judged by the appearance of an increased population of cells that had a sub-G0/G1 DNA content, and that stained positively with Annexin V. Moreover, HDP101 treatment caused the appearance of cleaved fragments of Caspase 9 and 3, and loss of the mitochondrial trans-membrane potential. H929, MM1.S, and MOLP-8 TP53 KO cells were more sensitive to both HDP101 and, less potently to a-Amanitin than were their isogenic TP53 WT parental cells. As H929 cells expressed high levels of BCMA regardless of TP53 or POLR2A status, these were further examined for their sensitivity to HDP101. Notably, the preferential impact upon TP53 KO cells was associated with increased expression of Activating transcription factors -4 and -6, suggesting enhanced induction of endoplasmic reticulum stress, and at least two arms of the unfolded protein response. Interestingly, shRNA-mediated knockdown (KD) of POLR2A expression alone was sufficient to increase baseline levels of apoptosis in both H929 TP53 WT and KO cells, with a greater impact in the latter, supporting the promise of this target. Importantly, KD of POLR2A expression to further model del 17p was also associated with enhanced sensitivity to HDP101 in both the TP53 WT and KO cells compared to the control treatments. Evaluation of HDP101 in primary samples and with in vivo models is underway, and will be presented at the Annual Meeting. These studies were supported by a Leukemia & Lymphoma Society Specialized Center of Research (SCOR-12206-17). Conclusions: Our preliminary data support the possibility that del 17p myeloma may have a therapeutic vulnerability to the POLR2A inhibitor a-Amanitin through loss of TP53, and that this sensitivity is further enhanced by decreased POLR2A expression, which is common among del 17p patients. Moreover, they suggest that HDP101 is a novel potent and specific therapeutic that could show enhanced activity in the clinic especially against high-risk multiple myeloma, where effective therapies are still needed to improve patient outcomes. Disclosures Pahl: Heidelberg Pharma AG: Employment. Orlowski:Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Janssen Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Consultancy; Genentech: Consultancy; Poseida: Research Funding; BioTheryX, Inc: Consultancy, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Millenium Pharmaceuticals: Consultancy, Research Funding.
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Singh, Ram Kumar, Richard J. Jones, Fazal M. Shirazi, Jianxuan M. Zou, Hua Wang, Hans C. Lee, Elisabet E. Manasanch, Isere Kuiatse, Andreas Pahl, and Robert Z. Orlowski. "The Anti-B-Cell Maturation Antigen (BCMA) Antibody-α-Amanitin Conjugate Hdp-101 Induces Immunogenic Cell Death and Immunologic Memory in Models of Multiple Myeloma." Blood 136, Supplement 1 (November 5, 2020): 9–10. http://dx.doi.org/10.1182/blood-2020-141615.
Full textAbstract:
Background: Patients with relapsed/refractory myeloma continue to represent an unmet medical need, and this is especially true for those with deletion (del) 17p, who have an inferior prognosis. We previously reported the ability of HDP-101 to induce anti-proliferative and pro-apoptotic effects in myeloma cell lines and primary patient samples. HDP-101 showed enhanced efficacy against del 17p models, which are characterized by concurrent haploinsufficiency of RNA polymerase II subunit A (POLR2A), which is the target of amanitin. This occurred in conjunction with activation of the endoplasmic reticulum stress, and at least two arms of the unfolded protein response. Also, a single dose of HDP-101 was sufficient to induce cures in murine models of myeloma both with and without TP53 and/or POLR2A. Therefore, we sought to better understand the mechanisms of action of HDP-101 and its potential immunologic sequelae. Methods: Pre-clinical studies were performed using HDP-101, the unconjugated BCMA antibody, free α-Amanitin, and a non-targeting control Amanitin antibody drug conjugate in myeloma cell line models and in vivo. These included H929, MM1.S, and MOLP-8 TP53 wild-type (WT) lines and isogenic cells in which TP53 had been knocked out (KO) using genome editing techniques. To further model del 17p and POLR2A haploinsufficiency, POLR2A expression was knocked down using sequence-specific shRNAs. Tumor rechallenge experiments were performed after 100 days of tumor free survival in NOD.CB17-Prkdcscid/J mice with longitudinal monitoring of tumor growth kinetics. Results: HDP-101 induced immunogenic cell death (ICD) in myeloma cell lines as determined by increased expression of Calreticulin (CRT), Heat shock protein (HSP)-70, and release of High mobility group box 1 (HMGB1) by flow cytometry and Western blotting. Microscopic analysis of ICD upon treatment with HDP-101 in MM1.S cells showed a speckled arrangement of both CRT and HMGB1 on the cell surface. In addition, an increased ADP/ATP ratio was observed across three different cell lines in a TP53-independent manner suggestive of an operational ICD pathway. Using our current knowledge of ER stress activation and induction of ICD, we evaluated the combination of bortezomib and HDP-101 in H929, MM1.S, and MOLP-8 cells and primary samples. A significant loss of cellular viability (p<0.01) was observed upon combination treatment across the cell lines, and primary samples also showed a similar effect but only in CD138+ve populations while sparing the CD138-ve fraction. NOD.CB17-Prkdcscid/J mice injected with MM1.S luciferase-transfected cells uniformly developed systemic disease, and one dose of HDP-101 induced cures with no evidence of relapse out to 100 days. These same mice were then rechallenged with MM1.S cells but none developed recurrent disease, suggesting the generation of immunologic memory. Natural killer (NK) cells isolated from these mice showed ex vivo activity against myeloma cell lines which was greatest against MM1.S cells. Finally, cured mice were treated with the anti-asialo GM1 antibody that depletes NK cells or control serum first, and then rechallenged with MM1.S cells. While MM1.S-based tumors could not become established in control serum treated mice, pre-treatment with the anti-asialo GM1 antibody did allow tumors to develop. Studies to compare the activity of HDP-101 with other antibody drug conjugates are currently ongoing. Conclusions: Our results support the statement that HDP-101 is a novel anti-BCMA antibody drug conjugate that shows potent activity against drug-naïve and -resistant models of myeloma, and has the potential to show enhanced anti-tumor activity against del 17 p myeloma. Moreover, its mechanism of action involves both a direct cytotoxic effect, as well as the induction of immunogenic cell death, with the latter potentially leading to immunologic memory. These studies were supported by a Leukemia & Lymphoma Society Specialized Center of Research (SCOR-12206-17). Disclosures Jones: Asylia Therapeutics, Inc.: Current equity holder in private company, Patents & Royalties. Lee:Amgen: Consultancy, Research Funding; Celgene: Consultancy, Research Funding; Janssen: Consultancy, Research Funding; Takeda: Consultancy, Research Funding; Genentech: Consultancy; GlaxoSmithKline: Consultancy, Research Funding; Sanofi: Consultancy; Daiichi Sankyo: Research Funding; Regeneron: Research Funding; Genentech: Consultancy. Manasanch:Sanofi: Honoraria, Research Funding; Novartis: Research Funding; JW Pharma: Research Funding; Merck: Research Funding; Quest Diagnostics: Research Funding; Takeda: Honoraria; BMS: Honoraria; Glaxo Smith Kline: Honoraria; Adaptive Biotechnologies: Honoraria. Pahl:Heidelberg Pharma AG: Current Employment. Orlowski:Sanofi-Aventis, Servier, Takeda Pharmaceuticals North America, Inc.: Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen, Inc., AstraZeneca, BMS, Celgene, EcoR1 Capital LLC, Forma Therapeutics, Genzyme, GSK Biologicals, Ionis Pharmaceuticals, Inc., Janssen Biotech, Juno Therapeutics, Kite Pharma, Legend Biotech USA, Molecular Partners, Regeneron Pharmaceuticals, Inc.,: Honoraria, Membership on an entity's Board of Directors or advisory committees; Laboratory research funding from BioTheryX, and clinical research funding from CARsgen Therapeutics, Celgene, Exelixis, Janssen Biotech, Sanofi-Aventis, Takeda Pharmaceuticals North America, Inc.: Research Funding; STATinMED Research: Consultancy; Founder of Asylia Therapeutics, Inc., with associated patents and an equity interest, though this technology does not bear on the current submission.: Current equity holder in private company, Patents & Royalties.
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King, Richard, Ann Friedman, Zesen Lin, and Rami Khoriaty. "Functional Overlap between the SEC23 Paralogs Suggests a Novel Treatment Paradigm for Congenital Dyserythropoietic Anemia Type II." Blood 134, Supplement_1 (November 13, 2019): 2221. http://dx.doi.org/10.1182/blood-2019-129669.
Full textAbstract:
Congenital dyserythropoietic anemia type II (CDAII), an autosomal recessive disease characterized by ineffective erythropoiesis and increased percentage of bi-nucleated erythroid precursors in the bone marrow (BM), results from loss of function mutations in SEC23B, which encodes a core component of COPII vesicles. Approximately 8,000 secretory proteins are transported from the endoplasmic reticulum to the Golgi apparatus via COPII vesicles, suggesting that a defect in this pathway would result in a profound systemic phenotype. However, CDAII patients exhibit a specific erythroid phenotype, with no other defects described. Mammals have 2 paralogs for SEC23, SEC23A and SEC23B. In contrast to SEC23B mutations, bi-allelic SEC23A loss of function mutations in humans result in cranio-lenticulo-sutural dysplasia, a disease characterized by skeletal defect but normal erythropoiesis. We previously demonstrated that a SEC23B-A chimeric protein composed of the first 122 amino acids of SEC23B followed by amino acids 123-765 of SEC23A overlaps in function with SEC23B, suggesting that the 2 SEC23 paralogs are functionally interchangeable. However, to rule out the possibility that the functional overlap was due to the first 122 amino acids of SEC23B, we generated a bacterial artificial chromosome (BAC) transgene that expresses the full Sec23a coding sequence from the endogenous genomic locus of Sec23b (Sec23b-a BAC). We crossed the Sec23b-a BAC to the Sec23b null allele (Sec23b-) and demonstrated that this BAC rescues the phenotype of mice deficient in Sec23b (Sec23b-/-). Therefore, we now conclusively demonstrate that the SEC23A protein functionally replaces SEC23B when expressed from the endogenous regulatory elements of Sec23b. We have previously shown that mice with erythroid-specific and pan-hematopoietic SEC23B deficiency exhibit a normal erythroid phenotype. In light of the functional overlap between SEC23A and SEC23B, we hypothesized that mice with erythroid-specific deficiency for SEC23A, alone or in combination with SEC23B, might exhibit an erythroid phenotype. First, we generated mice with erythroid-specific (EpoR-Cre) SEC23A deficiency. These mice were observed at the expected Mendelian ratios at weaning. Complete (or near complete) excision of the Sec23a floxed (Sec23afl) allele was confirmed in the erythroid cells. Peripheral blood counts, BM cellularity and morphology, and percent and distribution of BM erythroid cells among the 5 stages of maturation were indistinguishable between mice with erythroid SEC23A deficiency and wildtype littermate controls. Additionally, the percentage of bi-nucleated erythroid precursors were not increased in Sec23afl/flEpoR-Cre+ mice. Thus, mice with erythroid-specific SEC23A deficiency do not exhibit an erythroid phenotype. Similarly, mice with pan-hematopoietic SEC23A deficiency (Vav1-Cre) do not exhibit a hematologic phenotype. Next, we generated mice with Sec23a deletion and Sec23b haploinsufficiency in the erythroid compartments. These mice exhibited normal survival, a mild reduction in hemoglobin levels (p = 0.014), and a block in late erythroid maturation (Stage V erythroid cells were reduced to 22.6% compared to 30.3% in control mice; p=0.08). In contrast, mice with erythroid-specific deletion for all 4 Sec23 alleles (combined SEC23A/B deficiency) died at mid-embryogenesis exhibiting reduced size and appearing pale compared to wildtype littermate controls, with histologic evidence of dyserythropoiesis reminiscent of human CDAII. Overall, these results suggest a requirement for a threshold level of total SEC23 (combined SEC23A/B) expression in the erythroid compartment. These results also suggest that the defect in CDAII is intrinsic to the RBC. Finally, we generated K562 cells with either SEC23B or SEC23A deletion using CRISPR/Cas9 genome editing. SEC23B or SEC23A deletion alone was tolerated in the K562 cells. However, combined deletion of SEC23A and SEC23B was not tolerated. Taken together, the results summarized above demonstrate that SEC23A and SEC23B appear to compensate for one another's function in murine and human erythroid cells. This finding suggests a potential therapeutic role for increasing expression of SEC23A to compensate for SEC23B deficiency in CDAII. This work is currently ongoing. Disclosures No relevant conflicts of interest to declare.
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Ambrosini, Chiara, Eliana Destefanis, Eyemen Kheir, Francesca Broso, Federica Alessandrini, Sara Longhi, Nicolò Battisti, et al. "Translational enhancement by base editing of the Kozak sequence rescues haploinsufficiency." Nucleic Acids Research, September 27, 2022. http://dx.doi.org/10.1093/nar/gkac799.
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Abstract A variety of single-gene human diseases are caused by haploinsufficiency, a genetic condition by which mutational inactivation of one allele leads to reduced protein levels and functional impairment. Translational enhancement of the spare allele could exert a therapeutic effect. Here we developed BOOST, a novel gene-editing approach to rescue haploinsufficiency loci by the change of specific single nucleotides in the Kozak sequence, which controls translation by regulating start codon recognition. We evaluated for translational strength 230 Kozak sequences of annotated human haploinsufficient genes and 4621 derived variants, which can be installed by base editing, by a high-throughput reporter assay. Of these variants, 149 increased the translation of 47 Kozak sequences, demonstrating that a substantial proportion of haploinsufficient genes are controlled by suboptimal Kozak sequences. Validation of 18 variants for 8 genes produced an average enhancement in an expression window compatible with the rescue of the genetic imbalance. Base editing of the NCF1 gene, whose monoallelic loss causes chronic granulomatous disease, resulted in the desired increase of NCF1 (p47phox) protein levels in a relevant cell model. We propose BOOST as a fine-tuned approach to modulate translation, applicable to the correction of dozens of haploinsufficient monogenic disorders independently of the causing mutation.
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Fear, Vanessa S., Catherine A. Forbes, Denise Anderson, Sebastian Rauschert, Genevieve Syn, Nicole Shaw, Sarra Jamieson, Michelle Ward, Gareth Baynam, and Timo Lassmann. "CRISPR single base editing, neuronal disease modelling and functional genomics for genetic variant analysis: pipeline validation using Kleefstra syndrome EHMT1 haploinsufficiency." Stem Cell Research & Therapy 13, no. 1 (February 9, 2022). http://dx.doi.org/10.1186/s13287-022-02740-3.
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Abstract Background Over 400 million people worldwide are living with a rare disease. Next Generation Sequencing (NGS) identifies potential disease causative genetic variants. However, many are identified as variants of uncertain significance (VUS) and require functional laboratory validation to determine pathogenicity, and this creates major diagnostic delays. Methods In this study we test a rapid genetic variant assessment pipeline using CRISPR homology directed repair to introduce single nucleotide variants into inducible pluripotent stem cells (iPSCs), followed by neuronal disease modelling, and functional genomics on amplicon and RNA sequencing, to determine cellular changes to support patient diagnosis and identify disease mechanism. Results As proof-of-principle, we investigated an EHMT1 (Euchromatin histone methyltransferase 1; EHMT1 c.3430C > T; p.Gln1144*) genetic variant pathogenic for Kleefstra syndrome and determined changes in gene expression during neuronal progenitor cell differentiation. This pipeline rapidly identified Kleefstra syndrome in genetic variant cells compared to healthy cells, and revealed novel findings potentially implicating the key transcription factors REST and SP1 in disease pathogenesis. Conclusion The study pipeline is a rapid, robust method for genetic variant assessment that will support rare diseases patient diagnosis. The results also provide valuable information on genome wide perturbations key to disease mechanism that can be targeted for drug treatments.
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Ye, Tao, Yangyang Duan, Hayley W. S. Tsang, He Xu, Yuewen Chen, Han Cao, Yu Chen, Amy K. Y. Fu, and Nancy Y. Ip. "Efficient manipulation of gene dosage in human iPSCs using CRISPR/Cas9 nickases." Communications Biology 4, no. 1 (February 12, 2021). http://dx.doi.org/10.1038/s42003-021-01722-0.
Full textAbstract:
AbstractThe dysregulation of gene dosage due to duplication or haploinsufficiency is a major cause of autosomal dominant diseases such as Alzheimer’s disease. However, there is currently no rapid and efficient method for manipulating gene dosage in a human model system such as human induced pluripotent stem cells (iPSCs). Here, we demonstrate a simple and precise method to simultaneously generate iPSC lines with different gene dosages using paired Cas9 nickases. We first generate a Cas9 nickase variant with broader protospacer-adjacent motif specificity to expand the targetability of double-nicking–mediated genome editing. As a proof-of-concept study, we examine the gene dosage effects on an Alzheimer’s disease patient-derived iPSC line that carries three copies of APP (amyloid precursor protein). This method enables the rapid and simultaneous generation of iPSC lines with monoallelic, biallelic, or triallelic knockout of APP. The cortical neurons generated from isogenically corrected iPSCs exhibit gene dosage-dependent correction of disease-associated phenotypes of amyloid-beta secretion and Tau hyperphosphorylation. Thus, the rapid generation of iPSCs with different gene dosages using our method described herein can be a useful model system for investigating disease mechanisms and therapeutic development.
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Warren, Emily B., Juan A. Briano, Jacob Ellegood, Taylor DeYoung, Jason P. Lerch, and Eric M. Morrow. "Mouse Model of 17q12 deletion shows defects in craniofacial, brain and kidney development, and in glucose homeostasis." Disease Models & Mechanisms, November 14, 2022. http://dx.doi.org/10.1242/dmm.049752.
Full textAbstract:
17q12 deletion (17q12Del) syndrome is a copy number variant (CNV) disorder associated with neurodevelopmental disorders (NDDs) and renal cysts and diabetes syndrome (RCAD). Using CRISPR/Cas9 genome-editing, we generated a mouse model of 17q12Del syndrome on both inbred (C57BL/6N) and outbred (CD-1) genetic backgrounds. On C57BL/6N, the 17q12Del mouse has severe head development defects, potentially mediated by haploinsufficiency of Lhx1, a gene within the interval that controls head development. Phenotypes include brain malformations, particularly disruption of the telencephalon, and craniofacial defects. On the CD-1 background, the 17q12Del mouse survives to adulthood and shows milder craniofacial and brain abnormalities. We report postnatal brain defects using automated MRI-based morphometry. In addition, we demonstrate renal and blood glucose abnormalities relevant to RCAD. On both genetic backgrounds, we found sex-specific presentations, with male 17q12Del mice exhibiting higher penetrance and more severe phenotypes. Results from these experiments pinpoint specific developmental defects and pathways that guide clinical studies and a mechanistic understanding of the human 17q12Del syndrome. This mouse mutant represents the first and only experimental model to date for the 17q12 CNV disorder.