CRISPR-ERA for Switching Off (Onco) Genes

Genome-editing nucleases like the popular CRISPR/Cas9 enable the generation of knockout cell lines and null zygotes by inducing site-specific double-stranded breaks (DSBs) within a genome. In most cases, when a DNA template is not present, the DSB is repaired by nonhomologous end joining (NHEJ), resulting in small nucleotide insertions or deletions that can be used to construct knockout alleles. However, for several reasons, these mutations do not produce the desired null result in all cases, instead generating a similar protein with functional activity. This undesirable effect could limit the therapeutic efficiency of gene therapy strategies focused on abrogating oncogene expression by CRISPR/Cas9 and should be taken into account. This chapter reviews the irruption of CRISPR technology for gene silencing and its application in gene therapy

CRISPR-Cas9 Technology as a Tool to Target Gene Drivers in Cancer: Proof of Concept and New Opportunities to Treat Chronic Myeloid Leukemia

Chronic myeloid leukemia (CML) is a hematopoietic malignancy produced by a unique oncogenic event involving the constitutively active tyrosine-kinase (TK) BCR/ABL1. TK inhibitors (TKI) changed its prognosis and natural history. Unfortunately, ABL1 remains unaffected by TKIs. Leukemic stem cells (LSCs) remain, and resistant mutations arise during treatment. To address this problem, we have designed a therapeutic CRISPR-Cas9 deletion system targeting BCR/ABL1. The system was efficiently electroporated to cell lines, LSCs from a CML murine model, and LSCs from CML patients at diagnosis, generating a specific ABL1 null mutation at high efficiency and allowing the edited leukemic cells to be detected and tracked. The CRISPR-Cas9 deletion system triggered cell proliferation arrest and apoptosis in murine and human CML cell lines. Patient and murine-derived xenografts with CRISPR-edited LSCs in NOD SCID gamma niches revealed that normal multipotency and repopulation ability of CRISPR edited LSCs were fully restored. Normal hematopoiesis was restored, avoiding myeloid bias. To the best of our knowledge, we show for the first time how a CRISPR-Cas9 deletion system efficiently interrupts BCR/ABL1 oncogene in primary LSCs to bestow a therapeutic benefit. This study is a proof of concept for genome editing in all those diseases, like CML, sustained by a single oncogenic event, opening up new therapeutic opportunities

A novel nonsense variant in TPM4 caused dominant macrothrombocytopenia, mild bleeding tendency and disrupted cytoskeleton remodeling

[Background]: Rare inherited thrombocytopenias are caused by alterations in genes involved in megakaryopoiesis, thrombopoiesis and/or platelet release. Diagnosis is challenging due to poor specificity of platelet laboratory assays, large numbers of culprit genes, and difficult assessment of the pathogenicity of novel variants. [Objectives]: To characterize the clinical and laboratory phenotype, and identifying the underlying molecular alteration, in a pedigree with thrombocytopenia of uncertain etiology. [Patients/Methods]: Index case was enrolled in our Spanish multicentric project of inherited platelet disorders due to lifelong thrombocytopenia and bleeding. Bleeding score was recorded by ISTH‐BAT. Laboratory phenotyping consisted of blood cells count, blood film, platelet aggregation and flow cytometric analysis. Genotyping was made by whole‐exome sequencing (WES). Cytoskeleton proteins were analyzed in resting/spreading platelets by immunofluorescence and immunoblotting. [Results]: Five family members displayed lifelong mild thrombocytopenia with a high number of enlarged platelets in blood film, and mild bleeding tendency. Patient's platelets showed normal aggregation and granule secretion response to several agonists. WES revealed a novel nonsense variant (c.322C>T; p.Gln108*) in TPM4 (NM_003290.3), the gene encoding for tropomyosin‐4 (TPM4). This variant led to impairment of platelet spreading capacity after stimulation with TRAP‐6 and CRP, delocalization of TPM4 in activated platelets, and significantly reduced TPM4 levels in platelet lysates. Moreover, the index case displayed up‐regulation of TPM2 and TPM3 mRNA levels. [Conclusions]: This study identifies a novel TPM4 nonsense variant segregating with macrothrombocytopenia and impaired platelet cytoskeletal remodeling and spreading. These findings support the relevant role of TPM4 in thrombopoiesis and further expand our knowledge of TPM4‐related thrombocytopenia.This work was partially supported by grants from Instituto de Salud Carlos III (ISCIII) and Feder (PI17/01966, PI20/00926), Gerencia Regional de Salud (GRS2061A/19, GRS2135/A/2020, GRS2314/A/2021), Fundación Mutua Madrileña (FMM, AP172142019) and Sociedad Española de Trombosis y Hemostasia (SETHFETH; Premio López Borrasca 2019 and Ayuda a Grupos de Trabajo en Patología Hemorrágica 2020 and 2021).Peer reviewe

Environmental Effects on Cognitive Performance in a 6 to 14 Month-Old Child Population.

The use of the A not B task with delays is presented as an instrument for the evaluation of cognitive performance depending on the prefrontal dorsolateral cerebral cortex. This task was admnistered to 250 children from 6 to 14 months old, from different socio-economical rearing environments (as classified by the Instituto Nacional de Estadística y Censo, INDEC). Results showed an impact of environmental variables on child performance: as a group children from homes providing unsatisfied basic needs homes were less efficient than those who were reared in homes in which their basic needs were satisfied

Characterization of the platelet phenotype caused by a germline RUNX1 Variant in a CRISPR/Cas9-generated murine model

RUNX1-related disorder (RUNX1-RD) is caused by germline variants affecting the RUNX1 gene. This rare, heterogeneous disorder has no specific clinical or laboratory phenotype, making genetic diagnosis necessary. Although international recommendations have been established to classify the pathogenicity of variants, identifying the causative alteration remains a challenge in RUNX1-RD. Murine models may be useful not only for definitively settling the controversy about the pathogenicity of certain RUNX1 variants, but also for elucidating the mechanisms of molecular pathogenesis. Therefore, we developed a knock-in murine model, using the CRISPR/Cas9 system, carrying the RUNX1 p.Leu43Ser variant (mimicking human p.Leu56Ser) to study its pathogenic potential and mechanisms of platelet dysfunction. A total number of 75 mice were generated; 25 per genotype (RUNX1WT/WT, RUNX1WT/L43S, and RUNX1L43S/L43S). Platelet phenotype was assessed by flow cytometry and confocal microscopy. On average, RUNX1L43S/L43S and RUNX1WT/L43S mice had a significantly longer tail-bleeding time than RUNX1WT/WT mice, indicating the variant's involvement in hemostasis. However, only homozygous mice displayed mild thrombocytopenia. RUNX1L43S/L43S and RUNX1WT/L43S displayed impaired agonist-induced spreading and α-granule release, with no differences in δ-granule secretion. Levels of integrin αIIbβ3 activation, fibrinogen binding, and aggregation were significantly lower in platelets from RUNX1L43S/L43S and RUNX1WT/L43S using phorbol 12-myristate 13-acetate (PMA), adenosine diphosphate (ADP), and high thrombin doses. Lower levels of PKC phosphorylation in RUNX1L43S/L43S and RUNX1WT/L43S suggested that the PKC-signaling pathway was impaired. Overall, we demonstrated the deleterious effect of the RUNX1 p.Leu56Ser variant in mice via the impairment of integrin αIIbβ3 activation, aggregation, α-granule secretion, and platelet spreading, mimicking the phenotype associated with RUNX1 variants in the clinical setting.This work was partially supported by grants from Instituto de Salud Carlos III (ISCIII) and Feder (PI17/01311, PI17/01966, and CB15/00055), Fundación Séneca (19873/GERM/15), Gerencia Regional de Salud (GRS 2061A/19 and 1647/A/17), Fundación Mutua Madrileña (FMM, AP172142019), and Sociedad Española de Trombosis y Hemostasia (SETH-FETH; Premio López Borrasca 2019 and Ayuda a Grupos de Trabajo en Patología Hemorrágica 2019). The authors' research on IPDs is conducted in accordance with the aims of the Functional and Molecular Characterization of Patients with Inherited Platelet Disorders Project, which is supported by the Hemorrhagic Diathesis Working Group of the Spanish Society of Thrombosis and Haemostasis. A.M.-Q., C.F.-I., and L.H.-C. were supported by predoctoral grants from the Junta de Castilla y León, Spain. E.V. was supported by the predoctoral grant from the University of Salamanca, Spain. IG-T and RB were supported by "Contratos postdoctorales Programa II) from the University of Salamanca, Spain

Stroma-Mediated Resistance to S63845 and Venetoclax through MCL-1 and BCL-2 Expression Changes Induced by miR-193b-3p and miR-21-5p Dysregulation in Multiple Myeloma.

BH3-mimetics targeting anti-apoptotic proteins such as MCL-1 (S63845) or BCL-2 (venetoclax) are currently being evaluated as effective therapies for the treatment of multiple myeloma (MM). Interleukin 6, produced by mesenchymal stromal cells (MSCs), has been shown to modify the expression of anti-apoptotic proteins and their interaction with the pro-apoptotic BIM protein in MM cells. In this study, we assess the efficacy of S63845 and venetoclax in MM cells in direct co-culture with MSCs derived from MM patients (pMSCs) to identify additional mechanisms involved in the stroma-induced resistance to these agents. MicroRNAs miR-193b-3p and miR-21-5p emerged among the top deregulated miRNAs in myeloma cells when directly co-cultured with pMSCs, and we show their contribution to changes in MCL-1 and BCL-2 protein expression and in the activity of S63845 and venetoclax. Additionally, direct contact with pMSCs under S63845 and/or venetoclax treatment modifies myeloma cell dependence on different BCL-2 family anti-apoptotic proteins in relation to BIM, making myeloma cells more dependent on the non-targeted anti-apoptotic protein or BCL-XL. Finally, we show a potent effect of the combination of S63845 and venetoclax even in the presence of pMSCs, which supports this combinatorial approach for the treatment of MM

ICO-ICS Praxis para el tratamiento de la leucemia linfática crónica

Tractament mèdic; Tractament amb irradiació; Leucèmia limfàtica crònicaMedical treatment; Irradiation treatment; Chronic lymphocytic leukemiaTratamiento médico; Tratamiento con irradiación; Leucemia linfática crónicaLa leucèmia limfàtica crònica (LLC) és una alteració hematopoètica monoclonal caracteritzada per una expansió progressiva de limfòcits de la línia B. Aquests limfòcits, madurs des del punt de vista morfològic, però menys madurs des del punt de vista immunològic, s’acumulen a la sang, la medul·la òssia, els nòduls limfàtics i la melsa. Els principals objectius d’aquesta ICO-ICSPraxi són: - Desenvolupar, difondre, implementar i avaluar resultats de la ICO-ICSPraxi de la leucèmia limfàticacrònica (LLC). - Disminuir la variabilitat terapèutica entre els pacients tractats als diferents centres d'aquesta xarxa. - Implementar els resultats de la terapèutica en els pacients amb LLC tractats d'acord amb lesrecomanacions d'aquesta guia

ICO-ICS Praxis para el tratamiento médico y con irradiación del linfoma B difuso de célula grande

Tractament mèdic; Tractament amb irradiació; Limfoma B difús de cèl·lula granMedical treatment; Irradiation treatment; Diffuse large cell B lymphomaTratamiento médico; Tratamiento con irradiación; Linfoma B difuso de célula grandeEls limfomes no hodgkinians (LNH) són neoplàsies originades en cèl·lules limfoides en diferents estats maduratius, la qual cosa explica la gran heterogeneïtat biològica i clínica d’aquests tumors. Hi ha diversos sistemes de classificació, però el més utilitzat és la Classificació euroamericana revisada de limfomes de l'Organització Mundial de la Salut. El limfoma B difús de cèl·lula gran suposa aproximadament entre el 30-40% dels limfomes dels adults. Habitualment afecta adults amb una edat mitjana superior a 60 anys i el 60% dels pacients presenten els anomenats símptomes B (febre de 38 ºC o més, pèrdua de pes de més del 10% i/o sudoració nocturna. La majoria de casos són formes de novo però també poden ser deguts a la progressió o transformació d’una malaltia limfoproliferativa prèvia. Els objectius d'aquest document són: Desenvolupar, difondre, implementar i avaluar resultats de la ICO-ICSPraxi del limfoma B difús de cèl·lula gran. - Disminuir la variabilitat terapèutica entre els pacients tractats als diferents centres d'aquesta institució. - Implementar els resultats de la terapèutica en els pacients amb limfoma B difús de cèl·lula gran tractats d’acord amb les recomanacions d’aquesta guia