RESUMEN
Schwann cell programming during myelination involves transcriptional networks that activate gene expression but also repress genes that are active in neural crest/embryonic differentiation of Schwann cells. We previously found that a Schwann cell-specific deletion of the EED subunit of the Polycomb Repressive Complex (PRC2) led to inappropriate activation of many such genes. Moreover, some of these genes become re-activated in the pro-regenerative response of Schwann cells to nerve injury, and we found premature activation of the nerve injury program in a Schwann cell-specific knockout of Eed. Polycomb-associated histone modifications include H3K27 trimethylation formed by PRC2 and H2AK119 monoubiquitination (H2AK119ub1), deposited by Polycomb repressive complex 1 (PRC1). We recently found dynamic regulation of H2AK119ub1 in Schwann cell genes after injury. Therefore, we hypothesized that H2AK119 deubiquitination modulates the dynamic polycomb repression of genes involved in Schwann cell maturation. To determine the role of H2AK119 deubiquitination, we generated a Schwann cell-specific knockout of the H2AK119 deubiquitinase Bap1 (BRCA1-associated protein). We found that loss of Bap1 causes tomacula formation, decreased axon diameters and eventual loss of myelinated axons. The gene expression changes are accompanied by redistribution of H2AK119ub1 and H3K27me3 modifications to extragenic sites throughout the genome. BAP1 interacts with OGT in the PR-DUB complex, and our data suggest that the PR-DUB complex plays a multifunctional role in repression of the injury program. Overall, our results indicate Bap1 is required to restrict the spread of polycomb-associated histone modifications in Schwann cells and to promote myelin homeostasis in peripheral nerve.
Asunto(s)
Proteínas de Drosophila , Histonas , Histonas/metabolismo , Complejo Represivo Polycomb 2/genética , Células de Schwann/metabolismo , Proteínas de Drosophila/metabolismo , Homeostasis , Enzimas Desubicuitinizantes/metabolismoRESUMEN
Retroviral gene therapy has proved efficacious for multiple genetic diseases of the hematopoietic system, but roughly half of clinical gene therapy trial protocols using gammaretroviral vectors have reported leukemias in some of the patients treated. In dramatic contrast, 39 adenosine deaminase-deficient severe combined immunodeficiency (ADA-SCID) patients have been treated with 4 distinct gammaretroviral vectors without oncogenic consequence. We investigated clonal dynamics and diversity in a cohort of 15 ADA-SCID children treated with gammaretroviral vectors and found clear evidence of genotoxicity, indicated by numerous common integration sites near proto-oncogenes and by increased abundance of clones with integrations near MECOM and LMO2 These clones showed stable behavior over multiple years and never expanded to the point of dominance or dysplasia. One patient developed a benign clonal dominance that could not be attributed to insertional mutagenesis and instead likely resulted from expansion of a transduced natural killer clone in response to chronic Epstein-Barr virus viremia. Clonal diversity and T-cell repertoire, measured by vector integration site sequencing and T-cell receptor ß-chain rearrangement sequencing, correlated significantly with the amount of busulfan preconditioning delivered to patients and to CD34+ cell dose. These data, in combination with results of other ADA-SCID gene therapy trials, suggest that disease background may be a crucial factor in leukemogenic potential of retroviral gene therapy and underscore the importance of cytoreductive conditioning in this type of gene therapy approach.
Asunto(s)
Adenosina Desaminasa/deficiencia , Agammaglobulinemia/genética , Agammaglobulinemia/terapia , Antineoplásicos Alquilantes/uso terapéutico , Busulfano/uso terapéutico , Gammaretrovirus/genética , Terapia Genética/métodos , Vectores Genéticos/uso terapéutico , Inmunodeficiencia Combinada Grave/genética , Inmunodeficiencia Combinada Grave/terapia , Proteínas Adaptadoras Transductoras de Señales/genética , Adenosina Desaminasa/genética , Agammaglobulinemia/patología , Niño , Proteínas de Unión al ADN/genética , Vectores Genéticos/genética , Humanos , Proteínas con Dominio LIM/genética , Proteína del Locus del Complejo MDS1 y EV11 , Proteínas Proto-Oncogénicas/genética , Proto-Oncogenes/genética , Inmunodeficiencia Combinada Grave/patología , Linfocitos T/citología , Linfocitos T/efectos de los fármacos , Linfocitos T/metabolismo , Linfocitos T/patología , Factores de Transcripción/genéticaRESUMEN
The use of engineered nucleases combined with a homologous DNA donor template can result in targeted gene correction of the sickle cell disease mutation in hematopoietic stem and progenitor cells. However, because of the high homology between the adjacent human ß- and δ-globin genes, off-target cleavage is observed at δ-globin when using some endonucleases targeted to the sickle mutation in ß-globin. Introduction of multiple double-stranded breaks by endonucleases has the potential to induce intergenic alterations. Using a novel droplet digital PCR assay and high-throughput sequencing, we characterized the frequency of rearrangements between the ß- and δ-globin paralogs when delivering these nucleases. Pooled CD34+ cells and colony-forming units from sickle bone marrow were treated with nuclease only or including a donor template and then analyzed for potential gene rearrangements. It was observed that, in pooled CD34+ cells and colony-forming units, the intergenic ß-δ-globin deletion was the most frequent rearrangement, followed by inversion of the intergenic fragment, with the inter-chromosomal translocation as the least frequent. No rearrangements were observed when endonuclease activity was restricted to on-target ß-globin cleavage. These findings demonstrate the need to develop site-specific endonucleases with high specificity to avoid unwanted gene alterations.
Asunto(s)
Edición Génica , Variación Genética , Células Madre Hematopoyéticas/metabolismo , Globinas beta/genética , Conversión Génica , Reordenamiento Génico , Marcación de Gen , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Técnicas de Amplificación de Ácido Nucleico , Translocación GenéticaRESUMEN
Sickle cell disease (SCD) is characterized by a single point mutation in the seventh codon of the ß-globin gene. Site-specific correction of the sickle mutation in hematopoietic stem cells would allow for permanent production of normal red blood cells. Using zinc-finger nucleases (ZFNs) designed to flank the sickle mutation, we demonstrate efficient targeted cleavage at the ß-globin locus with minimal off-target modification. By co-delivering a homologous donor template (either an integrase-defective lentiviral vector or a DNA oligonucleotide), high levels of gene modification were achieved in CD34(+) hematopoietic stem and progenitor cells. Modified cells maintained their ability to engraft NOD/SCID/IL2rγ(null) mice and to produce cells from multiple lineages, although with a reduction in the modification levels relative to the in vitro samples. Importantly, ZFN-driven gene correction in CD34(+) cells from the bone marrow of patients with SCD resulted in the production of wild-type hemoglobin tetramers.
Asunto(s)
Anemia de Células Falciformes/genética , Anemia de Células Falciformes/terapia , Terapia Genética , Células Madre Hematopoyéticas/metabolismo , Mutación , Globinas beta/genética , Anemia de Células Falciformes/patología , Animales , Antígenos CD34/análisis , Secuencia de Bases , Células de la Médula Ósea/metabolismo , Células de la Médula Ósea/patología , Células Cultivadas , Endodesoxirribonucleasas/metabolismo , Sangre Fetal/trasplante , Sitios Genéticos , Trasplante de Células Madre Hematopoyéticas , Células Madre Hematopoyéticas/patología , Humanos , Ratones , Ratones Endogámicos NOD , Ratones SCID , Datos de Secuencia Molecular , Dedos de ZincRESUMEN
Although clonal studies of lineage potential have been extensively applied to organ specific stem and progenitor cells, much less is known about the clonal origins of lineages formed from the germ layers in early embryogenesis. We applied lentiviral tagging followed by vector integration site analysis (VISA) with high-throughput sequencing to investigate the ontogeny of the hematopoietic, endothelial and mesenchymal lineages as they emerge from human embryonic mesoderm. In contrast to studies that have used VISA to track differentiation of self-renewing stem cell clones that amplify significantly over time, we focused on a population of progenitor clones with limited self-renewal capability. Our analyses uncovered the critical influence of sampling on the interpretation of lentiviral tag sharing, particularly among complex populations with minimal clonal duplication. By applying a quantitative framework to estimate the degree of undersampling we revealed the existence of tripotent mesodermal progenitors derived from pluripotent stem cells, and the subsequent bifurcation of their differentiation into bipotent endothelial/hematopoietic or endothelial/mesenchymal progenitors. Stem Cells 2016;34:1239-1250.
Asunto(s)
Diferenciación Celular , Técnicas Genéticas , Mesodermo/citología , Células Madre Multipotentes/citología , Animales , Antígenos CD/metabolismo , Línea Celular , Linaje de la Célula , Separación Celular , Células Clonales , Citometría de Flujo , Humanos , Lentivirus/metabolismo , RatonesRESUMEN
BACKGROUND AIMS: Gene therapy by autologous hematopoietic stem cell transplantation (HSCT) represents a new approach to treat sickle cell disease (SCD). Optimization of the manufacture, characterization and testing of the transduced hematopoietic stem cell final cell product (FCP), as well as an in depth in vivo toxicology study, are critical for advancing this approach to clinical trials. METHODS: Data are shown to evaluate and establish the feasibility of isolating, transducing with the Lenti/ßAS3-FB vector and cryopreserving CD34+ cells from human bone marrow (BM) at clinical scale. In vitro and in vivo characterization of the FCP was performed, showing that all the release criteria were successfully met. In vivo toxicology studies were conducted to evaluate potential toxicity of the Lenti/ßAS3-FB LV in the context of a murine BM transplant. RESULTS: Primary and secondary transplantation did not reveal any toxicity from the lentiviral vector. Additionally, vector integration site analysis of murine and human BM cells did not show any clonal skewing caused by insertion of the Lenti/ßAS3-FB vector in cells from primary and secondary transplanted mice. CONCLUSIONS: We present here a complete protocol, thoroughly optimized to manufacture, characterize and establish safety of a FCP for gene therapy of SCD.
Asunto(s)
Anemia de Células Falciformes/terapia , Terapia Genética/métodos , Trasplante de Células Madre Hematopoyéticas/métodos , Células Madre Hematopoyéticas , Adulto , Animales , Antígenos CD34/metabolismo , Células de la Médula Ósea , Trasplante de Médula Ósea , Estudios de Casos y Controles , Ensayos Clínicos Fase I como Asunto , Vectores Genéticos , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/fisiología , Humanos , Lentivirus/genética , Ratones Endogámicos NOD , Transducción Genética , Trasplante Autólogo/métodosRESUMEN
Targeted genome editing technology can correct the sickle cell disease mutation of the ß-globin gene in hematopoietic stem cells. This correction supports production of red blood cells that synthesize normal hemoglobin proteins. Here, we demonstrate that Transcription Activator-Like Effector Nucleases (TALENs) and the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 nuclease system can target DNA sequences around the sickle-cell mutation in the ß-globin gene for site-specific cleavage and facilitate precise correction when a homologous donor template is codelivered. Several pairs of TALENs and multiple CRISPR guide RNAs were evaluated for both on-target and off-target cleavage rates. Delivery of the CRISPR/Cas9 components to CD34+ cells led to over 18% gene modification in vitro. Additionally, we demonstrate the correction of the sickle cell disease mutation in bone marrow derived CD34+ hematopoietic stem and progenitor cells from sickle cell disease patients, leading to the production of wild-type hemoglobin. These results demonstrate correction of the sickle mutation in patient-derived CD34+ cells using CRISPR/Cas9 technology.
Asunto(s)
Anemia de Células Falciformes/genética , Sistemas CRISPR-Cas , Edición Génica , Células Madre Hematopoyéticas/metabolismo , Mutación , Reparación del Gen Blanco , Globinas beta/genética , Anemia de Células Falciformes/terapia , Secuencia de Bases , Línea Celular , División del ADN , Marcación de Gen , Sitios Genéticos , Humanos , Unión Proteica , ARN Guía de Kinetoplastida , Nucleasas de los Efectores Tipo Activadores de la Transcripción/metabolismoRESUMEN
Lentiviral vectors almost universally use heterologous internal promoters to express transgenes. One of the most commonly used promoter fragments is a 1.2-kb sequence from the human ubiquitin C (UBC) gene, encompassing the promoter, some enhancers, first exon, first intron and a small part of the second exon of UBC. Because splicing can occur after transcription of the vector genome during vector production, we investigated whether the intron within the UBC promoter fragment is faithfully transmitted to target cells. Genetic analysis revealed that more than 80% of proviral forms lack the intron of the UBC promoter. The human elongation factor 1 alpha (EEF1A1) promoter fragment intron was not lost during lentiviral packaging, and this difference between the UBC and EEF1A1 promoter introns was conferred by promoter exonic sequences. UBC promoter intron loss caused a 4-fold reduction in transgene expression. Movement of the expression cassette to the opposite strand prevented intron loss and restored full expression. This increase in expression was mostly due to non-classical enhancer activity within the intron, and movement of putative intronic enhancer sequences to multiple promoter-proximal sites actually repressed expression. Reversal of the UBC promoter also prevented intron loss and restored full expression in bidirectional lentiviral vectors.
Asunto(s)
Vectores Genéticos , Intrones , Lentivirus/genética , Regiones Promotoras Genéticas , Ubiquitina C/genética , Elementos de Facilitación Genéticos , Exones , Expresión Génica , Células HEK293 , Humanos , Células K562 , Factor 1 de Elongación Peptídica/genética , Empalme del ARNRESUMEN
Autologous hematopoietic stem cell (HSC) gene therapy for sickle cell disease has the potential to treat this illness without the major immunological complications associated with allogeneic transplantation. However, transduction efficiency by ß-globin lentiviral vectors using CD34-enriched cell populations is suboptimal and large vector production batches may be needed for clinical trials. Transducing a cell population more enriched for HSC could greatly reduce vector needs and, potentially, increase transduction efficiency. CD34(+) /CD38(-) cells, comprising â¼1%-3% of all CD34(+) cells, were isolated from healthy cord blood CD34(+) cells by fluorescence-activated cell sorting and transduced with a lentiviral vector expressing an antisickling form of beta-globin (CCL-ß(AS3) -FB). Isolated CD34(+) /CD38(-) cells were able to generate progeny over an extended period of long-term culture (LTC) compared to the CD34(+) cells and required up to 40-fold less vector for transduction compared to bulk CD34(+) preparations containing an equivalent number of CD34(+) /CD38(-) cells. Transduction of isolated CD34(+) /CD38(-) cells was comparable to CD34(+) cells measured by quantitative PCR at day 14 with reduced vector needs, and average vector copy/cell remained higher over time for LTC initiated from CD34(+) /38(-) cells. Following in vitro erythroid differentiation, HBBAS3 mRNA expression was similar in cultures derived from CD34(+) /CD38(-) cells or unfractionated CD34(+) cells. In vivo studies showed equivalent engraftment of transduced CD34(+) /CD38(-) cells when transplanted in competition with 100-fold more CD34(+) /CD38(+) cells. This work provides initial evidence for the beneficial effects from isolating human CD34(+) /CD38(-) cells to use significantly less vector and potentially improve transduction for HSC gene therapy.
Asunto(s)
Terapia Genética , Trasplante de Células Madre Hematopoyéticas , Células Madre Hematopoyéticas/citología , Transducción Genética , ADP-Ribosil Ciclasa 1/metabolismo , Animales , Antígenos CD34/metabolismo , Diferenciación Celular , Línea Celular , Proliferación Celular , Separación Celular , Células Eritroides/citología , Vectores Genéticos/metabolismo , Células Madre Hematopoyéticas/metabolismo , Humanos , Lentivirus/genética , Ratones Endogámicos NOD , Receptores de LDL/metabolismoRESUMEN
Sulfur bonding interactions between organosulfur compounds and proteins were examined using crystal structures deposited to-date in the PDB. The data was analyzed as a function of sulfur-σ-hole-bonding (i.e., sulfur bonds) to main chain Lewis bases, viz. oxygen and nitrogen atoms of the backbone amide linkages. The analyses also included an examination of sulfur bonding to side chain Lewis bases (O, N, and S) and to the "non-classical" Lewis bases present in electron-rich aromatic amino acids as-well-as to donor-acceptor bond angle distributions. The interactions analyzed included those restricted to the sum of van der Waals radii of the respective atoms or to a distance of 4 Å. The surveyed data revealed that sulfur bonding tendencies (C-S-C bond angles) were impacted not only by steric effects but perhaps also by enthalpic features present in both the donor and acceptor participants. This knowledge is not only of fundamental interest but is also important in terms of materials and drug-design involving moieties incorporating the sulfur atom. Additionally, a new empirical scoring function was developed to address the anisotropy of sulfur in protein-ligand interactions. This newly developed scoring function is incorporated into AutoDock Vina molecular docking program and is valuable for modeling and drug design.
Asunto(s)
Diseño de Fármacos , Simulación del Acoplamiento Molecular , Proteínas/metabolismo , Compuestos de Azufre/química , Compuestos de Azufre/farmacología , Algoritmos , Bacterias/enzimología , Proteínas Bacterianas/metabolismo , Bases de Datos de Proteínas , Humanos , Hidrocarburos Aromáticos/química , Hidrocarburos Aromáticos/farmacología , Ligandos , Plasmodium falciparum/enzimología , Electricidad Estática , Azufre/química , Azufre/farmacología , TermodinámicaRESUMEN
Positron emission tomography (PET) reporter genes allow noninvasive whole-body imaging of transplanted cells by detection with radiolabeled probes. We used a human deoxycytidine kinase containing three amino acid substitutions within the active site (hdCK3mut) as a reporter gene in combination with the PET probe [(18)F]-L-FMAU (1-(2-deoxy-2-(18)fluoro-ß-L-arabinofuranosyl)-5-methyluracil) to monitor models of mouse and human hematopoietic stem cell (HSC) transplantation. These mutations in hdCK3mut expanded the substrate capacity allowing for reporter-specific detection with a thymidine analog probe. Measurements of long-term engrafted cells (up to 32 wk) demonstrated that hdCK3mut expression is maintained in vivo with no counter selection against reporter-labeled cells. Reporter cells retained equivalent engraftment and differentiation capacity being detected in all major hematopoietic lineages and tissues. This reporter gene and probe should be applicable to noninvasively monitor therapeutic cell transplants in multiple tissues.
Asunto(s)
Desoxicitidina Quinasa/metabolismo , Trasplante de Células Madre Hematopoyéticas/métodos , Células Madre Hematopoyéticas/diagnóstico por imagen , Tomografía de Emisión de Positrones/métodos , Animales , Arabinofuranosil Uracilo/análogos & derivados , Arabinofuranosil Uracilo/química , Arabinofuranosil Uracilo/metabolismo , Western Blotting , Línea Celular Tumoral , Desoxicitidina Quinasa/genética , Femenino , Radioisótopos de Flúor/química , Células Madre Hematopoyéticas/metabolismo , Inmunohistoquímica , Subunidad gamma Común de Receptores de Interleucina/deficiencia , Subunidad gamma Común de Receptores de Interleucina/genética , Estimación de Kaplan-Meier , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , Ratones Endogámicos , Ratones Noqueados , Ratones SCID , Mutación , Timo/diagnóstico por imagen , Timo/metabolismo , Factores de Tiempo , Trasplante HeterólogoRESUMEN
The relationship between the cells that initiate cancer and the cancer stem-like cells that propagate tumors has been poorly defined. In a human prostate tissue transformation model, basal cells expressing the oncogenes Myc and myristoylated AKT can initiate heterogeneous tumors. Tumors contain features of acinar-type adenocarcinoma with elevated eIF4E-driven protein translation and squamous cell carcinoma marked by activated beta-catenin. Lentiviral integration site analysis revealed that alternative histological phenotypes can be clonally derived from a common cell of origin. In advanced disease, adenocarcinoma can be propagated by self-renewing tumor cells with an androgen receptor-low immature luminal phenotype in the absence of basal-like cells. These data indicate that advanced prostate adenocarcinoma initiated in basal cells can be maintained by luminal-like tumor-propagating cells. Determining the cells that maintain human prostate adenocarcinoma and the signaling pathways characterizing these tumor-propagating cells is critical for developing effective therapeutic strategies against this population.
Asunto(s)
Adenocarcinoma/fisiopatología , Transformación Celular Neoplásica/metabolismo , Neoplasias Basocelulares/fisiopatología , Fenotipo , Neoplasias de la Próstata/fisiopatología , Transducción de Señal/fisiología , Western Blotting , Factor 4E Eucariótico de Iniciación/metabolismo , Citometría de Flujo , Regulación Neoplásica de la Expresión Génica/fisiología , Humanos , Inmunohistoquímica , Masculino , beta Catenina/metabolismoRESUMEN
Gene transfer into autologous hematopoietic stem cells by γ-retroviral vectors (gRV) is an effective treatment for adenosine deaminase (ADA)-deficient severe combined immunodeficiency (SCID). However, current gRV have significant potential for insertional mutagenesis as reported in clinical trials for other primary immunodeficiencies. To improve the efficacy and safety of ADA-SCID gene therapy (GT), we generated a self-inactivating lentiviral vector (LV) with a codon-optimized human cADA gene under the control of the short form elongation factor-1α promoter (LV EFS ADA). In ADA(-/-) mice, LV EFS ADA displayed high-efficiency gene transfer and sufficient ADA expression to rescue ADA(-/-) mice from their lethal phenotype with good thymic and peripheral T- and B-cell reconstitution. Human ADA-deficient CD34(+) cells transduced with 1-5 × 10(7) TU/ml had 1-3 vector copies/cell and expressed 1-2x of normal endogenous levels of ADA, as assayed in vitro and by transplantation into immune-deficient mice. Importantly, in vitro immortalization assays demonstrated that LV EFS ADA had significantly less transformation potential compared to gRV vectors, and vector integration-site analysis by nrLAM-PCR of transduced human cells grown in immune-deficient mice showed no evidence of clonal skewing. These data demonstrated that the LV EFS ADA vector can effectively transfer the human ADA cDNA and promote immune and metabolic recovery, while reducing the potential for vector-mediated insertional mutagenesis.
Asunto(s)
Adenosina Desaminasa/deficiencia , Adenosina Desaminasa/genética , Agammaglobulinemia/inmunología , Agammaglobulinemia/terapia , Vectores Genéticos/efectos adversos , Lentivirus/genética , Factor 1 de Elongación Peptídica/genética , Inmunodeficiencia Combinada Grave/inmunología , Inmunodeficiencia Combinada Grave/terapia , Adenosina Desaminasa/inmunología , Adenosina Desaminasa/metabolismo , Agammaglobulinemia/genética , Agammaglobulinemia/patología , Animales , Linfocitos B/inmunología , Células Cultivadas , Modelos Animales de Enfermedad , Femenino , Células HEK293 , Células HT29 , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Regiones Promotoras Genéticas , Inmunodeficiencia Combinada Grave/genética , Inmunodeficiencia Combinada Grave/patología , Linfocitos T/inmunología , Transducción Genética , Integración ViralRESUMEN
We conducted a gene therapy trial in 10 patients with adenosine deaminase (ADA)-deficient severe combined immunodeficiency using 2 slightly different retroviral vectors for the transduction of patients' bone marrow CD34(+) cells. Four subjects were treated without pretransplantation cytoreduction and remained on ADA enzyme-replacement therapy (ERT) throughout the procedure. Only transient (months), low-level (< 0.01%) gene marking was observed in PBMCs of 2 older subjects (15 and 20 years of age), whereas some gene marking of PBMC has persisted for the past 9 years in 2 younger subjects (4 and 6 years). Six additional subjects were treated using the same gene transfer protocol, but after withdrawal of ERT and administration of low-dose busulfan (65-90 mg/m(2)). Three of these remain well, off ERT (5, 4, and 3 years postprocedure), with gene marking in PBMC of 1%-10%, and ADA enzyme expression in PBMC near or in the normal range. Two subjects were restarted on ERT because of poor gene marking and immune recovery, and one had a subsequent allogeneic hematopoietic stem cell transplantation. These studies directly demonstrate the importance of providing nonmyeloablative pretransplantation conditioning to achieve therapeutic benefits with gene therapy for ADA-deficient severe combined immunodeficiency.
Asunto(s)
Agammaglobulinemia/terapia , Trasplante de Médula Ósea/métodos , Terapia Genética/métodos , Vectores Genéticos , Trasplante de Células Madre Hematopoyéticas/métodos , Inmunodeficiencia Combinada Grave/terapia , Adenosina Desaminasa/deficiencia , Adolescente , Antígenos CD34/metabolismo , Niño , Preescolar , Femenino , Humanos , Lactante , Masculino , Retroviridae/genética , Transducción Genética , Acondicionamiento Pretrasplante , Adulto JovenRESUMEN
Frequent and widespread testing of members of the population who are asymptomatic for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is essential for the mitigation of the transmission of the virus. Despite the recent increases in testing capacity, tests based on quantitative polymerase chain reaction (qPCR) assays cannot be easily deployed at the scale required for population-wide screening. Here, we show that next-generation sequencing of pooled samples tagged with sample-specific molecular barcodes enables the testing of thousands of nasal or saliva samples for SARS-CoV-2 RNA in a single run without the need for RNA extraction. The assay, which we named SwabSeq, incorporates a synthetic RNA standard that facilitates end-point quantification and the calling of true negatives, and that reduces the requirements for automation, purification and sample-to-sample normalization. We used SwabSeq to perform 80,000 tests, with an analytical sensitivity and specificity comparable to or better than traditional qPCR tests, in less than two months with turnaround times of less than 24 h. SwabSeq could be rapidly adapted for the detection of other pathogens.
Asunto(s)
ARN Viral/genética , SARS-CoV-2/patogenicidad , Saliva/virología , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , SARS-CoV-2/genética , Sensibilidad y EspecificidadRESUMEN
The rapid spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is due to the high rates of transmission by individuals who are asymptomatic at the time of transmission1,2. Frequent, widespread testing of the asymptomatic population for SARS-CoV-2 is essential to suppress viral transmission. Despite increases in testing capacity, multiple challenges remain in deploying traditional reverse transcription and quantitative PCR (RT-qPCR) tests at the scale required for population screening of asymptomatic individuals. We have developed SwabSeq, a high-throughput testing platform for SARS-CoV-2 that uses next-generation sequencing as a readout. SwabSeq employs sample-specific molecular barcodes to enable thousands of samples to be combined and simultaneously analyzed for the presence or absence of SARS-CoV-2 in a single run. Importantly, SwabSeq incorporates an in vitro RNA standard that mimics the viral amplicon, but can be distinguished by sequencing. This standard allows for end-point rather than quantitative PCR, improves quantitation, reduces requirements for automation and sample-to-sample normalization, enables purification-free detection, and gives better ability to call true negatives. After setting up SwabSeq in a high-complexity CLIA laboratory, we performed more than 80,000 tests for COVID-19 in less than two months, confirming in a real world setting that SwabSeq inexpensively delivers highly sensitive and specific results at scale, with a turn-around of less than 24 hours. Our clinical laboratory uses SwabSeq to test both nasal and saliva samples without RNA extraction, while maintaining analytical sensitivity comparable to or better than traditional RT-qPCR tests. Moving forward, SwabSeq can rapidly scale up testing to mitigate devastating spread of novel pathogens.
RESUMEN
Scalable, inexpensive, and secure testing for SARS-CoV-2 infection is crucial for control of the novel coronavirus pandemic. Recently developed highly multiplexed sequencing assays (HMSAs) that rely on high-throughput sequencing can, in principle, meet these demands, and present promising alternatives to currently used RT-qPCR-based tests. However, reliable analysis, interpretation, and clinical use of HMSAs requires overcoming several computational, statistical and engineering challenges. Using recently acquired experimental data, we present and validate a computational workflow based on kallisto and bustools, that utilizes robust statistical methods and fast, memory efficient algorithms, to quickly, accurately and reliably process high-throughput sequencing data. We show that our workflow is effective at processing data from all recently proposed SARS-CoV-2 sequencing based diagnostic tests, and is generally applicable to any diagnostic HMSA.
Asunto(s)
Prueba de Ácido Nucleico para COVID-19 , COVID-19 , Técnicas de Diagnóstico Molecular , Reacción en Cadena en Tiempo Real de la Polimerasa , SARS-CoV-2/genética , COVID-19/diagnóstico , COVID-19/genética , HumanosRESUMEN
X-linked hyper-immunoglobulin M (hyper-IgM) syndrome (XHIM) is a primary immunodeficiency due to mutations in CD40 ligand that affect immunoglobulin class-switch recombination and somatic hypermutation. The disease is amenable to gene therapy using retroviral vectors, but dysregulated gene expression results in abnormal lymphoproliferation in mouse models, highlighting the need for alternative strategies. Here, we demonstrate the ability of both the transcription activator-like effector nuclease (TALEN) and clustered regularly interspaced short palindromic repeats-associated protein 9 (CRISPR/Cas9) platforms to efficiently drive integration of a normal copy of the CD40L cDNA delivered by Adeno-Associated Virus. Site-specific insertion of the donor sequence downstream of the endogenous CD40L promoter maintained physiologic expression of CD40L while overriding all reported downstream mutations. High levels of gene modification were achieved in primary human hematopoietic stem cells (HSCs), as well as in cell lines and XHIM-patient-derived T cells. Notably, gene-corrected HSCs engrafted in immunodeficient mice at clinically relevant frequencies. These studies provide the foundation for a permanent curative therapy in XHIM.
Asunto(s)
Edición Génica , Enfermedades Genéticas Ligadas al Cromosoma X/genética , Células Madre Hematopoyéticas/metabolismo , Síndrome de Inmunodeficiencia con Hiper-IgM/genética , Animales , Antígenos CD34/metabolismo , Secuencia de Bases , Ligando de CD40/metabolismo , Proteína 9 Asociada a CRISPR/metabolismo , Sistemas CRISPR-Cas/genética , Diferenciación Celular , Línea Celular , Ensayo de Unidades Formadoras de Colonias , Reparación del ADN , ADN Complementario/genética , Humanos , Ratones , Linfocitos T/metabolismo , Nucleasas de los Efectores Tipo Activadores de la Transcripción/metabolismoRESUMEN
Wheat ( L.) spike characteristics determine the number of grains produced on each spike and constitute key components of grain yield. Understanding of the genetic basis of spike characteristics in wheat, however, is limited. In this study, genotyping-by-sequencing (GBS) and the iSelect 9K assay were used on a doubled-haploid (DH) soft red winter wheat population that showed a wide range of phenotypic variation for spike traits. A genetic map spanning 2934.1 cM with an average interval length of 3.4 cM was constructed. Quantitative trait loci (QTL) analysis involving additive effects, epistasis (QQ) and QTL × environment (QE), and epistasis × environment (QQE) interactions detected a total of 109 QTL, 13 QE, and 20 QQ interactions in five environments. Spike characteristics were mainly determined by additive effects and were fine-tuned by QQ, QE, and QQE. Major QTL / explained up to 30.9% of the phenotypic variation for spike length (SL) and fertile spikelet number, .1 explained up to 15.6% of the phenotypic variation of grain number per spikelet, and explained up to 80.2% of the phenotypic variation for spike compactness. Additionally, QTL for correlated spike characteristics formed QTL clusters on chromosomes 1A, 5A, 2B, 3B, 5B, 1D, and 5D. This study expands the understanding of the genetic basis of spike characteristics in hexaploid wheat. A number of stable QTL detected in this study have potential to be used in marker-assisted selection. Additionally, the genetic map generated in this study could be used to study other traits of economic importance.
Asunto(s)
Poliploidía , Sitios de Carácter Cuantitativo , Triticum/genética , Cromosomas de las Plantas , Epistasis Genética , Genes de Plantas , Haploidia , FenotipoRESUMEN
BACKGROUND: Autologous hematopoietic stem cell transplantation (HSCT) of gene-modified cells is an alternative to enzyme replacement therapy (ERT) and allogeneic HSCT that has shown clinical benefit for adenosine deaminase-deficient (ADA-deficient) SCID when combined with reduced intensity conditioning (RIC) and ERT cessation. Clinical safety and therapeutic efficacy were evaluated in a phase II study. METHODS: Ten subjects with confirmed ADA-deficient SCID and no available matched sibling or family donor were enrolled between 2009 and 2012 and received transplantation with autologous hematopoietic CD34+ cells that were modified with the human ADA cDNA (MND-ADA) γ-retroviral vector after conditioning with busulfan (90 mg/m2) and ERT cessation. Subjects were followed from 33 to 84 months at the time of data analysis. Safety of the procedure was assessed by recording the number of adverse events. Efficacy was assessed by measuring engraftment of gene-modified hematopoietic stem/progenitor cells, ADA gene expression, and immune reconstitution. RESULTS: With the exception of the oldest subject (15 years old at enrollment), all subjects remained off ERT with normalized peripheral blood mononuclear cell (PBMC) ADA activity, improved lymphocyte numbers, and normal proliferative responses to mitogens. Three of nine subjects were able to discontinue intravenous immunoglobulin replacement therapy. The MND-ADA vector was persistently detected in PBMCs (vector copy number [VCN] = 0.1-2.6) and granulocytes (VCN = 0.01-0.3) through the most recent visits at the time of this writing. No patient has developed a leukoproliferative disorder or other vector-related clinical complication since transplant. CONCLUSION: These results demonstrate clinical therapeutic efficacy from gene therapy for ADA-deficient SCID, with an excellent clinical safety profile. TRIAL REGISTRATION: ClinicalTrials.gov NCT00794508. FUNDING: Food and Drug Administration Office of Orphan Product Development award, RO1 FD003005; NHLBI awards, PO1 HL73104 and Z01 HG000122; UCLA Clinical and Translational Science Institute awards, UL1RR033176 and UL1TR000124.