Matthew Porteus, MD

Abstract

Sickle cell disease (SCD) is the most common serious monogenic disease with 300,000 births annually worldwide. SCD is an autosomal recessive disease resulting from a single point mutation in codon six of the beta-globin gene (HBB). Ex vivo beta-globin gene correction in autologous patient-derived hematopoietic stem and progenitor cells (HSPCs) may potentially provide a curative treatment for SCD. We previously developed a CRISPR-Cas9 gene targeting strategy that uses high-fidelity Cas9 precomplexed with chemically modified guide RNAs to induce recombinant adeno-associated virus serotype 6 (rAAV6)-mediated HBB gene correction of the SCD-causing mutation in HSPCs. Here, we demonstrate the preclinical feasibility, efficacy, and toxicology of HBB gene correction in plerixafor-mobilized CD34+ cells from healthy and SCD patient donors (gcHBB-SCD). We achieved up to 60% HBB allelic correction in clinical-scale gcHBB-SCD manufacturing. After transplant into immunodeficient NSG mice, 20% gene correction was achieved with multilineage engraftment. The long-term safety, tumorigenicity, and toxicology study demonstrated no evidence of abnormal hematopoiesis, genotoxicity, or tumorigenicity from the engrafted gcHBB-SCD drug product. Together, these preclinical data support the safety, efficacy, and reproducibility of this gene correction strategy for initiation of a phase 1/2 clinical trial in patients with SCD.

View details for DOI 10.1126/scitranslmed.abf2444

View details for PubMedID 34135108

View details for Web of Science ID 000645188700526

View details for Web of Science ID 000645188700095

View details for DOI 10.1007/s10875-021-01030-6

View details for PubMedID 33821398

Abstract

beta-Thalassemia pathology is due not only to loss of beta-globin (HBB), but also to erythrotoxic accumulation and aggregation of the beta-globin-binding partner, alpha-globin (HBA1/2). Here we describe a Cas9/AAV6-mediated genome editing strategy that can replace the entire HBA1 gene with a full-length HBB transgene in beta-thalassemia-derived hematopoietic stem and progenitor cells (HSPCs), which is sufficient to normalize beta-globin:alpha-globin messenger RNA and protein ratios and restore functional adult hemoglobin tetramers in patient-derived red blood cells. Edited HSPCs were capable of long-term and bilineage hematopoietic reconstitution in mice, establishing proof of concept for replacement of HBA1 with HBB as a novel therapeutic strategy for curing beta-thalassemia.

View details for DOI 10.1038/s41591-021-01284-y

View details for PubMedID 33737751

Abstract

X-linked chronic granulomatous disease is an immunodeficiency characterized by defective production of microbicidal reactive oxygen species (ROS) by phagocytes. Causative mutations occur throughout the 13 exons and splice sites of the CYBB gene, resulting in loss of gp91phox protein. Here we report gene correction by homology-directed repair in patient hematopoietic stem/progenitor cells (HSPCs) using CRISPR/Cas9 for targeted insertion of CYBB exon 1-13 or 2-13 cDNAs from adeno-associated virus donors at endogenous CYBB exon 1 or exon 2 sites. Targeted insertion of exon 1-13 cDNA did not restore physiologic gp91phox levels, consistent with a requirement for intron 1 in CYBB expression. However, insertion of exon 2-13 cDNA fully restored gp91phox and ROS production upon phagocyte differentiation. Addition of a woodchuck hepatitis virus post-transcriptional regulatory element did not further enhance gp91phox expression in exon 2-13 corrected cells, indicating that retention of intron 1 was sufficient for optimal CYBB expression. Targeted correction was increased ~1.5-fold using i53 mRNA to transiently inhibit nonhomologous end joining. Following engraftment in NSG mice, corrected HSPCs generated phagocytes with restored gp91phox and ROS production. Our findings demonstrate the utility of tailoring donor design and targeting strategies to retain regulatory elements needed for optimal expression of the target gene.

View details for DOI 10.1038/s41434-021-00251-z

View details for PubMedID 33712802

Abstract

Severe combined immune deficiency (SCID) caused by RAG1 or RAG2 deficiency is a genetically determined immune deficiency characterized by the virtual absence of T and B lymphocytes. Unless treated with hematopoietic stem cell transplantation (HSCT), patients with RAG deficiency succumb to severe infections early in life. However, HSCT carries the risk of graft-versus-host disease. Moreover, a high rate of graft failure and poor immune reconstitution have been reported after unconditioned HSCT. Expression of the RAG genes is tightly regulated, and preclinical attempts of gene therapy with heterologous promoters have led to controversial results. Using patient-derived induced pluripotent stem cells (iPSCs) and an in vitro artificial thymic organoid system as a model, here we demonstrate that gene editing rescues the progressive T cell differentiation potential of RAG2-deficient cells to normal levels, with generation of a diversified T cell repertoire. These results suggest that targeted gene editing may represent a novel therapeutic option for correction of this immunodeficiency.

View details for DOI 10.1007/s10875-021-00989-6

View details for PubMedID 33650026

Abstract

Lentivector gene therapy for X-linked chronic granulomatous disease (X-CGD) has proven to be a viable approach, but random vector integration and subnormal protein production from exogenous promoters in transduced cells remain concerning for long-term safety and efficacy. A previous genome editing-based approach using SpCas9 and an oligodeoxynucleotide donor to repair genetic mutations demonstrated the capability to restore physiological protein expression, but lacked sufficient efficiency in quiescent CD34+ hematopoietic cells for clinical translation. Here, we show transient inhibition of p53-binding protein 1 (53BP1) significantly increased (2.3-fold) long-term homology directed repair (HDR) to achieve highly efficient (80% gp91phox+ cells compared to healthy donor control) long-term correction of X-CGD CD34+ cells.

View details for DOI 10.1182/blood.2020008503

View details for PubMedID 33623984

Abstract

CRISPR/Cas9-mediated beta-globin (HBB) gene correction of sickle cell disease (SCD) patient-derived hematopoietic stem cells (HSCs) in combination with autologous transplantation represents a recent paradigm in gene therapy. Although several Cas9-based HBB-correction approaches have been proposed, functional correction of in vivo erythropoiesis has not been investigated previously. Here, we use a humanized globin-cluster SCD mouse model to study Cas9-AAV6-mediated HBB-correction in functional HSCs within the context of autologous transplantation. We discover that long-term multipotent HSCs can be gene corrected ex vivo and stable hemoglobin-A production can be achieved in vivo from HBB-corrected HSCs following autologous transplantation. We observe a direct correlation between increased HBB-corrected myeloid chimerism and normalized in vivo red blood cell (RBC) features, but even low levels of chimerism resulted in robust hemoglobin-A levels. Moreover, this study offers a platform for gene editing of mouse HSCs for both basic and translational research.

View details for DOI 10.1038/s41467-021-20909-x

View details for PubMedID 33514718

Abstract

Introduction On May 11, 2020, the National Institutes of Health (NIH) and the Bill & Melinda Gates Foundation (Gates Foundation) held an exploratory expert scientific roundtable to inform an NIH-Gates Foundation collaboration on the development of scalable, sustainable, and accessible HIV and sickle cell disease (SCD) therapies based on in vivo gene editing of hematopoietic stem cells (HSC). A particular emphasis was on how such therapies could be developed for low-resource settings in sub-Saharan Africa. Paula Cannon, Ph.D., of the University of Southern California and Hans-Peter Kiem, M.D., Ph.D., of the Fred Hutchinson Cancer Research Center served as roundtable co-chairs. Welcoming remarks were provided by the leadership of NIH, NHLBI, and BMGF, who cited the importance of assessing the state of the science and charting a path toward finding safe, effective, and durable gene-based therapies for HIV and sickle cell disease. These remarks were followed by three sessions in which participants heard presentations on and discussed the therapeutic potential of modified HSCs, leveraging HSC biology and differentiation, and in vivo HSC targeting approaches. This roundtable serves as the beginning of an ongoing discussion among NIH, the Gates Foundation, research and patient communities, and the public at large. As this collaboration progresses, these communities will be engaged as we collectively navigate the complex scientific and ethical issues surrounding in vivo HSC targeting and editing. Summarized excerpts from each of the presentations are below, reflecting the individual views and perspectives of each presenter.

View details for DOI 10.1089/hum.2020.263

View details for PubMedID 33427035

Abstract

Targeted DNA correction of disease-causing mutations in hematopoietic stem and progenitor cells (HSPCs) may enable the treatment of genetic diseases of the blood and immune system. It is now possible to correct mutations at high frequencies in HSPCs by combining CRISPR/Cas9 with homologous DNA donors. Because of the precision of gene correction, these approaches preclude clonal tracking of gene-targeted HSPCs. Here, we describe Tracking Recombination Alleles in Clonal Engraftment using sequencing (TRACE-Seq), a methodology that utilizes barcoded AAV6 donor template libraries, carrying in-frame silent mutations or semi-randomized nucleotides outside the coding region, to track the in vivo lineage contribution of gene-targeted HSPC clones. By targeting the HBB gene with an AAV6 donor template library consisting of ~20,000 possible unique exon 1 in-frame silent mutations, we track the hematopoietic reconstitution of HBB targeted myeloid-skewed, lymphoid-skewed, and balanced multi-lineage repopulating human HSPC clones in mice. We anticipate this methodology could potentially be used for HSPC clonal tracking of Cas9 RNP and AAV6-mediated gene targeting outcomes in translational and basic research settings.

View details for DOI 10.1038/s41467-020-20792-y

View details for PubMedID 33473139

Abstract

Adeno-associated virus is a highly efficient DNA delivery vehicle for genome editing strategies that employ CRISPR/Cas9 and a DNA donor for homology-directed repair. Many groups have used this strategy in development of therapies for blood and immune disorders such as sickle-cell anemia and severe-combined immunodeficiency. However, recent events have called into question the immunogenicity of AAV as a gene therapy vector and the safety profile dictated by the immune response to this vector. The target cells dictating this response and the molecular mechanisms dictating cellular response to AAV are poorly understood. Here, we will investigate the current known AAV capsid and genome interactions with cellular proteins during early stage vector transduction and how these interactions may influence innate cellular responses. We will discuss the current understanding of innate immune activation and DNA damage response to AAV, and the limitations of what is currently known. In particular, we will focus on pathway differences in cell line verses primary cells, with a focus on hematopoietic stem and progenitor cells (HSPCs) in the context of ex-vivo gene editing, and what we can learn from HSPC infection by other parvoviruses. Finally, we will discuss how innate immune and DNA damage response pathway activation in these highly sensitive stem cell populations may impact long-term engraftment and clinical outcomes as these gene-editing strategies move towards the clinic, with the aim to propose pathways relevant for improved hematopoietic stem cell survival and long-term engraftment after AAV-mediated genome editing.

View details for DOI 10.3389/fimmu.2021.660302

View details for PubMedID 34122418

Abstract

Cystic fibrosis (CF) is a monogenic disease caused by impaired production and/or function of the cystic fibrosis transmembrane conductance regulator (CFTR) protein. Although we have previously shown correction of the most common pathogenic mutation, there are many other pathogenic mutations throughout the CF gene. An autologous airway stem cell therapy in which the CFTR cDNA is precisely inserted into the CFTR locus may enable the development of a durable cure for almost all CF patients, irrespective of the causal mutation. Here, we use CRISPR/Cas9 and two adeno-associated viruses (AAV) carrying the two halves of the CFTR cDNA to sequentially insert the full CFTR cDNA along with a truncated CD19 (tCD19) enrichment tag in upper airway basal stem cells (UABCs) and human bronchial basal stem cells (HBECs). The modified cells were enriched to obtain 60-80% tCD19+ UABCs and HBECs from 11 different CF donors with a variety of mutations. Differentiated epithelial monolayers cultured at air-liquid interface showed restored CFTR function that was >70% of the CFTR function in non-CF controls. Thus, our study enables the development of a therapy for almost all CF patients, including patients who cannot be treated using recently approved modulator therapies.

View details for DOI 10.1016/j.ymthe.2021.03.023

View details for PubMedID 33794364

Abstract

Genome editing technologies that enable the introduction of precise changes in DNA sequences have the potential to lead to a new class of treatments for genetic diseases. Epidermolysis bullosa is a group of rare genetic disorders characterized by extreme skin fragility. The Recessive Dystrophic subtype of EB (RDEB), which has one of the most severe phenotypes, is caused by mutations in COL7A1. Here, we report a gene editing approach for ex vivo homology-directed repair (HDR)-based gene correction that uses the CRISPR/Cas9 system delivered as a ribonucleoprotein (RNP) complex in combination with donor DNA templates delivered by adeno-associated viral vectors (AAV). We demonstrate sufficient mutation correction frequencies to achieve therapeutic benefit in primary RDEB keratinocytes containing different COL7A1 mutations as well as efficient HDR-mediated COL7A1 modification in healthy cord blood-derived CD34+ cells and mesenchymal stem cells (MSCs). These results are a proof-of-concept for HDR-mediated gene correction in different cell types with therapeutic potential for RDEB.

View details for DOI 10.1016/j.ymthe.2021.02.019

View details for PubMedID 33609734

Abstract

Recombinant adeno-associated virus (rAAV) vectors have the unique property of being able to perform genomic targeted integration (TI) without inducing a double-strand break (DSB). In order to improve our understanding of the mechanism behind TI mediated by AAV and improve its efficiency, we performed an unbiased genetic screen in human cells usinga promoterless AAV-homologous recombination (AAV-HR) vector system. We identified that the inhibition of the Fanconi anemia complementation group M (FANCM) protein enhanced AAV-HR-mediated TI efficiencies in different cultured human cells by 6- to 9-fold. The combined knockdown of the FANCM and two proteins also associated with the FANCM complex, RecQ-mediated genome instability 1 (RMI1) and Bloom DNA helicase (BLM) from the BLM-topoisomerase III (TOP3A)-RMI (BTR) dissolvase complex (RMI1, having also been identified in our screen), led to the enhancement of AAV-HR-mediated TI up to 17 times. AAV-HR-mediated TI in the presence of a nuclease (CRISPR-Cas9) was also increased by 1.5- to 2-fold in FANCM and RMI1 knockout cells, respectively. Furthermore, knockdown of FANCM in human CD34+ hematopoietic stem and progenitor cells (HSPCs) increased AAV-HR-mediated TI by 3.5-fold. This study expands our knowledge on the mechanisms related to AAV-mediated TI, and it highlights new pathways that might be manipulated for future improvements in AAV-HR-mediated TI.

View details for DOI 10.1016/j.ymthe.2020.10.020

View details for PubMedID 33678249

View details for DOI 10.1182/bloodadvances.2020002891

View details for PubMedID 33136125

Abstract

Umbilical cord blood is an important graft source in the treatment of many genetic, hematologic, and immunologic disorders by hematopoietic stem cell transplantation. Millions of cord blood units have been collected and stored for clinical use since the inception of cord blood banking in 1989. However, the use of cord blood in biomedical research has been limited by access to viable samples. Here, we present a cost-effective, self-sustaining model for the procurement of fresh umbilical cord blood components for research purposes within hospital-affiliated academic institutions.

View details for DOI 10.1016/j.placenta.2020.10.018

View details for PubMedID 33075720

View details for Web of Science ID 000572509100387

View details for Web of Science ID 000655609700081

Abstract

Safeguard mechanisms can ameliorate the potential risks associated with cell therapies but currently rely on the introduction of transgenes. This limits their application owing to immunogenicity or transgene silencing. We aimed to create a control mechanism for human cells that is not mediated by a transgene. Using genome editing methods, we disrupt uridine monophosphate synthetase (UMPS) in the pyrimidine de novo synthesis pathway in cell lines, pluripotent cells and primary human T cells. We show that this makes proliferation dependent on external uridine and enables us to control cell growth by modulating the uridine supply, both in vitro and in vivo after transplantation in xenograft models. Additionally, disrupting this pathway creates resistance to 5-fluoroorotic acid, which enables positive selection of UMPS-knockout cells. We envision that this approach will add an additional level of safety to cell therapies and therefore enable the development of approaches with higher risks, especially those that are intended for limited treatment durations.

View details for DOI 10.1038/s41587-020-0580-6

View details for PubMedID 32661439

Abstract

Invivo tracking of retrovirus-tagged blood stem and progenitor cells is used to study hematopoiesis. Two techniques are used most frequently: sequencing the locus of retrovirus insertion, termed integration site analysis, or retrovirus DNA barcode sequencing. Of these, integration site analysis is currently the only available technique for monitoring clonal pools in patients treated with retrovirus-modified blood cells. A key question is how these two techniques compare in their ability to detect and quantify clonal contributions. In this study, we assessed both methods simultaneously in a clinically relevant nonhuman primate model of autologous, myeloablative transplantation. Our data demonstrate that both methods track abundant clones; however, DNA barcode sequencing is at least 5-fold more efficient than integration site analysis. Using computational simulation to identify the sources of low efficiency, we identify sampling depth as the major factor. We show that the sampling required for integration site analysis to achieve minimal coverage of the true clonal pool is likely prohibitive, especially in cases of low gene-modified cell engraftment. We also show that early subsampling of different blood cell lineages adds value to clone tracking information in terms of safety and hematopoietic biology. Our analysis demonstrates DNA barcode sequencing as a useful guide to maximize integration site analysis interpretation in gene therapy patients.

View details for DOI 10.1016/j.omtm.2020.03.021

View details for PubMedID 32355868

Abstract

Despite their rapidly-expanding therapeutic potential, human pluripotent stem cell (hPSC)-derived cell therapies continue to have serious safety risks. Transplantation of hPSC-derived cell populations into preclinical models has generated teratomas (tumors arising from undifferentiated hPSCs), unwanted tissues, and other types of adverse events. Mitigating these risks is important to increase the safety of such therapies. Here we use genome editing to engineer a general platform to improve the safety of future hPSC-derived cell transplantation therapies. Specifically, we develop hPSC lines bearing two drug-inducible safeguards, which have distinct functionalities and address separate safety concerns. In vitro administration of one small molecule depletes undifferentiated hPSCs >106-fold, thus preventing teratoma formation in vivo. Administration of a second small molecule kills all hPSC-derived cell-types, thus providing an option to eliminate the entire hPSC-derived cell product in vivo if adverse events arise. These orthogonal safety switches address major safety concerns with pluripotent cell-derived therapies.

View details for DOI 10.1038/s41467-020-16455-7

View details for PubMedID 32483127

Abstract

Human mesenchymal stromal cells (hMSCs) are a promising source for engineered cell-based therapies in which genetic engineering could enhance therapeutic efficacy and install novel cellular functions. Here, we describe an optimized Cas9-AAV6-based genome editing tool platform for site-specific mutagenesis and integration of up to more than 3 kilobases of exogenous DNA in the genome of hMSCs derived from the bone marrow, adipose tissue, and umbilical cord blood without altering their ex vivo characteristics. We generate safe harbor-integrated lines of engineered hMSCs and show that engineered luciferase-expressing hMSCs are transiently active in vivo in wound beds of db/db mice. Moreover, we generate PDGF-BB- and VEGFA-hypersecreting hMSC lines as short-term, local wound healing agents with superior therapeutic efficacy over wildtype hMSCs in the diabetic mouse model without replacing resident cells long-term. This study establishes a precise genetic engineering platform for genetic studies of hMSCs and development of engineered hMSC-based therapies.

View details for DOI 10.1038/s41467-020-16065-3

View details for PubMedID 32424320

View details for Web of Science ID 000530089301338

View details for Web of Science ID 000530089301274

View details for Web of Science ID 000530089300130

View details for Web of Science ID 000530089300107

View details for Web of Science ID 000530089302214

View details for Web of Science ID 000530089302407

View details for Web of Science ID 000530089302154

View details for Web of Science ID 000516887900438

View details for DOI 10.1016/j.ymgme.2019.11.150

View details for Web of Science ID 000510805200160

Abstract

Cortico-striatal projections are critical components of forebrain circuitry that regulate motivated behaviors. To enable the study of the human cortico-striatal pathway and how its dysfunction leads to neuropsychiatric disease, we developed a method to convert human pluripotent stem cells into region-specific brain organoids that resemble the developing human striatum and include electrically active medium spiny neurons. We then assembled these organoids with cerebral cortical organoids in three-dimensional cultures to form cortico-striatal assembloids. Using viral tracing and functional assays in intact or sliced assembloids, we show that cortical neurons send axonal projections into striatal organoids and form synaptic connections. Medium spiny neurons mature electrophysiologically following assembly and display calcium activity after optogenetic stimulation of cortical neurons. Moreover, we derive cortico-striatal assembloids from patients with a neurodevelopmental disorder caused by a deletion on chromosome 22q13.3 and capture disease-associated defects in calcium activity, showing that this approach will allow investigation of the development and functional assembly of cortico-striatal connectivity using patient-derived cells.

View details for DOI 10.1038/s41587-020-00763-w

View details for PubMedID 33273741

Abstract

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

View details for DOI 10.1038/s41467-020-18044-0

View details for PubMedID 32820153

Abstract

Intravenous infusion of mesenchymal stromal cells (MSCs) is thought to be a viable treatment for numerous disorders. Although the intrinsic immunosuppressive ability of MSCs has been credited for this therapeutic effect, their exact impact on endogenous tissue-resident cells following delivery has not been clearly characterized. Moreover, multiple studies have reported pulmonary sequestration of MSCs upon intravenous delivery. Despite substantial efforts to improve MSC homing, it remains unclear whether MSC migration to the site of injury is necessary to achieve a therapeutic effect. Using a murine excisional wound healing model, we offer an explanation of how sequestered MSCs improve healing through their systemic impact on macrophage subpopulations. We demonstrate that infusion of MSCs leads to pulmonary entrapment followed by rapid clearance, but also significantly accelerates wound closure. Using single-cell RNA sequencing of the wound, weshow that following MSC delivery, innate immune cells, particularly macrophages, exhibit distinctive transcriptional changes. We identify the appearance of a pro-angiogenic CD9+ macrophage subpopulation, whose induction is mediated by several proteins secreted by MSCs, including COL6A1, PRG4, and TGFB3. Our findings suggest that MSCs do not need to act locally to induce broad changes in the immune system and ultimately treat disease.

View details for DOI 10.1016/j.ymthe.2020.05.022

View details for PubMedID 32531238

Abstract

Allogeneic hematopoietic stem cell transplantation is an effective therapy for high-risk leukemias. In children, graft manipulation based on the selective removal of T cells and B cells has been shown to reduce the risk of acute and chronic graft-versus-host disease, thus allowing the use of haploidentical donors which expands the population that allogeneic hematopoietic stem cell transplantation can be used in. Leukemic relapse, however, remains a problem. T cells expressing chimeric antigen receptors can potently eliminate leukemia, including in the central nervous system. We hypothesized that by modifying donor T cells to simultaneously express a CD19-specific chimeric antigen receptors and inactivating the T cell receptor by genome editing, we could create a therapy that enhances the anti-leukemic efficacy of the stem cell transplant without increasing the risk of graft-versus-host disease. Using genome editing with Cas9 ribonucleoprotein and adeno-associated virus serotype 6, we integrate a CD19-specific chimeric antigen receptor in-frame into the TRAC locus. Greater than 90% of cells lost TCR expression, while >75% expressed the CAR. The product was further purified to ultimately have less than 0.05% residual TCR+ cells. In vitro, the CAR T cells efficiently eliminated target cells and produced high cytokine levels when challenged with CD19+ leukemia cells. In vivo, the gene modified T cells eliminated leukemia without causing xenogeneic graft-versus-host disease in a xenograft model. Gene editing was highly specific with no evidence of off-target effects. These data support the concept that the addition of T cell-derived, genome edited T cells expressing CD19-specific chimeric antigen receptors could enhance the anti-leukemic efficacy of T cell-depleted haploidentical hematopoietic stem cell transplantation without increasing the risk of graft-versus-host disease.

View details for DOI 10.3324/haematol.2019.233882

View details for PubMedID 32241852

View details for DOI 10.1038/s41467-020-17148-x

Abstract

Non-coding mutations at the far end of a large gene desert surrounding the SOX9 gene result in a human craniofacial disorder called Pierre Robin sequence (PRS). Leveraging a human stem cell differentiation model, we identify two clusters of enhancers within the PRS-associated region that regulate SOX9 expression during a restricted window of facial progenitor development at distances up to 1.45 Mb. Enhancers within the 1.45 Mb cluster exhibit highly synergistic activity that is dependent on the Coordinator motif. Using mouse models, we demonstrate that PRS phenotypic specificity arises from the convergence of two mechanisms: confinement of Sox9 dosage perturbation to developing facial structures through context-specific enhancer activity and heightened sensitivity of the lower jaw to Sox9 expression reduction. Overall, we characterize the longest-range human enhancers involved in congenital malformations, directly demonstrate that PRS is an enhanceropathy, and illustrate how small changes in gene expression can lead to morphological variation.

View details for DOI 10.1016/j.stem.2020.09.001

View details for PubMedID 32991838

Abstract

22q11.2 deletion syndrome (22q11DS) is a highly penetrant and common genetic cause of neuropsychiatric disease. Here we generated induced pluripotent stem cells from 15 individuals with 22q11DS and 15 control individuals and differentiated them into three-dimensional (3D) cerebral cortical organoids. Transcriptional profiling across 100 days showed high reliability of differentiation and revealed changes in neuronal excitability-related genes. Using electrophysiology and live imaging, we identified defects in spontaneous neuronal activity and calcium signaling in both organoid- and 2D-derived cortical neurons. The calcium deficit was related to resting membrane potential changes that led to abnormal inactivation of voltage-gated calcium channels. Heterozygous loss of DGCR8 recapitulated the excitability and calcium phenotypes and its overexpression rescued these defects. Moreover, the 22q11DS calcium abnormality could also be restored by application of antipsychotics. Taken together, our study illustrates how stem cell derived models can be used to uncover and rescue cellular phenotypes associated with genetic forms of neuropsychiatric disease.

View details for DOI 10.1038/s41591-020-1043-9

View details for PubMedID 32989314

View details for DOI 10.1182/blood-2019-129424

View details for Web of Science ID 000577164600285

View details for DOI 10.1016/j.ymthe.2019.11.004

View details for PubMedID 31735604

View details for Web of Science ID 000487707800198

View details for Web of Science ID 000490282100027

Abstract

Human neural stem cells (NSCs) offer therapeutic potential for neurodegenerative diseases, such as inherited monogenic nervous system disorders, and neural injuries. Gene editing in NSCs (GE-NSCs) could enhance their therapeutic potential. We show that NSCs are amenable to gene targeting at multiple loci using Cas9 mRNA with synthetic chemically modified guide RNAs along with DNA donor templates. Transplantation of GE-NSC into oligodendrocyte mutant shiverer-immunodeficient mice showed that GE-NSCs migrate and differentiate into astrocytes, neurons, and myelin-producing oligodendrocytes, highlighting the fact that GE-NSCs retain their NSC characteristics of self-renewal and site-specific global migration and differentiation. To show the therapeutic potential of GE-NSCs, we generated GALC lysosomal enzyme overexpressing GE-NSCs that are able to cross-correct GALC enzyme activity through the mannose-6-phosphate receptor pathway. These GE-NSCs have the potential to be an investigational cell and gene therapy for a range of neurodegenerative disorders and injuries of the central nervous system, including lysosomal storage disorders.

View details for DOI 10.1016/j.isci.2019.04.036

View details for PubMedID 31132746

Abstract

Genome editing of human pluripotent stem cells (hPSCs) provides powerful opportunities for invitro disease modeling, drug discovery, and personalized stem cell-based therapeutics. Currently, only small edits can be engineered with high frequency, while larger modifications suffer from low efficiency and a resultant need for selection markers. Here, we describe marker-free genome editing in hPSCs using Cas9 ribonucleoproteins (RNPs) in combination with AAV6-mediated DNA repair template delivery. We report highly efficient and bi-allelic integration frequencies across multiple loci and hPSC lines, achieving mono-allelic editing frequencies of up to 94% at the HBB locus. Using this method, we show robust bi-allelic correction of homozygous sickle cell mutations in a patient-derived induced PSC (iPSC) line. Thus, this strategy shows significant utility for generating hPSCs with large gene integrations and/or single-nucleotide changes at high frequency and without the need for introducing selection genes, enhancing the applicability of hPSC editing for research and translational uses.

View details for PubMedID 31051134

View details for DOI 10.1016/j.stem.2019.04.001

View details for Web of Science ID 000466726500019

View details for Web of Science ID 000463117000038

View details for DOI 10.1038/s41467-019-10080-9

View details for Web of Science ID 000465840000001

View details for Web of Science ID 000464381001006

View details for Web of Science ID 000464381000008

View details for Web of Science ID 000464381003155

View details for Web of Science ID 000464381005072

View details for Web of Science ID 000464381004151

View details for Web of Science ID 000464381002083

View details for Web of Science ID 000464381005062

View details for Web of Science ID 000464381005067

View details for DOI 10.1038/s41467-019-09614-y

View details for Web of Science ID 000463872400010

Abstract

Gene correction in human long-term hematopoietic stem cells (LT-HSCs) could be an effective therapy for monogenic diseases of the blood and immune system. Here we describe an approach for X-linked sSevere cCombined iImmunodeficiency (SCID-X1) using targeted integration of a cDNA into the endogenous start codon to functionally correct disease-causing mutations throughout the gene. Using a CRISPR-Cas9/AAV6 based strategy, we achieve up to 20% targeted integration frequencies in LT-HSCs. As measures of the lack of toxicity we observe no evidence of abnormal hematopoiesis following transplantation and no evidence of off-target mutations using a high-fidelity Cas9 as a ribonucleoprotein complex. We achieve high levels of targeting frequencies (median 45%) in CD34+ HSPCs from six SCID-X1 patients and demonstrate rescue of lymphopoietic defect in a patient derived HSPC population in vitro and in vivo. In sum, our study provides specificity, toxicity and efficacy data supportive of clinical development of genome editing to treat SCID-Xl.

View details for PubMedID 30967552

View details for PubMedID 30855744

View details for DOI 10.1038/s41591-018-0326-x

View details for Web of Science ID 000457842100021

Abstract

The CRISPR-Cas9 system is a powerful tool for genome editing, which allows the precise modification of specific DNA sequences. Many efforts are underway to use the CRISPR-Cas9 system to therapeutically correct human genetic diseases1-6. The most widely used orthologs of Cas9 are derived from Staphylococcus aureus and Streptococcus pyogenes5,7. Given that these two bacterial species infect the human population at high frequencies8,9, we hypothesized that humans may harbor preexisting adaptive immune responses to the Cas9 orthologs derived from these bacterial species, SaCas9 (S. aureus) and SpCas9 (S. pyogenes). By probing human serum for the presence of anti-Cas9 antibodies using an enzyme-linked immunosorbent assay, we detected antibodies against both SaCas9 and SpCas9 in 78% and 58% of donors, respectively. We also found anti-SaCas9 T cells in 78% and anti-SpCas9 T cells in 67% of donors, which demonstrates a high prevalence of antigen-specific T cells against both orthologs. We confirmed that these T cells were Cas9-specific by demonstrating a Cas9-specific cytokine response following isolation, expansion, and antigen restimulation. Together, these data demonstrate that there are preexisting humoral and cell-mediated adaptive immune responses to Cas9 in humans, a finding that should be taken into account as the CRISPR-Cas9 system moves toward clinical trials.

View details for PubMedID 30692695

View details for DOI 10.1016/j.ymthe.2018.10.008

View details for Web of Science ID 000454708300015

Abstract

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

View details for DOI 10.1038/s41467-019-13620-5

View details for PubMedID 31796738

View details for DOI 10.1038/s41467-019-13392-y

View details for DOI 10.1016/j.ymthe.2019.09.005

View details for PubMedID 31537456

Abstract

The functional role of U2AF1 mutations in lung adenocarcinomas (LUADs) remains incompletely understood. Here, we report a significant co-occurrence of U2AF1 S34F mutations with ROS1 translocations in LUADs. To characterize this interaction, we profiled effects of S34F on the transcriptome-wide distribution of RNA binding and alternative splicing in cells harboring the ROS1 translocation. Compared to its wild-type counterpart, U2AF1 S34F preferentially binds and modulates splicing of introns containing CAG trinucleotides at their 3' splice junctions. The presence of S34F caused a shift in cross-linking at 3' splice sites, which was significantly associated with alternative splicing of skipped exons. U2AF1 S34F induced expression of genes involved in the epithelial-mesenchymal transition (EMT) and increased tumor cell invasion. Finally, S34F increased splicing of the long over the short SLC34A2-ROS1 isoform, which was also associated with enhanced invasiveness. Taken together, our results suggest a mechanistic interaction between mutant U2AF1 and ROS1 in LUAD.

View details for DOI 10.1038/s41467-019-13392-y

View details for PubMedID 31836708

Abstract

Cystic fibrosis (CF) is a monogenic disorder caused by mutations in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene. Mortality in CF patients is mostly due to respiratory sequelae. Challenges with gene delivery have limited attempts to treat CF using invivo gene therapy, and low correction levels have hindered exvivo gene therapy efforts. We have used Cas9 and adeno-associated virus 6 to correct the F508 mutation in readily accessible upper-airway basal stem cells (UABCs) obtained from CF patients. On average, we achieved 30%-50% allelic correction in UABCs and bronchial epithelial cells (HBECs) from 10 CF patients andobserved 20%-50% CFTR function relative to non-CF controls in differentiated epithelia. Furthermore, we successfully embedded the corrected UABCs on an FDA-approved porcine small intestinal submucosal membrane (pSIS), and they retained differentiation capacity. This study supports further development of genetically corrected autologous airway stem cell transplant as a treatment for CF.

View details for DOI 10.1016/j.stem.2019.11.002

View details for PubMedID 31839569

Abstract

Chromosomal rearrangements involving the mixed lineage leukemia (MLL) gene, also known as KMT2A, are often observed in human leukemias and are generally associated with a poor prognosis. To model these leukemias, we applied clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 gene editing to induce MLL chromosomal rearrangements in human hematopoietic stem and progenitor cells purified from umbilical cord blood. Electroporation of ribonucleoprotein complexes containing chemically modified synthetic single guide RNAs and purified Cas9 protein induced translocations between chromosomes 9 and 11 [t(9;11)] at an efficiency >1%. Transplantation of gene-edited cells into immune-compromised mice rapidly induced acute leukemias of different lineages and often with multiclonal origins dictated by the duration of in vitro culture prior to transplantation. Breakpoint junction sequences served as biomarkers to monitor clonal selection and progression in culture and in vivo. High-dimensional cell surface and intracellular protein analysis by mass cytometry (CyTOF) revealed that gene-edited leukemias recapitulated disease-specific protein expression observed in human patients and showed that MLL-rearranged (MLLr) mixed phenotype acute leukemias (MPALs) were more similar to acute myeloid leukemias (AMLs) than to acute lymphoblastic leukemias (ALLs). Therefore, highly efficient generation of MLL chromosomal translocations in primary human blood stem cells using CRISPR/Cas9 reliably models human acute MLLr leukemia and provides an experimental platform for basic and translational studies of leukemia biology and therapeutics.

View details for DOI 10.1182/bloodadvances.2019000450

View details for PubMedID 31582391

Abstract

The original version of this Article omitted the following from the Acknowledgements:"G.B. acknowledges the support from the Cancer Prevention and Research Institute of Texas (RR140081 and RR170721)."This has now been corrected in both the PDF and HTML versions of the Article.

View details for PubMedID 31028274

Abstract

Sickle cell disease (SCD) is a monogenic disorder that affects millions worldwide. Allogeneic hematopoietic stem cell transplantation is the only available cure. Here, we demonstrate the use of CRISPR/Cas9 and a short single-stranded oligonucleotide template to correct the sickle mutation in the -globin gene in hematopoietic stem and progenitor cells (HSPCs) from peripheral blood or bone marrow of patients with SCD, with 24.5 7.6% efficiency without selection. Erythrocytes derived from gene-edited cells showed a marked reduction of sickle cells, with the level of normal hemoglobin (HbA) increased to 25.3 13.9%. Gene-corrected SCD HSPCs retained the ability to engraft when transplanted into non-obese diabetic (NOD)-SCID-gamma (NSG) mice with detectable levels of gene correction 16-19 weeks post-transplantation. We show that, by using a high-fidelity SpyCas9 that maintained the same level of on-target gene modification, the off-target effects including chromosomal rearrangements were significantly reduced. Taken together, our results demonstrate efficient gene correction of the sickle mutation in both peripheral blood and bone marrow-derived SCD HSPCs, a significant reduction in sickling of red blood cells, engraftment of gene-edited SCD HSPCs in vivo and the importance of reducing off-target effects; all are essential for moving genome editing based SCD treatment into clinical practice.

View details for DOI 10.1093/nar/gkz475

View details for PubMedID 31147717

Abstract

Lysosomal enzyme deficiencies comprise a large group of genetic disorders that generally lack effective treatments. A potential treatment approach is to engineer the patient's own hematopoietic system to express high levels of the deficient enzyme, thereby correcting the biochemical defect and halting disease progression. Here, we present an efficient ex vivo genome editing approach using CRISPR-Cas9 that targets the lysosomal enzyme iduronidase to the CCR5 safe harbor locus in human CD34+ hematopoietic stem and progenitor cells. The modified cells secrete supra-endogenous enzyme levels, maintain long-term repopulation and multi-lineage differentiation potential, and can improve biochemical and phenotypic abnormalities in an immunocompromised mouse model of Mucopolysaccharidosis type I. These studies provide support for the development of genome-edited CD34+ hematopoietic stem and progenitor cells as a potential treatment for Mucopolysaccharidosis type I. The safe harbor approach constitutes a flexible platform for the expression of lysosomal enzymes making it applicable to other lysosomal storage disorders.

View details for DOI 10.1038/s41467-019-11962-8

View details for PubMedID 31492863

Abstract

Scarless genome editing in human pluripotent stem cells (hPSCs) represents a goal for both precise research applications and clinical translation of hPSC-derived therapies. Here we established a versatile and efficient method that combines CRISPR-Cas9-mediated homologous recombination with positive-negative selection of edited clones to generate scarless genetic changes in hPSCs.

View details for PubMedID 30504872

View details for DOI 10.1038/s41592-018-0212-y

View details for Web of Science ID 000451826200029

View details for DOI 10.1182/blood-2018-99-111772

View details for Web of Science ID 000454842807199

View details for DOI 10.1182/blood-2018-99-117371

View details for Web of Science ID 000454837606172

View details for DOI 10.1182/blood-2018-99-117926

View details for Web of Science ID 000454837607164

View details for DOI 10.1182/blood-2018-99-119954

View details for Web of Science ID 000454837602132

View details for DOI 10.1126/science.aav2483

View details for Web of Science ID 000450441900035

Abstract

The Primary Immune Deficiency Treatment Consortium (PIDTC) performed a retrospective analysis of 662 patients with severe combined immunodeficiency (SCID) who received a hematopoietic cell transplantation (HCT) as first-line treatment between 1982 and 2012 in 33 North American institutions. Overall survival was higher after HCT from matched-sibling donors (MSDs). Among recipients of non-MSD HCT, multivariate analysis showed that the SCID genotype strongly influenced survival and immune reconstitution. Overall survival was similar for patients with RAG, IL2RG, or JAK3 defects and was significantly better compared with patients with ADA or DCLRE1C mutations. Patients with RAG or DCLRE1C mutations had poorer immune reconstitution than other genotypes. Although survival did not correlate with the type of conditioning regimen, recipients of reduced-intensity or myeloablative conditioning had a lower incidence of treatment failure and better T- and B-cell reconstitution, but a higher risk for graft-versus-host disease, compared with those receiving no conditioning or immunosuppression only. Infection-free status and younger age at HCT were associated with improved survival. Typical SCID, leaky SCID, and Omenn syndrome had similar outcomes. Landmark analysis identified CD4+ and CD4+CD45RA+ cell counts at 6 and 12 months post-HCT as biomarkers predictive of overall survival and long-term T-cell reconstitution. Our data emphasize the need for patient-tailored treatment strategies depending upon the underlying SCID genotype. The prognostic significance of CD4+ cell counts as early as 6 months after HCT emphasizes the importance of close follow-up of immune reconstitution to identify patients who may need additional intervention to prevent poor long-term outcome.

View details for PubMedID 30154114

View details for PubMedCentralID PMC6202916

Abstract

Editing the beta-globin locus in hematopoietic stem cells is an alternative therapeutic approach for gene therapy of beta-thalassemia and sickle cell disease. Using the CRISPR/Cas9 system, we genetically modified human hematopoietic stem and progenitor cells (HSPCs) to mimic the large rearrangements in the beta-globin locus associated with hereditary persistence of fetal hemoglobin (HPFH), a condition that mitigates the clinical phenotype of patients with beta-hemoglobinopathies. We optimized and compared the efficiency of plasmid-, lentiviral vector (LV)-, RNA-, and ribonucleoprotein complex (RNP)-based methods to deliver the CRISPR/Cas9 system into HSPCs. Plasmid delivery of Cas9 and gRNA pairs targeting two HPFH-like regions led to high frequency of genomic rearrangements and HbF reactivation in erythroblasts derived from sorted, Cas9+ HSPCs but was associated with significant cell toxicity. RNA-mediated delivery of CRISPR/Cas9 was similarly toxic but much less efficient in editing the beta-globin locus. Transduction of HSPCs by LVs expressing Cas9 and gRNA pairs was robust and minimally toxic but resulted in poor genome-editing efficiency. Ribonucleoprotein (RNP)-based delivery of CRISPR/Cas9 exhibited a good balance between cytotoxicity and efficiency of genomic rearrangements as compared to the other delivery systems and resulted in HbF upregulation in erythroblasts derived from unselected edited HSPCs.

View details for PubMedID 30424953

View details for DOI 10.1016/j.ymthe.2018.06.002

View details for Web of Science ID 000447758800012

Abstract

The Cas9/guide RNA (Cas9/gRNA) system is commonly used for genome editing. mRNA expressing Cas9 can induce innate immune responses, reducing Cas9 expression. First-generation Cas9 mRNAs were modified with pseudouridine and 5-methylcytosine to reduce innate immune responses. We combined four approaches to produce more active, less immunogenic second-generation Cas9 mRNAs. First, we developed a novel co-transcriptional capping method yielding natural Cap 1. Second, we screened modified nucleotides in Cas9 mRNA to identify novel modifications that increase Cas9 activity. Third, we depleted the mRNA of uridines to improve mRNA activity. Lastly, we tested high-performance liquid chromatography (HPLC) purification to remove double-stranded RNAs. The activity of these mRNAs was tested in cell lines and primary human CD34+ cells. Cytokines were measured in whole blood and mice. These approaches yielded more active and less immunogenic mRNA. Uridine depletion (UD) most impacted insertion or deletion (indel) activity. Specifically, 5-methoxyuridine UD induced indel frequencies as high as 88% (average SD= 79% 11%) and elicited minimal immune responses without needing HPLC purification. Our work suggests that uridine-depleted Cas9 mRNA modified with 5-methoxyuridine (without HPLC purification) or pseudouridine may be optimal for the broad use of Cas9 both invitro and invivo.

View details for PubMedID 30195789

View details for DOI 10.1016/j.omtn.2018.06.010

View details for Web of Science ID 000443860200045

View details for Web of Science ID 000487702805034

View details for Web of Science ID 000487702807129

Abstract

Smithies et al. (1985) and Jasin and colleagues (1994) provided proof of concept that homologous recombination (HR) could be applied to the treatment of human disease and that its efficiency could be improved by the induction of double-strand breaks (DSBs). A key advance was the discovery of engineered nucleases, such as zinc-finger nucleases (ZFNs) and transcription activator-like (TAL) effector nucleases (TALENs), that can generate site-specific DSBs. The democratization and widespread use of genome editing was enabled by the discovery of the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 nuclease system. While genome editing using ZFNs and TALENs has already reached clinical trials, the pace at which genome editing enters human trials is bound to accelerate in the next several years with multiple promising preclinical studies heralding cures for monogenic diseases that are currently difficult to manage or even incurable. Here we review recent advances and current limitations and discuss the path forward using genome editing to understand, treat, and cure genetic diseases.

View details for PubMedID 29908711

View details for Web of Science ID 000435342204102

View details for Web of Science ID 000435342203060

View details for Web of Science ID 000435342205041

View details for Web of Science ID 000435342203069

View details for Web of Science ID 000435342205038

View details for Web of Science ID 000435342200067

Abstract

Naturally occurring, large deletions in the -globin locus result in hereditary persistence of fetal hemoglobin, a condition that mitigates the clinical severity of sickle cell disease (SCD) and -thalassemia. We designed a clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (Cas9) (CRISPR/Cas9) strategy to disrupt a 13.6-kb genomic region encompassing the - and -globin genes and a putative - intergenic fetal hemoglobin (HbF) silencer. Disruption of just the putative HbF silencer results in a mild increase in -globin expression, whereas deletion or inversion of a 13.6-kb region causes a robust reactivation of HbF synthesis in adult erythroblasts that is associated with epigenetic modifications and changes in chromatin contacts within the -globin locus. In primary SCD patient-derived hematopoietic stem/progenitor cells, targeting the 13.6-kb region results in a high proportion of -globin expression in erythroblasts, increased HbF synthesis, and amelioration of the sickling cell phenotype. Overall, this study provides clues for a potential CRISPR/Cas9 genome editing approach to the therapy of -hemoglobinopathies.

View details for PubMedID 29519807

Abstract

Genome editing provides a potential approach to model de novo leukemogenesis in primary human hematopoietic stem and progenitor cells (HSPCs) through induction of chromosomal translocations by targeted DNA double-strand breaks. However, very low efficiency of translocations and lack of markers for translocated cells serve as barriers to their characterization and model development. Here, we used transcription activator-like effector nucleases to generate t(9;11) chromosomal translocations encoding MLL-AF9 and reciprocal AF9-MLL fusion products in CD34+ human cord blood cells. Selected cytokine combinations enabled monoclonal outgrowth and immortalization of initially rare translocated cells, which were distinguished by elevated MLL target gene expression, high surface CD9 expression, and increased colony-forming ability. Subsequent transplantation into immune-compromised mice induced myeloid leukemias within 48 weeks, whose pathologic and molecular features extensively overlap with de novo patient MLL-rearranged leukemias. No secondary pathogenic mutations were revealed by targeted exome sequencing and whole genome RNA-sequencing analyses, suggesting the genetic sufficiency of t(9;11) translocation for leukemia development from human HSPCs. Thus, genome editing enables modeling of human acute MLL-rearranged leukemia in vivo, reflecting the genetic simplicity of this disease, and provides an experimental platform for biological and disease-modeling applications.

View details for PubMedID 29650777

View details for PubMedCentralID PMC5916000

View details for Web of Science ID 000431311600087

View details for Web of Science ID 000431311600212

View details for DOI 10.1016/j.ymgme.2017.12.129

View details for Web of Science ID 000424963800122

Abstract

Genome-editing technologies are currently being translated to the clinic. However, cellular effects of the editing machinery have yet to be fully elucidated. Here, we performed global microarray-based gene expression measurements on human CD34+ hematopoietic stem and progenitor cells that underwent editing. We probed effects of the entire editing process as well as each component individually, including electroporation, Cas9 (mRNA or protein) with chemically modified sgRNA, and AAV6 transduction. We identified differentially expressed genes relative to control treatments, which displayed enrichment for particular biological processes. All editing machinery components elicited immune, stress, and apoptotic responses. Cas9 mRNA invoked the greatest amount of transcriptional change, eliciting a distinct viral response and global transcriptional downregulation, particularly of metabolic and cell cycle processes. Electroporation also induced significant transcriptional change, with notable downregulation of metabolic processes. Surprisingly, AAV6 evoked no detectable viral response. We also found Cas9/sgRNA ribonucleoprotein treatment to be well tolerated, in spite of eliciting a DNA damage signature. Overall, this data establishes a benchmark for cellular tolerance of CRISPR/Cas9-AAV6-based genome editing, ensuring that the clinical protocol is as safe and efficient as possible.

View details for PubMedID 30005866

View details for Web of Science ID 000526798400022

View details for PubMedID 30361364

Abstract

Engineered nuclease-mediated gene targeting through homologous recombination (HR) in hematopoietic stem and progenitor cells (HSPCs) has the potential to treat a variety of genetic hematologic and immunologic disorders. Here, we identify critical parameters to reproducibly achieve high frequencies of RNA-guided (single-guide RNA [sgRNA]; CRISPR)-Cas9 nuclease (Cas9/sgRNA) and rAAV6-mediated HR at the -globin (HBB) locus in HSPCs. We identified that by transducing HSPCs with rAAV6 post-electroporation, there was a greater than 2-fold electroporation-aided transduction (EAT) of rAAV6 endocytosis with roughly 70% of the cell population having undergone transduction within 2hr. When HSPCs are cultured at low densities (1 105 cells/mL) prior to HBB targeting, HSPC expansion rates are significantly positively correlated with HR frequencies invitro as well as in repopulating cells in immunodeficient NSG mice invivo. We also show that culturing fluorescence-activated cell sorting (FACS)-enriched HBB-targeted HSPCs at low cell densities in the presence of the small molecules, UM171 and SR1, stimulates the expansion of gene-edited HSPCs as measured by higher engraftment levels in immunodeficient mice. This work serves not only as an optimized protocol for genome editing HSPCs at the HBB locus for the treatment of -hemoglobinopathies but also as a foundation for editing HSPCs at other loci for both basic and translational research.

View details for PubMedID 30195800

Abstract

Translation of the CRISPR-Cas9 system to human therapeutics holds high promise. However, specificity remains a concern especially when modifying stem cell populations. We show that existing rationally engineered Cas9 high-fidelity variants have reduced on-target activity when using the therapeutically relevant ribonucleoprotein (RNP) delivery method. Therefore, we devised an unbiased bacterial screen to isolate variants that retain activity in the RNP format. Introduction of a single point mutation, p.R691A, in Cas9 (high-fidelity (HiFi) Cas9) retained the high on-target activity of Cas9 while reducing off-target editing. HiFi Cas9 induces robust AAV6-mediated gene targeting at five therapeutically relevant loci (HBB, IL2RG, CCR5, HEXB, and TRAC) in human CD34+ hematopoietic stem and progenitor cells (HSPCs) as well as primary T cells. We also show that HiFi Cas9 mediates high-level correction of the sickle cell disease (SCD)-causing p.E6V mutation in HSPCs derived from patients with SCD. We anticipate that HiFi Cas9 will have wide utility for both basic science and therapeutic genome-editing applications.

View details for PubMedID 30082871

View details for DOI 10.1016/j.omtn.2018.04.017

Abstract

Genome editing via homologous recombination (HR) (gene targeting) in human hematopoietic stem cells (HSCs) has the power to reveal gene-function relationships and potentially transform curative hematological gene and cell therapies. However, there are no comprehensive and reproducible protocols for targeting HSCs for HR. Herein, we provide a detailed protocol for the production, enrichment, and in vitro and in vivo analyses of HR-targeted HSCs by combining CRISPR/Cas9 technology with the use of rAAV6 and flow cytometry. Using this protocol, researchers can introduce single-nucleotide changes into the genome or longer gene cassettes with the precision of genome editing. Along with our troubleshooting and optimization guidelines, researchers can use this protocol to streamline HSC genome editing at any locus of interest. The in vitro HSC-targeting protocol and analyses can be completed in 3 weeks, and the long-term in vivo HSC engraftment analyses in immunodeficient mice can be achieved in 16 weeks. This protocol enables manipulation of genes for investigation of gene functions during hematopoiesis, as well as for the correction of genetic mutations in HSC transplantation-based therapies for diseases such as sickle cell disease, -thalassemia, and primary immunodeficiencies.

View details for PubMedID 29370156

View details for DOI 10.1056/NEJMe1713735

View details for Web of Science ID 000417201100012

View details for DOI 10.7554/eLife.27873.001

View details for Web of Science ID 000413722800001

Abstract

The CRISPR/Cas9 system has recently been shown tofacilitate high levels of precise genome editing using adeno-associated viral (AAV) vectors to serve as donor template DNA during homologous recombination (HR). However, the maximum AAV packaging capacity of 4.5 kb limits the donor size. Here, we overcome this constraint by showing that two co-transduced AAV vectors can serve as donors during consecutive HR events for the integration of large transgenes. Importantly, the method involves a single-step procedure applicable to primary cells with relevance to therapeutic genome editing. We use the methodology in primary human Tcells and CD34+ hematopoietic stem and progenitor cells to site-specifically integrate an expression cassette that, as a single donor vector, would otherwise amount to a total of 6.5 kb. This approach now provides an efficient way to integrate large transgene cassettes into the genomes of primary human cells using HR-mediated genome editing with AAV vectors.

View details for PubMedID 28723575

View details for DOI 10.1001/jama.2017.4548

View details for Web of Science ID 000400842400008

View details for PubMedID 28395003

View details for Web of Science ID 000401083600742

View details for Web of Science ID 000401083600356

View details for Web of Science ID 000401083600645

View details for Web of Science ID 000401083600746

View details for Web of Science ID 000401083600162

View details for Web of Science ID 000401083600357

View details for Web of Science ID 000401083600514

View details for DOI 10.1161/CIRCRESAHA.116.310197

View details for PubMedID 28254802

View details for PubMedCentralID PMC5394983

View details for DOI 10.1016/j.bbmt.2016.12.216

View details for Web of Science ID 000540635000408

View details for Web of Science ID 000540635000101

Abstract

Targeted genetic engineering using programmable nucleases such as transcription activator-like effector nucleases (TALENs) is a valuable tool for precise, site-specific genetic modification in the human genome.The emergence of novel technologies such as human induced pluripotent stem cells (iPSCs) and nuclease-mediated genome editing represent a unique opportunity for studying cardiovascular diseases in vitro.By incorporating extensive literature and database searches, we designed a collection of TALEN constructs to knockout 88 human genes that are associated with cardiomyopathies and congenital heart diseases. The TALEN pairs were designed to induce double-strand DNA break near the starting codon of each gene that either disrupted the start codon or introduced a frameshift mutation in the early coding region, ensuring faithful gene knockout. We observed that all the constructs were active and disrupted the target locus at high frequencies. To illustrate the utility of the TALEN-mediated knockout technique, 6 individual genes (TNNT2, LMNA/C, TBX5, MYH7, ANKRD1, and NKX2.5) were knocked out with high efficiency and specificity in human iPSCs. By selectively targeting a pathogenic mutation (TNNT2 p.R173W) in patient-specific iPSC-derived cardiac myocytes, we demonstrated that the knockout strategy ameliorates the dilated cardiomyopathy phenotype in vitro. In addition, we modeled the Holt-Oram syndrome in iPSC-cardiac myocytes in vitro and uncovered novel pathways regulated by TBX5 in human cardiac myocyte development.Collectively, our study illustrates the powerful combination of iPSCs and genome editing technologies for understanding the biological function of genes, and the pathological significance of genetic variants in human cardiovascular diseases. The methods, strategies, constructs, and iPSC lines developed in this study provide a validated, readily available resource for cardiovascular research.

View details for DOI 10.1161/CIRCRESAHA.116.309948

View details for PubMedID 28246128

Abstract

Precise and efficient manipulation of genes is crucial for understanding the molecular mechanisms that govern human hematopoiesis and for developing novel therapies for diseases of the blood and immune system. Current methods do not enable precise engineering of complex genotypes that can be easily tracked in a mixed population of cells. We describe a method to multiplex homologous recombination (HR) in human hematopoietic stem and progenitor cells and primary human T cells by combining rAAV6 donor delivery and the CRISPR/Cas9 system delivered as ribonucleoproteins (RNPs). In addition, the use of reporter genes allows FACS-purification and tracking of cells that have had multiple alleles or loci modified by HR. We believe this method will enable broad applications not only to the study of human hematopoietic gene function and networks, but also to perform sophisticated synthetic biology to develop innovative engineered stem cell-based therapeutics.

View details for PubMedID 28956530

View details for PubMedID 29211662

Abstract

Since the discovery two decades ago that programmable endonucleases can be engineered to modify human cells at single nucleotide resolution, the concept of genome editing was born. Now these technologies are being applied to therapeutically relevant cell types, including hematopoietic stem cells (HSC), which possess the power to repopulate an entire blood and immune system. The purpose of this review is to discuss the changing landscape of genome editing in hematopoietic stem cells (GE-HSC) from the discovery stage to the preclinical stage, with the imminent goal of clinical translation for the treatment of serious genetic diseases of the blood and immune system.With the discovery that the RNA-programmable (sgRNA) clustered regularly interspace short palindromic repeats (CRISPR)-Cas9 nuclease (Cas9/sgRNA) systems can be easily used to precisely modify the human genome in 2012, a genome-editing revolution of hematopoietic stem cells (HSC) has bloomed. We have observed that over the last 2 years, academic institutions and small biotech companies are developing HSC-based Cas9/sgRNA genome-editing curative strategies to treat monogenic disorders, including -hemoglobinopathies and primary immunodeficiencies. We will focus on recent publications (within the past 2 years) that employ different genome-editing strategies to 'hijack' the cell's endogenous double-strand repair pathways to confer a disease-specific therapeutic advantage.The number of genome-editing strategies in HSCs that could offer therapeutic potential for diseases of the blood and immune system have dramatically risen over the past 2 years. The HSC-based genome-editing field is primed to enter clinical trials in the subsequent years. We will summarize the major advancements for the development of novel autologous GE-HSC cell and gene therapy strategies for hematopoietic diseases that are candidates for curative allogeneic bone marrow transplantation.

View details for PubMedID 28806273

Abstract

The beta-hemoglobinopathies are diverse set of disorders caused by mutations in the beta-globin (HBB) gene. Because HBB protein is a critical component (along with alpha-globin, heme, and iron) of hemoglobin, the molecule essential for oxygen delivery to tissues, mutations in HBB can result in lethal diseases or diseases with multi-organ dysfunction. HBB mutations can be roughly divided into two categories: those that cause a dysfunctional protein (such as sickle cell disease but also including varied diseases caused by high-affinity hemoglobins, low-affinity hemoglobins, and methemoglobinemia) and those that cause the insufficient production of HBB protein (beta-thalassemia). Sickle cell disease and beta-thalassemia are both the most prevalent and the most devastating of the beta-hemoglobinopathies.

View details for PubMedID 29127682

View details for Web of Science ID 000394452302082

View details for Web of Science ID 000394452705070

View details for Web of Science ID 000394446800058

Abstract

The -haemoglobinopathies, such as sickle cell disease and -thalassaemia, are caused by mutations in the -globin (HBB) gene and affect millions of people worldwide. Ex vivo gene correction in patient-derived haematopoietic stem cells followed by autologous transplantation could be used to cure -haemoglobinopathies. Here we present a CRISPR/Cas9 gene-editing system that combines Cas9 ribonucleoproteins and adeno-associated viral vector delivery of a homologous donor to achieve homologous recombination at the HBB gene in haematopoietic stem cells. Notably, we devise an enrichment model to purify a population of haematopoietic stem and progenitor cells with more than 90% targeted integration. We also show efficient correction of the Glu6Val mutation responsible for sickle cell disease by using patient-derived stem and progenitor cells that, after differentiation into erythrocytes, express adult -globin (HbA) messenger RNA, which confirms intact transcriptional regulation of edited HBB alleles. Collectively, these preclinical studies outline a CRISPR-based methodology for targeting haematopoietic stem cells by homologous recombination at the HBB locus to advance the development of next-generation therapies for -haemoglobinopathies.

View details for DOI 10.1038/nature20134

View details for PubMedID 27820943

View details for Web of Science ID 000385523700029

Abstract

Membrane fusion is essential for eukaryotic life, requiring SNARE proteins to zipper up in an -helical bundle to pull two membranes together. Here, we show that vesicle fusion can be suppressed by phosphorylation of core conserved residues inside the SNARE domain. We took a proteomics approach using a PKCB knockout mast cell model and found that the key mast cell secretory protein VAMP8 becomes phosphorylated by PKC at multiple residues in the SNARE domain. Our data suggest that VAMP8 phosphorylation reduces vesicle fusion invitro and suppresses secretion in living cells, allowing vesicles to dock but preventing fusion with the plasma membrane. Markedly, we show that the phosphorylation motif is absent in all eukaryotic neuronal VAMPs, but present in all other VAMPs. Thus, phosphorylation of SNARE domains is a general mechanism to restrict how much cells secrete, opening the door for new therapeutic strategies for suppression of secretion.

View details for DOI 10.15252/embj.201694071

View details for PubMedID 27402227

Abstract

Gene editing is a rapidly developing area of biotechnology in which the nucleotide sequence of the genome of living cells is precisely changed. The use of genome-editing technologies to modify various types of blood cells, including hematopoietic stem cells, has emerged as an important field of therapeutic development for hematopoietic disease. Although these technologies offer the potential for generation of transformative therapies for patients suffering from myriad disorders of hematopoiesis, their application for therapeutic modification of primary human cells is still in its infancy. Consequently, development of ethical and regulatory frameworks that ensure their safe and effective use is an increasingly important consideration. Here, we review a number of issues that have the potential to impact the clinical implementation of genome-editing technologies, and suggest paths forward for resolving them such that new therapies can be safely and rapidly translated to the clinic.

View details for DOI 10.1182/blood-2016-01-678136

View details for Web of Science ID 000378334400009

View details for PubMedID 27053531

View details for PubMedID 27231391

View details for PubMedCentralID PMC4882800

View details for DOI 10.1016/S1525-0016(16)33371-8

View details for Web of Science ID 000375264200556

View details for DOI 10.1016/S1525-0016(16)32932-X

View details for Web of Science ID 000375264200124

View details for DOI 10.1016/S1525-0016(16)32937-9

View details for Web of Science ID 000375264200129

View details for DOI 10.1016/S1525-0016(16)33367-6

View details for Web of Science ID 000375264200552

Abstract

Oncogenes are activated through well-known chromosomal alterations such as gene fusion, translocation, and focal amplification. In light of recent evidence that the control of key genes depends on chromosome structures called insulated neighborhoods, we investigated whether proto-oncogenes occur within these structures and whether oncogene activation can occur via disruption of insulated neighborhood boundaries in cancer cells. We mapped insulated neighborhoods in T cell acute lymphoblastic leukemia (T-ALL) and found that tumor cell genomes contain recurrent microdeletions that eliminate the boundary sites of insulated neighborhoods containing prominent T-ALL proto-oncogenes. Perturbation of such boundaries in nonmalignant cells was sufficient to activate proto-oncogenes. Mutations affecting chromosome neighborhood boundaries were found in many types of cancer. Thus, oncogene activation can occur via genetic alterations that disrupt insulated neighborhoods in malignant cells.

View details for DOI 10.1126/science.aad9024

View details for Web of Science ID 000372756200050

View details for DOI 10.1158/1538-7445.PEDCA15-IA04

View details for Web of Science ID 000374168400076

View details for DOI 10.1038/ncomms10713

View details for Web of Science ID 000371035200014

Abstract

Neonatal cholestasis is a potentially life-threatening condition requiring prompt diagnosis. Mutations in several different genes can cause progressive familial intrahepatic cholestasis, but known genes cannot account for all familial cases. Here we report four individuals from two unrelated families with neonatal cholestasis and mutations in NR1H4, which encodes the farnesoid X receptor (FXR), a bile acid-activated nuclear hormone receptor that regulates bile acid metabolism. Clinical features of severe, persistent NR1H4-related cholestasis include neonatal onset with rapid progression to end-stage liver disease, vitamin K-independent coagulopathy, low-to-normal serum gamma-glutamyl transferase activity, elevated serum alpha-fetoprotein and undetectable liver bile salt export pump (ABCB11) expression. Our findings demonstrate a pivotal function for FXR in bile acid homeostasis and liver protection.

View details for DOI 10.1038/ncomms10713

View details for PubMedID 26888176

Abstract

Invasive fungal disease (IFD) remains a major cause of morbidity and mortality in pediatric patients after allogeneic hematopoietic stem cell transplant (HSCT). We analyzed the outcome of 152 consecutive pediatric patients who underwent allogeneic HSCT from 2005 to 2012: 126 of these without a history of IFD and 26 with IFD before HSCT. Antifungal prophylaxis agent was determined by the primary transplant attending. The rate of IFD after HSCT among patients with or without prior IFD was similar (7.7% with and 7.1% without a history of fungal disease before transplant). Mortality in these 2 populations did not differ (35% vs. 28%, P=0.48, ). Patients deemed at higher risk for IFD were generally placed on voriconazole prophylaxis; however, this did not affect rates of posttransplant IFD. All-cause mortality in patients with posttransplant IFD was significantly higher than those without posttransplant IFD (67% vs. 21%, P<0.0001,). Identifying risk factors for posttransplant IFD remains a high priority to improve outcome of HSCT.

View details for PubMedID 27658021

Abstract

Oncogenes are activated through well-known chromosomal alterations such as gene fusion, translocation, and focal amplification. In light of recent evidence that the control of key genes depends on chromosome structures called insulated neighborhoods, we investigated whether proto-oncogenes occur within these structures and whether oncogene activation can occur via disruption of insulated neighborhood boundaries in cancer cells. We mapped insulated neighborhoods in T cell acute lymphoblastic leukemia (T-ALL) and found that tumor cell genomes contain recurrent microdeletions that eliminate the boundary sites of insulated neighborhoods containing prominent T-ALL proto-oncogenes. Perturbation of such boundaries in nonmalignant cells was sufficient to activate proto-oncogenes. Mutations affecting chromosome neighborhood boundaries were found in many types of cancer. Thus, oncogene activation can occur via genetic alterations that disrupt insulated neighborhoods in malignant cells.

View details for PubMedID 26940867

Abstract

The ability to manipulate the genome with precise spatial and nucleotide resolution (genome editing) has been a powerful research tool. In the past decade, the tools and expertise for using genome editing in human somatic cells and pluripotent cells have increased to such an extent that the approach is now being developed widely as a strategy to treat human disease. The fundamental process depends on creating a site-specific DNA double-strand break (DSB) in the genome and then allowing the cell's endogenous DSB repair machinery to fix the break such that precise nucleotide changes are made to the DNA sequence. With the development and discovery of several different nuclease platforms and increasing knowledge of the parameters affecting different genome editing outcomes, genome editing frequencies now reach therapeutic relevance for a wide variety of diseases. Moreover, there is a series of complementary approaches to assessing the safety and toxicity of any genome editing process, irrespective of the underlying nuclease used. Finally, the development of genome editing has raised the issue of whether it should be used to engineer the human germline. Although such an approach could clearly prevent the birth of people with devastating and destructive genetic diseases, questions remain about whether human society is morally responsible enough to use this tool.

View details for PubMedID 26566154

View details for Web of Science ID 000385740200001

Abstract

The ability to manipulate the genome with precise spatial and nucleotide resolution (genome editing) has been a powerful research tool. In the past decade, the tools and expertise for using genome editing in human somatic cells and pluripotent cells have increased to such an extent that the approach is now being developed widely as a strategy to treat human disease. The fundamental process depends on creating a site-specific DNA double-strand break (DSB) in the genome and then allowing the cell's endogenous DSB repair machinery to fix the break such that precise nucleotide changes are made to the DNA sequence. With the development and discovery of several different nuclease platforms and increasing knowledge of the parameters affecting different genome editing outcomes, genome editing frequencies now reach therapeutic relevance for a wide variety of diseases. Moreover, there is a series of complementary approaches to assessing the safety and toxicity of any genome editing process, irrespective of the underlying nuclease used. Finally, the development of genome editing has raised the issue of whether it should be used to engineer the human germline. Although such an approach could clearly prevent the birth of people with devastating and destructive genetic diseases, questions remain about whether human society is morally responsible enough to use this tool.

View details for DOI 10.1146/annurev-pharmtox-010814-124454

View details for Web of Science ID 000368345700010

Abstract

Cystic fibrosis (CF) is a recessive inherited disease associated with multiorgan damage that compromises epithelial and inflammatory cell function. Induced pluripotent stem cells (iPSCs) have significantly advanced the potential of developing a personalized cell-based therapy for diseases like CF by generating patient-specific stem cells that can be differentiated into cells that repair tissues damaged by disease pathology. The F508del mutation in airway epithelial cell-derived CF-iPSCs was corrected with small/short DNA fragments (SDFs) and sequence-specific TALENs. An allele-specific PCR, cyclic enrichment strategy gave ~100-fold enrichment of the corrected CF-iPSCs after six enrichment cycles that facilitated isolation of corrected clones. The seamless SDF-based gene modification strategy used to correct the CF-iPSCs resulted in pluripotent cells that, when differentiated into endoderm/airway-like epithelial cells showed wild-type (wt) airway epithelial cell cAMP-dependent Cl ion transport or showed the appropriate cell-type characteristics when differentiated along mesoderm/hematopoietic inflammatory cell lineage pathways.

View details for DOI 10.1038/mtna.2015.43

View details for PubMedID 26730810

Abstract

Whether aged hematopoietic stem and progenitor cells (HSPCs) have impaired DNA damage repair is controversial. Using a combination of DNA mutation indicator assays, we observe a 2- to 3-fold increase in the number of DNA mutations in the hematopoietic system upon aging. Young and aged hematopoietic stem cells (HSCs) and hematopoietic progenitor cells (HPCs) do not show an increase in mutation upon irradiation-induced DNA damage repair, and young and aged HSPCs respond very similarly to DNA damage with respect to cell-cycle checkpoint activation and apoptosis. Both young and aged HSPCs show impaired activation of the DNA-damage-induced G1-S checkpoint. Induction of chronic DNA double-strand breaks by zinc-finger nucleases suggests that HSPCs undergo apoptosis rather than faulty repair. These data reveal a protective mechanism in both the young and aged hematopoietic system against accumulation of mutations in response to DNA damage.

View details for DOI 10.1016/j.celrep.2015.11.030

View details for Web of Science ID 000367101400012

View details for PubMedID 26686632

View details for PubMedCentralID PMC4691560

Abstract

Genome editing is the process of precisely modifying the nucleotide sequence of the genome. It has provided a powerful approach to research questions but, with the development of a new set of tools, it is now possible to achieve frequencies of genome editing that are high enough to be useful therapeutically. Genome editing is being developed to treat not only monogenic diseases but also infectious diseases and diseases that have both a genetic and an environmental component.

View details for DOI 10.1186/s13059-015-0859-y

View details for Web of Science ID 000366898900002

View details for PubMedID 26694713

View details for PubMedCentralID PMC4699361

View details for Web of Science ID 000368020104258

Abstract

Chromosomal rearrangements involving the mixed-lineage leukemia (MLL) gene occur in primary and treatment-related leukemias and confer a poor prognosis. Studies based primarily on mouse models have substantially advanced our understanding of MLL leukemia pathogenesis, but often use supraphysiological oncogene expression with uncertain implications for human leukemia. Genome editing using site-specific nucleases provides a powerful new technology for gene modification to potentially model human disease, however, this approach has not been used to re-create acute leukemia in human cells of origin comparable to disease observed in patients. We applied transcription activator-like effector nuclease-mediated genome editing to generate endogenous MLL-AF9 and MLL-ENL oncogenes through insertional mutagenesis in primary human hematopoietic stem and progenitor cells (HSPCs) derived from human umbilical cord blood. Engineered HSPCs displayed altered in vitro growth potentials and induced acute leukemias following transplantation in immunocompromised mice at a mean latency of 16 weeks. The leukemias displayed phenotypic and morphologic similarities with patient leukemia blasts including a subset with mixed phenotype, a distinctive feature seen in clinical disease. The leukemic blasts expressed an MLL-associated transcriptional program with elevated levels of crucial MLL target genes, displayed heightened sensitivity to DOT1L inhibition, and demonstrated increased oncogenic potential ex vivo and in secondary transplant assays. Thus, genome editing to create endogenous MLL oncogenes in primary human HSPCs faithfully models acute MLL-rearranged leukemia and provides an experimental platform for prospective studies of leukemia initiation and stem cell biology in a genetic subtype of poor prognosis leukemia.

View details for DOI 10.1182/blood-2015-05-646398

View details for PubMedID 26311362

Abstract

CRISPR-Cas-mediated genome editing relies on guide RNAs that direct site-specific DNA cleavage facilitated by the Cas endonuclease. Here we report that chemical alterations to synthesized single guide RNAs (sgRNAs) enhance genome editing efficiency in human primary T cells and CD34(+) hematopoietic stem and progenitor cells. Co-delivering chemically modified sgRNAs with Cas9 mRNA or protein is an efficient RNA- or ribonucleoprotein (RNP)-based delivery method for the CRISPR-Cas system, without the toxicity associated with DNA delivery. This approach is a simple and effective way to streamline the development of genome editing with the potential to accelerate a wide array of biotechnological and therapeutic applications of the CRISPR-Cas technology.

View details for DOI 10.1038/nbt.3290

View details for PubMedID 26121415

View details for DOI 10.1038/nbt.3290

View details for PubMedID 26121415

Abstract

T-cell Prolymphocytic Leukemia (T-PLL) is a rare entity, and to date has never been reported in children. Here, we describe the first pediatric case of T-PLL in a 16-year old male and review his clinical course through treatment. He underwent therapy with alemtuzumab and pentostatin, which was successful in inducing initial remission. He then underwent an allogeneic matched sibling stem cell transplant following a myeloablative conditioning regimen and remains disease-free 1.5 years after diagnosis. Pediatr Blood Cancer 2014 Wiley Periodicals, Inc.

View details for DOI 10.1002/pbc.25336

View details for PubMedID 25417638

View details for Web of Science ID 000352855600223

Abstract

Genome editing that results in humans with precisely modified germ cells may never become practical. Nonetheless, the implications are great enough that we strongly support the idea of starting the conversation now, providing time for a broad consensus to be developed. We are confident that if diverse voices are heard, a consensus can be reached on a strategy in which societal mores are respected, the desires of parents are integrated, and the health of future generations is maximized.

View details for DOI 10.1038/mt.2015.83

View details for PubMedID 26022625

View details for DOI 10.1038/mt.2015.54

View details for PubMedID 25943494

View details for PubMedCentralID PMC4427885

View details for DOI 10.1038/mt.2015.54

View details for PubMedID 28142000

Abstract

Genetic disorders resulting from defects in the adult globin genes are among the most common inherited diseases. Symptoms worsen from birth as fetal -globin expression is silenced. Genome editing could permit the introduction of beneficial single-nucleotide variants to ameliorate symptoms. Here, as proof of concept, we introduce the naturally occurring Hereditary Persistance of Fetal Haemoglobin (HPFH) -175T>C point mutation associated with elevated fetal -globin into erythroid cell lines. We show that this mutation increases fetal globin expression through de novo recruitment of the activator TAL1 to promote chromatin looping of distal enhancers to the modified -globin promoter.

View details for DOI 10.1038/ncomms8085

View details for Web of Science ID 000355531400011

View details for PubMedID 25971621

Abstract

The ability to remove blood cells, including hematopoietic stem cells (HSCs), from a person and then re-transplant them (hematopoietic stem cell transplantation (HSCT) is a well-established treatment paradigm that can be used in both the autologous setting or in the allogeneic setting. Using allogeneic HSCT can cure different genetic diseases of the blood but has significant limitations. An alternative to allogeneic HSCT is to transplant genetically modified HSCs instead. A powerful approach to the precision modification of HSCs is to use genome editing whereby the genome is modified with spatial precision (at an exact location) in the genome and sometimes with nucleotide precision (the exact nucleotide changes are introduced). The progress and challenges of genome editing of blood are discussed.

View details for PubMedID 26029496

Abstract

The purpose of this study is to evaluate the survival of pediatric patients undergoing autologous bone marrow transplantation (auBMT) for relapsed or refractory Hodgkin lymphoma (rrHL) and to identify factors that might contribute to their outcome. We reviewed the records and clinical course of 89 consecutive rrHL patients 21 years old who underwent auBMT at Stanford Hospitals and Clinics and the Lucile Packard Children's Hospital, Stanford between 1989 and 2012. We investigated, by multiple analyses, patient, disease, and treatment characteristics associated with outcome. Endpoints were 5-year overall and event-free survival. Our findings include that cyclophosphamide, carmustine, and etoposide (CBV) as a conditioning regimen for auBMT is effective for most patients 21 years old with rrHL (5-year overall survival, 71%). Transplantation after the year 2001 was associated with significantly improved overall survival compared with our earlier experience (80% compared with 65%). Patients with multiply relapsed disease or with disease not responsive to initial therapy fared less well compared with those with response to initial therapy or after first relapse. Administration of post-auBMT consolidative radiotherapy (cRT) also appears to contribute to improved survival. We are able to conclude that high-dose chemotherapy with CBV followed by auBMT is effective for the treatment of rrHL in children and adolescents. Survival for patients who undergo auBMT for rrHL has improved significantly. This improvement may be because of patient selection and improvements in utilization of radiotherapy rather than improvements in chemotherapy. Further investigation is needed to describe the role of auBMT across the entire spectrum of patients with rrHL and to identify the most appropriate preparative regimen with or without cRT therapy in the treatment of rrHL in young patients.

View details for DOI 10.1016/j.bbmt.2014.10.020

View details for PubMedID 25445024

Abstract

Genome editing with engineered nucleases is a rapidly growing field thanks to transformative technologies that allow researchers to precisely alter genomes for numerous applications including basic research, biotechnology, and human gene therapy. While the ability to make precise and controlled changes at specified sites throughout the genome has grown tremendously in recent years, we still lack a comprehensive and standardized battery of assays for measuring the different genome editing outcomes created at endogenous genomic loci. Here we review the existing assays for quantifying on- and off-target genome editing and describe their utility in advancing the technology. We also highlight unmet assay needs for quantifying on- and off-target genome editing outcomes and discuss their importance for the genome editing field.

View details for DOI 10.1016/j.tibtech.2014.12.001

View details for PubMedID 25595557

Abstract

Genome editing with engineered nucleases is a rapidly growing field thanks to transformative technologies that allow researchers to precisely alter genomes for numerous applications including basic research, biotechnology, and human gene therapy. While the ability to make precise and controlled changes at specified sites throughout the genome has grown tremendously in recent years, we still lack a comprehensive and standardized battery of assays for measuring the different genome editing outcomes created at endogenous genomic loci. Here we review the existing assays for quantifying on- and off-target genome editing and describe their utility in advancing the technology. We also highlight unmet assay needs for quantifying on- and off-target genome editing outcomes and discuss their importance for the genome editing field.

View details for DOI 10.1016/j.tibtech.2014.12.001

View details for Web of Science ID 000349504000011

View details for PubMedCentralID PMC4308725

Abstract

Site-specific gene addition can allow stable transgene expression for gene therapy. When possible, this is preferred over the use of promiscuously integrating vectors, which are sometimes associated with clonal expansion and oncogenesis. Site-specific endonucleases that can induce high rates of targeted genome editing are finding increasing applications in biological discovery and gene therapy. However, two safety concerns persist: endonuclease-associated adverse effects, both on-target and off-target; and oncogene activation caused by promoter integration, even without nucleases. Here we perform recombinant adeno-associated virus (rAAV)-mediated promoterless gene targeting without nucleases and demonstrate amelioration of the bleeding diathesis in haemophilia B mice. In particular, we target a promoterless human coagulation factor IX (F9) gene to the liver-expressed mouse albumin (Alb) locus. F9 is targeted, along with a preceding 2A-peptide coding sequence, to be integrated just upstream to the Alb stop codon. While F9 is fused to Alb at the DNA and RNA levels, two separate proteins are synthesized by way of ribosomal skipping. Thus, F9 expression is linked to robust hepatic albumin expression without disrupting it. We injected an AAV8-F9 vector into neonatal and adult mice and achieved on-target integration into 0.5% of the albumin alleles in hepatocytes. We established that F9 was produced only from on-target integration, and ribosomal skipping was highly efficient. Stable F9 plasma levels at 7-20% of normal were obtained, and treated F9-deficient mice had normal coagulation times. In conclusion, transgene integration as a 2A-fusion to a highly expressed endogenous gene may obviate the requirement for nucleases and/or vector-borne promoters. This method may allow for safe and efficacious gene targeting in both infants and adults by greatly diminishing off-target effects while still providing therapeutic levels of expression from integration.

View details for DOI 10.1038/nature13864

View details for PubMedID 25363772

View details for PubMedCentralID PMC4297598

View details for DOI 10.1038/nature13864

View details for PubMedID 25363772

Abstract

The power of genome editing is increasingly recognized as it has become more accessible to a wide range of scientists and a wider range of uses has been reported. Nonetheless, an important practical aspect of the strategy is develop methods to increase the frequency of genome editing or methods that enrich for genome-edited cells such that they can be more easily identified. This chapter discusses several different approaches including the use of cold-shock, exonucleases, surrogate markers, specialized donor vectors, and oligonucleotides to enhance the frequency of genome editing or to facilitate the identification of genome-edited cells.

View details for DOI 10.1007/978-1-4939-1862-1_16

View details for PubMedID 25408413

View details for DOI 10.1371/journal.pone.0136644

View details for PubMedID 26351841

Abstract

One of the challenging questions in cancer biology is how a normal cell transforms into a cancer cell. There is strong evidence that specific chromosomal translocations are a key element in this transformation process. Our studies focus on understanding the developmental mechanism by which a normal stem or progenitor cell transforms into leukemia. Here we used engineered nucleases to induce simultaneous specific double strand breaks in the MLL gene and two different known translocation partners (AF4 and AF9), which resulted in specific chromosomal translocations in K562 cells as well as primary hematopoietic stem and progenitor cells (HSPCs). The initiation of a specific MLL translocation in a small number of HSPCs likely mimics the leukemia-initiating event that occurs in patients. In our studies, the creation of specific MLL translocations in CD34+ cells was not sufficient to transform cells in vitro. Rather, a variety of fates was observed for translocation positive cells including cell loss over time, a transient proliferative advantage followed by loss of the clone, or a persistent proliferative advantage. These studies highlight the application of genome engineering tools in primary human HSPCs to induce and prospectively study the consequences of initiating translocation events in leukemia pathogenesis.

View details for DOI 10.1371/journal.pone.0136644

View details for PubMedID 26351841

View details for PubMedCentralID PMC4564237

Abstract

Genetic disorders resulting from defects in the adult globin genes are among the most common inherited diseases. Symptoms worsen from birth as fetal -globin expression is silenced. Genome editing could permit the introduction of beneficial single-nucleotide variants to ameliorate symptoms. Here, as proof of concept, we introduce the naturally occurring Hereditary Persistance of Fetal Haemoglobin (HPFH) -175T>C point mutation associated with elevated fetal -globin into erythroid cell lines. We show that this mutation increases fetal globin expression through de novo recruitment of the activator TAL1 to promote chromatin looping of distal enhancers to the modified -globin promoter.

View details for DOI 10.1038/ncomms8085

View details for PubMedID 25971621

View details for Web of Science ID 000349243504090

View details for Web of Science ID 000349242702103

Abstract

Editing the genome to create specific sequence modifications is a powerful way to study gene function and promises future applicability to gene therapy. Creation of precise modifications requires homologous recombination, a very rare event in most cell types that can be stimulated by introducing a double strand break near the target sequence. One method to create a double strand break in a particular sequence is with a custom designed nuclease. We used engineered nucleases to stimulate homologous recombination to correct a mutant gene in mouse "GS" (germline stem) cells, testicular derived cell cultures containing spermatogonial stem cells and progenitor cells. We demonstrated that gene-corrected cells maintained several properties of spermatogonial stem/progenitor cells including the ability to colonize following testicular transplantation. This proof of concept for genome editing in GS cells impacts both cell therapy and basic research given the potential for GS cells to be propagated in vitro, contribute to the germline in vivo following testicular transplantation or become reprogrammed to pluripotency in vitro.

View details for DOI 10.1371/journal.pone.0112652

View details for Web of Science ID 000345533200031

View details for PubMedCentralID PMC4237364

Abstract

Exogenous cytokines, such as platelet-derived growth factor (PDGF)-B, can augment wound healing, but sustained delivery to maintain therapeutic levels remains a problem. "Genome editing" is a new technology in which precise genome modifications are made within cells using engineered site-specific nucleases. Genome editing avoids many of the complications associated with traditional gene therapy and the use of viral vectors, including random integration, imprecise gene expression, and inadvertent oncogene activation.This study demonstrates site-specific nuclease-mediated integration of a PDGF-B transgene into a predefined locus within the genome of primary mouse fibroblasts. Engineered fibroblasts were applied to splinted mouse wounds and evaluated after 14 days and 5 months for the retention of engineered fibroblasts, wound healing morphology, angiogenesis, and systemic PDGF-B expression.The application of engineered PDGF-B-expressing fibroblasts enhanced wound healing compared with controls. Low-level, constitutive expression of PDGF-B was achieved without detectable levels of systemic PDGF-B. The mechanism of improved wound healing is, at least in part, the result of increased wound vascularization, as the wounds treated with PDGF-B fibroblasts had a blood vessel density 2.5 times greater than controls. After 5 months, the engineered fibroblasts persisted in the wound bed. No adverse effects were detected from the application of these fibroblasts after 5 months as assessed by hematoxylin and eosin staining of wounds and by mouse necropsy.These data support that site-specific genome editing allows for sustained cell-based cytokine delivery. Furthermore, sustained release of PDGF-B increases the speed and quality of wound healing after a single application.

View details for DOI 10.1097/PRS.0000000000000427

View details for PubMedID 25158716

Abstract

Newborn screening for severe combined immunodeficiency (SCID) using assays to detect T-cell receptor excision circles (TRECs) began in Wisconsin in 2008, and SCID was added to the national recommended uniform panel for newborn screened disorders in 2010. Currently 23 states, the District of Columbia, and the Navajo Nation conduct population-wide newborn screening for SCID. The incidence of SCID is estimated at 1 in 100,000 births.To present data from a spectrum of SCID newborn screening programs, establish population-based incidence for SCID and other conditions with T-cell lymphopenia, and document early institution of effective treatments.Epidemiological and retrospective observational study.Representatives in states conducting SCID newborn screening were invited to submit their SCID screening algorithms, test performance data, and deidentified clinical and laboratory information regarding infants screened and cases with nonnormal results. Infants born from the start of each participating program from January 2008 through the most recent evaluable date prior to July 2013 were included. Representatives from 10 states plus the Navajo Area Indian Health Service contributed data from 3,030,083 newborns screened with a TREC test.Infants with SCID and other diagnoses of T-cell lymphopenia were classified. Incidence and, where possible, etiologies were determined. Interventions and survival were tracked.Screening detected 52 cases of typical SCID, leaky SCID, and Omenn syndrome, affecting 1 in 58,000 infants (95% CI, 1/46,000-1/80,000). Survival of SCID-affected infants through their diagnosis and immune reconstitution was 87% (45/52), 92% (45/49) for infants who received transplantation, enzyme replacement, and/or gene therapy. Additional interventions for SCID and non-SCID T-cell lymphopenia included immunoglobulin infusions, preventive antibiotics, and avoidance of live vaccines. Variations in definitions and follow-up practices influenced the rates of detection of non-SCID T-cell lymphopenia.Newborn screening in 11 programs in the United States identified SCID in 1 in 58,000 infants, with high survival. The usefulness of detection of non-SCID T-cell lymphopenias by the same screening remains to be determined.

View details for DOI 10.1001/jama.2014.9132

View details for PubMedID 25138334

Abstract

The Primary Immune Deficiency Treatment Consortium was formed to analyze the results of hematopoietic-cell transplantation in children with severe combined immunodeficiency (SCID) and other primary immunodeficiencies. Factors associated with a good transplantation outcome need to be identified in order to design safer and more effective curative therapy, particularly for children with SCID diagnosed at birth.We collected data retrospectively from 240 infants with SCID who had received transplants at 25 centers during a 10-year period (2000 through 2009).Survival at 5 years, freedom from immunoglobulin substitution, and CD3+ T-cell and IgA recovery were more likely among recipients of grafts from matched sibling donors than among recipients of grafts from alternative donors. However, the survival rate was high regardless of donor type among infants who received transplants at 3.5 months of age or younger (94%) and among older infants without prior infection (90%) or with infection that had resolved (82%). Among actively infected infants without a matched sibling donor, survival was best among recipients of haploidentical T-cell-depleted transplants in the absence of any pretransplantation conditioning. Among survivors, reduced-intensity or myeloablative pretransplantation conditioning was associated with an increased likelihood of a CD3+ T-cell count of more than 1000 per cubic millimeter, freedom from immunoglobulin substitution, and IgA recovery but did not significantly affect CD4+ T-cell recovery or recovery of phytohemagglutinin-induced T-cell proliferation. The genetic subtype of SCID affected the quality of CD3+ T-cell recovery but not survival.Transplants from donors other than matched siblings were associated with excellent survival among infants with SCID identified before the onset of infection. All available graft sources are expected to lead to excellent survival among asymptomatic infants. (Funded by the National Institute of Allergy and Infectious Diseases and others.).

View details for DOI 10.1056/NEJMoa1401177

View details for Web of Science ID 000339556900008

View details for PubMedID 25075835

View details for Web of Science ID 000337231300732

View details for Web of Science ID 000337231300090

Abstract

Targeted genome editing with engineered nucleases has transformed the ability to introduce precise sequence modifications at almost any site within the genome. A major obstacle to probing the efficiency and consequences of genome editing is that no existing method enables the frequency of different editing events to be simultaneously measured across a cell population at any endogenous genomic locus.We have developed a method for quantifying individual genome-editing outcomes at any site of interest with single-molecule real-time (SMRT) DNA sequencing. We show that this approach can be applied at various loci using multiple engineered nuclease platforms, including transcription-activator-like effector nucleases (TALENs), RNA-guided endonucleases (CRISPR/Cas9), and zinc finger nucleases (ZFNs), and in different cell lines to identify conditions and strategies in which the desired engineering outcome has occurred. This approach offers a technique for studying double-strand break repair, facilitates the evaluation of gene-editing technologies, and permits sensitive quantification of editing outcomes in almost every experimental system used.

View details for DOI 10.1016/j.celrep.2014.02.040

View details for PubMedID 24685129

Abstract

Transcription activator-like effector nucleases (TALENs) have become a powerful tool for genome editing due to the simple code linking the amino acid sequences of their DNA-binding domains to TALEN nucleotide targets. While the initial TALEN-design guidelines are very useful, user-friendly tools defining optimal TALEN designs for robust genome editing need to be developed. Here we evaluated existing guidelines and developed new design guidelines for TALENs based on 205 TALENs tested, and established the scoring algorithm for predicting TALEN activity (SAPTA) as a new online design tool. For any input gene of interest, SAPTA gives a ranked list of potential TALEN target sites, facilitating the selection of optimal TALEN pairs based on predicted activity. SAPTA-based TALEN designs increased the average intracellular TALEN monomer activity by >3-fold, and resulted in an average endogenous gene-modification frequency of 39% for TALENs containing the repeat variable di-residue NK that favors specificity rather than activity. It is expected that SAPTA will become a useful and flexible tool for designing highly active TALENs for genome-editing applications. SAPTA can be accessed via the website at http://baolab.bme.gatech.edu/Research/BioinformaticTools/TAL_targeter.html.

View details for DOI 10.1093/nar/gkt1363

View details for PubMedID 24442582

View details for PubMedCentralID PMC3973288

View details for DOI 10.1016/j.bbmt.2013.12.007

View details for Web of Science ID 000331155400009

View details for DOI 10.1016/j.bbmt.2013.12.430

View details for Web of Science ID 000331155400402

Abstract

The Primary Immune Deficiency Treatment Consortium (PIDTC) is a network of 33 centers in North America that study the treatment of rare and severe primary immunodeficiency diseases. Current protocols address the natural history of patients treated for severe combined immunodeficiency (SCID), Wiskott-Aldrich syndrome, and chronic granulomatous disease through retrospective, prospective, and cross-sectional studies. The PIDTC additionally seeks to encourage training of junior investigators, establish partnerships with European and other International colleagues, work with patient advocacy groups to promote community awareness, and conduct pilot demonstration projects. Future goals include the conduct of prospective treatment studies to determine optimal therapies for primary immunodeficiency diseases. To date, the PIDTC has funded 2 pilot projects: newborn screening for SCID in Navajo Native Americans and B-cell reconstitution in patients with SCID after hematopoietic stem cell transplantation. Ten junior investigators have received grant awards. The PIDTC Annual Scientific Workshop has brought together consortium members, outside speakers, patient advocacy groups, and young investigators and trainees to report progress of the protocols and discuss common interests and goals, including new scientific developments and future directions of clinical research. Here we report the progress of the PIDTC to date, highlights of the first 2PIDTC workshops, and consideration of future consortium objectives.

View details for DOI 10.1016/j.jaci.2013.07.052

View details for PubMedID 24139498

Abstract

Tal-effector nucleases (TALENs) are engineered proteins that can stimulate precise genome editing through specific DNA double-strand breaks. Sickle cell disease and -thalassemia are common genetic disorders caused by mutations in -globin, and we engineered a pair of highly active TALENs that induce modification of 54% of human -globin alleles near the site of the sickle mutation. These TALENS stimulate targeted integration of therapeutic, full-length beta-globin cDNA to the endogenous -globin locus in 19% of cells prior to selection as quantified by single molecule real-time sequencing. We also developed highly active TALENs to human -globin, a pharmacologic target in sickle cell disease therapy. Using the -globin and -globin TALENs, we generated cell lines that express GFP under the control of the endogenous -globin promoter and tdTomato under the control of the endogenous -globin promoter. With these fluorescent reporter cell lines, we screened a library of small molecule compounds for their differential effect on the transcriptional activity of the endogenous - and -globin genes and identified several that preferentially upregulate -globin expression.

View details for DOI 10.1093/nar/gkt947

View details for PubMedID 24157834

View details for PubMedCentralID PMC3902937

Abstract

Editing the genome to create specific sequence modifications is a powerful way to study gene function and promises future applicability to gene therapy. Creation of precise modifications requires homologous recombination, a very rare event in most cell types that can be stimulated by introducing a double strand break near the target sequence. One method to create a double strand break in a particular sequence is with a custom designed nuclease. We used engineered nucleases to stimulate homologous recombination to correct a mutant gene in mouse "GS" (germline stem) cells, testicular derived cell cultures containing spermatogonial stem cells and progenitor cells. We demonstrated that gene-corrected cells maintained several properties of spermatogonial stem/progenitor cells including the ability to colonize following testicular transplantation. This proof of concept for genome editing in GS cells impacts both cell therapy and basic research given the potential for GS cells to be propagated in vitro, contribute to the germline in vivo following testicular transplantation or become reprogrammed to pluripotency in vitro.

View details for DOI 10.1371/journal.pone.0112652

View details for PubMedID 25409432

Abstract

Immune dysregulation, Polyendocrinopathy, Enteropathy, X-linked (IPEX) syndrome is a rare autoimmune disease due to mutations in the gene encoding for Forkhead box P3 (FOXP3), a transcription factor fundamental for the function of thymus-derived (t) regulatory T (Treg) cells. The dysfunction of Treg cells results in the development of devastating autoimmune manifestations affecting multiple organs, eventually leading to premature death in infants, if not promptly treated by hematopoietic stem cell transplantation (HSCT). Novel gene therapy strategies can be developed for IPEX syndrome as more definitive cure than allogeneic HSCT. Here we describe the therapeutic approaches, alternative to HSCT, currently under development. We described that effector T cells can be converted in regulatory T cells by LV-mediated FOXP3-gene transfer in differentiated T lymphocytes. Despite FOXP3 mutations mainly affect a highly specific T cell subset, manipulation of stem cells could be required for long-term remission of the disease. Therefore, we believe that a more comprehensive strategy should aim at correcting FOXP3-mutated stem cells. Potentials and hurdles of both strategies will be highlighted here.

View details for PubMedID 25274247

Abstract

Immune dysregulation, Polyendocrinopathy, Enteropathy, X-linked (IPEX) syndrome is a rare autoimmune disease due to mutations in the gene encoding for Forkhead box P3 (FOXP3), a transcription factor fundamental for the function of thymus-derived (t) regulatory T (Treg) cells. The dysfunction of Treg cells results in the development of devastating autoimmune manifestations affecting multiple organs, eventually leading to premature death in infants, if not promptly treated by hematopoietic stem cell transplantation (HSCT). Novel gene therapy strategies can be developed for IPEX syndrome as more definitive cure than allogeneic HSCT. Here we describe the therapeutic approaches, alternative to HSCT, currently under development. We described that effector T cells can be converted in regulatory T cells by LV-mediated FOXP3-gene transfer in differentiated T lymphocytes. Despite FOXP3 mutations mainly affect a highly specific T cell subset, manipulation of stem cells could be required for long-term remission of the disease. Therefore, we believe that a more comprehensive strategy should aim at correcting FOXP3-mutated stem cells. Potentials and hurdles of both strategies will be highlighted here.

View details for Web of Science ID 000345248000002

View details for PubMedCentralID PMC4443799

Abstract

Resection of DNA double-strand breaks (DSBs) is a pivotal step during which the choice between NHEJ and HR DNA repair pathways is made. Although CDKs are known to control initiation of resection, their role in regulating long-range resection remains elusive. Here we show that CDKs 1/2 phosphorylate the long-range resection nuclease EXO1 at four C-terminal S/TP sites during S/G2 phases of the cell cycle. Impairment of EXO1 phosphorylation attenuates resection, chromosomal integrity, cell survival and HR, but augments NHEJ upon DNA damage. In contrast, cells expressing phospho-mimic EXO1 are proficient in resection even after CDK inhibition and favour HR over NHEJ. Mutation of cyclin-binding sites on EXO1 attenuates CDK binding and EXO1 phosphorylation, causing a resection defect that can be rescued by phospho-mimic mutations. Mechanistically, phosphorylation of EXO1 augments its recruitment to DNA breaks possibly via interactions with BRCA1. In summary, phosphorylation of EXO1 by CDKs is a novel mechanism regulating repair pathway choice.

View details for DOI 10.1038/ncomms4561

View details for PubMedID 24705021

Abstract

Cell lines are often regarded as clonal, even though this simplifies what is known about mutagenesis, transformation and other processes that destabilize them over time. Monitoring these clonal dynamics is important for multiple areas of biomedical research, including stem cell and cancer biology. Tracking the contributions of individual cells to large populations, however, has been constrained by limitations in sensitivity and complexity.We utilize cellular barcoding methods to simultaneously track the clonal contributions of tens of thousands of cells. We demonstrate that even with optimal culturing conditions, common cell lines including HeLa, K562 and HEK-293 T exhibit ongoing clonal dynamics. Starting a population with a single clone diminishes but does not eradicate this phenomenon. Next, we compare lentiviral and zinc-finger nuclease barcode insertion approaches, finding that the zinc-finger nuclease protocol surprisingly results in reduced clonal diversity. We also document the expected reduction in clonal complexity when cells are challenged with genotoxic stress. Finally, we demonstrate that xenografts maintain clonal diversity to a greater extent than in vitro culturing of the human non-small-cell lung cancer cell line HCC827.We demonstrate the feasibility of tracking and quantifying the clonal dynamics of entire cell populations within multiple cultured cell lines. Our results suggest that cell heterogeneity should be considered in the design and interpretation of in vitro culture experiments. Aside from clonal cell lines, we propose that cellular barcoding could prove valuable in modeling the clonal behavior of heterogeneous cell populations over time, including tumor populations treated with chemotherapeutic agents.

View details for DOI 10.1186/gb-2014-15-5-r75

View details for Web of Science ID 000338981700009

View details for PubMedCentralID PMC4073073

Abstract

Resection of DNA double-strand breaks (DSBs) is a pivotal step during which the choice between NHEJ and HR DNA repair pathways is made. Although CDKs are known to control initiation of resection, their role in regulating long-range resection remains elusive. Here we show that CDKs 1/2 phosphorylate the long-range resection nuclease EXO1 at four C-terminal S/TP sites during S/G2 phases of the cell cycle. Impairment of EXO1 phosphorylation attenuates resection, chromosomal integrity, cell survival and HR, but augments NHEJ upon DNA damage. In contrast, cells expressing phospho-mimic EXO1 are proficient in resection even after CDK inhibition and favour HR over NHEJ. Mutation of cyclin-binding sites on EXO1 attenuates CDK binding and EXO1 phosphorylation, causing a resection defect that can be rescued by phospho-mimic mutations. Mechanistically, phosphorylation of EXO1 augments its recruitment to DNA breaks possibly via interactions with BRCA1. In summary, phosphorylation of EXO1 by CDKs is a novel mechanism regulating repair pathway choice.

View details for DOI 10.1038/ncomms4561

View details for PubMedID 24705021

Abstract

Genome-wide association studies (GWASs) have ascertained numerous trait-associated common genetic variants, frequently localized to regulatory DNA. We found that common genetic variation at BCL11A associated with fetal hemoglobin (HbF) level lies in noncoding sequences decorated by an erythroid enhancer chromatin signature. Fine-mapping uncovers a motif-disrupting common variant associated with reduced transcription factor (TF) binding, modestly diminished BCL11A expression, and elevated HbF. The surrounding sequences function in vivo as a developmental stage-specific, lineage-restricted enhancer. Genome engineering reveals the enhancer is required in erythroid but not B-lymphoid cells for BCL11A expression. These findings illustrate how GWASs may expose functional variants of modest impact within causal elements essential for appropriate gene expression. We propose the GWAS-marked BCL11A enhancer represents an attractive target for therapeutic genome engineering for the -hemoglobinopathies.

View details for DOI 10.1126/science.1242088

View details for Web of Science ID 000325475200047

View details for PubMedID 24115442

Abstract

Engineered nucleases, which incise the genome at predetermined sites, have a number of laboratory and clinical applications. There is, however, a need for better methods for controlled intracellular delivery of nucleases. Here, we demonstrate a method for ligand-mediated delivery of zinc finger nucleases (ZFN) proteins using transferrin receptor-mediated endocytosis. Uptake is rapid and efficient in established mammalian cell lines and in primary cells, including mouse and human hematopoietic stem-progenitor cell populations. In contrast to cDNA expression, ZFN protein levels decline rapidly following internalization, affording better temporal control of nuclease activity. We show that transferrin-mediated ZFN uptake leads to site-specific in situ cleavage of the target locus. Additionally, despite the much shorter duration of ZFN activity, the efficiency of gene correction approaches that seen with cDNA-mediated expression. The approach is flexible and general, with the potential for extension to other targeting ligands and nuclease architectures.

View details for DOI 10.1093/nar/gkt710

View details for Web of Science ID 000326044700005

View details for PubMedID 23956220

View details for PubMedCentralID PMC3799454

Abstract

Assay of T-cell receptor excision circles (TRECs) in dried blood spots obtained at birth permits population-based newborn screening (NBS) for severe combined immunodeficiency (SCID).We sought to report the first 2 years of TREC NBS in California.Since August 2010, California has conducted SCID NBS. Ahigh-throughput TREC quantitative PCR assay with DNA isolated from routine dried blood spots was developed. Samples with initial low TREC numbers had repeat DNA isolation with quantitative PCR for TRECs and a genomic control, and immunophenotyping was performed within the screening program for infants with incomplete or abnormal results. Outcomes were tracked.Of 993,724 infants screened, 50 (1/19,900 [0.005%]) had significant T-cell lymphopenia. Fifteen (1/66,250) required hematopoietic cell or thymus transplantation or gene therapy; these infants had typical SCID (n= 11), leaky SCID or Omenn syndrome (n= 3), or complete DiGeorge syndrome (n= 1). Survival to date in this group is 93%. Other T-cell lymphopenic infants had variant SCID or combined immunodeficiency (n= 6), genetic syndromes associated with T-cell impairment (n= 12), secondary T-cell lymphopenia (n= 9), or preterm birth (n= 8). All T-cell lymphopenic infants avoided live vaccines and received appropriate interventions to prevent infections. TREC test specificity was excellent: only 0.08% of infants required a second test, and 0.016% required lymphocyte phenotyping by using flow cytometry.TREC NBS in California has achieved early diagnosis of SCID and other conditions with T-cell lymphopenia, facilitating management and optimizing outcomes. Furthermore, NBS has revealed the incidence, causes, and follow-up of T-cell lymphopenia in a large diverse population.

View details for DOI 10.1016/j.jaci.2013.04.024

View details for Web of Science ID 000321052300019

View details for PubMedID 23810098

Abstract

Restriction factors constitute a newly appreciated line of innate immune defense, blocking viral replication inside of infected cells. In contrast to these antiviral proteins, some cellular proteins, such as the CD4, CCR5, and CXCR4 cell surface receptors, facilitate HIV replication. We have used zinc finger nucleases (ZFNs) to insert a cocktail of anti-HIV restriction factors into the CCR5 locus in a T-cell reporter line, knocking out the CCR5 gene in the process. Mirroring the logic of highly active antiretroviral therapy, this strategy provides multiple parallel blocks to infection, dramatically limiting pathways for viral escape, without relying on random integration of transgenes into the genome. Because of the combination of blocks that this strategy creates, our modified T-cell lines are robustly resistant to both CCR5-tropic (R5-tropic) and CXCR4-tropic (X4-tropic) HIV-1. While zinc finger nuclease-mediated CCR5 disruption alone, which mimics the strategy being used in clinical trials, confers 16-fold protection against R5-tropic HIV, it has no effect against X4-tropic virus. Rhesus TRIM5, chimeric human-rhesus TRIM5, APOBEC3G D128K, or Rev M10 alone targeted to CCR5 confers significantly improved resistance to infection by both variants compared with CCR5 disruption alone. The combination of three factors targeted to CCR5 blocks infection at multiple stages, providing virtually complete protection against infection by R5-tropic and X4-tropic HIV.

View details for DOI 10.1038/mt.2012.284

View details for Web of Science ID 000317110300010

View details for PubMedID 23358186

View details for PubMedCentralID PMC3616536

Abstract

Recent studies have shown that zinc finger nucleases (ZFNs) are powerful reagents for making site-specific genomic modifications. The generic structure of these enzymes includes a ZF DNA-binding domain and nuclease domain (Fn) are separated by an amino acid "linker" and cut genomic DNA at sites that have a generic structure (site1)-(spacer)-(site2) where the "spacer" separates the two binding sites. In this work, we compare the activity of ZFNs with different linkers on target sites with different spacer lengths. We found those nucleases with linkers' lengths of 2 or 4 amino acid (aa) efficiently cut at target sites with 5 or 6 base pair (bp) spacers, and that those ZFNs with a 5-aa linker length efficiently cut target sites with 6 or 7bp spacers. In addition, we demonstrate that the Oligomerized Pool ENgineering (OPEN) platform used for making three-fingered ZF proteins (ZFPs) can be modified to incorporate modular assembly fingers (including those recognizing ANNs, CNNs, and TNNs) and we were able to generate nucleases that efficiently cut cognate target sites. The ability to use module fingers in the OPEN platform at target sites of 5-7bp spacer lengths increases the probability of finding a ZFN target site to 1 in 4bp. These findings significantly expand the range of sites that can be potentially targeted by these custom-engineered proteins.Molecular Therapy - Nucleic Acids (2013) 2, e88; doi:10.1038/mtna.2013.13; published online 30 April 2013.

View details for DOI 10.1038/mtna.2013.13

View details for Web of Science ID 000332461900006

View details for PubMedID 23632390

View details for PubMedCentralID PMC3650245

Abstract

The ability to direct human telomerase reverse transcriptase (hTERT) expression through either genetic control or tunable regulatory factors would advance not only our understanding of the transcriptional regulation of this gene, but also potentially produce new strategies for addressing telomerase-associated disease. In this work, we describe the engineering of artificial zinc finger transcription factors (ZFTFs) and ZF nucleases (ZFNs) to target sequences within the hTERT promoter and exon-1. We were able to identify several active ZFTFs that demonstrate a broadly tunable response when screened by a cell-based transcriptional reporter assay. Using the same DNA-binding domains, we generated ZFNs that were screened in combinatorial pairs in cell-based extrachromosomal single-strand annealing (SSA) assays and in gene-targeting assays using stably integrated constructs. Selected ZFN pairs were tested for the ability to induce sequence changes in a Cel1 assay and we observed frequencies of genomic modification up to 18.7% at the endogenous hTERT locus. These screening strategies have pinpointed several ZFN pairs that may be useful in gene editing of the hTERT locus. Our work provides a foundation for using engineered ZF proteins (ZFPs) for modulation of the hTERT locus.Molecular Therapy - Nucleic Acids (2013) 2, e87; doi:10.1038/mtna.2013.12; published online 23 April 2013.

View details for DOI 10.1038/mtna.2013.12

View details for Web of Science ID 000332461900005

View details for PubMedID 23612114

View details for PubMedCentralID PMC3650244

View details for DOI 10.1038/mt.2013.46

View details for Web of Science ID 000317110300002

View details for PubMedCentralID PMC3616526

View details for DOI 10.1038/mt.2013.46

View details for PubMedID 23542565

View details for PubMedCentralID PMC3616526

Abstract

The ability to deliver a gene of interest into a specific cell type is an essential aspect of biomedical research. Viruses can be a useful tool for this delivery, particularly in difficult to transfect cell types. Adeno-associated virus (AAV) is a useful gene transfer vector because of its ability to mediate efficient gene transduction in numerous dividing and quiescent cell types, without inducing any known pathogenicity. There are now a number of natural for that designed AAV serotypes that each has a differential ability to infect a variety of cell types. Although transduction studies have been completed, the bulk of the studies have been done in vivo, and there has never been a comprehensive study of transduction ex vivo/in vitro.Each cell type was infected with each serotype at a multiplicity of infection of 100,000 viral genomes/cell and transduction was analyzed by flow cytometry + .We found that AAV1 and AAV6 have the greatest ability to transduce a wide range of cell types, however, for particular cell types, there are specific serotypes that provide optimal transduction.In this work, we describe the transduction efficiency of ten different AAV serotypes in thirty-four different mammalian cell lines and primary cell types. Although these results may not be universal due to numerous factors such as, culture conditions and/ or cell growth rates and cell heterogeneity, these results provide an important and unique resource for investigators who use AAV as an ex vivo gene delivery vector or who work with cells that are difficult to transfect.

View details for DOI 10.1186/1743-422X-10-74

View details for Web of Science ID 000316756700001

View details for PubMedID 23497173

Abstract

An emerging strategy for the treatment of monogenic diseases uses genetic engineering to precisely correct the mutation(s) at the genome level. Recent advancements in this technology have demonstrated therapeutic levels of gene correction using a zinc-finger nuclease (ZFN)-induced DNA double-strand break in conjunction with an exogenous DNA donor substrate. This strategy requires efficient nucleic acid delivery and among viral vectors, recombinant adeno-associated virus (rAAV) has demonstrated clinical success without pathology. However, a major limitation of rAAV is the small DNA packaging capacity and to date, the use of rAAV for ZFN gene delivery has yet to be reported. Theoretically, an ideal situation is to deliver both ZFNs and the repair substrate in a single vector to avoid inefficient gene targeting and unwanted mutagenesis, both complications of a rAAV co-transduction strategy. Therefore, a rAAV format was generated in which a single polypeptide encodes the ZFN monomers connected by a ribosome skipping 2A peptide and furin cleavage sequence. On the basis of this arrangement, a DNA repair substrate of 750 nucleotides was also included in this vector. Efficient polypeptide processing to discrete ZFNs is demonstrated, as well as the ability of this single vector format to stimulate efficient gene targeting in a human cell line and mouse model derived fibroblasts. Additionally, we increased rAAV-mediated gene correction up to sixfold using a combination of Food and Drug Administration-approved drugs, which act at the level of AAV vector transduction. Collectively, these experiments demonstrate the ability to deliver ZFNs and a repair substrate by a single AAV vector and offer insights for the optimization of rAAV-mediated gene correction using drug therapy.

View details for DOI 10.1038/gt.2011.211

View details for Web of Science ID 000313053900005

View details for PubMedID 22257934

Abstract

Primary immunodeficiencies (PIDs) are an often-devastating class of genetic disorders that can be effectively treated by hematopoietic stem cell transplantation, but the lack of a suitable donor precludes this option for many patients. Gene therapy overcomes this obstacle by restoring gene expression in autologous hematopoietic stem cells and has proven effective in clinical trials, but widespread use of this approach has been impeded by the occurrence of serious complications. In this review, we discuss recent advances in gene therapy with an emphasis on strategies to improve safety, including the emergence of gene targeting technologies for the treatment of PIDs.New viral vectors, including lentiviral vectors with self-inactivating long terminal repeats, have been shown to have improved safety profiles in preclinical studies, and clinical trials using these vectors are now underway. Preclinical studies using engineered nucleases to stimulate precise gene targeting have also demonstrated correction of disease phenotypes for X-linked severe combined immunodeficiency, chronic granulomatous disease, and other diseases.Advances in viral vector design and the development of new technologies that allow precise alteration of the genome have the potential to begin a new chapter for gene therapy where effective treatment of PIDs is achieved without serious risk for patients.

View details for DOI 10.1097/MOP.0b013e328359e480

View details for Web of Science ID 000311106800012

View details for PubMedID 23073463

View details for Web of Science ID 000314049603094

View details for DOI 10.1016/j.coi.2012.09.004

View details for Web of Science ID 000319248200011

Abstract

Genome engineering is an emerging strategy to treat monogenic diseases that relies on the use of engineered nucleases to correct mutations at the nucleotide level. Zinc finger nucleases can be designed to stimulate homologous recombination-mediated gene targeting at a variety of loci, including genes known to cause the primary immunodeficiencies (PIDs). Recently, these nucleases have been used to correct disease-causing mutations in human cells, as well as to create new animal models for human disease. Although a number of hurdles remain before they can be used clinically, engineered nucleases hold increasing promise as a therapeutic tool, particularly for the PIDs.

View details for DOI 10.1016/j.coi.2012.08.005

View details for PubMedID 22981684

View details for Web of Science ID 000303484600307

View details for DOI 10.1038/nmeth.1811

View details for PubMedID 22205513

View details for DOI 10.1038/nmeth.1811

View details for Web of Science ID 000298667000014

Abstract

Human embryonic stem cells (hESCs) are primed for rapid apoptosis following mild forms of genotoxic stress. A natural form of such cellular stress occurs in response to recombinant adeno-associated virus (rAAV) single-strand DNA genomes, which exploit the host DNA damage response for replication and genome persistence. Herein, we discovered a unique DNA damage response induced by rAAV transduction specific to pluripotent hESCs. Within hours following rAAV transduction, host DNA damage signaling was elicited as measured by increased gamma-H2AX, ser15-p53 phosphorylation, and subsequent p53-dependent transcriptional activation. Nucleotide incorporation assays demonstrated that rAAV transduced cells accumulated in early S-phase followed by the induction of apoptosis. This lethal signaling sequalae required p53 in a manner independent of transcriptional induction of Puma, Bax and Bcl-2 and was not evident in cells differentiated towards a neural lineage. Consistent with a lethal DNA damage response induced upon rAAV transduction of hESCs, empty AAV protein capsids demonstrated no toxicity. In contrast, DNA microinjections demonstrated that the minimal AAV origin of replication and, in particular, a 40 nucleotide G-rich tetrad repeat sequence, was sufficient for hESC apoptosis. Our data support a model in which rAAV transduction of hESCs induces a p53-dependent lethal response that is elicited by a telomeric sequence within the AAV origin of replication.

View details for DOI 10.1371/journal.pone.0027520

View details for Web of Science ID 000297555800024

View details for PubMedID 22114676

View details for PubMedCentralID PMC3219675

View details for DOI 10.1038/nm1011-1192

View details for PubMedID 21988995

View details for DOI 10.1038/nmeth.1656

View details for Web of Science ID 000293220600013

View details for PubMedID 21799496

View details for DOI 10.1038/mt.2011.14

View details for PubMedID 21358706

View details for PubMedCentralID PMC3048194

Abstract

The devastating nature of primary immunodeficiencies, the ability to cure primary immunodeficiencies by bone marrow transplantation, the ability of a small number of gene-corrected cells to reconstitute the immune system, and the overall suboptimal results of bone marrow transplantation for most patients with primary immunodeficiencies make the development of gene therapy for this class of diseases important. While there has been clear clinical benefit for a number of patients from viral-based gene therapy strategies, there have also been a significant number of serious adverse events, including the development of leukemia, from the approach. In this review, I discuss the development of nuclease-stimulated, homologous recombination-based approaches as a novel gene therapy strategy for the primary immunodeficiencies.

View details for DOI 10.1111/j.1749-6632.2011.06314.x

View details for Web of Science ID 000301519900013

View details for PubMedID 22236437

Abstract

The use of lentiviral vectors extends from the laboratory, where they are used for basic studies in virology and as gene transfer vectors gene delivery, to the clinic, where clinical trials using these vectors for gene therapy are currently underway. Lentiviral vectors are useful for gene transfer because they have a large cloning capacity and a broad tropism. Although procedures for lentiviral vector production have been standardized, simple methods to create higher titer virus during production would have extensive and important applications for both research and clinical use. Here we present a simple and inexpensive method to increase the titer by 3- to 8-fold for both integration-competent lentivirus and integration-deficient lentivirus. This is achieved during standard lentiviral production by the addition of caffeine to a final concentration of 2-4 mM. We find that sodium butyrate, a histone deacetylase inhibitor shown previously to increase viral titer, works only 50% as well as caffeine. We also show that the DNA-PKcs (DNA-dependent protein kinase catalytic subunit) inhibitor NU7026 can also increase viral titer, but that the combination of caffeine and NU7026 is not more effective than caffeine alone. We show that the time course of caffeine treatment is important in achieving a higher titer virus, and is most effective when caffeine is present from 17 to 41 hr posttransfection. Last, although caffeine increases lentiviral vector titer, it has the opposite effect on the titer of adeno-associated virus type 2 vector. Together, these results provide a novel, simple, and inexpensive way to significantly increase the titer of lentiviral vectors.

View details for DOI 10.1089/hum.2010.068

View details for Web of Science ID 000286453600012

View details for PubMedID 20626321

Abstract

Adeno-associated virus (AAV) mediates gene targeting in humans by providing exogenous DNA for allelic replacement through homologous recombination. In comparison to other methods of DNA delivery or alternative DNA substrates, AAV gene targeting is reported to be very efficient, perhaps due to its single-stranded DNA genome, the inverted terminal repeats (ITRs), and/or the consequence of induced cellular signals on infection or uncoating. These viral attributes were investigated in the presence and absence of an I-Sce endonuclease-induced double-strand break (DSB) within a chromosomal defective reporter in human embryonic kidney cells. Gene correction was evaluated using self-complementary (sc) AAV, which forms a duplexed DNA molecule and results in earlier and robust transgene expression compared with conventional single-strand (ss) AAV genomes. An scAAV repair substrate was modestly enhanced for reporter correction showing no dependency on ssAAV genomes for this process. The AAV ITR sequences were also investigated in a plasmid repair context. No correction was noted in the absence of a DSB, however, a modest inhibitory effect correlated with the increasing presence of ITR sequences. Similarly, signaling cascades stimulated upon recombinant AAV transduction had no effect on plasmid-mediated DSB repair. Noteworthy, was the 20-fold additional enhancement in reporter correction using scAAV vectors, over ss versions, to deliver both the repair substrate and the endonuclease. In this case, homologous recombination repaired the defective reporter in 4% of cells without any selection. This report provides novel insights regarding the recombination substrates used by AAV vectors in promoting homologous recombination and points to the initial steps in vector optimization that could facilitate their use in gene correction of genetic disorders.

View details for DOI 10.1038/gt.2010.65

View details for Web of Science ID 000281757900012

View details for PubMedID 20463753

Abstract

Zinc Finger nucleases (ZFNs) have been used to create precise genome modifications at frequencies that might be therapeutically useful in gene therapy. We created a mouse model of a generic recessive genetic disease to establish a preclinical system to develop the use of ZFN-mediated gene correction for gene therapy. We knocked a mutated GFP gene into the ROSA26 locus in murine embryonic stem (ES) cells and used these cells to create a transgenic mouse. We used ZFNs to determine the frequency of gene correction by gene targeting in different primary cells from this model. We achieved targeting frequencies from 0.17 to 6% in different cell types, including primary fibroblasts and astrocytes. We demonstrate that ex vivo gene-corrected fibroblasts can be transplanted back into a mouse where they retained the corrected phenotype. In addition, we achieved targeting frequencies of over 1% in ES cells, and the targeted ES cells retained the ability to differentiate into cell types from all three germline lineages. In summary, potentially therapeutically relevant frequencies of ZFN-mediated gene targeting can be achieved in a variety of primary cells and these cells can then be transplanted back into a recipient.

View details for DOI 10.1038/mt.2010.57

View details for Web of Science ID 000278545800008

View details for PubMedID 20389291

Abstract

We report here homologous recombination (HR)-mediated gene targeting of two different genes in human iPS cells (hiPSCs) and human ES cells (hESCs). HR-mediated correction of a chromosomally integrated mutant GFP reporter gene reaches efficiencies of 0.14%-0.24% in both cell types transfected by donor DNA with plasmids expressing zinc finger nucleases (ZFNs). Engineered ZFNs that induce a sequence-specific double-strand break in the GFP gene enhanced HR-mediated correction by > 1400-fold without detectable alterations in stem cell karyotypes or pluripotency. Efficient HR-mediated insertional mutagenesis was also achieved at the endogenous PIG-A locus, with a > 200-fold enhancement by ZFNs targeted to that gene. Clonal PIG-A null hESCs and iPSCs with normal karyotypes were readily obtained. The phenotypic and biological defects were rescued by PIG-A transgene expression. Our study provides the first demonstration of HR-mediated gene targeting in hiPSCs and shows the power of ZFNs for inducing specific genetic modifications in hiPSCs, as well as hESCs.

View details for DOI 10.1016/j.stem.2009.05.023

View details for Web of Science ID 000267879200014

View details for PubMedID 19540188

Abstract

Zinc finger nucleases (ZFNs) have been used successfully to create genome-specific double-strand breaks and thereby stimulate gene targeting by several thousand fold. ZFNs are chimeric proteins composed of a specific DNA-binding domain linked to a non-specific DNA-cleavage domain. By changing key residues in the recognition helix of the specific DNA-binding domain, one can alter the ZFN binding specificity and thereby change the sequence to which a ZFN pair is being targeted. For these and other reasons, ZFNs are being pursued as reagents for genome modification, including use in gene therapy. In order for ZFNs to reach their full potential, it is important to attenuate the cytotoxic effects currently associated with many ZFNs. Here, we evaluate two potential strategies for reducing toxicity by regulating protein levels. Both strategies involve creating ZFNs with shortened half-lives and then regulating protein level with small molecules. First, we destabilize ZFNs by linking a ubiquitin moiety to the N-terminus and regulate ZFN levels using a proteasome inhibitor. Second, we destabilize ZFNs by linking a modified destabilizing FKBP12 domain to the N-terminus and regulate ZFN levels by using a small molecule that blocks the destabilization effect of the N-terminal domain. We show that by regulating protein levels, we can maintain high rates of ZFN-mediated gene targeting while reducing ZFN toxicity.

View details for DOI 10.1371/journal.pgen.1000376

View details for Web of Science ID 000266320000016

View details for PubMedID 19214211

Abstract

Custom-made zinc-finger nucleases (ZFNs) can induce targeted genome modifications with high efficiency in cell types including Drosophila, C. elegans, plants, and humans. A bottleneck in the application of ZFN technology has been the generation of highly specific engineered zinc-finger arrays. Here we describe OPEN (Oligomerized Pool ENgineering), a rapid, publicly available strategy for constructing multifinger arrays, which we show is more effective than the previously published modular assembly method. We used OPEN to construct 37 highly active ZFN pairs which induced targeted alterations with high efficiencies (1%-50%) at 11 different target sites located within three endogenous human genes (VEGF-A, HoxB13, and CFTR), an endogenous plant gene (tobacco SuRA), and a chromosomally integrated EGFP reporter gene. In summary, OPEN provides an "open-source" method for rapidly engineering highly active zinc-finger arrays, thereby enabling broader practice, development, and application of ZFN technology for biological research and gene therapy.

View details for DOI 10.1016/j.molcel.2008.06.016

View details for Web of Science ID 000258083400014

View details for PubMedID 18657511

Abstract

Homologous recombination is a technique used for performing precise genomic manipulations, and this makes it potentially ideal for gene therapy. The rate of spontaneous homologous recombination in human cells has been too low to be used experimentally or therapeutically but, by inducing a DNA double-strand break (DSB) in the target gene this rate can be stimulated. Zinc finger nucleases (ZFNs) are synthetic fusion proteins that can induce DSBs at specific sequences of DNA and stimulate gene targeting. Although the success of ZFNs in this application has been demonstrated, several issues remain. First, an optimal, generalized method of making effective and safe ZFNs needs to be determined. Second, a systematic method of evaluating the efficiency and safety of ZFNs is needed. We compared the gene-targeting efficiencies and cytotoxicity of ZFNs made using modular-assembly and ZFNs made using a bacterial 2-hybrid (B2H) selection-based method, in each case targeting the same single site. We found that a ZFN pair made using the B2H strategy is more efficient at stimulating gene targeting and less toxic than a pair made using modular-assembly. We demonstrate that a pair of three-finger B2H ZFNs is as efficient at stimulating gene targeting as ZFNs with more fingers, and induce similar or lower rates of toxicity.

View details for DOI 10.1038/mt.2008.20

View details for Web of Science ID 000254929600014

View details for PubMedID 18334988

Abstract

The long-term production of billions of spermatozoa relies on the regulated proliferation and differentiation of spermatogonial stem cells (SSCs). To date only a few factors are known to function in SSCs to provide this regulation. Octamer-4 (OCT4) plays a critical role in pluripotency and cell survival of embryonic stem cells and primordial germ cells; however, it is not known whether it plays a similar function in SSCs. Here, we show that OCT4 is required for SSC maintenance in culture and for colonization activity following cell transplantation, using lentiviral-mediated short hairpin RNA expression to knock down OCT4 in an in vitro model for SSCs ("germline stem" [GS] cells). Expression of promyelocytic leukemia zinc-finger (PLZF), a factor known to be required for SSC self-renewal, was not affected by OCT4 knockdown, suggesting that OCT4 does not function upstream of PLZF. In addition to developing a method to test specific gene function in GS cells, we demonstrate that retinoic acid (RA) triggers GS cells to shift to a differentiated, premeiotic state lacking OCT4 and PLZF expression and colonization activity. Our data support a model in which OCT4 and PLZF maintain SSCs in an undifferentiated state and RA triggers spermatogonial differentiation through the direct or indirect downregulation of OCT4 and PLZF. The current study has important implications for the future use of GS cells as an in vitro model for spermatogonial stem cell biology or as a source of embryonic stem-like cells. Disclosure of potential conflicts of interest is found at the end of this article.

View details for DOI 10.1634/stemcells.2008-0134

View details for Web of Science ID 000261156500023

View details for PubMedID 18719224

Abstract

The concept of gene therapy has long appealed to biomedical researchers and clinicians because it promised to treat certain diseases at their origins. In the last several years, there have been several trials in which patients have benefited from gene therapy protocols. This progress, however, has revealed important problems, including the problem of insertional oncogenesis. In this review, which focuses on monogenic diseases, we discuss the problem of insertional oncogenesis and identify areas for future research, such as developing more quantitative assays for risk and efficacy, and ways of minimizing the genotoxic effects of gene therapy protocols, which will be important if gene therapy is to fulfill its conceptual promise.

View details for DOI 10.1371/journal.pgen.0020133

View details for Web of Science ID 000240867700002

View details for PubMedID 17009872

Abstract

The structural maintenance of chromosomes (SMC) family of proteins has been implicated in the repair of DNA double-strand breaks (DSBs) by homologous recombination (HR). The SMC1/3 cohesin complex is thought to promote HR by maintaining the close proximity of sister chromatids at DSBs. The SMC5/6 complex is also required for DNA repair, but the mechanism by which it accomplishes this is unclear. Here, we show that RNAi-mediated knockdown of the SMC5/6 complex components in human cells increases the efficiency of gene targeting due to a specific requirement for hSMC5/6 in sister chromatid HR. Knockdown of the hSMC5/6 complex decreases sister chromatid HR, but does not reduce nonhomologous end-joining (NHEJ) or intra-chromatid, homologue, or extrachromosomal HR. The hSMC5/6 complex is itself recruited to nuclease-induced DSBs and is required for the recruitment of cohesin to DSBs. Our results establish a mechanism by which the hSMC5/6 complex promotes DNA repair and suggest a novel strategy to improve the efficiency of gene targeting in mammalian somatic cells.

View details for DOI 10.1038/sj.emboj.7601218

View details for Web of Science ID 000239625900013

View details for PubMedID 16810316

Abstract

Engineered zinc finger nucleases can stimulate gene targeting at specific genomic loci in insect, plant and human cells. Although several platforms for constructing artificial zinc finger arrays using "modular assembly" have been described, standardized reagents and protocols that permit rapid, cross-platform "mixing-and-matching" of the various zinc finger modules are not available. Here we describe a comprehensive, publicly available archive of plasmids encoding more than 140 well-characterized zinc finger modules together with complementary web-based software (termed ZiFiT) for identifying potential zinc finger target sites in a gene of interest. Our reagents have been standardized on a single platform, enabling facile mixing-and-matching of modules and transfer of assembled arrays to expression vectors without the need for specialized knowledge of zinc finger sequences or complicated oligonucleotide design. We also describe a bacterial cell-based reporter assay for rapidly screening the DNA-binding activities of assembled multi-finger arrays. This protocol can be completed in approximately 24-26 d.

View details for DOI 10.1038/nprot.2006.259

View details for Web of Science ID 000251155400069

View details for PubMedID 17406455

Abstract

The ability to achieve site-specific manipulation of the mammalian genome has widespread implications for basic and applied research. Gene targeting is a process in which a DNA molecule introduced into a cell replaces the corresponding chromosomal segment by homologous recombination, and thus presents a precise way to manipulate the genome. In the past, the application of gene targeting to mammalian cells has been limited by its low efficiency. Zinc finger nucleases (ZFNs) show promise in improving the efficiency of gene targeting by introducing DNA double-strand breaks in target genes, which then stimulate the cell's endogenous homologous recombination machinery. Recent results have shown that ZFNs can be used to create targeting frequencies of up to 20% in a human disease-causing gene. Future work will be needed to translate these in vitro findings to in vivo applications and to determine whether zinc finger nucleases create undesired genomic instability.

View details for DOI 10.1038/nbt1125

View details for Web of Science ID 000231019900029

View details for PubMedID 16082368

Abstract

Custom-designed zinc finger nucleases (ZFNs), proteins designed to cut at specific DNA sequences, are becoming powerful tools in gene targeting--the process of replacing a gene within a genome by homologous recombination (HR). ZFNs that combine the non-specific cleavage domain (N) of FokI endonuclease with zinc finger proteins (ZFPs) offer a general way to deliver a site-specific double-strand break (DSB) to the genome. The development of ZFN-mediated gene targeting provides molecular biologists with the ability to site-specifically and permanently modify plant and mammalian genomes including the human genome via homology-directed repair of a targeted genomic DSB. The creation of designer ZFNs that cleave DNA at a pre-determined site depends on the reliable creation of ZFPs that can specifically recognize the chosen target site within a genome. The (Cys2His2) ZFPs offer the best framework for developing custom ZFN molecules with new sequence-specificities. Here, we explore the different approaches for generating the desired custom ZFNs with high sequence-specificity and affinity. We also discuss the potential of ZFN-mediated gene targeting for 'directed mutagenesis' and targeted 'gene editing' of the plant and mammalian genome as well as the potential of ZFN-based strategies as a form of gene therapy for human therapeutics in the future.

View details for DOI 10.1093/nar/gki912

View details for Web of Science ID 000233046100040

View details for PubMedID 16251401

View details for Web of Science ID 000182579800039

View details for PubMedID 12730593

Abstract

Gene targeting is the in situ manipulation of the sequence of an endogenous gene by the introduction of homologous exogenous DNA. Presently, the rate of gene targeting is too low for it to be broadly used in mammalian somatic cell genetics or to cure genetic diseases. Recently, it has been demonstrated that infection with recombinant adeno-associated virus (rAAV) vectors can mediate gene targeting in somatic cells, but the mechanism is unclear. This paper explores the balance between random integration and gene targeting with rAAV. Both random integration and spontaneous gene targeting are dependent on the multiplicity of infection (MOI) of rAAV. It has previously been shown that the introduction of a DNA double-stranded break (DSB) in a target gene can stimulate gene targeting by several-thousand-fold in somatic cells. Creation of a DSB stimulates the frequency of rAAV-mediated gene targeting by over 100-fold, suggesting that the mechanism of rAAV-mediated gene targeting involves, at least in part, the repair of DSBs by homologous recombination. Absolute gene targeting frequencies reach 0.8% with a dual vector system in which one rAAV vector provides a gene targeting substrate and a second vector expresses the nuclease that creates a DSB in the target gene. The frequencies of gene targeting that we achieved with relatively low MOIs suggest that combining rAAV vectors with DSBs is a promising strategy to broaden the application of gene targeting.

View details for DOI 10.1128/MCB.23.10.3558-3565.2003

View details for Web of Science ID 000182696100017

View details for PubMedID 12724414

Abstract

There are at least five murine Dlx genes that are related to the Drosophila Distal-less homeobox gene. The Dlx genes are primarily expressed in the developing forebrain, derivatives of the cranial neural crest and restricted epidermal craniofacial and limb domains. Dlx-2 is required for differentiation of subsets of cranial neural crest and forebrain cells. Previous genomic studies have shown that Dlx-1 and Dlx-2 are linked on mouse chromosome 2, near the HoxD cluster. Here we report a detailed analysis of the nucleotide sequence (approximately 14 kb), organization, and transcription of the murine Dlx-1 and Dlx-2 locus. In addition, we show that Dlx-1 makes multiple sense transcripts and at least one antisense transcript, whereas Dlx-2 makes one major transcript. The sequence of the human Dlx-2 gene is reported and is compared to that of the murine gene. Finally, sequence analysis of the deduced protein sequences reveals several candidate functional domains.

View details for Web of Science ID A1996VB75800009

View details for PubMedID 8812481

Abstract

CSF lymphocytes from patients with Coccidioides immitis meningitis exhibited a significant antigen-specific response to in vitro stimulation with C. immitis antigens. In some patients, lesser responses to control antigens (Candida and PPD) were also detected. Antigen-specific responses by CSF lymphocytes were seen early in the course of this disease as well as several years after patients had entered remission. When compared to CSF cells, the response of autologous peripheral blood mononuclear cells was similar but of a much smaller magnitude and at times undetectable. Fluorescence activated cell sorting revealed an increased percentage of CD3+ (T-cells), CD4+ (helper/inducer) and CD3+/HLA-DR+ (activated T-cell) cells in the CSF of C. immitis meningitis patients compared to their blood. Most of the antigen-specific proliferative response resided in the CD4+ lymphocyte subset. CSF T-cell proliferation assays may have a role in the diagnosis of C. immitis meningitis.

View details for Web of Science ID A1995QZ83200008

View details for PubMedID 7544405

Abstract

The spatial distribution of Dlx-2 protein during murine tooth development has been investigated using immunohistochemistry with Dlx-2 antibodies. In common with several other homeobox genes expressed in toothgerms, Dlx-2 shows a multiphasic distribution in both epithelially and mesenchymally derived structures. This localization shows a number of similarities with the expression of Msx-2 and suggests a role for Dlx-2 in tooth initiation and tissue patterning.

View details for Web of Science ID A1995VT03000005

View details for PubMedID 7554927

Abstract

Recently, the Dlx family of homeobox genes have been identified as candidates for regulating patterning and differentiation of the forebrain. We have made a polyclonal antiserum to the protein product of the Dlx-2 gene. Using this antiserum, we have characterized the spatial and temporal pattern of DLX-2 protein expression during murine development and in the adult mouse brain. These studies demonstrate that, like the mRNA from the Dlx-2 gene, DLX-2 protein is expressed in mouse embryonic forebrain, limbs, tail, genital tubercle, and branchial arches. Within the embryonic forebrain, DLX-2 protein is expressed within specific transverse and longitudinal domains. Analysis of expression within the wall of the forebrain shows that DLX-2 is expressed in proliferative regions including the ventricular and subventricular zones. DLX-2 is expressed in the same cells as MASH-1, a marker of relatively undifferentiated cells, but in a reciprocal fashion to MAP-2, a marker of terminal neuronal differentiation. A number of DLX-2-expressing cells, but not all, can be labeled with bromodeoxyuridine (BrdU). Using the patterns of DLX-2, MASH-1, MAP-2 expression, and bromodeoxyuridine incorporation, we identify four molecularly distinct populations of cells that may correspond to different stages of neuronal differentiation in the mouse basal forebrain, in which DLX-2 is expressed at the transition from proliferation to terminal differentiation.

View details for Web of Science ID A1994PQ38100006

View details for PubMedID 7965042

Abstract

The expression patterns of four genes that are potential regulators of development were examined in the CNS of the embryonic day 12.5 mouse embryo. Three of the genes, Dlx-1, Dlx-2 (Tes-1), and Gbx-2, encode homeodomain-containing proteins, and one gene, Wnt-3, encodes a putative secreted differentiation factor. These genes are expressed in spatially restricted transverse and longitudinal domains in the embryonic neural tube, and are also differentially expressed within the wall of the neural tube. Dlx-1 and Dlx-2 are expressed in two separate regions of the forebrain in an identical pattern. The Gbx-2 gene is expressed in four domains, two of which share sharp boundaries with the domains of the Dlx genes. One boundary is in the basal telecephalon between deep and superficial strata of the medial ganglionic eminence; the other boundary is in the diencephalon at the zona limitans intrathalamica. The Wnt-3 gene is expressed in a dorsal longitudinal zone extending from the hindbrain into the diencephalon, where its expression terminates at the zona limitans intrathalamica. Reciprocal patterns of expression are found within the dorsal thalamus for the Gbx-2 and Wnt-3 genes. These findings are consistent with neuromeric theories of forebrain development, and based upon them we suggest a model for forebrain segmentation.

View details for Web of Science ID A1993LM27300037

View details for PubMedID 7687285

Abstract

The pattern of RNA expression of the murine Dlx-2 (Tes-1) homeobox gene is described in embryos ranging in age from E8.5 through E11.5. Dlx-2 is a vertebrate homologue of the Drosophila Distal-less (Dll) gene. Dll expression in the Drosophila embryo is principally limited to the primordia of the brain, head and limbs. Dlx-2 is also expressed principally in the primordia of the forebrain, head and limbs. Within these regions it is expressed in spatially restricted domains. These include two discontinuous regions of the forebrain (basal telencephalon and ventral diencephalon), the branchial arches, facial ectoderm, cranial ganglia and limb ectoderm. Several mouse and human disorders have phenotypes which potentially are the result of mutations in the Dlx genes.

View details for Web of Science ID A1993KW17900001

View details for PubMedID 8098616

Abstract

Dlx-2 (also called Tes-1), a mammalian member of the Distal-less family of homeobox genes, is expressed during murine fetal development in spatially restricted domains of the forebrain. Searching for a candidate neurological mutation that might involve this gene, we have assigned the human and mouse loci to regions of conserved synteny on human chromosome 2, region cen--q33, and mouse chromosome 2 by Southern analysis of somatic cell hybrid lines. An EcoRI dimorphism, discovered in common inbred laboratory strains, was used for recombinant inbred strain mapping. The results place Dlx-2/Tes-1 near the Hox-4 cluster on mouse chromosome 2.

View details for Web of Science ID A1992JH14800031

View details for PubMedID 1354641

Abstract

In order to isolate genes involved in development of the mammalian telencephalon we employed an efficient cDNA library procedure. By subtracting an adult mouse telencephalic cDNA library from an embryonic day 15 (E15) mouse telencephalic cDNA library we generated two subtracted libraries (ES1 and ES2). We estimate that ES1 contains between 200 and 600 different cDNA clones, which approximates the number of genes that are preferentially expressed in the E15 telencephalon, compared to the adult telencephalon. Northern analysis of 20 different cDNA clones shows that 14 of these are expressed at least 5-fold more in the E15 telencephalon than the adult telencephalon. Limited sequencing of the 14 differentially expressed clones reveals that 10 have no significant identity to sequences in GenBank and EMBL databases, whereas the other 4 have significant sequence identity to vimentin, histone 3.3, topoisomerase I and the B2 repeat element. In situ hybridization using one of the differentially expressed cDNAs, TES-1, demonstrates that it is transiently expressed in the anlage of the basal ganglia. In situ hybridization with another differentially expressed cDNA clone, TES-4, shows that it is specifically expressed in differentiating cells of the neural axis with a distinctive rostral-caudal temporal pattern. These findings, and the methods that we have developed, provide a framework for future investigations of the genetic control of telencephalon development.

View details for Web of Science ID A1992GZ10000002

View details for PubMedID 1372074

Abstract

A complementary DNA, Tes-1, of a novel homeodomain protein has been cloned, and its pattern of expression has been characterized. It is a structural homolog of Distal-less, a homeodomain-encoding gene in D. melanogaster. Its expression is developmentally regulated and is limited to structures in the head. Within the central nervous system of the midgestation mouse embryo, it is expressed exclusively in the ventral forebrain. It is likely that Tes-1 plays a regulatory role in the development of this complex neural structure.

View details for Web of Science ID A1991GB93300005

View details for PubMedID 1678612

Abstract

We describe a subtractive hybridization protocol which is designed to permit subtractions between cDNA libraries. The method uses single-stranded phagemids with directional inserts as both the driver and the target. We modified the M13 phagemid vector pBluescript for the directional cDNA cloning and subtractive hybridization. Two simplified methods for efficient construction of directional cDNA libraries are also described. Using a model system, we found that one round of subtractive hybridization results in a 5,000-fold specific subtraction of abundant molecules. We used two methods to quantify the efficiency and verify the specificity of the subtraction. In order to obtain these subtraction efficiencies, it was necessary to develop a method to purify the single-stranded DNA to homogeneity. The single-stranded purification involved using potassium iodide (KI) density centrifugation, restriction endonuclease digestion and phenol extraction in the presence of magnesium. We describe the several advantages of using directional inserts for the subtraction procedure.

View details for Web of Science ID A1990DX66200027

View details for PubMedID 2168539

Abstract

To study the movement of subretinal fluid, we have injected fluid into the subretinal space through a glass micropipette and monitored its resorption. This technique has been criticized as a model of non-rhegmatogenous detachment because the small retinal hole made by the micropipette might allow efflux of subretinal fluid into the vitreous. The present experiments answer this criticism: we found that sealing the micropipette hole with cyanoacrylate, mucilage or an air bubble had no effect on the rate of subretinal fluid resorption, and detachments with two to five micropipette holes did not resorb faster than those with only one.

View details for Web of Science ID A1984SC67800016

View details for PubMedID 6697753