The promising ability to genetically modify hematopoietic stem and progenitor cells by precise gene editing remains challenging due to their sensitivity to in vitro manipulations and poor ...efficiencies of homologous recombination. This study represents the first evidence of implementing a gene editing strategy in a murine safe harbor locus site that phenotypically corrects primary cells from a mouse model of Fanconi anemia A. By means of the co-delivery of transcription activator-like effector nucleases and a donor therapeutic FANCA template to the Mbs85 locus, we achieved efficient gene targeting (23%) in mFA-A fibroblasts. This resulted in the phenotypic correction of these cells, as revealed by the reduced sensitivity of these cells to mitomycin C. Moreover, robust evidence of targeted integration was observed in murine wild type and FA-A hematopoietic progenitor cells, reaching mean targeted integration values of 21% and 16% respectively, that were associated with the phenotypic correction of these cells. Overall, our results demonstrate the feasibility of implementing a therapeutic targeted integration strategy into the mMbs85 locus, ortholog to the well-validated hAAVS1, constituting the first study of gene editing in mHSC with TALEN, that sets the basis for the use of a new safe harbor locus in mice.
Abstract Background X-linked lymphoproliferative disease is an immunodeficiency arising from mutations in the SH2D1A gene encoding SAP, a key regulator of immune function expressed in T cells, ...natural killer cells, and natural killer T cells. Haemopoietic stem-cell transplantation is curative, and we have shown proof of concept of haemopoietic stem-cell gene therapy in a mouse model. Preliminary data suggest that adoptive transfer of gene-corrected T cells can correct immune defects. Using gene editing techniques, we aimed to correct the SH2D1A locus in situ allowing physiologically regulated SAP expression. Methods We constructed SAP-specific TALE-effector nucleases (TALENs) capable of generating a site-specific double-stranded break close to the translation start codon, allowing correction of most described SAP mutations. TALENs were validated with a surveyor assay in HEK293T cells. To facilitate optimisation of delivery methods, we designed a fluorescent reporter assay that, when stably integrated into a cellular DNA, provides a fluorescence signal when TALEN-mediated DNA double-stranded break repair occurs. Findings The ability of TALENs to cleave DNA at the SH2D1A locus was validated with both surveyor and fluorescent reporter assays in HEK293T cells by transfection of plasmid DNA. Using the reporter assay in Jurkat cells we observed reduced toxicity and efficient nuclease activity in an mRNA nucleofection delivery platform. We therefore proceeded to deliver TALEN mRNA to human primary T cells, and demonstrated the generation of double-stranded breaks. We constructed a homologous recombination donor in a non-integrating lentiviral vector format to mediate delivery of corrective cDNA with low toxicity. Combined with TALEN mRNA nucleofection, homologous recombination-driven incorporation of donor in T cells was possible. Interpretation Our data suggest that SAP-specific TALENs can efficiently target the SH2D1A locus and induce double-stranded breaks, which can be used to incorporate donor template SH2D1A cDNA. Targeted gene editing allows us to harness a greater amount of genomic regulatory elements and therefore offers an attractive approach to treat X-linked lymphoproliferative disease. This methodology could be applied to an autologous T cell gene therapy strategy, although the low level of homologous recombination remains a limiting factor. We plan to test our strategy in patient cell lines to restore SAP expression and cytotoxic function of cytotoxic T lymphocytes. Funding Wellcome Trust, Academy of Medical Sciences.
Genome editing (GE) tools and RNA interference technology enable the modulation of gene expression in cancer research. While GE mediated by clustered regularly interspaced short palindromic repeats ...(CRISPR)/Cas9 or transcription activator-like effector nucleases (TALEN) activity can be used to induce gene knockouts, shRNA interacts with the targeted transcript, resulting in gene knockdown. Here, we compare three different methods for
knockout or knockdown in rhabdomyosarcoma (RMS) cells. RMS is the most common sarcoma in children and its development has been previously associated with SNAI1 transcription factor activity. To investigate the role of SNAI1 in RMS development, we compared CRISPR/Cas9, TALEN, and shRNA tools to identify the most efficient tool for the modulation of SNAI1 expression with biological effects. Subsequently, the genome sequence, transcript levels, and protein expression of SNAI1 were evaluated. The modulation of SNAI1 using three different approaches affected the morphology of the cells and modulated the expression of myogenic factors and HDAC1. Our study revealed a similar effectiveness of the tested methods. Nevertheless, the low efficiency of the GE tools was a limiting factor in obtaining biallelic gene knockouts. To conclude, we established and characterized three different models of
knockout and knockdown that might be used in further studies investigating the role of SNAI1 in RMS.
X-linked lymphoproliferative disease is a rare inherited immune disorder, caused by mutations or deletions in the
gene that encodes an intracellular adapter protein SAP (Slam-associated protein). SAP ...is essential for mediating several key immune processes and the immune system - T cells in particular - are dysregulated in its absence. Patients present with a spectrum of clinical manifestations, including haemophagocytic lymphohistiocytosis (HLH), dysgammaglobulinemia, lymphoma and autoimmunity. Treatment options are limited, and patients rarely survive to adulthood without an allogeneic haematopoietic stem cell transplant (HSCT). However, this procedure can have poor outcomes in the mismatched donor setting or in the presence of active HLH, leaving an unmet clinical need. Autologous haematopoeitic stem cell or T cell therapy may offer alternative treatment options, removing the need to find a suitable donor for HSCT and any risk of alloreactivity. SAP has a tightly controlled expression profile that a conventional lentiviral gene delivery platform may not be able to fully replicate. A gene editing approach could preserve more of the endogenous regulatory elements that govern SAP expression, potentially providing a more optimum therapy. Here, we assessed the ability of TALEN, CRISPR-Cas9 and CRISPR-Cas12a nucleases to drive targeted insertion of
cDNA at the first exon of the
locus using an adeno-associated virus serotype 6 (AAV6)-based vector containing the donor template. All nuclease platforms were capable of high efficiency gene editing, which was optimised using a serum-free AAV6 transduction protocol. We show that T cells from XLP patients corrected by gene editing tools have restored physiological levels of
gene expression and restore SAP-dependent immune functions, indicating a new therapeutic opportunity for XLP patients.
Genome editing tools, such as CRISPR/Cas, TALE nucleases and, more recently, double-strand-break-independent editors, have been successfully used for gene therapy and reverse genetics. Among various ...challenges in the field, tolerable and efficient delivery of editors to target cells and sites, as well as independence from commercially available tools for flexibility and fast adoption of new editing technology are the most pressing. For many hematopoietic research applications, primary CD34
cells and the human umbilical cord-derived progenitor erythroid 2 (HUDEP-2) cell line are highly informative substrates and readily accessible for
manipulation. Moreover,
editing of CD34
cells has immediate therapeutic relevance. Both cell types are sensitive to standard transfection procedures and reagents, such as lipofection with plasmid DNA, calling for more suitable methodology in order to achieve high efficiency and tolerability of editing with editors of choice. These challenges can be addressed by RNA delivery, either as a mixture of guide RNA and mRNA for CRISRP/Cas-based systems or as a mixture of mRNAs for TALENs. Compared to ribonucleoproteins or proteins, RNA as vector creates flexibility by removing dependence on commercial availability or laborious in-house preparations of novel editor proteins. Compared to DNA, RNA is less toxic and by obviating nuclear transcription and export of mRNA offers faster kinetics and higher editing efficiencies.
Here, we detail an
transcription protocol based on plasmid DNA templates with the addition of Anti-Reverse Cap Analog (ARCA) using T7 RNA polymerase, and poly (A) tailing using poly (A) polymerase, combined with nucleofection of HUDEP-2 and patient-derived CD34
cells. Our protocol for RNA-based delivery employs widely available reagents and equipment and can easily be adopted for universal
delivery of genome editing tools.
Drawing on a common use case, we employ the protocol to target a β-globin mutation and to reactivate γ-globin expression as two potential therapies for β-hemoglobinopathies, followed by erythroid differentiation and functional analyses. Our protocol allows high editing efficiencies and unimpaired cell viability and differentiation, with scalability, suitability for functional assessment of editing outcomes and high flexibility in the application to different editors.
In vitro disease modeling based on induced pluripotent stem cells (iPSCs) provides a powerful system to study cellular pathophysiology, especially in combination with targeted genome editing and ...protocols to differentiate iPSCs into affected cell types. In this study, we established zinc-finger nuclease-mediated genome editing in primary fibroblasts and iPSCs generated from a mouse model for radiosensitive severe combined immunodeficiency (RS-SCID), a rare disorder characterized by cellular sensitivity to radiation and the absence of lymphocytes due to impaired DNA-dependent protein kinase (DNA-PK) activity. Our results demonstrate that gene editing in RS-SCID fibroblasts rescued DNA-PK dependent signaling to overcome radiosensitivity. Furthermore, in vitro T-cell differentiation from iPSCs was employed to model the stage-specific T-cell maturation block induced by the disease causing mutation. Genetic correction of the RS-SCID iPSCs restored T-lymphocyte maturation, polyclonal V(D)J recombination of the T-cell receptor followed by successful beta-selection. In conclusion, we provide proof that iPSC-based in vitro T-cell differentiation is a valuable paradigm for SCID disease modeling, which can be utilized to investigate disorders of T-cell development and to validate gene therapy strategies for T-cell deficiencies. Moreover, this study emphasizes the significance of designer nucleases as a tool for generating isogenic disease models and their future role in producing autologous, genetically corrected transplants for various clinical applications.
Chronic hepatitis B virus (HBV) infection remains an important global health problem. Stability of the episomal covalently closed circular HBV DNA (cccDNA) is largely responsible for the modest ...curative efficacy of available therapy. Since licensed anti-HBV drugs have a post-transcriptional mechanism of action, disabling cccDNA is potentially of therapeutic benefit. To develop this approach, we engineered mutagenic transcription activator-like effector nucleases (TALENs) that target four HBV-specific sites within the viral genome. TALENs with cognate sequences in the S or C open-reading frames (ORFs) efficiently disrupted sequences at the intended sites and suppressed markers of viral replication. Following triple transfection of cultured HepG2.2.15 cells under mildly hypothermic conditions, the S TALEN caused targeted mutation in ~35% of cccDNA molecules. Markers of viral replication were also inhibited in vivo in a murine hydrodynamic injection model of HBV replication. HBV target sites within S and C ORFs of the injected HBV DNA were mutated without evidence of toxicity. These findings are the first to demonstrate a targeted nuclease-mediated disruption of HBV cccDNA. Efficacy in vivo also indicates that these engineered nucleases have potential for use in treatment of chronic HBV infection.
Manipulation of gene expression can be facilitated by editing the genome or the epigenome. Precise genome editing is traditionally achieved by using designer nucleases which are generally exploited ...to eliminate a specific gene product. Upon the introduction of a site-specific DNA double-strand break (DSB) by the nuclease, endogenous DSB repair mechanisms are in turn harnessed to induce DNA sequence changes that can result in target gene inactivation. Minimal off-target effects can be obtained by endowing designer nucleases with the highly specific DNA-binding domain (DBD) derived from transcription activator-like effectors (TALEs). In contrast, epigenome editing allows gene expression control without inducing changes in the DNA sequence by specifically altering epigenetic marks, as histone tails modifications or DNA methylation patterns within promoter or enhancer regions. Importantly, this approach allows both up- and downregulation of the target gene expression, and the effect is generally reversible. TALE-based designer epigenome modifiers combine the high specificity of TALE-derived DBDs with the power of epigenetic modifier domains to induce fast and long-lasting changes in the epigenetic landscape of a target gene and control its expression. Here we provide a detailed description for the generation of TALE-based designer epigenome modifiers and of a suitable reporter cell line to easily monitor their activity.
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