Impaired mammary tumor formation and metastasis by the point mutation of a Smad3 linker phosphorylation site

Triple-negative breast cancer (TNBC) is often aggressive and metastatic. Transforming growth factor-β acts as a tumor-promoter in TNBC. Smad3, a major downstream effector protein in the TGF-β signaling pathway, is regulated by phosphorylation at several sites. The functional significance of the phosphorylation of the linker region in Smad3 is poorly understood for TNBC. Among the four sites in the Smad3 linker region, threonine-179 (T179) appears to be unique as it serves as the binding site for multiple WW-domain-containing proteins upon phosphorylation, suggesting that this phosphorylation is a key for Smad3 to engage other pathways.Using genome editing, we introduced for the first time a knock-in (KI) mutation in the endogenous Smad3 gene in IV2, a lung-tropic subline of the human MDA-MB-231 TNBC cell line. In the resulting cell line, the Smad3 T179 phosphorylation site is replaced by non-phosphorylatable valine (T179V) with the mutation in both alleles.The T179V KI reduced cell growth rate and mammosphere formation. These phenomena were accompanied by a significant upregulation of p21^Cip1 and downregulation of c-Myc. The T179V KI also reduced cell migration and invasion in vitro. In the mouse xenograft models, the T179V KI markedly reduced the establishment of primary tumor in the mammary fat pad and the lung metastasis.Our results using gene editing indicate the cancer-promoting role of Smad3 T179 phosphorylation in the human TNBC cells. Our findings highly suggest that controlling this phosphorylation may have therapeutic potential for TNBC.     

Highly efficient homology-driven genome editing in human T cells by combining zinc-finger nuclease mRNA and AAV6 donor delivery

The adoptive transfer of engineered T cells for the treatment of cancer, autoimmunity, and infectious disease is a rapidly growing field that has shown great promise in recent clinical trials. Nuclease-driven genome editing provides a method in which to precisely target genetic changes to further enhance T cell function in vivo. We describe the development of a highly efficient method to genome edit both primary human CD8 and CD4 T cells by homology-directed repair at a pre-defined site of the genome. Two different homology donor templates were evaluated, representing both minor gene editing events (restriction site insertion) to mimic gene correction, or the more significant insertion of a larger gene cassette. By combining zinc finger nuclease mRNA delivery with AAV6 delivery of a homologous donor we could gene correct 41% of CCR5 or 55% of PPP1R12C (AAVS1) alleles in CD8⁺ T cells and gene targeting of a GFP transgene cassette in >40% of CD8⁺ and CD4⁺ T cells at both the CCR5 and AAVS1 safe harbor locus, potentially providing a robust genome editing tool for T cell-based immunotherapy.     

The emerging role of RNA and DNA editing in cancer

Nucleotide sequence modification through single base editing in animals is emerging as an important player in tumorigenesis. RNA editing especially has increased greatly during mammalian evolution and modulates diverse cellular functions presumably in a context-dependent manner. Sequence editing impacts development, including pluripotency and hematopoiesis, and multiple recent studies have shown that dysregulation of editing is associated with tumor biology. Much is yet to be learned about the role of sequence editing in human biology but this process is a critical modulator of cell regulation and may present an attractive option for therapeutic intervention in cancer in the future.SignificanceSequence editing provides an additional regulatory layer of cancer initiation and progression that may be amenable to therapeutic design. Although editing of both RNA and DNA substrates has been known to occur for some time, the extent and implications of these modifications have been grossly underappreciated until recent genome-wide and disease-association studies were reported. This review highlights the cellular processes controlled by sequence editing, their implications in normal and cancerous states and considers potential targeted therapeutic strategies.     

Role of receptor editing and revision in shaping the B and T lymphocyte repertoire

B and T lymphocytes that carry antigen receptors are able to change specificity through subsequent receptor gene rearrangements. Receptor editing and receptor revision are terms used to distinguish those rearrangements occurring, respectively, in central lymphoid organs and the periphery. Secondary rearrangement appears to be a major player at two levels in the life of B lymphocytes. First, editing preserves a diverse repertoire without compromising self-tolerance, and revision further increases this repertoire once B cells have been engaged in an immune response, most likely for a better interaction with microbes. Recent studies have likewise suggested a role for receptor editing and revision in shaping the T cell repertoire during development and tolerance.     

Hepatitis D virus RNA editing: specific modification of adenosine in the antigenomic RNA.

RNA editing plays a central role in the life cycle of hepatitis D virus (HDV), a subviral human pathogen. Previous studies (J.L. Casey, K.F. Bergmann, T.L. Brown, and J.L. Gerin, Proc. Natl. Acad. Sci USA 89:7149-7153, 1992; H. Zheng, T.-B. Fu, D. Lazinski, and J. Taylor, J. Virol. 66:4693-4697, 1992) had concluded that the genomic RNA of HDV was the target for RNA editing and that the editing reaction was a conversion of U to C. However, we show here that the antigenomic RNA of HDV is in fact the target for HDV RNA editing, which is therefore a conversion of A to G. This result is verified by using an assay specific for editing on the antigenomic RNA and by analyzing the editing of site-directed mutant RNAs in transfected cells and in cell extracts. Because editing occurs in the absence of viral antigens and the specificity for the HDV editing target site is present even in extracts from Drosophila cells, it is likely that HDV RNA is edited by one or more cellular factors that are conserved among higher eukaryotes. These results raise the likelihood that double-stranded RNA adenosine deaminase specifically edits HDV antigenomic RNA.     

The central tolerance response to male antigen in normal mice is deletion and not receptor editing

It is widely accepted that developing T cells can undergo clonal deletion in the thymus in response to a high affinity self-Ag. This is largely based on studies of TCR transgenics. However, encounter with high affinity self-Ag can also result in receptor editing in TCR transgenic models. Because all TCR transgenics display ectopic receptor expression, the tolerance mechanism that predominates in normal mice remains an open question. When self-Ag drives receptor editing during T cell development, one expects to find in-frame, self-reactive TCRalpha joins on TCR excision circles (TRECs), which are the products of secondary V/J recombination in the TCRalpha locus. Such joins are not expected if clonal deletion occurs, because the progenitor cell would be eliminated by apoptosis. To test the relative utilization of receptor editing vs clonal deletion, we determined the frequency of in-frame, male-specific joins on TRECs in male and female HYbeta transgenic mice. In comparison with female HYbeta transgenic mice, our analysis showed a lower frequency of TRECs with male-reactive V17J57 joins in male mice. Thus, it would appear that receptor editing is not a predominant tolerance mechanism for this self-Ag.     

Sequential immune escape and shifting of T cell responses in a long-term survivor of melanoma

Immune-mediated control of tumors may occur, in part, through lysis of malignant cells by CD8(+) T cells that recognize specific Ag-HLA class I complexes. However, tumor cell populations may escape T cell responses by immune editing, by preventing formation of those Ag-HLA complexes. It remains unclear whether the human immune system can respond to immune editing and recognize newly arising escape variants. We report an example of shifting immune responses to escape variants in a patient with sequential metastases of melanoma and long-term survival after surgery alone. Tumor cells in the first metastasis escaped immune recognition via selective loss of an HLA haplotype (HLA-A11, -B44, and -Cw17), but maintained expression of HLA-A2. In the second metastasis, immune escape from an immunodominant MART-1-specific T cell response was mediated by HLA class I down-regulation, resulting in a failure to present this epitope, but persistent presentation of a tyrosinase-derived epitope. Consequent to this modification in tumor Ag presentation, the dominant CTL response shifted principally toward a tyrosinase-targeted response, even though tyrosinase-specific CTL had been undetectable during the initial metastatic event. Thus, in response to immune editing of tumor cells, a patient's spontaneous T cell response adapted, gaining the ability to recognize and to lyse "edited" tumor targets. The observation of both immune editing and immune adaptation in a patient with long-term survival after surgery alone demonstrates an example of immune system reactivity to counteract the escape mechanism(s) developed by tumor cells, which may contribute to the clinical outcome of malignant disease.     

Amino-terminal flanking residues determine the conformation of a peptide-class II MHC complex

The peptide spanning residues 48-62 of hen egg white lysozyme presented by I-A(k) molecules gives rise to two T cell populations, types A and B, that recognize distinct conformers of the complex generated in late and recycling endosomes. The class II-like accessory molecule H2-DM functions as a conformational editor, eliminating the type B conformer in late endosomes. Here, we show that the conformation of the complex, and its susceptibility to editing by H2-DM, are determined by peptide amino-terminal flanking residues. Elimination of these residues abolished editing, permitting formation of the type B conformer in late endosomes. Substitutions at P(-2) affected the stability of the type B conformer, preventing its formation and/or editing, without hindering peptide binding or formation of the type A conformer of the complex. We conclude that interactions involving amino-terminal flanking residues stabilize peptide-MHC conformers and confer resistance to editing by H2-DM, influencing the nature of the T cell repertoire.     

Central tolerance to self-antigen expressed by cortical epithelial cells

The exposure of developing thymocytes to high-affinity self-Ag results in T cell tolerance. A predominant mechanism for this is clonal deletion; though receptor editing, anergy induction, and positive selection of regulatory T cells have also been described. It is unclear what signals are involved in determining different tolerance mechanisms. In particular, OT-I mice displayed receptor editing when the high-affinity self-Ag was expressed in cortical epithelial cells (cEC) using the human keratin 14 promoter. To test the hypothesis that receptor editing is a consequence of a unique instruction given by cEC presenting self-Ag, we created mice expressing the 2C and HY ligands under control of the keratin 14 promoter. Alternatively, we studied the fate of developing T cells in OT-I mice where Ag was presented by all thymic APC. Surprisingly, we found that the tolerance mechanism was not influenced by the APC subset involved in presentation. Clonal deletion was observed in 2C and HY models even when Ag was presented only by cEC; and receptor editing was observed in OT-I mice even when Ag was presented by all thymic APC. These results suggest that different TCRs show intrinsic differences in thymic tolerance mechanism.     

利用电转的方法对T细胞TET2基因敲除并探讨TET2对T细胞增殖的影响

近年来,利用重组病毒对T细胞进行基因编辑用于免疫治疗,受到了广泛重视。然而,重组病毒因存在随机整合,制备耗时长且昂贵的缺点制约了其应用。与此同时,电转染技术的应用能够快速将外源DNA带入细胞内,有助于提高T细胞基因编辑效率。TET(Ten-eleven translocation)家族蛋白可以催化5-甲基胞嘧啶(5mC)转化为5-羟甲基胞嘧啶(5hmC),5hmC作为细胞中的DNA去甲基化酶,在细胞基因组表观遗传学中起着重要调控作用。研究表明TET2基因的缺失能够促进CAR-T细胞的快速繁殖,产生强力的CAR-T细胞。该研究利用CRISPR/Cas9基因编辑技术对TET2基因进行敲除。首先对sgRNA进行体外转录,与大肠杆菌诱导表达的Cas9蛋白孵育形成Cas9:gRNA核糖核蛋白复合物(RNP),并在体外酶切验证sgRNA的活性。接着利用电转染技术将Cas9:gRNA核糖核蛋白复合物(RNP)带入细胞内,并检测T细胞基因编辑效率。最后利用流式细胞分析技术检测T细胞的增殖情况。基因测序与T7EⅠ酶切结果表明,T细胞中的TET2基因被成功敲除,T细胞活力和功能并未受到影响。流式细胞技术,CCK-8以及台盼蓝细胞活率检测结果显示缺失Tet2蛋白后,T细胞的增殖速率明显快于野生型T细胞。该研究为非病毒载体替代传统的慢病毒载体构建携带嵌合抗原T细胞奠定了基础,具有制备周期短,安全性高的特点。同时TET2缺失促进了CAR-T细胞的增殖,使其能够引发有效的抗肿瘤反应,为CAR-T细胞免疫治疗提供了新的思路。     

Altered expression and editing of miRNA-100 regulates iTreg differentiation

RNA editing of miRNAs, especially in the seed region, adds another layer to miRNA mediated gene regulation which can modify its targets, altering cellular signaling involved in important processes such as differentiation. In this study, we have explored the role of miRNA editing in CD4⁺ T cell differentiation. CD4⁺ T cells are an integral component of the adaptive immune system. Naïve CD4⁺ T cells, on encountering an antigen, get differentiated either into inflammatory subtypes like Th1, Th2 or Th17, or into immunosuppressive subtype Treg, depending on the cytokine milieu. We found C-to-U editing at fifth position of mature miR-100, specifically in Treg. The C-to-U editing of miR-100 is functionally associated with at least one biologically relevant target change, from MTOR to SMAD2. Treg cell polarization by TGFβ1 was reduced by both edited and unedited miR-100 mimics, but percentage of Treg in PBMCs was only reduced by edited miR-100 mimics, suggesting a model in which de-repression of MTOR due to loss of unedited mir-100, promotes tolerogenic signaling, while gain of edited miR-100 represses SMAD2, thereby limiting the Treg. Such delicately counterbalanced systems are a hallmark of immune plasticity and we propose that miR-100 editing is a novel mechanism toward this end.     

DM peptide-editing function leads to immunodominance in CD4 T cell responses in vivo

DM functions as a peptide editor for MHC class II-bound peptides. We examined the hypothesis that DM peptide editing plays a key role in focusing the in vivo CD4 T cell responses against complex pathogens and protein Ags to only one, or at most a few, immunodominant peptides. Most CD4 T cells elicited in the wild-type BALB/c (H-2d) mice infected with Leishmania major predominantly recognize a single epitope 158-173 within Leishmania homologue of activated receptor for c-kinase (LACK), as is the case when these mice are immunized with rLACK. Using DM-deficient (DM-/-) H-2d mice, we now show that in the absence of DM, the in vivo CD4 T cell responses to rLACK are skewed away from the immunodominant epitopes and are diversified to include two novel epitopes (LACK 33-48 and 261-276). DM-/- B10.BR (H-2k) mice showed similar results. These results constitute the first demonstration of the role of DM peptide editing in sculpting the specificity and immunodominance in in vivo CD4 T cell responses.     

Dynamics and competition of CRISPR–Cas9 ribonucleoproteins and AAV donor-mediated NHEJ,MMEJ and HDR editing

Investigations of CRISPR gene knockout editing profiles have contributed to enhanced precision of editing outcomes. However, for homology-directed repair (HDR) in particular, the editing dynamics and patterns in clinically relevant cells, such as human iPSCs and primary T cells, are poorly understood. Here, we explore the editing dynamics and DNA repair profiles after the delivery of Cas9-guide RNA ribonucleoprotein (RNP) with or without the adeno-associated virus serotype 6 (AAV6) as HDR donors in four cell types. We show that editing profiles have distinct differences among cell lines. We also reveal the kinetics of HDR mediated by the AAV6 donor template. Quantification of T₅₀ (time to reach half of the maximum editing frequency) indicates that short indels (especially +A/T) occur faster than longer (>2 bp) deletions, while the kinetics of HDR falls between NHEJ (non-homologous end-joining) and MMEJ (microhomology-mediated end-joining). As such, AAV6-mediated HDR effectively outcompetes the longer MMEJ-mediated deletions but not NHEJ-mediated indels. Notably, a combination of small molecular compounds M3814 and Trichostatin A (TSA), which potently inhibits predominant NHEJ repairs, leads to a 3-fold increase in HDR efficiency.     

Editing T cell specificity towards leukemia by zinc finger nucleases and lentiviral gene transfer

The transfer of high-avidity T cell receptor (TCR) genes isolated from rare tumor-specific lymphocytes into polyclonal T cells is an attractive cancer immunotherapy strategy. However, TCR gene transfer results in competition for surface expression and inappropriate pairing between the exogenous and endogenous TCR chains, resulting in suboptimal activity and potentially harmful unpredicted antigen specificities of the resultant TCRs. We designed zinc-finger nucleases (ZFNs) that promoted the disruption of endogenous TCR β- and α-chain genes. Lymphocytes treated with ZFNs lacked surface expression of CD3-TCR and expanded with the addition of interleukin-7 (IL-7) and IL-15. After lentiviral transfer of a TCR specific for the Wilms tumor 1 (WT1) antigen, these TCR-edited cells expressed the new TCR at high levels, were easily expanded to near purity and were superior at specific antigen recognition compared to donor-matched, unedited TCR-transferred cells. In contrast to unedited TCR-transferred cells, the TCR-edited lymphocytes did not mediate off-target reactivity while maintaining their anti-tumor activity in vivo, thus showing that complete editing of T cell specificity generates tumor-specific lymphocytes with improved biosafety profiles.     

Targeted Genome Editing by Recombinant Adeno-Associated Virus （rAAV） Vectors for Generating Genetically Modified Pigs

Recombinant adeno-associated virus(rAAV) vectors have been extensively used for experimental gene therapy of inherited human diseases.Several advantages,such as simple vector construction,high targeting frequency by homologous recombination,and applicability to many cell types,make rAAV an attractive approach for targeted genome editing.Combined with cloning by somatic cell nuclear transfer(SCNT),this technology has recently been successfully adapted to generate gene-targeted pigs as models for cystic fibrosis, hereditary tyrosinemia type 1,and breast cancer.This review summarizes the development of rAAV for targeted genome editing in mammalian cells and provides strategies for enhancing the rAAV-mediated targeting frequency by homologous recombination.We discuss current development and application of the rAAV vectors for targeted genome editing in porcine primary fibroblasts,which are subsequently used as donor cells for SCNT to generate cloned genetically designed pigs and provide positive perspectives for the generation of gene-targeted pigs with rAAV in the future.