ABSTRACT
Off-target effects present a significant impediment to the safe and efficient use of CRISPR-Cas genome editing. Since off-target activity is influenced by the genomic sequence, the presence of sequence variants leads to varying on- and off-target profiles among different alleles or individuals. However, a reliable tool that quantifies genome editing activity in an allelic context is not available. Here, we introduce CRISPECTOR2.0, an extended version of our previously published software tool CRISPECTOR, with an allele-specific editing activity quantification option. CRISPECTOR2.0 enables reference-free, allele-aware, precise quantification of on- and off-target activity, by using de novo sample-specific single nucleotide variant (SNV) detection and statistical-based allele-calling algorithms. We demonstrate CRISPECTOR2.0 efficacy in analyzing samples containing multiple alleles and quantifying allele-specific editing activity, using data from diverse cell types, including primary human cells, plants, and an original extensive human cell line database. We identified instances where an SNV induced changes in the protospacer adjacent motif sequence, resulting in allele-specific editing. Intriguingly, differential allelic editing was also observed in regions carrying distal SNVs, hinting at the involvement of additional epigenetic factors. Our findings highlight the importance of allele-specific editing measurement as a milestone in the adaptation of efficient, accurate, and safe personalized genome editing.
ABSTRACT
Cancer exploits different mechanisms to escape T-cell immunosurveillance, including overexpression of checkpoint ligands, secretion of immunosuppressive molecules, and aberrant glycosylation. Herein, we report that IFNγ, a potent immunomodulator secreted in the tumor microenvironment, can induce α2,6 hypersialylation in cancer cell lines derived from various histologies. We then focused on Siglec-9, a receptor for sialic acid moieties, and demonstrated that the Siglec-9+ T-cell population displayed reduced effector function. We speculated that Siglec-9 in primary human T cells can act as a checkpoint molecule and demonstrated that knocking out Siglec-9 using a CRISPR/Cas9 system enhanced the functionality of primary human T cells. Finally, we aimed to augment cancer-specific T-cell activity by taking advantage of tumor hypersialylation. Thus, we designed several Siglec-9-based chimeric switch receptors (CSRs), which included an intracellular moiety derived from costimulatory molecules (CD28/41BB) and different hinge regions. In an antigen specific context, T cells transduced with Siglec-9 CSRs demonstrated increased cytokine secretions and upregulation of activation markers. Moreover, T cells equipped with specific Siglec-9 CSRs mediated robust antitumor activity in a xenograft model of human tumors. Overall, this work sheds light on tumor evasion mechanisms mediated by sialylated residues and exemplifies an approach to improve engineered T cell-based cancer treatment.
ABSTRACT
This Viewpoint reviews the history and current state of gene therapy for inborn errors of immunity.
Subject(s)
Genetic Therapy , Immune System Diseases , Humans , Genetic Therapy/methods , Immune System Diseases/geneticsABSTRACT
Natural killer (NK) cells are a vital component of cancer immune surveillance. They provide a rapid and potent immune response, including direct cytotoxicity and mobilization of the immune system, without the need for antigen processing and presentation. NK cells may also be better tolerated than T cell therapy approaches and are susceptible to various gene manipulations. Therefore, NK cells have become the focus of extensive translational research. Gamida Cell's nicotinamide (NAM) platform for cultured NK cells provides an opportunity to enhance the therapeutic potential of NK cells. CD38 is an ectoenzyme ubiquitously expressed on the surface of various hematologic cells, including multiple myeloma (MM). It has been selected as a lead target for numerous monoclonal therapeutic antibodies against MM. Monoclonal antibodies target CD38, resulting in the lysis of MM plasma cells through various antibody-mediated mechanisms such as antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity, and antibody-dependent cellular phagocytosis, significantly improving the outcomes of patients with relapsed or refractory MM. However, this therapeutic strategy has inherent limitations, such as the anti-CD38-induced depletion of CD38-expressing NK cells, thus hindering ADCC. We have developed genetically engineered NK cells tailored to treat MM, in which CD38 was knocked-out using CRISPR-Cas9 technology and an enhanced chimeric antigen receptor (CAR) targeting CD38 was introduced using mRNA electroporation. This combined genetic approach allows for an improved cytotoxic activity directed against CD38-expressing MM cells without self-inflicted NK-cell-mediated fratricide. Preliminary results show near-complete abolition of fratricide with a 24-fold reduction in self-lysis from 19% in mock-transfected and untreated NK cells to 0.8% of self-lysis in CD38 knock-out CAR NK cells. Furthermore, we have observed significant enhancements in CD38-mediated activity in vitro, resulting in increased lysis of MM target cell lines. CD38 knock-out CAR NK cells also demonstrated significantly higher levels of NK activation markers in co-cultures with both untreated and αCD38-treated MM cell lines. These NAM-cultured NK cells with the combined genetic approach of CD38 knockout and addition of CD38 CAR represent a promising immunotherapeutic tool to target MM.
Subject(s)
Hematopoietic Stem Cell Transplantation , Multiple Myeloma , Receptors, Chimeric Antigen , Humans , Multiple Myeloma/therapy , Multiple Myeloma/drug therapy , Killer Cells, Natural , Antibody-Dependent Cell CytotoxicityABSTRACT
The European Cooperation in Science and Technology (COST) is an intergovernmental organization dedicated to funding and coordinating scientific and technological research in Europe, fostering collaboration among researchers and institutions across countries. Recently, COST Action funded the "Genome Editing to treat Human Diseases" (GenE-HumDi) network, uniting various stakeholders such as pharmaceutical companies, academic institutions, regulatory agencies, biotech firms, and patient advocacy groups. GenE-HumDi's primary objective is to expedite the application of genome editing for therapeutic purposes in treating human diseases. To achieve this goal, GenE-HumDi is organized in several working groups, each focusing on specific aspects. These groups aim to enhance genome editing technologies, assess delivery systems, address safety concerns, promote clinical translation, and develop regulatory guidelines. The network seeks to establish standard procedures and guidelines for these areas to standardize scientific practices and facilitate knowledge sharing. Furthermore, GenE-HumDi aims to communicate its findings to the public in accessible yet rigorous language, emphasizing genome editing's potential to revolutionize the treatment of many human diseases. The inaugural GenE-HumDi meeting, held in Granada, Spain, in March 2023, featured presentations from experts in the field, discussing recent breakthroughs in delivery methods, safety measures, clinical translation, and regulatory aspects related to gene editing.
ABSTRACT
RAG2-SCID is a primary immunodeficiency caused by mutations in Recombination-activating gene 2 (RAG2), a gene intimately involved in the process of lymphocyte maturation and function. ex-vivo manipulation of a patient's own hematopoietic stem and progenitor cells (HSPCs) using CRISPR-Cas9/rAAV6 gene editing could provide a therapeutic alternative to the only current treatment, allogeneic hematopoietic stem cell transplantation (HSCT). Here we show an innovative RAG2 correction strategy that replaces the entire endogenous coding sequence (CDS) for the purpose of preserving the critical endogenous spatiotemporal gene regulation and locus architecture. Expression of the corrective transgene leads to successful development into CD3+TCRαß+ and CD3+TCRγδ+ T cells and promotes the establishment of highly diverse TRB and TRG repertoires in an in-vitro T-cell differentiation platform. Thus, our proof-of-concept study holds promise for safer gene therapy techniques of tightly regulated genes.
Subject(s)
CRISPR-Cas Systems , Hematopoietic Stem Cell Transplantation , Humans , CRISPR-Cas Systems/genetics , Hematopoietic Stem Cells/metabolism , Gene Editing/methods , Gene Expression Regulation , DNA-Binding Proteins/metabolism , Nuclear Proteins/metabolismABSTRACT
Genome engineering via targeted nucleases, specifically CRISPR-Cas9, has revolutionized the field of gene therapy research, providing a potential treatment for diseases of the blood and immune system. While numerous genome editing techniques have been used, CRISPR-Cas9 homology-directed repair (HDR)-mediated editing represents a promising method for the site-specific insertion of large transgenes for gene knock-in or gene correction. Alternative methods, such as lentiviral/gammaretroviral gene addition, gene knock-out via non-homologous end joining (NHEJ)-mediated editing, and base or prime editing, have shown great promise for clinical applications, yet all possess significant drawbacks when applied in the treatment of patients suffering from inborn errors of immunity or blood system disorders. This review aims to highlight the transformational benefits of HDR-mediated gene therapy and possible solutions for the existing problems holding the methodology back. Together, we aim to help bring HDR-based gene therapy in CD34+ hematopoietic stem progenitor cells (HSPCs) from the lab bench to the bedside.
ABSTRACT
Severe combined immunodeficiency (SCID) is a group of disorders caused by mutations in genes involved in the process of lymphocyte maturation and function. CRISPR-Cas9 gene editing of the patient's own hematopoietic stem and progenitor cells (HSPCs) ex vivo could provide a therapeutic alternative to allogeneic hematopoietic stem cell transplantation, the current gold standard for treatment of SCID. To eliminate the need for scarce patient samples, we engineered genotypes in healthy donor (HD)-derived CD34+ HSPCs using CRISPR-Cas9/rAAV6 gene-editing, to model both SCID and the therapeutic outcomes of gene-editing therapies for SCID via multiplexed homology-directed repair (HDR). First, we developed a SCID disease model via biallelic knockout of genes critical to the development of lymphocytes; and second, we established a knockin/knockout strategy to develop a proof-of-concept single-allelic gene correction. Based on these results, we performed gene correction of RAG2-SCID patient-derived CD34+ HSPCs that successfully developed into CD3+ T cells with diverse TCR repertoires in an in vitro T cell differentiation platform. In summary, we present a strategy to determine the optimal configuration for CRISPR-Cas9 gene correction of SCID using HD-derived CD34+ HSPCs, and the feasibility of translating this gene correction approach in patient-derived CD34+ HSPCs.
ABSTRACT
The TP53 gene is mutated in approximately 60% of all colorectal cancer (CRC) cases. Over 20% of all TP53-mutated CRC tumors carry missense mutations at position R175 or R273. Here we report that CRC tumors harboring R273 mutations are more prone to progress to metastatic disease, with decreased survival, than those with R175 mutations. We identify a distinct transcriptional signature orchestrated by p53R273H, implicating activation of oncogenic signaling pathways and predicting worse outcome. These features are shared also with the hotspot mutants p53R248Q and p53R248W. p53R273H selectively promotes rapid CRC cell spreading, migration, invasion and metastasis. The transcriptional output of p53R273H is associated with preferential binding to regulatory elements of R273 signature genes. Thus, different TP53 missense mutations contribute differently to cancer progression. Elucidation of the differential impact of distinct TP53 mutations on disease features may make TP53 mutational information more actionable, holding potential for better precision-based medicine.
Subject(s)
Colorectal Neoplasms , Tumor Suppressor Protein p53 , Colorectal Neoplasms/genetics , Genes, p53 , Humans , Mutation , Phenotype , Tumor Suppressor Protein p53/genetics , Tumor Suppressor Protein p53/metabolismABSTRACT
Establishing causal relationships between genetic alterations of human cancers and specific phenotypes of malignancy remains a challenge. We sequentially introduced mutations into healthy human melanocytes in up to five genes spanning six commonly disrupted melanoma pathways, forming nine genetically distinct cellular models of melanoma. We connected mutant melanocyte genotypes to malignant cell expression programs in vitro and in vivo, replicative immortality, malignancy, rapid tumor growth, pigmentation, metastasis, and histopathology. Mutations in malignant cells also affected tumor microenvironment composition and cell states. Our melanoma models shared genotype-associated expression programs with patient melanomas, and a deep learning model showed that these models partially recapitulated genotype-associated histopathological features as well. Thus, a progressive series of genome-edited human cancer models can causally connect genotypes carrying multiple mutations to phenotype.
Subject(s)
Melanoma , Skin Neoplasms , Humans , Melanocytes/metabolism , Melanoma/pathology , Mutation , Skin Neoplasms/genetics , Skin Neoplasms/pathology , Tumor Microenvironment/geneticsABSTRACT
BACKGROUND: Implementation of newborn screening (NBS) programs for severe combined immunodeficiency (SCID) have advanced the diagnosis and management of affected infants and undoubtedly improved their outcomes. Reporting long-term follow-up of such programs is of great importance. OBJECTIVE: We report a 5-year summary of the NBS program for SCID in Israel. METHODS: Immunologic and genetic assessments, clinical analyses, and outcome data from all infants who screened positive were evaluated and summarized. RESULTS: A total of 937,953 Guthrie cards were screened for SCID. A second Guthrie card was requested on 1,169 occasions (0.12%), which resulted in 142 referrals (0.015%) for further validation tests. Flow cytometry immune-phenotyping, T cell receptor excision circle measurement in peripheral blood, and expression of TCRVß repertoire for the validation of positive cases revealed a specificity and sensitivity of 93.7% and 75.9%, respectively, in detecting true cases of SCID. Altogether, 32 SCID and 110 non-SCID newborns were diagnosed, making the incidence of SCID in Israel as high as 1:29,000 births. The most common genetic defects in this group were associated with mutations in DNA cross-link repair protein 1C and IL-7 receptor α (IL-7Rα) genes. No infant with SCID was missed during the study time. Twenty-two SCID patients underwent hematopoietic stem cell transplantation, which resulted in a 91% survival rate. CONCLUSIONS: Newborn screening for SCID should ultimately be applied globally, specifically to areas with high rates of consanguineous marriages. Accumulating data from follow-up studies on NBS for SCID will improve diagnosis and treatment and enrich our understanding of immune development in health and disease.
Subject(s)
Severe Combined Immunodeficiency , DNA , Humans , Infant, Newborn , Israel/epidemiology , Neonatal Screening/methods , Receptors, Antigen, T-Cell/genetics , Receptors, Interleukin-7 , Severe Combined Immunodeficiency/diagnosis , Severe Combined Immunodeficiency/epidemiology , Severe Combined Immunodeficiency/geneticsABSTRACT
CRISPR-Cas technology has revolutionized gene editing, but concerns remain due to its propensity for off-target interactions. This, combined with genotoxicity related to both CRISPR-Cas9-induced double-strand breaks and transgene delivery, poses a significant liability for clinical genome-editing applications. Current best practice is to optimize genome-editing parameters in preclinical studies. However, quantitative tools that measure off-target interactions and genotoxicity are costly and time-consuming, limiting the practicality of screening large numbers of potential genome-editing reagents and conditions. Here, we show that flow-based imaging facilitates DNA damage characterization of hundreds of human hematopoietic stem and progenitor cells per minute after treatment with CRISPR-Cas9 and recombinant adeno-associated virus serotype 6. With our web-based platform that leverages deep learning for image analysis, we find that greater DNA damage response is observed for guide RNAs with higher genome-editing activity, differentiating even single on-target guide RNAs with different levels of off-target interactions. This work simplifies the characterization and screening process of genome-editing parameters toward enabling safer and more effective gene-therapy applications.
Subject(s)
Dependovirus , Gene Editing , CRISPR-Cas Systems/genetics , DNA Damage/genetics , Dependovirus/genetics , Gene Editing/methods , Humans , Stem CellsABSTRACT
RNA guided nucleases are regarded as the future genome editing technologies. As such, they need to meet strong safety margins. Two major challenges in incorporating CRISPR technologies into the clinical world are off-target activity and editing efficiency. The common way to tackle such issues is to measure the binding and cleavage kinetics of the CRISPR enzyme. This can be challenging since, for example, DNA is not released from the CAS9 protein post cleavage. Here a promising new microfluidic approach to characterizing Enzymatic Interaction and Function of CRISPR complexes on a microfluidic platform (EnzyMIF) is presented. The method can rapidly detect the kd, koff, km and kcat for various RNA guided nucleases. In this work, two single guide RNAs with significantly different in-cell cleavage efficiency, RAG2 and RAG1, are used as proof-of-concept. The EnzyMIF assay results provide biochemical characterization of these guide RNAs that can explain the difference in cleavage using both wild type (WT) CAS9 and HiFi CAS9. Notably, it is shown that EnzyMIF characterization correlates with cell culture genomic editing efficiency results. It is suggested that EnzyMIF can predict the quality of cleavage rapidly and quantitatively.
Subject(s)
CRISPR-Cas Systems , Microfluidics , CRISPR-Cas Systems/genetics , Gene Editing , Genomics , RNA, Guide, Kinetoplastida/genetics , RNA, Guide, Kinetoplastida/metabolismABSTRACT
Controlling off-target editing activity is one of the central challenges in making CRISPR technology accurate and applicable in medical practice. Current algorithms for analyzing off-target activity do not provide statistical quantification, are not sufficiently sensitive in separating signal from noise in experiments with low editing rates, and do not address the detection of translocations. Here we present CRISPECTOR, a software tool that supports the detection and quantification of on- and off-target genome-editing activity from NGS data using paired treatment/control CRISPR experiments. In particular, CRISPECTOR facilitates the statistical analysis of NGS data from multiplex-PCR comparative experiments to detect and quantify adverse translocation events. We validate the observed results and show independent evidence of the occurrence of translocations in human cell lines, after genome editing. Our methodology is based on a statistical model comparison approach leading to better false-negative rates in sites with weak yet significant off-target activity.
Subject(s)
CRISPR-Cas Systems , Computational Biology/methods , Gene Editing/methods , Algorithms , DNA-Binding Proteins/genetics , HEK293 Cells , Homeodomain Proteins/genetics , Humans , Nuclear Proteins/genetics , Software , Transcription Factors/geneticsABSTRACT
Genetic diagnostic tools including whole-exome sequencing (WES) have advanced our understanding in human diseases and become common practice in diagnosing patients with suspected primary immune deficiencies. Establishing a genetic diagnosis is of paramount importance for tailoring adequate therapeutic regimens, including identifying the need for hematopoietic stem cell transplantation (HSCT) and genetic-based therapies. Here, we genetically studied two adult patients who were clinically diagnosed during infancy with severe combined immune deficiency (SCID). Two unrelated patients, both of consanguineous kindred, underwent WES in adulthood, 2 decades after their initial clinical manifestations. Upon clinical presentation, immunological workup was performed, which led to a diagnosis of SCID. The patients presented during infancy with failure to thrive, generalized erythematous rash, and recurrent gastrointestinal and respiratory tract infections, including episodes of Pneumocystis pneumonia infection and Candida albicans fungemia. Hypogammaglobulinemia and T-cell lymphopenia were detected. Both patients were treated with a 10/10 HLA matched sibling donor unconditioned HSCT. Retrospective genetic workup revealed homozygous bi-allelic mutations in IL7RA in one patient and in RAG2 in the other. Our study exemplifies the impact of retrospectively establishing a genetic diagnosis. Pinpointing the genetic cause raises several issues including optimized surveillance and treatment, understanding disease mechanisms and outcomes, future family planning, and social and psychological considerations.
Subject(s)
Severe Combined Immunodeficiency/genetics , Adult , DNA-Binding Proteins/genetics , Female , Hematopoietic Stem Cell Transplantation , Humans , Infant , Male , Mutation , Nuclear Proteins/genetics , Receptors, Antigen, T-Cell/immunology , Receptors, Interleukin-17/genetics , Severe Combined Immunodeficiency/diagnosis , Severe Combined Immunodeficiency/immunology , Severe Combined Immunodeficiency/therapy , Young AdultABSTRACT
Human CD34+ hematopoietic stem and progenitor cells (HSPCs) have the unique ability to repopulate the entire hematopoietic system and thus are at the center of diverse, therapeutically relevant studies. The recent development of the CRISPR/Cas9 tool made the powerful research technique of genome editing highly accessible. Our previous studies demonstrated that high editing efficiency is reached when the CRISPR/Cas9 is introduced to CD34+ HSPCs as a ribonucleoprotein (RNP) complex with chemically modified guide RNAs (gRNAs). The current protocol details a quick 4-day procedure for ex vivo genome editing in CD34+ HSPCs by RNP complexes that are targeted to a specific locus by either a single gRNA (sgRNA) or a 2-part gRNA. The delivery of RNP complexes is performed by electroporation in the presence of a nonspecific, ssDNA electroporation enhancer, which highly improves editing efficiency under the described conditions. This approach is simple and effective with the potential to accelerate many biotechnological and therapeutic applications of the CRISPR/Cas9 technology.
Subject(s)
CRISPR-Cas Systems/genetics , Electroporation/methods , Gene Editing/methods , RNA, Guide, Kinetoplastida/genetics , Antigens, CD34/genetics , DNA, Single-Stranded/genetics , Hematopoietic Stem Cells , HumansABSTRACT
The T cell receptor (TCR) signaling pathway is an ensemble of numerous proteins that are crucial for an adequate immune response. Disruption of any protein involved in this pathway leads to severe immunodeficiency and unfavorable clinical outcomes. Here, we describe an infant with severe immunodeficiency who was found to have novel biallelic mutations in SLP76. SLP76 is a key protein involved in TCR signaling and in other hematopoietic pathways. Previous studies of this protein were performed using Jurkat-derived human leukemic T cell lines and SLP76-deficient mice. Our current study links this gene, for the first time, to a human immunodeficiency characterized by early-onset life-threatening infections, combined T and B cell immunodeficiency, severe neutrophil defects, and impaired platelet aggregation. Hereby, we characterized aspects of the patient's immune phenotype, modeled them with an SLP76-deficient Jurkat-derived T cell line, and rescued some consequences using ectopic expression of wild-type SLP76. Understanding human diseases due to SLP76 deficiency is helpful in explaining the mixed T cell and neutrophil defects, providing a guide for exploring human SLP76 biology.
Subject(s)
Adaptor Proteins, Signal Transducing/deficiency , Blood Platelets/pathology , Neutrophils/pathology , Phosphoproteins/deficiency , Severe Combined Immunodeficiency/metabolism , Severe Combined Immunodeficiency/pathology , Adaptor Proteins, Signal Transducing/chemistry , Adaptor Proteins, Signal Transducing/genetics , Adaptor Proteins, Signal Transducing/metabolism , Amino Acid Sequence , Base Sequence , Blood Platelets/metabolism , Fatal Outcome , Humans , Infant , Infant, Newborn , Jurkat Cells , Mutation/genetics , Neutrophils/metabolism , Phenotype , Phosphoproteins/chemistry , Phosphoproteins/genetics , Phosphoproteins/metabolism , Receptors, Antigen, B-Cell/metabolism , Receptors, Antigen, T-Cell/metabolism , Severe Combined Immunodeficiency/immunology , Signal TransductionABSTRACT
Genome editing of human cluster of differentiation 34+ (CD34+) hematopoietic stem and progenitor cells (HSPCs) holds great therapeutic potential. This study aimed to optimize on-target, ex vivo genome editing using the CRISPR-Cas9 system in CD34+ HSPCs and to create a clear workflow for precise identification of off-target effects. Modified synthetic guide RNAs (gRNAs), either 2-part gRNA or single-guide RNA (sgRNA), were delivered to CD34+ HSPCs as part of ribonucleoprotein (RNP) complexes, targeting therapeutically relevant genes. The addition of an Alt-R electroporation enhancer (EE), a short, single-stranded oligodeoxynucleotide (ssODN), significantly increased editing efficiency in CD34+ HSPCs. Notably, similar editing improvement was observed when excess gRNA over Cas9 protein was used, providing a DNA-free alternative suitable for therapeutic applications. Furthermore, we demonstrated that sgRNA may be preferable over 2-part gRNA in a locus-specific manner. Finally, we present a clear experimental framework suitable for the unbiased identification of bona fide off-target sites by Genome-Wide, Unbiased Identification of Double-Strand Breaks (DSBs) Enabled by Sequencing (GUIDE-seq), as well as subsequent editing quantification in CD34+ HSPCs using rhAmpSeq. These findings may facilitate the implementation of genome editing in CD34+ HSPCs for research and therapy and can be adapted for other hematopoietic cells.
ABSTRACT
Adenosine-to-inosine (A-to-I) RNA editing is a common post transcriptional modification. It has a critical role in protecting against false activation of innate immunity by endogenous double stranded RNAs and has been associated with various regulatory processes and diseases such as autoimmune and cardiovascular diseases as well as cancer. In addition, the endogenous A-to-I editing machinery has been recently harnessed for RNA engineering. The study of RNA editing in humans relies heavily on the usage of cell lines as an important and commonly-used research tool. In particular, manipulations of the editing enzymes and their targets are often developed using cell line platforms. However, RNA editing in cell lines behaves very differently than in normal and diseased tissues, and most cell lines exhibit low editing levels, requiring over-expression of the enzymes. Here, we explore the A-to-I RNA editing landscape across over 1000 human cell lines types and show that for almost every editing target of interest a suitable cell line that mimics normal tissue condition may be found. We provide CLAIRE, a searchable catalogue of RNA editing levels across cell lines available at http://srv00.recas.ba.infn.it/atlas/claire.html, to facilitate rational choice of appropriate cell lines for future work on A-to-I RNA editing.