-
Nature Mar 2023T cell receptors (TCRs) enable T cells to specifically recognize mutations in cancer cells. Here we developed a clinical-grade approach based on CRISPR-Cas9...
T cell receptors (TCRs) enable T cells to specifically recognize mutations in cancer cells. Here we developed a clinical-grade approach based on CRISPR-Cas9 non-viral precision genome-editing to simultaneously knockout the two endogenous TCR genes TRAC (which encodes TCRα) and TRBC (which encodes TCRβ). We also inserted into the TRAC locus two chains of a neoantigen-specific TCR (neoTCR) isolated from circulating T cells of patients. The neoTCRs were isolated using a personalized library of soluble predicted neoantigen-HLA capture reagents. Sixteen patients with different refractory solid cancers received up to three distinct neoTCR transgenic cell products. Each product expressed a patient-specific neoTCR and was administered in a cell-dose-escalation, first-in-human phase I clinical trial ( NCT03970382 ). One patient had grade 1 cytokine release syndrome and one patient had grade 3 encephalitis. All participants had the expected side effects from the lymphodepleting chemotherapy. Five patients had stable disease and the other eleven had disease progression as the best response on the therapy. neoTCR transgenic T cells were detected in tumour biopsy samples after infusion at frequencies higher than the native TCRs before infusion. This study demonstrates the feasibility of isolating and cloning multiple TCRs that recognize mutational neoantigens. Moreover, simultaneous knockout of the endogenous TCR and knock-in of neoTCRs using single-step, non-viral precision genome-editing are achieved. The manufacture of neoTCR engineered T cells at clinical grade, the safety of infusing up to three gene-edited neoTCR T cell products and the ability of the transgenic T cells to traffic to the tumours of patients are also demonstrated.
Topics: Humans; Antigens, Neoplasm; Biopsy; Cell- and Tissue-Based Therapy; Cytokine Release Syndrome; Disease Progression; Encephalitis; Gene Editing; Gene Knock-In Techniques; Gene Knockout Techniques; Genes, T-Cell Receptor alpha; Genes, T-Cell Receptor beta; Mutation; Neoplasms; Patient Safety; Precision Medicine; Receptors, Antigen, T-Cell; T-Lymphocytes; Transgenes; HLA Antigens; CRISPR-Cas Systems
PubMed: 36356599
DOI: 10.1038/s41586-022-05531-1 -
Genetics Mar 2017females are larger than males. In this article, we describe how -chromosome dosage drives sexual dimorphism of body size through two means: first, through unbalanced...
females are larger than males. In this article, we describe how -chromosome dosage drives sexual dimorphism of body size through two means: first, through unbalanced expression of a key -linked growth-regulating gene, and second, through female-specific activation of the sex-determination pathway. -chromosome dosage determines phenotypic sex by regulating the genes of the sex-determining pathway. In the presence of two sets of -chromosome signal elements (XSEs), () is activated in female () but not male () animals. Sxl activates (), a gene that encodes a splicing factor essential for female-specific development. It has previously been shown that null mutations in the gene result in only a partial reduction of body size of animals, which shows that other factors must contribute to size determination. We tested whether dosage directly affects animal size by analyzing males with duplications of -chromosomal segments. Upon tiling across the chromosome, we found four duplications that increase male size by >9%. Within these, we identified several genes that promote growth as a result of duplication. Only one of these, , was found not to be dosage compensated. Together, our results indicate that both dosage and expression play crucial roles in determining sex-specific size in larvae and adult tissue. Since also acts as an XSE that contributes to activation in early development, a double dose of in females serves at least twice in development to promote sexual size dimorphism.
Topics: Animals; Body Size; Chromosomes, Insect; DNA-Binding Proteins; Dosage Compensation, Genetic; Drosophila; Drosophila Proteins; Female; Gene Duplication; Male; Nuclear Proteins; RNA-Binding Proteins; Sex Characteristics; Transcription Factors; X Chromosome
PubMed: 28064166
DOI: 10.1534/genetics.116.192260 -
Immunology and Cell Biology Feb 1998We describe the generation of ovalbumin (OVA)-specific, MHC class II-restricted alpha beta T cell receptor (TCR) transgenic mice. Initial attempts at generating these...
We describe the generation of ovalbumin (OVA)-specific, MHC class II-restricted alpha beta T cell receptor (TCR) transgenic mice. Initial attempts at generating these transgenic mice utilized heterologous regulatory elements to drive the expression of cDNA genes encoding the separate alpha- and beta-chains of the TCR. Unexpectedly, T cells bearing the transgenic alpha beta TCR failed to emerge from the thymus in these mice, although the transgenes did modify endogenous TCR expression. However, subsequent modification of the approach which enabled expression of the TCR beta-chain under the control of its natural regulatory elements generated mice whose peripheral T cells expressed the transgenic TCR and were capable of antigen-dependent proliferation. These results show that successful generation of MHC class II-restricted, OVA-specific alpha beta TCR transgenic mice was dependent upon combining cDNA- and genomic DNA-based constructs for expression of the respective alpha- and beta-chains of the TCR.
Topics: Animals; Cloning, Molecular; DNA, Complementary; Gene Expression; Genes, T-Cell Receptor alpha; Genes, T-Cell Receptor beta; Mice; Mice, Inbred C57BL; Mice, Transgenic; Ovalbumin; Phenotype; Promoter Regions, Genetic; Tumor Cells, Cultured
PubMed: 9553774
DOI: 10.1046/j.1440-1711.1998.00709.x -
Frontiers in Cellular and Infection... 2014Bacteria frequently acquire novel genes by horizontal gene transfer (HGT). HGT through the process of bacterial conjugation is highly efficient and depends on the... (Review)
Review
Bacteria frequently acquire novel genes by horizontal gene transfer (HGT). HGT through the process of bacterial conjugation is highly efficient and depends on the presence of conjugative plasmids (CPs) or integrated conjugative elements (ICEs) that provide the necessary genes for DNA transmission. This review focuses on recent advancements in our understanding of ssDNA transfer systems and regulatory networks ensuring timely and spatially controlled DNA transfer (tra) gene expression. As will become obvious by comparing different systems, by default, tra genes are shut off in cells in which conjugative elements are present. Only when conditions are optimal, donor cells-through epigenetic alleviation of negatively acting roadblocks and direct stimulation of DNA transfer genes-become transfer competent. These transfer competent cells have developmentally transformed into specialized cells capable of secreting ssDNA via a T4S (type IV secretion) complex directly into recipient cells. Intriguingly, even under optimal conditions, only a fraction of the population undergoes this transition, a finding that indicates specialization and cooperative, social behavior. Thereby, at the population level, the metabolic burden and other negative consequences of tra gene expression are greatly reduced without compromising the ability to horizontally transfer genes to novel bacterial hosts. This undoubtedly intelligent strategy may explain why conjugative elements-CPs and ICEs-have been successfully kept in and evolved with bacteria to constitute a major driving force of bacterial evolution.
Topics: Bacterial Physiological Phenomena; Biofilms; Biological Evolution; Conjugation, Genetic; DNA Transposable Elements; DNA, Bacterial; DNA, Single-Stranded; Gene Transfer, Horizontal; Gram-Negative Bacteria; Gram-Positive Bacteria; Host-Pathogen Interactions; Plasmids; Quorum Sensing
PubMed: 24809026
DOI: 10.3389/fcimb.2014.00054 -
Insect Science Jun 2022Zeugodacus tau (Walker) is an invasive pest. The sterile insect technique is an environment-friendly method for pest control. Understanding the mechanism of sex... (Review)
Review
Zeugodacus tau (Walker) is an invasive pest. The sterile insect technique is an environment-friendly method for pest control. Understanding the mechanism of sex determination will contribute to improving efficiency of this technique. In this study, we identified the transformer (tra) gene in Z. tau. One female-specific and two male-specific isoforms of tra were found in Z. tau, and the male-specific splicing pattern of tra was found to occur 5 h after egg laying. We performed transcriptome sequencing at 1 h (E1), 5 h (E5), and 9 h (E9) after egg laying and obtained high-quality transcriptome libraries of early embryo development. We identified 13 297 and 11 713 differentially expressed genes (DEGs) from E5 versus E1 and E9 versus E1 comparisons, respectively. To explore the potential functions of the DEGs during embryonic development, Gene Ontology, Clusters of Orthologous Groups of proteins, and Kyoto Encyclopedia of Genes and Genomes analyses were performed. Twenty-six genes potentially involved in sex determination or differentiation, including Maleness-on-the-Y (MoY), were identified in Z. tau. To verify the transcriptome results, 15 genes were selected for quantitative real-time PCR validation. The results were consistent with the transcriptome sequencing results. Moreover, U2 small nuclear riboprotein auxiliary factor (U2AF-50), female lethal d (fl(2)d), and virilizer (vir) were highly expressed at E5, indicating that they may be related to the sex-specific splicing of tra. Further functional analysis is needed to confirm this speculation. Our data provide an insight into the mechanism underlying sex determination and differentiation in tephritid species.
Topics: Animals; Female; Male; Tephritidae; Transcriptome
PubMed: 34553826
DOI: 10.1111/1744-7917.12974 -
American Journal of Respiratory and... Oct 1997
Review
Topics: Allergens; Asthma; Chromosome Mapping; Chromosomes, Human, Pair 11; Chromosomes, Human, Pair 14; Genes, T-Cell Receptor alpha; Genetic Linkage; HLA-DR Antigens; Humans; Hypersensitivity; Polymorphism, Genetic; Receptors, IgE; Tumor Necrosis Factor-alpha
PubMed: 9351589
DOI: 10.1164/ajrccm.156.4.12-t-6 -
Genes & Development May 1990The transformer-2 (tra-2) gene of Drosophila melanogaster plays essential roles in both sexual differentiation in the female soma and spermatogenesis in the male germ...
The transformer-2 (tra-2) gene of Drosophila melanogaster plays essential roles in both sexual differentiation in the female soma and spermatogenesis in the male germ line. In the female soma, tra-2 is known to act with other genes in the sex determination regulatory cascade to control the sex-specific alternative splicing of transcripts from the doublesex gene. Here, we determine whether or not any sex-specific tra-2 products are expressed that may account for either of these sex-specific activities. Sequence analysis of the tra-2 gene and 10 tra-2 cDNA clones coupled with nuclease protection analysis reveals a variety of alternatively spliced tra-2 mRNAs that each encode one of four distinct but overlapping polypeptides. Three of the encoded polypeptides contain both a ribonucleoprotein consensus sequence and arginine/serine-rich regions, suggesting a direct role for these products in RNA splicing. We show that although two transcripts are expressed male specifically in the germ line, the tra-2 transcripts expressed in the soma are not sex-specific. The translation of products from a tra-2-lacZ fusion gene in both sexes suggests that the female-specific functioning of tra-2 in somatic tissues is not attributable to a translational mechanism. We suggest that tra-2 activity in somatic tissues is regulated through a post-translational sex-specific interaction with the product of the tra gene rather than through the expression of a female-specific tra-2 polypeptide.
Topics: Amino Acid Sequence; Animals; Base Sequence; Drosophila melanogaster; Female; Gene Expression Regulation; Genes; Germ Cells; Male; Molecular Sequence Data; Protein Biosynthesis; RNA Splicing; RNA, Messenger; Recombinant Fusion Proteins; Sex Determination Analysis; Transcription, Genetic
PubMed: 2116360
DOI: 10.1101/gad.4.5.789 -
Immunologic Research 2000The joining of T cell receptor (TCR) and immunoglobulin (Ig) gene segments through the process of V(D)J recombination occurs in a lineage-specific and... (Review)
Review
The joining of T cell receptor (TCR) and immunoglobulin (Ig) gene segments through the process of V(D)J recombination occurs in a lineage-specific and developmental-stage-specific way during the early stages of lymphocyte development. Such developmental regulation is thought to be mediated through the control of gene segment accessibility to the recombinase. We have studied the regulation of V(D)J recombination at the TCR alpha/delta locus, because this locus provides a fascinating model in which distinct sets of gene segments are activated at different stages of T cell development. The transcriptional enhancers Edelta and Ealpha have been implicated as critical regulators that, in conjunction with other cis-acting elements, confer region-specific and developmental-stage-specific changes in gene segment accessibility within TCR alpha/delta locus chromatin. Current work suggests that they may do so by functioning as regional modulators of histone acetylation.
Topics: Acetylation; Animals; Cell Differentiation; Chromatin; Enhancer Elements, Genetic; Gene Rearrangement, alpha-Chain T-Cell Antigen Receptor; Gene Rearrangement, delta-Chain T-Cell Antigen Receptor; Genes, T-Cell Receptor delta; Histones; Humans; Receptors, Antigen, T-Cell, alpha-beta; T-Lymphocytes; Thymus Gland
PubMed: 11339350
DOI: 10.1385/IR:22:2-3:127 -
Genetics May 1992The transformer (tra) gene of Drosophila melanogaster occupies an intermediate position in the regulatory pathway controlling all aspects of somatic sexual... (Comparative Study)
Comparative Study
The transformer (tra) gene of Drosophila melanogaster occupies an intermediate position in the regulatory pathway controlling all aspects of somatic sexual differentiation. The female-specific expression of this gene's function is regulated by the Sex lethal (Sxl) gene, through a mechanism involving sex-specific alternative splicing of tra pre-mRNA. The tra gene encodes a protein that is thought to act in conjunction with the transformer-2 (tra-2) gene product to control the sex-specific processing of doublesex (dsx) pre-mRNA. The bifunctional dsx gene carries out opposite functions in the two sexes, repressing female differentiation in males and repressing male differentiation in females. Here we report the results from an evolutionary approach to investigate tra regulation and function, by isolating the tra-homologous genes from selected Drosophila species, and then using the interspecific DNA sequence comparisons to help identify regions of functional significance. The tra-homologous genes from two Sophophoran subgenus species, Drosophila simulans and Drosophila erecta, and two Drosophila subgenus species, Drosophila hydei and Drosophila virilis, were cloned, sequenced and compared to the D. melanogaster tra gene. This comparison reveals an unusually high degree of evolutionary divergence among the tra coding sequences. These studies also highlight a highly conserved sequence within intron one that probably defines a cis-acting regulator of the sex-specific alternative splicing event.
Topics: Amino Acid Sequence; Animals; Base Sequence; Biological Evolution; Blotting, Southern; DNA; Drosophila melanogaster; Genes, Lethal; Molecular Sequence Data; Nucleic Acid Hybridization; Phenotype; RNA Precursors; RNA Splicing; RNA, Messenger; Restriction Mapping; Sequence Homology, Nucleic Acid; Sex Differentiation; Species Specificity; Transcription, Genetic; Transformation, Genetic
PubMed: 1592233
DOI: 10.1093/genetics/131.1.113 -
PloS One 2013Transformer (TRA) promotes female development in several dipteran species including the Australian sheep blowfly Lucilia cuprina, the Mediterranean fruit fly, housefly...
Transformer (TRA) promotes female development in several dipteran species including the Australian sheep blowfly Lucilia cuprina, the Mediterranean fruit fly, housefly and Drosophila melanogaster. tra transcripts are sex-specifically spliced such that only the female form encodes full length functional protein. The presence of six predicted TRA/TRA2 binding sites in the sex-specific female intron of the L. cuprina gene suggested that tra splicing is auto-regulated as in medfly and housefly. With the aim of identifying conserved motifs that may play a role in tra sex-specific splicing, here we have isolated and characterized the tra gene from three additional blowfly species, L. sericata, Cochliomyia hominivorax and C. macellaria. The blowfly adult male and female transcripts differ in the choice of splice donor site in the first intron, with males using a site downstream of the site used in females. The tra genes all contain a single TRA/TRA2 site in the male exon and a cluster of four to five sites in the male intron. However, overall the sex-specific intron sequences are poorly conserved in closely related blowflies. The most conserved regions are around the exon/intron junctions, the 3' end of the intron and near the cluster of TRA/TRA2 sites. We propose a model for sex specific regulation of tra splicing that incorporates the conserved features identified in this study. In L. sericata embryos, the male tra transcript was first detected at around the time of cellular blastoderm formation. RNAi experiments showed that tra is required for female development in L. sericata and C. macellaria. The isolation of the tra gene from the New World screwworm fly C. hominivorax, a major livestock pest, will facilitate the development of a "male-only" strain for genetic control programs.
Topics: Animals; Base Sequence; Conserved Sequence; Diptera; Female; Genes, Insect; Male; Molecular Sequence Data; Nucleotide Motifs; RNA Splicing; RNA, Messenger; Sex Characteristics
PubMed: 23409170
DOI: 10.1371/journal.pone.0056303