RESUMO
Microsatellite expansions are the cause of >20 neurological or developmental human disorders. Shortening expanded repeats using specific DNA endonucleases may be envisioned as a gene editing approach. Here, we measured the efficacy of several CRISPR-Cas nucleases to induce recombination within disease-related microsatellites, in Saccharomyces cerevisiae. Broad variations in nuclease performances were detected on all repeat tracts. Wild-type Streptococcus pyogenes Cas9 (SpCas9) was more efficient than Staphylococcus aureus Cas9 on all repeats tested, except (CAG)33. Cas12a (Cpf1) was the most efficient on GAA trinucleotide repeats, whereas GC-rich repeats were more efficiently cut by SpCas9. The main genetic factor underlying Cas efficacy was the propensity of the recognition part of the sgRNA to form a stable secondary structure, independently of its structural part. This suggests that such structures form in vivo and interfere with sgRNA metabolism. The yeast genome contains 221 natural CAG/CTG and GAA/CTT trinucleotide repeats. Deep sequencing after nuclease induction identified three of them as carrying statistically significant low frequency mutations, corresponding to SpCas9 off-target double-strand breaks.
Assuntos
Sistemas CRISPR-Cas/genética , Endonucleases/genética , Doenças Genéticas Inatas/genética , Repetições de Microssatélites/genética , Edição de Genes , Humanos , Mutação/genética , Streptococcus pyogenes/enzimologia , Streptococcus pyogenes/genética , Repetições de Trinucleotídeos/genéticaRESUMO
Colibactin-producing E. coli (CoPEC) are frequently detected in colorectal cancer (CRC) and exhibit procarcinogenic properties. Because increasing evidence show the role of immune environment and especially of antitumor T-cells in CRC development, we investigated the impact of CoPEC on these cells in human CRC and in the APCMin/+ mice colon. T-cell density was evaluated by immunohistochemistry in human tumors known for their CoPEC status. APCmin/+ mice were chronically infected with a CoPEC strain (11G5). Immune cells (neutrophils and T-cell populations) were then quantified by immunofluorescent staining of the colon. The quantification of lymphoid populations was also performed in the mesenteric lymph nodes (MLNs). Here, we show that the colonization of CRC patients by CoPEC is associated with a decrease of tumor-infiltrating T lymphocytes (CD3+ T-cells). Similarly, we demonstrated, in mice, that CoPEC chronic infection decreases CD3+ and CD8+ T-cells and increases colonic inflammation. In addition, we noticed a significant decrease in antitumor T-cells in the MLNs of CoPEC-infected mice compared to that of controls. Moreover, we show that CoPEC infection decreases the antimouse PD-1 immunotherapy efficacy in MC38 tumor model. Our findings suggest that CoPEC could promote a procarcinogenic immune environment through impairment of antitumor T-cell response, leading to tumoral resistance to immunotherapy. CoPEC could thus be a new biomarker predicting the anti-PD-1 response in CRC.
Assuntos
Neoplasias do Colo/terapia , Resistencia a Medicamentos Antineoplásicos/imunologia , Escherichia coli/metabolismo , Linfócitos do Interstício Tumoral/imunologia , Peptídeos/metabolismo , Policetídeos/metabolismo , Animais , Linfócitos T CD8-Positivos/imunologia , Neoplasias do Colo/patologia , Feminino , Humanos , Imunoterapia/métodos , Contagem de Linfócitos , Camundongos , Camundongos Endogâmicos C57BL , Neutrófilos/imunologia , Receptor de Morte Celular Programada 1 , Microambiente Tumoral/imunologiaRESUMO
The search for new antibodies is a major field of pharmaceutical research that remains lengthy and costly due to the need for successive library screenings. Existing in vitro and in vivo antibody discovery processes require that libraries are repeatedly subcloned to switch the antibody format or the secretory host, a resource-intensive process. There is an urgent need for an antibody identification platform capable of screening large antibody libraries in their final soluble format. Previous attempts to develop such a platform have struggled to combine large antibody libraries with screening of high specificity, while retaining sufficient library diversity coverage (ability to detect rare events). Here, we describe a new antibody screening platform based on the encapsulation of antibody secreting yeast cells into picoreactor droplets. We developed and optimized a Yarrowia lipolytica yeast strain capable of growing and secreting full-length human IgGs in picoreactors, and applied a microfluidics-based high-throughput screening approach to sort and recover target-specific antibody-secreting yeasts. Critically, the direct recovery of secretory yeasts allows for downstream screening and antibody characterization, without the need to reformat or subclone the coding sequences. We successfully increased the diversity coverage of sorting the antibody library without compromising sorting specificity by developing a new fluorescence signal processing methodology. By combining this drastically enhanced sorting efficiency with the high-throughput capability of droplet microfluidics, and the rapid growth of Y. lipolytica, our new platform is capable of screening millions of antibodies per day and enriching for target-specific ones in 4 days. This platform will enable the efficient screening of antibody libraries in a variety of contexts, including primary screening of synthetic libraries, affinity maturation, and identification of multi-specific or cross-reactive antibodies.
Assuntos
Imunoglobulina G , Microfluídica , Humanos , Transporte Proteico , Microfluídica/métodos , Células Clonais , Biblioteca GênicaRESUMO
Cells can repair a double-strand break (DSB) by homologous recombination if a homologous sequence is provided as a template. This can be achieved by classical gene conversion (with or without crossover) or by single-strand annealing (SSA) between two direct repeat sequences flanking the DSB. To initiate SSA, single-stranded regions are needed adjacent to the break, extending up to the direct repeats in such a way that complementary strands can anneal to each other to repair the DSB. In the present protocol, we describe a GFP reporter assay in Saccharomyces cerevisiae allowing for the quantification of nuclease efficacy at inducing a DSB, by monitoring the reconstitution of a functional GFP gene whose expression can be rapidly quantified by flow cytometry.
Assuntos
Proteínas de Fluorescência Verde/metabolismo , Saccharomyces cerevisiae/crescimento & desenvolvimento , Repetições de Trinucleotídeos , Quebras de DNA de Cadeia Dupla , Citometria de Fluxo , Genes Reporter , Proteínas de Fluorescência Verde/genética , Reparo de DNA por Recombinação , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismoRESUMO
Because of their favorable properties as macromolecular drugs, antibodies are a very successful therapeutic modality for interfering with disease-relevant targets in the extracellular space or at the cell membrane. However, a large number of diseases involve cytosolic targets and designing antibodies able to efficiently reach intracellular compartments would expand the antibody-tractable conditions. Here, we genetically fused cell penetrating peptides (CPPs) at various positions to an antibody targeting cancer cells, evaluated the developability features of the resulting antibody-peptide fusions and the ability of selected constructs to reach the cytosol. We first determined positions in the IgG structure that were permissive to CPP incorporation without destabilizing the antibody. Fusing CPPs to the C-terminus of the light chain and either before or after the hinge had the least effect on antibody developability features. These constructs were further evaluated for cell penetration efficiency. Two out of five tested CPPs significantly enhanced antibody penetration into the cytosol, in particular when fused before or after the hinge. Finally, we demonstrate that specific antibody binding to the cell surface target is necessary for efficient cell penetration of the CPP-antibody fusions. This study provides a solid basis for further exploration of therapeutic antibodies for intracellular targets.
Assuntos
Anticorpos/administração & dosagem , Peptídeos Penetradores de Células/administração & dosagem , Sistemas de Liberação de Medicamentos , Animais , Antígeno Carcinoembrionário/química , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Separação Celular , Citoplasma/metabolismo , Citosol/metabolismo , Espaço Extracelular , Citometria de Fluxo , Proteínas Ligadas por GPI/química , Proteínas de Fluorescência Verde/química , Células HEK293 , Humanos , Imunoglobulina G/administração & dosagem , Camundongos , Microscopia de Fluorescência , Ligação Proteica , Domínios Proteicos , Transporte Proteico , Proteínas Recombinantes de Fusão/administração & dosagem , Ressonância de Plasmônio de SuperfícieRESUMO
We report on the production of hydrocortisone, the major adrenal glucocorticoid of mammals and an important intermediate of steroidal drug synthesis, from a simple carbon source by recombinant Saccharomyces cerevisiae strains. An artificial and fully self-sufficient biosynthetic pathway involving 13 engineered genes was assembled and expressed in a single yeast strain. Endogenous sterol biosynthesis was rerouted to produce compatible sterols to serve as substrates for the heterologous part of the pathway. Biosynthesis involves eight mammalian proteins (mature forms of CYP11A1, adrenodoxin (ADX), and adrenodoxin reductase (ADR); mitochondrial forms of ADX and CYP11B1; 3beta-HSD, CYP17A1, and CYP21A1). Optimization involved modulating the two mitochondrial systems and disrupting of unwanted side reactions associated with ATF2, GCY1, and YPR1 gene products. Hydrocortisone was the major steroid produced. This work demonstrates the feasibility of transfering a complex biosynthetic pathway from higher eukaryotes into microorganisms.
Assuntos
Carbono/metabolismo , Engenharia Genética/métodos , Hidrocortisona/biossíntese , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Animais , Bovinos , Clonagem Molecular , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Etanol/metabolismo , Regulação Fúngica da Expressão Gênica , Glucose/metabolismo , Humanos , Hidrocortisona/genética , Complexos Multienzimáticos/genética , Complexos Multienzimáticos/metabolismo , Controle de Qualidade , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Recombinação Genética , Saccharomyces cerevisiae/classificação , Especificidade da EspécieRESUMO
Various important biological pathways are modulated by TGFß isoforms; as such they are potential targets for therapeutic intervention. Fresolimumab, also known as GC1008, is a pan-TGFß neutralizing antibody that has been tested clinically for several indications including an ongoing trial for focal segmental glomerulosclerosis. The structure of the antigen-binding fragment of fresolimumab (GC1008 Fab) in complex with TGFß3 has been reported previously, but the structural capacity of fresolimumab to accommodate tight interactions with TGFß1 and TGFß2 was insufficiently understood. We report the crystal structure of the single-chain variable fragment of fresolimumab (GC1008 scFv) in complex with target TGFß1 to a resolution of 3.00 Å and the crystal structure of GC1008 Fab in complex with TGFß2 to 2.83 Å. The structures provide further insight into the details of TGFß recognition by fresolimumab, give a clear indication of the determinants of fresolimumab pan-specificity and provide potential starting points for the development of isoform-specific antibodies using a fresolimumab scaffold.
Assuntos
Anticorpos Monoclonais/química , Anticorpos Monoclonais/imunologia , Especificidade de Anticorpos/imunologia , Reações Antígeno-Anticorpo/imunologia , Fator de Crescimento Transformador beta/química , Fator de Crescimento Transformador beta/imunologia , Anticorpos Monoclonais Humanizados , Anticorpos Neutralizantes/química , Anticorpos Neutralizantes/imunologia , Cristalografia por Raios X , Humanos , Fragmentos Fab das Imunoglobulinas/química , Fragmentos Fab das Imunoglobulinas/imunologia , Modelos Moleculares , Conformação Proteica , Isoformas de Proteínas/química , Isoformas de Proteínas/imunologia , Anticorpos de Cadeia Única/química , Anticorpos de Cadeia Única/imunologiaAssuntos
Hidrocortisona/biossíntese , Organismos Geneticamente Modificados/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/fisiologia , Animais , Vias Biossintéticas/genética , Humanos , Mitocôndrias/genética , Mitocôndrias/metabolismo , Modelos Biológicos , Fenômenos Fisiológicos Vegetais , Engenharia de Proteínas/tendências , Biologia de SistemasRESUMO
Inspired by the successful work of converting Saccharomyces cerevisiae into an microorganism capable of synthesizing hydrocortisone, a 27-carbon molecule, from ethanol, a 2-carbon molecule, this review provides an overview of the potential of yeast as a recombinant organism in the 21st century. Yeast has been used by man for more than 6,000 years, and is still paving the way to new discoveries. It was the first eukaryotic organism to be sequenced, in 1996, and the first to produce hydrocortisone in 2003. In addition, extensive genome-wide analyses have been performed with yeast. In this review, we discuss the pros and cons of using yeast to produce small therapeutic molecules. It is obvious that S. cerevisiae has a cutting edge advantage of being a well-known organism and time will tell if yeast "biohydrocortisone" is a unique example or the beginning of a long list of yeast bioproducts. Other organisms, such as plants and bacteria, are competing with yeast. Bacteria produce a wealth of marketed molecules and plants are capable of producing extremely complex molecules with an unbeatable yield. However, S. cerevisiae offers a unique mix of the simplicity of a recombinant organism combined with the complexity of a eukaryote.
Assuntos
Biotecnologia/tendências , Desenho de Fármacos , Indústria Farmacêutica/tendências , Engenharia Genética/métodos , Hidrocortisona/genética , Hidrocortisona/metabolismo , Saccharomyces cerevisiae/metabolismo , Biotecnologia/métodos , Indústria Farmacêutica/métodos , Microbiologia Industrial/métodos , Microbiologia Industrial/tendências , Saccharomyces cerevisiae/genéticaRESUMO
Engineered yeast cells efficiently convert ergosta-5-eneol to pregnenolone and progesterone provided that endogenous pregnenolone acetylase activity is disrupted and that heterologous sterol delta7-reductase, cytochrome P450 side chain cleavage (CYP11A1) and 3beta hydroxysteroid dehydrogenase/isomerase (3beta-HSD) activities are present. CYP11A1 activity requires the expression of the mammalian NADPH-adrenodoxin reductase (Adrp) and adrenodoxin (Adxp) proteins as electron carriers. Several parameters modulate this artificial metabolic pathway: the effects of steroid products; the availability and delivery of the ergosta-5-eneol substrate to cytochrome P450; electron flux and protein localization. CYP11A1, Adxp and Adrp are usually located in contact with inner mitochondrial membranes and are directed to the outside of the mitochondria by the removal of their respective mitochondrial targeting sequences. CYP11A1 then localizes to the plasma membrane but Adrp and Adxp are detected in the endoplasmic reticulum and cytosol as expected. The electron transfer chain that involves several subcellular compartments may control side chain cleavage activity in yeast. Interestingly, Tgl1p, a potential ester hydrolase, was found to enhance steroid productivity, probably through both the availability and/or the trafficking of the CYP11A1 substrate. Thus, the observation that the highest cellular levels of free ergosta-5-eneol are found in the plasma membrane suggests that the substrate is freely available for pregnenolone synthesis.
Assuntos
Membrana Celular/metabolismo , Enzima de Clivagem da Cadeia Lateral do Colesterol/metabolismo , Mitocôndrias/enzimologia , Saccharomyces cerevisiae/metabolismo , Esteroides/metabolismo , 3-Hidroxiesteroide Desidrogenases/genética , 3-Hidroxiesteroide Desidrogenases/metabolismo , Adrenodoxina/genética , Adrenodoxina/metabolismo , Animais , Arabidopsis/enzimologia , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Bovinos , Compartimento Celular/fisiologia , Colesterol/análogos & derivados , Colesterol/metabolismo , Enzima de Clivagem da Cadeia Lateral do Colesterol/genética , Ativação Enzimática/fisiologia , Ergosterol/análogos & derivados , Ergosterol/metabolismo , Esterases/metabolismo , Ferredoxina-NADP Redutase/genética , Ferredoxina-NADP Redutase/metabolismo , Humanos , Mamíferos , Oxirredução , Oxirredutases/genética , Oxirredutases/metabolismo , Reguladores de Crescimento de Plantas/genética , Reguladores de Crescimento de Plantas/metabolismo , Pregnenolona/biossíntese , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
The A variant of bovine alpha(s1) casein is devoid of the segment of 13 amino acid residues which occupies the 14th to 26th position from the NH(2)-terminal in the polypeptide chain (198 residues) of the B and C variants.