RESUMO
BACKGROUND: Chimeric Antigen Receptor (CAR) T cells are now standard of care (SOC) for some patients with B cell and plasma cell malignancies and could disrupt the therapeutic landscape of solid tumors. However, access to CAR-T cells is not adequate to meet clinical needs, in part due to high cost and long lead times for manufacturing clinical grade virus. Non-viral site directed CAR integration can be accomplished using CRISPR/Cas9 and double-stranded DNA (dsDNA) or single-stranded DNA (ssDNA) via homology-directed repair (HDR), however yields with this approach have been limiting for clinical application (dsDNA) or access to large yields sufficient to meet the manufacturing demands outside early phase clinical trials is limited (ssDNA). METHODS: We applied homology-independent targeted insertion (HITI) or HDR using CRISPR/Cas9 and nanoplasmid DNA to insert an anti-GD2 CAR into the T cell receptor alpha constant (TRAC) locus and compared both targeted insertion strategies in our system. Next, we optimized post-HITI CRISPR EnrichMENT (CEMENT) to seamlessly integrate it into a 14-day process and compared our knock-in with viral transduced anti-GD2 CAR-T cells. Finally, we explored the off-target genomic toxicity of our genomic engineering approach. RESULTS: Here, we show that site directed CAR integration utilizing nanoplasmid DNA delivered via HITI provides high cell yields and highly functional cells. CEMENT enriched CAR T cells to approximately 80% purity, resulting in therapeutically relevant dose ranges of 5.5 × 108-3.6 × 109 CAR + T cells. CRISPR knock-in CAR-T cells were functionally comparable with viral transduced anti-GD2 CAR-T cells and did not show any evidence of off-target genomic toxicity. CONCLUSIONS: Our work provides a novel platform to perform guided CAR insertion into primary human T-cells using nanoplasmid DNA and holds the potential to increase access to CAR-T cell therapies.
Assuntos
DNA , Linfócitos T , Humanos , Reparo de DNA por Recombinação , Imunoterapia AdotivaRESUMO
Murine infection models are needed to develop therapeutics and vaccines to combat the Plasmodium parasites causing malaria. Herein, we describe an easy to perform flow cytometry-based methodology for detecting green fluorescent protein-expressing Plasmodium berghei in the peripheral red blood cells (RBC) of mice. This methodology uses one-step staining and simplified gating strategies to streamline the process of Plasmodium quantification and can detect parasitemia at an earlier time point after infection compared to traditional light microscopy-based techniques.
Assuntos
Malária , Parasitemia , Animais , Citometria de Fluxo/métodos , Malária/diagnóstico , Camundongos , Parasitemia/diagnóstico , Parasitemia/parasitologia , Plasmodium berghei , Coloração e RotulagemRESUMO
Recently, the field of stem cell-based regeneration has turned its attention toward chemical approaches for controlling the pluripotency and differentiation of embryonic stem cells (ESCs) using drug-like small molecule modulators. Growth factor receptors or their associated downstream kinases that regulate intracellular signaling pathways during differentiation are typically the targets for these molecules. The glycocalyx, which plays an essential role in actuating responses to growth factors at the cellular boundary, offers an underexplored opportunity for intervention using small molecules to influence differentiation. Here, we show that surfen, an antagonist of cell-surface glycosaminoglycans required for growth factor association with cognate receptors, acts as a potent and general inhibitor of differentiation and promoter of pluripotency in mouse ESCs. This finding shows that drugging the stem cell Glycome with small molecules to silence differentiation cues can provide a powerful new alternative to existing techniques for controlling stem cell fate. Stem Cells 2018;36:45-54.
Assuntos
Glicosaminoglicanos/metabolismo , Células-Tronco Embrionárias Murinas/metabolismo , Células-Tronco Pluripotentes/metabolismo , Animais , Diferenciação Celular , CamundongosRESUMO
Current drugs against the influenza A virus (IAV) act by inhibiting viral neuraminidase (NA) enzymes responsible for the release of budding virions from sialoglycans on infected cells. Here, we describe an approach focused on a search for inhibitors that reinforce the protective functions of mucosal barriers that trap viruses en route to the target cells. We have generated mimetics of sialo-glycoproteins that insert into the viral envelope to provide a well-defined mucus-like environment encapsulating the virus. By introducing this barrier, which the virus must breach using its NA enzymes to infect a host cell, into a screening platform, we have been able to identify compounds that provide significant protection against IAV infection. This approach may facilitate the discovery of potent new IAV prophylactics among compounds with NA activities too weak to emerge from traditional drug screens.
RESUMO
The initial engagement of host cells by pathogens is often mediated by glycan structures presented on the cell surface. Various components of the glycocalyx can be targeted by pathogens for adhesion to facilitate infection. Glycans also play integral roles in the modulation of the host immune response to infection. Therefore, understanding the parameters that define glycan interactions with both pathogens and the various components of the host immune system can aid in the development of strategies to prevent, interrupt, or manage infection. Glycomaterials provide a unique and powerful tool with which to interrogate the compositional and functional complexity of the glycocalyx. The objective of this review is to highlight some key contributions from this area of research in deciphering the mechanisms of pathogenesis and the associated host response.
Assuntos
Interações Hospedeiro-Patógeno , Imunidade Inata , Polissacarídeos/imunologia , Polissacarídeos/metabolismo , Animais , HumanosRESUMO
Influenza A viruses (IAVs) utilize sialylated host glycans as ligands for binding and infection. The glycan-binding preference of IAV hemagglutinin (HA) is an important determinant of host specificity. Propagation of IAV in embryonated chicken eggs and cultured mammalian cells yields viruses with amino acid substitutions in the HA that can alter the binding specificity. Therefore, it is important to determine the binding specificity of IAV directly in primary samples since it reflects the actual tropism of virus in nature. We developed a novel platform for analysis of IAV binding specificity in samples that contain very low virus titers. This platform consists of a high-density flexible glycan display on magnetic beads, which promotes multivalent interactions with the viral HA. Glycan-bound virus is detected by quantifying the viral neuraminidase activity via a fluorogenic reporter, 2'-(4-methylumbelliferyl)-α-d-N-acetylneuraminic acid. This method eliminates the need for labeling the virus and significantly enhances the sensitivity of detection.
Assuntos
Cloaca/virologia , Vírus da Influenza A/isolamento & purificação , Microesferas , Polissacarídeos/metabolismo , Animais , Cães , Patos/virologia , Vírus da Influenza A/metabolismo , Células Madin Darby de Rim Canino , Magnetismo , Mucinas , Neuraminidase/metabolismo , Sensibilidade e Especificidade , Proteínas Virais/metabolismoRESUMO
Influenza viruses bind to mucosal glycans to gain entry into a host organism and initiate infection. The target glycans are often displayed in multivalent arrangements on proteins; however, how glycan presentation influences viral specificity is poorly understood. Here, we report a microarray platform approximating native glycan display to facilitate such studies.
Assuntos
Análise em Microsséries/métodos , Orthomyxoviridae/química , Orthomyxoviridae/metabolismo , Polissacarídeos/análise , Polissacarídeos/metabolismo , Estrutura Molecular , Polissacarídeos/químicaRESUMO
Through its transcriptional activities, the proto-oncoprotein c-Jun can regulate cellular proliferation, survival, and differentiation. We have established a novel yeast assay that screens for repressors of c-Jun transcriptional activity. This screen led to the identification of a ubiquitously expressed novel RING zinc finger protein, termed Makorin RING zinc finger protein 1 (MKRN1), recently shown to act as an E3 ubiquitin ligase. Overexpression of MKRN1 in mammalian cells inhibited the transcriptional activities of not only c-Jun, but also the nuclear receptors, the androgen receptor, and the retinoic acid receptors. Truncation analysis indicates that both the amino and carboxy termini are required for this transrepression activity. Surprisingly, when fused to the heterologous DNAbinding domain of GAL4, MKRN1 activates, rather than inhibits, a GAL4-responsive reporter plasmid. In addition, truncation of either the amino- or carboxy-terminal half of MKRN1 disrupts its transactivation activity, the same observation that was made on its transrepression activity. These results demonstrate that MKRN1 has transcriptional activity and suggest that its transrepression and transactivation functions are mediated by the same mechanism. Interestingly, disruption of MKRN1's ubiquitin ligase activity does not affect its inhibitory transcriptional activity. Thus, MKRN1 may represent a nuclear protein with multiple nuclear functions, including regulating RNA polymerase II-catalyzed transcription.
Assuntos
Proteínas do Tecido Nervoso/metabolismo , RNA Polimerase II/metabolismo , Ribonucleoproteínas/metabolismo , Transcrição Gênica , Animais , Antígenos/metabolismo , Células COS , Chlorocebus aethiops , Proteínas de Ligação a DNA/metabolismo , Regulação para Baixo , Células HeLa , Humanos , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas c-jun/metabolismo , Receptores do Ácido Retinoico/antagonistas & inibidores , Saccharomyces cerevisiae/genética , Fator de Transcrição AP-1/antagonistas & inibidores , Técnicas do Sistema de Duplo-Híbrido , Ubiquitina-Proteína Ligases/metabolismoRESUMO
We describe a patient with chronic progressive external ophthalmoplegia (CPEO) due to a deletion of mitochondrial DNA (mtDNA) who had electromyographic evidence of myotonic discharges. Myotonia has not previously been described in association with mitochondrial disease and this report extends the known phenotypic expression of these disorders.