Gene editing strategies for cystic fibrosis are challenged by the complex barrier properties of airway epithelia. We previously reported that the amphiphilic S10 shuttle peptide non-covalently ...combined with CRISPR-associated (Cas) ribonucleoprotein (RNP) enabled editing of human and mouse airway epithelial cells. Here, we derive the S315 peptide as an improvement over S10 in delivering base editor RNP. Following intratracheal aerosol delivery of Cy5-labeled peptide in rhesus macaques, we confirm delivery throughout the respiratory tract. Subsequently, we target CCR5 with co-administration of ABE8e-Cas9 RNP and S315. We achieve editing efficiencies of up-to 5.3% in rhesus airway epithelia. Moreover, we document persistence of edited epithelia for up to 12 months in mice. Finally, delivery of ABE8e-Cas9 targeting the CFTR R553X mutation restores anion channel function in cultured human airway epithelia. These results demonstrate the therapeutic potential of base editor delivery with S315 to functionally correct the CFTR R553X mutation in respiratory epithelia.
Natural Killer (NK) cells hold the potential to shift cell therapy from a complex autologous option to a universal off-the-shelf one. Although NK cells have demonstrated efficacy and safety in the ...treatment of leukemia, the limited efficacy of NK cell-based immunotherapies against solid tumors still represents a major hurdle. In the immunosuppressive tumor microenvironment (TME), inhibitory interactions between cancer and immune cells impair antitumoral immunity.
gene encodes the NK cell inhibitory receptor NKG2A, which is a potent NK cell immune checkpoint. NKG2A specifically binds HLA-E, a non-classical HLA class I molecule frequently overexpressed in tumors, leading to the transmission of inhibitory signals that strongly impair NK cell function.
To restore NK cell cytotoxicity against HLA-E
tumors, we have targeted the NKG2A/HLA-E immune checkpoint by using a CRISPR-mediated
gene editing.
knockout resulted in a reduction of 81% of NKG2A
cell frequency in
expanded human NK cells post-cell sorting.
, the overexpression of HLA-E by tumor cells significantly inhibited wild-type (WT) NK cell cytotoxicity with
-values ranging from 0.0071 to 0.0473 depending on tumor cell lines. In contrast,
NK cells exhibited significantly higher cytotoxicity when compared to WT NK cells against four different HLA-E
solid tumor cell lines, with
-values ranging from<0.0001 to 0.0154. Interestingly, a proportion of 43.5% to 60.2% of NKG2A
NK cells within the edited NK cell population was sufficient to reverse at its maximum the HLA-E-mediated inhibition of NK cell cytotoxicity. The expression of the activating receptor NKG2C was increased in
NK cells and contributed to the improved NK cell cytotoxicity against HLA-E
tumors.
, the adoptive transfer of human
NK cells significantly delayed tumor progression and increased survival in a xenogeneic mouse model of HLA-E
metastatic breast cancer, as compared to WT NK cells (
= 0.0015).
Our results demonstrate that
knockout is an effective strategy to improve NK cell antitumor activity against HLA-E
tumors and could be applied in the development of NK cell therapy for solid tumors.
The androgen receptor (AR) is an established orchestrator of cell metabolism in prostate cancer (PCa), notably by inducing an oxidative mitochondrial program. Intriguingly, AR regulates cytoplasmic ...isocitrate dehydrogenase 1 (IDH1), but not its mitochondrial counterparts IDH2 and IDH3. Here, we aimed to understand the functional role of IDH1 in PCa. Mouse models, in vitro human PCa cell lines, and human patient-derived organoids (PDOs) were used to study the expression and activity of IDH enzymes in the normal prostate and PCa. Genetic and pharmacological inhibition of IDH1 was then combined with extracellular flux analyses and gas chromatography-mass spectrometry for metabolomic analyses and cancer cell proliferation in vitro and in vivo. In PCa cells, more than 90% of the total IDH activity is mediated through IDH1 rather than its mitochondrial counterparts. This profile seems to originate from the specialized prostate metabolic program, as observed using mouse prostate and PDOs. Pharmacological and genetic inhibition of IDH1 impaired mitochondrial respiration, suggesting that this cytoplasmic enzyme contributes to the mitochondrial tricarboxylic acid cycle (TCA) in PCa. Mass spectrometry-based metabolomics confirmed this hypothesis, showing that inhibition of IDH1 impairs carbon flux into the TCA cycle. Consequently, inhibition of IDH1 decreased PCa cell proliferation in vitro and in vivo. These results demonstrate that PCa cells have a hybrid cytoplasmic-mitochondrial TCA cycle that depends on IDH1. This metabolic enzyme represents a metabolic vulnerability of PCa cells and a potential new therapeutic target.
The non-nucleoside reverse transcriptase inhibitor efavirenz (EFV) is directly conjugated by the UDP-glucuronosyltransferase (UGT) pathway to form EFV-N-glucuronide (EFV-G), but the enzyme(s) ...involved has not yet been identified. The glucuronidation of EFV was screened with UGT1A and UGT2B enzymes expressed in a heterologous system, and UGT2B7 was shown to be the only reactive enzyme. The apparent K(m) value of UGT2B7 (21 microM) is similar to the value observed for human liver microsomes (24 microM), whereas the variant allozyme UGT2B7*2 (Tyr(268)) displayed similar kinetic parameters. Because 3'-azido-3'-deoxythymidine (AZT), one of the most current nucleotide reverse transcriptase inhibitors prescribed in combination with EFV, is also conjugated by UGT2B7, the potential metabolic interaction between EFV and AZT has been studied using human liver microsomes. Glucuronidation of both drugs was inhibited by one another, in a concentration-dependent manner. At K(m) values (25 and 1000 microM for EFV and AZT, respectively), EFV inhibited AZT glucuronidation by 47%, whereas AZT inhibited EFV glucuronidation by 23%. With a K(i) value of 17 microM for AZT-glucuronide formation, EFV appears to be one of the most selective and potent competitive inhibitor of AZT glucuronidation in vitro. Moreover, assuming that concentrations of EFV achieved in plasma (C(max) = 12.9 microM) are in a range similar to its K(i) value, it was estimated that EFV could produce a theoretical 43% inhibition of AZT glucuronidation in vivo. We conclude that UGT2B7 has a major role in EFV glucuronidation and that EFV could potentially interfere with the hepatic glucuronidation of AZT.
The prostate is metabolically unique: it produces high levels of citrate for secretion via a truncated tricarboxylic acid (TCA) cycle to maintain male fertility. In prostate cancer (PCa), this ...phenotype is reprogrammed, making it an interesting therapeutic target. However, how the truncated prostate TCA cycle works is still not completely understood.
We optimized targeted metabolomics in mouse and human organoid models in ex vivo primary culture. We then used stable isotope tracer analyses to identify the pathways that fuel citrate synthesis.
First, mouse and human organoids were shown to recapitulate the unique citrate-secretory program of the prostate, thus representing a novel model that reproduces this unusual metabolic profile. Using stable isotope tracer analysis, several key nutrients were shown to allow the completion of the prostate TCA cycle, revealing a much more complex metabolic profile than originally anticipated. Indeed, along with the known pathway of aspartate replenishing oxaloacetate, glutamine was shown to fuel citrate synthesis through both glutaminolysis and reductive carboxylation in a GLS1-dependent manner. In human organoids, aspartate entered the TCA cycle at the malate entry point, upstream of oxaloacetate. Our results demonstrate that the citrate-secretory phenotype of prostate organoids is supported by the known aspartate–oxaloacetate–citrate pathway, but also by at least three additional pathways: glutaminolysis, reductive carboxylation, and aspartate–malate conversion.
Our results add a significant new dimension to the prostate citrate-secretory phenotype, with at least four distinct pathways being involved in citrate synthesis. Better understanding this distinctive citrate metabolic program will have applications in both male fertility as well as in the development of novel targeted anti-metabolic therapies for PCa.
•Targeted metabolomics and stable isotope tracer analysis were optimized in mouse and human prostate organoids.•Organoids recapitulate the unique citrate-secretory phenotype of the prostate.•Glutamine fuels citrate synthesis for secretion by glutaminolysis and reductive carboxylation.•Aspartate enters the TCA cycle at different entry points in mouse and human prostate organoids for citrate production.•We revealed a much more complex TCA cycle in the prostate than originally anticipated.
UDP-glucuronosyltransferase 1A1 (UGT1A1) is a pivotal enzyme involved in metabolism of SN-38, the active metabolite of irinotecan commonly used to treat metastatic colorectal cancer. We previously ...demonstrated aberrant methylation of specific CpG dinucleotides in UGT1A1-negative cells, and revealed that methylation state of the UGT1A1 5'-flanking sequence is negatively correlated with gene transcription. Interestingly, one of these CpG dinucleotides (CpG -4) is found close to a HNF1 response element (HRE), known to be involved in activation of UGT1A1 gene expression, and within an upstream stimulating factor (USF) binding site.
Gel retardation assays revealed that methylation of CpG-4 directly affect the interaction of USF1/2 with its cognate sequence without altering the binding for HNF1-alpha. Luciferase assays sustained a role for USF1/2 and HNF1-alpha in UGT1A1 regulation in colon cancer cells. Based on the differential expression profiles of HNF1A gene in colon cell lines, we also assessed whether methylation affects its expression. In agreement with the presence of CpG islands in the HNF1A promoter, treatments of UGT1A1-negative HCT116 colon cancer cells with a DNA methyltransferase inhibitor restore HNF1A gene expression, as observed for UGT1A1.
This study reveals that basal UGT1A1 expression in colon cells is positively regulated by HNF1-alpha and USF, and negatively regulated by DNA methylation. Besides, DNA methylation of HNF1A could also play an important role in regulating additional cellular drug metabolism and transporter pathways. This process may contribute to determine local inactivation of drugs such as the anticancer agent SN-38 by glucuronidation and define tumoral response.
This study investigated the molecular mechanisms underlying the regulatory effect of the newly discovered 45-kDa enzymatically inactive UGT1A spliced polypeptides, named isoform i2, upon ...UGT1A-mediated glucuronidation. Initially, using an inducible system that mimics the relative abundance of isoforms 1 and 2 of UGT1A1 in human tissues, the rates of formation of glucuronides were significantly reduced. We then used a heterologous system constitutively expressing both isoforms i1 and i2 for an in-depth investigation of the presence of spliced i2 on glucuronidation kinetics. UGT1A1, UGT1A7, and UGT1A8 were selected as candidates for these studies. In all cases, co-expression of i1 and i2 in HEK293 cells leads to a significant reduction of the velocity of the glucuronidation reaction without affecting the affinity (Kmapp) for all substrates tested and the Km for the co-substrate, UDP-glucuronic acid. The data are consistent with a dominant-negative model of inhibition but do not sustain with an UGT1A_i2-mediated inhibition by competitive binding for substrate or the co-substrate. In contrast, the data from the co-immunoprecipitation experiments are indicative of the existence of a mixture homo-oligomeric (i1-i1 or i2-i2) and hetero-oligomeric (i1-i2) complexes in which the i2-i2 and i1-i2 subunits would be inactive. Thus, protein-protein interactions are likely responsible for the inhibition of active UGT1A_i1 by i2 spliced polypeptides. This new regulatory mechanism may alternatively modulate cellular response to endo/xeno stimulus.
In the way of development Blaser, Mario; Feit, Harvey A; McRae, Glenn
In the way of development,
2004, 2014, 2006, 2013-07-04
eBook
Indigenous peoples today are enmeshed in the expanding modern economy, subject to the pressures of both market and government. This book takes indigenous peoples as actors, not victims, as its ...starting point in analysing this interaction. It assembles a rich diversity of statements, case studies and wider thematic explorations, primarily from North America, and particularly the Cree, the Haudenausaunee (Iroquois) and Chippewa-Ojibwe peoples who straddle the US/Canadian border, but also from South America and the former Soviet Union. It explores the complex relationships between indigenous peoples, civil society, and the environment. It shows how the boundaries between indigenous peoples' organizations, civil society, the state, markets, development and the environment are ambiguous and constantly changing. These complexities create both opportunities and threats for local agency. People resist or react to the pressures of market and state, while sustaining 'life projects' of their own, embodying their own local history, visions and strategies.
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