Showing papers by "University of California published in 2023"

Equilibrated Gas and Carbonate Standard‐Derived Dual (Δ ₄₇ and Δ ₄₈ ) Clumped Isotope Values

Abstract: Carbonate clumped isotope geochemistry has primarily focused on mass spectrometric determination of m/z 47 CO2 for geothermometry, but theoretical calculations and recent experiments indicate paired analysis of the m/z 47 (13C18O16O) and m/z 48 (12C18O18O) isotopologues (referred to as Δ47 and Δ48) can be used to study non-equilibrium isotope fractionations and refine temperature estimates. We utilize 5,448 Δ47 and 3,400 Δ48 replicate measurements of carbonate samples and standards, and 183 Δ47 and 195 Δ48 replicate measurements of gas standards from 2015 to 2021 from a multi-year and multi-instrument data set to constrain Δ47 and Δ48 values for 27 samples and standards, including Devils Hole cave calcite, and study equilibrium Δ47-Δ48, Δ47-temperature, and Δ48-temperature relationships. We compare results to previously published findings and calculate equilibrium regressions based on data from multiple laboratories. We report acid digestion fractionation factors, Δ*63-47 and Δ*64-48, and account for their dependence on the initial clumped isotope values of the mineral.

Electrochemical Oxidation of Methane to Methanol on Electrodeposited Transition Metal Oxides

Abstract: Electrochemical partial oxidation of methane to methanol is a promising approach to the transformation of stranded methane resources into a high-value, easy-to-transport fuel or chemical. Transition metal oxides are potential electrocatalysts for this transformation. However, a comprehensive and systematic study of the dependence of methane activation rates and methanol selectivity on catalyst morphology and experimental operating parameters has not been realized. Here, we describe an electrochemical method for the deposition of a family of thin-film transition metal (oxy)hydroxides as catalysts for the partial oxidation of methane. CoOx, NiOx, MnOx, and CuOx are discovered to be active for the partial oxidation of methane to methanol. Taking CoOx as a prototypical methane partial oxidation electrocatalyst, we systematically study the dependence of activity and methanol selectivity on catalyst film thickness, overpotential, temperature, and electrochemical cell hydrodynamics. Optimal conditions of low catalyst film thickness, intermediate overpotentials, intermediate temperatures, and fast methanol transport are identified to favor methanol selectivity. Through a combination of control experiments and DFT calculations, we show that the oxidized form of the as-deposited (oxy)hydroxide catalyst films are active for the thermal oxidation of methane to methanol even without the application of bias potential, demonstrating that high valence transition metal oxides are intrinsically active for the activation and oxidation of methane to methanol at ambient temperatures. Calculations uncover that electrocatalytic oxidation enables reaching an optimum potential window in which methane activation forming methanol and methanol desorption are both thermodynamically favorable, methanol desorption being favored by competitive adsorption with hydroxide anion.

Treatment of prostate cancer with CD46 targeted 225Ac alpha particle radioimmunotherapy

Abstract: Radiopharmaceutical therapy is changing the standard of care in prostate cancer and other malignancies. We previously reported high CD46 expression in prostate cancer and developed an antibody-drug conjugate and immunoPET agent based on the YS5 antibody, which targets a tumor-selective CD46 epitope. Here, we present the preparation, preclinical efficacy, and toxicity evaluation of [225Ac]DOTA-YS5, a radioimmunotherapy agent based on the YS5 antibody.[225Ac]DOTA-YS5 was developed, and its therapeutic efficiency was tested on cell-derived (22Rv1, DU145), and patient-derived (LTL-545, LTL484) prostate cancer xenograft models. Biodistribution studies were carried out on 22Rv1 tumor xenograft models to confirm the targeting efficacy. Toxicity analysis of the [225Ac]DOTA-YS5 was carried out on nu/nu mice to study short-term (acute) and long-term (chronic) toxicity.Biodistribution study shows that [225Ac]DOTA-YS5 agent delivers high levels of radiation to the tumor tissue (11.64% ± 1.37%ID/g, 28.58% ± 10.88%ID/g, 29.35% ± 7.76%ID/g, and 31.78% ± 5.89%ID/g at 24, 96, 168, and 408 hours, respectively), compared with the healthy organs. [225Ac]DOTA-YS5 suppressed tumor size and prolonged survival in cell line-derived and patient-derived xenograft models. Toxicity analysis revealed that the 0.5 μCi activity levels showed toxicity to the kidneys, likely due to redistribution of daughter isotope 213Bi.[225Ac]DOTA-YS5 suppressed the growth of cell-derived and patient-derived xenografts, including prostate-specific membrane antigen-positive and prostate-specific membrane antigen-deficient models. Overall, this preclinical study confirms that [225Ac]DOTA-YS5 is a highly effective treatment and suggests feasibility for clinical translation of CD46-targeted radioligand therapy in prostate cancer.

Data from Treatment of Prostate Cancer with CD46-targeted <sup>225</sup>Ac Alpha Particle Radioimmunotherapy

Abstract: <div>AbstractPurpose:<p>Radiopharmaceutical therapy is changing the standard of care in prostate cancer and other malignancies. We previously reported high CD46 expression in prostate cancer and developed an antibody–drug conjugate and immunoPET agent based on the YS5 antibody, which targets a tumor-selective CD46 epitope. Here, we present the preparation, preclinical efficacy, and toxicity evaluation of [²²⁵Ac]DOTA-YS5, a radioimmunotherapy agent based on the YS5 antibody.</p>Experimental Design:<p>[²²⁵Ac]DOTA-YS5 was developed, and its therapeutic efficiency was tested on cell-derived (22Rv1, DU145), and patient-derived (LTL-545, LTL484) prostate cancer xenograft models. Biodistribution studies were carried out on 22Rv1 tumor xenograft models to confirm the targeting efficacy. Toxicity analysis of the [²²⁵Ac]DOTA-YS5 was carried out on nu/nu mice to study short-term (acute) and long-term (chronic) toxicity.</p>Results:<p>Biodistribution study shows that [²²⁵Ac]DOTA-YS5 agent delivers high levels of radiation to the tumor tissue (11.64% ± 1.37%ID/g, 28.58% ± 10.88%ID/g, 29.35% ± 7.76%ID/g, and 31.78% ± 5.89%ID/g at 24, 96, 168, and 408 hours, respectively), compared with the healthy organs. [²²⁵Ac]DOTA-YS5 suppressed tumor size and prolonged survival in cell line–derived and patient-derived xenograft models. Toxicity analysis revealed that the 0.5 μCi activity levels showed toxicity to the kidneys, likely due to redistribution of daughter isotope ²¹³Bi.</p>Conclusions:<p>[²²⁵Ac]DOTA-YS5 suppressed the growth of cell-derived and patient-derived xenografts, including prostate-specific membrane antigen–positive and prostate-specific membrane antigen–deficient models. Overall, this preclinical study confirms that [²²⁵Ac]DOTA-YS5 is a highly effective treatment and suggests feasibility for clinical translation of CD46-targeted radioligand therapy in prostate cancer.</p></div>

Decision letter: Engineering of the endogenous HBD promoter increases HbA2

Abstract: Full text Figures and data Side by side Abstract Editor's evaluation Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract The β-hemoglobinopathies, such as sickle cell disease and β-thalassemia, are one of the most common genetic diseases worldwide and are caused by mutations affecting the structure or production of β-globin subunits in adult hemoglobin. Many gene editing efforts to treat the β-hemoglobinopathies attempt to correct β-globin mutations or increase γ-globin for fetal hemoglobin production. δ-globin, the subunit of adult hemoglobin A2, has high homology to β-globin and is already pan-cellularly expressed at low levels in adult red blood cells. However, upregulation of δ-globin is a relatively unexplored avenue to increase the amount of functional hemoglobin. Here, we use CRISPR-Cas9 to repair non-functional transcriptional elements in the endogenous promoter region of δ-globin to increase overall expression of adult hemoglobin 2 (HbA2). We find that insertion of a KLF1 site alone is insufficient to upregulate δ-globin. Instead, multiple transcription factor elements are necessary for robust upregulation of δ-globin from the endogenous locus. Promoter edited HUDEP-2 immortalized erythroid progenitor cells exhibit striking increases of HBD transcript, from less than 5% to over 20% of total β-like globins in clonal populations. Edited CD34 +hematopoietic stem and progenitors (HSPCs) differentiated to primary human erythroblasts express up to 46% HBD in clonal populations. These findings add mechanistic insight to globin gene regulation and offer a new therapeutic avenue to treat β-hemoglobinopathies. Editor's evaluation This study presents the important finding that gene editing could be used to activate δ-globin expression to treat disorders of red blood cell synthesis. The evidence supporting the claims of the authors is convincing, particularly in the clonal cell lines. The data show this approach to have promise and identify avenues of effort that could be pursued to advance it to a clinical strategy for hemoglobinopathy treatment. https://doi.org/10.7554/eLife.85258.sa0 Decision letter Reviews on Sciety eLife's review process Introduction Red blood cells, also known as erythrocytes, are packed with hemoglobin and circulate the body to supply all organs with oxygen. Hemoglobin is a hetero-tetrameric protein made up of two α-like and two β-like subunits. Hemoglobin A1 (HbA1) accounts for approximately 97% of the hemoglobin expressed in adults and is composed of two α-globin subunits (HBA) and two β−globin (HBB) subunits. Hemoglobin A2 (HbA2) accounts for the remaining 2–3% of hemoglobin expressed in adults and is composed of two α-globin subunits and two δ-globin (HBD) subunits Steinberg and Rodgers, 2015. The β-hemoglobinopathies, such as sickle cell disease (SCD) and β-thalassemia, are caused by mutations in HBB that effect the structure or expression of β-globin. The clinical hallmarks include hemolytic anemia and vaso-occlusion, which can lead to acute and chronic pain and organ damage. Clinical management is limited to frequent blood transfusions and life-long treatment of anemia and pain crises. The only curative approach is allogeneic stem cell transplantation, which is dependent upon HLA-identical donor availability Locatelli et al., 2013. Fetal hemoglobin (HbF), which is the predominant hemoglobin expressed before birth, has anti-sickling properties and its re-expression is frequently pursued as a treatment for β-hemoglobinopathies Wienert et al., 2018. While increasing HbF has shown to be clinically effective to combat SCD, studies have also validated the in vitro and in vivo anti-sickling abilities of δ-globin using a humanized mouse model of SCD Nagel et al., 1979; Poillon et al., 1993; Porcu et al., 2021; Waterman et al., 1979. Increased HbA2 expression has some potential advantages over HbF that suggests it could provide an alternative avenue for ameliorating the β-hemoglobinopathies. HbA2 is weakly transcriptionally active and expressed pancellularly in all adult red blood cells Heller and Yakulis, 1969; Steinberg, 2021. Additionally, δ-globin shares 93% amino acid homology to β-globin, suggesting that HbA2 may be a better replacement relative to HbF, whose γ-globin subunit shares 73% amino acid homology to β-globin. Finally, HbF is known to bind oxygen more tightly than HbA1, an evolutionary advantage for the fetus to draw oxygen from the maternal blood source, while HbA2 has a similar oxygen-binding capacity as HbA1 Di Cera et al., 1989; Inagaki et al., 2000. On-going clinical trials re-express HbF to extremely high levels not commonly seen even in individuals with Hereditary Persistence of Fetal Hemoglobin Frangoul et al., 2021. The effect of extreme maternal HbF re-expression during pregnancy is currently unknown. The genes for β−globin (HBB) and δ-globin (HBD) are located in the β-like globin cluster and regulated by the same control region. The β-like globin cluster is located on chromosome 11, and harbors the five β-like genes: HBB (β-globin gene), HBD (δ-globin gene), HBG1 and HBG2 (γ-globin genes), and HBE (ε-globin gene). β-globin and δ-globin both comprise 147 amino acids and differ at only 10 positions Moleirinho et al., 2013. The extreme difference in expression levels between these two globins is not due to protein instability or differences in translation, but instead results from a lower transcription rate Steinberg and Rodgers, 2015. The globin genes are arranged in order of their expression during development and regulated by contact to a distal enhancer called the Locus Control Region that contains five active DNase Hypersensitivity Sites Li et al., 2002. A comparative genomics study has shown that, compared to the HBB promoter, the HBD promoter has mutations in multiple transcriptional elements including a KLF1, NF-Y, β-DRF, and TFIIB binding site (Figure 1A) Zaldívar-López et al., 2017. Figure 1 with 1 supplement see all Download asset Open asset Targeting and design of the endogenous HBD promoter. (A) Alignment of the HBB and HBD promoter sequences. Transcription factor binding sequences for KLF1, NF-Y, β-DRF, TFIIb, and TATA are shown in boxes and base pair mismatches between HBB and HBD are highlighted in red. (B) The repair template designs for insertions of KLF1 (K), β-DRF and TFIIb (DT), and KLF1, β-DRF, and TFIIb (KDT) directly compared to the HBD promoter. The inserted transcription factor binding sequences are highlighted in blue. Any base pair changes are in bold. The gRNA is indicated by a black horizontal line and the cut site is indicated by a red vertical line. (C) HUDEP-2 editing efficiencies showing percentages of unmodified, NHEJ, or HDR alleles. Conditions tested were Cas9 and sgRNA RNP with no repair template (no RT), K, DT, and KDT repair templates. This experiment was performed three times and the data is presented as mean ± SD of three biological replicates. (D) qRT-PCR of HBD after pooled editing of HUDEP-2 cells with no RT, K, DT, and KDT and 5 days of differentiation. Data is plotted as % of all β-like globins (HBB, HBG1/2, HBD). The three biological replicates from the editing experiment in (C) were each differentiated and the data is presented as mean ± SD of three biological replicates. p Value indicates paired, two-tailed student t test (ns, non-significant; *, p≤0.05; **, p≤0.01). Previous studies using transgenic approaches have shown that inclusion of a KLF1 motif in the HBD promoter can drive exogenous expression of δ-globin Porcu et al., 2021; Donze et al., 1996; Ristaldi et al., 1999. However, these studies do not reflect the complex chromosomal context and extensive epigenetic regulation at the β-like globin cluster. Due to the large size of the β-like globin locus, transgenic studies have mostly included only a subset of the genes of the β-like globin locus and a minimal region of the LCR. They therefore do not necessarily predict the biological outcomes of perturbations at the native β-globin locus Woodard et al., 2022. The expression of globin genes is a tightly regulated developmental process. There are currently no drugs or therapeutic approaches to increase HbA2 for the β-hemoglobinopathies. Using CRISPR-Cas9 genome editing, we used homology directed repair at the endogenous HBD promoter to engineer the transcriptional elements present in HBB. We find that insertion of single transcriptional elements to the endogenous promoter is insufficient for δ-globin upregulation. However, insertion of KLF1, β-DRF, and TFIIB motifs drive high expression of δ-globin from the endogenous locus in clonal populations of HUDEP-2 cells and primary erythroblasts. This leads to reconstitution of high levels of HbA2, over 10-fold increase compared to WT unedited cells. Our work adds mechanistic insight to the globin gene regulation at the β-like globin cluster and suggests a potential therapeutic avenue to upregulate HbA2 for the β-hemoglobinopathies. Results Targeting the endogenous HBD promoter to introduce functional HBB promoter elements We aligned the promoter sequences of HBB and HBD to identify transcriptional elements missing in the HBD promoter (Figure 1a). This highlighted multiple mutations and deletions in the KLF1, NF-Y, β-DRF, and TFIIB binding motifs. To re-engineer the endogenous HBD promoter, we employed CRISPR-Cas9 induced homology directed repair (HDR) gene editing Jinek et al., 2012; Yeh et al., 2019. We designed and tested three sgRNAs targeting the HBD promoter (Supplementary file 1) and three HDR templates that would incorporate base pairs needed to complete the transcriptional element motifs (Figure 1b). The HDR templates were designed as single stranded oligo donor nucleotides (ssODN) to either insert a KLF1 (K) sequence, a β-DRF and TFIIB (DT) sequence, or all three elements (KDT). A previous transgenic study showed that the NF-Y site has a lesser impact on HBB transcription in comparison to KLF1 Tang et al., 1997. Therefore, in order to minimize the number of transcription element iterations for testing, we omitted NF-Y from our designs. For the K repair template, the KLF1 sequence was inserted adjacent to the cut site, after the TATA box sequence, in order to disrupt the seed sequence of the sgRNA to reduce subsequent cutting post HDR repair. For the DT repair template, β-DRF and TFIIB motifs were designed into a single template as they are separated by only 2 base pairs. For the KDT repair template, we maintained the ordering between motifs KLF1, β-DRF and TFIIB to mimic the HBB promoter and limited the number of mutated base pairs to increase the likelihood of successful HDR editing. For both the K and KDT repair template, the KLF1 motifs inserted are not designed in the homologous position to the KLF1 site in the HBB promoter. The homologous site is considerably upstream of the sgRNA cut site and therefore more difficult to integrate given the relatively short conversion tracts of HDR in human cells. We performed editing in HUDEP-2 cells, an immortalized cell line capable of differentiating into hemoglobin-producing erythroid cells Kurita et al., 2013, using Cas9 ribonucleoprotein (RNP) and an ssODN. After optimizing conditions and testing multiple sgRNAs (Supplementary file 1; data not shown), we found that the sgRNA g1 had the highest editing efficiency, and proceeded to use sgRNA g1 for all further experiments. Editing resulted in NHEJ rates ranging from 56% to 74% and HDR efficiencies from 13% to 27% as measured by next generation sequencing (NGS; Figure 1c). The edited pools were differentiated to erythroblasts and qRT-PCR measurements were taken to assess the effects on HBD expression (Figure 1d). Despite roughly equal HDR rates between all promoter designs, we observed a statistically significant increase in HBD only for the KDT design with three elements. Homozygous knock-in of KLF1, TFIIB, and β-DRF leads to robust increase of HBD in HUDEP-2 cells Heterogeneously edited pools of cells, containing a mixture of alleles, can mask large effects at a clonal level. We isolated heterozygous and homozygous HDR clones to more accurately assess the effect of each motif edit on HBD expression. We obtained at least three heterozygotes and homozygotes for each knock-in construct, as verified by amplicon NGS sequencing of the HBD promoter (Figure 1—figure supplement 1). We used ChIP-qPCR to test whether the inserted motifs were functional in recruiting KLF1 and RNA Pol II to the HBD promoter, comparing to the HBB promoter and VEGFA as a positive and negative control, respectively. Due to the unavailability of a TFIIB antibody suitable for ChIP, we performed ChIP for RNA Pol II (Figure 2a). While Pol II is a binding partner of TFIIB in the pre-initiation complex, this ChIP does have the limitation of indirectly immunoprecipitating other factors in the transcriptional pre-initiation complex and might also reflect general promoter activity. The β-DRF motif has been shown to be important for high transcription of HBB, but its bona fide binding factor has not yet been identified Stuve and Myers, 1990. In the homozygous clones K and KDT, which harbor the edited KLF1 sequence, we observed increased binding of KLF1 to the HBD promoter relative to unedited WT HUDEP-2 cells. However, we found RNA Pol II binding at the HBD promoter binding only in the homozygous KDT clone. The homozygous DT clone, which harbors a TFIIB site, did not show significant binding of RNA Pol II. Taken together, our data show that three transcription factor binding sites are necessary to recruit the transcriptional machinery to the HBD promoter Gillinder et al., 2018. Figure 2 with 1 supplement see all Download asset Open asset Characterization of HUDEP-2 clones with heterozygous or homozygous knock-ins of KLF1, β-DRF, and TFIIb sequences. (A) ChIP-qPCR of KLF1 and RNA Pol II performed on WT HUDEP-2 cells and homozygous clones of K, DT, and KDT. Data is shown as relative fraction of input and normalized to SP1. The genes targeted are HBD, HBB, and VEGFA as a negative control. Cells from WT, one K homozygous clone, one DT homozygous clone, and one KDT homozygous clone were grown and harvested separately and on different days for each biological replicate. The data is presented as mean ± SD of three biological replicates. P value indicates paired, two-tailed student t test (ns, non-significant; *, p≤0.05; **, p≤0.01). (B) qRT-PCR of HBD of HUDEP-2 heterozygous and homozygous clones with K, DT, and KDT knock-in and 5 days of differentiation. Each dot represents an individual clonal population, each validated by NGS. Data is plotted as % of β-like globins (HBB, HBG1/2, HBD). Each clone was differentiated and the data is presented as one replicate for each clonal population. p Value indicates paired, two-tailed student t test (ns, non-significant; *, p≤0.05; **, p≤0.01). (C) HPLC of of HUDEP-2 homozygous clones with K, DT, and KDT knock-in and 5 days of differentiation. Hemoglobin A (HbA) and Hemoglobin A2 (HbA2) peaks are annotated. HPLC of one homozygous clone of K, DT, and KDT was performed in triplicate, with a representative dataset of one replicate shown. We next performed qRT-PCR on all differentiated heterozygous and homozygous clones to look at percentage of HBD produced compared to total β-like globins (Figure 2b). HBB expression was unaffected by the K, DT, and KDT knock-ins (Figure 2—figure supplement 1). Like the pooled editing results, we found that clones with single knock-ins of KLF1 or β-DRF and TFIIB motifs alone did not show increased HBD. Strikingly, we observed a significant increase in HBD for KDT clones harboring all three motifs. Wildtype clones showed HBD expression less than 5% of β-like globins, while heterozygous KDT clones had increased HBD expressions ranging from 5% to 19% and homozygous KDT clones showed increases in HBD to ranges of 20–30% of β-like globins. Next, hemoglobin protein levels were measured using high pressure liquid chromatography (HPLC) for the differentiated homozygous clones (Figure 2c). Peaks were assigned to hemoglobin complexes based on previous work that performed mass-spectrometry on each globin peak fraction Boontanrart et al., 2020. We found that the transcript-level results are further supported at the protein level. The KLF1 and the β-DRF and TFIIB clones did not show any measurable increase in HbA2, while the KDT homozygous showed a large increase in HbA2. Endogenous editing of the HBD promoter increases HBD in CD34+ derived erythroblasts To test the effects of HBD promoter engineering in a more clinically relevant cell type for a potential ex vivo therapy to treat β-hemoglobinopathies, we edited human CD34 +derived erythroblasts. We used CRISPR-Cas9 to perform pooled knock-in at the HBD promoter in mobilized peripheral blood CD34 +human hematopoietic stem and progenitor cells (HSPCs) with the K, DT, and KDT RNP and ssODNs. We found HDR rates ranging from 18% to 25% for all promoter knock-ins (Figure 3a). We differentiated the edited HSPC pools and performed qRT-PCR to measure HBD expression levels. As in HUDEP-2 cells, there was no statistically significant increase in HBD levels for K and DT, and a slight increase in four different donors in the KDT knock-in condition (Figure 3b). Figure 3 with 1 supplement see all Download asset Open asset Endogenous editing of the HBD promoter in HSPCs. (A) Editing efficiencies showing percentages of unmodified, NHEJ, or HDR alleles. Conditions tested were Cas9 and sgRNA RNP with no repair template (no RT), K, DT, and KDT repair templates. The data is presented as independent editing experiments with four different donor samples. (B) qRT-PCR of HBD after pooled editing of HSPCs with no RT, K, DT, and KDT. Cells were expanded in erythroid expansion conditions and differentiated for 5 days. Data is plotted as % of all β-like globins (HBB, HBG1/2, HBD). The data is presented as independent editing experiments with four different donor samples. (C) qRT-PCR of HBD of clonal erythroblast populations after 5 days of differentiation. Genotypes were determined by NGS. Data is plotted as % of β-like globins (HBB, HBG1/2, HBD). The data is presented as independent editing experiments with four different donor samples and each dot denotes an individual clonal population. (D) Editing efficiencies showing percentages of unmodified, NHEJ, or HDR alleles. Conditions tested were Cas9 and sgRNA RNP with no repair template (no RT), KDT repair template, and KDT repair template with AZD-7648 (KDT +enh). The data is presented as one editing experiment with three different donor samples. (E) qRT-PCR of HBD after pooled editing of HSPCs with no RT, KDT, or KDT +enh. Cells were expanded in erythroid expansion conditions and differentiated for 5 days. Data is plotted as % of all β-like globins (HBB, HBG1/2, HBD). The data is presented as one editing experiment with three different donor samples. Overall, we observed high editing rates in HSPCs using our CRISPR-Cas9 editing reagents. Cas9 editing can sometimes be accompanied by large deletions Shin et al., 2017, which in the case of editing the HBD promoter might impinge upon the neighboring gene for β-globin. To test if editing at the HBD promoter leads to decrease of other β-like globins, we edited four different HSPC donors with Cas9 and the HBD gRNA alone, mimicking a ‘worst case’ scenario of only NHEJ with no HDR alleles. We observed indel efficiencies ranging from 61% to 78% in the various donors (Figure 3—figure supplement 1a). We differentiated these edited pools into erythroblasts and performed qRT-PCR on the β-like globins HBB, HBD, and HBG1/2 and normalized their expressions to HBA (Figure 3—figure supplement 1b). The HBA gene, which is present in a different locus from the β-like genes, should be unaffected by editing at the β-globin locus. We observed that HBD expression decreased by approximately 50%, while the expression of HBB and HBG1/2 were not significantly changed. While we cannot rule out a small percentage of large deletions among the indel alleles, we do conclude that indel-based targeting of the HBD promoter decreases HBD, as expected, without grossly affecting the expression of the other β-like globins. To test the effect of KDT editing on a single-cell level, we isolated and grew colonies from HSPCs under erythroid expansion conditions. Each colony was genotyped and their alleles classified as either unmodified, NHEJ, or HDR (Figure 3—figure supplement 1c). These CD34 +derived clonal erythroblasts were differentiated and qRT-PCR was performed to determine HBD expression relative to total β-like globins (Figure 3D). HBB transcript levels remain similar between unedited or NHEJ clones and KDT knock-in clones (Figure 3—figure supplement 1d). We observed that colonies with unedited or all NHEJ alleles had an average expression of lower than 3% HBD of total β-like globins. Heterozygous KDT knock-ins from multiple donors increased HBD expression dramatically, between 15–35% of total β-like globins in heterozygous knock-ins and between 17–46% of total β-like globins in homozygous knock-ins. Clonal erythroblasts harboring either a heterozygous or homozygous knock-in of KDT expressed significantly increased HBD. However, the edited pools of HSPCs had only a slight increase in HBD, far below the level required to be clinically relevant. We hypothesized that increased HDR efficiency in the pool could lead to higher HBD levels. Small molecule drugs, most notably DNA-PKcs inhibitors that effectively inhibit NHEJ, have shown to shift editing outcomes in CRISPR-Cas9 induced double-stranded break to favor HDR Lee et al., 2022; Peterka et al., 2022. We tested the DNA-PKcs inhibitor AZD-7648 on HSPCs using our editing conditions for KDT knock-in. In three different HSPC donors, we observed an increase from 20% to 24% HDR alleles to 47–65% when we used AZD-7648. We differentiated the edited HSPC pools and performed qRT-PCR to measure HBD expression levels. In these edited erythroblast pools, the KDT-editing pool expressed 6–7% HBD of total β-like globins, while the pools edited with KDT along with HDR enhancer showed increased HBD expression to 17–19%, confirming that increasing the HDR efficiency of KDT knock-in can increase HBD levels within edited pools of HSPCs. Discussion Current gene editing efforts to treat the β-hemoglobinopathies include correcting individual HBB mutations DeWitt et al., 2016, a method which would be limited to specific types of patient mutations, or increasing fetal hemoglobin Wienert et al., 2018, which has different oxygen-binding capacities than HbA1. In this study, we describe a path to upregulating HbA2, which shares high similarity to HbA1 and is applicable to all β-hemoglobinopathy disease mutations. Previous transgenic studies have shown that insertion of KLF1 alone to the HBD promoter sequence is sufficient to drive expression of HBD Porcu et al., 2021; Donze et al., 1996. In our study, we found that endogenous insertion of a KLF1 motif is insufficient to drive HBD expression. One explanation of this discrepancy could be that previous transgenic studies do not reflect the complex chromatin context and regulation at the β-like globin locus. Another explanation could be the importance of the KLF1 motif placement. To ablate re-recognition of the HDR alleles by Cas9, we inserted the KLF1 motif in the K repair template after the TATA box, while the DT and KDT repair templates inserted all motifs upstream of the TATA box. In the HBB promoter, the KLF1 motif is upstream of the TATA box. In a previous study using a HBD promoter luciferase reporter system, they found the highest luciferase activity when the KLF1 motif was upstream of the TATA box, in the homologous position to the HBB promoter Donze et al., 1996. In another study, it was shown that insertion of the KLF1 motif in varying positions resulted in differences in luciferase activity Ristaldi et al., 1999. The placement of transcription factor binding sites can play a crucial role in promoter activity and follow-up studies of our work should further test the spacing and positioning of these various promoter elements, as well as include the NF-Y motif that was not explored in this study. We have shown that insertion of three motifs, KLF1, β-DRF, and TFIIB, is necessary to recruit RNA Pol II and induce high expression of HBD. In CD34 + derived erythroblasts, homozygous knock-in of KLF1, β-DRF, and TFIIB leads to increases in HBD up to 46% of total β-like globins in clonal populations. To our knowledge, this is the first genomic editing of the HBD promoter that results in increased HbA2. Bulk editing of HBD through insertion of multiple transcriptional elements will rely on high levels of HDR. Increasing HDR outcomes might be achieved in CD34 + cells by a variety of methods such as controlled cell-cycling or modulation of DNA repair factors Howden et al., 2016; Charpentier et al., 2018; Schiroli et al., 2019; Shin et al., 2018. In this study, we found that AZD-7648 increased HDR outcomes in pooled edited HSPCs with concomitant increases in HBD expression. Further work should assess the safety and the effects of the AZD-7648 or other HDR-enhancing in HSPCs if they are to be pursued in a clinical context. We observed high editing efficiencies when targeting CD34 + HSPCs. When editing targets HBB, NHEJ alleles that co-occur with HDR alleles can cause reductions in overall β-globin levels DeWitt et al., 2016. When targeting HBD, HBB might also be inadvertently affected by large deletions that extend into neighboring β-like globin genes. However, we using qRT-PCR experiment we found that HBB and HBG1/2 mRNA expression were unaffected in our edited populations of HSPCs, suggesting that large deletions were not occurring with enough frequency to grossly affect β-like globin expression. However, some studies have shown that large deletions can occur at low levels, which might be detected by very sensitive tests such as long-range next-generation sequencing or ddPCR. It is estimated that roughly 30% of beneficial hemoglobin (10 pg per cell) is sufficient to ameliorate β-hemoglobinopathy symptoms Steinberg, 2021. There are currently many gene editing approaches being explored for the β-hemoglobinopathies, mainly focused on increasing fetal hemoglobin or directly correcting the SCD mutation Wienert et al., 2018. A recent study reversed the SCD mutation using CRISPR-Cas9 prime-editing Everette et al., 2023. They showed SCD mutation reversion rates of 17–41% in 4 SCD patient donors and resulted in expression of HbA1 to 10–45% of total hemoglobins. Sickling decreased proportionally to editing efficiencies, with an average of 63% sickled cells for untreated samples and decreasing to 37% in treated samples. Another strategy utilizes CRISPR-Cas9 base-editing to disrupt the LRF repressor binding site within HBG1/2 and observed an upregulation of HbF to over 20% of total hemoglobins in SCD HSPCs Antoniou et al., 2022. When disrupting the LRF site by insertion of a KLF1 site, HbF levels increased to 65–77%. All edited cell populations showed a therapeutically relevant reduction in the number of sickling cells. Another approach utilizes CRISPR-Cas9 to disrupt the enhancer region of BCL11A, a repressor of fetal hemoglobin Frangoul et al., 2021. In an on-going clinical trial, BCL11A enhancer targeting yields editing efficiencies of 80–93% in four SCD patients and HbF upregulation to over 30% of total hemoglobins, with no vaso-occlusive crises 3 months post-transfusion of edited cells. These studies support the curative effect of increasing functional hemoglobin levels on β-hemoglobinopathies. In our study using CRISPR-Cas9 and a DNAPKcs inhibitor to knock the KDT promoter into HBD, we observed increased HbA2 to 17–19% of total hemoglobins in CD34+-derived erythroblasts. Further studies will need to be done to achieve even higher levels of HbA2 expression or test whether this lower expression of HbA2 is sufficient to ameliorate a disease phenotype. In our experiments, heterozygous and homozygous knock-in of KDT in CD34+ erythroblasts led to 15–46% HBD relative to total β-like globins. Interestingly, in edited CD34+ erythroblasts, we observed that heterozygous and homozygous KDT populations expressed similar increases in HBD. Further studies should investigate whether heterozygous knock-in of KDT in β-hemoglobinopathy cells is sufficient to ameliorate disease phenotypes. For example, one could knock-in KDT to the HBD promoter of SCD patient HSPCs and perform HPLC or microscopy assays to determine the anti-sickling effects of a heterozygous or homozygous knock-in. If heterozygous knock-in of KDT is sufficient, one would see a decrease in sickle hemoglobin produced, or a decreased percentage of sickled red blood cells. In summary, our presented study provides a novel strategy to increase levels of HbA2 from the endogenous HBD locus that could potentially be applicable as an ex vivo gene therapy. In pre-clinical studies, it will be important to quantitatively investigate the amount of HBD that is optimal for improving the function and health of patient red blood cells and explore the overall potential and safety of increasing HbA2 as a therapeutic option for the β-hemoglobinopathies. Materials and methods Cas9 RNP nucleofection Request a detailed protocol Cas9 RNP was performed as described previously (Lingeman et al., 2017). Briefly, either IVT guides are purified or chemically protected guides were ordered from Synthego and complexed with purified Cas9-NLS protein. The nucleofection was perform

Round Table comment

Abstract: Trying to comprehend and then to describe the process of technological change and its impact is a difficult task. Explaining it is then extremely demanding. Many of the contributions in the papers offered indicate the value of thick description, of looking closely at relevant documentation, piecing together events from those documents and presenting the information in a comprehensible way. Placing that description within the context of time and place, making the small picture part of the big picture is, after all, doing good history

Data from Treatment of Prostate Cancer with CD46-targeted &lt;sup&gt;225&lt;/sup&gt;Ac Alpha Particle Radioimmunotherapy

Abstract: <div>AbstractPurpose:<p>Radiopharmaceutical therapy is changing the standard of care in prostate cancer and other malignancies. We previously reported high CD46 expression in prostate cancer and developed an antibody–drug conjugate and immunoPET agent based on the YS5 antibody, which targets a tumor-selective CD46 epitope. Here, we present the preparation, preclinical efficacy, and toxicity evaluation of [²²⁵Ac]DOTA-YS5, a radioimmunotherapy agent based on the YS5 antibody.</p>Experimental Design:<p>[²²⁵Ac]DOTA-YS5 was developed, and its therapeutic efficiency was tested on cell-derived (22Rv1, DU145), and patient-derived (LTL-545, LTL484) prostate cancer xenograft models. Biodistribution studies were carried out on 22Rv1 tumor xenograft models to confirm the targeting efficacy. Toxicity analysis of the [²²⁵Ac]DOTA-YS5 was carried out on nu/nu mice to study short-term (acute) and long-term (chronic) toxicity.</p>Results:<p>Biodistribution study shows that [²²⁵Ac]DOTA-YS5 agent delivers high levels of radiation to the tumor tissue (11.64% ± 1.37%ID/g, 28.58% ± 10.88%ID/g, 29.35% ± 7.76%ID/g, and 31.78% ± 5.89%ID/g at 24, 96, 168, and 408 hours, respectively), compared with the healthy organs. [²²⁵Ac]DOTA-YS5 suppressed tumor size and prolonged survival in cell line–derived and patient-derived xenograft models. Toxicity analysis revealed that the 0.5 μCi activity levels showed toxicity to the kidneys, likely due to redistribution of daughter isotope ²¹³Bi.</p>Conclusions:<p>[²²⁵Ac]DOTA-YS5 suppressed the growth of cell-derived and patient-derived xenografts, including prostate-specific membrane antigen–positive and prostate-specific membrane antigen–deficient models. Overall, this preclinical study confirms that [²²⁵Ac]DOTA-YS5 is a highly effective treatment and suggests feasibility for clinical translation of CD46-targeted radioligand therapy in prostate cancer.</p></div>

Chapter 4. Cross-language influences in the acquisition of L2 and L3 phonology

Abstract: This chapter synthesises some of the main findings from research investigating cross-language influence (CLI) in Second Language (L2) and Third Language (L3) speech learning. In addition to an overview of the findings that have gathered growing consensus in the fields of L2 and L3 phonology, and how these results have been tested and explained in various theories of L2 speech, this chapter points to methodological and theoretical considerations discussed in three areas that hold promise for future research in L2 and L3 speech: (i) the relationship between L2/L3 speech perception and L2/L3 speech production, (ii) the acquisition of phonological processes vs. phonological contrasts, and (iii) the distinction between static and dynamic phonetic interactions in L2 and L3 speech.

Ships, shipping, technological change and global economic growth, 1400-1800

Abstract: The major breakthrough in ship design around 1400 creating the full-rigged ship constituted a general purpose technology. It had far-reaching effects on shipping, trade volume, orientation of trade routes, location of production, settlement patterns and many other aspects of life throughout the globe from 1400 to1800. The greater efficiency of the type in a number of uses led to its dissemination, to a limited degree, throughout the world. Spillovers from the success of the design were extensive and included for example a literature on designing and building ships, improvements in navigation and in government practices. Advances in shipbuilding were one of the very few technologies in the period that qualified as a technological advance with massive consequences.

<i>Sapho Kiss</i>: Queer Reproduction in Early Cinema

Abstract: In 1896 ‘the Nethersole kiss’ revolutionized the stage kiss and made legitimate-stage actress Olga Nethersole a household name. This article considers Olga Nethersole’s queer influence on the early erotic genres of the kissing film and the ‘stag’ film, arguing that she originated the mainstream Sapphic tropes of the twentieth century. Leaning on foundational texts by Shelley Stamp, Charles Musser and Linda Williams and drawing on recent work by Susan Potter and Russell Sheaffer, this article intervenes in the presumed ‘male only’ discourse of ‘stag’ during the early silent film era (1896–1907). Using original archival research including newspapers, catalogues, scrapbooks, and other ephemera such as erotic ‘French’ postcards, I reconsider a cluster of films that engage Nethersole’s onstage and offstage ‘character’, including The Kiss (Edison, 1896), Something Good – Negro Kiss (Selig, 1898), Sapho (Lubin, 1900), The New Kiss (Edison, 1900), Sapho Kiss (Lubin, 1900), In a Massage Parlor (American Mutoscope & Biograph, 1902), The Seven Ages (Edison, 1905), A Modern Sapho (American Mutoscope and Biograph, 1905), and Under the Old Apple Tree (American Mutoscope and Biograph, 1907). I demonstrate that these films constituted a racialized Sapphic authorial discourse that produced queer pleasures for early audiences by engaging Nethersole’s scandalous stardom. As a progenitor of early erotic screen genres, Nethersole must be contextualized in relation to the spectrum of erotic entertainment at the turn of the century from the ‘unobjectionable’ to the pornographic. In this context, I demonstrate how the intermediality of early cinema, so often understood through the heterosexualized metaphor of ‘birth’, is better understood as a form of queer reproduction.

Justice-involved youth

Abstract: Youth can become involved in the juvenile justice system through an arrest for a law or criminal violation or referral to the juvenile court for a status offense (crimes based on the youth's age such as truancy or curfew violations). Once involved in the juvenile justice system, there are several individuals and agencies that have decision-making power over youth's trajectory through the system (e.g., juvenile court judges, attorneys, probation officers). Justice-involved youth tend to have histories of trauma exposure and child welfare involvement. Once involved in the justice system, youth are often involved in multiple service systems such as mental health and education and experience a range of collateral consequences due to their involvement these system. As a result, there are several evidence-based programs that should be used to support justice-involved youth.

Resistin Pathway as Novel Mechanism of Post-lung Transplantation Bronchial Stenosis

Abstract: Background: Bronchial stenosis remains a significant source of morbidity among lung transplant recipients. Though infection and anastomotic ischemia have been proposed etiologies of the development of bronchial stenosis, the pathophysiologic mechanism has not been well elucidated. Methods: In this single-centered prospective study, from January 2013 through September 2015, we prospectively collected bronchoalveolar lavage (BAL) and endobronchial epithelial brushings from the direct anastomotic site of bronchial stenosis of bilateral lung transplant recipients who developed unilateral post-transplant bronchial stenosis. Endobronchial epithelial brushings from the contralateral anastomotic site without bronchial stenosis and BAL from bilateral lung transplant recipients who did not develop post-transplant bronchial stenosis were used as controls. Total RNA was isolated from the endobronchial brushings and real-time polymerase chain reaction reactions were performed. Electrochemiluminescence biomarker assay was used to measure 10 cytokines from the BAL. Results: Out of 60 bilateral lung transplant recipients, 9 were found to have developed bronchial stenosis with 17 samples adequate for analysis. We observed a 1.56 to 70.8 mean-fold increase in human resistin gene expression in the anastomotic bronchial stenosis epithelial cells compared with nonstenotic airways. Furthermore, IL-1β (21.76±10.96 pg/mL; control 0.86±0.44 pg/mL; P<0.01) and IL-8 levels (990.56±326.60 pg/mL; control 20.33±1.17 pg/mL; P<0.01) were significantly elevated in the BAL of the lung transplant patients who developed anastomotic bronchial stenosis. Conclusion: Our data suggest that the development of postlung transplantation bronchial stenosis may be in part mediated through the human resistin pathway by IL-1β induced transcription factor nuclear factor-κβ activation and downstream upregulation of IL-8 in alveolar macrophages. Further study is needed in the larger patient cohorts and to determine its potential therapeutic role in the management of post-transplant bronchial stenosis.

Data from CUB Domain-Containing Protein 1 (CDCP1) Is a Target for Radioligand Therapy in Castration-Resistant Prostate Cancer, including PSMA Null Disease

Abstract: <div>AbstractPurpose:<p>With the improvement in overall survival with 177Lu-PSMA 617, radioligand therapy (RLT) is now a viable option for patients with metastatic castration-resistant prostate cancer (mCRPC). However, responses are variable, in part due to low PSMA expression in 30% of patients. Herein, we evaluated whether the cell surface protein CUB domain-containing protein 1 (CDCP1) can be exploited to treat mCRPC with RLT, including in PSMA-low subsets.</p>Experimental Design:<p>CDCP1 levels were evaluated using RNA sequencing from 119 mCRPC biopsies. CDCP1 levels were assessed in 17 post–enzalutamide- or abiraterone-treated mCRPC biopsies, 12 patient-derived xenografts (PDX), and prostate cancer cell lines. 4A06, a recombinant human antibody that targets the CDCP1 ectodomain, was labeled with Zr-89 or Lu-177 and tested in tumor-bearing mice.</p>Results:<p>CDCP1 expression was observed in 90% of mCRPC biopsies, including small-cell neuroendocrine (SCNC) and adenocarcinomas with low FOLH1 (PSMA) levels. Fifteen of 17 evaluable mCRPC biopsies (85%) demonstrated membranous CDCP1 expression, and 4 of 17 (23%) had higher CDCP1 H-scores compared with PSMA. CDCP1 was expressed in 10 of 12 PDX samples. <i>B</i>_max values of approximately 22,000, 6,200, and 2,800 fmol/mg were calculated for PC3, DU145, and C4–2B human prostate cancer cells, respectively. ⁸⁹Zr-4A06 PET detected six human prostate cancer xenografts, including PSMA-low tumors. ¹⁷⁷Lu-4A06 significantly suppressed growth of DU145 and C4–2B xenografts.</p>Conclusions:<p>The data provide the first evidence supporting CDCP1-directed RLT to treat mCRPC. Expanded studies are warranted to determine whether CDCP1 is a viable drug target for patients with mCPRC.</p></div>

Treatment of pancreatic cancer with irreversible electroporation and intratumoral CD40 antibody stimulates systemic immune responses that inhibit liver metastasis in an orthotopic model

Abstract: Background Pancreatic cancer (PC) has a poor prognosis, and most patients present with either locally advanced or distant metastatic disease. Irreversible electroporation (IRE) is a non-thermal method of ablation used clinically in locally advanced PC, but most patients eventually develop distant recurrence. We have previously shown that IRE alone is capable of generating protective, neoantigen-specific immunity. Here, we aim to generate meaningful therapeutic immune effects by combining IRE with local (intratumoral) delivery of a CD40 agonistic antibody (CD40Ab). Methods KPC46 organoids were generated from a tumor-bearing male KrasLSL-G12D-p53LSL-R172H-Pdx-1-Cre (KPC) mouse. Orthotopic tumors were established in the pancreatic tail of B6/129 F1J mice via laparotomy. Mice were randomized to treatment with either sham laparotomy, IRE alone, CD40Ab alone, or IRE followed immediately by CD40Ab injection. Metastatic disease and immune infiltration in the liver were analyzed 14 days postprocedure using flow cytometry and multiplex immunofluorescence imaging with spatial analysis. Candidate neoantigens were identified by mutanome profiling of tumor tissue for ex vivo functional analyses. Results The combination of IRE+CD40 Ab improved median survival to greater than 35 days, significantly longer than IRE (21 days) or CD40Ab (24 days) alone (p<0.01). CD40Ab decreased metastatic disease burden, with less disease in the combination group than in the sham group or IRE alone. Immunohistochemistry of liver metastases revealed a more than twofold higher infiltration of CD8+T cells in the IRE+CD40 Ab group than in any other group (p<0.01). Multiplex immunofluorescence imaging revealed a 4–6 fold increase in the density of CD80+CD11c+ activated dendritic cells (p<0.05), which were spatially distributed throughout the tumor unlike the sham group, where they were restricted to the periphery. In contrast, CD4+FoxP3+ T-regulatory cells (p<0.05) and Ly6G+myeloid derived cells (p<0.01) were reduced and restricted to the tumor periphery in the IRE+CD40 Ab group. T-cells from the IRE+CD40 Ab group recognized significantly more peptides representing candidate neoantigens than did T-cells from the IRE or untreated control groups. Conclusions IRE can induce local tumor regression and neoantigen-specific immune responses. Addition of CD40Ab to IRE improved dendritic cell activation and neoantigen recognition, while generating a strong systemic antitumor T-cell response that inhibited metastatic disease progression.

Data from CUB Domain-Containing Protein 1 (CDCP1) Is a Target for Radioligand Therapy in Castration-Resistant Prostate Cancer, including PSMA Null Disease

Abstract: <div>AbstractPurpose:<p>With the improvement in overall survival with 177Lu-PSMA 617, radioligand therapy (RLT) is now a viable option for patients with metastatic castration-resistant prostate cancer (mCRPC). However, responses are variable, in part due to low PSMA expression in 30% of patients. Herein, we evaluated whether the cell surface protein CUB domain-containing protein 1 (CDCP1) can be exploited to treat mCRPC with RLT, including in PSMA-low subsets.</p>Experimental Design:<p>CDCP1 levels were evaluated using RNA sequencing from 119 mCRPC biopsies. CDCP1 levels were assessed in 17 post–enzalutamide- or abiraterone-treated mCRPC biopsies, 12 patient-derived xenografts (PDX), and prostate cancer cell lines. 4A06, a recombinant human antibody that targets the CDCP1 ectodomain, was labeled with Zr-89 or Lu-177 and tested in tumor-bearing mice.</p>Results:<p>CDCP1 expression was observed in 90% of mCRPC biopsies, including small-cell neuroendocrine (SCNC) and adenocarcinomas with low FOLH1 (PSMA) levels. Fifteen of 17 evaluable mCRPC biopsies (85%) demonstrated membranous CDCP1 expression, and 4 of 17 (23%) had higher CDCP1 H-scores compared with PSMA. CDCP1 was expressed in 10 of 12 PDX samples. <i>B</i>_max values of approximately 22,000, 6,200, and 2,800 fmol/mg were calculated for PC3, DU145, and C4–2B human prostate cancer cells, respectively. ⁸⁹Zr-4A06 PET detected six human prostate cancer xenografts, including PSMA-low tumors. ¹⁷⁷Lu-4A06 significantly suppressed growth of DU145 and C4–2B xenografts.</p>Conclusions:<p>The data provide the first evidence supporting CDCP1-directed RLT to treat mCRPC. Expanded studies are warranted to determine whether CDCP1 is a viable drug target for patients with mCPRC.</p></div>