A-to-G/C/T and C-to-T/G/A dual-function base editor for creating multi-nucleotide variants

Redefining “Gold Standard”: Ultra-Sensitive Characterization of Commercial DNA Standards with Duplex Sequencing

Molecular inversion probes equipped with discontinuous rolling cycle amplification for targeting nucleotide variants: Determining SMN1 and SMN2 genes in diagnosis of spinal muscular atrophy

3105 Background: The KDM4 family of histone lysine demethylases consists of four main isoforms (KDM4A, B, C, D), all of which have been identified as key oncogenic drivers. They function as epigenetic regulators and control transitions between transcriptionally silent and active chromatin states via removal of methyl marks on histone H3K9 and histone H3K36. KDM4 isoforms play an important role in the epigenetic dysregulation in various cancers and is linked to more aggressive disease and poorer clinical outcomes. Functional redundancy and cross-activity have been observed across KDM4 family members, thus, selective inhibition of one isoform appears to not be effective. TACH101 is a novel, first-in-class pan inhibitor of KDM4 that simultaneously targets multiple isoforms of KDM4. Here we present data that show TACH101 has promising pre-clinical and pharmacologic properties as a cancer therapeutic. Methods: TACH101 was evaluated in in vitro and in vivo studies including cell-proliferation assays in multiple cancer cell lines, apoptotic and cell cycle analyses, and efficacy studies in various xenograft tumor models and patient-derived organoid models. Results: In vitro, TACH101 was broadly effective in killing 67% (200 out of 300) of cancer cell lines screened. TACH101 demonstrated potent increase of H3K36me3 levels (EC50 < 0.001 mM, HTRF) in KYSE-150 cell line engineered to overexpress KDM4C and potent anti-proliferative activity in multiple cell lines in OncoPanel. TACH101 treatment increased cancer cell population in S-phase in multiple cancer cell lines indicating cell-cycle arrest. TACH101 induced apoptosis in human colorectal (HT-29), esophageal (KYSE-150), and triple negative breast cancer (MDA-MB-231) cell lines with EC50s ranging from 0.033-0.092 µM. In vivo, TACH101 triggered effective tumor control in xenograft models including colorectal, esophageal, gastric, breast, and lymphoma with tumor growth inhibition of up to 100%. Further evaluation using a panel of patient-derived colorectal models and patient-derived organoids showed a strong correlation of TACH101 sensitivity with MSI-H status (IC50 ranges 1-150 nM). TACH101 also reduced tumorigenic potential by 4.4-fold as determined by FACS analysis using sorted CD44High EpCAM+ population in Limiting Dilution Assays in vivo, suggesting that reduction of cancer stem cells by TACH101 may be effective in therapy-resistant settings. Pharmacologic studies showed TACH101 demonstrated favorable cell permeability, good oral bioavailability, and high metabolic stability. Conclusions: Extensive preclinical work on TACH101 KDM4 inhibitor shows compelling data and broad applicability as a potential anti-cancer agent. Further evaluation is ongoing to advance the molecule into clinical trials.

Aberrant functions of fibroblast growth factor receptors (FGFR) have been implicated in the tumor progression of pancreatic cancer (PaCA). However, the mechanisms of FGFR action and the therapeutic utilities of FGFR inhibitors (FGFRi) in PaCA remain largely under-investigated. Here the mechanisms of FGFR inhibition in PaCA were investigated using pan-FGFRi that are under clinical development and specific shRNA against individual FGFRs. FGFRi treatment, such as BGJ398 and AZD4547, significantly inhibited cell proliferation, migration, and invasion, and cell expression of key markers associated with cell survival and invasiveness was suppressed, in multiple PaCA cell lines. Gemcitabine (Gem) is the standard-of-care for PaCA, and the development of Gem resistance (GemR) compromises its efficacy, thereby Gem only provides modest survival benefits. We found that FGFRi was effective in inhibiting cell proliferation, migration of strongly GemR cells and tumors (named as MIA-GR8 cells and MIA-GR8 tumors) that were all derived by increasing Gem exposure from MIA PaCa-2 cells in vitro and selected by Gem in vivo. These results suggest its potential clinical utility as a class of alternative drug candidates in tumors that acquired GemR. Downregulation of GemR markers mediated by FGFRi treatment in GemR cells also suggested its therapeutic application of FGFRi in combination with Gem, in order to overcome GemR. Mechanistically, the molecular mechanisms of individual FGFR were investigated. shRNA-mediated inhibition of FGFR2 or FGFR3 did not decrease cell growth, and the overexpression of FGFR1 promoted cell migration rather than proliferation. Furthermore, the efficacy of FGFRi treatment was investigated in multiple cell lines and patient-derived xenograft. The inhibitory effects of BGJ398 in PANC-1 cell growth and migration were compromised by exogenic overexpression of FGFR1, and its efficacy on cell viability suppression was correlated with FGFR1 in multiple cell lines. In patient-derived xenografts, the efficacy of FGFRi on tumor growth inhibition was also correlated with FGFR1 expression. These data provide evidence that precision medicine which targets FGFR using FGFRi in PaCA tumors which express relatively modest or low amount of FGFR1 could be more effective than that with a high FGFR1 expression. Citation Format: Qingxiang Lin, Andrea Serratore, Eugene Kandel, Robert M. Straubinger. FGFR1 expression correlates inversely with the in vitro and in vivo efficacy of FGFR inhibitors in pancreatic cancer [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2020 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2020;80(22 Suppl):Abstract nr PO-042.

Glioblastoma multiforme (GBM) is the most common and aggressive malignant brain tumor, characterized by infiltrative growth, resistance to chemotherapy and radiotherapy, and an immune-suppressive microenvironment. Interstitial Photodynamic Therapy (iPDT), which involves oral ALA administration and localized light activation, offers a promising approach by killing tumor cells, disrupting tumor vasculature, and stimulating the immune response. However, iPDT faces limitations due to the blood-brain barrier (BBB), insufficient irradiation of satellite and margin tumors, hypoxia within the tumor microenvironment, and glioma cell repair mechanisms. These factors collectively contribute to the suboptimal efficacy of iPDT, emphasizing the need for combined therapeutic strategies to improve outcomes. Some in-vitro research on D, L-methadone, which enhances chemotherapy and ALA-PDT, has caught our interest, although it remains controversial. The levels of the μ-opioid receptor (MOR) were measured in multiple GBM cell lines. Multiple GBM cell lines (A172, U251, GB14) were treated with 2.5µg/ml and 10µg/ml D, L-methadone in combination with sublethal-dose ALA-PDT to mimic the limitations of PDT. A MOR antagonist, naloxone (NAL), was used to inhibit the potential role of methadone. Cell viability, apoptosis, PpIX, and ROS were examined. The expression level of MOR in GBM cell lines was low. In the A172 cell line, 10µg/ml methadone enhanced sublethal-dose ALA-PDT-induced apoptosis; however, in the U251 and GB14 cell lines, neither 2.5µg/ml methadone nor 10µg/ml methadone could enhance ALA-PDT-induced apoptosis. The viability of the three cell lines was unaffected by methadone combined with ALA-PDT. No reversal effect of naloxone on methadone was observed. Data show that methadone did not significantly enhance PpIX accumulation or ROS generation when combined with ALA-PDT. A general sensitizing effect of methadone on ALA-PDT was not observed in this in vitro experiment involving multiple GBM cell lines. The sensitizing effect of methadone on ALA-PDT may depend on the specific cell line used. Therefore, the recommendation to use methadone as a sensitizer for ALA-PDT in the treatment of GBM should be made with caution.

Precise genome editing requires the resolution of nuclease-induced DNA double strand breaks (DSBs) via the homology-directed repair (HDR) pathway. In mammals, this is typically outcompeted by non-homologous end-joining (NHEJ) that can generate potentially genotoxic insertion/deletion mutations at DSB sites. Because of higher efficacy, clinical genome editing has been restricted to imperfect but efficient NHEJ-based approaches. Hence, strategies that promote DSB resolution via HDR are essential to facilitate clinical transition of HDR-based editing strategies and increase safety. Here we describe a novel platform that consists of a Cas9 fused to DNA repair factors to synergistically inhibit NHEJ and favor HDR for precise repairing of Cas-induced DSBs. Compared to canonical CRISPR/Cas9, the increase in error-free editing ranges from 1.5-fold to 7-fold in multiple cell lines and in primary human cells. This novel CRISPR/Cas9 platform accepts clinically relevant repair templates, such as oligodeoxynucleotides (ODNs) and adeno-associated virus (AAV)-based vectors, and has a lower propensity to induce chromosomal translocations as compared to benchmark CRISPR/Cas9. The observed reduced mutational burden, resulting from diminished indel formation at on- and off-target sites, provides a remarkable gain in safety and advocates this novel CRISPR system as an attractive tool for therapeutic applications depending on precision genome editing.

Background:Although trastuzumab and other HER2-targeted therapies have significantly improved survival in patients with HER2 overexpressed or amplified (HER2+) breast cancer, a significant proportion of patients do not respond or eventually develop clinical resistance. Strategies to reverse trastuzumab resistance remain a high clinical priority. We were the first to report the role of CXCR4 in trastuzumab resistance. The present study aims to explore the therapeutic potential of targeting CXCR4 and better understand the associated mechanisms.Methods:Immunofluorescent staining, confocal microscopy analysis, and immunoblotting were used to analyze CXCR4 expression. BrdU incorporation assays and flow cytometry were used to analyze dynamic CXCR4expression. Three-dimensional co-culture (tumor cells/ breast cancer-associated fibroblasts / human peripheral blood mononuclear cells) or antibody-dependent cellular cytotoxicity assay was used to mimic human tumor microenvironment, which is necessary for testing therapeutic effect of CXCR4 inhibitor or trastuzumab. The FDA-approved CXCR4 antagonist AMD3100, trastuzumab, and docetaxel chemotherapy were used to evaluate therapeutic efficacy in vitro and in vivo. Reverse phase protein array and immunoblotting were used to discern the associated molecular mechanisms.Results:Using multiple cell lines and patient breast cancer samples we confirmed CXCR4 drives trastuzumab resistance in HER2+ breast cancer and further demonstrated that the increased CXCR4 expression in trastuzumab-resistant cells is associated with cell cycle progression with a peak in the G2/M phases. Blocking CXCR4 with AMD3100 inhibits cell proliferation by downregulating mediators of G2-M transition, leading to G2/M arrest and abnormal mitosis. Using multiple trastuzumab-resistant cell lines and an in vivo established trastuzumab-resistant xenograft mouse model, we demonstrated that targeting CXCR4 with AMD3100 suppresses tumor growth in vitro and in vivo, and synergizes with docetaxel.Conclusions:Our findings support CXCR4 as a novel therapeutic target and a predictive biomarker for trastuzumab resistance in HER2+ breast cancer.

We recently identified AHNAK as a binding partner for the C type natriuretic peptide receptor (NPR‐C) in normal rat gastric mucosa (RGM1) cells. To investigate the role of AHNAK in NPR‐C function in different cell types and species we examined the expression and interaction of both proteins in multiple cell lines. Antibodies to NPR subtypes and AHNAK domains were used to examine expression by immunoblotting, colocalization by immunohistochemistry, and interaction by immunoprecipitation in multiple mammalian cell lines. Mass spectrometry was used to confirm the identity of the proteins from the immunoprecipitation studies. GST fusion proteins of the N‐terminal, internal repeating, and C‐terminal domains of AHNAK were purified and used to perform GST pulldown assays to verify the association of the two proteins and determine specificity. Coding sequences of human NPR‐C and AHNAK were subcloned into mammalian two hybrid expression vectors to ascertain the interaction in situ. NPR‐C was found to be the most abundantly expressed NPR subtype and AHNAK was found to also be expressed in human aortic vascular smooth muscle cells (AoVSMCs). NPR‐C and AHNAK colocalized to the plasma membrane of AoVSMCs. NPR‐C bound to AHNAK isoform 1 in vitro. The interaction was attributed to a single AHNAK domain. This research was supported by an American Heart Association Predoctoral fellowship and a Veterans Affairs Merit grant.

Genome editing facilitates alterations to DNA, large or subtle, in a precise fashion. In its most popular form it uses the programmable endonuclease system, CRISPR/Cas9. Edits can be made to any genome, including the human genome. This raises the possibility of genome editing in human embryos in both a research and reproductive context. All reports of genome editing in human embryos are included here, along with key papers examining the science and ethics of human genome editing. As a basic research tool, genome editing promises to accelerate our understanding of genome biology. It also shows great promise as a means of combatting disease through so-called somatic genome editing. Genome editing could be used to prevent human disease transmission in a reproductive context. Such germ line interventions are opposed by some, for a number of reasons. Some of these reasons are discussed and a comparison is made with preimplantation genetic diagnosis (PGD). It is important that scientists, clinicians, bioethicists and other stakeholders engage widely with all those with an interest in genome editing. In addition to offering new insights into human biology, basic (fundamental) research will deliver expertise allowing ever more precise and controllable genome editing methodologies and allied technologies. A range of clear and accessible ethical frameworks must be developed and scrutinized as part of a wider societal debate about possible applications of genome editing. In the UK, human reproductive genome editing can only take place if a change to primary legislation occurs. Inclusive discussions and assessments, involving difficult scientific and ethical concepts, must form part of any democratic decision.

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 is a leading genomic editing tool, but its effectiveness is limited by considerable heterogeneity in target efficiency among different single guide RNAs (sgRNA). This study presents RNAS-sgRNA, a hybrid model that integrates neural architecture search (NAS) with recurrent neural networks (RNN) to evaluate the on-target efficacy of CRISPR/Cas9 sgRNA. The RNAS-sgRNA model automates architectural discovery, improving sgRNA sequence categorization without considerable manual adjustment. The NAS component improves the RNN architecture, which analyzes sgRNA sequences represented as binary matrices and produces a classification score. Upon evaluation across several datasets, RNAS-sgRNA exhibits substantial performance enhancements with multiple cell lines, comparing its area under the receiver operating characteristic curve (AUROC) performance to the baseline CRISPRpred(SEQ) and DeepCRISPR models. RNAS-sgRNA demonstrated substantial improvements in AUROC performance in several cell lines compared with existing models. Notable improvements include enhancements of 8.62% for HCT116, 121.57% for HEK293T, 13.40% for HeLa, and 20.78% for HL60 cell lines, resulting in an overall improvement of 13.46%. Compared with DeepCRISPR, the model achieved additional AUROC gains in all cell lines tested, with an average improvement of 14.74%. The study also highlighted the ability of the model to deliver superior performance on smaller datasets through transfer learning, underscoring its potential applications in personalized medicine and genetic research. RNAS-sgRNA introduces a novel integration of NAS with RNN to evaluate the efficacy of CRISPR/Cas9 sgRNA. Unlike traditional methods that require significant manual adjustments, this model automates architectural discovery, optimizing the RNN structure for sgRNA sequence analysis. Furthermore, the application of transfer learning to fine-tune the pretrained model on small cell-line datasets represents a pioneering approach in the domain. The model's demonstrated ability to significantly outperform existing algorithms, including CRISPRpred(SEQ) and DeepCRISPR, across multiple cell lines highlights its innovative contribution to genomic editing research and personalized medicine.

Background: Prostate cancer (PC) is the single most common and second most lethal cancer in men. Despite therapeutic advances in androgen receptor (AR)-targeting agents, progression to lethal, drug-resistant, castration-resistant prostate cancer (CRPC) remains a major clinical problem. Thus, there is an unmet need for novel treatment approaches and novel, druggable therapeutic targets in metastatic CRPC. Profiling of PC patient samples shows increases in two master regulators of lipogenesis-sterol regulatory binding proteins 1 and 2 (SREBP1 and SREBP2)-and their transcriptional targets. Overexpression of SREBP1 and SREBP2 and their target genes has been associated with tumor aggressiveness, poor clinical outcomes, and drug resistance in PC. The activation and nuclear translocation of SREBPs is tightly regulated by SREBP cleavage-activating protein (SCAP). We propose targeting SCAP will simultaneously block the activation and subsequent activity of all three SREBPs and that this represents a promising therapeutic strategy to treat CRPC. Methods: To increase our understanding of SREBP biology in PC cells, we defined the cistrome of SREBPs across multiple PC cell lines via ChIP-Seq. We compared the transcriptomic profiles of genetic targeting of SREBP via siRNA and pharmacological targeting via the novel SCAP inhibitor, SCAPi. We evaluated the effect of SCAPi on cell viability, cell invasion, and AR-signaling in multiple PC cell lines using immunoblotting, luciferase reporters, flow cytometry, MTT, and invasion assays. Additionally, we will evaluate the anticancer activity and safety profile of our novel SCAPi using CDX mouse models. Results: We found that inhibition of SCAP via siRNA and via SCAPi both yielded similar gene signatures, particularly the perturbation of cell cycle and lipogenesis genes. SCAPi dramatically reduces cell viability, migration, invasion, and expression of both AR- and SREBP-target genes in multiple PC cell lines. Through flow cytometry (Annexin V) and western blots (cleaved PARP) we found SCAPi induces apoptosis within 48 hours in PC cells. Pilot studies in tumor-bearing mice show SCAPi significantly increases survival without inducing animal weight loss. Conclusions: There is a critical need for novel targets in PC, and dysregulated lipogenesis is an untargeted oncogenic pathway. Our work furthers the field’s understanding of hijacked lipogenesis in PC and paves the way for a first-in-class inhibitor of lipogenesis to treat lethal PC. Citation Format: Dallin Lowder, Jin Na Shin, Maria E. Ruiz-Echartea, Darlene Skapura, Jenny Deng, Aleksandra Rusin, Christel Davis, Erik Ehli, Cristian Coarfa, Michael Ittmann, Salma Kaochar. Developing a lipogenesis inhibitor for prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 442.

Previously reported shRNA knockdown studies have established that expression of the DNA repair enzyme human uracil DNA glycosylase (hUNG2) is protective against 5-flourodeoxyuridine (5FdUrd) cell killing in p53 mutated cancer cell lines, but not in cell lines with functional p53. Here we take a different approach to understanding the role of hUNG2 in 5FdUrd chemotherapy by selectively inhibiting the catalytic activity of hUNG2 using the small bacterial inhibitor protein UGI. UGI binds extremely tightly to the DNA binding and catalytic site of hUNG2, effectively mimicking the impact of a small molecule inhibitor and allowing us to explore the potential therapeutic vulnerabilities that loss of hUNG2 activity produces in p53 mutated and wild-type cancer cells. Accordingly, we used lentiviral transduction to introduce UGI, under control of a doxycycline-inducible promoter, into a large panel of cancer cell lines. In vitro hUNG2 activity measurements using cell extracts established that all lines had robust activity before induction of UGI and undetectable activity after induction. We find that some wild-type and mutant p53 cancer lines from different tissues show significant increases in drug sensitivity. For instance, the p53 wild-type MCF7 breast cancer line showed a 10,000-fold increase in sensitivity, while the p53 mutant HT29 colorectal cancer line showed a 50-fold increased sensitivity. Overall, the results do not support the proposal that p53 status itself determines whether a cancer cell will be sensitized to 5FdUrd in the presence of hUNG2 inhibition. Ongoing studies are focused on identifying the cellular factors that contribute to drug sensitivity under conditions of hUNG2 inhibition. Realizing the full therapeutic benefit of 5FdUrd requires identification of the cancer cell phenotypes that are sensitized upon hUNG2 inhibition. Citation Format: Eric S. Christenson, Junru Cui, Anthony Gizzi, Mesfin Meshesha, James T. Stivers. Inhibition of the catalytic activity of hUNG2 sensitizes multiple cancer cell lines to 5-FdUrd killing [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1390.

Genome editing with CRISPR-Cas systems hold promise for treating a wide range of genetic disorders and cancers. However, efficient delivery of genome editors remains challenging due to the requirement for the simultaneous delivery or intracellular generation of Cas proteins, guide RNAs, and, in some applications, donor DNAs. Furthermore, the immunogenicity and toxicity of delivery vehicles can limit the safety and efficacy of genetic medicines. Here, we combine two nucleic acid delivery approaches to create CRISPR lipid nanoparticle-spherical nucleic acids (LNP-SNAs) that are both efficient and biocompatible. Compared to lipid nanoparticles (LNPs) lacking a surface-bound DNA shell, CRISPR LNP-SNAs exhibit two- to three-fold higher cellular uptake, reduced cytotoxicity, and improved gene transfection efficiency. Across multiple cell lines and genomic loci, CRISPR LNP-SNAs induce insertion-deletion mutations at average frequencies two- to three-fold higher than those observed with LNPs. When codelivered with donor templates, CRISPR LNP-SNAs enable homology-directed repair at an average efficiency of 21 ± 7%, a 2.5-fold improvement over LNPs (8 ± 4%). The ease of synthesis and biocompatibility of CRISPR LNP-SNAs highlight their potential as a versatile delivery platform for CRISPR-Cas and other gene therapies.

Lenalidomide Induces Ubiquitination and Degradation of CSNK1A1 in MDS with Del(5q)

In this review article, we analyzed the literature on the creation of cultures containing mutations associated with cardiovascular diseases (CVD) using transfection, transduction and editing of the human genome. We described different methods of transfection, transduction and editing of the human genome, used in the literature. We reviewed the researches in which the creation of сell cultures containing mutations was described. According to the literature, system CRISPR/Cas9 proved to be the most preferred method for editing the genome. We found rather promising and interesting a practically undeveloped direction of mitochondria transfection using a gene gun. Such a gun can direct a genetically-engineered construct containing human DNA mutations to the mitochondria using heavy metal particles. However, in human molecular genetics, the transfection method using a gene gun is unfairly forgotten and is almost never used. Ethical problems arising from editing the human genome were also discussed in our review. We came to a conclusion that it is impossible to stop scientific and technical progress. It is important that the editing of the genome takes place under the strict control of society and does not bear dangerous consequences for humanity. To achieve this, the constant interaction of science with society, culture and business is necessary. The most promising methods for the creation of cell cultures containing mutations linked with cardiovascular diseases, were system CRISPR/Cas9 and the gene gun.