Human Gene Sequences Research Articles

MiCK: a database of gut microbial genes linked with chemoresistance in cancer patients.

Cancer remains a global health challenge, with significant morbidity and mortality rates. In 2020, cancer caused nearly 10 million deaths, making it the second leading cause of death worldwide. The emergence of chemoresistance has become a major hurdle in successfully treating cancer patients. Recently, human gut microbes have been recognized for their role in modulating drug efficacy through their metabolites, ultimately leading to chemoresistance. The currently available databases are limited to knowledge regarding the interactions between gut microbiome and drugs. However, a database containing the human gut microbial gene sequences, and their effect on the efficacy of chemotherapy for cancer patients has not yet been developed. To address this challenge, we present the Microbial Chemoresistance Knowledgebase (MiCK), a comprehensive database that catalogs microbial gene sequences associated with chemoresistance. MiCK contains 1.6 million sequences of 29 gene types linked to chemoresistance and drug metabolism, curated manually from recent literature and sequence databases. The database can support downstream analysis as it provides a user-friendly web interface for sequence search and download functionalities. MiCK aims to facilitate the understanding and mitigation of chemoresistance in cancers by serving as a valuable resource for researchers. Database URL: https://microbialchemreskb.com/.

Generative language models on nucleotide sequences of human genes.

Language models, especially transformer-based ones, have achieved colossal success in natural language processing. To be precise, studies like BERT for natural language understanding and works like GPT-3 for natural language generation are very important. If we consider DNA sequences as a text written with an alphabet of four letters representing the nucleotides, they are similar in structure to natural languages. This similarity has led to the development of discriminative language models such as DNABERT in the field of DNA-related bioinformatics. To our knowledge, however, the generative side of the coin is still largely unexplored. Therefore, we have focused on the development of an autoregressive generative language model such as GPT-3 for DNA sequences. Since working with whole DNA sequences is challenging without extensive computational resources, we decided to conduct our study on a smaller scale and focus on nucleotide sequences of human genes, i.e. unique parts of DNA with specific functions, rather than the whole DNA. This decision has not significantly changed the structure of the problem, as both DNA and genes can be considered as 1D sequences consisting of four different nucleotides without losing much information and without oversimplification. First of all, we systematically studied an almost entirely unexplored problem and observed that recurrent neural networks (RNNs) perform best, while simple techniques such as N-grams are also promising. Another beneficial point was learning how to work with generative models on languages we do not understand, unlike natural languages. The importance of using real-world tasks beyond classical metrics such as perplexity was noted. In addition, we examined whether the data-hungry nature of these models can be altered by selecting a language with minimal vocabulary size, four due to four different types of nucleotides. The reason for reviewing this was that choosing such a language might make the problem easier. However, in this study, we found that this did not change the amount of data required very much.

High-Throughput Expression and Purification of Human Solute Carriers for Structural and Biochemical Studies.

Solute carriers (SLCs) are membrane transporters that import and export a range of endogenous and exogenous substrates, including ions, nutrients, metabolites, neurotransmitters, and pharmaceuticals. Despite having emerged as attractive therapeutic targets and markers of disease, this group of proteins is still relatively underdrugged by current pharmaceuticals. Drug discovery projects for these transporters are impeded by limited structural, functional, and physiological knowledge, ultimately due to the difficulties in the expression and purification of this class of membrane-embedded proteins. Here, we demonstrate methods to obtain high-purity, milligram quantities of human SLC transporter proteins usingcodon-optimized gene sequences. In conjunction with a systematic exploration of construct design and high-throughput expression, these protocols ensure the preservation of the structural integrity and biochemical activity of the target proteins. We also highlight critical steps in the eukaryotic cell expression, affinity purification, and size-exclusion chromatography of these proteins. Ultimately, this workflow yields pure, functionally active, and stable protein preparations suitable for high-resolution structure determination, transport studies, small-molecule engagement assays, and high-throughput in vitro screening.

A Study and Analysis of Disease Identification using Genomic Sequence Processing Models: An Empirical Review.

Human gene sequences are considered a primary source of comprehensive information about different body conditions. A wide variety of diseases including cancer, heart issues, brain issues, genetic issues, etc. can be pre-empted via efficient analysis of genomic sequences. Researchers have proposed different configurations of machine learning models for processing genomic sequences, and each of these models varies in terms of their performance & applicability characteristics. Models that use bioinspired optimizations are generally slower, but have superior incremental-performance, while models that use one-shot learning achieve higher instantaneous accuracy but cannot be scaled for larger disease-sets. Due to such variations, it is difficult for genomic system designers to identify optimum models for their application-specific & performance-specific use cases. To overcome this issue, a detailed survey of different genomic processing models in terms of their functional nuances, application-specific advantages, deployment-specific limitations, and contextual future scopes is discussed in this text. Based on this discussion, researchers will be able to identify optimal models for their functional use cases. This text also compares the reviewed models in terms of their quantitative parameter sets, which include, the accuracy of classification, delay needed to classify large-length sequences, precision levels, scalability levels, and deployment cost, which will assist readers in selecting deployment-specific models for their contextual clinical scenarios. This text also evaluates a novel Genome Processing Efficiency Rank (GPER) for each of these models, which will allow readers to identify models with higher performance and low overheads under real-time scenarios.

Combining In Vitro and In Silico New Approach Methods to Investigate Type 3 Iodothyronine Deiodinase Chemical Inhibition Across Species.

New approach methodologies (NAMs) are being developed to reduce and replace vertebrate animal testing in support of ecotoxicology and risk assessment. The US Environmental Protection Agency's Sequence Alignment to Predict Across Species Susceptibility (SeqAPASS) bioinformatic tool was used to evaluate amino acid sequence conservation of the type 3 iodothyronine deiodinase (DIO3) enzyme across species to demonstrate NAM applications for understanding effects of chemical interactions with a specific protein target. Existing literature was used to identify critical amino acids for thyroid hormone binding and interaction with a reducing cofactor. The SeqAPASS tool identifies whether known critical amino acids involved in ligand binding are exact, partial, or not matches across species compared with a template species based on molecular weight and side chain classification. This evaluation guided the design of variant proteins representing critical amino acid substitutions found in various species. Site-directed mutagenesis of the wild-type (WT) human DIO3 gene sequence was used to create six variant proteins expressed in cell culture, which were then tested in vitro for chemical inhibition. Significant differences in in vitro median inhibitory concentration results were observed among variants for potential competitive inhibitors. A molecular model representing the WT human DIO3 was constructed using Molecular Operating Environment (MOE) software and mutated in silico to create the six variants. The MOE Site Finder tool identified the proposed catalytic and cofactor sites and potential alternative binding sites. Virtual docking did not provide affinity scores with sufficient resolution to rank the potency of the chemical inhibitors. Chemical characteristics, function and location of substituted amino acids, and complexities of the protein target are important considerations in developing NAMs to evaluate chemical susceptibility across species. Environ Toxicol Chem 2023;42:1032-1048. © 2023 University of Wisconsin-Madison. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.

Abstract 5208: Development of immunodeficient mice expressing human IL3, GM-CSF, CSF1 and THPO for improved human myeloid and lymphoid cell reconstitution

Abstract Mice reconstituted with human immune cells are essential tools to study human immune reactions in vivo, including those involving cancer immunotherapies. A major limitation of these models is the poor development of human myeloid cells to mediate effective human immune cell-tumor interactions. This is because hematopoietic cells are highly dependent on several human cytokines for efficient differentiation, maturation, and survival. THPO (thrombopoietin) can enhance the maintenance of functional human hematopoietic stem cells, and stimulates the production and differentiation of megakaryocytes, which produce large numbers of platelets in the bone marrow. IL3 (interleukin 3), GM-CSF (granulocyte-macrophage colony-stimulating factor, CSF2) and CSF1 (macrophage colony-stimulating factor, M-CSF) are cytokines that promote the proliferation and differentiation of a variety of myeloid cells, including monocytes, macrophages, granulocytes, dendritic cells, and platelets. Because these cytokines lack sufficient cross-reactivity between human and mice, it is difficult to reconstitute human immune cells of these lineages in immunodeficient mice. Therefore, we predict that genetic humanization of these four cytokines will improve the reconstitution of human myeloid cells in immunodeficient mice. We engineered an M-CSF, GM-CSF, THPO, and IL3 humanized strain in the background of B-NDG mice (NOD.CB17-PrkdcscidIl2rgtm1/Bcgen), termed B-NDG MGMT3 mice. The full sequences of mouse Il3, Csf2, Csf1 and Thpo genes (except the UTRs) were respectively replaced with the coding sequences (CDS) of human IL3, CSF2, CSF1 and THPO genes. Human GM-CSF, CSF1 and THPO proteins were confirmed to be expressed by ELISA in B-NDG MGMT3 mice. Human CD34+ HSCs (3E4) were intravenously engrafted into wild-type B-NDG mice and homozygous B-NDG MGMT3 male and female newborn mice via the temporal vein. B-NDG mice were treated with 1.0 Gy irradiation, while B-NDG MGMT3 mice were not irradiated. The survival rate of B-NDG MGMT3 mice was similar to B-NDG mice until 16 weeks post-CD34+ engraftment. However, the body weight of B-NDG MGMT3 mice was significantly higher than that of B-NDG mice. Analysis of peripheral blood lymphocytes showed that the proportion of hCD45+ cells in B-NDG MGMT3 mice reached 25% starting from 12 weeks after engraftment and continued to rise; levels were significantly higher than that observed in B-NDG mice. The proportions and cell number of T cells, NK cells, monocytes, MDSCs, DCs and Tregs engrafted in B-NDG MGMT3 mice were higher than B-NDG mice. The results indicate that B-NDG MGMT3 mice are a novel humanized model for human myeloid and lymphocytic cell reconstitution that does not require preconditioning. Citation Format: Ruili Lv, Yanhui Nie, Jing Zhang, Jenna Frame, Qiang Liu, James Jin. Development of immunodeficient mice expressing human IL3, GM-CSF, CSF1 and THPO for improved human myeloid and lymphoid cell reconstitution. [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 5208.

Abstract 3233: A novel triple humanized B-hPD-1/hPD-L1/hCCR8 mouse model to evaluate in vivo efficacy of combination therapies

Abstract Regulatory T cells (Tregs) play an immunosuppressive role in the tumor microenvironment (TME), such that altering or depleting Treg function has emerged as an important anti-tumor strategy. Targeting CCR8, which is highly expressed in tumor-infiltrating Tregs, has shown its potency in reducing tumor burden and increasing responsiveness to immune-checkpoint blockade therapy. Collectively, this suggests a need for preclinical models that support Treg-targeted therapies. To address this, Biocytogen developed a triple humanized (B-hPD-1/hPD-L1/hCCR8) mouse model to evaluate in vivo efficacy of combination therapies. In this model, the full coding sequence of murine Ccr8 was replaced by the full coding sequence of human CCR8, while murine Pd-1 and Pd-l1 IgV domains were replaced by their human counterpart gene sequences. Human PD-1 and PD-L1 protein expression was confirmed in B-hPD-1/hPD-L1/hCCR8 mice by flow cytometry, and human CCR8 protein expression was observed in MC38 tumors harvested from B-hPD-1/hPD-L1/hCCR8 mice. Finally, combination therapy using anti-human PD-1 and anti-human CCR8 antibodies were effective in controlling B-hPD-L1 MC38 tumor growth in B-hPD-1/hPD-L1/hCCR8 mice. Altogether, this line provides a powerful preclinical model for in vivo evaluation of therapeutic antibodies. Citation Format: Linlin Wang, Xiao Liu, James Jin, Yuxiang Yu. A novel triple humanized B-hPD-1/hPD-L1/hCCR8 mouse model to evaluate in vivo efficacy of combination therapies [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 3233.

Low-scale production and purification of a biologically active optimized form of the antitumor protein growth arrest specific 1 (GAS1) in a mammalian system for post-translational analysis

Growth arrest specific 1 (GAS1) is a protein involved in cell arrest that induces quiescence and apoptosis, when it is overexpressed in tumor cells. We have previously reported the generation of a soluble truncated protein known as tGAS1, with antitumoral capacity. In this paper two lentiviral constructs were developed, containing: the native human gene sequence fused to a Hisx6-tag (tG1Hc); or a synthetic and optimized DNA sequence (tG1Hs). Both transgenes encode for a version of the human tGAS1 protein with the addition of a Hisx6-tag in the C-terminus (tG1H). Resultant VSV-G pseudotyped lentiviruses were used to generate HEK-293FT and U87-MG cell cultures stable for the constitutive production of tG1H proteins. The data show that only U87-MG expressed the recombinant proteins when infected. In fact, conditioned medium obtained from U87-MG cells producing tG1Hs mRNA preserves anti-proliferative and pro-apoptotic properties. Noteworthy, viral particles carrying the tG1Hs transgene inhibit tumor growth in a heterotopic xenograft mice model. Additionally, the recombinant tG1H protein was purified by multiple affinity protocols and used to perform further experiments to analyze post-translational processing or affinity co-precipitation protocols. In the present work we have studied the N-glycosylation of GAS1 in the purified protein.

Solution structure of poly(UG) RNA reveals a new fold that drives gene silencing

The human genome is extraordinarily abundant in polymorphic (GT)n microsatellite sequences, which code for poly(UG) dinucleotide repeats in RNA (hereafter, “pUGs”). Expansion of pUG sequences have been correlated with diseases such as cancer and cystic fibrosis. In contrast, C. elegans lacks genomic pUGs but possesses a ribonucleotidyl transferase that can add “pUG tails” to RNA 3’ ends. These pUG tails turn RNAs into potent vectors for gene silencing. We therefore hypothesized that pUGs may form RNA structures responsible for gene silencing and possibly, human disease. We report the solution structure of the pUG sequence (GU)12 using solution NMR in combination with SAXS-WAXS scattering data at 0.75 Å r.m.s.d. The RNA folds into a newly identified single-stranded quadruplex characterized by its unique left-handed structure which we define as the “pUG fold”. This structure has four-fold symmetry with non-sequential stacking of 3 G quartets and a U quartet. Other U nucleotides are either budged or in propeller loops. Interestingly, the backbone presents alternate inversions at the quartets, a feature that closely resembles Z-RNA. NMR H/D exchange measurements indicate the pUG fold is kinetically stable for days. We also determined the x-ray crystal structure of (GU)12 bound to the quadruplex ligand N-methyl meosporphyrin IX (NMM). The structures have an r.m.s.d of 1.7 Å, indicating that NMM docks to a pre-folded pUG structure. NMR revealed features of the RNA that were poorly resolved by the electron density maps. The importance of the pUG fold for gene silencing in C. elegans was demonstrated by incorporation of the modified nucleotide 7-deaza guanosine into pUG tailed RNAs. The modified nucleotides disrupt RNA folding and potently inhibit gene silencing. We find ∼20,000 pUG sequences in human genes, suggesting roles beyond gene silencing.

Integrated mRNA sequence optimization using deep learning.

The coronavirus disease of 2019 pandemic has catalyzed the rapid development of mRNA vaccines, whereas, how to optimize the mRNA sequence of exogenous gene such as severe acute respiratory syndrome coronavirus 2 spike to fit human cells remains a critical challenge. A new algorithm, iDRO (integrated deep-learning-based mRNA optimization), is developed to optimize multiple components of mRNA sequences based on given amino acid sequences of target protein. Considering the biological constraints, we divided iDRO into two steps: open reading frame (ORF) optimization and 5' untranslated region (UTR) and 3'UTR generation. In ORF optimization, BiLSTM-CRF (bidirectional long-short-term memory with conditional random field) is employed to determine the codon for each amino acid. In UTR generation, RNA-Bart (bidirectional auto-regressive transformer) is proposed to output the corresponding UTR. The results show that the optimized sequences of exogenous genes acquired the pattern of human endogenous gene sequence. In experimental validation, the mRNA sequence optimized by our method, compared with conventional method, shows higher protein expression. To the best of our knowledge, this is the first study by introducing deep-learning methods to integrated mRNA sequence optimization, and these results may contribute to the development of mRNA therapeutics.

Recognition of 5-methyl-CG and CG base pairs in duplex DNA with high stability using antiparallel-type triplex-forming oligonucleotides with 2-guanidinoethyl-2'-deoxynebularine.

The formation of triplex DNA is a site-specific recognition method that directly targets duplex DNA. However, triplex DNA formation is generally formed for the GC and AT base pairs of duplex DNA, and there are no natural nucleotides that recognize the CG and TA base pairs, or even the 5-methyl-CG (5mCG) base pair. Moreover, duplex DNA, including 5mCG base pairs, epigenetically regulates gene expression in vivo, and thus targeting strategies are of biological importance. Therefore, the development of triplex-forming oligonucleotides (TFOs) with artificial nucleosides that selectively recognize these base pairs with high affinity is needed. We recently reported that 2'-deoxy-2-aminonebularine derivatives exhibited the ability to recognize 5mCG and CG base pairs in triplex formation; however, this ability was dependent on sequences. Therefore, we designed and synthesized new nucleoside derivatives based on the 2'-deoxy-nebularine (dN) skeleton to shorten the linker length connecting to the hydrogen-bonding unit in formation of the antiparallel motif triplex. We successfully demonstrated that TFOs with 2-guanidinoethyl-2'-deoxynebularine (guanidino-dN) recognized 5mCG and CG base pairs with very high affinity in all four DNA sequences with different adjacent nucleobases of guanidino-dN as well as in the promoter sequences of human genes containing 5mCG base pairs with a high DNA methylation frequency.

Spatial and Temporal Expression Characteristics of the HBB Gene Family in Six Different Pig Breeds.

Simple Summaryβ-Thalassemia is one of the most prevalent inherited diseases in China. It is important to develop animal models to accurately simulate human β-thalassemia and there are unique advantages to studying β-thalassemia in pigs. However, there are only few reports on the systematic analysis of the β-thalassemia-related genes and their expression pattern in pigs so far. Therefore, in this study, we firstly predicted 11 porcine hemoglobin-encoding genes and found that there was no HBG gene in pigs, indicating that the globin switches might not exist in pigs. A new hemoglobin-encoding gene, ‘HBB-like’, was found in pigs, which showed high conservation in its amino sequences between pigs and humans. Then, we studied the evolutionary relationship of hemoglobin-encoding genes in human, pig and mouse. The results showed that the β-chain structure of pig and human was highly similar. In addition, we analyzed the hemoglobin-encoding gene expressions by using the iswine database and qPCR. Our results showed significant differences in the spatiotemporal expression patterns among the four genes (HBA, HBB, HBB-like and HBE) in three developmental stages of six different pig breeds. Our study provides an important theoretical basis for further construction of a gene-edited β-thalassemia miniature pig model to assess β--thalassemia treatments.β-Thalassemia induces hemolytic anemia caused by mutations in the β-chain gene locus. As humans progress from embryo to adulthood, hemoglobin recombines twice. To test whether similar hemoglobin reassembly occurs in pigs, bioinformatics tools were used to predict the pig hemoglobin-encoding gene. We then systematically analyzed the expression patterns of the HBB gene family in three developmental stages (weaning, sexual maturity and physical maturity) of six different pig breeds (Landrace, Yorkshire, Wuzhishan, Songliao black, Meishan and Tibetan). The results showed that the new hemoglobin coding gene ‘HBB-like’ was found in pigs, while the HBG gene did not exist in pigs, indicating that human-like reassembly might not exist in pigs. The HBB and HBB-like genes shared highly similar amino acid sequences and gene sequences. The genes on the β-chain were highly similar between humans and pigs and the amino acid sequences of human and pig HBB genes at position 26 and positions 41–42 were identical. qPCR results showed that there were significant differences in the spatiotemporal expression patterns of the four genes (HBA, HBB, HBB-like and HBE) across breeds. Our results provide a foundation for follow-up studies assessing the relationship between the gene-encoding hemoglobin and β-thalassemia disease, as well as the construction of a gene-edited β-thalassemia miniature pig model to assess β-thalassemia treatments.

Translational enhancement by base editing of the Kozak sequence rescues haploinsufficiency.

A variety of single-gene human diseases are caused by haploinsufficiency, a genetic condition by which mutational inactivation of one allele leads to reduced protein levels and functional impairment. Translational enhancement of the spare allele could exert a therapeutic effect. Here we developed BOOST, a novel gene-editing approach to rescue haploinsufficiency loci by the change of specific single nucleotides in the Kozak sequence, which controls translation by regulating start codon recognition. We evaluated for translational strength 230 Kozak sequences of annotated human haploinsufficient genes and 4621 derived variants, which can be installed by base editing, by a high-throughput reporter assay. Of these variants, 149 increased the translation of 47 Kozak sequences, demonstrating that a substantial proportion of haploinsufficient genes are controlled by suboptimal Kozak sequences. Validation of 18 variants for 8 genes produced an average enhancement in an expression window compatible with the rescue of the genetic imbalance. Base editing of the NCF1 gene, whose monoallelic loss causes chronic granulomatous disease, resulted in the desired increase of NCF1 (p47phox) protein levels in a relevant cell model. We propose BOOST as a fine-tuned approach to modulate translation, applicable to the correction of dozens of haploinsufficient monogenic disorders independently of the causing mutation.

Effects of silencing farnesyltransferase on the migration, invasion, and epithelial-mesenchymal transition of salivary adenoid cystic carcinoma cells.

This study aimed to investigate the effects of farnesyltransferase (FTase) on the migration, invasion, and epithelial-mesenchymal transition (EMT) of SACC-LM and SACC-83 cells in salivary adenoid cystic carcinoma and determine the relative mechanism. Three small interfering RNA (siRNA) sequences were designed and constructed based on the human FTase gene sequence. The SACC-LM and SACC-83 cells in the logarithmic growth period were used, and the expression of FTase was suppressed by liposomal transient transfection. The tested cells were categorized as the FTase-siRNA-1, FTase-siRNA-2, and FTase-siRNA-3 groups. Both negative control group (NC-siRNA) and blank control group (only transfection reagent was added) were set. The mRNA expression of FTase and HRAS was detected by quantitive real-time polymerase chain reaction, and the silencing efficiency was determined. The expression levels of FTase, HRAS, protein kinase B (AKT), phospho-AKT, p65, phospho-p65 (Ser563), E-cadherin, vimentin, matrix metalloproteinase (MMP)-9 protein, and HRAS membrane protein were detected by Western blot. Transwell assay and wound healing assay were used to detect the invasion and migration abilities of cells. The relative expression of FTase mRNA and protein in the FTase-siRNA-1 group decreased compared with those in the control group (P<0.05). HRAS mRNA and total protein expression had no significant difference (P>0.05), and the relative expression of HRAS membrane protein decreased (P<0.05). The relative expression of E-cadherin increased (P<0.05), vimentin decreased (P<0.05), and MMP-9 decreased (P<0.05). There was no significant difference in the relative expression levels of the RAS/PI3K/AKT/nuclear factor-κB signaling pathway-related proteins AKT and p65 (P>0.05), but the relative expression levels of phospho-AKT and phospho-p65 decreased. The invasion and migration ability of the FTase-siRNA-1 group significantly decreased compared with that in the control group (P<0.05). Silencing FTase in vitro could effectively inhibit the invasion and migration of SACC-LM and SACC-83 cells by interfering with the localization of the HRAS membrane protein and regulating the RAS/PI3K/AKT/nuclear factor-κB signaling pathway to mediate EMT.

Abstract 1643: B-hIL12A/hIL12B/hIL12RB1/hIL12RB2: A novel animal model for generation of IL12 therapies

Abstract The pro-inflammatory cytokine, interleukin 12 (IL12), is primarily responsible for cell-mediated immunity and is chiefly produced by antigen presenting cells. Prior preclinical studies elucidated a promising role for IL12 mediating anti-tumor effects, however, clinical studies using cancer patients showed disappointing efficacy results, as well as severe adverse effects, with administration of systemic IL12 therapies. Intriguingly, progress in targeted delivery to the tumor microenvironment renewed high expectations on IL12 as a promising anti-tumor drug either alone or in combination due to its ability to inhibit and/or reprogram immunosuppressive cell subsets. Therefore, to evaluate the efficacy of IL12 and IL12 analogs in preclinical studies, Biocytogen has successfully generated a novel hIL12A/hIL12B/hIL12RB1/hIL12RB2 knock-in mouse strain. In this mouse model, the full coding sequences (CDS) of the mouse Il12a or Il12b gene were replaced by their human counterpart IL12A or IL12B gene sequences respectively. In addition, chimeric CDS, composed of human IL12RB1 or IL12RB2 extracellular region plus mouse Il12rb1 or Il12rb2 cytoplasmic region, was inserted into the mouse endogenous gene locus in in these animals, to express an IL12 receptor (IL12R) capable of recognizing human IL12 signaling. Expression analysis by RT-PCR detected human IL12A/IL12B/IL12RB1/IL12RB2 mRNA in humanized mice but not in wild-type mice. Additionally, human IL12 p70 was successfully detected in the serum of humanized mice by ELISA. The immune cell subpopulations in spleen, blood, and lymph node did not differ between humanized mice and wild-type mice suggesting no impact on cell differentiation and maturation. Anti-tumor activity of a modified human IL12 was assessed in humanized mice bearing colon cancer MC38 cells and showed tumor inhibitory effects. Moreover, the tumor growth recovered when the administration of the drug was stopped. Altogether, B-hIL12A/hIL12B/hIL12RB1/hIL12RB2 humanized mice provide a promising preclinical efficacy model for human IL12 therapy alone or in combination. Keywords: IL12, humanized mice, antibody efficacy Citation Format: Linlin Wang, Chang Liu, Veronika Chromikova, Ruili Lyu, Tianquan Jin, Mari Kuraguchi. B-hIL12A/hIL12B/hIL12RB1/hIL12RB2: A novel animal model for generation of IL12 therapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1643.

Pathophysiology and system biology of rat c-BSA induced immune complex glomerulonephritis and pathway comparison with human gene sequencing data

Immune-complex glomerulonephritis (ICGN) is a major cause of nephrotic syndrome in adults and children. Cationic BSA (c-BSA) intravenous injection could produce significant albuminuria within a short time, and is a suitable in vivo experimental animal model to investigate the pathophysiology of ICGN and for drug screening, but lack of thorough study to clarify its dynamic pathophysiological alteration so far, as well as detailed changes in mRNA and LncRNA levels. The purpose of this study is to investigate the dynamic alteration in renal function, lipid metabolism and histopathology during the progress of c-BSA induced ICGN. RNA sequencing was used to identified differentially expressed mRNA and LncRNA in kidney cortex of ICGN. Results demonstrated that c-BSA induced ICGN model could completely exhibit clinical features of immune-mediated nephrotic syndrome with gradual declining renal function, and increased albuminuria and deteriorated histopathological injuries. The correlation analysis suggested that complement activation was the most key element in mediating of ICGN. RNA sequencing using rat kidney tissues combined with Gene Expression Omnibus (GEO) data of human glomerulonephritis showed the most enriched KEGG pathways in ICGN were Toll-like receptor signaling pathway, B cell receptor and Focal adhesion. The differential lncRNAs in ICGN rats were also screened, and the lncRNA-mRNA co-expression network was constructed to clarify lncRNA role in molecular mechanism of ICGN progression. Their human homogenous lncRNAs were also identified, such as ST3GAL5-AS1 and DIO3OS, which provide the potential lncRNA targets to treat ICGN. All the differential LncRNAs in ICGN kidneys caused by MMF were also identified and provided another possible pharmacological mechanism of MMF through lncRNA regulation. In summary, the current study firstly described the dynamic physiological changes of c-BSA induced ICGN, identified most key KEGG pathways, and provided lncRNA-mRNA regulatory network in ICGN.

Modular Cloning of MUC1 Recombinant Antibodies by Assembly of Synthetic Domain Genes

Antibody immunotherapy has emerged as a constructive way to fight cancer. New immunotherapies require the identification of a tumor‐specific antigen. The protein mucin 1 (MUC1) has long been recognized as an important specific antigen and a potential immunotherapy target. In healthy human tissues, MUC1 is responsible for protecting the epithelial lining. However, in cancer cells, MUC1 displays truncated glycosylation, revealing tumor‐specific epitopes. Recombinant antibodies are preferred for therapeutic development, and in some cases, it is desirable to produce different antibody formats, including intact IgG or Fab fragments. Using the MUC1 specific antibody 4H5 as a model, we have developed a modular cloning approach that can rapidly produce different antibody modalities to facilitate the rapid production of both IgG and Fab antibody formats. The 4H5 variable domains were PCR amplified to contain 20 base‐pair (bp) overlaps with the pcDNA3.1 vector on the 5’ end, and 20 bp overlaps to either the human CH1 or Ck domains on the 3’end of the genes. To produce the Fab, a His‐tag and a 20 base‐pair (bp) overlap to the vector was added to the 3’ end of a human CH1 domain gene sequence. For IgG production, human CH1‐CH2‐CH3 domains were PCR amplified, and the same vector overlap sequence was added to the 3’ end of the sequence. Finally, a 20 bp overlap to the vector was added to the 3’ end of a human Ck domain. The PCR products were purified, and the constructs were assembled using the NEBuilder®HiFi DNA Assembly Cloning Kit. DNA sequencing confirmed the successful assembly of the coding sequence, and the 4H5 Fab fragments and IgG were produced by transient transfection in CHO cells. The antibodies (Fab and IgG) were purified by affinity chromatography and binding to recombinant MUC1 antigens confirmed by ELISA. The cloning method presented here simultaneously produces multiple antibody formats. This approach will permit rapid assembly and screening of different antibody formats and isotypes for therapeutic antibody discovery and analysis.

A universal in silico V(D)J recombination strategy for developing humanized monoclonal antibodies

BackgroundHumanization of mouse monoclonal antibodies (mAbs) is crucial for reducing their immunogenicity in humans. However, humanized mAbs often lose their binding affinities. Therefore, an in silico humanization method that can prevent the loss of the binding affinity of mAbs is needed.MethodsWe developed an in silico V(D)J recombination platform in which we used V(D)J human germline gene sequences to design five humanized candidates of anti-tumor necrosis factor (TNF)-α mAbs (C1–C5) by using different human germline templates. The candidates were subjected to molecular dynamics simulation. In addition, the structural similarities of their complementarity-determining regions (CDRs) to those of original mouse mAbs were estimated to derive the weighted interatomic root mean squared deviation (wRMSDi) value. Subsequently, the correlation of the derived wRMSDi value with the half maximal effective concentration (EC50) and the binding affinity (KD) of the humanized anti-TNF-α candidates was examined. To confirm whether our in silico estimation method can be used for other humanized mAbs, we tested our method using the anti-epidermal growth factor receptor (EGFR) a4.6.1, anti-glypican-3 (GPC3) YP9.1 and anti-α4β1 integrin HP1/2L mAbs.ResultsThe R2 value for the correlation between the wRMSDi and log(EC50) of the recombinant Remicade and those of the humanized anti-TNF-α candidates was 0.901, and the R2 value for the correlation between wRMSDi and log(KD) was 0.9921. The results indicated that our in silico V(D)J recombination platform could predict the binding affinity of humanized candidates and successfully identify the high-affinity humanized anti-TNF-α antibody (Ab) C1 with a binding affinity similar to that of the parental chimeric mAb (5.13 × 10−10). For the anti-EGFR a4.6.1, anti-GPC3 YP9.1, and anti-α4β1 integrin HP1/2L mAbs, the wRMSDi and log(EC50) exhibited strong correlations (R2 = 0.9908, 0.9999, and 0.8907, respectively).ConclusionsOur in silico V(D)J recombination platform can facilitate the development of humanized mAbs with low immunogenicity and high binding affinities. This platform can directly transform numerous mAbs with therapeutic potential to humanized or even human therapeutic Abs for clinical use.Graphical

New polymorphic DNA marker to determine a person's sex from biological material

The objective of the study was to pre-evaluate the applicability of gender-specific nucleotide sequences in human neuroligin genes as alternative DNA markers of sex. A new polymorphic locus based on NLGNX and NLGNY genes was proposed to establish the sex attribute of human biomaterials. The significant difference in the location of these loci relative to the pseudoautosomal region (PAR), as well as the combination of different types of polymorphism on the one hand, and the possibility of using gender-specific primers «in one assay» on the other hand, warrants their use as an additional marker of human sex attribute, including utilization as part of systems for DNA registration in the population. The introduction of a new polymorphic locus based on the NLGNX and NLGNY genes will make it possible to reliably identify the sex attribute of biological material recovered from crime scenes.

Diversity Control in Early Transcribed ROS/RNS-balancing Genes: A Common Mechanism for Healthy Resilience?

Diversity Control in Early Transcribed ROS/RNS-balancing Genes: A Common Mechanism for Healthy Resilience?