130 Engineered natural killer cells reactively block TIGIT and CD73 in the GBM microenvironment

BackgroundNatural killer (NK) cells have emerged as promising effectors to target GBM and other solid tumors through genetic modifications and ex vivo manipulation. However, immunosuppressive conditions within the tumor microenvironment (TME) and interactions between NK cell activating and inhibitory receptors further complicate NK cell-based treatments. In particular, the T cell immunoreceptor with Ig and ITIM domains (TIGIT) is expressed on NK cells and interacts with CD155 to induce immunosuppression of NK cell cytolytic functions.1 2 Although CD155 also binds with activating receptors DNAM-1 and CD96 on NK cells, spurring NK cell activity, TIGIT has predominantly been reported as having an inhibitory effect on NK cells.3–5 Further, tumor cells release of high levels of ATP extracellularly. While intracellular ATP is necessary for cell metabolism, extracellular ATP is converted into adenosine (ADO) by ectonucleotidases CD39 and CD73, both overexpressed on GBM and other solid tumors.6 Extracellular ADO induces immunometabolic suppression of NK cells through binding with A2A adenosine receptors (A2ARs) on NK cells, suppressing cytokine secretion, proliferation, and other functional activities.7–9 We found that TIGIT and CD73 are effective combination targets in GBM for both primary and iPSC-derived NK cells.MethodsIn order to effectively target immunometabolic reprogramming induced by CD73-produced adenosine and the immunosuppressive TIGIT-CD155 axis, we have engineered NK cells to concomitantly target CD155 and CD73-induced immunosuppression on GBM using a tumor-responsive genetic construct based on the synNotch signaling system. The construct is capable of blocking the immunosuppressive CD155/TIGIT interaction, and, upon binding, release a CD73-blocking scFv to inhibit the accumulation of extracellular ADO and mitigate immunosuppression of NK cells. Such localized response enhances specificity and reduces off-target effects of NK-based targeting.ResultsPrimary NK cells and iPSC-derived NK cells were successfully engineered to express the synthetic TIGIT-synNotch construct, measured through expression of TIGIT. To evaluate the functionality of engineered NK cells against GBM targets, we tested the cytotoxicity of our engineered NK cells against a primary, patient-derived GBM cell line, GBM43. Overall, cytolytic function of engineered NK cells against GBM was significantly higher than that of non-engineered NK cells, with or without CD73 (10 ug/mL) and TIGIT (50 ug/mL) antibodies, for E:T ratios of 5:1 and 10:1, demonstrating the functional efficacy of our genetic construct.ConclusionsOverall, we have shown that co-targeting CD155 and CD73 in a localized, responsive manner can dampen immunosuppression and significantly enhance the killing potential of engineered NK cells against aggressive patient-derived GBM tumors.ReferencesZhang B, et al. Immunoreceptor TIGIT inhibits the cytotoxicity of human cytokine-induced killer cells by interacting with CD155. Cancer Immunol Immunother 2016;65:305–314.Lupo KB & Matosevic S. CD155 immunoregulation as a target for natural killer cell immunotherapy in glioblastoma. J Hematol Oncol 2020;13:76.Hung AL, et al. TIGIT and PD-1 dual checkpoint blockade enhances antitumor immunity and survival in GBM. OncoImmunology 2018; e1466769. doi:10.1080/2162402X.2018.1466769.Mahnke K & Enk, AH. TIGIT-CD155 Interactions in Melanoma: A Novel Co-Inhibitory Pathway with Potential for Clinical Intervention. Journal of Investigative Dermatology 2016; 136, 9–11.Stanietsky N, et al. Mouse TIGIT inhibits NK-cell cytotoxicity upon interaction with PVR: Innate immunity. Eur J Immunol 2013; 43:2138–2150.Chambers AM, et al. Adenosinergic Signaling Alters Natural Killer Cell Functional Responses. Front Immunol 2018;9:2533.Chambers AM, Lupo KB & Matosevic S. Tumor Microenvironment-Induced Immunometabolic Reprogramming of Natural Killer Cells. Front Immunol 2018;9:2517.Chambers AM. et al. Adenosinergic Signaling Alters Natural Killer Cell Functional Responses. Front Immunol 2018;9:2533.Wang J, Lupo KB, Chambers AM & Matosevic S. Purinergic targeting enhances immunotherapy of CD73+ solid tumors with piggyBac-engineered chimeric antigen receptor natural killer cells. J Immunotherapy Cancer 2018;6:136.Ethics ApprovalPrimary human NK cells were obtained from healthy adult donors approved under Purdue University’s Institutional Review Board (IRB) (IRB-approved protocol #1804020540). Donors gave written informed consent prior to taking part in the study.

Reconstructing Sociogenomics Research: Dismantling Biological Race and Genetic Essentialism Narratives

We detail the implications of sociogenomics for social determinants research. We focus on education and race because of how early twentieth-century scientific eugenic thinking facilitated a range of racist and eugenic policies, most of which helped justify and pattern racial and educational morbidity and mortality disparities that remain today, and are central to sociological research. Consequently, we detail the implications of sociogenomics research by unpacking key controversies and opportunities in sociogenomics as they pertain to the understanding of racial and educational inequalities. We clarify why race is not a valid biological or genetic construct, the ways that environments powerfully shape genetic influence, and risks linked to this field of research. We argue that sociologists can usefully engage in genetics research, a domain dominated by psychologists and behaviorists who, given their focus on individuals, have mostly not examined the role of history and social structure in shaping genetic influence.

An increased proportion of transgenic plants in the progeny of rapeseed (Brassica napus L.) transformants

Cotyledon and leaf explants of two spring rapeseed varieties were transformed with Agrobacterium tumefaciens harboring a genetic construct with the gfp marker gene. In order to reduce the proportion of hyperhydrated shoots, which appeared during regenerant formation, we optimized sucrose content in the regeneration media. Analysis of the progeny obtained from T0 regenerants showed that in a number of lines the distribution of the gfp marker did not follow Mendelian segregation of a monogenic trait in self-pollinated plants, while in the progeny of the other lines of transgenic plants, the gfp marker was completely absent, although its presence had been confirmed in all selected T0 plants. We also found that in individual transformants gfp is randomly inherited throughout the central peduncle; its presence in the genome of seedlings does not depend on the location of the pod. Thus, both transformed and non-transformed cells were involved in the formation of gametes in T0 plants. In addition, marker segregation was different in plants of the T1 line obtained by nodal cuttings of a primary transformant, depending on the location of the cuttings on the stem of the original plant, indicating that the nature of T1 generation plants was also chimeric. Furthermore, we showed that propagation of plants by cutting followed by propagation by seeds formed as a result of self-pollination led to an increase in the proportion of transgenic plants in subsequent generations. The results obtained during the course of this study show that the transformants were chimeric, i. e. their tissues contained both transgenic and non-transgenic cells, and this chimeric nature was passed on to subsequent generations. We found that, in addition to nutrient media composition, other factors such as plant genotype and explant type also contribute to the rising of chimeric plants during transformation. Based on these results, we developed a simplified method, which consists of several rounds of a combination of cutting, seed production by self-pollination, and subsequent culling of wild-type plants, which significantly enriched descendent populations of the original rapeseed transformants with plants transgenic for the gfp marker.

Positive symptom phenotypes appear progressively in “EDiPS”, a new animal model of the schizophrenia prodrome

An increase in dopamine (DA) synthesis capacity in the dorsal striatum (DS) during the prodromal stage of schizophrenia becomes more pronounced as patients progress to the full disorder. Understanding this progression is critical to intervening in disease course. We developed an animal model—Enhanced Dopamine in Prodromal Schizophrenia (EDiPS)—which uses a genetic construct to increase DA synthesis capacity in the DS of male rats. We assessed pre-pulse inhibition (PPI) and amphetamine (AMPH)-induced locomotion (0.6 mg/kg) in EDiPS animals longitudinally after post-natal day 35 (when the EDiPS construct is administered). We also assessed their response to repeated acute restraint stress. In adult EDiPS animals, we measured baseline and evoked extracellular DA levels, and their stereotyped responses to 5 mg/kg AMPH. AMPH-induced hyperlocomotion was apparent in EDiPS animals 6-weeks after construct administration. There was an overall PPI deficit in EDiPS animals across all timepoints, however the stress response of EDiPS animals was unaltered. Adult EDiPS animals show normal baseline and potassium-evoked DA release in the DS. These findings suggest that key behavioural phenotypes in EDiPS animals show a progressive onset, similar to that demonstrated by patients as they transition to schizophrenia. The EDiPS model could therefore be used to investigate the molecular mechanisms underlying the prodrome of schizophrenia.

RNAi-Mutants of Sorghum bicolor (L.) Moench with Improved Digestibility of Seed Storage Proteins

Modification of the composition of grain storage proteins is an intensively developing area of plant biotechnology, which is of particular importance for sorghum – high-yielding drought tolerant crop. Compared to other cereals, the majority of sorghum cultivars and hybrids are characterized by reduced nutritional value that is caused by a low content of essential amino acids in the seed storage proteins (kafirins), and resistance of kafirins to protease digestion. RNA interference (RNAi) by suppressing synthesis of individual kafirin subclasses may be an effective approach to solve this problem. In this chapter, we review published reports on RNAi silencing of the kafirin-encoding genes. In addition, we present new experimental data on phenotypic effects of RNAi-silencing of γ-KAFIRIN-1 gene in sorghum cv. Avans. To obtain RNAi mutants with γ-KAFIRIN-1 gene silencing we used Agrobacterium-mediated genetic transformation. Transgenic kernels had modified endosperm type with reduced vitreous layer and significantly improved in vitro protein digestibility (93% vs. 57%, according to the densitometry of SDS-PAGE patterns). SDS-PAGE of transgenic kernels showed lowered level of kafirins and appearance of globulin proteins, which were not observed in the original cultivar. For the first time, the cases of instability of inserted genetic construct were identified: elimination of ubi1-intron that is a constituent part of the genetic construct for RNAi silencing, or nos-promotor governing expression of the marker gene (bar) (in the RNAi mutants of cv. Zheltozernoe 10). The research findings presented in this chapter provide strong evidence that RNA interference can be used for improvement of the nutritional properties of sorghum grain.

Expression and Isolation of N-Terminal Truncated Human Recombinant Renalase in Prokaryotic Cells

Renalase (RNLS) is a flavoproteinin which its N-terminal peptide (residues 1-17) has several important functions. In cells, it participates in the formation of the so-called Rossmanfold (residues 2-35), needed for «accommodation» of the FAD cofactor and for performing the catalytic functions of RNLS as a FAD-dependent oxidoreductase (EC 1.6.3.5). RNLS secretion into the extracellular space is accompanied by cleavage of this peptide. The resultant truncated extracellular RNLS cannot bind FAD and therefore performs various noncatalytic functions. In this work, we have performed expression the genetic construct encoding RNLS lacking its N-terminal signal peptide (tRNLS) in E. coli Rosetta (DE3) cells. The recombinant protein was accumulated in inclusion bodies in an insoluble form, which could be solubilized in the presence of a high concentration of urea or guanidine chloride. In contrast to full-length RNLS, which was effectively solubilized in the presence of 8 M urea, tRNLS was preferentially solubilized in the presence of 6 M guanidine chloride.

123 Natural killer cells engineered with an inducible, responsive genetic construct targeting TIGIT and CD73 to relieve immunosuppression within the GBM microenvironment

BackgroundSolid tumors such as GBM are particularly difficult to treat, being largely resistant to traditional treatments, fueling interest in alternative treatment approaches, including cell-based immunotherapy. Natural killer (NK) cells have emerged as promising effectors to target GBM through genetic modifications and ex vivo manipulation. However, immunosuppressive conditions within the tumor microenvironment (TME) further complicate NK cell-based treatments. Specifically, within the TME tumor cells release of high levels of ATP extracellularly. While intracellular ATP is necessary for cell metabolism, extracellular ATP is converted into adenosine (ADO) by ectonucleotidases CD39 and CD73, both overexpressed on GBM.1 Extracellular ADO induces immunometabolic suppression of NK cells through binding with A2A adenosine receptors (A2ARs) on NK cells, suppressing cytokine secretion, proliferation, and other functional activities. 2–4 Adding to the suppression of NK cells is the interaction between CD155, expressed highly on GBM and other solid tumors, and T cell immunoreceptor with Ig and ITIM domains (TIGIT) expressed on NK cells. This interaction signals inhibition of NK cell cytolytic function, allowing for cancer cell immune-evasion.5,6MethodsTo restore impaired NK cell anti-tumor activity, we have engineered NK cells to concomitantly target CD155 and CD73-induced immunosuppression on GBM using a tumor-responsive genetic construct. The construct is capable of blocking the immunosuppressive CD155/TIGIT interaction, and, upon binding, release a CD73-blocking scFv to inhibit the accumulation of extracellular ADO and mitigate immunosuppression of NK cells. Such localized response enhances specificity and reduces off-target effects of NK-based targeting.ResultsPrimary NK cells were successfully electroporated to express our synthetic TIGIT-synNotch construct, as evidenced by increased expression levels of TIGIT (% and MFI) (figure 1). To evaluate the functionality of engineered NK cells against GBM targets, we tested the cytotoxicity of our engineered NK cells against a primary, patient-derived GBM cell line, GBM43. Overall, cytolytic function of engineered NK cells against GBM was significantly higher than that of non-engineered NK cells, with or without CD73 (10 ug/mL) and TIGIT (50 ug/mL) antibodies, for E:T ratios of 5:1 and 10:1 (figure 2), demonstrating the functional efficacy of our genetic construct. Further, engineered NK cells (T-PNK) expressed significantly higher levels of CD107a in response to GBM43 stimulation than non-engineered PNK at E:T ratios 2.5:1 and 10:1 (figure 3).Abstract 123 Figure 1TIGIT-synNotch gene expressionGene expression (left: %, right, MFI) of electroporated NK cells engineered with anti-CD73 and TIGIT blocking mRNAAbstract 123 Figure 2Engineered NK cell cytotoxicityCytotoxicity of NK cells against GBM43 cells at E:T ratios of 2.5:1, 5:1, and 10:1. NK cells were either un-transfected (with and without CD73 and TIGIT mAbs), transfected with the TIGIT-synNotch construct, or transfected with the TIGIT-synNotch and CD73 genetic constructsAbstract 123 Figure 3Engineered NK cell degranulationCD107a expression measured on transfected and non-transfected NK cells stimulated with GBM43 at E:T ratios of 2.5:1, 5:1, and 10:1ConclusionsOverall, we have shown that co-targeting CD155 and CD73 in a localized, responsive manner can dampen immunosuppression and significantly enhance the killing potential of engineered NK cells against aggressive patient-derived GBM tumors.ReferencesChambers AM, et al. Adenosinergic Signaling Alters Natural Killer Cell Functional Responses. Front. Immunol 2018;9:2533.Chambers AM, Lupo KB & Matosevic S. Tumor microenvironment-induced immunometabolic reprogramming of natural killer cells. Front Immunol 2018;9:2517.Chambers AM, et al. Adenosinergic signaling alters natural killer cell functional responses. Front. Immunol 2018;9:2533.Wang, J., Lupo, K. B., Chambers, A. M. & Matosevic, S. Purinergic targeting enhances immunotherapy of CD73+ solid tumors with piggyBac-engineered chimeric antigen receptor natural killer cells. J. immunotherapy cancer 2018;6:136.Zhang B, et al. Immunoreceptor TIGIT inhibits the cytotoxicity of human cytokine-induced killer cells by interacting with CD155. Cancer Immunol Immunother 2016;65:305–314.Lupo KB & Matosevic S. CD155 immunoregulation as a target for natural killer cell immunotherapy in glioblastoma. J Hematol Oncol 2020;13:76.

Strategy for modification of the bovine beta-lactoglobulin geneusing components of the CRISPR/Cas9 system in plasmid form

Using on-line programs, sites were selected for obtaining double-stranded breaks in the BLG gene of cattle. The strategy for making double-stranded cuts in the BLG gene was developed taking into account the polymorphic variant of the gene (A-allele): DNA was isolated from bovine sperm used for fertilization of cow eggs in vitro. Four pX330 plasmids encoding Cas9 endonuclease and gRNAs specific to the selected BLG gene sequences were obtained. A strategy was developed for analyzing possible genetic modifications resulting from the operation of the CRISPR/Cas9 system components and the genetic construct microinjected into zygotes (NHEJ, HDR). The pBLGcmvEGFP plasmid containing the green fluorescent protein gene under the cytomegalovirus promoter was proposed as a model genetic construct for replacing the BLG gene. The use of a plasmid containing the reporter protein gene under its own regulatory sequences, flanked by homology arms to the beta-lactoglobulin gene, can be useful for evaluating the effectiveness of site-specific activity of the CRISPR/Cas9 system components in vitro.

Creation of a genetic vector carrying a synthesis bacterial protein gene CAS9 for plant genome editing

Aim. To create a genetic construct carrying the bacterial protein Cas9 gene, the reporter β-glucuronidase gus gene, as well as the marker phosphinotricin-N-acetyltransferase bar gene for plant genome editing. Methods. Molecular-biological, biotechnological, microbiological and bioinformatic methods were used in the study; Golden Gate molecular cloning method was used to create genetic constructs. Results. The genetic construct pSPE2053 which carries the Cas9 endonuclease gene, the gus and bar genes was created; the assembly correctness of all vector elements was confirmed by polymerase chain reaction; the construct was transferred to Escherichia coli and Agrobacterium tumefaciens cells; β-glucuronidase gene expression was verified by histochemical analysis after Nicotiana rustica L transient genetic transformation. Conclusions. The created genetic construct can be used to edit the plant genome for both stable and transient genetic transformation to accumulate recombinant Cas9 protein. The guide RNA sequences may be subsequently transferred into such plants using either stable or transient genetic transformation or traditional crossing methods. Keywords: cloning, genetic construction, gus and bar genes, Cas9 endonuclease protein, transient expression.

Genetic transformation of new perspective winter wheat genotypes in vitro

Aim. Optimization of conditions for genetic transformation of new perspective winter wheat genotypes and production of transgenic plants. Methods. Agrobacterium-mediated transformation in vitro culture using callus cultures. Results. The influence of the optical density of cells of agrobacterial suspension, the concentration of the antibiotic cefotaxime, the duration of coculture on the frequency of obtaining kanamycin-resistant regenerants of new winter wheat genotypes by genetic transformation of callus cultures were investigated. By Agrobacterium-mediated transformation of morphogenic calluses of new perspective winter wheat genotypes were obtained plant-regenerants in the genome of which revealed the complete incorporation of a genetic construct containing oat and nptII transgenes. Conclusions. Agrobacterium-mediated transformation of callus cultures of new perspective winter wheat genotypes was optimized, and transgenic plants with the target gene of ornithine-δ-aminotransferase were obtained. Keywords: Triticum aestivum, Agrobacterium-mediated transformation, callus cultures, ornithine-δ-aminotransferase gene.