[Enhanced Natural Killers with CISH and B2M Gene Knockouts Reveal Increased Cytotoxicity in Glioblastoma Primary Cultures].

In an experimental study using the CRISPR/Cas9 system, "enhanced" NK cell lines with knockout of CISH, the gene for the CIS protein (a negative regulator of NK cytotoxicity), as well as two lines with a knocked-out ß2-microglobulin gene, which provides membrane exposure of MHC class I, were obtained from two parental lines of human natural killers (YT wild type and YT-VAV1^(+) overexpressing the VAV1 cytotoxicity enhancing protein). The knockout efficiency was determined by real-time PCR as well as by flow cytometry with specific antibodies. The resulting CISH^(-/-) or B2M^(-/-) knockout lines were tested for cytotoxicity in primary monolayer cultures of human glioblastoma multiforme. The cytotoxicity of the lines was assessed using a cell analyzer that records the cell index based on cell impedance. YT-CISH^(-/-) has been shown to be significantly more effective than wild-type YT in eliminating primary glioblastoma cells in an in vitro cell monolayer experiment. The cytotoxicity of the YT-VAV1^(+)-CISH^(-/-) and YT-VAV1^(+)B2M^(-/-) lines against glioblastoma cells was the highest, but overall, it did not significantly differ from the initially increased cytotoxicity of the YT-VAV1^(+) line. The lines of NK-like cells obtained may serve as a prototype for the creation of "enhanced" allogeneic and autologous NK- and CAR-NK cells for the immunotherapy of glioblastoma multiforme.

[Combination of Oncolytic Virotherapy and CAR T/NK Cell Therapy for the Treatment of Cancer].

Multiple lines of evidence indicate that CAR-T cell based therapy and oncolytic virotherapy display robust performance in both immunocompetent and immunodeficient mouse models. Rare, yet highly successful attempts to combine these therapeutic platforms have also been reported. Interestingly, both approaches have shown pronounced efficacy in human trials, albeit these were limited to just a handful of malignancies. Specifically, CD19-specific CAR-T cell products (Kymriah and Yescarta) have been highly effective against B cell lymphomas and leukemias, whereas administering oncolytic viruses resulted in pronounced responses in melanoma (Imlygic and Rigvir) and nasopharyngeal carcinoma (Oncorine) patients. It is well established that efficacy of virotherapy as a standalone approach is largely restricted by the pre-existing and mounting immune response against viral antigens, and requires a relatively functional immune system, which is not typical for cancer patients, with the current antitumor therapy schemes. On the other hand, the most important challenges faced by the current CAR-T cell therapy formats include the lack of targetable tumor-specific surface antigens, tumor cell heterogeneity, and immunosuppressive tumor microenvironment, not to mention the unacceptably high costs. Remarkably, combining the two approaches may help address their individual bottlenecks. Namely, local acute inflammatory reaction induced by the viral infection may reverse tumor-associated immunosuppression and lead to more efficient homing and penetration of CAR-expressing lymphocytes into the tumor stroma; combined viral and CAR-mediated cytotoxicity may ensure the production of immunogenic cell debris and efficient presentation of tumor neoantigens, and potently recruit the patient's own bystander immune cells to attack cancer cells. Thus, testing the combinations of CAR-based and virolytic approaches in the clinical setting appears both logical and highly promising.

shp-2 gene knockout upregulates CAR-driven cytotoxicity of YT NK cells.

In Russia, cancer is the second leading cause of death following cardiovascular diseases. Adoptive transfer of NK cells is a promising approach to fight cancer; however, for their successful use in cancer treatment, it is necessary to ensure their robust accumulation at tumor foci, provide resistance to the immunosuppressive tumor microenvironment, and to engineer them with higher cytotoxic activity. NK lymphocytes are known to kill cancer cells expressing a number of stress ligands; and the balance of signals from inhibitory and activating receptors on the surface of the NK cell determines whether a cytotoxic reaction is triggered. We hypothesized that stronger cytotoxicity of NK cells could be achieved via gene editing aimed at enhancing the activating signaling cascades and/or weakening the inhibitory ones, thereby shifting the balance of signals towards NK cell activation and target cell lysis. Here, we took advantage of the CRISPR/Cas9 system to introduce mutations in the coding sequence of the shp-2 (PTPN11) gene encoding the signaling molecule of inhibitory pathways in NK cells. These shp-2 knock-out NK cells were additionally transduced to express a chimeric antigen receptor (CAR) that selectively recognized the antigen of interest on the target cell surface and generated an activating signal. We demonstrate that the combination of shp-2 gene knockout and CAR expression increases the cytotoxicity of effector NK-like YT cells against human prostate cancer cell line Du-145 with ectopic expression of PSMA protein, which is specifically targeted by the CAR.

Engineering Chimeric Antigen Receptors.

Chimeric antigen receptors (CARs) are recombinant protein molecules that redirect cytotoxic lymphocytes toward malignant and other target cells. The high feasibility of manufacturing CAR-modified lymphocytes for the therapy of cancer has spurred the development and optimization of new CAR T cells directed against a broad range of target antigens. In this review, we describe the main structural and functional elements constituting a CAR, discuss the roles of these elements in modulating the anti-tumor activity of CAR T cells, and highlight alternative approaches to CAR engineering.

[CAR T-cell therapy: Balance of efficacy and safety].

Early results from clinical trials of autologous chimeric antigen receptor (CAR)-expressing T cells for the therapy of B-cell malignancies have encouraged extending the potency of this therapy to other cancers. However, the success of using CAR T-cells to treat patients with solid tumors has been limited. In this review, we summarize current knowledge on the design and applications of CARs for the targeted therapy of cancer. We describe existing issues that limit the widespread application of CAR T cells and discuss the optimization steps needed to further improve safety and efficacy of this therapeutic platform.

[Dosage compensation in drosophila: sequence-specific initiation and sequence-independent spreading of MSL complex to the active genes on the male X chromosome].

For the dosage compensation to occur, genes on the single male X chromosomes in Drosophila must be selectively bound and acetylated by the ribonucleoprotein complex called MSL complex. It remained unknown how such exquisite specificity is achieved, and whether specific DNA sequences were involved. In the present work we demonstrate that it is transcription of the gene on the X chromosome that is important for MSL targeting, irrespective of gene origin and DNA sequence.

[Construction of pMH, a convenient Escherichia coli protein expression vector]. / pMH--udobnyi vektor dlia produktsii belkov v Escherichia coli.

Here we describe the construction of a new vector, pMH, designed for protein expression in E. coli. The vector provides inducible and powerful T7 RNA polymerase driven transcription of the sequences introduced, and a polylinker comprising now 10 most widely used restriction sites, which allows virtually any sequence to be cloned. Cloning in-frame with the N-terminal (c-myc)3-(His)6-tag makes it possible, first, to easily affinity purify the proteins being expressed and, second, to detect the recombinant proteins with the antibodies specific for any of the tags when protein-specific antibodies are unavailable. General utility of pMH was demonstrated by successful expression in E. coli and further purification of Drosophila melanogaster Chriz (CG10712) product and of a number of its C-terminal truncations, with the approximate protein yeild constituting 10 mg/l culture.

[Creation of a new construct for cloning DNA and modeling the structure of Drosophila polytene chromosomes]. / Sozdanie novoi konstruktsii dlia klonirovaniia DNK i modelirovaniia struktur politennykh khoromsom drozofily.

Modification of P element-based transformation vector pCaSpeR3New yielded a new construct, pICon, which contains the structural region of the Escherichia coli lacZ, the adjacent 5' and 3' regulatory regions of hsp70, pUC19, and two tandem FRTs. Owing to the hsp70 promoter, the pICon insertion site may be localized on polytene chromosomes after heat shock by light or electron microscopy. The pUC19 sequence with a polylinker allows cloning of the genomic sequence adjacent to the 3' end of pICon by the rescue of the P-element target. Functional FRTs allow insertion or deletion of various DNA fragments. The construct is large (22046 bp), forms easily detectable structures in polytene chromosomes, and may be used to study the structural and functional organization of the Drosophila melanogaster genome, in particular, to elucidate the causes of banding pattern formation. To map the molecular boundaries of interband 3C6/C7, the DNA sequence of this region was cloned between the two FRTs.

[Creation of a convenient vector for transformation of Drosophila with functional FRT-pFRT sites]. / Sozdanie udobnogo vektora transformatsii drozofily s funktsional'nymi saitami FRT-pFRT.

Modification of Drosophila transformation vector pCaSper3 with the P element was used to construct a new vector, pFRT. The vector contains two tandem FRT sites flanked with several unique restriction sites and separated by a polylinker of five restriction sites, and allows easy cloning of DNA fragments between or close to the FRT sites. FRT-mediated excision of DNA sequences cloned between the FRT sites was demonstrated in vivo. The vector was proposed for molecular genetic studies of the position effect variegation, structural and molecular organization of Drosophila polytene chromosomes, etc.

[Analysis of DNA interband regions 3A5/A6, 3C5-6/c7 and 60E8-9/E10 of Drosophila melanogaster polytene chromosomes]. / Analiz DNK mezhdiskovykh raionov 3A5/A6, 3C5-6/C7 i 60E8-9/E10 politennykh khromosom.

Using electron microscopic (EM) data on the formation of a novel band from the P-element material after its insertion in the interband and the procedure of P-target rescue, DNA interband regions 3A5/A6, and 60E8-9/E10 of Drosophila melanogaster polytene chromosomes were cloned and sequenced. EM analysis of the 3C region have shown that the formation of the full-size 3C5-6/C7 interband requires a 880-bp DNA sequences removed by deletion Df(1)faswb. A comparison of DNA sequences of six bands, two of which were obtained in the present work and four were described earlier, demonstrated the uniqueness of each of them in the Drosophila genome and heterogeneity of their molecular organization. Interband 60E8-9/E10 contains gene rpl19 transcribed throughout the development, in particular in salivary glands. In the other interbands examined 5' and 3' nontranslated gene regions are located. These results suggest that Drosophila interbands may contain both housekeeping genes and regulatory sequences of currently inactive genes from adjacent bands.