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Background: Immune checkpoint blockade (ICB) is rapidly becoming a standard of care in the treatment of many cancer types. However, the subset of patients who respond to this type of therapy is limited. Anotherway to promote antitumoral immunity is the use of immunostimulatory molecules, such as cytokines or T cell co-stimulators. The systemic administration of immunotherapeutics leads to significant immune-related adverse events (irAEs), therefore, the localized antitumoral action is needed. One way to achieve this is intratumoral non-viral gene-immune therapy, which allows for prolonged and localized gene expression, and multiple drug administration. In this study, we combined the previously described non-viral gene delivery system, PEG-PEI-TAT copolymer, PPT, with murine OX40L-encoding plasmid DNA. Methods: The resulting OX40L/PPT nanoparticles were characterized via gel mobility assay, dynamic light scattering analysis and in vitro transfection efficiency evaluation. The antitumoral efficacy of intratumorally (i.t.) administered nanoparticles was estimated using subcutaneously (s.c.) implanted CT26 (colon cancer), B16F0 (melanoma) and 4T1 (breast cancer) tumor models. The dynamics of stromal immune cell populations was analyzed using flow cytometry. Weight loss and cachexia were used as irAE indicators. The effect of combination of i.t. OX40L/PPT with intraperitoneal PD-1 ICB was estimated in s.c. CT26 tumor model. Results: The obtained OX40L/PPT nanoparticles had properties applicable for cell transfection and provided OX40L protein expression in vitro in all three investigated cancer models. We observed that OX40L/PPT treatment successfully inhibited tumor growth in B16F0 and CT26 tumor models and showed a tendency to inhibit 4T1 tumor growth. In B16F0 tumor model, OX40L/PPT treatment led to the increase in antitumoral effector NK and T killer cells and to the decrease in pro-tumoral myeloid cells populations within tumor stroma. No irAE signs were observed in all 3 tumor models, which indicates good treatment tolerability in mice. Combining OX40L/PPT with PD-1 ICB significantly improved treatment efficacy in the CT26 subcutaneous colon cancer model, providing protective immunity against CT26 colon cancer cells. Conclusion: Overall, the anti-tumor efficacy observed with OX40L non-viral gene therapy, whether administered alone or in combination with ICB, highlights its potential to revolutionize cancer gene therapy, thus paving the way for unprecedented advancements in the cancer therapy field. [ABSTRACT FROM AUTHOR]

We spliced the promoters of the human telomerase and human survivin genes (PhTERT and PhSurv, respectively) widely used for gene therapy and known to have the broadest cancer type spectrum of activity. Two head-to-tail constructs were obtained: the PhTERT-PhSurv and PhSurv-PhTERT tandems. The splicing caused quantitative and qualitative changes in the promoter features. In both constructs, only the promoter proximal to the transcribed gene retained its ability to initiate transcription, whereas the distal promoter was silent, the phenomenon never reported before. However, the distal promoter modulated the activity of the proximal one by increasing its strength and causing an appearance of additional transcription start sites. We suggested that this suppression might be due to the presence of Sp1 transcription factor binding sites in both promoters and Sp1-bridges between these sites. Such Sp1-bridges might convert the tandem promoter linear DNA into a stem-loop structure. If localized inside the formed loop, the distal promoter could lose its ability to initiate transcription. To test this hypothesis, we constructed two modified double promoters, where the proximal PhSurv promoter was replaced either by a shortened variant of the survivin promoter (PhSurv269) or by the mouse survivin promoter. Both PhSurv substitutes were considerably shorter than PhSurv and had different numbers and/or positions of Sp1 sites. In modified tandems, transcription was initiated from both promoters. We also prepared two mutant forms of the PhSurv- PhTERT tandem with two or four Sp1 sites removed from the distal "long" PhSurv promoter. In the first case, the distal PhSurv promoter remained silent, whereas the removal of four Sp1 binding sites restored its activity. In the majority of studied cancer cell lines the efficiency of transcription from the hTERT-(shortened hSurv269) promoter tandem was markedly higher than from each constituent promoter. In normal lung fibroblast cells, the tandem promoter activity was considerably lower. [ABSTRACT FROM AUTHOR]

Abstract: Prominin 1 (PROM1, CD133) is a unique transmembrane glycoprotein encoded by the PROM1 gene. It is a cell surface marker of various stem cells including hematopoietic, prostatic epithelial, pancreatic, leukemic, liver cancer, and colorectal cancer stem cells. Here, we studied tissue specificity of PROM1 transcription isoforms and the methylation level of its two main promoters (P1 and P2) in different human cell lines. Only transcripts lacking the 4th exon (the CD133.s1 form) were expressed in cell lines studied. Moreover, these transcripts, if sufficiently abundant, were initiated simultaneously and independently from both promoters P1 and P2. In cell lines with low levels of the total PROM1 transcript, the transcription was likely initiated from other promoters. Promoter P1 was hypermethylated in all cell lines under study, and therefore, methylation can hardly play an important role in its regulation. In contrast, the methylation of promoter P2 was tissue specific, and hypomethylation of this promoter is probably necessary but not sufficient for efficient transcription of the PROM1 gene. Therefore, we report an unusual instance of different mechanisms of transcription activity regulation for two closely located promoters of the same gene. [Copyright &y& Elsevier]

Prominin 1 (PROM1, CD133) is a unique transmembrane glycoprotein encoded by the PROM1 gene. It is a cell surface marker of various stem cells including hematopoietic, prostatic epithelial, pancreatic, leukemic, liver cancer, and colorectal cancer stem cells. Here, we studied tissue specificity of PROM1 transcription isoforms and the methylation level of its two main promoters (P1 and P2) in different human cell lines. Only transcripts lacking the 4th exon (the CD133.s1 form) were expressed in cell lines studied. Moreover, these transcripts, if sufficiently abundant, were initiated simultaneously and independently from both promoters P1 and P2. In cell lines with low levels of the total PROM1 transcript, the transcription was likely initiated from other promoters. Promoter P1 was hypermethylated in all cell lines under study, and therefore, methylation can hardly play an important role in its regulation. In contrast, the methylation of promoter P2 was tissue specific, and hypomethylation of this promoter is probably necessary but not sufficient for efficient transcription of the PROM1 gene. Therefore, we report an unusual instance of different mechanisms of transcription activity regulation for two closely located promoters of the same gene.