Non-viral-mediated gene transfer of OX40 ligand for tumor immunotherapy.

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. Another way 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.

Cell-specific expression of the FAP gene is regulated by enhancer elements.

Fibroblast activation protein (FAP) is an integral membrane serine protease that acts as both dipeptidyl peptidase and collagenase. In recent years, FAP has attracted considerable attention due to its specific upregulation in multiple types of tumor cell populations, including cancer cells in various cancer types, making FAP a potential target for therapy. However, relatively few papers pay attention to the mechanisms driving the cell-specific expression of the FAP gene. We found no correlation between the activities of the two FAP promoter variants (short and long) and the endogenous FAP mRNA expression level in several cell lines with different FAP expression levels. This suggested that other mechanisms may be responsible for specific transcriptional regulation of the FAP gene. We analyzed the distribution of known epigenetic and structural chromatin marks in FAP-positive and FAP-negative cell lines and identified two potential enhancer-like elements (E1 and E2) in the FAP gene locus. We confirmed the specific enrichment of H3K27ac in the putative enhancer regions in FAP-expressing cells. Both the elements exhibited enhancer activity independently of each other in the functional test by increasing the activity of the FAP promoter variants to a greater extent in FAP-expressing cell lines than in FAP-negative cell lines. The transcription factors AP-1, CEBPB, and STAT3 may be involved in FAP activation in the tumors. We hypothesized the existence of a positive feedback loop between FAP and STAT3, which may have implications for developing new approaches in cancer therapy.

Searching for Promoters to Drive Stable and Long-Term Transgene Expression in Fibroblasts for Syngeneic Mouse Tumor Models.

Tumor is a complex system of interactions between cancer cells and other cells of the tumor microenvironment. The cancer-associated fibroblasts (CAFs) of the tumor microenvironment remain in close contact with the cancer cells and play an important role in cancer progression. Genetically, CAFs are more stable than cancer cells, making them an attractive target for genetic modification in gene therapy. However, the efficiency of various promoters for transgene expression in fibroblasts is scarcely studied. We performed a comparative analysis of transgene long-term expression under the control of strong cytomegalovirus promoter (pCMV), constitutive cell promoter of the PCNA gene (pPCNA), and the potentially fibroblast-specific promoter of the IGFBP2 gene (pIGFBP2). In vitro expression of the transgene under the control of pCMV in fibroblasts was decreased soon after transduction, whereas the expression was more stable under the control of pIGFBP2 and pPCNA. The efficiency of transgene expression was higher under pPCNA than that under pIGFBP2. Additionally, in a mouse model, pPCNA provided more stable and increased transgene expression in fibroblasts as compared to that under pCMV. We conclude that PCNA promoter is the most efficient for long-term expression of transgenes in fibroblasts both in vitro and in vivo.

Microdistribution of MC1R-targeted polyplexes in murine melanoma tumor tissue.

Targeted sodium-iodide symporter (NIS) gene transfer can be considered as a promising approach for diagnostics of specific types of cancer. For this purpose we used targeted polyplexes based on PEI-PEG-MC1SP block-copolymer containing MC1SP-peptide, a ligand specific for melanocortin receptor-1 (MC1R) overexpressed on melanoma cells. Targeted polyplexes demonstrated enhanced NIS gene transfer compared to non-targeted (lacking MC1SP) ones in vitro. Using dorsal skinfold chamber and intravital microscopy we evaluated accumulation and microdistribution of quantum dot-labeled polyplexes in tumor and normal subcutaneous tissues up to 4 h after intravenous injection. Polyplexes demonstrated significantly higher total accumulation in tumor tissue in comparison with subcutaneous ones (control). Targeted and non-targeted polyplexes extravasated and penetrated into the tumor tissue up to 20 µm from the vessel walls. In contrast, in normal subcutaneous tissue polyplexes penetrated not more than 3 µm from the vessel walls with the level of extravasated polyplexes 400-fold less than in tumor. Accumulated polyplexes in tumor tissue caused NIS gene expression. Subsequent (123)I(-) intravenous injection resulted in 6.8 ± 1.1 and 4.5 ± 0.8% ID/g (p < 0.001) iodide accumulation in tumors in the case of targeted and non-targeted polyplexes, respectively, as was shown using SPECT/CT.