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

BackgroundImmune 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.MethodsThe 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.ResultsThe 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.ConclusionOverall, 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

Possibility for Transcriptional Targeting of Cancer-Associated Fibroblasts—Limitations and Opportunities

Cancer-associated fibroblasts (CAF) are attractive therapeutic targets in the tumor microenvironment. The possibility of using CAFs as a source of therapeutic molecules is a challenging approach in gene therapy. This requires transcriptional targeting of transgene expression by cis-regulatory elements (CRE). Little is known about which CREs can provide selective transgene expression in CAFs. We hypothesized that the promoters of FAP, CXCL12, IGFBP2, CTGF, JAG1, SNAI1, and SPARC genes, the expression of whose is increased in CAFs, could be used for transcriptional targeting. Analysis of the transcription of the corresponding genes revealed that unique transcription in model CAFs was characteristic for the CXCL12 and FAP genes. However, none of the promoters in luciferase reporter constructs show selective activity in these fibroblasts. The CTGF, IGFBP2, JAG1, and SPARC promoters can provide higher transgene expression in fibroblasts than in cancer cells, but the nonspecific viral promoters CMV, SV40, and the recently studied universal PCNA promoter have the same features. The patterns of changes in activity of various promoters relative to each other observed for human cell lines were similar to the patterns of activity for the same promoters both in vivo and in vitro in mouse models. Our results reveal restrictions and features for CAF transcriptional targeting

Intragenic Locus in Human PIWIL2 Gene Shares Promoter and Enhancer Functions.

Recently, more evidence supporting common nature of promoters and enhancers has been accumulated. In this work, we present data on chromatin modifications and non-polyadenylated transcription characteristic for enhancers as well as results of in vitro luciferase reporter assays suggesting that PIWIL2 alternative promoter in exon 7 also functions as an enhancer for gene PHYHIP located 60Kb upstream. This finding of an intragenic enhancer serving as a promoter for a shorter protein isoform implies broader impact on understanding enhancer-promoter networks in regulation of gene expression

Alterations in gene expression of proprotein convertases in human lung cancer have a limited number of scenarios.

Proprotein convertases (PCs) is a protein family which includes nine highly specific subtilisin-like serine endopeptidases in mammals. The system of PCs is involved in carcinogenesis and levels of PC mRNAs alter in cancer, which suggests expression status of PCs as a possible marker for cancer typing and prognosis. The goal of this work was to assess the information value of expression profiling of PC genes. Quantitative polymerase chain reaction was used for the first time to analyze mRNA levels of all PC genes as well as matrix metalloproteinase genes MMP2 and MMP14, which are substrates of PCs, in 30 matched pairs of samples of human lung cancer tumor and adjacent tissues without pathology. Significant changes in the expression of PCs have been revealed in tumor tissues: increased FURIN mRNA level (p<0.00005) and decreased mRNA levels of PCSK2 (p<0.007), PCSK5 (p<0.0002), PCSK7 (p<0.002), PCSK9 (p<0.00008), and MBTPS1 (p<0.00004) as well as a tendency to increase in the level of PCSK1 mRNA. Four distinct groups of samples have been identified by cluster analysis of the expression patterns of PC genes in tumor vs. normal tissue. Three of these groups covering 80% of samples feature a strong elevation in the expression of a single gene in cancer: FURIN, PCSK1, or PCSK6. Thus, the changes in the expression of PC genes have a limited number of scenarios, which may reflect different pathways of tumor development and cryptic features of tumors. This finding allows to consider the mRNAs of PC genes as potentially important tumor markers

Non-polyadenylated transcription across exon-exon junctions of <i>PIWIL2</i> gene.

<p>qRT-PCR was used to assess the level of total RNA and its polyA+ fraction and the ratio of total RNA to polyA+ fraction was calculated. Seminoma and nonseminoma testicular cancer samples as well as adjacent normal testis tissues were assayed. P-value summary of Mann-Whitney non-paired U test is presented for 4–5 and 8–9 exon-exon junction peaks (ns–non-significant, ***—p-value<0.001).</p

<i>PIWIL2</i> alternative promoter in exon 7 acts as an enhancer in luciferase reporter constructs.

<p>Relative promoter activity of SV40 and CMV promoters was assessed in luciferase reporter vectors from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0156454#pone.0156454.g003" target="_blank">Fig 3</a> in two cell lines: TERA1 and NT2D1 (embryonal carcinoma, panel A). <i>PPP3CC</i> and <i>PHYHIP</i> (panel B) promoters were assessed in luciferase reporter vectors from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0156454#pone.0156454.g003" target="_blank">Fig 3</a> in four cell lines: TERA1 and NT2D1 –embryonal carcinoma, Tcam2 –seminoma, and A549 –lung carcinoma. P-value summary of Mann-Whitney non-paired U test is presented for peaks showing more than two-fold increase (above horizontal dashed lines) for both forward and reverse orientation of the candidate enhancer compared to “no enhancer” controls (*—p-value<0.05, **—p-value<0.01).</p

Chromatin modifications around <i>PIWIL2</i> canonical and alternative promoters.

<p>Relative level of H3K4me1 (Histone 3 lysine 4 monomethylated, enhancer mark), H3K4me3 (Histone 3 lysine 4 trimethylated, active promoter mark), H3K27ac (Histone 3 lysine 27 acetylated, active regulatory element mark) and H3K27me3 (Histone 3 lysine 27 trimethylated, facultative heterochromatin mark) histone modifications assessed by ChIP-PCR with two sets of primer pairs around <i>PIWIL2</i> canonical promoter in exon 1 and alternative promoters in exons 5 and 7. Results are are shown for four cell lines: TERA1 and NT2D1 –embryonal carcinoma, TCam2 –seminoma, and A549 –lung carcinoma. The negative controls are <i>PIWIL2</i> intron 8 and an intergenic locus on chromosome 1, the positive control is <i>GAPDH</i> promoter. P-value summary of Mann-Whitney non-paired U test is presented for some peaks (ns–non-significant, *—p-value<0.05, **—p-value<0.01).</p

Structure of luciferase reporter vectors used in the assays.

<p>pGL4.10 plasmid was used to construct vectors with <i>luc</i> gene expression driven by various promoters (upper part in brackets) and a candidate enhancer in either orientation (lower part). Because the candidate enhancers also possess promoter activity, to discern between those, the vectors were linearized by cutting at the sites shown with red arrows. Note that after such linearization the candidate enhancer will be located either upstream (reverse orientation) or downstream (forward orientation) of the <i>luc</i> gene and its promoter.</p

Description of the specimens studied and heat map presentation of the ratio values of gene expression in tumor vs. adjacent tissues without histological pathology.

<p>SCC, squamous cell lung carcinoma; AdC, adenocarcinoma; AdC/SCC, both AdC and SCC cells were found in the tumor tissue; SCLC, small cell lung carcinoma; P, peripheral tumor location; C, central tumor location; Y, tumor with keratinization; N, tumor without keratinization; ‘-’, no data. The heat map is shown in log₂ scale. Gray cells indicate specimens with undetectable mRNA in both tumor and normal tissues.</p

Gene designations and TaqMan Gene Expression Assays used in real-time PCR.

*<p>HGNC – HUGO Gene Nomenclature Committee (<a href="http://www.genenames.org" target="_blank">www.genenames.org</a>).</p