Polypurine reverse Hoogsteen (PPRH) molecules are DNA hairpins formed by two polypurine strands running in an antiparallel orientation and containing no nucleotide modifications. The two strands, ...linked by a pentathymidine loop, are bound through intramolecular reverse Hoogsteen bonds. Then, PPRHs can bind by Watson-Crick bonds to their corresponding polypyrimidine target in the dsDNA provoking a displacement of the polypurine strand of the duplex. We described the effect and mechanisms of action of PPRHs in cells using PPRHs designed against the template and coding strands of the dhfr gene. The proof of principle of PPRHs as a therapeutic tool was established using a PPRH against survivin in a xenograft prostate cancer tumor model. To improve the PPRHs effect, the influence of the length was studied obtaining a higher efficiency with longer molecules. To decrease the possible offtarget effect, when a purine interruption is found in the pyrimidine target, the PPRH sequence should contain both strands of the complementary base opposite to the interruption. Furthermore, the stability of PPRHs is higher than that of siRNAs, as evidenced by the longer halflife of the former in different types of serum and in PC3 cells. PPRHs do not induce the levels of the transcription factors nor the proinflammatory cytokines involved in the Toll-like Receptor pathway and they do not trigger the formation of the inflammasome complex. PPRHs can be used as therapeutic tools to target genes related to cancer progression, resistance to drugs or immunotherapy approaches.
Methotrexate is a chemotherapeutic drug that is used in therapy of a wide variety of cancers. The efficiency of treatment with this drug is compromised by the appearance of resistance. Combination ...treatments of MTX with other drugs that could modulate the expression of genes involved in MTX resistance would be an adequate strategy to prevent the development of this resistance.
The differential expression pattern between sensitive and MTX-resistant cells was determined by whole human genome microarrays and analyzed with the GeneSpring GX software package. A global comparison of all the studied cell lines was performed in order to find out differentially expressed genes in the majority of the MTX-resistant cells. S100A4 mRNA and protein levels were determined by RT-Real-Time PCR and Western blot, respectively. Functional validations of S100A4 were performed either by transfection of an expression vector for S100A4 or a siRNA against S100A4. Transfection of an expression vector encoding for beta-catenin was used to inquire for the possible transcriptional regulation of S100A4 through the Wnt pathway.
S100A4 is overexpressed in five out of the seven MTX-resistant cell lines studied. Ectopic overexpression of this gene in HT29 sensitive cells augmented both the intracellular and extracellular S100A4 protein levels and caused desensitization toward MTX. siRNA against S100A4 decreased the levels of this protein and caused a chemosensitization in combined treatments with MTX. beta-catenin overexpression experiments support a possible involvement of the Wnt signaling pathway in S100A4 transcriptional regulation in HT29 cells.
S100A4 is overexpressed in many MTX-resistant cells. S100A4 overexpression decreases the sensitivity of HT29 colon cancer human cells to MTX, whereas its knockdown causes chemosensitization toward MTX. Both approaches highlight a role for S100A4 in MTX resistance.
As a new approach for gene therapy, we recently developed a new type of molecule called polypurine reverse Hoogsteen hairpins (PPRHs). We decided to explore the in vitro and in vivo effect of PPRHs ...in cancer choosing survivin as a target since it is involved in apoptosis, mitosis and angiogenesis, and overexpressed in different tumors. We designed four PPRHs against the survivin gene, one of them directed against the template strand and three against different regions of the coding strand. These PPRHs were tested in PC3 prostate cancer cells in an in vitro screening of cell viability and apoptosis. PPRHs against the promoter sequence were the most effective and caused a decrease in survivin mRNA and protein levels. We confirmed the binding between the selected PPRHs and their target sequences in the survivin gene. In addition we determined that both the template- and the coding-PPRH targeting the survivin promoter were interfering with the binding of transcription factors Sp1 and GATA-3, respectively. Finally, we conducted two in vivo efficacy assays using the Coding-PPRH against the survivin promoter and performing two routes of administration, namely intratumoral and intravenous, in a subcutaneous xenograft tumor model of PC3 prostate cancer cells. The results showed that the chosen Coding-PPRH proved to be effective in decreasing tumor volume, and reduced the levels of survivin protein and the formation of blood vessels. These findings represent the preclinical proof of principle of PPRHs as a new silencing tool for cancer gene therapy.
Gene silencing by either small-interference RNAs (siRNA) or antisense oligodeoxynucleotides (aODN) is widely used in biomedical research. However, their use as therapeutic agents is hindered by two ...important limitations: their low stability and the activation of the innate immune response. Recently, we developed a new type of molecule to decrease gene expression named polypurine reverse Hoogsteen hairpins (PPRHs) that bind to polypyrimidine targets in the DNA. Herein, stability experiments performed in mouse, human, and fetal calf serum and in PC3 cells revealed that the half-life of PPRHs is much longer than that of siRNAs in all cases. Usage of PPRHs with a nicked-circular structure increased the binding affinity to their target sequence and their half-life in FCS when bound to the target. Regarding the innate immune response, we determined that the levels of the transcription factors IRF3 and its phosphorylated form, as well as NF-κB were increased by siRNAs and not by PPRHs; that the expression levels of several proinflammatory cytokines including IL-6, TNF-α, IFN-α, IFN-ß, IL-1ß, and IL-18 were not significantly increased by PPRHs; and that the cleavage and activation of the proteolytic enzyme caspase-1 was not triggered by PPRHs. These determinations indicated that PPRHs, unlike siRNAs, do not activate the innate inflammatory response.
We studied the ability of polypurine reverse Hoogsteen hairpins (PPRHs) to silence a variety of relevant cancer-related genes in several human cell lines. PPRHs are hairpins formed by two ...antiparallel polypurine strands bound by intramolecular Hoogsteen bonds linked by a pentathymidine loop. These hairpins are able to bind to their target DNA sequence through Watson-Crick bonds producing specific silencing of gene expression. We designed PPRHs against the following genes: BCL2, TOP1, mTOR, MDM2, and MYC and tested them for mRNA levels, cytotoxicity, and apoptosis in prostate, pancreas, colon, and breast cancer cell lines. Even though all PPRHs were effective, the most remarkable results were obtained with those against BCL2 and mammalian target of rapamycin (mTOR) in decreasing cell survival and mRNA levels and increasing apoptosis in prostate, colon, and pancreatic cancer cells. In the case of TOP1, MDM2, and MYC, their corresponding PPRHs produced a strong effect in decreasing cell viability and mRNA levels and increasing apoptosis in breast cancer cells. Thus, we confirm that the PPRH technology is broadly useful to silence the expression of cancer-related genes as demonstrated using target genes involved in metabolism (DHFR), proliferation (mTOR), DNA topology (TOP1), lifespan and senescence (telomerase), apoptosis (survivin, BCL2), transcription factors (MYC), and proto-oncogenes (MDM2).
Nowadays, the modulation of gene expression by nucleic acids has become a routine tool in biomedical research for target validation and it is also used to develop new therapeutic approaches. ...Recently, we developed the so-called polypurine reverse Hoogsteen hairpins (PPRHs) that show high stability and a low immunogenic profile and we demonstrated their efficacy both in vitro and in vivo. In this work, we explored different characteristics of PPRHs to improve their usage as a tool for gene silencing. We studied the role of PPRH length in the range from 20 to 30 nucleotides. We also proved their higher affinity of binding and efficacy on cell viability compared to nonmodified TFOs. To overcome possible off-target effects, we tested wild-type PPRHs, which proved to be capable of binding to their target sequence with more affinity, displaying a higher stability of binding and a higher effect in terms of cell viability. Moreover, we developed a brand new molecule called Wedge-PPRH with the ability to lock the ds-DNA into the displaced structure and proved its efficacy in prostate and breast cancer cell lines.
Digitalizing clinical trials provide an opportunity to address challenges faced in the Phase I trial settings, where near real-time data capture and data interpretation are prerequisites for ...iterative decision-making to rapidly adapt trial designs based on emerging insights. Although digital technologies have driven significant improvements in many businesses and organizations, the adoption of digital technologies in clinical trials has been slow. In recognition of this lag, the UpSMART consortium, a 5-year funded program (2020-2024), has been established in Europe between the UK, Spain, and Italy to embrace digital technologies and drive benefits to patients. The consortium, led by the Cancer Research UK Manchester Institute Cancer Biomarker Centre, aims to ’digitalize’ Experimental Cancer Medicine Centres in the UK and Early Drug Development Units in Spain and Italy by open-sourcing and sharing digital healthcare products between participating centers across the consortium. The goal is to optimize data capture and interpretation thus accelerating Phase I clinical research to ultimately benefit patients by allowing faster access to tomorrow’s medicines.
Digitalizing clinical trials provide an opportunity to address challenges faced in the Phase I trial settings, where near real-time data capture and data interpretation are prerequisites for ...iterative decision-making to rapidly adapt trial designs based on emerging insights. Although digital technologies have driven significant improvements in many businesses and organizations, the adoption of digital technologies in clinical trials has been slow. In recognition of this lag, the UpSMART consortium, a 5-year funded program (2020-2024), has been established in Europe between the UK, Spain, and Italy to embrace digital technologies and drive benefits to patients. The consortium, led by the Cancer Research UK Manchester Institute Cancer Biomarker Centre, aims to 'digitalize' Experimental Cancer Medicine Centres in the UK and Early Drug Development Units in Spain and Italy by open-sourcing and sharing digital healthcare products between participating centers across the consortium. The goal is to optimize data capture and interpretation thus accelerating Phase I clinical research to ultimately benefit patients by allowing faster access to tomorrow's medicines.
Polypurine reverse Hoogsteen hairpins (PPRHs) are formed by two intramolecularly bound antiparallel homopurine domains linked by a five-thymidine loop. One of the homopurine strands binds with ...antiparallel orientation by Watson-Crick bonds to the polypyrimidine target sequence, forming a triplex. We had previously reported the ability of PPRHs to effectively bind dsDNA displacing the fourth strand away from the newly formed triplex. The main goal of this work was to explore the possibility of repairing a point mutation in mammalian cells using PPRHs as tools. These repair-PPRHs contain different combinations of extended sequences of DNA with the corrected nucleotide to repair the point mutation. As a model we used the dihydrofolate reductase gene. On the one hand, we demonstrate in vitro that PPRHs bind specifically to their polypyrimidine target sequence, opening the two strands of the dsDNA, and allowing the binding of a given repair oligonucleotide to the displaced strand of the DNA. Subsequently, we show at a cellular level (Chinese ovary hamster cells) that repair-PPRHs are able to correct a single-point mutation in a dihydrofolate reductase minigene bearing a nonsense mutation, both in an extrachromosomal location and when the mutated plasmid was stably transfected into the cells. Finally, this methodology was successfully applied to repair a single-point mutation at the endogenous locus, using the DA5 cell line with a deleted nucleotide in exon six of the dhfr gene.
MicroRNAs are small non-coding RNAs involved in gene expression regulation by targeting specific regions in the 3'-UTR of the mRNA of their target genes. This binding leads to a decrease in the ...protein levels of such genes either by mRNA degradation or mRNA destabilization and translation inhibition. The interaction between a miRNA and its target mRNAs is usually studied by co-transfection of a reporter expression vector containing the 3'-UTR region of the mRNA and an inhibitory or precursor molecule for the miRNA. This approach, however, does not measure the direct and physical interaction between a miRNA and a specific mRNA.
RNA molecules corresponding to miR-224 and to the 3'-UTR of SLC4A4 were incubated together and their interaction studied under different binding conditions using electrophoretic mobility shift assays. A direct and specific interaction between miR-224 and SLC4A4 mRNA was observed. This interaction was abolished in the presence of competitors.
In this study, we explored a new application for the electrophoretic mobility shift assay and we demonstrated that it is a useful alternative method to assess, in a direct and specific manner, whether a miRNA binds to a specific predicted target mRNA.
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