Reverse Hoogsteen Hairpins Research Articles

Combinatorial Anti-Cancer Effect of Polypurine Reverse Hoogsteen Hairpins against KRAS and MYC Targeting in Prostate and Pancreatic Cancer Cell Lines

Introduction: KRAS and MYC are proto-oncogenes that are strictly regulated in healthy cells that have key roles in several processes such as cell growth, proliferation, differentiation, or apoptosis. These genes are tightly interconnected, and their dysregulation can lead to cancer progression. We previously individually targeted these oncogenes using Polypurine Reverse Hoogsteen (PPRH) hairpins, mostly targeting the complementary strand of G-quadruplex-forming sequences. We validated them in vitro in different cancer cell lines with deregulated KRAS and/or MYC. In this work we focused on our understanding of the cooperative dynamics between these oncogenes, by investigating the combined impact of PPRHs targeting KRAS and MYC in pancreatic and prostate cancer cells. Results: The combinations had a modulatory impact on the expression of both oncogenes, with transcriptional and translational downregulation occurring five days post-treatment. Out of the four tested PPRHs, MYC-targeting PPRHs, especially HpMYC-G4-PR-C directed against the promoter, showed a greater cytotoxic and expression modulation effect. When both KRAS- and MYC-targeting PPRHs were applied in combination, a synergistic reduction in cell viability was observed. Conclusion: The simultaneous targeting of KRAS and MYC demonstrates efficacy in gene modulation, thus in decreasing cell proliferation and viability.

Polypurine Reverse Hoogsteen hairpins as a therapeutic tool for SARS-CoV-2 infection

Although COVID-19 pandemic was declared no longer a global emergency by the World Health Organization in May 2023, SARS-CoV-2 is still infecting people across the world. Many therapeutic oligonucleotides such as ASOs, siRNAs or CRISPR-based systems emerged as promising antiviral strategies for the treatment of SARS-CoV-2. In this work we explored the inhibitory potential on SARS-CoV-2 replication of Polypurine Reverse Hoogsteen Hairpins (PPRHs), CC1-PPRH and CC3-PPRH, targeting specific polypyrimidine sequences within the replicase and Spike regions, respectively, and previously validated for COVID-19 diagnosis. Both PPRHs bound to their target sequences in the viral genome with high affinity in the order of nM. In vitro, both PPRHs reduced viral replication by more than 92% when transfected into VERO-E6 cells 24 hours prior infection with SARS-CoV-2. In vivo intranasal administration of CC1-PPRH in K18-hACE2 mice expressing the human ACE receptor protected all the animals from SARS-CoV-2 infection. The properties of PPRHs position them as promising candidates for the development of novel therapeutics against SARS-CoV-2 and other viral infections.

Synthesis and Validation of TRIFAPYs as a Family of Transfection Agents for Therapeutic Oligonucleotides.

Transfection agents play a crucial role in facilitating the uptake of nucleic acids into eukaryotic cells offering potential therapeutic solutions for genetic disorders. However, progress in this field needs the development of improved systems that guarantee efficient transfection. Here, we describe the synthesis of a set of chemical delivery agents (TRIFAPYs) containing alkyl chains of different lengths based on the 1,3,5-tris[(4-alkyloxy-1pyridinio)methyl]benzene tribromide structure. Their delivery properties for therapeutic oligonucleotides were evaluated using PolyPurine Reverse Hoogsteen hairpins (PPRHs) as a silencing tool. The binding of liposomes to PPRHs was evaluated by retardation assays in agarose gels. The complexes had a size of 125 nm as determined by DLS, forming well-defined concentrical vesicles as visualized by Cryo-TEM. The prostate cancer cell line PC-3 was used to study the internalization of the nanoparticles by fluorescence microscopy and flow cytometry. The mechanism of entrance involved in the cellular uptake was mainly by clathrin-mediated endocytosis. Cytotoxicity analyses determined the intrinsic toxicity caused by each TRIFAPY and the effect on cell viability upon transfection of a specific PPRH (HpsPr-C) directed against the antiapoptotic target survivin. TRIFAPYs C12-C18 were selected to expand these studies in the breast cancer cell line SKBR-3 opening the usage of TRIFAPYs for both sexes and, in the hCMEC/D3 cell line, as a model for the blood-brain barrier. The mRNA levels of survivin decreased, while apoptosis levels increased upon the transfection of HpsPr-C with these TRIFAPYs in PC-3 cells. Therefore, TRIFAPYs can be considered novel lipid-based vehicles for the delivery of therapeutic oligonucleotides.

Post- and Pre-Radiolabeling Assays for anti Thymidine Cyclobutane Dimers as Intrinsic Photoprobes of Various Types of G-Quadruplexes, Reverse Hoogsteen Hairpins, and Other Non-B DNA Structures.

G-quadruplexes are thought to play an important role in gene regulation and telomere maintenance, but developing probes for their presence and location is challenging due to their transitory and highly dynamic nature. The majority of probes for G-quadruplexes have relied on antibody or small-molecule binding agents, many of which can also alter the dynamics and relative populations of G-quadruplexes. Recently, it was discovered that ultraviolet B (UVB) irradiation of human telomeric DNA and various G-quadruplex forming sequences found in human promoters, as well as reverse Hoogsteen hairpins, produces a unique class of non-adjacent anti cyclobutane pyrimidine dimers (CPDs). Therefore, one can envision using a pulse of UVB light to irreversibly trap these non-B DNA structures via anti CPD formation without perturbing their dynamics, after which the anti CPDs can be identified and mapped. As a first step toward this goal, we report radioactive post- and pre-labeling assays for the detection of non-adjacent CPDs and illustrate their use in detecting trans,anti T=(T) CPD formation in a human telomeric DNA sequence. Both assays make use of snake venom phosphodiesterase (SVP) to degrade the trans,anti T=(T) CPD-containing DNA to the tetranucleotide pTT=(pTT) corresponding to CPD formation between the underlined T's of two separate dinucleotides while degrading the adjacent syn TT CPDs to the trinucleotide pGT=T. In the post-labeling assay, calf intestinal phosphodiesterase is used to dephosphorylate the tetranucleotides, which are then rephosphorylated with kinase and [32P]-ATP to produce radiolabeled mono- and diphosphorylated tetranucleotides. The tetranucleotides are confirmed to be non-adjacent CPDs by 254 nm photoreversion to the dinucleotide p*TT. In the pre-labeling assay, radiolabeled phosphates are introduced into non-adjacent CPD-forming sites by ligation prior to irradiation, thereby eliminating the dephosphorylation and rephosphorylation steps. The assays are also demonstrated to detect the stereoisomeric cis,anti T=(T) CPD.

In Vitro and In Vivo Effects of the Combination of Polypurine Reverse Hoogsteen Hairpins against HER-2 and Trastuzumab in Breast Cancer Cells.

Therapeutic oligonucleotides are powerful tools for the inhibition of potential targets involved in cancer. We describe the effect of two Polypurine Reverse Hoogsteen (PPRH) hairpins directed against the ERBB2 gene, which is overexpressed in positive HER-2 breast tumors. The inhibition of their target was analyzed by cell viability and at the mRNA and protein levels. The combination of these specific PPRHs with trastuzumab was also explored in breast cancer cell lines, both in vitro and in vivo. PPRHs designed against two intronic sequences of the ERBB2 gene decreased the viability of SKBR-3 and MDA-MB-453 breast cancer cells. The decrease in cell viability was associated with a reduction in ERBB2 mRNA and protein levels. In combination with trastuzumab, PPRHs showed a synergic effect in vitro and reduced tumor growth in vivo. These results represent the preclinical proof of concept of PPRHs as a therapeutic tool for breast cancer.

Targeting MYC Regulation with Polypurine Reverse Hoogsteen Oligonucleotides.

The oncogene MYC has key roles in transcription, proliferation, deregulating cellular energetics, and more. Modulating the expression or function of the MYC protein is a viable therapeutic goal in an array of cancer types, and potential inhibitors of MYC with high specificity and selectivity are of great interest. In cancer cells addicted to their aberrant MYC function, suppression can lead to apoptosis, with minimal effects on non-addicted, non-oncogenic cells, providing a wide therapeutic window for specific and efficacious anti-tumor treatment. Within the promoter of MYC lies a GC-rich, G-quadruplex (G4)-forming region, wherein G4 formation is capable of mediating transcriptional downregulation of MYC. Such GC-rich regions of DNA are prime targets for regulation with Polypurine Reverse Hoogsteen hairpins (PPRHs). The current study designed and examined PPRHs targeting the G4-forming and four other GC-rich regions of DNA within the promoter or intronic regions. Six total PPRHs were designed, examined in cell-free conditions for target engagement and in cells for transcriptional modulation, and correlating cytotoxic activity in pancreatic, prostate, neuroblastoma, colorectal, ovarian, and breast cancer cells. Two lead PPRHs, one targeting the promoter G4 and one targeting Intron 1, were identified with high potential for further development as an innovative approach to both G4 stabilization and MYC modulation.

Adjacent and Nonadjacent Dipyrimidine Photoproducts as Intrinsic Probes of DNA Secondary and Tertiary Structure†.

While the photochemistry of duplex DNA has been extensively studied, the photochemistry of nonduplex DNA structures is largely unexplored. Because the structure and stereochemistry of DNA photoproducts depend on the secondary structure and conformation of the DNA precursor, they can serve as intrinsic probes of DNA structure. This review focuses on the structures and stereoisomers of pyrimidine dimer photoproducts arising from adjacent and nonadjacent pyrimidines in A, B and denatured DNA, bulge loops, G-quadruplexes and reverse Hoogsteen hairpins and methods for their detection.

Targeting KRAS Regulation with PolyPurine Reverse Hoogsteen Oligonucleotides.

KRAS is a GTPase involved in the proliferation signaling of several growth factors. The KRAS gene is GC-rich, containing regions with known and putative G-quadruplex (G4) forming regions. Within the middle of the G-rich proximal promoter, stabilization of the physiologically active G4mid structure downregulates transcription of KRAS; the function and formation of other G4s within the gene are unknown. Herein we identify three putative G4-forming sequences (G4FS) within the KRAS gene, explore their G4 formation, and develop oligonucleotides targeting these three regions and the G4mid forming sequence. We tested Polypurine Reverse Hoogsteen hairpins (PPRHs) for their effects on KRAS regulation via enhancing G4 formation or displacing G-rich DNA strands, downregulating KRAS transcription and mediating an anti-proliferative effect. Five PPRH were designed, two against the KRAS promoter G4mid and three others against putative G4FS in the distal promoter, intron 1 and exon 5. PPRH binding was confirmed by gel electrophoresis. The effect on KRAS transcription was examined by luciferase, FRET Melt2, qRT-PCR. Cytotoxicity was evaluated in pancreatic and ovarian cancer cells. PPRHs decreased activity of a luciferase construct driven by the KRAS promoter. PPRH selectively suppressed proliferation in KRAS dependent cancer cells. PPRH demonstrated synergistic activity with a KRAS promoter selective G4-stabilizing compound, NSC 317605, in KRAS-dependent pancreatic cells. PPRHs selectively stabilize G4 formation within the KRAS mid promoter region and represent an innovative approach to both G4-stabilization and to KRAS modulation with potential for development into novel therapeutics.

PolyPurine Reverse Hoogsteen Hairpins Work as RNA Species for Gene Silencing.

PolyPurine Reverse Hoogsteen Hairpins (PPRHs) are gene-silencing DNA-oligonucleotides developed in our laboratory that are formed by two antiparallel polypurine mirror repeat domains bound intramolecularly by Hoogsteen bonds. The aim of this work was to explore the feasibility of using viral vectors to deliver PPRHs as a gene therapy tool. After treatment with synthetic RNA, plasmid transfection, or viral infection targeting the survivin gene, viability was determined by the MTT assay, mRNA was determined by RT-qPCR, and protein levels were determined by Western blot. We showed that the RNA-PPRH induced a decrease in cell viability in a dose-dependent manner and an increase in apoptosis in PC-3 and HeLa cells. Both synthetic RNA-PPRH and RNA-PPRH intracellularly generated upon the transfection of a plasmid vector were able to reduce survivin mRNA and protein levels in PC-3 cells. An adenovirus type-5 vector encoding the PPRH against survivin was also able to decrease survivin mRNA and protein levels, leading to a reduction in HeLa cell viability. In this work, we demonstrated that PPRHs can also work as RNA species, either chemically synthesized, transcribed from a plasmid construct, or transcribed from viral vectors. Therefore, all these results are the proof of principle that viral vectors could be considered as a delivery system for PPRHs.

Synthesis and validation of DOPY: A new gemini dioleylbispyridinium based amphiphile for nucleic acid transfection

Nucleic acids therapeutics provide a selective and promising alternative to traditional treatments for multiple genetic diseases. A major obstacle is the development of safe and efficient delivery systems. Here, we report the synthesis of the new cationic gemini amphiphile 1,3-bis[(4-oleyl-1-pyridinio)methyl]benzene dibromide (DOPY). Its transfection efficiency was evaluated using PolyPurine Reverse Hoogsteen hairpins (PPRHs), a nucleic acid tool for gene silencing and gene repair developed in our laboratory. The interaction of DOPY with PPRHs was confirmed by gel retardation assays, and it forms complexes of 155 nm. We also demonstrated the prominent internalization of PPRHs using DOPY compared to other chemical vehicles in SH-SY5Y, PC-3 and DF42 cells. Regarding gene silencing, a specific PPRH against the survivin gene delivered with DOPY decreased survivin protein levels and cell viability more effectively than with N-[1-(2,3-Dioleoyloxy)propyl]-N,N,N-trimethylammonium methylsulfate (DOTAP) in both SH-SY5Y and PC-3 cells. We also validated the applicability of DOPY in gene repair approaches by correcting a point mutation in the endogenous locus of the dhfr gene in DF42 cells using repair-PPRHs. All these results indicate both an efficient entry and release of PPRHs at the intracellular level. Therefore, DOPY can be considered as a new lipid-based vehicle for the delivery of therapeutic oligonucleotides.

Gene Correction of Point Mutations Using PolyPurine Reverse Hoogsteen Hairpins Technology.

Monogenic disorders are often the result of single point mutations in specific genes, leading to the production of non-functional proteins. Different blood disorders such as ß-thalassemia, sickle cell disease, hereditary spherocytosis, Fanconi anemia, and Hemophilia A and B are usually caused by point mutations. Gene editing tools including TALENs, ZFNs, or CRISPR/Cas platforms have been developed to correct mutations responsible for different diseases. However, alternative molecular tools such as triplex-forming oligonucleotides and their derivatives (e.g., peptide nucleic acids), not relying on nuclease activity, have also demonstrated their ability to correct mutations in the DNA. Here, we review the Repair-PolyPurine Reverse Hoogsteen hairpins (PPRHs) technology, which can represent an alternative gene editing tool within this field. Repair-PPRHs are non-modified single-stranded DNA molecules formed by two polypurine mirror repeat sequences linked by a five-thymidine bridge, followed by an extended sequence at one end of the molecule which is homologous to the DNA sequence to be repaired but containing the corrected nucleotide. The two polypurine arms of the PPRH are bound by intramolecular reverse-Hoogsteen bonds between the purines, thus forming a hairpin structure. This hairpin core binds to polypyrimidine tracts located relatively near the target mutation in the dsDNA in a sequence-specific manner by Watson-Crick bonds, thus producing a triplex structure which stimulates recombination. This technology has been successfully employed to repair a collection of mutants of the dhfr and aprt genes within their endogenous loci in mammalian cells and could be suitable for the correction of mutations responsible for blood disorders.

Detection of a G-Quadruplex as a Regulatory Element in Thymidylate synthase for Gene Silencing Using Polypurine Reverse Hoogsteen Hairpins.

Thymidylate synthase (TYMS) enzyme is an anti-cancer target given its role in DNA biosynthesis. TYMS inhibitors (e.g., 5-Fluorouracil) can lead to drug resistance through an autoregulatory mechanism of TYMS that causes its overexpression. Since G-quadruplexes (G4) can modulate gene expression, we searched for putative G4 forming sequences (G4FS) in the TYMS gene that could be targeted using polypurine reverse Hoogsteen hairpins (PPRH). G4 structures in the TYMS gene were detected using the quadruplex forming G-rich sequences mapper and confirmed through spectroscopic approaches such as circular dichroism and NMR using synthetic oligonucleotides. Interactions between G4FS and TYMS protein or G4FS and a PPRH targeting this sequence (HpTYMS-G4-T) were studied by EMSA and thioflavin T staining. We identified a G4FS in the 5’UTR of the TYMS gene in both DNA and RNA capable of interacting with TYMS protein. The PPRH binds to its corresponding target dsDNA, promoting G4 formation. In cancer cells, HpTYMG-G4-T decreased TYMS mRNA and protein levels, leading to cell death, and showed a synergic effect when combined with 5-fluorouracil. These results reveal the presence of a G4 motif in the TYMS gene, probably involved in the autoregulation of TYMS expression, and the therapeutic potential of a PPRH targeted to the G4FS.

Targeting replication stress response using polypurine reverse hoogsteen hairpins directed against WEE1 and CHK1 genes in human cancer cells

In response to DNA damage, cell cycle checkpoints produce cell cycle arrest to repair and maintain genomic integrity. Due to the high rates of replication and genetic abnormalities, cancer cells are dependent on replication stress response (RSR) and inhibitors of this pathway are being studied as an anticancer approach. In this direction, we investigated the inhibition of CHK1 and WEE1, key components of RSR, using Polypurine Reverse Hoogsteen hairpins (PPRHs) as gene silencing tool. PPRHs designed against WEE1 or CHK1 reduced the viability of different cancer cell lines and showed an increase of apoptosis in HeLa cells. The effect of the PPRHs on cell viability were dose- and time-dependent in HeLa cells. Both the levels of mRNA and protein for each gene were decreased after treatment with the PPRHs. When analyzing the levels of the two CHK1 mRNA splicing variants, CHK1 and CHK1-S, there was a proportional decrease of the two forms, thus maintaining the same expression ratio. PPRHs targeting WEE1 and CHK1 also proved to disrupt cell cycle after 15 h of treatment. Moreover, PPRHs showed a synergy effect when combined with DNA damaging agents, such as methotrexate or 5-Fluorouracil, widely used in clinical practice. This work validates in vitro the usage of PPRHs as a silencing tool against the RSR genes WEE1 and CHK1 and corroborates the potential of inhibiting these targets as a single agent therapy or in combination with other chemotherapy agents in cancer research.

Correction of the aprt Gene Using Repair-Polypurine Reverse Hoogsteen Hairpins in Mammalian Cells

In this study, we describe the correction of single-point mutations in mammalian cells by repair-polypurine reverse Hoogsteen hairpins (repair-PPRHs). These molecules consist of (1) a PPRH hairpin core that binds to a polypyrimidine target sequence in the double-stranded DNA (dsDNA), producing a triplex structure, and (2) an extension sequence homologous to the DNA sequence to be repaired but containing the wild-type nucleotide instead of the mutation and acting as a donor DNA to correct the mutation. We repaired different point mutations in the adenosyl phosphoribosyl transferase (aprt) gene contained in different aprt-deficient Chinese hamster ovary (CHO) cell lines. Because we had previously corrected mutations in the dihydrofolate reductase (dhfr) gene, in this study, we demonstrate the generality of action of the repair-PPRHs. Repaired cells were analyzed by DNA sequencing, mRNA expression, and enzymatic activity to confirm the correction of the mutation. Moreover, whole-genome sequencing analyses did not detect any off-target effect in the repaired genome. We also performed gel-shift assays to show the binding of the repair-PPRH to the target sequence and the formation of a displacement-loop (D-loop) structure that can trigger a homologous recombination event. Overall, we demonstrate that repair-PPRHs achieve the permanent correction of point mutations in the dsDNA at the endogenous level in mammalian cells without off-target activity.

Silencing PD-1 and PD-L1: the potential of PolyPurine Reverse Hoogsteen hairpins for the elimination of tumor cells.

Silencing PD-1 and PD-L1: the potential of PolyPurine Reverse Hoogsteen hairpins for the elimination of tumor cells.

Cancer immunotherapy using PolyPurine Reverse Hoogsteen hairpins targeting the PD-1/PD-L1 pathway in human tumor cells.

Immunotherapy approaches stand out as innovative strategies to eradicate tumor cells. Among them, PD-1/PD-L1 immunotherapy is considered one of the most successful advances in the history of cancer immunotherapy. We used our technology of Polypurine reverse Hoogsteen hairpins (PPRHs) for silencing both genes with the aim to provoke the elimination of tumor cells by macrophages in co-culture experiments. Incubation of PPRHs against PD-1 and PD-L1 decreased the levels of mRNA and protein in THP-1 monocytes and PC3 prostate cancer cells, respectively. Viability of THP-1 cells and macrophages obtained by PMA-differentiation of THP-1 cells was not affected upon incubation with the different PPRHs. On the other hand, PC3 cell survival was partially decreased by PPRHs against PD-L1. The greatest effect in decreasing cell viability was obtained in macrophages/PC3 co-culture experiments by combining PPRHs against PD-1 and PD-L1. This effect was also observed in other cancer cell lines: HeLa, SKBR3 and to a minor extent in M21. Apoptosis was not detected when macrophages were treated with the different PPRHs. However, co-cultures of macrophages with the four cancer cell lines treated with PPRHs showed an increase in apoptosis. The order of fold-increase in apoptosis was HeLa > PC3 > SKBR3 > M21. This study demonstrates that PPRHs could be powerful pharmacological agents to use in immunotherapy approaches for the inhibition of PD-1 and PD-L1.

Functional pharmacogenomics and toxicity of PolyPurine Reverse Hoogsteen hairpins directed against survivin in human cells

PolyPurine Reverse Hoogsteen (PPRH) hairpins constitute a relatively new pharmacological agent for gene silencing that has been applied for a growing number of gene targets. Previously we reported that specific PPRHs against the antiapoptotic gene survivin were able to decrease viability of PC3 prostate cancer cells by increasing apoptosis, while not acting on HUVEC non-tumoral cells. These PPRHs were efficient both in vitro and in vivo. In the present work, we performed a functional pharmacogenomics study on the effects of specific and unspecific hairpins against survivin. Incubation of PC3 cells with the specific HpsPr-C-WT led to 244 differentially expressed genes when applying the p < 0.05, FC > 2, Benjamini-Hochberg filtering. Importantly, the unspecific or control Hp-WC did not originate differentially expressed genes using the same settings. Gene Set Enrichment Analysis (GSEA) revealed that the differentially expressed genes clustered very significantly within the gene sets of Regulation of cell proliferation, Cellular response to stress, Apoptosis and Prostate cancer. Network analyses using STRING identified important interacting gene-nodes within the response of PC3 cells to treatment with the PPRH against survivin, mainly POLR2G, PAK1IP1, SMC3, SF3A1, PPARGC1A, NCOA6, UGT2B7, ALG5, VAMP7 and HIST1H2BE, the former six present in the Gene Sets detected in the GSEA. Additionally, HepG2 and 786-O cell lines were used to carry out in vitro experiments of hepatotoxicity and nephrotoxicity, respectively. The unspecific hairpin did not cause toxicity in cell survival assays (MTT) and produced minor changes in gene expression for selected genes in RT-qPCR arrays specifically developed for hepatic and renal toxicity screening.

Evidence for Reverse Hoogsteen Hairpin Intermediates in the Photocrosslinking of Human Telomeric DNA Sequences.

UVB irradiation of human telomeric d(GGGTTA)3 GGG sequences in potassium ion solution crosslinks the first and third TTA segments through anti cyclobutane pyrimidine dimer (CPD) formation. The photocrosslinking reaction was first proposed to occur through a form 3 two-tetrad G-quadruplex in which the lateral four-nucleotide GTTA loop can interact with an adjacent TTA loop. Curiously, the reaction does not occur with sodium ion, which was explained by the formation of a basket structure which only has three-nucleotide TTA loops that cannot interact. Sequences known or expected to favor the two-tetrad basket did not show enhanced photocrosslinking, suggesting that some other structure was the reactive intermediate. Herein, we report that anti CPDs form in human telomeric DNA sequences with lithium ion that is known to disfavor G-quadruplex formation, as well as with potassium ion when the bases are modified to interfere with G-quartet formation. We also show that anti CPDs form in sequences containing A's in place of G's that cannot form Hoogsteen hairpins, but can form reverse Hoogsteen hairpins. These results suggest that reverse Hoogsteen hairpins may play a hitherto unrecognized role in the biology and photoreactivity of DNA in telomeres, and possibly in other purine-rich sequences found in regulatory regions.

Polypurine Reverse Hoogsteen Hairpins as a Gene Silencing Tool for Cancer.

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.

Silencing of Foxp3 enhances the antitumor efficacy of GM-CSF genetically modified tumor cell vaccine against B16 melanoma.

The antitumor response after therapeutic vaccination has a limited effect and seems to be related to the presence of T regulatory cells (Treg), which express the immunoregulatory molecules CTLA4 and Foxp3. The blockage of CTLA4 using antibodies has shown an effective antitumor response conducing to the approval of the human anti-CTLA4 antibody ipilimumab by the US Food and Drug Administration. On the other hand, Foxp3 is crucial for Treg development. For this reason, it is an attractive target for cancer treatment. This study aims to evaluate whether combining therapeutic vaccination with CTLA4 or Foxp3 gene silencing enhances the antitumor response. First, the “in vitro” cell entrance and gene silencing efficacy of two tools, 2′-O-methyl phosphorotioate-modified oligonucleotides (2′-OMe-PS-ASOs) and polypurine reverse Hoogsteen hairpins (PPRHs), were evaluated in EL4 cells and cultured primary lymphocytes. Following B16 tumor transplant, C57BL6 mice were vaccinated with irradiated B16 tumor cells engineered to produce granulocyte-macrophage colony-stimulating factor (GM-CSF) and were intraperitoneally treated with CTLA4 and Foxp3 2′-OMe-PS-ASO before and after vaccination. Tumor growth, mice survival, and CTLA4 and Foxp3 expression in blood cells were measured. The following results were obtained: 1) only 2′-OMe-PS-ASO reached gene silencing efficacy “in vitro”; 2) an improved survival effect was achieved combining both therapeutic vaccine and Foxp3 antisense or CTLA4 antisense oligonucleotides (50% and 20%, respectively); 3) The blood CD4+CD25+Foxp3+ (Treg) and CD4+CTLA4+ cell counts were higher in mice that developed tumor on the day of sacrifice. Our data showed that tumor cell vaccine combined with Foxp3 or CTLA4 gene silencing can increase the efficacy of therapeutic antitumor vaccination.