Construction of charge-reversible coordination-crosslinked spherical nucleic acids to deliver dual anti-cancer genes and ferroptosis payloads.

Spherical nucleic acids (SNAs) are nanostructures with the DNA arranged radially on the surface, thus allowing specific binding with cancer cells expressing high levels of scavenger receptor-A to enhance cellular uptake. However, conventional carriers for SNAs are cytotoxic, not degradable and difficult to deliver multiple payloads. In this study, we developed charge-reversible coordination-crosslinked SNAs to deliver dual anti-cancer genes and ferroptosis payload for anti-cancer purposes. To this end, we modified poly(lactic acid) (PLA) with functionalized side chains to allow its binding with antisense oligonucleotides (ASOs) and siRNA, annealed two single-stranded RNAs to obtain double-stranded RNA, and introduced a polyethylene glycol (PEG) shell to enhance the circulation time. Additionally, the ferroptosis payload imidazole was coordinated with iron ions as a core-crosslinked group to enhance the stability of SNAs and efficiency to kill cancer cells. We demonstrated that this novel nanocomplex efficiently internalized and killed CT-26 cells in vitro. In vivo data confirmed that the dual gene delivery system successfully targeted CT-26 tumors in tumor-bearing BALB/c mice, and exhibited strong tumor suppression ability, without inducing adverse toxic effects. Taken together, our dual gene therapy system offered an enhanced anti-tumor solution by simultaneously delivering dual anti-cancer genes and ferroptosis payload in tumor microenvironment.

KCTD17-mediated Ras stabilization promotes hepatocellular carcinoma progression.

Background/Aims: Potassium channel tetramerization domain containing 17 (KCTD17) protein, an adaptor for the cullin3 (Cul3) ubiquitin ligase complex, has been implicated in various human diseases; however, its role in hepatocellular carcinoma (HCC) remains elusive. Here, we aimed to elucidate the clinical features of KCTD17, and investigate the mechanisms by which KCTD17 affects HCC progression. Methods: We analyzed transcriptomic data from patients with HCC. Hepatocyte-specific KCTD17 deficient mice were treated with diethylnitrosamine (DEN) to assess its effect on HCC progression. Additionally, we tested KCTD17-directed antisense oligonucleotides for their therapeutic potential in vivo. Results: Our investigation revealed the upregulation of KCTD17 expression in both tumors from patients with HCC and mouse models of HCC, in comparison to non-tumor controls. We identified the leucine zipper-like transcriptional regulator 1 (Lztr1) protein, a previously identified Ras destabilizer, as a substrate for KCTD17-Cul3 complex. KCTD17-mediated Lztr1 degradation led to Ras stabilization, resulting in increased proliferation, migration, and wound healing in liver cancer cells. Hepatocyte-specific KCTD17 deficient mice or liver cancer xenograft models were less susceptible to carcinogenesis or tumor growth. Similarly, treatment with KCTD17-directed antisense oligonucleotides (ASO) in a mouse model of HCC markedly lowered tumor volume as well as Ras protein levels, compared to those in control ASO-treated mice. Conclusions: KCTD17 induces the stabilization of Ras and downstream signaling pathways and HCC progression and may represent a novel therapeutic target for HCC.

The roles of CD4+ T cell help, sex, and dose in the induction of protective CD8+ T cells against a lethal poxvirus by mRNA-LNP vaccines.

The role of CD4+ T cells in the induction of protective CD8+ T cells by mRNA lipid nanoparticle (LNP) vaccines is unknown. We used B6 or Tlr9 -/- mice depleted or not of CD4+ T cells and LNP vaccines loaded with mRNAs encoding the ectromelia virus (ECTV) MHC class I H-2 Kb-restricted immunodominant CD8+ T cell epitope TSYKFESV (TSYKFESV mRNA-LNPs) or the ECTV EVM158 protein, which contains TSYKFESV (EVM-158 mRNA-LNPs). Following prime and boost with 10 µg of either vaccine, Kb-TSYKFESV-specific CD8+ T cells fully protected male and female mice from ECTV at 29 (both mRNA-LNPs) or 90 days (EVM158 mRNA-LNPs) post boost (dpb) independently of CD4+ T cells. However, at 29 dpb with 1 µg mRNA-LNPs, males had lower frequencies of Kb-TSYKFESV-specific CD8+ T cells and were much less well protected than females from ECTV, also independently of CD4+ T cells. At 90 dpb with 1 µg EVM158 mRNA-LNPs, the frequencies of Kb-TSYKFESV-specific CD8+ T cells in males and females were similar, and both were similarly partially protected from ECTV, independently of CD4+ T cells. Therefore, at optimal or suboptimal doses of mRNA-LNP vaccines, CD4+ T cell help is unnecessary to induce protective anti-poxvirus CD8+ T cells specific to a dominant epitope. At suboptimal doses, protection of males requires more time to develop.

Development of polymerase chain reaction-lateral flow dipstick assay for detection of Mycoplasma bovis in cattle.

Mycoplasma bovis (M. bovis) is capable of causing a range of diseases in cattle, encompassing calf pneumonia, arthritis, conjunctivitis, meningitis, and mastitis. It is widely recognized as one of the predominant pathogens posing a significant threat to the global cattle industry. Therefore, accurate and sensitive methods are urgently needed to detect M. bovis. This study aims to detect M. bovis by combining colloidal gold with biotin-labeled oligonucleotides to improve detection sensitivity and form a chromogenic detection probe based on signal amplification technology. Here, we developed a sensitive and specific polymerase chain reaction-lateral flow dipstick assay (PCR-LFD) strip for efficient nucleic acid detection of M. bovis. A pair of specific primers with 5' ends labeled with biotin and digoxigenin probes was designed for PCR experiments. Colloidal gold particles-labeled anti-digoxigenin IgG coated gold-labeled test strip was prepared, streptavidin was used as the detection probe, and nitrocellulose membrane coated goat anti-mouse IgG was used as the control line. Our results showed that the detection limit of the PCR-LFD was 89 fg/µL for the M. bovis DNA. The results from the test strip were highly consistent with those from real-time qPCR. This assay were highly specific for M. bovis, as there were no cross-reactions with other microorganisms tested and the detection sensitivity of the test was also relatively high (97.67%). The novel strips present a promising tool for the cost-effective and sensitive diagnosis of M. bovis.

FLI1 in PBMCs contributes to elevated inflammation in combat-related posttraumatic stress disorder.

Post-traumatic stress disorder (PTSD) is a debilitating psychiatric condition with significant public health implications that arise following exposure to traumatic events. Recent studies highlight the involvement of immune dysregulation in PTSD, characterized by elevated inflammatory markers. However, the precise mechanisms underlying this immune imbalance remain unclear. Previous research has implicated friend leukemia virus integration 1 (FLI1), an erythroblast transformation-specific (ETS) transcription factor, in inflammatory responses in sepsis and Alzheimer's disease. Elevated FLI1 levels in peripheral blood mononuclear cells (PBMCs) have been linked to lupus severity. Yet, FLI1's role in PTSD-related inflammation remains unexplored. In our study, PBMCs were collected from Veterans with and without PTSD. We found significantly increased FLI1 expression in PBMCs from PTSD-afflicted Veterans, particularly in CD4+ T cells, with no notable changes in CD8+ T cells. Stimulation with LPS led to heightened FLI1 expression and elevated levels of inflammatory cytokines IL-6 and IFNγ in PTSD PBMCs compared to controls. Knockdown of FLI1 using Gapmers in PTSD PBMCs resulted in a marked reduction in inflammatory cytokine levels, restoring them to control group levels. Additionally, co-culturing PBMCs from both control and PTSD Veterans with the human brain microglia cell line HMC3 revealed increased inflammatory mediator levels in HMC3. Remarkably, HMC3 cells co-cultured with PTSD PBMCs treated with FLI1 Gapmers exhibited significantly lower inflammatory mediator levels compared to control Gapmer-treated PTSD PBMCs. These findings suggest that suppressing FLI1 may rebalance immune activity in PBMCs and mitigate microglial activation in the brain. Such insights could provide novel therapeutic strategies for PTSD.

Synthetic RIG-I agonist-mediated cancer immunotherapy synergizes with MAP kinase inhibition against BRAF-mutated melanoma.

The implementation of targeted molecular therapies and immunotherapy in melanoma vastly improved the therapeutic outcome in patients with limited efficacy of surgical intervention. Nevertheless, a large fraction of patients with melanoma still remain refractory or acquire resistance to these new forms of treatment, illustrating a need for improvement. Here, we report that the clinically relevant combination of mitogen-activated protein (MAP) kinase pathway inhibitors dabrafenib and trametinib synergize with RIG-I agonist-induced immunotherapy to kill BRAF-mutated human and mouse melanoma cells. Kinase inhibition did not compromise the agonist-induced innate immune response of the RIG-I pathway in host immune cells. In a melanoma transplantation mouse model, the triple therapy outperformed individual therapies. Our study suggests that agonist-induced activation of RIG-I with its synthetic ligand 3pRNA could vastly improve tumor control in a substantial fraction of patients with melanoma receiving MAP kinase inhibitors.

Robust Sequence Design Space for the Isothermal Exponential Amplification of Short Oligonucleotides.

Advances in isothermal amplification techniques have accelerated development in biosensing applications and the design of complex molecular devices. The exponential amplification reaction technique, or EXPAR, is uniquely positioned to process molecular information from short oligonucleotide strands (≈10 nucleotides length) typically encountered in molecular computing or microRNA detection. Despite its conceptual simplicity (requiring only a template strand and two enzymes), the issue of nonspecific background amplification has hindered broader adoption. In this work, a new system configuration is established at 37 °C to achieve significantly improved performance. Critical sequence motifs responsible for the excellent signal-to-background profile are identified and generalized as a universal adapter design framework. Orthogonal template sequences generated from the framework are implemented for a triplex reaction and successfully evaluated mixtures of multiple-target inputs in a single-step, one-pot format without the need for exogenous agents.

Multiantigen T-Cell Hybridizers: A Two-Component T-Cell-Activating Therapy.

Multispecific T-cell-engaging scaffolds have emerged as effective anticancer therapies for the treatment of hematological malignancies. Approaches that modulate cancer cell targeting and provide personalized, multispecific immunotherapeutics are needed. Here, we report on a modular, split antibody-like approach consisting of Fab' fragments modified with complementary morpholino oligonucleotides (MORFs). We synthesized a library of B-cell-targeting Fab'-MORF1 conjugates that self-assemble, via a Watson-Crick base pairing hybridization, with a complementary T-cell-engaging Fab'-MORF2 conjugate. We aptly titled our technology multiantigen T-cell hybridizers (MATCH). Using MATCH, cancer-specific T-cell recruitment was achieved utilizing four B-cell antigen targets: CD20, CD38, BCMA, and SLAMF7. The antigen expression profiles of various malignant B-cell lines were produced, and using these distinct profiles, cell-specific T-cell activation was attained on lymphoma, leukemia, and multiple myeloma cell lines in vitro. T-cell rechallenge experiments demonstrated the modular approach of MATCH by sequentially activating the same T-cell cohort against three different cancers using cancer antigen-specific Fab'-MORF1 conjugates. Furthermore, MATCH's efficacy was demonstrated in vivo by treating xenograft mouse models of human non-Hodgkin's lymphoma with CD20-directed MATCH therapy. In the pilot study, a single dose of MATCH allowed for long-term survival of all treated mice compared to saline control. In a second in vivo model, insights regarding optimal T-cell-to-target cell ratio were gleaned when a ratio of 5:1 T-cell-to-target cell MATCH-treated mice significantly delayed the onset of disease compared to higher and lower ratios.

Long-term and Continuous Plasmonic Oligonucleotide Monitoring Enabled by Regeneration Approach.

The demand for continuous monitoring of biochemical markers for diagnostic purposes is increasing as it overcomes the limitations of traditional intermittent measurements. This study introduces a method for long-term, continuous plasmonic biosensing of oligonucleotides with high temporal resolution. Our method is based on a regeneration-based reversibility approach that ensures rapid reversibility in less than 1 minute, allowing the sensor to fully reset after each measurement. We investigated label-free and AuNP enhancements for different dynamic ranges and sensitivities, achieving a limit of detection down to pM levels. We developed a regeneration-based reversibility approach for continuous biosensing, optimizing buffer conditions using the Taguchi method to achieve rapid, consistent reversibility, ensuring reliable performance for long-term monitoring. We detected oligonucleotides in buffered and complex solutions, including undiluted and unfiltered human serum, for up to 100 sampling cycles in a day. Moreover, we showed the long-term stability of the sensor for monitoring capabilities in buffered solutions and human serum, with minimal signal value drift and excellent sensor reversibility for up to 9 days. Our method opens the door to new prospects in continuous biosensing by providing insights beyond intermittent measurements for numerous analytical and diagnostic applications.

Emerging therapies in hereditary ataxias.

Recent investigations have defined the pathophysiological basis of many hereditary ataxias (HAs), including loss-of-function as well as gain-of-function mechanisms at either the RNA or protein level. Preclinical studies have assessed gene editing, gene and protein replacement, gene enhancement, and gene knockdown strategies. Methodologies include viral vector delivery of genes, oligonucleotide therapies, cell-penetrating peptides, synthetic transcription factors, and technologies to deliver therapies to defined targets. In this review, we focus on Friedreich ataxia (FRDA) and the polyglutamine ataxias in which translational research is active. However, much remains to be done to identify safe and effective molecules, create ideal delivery methods, and perform innovative clinical trials to prove the safety and efficacy of treatments for these rare but devastating diseases.

CpG oligonucleotides induce an acute murine thrombocytopenia dependent on toll-like receptor 9 and spleen tyrosine kinase pathways.

BACKGROUND: CpG ODN are synthetic single stranded DNA sequences that act as immunostimulants. They have been increasingly used to treat several cancers, however, thrombocytopenia is a potential recognized side effect of some sequences. OBJECTIVES: We tested the ability of two CpG ODN (ODN 2395 and ISIS 120704) to induce thrombocytopenia when administered to BALB/c mice and determined mechanisms associated with thrombocytopenia. METHODS: BALB/c mice were pre-bled and then injected with titrated doses of CpG ODNs and platelet counts were determined. The mice were treated IVIg or with various inhibitors and antagonists of toll-like receptor 9 (TLR9) and spleen tyrosine kinase (Syk) to determine their effects on the thrombocytopenia. RESULTS AND CONCLUSIONS: Compared with saline-treated mice or mice treated with 2'-O-methoxyethyl (MOE)-modified antisense (ASO) ODN, both ODN 2395 and ISIS 120704 induced an acute dose-dependent thrombocytopenia within 3 and 24 hours respectively. The thrombocytopenia was associated with significant increases in plasma monocyte chemoattractant protein 1 (MCP1). Intravenous immunoglobulin (IVIg) administration significantly rescued the CpG ODN-induced thrombocytopenia, as did treatment with either a Syk-inhibitor or TLR9 antagonists. In vitro, CpG ODN could activate human platelets and this correlated significantly with enhanced IVIg- and Syk-dependent phagocytosis by THP-1 monocytes. These results suggest that CpG ODN induce an acute inflammatory-associated (IVIg-sensitive) thrombocytopenia that can be alleviated by Syk- or TLR9-blockade, and an IVIg- and Syk-dependent platelet clearance pathway appears primarily responsible for the thrombocytopenia. Whether these results are applicable to humans still has to be elucidated.

Emerging DNA & RNA editing strategies for the treatment of epidermolysis bullosa.

Background: Epidermolysis bullosa (EB) is a clinically-heterogeneous genodermatosis with severe manifestations in the skin and other organs. The significant burden this condition places on patients justifies the development of gene therapeutic strategies targeting the genetic cause of the disease.Methods: Emerging RNA and DNA editing tools have shown remarkable advances in efficiency and safety. Applicable both in ex vivo- and in vivo settings, these gene therapeutics based on gene replacement or editing are either at the pre-clinical or clinical stage.Results: The recent landmark FDA approvals for gene editing based on CRISPR/Cas9, along with the first FDA-approved redosable in vivo gene replacement therapy for EB, will invigorate ongoing research efforts, increasing the likelihood of achieving local cure via CRISPR-based technologies in the near future.Conclusions: This review discusses the status quo of current gene therapeutics that act at the level of RNA or DNA, all with the common aim of improving the quality of life for EB patients.

Optimising in-cell NMR acquisition for nucleic acids.

Understanding the structure and function of nucleic acids in their native environment is crucial to structural biology and one focus of in-cell NMR spectroscopy. Many challenges hamper in-cell NMR in human cell lines, e.g. sample decay through cell death and RNA degradation. The resulting low signal intensities and broad line widths limit the use of more complex NMR experiments, reducing the possible structural and dynamic information that can be extracted. Here, we optimize the detection of imino proton signals, indicators of base-pairing and therefore secondary structure, of a double-stranded DNA oligonucleotide in HeLa cells, using selective excitation. We demonstrate the reproducible quantification of in-cell selective longitudinal relaxation times (selT1), which are reduced compared to the in vitro environment, as a result of interactions with the complex cellular environment. By measuring the intracellular selT1, we optimize the existing proton pulse sequences, and shorten measurement time whilst enhancing the signal gained per unit of time. This exemplifies an advantage of selective excitation over conventional methods like jump-return water suppression for in-cell NMR. Furthermore, important experimental controls are discussed, including intracellular quantification, supernatant control measurements, as well as the processing of lowly concentrated in-cell NMR samples. We expect that robust and fast in-cell NMR experiments of nucleic acids will facilitate the study of structure and dynamics and reveal their functional correlation.

Molecular mechanisms and antisense oligonucleotide therapies of familial amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS), a progressive neurodegenerative disease, presents considerable challenges in both diagnosis and treatment. It is categorized into sporadic and familial amyotrophic lateral sclerosis (fALS); the latter accounts for approximately 10% of cases and is primarily inherited in an autosomal dominant manner. This review summarizes the molecular genetics of fALS, highlighting key mutations that contribute to its pathogenesis, such as mutations in SOD1, FUS, and C9orf72. Central to this discourse is exploring antisense oligonucleotides (ASOs) that target these genetic aberrations, providing a promising therapeutic strategy. This review provides a detailed overview of the molecular mechanisms underlying fALS and the potential therapeutic value of ASOs, offering new insights into treating neurodegenerative diseases.

Splice modulation strategy applied to deep intronic variants in COL7A1 causing recessive dystrophic epidermolysis bullosa.

Recessive dystrophic epidermolysis bullosa (RDEB) is a rare and most often severe genetic disease characterized by recurrent blistering and erosions of the skin and mucous membranes after minor trauma, leading to major local and systemic complications. The disease is caused by loss-of-function variants in COL7A1 encoding type VII collagen (C7), the main component of anchoring fibrils, which form attachment structures stabilizing the cutaneous basement membrane zone. Alterations in C7 protein structure and/or expression lead to abnormal, rare or absent anchoring fibrils resulting in loss of dermal-epidermal adherence and skin blistering. To date, more than 1,200 distinct COL7A1 deleterious variants have been reported and 19% are splice variants. Here, we describe two RDEB patients for whom we identified two pathogenic deep intronic pathogenic variants in COL7A1. One of these variants (c.7795-97C > G) promotes the inclusion of a pseudoexon between exons 104 and 105 in the COL7A1 transcript, while the other causes partial or complete retention of intron 51. We used antisense oligonucleotide (ASO) mediated exon skipping to correct these aberrant splicing events in vitro. This led to increased normal mRNA splicing above 94% and restoration of C7 protein expression at a level (up to 56%) that should be sufficient to reverse the phenotype. This first report of exon skipping applied to counteract deep intronic variants in COL7A1 represents a promising therapeutic strategy for personalized medicine directed at patients with intronic variants at a distance of consensus splice sites.

Is Exon Skipping a Viable Therapeutic Approach for Vascular Ehlers-Danlos Syndrome with Mutations in COL3A1 Exon 10 or 15?

Vascular Ehlers-Danlos syndrome or Ehlers-Danlos syndrome type IV (vEDS) is a connective tissue disorder characterised by skin hyperextensibility, joint hypermobility and fatal vascular rupture caused by COL3A1 mutations that affect collagen III expression, homo-trimer assembly and secretion. Along with collagens I, II, V and XI, collagen III plays an important role in the extracellular matrix, particularly in the inner organs. To date, only symptomatic treatment for vEDS patients is available. Fibroblasts derived from vEDS patients carrying dominant negative and/or haploinsufficiency mutations in COL3A1 deposit reduced collagen III in the extracellular matrix. This study explored the potential of an antisense oligonucleotide (ASO)-mediated splice modulating strategy to bypass disease-causing COL3A1 mutations reported in the in-frame exons 10 and 15. Antisense oligonucleotides designed to redirect COL3A1 pre-mRNA processing and excise exons 10 or 15 were transfected into dermal fibroblasts derived from vEDS patients and a healthy control subject. Efficient exon 10 or 15 excision from the mature COL3A1 mRNA was achieved and intracellular collagen III expression was increased after treatment with ASOs; however, collagen III deposition into the extracellular matrix was reduced in patient cells. The region encoded by exon 10 includes a glycosylation site, and exon 15 encodes hydroxyproline and hydroxylysine-containing triplet repeats, predicted to be crucial for collagen III assembly. These results emphasize the importance of post-translational modification for collagen III homo-trimer assembly. In conclusion, while efficient skipping of target COL3A1 exons was achieved, the induced collagen III isoforms generated showed defects in extracellular matrix formation. While therapeutic ASO-mediated exon skipping is not indicated for the patients in this study, the observations are restricted to exons 10 and 15 and may not be applicable to other collagen III in-frame exons.

Identifying Cell-Penetrating Peptides for Effectively Delivering Antimicrobial Molecules into Streptococcus suis.

Cell-penetrating peptides (CPPs) are promising carriers to effectively transport antisense oligonucleotides (ASOs), including peptide nucleic acids (PNAs), into bacterial cells to combat multidrug-resistant bacterial infections, demonstrating significant therapeutic potential. Streptococcus suis, a Gram-positive bacterium, is a major bacterial pathogen in pigs and an emerging zoonotic pathogen. In this study, through the combination of super-resolution structured illumination microscopy (SR-SIM), flow cytometry analysis, and toxicity analysis assays, we investigated the suitability of four CPPs for delivering PNAs into S. suis cells: HIV-1 TAT efficiently penetrated S. suis cells with low toxicity against S. suis; (RXR)4XB had high penetration efficiency with inherent toxicity against S. suis; (KFF)3K showed lower penetration efficiency than HIV-1 TAT and (RXR)4XB; K8 failed to penetrate S. suis cells. HIV-1 TAT-conjugated PNA specific for the essential gyrase A subunit gene (TAT-anti-gyrA PNA) effectively inhibited the growth of S. suis. TAT-anti-gyrA PNA exhibited a significant bactericidal effect on serotypes 2, 4, 5, 7, and 9 strains of S. suis, which are known to cause human infections. Our study demonstrates the potential of CPP-ASO conjugates as new antimicrobial compounds for combating S. suis infections. Furthermore, our findings demonstrate that applying SR-SIM and flow cytometry analysis provides a convenient, intuitive, and cost-effective approach to identifying suitable CPPs for delivering cargo molecules into bacterial cells.

Targeting alternative splicing of fibronectin in human renal proximal tubule epithelial cells with antisense oligonucleotides to reduce EDA+ fibronectin production and block an autocrine loop that drives renal fibrosis.

TGFß1 is a powerful regulator of fibrosis; secreted in a latent form, it becomes active after release from the latent complex. During tissue fibrosis, the EDA + isoform of cellular fibronectin is overexpressed. In pulmonary fibrosis it has been proposed that the fibronectin splice variant including an EDA domain (FN EDA+) activates latent TGFß. Our work investigates the potential of blocking the 'splicing in' of EDA with antisense oligonucleotides to inhibit TGFß1-induced EDA + fibronectin and to prevent the cascade of events initiated by TGFß1 in human renal proximal tubule cells (PTEC). Human primary PTEC were treated with TGFß1 for 48 h, medium removed and the cells transfected with RNase H-independent antisense oligonucleotides (ASO) designed to block EDA exon inclusion (ASO5). The efficacy of ASO to block EDA exon inclusion was assessed by EDA + fibronectin RNA and protein expression; the expression of TGFß, αSMA (α smooth muscle actin), MMP2 (matrix metalloproteinse-2), MMP9 (matrix metalloproteinse-9), Collagen I, K Cadherin and connexin 43 was analysed. Targeting antisense oligonucleotides designed to block EDA exon inclusion in fibronectin pre mRNA were effective in reducing the amount of TGFß1 -induced cellular EDA + fibronectin RNA and secreted EDA + fibronectin protein (assessed by western immunoblotting and immunocytochemistry) in human proximal tubule cells in an in vitro cell culture model. The effect was selective for EDA + exon with no effect on EDB + fibronectin RNA and total fibronectin mRNA. Exogenous TGFß1 induced endogenous TGFß, αSMA, MMP2, MMP9 and Col I mRNA. TGFß1 treatment for 48h reduced the expression of K-Cadherin and increased the expression of connexin-43. These TGFß1-induced pro-fibrotic changes were attenuated by ASO5 treatment. 48 h after the removal of exogenous TGFß, further increases in αSMA, MMP2, MMP9 was observed; ASO5 significantly inhibited this subsequent increase. ASO5 treatment also significantly inhibited ability of the cell culture medium harvested at the end of the experiment (96h) to stimulate SMAD3 reporter cells. The role of endogenous TGFß1 was confirmed by the use of a TGFß receptor inhibitor. Our results demonstrate a critical role of FN EDA+ in a cycle of TGFß driven pro-fibrotic responses in human PTEC and blocking its production with ASO technology offers a potential therapy to interrupt this vicious circle and hence limit the progression of renal fibrosis.

Bi-functional CpG-STAT3 decoy oligonucleotide triggers multilineage differentiation of acute myeloid leukemia in mice.

Acute myeloid leukemia (AML) cells resist differentiation stimuli despite high expression of innate immune receptors, such as Toll-like receptor 9 (TLR9). We previously demonstrated that targeting Signal Transducer and Activator of Transcription 3 (STAT3) using TLR9-targeted decoy oligodeoxynucleotide (CpG-STAT3d) increases immunogenicity of human and mouse AML cells. Here, we elucidated molecular mechanisms of inv(16) AML reprogramming driven by STAT3-inhibition/TLR9-activation in vivo. At the transcriptional levels, AML cells isolated from mice after intravenous administration of CpG-STAT3d or leukemia-targeted Stat3 silencing and TLR9 co-stimulation, displayed similar upregulation of myeloid cell differentiation (Irf8, Cebpa, Itgam) and antigen-presentation (Ciita, Il12a, B2m)-related genes with concomitant reduction of leukemia-promoting Runx1. Single-cell transcriptomics revealed that CpG-STAT3d induced multilineage differentiation of AML cells into monocytes/macrophages, erythroblastic and B cell subsets. As shown by an inducible Irf8 silencing in vivo, IRF8 upregulation was critical for monocyte-macrophage differentiation of leukemic cells. TLR9-driven AML cell reprogramming was likely enabled by downregulation of STAT3-controlled methylation regulators, such as DNMT1 and DNMT3. In fact, the combination of DNA methyl transferase (DNMT) inhibition using azacitidine with CpG oligonucleotides alone mimicked CpG-STAT3d effects, resulting in AML cell differentiation, T cell activation, and systemic leukemia regression. These findings highlight immunotherapeutic potential of bi-functional oligonucleotides to unleash TLR9-driven differentiation of leukemic cells by concurrent STAT3 and/or DNMT inhibition.

Allele-Selective Thiomorpholino Antisense Oligonucleotides as a Therapeutic Approach for Fused-in-Sarcoma Amyotrophic Lateral Sclerosis.

Pathogenic variations in the fused in sarcoma (FUS) gene are associated with rare and aggressive forms of amyotrophic lateral sclerosis (ALS). As FUS-ALS is a dominant disease, a targeted, allele-selective approach to FUS knockdown is most suitable. Antisense oligonucleotides (AOs) are a promising therapeutic platform for treating such diseases. In this study, we have explored the potential for allele-selective knockdown of FUS. Gapmer-type AOs targeted to two common neutral polymorphisms in FUS were designed and evaluated in human fibroblasts. AOs had either methoxyethyl (MOE) or thiomorpholino (TMO) modifications. We found that the TMO modification improved allele selectivity and efficacy for the lead sequences when compared to the MOE counterparts. After TMO-modified gapmer knockdown of the target allele, up to 93% of FUS transcripts detected were from the non-target allele. Compared to MOE-modified AOs, the TMO-modified AOs also demonstrated reduced formation of structured nuclear inclusions and SFPQ aggregation that can be triggered by phosphorothioate-containing AOs. How overall length and gap length of the TMO-modified AOs affected allele selectivity, efficiency and off-target gene knockdown was also evaluated. We have shown that allele-selective knockdown of FUS may be a viable therapeutic strategy for treating FUS-ALS and demonstrated the benefits of the TMO modification for allele-selective applications.