Tumor cell derived osteopontin and prostaglandin E2 synergistically promote the expansion of myeloid derived suppressor cells during the tumor immune escape phase.

The immune escape stage in cancer immunoediting is a pivotal feature, transitioning immune-controlled tumor dormancy to progression, and augmenting invasion and metastasis. Tumors employ diverse mechanisms for immune escape, with generating immunosuppressive cells from skewed hematopoiesis being a crucial mechanism. This led us to suggest that tumor cells with immune escape properties produce factors that induce dysregulations in hematopoiesis. In support of this suggestion, this study found that mice bearing advanced-stage tumors exhibited dysregulated hematopoiesis characterized by the development of splenomegaly, anemia, extramedullary hematopoiesis, production of immunosuppressive mediators, and expanded medullary myelopoiesis. Further ex vivo studies exhibited that conditioned medium derived from EL4lu2 cells could mediate the expansion of myeloid derived suppressor cells (MDSCs) in bone marrow cell cultures. The protein array profiling results revealed the presence of elevated levels of osteopontin (OPN), prostaglandin E2 (PGE2) and interleukin 17 (IL-17) in the culture medium derived from EL4luc2 cells. Accordingly, substantial levels of these factors were also detected in the sera of mice bearing EL4luc2 tumors. Among these factors, only PGE2 alone could increase the number of MDSCs in the BM cell cultures. This effect of PGE2 was significantly potentiated by the presence of OPN but not IL-17. Finally, in vitro treatment of EL4luc2 cells with pioglitazone, a modulator of OPN and cyclooxygenase 2 (COX-2) resulted in a significant reduction in cell proliferation in EL4luc2 cells. Our findings highlight the significant role played by tumor cell-derived OPN and PGE2 in fostering the expansion of medullary MDSCs and in promoting tumor cell proliferation. Furthermore, these intertwined cancer processes could be key targets for pioglitazone intervention.

The cellular response to extracellular vesicles is dependent on their cell source and dose.

Extracellular vesicles (EVs) have been established to play important roles in cell-cell communication and shown promise as therapeutic agents. However, we still lack a basic understanding of how cells respond upon exposure to EVs from different cell sources at various doses. Thus, we treated fibroblasts with EVs from 12 different cell sources at doses between 20 and 200,000 per cell, analyzed their transcriptional effects, and functionally confirmed the findings in various cell types in vitro, and in vivo using single-cell RNA sequencing. Unbiased global analysis revealed EV dose to have a more significant effect than cell source, such that high doses down-regulated exocytosis and up-regulated lysosomal activity. However, EV cell source-specific responses were observed at low doses, and these reflected the activities of the EV's source cells. Last, we assessed EV-derived transcript abundance and found that immune cell-derived EVs were most associated with recipient cells. Together, this study provides important insights into the cellular response to EVs.

Chemical Modifications and Design Influence the Potency of Huntingtin Anti-Gene Oligonucleotides.

Huntington's disease is a neurodegenerative, trinucleotide repeat (TNR) disorder affecting both males and females. It is caused by an abnormal increase in the length of CAG•CTG TNR in exon 1 of the Huntingtin gene (HTT). The resultant, mutant HTT mRNA and protein cause neuronal toxicity, suggesting that reduction of their levels would constitute a promising therapeutic approach. We previously reported a novel strategy in which chemically modified oligonucleotides (ONs) directly target chromosomal DNA. These anti-gene ONs were able to downregulate both HTT mRNA and protein. In this study, various locked nucleic acid (LNA)/DNA mixmer anti-gene ONs were tested to investigate the effects of varying ON length, LNA content, and fatty acid modification on HTT expression. Altering the length did not significantly influence the ON potency, while LNA content was critical for activity. Utilization of palmitoyl-modified LNA monomers enhanced the ON activity relatively to the corresponding nonmodified LNA under serum starvation conditions. Furthermore, the number of palmitoylated LNA monomers and their positioning greatly affected ON potency. In addition, we performed RNA sequencing analysis, which showed that the anti-gene ONs affect the "immune system process, mRNA processing, and neurogenesis." Furthermore, we observed that for repeat containing genes, there is a higher tendency for antisense off-targeting. Taken together, our findings provide an optimized design of anti-gene ONs that could potentially be developed as DNA-targeting therapeutics for this class of TNR-related diseases.

Novel endosomolytic compounds enable highly potent delivery of antisense oligonucleotides.

The therapeutic and research potentials of oligonucleotides (ONs) have been hampered in part by their inability to effectively escape endosomal compartments to reach their cytosolic and nuclear targets. Splice-switching ONs (SSOs) can be used with endosomolytic small molecule compounds to increase functional delivery. So far, development of these compounds has been hindered by a lack of high-resolution methods that can correlate SSO trafficking with SSO activity. Here we present in-depth characterization of two novel endosomolytic compounds by using a combination of microscopic and functional assays with high spatiotemporal resolution. This system allows the visualization of SSO trafficking, evaluation of endosomal membrane rupture, and quantitates SSO functional activity on a protein level in the presence of endosomolytic compounds. We confirm that the leakage of SSO into the cytosol occurs in parallel with the physical engorgement of LAMP1-positive late endosomes and lysosomes. We conclude that the new compounds interfere with SSO trafficking to the LAMP1-positive endosomal compartments while inducing endosomal membrane rupture and concurrent ON escape into the cytosol. The efficacy of these compounds advocates their use as novel, potent, and quick-acting transfection reagents for antisense ONs.

Growth Media Conditions Influence the Secretion Route and Release Levels of Engineered Extracellular Vesicles.

Extracellular vesicles (EVs) are nanosized cell-derived vesicles produced by all cells, which provide a route of intercellular communication by transmitting biological cargo. While EVs offer promise as therapeutic agents, the molecular mechanisms of EV biogenesis are not yet fully elucidated, in part due to the concurrence of numerous interwoven pathways which give rise to heterogenous EV populations in vitro. The equilibrium between the EV-producing pathways is heavily influenced by factors in the extracellular environment, in such a way that can be taken advantage of to boost production of engineered EVs. In this study, a quantifiable EV-engineering approach is used to investigate how different cell media conditions alter EV production. The presence of serum, exogenous EVs, and other signaling factors in cell media alters EV production at the physical, molecular, and transcriptional levels. Further, it is demonstrated that the ceramide-dependent EV biogenesis route is the major pathway to production of engineered EVs during optimized EV-production. These findings suggest a novel understanding to the mechanisms underlying EV production in cell culture which can be applied to develop advanced EV production methods.

Oligonucleotides Targeting DNA Repeats Downregulate Huntingtin Gene Expression in Huntington's Patient-Derived Neural Model System.

Huntington's disease (HD) is one of the most common, dominantly inherited neurodegenerative disorders. It affects the striatum, cerebral cortex, and other subcortical structures leading to involuntary movement abnormalities, emotional disturbances, and cognitive impairments. HD is caused by a CAG•CTG trinucleotide-repeat expansion in exon 1 of the huntingtin (HTT) gene leading to the formation of mutant HTT (mtHTT) protein aggregates. Besides the toxicity of the mutated protein, there is also evidence that mtHTT transcripts contribute to the disease. Thus, the reduction of both mutated mRNA and protein would be most beneficial as a treatment. Previously, we designed a novel anti-gene oligonucleotide (AGO)-based strategy directly targeting the HTT trinucleotide-repeats in DNA and reported downregulation of mRNA and protein in HD patient fibroblasts. In this study, we differentiate HD patient-derived induced pluripotent stem cells to investigate the efficacy of the AGO, a DNA/Locked Nucleic Acid mixmer with phosphorothioate backbone, to modulate HTT transcription during neural in vitro development. For the first time, we demonstrate downregulation of HTT mRNA following both naked and magnetofected delivery into neural stem cells (NSCs) and show that neither emergence of neural rosette structures nor self-renewal of NSCs is compromised. Furthermore, the inhibition potency of both HTT mRNA and protein without off-target effects is confirmed in neurons. These results further validate an anti-gene approach for the treatment of HD.

Novel Orthogonally Hydrocarbon-Modified Cell-Penetrating Peptide Nanoparticles Mediate Efficient Delivery of Splice-Switching Antisense Oligonucleotides In Vitro and In Vivo.

Splice-switching therapy with splice-switching oligonucleotides (SSOs) has recently proven to be a clinically applicable strategy for the treatment of several mis-splice disorders. Despite this, wider application of SSOs is severely limited by the inherently poor bioavailability of SSO-based therapeutic compounds. Cell-penetrating peptides (CPPs) are a class of drug delivery systems (DDSs) that have recently gained considerable attention for improving the uptake of various oligonucleotide (ON)-based compounds, including SSOs. One strategy that has been successfully applied to develop effective CPP vectors is the introduction of various lipid modifications into the peptide. Here, we repurpose hydrocarbon-modified amino acids used in peptide stapling for the orthogonal introduction of hydrophobic modifications into the CPP structure during peptide synthesis. Our data show that α,α-disubstituted alkenyl-alanines can be successfully utilized to introduce hydrophobic modifications into CPPs to improve their ability to formulate SSOs into nanoparticles (NPs), and to mediate high delivery efficacy and tolerability both in vitro and in vivo. Conclusively, our results offer a new flexible approach for the sequence-specific introduction of hydrophobicity into the structure of CPPs and for improving their delivery properties.

Lipophilic Peptide Dendrimers for Delivery of Splice-Switching Oligonucleotides.

Non-viral transfection reagents are continuously being developed in attempt to replace viral vectors. Among those non-viral vectors, dendrimers have gained increasing interest due to their unique molecular structure and multivalency. However, more improvements are still needed to achieve higher efficacy and lower toxicity. In this study, we have examined 18 peptide dendrimers conjugated to lipophilic moieties, such as fatty acids or hydrophobic amino acids, that were previously explored for siRNA. Reporter cells were employed to investigate the transfection of single strand splice-switching oligonucleotides (ONs) using these peptide dendrimers. Luciferase level changes reflecting efficiency varied with amino acid composition, stereochemistry, and complexation media used. 3rd generation peptide dendrimers with D-amino acid configuration were superior to L-form. Lead formulations with 3rd generation, D-amino acid peptide dendrimers increased the correction level of the delivered ON up to 93-fold over untreated HeLa Luc/705 cells with minimal toxicity. To stabilize the formed complexes, Polyvinyl alcohol 18 (PVA18) polymer was added. Although PVA18 addition increased activity, toxicity when using our best candidates G 2,3KL-(Leu)4 (D) and G 2,3KL-diPalmitamide (D) was observed. Our findings demonstrate the potential of lipid-conjugated, D-amino acid-containing peptide dendrimers to be utilized as an effective and safe delivery vector for splice-switching ONs.

Quantification of extracellular vesicles in vitro and in vivo using sensitive bioluminescence imaging.

Extracellular vesicles (EVs) are naturally occurring nano-sized carriers that are secreted by cells and facilitate cell-to-cell communication by their unique ability to transfer biologically active cargo. Despite the pronounced increase in our understanding of EVs over the last decade, from disease pathophysiology to therapeutic drug delivery, improved molecular tools to track their therapeutic delivery are still needed. Unfortunately, the present catalogue of tools utilised for EV labelling lacks sensitivity or are not sufficiently specific. Here, we have explored the bioluminescent labelling of EVs using different luciferase enzymes tethered to CD63 to achieve a highly sensitive system for in vitro and in vivo tracking of EVs. Using tetraspanin fusions to either NanoLuc or ThermoLuc permits performing highly sensitive in vivo quantification of EVs or real-time imaging, respectively, at low cost and in a semi-high throughput manner. We find that the in vivo distribution pattern of EVs is determined by the route of injection, but that different EV subpopulations display differences in biodistribution patterns. By applying this technology for real-time non-invasive in vivo imaging of EVs, we show that their distribution to different internal organs occurs just minutes after administration.

Novel mouse model resistant to irreversible BTK inhibitors: a tool identifying new therapeutic targets and side effects.

Pharmacological inhibitors of Bruton tyrosine kinase (BTK) have revolutionized treatment of B-lymphocyte malignancies and show great promise for dampening autoimmunity. The predominant BTK inhibitors tether irreversibly by covalently binding to cysteine 481 in the BTK catalytic domain. Substitution of cysteine 481 for serine (C481S) is the most common mechanism for acquired drug resistance. We generated a novel C481S knock-in mouse model and, using a battery of tests, no overt B-lymphocyte phenotype was found. B lymphocytes from C481S animals were resistant to irreversible, but sensitive to reversible, BTK inhibitors. In contrast, irreversible inhibitors equally impaired T-lymphocyte activation in mice, mimicking the effect of treatment in patients. This demonstrates that T-lymphocyte blockage is independent of BTK. We suggest that the C481S knock-in mouse can serve as a useful tool for the study of BTK-independent effects of irreversible inhibitors, allowing for the identification of novel therapeutic targets and pinpointing potential side effects.

Sugar and Polymer Excipients Enhance Uptake and Splice-Switching Activity of Peptide-Dendrimer/Lipid/Oligonucleotide Formulations.

Non-viral transfection vectors are commonly used for oligonucleotide (ON) delivery but face many challenges before reaching the desired compartments inside cells. With the support of additional compounds, it might be more feasible for a vector to endure the barriers and achieve efficient delivery. In this report, we screened 18 different excipients and evaluated their effect on the performance of peptide dendrimer/lipid vector to deliver single-stranded, splice-switching ONs under serum conditions. Transfection efficiency was monitored in four different reporter cell lines by measuring splice-switching activity on RNA and protein levels. All reporter cell lines used had a mutated human ß-globin intron 2 sequence interrupting the luciferase gene, which led to an aberrant splicing of luciferase pre-mRNA and subsidence of luciferase protein translation. In the HeLa Luc/705 reporter cell line (a cervical cancer cell line), the lead excipients (Polyvinyl derivatives) potentiated the splice-switching activity up to 95-fold, compared to untreated cells with no detected cytotoxicity. Physical characterization revealed that lead excipients decreased the particle size and the zeta potential of the formulations. In vivo biodistribution studies emphasized the influence of formulations as well as the type of excipients on biodistribution profiles of the ON. Subsequently, we suggest that the highlighted impact of tested excipients would potentially assist in formulation development to deliver ON therapeutics in pre-clinical and clinical settings.

The viral protein corona directs viral pathogenesis and amyloid aggregation.

Artificial nanoparticles accumulate a protein corona layer in biological fluids, which significantly influences their bioactivity. As nanosized obligate intracellular parasites, viruses share many biophysical properties with artificial nanoparticles in extracellular environments and here we show that respiratory syncytial virus (RSV) and herpes simplex virus type 1 (HSV-1) accumulate a rich and distinctive protein corona in different biological fluids. Moreover, we show that corona pre-coating differentially affects viral infectivity and immune cell activation. In addition, we demonstrate that viruses bind amyloidogenic peptides in their corona and catalyze amyloid formation via surface-assisted heterogeneous nucleation. Importantly, we show that HSV-1 catalyzes the aggregation of the amyloid ß-peptide (Aß42), a major constituent of amyloid plaques in Alzheimer's disease, in vitro and in animal models. Our results highlight the viral protein corona as an acquired structural layer that is critical for viral-host interactions and illustrate a mechanistic convergence between viral and amyloid pathologies.

Oligonucleotide⁻Palladacycle Conjugates as Splice-Correcting Agents.

2'-O-Methylribo phosphorothioate oligonucleotides incorporating cyclopalladated benzylamine conjugate groups at their 5'-termini have been prepared and their ability to hybridize with a designated target sequence was assessed by conventional UV melting experiments. The oligonucleotides were further examined in splice-switching experiments in human cervical cancer (HeLa Luc/705), human liver (HuH7_705), and human osteosarcoma (U-2 OS_705) reporter cell lines. Melting temperatures of duplexes formed by the modified oligonucleotides were approximately 5 °C lower than melting temperatures of the respective unmodified duplexes. The cyclopalladated oligonucleotides functioned as splice-correcting agents in the HeLa Luc/705 cell line somewhat more efficiently than their unmodified counterparts. Furthermore, the introduction of this chemical modification did not induce toxicity in cells. These results demonstrate the feasibility of using covalently metalated oligonucleotides as therapeutic agents.

Novel peptide-dendrimer/lipid/oligonucleotide ternary complexes for efficient cellular uptake and improved splice-switching activity.

Despite the advances in gene therapy and in oligonucleotide (ON) chemistry, efficient cellular delivery remains an obstacle. Most current transfection reagents suffer from low efficacy or high cytotoxicity. In this report, we describe the synergism between lipid and dendrimer delivery vectors to enhance the transfection efficiency, while avoiding high toxicity. We screened a library of 20 peptide dendrimers representing three different generations and evaluated their capability to deliver a single-stranded splice-switching ON after formulating with lipids (DOTMA/DOPE). The transfection efficiency was analyzed in 5 reporter cell lines, in serum-free and serum conditions, and with 5 different formulation protocols. All formulations displayed low cytotoxicity to the majority of the tested cell lines. The complex sizes were < 200 nm; particle size distributions of effective mixtures were < 80 nm; and, the zeta potential was dependent on the formulation buffer used. The best dendrimer enhanced transfection in a HeLa reporter cell line by 30-fold compared to untreated cells under serum-free conditions. Interestingly, addition of sucrose to the formulation enabled - for the first time - peptide dendrimers/lipid complexes to efficiently deliver splice-switching ON in the presence of serum, reaching 40-fold increase in splice switching. Finally, in vivo studies highlighted the potential of these formulae to change the biodistribution pattern to be more towards the liver (90% of injected dose) compared to the kidneys (5% of injected dose) or to unformulated ON. This success encourages further development of peptide dendrimer complexes active in serum and future investigation of mechanisms behind the influence of additives on transfection efficacy.

Preparation and in vitro/in vivo evaluation of antimicrobial ocular in situ gels containing a disappearing preservative for topical treatment of bacterial conjunctivitis.

The study aimed to formulate and evaluate levofloxacin hemihydrate ocular in situ gels along with freshly prepared disappearing preservative reported to be safer to human eyes. Formulae were prepared using thermosensitive (PF127 and PF68) or ion-activated (Gelrite) polymers. They were evaluated for gelation temperature (GT), capacity, content uniformity, pH, rheological behavior, in vitro drug release with kinetic analysis. Best formulae were exposed to storage effect to select the optimum formula that was subjected to different sterilization methods and in vivo evaluation. The prepared disappearing preservative (sodium perborate monohydrate) proved to be active oxidative preservative and compatible with our formulae. F9 (24% PF127, 15% PF 68, 0.5% levofloxacin hemihydrate, and 0.0025% sodium perborate monohydrate) showed prolonged drug release (12 h), acceptable GT, viscosity, and pH. It remained stable over 3 months at two temperatures and was best sterilized by filtration. It showed longer residence time (12 h) in rabbits' eye fluids compared with the Levoxin® eye drops (4 h). This successful attempt of using thermo-gelling system along with a disappearing type of preservatives would allow the use of these systems to achieve sustained release of antimicrobial drugs with minimum risk of eye damage improving patient compliance and treatment efficacy.