HaiFang Yin

Tumor‐derived exosomes elicit tumor suppression in murine hepatocellular carcinoma models and humans in vitro

Hepatocellular carcinoma (HCC) remains a global challenge due to high morbidity and mortality rates and poor response to treatment. Immunotherapy, based on introduction of dendritic cells (DCs) activated by tumor cell lysates as antigens ex vivo, shows limited response rates in HCC patients. Here, we demonstrate that tumor cell–derived exosomes (TEXs), displaying an array of HCC antigens, can elicit a stronger immune response than cell lysates in vitro and in vivo. Significant tumor growth inhibition was achieved in ectopic and orthotopic HCC mice treated with TEX‐pulsed DCs. Importantly, the tumor immune microenvironment was significantly improved in orthotopic HCC mice treated by TEX‐pulsed DCs, demonstrated by increased numbers of T lymphocytes, elevated levels of interferon‐γ, and decreased levels of interleukin‐10 and tumor growth factor‐β in tumor sites. As expected, T cells played an essential role in the TEX‐pulsed DC‐mediated immune response. Notably, exosomes from HCC cells not only promoted HCC‐specific cytolysis but also provided cross‐protective effects against pancreatic cancer cells. Moreover, HCC‐specific cytolysis, elicited by DCs pulsed with human HepG2 cell–derived exosomes, was observed across different human HCC cells irrespective of human leukocyte antigen types. Conclusion: HCC TEXs can potently carry HCC antigens, trigger a strong DC‐mediated immune response, and improve the HCC tumor microenvironment. (Hepatology 2016;64:456‐472)

Dendritic cell-derived exosomes elicit tumor regression in autochthonous hepatocellular carcinoma mouse models

BACKGROUND & AIMS Dendritic cell (DC)-derived exosomes (DEXs) form a new class of vaccines for cancer immunotherapy. However, their potency in hepatocellular carcinoma (HCC), a life-threatening malignancy with limited treatment options in the clinic that responds poorly to immunotherapy, remains to be investigated. METHODS Exosomes derived from α-fetoprotein (AFP)-expressing DCs (DEXAFP) were investigated in three different HCC mouse models systemically. Tumor growth and microenvironment were monitored. RESULTS DEXAFP elicited strong antigen-specific immune responses and resulted in significant tumor growth retardation and prolonged survival rates in mice with ectopic, orthotopic and carcinogen-induced HCC tumors that displayed antigenic and pathological heterogeneity. The tumor microenvironment was improved in DEXAFP-treated HCC mice, demonstrated by significantly more γ-interferon (IFN-γ)-expressing CD8+ T lymphocytes, elevated levels of IFN-γ and interleukin-2, and fewer CD25+Foxp3+ regulatory T (Treg) cells and decreased levels of interleukin-10 and transforming growth factor-β in tumor sites. Lack of efficacy in athymic nude mice and CD8+ T cell-depleted mice showed that T cells contribute to DEXAFP-mediated antitumor function. Dynamic examination of the antitumor efficacy and the immune microenvironment in DEXAFP-treated orthotopic HCC mice at different time-points revealed a positive correlation between tumor suppression and immune microenvironment. CONCLUSIONS Our findings provide evidence that AFP-enriched DEXs can trigger potent antigen-specific antitumor immune responses and reshape the tumor microenvironment in HCC mice and thus provide a cell-free vaccine option for HCC immunotherapy. Lay summary: Dendritic cell (DC)-derived exosomes (DEXs) form a new class of vaccines for cancer immunotherapy. However, their potency in hepatocellular carcinoma (HCC) remains unknown. Here, we investigated exosomes from HCC antigen-expressing DCs in three different HCC mouse models and proved their feasibility and capability of treating HCC, and thus provide a cell-free vaccine for HCC immunotherapy.

Effective dystrophin restoration by a novel muscle-homing peptide-morpholino conjugate in dystrophin-deficient mdx mice.

Antisense oligonucleotide (AO)-mediated splice correction therapy for Duchenne muscular dystrophy has shown huge promise from recent phase 2b clinical trials, however high doses and costs are required and targeted delivery can lower both of these. We have previously demonstrated the feasibility of targeted delivery of AOs by conjugating a chimeric peptide, consisting of a muscle-specific peptide and a cell-penetrating peptide, to AOs in mdx mice. Although increased uptake in muscle was observed, the majority of peptide-AO conjugate was found in the liver. To search for more effective muscle-homing peptides, we carried out in vitro biopanning in myoblasts and identified a novel 12-mer peptide (M12) showing preferential binding to skeletal muscle compared to the liver. When conjugated to phosphorodiamidate morpholino oligomers, ~25% of normal level of dystrophin expression was achieved in body-wide skeletal muscles in mdx mice with significant recovery in grip strength, whereas <2% in corresponding tissues treated with either muscle-specific peptide-phosphorodiamidate morpholino oligomer or unmodified phosphorodiamidate morpholino oligomer under identical conditions. Our data provide evidences for the first time that a muscle-homing peptide alone can enhance AO delivery to muscle without appreciable toxicity at 75 mg/kg, suggesting M12-phosphorodiamidate morpholino oligomer can be an alternative option to current AOs.

Effective Exon Skipping and Dystrophin Restoration by 2′-O-Methoxyethyl Antisense Oligonucleotide in Dystrophin-Deficient Mice

Antisense oligonucleotide (AO)–mediated exon-skipping therapy is one of the most promising therapeutic strategies for Duchenne Muscular Dystrophy (DMD) and several AO chemistries have been rigorously investigated. In this report, we focused on the effect of 2′-O-methoxyethyl oligonucleotides (MOE) on exon skipping in cultured mdx myoblasts and mice. Efficient dose-dependent skipping of targeted exon 23 was achieved in myoblasts with MOE AOs of different lengths and backbone chemistries. Furthermore, we established that 25-mer MOE phosphorothioate (PS) AOs provided the greatest exon-skipping efficacy. When compared with 2′O methyl phosphorothioate (2′OmePS) AOs, 25-mer MOE (PS) AOs also showed higher exon-skipping activity in vitro and in mdx mice after intramuscular injections. Characterization of uptake in vitro corroborated with exon-skipping results, suggesting that increased uptake of 25-mer MOE PS AOs might partly contribute to the difference in exon-skipping activity observed in vitro and in mdx mice. Our findings demonstrate the substantial potential for MOE PS AOs as an alternative option for the treatment of DMD.

Fructose Promotes Uptake and Activity of Oligonucleotides With Different Chemistries in a Context-dependent Manner in mdx Mice

Antisense oligonucleotide (AO)-mediated exon-skipping therapeutics shows great promise in correcting frame-disrupting mutations in the DMD gene for Duchenne muscular dystrophy. However, insufficient systemic delivery limits clinical adoption. Previously, we showed that a glucose/fructose mixture augmented AO delivery to muscle in mdx mice. Here, we evaluated if fructose alone could enhance the activities of AOs with different chemistries in mdx mice. The results demonstrated that fructose improved the potency of AOs tested with the greatest effect on phosphorodiamidate morpholino oligomer (PMO), resulted in a 4.25-fold increase in the number of dystrophin-positive fibres, compared to PMO in saline in mdx mice. Systemic injection of lissamine-labeled PMO with fructose at 25 mg/kg led to increased uptake and elevated dystrophin expression in peripheral muscles, compared to PMO in saline, suggesting that fructose potentiates PMO by enhancing uptake. Repeated intravenous administration of PMO in fructose at 50 mg/kg/week for 3 weeks and 50 mg/kg/month for 5 months restored up to 20% of wild-type dystrophin levels in skeletal muscles with improved functions without detectable toxicity, compared to untreated mdx controls. Collectively, we show that fructose can potentiate AOs of different chemistries in vivo although the effect diminished over repeated administration.Antisense oligonucleotide (AO)-mediated exon-skipping therapeutics shows great promise in correcting frame-disrupting mutations in the DMD gene for Duchenne muscular dystrophy. However, insufficient systemic delivery limits clinical adoption. Previously, we showed that a glucose/fructose mixture augmented AO delivery to muscle in mdx mice. Here, we evaluated if fructose alone could enhance the activities of AOs with different chemistries in mdx mice. The results demonstrated that fructose improved the potency of AOs tested with the greatest effect on phosphorodiamidate morpholino oligomer (PMO), resulted in a 4.25-fold increase in the number of dystrophin-positive fibres, compared to PMO in saline in mdx mice. Systemic injection of lissamine-labeled PMO with fructose at 25 mg/kg led to increased uptake and elevated dystrophin expression in peripheral muscles, compared to PMO in saline, suggesting that fructose potentiates PMO by enhancing uptake. Repeated intravenous administration of PMO in fructose at 50 mg/kg/week for 3 weeks and 50 mg/kg/month for 5 months restored up to 20% of wild-type dystrophin levels in skeletal muscles with improved functions without detectable toxicity, compared to untreated mdx controls. Collectively, we show that fructose can potentiate AOs of different chemistries in vivo although the effect diminished over repeated administration.

Peptide Nucleic Acid Promotes Systemic Dystrophin Expression and Functional Rescue in Dystrophin-deficient mdx Mice.

Antisense oligonucleotide (AO)-mediated exon-skipping therapeutics shows great promise for Duchenne muscular dystrophy (DMD) patients. However, recent failure with drisapersen, an AO candidate drug in phase 3 trial, highlights the importance of exploring other effective AO chemistries for DMD. Previously, we demonstrated the appreciable biological activity of peptide nucleic acid (PNA) AOs in restoring dystrophin expression in dystrophin-deficient mdx mice intramuscularly. Here, we further explore the systemic potential and feasibility of PNA AOs in mediating exon skipping in mdx mice as a comprehensive systemic evaluation remains lacking. Systemic delivery of PNA AOs resulted in therapeutic level of dystrophin expression in body-wide peripheral muscles and improved dystrophic pathology in mdx mice without any detectable toxicity. Up to 40% of dystrophin restoration was achieved in gastrocnemius, to a less extent with other skeletal muscles, with no dystrophin in heart. Notably, comparable systemic activity was obtained between PNA AOs and phosphorodiamidate morpholino oligomer, a DMD AO chemistry in phase 3 clinical trial, under an identical dosing regimen. Overall, our data demonstrate that PNA is viable for DMD exon-skipping therapeutics with 20 mer showing the best combination of activity, solubility, and safety and further modifications to increase PNA aqueous solubility can enable longer, more effective therapeutics without the associated toxicity.

HES1 promotes extracellular matrix protein expression and inhibits proliferation and migration in human trabecular meshwork cells under oxidative stress

Glaucoma is the leading cause of irreversible blindness. The most prevalent form of glaucoma is primary open-angle glaucoma (POAG). Oxidative stress is one of the major pathogenic factors of the POAG, and can elicit molecular and functional changes in trabecular meshwork cells, causing increased aqueous humor outflow resistance and elevated intraocular pressure. However, the regulatory mechanisms underlying oxidative stress-induced cell phenotypic changes remain elusive. Herein, we exposed primary human trabecular meshwork cells to the oxidative stress induced by 300 μM H2O2 for 2 h, and found significantly up-regulated expression of extracellular matrix proteins and a transcription factor, hairy and enhancer of split-1 (HES1). The cell functions, including migration and proliferation, were impaired by the oxidative stress. Furthermore, HES1 shRNA abrogated the extracellular matrix protein up-regulation and rescued the functional defects caused by the oxidative stress; conversely, HES1 overexpression resulted in the molecular and functional changes similar to those induced by H2O2. These results suggest that HES1 promotes extracellular matrix protein expression and inhibits proliferative and migratory functions in the trabecular meshwork cells under oxidative stress, thereby providing a novel pathogenic mechanism underlying and a potential therapeutic target to the POAG.

Characterization of the polycystic kidney disease 2 gene promoter.

The key regulatory elements for PKD2 transcription remain unclear. To identify these core elements, we characterized porcine PKD2 promoter with bioinformatics and molecular tools and found porcine PKD2 promoter bearing typical features of enriched CpG and less TATA. Further studies demonstrated that the core region was located in fragment -483 to +100. Subsequent biophysical binding assays and mutation experiments revealed that G4 motif and Sp1 are critical regulators for mediating the transcription of porcine PKD2. Moreover, the same regulatory pattern was reproduced in human PKD2 promoter region, indicating the critical role of G4 and Sp1 in regulating PKD2.

Fluorescent peptide highlights micronodules in murine hepatocellular carcinoma models and humans in vitro

Early detection and clear delineation of microscopic lesions during surgery are critical to the prognosis and survival of patients with hepatocellular carcinoma (HCC), a devastating malignancy without effective treatments except for resection. Tools to specifically identify and differentiate micronodules from normal tissue in HCC patients can have a positive impact on survival. Here, we discovered a peptide that preferentially binds to HCC cells through phage display. Significant accumulation of the fluorescence‐labeled peptide in tumor from ectopic and orthotopic HCC mice was observed within 2 hours of systemic injection. Contrast between tumor and surrounding liver is up to 6.5‐fold, and useful contrast lasts for 30 hours. Micronodules (0.03 cm in diameter) in liver and lung can clearly be distinguished from normal tissue with this fluorescence‐labeled peptide in orthotopic HCC mice and HCC patients. Compared to indocyanine green, a Food and Drug Administration–approved imaging contrast agent, an up to 8.7‐fold higher differentiation ratio of tumor to fibrosis is achieved with this fluorescence‐labeled peptide. Importantly, this peptide enables up to 10‐fold differentiation between HCC and peritumoral tissue in human tissues and the complete removal of tumor in HCC mice with surgical navigation. No abnormalities in behavior or activity are observed after systemic treatment, indicating the absence of overt toxicity. The peptide is metabolized with a half‐life of approximately 4 hours in serum. Conclusion: Our findings demonstrate that micronodules can be specifically differentiated with high sensitivity from surrounding tissue with this molecule, opening clinical possibilities for early detection and precise surgery of HCC. (Hepatology 2018).

Wild-Type Mouse Models to Screen Antisense Oligonucleotides for Exon-Skipping Efficacy in Duchenne Muscular Dystrophy

A readily available animal model is essential for rapidly identifying effective treatments for Duchenne muscular dystrophy (DMD), a devastating neuromuscular disorder caused by the lack of dystrophin protein, which results from frame-disrupting mutations in the DMD gene. Currently, the mdx mouse is the most commonly used model for antisense oligonucleotide (AO)-mediated exon skipping pre-clinical studies, with a mild phenotype. However, the accessibility of mdx mouse colonies particularly in developing countries can constrain research. Therefore in this study we explore the feasibility of using wild-type mice as models to establish exon-skipping efficiency of various DMD AO chemistries and their conjugates. Four different strains of wild-type mice and six different AO chemistries were investigated intramuscularly and the results indicated that the same exon-skipping efficiency was achieved for all tested AOs as that from mdx mice. Notably, levels of exon-skipping obtained in C57BL6 and C3H and mdx mice were most closely matched, followed by ICR and BALB/C mice. Systemic validation revealed that wild-type mice are less responsive to AO-mediated exon skipping than mdx mice. Our study provides evidence for the first time that wild-type mice can be appropriate models for assessing DMD AO exon-skipping efficiency with similar sensitivity to that of mdx mice and this finding can further accelerate the development of effective DMD AOs.