Treating large established tumors is challenging for dendritic cell (DC)-based immunotherapy. DC activation with tumor cell-derived exosomes (TEXs) carrying multiple tumor-associated antigen can ...enhance tumor recognition. Adding a potent adjuvant, high mobility group nucleosome-binding protein 1 (HMGN1), boosts DCs' ability to activate T cells and improves vaccine efficiency. Here, we demonstrate that TEXs painted with the functional domain of HMGN1 (TEX-N1ND) via an exosomal anchor peptide potentiates DC immunogenicity. TEX-N1ND pulsed DCs (DC
) elicit long-lasting antitumor immunity and tumor suppression in different syngeneic mouse models with large tumor burdens, most notably large, poorly immunogenic orthotopic hepatocellular carcinoma (HCC). DC
show increased homing to lymphoid tissues and contribute to augmented memory T cells. Importantly, N1ND-painted serum exosomes from cancer patients also promote DC activation. Our study demonstrates the potency of TEX-N1ND to strengthen DC immunogenicity and to suppress large established tumors, and thus provides an avenue to improve DC-based immunotherapy.
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)
To realize the therapeutic potential of RNA drugs, efficient, tissue-specific and nonimmunogenic delivery technologies must be developed. Here we show that exosomes-endogenous nano-vesicles that ...transport RNAs and proteins-can deliver short interfering (si)RNA to the brain in mice. To reduce immunogenicity, we used self-derived dendritic cells for exosome production. Targeting was achieved by engineering the dendritic cells to express Lamp2b, an exosomal membrane protein, fused to the neuron-specific RVG peptide. Purified exosomes were loaded with exogenous siRNA by electroporation. Intravenously injected RVG-targeted exosomes delivered GAPDH siRNA specifically to neurons, microglia, oligodendrocytes in the brain, resulting in a specific gene knockdown. Pre-exposure to RVG exosomes did not attenuate knockdown, and non-specific uptake in other tissues was not observed. The therapeutic potential of exosome-mediated siRNA delivery was demonstrated by the strong mRNA (60%) and protein (62%) knockdown of BACE1, a therapeutic target in Alzheimer's disease, in wild-type mice.
DNA Cage Delivery to Mammalian Cells Walsh, Anthony S; Yin, HaiFang; Erben, Christoph M ...
ACS nano,
07/2011, Letnik:
5, Številka:
7
Journal Article
Recenzirano
DNA cages are nanometer-scale polyhedral structures formed by self-assembly from synthetic DNA oligonucleotides. Potential applications include in vivo imaging and the targeted delivery of ...macromolecules into living cells. We report an investigation of the ability of a model cage, a DNA tetrahedron, to enter live cultured mammalian cells. Cultured human embryonic kidney cells were treated with a range of fluorescently labeled DNA tetrahedra and subsequently examined using confocal microscopy and flow cytometry. Substantial uptake of tetrahedra into cells was observed both when the cells were treated with tetrahedra alone and when the cells were treated with a mixture of tetrahedra and a transfection reagent. Analysis of the subcellular localization of transfected tetrahedra using confocal microscopy and organelle staining indicates that the cages are located in the cytoplasm. FRET experiments indicate that the DNA cages remain substantially intact within the cells for at least 48 h after transfection. This is a first step toward the use of engineered DNA nanostructures to deliver and control the activity of cargoes within cells.
The need to distribute therapy evenly systemically throughout the large muscle volume within the body makes Duchenne muscular dystrophy (DMD) therapy a challenge. Cell and exon-skipping therapies are ...promising but have limited effects, and thus enhancing their therapeutic potency is of paramount importance to increase the accessibility of these therapies to DMD patients. In this study, we demonstrate that co-administered glycine improves phosphorodiamidate morpholino oligomer (PMO) potency in mdx mice with marked functional improvement and an up to 50-fold increase of dystrophin in abdominal muscles compared to PMO in saline. Glycine boosts satellite cell proliferation and muscle regeneration by increasing activation of mammalian target of rapamycin complex 1 (mTORC1) and replenishing the one-carbon unit pool. The expanded regenerating myofiber population then results in increased PMO uptake. Glycine also augments the transplantation efficiency of exogenous satellite cells and primary myoblasts in mdx mice. Our data provide evidence that glycine enhances satellite cell proliferation, cell transplantation, and oligonucleotide efficacy in mdx mice, and thus it has therapeutic utility for cell therapy and drug delivery in muscle-wasting diseases.
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Yin and colleagues demonstrate that intravenous or dietary glycine enhances satellite cell proliferation, cell transplantation efficiency, and oligonucleotide-mediated dystrophin restoration in a Duchenne muscular dystrophy (DMD) mouse model, portending more efficacious cell and exon-skipping therapies for DMD.
Personalized immunotherapy utilizing cancer vaccines tailored to the tumors of individual patients holds promise for tumors with high genetic heterogeneity, potentially enabling eradication of the ...tumor in its entirety.
Here, we demonstrate a general strategy for biological nanovaccines that trigger tailored tumor-specific immune responses for hepatocellular carcinoma (HCC). Dendritic cell (DC)-derived exosomes (DEX) are painted with a HCC-targeting peptide (P47-P), an α-fetoprotein epitope (AFP212-A2) and a functional domain of high mobility group nucleosome-binding protein 1 (N1ND-N), an immunoadjuvant for DC recruitment and activation, via an exosomal anchor peptide to form a "trigger" DEX vaccine (DEX
).
DEX
specifically promoted recruitment, accumulation and activation of DCs in mice with orthotopic HCC tumor, resulting in enhanced cross-presentation of tumor neoantigens and de novo T cell response. DEX
elicited significant tumor retardation and tumor-specific immune responses in HCC mice with large tumor burdens. Importantly, tumor eradication was achieved in orthotopic HCC mice when antigenic AFP peptide was replaced with the full-length AFP (A) to form DEX
. Supplementation of Fms-related tyrosine kinase 3 ligand greatly augmented the antitumor immunity of DEX
by increasing immunological memory against tumor re-challenge in orthotopic HCC mice. Depletion of T cells, cross-presenting DCs and other innate immune cells abrogated the functionality of DEX
.
These findings demonstrate the capacity of universal DEX vaccines to induce tumor-specific immune responses by triggering an immune response tailored to the tumors of each individual, thus presenting a generalizable approach for personalized immunotherapy of HCC, by extension of other tumors, without the need to identify tumor antigens.
Duchenne muscular dystrophy (DMD) is the most common and severe form of muscular dystrophy, arising from mutations in the dystrophin gene that preclude the synthesis of functional protein. Antisense ...oligonucleotides (AOs) have been shown to induce specific exon skipping and thereby restore the reading frame and expression of functional dystrophin. In this report, we examine the effects of peptide nucleic acid (PNA) oligonucleotides and PNAs conjugated with peptides including TAT, muscle-specific peptide (MSP), adeno-associated virus 6 (AAV6) functional domain (AAV6), and AAV8 functional domain (AAV8), on exon skipping in vitro and in vivo. Efficient skipping of targeted exon 23 was achieved in cultured mdx myoblasts with PNA and PNA–peptide conjugates. Furthermore, single intramuscular injections of PNA and all PNA–peptide conjugates resulted in significant numbers of dystrophin-positive fibers in the injected tibialis anterior (TA) muscles of mdx mice, with no apparent local toxicity. Similar effects of exon skipping and dystrophin expression were obtained in mice of all ages. PNA and PNA-AAV6, PNA-AAV8 conjugates induced dystrophin expression in a dose-dependent manner. Our results demonstrate that PNAs have a higher efficiency of exon skipping than 2′O methyl phosphorothioate AOs do, and have a potential use in AO chemistry for antisense therapy of DMD.
Clinical deployment of oligonucleotides requires delivery technologies that improve stability, target tissue accumulation and cellular internalization. Exosomes show potential as ideal delivery ...vehicles. However, an affordable generalizable system for efficient loading of oligonucleotides on exosomes remain lacking. Here, we identified an Exosomal Anchor DNA Aptamer (EAA) via SELEX against exosomes immobilized with our proprietary CP05 peptides. EAA shows high binding affinity to different exosomes and enables efficient loading of nucleic acid drugs on exosomes. Serum stability of thrombin inhibitor NU172 was prolonged by exosome-loading, resulting in increased blood flow after injury in vivo. Importantly, Duchenne Muscular Dystrophy PMO can be readily loaded on exosomes via EAA (EXO
EAA-PMO
). EXO
EAA-PMO
elicited significantly greater muscle cell uptake, tissue accumulation and dystrophin expression than PMO in vitro and in vivo. Systemic administration of EXO
EAA-PMO
elicited therapeutic levels of dystrophin restoration and functional improvements in
mdx
mice. Altogether, our study demonstrates that EAA enables efficient loading of different nucleic acid drugs on exosomes, thus providing an easy and generalizable strategy for loading nucleic acid therapeutics on exosomes.
Synopsis
This study identifies an exosome-binding DNA aptamer (Exosomal Anchor Aptamer—EAA) and demonstrates that EAA binds to exosomes of different origins effectively and enables efficient loading of different nucleic acid drugs on exosomes. This study provides an easy generalizable strategy for loading nucleic acid therapeutics on exosomes orthogonal to CD63-binding peptides, which are better suited for protein and peptide loading.
EAA showed high binding affinity to exosomes irrespective of origin.
EAA enabled loading of different nucleic acid drugs on exosomes, with thrombin DNA aptamer inhibitor NU172 loaded on exosomes extended the serum stability.
Duchenne Muscular Dystrophy (DMD) phosphorodiamidate morpholino oligomers (PMOs) were efficiently loaded on exosomes via EAA to form EXO
EAA-PMO
and systemic administration EXO
EAA-PMO
at low doses improved muscle function and pathologies in dystrophic mice.
This study identifies an exosome-binding DNA aptamer (Exosomal Anchor Aptamer - EAA) and demonstrates that EAA binds to exosomes of different origins effectively and enables efficient loading of different nucleic acid drugs on exosomes. This study provides an easy generalizable strategy for loading nucleic acid therapeutics on exosomes orthogonal to CD63-binding peptides, which are better suited for protein and peptide loading.
Carbohydrate-based infusion solutions are widely used in the clinic. Here we show that co-administration of phosphorodiamidate morpholino oligomers (PMOs) with glucose enhances exon-skipping activity ...in Duchenne muscular dystrophy (DMD) mdx mice. We identify a glucose-fructose (GF) formulation that potentiates PMO activity, completely corrects aberrant Dmd transcripts, restores dystrophin levels in skeletal muscles and achieves functional rescue without detectable toxicity. This activity is attributed to enhancement of GF-mediated PMO uptake in the muscle. We demonstrate that PMO cellular uptake is energy dependent, and that ATP from GF metabolism contributes to enhanced cellular uptake of PMO in the muscle. Collectively, we show that GF potentiates PMO activity by replenishing cellular energy stores under energy-deficient conditions in mdx mice. Our findings provide mechanistic insight into hexose-mediated oligonucleotide delivery and have important implications for the development of DMD exon-skipping therapy.
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