Generalizable anchor aptamer strategy for loading nucleic acid therapeutics on exosomes.

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 (EXOEAA-PMO). EXOEAA-PMO elicited significantly greater muscle cell uptake, tissue accumulation and dystrophin expression than PMO in vitro and in vivo. Systemic administration of EXOEAA-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.

Simultaneous and rapid colorimetric detection of distinct miRNAs using Split-LAMP.

Introduction: Aberrant microRNA (miRNA) expressions are often discovered in many life threatening diseases such as cancer. In particular, recent studies show combinations of miRNA levels have greater diagnostic accuracy as opposed to single miRNA levels. For point-of-care applications, rapid and sensitive isothermal amplification with loop-mediated isothermal amplification (LAMP) has gained significant interest. Method: We developed a cost-effective point-of-care testing (POCT) device for multiple miRNAs that can integrate miRNA signals into a single output. Results and Discussion: We demonstrate that the loop primers for LAMP can be broken and be used for miRNA detection. This split-LAMP approach provides a logic AND-gate output for two distinct miRNA inputs. We then show that this is potentially useable in point-of-care testing using pH-sensitive dye to give a rapid, colorimetric endpoint readout within 30 min. This novel logic gate approach can potentially be extended to multiple miRNAs such that there can be a powerful diagnostic concept for multiple short RNAs in a point-of-care rapid test.

Complete remission of tumors in mice with neoantigen-painted exosomes and anti-PD-1 therapy.

Neoantigen-based cancer vaccines are emerging as promising tumor therapies, but enhancement of immunogenicity can further improve therapeutic outcomes. Here, we demonstrate that anchoring different peptide neoantigens on subcutaneously administered serum exosomes promote lymph node homing and dendritic cell uptake, resulting in significantly enhanced antigenicity in vitro and in vivo. Exosomes anchoring of melanoma peptide neoantigens augmented the magnitude and breadth of T cell response in vitro and in vivo, to a greater extent with CD8+ T cell responses. Simultaneous decoration of different peptide neoantigens on serum exosomes induced potent tumor suppression and neoantigen-specific immune responses in mice with melanoma and colon cancer. Complete tumor eradication and sustainable immunological memory were achieved with neoantigen-painted serum exosome vaccines in combination with programmed cell death protein 1 (PD-1) antibodies in mice with colon cancer. Importantly, human serum exosomes loaded with peptide neoantigens elicited significant tumor growth retardation and immune responses in human colon cancer 3-dimensional (3D) multicellular spheroids. Our study demonstrates that serum exosomes direct in vivo localization, increase dendritic cell uptake, and enhance the immunogenicity of antigenic peptides and thus provides a general delivery tool for peptide antigen-based personalized immunotherapy.

Towards a Rapid-Turnaround Low-Depth Unbiased Metagenomics Sequencing Workflow on the Illumina Platforms.

Unbiased metagenomic sequencing is conceptually well-suited for first-line diagnosis as all known and unknown infectious entities can be detected, but costs, turnaround time and human background reads in complex biofluids, such as plasma, hinder widespread deployment. Separate preparations of DNA and RNA also increases costs. In this study, we developed a rapid unbiased metagenomics next-generation sequencing (mNGS) workflow with a human background depletion method (HostEL) and a combined DNA/RNA library preparation kit (AmpRE) to address this issue. We enriched and detected bacterial and fungal standards spiked in plasma at physiological levels with low-depth sequencing (<1 million reads) for analytical validation. Clinical validation also showed 93% of plasma samples agreed with the clinical diagnostic test results when the diagnostic qPCR had a Ct < 33. The effect of different sequencing times was evaluated with the 19 h iSeq 100 paired end run, a more clinically palatable simulated iSeq 100 truncated run and the rapid 7 h MiniSeq platform. Our results demonstrate the ability to detect both DNA and RNA pathogens with low-depth sequencing and that iSeq 100 and MiniSeq platforms are compatible with unbiased low-depth metagenomics identification with the HostEL and AmpRE workflow.

Sub genomic analysis of SARS-CoV-2 using short read amplicon-based sequencing.

The novel coronavirus disease 2019 (COVID-19) pandemic poses a serious public health risk. In this report, we present a modified sequencing workflow using short tiling (280bp) amplicons library preparation method paired with Illumina's iSeq100 desktop sequencer. We demonstrated the utility of our workflow in identifying gapped reads that capture characteristics of subgenomic RNA junctions within our patient cohort. These analytical and library preparation approaches allow a versatile, small footprint and decentralized deployment that can facilitate comprehensive genetics characterizations during outbreaks. Based on the sequencing data, Taqman assays were designed to accurately capture the quantity of subgenomic ORF5 and ORF7a RNA from patient samples and demonstrated utility in tracking subgenomic titres in patient samples when combined with a standard COVID-19 qRT-PCR assay.

Functional imaging and targeted drug delivery in mice and patient tumors with a cell nucleolus-localizing and tumor-targeting peptide.

Tumor-targeting peptides have profound clinical implications in early detection and delineation of microscopic lesions for surgical resection, and also delivery of therapeutics with reduced systemic toxicity. Here, we demonstrate that a peptide (RS), evolved from a previously reported hepatocellular carcinoma (HCC)-targeting peptide P47, enables improved HCC micrometastasis discrimination and delineation from noncancerous tissues in murine orthotopic mice and patient biopsies, with up to 21-fold contrast. Importantly, RS targets non-small cell lung (NSCLC) and colon cancers in mice and patient biopsies, with higher selectivity for highly proliferative tumor nodules. Moreover, RS localizes to cell nucleoli of HCC, NSCLC, breast, colon and cervical cancer cells and induces nucleolar stress when conjugated with chemotherapeutic Oxaliplatin (OXA) (RS-OXA), demonstrating both cellular and subcellular targeting. RS-delivered OXA elicits significant tumor retardation in orthotopic HCC mice with markedly reduced systemic toxicity compared to OXA alone. Injection of fluorescence-labeled RS enables dynamic visualization of tumor growth in RS-OXA-treated subcutaneous HCC mice. Our study demonstrates that RS targets a spectrum of tumors and localizes to cell nucleolus, thus enabling functional imaging and targeted delivery of OXA in HCC mice, and consequently provides a versatile tool for tumor imaging and targeted therapeutics.

Cardio-respiratory and phenotypic rescue of dystrophin/utrophin-deficient mice by combination therapy.

Duchenne muscular dystrophy (DMD) is a systemic progressive muscular disease caused by frame-disrupting mutations in the DMD gene. Although exon-skipping antisense oligonucleotides (AOs) are clinically approved and can correct DMD, insufficient muscle delivery limits efficacy. If AO activity can be enhanced by safe dietary supplements, clinical trials for efficacy can be undertaken rapidly to benefit patients. We showed previously that intravenous glycine enhanced phosphorodiamidate morpholino oligomer (PMO) delivery to peripheral muscles in mdx mice. Here, we demonstrate that the combination of oral glycine and metformin with intravenous PMO enhances PMO activity, dystrophin restoration, extends lifespan, and improves body-wide function and phenotypic rescue of dystrophin /utrophin double knock-out (DKO) mice without any overt adverse effects. The DKO mice treated with the combination without altering the approved administration protocol of PMO show improved cardio-respiratory and behavioral functions. Metformin and glycine individually are ineffective in DMD patients, but the combination of PMO with clinically-approved oral glycine and metformin might improve the efficacy of the treatment also in DMD patients. Our data suggest that this combination therapy might be an attractive therapy for DMD and potentially other muscle diseases requiring systemic treatment with AOs.

Universal immunotherapeutic strategy for hepatocellular carcinoma with exosome vaccines that engage adaptive and innate immune responses.

BACKGROUND: 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. METHODS: 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 (DEXP&A2&N). RESULTS: DEXP&A2&N 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. DEXP&A2&N 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 DEXP&A&N. Supplementation of Fms-related tyrosine kinase 3 ligand greatly augmented the antitumor immunity of DEXP&A&N 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 DEXP&A&N. CONCLUSIONS: 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.

Amelioration of systemic inflammation via the display of two different decoy protein receptors on extracellular vesicles.

Extracellular vesicles (EVs) can be functionalized to display specific protein receptors on their surface. However, surface-display technology typically labels only a small fraction of the EV population. Here, we show that the joint display of two different therapeutically relevant protein receptors on EVs can be optimized by systematically screening EV-loading protein moieties. We used cytokine-binding domains derived from tumour necrosis factor receptor 1 (TNFR1) and interleukin-6 signal transducer (IL-6ST), which can act as decoy receptors for the pro-inflammatory cytokines tumour necrosis factor alpha (TNF-α) and IL-6, respectively. We found that the genetic engineering of EV-producing cells to express oligomerized exosomal sorting domains and the N-terminal fragment of syntenin (a cytosolic adaptor of the single transmembrane domain protein syndecan) increased the display efficiency and inhibitory activity of TNFR1 and IL-6ST and facilitated their joint display on EVs. In mouse models of systemic inflammation, neuroinflammation and intestinal inflammation, EVs displaying the cytokine decoys ameliorated the disease phenotypes with higher efficacy as compared with clinically approved biopharmaceutical agents targeting the TNF-α and IL-6 pathways.

A fluid-supported 3D hydrogel bioprinting method.

Hydrogels are used in many biomedical applications, including regenerative medicine and surgical training phantoms. However, the ability to shape these materials into complex anatomical structures using additive manufacturing is limited in part by their low mechanical stiffness. We developed a hydrogel 3D printer, that projects patterns directly onto a thin layer of fluid-supported hydrogel precursor, which serves as a floating, liquid projection screen. This approach avoids inadvertent adhesion that affects typical resin-based 3D printers, and enables fast, continuous printing. As a consequence, we can print smooth objects free of layering artifacts, at rates of 200 mm/h along the Z-axis. We demonstrate the versatility of our approach by printing various complex structures, including free-standing channel networks with 500 µm-thick walls, using hydrogels with a wide range of stiffness from 7 kPa to more than 4 MPa. Lastly, because the printer features a free surface, we combined it with an extruder to perform multi-material printing. We use this strategy to create centimeter-scale, cell-laden hydrogels containing channels, that help address the key nutrient supply problem in bioprinting.

Clinical performance of Roche cobas 6800, Luminex ARIES, MiRXES Fortitude Kit 2.1, Altona RealStar, and Applied Biosystems TaqPath for SARS-CoV-2 detection in nasopharyngeal swabs.

We compared the performance of five assays for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) detection on nasopharyngeal swab samples: Roche "cobas," Luminex "ARIES," MiRXES "Fortitude," Altona "RealStar," and Thermo Fisher Scientific "TaqPath." A total of 94 nasopharyngeal swab samples were obtained from 80 confirmed coronavirus disease 2019 cases in the first 2 weeks of illness (median, 7 days; range, 2-14 days) and 14 healthy controls. After collection, all samples were transported to the hospital clinical laboratory within 24 h. These samples were tested on all five assays within 3 days of sample receipt. Of the 94 samples, 69 yielded the same result on all platforms, resulting in an agreement of 73.4% (69 of 94). Of these, 14 were the healthy control swabs which all tested negative, demonstrating good specificity across all platforms. The ARIES assay had the lowest detection rate (68.8%), followed by Fortitude (85.0%), RealStar (86.3%), cobas (95.0%), and TaqPath (100%). Statistically significant differences were observed for ARIES, Fortitude, and RealStar when compared against the best performing TaqPath using McNemar's χ2 test. A consensus result was established based on the results obtained by the cobas, Fortitude, RealStar, and TaqPath. Six discrepancies had failed to reach a consensus and were adjudicated using the Cepheid Xpert Xpress SARS-CoV-2. Overall, the TaqPath and cobas assays were the most sensitive at detecting their designated SARS-CoV-2 gene targets. On the other hand, the ARIES assay was the least sensitive, thus warranting the need for assay re-optimization before go-live at the testing laboratory.

Erratum: Fructose Promotes Uptake and Activity of Oligonucleotides with Different Chemistries in a Context-Dependent Manner in mdx Mice.

[This corrects the article DOI: 10.1038/mtna.2016.46.].

Smad­binding decoy reduces extracellular matrix expression in human hypertrophic scar fibroblasts.

The exact mechanisms underlying hypertrophic scarring is yet to be fully understood. However, excessive collagen deposition by fibroblasts has been demonstrated to result in hypertrophic scar formation, and collagen synthesis in dermal fibroblasts is regulated by the transforming growth factor­ß1/Smad signaling pathway. In view of this, a Smad­binding decoy was designed and its effects on hypertrophic scar­derived human skin fibroblasts was evaluated. The results of the present study revealed that the Smad decoy attenuates the total amount of collagen, collagen I and Smad2/3 expression in scar fibroblasts. Data from RNA sequencing indicated that the Smad decoy induced more than 4­fold change in 178 genes, primarily associated with to the extracellular matrix, compared with the untreated control. In addition, results from quantitative real­time polymerase chain reaction further confirmed that the Smad decoy significantly attenuated the expression of extracellular matrix­related genes, including COL1A1, COL1A2 and COL3A1. Furthermore, the Smad decoy reduced transforming growth factor­ß1­induced collagen deposition in scar fibroblasts. Data generated from the present study provide evidence supporting the use of the Smad decoy as a potential hypertrophic scar treatment.

Glycine Enhances Satellite Cell Proliferation, Cell Transplantation, and Oligonucleotide Efficacy in Dystrophic Muscle.

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.

Systemic Exosomal Delivery of shRNA Minicircles Prevents Parkinsonian Pathology.

The development of new therapies to slow down or halt the progression of Parkinson's disease is a health care priority. A key pathological feature is the presence of alpha-synuclein aggregates, and there is increasing evidence that alpha-synuclein propagation plays a central role in disease progression. Consequently, the downregulation of alpha-synuclein is a potential therapeutic target. As a chronic disease, the ideal treatment will be minimally invasive and effective in the long-term. Knockdown of gene expression has clear potential, and siRNAs specific to alpha-synuclein have been designed; however, the efficacy of siRNA treatment is limited by its short-term efficacy. To combat this, we designed shRNA minicircles (shRNA-MCs), with the potential for prolonged effectiveness, and used RVG-exosomes as the vehicle for specific delivery into the brain. We optimized this system using transgenic mice expressing GFP and demonstrated its ability to downregulate GFP protein expression in the brain for up to 6 weeks. RVG-exosomes were used to deliver anti-alpha-synuclein shRNA-MC therapy to the alpha-synuclein preformed-fibril-induced model of parkinsonism. This therapy decreased alpha-synuclein aggregation, reduced the loss of dopaminergic neurons, and improved the clinical symptoms. Our results confirm the therapeutic potential of shRNA-MCs delivered by RVG-exosomes for long-term treatment of neurodegenerative diseases.

Exosome-Mediated miR-29 Transfer Reduces Muscle Atrophy and Kidney Fibrosis in Mice.

Our previous study showed that miR-29 attenuates muscle wasting in chronic kidney disease. Other studies found that miR-29 has anti-fibrosis activity. We hypothesized that intramuscular injection of exosome-encapsulated miR-29 would counteract unilateral ureteral obstruction (UUO)-induced muscle wasting and renal fibrosis. We used an engineered exosome vector, which contains an exosomal membrane protein gene Lamp2b that was fused with the targeting peptide RVG (rabies viral glycoprotein peptide). RVG directs exosomes to organs that express the acetylcholine receptor, such as kidney. The intervention of Exo/miR29 increased muscle cross-sectional area and decreased UUO-induced upregulation of TRIM63/MuRF1 and FBXO32/atrogin-1. Interestingly, renal fibrosis was partially depressed in the UUO mice with intramuscular injection of Exo/miR29. This was confirmed by decreased TGF-ß, alpha-smooth muscle actin, fibronectin, and collagen 1A1 in the kidney of UUO mice. When we used fluorescently labeled Exo/miR29 to trace the Exo/miR route in vivo and found that fluorescence was visible in un-injected muscle and in kidneys. We found that miR-29 directly inhibits YY1 and TGF-ß3, which provided a possible mechanism for inhibition of muscle atrophy and renal fibrosis by Exo/miR29. We conclude that Exo/miR29 ameliorates skeletal muscle atrophy and attenuates kidney fibrosis by downregulating YY1 and TGF-ß pathway proteins.