Quality and Safety Considerations for Therapeutic Products Based on Extracellular Vesicles.

Extracellular vesicles (EVs) serve as an intrinsic system for delivering functional molecules within our body, playing significant roles in diverse physiological phenomena and diseases. Both native and engineered EVs are currently the subject of extensive research as promising therapeutics and drug delivery systems, primarily due to their remarkable attributes, such as targeting capabilities, biocompatibility, and low immunogenicity and mutagenicity. Nevertheless, their clinical application is still a long way off owing to multiple limitations. In this context, the Science Board of the Pharmaceuticals and Medical Devices Agency (PMDA) of Japan has conducted a comprehensive assessment to identify the current issues related to the quality and safety of EV-based therapeutic products. Furthermore, we have presented several examples of the state-of-the-art methodologies employed in EV manufacturing, along with guidelines for critical processes, such as production, purification, characterization, quality evaluation and control, safety assessment, and clinical development and evaluation of EV-based therapeutics. These endeavors aim to facilitate the clinical application of EVs and pave the way for their transformative impact in healthcare.

Multivalent dendritic DNA aptamer molecules for the enhancement of therapeutic effects.

Dendritic DNA molecules, referred to as DNA dendrons, consist of multiple covalently linked strands and are expected to improve the cellular uptake and potency of therapeutic oligonucleotides because of their multivalency. In this study, we developed an efficient synthetic method for producing DNA dendrons using strain-promoted azide-alkyne cycloaddition. Integration of the antitumor aptamer AS1411 into DNA dendrons enhanced cellular uptake and antiproliferative activity in cancer cells. These findings demonstrate that the incorporation of multivalent aptamers into DNA dendrons can effectively boost their therapeutic effects.

Tuning CpG motif position in nanostructured DNA for efficient immune stimulation.

It was previously demonstrated that polypod-like nanostructured DNA (polypodna) comprising three or more oligodeoxynucleotides (ODNs) were useful for the delivery of ODNs containing cytosine-phosphate-guanine (CpG) motifs, or CpG ODNs, to immune cells. Although the immunostimulatory activity of single-stranded CpG ODNs is highly dependent on CpG motif sequence and position, little is known about how the position of the motif affects the immunostimulatory activity of CpG motif-containing nanostructured DNAs. In the present study, four series of polypodna were designed, each comprising a CpG ODN with one potent CpG motif at varying positions and 2-5 CpG-free ODNs, and investigated their immunostimulatory activity using Toll-like receptor-9 (TLR9)-positive murine macrophage-like RAW264.7 cells. Polypodnas with the CpG motif in the 5'-overhang induced more tumor necrosis factor-α release than those with the motif in the double-stranded region, even though their cellular uptake were similar. Importantly, the rank order of the immunostimulatory activity of single-stranded CpG ODNs changed after their incorporation into polypodna. These results indicate that the CpG ODN sequence as well as the motif location in nanostructured DNAs should be considered for designing the CpG motif-containing nanostructured DNAs for immune stimulation.

Development of a Cytosolic DNA Sensor Agonist Using GALA Peptide-Conjugated DNA and Long Single-Stranded DNA.

Cytosolic DNA sensors (CDSs) recognize DNA molecules that are abnormally located in the cytosol, thus leading to the activation of the stimulator of interferon genes (STING) and the induction of type 1 interferon. In turn, type 1 interferon evokes defensive reactions against viral infections and activates the immune system; therefore, the use of agonists of CDSs as cancer therapeutics and vaccine adjuvants is expected. Double-stranded DNA molecules with dozens to thousands of bases derived from bacteria and viruses are agonists of CDSs. However, DNA is a water-soluble molecule with a high molecular weight, resulting in poor cellular uptake and endosomal escape. In contrast, long single-stranded DNA (lssDNA) obtained by rolling circle amplification is efficiently taken up and localized to endosomes. Here we constructed a CDS-targeting lssDNA via the facilitation of its intracellular transport from endosomes to the cytosol. An endosome-disrupting GALA peptide was used to deliver the lssDNA to the cytosol. A peptide-oligonucleotide conjugate (POC) was successfully obtained via the conjugation of the GALA peptide with an oligonucleotide complementary to the lssDNA. By hybridization of the POC to the complementary lssDNA (POC/lssDNA), the CDS-STING pathway in dendritic cells was efficiently stimulated. GALA peptide-conjugated DNA seems to be a helpful tool for the delivery of DNA to the cytosol.

Development of potent unmethylated CpG DNA hydrogel by introducing i-motifs into long single-stranded DNA.

Unmethylated cytosine-phosphate-guanine (CpG) DNA is recognized by Toll-like receptor 9, expressed in the endosomes of immune cells, and induces the secretion of proinflammatory cytokines. CpG DNA is, therefore, expected to be used as vaccine adjuvants, but there are many obstacles for its therapeutic application, such as poor cellular uptake and biostability. Long single-stranded DNA (lssDNA) synthesized by rolling circle amplification can be a useful delivery carrier for CpG DNA because of its cellular uptake efficiency, but the immunostimulatory effect is transient because it is easily degraded in endosomes. To improve its stability, we constructed lssDNA which forms hydrogel by i-motifs in an acidic environment mimicking endosome, and incorporated CpG DNA into lssDNA (i-CpG-lssDNA). We synthesized lssDNA containing the optimized i-motif sequence, and confirmed the formation of a DNA hydrogel in an acidic environment. The i-CpG-lssDNA elicited a potent proinflammatory cytokine production in murine macrophages, compared to CpG DNA-containing lssDNA without i-motifs. Consistently, its intradermal administration induced potent inflammatory cytokines at the regional lymph nodes. These results suggested that i-CpG-lssDNA could serve as a novel type of adjuvant for the induction of a potent immune response.

Formation of DNA nanotubes increases uptake into fibroblasts via enhanced affinity for collagen.

DNA nanostructures are promising delivery carriers because of their flexible structural design and high biocompatibility. Selectivity in cellular uptake is an important factor in the development of DNA-nanostructure-based delivery carriers. In this study, DNA nanotubes were selected as the DNA structures, and their selectivity for cellular uptake and the mechanisms involved were investigated. Unlike DNA nanostructures such as polypod-like nanostructured DNA or DNA tetrahedrons, which are easily taken up by macrophages, the formation of DNA nanotubes increases uptake by fibroblasts and fibroblast-like cells. We focused on the collagen expressed in cells as a factor in this process, and found DNA nanotube formation increased the affinity for type I collagen compared with that of single-stranded DNA. Collagenase treatment removes collagen from fibroblasts and reduces the uptake of DNA nanotubes by fibroblasts. We directly observed DNA nanotube uptake by fibroblasts using transmission electron microscopy, whereby the nanotubes were distributed on the cell surface, folded, fragmented, and taken up by phagocytosis. In conclusion, we demonstrated a novel finding that DNA nanotubes are readily taken up by fibroblasts and myoblasts.

Development of nucleic acid medicines based on chemical technology.

Oligonucleotide-based therapeutics have attracted attention as an emerging modality that includes the modulation of genes and their binding proteins related to diseases, allowing us to take action on previously undruggable targets. Since the late 2010s, the number of oligonucleotide medicines approved for clinical uses has dramatically increased. Various chemistry-based technologies have been developed to improve the therapeutic properties of oligonucleotides, such as chemical modification, conjugation, and nanoparticle formation, which can increase nuclease resistance, enhance affinity and selectivity to target sites, suppress off-target effects, and improve pharmacokinetic properties. Similar strategies employing modified nucleobases and lipid nanoparticles have been used for developing coronavirus disease 2019 mRNA vaccines. In this review, we provide an overview of the development of chemistry-based technologies aimed at using nucleic acids for developing therapeutics over the past several decades, with a specific emphasis on the structural design and functionality of chemical modification strategies.

Pharmacokinetic Approach for the Elucidation of Elevated Plasma Small Extracellular Vesicle (sEV) Concentration in Cancer.

The abundance of circulating plasma small extracellular vesicles (sEVs) has been reported to be elevated in cancer; however, the underlying mechanism remains unclear. In this study, a pharmacokinetic approach was used to determine the factors contributing to elevated plasma sEV levels during cancer in a tumor-bearing mouse model. Mouse plasma-derived sEVs (MP-sEVs) isolated from tumor-bearing mice showed increased protein concentrations and physicochemical characteristics comparable to MP-sEVs isolated from healthy mice. The steady-state concentration of sEVs is determined by the balance between the MP-sEV production and clearance. Thus, to determine whether tumorigenesis influences sEV clearance, isolated MP-sEVs were intravenously administered to either tumor-bearing or healthy mice. The results showed minimal differences in sEV clearance rates, suggesting that sEV production is the driving force of elevated MP-sEV concentrations. Lastly, CD63-gLuc stably expressing B16BL6-bearing mice were used to estimate the contribution of tumor cell-derived sEVs in the plasma. The gLuc activity of the MP-sEVs isolated was below the limit of detection, and it was estimated that the tumor cell-derived sEVs comprised at most 0.5% of the total MP-sEVs. Taken together, these results suggest that cells other than tumor cells contribute to elevated plasma sEV levels in cancer.

Extracellular vesicle-based therapeutics: Extracellular vesicles as therapeutic targets and agents.

Extracellular vesicles (EVs) are cell-derived membrane vesicles composed of a lipid bilayer. EVs contain biological molecules, such as nucleic acids, lipids, and proteins. As these molecules are transferred to cells that receive EVs, EVs function as intercellular communication tools. EV-mediated intercellular communication is involved in various diseases, such as cancer and neurodegenerative diseases, and biological events, such as immune reactions and inflammation. Therefore, EVs are suggested to be useful as therapeutic targets for various diseases. However, an EV-based drug delivery system (DDS) that utilizes its therapeutic properties has not yet been reported. The biological activities of EVs are derived from their endogenous components; hence, they can be directly applied as drugs. In this review, the basic aspects of EVs, such as their types, methods of isolation, and in vivo behavior, are briefly summarized. Moreover, the potential of using therapeutics targeting EVs has been discussed in cancer and neurodegenerative diseases. Various therapeutics using EVs, including DDSs, are listed and their associated advantages and challenges are discussed.

Development of Enzymatic Depletion Methods for Preparation of Small Extracellular Vesicles with Long Blood-Circulation Half-Life.

PURPOSE: Phosphatidylserine (PS)-deficient small extracellular vesicle (sEV) subpopulations (PS(-) sEVs) circulate in blood for long periods; hence, they are expected to have therapeutic applications. However, limited production of PS(-) sEVs makes their application difficult. In this study, a method for the preparation of such populations using an enzymatic reaction was developed. METHODS: Bulk sEVs collected from a cell culture supernatant via ultracentrifugation were subjected to an enzymatic reaction using phosphatidylserine decarboxylase (PSD). The yield of PS(-) sEVs was estimated using magnetic beads that bind to PS(+) sEVs. Then, the physical properties and pharmacokinetics (PK) of the sEVs were evaluated. RESULTS: Enzymatic depletion of PS exposed on sEV surfaces using PSD increased the yield of PS(-) sEVs. PSD treatment hardly changed the physicochemical properties of PS(-) sEVs. Moreover, the serum concentration profile and PK parameters of the PS(-) sEVs derived from PSD-treated bulk sEVs indicated a long blood-circulation half-life. CONCLUSIONS: Treatment of sEVs with PSD successfully reduced surface PS levels and increased the amount of the PS(-) sEV subpopulation among bulk sEVs. This protocol of efficient preparation of PS(-) sEVs based on PSD treatment, as well as information on the basic PK, can be foundational for the therapeutic application of sEVs.

Strategies for persistent retention of macromolecules and nanoparticles in the blood circulation.

The enhanced permeability and retention (EPR) effect has been the gold standard in developing drug delivery systems for passive tumor targeting. Although the importance of this concept remains unchanged, some controversies have arisen. In this review, various strategies for tumor targeting using macromolecules and nanoparticles based on the EPR effect are discussed from the viewpoint of pharmacokinetics. Overall, such strategies seek to retain therapeutic material in the blood circulation, which is a key factor for successful targeting. Strategies using macromolecules, including antibody-drug conjugates, serum albumin-based delivery systems, PEGylated recombinant proteins, and stealth liposomes as well as nanoparticle-based strategies such as those based on lipid nanoparticles, and polymeric micelles, have been discussed. The feasibility of small extracellular vesicles, a new class of nanosized delivery carriers, is also discussed.

Development of allergic rhinitis immunotherapy using antigen-loaded small extracellular vesicles.

Allergic rhinitis is caused by a breakdown of the Th1/Th2 balance, in which the allergen-induced Th2 immune response predominates over the Th1 immune response, culminating in IgE-mediated anaphylaxis. In this study, we used small extracellular vesicles (sEVs), cell-derived membrane vesicles with a particle size of 100 nm, as simultaneous delivery carriers for allergens (ovalbumin, OVA) and CpG DNA, an adjuvant that can induce a Th1 immune response, for the treatment of allergic rhinitis. sEVs loaded with CpG DNA and OVA(CpG-OVA-sEVs) were successfully prepared. CpG-OVA-sEVs possessed an average particle size of 90 nm and average zeta potential of -30 mV. CpG DNA modification did not influence the uptake of sEVs by dendritic cells and CpG-OVA-sEV can activate dendritic cells. The CpG-OVA-sEVs were delivered to the nasopharynx-associated lymphoid tissue (NALT) of mice and were primarily taken up by the CD11c positive cells after intranasal administration. Intranasally administering CpG-OVA-sEVs significantly enhanced OVA-specific IgG antibody titers in mice models of allergic rhinitis, suggesting a transformed Th1/2 balance. Moreover, The CpG-OVA-sEV administration alleviated allergic symptoms compared to the control group. Further, the amount of IgE secreted in mouse serum decreased. Thus, CpG-OVA-sEVs could be a useful therapeutic method for treating allergic rhinitis.

Enhanced Immunostimulatory Activity of Covalent DNA Dendrons.

The present study focused on the design and synthesis of covalent DNA dendrons bearing multivalent cytosine-phosphate-guanine oligodeoxynucleotides (CpG ODNs) that can stimulate the immune system through the activation of TLR9. These dendrons were synthesized using branching trebler phosphoramidite containing three identical protecting groups that enabled the simultaneous synthesis of multiple strands on a single molecule. Compared with linear ODNs, covalent DNA dendrons were found to be more resistant to nuclease degradation and were more efficiently taken up by macrophage-like RAW264.7 cells. Cellular uptake was suggested to be mediated by macrophage scavenger receptors. The covalent DNA dendrons composed of multivalent immunostimulatory branches enhanced the secretion of proinflammatory cytokines TNF-α and IL-6 from RAW264.7 cells, and 9-branched DNA dendrons showed the highest enhancement. Given their enhanced efficacy, we expect covalent DNA dendrons to be useful structures of oligonucleotide medicines.

Development of Hydrophobic Interaction-based DNA Supramolecules as Efficient Delivery Carriers of CpG DNA to Immune cells.

Unmethylated cytosine-phosphate-guanine (CpG) DNA stimulates mammalian immune cells through recognition by Toll-like receptor 9 (TLR9). Therefore, CpG DNA is expected to be an effective adjuvant for the treatment of immune and allergic diseases. However, challenges, such as low stability against DNase and low delivery efficiency for immune cells, still need to be resolved for the application of CpG DNA. To overcome these challenges, we developed DNA supramolecules consisting of long single-stranded DNA (lss-DNA) synthesized using rolling circle amplification (RCA) and cholesterol-modified DNA (chol-DNA). Lss-DNAs containing multiple CpG motifs were annealed with complementary chol-DNAs to form DNA supramolecules through hydrophobic interactions. Transmission electron microscopy revealed that lss-DNA mixed with chol-DNA formed micrometer-sized DNA supramolecules. The formation of DNA supramolecules increased their stability against DNase compared to lss DNA, which was evaluated using FBS. Furthermore, DNA supramolecules induced three-times higher TNF-α release from RAW264.7 cells than lss-DNA alone. These results demonstrate that DNA supramolecules are efficient delivery carriers of CpG DNA to immune cells.

The identification of novel small extracellular vesicle (sEV) production modulators using luciferase-based sEV quantification method.

Small extracellular vesicles (sEVs) are nano-sized vesicles secreted from various cells that contain bioactive metabolites and function as key regulators for intercellular communication. sEVs modulate diverse biological and pathological processes in the body, and the amount of circulating sEVs has been reported to correlate with certain disease progression. Therefore, the identification of small molecular compounds that can control sEV production may become a novel therapeutic strategy. In this study, a rapid, highly sensitive sEV quantification method utilizing fusion proteins consisting of Gaussia luciferase (gLuc) reporter protein and sEV markers (CD63 and CD82) was developed. A total of 480 compounds were screened to identify potent inducers and inhibitors of gLuc activity. Two novel compounds, KPYC08425 and KPYC12163, showed significant and dose-dependent changes in gLuc activity with minimal cytotoxicity based on the LDH assay. The efficacy of these two compounds was further evaluated by protein quantification of the isolated sEVs. Further evaluation of KPYC12163 suggested that the autolysosomal pathway may be involved in its inhibitory effect on sEV production.

Calcium Peroxide-Containing Polydimethylsiloxane-Based Microwells for Inhibiting Cell Death in Spheroids through Improved Oxygen Supply.

Multicellular spheroids are expected to be used for in vivo-like tissue models and cell transplantation. Microwell devices are useful for the fabrication of multicellular spheroids to improve productivity and regulate their size. However, the high cell density in microwell devices leads to accelerated cell death. In this study, we developed O2-generating microwells by incorporating calcium peroxide (CaO2) into polydimethylsiloxane (PDMS)-based microwells. The CaO2-containing PDMS was shown to generate O2 for 3 d. Then, CaO2-containing PDMS was used to fabricate O2-generating microwells using a micro-molding technique. When human hepatocellular carcinoma (HepG2) spheroids were prepared using the conventional microwells, the O2 concentration in the culture medium reduced to approx. 67% of the cell-free level. In contrast, the O2-generating microwells maintained O2 at constant levels. The HepG2 spheroids prepared using the O2-generating microwells had a larger number of live cells than those prepared using the conventional microwells. In addition, the O2-generating microwells rescued hypoxia in the HepG2 spheroids and increased cell viability. Lastly, the O2-generating microwells were also useful for the preparation of multicellular spheroids of other cell types (i.e., MIN6, B16-BL6, and adipose-derived stem cells) with high cell viability. These results showed that the O2-generating microwells are useful for preparing multicellular spheroids with high cell viability.

Interleukin-4-carrying small extracellular vesicles with a high potential as anti-inflammatory therapeutics based on modulation of macrophage function.

Interleukin-4 (IL4), a Th2-type cytokine that can drive M2 macrophage polarization, is expected to be used as an anti-inflammatory therapy agent as M2 polarization of macrophages can ameliorate chronic inflammation. However, several problems, such as the low effectiveness and side effects, have hampered the clinical application. To safely and effectively use IL4, an efficient delivery of IL4 to target cells, macrophages, is necessary. Small extracellular vesicles (sEVs) are promising candidates as macrophage delivery carriers because they are efficiently recognized by macrophages. In addition, considering the property of IL4 signaling, for which the internalization of IL4 receptor into the cellular compartment is important, and sEV uptake mechanism by macrophages, sEVs are expected to amplify IL4 signaling. In this study, we developed IL4-carrying sEVs (IL4-sEVs) by genetically engineering sEV-producing cells. We investigated the bioactivity of IL4-sEVs using RAW264.7 macrophages and their potential for therapeutic application to the treatment of an inflammatory disease using collagen-induced arthritis model mice. IL4-sEVs exhibited stronger anti-inflammatory effects on M1-polarized macrophages through M2 polarization of macrophages than those of soluble IL4 proteins. Moreover, IL4-sEVs exhibited more effective therapeutic effects on rheumatoid arthritis than those of IL4. These results indicate that IL4-carrying sEVs are promising anti-inflammatory therapeutics.

Phosphatidylserine-deficient small extracellular vesicle is a major somatic cell-derived sEV subpopulation in blood.

Small extracellular vesicles (sEVs) are important mediators of intercellular communication with respect to diverse pathophysiological processes. Here, we determined novel phosphatidylserine (PS)-deficient sEV subpopulations as a major somatic cell-derived sEV subpopulation in blood because of long blood circulation half-life through escape from macrophage uptake. PS(-)-sEVs were identified in various cultured cells as a minor population. However, as a result of rapid uptake of PS(+)-sEVs by macrophages, circulating somatic cell-derived sEVs in the blood were found to be mainly PS(-)-sEVs. These results suggest that endogenous PS(-)-sEVs could indeed be the key player in sEV-mediated intercellular communication, a good target for sEV-based diagnosis, and a potent candidate for sEV-based drug delivery. Our findings bring a paradigm shift in the understanding of the biology and translational applications of sEVs.

Determining The Role of Surface Glycans in The Pharmacokinetics of Small Extracellular Vesicles.

Small extracellular vesicles (sEVs) are important mediators of intercellular communication and are thereby expected to be promising carriers for drug delivery. Understanding the factors that affect sEV pharmacokinetics is crucial for its application as a drug delivery carrier. In this study, the role of sEV surface glycans was investigated by evaluating the effects of enzymatic deglycosylation treatment on sEV pharmacokinetics. First, control glycoprotein fetuin was used to optimize the glycosidase treatment conditions. B16-BL6-derived sEVs labeled with fusion proteins comprising Gag protein and Gaussia luciferase (gLuc) (Gag-gLuc) were then treated with glycosidases, Peptide-N-Glycosidase F or O-glycosidase, which cleaves N- and O-glycans, respectively. Glycosidase-treated sEVs showed physicochemical characteristics comparable to those of the untreated sEVs. However, removal of N-glycans from B16-BL6 sEVs enhanced cellular uptake by the peritoneal macrophages, while the removal of O-glycans had minimal impact, as evaluated by flow cytometry. To determine the effect of surface glycans on the sEV pharmacokinetics, Gag-gLuc labeled B16-BL6 sEVs treated with or without glycosidases were then intravenously administered to mice. Glycosidase-treated sEVs showed almost identical clearance from the blood circulation as that of the untreated sEVs. These results suggest minimal impact of surface glycans on sEV pharmacokinetics, despites its effect on cellular uptake.