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.

Umbilical cord-derived mesenchymal stromal cells preserve endogenous insulin production in type 1 diabetes: a Phase I/II randomised double-blind placebo-controlled trial.

AIM/HYPOTHESIS: This study aimed to investigate the safety and efficacy of treatment with allogeneic Wharton's jelly-derived mesenchymal stromal cells (MSCs) in recent-onset type 1 diabetes. METHODS: A combined Phase I/II trial, composed of a dose escalation followed by a randomised double-blind placebo-controlled study in parallel design, was performed in which treatment with allogeneic MSCs produced as an advanced therapy medicinal product (ProTrans) was compared with placebo in adults with newly diagnosed type 1 diabetes. Inclusion criteria were a diagnosis of type 1 diabetes <2 years before enrolment, age 18-40 years and a fasting plasma C-peptide concentration >0.12 nmol/l. Randomisation was performed with a web-based randomisation system, with a randomisation code created prior to the start of the study. The randomisation was made in blocks, with participants randomised to ProTrans or placebo treatment. Randomisation envelopes were kept at the clinic in a locked room, with study staff opening the envelopes at the baseline visits. All participants and study personnel were blinded to group assignment. The study was conducted at Karolinska University Hospital, Stockholm, Sweden. RESULTS: Three participants were included in each dose cohort during the first part of the study. Fifteen participants were randomised in the second part of the study, with ten participants assigned to ProTrans treatment and five to placebo. All participants were analysed for the primary and secondary outcomes. No serious adverse events related to treatment were observed and, overall, few adverse events (mainly mild upper respiratory tract infections) were reported in the active treatment and placebo arms. The primary efficacy endpoint was defined as Δ-change in C-peptide AUC for a mixed meal tolerance test at 1 year following ProTrans/placebo infusion compared with baseline performance prior to treatment. C-peptide levels in placebo-treated individuals declined by 47%, whereas those in ProTrans-treated individuals declined by only 10% (p<0.05). Similarly, insulin requirements increased in placebo-treated individuals by a median of 10 U/day, whereas insulin needs of ProTrans-treated individuals did not change over the follow-up period of 12 months (p<0.05). CONCLUSIONS/INTERPRETATION: This study suggests that allogeneic Wharton's jelly-derived MSCs (ProTrans) is a safe treatment for recent-onset type 1 diabetes, with the potential to preserve beta cell function. TRIAL REGISTRATION: ClinicalTrials.gov NCT03406585 FUNDING: The sponsor of the clinical trial is NextCell Pharma AB, Stockholm, Sweden.

Nanotechnology approaches for gene transfer.

In both basic research as well as experimental gene therapy the need to transfer genetic material into a cell is of vital importance. The cellular compartment, which is the target for the genetic material, depends upon application. An siRNA that mediates silencing is preferably delivered to the cytosol while a transgene would need to end up in the nucleus for successful transcription to occur. Furthermore the ability to regulate gene expression has grown substantially since the discovery of RNA interference. In such diverse fields as medical research and agricultural pest control, the capability to alter the genetic output has been a useful tool for pushing the scientific frontiers. This review is focused on nanotechnological approaches to assemble optimised structures of nucleic acid derivatives to facilitate gene delivery as well as promoting down regulation of endogenous genes.

Building biologically active nucleic acid nanocomplexes.

The Bioplex technology allows the hybridization of functional entities to various forms of nucleic acids by the use of synthetic nucleic acid analogs. Such supramolecular assemblies can be made in a predetermined fashion and can confer new properties. The Zorro technology is based on a novel construct generated to simultaneously bind to both DNA strands. Such compounds may have gene silencing activity.

Sequence-specific inhibition of RNA polymerase III-dependent transcription using Zorro locked nucleic acid (LNA).

BACKGROUND: RNA polymerase III (pol III)-dependent transcripts are involved in many fundamental activities in a cell, such as splicing and protein synthesis. They also regulate cell growth and influence tumor formation. During recent years vector-based systems for expression of short hairpin (sh) RNA under the control of a pol III promoter have been developed as gene-based medicines. Therefore, there is an increasing interest in means to regulate pol III-dependent transcription. Recently, we have developed a novel anti-gene molecule 'Zorro LNA (Locked Nucleic Acid)', which simultaneously hybridizes to both strands of super-coiled DNA and potently inhibits RNA polymerase II-derived transcription. We have now applied Zorro LNA in an attempt to also control U6 promoter-driven expression of shRNA. METHODS: In this study, we constructed pshluc and pshluc2BS plasmids, in which U6 promoter-driven small hairpin RNA specific for luciferase gene (shluc) was without or with Zorro LNA binding sites, respectively. After hybridization of Zorro LNA to pshluc2BS, the LNA-bound plasmid was cotransfected with pEGFPluc into mammalian cells and into a mouse model. In cellular experiments, cotransfection of unhybridized pshluc2BS, Zorro LNA and pEGFPluc was also performed. RESULTS: The results showed that the Zorro LNA construct efficiently inhibited pol III-dependent transcription as an anti-gene reagent in a cellular context, including in vivo in a mouse model. CONCLUSIONS: Thus, this new form of gene silencer 'Zorro LNA' could potentially serve as a versatile regulator of pol III-dependent transcription, including various forms of shRNAs.

Evaluating coupling procedures for synthesis of lna-Peptide conjugates.

As initial studies of block coupling of peptides with C-terminal glycines, we have evaluated of coupling of glycine building blocks under various conditions to a preassembled solid support bound LNA-fragment. Potentially competing side reaction have been studied and we have worked out a procedure for trifluoroacetyl protection of peptides.

Self-assembling supramolecular complexes by single-stranded extension from plasmid DNA.

Self-assembling supramolecular complexes are of great interest for bottom-up research like nanotechnology. DNA is an inexpensive building block with sequence-specific self-assembling capabilities through Watson-Crick and/or Hoogsteen base pairing and could be used for applications in surface chemistry, material science, nanomechanics, nanoelectronics, nanorobotics, and of course in biology. The starting point is usually single-stranded DNA, which is rather easily accessible for base pairing and duplex formation. When long stretches of double-stranded DNA are desirable, serving either as genetic codes or electrical wires, bacterial expansion of plasmids is an inexpensive approach with scale-up properties. Here, we present a method for using double-stranded DNA of any sequence for generating simple structures, such as junctions and DNA lattices. It is known that supercoiled plasmids are strand-invaded by certain DNA analogs. Here we add to the complexity by using "Self-assembling UNiversal (SUN) anchors" formed by DNA analog oligonucleotides, synthesized with an extension, a "sticky-end" that can be used for further base pairing with single-stranded DNA. We show here how the same set of SUN anchors can be utilized for gene therapy, plasmid purification, junction for lattices, and plasmid dimerization through Watson-Crick base pairing. Using atomic force microscopy, it has been possible to characterize and quantify individual components of such supra-molecular complexes.

Zorro locked nucleic acid induces sequence-specific gene silencing.

Locked nucleic acids (LNAs) are synthetic analogs of nucleic acids that contain a bridging methylene carbon between the 2' and 4' positions of the ribose ring. In this study, we generated a novel sequence-specific antigene molecule "Zorro LNA", which simultaneously binds to both strands, and that induced effective and specific strand invasion into DNA duplexes and potent inhibition of gene transcription, also in a cellular context. By comparing the Zorro LNA with linear LNA as well as an optimized bisPNA (peptide nucleic acid) oligonucleotide directed against the same target sites, respectively, we found that the Zorro LNA construct was unique in its ability to arrest gene transcription in mammalian cells. To our knowledge, this is the first time that in mammalian cells, gene transcription was blocked by a nucleic acid analog in a sequence-specific way using low but saturated binding of a blocking agent. This offers a novel type of antigene drug that is easy to synthesize.

Bioplex technology: novel synthetic gene delivery pharmaceutical based on peptides anchored to nucleic acids.

Non-viral gene delivery is an important approach in order to establish safe in vivo gene therapy in the clinic. Although viral vectors currently exhibit superior gene transfer efficacy, the safety aspect of viral gene delivery is a concern. In order to improve non-viral in vivo gene delivery we have designed a pharmaceutical platform called Bioplex (biological complex). The concept of Bioplex is to link functional entities via hybridising anchors, such as Peptide Nucleic Acids (PNA), directly to naked DNA. In order to promote delivery functional entities consisting of biologically active peptides or carbohydrates, are linked to the PNA anchor. The PNA acts as genetic glue and hybridises with DNA in a sequence specific manner. By using functional entities, which elicit receptor-mediated endocytosis, improved endosomal escape and enhance nuclear entry we wish to improve the transfer of genetic material into the cell. An important aspect is that the functional entities should also have tissue-targeting properties in vivo. Examples of functional entities investigated to date are the Simian virus 40 nuclear localisation signal to improve nuclear uptake and different carbohydrate ligands in order to achieve receptor specific uptake. The delivery system is also endowed with regulatory capability, since the release of functional entities can be controlled. The aim is to create a safe, pharmaceutically defined and stable delivery system for nucleic acids with enhanced transfection properties that can be used in the clinic.

Increased stability and specificity through combined hybridization of peptide nucleic acid (PNA) and locked nucleic acid (LNA) to supercoiled plasmids for PNA-anchored "Bioplex" formation.

Low cellular uptake and poor nuclear transfer hamper the use of non-viral vectors in gene therapy. Addition of functional entities to plasmids using the Bioplex technology has the potential to improve the efficiency of transfer considerably. We have investigated the possibility of stabilizing sequence-specific binding of peptide nucleic acid (PNA) anchored functional peptides to plasmid DNA by hybridizing PNA and locked nucleic acid (LNA) oligomers as "openers" to partially overlapping sites on the opposite DNA strand. The PNA "opener" stabilized the binding of "linear" PNA anchors to mixed-base supercoiled DNA in saline. For higher stability under physiological conditions, bisPNA anchors were used. To reduce nonspecific interactions when hybridizing highly cationic constructs and to accommodate the need for increased amounts of bisPNA when the molecules are uncharged, or negatively charged, we used both PNA and LNA oligomers as "openers" to increase binding kinetics. To our knowledge, this is the first time that LNA has been used together with PNA to facilitate strand invasion. This procedure allows hybridization at reduced PNA-to-plasmid ratios, allowing greater than 80% hybridization even at ratios as low as 2:1. Using significantly lower amounts of PNA-peptides combined with shorter incubation times reduces unspecific binding and facilitates purification.

Protease-induced release of functional peptides from bioplexes.

Linking peptide functions directly to nucleic acids can be used to improve transfection. We have previously demonstrated this by sequence-specific hybridization of a bifunctional peptide nucleic acid (PNA) consisting of a nucleic acid binding moiety conjugated to a peptide. The resulting biological complex of PNA/DNA is called a Bioplex. The bifunctional PNA is continuously synthesized with one or more functional entities. For certain applications, it might be preferable to eliminate a functional entity after it has served its purpose. We have addressed this issue by adding a specific protease cleavage site to the construct. In this first approach, cathepsin L was used to cleave a linker sequence including a cathepsin L site: afrsaaq, thereby releasing the tri-peptide Arg-Gly-Asp (RGD) from the PNA anchor. In vitro and in vivo experiments showed an efficient cleavage of the peptide. Moreover, bifunctional PNA constructs were shown to retain activity of the second entity following removal of the first function. Since cathepsin L is ubiquitously expressed in eukaryotic cells and becomes active as the endosomal pH drops, inclusion of cathepsin sites makes it possible to remove functional entities in late endosomes/early lysosomes.

Cooperative strand invasion of supercoiled plasmid DNA by mixed linear PNA and PNA-peptide chimeras.

Peptide nucleic acid (PNA) is a DNA analog with broad biotechnical applications, and possibly also treatment applications. Its suggested uses include that of a specific anchor sequence for biologically active peptides to plasmids in a sequence-specific manner. Such complexes, referred to as Bioplex, have already been used to enhance non-viral gene transfer in vitro. To investigate how hybridization of PNAs to supercoiled plasmids would be affected by the binding of multiple PNA-peptides to the same strand of DNA, we have developed a method of quantifying the specific binding of PNA using a PNA labeled with a derivative of the fluorophore thiazole orange (TO). Cooperative effects were found at a distance of up to three bases. With a peptide present at the end of one of the PNAs, steric hindrance occurred, reducing the increase in binding rate when the distance between the two sites was less than two bases. In addition, we found increased binding kinetics when two PNAs binding to overlapping sites on opposite DNA strands were used, without the use of chemically modified bases in the PNAs.