In vitro investigation of an intracranial flow diverter with a fibrin-based, hemostasis mimicking, nanocoating.

Flow diversion aims at treatment of intracranial aneurysms via vessel remodeling mechanisms, avoiding the implantation of foreign materials into the aneurysm sack. However, complex implantation procedure, high metal surface and hemodynamic disturbance still pose a risk for thromboembolic complications in the clinical praxis. A novel fibrin and heparin based nano coating considered as a hemocompatible scaffold for neointimal formation was investigated regarding thrombogenicity and endothelialization. The fibrin-heparin coating was compared to a bare metal as well as fibrin- or heparin-coated flow diverters. The implants were tested separately in regard to inflammation and coagulation markers in two different in vitro hemocompatibility models conducted with human whole blood (n = 5). Endothelialization was investigated through a novel dynamic in vitro cell seeding model containing primary human cells with subsequent viability assay. It was demonstrated that platelet loss and platelet activation triggered by presence of a bare metal stent could be significantly reduced by applying the fibrin-heparin, fibrin and heparin coating. Viability of endothelial cells after proliferation was similar in fibrin-heparin compared to bare metal implants, with a slight, non-significant improvement observed in the fibrin-heparin group. The results suggest that the presented nanocoating has the potential to reduce thromboembolic complications in a clinical setting. Though the new model allowed for endothelial cell proliferation under flow conditions, a higher number of samples is required to assess a possible effect of the coating.

Hemocompatibility Testing of Blood-Contacting Implants in a Flow Loop Model Mimicking Human Blood Flow.

The growing use of medical devices (e.g., vascular grafts, stents, and cardiac catheters) for temporary or permanent purposes that remain in the body's circulatory system demands a reliable and multiparametric approach that evaluates the possible hematologic complications caused by these devices (i.e., activation and destruction of blood components). Comprehensive in vitro hemocompatibility testing of blood-contacting implants is the first step towards successful in vivo implementation. Therefore, extensive analysis according to the International Organization for Standardization 10993-4 (ISO 10993-4) is mandatory prior to clinical application. The presented flow loop describes a sensitive model to analyze the hemostatic performance of stents (in this case, neurovascular) and reveal adverse effects. The use of fresh human whole blood and gentle blood sampling are essential to avoid the preactivation of blood. The blood is perfused through a heparinized tubing containing the test specimen by using a peristaltic pump at a rate of 150 mL/min at 37 °C for 60 min. Before and after perfusion, hematologic markers (i.e., blood cell count, hemoglobin, hematocrit, and plasmatic markers) indicating the activation of leukocytes (polymorphonuclear [PMN]-elastase), platelets (ß-thromboglobulin [ß-TG]), the coagulation system (thombin-antithrombin III [TAT]), and the complement cascade (SC5b-9) are analyzed. In conclusion, we present an essential and reliable model for extensive hemocompatibility testing of stents and other blood-contacting devices prior to clinical application.

Efficient reduction of synthetic mRNA induced immune activation by simultaneous delivery of B18R encoding mRNA.

The application of synthetic modified messenger RNA (mRNA) is a promising approach for the treatment of a variety of diseases and vaccination. In the past few years, different modifications of synthetic mRNA were applied to render the mRNA more stable and less immunogenic. However, the repeated application of synthetic mRNA still requires the suppression of immune activation to avoid cell death and to allow a sufficient production of exogenous proteins. Thus, the addition of type I interferon (IFN) inhibiting recombinant protein B18R is often required to avoid IFN response. In this study, the ability of B18R encoding mRNA to prevent the immune response of cells to the delivered synthetic mRNA was analyzed. The co-transfection of enhanced green fluorescent protein (eGFP) mRNA transfected fibroblasts with B18R encoding mRNA over 7-days resulted in comparable cell viability and eGFP protein expression as in the cells transfected with eGFP mRNA and incubated with B18R protein. Using qRT-PCR, significantly reduced expression of interferon-stimulated gene Mx1 was detected in the cells transfected with B18R mRNA and stimulated with IFNß compared to the cells without B18R mRNA transfection. Thereby, it was demonstrated that the co-transfection of synthetic mRNA transfected cells with B18R encoding mRNA can reduce the IFN response-related cell death and thus, improve the protein expression.

Generation of Cationic Nanoliposomes for the Efficient Delivery of In Vitro Transcribed Messenger RNA.

The development of messenger RNA (mRNA)-based therapeutics for the treatment of various diseases becomes more and more important because of the positive properties of in vitro transcribed (IVT) mRNA. With the help of IVT mRNA, the de novo synthesis of a desired protein can be induced without changing the physiological state of the target cell. Moreover, protein biosynthesis can be precisely controlled due to the transient effect of IVT mRNA. For the efficient transfection of cells, nanoliposomes (NLps) may represent a safe and efficient delivery vehicle for therapeutic mRNA. This study describes a protocol to generate safe and efficient cationic NLps consisting of DC-cholesterol and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) as a delivery vector for IVT mRNA. NLps having a defined size, a homogeneous distribution, and a high complexation capacity, and can be produced using the dry-film method. Moreover, we present different test systems to analyze their complexation and transfection efficacies using synthetic enhanced green fluorescent protein (eGFP) mRNA, as well as their effect on cell viability. Overall, the presented protocol provides an effective and safe approach for mRNA complexation, which may advance and improve the administration of therapeutic mRNA.

Nanoliposomes for Safe and Efficient Therapeutic mRNA Delivery: A Step Toward Nanotheranostics in Inflammatory and Cardiovascular Diseases as well as Cancer.

Rationale: Genetic therapy using modified mRNA for specific therapeutic protein expression for disease treatment and vaccination represents a new field of therapeutic and diagnostic medicine. Non-viral vectors transfection using biocompatible nanoliposomes enables safe and efficient delivery of therapeutic mRNA. Objective: Generation of non-toxic, cell-compatible cationic nanoliposomes as nanotheranostic agents to successfully deliver therapeutic mRNA. Methods and results: Cationic nanoliposomes (DC-Cholesterol/DOPE) were generated as transfection vehicles for either eGFP mRNA or the therapeutic anti-inflammatory, CD39 mRNA. We observed no toxicity using these nanoplexes and noted high cell viability after transfection. Nanoplexes for the transfection of eGFP mRNA showed an increase in fluorescence signals on microscopy as compared to the mRNA control after 24 hours in Chinese hamster ovary (CHO) cells (14.29 ± 5.30 vs. 1.49 ± 0.54; mean ± SD respectively; p<0.001) and flow cytometry (57.29 ± 14.59 vs 1.83 ± 0.34; % mean ± SD; p<0.001). Nanoplexes for the transfection of CD39 mRNA showed increased CD39 expression in flow cytometry (45.64 ± 15.3 vs. 3.94 ± 0.45; % mean ± SD; p<0.001) as compared to the mRNA control after 24 hours using CHO cells. We also demonstrated efficient transfection across several cell lines (CHO, HEK293, and A549), as well as long-term protein expression (120 h and 168 h) using these nanoplexes. Conclusions: We have developed and tested non-toxic, safe, and efficient nanoliposome preparations for the delivery of therapeutic mRNA that hold promise for novel therapies in diseases such as inflammatory and cardiovascular diseases, as well as cancer. We have also demonstrated that this approach provides a reliable technology to deliver CD39 mRNA as an anti-inflammatory therapeutic for future nanotheranostics approaches.

Cationic Nanoliposomes Meet mRNA: Efficient Delivery of Modified mRNA Using Hemocompatible and Stable Vectors for Therapeutic Applications.

Synthetically modified mRNA is a unique bioactive agent, ideal for use in therapeutic applications, such as cancer vaccination or treatment of single-gene disorders. In order to facilitate mRNA transfections for future therapeutic applications, there is a need for the delivery system to achieve optimal transfection efficacy, perform with durable stability, and provide drug safety. The objective of our study was to comprehensively analyze the use of 3ß-[N-(N',N'-dimethylaminoethane) carbamoyl](DC-Cholesterol)/dioleoylphosphatidylethanolamine (DOPE) liposomes as a potential transfection agent for modified mRNAs. Our cationic liposomes facilitated a high degree of mRNA encapsulation and successful cell transfection efficiencies. More importantly, no negative effects on cell viability or immune reactions were detected posttransfection. Notably, the liposomes had a long-acting transfection effect on cells, resulting in a prolonged protein production of alpha-1-antitrypsin (AAT). In addition, the stability of these mRNA-loaded liposomes allowed storage for 80 days, without the loss of transfection efficacy. Finally, comprehensive analysis showed that these liposomes are fully hemocompatible with fresh human whole blood. In summary, we present an extensive analysis on the use of DC-cholesterol/DOPE liposomes as mRNA delivery vehicles. This approach provides the basis of a safe and efficient therapeutic strategy in the development of successful mRNA-based drugs.

In Vitro Evaluation of a Novel mRNA-Based Therapeutic Strategy for the Treatment of Patients Suffering from Alpha-1-Antitrypsin Deficiency.

In single-gene disorders, like alpha-1-antitrypsin deficiency (AATD), a gene mutation causes missing or dysfunctional protein synthesis. This, in turn, can lead to serious complications for the patient affected. Furthermore, single-gene disorders are associated with severe early-onset conditions and necessitate expensive lifelong care. Until nowadays, therapeutic treatment options are still limited, cost-intensive, or lack effectiveness. For these reasons, we aim to develop a novel mRNA-based therapeutic strategy for the treatment of single-gene disorders, such as AATD, which is based on the induction of de novo synthesis of the functional proteins. Therefore, an alpha-1-antitrypsin (AAT) encoding mRNA was generated by in vitro transcription. After in vitro delivery of the mRNA to different cells, protein expression and functionality, as well as adverse effects and mRNA serum stability, were analyzed. Our results show that the AAT mRNA-transfected cells express the AAT protein in high amounts within the first 24 h. Moreover, the expressed AAT protein is highly functional, since the activity of elastase is significantly inhibited. Our data also show that mRNA concentrations up to 1 µg per 150,000 cells have no adverse effects on cell viability and immune activation. Furthermore, the encapsulated AAT encoding mRNA is stable and functional in human serum for up to 30 min. Overall, the proposed project provides an innovative, highly promising, and safe therapeutic approach and, thus, promises a novel progress in the treatment of single-gene disorders, whereby affected patients could greatly benefit.

In vitro test system for evaluation of immune activation potential of new single-stranded DNA-based therapeutics.

Aptamers are synthetic single-stranded DNA (ssDNA) molecules with the ability to fold into complex three-dimensional structures. They can bind their targets with a high selectivity and affinity, thus they have an enormous potential as therapeutic agents. However, since aptamers are synthetic and especially since certain sequences can increasingly bind to the pattern recognition receptors of the immune cells when applied in vivo, they can induce an immune activation. Here, we established a real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) based assay to evaluate aptamers-induced immune activation prior to in vivo studies. Human whole blood or plasmacytoid dendritic cell line (PMDC05) were incubated with CpG, R10-60 aptamer, start library, or a CpG containing aptamer. After 2 and 4 h, cytokine expression was measured using qRT-PCR to determine immune reaction against different aptamers. CpG containing a phosphorothioate backbone led to a significant up-regulation of CCL-7, IFN-1α, IFN-1ß in whole blood after 4 h. Compared to the samples without ssDNA, significantly higher TNF-α expression was detected after the R10-60 aptamer incubation for 4 h. The stimulation of PMDC05 cells with different ssDNA enabled more sensitive detection of aptamer sequence specific immune activation. After 4 h, CpG led to a significantly higher expression of CCL-8, CXCL-10, IL-1ß, IL-6, IL-8, IFN-1ß, and TNF-α. R10-60 aptamer caused a significant up-regulation of IL-1ß, IFN-1ß, and TNF-α. Negative control aptamers did not induce an immune activation. The use of this assay before starting with in vivo studies will facilitate the in vitro prediction of immune activation potential of aptamers.

Potential capacity of aptamers to trigger immune activation in human blood.

Target specific short single-stranded DNA (ssDNA) molecules, called aptamers, are auspicious ligands for numerous in vivo applications. However, aptamers are synthetic molecules, which might be recognized by the immune cells in vivo and induce an activation of the innate immune system. Thus, immune activation potential of synthetic ssDNA oligonucleotides (ODNs) was determined using a well established closed-loop circulation model. Fresh human blood was incubated at 37°C for 2 or 4 hours with ssDNA ODNs (SB_ODN) or CpG ODN as positive control. Transcriptional changes were determined by microarray analyses. Blood samples containing SB_ODN demonstrated after 4 hours a significant regulation of 295 transcripts. Amongst others, CCL8, CXCL10, CCL7 and CXCL11 were highest regulated genes. Gene Ontology terms and KEGG pathway analyses exhibited that the differentially expressed genes belong to the transcripts that are regulated during an immune and inflammatory response, and were overrepresented in TLR signaling pathway. This study shows for the first time the potential of aptamers to activate immune system after systemic application into the human blood. Thus, we highly recommend performing of these preclinical tests with potential aptamer-based therapeutics.