Publications by authors named "Carsten Rudolph"

64 Publications

Alternative oxidase encoded by sequence-optimized and chemically-modified RNA transfected into mammalian cells is catalytically active.

Gene Ther 2021 Mar 4. Epub 2021 Mar 4.

Faculty of Medicine and Health Technology, FI-33014 Tampere University, Tampere, Finland.

Plants and other organisms, but not insects or vertebrates, express the auxiliary respiratory enzyme alternative oxidase (AOX) that bypasses mitochondrial respiratory complexes III and/or IV when impaired. Persistent expression of AOX from Ciona intestinalis in mammalian models has previously been shown to be effective in alleviating some metabolic stresses produced by respiratory chain inhibition while exacerbating others. This implies that chronic AOX expression may modify or disrupt metabolic signaling processes necessary to orchestrate adaptive remodeling, suggesting that its potential therapeutic use may be confined to acute pathologies, where a single course of treatment would suffice. One possible route for administering AOX transiently is AOX-encoding nucleic acid constructs. Here we demonstrate that AOX-encoding chemically-modified RNA (cmRNA), sequence-optimized for expression in mammalian cells, was able to support AOX expression in immortalized mouse embryonic fibroblasts (iMEFs), human lung carcinoma cells (A549) and primary mouse pulmonary arterial smooth muscle cells (PASMCs). AOX protein was detectable as early as 3 h after transfection, had a half-life of ~4 days and was catalytically active, thus supporting respiration and protecting against respiratory inhibition. Our data demonstrate that AOX-encoding cmRNA optimized for use in mammalian cells represents a viable route to investigate and possibly treat mitochondrial respiratory disorders.
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http://dx.doi.org/10.1038/s41434-021-00235-zDOI Listing
March 2021

Preclinical Evaluation of a Novel TALEN Targeting CCR5 Confirms Efficacy and Safety in Conferring Resistance to HIV-1 Infection.

Biotechnol J 2021 Jan 16;16(1):e2000023. Epub 2020 Nov 16.

Institute for Transfusion Medicine and Gene Therapy, Center for Chronic Immunodeficiency, Medical Center-University of Freiburg, Freiburg, 79106, Germany.

Therapies to treat patients infected with human immunodeficiency virus (HIV) aim at preventing viral replication but fail to eliminate the virus. Although transplantation of allogeneic CCR5Δ32 homozygous stem cell grafts provided a cure for a few patients, this approach is not considered a general therapeutic strategy because of potential side effects. Conversely, gene editing to disrupt the C-C chemokine receptor type 5 (CCR5) locus, which encodes the major HIV coreceptor, has shown to confer resistance to CCR5-tropic HIV strains. Here, an engineered transcription activator-like effector nuclease (TALEN) that enables efficient CCR5 editing in hematopoietic cells is presented. After transferring TALEN-encoding mRNA into primary CD4+ T cells, up to 89% of CCR5 alleles are disrupted. Genotyping confirms the genetic stability of the CCR5-edited cells, and genome-wide off-target analyses established the absence of relevant mutagenic events. When challenging the edited T cells with CCR5-tropic HIV, protection in a dose-dependent manner is observed. Functional assessments reveal no significant differences between edited and control cells in terms of proliferation and their ability to secrete cytokines upon exogenous stimuli. In conclusion, a highly active and specific TALEN to disrupt CCR5 is successfully engineered, paving the way for its clinical application in hematopoietic stem cell grafts.
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http://dx.doi.org/10.1002/biot.202000023DOI Listing
January 2021

Self-assembled peptide-poloxamine nanoparticles enable in vitro and in vivo genome restoration for cystic fibrosis.

Nat Nanotechnol 2019 03 28;14(3):287-297. Epub 2019 Jan 28.

Department of Pediatrics, Ludwig Maximilian University of Munich, Munich, Germany.

Developing safe and efficient non-viral delivery systems remains a major challenge for in vivo applications of gene therapy, especially in cystic fibrosis. Unlike conventional cationic polymers or lipids, the emerging poloxamine-based copolymers display promising in vivo gene delivery capabilities. However, poloxamines are invalid for in vitro applications and their in vivo transfection efficiency is still low compared with viral vectors. Here, we show that peptides developed by modular design approaches can spontaneously form compact and monodisperse nanoparticles with poloxamines and nucleic acids via self-assembly. Both messenger RNA and plasmid DNA expression mediated by peptide-poloxamine nanoparticles are greatly boosted in vitro and in the lungs of cystic fibrosis mice with negligible toxicity. Peptide-poloxamine nanoparticles containing integrating vectors enable successful in vitro and in vivo long-term restoration of cystic fibrosis transmembrane conductance regulator deficiency with a safe integration profile. Our dataset provides a new framework for designing non-viral gene delivery systems qualified for in vivo genetic modifications.
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http://dx.doi.org/10.1038/s41565-018-0358-xDOI Listing
March 2019

Delivery of mRNA Therapeutics for the Treatment of Hepatic Diseases.

Mol Ther 2019 04 22;27(4):794-802. Epub 2018 Dec 22.

Department of Pediatrics, Ludwig Maximilian University of Munich, 80337 Munich, Germany; Ethris GmbH, RNA Biology, 82152 Planegg, Germany. Electronic address:

Promising improvements in the field of transcript therapeutics have clearly enhanced the potential of mRNA as a new pillar for protein replacement therapies. Synthetic mRNAs are engineered to replace mutated mRNAs and to be immunologically inconspicuous and highly stable while maximizing protein expression. Approaches to deliver mRNA into the cellular cytoplasm safely and efficiently have been further developed so that two mRNA-based approaches replacing vascular endothelial growth factor (VEGF) and cystic fibrosis transmembrane conductance regulator (CFTR) have now made it into clinical trials. These studies bring mRNA therapeutics for protein replacement therapy closer to clinical realization. Herein, we provide an overview of preclinical and clinical developments of mRNA therapeutics for liver diseases.
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http://dx.doi.org/10.1016/j.ymthe.2018.12.012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6453508PMC
April 2019

Segmented poly(A) tails significantly reduce recombination of plasmid DNA without affecting mRNA translation efficiency or half-life.

RNA 2019 04 15;25(4):507-518. Epub 2019 Jan 15.

Department of Pediatrics, Ludwig-Maximilian-University of Munich, 80337 Munich, Germany.

Extensive research in the past decade has brought mRNA closer to the clinical realization of its therapeutic potential. One common structural feature for all cellular messenger RNAs is a poly(A) tail, which can either be brought in cotranscriptionally via the DNA template (plasmid- or PCR-based) or added to the mRNA in a post-transcriptional enzymatic process. Plasmids containing poly(A) regions recombine in , resulting in extensive shortening of the poly(A) tail. Using a segmented poly(A) approach, we could significantly reduce recombination of plasmids in without any negative effect on mRNA half-life and protein expression. This effect was independent of the coding sequence. A segmented poly(A) tail is characterized in that it consists of at least two A-containing elements, each defined as a nucleotide sequence consisting of 40-60 adenosines, separated by a spacer element of different length. Furthermore, reducing the spacer length between the poly(A) segments resulted in higher translation efficiencies compared to homogeneous poly(A) tail and reduced recombination (depending upon the choice of spacer nucleotide). Our results demonstrate the superior potential of segmented poly(A) tails compared to the conventionally used homogeneous poly(A) tails with respect to recombination of the plasmids and the resulting mRNA performance (half-life and translational efficiency).
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http://dx.doi.org/10.1261/rna.069286.118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6426288PMC
April 2019

Conversion of adult human fibroblasts into neural precursor cells using chemically modified mRNA.

Heliyon 2018 Nov 8;4(11):e00918. Epub 2018 Nov 8.

Department of Pharmacology & Clinical Pharmacology, Centre for Brain Research, School of Medical Science, Faculty of Medical and Health Sciences, University of Auckland, Auckland, New Zealand.

Direct reprogramming offers a unique approach by which to generate neural lineages for the study and treatment of neurological disorders. Our objective is to develop a clinically viable reprogramming strategy to generate neural precursor cells for the treatment of neurological disorders through cell replacement therapy. We initially developed a method for directly generating neural precursor cells (iNPs) from adult human fibroblasts by transient expression of the neural transcription factors, and using plasmid DNA. This study advances these findings by examining the use of chemically modified mRNA (cmRNA) for direct-to-iNP reprogramming. Chemically modified mRNA has the benefit of being extremely stable and non-immunogenic, offering a clinically suitable gene delivery system. The use of and cmRNA resulted in high co-transfection efficiency and cell viability compared with plasmid transfection. Neural positioning and fate determinant genes were observed throughout reprogramming with ion channel and synaptic marker genes detected during differentiation. Differentiation of cmRNA-derived iNPs generated immature GABAergic or glutamatergic neuronal phenotypes in conjunction with astrocytes. This represents the first time a cmRNA approach has been used to directly reprogram adult human fibroblasts to iNPs, potentially providing an efficient system by which to generate human neurons for both research and clinical application.
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http://dx.doi.org/10.1016/j.heliyon.2018.e00918DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6226601PMC
November 2018

Exploring Cytotoxic mRNAs as a Novel Class of Anti-cancer Biotherapeutics.

Mol Ther Methods Clin Dev 2018 Mar 24;8:141-151. Epub 2017 Dec 24.

Ethris GmbH, Planegg 82152, Germany.

New treatments to overcome the obstacles of conventional anti-cancer therapy are a permanent subject of investigation. One promising approach is the application of toxins linked to cell-specific ligands, so-called immunotoxins. Another attractive option is the employment of toxin-encoding plasmids. However, immunotoxins cause hepatoxicity, and DNA therapeutics, among other disadvantages, bear the risk of insertional mutagenesis. As an alternative, this study examined chemically modified mRNAs coding for diphtheria toxin, subtilase cytotoxin, and abrin-a for their ability to reduce cancer cell growth both and . The plant toxin abrin-a was the most promising candidate among the three tested toxins and was further investigated. Its expression was demonstrated by western blot. Experiments with firefly luciferase in reticulocyte lysates and co-transfection experiments with EGFP demonstrated the capability of abrin-a to inhibit protein synthesis. Its cytotoxic effect was quantified employing viability assays and propidium iodide staining. By studying caspase-3/7 activation, Annexin V-binding, and chromatin condensation with Hoechst33258 staining, apoptotic cell death could be confirmed. In mice, repeated intratumoral injections of complexed abrin-a mRNA resulted in a significant reduction (89%) of KB tumor size compared to a non-translatable control mRNA.
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http://dx.doi.org/10.1016/j.omtm.2017.12.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5908148PMC
March 2018

Chemically Modified Messenger RNA: Modified RNA Application for Treatment of Achilles Tendon Defects.

Tissue Eng Part A 2019 01 24;25(1-2):113-120. Epub 2018 May 24.

1 Department of Orthopaedic Sports Medicine, Technical University of Munich, Munich, Germany.

Different regenerative medicine approaches for tendon healing exist. Recently, especially gene therapy gained popularity. However, potential mutagenic and immunologic effects might prevent its translation to clinical research. Chemically modified mRNA (cmRNA) might bypass these limitations of gene therapy. Therefore, the purpose of this study was to evaluate the early healing properties of Achilles tendon defects in rats treated with basic fibroblast growth factor (bFGF) cmRNA. Forty male Lewis rats were used for the study and randomly assigned to two study groups: (1) treatment with cmRNA coding for bFGF and (2) noncoding cmRNA control. Protein expression was measured using in vivo bioluminescence imaging at 24, 48, and 72 h, as well as 14 days. Animals were euthanized 2 weeks following surgery. Biomechanical, histological, and immunohistological analyses were performed with the significance level set at p < 0.05. Protein expression was evident for 3 days. At 14 days, bioluminescence imaging revealed only little protein expression. Biomechanically, tendons treated with bFGF cmRNA showed a construct stiffness closer to the healthy contralateral side when compared with the control group (p = 0.034), without any significant differences in terms of load to failure. Hematoxylin and eosin staining detected no side effects of the treatment, as signs of inflammation, or necrosis. Furthermore, it revealed the shape of the nuclei to be more oval in the bFGF group in the tendon midsubstance (p = 0.043) with a reduced cell count (p = 0.035). Immunohistological staining for type I, II, III, and IV collagen did not differ significantly between the two groups. In conclusion, this pilot study demonstrates the feasibility of a novel messenger RNA (mRNA)-based therapy for Achilles tendon defects using chemically modified mRNA coding for bFGF.
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http://dx.doi.org/10.1089/ten.TEA.2017.0443DOI Listing
January 2019

Maximizing the Translational Yield of mRNA Therapeutics by Minimizing 5'-UTRs.

Tissue Eng Part A 2019 01 17;25(1-2):69-79. Epub 2018 Nov 17.

1 Department of Pediatrics, Ludwig-Maximilian-University of Munich, Munich, Germany.

The 5'-untranslated region (5'-UTR) of mRNA contains structural elements, which are recognized by cell-specific RNA-binding proteins, thereby affecting the translation of the molecule. The activation of an innate immune response upon transfection of mRNA into cells is reduced when the mRNA comprises chemically modified nucleotides, putatively by altering the secondary structure of the molecule. Such alteration in the 5'-UTR in turn may affect the functionality of mRNA. In this study, we report on the impact of seven synthetic minimalistic 5'-UTR sequences on the translation of luciferase-encoding unmodified and different chemically modified mRNAs upon transfection in cell culture and in vivo. One minimalistic 5'-UTR, consisting of 14 nucleotides combining the T7 promoter with a Kozak consensus sequence, yielded similar or even higher expression than a 37 nucleotides human alpha-globin 5'-UTR containing mRNA in HepG2 and A549 cells. Furthermore, also the kind of modified nucleotides used in in vitro transcription, affected mRNA translation when using different translation regulators (Kozak vs. translation initiator of short UTRs). The in vitro data were confirmed by bioluminescence imaging of expression in mouse livers, 6 h postintravenous injection of a lipidoid nanoparticle-formulated RNA in female Balb/c mice. Luciferase measurements from liver and spleen showed that minimal 5'-UTRs (3 and 7) were either equally effective or better than human alpha-globin 5'-UTR. These findings were confirmed with a human erythropoietin (hEPO)-encoding mRNA. Significantly, higher levels of hEPO could be quantified in supernatants from A549 cells transfected with minimal 5'-UTR7 containing RNA when compared to commonly used benchmarks 5'-UTRs. Our results demonstrate the superior potential of synthetic minimalistic 5'-UTRs for use in transcript therapies.
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http://dx.doi.org/10.1089/ten.TEA.2017.0485DOI Listing
January 2019

Efficient Non-viral Gene Delivery into Human Hematopoietic Stem Cells by Minicircle Sleeping Beauty Transposon Vectors.

Mol Ther 2018 04 31;26(4):1137-1153. Epub 2018 Jan 31.

Transposition and Genome Engineering, Division of Medical Biotechnology, Paul Ehrlich Institute, Langen, Germany. Electronic address:

The Sleeping Beauty (SB) transposon system is a non-viral gene delivery platform that combines simplicity, inexpensive manufacture, and favorable safety features in the context of human applications. However, efficient correction of hematopoietic stem and progenitor cells (HSPCs) with non-viral vector systems, including SB, demands further refinement of gene delivery techniques. We set out to improve SB gene transfer into hard-to-transfect human CD34 cells by vectorizing the SB system components in the form of minicircles that are devoid of plasmid backbone sequences and are, therefore, significantly reduced in size. As compared to conventional plasmids, delivery of the SB transposon system as minicircle DNA is ∼20 times more efficient, and it is associated with up to a 50% reduction in cellular toxicity in human CD34 cells. Moreover, providing the SB transposase in the form of synthetic mRNA enabled us to further increase the efficacy and biosafety of stable gene delivery into hematopoietic progenitors ex vivo. Genome-wide insertion site profiling revealed a close-to-random distribution of SB transposon integrants, which is characteristically different from gammaretroviral and lentiviral integrations in HSPCs. Transplantation of gene-marked CD34 cells in immunodeficient mice resulted in long-term engraftment and hematopoietic reconstitution, which was most efficient when the SB transposase was supplied as mRNA and nucleofected cells were maintained for 4-8 days in culture before transplantation. Collectively, implementation of minicircle and mRNA technologies allowed us to further refine the SB transposon system in the context of HSPC gene delivery to ultimately meet clinical demands of an efficient and safe non-viral gene therapy protocol.
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http://dx.doi.org/10.1016/j.ymthe.2018.01.012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6079369PMC
April 2018

Translation of Angiotensin-Converting Enzyme 2 upon Liver- and Lung-Targeted Delivery of Optimized Chemically Modified mRNA.

Mol Ther Nucleic Acids 2017 Jun 13;7:350-365. Epub 2017 Apr 13.

Ethris GmbH, 82152 Planegg, Germany. Electronic address:

Changes in lifestyle and environmental conditions give rise to an increasing prevalence of liver and lung fibrosis, and both have a poor prognosis. Promising results have been reported for recombinant angiotensin-converting enzyme 2 (ACE2) protein administration in experimental liver and lung fibrosis. However, the full potential of ACE2 may be achieved by localized translation of a membrane-anchored form. For this purpose, we advanced the latest RNA technology for liver- and lung-targeted ACE2 translation. We demonstrated in vitro that transfection with ACE2 chemically modified messenger RNA (cmRNA) leads to robust translation of fully matured, membrane-anchored ACE2 protein. In a second step, we designed eight modified ACE2 cmRNA sequences and identified a lead sequence for in vivo application. Finally, formulation of this ACE2 cmRNA in tailor-made lipidoid nanoparticles and in lipid nanoparticles led to liver- and lung-targeted translation of significant amounts of ACE2 protein, respectively. In summary, we provide evidence that RNA transcript therapy (RTT) is a promising approach for ACE2-based treatment of liver and lung fibrosis to be tested in fibrotic disease models.
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http://dx.doi.org/10.1016/j.omtn.2017.04.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5423349PMC
June 2017

cmRNA/lipoplex encapsulation in PLGA microspheres enables transfection via calcium phosphate cement (CPC)/PLGA composites.

J Control Release 2017 03 2;249:143-149. Epub 2017 Feb 2.

Technische Universität München, Institute of Molecular Immunology & Experimental Oncology, Germany. Electronic address:

In this study lipoplexes containing chemically modified messenger RNA (cmRNA) were incorporated into poly (lactic-co-glycolic acid) (PLGA) microspheres via water-in-oil-in-water (W/O/W) double emulsion solvent evaporation technique. The nanoparticle encapsulation by microparticle formation was optimized to achieve lipoplex release and maximum transfection efficiency in surrounding cells. It was possible to adjust characteristic features in surface topology and size of the PLGA-microspheres by varying the extent of lipoplex loading into the polymer matrix. The partial release of lipids and mRNA out of the microparticle system, their accumulation in cells and the production of encoded protein were visualized via fluorescence microscopy. These bioactive microspheres, containing cmRNA bearing lipoplexes, were developed for the incorporation of a therapeutic component into injectable calcium phosphate cements (CPC). Due to the incorporation of PLGA/lipoplex microspheres as a degradable entity, the porosity of the cement phase could additionally be adjusted. This approach of complex nanoparticle incorporation into polymer/cement composites represents a promising example for combining transcript therapy with biomechanical engineering.
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http://dx.doi.org/10.1016/j.jconrel.2017.01.045DOI Listing
March 2017

Human cellular CYBA UTR sequences increase mRNA translation without affecting the half-life of recombinant RNA transcripts.

Sci Rep 2016 12 15;6:39149. Epub 2016 Dec 15.

Institute of Molecular Immunology- Experimental Oncology, Klinikum rechts der Isar, Technische Universität München, Munich, 81675, Germany.

Modified nucleotide chemistries that increase the half-life (T) of transfected recombinant mRNA and the use of non-native 5'- and 3'-untranslated region (UTR) sequences that enhance protein translation are advancing the prospects of transcript therapy. To this end, a set of UTR sequences that are present in mRNAs with long cellular T were synthesized and cloned as five different recombinant sequence set combinations as upstream 5'-UTR and/or downstream 3'-UTR regions flanking a reporter gene. Initial screening in two different cell systems in vitro revealed that cytochrome b-245 alpha chain (CYBA) combinations performed the best among all other UTR combinations and were characterized in detail. The presence or absence of CYBA UTRs had no impact on the mRNA stability of transfected mRNAs, but appeared to enhance the productivity of transfected transcripts based on the measurement of mRNA and protein levels in cells. When CYBA UTRs were fused to human bone morphogenetic protein 2 (hBMP2) coding sequence, the recombinant mRNA transcripts upon transfection produced higher levels of protein as compared to control transcripts. Moreover, transfection of human adipose mesenchymal stem cells with recombinant hBMP2-CYBA UTR transcripts induced bone differentiation demonstrating the osteogenic and therapeutic potential for transcript therapy based on hybrid UTR designs.
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http://dx.doi.org/10.1038/srep39149DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5156912PMC
December 2016

Efficient ex vivo delivery of chemically modified messenger RNA using lipofection and magnetofection.

Biochem Biophys Res Commun 2017 Jan 22;482(4):796-801. Epub 2016 Nov 22.

Institute of Molecular Immunology-Experimental Oncology, Technische Universität München, Munich, Germany; Ethris GmbH, Planegg, Germany.

Recently, chemically modified mRNA (cmRNA) therapeutics have been the subject of extensive application-oriented research in both academia and industry as a safer alternative for gene and recombinant protein therapies. However, the lack of an efficient delivery system hinders widespread application. Here we used ∼100-nm lipoplexes and magnetic lipoplexes that can protect cmRNA from RNases and efficiently deliver it into muscle and fat tissues as well as to the endothelium of the carotid artery. Establishing magnetofection for ex vivo cmRNA delivery for the first time, we suggest this method for potential enhanced and targeted delivery of cmRNA. This study introduces optimal cmRNA complexes with high ex vivo efficiency as good candidates for further in vivo cmRNA delivery.
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http://dx.doi.org/10.1016/j.bbrc.2016.11.113DOI Listing
January 2017

Modified mRNA for BMP-2 in Combination with Biomaterials Serves as a Transcript-Activated Matrix for Effectively Inducing Osteogenic Pathways in Stem Cells.

Stem Cells Dev 2017 01 24;26(1):25-34. Epub 2016 Oct 24.

2 Ethris GmbH , Planegg, Germany .

Bone regeneration using stem cells and growth factors has disadvantages while needing to use supraphysiological growth factors concentrations. Gene therapy has been proposed as alternative, but also has limitation. Messenger RNA (mRNA)-based transcript therapy is a novel approach that may solve plasmid DNA-based gene therapy limitations. Although much more efficient in delivering genes into the cell, mRNA is unfortunately unstable and immunogenic. However, recent reports indicated that chemical modifications of the mRNA molecule can improve stability and toxicity. In this study, we have combined biomaterials and chemically modified mRNA (cmRNA) encoding Metridia luciferase, eGFP, and bone morphogenetic protein (BMP)-2 to develop transcript-activated matrices (TAMs) for gene transfer to stem cells. BMP-2 cmRNA was produced to evaluate its feasibility in stimulating osteogenic differentiation. Fibrin gel and micro-macro biphasic calcium phosphate (MBCP) granules were used as biomaterials. A sustained release of hBMP-2 cmRNA from both biomaterials was observed during 7 days. This occurred significantly faster from the MBCP granules compared to fibrin gels (92% from MBCP and 43% from fibrin after 7 days). Stem cells cultured in hBMP-2 cmRNA/fibrin or on hBMP-2 cmRNA/MBCP were transfected and able to secrete significant amounts of hBMP-2. Furthermore, transfected cells expressed osteogenic markers in vitro. Interestingly, although both TAMs promoted gene expression at the same level, hBMP-2 cmRNA/MBCP granules induced significantly higher collagen I and osteocalcin gene expression. This matrix also induced more mineral deposition. Overall, our results demonstrated the feasibility of developing efficient TAMs for bone regeneration by combining biomaterials and cmRNAs. MBCP synergistically enhances the hBMP-2 cmRNA-induced osteogenic pathway.
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http://dx.doi.org/10.1089/scd.2016.0171DOI Listing
January 2017

Transcript-activated collagen matrix as sustained mRNA delivery system for bone regeneration.

J Control Release 2016 10 30;239:137-48. Epub 2016 Aug 30.

Institute of Molecular Immunology - Experimental Oncology, Technische Universität München, Munich, Germany; Ethris GmbH, Planegg, Germany. Electronic address:

Transcript therapies using chemically modified messenger RNAs (cmRNAs) are emerging as safe and promising alternatives for gene and recombinant protein therapies. However, their applications have been limited due to transient translation and relatively low stability of cmRNAs compared to DNA. Here we show that vacuum-dried cmRNA-loaded collagen sponges, termed transcript activated matrices (TAMs), can serve as depots for sustained delivery of cmRNA. TAMs provide steady state protein production for up to six days, and substantial residual expression until 11days post transfection. Another advantage of this technology was nearly 100% transfection efficiency as well as low toxicity in vitro. TAMs were stable for at least 6months at room temperature. Human BMP-2-encoding TAMs induced osteogenic differentiation of MC3T3-E1 cells in vitro and bone regeneration in a non-critical rat femoral bone defect model in vivo. In summary, TAMs are a promising tool for bone regeneration and potentially also for other applications in regenerative medicine and tissue engineering.
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http://dx.doi.org/10.1016/j.jconrel.2016.08.037DOI Listing
October 2016

A Single Methylene Group in Oligoalkylamine-Based Cationic Polymers and Lipids Promotes Enhanced mRNA Delivery.

Angew Chem Int Ed Engl 2016 08 4;55(33):9591-5. Epub 2016 Jul 4.

ethris GmbH, Semmelweisstrasse 3, 82152, Planegg, Germany.

The development of chemically modified mRNA holds great promise as a new class of biologic therapeutics. However, the intracellular delivery and endosomal escape of mRNA encapsulated in nanoparticles has not been systematically investigated. Here, we synthesized a diverse set of cationic polymers and lipids from a series of oligoalkylamines and subsequently characterized their mRNA delivery capability. Notably, a structure with an alternating alkyl chain length between amines showed the highest transfection efficiency, which was linked to a high buffering capacity in a narrow range of pH 6.2 to 6.5. Variation in only one methylene group resulted in enhanced mRNA delivery to both the murine liver as well as porcine lungs after systemic or aerosol administration, respectively. These findings reveal a novel fundamental structure-activity relationship for the delivery of mRNA that is independent of the class of mRNA carrier and define a promising new path of exploration in the field of mRNA therapeutics.
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http://dx.doi.org/10.1002/anie.201603648DOI Listing
August 2016

Chemically modified RNA induces osteogenesis of stem cells and human tissue explants as well as accelerates bone healing in rats.

Biomaterials 2016 May 19;87:131-146. Epub 2016 Feb 19.

Institute of Molecular Immunology and Experimental Oncology, Technical University Munich, Ismaninger Str. 22, 81675 Munich, Germany; Ethris GmbH, Semmelweisstr. 3, 82152 Planegg, Germany. Electronic address:

Limitations associated to the use of growth factors represent a major hurdle to musculoskeletal regeneration. On the one hand, they are needed to induce neo-tissue formation for the substitution of a necrotic or missing tissue. On the other hand, these factors are used in supraphysiological concentrations, are short lived and expensive and result in many side effects. Here we develop a gene transfer strategy based on the use of chemically modified mRNA (cmRNA) coding for human bone morphogenetic protein 2 (hBMP-2) that is non-immunogenic and highly stable when compared to unmodified mRNA. Transfected stem cells secrete hBMP-2, show elevated alkaline phosphatase levels and upregulated expression of RunX2, ALP, Osterix, Osteocalcin, Osteopontin and Collagen Type I genes. Mineralization was induced as seen by positive Alizarin red staining. hBMP-2 cmRNA transfected human fat tissue also yielded an osteogenic response in vitro as indicated by expression of hBMP-2, RunX2, ALP and Collagen Type I. Delivering hBMP-2 cmRNA to a femur defect in a rat model results in new bone tissue formation as early as 2 weeks after application of very low doses. Overall, our studies demonstrate the feasibility and therapeutic potential of a new cmRNA-based gene therapy strategy that is safe and efficient. When applied clinically, this approach could overcome BMP-2 growth factor associated limitations in bone regeneration.
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http://dx.doi.org/10.1016/j.biomaterials.2016.02.018DOI Listing
May 2016

Stability analysis of chemically modified mRNA using micropattern-based single-cell arrays.

Lab Chip 2015 Sep 23;15(17):3561-71. Epub 2015 Jul 23.

Institute of Molecular Immunology - Experimental Oncology, Technische Universität München, Munich, 81675 Germany.

The measurement of mRNA turnover in living cells plays an important role in the search for stable mRNA constructs for RNA-based therapies. Here we show that automated time-lapse microscopy combined with micropatterned arrays allows for efficient high-throughput monitoring of fluorescent reporter protein expression at the single-cell level. The fluorescence time courses after mRNA transfection yield the distribution of individual mRNA expression and degradation rates within a population. We compare mRNA constructs with combinations of 5' and 3' UTR sequences and find a systematic broadening and shift towards longer functional half-lives for UTR stabilized mRNA. At the same time the life time distribution of the destabilized EGFP reporter protein was found to be constant and narrowly distributed. Using mathematical modeling, we show that mRNA functional life-time predicts the time-integrated protein level, i.e. the area under the curve (AUC) of mRNA translation. Our approach paves the way for quantitative assessment of hitherto unexplored mRNA functional life time heterogeneity, possibly predicated on multiple mRNA secondary structures and its dependence on UTR sequences.
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http://dx.doi.org/10.1039/c5lc00749fDOI Listing
September 2015

Cystic fibrosis transmembrane conductance regulator-mRNA delivery: a novel alternative for cystic fibrosis gene therapy.

J Gene Med 2013 Nov-Dec;15(11-12):414-26

Institute of Animal Physiology, University of Muenster, Muenster, Germany.

Background: Cystic fibrosis (CF) is the most frequent lethal genetic disease in the Caucasian population. CF is caused by a defective gene coding for the cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP- and ATP-dependent Cl(-) channel and central regulatory protein in epithelia. CFTR influences the fluid composition of the mucus in the respiratory tract. The most common mutation inducing CF, ΔF508, impairs CFTR processing within the cell and thus prevents functional CFTR expression in the apical membrane. The present study aimed to investigate the functional restoration of CFTR in human CF airway epithelia after transfection with optimized wild-type (wt)CFTR-mRNA.

Methods: We used primary cultured human nasal epithelial (HNE) cells and the human bronchial epithelial cell line CFBE41o(-) that stably expresses ΔF508-CFTR and carried out transepithelial Ussing chamber measurements after transfection with optimized wtCFTR-mRNA. We confirmed the data obtained using immunofluorescence and protein biochemical approaches.

Results: Transfection of the CFBE41o(-) cells with wtCFTR-mRNA restored cAMP-induced CFTR currents similar to the values seen in control cells (16HBE14o(-)). Using immunofluorescence approaches, we demonstrated that a considerable amount of CFTR is located at the apical surface in the CF cells after transfection. Western blot analyses of wtCFTR-mRNA transfected CFBE41o(-) cells confirmed these findings. Furthermore, we demonstrated physiological relevance by using primary cultured HNE cells and showed an almost two-fold increase in the cAMP-stimulated CFTR current after transfection.

Conclusions: From these data, we conclude that CFTR-mRNA transfection could comprise a novel alternative for gene therapy to restore impaired CFTR function.
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http://dx.doi.org/10.1002/jgm.2748DOI Listing
August 2014

Formation of solid lipid nanoparticle (SLN)-gene vector complexes for transfection of mammalian cells in vitro.

Cold Spring Harb Protoc 2012 Mar 1;2012(3):357-60. Epub 2012 Mar 1.

Solid lipid nanoparticles (SLNs) offer several technological advantages over standard DNA carriers such as cationic lipids or cationic polymers. However, in the absence of endosomolytic agents such as chloroquine, gene-transfer efficiency mediated by SLN-derived gene vectors consisting of optimized lipid composition remains lower compared to those achieved with standard transfection agents. This protocol describes the incorporation of a dimeric human immunodeficiency virus type-1 (HIV-1) TAT peptide into SLN gene vectors to increase gene-transfer efficiency. This results in higher transfection rates than for standard transfection agents in vitro; the ternary SLN-gene vector complexes usually result in transfection levels equal to or higher than those observed with gene vector complexes formulated with branched polyethylenimine (PEI) 25 kDa. One significant advantage of using this method is the low cytotoxicity of the SLN gene vectors. The application of the gene-transfer technique is limited to relatively low plasmid DNA (pDNA) concentrations of the resulting complexes (10 µg/mL). At higher concentrations, the particles tend to aggregate and precipitate. Therefore, their use for in vivo application, which generally requires high pDNA concentrations, is limited.
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http://dx.doi.org/10.1101/pdb.prot068122DOI Listing
March 2012

Characterization of Ku70(2)-NLS as bipartite nuclear localization sequence for non-viral gene delivery.

PLoS One 2012 8;7(2):e24615. Epub 2012 Feb 8.

Department of Pediatrics, Ludwig-Maximilians University, Munich, Germany.

Several barriers have to be overcome in order to achieve gene expression in target cells, e.g. cellular uptake, endosomal release and translocation to the nucleus. Nuclear localization sequences (NLS) enhance gene delivery by increasing the uptake of plasmid DNA (pDNA) to the nucleus. So far, only monopartite NLS were analysed for non-viral gene delivery. In this study, we examined the characteristics of a novel bipartite NLS like construct, namely NLS Ku70. We synthesized a dimeric structure of a modified NLS from the Ku70 protein (Ku70(2)-NLS), a nuclear transport active mutant of Ku70(2)-NLS (s1Ku70(2)-NLS) and a nuclear transport deficient mutant of Ku70(2)-NLS (s2Ku70(2)). We examined the transfection efficiency of binary Ku70(2)-NLS/DNA and ternary Ku70(2)-NLS/PEI/DNA gene vector complexes in vitro by using standard transfection protocols as well as the magnetofection method. The application of Ku70(2)-NLS and s1Ku70(2)-NLS increased gene transfer efficiency in vitro and in vivo. This study shows for the first time that the use of bipartite NLS compounds alone or in combination with cationic polymers is a promising strategy to enhance the efficiency of non-viral gene transfer.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0024615PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3275586PMC
July 2012

Magnetized aerosols comprising superparamagnetic iron oxide nanoparticles improve targeted drug and gene delivery to the lung.

Pharm Res 2012 May 21;29(5):1308-18. Epub 2012 Jan 21.

Department of Pediatrics, Ludwig-Maximilians-University, Lindwurmstr 2a, 80337 Munich, Germany.

Purpose: Targeted delivery of aerosols could not only improve efficacy of inhaled drugs but also reduce side effects resulting from their accumulation in healthy tissue. Here we investigated the impact of magnetized aerosols on model drug accumulation and transgene expression in magnetically targeted lung regions of unanesthetized mice.

Methods: Solutions containing superparamagnetic iron oxide nanoparticles (SPIONs) and model drugs (fluorescein or complexed plasmid DNA) were nebulized to unanesthetized mice under the influence of an external magnetic gradient directed to the lungs. Drug accumulation and transgene expression was subsequently measured at different time points.

Results: We could demonstrate 2-3 fold higher accumulation of the model drug fluorescein and specific transgene expression in lung regions of mice which had been exposed to an external magnetic gradient during nebulization compared to the control mice without any exposure to magnetic gradient.

Conclusions: Magnetized aerosols present themselves as an efficient approach for targeted pulmonary delivery of drugs and gene therapeutic agents in order to treat localized diseases of the deeper airways.
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http://dx.doi.org/10.1007/s11095-012-0682-zDOI Listing
May 2012

Aerosolized BC-819 inhibits primary but not secondary lung cancer growth.

PLoS One 2011 8;6(6):e20760. Epub 2011 Jun 8.

Department of Pediatrics, Ludwig-Maximilians-University, Munich, Germany.

Despite numerous efforts, drug based treatments for patients suffering from lung cancer remains poor. As a promising alternative, we investigated the therapeutic potential of BC-819 for the treatment of lung cancer in mouse tumor models. BC-819 is a novel plasmid DNA which encodes for the A-fragment of Diphtheria toxin and has previously been shown to successfully inhibit tumor growth in human clinical study of bladder carcinoma. In a first set of experiments, we examined in vitro efficacy of BC-819 in human lung cancer cell-lines NCI-H460, NCI-H358 and A549, which revealed >90% reduction of cell growth. In vivo efficacy was examined in an orthotopic mouse xenograft lung cancer model and in a lung metastasis model using luminescent A549-C8-luc adenocarcinoma cells. These cells resulted in peri- and intra-bronchiolar tumors upon intrabronchial application and parenchymal tumors upon intravenous injection, respectively. Mice suffering from these lung tumors were treated with BC-819, complexed to branched polyethylenimine (PEI) and aerosolized to the mice once per week for a period of 10 weeks. Using this regimen, growth of intrabronchially induced lung tumors was significantly inhibited (p = 0.01), whereas no effect could be observed in mice suffering from lung metastasis. In summary, we suggest that aerosolized PEI/BC-819 is capable of reducing growth only in tumors arising from the luminal part of the airways and are therefore directly accessible for inhaled BC-819.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0020760PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3110766PMC
October 2011

Dry powder aerosols of polyethylenimine (PEI)-based gene vectors mediate efficient gene delivery to the lung.

J Control Release 2011 Aug 10;154(1):69-76. Epub 2011 May 10.

Department of Pediatrics, Ludwig-Maximilians University, 80337 Munich, Germany.

Aerosol gene delivery holds great therapeutical potential for many inherited and acquired pulmonary diseases. The physical instability of aqueous suspensions of non-viral vector complexes is a major limitation for their successful application. In this study, we investigated dry powder aerosols as novel gene vector formulations for gene transfer in vitro and murine lungs in vivo. Lyophilization was used to produce dry powder cakes followed by powderization to produce dry powder aerosols. Different sugars, namely lactose, sucrose and trehalose, were tested as lyoprotectants for gene delivery complexes consisting of branched polyethylenimine 25 kDa and plasmid DNA. Biophysical particle characterization demonstrated that lyophilization and powderization in the presence of lyoprotectants were well tolerated. In vitro transfection efficiency remained unaffected by the choice of lyoprotectant and subsequent lyophilization and/or powderization. In vivo screening of powderized samples, by applying the powder with an insufflator, resulted in highest gene expression with lactose as lyoprotectant. Delivering a plasmid coding for murine erythropoietin together with lactose as lyoprotectant resulted in increased blood hematocrit values post application thereby demonstrating the potential of dry powder aerosol as a promising method for pulmonary gene delivery.
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http://dx.doi.org/10.1016/j.jconrel.2011.05.006DOI Listing
August 2011

PEGylation improves nanoparticle formation and transfection efficiency of messenger RNA.

Pharm Res 2011 Sep 19;28(9):2223-32. Epub 2011 May 19.

Division of Molecular Pulmonology, Department of Pediatrics, Ludwig Maximilians University of Munich, Lindwurmstr. 2a, 80337 Munich, Germany.

Purpose: Cationic polymers have been intensively investigated for plasmid-DNA (pDNA), but few studies addressed their use for messenger-RNA (mRNA) delivery. We analyzed two types of polymers, linear polyethylenimine (l-PEI) and poly-N,N-dimethylaminoethylmethacrylate P(DMAEMA), to highlight specific requirements for the design of mRNA delivery reagents. The effect of PEGylation was investigated using P(DMAEMA-co-OEGMA) copolymer.

Methods: The influence of polymer structure on mRNA binding and particle formation was assessed in a side-by-side comparison with pDNA by methods such as agarose-retardation assay and scanning probe microscopy. Transfection studies were performed on bronchial epithelial cells.

Results: Binding of cationic polymers inversely correlated with type of nucleic acid. Whereas P(DMAEMA) bound strongly to pDNA, only weak mRNA binding was observed, which was vice versa for l-PEI. Both polymers resulted in self-assembled nanoparticles forming pDNA complexes of irregular round shape; mRNA particles were significantly smaller and more distinct. Surprisingly, PEGylation improved mRNA binding and transfection efficiency contrary to observations made with pDNA. Co-transfections with free polymer improved mRNA transfection.

Conclusions: Gene delivery requires tailor-made design for each type of nucleic acid. PEGylation influenced mRNA-polymer binding efficiency and transfection and may provide a method of further improving mRNA delivery.
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http://dx.doi.org/10.1007/s11095-011-0464-zDOI Listing
September 2011

Expression of therapeutic proteins after delivery of chemically modified mRNA in mice.

Nat Biotechnol 2011 Feb 9;29(2):154-7. Epub 2011 Jan 9.

Department of Pediatrics, Ludwig Maximilian's University Munich, Munich, Germany.

Current viral vectors for gene therapy are associated with serious safety concerns, including leukemogenesis, and nonviral vectors are limited by low gene transfer efficiency. Here we investigate the therapeutic utility of chemically modified mRNA as an alternative to DNA-based gene therapy. A combination of nucleotide modifications abrogates mRNA interaction with Toll-like receptor (TLR)3, TLR7, TLR8 and retinoid-inducible gene I (RIG-I), resulting in low immunogenicity and higher stability in mice. A single intramuscular injection of modified murine erythropoietin mRNA raises the average hematocrit in mice from 51.5% to 64.2% after 28 days. In a mouse model of a lethal congenital lung disease caused by a lack of surfactant protein B (SP-B), twice weekly local application of an aerosol of modified SP-B mRNA to the lung restored 71% of the wild-type SP-B expression, and treated mice survived until the predetermined end of the study after 28 days.
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http://dx.doi.org/10.1038/nbt.1733DOI Listing
February 2011

Efficient, specific and targeted delivery of genes to the lung.

Ther Deliv 2010 Jul;1(1):133-48

Division of Molecular Pulmonology, Department of Pediatrics, Ludwig-Maximilians University, Lindwurmstrasse 2A, 80337 Munich, Germany.

Many inherited and acquired pulmonary disorders without satisfactory therapies may be amenable to gene therapy. Despite numerous advances, efficient delivery and expression of the therapeutic transgene at physiological levels for phenotypic correction of disease has proved elusive. This article focuses on various strategies aimed at achieving targeted delivery to the lungs. Both physical methods and biological targeting have been successfully applied in various gene delivery systems. Targeting of different cell types has been achieved by pseudotyping of viral vectors with capsids from different serotypes and modification of nonviral vectors with targeting ligands. Both classes of vectors are discussed with respect to their gene delivery and expression efficiencies, longevity of expression and immunogenicity. Moreover, gene therapy clinical trials for different lung diseases are discussed.
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http://dx.doi.org/10.4155/tde.10.11DOI Listing
July 2010

First theoretic analysis of magnetic drug targeting in the lung.

IEEE Trans Biomed Eng 2010 Sep 27;57(9):2115-21. Epub 2010 May 27.

Technical University Munich, Nanoelectronics and Biosensors Division, Munich D-80333, Germany.

Magnetic drug targeting in the bloodstream has been intensively studied recently, but also other interesting access and transport pathways fulfill the basic conditions for this method. More recently, magnetic drug targeting has even been accomplished for aerosol application in mouse models and indicated unmistakable advantages over all currently available therapies for lung tumors and any other very localized lung disease. In this paper, the application of magnetically labeled aerosols to the lung via the airways is theoretically highlighted for the first time in the literature. The fundamental difference compared to targeting via the bloodstream lies in the medium and the presence of the bronchial surface: When touching the epithelium of the lung outside the target region, a particle will deposit on the surface and not enter the air stream again. We are the first to compose a comprehensive physical description and provide a fundamental understanding of this potential expedient for treating cancer and other localized diseases. With our approach, we found optimal conditions for this sort of therapy. As a main parameter for optimization, the droplet size could be identified by minimizing unwanted deposition outside the target due to secondary effects, which compete with the magnetic forces. This may improve therapeutic efficiency and reduce side effects of otherwise not well-tolerated compounds, such as chemotherapeutics at the same time.
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http://dx.doi.org/10.1109/TBME.2010.2051032DOI Listing
September 2010

Repeated siRNA application is a precondition for successful mRNA gammaENaC knockdown in the murine airways.

Eur J Pharm Biopharm 2010 Aug 18;75(3):305-10. Epub 2010 Apr 18.

Department of Pediatrics, Ludwig-Maximilians University, Munich, Germany.

The volume of the airway surface liquid is regulated by Na(+) absorption and Cl(-) secretion by the respiratory epithelium. In cystic fibrosis, Na(+) hyperabsorption caused by the absence of functional CFTR protein leads to an altered airway surface liquid composition and finally to a deteriorated mucociliary clearance. It has been suggested that down regulation or inhibition of the amiloride-sensitive epithelial Na(+) channel (ENaC) could restore the disrupted airway hydration. Therefore, targeting ENaC by RNA interference could be of therapeutic relevance. In this context, we investigated whether RNAi could lead to a reduction in gammaENaC expression in epithelia in vitro and in vivo in mice. Transfection of cells with specific siRNA sequences for gammaENaC subunit reduced expression to approximately 10% relative to control. For in vivo experiments, siRNA sequences specific for the gammaENaC subunit were administered to the murine nasal cavity and, 72h later the animals were killed. In the first approach, only a single application of naked siRNA was given. In the second approach, repeated siRNA applications were performed. The single application of siRNA sequences had no effect on mRNA content of the targeted gammaENaC subunit, whereas repeated siRNA application resulted in a significant reduction in gammaENaC mRNA in the respiratory tissue. We conclude that repeated siRNA application is necessary for gammaENaC knockdown in the murine airways.
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http://dx.doi.org/10.1016/j.ejpb.2010.04.005DOI Listing
August 2010