A computationally designed antigen eliciting broad humoral responses against SARS-CoV-2 and related sarbecoviruses.

The threat of spillovers of coronaviruses associated with the severe acute respiratory syndrome (SARS) from animals to humans necessitates vaccines that offer broader protection from sarbecoviruses. By leveraging a viral-genome-informed computational method for selecting immune-optimized and structurally engineered antigens, here we show that a single antigen based on the receptor binding domain of the spike protein of sarbecoviruses elicits broad humoral responses against SARS-CoV-1, SARS-CoV-2, WIV16 and RaTG13 in mice, rabbits and guinea pigs. When administered as a DNA immunogen or by a vector based on a modified vaccinia virus Ankara, the optimized antigen induced vaccine protection from the Delta variant of SARS-CoV-2 in mice genetically engineered to express angiotensin-converting enzyme 2 and primed by a viral-vector vaccine (AZD1222) against SARS-CoV-2. A vaccine formulation incorporating mRNA coding for the optimized antigen further validated its broad immunogenicity. Vaccines that elicit broad immune responses across subgroups of coronaviruses may counteract the threat of zoonotic spillovers of betacoronaviruses.

Cellular uptake of modified mRNA occurs via caveolae-mediated endocytosis, yielding high protein expression in slow-dividing cells.

Nucleic acids have clear clinical potential for gene therapy. Plasmid DNA (pDNA) was the first nucleic acid to be pursued as a therapeutic molecule. Recently, mRNA came into play as it offers improved safety and affordability. In this study, we investigated the uptake mechanisms and efficiencies of genetic material by cells. We focused on three main variables (1) the nucleic acid (pDNA, or chemically modified mRNA), (2) the delivery vector (Lipofectamine 3000 or 3DFect), and (3) human primary cells (mesenchymal stem cells, dermal fibroblasts, and osteoblasts). In addition, transfections were studied in a 3D environment using electrospun scaffolds. Cellular internalization and intracellular trafficking were assessed by using enhancers or inhibitors of endocytosis and endosomal escape. The polymeric vector TransIT-X2 was included for comparison purposes. While lipoplexes utilized several entry routes, uptake via caveolae served as the main route for gene delivery. pDNA yielded higher expression levels in fast-dividing fibroblasts, whereas, in slow-dividing osteoblasts, cmRNA was responsible for high protein production. In the case of mesenchymal stem cells, which presented an intermediate doubling time, the combination vector/nucleic acid seemed more relevant than the nucleic acid per se. In all cases, protein expression was higher when the cells were seeded on 3D scaffolds.

Efficient healing of large osseous segmental defects using optimized chemically modified messenger RNA encoding BMP-2.

Large segmental osseous defects heal poorly. Recombinant, human bone morphogenetic protein-2 (rhBMP-2) is used clinically to promote bone healing, but it is applied at very high doses that cause adverse side effects and raise costs while providing only incremental benefit. We describe a previously unexplored, alternative approach to bone regeneration using chemically modified messenger RNA (cmRNA). An optimized cmRNA encoding BMP-2 was delivered to critical-sized femoral osteotomies in rats. The cmRNA remained orthotopically localized and generated BMP locally for several days. Defects healed at doses ≥25 µg of BMP-2 cmRNA. By 4 weeks, all animals treated with 50 µg of BMP-2 cmRNA had bridged bone defects without forming the massive callus seen with rhBMP-2. Moreover, such defects recovered normal mechanical strength quicker and initiated bone remodeling faster. cmRNA regenerated bone via endochondral ossification, whereas rhBMP-2 drove intramembranous osteogenesis; cmRNA provides an innovative, safe, and highly translatable technology for bone healing.

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

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.

In Vitro Evaluation of a Nanoparticle-Based mRNA Delivery System for Cells in the Joint.

Biodegradable and bioresponsive polymer-based nanoparticles (NPs) can be used for oligonucleotide delivery, making them a promising candidate for mRNA-based therapeutics. In this study, we evaluated and optimized the efficiency of a cationic, hyperbranched poly(amidoamine)s-based nanoparticle system to deliver tdTomato mRNA to primary human bone marrow stromal cells (hBMSC), human synovial derived stem cells (hSDSC), bovine chondrocytes (bCH), and rat tendon derived stem/progenitor cells (rTDSPC). Transfection efficiencies varied among the cell types tested (bCH 28.4% ± 22.87, rTDSPC 18.13% ± 12.07, hBMSC 18.23% ± 14.80, hSDSC 26.63% ± 8.81) and while an increase of NPs with a constant amount of mRNA generally improved the transfection efficiency, an increase of the mRNA loading ratio (2:50, 4:50, or 6:50 w/w mRNA:NPs) had no impact. However, metabolic activity of bCHs and rTDSPCs was significantly reduced when using higher amounts of NPs, indicating a dose-dependent cytotoxic response. Finally, we demonstrate the feasibility of transfecting extracellular matrix-rich 3D cell culture constructs using the nanoparticle system, making it a promising transfection strategy for musculoskeletal tissues that exhibit a complex, dense extracellular matrix.

Transcript-Activated Coatings on Titanium Mediate Cellular Osteogenesis for Enhanced Osteointegration.

Osteointegration is one of the most important factors for implant success. Several biomolecules have been used as part of drug delivery systems to improve implant integration into the surrounding bone tissue. Chemically modified mRNA (cmRNA) is a new form of therapeutic that has been used to induce bone healing. Combined with biomaterials, cmRNA can be used to develop transcript-activated matrices for local protein production with osteoinductive potential. In this study, we aimed to utilize this technology to create bone morphogenetic protein 2 (BMP2) transcript-activated coatings for titanium (Ti) implants. Therefore, different coating methodologies as well as cmRNA incorporation strategies were evaluated. Three different biocompatible biomaterials were used for the coating of Ti, namely, poly-d,l-lactic acid (PDLLA), fibrin, and fibrinogen. cmRNA-coated Ti disks were assayed for transfection efficiency, cmRNA release, cell viability and proliferation, and osteogenic activity in vitro. We found that cmRNA release was significantly delayed in Ti surfaces previously coated with biomaterials. Consequently, the transfection efficiency was greatly improved. PDLLA coating improved the transfection efficiency in a concentration-dependent manner. Lower PDLLA concentration used for the coating of Ti resulted in higher transfection efficiency. Fibrin and fibrinogen coatings showed even higher transfection efficiencies compared to all PDLLA concentrations. In those disks, not only the expression was up to 24-fold higher but also the peak of maximal expression was delayed from 24 h to 5 days, and the duration of expression was also extended until 7 days post-transfection. For fibrin, higher transfection efficiencies were obtained in the coatings with the lowest thrombin amounts. Accordingly, fibrinogen coatings gave the best results in terms of cmRNA transfection. All biomaterial-coated Ti surfaces showed improved cell viability and proliferation, though this was more noticeable in the fibrinogen-coated disks. The latter was also the only coating to support significant amounts of BMP2 produced by C2C12 cells in vitro. Osteogenesis was confirmed using BMP2 cmRNA fibrinogen-coated Ti disks, and it was dependent of the cmRNA amount present. Alkaline phosphatase (ALP) activity of C2C12 increased when using fibrinogen coatings containing 250 ng of cmRNA or more. Similarly, mineralization was also observed that increased with increasing cmRNA concentration. Overall, our results support fibrinogen as an optimal material to deliver cmRNA from titanium-coated surfaces.

Magnetic and Acoustically Active Microbubbles Loaded with Nucleic Acids for Gene Delivery.

Targeted gene or drug delivery aims to locally accumulate the active agent and achieve the maximum local therapeutic effect at the target site while reducing unwanted effects at nontarget sites. A further development of the magnetic drug-targeting concept is combining it with an ultrasound-triggered delivery using magnetic microbubbles as a carrier for gene or drug delivery. For this purpose, selected magnetic nanoparticles (MNPs), phospholipids, and nucleic acid are assembled in the presence of perfluorocarbon gas into flexible formulations of magnetic lipospheres or microbubbles. This chapter describes the protocols for preparation of magnetic lipospheres and microbubbles for nucleic acid delivery, and it also describes the procedures for labeling the components of the bubbles (lipids, MNPs, and nucleic acids) for the visualization of the vectors and their characterization, such as magnetic responsiveness and ultrasound contrast effects. Protocols are given for the transfection procedure in adherent cells, evaluation of the association of the magnetic vectors with the cells, reporter gene expression analysis, and cell viability assessment.

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

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 E. coli, resulting in extensive shortening of the poly(A) tail. Using a segmented poly(A) approach, we could significantly reduce recombination of plasmids in E. coli 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).

Delivery of mRNA Therapeutics for the Treatment of Hepatic Diseases.

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.

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

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.

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

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.

An Improved, Chemically Modified RNA Encoding BMP-2 Enhances Osteogenesis In Vitro and In Vivo.

IMPACT STATEMENT: The use of chemically modified RNA (cmRNA) with increased stability using translation initiator of short untranslated regions (TISU) offers the prospect of finally allowing us to unlock the potent osteogenic properties of BMP-2 in a clinically expedient manner. As noted, delivery of recombinant BMP-2 protein has had modest clinical efficacy, whereas gene delivery is effective but very difficult to translate into human clinical use. This study shows the great potential of cmRNA encoding BMP-2 with TISU in a long-bone critical-sized rat model.

Comparative analysis of bone regeneration behavior using recombinant human BMP-2 versus plasmid DNA of BMP-2.

Bone regeneration and the osteoinductive capacity of implants are challenging issues in clinical medicine. Currently, recombinant growth factors and nonviral gene transfer are the most frequently investigated methods for bone growth enhancement, although the more favorable method remains unclear. There is a lack of knowledge in literature about the in vivo comparison of these methods for bone regeneration. BMP-2, which is the most commonly used growth factor for osteogenesis, was applied at its most efficient dose as a recombinant growth factor (rhBMP-2) and as a growth-factor-encoding copolymer protected gene vector (pBMP-2) in a critical size bone defect (CSD) model to determine the most suitable method for bone regeneration. CSDs were induced bilaterally in 32 Sprague-Dawley rats. RhBMP-2 (62.5 µg) or pBMP-2 (2.5 µg) was embedded in poly(d,l-)lactide-coated titanium discs. Survival times were set at 14, 28, 56, and 112 days. After euthanasia, samples were analyzed via micro-computed tomography, polychrome sequential fluorescent labeling, and immunohistochemistry. Whereas defects in both groups were bridged with new bone after 56 days, rhBMP-2 initially induced ectopic new bone formation that was later remodeled in an unorganized hypodense manner. In contrast, pBMP-2 led to slower but steady bone regeneration with physiological tissue morphology, as confirmed by high osteoblast activity shown by osteocalcin staining. CD68 and TRAP staining verified high osteoclast activity for the rhBMP-2 group. pBMP-2 successfully induced locally controlled physiological bone regeneration, whereas rhBMP-2 triggered rapid and ectopic but insufficient bone formation. Thus, nonviral gene transfer appears to be more favorable for clinical applications. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 107A: 163-173, 2019.

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

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 in vitro and in vivo. 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.

Improved heart repair upon myocardial infarction: Combination of magnetic nanoparticles and tailored magnets strongly increases engraftment of myocytes.

Cell replacement in the heart is considered a promising strategy for the treatment of post-infarct heart failure. Direct intramyocardial injection of cells proved to be the most effective application route, however, engraftment rates are very low (<5%) strongly hampering its efficacy. Herein we combine magnetic nanoparticle (MNP) loading of EGFP labeled embryonic cardiomyocytes (eCM) and embryonic stem cell-derived cardiomyocytes (ES-CM) with application of custom designed magnets to enhance their short and long-term engraftment. To optimize cellular MNP uptake and magnetic force within the infarct area, first numerical simulations and experiments were performed in vitro. All tested cell types could be loaded efficiently with SOMag5-MNP (200 pg/cell) without toxic side effects. Application of a 1.3 T magnet at 5 mm distance from the heart for 10 min enhanced engraftment of both eCM and ES-CM by approximately 7 fold at 2 weeks and 3.4 fold (eCM) at 8 weeks after treatment respectively and also strongly improved left ventricular function at all time points. As underlying mechanisms we found that application of the magnetic field prevented the initial dramatic loss of cells via the injection channel. In addition, grafted eCM displayed higher proliferation and lower apoptosis rates. Electron microscopy revealed better differentiation of engrafted eCM, formation of cell to cell contacts and more physiological matrix formation in magnet-treated grafts. These results were corroborated by gene expression data. Thus, combination of MNP-loaded cells and magnet-application strongly increases long-term engraftment of cells addressing a major shortcoming of cardiomyoplasty.

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

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.

HIF-1α Dependent Wound Healing Angiogenesis In Vivo Can Be Controlled by Site-Specific Lentiviral Magnetic Targeting of SHP-2.

Hypoxia promotes vascularization by stabilization and activation of the hypoxia inducible factor 1α (HIF-1α), which constitutes a target for angiogenic gene therapy. However, gene therapy is hampered by low gene delivery efficiency and non-specific side effects. Here, we developed a gene transfer technique based on magnetic targeting of magnetic nanoparticle-lentivirus (MNP-LV) complexes allowing site-directed gene delivery to individual wounds in the dorsal skin of mice. Using this technique, we were able to control HIF-1α dependent wound healing angiogenesis in vivo via site-specific modulation of the tyrosine phosphatase activity of SHP-2. We thus uncover a novel physiological role of SHP-2 in protecting HIF-1α from proteasomal degradation via a Src kinase dependent mechanism, resulting in HIF-1α DNA-binding and transcriptional activity in vitro and in vivo. Excitingly, using targeting of MNP-LV complexes, we achieved simultaneous expression of constitutively active as well as inactive SHP-2 mutant proteins in separate wounds in vivo and hereby specifically and locally controlled HIF-1α activity as well as the angiogenic wound healing response in vivo. Therefore, magnetically targeted lentiviral induced modulation of SHP-2 activity may be an attractive approach for controlling patho-physiological conditions relying on hypoxic vessel growth at specific sites.

Functional analysis of bioactivated and antiinfective PDLLA - coated surfaces.

Common scaffold surfaces such as titanium can have side effects; for example, infections, cytotoxicity, impaired osseointegration, or low regeneration rates for bone tissue. These effects lead to poor implant integration or even implant loss. Therefore, bioactive implants are promising instruments in tissue regeneration. Osteoinductive elements-such as growth factors and anti-infectives-support wound healing and bone growth and thereby enable faster osseointegration, even in elderly patients. In this study, titanium surfaces were coated with a poly-(d,l-lactide) (PDLLA) layer containing different concentrations of copolymer-protected gene vectors (COPROGs) to locally provide bone morphogenetic protein-2 (BMP-2) or activated anti-infective agents, such as chlorhexidine gluconate, triclosan, and metronidazole, to prevent peri-implantitis. The coated titanium implants were then loaded with osteoblasts, NIH 3T3 fibroblasts, and human mesenchymal stem cells in 96-well plates. When shielded by COPROGs as a protective layer and resuspended in PDLLA, BMP-2-encoding pDNA at relatively low doses (5.63 µg/implant) induced the local expression of BMP-2. A linear dose dependence, which is common for recombinant growth factors, was not found, probably due to the retention property of the PDLLA surface. PDLLA, in general, successfully retains additional elements, such as osteoconductive growth factors (BMP-2) and anti-infective agents, which was demonstrated using metronidazole, and thus prevents the systemic application of excessive doses. These bioactive implant surfaces that provide the local release of therapeutic gene vectors or anti-infective agents allow the controlled stimulation of the implant and scaffold osseointegration. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 1672-1683, 2017.

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

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.