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.

The present study was conducted to investigate the effect of decomposition site and plant litter species on the colonizing microbial communities. For this, litter bag technique using beech and spruce litter was combined with RNA-based fingerprinting and cloning. Litter bags were incubated for 2 and 8 weeks in the Ah horizon of beech and beech-spruce mixed forest sites. Although sugars and starch were rapidly lost, lignin content increased by more than 40% for beech and more than doubled for spruce litter at both soil sites at the end of the experiment. Denaturing gradient gel electrophoresis analysis of 16S and 18S rRNA RT-PCR products was used for screening of differences between bacterial and fungal communities colonizing the two litter types. Development of the microbial community over time was observed to be specific for each litter type and decomposition site. RT-PCR products from both litter types incubated in beech-spruce mixed forest site were also cloned to identify the bacterial and fungal colonizers. The 16S rRNA clone libraries of beech litter were dominated by gamma-proteobacterial members, whereas spruce libraries were mainly composed of alpha-, beta-, and gamma-proteobacterial members. Ascomycota members dominated the 18S rRNA clone libraries. Clones similar to Zygomycota were absent from spruce, whereas those similar to Basidiomycota and Glomeromycota were absent from beech libraries. Selective effects of litter quality were observed after 8 weeks. The study provides an insight into the bacterial and fungal communities colonizing beech and spruce litter, and the importance of litter quality and decomposition site as key factors in their development and succession.

No abstract

No abstract

No abstract

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.

The first therapeutic application of messenger RNA (mRNA) was suggested more than two decades ago. However, its application was constrained by the ability of mRNA to activate the innate immune response, cytotoxicity, and poor potency. We and others recently demonstrated that these undesirable properties of mRNA may be overcome by alterating its structure. In this study, we developed a new chemically modified mRNA coding for BMP-2 with improved osteogenic features. To develop this new construct, we removed from the mRNA sequence the following undesirable elements: an upstream open reading frame in the 5'-untranslated region (UTR) and a polyadenylation element together with an AU-rich tract in the 3'UTR. In addition, a translation initiator of short UTRs (TISU) was introduced together with 5-iodo modified pyrimidine nucleotides. The new TISU BMP-2 chemically modified RNA (cmRNA) showed robust BMP-2 production in vitro in cell lines (HEK293 and MC3T3) and primary cells (muscle-derived mesenchymal stem cells). Stem cells additionally showed upregulation of osteogenic and angiogenic genes as a result of the TISU BMP-2 cmRNA transfection. The in vivo osteogenic properties of TISU BMP-2 cmRNA were explored in a critical-sized femoral defect in the rat. For this, the TISU BMP-2 cmRNA was loaded into collagen sponges to form transcript-activated matrices. Animals treated with TISU BMP-2 cmRNA showed superior bone formation that seemed to recapitulate endochondral ossification. The higher of the two doses examined in this model showed more robust new tissue formation. Finally, improved vascularization was detected in the healing area for animals treated with TISU BMP-2 cmRNA.

Transcription of the nirK and nirS genes coding for dissimilatory bacterial nitrite reductases was analyzed by reverse transcription PCR (RT-PCR) of mRNA isolated from rhizosphere samples of three economically important grain legumes at maturity: Vicia faba, Lupinus albus, and Pisum sativum. The nirK gene and transcripts could be detected in all the rhizosphere samples. In contrast, nirS could not be detected. Sampling variations were analyzed by comparing denaturing gradient gel electrophoresis profiles derived from nirK RT-PCR products. High similarity was observed between the replicates, and so one representative product per legume was cloned. Clones with the correct insert size were screened by restriction fragment length polymorphism by using the restriction enzyme MspI. The clones could be distributed into 12 different patterns. Patterns 1, 3, 4, 5, and 7 were common in clone libraries of the three rhizosphere types under study. Patterns 2, 9, 10, and 11 were absent from Pisum rhizospheres, while patterns 6, 8, and 12 were absent from the Vicia library. Pattern 1, which was the most dominant in the Vicia and Lupinus libraries, constituted about 25% of all clones. The Lupinus library had clones representing all 12 patterns, indicating it to be the most diverse among the three. Clones representative of each pattern were sequenced. All patterns grouped together forming a distinct cluster, which was divergent from previously described nirK sequences in the database. The study revealed a hitherto unknown diversity of denitrifiers in legume rhizospheres. A plant-dependent rhizosphere effect on the transcripts of a gene was evident.