Characterization of the ribonuclease activity on the skin surface
© Probst et al; licensee BioMed Central Ltd. 2006
Received: 28 February 2006
Accepted: 29 May 2006
Published: 29 May 2006
The rapid degradation of ribonucleic acids (RNA) by ubiquitous ribonucleases limits the efficacy of new therapies based on RNA molecules. Therefore, our aim was to characterize the natural ribonuclease activities on the skin and in blood plasma i.e. at sites where many drugs in development are applied. On the skin surfaces of Homo sapiens and Mus musculus we observed dominant pyrimidine-specific ribonuclease activity. This activity is not prevented by a cap structure at the 5'-end of messenger RNA (mRNA) and is not primarily of a 5'- or 3'-exonuclease type. Moreover, the ribonuclease activity on the skin or in blood plasma is not inhibited by chemical modifications introduced at the 2'OH group of cytidine or uridine residues. It is, however, inhibited by the ribonuclease inhibitor RNasin® although not by the ribonuclease inhibitor SUPERase· In™. The application of our findings in the field of medical science may result in an improved efficiency of RNA-based therapies that are currently in development.
The presence of ribonucleases on human and rodent skin surfaces was described more than 40 years ago.[1, 2] Subsequently their distribution within different skin layers was studied by different techniques.[3–5] However, the diversity, specificity and activity of extracellular (i.e. secreted or originating from dead cells) ribonucleases present on skin was never investigated.
However, information is available on extracellular ribonucleases expressed in internal human organs. These enzymes belong to the RNaseA protein superfamily. Based on structural, catalytic and/or biological characteristics they can be classified into two major groups: the pancreatic type (pt) and the non-pancreatic type (npt) ribonucleases. Human pt ribonucleases are similar to bovine pancreas RNaseA. They are active on poly(A) and double stranded RNA (dsRNA) and prefer as substrate poly(C) over poly(U). In contrast, npt ribonucleases are not active on poly(A) nor on dsRNA substrates and prefer poly(U) rather than poly(C) as substrate. At present, eight distinct human extracellular ribonucleases have been described at the genetic level. All of them are encoded by genes located on the long arm of chromosome14. At the protein level, five different ribonuclease activities have been described for human blood plasma. These ribonucleases range in size between 14 and 31 kDa.
Extracellular ribonucleases are important in the formation of new blood vessels and thus tumor progression . Indeed, Angiogenin that is the first identified tumor derived secreted angiogenic factor is an extracellular protein with a pt ribonucleolytic activity. This nuclease feature is necessary but not sufficient for angiogenin's angiogenic activity. However, the mechanisms of action of angiogenin and related poteins (angiogenins) on angiogenesis and in particular the role of the intrinsic RNAse activity, is still not clearly deciphered (for review see Strydom et al. ). For other extracellular ribonucleases it is suggested that they play a role in the prevention of infection by microbes [11, 12] or RNA-viruses. They might also control the hypothesized cell-to-cell communication mediated by the release and uptake of RNA by neighboring cells. Finally, they may block unwanted activation of the immune system by dead cells which release RNA that, if not degraded, would stimulate antigen presenting cells (APC) through TLR-3, TLR-7 or TLR-8.[15–18]
The characterization of the extracellular ribonuclease activity has become again an attractive topic at the post-genomic era, where the development of safe gene therapies is needed for the transfer of basic research to the clinic. Plasmid DNA or recombinant viruses that were proposed as delivery vehicles for gene therapy approaches are associated to potential side effects and have uncontrolled half life.[19, 20] As an alternative, mRNA, a nucleic acid with a controlled half life, is being evaluated in pre-clinical and clinical trials. Several mRNA-based immunization methods have been developed (reviewed in ): mRNA injected intradermally [22–26], mRNA entrapped in liposomes and injected subcutaneously or intravenously [27, 28], mRNA loaded on gold particles and delivered intradermally by Gene-Gun  and mRNA transfected in vitro into APC.[30–33]
The quick degradation of mRNA by ubiquitous ribonucleases is one of the safety features of mRNA-based therapies. This process guaranties that the injected genetic information will be completely degraded and cleared from the body in a short time. The instability, however, puts an obvious limit on efficacy. Therefore, all mRNA-based therapies would benefit from the utilization of stabilized mRNA that have enhanced resistance towards ribonucleases contained in physiologic fluids, cell culture media and on the surface of the skin.
In order to gain more insights into the fundamental functions of extracellular ribonucleases, we investigated their diversity, their activity and their specificity. With the goal to enhance mRNA-based therapies, we also tested different strategies to stabilize the mRNA with regard to extracellular ribonuclease activity. We report here the characterization of the ribonuclease activity contained on the skin surface and in blood plasma and methods to inhibit them. Our results are relevant for applications in the field of mRNA-based therapies.
BALB/c mice were purchased from Charles River (Sulzfeld, Germany). The mice were not kept under special pathogen free conditions. All animal experiments were performed according to institutional and national guidelines.
Preparation of ribonucleases
Homo sapiens skin surface ribonucleases were repeatedly isolated from one healthy individual by wetting an area of ~10 cm2 pre-cleaned skin (sterilized and subsequently washed with soap and water) with 200–300 μl water for ~3 min. During this time the drop of water was several times pipetted up and down. For Mus musculus, skin surface ribonucleases were isolated by incubating an ear over night at 4°C in 200–300 μl water. Contact of water with the cut zone was avoided.
Protein content of skin surface preparations of both origins was below the detection limit for protein quantification by photometric measurements (Roti®-Nanoquant, Carl Roth, Karlsruhe, Germany). We observed only little variations in ribonuclease activity of different preparations as determined in degradation assays.
Peripheral blood from Homo sapiens and Mus musculus was collected in EDTA containing tubes to avoid coagulation. Blood plasma was separated by centrifugation for 6 min at 600 g and collected.
RibonucleaseA from Bos taurus pancreas was purchased from Roche (Mannheim, Germany) and dissolved in water to 10 mg/ml.
All preparations were aliquoted immediately and stored at -80°C.
mRNA was produced by in vitro transcription with T7 RNA polymerase (T7-Opti mRNA kits, CureVac, Tübingen, Germany). Modified nucleotides were purchased from TriLink (San Diego, USA). All transcripts contained a poly(A) tail (70 bases long) and if not otherwise stated a 5'-cap structure. This cap structure was introduced during in vitro transcription: a fourfold excess of synthetic N7-Methyl-Guanosine-5'-Triphosphate-5'-Guanosine compared to GTP was used to guaranty that approximately 80% of the synthesized mRNA molecules started with a cap (whereas the remaining approximately 20% of the mRNA molecules started with GTP). Synthetic 18-mer RNA homopolymers were produced by CureVac using the phosphoramidite method. Poly(C) was purchased from Amersham (Freiburg, Germany)
After denaturation at 95°C for 2 min in 1 × Laemmli loading buffer, samples were loaded on a SDS-PAGE where the 12, 5% stacking gel contained ~0, 6 mg/ml poly(C). Subsequently to electrophoresis (~2 h at 150 V), the gel was washed twice for 10 min with 25% (v/v) 2-propanol, 50 mM TrisHCl (pH7, 4) and 5 mM EDTA. The gel was scanned to document the position of the pre-stained molecular weight marker proteins (SeeBlue® Plus2, Invitrogen, Karlsruhe, Germany). Then, it was further washed four times for 10 min with 50 mM TrisHCl (pH7, 4) and 5 mM EDTA (washing buffer). Thereafter, the gel was incubated at 37°C for 17 h in washing buffer supplemented with 150 mM NaCl. Ribonuclease activity was visualized by staining the gel with washing buffer supplemented with 0, 2%(w/v) toluidine blue O (Sigma, Munich, Germany) and destaining with washing buffer. For documentation the gel was scanned (GS-700, Biorad, Munich, Germany).
Ribonuclease activity assay
Ribonuclease activity was assayed at 37°C in PBS (pH7, 2) by co-incubation of 0, 16 μg/ml of mRNA or 166 nM of 18-mer homopolymers and the indicated final dilution of ribonuclease preparations.
Reaction products were analyzed according to the following protocols: For mRNA, 6 μl samples were transferred to 6 μl formaldehyde loading buffer containing ethidium bromide (0, 01 mg/ml) and heat-denatured for 5 min at 80°C. The extend of mRNA digestion was analyzed by electrophoresis on formaldehyde agarose (FA) gels (1, 2%(w/v) agarose and 0, 65%(w/v) formalin in 1× FA buffer).
For RNA 18-mer homopolymers 6 μl samples were transferred to 6 μl formamide, heated for 5 min at 55°C, separated by urea-PAGE (42%(w/v) urea and 20%(w/v) acrylamide(29:1) in 1× TBE) and visualized by epiillumination of the gels on top of a thin layer chromatography plate.
The content of FA gels was blotted over night onto Hybond-N+ membranes (Amersham, Freiburg, Germany) by the capillary blot technique with 20× SSC as transfer buffer. After fixation (UV 1300 J/cm2 plus backing 80°C for 2 h), membranes were equilibrated with hybridization buffer (5×SSC, 5×Denhardt's and 0, 5%(w/v) SDS) for 30 min at 50°C before the [γ-32P]-labeled 3'-probe (5'-GCA AGG AGG GGA GGA GGG-3', MWG-Biotech, Ebersberg, Germany) was added and incubation was continued over night. After repeated washing with decreasing salt (SSC) concentrations, the blot was exposed to a phosphor imager (PI) plate (Fujifilm, Düsseldorf, Germany). After documentation by scanning the PI plate (BAS-1500, Fujifilm) the blot was stripped by boiling in 0, 1% SDS (w/v) and then hybridized to the [γ-32P]-labeled 5'-probe (5'-TGA GCG TTT ATT CTG AGC TTC TGC-3', Thermo, Ulm, Germany). Some experiments were also carried out using first the 5'-probe and subsequently the 3'-probe. Densitograms were calculated with the Tina2.09d software (Raytest, Straubenhardt, Germany).
Electroporation and FACS
Baby hamster kidney (BHK21) cells were grown to 80% confluence in cell culture medium (RPMI1640 supplemented with 100 U/μg/ml penicillin/streptomycin, 2 mM L-glutamine and 10%(v/v) FCS). Cells were harvested by trypsin-EDTA, washed once with cell culture medium and resuspended in PBS. Electroporation of 1–2 × 106 BHK cells in 4 mm cuvettes was performed at 250 V and 1050 μF in 200 μl PBS with 10 μg mRNA. After transfection, cells were immediately transferred to a cell culture vessel and allowed to grow for 15 h. They were harvested with trypsin-EDTA, fixed with 1%(w/v) formalin in PBS and analyzed by a FACSCalibur (BD, Heidelberg, Germany) flow cytometer and the CellQuest™ Pro software (BD).
The ribonuclease inhibitors RNasin® and SUPERase· In™ were purchased from Promega (Mannheim, Germany) and Ambion (Huntingdon, UK). These inhibitors were added to ribonuclease activity assays before the addition of ribonucleases.
Diversity of extracellular ribonucleases
The ribonuclease activity on the skin surface is independent of the 5'-cap
The ribonuclease activity on the skin surface is not dominantly of the exonuclease type
The ribonuclease activity at the skin surface is pyrimidine-specific
2' modified mRNA have no increased resistance towards extracellular ribonucleases
Alternatively to 2' modified nucleotides, sulfur substitutions at the phosphate group (R1, Fig. 5A) of pyrimidines might enhance mRNA stability. However, we obtained similar results as for 2' modified nucleotides (data not shown): poor transcription and no enhanced stability towards ribonucleases when using one or a combination of phosphorothioate nucleotide triphosphates.
RNasin®but not SUPERase· In™ protects mRNA from ribonuclease activity of skin surfaces
Towards the characterization of the concerted extracellular ribonuclease activity (i.e. secreted or originating from dead cells), we first evaluated the number of proteins with different sizes capable of ribonuclease activity on the skin surface or in blood plasma (Fig. 1). Zymograms indicated that the skin surface contains one dominant ribonuclease activity mediated by a ~13 kDa protein. In humans, several sub-dominant ribonuclease activities are performed by 7 larger and one smaller protein. The ribonuclease activity of blood plasma is dominantly mediated by a protein of ~12 kDa in mice and ~26 kDa in humans. Further characterization of the ribonuclease activities on the skin surface indicated that they are not dominantly of a specific exonuclease type (5'-exo or 3'-exo, Fig. 3), are not impaired when the substrate contains a 5'-cap structure (Fig. 2), are specific for pyrimidines (Fig. 4) and can be efficiently inhibited by RNasin® but not SUPERase· In™ (Fig. 6).
Moreover, using homopolymers as substrates, we found in all cases (mouse and human, skin surface and blood plasma) that the extracellular ribonucleases are specific for pyrimidines and that C is their preferred substrate (except for ribonucleases contained in mouse blood plasma where U is preferred, Fig. 4). This result has a great impact on the development of RNA-based drugs. Since a similar specificity was observed for the major ribonuclease extracted from mammalian's epidermis [38, 39] we anticipate that the utilization of C-low RNA may be a method to increase the efficacy of RNA-therapies delivered transcutaneously, intradermally or subcutaneously.
We investigated whether the preference of extracellular ribonucleases for pyrimidines was exploited by viruses: a low U and C content in their transcriptome would be an advantage for their mRNA half life (especially when the genome is a RNA molecule). Comparing the mean (± standard deviation) C content of human mRNA (26, 5 ± 4, 3%) to the mean C content of RNA viruses (25, 1 ± 7, 3% for retro, 23, 1 ± 5, 4% for plus ssRNA and 19, 6 ± 2, 0% for minus ssRNA viruses) we cannot detect a clear tendency for a lower C content in RNA viruses. Thus, viruses do not appear to have evolved in order to resist extracellular ribonucleases.
In the context of mRNA-based therapies, a possible method to protect the nucleic acid against degradation by extracellular ribonucleases would be to modify pyrimidines, rendering them resistant to ribonucleases. Unfortunately, in our reaction conditions, most available UTP or CTP with a 2' modification were no substrates for in vitro polymerization with T7 or SP6 polymerase. 2'-fluoro substitutions were shown to be compatible with in vitro polymerization  but we failed to produce long mRNA containing modified U and modified C together. A single 2'-modified nucleotide (U or C) could not stabilize the mRNA sufficiently to resist extracellular ribonucleases while it abrogated translation in vivo (in transfected cells, Fig. 5). Thus, the available 2'-modified pyrimidines do not allow the generation of functional mRNA resistant to extracellular ribonucleases. Moreover (data not shown), neither the use of phosphorothioate modified cytidine  (sulfur for oxygen substitution at the phosphate residue, position R1 in Fig. 5A) nor the addition of poly(C) to the ribonuclease mixture (as a competitor for ribonuclease activity) did improve mRNA stability.
In contrast, the natural ribonuclease inhibitor RNasin® was efficient in preventing the degradation of mRNA by extracellular ribonucleases (Fig. 6). RNasin® was also more effective than SUPERase· In™ for the inhibition of the ribonucleases present on the skin surface. This result was unexpected since SUPERase· In™ has a larger reported spectrum of ribonuclease inhibition compared to RNasin®. Indeed, SUPERase· In™ may be more efficient than RNasin to inhibit ribonucleases in other applications. In the case of RNA protection against skin surface ribonucleases, RNasin® might have some unknown relevant ribonuclease-specificity.
Our data suggest that mRNA used for therapies as an injected drug should be delivered together with RNasin®. RNasin® being a human self protein, is not expected to have side effects: It should be catabolized naturally in a relatively short time, it should be not toxic for cells and, because it is a conserved self protein that is expressed in several organs , it should not trigger an immune response.
Although our studies document the activities of extracellular ribonucleases present on the skin, they do not provide an explanation for the role of such molecules. Some of the extracellular ribonucleases may originate from the cytosol of dead keratinocytes that constitute the skin surface. This seems to be unlikely since intracellular ribonucleases are mainly of the exonuclease type  and we could demonstrate that this is not the case for extracellular ribonucleases (Fig. 3). Besides, the characterization at the DNA level of genes coding for secreted (defined by the presence of a leader sequence) ribonucleases demonstrates that there must be a need in higher organisms for such activities at their surface. All three hypothesized roles of these ribonucleases on the skin (protection against foreign pathogens like RNA-viruses, prevention of the activation of the immune system by RNA released from dead cells or inhibition of cell-to-cell interactions through release-capture of RNA by neighboring cells) are not mutually exclusive. A role for RNA in cell-to-cell communication mediated by secretion and recapture of RNA by neighboring cells was originally suggested by Benner. In line with this hypothesis we observed a lower content of ribonuclease activity in fast dividing tissues like tumors (data not shown and ).
Further studies are required to prove whether extracellular ribonucleases play indeed a role in the control of cell growth.
RNases present at the skin surfaces recognize pyrimidines and are not inhibited by a 5'cap structure. As far as enzymatically produced messenger RNA are concerned, the replacement of natural nucleotides by chemically substituted ones is limited by the poor utilization of such analogs by RNA polymerases. Moreover, chemical modifications did not decrease RNase-sensitivity and they impaired translation. For protecting exogenous mRNA from RNases and keeping an efficient mRNA translation, we found that the best method is to mix non-modified, natural mRNA together with the protein RNAsin®. This is a simple method that can protect the extracellular therapeutic mRNA. Particularly in the context of mRNA-based vaccination, such a trans-protection of the mRNA thanks to additional RNAsin® can be foreseen as safe method to improve the mRNA'as half life, thus its penetration in cells and thereby the efficacy of the vaccine.
J.P. was supported by the "Deutsche Forschungsgemeinschaft, DFG" (graduate college 685) and J-P.C. by a "Fortüne" grant from the University of Tübingen. BS and SP are supported by the Fritz Bender Stiftung.
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