SUBSTITUTION OF THE MESSENGER RNA CAP WITH TWO RNA SEQUENCES INTRODUCED AT THE 5-PRIME END THEREOF
20210214729 ยท 2021-07-15
Inventors
Cpc classification
C12N7/00
CHEMISTRY; METALLURGY
C12N15/67
CHEMISTRY; METALLURGY
C12P19/34
CHEMISTRY; METALLURGY
C12N2770/32222
CHEMISTRY; METALLURGY
C12N15/115
CHEMISTRY; METALLURGY
C12P21/02
CHEMISTRY; METALLURGY
International classification
C12N15/115
CHEMISTRY; METALLURGY
Abstract
The invention relates to a messenger ribonucleic acid (mRNA) molecule lacking a cap molecule, for which the cost of in vitro transcription synthesis is greatly reduced, comprising, from 5 to 3, at least one copy of a GUCAGRYC(N.sub.7-19)GCCA(N.sub.12-19)UGCNRYCUG consensus sequence which is resistant to the Xrn1 exoribonuclease, a copy of an internal ribosome entry site (IRES) RNA sequence, and an open reading frame.
Claims
1. A messenger ribonucleic acid (mRNA) molecule lacking a cap molecule comprising from 5 to 3: a 5-UTR region comprising at least one copy of a consensus sequence GUCAGRYC(N.sub.7-19)GCCA(N.sub.12-19)UGCNRYCUG (xrRNA); a copy of an internal ribosome entry site (IRES) RNA sequence; and at least one open reading frame.
2. The mRNA molecule according to claim 1, comprising two copies of xrRNA.
3. The mRNA molecule according to claim 1, comprising at least one sequence having the SEQ ID NO of any one of SEQ ID NO: 1 to 44.
4. The mRNA molecule according to claim 1, comprising the IRES sequence of encephalomyocarditis virus.
5. The mRNA molecule according to claim 1, comprising a stem-loop, wherein the stem-loop is located at the 5 end of the 5-UTR region.
6. The mRNA molecule according to claim 1, comprising at least one aptamer selected from aptamer A having the sequence of SEQ ID NO: 64 and aptamer C having the sequence of SEQ ID NO: 66.
7. The mRNA molecule comprising aptamer A according to claim 6, wherein the mRNA molecule is bound to a cell-penetrating peptide (CPP) fused to a poly-histidine tag.
8. The mRNA molecule according claim 1, wherein an open reading frame codes for the 2Apro protein of the HRV2 virus.
9. A deoxyribonucleic acid (DNA) molecule comprising a sequence coding the mRNA according to claim 1.
10. A vector comprising the mRNA molecule according to claim 1.
11. An in vitro method for producing at least one mRNA comprising contacting the DNA molecule according to claim 9 with at least one RNA polymerase.
12. The method according to claim 11, comprising a step of purifying the mRNA.
13. A pharmaceutically acceptable composition comprising the mRNA according to claim 1 and a physiologically acceptable excipient and/or adjuvant.
14. A method of enhancing or inducing an immune response to a polypeptide in a subject comprising administering the composition according to claim 13.
15. The composition according to claim 13, comprising a second mRNA molecule lacking a cap molecule comprising from 5 to 3: a 5-UTR region comprising at least one copy of a consensus sequence GUCAGRYC(N.sub.7-19)GCCA(N.sub.12-19)UGCNRYCUG (xrRNA); a copy of an internal ribosome entry site (IRES) RNA sequence; and at least one open reading frame, wherein in the second mRNA molecule, an open reading frame codes the 2Apro protein.
16. The mRNA molecule according to claim 3, comprising SEQ ID NO: 11 and SEQ ID NO: 26.
17. The mRNA molecule according to claim 5, wherein the stem-loop has the sequence of SEQ ID NO: 87.
18. The mRNA molecule comprising aptamer A according to claim 7, wherein the CPP is selected from M12-H6 having the sequence of SEQ ID NO: 75, CPP1-H6 having the sequence of SEQ ID NO: 76, CPP2-H6 having the sequence of SEQ ID NO: 77, and CPP3-H6 having the sequence of SEQ ID NO: 78.
19. The DNA molecule according to claim 9, comprising a promoter recognized by the T7 RNA polymerase, the promoter comprising a sequence represented by SEQ ID NO: 46; a 5-UTR region comprising a sequence represented by SEQ ID NO: 50, 71, 72, 73, 85, or 86; an open reading frame; and a 3-UTR region comprising a sequence selected from the sequences represented by SEQ ID NOs: 53, 54, 55, and 56.
20. A vector comprising the DNA molecule according to claim 9.
Description
FIGURES
[0173]
[0174] (A) The capped MB5-luc2 mRNA corresponds to a traditional messenger RNA coding luciferase, with a cap analog at its 5 end and a 3-UTR region comprising a poly(A) tail. (B) The MB5-luc2 mRNA is identical to (A) except that it lacks a cap analog. (C) The MB7-luc2 mRNA lacks a cap and has a stem-loop and the internal ribosome entry site (IRES) of the EMCV virus at its 5 end. (D) The MB8-luc2 mRNA lacks a cap and has both a stem-loop, two Xrn1 resistant sequences (xrRNA1 and xrRNA2) from West Nile virus (WNV) and the IRES from the EMCV virus in the 5 UTR region. (E) The MB9-luc2 mRNA is similar to (D), except that the 3-UTR and poly(A) of the latter have been replaced by the 3-UTR from the Kunjin virus (KUN). (F) The uncapped MB11-luc2 mRNA is similar to (D), except that aptamer A has been added in the 5 region, upstream of the two Xrn1 resistant sequences (xrRNA1 and xrRNA2) from West Nile virus (WNV) and the IRES of the EMCV virus and that it does not have a stem-loop. The MB13-luc2 (G) and MB14-luc2 (H) mRNAs have this same configuration, but comprise a 5 stem-loop followed by the RNA aptamers B and C, respectively. (I) The capped MB15-luc2 mRNA is similar to (A), except that the aptamer A has been added in the 5 region after the cap. (J) The MB17-luc2 mRNA is similar to (D), except the aptamer A has been added 5 between the stem-loop and the xrRNA sequences. (K) The MB18-luc2 mRNA is similar to (J), except that the A aptamer is placed in the 5 region downstream of the xrRNA sequences and upstream of the IRES sequence.
[0175]
[0176] Confluent Caco-2 cells were transfected with the MB5-luc2 capped (diamond) and uncapped (circle) mRNAs, as well as the MB7-luc2 (triangle) and MB8-luc2 (square) mRNAs. Expression kinetics were followed for four days.
[0177]
[0178] Confluent Caco-2 cells were transfected with the capped MB5-luc2 (diamond), MB8-luc2 (square) and MB9-luc2 (triangle) mRNAs. Expression kinetics were followed for ten days.
[0179]
[0180] Mesenchymal stem cells were transfected with 2 g of MB8-luc2 mRNA complexed with pepMB1 peptide at a ratio of pepMB1 peptide positive charge:mRNA negative charge of about 2.2:1 per well in 48 well plates for 1 hour. Luciferase activity was then monitored for approximately 48 days.
[0181]
[0182] The muscle and dermis of male BALB/cByJ mice were transfected with 10 g and 5 g, respectively, of uncapped MB8-luc2 mRNA or capped MB5-luc2 mRNA. Luciferase activity was measured in muscle and skin 16 hours and 18 hours, respectively, after the injections.
[0183]
[0184] C2C12 cells were transfected with MB8-luc2 mRNA alone or MB8-luc2 mRNA mixed with MB8-2Apro mRNA at a ratio of 465:1 or 9:1, in order to assess the cytotoxic effect of 2Apro protein expression. Cytotoxicity was determined by measuring lactate dehydrogenase (LDH) released into the extracellular medium. LDH activity is determined by measuring the absorbance at 490 nm. Negative control: Untransfected cells or cells transfected with MB8-luc2 mRNA alone without lysis. Positive control: Untransfected cells lysed with Triton X-100.
[0185]
[0186] C2C12 cells were transfected with MB8-luc2 mRNA in the presence of an increasing amount of MB8-2Apro mRNA. The molar ratio of MB8-luc2 mRNA:MB8-2Apro mRNA is from 540:1 to 240:1. The total amount of transfected mRNA was 750 ng per well. Luciferase activity was measured 18 hours after transfection.
[0187]
[0188] Confluent C2C12 cells were transfected with MB8-luc2 mRNA alone (black line) or in combination with MB8-2Apro mRNA (gray dotted line). The molar ratio of MB8-luc2 mRNA:MB8-2Apro mRNA is 465:1. Expression kinetics, measured by the level of luciferase activity, were followed for seven days.
[0189]
[0190] The effect of the 2Apro protein on luciferase expression from different messenger RNAs was evaluated. In each case, the mRNA coding luciferase was transfected alone () or co-transfected with a second mRNA coding the 2Apro protein (+) and having the same characteristics at the optimal molar ratio of 465:1. (1) MB8-luc2 mRNA alone; (2) co-transfection of MB8-luc2 mRNA with MB8-2Apro mRNA; (3) Capped MB5-luc2 mRNA alone; (4) co-transfection of capped MB5-luc2 mRNA with uncapped MB8-2Apro mRNA; (5) MB5-luc2 mRNA lacking a cap analog alone; (6) co-transfection of MB5-luc2 mRNA lacking a cap analog with MB8-2Apro mRNA lacking a cap analog; (7) MB7-luc2 mRNA alone; (8) co-transfection of MB7-luc2 mRNA with MB8-2Apro mRNA. Luciferase activity was measured 18 hours after transfection.
[0191]
[0192] Muscle of male BALB/cByJ mice was transfected with: 5 g of MB8-luc2 mRNA, MB13-luc2 mRNA, MB14-luc2 mRNA, MB11-luc2 mRNA complexed with M12-H6 peptide, or of MB11-luc2 mRNA complexed with the M12-H6 peptide in the presence of 5 g of chloroquine. Luciferase activity was measured 16 hours after the injections.
[0193]
[0194] The dermis of male OF1 mice was transfected with 5.6 g of MB11-luc2 mRNA complexed with the CPP3-H6 peptide at increasing molar ratios (molar ratios between 1:8 and 1125:1 of CPP3-H6:MB11-luc2 mRNA). Luciferase activity was measured 18 hours after the injections. The molar ratio allowing for optimal transfection was 1 CPP3-H6:4 MB11-luc2 mRNA.
[0195]
[0196] The dermis of male OF1 mice was transfected with 5.6 g of MB11-luc2 mRNA complexed with the CPP1-H6 peptide at increasing molar ratios (molar ratios between 1:8 and 3:1 of CPP1-H6:MB11-luc2 mRNA). Luciferase activity was measured 18 hours after the injections. The molar ratio allowing for optimal transfection was 1 CPP1-H6:4 MB11-luc2 mRNA.
[0197]
[0198] The dermis of male OF1 mice was transfected with 5.6 g of MB11-luc2 mRNA complexed with the CPP2-H6 peptide at increasing molar ratios (molar ratios between 1:4 and 2.75:1 of CPP2-H6:MB11-luc2 mRNA). Luciferase activity was measured 18 hours after the injections. The molar ratio allowing for optimal transfection was 2 CPP2-H6:1 MB11-luc2 mRNA.
[0199]
[0200] RNA aptamer A was inserted in the 5-UTR of the conventional capped MB5-luc2 mRNA to generate the capped MB15-luc2 mRNA. The dermis of male OF1 mice was transfected with 5.6 g of capped MB15-luc2 mRNA complexed with the CPP2-H6 peptide at a molar ratio of 2 CPP2-H6:1 MB15-luc2 mRNA or not, in view of the results obtained previously (see
[0201]
[0202] The effect of a stem-loop (here 5-SL having the sequence of SEQ ID NO: 87) on transfection efficiency when the stem-loop is followed by the xrRNA1 sequence, 5 nucleotides downstream (MB8-luc2 and MB18-luc2 mRNA), as compared to an mRNA lacking a stem-loop (MB11-luc2 mRNA) and an mRNA whose stem-loop is located more than 70 nucleotides upstream of xrRNA1 (MB17-luc2 mRNA). The conventional MB5-luc2 capped mRNA serves as a control for the efficacy of 5-SL followed by two xrRNA sequences. The dermis of male OF1 mice was transfected with 5.6 g of each of the different mRNAs and luciferase activity was measured 18 hours after the injections. Aptamer A of MB11-luc2 is not linked to a peptide, and therefore has no effect on the transfection efficiency of this mRNA.
EXAMPLES
[0203] The invention is illustrated by the following non-limiting examples. These teachings include alternatives, modifications and equivalents, as will be appreciated by one skilled in the art.
Example 1: Plasmid Construction
[0204] DNA sequences corresponding to 5 non-coding sequences of SEQ ID NO: 49, SEQ ID NO: 50, or SEQ ID NO: 51 were chemically synthesized, integrated into a DNA vector and sequenced by ProteoGenix. The DNA fragments were excised by restriction enzymes. Plasmids of the pMBx-luc2 series were digested with the same restriction enzymes and the DNA fragments were integrated into these plasmids by the action of T4 DNA ligase. These plasmids contain the gene coding the luciferase enzyme (SEQ ID NO: 62), inserted downstream of the bacteriophage T7 promoter. A short non-coding 3-UTR sequence followed by a transcriptional polyadenylation sequence, according to SEQ ID NO: 53, is located downstream of the luciferase gene. The different non-coding 5-UTR sequences separate the promoter from the luciferase gene. The plasmids thus constructed were amplified, verified and linearized downstream of the transcriptional polyadenylation sequence by a restriction enzyme.
Example 2: Plasmid Linearization and In Vitro Transcription of mRNAs
[0205] An Ssp1 restriction site is located immediately downstream of the poly(A) of each plasmid (cf. SEQ ID NO: 54). Ten micrograms of plasmid were digested with twenty units of the
[0206] Ssp1-HF restriction enzyme (New England Biolabs) in 1 CutSmart buffer for four hours at 37 C.
[0207] Eight l of plasmid linearized by Ssp1-HF were then mixed with 2 l of 10T7 RNA polymerase reaction buffer, 2 l each of the four nucleotide triphosphates (ATP, GTP, CTP and UTP) and 2 l of T7 RNA polymerase solution (New England Biolabs). A cap analog was included in this reaction mixture for the synthesis of capped mRNAs. It was omitted for the synthesis of uncapped mRNAs.
[0208] Transcription took three to ten hours and was performed in a 37 C. block heater. Then, 1 L of TURBO DNase (Thermo Fisher) was added to degrade the plasmid and the mixture was incubated at 37 C. for 15 minutes.
[0209] MB5-luc2 mRNA is a conventional mRNA coding luciferase. It has a 5 non-coding sequence (UTR) according to SEQ ID NO: 57, which has been selected for optimal initiation of translation as well as a 3-UTR region comprising a poly(A) tail according to SEQ ID NO: 60. This mRNA was synthesized with or without a cap (cf.
[0210] Uncapped MB7-luc2 mRNA has at its 5 end, in place of the 5-UTR of the MB5-luc2 mRNA, a stem-loop at the 5 end, followed by the IRES sequence of the EMCV virus. The latter would thus allow it to efficiently recruit ribosomes, despite the absence of a cap. However, this mRNA would be sensitive to the Xrn1 enzyme (cf.
[0211] Uncapped MB8-luc2 mRNA has a stem-loop at its 5 end, followed, in the 5-UTR region, by two successive sequences resistant to Xrn1, derived from the 3-UTR of Flavivirus WNV, called xrRNA1 and xrRNA2 (Kieft et al., 2015). These sequences are followed by that of the IRES of the EMCV virus, which is thus protected by the two sequences resistant to Xrn1 (cf.
[0212] Uncapped MB9-luc2 mRNA differs from uncapped MB8-luc2 mRNA only by its 3 end. Indeed, the 3-UTR and the poly(A) sequence of MB8-luc2 have been replaced by the 3-UTR region of the Kunjin virus. This region does not have a poly(A) sequence (cf.
[0213] Uncapped MB11-luc2 mRNA differs from uncapped MB8-luc2 mRNA only by the absence of a stem-loop at the 5 end, and the presence of an aptamer in the 5 region upstream of the two successive sequences resistant to Xrn1 (cf.
[0214] Uncapped MB13-luc2 and MB14-luc2 mRNAs differ from uncapped MB8-luc2 mRNA only by the presence of an aptamer in the 5 region upstream of the two successive sequences resistant to Xrn1 (cf.
[0215] MB15-luc2 mRNA corresponds to the capped MB5-luc2 mRNA, in which the A aptamer (SEQ ID NO: 64) has been inserted into the 5-UTR region (cf.
[0216] MB17-luc2 mRNA differs from uncapped MB11-luc2 mRNA only by the presence of a stem-loop at the 5 end upstream of aptamer A (cf.
[0217] Uncapped MB18-luc2 mRNA differs from uncapped MB8-luc2 mRNA only by the presence of an aptamer in the 5 region between the two successive sequences resistant to Xrn1 and the IRES sequence (cf.
Example 3: Purification of Messenger RNAs
[0218] Purification of the different luciferase mRNAs was carried out using the MegaClear kit (Ambion). Seventy-nine l of Elution Solution, 350 l of Binding Solution Concentrate and 250 l of 100% ethanol were added to the 21 l of the previous mixture. These 700 l were placed on a Filter Cartridge and centrifuged at 10,000g for one minute. The filter retained the messenger RNA. Two washes were performed with 500 l of Wash Solution, centrifuging at 10,000g, for one minute. RNA was then eluted from the filter by adding 50 l of Elution Solution twice and heating to 70 C. for ten minutes in a block heater. Elution was obtained by centrifugation at 10,000g for one minute.
[0219] A second purification step was performed by precipitation with lithium chloride. Sixty l of LiCl Precipitation Solution was added to the 100 l of eluate. The mixture was cooled at 20 C. for one hour, before being centrifuged at maximum speed at four degrees, for 15 minutes. The pellet was washed with 500 l of 70% ethanol and a final centrifugation was performed at maximum speed at four degrees, for 5 minutes. The messenger RNA pellet, air dried for a few minutes, was resuspended in sterile deionized water. The concentration of the mRNA solution was determined by measuring the absorbance at 260 nm, using a spectrophotometer.
Example 4: Assembly of Messenger RNA/pepMB1 Complexes
[0220] The cationic peptide pepMB1 was synthesized, purified and lyophilized by ProteoGenix. Its amino acid sequence is as follows: CRRRRRRRRC. The lyophilizate was resuspended in sterile deionized water.
[0221] Five g of luciferase mRNA was mixed with 5 g of pepMB1 at a final RNA concentration of 20 g/ml. Mixtures were incubated at room temperature (20-25 C.) for 15 minutes before being frozen at 80 C. mRNA/pepMB1 complexes were then lyophilized for about 20 hours.
Example 5: Transfection of Caco-2 or C2C12 Cells
Materials and Methods
a) Culture and Seeding of Cells of the Caco-2 or C2C12 Line
[0222] All cell manipulations were performed under a laminar flow hood. The Caco-2 cell line (ECACC) was cultured in DMEM (Gibco) supplemented with non-essential amino acids, a mixture of antibiotics and an antimycotic, and fetal calf serum (15% final). Culture was performed at 37 C. in 75 cm.sup.2 flasks (Corning).
[0223] When the number of cells needed to seed a 48-well plate (Corning) was reached, the cells were detached from the bottom of the flask using 3 ml of TryPLE Select 1 (Gibco) at 37 C., for 5 minutes. 7 ml of DMEM was added to neutralize the TryPLE Select 1. The cells were centrifuged at 100g, for 10 minutes at room temperature. The cell pellet was then resuspended in 10 ml of culture medium. 250 l of this cell suspension was introduced into each well of a 48-well plate and the latter was placed in an incubator at 37 C. containing 5% CO.sub.2.
[0224] C2C12 cells can be kept at confluence in the wells of a 48-well plate for about a dozen days after seeding. Beyond this period, these cells differentiate into intestinal epithelium, which affects mRNA translation. In contrast, human mesenchymal stem cells can be stored in confluent culture for more than 7 weeks. Mesenchymal stem cells (Millipore, Human Mesenchymal Stem Cell (Bone Marrow)) were thus cultured in 48-well plates (Corning) in a ready-to-use medium (Millipore, Mesenchymal Stem Cell Expansion Medium) for up to 48 days. For cells that will be lysed more than five days after transfection, the culture medium was changed three times per week.
b) Transfection of Caco-2 or C2C12 Cells
[0225] For Caco-2 cells, transfection with each mRNA was performed in five different wells. Lyophilizates of mRNA/pepMB1 complexes (Proteogenix) were resuspended in 750 l of transfection buffer (20 mM Hepes, 40 mM KCl and 100 mM trifluoroacetate).
[0226] For C2C12 cells, transfection with MB8 mRNA is carried out as indicated below. MB8-luc2 mRNA/pepMB1 complexes (Proteogenix) are assembled by incubating the mRNA in presence of the peptides at a ratio of positively-charged peptide:negatively charged mRNA of approximately 2.2 for 30 min at room temperature. The solution is then diluted with 3DMEM to obtain a final DMEM at 1.
[0227] In both cases, wells were emptied of the culture medium they contained in order to introduce 150 l of RNA/pepMB1 complex solution (corresponding to 1 g of mRNA per well for the Caco-2 cells and to 2 g per well for C2C12 cells). Cells were incubated for 30 minutes (Caco-2 cells) or 1 hour (C2C12 cells) at 37 C. in a CO.sub.2 incubator. The mRNA/pepMB1 complex solution was then aspirated and replaced with 250 l of culture medium. Cells were then incubated, for 6 hours to 48 days depending on the cell type, at 37 C. in a CO.sub.2 incubator.
c) Lysis of Caco-2 or C2C12 Cells and Measurement of Luciferase Activity
[0228] Six hours to 48 days after transfection, cells were lysed in order to conduct expression kinetics of the luciferase protein. Culture medium was aspirated and replaced with 250 l of lysis buffer (Luciferase Assay System, Promega). 20 l of each cell lysate was placed in a tube suitable for the luminometer (Berthold Technologies). 100 l of luciferase substrate (Promega) was added to the cell lysate by the luminometer. The latter then measured the amount of light emitted by the enzymatic reaction catalyzed by luciferase. Results are expressed in relative light units (RLU). The amount of luciferase protein produced by Caco-2 cells or C2C12 cells, by means of the luciferase mRNA, was normalized by assaying for total cell proteins with the 660 nm Protein Assay kit (Pierce). For this, 100 l of cell lysate was mixed with 1.5 ml of reagent and absorbance was measured at 660 nm. A calibration range was performed using solutions of bovine serum albumin. Luciferase activity is therefore expressed in RLU per milligram of protein.
Results
[0229] Results are illustrated in
[0230] Uncapped MB7-luc2 mRNA gives stronger and more durable expression of the luciferase protein in Caco-2 cells than uncapped MB5-luc2 mRNA. The IRES region recruits ribosomes, but does not provide significant resistance to Xrn1 (cf.
[0231] Uncapped MB8-luc2 mRNA induces luciferase expression kinetics similar to that obtained with capped MB5-luc2 mRNA (cf.
[0232] MB9-luc2 mRNA differs from MB8-luc2 mRNA by its 3-UTR end which lacks poly(A). The luciferase expression it induces in Caco-2 cells is notably lower and less durable than that induced by MB8-luc2 mRNA (cf.
[0233] In human mesenchymal stem cells, MB8-luc2 mRNA surprisingly and advantageously induces luciferase expression which persists very long term. Indeed, even if the expression decreases over time, it remains detectable even 48 days after transfection (cf.
Example 6: Transfection of Mouse Muscle and Dermis
Materials and Methods
a) Animal Housing
[0234] For muscle, 8-week-old male BALB/cByJ mice, were housed in open cages with five animals per cage. The day/night cycles were managed by an automatic device (12 h day/12 h night). They were fed and had access to filtered water ad libitum. For skin, 6-week-old male OF1 mice, were housed in open cages with four animals per cage.
b) Extemporaneous Preparation of mRNA Samples
[0235] For each intramuscular injection, 100 l of a solution of naked mRNA containing 230 mM NaCl was prepared.
[0236] For each intradermal injection, 17 l of a solution of naked mRNA containing 160 mM NaCl was prepared.
[0237] When a CPP-H6 peptide was used, this was mixed with mRNA and incubated for 30 minutes at room temperature in presence of 5 mM Hepes pH 7.5 and 0.7 mM MgCl.sub.2.
c) Intradermal and Intramuscular Injections of mRNA
[0238] Anesthesia of mice was achieved by the use of isoflurane. For intramuscular injections, analgesia was given by injection of buprenorphine. For intradermal injections, the skin on the back was shaved three to four days earlier (see Example 10 for details).
[0239] 100 l and 17 l of mRNA solution was injected into the biceps femoris and skin, respectively. The animals were returned to their cages until the next morning.
d) Skin and Muscle Samples
[0240] For muscle, mice were euthanized with CO.sub.2 16 hours after the injections. For skin, mice were anesthetized with isoflurane and euthanized by cervical dislocation 18 hours after the injections. The skin and muscle injection sites were harvested. These biopsies were washed with physiological saline, cut into fine pieces and placed in tubes containing lysis buffer (Promega). These tubes were immediately frozen in liquid nitrogen.
e) Lysis of Cells From Skin and Muscle Biopsies
[0241] Each skin and muscle biopsy underwent three freeze/thaw cycles. Indeed, tubes containing the biopsies and lysis buffer were frozen at 80 C. for 10 minutes. Then, they were thawed in a water bath at room temperature for two minutes and mixed briefly using a vortex. Tubes were then centrifuged at 5000g at 20 C. for 5 minutes in order to precipitate tissue debris and obtain a clarified supernatant of cell lysate.
f) Measurement of Luciferase Activity
[0242] 20 l of each cell lysate was used for measurement of luciferase expression in each biopsy. A tube luminometer added 100 l of luciferase substrate (Promega) to each sample and measured the amount of light emitted for 10 seconds. Results are expressed in relative light units or RLUs.
[0243] Cell lysates were then diluted 8 to 20-fold for protein assay, using the 660 nm Protein Assay kit (Pierce). 100 l of diluted cell lysate was mixed with 1.5 ml of reagent for 6 minutes and the absorbance was measured at 660 nm. A calibration range was performed with bovine serum albumin from 0 to 750 g/ml.
Results
[0244] Results are shown in
[0245] The MB8-luc2 mRNA can therefore fully replace the capped MB5-luc2 mRNA, and would even be more advantageous.
Example 7: Cellular Toxicity of the MB8-2Apro mRNA
Materials and Methods
[0246] Expression of the 2Apro protein in a mammalian cell can induce toxicity to the point of causing cell death by apoptosis or necrosis (Goldstaub et al., 2000). During these processes, cells release lactate dehydrogenase (LDH) into the extracellular environment. This LDH activity can be measured using a commercial kit, CytoTox96 Non-Radioactive Cytotoxicity Assay, Promega.
[0247] The MB8-2Apro mRNA (SEQ ID NO: 80) is an mRNA coding the 2A nonstructural protein (2Apro, having the sequence of SEQ ID NO: 81) from the genome of human rhinovirus 2 (HRV2). It has a 5 non-coding sequence (UTR) according to SEQ ID NO: 57, which is identical to that of the MB8-luc2 mRNA, as well as a 3-UTR region comprising a poly(A) tail according to SEQ ID NO: 60.
[0248] 750 ng of MB8-luc2 mRNA alone or of a mixture of MB8-luc2 mRNA and MB8-2Apro mRNA were complexed with the pepMB1 peptide, as described in Example 4.
[0249] MB8-luc2 mRNA alone or MB8-luc2 mRNA mixed with MB8-2Apro mRNA, at two different molar ratios, were transfected into C2C12 cells. Specifically, C2C12 cells from a well of a 48-well plate were incubated for one hour with the mRNA/pepMB1 complexes. 18 hours later, luciferase activity was measured (
Results
[0250] mRNA mixtures did not induce cytotoxicity, even at the highest molar ratio (MB8-luc2 mRNA:MB8-2Apro mRNA ratio of 9:1). This indicates that the expression of the viral protease does not induce cytotoxicity (
Example 8: Luciferase Expression Kinetics Optimized by Co-Transfection of MB8-luc2 and MB8-2Apro mRNA
Materials and Methods
[0251] In the absence of toxicity, the MB8-luc2 and MB8-2Apro mRNAs were next co-transfected into C2C12 cells at different ratios, according to the methods described above. 18 hours later, luciferase activity was measured (
Results
[0252] Surprisingly, co-transfection of MB8-luc2 and MB8-2Apro mRNAs increased luciferase expression of MB8-luc2 mRNA by at least 2.5-fold in C2C12 cells at all ratios tested. The best MB8-luc2 mRNA:MB8-2Apro mRNA molar ratio is 465:1, and increases luciferase expression by 3.4-fold.
[0253] Kinetics conducted from 6 hours to 7 days post-transfection surprisingly demonstrate that improvement in luciferase expression is observed for at least a week. Indeed, luciferase expression is on average increased by 2.4-fold (
Example 9: Effect of RNA Aptamers on the Efficiency of mRNA Transfection in Muscle
[0254] In order to improve the internalization of the mRNA molecule according to the invention, different aptamers have been incorporated into the RNA molecule, as detailed below. The effect of these aptamers was then evaluated in vivo, to determine if an improvement in luciferase expression could be observed.
Materials and Methods
Aptamer Selection
[0255] Aptamers penetrating C2C12 cells were selected. First, double-stranded DNA was generated by hybridizing a 5 primer followed by extension of a single-stranded DNA from a single-stranded DNA library. Double-stranded DNA thus obtained was then precipitated and purified according to methods well-known to the person skilled in the art.
[0256] An RNA aptamer library was then obtained by transcription of the purified fragments using the T7 DuraScribe transcription kit (20 l/run) followed by RNA purification using a ssDNA and RNA purification kit. Finally, the solution is treated with DNAsel to remove the contaminating DNA. Aptamers are dissolved in 1DMEM+ITS at 8 M RNA (1288 g/5 ml). To select the aptamers, 5 ml of the DMEM/ITS/RNA solution is added to cells previously washed twice with DMEM without antibiotics or serum. Cells are incubated at 37 C. for 1 hour, with the flask containing the mixture being briefly shaken every 15 minutes. The flask containing the cells is then placed on ice and cells are washed 5 times with 15 mL of cold 1PBS to remove the RNA aptamers which have not penetrated cells. Cells are then lysed with TRIzol (Invitrogen) and total RNA extracted by the phenol-chloroform method.
[0257] Endogenous RNAs are digested with RNAse A and remaining RNAs hybridized with 3 primers and reverse transcribed by the Superscript III enzyme (ThermoFisher) before PCR amplification in the presence of 5 primer (100 M), 3 primer (100 M), Q5 High Fidelity DNA polymerase (NEB) and the Q5 High-Fidelity Master Mix buffer at a 1 concentration. All of these steps allowing for the obtention of an aptamer RNA library are repeated. Thus, two cycles of selection of RNA aptamers penetrating C2C12 cells are performed.
[0258] Two RNA aptamers (B and C) penetrating C2C12 cells were selected and sequenced (SEQ ID NO: 65 and 66, respectively). RNA aptamers B and C were then inserted into the 5-UTR of the MB8-luc2 mRNA, upstream of xrRNA1, to generate the MB13-luc2 and MB14-luc2 mRNAs, respectively.
Aptamer Which Strongly Binds the Poly-Histidine Peptide Motif
[0259] A second strategy aimed at improving mRNA internalization consisted of inserting another RNA aptamer (aptamer A) capable of strongly binding the poly-histidine peptide motif in the presence of magnesium into the 5-UTR of the MB8-luc2 mRNA, upstream of xrRNA1. The mRNA incorporating RNA aptamer A was named MB11-luc2. A mouse muscle fiber penetrating peptide, M12 (see Gao et al., 2014), was linked via a spacer (here comprising the glycine and serine amino acids) to the hexahistidine motif. Different spacers are illustrated by way of example in the sequences of
TABLE-US-00001 SEQIDNO:75 (RRQPPRSISSHPGGGGSGGGGSGGGGSGGGGSGGHHHHHH), SEQIDNO:76 (PQRDTVGGRTTPPSWGPAKAGGGGSGGGGSGGGGHHHHHH), SEQIDNO:77 (GPFHFYQFLFPPVGGGGSGGGGSGGGGSGGGGSGHHHHHH) SEQIDNO:78 (GSPWGLQHHPPRTGGGGSGGGGSGGGGSGGGGSGHHHHHH) (spacersequenceunderlined).
The peptide thus formed was named M12-H6 (SEQ ID NO: 75). The MB11-luc2 mRNA was incubated for 30 minutes at room temperature with the M12-H6 peptide, before being injected into the biceps femoris of mice.
In Vivo Transfection
[0260] The biceps femoris muscle of male BALB/cByJ mice was transfected with 5 g of MB8-luc2 mRNA, MB13-luc2 mRNA, MB14-luc2 mRNA, or MB11-luc2 mRNA complexed with M12-H6 peptide in the presence or absence of 5 g of chloroquine. Luciferase activity was measured 16 hours after the injections.
Results
[0261] MB13-luc2 mRNA transfected the muscle just as well as MB8-luc2 mRNA (
[0262] Transfection efficiency of MB11-luc2 mRNA was modest (
[0263] Without being bound by theory, one could suppose that the high transfection efficiency of MB11-luc2 RNA complexed with the M12-H6 peptide and in the presence of chloroquine was favored by a slowing of endosome acidification by chloroquine following the penetration of mRNA into cells. Chloroquine could slow hexahistidine protonation at acidic pH and therefore prevent destabilization of the complexes between MB11-luc2 mRNA and the M12-H6 peptide. As a result, mRNA escape from the endosome could be enhanced, by crossing the endosomal membrane with the help of M12-H6, to which it is still complexed, to enter the cytosol where mRNA is then translated.
Example 10: Intradermal Injection Protocol in Mice
aSolution Preparation
[0264] A 60 l solution containing 20 g of mRNA was prepared for each mouse. For this, demineralized water, 50 mM Hepes (1/8 of Hepes, 7/8 of sodium Hepes), NaCl (160 mM final), MgCl.sub.2, an mRNA, and, optionally, a peptide, were mixed. A 30-minute incubation at room temperature allowed the peptide to bind to the RNA. There was no incubation when mRNA was not complexed to a peptide. mRNA solutions were frozen at 80 C. for conservation until they were injected.
bIntradermal Injection and Biopsy Sampling
[0265] 6-week-old male OF1 mice were used (Charles River). They were shaved three to four days before intradermal injection. Anesthesia was performed using a mask. Induction of anesthesia was achieved with 4% isoflurane (Piramal Heathcare). Anesthesia was maintained at a percentage of isoflurane of 2%. Before injection, previously shaved skin of the back was cleaned with an alcohol wipe. mRNA solutions were slowly thawed at room temperature in order to fill 0.3mm8 mm U-100 (30 G) insulin syringes (Becton-Dickinson). Three injections of approximately 17 l of RNA solution were performed in the skin of the shaved back of each mouse. Papules formed before resolving. These were delimited using a permanent marker to allow the area of skin to be biopsied to be identified the next day. Tail tagging was also performed using a marker to distinguish between animals from the same cage.
[0266] 18 hours after injections, skin biopsies were taken from the injected areas. For this, the mice were anesthetized, then euthanized by cervical dislocation. Biopsies were cut into small pieces using a pair of scissors, to facilitate cell lysis, and introduced into tubes containing 500 l of 1 lysis buffer (5 Luciferase Cell Culture Lysis (Promega), diluted in water). Each tube was frozen at 20 C. until the next step.
c-Cell Lysis, Luciferase and Protein Assays
[0267] Lysis of the collected tissues was obtained by performing three freeze/thaw cycles: 80 C. for 10 minutes, 3 minutes in a water bath at room temperature and mixing using a vortex mixer for a few seconds. Tubes were then centrifuged at 5000g for 5 minutes at 20 C. to sediment tissue debris. Supernatant was transferred to another tube.
[0268] The luciferase assay was performed using the Luciferase Assay System kit (Promega). 20 l of each sample was introduced into dedicated tubes for the luminometer (Berthold AutoLumat Plus LB 953). The device injected 100 l of substrate and measured the amount of light emitted (RLU).
[0269] The protein assay was performed using the Pierce 660 nm Protein Assay Kit (Thermo scientific). A calibration range was performed with bovine serum albumin (Thermo scientific) and 1 lysis buffer as diluent. It covered a range of 100 to 500 g of protein per ml. The blank was obtained using 100 l of 1 lysis buffer. Lysates were in some cases diluted with 1 lysis buffer. 100 l of each sample was used for the protein assay. 1.5 ml of reagent was added to the blank and samples. After exactly 5 minutes of incubation at room temperature and in the dark, the absorbance of each sample was measured at 660 nm using a spectrophotometer.
Example 11: Effect of CPPs on the Efficiency of mRNA Transfection in the Skin
[0270] The strategy to improve internalization of naked mRNA by binding a cell penetrating peptide (CPP) to RNA aptamer A is not restricted to muscle, as described in Example 9 above. This strategy has been applied here to the skin of mice using different CPPs.
Materials and Methods
[0271] Three CPPs were used here: CPP1, CPP2 and CPP3 (see Kamada et al., 2007 and Lee et al., 2012). They were connected to hexahistidine by a spacer constituted of glycine and serine to form the peptides CPP1-H6, CPP2-H6 and CPP3-H6 having the sequences of SEQ ID NO: 76, 77, and 78, respectively.
[0272] MB11-luc2 mRNA was incubated with increasing amounts of CPP3-H6, CPP1-H6, or CPP2-H6 peptide for 30 minutes in previously optimized buffer comprising 160mM NaCl, 0.7 mM MgCl.sub.2 and 5 mM Hepes. MB11-luc2 mRNA alone and MB8-luc2 mRNA diluted in sterile, ultrapure deionized water, supplemented with 160 mM NaCl, were also injected, and are used here as controls.
[0273] An intradermal injection of 5.6 g of MB8-luc2 mRNA or of MB11-luc2 was performed in OF1 mice according to the protocol detailed in Example 10. Mouse skin was also injected with a mixture of MB11-luc2 mRNA and MB8-luc2 mRNA at a ratio of 1:1, and the same amount of CPP3-H6 as that of the 0.5 molar ratio to determine if the CPP3-H6 peptide can dissociate from RNA aptamer A, present in MB11-luc2 mRNA, after intradermal injection. Luciferase activity was measured 18 hours after injections.
Results
[0274] As expected, injection of MB8-luc2 mRNA resulted in efficient skin transfection. Co-injection of CPP3-H6, 0.7 mM MgCl.sub.2 and 5 mM Hepes with MB8-luc2 mRNA did not significantly affect transfection efficiency (
[0275] The optimal amount of CPP3-H6 peptide, which advantageously increases transcription efficiency by 9.2-fold as compared to MB11-luc2 mRNA alone, corresponds to a molar ratio of 1 CPP3-H6 peptide:2 mRNAs (
[0276] When one out of every two mRNAs does not bind CPP3-H6, transfection drops 12.3-fold (
[0277] MB11-luc2 mRNA was also incubated with the CPP1-H6 peptide at different molar ratios. The best molar ratio was 1 CPP1-H6 peptide:4 MB11-luc2 mRNAs (
[0278] CPP1-H6 therefore gave a poorer result than CPP3-H6. This can be explained by the number of copies of the receptor(s) of each CPP present in the plasma membrane of skin cells and the affinity of each CPP for its receptor(s).
[0279] Finally, MB11-luc2 mRNA was also incubated with CPP2-H6 at various molar ratios. The best molar ratio was 2 CPP2-H6 peptides:1 MB11-luc2 mRNA (
Example 12: CPP-H6 Effect on Transfection Efficiency of a capped mRNA in the Skin
Materials and Methods
[0280] In order to evaluate the effect of a CPP (here CPP2-H6) on the transfection efficiency of a capped mRNA, aptamer A was inserted into the 5-UTR of the conventional capped MB5-luc2 mRNA to generate the capped MB15-luc2 mRNA having the sequence of SEQ ID NO: 70. MB15-luc2 mRNA was incubated with CPP2-H6 for 30 minutes in buffer comprising 0.7 mM MgCl.sub.2 and 5 mM Hepes, as described above in Example 11.
[0281] An intradermal injection of 5.6 g of MB15-luc2 mRNA was performed in OF1 mice according to the protocol detailed in Example 10. Luciferase activity was measured 18 hours after injections.
Results
[0282] In the absence of the CPP2-H6 peptide, luciferase expression obtained with the capped MB15-luc2 mRNA is 1.68-fold lower than that obtained with the capped MB5-luc2 mRNA.
[0283] Co-injection of the capped MB15-luc2 mRNA and CPP2-H6 peptide, at a molar ratio of 2 peptides per mRNA, only improved transfection efficiency by 1.48-fold as compared to MB15-luc2 mRNA alone. This indicates that the insertion of the RNA aptamer A into the 5-UTR of a conventional capped mRNA and the attachment of a CPP-H6 peptide to this RNA aptamer does not improve transfection efficiency as compared to that observed with the capped mRNA, MB11-luc2 (10.6-fold). Regardless of the capped mRNA molecule (with or without aptamer, linked or not to the CPP2-H6 CPP), transfection efficiency remains much lower than that observed for the MB8-luc2 and MB11-luc2 mRNAs. It is therefore highly advantageous to use the mRNA molecules of the invention rather than capped mRNA molecules.
Example 13: Effect of 5-SL on Luciferase Expression In Vivo in the Skin
Materials and Methods
[0284] The following mRNAs: MB8-luc2, MB11-luc2, MB17-luc2, MB18-luc2, and capped MB5-luc2 were injected into the skin and luciferase activity measured 18 hours after injection, according to the protocol detailed in Example 10 above.
Results
[0285] As illustrated in
[0286] However, when the stem-loop is located approximately 70 nucleotides upstream of the xrRNA sequence, the stem-loop does not improve efficiency beyond what is already observed in the absence of the stem-loop. Indeed, translation efficiency is similar for the MB11-luc2 and MB17-luc2 mRNAs. The placement of aptamer A between the xrRNA sequences and the IRES sequence (MB18-luc2) increases translation efficiency even more as compared to the MB11-luc2 and MB17-luc2 mRNAs in a very surprising way. MB8-luc2 and MB18-luc2 mRNAs thus have a similar translation efficiency.
Conclusion
[0287] An mRNA molecule according to the invention has a cost of synthesis which is reduced by approximately 30-fold as compared to a capped mRNA molecule, while having a yield in protein expression that is increase by at least 2-fold, preferably about 10-fold. In addition, the mRNA of the invention is at least as stable as a capped mRNA, and its production by in vitro transcription is simplified by the absence of a cap molecule or an analog thereof. An mRNA of the invention coding the 2A protease of HRV2 makes it possible to increase the translation of an mRNA coding a protein of interest when these two molecules are co-transfected. In addition, the efficiency of transfection in a tissue, such as muscle or skin, can be improved, e.g., by inserting into the 5-UTR region of the mRNA molecule according to the invention an RNA aptamer directly penetrating cells, a CPP (attached to an RNA aptamer as described above), and/or by the addition of a stem-loop at the 5 end of the 5-UTR region upstream of the xrRNA sequence(s).
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