阅读说明：本技术 非黑素瘤皮肤癌(nmsc)的预防和治疗 (Prevention and treatment of non-melanoma skin cancer (NMSC) ) 是由 M·曼德勒 A·施尼博格 W·施密特 F·玛特纳 于 2018-03-29 设计创作，主要内容包括：本发明公开了用于预防和治疗非黑素瘤皮肤癌(NMSC)的干扰素α(IFN-α)信使RNA(mRNA)以及用于向人患者施用该IFN-αmRNA的试剂盒,其中所述mRNA具有5’帽区、5’非翻译区(5’-UTR)、编码区、3’非翻译区(3’-UTR)和多聚腺苷尾部(多聚A尾部)。(The present invention discloses interferon alpha (IFN- α) messenger rna (mRNA) for the prevention and treatment of non-melanoma skin cancer (NMSC) and a kit for administering the IFN- α mRNA to a human patient, wherein the mRNA has a 5' cap region, a 5' untranslated region (5' -UTR), a coding region, a 3' untranslated region (3' -UTR), and a polyadenylic tail (polya tail).)

1. Interferon alpha (IFN- α) messenger rna (mRNA) for the prevention and treatment of non-melanoma skin cancer (NMSC) in a human patient, wherein said mRNA has a 5' cap region, a 5' untranslated region (5' -UTR), a coding region encoding human IFN- α, a 3' untranslated region (3' -UTR) and a polyadenylic tail (polya tail), in particular wherein NMSC is Actinic Keratosis (AK), Basal Cell Carcinoma (BCC) and Squamous Cell Carcinoma (SCC), in particular AK.

2. The IFN- α mRNA for use according to claim 1, wherein the IFN- α mRNA is selected from the group consisting of IFN- α type 1 mRNA (IFNa1), IFN- α type 2a mRNA (IFNa2a) and IFN- α type 2b mRNA (IFNa2 b).

3. The IFN- α mRNA for use according to claim 1 or 2, wherein the poly-a tail comprises at least 100 adenosine monophosphate, preferably at least 120 adenosine monophosphate.

4. IFN-alpha mRNA for use according to any one of claims 1-3, wherein the 5' -UTR or the 3' -UTR or the 5' -UTR and the 3' -UTR are different from the native IFN-alpha mRNA, preferably wherein the 5' -UTR or the 3' -UTR or the 5' -UTRAnd the 3' -UTR comprises at least one stabilizing sequence, preferably having the formula (C/U) CCAN_xCCC(U/A)Py_xUC (C/U) CC (SEQ ID NO:38) wherein "x" is independently at N_xAnd Py_xIs an integer from 0to 10, preferably an integer from 0to 5, in particular 0, 1,2, 4 and/or 5.

5. IFN-alpha mRNA for use according to any one of claims 1 to 4, wherein the 5'-UTR or the 3' -UTR or the 5'-UTR and the 3' -UTR are different from native IFN-alpha mRNA, wherein the 5'-UTR and/or the 3' -UTR are 5'-UTR and/or 3' -UTR of human mRNA different from IFN-alpha, preferably selected from the group consisting of alpha globin, beta globin, albumin, lipoxygenase, ALOX15, alpha (1) collagen, tyrosine hydroxylase, ribosomal protein 32L, eukaryotic elongation factor 1a (EEF1A1), 5 □ -UTR elements present in orthopoxviruses and mixtures thereof, in particular selected from the group consisting of alpha globin, beta globin, alpha (1) collagen and mixtures thereof.

6. The IFN-alpha mRNA for use according to any one of claims 1 to 5, wherein in the IFN-alpha mRNA,

at least 5%, preferably at least 10%, preferably at least 30%, in particular at least 50% of all cytidine residues are substituted by 5-methylcytidine residues, and/or

At least 5%, preferably at least 10%, preferably at least 30%, in particular at least 50% of all cytidine residues are replaced by 2-amino-2-deoxycytidine residues, and/or

At least 5%, preferably at least 10%, preferably at least 30%, in particular at least 50% of all cytidine residues are replaced by 2-fluoro-2-deoxycytidine residues, and/or

At least 5%, preferably at least 10%, preferably at least 30%, in particular at least 50% of all cytidine residues are replaced by 2-thiocytidine residues, and/or

At least 5%, preferably at least 10%, preferably at least 30%, in particular at least 50% of all cytidine residues are replaced by 5-iodocytidine residues, and/or

At least 5%, preferably at least 10%, preferably at least 30%, in particular at least 50% of all uridine residues are substituted by pseudouridine residues, and/or

At least 5%, preferably at least 10%, preferably at least 30%, in particular at least 50% of all uridine residues are substituted by 1-methylpseudouridine residues, and/or

At least 5%, preferably at least 10%, preferably at least 30%, in particular at least 50% of all uridine residues are substituted by 2-thiouridine residues, and/or

At least 5%, preferably at least 10%, preferably at least 30%, in particular at least 50% of all uridine residues are substituted by 5-methyluridine residues, and/or

At least 5%, preferably at least 10%, preferably at least 30%, in particular at least 50% of all adenosine residues are substituted by N6-methyladenosine residues.

7. The IFN- α mRNA for use according to any one of claims 1 to 6, wherein the IFN- α mRNA has a GC to AU ratio of at least 49.5%, preferably at least 49.6%, more preferably 50%, even more preferably at least 55%, in particular at least 60%.

8. The IFN- α mRNA for use according to any one of claims 1 to 7, wherein the IFN- α mRNA has a Codon Adaptation Index (CAI) of at least 0.8, preferably at least 0.9.

9. The IFN-alpha mRNA for use according to any one of claims 1 to 8, wherein the coding region of the IFN-alpha mRNA encodes

-human IFNa2a, and preferably SEQ ID NO: 12, in particular SEQ ID NO: 2. 3, 5, 6, 7, 8, 9, 10 or 11; and/or

-human IFNa2b, and preferably SEQ ID NO: 26, in particular SEQ ID NO: 19. 20, 22 or 25, and/or

-human IFN α 1, and preferably SEQ ID NO: 36, in particular SEQ ID NO: 29. 30, 31, 32, 34 or 35.

10. The IFN- α mRNA for use according to any one of claims 1 to 9, wherein the IFN- α mRNA is administered subcutaneously, intradermally, transdermally, epicutaneously or topically, in particular epicutaneously.

11. Pharmaceutical formulation for the prevention and treatment of NMSC, preferably AK, BCC and SCC, in particular AK, comprising IFN- α mRNA as defined in any of claims 1 to 10.

12. The pharmaceutical formulation for use according to claim 11, comprising a pharmaceutically acceptable carrier, preferably a polymer-based carrier, in particular a cationic polymer, including linear and branched PEI and viromer; lipid nanoparticles and liposomes, nanoliposomes, ceramide-containing nanoliposomes, proteoliposomes, cationic amphiphilic lipids, such as: SAINT-lipids, exosomes of natural and synthetic origin, lamellar bodies of natural, synthetic and semi-synthetic origin, nanoparticles, calcium phosphosilicate nanoparticles, calcium phosphate nanoparticles, silica nanoparticles, nanocrystal particles, semiconductor nanoparticles, dry powders, poly (D-arginine), nanotreeds, starch-based delivery systems, micelles, emulsions, sol-gels, non-ionic surfactant vesicles (niosomes), plasmids, viruses, calcium phosphate nucleotides, aptamers, peptides, peptide conjugates, carrier tags, preferably small molecule targeting conjugates, or viral capsid proteins, preferably biological nanocapsules.

13. A kit for administering IFN- α mRNA for use according to any one of claims 1 to 10to a patient, comprising

-IFN- α mRNA as defined in any one of claims 1 to 10, and

-a skin delivery device.

14. The kit of claim 13, wherein the dermal delivery device is

An intradermal delivery device, preferably selected from a needle-based injection system and a needle-free injection system, or

A transdermal delivery device, preferably selected from transdermal patches, hollow and solid microneedle systems, microstructured transdermal systems, electrophoretic systems and iontophoretic systems, or

-an epidermal delivery device, preferably selected from the group consisting of needleless injection systems, laser-based systems, in particular erbium YAG laser systems and gene gun systems.

15. Method for the treatment and prevention of NMSC, preferably AK, BCC and SCC, wherein an effective amount of IFN- α mRNA as defined in any of claims 1 to 10 is administered to a patient in need thereof.

Example (b):

the material and the method are as follows:

transfection of murine 3T3 fibroblasts and human b.j. skin fibroblasts

For transfection, murine 3T3 fibroblasts and human b.j. skin fibroblasts were plated at 4-6 × 10⁴Individual cells/well were seeded in 12-well plates. After 24 hours incubation in complete EMEM or DMEM medium (Gibco, Thermo Fisher, USA), the medium was changed. Different preparations of IVT mRNA complexed with TransIT mRNA transfection reagent were prepared (Mirus Bio; complexes were formed according to the manufacturer's instructions) and added to the cells. 24 hours after transfection, the medium was replaced with complete DMEM. Cells were further cultured under standard conditions for up to 5 days with daily medium changes until the results were evaluated.

Isolation and transfection of intact pigskin biopsy:

full thickness pigskin flaps were isolated from the carcasses of pigs (samples were obtained under full compliance with the current national regulations (i.e. tioversucsgesetz 2012, TVG 2012)) and usedDisinfectants (Schuelke + Mayr GmbH, Germany) were used for disinfection.

Transfection of intact pig skin was accomplished by direct intradermal injection of IVT-mRNA solutions (1-10. mu.g mRNA/dose). Used according to the manufacturer's instructions (slightly modified according to Kariko et al; mol. ther.2012.20(5): 948-53)mRNA transfection kit (Mirus Bio)^TM) Or LacZ IVTmRNA was formulated using DOTAP-based liposomal formulations (Sigma Aldrich, USA) (fully modified with 5-methylcytidine, pseudouridine; trilink inc., USA). DOTAP-based formulations were prepared using a lipid/RNA ratio of 5/1(μ g/μ g). In addition, the mRNA complexes were also supplemented with RNase inhibitors (5U/dose, RNase, Promega, USA). The injection amount is 20. mu.l to 30. mu.l.

Alternatively, whole pigskin was transfected by direct intradermal injection of eGFP IVT-mRNA solution (0.5-25 μ g mRNA/dose). Used according to the manufacturer's instructions (modified according to Kariko et al, 2012)mRNA transfection kit (Mirus Bio)^TM) Or eGFP IVTmRNA (AMPTec, Germany) was formulated using DOTAP-based liposomal formulations (Sigma Aldrich, USA) or using lipid-nanoparticle formulations (Polymun, Austria) or using SAINT-based liposomal formulations (Synvolux, Netherlands). DOTAP-based formulations were prepared using a lipid/RNA ratio of 5/1(μ g/μ g). lipid/RNA ratios of 2.5-4/1(μ g/. mu.g) were used to prepare SAINT lipid-based formulations. In addition, uncomplexed mRNA in physiological buffer was also injected intradermally. The injection amount is 20. mu.l to 30. mu.l.

After injection, a needle biopsy of the injection area (8 mm diameter) was taken, subcutaneous fat was removed, and the biopsy was transferred epicutaneously up into standard complete medium (5 mL; containing: Dulbecco's Modified Eagle Medium (DMEM) with GlutaMAX, 10% FCS, 1 XPicillin-streptomycin-amphotericin B; obtained from Gibco, Life Tec) in petri dishesTechnologies). Subsequent incubations were performed at 37 deg.C/5% CO₂The reaction was carried out for 24 hours. Biopsies are typically harvested 24 hours after transfection.

Isolation and transfection of porcine epidermal sheets

Full thickness pigskin flaps were isolated from the carcasses of pigs (samples were obtained under full compliance with the current national regulations (i.e. tioversucsgesetz 2012, TVG 2012)) and usedDisinfectants (Schuelke + Mayr GmbH, Germany) were used for disinfection. A needle biopsy (6 or 8mm in diameter) was taken from a full thickness flap, subcutaneous fat was removed, and the biopsy was cut in two parts. Immediately thereafter, the dissected biopsy was transferred epicutaneously up to a 9cm (diameter) petri dish containing 5mL of Dispase II digest (approximately 2.5 units/mL; Dispase II; Sigma Aldrich, USA). Subsequent digestions were carried out overnight at 4 ℃. The samples were prepared by mixing with 1 × DMEM (Gibco) at 1: dispase II stock solution (10 mg/mL in 50mM HEPES/150mM NaCl; pH-7.4) was diluted 2 and Dispase II digest was prepared by addition of 1 Xpenicillin/streptavidin. The next day, the epidermal sheets were removed from the underlying dermis with forceps and transferred to DMEM for a short (5 min) washing step. The sheets were then placed in complete DMEM medium and incubated at 37 deg.C/5% CO₂Incubate (6 to 8 hours) until transfection in 24 well dishes. Transfection of porcine epidermal sheets was performed using IVT mRNA constructs of eGFP IVTmRNA (Amptec, Germany) or IFNa (e.g.: SEQ ID NOs: 1-5 and NO: 53). Use according to manufacturer's instructionsTransfection kit (Mirus Bio)^TM) Or the mRNA is formulated from liposome preparations (Polymun, Austria). Liposome formulations were prepared using a lipid/RNA ratio of 5/1(μ g/μ g). All lipoplex solutions used for transfection contained 0.1. mu.g to 10. mu.g mRNA/mL DMEM medium and the epidermal sheets were cultured for one to three days.

For the analysis, tissue culture supernatants were collected for subsequent ELISA analysis. The sheets were harvested for RNA and protein extraction and then analyzed by qPCR and ELISA, respectively. In addition, eGFP-transfected epidermal sheets were analyzed for eGFP expression by direct fluorescence microscopy and immunohistochemistry to detect eGFP in situ.

RT-PCR analysis of cells transfected with IVT mRNA preparations

Human b.j. cells and murine 3T3 fibroblasts were transfected with 0.1 to 1 μ g IFNa2 IVTmRNA complexed with TransIT mRNA transfection reagent. Total cellular RNA was isolated from murine and human fibroblasts or porcine epidermal sheets at various time points after transfection using a Tri-Reagent (Thermo Fisher, USA, manufacturer's instructions) and mRNA was reverse transcribed to cDNA by conventional RT-PCR (Protoscript First Strand cDNA Synthesis kit, NewEngland Biolabs, according to manufacturer's instructions). The cDNA samples were then subjected to conventional PCR and qPCR. Primers used were obtained from Invitrogen.

PCR analysis to detect IFNa2 variants was performed using Platinum Taq polymerase (Invitrogen, USA) and IFNa2 variant specific primers (Invitrogen, USA) from cDNA obtained from cells/sheets transfected with different IFNa2 variants. Human RPL4 and murine actb (eurofins genomics) were used as positive controls. The PCR products were analyzed using conventional agarose gel electrophoresis.

Table 4: PCR primer

Primers (manufacturer, SEQ ID NO:)	Sequence of
		huRPL4_fw(EG,39)	5’-AGC GTG GCT GTC TCC TCT C-3’
huRPL4_rev(EG,40)	5’-GAG CCT TGA ATA CAG CAG GC-3’
		hu_IFNA2_v2_fw(IVG,41)	5’-GCT TGG GAT GAG ACC CTC CTA-3’
hu_IFNA2_v2_rev(IVG,42)	5’-CCC ACC CCC TGT ATC ACA C-3’
		hu_IFNA2_ACC1_fw(IVG,43)	5’-CAC GAG ATG ATC CAG CAG AT-3’
hu_IFNA2_ACC1_rev(IVG,44)	5’-CTT GTC CAG CAG TGT CTC GT-3’
		huIFNA2_AMP_humod_f(IVG,45)	5’-CTG CTC TGT TGG CTG TGA TT-3’
huIFNA2_AMP_humod_r(IVG,46)	5’-CAG GCA TGA GAA CAG GCT AA-3’
		hu_IFNA2_AMP_AU_fw(IVG,47)	5’-TGA TGC TTC TTG CAC AAA TG-3’
hu_IFNA2_AMP_AU_rev(IVG,48)	5’-AGG ACA GGA ATG GTT TCA GC-3’
		hu_IFNA2_AMP_GC_fw(IVG,49)	5’-CAA GGA GTC GGA GTG ACT GA-3’
hu_IFNA2_AMP_GC_rev(IVG,50)	5’-CAG GGT GAT CCT CTG GAA GT-3’
		muACTB_fw(EG,51)	5‘-GGC TGT ATT CCC CTC CAT CG-3‘
muACTB_rev(EG,52)	5‘-CCA GTT GGT AAC AAT GCC ATG T-3‘

EG:Eurofins Genomics,IVG:Invitrogen

Analysis of human IFNa2 protein induced by IVT mRNA

Human b.j. cells and porcine epidermal sheets were transfected with 0.1-1 μ g IVT mRNA for different IFNa2 variants complexed with TransIT mRNA transfection reagents and cultured for 120 hours post transfection. Supernatants were obtained from transfected cells and epidermal sheets at several time points after transfection. Likewise, cells were harvested and proteins were extracted at the same time point. Proteins were extracted using cell extraction buffer (10mM HEPES, 10mM KCl, 0.1 μ M EDTA, 0.3% NP40 and Roche protease inhibitor, according to the manufacturer's protocol). The determination of IFN-. alpha.in supernatants and cell extracts was carried out using the human IFN-. alpha. (subtype 2; IFNa2) ELISA development kit (MABTECH AB, Sweden, according to the manufacturer's instructions) and the measurements were carried out on an Infinite 200PRO multimodal reader (Tecan AG, Switzerland).

Analysis of IVT mRNA-induced eGFP protein

Intact porcine skin explants and porcine epidermal sheets were transfected with 0.1-10 μ g of eGFP IVT mRNA, complexed or uncomplexed (` naked ` in physiological buffer) with TransIT mRNA transfection reagents or different liposome vectors and cultured for 24h post transfection. Samples were collected and proteins were extracted using cell extraction buffer (10mM HEPES, 10mM KCl, 0.1 μ M EDTA, 0.3% NP40 and Roche protease inhibitor, according to the manufacturer's protocol). SimpleStep in vitro Using GFPeGFP assay was performed with the kit (Abcam plc., UK, according to manufacturer's instructions) and read multimodal at Infine 200PROMeasurements were performed on a reader (Tecan AG, Switzerland).

Detection of beta-galactosidase Activity in porcine tissue

The total amount of beta-galactosidase (bGal) staining of the biopsy was performed in 24-well plates at 37 ℃ for 24 or 48 hours. Positive staining controls were generated by intradermal injection of bGal enzyme (1U recombinant bGal protein/injection) into porcine skin. The biopsies were incubated at room temperature in 4% formaldehyde solution (PBS) for 1 hour prior to starting the staining procedure. After fixation, samples were washed in PBS (3 ×), then LacZ wash buffer (2 mM MgCl in PBS₂0.01% sodium deoxycholate and 0.02% NP-40). After equilibration in LacZ buffer, samples were stored overnight (4 ℃). Subsequently, the samples were incubated in a staining solution at 37 ℃ and the color reaction was monitored. Staining solution was freshly prepared (5 mM K in LacZ buffer)₄Fe(CN)₆And 5mM K₃Fe(CN)₆) And 1mg/mL of 5-bromo-3-indolyl beta-D-galactopyranoside (Bluo-Gal) was added as a colored substrate. If the staining is carried out for 48 hours, the staining solution is replaced after 24 hours. The staining volume is typically 0.5 mL/well. Staining was stopped by washing in LacZ wash buffer and 3x PBS. The samples were then fixed in 4% formaldehyde buffer overnight and then further processed for standard histology or frozen in OCT for subsequent histological analysis.

Isolation and biolistic transfection of a complete human skin biopsy:

obtaining full thickness human skin flaps (obtaining samples in full compliance with current national regulations) by standard aesthetic and reconstructive surgical procedures, and usingDisinfectant (Germany Schuelke + Mayr GmbH).

For the biolar mRNA transfection, the BioRad helicos gene gun system was used. The system was loaded with IVT mRNA coated gold particles (1.6 μm gold microcarriers loaded with 1 μ g/. mu.l IVT-mRNA; Biorad; according to manufacturer's protocol). Gene gun transfection using 400psi helium pressure at a distance of 2.5cm from human skin explants. After transfection, the transfected area was punch biopsied (8 mm diameter), subcutaneous fat was removed, and the biopsied epidermis was transferred up into standard complete medium in a petri dish. Biopsy in 5% CO₂Next, the mixture was maintained in the α MEM + 10% pHPL medium at the gas-liquid interface for 24 hours. Biopsies are typically harvested 24 hours after transfection.

In situ analysis of eGFP protein in human skin

For the particle gun transfection, a BioRad Helios particle gun system loaded with eGFP-mRNA coated gold particles (1.6 μm gold microcarriers loaded with 1 μ g/. mu.l eGFP-mRNA) was used, using a helium pressure of 400psi, at a distance of 2.5cm from 8mm human skin explants. Explants were incubated at 5% CO₂Next, the mixture was maintained in the α MEM + 10% pHPL medium at the gas-liquid interface for 24 hours. The biopsy was fixed in 4% paraformaldehyde overnight at 4 ℃ and a 10 μm frozen section was obtained. GFP antibodies (anti-GFP; chicken IgY) were used and detection was performed by Alexa Fluor 488-conjugated donkey anti-chicken IgY antibodies. Cytoskeleton was examined by staining for F-actin with Alexa Fluor 568 phalloidin and slides were counterstained in Roti-mount Fluor Care DAPI with DAPI (4', 6-diamidino-2-phenylindole). The slides were scanned at 20 x magnification by an olympus slidescanner VS-120-L100-W system. The scale bar in (a, B) is 500 μm; scale bar in (C-L) 50 μm

In vivo use of firefly luciferase IVT mRNA and analysis of IVT mRNA-induced FLuc protein

All animal experiments were carried out exclusively on University Clinic for Swine (University of Veterinary Medicine Vienna) and according to Austria animal test method (TVG2012) using pigs (mixed breed; Edelschwein x Pietrain). The experiments were approved by the university of vienna veterinary animal experimentation ethics committee and the federal department of education, science and research, austria, and were conducted under approval number GZ 68.205/0192-WF/V/3 b/2017. The weight of the pigs at the beginning of the experiment was 30kg to 50 kg. The flanks of all pigs were carefully shaved the day before the planned injection to avoid skin irritation as much as possible. On the day of injection, pigs were anesthetized. Thoroughly cleaning the shaved skin area with warm water and applying O before injectionctenisept (Schuelke + Mayr GmbH, Germany) was sterilized twice. For intradermal injection, 30 μ L was administered using an insulin syringe (BD Micro-FineTM +). Suitable markers for injection sites (Laboratory Markers) and labeled according to the injection protocol. For sample analysis, pigs were euthanized by professionals at 24h and 48h post-injection. Skin flaps containing all injection sites were excised and immediately placed on ice. Porcine skin biopsies from the marked area were collected using 10mm puncture biopsies. In white 96-well plates (MicroWell)^TMNunc) was collected in 100uL Dulbecco's modified Eagle Medium high glucose DMEM (Gibco). Direct luciferase activity measurements were performed on the samples. Measurements were performed using the Firefly Luc One-Step Glow assay kit (Thermo Scientific, USA, according to the manufacturer's instructions) and analyzed on an Infinite 200PRO multimodal reader (Tecan AG, Switzerland).