Novel RNA Composition and Production Method for Use in iPS Cell Generation

Abstract

This invention generally relates to a novel RNA composition and its production method useful for generating and expanding induced pluripotent stem cells (iPS cells; iPSC) as well as adult stem cells (ASC). The RNA composition so defined can be used for producing not only non-transgenic but also tumor-free iPS cells. The defined RNA composition contans at least two types of different RNA constructs; one is “miR-302 precursor RNA (pre-miR-302)” and the other is “RNA-dependent RNA polymerase (RdRp)” mRNA. Both of pre-miR-302 and RdRp mRNA contain highly structured RNA comformations, such as hairpin and stem-loop structures. To produce highly structured RNAs, a novel PCR-IVT methodology has been developed and used with a specially designed RNA polymerase-helicase mixture activity.

Claims

1. A novel RNA composition for use in induced pluripotent stem cell (iPSC) generation, comprising: A mixture of at least a miR-302 precursor RNA (pre-miR-302) construct and at least an RNA-dependent RNA polymerase (RdRp) mRNA, wherein the pre-miR-302 construct contains at least an RdRp binding site in its 5′-end or 3′-end region, or both, and wherein the RdRp mRNA is isolated or modified from RNA virus.

2. The composition as defined in claim 1, wherein the ratio of said pre-miR-302 and RdRp mRNA mixture is ranged from 20:1 to 1:20.

3. The composition as defined in claim 1, wherein said 5′-end RdRp binding site contains a sequence of either SEQ.ID.NO.1 or SEQ.ID.NO.2.

4. The composition as defined in claim 3, wherein said 5′-end RdRp binding site is selected from a sequence containing SEQ.ID.NO.3, SEQ.ID.NO.4, SEQ.ID.NO.5, or SEQ.ID.NO.6, or a combination thereof.

5. The composition as defined in claim 1, wherein said 3′-end RdRp binding site contains a sequence of either SEQ.ID.NO.7 or SEQ.ID.NO.8.

6. The composition as defined in claim 5, wherein said 3′-end RdRp binding site is selected from a sequence containing SEQ.ID.NO.9, SEQ.ID.NO.10, SEQ.ID.NO.11, or SEQ.ID.NO.12, or a combination thereof.

7. The composition as defined in claim 1, wherein said pre-miR-302 is selected from at least a sequence containing SEQ.ID.NO.13, SEQ.ID.NO.14, SEQ.ID.NO.15, or SEQ.ID.NO.16, or a combination thereof.

8. The composition as defined in claim 1, wherein said RdRp mRNA is isolated from RNA virus.

9. The composition as defined in claim 1, wherein said RdRp mRNA is coronaviral or hepatitis C viral RNA-dependent RNA polymerase mRNA.

10. The composition as defined in claim 1, wherein said pre-miR-302 is produced using a novel polymerase chain reaction-in-vitro transcription (PCR-IVT) methodology with an RNA polymerase and helicase mixture activity.

11. The composition as defined in claim 1, wherein said RdRp mRNA is produced using a novel polymerase chain reaction-in-vitro transcription (PCR-IVT) methodology with an RNA polymerase and helicase mixture activity.

12. The composition as defined in claim 10, wherein said helicase is an enzyme capable of unwinding both DNA and RNA secondary structures.

13. The composition as defined in claim 10, wherein the IVT reaction of said PCR-IVT methodology is performed in an improved buffer system containing 1× transcription buffer with additional 0.001˜10 mM of betaine (trimethylglycine, TMG), dimethylsulfoxide (DMSO), or 3-(N-morpholino)propane sulfonic acid (MOPS), or a combination thereof.

14. The composition as defined in claim 1, wherein said pre-miR-302 and RdRp mRNA mixture is further formulated with at least a delivery agent for facilitating intracellular transfection in vitro, ex vivo and/or in vivo.

15. The composition as defined in claim 14, wherein said delivery agent includes glycylglycerins, liposomes, nanoparticles, liposomal nanoparticles, conjugating molecules, infusion chemicals, gene gun materials, electroporation agents, transposon, and a combination thereof.

16. The composition as defined in claim 1, wherein said iPSCs can differentiate into various tissue cells derived from all three germ layers of ectoderm, mesoderm and endoderm.

17. The composition as defined in claim 16, wherein said iPSC-derived tissue cells are used for developing cell-based therapies.

18. The composition as defined in claim 1, wherein said iPSCs is used for developing stem cell-based therapies.

19. The composition as defined in claim 1, wherein said iPSCs is used for searching and/or producing new medicine materials.

20. The composition as defined in claim 1, wherein said pre-miR-302 and RdRp mRNA mixture is used for developing reprogramming-associated therapies and medicines.

21. The composition as defined in claim 1, wherein said pre-miR-302 and RdRp mRNA mixture is used as an ingredient in medicines or therapies.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0072] Referring particularly to the drawings for the purpose of illustration only and not limitation, there is illustrated:

[0073] FIG. 1 depicts the step-by-step procedure of the PCR-IVT methodology. For RNA production, a part or whole procedure of this PCR-IVT method can be adopted for either single or multiple cycle amplification of desired RNA products.

[0074] FIG. 2 depicts one of the preferred designs of the miR-302 precursor RNA (pre-miR-302) construct. At least an RNA-dependent RNA polymerase (RdRp)-binding site or more can be placed in either 5′-end or 3′-end region, or both, of the pre-miR-302 construct.

[0075] FIG. 3 shows markedly increased expressions of miR-302s (i.e. top to bottom: b, c, d, a) and RdRp mRNA in reprogrammed cells after co-transfection of both pre-miR-302 and RdRp mRNA (right) compared to that of transfection of only pre-miR-302 (middle), using Northern blot analysis.

[0076] FIG. 4 shows elevated expression of standard embryonic stem cell (ESC) markers such as Oct3/4, Sox2 and Nanog found in reprogrammed cells three days after co-transfection of pre-miR-302 and RdRp mRNA, using Western blot analysis.

[0077] FIG. 5 shows iPSC-derived embryoid body formation after co-transfection of pre-miR-302 and RdRp mRNA.

[0078] FIG. 6 shows global genomic DNA demethylation observed in the reprogrammed cells, similar to the status of human ESC genome.

[0079] FIG. 7 shows >92% high similarity of genome-wide gene expression patterns compared to those of human ESC WA01 (H1) and WA09 (H9) cell lines, using microarray analysis.

[0080] FIG. 8 shows iPSC-derived teratoma formation containing tissues derived from all three embryonic germ layers (ectoderm, mesoderm and definitive endoderm), using immunohisto-chemical staining with respective tissue cell markers.

EXAMPLES

1. Human Cell Isolation and Cultivation

[0081] Starting tissue cells can be obtained from either enzymatically dissociated skin cells using Aasen's protocol (Nat. Protocols 5, 371-382, 2010) or simply from the buffy coat fraction of heparin-treated peripheral blood cells. The isolated tissue samples must be kept fresh and used immediately by mixing with 4 mg/mL collagenase I and 0.25% TrypLE for 15-45 min, depending on cell density, and rinsed by HBSS containing trypsin inhibitor two times and then transferred to a new sterilized microtube containing 0.3 mL of feeder-free SFM culture medium (IrvineScientific, CA). After that, cells were further dissociated by shaking in a microtube incubator for 1 min at 37° C. and then transferred the whole 0.3 mL cell suspension to a 35-mm Matrigel-coated culture dish containing 1 mL of feeder-free SFM culture medium supplemented with formulated pre-miR-302+RdRp mRNA mixture, LIF, and bFGF/FGF2, or other optional defined factors. The used concentrations of pre-miR-302+RdRp mRNA mixture, LIF, bFGF/FGF2, and other optional defined factors is ranged from 0.1 to 500 microgram (μg)/mL, respectively, in the cell culture medium. The cell culture medium and all of the supplements must be refreshed every 2˜3 days and the cells are passaged at about 50%˜60% confluence by exposing the cells to trypsin/EDTA for 1 min and then rinsing two times in HBSS containing trypsin inhibitor. For ASC expansion, the cells were replated at 1:5˜1:500 dilution in fresh feeder-free MSC Expansion SFM culture medium supplemented with formulated pre-miR-302+RdRp mRNA mixture, LIF, bFGF/FGF2, and/or other optional defined factors. For culturing keratinocytes, cells are isolated from skin tissues and cultivated in EpiLife serum-free cell culture medium supplemented with human keratinocyte growth supplements (HKGS, Invitrogen, Carlsbad, Calif.) in the presence of proper antibiotics at 37° C. under 5% CO.sub.2. Culture cells are passaged at 50%-60% confluency by exposing cells to trypsin/EDTA solution for 1 min and rinsing once with phenol red-free DMEM medium (Invitrogen), and the detached cells are replated at 1:10 dilution in fresh EpiLife medium with HKGS supplements. Human cancer and normal cell lines A549, MCF7, PC3, HepG2, Colo-829 and BEAS-2B were obtained either from the American Type Culture Collection (ATCC, Rockville, Md.) or our collaborators and then maintained according to manufacturer's or provider's suggestions. After reprogramming, the resulting iPS cells (iPSCs) were cultivated and maintained following either Lin's feeder-free or Takahashi's feeder-based iPSC culture protocols (Lin et al., RNA 14:2115-2124, 2008; Lin et al., Nucleic Acids Res. 39:1054-1065, 2011; Takahashi K and Yamanaka S, Cell 126:663-676, 2006).

2. In-Vitro RNA Transfection

[0082] For RNA transfection, 0.5˜500 μg of isolated pre-miR-302 and RdRp mRNA mixture (ratio ranged from about 20:1 to 1:20) is dissolved in 0.5 ml of fresh cell culture medium and mixed with 1˜50 μl of In-VivoJetPEI or other similar transfection reagents. After 10˜30 min incubation, the mixture is then added into a cell culture containing 50%-60% confluency of the cultivated cells. The medium is reflashed every 12 to 48 hours, depending on cell types. This transfection procedure may be performed repeatedly to increase transfection efficiency. The transfection results are further analyzed by various detection methods and shown in FIG. 3 to FIG. 8, respectively.

3. DNA Demethylation Assay

[0083] Genomic DNAs were isolated from about two million cells with a Roche DNA isolation kit (Sigma-Aldrich, MO, USA). About 2 microgram (μg) of the isolated genomic DNAs was digested with a CCGG-cutting restriction enzyme, HpaII, and then assessed with 1% agarose gel electrophoresis to determine genome-wide demethylation (FIG. 4).

4. Genome-Wide Microarray Analysis of Global Cellular Gene Expression Patterns

[0084] Human genome GeneChip arrays (Affymetrix, CA) were used to detect the expression patterns of genome-wide 47,000 human gene transcripts in iPS cells, as shown in FIG. 7. Each sample was tested in triplicate and the same experiment was repeated for four times. Total RNAs from each tested sample were isolated using RNeasy spin columns (Qiagen, MD). To prepare labeled probes for microarray hybridization, the extracted total RNAs (2 μg) were converted into cDNAs, using a T7 promoter-containing oligo(dT).sub.24 primer and SuperScript double-stranded cDNA synthesis kit (ThermoFisher Scientific, MA), following manufacturer's protocols. The resulting cDNAs were purified by phenol-chloroform extractions, precipitated with ethanol, and resuspended at a concentration of 0.5 μg/μl in diethyl pyrocarbonate (DEPC)-treated ddH.sub.2O. Then, in-vitro transcription was performed, containing 1 μg of the dsDNAs, 7.5 mM unlabeled ATP and GTP, 5 mM unlabeled UTP and CTP, and 2 mM biotin-labeled CTP and UTP (biotin-11-CTP, biotin-16-UTP, Enzo Diagnostics), and 20 U of T7 RNA polymerase. Reactions were carried out for 4 hours at 37° C., and the resulting cRNAs were purified by RNeasy spin columns (Qiagen). A part of the cRNA sample was separated on a 1% agarose gel to check the size range, and then 10 μg of the cRNAs were fragmented randomly to an average size of 50 bases by heating at 94° C. for 35 min in 40 mM Tris-acetate, pH 8.0, 100 mM KOAc/30 mM MgOAc. Hybridizations were completed in 200 μl of AFFY buffer (Affymetrix, CA) at 40° C. for 16 hours with constant mixing. After hybridization, arrays were rinsed three times with 200 μl of 6×SSPE-T buffer (1×0.25 M sodium chloride/15 mM sodium phosphate, pH 7.6/1 mM EDTA/0.005% Triton) and then washed with 200 μl of 6×SSPE-T for 1 hour at 50° C. The arrays were further rinsed twice with 0.5×SSPE-T and washed with 0.5×SSPE-T at 50° C. for 15 min. Then, staining assays were done with 2 μg/ml streptavidin-phycoerythrin (Invitrogen) and 1 mg/ml acetylated BSA (Sigma-Aldrich) in 6×SSPE-T (pH 7.6). The arrays were read at 7.5 μm with a confocal scanner (Molecular Dynamics).

[0085] To identify the background variations, we duplicated the microarray tests using the same sample and selected two hundred genes, which were slightly presented in one side of the tests, for further comparison. The sample signals were normalized using the total average difference between perfectly matched probes and mismatched probes. Then, alterations of overall genome-wide gene expression patterns were analyzed using Affymetrix Microarray Suite version 5.0, Expression Console™ (Affymetrix) and Genesprings (Silicon Genetics) softwares. Changes in gene expression rates more than 1-fold were considered as positive differential genes. In gene clustering assays, a plug-in program Genetrix (Epicenter Software) was used in conjunction with the Affymetrix softwares. Signals of the sample were normalized with the internal house-keeping control average in each microarray. After normalization, as signal intensity increased from level 1 to level 65,535, the corresponding color changed from green to black, and to red. The level above 23,000 was considered to be a positive call in that a Northern blotting assay could positively detect.

5. Teratoma Formation and Guided iPSC Differentiation

[0086] Xenograft transplantation of iPS-derived embryoid bodies into the uterus or peritoneal cavity of a 6-week-old female pseudopregnant immunocompromised SCID-beige mouse formed teratoma-like cysts, containing tissues derived from all three embryonic germ layers (ectoderm, mesoderm and definitive endoderm) (FIG. 8). The use of immunocompromised nude mice was to provide an in vivo environment mimicking transplantation therapy. The pseudopregnant mice were made by intraperitoneally injection of 1 IU human menopausal gonadotrophin (HMG) for two days and then human chorionic gonadotrophin (hCG) for one more day. For In vitro guidance of stem cell differentiation into the germ line lineage, iPS cells were maintained on polyornithine/laminin-coated dishes in DMEM/F12 (1:1; high glucose) medium supplemented with charcoal-stripped 10% FBS, 4 mM L-glutamine, 1 mM sodium pyruvate, 5 ng/ml activin and 50 ng/ml dihydrotestosterone (DHT) for 12 hours, at 37° C. under 5% CO.sub.2. Then the cells were trypsinized, washed with 1×PBS, and collected in four aliquots of chilled Matrigel (100 μl each) and one aliquots of 100 μl 1×PBS. Immediately after that, we transplanted the cells into the hind limb muscle, peritoneum, uterus, subcutaneous neck skin (with Matrigel) and tail vein (with PBS) of 6-week-old immunocompromised SCID-beige nude mice. The mice were anesthetized with diethyl ether during experimental processing.

6. Immunostaining Assay

[0087] Cell/Tissue samples are fixed in 100% methanol for 30 min at 4° C. and then 4% paraformaldehyde (in 1×PBS, pH 7.4) for 10 min at 20° C. After that, the samples are incubated in 1×PBS containing 0.1%˜0.25% Triton X-100 for 10 min and then washed in 1×PBS three times for 5 min. For immunostaining, primary antibodies were purchased from Invitrogen (CA, USA) and Sigma-Aldrich (MO, USA), respectively. Dye-labeled goat anti-rabbit or horse anti-mouse antibody are used as the secondary antibody (Invitrogen, CA, USA). Results are examined and analyzed at 100× or 200× magnification under a fluorescent 80i microscopic quantitation system with a Metamorph imaging program (Nikon).

7. Novel PCR-IVT Protocol

[0088] Reverse transcription (RT) of desired mRNA is performed by adding about 0.01 ng-10 microgram (μg) of isolated mRNA into a 20˜50 μL RT reaction (SuperScript III cDNA RT kit, ThermoFisher Scientific, MA, USA), following the manufacturer's suggestions. Depending on the mRNA amount, the RT reaction mixture contains the mRNA, about 0.01˜20 nmole RT primer, a proper amount of dNTPs and reverse transcriptase in 1×RT buffer. Then, the RT reaction is incubated at 46˜65° C. for 1˜3 hours (hr), depending on the structure and length of the desired mRNA, so as to make at least a complementary DNA (cDNA) template for the next step of PCR. For cloning of viral RdRp cDNA, we design and use an RT-reverse primer 5′-GACAACAGGT GCGCTCAGGT CCT-3′ (SEQ.ID.NO.17) to perform RT of coronaviral COVID-19 mRNA into cDNA.

[0089] Next, polymerase chain reaction (PCR) is performed by adding about 0.01 pg˜10 μg of the RT-derived cDNAs into a 20˜50 μL PCR preparation mixture (High-Fidelity PCR kit, ThermoFisher Scientific, MA, USA), following the manufacturer's suggestions. Then, the PCR mixture is first incubated in five to twenty (5˜20) cycles of denaturation at 94° C. for 1 mim, annealing at 30˜55° C. for 30 sec-1 min, and then extension at 72° C. for 1-3 min, depending on the structure and length of the desired DNA and primers. After that, another ten to twenty (10˜20) cycles of PCR are performed with a series of sequential steps of denaturation at 94° C. for 1 mim, annealing at 50˜55° C. for 30 sec, and then extension at 72° C. for 1-3 min, depending on the structure and length of the resulting PCR products. Finally, the resulting PCR products are used as templates for IVT. For IVT template preparation, we design and use a specific pair of PCR primers for amplifying RNA promoter-containing RdRp cDNA templates from coronaviral mRNA, including SEQ.ID.NO.17 and 5′-GATATCTAAT ACGACTCACT ATAGGGAGAG GTATGGTACT TGGTAGTT-3′ (SEQ.ID.NO.18). Later, a 5′-cap nucleotide may be further incorporated in the resulting mRNA products during or after IVT. On the other hand, we also design and use another pair of PCR primers for amplifying RNA promoter-containing miR-302 familial cluster cDNA templates from isolated human cell genome DNAs, including 5′-GATATCTAAT ACGACTCACT ATAGGGAGAT CTGTGGGAAC TAGTTCAGGA AGGTAA-3′ (SEQ.ID.NO.19) and 5′-GTTCTCCTAA GCCTGTAGCC AAGAACTGCA CA-3′ (SEQ.ID.NO.20). In the designed primers, various RNA promoter sequences can be used, such as T7, T3 and/or SP6 promoter, and at least a RdRp initiation/binding site has been incorporated in the 5′-forward and 3′-reverse primer sequences, respectively. Also, the resulting miR-302 familial cluster template contains the cDNA sequences of at least one pre-miR-302a (SEQ.ID.NO.13), pre-miR-302b (SEQ.ID.NO.14), pre-miR-302c (SEQ.ID.NO.15), and/or pre-miR-302d (SEQ.ID.NO.16). Multiple same or different pre-miR-302 sequences can be repeatedly placed in the miR-302 familial cluster up to 9 hairpin-structured sequences.

[0090] For mRNA production, since a promoter-primer has been incorporated into the resulting PCR products, an improved IVT reaction can be performed to amplify desired mRNA sequences, using the PCR products as templates. The IVT reaction mixture contains 0.01 ng-10 μg of the PCR product, 0.1˜10 U of isolated coronaviral helicase (i.e. from COVID-19), a proper amount of NTPs and RNA polymerase (i.e. T7, T3, or SP6) in 1× transcription buffer. The contents of 1× transcription buffer may be adjusted according to the used RNA polymerase, following the manufacturer's suggestions. Additionally, the 1× transcription buffer may further conatin 0.001˜10 mM of betaine (trimethylglycine, TMG), dimethylsulfoxide (DMSO), and/or 3-(N-morpholino)propane sulfonic acid (MOPS), and/or a combination thereof, which facilitates the denaturation of highly structured RNA/DNA sequences, such as hairpins and stem-loop structures. Then, the IVT reaction is incubated at 37° C. for 1-6 hr, depending on the stability and activity of the used RNA polymerase(s). In this improved novel IVT reaction, at least an additional helicase enzyme is added in order to facilitate the unwinding of RNA/DNA secondary structures, such as hairpin-like stem-loop structures, so as to overcome the low efficiency problem of hairpin-like RNA production in vitro. Notably, the helicase enzyme can unwind the secondary structures in both DNA and RNA strands.

8. RNA Purification and Northern Blot Analysis

[0091] Desired mRNAs (10 μg) are isolated with a mirVana™ RNA isolation kit (Ambion, Austin, Tex.), following the manufacturer's protocol, and then further purified by using either 15% TBE-urea polyacrylamide gel or 3.5% low melting point agarose gel electrophoresis. For Northern blot analysis, the gel-fractionated mRNAs are electroblotted onto a nylon membrane. Detection of the mRNA and its IVT template (the PCR product) is performed with a labeled [LNA]-DNA probe complementary to a desired target sequence of the mRNA. The probe is further purified by high-performance liquid chromatography (HPLC) and tail-labeled with terminal transferase (20 units) for 20 min in the presence of either a dye-labeled nucleotide analog or [.sup.32P]-dATP (>3000 Ci/mM, Amersham International, Arlington Heights, Ill.).

9. Protein Extraction and Western Blot Analysis

[0092] Cells (10.sup.6) are lysed with a CelLytic-M lysis/extraction reagent (Sigma) supplemented with protease inhibitors, Leupeptin, TLCK, TAME and PMSF, following the manufacturer's suggestion. Lysates are centrifuged at 12,000 rpm for 20 min at 4° C. and the supernatant is recovered. Protein concentrations are measured using an improved SOFTmax protein assay package on an E-max microplate reader (Molecular Devices, CA). Each 30 μg of cell lysate are added to SDS-PAGE sample buffer under reducing (+50 mM DTT) and non-reducing (no DTT) conditions, and boiled for 3 min before loading onto a 6-8% polyacylamide gel. Proteins are resolved by SDS-polyacrylamide gel electrophoresis (PAGE), electroblotted onto a nitrocellulose membrane and incubated in Odyssey blocking reagent (Li-Cor Biosciences, Lincoln, NB) for 2 hr at room temperature. Then, a primary antibody is applied to the reagent and incubated the mixture at 4° C. After overnight incubation, the membrane is rinsed three times with TBS-T and then exposed to goat anti-mouse IgG conjugated secondary antibody to Alexa Fluor 680 reactive dye (1:2,000; Invitrogen—Molecular Probes), for 1 hr at the room temperature. After three additional TBS-T rinses, fluorescent scanning of the immunoblot and image analysis are conducted using Li-Cor Odyssey Infrared Imager and Odyssey Software v.10 (Li-Cor).

10. In Vivo Transfection Assay

[0093] Isolated pre-miR-302 and RdRp mRNA mixture is mixed well with a proper amount of delivery agent, such as an In-VivoJetPEI transfection reagent, following the manufacturer's protocol, and then injected into blood veins or muscles of an animal, depending the purpose of applications. The delivery agent is used for mixing, conjugating, encapsulating or formulating the isolated pre-miR-302 and RdRp mRNA mixture, so as to not only protect the RNA contents from degradation but also facilitate the delivery of the isolated pre-miR-302 and RdRp mRNA mixture into specific target cells of interest in vitro, ex vivo and/or in vivo.

11. Statistic Analysis

[0094] All data were shown as averages and standard deviations (SD). Mean of each test group was calculated by AVERAGE of Microsoft Excel. SD was performed by STDEV. Statistical analysis of data was performed by One-Way ANOVA. Tukey and Dunnett's t post hoc test were used to identify the significance of data difference in each group. p<0.05 was considered significant (SPSS v12.0, Claritas Inc).

REFERENCES

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