Induced pluripotent stem cells with synthetic modified RNAs

• US 9,803,177 B2
• Filed: 06/23/2014
• Issued: 10/31/2017
• Est. Priority Date: 04/16/2010
• Status: Active Grant

• Current Assignee: Children's Medical Center Corporation
• Original Assignee: Children's Medical Center Corporation
• Inventors: Rossi, Derrick, Warren, Luigi
• Primary Examiner(s): WILSON, MICHAEL C
• Application Number:
  US14/311,545
• Publication Number:
  US 20140308746A1
• Time in Patent Office: 1,226 Days
• Field of Search: 435325, 435455
• CPC Class Codes: A61P 19/10 for osteoporosis
• CPC Class Codes: A61P 25/00 Drugs for disorders of the nervous system
• CPC Class Codes: A61P 25/08 Antiepileptics; Anticonvulsants
• CPC Class Codes: A61P 27/02 Ophthalmic agents
• CPC Class Codes: A61P 3/00 Drugs for disorders of the metabolism of the blood or the extracellular fluid A61P7/00
• CPC Class Codes: A61P 3/10 for hyperglycaemia, e.g. antidiabetics
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 35/02 specific for leukemia
• CPC Class Codes: A61P 7/00 Drugs for disorders of the blood or the extracellular fluid
• CPC Class Codes: A61P 7/06 Antianaemics
• CPC Class Codes: A61P 9/04 Inotropic agents, i.e. stimulants of cardiac contraction; Drugs for heart failure
• CPC Class Codes: A61P 9/10 for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
• CPC Class Codes: C07K 14/435 from animals; from humans
• CPC Class Codes: C12N 15/11 DNA or RNA fragments; Modified forms thereof DNA or RNA not used in recombinant technology, C07H21/00; Non-coding nucleic acids having a biological activity
• CPC Class Codes: C12N 15/111 General methods applicable to biologically active non-coding nucleic acids
• CPC Class Codes: C12N 2310/14 interfering N.A.
• CPC Class Codes: C12N 2320/30 Special therapeutic applications
• CPC Class Codes: C12N 5/0696 Artificially induced pluripotent stem cells, e.g. iPS

Abstract

Described herein are synthetic, modified RNAs for changing the phenotype of a cell, such as expressing a polypeptide or altering the developmental potential. Accordingly, provided herein are compositions, methods, and kits comprising synthetic, modified RNAs for changing the phenotype of a cell or cells. These methods, compositions, and kits comprising synthetic, modified RNAs can be used either to express a desired protein in a cell or tissue, or to change the differentiated phenotype of a cell to that of another, desired cell type.

Claims

1. An isolated, human induced pluripotent stem (iPS) cell comprising a transfection mixture comprising synthetic modified RNAs encoding octamer-binding transcription factor 4 (Oct4), sex determining region Y-box 2 (Sox2), and Kruppel-like factor 4 (K1f4), wherein each occurrence of a cytosine is replaced with 5-methylcytosine (5mC) and each occurrence of a uracil is replaced with pseudouracil;

wherein the iPS cell is produced by contacting an isolated human somatic fibroblast cell with the transfection mixture for a time sufficient to obtain the iPS cell.

2. The iPS cell of claim 1, wherein the somatic fibroblast cell further comprises one or more synthetic modified RNAs encoding NANOG, c-MYC (V-Myc Avian Myelocytomatosis Viral Oncogene Homolog), and LIN28 (Zinc Finger CCHC Domain-Containing Protein 1), wherein each occurrence of a cytosine is replaced with 5-methylcytosine (5mC) and each occurrence of a uracil is replaced with pseudouracil.

3. The iPS cell of claim 1, wherein said cell has a reduced expression of a Type I or Type II IFN (interferon) relative to a cell subjected to at least three consecutive rounds of contacting with an exogenously introduced non-modified synthetic RNA encoding the reprogramming factors.

4. The iPS cell of claim 1, wherein said cell has a reduced expression of at least one IFN-signature gene relative to a somatic fibroblast cell subjected to at least three consecutive rounds of contacting with an exogenously introduced non-modified synthetic RNA encoding the reprogramming factors, and wherein the IFN-signature gene is selected from the group consisting of IFNα

(interferon-α

), IFNβ

1 (interferon-β

1), IFIT (Interferon Induced protein with Tetratricopeptide repeats), OAS1 (2′

-5′

-Oligoadenylate Synthetase-Like), PKR (Protein Kinase RNA-Activated), RIGI (Retinoic Acid-Inducible Gene 1 Protein), CCL5 (Chemokine (C-C Motif) Ligand

     5), RAP 1A (Ras-Related Protein Rap-1A), CXCL10 (Chemokine (C-X-C Motif) Ligand

     10), IFIT1 (Interferon Induced protein with Tetratricopeptide repeats-1), CXCL11 (Chemokine (C-X-C Motif) Ligand

     11), MX1, RP11-167P23.2, HERC5 (HECT and RLD Domain Containing E3 Ubiquitin Protein Ligase

     5), GALR3 (Galanin Receptor

     3), IFIT3 (Interferon Induced protein with Tetratricopeptide repeats-3), IFIT2 (Interferon Induced protein with Tetratricopeptide repeats-2), RSAD2 (Radical S-Adenosyl Methionine Domain Containing

     2), and CDC20 (Cell Division Cycle

     20).

5. The iPS cell of claim 1, wherein the synthetic, modified RNA further comprises a 5′

cap.

6. The iPS cell of claim 5, wherein the 5′

cap is a 5′

cap analog.

7. The iPS cell of claim 6, wherein the 5′

cap analog is a 5′

diguanosine cap.

8. The iPS cell of claim 1, wherein the synthetic, modified RNA does not comprise a 5′

triphosphate.

9. The iPS cell of claim 1, wherein the synthetic, modified RNAs further comprise a poly(A) tail, a Kozak sequence, a 3′

untranslated region, a 5′

untranslated region, or any combination thereof.

10. The iPS cell of claim 1, wherein the synthetic, modified RNAs are formulated in a lipid or lipid-based molecule.