Kits comprising linear DNAs for sustained polypeptide expression using synthetic, modified RNAs

• US 8,883,506 B2
• Filed: 08/21/2012
• Issued: 11/11/2014
• 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:
  US13/590,902
• Publication Number:
  US 20120322865A1
• Time in Patent Office: 812 Days
• Field of Search: 536/23.1, 435/455
• US Class Current: 435/455
• 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. A kit for making synthetic mRNAs for the production of induced pluripotent stem (iPS) cells comprising the following components:

(a) a linear DNA template for the generation of a synthetic modified RNA, the template encoding an OCT4 mRNA of SEQ ID NO;

788;

(b) a linear DNA template for the generation of a synthetic modified RNA, the template encoding a SOX2 mRNA of SEQ ID NO;

941 or SEQ ID NO;

1501;

(c) a linear DNA template for the generation of a synthetic modified RNA, the template encoding a KLF4 mRNA of SEQ ID NO;

501;

(d) a linear DNA template for the generation of a synthetic modified RNA, the template encoding a c-MYC mRNA of SEQ ID NO;

636; and

(e) a ribonucleoside mixture component comprising 5-methylcytidine and pseudouridine.

2. The kit of claim 1, wherein a T7 promoter is operably linked upstream of each of the linear DNAs encoding the OCT4 mRNA, SOX2 mRNA, KLF4 mRNA, and c-MYC mRNA.

3. The kit of claim 1, wherein the ribonucleoside mixture further comprises a 5′

cap analog nucleoside component.

4. The kit of claim 3, wherein the 5′

cap analog is 3″

-0-Me-m⁷G(5′

)ppp(5′

)G.

5. The kit of claim 1, further comprising a phosphatase enzyme, an RNA polymerase enzyme, or both.

6. The kit of claim 1, further comprising a buffer.

7. The kit of claim 1, further comprising a linear DNA template for the generation of a synthetic modified RNA, the template encoding a sequence encoding a soluble interferon receptor.

8. The kit of claim 7, wherein the soluble interferon receptor is B18R.