RNA preparations comprising purified modified RNA for reprogramming cells

Patent metadata

Field	Value
Publication number	US-11028370-B2
Application number	US-201815994093-A
Country	US
Kind code	B2
Filing date	May 31, 2018
Priority date	Dec 7, 2009
Publication date	Jun 8, 2021
Grant date	Jun 8, 2021

Abstract

The present invention provides compositions and methods for reprogramming somatic cells using purified RNA preparations comprising single-strand mRNA encoding an iPS cell induction factor. The purified RNA preparations are preferably substantially free of RNA contaminant molecules that: i) would activate an immune response in the somatic cells, ii) would decrease expression of the single-stranded mRNA in the somatic cells, and/or iii) active RNA sensors in the somatic cells. In certain embodiments, the purified RNA preparations are substantially free of partial mRNAs, double-stranded RNAs, un-capped RNA molecules, and/or single-stranded run-on mRNAs.

First claim

We claim:

1. A purified RNA preparation comprising in vitro-synthesized single-stranded mRNA molecules that:
A) encode at least one iPSC induction factor selected from the group consisting of OCT4, SOX2, KLF4, LIN28, NANOG, and a MYC protein selected from c-MYC, L-MYC and N-MYC;
B) comprise (i) guanosine, (ii) adenosine, (iii) cytidine, (iv) pseudouridine (Ψ) or 1-methyl-pseudouridine (m 1 Ψ) in place of uridine, (v) a 5′ cap, and (vi) a 3′ poly(A) tail; and
C) have been purified using a purification process that removes RNA contaminant molecules that are immunogenic and toxic to mammalian cells by inducing an innate immune response, such that said mRNA molecules lack immunogenicity, as can be determined using an in vitro monocyte-derived dendritic cell (MDDC) immunogenicity assay by measuring an amount of IFN-α or TNF-α cytokine secreted from human or murine MDDCs transfected with said purified RNA preparation that is no greater than the amount of IFN-α or TNF-α secreted by MDDCs transfected with negative controls that do not contain RNA.

2. The purified RNA preparation of claim 1 , wherein said in vitro-synthesized single-stranded mRNA molecules encode 2 or more, 3 or more, 4 or more, 5 or more, or 6 iPSC induction factors selected from the group consisting of OCT4, SOX2, KLF4, LIN28, NANOG, and a MYC protein selected from c-MYC, L-MYC and N-MYC.

3. The purified RNA preparation of claim 1 wherein said in vitro-synthesized single-stranded mRNA molecules encode the iPSC induction factors:
A) OCT4, SOX2, KLF4 and MYC; or
B) OCT4, SOX2, KLF4, MYC and LIN28; or
C) OCT4, SOX2, KLF4, MYC and NANOG; or
D) OCT4, SOX2, KLF4, MYC, LIN28 and NANOG;
wherein said MYC in A) D) is selected from c-MYC, L-MYC and N-MYC.

4. The purified RNA preparation of claim 1 , wherein said purification comprises at least one purification process selected from the group consisting of:
A) HPLC or gravity flow column chromatography;
B) treating a composition comprising said in vitro-synthesized RNA molecules with one or more enzymes that specifically digest one or more RNA contaminant molecules or contaminant DNA molecules; and
C) treating a composition comprising said in vitro-synthesized RNA molecules with a ribonuclease III (RNase III) enzyme such that short RNase III digestion products are generated, and purifying said short RNase III digestion products away from said mRNA molecules.

5. The purified RNA preparation of claim 4 , wherein said purification further comprises collecting and analyzing fractions from said purification processes for:
(i) RNA contaminant molecules,
(ii) DNA contaminant molecules, and
(iii) reduced immunogenicity using an in vitro MDDC immunogenicity assay, by measuring less secretion of IFN-α or TNF-α from MDDCs transfected with in vitro-synthesized RNA molecules in more purified fractions.

6. The purified RNA preparation of claim 4 , wherein the result of said purification process is that said in vitro-synthesized mRNA molecules can be repeatedly administered without eliciting an immune response sufficient to eliminate detectable expression of the recombinant protein or that said in vitro-synthesized mRNA lacks immunogenicity, enabling repeated delivery without generation of inflammatory cytokines.

7. The purified RNA preparation of claim 1 , wherein at least one of the following applies:
(i) greater than 98% of said mRNA molecules comprise a 5′ cap;
(ii) said mRNA molecules comprise a cap with a cap0 structure;
(iii) said mRNA molecules comprise a cap with a cap1 structure;
(iv) all or substantially all said mRNA molecules comprise a poly(A) tail having 50-200 or greater than 150 nucleotides;
(v) said mRNA molecules comprise at least one particular sequence selected from the group consisting of: a heterologous 5′ UTR sequence, a Kozak sequence, an IRES sequence, and a 3′ UTR sequence;
(vi) said 5′ UTR sequence or 3′ UTR sequence is from a Xenopus or human alpha globin or beta globin mRNA, or wherein said 5′ UTR sequence is a sequence from a tobacco etch virus (TEV) RNA;
(vii) said RNA contaminant molecules that are removed from said in vitro-synthesized mRNA molecules using said purification process are selected from the group consisting of double-stranded RNA (dsRNA) molecules and un-capped mRNA molecules; or
(viii) less than 0.01% of the total RNA in said purified RNA preparation consists of double-stranded RNA contaminant molecules.

8. The purified RNA preparation of claim 1 , further comprising a transfection reagent.

9. The purified RNA preparation of claim 1 , further comprising a mammalian somatic cell, wherein at least some of said in vitro-synthesized single-stranded mRNA molecules are inside said cell.

10. The purified RNA preparation of claim 3 , further comprising a mammalian somatic cell, wherein at least some of said in vitro-synthesized single-stranded mRNA molecules are inside said cell.

11. The purified RNA preparation of claim 7 , further comprising a mammalian somatic cell, wherein at least some of said in vitro-synthesized single-stranded mRNA molecules are inside said cell.

12. A purified RNA preparation comprising a plurality of in vitro-synthesized single-stranded mRNA molecules that encode 2 or more, 3 or more, 4 or more, 5 or more, or 6 iPSC induction factors selected from the group consisting of OCT4, SOX2, KLF4, LIN28, NANOG, and a MYC protein selected from c-MYC, L-MYC and N-MYC;
wherein said in vitro-synthesized mRNA molecules:
a) comprise a modified nucleotide comprising a modified nucleoside in place of uridine that is selected from the group consisting of pseudouridine that is not further modified (Ψ), 1-methyl-pseudouridine (m 1 Ψ), 5-methyluridine (m 5 U), 5-methoxyuridine (mo 5 U) and 2-thiouridine (s 2 U); and
b) have been purified using a purification process that removes RNA contaminant molecules that are immunogenic and toxic to a mammalian cell by inducing an innate immune response, such that said purified in vitro-synthesized mRNA lacks immunogenicity, as can be determined using an in vitro monocyte-derived dendritic cell (MDDC) assay by measuring an amount of IFN-α or TNF-α secreted by MDDCs transfected with said purified in vitro-synthesized mRNA molecules that is no greater than the mean+the standard error of the mean (SEM) amount of IFN-α or TNF-α secreted by MDDCs transfected with negative controls that do not contain RNA.

13. The purified RNA preparation of claim 12 , wherein said in vitro-synthesized mRNA molecules encode the iPSC induction factors:
A) OCT4, SOX2, KLF4 and MYC; or
B) OCT4, SOX2, KLF4, MYC and LIN28; or
C) OCT4, SOX2, KLF4, MYC and NANOG; or
D) OCT4, SOX2, KLF4, MYC, LIN28 and NANOG;
wherein said MYC in A) D) is selected from c-MYC, L-MYC and N-MYC.

14. The purified RNA preparation of claim 12 , wherein said purification comprises at least one purification process selected from the group consisting of:
A) HPLC or gravity flow column chromatography;
B) treating a composition comprising said in vitro-synthesized RNA molecules with one or more enzymes that specifically digest one or more RNA contaminant molecules or contaminant DNA molecules; and
C) treating a composition comprising said in vitro-synthesized RNA molecules with a ribonuclease III (RNase III) enzyme such that short RNase III digestion products are generated, and purifying said short RNase III digestion products away from said mRNA molecules,
wherein, said purification comprises collecting and analyzing fractions from said purification processes (i) for RNA contaminant molecules, (ii) for DNA contaminant molecules, and (iii) for reduced immunogenicity using an in vitro

Assignees

• Univ Pennsylvania

Inventors

• Kariko Katalin
• Weissman Drew
• Dahl Gary
• Person Anthony
• Meis Judith
• Jendrisak Jerome

Classifications

• C12N2310/335

  Modified T or U · CPC title

• A61K38/00

  Medicinal preparations containing peptides (peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00) · CPC title

• A61K48/0091

  Purification or manufacturing processes for gene therapy compositions · CPC title

• C12N9/22

  Ribonucleases {[RNase]; Deoxyribonucleases [DNase]} · CPC title

• C12N15/10 Primary

  Processes for the isolation, preparation or purification of DNA or RNA (chemical preparation of DNA or RNA C07H21/00; preparation of non-structural polynucleotides from microorganisms or with enzymes C12P19/34) · CPC title