# US 11,028,370 B2 · RNA preparations comprising purified modified RNA for reprogramming cells

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- Number: US 11,028,370 B2
- Document ID: US11028370B2
- Status: Granted
- Kind: B2
- Application number: 15/994,093
- Filed: 2018-05-31
- Published: 2018-09-20
- Granted: 2021-06-08
- Earliest claimed: 2009-12-07
- Applicants: The Trustees of the University of Pennsylvania
- Inventors: Katalin Kariko; Drew Weissman; Gary Dahl; Anthony Person; Judith Meis; Jerome Jendrisak
- Web page: https://patentgrep.com/patent/US11028370B2

## CPC

- C12N 5/0696: {Artificially induced pluripotent stem cells, e.g. iPS}
- A61K 38/1709: {from mammals}
- A61K 38/1816: {Erythropoietin [EPO]}
- A61K 38/44: Oxidoreductases (1)
- A61K 38/465: {acting on ester bonds (3.1), e.g. lipases, ribonucleases}
- A61K 38/50: acting on carbon-nitrogen bonds, other than peptide bonds (3.5), e.g. asparaginase
- A61K 48/005: {characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered}
- A61K 48/0075: {characterised by an aspect of the delivery route, e.g. oral, subcutaneous}
- A61P 7/04: Antihaemorrhagics; Procoagulants; Haemostatic agents; Antifibrinolytic agents
- A61P 9/00: Drugs for disorders of the cardiovascular system
- A61P 11/00: Drugs for disorders of the respiratory system
- A61P 17/14: for baldness or alopecia
- A61P 25/28: for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
- A61P 37/04: Immunostimulants
- A61P 37/06: Immunosuppressants, e.g. drugs for graft rejection
- C07K 14/47: from mammals
- C07K 14/4712: {Cystic fibrosis}
- C07K 14/505: Erythropoietin [EPO]
- C12N 9/0075: {Nitric-oxide synthase (1.14.13.39)}
- C12N 9/22: Ribonucleases {[RNase]; Deoxyribonucleases [DNase]}
- C12N 9/78: acting on carbon to nitrogen bonds other than peptide bonds (3.5)
- C12N 15/117: Nucleic acids having immunomodulatory properties, e.g. containing CpG-motifs
- C12N 15/85: for animal cells
- C12N 15/87: Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
- C12N 2310/17: Immunomodulatory nucleic acids
- C12N 2310/335: Modified T or U
- C12N 2320/30: Special therapeutic applications
- C12N 2501/602: Sox-2
- C12N 2501/603: Oct-3/4
- C12N 2501/604: Klf-4
- C12N 2501/605: Nanog
- C12N 2501/606: c-Myc
- C12N 2501/608: Lin28
- C12N 2506/02: from embryonic cells
- C12Y 114/13039: Nitric-oxide synthase (NADPH dependent) (1.14.13.39)
- C12Y 301/04012: Sphingomyelin phosphodiesterase (3.1.4.12)
- C12Y 305/04004: Adenosine deaminase (3.5.4.4)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)
- C12Y: ENZYMES

## 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.

## Claims

### 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 (m1Ψ) 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.

### Claim 2 (depends on 1)

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.

### Claim 3 (depends on 1)

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.

### Claim 4 (depends on 1)

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.

### Claim 5 (depends on 4)

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.

### Claim 6 (depends on 4)

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.

### Claim 7 (depends on 1)

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.

### Claim 8 (depends on 1)

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

### Claim 9 (depends on 1)

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.

### Claim 10 (depends on 3)

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.

### Claim 11 (depends on 7)

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.

### Claim 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 (m1Ψ), 5-methyluridine (m5U), 5-methoxyuridine (mo5U) and 2-thiouridine (s2U); 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.

### Claim 13 (depends on 12)

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.

### Claim 14 (depends on 12)

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 monocyte-derived dendritic cell (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.

### Claim 15 (depends on 12)

The purified RNA preparation of claim 12, 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;
(viii) less than 0.01% of the total RNA in said purified RNA preparation consists of double-stranded RNA contaminant molecules;
(ix) said in vitro-synthesized mRNA molecules further comprise m5C (5-methylcytidine) in place of cytidine; or
(x) said composition further comprises a transfection reagent.

### Claim 16 (depends on 12)

The purified RNA preparation of claim 12, further comprising a mammalian somatic cell, wherein at least some of said in vitro-synthesized single-stranded mRNA molecules are inside said cell.

### Claim 17 (depends on 13)

The purified RNA preparation of claim 13, further comprising a mammalian somatic cell, wherein at least some of said in vitro-synthesized single-stranded mRNA molecules are inside said cell.

### Claim 18

A composition comprising a mammalian somatic cell containing in vitro-synthesized mRNA molecules,
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; and
wherein said in-vitro synthesized mRNA molecules comprise:
(i) guanosine,
(ii) adenosine,
(iii) cytidine, and
(iv) pseudouridine in place of uridine.

### Claim 19 (depends on 18)

The composition of claim 18, further comprising a transfection reagent.

### Claim 20 (depends on 19)

The composition of claim 19, wherein the transfection agent is selected from a liposome, a micelle, a nanoparticle, a nanotube, and a cationic compound.