# US 2019/0169235 A1 · Modified protein encoding sequences having increased rare hexamer content

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- Number: US 2019/0169235 A1
- Document ID: US20190169235A1
- Status: Published application
- Kind: A1
- Application number: 15/773,685
- Filed: 2016-11-07
- Published: 2019-06-06
- Earliest claimed: 2015-11-05
- Assignees: The Research Foundation for The State University of New York
- Applicants: The Research Foundation for the State University of New York
- Inventors: Bruce Futcher; Justin Gardin; Steven Skiena; Alisa Yurovsky; Eckard Wimmer; Steffen Mueller
- Web page: https://patentgrep.com/patent/US20190169235A1

## CPC

- C07K 14/005: from viruses
- A61P 31/12: Antivirals
- C12N 7/00: Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof (preparing medicinal viral antigen or antibody compositions, e.g. virus vaccines, A61K39/00)
- C12N 2770/32621: Viruses as such, e.g. new isolates, mutants or their genomic sequences
- C12N 2770/32622: New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
- 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)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

This invention provides a modified protein encoding sequence containing nucleotide substitutions at multiple locations in the protein encoding sequence, wherein the substitutions introduce rare hexamers. These hexamers may be Frame Dependent, or depleted in only the reading frame, or Frame Independent, or depleted in all three frames. Modified protein encoding se quences of the present invention may include modified viruses useful for vaccines.

## Claims

### Claim 1

A modified protein encoding sequence comprising a polynucleotide sequence derived from a target protein encoding sequence, wherein the modified protein encoding sequence encodes a polypeptide having substantially the same amino acid sequence as the polypeptide encoded by the target protein encoding sequence and comprises a plurality of additional hexamers selected from one or more of the group consisting of SEQ ID NO:19 to SEQ ID NO:418 compared to the target protein encoding sequence.

### Claim 2 (depends on 1)

The modified protein encoding sequence of claim 1, wherein the plurality of hexamers comprises frame independent hexamers.

### Claim 3 (depends on 1)

The modified protein encoding sequence of claim 1, wherein the plurality of hexamers comprises frame dependent hexamers.

### Claim 4 (depends on 1)

The modified protein encoding sequence of claim 1, wherein the modified protein encoding sequence comprises more than about 50 additional hexamers selected from one or more of the group consisting of SEQ ID NO:19 to SEQ ID NO:418 compared to the target protein encoding sequence.

### Claim at position 5

5-9. (canceled)

### Claim 10 (depends on 1)

The modified protein encoding sequence of claim 1, wherein the modified protein encoding sequence has reduced expression in mammalian cells compared to the unmodified protein encoding sequence.

### Claim at position 7

11-12. (canceled)

### Claim 13 (depends on 1)

The modified protein encoding sequence of claim 1, wherein the hexamers are introduced by rearranging synonymous codons of the target protein encoding sequence.

### Claim 14 (depends on 1)

The modified protein encoding sequence of claim 1, wherein the hexamers are introduced by substituting synonymous codons of the target protein encoding sequence.

### Claim 15 (depends on 1)

The modified protein encoding sequence of claim 1, wherein the target protein encoding sequence encodes a viral protein.

### Claim 16 (depends on 15)

A modified virus comprising the modified protein encoding sequence of claim 15.

### Claim 17

A method for reducing the expression of a target protein comprising introducing into the target protein encoding sequence a plurality of hexamers selected from one or more of the group consisting of SEQ ID NO: 19 to SEQ ID NO: 418 without altering the polypeptide sequence encoded by the target protein encoding sequence.

### Claim 18 (depends on 17)

The method of claim 17, wherein the plurality of hexamers comprises frame dependent hexamers.

### Claim 19 (depends on 17)

The method of claim 17, wherein the plurality of hexamers comprises frame independent hexamers.

### Claim 20 (depends on 17)

The method of claim 17, wherein greater than about 50 hexamers are introduced into the target protein encoding sequence.

### Claim 21

(canceled)

### Claim 22 (depends on 17)

The method of claim 17, wherein hexamers are introduced by rearranging synonymous codons.

### Claim 23 (depends on 17)

The method of claim 17, wherein hexamers are introduced by substituting synonymous codons.

### Claim 24 (depends on 17)

The method of claim 17, wherein the target protein encoding sequence is a viral gene.

### Claim 25

A modified protein encoding sequence comprising a polynucleotide sequence derived from a target protein encoding sequence, wherein the modified protein encoding sequence encodes a polypeptide having substantially the same amino acid sequence as the polypeptide encoded by the target protein encoding sequence and comprises at least one of: a plurality of frame dependent hexamers each having a frame dependent score less than about −0.51 and a plurality of frame independent hexamers each having a frame independent score less than about −0.33.

### Claim at position 21

26-28. (canceled)

### Claim 29 (depends on 25)

The modified protein encoding sequence of claim 25, wherein the modified protein encoding sequence comprises more than about 50 additional hexamers selected from one or more of the group consisting SEQ ID NO:19 to SEQ ID NO:418 compared to the target protein encoding sequence.

### Claim at position 23

30-35. (canceled)

### Claim 36 (depends on 25)

The modified protein encoding sequence of claim 25, wherein the modified protein encoding sequence has reduced expression in mammalian cells compared to the unmodified protein encoding sequence. 37-38. (cancelled).

### Claim 39 (depends on 25)

The modified protein encoding sequence of claim 25, wherein the hexamers are introduced by rearranging synonymous codons of the target protein encoding sequence.

### Claim 40 (depends on 25)

The modified protein encoding sequence of claim 25, wherein the hexamers are introduced by substituting synonymous codons of the target protein encoding sequence.

### Claim 41 (depends on 25)

The modified protein encoding sequence of claim 25, wherein the target protein encoding sequence encodes a viral protein.