# US 2023/0054471 A1 · Long poly(a) plasmids and methods for introduction of long poly(a) sequences into the plasmid

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- Number: US 2023/0054471 A1
- Document ID: US20230054471A1
- Status: Published application
- Kind: A1
- Application number: 17/811,685
- Filed: 2022-07-11
- Published: 2023-02-23
- Earliest claimed: 2014-10-09
- Assignees: Seattle Children's Hospital (dba Seattle Children's Research Institute)
- Applicants: Seattle Children's Hospital (dba Seattle Children's Research Institute)
- Inventors: Andrew M. Scharenberg; Kyle Jacoby; Alexandra E. Grier
- Web page: https://patentgrep.com/patent/US20230054471A1

## CPC

- A61K 48/0066: {Manipulation of the nucleic acid to modify its expression pattern, e.g. enhance its duration of expression, achieved by the presence of particular introns in the delivered nucleic acid}
- A61K 41/0047: {Sonopheresis, i.e. ultrasonically-enhanced transdermal delivery, electroporation of a pharmacologically active agent}
- C12N 15/63: Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N 15/67: General methods for enhancing the expression
- C12P 19/34: Polynucleotides, e.g. nucleic acids, oligoribonucleotides
- C12N 2710/10343: viral genome or elements thereof as genetic vector
- C12N 2750/14143: viral genome or elements thereof as genetic vector
- 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)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C12P: FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE {(brewing of beer C12C; producing vinegar C12J; producing specific peptides or proteins C07K; producing enzymes C12N9/00; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; measuring or testing processes involving enzymes or microorganisms C12Q; measuring or testing processes involving nucleic acid amplification reactions C12Q1/6844; fermentation processes to form a food composition, A21 or A23; compounds in general, see the relevant compound class, e.g. C01, C07)}

## Abstract

Disclosed herein are polynucleotides having a plurality of thymine nucleotides and an endonuclease recognition site inserted therein, methods of engineering the polynucleotides having a plurality of thymine nucleotides and an endonuclease recognition site inserted therein, and methods of enhancing transcription, translation, and increasing stability of a polynucleotide.

## Claims

### Claim 1

(canceled)

### Claim 2

An isolated polynucleotide encoding a polypeptide, the polynucleotide comprising:
(a) a first nucleic acid encoding the polypeptide; and
(b) a second nucleic acid comprising a plurality of thymine nucleotides of at least 130 consecutive thymine nucleotides, and an endonuclease cleavage site located within the plurality of thymine nucleotides or within 40 consecutive nucleotides from the 5′ terminal thymine of the plurality of thymine nucleotides, and
(c) a third nucleic acid comprising an endonuclease recognition site, wherein the endonuclease recognition site is within 55 consecutive nucleotides of the endonuclease cleavage site, wherein the second nucleic acid is covalently linked to the third nucleic acid at one end of the third nucleic acid.

### Claim 3 (depends on 2)

The isolated polynucleotide of claim 2, wherein the polypeptide comprises a nuclease, a chimeric antigen receptor (CAR), an antigen for a cancer vaccine, an antigen for an infectious disease, or a protein for a replacement protein therapy.

### Claim 4 (depends on 3)

The isolated polynucleotide of claim 3, wherein the polypeptide comprises an antigen for an infectious disease vaccine.

### Claim 5 (depends on 2)

The isolated polynucleotide of claim 2, wherein the first nucleic acid is codon optimized for expression in a human cell.

### Claim 6 (depends on 2)

The isolated polynucleotide of claim 2, further comprising a promoter operably linked to the first nucleic acid.

### Claim 7 (depends on 2)

The isolated polynucleotide of claim 2, wherein the endonuclease recognition site is a Type II restriction endonuclease recognition site.

### Claim 8 (depends on 7)

The isolated polynucleotide of claim 7, wherein the endonuclease recognition site is a Type II restriction endonuclease recognition site is a BsaI recognition site or a StuI recognition site.

### Claim 9 (depends on 2)

The isolated polynucleotide of claim 2, further comprising a telomere.

### Claim 10 (depends on 2)

The isolated polynucleotide of claim 2 prepared by a method comprising:
(i) obtaining a linear plasmid comprising a telomere, and a double-stranded nucleic acid comprising at least 130 consecutive thymine nucleotides; and
(ii) inserting the double-stranded nucleic acid into the vector, thereby preparing the isolated polynucleotide.

### Claim 11 (depends on 2)

A linear vector comprising the isolated polynucleotide of claim 2.

### Claim 12 (depends on 2)

A method for preparing a therapeutic polynucleotide, comprising:
contacting the isolated polynucleotide of claim 2 with a polymerase and a plurality of nucleotides, thereby obtaining the therapeutic polynucleotide.

### Claim 13 (depends on 12)

The method of claim 12, further comprising preparing the isolated polynucleotide comprising:
(i) obtaining a linear plasmid comprising a telomere, and a double-stranded nucleic acid comprising at least 130 consecutive thymine nucleotides; and
(iii) inserting the double-stranded nucleic acid into the vector, thereby preparing the isolated polynucleotide.

### Claim 14 (depends on 12)

The method of claim 12, wherein the therapeutic polynucleotide is RNA.

### Claim 15 (depends on 12)

A pharmaceutical composition comprising a therapeutic polynucleotide prepared according to the method of claim 12, and a pharmaceutically acceptable vehicle.

### Claim 16 (depends on 12)

A method for treating, ameliorating or inhibiting a disorder in a subject, comprising: administering a therapeutic polynucleotide to the subject, wherein the therapeutic polynucleotide is prepared according to the method of claim 12.

### Claim 17 (depends on 16)

The method of claim 16, further comprising preparing the therapeutic polynucleotide.

### Claim 18 (depends on 16)

The method of claim 16, wherein the polypeptide is a nuclease, a chimeric antigen receptor (CAR), an antigen for a cancer vaccine, an antigen for an infectious disease, or a protein for a replacement protein therapy.

### Claim 19 (depends on 18)

The method of claim 18, wherein the polypeptide comprises an antigen for an infectious disease.

### Claim 20 (depends on 16)

The method of claim 16, wherein the disorder comprises an infectious disorder.

### Claim 21 (depends on 16)

The method of claim 16, wherein the subject is human.