# US 10,308,705 B2 · Optimized human clotting factor VIII gene expression cassettes and their use

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- Number: US 10,308,705 B2
- Document ID: US10308705B2
- Status: Granted
- Kind: B2
- Application number: 15/017,015
- Filed: 2016-02-05
- Published: 2016-08-11
- Granted: 2019-06-04
- Earliest claimed: 2015-02-06
- Assignees: The University of North Carolina at Chapel Hill
- Applicants: The University of North Carolina at Chapel Hill
- Inventors: Xiao Xiao; Juan Li; Zhenhua Yuan
- Web page: https://patentgrep.com/patent/US10308705B2

## CPC

- C07K 14/755: Factors VIII {, e.g. factor VIII C (AHF), factor VIII Ag (VWF)}
- A61P 7/04: Antihaemorrhagics; Procoagulants; Haemostatic agents; Antifibrinolytic agents
- C12N 15/63: Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12P 21/02: having a known sequence of two or more amino acids, e.g. glutathione
- A61K 38/00: 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)
- 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)
- 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)}
- 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)

## Abstract

The invention relates to synthetic liver-specific promoters and expression constructs for producing polypeptides and functional nucleic acids in the liver of a subject. The invention further relates to Factor VIII proteins containing modifications in the amino acid sequence of the Factor VIII protein, as well as nucleic acid constructs encoding the Factor VIII proteins and methods of using these compositions to treat a bleeding disorder.

## Claims

### Claim 1

An isolated polynucleotide comprising a synthetic liver specific promoter, wherein the promoter comprises the nucleotide sequence as set forth in SEQ ID NO: 1.

### Claim 2 (depends on 1)

The polynucleotide of claim 1, wherein the promoter is operably linked to an intron.

### Claim 3 (depends on 2)

The polynucleotide of claim 2, wherein the intron is from heavy chain variable region (VH) 4 (VH4).

### Claim 4 (depends on 3)

The polynucleotide of claim 3, wherein the promoter and the intron together comprise the nucleotide sequence as set forth in SEQ ID NO: 2.

### Claim 5 (depends on 1)

The polynucleotide of claim 1, wherein the promoter is operably linked to a polynucleotide of interest.

### Claim 6 (depends on 5)

The polynucleotide of claim 5, wherein the polynucleotide of interest encodes a polypeptide or a functional nucleic acid.

### Claim 7 (depends on 6)

The polynucleotide of claim 6, wherein the polynucleotide of interest encodes Factor VIII.

### Claim 8 (depends on 1)

A vector comprising the polynucleotide of claim 1.

### Claim 9 (depends on 1)

An isolated transformed cell comprising the polynucleotide of claim 1.

### Claim 10 (depends on 6)

The polynucleotide of claim 6, wherein the polynucleotide of interest encodes a clotting factor.

### Claim 11 (depends on 7)

The polynucleotide of claim 7, wherein the polynucleotide interest encodes a B domain-deleted Factor VIII.

### Claim 12 (depends on 11)

The polynucleotide of claim 11, wherein the polynucleotide of interest comprises the nucleotide sequence as set forth in SEQ ID NO: 3.

### Claim 13 (depends on 11)

The polynucleotide of claim 11, wherein the promoter and the polynucleotide of interest comprises the nucleotide sequence as set forth in SEQ ID NO: 4.

### Claim 14 (depends on 1)

The polynucleotide of claim 1, further comprising a polyadenylation site downstream of the polynucleotide of interest.

### Claim 15 (depends on 8)

The vector of claim 8, wherein the vector is a viral vector.

### Claim 16 (depends on 14)

The vector of claim 14, wherein the vector is an adeno-associated virus vector.