# US 11,110,153 B2 · Modified factor IX, and compositions, methods and uses for gene transfer to cells, organs, and tissues

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- Number: US 11,110,153 B2
- Document ID: US11110153B2
- Status: Granted
- Kind: B2
- Application number: 17/014,782
- Filed: 2020-09-08
- Published: 2021-01-14
- Granted: 2021-09-07
- Earliest claimed: 2015-06-23
- Assignees: The Children's Hospital of Philadelphia
- Applicants: The Children's Hospital of Philadelphia
- Inventors: Katherine A. High; Xavier Anguela
- Web page: https://patentgrep.com/patent/US11110153B2

## CPC

- A61K 38/4846: {Factor VII (3.4.21.21); Factor IX (3.4.21.22); Factor Xa (3.4.21.6); Factor XI (3.4.21.27); Factor XII (3.4.21.38)}
- A61P 7/04: Antihaemorrhagics; Procoagulants; Haemostatic agents; Antifibrinolytic agents
- C12N 9/644: {Coagulation factor IXa (3.4.21.22)}
- C12N 15/86: Viral vectors
- C12Y 304/21022: Coagulation factor IXa (3.4.21.22)
- 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)
- 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)
- C12Y: ENZYMES

## Abstract

The invention relates to modified Factor IX coding sequence, expression cassette, vectors such as viral (e.g., lenti- or adeno-associated viral) vectors, and gene transfer methods and uses. In particular, to target Factor IX nucleic acid to cells, tissues or organs for expression (transcription) of Factor IX.

## Claims

### Claim 1

A recombinant adeno-associated virus (rAAV) vector comprising a vector genome encapsidated by an AAV capsid, wherein said vector genome comprises an AAV inverted terminal repeat (ITR) and a nucleic add sequence encoding human Factor IX (FIX) protein, wherein said nucleic add sequence encoding human FIX protein is at least 90% identical to SEQ ID NO:10, has a reduced number of CpG di-nucleotides compared to wild-type nucleic add sequence encoding human FIX protein and encodes the same human FIX protein encoded by SEQ ID NO: 10.

### Claim 2 (depends on 1)

The rAAV vector of claim 1, wherein said nucleic acid sequence encoding human FIX protein has 1-55 fewer CpG di-nucleotides compared to wild-type nucleic acid sequence encoding human FIX protein.

### Claim 3 (depends on 1)

The rAAV vector of claim 1, wherein said nucleic acid sequence encoding human FIX protein is devoid of any CpG di-nucleotides.

### Claim 4 (depends on 1)

The rAAV vector of claim 1, wherein said vector genome further comprises an expression control element operably linked with said nucleic acid sequence encoding human FIX protein.

### Claim 5 (depends on 4)

The rAAV vector of claim 4, wherein said expression control element comprises an enhancer or promoter conferring liver tissue-specific expression.

### Claim 6 (depends on 4)

The rAAV vector of claim 4, wherein said vector genome further comprises a polyadenylation signal sequence.

### Claim 7 (depends on 6)

The rAAV vector of claim 6, wherein said vector genome further comprises at least two AAV ITRs.

### Claim 8 (depends on 1)

The rAAV vector of claim 1, wherein said vector genome further comprises an intron.

### Claim 9 (depends on 8)

The rAAV vector of claim 8, wherein said intron is positioned within said nucleic acid sequence encoding human FIX protein.

### Claim 10 (depends on 1)

The rAAV vector of claim 1, wherein said vector genome further comprises an enhancer and promoter operably linked with said nucleic acid sequence encoding human FIX protein, a polyadenylation signal sequence and at least two AAV ITRs.

### Claim 11 (depends on 10)

The rAAV vector of claim 10, wherein said vector genome comprises, in 5′ to 3′ order, a first AAV ITR, said enhancer, said promoter, said nucleic acid sequence encoding human FIX protein, said polyadenylation signal sequence and a second AAV ITR.

### Claim 12 (depends on 10)

The rAAV vector of claim 10, wherein said enhancer is a human apolipoprotein HCR enhancer.

### Claim 13 (depends on 12)

The rAAV vector of claim 12, wherein said enhancer comprises the nucleic acid sequence of SEQ ID NO:14.

### Claim 14 (depends on 10)

The rAAV vector of claim 10, wherein said promoter is a human alpha-1-antitrypsin gene promoter.

### Claim 15 (depends on 14)

The rAAV vector of claim 14, wherein said promoter comprises the nucleic acid sequence of SEQ ID NO:15.

### Claim 16 (depends on 10)

The rAAV vector of claim 10, wherein said vector genome further comprises an intron.

### Claim 17 (depends on 16)

The rAAV vector of claim 16, wherein said intron is positioned within said nucleic acid sequence encoding human FIX protein.

### Claim 18 (depends on 10)

The rAAV vector of claim 10, wherein a first AAV ITR from AAV2 and a least second AAV ITR from AAV2 are respectively positioned at the 5′ and 3′ termini of the vector genome.

### Claim 19 (depends on 10)

The rAAV vector of claim 10, wherein said vector genome is single-stranded DNA.

### Claim 20 (depends on 10)

The rAAV vector of claim 10, wherein said AAV capsid is selected from the group consisting of:
(i) an AAV capsid of serotype AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, Rh10 or AAV-2i8;
(ii) an AAV capsid comprising a VP1 protein consisting of the amino acid sequence of SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8 or SEQ ID NO:9;
(iii) an AAV capsid comprising a VP1 protein consisting of the amino acid sequence of SEQ ID NO:1 with an amino acid substitution at any one or more of amino acid positions 195, 199, 201 or 202 in SEQ ID NO:1; and
(iv) an AAV capsid comprising a VP1 protein consisting of the amino acid sequence of SEQ ID NO:1 with an amino acid substitution of arginine for any one or more lysines in SEQ ID NO:1.

### Claim 21 (depends on 10)

The rAAV vector of claim 10, wherein said vector genome further comprises an intron between said promoter and said nucleic acid sequence encoding human FIX protein, wherein said enhancer is a human apolipoprotein HCR enhancer , wherein said promoter is a human alpha-1-antitrypsin gene promoter , wherein a first AAV ITR from AAV2 and a second AAV ITR from AAV2 are respectively positioned at the 5′ and 3′ termini of the vector genome, and wherein said capsid is AAV5.

### Claim 22 (depends on 10)

The rAAV vector of claim 10, wherein said capsid is AAV5.

### Claim 23 (depends on 22)

The rAAV vector of claim 22, wherein said promoter is a human alpha-1-antitrypsin gene promoter.

### Claim 24 (depends on 23)

The rAAV vector of claim 23, wherein said enhancer is a human apolipoprotein HCR enhancer.

### Claim 25 (depends on 10)

A method of treating a human subject with hemophilia B comprising administering to said subject a therapeutically effective amount of the rAAV vector of claim 10.

### Claim 26 (depends on 25)

The method of claim 25, wherein the therapeutically effective amount of said rAAV vector is a dose ranging from 1×1011 to 1×1014 vector genomes per kilogram of subject body weight.

### Claim 27 (depends on 25)

The method of claim 25, wherein said human subject has severe hemophilia B and wherein said treatment is effective to reduce the hemophilia symptoms from severe to those of moderate or mild hemophilia B.

### Claim 28 (depends on 25)

The method of claim 25, wherein said human subject has severe hemophilia B and wherein said treatment is effective to produce FIX activity that is at least 20% of normal for a sustained period of at least 12 months.