# US 2023/0055020 A1 · Adeno associated virus based gene therapy for phenylketonuria

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- Number: US 2023/0055020 A1
- Document ID: US20230055020A1
- Status: Published application
- Kind: A1
- Application number: 17/792,100
- Filed: 2021-01-15
- Published: 2023-02-23
- Earliest claimed: 2020-01-16
- Assignees: Takeda Pharmaceutical Company Limited
- Applicants: Takeda Pharmaceutical Company Limited
- Inventors: Matthias KLUGMANN; Hanspeter ROTTENSTEINER; Franziska HORLING; Johannes LANGLER
- Web page: https://patentgrep.com/patent/US20230055020A1

## CPC

- C12N 15/86: Viral vectors
- A61K 48/005: {characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered}
- C12N 9/0071: {acting on paired donors with incorporation of molecular oxygen (1.14)}
- C12Y 114/16001: Phenylalanine 4-monooxygenase (1.14.16.1)
- A61K 48/0058: {Nucleic acids adapted for tissue specific expression, e.g. having tissue specific promoters as part of a contruct}
- 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}
- C12N 2750/14122: New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
- C12N 2750/14143: viral genome or elements thereof as genetic vector
- C12N 2750/14145: Special targeting system for viral vectors
- C12N 2800/22: Vectors comprising a coding region that has been codon optimised for expression in a respective host
- C12N 2830/008: cell type or tissue specific enhancer/promoter combination
- C12N 2830/42: being an intron or intervening sequence for splicing and/or stability of RNA
- C12N 2830/48: regulating transport or export of RNA, e.g. RRE, PRE, WPRE, CTE
- 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)
- C12Y: ENZYMES

## Abstract

The present disclosure provides, among other things, a recombinant adeno-associated virus (rAAV) vector comprising an AAV8 capsid and a codon-optimized sequence encoding a human phenylalanine hydroxylase (PAH) enzyme. The disclosure also provides a method of treating a subject having phenylketonuria (PKU), comprising administering to the subject in need thereof a recombinant adeno-associated vims (rAAV) vector comprising an AAV8 capsid, and a promoter operably linked to a nucleic acid sequence that encodes PAH, and wherein administering results in a decrease in phenylalanine level in the subject.

## Claims

### Claim 1

A recombinant adeno-associated virus vector (rAAV) comprising a codon-optimized sequence encoding a human phenylalanine hydroxylase (PAH), wherein the codon-optimized sequence has at least 70% identity to one of SEQ ID Nos: 11-27.

### Claim 2 (depends on 1)

The rAAV of claim 1, wherein the codon-optimized sequence has at least 75%, 80%, 85%, 90%, 95% or 99% identity to one of SEQ ID Nos: 11-27.

### Claim 3 (depends on 1, 2)

The rAAV of claim 1 or 2, wherein the codon-optimized sequence is identical to one of SEQ ID Nos: 11-27.

### Claim 4

The rAAV of any one of the preceding claims, wherein the rAAV encodes an AAV8 capsid.

### Claim 5 (depends on 4)

The rAAV of claim 4, wherein the AAV8 capsid is a modified AAV8 capsid with improved liver tropism compared to the wild-type AAV8 capsid.

### Claim 6 (depends on 5)

The rAAV of claim 5, wherein the AAV8 capsid has at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identity to the wild-type AAV8 capsid.

### Claim 7

The rAAV of any one of the preceding claims, wherein the rAAV further comprises a WPRE sequence.

### Claim 8 (depends on 7)

The rAAV of claim 7, wherein the WPRE sequence is a naturally-occurring WPRE sequence.

### Claim 9 (depends on 7)

The rAAV of claim 7, wherein the WPRE sequence is a modified WPRE sequence.

### Claim 10

The rAAV of claim of any one of the preceding claims, wherein the rAAV further comprises a liver-specific promoter.

### Claim 11 (depends on 10)

The rAAV of claim 10, wherein the liver-specific promoter is a transthyretin promotor (TTR).

### Claim 12

The rAAV of any one of the preceding claims, wherein the rAAV comprises a cis-acting regulatory module (CRM).

### Claim 13 (depends on 12)

The rAAV of claim 12, wherein the vector comprises one, two, three, four, five or more CRM repeats.

### Claim 14 (depends on 12, 13)

The rAAV of claim 12 or 13, wherein the CRM is CRM8.

### Claim 15

The rAAV of any one of the preceding claims, wherein the rAAV further comprises an intron upstream of the PAH sequence.

### Claim 16 (depends on 15)

The rAAV of claim 15, wherein the intron is a minute virus of mice (MVM) intron.

### Claim 17

A method of treating phenylketonuria (PKU), comprising administering to a subject in need of treatment a recombinant adeno-associated virus (rAAV) comprising a codon-optimized sequence encoding a human phenylalanine hydroxylase (PAH), wherein the codon-optimized sequence has at least 70% identity to one of SEQ ID Nos: 11-27.

### Claim 18 (depends on 17)

The method of claim 17, wherein administering the rAAV results in a decrease in plasma phenylalanine (Phe) level in the subject compared to a control.

### Claim 19 (depends on 17, 18)

The method of claim 17 or 18, wherein administering the rAAV results in an increase in plasma tyrosine level in the subject compared to a control.

### Claim 20 (depends on 17, 18, 19)

The method of any one of claims 17-19, wherein administering the rAAV results in an increase in plasma tryptophan level in the subject compared to a control.

### Claim 21 (depends on 18, 19, 20)

The method of any one of claims 18-20, wherein the control is the pre-treatment level in the subject.

### Claim 22 (depends on 18, 19, 20)

The method of any one of claims 18-20, wherein the control is a reference level based on historical data.

### Claim 23 (depends on 17, 18, 19, 20, 21, 22)

The method of any one of claims 17-22, wherein the codon-optimized sequence has at least 75%, 80%, 85%, 90%, 95% or 99% identity to one of SEQ ID Nos: 11-27.

### Claim 24 (depends on 17, 18, 19, 20, 21, 22, 23)

The method of any one of claims 17-23, wherein the codon-optimized sequence is identical to one of SEQ ID Nos: 11-27.

### Claim 25 (depends on 17, 18, 19, 20, 21, 22, 23, 24)

The method of any one of claims 17-24, wherein the rAAV encodes an AAV8 capsid.

### Claim 26 (depends on 25)

The method of claim 25, wherein the AAV8 capsid is a modified AAV8 capsid with improved liver tropism compared to the wild-type AAV8 capsid.

### Claim 27 (depends on 25)

The method of claim 25, wherein the AAV8 capsid has at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identity to the wild-type AAV capsid.

### Claim 28 (depends on 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27)

The method of any one of claims 17-27, wherein the rAAV further comprises a WPRE sequence.

### Claim 29 (depends on 28)

The method of claim 28, wherein the WPRE sequence is a naturally-occurring WPRE sequence.

### Claim 30 (depends on 28)

The method of claim 28, wherein the WPRE sequence is a modified WPRE sequence.

### Claim 31 (depends on 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30)

The method of any one of claims 17-30, wherein the rAAV further comprises a liver-specific promoter.

### Claim 32 (depends on 31)

The method of claim 31, wherein the liver-specific promoter is a transthyretin promotor (TTR).

### Claim 33 (depends on 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32)

The method of any one of claims 17-32, wherein the rAAV comprises a cis-acting regulatory module (CRM).

### Claim 34 (depends on 33)

The method of claim 33, wherein the vector comprises one, two, three, four, five or more CRM repeats.

### Claim 35 (depends on 33, 34)

The method of claim 33 or 34, wherein the CRM is CRM8.

### Claim 36 (depends on 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35)

The method of any one of claims 17-35, wherein the rAAV further comprises an intron upstream of the PAH sequence.

### Claim 37 (depends on 36)

The method of claim 36, wherein the intron is a minute virus of mice (MVM) intron.

### Claim 38 (depends on 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37)

The method of any one of claims 17-37, wherein the rAAV is administered at dose of about 1×1010 vg/kg, about 1×1011 vg/kg, about 1×1012 vg/kg, about 1×1013 vg/kg, about 1×1014 vg/kg, or about 1×1015 vg/kg.

### Claim 39 (depends on 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38)

The method of any one of claims 17-38, wherein the rAAV is administered systemically.

### Claim 40 (depends on 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38)

The method of any one of claims 17-38, wherein the rAAV is administered intravenously.