# US 11,471,539 B2 · Genetic construct

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- Number: US 11,471,539 B2
- Document ID: US11471539B2
- Status: Granted
- Kind: B2
- Application number: 16/436,676
- Filed: 2019-06-10
- Published: 2020-02-06
- Granted: 2022-10-18
- Earliest claimed: 2015-10-26
- Assignees: Quethera Limited
- Applicants: Quethera Limited
- Inventors: Peter Widdowson; Keith Martin
- Web page: https://patentgrep.com/patent/US11471539B2

## 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 38/185: {Nerve growth factor [NGF]; Brain derived neurotrophic factor [BDNF]; Ciliary neurotrophic factor [CNTF]; Glial derived neurotrophic factor [GDNF]; Neurotrophins, e.g. NT-3}
- A61K 48/0058: {Nucleic acids adapted for tissue specific expression, e.g. having tissue specific promoters as part of a contruct}
- A61P 9/14: Vasoprotectives; Antihaemorrhoidals; Drugs for varicose therapy; Capillary stabilisers
- A61P 17/02: for treating wounds, ulcers, burns, scars, keloids, or the like
- A61P 25/00: Drugs for disorders of the nervous system
- A61P 25/02: for peripheral neuropathies
- A61P 27/00: Drugs for disorders of the senses
- A61P 27/02: Ophthalmic agents
- A61P 27/06: Antiglaucoma agents or miotics
- A61P 27/16: Otologicals
- C07K 14/48: Nerve growth factor [NGF]
- C12N 9/12: transferring phosphorus containing groups, e.g. kinases (2.7)
- C12N 15/1027: {by DNA shuffling, e.g. RSR, STEP, RPR}
- C12N 15/62: DNA sequences coding for fusion proteins
- C12N 15/85: for animal cells
- C12N 15/86: Viral vectors
- C12N 15/8645: {Adeno-associated virus}
- 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)
- A61K 48/00: Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
- C12N 2750/14132: Use of virus as therapeutic agent, other than vaccine, e.g. as cytolytic agent
- C12N 2750/14143: viral genome or elements thereof as genetic vector
- C12N 2830/008: cell type or tissue specific enhancer/promoter combination
- C12N 2840/203: having an IRES
- 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
- 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)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

The invention provides genetic constructs and recombinant vectors comprising such constructs. The constructs and vectors can be used in gene therapy methods for treating a range of disorders, including glaucoma and deafness, or for promoting nerve regeneration and/or survival.

## Claims

### Claim 1

A genetic construct comprising a promoter operably linked to a first coding sequence, which encodes the tyrosine kinase receptor B (TrkB), and a second coding sequence, which encodes an agonist of the TrkB receptor, wherein the agonist is mature BDNF and wherein the genetic construct comprises a spacer sequence disposed between the first and second coding sequences, which spacer sequence encodes a peptide spacer that is configured to be digested to thereby produce the TrkB receptor and agonist as separate molecules.

### Claim 2 (depends on 1)

A genetic construct according to claim 1, wherein the promoter is the human synapsin I (SYN I) promoter or the CAG promoter.

### Claim 3 (depends on 1)

A genetic construct according to claim 1, wherein the spacer sequence comprises and encodes a viral peptide spacer sequence.

### Claim 4 (depends on 1)

A genetic construct according to claim 1, wherein the peptide spacer sequence comprises an amino acid sequence substantially as set out in SEQ ID NO. 4, or a fragment or variant thereof; or (ii) an amino acid sequence substantially as set out in SEQ ID NO. 6, or a fragment or variant thereof; or (iii) an amino acid sequence substantially as set out in SEQ ID NO. 8, or a fragment or variant thereof.

### Claim 5 (depends on 1)

A genetic construct according to claim 1, wherein the spacer sequence comprises: (i) a nucleotide sequence substantially as set out in SEQ ID NO. 5, or a fragment or variant thereof; or (ii) a nucleotide sequence substantially as set out in SEQ ID NO. 7, or a fragment or variant thereof.

### Claim 6 (depends on 1)

A genetic construct according to claim 1, wherein the first coding sequence comprises a nucleotide sequence encoding the human canonical isoform of TrkB comprising an amino acid sequence substantially as set out in SEQ ID NO. 9, or a fragment or variant thereof, and/or wherein the first coding sequence comprises a nucleotide sequence substantially as set out in SEQ ID NO. 10, or a fragment or variant thereof.

### Claim 7 (depends on 1)

A genetic construct according to claim 1, wherein the first coding sequence comprises a nucleotide sequence which encodes isoform 4 of TrkB, and (i) wherein isoform 4 of TrkB comprises an amino acid sequence substantially as set out in SEQ ID NO. 11, or a fragment or variant thereof, and/or (ii) wherein the first coding sequence comprises a nucleotide sequence substantially as set out in SEQ ID NO. 12, or a fragment or variant thereof.

### Claim 8 (depends on 1)

A genetic construct according to claim 1, wherein the first coding sequence comprises a nucleotide sequence encoding a mutant form of TrkB receptor, wherein one or more tyrosine residue at position 516, 701, 705, 706 and/or 816 of SEQ ID No: 9 is modified or mutated, and/or wherein at least two, three or four tyrosine residues at position 516, 701, 705, 706 and/or 816 of SEQ ID No: 9 are modified, and/or wherein all five tyrosine residues at position 516, 701, 705, 706 and/or 816 of SEQ ID No: 9 are modified, and/or wherein the or each tyrosine residue is modified to a glutamic acid.

### Claim 9 (depends on 8)

A genetic construct according to claim 8, wherein the modified form of the TrkB receptor comprises an amino acid sequence substantially as set out in SEQ ID NO. 13, or a fragment or variant thereof, and/or wherein the first coding sequence comprises a nucleotide sequence substantially as set out in SEQ ID NO. 14, or a fragment or variant thereof.

### Claim 10 (depends on 1)

A genetic construct according to claim 1, wherein the second coding sequence comprises a nucleotide sequence which encodes mature BDNF, and/or wherein mature BDNF comprises an amino acid sequence substantially as set out in SEQ ID NO. 18, or a fragment or variant thereof, and/or wherein the second coding sequence comprises a nucleotide sequence substantially as set out in SEQ ID NO. 19, or a fragment or variant thereof.

### Claim 11 (depends on 1)

A genetic construct according to claim 1, wherein the second coding sequence comprises a nucleotide sequence encoding a signal peptide for the agonist of the TrkB receptor.

### Claim 12 (depends on 11)

A genetic construct according to claim 11, wherein the second coding sequence comprises a nucleotide sequence which encodes a signal peptide comprising an amino acid sequence substantially as set out in SEQ ID NO. 20, or a fragment or variant thereof, and/or wherein the second coding sequence comprises a nucleotide sequence substantially as set out in SEQ ID NO. 21, or a fragment or variant thereof.

### Claim 13 (depends on 1)

A genetic construct according to claim 1, wherein the second coding sequence comprises a nucleotide sequence encoding a signal sequence peptide substantially as set out in any one of SEQ ID NO. 23, 25, 27 or 29, or wherein the signal peptide comprises an amino acid sequence substantially as set out in any one of SEQ ID NO. 22, 24, 26 or 28, and/or wherein the second coding sequence comprises a nucleotide sequence encoding a signal sequence peptide substantially as set out in any one of SEQ ID NO. 31, 33, 35, 37, 39, 41, 43, 45, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101 or 103; or wherein the signal peptide comprises an amino acid sequence substantially as set out in any one of SEQ ID NO. 30, 32, 34, 36, 38, 40, 42, 44, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100 or 102.

### Claim 14 (depends on 1)

A genetic construct according to claim 1, wherein the construct comprises a nucleotide sequence substantially as set out in SEQ ID No: 107 or 108, or a fragment or variant thereof.

### Claim 15 (depends on 1)

A recombinant vector comprising the genetic construct according to claim 1, wherein the vector is a recombinant AAV (rAAV) vector, optionally wherein the rAAV is AAV-1, AAV-2, AAV-3A, AAV-3B , AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10 or AAV-11.

### Claim 16 (depends on 1)

A pharmaceutical composition comprising the genetic construct according to claim 1, and a pharmaceutically acceptable vehicle.

### Claim 17 (depends on 1)

A method of treating, preventing or ameliorating an optic nerve disorder or a cochlear disorder in a subject, or for promoting nerve regeneration and/or survival in a subject, the method comprising administering, to a subject in need of such treatment, a therapeutically effective amount of the genetic construct according to claim 1.

### Claim 18 (depends on 17)

The method according to claim 17, wherein the optic nerve disorder that is treated is any pathophysiological condition which results in loss of RGCs, optionally wherein the optic nerve disorder that is treated is glaucoma, or wherein the cochlear disorder which is treated is hearing loss or deafness.

### Claim 19 (depends on 3)

A genetic construct according to claim 3, wherein the viral peptide spacer sequence is a viral 2A peptide spacer sequence.

### Claim 20 (depends on 11)

A genetic construct according to claim 11, wherein signal peptide for BDNF, optionally wherein the nucleotide sequence encodes the canonical signal peptide for BDNF.

### Claim 21 (depends on 18)

The method according to claim 18, wherein the pathophysiological condition is trauma to the head or face or vascular insult.