# US 10,342,880 B2 · Genetic construct

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- Number: US 10,342,880 B2
- Document ID: US10342880B2
- Status: Granted
- Kind: B2
- Application number: 15/762,089
- Filed: 2016-10-25
- Published: 2018-12-13
- Granted: 2019-07-09
- Earliest claimed: 2015-10-26
- Assignees: Quethera Limited
- Applicants: Quethera Limited
- Inventors: Peter Widdowson; Keith Martin
- Web page: https://patentgrep.com/patent/US10342880B2

## 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 recombinant vector comprising 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 or mature NT-4, wherein the second coding sequence comprises a nucleotide sequence encoding a signal peptide which boosts secretion of the agonist of the TrkB receptor, 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 recombinant vector 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 recombinant vector according to claim 1, wherein the spacer sequence comprises and encodes a viral peptide spacer sequence, optionally a viral 2A peptide spacer sequence.

### Claim 4 (depends on 1)

A recombinant vector according to claim 1, wherein the peptide spacer sequence comprises: (i) an amino acid sequence substantially as set out in SEQ ID NO. 4, or a fragment or variant with at least 65% sequence identity to SEQ ID No: 4; or (ii) an amino acid sequence substantially as set out in SEQ ID NO. 6, or a fragment or variant with at least 65% sequence identity to SEQ ID No: 6; or (iii) an amino acid sequence substantially as set out in SEQ ID NO. 8, or a fragment or variant with at least 65% sequence identity to SEQ ID No: 8.

### Claim 5 (depends on 1)

A recombinant vector 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 with at least 65% sequence identity to SEQ ID No: 5; or (ii) a nucleotide sequence substantially as set out in SEQ ID NO. 7, or a fragment or variant with at least 65% sequence identity to SEQ ID No: 7.

### Claim 6 (depends on 1)

A recombinant vector according to claim 1, wherein the first coding sequence comprises a nucleotide sequence encoding the human canonical isoform of TrkB, wherein the canonical isoform of TrkB comprises an amino acid sequence as set out in SEQ ID NO. 9, or a fragment or variant with at least 65% sequence identity to SEQ ID No: 9.

### Claim 7 (depends on 1)

A recombinant vector according to claim 1, wherein the first coding sequence comprises a nucleotide sequence substantially as set out in SEQ ID NO. 10, or a fragment or variant with at least 65% sequence identity to SEQ ID No: 10.

### Claim 8 (depends on 1)

A recombinant vector according to claim 1, wherein the first coding sequence comprises a nucleotide sequence which encodes isoform 4 of TrkB, and wherein isoform 4 of TrkB comprises an amino acid sequence substantially as set out in SEQ ID NO. 11, or a fragment or variant with at least 65% sequence identity to SEQ ID No: 11.

### Claim 9 (depends on 1)

A recombinant vector according to claim 1, wherein the first coding sequence comprises a nucleotide sequence substantially as set out in SEQ ID NO. 12, or a fragment or variant with at least 65% sequence identity to SEQ ID No: 12.

### Claim 10 (depends on 1)

A recombinant vector according to claim 1, wherein the first coding sequence comprises an amino acid sequence as set out in SEQ ID NO: 9, wherein one or more tyrosine residue at position 516, 701, 705, 706 and/or 816 of SEQ ID No: 9 is modified to a different amino acid residue, optionally wherein each tyrosine residue is modified to a glutamic acid.

### Claim 11 (depends on 10)

A recombinant vector according to claim 10, 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 with at least 65% sequence identity to SEQ ID No: 13.

### Claim 12 (depends on 1)

A recombinant vector according to claim 1, wherein the second coding sequence encodes neurotrophin-4 (NT-4), which comprises an amino acid sequence substantially as set out in SEQ ID NO: 49 or 55, or a fragment or variant with at least 65% sequence identity to SEQ ID No: 49 or 55, and/or the second coding sequence comprises a nucleotide sequence substantially as set out in SEQ ID No: 50 or 56, or a fragment or variant with at least 65% sequence identity to SEQ ID No: 50 to 56.

### Claim 13 (depends on 1)

A recombinant vector according to claim 1, wherein the second coding sequence comprises a nucleotide sequence which encodes mature BDNF comprising an amino acid sequence substantially as set out in SEQ ID NO. 18, or a fragment or variant with at least 65% sequence identity to SEQ ID No: 18.

### Claim 14 (depends on 1)

A recombinant vector according to claim 1, wherein the second coding sequence comprises a nucleotide sequence substantially as set out in SEQ ID NO. 19, or a fragment or variant with at least 65% sequence identity to SEQ ID No: 19.

### Claim 15 (depends on 1)

A recombinant vector according to claim 1, wherein the second coding sequence comprises a nucleotide sequence encoding a signal peptide for the agonist of the TrkB receptor, optionally a signal peptide for BDNF.

### Claim 16 (depends on 15)

A recombinant vector according to claim 15, wherein the nucleotide sequence encodes the canonical signal peptide for BDNF, wherein the second coding sequence comprises a nucleotide sequence which encodes a signal peptide comprising an amino acid sequence as set out in SEQ ID NO. 20, or a fragment or variant with at least 65% sequence identity to SEQ ID No: 20.

### Claim 17 (depends on 1)

A recombinant vector according to claim 1, wherein the vector is a recombinant AAV (rAAV) vector, optionally rAAV serotype-2.

### Claim 18 (depends on 1)

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

### Claim 19 (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 recombinant vector according to claim 1.

### Claim 20 (depends on 19)

The method according to claim 19, wherein the optic nerve disorder that is treated is any pathophysiological condition which results in loss of RGCs, such as trauma to the head or face or vascular insults, for example partial or complete loss in blood supply to the ocular structures or regions of the brain which receive input from the optic nerve, or is used to support replacement of RGCs through introduction of untransformed or transformed stem cell into the eye or regions associated with vision in patients.

### Claim 21 (depends on 20)

The method according to claim 20, wherein the optic nerve disorder that is treated is glaucoma, or wherein the cochlear disorder which is treated is hearing loss or deafness.