# US 2016/0074533 A1 · Vector encoding therapeutic polypeptide and safety elements to clear transduced cells

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- Number: US 2016/0074533 A1
- Document ID: US20160074533A1
- Status: Published application
- Kind: A1
- Application number: 14/949,639
- Filed: 2015-11-23
- Published: 2016-03-17
- Earliest claimed: 2007-03-27
- Assignees: University Health Network
- Applicants: University Health Network
- Inventors: Jeffrey A. Medin
- Web page: https://patentgrep.com/patent/US20160074533A1

## CPC

- A61K 48/005: {characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered}
- A61K 47/6825: {Ribosomal inhibitory proteins, i.e. RIP-I or RIP-II, e.g. Pap, gelonin or dianthin}
- A61K 47/6849: {the antibody targeting a receptor, a cell surface antigen or a cell surface determinant}
- A61K 48/0083: {characterised by an aspect of the administration regime}
- A61P 3/00: Drugs for disorders of the metabolism (of the blood or the extracellular fluid A61P7/00)
- A61P 35/00: Antineoplastic agents
- A61P 37/04: Immunostimulants
- A61P 37/06: Immunosuppressants, e.g. drugs for graft rejection
- C07K 16/2866: {against receptors for cytokines, lymphokines, interferons}
- C12N 9/1211: {Thymidine kinase (2.7.1.21)}
- C12N 15/86: Viral vectors
- A61K 2039/505: {comprising antibodies}
- C07K 2319/55: containing a fusion with a toxin, e.g. diphteria toxin
- C07K 2319/74: containing a fusion for binding to a cell surface receptor
- C12N 2740/15033: Use of viral protein as therapeutic agent other than vaccine, e.g. apoptosis inducing or anti-inflammatory
- C12N 2740/15043: viral genome or elements thereof as genetic vector
- C12N 2740/17033: Use of viral protein as therapeutic agent other than vaccine, e.g. apoptosis inducing or anti-inflammatory
- C12N 2740/17043: viral genome or elements thereof as genetic vector
- C12N 2750/14133: Use of viral protein as therapeutic agent other than vaccine, e.g. apoptosis inducing or anti-inflammatory
- C12N 2750/14143: viral genome or elements thereof as genetic vector
- C12N 2799/027: where the vector is derived from a retrovirus
- 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

A composition comprising: a stably integrating delivery vector; a modified mammalian thymidylate kinase (tmpk) activator polynucleotide wherein the modified mammalian tmpk polynucleotide encodes a modified mammalian tmpk polypeptide that increases phosphorylation of a prodrug relative to phosphorylation of the prodrug by wild-type mammalian tmpk polypeptide to a drug; and/or a targeting polynucleotide encoding a cell surface polypeptide that selectively binds a toxic binding agent. The disclosure also relates to use of these compositions in methods of treatment of diseases such as Fabry disease.

## Claims

### Claim 1

A suicide gene system comprising:
a. a stably integrating delivery vector;
b. an activator polynucleotide encoding a polypeptide that converts a prodrug to a drug;
c. a docking polynucleotide encoding a cell surface polypeptide that selectively binds a toxic binding agent; and
d. a therapeutic α-galactosidase A polynucleotide;
wherein the suicide gene system induces death in a cell expressing the activator polynucleotide and/or docking polynucleotide when the cell is contacted with the prodrug and/or the toxic binding agent.

### Claim 2

A composition comprising:
a. a stably integrating delivery vector;
b. an activator polynucleotide encoding a polypeptide that converts a prodrug to a drug;
c. a docking polynucleotide encoding a docking polypeptide that selectively binds an antibody or an antibody conjugated with a toxic agent; and
d. a therapeutic α-galactosidase A polynucleotide.

### Claim 3 (depends on 2)

The composition of claim 2, wherein the activator polynucleotide comprises a tmpk polynucleotide with at least 80% sequence identity to a modified tmpk polynucleotide.

### Claim 4 (depends on 2, 3)

The composition of claim 2 or 3, wherein the polynucleotide comprises a mammalian polynucleotide, optionally a human polynucleotide, and the polypeptide comprise a mammalian polypeptide, optionally a human polypeptide.

### Claim 5 (depends on 2, 3, 4)

The composition any one of claims 2 to 4, wherein the activator polynucleotide comprises a modified mammalian tmpk polynucleotide encoding a modified mammalian tmpk polypeptide that increases phosphorylation of a prodrug relative to phosphorylation of the prodrug by wild-type mammalian tmpk polypeptide, optionally the modified mammalian tmpk polynucleotide comprises a mammalian tmpk polynucleotide with a point mutation or multiple mutations.

### Claim 6 (depends on 5)

The composition of claim 5, wherein the point mutation comprises a mutation in a codon of the polynucleotide selected from the group consisting of a mutation that encodes a F to Y mutation at amino acid position 105, a mutation that encodes a R to G point mutation at amino acid position 16, and a mutation that encodes a R to A mutation at amino acid position 200 or combinations of the above.

### Claim 7 (depends on 6)

The composition of claim 6, wherein the polynucleotide further comprises all or part of the large lid or small lid domain of E. coli thymidine kinase.

### Claim 8 (depends on 2, 3, 4, 5)

The composition of any one of claims 2 to 5, wherein the modified mammalian tmpk polynucleotide has been modified by substituting a portion of wild-type tmpk polynucleotide sequence with an exogenous polynucleotide sequence.

### Claim 9 (depends on 8)

The composition of claim 8, wherein the substituted portion comprises all or part of a large lid or small lid domain from E. coli thymidine kinase.

### Claim 10 (depends on 2, 3, 4, 5, 6, 7, 8, 9)

The composition of any one of claims 2 to 9, wherein the activator polynucleotide and docking polynucleotide are fused and encode an activator/docking fusion.

### Claim 11 (depends on 2, 3, 4, 5, 6, 7, 8, 9)

The composition of any one of claims 2 to 9, further comprising a detection cassette comprising a polynucleotide sequence different than the docking polynucleotide.

### Claim 12 (depends on 2, 3, 4, 5, 6, 7, 8, 9, 10)

The composition of anyone of claims 2 to 10, wherein the docking polynucleotide encodes HSA, CD24, CD34, LNGFR, EpoR, CD19, CD25 or CD20, or a fragment thereof that binds an antibody or the toxic binding agent directly.

### Claim 13 (depends on 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12)

The composition of any one of claims 2 to 12, wherein the toxic binding agent comprises an antibody conjugated to a toxin.

### Claim 14 (depends on 13)

The composition of claim 13, wherein the antibody comprises an anti-CD19 antibody, anti-CD20 antibody or anti-CD25 antibody and the toxin comprises saporin.

### Claim 15 (depends on 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13)

The composition of any one of claims 2 to 13, wherein the delivery vector comprises a retroviral vector, an adenoviral vector, an adeno-associated viral vector, spumaviral vector, a lentiviral vector or a plasmid or other vector described in the application.

### Claim 16 (depends on 15)

The composition of claim 15, wherein the delivery vector comprises a lentiviral vector that has a pHR′ backbone and comprises 5′-Long terminal repeat (LTR), HIV signal sequence, HIV Psi signal 5′-splice site (SD), delta-GAG element, Rev Responsive Element (RRE), 3′-splice site (SA), Elongation factor (EF) 1-alpha promoter and 3′-Self inactivating LTR (SIN-LTR).

### Claim 17 (depends on 15)

The composition of claim 15, wherein the delivery vector comprises a lentiviral vector that has a pCCL backbone and comprises 5′-Long terminal repeat (LTR), HIV signal sequence, HIV Psi signal 5′-splice site (SD), delta-GAG element, Rev Responsive Element (RRE), 3′-splice site (SA), Elongation factor (EF) 1-alpha promoter and 3′-Self inactivating LTR (SIN-LTR).

### Claim 18 (depends on 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17)

A method of expressing an activator polynucleotide, a docking polynucleotide and a therapeutic α-galactosidase A polynucleotide in a mammalian cell comprising contacting the mammalian cell with the composition of anyone of claims 2 to 17.

### Claim 19 (depends on 18)

The method of claim 18, further comprising isolating the cells.

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

The method of any one of claim 18 or 19, wherein the mammalian cell is a an embryonic stem cell, a stem cell, a hematopoietic cell, an iPS cell, a marrow stroma cell, a mesenchymal stem cell, an endothelia progenitor cell, a T cell or a human cell.

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

The method of any one of claims 18 to 20, wherein the mammalian cell is a tumor cell.

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

The method of any one of claims 18 to 21, further comprising a step wherein the isolated mammalian cell is transplanted into a mammal.

### Claim 23 (depends on 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17)

A method of killing a mammalian cell expressing the activator polynucleotide, a docking polynucleotide and the therapeutic α-galactosidase A polynucleotide of any one of claims 2 to 17, comprising contacting the cell with an effective amount of a prodrug and/or a toxic binding agent to kill the cell.

### Claim 24 (depends on 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17)

A method of killing a mammalian cell expressing an activator polynucleotide and/or a docking polynucleotide comprising:
a. contacting the mammalian cell with a composition of any one of claims 2 to 17;
b. isolating the cell; and
c. contacting the cell with an effective amount of a prodrug and/or a toxic binding agent to kill the cell.

### Claim 25 (depends on 23, 24)

The method of claim 23 or 24, wherein the prodrug is selected from the group consisting of thymidine analog, uracil analog, AZT, dT4 and 5-FU.

### Claim 26 (depends on 23, 24)

The method of claim 23 or 24, wherein the toxic binding agent comprises an antibody or an immunotoxin that binds CD19, truncated CD19, CD20, or CD25.

### Claim 27

An actuable cell destruction component of an expression vector construct comprising:
a. an activator polynucleotide encoding a polypeptide that converts a prodrug to a drug;
b. a docking polynucleotide encoding a cell surface polypeptide that selectively binds a toxic binding agent; and
c. a therapeutic α-galactosidase A polynucleotide.

### Claim 28 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17)

The suicide gene system of claim 1, wherein the activator polynucleotide comprises the activator polynucleotide of any one of claims 2 to 17 and the docking polynucleotide comprises the docking polynucleotide of any one of claims 2 to 17.

### Claim 29 (depends on 1, 28)

The suicide system of claim 1 or 28, for use in gene therapy treatment of a subject.

### Claim 30 (depends on 1, 28)

The suicide system of claim 1 or 28, for use in cells for transplant into a subject.

### Claim 31 (depends on 1, 23, 28)

The suicide system of any one of claim 1, 23 or 28, wherein the cells are killed if the subject develops or is suspected of developing GVHD.

### Claim 32 (depends on 1, 23, 28, 31)

A mammalian cell comprising the suicide gene system of any one of claim 1, 23, 28 or 31.

### Claim 33 (depends on 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17)

A kit comprising the composition of any one of claims 2 to 17, a toxic binding agent such as an immunotoxin and/or a prodrug.

### Claim 34 (depends on 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 32)

A method of medical treatment of Fabry disease in a subject in need thereof, comprising administering to the subject in need thereof the composition of any one of claims 2 to 17 or the cell of claim 32.

### Claim 35 (depends on 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17)

A method of gene therapy of a subject with Fabry disease, comprising administering to the subject in need thereof the composition of any one of claims 2 to 17.