# US 12,195,766 B2 · Tumor-selective E1a and E1b mutants

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- Number: US 12,195,766 B2
- Document ID: US12195766B2
- Status: Granted
- Kind: B2
- Application number: 17/993,182
- Filed: 2022-11-23
- Published: 2023-08-31
- Granted: 2025-01-14
- Earliest claimed: 2009-03-02
- Assignees: The Regents of the University of California
- Applicants: The Regents of the University of California
- Inventors: Tony Reid; Farah Hedjran; Shantanu Kumar
- Web page: https://patentgrep.com/patent/US12195766B2

## CPC

- C12N 7/00: Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof (preparing medicinal viral antigen or antibody compositions, e.g. virus vaccines, A61K39/00)
- A61K 35/761: Adenovirus
- A61P 35/00: Antineoplastic agents
- C07K 14/005: from viruses
- C12N 15/86: Viral vectors
- C12N 15/861: Adenoviral vectors
- A61K 48/00: Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
- C12N 2710/10021: Viruses as such, e.g. new isolates, mutants or their genomic sequences
- C12N 2710/10022: New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
- C12N 2710/10032: Use of virus as therapeutic agent, other than vaccine, e.g. as cytolytic agent
- C12N 2710/10321: Viruses as such, e.g. new isolates, mutants or their genomic sequences
- C12N 2710/10322: New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
- C12N 2710/10332: Use of virus as therapeutic agent, other than vaccine, e.g. as cytolytic agent
- C12N 2710/10341: Use of virus, viral particle or viral elements as a vector
- C12N 2710/10343: viral genome or elements thereof as genetic vector
- C12N 2830/00: Vector systems having a special element relevant for transcription
- 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)
- 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)

## Abstract

Modified E1a regulatory sequences are provided, wherein at least one Pea3 binding site, or a functional portion thereof, is deleted. Also provided are modified E1a sequences that selectively express particular isoforms. Also provided is an E1b-19K clone insertion site. These modified sequences can be used individually, or in combination with one another, to provide tumor-selective expression of proteins.

## Claims

### Claim 1

A method of transfecting a cell, said method comprising contacting said cell with a recombinant adenovirus comprising an E1a regulatory sequence, wherein at least one Pea3 binding site, or functional portion thereof, is modified, and a DNA sequence inserted into an E1b-19K insertion site, wherein said insertion site is located between the start site of E1b-19K and the start site of E1b 55K.

### Claim 2 (depends on 1)

The method of claim 1, wherein said E1b-19K insertion site comprises a deletion of at least 100 base pairs.

### Claim 3 (depends on 2)

The method of claim 2, wherein said insertion site comprises a deletion of 202 base pairs following said start site of E1b-19K.

### Claim 4 (depends on 1)

The method of claim 1, wherein said DNA sequence encodes a transgene, a cancer gene, or a peptide.

### Claim 5 (depends on 4)

The method of claim 4, wherein said transgene is a tumor suppressor gene, a toxin gene, a cytokine gene, a reporter gene, a pro-drug activating gene, or an apoptotic gene.

### Claim 6 (depends on 1)

The method of claim 1, wherein at least one nucleotide in the range of −305 to −141 is retained.

### Claim 7 (depends on 1)

The method of claim 1, wherein at least one of Pea3 II, Pea3 III, Pea3 IV, and Pea3 V, or a functional portion thereof is deleted.

### Claim 8 (depends on 1)

The method of claim 1, wherein Pea3 II or a functional portion thereof, and Pea3 III or a functional portion thereof is deleted.

### Claim 9 (depends on 1)

The method of claim 1, wherein Pea3 IV or a functional portion thereof, and Pea3 V or a functional portion thereof is deleted.

### Claim 10 (depends on 1)

The method of claim 1, wherein said recombinant adenovirus comprises deletion of nucleotides located upstream of a E1a initiation site, wherein the deletion comprises nucleotide −393 to −304, nucleotide −305 to −255, nucleotide −270 to −240, nucleotide −299 to −293, or nucleotide −270 to −265.

### Claim 11 (depends on 1)

The method of claim 1, wherein said recombinant virus selectively expresses an E1a isoform, wherein the sequence encoding the E1a isoform is operably linked to said modified E1a regulatory sequence.

### Claim 12 (depends on 1)

The method of claim 1, wherein said recombinant adenovirus selectively expresses E1a-12S or E1a-13S.

### Claim 13 (depends on 1)

The method of claim 1, wherein said recombinant adenovirus substantially excludes expression of an E1a isoform, wherein said sequence encoding said E1a isoform is operably linked to said modified E1a regulatory sequence.

### Claim 14 (depends on 13)

The method of claim 13, wherein said excluded E1a isoform is E1a-12S or E1a-13S.

### Claim 15 (depends on 1)

The method of claim 1, wherein said modification comprises a deletion, substitution, or addition of one or more nucleotides compared to the wild-type sequence.

### Claim 16 (depends on 1)

The method of claim 1, wherein said modification reduces affinity for a corresponding transcription factor.

### Claim 17 (depends on 1)

The method of claim 1, wherein said cell is in vitro.

### Claim 18 (depends on 1)

The method of claim 1, wherein said cell is a neoplastic cell.

### Claim 19 (depends on 18)

The method of claim 18, wherein the neoplastic cell is a lung, pancreas, prostate, or cervical cancer cell.

### Claim 20 (depends on 1)

The method of claim 1, wherein said cell is a normal cell.