# US 9,636,354 B2 · Methods for sustained and regulatable gene expression using viral based expression vectors

> Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt

- Number: US 9,636,354 B2
- Document ID: US9636354B2
- Status: Granted
- Kind: B2
- Application number: 14/686,541
- Filed: 2015-04-14
- Published: 2015-08-20
- Granted: 2017-05-02
- Earliest claimed: 2004-02-17
- Assignees: University of Florida Research Foundation, Inc.
- Applicants: University of Florida Research Foundation, Inc.
- Inventors: David C. Bloom; Antonio L. Amelio
- Web page: https://patentgrep.com/patent/US9636354B2

## CPC

- A61K 31/713: Double-stranded nucleic acids or oligonucleotides
- C12N 15/85: for animal cells
- C12N 15/86: Viral 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 2710/16611: Simplexvirus, e.g. human herpesvirus 1, 2
- C12N 2710/16622: New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
- C12N 2710/16633: Use of viral protein as therapeutic agent other than vaccine, e.g. apoptosis inducing or anti-inflammatory
- C12N 2710/16642: virus or viral particle as vehicle, e.g. encapsulating small organic molecule
- C12N 2710/16643: viral genome or elements thereof as genetic vector
- C12N 2830/007: cell cycle specific enhancer/promoter combination
- C12N 2830/40: being an insulator
- C12N 2830/60: from viruses
- 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)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

Disclosed are genetic expression cassettes, and vectors comprising them useful for the delivery of isolated nucleic acid segments including those expressing or encoding one or more selected therapeutic constructs (including, without limitation, therapeutic peptides, polypeptides, ribozymes, or catalytic RNA molecules), to one or more selected cells or tissues of a vertebrate animal. Methods employing the disclosed genetic constructs in the development of gene therapy-based viral vector systems are also disclosed. The expression cassettes and viral vectors disclosed herein provide new tools for methods of treating mammalian, and in particular, human diseases, disorders, and/or dysfunctions. The disclosed compositions and methods find particular utility in a variety of investigative, diagnostic, and therapeutic regimens, including, for example, in the treatment or amelioration of symptoms of a variety of mammalian, and particularly, human conditions.

## Claims

### Claim 1

A method for expressing a heterologous nucleic acid segment of interest in a mammalian host cell, comprising providing to a population of mammalian cells, an expression vector comprising, in 5′ to 3′ order,
(a) a first isolated HSV LAT insulator/boundary region that consists essentially of a contiguous nucleotide sequence from about nucleotide 8365 to about nucleotide 9273 of SEQ ID NO:102, SEQ ID NO:103, or SEQ ID NO:104, operably positioned 5′ of a first polynucleotide comprising the heterologous nucleic acid segment of interest operably linked to a first promoter that expresses the heterologous nucleic acid segment in one or more cells of the population; and
(b) a second isolated HSV LAT insulator/boundary region that consists essentially of a contiguous nucleotide sequence from about nucleotide 120,208 to about nucleotide 120,940 of SEQ ID NO:102, SEQ ID NO:103, or SEQ ID NO:104, operably positioned 3′ of the heterologous nucleic acid segment of interest;
in an amount and for a time effective to express the heterologous nucleic acid segment in one or more cells of the population.

### Claim 2 (depends on 1)

The method of claim 1, wherein the first polynucleotide further comprises:
(c) a first enhancer operably linked to the first nucleic acid segment.

### Claim 3 (depends on 1)

The method of claim 1, wherein the first enhancer comprises an isolated HSV LAT enhancer, or the first promoter comprises an isolated HSV LAP1 promoter.

### Claim 4 (depends on 1)

The method of claim 1, wherein the first promoter comprises an isolated HSV LAP1 promoter that consists essentially of a sequence region of from about nucleotide 117,938 to about 118,843 of any one of SEQ ID NO:102, SEQ ID NO:103, and SEQ ID NO:104.

### Claim 5 (depends on 1)

The method of claim 1, wherein the first polynucleotide further comprises a multiple cloning region operably positioned 5′ or 3′ of the first nucleic acid segment, comprising a nucleotide segment that expresses or that encodes a first diagnostic agent, or a nucleotide segment that expresses or that encodes a second therapeutic agent.

### Claim 6 (depends on 1)

The method of claim 1, wherein the first promoter comprises an HSV-1 latency active promoter 1, and the population of mammalian cells comprises neurons.

### Claim 7 (depends on 6)

The method of claim 6, wherein the HSV-1 latency active promoter 1 comprises from about nucleotide 117,938 to about nucleotide 118,843 of the sequence as set forth in SEQ ID NO:102.

### Claim 8 (depends on 1)

The method of claim 1, wherein the expression vector further comprises a LAT enhancer region that is flanked by splice donor and splice acceptor sites, and is operably positioned between the first and the second isolated HSV LAT insulator/boundary regions.

### Claim 9 (depends on 8)

The method of claim 8, wherein the LAT enhancer region comprises from about nucleotide 118,975 to about nucleotide 120,471 of the sequence as set forth in SEQ ID NO:102.

### Claim 10 (depends on 1)

The method of claim 1, wherein the presence of the first and the second isolated HSV LAT insulator/boundary regions in the expression vector effectively maintains permanent regulatable expression or silencing-resistant expression of the heterologous nucleic acid segment of interest in the one or more cells.

### Claim 11 (depends on 1)

The method of claim 1, wherein expression of the heterologous nucleic acid segment of interest in the one or more cells is unaffected over time by chromatin from a genomic DNA sequence into which the expression vector has been integrated.

### Claim 12 (depends on 1)

The method of claim 1, wherein the expression vector is a gutless HSV vector, a gutless AV vector, a gutless AAV vector, a recombinant HSV vector, a recombinant AV vector, a recombinant AAV vector, or an insulated viral artificial chromosome (IVAC).

### Claim 13 (depends on 1)

The method of claim 1, wherein the expression vector is an HSV-1 vector, deleted in one or more essential genes.

### Claim 14

A method of expressing a heterologous nucleic acid segment in a population of selected mammalian host cells, comprising providing to the population of cells, a recombinant viral expression cassette that comprises, in 5′ to 3′ order,
(a) a first isolated HSV LAT insulator/boundary region that consists essentially of a contiguous nucleotide sequence from about nucleotide 8365 to about nucleotide 9273 of SEQ ID NO:102, SEQ ID NO:103, or SEQ ID NO:104;
(b) a first isolated nucleic acid segment comprising a first multiple cloning region operably linked to a first nucleic acid segment comprising a first selected gene of interest operably linked to a first promoter that expresses the first gene of interest in the population of selected mammalian host cells;
(c) a first enhancer element operably linked to the first nucleic acid segment, wherein the enhancer element consists essentially of a contiguous nucleotide sequence from about nucleotide 118,975 to about nucleotide 120,471 of SEQ ID NO:102, SEQ ID NO:103, or SEQ ID NO:104;
(d) a second nucleic acid segment comprising a second multiple cloning region operably linked to a second nucleic acid segment comprising a second selected gene of interest operably linked to a second promoter that expresses the second gene of interest in a selected host cell; and
(e) a second isolated HSV LAT insulator/boundary region that consists essentially of a contiguous nucleotide sequence from about nucleotide 120,208 to about nucleotide 120,940 of SEQ ID NO:102, SEQ ID NO:103, or SEQ ID NO:104.

### Claim 15 (depends on 14)

The method of claim 14, wherein the population of cells is comprised within a mammal that is diagnosed with, or suffers one or more symptoms of, a disease, a disorder, a dysfunction, a deficiency, a defect, an injury, or trauma.

### Claim 16 (depends on 14)

The method of claim 14, wherein the first or the second gene of interest encodes a compound selected from the group consisting of a peptide, a polypeptide, a protein, an antibody, an antigen-binding fragment, a regulatory element, a ribozyme, a catalytic RNA molecule, an shRNA, an siRNA, an antisense oligonucleotide, an antisense polynucleotide, and any combination thereof.

### Claim 17 (depends on 16)

The method of claim 16, wherein the first or the second gene of interest encodes a growth factor, a neurotrophic factor, a transcription factor, an anti-apoptotic factor, a proliferation factor, an enzyme, a cytotoxin, a transcription factor, an apoptotic factor, a tumor suppressor, a kinase, a cytokine, a lymphokine, a protease, or any combination thereof.

### Claim 18 (depends on 16)

The method of claim 16, wherein the catalytic RNA molecule specifically cleaves an mRNA encoding a transcription factor, an anti-apoptotic factor, a proliferation factor, a hormone receptor, a growth factor, an oncogenic peptide, a growth factor polypeptide, or any combination thereof.

### Claim 19 (depends on 1, 14)

The method of claim 1 or claim 14, wherein the expression vector or the recombinant viral expression cassette is first packaged into a virion or a viral capsid, and the resulting virion or viral particle is then used to infect the population of mammalian host cells.

### Claim 20 (depends on 19)

The method of claim 19, wherein the virion or the viral particle is of retroviral, adenoviral (AV), adeno-associated viral (AAV), lentiviral (LV), or herpes viral (HSV) origin.