# US 6,776,985 B1 · Encapsulated cells producing viral particles

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- Number: US 6,776,985 B1
- Document ID: US6776985B1
- Status: Granted
- Kind: B1
- Application number: 08/996,460
- Filed: 1997-12-23
- Granted: 2004-08-17
- Earliest claimed: 1995-06-27
- Assignees: Bavarian Nordic A/S; GSF-Gesellschaft fur Umwelt und Gesundheit GmbH
- Inventors: Robert Michael Saller; Walter H. Günzburg; Brian Salmons
- Web page: https://patentgrep.com/patent/US6776985B1

## CPC

- C12N 15/86: Viral vectors
- A61K 9/5026: {obtained by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyvinyl pyrrolidone, poly(meth)acrylates}
- A61K 9/5042: {Cellulose; Cellulose derivatives, e.g. phthalate or acetate succinate esters of hydroxypropyl methylcellulose}
- A61K 48/00: Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
- A61P 31/12: Antivirals
- A61P 35/00: Antineoplastic agents
- A61P 43/00: Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
- C12N 7/00: Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof (preparing medicinal viral antigen or antibody compositions, e.g. virus vaccines, A61K39/00)
- C12N 11/04: entrapped within the carrier, e.g. gel or hollow fibres
- C12N 2740/13021: Viruses as such, e.g. new isolates, mutants or their genomic sequences
- C12N 2740/13043: viral genome or elements thereof as genetic vector
- 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

## Abstract

The present invention relates to encapsulated cells producing viral particles, especially retroviral particles containing the genome of a retroviral vector carrying therapeutic genes, to methods for the preparation of such encapsulated cells, as well as to the use of such encapsulated cells for the delivery of genes, especially therapeutic genes, to target organs/cells.

## Claims

### Claim 1

Encapsulated retroviral packaging cells producing retroviral particles, comprising capsules having a porous capsule wall which is permeable to said retroviral particles.

### Claim 2 (depends on 1)

The encapsulated cells according to claim 1 wherein said porous capsule wall comprises of a polyelectrolyte complex formed from counter-charged polyelectrolytes.

### Claim 3 (depends on 1)

The encapsulated cells according to claim 1 wherein said porous capsule wall comprises a polyelectrolyte complex formed from sulphate group-containing polysaccharides or polysaccharide derivatives or sulphonate group-containing synthetic polymers and polymers with quaternary ammonium groups.

### Claim 4 (depends on 3)

The encapsulated cells according to claim 3 wherein the sulphate group-containing polysaccharides or polysaccharide derivative are selected from one or more elements of the group consisting of: cellulose sulphate, cellulose acetate sulphate, carboxymethylcellulose sulphate, dextran sulphate or starch sulphate, and wherein the sulphonate group-containing synthetic polymer is a polystyrene sulphonate.

### Claim 5 (depends on 3)

The encapsulated cells according to claim 3 wherein the polymer with quaternary ammonium groups is polydimethydiallylammonium or polyvinylbenzyl-trimethylammonium.

### Claim 6 (depends on 1)

The encapsulated cells according to claim 1 wherein the porous capsule wall comprises a complex formed from cellulose sulphate and polydimethydiallyl-ammonium.

### Claim 7 (depends on 1)

The encapsulated cells according to claim 1 having the form of microcapsules with a diameter between 0.01 and 5 mm, preferably between 0.1 and 1 mm.

### Claim 8 (depends on 1)

The encapsulated cells according to claim 1 wherein said capsule comprises a spongy matrix forming the interior of the capsule surrounded by the capsule wall containing pores; said spongy matrix being filled with said cells.

### Claim 9 (depends on 1)

The encapsulated cells according to claim 1 wherein the surface pore size of the porous capsule wall is between 80 and 150 nm, preferably between 100-120 nm.

### Claim 10 (depends on 1)

The encapsulated cells according to claim 1 wherein the retroviral particle produced by the encapsulated cells is a retroviral particle containing the genome of a retroviral vector.

### Claim 11 (depends on 1)

The encapsulated cells according to claim 1 wherein the encapsulated cells producing retroviral particles are a packaging cell line transfected with an expression vector, said expression vector carrying a retroviral vector construct capable of infecting and directing the expression in targets cells of one or more coding sequences carried by said retroviral vector construct; said packaging cell line harboring at least one expression vector carrying genes coding for the proteins required for the genome of said retroviral vector construct to be packaged.

### Claim 12 (depends on 11)

The encapsulated cells according to claim 11 wherein at least one of said coding sequences code for heterologous peptides selected from marker genes, therapeutic genes, antiviral genes, antitumour genes, and cytokine genes.

### Claim 13 (depends on 12)

The encapsulated cells according to claim 12 wherein said marker gene codes for a protein selected from the group consisting of: β-galactosidase, neomycin, alcohol dehydrogenase, puromycin, hypoxanthine phosphoribosyl transferase (HPRT), hygromycin and secreted alkaline phosphatase, and said therapeutic gene codes for a protein selected from the group consisting of: Herpes Simplex Virus thymidine kinase, cytosine deaminase, guanine phosphoribosyl transferase (gpt), cytochrome P450, a cell cycle regulatory protein, SDI, a tumour suppressor protein, p53, an antiproliferation protein, melittin, cecropin, a cytokine and IL-2.

### Claim 14 (depends on 11)

The encapsulated cells according to claim 11 wherein the packaging cell line is selected from psi-2, psi-crypt, psi-AM, GP+E-86, PA317, and GP+envAM-12.

### Claim 15 (depends on 11)

The encapsulated cells according to claim 11 wherein the expression vector is selected from the group consisting of: pBAG, pLXSN, p125LX, pLX2B1, pc3/2B1.

### Claim 16

A process for preparing encapsulated retroviral packaging cells which produce retroviral particles, comprising capsules having a porous capsule wall which is permeable to said retroviral particles, comprising suspending the cells producing retroviral particles in an aqueous solution of polyelectrolyte, whereafter the suspension in the form of preformed particles is introduced into a precipitation bath containing an aqueous solution of a counter-charged polyelectrolyte.

### Claim 17 (depends on 16)

A process according to claim 16 wherein particle formation takes place by spraying.

### Claim 18 (depends on 16)

A process according to claim 16 wherein the cells are suspended in an aqueous solution of a sulphate group-containing polysaccharide or polysaccharide derivative, or a sulphonate group-containing synthetic polymer.

### Claim 19 (depends on 18)

A process according to claim 18 wherein the sulphate group-containing polysaccharide or polysaccharide derivative is selected from cellulose sulphate, cellulose acetate sulphate, carboxymethylcellulose sulphate, dextran sulphate or starch sulphate, and the sulphonate group-containing synthetic polymer is a polystyrene sulphonate.

### Claim 20 (depends on 16)

A process according to claim 16 wherein the precipitation bath contain an aqueous solution of a polymer with quaternary ammonium groups.

### Claim 21 (depends on 20)

A process according to claim 20 wherein the polymer with quaternary ammonium group is polydimethyldiallylammonium or polyvinylbenzyl-trimethylammonium.

### Claim 22 (depends on 16)

A process according to claim 16 wherein the cells are suspended in an aqueous solution of sodium cellulose sulphate, and introduced into a precipitation bath containing an aqueous solution of polydimethyldiallylammonium chloride.

### Claim 23 (depends on 22)

A method according to claim 22 wherein the aqueous solution of sodium cellulose sulphate is composed of 0.5%-50%, preferably 2-5% sodium cellulose sulphate and 2-10%, preferably 5%, fetal calf serum in buffered saline.

### Claim 24 (depends on 22)

A method according to claim 22 wherein the aqueous solution in the precipitation bath is composed of 0.5-50%, preferably 2-10%, or more preferred 3% polydimethyldiallylammonium chloride in buffered saline.

### Claim 25 (depends on 16)

Encapsulated cells produced by a process according to claim 16.

### Claim 26

A method for delivery of a gene into or close to the site of a target organ or cell wherein the gene is expressed comprising:
a) culturing encapsulated retroviral packaging cells which produce retroviral particles, comprising capsules having a porous capsule wall which is permeable to said retroviral particles wherein the genome of the retroviral particles express the expression product of the gene, in a suitable medium, and
b) locally administering the encapsulated cells into or near the site of the target organ or cell of a living animal body, including a human
wherein infectious retroviral particles are produced and released from the capsules and the retroviral particles infect the target organ or cell, thereby delivering the gene to the target organ or cell wherein the gene is expressed.

### Claim 27 (depends on 26)

The method according to claim 26 wherein the target organ or cell is a mammary gland, or a pancreas.

### Claim 28 (depends on 26)

The method according to claim 26 wherein the target organ or cell are smooth muscle cells surrounding arteries.