AU732445B2

AU732445B2 - Stabilized transient gene expression

Info

Publication number: AU732445B2
Application number: AU51601/98A
Authority: AU
Inventors: Adeelia S. Goffe; Randal A. Goffe
Original assignee: Genespan Corp
Current assignee: Genespan Corp
Priority date: 1996-11-01
Filing date: 1997-10-31
Publication date: 2001-04-26
Anticipated expiration: 2017-10-31
Also published as: JP2000509998A; EP0941356A2; WO1998020146A3; CA2270137A1; AU5160198A; CN1238807A; WO1998020146A2

Description

Stabilised Transient Gene Expression Field of the Invention This invention relates to methods and agents that enhance the transient expression of foreign genes that have been introduced into cultured eukaryotic cells.

Background of the Invention The introduction of foreign DNA into eukaryotic host cells can serve many purposes. For example, this technique can provide a means of genetic complementation for identifying specific genes, a gene expressing an enzyme critical to a metabolic pathway can be identified by virtue of its ability to rescue cells defective in that pathway.

lo Also, exogenous genes can be introduced for the purpose of exposing a recipient cell to a high dose of a protein not normally native to that cell, as for example, a cytotoxic protein introduced into a malignant cell for the purpose of killing it. Alternatively, foreign genes S may be introduced into host cells to obtain the protein product of the foreign gene in sufficiently large amounts so that the protein can be harvested for further study or used as o.15 a pharmaceutical. In addition, the introduction of foreign genes is viewed as a promising *000 avenue for somatic gene therapy. The goal of gene therapy is to cure inborn genetic defects by providing patients with a working copy of a missing or defective gene, or, 966 alternatively, to provide a foreign gene product on a temporary basis for therapeutic purposes. One approach to somatic gene therapy is the ex vivo strategy, wherein cells are removed se 0S •0 0 S 0o 0 a0 005

S

5555 S S 5 05 0 0 [N:\LIBM22971 :MER WO 98/20146 PCT/US97/19860 -2from the body, transgenic DNA is inserted into the cells, and the cells are then returned to the body. In another approach, cells in vivo are targeted by foreign DNA that is introduced directly into the patient.

Foreign genes can be introduced into living eukaryotic cells by a variety of methods. These include, for example, the use of viral vectors into which the foreign gene has been ligated. Typically, viral vectors target only actively dividing cells hematopoietic stem cells). Another method useful for the introduction of foreign DNA is electroporation, wherein cells take up DNA in response to a brief high voltage electrical pulse administered to the cells in the presence of a DNA solution. Other frequently used methods for introducing foreign DNA into cells include lipofection, in which the DNA is associated with liposomes, which are liquid-filled sacs formed by lipid molecules that aggregate to form a membrane structure. DNA molecules can become encapsulated in liposomes or can be associated with liposomal membranes.

The liposomes are fused with recipient cells as a means of introducing foreign genes into the cells. Although widely used, one limitation of lipofection is that liposomes are somewhat toxic to living eukaryotic cells. In other commonly-used methods, DNA is co-precipitated with CaPO 4 before being applied to cells, or the entry of foreign DNA can be mediated by DEAE-dextran, a polymer that forms an electrostatic complex with DNA, the complex being internalized into cells by endocystosis (Kormis and Wu, Seminars Liv. Dis., 15:257-267 (1995)). Exemplary transfection protocols are widely available (see, Sambrook et al., Molecular Cloning, 2d ed., (1989), which is hereby incorporated by reference; Gorman, C., "High Efficiency Gene Transfer into Mammalian Cells", Chap. 6, pp. 143-190 from DNA Cloning II A Practical Approach, IRL Press, Oxford (1985), Ed. Glover, Wynshaw-Boris et al., BioTechniques, 4:104-119 (1986); Chang, P.L. (Ed), Somatic Gene Therapy, CRC Press, 1995; and Guide to Eukaryotic Transfection with Cationic LipidReagents, Life Technologies (Gibco-BRL)).

"Transfection" is a general term often used to describe processes by which foreign genes ("transgenes") are introduced into a living host cell. Another term for this process is "transduction," this latter term being most commonly used when referring to viral vector-mediated gene transfer. Host cells that express or incorporate the foreign DNA are known as "transformed cells," and the process by which they become transformed is called "transformation" or "transduction." Different types of cells vary in their susceptibility to transformation, and protocols for introducing the foreign DNA are typically optimized by the adjustment of various parameters such as WO 98/20146 PCTIUS97/19860 -3pH, type of culture medium, amount of DNA, CO 2 concentration, or method of DNA introduction (see, Chen and Okayama, Mol. Cell. Biol., 7:2745-2752 (1987)).

When foreign DNA is introduced into cells by the CaPO 4 or by the DEAEdextran method, it has been observed most of the cells initially take up the DNA, but only a fraction of them will express the DNA during the first few days after it has been introduced (see, Gorman (1985)). Early expression of transfected DNA is typically short-lived, and is referred to as "transient expression." A still smaller fraction of the recipient cells (0.1-0.001%) will stably incorporate the transfected DNA by covalently linking it into the host genome (see, Wynshaw-Boris et al.

(1986)). When transfected foreign DNA becomes covalently inserted into the host DNA in a fashion that supports expression of the foreign gene, the resulting cells are said to be "stably transformed." A likely reason for the observed low level of covalent integration is that active DNA synthesis must occur in order for integration of the foreign DNA to take place. Thus, cells are considered to be susceptible to stable transformation only during the S phase of the cell cycle.

In stably transformed cells, the foreign gene is reproduced each time the cell divides, and the protein encoded by the foreign gene is expressed by the same enzymatic machinery that transcribes the endogenous cellular chromosomal genes.

However, after the initial wave of transient expression, only a tiny fraction of the cells in a transfected culture typically contain integrated copies of the transfected gene.

Thus, the amount of foreign protein expressed in such a culture will be small, and may even be undetectable. Accordingly, stable transformation protocols generally rely on a post-transfection selection step to provide a selective growth advantage to the few stably transformed cells that are present following the addition of foreign DNA.

Homogenous cultures of stably transformed cells can be selectively isolated under a variety of experimental conditions to obtain lines of cells that have integrated the foreign gene, and that continue to express it. To accomplish this, a selectable gene, usually one that confers drug resistance or that encodes a chromogenic protein, must be introduced into the host cell concurrently with the introduction of the DNA encoding the desired protein. Examples of reporter genes suitable for this purpose include bacterial chloramphenicol acetyltransferase, luciferase, alkaline phosphatase, bacterial P-galactosidase, and others (see, Alam and Cook, Anal. Biochem., 188:245-254 (1990)). If a drug-resistance marker is being used, drug resistant cells must be selected by long-term exposure to the relevant drug in order that the stably transformed cells can become predominant in the culture. Alternatively, cells WO 98/20146 PCT/US97/19860 -4containing integrated DNA may be identified by their expression of a co-transfected gene that is capable of converting a chromogenic substrate into a colored substance that permits the identification and manual cloning of individual stably transformed cells. In some instances, the desired gene itself may confer a selectable trait on the stably transformed cells, but this circumstance is rare. In any case, the creation and isolation of stably transformed cell lines can take one to three months to accomplish.

(Wynshaw-Boris et al. (1986)).

In contrast to stable transformation, the transient expression of transfected DNA does not depend on the integration of the foreign DNA into host cell chromosomes. Although the majority of DNA applied to a cell is believed to be rapidly transported into the nucleus, in some systems expression can be detected for up to 80 hours post-transfection in the absence of any detectable integration (see, e.g., Gorman (1985)); Wynshaw-Boris et al. (1986)). No selection step is required before transient expression can be detected. However, only about 1-10% of cells that take up foreign DNA typically transcribe mRNA from a transfected foreign gene (see, e.g., Gorman et al., Nucl. Ac. Res., 11:7631-7648 (1983)). Although the vast majority of transfected DNA in transiently transfected cells does not become incorporated into the host DNA, it does become incorporated in about 0.001-1% of these cells (Alam and Cook (1990)). This small stably transfected fraction of cells is believed to play no significant or useful role in the foreign gene expression profile observed immediately after transfection.

Without a selection step, the expression of foreign genes quickly disappears from cultures of transfected cells. Typically, transient expression in cultured cells peaks in about 48 hours, and is detectable for only 24-80 hours. (Gorman (1985); Wynshaw-Boris et al. (1986)). It is widely believed that most of the DNA taken up by transfected cells becomes rapidly catabolized by nucleases or becomes diluted by cell division (see, Gorman (1985); Guide to Eukaryotic Transfection with Cationic Lipid Reagents Life Technologies).

Because transient expression does not require that the target cells are actively dividing, it can be achieved in terminally differentiated cells that do not normally divide, although susceptibility to transfection varies dramatically among such cells.

For example, naked DNA can be expressed when injected directly into mouse skeletal muscle (Wolff, et al., Science, 247, 1465-1468 (1990)). In other studies, naked DNA was used as a vaccine (Cohen, Science, 259, 1691-1692 (1993)).

WO 98/20146 PCT/US97/19860 Many studies have focused on the liposomal delivery of foreign DNA in vivo to hepatocytes (see, Wu and Wu, Biol. Chem. 263:14621-14624 (1988); Chow et al., J. Pharmacol. Exp. Ther., 248:506-13 (1989); Wu et al., J. Biol. Chem, 264:16985-16987 (1989); Kaneda et al., J. Biol. Chem., 264:12126-12129 (1989a); Kaneda etal., Science, 243:375-378 (1989b); Wilson etal., J. Biol. Chem., 267:963-967 (1992); Wilson etal., J. Biol. Chem., 267:11483-11489 (1992b); Chowdhury et al., J. Biol. Chem., 268:11265-11271 (1993); Perales et al., Proc. Natl.

Acad. Sci. USA, 91:4086-4090 (1994); Kormis and Wu, (1995)). One approach to targeting foreign DNA to specific tissues in vivo is receptor-mediated liposomal delivery (reviewed in Kormis and Wu (1995)). In applying this strategy to liver, Wu and his colleagues exploited the presence of asialoglycoprotein receptors on hepatocyte surfaces to target injected liposomes to the liver. This delivery system is characterized in a series of publications (Wu and Wu (1988); Wu et al. (1989); Wilson et al. (1992a); Wilson et al. (1992b); Chowdhury et al. (1993); Perales et al. (1994)).

The asialoglycoprotein was packaged into liposomes together with DNA that had formed an electrostatic complex with polylysine. When initial efforts were successful, this group attempted to maximize the stable integration of the foreign DNA by performing partial hepatectomies in the recipient rats. As regenerating liver cells provide a higher proportion of cells in S phase than are present in normal liver, this tactic was expected to increase the proportion of liver cells into which foreign DNA could integrate. After partial hepatectomy, the transgenic protein was detectable in the blood for as long as 11 weeks post-transfection (Wu et al. (1989)). At first, these investigators believed that the injected DNA had become integrated, but later experiments revealed no detectable integrated DNA, showing instead that the preserved foreign DNA resided in the plasma membrane/endosome fraction (Wilson et al. (1992b); Chowdhury et al. (1993)). This surprising observation indicated that partial hepatectomy leads to the persistence of transgenic DNA by a mechanism that is independent of DNA synthesis per se.

Another group also has employed a targeting strategy for directing injected DNA to the liver (Kaneda et al. (1989a); Kaneda et al. (1989b)). Here, transgenic DNA was packaged in liposomes with proteins normally found in the nucleus, the non-histone chromosomal proteins. They observed transport of the injected vesicles to the nuclei of liver cells, and detected measurable transgene expression for up to 7 or 8 days after injection. However, this DNA did not become integrated into the liver cell chromosomes. Others have reported the successful in vivo expression of foreign WO 98/20146 PCTIUS97/19860 -6- DNA following the injection of CaPO 4 -DNA precipitates directly into the liver, spleen, or peritoneum (see Kaneda et al. (1989a)).

A number of reagents have been shown to increase the efficiency in vitro of stable transformation. One group has reported that by controlling the pH in the culture medium during CaPO 4 mediated transfection, stable transformation efficiencies as high as 50% can be achieved (Chen and Okayama (1987)).

Another reagent reported to enhance the expression of transfected DNA is butyric acid or its sodium salt (Gorman et al. (1983)). Because of its known ability to alter chromatin structure, Gorman et al. (1983) tested the effects of butyric acid on the expression of foreign genes introduced into cells by transfection. In these studies, cells were transfected, then exposed to a single 12 hour dose of sodium butyrate.

When transient expression was monitored over the next five days, they observed a 2-4-fold increase in the percentage of recipient cells that expressed the transfected gene. They also noted 25-100-fold increases in the foreign gene expression levels when the transfected construct included an SV40 enhancer element. When other cultures transfected in the presence of butyrate were selected for stable transformants, they observed a significant increase over controls in the percentage of transfected cells that gave rise to stable transformants. However, Palermo et al. Biotech., 19:35-48 (1991)) found that butyrate could induce increased transgene expression in stable transformants whether or not butyrate had been present during the transfection step.

Indeed, many reports have documented butyrate's ability to induce the synthesis of certain proteins or to increase cell differentiation in vitro. (Boffa, et al., J Biol.

Chem., 256:9612-9621 (1981); Kruh, Mol. Cell. Biochem. 42:65-82 (1982); Chabanas, et al., J.Mol. Biol., 183:141-151 (1985); Parker, J Biol. Chem., 261:2786-2790 (1986); Kooistra, et al., Biochem. 247:605-612 (1987); Kaneko, et al., Canc. Res., 50:3101-3105 (1990); Nathan, etal., Exp. Cell Res., 190:76-84 (1990); Palermo, J. Biotech., 19:35-48 (1991); Kosaka, et al., Exp. Cell Res., 192:46-51 (1991); and Oh, et al. Biotechnol. Bioeng., 42:601-610 (1993)). Optimal concentrations of butyrate for gene induction vary from cell type to cell type, and a suitable concentration range that minimizes its cytotoxic effects must be empirically determined for each type of target cell (see, Gorman (1985); Parker et al. (1986); Oh et al. (1993)).

The effects in living cells of butyric acid (or butyrate) are not limited to the modification of chromosome proteins. One of this compound's most striking manifestations is its ability to reversibly suppress the growth of cultured cells (see, Boffa et al (1981), reporting that most cells exposed to butyrate are arrested at the GuS boundary). Moreover, butyrate enhances the antitumor action of interferon (Kruh (1982)). This antitumor activity is particularly interesting, as it was achieved by injecting 0.5 mL of a 50 mM butyrate solution directly into live mice.

There are many advantages to using transient expression rather than stable transformation for the expression of foreign genes. First, by using transient expression, one can quickly analyse a relatively large number of constructs. Also, it may be the method of choice for delivering therapeutic proteins whose presence in the body is desired only for the duration of the disease.

Furthermore, transient expression avoids the danger of mutagenesis or cell death that may occur to as the result of foreign DNA being inserted into a critical cell gene. In addition, transient expression can be achieved in primary cell lines that are not immortalised, whereas stable transformants can be established only from cells that can survive and divide in cultures for long periods of time.

However, with the exception of the liver hepatectomy model, a major drawback of currently known transient expression methods continues to be the relatively short-lived expression of the foreign gene, and the tendency of transfection reagents, including purified DNA itself to be toxic to living cells. Hepatectomy or other surgical excisions are too drastic an approach for most practical purposes. Thus, the availability of other means of stabilising transient expression would widen the potential range of applications for this technique.

Summary of the Invention 20 This invention provides methods and agents that significantly enhance the expression of foreign DNA that has been introduced into a host eukaryotic cell. The agents described herein increase both the amount and duration of transient expression.

The chemical compounds that comprise these agents are demonstrated to be efficacious in both growing cells and in static cultures of non-dividing cells. This circumstance indicates that the 25 enhanced transgenic expression observed with these compounds does not involve integration of the foreign DNA into the genome of the recipient host cell.

Thus, according to a first embodiment of the invention, there is provided a method of enhancing the transient expression of a foreign gene in a eukaryotic cell comprising: introducing into the cell a molecule of foreign DNA that encodes a protein in a form capable of being expressed in the cell; and, contacting the cell before, during, or after introducing the DNA with a transient expression enhancing agent; wherein the concentration of the transient expression enhancing agent is 1-15 mM and the transient expression enhancing agent comprises at least one carboxylic acid derivative having the formula:

O

R

1

C-OR

2 wherein R 1 is:

CHNH

2

R

3 wherein R 3 is the side chain of an amino acid or amino acid derivative selected from the group consisting of glycine, alanine, valine, leucine, isoleucine, aspartic acid, R A4,o glutamic acid, glutamine, serine, threonine, methionine, arginine, lysine, histidine, proline, Il:\DayLib\LibW]03734a.doc:mrr 8 tryptophan, phenylalanine, tyrosine, their unnatural optical isomers, 3-methyl-L-histidine, c-ketoglutaric acid, p-alanine, carnosine, citrulline, creatine, folic acid, glutathione, hippuric acid, homoserine, N-carbamyl aspartic acid, N-formyl-L-methionine, ornithine and optical isomers thereof; s CsH 4

R

4 wherein R 4 is H, NH 2

COCH

3

CO(CH

2 )nCH 3

C(CH

3 3

CH(CH

3 2

(CH

2 )nCH(CH 3 2

(CH

2 )nCOCH 3

OCH

3 or O(CH 2 )nCH 3 wherein n 1-3;

CHNH

2

(CH

2 )nR 5 wherein n 1-7 and Rs is CH 3 OH, CONH 2

C

6

H

4 OH, or

CONHNH

2

(CH

2 )nR 6 wherein n 3-9 and R6 is an indole group, NCH3C(=NH)NH 2

SCH

3 o0 NH 2

CH

3

CO

2 H, CONH 2 or NHC(=NH)NH 2 provided that when n 2 and R 2 is H or M, R 6 is not

CH

3

(CH

2 )nCHNH 2

CO

2 H, wherein n 1-8; CH(C0 2

H)NHCONH

2 or CsH 4 N; and is wherein R 2 is selected from H, (CH 2 )xO(CH 2 )yCH 3 or (CH2)xCO(CH 2 )yCH 3 wherein x y 2-7, or M, wherein M is a metal counterion or ammonium; maintaining the cell in a non-selective medium after introducing the DNA; and detecting the foreign protein in the cell after maintaining the cell for at least five days in the 2 non-selective medium.

20 According to a second embodiment of the invention, there is provided a method of enhancing the transient expression of a foreign gene in a eukaryotic cell comprising: introducing into the cell a molecule of foreign DNA that encodes a protein in a form capable of being expressed in the cell; and, contacting the cell before, during, or after introducing the DNA with a transient expression 25 enhancing agent; wherein the concentration of the transient expression enhancing agent is 1-15 mM and the transient expression enhancing agent comprises a sulfonic acid derivative having the formula:

R

7 -S0 2

-OR

8 wherein R 7 is a straight chain or branched C1-C5 lower alkyl, aryl, substituted lower alkyl, or substituted aryl; and

R

8 is a hydrogen, a metal counterion, or ammonium; maintaining the cell in a non-selective medium after introducing the DNA; and detecting the foreign protein in the cell after maintaining the cell for at least five days in the non-selective medium.

According to a third embodiment of the invention, there is provided a method of enhancing the transient expression of a foreign gene in a eukaryotic cell comprising: introducing into the cell a molecule of foreign DNA that encodes a protein in a form capable of being expressed in the cell; and, contacting the cell before, during, or after introducing the DNA with a transient expression enhancing agent; Awherein the transient expression enhancing agent comprises a sulfonated amino S polysaccharide and the concentration of the transient expression enhancing agent is 0.01-0.5 mM; [I:\DayLib\LibW]03734a.doc:mrr maintaining the cell in a non-selective medium after introducing the DNA; and detecting the foreign protein in the cell after maintaining the cell for at least five days in the non-selective medium.

According to a fourth embodiment of the invention, there is provided a method of enhancing the transient expression of a foreign gene in a eukaryotic cell comprising: introducing into the cell a molecule of foreign DNA that encodes a protein in a form capable of being expressed in the cell; and, contacting the cell before, during, or after introducing the DNA with a first and second transient expression enhancing agent wherein the concentration of each of said first and second io transient expression enhancing agents is 1-15 mM; wherein the cell is contacted with said first transient expression enhancing agent prior to, during, and following the introduction into the cell of the foreign DNA, wherein the agent comprises at least one compound having the formula:

O

II

R

1

C-OR

2 5 wherein Ri is:

CHNH

2

R

3 wherein R 3 is the side chain of an amino acid or amino acid derivative selected from the group consisting of glycine, alanine, valine, leucine, isoleucine, aspartic acid, glutamic acid, glutamine, serine, threonine, methionine, arginine, lysine, histidine, proline, tryptophan, phenylalanine, tyrosine, their unnatural optical isomers, 3-methyl-L-histidine, a-ketoglutaric acid, p-alanine, carnosine, citrulline, creatine, folic acid, glutathione, hippuric acid, homoserine, N-carbamyl aspartic acid, N-formyl-L-methionine, ornithine and optical isomers thereof; CsH 4

R

4 wherein R 4 is H, CH 3

(CH

2 )nCH 3

NH

2

COCH

3 CO(CH2)nCH 3

C(CH

3 3

CH(CH

3 2

(CH

2 )nCH(CH3)2, (CH2)nCOCH 3

OCH

3 or O(CH2)nCH 3 wherein n 1-3;

CHNH

2

(CH

2 )nR 5 wherein n 1-7 and R 5 is CH 3 OH, CONH 2 CsH 4 OH, or

*"CONHNH

2

(CH

2 )nR 6 wherein n 1-9 and Re is an indole group, NCH 3

C(=NH)NH

2

SCH

3

NH

2

CH

3 C02H, CONH 2 or NHC(=NH)NH 2 provided that when n 2 and R 2 is H or M, R 6 is not

CH

3

(CH

2 )nCHNH 2

CO

2 H, wherein n 1-8;

CH(CO

2

H)NHCONH

2 or 4 N;and wherein R 2 is H, CH 3

(CH

2 )nCH 3 wherein n 1-8, or (CH2)xO(CH 2 )yCH 3 or (CH2)xCO(CH 2 )yCH 3 wherein x y 2-7, or M, wherein M is a metal counterion or ammonium, provided that when R 1 is (CH 2 )2CH 3

R

2 is not H; or the first transient expression enhancing agent comprises a compound having the formula: 2

-OR

8 wherein R 7 is a straight chain or branched CI-C5 lower alkyl, aryl, substituted lower alkyl, or .0 substituted aryl; and [l:\DayLib\LibW]03734a.doc:mrr

R

8 is a hydrogen, a metal counterion, ammonium; and following the introduction of the foreign DNA, the cell is contacted with said second transient expression enhancing agent, wherein the second agent comprises a sulfonated amino polysaccharide; maintaining the cell in a non-selective medium after introducing the DNA; and detecting the foreign protein in the cell after maintaining the cell for at least five days in the non-selective medium.

According to a fifth embodiment of the invention, there is provided a method of enhancing the transient expression of a foreign gene in a eukaryotic cell comprising: introducing into the cell a molecule of foreign DNA that encodes a protein in a form capable of being expressed in the cell; and, contacting the cell with a first agent prior to, during, and following the introduction into the cell of the foreign DNA, wherein the concentration of the first agent is 1-15 mM and the first agent comprises at least one chemical compound having the formula: 0 SII 15 R- C-OR 2 .o wherein Ri is:

CHNH

2

R

3 wherein R 3 is the side chain of an amino acid or amino acid derivative selected from the group consisting of glycine, alanine, valine, leucine, isoleucine, aspartic acid, glutamic acid, glutamine, serine, threonine, methionine, arginine, lysine, histidine, proline, tryptophan, phenylalanine, tyrosine, their unnatural optical isomers, 3-methyl-L-histidine, -ketoglutaric acid, p-alanine, carnosine, citrulline, creatine, folic acid, glutathione, hippuric acid, homoserine, N-carbamyl aspartic acid, N-formyl-L-methionine, ornithine and optical isomers thereof;

C

6

H

4

R

4 wherein R 4 is H, CH 3

(CH

2 )nCH 3

NH

2

COCH

3

CO(CH

2 )nCH 3

C(CH

3 3 25 CH(CH 3 2

(CH

2 )nCH(CH 3 2

(CH

2 )nCOCH 3

OCH

3 or O(CH 2 )nCH 3 wherein n 1-3; "N CHNH 2

(CH

2 )nR 5 wherein n 1-7 and R 5 is CH 3 OH, CONH 2 CsH 4 OH, or S CONHNH2;

(CH

2 )nRe, wherein n 1-9 and R 6 is an indole group, NCH 3

C(=NH)NH

2

SCH

3

NH

2

CH

3

CO

2 H, CONH 2 or NHC(=NH)NH 2

(CH

2 )nCHNH 2

CO

2 H, wherein n 1-8;

CH(CO

2

H)NHCONH

2 or CsH 4 N; and wherein R 2 is H, CH 3

(CH

2 )nCH 3 wherein n 1-8, (CH 2 )xO(CH 2 )yCH 3 or (CH2)xCO(CH 2 )yCH 3 wherein x y 2-7, or M, wherein M is a metal counterion or ammonium, provided that when R 1 is (CH2) 2

CH

3

R

2 is not H; or the first agent comprises at least one chemical compound having the formula:

R

7 -S0 2

-OR

8 wherein R 7 is a straight chain or branched C1-C5 lower alkyl, aryl, substituted lower alkyl, or substituted lower aryl; and

R

8 is a hydrogen atom, a metal counterion, or ammonium; [I:\DayLib\LibW]03734a.doc:mrr 9b and following the introduction of the foreign DNA, the cell is contacted with a second agent, wherein the second agent comprises at least one sulfonated amino polysaccharide at a concentration of 0.01-0.5 mM or wherein the second agent comprises, at a concentration of 1-15 mM, at least one chemical compound having the formula:

O

R- C -OR 2 wherein R 1 is:

CHNH

2

R

3 wherein R 3 is the side chain of an amino acid or amino acid derivative selected from the group consisting of glycine, alanine, valine, leucine, isoleucine, aspartic acid, glutamic acid, glutamine, serine, threonine, methionine, arginine, lysine, histidine, proline, to tryptophan, phenylalanine, tyrosine, their unnatural optical isomers, 3-methyl-L-histidine, ca-ketoglutaric acid, p-alanine, carnosine, citrulline, creatine, folic acid, glutathione, hippuric acid, homoserine, N-carbamyl aspartic acid, N-formyl-L-methionine, ornithine and optical isomers thereof; CsH 4

R

4 wherein R 4 is H, CH 3

(CH

2 )nCH 3

NH

2

COCH

3

CO(CH

2 )nCH 3

C(CH

3 3 5i CH(CH 3 2

(CH

2 )nCH(CH 3 2 (CH2)nCOCH 3

OCH

3 or O(CH 2 )nCH 3 wherein n 1-3; NHNH CHNH 2

(CH

2 )nR 5 wherein n 1-7 and R 5 is CH 3 OH, CONH 2 CsH 4 0H, or CONHNH2;

(CH

2 )nR 6 wherein n 1-9 and R 6 is an indole group, NCH 3

C(=NH)NH

2

SCH

3

.NH

2

CH

3

CO

2 H, CONH 2 or NHC(=NH)NH 2

(CH

2 )nCHNH 2

CO

2 H, wherein n 1-8; CH(C02H)NHCONH 2 or CsH 4 N; and wherein R 2 is H, CH 3

(CH

2 )nCH 3 wherein n 1-8, (CH2)xO(CH 2 )yCH 3 or (CH2)xCO(CH 2 )yCH 3 wherein x y 2-7, or M, wherein M is a metal counterion or ammonium, provided that when R 1 is (CH 2 2

CH

3

R

2 is not H; or the second agent comprises at least one chemical compound having the formula:

R

7 -S0 2

-OR

R

8 is a hydrogen atom, a metal counterion, or ammonium.

Furthermore, it is shown that the compounds of this invention depress the consumption by cultured cells of glucose present in the culture medium, thus forcing the cells to rely for energy on alternative carbon sources, such as lipids or proteins. These same cells exhibit increased production of ammonia, thus suggesting that protein is being used as a source of energy. Their effect on glucose consumption suggests that the principal site of action of these compounds is the mitochondria. This observation implicates the mitochondria as playing a critical role in sustained expression of non-integrated foreign DNA both in vitro and in vivo. In addition, cells grown in the presence of these compounds become induced to express and secrete an endogenous alkaline phosphatase activity.

Thus, according to a sixth embodiment of the invention, there is provided a method of [l:\DayLib\LibW]03734a.doc:mrr 9c manipulating the metabolism of a cell to reduce the cell's consumption of glucose, comprising the step of contacting the cell with an agent that induces the cell to use proteins or amino acids as their primary energy source, wherein the concentration of said agent is 1-15 mM, provided that if the agent is a sulfonated amino polysaccharide the concentration of said sulfonated amino s polysaccharide is 0.01-0.5 mM, wherein said concentration of said agent is in addition to the concentration of said agent present in any cell culture medium used to culture said cell.

According to a seventh embodiment of the invention, there is provided a method of manipulating the metabolism of a cell to reduce the cell's consumption of glucose, comprising the step of contacting the cell with an agent, wherein the concentration of said agent is from 1-15 mM, said concentration of said agent being in addition to the concentration of said agent present in any cell culture medium used to culture said cell and the agent comprises at least one chemical compound having the formula:

O

R- C- OR 2 wherein R 1 is: I: CHNH 2

R

3 wherein R 3 is the side chain of an amino acid or amino acid derivative selected from the group consisting of glycine, alanine, valine, leucine, isoleucine, aspartic acid, glutamic acid, glutamine, serine, threonine, methionine, arginine, lysine, histidine, proline, tryptophan, phenylalanine, tyrosine, their unnatural optical isomers, 3-methyl-L-histidine, oa-ketoglutaric acid, p-alanine, carnosine, citrulline, creatine, folic acid, glutathione, hippuric acid, homoserine, N-carbamyl aspartic acid, N-formyl-L-methionine, ornithine and optical isomers thereof;

CH

4

R

4 wherein R 4 is H, CH 3

(CH

2 )nCH 3

NH

2

COCH

3

CO(CH

2 )nCH 3

C(CH

3 3 *i CH(CH3) 2

(CH

2 )nCH(CH 3 2

(CH

2 )nCOCH 3 OCH3, or O(CH 2 )nCH 3 wherein n 1-3;

CHNH

2

(CH

2 )nR 5 wherein n 1-7 and Rs is CH 3 OH, CONH 2 CsH 4 0H, or

CONHNH

2 N H. (CH 2 )nR 6 wherein n 1-9 and R 6 is an indole group, NCH 3

C(=NH)NH

2

SCH

3 NH2, C0 2 H, CONH 2 or NHC(=NH)NH 2 provided that when n 2 and R 2 is H or M, R 6 is not CH 3 (CH2)nCHNH 2

CO

2 H, wherein n 1-8;

CH(CO

2

H)NHCONH

2 or CsH 4 N; and wherein R 2 is H, CH 3

(CH

2 )nCH 3 wherein n 1-8, (CH 2 )xO(CH 2 )yCH 3 or

(CH

2 )xCO(CH 2 )yCH 3 wherein x y 2-7, or M, wherein M is a metal counterion or ammonium, provided that when R 1 is (CH 2 2

CH

3

R

2 is not H; or the group consisting of a sulfonic acid derivative having the formula:

R

7 -S0 2

-OR

8 wherein R 7 is a straight chain or branched C 1

-C

5 lower alkyl, aryl, substituted lower alkyl, or substituted lower aryl; and

R

8 is a hydrogen atom, a metal counterion, or ammonium; or a sulfonated amino polysaccharide.

[I:\DayLib\LibW]03734a.doc:mrr The invention further provides long-term transient expression of foreign genes that have been introduced into target cells by a variety of delivery systems, including but not limited to cationic lipids liposomes) and various synthetic polymers such as dendrimers (also known as "starburst" polymers). Many of the subject chemical compounds influence the fate of foreign gene expression well after the foreign DNA has been introduced into the cell, thus act independently of the method by which the DNA is introduced. Some of the subject compounds are especially effective in increasing the degree of expression during the first four days following the introduction of foreign DNA, thus appear to enhance the initial amount of DNA taken into the cells, or to increase the proportion of cells that express the DNA, or both.

1o The compounds of this invention have a hydrophobic moiety and an acidic moiety, and the latter may take the form of a salt or an ester. Moreover, they are biocompatible, when applied to cells at appropriate concentrations, greater than 50% of the cells remain viable.

The methods of the subject invention involve categorising the compounds into 20 "Type A" formulations, which primarily increase the degree of transient expression during the first four days 5 after foreign DNA is added to the cells, and "Type B" formulations, which primarily stabilise transient expression after foreign DNA has entered the cell. Thus, the designations "Type A" and "Type B" reflect when the compounds are added to the cultures. Optimal expression is obtained by treating cells with a Type A compound before, during, and/or after the transfection step (first phase 20 of transient expression), and by further adding a Type B compound within hours or days within 12-60 hours) of introducing the foreign DNA, and leaving it in contact with the cells thereafter (second phase of transient expression). Optimally, the Type A compound is maintained in the medium throughout both phases of transient expression. The invention further provides an assay for determining the efficacy of individual chemical compounds and formulations of two or more compounds for their use in both phases of transient expression.

o 25 According to an eighth embodiment of the invention, there is provided a method of screening an agent comprising at least one chemical compound to determine whether the agent is capable of enhancing the transient expression of a foreign gene in a eukaryotic cell, wherein the agent is biocompatible and contains at least one hydrophobic moiety and at least one acidic moiety, the method comprising the steps of: introducing into a first and a second SW480 P3 cell on day 0 a molecule of foreign DNA that encodes a protein in a form capable of being expressed in the cells; before, during, or after introducing the DNA, contacting the second cell with the agent; cumulatively measuring in both cells between days 0 and 4, or between days 4 and 7, or between days 4 and 14 the amount of protein expressed from the foreign DNA, and using these amounts to determine, respectively, a value for X, G7, or G 14 according to the formula: (A x 100) X, orG7 orG 14 100wherein is the amount of the protein encoded by the foreign gene expressed in the first cell and, is the amount of protein expressed in the second cell; and, determining that the agent is capable of enhancing transient expression if X or G 7 or G 14 is greater than [I :\DayLib\ibW]03734a doc:mrr The chemical compounds of the subject invention create profound metabolic changes in living cells, resulting in their being able to sustain the transient expression of foreign DNA for periods far longer than previously observed. Moreover,following the addition of these chemical substances, cultured cells surprisingly reduce their consumption of glucose, and concomitantly increase their use of alternative energy sources, such as proteins and possibly lipids.

In other embodiments, this invention provides methods for culturing 5 hepatocytes in the absence of feeder cells and without the need to pre-coat cell culture substrata with proteinaceous or other adhesion-promoting molecules.

Thus, according to a ninth embodiment of the invention, there is provided a method of 1o enhancing the adhesion of a cell to a culture substratum, wherein a sulfonated amino polysaccharide having a molecular weight of about 4,000 daltons is added to the medium in which the cells are cultured.

Brief Description of the Drawings The foregoing aspects and many of the attendant advantages of this invention will become more is readily appreciated as the same becomes better understood by reference to the following detailed description, when taken in conjunction with the accompanying drawings, wherein: FIGURE 1 shows a cell growth curve for cells exposed to several different compounds of the subject invention. FIGURE 1 graphically depicts the amount of cell growth for some of the plates °oooo described in Example 4 and Table 7. The numbers in the inset boxes of FIGURE 1 correspond to 20 the plate numbers listed in Table 7; FIGURE 2 graphically illustrates the cytoxicity of some of the compounds whose test results are presented in Table 7. The numbers in the inset boxes of FIGURE 2 correspond to the plate numbers listed in Table 7; and FIGURE 3 graphically illustrates the results of the experiments of Example 6. 25 These experiments involved transient expression in differentiated porcine PICM-19 3BT cells, which resemble hepatocytes, in the presence of various chemical compounds that :°-!prolong the duration of transient expression.

FIGURE 4 is a graphic illustration of the amounts of 1-galactosidase measured in the samples harvested daily during the experiment described in Example 8, and illustrates the longterm stabilisation 32 days) of transgene expression in transfected cells cultured in a bioreactor device in the presence of transient expression-stabilising compounds.

FIGURES 5A-5C graphically illustrate the concentrations of ammonia, glucose, and lactate in the culture medium sampled daily during the experiment described in Example 8. FIGURE indicates the concentration of ammonia measured in each sample; FIGURE 5B indicates the concentration of glucose measured in each sample; and FIGURE 5C indicates the concentration of lactate measured in each sample.

[:\DayLib\LibW]03734a.doc:mrr WO 98/20146 PCTIS97/19860 Detailed Description of the Preferred Embodiment Definitions: Transfection: For purposes of the following disclosure, "transfection" will refer to any means of introducing foreign DNA into a recipient cell, including liposome-mediated methods, viral vectors, CaPO 4 -DNA coprecipitates,

DEAE-

dextran, naked DNA, DNA complexed with proteins transfection in the presence of starburst polymers, or other means of introducing the DNA into the recipient cell.

Foreign DNA/transgenic DNA: Genetic material that has been appropriately modified for expression in recipient eukaryotic cells, typically but not necessarily originating from an organism other than the recipient cell. Transgenic DNA typically will contain the coding region for a biologically active protein or protein domain. By "appropriate modifications," it is meant that the transgenic DNA is operably linked with a eukaryotic promoter and any other regulatory sequences required to ensure functional transcription of the foreign gene in the host cell. The DNA usually is in circular form, and contains the coding region for a polypeptide operably linked to the regulatory signals necessary for transcription and the subsequent translation of the resulting mRNA. Such signals may include a promoter for binding RNA polymerase, an enhancer, transcription termination signals, ribosome binding sites, translation start and stop signals, poly(A) addition signals, and so on. The enhancer may be tissuespecific.

3-galactosidase (P-gal): A bacterial enzyme able to convert a colorless substrate into an easily detectable colored product. This gene is used in the Examples described below as a representative foreign gene for the purposes of demonstrating the efficacy of this invention.

The subject invention provides methods and agents for enhancing the transient expression of foreign genes in eukaryotic cells. The methods have been shown to be efficacious in human colon carcinoma cells, mouse melanoma cells, porcine primary hepatocytes, and in a porcine cell line that resembles differentiated hepatocytes. This method involves first introducing into the cell a molecule of foreign DNA that encodes a protein in a form capable of being expressed in the cell.

The foreign DNA may be introduced into the cell by any convenient method, including but not limited to, lipofection, electroporation, incubation with CaPO 4

-DNA

coprecipitates, incubation with DEAE-dextran, ligation of the DNA into a viral vector, and so on. Vectors derived from retroviruses or adenoviruses are useful for introducing foreign DNA into eukaryotic host cells. If desired, the plasmid or viral WO 98/20146 PCT[US97/19860 -11vector containing the foreign DNA may provide nucleotide sequences positioned between the promoter and the insertion site, or alternatively, positioned following the insertion site, such that one or more amino acids encoded by the vector-provided nucleotide sequences become fused to the protein encoded by the foreign DNA. Such fusion sequences can provide peptides that direct desired post-translational modifications, such as signal peptides for secretion, or sites for attachment of carbohydrate moieties.

The subject invention provides methods and agents for enhancing the transient expression of a foreign gene in a cell. Before, during, or after the introduction of the foreign DNA into the cell, the cell is contacted with one or more of the chemical compounds described below, whereafter the expression of the foreign DNA becomes substantially enhanced as compared with cells transfected in the absence of these compounds. By "enhancing transient expression," it is meant here that when the subject methods are used, the amount of transgene expression during the first few days following transfection is increased as compared with controls, or that the period during which transient expression occurs is prolonged as compared with controls, or both. In the subject methods, cells into which foreign DNA is introduced are contacted with a chemical agent that increases the efficiency of initial DNA uptake or expression, or that prolongs the effective half-life of the foreign DNA after it has entered the cell. Individual compounds may increase the efficiency of initial expression, and also prolong the period of transient expression. The term "effective half-life of the foreign DNA" is used here in a chronological sense to refer to the length of the period during which protein encoded by the foreign DNA can be detected in the cells. When conventional transfection protocols are used, when transfection is performed without using the below-described chemical compounds according to the disclosed methods, transient expression typically decays to an undetectable level within 3-4 days after the foreign DNA has been introduced into the cells. However, using the subject methods, easily detectable levels of expression are typically observed as long as 14 days after the transfection step, and have been observed for as long as 32 days after transfection. DNA and RNA homologous to the transfected foreign DNA has been detected 32 days post-transfection in transfected cells that were treated according to the subject methods. Using the subject methods, transient expression typically peaks within about 2-3 days, then drops off to a level of about one-third the initial level, thereafter remaining stable for several days to several weeks.

WO 98/20146 PCTIUS97/19860 -12- Agents useful for the subject methods include a large number of chemical compounds that are described more fully below. An "agent" may consist of a single chemical compound, or a combination of two or more compounds. Moreover, the agent may include one or more compounds administered during the early phases of the transfection protocol, and an additional compound or compounds added after the foreign DNA has entered the cell. These transient expression enhancing agents may be present before, during, and after the introduction of foreign DNA. When added to the cells after introducing the DNA, the agent typically remains in contact with the cell for at least 24 hours, or longer.

Generally, chemical compounds useful as agents in the subject method include at least one hydrophobic moiety and at least one acidic moiety. The acidic moiety may also be hydrophobic and organic. For certain of these chemical compounds, the acidic moiety has been modified as a salt or an ester. In one embodiment, the chemical compounds are carboxylic acid derivatives represented by the general formula Ri-C(=O)-OR 2 and in another embodiment, the chemical compounds are sulfonic acid derivatives represented by the general formula R 7 -S0 2 -ORs.

Suitable carboxylic acid derivatives RI-C(=O)-OR 2 include naturally occurring amino acids glycine, alanine, valine, leucine, isoleucine, aspartic acid, glutamic acid, glutamine, serine, threonine, methionine, arginine, lysine, histidine, proline, tryptophan, phenylalanine, tyrosine), their unnatural optical isomers, and certain amino acid derivatives 3-methyl-L-histidine, a-ketoglutaric acid, 3alanine, carnosine, citrulline, creatine, folic acid, glutathione, hippuric acid, homoserine, N-carbamyl aspartic acid, N-formyl-L-methionine, and ornithine).

Referring to the general carboxylic acid derivative formula, Ri-C(=O)-OR 2 for the amino acids, Ri is CHNH 2

R

3 wherein R 3 is the side chain of a naturally occurring amino acid. Other amino acid derivatives that are useful in the method of the present invention include amino acids that further include alkyl substituents and alkyl substituents having additional functional groups. These amino acid derivatives are represented by the above carboxylic acid derivative formula where R 1 is

CHNH

2

(CH

2

),R

5 wherein n 1-7 and Rs is selected from CH 3 OH, CONH 2

C

6

H

4 OH, and CONHNH 2 Alternatively, R 1 is -(CH 2 )nCHNH 2 CO2H, wherein n 1- 8; -CH(CO 2

H)NHCONH

2 or R 1 is -CsH 4 N nicotinic acid and derivatives).

In addition to amino acids, the carboxylic acid derivatives useful in the present method include alkyl, aryl, and substituted alkyl and aryl carboxylic acid derivatives.

Preferred alkyl and substituted alkyl carboxylic acid derivatives are represented by the WO 98/20146 PCT/US97/19860 -13general formula above where R 1 is -(CH 2 wherein n 1-9 and R 6 is selected from an indole group, NCH 3

C(=NH)NH

2

SCH

3

NH

2

CH

3

CO

2 H, CONH 2 and

NHC(=NH)NH

2 Preferable aryl carboxylic acid derivatives include benzoic acid and its derivatives. The benzoic acid derivatives are represented by the formula above where Ri is -C 6

H

4 IR, wherein R 4 is selected from H, CH 3

(CH

2 )nCH 3

NH

2

COCH

3

CO(CH

2 )nCH 3

C(CH

3 3

CH(CH

3 2

(CH

2

),CH(CH

3 2

(CH

2

)COCH

3

OCH

3 and

O(CH

2

).CH

3 wherein n 1-3. It has been found that branched chains often are more efficacious than linear drains.

The carboxylic acid derivatives useful in the present invention include carboxylic acids R 2 is carboxylic acid esters R 2 is CH 3 and (CH 2

),CH

3 wherein n 1-8) including esters having additional functional groups such as ether and ketone groups R 2 is (CH 2 )xO(CH 2 )yCH 3 and (CH 2 )xCO(CH 2 )yCH 3 wherein x y and carboxylate salts including metallic salts lithium, sodium, potassium, calcium, and magnesium) as well as relatively low molecular weight cations ammonium).

Suitable sulfonic acid derivatives, represented by the general formula

R

7 -S0 2 -ORs, include alkyl, aryl, substituted alkyl and aryl sulfonic acid derivatives

R

7 is an alkyl, aryl, or substituted alkyl or aryl group). Preferably, the sulfonic acid derivative is a lower alkyl straight chain or branched C 1

-C

5 alkyl group) sulfonate, and more preferably, an amino substituted lower alkyl sulfonate, for example, taurine. Preferably, the aryl sulfonic acid derivative is a benzene sulfonic acid derivative, and more preferably, an amino substituted benzene sulfonate, for example, 3-aminobenzene sulfonic acid. The sulfonic acid derivatives useful in the present invention include sulfonic acids Rs is and sulfonic acid salts including metallic salts Ra is lithium, sodium, potassium, calcium, or magnesium) as well as relatively low molecular weight organic cations Rs is ammonium ion).

In another embodiment, the chemical compounds useful as agents in the subject methods include sulfonated amino polysaccharides. Preferably, the polysaccharide is a sulfonated N-acetylated amino polysaccharide. Suitable polysaccharides typically contain from about 4 to about 200 sugar residues.

Preferred sulfonated N-acetylated amino polysaccharides include chondroitin- 6-sulfate and sulfonated guarans. Guarans are isolated from the endosperm of Cyamopsis tetragonolobus seeds and are high molecular weight 3-1,4 Dgalactomannans up to about 1,200,000 daltons) having a-1,6-linked D- WO 98/20146 PCT/US97/19860 -14galactose residues attached to a mannan backbone. Suitable guarans include 2hydroxypropyl ether derivatives commonly referred to as hydroxypropyl guarans.

Other chemicals compounds that are useful in the method of the present invention include adrenaline (epinephrine), coenzyme B12, and methylcobalamin.

An especially effective example of a sulfonated amino polysaccharide identified thus far is chondroitin-6-sulfate (type C chondroitin sulfate), a polyanionic glycosaminogycan. Chondroitin sulfate is a naturally-occurring mucopolysaccharide found in cartilage and soft connective tissue throughout the body. Type C chondroitin sulfate is a polymeric molecule that varies in its molecular weight, degree of sulfonation at the N-acetylgalactosamine residue of the repeat disaccharide, and in the relative distribution of sulfated to unsulfated repeat units. Chondroitin-6-sulfate having a molecular weight of about 4000 daltons is particularly effective. Commercial preparations of type C chondroitin-6-sulfate typically contain a variable proportion of type A chondroitin sulfate, which is chondroitin-4-sulfate. While preparations containing predominantly type C chondroitin sulfate are highly effective for stabilizing transient expression, preparations that contain predominantly the type A form of this polymer are ineffective. Thus, the relative amount of 4-sulfate to 6-sulfate substitution sites occupying the N-acetylgalactosamine residue of the repeat disaccharide in a given polymer preparation is critical to the polymer's activity in the subject method, as only the 6-sulfate form appears to be active in enhancing transient expression.

Chemical compounds effective in enhancing transient expression have the following desirable characteristics in common: 1. Little or no cytotoxicity when added to cells in culture within concentration ranges effective for enhancing transient expression. For the sulfonated amino polysaccharides, this range is about 0.01-0.5 mM. For the remaining compounds effective in enhancing transient expression, this range is about between 1- 15 mM. These optimal amounts are in addition to quantities of these substances that may already be present as a cell culture media component certain amino acids).

Compounds that are not cytotoxic according to this assay are defined here as "biocompatible." For the purposes of this invention, a cytotoxic substance may be defined as one that, at a given concentration, results in >50% decline in the number of viable cells within 4 days post-transfection in an 8 day static culture of SW480 cells, with continuous exposure to the substance, and wherein no net expansion of the cells occurs by the end of the 8 day period. However, it should be understood that various WO 98/20146 PCT/US97/19860 types of cells vary in their sensitivity to different chemical compounds. Thus, while SW480 cells may be used as a convenient tool for determining biocompatible concentrations of the chemical compounds, it may be necessary to empirically adjust the concentrations determined with SW480 cells in order to optimize biocompatibility with other types of cells. Assays for cytotoxicity are described in greater detail in Example 4.

2. An acidic functional group is always present carboxylic, sulfonic, and the like), and may be modified to reduce cytotoxicity. Preferred modifications are ester and salt formation (including salt based on organic cations), as salt and ester bonds are readily cleaved by metabolic processes after the compound has entered the target cell. In a preferred embodiment, the chemical compound in aqueous solution has a pH of 4.5-9.0.

3. To the acidic group is linked a relatively hydrophobic organic group.

For compounds other than the sulfonated polysaccharides, this portion of the molecule is preferably non-polar and hydrophobic. Similarly, the sulfonated polysaccharides have their natural hydrophilic character modified by the presence-of a relatively hydrophobic functional group the N-acetyl group in chondroitin-6sulfate in the 2-substitution position). Many of the compounds that have proven efficacious for the subject invention contain an acidic group that is organic and hydrophobic.

4. Several of the most effective agents a 50/50 mixture of benzoic acid and sodium benzoate (benzoate buffer), and chondroitin-6-sulfate) possess antioxidant and free radical scavenging character see Merck Index).

For convenience, the compounds other than the sulfonated amino polysaccharides will hereafter be referred to as "Group while the sulfonated amino polysaccharides will hereafter be called "Group II." In a preferred embodiment of the invention, the cells are contacted with a chemical compound selected from Group I prior to and during the introduction into the cell of the foreign DNA, and are further contacted with a chemical compound selected from Group II following the introduction of the foreign DNA. The chemical compounds of the subject invention are efficacious whether they are added to the cells prior to, during, or after the transfection step.

The subject invention provides methods useful for prolonging transient expression in cultured cells, including primary cultures, established cell lines, stable cultures of differentiated cells, normal cell lines maintained by exposure to growth WO 98/20146 PCTfUS97/19860 -16factors, and transformed cells, such as cultures established from various tumors, including hybridoma cells and the SW480 P3 human colon carcinoma cell line (ATCC #CCL228; hereafter referred to as "SW480 cells").

The subject methods are useful also for introducing foreign DNA into cells in vivo. The compounds can be administered by any convenient means, including orally, topically, by perfusion, or by injection. If it is desired to confine the host's exposure to the chemical compound to the tissues that will receive foreign DNA, the chemical compound can be introduced by localized injection, such as, for example, injection directly into a solid tumor mass, or by incorporating into liposomes a protein that targets them to specific tissues. Injections of the chemical or combination of chemicals can be accompanied by or followed by injection at the same site of a vehicle for delivering the foreign DNA. Alternatively, the compounds can be applied in a vehicle that provides for the slow release of the compounds at the target site, as, for example, by the dissolution of an inert solid carrier.

The method of the subject invention contemplates the harvesting of proteins expressed by transgenic DNA introduced into cells in the presence of the abovedescribed compounds. The protein can be harvested by any convenient means, such as, for example, by extracting the transfected cells, or by extracting the culture medium in which the cells are grown. If desired, the transgenic protein can be expressed using a vector that provides signal peptides that direct the secretion of the transgenic protein into the culture medium. Secreted protein also can be assayed over a period of days to determine the relative or absolute amount of protein produced, thus providing a means for evaluating the effectiveness of variations in the transfection protocol. Crude cell extracts can be assayed for enzymatic or other biological activity of the harvested protein, or the protein can be further purified using standard procedures before performing functional assays for the protein's activity. If the transgene is expressed in vivo, the protein can be harvested from body fluids of the host, such as milk, or other body tissues. The purification procedure used for a given protein will depend on the physical properties of the protein, such as its size, shape, hydrophobicity, stability, and so on. Also, the harvested protein may be detected or quantified by physical means, such as, for example, gel electrophoresis, isoelectric focusing, or by chromatographic methods such as high-pressure liquid chromatography, or the like.

The methods of the subject invention result in the rapid production in cultured eukaryotic cells or in transiently expressing mammalian hosts of milligram quantities WO 98/20146 PCT/US97/19860-- -17of the protein product of the expressed transfected genes. Protein production in transfected eukaryotic cells provides an advantage over bacterial expression systems in that expression in eukaryotic cells can support post-translational modifications that may be required for the biological activity of many proteins. Moreover, by using eukaryotic instead of prokaryotic host cells, one avoids having to eliminate potentially toxic bacterial proteins from final preparations of the transgenic protein.

The subject methods provide a means for using eukaryotic host cells to obtain commercially useful amounts of biologically active protein, growth factors, hormones, antibiotics, and the like, in a eukaryotic host cell without having to establish permanent cell lines containing stably integrated foreign DNA. These methods can be used for rapidly obtaining biologicals to be tested for their pharmaceutical properties.

Contacting cells with the chemical compounds selected from Group II of the subject invention results in increased cell adhesion and cell-to-cell contact and communication, thus administration of these compounds provides a means for enhancing these cell-cell interactions. Thus, the subject invention includes methods for enhancing the adhesion of a cell to a culture substratum by growing the cells in the presence of a sulfonated amino polysaccharide that has been added to the culture medium, thus promoting the longevity of the cells in culture. For example, chondroitin-6-sulfate is effective in promoting the long-term growth in culture of a cell line that has the characteristics of differentiated hepatocytes. Ordinarily, hepatocytes will not survive in culture unless feeder cells are provided or the culture substratum is first coated with a substance to promote hepatocyte adhesion (see, e.g., Sidhu and Omiecinski, Pharmacogenetics, 5:24-36 (1995)).

When agents of the subject invention were contacted with cultured cells, the cells exhibited altered metabolic processes, including reduced glucose consumption and lactate production, as well as increased ammonia production. Thus, the subject methods are useful for manipulating the metabolism of a cell such that the cell utilizes alternative carbon sources such as proteins or lipids. In addition, the agents induce cells to express elevated levels of an endogenous alkaline phosphatase activity.

Compounds of both Groups I and II are useful for this purpose. An agent especially useful for manipulating a cell's utilization of energy sources is a combination of benzoic acid, 4-ethylbenzoic acid, chondroitin-6-sulfate, and benzoate buffer, wherein benzoate buffer is an equimolar mixture of benzoic acid and sodium benzoate. The WO 98/20146 PCTIUS97/19860 -18subject methods are useful for manipulating cell metabolism either in vitro or in vivo, to treat mammals for obesity.

Because of their hydrophobic nature, the chemical compounds of the described methods may be capable of crossing the highly hydrophobic mitochondrial outer membrane. As these agents affect mitochondrial-based metabolic processes, i.e., glucose metabolism, it seems possible that the observed enhanced expression results from the transcription of the foreign DNA inside of mitochondria. As the foreign DNA is circular, it may even be replicated by the machinery normally used for replicating the circular mitochondrial DNA, thus increasing the amount of template available for expressing the transgene.

The subject invention provides for the use of these chemical compounds as agents for enhancing transient expression both in vitro and in vivo. When employed either in vitro or in vivo, the compounds are optimally used before, during, and after the introduction of foreign DNA. On removal of the chemical compounds from the cell culture media, their effect on long-term expression gradually disappears and the previous behavior of the cell resumes. When employed in vivo, the compounds may be injected as a primer into the recipient tissue or intravenously admixed with the transgenic DNA solution, and administered after introduction of the foreign gene by injection, or may be administered as a dietary supplement. For example, a tumor could be primed by direct injection of a chemical compound followed by later injection of the DNA, followed still later by an oral supplement of the same or different compounds.

In other embodiments, the invention provides methods for obtaining stabilized transient expression of foreign genes in a system of cell culture that perpetuates cells in semi-solid masses thai-simulate solid tumors, cells grown in a bioreactor.

Protocols developed in this model tumor system can be used to transfect genes expressing anti-tumor compounds, IL-2, directly into solid tumors.

The mechanism by which the subject agents enhance transgenic expression has not been determined. They may directly stabilize the transfected DNA or its transcripts, or may even stabilize the expressed protein, though this latter possibility is unlikely. Moreover, Group I and Group II compounds appear to act through different mechanisms to enhance transient expression, since combinations of compounds from the two groups are often more effective than when the compounds are used separately (see, Example In a preferred embodiment of the subject invention, the cells are contacted before, during, and after transfection with one or more compounds from WO 98/20146 PCTfUS97/19860 -19- Group I, and are contacted following the transfection step with a compound of Group II, a sulfonated polysaccharide.

The following parameters have been defined to facilitate and characterize chemical compounds that are useful as agents for prolonging the duration of transient expression. These parameters are called the factor, the factor, and the "K" factor.

1. Xfactor: X=100-(Axl00)) C where is the amount of protein expressed in the control transfected cells during the chosen time period, and is the amount of protein made in cells to which the chemical compound has been added.

This factor reflects the extent to which a chemical compound added to a transfected cell enhances stabilizes transient expression for the first four days following transfection. For chemical compounds active in stabilizing transient expression, the value for X will be For example, if expression is doubled in the presence of a compound, X= 50. Preferred compounds will have X>10, and most preferred compounds will have X>25. This factor provides a way of comparing the amount of foreign gene expression observed when a chemical compound of the present invention is present in the culture medium for the first four days after transfection, as compared with the amount of expression observed in control cultures lacking the compound. Thus, the Xfactor is related to the ratio between the amount of expression observed in the presence and absence of the compound. X may be calculated similarly when the agent in question is a mixture of more than one chemical compound.

Cumulative protein expression, the values for and is measured by summing the values measured daily in aliquots of the cultured cells.

2. G factors. G factors differ from the X factor only with respect to the time period evaluated. For calculating a G factor, the amount of protein expressed is measured from days 4-7 or days 4-14, where day 0 is the day on which the foreign DNA is added to the cells. The subscripts denote which of the two time periods provided the basis for measurement. Thus, "G 7 indicates that the measurements were made between days 4-7, and "G 14 indicates that measurements were made between days 4-14. As for theXfactor, WO 98/20146 PCTIUS97/19860 A x 100

G

7 or G 14 10 0

C

where and are defined as for the Xfactor.

It is useful to characterize compounds according to both the X and G factors, because some compounds having low or negative values for X may have high or positive values for the G factors. Compounds with high values for G 7 or G 14 are especially useful for transient expression where one or more compounds are added to the cultures after the DNA has already entered the cells, during the second phase of transient expression. Preferred compounds have values for G More preferably, G >10, and most preferably, G 3. K factor. The K factor is the ratio of the rate constants for the decay of the foreign DNA expression in control transfected cells and in cells exposed to a chemical compound of the subject invention. K is determined according to the following equation: K k(DNA)control k(DNA)compound wherein "kDNA)" is the first order rate constant for the decay of expression of the transgenic DNA which is expressing protein as a function of time, i.e., d (DNA) dt k(DNA).

kt which is equivalent to log(DNA) 2.0 log(DNA) For convenience, the term "d(DNA)" is used as if it reflected changes in the effective concentration of transfected DNA, though it remains possible that the observed changes in expression are a function of some parameter other than simply the concentration of foreign DNA in the cells. Hence, the first order reaction rate in control transfected cultures can be expressed as k(DNA) d(DNA)/dt orlog(DNA) kt/2.303 log(DNA)0. Thus, when log(DNA) is plotted against time, the intercept with the Y axis, or log(DNA)0 reflects the initial concentration of transfected DNA that is expressed. Moreover, the slope of this line equals -k(NA/ 2 .303.

Values for kDNA) are derived by using a computer program that plots the log of the foreign protein concentration against time. The program determines the slope of that portion of the resulting line that corresponds to the period during which WO 98/20146 PCTiUS97/19860 -21protein production is decreasing. Typically, the optimal amount of protein synthesis occurs during the 48 hour period following transfection. Thereafter, the rate of expression declines at a rate that is subject to manipulation by contacting the cells before, during, and/or after transfection with the various chemical compounds of the invention. Thus, the slope is calculated during this period of decline to provide values for k(DNA) and K that can be compared for the purpose of comparing the efficacy of different chemical compounds. For especially effective formulations of the subject chemical compounds, the initial decline in rate of expression may be followed by an increase in the rate.

The K factor thus reflects the effects of chemical compounds on the stability of the foreign gene expression after it is already inside the cell, and not the effects of these compounds on initial DNA uptake. The K factor is important because the advantages of this invention, in contrast with other reported methods for improving transient expression, derive primarily from providing a means for stabilizing transient expression after the transfection step, rather than on the traditional approach of trying to improve the efficiency of DNA uptake. However, some of that the chemical compounds of the subject invention have their maximal effectiveness during the first 4 days post-transfection, thus suggesting that they may act by inducing cells to take up increased amounts of the transfected DNA. Such compounds may act as well to prolong the effective half-life of gene expression once the foreign DNA is inside the cell. A positive value for K indicates that a compound or combination of compounds is effective in stabilizing foreign DNA expression post-transfection, thus the preferred chemical compounds and formulations of the invention will have values of K >0.

In the absence of the chemical compounds of the invention, the decay of transgenic DNA expression, k(DNA), is a first order reaction. When the chemical compounds of the invention are added to the cultures, the kinetics for expression of the foreign DNA change dramatically, as compared with control cultures. In the presence of these compounds, k(DNA) becomes increasingly more positive as the production of foreign protein is extended for long periods of time. Indeed, the changes are so dramatic for some of the most preferred formulations, those in which K>40, that conventional first order kinetics cannot adequately represent the results. Thus, it appears that the preferred compounds/formulations change the kinetics to either a pseudo-first order or a second order reaction. This result is not predicted by conventional wisdom.

WO 98/20146 PCT/US97/19860 -22- Many compounds and formulations useful in the subject methods are discussed in the Examples and are included among those listed in these Tables 1, 8, 9, and 10, in which values for X, G, and K are presented.

This invention further provides a method based on the SW480 cell line for screening chemical agents to determine whether they are capable of stabilizing transient expression. For this method, candidate chemical compounds for screening are those that are biocompatible and that contain at least one hydrophobic moiety and at least one acidic moiety. The test compound or group of compounds is introduced into a culture of SW480 cells before, during, and/or after the introduction on day zero of foreign DNA that encodes a protein capable of being detected if it is expressed in the cells. For a period of days following the transfection step, samples of the culture are harvested and the amount of foreign protein therein is determined. The amount of the protein expressed cumulatively in the culture is determined by summing the amounts measured in the daily samples, and these sums are compared between test cultures, those that are contacted with the test compound, and parallel control cultures that are not contacted with the compound. Aliquots for protein measurement may be harvested daily between days 0 and 4, or between days 4 and 7, or between days 4 and 14, and the amounts of protein measured are used to determine, respectively, a value for X, G 7 or G 14 according to the formulae given above. If the value thus determined for X or G 7 or G 14 it may be concluded that the agent is capable of enhancing transient expression. Preferably, X, G 7 or G 14 >10, and most preferably, are >25. Chemical compounds so identified may be used to enhance the transient expression of foreign genes in the procedures described above. Other cell lines can be substituted for SW480 cells in this screening assay.

Preferred product formulations are selected from among those compounds and combinations of compounds that exhibit the highest (or most positive) values for the X, G and K factors. Furthermore, the various chemical compounds of the invention can be used together to maximize the enhancement of transgene expression. For example, the various compounds can be used to treat the same culture at different times during the procedure. Different formulations require different combinations of properties. Two distinctly different types of preferred formulations are: Type A Formulations: These are compounds having a high value for X These compounds are highly active immediately following the transfection step, and thus may act during the first phase of transient expression by enhancing the efficiency of DNA uptake. Therefore, the logDNA), or Y intercept, from a semi-log plot as WO 98/20146 PCTIUS97/19860 -23described above, is higher in the case of a Type A compound or formulation than for the control culture without these compounds. Such compounds are assumed to affect the efficiency of DNA uptake because the Y intercept is a rough measure of the concentration of active foreign DNA inside the cell immediately following its introduction to the cell. Many of the compounds tested have positive values for the Xfactor see Table Thus, this invention not only provides chemical compounds for stabilizing transfected DNA, but also provides compounds that appear to enhance initial DNA uptake into the cell. Many of the treated compounds had high values for G 7 or G14 or K as well as high values for X, thus are efficacious during both phases of transient expression see Tables 1, 3, and 9).

Type B Formulations: Compounds useful in this category require both a high G and K factor. A high value for X is desirable, but is not required. Values for K that are greater than zero are characteristic of compounds capable of stabilizing transfected DNA. The most highly preferred Type B stabilizers have values of K >1, or more preferably, K>10, and values for G 7 or G 14 >25. Furthermore, replicate experiments exhibiting X and/or G factors >25 are required before a particular agent is considered a highly preferred compound in either a Type A or Type B formulation.

For both in vitro and in vivo applications, transient expression is best maximized by the use of both Type A and Type B formulations. For example, a preferred method involves first priming the cells by exposing them prior to transfection to one or more Type A compounds that have a value ofX>25. The Type A compounds are also present during transfection, and optimally remain present throughout the period of transient expression. After the transfection step, the cells are contacted for the remainder of the period of transient expression with one or more Type B compounds each of which preferably has a value for G 7 or G1 4 >25. In a preferred embodiment, the Type A compound is benzoate buffer, and the Type B compound is chondroitin-6-sulfate. In another preferred embodiment, the Type A compounds are benzoic acid and 4-ethylbenzoic acid, and the Type B compounds are benzoate buffer and chondroitin-6-sulfate. In yet another preferred embodiment, the Type A compounds are benzoate buffer and glutamic acid, and the Type B compound is chondroitin-6-sulfate.

For in vitro applications, the best results are achieved when cells are cultured in the presence of a Type A formulation for several hours, about 20-24 hours, prior to the transfection step. Note that priming with a Type A formulation in the form of a dietary (or oral application) is a realistic option in vivo. Many of the WO 98/20146 PCTIUS97/19860 -24compounds effective for enhancing transient infection are known to be non-toxic (see Table 1, below). Cultured cells are optimally maintained in the presence of the Type A formulation for at least 48 hours post-transfection. If desired, the Type A formulation can be removed after about >90% of the cells have taken up the foreign DNA, several days after the DNA is added to the culture, or this formulation can remain in contact with the cells during the second phase of transient expression. The Type B formulation is optimally added to the cells at the peak oftransgene expression, which typically occurs 24-48 hours post-transfection. Optimally, feeding with medium containing a Type B formulation is repeated periodically for the duration of the experiment.

It should be apparent that the subject methods can be used in vivo animal studies and clinical procedures). In particular, in the case of gene therapy involving a solid tumor, a Type A formulation may be co-administered with the DNA delivery vehicle, where the recipient tissue is "primed" by injection of a Type A formulation prior to administering the DNA. Thereafter, the Type B formulation is administered.

The useful concentration ranges for individual compounds may vary, and the upper limits of useful ranges may be limited by cytotoxic effects. Direct injection of the DNA/Type A formulation into a tumor would involve only routine procedures, as a variety of pharmaceutical carriers are well-known in the art. Direct injection would avoid exposing non-target tissues to the transfection reagents. Choline, liposomal formulations, or controlled release formulations can be combined with a Type B formulation to prolong the localized effect on the transfected tumor cells. In addition, a Type B formulation can be fed to the patient as a dietary supplement (or additive) for extended periods of time. Both injection and dietary feeding can be combined for optimal effectiveness according to factors such as toxicity, and the like. This approach offers the advantage of delivering high doses of a cytotoxic protein to a tumor without damaging other body tissues. Using this strategy, the tumor cells themselves would be induced to continuously produce the cytotoxic protein over a period of days, thus providing a far more effective means of delivery than simply injecting a dose of the protein itself into the tumor. This approach would be particularly helpful in cases where the protein in question is one that could not easily cross the plasma membrane if applied externally, or in cases where a therapeutic protein has a short half-life once inside the target cell.

WO 98/20146 PCTIUS97/19860- In other manifestations of the invention, a compound selected from Group I may be linked covalently or non-covalently to a compound selected from Group II, chondroitin-6-sulfate.

Example 1.

Screening Assay for Enhancement of Transient Expression Protocolfor control transfections: The following procedures were used to provide transient expression: SW480 P3 (ATCC CCL228) human colon carcinoma cells (typically, 1 x 106 cells) were plated in the wells of a 6-well tissue culture plate. The number of wells plated reflected the number of days post-transfection during which the experiment would proceed. Each well contained 1 ml of complete media from a 30 ml stock solution containing: 26.4 ml RPMI tissue culture medium, 4 mM L-glutamine, ml fetal bovine serum, and 10 ig/ml gentamicin. Cells were cultured at 37 0 C in a

CO

2 incubator with 10% CO 2 for 24 hours after being plated, during which time the cells adhered to the plates.

After the 24 hour pre-incubation step, the transfection step was carried out by removing the RPMI and adding 900 jiL OPTI-MEM® (Gibco) medium containing 2 lg of VR1412 DNA, (Vical, Inc., San Diego, CA), which expresses the bacterial P-galactosidase gene under the control of a cytomegalovirus promoter, and 8 g of a mixture of cationic lipid (1,2-dimyristyloxyproply-3-dimethyl-hydroxyethyl ammonium bromide "DMRIE/DOPE") mixed in equimolar proportions with dioleoylphosphatidylethanolamine) to yield a lipid:DNA molar ratio of 0.99:1. It should be noted that typical transient transfection protocols employ 10 Pg DNA per 106 cells, but the protocol described here uses less DNA in order to reduce toxicity to the cells. The plates were then incubated for 4 hours at 37"C.

After the 4 hour incubation step, 100 4l of heat deactivated fetal bovine serum (to stop transfection), plus 12.0 jil of 50 mg/ml gentamicin were added to each well.

Twenty-four hours after the addition of foreign DNA to the wells, all of the cells from one well were trypsinized and counted, then 2 x 10 4 cells from each well were lysed and stored in liquid N 2 until being used at a later time to determine 0-galactosidase concentration. At that time, each of the unharvested wells received 1 ml of the previously defined OPTI-MEM medium (without L-glutamine added). For the remainder of the experiment, one additional well was harvested at 24 hour intervals, and unharvested wells were fed 1 ml of OPTI-MEM (without L-glutamine, and containing the test compounds) every 48 hours.

WO 98/20146 PCT/US97/19860 -26- Protocolfor test compounds: To test various compounds for their efficacy in enhancing transient expression, the protocol described above for control cultures was modified by incorporating thie candidate chemical compound(s) into the culture media. The rest of the procedure remained unaltered with respect to the protocol for the control cultures.

Lysed samples from 2 x 10 4 cells were retained for each P-galactosidase assay, and the remaining cells from each well sacrificed daily. The lysates were frozen and maintained in liquid nitrogen until 1-galactosidase assays could be conducted. The thawed samples were assayed for 1-galactosidase using a chlorophenol red-based procedure based on chlorophenol red, wherein the colored product was quanitated at 580nm using an ultraviolet/visible light spectrophotometer.

The results from this assay for a large number of chemical compounds are presented below in Table 1. Table 1 gives values of X determined in experiments in which cultured SW480 human colon carcinoma cells were cultured and transfected with a bacterial P-galactosidase gene using the methods described in the Examples.

For purposes of comparison, Table 1 includes compounds that tested negative in the assay as well as a large number of compounds that tested positive. The pH values shown in Table 1 were determined in aqueous solutions made by diluting stock solutions prepared in culture media with deionized water. Compounds ranging from about pH 3 (melanin) to pH 10 (adrenaline) were observed to be effective for prolonging the duration of transient expression. The preferred pH range is about pH 4.5-9.0.

A test result was considered positive if the value calculated for any one of X, G, or K exceeded zero.

WO 98/20146 WO 9820146PCTIUS97/19860 -27- Table 1 GROUP I CHEMICAL COMPOUND mM pH (1120) X Factor G 14 Factor K Factor '3-[BIS(2HYDROXYETHYL 1 6.81 -55 AMINO0)]-2-HYDROXYL-' 1- PROPANE SULFONIC ACID 3-AMI1NO BENZENE 1 3.8 -19 32 SULFONIC ACIOD 3-METHYL-L-H1ISTIDINE 4 7.39 -15 4-AMIfNO-BENZOIC ACID 1 7.67 52, 52 96*, -12 1, 1 4-BUTYLBENZOIC ACID 1 5.94 -26 165 9 4-ETHYLBENZOIC ACID 1 6 42, 46 43, 63 1, 2 4-HEXYLBENZOIC ACID 1 6.13 -68 -26 4-OCTYL BENZOIC ACID 1 7.45 -2314 1-665 4-PENTYLBENZOIC ACID 1 1 -96 -88 ct-AMINO-n-BUTYRIC ACID 4 7.58 19 35 1 o,-KETOGLUTARIC ACID 1 3.75 7.28 53 ADRENALINE 1 10.29 49 68 ASPARTIC ACID 4 5.75 40,-13 -45, -17* f3-ALANINE 4 8.38 .2 33 1 ax-ALANINE 4 7.27 31 BENZOATE /HEPAIN 2.5,0.1 5.51 -3 22 BENZOATE BUTFFER 4 4.74 29, 17, 3, 41*, 58, 27, 2, 2, 1, 1, (equimolar benzoic acid/sodium 2, -16, 30 38, 44, 41* 1, 3* benzoate)III BENZOIC ACID 1 14.21 1-3.1 128 1 WO 98/20146 WO 9820146PCTJLJS97/19860 GROUP I CHEMICAL COMPOUND mM pH (11 2 0) Factor G14 Factor K Factor BENZOIC ACID &4 1,1 5.82 41 80 2 ETHYLBENZOIC ACI1D BES 1 6.64 177,57 3 2,-24 1, 1 BUTYRATE BUFFER 2.5 6.12 -169, -275 81*, 69* -9,8 CARNOSINE 4 8.32 -8 CITRULLINE 1 7.71 139 46 1 COENZYME B12 N/A N/A -17 51 CREATINE -4 7.54 .34 28 CYSTELNE 4 7.24 -35 DR[ODOTYROSINE 4 7.23 -4 8 -8 ETHYL 4-ACETYLBENZOATE 1 7.59 49 42 1 ETHYL 4-ACETYLBUTYRATE 1 5.98 51 58 1 FOLIC ACID 1 6 -1.4 24 GLUTAMIC ACI1D 1 4.2 -17,8 31* 2 GLUTANHIC ACI1D WITH 1 4.91 44, 45, 3, 69, 65, 27, 1, 1,1, 2, BENZOATE BUFFER 44,42, 53, 69, 76, 10, 6* __44 69* GLUTARIC ACI1D 1 3.85 22 -77 GLUTATIHONE 2 3.58 2 34 1 GLYCINE 4 7.2-7 1-34 40 2 HIPPURIC ACID 2 6.46 2 33 1 HISTIDINE 4 6.75 26, -30 54*, 6 1, 1 HOMOSERINE 11 7.4 177 39 ISOLEUCINE 14. 16.99 -19 3 WO 98/20146 WO 9820146PCT/US97/19860 -29- GROUP I CHEMICAL COMPOUND mM pH1(H1 2 0) XFactor G.1 4 Factor KFactor L-ARGINJNE 4 8.66 15,54 11*, 57 L-GLUTTAN4INE 4 7.14 -13 23 L-THREONINE 4 8.14 28 401 LEUCINE 4 7.88 -13 32 L-LYSINE 4 8.34 -39, 18 -10, 9* MELANIN 0.1 3.45 -155,0 -453,-173 METHYLCOBALAMIN N/A N/A 1 MEIHONINE 1 7.41 2 36 0 N-(4-AMIINOBENZYL)-L- 1 6.44 -35 8 GLUTTAM[IC DIETHYLESTER N-CARBAMYL-DL-ASPARTIC 4 4.19 26 115

ACID

N-FORMYL-L-METIONINE 1 4.34 26 63 NICOTINIC ACIID 1 6.91 12 87 1 ORNITHINE 1 7.37 116 28 PIENYLALANJNE 4 6.97 -12 53 3 PROLINE 4 7.71 -22 21 S-CARBAMYL-L-CYSTEINE 1 6.52 -76 -74 SERINE 1 7.4 43 104 1 SODIUMBENZOATE 1 8.14 -15 -6 1 TAURINE 4 7.88 -20 -34 2 TRYPTOPITAN 4 6.25 55 67 2 .TYROSINE 14 17.88 136 152 2 -WO 98/20146 PCT/US97/19860 GROUP I CHEMICAL COMPOUND mM pH (H 2 0) XFactor G1 4 Factor K Factor VALINE 4 8.12 -18 40 2 *Values for G denoted by an asterisk indicate values for G 7 while G values without an asterisk are values for G 1 4 Note that those compounds in Table 1 for which X >1 are compounds that increase the degree of transient expression during the first few days post-transfection.

Such compounds may influence cells to take up larger amounts ofDNA per cell than they otherwise would, or alternatively, may cause a higher proportion of transfected cells to express the foreign DNA than otherwise would have expressed it. It remains possible also that these compounds enhance early transcription or expression. These compounds have not been previously reported to have this effect on transfection.

Interestingly, melanin was noted to significantly suppress transient expression.

In addition to those compounds listed in Table 1, additional compounds that have been tested and found capable of prolonging transient expression include t-butyl benzoic acid, ethyoxy benzoic acid, iso-propyl benzoic acid, methoxy benzoic acid, isobutyl benzoic acid, chondroitin-6-sulfate (type and guarans, particularly hydroxypropyl guaran.

Example 2.

Chemical Compounds Enhance Transient Expression and Reduce Glucose Consumption Additional experiments were performed to further characterize the enhanced transient expression method. For these experiments, the five culture conditions described in Table 2 were tested using the transient expression protocol described in Example 1. Six-well plates were used, and a sufficient number of wells were seeded with SW480 cells so that the cells from individual wells could be harvested as described below.

WO 98/20146 PCTIUS97/19860-- -31- Table 2 Plate Chemical Compounds Concentration Media Type 1A Control with gentamicin see above lB Control with no gentamicin see above see above 2 Benzoate buffer 2.5 mM Pre-transfection L-glutamine 4 mM Transfection Post-transfection feeding 3 Chondroitin sulfate (type C) 0.1 mM Pre-transfection Benzoate buffer 2.5 mM Transfection L-glutamine 4 mM Post-transfection Feeding 4 Glutamic acid 4 mM Pre-transfection Benzoate buffer 2.5 mM Transfection L-glutamine 4 mM Post-transfection Feeding Each day, one well was harvested for counting, and 2 x 10 4 cells from each harvested well were lysed, and the lysates retained for a P-galactosidase assay.

Supernatants from these same wells were retained frozen and used later for evaluation of pH, glucose consumption, and the production of lactate and ammonia. As seen in Table 3 below, the various combinations of chemical compounds used in plates 2, 3, and 4 differed in their ability to enhance and sustain gene expression. Plate 4 had the best overall performance in this experiment, with high X and G factors. Plate 3, the only plate in this experiment that included a Group II compound, clearly showed signs of reduced transfection efficiency a low X factor) but showed promise for sustained expression a relatively high G factor).

WO 98/20146 PCTIUS97/19860 -32- Table 3 Plate Number 1A 1B 2 3 4 Parameter gentamicin no gentamicin Xfactor n/a 9 30 -26 44 Gfactor n/a 32 41 51 69 Time elapsed X-gal (blue) 24 hr 60-70 85-90 80-90 85-95 95-100 48 hr 40-50 60 50-60 70 72 hr 40-50 60 60-70 60 70-75 96 hr 10-20 30 50-60 55-60 120 hr 10-20 20-30 30-40 20-30 144 hr 10-15 10-20 20 25 20-30 168 hr 10-15 2-5 10 10 Cell culture experiments typically show a standard deviation in the range of For this reason, X and G factors less than 25 were not considered to be significant improvements over the control.

A control experiment (plates 1A and 1B, Table 3) was included in the abovedescribed experimental scheme to determine whether gentamicin, an antibiotic present in the culture media, may have influenced the outcome of the experiments described above. From comparing the results for control plates #s 1A and 1B, it is evident that gentamicin somewhat suppressed protein production. This is suggested by the slightly lower values for X and G factors in controls with gentamicin, plate lA, as compared with plate 1B, the control without gentamicin. Furthermore, results from the P-galactosidase assays supported this conclusion.

Glucose consumption and lactate production, as well as ammonia production, in these same cell samples were analyzed. Glucose and lactate were measured using a Kodak Ektachem DT60 II Analyzer according to standard protocols provided by WO 98/20146 PCTIUS97/19860 -33- Kodak and routinely used for measuring serum glucose and lactate levels in clinical laboratories. The analyses are conducted by applying 10 Pl of each test sample to a well on a plastic slide covered with a film containing all the reagents necessary foT measuring either glucose or lactate (Ektachem DT slide (GLU) or Ektachem DT Slide For measuring glucose,- the analysis is based on the glucose oxidasecatalyzed reaction of glucose with molecular oxygen, followed by a second reaction that produces a red dye whose intensity is proportional to the amount of glucose in the sample. The slide for measuring lactate similarly provides enzymes and substrates capable of producing a red dye in an amount proportional to the amount of lactate applied to the slide. Slides are placed in the Ektachem DT60 II Analyzer in which the red color is read by reflectance spectrophotometry. Ammonia analysis was performed similarly, using Ektachem DT slides (NH 3 based on a reaction wherein NH 3 reacts with bromphenol blue to yield a blue dye detectable with the same instrument.

The results of measuring glucose and lactate concentrations as a function of time are presented in Table 4. Table 4 indicates, surprisingly, that the control with gentamicin (plate 1A) consumed more glucose and produced more lactate than any of the experimental samples, which also contained gentamicin (note that the control without gentamicin, plate 1B, is not included in Table The data of Table 4 provide a clear indication that relative to the control the cells that received the chemical compounds described in Table 2 experienced a profound shift in metabolism that corresponded with a substantially higher level of expression of the foreign gene.

In addition to the data in Table 4, a combination of benzoic acid and 4-ethylbenzoic acid also have been observed to result in reduced glucose consumption. Here, an experiment was conducted in which a Type A formulation was first applied to SW480 cells before and during the transfection step, and a Type B formulation added one day after the DNA was introduced into the cells. The Type A formulation consisted of OPTI-MEM containing 1 mM benzoic acid, 1 mM 4-ethylbenzoate, and 4 mM L-glutamine, while the Type B formulation contained these same components and in addition contained 0.1 mM type C chondroitin-6-sulfate. Gentomicin was also present throughout the experiment. In this experiment, essentially no glucose consumption was observed in cells cultured in 6-well plates for as long as 14 days post-transfection, or for as long as 32 days posttransfection in bioreactors, during which time the cells continued to express protein from the transfected DNA.

WO 98/20146 PCT/US97/19860 -34- Table 4 Glucose Lactate Concentration Concentration Plate Day (mg/dL) (mmol/L) 1A. 0 218 2 180 4 141 9.6 6 38 >12.0 2. 0 209 1.8 2 188 4 175 6 6 3. 0 213 1.6 2 195 4.3 4 185 6.1 6 4. 0 209 1.6 2 199 3.8 4 181 5.7 6 140 It has been previously reported -that butyrate, a Group I compound, when administered to cultured hepatocytes compensates for the effects of glucose-starvation on post-translational glycosylation, most likely increasing the intracellular glucose pool (Morrow etal.,_Biochem. Biophys. Res. Comm. 112:115-125 (1983)).

However, Morrow et al. did not assay the consumption of glucose in their cultures, thus did not observe the shift in metabolism that is noted here in the presence of Group I compounds. The observed shift in glucose metabolism is a highly significant feature of this invention. Not only does it correlate with the enhanced efficacy of WO 98/20146 PCT/US97/19860 chemical compounds relevant to gene therapy methods (as is evident from this example), but suggests that the ability to selectively and non-toxically redirect cellular metabolic processes with these same chemical compounds could be applied to a wide range of therapies, including, for example, the modulation of fat/lipid metabolism in treating obesity.

Example 3.

Enhanced Transient Expression in Bioreactors A series of four lipofection-based gene transfection experiments were -conducted in a high performance hollow fiber perfusion prototype bioreactor device (hereafter referred to as the "HPBr" device) in a Genespan prototype incubator instrument. The device consists essentially of a sterile chamber through which two sets of hollow fibers are passed. Culture medium is continuously circulated through one set of fibers, while gases required oxygen and carbon dioxide) for cell growth are passed through the second set of fibers. The fibers are composed of a porous material through which gases and nutrients can pass in one direction, while waste molecules produced by the cells growing within the chamber can pass in the other direction. Cells growing in the device may remain in suspension, or may attach to the outer surfaces of both sets of hollow fibers.

A useful feature of the HPBr device is that the cells can be agitated by rotating the chamber through which the tubes pass. When the chamber is rotated 120° in one direction around its longitudinal axis, then 120° in the other direction, this constitutes one "cycle" of rotation. Alternatively, cultures can be grown under "static" conditions, using no rotation.

The HPBr device was used to conduct a series of experiments using SW480 cells. Each experiment included a parallel control in which the cells were plated in a conventional 6-well plate that was placed in a conventional 10% CO 2 incubator. The control 6-well plates were cultured and transfected using the protocol .described above for the control plates in Example 1, while the following experimental procedures were employed for the bioreactor devices.

HPBr Device Experiments Four 3-galactosidase reporter gene transfection experiments were conducted in HPBr devices using a protocol similar to that described in Example 1 for 6-well plates, although volumes of the various reagents had to be adjusted proportionately to accommodate the larger volumes and higher number of cells in the bioreactors. Due WO 98/20146 PCTIUS97/19860 -36to the perfusion mode of cell culture which is characteristic of the HPBr continuous feeding), there was no requirement for periodic feeding by hand.

Procedures for the bioreactor experiments differed in the following ways from the procedures described in Example 1. Sufficient Cytodex® I microcarriers- microspheres composed of crosslinked dextran with positively charged. quaternary ammonium functional groups on the surface for cell attachment; Sigma, St. Louis, MO) were pre-swollen in phosphate-buffered saline and introduced into the side ports of the HPBr. Approximately 1 microcarrier bead per 10 cells were used. At the onset of the experiment, 1 x 10 7 viable SW480 cells and 1 x 106 beads were co-injected into the device. The media described in Table 5 were present when the cells were seeded into the device. Table 5 identifies the rotational parameter corresponding to cycles per minute) employed in this study. A volume of 839 ml of medium were added to each bioreactor. Type C chondroitin sulfate at-0.1 mM was included in the OPTI-MEM transfection media for runs 2, 3, and 4 ("runs" refer to separate experiments). Following the transfection step, the recirculating OPTI-MEM medium the medium inside the tubes) was replaced, but the medium in the compartment containing the cells (the extracapillary space) was not replaced. The replacement medium included the compounds listed in Table 5. Liposomes containing the foreign DNA were added to the extracapillary space 24 hours after the cells were seeded into the bioreactors. This space has a small volume (17 ml) as compared with the volume inside the tubes (839 ml).

WO 98/20146 PCTIS97/19860 -37- Table RUN TYPE CONDITION MEDIA COMPOSITION 1. Plate CO 2 Incubator OPTI-MEM (see Example 1) [control] 2. IIPBr 30 cpm OPTI-MEM; 10% fetal bovine serum; 4 mM Lglutamine; 10 g/ml gentamicin; 2.5 mM benzoate buffer; 0.1 mM chondroitin sulfate (type C) [also present in OPTI-MEM transfection media].

3. HPBr Static Same as run #2.

4. HPBr 30 cpm for first Same as run #2.

48 hr., then static HPBr Static [control] OPTI-MEM Daily samples (about 1.5 ml) of cells and supernatant were taken from the cell compartment of each bioreactor and an equal volume of fresh media was added to replace it. Cell counts and viabilities were determined, and 2 x 10 4 viable cells were lysed and retained for 0-galactosidase determination using the spectrophotometric method described in Example 1.

Table 6 contains data comparing the results from four perfusion device experiments (runs with a plate control (run In Table 6, the column labeled "area under the curve" refers to the area under a curve in which the amount of 1-galactosidase produced in the daily aliquots of harvested cells were plotted as a function of time for the two-week duration of this experiment. Thus, the values in the "area under the curve" column thus are expressed in arbitrary units, cm 2 and reflect the total amount of 1-galactosidase produced on a per cell basis for the duration of the experiment. The last column in Table 6 shows for each run, each plate or bioreactor, the sum amount of 0-galactosidase present at day 13 in all of the viable cells remaining at that time.

It is evident that the perfusion bioreactor can be employed to scale-up gene transfection and harvesting transfected cells, which is advantageous for therapeutic applications for creating large numbers of T-lymphocytes and hematopoietic WO 98/20146 PCT/US97/19860-- -38stem cells expressing foreign genes either stably or transiently, to be used in somatic cell therapy). This system can also be utilized as an artificial organ so that the long-term expression of the foreign gene can be easily and realistically studied; in a way, this is equivalent to taking a biopsy from an intact organ in vivo.

Table 6: 2-Week O-Galactosidase Production in Plate and HPBr Device 0 00 0 0% Run Experimental Total %Area 1-Galactosidase ng/mi 1-Gal Total Conditions Cells Viability Under Expression per per 2x10 4 Expression (13 Days9 Curve 2x10 4 Cells as Cells at Based on Viable 1 (cm 2 of Control Day 13 Cells at Day 13 1 Plate Control 6.5 x 106 97% 70 0.084 33 2 30 cpm I x10 7 86% 84 20% 0.120 3 Static 2.3 x 17 42% 105 50% 0.602 364 4 30 cpmn/48 hr 7.3 X10 7 77% 180 157% 0.357 1254 then static Bioreactor 29.3 x 10 7 34% 76 9% 0.040 249 Control (Static) WO 98/20146 PCTIUS97/19860 The data in Table 6 show that manipulating the rotational parameter of the bioreactor provides a unique and convenient means for enhancing transfection efficiency and sustained transient expression in using this device.

It is notable that the presence of type C chondroitin sulfate, a polyanionic carbohydrate, allowed the transfection to proceed unimpeded, and it also resulted in a substantial improvement in gene expression. Some of the other polyanionic carbohydrates tested actively blocked the transfection process. These included Type A chondroitin sulfate, dermatan sulfate, heparin sulfate, heparin, carboxymethylcellulose, and N-carboxymethylchitosan N,S-sulfate (Table 1).

Therefore, the tolerance of cells to type C chondroitin sulfate is not typical of their tolerance in general for polysaccharides in the culture medium.

As discussed above, microspheres can be introduced into the chamber in order to provide attachment sites for cells. It has been observed, for example, that when an immortal mouse melanoma cell ATCC #B16-FO) is introduced into the chamber with microspheres present, the microspheres act as "seeds" for the accumulation of large masses of cells. It was further observed that these masses of cells could be transfected and that thereafter the cells in these masses transiently express the transfected DNA. Samples are readily obtainable from such cultures by sampling the medium within the chamber. This sampling is accomplished by directing a flow of fresh media from a syringe against the cell mass, which results in a number of cells sufficient for sampling becoming suspended in the medium. The masses of cells resemble a solid tumor and provide a model system for developing therapeutic methods effective in delivering therapeutic proteins to tumors in vivo.

Using the same protocols that are effective for cell masses growing inside the bioreactor, melanoma cells will be injected subcutaneously into mice, allowed to develop into tumors at the site of injection, and then liposomes containing the P-galactosidase vector DNA will be introduced directly into the tumors to achieve transient expression of the P-galactosidase. The methods effective for expression of 0-galactosidase are expected to be effective for other proteins as well, and similar experiments will be conduced to evaluate the effects of delivering various proteins, DNA encoding therapeutic proteins, directly into solid cell masses in vivo.

The bioreactor system used with the methods of the subject invention is useful for creating large numbers of cells genetically modified to express a foreign protein.

Such cells can be administered to patients for therapeutic purposes and maintained thereafter in an active state only for as long as the therapeutic regimen dictates. Thus, WO 98/20146 PCTIUS97/19860 -41the subject invention provides a unique form of gene therapy wherein the introduced gene can be turned off simply by restricting its access to the stabilizing substances, by administering cells transiently expressing a therapeutic protein, then administering the enhancing compounds for only so long as continued transgene expression is desired.

Finally, it should be noted that the use of chondroitin-sulfate (type C) is important as it enabled the anchorage-dependent cells to adhere well to the microcarrier despite the relatively high rotational speed. This results indicates that compounds of Group II are useful for providing anchorage to solid substrata for cultured cells. Chondroitin sulfate has been proposed as a compound for providing a cell adhesive surface in a device for controlling the pattern of cells on a surface 5,593,814). However, the method of U.S. 5,593,814, in contrast to the subject procedure, requires that the chondroitin sulfate be bonded to the solid substratum, rather than being added to the culture medium. Others have reported using chondroitin sulfate in conjunction with other compounds in order to promote cell adhesion in culture or in vivo. 5,593,814; U.S. 4,458,678; U.S. 4,418,691; U.S. 4,711,780; U.S. 5,545,722).

Example 4.

Assay for Cvtotoxicity A number of chemical compounds were tested in 6-well plates according to the protocol described in Example 1 to determine the relationship between their cytotoxicity and their ability to promote the uptake and expression of foreign genes in SW480 cells. Unless otherwise noted, except for the control, all plates contained 4 mM L-glutamine as well as gentamicin to retard bacterial growth.

Cytotoxicity assays were performed as follows. SW480 cells (approximately 1 x 106 cells per well) were plated in 1 ml of RPMI in 6-well culture dishes at day zero in the presence of the chemical compound whose cytotoxicity was being tested.

Twenty-four hours after seeding the wells, the RPMI medium was removed, and liposomes containing foreign DNA were added to the culture in 1 ml of OPTI-MEM medium, as described in Example 1. The transfection medium also contained the chemical compounds whose cytotoxicity was being tested. Control plates were included that were identical to the test plates, except that the test compounds were not present in the culture medium. Test and control cultures were grown under "static" conditions, the plates were not shaken, rotated, or otherwise agitated.

Each day for a total of 8 days, the cells from one test well and one control well were WO 98/20146 PCTIS97/19860 -42harvested and viability assessed by staining with trypan blue. In control cultures exposed to liposomal DNA, the cell number remained fairly constant or increased only slightly for the first 4 days post-transfection, then increased to about 1 x 10 7 per well by the end of 8 days. The retardation of growth of control cultures during the first 4 days was presumably due to-the mild cytotoxic effects of the liposomal DNA itself. A compound being tested was considered "cytotoxic" at the test concentration if a decline in the number of viable cells was observed within 4 days after the introduction of foreign DNA, and furthermore, there was no net expansion of the cells at the end of -8 days.

By applying this test protocol, it was possible in many cases to manipulate the concentrations of individual compounds or formulations of compounds to arrive at concentrations well-tolerated by SW480 cells yet also capable of enhancing the levels of transient expression in these cells. Other cell types also were tested for their ability to tolerate some of the chemical compounds of the subject invention. For example, human melanoma cells, mouse melanoma cells, and COS-7 cells (ATCC CRL 1651) were tested for their ability to tolerate the formulations applied to plate #6 in Table 9.

The cells differed somewhat in their sensitivity to the tested compounds, but a set of concentrations was identified that could be tolerated by all of these cell types, at these concentrations the compounds were not cytotoxic according to the above-described assay.

Sulfonated amino polysaccharides that enhanced transient expression were all found capable of supporting normal cell growth, they were not too toxic to be tolerated by the cells. The cell growth and cytotoxicity curves for the cells exposed to the various chemical compounds and formulations in Table 7 are presented in FIGURES 1 and 2, in which the numbers describing each plot correspond to the plate numbers in Table 7. Table 7 illustrates that the polysaccharide heparin blocks transient expression, but that type C chondroitin-6-sulfate did enhance transient expression. Although not shown in Table 7, it has also been observed that guarans also enhance transient expression. The heparin-mediated suppression of gene expression may have resulted from the formation of complexes between heparin and the cationic lipid in the liposomes, thus leaving the DNA without a carrier to deliver it to the cells. In light of this inference, the ability of chondroitin-6-sulfate to support both gene expression and cell growth is surprising.

WO 98/20146 PCTIUS97/19860- -43- Table 7 Plate Compound/Formulation Group Transgene Cytotoxic Expression 1. Control n/a Yes. No 4mM L-glutamine 2. 2.5 mM benzoate buffer I II Yes No -0.1 mM chondroitin-6-sulfate (type C) ImM L-glutamine L-glutamine 3. 2.5 mM benzoate buffer I II No No -0.1 mM heparin 1mM L-glutamine 4mM L-glutamine 4. -0.1 mM heparin II No No-- ImM L-glutamine 4mM L-glutamine 0.1 mM chondroitin-6-sulfate (type C) II Yes No 1mM L-glutamine 4mM L-glutamine 6. 2.5 mM butyrate buffer I Yes Yes 7. 2.5 mM butyrate buffer I Yes Yes ImM L-glutamine 4mM L-glutamine The plates containing butyrate buffer expressed the transfected gene, however, this buffer was cytotoxic to the SW480 cells under the experimental conditions used for this set of experiments.

Example Transfection with Starburst Polymers This set of experiments addressed the issue of whether the efficacy of the subject methods for enhancing transient expression were dependent on the means by which the DNA is delivered to the cells. Two different combinations of chemical WO 98/20146 PCT/US97/19860 -44compounds (see Table 8) were employed in transfecting SW480 cells using a protocol similar to that in Example 1, except that here the DNA was introduced into the cells in the presence of polymeric dendrimers instead of using liposomal delivery. These dendrimers are microscopic synthetic polymer spheres (first commercialized by Dow Chemicals as "starburst" polymeric bead standards to be used for sizing), which can be chemically derivatized to play the role of a cationic lipid. The dendrimers employed in this example were provided by F.C. Szoka, Jr., Department of Pharmacy/Pharmaceutical Chemistry, University of California, San Francisco, CA.

While the detailed mechanism of gene delivery for either lipofection or dendrimermediated processes is not known, based on physicochemical properties such as their shape and distribution of chemical moieties, they are highly likely to be quite different.

The procedure used deviated from that of Example 1 in the following steps.

Fourteen jig DNA were diluted into 397 1 deionized water, and 56 gg of the dendrimer was diluted into 393 p1 deionized water. The DNA solution and dendrimer suspension were combined no more than one hour before use. OPTI-MEM medium (733 pl) and the DNA/dendrimer mix (167ll) were added to each well, and the 6-well plates were swirled by hand to ensure thorough mixing. After incubating for 5 hours, the DNA/dendrimer-containing media was removed and 1.0 ml of culture medium was added. The remaining steps in the procedure were as described in Example 1.

As illustrated in Table 8, the tested compounds were efficacious when dendrimers were used as the method for delivering the foreign DNA to the cells.

These findings strongly suggest that the formulation of chemical compounds shown in Table 8 exert their effect after the DNA enters the cell, and thus are effective regardless of the method used to introduce the DNA.

WO 98/20146 PCTIUS9719860 Table 8 Plate Compound/Formulation XFactor GFactor KFactor 1. Control n/a n/a n/a 2. 2.5 mM benzoate buffer 14 63 2 0.1 mM chondroitin-6-sulfate (type C) 4 mM L-glutamine 3. 2.5 mM benzoate buffer 4 42 34 -2 mM glutamic acid 4 mM L-glutamine Example 6.

Protein Production During Transient Expression The following experiment illustrates that the subject transient expression system is useful for the rapid production of large amounts of a protein product expressed by a foreign gene that is introduced into recipient cells using the methods described in the preceding examples.

A 15 plate experiment was conducted in which the chemical compounds indicated in Table 9 were added to the culture medium of SW480 cells that were transfected in 6-well plates as detailed in Example 1. The X, G 14 and K factors, plus the cumulative amount of protein produced in 14 days in 2 x 104 cells, were calculated and are shown in the last column of Table 9. The data presented in Table 9 illustrate that all of the listed compositions were superior to the control with respect to the amount of protein produced in their presence. The most efficacious formulations, in order of their effectiveness, were those used in plates 6, 3, 13 and 14. Superior results were observed in the plates that received both Type A and B formulations, thus, these combinations are especially useful for animal testing, as in treating tumors with toxic proteins, delivering hormones to specific tissues, or other pathological conditions where local delivery of a bioactive protein may be desirable.

WO 98/20146 PCT/US97/19860 -46- Table 9 X G.

14 K Total Protein Plate Compound/Formulation Factor Factor Factor (ng per 2 x cells) 1. control (DNA but no n/a n/a n/a 10.2 compounds) 2. 2.5 iM benzoate buffer -16 43 1 11.7 3. 2.5 mM benzoate buffer -14 63 2 28.7 Cells fed after 48 hr with Type B Formulation: mM benzoate buffer 0. 1 m.M chondroitin-6-sulfate 4. 4niM tryptophan 55 67 2 34.2 1 mM benzoic acid 41 80 2 27.5 1 m.M 4-ethylbenzoic acid 6. Type A Formulation 20 82 42 26.2 1 mM benzoic acid 1 m.M 4-ethylbenzoic acid Cells fed after 48 hr with Type B Formulation: mM benzoate buffer 0.1 mM chondroitih-6-sulfate 7. 1 mM 4-ethylbenzoic acid 47 64 2 21.9 1 mrM 4-butylbenzoic acid -26 65 9 14.6 9. 4mrM L-glutamine -12 42 11.7.

4 m.M citrulline 40 46 1 17.5 11. 4 .M benzoate buffer 54 72 2 26.1 0. 1 MM chondroitin-6-sulfate WO 98/20146 PCT/US97/19860- -47- X G14 K Total Protein Plate Compound/Formulation Factor Factor Factor (ng per 2 x 4 cells) 12. 2.5 mM benzoate buffer 42 76 6 25.1 4 mM glutamic acid 13. Type A Formulation 49 78 28.2 mM benzoate buffer 4 mM glutamic acid Cells fed after 48 hr with Type B Formulation: mM benzoate buffer 0.1 mM chondroitin-6-sulfate 14. Type A Formulation: 57 77 1 29.6 1 mM glutathione 1 mM methionine 4 mM glycine 4 mM a-amino-n-butyric acid 1 mM taurine 4 mM phenylalanine mM benzoate buffer 4 mM alanine 1 mM ethyl-4-acetylbutyrate 51 59 1 22.0 Note: Medium in all plates included gentamicin, and except for the control, also contained 4 mM L-glutamine; the chondroitin-6-sulfate was type C.

These experiments illustrate also the utility of enhanced transient expression for very rapidly producing milligram quantities of protein without the need to first establish cell lines into which the foreign gene has become stably integrated. Thus, enhanced transient expression provides a new means by which candidate biopharmaceuticals can be efficaciously expressed in sufficient quantities to be recovered and rapidly screened for pharmaceutical activity. Thus, the subject invention provides a means for implementing an accelerated drug discovery program.

Plate 6, for example, produced about 26 ng P-galactosidase per 2x 104 cells in 14 days (see Table Scaled up to a conventional culture containing around 2 x WO 98/20146 PCTIUS97/19860 -48- 106 cells, the cumulative protein production using this formulation would be about 26 mg. In the HPBr device employed in Example 2, as many as 10 9 cells are routinely grown, thus in such a culture, tens or even hundreds of milligrams of a novel or interesting protein could be obtained within a matter of a few days.

Example 7.

Transient Expression in Hepatocytes A totipotent (stem-cell like) clonal nontransformed cell line (PICM-19 3BT cells; hereafter referred to as "PICM-19 cells") derived from pig embryonic cells (epiblast stage), was obtained from Dr. N. Talbots Beltsville, MA). These cells behave like hepatic stem cells, showing self-renewing properties for many months when cultured in the presence of 5% or less CO 2 At higher levels of C0 2 up to about these cells begin to differentiate. At least two different differentiated cell phenotypes have been isolated from differentiated PICM-19 cells, namely, mature hepatocytes and liver ductile cells, which produce bile. PICM-19 cells that had been induced to differentiate were used as a means for determining the transfection characteristics of primary hepatocytes, a cell type that they strongly resemble. In earlier experiments with primary pig liver cultures, results were obtained that mirrored those described above for the SW480 cells. Because the primary liver cultures contained cell types other than hepatocytes, the experiments were repeated with PICM-19 cells providing a homogeneous source of hepatocyte-like cells.

The protocol employed was identical to that described in Example 1 used for transfecting SW480 cells, using 1 x 107 cells per well, except that the PICM-19 cells were plated on a layer of mytomicin C-inactivated STO mouse fibroblast feeder cells (CRL 1503), without which PICM-19 cells normally will not grow. In preparing liposomes, the DNA/lipid to cell ratio was as in Example 1. The incubator was maintained at 10% CO 2 throughout these experiments. The PICM-19 cells expanded and under these culture conditions differentiated into mature hepatocytes. To ensure that the differentiation was complete, the cultures were maintained in 10% CO 2 for 3 weeks prior to the transfection step.

Table 10 describes the media that were used in a transfection study using these cells, as well as the X, G 7 and K factors measured in these cultures. The results shown in Table 10 are consistent with the findings for SW480 cells and the results observed when primary isolates from adult pig liver were transfected under similar conditions.

WO 98/20146 PCT/US97/19860 -49- Surprisingly, it was observed also that the plates lacking feeder cells were capable of supporting differentiated PICM-19 cells for at least 4 weeks. These cells moreover expressed the transfected DNA, as illustrated in FIGURE 1. This result was extremely surprising, as there are no reports of hepatocytes being grown or maintained in culture for more than a few days without either a feeder layer or a proteinaceous-coating collagen) having been applied to the plates prior to adding the cells. Remarkably, the cells in plate #4 adhered as well as did cells in plates containing feeder cells, suggesting that the type C chondroitin-6-sulfate created in vivo-like conditions for both cell growth and maintenance. Thus, these experiments demonstrate for the first time the utility of chondroitin sulfate for culturing hepatocytes without feeder cells in a low cost medium composition while maintaining a phenotype similar to that observed for hepatocytes in vivo.

WO 98/20146 PCT[US97/19860 Table Plate Compound/Formulation XFactor G 7 Factor K Factor 1. Control n/a n/a n/a 2. 2.5 mM benzoic acid -17 99 1 4 mM L-glutamine 3. 2.5 mM benzoate buffer -41 102 0.0 0.1 mM chondroitin-6-sulfate (type C) 4 mM L-glutamine 4. 2.5 mM benzoate buffer -357 103 2 0.1 mM chondroitin-6-sulfate (type C) 4 mM L-glutamine NO FEEDER CELLS 2.5 mM benzoate buffer -46 103 0.0 4 mM glutamic acid 4 mM L-glutamine Example 8.

Recovery of Transgenic mRNA and DNA from Transfected Cells Grown in a Bioreactor The high performance bioreactor device (HPBr) described in Example 3 was used in a 32-day experiment in which SW480 cells were transfected and propagated as described in Example 3 and in Table 5. Except as described otherwise below, the conditions and assays used were the same as described in Example 3. At the onset of the experiment, 1 x 107 SW480 cells freshly harvested from tissue culture flasks were injected into the HPBr device-concurrently with 1 x 106 preswollen microspheres.

The cells were then cultured for 24 hours without rotation in medium containing 1 mM benzoic acid and 1 mM 4-ethylbenzoic acid (a Type A formulation). At the end of 24 hours, plasmid DNA encoding P-galactosidase was added, and the bioreactor was rotated at a rate of 30 cpm for 4 hours. The medium containing the DNA was then removed from the extra-capillary space (ECS) of the bioreactor by flushing three times with feeding medium containing 1 mM benzoic acid, 1 mM 4-ethlybenzoic acid, -WO 98/20146 PCTIUS97/19860 -51and 0.1 mM chondroitin-6-sulfate (a Type B formulation). Thereafter, the 1 liter bottle of culture medium for circulating through the bioreactor was replaced with a 1 liter bottle of feeding medium containing the same Type B formulation. For the remainder of the experiment, the medium circulating through the bioreactor was replaced every 7 days with a fresh 1 liter bottle of feeding medium containing the Type B formulation. The device was not rotated after the DNA was removed so that the cells could form a tumor-like solid mass.

Beginning 24 hours after removing the DNA from the bioreactor, aliquots of cells and culture supernatant from the ECS were removed daily for 32 days. Cell sampling was accomplished by directing a stream of culture medium against the cell mass to dislodge some of the cells, then withdrawing a small volume of the resulting cell suspension. The cells and culture medium in each sample were separated by brief centrifugation. A total of 2 x 104 cells from each daily aliquot were analyzed for P-galactosidase and each supernatant was analyzed for its metabolic signature, its concentrations of glucose, lactate, and ammonia. After collecting the daily sample on day 32, the remaining cells were harvested from the ECS by trypsinization, and 2.8 x 105 of the harvested cells were used for the extraction of RNA and DNA.

Beta-galactosidase in the daily cell samples was assayed as described in Example 3, and the results of these assays are illustrated in FIGURE 4. FIGURE 4 shows that the peak level of expression of P-galactosidase occurred at day 4, and remained virtually unchanged until about day 12, whereafter the values became less consistent but nonetheless remained relatively high. The final data point, corresponding to cells collected by trypsinization at the end of the experiment, is indicated in FIGURE 4 by a square-shaped symbol, and its value corresponded to roughly 60% of the peak value. Thus, a relatively high level of P-galactosidase production took place in this culture throughout the entire 32-day period.

The procedures described in Example 2 were used to measure the concentrations of glucose, lactate, and ammonia in the supernatants, and the results of these measurements are presented in FIGURES 5A-5C. It is apparent from FIGURES 5A and 5B that neither the glucose nor the lactate concentrations changed to a significant extent throughout the course of the experiment (the fluctuations in lactate were not considered significant in view of the low amounts of lactate present in these samples and in view of the relatively constant amounts measured past day In contrast, the ammonia concentration increased over two-fold by the end of each seven-day period between media changes, before dropping back to the base value WO 98/20146 PCTIS97/19860 -52each time fresh medium was provided. This repeated accumulation of ammonia after each- medium change strongly supports the notion that exposure to transfectionstabilizing compounds causes cells to shift their metabolism from using glucose (glycolysis) to using proteins or amino acids instead as their primary carbon source (tricarboxylic acid cycle). Had the cells in this experiment used glucose for their primary source of energy, one would have expected lactate and not ammonia to increase in concentration during each 7-day period (note that FIGURE 5B suggests that some glycolysis may have occurred during the first 7-day period).

Ammonia is a byproduct of the deamination that is an early step in the entry of amino acid metabolites into the tricarboxylic acid cycle. Accordingly, the most likely explanation for the accumulation of ammonia in the culture media is that the cells used amino acids, or possibly peptides or proteins, as their source of energy during their exposure to the compounds used to stabilize transient expression. These amino acids may have originated, for example, from peptides present in the culture medium. Such peptides could have been created by the heat-induced breakdown of serum proteins during the heat inactivation of the serum present in the culture medium.

Their ability to cause cells to shift from the use of glucose to the use of amino acids as an energy source has significant implications for the use of transient expression-stabilizing compounds. For example, the tricarboxylic acid cycle by which amino acids are metabolized is critical also in the metabolism of fats and lipids. Thus, treating cells or a human subject with transient expression-inducing compounds may result also in the increased metabolism of fats and lipids by virtue of activating the tricarboxylic acid cycle. Thus, the compounds could serve, for example, as agents for controlling weight. These results also illustrate an association between the unique metabolic signature seen in FIGURES 5A-5C and the physiological state in which the transient expression oftransfected genes is enhanced and stabilized.

To prepare nucleic acids, 2.8 x 105 trypsinized cells harvested at the end of the 32-day incubation were pelleted by centrifugation, washed with 5 ml of calcium-free and magnesium-free PBS, and mixed with 1 ml of TRIZOLTM (Life Technologies) reagent at room temperature. The cells suspended in TRIZOL T M were then incubated at 4 0 C for ten minutes. At this point, the sample was stored frozen at -70 0 C. After being thawed, the sample was permitted to stand at room temperature for 20 minutes before adding 200 .1l of chloroform, mixing vigorously for 15 seconds, and incubating at room temperature for 5-20 minutes. Next, the sample was centrifuged at 2,000 x g for 15 minutes at 4 0 C to separate the emulsion into two phases.

WO 98/20146 PCT/US97/19860 -53- For isolating RNA, the upper aqueous phase was carefully collected without including any portion of the interphase, and transferred to another tube to precipitate the RNA, 0.5 ml of isopropanol was mixed with this aqueous phase, the tube was incubated at room temperature for 10-20 minutes, and then was centrifuged at 12,000 x g at 4 0 C to collect the RNA pellet. The pellet was carefully washed with 1 ml of 70% ethanol, air-dried for 5-10 minutes at room temperature, and resuspended in 30 pl of RNAse-free water (Five Prime Three Prime).

To isolate the DNA, the lower phase and organic layers described above were collected and mixed by inversion with 300 pl of 100% ethanol, then allowed to stand at room temperature for 2-3 minutes to precipitate the DNA. The DNA pellet was collected by centrifugation at 2,000 x g for five minutes at 4 0 C, then washed twice with 0.1-.M sodium citrate containing 10% ethanol. After the second wash, the DNA pellet was again collected by centrifugation at 2,000 g for five minutes at 4°C, and washed by being resuspended in 75% ethanol for 10-20 minutes at room temperature.

The pellet was again collected by centrifugation, briefly dried, and resuspended and dissolved in 8 mM sodium hydroxide.

To detect the presence of 0-galactosidase sequences, the concentration of the RNA was determined by reading the absorbence at 260 nm, then the RNA solution was diluted with RNAse-free water to a final concentration of 100 pg/ml. Fifty p1 of the diluted RNA solution was then mixed with 150 pl of a 50:50 solution of 37% formaldehyde and 20 x SSC. Samples were heated to 55-60 0 C for 20 minutes to denature the target nucleic acid, placed on ice, and 200 pl RNA-free water were added. Samples were shaken and briefly centrifuged to pellet debris, then loaded into the wells of a slot-blot apparatus under light vacuum to collect the RNA onto a GeneScreen PlusM membrane (New England Nuclear). Wells were washed with pl of 10 x SSC, and the membrane was exposed to ultraviolet light to crosslink the RNA to the membrane, then was baked for one hour at about 90 0 C to remove the formaldehyde. DNA samples were slot-blotted using the same procedure, except no vacuum was used.

The presence of 0-galactosidase DNA or mRNA on the slot-blot membranes was determined by hybridization with a 32 P-labeled oligonucleotide corresponding to a portion of the 0-galactosidase gene present in the plasmid used for transfection.

The nucleotide sequence of this oligonucleotide was CTCCAACGCAGCACCATCAC 3' (SEQ ID NO:1). For hybridization, 10 ml of hybridization buffer (1 ml 50 x Denhardt's solution, 10 pl of 10 mg/ml polyadenylic WO 98/20146 PCTIUS97/19860 -54acid, 12.5 ml of 20 x SSC, 5 ml of 10% sodium dodecyl sulfate, and 2.5 ml of 0.5 M NaP0 4 (pH 6.5) in a final volume of 50 ml) were placed in a plastic bag with the loaded slot-blot membrane and 1 x 106 counts/ml of 32 P-labeled probe. Bags were sealed and incubated overnight at 52-53 0 C. After hybridization, the membranes were washed twice with buffer containing 5 x SSC and 0.1% sodium dodecyl sulfate for 5-10 minutes at room temperature, then twice more with the same buffer at 52-53 0

C

for 20-30 minutes per wash, then exposed to x-ray film.

On the resulting autoradiograms, a signal was present indicating the presence oftransfected DNA containing the P-galactosidase gene in the cells harvested 32 days after transfection. Thus, the DNA evidently had persisted in relatively high amounts throughout the 32 day test period. Also, the autoradiogram of the RNA slot-blot showed a surprisingly strong signal after hybridization with the P-galactosidase probe.

In numerous previous experiments, it was shown that production of P-galactosidase declined and disappeared from cells within 2-3 days after removing the inducing compounds from the culture medium. Thus, it was clear that the observed persistence of detectable 1-galactosidase DNA and mRNA in this experiment did not result from the outgrowth of cells in which the foreign DNA had become integrated. Moreover, the typical half-life for an mRNA is only about 1-3 days, thus the presence of 0-galactosidase mRNA at the end of the 32-day incubation period suggests that this mRNA was recently transcribed and that the transfected foreign DNA thus must have persisted throughout the 32-day experiment.

The detection of P-galactosidase DNA after 32 days of incubation suggests furthermore that the foreign DNA may have replicated and increased in amount during this period. Because the cells continued to grow and divide during the experiment, one would have expected the plasmid DNA added at day 0 to have become diluted, and therefore that cells analyzed 32 days later would contain very little P-galactosidase DNA. Thus, the surprising presence of easily detectable amounts of P-galactosidase mRNA and DNA suggests that the transfected DNA may have replicated during the experiment, possibly within the mitochondria.

Example 9.

Induction of Alkaline Phosphatase in Cells Treated with Transient-Expression Stabilizing Compounds The results of the following experiment indicated that, in addition to inducing the tricarboxylic acid cycle, the metabolic signature of cells treated as described in Example 8 also includes the induction of an endogenous alkaline phosphatase activity WO 98/20146 PCT/US97/19860 that normally is barely detectable in SW480 cells. Cells were grown in plastic tissue culture dishes, and were transfected and propagated using the same culture media described in Example 8. The cells were fed every few days by the addition of a few ml of feeding medium. Aliquots of the culture medium from these plates were harvested daily for 14 days, beginning with the first day post-transfection, and analyzed for concentrations of glucose, lactate, and ammonia as described in Example 8.

Unexpectedly, when these same samples were analyzed for endogenous alkaline phosphatase activity, high amounts were found to be present. The observed degree of elevation ranged from about 2-fold to about 20-fold, as compared with conventionally-grown SW480 cells.

The assay used to measure the SEAP activity was as follows. One-half ml of each sample was mixed with 2 x SEAP buffer (1 x SEAP buffer 1 M diethanolamine, 0.50 mM magnesium chloride, pH9.8). As a control, bovine intestinal mucosal alkaline phosphatase was assayed concurrently. The bovine alkaline phosphatase was made up in 1 x SEAP. The chromogenic substrate for these assays was 0.15 M p-nitrophenylphosphate which yields a product detectable at 405 nm after being cleaved by alkaline phosphatase. The substrate (100 P1) was added to each assay tube, then the tubes were placed at 37 0 C. Thereafter, the absorbance of the control sample was read each minute for 10 minutes, and that of each test sample at 1 and 6 minutes. The units of alkaline phosphatase/ml of test sample were determined using the formula: AA 405nm sample- AA 405nm) blank Vxdf S mm mm units enzyme/ml m 1 x mi n 18.5 x VE, where: A405nm absorbance at 405nm, V volume in the assay tube, df= dilution factor, VE volume of sample added to the assay tube.

For this set of assays, V=1.1 ml, df=2.2, and VE=0.5 ml.

To determine whether the induced alkaline phosphatase activity was heatsensitive, a second set of assays was run on the same samples using assay buffer identical to the SEAP buffer described above, but containing 0.01 M L-homoarginine.

The control enzyme samples and samples of culture medium were heated in this buffer to 65 0 C for 5-10 minutes before adding the substrate. This heat treatment is known WO 98/20146 PCT/US97/19860 -56to destroy the alkaline phosphatase that is found in most mammalian cells that express the enzyme. This pre-treatment indeed destroyed the alkaline phosphatase activity in these samples, as well as that in the control enzyme samples, thus indicating that the induced alkaline phosphatase corresponded to the type of alkaline phosphatase most commonly detected in animal cells, rather than to the heat-resistant variety known to be present in placenta.

While the preferred embodiment of the invention has been illustrated and described, it will be appreciated that various changes can be made therein without departing from the spirit and scope of the invention.

WO 98/20146 PCT/US97/19860- -57- SEQUENCE LISTING GENERAL INFORMATION: APPLICANT: Goffe, Randall A.

Goffe, Adeelia S.

(ii) TITLE OF INVENTION: Stabilized Transient Gene Expression (iii) NUMBER OF SEQUENCES: 1 (iv) CORRESPONDENCE ADDRESS: ADDRESSEE: Christensen O'Connor Johnson Kindness STREET: 1420 5th Ave., Suite 2800 CITY: Seattle STATE: WA COUNTRY: US ZIP: 98101-2347 COMPUTER READABLE FORM: MEDIUM TYPE: Floppy disk COMPUTER: IBM PC compatible OPERATING SYSTEM: PC-DOS/MS-DOS SOFTWARE: PatentIn Release Version #1.30 (vi) CURRENT APPLICATION DATA: PCT APPLICATION NUMBER: FILING DATE:

CLASSIFICATION:

(viii) ATTORNEY/AGENT INFORMATION: NAME: Sheiness, Diana K.

REGISTRATION NUMBER: 35,356 REFERENCE/DOCKET NUMBER: GSPN-1-11156 (ix) TELECOMMUNICATION INFORMATION: TELEPHONE: (206) 682-8100; (206) 224-0735, direct TELEFAX: (206) 224-0779 INFORMATION FOR SEQ ID NO:1: SEQUENCE CHARACTERISTICS: LENGTH: 20 nucleotides TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: oligonucleotide (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: YES (vi) ORIGINAL SOURCE: ORGANISM: Escherichia coli (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: CTCCAACGCA GCACCATCAC

Claims

1. A method of enhancing the transient expression of a foreign gene in a eukaryotic cell comprising: introducing into the cell a molecule of foreign DNA that encodes a protein in a form capable of being expressed in the cell; and, contacting the cell before, during, or after introducing the DNA with a transient expression enhancing agent; wherein the concentration of the transient expression enhancing agent is 1-15 mM and the transient expression enhancing agent comprises at least one carboxylic acid derivative having the o1 formula: O R 1 C-OR 2 wherein R 1 is: CHNH 2 R 3 wherein R 3 is the side chain of an amino acid or amino acid derivative selected from the group consisting of glycine, alanine, valine, leucine, isoleucine, aspartic acid, 15 glutamic acid, glutamine, serine, threonine, methionine, arginine, lysine, histidine, proline, tryptophan, phenylalanine, tyrosine, their unnatural optical isomers, 3-methyl-L-histidine, a-ketoglutaric acid, (3-alanine, carnosine, citrulline, creatine, folic acid, glutathione, hippuric acid, homoserine, N-carbamyl aspartic acid, N-formyl-L-methionine, ornithine and optical isomers thereof; C 6 H 4 R 4 wherein R 4 is H, NH 2 COCH 3 CO(CH 2 )nCH 3 C(CH3)3, CH(CH3) 2 20 (CH 2 )nCH(CH 3 2 (CH 2 )nCOCH 3 OCH 3 or O(CH 2 )nCH 3 wherein n 1-3; CHNH 2 (CH 2 )nR 5 wherein n 1-7 and R 5 is CH 3 OH, CONH 2 C 6 H 4 0H, or CONHNH 2 (CH 2 )nR 6 wherein n 3-9 and R 6 is an indole group, NCH 3 C(=NH)NH 2 SCH 3 NH 2 CH3, C0 2 H, CONH 2 or NHC(=NH)NH 2 provided that when n 2 and R 2 is H or M, R 6 is not CH 3 (CH 2 )nCHNH 2 CO 2 H, wherein n 1-8; 25 CH(CO 2 H)NHCONH 2 or C 5 H 4 N; and wherein R 2 is selected from H, (CH2)xO(CH 2 )yCH 3 or (CH 2 )xCO(CH 2 )yCH 3 wherein x y 2-7, or M, wherein M is a metal counterion or ammonium, maintaining the cell in a non-selective medium after introducing the DNA; and detecting the foreign protein in the cell after maintaining the cell for at least five days in the non-selective medium.

2. The method of Claim 1, wherein said agent induces the cell to use proteins or amino acids as its primary energy source.

3. The method of Claim 1 wherein the transient expression enhancing agent comprises an amino acid derivative selected from the group consisting of 3-methyl-L-histidine, a-ketoglutaric acid, 3-alanine, carnosine, citrulline, creatine, folic acid, glutathione, hippuric acid, homoserine, N-(4- aminobenzyl)-L-glutamic diethylester, N-carbamyl aspartic acid, N-formyl-L-methionine, and ornithine.

4. The method of Claim 1, wherein R 1 is non-polar and hydrophobic. RA c 5. A method of enhancing the transient expression of a foreign gene in a eukaryotic cell comprising: IR:\LI BA]32082do:MSOffice -59- introducing into the cell a molecule of foreign DNA that encodes a protein in a form capable of being expressed in the cell; and, contacting the cell before, during, or after introducing the DNA with a transient expression enhancing agent; wherein the concentration of the transient expression enhancing agent is 1-15 mM and the transient expression enhancing agent comprises a sulfonic acid derivative having the formula: R 7 -S0 2 -ORs wherein R 7 is a straight chain or branched C 1 -C 5 lower alkyl, aryl, substituted lower alkyl, or substituted aryl; and R 8 is a hydrogen, a metal counterion, or ammonium; maintaining the cell in a non-selective medium after introducing the DNA; and detecting the foreign protein in the cell after maintaining the cell for at least five days in the non-selective medium.

6. The method of Claim 5 wherein R 7 is an amino substituted straight chain or branched 15 C1-C5 lower alkyl group or an amino substituted aryl group.

7. The method of Claim 5 wherein the sulfonic acid derivative is selected from the group consisting of 3-aminobenzene sulfonic acid, taurine, and salts thereof.

8. A method of enhancing the transient expression of a foreign gene in a eukaryotic cell comprising: 20 introducing into the cell a molecule of foreign DNA that encodes a protein in a form capable of being expressed in the cell; and, contacting the cell before, during, or after introducing the DNA with a transient expression on: enhancing agent; wherein the transient expression enhancing agent comprises a sulfonated amino 25 polysaccharide and the concentration of the transient expression enhancing agent is 0.01-0.5 mM; maintaining the cell in a non-selective medium after introducing the DNA; and detecting the foreign protein in the cell after maintaining the cell for at least five days in the non-selective medium.

9. The method of Claim 8, wherein the sulfonated amino polysaccharide comprises an N- acetylated amino polysaccharide. The method of Claim 9, wherein the N-acetylated amino polysaccharide is selected from the group consisting of chondroitin-6-sulfate and a guaran.

11. The method of Claim 10 wherein the guaran is a hydroxypropyl guaran.

12. The method of Claim 1 wherein the transient expression enhancing agent comprises a compound selected from the group consisting of adrenaline, coenzyme B12, and methylcobalamin.

13. The method of Claim 10, wherein the N-acetylated amino polysaccharide is chondroitin-6-sulfate having a molecular weight of about 4000 daltons.

14. The method of Claim 1, wherein the transient expression enhancing agent comprises an aqueous solution having a pH of 4.5-9.0.

15. A method of enhancing the transient expression of a foreign gene in a eukaryotic cell I comprising: [R:\IBA32082.doc:MSOffie introducing into the cell a molecule of foreign DNA that encodes a protein in a form capable of being expressed in the cell; and, contacting the cell before, during, or after introducing the DNA with a first and second transient expression enhancing agent wherein the concentration of each of said first and second transient expression enhancing agents is 1-15 mM; wherein the cell is contacted with said first transient expression enhancing agent prior to, during, and following the introduction into the cell of the foreign DNA, wherein the agent comprises at least one compound having the formula: O R- C- OR2, o0 wherein R 1 is: CHNH 2 R 3 wherein R 3 is the side chain of an amino acid or amino acid derivative selected from the group consisting of glycine, alanine, valine, leucine, isoleucine, aspartic acid, Sglutamic acid, glutamine, serine, threonine, methionine, arginine, lysine, histidine, proline, tryptophan, phenylalanine, tyrosine, their unnatural optical isomers, 3-methyl-L-histidine, c-ketoglutaric acid, 15 3-alanine, carnosine, citrulline, creatine, folic acid, glutathione, hippuric acid, homoserine, N-carbamyl aspartic acid, N-formyl-L-methionine, ornithine and optical isomers thereof; C 6 H 4 R 4 wherein R 4 is H, CH 3 (CH2)nCH 3 NH 2 COCH 3 CO(CH 2 )nCH 3 C(CH 3 3 CH(CH 3 2 (CH 2 )nCH(CH 3 2 (CH 2 )nCOCH 3 OCH 3 or O(CH 2 )nCH 3 wherein n 1-3; CHNH 2 (CH 2 )nR 5 wherein n 1-7 and R 5 is CH 3 OH, CONH 2 C6H 4 0H, or CONHNH 2 20 (CH 2 )nR 6 wherein n 1-9 and Re is an indole group, NCH 3 C(=NH)NH 2 SCH 3 NH 2 CH 3 CO 2 H, CONH 2 or NHC(=NH)NH 2 provided that when n 2 and R 2 is H or M, R 6 is not CH 3 (CH 2 )nCHNH 2 CO 2 H, wherein n 1-8; *I CH(C0 2 H)NHCONH 2 or :oo: C5H4N;and 25 wherein R 2 is H, CH 3 (CH 2 )nCH 3 wherein n 1-8, or (CH 2 )xO(CH 2 )yCH 3 or (CH 2 )xCO(CH 2 )yCH 3 wherein x y 2-7, or M, wherein M is a metal counterion or ammonium, provided that when R 1 is (CH2)2CH3 R 2 is not H; or the first transient expression enhancing agent comprises a compound having the formula: R 7 -S0 2 -OR 8 wherein R 7 is a straight chain or branched C1-C5 lower alkyl, aryl, substituted lower alkyl, or substituted aryl; and R 8 is a hydrogen, a metal counterion, ammonium; and following the introduction of the foreign DNA, the cell is contacted with said second transient expression enhancing agent, wherein the second agent comprises a sulfonated amino polysaccharidel maintaining the cell in a non-selective medium after introducing the DNA; and detecting the foreign protein in the cell after maintaining the cell for at least five days in the non-selective medium. R 16. The method of Claim 1, wherein the cell is contacted with the agent prior to introduction of the foreign DNA into the cell. I[I:\LIBA]32082.doc:MSOffice -61-

17. The method of Claim 1, wherein the cell is contacted with the agent during the introduction of the foreign DNA into the cell.

18. The method of Claim 1, wherein thecell is continuously exposed to the agent after the introduction of the foreign DNA into the cell.

519. The method of Claim 1, wherein the cell is a cultured cell. The method of Claim 19, wherein the cultured cell is a primary culture. 21. The method of Claim 20, wherein the cultured cell is selected from the group consisting of a stably transformed cell, a tumor cell line, and a hybridoma cell. 22. The method of Claim 21, wherein the cultured cell is a SW480 P3 cell. o 23. The method of Claim 1, wherein the agent is biocompatible. 24. The method of Claim 1, wherein the protein encoded by the foreign gene is harvested. The method of Claim 1, wherein the cell is present in a live host, and the transient expression enhancing agent is introduced into the host orally or by injection. 26. The method of Claim 1, wherein the foreign DNA is introduced into the cell by a 0° 15 method selected from the group consisting of lipofection, a viral vector, exposure of cells to coprecipitates of calcium phosphate, and transfection in the presence of starburst polymers. 27. The method of Claim 26, wherein the DNA is introduced into the cell by a viral vector, and the viral vector is derived from an adenovirus. eo*:9 .06 28. The method of Claim 23, wherein the agent contains at least one hydrophobic moiety 2o and at least one acidic moiety, and wherein the acidic group may be modified to form a salt or an ester. S29. The method of Claim 28, wherein the acidic moiety is hydrophobic and organic. 30. A method of screening an agent comprising at least one chemical compound to 2 determine whether the agent is capable of enhancing the transient expression of a foreign gene in a 25 eukaryotic cell, wherein the agent is biocompatible and contains at least one hydrophobic moiety and at least one acidic moiety, the method comprising the steps of: introducing into a first and a second SW480 P3 cell on day 0 a molecule of foreign DNA that encodes a protein in a form capable of being expressed in the cells; before, during, or after introducing the DNA, contacting the second cell with the agent; cumulatively measuring in both cells between days 0 and 4, or between days 4 and 7, or between days 4 and 14 the amount of protein expressed from the foreign DNA, and using these amounts to determine, respectively, a value for X, G 7 or G 1 4 according to the formula: X, or G 7 or G 14 100 (A x 100) C wherein is the amount of the protein encoded by the foreign gene expressed in the first cell and, is the amount of protein expressed in the second cell; and, determining that the agent is capable of enhancing transient expression if X or G 7 or G 14 is greater than 31. The method of Claim 30, wherein X or G 7 or G 14 is greater than 32. A method of enhancing the transient expression of a foreign gene in a cell comprising: IR:\L BA]32082.doc:MSSffice introducing into the cell a molecule of foreign DNA that encodes a protein in a form capable of being expressed in the cell; and, contacting the cell with an agent for which X or G 7 or G 14 is greater than 25 when the agent is evaluated according to the assay of Claim 33. A method of manipulating the metabolism of a cell to reduce the cell's consumption of glucose, comprising the step of contacting the cell with an agent that induces the cell to use proteins or amino acids as their primary energy source, wherein the concentration of said agent is 1-15 mM, provided that if the agent is a sulfonated amino polysaccharide the concentration of said sulfonated amino polysaccharide is 0.01-0.5 mM, wherein said concentration of said agent is in addition to the to concentration of said agent present in any cell culture medium used to culture said cell. 34. The method of Claim 33, wherein the agent further induces the cell to express an endogenous alkaline phosphatase activity. A method of manipulating the metabolism of a cell to reduce the cell's consumption of glucose, comprising the step of contacting the cell with an agent, wherein the concentration of said 15 agent is from 1-15 mM, said concentration of said agent being in addition to the concentration of said agent present in any cell culture medium used to culture said cell and the agent comprises at least one chemical compound having the formula: 0 0 R 1 I C-OR2 wherein R 1 is: 20 CHNH 2 R 3 wherein R 3 is the side chain of an amino acid or amino acid derivative selected from the group consisting of glycine, alanine, valine, leucine, isoleucine, aspartic acid, glutamic acid, glutamine, serine, threonine, methionine, arginine, lysine, histidine, proline, tryptophan, phenylalanine, tyrosine, their unnatural optical isomers, 3-methyl-L-histidine, a-ketoglutaric acid, fp-alanine, carnosine, citrulline, creatine, folic acid, glutathione, hippuric acid, homoserine, N-carbamyl 25 aspartic acid, N-formyl-L-methionine, ornithine and optical isomers thereof; CsH 4 R 4 wherein R 4 is H, CH 3 (CH 2 )nCH 3 NH 2 COCH 3 CO(CH 2 )nCH 3 C(CH 3 3 CH(CH 3 2 (CH 2 )nCH(CH 3 2 (CH2)nCOCH 3 OCH 3 or O(CH 2 )nCH 3 wherein n 1-3; CHNH 2 (CH 2 )nR 5 wherein n 1-7 and Rs is CH 3 OH, CONH 2 C 6 H 4 OH, or CONHNH 2 (CH 2 )nR 6 wherein n 1-9 and R 6 is an indole group, NCH 3 C(=NH)NH 2 SCH 3 NH 2 CO 2 H, CONH 2 or NHC(=NH)NH 2 provided that when n 2 and R 2 is H or M, R 6 is not CH 3 (CH 2 )nCHNH 2 CO 2 H, wherein n 1-8; CH(CO 2 H)NHCONH 2 or C 5 H 4 N; and wherein R 2 is H, CH 3 (CH 2 )nCH 3 wherein n 1-8, (CH 2 )xO(CH 2 )yCH 3 or (CH 2 )xCO(CH 2 )yCH 3 wherein x y 2-7, or M, wherein M is a metal counterion or ammonium, provided that when R 1 is (CH 2 2 CH 3 R 2 is not H; or the group consisting of a sulfonic acid derivative having the formula: R 7 -SO 2 -OR 8 A W wherein R 7 is a straight chain or branched Ci-C 5 lower alkyl, aryl, substituted lower alkyl, or S 4 substituted lower aryl; and IR:\LIBA32082.doc:MSOffice -63- R 8 is a hydrogen atom, a metal counterion, or ammonium; or a sulfonated amino polysaccharide. 36. The method of Claim 35, wherein the agent comprises a chemical compound selected from the group consisting of benzoic acid, 4-ethylbenzoic acid, benzoate buffer, and 5 chondroitin-6-sulfate. 37. The method of Claim 35, wherein the agent is administered in vivo to a mammal. 38. A method of enhancing the adhesion of a cell to a culture substratum, wherein a sulfonated amino polysaccharide having a molecular weight of about 4,000 daltons is added to the medium in which the cells are cultured. So 39. The method of Claim 38, wherein the cell is a cell that normally requires a feeder layer in order to grow in culture. The method of Claim 38, wherein the cell is a hepatocyte, and the sulfonated amino polysaccharide is chondroitin-6-sulfate. 41. A method of enhancing the transient expression of a foreign gene in a eukaryotic cell 15 comprising: introducing into the cell a molecule of foreign DNA that encodes a protein in a form capable of being expressed in the cell; and, contacting the cell with a first agent prior to, during, and following the introduction into the cell 0. of the foreign DNA, wherein the concentration of the first agent is 1-15 mM and the first agent f* 20 comprises at least one chemical compound having the formula: O R I C- OR 2 wherein R 1 is: CHNH 2 R 3 wherein R 3 is the side chain of an amino acid or amino acid derivative selected from the group consisting of glycine, alanine, valine, leucine, isoleucine, aspartic acid, 25 glutamic acid, glutamine, serine, threonine, methionine, arginine, lysine, histidine, proline, tryptophan, phenylalanine, tyrosine, their unnatural optical isomers, 3-methyl-L-histidine, a-ketoglutaric acid, 13-alanine, carnosine, citrulline, creatine, folic acid, glutathione, hippuric acid, homoserine, N-carbamyl aspartic acid, N-formyl-L-methionine, ornithine and optical isomers thereof; CeH 4 R 4 wherein R 4 is H, CH 3 (CH 2 )nCH 3 NH 2 COCH 3 CO(CH 2 )nCH 3 C(CH 3 3 CH(CH 3 2 (CH 2 )nCH(CH 3 2 (CH2)nCOCH 3 OCH 3 or O(CH 2 )nCH 3 wherein n 1-3; CHNH 2 (CH 2 )nR 5 wherein n 1-7 and R 5 is CH 3 OH, CONH 2 C 6 H 4 0H, or CONHNH 2 (CH 2 )nR 6 wherein n 1-9 and R 6 is an indole group, NCH 3 C(=NH)NH 2 SCH 3 NH 2 CH 3 CO 2 H, CONH 2 or NHC(=NH)NH 2 (CH 2 )nCHNH 2 CO 2 H, wherein n 1-8; r 35 CH(CO 2 H)NHCONH 2 or C 5 H 4 N; and wherein R 2 is H, CH 3 (CH 2 )nCH 3 wherein n 1-8, (CH 2 )xO(CH 2 )yCH 3 or (CH 2 )xCO(CH 2 )yCH 3 wherein x y 2-7, or M, wherein M is a metal counterion or ammonium, provided that when Ri is (CH 2 2 CH 3 R 2 is not H; S or the first agent comprises at least one chemical compound having the formula: 1-:\LIBA]3 2082.doc MSOffice -64- R 7 -S0 2 -OR 8 wherein R 7 is a straight chain or branched C1-C5 lower alkyl, aryl, substituted lower alkyl, or substituted lower aryl; and R 8 is a hydrogen atom, a metal counterion, or ammonium; and following the introduction of the foreign DNA, the cell is contacted with a second agent, wherein the second agent comprises at least one sulfonated amino polysaccharide at a concentration of 0.01-0.5 mM or wherein the second agent comprises, at a concentration of 1-15 mM, at least one chemical compound having the formula: O II R 1 C-OR 2 ;o wherein R 1 is: CHNH 2 R 3 wherein R 3 is the side chain of an amino acid or amino acid derivative selected from the group consisting of glycine, alanine, valine, leucine, isoleucine, aspartic acid, glutamic acid, glutamine, serine, threonine, methionine, arginine, lysine, histidine, proline, tryptophan, phenylalanine, tyrosine, their unnatural optical isomers, 3-methyl-L-histidine, ao-ketoglutaric acid, 15 p-alanine, carnosine, citrulline, creatine, folic acid, glutathione, hippuric acid, homoserine, N-carbamyl aspartic acid, N-formyl-L-methionine, ornithine and optical isomers thereof; C 6 H 4 R 4 wherein R 4 is H, CH 3 (CH 2 )nCH 3 NH 2 COCH 3 CO(CH 2 )nCH 3 C(CH 3 3 CH(CH 3 2 (CH 2 )nCH(CH 3 2 (CH 2 )nCOCH 3 OCH 3 or O(CH 2 )nCH 3 wherein n 1-3; CHNH 2 (CH 2 )nR 5 wherein n 1-7 and R 5 is CH 3 OH, CONH 2 C 6 H 4 OH, or CONHNH 2 20 (CH 2 )nR 6 wherein n 1-9 and R 6 is an indole group, NCH 3 C(=NH)NH 2 SCH 3 NH 2 CH 3 CO 2 H, CONH 2 or NHC(=NH)NH 2 (CH 2 )nCHNH 2 CO 2 H, wherein n 1-8; CH(C0 2 H)NHCONH 2 or CsH 4 N; and 25 wherein R 2 is H, CH 3 (CH 2 )nCH 3 wherein n 1-8, (CH 2 )xO(CH 2 )yCH 3 or (CH 2 )xCO(CH 2 )yCH 3 wherein x y 2-7, or M, wherein M is a metal counterion or ammonium, provided that when Ri is (CH 2 2 CH 3 R 2 is not H; or the second agent comprises at least one chemical compound having the formula: R 7 -S0 2 -OR 8 wherein R 7 is a straight chain or branched C1-C5 lower alkyl, aryl, substituted lower alkyl, or substituted lower aryl; and R 8 is a hydrogen atom, a metal counterion, or ammonium. 42. The method of Claim 41, wherein the first agent is benzoate buffer, and the second agent is chondroitin-6-sulfate. 3 5 43. The method of Claim 41, wherein the first agent comprises benzoic acid and 4-ethylbenzoic acid, and the second agent comprises benzoate buffer and chondroitin-6-sulfate. 44. The method of Claim 41, wherein the first agent comprises benzoate buffer and glutamic acid, and the second agent comprises chondroitin-6-sulfate. The method of Claim 42, wherein the cell is contacted with the first agent for about 4 24 hours prior to the introduction into the cell of the foreign DNA. [R:\LIBA]32082doc:MSOffice 46. The method of Claim 1, wherein the agent is: benzoic acid and 4-ethylbenzoic acid; or benzoate buffer and chondroitin-6-sulfate; or benzoate buffer and glutamic acid; or glutathione, methionine, glycine, a-amino-n-butyric acid, taurine, phenylalanine, benzoate buffer, and alanine. 47. The method of Claim 15, wherein the sulfonated amino polysaccharide comprises an N-acetylated amino polysaccharide. 48. The method of Claim 47, wherein the N-acetylated amino polysaccharide is chondroitin-6-sulfate or a guaran. 49. A method of enhancing the transient expression of a foreign gene in a eukaryotic cell, substantially as hereinbefore described with reference to any one of the examples, but excluding So reference to any comparative examples. A method of screening an agent comprising at least one chemical compound to determine whether the agent is capable of enhancing the transient expression of a foreign gene in a eukaryotic cell, wherein the agent is biocompatible and contains at least one hydrophobic moiety and at least one acidic moiety, substantially as hereinbefore described with reference to any one of the examples, but excluding reference to any comparative examples. 51. A method of manipulating the metabolism of a cell to reduce the cell's consumption of glucose, substantially as hereinbefore described with reference to any one of the examples, but excluding reference to any comparative examples. 52. A method of enhancing the adhesion of a cell to a culture substratum, substantially as hereinbefore described with reference to any one of the examples, but excluding reference to any comparative examples. Dated 21 February, 2001 Genespan Corporation Patent Attorneys for the Applicant/Nominated Person SPRUSON FERGUSON [R:\LIBA]32082.doc:MSffice