Protamine Enhances the Efficiency of Liposome-Mediated Gene Transfer in a Cultur ed Human Hepatoma Cell Line
³½ºë³J¥Õ¥i¼W±j·L¯×ÅéÄâ±a°ò¦]¶i¤J¨xÀù²Ó-M®è¤§®Ä¯à -Ù-l¥È ¥x¤jÂå°|¤p¨à¬ì
ºK-n
³½ºë³J¥Õ¬O¤@ºØ´I§tºë®ò»Äªº¤p³J¥Õ½è¡A¥¦¥i¥H¨ÏDNA±²¦±¥HÁY¤pÅé¿n¡C¦]¦¹DNA¦p ¥Ñ³½ºë³J¥Õ¦ñÀH¶i¤J²Ó-M¤º¡A·|§Î¦¨¤@ºò±Kµ²ºc¡A¦Ó´î¤ÖDNA³Q-M¤º»Ã¯À¤À¸Ñªº¾÷·|¡C ¦]¦¹³½ºë³J¥Õ·¥¥i¯à¥i¥H«P¶i°ò¦]Âà´Þ¡C¥»¬ã¨s¤¤§Y§Q¥Î³½ºë³J¥Õ¨Ó«P¶i·L¯×ÅéÄâ±a°ò ¦]Âà´Þ¶i¤J¨xÀù²Ó-M®èªº¯à¤O¡C¥ý§Q¥Î¤@¬vµæ½¦¿ðº¢¤ÏÀ³¨M©w³½ºë³J¥Õ»PDNA°t¦X¤§ ³Ì¤p¥Î¶q¡AµM«á¥H¤£¦P¾¯¶qªº³½ºë³J¥Õ°t¦X·L¯×ÅéÄâ±aºñ¿Ã¥ú°ò¦]¶i¤J¨xÀù²Ó-M®è¡C°£ ¤F¥Î¿Ã¥úÅã·LÃèÆ[¹îºñ¿Ã¥ú¤§¥~¡A¦A¥Î¬y¦¡²Ó-M»ö-pºâÂà´Þ¦¨¥\¤§®Ä¯à¡Cµ²ªGÅã¥Ü¥[¤J ³½ºë³J¥Õ¤§«á¡A·L¯×ÅéÄâ±a°ò¦]Âà´Þ¶i¤J¨xÀù²Ó-M®èªº¯à¤OªGµM¤W¤É¡A¦¹¶µ¦¨ªG¥¼¨Ó±N ¥iÀ³¥Î¦Ü°ò¦]ªvÀø¡C
ABSTRACT
Protamine, clinically an antidote for heparin, is a small protein with a high arginine content and is potent in folding DNA. Protamine and DNA can form a compact structure, thus protecting DNA from digestion by intracellular enzymes. Protamine may therefore enhance the efficiency of gene transfer. In this study, we tested the ability of protamine to improve liposome-mediated gene transfer efficiency in a human hepatoma cell line. The results of a preliminary gel retardation assay indicated that 10 £gg was the minimal amount of protamine sulfate needed to completely bind 5 £gg of a plasmid containing a reporter gene, green fluorescent protein (GFP). For transfection assays, protamine (0, 10, 50, 100, and 500 £gg) was added to a DNA-liposome mixture (5£gg DNA and 20£gg lipofectamine) to transfect cultured Huh7 cells. Transfected cells (those expressing GFP) were counted by means of flow cytometry. The expression index (EI) was calculated as the transfection efficiency (% transfected cells) with protamine divided by the transfection efficiency with DNA and liposome only. The addition of 10, 50, or 100£gg of protamine to the liposome-DNA mixture significantly increased the EI, with the greatest increase noted when 10£gg was used. The results of this study show that an appropriate amount (10-100 £gg ) of protamine sulfate increases the transfection efficiency of GFP in Huh7 cells.
Key Words: gene transfection, protamine, liposome, green fluorescent protein INTRODUCTION
Liposomes are a useful non-viral vector that can carry foreign DNA for gene therapy [1]. They resemble animal cells in that the outer membrane consists of a double layer of lipid molecules. When liposomes fuse with cells, they are taken up by endocytosis, thereby delivering their contents (eg, targeted genes) into the cells[2,3].
Cationic liposomes may be complexed to negatively charged, naked DNA by simply mixing the liposomes and DNA together [4]. The complex is easily taken up by the cells and results in an acceptable level of gene expression [5]. The advantages of this vector are 1) it is non-immunogenic; 2) there is almost no limitation in the size of gene of interest; 3) it can be administered repeatedly; and 4) it is commercially available and ready to use [6,7]. However, its disadvantages are the low transfection efficiency in certain types of cells, lack of tissue targeting ability in standard preparation, and inability to yield a long-term stable expression in vivo [8].
For ex vivo hepatocyte gene therapy, liposomes may be a good vector because of their availability and lack of limitation in gene size [9]. However, the internal diameter of a liposome is about 0.025 to 0.1 £gm, which is much less than the longest dimension of plasmid DNA (about 2 £gm) [10]. This means that the expression vector needs to be
compacted inside the liposome.
A possible explanation for the low transfection efficiency of liposome-DNA complex is the relatively linear shape of the DNA in the complex [11]. Linear DNA is highly exposed and is therefore subject to digestion by intracellular DNAase. Thus, methods to deliver DNA in a more compact form might improve transfection efficiency. Protamines are small basic proteins (MW 4000-4250) with a high arginine content, and potent DNA folding ability. In salmon and herring, as well as mammals, sperm DNA is packed in a highly condensed state by protamines. Salmon sperm protamine has been sequenced and shown to contain 32 amino acid residues, 21 of which are arginine [12]. Although the exact mechanism of DNA-protamine complex formation remains unknown, the complex is speculated to acquire a very condensed, almost crytsalline form. In this compact structure, DNA is well protected from enzymatic hydrolysis and may reach the nucleus in an intact form. Indeed, Sorgi et al. demonstrated that protamine enhances liposome-mediated gene transfer to several different types of cells in vitro [13], but not specifically in hepatocytes.
Improving gene transfer efficiency may be critical for ex vivo hepatocyte gene therapy, especially for autologous hepatocyte transplantation. When hepatocytes are procured from donors and cultured ex vivo, a certain fraction of hepatocytes will not survive. A more efficient gene transfer is needed to compensate for the cell loss, and to achieve effective hepatocyte transplantation [14].
In this study, we tested the ability of protamine to enhance the efficiency of liposome-mediated gene transfer in cultured human hepatoma cells. We selected the green fluorescent protein (GFP) gene cloned from jellyfish [15], altered to allow expression of GFP in mammalian cells [16], as the reporter gene. The efficiency of gene transfer could then be estimated by using FACscan to determine the percentage of cells successfully transfected [17].
MATERIALS AND METHODS
Plasmids and Cell Culture The GFP gene was inserted into a commercially available expression vector (pGL-1,Gibco Life Technology, Gaithersburg, MD, USA). This vector is a 5030 bp plasmid containing the cytomegalovirus (CMV) immediate early promoter and an SV40 polyadenylation signal downstream of the GFP gene to direct the processing of mRNA. A conventional large-scale cesium chloride plasmid preparation was performed to amplify and purify the plasmid [18]. Huh7 hepatoma cells (a gift from Dr. Lee PI) [19,20] were cultured and maintained in Dulbecco's modified essential medium (DMEM, Gibco Life Technology) containing 5% fetal bovine serum (FBS, Gibco, Life Technology) in a 37° C incubator with 5% CO2.
Simplified Gel Retardation Assay to Determine DNA-Protamine Binding Curve Normal saline was used to serially dilute protamine sulfate (Sigma, St. Louis, MO, USA) to 0.01, 0.1, 1, and 10 £gg/£gL. Various amounts of normal saline-diluted protamine were mixed with 5 £gg of pGL-1 DNA in a total volume of 10 £gl followed by incubation at room temperature for 15 to 30 minutes. The mixtures then subjected to electrophoresis (100Volts, 30 minutes) through a 1% agarose gel with 1X TBE and stained with ethidium bromide to see if the DNA was retained in the well. The endpoint was the lowest amount of protamine that could bind 5 £gg of DNA completely, such that no DNA ran into the gel: this was used as the starting point for the transfection experiments.
Transfection experiments Forty-eight hours after transfection, the cultured Huh7 cells were harvested and the number of viable cells were counted with a hemacytometer (Reichert, Buffalo, NY, USA) after trypan blue (0.04%) (Gibco) exclusion. LipofectAMINE (Gibco), a mixed formulation of the polycationic lipid 2,3-dioleyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanaminium trifluoroacetate (DOSPA)
and the neutral phospholipid dioleoylphosphatidylethanolamine (DOPE)[14] (DOSPA: DOPE=3:1 w/w) was used for the transfection experiments. On the day before transfection, the Huh7 cells were dispensed into 6-well culture plates (Falcon, Becton Dickinson Labware, Lincoln Park, NJ, USA) at a density of 6x105 cell per well and incubated overnight at 37¢J in 5% CO2. On the experiment day, the cultured cells were then rinsed twice with 1ml serum-free DMEM to remove the previous culture media. For the liposome-DNA-protamine mixtures, 10£gl LipofectAMINE (L) ( 2£gg/£gL ) in 100£gl serum- free DMEM was mixed with 5£gg GFP plasmid DNA (G) (10 £gL, 0.5 £gg/£gL) in 100£gL serum-free DMEM in 3.5 mm cell culture plates. Protamine sulfate (10 mg/mL) (0, 10, 50, 100, or 500 £gg ) was then immediately added to the mixture, followed by incubation at room temperature for 30 minutes. After incubation, 800 £gL serum-free DMEM was added to the transfection mixtures. The final transfection mixtures (total 1 ml for each experiment) were added to the Huh7 cells, followed by incubation at 37°C for 6 to 8 hours. Toxicity (20 £gg lipofectAMINE in 1 mL serum-free DMEM) and negative ( 5£gg GFP plasmid in 1 mL serum-free DMEM without lipofectAMINE) control experiments were also performed. Equal volumes of DMEM+10%FBS were then added to each well after this incubation, without removing the transfection mixture. The culture media was replaced with 2 mL DMEM+5%FBS the next day, and transfection efficiency was analyzed 48 hours later, as described below. All experiments were performed in quadruplicate.
F ACscan counting: Cells from culture plate were detached by digestion with trypsin-EDTA 1ml (Gibco) and incubated at 37° C for 5 minutes. Cell suspensions were collected in 1.5 ml microcentrifuge tube and centrifuged 1500 rpm for 2-3 min. Decanted supernatant and then resuspended the cell pellet with 1X phosphate buffer saline (PBS). The cell suspension was then transferred to the counting tube and subjected to the FACscan (Becton Dickinson, Mountain View, CA). The efficiency of transfection was represented by the percentage of number of cells emitting fluorescence. It was calculated by the software CellQuest software (Becton Dickinson). The expression index (EI) is defined below:
% of cells emitting fluorescence after transfection with x £gg of protamine EI =
% of cells emitting fluorescence after transfection without protamine,
where x= 0, 10, 50, 100, or 500 £gg of protamine. Data are expressed as the mean¡Ó standard deviation where appropriate. Differences in the GFP EI between experiments were assessed with student’s t test. P value of less than 0.05 were considered statistically significant.
RESULTS
Minimal Amount of Protamine needed to Completely Bind 5 £gg GF P Plasmid. Ten £gg of protamine sulfate could retain almost all DNA in the well (Fig. 1), while smaller amounts could not. Therefore, the minimal amount of protamine used in transfection assay was 10 £gg. We also used larger amounts of protamine (50, 100, or 500 £gg) in other experiments, to check the dose-responsive curve.
Transfection of Huh7 cells with GF P Forty-eight hours after transfection, the cultured Huh7 cells were harvested and the number of viable cells were counted. The numbers of cells after transfection were about the same in each experiment. (G+L: 2.1±0.4X106 cells/well; G+L+10 £gg protamine: 1.9±0.3X106 cells/well; G+L+50 £gg protamine: 2.1±0.3X106 cells/well; G+L+100 £gg protamine:1.8±0.2X106 cells/well;
G+L+500£gg protamine: 1.7±0.1X106 cells/well; negative control: 2.1±0.3X106 cells/well; and toxicity control: 1.9+0.1X106 cells/well). There was no statistically significant difference in cell viability among the experimental groups (all p>0.05, student’s t test).
Percentage of Cells Emitting Green F luorescence Counted by F ACscan and Expression Index. Forty-eight hours after transfection, the cell culture plates were photographed under fluorescent microscope to check for green light emission (Fig. 2). There was no green fluorescence emission observed or the negative control plate. After each plate was photographed, the cells were harvested and subjected to FACscan cell counting. The results of quadruplicate experiments showed that the tranfection mixture containing G+L + 10£gg protamine resulted in the highest EI (EI=3.4¡Ó2.3), followed by G+L+50£gg protamine (EI=1.9±0.6), G+L+100£gg protamine (EI=1.6±0.9), and G+L+500 £gg protamine (EI= 0.9±0.9). All mixtures except G+L+500£gg protamine yielded significantly higher EI than the control (G+L) (Fig. 3).
DISCUSSION
DNA transfection efficiency with liposome vectors is not high. One paper reported only 80 pmol out of 1£gmol of oligonucleotide was uptaken in 106 cultured HepG2 cells [21]. However, the efficiency can be enhanced by altering the composition, size, and charge of liposome-DNA complex for in vitro gene delivery [22]. Not much effort has been directed at investigating the use of molecules to modify DNA and improve transfection efficiency. In a previous study of histone H1, investigators set up a model of positively charged protein that can drastically affect the DNA binding activity of specific transcription factors [23]. Because of its positive charge, protamine sulfate can help to fold DNA and increase the transfection efficiency in the liposome-mediated gene transfer system[13]. In this study, we successfully demonstrated that protamine sulfate can indeed increase gene transfer efficiency in liposome mediated transfection.
The other advantage of using protamine is that it is already used clinically as an antidote for heparin and the adverse drug effects and pharmacology are known. This should facilitate the clinical accessibility of this agent if it is eventually applied in human gene therapy.
The results of the simplified gel retardation assay showed that 10 £gg of protamine is the minimal amount needed to completely bind 5 £gg of GFP plasmid. The minimal amount of protamine also seemed to be the optimal amount for in vitro transfection as adding more protamine did not increase the gene transfer efficiency. It is possible that, because of its high affinity for DNA, protamine hinders binding of transcription factors to DNA and thereby inhibits expression when high levels are used for transfection.
In the future, we may apply this method to improve transfection efficiency for primary rat hepatocytes and even human hepatocytes. Cultured cells can be transfected with wild type foreign genes, and stable transfectants can then be selected, and reintroduced back to the hosts. This may provide a good model for gene therapy for treatment of inherited diseases, cancer, and even infectious diseases. Furthermore, protamine can be conjugated with various liver-specific ligands, such as asialoglycoproteins like asialoorosomucoid [24], or asialofetuin [25], and therefore has a potential application in in vivo gene therapy. Asialoglycoprotein/polylysine/DNA complexes have been used for targeted in vivo gene delivery in mice[26] and rats[27]. Similarly, protamine may be used to bind foreign DNA and a ligand to target the complex to a specific organ, such as the liver. Once the ligand is taken up by hepatocytes, protamine may help to fold the foreign DNA and transport it to the nucleus for transcription and expression in an intact form. Furhter studies are needed to examine the feasibility of this approach.
1. Felgner PL: Improvements in cationic liposomes for in vivo gene transfer. Hum Gene Ther 1996;7:1791-3.
2. Chang AGY, Wu GY: Gene therapy: applications to the treatment of gastrointestinal and liver diseases. Gastroenterology 1994;106:1076-84.
3. Nicolau C, Legrand A, Grosse E: Liposomes as carriers for in vivo gene transfer and expression. Methods Enzymol 1987;149:157-76.
4. Felgner PL, Gadek TR, Holm R, et al: Lipofection: a highly efficient, lipid-mediated DNA transfection procedure. Proc Natl Acad Sci USA 1987;84:7413-7.
5. Farhood H, Gao X, Son K, et al: Cationic liposomes for direct gene transfer in therapy of cancer and other disease. Ann NY Acad Sci 1994;716:23-34.
6. Gao X, Huang L: Cationic liposome-mediated gene transfer. Gene Ther 1995;2:710-22. 7. Ledley FD: Nonviral gene therapy: the promise of genes as pharmaceutical products. Hum
Gene Ther 1995;6:1129-44.
8. Morgan RA, Anderson WF: Human gene therapy. Annu Rev Biochem 1993;62:191-217. 9. Yu HY, Lin CY: Uptake of charged liposomes by the rat liver. J Formos Med Assoc
1997;96:409-13.
10. Felgner PL: Nonviral strategies for gene therapy. Sci Am 1997;276:102-6.
11. Bianchi F, Rousseaux-Prevost R, Bailly C, et al: Interaction of human p1 and p2 protamines with DNA. Biochem Biophys Res Comm 1994;201:1197-204.
12. Warrant RW, Kim SH:£\-Helix double helix interaction shown in the structure of a protamine-transfer RNA complex and a nucleoprotamine model. Nature 1978;271:130-5. 13. Sorgi FL, Bhattacharya S, Huang L: Protamine sulfate enhances lipid-mediated gene
transfer. Gene Ther 1997;4:961-8.
14. Kormis KK, Wu GY: Prospects of therapy of liver diseases with foreign genes. Semin Liver Dis 1995;15:257-67.
15. Chalfie M, Tu Y, Euskirchen G, et al: Green fluorescent protein as a marker for gene expression. Science 1994;263:802-5.
16. Cheng L, Fu J, Tsukamoto A, et al: Use of green fluorescent protein variants to monitor gene transfer and expression in mammalian cells. Nature Biotechnol 1996;14: 606-9.
17. Cormack BP, Valdivia R, Falkow S: FACS-optimized mutants of the green fluorescent protein (GFP). Gene 1996;173:33-8.
18. Sambrook J, Fritsch EF, Maniatis T: Molecular cloning. 2nd
ed. Cold Spring Harbor Press, 1989:1.33-8.
19. Nakabayashi H, Taketa K, Miyano K, et al: Growth of human hepatoma cells lines with differentiated functions in chemically defined medium. Cancer Res 1982;42:3858-63. 20. Wu CH, Wu GY: Targeted inhibition of hepatitis C virus-directed gene expression in
human hepatoma cell lines. Gastroenterology 1998;114:1304-12.
21. Tu GC, Cao QN, Israel Y: Inhibition of gene expression by triple helix formation in hepatoma cells. J Biol Chem 1995;270:28402-7.
22. Felgner JH, Kumar R, Sridhar CN, et al: Enhanced gene delivery and mechanism studies with a novel series of cationic lipid formulations. J Biol Chem 1994;269:2550-61. 23. Schultz TF, Spiker S, Quatran RS: Histone H1 enhances the DNA binding activity of
the transcription factor EmBP-1. J Biol Chem 1996;271:25742-5.
24. Wu GY, Wu CH. Receptor-mediated gene delivery and expression in vivo. J Biol Chem 1988; 263:14621-4.
25. Kaneo Y, Tanaka T, Iguchi S: Targeting of mitomycin C to the liver by the use of asialofetuin as a carrier. Chem Pharm Bull 1991;39:999-1003.
26. Stankovics J, Crane AM, Andrews E, et al: Overexpression of human methylmalonyl CoA mutase in mice after in vivo gene transfer with asialoglycoprotein/ polylysine/DNA
complexes. Hum Gene Ther 1994;5: 1095-104.
27. Wu GY, Wilson JM, Shalaby F, et al: Receptor-mediated gene delivery in vivo. Partial correction of genetic analbuminemia in Nagase rats. J Biol Chem 1991;266:14338-42.
F ig. 1 Green fluorescence emission in (a) G+L , (b) G+L+10p, (c) G+L+50p, (d) G+L+100p,(e) G+L+500p, (f) negative control groups. For the liposome-DNA-protamine mixtures, 10£gl LipofectAMINE (L) ( 2£gg/£gL ) in 100£gl serum- free DMEM was mixed with 5£gg GFP plasmid DNA (G) (10 £gL, 0.5 £gg/£gL) in 100£gL serum-free DMEM in 3.5 mm cell culture plates. Protamine sulfate (P) (10 mg/mL) (0, 10, 50, 100, or 500 £gg ) was then immediately added to the mixture. (100X magnification)
0 10 50 100 500 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 G F P e x p re s s io n i n d e x Amount of Protamine (µg) added
