gene delivery to mammalian cells, volume 1

Methods in Molecular Biology TM Methods in Molecular Biology TM Edited by William C. Heiser Gene Delivery to Mammalian Cells VOLUME 245 Volume 1: Nonviral Gene Transfer Techniques Edited by William C. Heiser Gene Delivery to Mammalian Cells Volume 1: Nonviral Gene Transfer Techniques 1 Chemical Methods for DNA Delivery An Overview Dexi Liu, Evelyn F. Chiao, and Hui Tian 1. Introduction Introduction of DNA into mammalian cells is a powerful tool for studying the function of various DNA sequences, and for gene therapy. The process of intro- ducing DNA into cells for the purpose of gene expression is called transfection or gene delivery. Synthetic compounds used to facilitate DNA transfer are often named synthetic vectors or transfection reagents. Compared with biological (viral vectors) and physical methods that are covered elsewhere in this volume and in the next volume, the major advantages of synthetic vectors (or chemical methods) are their simplicity, ease of production, and relatively low toxicity. Many synthetic compounds have been developed since DEAE-dextran was first used in transfection experiments more than 35 years ago. Rapid progress in de- veloping more efficient synthetic vectors has led to successful DNA delivery into a variety of cell types in vitro and in vivo. More importantly, in the last few years, we have witnessed significant efforts and progress in elucidating the mechanisms underlying synthetic vector-mediated DNA delivery. With the con- tinuous effort to meet the need for safe and efficient gene-delivery methods for human gene therapy, it is foreseeable that significant advances will be made in the future. This article concentrates on four major types of chemical reagents that are available to most investigators: calcium phosphate, DEAE-dextran, cationic lipid, and cationic polymer. Each of these types of reagents has its advantages and disadvantages, some of which we briefly outline in this overview chapter. From: Methods in Molecular Biology, vol. 245: Gene Delivery to Mammalian Cells: Vol. 1: Nonviral Gene Transfer Techniques Edited by: W. C. Heiser © Humana Press Inc., Totowa, NJ 3 One type of compound that is not covered in this section is biopolymers. These compounds, including polylysine (1–7), histone (8,9), chitosan (10,11), and peptides (12–17), have shown relatively low transfection efficiency when used alone. Although they might become relatively more important in the fu- ture, we feel their utility has not, at present, been demonstrated to be broad enough to recommend their routine use for transfection. For those who are in- terested in obtaining more information about the properties and activity of this group of polymers in DNA delivery, relevant information can be found in the references cited at the end of this chapter or in relevant chapters in this volume. 2. For What Purpose Are Synthetic Vectors Needed? Cell membranes are sheetlike assemblies of amphipathic molecules that sep- arate cells from their environment and form the boundaries of different or- ganelles inside the cells. However, these physical structures, allow only the con- trolled exchange of materials among the different parts of a cell and with its immediate surroundings. DNA, on the other hand, is an anionic polymer, large in molecular weight, hydrophilic, and sensitive to nucleases in biological ma- trices. Unless special means are used, DNA molecules are not able to cross the physical barrier of membrane and enter the cells. In theory, for a successful in- troduction of DNA into cells and expression of the encoded gene, one needs to overcome three major hurdles. These include: (1) transfer of DNA from the site of DNA administration to the surface of target cells; (2) transfer of DNA across the plasma membrane into the cytoplasm; and (3) transfer of the DNA across the nuclear membrane into the nucleus to initiate gene expression. Thus, the corresponding properties for an ideal synthetic vector should include protecting DNA against nuclease degradation; transporting DNA to the target cells; facil- itating transport of DNA across the plasma membrane; and finally promoting the import of DNA into the nucleus. 3. Properties of the Synthetic Compounds Most Commonly Used for DNA Delivery 3.1. Calcium Phosphate Transfection with calcium phosphate was developed by Graham and Van der Eb in 1973 (18) and has been one of the most commonly used methods for DNA delivery into mammalian cells. This method takes advantage of the formation of small, insoluble, calcium-phosphate-DNA precipitates that can be adsorbed onto the cell surface and be taken up by cells through endocytosis. The proce- dure requires mixing of DNA with calcium ions, subsequent addition of phos- phate to the mixture, and presentation of the final solution to cells in culture. Various types of cells have been transfected using this procedure. Transfection 4 Liu et al. efficiency can be as high as 50%, depending on the cell type and the size and quality of the precipitate. The variation in the composition and particle size of the calcium-phosphate-DNA precipitates results in poor reproducibility. This method does not seem to work on cells in primary culture or in animals. 3.2. DEAE-Dextran DEAE-dextran was the very first chemical reagent used for DNA delivery. It was initially reported by Vaheri and Pagano in 1965 (19) for enhancing the viral infectivity of cells. Similar to cationic polymers (see Subheading 3.4), DNA and DEAE-dextran form aggregates through electrostatic interaction. A slight excess of DEAE-dextran in the mixture results in net positive charge in the DEAE-dex- tran/DNA complexes formed. These complexes, when added to cells, bind to the negatively charged plasma membrane and then are internalized through endocy- tosis. Compared to calcium phosphate, the transfection efficiency of DEAE-dex- tran is much higher, although it varies with the type of cells and other experi- mental conditions. Transfection efficiency as high as 80% has been reported with DEAE-dextran/DNA complexes. The method is relatively simple and re- producible, but requires low or no serum during transfection. DEAE-dextran is toxic to cells at high concentration. 3.3. Cationic Lipids The use of cationic lipids for DNA delivery was pioneered by Felgner and col- leagues (20). The first cationic lipid synthesized for the purpose of DNA delivery was N-(2,3-dioleyloxypropyl) N, N, N-trimethylamonium chloride (DOTMA). When hydrated, DOTMA forms liposomes with or without additional lipid com- ponents. When mixed with DNA, the positive charges at the liposome surface electrostatically interact with the negative charges on the phosphate backbone of the DNA to form DNA/liposome complexes (lipoplexes). Addition of lipoplexes to cells in culture normally results in significant levels of gene expression, with an efficiency ranging from 5% to more than 90% depending on the type of cell line used. A broad spectrum of transfection activity among many cell types, low toxi- city, and high efficiency are the major advantages of cationic lipids. Major efforts have been made over the past decade to optimize the transfec- tion activity of cationic lipids. Results from in vitro and in vivo studies have re- vealed that, among the many physicochemical properties that affect transfection activity of cationic liposomes, the most important one is the cationic lipid struc- ture. The common features shared by all transfection-active cationic lipids de- veloped so far include three structural domains: (1) a positively charged head group, (2) a hydrophobic anchor, and (3) a linker connecting the head group and the hydrophobic anchor. The structure of the head group varies from one to four ammonium groups, which can be primary, secondary, tertiary, or quaternary for Chemical Methods for DNA Delivery 5 multivalent cationic lipids (21,22). The most common structure for the hy- drophobic anchor consists of either two hydrocarbon chains or cholesterol. With a few exceptions, glycerol is the linker in cationic lipids with a double-chain an- chor, whereas a variety of linkers have been used in cationic lipids with a cho- lesterol anchor (21,22). Our studies on structure–function relationships for in- travenous transfection revealed that the optimal lipid structure should include the following: (1) a cationic head group and its neighboring hydrocarbon chain being in a 1,2-relationship on the backbone, (2) an ether bond to link the hy- drocarbon chain to the backbone, and (3) paired oleyl chains as the hydropho- bic anchor (25). Cationic lipids without these structural features had lower in- travenous transfection activity in mice. For in vitro transfection, structure-based rules that allow for prediction of transfection activity of cationic lipids have yet to be established. The transfection activity of cationic lipids varies significantly with the type of cells used. Inclusion of specific lipids (helper lipids) such as dioleoylphosphatidylethanolamine (DOPE) into cationic liposomes enhances transfection activity in some cell types but not others (23). For transfection of epithelial cells by airway administration, Lee et al. have shown that cholesterol-based cationic lipids (lipid-67) exhibit much higher activity than their noncholesterol-based counterparts (24). The mechanism of lipid-67-based gene transfer into lung epithelial cells is not known. Although many complex structures between DNA and cationic liposomes have been identified (26–29), the most important parameters affecting the trans- fection activity of cationic liposomes appear to be the particle size of the com- plexes (30) and the charge ratio (amines to DNA phosphates ratio, ϩ/Ϫ) (20–25). Complexes with larger particle size (Ͼ200 nm) and with charge ratio of slightly greater than 1 appear to be optimal for in vitro transfection. For in- travenous transfection, however, a charge ratio (ϩ/Ϫ) of greater than 12 is re- quired for an optimal transfection activity (31,32). Cholesterol generally func- tions as a better helper lipid than DOPE for systemic transfection (31,33,34). 3.4. Cationic Polymers Different from the hydrophobic cationic lipids, cationic polymers are a group of highly water-soluble molecules. Three general types of cationic polymers have been used for transfection: linear (polylysine, spermine, and histone), branched, and spherical. The most extensively studied cationic polymers for DNA delivery are polyethyleneimine (PEI) (35,36) and dendrimers (37–39). PEIs are highly branched organic polymers produced by polymerizing aziridine (40). In princi- ple, PEI, with every third atom in the polymer being amino nitrogen, is a type of organic compound that contains the highest density of potential positive charges. It has been estimated that about 25, 50, and 25% of nitrogens in branched PEIs are primary, secondary, and tertiary amines, respectively. The enriched nitrogen 6 Liu et al. atoms and the diversified nitrogen forms provide considerable buffer capacity for the PEIs over a wide pH range (41,42). Both branched and linear types of PEIs have been used for transfection. The in vitro transfection efficiency of PEI 800 KDa on a large variety of cell lines and primary culture is comparable to that of cationic lipids. Dendrimers are a new class of branched, spherical, and starburst molecules. Dendrimers differ in their initiator structure and in the number of layers of the building blocks in each molecule. (The number of layers is also called the num- ber of generations.) The common initiators include ammonia (NH 3 ) as trivalent initiator and ethylenediamine as a tetravalent initiator. Polymerization takes place in a geometrically outward fashion, resulting in branched polymer with spherical geometry and containing interior tertiary and exterior primary amines. The defined structure and large number of surface amino groups of dendrimers have led to these polymers being employed as a carrier for DNA delivery. Dif- ferent forms of dendrimers have been shown to be active in transfection (37–39). Their precisely controlled size and shape provide them with the potential advan- tage of forming more homogeneous and highly reproducible DNA complexes. When mixed with DNA, cationic polymers readily self-assemble with DNA and generate small tortoidal or spherical structures of approx 40–100 nm (42), depending on polymer size, structure, DNA to polymer ratio, and the type and concentration of ions in the buffer. The complexes formed between DNA and cationic polymers are called polyplexes. When added to cells in culture, poly- plexes are taken up by the cells. Similar to that of cationic lipids, transfection activity of cationic polymers varies with cell type, structure, and size of the polymer, and polymer to DNA ratio. Compared to cationic lipids, the major drawback of cationic polymers is their relatively high toxicity. 3.5. Combined Systems Transfection activity of combined synthetic compounds has been explored. Depending on the transfection reagents selected, a significant enhancement in transfection activity can be achieved. In fact, much higher transfection activity of cationic liposomes was reported when mixed with polylysine (43), protamine sulfate (44), peptides (45,46), or PNA (Chapter 6 in this volume). The mecha- nisms for such synergistic effect between cationic liposomes and polymers are not known, but it is believed that the structure of DNA complexes in the com- bined system may be more effective in escaping the endosomal degradation and/or more efficient in facilitating DNA transfer into the nucleus. 4. Mechanisms of DNA Delivery by Synthetic Vectors A central tenet of DNA delivery by synthetic compounds is that DNA mole- cules can only be delivered into a cell when they are converted into a particu- Chemical Methods for DNA Delivery 7 late form. This appears to be true for all of the synthetic compounds that have been developed for transfection thus far. Obviously, synthetic compounds such as calcium phosphate, DEAE-dextran, cationic lipids, and cationic polymers are all capable of forming particles with DNA. With the exception of the calcium phosphate method that forms calcium-phosphate-DNA precipitates, other syn- thetic compounds form complexes with DNA through electrostatic interaction between DNA and the synthetic vectors. Once complexed with a synthetic vec- tor, DNA molecules are protected against nuclease-mediated degradation. To accomplish DNA delivery, these complexes need to (1) bind to the cell surface, (2) cross the plasma membrane, (3) release DNA into the cytoplasm, and finally, (4) transport the DNA into the nucleus. The following is a brief summary of the current understanding on these events as they are involved in synthetic com- pound-mediated DNA delivery. 4.1. Binding to the Cell Surface Without exception, binding of DNA complexes to the cell surface with the most commonly used synthetic compounds is accomplished by electrostatic in- teraction between the positively charged complexes and the negatively charged cell surface. It has been shown that cell surface binding of DNA complexes can be significantly enhanced by centrifugation of the transfection reagents onto cell surface (47). Another approach to enhance complex binding to the cell surface is to use DNA complexes of larger size (30). Enhanced binding usually corre- lates with higher transfection efficiency. 4.2. Crossing the Plasma Membrane and Entering the Cytoplasm The fact that particle structure of DNA complexes is required for DNA deliv- ery into cells stimulates the strong notion that endocytosis is the major pathway, through which DNA molecules are internalized (48–51). Because the endocy- totic process ends at the lysosome where DNA is degraded, escape of DNA from the endosome at an early stage of endocytosis is believed to be critical for cy- tosolic DNA delivery and is considered to be one of the most important rate-lim- iting steps that determine overall transfection efficiency. The mechanism of how DNA molecules leave the endosome and enter the cytoplasm is unknown. How- ever, a number of strategies have been explored to enhance endosomal release. The first of these involves the use of DOPE as a helper lipid for liposome-based DNA delivery. It is believed that DOPE is capable of inducing membrane fusion between the endosome and the liposome through the formation of an inverted hexagonal structure, which, in turn, results in membrane destabilization and re- lease of DNA into the cytoplasm (52). The second strategy is inclusion of mem- brane fusion proteins or peptides into DNA complexes (53). The mechanism of action of these compounds is similar to that of DOPE: low pH in the early endo- 8 Liu et al. some induces the proteins or peptides to fuse with membrane, releasing the DNA into the cytoplasm (53). Finally, buffering capacity of charged groups such as the primary, secondary, and tertiary amines in PEIs and polyamidoamine in den- drimers may play an important role in endosomal DNA release in polyplex-me- diated DNA delivery. Protonation of these groups in the endosome may create sufficient osmotic pressure to induce endosomal lysis (35,36). 4.3. DNA Release from Complexes Although formation of complexes between DNA and synthetic compounds is essential for cell entry, dissociation of DNA from the complexes after the com- plex has entered the cell appears to be essential for successful transfection. The necessity for DNA release from the complexes was demonstrated by Zanber et al. (54). In their experiments, lipoplexes or free DNA was injected directly into the nucleus of oocytes and the level of gene expression in these injected oocytes was analyzed at a later time. No gene expression was detected in oocytes injected with lipoplexes as compared to a significant level of gene expression in those in- jected with free DNA. With respect to cationic liposome-based transfection, work by Xu and Szoka (55) suggested that DNA release from the complexes re- sult from fusion of liposomes and cell membranes. Neutralization of positive charges of synthetic vector in DNA complexes by negatively charged cellular lipids and other components has been proposed as the mechanism of cytosolic DNA release from the complexes (55). 4.4. Nuclear Transport of DNA Once DNA molecules are in the cytosol, they must still enter the nucleus. How this occurs is largely unknown, but the transport of DNA from the cytosol into the nucleus does seem to occur because transgene expression is achieved. Mi- croinjection of plasmid DNA directly into cytoplasm revealed that transport of DNA from the cytoplasm into the nucleus was of extremely low efficiency (54,56). An additional factor affecting the transfer of DNA into the nucleus is nu- clease activity (56). Cytoplasmic nuclease activity can reduce the amount of in- tact DNA molecules available for nuclear import and gene expression, support- ing the notion that movement of DNA from cytoplasm to the nucleus is one of the most important limitations to a successful gene transfer. Interestingly, studies by Pollard et al. (57) demonstrated that PEI of 25KDa is able to promote gene de- livery from the cytoplasm into the nucleus. Such activity, however, is cell type dependent. Different from cationic liposomes, dissociation of PEI/DNA com- plexes to allow transcription does not seem to be a problem because injection of these complexes into the nucleus produces a level of gene product comparable to injection of plasmid DNA alone (57). Chemical Methods for DNA Delivery 9 Dean et al. (58) showed that the efficiency of nuclear transport of DNA is de- pendent on the DNA sequence. Cytosolic injection of DNA containing replica- tion origin and SV40 promoter sequences resulted in a significantly higher level of DNA importation into the nucleus than that observed with plasmids without these specific sequences (58). Inclusion of a nuclear localization signal, nor- mally a short lysine- and arginine-rich peptide, as part of synthetic vector has been considered as one of the promising strategies to enhance DNA import into the nucleus (59). 5. Optimizing Transfection In general, expression of the genetically coded information in DNA in a par- ticular type of cell presents its own particular set of problems that must be over- come to achieve a high level of expression. Unfortunately, selection of synthetic compounds for a given type of cell is still largely empirical. There is not a set of hard-and-fast rules to follow. In fact, a particular synthetic compound is al- most as likely to be the exception as it is to follow any set of rules. Keeping this caveat in mind, we will make some general comments that we hope will aid readers in selecting an initial transfection reagent. Selecting an appropriate synthetic vector for DNA delivery may require some homework on the researcher’s part. The first piece of advice is to consult with the technical service department of commercial resources (Table 1) for an es- tablished protocol, because many companies have already optimized the ex- perimental condition for their products in selected cell types. Researchers could adopt these conditions if the types of cells to be used in their experiments are the same or similar. This may significantly reduce the effort required to opti- mize the experimental conditions. In cases where there is not enough information available to make a selection, researchers may wish to obtain transfection reagents from a number of com- mercial sources to identify the one that gives the best results. Many companies sell kits that contain multiple reagents. Alternatively, if researchers are new to this type of study or are dealing with new types of cells, they will have to opti- mize the experimental conditions themselves following the protocols and sug- gestions described in the following chapters of this section. As a standard procedure, the most convenient way to do optimization work is to use a reporter system. The most commonly used reporter genes for this pur- pose include those that code for luciferase (Luc), green fluorescence protein (GFP), b-galactosidase (b-gal), chloramphenicol acetyltransferase (CAT), or se- creted alkaline phosphatase (SEAP). Most reporter gene-containing plasmids and related reagents for gene-expression analysis are commercially available from many resources (Table 2). The following is a brief description of the ad- vantages and disadvantages of the most commonly used reporter assay systems. 10 Liu et al. Chemical Methods for DNA Delivery 11 Table 1 Commercial Sources for Transfection Reagents a Company Product Composition Amersham-Biosciences CellPhect Transfection Kit CaPO4 or DEAE-Dextran 800-526-3593 www.amershambiosciences.com Bio-Rad Labs CytoFectene Transfection Reagent Cationic lipid 800-424-6723 www.bio-rad.com BD Biosciences–CLONTECH CLONfectin ΤΜ Cationic lipid 800-662-2566 CalPhos ΤΜ Calcium phosphate www.clontech.com CPG Inc. GeneLimo ΤΜ Transfection Reagent Plus Polycationic lipids ϩ lipid compound 800-362-2740 GeneLimo ΤΜ Transfection Reagent Super Polycationic lipids ϩ lipid compound www.cpg-biotech.com Gene Therapy Systems GenePORTER ΤΜ DOPE ϩ Proprietary compounds 888-428-0558 GenePORTER ΤΜ 2Proprietary material www.genetherapysystems.com BoosterExpress ΤΜ Reagent Kit Proprietary material PGene Grip ΤΜ Vector/Transfection Systems GenePORTER ϩ plasmid vector Glen Research Cytofectin GS Cationic lipid 703-437-6191 www.glenres.com Invitrogen LipofectAMINE 2000 TM Reagent Polycationic lipid 800-955-6288 LipofectAMINE PLUS ΤΜ Reagent Polycationic lipid (DOSPA:DOPE) www.invitrogen.com Lipofectin ® Reagent Cationic lipid (DOTMA:DOPE) Transfection Reagent Optimization System LipofectAMINE PLUS ΤΜ ϩ Lipofectin ® ϩ CellFectin ® ϩDMRIE CellFectin ® Reagent Cationic lipopolyamine OligofectAMINE ΤΜ Reagent Proprietary [...]... et al (19 93) Direct gene transfer with DNA-liposome complexes in melanoma: expression, biologic activity, and lack of toxicity in humans Proc Natl Acad Sci USA 90, 11 307 11 311 67 Ostresh, M (19 99) No barriers to entry, transfection tools get biomolecules in the door The Scientists 11 , 21 2 Gene Transfer into Mammalian Cells Using Calcium Phosphate and DEAE-Dextran Gregory S Pari and Yiyang Xu 1 Introduction... 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Stevenson, B J., et al (19 96) Laboratory and clinical studies in support of cystic fibrosis gene therapy using pCMV-CFTR-DOTAP Gene Ther 3, 11 13 11 23 65 Gill, D R., Southern, K W., Mofford, K A., Seddon, T., Huang, L., Sorgi, F., et al (19 97) A placebo-controlled study of liposome-mediated gene transfer to the nasal epithelium of patients with cystic fibrosis Gene Ther 4, 19 9–209 66 Nabel, G.J., Nabel, E... Gene Ther 10 , 319 -332 20 Liu et al 16 McKenzie, D L., Kwok, K Y., and Rice, K G (2000) A potent new class of reductively activated peptide gene delivery agents J Biol Chem 275, 9970–9977 17 Niidome, T., Takaji, K., Urakawa, M., Ohmori, N., Wada, A., Hirayama, T., and Aoyagi, H (19 99) Chain length of cationic alpha-helical peptide sufficient for gene delivery into cells Bioconjug Chem 10 , 773–780 18 . by William C. Heiser Gene Delivery to Mammalian Cells VOLUME 245 Volume 1: Nonviral Gene Transfer Techniques Edited by William C. 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