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Bioconjugate Chem. 1994,5,382-389
REVIEWS Gene Transfer with Synthetic Cationic Amphiphiles: Prospects for Gene Therapy Jean-Paul Behr Laboratoire de Chimie Genetique, CNRS URA 1386, Faculte de Pharmacie de Strasbourg, B.P. 24, F-67401 Illkirch Cedex, France. Received February 9, 1994 The introduction of genes into cells of various origins is a major technique of cell biology research. Gene transfer indeed is the most straightforward way to study gene and protein function and regulation, from a n in vitro isolated cell context up to in vivo multicellular processes such as embryogenesis or through the creation of animal models of human diseases. Besides being a powerful research tool, transfection also has important economic implications through the genetic engineering of microorganisms, plants, and animals, for the production of proteins as well as for crop and livestock improvement. The diversity of gene transfer uses has resulted in the development of a variety of artificial techniques, such as direct DNA microinjection, DNA coprecipitation with inorganic salts or with polycations, DNA encapsulation into liposomes, and cell membrane perturbation by chemicals (organic solvents, detergents, polymers, enzymes) or by physical means: mechanic (particle gun), osmotic, thermic, and electric (electroporation) shocks. This sustained interest in gene transfer techniques has recently exploded with the advent of gene therapy. As a conceptually new therapeutic approach, gene therapy, besides raising a n unprecedented media interest, is intellectually appealing to a very widespread population of scientists and clinicians, who join their efforts for the development of techniques that can be used in humans. Indeed, the weak link of gene therapy paradoxically is the vehicle rather than the “drug’’itself. Many ongoing clinical protocols (1) make use of recombinant retroviruses which are by far the most efficient vehicles to integrate foreign DNA into the genome of dividing cells. On the other hand, adenoviral vectors have been shown recently to efficiently transfect a large variety of postmitotic cells. These and other currently developed biological vectors (HSV, AAV), however, raise unassessable long term risks and have a limited capacity to carry foreign genetic material. In addition to these natural vectors, designed synthetic vectors are being developed (2-41, which in principle could solve the aforementioned questions, provided they are efficient enough in vivo. Two classes of molecules have been described so far: Cationic polypeptides chemically linked to cell surfacebinding ligands such as asialoorosomucoid, insulin, or transferrin bind ionically to DNA and trigger targeted cell entry of the complex ( 4 ) . Receptor-mediated endocytosis, however, leads to low transfection efficiencies since DNA remains essentially entrapped (or is degraded) in endosomes. This technique has greatly improved with the addition of endosomolytic components such as inactivated viruses or fusogenic peptides, leading to synthetic virus-like particles ( 4 ) . Other related cationic peptides ( 5 ) or dendrimeric polymers ( 6 )also have been used to efficiently transfect eucaryotic cells in vitro. Cationic amphiphiles do not require a specific cell surface receptor. These transfection vehicles are most often erroneously called “cationic liposomes”, and al1043-160219412905-0382$04.50/0
though many true liposome-based DNA delivery systems have been described (7, 81, the latter do not compare favorably with cationic amphiphiles (9)unless viral fusion proteins are included (10). “Cationic liposomes’’ is a confusing term, since it implicitly suggests both the occurrence during transfection of lipid particles with an aqueous interior and a DNA encapsulation step, none of which is needed. Rather, cationic amphiphiles bind cooperatively to DNA and coat it with a cationic layer which in turn interacts with anionic residues on the cell surface. Subsequent cell membrane destabilization is an intrinsic property of the amphiphilic gene carrier. This mechanism will be discussed in detail below, following a chronological description of the various systems which have been developed up to now.
Various Cationic Amphiphile Structures Transfect Cells. The rational design of such synthetic gene carriers is recent (3, 41, and within a few years, several groups reported on the synthesis or the use of already commercially available cationic, DNA binding, amphiphiles for gene transfer purposes. Felgner and co-workers synthesized the quaternary ammonium amphiphile DOTMA ((dioleoyloxypropy1)trimethylammonium bromide, see Chart 1)and were the first to describe DNA (11)as well as RNA (12)transfer into eukaryotic cell lines following mixing of the nucleic acid with a cationic lipid (as opposed to encapsulation into a liposome). This new technique was shown to be up to 100-fold more efficient than calcium phosphate or DEAE-dextran coprecipitation. DOTMA was commercialized (Lipofectin, GIBCO-BRL) as a one to one mixture with dioleoylphosphatidylethanolamine (DOPE) and has been widely used since to transfect a large variety of animal and plant eukaryotic cells (13-31). Our own Chart 1 0-
- A 3 ; h 3
-
DOTMA H3C
efforts were directed toward the design of cationic amphiphiles able to compact genomic DNA, namely, lipopolyamines (32). The metabolizable parent lipids DOGS and DPPES (see Chart 2) were shown to transfect (2 orders of magnitude better than Ca phosphate) established cell lines as well as primary neuronal cultures. Most importantly, DNA coating with excess cationic lipid (rather than liposome binding to the nucleic acid) and subsequent binding of the resulting particle to the negatively charged cell surface via ionic forces was inferred (33). Such a n excess of lipid cationic charges over the DNA anionic phosphates is a general requirement for optimal in vitro transfection with cationic lipids (see below). DOGS also has been commercialized (Transfectam, Promega) and has been shown to transfect many animal cells very efficiently (33-53). 1994 American Chemical Society
Bioconjugafe Chem., Vol. 5, No. 5, 1994 383
Reviews
Chart 2
Chart 3
C ,,GluPhC,,N+
0
-0
1
iv
Kunitake et al. (54) synthesized a whole series of lipophilic glutamate diesters with pendent trimethylammonium heads (Chart 3). Whereas transfection properties were similar for dodecyl and tetradecyl esters, they decreased drastically as the link between the lipidic and cationic moieties increased. C12GluPhCZN+ was as efficient as Lipofectin. In an effort to reduce the cytotoxicity of DOTMA, Silvius et al. (55) synthesized a series of metabolizable quaternary ammonium salts, some of which (DOTB, DOTAP dioleoyl esters, Chart 4)had efficiencies comparable to that of Lipofectin when dispersed with DOPE. Mixtures with cholesterol-derived cations (ChoTB, ChoSC) and, most surprisingly, the dioleoyl ester with a slightly longer head group spacer (DOSC)were much less efficient. DOTAP also is now available (Boehringer, Mannheim). Huang et al. (56) thought along the same lines and synthesized DC-Chol (Chart 5) having a hydrolyzable dimethylethylenediamine headgroup. DOPEDC-Chol one to one mixtures were able to transfect several cell lines, with efficiencies slightly better than with Lipofectin. The same group also reported about lipophilic polylysines (LPLL) (57), which were up to %fold more
0
C14GluC,Nt n=2,6,11
efficient than Lipofectin, but only when the fibroblast cells were mechanically scraped following the transfection period. Meanwhile, biologists had discovered that some commercial cationic detergents, when mixed with true lipids, could also function as transfection agents. Loyter et al. (58) showed that DEBDA hydroxide (Chart 6) added to excess phosphatidylcholine/cholesterol is able to introduce tobacco mosaic virus-RNA into tobacco and petunia protoplasts. Unfortunately, no comparison was made with the classical calciudpoly(ethy1ene glycol) technique for transfection of plant protoplasts. Huang et al. (59) compared the transfection properties of several quaternary ammonium detergents (Chart 7) on fibroblasts and found that CTABDOPE mixtures were the most eflicient, although somewhat less than Lipofectin. Yagi et al. (60)used a lipophilic diester of glutamic acid (TMAG, Chart 8) with DOPE and found i t to be as efficient as calcium phosphate for fibroblast transfection. Rose et al. (61) compared commonly used detergents of diverse structures: CTAB, DEBDA (see structures above), DDAB (Chart 9), and stearylamine in admixture with phosphatidylethanolamine. Comparative transfec-
Behr
384 Bioconjugate Chem., Vol. 5, No. 5, 1994
Chart 4
A
Cho
DO
X= N+Me3
DOTAP
OOC( CH2),N+Me3
DOTB ChoTB
00CH2CH2COOCH2CH2N+Me3 DOSC ChoSC
Chart 5
DC-Chol
Chart 6
Chart 9
H
DTAB
Chart 8
tion efficiencies were highly variable with the cell line; DDAB (which was shown earlier to be efficient (33),albeit very toxic) seemed to be the most promising, and the DDABDOPE formulation was patented (TransfectACE, GIBCO BRL).
ComparativeEfficiencies. The level of transfection is best assessed by using a gene which is absent from the cells to be transduced (reporter gene), driven by a strong viral promotedenhancer element. Normally, this exo-gene must reach the eukaryotic cell nucleus where it is transiently transcribed; alternatively, “stable” expression may be selected out from a subpopulation of daughter cells. In any case the net result is the synthesis of a foreign protein, which may be detected immunologically or enzymatically. Widely used reporter genes include those for the following enzymes: (1) the bacterial chloramphenicol-acetyltransferase (CAT)with either radioactive or fluorescent detection of acetylchloramphenicol or immunochemical ELISA quantitation of the CAT protein; (2) the bacterial P-galactosidase (P-Gal), sometimes bearing a nuclear localization signal, which is mostly used for in vivo histochemical visualization of transfected cells: a synthetic substrate (X-Gal) is diffused into the fixed tissue, leading to precipitation of a deep blue product in cells expressing high levels of P-Gal; and (3) the firefly luciferase (Luc) which is quantitated in the cell extract by photon
Bioconjugafe Chem., Vol. 5, No. 5, 1994 385
Reviews Table 1. Gene Transfer Efficiencies of Various Cationic Amphiphiles cationic amphiphile
reporter genen
lipid DOTMA DOGS DDAB CiZGluC2N+ DOTAP LPLL detergenb'neutral lipid DEBDA 1Aecithin 1.3/Chol. 0.7 DC-Chol l/DOPE 1 CTAB l/DOPE 4 TMAG 1Aecithin 4 DDAB 1 P E 2
cell type
f ratiob
CAT Neo CAT DHFR CAT c y t c5 CAT CAT
CV1, COS7... LMTK, ~2 ... primary neurons, CHO, S49... CHO- ... primary neurons
TMV-RNA CAT CAT P-Gal VSV-G protein
tobacco, petunia protoplasts A431, L929... L929
cos1
CV1,3T3 L929, HeLa ...
cos1
'2 4 3 22 2.5 6
VTF 7-3 infected HeLa, BHK'
efficiency
ref
5-1OOx DEAE dextran 10-3Ox calcium phosphate > l o x calcium phosphate 10-1OOx calcium phosphate
DNA
Figure 5. Self-assembly of several “programmed lipids with DNA leads to a transfecting particle having the useful properties of a virus. N-t-HA, N-terminal peptide of hemagglutinin; NLSSV40,nuclear localization signal of SV40 large T antigen. penetrate a tissue. In spite of several successful reports (35,40, 68-80), real advances in this field will probably arise from novel formulations of already existing molecules, or from vectors based on a different principle. The several orders of magnitude wide gap between the efficiencies of viruses and that of synthetic vectors remains a challenge to chemists ( 3 , 4 ) . One way to take up this challenge is to develop a modular transfection system, where each molecular component is in charge of a key step of viral entry. Such a system is still based on a (neutral)lipopolyamind DNA core particle (Figure 5 ) . Self-aggregation and intermixing of lipids allows one to add new properties to this particle via other lipids: Hepatic cell targeting has been achieved recently with an additional oligogalactosebearing lipid (81). Virus-derived fusion and nuclear localization peptide headgroups should help the particles to cross the cytoplasmic and nuclear membranes. Thus, upon mixing these various lipids with a gene, only the lipopolyamine component will interact with DNA and condense it. Concentrated a t the resulting particle’s surface will be molecular signals for cell targeting, entry into the cytoplasm, and finally trafficking toward the nucleus, a process reminiscent of programmed molecular self-organization (82)leading to a virus. ACKNOWLEDGMENT
I thank my collegues V. Mordvinov, H. Perron, J. S. Remy, and C. Sirlin for references to unpublished experiments and Prof. B. Spiess for pK measurements. This work was supported by the Centre National de la Recherche Scientifique, Association Francaise contre les Myopathies, Association Francaise de Lutte contre la Mucoviscidose, and Association pour la Recherche contre le Cancer.
388 Bioconjugate Chem., Vol. 5, No. 5, 1994 LITERATURE CITED
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