Advances in cationic lipid-mediated gene delivery

origin are open to treatment in a most fundamental sense

I. Introduction

(Mulligan, 1993; Anderson, 1998). Those working in theThe use of genes as medicines remains both a field of gene delivery have much to learn from virusescaptivating goal and a formidable challenge. By deliberate which achieve efficient levels of gene transductionintroduction of either a functional gene or a sequence commensurate both with their need to deliver their geneticcapable of interfering with the functioning of a cellular material into host cells for the purpose of reproduction andgene, a wide variety of diseases of inherited and acquired

with the evolutionary time-scale upon which these abilities have been honed. Although it seemed therefore natural to harness viruses (among them adenoviruses, adenoassociated viruses and retroviruses) for therapeutic gene delivery (Mulligan, 1993; Kootstra and Verma, 2003), it can be contested that much of their inconveniences are yet to be discarded. Problems with immunogenicity and toxicity remain, added to the practical issues of large scale production and quality control.

Focus has therefore shifted to a de novo approach in vector design, where synthetic organic molecules are used to bind the transgene and facilitate its passage across the significant extracellular and intracellular barriers that separate it from the cell nucleus where expression takes place via the cellular transcription machinery (Crystal, 1995; Lehn et al, 1998). Such carriers are termed non-viral vectors and generally take the form of cationic lipids or cationic polymers. In addition to avoiding problems associated with the use of recombinant viruses, an advantage of using synthetic vectors is that there is no limit on the size of DNA to be delivered. A large number and wide variety of synthetic non-viral vectors have been prepared and their transfection efficiency assessed not only in in vitro and in vivo experimental studies, but further, into the clinical setting for treatment in particular of cancer (Roth and Cristiano, 1997; Hersh and Stopeck, 1998) and cystic fibrosis (Alton et al, 1999; Boucher, 1999; Griesenbach et al, 1999; Davies et al, 2001). An exhaustive list of clinical gene therapy trials is available at www.wiley.co.uk/genmed/clinical.

Despite some positive results, the overall outcome indicates that a critical requirement for successful gene therapy is the use of more efficient gene delivery systems,

i.e. systems leading to a higher percentage of transfected cells or an increased amount of transgene protein in the transfected cells according to the given experimental or clinical situation (Crystal, 1995; Aissaoui et al, 2002; Miller, 2003). This review aims to highlight the recent advances in improving cationic lipid-mediated gene delivery in terms of overcoming both intracellular and extracellular barriers to gene transfer. This will be dealt with by firstly surveying the progress made in vector design at the molecular level. Structure and functionality of the cationic lipid/DNA complexes will then be described with special focus placed on our own work with novel lipids. Finally, the stability of the lipoplex in the extracellular medium and the features of its intracellular trafficking towards the cell nucleus will be discussed, as well as the proposal of creating sophisticated modular self-assembling gene delivery systems incorporating various functional elements to face the barriers encountered. In short, the goal is the development of ‘virus-like’ systems, which share the levels of gene delivery efficiency of viral counterparts, but coupled with the safety of purpose-made organic molecules.

II. Basic principles

The first stage in the preparation of particles suitable for gene delivery is the condensation of the large DNA molecules by the vectors. The general structure of a cationic lipid vector is shown in Figure 1. The cationic nature of the amphiphilic vector drives an electrostatic interaction in the presence of negatively charged DNA, spontaneously self-assembling into nanometric vector/DNA complexes termed lipoplexes (stage 1, Figure 2). This initial compaction step enables protection of the DNA from nucleases which are found in the extracellular medium. Use of an excess of cationic vector (quantified by the lipid/DNA ratio resulting in a mean theoretical charge ratio of the lipoplex (+/-)) conveniently decorates the outer surface of the lipoplex with a net positive charge which is generally considered to facilitate subsequent cellular uptake by interaction with negative cell surface residues such as proteoglycans (Friend et al, 1996; Labat-Moleur et al, 1996). Non-specific endocytosis ensues, encapsulating the lipoplex in intracellular vesicular compartments (Zabner et al, 1995) (stage 2), though fusion-based uptake cannot be entirely ruled out (Gao and Huang, 1995). Internalisation achieved, the DNA must avoid degradation in the late endosome and lysosome (barred arrow) by escaping the endosome to the cytoplasm (stage 3) (Zabner et al, 1995; Mukherjee et al, 1997). Trafficking of the DNA to the perinuclear region precedes passage across the nuclear membrane (stage 4) and subsequent expression of the transgene (stage 5). When localised within the nucleus, the DNA is already separated from its vector (Hasegawa et al, 2001) and it has been shown by microinjection experiments (Zabner et al, 1995) that gene expression does not occur if the complex remains intact.

Cationic lipids designed to achieve the ambitious task of gene delivery were first introduced by Felgner et al, whose work was founded on initial attempts to transfer nucleic acids via encapsulation into classical liposomes (Nicolau and Sene, 1982; Nicolau et al, 1983).

The lipid DOTMA (N-(1-(2,3-dioleyloxy)propyl)N,N,N-trimethyl ammonium chloride) resulted, consisting of a quaternary amine connected to two unsaturated aliphatic hydrocarbon chains via ether groups (Felgner et al, 1987) (Figure 3).

A multivalent lipid soon followed in the form of the lipopolyamine DOGS (dioctadecylamido-glycylspermine) (Behr et al, 1989) and use of cholesterol as the hydrophobic portion was subsequently validated by the

vector DC-Chol (3‚-[_N-(-(N’,N’dimethylaminoethyl)carbamoyl] cholesterol) (Gao and Huang, 1991) (Figure 3). It is of particular importance that the transfection efficiencies of many cationic lipids can be enhanced by their formulation as stable cationic liposomes (Farhood et al, 1995). This is achieved by mixing cationic lipids, especially those which are incapable of forming bilayers alone, with the neutral colipid DOPE (dioleoyl phosphatidylethanolamine) prior to complexation with DNA. For example, DOTMA/DOPE liposomes are commercially available as Lipofectin (Gibco BRL). Also, the structural analogues DDAB (dimethyldioctadecyl ammonium bromide) (Gibco BRL) and DOTAP (1,2-dioleoyloxy-3-[trimethylammonio]propane, where ester groups replace ethers of DOTMA) (Boehringer Mannheim) (Figure 4), are commercially available alone, as well as formulated with DOPE.

In addition to stabilisation properties, DOPE is also thought to have fusogenic properties which are expected to play a role in endosomal membrane disruption and so enhance escape of the lipoplexes into the cytoplasm (Ellens et al, 1986; Farhood et al, 1995; Vidal and Hoekstra, 1995). However, because a selection of lipoplexes formed in the absense of DOPE are also able to escape the endosome (Behr et al, 1989; Vigneron et al, 1996), these cationic lipids are also credited with intrinsic membrane destabilisation properties.

Since the initial ‘proof of principle’ period which confirmed the ability of cationic lipids to protect, transfer and release DNA for cellular expression, a challenging period has followed. Indeed slow progress has been made in improving the level of transfection efficiency up to that required for the potential therapeutic use of non-viral vectors, and this is largely attributed to an unclear structure-activity relationship in vector design. Thus, the development of novel vectors is justified as the highly complex series of steps that connect the DNA outside the cell to its expression in the nucleus are not fully understood and so a novel cationic lipid may not just be a ‘me too’ addition to an already extended list, but rather open new possibilites for differently influencing those steps (Lehn, 1999). As a result, a diverse library of vectors exists, representing a wide variety in structures and thus numerous potential mechanisms by which better transfection levels might be obtained. Highlights of its contents will be discussed in the following section.

III. Design of the basic domains of a cationic lipid

All cationic lipids are positively charged amphiphiles containing the three following functional domains: i) a polar hydrophilic headgroup which is positively charged, generally via the protonation of one (monovalent lipid) or several (multivalent lipid) amino groups; ii) a linker whose length and nature may influence the stability and the biodegradability of the vector; and iii) a hydrophobic portion composed of alkyl chains (saturated or unsaturated) or of a steroid (Figure 1).

Progress has been made in the design of each of these components. In particular, the choice of headgroup has expanded into the use of natural architectures and functional groups with recognised DNA binding modes. Linkers have been developed which are sensitive to biological stimuli, inducing DNA release from the lipoplex at defined time-points.

Finally, modifications of the hydrophobic portion have revealed that optimal vector design is often dependant on this moiety. Accordingly, the following sections will deal with the advances made to the three functional domains.

Figure 5: Advances made in headgroup design: multivalent lipids with recognised modes of nucleic acid binding.

V. Inclusion of ligands for nuclear uptake

As vectors have become increasingly sophisticated, it has become clearer that significant advances can only be made if cationic lipid design also addresses the most considerable barrier to gene delivery: the passage across the nuclear membrane. Indeed, experiments comparing transgene expression after microinjection of plasmid DNA either into the cytoplasm or directly into the nucleus have identified the nuclear membrane as being a severely limiting step in transgene delivery (Capecchi, 1980; Zabner et al, 1995; Escriou et al, 1998). Accordingly, the success of the current non-viral vectors may at least partly rely on the disappearance of the nuclear membrane during mitosis, as transfection is generally found to depend on the proliferation status of the cells (Fasbender et al, 1997; Oudrhiri et al, 1997; Mortimer et al, 1999; Escriou et al, 2001). As such it is suggested that gene delivery during interphase is likely to be low. When adding to this the relatively instability of plasmid DNA in the cytoplasm (probably due to degradation by Ca²⁺-sensitive nucleases (Lechardeur et al, 1999; Pollard et al, 2001), the need for an efficient method of crossing the nuclear membrane becomes evident.

The nuclear membrane consists of two concentric lipid bilayers which make contact at numerous points, forming aqueous pores through the two membranes, which are termed the nuclear pore complexes (NPCs) and have an internal diameter of roughly 9 nm (Mazzanti et al, 2001). These are the only known route for direct exchange of substances between the cytoplasm and the intranuclear compartment. Consequently, the transport of large molecules such as plasmid DNA is non-passive and requires signal mediation by shuttling molecules (importins) (Boulikas, 1997; Escriou et al, 2003). The basic peptide from the SV40 large tumor antigen characterised by a PKKKRL amino acid sequence acts as a nuclear localisation signal (NLS) and has been used in attempts to induce nuclear uptake of plasmid DNA. Binding of multiple NLSs to plasmid DNA (Collas et al, 1996; Ciolina et al, 1999; Neves et al, 1999) was found to be bettered by attachment of just a single NLS peptide to one end of a capped linear DNA fragment (Zanta et al, 1999). However, although transfection (mediated by PEI or DOGS) was enhanced by an impressive 10-1000 fold with the single NLS-DNA conjugate, the technique remains only a proof of principle as preparation of such a DNA fragment requires complex engineering and is therefore relatively impractical for gene therapy applications.

An alternative approach to the use of NLSs for nuclear uptake of plasmids is steroid-mediated gene delivery. This technique relies on binding of the transfected plasmid to the glucocorticoid receptors (GRs), which thus stimulated, actively transport the bound plasmid into the nucleus. The steroid dexamethasone was used as a GR-binding motif and so attached to a DNA binder (psoralen) via a short spacer (Rebuffat et al, 2001). The small increase in transfection activity seen in comparison to lipofection of unmodified DNA in dividing cells, became more pronounced (15-40 fold) in nondividing cells. Importantly, increasing the number of GR targeting molecules attached to the DNA (via psoralen coupling and thus without regiospecificity) led to loss of transgene expression probably due to covalent damage to the reporter gene sequence in the plasmid. These studies were therefore extended, and the preparation of a peptide nucleic acid (PNA) ‘clamp’ followed, which was demonstrated to link dexamethasone to the DNA at a defined position where transgene expression remained unhindered (Rebuffat et al, 2002).

VI. Extracellular barriers and requirements of in vivo lipofection

The applicability of cationic lipids for in vivo gene delivery was investigated following their proven efficiency for in vitro gene transfection into a great diversity of established cell lines and primary cell cultures. Although some promising results have been reported, such as the transfection of the airway epithelium via the airway passages, numerous studies in animals yielded much less satisfactory results, especially as regards systemic administration (Gao and Huang, 1995; Miller, 1998). Accordingly, in the first gene therapy clinical trials in man with cationic lipids, the lipoplexes were applied to the patients via in situ administration such as intranasal instillation or direct intratumoral injection (Hersh and Stopeck, 1998; Alton et al, 1999; Boucher, 1999; Griesenbach et al, 1999; Davies et al, 2001).

Identifying the numerous environments that the lipoplex is to encounter on passage to the target cells and highlighting the transfection-limiting barriers therein has been a particular priority. Firstly, serum-associated inhibition of lipofection has been reported (Li and Huang, 1996). Indeed, binding of the positive lipoplexes to the negatively charged molecules found in the serum may lead to their neutralisation, thereby hindering the non-specific electrostatic interaction of the lipoplex with the plasma membrane of the target cells. However, serum sensitivity of lipofection was found to be dependent on the type of cationic lipid, as different observations were noted by different investigators including serum resistance and lipoplex charge ratio and size dependence of the inhibitory effect of serum (Brunette et al, 1992; Yang and Huang, 1997; Turek et al, 2000; Almofti et al, 2003). Our own results with guanidinium-based cationic lipids have shown for example that the in vitro transfection activity of lipid BGTC, of a bisguanidinylated diacetylene lipid and of an acylhydrazone linked bisguanylated lipid were not decreased in the presence of serum (Patel et al, 2001; Aissaoui et al, submitted for publication).

The second barrier to lipofection in vivo is the presence of opsonins in the blood stream which may bind to the positive lipoplexes and trigger their rapid clearance from the blood via uptake by the macrophages of the reticuloendothelial system, thereby leading to a decreased circulation of the lipoplexes and hindering DNA uptake by the target tissues (Gao and Huang, 1995; Li and Huang, 1996; Aissaoui et al, 2002). The components of the complement system are likely to be involved in opsonisation of intravenously administered lipoplexes (Plank et al, 1996). The degree of complement activation has been shown to depend on the type of cationic lipid used, with monovalent lipids being weak activators. However, incubation of the cationic vectors with DNA to form complexes was found to reduce complement activation, as was coating the DNA complexes with polyethyleneglycol (PEG) (Plank et al, 1996). Thus by limiting the half-life and the targetability of the lipoplexes, complement activation appears to be a potential barrier for intravenous gene delivery, although it may be minimised by appropriate vector formulation.

A third barrier to in vivo lipofection is the extracellular matrix which is present between cells and protects the plasma membrane of the target cells (Felgner, 1999). The extracellular matrix contains negatively charged glycosaminoglycans capable of interacting with, and limiting the diffusion of, positive lipoplexes. It is also noteworthy that DNases present in the serum and the extracellular space could rapidly degrade unprotected DNA.

Finally, it should be stressed here that in vivo lipofection usually necessitates the administration of highly concentrated solutions of lipoplexes which should ideally be electrically neutral and carry a specific ligand for receptor-mediated uptake when cell targeting is required. Unfortunately, it is common knowledge that the preparation of such lipoplexes is problematic, as high DNA concentrations and electroneutrality lead to colloidal instability resulting in the flocculation and precipitation of the lipoplexes (Lee and Huang, 1997; Lasic et al, 1998). We and others have reported the use of lipophilic PEG derivatives to sterically stabilise lipoplexes formed at high DNA concentration (Hong et al, 1997; Wheeler et al, 1999; Pitard et al, 2001). Indeed, it has been reported that incorporation of PEG derivatives into liposomes (thereby creating so-called ‘stealth’ liposomes) resulted in prolonged circulation times in blood, the PEG polymer forming an exclusion barrier around the liposome which hinders its aggregation as well as opsonisation in the presence of serum components (Klibanov et al, 1990; Lasic et al, 1991; Hong et al, 1997; Martin and Boulikas, 1998; Zhang et al, 1999). However, although PEG coatings have been undoubtably useful for the preparation of stabilised particles, recent reports state that the protection offered by the polymer can inhibit both binding to the target cell and, upon internalisation, release of the lipoplex from the endosomal compartment causing PEG concentration-dependent decreases in levels of transfection (Harvie et al, 2000; Shi et al, 2002; Song et al, 2002; Keller et al, 2003). Although careful choice of PEG length is likely to help avoid such inhibition (Mori et al, 1991), alternative options are also being sought such as the preparation of stabilised liposomes which are able to lose their protective coat, in particular after their accumulation at tumor sites (Martin and Boulikas, 1998). Disuphidelinked PEG chains have been reported, which, once cleaved intracellularly, have been shown to rapidly release the liposomal contents and re-enable membrane fusion properties (Kirpotin et al, 1996). Recovered transfection activity has also been achieved by varying the length of the acyl chain connecting a PEG polymer to a ceramide group used to anchor it to the lipsosome, short acyl chains being found to cause rapid dissociation of the PEG coating (Wheeler et al, 1999). In addition, entirely different stabilising agents are proposed based on a single carbohydrate linked to cholesterol. These neoglycolipids have been reported to confer the stabilisation properties of PEGylated systems, but without impairing transfection efficiency (Perouzel et al, 2003).

Finally, targeting ligands such as monoclonal antibodies (Trubetskoy et al, 1992), the iron-carrying protein transferrin (Zenke et al, 1990) and sugars (Wagner, 1998; Fajac et al, 1999) have been shown to confer selective delivery to target cells. For example, lipopolyamine-condensed DNA particles decorated with a triantennary galactose ligand have allowed targeted gene transfer into hepatoma cells (Remy et al, 1995). An interesting concept is use of the PEG coating as a linker to equip the lipoplexes with specific ligands for targeted receptor-mediated gene delivery. It has been shown that coupling of plasminogen to the ends of PEG chains led to long circulation times and effective target binding of the PEG-modified liposomes (Blume et al, 1993). In an elegant study, Behr and coworkers introduce, as an outer envelope to a nanometric pre-condensed lipoplex, a PEG chain which acts as linker between the targeting ligand folic acid at one end and DNA binding agents at the other (Zuber et al, 2003). However, although stability and folate targeting was confirmed, this supramolecular assembly was not efficient. The inclusion of endosomolytic and nuclear uptake agents to the lipid-bearing nanoparticles is expected to be required for optimisation of such a ‘viruslike’ gene delivery system (Zuber et al, 2001).

VII. Conclusions

Cationic lipid-mediated gene delivery has now passed from the in vitro stage of validation onto the in vivo stage, and this with early clinical data emerging. It is clear though that the future of non-viral systems will require an increase in efficiency of gene delivery such that therapeutic levels of gene expression can be attained (Li and Huang, 2000). Because the multiple barriers to non-viral gene delivery are only just becoming clear at the molecular level, the advances in vector design, formulation and modular assembly are at present focused on surmounting single, at most a few, of these barriers in any distinct study. This article was intended to alert the reader to research efforts which have included stabilisation of the lipoplex in the extracellular medium, targeting of a particular cell type by pendant motifs, decomplexation of the transgene in response to the drop in endosomal pH or the reducing environment of the cytosol, and finally trafficking of the transgene to the perinuclear region with ultimately active passage across the nuclear membrane. The future will require the difficult task of incorporating the functions capable of conferring the series of actions described above into a single system, capable of working in a chronological order with unwanted inter-reactivity of the individual functions avoided. Certainly such a multimodular assembly is imaginable as viruses form such highly complex systems. For example, the human adenovirus is of about 90 nm in diameter and infects respiratory epithelial cells. Pendant targeting ligands in the knob of the viral fiber bind to cellular CAR receptors and locally activate αV integrins which trigger endocytosis. The virus is then delivered to an intracellular compartment, whereupon it rapidly escapes on drop in pH via interactions including the binding of the penton base protein to αVβ5 integrin. The virus is subsequently transported to the perinuclear region by exploitation of microtubule- and dynein/dynactin-dependent mechanisms, upon which it docks with the nuclear pore receptor CAN/Nup214 and disassembles to allow entry of the 36kb linear DNA into the nucleus (Meier and Greber, 2003). However, reasonably, the required non-viral gene delivery system is more likely to resemble something between a lipoplex and such a virus, together in structure, complexity and efficacy. The system will be designed to avoid the immunological and toxicological responses which impede virus-mediated transfer and the gap in efficiencies between the two methods may be further narrowed by using a greater number of non-viral transfecting particles of somewhat lesser efficiency. Clearly this does not obviate the advances that need to be made to bring non-viral gene transfer into the therapeutic mainstream, a gradual combination of the current approaches being an indispensable first step. Finally, one might also predict there will be no ultimate ‘all purpose’ vector, rather each vector having to be individualised according to the clinical setting.

Acknowledgements

This work was supported by the Marie Curie Individual Fellowship Program and grants from Vaincre la Mucoviscidose (Paris, France) and the Association Française contre les Myopathies (Evry, France).

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Dr. Benjamin Martin