Issues in Non Viral Gene Delivery

Advanced Drug Delivery Reviews 46 (2001) 187203
www.elsevier.com / locate / drugdeliv
Key issues in non-viral gene delivery q

Colin W. Pouton a , *, Leonard W. Seymour b
b
Department of Pharmacy and Pharmacology, University of Bath, Bath BA2 7 AY, UK CRC Institute for Cancer Studies, University of Birmingham, Birmingham B15 2 TT, UK
Abstract The future of non-viral gene therapy depends on a detailed understanding of the barriers to delivery of polynucleotides. These include physicomechanical barriers, which limit the design of delivery devices, physicochemical barriers that inuence self-assembly of colloidal particulate formulations, and biological barriers that compromise delivery of the DNA to its target site. It is important that realistic delivery strategies are adopted for early clinical trials in non-viral gene therapy. In the longer term, it should be possible to improve the efciency of gene delivery by learning from the attributes which viruses have evolved; attributes that enable translocation of viral components across biological membranes. Assembly of stable, organized virus-like particles will require a higher level of control than current practice. Here, we summarize present knowledge of the biodistribution and cellular interactions of gene delivery systems and consider how improvements in gene delivery will be accomplished in the future. 2001 Elsevier Science B.V. All rights reserved.
Keywords: Gene therapy; DNA delivery; Virus-like particles; DNA condensation; Biopharmaceutics
Contents 1. Introduction ............................................................................................................................................................................ 2. Biodistribution ........................................................................................................................................................................ 2.1. Routes of administration ................................................................................................................................................... 2.2. Stability in blood circulation ............................................................................................................................................. 2.3. Extravasation ................................................................................................................................................................... 2.4. Intramuscular injection ..................................................................................................................................................... 2.5. Intratumoural injection ..................................................................................................................................................... 2.6. Subcutaneous administration ............................................................................................................................................. 2.7. Pulmonary biodistribution................................................................................................................................................. 2.8. Feasibility of local injection to other sites .......................................................................................................................... 3. Cellular interactions ................................................................................................................................................................ 3.1. Major barriers .................................................................................................................................................................. 3.2. Viral models .................................................................................................................................................................... 3.3. Binding and internalisation ............................................................................................................................................... 3.3.1. Molecular complexes.............................................................................................................................................. 188 188 188 189 190 192 192 192 193 194 194 194 194 195 195
q PII of original article: S0169-409X(98)00048-9. The article was originally published in Advanced Drug Delivery Reviews 34 (1998) 319. *Corresponding author. Tel.: 1 44-1225-826786; fax: 1 44-1225-826114. E-mail address: c.w.pouton@bath.ac.uk (C.W. Pouton).
0169-409X / 01 / $ see front matter 2001 Elsevier Science B.V. All rights reserved. PII: S0169-409X( 00 )00133-2
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C.W. Pouton, L.W. Seymour / Advanced Drug Delivery Reviews 46 (2001) 187 203 195 196 196 196 197 197 198 198 198 198 198 199
3.3.2. Liposome complexes .............................................................................................................................................. 3.4. Intracellular trafcking ..................................................................................................................................................... 3.4.1. Escape from the endosome...................................................................................................................................... 3.4.2. Involvement of the cytoskeleton in translocation ...................................................................................................... 3.5. Uncoupling of complexes ................................................................................................................................................. 3.6. Nuclear uptake................................................................................................................................................................. 4. Delivery devices ..................................................................................................................................................................... 4.1. Inhalation systems............................................................................................................................................................ 4.2. Particle bombardment....................................................................................................................................................... 4.3. Sustained delivery ............................................................................................................................................................ 5. Concluding remarks ................................................................................................................................................................ References ..................................................................................................................................................................................
1. Introduction The aim of this issue of Advanced Drug Delivery Reviews is to review the fundamental factors that affect the transport of macromolecules and particles in biological systems. The biodistribution of macromolecules has been of interest for many years in the eld of drug targeting and in the design of macromolecular prodrugs. The desire to develop experimental gene delivery systems towards pharmaceutical products is further reason to understand the fundamental transport processes that will determine their biodistribution. In this article, we introduce the challenge of gene delivery, in the extracellular and intracellular environments. There are many factors that inuence the extent and duration of gene expression following administration of a gene medicine. Delivery is an essential component of activity, which we review here, but the design and optimization of the expression system will also be a major part of the development of a successful gene medicine. The latter issues are addressed within a recent review of pharmaceutical issues in gene delivery, edited by Rolland and Felgner (Volume 31 of this journal) [1]. Other recent texts are recommended for more detailed discussion [2,3] Gene delivery presents an irresistible challenge to drug delivery scientists. It is clear that passive transport of the drug (usually a DNA plasmid) to its target site (the appropriate cell nucleus) is extremely inefcient. Yet, delivery of a few molecules to each cell nucleus is likely to be sufcient to achieve the desired outcome. Thus, gene delivery represents a very different challenge to delivery of low molecular weight drugs. Gene delivery can be
envisaged as a series of hurdles (both extracellular and intracellular) that successively deplete the mass of DNA that progresses towards the target site. Improvement in the efciency of any individual step would be expected to improve the overall efciency. Thus, it is necessary to break down gene delivery into individual steps, which can be studied separately and improved systematically. A major determinant of the feasibility of gene therapy, for each clinical indication, will be the mass of protein that needs to be expressed to obtain the desired therapeutic activity. This will be specic to each clinical indication, and should be considered at an early stage in the context of the available delivery strategies. In some cases, it will be appropriate to defer an attempt at gene therapy until such time as the efciency of gene delivery has been improved.
2. Biodistribution
2.1. Routes of administration

In practice, the signicance of each biological barrier will depend on the intended route of administration of the gene medicine and on the location of the target cells. Therefore, it is sensible to consider biodistribution, and the signicance of barriers to biodistribution, in the context of each clinical indication. A key issue is whether administration is required to be intravenous or by other parenteral routes. Direct injection of gene medicines into a target tissue represents a far simpler task than targeting delivery to a specic tissue from the systemic circulation. There are several reports describing the location
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and extent of gene expression after intravenous administration of DNA complexes. Unfortunately, the complexes used have often been poorly characterized, making it difcult to draw conclusions about their biodistribution. It may yet be possible to produce long-circulating DNA delivery systems, but this has not been achieved with existing DNA delivery systems. Thus, the prospects for widespread distribution from the systemic circulation are limited at present. However, administration by other routes for direct delivery to target tissues may well be feasible, particularly given that the treatment may have a therapeutic lifetime of several days or weeks. The inuence of each route of administration is discussed below, beginning with issues relating to intravenous administration.
2.2. Stability in blood circulation

Given the interest in ligand-mediated targeting, it is perhaps surprising that there are no systems yet available for efcient tissue targeting following systemic delivery. In fact, very little attention has been paid to the pharmacokinetic analysis of vectors for systemic delivery, possibly reecting the difculty in designing a material that is capable of surviving in the bloodstream for any signicant length of time. In fact, the kinetics of systemically administered naked DNA have been elucidated only comparatively recently, using expression vectors that were radiolabelled following nicking and template extension with [ 32 P]ATP. Hashida, Takakura et al. [4,5] have focused much effort on understanding the biodistribution of macromolecules and particles from the systemic circulation. Recently, these authors turned their attention to gene delivery systems [611], and contrasted the clearance of naked DNA and cationic lipidDNA complexes (lipoplexes) with other macromolecules. The hepatic clearance of naked DNA was very close to the plasma ow rate through this organ, which indicates that DNA is cleared substantially on rst-pass of the liver. The uptake of naked DNA was thought to be by non-parenchymal cells, probably Kupffer cells, following interaction of DNA with scavenger receptors for polyanions [10]. These data suggest that it will be necessary to neutralize the polyanionic nature of DNA to avoid uptake by non-parenchymal cells. LiposomeDNA
complexes were also cleared rapidly by the liver, in this case, by phagocytosis by Kupffer cells. This result could be explained by the instability of cationic particulate systems, which is discussed below. In addition, however, there is rapid degradation of the DNA by plasma nucleases, giving rise to signicant rates of urinary excretion of partially degraded DNA [10]. Cationic polymer / DNA complexes also show disappointingly short plasma circulation times, again with rapid hepatic uptake [10], but also with evidence of accumulation or deposition in organs such as the skin and intestine [11]. Since these organs are known to possess very ne capillary structures, it is possible that the deposition results from physical trapping of the complexes, perhaps following their aggregation or binding to serum components such as albumin. The pattern of deposition and of plasma clearance is partially inuenced by the charge ratio of complex formation. Many workers have studied DNA complexes that were administered systemically, monitoring the biological activity of the DNA as reporter gene activity determined in transfected tissues, or by polymerase chain reaction (PCR) of the gene delivered [12,13]. PCR can determine extremely small amounts of DNA, but it is extremely hard to quantify, meaning that, in general, very little is known of the quantitative pharmacokinetics of the materials administered. The single exception is targeted delivery to the liver, perhaps the easiest site to reach because of the sinusoidal endothelium, coupled with its natural DNA-scavenging activities. Following on from the pioneering studies by Stankovics et al. [14] and Wu et al. [15], Lollo and colleagues at the Immune Response Corporation have been conducting quantitative in vivo targeting of poly( L)lysine (pLL) / DNA complexes to the hepatocyte asialoglycoprotein receptor, by incorporation of asialoorosomucoid into the conjugate structure [16]. This strategy has been relatively successful, and intravenous administration of asialoorosomucoid-bearing net-negatively charged complexes to rodents has given enhanced liver uptake [16]. Although many stages of this process are still probably highly inefcient, targeting of gene constructs administered systemically has at last been quantied. Many other research groups have attempted to
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target macromolecules or particles to hepatocytes using galactose or galactosamine residues [17], and other strategies, such as antibody-mediated targeting of particles, could be exploited. The endothelium itself, being accessible, could be a useful target for some clinical applications [18]. However, before such mechanisms can be exploited, it will be necessary to prevent unwanted interactions between particles and the dynamic environment of the blood circulation. This challenge should not be underestimated. In the absence of a hydrophilic surface, many particles interact with, and become coated by, specic plasma proteins, which have evolved to participate in clearance of particles from the blood. The coating of particles is referred to as opsonization, a process that is difcult to study, and is often specic to each particles surface. Opsonization, reviewed in the context of liposome clearance by Patel [19], prepares a particle for uptake by xed macrophages of the mononuclear phagocytic system (MPS). The MPS is a collection of phagocytic cells that are present in tissues of the reticulo-endothelial system and are collectively responsible for clearance of particles from the circulation. The activity of this system is not restricted to clearance of charged particles. Typically in practice 8090% of hydrophobic particles are opsonized and taken up by xed macrophages of the liver and spleen, often within a few minutes of intravenous administration. Opsonization represents a major biological barrier to the delivery of DNA using condensed particles, and more effort will need to be directed at detailing these interactions in the context of gene delivery. Plank et al. [20] investigated the activation of complement by cationic materials and their complexes with DNA. Polycations, including polylysine, dendrimers and polyethyleneimine, all activated complement, and the potency of activation was chain-length specic for polylysine. Cationic lipids were weaker activators than polycations, but short chain oligolysines had similar properties to the lipids. Condensation with DNA reduced the activation of complement in all cases, although this effect was dependent on the charge ratio of complexes, particularly for polycations. Coating particles with polyoxyethylene prevented activation of complement, which suggested that formulations could be
developed with a low tendency to activate complement. More systematic work of this type will be required as new gene delivery systems emerge.
2.3. Extravasation
Extravasation of macromolecules, conjugates or particles is a function of their overall size and the permeabilities of the different vascular layers that they encounter. The tightest endothelial barrier in the body is the bloodbrain barrier. Here, there is no evidence for non-specic uid diacytosis, and the gaps at the sites of inter-endothelial cell junctions are only a few nanometres wide, even in post-capillary venules, which, in other tissues, are generally relatively permeable. Organs such as the skin, muscle and lung also possess tight endothelial layers, but with slightly larger interendothelial junctions in the post-capillary venules. Fenestrated endothelium, such as that found in the kidney, is much more permeable, reecting its professional function of ltration. Finally, tissues of the reticulo-endothelial system, the liver, spleen and bone marrow possess a sinusoidal endothelial structure that permits relatively free passage of materials without size restrictions, up to 100 nm [21]. The implication is that one would not expect particulate systems to be distributed from the blood circulation to peripheral organs, with the possible exception of the liver and other organs with sinusoidal capillaries. In practice, even delivery of 50100 nm particles to the liver is proving to be elusive and poorly reproducible after intravenous administration. Pathological vasculature is often relatively leaky to blood-borne macromolecules. For example, oedematous accumulation of protein-rich extravascular uid is common in sites of inammation and in tumours. The vasculature in tumours often shows a variety of structures, with regions of hyperpermeable endothelium, particularly in the post-capillary venules. The exact reason for this is still unclear, but may reect the activities of tumour-secreted permeability factors (see the accompanying article in this issue [22]). Wu and colleagues [14,15,2326] established that gene transfer to the liver is feasible using particles of DNA condensed with polypeptides. However, transfection by such methods is variable and delivery
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systems that give reproducible activity have been difcult to establish. The particle size of complexes is likely to be a major determinant of activity, since access to the liver probably requires particles that are smaller than 100 nm in diameter. Such particles have been described [27] but often DNApolylysine particles are reported to be greater than 100 nm in diameter. For 100 nm particles, extravasation is likely to be highly dependent on the blood pressure within the liver. If an animal model is used and the volume injected is large, then elevated blood pressure may result, leading to temporary, high levels of extravasation. Indeed, pressure-dependent effects have been reported to occur with naked DNA. Zhang et al. [28] demonstrated, by clamping the venous blood ow from the liver, that local injection into a clamped tissue (by way of the portal vein, vena cava or bile duct) can result in transfection of up to 10% of hepatocytes throughout the liver mass. These authors were able to plot the expression of reporter gene against the parenchymal pressure in the liver. This is presumably one reason why many groups have had difculty in reproducing intravenous experiments using either naked DNA or condensed formulations of DNA. It is likely that aggregation of cationic particles, such as cationic lipid / DNA complexes, can occur following interaction with blood components. Albumin is known to bind to cationic particles. This would reduce the zeta potential and promote aggregation of polyplexes. However, at present, it is only possible to speculate about the dynamic interactions of polycationDNA complexes with blood. There is a need for more fundamental research in this area. A comprehensive imaging study in mice revealed that lipoplexes are taken up widely by endothelia, but in a complex vessel- and tissue-specic manner [29]. The bulk of the plasma clearance after intravenous injection was due to uptake by lung endothelia, xed macrophages (Kupffer cells) in the liver sinusoids and xed macrophages in the spleen. The only organ in which the lipoplexes were observed to leave the vasculature was the spleen. It can be assumed that the high levels of gene expression found in the lung, by other authors [3032], were due to expression by lung endothelia. At present, an explanation for the uptake by lung endothelia has not been established. It seems likely that blood com-
ponents will bind to cationic lipoplexes. Albumin binds to cationic lipoplexes, which will change their physical properties substantially. However, it may be that small quantities of specic opsonins are more important. The binding of albumin may be enough to cause aggregation of particles in the bloodstream, by reducing their zeta potential and, hence, reducing the charge repulsion between particles. Extensive aggregation could lead to physical deposition in the capillary bed. However, the possibility of capillary blockade was not supported by the work of McLean et al. [29]. These authors observed that, although clustering of lipoplexes denitely occurred in the bloodstream, the aggregates were generally smaller than erythrocytes, and often less than 1 mm in diameter. Uptake was not only observed in the smallest arterial capillaries, but was often observed in the high endothelial venules, sometimes specically in certain organs (such as lymph nodes). It seems more likely that aggregated lipoplexes are taken up by adsorptive endocytosis, possibly involving a receptor for which they are a ligand. One possibility is that albuminlipidDNA complexes interact with scavenger receptors for denatured albumin, which are commonly expressed by endothelia [3335]. From a practical point of view, the important question is whether expression in the lung endothelium is reproducible and how sensitive is lung expression to the route of administration, the volume of injection and to inter-species variation. A key experiment, which was suggested by McLean et al. [29], will be to compare biodistribution and expression observed after injection into the left- versus the right side of the heart. If the pattern of uptake and expression by endothelia is similar after both injections, then there may be scope for use of this pattern of expression in man. Until such a time, it may be wiser to explore the opportunities for ligandmediated uptake of neutral or negatively charged complexes from the vasculature. In summary, the biodistribution of DNA or condensed particles of DNA from the systemic circulation is complex, dependent on the colloidal properties of the materials and is highly dependent on their specic interactions with blood components. Each particulate delivery system will have a passive distribution pattern, which will be sensitive to formulation and could be modied signicantly by grafting
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a hydrophilic polymer to the surface of the particle. There are opportunities for expression of genes by the endothelia, either for secretion into the systemic distribution, but also for local effect [13,36,37]. To obtain specic uptake of particles by a tissue that is not normally involved in clearance of particles from the blood will be a considerable challenge. It will certainly be necessary to rst establish a long-circulating form of the delivery system, using steric stabilization, before attempting a targeting strategy. There is scope for delivery of gene expression systems to the livers parenchymal cells, but current data indicate that it will be necessary to form stable particles that are 100 nm or less in diameter to achieve passive distribution to the parenchymal space of the liver.
DNA to the nucleus appears to be unusual in myocytes [42]. Although the relatively high activity after intramuscular injection of naked DNA is surprising, it should not be forgotten that the overall efciency of transfection is very low, usually with | 1% of cells being transfected after an intramuscular injection. It is conceivable that this level of activity could be the result of passive processes. The mechanism of uptake needs to be determined using uorescent or radioactive analogues of DNA. However, the important issue for gene delivery is whether active processes, involving internalisation or intracellular trafcking, can be exploited to gain a signicant improvement in the efciency of transfection.
2.5. Intratumoural injection

There are some similarities between expression of DNA in muscle and tumour tissue, although, in general, the levels of expression after intratumoural injection are much lower. Naked DNA appears to be as active as lipid formulations [48], presumably due to poor dispersion of particles within solid tumour tissue. Solid tumours lack effective lymphatic drainage, thus, gene delivery systems are likely to be forced towards the perimeter of the tumour tissue, where the material may become immobilized, unless extracellular uid is able to drain into the surrounding tissue. The high hydrostatic pressure within solid tumours presents a serious barrier to dispersion. In animal models, uid may be forced away from the injection site after intratumoural administration. Nevertheless, dispersion of particulate systems occurs to some extent, in some tumours, since small viral vectors are able to transfect some tumours on a widespread basis after a single injection.
2.4. Intramuscular injection

Although naked DNA plasmid has little or no activity in a typical cell culture transfection experiment, it has been known for some time that naked DNA can be active in vivo after direct injection in muscle [3846] and other solid tissues. Staining of muscle or tumour tissues suggests that expression following direct injection of lipoplexes is often localized in regions that are close to the injection site. This implies that the dispersion of colloidal particles within muscle is a critical issue and there is a need for basic studies of the effect of formulation on dispersion within solid tissues. Formulations of plasmids with hydrophilic polymers appear to be more effective than particulate systems at promoting transfection in solid tissues [47], and the pattern of expression is more uniform and widespread, which supports the hypothesis that dispersion is a critical factor. As yet, it is not clear what the relative contributions of diffusion and convection are in the dispersion of DNA formulations within a solid tissue. Convection will be an inuence as a result of the ow of interstitial uids into the lymphatic system. Albumin (hydrodynamic radius, 3.7 nm) circulates widely by convection through tissues. It is not yet known how myocytes take up plasmids in vivo, or how the DNA escapes lysosomal degradation. The unusual persistence of expression in muscle tissue has been attributed to the slow turnover of myober nuclei. In addition, access of
2.6. Subcutaneous administration

Subcutaneous administration of membrane-impermeable agents is a useful means of delivering materials into the lymphatic system [49]. In determining the inuence of molecular size on the efciency of lymphatic drainage, however, Seymour et al. [50] found that exible hydrophilic polymers with molecular weights greater than 500,000 were retained subcutaneously for a period of several weeks, although the exact mechanism of retention
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was not determined. Other workers have shown repeatedly that hydrophilic polymers and small liposomes, when administered intravenously, often display an accumulation in the skin [51]. At least in the case of doxorubicin-containing liposomes (Doxil), this appears to contribute to supercial toxicity, described as hand and foot syndrome [52]. Hence, it seems likely that subcutaneous administration of vectors for gene delivery would lead to substantial retention of the material at or near the site of injection, and the possibility should therefore be approached with caution. This phenomenon may be used to advantage for local effect, such as for peritumoural expression of immune modulators.
2.7. Pulmonary biodistribution

There is considerable interest in gene delivery to the lung, due to its immediate accessibility by inhalation, and the wide variety of conditions that could be treated by gene therapy [5356]. Several preliminary clinical trials in relation to gene therapy of cystic brosis have been completed and more studies are in progress [5763]. Given the wealth of experience with delivery of drugs to the lung, it should be possible to overcome the technical problems associated with deposition of gene delivery systems in the lung. The critical issue in delivery to the lung will be the interaction of the formulation with the biological barriers presented by the lung epithelium. Although it has been possible using gene therapy to treat cystic brosis in mouse models of the disease [64,65], the results of trials in humans have been less encouraging. Gene expression systems have been introduced to the lungs of animals either by inhalation [6670] or by aerosolization [64,7173]. Extensive studies have aimed at optimizing the formulation of lipoplexes for gene delivery to airway epithelia [74,75]. Despite these efforts, the lung epithelium in vivo is difcult to transfect at doses of cationic lipid that are well-tolerated [76]. The apical surface of an epithelial barrier has a number of features that discourage the uptake of particles. Epithelial monolayers are polarized cells with characteristic tight junctions between cells. The junctions are formed between neighbouring cytoskeletal networks through desmosomes, preventing paracellular uptake of all but very small polar
molecules. The apical surfaces are tough, strengthened by actin laments close to the plasma membrane, and do not have extensive endocytic capacity. Mucus is secreted by goblet cells within the epithelium, which acts as a primary mechanical and diffusional barrier, preventing access to the plasma membrane of the epithelium. Nutrients are taken up into epithelia from the basolateral surfaces, which are not generally exposed to the external environment, unless the tissue is damaged. In the bronchiolar regions of the lung (the conducting airways), the apical surface is coated with lamentous projections, i.e. cilia, which beat constitutively and move particulate material upwards until eventually it is expelled from the lung and swallowed into the oesophagus. Particulate material is cleared from the deeper, respiratory airways by alveolar macrophages. Phagocytosis is the likely fate of any lipoplexes (or viral particles) that reach the alveoli. Should there be a clinical indication for gene delivery to alveolar macrophages, phagocytosis may be regarded as a passive process that could be exploited. There have been attempts to compromise the tightness of cooperation between cells to allow uptake of lipoplexes via the basolateral surface. One approach, described by Sawa et al. [77], was to administer the formulation in water, to create a local, temporary hypotonic environment. It was postulated that this would increase the permeability of the barrier and lead to higher levels of expression. There was a signicant effect, but whether this could be applied in practice is not clear. Freeman and Niven [78] tested the ability of sodium glycocholate, a penetration enhancer, to enhance transfection of rat lung. Formulation of plasmid in solution with 1% glycocholate produced a 100-fold increase in gene expression, however, at 10% glycocholate, the animals died, indicating that this agent is probably unacceptable. Another factor that has been considered is the spreading of the formulation within the lung, since instilled suspensions do not necessarily make contact with an adequate surface area. Transfection by an adenoviral reporter vector was enhanced by co-administration of synthetic lung surfactant (Survanta) [79]. Others point out that in vitro transfection of lung epithelia with lipoplexes is retarded by the presence of lung surfactant [80,81], suggesting that the coating of lung surfactant, which
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is present in the deep lung, is likely to be a barrier to the uptake of lipoplexes. The barriers to gene transfer discussed above relate to access of DNA to the epithelial cell surface. The considerable interest in gene delivery to the lung has also highlighted the difference between transfection of cells in culture and transfection of differentiated, non-dividing cells in vivo. Grubb et al. [82] reported the inefciency of adenoviral vectors in vivo and, recently, two studies have appeared making use of lterculture techniques to compare the transfection of differentiated cells with dividing cells [83,84]. One problem with cell culture systems is that most models involve dividing cells that are metabolizing rapidly. In addition, the nuclear membrane will be compromised during mitosis, and many cells are likely to undergo mitosis during the time course of a transfection experiment. In contrast, differentiated cells in vivo are unlikely to divide and may not be in a highly metabolic state. Matsui et al. [83] reported that lung epithelia were heterogeneous in their ability to take up and process DNA, and that undifferentiated cells were responsible for most of the activity of the epithelium. Fasbender et al. [84] were able to demonstrate, using BrdU-treated cultures, that gene expression was predominantly associated with cells that had undergone DNA synthesis. These reports emphasize the role of mitosis in the transfection of cells in growth phase culture, and explain the poor correlation between cell culture systems and transfection in vivo. One potential way to improve the correlation is to use a fully differentiated, non-dividing monolayer as the cell culture model. The Caco-2 lterculture model is a promising option in this context [85,86]. Poor transfection in vivo also indicates the importance of improving nuclear uptake in non-dividing cells.
ment of local disorders, e.g. inammatory bowel syndrome or ulcers, where resection is not a preferred option. The approach may also contribute towards the objective of restoring gene function in the appropriate defective tissue, rather than inducing expression in surrogate tissues for gene products that are secreted and act systemically. However, every organ and tissue in the body is composed of multiple different cells types, and simple direct injection of replacement genes will not mediate transfection selectively in the target population unless additional targeting strategies are used. In the short term, this will probably rely on the regulation of gene expression by tissue-associated promoters, although in the longer term, the use of targeted delivery systems will further increase specicity. Ultimately, a combination of intracellular and extracellular targeting mechanisms may be combined with direct injection techniques for exquisite selectivity of action.
3. Cellular interactions
3.1. Major barriers

The arrival of a DNA plasmid in the nucleus by a passive process is a rare event, due to the barrier effect of lipid membranes, and diffusional barriers within the cell. Eukaryotic cells rely on membrane compartmentalization of macromolecules for many functions, and intercompartmental transfer of a DNA plasmid, in a controlled manner, will be a formidable challenge to delivery scientists. An effective gene delivery system will need to bind to an appropriate cell, be internalised by endocytosis, escape the degradative pathway and ultimately allow delivery of the expression plasmid to the nucleus. Current gene delivery systems have some of these requirements, but often make use of non-specic mechanisms. To improve the overall efciency of non-viral gene delivery, it may be necessary to assemble particles with the precision that is achieved by viruses, and this aim will take some time to come to fruition.
2.8. Feasibility of local injection to other sites

Advances in surgical technology, particularly endoscopic and keyhole techniques, now affords the possibility of local administration of genes to virtually any organ or tissue in the body, provided that the benets can be shown to be likely to justify the procedure. Hence, for well-dened target sites that defy access via systemic routes, direct injection is now a real possibility. This may afford signicant advance in gene therapeutic strategies for the treat-
3.2. Viral models

Many viruses have evolved mechanisms to overcome the intracellular barriers of eukaryotic cells. Cellular entry of enveloped viruses is reviewed in an
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accompanying article in this volume [87]. Progress has been made in the design and assembly of viruslike particles, particularly in relation to escape from the degradative pathway. However, another quantum leap will be required to make a signicant impact on delivery to the nucleus. Research on viral tropism suggests that some viruses have evolved means of interaction with nuclear transport systems [8891]. There is a good chance that, with a focused effort, particles can be assembled synthetically to allow active nuclear transport to be utilized. This approach could be critical for the success of non-viral gene delivery and is discussed in another accompanying article [92].
3.3. Binding and internalisation 3.3.1. Molecular complexes The most primitive molecular complexes consist of simple interpolyelectrolyte conjugates containing DNA and a cationic macromolecule, often a polymer. When applied to cells, the complexes are thought to mediate transfection via a multistage process that includes either hydrophobic or cationic binding to the cell membrane and subsequent entry into the cytoplasm. With simple poly( L)lysine / DNA complexes, it is noticeable that transfection activity parallels the membrane toxicity of the parent poly( L)lysine, with complexes based on low molecular weight pLL showing no toxicity and no spontaneous transfection activity [93]. A general association between cytotoxicity and transfection suggests that a degree of membrane-damage must be mediated for the DNA to gain access to the cytoplasm. Successful transfection relies on achieving the correct balance between gaining adequate access of DNA into the cytoplasm and causing excessive and lethal damage to the cell. The ability of chloroquine to promote transfection activity of pLL / DNA complexes against a variety of target cells suggests that a vesicular stage with acidication is probably involved. The mechanism of chloroquine action is not understood (see below), but most of the proposed mechanisms of action depend on low pH. In addition, the relatively high transfection activity of molecular complexes based on cationic polymers with pKa values in the range 5.57.0 [e.g. poly(ethyleneimine) or starburst dendrimers] also suggests that a low pH vesicular stage may be
involved. How access is gained to the vesicle remains unclear, although simple binding of such large polyelectrolyte complexes to the surfaces of many cells may promote a form of adsorptive endocytic internalisation. More sophisticated molecular complexes are designed to usurp physiological (or pathological) mechanisms of endocytosis, to increase the efciency of cell transfection. The simplest form of receptormediated transfection makes use of the binding of aV- and a5 integrins to the tripeptide ArgGlyAsp (RGD). Incorporation of the RGD sequence, in an appropriate conguration, into pLL / DNA complexes causes increased transfection of cells in vitro, probably by promoting internalisation following integrin binding, in the same pathway as that followed by adenoviruses [94]. Incorporation of transferrin into the structure of the polyelectrolyte complex can signicantly increase transfection of transferrin receptor-positive cells in vitro. Although the mechanism is not clear, it is assumed that endocytosis following receptor binding leads to routing of the pLL / DNA / transferring complex into the endosomal compartment. Whatever the mechanism, complexes targeted in this way show good transfecting activity against receptor-positive cells in vitro [95]. Attempts to exploit other pathways of receptor-mediated endocytosis (e.g. FGF receptor, VEGF receptor) for targeted transfection are now receiving considerable attention.
3.3.2. Liposome complexes The formation of complexes between DNA and cationic liposomes leads to molecular reorganization yielding a poorly dened complex based on the polyelectrolyte interaction, with the hydrophobic lipid groups oriented outwards into the solvent. This complex is therefore very hydrophobic, poorly soluble in water and is able to interact effectively with cell membranes. In general, lipoplexes appear to be taken up into membrane vesicles, but it is anticipated that the presence of membrane-active oleyl groups in the lipid component endow the complex with a fusogenic, or membrane-disrupting activity, which is important for transfection [96,97]. Although efciency and specicity can be further increased by incorporation of ligands that recognize cell surface receptors, the key requirements of lipoplexes appear to be: (a) their net cationic charge and (b) the
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presence of bilayers of membrane lipids above their melting transition. Hagstrom et al. [98] and Fritz et al. [99] have demonstrated that the lipid itself does not need to be cationic, provided that a cationic component is present within the complex [98,99]. Chloroquine does not usually have a benecial effect on the performance of lipoplexes [85]. This indicates that escape from the endosome is either achieved with adequate efciency or is not necessary, perhaps due to uptake of lipoplexes into a population of vesicles that are independent of the endosomal compartment.
3.4. Intracellular trafcking 3.4.1. Escape from the endosome Complexes routed via an acidied compartment, loosely assumed to be endosomal, frequently exploit the falling pH to trigger membrane activity and permit their entry into the cytoplasm. Viruses, notably the inuenza virus, make efcient use of the falling pH to induce a conformational change in proteins to yield an amphipathic a-helix structure that is capable of mediating membrane fusion [87]. Plank et al. [100] showed that fragments of the virus haemagglutinin could mediate pH-dependent lysis of liposomes and erythrocytes, but also increased the frequency of transfection when incorporated into pLL / DNA complexes, provided that a sufciently high cationic charge ratio was employed. They also showed that the introduction of more glutamic acid residues into the peptide structure could further promote transfection. Although the accepted wisdom is that these pH-responsive amphipathic helices act via relatively subtle membrane disruption, there are several other possible explanations. One well-dened suggestion is popularly referred to as the proton sponge hypothesis. While this hypothesis has been built essentially around the remarkable transfecting activities of cationic polymers with pKa values in the region 5.57.0 [e.g. poly(ethyleneimine) (PEI), or starburst dendrimers], the same principle could apply to anions (such as glutamic acid in the pH-responsive amphipathic helices, described above), with pKa values in the same region. The hypothesis suggests that high concentrations of protonatable groups within the endosome will effectively buffer the falling pH, increasing the length of time before the DNA reaches the lysosome and increasing the likeli-
hood of its spontaneous translocation into the cytoplasm. The proton sponge hypothesis [101] is based essentially on the perceived mechanism of action of the weak base chloroquine, which is known to increase the transfecting activity of polyelectrolyte complexes against many cell lines. Chloroquine is a weak base, non-charged at neutral pH but charged at pH 5.5. It is able to pass easily through membranes in its uncharged form, becoming protonated and accumulating within acidic vacuoles in its positively charged, membrane-impermeable, form. Again, the accepted wisdom is that preventing endosome acidication increases the residence time of DNA within the endosomes and increases the chances of transfer to the cytoplasm. It has also been suggested that the increasing osmotic concentration within the endosome may cause swelling and eventually lysis, releasing the entrapped DNA into the cytoplasm; however, there is currently no evidence to support this idea. Contradictory evidence comes from the observation that other lysosomotropic weak bases, such as ammonium sulphate, which are similarly capable of buffering the endosomal pH, do not promote improved transfection. This latter observation tends to undermine a central tennet of the proton sponge hypothesis, although precise values of the pKa s of the bases may be important. Nevertheless, an alternative suggestion, based on the observation that high concentrations of chloroquine can promote disassembly of polyelectrolyte DNA complexes, seems increasingly plausible [102]. If transfection enhancement by chloroquine is not primarily due to its endosomal buffering capacity, the proposed mechanism of action of the pH-responsive cationic polymers (such as PEI) must be brought into question and more sophisticated mechanisms sought. While the authors are not aware of any further insights into this question, it is interesting that chloroquine can enhance the transfecting activities of pLL / DNA complexes against many cell types, but has no benecial effects on the activities of PEI / DNA complexes.
3.4.2. Involvement of the cytoskeleton in translocation Much research activity has focused on mechanisms by which complexes may escape the endosome, the implication being that delivery to the
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cytoplasm would enable the particle to approach the nuclear envelope. However, the cytoplasm is a viscous uid and diffusion of macromolecular or particulate systems within this medium may be extremely slow [103]. This factor was discussed in detail by Meyer et al. [104]. Escape of the endosome may leave a condensed particle of DNA stranded at a site some distance from the nuclear envelope. The transport of colloidal systems from one site to another is a vital cellular process, but it is not a passive process. Vesicles, organelles and other colloidal structures are transported actively using molecular motors associated with the microtubule network or actin microlaments [105108]. Movement along microtubules is mediated by two classes of motor proteins in an oriented manner. Kinesins direct movement in an outward direction, usually linked to the secretory pathway. Dyneins move vesicles in the inward direction, normally associated with the endocytic pathway. Myosins are motors that can direct movement of vesicles using actin as a substrate. Accessory molecules are required; kinesin-associated proteins (for kinesin motors) and dynactin (for dynein motors). This is a complex system of active transport that is partially understood at the molecular level, with new insights appearing regularly. The signicance of these processes for gene delivery is clear. It will probably be necessary for a condensed DNA particle to utilize active cytoplasmic transport systems to approach the vicinity of the nucleus. In general terms, the dynein system directs vesicles from the endosome towards the perinuclear region. If the gene delivery system escapes the endosome too early, it may never reach the deeper regions of the cell. It may be necessary to ensure that endosomal escape is delayed until the nucleus is approached. Alternately, an appropriate ligand for binding dynein could be incorporated into a particle. It seems likely that cytoplasmic transport is a limitation in non-viral gene delivery, but these issues have not been resolved adequately as yet.
Experimental data to hand suggest that this is probably not the case, since microinjection of pLL / DNA complexes directly into the cytoplasm of Xenopus oocytes in the absence of chloroquine results in signicantly higher levels of gene expression than equivalent injections of free DNA [109]. Direct intranuclear injection of pLL / DNA complexes, on the other hand, results in levels of expression comparable to those of free DNA. These results suggest that the cationic component of pLL / DNA complexes may exert some nuclear-localizing effects, not an unexpected result in light of the cationic nature of the many nuclear-homing sequences already identied. In addition, however, it is clear that, following arrival within the nucleus, the enzymes associated with transcription are readily able to release the DNA from the cationic polymer to act as a template for RNA polymerization. Hence pLL / DNA complexes reaching the cytoplasm are more efcient at transfection than free DNA, and the mechanism of action of chloroquine proposed by Erbacher et al. [102] seems unlikely. In contrast, it is clear that complexes between cationic lipids and DNA do require uncoupling prior to delivery into the nucleus. The direct injection of cationic lipid / DNA complexes into the cytoplasm or even into the nucleus of Xenopus oocytes [96] resulted in negligible levels of gene expression, implying that the normal endosomal route of entry into the cell includes an uncoupling stage where the DNA is released from the cationic lipid. Although still unproven, it is generally assumed that this occurs at lipid membranes, probably at the endosome membrane. Clearly, the possibility of using cationic lipids to promote cell entry, with uncoupling of lipid, could be combined with the presence of residual cationic polymer. This may enhance nuclear accumulation and produce a more efcient delivery system. The recent description of systems prepared by partial condensation of DNA with pLL prior to the addition of cationic lipid [110] might well full this role.
3.5. Uncoupling of complexes

It has been suggested that part of the mechanism of action of chloroquine may be to promote the uncoupling of DNA and pLL within the endosome [102], resulting in improved translocation of DNA to the nucleus, or improved rates of transcription.
3.6. Nuclear uptake

The nuclear membrane is a barrier that prevents the uptake of most macromolecules greater than 70 kDa into the nucleus, unless they are able to interact with the nuclear pore active transport system [111 113]. It is clear that poor access of DNA plasmids to
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the nucleus represents a major barrier to the success of non-viral gene therapy [96,114]. The mechanisms of nuclear import are discussed in another article in this volume [92].
is scope for improvement in the efciency of the technique.
4.3. Sustained delivery

Given the popularity of sustained release technology, and the general nding that gene expression in vivo is short-lived in most tissues, it is rather surprising that sustained release DNA formulations have only been considered in recent years. This is set to change as various groups have begun to explore the use of polyesters for sustained release of plasmids [123,124]. This is a good opportunity for drug delivery scientists to apply familiar technology to the problem of gene delivery and, in the case of intratumoural or intramuscular applications, there could be signicant benet from sustained release.
4. Delivery devices
4.1. Inhalation systems

Formulations of lipoplexes have been successfully developed for aerosol formation by nebulization [115,116]. It is necessary to select an appropriate nebulizer, which does not create too much force of shear, and also to screen formulations for stability under conditions of shear. High concentrations of DNA in the formulation were achieved, in the studies of Eastman et al. [116], by incorporation of some polyethylene glycolated lipids. It seems likely that existing nebulization systems will allow clinical evaluation of gene delivery to the lung, but improvements in gene delivery to the lung will be desirable. The design of devices for drug delivery to the lung is progressing signicantly at present, due to the desire to phase-out the use of chlorouorocarbon and hydrouorocarbon propellants, and the need to improve delivery of biopharmaceuticals to the lung. Several dry-powder inhalation devices are in development. Others are developing unit-dose aqueous aerosol devices that would allow the formulation of gene delivery systems as colloidal suspensions [117].
5. Concluding remarks The development of successful gene delivery systems will depend on the selection of realistic approaches, and much depends on the proportion of cells that need to be transfected for each clinical indication. This is illustrated clearly by the variety of approaches to cancer gene therapy. Immunotherapeutic approaches are attractive because they do not depend on transfection of all tumour cells. At the other extreme, an ablative approach based on expression of a glycoprotein toxin, or a growth inhibitory approach induced by expression of a tumour-suppressor, are likely to require transfection of all dividing cells. This level of efciency cannot be achieved at present with non-viral systems and will rely on major improvements in the design of viruslike particles. However, it is conceivable that an ablative strategy may still be valuable as part of a combination therapy, even with existing delivery systems. The proportion of cells that are required to be transfected to achieve clinical efcacy is a key issue in the gene therapy of cystic brosis, which aims to replace the defective CFTR protein in airway epithelia. Current estimates suggest that approximately 10% of epithelia in affected areas need to express the gene product. This is proving difcult to achieve with present technology. In vivo experiments in non-viral gene delivery have emphasized how
4.2. Particle bombardment

Particle-mediated gene delivery was introduced in the late 1980s [118] and has been developed for in vivo transfection experiments in mammalian species [119,120]. DNA is coated onto the surface of gold microparticles, which are then red at the surface of a tissue using high gaseous pressure. This approach is particularly promising for genetic vaccine development using the direct introduction of DNA to antigen-presenting cells, such as the Langerhans cells in the skin [121,122]. The technology will need to be developed for widespread use in humans, but the general principle of the technique of particle bombardment may be popular for vaccination, and there
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difcult the transfection of differentiated, non-dividing cells is. It is likely that efforts to control the assembly of gene delivery systems with higher precision, and higher complexity, will be rewarded with success. This represents a great opportunity for drug delivery scientists.
[14]
[15]
References
[16] [1] A. Rolland, P. Felgner (Eds.), Non-viral gene delivery systems, Adv. Drug Deliv. Rev. 30 (1998). [2] A.V. Kabanov, L.W. Seymour, P. Felgner (Eds.), Self-assembling Complexes for Gene Delivery: From Laboratory to Clinic, John Wiley, Chichester, UK, 1998, p. 442. [3] A. Rolland (Ed.), Advanced Gene Delivery: From Concepts to Pharmaceutical Products, Harwood Academic, New York, in press. [4] Y. Takakura, M. Hashida, Macromolecular carrier systems for targeted drug delivery: pharmacokinetic considerations on biodistribution, Pharm. Res. 13 (1996) 820831. [5] Y. Takakura, R.I. Mahato, M. Nishikawa, M. Hashida, Control of pharmacokinetic proles of drugmacromolecule conjugates, Adv. Drug Deliv. Rev. 19 (1996) 377 399. [6] K. Kawabata, Y. Takakura, M. Hashida, The fate of plasmid DNA after intravenous injection in mice: involvement of scavenger receptors in its hepatic uptake, Pharm. Res. 12 (1995) 825830. [7] R.I. Mahato, K. Kawabata, T. Nomura, Y. Takakura, M. Hashida, Physicochemical and pharmacokinetic characteristics of plasmid DNA / cationic liposome complexes, J. Pharm. Sci. 84 (1995) 12671271. [8] M. Yoshida, R.I. Mahato, K. Kawabata, Y. Taakakura, M. Hashida, Disposition characteristics of plasmid DNA in the single-pass rat liver perfusion, Pharm. Res. 13 (1996) 597 603. [9] R.I. Mahato, Y. Takakura, M. Hashida, Nonviral vectors for in vivo gene delivery: physicochemical and pharmacokinetic considerations, Crit. Rev. Ther. Drug Carrier Syst. 14 (1997) 133172. [10] M. Hashida, R.I. Mahato, K. Kawabata, T. Miyao, M. Nishikawa, Y. Takakura, Pharmacokinetics and targeted delivery of proteins and genes, J. Control. Release 41 (1996) 9197. [11] Seymour et al., unpublished. [12] T.H. Welling, B.L. Davidson, J.A. Zelenock, J.C. Stanley, D. Gordon, B.J. Roessler, L.M. Messina, Systemic delivery of the interleukin-1 receptor antagonist protein using a new strategy of direct adenoviral-mediated gene transfer to skeletal muscle capillary endothelium in the isolated rat hindlimb, Hum. Gene Ther. 7 (1996) 17951802. [13] D.J. Stephan, Z.Y. Yang, H. San, R.D. Simari, C.J. Wheeler, P.L. Felgner, D. Gordon, G.J. Nabel, E.G. Nabel, A new cationic liposome DNA complex enhances the efciency of
[17]
[18]
[19]
[20]
[21]
[22] [23]
[24] [25]
[26]
[27]
[28]
arterial gene transfer in vivo, Hum. Gene Ther. 7 (1996) 18031812. J. Stankovics, A.M. Crane, E. Andrews, C.H. Wu, G.Y. Wu, F.F. Ledley, Over-expression of human methylmalonyl CoA mutase in mice after in vivo gene transfer with asialoglycoprotein / polylysine / DNA complexes, Hum. Gene Ther. 5 (1994) 10951104. G.Y. Wu, J.M. Wilson, F. Shalaby, M. Grosman, D.A. Shafritz, C.H. Wu, Receptor-mediated gene delivery in vivo. Partial correction of genetic analbumenia in Nagase rats, J. Biol. Chem. 266 (1991) 1433814342. C.P. Lollo, Formulation improvements for nonviral gene delivery, Proceedings of Cambridge Healthtech Institutes 3rd Annual Meeting on Articial Self-assembling Systems for Gene Delivery, Coronado, CA, November 1718, 1996. M. Monsigny, A.-C. Roche, P. Midoux, R. Mayer, Glycoconjugates as carriers for specic delivery of therapeutic drugs and genes, Adv. Drug Deliv. Rev. 14 (1994) 124. P.E. Thorpe, F.J. Burrows, Antibody-directed targeting of the vasculature of solid tumours, Breast Cancer Res. Treat. 36 (1995) 237251. H.M. Patel, Serum opsonins and liposomes: their interaction and opsonophagocytosis, Crit. Rev. Ther. Drug Carrier Syst. 9 (1992) 3990. C. Plank, K. Mechtler, F.C. Szoka, E. Wagner, Activation of the complement system by synthetic DNA complexes: a potential barrier for intravenous gene delivery, Hum. Gene Ther. 7 (1996) 14371446. L.W. Seymour, Passive tumour-targeting of soluble macromolecules and drug conjugates CRC, Crit. Rev. Ther. Drug Carrier Syst. 9 (1992) 135187. D.F. Baban, L.W. Seymour, Control of tumour vascular permeability, Adv. Drug Deliv. Rev. 34 (1998) 109119. G.Y. Wu, C.H. Wu, Receptor-mediated gene delivery and expression in vivo, J. Biol. Chem. 263 (1988) 14621 14624. G.Y. Wu, C.H. Wu, Liver-directed gene delivery, Adv. Drug Deliv. Rev. 12 (1993) 159167. C.H. Wu, J.M. Wilson, G.Y. Wu, Targeting genes: delivery and persistent expression of a foreign gene driven by mammalian regulatory elements in vivo, J. Biol. Chem. 264 (1989) 1698516987. G.Y. Wu, J.M. Wilson, F. Shalaby, M. Grossman, D.A. Shafritz, C.H. Wu, Receptor-mediated gene delivery in vivo. Partial correction of genetic analbuminemia in Nagase rats, J. Biol. Chem. 266 (1991) 1433814342. J.C. Perales, T. Ferkol, H. Beegen, O.D. Ratnoff, R.W. Hanson, Gene transfer in vivo: sustained expression and regulation of genes introduced into the liver receptortargeted uptake, Proc. Natl. Acad. Sci. USA 91 (1994) 40864090. G. Zhang, D. Vargo, V. Budker, N. Armstrong, S. Knechtle, J.A. Wolff, Expression of naked plasmid DNA injected into the afferent and efferent vessels of rodent and dog livers, Hum. Gene Ther. 8 (1997) 17631772.
200
C.W. Pouton, L.W. Seymour / Advanced Drug Delivery Reviews 46 (2001) 187 203 [44] H.L. Davis, R.G. Whalen, B.A. Demeneix, Direct gene transfer into skeletal muscle in vivo: Factors affecting efciency of transfer and stability of expression, Hum. Gene Ther. 4 (1993) 151159. [45] M.Y. Levy, K.B. Meyer, L. Barron, F.C. Szoka Jr., Mechanism of gene uptake and expression in adult mouse skeletal muscle, Pharm. Res. 11 (1994) 317. [46] M.Y. Levy, L.G. Barron, K.B. Meyer, F.C. Szoka Jr., Characterization of plasmid DNA transfer into mouse skeletal muscle: evaluation of uptake mechanism, expression and secretion of gene products into blood, Gene Ther. 3 (1996) 201211. [47] R.J. Mumper, J.G. Duguid, K. Anwer, M.K. Barron, H. Nitta, A.P. Rolland, Polyvinyl derivatives as novel interactive polymers for controlled gene delivery to muscle, Pharm. Res. 13 (1996) 701709. [48] J.-P. Yang, L. Huang, Direct gene transfer to mouse melanoma by intratumour injection of free DNA, Gene Ther. 3 (1996) 542548. [49] A.W. Segal, G. Gregoriadis, C.D.V. Black, Liposomes as vehicles for local release of drugs, Clin. Sci. Mol. Med. 49 (1975) 99104. [50] L.W. Seymour, R. Duncan, J. Strohalm, J. Kopecek, Effect of molecular weight (Mw) of N-(2-hydroxypropyl)methacrylamide copolymers on body distribution and rate of excretion after subcutaneous, intraperitoneal and intravenous administration to rats, J. Biomed. Mater. Res. 21 (1987) 13411358. [51] P. Goddard, I. Williamson, J. Brown, L.E. Hutchinson, J. Nicholls, K. Petrak, Soluble polymeric carriers for drug delivery. 4. Tissue autoradiography and whole body tissue distribution in mice of N-(2-hydroxypropyl)methacrylamide copolymers following intravenous administration, J. Bioact. Compat. Polym. 6 (1991) 424. [52] A. Gabizon, R. Isacson, E. Libson, B. Kaufman, B. Uziely, R. Catane, C.G. Bendor, E. Rabello, Y. Cass, T. Peretz, A. Sulkes, R. Chisin, Y. Barenholz, Clinical studies of liposome-encapsulated doxorubicin, Acta Oncol. 33 (1994) 779786. [53] D.T. Curiel, J.M. Pilewski, S.M. Albelda, Gene therapy approaches for inherited and acquired lung diseases, Am. J. Respir. Cell Mol. Biol. 14 (1996) 118. [54] P. Demoly, M. Mathieu, D.T. Curiel, Ph. Godard, J. Bousqquet, F.B. Michel, Gene therapy strategies for asthma, Gene Ther. 4 (1997) 507516. [55] E.W.F.W. Alton, D.M. Geddes, Gene therapy for cystic brosis: a clinical perspective, Gene Ther. 2 (1995) 8895. [56] K.L. Brigham (Ed.), Gene Therapy for Diseases of the Lung (Lung Biology in Health and Disease, Vol. 104). Marcel Dekker, New York, 1997, pp. 1408. [57] N.J. Caplen, X. Gao, P. Hayes, R. Elaswarapu, G. Fisher, E. Kinrade, A. Chakera, J. Schorr, B. Hughes, J.R. Dorin, D.J. Porteous, E.W.F.W. Alton, D.M. Geddes, C. Coutelle, R. Williamson, L. Huang, C. Gilchrist, Gene therapy for cystic brosis in humans by liposome-mediated DNA transfer: the production of resources and the regulatory process, Gene Ther. 1 (1994) 139147.
[29] J.W. McLean, E.A. Fox, P. Baluk, P.B. Bolton, A. Haskell, R. Pearlman, G. Thurston, E.Y. Umemoto, D.M. McDonald, Organ-specic endothelial cell uptake of cationic liposome DNA complexes in mice, Am. J. Physiol. 273 (Heart Circ. Physiol. 42) (1997) H387H404. [30] F. Liu, H. Qi, L. Huang, D. Liu, Factors controlling the efciency of cationic lipid-mediated transfection in vivo via intravenous administration, Gene Ther. 4 (1997) 517523. [31] G. Osaka, K. Carey, A. Cuthbertson, P. Godowski, T. Patapoff, A. Ryan, T. Gadek, J. Mordenti, Pharmacokinetics, tissue distribution, and expression efciency of plasmid [ 33 P]DNA following intravenous administration of DNA / cationic lipid complexes in mice: use of a novel radionuclide approach, J. Pharm. Sci. 85 (1996) 612618. [32] A.R. Thierry, P. Rabinovich, B. Peng, L.C. Mahan, J.L. Bryant, R.C. Gallo, Characterization of liposome-mediated gene delivery: expression stability and pharmacokinetics of plasmid DNA, Gene Ther. 4 (1997) 226237. [33] J.E. Schnitzer, Update on the cellular and molecular basis of capillary permeability, Cardiovasc. Med. 3 (1993) 124 130. [34] J.E. Schnitzer, J. Bravo, High afnity binding, endocytosis, and degradation of conformationally modied albumins, J. Biol. Chem. 268 (1993) 75627570. [35] J.E. Schnitzer, P. Oh, Albondin-mediated capillary permeability to albumin, J. Biol. Chem. 269 (1994) 6072 6082. [36] E.G. Nabel, Gene therapy for cardiovascular disease, Circulation 91 (1995) 541548. [37] M.-C. Keogh, D. Chen, F. Lupu, N. Shaper, J.F. Schmitt, V.V. Kakkar, N.R. Lemoine, High efciency reporter gene transfection of vascular tissue in vitro and in vivo using a cationic lipidDNA complex, Gene Ther. 4 (1997) 162 171. [38] J.A. Wolff, R.W. Malone, P. Williams, W. Chong, G. Acsadi, A. Jani, P.L. Felgner, Direct gene transfer into mouse muscle in vivo, Science 247 (1990) 14651468. [39] J.A. Wolff, J.J. Ludtke, G. Acsadi, P. Williams, A. Jani, Long-term persistence of plasmid DNA and foreign gene expression in mouse muscle, Hum. Mol. Genet. 1 (1992) 363369. [40] J.A. Wolff, M.E. Dowty, S. Jiao, G. Repetto, R.K. Berg, J.J. Ludtke, P. Williams, Expression of naked plasmids by cultured myotubes and entry of plasmids into T tubules and caveolae of mammalian skeletal muscle, J. Cell Sci. 103 (1992) 12491269. [41] S. Jiao, P. Williams, R.K. Berg, B.A. Hodgeman, L. Liu, G. Repetto, J.A. Wolff, Direct gene transfer into nonhuman primate myobers in vivo, Hum. Gene Ther. 3 (1992) 2133. [42] M.E. Dowty, P. Williams, G. Zhang, J.E. Hagstrom, J.A. Wolff, Plasmid DNA entry into postmitotic nuclei of primary rat myotubes, Proc. Natl. Acad. Sci. USA 92 (1995) 45724576. [43] H.L. Davis, B.A. Demeneix, B. Quantin, J. Coulombe, R.G. Whalen, Plasmid DNA is superior to viral vectors for direct gene transfer into adult mouse skeletal muscle, Hum. Gene Ther. 4 (1993) 733740.
C.W. Pouton, L.W. Seymour / Advanced Drug Delivery Reviews 46 (2001) 187 203 [58] N.J. Caplen, E.W. Alton, P.G. Middleton, J.R. Dorin, B.J. Stevenson, X. Gao, S.R. Durham, P.K. Jeffery, M.E. Hodson, C. Coutelle, L. Huang, D.J. Porteous, R. Williamson, D.M. Geddes, Liposome mediated CFTR gene transfer to the nasal epithelium of patients with cystic brosis, Nat. Med. 1 (1995) 3946. [59] J. Zabner, L.A. Couture, R.J. Gregory, S.M. Graaham, A.E. Smith, M.J. Welsh, Adenovirus-mediated gene transfer transiently corrects the chloride transport defect in nasal epithelia of patients with cystic brosis, Cell 75 (1993) 207216. [60] J. Zabner, B.W. Ramsey, D.P. Meeker, M.L. Aitken, R.P. Balfour, R.L. Gibson, J. Launspach, R.A. Moscicki, S.M. Richards, T.A. Standaert, J. Williams-Warren, S.C. Wadsworth, A.E. Smith, M.J. Welsh, Repeat administration of an adenovirus vector encoding cystic brosis transmembrane conductance regulator to the nasal epithelium of patients with cystic brosis, J. Clin. Invest. 97 (1996) 15041511. [61] R.G. Crystal, N.G. McElvaney, M.A. Rosenfeld, C.S. Chu, A. Mastrangeli, J.G. Hay, S.L. Brody, H.A. Jaffee, N.T. Eissa, C. Danel, Administration of an adenovirus containing the human CFTR cDNA to the respiratory tract of individuals with cystic brosis, Nat. Genet. 8 (1994) 4251. [62] D.J. Porteous, J.R. Dorin, G. McLaughlan, H. DavidsonSmith, H. Davidson, B.J. Stevenson, A.D. Carothers, W.A.H. Wallace, S. Moralee, C. Hoenes, G. Kallmeyer, U. Michaelis, K. Naujoks, L.-P. Ho, J.M. Samways, M. Imrie, A.P. Greening, J.A. Innes, Evidence for safety and efcacy of DOTAP cationic liposome mediated CFTR gene transfer to the nasal epithelium of patients with cystic brosis, Gene Ther. 4 (1997) 210218. [63] D.R. Gill, K.W. Southern, K.A. Mofford, T. Seddon, L. Huang, F. Sorgi, A. Thomson, L.J. MacVinish, R. Ratcliff, D. Bilton, D.J. Lane, J.M. Littlewood, A.K. Webb, P.G. Middleton, W.H. Colledge, A.W. Cuthbert, M.J. Evans, C.F. Higgins, S.C. Hyde, A placebo-controlled study of liposome-mediated gene transfer to the nasal epithelium of patients with cystic brosis, Gene Ther. 4 (1997) 199209. [64] E.W. Alton, P.G. Middleton, N.J. Caplen, S.N. Smith, D.M. Steel, F.M. Munkonge, P.K. Jeffery, D.M. Geddes, S.L. Hart, R. Williamson, K.I. Fasold, A.D. Miller, P. Dickinson, B.J. Stevenson, G. MacLachlan, J.R. Dorin, D.J. Porteous, Non-invasive liposome-mediated gene delivery can correct the ion transport defect in cystic brosis mutant mice, Nat. Genet. 5 (1993) 135142. [65] J.R. Dorin, R. Farley, S. Webb, S.N. Smith, E. Farini, S.J. Delaney, B.J. Wainwright, E.W.F.W. Alton, D.J. Porteous, A demonstration using mouse models that successful gene therapy for cystic brosis requires only partial gene correction, Gene Ther. 3 (1996) 797801. [66] K.L. Brigham, B. Meyrick, B. Christman, M. Magnuson, G. King, L.C. Berry Jr., In vivo transfection of murine lungs with a functioning prokaryotic gene using a liposome vehicle, Am. J. Med. Sci. 298 (1989) 278281. [67] K.L. Brigham, B. Meyrick, B. Christman, J.T. Conary, G. King, L.C. Berry Jr., M.A. Magnuson, Expression of human growth hormone fusion genes in cultured lung endothelial
201
[68]
[69]
[70]
[71]
[72]
[73]
[74]
[75]
[76]
[77]
[78]
[79]
[80]
cells and in the lungs of mice, Am. J. Respir. Cell Mol. Biol. 8 (1993) 209213. T.A. Hazinski, P.A. Ladd, C.A. Dematteo, Localization and induced expression of fusion genes in the rat lung, Am. J. Respir. Cell Mol. Biol. 4 (1991) 206209. K. Yoshimura, M.A. Rosenfeld, H. Nakamura, E.M. Scherer, A. Pavirani, J.P. Lecocq, R.G. Crystal, Expression of the human cystic brosis transmembrane conductance gene in the mouse lung after in vivo intratracheal plasmidmediated gene transfer, Nucleic Acids Res. 20 (1992) 32333240. J.J. Logan, Z. Bebok, L.C. Walker, S. Peng, P.L. Felgner, G.P. Siegal, R.A. Frizzell, J. Dong, M. Howard, S. Matalon, J.R. Lindsey, M. DuVall, E.J. Sorscher, Cationic lipids for reporter gene and CFTR transfer to rat pulmonary epithelium, Gene Ther. 2 (1995) 3849. R. Stribling, E. Brunette, D. Liggit, K. Gaensler, R. Debs, Aerosol gene delivery in vivo, Proc. Natl. Acad. Sci. USA 89 (1992) 1127711281. A.E. Canonico, J.T. Conary, B.O. Meyrick, K.L. Brigham, Aerosol and intravenous transfection of human alpha 1antitrypsin gene to lungs of rabbits, Am. J. Respir. Cell Mol. Biol. 10 (1994) 2429. G. McLachlan, H. Davidson, D. Davison, P. Dickinson, J. Dorin, D. Porteous, DOTAP as a vehicle for efcient gene delivery in vitro and in vivo, Biochemistry 11 (1994) 1921. A.J. Fasbender, J. Zabner, M.J. Welsh, Optimization of cationic lipid-mediated gene transfer to airway epithelia, Am. J. Physiol. 269 (Lung Cell. Mol. Physiol. 13) (1995) L45L51. E.R. Lee, J. Marshall, C.S. Siegel, C. Jiang, N.S. Yew, M.R. Nichols, J.B. Nietupski, R.J. Ziegler, M.B. Lane, K.X. Wang, N.C. Wan, R.K. Scheule, D.J. Harris, A.E. Smith, S.H. Cheng, Detailed analysis of structures and formulations of cationic lipids for efcient gene transfer to the lung, Hum. Gene Ther. 7 (1996) 17011717. R.K. Scheule, J.A. St. George, R.G. Bagley, J. Marshall, J.M. Kaplan, G.Y. Akita, K.X. Wang, E.R. Lee, D.J. Harris, C. Jiang, N.S. Yew, A.E. Smith, S.H. Cheng, Basis of pulmonary toxicity associated with cationic lipid-mediated gene transfer to the mammalian lung, Hum. Gene Ther. 8 (1997) 689707. T. Sawa, H. Miyazaki, J.-F. Pittet, J.H. Widdicombe, M.A. Gropper, S. Hasshimoto, D.J. Conrad, H.G. Folkesson, R. Debs, J.R. Forsaayeth, B. Foxx, J.P. Wiener-Kronish, Intraluminal water increases expression of plasmid DNA in rat lung, Hum. Gene Ther. 7 (1996) 933941. D.J. Freeman, R.W. Niven, The inuence of sodium glycocholate and other additives on the in vivo transfection of plasmid DNA in the lungs, Pharm. Res. 13 (1996) 202209. A.H. Jobe, T. Ueda, J.A. Whitsett, B.C. Trapnell, M. Ikegami, Surfactant enhances adenovirus-mediated gene expression in rabbit lungs, Gene Ther. 3 (1996) 775779. M.-F. Tsan, G.L. Tsan, J.E. White, Surfactant inhibits cationic liposome-mediated gene transfer, Hum. Gene Ther. 8 (1997) 817825.
202
C.W. Pouton, L.W. Seymour / Advanced Drug Delivery Reviews 46 (2001) 187 203 M.J. Welsh, Cellular and molecular barriers to gene transfer by a cationic lipid, J. Biol. Chem. 270 (1995) 18997 19007. Y. Xu, F.C. Szoka Jr., Mechanism of DNA release from cationic liposome / DNA complexes used in cell transfection, Biochemistry 35 (1996) 56165623. J.E. Hagstrom, M.G. Sebestyen, V. Budker, J.J. Ludtke, J.D. Fritz, J.A. Wolff, Complexes of non-cationic liposomes and histone H1 mediate efcient transfection of DNA without encapsulation, Biochim. Biophys. Acta 1284 (1996) 4755. J.D. Fritz, H. Herweijer, G. Zhang, J.A. Wolff, Gene transfer into mammalian cells using histone-condensed plasmid DNA, Hum. Gene Ther. 7 (1996) 13951404. C. Plank, B. Oberhauser, K. Mechtler, C. Koch, E. Wagner, The inuence of endosome-disruptive peptides on gene transfer using synthetic virus-like gene-transfer systems, J. Biol. Chem. 269 (1994) 1291812924. J.P. Behr, The proton sponge, a means to enter cells viruses never thought of, Med. Sci. 12 (1996) 5658. P. Erbacher, A.C. Roche, M. Monsigny, P. Midoux, Putative role of chloroquine in gene transfer into a human hepatoma cell line by DNA lactosylated polylysine complexes, Exp. Cell Res. 225 (1996) 186194. K. Luby-Phelps, P.E. Castle, D.L. Taylor, F. Lanni, Hindered diffusion of inert tracer particles in the cytoplasm of mouse 3T3 cells, Proc. Natl. Acad. Sci. USA 84 (1987) 49104913. K.E.B. Meyer, L.S. Uyechi, F.C. Szoka, Manipulating the intracellular trafcking of nucleic acids. In: K.L. Brigham (Ed.), Gene Therapy for Diseases of the Lung (Lung Biology in Health and Disease, Vol. 104), Marcel Dekker, New York, 1997, pp. 135180. R.A. Walker, M.P. Sheetz, Cytoplasmic microtubule-associated motors, Annu. Rev. Biochem. 62 (1993) 429451. N.B. Cole, J. Lippincott-Schwartz, Organization of organelles and membrane trafc by microtubules, Curr. Opin. Cell Biol. 7 (1995) 5564. T.A. Schroer, S.R. Gill, Interactions between microtubules and intracellular membranes: the roles of microtubule-based motors and accessory proteins, Curr. Top. Membr. 43 (1996) 2752. S.F. Hamm-Alvarez, Molecular motors and their role in membrane trafc, Adv. Drug Deliv. Rev. 29 (1998) 229 242. Wolfert et al., 1997, unpublished. X. Gao, L. Huang, Potentiation of cationic liposome-mediated gene delivery by polycations, Biochemistry 35 (1996) 10271036. N. Pante, U. Aebi, Toward a molecular understanding of the structure and function of the nuclear pore complex, Int. Rev. Cytol. 162B (1995) 225255. D. Gorlich, I.W. Mattaj, Nucleocytoplasmic transport, Science 271 (1996) 15131518. D.A. Jans, S. Hubner, Regulation of protein transport to the nucleus: central role of phosphorylation, Physiol. Rev. 76 (1996) 651685. M. Wilke, E. Fortunati, M. van den Broek, A.T. Hoogeveen,
[81] J.E. Duncan, J.A. Whitsett, D. Horowitz, Pulmonary surfactant inhibits cationic liposome-mediated gene delivery to respiratory epithelial cell in vitro, Hum. Gene Ther. 8 (1997) 431438. [82] B.R. Grubb, R.J. Pickles, H. Ye, J.R. Yankaskas, R.N. Vick, J.F. Engelhardt, J.M. Wilson, L.G. Johnson, R.C. Boucher, Inefcient gene transfer by adenovirus vector to cystic brosis airway epithelia of mice and humans, Nature 371 (1994) 802806. [83] H. Matsui, L.G. Johnson, S.H. Randell, R.C. Boucher, Loss of binding and entry of liposomeDNA complexes decreases transfection efciency in differentiated airway epithelial cells, J. Biol. Chem. 272 (1997) 11171126. [84] A. Fasbender, J. Zabner, B.G. Zeiher, M.J. Welsh, A low rate of cell proliferation and reduced DNA uptake limit cationic lipid-mediated gene transfer to primary cultures of ciliated human airway epithelia, Gene Ther. 4 (1997) 1173 1180. [85] C.W. Pouton, P. Lucas, B.J. Thomas, A.N. Uduehi, D.A. Milroy, S.H. Moss, PolycationDNA complexes for gene delivery: a comparison of the biopharmaceutical properties of cationic polypeptides and cationic lipids, J. Control. Release 53 (1998) 289299. [86] A.N. Uduehi, S.H. Moss, J. Nuttall, C.W. Pouton, Cationic lipid-mediated gene transfer of differentiated Caco-2 cells: a lterculture model of polarized epithelium, 1998, in preparation. [87] P.J. Klasse, R. Bron, M. Marsh, Mechanisms of enveloped virus entry into animal cells. Adv. Drug Deliv. Rev. 34 (1998) 6591. [88] J. Clever, M. Yamada, H. Kasamatsu, Import of simian virus 40 through nuclear pore complexes, Proc. Natl. Acad. Sci. USA 88 (1991) 73337337. [89] M. Yamada, H. Kasamatsu, Role of nuclear pore complex in simian virus 40 nuclear targeting, J. Virol. 67 (1993) 119 130. [90] G. Whittaker, M. Bui, A. Helenius, The role of nuclear import and export in inuenza virus infection, Trends Cell Biol. 6 (1996) 6771. [91] U.F. Greber, H. Kasamatsu, Nuclear targeting of SV40 and adenovirus, Trends Cell Biol. 6 (1996) 189195. [92] C.W. Pouton, Nuclear impart of polypeptides, polynucleotides and supramolecular complexes, Adv. Drug Deliv. Rev. 34 (1998) 5164. [93] M.A. Wolfert, L.W. Seymour, Atomic force microscopy analysis of the inuence of the molecular weight of poly( L)lysine on the size of polyelectrolyte complexes formed with DNA, Gene Ther. 3 (1996) 269273. [94] S.L. Hart, R.P. Harbottle, R. Cooper, A. Miller, R. Williamson, C. Coutelle, Gene delivery and expression mediated by an integrin-binding peptide, Gene Ther. 3 (1996) 1032 1033. [95] E. Wagner, M. Cotten, R. Foisner, M.L. Birnstiel, TransferrinpolycationDNA complexes The effect of polycations on the structure of the complex and DNA delivery to cells, Proc. Natl. Acad. Sci. USA 88 (1991) 42554259. [96] J. Zabner, J. Fasbender Al, T. Moninger, K.A. Poellinger,
[97]
[98]
[99]
[100]
[101] [102]
[103]
[104]
[105] [106]
[107]
[108]
[109] [110]
[111]
[112] [113]
[114]
C.W. Pouton, L.W. Seymour / Advanced Drug Delivery Reviews 46 (2001) 187 203 B.J. Scholte, Efcacy of a peptide-based gene delivery system depends on mitotic activity, Gene Ther. 3 (1996) 11331142. L.A. Schwarz, J.L. Johnson, M. Black, S.H. Cheng, M.E. Hogan, J.C. Waldrep, Delivery of DNAcationic liposome complexes by small-particle aerosol, Hum. Gene Ther. 7 (1996) 731741. S.J. Eastman, J.D. Tousignant, M.J. Lukason, H. Murray, C.S. Siegel, P. Constantino, D.J. Harris, S.H. Cheng, R.K. Scheule, Optimization of formulations and conditions for the aerosol delivery of functional cationic lipid:DNA complexes, Hum. Gene Ther. 8 (1997) 313322. F.L. Sorgi, L. Gagne, S. Sharif, D. Cipollla, S.J. Farr, I. Gonda, H. Schreier, Aerosol delivery using the AERx system, Proc. Intl. Symp. Control. Rel. Bioact. Mater. 25 (1998) 184185. N.-S. Yang, W.H. Sun, Gene gun and other non-viral approaches for cancer gene therapy, Nat. Med. 1 (1995) 481483. W.H. Sun, J.K. Burkholder, J. Sun, J. Culp, X.G. Lu, T.D. Pugh, W.B. Ershler, N.S. Yang, In vivo cytokine gene
203
[120]
[115]
[121]
[116]
[122]
[117]
[123]
[118]
[124]
[119]
transfer by gene gun reduces tumour growth in mice, Proc. Natl. Acad. Sci. USA 92 (1995) 1130711311. N.S. Yang, J. Burkholder, B. Roberts, B. Martinell, D. McCabe, In vivo and in vitro gene transfer to mammalian somatic cells by particle bombardment, Proc. Natl. Acad. Sci. USA 87 (1990) 95689572. D.C. Tang, M. DeVit, S.A. Johnston, Genetic immunization is a simple method for eliciting an immune response, Nature 356 (1992) 152154. C. Condon, S.C. Watkins, C.M. Celluzzi, K. Thompson, L.D. Falo Jr., DNA-based immunization by in vivo transfection of dendritic cells, Nat. Med. 2 (1996) 11221128. H.-Q. Mao, B.S. Hendriks, K.Y. Lin, K.W. Leong, Intramuscular delivery of LacZ plasmid encapsulated in microspheres composed of biodegradable phosphate chainextended poly( L-lactide), Proc. Intl. Symp. Control. Rel. Bioact. Mater. 25 (1998) 203204. V. Labhasetwar, W. Chen, S. Goldstein, J. Bonadio, R. Levy, DNA coating for gene transfer, Proc. Intl. Symp. Control. Rel. Bioact. Mater. 25 (1998) 372373.

Issues in Non Viral Gene Delivery

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Advanced Drug Delivery Reviews 46 (2001) 187203

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Key issues in non-viral gene delivery q

2.1. Routes of administration

2.2. Stability in blood circulation

2.5. Intratumoural injection

2.4. Intramuscular injection

2.6. Subcutaneous administration

2.7. Pulmonary biodistribution

3.1. Major barriers

2.8. Feasibility of local injection to other sites

3.2. Viral models

3.5. Uncoupling of complexes

3.6. Nuclear uptake

is scope for improvement in the efciency of the technique.

4.3. Sustained delivery

4.1. Inhalation systems

4.2. Particle bombardment

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