Toward development of a non-viral gene therapeutic

LAdvanced Drug Delivery Reviews 26 (1997) 135–150

Toward development of a non-viral gene therapeutic

*Janet Smith , Yilin Zhang, Ralph NivenMegabios Corp., 863A Mitten Road, Burlingame, CA 94010, USA

Received 11 November 1996; accepted 6 March 1997

Abstract

Gene therapy is an emerging field that has reached the early clinical stages of development for some disease states.However, the demonstration of safety in animals and the introduction of gene-based formulations in humans hides the factthat numerous developmental and basic research questions remain. This article highlights progress and emerging issues in thearea of liposome-based non-viral gene delivery. The colloidal nature of these formulations render them complicated at thephysico-chemical and biological levels. Instrumentation and methodologies need to be developed to better understand thesubtleties of plasmid DNA, complexing agents, delivery mode and the route of entry into the cell and the nucleus. Majorhurdles to entry include membrane binding, endosomal release, nuclear uptake and decomplexation. Each ‘stage’ is poorlyunderstood but numerous approaches are being directed to increase cellular delivery. These research efforts, coupled withsensible formulation research and a multi-disciplinary, long-term effort, are necessary for success. 1997 Elsevier ScienceB.V.

Keywords: Cancer; DNA; Drug delivery; Formulation; Gene therapy; Lipids; Liposomes

Contents

1. Introduction ............................................................................................................................................................................ 1362. Perspective: cancer and gene therapy ........................................................................................................................................ 1363. Formulation ............................................................................................................................................................................ 137

3.1. Components .................................................................................................................................................................... 1373.1.1. DNA ..................................................................................................................................................................... 1373.1.2. Lipids and liposome ............................................................................................................................................... 1383.1.3. Lipopolyamines ..................................................................................................................................................... 1383.1.4. Buffers .................................................................................................................................................................. 139

3.2. Complexes ...................................................................................................................................................................... 1393.2.1. Preparation ............................................................................................................................................................ 1393.2.2. Purification ............................................................................................................................................................ 140

3.3. Characteristics ................................................................................................................................................................. 1413.4. Delivery .......................................................................................................................................................................... 1423.5. Development ................................................................................................................................................................... 142

4. Subcellular delivery................................................................................................................................................................. 1424.1. Membrane binding, internalization and cytoplasmic localization .......................................................................................... 1434.2. Nuclear localization ......................................................................................................................................................... 1444.3. Decomplexation ............................................................................................................................................................... 145

5. Summary ................................................................................................................................................................................ 146

*Corresponding author. Tel.: 1 1 415 6971900; fax: 1 1 415 6521999.

0169-409X/97/$32.00 1997 Elsevier Science B.V. All rights reservedPII S0169-409X( 97 )00031-8

136 J. Smith et al. / Advanced Drug Delivery Reviews 26 (1997) 135 –150

Acknowledgments ....................................................................................................................................................................... 146References .................................................................................................................................................................................. 146

1. Introduction 2. Perspective: cancer and gene therapy

Gene therapy offers the promise of treating disease Cancer is a progressive disease characterized bythrough the production of therapeutic proteins within loss of cellular growth control. Many of the physio-cells. This requires targeting, delivery of the DNA to logical changes that take place upon tumor formationsufficient cells, and expression of the gene at high can be utilized in targeting strategies and thus mayenough concentrations and with sufficient persistence make this disease an ideal target for gene therapyto produce a pharmacological effect. The manner in [16]. As the mechanisms involved in cell transforma-which the genes are packaged for delivery can take tion are better understood, genes active against tumorseveral forms, usually involving either viruses or activity will become available. But having identifiedlipids. Adenoviruses have demonstrated that high and produced genes is not sufficient for therapy, andexpression can be achieved in vivo [1–3]. However effective cancer treatment will require accurateviral vectors can cause nonspecific inflammation and targeting of the delivery vehicle to the affected tissueanti-vector immune responses [4–7]. In addition, as well as an appropriate level of therapeutic genethere are concerns regarding the ability of the agent expression. To illustrate, a moderate tumor burden of

11to develop replication competent virus, or activate 100 g represents 10 cells, and therefore a largeoncogenes, once delivered [8]. Retroviral vectors are number of in vivo ‘hits’ will be required from anylimited to accepting a maximum of 8 kbp of non- gene delivery system that is used [16].viral sequence [3,9], whereas the parvovirus minute Vile and Russell have described two broadvirus of mice, can accept only 2 kbp of foreign DNA categories of cancer gene therapy strategy depending[10,11]. Non-viral alternatives include the use of on whether the treatment is devised to correct thecationic liposomes complexed to DNA or administra- genetic abnormality in the abnormal tumor cellstion of the DNA alone directly by injection [12,13]. (corrective gene therapy), or to completely destroyThe use of cationic liposomes is not limited by these cells (cytotoxic gene therapy or immuno-vector size and, for example, lipid-based systems therapy depending on whether the cells are killed byhave been used to deliver vectors as large as yeast direct toxic effects or by activating the host immuneartificial chromosome (650 kbp) in mice [14]. Addi- system). Clearly, the therapeutic gene and its co-tionally, the low immunogenicity of the active acting elements are key to the choice of strategy,complex allows it to be administered repeatedly in therefore it is worth reiterating here that liposomalvivo [15]. Non-viral gene therapy therefore promises gene therapy offers no limitation to the vector size,to provide a relatively non-toxic means to administer and has the potential to deliver multiple vectors togenes of a sizable range and, if required, using a the same site for a multi-directed delivery. Complex-multiple dosing regime. es of lipid and DNA have already shown success in

In this article, the requirements for an effective delivery of genes to humans [15,17,18]. This in-non-viral gene therapeutic are described. The intent cludes nasal delivery of the cDNA for cystic fibrosisis to provide a developmental overview of non-viral transmembrane conductance regulator (CFTR) com-gene therapy. Formulation, development and delivery plexed with lipid, resulting in the expression ofissues are considered in that order. Only a cursory CFTR mRNA in the epithelium [19,20]. Therapeuticmention will be given to the effects of the delivery gene products have also been delivered to colorectalroute and more effort is directed to subcellular and renal cell carcinomas, and melanomas, usingdelivery where the ‘attrition’ of the complex as it lipid-DNA delivery vehicles reviewed by Crystalprogresses into and within the cell is elaborated. [21].

J. Smith et al. / Advanced Drug Delivery Reviews 26 (1997) 135 –150 137

Clearly, there are experimental precedents indicat- and protein removal are difficult, hence processing that gene therapy may have a role in treating engineers must develop methods to ensure bothcancer. Assuming that evidence of efficacy in models purity and yield during manufacture. Further, ascontinues and that any preliminary toxicity concerns alluded to above, a batch of ‘purified’ DNA maycan been addressed, a developmental pathway must contain hitherto undetected contaminants [31] andfollow to produce and scale up a formulation for the effect of impurities on the physico-chemicalclinical use. No matter how efficacious an anti- properties of DNA could be significant. For example,cancer gene therapeutic proves to be in vivo, the the superhelical conformation of circular DNA maypotential of the therapy is limited unless an accept- be influenced; the susceptibility to nicking andable formulation can be developed and scaled econ- linearization on storage may be enhanced; the extentomically. The following section discusses some of of lipid binding and the structures formed could bethe current issues facing development of formula- altered and so forth. These types of questions havetions for non-viral delivery systems. not been answered and although their impact may be

minor, there is presently insufficient evidence todetermine their influence on efficacy.

3. Formulation Although the majority of the eukaryotic genome ismaintained in the duplex b-form, the duplex struc-

3.1. Components ture itself is flexible, allowing DNA the ability tochange its organization. A number of parameters can

The most common materials used in current non- be changed, including the number of base pairs perviral preparations are purified, supercoiled plasmid turn of DNA, discontinuities in the structure such asDNA, lipids (usually a mixture of cationic and sequences that induce bends, supercoiling of closedneutral lipids) and an appropriate buffer [22–26]. In DNA and temporary separation of the double helixsome instances the cationic lipid has been substituted strands to allow replication or expression. Thisby other reagents such as synthetic polymers, includ- flexibility is therefore crucial to DNA functionality.ing polylysine [27], polyvinylpyrrolidone [28] and Further, the base pair composition is known todextran derivatives [29,30], or bile salts [31], other influence the stability and properties of the DNAmicellar systems [32] and even alginate microspheres molecule [37]. Fortunately, eukaryotic genes do not[33]. In others, DNA without additional condensation normally contain a heavy weighting toward CGs oror complexation agents has met with partial success ATs. However, the length of plasmids may range[34]. Typically, though, an additional agent is re- from 2 to over 20 and there will be many morequired to protect and perhaps condense the DNA. molecules in a milligram of DNA of 2 kbp than forThe buffer will play a significant role in the electro- 20 kbp. The conformational shape and solutionstatic interaction between the various components of dynamics of the plasmids are therefore likely to bethe formulation and DNA, and in the condensation very different, and hence the time-averaged exposurestate of the DNA and thus can directly influence of ‘charge’ to the surrounding medium may vary,efficacy as well as stability. Its importance should affecting the interactions between ligands and DNA.therefore not be neglected. Despite the above concerns, plasmid DNA has

unique features that make it an attractive formulation3.1.1. DNA candidate. The molecule is inherently stable and can

Current techniques are being maximized to assess withstand environmental conditions that would readi-the purity of manufactured DNA. Despite numerous ly denature many therapeutic proteins. Consequently,means whereby ‘purified’ plasmid DNA can be subjecting DNA to varying environmental parame-prepared, there is no ‘standardized’ approach and ters such as pH and ionic strength to accommodatemany novel methods are proprietary. DNA is also a other components of a formulation is quite feasible.notoriously difficult molecule to purify [35]. The Modifications of the chemical nature of the DNA canpolymeric DNA is highly negatively charged and also be made in an effort to reduce enzymatic attack,thus can bind a variety of molecules readily via or increase tissue specificity, without necessarilyhydrophobic and electrostatic forces. Endotoxin [36] compromising transcription fidelity. In this respect,


the molecule can be ‘tailored’ far more than any phatidylethanolamine (DOPE) [50,51] or cholesterolprotein therapeutic in development. There is also the [52–54]. DOPE is interesting because of its sensitivi-potential to formulate multiple DNAs in a single vial ty to pH and ability to form hexagonal-phase struc-because of the simplicity of the building blocks (four tures in an aqueous environment [55]. Researchersnucleotides). Incompatibilities are therefore unlikely. have tried to take advantage of this phenomenon and,If the formulation can be targeted to the disease or when exposed to lower pH such as is generated inregion of interest, then multiple genes encoding for endosomes, DOPE can cause the disruption ofmolecules that target different susceptible points of a membranes as has been demonstrated in vitromanifested disease can be delivered. This increases [56,57]. In contrast, cholesterol is employed tothe chances of therapeutic success and possibly provide structural stability and protection and there isavoids the need for multiple injections. also evidence that cholesterol can influence targeting

in vivo via scavenger receptors [52–54,58]. This3.1.2. Lipid and liposomes latter observation also raises the point that many

Liposomes are the vehicle by which lipids can be lipids can have pharmacological activity and there-complexed to DNA in an aqueous environment. fore could actively contribute, in addition to playingWhen reduced in size (50–100 nm), the relatively a carrier role, in the transfection of DNA.large surface area (and surface charge) generatedenables complex formation in an aqueous medium.The lipids protect DNA and can provide tissue 3.1.3. Lipopolyaminestargeting when injected intravenously. Liposomes are Synthetic polymers [59,60] are also being evalu-also well characterized and widely used in drug ated as DNA carriers. Polylysine has been widelydelivery research and can now be found as an used to couple DNA to specific target ligands forintegral component of certain prescription drugs. For receptor-mediated uptake during gene therapyexample, AmBisome and DaunoXome (NeXstar [61,62], or when covalently linked to phospholipid,Pharmaceuticals) are proprietary liposomal formula- to introduce positive charge [63,64]. Polylysine hastions of amphotericin B and daunorubicin, respec- also been used to condense DNA prior to associationtively, also Doxil (Sequus Pharmaceuticals) is a with cationic liposomes, producing much smallerliposomal form of the chemotherapy drug doxorubi- particles than the lipid-DNA alone. When comparedcin. Liposomal delivery of drugs was recently re- with Lipofectamine alone, a 2–3-fold increase inviewed in a thematic issue of Advanced Drug luciferase expression was found using the polylysine-Delivery Reviews [38–42]. lipofectamine complexes following delivery to

Numerous lipids have been screened and many C C myoblast cells [27]. When the relative sizes of2 12

more are currently being evaluated for use in gene poly (L-lysine)-plasmid DNA complexes, formulatedtherapy [43–48]. No one type or class of lipid has, to with varying molecular weight average poly (L-date, enabled DNA to be efficiently transfected and lysine) molecules were compared by atomic forceexpressed in vivo relative to their adenoviral counter- microscopy, the lower molecular weight poly (L-parts, although significant improvements in transfec- lysine) complexes (3970 Da, | 20–30 nm complex-tion have been made over the past five years [49]. In es) demonstrated a more uniform distribution thanstudying these various lipids one general rule has those of a higher molecular weight (224 500 Da,emerged. At least one of the lipids should be maximum complex diameter 300 nm). The lowercationic. It is also known that the nature of the lipid molecular weight complexes were also less toxichead group, linker and hydrocarbon tail may impact when applied to human ovarian carcinoma A2780transfection, although a structure-function relation- cells. Thus, the smaller poly (L-lysine) may beship is not yet established. The approach to syn- preferable for in vivo gene therapy [27]. Similarly,thesizing candidate molecules therefore remains block-copolymer micelles, typically consisting oflargely empirical. polyoxyethylene and polyspermine chains have also

Many cationic molecules cannot form liposomes been developed to be water soluble [65] and there-alone, and are normally accompanied by a neutral fore may provide a suitable vehicle in which tolipid (or lipid-like molecule) such as dioleyl phos- deliver DNA.


The lipospermines DOGS and DPPES have been 3.2. Complexesused to transfect numerous cell lines with greaterefficiency than calcium phosphate precipitation or 3.2.1. Preparationmonovalent cationic surfactants [63,66]. In all cases, Once liposomes have been prepared they arethe transfection efficiency depended on the polyca- typically directly admixed with an aqueous solutiontion /DNA ratio. This effect was only seen when the of DNA, resulting in a heterogeneous colloidalcomplexes were positively charged, and it was dispersion. Two-phase colloid dispersions aresuggested that the charge increases binding to thermodynamically unstable and any form of hetero-anionic membrane surfaces, and the resulting per- geneity associated with the dispersion will presumab-turbation of the membrane, induces endocytosis and ly accelerate instability. There is also uncertaintyuptake of the DNA. However, release from the about batch-to-batch consistency and to date theendosome has proven to be a difficult barrier to tools (instrumentation) and methodology have notunderstand and overcome (see below). evolved to the point where these complexes can be

characterized to the level of detail necessary. Fortu-nately, investigators are recognizing the importance

3.1.4. Buffers of the complex and the way in which it is prepared.Scant attention has been given to the type of Clearly if made inappropriately, not only will this

buffer employed with non-viral DNA complexes. result in a poor formulation, in vivo activity will beThe buffer plays the normal expected roles of affected.maintaining the pH of the formulation over time and One approach to prepare complexes that hasminimizing instability of the components during recently been used involves the use of octylglucosidestorage. The buffer components also have the po- to solubilize the lipids and the DNA which is thentential to influence the nature of the complex that is dialyzed against buffer to remove the surfactant andformed. For example, the condensed state of DNA is induce the formation of the complexes (i.e., themarkedly influenced by the valency of the ions in formation of two phases) [73]. This is a ‘gentle’,solution [67]. Electrostatic neutralization of the albeit lengthy, approach to preparing complexes andphosphate groups on DNA by associated cationic thus has some practical advantages to that of thesubstances can convert a curved segment of DNA turbulent process associated with direct mixing. Onfrom an unstable to a stable structure [68]. The shape the other hand, applying an approach that can beof the structure achieved, be it rod-like, torroidal or better controlled and is easier to scale-up mayrandom depends on the extent of condensation. This improve batch reproducibility. Thus, rather than anelectrostatic compaction was found to be dependent ad-hoc approach to combination, with careful consid-on the external salt concentration, the valence of the eration given to the concentration, temperature,salt (higher valencies can induce the same level of environment (buffer, package, etc.), the kinetics ofDNA condensation at lower concentrations) and the the mixing process and how the liquids are com-amount of alcohol present [69–71]. bined, the final colloid may be distinctly improved in

Lipids are also sensitive to environmental salt terms of stability and efficacy.conditions and thus the stability of the colloid will These comments can be further justified by con-also be impacted by the buffer. If present at suffi- sidering the nature of the colloid interface. Sub-cient ionic strength, aggregation may be induced micron particles have large surface to volume ratiosthrough compression of the diffuse region of the and important surface characteristics such as chargeelectric double layer, as will be discussed. The and charge density which need to be addressed whentemperature vs. pH stability of the buffer is another formulating them. The theory developed by De-issue. Depending upon the storage conditions, the ryagin-Landau and Verney-Overbeek (DLVO) quan-buffer pH may alter by several units if stored at titatively describes colloid stability by consideringlower temperatures; even in frozen conditions at the combined charge-related repulsive forces and van2 208C [72]. Consequently, detrimental chemical der Waals forces of attraction between two approach-changes, such as hydrolysis of lipids and nicking of ing spheres. Each charged sphere develops an elec-DNA, may occur during storage. tric double layer when placed in an aqueous medium.


In the case of lipid-DNA complexes, the sign of the dividual molecules tend to have extremely shortnet charge on a complex will depend on the ratio of range van der Waals forces (varying with distanceDNA combined with cationic lipid. The double layer from the surface by the inverse of the 6th power),consists of an inner boundary of partially hydrated within a collection of molecules such as a colloid,and tightly bound ions (Stern layer) and a diffuse the range of attraction can be significantly extended.layer consisting of an asymmetric distribution of Consequently, the diffuse layer is sensitive to manycounter and co-ions as depicted in Fig. 1. This factors as described in Table 1. For example, anboundary layer tends to repel other similar approach- increase in temperature increases the potential energying particles. However, under some circumstances of particles and the frequency and force of collisionsthe attractive forces may dominate, resulting in the will increase, whereas cooling or an increase inirreversible coagulation of particles. Although in- viscosity will produce the opposite effect. Further-

more, an increase in ion concentration or ionicstrength will tend to compress the interfacial layer,as will agitation or mixing. These points indicatewhy the final product is likely to be highly dependenton the method, conditions and environment in whichcomplexation takes place.

3.2.2. PurificationImprovements in the preparative method should

increase the homogeneity of the complex, but one ormore purification steps may be necessary to improvethe final product. Since the DNA and liposomes canindividually be transferred through a series of elabo-rate steps to produce material that is of sufficient‘purity’ for in vivo use, the same thinking should beapplied to the complex. Thus filtration, centrifuga-tion, dialysis, etc., may be considered processingsteps following initial preparation. The combinationof liposomes and DNA in absolute terms may consistof a fixed ratio, but the complex at the colloidalparticle scale will probably consist of a dynamic-average ratio and some particles may consist ofpredominantly DNA and others predominantly lipid.Hence, additional means to ‘separate’ the desiredfractions from the initial complex may lead to a morehomogeneous and reproducible product. Despite the

Table 1Factors influencing aggregation of complexes

Factor Change Effect on coagulationFig. 1. An illustration of the basic models proposed to describe the

Ion concentration ↑ ↑diffuse double layer. The Gouy Chapman model considers a flat

Temperature ↑ ↑surface and point charge. The Stern model adapted this to include

Agitation /mixing ↑ ↑a layer of tightly adsorbed ions that can ‘shear’ at a distance from

Co-ion valency ↑ ↓?the surface of a particle and the zeta potential (z ) is an

Counter-ion valency ↑ ↑approximate, but experimental, measure of this theoretical shear

Viscosity ↑ ↓plane. The inserts also illustrate that the z can carry a greater, or

Colloid concentration ↑ ↑opposite charge than would be predicted from the surface charge

Colloid size ↑ ↑of the particle.


fact that an additional processing step adds time and headgroups, or formulated with different neutraleffort to the production process, the purification compounds, interact with DNA in different ways andstep(s) may ease restrictions on specification and with different binding energies. The resulting com-therefore reduce the overall cost and perhaps even plexes may take a variety of structural forms.the time involved in production. A more convenient indirect approach to measuring

size involves the use of photon correlation spec-3.3. Characteristics troscopy (PCS) which indirectly measures size by

employing light scattering to gauge the diffusion ofThe characteristics of the complexes can be mea- the particles in the dispersions and then derives

sured by techniques currently employed to character- hydrodynamic size using the Stokes-Einstein rela-ize liposomes as both represent examples of two- tionship. This is a useful tool for quality control but,phase colloidal systems. Routine measurements in- unlike microscopy, does not provide much infor-clude pH, osmolarity, turbidity and HPLC of the mation about the nature of individual particles.lipids and the DNA. Newer chromatographic ap- Zeta potential is a measure of the adsorbed surfaceproaches like capillary electrophoresis may provide potential at the double layer (described above) or netsignificant detail about the state of the DNA in stored charge of the overall colloid dispersion. From thesamples [74,75]. However, the study of intact plas- charge information, inferences can be made aboutmid DNA by this technique is still under develop- stability on storage and the interaction with bio-ment. logical fluids and tissues. This is a useful tool to

Size measurements can be made directly through monitor stability but, given the heterogeneous andthe use of electron microscopy (EM) [76] as com- possibly dynamic nature of the complexes, signifi-plexes cannot normally be resolved by light micro- cant variability in data is possible and results canscopy. Unfortunately, this is an expensive and only be generated in low ionic strength media [78].lengthy approach depending on the level of process- Differential scanning calorimetric data on theing involved, and may result in artifacts as a purity of lipids will probably be a regulatory require-consequence of simple manipulation. It is therefore ment. But microcalorimetry should also provideimpractical to monitor batches routinely and tedious information about the influence of formulation on theto perform statistical validation. Microscopy is better melting transition temperatures. Titration calorimetrysuited to providing morphological information and, may prove useful by interpreting the thermal changesin this respect, cryo-EM, freeze-fracture and nega- occurring upon mixing of liposomes and DNA [79].tive-stain transmission EM and atomic force micro- The above-mentioned techniques provide charac-scopy have all been used [48,76]. Results must be teristic information on the macroscopic properties ofviewed with caution, however, because of the diffi- colloids. Obtaining information about individualculty in establishing whether photomicrographs re- particles and their composition requires extensiveveal structures that are real or artifact. For example, study and the use of multiple techniques. NMR [80],using atomic force microscopy [48] the interaction of ESR [79], X-ray scattering, and circular dichroismcomplexes with mica substrates, drying of sample [81] all have utility. However, these techniques areand positioning of the probe can readily cause not widely used, due to a lack of development of theartifacts [77]. Despite these issues, microscopic methodologies to study lipid-based complexes. Fur-analysis has provided useful information about com- ther, these techniques are expensive and the dataplexes. Some lipids, notably DOGS, are seen to coat generated typically requires expert interpretation.the DNA [63] and freeze-fracture micrographs have Methodologies are also being adapted to specifi-revealed a more heterogeneous population of struc- cally study DNA complexes. Density gradient cen-tures in the lipid-DNA complex than previously trifugation has indicated that complexes can bethought. This population includes so-called spaghetti separated from free liposomes by centrifugation(rod-like structures, supposedly a DNA duplex sur- through sucrose gradients [62,82], and that therounded by a single lipid leaflet) and meatballs (a resulting profiles may contribute towards our under-conglomerate of lipid or liposomal aggregates) [76]. standing of size /packing relationships through anIt is not unlikely that cationic lipids with varying analysis of the size and density of the separated


particles. Equilibrium dialysis studies can also be scrutiny. Regulatory guidelines must be followed,used to determine vesicle aggregation, leakage, lipid recommendations must be carried out and docu-mixing, as well as nonspecific ligand-nucleic acid mentation requirements must be met. The effects ofbinding [83,84]. time, temperature, light, excipients, pH, ionic

It can readily be concluded from the above strength, container and closure materials, bacterialdiscussion, that in order to characterize and under- contamination, endotoxin, degradation, and a multi-stand these colloidal systems and quantitate their tude of other factors must be accounted for. Thesefeatures, an integrated approach involving many topics are well beyond the scope of this discussiontechniques is required. and are discussed elsewhere [86,87], but the point

must be made that success at the early stage in the3.4. Delivery clinic is no guarantee that a commercial product can

be ultimately developed. In addition, DNA-derivedThe final composition of the formulation depends products are a novel concept and some of the

on the target tissue and route of administration. For problems, for scientists and regulators alike, have notexample, the requirements for injection directly into yet been encountered.a solid tumor may be very different in terms ofcontent, dose, volume, etc., than those needed for aformulation to be infused intravenously. This situa- 4. Subcellular deliverytion of course pertains to any form of drug therapy.Unfortunately, since the DNA is so dependent upon The final target site for DNA delivery is thethe accompanying reagents to maximize transfection, nucleus, as this is the site of the transcriptionalany necessary change in formulation to accommo- machinery. Understanding the steps involved indate the mode of delivery may nullify transfection. subcellular delivery is critical to identifying the rate-These future problems are already hinted at in the limiting steps of the delivery process, and will formliterature. The work of Felgner and associates, for the basis of strategies to overcome such bottle-necks.example, has shown that uncomplexed DNA expres- The subcellular process can be crudely separatedses b-galactosidase in muscle more efficiently than into four stages: (1) membrane binding and internali-does DNA complexed with lipids [50]. Yet, most zation; (2) cytoplasmic localization; (3) nuclearpublished in vivo studies by intravenous injection localization; and (4) decomplexation (indicated inhave required the presence of a cationic lipid to Fig. 2). All of these steps impact final gene expres-effect any observable DNA expression. Consequent- sion although their relative importance is unknown.ly, it will be surprising if a single non-viral gene Transfection using ‘physical’ methods such as cal-delivery vehicle can be developed for all applica- cium precipitation can produce successful transfec-tions. Assuming that some degree of targeted gene tion, indicating that there must be some ‘leakage’ indelivery can be achieved, efforts must be directed each of the four steps described, resulting in a basetoward a better understanding of the mechanism of level of transfection of low efficiency. Theoretically,cellular uptake and processing of the DNA. only one copy of the plasmid is needed in the

nucleus to achieve protein expression but its time3.5. Development there may be limited before it is incapacitated by

nuclear enzymes or by translocation, and thereforePresently, the emphasis is on designing and de- its ‘opportunity’ to employ or borrow the transcrip-

veloping complexes that function optimally in vivo tional machinery from the host DNA will be finite.and only recently have formulations been tested in The presence of multiple copies or the continualthe clinic [15,17,18,85]. For an industrially spon- appearance of new plasmid being delivered to thesored investigational new drug this requires that nucleus should increase the probability of success. Inaround 3 months stability data be available as practice, unfortunately, expression levels are typical-supportive documentation. Assuming that phase I ly low or nonexistent even though millions of copiestrials are successful and development continues, reach the external cell surface [88]. Consequently,formulation and stability come under much greater millions must be ‘lost’ during the transition into the


Fig. 2. Steps towards cellular delivery: (1) binding, (2) internalization, (3) endosomal release, (4) nuclear localization, (5) decomplexation.

cell and delivery to the nucleus. Improvement of any bind with receptors on the HepG2 cell surface. Thus,of the described steps of subcellular delivery, by a second binding force in addition to a non-specificeven a minute fraction, should lead to improved electrostatic interaction may retain complexes at theexpression efficiency and intensity. cell surface. Receptor-mediated gene delivery has

also been used with strategies utilizing ligand-poly-4.1. Membrane binding, internalization and cation conjugates. Wu’s group has used asialoglycop-cytoplasmic localization rotein-polylysine conjugates to mediate gene delivery

to the liver in vivo [90,91]. However, hepatectomyThe binding force of cationic lipid-mediated deliv- was needed to obtain high levels of expression for

ery is thought to be the electrostatic interaction long periods.between the positive charges of the lipid-DNA The uptake of DNA in skeletal muscle appears tocomplex and the negative charges of the cellular be receptor mediated and naked plasmid DNA ismembrane. However, not all of the complexed lipid- superior to viral vectors for direct gene transfer intoDNA particles currently used have a net positive adult mouse skeletal muscle [92,93]. However, genecharge. Although a large number of copies of expression in muscle can be ‘saturated’ by theplasmid DNA are internalized, the efficiency is still introduction of high doses of plasmid DNA [34,92–low and a small fraction of the DNA molecules in 94], or inhibited by an excess of non-coding DNA orthe extracellular space are internalized [88]. To dextran sulfate [95]. These data suggest that animprove internalization Remy et al. incorporated a uptake mechanism mediated by a receptor-like pro-ligand-receptor binding component into their cationic cess involving multivalent negative charges is in-lipid-DNA complex [89]. A galactose-containing volved.lipid was used to facilitate the transfection of the Physical parameters, in addition to the chemicallygalactose-receptor containing HepG2 cells. The addi- driven events described above also play a role andtion of 25% galactose-containing lipid increased particle size is an important factor in the targetingtransfection 1000-fold. This effect may be due to an and internalization processes. Lactosylated high den-improved efficiency of uptake of the complexes sity lipoprotein (10 nm) has been found to be almostthrough a receptor-mediated mechanism where the exclusively taken up by liver parenchymal cells [96].galactose molecules of the lipid-DNA complex may In the case of the larger size lactosylated low density


lipoprotein (22 nm), these particles were mainly tion site is the cytosol, and is responsible for thetaken up by the Kupffer cells of the liver [97]. Using translocation of the intracellular pathogen from thea high salt ( . 1 M NaCl) procedure, Perales et al. endosome/phagosome to the cytosolic space of thehave obtained a DNA/galactosylated-polylysine host cells. When used to deliver ovalbumin, lis-complex of a very small size (10–12 nm). Such teriolysin O increases the efficiency of the cyto-particles led to high level long-term expression in plasmic delivery. Without this protein, very littlehepatocytes, rather than Kupffer cells, of whole cytoplasmic delivery was observed for pH-sensitiveanimals [98]. Approaches using different DNA bind- liposomes alone. Fusogenic peptides have also beening components, such as galactosylated-histone [99], used to increase the efficiency of endosomal release.or different receptor ligands, such as transferrin- A peptide derived from the N-terminal sequence ofpolylysine [100–104], have also been devised. the influenza virus hemagglutinin subunit HA-2

The mechanism of internalization and cytoplasmic augmented the transferrin-polylysine-mediated genelocalization mediated by cationic lipids is not well transfer substantially [103,104]. pH-sensitive pep-understood. Although membrane fusion at the cyto- tides were also used as endosomal-releasing agentsplasmic membrane level can occur [29,93], most for DNA-peptide complexes [109].recent experimental evidence suggests that endo-cytosis is the dominant mechanism of uptake 4.2. Nuclear localization[64,105,106]. For example, when the endocytosisinhibitor cytochalasin B was added to the medium It is still not clear how the DNA plasmid isduring transfection in vitro, transfection was in- translocated from the cytoplasm into the nucleushibited [106]. In addition, EM images of gold- during cationic lipid-mediated delivery. The nuclearlabeled plasmid have revealed an entry process envelope has nuclear pores with a passive transporttypical of endocytosis [88]. Based on DNA replace- limit of about 70 kDa molecular weight or 10 nmment experiments, it has been proposed that DNA is diameter [110]. A 5-kb plasmid has a molecularsubsequently released from the lipid-DNA complex weight of about 3.3 million daltons and hydro-following charge neutralization of the cationic lipids dynamic dimensions that are far above the ‘cut-off’by the negatively charged lipids located at the for passive transport through the nuclear pore. Ascytoplasmic-facing leaflet of plasmid membrane evidence of this point, microinjection of a plasmid[82]. The flip-flop of the anionic phospholipids containing the herpes simplex virus thymidine kinaseinduces membrane destabilization, which may lead gene into the cytoplasm of LMTK-cells led to noto further flip-flop of the anionic lipids. When located expression of the thymidine kinase. Yet, nuclearat the facing leaflet, the anionic lipids may migrate injection of the same plasmid resulted in a high levelonto the cationic leaflet, form a charge neutral ion of thymidine kinase activity [12]. Gao and Huangpair with cationic lipids and release DNA from the co-delivered purified T7 RNA polymerase, DC-cholcationic lipids. Unfortunately, this mechanism does cationic liposomes and a plasmid containing thenot distinguish between membrane fusion at the chloramphenicol acetyltransferase (CAT) genecytoplasmic membrane or the endosomal membrane. driven by the T7 promoter [111]. This approach

Cytoplasmic localization may be achieved by pH- brings the transcription step out of the nucleus intosensitive liposomes [105,107] but the transfection the cytoplasm. Faster and higher CAT expressionefficiency is 3–150-fold less than that achieved using was observed compared to delivery using cationiccationic lipids, perhaps due to the lack of positive liposomes without the polymerase.charge, and thus electrostatic binding to the cyto- In apparent contradiction to the above data indicat-plasmic membrane. Consequently, although the net ing that the nuclear envelope acts as a significantendosomal release efficiency of the pH-sensitive barrier, the use of nuclear localization peptides canliposome is low, it cannot strictly be compared to facilitate the nuclear localization of proteins of largerthat of cationic lipids. Another approach has in- than 70 kDa (e.g., BSA, 7 nm). Colloidal goldvolved the use of the pH-sensitive hemolytic protein, particles coated with nucleoplasmin have shown thatlisteriolysin O [108]. It is a soluble protein isolated particles as large as 15 nm in size may be transportedfrom Listeria monocytogenes, whose natural infec- across the nuclear envelope [112]. One explanation


for these observations is that the nuclear localization croinjection data obtained by Zabner et al. [88].peptides mediate a receptor-mediated process When lipid-DNA complexes with a high lipid con-[110,113]. Peptides bind with a soluble cytoplasmic centration were injected into the nucleus, transgenereceptor, which initiates the active process and expression was inhibited. The same experimentstransports the conjugates through the nuclear pore. conducted using low concentrations of lipid showedWhether such a peptide is sufficient to transport a comparable expression to naked DNA. This impliesplasmid or plasmid/ lipid complex is still not clear. that decomplexation in the nucleus is not veryHowever, a nuclear localization sequence located on efficient. Further, DNA/lipid complexes injected intothe N-terminus of the HIV-1 gag matrix protein is the cytosol showed very little expression, whichresponsible for the nuclear localization of the retro- implies that the cationic lipid does not significantlyviral pre-integration complex (diameter | 30 nm) facilitate the penetration of the nuclear membrane.[114]. A single mutation of the sequence led to a However, microinjection of DNA alone into cytosoldramatic decrease in the infection efficiency of the also showed that naked plasmid passes into theHIV virus in non-dividing cells. nucleus very poorly [12,88]. One possibility, albeit

Cell division is an important factor for the nuclear speculative, which does not conflict with any of thelocalization of transgenes. During mitosis, the in- experimental data, is that the free DNA dissociatedtegrity of the nuclear membrane is lost. Therefore, from the complex passes through the nuclear mem-the nuclear envelope is not a significant barrier for brane only if oriented longitudinally. The probabilitydividing cells. This is the case for all in vitro of this kind of orientation (of the supercoiled plas-transfection. However, during in vivo transfection, mid?) is presumably low and hence consistent withmany of the targets are terminally differentiated the observations of low transfection. Another possi-non-dividing cells. In such cases, the nuclear en- bility, as hypothesized earlier, is that there is a basalvelope cannot be neglected. Evidence of the impor- leakage through the nuclear envelope (by an un-tance of the nuclear envelope is furthered by experi- known mechanism) and partial decomplexation oc-ments showing that mutant HIV with a dysfunctional curs during cytoplasmic localization. Cationic lipidsnuclear localization peptide sequence can still infect remaining from the first membrane penetration stepdividing CD-4 1 MT-4 cells. When the MT-4 cells may retain the DNA in a partially condensed statewere arrested at the G1/S interface of the cell cycle and the smaller hydrodynamic size of the condensedby aphidicolin, no infection was observed with the DNA may help nuclear translocation through basalmutant HIV. Yet, wild-type HIV could effectively leakage of the nuclear envelope.infect MT-4 cells in both cases [114]. The difference Microinjection of DNA/lipid complexes into nu-between nuclear transport in dividing and non-divid- clei results in poor transgene expression compared toing cells may, in part, contribute to the differences in nuclear injection of naked DNA plasmid [88]. In aactivity observed between in vitro and in vivo physiological situation, the association and dissocia-experiments. tion between genomic DNA and DNA binding

proteins such as histones are highly regulated. It is4.3. Decomplexation speculated that plasmid may dissociate from the

complex in the nucleus due to displacement of theDecomplexation is another important step for gene plasmid DNA from the cationic lipids by genomic

expression. For transcription factors to bind with the DNA [89]. However, as discussed above such in-plasmid DNA, it is assumed that the cationic agents tranuclear decomplexation may not be very effective.need to be removed. Although high levels of trans- It has been proposed therefore that decomplexation isgene expression can be achieved using cationic lipid- completed during cytoplasmic entry [82]. Xu andmediated delivery, it is not known whether the Szoka suggest that penetration of the endosomalcomplex dissociates before reaching the nuclear membrane barrier neutralizes the cationic lipidenvelope or after. Xu and Szoka have hypothesized charges, and the lipid is retained by this membranethat plasmid DNA is released from the cationic lipids as the DNA is released into the cytoplasm. Combin-during cytoplasmic localization, leaving naked DNA ing this idea with the experimental observationsinside the cytosol [82]. This agrees with the mi- occurring in the nuclear localization process, another


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Toward development of a non-viral gene therapeutic

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