recent developments in cationic lipid-mediated gene delivery and gene therapy

Review

2001 © Ashley Publications Ltd ISSN 1354-3776 1729

Ashley Publicationswww.ashley-pub.com

1. Introduction

2. Novel cationic lipids

3. Physicochemical characteristics

of lipoplexes and their

correlation with the

transfection efficiency

4. Gene transfer: delivery barriers

5. Therapeutic applications

6. Conclusions

7. Expert opinion

Recent developments in cationic lipid-mediated gene delivery and gene therapyMarc Antoniu Ilies & Alexandru T BalabanTexas A&M University at Galveston, Department of Oceanography and Marine Sciences, 5007 Avenue U, Galveston, TX 7755, USA

Gene therapy will revolutionize medicine, helping us to cure and prevent dis-eases at their core level. Until becoming a widespread reality, the problem ofefficient gene transfer and expression (transfection) must be solved. Cationiclipids represent a safer alternative than viral vectors, which, although moreefficient, have the drawback of immunogenicity and propagation risks. Addi-tionally, cationic lipids and cationic liposomes allow the delivery of largerplasmids and may be GMP manufactured and stored in bulk quantities. How-ever, their specific transfection efficiency must be improved in order to reachthe performance of biological vectors. In recent years, new structures havebeen released and tested, with designs adapted to recent findings in lipid-mediated transfection mechanisms. Another trend is the increased use of nat-ural, biodegradable, building blocks in the backbone of these compounds.Here we review the very recent developments in the field of cationic lipids,both from industry and academia. Physicochemical characteristics, insights oftransfection mechanisms, as well as therapeutic applications are also pre-sented. Finally, some future prospects and trends are proposed.

Keywords: cationic lipid, gene delivery, gene therapy, lipoplexes, plasmid DNA, transfection

Expert Opin. Ther. Patents (2001) 11(11):1729-1752

1. Introduction

The completion of the human genome charting [1,2], correlated with the progress inmolecular biology on understanding the involvement of different genes in severalsevere maladies [3,4], constitutes an excellent premise for the development of a newmethod of curing and preventing diseases: gene therapy. This new form of therapyconsists of the transfer and expression (transfection) of a correct copy of a deficient(abnormal) gene in cells, tissues or organs affected by specific hereditary or acquireddiseases [3-7]. Gene therapy is a conceptual revolution in medicine for two main rea-sons: DNA can be considered (and used) as a drug for the first time, and the cause ofdisease, not just the symptoms, can be treated or eliminated [7, 8].

Despite the simple concept, implementation of gene therapy is rather difficult.Again, in contrast to conventional therapies, the drug itself (DNA) is less important,the limiting factor is the efficiency of the in vivo transfection of genetic material.Therefore, developing efficient vectors for gene delivery is the goal of many researchteams, both from academia and industry [9].

The available delivery technologies fall into three main categories: physical, bio-logical and chemical methods [7,9]. Physical methods include naked DNA muscleand skin (micro)injection [10-12], electroporation [13-17] and DNA-coated particlebombardment - gene gun technology [18-20]. Despite some recent developments,their application in human gene therapy remains limited.

Modified viruses are the main exponents of biological methods, being themost efficient transfection vectors [21-24]. However, they have major drawbacks

Recent developments in cationic lipid-mediated gene delivery and gene therapy

1730 Expert Opin. Ther. Patents (2001) 11(11)

4

N+

O

O

O

F F

F F

F F

F F

F F

F F

F F

F

FF

1a m = 1, n = 1 (DOTMA)1b - e m = 1 - 4, n = 1 - 4

2

3

N+

OO

O

OO

O

OO

OON

+

O

O

OH

OH

OH

OH

OH

OHOH

OH

m nN+

O

O

such as immunogenicity, difficulties associated with GMPproduction and storage and a maximum size of insertedDNA limited to about 40,000 base pairs [7].

Chemical methods provide a safer alternative to viral vec-tors, by means of synthetic transfection agents. These arechemical entities capable of co-operative DNA binding, form-ing supramolecular complexes in which the plasmid is highlycompacted and thus able to be introduced into the living cell.Also, these self-assembling systems are able to mediate boththe penetration of the cell membrane and the release of thegenetic material into the cytosol [25-27]. Although less efficientthan viruses, synthetic DNA delivery agents possess severaladvantages, such as low immunogenicity and toxicity, lack ofpropagation risks, unlimited size of the plasmid that can betransfected, easy (GMP-compliant) bulk production, storageand quality control assessment, that recommend them as a via-ble alternative to biological methods of transfection [7].

There are two main categories of chemical transfectionagents, namely cationic lipids and cationic polymers. They areused neat or co-formulated with various additives that eitheramplify the transfection efficiency by different mechanisms[28] or improve their physical stability [29,30].

Both cationic lipids and cationic polymers interact electro-statically with the anionic groups of DNA, forming DNA-lipid complexes (lipoplexes) or DNA-polycation complexes(polyplexes) [31]. The association is favoured by the release ofthe counterions of the lipids or polymers, a process with sub-stantial entropy gain (see later). In the case of lipoplex forma-tion, the self-assembly process requires both interaction with

the DNA and association of the lipid molecules themselves incationic liposomes [25,32-36].

The use of cationic lipids as gene delivery agents was initi-ated by the pioneering work of the Felgner, Behr and Huanggroups [37-44]. Almost simultaneously, cationic polymers wereintroduced as alternative DNA transfection systems [45-48].Both fields evolved rapidly, a large number of vectors weresynthesised and tested with respect to their transfection effi-ciency. Several excellent reviews are available in both fields[6,9,49-62], including patent literature [63,64].

The present review is focused on the use of cationic lipids astransfection agents, covering the very recent literature from astructural point of view.

2. Novel cationic lipids

All these synthetic lipids consist of three parts, a hydrophobicanchor region linked via a spacer group to a polar head. Thepolar head associates electrostatically with negatively-chargedphosphate groups of the DNA, while the hydrophobic regionleads to the formation of a lipid bilayer (by co-operative inter-molecular binding) that self-closes into a liposome. Theentrapped genetic material is compacted and protected fromthe external medium, thus being able to reach the target cells.

A substantial synthetic effort was made to improve the effi-cacy of cationic liposome-mediated gene delivery, generating alarge variety of active structures. According to the structure ofthe polar head, cationic lipids could be divided into fourclasses: quaternary ammonium salt lipids (and phosphonium/

Ilies & Balaban

Expert Opin. Ther. Patents (2001) 11(11) 1731

arsonium congeners), lipopolyamines, cationic lipids bearingboth quaternary ammonium and polyamine moieties andamidinium, guanidinium and heterocyclic salt lipids.

2.1 Quaternary ammonium salt lipids and congenersThere has been a tremendous development in this field [53,63]

since the introduction of 2,3-dioleoyloxypropyl-1-trimethyl-ammonium bromide (DOTMA, 1a), co-formulated with dio-leoylphosphatidylethanolamine (DOPE) as Lipofectin™ bySyntex [37,301]. Recently, Ren and co-workers synthesised aseries of DOTMA analogues 1b - e [65,66] and used them toevaluate the main structural characteristics responsible for thehigh in vivo transfection activity of this cationic lipid. Theypresented evidence that paired oleoyl chains as lipid anchor,attached to a cationic head group by ether linkage bonds tothe (pseudo)glyceryl backbone in a 1,2-relationship wereresponsible for the high in vivo transfection efficiency [67].They also found that in vitro transfection activity was deter-mined by the lipoplex structure and by the outcome of theinteractions between lipoplexes and blood components fol-lowing iv. administration. The same group introduced a newtype of cationic lipids, using as a backbone 3,5-dihydroxyben-zyl amine [68], which proved to be efficient both in vitro(against murine melanoma BL-6 cells, human embryonic kid-ney 293 cells, human hepatocarcinoma HepG2 and humancervical carcinoma HeLa cells) and in vivo (in mice and ani-mal models), where these cationic lipids transfect preferen-tially the lung cells. To improve cell targeting, based on theconcept of receptor-mediated endocytosis [69-71], a galactose orglucose moiety was attached at the ω position of the hydro-carbon chains, resulting in compounds of type 2 [72]. Anotherchange at the level of the lipophilic anchor was proposed byBhattacharya et al., who inserted oligo-oxyethylene units at

the linkage site of the hydrophobic tails [73]. The resultingDOTMA-like structures 3 were tested for their vesicle-form-ing properties. However, no transfection properties have yetbeen reported.

Based on a similar concept of improving transfectionproperties by hydrophobic anchor (and backbone) modifica-tion, Sigma Tau introduced a series of carnitine perfluori-nated esters 4 that proved efficient to transfect HeLa,human mammary carcinoma MCF-7, human colon adeno-carcinoma Caco-2 and human glioblastoma T98-G cell linesin vitro [302]. The idea of using natural, biodegradable build-ing blocks to construct the cationic lipid structure also liesbehind this approach. Similar structures are also found inthe design proposed by Life Technologies [303] for DOTMAanalogues 5, as an alternative to the well-known 1,2-bis(ole-oyloxy)-3-(trimethylammonium)propane DOTAP 6 [43].This seems a general trend for countering the fact that cati-onic lipids with ether linkage between the lipophilic anchorand backbone tend to accumulate in the fatty tissues of theliving organisms after repeated transfections.

Another design used the cholesteryl moiety as a hydropho-bic anchor, first introduced by Huang’s group in the synthesisof DC-Chol 7, which has remained a standard cationic lipidsince [44,304]. In a related approach, Université Paris Nordrecently proposed 3-b[N-(N′,N′,N′-triethylaminopropane)-carbamoyl]cholesterol (TEAPC-Chol) 8 and other deriva-tives, which proved to have good transfection efficiencyagainst MCF7, A549, 9L, U37MG and HUH7 cell lines [305].

Haces used a different head group modification with a 1,4-diazabicyclo[2.2.2]octane moiety (DABCO™) in the prepa-ration of a DOTAP-like cationic lipid 9 [306]. In the samestudy [306], two series of bis-quaternary ammonium com-pounds of type 10 and 11 were reported; these resulted from

6 DOTAP

N+

O

O

O

O

7 DC-Chol

H

O

H HO

NHN

8 TEAPC-Chol

H H

H

OC

NH

O

N+

5

N+

O

O

O

O

NH

NH



two different synthetic strategies. When co-formulated withDOPE, at a determined optimal molar ratio, these com-pounds exhibited good in vitro transfection profiles againstseveral cell lines, including Hep G2, HeLa S3, Jurkat cells (T-cell leukaemia), COS and CHO cell lines. The polyether-typederivative 11 was particularly effective to transfect humanbladder carcinoma T-24 and human kidney 293 cell lines, act-ing without diminished activity in the presence of 10%serum, a promising characteristic for in vivo applications.

In the same time frame, other bis-quaternary ammoniumsalt lipids were proposed by Vical [307] using a variable lengthspacer to connect two DMRIE ((±)-N-(2-hydroxyethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-1-propanaminium bromide[74]) or GAP-DLRIE ((±)-N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(dodecyloxy)-1-propanaminium bromide [75]) units bydifferent type linkages. Ethereal linkages were employed in thesynthesis of PEG derivative PentaEG-bis-DMRIE 12,whereas a bis-ureyl linkage generated 1,4-bis-(N′-butyl-(4-(N,N-dimethyl-2,3-bis(tetradecyloxy)-1-propanaminium))-ureyl-butane (SBDU-DMRIE) 13 and related compounds.Bis-amide-linked DMRIE-type compounds were also dis-closed. These novel compounds presented a good capacity toenhance the immune response of a pDNA encoding influenzanuclear protein (in mice) or haemaglutinin (in humans), aswell as for their in vivo transfection properties using an sc.melanoma model.

An original approach was proposed by Le Bloc’h, Ferec,Floch and co-workers, by introducing cationic phosphonolipids

of type 14 (Z = N), coformulated with DOPE or cholesterol,as efficient transfection vectors for in vitro and in vivo applica-tions [76-78]. Recently, the same group reported that cationsubstitution (N, P or As) on the polar domain of this type ofcompounds results in significant increase in transfection activ-ity for both in vitro (against K562, CFT1, HT29 and HeLacell lines) or in vivo assays and in a decrease of cellular toxicity[79-81].

2.2 LipopolyaminesThe smallest natural polycations able to compact DNA arethe polyamines spermidine and spermine. By attaching ahydrophobic anchor to these molecules via a linker, Behrintroduced DOGS 15 (Transfectam™), as the first represent-ative of a new class of transfection vectors [40,41,308]. Subse-quent developments followed [9,53,63].

In addition to the existing structures [63], the Max DelbrückCentrum fur Molekulare Medizin recently introduced [309]

new representatives derived from spermine, putresceine andornithine 16 - 20 with distearoyl moieties attached to glycerolor serinol backbones, or bearing a cholesteryl carbamategroup as hydrophobic anchor. Some of these compounds (SP-Chol 17, O-Chol 19, Put-Chol 20), exhibit comparable orhigher in vitro transfection profiles against MaTu, F98,CC531 and N64 cell lines when coformulated with DOPE indifferent proportions, as compared with the DC-Chol/DOPEclassical transfection system. This behaviour is retained evenin presence of 5% serum.

12 PentaEG-bis-DMRIE

4

N+

O

O

O

O

O

O

ON

+

10

N+

N+

O

11

2

N+

N+

O

O

O

9

N+

O

O

O

O

N

Ilies & Balaban


18

O

O

NH

O

OONH2

NH2

19 O-Chol

H

O

H HO

NH2

NH2

20 Put-Chol

H

O

H HO

NHNH2

15 DOGS

N

O

NH

O

NH

NH2

NH

NH2

14 Z = N, P, As. n = 1 - 3

13 SBDU-DMRIE

N+

O

O

N+

O

O

NH

O NH

NH O

NH

nP

O

O

O

Z+

16

O

O

O

O

OO

N

NH2

NH

NH2

H

O

H HO

N

NH

NH2

NH2

17



The use of natural/biocompatible building blocks in thedesign of lipopolyamines is also found in Transgene’sapproach [310], which proposed spermidine derivativespcTG37 21 pcTG39 (similar with 21 but with stearoyl lipidchains) and the T-shaped analogues pcTG89 22, all derivedfrom diaminopropionic acid. The compounds were effective(neat or co-formulated with DOPE at different ratios) bothin vitro (against dog myoblast and human pulmonary epithe-lial carcinoma A549 cells) and in vivo on C57BL/6 mice. Par-ticularly important for the transfection properties are thephysicochemical characteristics of the complexes obtainedfrom neat or DOPE-coformulated compounds. Both totalcomplexation of the DNA and a diameter of the complexes

< 500 nm are critical for obtaining good biological effects;several different methods of formulation were also presented.Also, in a more recent work, the same group introduced apentaammonio analogue of 21 that exhibited high levels oftransfection in vitro and in vivo [82,83].

Another amide-based approach has recently been proposedby Genta [311], using amino acid/peptide-based spacers to con-nect the polyamine head group with the hydrophobic tails.The resulting compounds were tested for their ability to trans-fect COS-7, SNB-19, C8161 and RD cells, with or withouthelper lipids, as compared with standard transfection systems(Transfectam™, Lipofectamine™, Lipofectin™). Com-pounds 23 - 25 were found to be the most active; their activity

21 pcTG37

23

NH2

NH

NH

NH

NH2

O

O

O

25

NHNH

N

NH

O

NH2

NH2

O

OOH

24

NHNH

NH

NH

NH

O

O

O

NH2

NH2

22 pcTG89

NH

NH

O

O

NH

O

NH

NH2

NH

NH

O

O

N

O

NH2

NH2

Ilies & Balaban


surpassed the above-mentioned standard transfection systemsfor specific cell lines when used neat or when coformulatedwith DOPE or Genta’s proprietary phosphonic co-lipid 26.This pro-cationic lipid was synthesised within a larger series ofphosphonic cationic lipids [312], also tested in vitro on thesame cell lines under similar experimental conditions andstandards as the compounds 23 - 25. It was found that a mix-ture of ornithine-based lipid 27 and DC-Chol-related phos-phonic co-lipid 28 was able to transfect COS-7 cells betterthan Lipofectamine™.

Park et al. developed a solid-phase synthesis method forobtaining oligo-lysine polycationic lipids of type 29 (n = 1 -10) [84,313]. Oligo-ornithine analogues were also disclosed, as

well as toxicity and physicochemical data for the cationic lipo-somes formed by these compounds, which proved to be ableto transfect mouse embryonic fibroblast NIH3T3, HepG2and 293T cell lines when coformulated with DOPE, withsimilar or superior efficiency comparatively with classical DC-Chol/DOPE, Lipofectin™ or polyethyleneimide transfectionsystems.

In a related study, Ren et al. [85] used the carbamoyl groupto link a structure-variant polyamine head group with a vita-min-D2/3-based hydrophobic anchor, generating biocompat-ible cationic lipids that were tested (co-formulated with

DOPE 1:1 mol/mol) as transfection vectors against Lewislung carcinoma 3LL, BL-6, NIH3T3, 293 and HeLa cells.The best results were obtained with compounds 30 and 31,which exhibited superior transfection activity in a broaderrange of lipid to DNA ratio in BL-6 cells, as compared withDC-Chol/DOPE system.

Other lipopolyamines with a globular head group, madefrom biodegradable building blocks, were introduced by Bes-sodes et al. [86]. In this approach a glycosidic scaffold was usedas backbone, several aminoalkyl groups being attached via anether or ester linkage to generate cationic lipids of type 32.The organised spatial density of positive charges confers goodin vitro transfection profiles against HeLa and NIH3T3 cells,comparable to other cationic lipids previously described bythe same authors [63,87].

An important innovation in the field of cationic lipid-mediated transfection is the use of reduction-sensitive lipopol-yamines harbouring a disulfide bridge, for modulated releaseof DNA from cationic lipid/DNA complexes. This approachis based on the idea that modulated degradation of the lipidsduring or after penetration of the cell (by natural occurringreduction systems) could improve the trafficking of thegenetic material to the nucleus, resulting in increased trans-gene expression.

26

OP

ON

O

27

NH2

NH

NH

NH

NH2

O

O

O

28

H H

H

OP

ON

O

29

30

O

H

NH

O

NNH2

NH2

31

O

H

NH

O

NNH2

NH2

n

H H

H

OC

NH

NH2

O

NH2

O



Within the same timeframe, two groups proposed differentstructures to materialise this concept [88-91,314-316]. Rhône-Pou-lenc Rorer [91,314] proposed a series of lipopolyamines of type33 - 37, having a disulfide bridge in different positions in thebackbone, which were biologically tested in respect of theirtransfection properties (in vitro, on HepG2 and HeLa cells).Extensive SAR studies established that the best gene expressionwas achieved when the disulfide bridge was inserted betweenone lipid chain and the residue of lipopolyamine. When thedisulfide bridge was positioned between the fatty chains and thepolyamine (such as in 33), a total loss of activity is observed,although multilamellar complexes with DNA are formed priorto transfection, similar to other related non-reducible, in vivo-efficient structures. The impact of physicochemical characteris-tics of the lipoplexes formed by these compounds and the effectof coformulation with helper lipids, like DOPE, on transfec-tion efficiency were recently presented [92].

On the other hand, the University of Florida [88-90,315,316]

introduced DOGSDSO 38, CHDTAEA 39 and related com-pounds as prototypes of the same approach. Their in vitrotransfection properties were tested comparatively to theirstructural analogues lacking the disulfide bridge (CHSTAEA)and to classical transfection systems like DOTAP/DOPE andDC-Chol/DOPE, on SKnSH and CHO cells. The resultsrevealed a superior transfection activity of CHDTAEA/DOPE lipoplexes compared to CHSTAEA/DOPE and DC-Chol/DOPE systems for a large range of cationic lipid/DNAratio, doubled by a diminished toxicity against both cell lines.Surprisingly, CHDTAEA/DOPE liposomes delivered lessDNA to cells than either the CHSTAEA/DOPE or DC-

Chol/DOPE liposomes, even though it showed better trans-gene expression.

Recently, Isis Pharmaceuticals introduced a series offusogenic lipids of type 40 [317], possessing both positively andnegatively charged groups in the molecule, separated by aspacer which contains a disulfide bond. These structures alsoexploited the reductant capacity of the intracellular systems,being defined as pro-cationic: after penetrating the mem-brane, the amphiphilic compounds are cleaved at the level ofthe disulfide bond, becoming cationic and being able to medi-ate fusion with the negatively charged plasma membrane, thusserving as vehicles for delivering pharmaceutical agentsincluding genetic material.

A different approach in the field of polycationic lipids wasproposed by Celltech [318], by using bipolar polycationic lipidsof type 41 and 42, comprising a polycationic head linked with along alkyl hydrophobic spacer to a hydrophilic tail. These com-pounds are capable of self-assembling and they compact DNAefficiently to give particle sizes < 500 nm, as proved by physico-chemical characterisation studies. They were successfully testedas transfection vectors against CHO cells.

2.3 Cationic lipids bearing both quaternary ammonium and polyamine moietiesThe first cationic lipid of this type was 2,3-dioleoyloxy-N-[2-sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanamin-ium trifluoroacetate (DOSPA, 43), co-formulated withDOPE to give the Lipofectamine™ transfection reagent[93,319]. Although highly efficient, its relatively high toxicityhindered further clinical developments. Several years later,

34 RPR 132535

NH2 NH

NH

NH

NH

NH

OS

S

O

35 RPR 132688

NH2 NH

NH

NH

NH

N

OS

O

S

32

OO

ONH2

O

NH2

O

NH2

O

O

O

N

33 RPR 128522

16

17NH2 N

H

NH

NH

NH

SS

N

O

O

Ilies & Balaban


37 RPR

38 DOGSDSO

39 CHDTAEA

H H

H

OCS

S

O

O

NH

NNH2

NH2

40

H H

H

OC

NH

NH

O

NH

SS

O

OH

41

23

OHNH

NH

NH

NH

NH

NH2

OH

OH

OH

OH

O

O

NH2 NH

NH

NH

NH

NH

OS

O

S

NH

NH

NH

NH

NH

NH2

O

O

17

17

36 RPR 202065

NH2 NH

NH

NH

NH

NH

OS

O

S

H H

H

16

42

24

OHNH

NH

NH

NH

NH2

OH

OH

OH

OH

O

O

O

O

O

OO

SS

O

O

O

O

NH2

NH2



Wheeler et al. introduced GAP-DLRIE 44, which coupleshigh levels of transfection with low cytotoxicity [75].

Within the frame of their programme to improve bioavaila-bility of proprietary classical vectors, Life Technologies [303]

introduced the DOSPA-DLRIE and GAP-DLRIE-relatedcompounds 45 and 46, containing carbamate linker bondsthat are claimed to have low toxicity when administeredin vitro or in vivo.

Vical proposed a more sophisticated approach, using apiperazine backbone to obtain a large variety of mixed polar-head cytofectins such as 47 - 49 [320,321]. When coformu-lated with DOPE in different proportions, these compoundswere able to transfect mouse myoblast muscle C2C12 andCOS-7 cells with similar or superior efficiency as comparedwith the classical DMRIE/DOPE system. When adminis-tered in vivo, GA-LOE-BP 47 yielded greater transfectionefficiency than GAP-DLRIE in a mouse sc. tumour assay, aswell as in an intralung transfection assay. Also, the resultsobtained for the ip. transfection assay in C57/B16 mice weresatisfactory in some cases, although inferior to thoseobtained for GAP-DLRIE.

In addition to the structures presented in previous chapters,the patent application by Haces [306] depicts the synthesis andbiological properties of a diamino-bis-quaternary ammoniumsalt lipid, 50, obtained from a spermine scaffold. This com-pound (coformulated with DOPE) showed a 2 to 2.4-foldefficiency to transfect HepG2 and HeLa cell lines as com-pared with the N,N,N,N-tetramethyltetrapalmitylspermine(TMTPS) taken as control [322]. The transfection efficiency ofcompound 50 was comparable to TMTPS against Jurkat cellsbut more than five times higher than Lipofectin™ in primaryhuman tracheobronchial cells. Finally, the same substanceproved particularly active to transfect primary human epider-mal keratinocytes, a potential target for genetic therapy [94].

2.4 Amidinium, guanidinium and heterocyclic salt lipidsRelated to the above-mentioned compound 50, is the bis-amidinium-bis-quaternary ammonium salt lipid 51. Bothcompounds share similar in vitro transfection properties andreduced cytotoxicity [306]. Also, Frederic et al. [95] recentlymanaged to introduce cyclic guanidine moieties into lipopolyamide

43 DOSPA

44 GAP-DLRIE

O

O

NH2N

+

45

N+

O

O

O

O

NH

NH

NH

O

NH

NH2

NH

NH2

46

N+

O

O

O

O

NH

NH

NH2

NH

O

ON

H2+

O

NH

NH2

NH

NH2

Ilies & Balaban


molecules to obtain compounds of type 52 and 53 with linearor globular polar head groups.

In addition to the cholesteryl-based derivatives syntheticprogramme, the Max-Delbrück Centrum für MolekulareMedizin also proposed the guanidyl derivative of serinolDOSGA 54 [309], which proved able to transfect F98 and N64cells when coformulated with DOPE. At an optimum molarratio DOSGA/DOPE of 30/70, this system was 2.5 - 5 timesmore active than the DC-Chol/DOPE classical transfectioncomplex against the above-mentioned cell lines.

A similar approach was recently proposed by Syntex [323]

after extensively investigating the field of guanidino cationiclipids useful in gene therapy. A large series of derivatives wassynthesised and biologically tested, both in vitro (againstmurine fibroblast, 3T3, human bronchial epithelial, 16HBEand vascular endothelial, IVEC cells) and in vivo by intratra-cheal instillation of the lipoplexes in rat lungs. The most

active compounds were found to be derivatives 55, 56 andtheir lauroyl analogues.

In extension to these widely used polar head groups, a fewnitrogen containing cationic heterocycles were introduced inthe structure of lipidic vectors.

Solodin et al. [96] synthesised and tested several imidazo-lium-based cationic lipids of type 57. Megabios developedthis class of transfection agents, by introducing a large seriesof low-toxicity imidazolium derivatives [324], which weretested on mice for their in vivo transfection efficiency, in com-parison with DOTIM 57. The best results were obtained withcompound 58, coformulated with cholesterol or dilauroyl-phosphatidylethanolamine (DLPE), which retained thein vivo activity of DOTIM. Another contribution to this classof transfection agents was proposed recently by Alza [325]

using the glyceryl derivative 59 for in vitro transfection of thehuman lung tumour 2E9 cell line.

47 GA-LOE-BP

N

N+

NH2

O

O

48 CH3,GA-LOE-BP

N+

N+

NH2

O

O

49 Gly-G-LOE

N

N+

NH

O

O

NH2O

50

NNH2

O

N+

N+

N

O

NH2



51

N+

NH

NH

O

N+

NH

NH2

NH2

NH

52

NH

NNH

NH

NH

O

O

N

NH

53

NH

NNH

NNH

O

N

NH

NH

NNH

O

54 DOSGA

55

56

O

O

NH

O

OON

H

NH2

NH

NH2 NH

N

ONH

NH NH

N

ON

57 DOTIM

N

N

OH

O

O

+

Ilies & Balaban


New pyridinium-based cationic lipids were synthesised byMeekly et al. in a comprehensive study [97]. The resulting com-pounds, coformulated with DOPE (1:1), were tested for theirin vitro transfection capacity against COS-7 cells. The mostactive compound was the dioleoylmethyl-N-methylpyridiniumsalt 60. The characteristics of the pyridinium amphiphile/DNAcomplexes have been also extensively studied.

The two authors of the present review, together with Dr.WA Seitz (Texas A&M University at Galveston), Dr. MWentz and Dr. RE Garfield (University of Texas MedicalBranch at Galveston) devised a novel approach for pyridin-ium-containing cationic lipids, avoiding the expensive quater-nisation reaction. Encouraging results were obtained bytesting the compounds on HeLa cells [326].

3. Physicochemical characteristics of lipoplexes and their correlation with the transfection efficiency

A recent success in the field of cationic liposome-mediatedtransfection was the transfer into cells of partial sections ofhuman artificial chromosomes with sizes around 1Mbp [98].Although extremely inefficient, this achievement emphasisesthe potential of these systems to transfect large pieces ofgenetic material into cells.

In order to improve the transfection performance of lipo-plexes, as well as to correlate and compare the results obtainedwith different cationic liposome formulations, it is essential toknow in detail the physicochemical characteristics and the

mode of action of the cationic lipid-DNA complexes. Conse-quently, an appreciable amount of work was done regardingthese aspects. Recent reviews of this area are available [58,99].

Among the factors known to determine the physicochem-ical properties of the lipoplexes, the most important are:

• The nature and properties of the cationic lipid (chemistry,stability, counterions influence, toxicity) [25,30,81,100-124]

• The use of a co-lipid (‘helper lipid’), its nature and proper-ties [25,30,33,81,105,110,112,113,116-118,123-132].

• The nature and properties of the DNA (chemistry, stability,secondary and tertiary structure) [118,132-135].

• The molar ratio of different components in the final com-plex [25,30,33,100,105,107,110,112,116,119,120,122,124,127,132,136-139].

• The protocol of bringing together the components of thelipoplex [29,30,33,122,139-141].

• The environment in which the lipid-DNA complexes aregenerated (the influence of pH, temperature, ionicstrength) [81,106,141-143].

It is beyond the scope of this review to detail all these aspects.In what will follow, only some key issues will be presented.

The interaction between DNA and cationic lipids in lipo-somal form with DNA is a complex process that takes place atthe aqueous/organic interface. This is the result of co-operativeinteractions, triggered by the DNA-mediated fusion of lipo-somes [33,112,120,144,145]. As stated, it involves large scale lipidrearrangement, driven by the electrostatic attraction betweenthe positively charged lipid head groups and the negativelycharged phosphate groups on the DNA, as well as by co-operative

58

59

O O

O

O

NH

N

NH

O

O

60

N

N

OH

N+

+



hydrophobic association between the hydrophobic tails of thecationic lipids and also by the entropically-favoured release ofthe counterions of both DNA and cationic lipids[106,121,127,146,147]. In recent years, new insights into the lipoplexstructure and formation has been revealed by means of isother-mal titration calorimetry [107,120,124], differential scanning calor-imetry [104,106,110,111,120,136,148], circular dichroism [133], molecularprobes [25,105,112,113,119-121,127,134,136,144,149,150], microelectrophore-sis [112,121,137], Raman microscopy [131] and especially by syn-chrotron small angle x-ray scattering (SAXS)[25,110,113,141,142,151-155] and atomic force microscopy[119,122,156].

These results clearly showed that the actual structure ofthe lipoplexes is different from the bead-on-string structureinitially proposed by Felgner et al. [37,38]. Two types of peri-odic structures were revealed [151-153]. Thus, by adding linearλ-phage DNA to DOTAP/dioleoylphosphatidylcholine(DOPC) cationic liposomes, Radler et al. [151] obtained liq-uid-crystalline globules with sizes ∼ 1 µm, which exhibitedhigher-ordered multilamellar structure with DNA sand-wiched between the cationic bilayers, the lamellar Lα

C phaseof lipoplexes. This is the most common and stable [157]

supramolecular assembly formed by association of DNAwith cationic liposomes. Oligolamellar structures had beenreported in cryo-transmission electron microscopy [158] andfreeze-fracture electron microscopy [100].

A different periodic structure was revealed by Koltoveret al., [153,154], using DOTAP/DOPE liposomes. As previouslystated, DOPE is a neutral helper lipid that was empiricallyfound [125,159] to improve the efficiency of cationic lipid-mediated transfection. It was shown [153] that an increase ofthe ratio of DOPE in DOTAP/DOPE-based lipoplexesinduced a structure change from the multilamellar structureLα

C to a columnar inverted-hexagonal HIIC liquid-crystalline

state. Moreover, in the same study [153] it was proved that thetwo supramolecular structures had different fusogenic proper-ties against giant anionic vesicles (G-vesicles), which are mod-els of cellular membranes. The HII

C complex rapidly fusedwith the G-vesicles spreading and releasing the DNA, whilethe Lα

C complexes remained stable in contact with the G-ves-icles. Recently, this interaction was confirmed by in vitrotransfection studies on mouse fibroblast L-cells by means offluorescent optical microscopy, using DOTAP/DOPE (72%DOPE) versus DOTAP/DOPC (72% DOPC)-based lipo-plexes. The images revealed that in contrast to Lα

C complexes,which remained stable inside cells, HII

C complexes fused withthe endosomal or plasma membrane, releasing the geneticmaterial inside the cell [160], thus confirming the conclusionsof the previous studies [125,161,162]. This finding put a scientificbasis to the empirical use of DOPE as a transfection-enhanc-ing helper lipid. Mention should be made of the fact that thepresence of DOPE in the formulation of cationic lipid vectorsis not a sine-qua-non condition of achieving good levels oftransfection, since it was proved that liposomes containingonly DOTAP were effective for transfecting A549 and COS-7

cell lines at appropriate lipid/DNA ratio with similar or betterresults than other (DOPE-containing) cationic lipid formula-tions [112]. In another study [132] it was demonstrated that evenfor DOPE-containing lipoplexes the transfection efficiencycould be heavily dependent on the pDNA promoter, so thatfor a specific plasmid promoter other helper lipids, e.g., cho-lesterol [126], may enhance the biological activity at higher lev-els than DOPE, for the same cationic lipid. The issue ofoptimal helper lipid remains controversial [129,132].

Lipopolyamides were also found to form lipoplexes with alamellar organisation (even when coformulated with DOPE),with plasmid DNA intercalated between alternating lipidbilayers [25,113,141,142]. They are able to efficiently condense thenucleic material, forming virus-sized spherical particles (downto 50 nm in diameter), which were proved highly effective totransfect different cell lines [25,113]. The studies also presentedthe influence of helper lipids [25,113], lipid:DNA ratio[25,113,141,142] and environment [141,142] on the morphologicalcharacteristics, stability and biological activity of the corre-sponding lipoplexes. It was also claimed that structural fea-tures of some lipoplexes were independent of theconformation and the size of the DNA [25].

4. Gene transfer: delivery barriers

As mentioned in the introduction, a general drawback in thedevelopment of cationic lipids as vectors for gene delivery isthe relatively low level of transfection achieved by these sys-tems in vitro and in vivo, as compared with biological meth-ods [7,163]. Also, the lack of correlation between in vitro andin vivo activity often accounts for drug development failure;compounds that had proved to be very active in vitro fre-quently exhibited low levels of transfection in vivo. To over-come this issue, a better understanding of the delivery barriersand their relationship with the structure of the lipoplexes isrequired. This field is extensively investigated and is periodi-cally reviewed [27,54,58,61,62,164-167].

Several routes of administration are usually available forreaching a specific tissue. Selection of the best one depends onthe localisation of the target tissue and on specific propertiesof the vector used. Thus, assessing the biodistribution andpharmacokinetic properties of the lipoplexes in connectionwith a specific route of administration is the first step to beelucidated in the long way to superior in vivo results[4,26,27,61,164,168-170].

Cationic lipid-DNA complexes can be administered by thesame routes as liposomal formulations [164], namely by sys-temic [26,171-174], local (im. [175,176], intratumour [177,178], sc.[179,180], percutaneous [181], intracerebral [182,183], intraarticular[184] and ip. [116,179]) injection, as well as by topical (skin[177,185], cornea [186,187], inhalation of an aerosol [81,177,188-190],intranasal/intratracheal instillation [172]) application. Theirbiodistribution always differs.

For example, when administered in blood circulation, thelipoplexes interact with serum proteins and blood cells, which

Ilies & Balaban


can change their physicochemical characteristics and drasti-cally reduce their transfection potency [165,191,192]. It has beenproved [193] that negatively charged proteins like albumincould effectively neutralise the cationic charge of the lipo-plexes which become unstable, significantly increasing in sizeand eventually disintegrating and exposing the plasmid tonucleases and other DNA-scavenger systems [61,194]. The sta-bility of the cationic lipid-DNA complexes in the serumdepends on the lipid composition and on the lipid-to-DNAratio, which controls the structure, size and the net charge ofthe lipoplexes (see above) [119,127,129,132,167,192,195,196]. Thus,lipoplexes with a moderate excess of positive charge werefound to be more efficient in vitro and in vivo than ones witha charge ratio near neutrality [119]. As stated, size and size dis-persion are other important parameters influencing transfec-tion capacity [132,191,197]. Consequently, efficient methods forproduction [26,29,164], purification [29], size-fractionation [139],physical stabilisation [30] and analysis [139,164] of the lipoplexeswere developed.

Along with rigorous physicochemical property control, sev-eral innovations were introduced in order to avoid prematuredegradation of the cationic lipid-DNA complexes, includingsurface modification by means of polyethyleneglycol (PEG)derivatives (PEG-ylated liposomes) [164,198] and shortenedpathways of administration [177].

Once the target tissue is reached, three barriers remain to beovercome in order to express the nucleic material, namely bind-ing and internalisation of the lipoplex, escape of the DNA intothe cytoplasm and entry of DNA into the nucleus [27,54,58].

The interaction of the lipoplexes with the cell membrane is amultistage process [27,170]. It begins with either hydrophobic orcationic binding to the negatively charged membrane surface,which is rich in glycoproteins and glycolipids containing sialicacid residues [137,195]. Proteoglycan molecules have also beenshown to mediate lipoplex binding and subsequent entry intothe cytoplasm [199-201]. It is generally accepted that the primarymethod of internalisation of lipoplexes is through endocytosis[125,161,162,202-206], although phagocytosis [207,208], membranefusion [205] and lipid-mediated poration are also possible inspecific cases [164]. Receptor-mediated endocytosis has beenalso exploited for transfection purposes [71,167,187,206,209].

The entry of a cationic lipid-DNA complex into cells is anefficient process, despite the relatively long time required[125,202,210]. On the contrary, it was proved that only a smallfraction of the lipoplexes escape from the endosomal com-partment into the cytoplasm [161,202]. Facilitation of nucleicacid release from endosomes into the cytosol significantlyincreases the transfection efficiency. Intracellular trafficking ofthe lipoplexes is also correlated with their nature and compo-sition [119], as well as with the cell type [119, 170]. As previouslymentioned, the use of optimal levels of DOPE as helper lipidin lipoplex formulation induces endosomal membranedestruction by undergoing a structural change to invertedhexagonal phase HII

C in the acidic medium of the vesicle[74,125,160,202]. This explains the superior in vitro transfection

efficiency of DOPE-containing lipoplexes as compared withcationic lipid-DNA complexes formulated with other helperlipids. However, very recent studies [192] demonstrated thatlipoplexes formulated with DOPE strongly interact witherythrocytes, significantly reducing their transfection efficacyin vivo. This could explain the lack of correlation frequentlyobserved between in vitro and in vivo experiments withDOPE-containing lipoplexes.

Wattiaux et al. [211] studied the impact of cationic lipids(DOTAP) on the endosomal membrane and found that thedestabilisation was due to the interaction of cationic lipidswith anionic residues in the membrane. Xu and Szoka [212]

hypothesised that destabilisation induced a flip-flop process ofanionic lipids from the cytoplasm-facing monolayer and sub-sequent formation of a charge-neutral ion pair with the cati-onic lipids. Other works confirmed this hypothesis[195,205,213]. In conjunction with this explanation it wasobserved that cationic lipids with short alkyl chains formedlipoplexes with enhanced efficacy, as compared with theirlonger-chain analogues [74,205,214]. Recently, it was demon-strated that mixtures of cationic lipids with anionic (phos-pho)lipids preferentially adopted the inverted hexagonal phase(HII

C), destabilising the lipid bilayer after a mechanism simi-lar to that involving DOPE [196,215].

The migration of DNA from the cytoplasm to the nucleusis one of the most important limitations to successful lipo-plex-mediated gene transfer [202,216,217]. This fact was evi-denced by Capecchi [218], who showed that nuclear-injectedDNA led to a much higher level of gene expression thanmicroinjection of DNA into the cytoplasm. These resultswere also confirmed by Zabner et al. [202].

On the other hand, injection of lipoplexes directly into thenucleus gives no detectable levels of expression, proving thatDNA is not available for transfection in this form [202]. Thus,in order to be expressed, the DNA must be completelyreleased from the lipid [202]. This finding lies behind the ideaof designing reduction-sensitive cationic lipids, like 33 - 39,with excellent transfection profiles. These vectors undergosevere structural changes under the influence of the cytoplas-mic reductive systems, which destabilise the lipoplex andrelease the plasmid into the cytosol [88-91,214-216].

Cytoplasmic nucleases rapidly degrade naked DNA within50 - 90 min [219]. Thus, in order to be expressed, the plasmidmust be transported actively from the cytoplasm to thenucleus. Although it was established that vesicles, organellesand other colloidal structures are transported actively by themolecular motors associated with the micotubule network oractin filaments, there is a lack of knowledge about activeDNA transport within the cytoplasm [27].

An important factor in DNA expression is the size of the pla-mid. Oligonucleotides comprising 20 - 30 base pairs rapidly andpreferentially accumulate into the nucleus when delivered vialipoplexes [220] or cytoplasmic injection [221]. The diffusion-pas-sage through the nuclear membrane is not possible for largerpieces of DNA, due to the structure of the nuclear pores [222],



which are freely permeable only to solutes < ∼ 9 nm (40 - 60KDa proteins) [223-225]. This problem may be solved by [70,225-227]

following the eukaryotic DNA virus pathway of replication,which uses the cell’s nuclear import machinery [224,228]. In arecent study, Zanta et al. [225] showed that linking an oligonucle-otide to a peptide containing the nuclear localisation signal(NLS) could enhance transfection up to 1000-fold. Other NLS-based approaches followed this work [229,230]. Histones [231,232],

or histone fragments [233] were also used in conjunction withcationic lipids in order to improve the transfection efficiency.

It was also demonstrated that the import of the plasmidDNA into the nucleus is a sequence-specific process [234] andthat it is also dependent on the cell cycle [235, 236].

5. Therapeutic applications

A recent overview of gene therapy clinical trials revealed thatcationic lipid mediated transfection protocols account for aboutone fifth of the total ongoing studies in this field [401]. Detailedpresentation of the gene therapy protocols, including targeteddiseases and protocols levels, are also available [177,235,237].

Lipoplex-mediated transfection has been successfully usedin the treatment of cancer [180,238-240], monogenic diseasesespecially cystic fibrosis [174,179,189,241,242], coronary andperipheral artery diseases [171,243], neurological diseases [183],renal [173], vision [186,244], articular [184] impairments etc. [7].

An important aspect associated with the use of lipoplexesin vivo is their cytotoxicity [190,240,245]. Although the subjectwas intensively studied, the results were controversial[119,190,246]. It is certain that the structure of all the compo-nents of the lipoplex influence the overall toxicity of the for-mulation [130,245-247]. In respect with the cationic lipids,sustained efforts were made to overcome this problem, byusing biodegradable building blocks in their structure (seeabove), non-toxic counterions [102], appropriate dose [248],delivery methods [178,249] and lipoplex generation techniques[29], as well as by correlating the structure of the vector withthe desired biodistribution of the lipoplex [80,170,186].

6. Conclusions

The contest for designing the magic lipid continues, poweredby recent progress in understanding the factors affecting lipo-some-mediated gene transfer [27,54,58,61,62,164-167]. Based on

structures previously assessed, more sophisticated transfer vec-tors were introduced, their designs targeting specific mecha-nisms of cellular intake. Also, new types of cationic lipids wereintroduced, thus expanding the structure database availablefor non-viral transfection agents.

7. Expert opinion

New insights into different transfection mechanisms will gen-erate new working hypotheses, so that the structure-to-mech-anism adapting process will continue to generate increasingefficiency for transfection mediated by cationic lipids. Bioa-vailability of the chemical vectors in general and cationic lip-ids in particular, remains an important factor to beconsidered.

In the case of cationic lipid vectors, as previously men-tioned, the transfection efficiency is usually influenced by thecell line. Although the discovery of a generally-efficient andideal cationic lipid constitutes the goal of many researchgroups, one is left with the impression that different targetswill require different vectors. Therefore, expanding the struc-tural diversity of the delivering agents will be appropriate.

Although competition between biological and chemicaltransfection systems and even between the different chemi-cal methods of gene delivery, lipoplex or polyplex-based,appears to be very challenging, it is likely that the ideal vec-tor (complex) will carry the characteristics of more than onegroup [70,250,251]. Thus, the highly compacted complexesbetween DNA and protamine, a natural cationic peptide,have been entrapped in cationic liposomes forming cationiclipid-protamine-DNA (LPD) complexes [252,253], whichexhibited enhanced in vivo transfection efficiency comparedto the corresponding lipoplexes [254,255]. Similar results havebeen reported by Schwartz et al., using short peptidesderived from human histone or protamine as DNA-pre-compacting agents [233]. Successful gene therapy for Canavandisease was first accomplished with LPD systems [256] andonly afterwards with recombinant viruses. A step forward toartificial viruses has been reported recently: cationic viralpeptides enhanced classical lipoplex-mediated gene delivery[257], confirming that interaction, competition and collabo-ration between researchers in various fields, from virologiststo chemists, remains particularly important for obtainingthe ultimate transfection vector.

Bibliography

1. LANDER ES, LINTON LM, BIRREN B, NUSBAUM C: Initial sequencing and analysis of the human genome. Nature (2001) 409:860-921.

2. VENTER JC, ADAMS MD, MYERS EW et al.: The sequence of the human genome. Science (2001) 291:1304-1351.

3. Gene Therapy: Lemoine NR, Cooper DN

(Eds.), Oxford, BIOS Scientific Publishers, (1996).

4. Gene Therapy. Therapeutical Mechanisms and Strategies: Templeton NS, Lasic DD (Eds.), New York, Basel, Marcel Dekker, (2000).

5. FRIEDMANN T: Gene Therapy: Delivering the Medicines of the 21st Century. Nat. Med. (1996) 2:144-147.

6. SCHERMAN D, BESSODES M,

CAMERON B et al.: Application of lipids and plasmid design for gene delivery to mammalian cells. Curr. Opin. Biotechnol (1998) 9:480-485.

7. MOUNTAIN A: Gene therapy: the first decade. Trends Biotechnol. (2000) 18:119-128.

8. BEHR JP: Synthetic gene-transfer vectors. Acc. Chem. Res. (1993) 26:274-278.

9. DESHMUKH HM, HUANG L: Liposome

Ilies & Balaban


and polylysine mediated gene transfer. New J. Chem. (1997) 21:113-124.

10. WOLFF JA, TRUBETSKOY VS: The Cambrian period of nonviral gene delivery. Nat. Biotechnol. (1998) 16:421-422.

11. ANGELOVA MI, HRISTOVA N, TSONEVA I: DNA-induced endocytosis upon local microinjection to giant unilamellar cationic vesicles. Eur. Biophys. J. (1999) 28:142-150.

12. HENGGE UR, TAICHMAN LB, KAUR P et al.: How realistic is cutaneous gene therapy? Exp. Dermatol. (1999) 8:419-431.

13. WEAVER JC: Electroporation: a general phenomenon for manipulating cells and tissues. J. Cell Biochem. (1993) 51:426-435.

14. ZHANG L, LI L, HOFFMANN GA, HOFFMAN RM: Depth-targeted efficient gene delivery and expression in the skin by pulsed electric fields: an approach to gene therapy of skin aging and other diseases. Biochem. Biophys. Res. Commun. (1996) 220:633-636.

15. JAROSZESKI MJ, GILBERT R, NICOLAU C, HELLER R: In vivo gene delivery by electroporation. Adv. Drug Deliv. Rev. (1999) 35:131-137.

16. VANBEVER R, PREAT VV: In vivo efficacy and safety of skin electroporation. Adv. Drug Deliv. Rev. (1999) 35:77-88.

17. WELLS JM, LI LH, SEN A, JAHREIS GP, HUI SW: Electroporation-enhanced gene delivery in mammary tumors. Gene Ther. (2000) 7:541-547.

18. TANELIAN DL, BARRY MA, JOHNSTON SA, LE T, SMITH G: Controlled gene gun delivery and expression of DNA within the cornea. Biotechniques (1997) 23:484-488.

19. UDVARDI A, KUFFERATH I, GRUTSCH H, ZATLOUKAL K, VOLC-PLATZER B: Uptake of exogenous DNA via the skin. J. Mol. Med. (1999) 77:744-750.

20. SATO H, HATTORI S, KAWAMOTO S et al.: In vivo gene gun-mediated DNA delivery into rodent brain tissue. Biochem. Biophys. Res. Commun. (2000) 270:163-170.

21. ROBBINS PD, GHIVIZZANI SC: Viral vectors for gene therapy. Pharmacol. Ther. (1998) 80:35-47.

22. STRAYER DS: Viral gene delivery. Expert Opin. Investig. Drugs (1999) 8:2159-2172.

23. WALTHER W, STEIN U: Viral vectors for gene transfer: a review of their use in the

treatment of human diseases. Drugs (2000) 60:249-271.

24. KAY MA, GLORIOSO JC, NALDINI L: Viral vectors for gene therapy: the art of turning infectious agents into vehicles of therapeutics. Nat. Med. (2001) 7:33-40.

25. PITARD B, AGUERRE O, AIRIAU M et al.: Virus-sized self-assembling lamellar complexes between plasmid DNA and cationic micelles promote gene transfer. Proc. Natl. Acad. Sci. USA (1997) 94:14412-14417.

26. CHONN A, CULLIS P: Recent advances in liposome technologies and their applications for systemic gene delivery. Adv. Drug Deliv. Rev. (1998) 30:73-83.

27. POUTON CW, SEYMOUR LW: Key issues in non-viral gene delivery. Adv. Drug Deliv. Rev. (1998) 34:3-19.

28. BALLY MB, HARVIE P, WONG FM, KONG S, WASAN EK, REIMER DL: Biological barriers to cellular delivery of lipid-based DNA carriers. Adv. Drug Deliv. Rev. (1999) 38:291-315.

29. HOFLAND HE, SHEPHARD L, SULLIVAN SM: Formation of stable cationic lipid/DNA complexes for gene transfer. Proc. Natl. Acad. Sci. USA (1996) 93:7305-7309.

30. ANCHORDOQUY TJ, ALLISON SD, MOLINA MC, GIROUARD LG, CARSON TK: Physical stabilisation of DNA-based therapeutics. Drug Discov. Today (2001) 6:463-470.

31. FELGNER PL, BARENHOLZ Y, BEHR JP et al.: Nomenclature for synthetic gene delivery systems. Hum. Gene Ther. (1997) 8:511-512.

32. EASTMAN SJ, SIEGEL C, TOUSIGNANT J, SMITH AE, CHENG SH, SCHEULE RK: Biophysical characterization of cationic lipid: DNA complexes. Biochim. Biophys. Acta (1997) 1325:41-62.

33. WONG FM, REIMER DL, BALLY MB: Cationic lipid binding to DNA: characterization of complex formation. Biochemistry (1996) 35:5756-5763.

34. BATTERSBY BJ, GRIMM R, HUEBNER S, CEVC G: Evidence for three-dimensional interlayer correlations in cationic lipid-DNA complexes as observed by cryo-electron microscopy. Biochim. Biophys. Acta (1998) 1372:379-383.

35. HARRIES D, MAY S, GELBART WM, BEN-SHAUL A: Structure, stability, and thermodynamics of lamellar DNA-lipid

complexes. Biophys. J. (1998) 75:159-173.

36. HUEBNER S, BATTERSBY BJ, GRIMM R, CEVC G: Lipid-DNA complex formation: reorganization and rupture of lipid vesicles in the presence of DNA as observed by cryoelectron microscopy. Biophys. J. (1999) 76:3158-3166.

37. FELGNER PL, GADEK TR, HOLM M et al.: Lipofection: a highly efficient, lipid-mediated DNA-transfection procedure. Proc. Natl. Acad. Sci. USA (1987) 84:7413-7417.

38. FELGNER PL, RINGOLD GM: Cationic liposome-mediated transfection. Nature (1989) 337:387-388.

39. MALONE RW, FELGNER PL, VERMA IM: Cationic liposome-mediated RNA transfection. Proc. Natl. Acad. Sci. USA (1989) 86:6077-6081.

40. BEHR JP: DNA strongly binds to micelles and vesicles containing lipopolyamines or lipointercalants. Tetrahedron Lett. (1986) 27:5861-5864.

41. BEHR JP, DEMENEIX B, LOEFFLER JP, PEREZ-MUTUL J: Efficient gene transfer into mammalian primary endocrine cells with lipopolyamine-coated DNA. Proc. Natl. Acad. Sci. USA (1989) 86:6982-6986.

42. PINNADUWAGE P, SCHMITT L, HUANG L: Use of a quaternary ammonium detergent in liposome mediated DNA transfection of mouse L-cells. Biochim. Biophys. Acta (1989) 985:33-37.

43. LEVENTIS R, SILVIUS JR: Interactions of mammalian cells with lipid dispersions containing novel metabolizable cationic amphiphiles. Biochim. Biophys. Acta (1990) 1023:124-132.

44. GAO X, HUANG L: A novel cationic liposome reagent for efficient transfection of mammalian cells. Biochim. Biophys. Acta (1991) 179:280-285.

45. WU GY, WU CH: Receptor-mediated in vitro gene transformation by a soluble DNA carrier system. J. Biol. Chem. (1987) 262:4429-4432.

46. WU GY, WU CH: Receptor-mediated gene delivery and expression in vivo. J. Biol. Chem. (1988) 263:14621-14624.

47. WAGNER E, ZENKE M, COTTEN M, BEUG H, BIRNSTIEL ML: Transferrin-polycation conjugates as carriers for DNA uptake into cells. Proc. Natl. Acad. Sci. USA (1990) 87:3410-3414.

48. ZHOU XH, KLIBANOV AL, HUANG L: Lipophylic polylysines mediate efficient DNA transfection in mammalian cells.



Biochim. Biophys. Acta (1991) 1065:8-14.

49. KONSTANTINOVA ID, SEREBRENNIKOVA GA: Positively charged lipids: structure, methods of synthesis and applications. Russ. Chem. Rev. (1996) 65:537-553.

50. LEE RJ, HUANG L: Lipidic vector systems for gene transfer. Crit. Rev. Ther. Drug Carrier Syst. (1997) 14:173-206.

51. ZABNER J: Cationic lipids used in gene transfer. Adv. Drug Deliv. Rev. (1997) 27:17-28.

52. FELGNER PL: Nonviral strategies for gene therapy. Sci. Am. (1997) 276:102-106.

53. MILLER AD: Cationic liposomes for gene therapy. Angew. Chem. Int. Ed. (1998) 37:1768-1785.

54. TSENG WC, HUANG L: Liposome-based gene therapy. Pharm. Sci. Tech. Today (1998) 1:206-213.

55. REMY JS, ABDALLAH B, ZANTA MA, BOUSSIF O, BEHR JP, DEMENEIX B: Gene transfer with lipospermines and polyethylenimines. Adv. Drug Deliv. Rev. (1998) 30:85-95.

56. KABANOV AV, KABANOV VA: Interpolyelectrolyte and block ionomer complexes for gene delivery: physico-chemical aspects. Adv. Drug Deliv. Rev. (1998) 30:49-60.

57. KABANOV AV: Taking polycation gene delivery systems from in vitro to in vivo. Pharm. Sci. Tech. Today (1999) 2:365-372.

58. CHESNOY S, HUANG L: Structure and function of lipid-DNA complexes for gene delivery. Ann. Rev. Biophys. Biomol. Struct. (2000) 29:27-47.

59. MADSEN SK, MOONEY DJ: Delivering DNA with polymer matrices: application in tissue engineering and gene therapy. Pharm. Sci. Tech. Today (2000) 3:381-384.

60. ESFAND R, TOMALIA DA: Poly(amidoamine) (PAMAM) dendrimers: from biomimicry to drug delivery and biomedical applications. Drug Discov. Today (2001) 6:427-436.

61. NISHIKAWA M, HUANG L: Nonviral vectors in the new millennium: delivery barriers in gene transfer. Hum. Gene Ther. (2001) 12:861-870.

62. PEDROSO DE LIMA MC, SIMOES S, PIRES P, FANECA H, DUZGUNES N: Cationic lipid-DNA complexes in gene delivery: from biophysics to biological applications. Adv. Drug Deliv. Rev. (2001) 47:277-294.

63. BYK G, SCHERMAN D: Novel cationic lipids for gene delivery and gene therapy. Expert Opin. Ther. Patents (1998) 8:1125-1141.

64. DEONARAIN MP: Ligand-targeted receptor-mediated vectors for gene delivery. Expert Opin. Ther. Patents (1998) 8:53-69.

65. REN T, LIU D: Synthesis of cationic lipids from 1,2,4-butanetriol. Tetrahedron Lett. (1999) 40:209-212.

66. REN T, LIU D: Synthesis of diether-linked cationic lipids for gene delivery. Bioorg. Med. Chem. Lett. (1999) 9:1247-1250.

67. REN T, SONG YK, ZHANG G, LIU D: Structural basis of DOTMA for its high intravenous transfection activity in mouse. Gene Ther. (2000) 7:764-768.

68. REN T, ZHANG G, SONG YK, LIU D: Synthesis and characterization of aromatic ring-based cationic lipids for gene delivery in vitro and in vivo. J. Drug Targets (1999) 7:285-292.

69. GAO X, HUANG L: Cationic liposome-mediated gene transfer. Gene Ther. (1995) 2:710-722.

70. REMY JS, KICHLER A, MORDVINOV V, SCHUBER F, BEHR JP: Targeted gene transfer into hepatoma cells with lipopolyamine-condensed DNA particles presenting galactose ligands: a stage toward artificial viruses. Proc. Natl. Acad. Sci. USA (1995) 92:1744-1748.

71. KAWAKAMI S, FUMOTO S, NISHIKAWA M, YAMASHITA F, HASHIDA M: In vivo gene delivery to the liver using novel galactosylated cationic liposomes. Pharm. Res. (2000) 17:306-313.

72. REN T, LIU D: Synthesis of targetable cationic amphiphiles. Tetrahedron Lett. (1999) 40:7621-7625.

73. BHATTACHARYA S, DILEEP PV: Synthesis of novel cationic lipids with oxyethylene spacers at the linkages between hydrocarbon chains and pseudoglyceryl backbone. Tetrahedron Lett. (1999) 40:8167-8171.

74. FELGNER JH, KUMAR R, SRIDHAR CN et al.: Enhanced gene delivery and mechanism studies with a novel series of cationic lipid formulations. J. Biol. Chem. (1994) 269:2550-2561.

75. WHEELER CJ, FELGNER PL, TSAI YJ et al.: A novel cationic lipid greatly enhances plasmid DNA delivery and expression in mouse lung. Proc. Natl. Acad. Sci. USA (1996) 93:11454-11459.

76. LE BOLC’H G, LEBRIS N, YAOUANC

JJ, CLEMENT JC, DES ABBAYES H: Cationic phosphonolipids as non viral vectors for DNA transfection. Tetrahedron Lett. (1995) 36:6681-6684.

77. FLOCH V, DELEPINE P, GUILLAUME C et al.: Systemic administration of cationic phosphonolipids/DNA complexes and the relationship between formulation and lung transfection efficiency. Biochim. Biophys. Acta (2000) 1464:95-103.

78. DELEPINE P, GUILLAUME C, FLOCH V et al.: Cationic phosphonolipids as nonviral vectors: in vitro and in vivo applications. J. Pharm. Sci. (2000) 89:629-638.

79. FLOCH V, LEGROS N, LOISEL S et al.: New biocompatible cationic amphiphiles derivative from glycine betaine: a novel family of efficient nonviral gene transfer agents. Biochem. Biophys. Res. Commun. (1998) 251:360-365.

80. FLOCH VV, LOISEL S, GUENIN E et al.: Cation substitution in cationic phosphonolipids: a new concept to improve transfection activity and decrease cellular toxicity. J. Med. Chem (2000) 43:4617-4628.

81. GUILLAUME C, DELEPINE P, DROAL C, MONTIER T, TYMEN G, CLAUDE F: Aerosolization of cationic lipid-DNA complexes: lipoplex characterization and optimization of aerosol delivery conditions. Biochem. Biophys. Res. Commun. (2001) 286:464-471.

82. NAZIH A, CORDIER Y, BISCHOFF R, KOLBE HVJ, HEISSLER D: Synthesis and stability study of the new pentammonio lipid pcTG90, a gene transfer agent. Tetrahedron Lett. (1999) 40:8089-8091.

83. SCHUGHART K, BISCHOFF R, ALI HADJI D et al.: Effect of liposome-encapsulated clodronate pretreatment on synthetic vector-mediated gene expression in mice. Gene Ther. (1999) 6(3):448-453.

84. CHOI JS, LEE EJ, JANG HS, PARK JS: New cationic liposomes for gene transfer into mammalian cells with high efficiency and low toxicity. Bioconjug. Chem. (2001) 12:108-113.

85. REN T, ZHANG G, LIU F, LIU D: Synthesis and evaluation of vitamin D-based cationic lipids for gene delivery in vitro. Bioorg. Med. Chem. Lett. (2000) 10:891-894.

86. BESSODES M, DUBERTRET C, JASLIN G, SCHERMAN D: Synthesis and

Ilies & Balaban


biological properties of new glycosidic cationic lipids for DNA delivery. Bioorg. Med. Chem. Lett. (2000) 10:1393-1395.

87. BYK G, DUBERTRET C, ESCRIOU V et al.: Synthesis, activity, and structure-activity relationship studies of novel cationic lipids for DNA transfer. J. Med. Chem. (1998) 41:229-235.

88. TANG F, HUGHES JA: Introduction of a disulfide bond into a cationic lipid enhances transgene expression of plasmid DNA. Biochem. Biophys. Res. Commun. (1998) 242:141-145.

89. TANG F, WANG W, HUGHES JA: Cationic liposomes containing disulfide bonds in delivery of plasmid DNA. J. Lipid Res. (1999) 9:331-347.

90. TANG F, HUGHES JA: Use of dithiodiglycolic acid as a tether for cationic lipids decreases the cytotoxicity and increases transgene expression of plasmid DNA in vitro. Bioconjug. Chem. (1999) 10:791-796.

91. BYK G, WETZER B, FREDERIC M et al.: Reduction-sensitive lipopolyamines as a novel nonviral gene delivery system for modulated release of DNA with improved transgene expression. J. Med. Chem. (2000) 43:4377-4387.

92. WETZER B, BYK G, FREDERIC M et al.: Reducible cationic lipids for gene transfer. Biochem. J. (2001) 356:747-756.

93. HAWLEY-NELSON P, CICCARONE V, GEBEYEHU G, JESSEE J, FELGNER PL: Lipofectamine reagent: a new higher efficiency polycationic liposome transfection reagent. Focus (1993) 15:73-79.

94. FENJVES ES, SMITH J, ZARADIC S, TAICHMAN LB: Systemic delivery of secreted protein by grafts of epidermal keratinocytes: prospects for keratinocyte gene therapy. Hum. Gene Ther. (1994) 5:1241-1248.

95. FREDERIC M, SCHERMAN D, BYK G: Introduction of cyclic guanidines into cationic lipids for non-viral gene delivery. Tetrahedron Lett. (2000) 41:675-679.

96. SOLODIN I, BROWN CS, BRUNO MS et al.: A novel series of amphiphilic imidazolinium compounds for in vitro and in vivo gene delivery. Biochemistry (1995) 34:13537-13544.

97. MEEKEL AAP, WAGENAAR A, SMISTEROVA J et al.: Synthesis of pyridinium amphiphiles used for transfection and some characteristics of

amphiphile/DNA complex formation. Eur. J. Org. Chem. (2000):665-673.

98. HARRINGTON JJ, VAN BOKKELEN G, MAYS RW, GUSTASHAW K, WILLARD HF: Formation of de novo centromeres and construction of first-generation human artificial microchromosomes. Nat. Genet. (1997) 15:345-355.

99. SAFINYA CR: Structures of lipid-DNA complexes: supramolecular assembly and gene delivery. Curr. Opin. Struct. Biol. (2001) 11:440-448.

100. STERNBERG B, SORGI FL, HUANG L: New structures in complex formation between DNA and cationic liposomes visualized by freeze-fracture electron microscopy. FEBS Lett (1994) 356:361-366.

101. COLLINS KD: Sticky ions in biological systems. Proc. Natl. Acad. Sci. USA (1995) 92:5553-5557.

102. ABERLE AM, BENNETT MJ, MALONE RW, NANTZ MH: The counterion influence on cationic lipid-mediated transfection of plasmid DNA. Biochim. Biophys. Acta (1996) 1299:281-283.

103. COLLINS KD: Charge density-dependent strength of hydration and biological structure. Biophys. J. (1997) 72:65-76.

104. BENNETT MJ, ABERLE AM, BALASUBRAMANIAM RP, MALONE JG, MALONE RW, NANTZ MH: Cationic lipid-mediated gene delivery to murine lung: correlation of lipid hydration with in vivo transfection activity. J. Med. Chem. (1997) 40:4069-4078.

105. ZUIDAM NJ, BARENHOLZ Y: Electrostatic parameters of cationic liposomes commonly used for gene delivery as determined by 4-heptadecyl-7-hydroxycoumarin. Biochim. Biophys. Acta (1997) 1329:211-222.

106. FANG Y, YANG J: Two-dimensional condensation of DNA molecules on cationic lipid membranes. J. Phys. Chem. B. (1997) 101:441-449.

107. SPINK CH, CHAIRES JB: Thermodinamics of the binding of a cationic lipid to DNA. J. Am. Chem. Soc. (1997) 119:10920-10928.

108. SARAPUK J, KLESZCZYNSKA H, ROZYCKA-ROSZAK B: The role of counterions in the interaction of bifunctional surface active compounds with model membranes. Biochem. Mol. Biol. Int. (1998) 44:1105-1110.

109. SARAPUK J, KLESZCZYNSKA H,

PERNAK J, KALEWSKA J, ROZYCKA-ROSZAK B: Influence of counterions on the interaction of pyridinium salts with model membranes. Z. Naturforsch [C] (1999) 54:952-955.

110. ZANTL R, BAICU L, ARTZNER F, SPRENGER I, RAPP G, RADLER JO: Thermotropic phase behavior of cationic lipid-DNA complexes compared to binary lipid mixtures. J. Phys. Chem. B. (1999) 103:10300-10310.

111. HIRSCH-LERNER D, BARENHOLZ Y: Hydration of lipoplexes commonly used in gene delivery: follow-up by laurdan fluorescence changes and quantification by differential scanning calorimetry. Biochim. Biophys. Acta (1999) 1461:47-57.

112. BIRCHALL JC, KELLAWAY IW, MILLS SN: Physico-chemical characterisation and transfection efficiency of lipid-based gene delivery complexes. Int. J. Pharm. (1999) 183:195-207.

113. PITARD B, OUDRHIRI N, VIGNERON JP et al.: Structural characteristics of supramolecular assemblies formed by guanidinium-cholesterol reagents for gene transfection. Proc. Natl. Acad. Sci. USA (1999) 96:2621-2626.

114. ROZYCKA-ROSZAK B, PRUCHNIK H: Effect of counterions on the influence of dodecyltrimethylammonium halides on thermotropic phase behaviour of phosphatidylcholine bilayers. Z. Naturforsch (2000) 55c:240-244.

115. ROZYCKA-ROSZAK B, ZYLKA R, SARAPUK J: Hydration of alkylammonium salt micelles - influence of bromide and chloride counterions. Z. Naturforsch (2000) 55c:413-417.

116. ISHIWATA H, SUZUKI N, ANDO S, KIKUCHI H, KITAGAWA T: Characteristics and biodistribution of cationic liposomes and their DNA complexes. J. Control Release (2000) 69:139-148.

117. BHATTACHARYA S, HALDAR S: Interactions between cholesterol and lipids in bilayer membranes. Role of lipid headgroup and hydrocarbon chain-backbone linkage. Biochim. Biophys. Acta (2000) 1467:39-53.

118. EVEN-CHEN S, BARENHOLZ Y: DOTAP cationic liposomes prefer relaxed over supercoiled plasmids. Biochim. Biophys. Acta (2000) 1509:176-188.

119. SAKURAI F, INOUE R, NISHINO Y et al.: Effect of DNA/liposome mixing ratio



on the physicochemical characteristics, cellular uptake and intracellular trafficking of plasmid DNA/cationic liposome complexes and subsequent gene expression. J. Control Release (2000) 66:255-269.

120. PECTOR V, BACKMANN J, MAES D, VANDENBRANDEN M, RUYSSCHAERT JM: Biophysical and structural properties of DNA.diC(14)-amidine complexes. Influence of the DNA/lipid ratio. J. Biol. Chem. (2000) 275:29533-29538.

121. KIKUCHI IS, CARMONA-RIBEIRO AM: Interactions between DNA and synthetic cationic liposomes. J. Phys. Chem. B. (2000) 104:2829-2835.

122. WANGEREK LA, DAHL HH, SENDEN TJ et al.: Atomic force microscopy imaging of DNA-cationic liposome complexes optimised for gene transfection into neuronal cells. J. Gene Med. (2001) 3:72-81.

123. STEWART L, MANVELL M, HILLERY E et al.: Physico-chemical analysis of cationic liposome-DNA complexes (lipoplexes) with respect to in vitro and in vivo gene delivery efficiency. J. Chem. Soc. Perkin. Trans. (2001) 2:624-632.

124. LOBO BA, DAVIS A, KOE G, SMITH JG, MIDDAUGH CR: Isothermal titration calorimetric analysis of the interaction between cationic lipids and plasmid DNA. Arch. Biochem. Biophys. (2001) 386:95-105.

125. FARHOOD H, SERBINA N, HUANG L: The role of dioleoyl phosphatidylethanolamine in cationic liposome mediated gene transfer. Biochim. Biophys. Acta (1995) 1235:289-295.

126. BHATTACHARYA S, HALDAR S: The effects of cholesterol inclusion on the vesicular membranes of cationic lipids. Biochim. Biophys. Acta (1996) 1283:21-30.

127. ZUIDAM NJ, BARENHOLZ Y: Electrostatic and structural properties of complexes involving plasmid DNA and cationic lipids commonly used for gene delivery. Biochim. Biophys. Act. (1998) 1368:115-128.

128. KOYNOVA R, CAFFREY M: Phases and phase transitions of the phosphatidylcholines. Biochim. Biophys. Acta (1998) 1376:91-145.

129. ZUIDAM NJ, HIRSCH-LERNER D, MARGULIES S, BARENHOLZ Y: Lamellarity of cationic liposomes and mode of preparation of lipoplexes affect transfection efficiency. Biochim. Biophys. Acta (1999) 1419:207-220.

130. AUDOUY S, MOLEMA G, DE LEIJ L, HOEKSTRA D: Serum as a modulator of lipoplex-mediated gene transfection: dependence of amphiphile, cell type and complex stability. J. Gene Med. (2000) 2:465-476.

131. MATSUI H, PAN S: Distribution of DNA in cationic liposome complexes probed by Raman microscopy. Langmuir (2001) 17:571-573.

132. KERNER M, MEYUHAS O, HIRSCH-LERNER D, ROSEN LJ, MIN Z, BARENHOLZ Y: Interplay in lipoplexes between type of pDNA promoter and lipid composition determines transfection efficiency of human growth hormone in NIH3T3 cells in culture. Biochim. Biophys. Acta (2001) 1532:128-136.

133. ZUIDAM NJ, BARENHOLZ Y, MINSKY A: Chiral DNA packaging in DNA-cationic liposome assemblies. FEBS Lett. (1999) 457:419-422.

134. MEIDAN VM, COHEN JS, AMARIGLIO N, HIRSCH-LERNER D, BARENHOLZ Y: Interaction of oligonucleotides with cationic lipids: the relationship between electrostatics, hydration and state of aggregation. Biochim. Biophys. Acta (2000) 1464:251-261.

135. BERGAN D, GALBRAITH T, SLOANE DL: Gene transfer in vitro and in vivo by cationic lipids is not significantly affected by levels of supercoiling of a reporter plasmid. Pharm. Res. (2000) 17:967-973.

136. HIRSCH-LERNER D, BARENHOLZ Y: Probing DNA-cationic lipid interactions with the fluorophore trimethylammonium diphenyl-hexatriene (TMADPH). Biochim. Biophys. Acta (1998) 1370:17-30.

137. WONG FM, BALLY MB, BROOKS DE: Electrostatically mediated interactions between cationic lipid-DNA particles and an anionic surface. Arch. Biochem. Biophys. (1999) 366:31-39.

138. SON KK, TKACH D, HALL KJ: Efficient in vivo gene delivery by the negatively charged complexes of cationic liposomes and plasmid DNA. Biochim. Biophys. Acta (2000) 1468:6-10.

139. LEE H, WILLIAMS SK, ALLISON SD, ANCHORDOQUY TJ: Analysis of self-assembled cationic lipid-DNA gene carrier complexes using flow field-flow fractionation and light scattering. Anal. Chem. (2001) 73:837-843.

140. SEMPLE SC, KLIMUK SK, HARASYM TO et al.: Efficient encapsulation of

antisense oligonucleotides in lipid vesicles using ionizable aminolipids: formation of novel small multilamellar vesicle structures. Biochim. Biophys. Acta (2001) 1510:152-166.

141. RASPAUD E, PITARD B, DURAND D et al.: Polymorphism of DNA/multi-cationic Lipid complexes driven by temperature and salts. J. Phys. Chem. B. (2001) 105:5291-5297.

142. BOUKHNIKACHVILI T, AGUERRE-CHARIOL O, AIRIAU M, LESIEUR S, OLLIVON M, VACUS J: Structure of in-serum transfecting DNA-cationic lipid complexes. FEBS Lett. (1997) 409:188-194.

143. MEL’NIKOV SM, LINDMAN B: Solubilisation of DNA-cationic lipid complexes in hydrophobic solvents. A single molecule visualization by fluorescence microscopy. Langmuir (1999) 15:1923-1928.

144. GERSHON H, GHIRLANDO R, GUTTMAN SB, MINSKY A: Mode of formation and structural features of DNA-cationic liposome complexes used for transfection. Biochemistry (1993) 32:7143-7151.

145. MOK KW, CULLIS PR: Structural and fusogenic properties of cationic liposomes in the presence of plasmid DNA. Biophys. J. (1997) 73:2534-2545.

146. REIMER DL, ZHANG Y, KONG S, WHEELER JJ, GRAHAM RW, BALLY MB: Formation of novel hydrophobic complexes between cationic lipids and plasmid DNA. Biochemistry (1995) 34:12877-12883.

147. WAGNER K, HARRIES D, MAY S, KAHL V, RADLER JO, BEN-SHAUL A: Direct evidence for counterion release upon cationic lipid-DNA condensation. Langmuir (2000) 16:303-306.

148. SAMUNI AM, CROMMELIN DJA, ZUIDAM NJ, BARENHOLZ Y: Differential scanning calorimetry - a tool to assess physical and chemical alterations in liposomes. J. Therm. Anal. Calorim. (1998) 51:37-48.

149. BHATTACHARYA S, MANDAL SS: Interaction of surfactants with DNA. Role of hydrophobicity and surface charge on intercalation and DNA melting. Biochim. Biophys. Acta (1997) 1323:29-44.

150. ZUIDAM NJ, BARENHOLZ Y: Characterization of DNA-lipid complexes commonly used for gene delivery. Int. J. Pharm. (1999) 183:43-46.

Ilies & Balaban


151. RADLER JO, KOLTOVER I, SALDITT T, SAFINYA CR: Structure of DNA-cationic liposome complexes: DNA intercalation in multilamellar membranes in distinct interhelical packing regimes [see comments]. Science (1997) 275:810-814.

152. LASIC DD, STREY H, STUART MCA, PODGORNIK R, FREDERIC PM: The structure of DNA-liposome complexes. J. Am. Chem. Soc. (1997) 119:832-833.

153. KOLTOVER I, SALDITT T, RADLER JO, SAFINYA CR: An inverted hexagonal phase of cationic liposome-DNA complexes related to DNA release and delivery. Science (1998) 281:78-81.

154. RADLER JO, KOLTOVER I, JAMIESON A, SALDITT T, SAFINYA CR: Structure and interfacial aspects of self-assembled cationic lipid-DNA gene carrier complexes. Langmuir (1998) 14:4272-4283.

155. KOLTOVER I, WAGNER K, SAFINYA CR: DNA condensation in two dimensions. Proc. Natl. Acad. Sci. USA (2000) 97:14046-14051.

156. THOMSON NH, COLLIN I, DAVIES MC et al.: Atomic force microscopy of cationic liposomes. Langmuir (2000) 16:4813-4818.

157. DAN N: The structure of DNA complexes with cationic liposomes-cylindrical or flat bilayers? Biochim. Biophys. ActaActa (1998) 1369:34-38.

158. GUSTAFSSON J, ARVIDSON G, KARLSSON G, ALMGREN M: Complexes between cationic liposomes and DNA visualized by cryo-TEM. Biochim. Biophys. Acta (1995) 1235:305-312.

159. LITZINGER DC, HUANG L: Phosphatidylethanolamine liposomes: drug delivery, gene transfer and immunodiagnostic applications. Biochim. Biophys. Acta (1992) 1113:201-227.

160. LIN AJ, SLACK NL, AHMAD A et al.: Structure and structure-function studies of lipid/plasmid DNA complexes. J. Drug Targets (2000) 8:13-27.

161. ZHOU X, HUANG L: DNA transfection mediated by cationic liposomes containing lipopolylysine: characterization and mechanism of action. Biochim. Biophys. Acta (1994) 1189:195-203.

162. WROBEL I, COLLINS D: Fusion of cationic liposomes with mammalian cells occurs after endocytosis. Biochim. Biophys. Acta (1995) 1235:296-304.

163. SIMBERG D, HIRSCH-LERNER D, NISSIM R, BARENHOLZ Y: Comparison

of different commercially available cationic lipid-based transfection kits. J. Liposome Res. (2000) 10:1-13.

164. LASIC DD: Liposomes in Gene Delivery:. CRC Press, Boca Raton, (1997).

165. YANG JP, HUANG L: Overcoming the inhibitory effect of serum on lipofection by increasing the charge ratio of cationic liposome to DNA. Gene Ther. (1997) 4:950-960.

166. LI S, HUANG L: Nonviral gene therapy: promises and challenges. Gene Ther. (2000) 7:31-34.

167. DA CRUZ MT, SIMOES S, PIRES PP, NIR S, DE LIMA MC: Kinetic analysis of the initial steps involved in lipoplex--cell interactions: effect of various factors that influence transfection activity. Biochim. Biophys. Acta (2001) 1510:136-151.

168. BRAGONZI A, DINA G, VILLA A et al.: Biodistribution and transgene expression with nonviral cationic vector/DNA complexes in the lungs. Gene Ther. (2000) 7:1753-1760.

169. TAKAKURA Y, NISHIKAWA M, YAMASHITA F, HASHIDA M: Development of gene drug delivery systems based on pharmacokinetic studies. Eur. J. Pharm. Sci. (2001) 13:71-76.

170. UYECHI LS, GAGNE L, THURSTON G, SZOKA FC, Jr.: Mechanism of lipoplex gene delivery in mouse lung: binding and internalization of fluorescent lipid and DNA components. Gene Ther. (2001) 8:828-836.

171. MANN MJ: Gene therapy for peripheral arterial disease. Mol. Med. Today (2000) 6:285-291.

172. JAFFE A, JUDD D, RATCLIFFE C et al.: Cationic lipid-mediated gene transfer to the growing murine and human airway. Gene Ther. (2000) 7:273-278.

173. MADRY H, RESZKA R, BOHLENDER J, WAGNER J: Efficacy of cationic liposome-mediated gene transfer to mesangial cells in vitro and in vivo. J. Mol. Med. (2001) 79:184-189.

174. KOEHLER DR, HANNAM V, BELCASTRO R et al.: Targeting transgene expression for cystic fibrosis gene therapy. Mol. Ther. (2001) 4:58-65.

175. GOWDAK LH, POLIAKOVA L, LI Z, GROVE R, LAKATTA EG, TALAN M: Induction of angiogenesis by cationic lipid-mediated VEGF165 gene transfer in the rabbit ischemic hindlimb model. J. Vasc. Surg. (2000) 32:343-352.

176. HELLGREN I, DRVOTA V, PIEPER R et al.: Highly efficient cell-mediated gene transfer using non-viral vectors and FuGene6: in vitro and in vivo studies. Cell Mol. Life Sci. (2000) 57:1326-1333.

177. ROBBINS PD (Ed.): Gene Therapy Protocols. Humana Press, Totowa, USA (1997).

178. MOHR L, YOON SK, EASTMAN SJ et al.: Cationic liposome-mediated gene delivery to the liver and to hepatocellular carcinomas in mice. Hum. Gene Ther. (2001) 12:799-809.

179. ENNIST DL: Gene therapy for lung disease. Trends Pharm. Sci. (1999) 20:260-266.

180. CLARK PR, STOPECK AT, FERRARI M, PARKER SE, HERSH EM: Studies of direct intratumoral gene transfer using cationic lipid-complexed plasmid DNA. Cancer Gene Ther. (2000) 7:853-860.

181. SAITO H, NAKAMURA H, KATO S et al.: Percutaneous in vivo gene transfer to the peripheral lungs using plasmid-liposome complexes. Am. J. Physiol. Lung Cell Mol. Physiol. (2000) 279:L651-657.

182. HECKER JG, HALL LL, IRION VR: Nonviral gene delivery to the lateral ventricles in rat brain: initial evidence for widespread distribution and expression in the central nervous system. Mol. Ther. (2001) 3:375-384.

183. COSTANTINI LC, BAKOWSKA JC, BREAKEFIELD XO, ISACSON O: Gene therapy in the CNS. Gene Ther. (2000) 7:93-109.

184. MADRY H, TRIPPEL SB: Efficient lipid-mediated gene transfer to articular chondrocytes. Gene Ther. (2000) 7:286-291.

185. SPIRITO F, MENEGUZZI G, DANOS O, MEZZINA M: Cutaneous gene transfer and therapy: the present and the future. J. Gene Med. (2001) 3:21-31.

186. PLEYER U, GROTH D, HINZ B et al.: Efficiency and toxicity of liposome-mediated gene transfer to corneal endothelial cells. Exp. Eye Res. (2001) 73:1-7.

187. TAN PH, KING WJ, CHEN D et al.: Transferrin receptor-mediated gene transfer to the corneal endothelium. Transplantation (2001) 71:552-560.

188. STRIBLING R, BRUNETTE E, LIGGITT D, GAENSLER K, DEBS R: Aerosol gene delivery in vivo. Proc. Natl. Acad. Sci. USA (1992) 89:11277-11281.



189. DAVIES JC, GEDDES DM, ALTON EW: Prospects for gene therapy for cystic fibrosis. Mol. Med. Today (1998) 4:292-299.

190. RUIZ FE, CLANCY JP, PERRICONE MA et al.: A clinical inflammatory syndrome attributable to aerosolized lipid-DNA administration in cystic fibrosis. Hum. Gene Ther. (2001) 12:751-761.

191. ESCRIOU V, CIOLINA C, LACROIX F, BYK G, SCHERMAN D, WILS P: Cationic lipid-mediated gene transfer: effect of serum on cellular uptake and intracellular fate of lipopolyamine/DNA complexes. Biochim. Biophys. Acta (1998) 1368:276-288.

192. SAKURAI F, NISHIOKA T, SAITO H et al.: Interaction between DNA-cationic liposome complexes and erythrocytes is an important factor in systemic gene transfer via the intravenous route in mice: the role of the neutral helper lipid. Gene Ther. (2001) 8:677-686.

193. LI S, TSENG WC, STOLZ DB, WU SP, WATKINS SC, HUANG L: Dynamic changes in the characteristics of cationic lipidic vectors after exposure to mouse serum: implications for intravenous lipofection. Gene Ther. (1999) 6:585-594.

194. HASHIDA M, MAHATO RI, KAWABATA K, MIYAO T, NISHIKAWA M, TAKAKURA Y: Pharmacokinetics and targeted delivery of proteins and genes. J. Control Release (1996) 41:91-97.

195. BAILEY AL, CULLIS PR: Membrane fusion with cationic liposomes: effects of target membrane lipid composition. Biochemistry (1997) 36:1628-1634.

196. HAFEZ IM, MAURER N, CULLIS PR: On the mechanism whereby cationic lipids promote intracellular delivery of polynucleic acids. Gene Ther. (2001) 8:1188-1196.

197. ROSS PC, HUI SW: Lipoplex size is a major determinant of in vitro lipofection efficiency. Gene Ther. (1999) 6:651-659.

198. JANOFF AS: Liposomes: Rational Design New York, Basel, Marcel Dekker, (1999).

199. MISLICK KA, BALDESCHWIELER JD: Evidence for the role of proteoglycans in cation-mediated gene transfer. Proc. Natl. Acad. Sci. USA (1996) 93:12349-12354.

200. LABAT-MOLEUR F, STEFFAN AM, BRISSON C et al.: An electron microscopy study into the mechanism of gene transfer with lipopolyamines. Gene Ther. (1996) 3:1010-1017.

201. MOUNKES LC, ZHONG W, CIPRES-PALACIN G, HEATH TD, DEBS RJ:

Proteoglycans mediate cationic liposome-DNA complex-based gene delivery in vitro and in vivo. J. Biol. Chem. (1998) 273:26164-26170.

202. ZABNER J, FASBENDER AJ, MONINGER T, POELLINGER KA, WELSH MJ: Cellular and molecular barriers to gene transfer by a cationic lipid. J. Biol. Chem. (1995) 270:18997-19007.

203. ZELPHATI O, SZOKA FC, JR.: Intracellular distribution and mechanism of delivery of oligonucleotides mediated by cationic lipids. Pharm. Res. (1996) 13:1367-1372.

204. FRIEND DS, PAPAHADJOPOULOS D, DEBS RJ: Endocytosis and intracellular processing accompanying transfection mediated by cationic liposomes. Biochim. Biophys. Acta (1996) 1278:41-50.

205. EL OUAHABI A, THIRY M, PECTOR V, FUKS R, RUYSSCHAERT JM, VANDENBRANDEN M: The role of endosome destabilizing activity in the gene transfer process mediated by cationic lipids. FEBS Lett. (1997) 414:187-192.

206. COLIN M, MAURICE M, TRUGNAN G et al.: Cell delivery, intracellular trafficking and expression of an integrin-mediated gene transfer vector in tracheal epithelial cells. Gene Ther. (2000) 7:139-152.

207. MATSUI H, JOHNSON LG, RANDELL SH, BOUCHER RC: Loss of binding and entry of liposome-DNA complexes decreases transfection efficiency in differentiated airway epithelial cells. J. Biol. Chem. (1997) 272:1117-1126.

208. SIMOES S, SLEPUSHKIN V, PIRES P, GASPAR R, PEDROSO DE LIMA MC, DUZGUNES N: Human serum albumin enhances DNA transfection by lipoplexes and confers resistance to inhibition by serum. Biochim. Biophys. Acta (2000) 1463:459-469.

209. KAWAKAMI S, SATO A, NISHIKAWA M, YAMASHITA F, HASHIDA M: Mannose receptor-mediated gene transfer into macrophages using novel mannosylated cationic liposomes. Gene Ther. (2000) 7:292-299.

210. VAN DER WOUDE I, VISSER HW, TER BEEST MB et al.: Parameters influencing the introduction of plasmid DNA into cells by the use of synthetic amphiphiles as a carrier system. Biochim. Biophys. Acta (1995) 1240:34-40.

211. WATTIAUX R, JADOT M, WARNIER-PIROTTE MT, WATTIAUX-DE CONINCK S: Cationic lipids destabilize

lysosomal membrane in vitro. FEBS Lett. (1997) 417:199-202.

212. XU Y, SZOKA FC, Jr.: Mechanism of DNA release from cationic liposome/DNA complexes used in cell transfection. Biochemistry (1996) 35:5616-5623.

213. ZELPHATI O, SZOKA FC, Jr.: Mechanism of oligonucleotide release from cationic liposomes. Proc. Natl. Acad. Sci. USA (1996) 93:11493-11498.

214. BALASUBRAMANIAM RP, BENNETT MJ, ABERLE AM, MALONE JG, NANTZ MH, MALONE RW: Structural and functional analysis of cationic transfection lipids: the hydrophobic domain. Gene Ther. (1996) 3:163-172.

215. HAFEZ IM, ANSELL S, CULLIS PR: Tunable pH-sensitive liposomes composed of mixtures of cationic and anionic lipids. Biophys. J. (2000) 79:1438-1446.

216. BRISSON M, HUANG L: Liposomes: Conquering the nuclear barrier. Curr. Opin. Mol. Ther. (1999) 1:140-146.

217. NAKANISHI M, AKUTA T, NAGOSHI E, EGUCHI A, MIZUGUCHI H, SENDA T: Nuclear targeting of DNA. Eur. J. Pharm. Sci. (2001) 13:17-24.

218. CAPECCHI MR: High efficiency transformation by direct microinjection of DNA into cultured mammalian cells. Cell (1980) 22:479-488.

219. LECHARDEUR D, SOHN KJ, HAARDT M et al.: Metabolic instability of plasmid DNA in the cytosol: a potential barrier to gene transfer. Gene Ther. (1999) 6:482-497.

220. BENNETT CF, CHIANG MY, CHAN H, SHOEMAKER JE, MIRABELLI CK: Cationic lipids enhance cellular uptake and activity of phosphorothioate antisense oligonucleotides. Mol. Pharmacol. (1992) 41:1023-1033.

221. CHIN DJ, GREEN GA, ZON G, SZOKA FC, Jr., STRAUBINGER RM: Rapid nuclear accumulation of injected oligodeoxyribonucleotides. New Biol. (1990) 2:1091-1100.

222. STOFFLER D, FAHRENKROG B, AEBI U: The nuclear pore complex: from molecular architecture to functional dinamics. Curr. Opin. Cell Biol. (1999) 11:391-401.

223. LANG I, SCHOLZ M, PETERS R: Molecular mobility and nucleocytoplasmic flux in hepatoma cells. J. Cell Biol. (1986) 102:1183-1190.

224. ADAM SA: Transport pathways of macromolecules between the nucleus and

Ilies & Balaban


the cytoplasm. Curr. Opin. Cell Biol. (1999) 11:402-406.

225. ZANTA MA, BELGUISE-VALLADIER P, BEHR JP: Gene delivery: A single nuclear localisation signal peptide is sufficient to carry DNA to the cell nucleus. Proc. Natl. Acad. Sci. USA (1999) 96:91-96.

226. KANEDA Y, IWAI K, UCHIDA T: Increased expression of DNA cointroduced with nuclear protein in adult rat liver. Science (1989) 243:375-378.

227. ARONSOHN AI, HUGHES JA: Nuclear localisation signal peptides enhance cationic liposome-mediated gene therapy. J. Drug Targets (1997) 5:163-169.

228. CSERMELY P, SCHNAIDER T, SZANTO I: Signalling and transport through the nuclear membrane. Biochim. Biophys. Acta (1995) 1241:425-452.

229. BRANDEN LJ, MOHAMED AJ, SMITH CI: A peptide nucleic acid-nuclear localization signal fusion that mediates nuclear transport of DNA. Nat. Biotechnol. (1999) 17:784-787.

230. SUBRAMANIAN A, RANGANATHAN P, DIAMOND SL: Nuclear targeting peptide scaffolds for lipofection of nondividing mammalian cells. Nat. Biotechnol. (1999) 17:873-877.

231. HAGSTROM JE, SEBESTYEN MG, BUDKER V, LUDTKE JJ, FRITZ JD, WOLFF JA: Complexes of non-cationic liposomes and histone H1 mediate efficient transfection of DNA without encapsulation. Biochim. Biophys. Acta (1996) 1284:47-55.

232. HABERLAND A, KNAUS T, ZAITSEV SV et al.: Histone H1-mediated transfection: serum inhibition can be overcome by Ca2+ ions. Pharm. Res. (2000) 17:229-235.

233. SCHWARTZ B, IVANOV MA, PITARD B et al.: Synthetic DNA-compacting peptides derived from human sequence enhance cationic lipid-mediated gene transfer in vitro and in vivo. Gene Ther. (1999) 6:282-292.

234. DEAN DA: Import of plasmid DNA into the nucleus is sequence specific. Exp. Cell Res. (1997) 230:293-302.

235. MORTIMER I, TAM P, MACLACHLAN I, GRAHAM RW, SARAVOLAC EG, JOSHI PB: Cationic lipid-mediated transfection of cells in culture requires mitotic activity. Gene Ther. (1999) 6:403-411.

236. BRUNNER S, SAUER T, CAROTTA S, COTTEN M, SALTIK M, WAGNER E: Cell cycle dependence of gene transfer by

lipoplex, polyplex and recombinant adenovirus. Gene Ther. (2000) 7:401-407.

237. Summary Table: Human Gene Therapy Protocols: Hum. Gene Ther. (2001) 12(12):1585.

238. ANWER K, MEANEY C, KAO G et al.: Cationic lipid-based delivery system for systemic cancer gene therapy. Cancer Gene Ther. (2000) 7:1156-1164.

239. ANWER K, KAO G, PROCTOR B, ROLLAND A, SULLIVAN S: Optimization of cationic lipid/DNA complexes for systemic gene transfer to tumor lesions. J. Drug Targets (2000) 8:125-135.

240. SPACK EG, SORGI FL: Developing non-viral DNA delivery systems for cancer and infectious disease. Drug Discov. Today (2001) 6:186-197.

241. GEDDES D, ALTON E: Cystic fibrosis clinical trials. Adv. Drug Deliv. Rev. (1998) 30:205-217.

242. DAVIES JC, GEDDES DM, ALTON EWFW: Prospects for gene therapy in lung disease. Curr. Opin. Pharmacol. (2001) 1: 272-277.

243. SCHOLL FG, SEN L, DRINKWATER DC et al.: Effects of human tissue plasminogen gene transfer on allograft coronary atherosclerosis. J. Heart Lung Transplant. (2001) 20:322-329.

244. ABUL-HASSAN K, WALMSLEY R, BOULTON M: Optimisation of non-viral gene transfer to human primary retinal pigment epithelial cells. Curr. Eye Res. (2000) 20:361-366.

245. BARENHOLZ Y: Liposome application: problems and prospects. Curr. Opin. Coll. Interf. Sci. (2001) 6:66-77.

246. MCLACHLAN G, STEVENSON BJ, DAVIDSON DJ, PORTEOUS DJ: Bacterial DNA is implicated in the inflammatory response to delivery of DNA/DOTAP to mouse lungs. Gene Ther. (2000) 7:384-392.

247. LOISEL S, LE GALL C, DOUCET L, FEREC C, FLOCH V: Contribution of plasmid DNA to hepatotoxicity after systemic administration of lipoplexes. Hum. Gene Ther. (2001) 12:685-696.

248. NAGAHIRO I, MORA BN, BOASQUEVISQUE CH, SCHEULE RK, PATTERSON GA: Toxicity of cationic liposome-DNA complex in lung isografts. Transplantation (2000) 69:1802-1805.

249. TAN Y, LIU F, LI Z, LI S, HUANG L: Sequential injection of cationic liposome

and plasmid DNA effectively transfects the lung with minimal inflammatory toxicity. Mol. Ther. (2001) 3:673-682.

250. LEHN P, FABREGA S, OUDRHIRI N, NAVARRO J: Gene delivery systems: Bridging the gap between recombinant viruses and artificial vectors. Adv. Drug Deliv. Rev. (1998) 30:5-11.

251. BLESSING T, REMY JS, BEHR JP: Monomolecular collapse of plasmid DNA into stable virus-like particles. Proc. Natl. Acad. Sci. USA (1998) 95:1427-1431.

252. GAO X, HUANG L: Potentiation of cationic liposome-mediated gene delivery by polycations. Biochemistry (1996) 35:1027-1036.

253. SORGI FL, BHATTACHARYA S, HUANG L: Protamine sulfate enhances lipid-mediated gene transfer. Gene Ther. (1997) 4:961-968.

254. LI S, HUANG L: In vivo gene transfer via intravenous administration of cationic lipid- protamine-DNA (LPD) complexes. Gene Ther. (1997) 4:891-900.

255. LI S, RIZZO MA, BHATTACHARYA S, HUANG L: Characterization of cationic lipid-protamine-DNA (LPD) complexes for intravenous gene delivery. Gene Ther. (1998) 5:930-937.

256. LEONE P, JANSON CG, BILIANUK L et al.: Aspartoacylase gene transfer to the mammalian central nervous system with therapeutic implications for Canavan disease. Ann. Neurol. (2000) 48: 27-38.

257. MURRAY KD, ETHERIDGE CJ, SHAH SI et al.: Enhanced cationic liposome-mediated transfection using the DNA-binding peptide mu (mu) from the adenovirus core. Gene Ther. (2001) 8:453-460.

PatentsPatents of special note have been highlighted as either of interest (•) or of considerable interest (••) to readers.

301. SYNTEX, INC.: EP-172007 (1986).

302. SIGMA-TAU IND. FARMACHEUTICHE RIUNITE SPA: WO9957094 (1999).

303. LIFE TECH., INC.: US6075012 (2000).

304. UNIV. OF PITTSBURGH: WO9622765 (1996).

305. UNIV. PARIS NORD: WO0111068 (2001).

306. A HACES: WO0012454 (2000).



307. VICAL, INC.: WO0073263 (2000).

308. CENTRE NATIONAL DE LA RECHERCHE SCIENTIFIQUE: EP-394111 (1990).

•• The present invention discloses the first lipopolyamine active in gene delivery.

309. MAX-DELBRÜCK-CENTRUM FÜR MOLEKULARE MEDIZIN: WO9805678 (1998).

310. TRANSGENE SA: WO9837916 (1998).

311. GENTA, INC.: US6020526 (2000).

312. GENTA, INC.: US5958901 (1999).

313. PARK JS, CHOI JS, LEE EJ, JANG HK: WO0073471 (2000).

314. RHÔNE-POULENC RORER SA: WO9938821 (1999).

•• The present invention describes a reduction-sensitive lipopolyamines, for modulated release of DNA from cationic lipid/DNA complexes.

315. UNIV. OF FLORIDA: WO9958152 (1999).

•• This patent also presents prototypes for reduction-sensitive cationic lipids useful in modulated DNA release from lipoplexes.

316. UNIV. OF FLORIDA: US6153434 (2000).

317. ISIS PHARM., INC.: WO0059474 (2000).

318. CELLTECH THERAPEUTICS LTD.: WO0064858 (2000).

• The present invention introduced bipolar polycationic lipids as a new class of lipidic transfection vectors.

319. LIFE TECHNOLOGIES, INC.: WO9427435 (1994).

320. VICAL, INC.: WO9814439 (1998).

321. VICAL, INC.: US6022874 (2000).

322. LIFE TECHNOLOGIES, INC.: WO99517373 (1995).

323. SYNTEX, INC.: US6034137 (2000).•• The present invention systematically

investigates the field of guanidino cationic lipids for gene delivery.

324. MEGABIOS CORP.: WO9925342 (1999).

325. ALZA CORP.: WO0126629 (2001).

326. TEXAS A&M UNIV.: US Provisional Appl. 60/304249 (2001).

Websites

401. Clinical trial internet site. www.wiley.co.uk/wileychi/genmed/clinical/(Charts and Statistics), last updated september 2001.

AffiliationMarc Antoniu Ilies1 & Alexandru T BalabanTexas A&M University at Galveston, Department of Oceanography and Marine Sciences, 5007 Avenue U, Galveston, TX 7755, USA. Fax: 409-740-4787;E-mail: [email protected], [email protected] leave from Department of Chemistry, Faculty of Biotechnologies, University of Agricultural Sciences and Veterinary Medicine, Bucharest, Romania

recent developments in cationic lipid-mediated gene delivery and gene therapy

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