Table Of Content1
Recent Advances in Non-viral
Gene Delivery
Christine C. Conwell and Leaf Huang
CenterforPharmacogenetics, School ofPharmacy
UniversityofPittsburgh
Pittsburgh,Pennsylvania 15261
I. Introduction
II. Cationic Lipids
A. Ligand-mediated targeting
B. Toxicity
III. Cationic Polymers
A. Novel polymer mixtures
B. Toxicity
IV. Triggered Release
A. pH sensitivity
B. Redox sensitivity
V. Physical Delivery Methods for Naked DNA
A. High pressure delivery methods
B. Electroporation
C. Laser beam gene transduction
D. Ultrasound
E. Magnetofection
F. Photochemical internalization
VI. Prospects
Acknowledgment
References
AdvancesinGenetics,Vol.53 0065-2660/05$35.00
Copyright2005,ElsevierInc.Allrightsreserved. DOI:10.1016/S0065-2660(05)53001-3
4 Conwell and Huang
ABSTRACT
Genetherapyhasbeendeemedthemedicineofthefutureduetoitspotentialto
treat many types of diseases. However, many obstacles remain before gene
deliveryisoptimizedtospecifictargetcells.Overthelastseveraldecades,many
approachestogenedeliveryhavebeencloselyexamined.Byunderstandingthe
factors that determine the efficiency of gene uptake and expression as well as
thosethatinfluencethetoxicityofthevector,wearebetterabletodevelopnew
vectorsystems.Thischapterwillprovideabriefoverviewofrecentadvancesin
gene delivery, specifically on the development of novel non-viral vectors. The
following chapters will provide additional details regarding the evolution of
non-viralgene delivery systems. (cid:1)2005,ElsevierInc.
I. INTRODUCTION
Genedeliveryholdsgreatpromiseasatherapeuticagentforavastarrayofmedical
ailmentsincludingcancer,geneticdisordersandacquireddiseases.Theidealgene
deliveryvehiclewouldexhibitcellspecificity,minimalimmuneresponse,efficient
releaseofDNAintocells,andhavealargeDNAcapacity.Viralvectorssuchas
attenuated viruses, adenoviruses and retroviruses, have thus far proven to have
significantly more efficient gene expression than most non-viral vectors. The
successofviralvectorshasbeenseverelylimitedduetothepotentialforaspecific
immuneresponsetothevectorthatcouldhindergenedeliveryaswellaselicita
severeinflammatoryreactionandcausenonspecificgeneintegrationintothehost
genome.Non-viralvectorsarenotexpectedtoelicitaspecificimmuneresponseor
randomly integrate DNA into the genomic DNA of the host and therefore are
lookedtoasthefutureofgenedeliverysystems.Non-viralvectorsincludecationic
polymer and lipid-based encapsulation of DNA as well as the delivery of naked
DNAbyphysicalmechanisms.Althoughnon-viralvectorsareconsideredsuperior
vehiclesforgenedeliveryduetotheirdecreasedimmunogenicity,theirsuccesshas
been severely limited by inefficient cellular uptake and gene expression. Recent
advances made in the field of non-viral gene delivery, specifically strategies to
improvetargetspecificityandgeneuptakeandrelease,andtofurtherreducethe
nonspecificimmuneresponse,areaddressedbrieflybelow.
II. CATIONIC LIPIDS
Felgneretal.reportedtheuseoflipid-basedvectorsinthelate 1980sandsince
thenthesevesicleshavebeenconsideredoneofthemostpromisingmethodsfor
non-viral gene delivery (Felgner et al., 1987). The cationic head groups make
1. RecentAdvancesinNonviralGeneDelivery 5
strong electrostatic associations with the DNA, eventually leading to the col-
lapse of the anionic polymer. The length and degree of saturation of the lipid
chainissignificantindeterminingthestabilityandtoxicityoftheliposome.To
formastablecomplex,thecationiclipidisoftencombinedwithaneutrallipid
and/or a helper lipid to form a liposome stable under physiological conditions.
PlasmidDNAcontainingthegeneofinterestisincorporatedintotheliposome
toform liposome/DNA complexes, orlipoplexes.
A. Ligand-mediated targeting
Significant efforts have been made to improve gene expression by modifying
lipoplexestotarget-specificcelltypes.Byassuringdeliveryofthevectorstothe
specific tissues, the probability of successful delivery is substantially increased.
Significant advances have been made towards the targeting of tumor cells
in vivo. Since cancer cells are known to over-express receptors (e.g., folate and
transferrin), lipoplexes have been modified to contain ligands that are recog-
nizedbytumorcells.Folatemoleculeshavebeentaggedtolipidsforincorpora-
tion into the lipoplexes (Dauty et al., 2002; Zuber et al., 2003). In addition to
target-specific molecules, short chain fragments of antibodies have also been
used to target tumor cells. Specifically, fragments of short chain antibodies for
transferrin have been covalently conjugated to the liposome to form immuno-
lipoplexes (Xu et al., 2002). Both systems showed an increased affinity for
binding tumor tissues in vitro and the immunolipoplex binding was also
increased in vivo (Dauty et al., 2002; Xu et al., 2002; Zuber et al., 2003). To
further enhance the efficiency of these targeted vectors, polyethylene glycol
(PEG) was attached to the vectors prior to delivery (i.e., PEGylation). This
modification was expected to increase lipoplex solubility as well as improve
circulation of the vector (Ogris et al., 1999). The PEG-modified vectors were
found to have enhanced binding of lipoplexes to the target cancer cells as
compared tounmodified lipoplexes(Yu et al.,2004;Zuber et al., 2003).
B. Toxicity
The toxicity of lipoplexes has been a major limitation for their use as in vivo
gene delivery systems. As mentioned above, lipid-based vectors do not trigger
a cellular immune response (i.e., specific recognition), however these vectors
may be recognized as foreign and initiate the production of cytokines such as
tumor necrosis factor-(cid:1) (TNF-(cid:1)), interferon-(cid:2) (INF-(cid:2)), interleukin-6 (IL-6),
and IL-12. The toxicity of the lipoplexes may be largely attributed to the
composition of the liposomes. Optimization of lipid-based vectors (e.g., incor-
poration of various concentrations of helper lipids) has been relatively
exhausted in recent years, as has the development of novel cationic lipids.
6 Conwell and Huang
Therefore, it has been necessary to address the toxicity of lipoplexes by other
methods. Tan et al. have shown that the sequential injection of liposome and
plasmid DNA can significantly reduce the inflammatory response induced by
systemic gene delivery (Tan et al., 2001). By first injecting liposome, then
waiting a short time and injecting the plasmid DNA, the authors were able to
decrease the levels of the cytokines, TNF-(cid:1) and IL-12 by greater than 80% as
compared to lipoplex delivery (Tan et al., 2001). More recently, Liu et al.
described the preparation of a nonimmunostimulatory lipid-based vector. In
contrast to the standard lipoplex, this vector contains lipid, DNA and an
inflammatory suppressor molecule that specifically inhibits the production of
the cytokine, NF-(cid:3) B (Liu et al., 2004b). These “safeplexes” successfully delivered
DNA to a number of tissues, all of which displayed a significant decrease in
TNF-(cid:1)ascomparedtolipoplexdelivery.Additionally,deliveryofsafeplexesdid
nottriggerasignificantincreaseinthelevelsofIL-12andINF-(cid:2),whichalsoact
asindicatorsoftheseverityoftheinducedimmuneresponse.Thus,theaddition
of an immunosuppressor molecule within the lipoplex can significantly reduce
the toxicity associated with lipid-based non-viral vectors. These advances
amongothers will beaddressed indetail inChapter8.
III. CATIONIC POLYMERS
Cationic polymers condense DNA into compact structures by neutralizing the
anionic charge on the DNA. The resulting cationic polymer/DNA complexes,
or polyplexes, encapsulate the DNA into small particles for gene delivery.
Commonpolycationsincludepolylysine,polyaminessuchaspolyethylenimine,
histoneproteins,polyarginine-containingproteins(i.e.,protamine,HIV-TAT),
andcationicdendrimers.Aswithlipids,notallcationicpolymersareoptimalfor
gene delivery and issues such as efficiency and toxicity must be considered.
Many cationic polymers that condense DNA can not withstand the stringent
conditionsofdelivery,suchashighionicstrength,thereforeitisessentialtouse
polymers thatbindtightly, butreversibly, to the DNA.
A. Novel polymer mixtures
Polyethylenimines (PEI) have been shown tohave oneof the highest transfec-
tion efficiencies of all cationic polymers and have become a favorite non-viral
genedeliveryvectorinthelastdecade(Boussifetal.,1995).PEIisappealingasa
deliveryvectorbecauseithasahighchargedensityforoptimalDNAcondensa-
tion as well as the ability to act as a “proton sponge”, which promotes release
fromendosomalcompartments(Kircheisetal.,2001).Toxicityissues,however,
havelimitedtheoverallsuccessofthiscationicpolymerforinvivogenedelivery.
1. RecentAdvancesinNonviralGeneDelivery 7
In an effort to circumvent toxicity issues while maintaining the transfection
efficiency, modified PEI molecules have been designed. These modifications
include investigating various molecular weights of branched and linear PEI,
conjugation of PEI with PEG (Hong et al., 2004), methylation to a charged
quaternary ammonium derivative ( Brownlie et al., 2004 ), and cholesterol–PEI
conjugates (Furgeson et al., 2002, 2003 ), among others (Kichler, 2004). A recent
study by Brownlie et al. used modular modifications of PEI to create several new
variations of the molecule. The presence of PEG decreased toxicity of the PEI
vector nearly 10-fold, whereas the quaternary ammonium derivate decreased
the toxicity up to 4-fold (Brownlie et al., 2004). By modifying the PEI to create
more biocompatible complexes, the toxicity of this vector may be reduced,
providing a superior polymer for gene delivery. These alternative molecules will
be addressed further in Chapter 9.
Combining lipids and polymers to form new vectors (i.e., lipopoly-
plexes) has provided additional options for more efficient cationic polymer-based
vector delivery. Specifically, complexes containing lipid-protamine-DNA (LPD)
as a modified gene delivery system were investigated for toxicity and efficiency as
compared to standard lipid and polymer vectors. LPD was shown to have
increased efficiency of gene delivery as compared to cationic liposomes ( Li and
Huang, 1997; Li et al., 1998). A recent study by Arangoa et al. described LPD
complexed with asialofetuin (AF), a target ligand-specific for the receptor found
on hepatocytes in large numbers ( Arangoa et al ., 2003 ). The LPD-AF vectors had
enhanced gene expression by approximately 3-fold over lipoplex-AF vectors
and a 10-fold increase over the unmodified lipoplex, indicating that the presence
of protamine significantly promoted gene delivery under these conditions. Fur-
thermore, modified LPD vectors have been explored for treatment of tumors
(Dileo et al., 2003a; Whitmore et al., 2001). Complexes containing CpG oligo-
nucleotides wereshowntoelicit astrongimmuneresponse andincreasedcyto-
kine production, leading to the reduction in tumor activity in vivo (Whitmore
et al., 2001). The increased efficiency of LPD vectors as compared with other
non-viralvectors,alongwithrecentadvancesintargetingofthecomplexes,has
providedapromisingsystemfornon-viralgenedelivery.
Block co-polymers have recently been developed as an adaptation of
polymer-based gene delivery systems. Many of the modified vectors contain a
frequentlyusedcationicpolymer(e.g.,PEI,poly-L-lysine(PLL),poly-histidine)
that condenses DNA in combination with a stabilizing polymer such as PEG
(Ahn et al., 2004; Miyata et al., 2004; Putnam et al., 2003). The production of
copolymers allows for combinations of valuable characteristics in individual
monomers to be combined to form a novel polymer. Recently, Li and Huang
investigatedthecopolymer,poly(D,L-lactide-co-4-hydroxyl-L-proline)(PHLP).
The polymer hydroxyproline is a component of naturally occurring substances
such as gelatin and collagen, and therefore, should minimize toxicity of the
8 Conwell and Huang
vector.Thisbiocompatiblecopolymerwasfoundtohaveincreasedgeneexpres-
sionoverlongerperiodsoftimeandlowertoxicityascomparedtoPEIandPLL,
making it an excellent candidate for further investigations (Li and Huang,
2004). Additionally, by creating biodegradable linkages between the polymers
(i.e.,incorporationofestersorthiols),vectorsmaybedesignedtobeincreasingly
biocompatible and have a higher propensity to release DNA under specific
intracellular conditions (discussed below) (Miyata et al., 2004). Block copoly-
mershavealsobeendesignedusingmoleculesthatarenotcationicinnature.A
prominentexampleisthePluronic1polymers,whichconsistofvariousratiosof
ethyleneoxideandpropyleneoxide(Kabanovetal.,2002).Thesepolymershave
been shown to have increased gene expression as compared to naked DNA in
skeletal muscle tissue. Additionally, efficient delivery of DNA was found with
low concentrations of the copolymer, which further reduces toxicity issues
(Lemieux et al., 2000). The use of copolymers as non-viral vectors allows for
the adjustment of specific characteristics of the vector by manipulating the
individual molecules, ratios and linkages incorporated, providing many oppor-
tunitiestooptimizeencapsulationanddelivery.
The use ofnatural molecules (e.g., albumin, chitosan, and gelatin) for
theencapsulationofDNAforgenedeliveryisalsoapromisingoptionfornon-
viral gene delivery. One advantage to using natural molecules is the reduced
toxicity of the vector, partially due to the biodegradability of the polymer.
Chitosan, a natural polysaccharide, has been successfully used to deliver DNA
bothinvitroandinvivo(Chellatetal.,2005;Koping-Hoggardetal.,2004;Kumar
et al., 2002; Mansouri et al., 2004). Nanospheres made from human serum
albumin have also been shown to have improved transfection efficiency over
naked DNA. Additionally, these vectors have minimized interactions with
other intercellular components due to the presence of the albumin protein
(Brzoska et al., 2004; Simoes et al., 2004). The advantages to investigating
naturallyoccurringpolymersfornanospheresforgenedeliverywillbediscussed
further inChapter12.
B. Toxicity
Thetoxicityofcationicpolymersisfrequentlyaresultofthequantityofpolymer
required to achieve the optimal þ/(cid:1) charge ratio for the polyplex. In many
instances, such as with PEI, the charge ratio that provides optimal efficiency is
very near that which induces severe toxicity (Chollet et al., 2004). Polymer
length/molecular weight has also been found to influence the toxicity of the
polyplexes (Ahn et al., 2004; Fischer et al., 1999; Kramer et al., 2004; Wadhwa
et al., 1997). Recent investigations have focused on creating new biodegrad-
able polymers such as poly[(cid:1)-(4-aminobutyl)-L-glycolic acid] (PAGA) and a
network of poly(amino ester) (n-PAE), which are expected to have reduced
1. RecentAdvancesinNonviralGeneDelivery 9
immunogenicity since they can be easily degraded within the host (Anderson
et al., 2003; Lim et al., 2000, 2002). n-PAE has been found to elicit a reduced
immuneresponsebutequivalenttransfectionefficiencyascomparedtoPEI(Lim
et al., 2002). The development of new biodegradable polymers holds great
promise for reducingthe toxicity ofcationic polymer-based delivery vectors.
IV. TRIGGERED RELEASE
A recurring issue with both lipid and cationic polymer-based non-viral vectors
isthereleaseofDNAoncetheparticleistakenintothecell.Manyvectorsare
able to efficiently bind to the target cells; however, the gene expression
was lower than expected. A primary example of this is the cationic lipid
containing the folate ligand. Particles were small (i.e., less than 50nm) and
associationwithtargetcellswasobserved,yetgeneexpressionwasnotefficient
(Dauty et al., 2002; Zuber et al., 2003). Some polymers bind DNA very tightly,
whichprotectsitfromnucleasedegradationintheserum,buttheassociationis
not easily reversible once the vector is inside the cell matrix. In order to
enhance the escape of DNA from the vector, new polymers and lipids have
been designed that are sensitive to intracellular conditions, such as decreasing
pHanddenaturingconditions(AsokanandCho,2002;GuoandSzoka,2003).
When the engineered vectors are exposed to specific conditions, the particles
become unstable and develop defects that result in DNA leaking out of the
complex.
A. pH sensitivity
VariationsinpHlevelshavebeenobservedinseveralcellularpathwaysaswell
as within specific cellular compartments (i.e., endosomes) (Drummond et al.,
2000;GuoandSzoka,2003).Forovertwodecades,thepHdecreaseobservedin
the endosome has been exploited to enhance DNA release from non-viral
vectors (Asokan and Cho, 2002; Yatvin et al., 1980). Destabilization of the
vectors largely stems from protonation of neutral or negative components of
the complex, which affects the overall structure and molecular interactions
of the vector (Guo and Szoka, 2003; Thomas and Tirrell, 2000). Lipids have
also been designed to contain an acid-sensitive linker region that is hydro-
lyzed upon exposure to acidic conditions, creating discontinuity throughout
the particle andpermitting the leakage ofDNA from the complex (Gerasimov
etal.,1999;Thompsonetal.,1996).DevelopmentofpH-sensitivemoleculeshas
beenshowntoincreasetheefficiencyofgeneexpressionbytheenhancedrelease
of DNA into the endosomal compartment. Several pH-sensitive molecules are
showninTable 1.1.
10 Conwell and Huang
Table1.1. ExamplesofpH-SensitiveMoleculesUsedinNonviralGeneDelivery
Modifiedfrom(AsokanandCho,2002;GuoandSzoka,2003).
1. RecentAdvancesinNonviralGeneDelivery 11
B. Redox sensitivity
Thecytoplasmofthecellhasareducingenvironmentthatisusefulindenatur-
ing disulfide bonds. Naturally occurring cationic polymers (i.e., protamines)
active in DNA compaction in vivo have been shown to be stable at high ionic
strengthsduetothepresenceofdisulfidelinkages.Inanenvironmentwithfree
sulfhydryl groups, the linkages are reduced and DNA release was observed
(Vilfan et al., 2004). Novel lipids and cationic polymers have cysteine residues
incorporatedintotheircore,andfrequentlythelinkageofligandstothelipidsor
polymersexistsviaareduciblemoiety(Dautyetal.,2001;GuoandSzoka,2001,
2003; Kwok et al., 2003). These molecules rely on basic redox chemistry to
establish stable disulfide linkages intended to aid in vector efficiency (i.e.,
attaching target-specific ligand) and stability while also providing a method to
increase DNA release from the complexonce inside the cell.
V. PHYSICAL DELIVERY METHODS FOR NAKED DNA
Delivery of naked DNA to cells elicits minimal immune response as compared
toDNAencapsulatedinlipidsorcationicpolymers.Thelackofimmunogenici-
ty of naked DNA makes it a good prospect for gene therapy. The limitations
with this approach arise in that naked DNA is unprotected against nuclease
degradation and the DNA does not have target specificity. Thus, the actual
physicaldeliveryofnakedDNAmustbedirectedtowardsthetissuesofinterest
sincenotargetligandsareattachedtotheDNA.Advanceshavebeenmadein
thedevelopmentofmethodologiestoimprovetargeteddeliveryofnakedDNA.
Many of the methods described briefly below will be examined in detail in
Part IV.NakedDNA, Oligonucleotides andPhysical Methods.
A. High pressure delivery methods
Manymethodsforthe delivery ofnakedDNA involvethe useofhigh-pressure
methods to force the DNA into the desired tissues. Particle bombardment
methodology(i.e.,genegun)providesapromisingmechanismforgenedelivery
due to the small quantities of DNA required and the minimal toxicity. To
deliverDNAbygenegun,theDNAmustfirstbecoatedontogoldortungsten
microparticles and then delivered to the cytoplasm of target cells by a pressur-
ized blast from the gene gun (Yang et al., 1990). The success of this method
has been severely limited by the small area of tissue to which vector is
delivered.Anewhigh-pressuregenegunhasrecentlybeendevelopedthatuses
helium gas to initiate a high-pressure blast, which delivers gold particles sus-
pended in ethanol into target tissue. Delivery with the improved gene gun
12 Conwell and Huang
increased gene delivery to muscle tissue by several orders of magnitude as
compared to the conventional gene gun (Dileo et al., 2003b). Penetration of
subdermaltissues(i.e.,muscle)morethandoubledthelengthofgeneexpression
previously observed.
HydroporationmethodsdeliverDNAtothetargetbyinjectingalarge
volume of solution with significant force (i.e., a burst of DNA-containing
solution). Recent studies by Zhang et al. have shown that by hydrodynamic
injection, delivery vectors were able to reach the target tissue (i.e., liver hepa-
tocytes) via tail vein injection by traveling through the hepatic vein (Zhang
et al., 2004). The increased pressure from the injection increases membrane
permeability by creating defects in the membrane of the hepatocytes, thereby
increasing vector uptake. Hydroporation has also recently been explored for
gene delivery tothe kidneys as wellas tomuscletissue(Maruyamaet al.,2002;
Zhangetal.,2001).Althoughincreasedgenedeliveryandexpressionhavebeen
observedbythisdeliverymethod,itremainsaveryinvasivetechniquethathas
beenfoundtoincreasebloodpressureanddecreaseheartrateduetothevolume
introduced into the system.
Jet injection, like hydroporation, delivers a DNA-containing solution
totargettissueviaahigh-pressuremechanism.Thedevelopmentofanovellow
volume‘highspeedjetinjector’allowsfortheefficientdeliveryofsmallvolumes
of DNA and is not affected by the length of DNA used (Walther et al., 2001).
The modified jet injector can penetrate tissues up to 10 mm deep and has
displayed transfection efficiencies similar to those observed with particle bom-
bardmenttechniques.Genedeliverybyjetinjectionisanappealingmechanism
becauseDNAremainedintactthroughoutthedeliveryprocess(i.e.,notsheared
bythe pressure) with no tissuedamage orimmuneresponse observed.
Another alternative to hydrodynamic injection involves manually
massaging liver tissue after intravenous injection of naked DNA (Liu and
Huang, 2002a). Mechanical massage of the liver (MML) by applying pressure
totheabdomenofthemouseinfourshortintervals,increasedgeneuptakeinto
theliverascomparedtodeliveryofnakedDNAalone;however,itwasapproxi-
mately an order of magnitude lower than that observed by hydrodynamic
injection. Further investigations of the mechanism by which gene expression
was enhanced suggest that permeability of liver tissue is increased for several
minutes after mechanical massage (Liu et al., 2004a). Delivery of DNA by MML
did not have increased toxicity as compared to the control. In vivo studies of
miceinhepaticfailurerevealedthatthosemicetreatedwithhepatocytegrowth
factor(HGF)byintravenousinjectionfollowedbyMMLsurvived,whereasmice
treatedwithemptyplasmidorleftuntreatedexpiredwithinapproximately20hr
of the diagnosis (Liu and Huang, 2002a). Thus, this less invasive technique may
beusedtoeffectivelydeliverDNAtotheliverwithreducedtoxicitycompared
tohydrodynamic injection.
