Gene Therapy and Gene Delivery Systems

255 Pages·2005·3.765 MB·English

by  David V. Schaffer, Weichang Zhou (auth.), David V. Schaffer, Weichang Zhou (eds.)

AdvBiochemEngin/Biotechnol(2005)99:1–5
DOI10.1007/10_001
© Springer-VerlagBerlinHeidelberg2005
Publishedonline:8November2005
GeneTherapyandGeneDeliverySystems
asFutureHumanTherapeutics
DavidV.Schaffer1 ((cid:1))·WeichangZhou2
1DepartmentofChemicalEngineeringandHelenWillsNeuroscienceInstitute,
UniversityofCalifornia,201GilmanHall,Berkeley,CA94720-1462,USA
[email protected]
2ProcessSciencesandEngineeringProteinDesignLabs,Inc.,34801CampusDrive,
Fremont,CA94555,USA
[email protected]
Although its underlying concepts date back to the 1960s, gene therapy as
a modern molecular medicine is a relatively young field that was born in
1989 with the transfer of a drug resistance gene marker to a patient’s lym-
phocytesinthefirstgenetransferclinicaltrial[1].Thisandotherearlytrials,
which utilized early generation retroviral vectors, provided evidence of safe
in vivo gene transfer for potential therapeutic benefit but also highlighted
the need for progress on many fronts, including deeper molecular know-
ledgeofdiseasetargetpathologies,thedevelopmentofenhancedtherapeutic
cargoes, further insights into gene delivery mechanisms, engineering of en-
hancedvectorsystems,andtheimprovementofvectorproductionprocesses.
This special volume of Advances in Biochemical Engineering and Biotech-
nology provides a broad, state of the art view of the modern field of gene
therapyfromacademicandindustrialviewpoints.Itdemonstratesthatmajor
progress has been made in many aspects including large scale GMP manu-
facturing technologies in the past decade and a half, which enable not only
clinical trials, but also potentially commercialization of these vectors for
widespreadapplicationsinhumans.
Genetherapy,thedeliveryoftherapeuticgenestoapatientfortherapeutic
benefit, offers the potential for permanent cures of diseases. Since its incep-
tion,however,thebiggestchallengeinthefieldhasrevolvedaroundtheword
“delivery”.Nucleicacidsarelabilemacromoleculesthatarechallengingtode-
liver tothe insides ofcells, and abroadspectrum of vectorshas accordingly
been engineered to facilitate delivery. On one end are in a sense the most
syntheticvectors,nakedorunformulatedplasmidDNA,whichareunderde-
velopment for various targetsincluding DNA vaccines as well as cancer and
cardiovascular applications. Manthorpe et al. (pp.) discuss how, due to its
relative simplicity, plasmid DNA systems have rapidly advanced, with major
progress in the optimization of genetic elements for transgene expression,
2 D.V.Schaffer·W.Zhou
largescaleGMPproduction,andvectoradministration.These plasmidDNA
systemsarenowinclinicaltrialsfornumerousapplications.
To expand the applicability of plasmid DNA to other tissues and disease
targets,theDNAiscomplexedwithsynthetic componentssuchaslipidsand
polymers, conceptually similar to the packaging of nucleic acids into a viral
particle. Heidel et al. (pp.) discuss the basic mechanisms of DNA transport
through cells and describe how polymeric systems can be designed to sur-
mount barriers in cellular targeting and intracellular delivery. Furthermore,
they raise the very important concept that as different modules or activi-
ties are added to a vector, from targeting to endosomal escape to nuclear
transport, the vector must be assembled as a functionally integrated system
that presents thecorrectcapabilities at thecorrectjunctures asitprogresses
inside a cell. In a sense, this approach will bring synthetic systems func-
tionally closer to their viral counterparts. Furthermore, Little and Langer
(pp.) discuss the application of synthetic systems to a very important class
of applications, cancer vaccines. This application builds upon fundamental
knowledge of immunology and antigen presentation to develop vaccine sys-
temsthatharnesscellularimmunity,allowingthebodytorecognizeacancer
as foreign and then eliminate it. Furthermore, this application perhaps best
harnesses the current advantages of synthetic systems: high-level, safe, and
transientgeneexpression.
At the other end of the vector spectrum, viral vectors have made signifi-
cant progress in safety and efficiency. Adenoviral vectors, developed in the
early 1990s, have progressed for a broad number of applications described
byAltarasetal.(pp.).Highlyadvancedmanufacturing processesusingmod-
ern mammalian cell and virus propagation and sophisticated purification
technologies,stableliquidformulation,andvectorproductreleaseandchar-
acterization methods have been developed for first generation adenoviral
vectors,servingasmodelsforthedevelopmentoflargescaleGMPproduction
systems for other viral vectors. As a result, the extremely efficient delivery
capabilities of these vectors can be harnessed towards potential commercial
use foranumber ofwidespread applications, including genetic vaccines and
cancertherapeutics.
Inparallel,viralvectorsystemshavedeveloped, becomingmoresynthetic
in nature. In addition to adenoviral vectors “gutted” of all cargo, which Al-
taras et al discuss, new lentiviral and adeno-associated viral vector systems
lacking all viral genes in the vector have advanced significantly during the
late1990sandearly2000s.AsLoewenandPoeschladiscuss,lentiviralvectors
have emerged as an important vector system that exhibits the capability of
very high efficiency for gene delivery to a broad range of cell types and tis-
sues resulting in sustained gene expression. Furthermore, a number of safer
vectors derived from nonhuman lentiviruses have been developed, and the
resulting gene expression from integrated genomes does not appear to suf-
fer fromthe transcriptional silencing that affected simple retroviral vectors.
GeneTherapyandGeneDeliverySystems 3
These vectors have enjoyed success in a number of animal models and have
enteredintoclinicaltrials.
Likewise,asdiscussedbyGriegerandSamulski(pp.),sincetheirinception
in thelate 1980s, adeno-associated viralvectorshaveenjoyed rapid develop-
mentintoveryhighefficiencyvectorsinnumeroustissuesandseveralclinical
trials. The development of enhanced production and purification technolo-
gies for these vectors has facilitated its clinicaldevelopment. The recent dis-
coveryofalargenumberofalternateadeno-associatedvirus(AAV)serotypes
withdifferentpropertiespromisestoyieldacollectionofvectorswithdiffer-
entcapabilitiesforapplicationtoanumberofdiseasetargets.Finally,sinceit
is composed ofDNA surrounded by a relatively simple protein shell, AAV is
perhaps the mammalian virus conceptually most related to a synthetic par-
ticle.
As discussed by both Grieger and Samulski as well as Yu and Schaffer
(pp.), it is not necessary to always “settle” for what nature has provided
during viral vector development. To optimize viruses for human therapeu-
ticapplications,thevectorsoftenrequirere-engineering. Forexample, isthe
development ofrationalapproachesfortargetedgene delivery, aneffortthat
hasbenefited fromimprovedbasic knowledgeofviralstructure-functionre-
lationships. Furthermore, the development of library and directed evolution
approaches mimics the natural process of viral evolution to create vectors
withnovelandattractiveproperties.Collectively,byengineering ormanipu-
latingthepropertiesofviralsystemsatthemolecularlevel,theseapproaches
bring viruses closer to the level of molecular control over vector properties
enjoyed by synthetic systems, making the spectrum between synthetic and
viralsystemscontinuous.
There are a number ofchallenges that remain in vector development and
commercial manufacturing. Targeted gene delivery promises to minimize
side effects and enhance therapeutic efficacy. Furthermore, to extend the
expression duration of both synthetic and viral vector systems, vector inte-
grationorstableepisomalmaintenance isrequired, andprogressintargeted
integration and episome engineering will enhance the safety ofthis process.
Finally,furtherenhancements invectorproductionsystemswillimprovethe
safetyandeconomicsofgenemedicines.
In parallel to vector development, there has been significant progress
in the development of enhanced cargos. With the completion of the hu-
man genome sequence, gene therapy can serve as a direct conduit to
translate basic knowledge of the molecular pathology underlying disease
into direct therapeutic benefit. For example, as alluded to by Heidel et al,
the delivery of nucleic acids to induce RNA interference promises to ex-
pand the therapeutic potential of clinical genetic medicines even further.
To date, nearly 1000 gene therapy clinical trials have been conducted
(http://www.wiley.co.uk/wileychi/genmed/clinical/). These have established
that the vast majority of vectors and gene products are extremely safe for
4 D.V.Schaffer·W.Zhou
human use. Inaddition, there hasbeen preliminary success inseveral trials,
including trials for hemophilia, cancer and cardiovascular disease. Further-
more,thefirstgenetherapyproduct,adenoviralvectordeliveryofthetumor
suppressorp53forcancertherapy,hasbeenclinicallyapprovedinChina.
Finally, fundamental advances in vector and cargo development promise
to yield further successes as they are translated to the clinic. Consequently,
considerableproduct,process,analytical,andformulationdevelopmentchal-
lenges remain towards large scale GMP manufacturing of these vectors for
clinicaltrialsandpotentialcommercialapplications.Eventhoughmanycrit-
ical development issues for manufacturing of first generation Adenovirus
type5vectorsandDNAplasmidshavebeensuccessfullysolved,asdescribed
in this special volume, efforts need to be focused on many next generation
adenoviralandotherviralvectorsindevelopment toenablelargescaleGMP
production. In addition, more sophisticated analytical techniques need to
be developed to enable the characterization of these viral vector productsat
the same level as well-characterized biologics such as monoclonal antibod-
ies. These technologieswouldensure abetter fundamentalunderstanding of
theseviralvectorproductcharacteristicsandaidtheir productionwithcon-
sistentqualityattributes.
There has sometimes been speculation that the field of gene therapy
could be progressing faster. This is a healthy question to pose, and one
for which deeper insights can be gained by examining the progression of
other fields in biotechnology and molecular medicine. Since they were first
derived thirty years ago in 1975, monoclonalantibodies (mAbs) have estab-
lished themselves as a major new generation of human therapeutics, with
18 approved antibodies (one of which was withdrawn from the market in
February,2005)andtwoantibodyfusionproteinstodateintheUnitedStates
by the FDA and a projected 2005 sales of more than 10billion US dollars
(http://www.fda.gov/cder/biologics/biologics_table.htm). However, hindsight
reveals that the road to their development was not smooth. A recent re-
view article discusses the fact that “By the end of the 1980s enthusiasm for
therapeutic mAbs was waning. It was further eroded by the pharmaceutical
problems of mAbs as they were expensive to produce, needed specialist ex-
pertise to administer, and were often associated with considerable toxicity.”
(GlennieandJohnson2000)Fundamentaladvancesinmolecularengineering
(e.g. humanization) and bioprocess development were successfully achieved
toovercomemanyoftheseproblemsandtransformearlypromisesintocom-
mercial human therapeutics, which have been shown to be highly clinically
beneficialinthetreatmentofvariousdiseases.
Gene therapy product candidates, with their many differences from
protein-based therapeutics, will be a new class of human therapeutics for
clinical needs that are currently not being met. They promise a great future
andinthemeantimeoffernewanddistinctchallengesrangingfromdelivery
toproduction.Thecreativeandcomprehensiveworkdiscussedinthisspecial
GeneTherapyandGeneDeliverySystems 5
volume shows strong potential to provide the fundamental biology and en-
gineering advances needed to overcome these challenges and deliver on the
promiseofclinicalgenetherapy.
References
1. Rosenberg SA, Aebersold P, Cornetta K, Kasid A, Morgan RA, Moen R, Karson EM,
LotzeMT,YangJC,TopalianSLetal(1990)Genetransferintohumans–immunother-
apyofpatientswithadvancedmelanoma,usingtumor-infiltratinglymphocytesmodi-
fiedbyretroviralgenetransduction.NEnglJMed323(9):570–578
2. Glennie MJ, Johnson PW (2000) Clinical trials of antibody therapy. Immunol Today
21(8):403–410
AdvBiochemEngin/Biotechnol(2005)99:7–39
DOI10.1007/10_002
© Springer-VerlagBerlinHeidelberg2005
Publishedonline:1November2005
MolecularConjugates
JeremyHeidel·SwaroopMishra·MarkE.Davis((cid:1))
ChemicalEngineering,210-41, CaliforniaInstituteofTechnology,Pasadena,CA91125,
USA
[email protected]
1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
2 Formulationbarriers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
3 Extracellularbarriers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
3.1 Stabilityandimmunogenicity . . . . . . . . . . . . . . . . . . . . . . . . . 14
3.2 Targetedsystemsforreceptor-mediateddelivery . . . . . . . . . . . . . . . 15
3.2.1 Transferrinreceptor(TfR) . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
3.2.2 Folatereceptor(FR) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
3.2.3 Lectins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
3.3 Futuredirections:Controlandcharacterizationofcomplexes . . . . . . . . 20
3.3.1 Complexsize . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
3.3.2 Uncomplexedmaterial . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
3.3.3 Liganddensity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
4 Intracellularbarriers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
4.1 Traffickingwithinandescapefromendocyticvesicles . . . . . . . . . . . . 22
4.2 Vectorunpackaging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26
4.3 Cytoplasmicpersistenceandmobility . . . . . . . . . . . . . . . . . . . . . 29
4.4 Nucleardelivery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30
4.5 Futuredirections:Intracellularbarriers . . . . . . . . . . . . . . . . . . . . 35
5 Overallsummary:Asystemsapproach . . . . . . . . . . . . . . . . . . . . 36
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36
Abstract Molecularconjugatesarenanometer-sizedentitiesconsistingofsyntheticmate-
rials(lipids,polycations,targetingagents,andsoon)andnucleicacids.Thesecomposites
aredeliveryvehiclesthatfunctiontoprovidethetransportofnucleicacidstositesofac-
tion.Recently,greatprogresshasbeenmadeintheconstructionofthesenonviraldelivery
vehiclesandtheunderstandingofhowtheyfunctionincellsandanimals.Here,wereview
someoftheimportantissuesinassemblingmolecularconjugatesandunderstandingtheir
behaviorinbiologicalfluids,cells,andanimals.Oneofthelargestchallengesinthefield
ofmolecularconjugatesishowtointegratethecomponents intoaworkablesystemthat
exploits the combined attributes of the components without suffering losses due to the
assembly of the system. We discuss some of the difficulties involvedin the assembly of
afunctioningdeliverysystemforinvivouse.
Keywords Nonviral·Polycations·Liposomes·Targeting·
Nuclearlocalizationsequence(NLS)
8 J.Heideletal.
Abbreviations
ASGPR asialoglycoproteinreceptor
bp basepairs
CD cyclodextrin
EGFR epidermalgrowthfactorreceptor
FR folatereceptor
h hour(s)
IFN-γ interferon-γ
IL-6 interleukin-6
IL-12 interleukin-12
i.v. intravenous
min minute(s)
NAD(P)H dihydronicotinamideadeninedinucleotide(phosphate)
NF-κB nuclearfactor-κB
NLS nuclearlocalizationsignal
nm nanometer(s)
PBS phosphate-bufferedsaline
pDNA plasmidDNA
PEG polyethyleneglycol
PEI polyethylenimine
shRNA smallhairpinRNA
siRNA smallinterferingRNA
Tf transferrin
TfR transferrinreceptor
1
Introduction
Nonviralgenedeliveryvectorsmustexhibitavarietyofpropertiestoachieve
their function. First, they must condense nucleic acids into small particles
approximately 100nm or less in diameter, and confer protection from de-
grading factors that exist in serum and in cells. The gene delivery particles
must betaken upbycellsthathavebeen targeted, directthenucleic acidsto
anappropriateintracellulardestination(cytoplasmforsmallinterferingRNA
(siRNA) and nucleus forplasmid DNA (pDNA)), release the nucleic acids to
allowtheiraction,andexhibitminimaltoxicity(Fig.1).Initially,manyinves-
tigations addressed these objectives in a serial fashion. This series approach
to the development of non-viral vectors has produced an array of different
materialsthatcombinewithnucleicacidstoformsmallparticlesthatareca-
pableofenteringcells.Measuredintermsoftransfectionefficiency,however,
these materials have yet to rival viruses as gene delivery agents. Although
thelimitingintracellularbarrier(s)tononviralgenedeliveryremain(s)poorly
defined, many recent approaches have sought to better understand and en-
hance: i)the escape fromthe endocytic pathway, ii) vector unpackaging, iii)
cytoplasmicpersistenceofnucleicacids,andiv)nucleardelivery.Apotential
intracellularbarrierthathasnotbeenextensivelyaddressedisthepoorcyto-
MolecularConjugates 9
Fig.1 Functionalitiesofanonviralvector.Nonviralgenedeliveryvectors(cationicpoly-
mersandliposomes)mustbindandcondense nucleicacidpayloads,protect themfrom
nuclease degradation, allow for cellular uptake, permit escape from endocytic vesicles,
andreleasethenucleicacidstopermittheirfunction.ItremainsunclearwhetherpDNA
mustbereleasedfromthevectorpriortonuclearentryorshouldbedirectedtothecell
nucleuswithinintactvector-pDNAcomplexes
plasmicmobilityofnucleicacidsabove2000bpinlength,andwediscussthis
issueinmoredetailbelow.
The synthetic nature of nonviral systems allows for facile, well-defined
modification. Although readily-available materials have not yet provided an
ideal vector, identification of their inadequacies has prompted rationally-
10 J.Heideletal.
designed modifications as well as the development of new materials. These
changes have generally been made in a modular fashion, with new compo-
nents incorporated toaddressspecific barriers. These combinations offunc-
tional components point to the eventual construction of an engineered sys-
temfornonviralgenedelivery.Theserialapproachisgivingwaytoasystems
approach where the various barriers to delivery are simultaneously consid-
eredinthematerialsdesign.Forexample,theinitialemphasisonidentifying
materials that bind and condense nucleic acids may have underappreciated
the importance of their subsequent intracellular release [1]. Attention has
now turned to vectors with a weaker binding strength [2] or whose binding
ofnucleicacidsisdisruptedfollowingcellularuptake[3–6].
Duetoshortcomingssuchashightoxicity,instabilitytophysiologicalsalt
and serum and poor transfection efficiency, early nonviral systems showed
little in vivo applicability. Modifications of existing systems to address these
problems have brightened the prospects for effective in vivo gene delivery.
However, these alterations, often involving changes to the chemical struc-
ture of the delivery vectors or nucleic acids, have also affected gene deliv-
ery performance in unanticipated ways. For example, Mishra et al. demon-
stratedthatconferringsaltstabilitytopolyethylenimine-DNAcomplexeswith
a poly(ethylene glycol) coating dramatically changes the morphology of the
endocytosed entities and significantly reduces the resulting gene expression
in vitro [7]. The need for a systems approach extends to in vitro investi-
gations, as modifications intended for in vivo applicability can significantly
affectbothinvitroandinvivoperformance.
Toachieveinvivoapplicability,non-viralvectorsmustbedevelopedthatcan
bepreparedinareproduciblemannerwithdefinedcompositionandproper-
ties.Foralmostallnonviralvectors,therearenowell-establishedformulations
that give homogeneous vehicles, and the product characteristics are not de-
fined in a consistent and quantitative manner. For example, it is likely that
polycation-nucleicacidcomplexesformulatedatahighchargeratio(ratioof
positive charge centers on the polycation to negative charge centers to the
nucleicacids)donotcontainallthepolycationinthecomplexedstate[8].Un-
complexedpolycationmaywellcontributetotheinvitrotransfectionefficiency
ofnonviralvectors,butcannotbeexpectedtodosoinvivo,whereitwouldnot
necessarilybedistributedinassociationwiththecomplexes.
Humanmedicalapplicationsarelikelypossiblewithnonviralgenedelivery
vectors, provided thatimprovements canbemade togiveappropriate thera-
peuticindices.Theadvantagesofthesesystemsincludeanabilitytoavoidthe
DNA size limitations and immunogenicity that are possible with some viral
vectors,andalsostraightforwardformulationwitheasily-manufactured,rela-
tively low-costmaterials that willassist inproviding large-scaletherapeutics
atacceptablecosts.
Nonviral gene delivery remains an area of steady progress. Here, we dis-
cuss issues that need to be addressed in order to reach the ultimate goal
MolecularConjugates 11
of molecular conjugates that are true human therapeutics. We discuss bar-
riers in formulation, extracellular transport, and intracellular transport. We
closewithadiscussionthatemphasizesasystemsapproachtothecreationof
these complex nanometer-sized assemblies. While molecular conjugates can
involvelipidsandpolycations,ourdiscussionprimarilyconcernspolycations.
2
Formulationbarriers
As with viruses, scale-up of the production of nonviral gene delivery par-
ticles to the commercial scale faces significant hurdles. Viral gene delivery
particles can be prepared in large quantities using “natural” cellular pro-
duction systems. For virus production, plasmids containing genes for viral
assemblyandthedesiredDNApayloadaretypicallycotransfectedinto“pack-
agingcells”invitro,wherein thevirusesareassembled andsecretedinlarge
quantity. This preparation scheme allows for scale-up, but has the poten-
tial to be complicated by the introduction of contamination and/or genetic
mutation. However, a key issue is that the synthesis and assembly processes
are all performed by biological entities that naturally perform these com-
plex steps. Nonviral vectors must be prepared using synthetic materials by
relying upon traditional reaction chemistry and purification (such as chro-
matography, filtration) schemes to separate the desired product from other
components that may be present within the reaction mixture; similar issues
are faced for production (such as extrusion with liposomes) and purifica-
tion of the final assembled product. These processes create the potential for
greater batch-to-batch variability, and variations in properties such as poly-
cation molecular weight distribution and complex size have been shown to
affectgenedeliveryefficiency[9]andtoxicity[10].
The mechanism of viral production within packaging cells ensures that
eachvirioncontainspreciselythesamenumber ofcopiesofthegeneticpay-
load. Because nonviral complex formation relies upon self-assembly, there
is a potential for greater heterogeneity within the resulting formulations.
Factors such as order-of-addition,concentration of components, and charge
ratioallaffectthemeanandpolydispersityofthecomplexdiameter.Further,
as will be discussed later in this chapter, many nonviral formulations con-
tain multiplecomponents inadditiontothevector andnucleic acid, suchas
stabilizing polymers and cell-targeting ligands. Additional interformulation
heterogeneity may be introduced if the method of assembling these con-
stituentsdoesnotprovidesufficientcontrol.
Non-uniformity within a single nonviral formulation presents a separate
concern. An increasing body of evidence suggests that significant quantities
of vector within a formulation may remain unbound to the nucleic acid [8].
Although a positive (greater than 1:1) charge ratio is required for com-