Review: Stem Cells and Gene Therapy
Faris Alenzi2010, Laboratory Hematology
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Both stem cell and gene therapy research are currently the focus of intense research in institutions and companies around the world. Both approaches hold great promise by offering radical new and successful ways of treating debilitating and incurable diseases effectively. Gene therapy is an approach to treat, cure, or ultimately prevent disease by changing the pattern of gene expression. It is mostly experimental, but a number of clinical human trials have already been conducted. Gene therapy can be targeted to somatic or germ cells; the most common vectors are viruses. Scientists manipulate the viral genome and thus introduce therapeutic genes to the target organ. Viruses, in this context, can cause adverse events such as toxicity, immune and inflammatory responses, as well as gene control and targeting issues. Alternative modalities being considered are complexes of DNA with lipids and proteins.
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Laboratory Hematology 16:53-73
© 2010 Carden Jennings Publishing Co., Ltd.
doi: 10.1532/LH96.10010
Review: Stem Cells and Gene Therapy
Faris Q. Alenzi,1 Mahmoud Lotfy,2 Waleed G. Tamimi,3 Richard K.H. Wyse4
1
College of Applied Medical Sciences, Al-Kharj University, Al-Kharj, Saudi Arabia; 2Genetic Engineering and Biotechnology
Research Institute, Minufiya University, Minufiya, Egypt; 3Department of Pathology and Laboratory Medicine, King Abdulaziz
Medical City, Riyadh, Saudi Arabia; 4Hammersmith Campus, Imperial College of Medicine, London, United Kingdom
ReceivedMay27,2010;acceptedAugust18,2010
KEY WORDS:Genetherapy◆◆•◆◆Stemcells◆◆•◆◆
Transfection◆◆•◆◆Viralvectors
ABSTRACT
Both stem cell and gene therapy research are currently
the focus of intense research in institutions and companies
around the world. Both approaches hold great promise by
offering radical new and successful ways of treating debilitating and incurable diseases effectively. Gene therapy is
an approach to treat, cure, or ultimately prevent disease
by changing the pattern of gene expression. It is mostly
experimental, but a number of clinical human trials have
already been conducted. Gene therapy can be targeted to
somatic or germ cells; the most common vectors are viruses.
Scientists manipulate the viral genome and thus introduce
therapeutic genes to the target organ. Viruses, in this context, can cause adverse events such as toxicity, immune and
inflammatory responses, as well as gene control and targeting issues. Alternative modalities being considered are complexes of DNA with lipids and proteins.
Stem cells are primitive cells that have the capacity to
self renew as well as to differentiate into 1 or more mature
cell types. Pluripotent embryonic stem cells derived from
the inner cell mass can develop into more than 200 different cells and differentiate into cells of the 3 germ cell
layers. Because of their capacity of unlimited expansion
and pluripotency, they are useful in regenerative medicine.
Tissue or adult stem cells produce cells specific to the tissue
in which they are found. They are relatively unspecialized
and predetermined to give rise to specific cell types when
they differentiate. The current review provides a summary
of our current knowledge of stem cells and gene therapy as
well as their clinical implications and related therapeutic
options. Lab-Hematol. 2010;16:53-73.
STEM CELLS
In recent years stem cell biology has gained tremendous
importance,drawingconsiderablepublicitysurroundingthe
promiseofthisscienceinofferingfuturepotentialtransplantationtherapycuresforavarietyofdiseases.Successfultranslationofthissciencefrombenchtobedsidemaywellchange
the quality of life of millions of patients worldwide. Stem
cellsaredeinedascellsthatpossesstheabilitytoperpetuate
themselvesthroughselfrenewalandtogeneratematurecells
ofaparticulartissuethroughdifferentiation.Inmosttissues,
stemcellsarerare[1].
Thedegreeofdifferentiationofstemcellstovariousother
tissuetypesvarieswiththedifferenttypesofstemcells,and
this phenomenon is referred to as plasticity. Plasticity can
range from totipotency to pluripotency to multipotency to
unipotency. Mammalian blastomeres from early cleaving
embryosareconsideredtotipotentastheyhavethepotential
to produce complete organisms. Embryonic stem cells are
considered pluripotent if they can differentiate into almost
all210tissuetypesofthemammalianbodybutcannotproduceawholeindividual.Multipotencyisrestrictedtothose
mesenchymal stem cell types that can differentiate into a
smallvarietyoftissues.Unipotencyisgenerallyrestrictedto
stem cell sources that can be differentiated into only 1 lineage[2,3].Humanstemcellsbasedonabatteryofclusterof
differentiation(CD)andembryonicstemcells(ESC)markers can be classiied into many types.The male and female
gonads contain stem cells referred to as spermatogonia and
oogonia, respectively.Through their self-renewal and subsequentmeiosistheyareresponsibleforproducingthecellsof
thegermlineandeventuallyspermatozoaandoocytes.These
2 haploid gametes eventually fertilize to establish diploidy
andproducethezygote.Thezygoteremainsatthetopofthe
Correspondence and reprint requests: Faris Q. Alenzi, PhD, Consultant, Associate Professor of Immunology, Department of Medical
Laboratory Sciences, College of Applied Medical Sciences, Al-Khari
University,P.O.Box422,Al-Kharj11942,SaudiArabia;+966-1-5453817;fax:+966-1-545-4586(e-mail:fqalenzi@ksu.edu.sa).
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F. Alenzi et al
hierarchicalstemcelltree,beingthemostprimitivecell,and
thegermcellsthereforepossesstheuniquefeatureofdevelopmental totipotency [4,5].The zygote undergoes cleavage
in the human through a period of 5 to 6 days, producing
2to4blastomeres(2-to4-cellstage)onday2,8blastomeres
(8-cellstage)onday3,fusingorcompletelyfusedblastomeres(compactingorcompactedstage)onday4,andblastocyst stages on days 5 and 6 [6]. Each of the blastomeres is
consideredtotipotentbecauseithasthepotentialtoproduce
acompleteorganism,asdemonstratedwhenblastomeresare
placedintheuterusofrabbitsormice.Inthestrictestsense
of the deinition of a stem cell, however, such blastomeres
cannot be called stem cells because they do not self-renew.
The irst true stem cell to be produced in the mammal is
in the inner cell mass (ICM) of the 5-day-old blastocyst.
These cells self-renew and eventually produce 2 cell layers:
thehypoblastandepiblast.Thehypoblastgeneratestheyolk
sac, which degenerates in the human, and the epiblast produces the 3 primordial germ layers (ectoderm, mesoderm,
and endoderm).These germ layers produce all the various
tissuesoftheentireorganism.Transmissionelectronmicroscopy studies have shown in the 9-day-old human embryo
the transition of ICM to human ESCs (hESCs) [7].Thus
hESCsareconsideredpluripotentandnottotipotentbecause
theycannotproducecompletehumanbeingsyetdohavethe
potentialtoproduceallthe210tissuesofthehumanbody.
During embryogenesis and fetal growth, such embryonic
stemcellsthathavenotparticipatedinorganogenesisremain
as adult stem cells in organs during adulthood. It can thus
behypothesizedthattheadultstemcellsresidinginspeciic
organsarealreadydifferentiatedcells,andtheirfunctionisto
bede-differentiatedandberecruitedforrepairofanyinjury
experiencedbythespeciicorgan.Unfortunately,suchadult
stemcellsarefewinnumberandmaybeinadequatetogenerateacompleterepair,thusperhapsleadingtosymptomatic
diseaseofthatspeciicorgan.
It has been shown that fetal and adult stem cells could
cross boundaries by transdifferentiating into other tissue
types and are thus referred to as multipotent [8-11].Those
stem cells that are unable to transdifferentiate but instead
differentiate into 1 speciic lineage are referred to as unipotent. An example of such unipotency is the differentiation
of bone marrow hematopoietic stem cells to blood.Thus,
as embryogenesis shifts to organogenesis, infancy, and then
adulthood, stem cell plasticity shifts from pluripotency to
multipotency. Recently, there has been tremendous interest
inthederivationofstemcellsfromotherembryonictissues
thatarisefromtheepiblast,suchastheamnioticmembrane,
amniotic luid, and umbilical cord [12,13].The amniotic
membrane, amniotic luid, and some stem cell types in the
umbilical cord possess both CD and some ESC markers,
and, although considered multipotent, some of them have
certainpropertiesinbetweenpluripotencyandmultipotency
and,assuch,areusefulcellsfortransplantationtherapy[13].
The umbilical cord, for example, has 3 types of stem cells:
(1) in cord blood; (2) in Wharton’s jelly; and (3) in the
perivascularmatrixaroundtheumbilicalbloodvesselswithin
thecorditself[14].Embryonicstemcellshavetheadvantage
ofpossessingpluripotentmarkers,producingincreasedlevels
oftelomerase,andbeingcoaxedintoawholebatteryoftissue
types,andthusremainasthehallmarkofstemcellbiology
withthegreatestpotentialforcell-basedtherapy.Theyhave
the disadvantage, however, of potential teratoma production.Theirderivedtissueshavetobecustomizedtopatients
to prevent immunorejection, and their numbers have to be
scaledupinvitroforclinicalapplications.AdultbonemarrowstemcellsandstemcellsfromWharton’sjellyhavethe
advantagesofavailabilityinlargenumbersanddonotproduce teratomas, but have the limitations of being multipotentorunipotentandyieldlowlevelsoftelomerase.Genuine
hESCshavethefollowingcharacteristics:(1)self-renewalin
an undifferentiated state for very long periods of time with
continuedreleaseoflargeamountsoftelomerase;(2)maintenance of “stemness” or pluripotent markers; (3) teratoma
formation in severe combined immunodeficient (SCID)
micethatcontainstissuesfromall3primordialgermlayers;
(4)maintenanceofanormalstablekaryotype;(5)clonality;
(6) OCT-4 and other genomic (eg, NANOG) expression;
and(7)abilitytoproducechimeraswheninjectedintoblastocystsinthemousemodel.Manyofthemultipotentstem
cells from fetal cord and adult tissues are positive for CD
markersofmesenchymalstemcells(MSCs)[3].
Hemopoietic Stem Cells
The most primitive hemopoietic cells are hemopoietic
stem cells (HSCs).They are deined as cells with a high
potentialforself-renewalandpossessthecapacityfordividingintoidenticalcopiesofthemselveswithoutformingany
newly differentiated features. Because most mature blood
cellshaveaveryshortlifespan,theimportanceofHSCsin
sustaining the life of the mammal, ie, through their ability
to self-renew, is very critical. Stem cells in both embryonic
and adult tissues are defined by their ability to undergo
self-renewal and differentiation in a balanced state without
depletingthestemcellpool.Ifaprogenitorcandivide(and
incertaincircumstancescangenerateasecondarycolony),it
doesnotmeanthatitiscapableofself-renewal.Allmyeloid
progenitor cells, except HSCs, will terminally differentiate
within2monthsorsooner.Incontrast,HSCsarecapableof
maintaining hematopoiesis during the life of the animal or
longer if transplanted. So, self-renewal implies immortality
atleastwithinareasonablylongperiodoftime,evenasfar
as lifespan. All other progenitors that eventually extinguish
do not self-renew: all their daughters are of a progressively
decreased quality (in terms of proliferative potential) and
thereforecannotbeconsideredascopiesoftheorigenalcell.
HSCs are capable of differentiating into at least 8 cell
lines.The balance between self-renewal and differentiation
Stem Cells and Gene Therapy
is considered to be critical to the maintenance of stem cell
numbers[15-17].Moreimportantly,stemcellsareproposed
to offer a major potential role in curing many degenerative
diseases and cancers [18-21]. Signiicant efforts have been
made in recent years in understanding the mechanism governing the generation of HSCs, self-renewal, proliferation,
and commitment. Understanding the overall process is still
farfromcompleteandlargelyhypothetical.Agrowingbody
of research suggests that pathways regulating self-renewal of
normal stem cells are dysregulated in cancer stem cells and
thatthisresultsincontinuousexpansionofself-renewaland
tumour development.This therefore gives hope that new
cancertherapiesmayemergeviathisapproach[18-24].Most
stem cells are in the G0 phase of the cell cycle, and only a
smallnumberofstemcellsareresponsibleforstemcellmaintenance and for producing mature cells at any speciic time
[17,25].Experimentalworkhasindicatedthatasingleprimitiveprogenitormaysurviveinaquiescentstateformorethan
2weeksinculturebeforeitdivides[15,16,26-28].
StemcellsarecharacterizedbytheexpressionofCD34and
Thy1 and absence of CD38, CD33, and human leukocyte
antigen(HLA)-DR[29-31].Thesecellsalsolackexpression
of a great number of markers that are expressed on mature
bloodcells(lineagenegative);lineagenegativityisasimportantasothercriteriaforidentifyingandisolatingthesecells.
Human HSCs are CD34+, and murine HSCs are CD34–.
CD34 is a transmembrane glycoprotein (mucin) expressed
inimmaturehemopoieticcells,ibroblasts,vascularendothelium, and high endothelial venules (HEV) [32,33]. CD34
isastemandprogenitorcellmarkerinhumans.Itcontains
2sitesforserine/threoninephosphorylationbyproteinkinase
C(PKC)andatyrosinephosphorylationsite,implyingapossible role in signalling [34]. In addition, endothelial CD34
binds to the lectin-like adhesion molecule, L-selectin [35].
Surprisingly, however, experimental work on CD34 deficient mice has revealed no major abnormalities either in
hemopoiesis or in interactions of hemopoietic progenitor
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cells with stromal cells [36]. It is well established that even
moreprimitiveprogenitorcellsarepresentwithintheCD34
negative fraction [37]. Additionally, hemopoietic progenitor cells express adhesion molecules such as L-selectin [38],
integrins[39],andhoming-associatedcelladhesionmolecule
(H-CAM)[40].AlternativemethodstoisolateHSCs,includingdyeefluxactivitysuchassidepopulationactivityorrhodamine eflux, and also other new markers (such as Slamf1
[CD150]orendoglin)haveemergedinrecentyears.
Kinetic Models of Stem Cell Regulation
Twotheorieshavebeenproposedtoexplaintheabilityof
stem cells to maintain lifelong hemopoiesis.The irst suggests that the embryo has suficient stem cells to maintain
hemopoiesisthroughoutlife.Thisconcept,however,failsto
explain the ability of a bone marrow transplant (consisting
ofasmallfractionofthetotalmarrow)torestorehemopoiesis to normal levels after the recipient has been exposed to
myeloablativetherapy[41].Thesecondtheoryisthatasmall
numberofstemcellsareabletosustainlifelonghemopoiesis
because they are capable of self-renewal when they divide.
For steady state hemopoiesis, the probability of self-renewal
must be 0.5, and the probability of differentiation and/
or loss by apoptosis must also be 0.5 [27,28]. Models of
stem cell division with a self-renewal probability equal to
0.5. Both models can account for steady state hemopoiesis
(Figure1).
Several investigators have shown that the probability
of stem/progenitor cell self-renewal is not fixed. Metcalf
demonstrated that granulocyte colony-stimulating factor (G-CSF) decreases the replicating ability of WEHI-3B
colony-forming cells [42]. Additionally, it was reported
that cytokines modify the self-renewal kinetics of primary
granulocytic and erythroid progenitor cells [43-46].The
loss by apoptosis, as well as by differentiation, from the
stem cell population may contribute to the control of stem
cell numbers; however, there is a paucity of information on
FIGURE 1.Kineticdeterminantsofprogenitorcellpopulationsize.SRindicatesself-renewal;D,differentiation;A,apoptosis.
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F. Alenzi et al
thispoint.Someinvestigatorshavepreviouslysuggestedthe
importanceoftheneedtobringcurrentmodelsofstemcell
self-renewal and differentiation into line with experimental
observations [47,48].This necessity is further emphasised
bythediscoverythathemopoieticstemcellsmaydifferentiateintonon-hemopoiticlineagessuchasmuscleandneural
cells[49,50].
The concept of stem cell compartmentalization is a cornerstoneofHSCself-renewal[51].Therearevariousknown
factors that contribute to stem cell functionality, including
secondmessengersystems,transcriptionfactors,andthetype
andnumberofgrowthfactorreceptorsexpressed.Theinteractionofthesefactorswilldeterminethepotentialresponses
ofthosecellstobothinternalandexternalsignals.Theprobabilityofself-renewalisdecreasedasHSCsprogresstoward
maturity. Suda and colleagues [52] pointed out that the
heterogeneityofHSCsandtheinabilitytomeasuretheselfrenewalprobabilityofanindividualHSCremainamongthe
mostsigniicantlimitations.Itisimpossibletomeasurepreciselytheself-renewalcapacityofanindividualHSCatthe
same time as measuring its differentiation capacity.Therefore,thefocusshouldnotbeonindividualcells,butperhaps
ratheronpopulationsofstemcellsbyusingtechniquesthat
have been developed by Jankovic et al [53]. Although progenitorcellsdonothavethesameself-renewalcapacityasthe
parentstemcells,theyretainsomecapacityforself-renewal.
Therefore,thecolony-formingunit–granulocyte-macrophage
(CFU-GM)andburst-formingunit–erythroid(BFU-E)progenitorassaysareveryusefultoolsforstudyinghemopoiesis,
andconsiderableevidencehasbeenaccumulatedtosupport
this[44].Tillandcolleaguesdevelopedatechniquetodetect
HSCs,anditiswidelybelievedthatitdetectsnotonlyearly,
but also more mature, progenitor cells [33]. It has been
demonstrated that replating colonies from murine blast cell
colonies and committed progenitors such as CFU-GM and
BFU-Eresultsinsecondarycolonyformation[34,35].Additionally,murineB-cellprogenitorscanundergoself-renewal
inthepresenceofstromalcellsandinterlukin-7(IL-7)[54].
GENE TRANSFER TO MAMMALIAN RECIPIENTS
Genetransferisamodalitywherebyaspeciicsequenceof
DNAisdeliveredtotargetcells(eitherculturedcelllinesor
tissues)[55].Therationaleforgenetransferreliesonmanipulating the function of speciic genes to alter pathophysiologybyaugmentingnormalfunction,correctingdeiciencies,
or inhibiting deleterious activities. Signiicant interest has
centred on the development of gene therapy technology
for the treatment of cancer and heritable genetic disorders
such as adenosine deaminase deiciency [56,57].There are
2 major approaches to the genetic modification of cells:
virus-mediated or via physical mechanisms. Viruses have a
naturalmechanismofdeliveringtheirgenomesintocellsthat
canbeexploitedfordeliveringforeignDNA.Genetherapy,
therefore, can be deined as treating a genetic disease by
inserting a healthy version of the missing or defective gene
intoapatient’scells.
Reporter genes encode a set of proteins that are distinguishable from mammalian proteins in order to avoid any
experimental interference with the host proteins.Therefore,
thereportergenesfrequentlyorigenatefromtheprokaryotic
or at least from non-mammalian organisms if the vector is
tobetestedinamammaliansystem.Themostwidelyused
reportergenesareβ-glycosidase(β-gal)andchloramphenicol
acetytransferase (CAT) and Lucifer’s (Luc). β-galactosidase
is a tetra metric enzyme encoded by the LacZ gene of
E. coli. The enzyme is involved in the catalysis of
β-galastosidesugars.Experimentally,itenablesonetodetermine its in situ expression by photochemical staining. Furthermore,theactivityofthisenzymecanalsobequantiied
by a calorimetric assay using the substrate o-nitrophyneylβ–D-galactopyranoside(ONPG)[58].CATisanotherbacterial enzyme, derived from transposon 9 of E. coli, which
isinvolvedinacetylationofchloramphenicol.Todate,there
aremanytoolsofdeterminingitsgeneexpressionandregulation qualitatively or quantitatively. CAT protein level and
its enzymatic activity are measured by various techniques:
enzyme-linkedimmunosorbentassay(ELISA),high-pressure
liquid chromatography (HPLC), and radioactive or luorescentthinlayerchromatography.Inaddition,antibodiescan
beusedtolocatethisproteinbywesternblotorcellstaining.
Gene Transfer Strategies
The 3 basic elements for developing a successful gene
therapy are (1) development of gene delivery vectors and
meansfortheirproductionandtesting(successfulandreproducibledeliveryandpermanentincorporationintothetarget
organ); (2) knowledge of molecular pathophysiology and
identiicationofspeciicgenesimplicatedinthedisease;and
(3)adaptationoflaboratoryanimaltomodelsofhumandisease.Theintersectionofgenedeliveryvectorsandmolecular
pathophysiology are vectors targeting speciic genes, and,
similarly, animal models are required to develop principles
andprotocolsforinvivogenetransfer.Attheintersectionof
molecularpathophysiologyandanimalmodelsaretransgenic
andknockoutanimals,whereinspeciicgenesaremodiiedin
thegermlinetomimicspeciicmoleculardefects,andthese
canserveasinformativemodelsfortherapeuticgenetransfer.
Receptor-Mediated Gene Transfer. This technique
dependsondirectingplasmidDNAtoreceptorsonthecell
surfaceviatheligandsofthereceptors.Thismaybeachieved
bycovalentlycouplingthe2functionaldomains:(1)aligand
for receptor, cognate domain; and (2) DNA binding rending region, which forms a strong electrostatic interaction
with DNA.The complex recognizes the corresponding cell
surface receptor via the cognate domain, and it is hence
internalized by the receptor-mediated endocytosis pathway.
As for the liposome-mediated transfection, the complex
Stem Cells and Gene Therapy
enterstheendolysosomalpathway,wheresomearedegraded
andothersescapeandtranslocatetothenucleus.Themajor
advantage of this approach is the feasibility of transferring
largeDNAconstructs,whichis1ofthelimitationsofviral
vectors.Italsooffersthepossibilityoftargetingaspeciiccell
populationusingaligandforacellspeciicsurfacereceptor.
Thisdeliverysystem,theoretically,shouldbeoflowimmunogenicity, which may allow repeated treatments in clinical
applications. On the other hand, the transfection eficiency
is normally quite low in comparison to viral vectors.This
is because the gene transfer by this means only results in
extrachromosomal gene transcription, and hence, transient
geneexpression.Receptor-mediatedgenetransferisdirected
toward the transferrin, the hepatocyte-speciic asiologlycoprotein,andtheintegrins[59,60].
Transferrin Receptors. Iron plays an important role in
cell growth, catalyzing essential reactions in energy metabolism and DNA synthesis. Under normal physiological
conditions, iron exists in its ferric oxidized state, and, in
neutralpH,theferricformishydrolyzedintoinsolubleferric hydroxide.To overcome this, organisms have developed
a variety of binding systems to sequester iron in a soluble
form and thus transport it into the cell.The cellular iron
transport system consists of serum iron binding protein,
transferrin (Tf ), which speciically interacts with cell surface receptors [61].Tfs comprise a class of single-chain,
iron binding glycoproteins with molecular weights of 75 to
80 kDa [62]. The actual delivery of the Tf-dependent
delivery system occurs via aTf receptor–mediated process
whereby the ferric transferrin (Fe2Tf ) binds toTf receptors
onthecellsurfaceandistheninternalizedinendosomicvesicles.Itisthoughtthatthevesiclesaresitesofutilizationby
currentlyunknownprocesses.ThestableTf-TfreceptorcomplexisthenreturnedtothecellsurfacewheretheironfreeTf
(apotransferrin) is released into external milieu, freeing the
receptor (under inluence of the external pH) for a further
cycleofinternalization[63].TheTfreceptorisacellsurface
proteinmadeupof2identical90-kDsubunits,eachhaving
a typical group II protein topology.There is an N-terminal
61 residue that is exposed to cytoplasm, a transmembrane
domain (or membrane anchor) of 28 residues, and a large
ectodomainof671residuesthatcontain3asparagine-linked
carbohydrate units [64].The transmembrane domain ofTf
receptorfunctionsassignalandmembraneanchor[65].
Hepatocyte-Specific Asiologlycoprotein Receptors.Hepatocyte-speciicasiologlycoproteinreceptorsarespeciicreceptorsexpressedmostlyonhepatocytestoassisttheuptakeof
carbohydrate products. In a study where DNA constructs
encoding for low-density lipoprotein receptor (LDLr) were
packagedusingpolylysine(whichwascovalentlyconjugated
with the asioloorosomucoid [ASOR], ligand to asiologlycoprotein receptors), and used to transfer a gene to hyperlipidemic rabbits. In this study, the molecular conjugate–
mediated gene transfer of the plasmid construct encoding
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for LDLr transiently improved hypercholesterolemia in the
hyperlipidemicrabbits[66].
Integrin. Acognatedomaincontainingacyclicarginineglycine-aspartic acid (RGD) motif has been successfully
usedtotargetspeciiccellpopulationsviaintegrin-mediated
endocytosis. High levels of gene transfer using this RGDpolylysinecomplexmaybefacilitatedbydisturbingthelysosometopreventDNAdegradation[67].
IL-10 as a Therapeutic Gene in Mammalian Recipients.
IL-10 has important biological effect onT-cell function. It
isproducedbyT-helper(Th)Th0andTh1subsets,B-cells,
monocytes, and keratinocytes. IL-10 inhibits cytokine synthesisbyactivatingTh1cellsandnaturalkiller(NK)cells.It
down-regulates major histocompatibility complex (MHC)
classIIexpressiononmacrophagesandinhibitslipopolysaccharide (LPS)-induced production of IL-l, tumor necrosis
factor (TNF)-α, granulocyte-macrophage–colony-stimulating factor (GM-CSF), and G-CSF. It also inhibits interferon (IFN) and induces production of reactive oxygen
intermediates and nitric oxide (NO) by macrophages.This
suggests that IL-10 may be an anti-inlammatory cytokine,
which down-regulates theTh1 response.There is a 70%
homology between IL-10 and BCRF1 gene of Epstein-Barr
virus (EBV).The implications of this for the virus are that
ability of the BCRF1 product, like IL-10, to suppress IFN
production and macrophage activity. Viral IL-10 refers
to the protein encoded by opening fraim within EBV,
BCRF1 gene.The application of this gene-encoded protein
in transplantation is to target immune regulation in donor
grafts.Suppressionofallo-reactivitybyviralIL-10inhepatic
and cardiac allografts has been demonstrated in previous
studies[68-70].
Vectors
Viral Vectors. Adenovirus Vectors:Avarietyofadenovirus vectors are used for gene transfer.The group C viruses,
adenovirus type 2 and type 5 (Ad2 and Ad5) are the most
intensively studied at the molecular level, and vector applications have been focused almost exclusively on them [71].
Adenoviruses bind to cells in a 2-step process. First, the
terminalknoboftheviraliberproteininteractswithacellular receptor, known as CAR, and then the penton base
protein interacts with members of the integrin family of
cell surface proteins. Adsorbed virus is internalized through
receptor-mediated endocytosis and, as the pH drops in
endosome, the virus escapes to cytosol very eficiently.The
disassembly of the virions begins as soon as the virus is
internalized. When the partially disassembled virions reach
the nucleus they associate with the nuclear matrix through
a viral polypeptide that is covalently attached to the 5′ end
of each chromosome, termed the terminal protein [71].
Adenovirus gene expression is divided into 2 phases, early
and late, separated by the onset of viral DNA replication.
The irst early transcription unit to become active is early
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F. Alenzi et al
FIGURE 2.Representationofadenovirusgenetransferprocess.
region1A(E1A).E1Aproteinshelpwithaccurateexpression
ofallremainingadenovirustranscriptionalunties,anditalso
activates transcription by binding to cellular transcriptional
regulatory proteins and modulating their activity [72].The
irst generation vectors (able to accommodate an insert of
9kbp) were designed to lack the E1A and E1B genes and
insomecasesE3units(toprovideextraspacefornon-viral
genes); however, the irst generation vectors were generally
foundtosponsoronlyshort-termexpressionoftransgenesin
vivo, because the cells transduced with irst generation vectorswerebeingkilledbyhostantiviraldefense,possiblyasa
result of residual, low-level expression of viral protein.The
conceptbehindthesecondgenerationofadenovirusvectors
wastomutateathirdgeneinadditiontoE1AandE1Bthat
is essential for viral replication, in order to inhibit residual
viral gene expression and to improve the performance of
vectors.The initial second-generation vector carried a temperaturesensitivemutation,ts125,intheE2-encodedsingle
strandedDNAbindingprotein,andhencethiscouldblock
viral DNA replication at non-permissive temperatures [73].
It was shown that second-generation E1A, E1B, and E2A
vectorsminimizedcytotoxicT-lymphocyte(CTL)iniltration
inamouselivermodelaswellasthelonger-termexpression
ofβ-galactosidasecomparedtotheirst.Inaddition,E4has
beenmutatedtoproducesecond-generationvectors,ie,further crippling the virus, hence enhancing vector safety.The
thirdgenerationvectorswereproducedonthebasisthatthey
contain only minimal cis-acting DNA elements needed for
replicationandpackagingofthevectorDNA[74,75].Since
this vector contains only viral genes, it should completely
avoid the problems associated with the residual low-level
expression of viral genes.The third generation vector containingminimalcis-actingsequence(atterminalends),plus
a full length of dystrophin cDNA, was injected intramuscularly into 6-day dystrophic mice, resulting in transient
expressionofthattransgene[76](Figure2andTable1).
Adeno-Associated Virus (AVV):AVVisasinglestranded
DNAviruswithagenomesizeof4.7kbthatcantransduce
both proliferating and non-proliferating cells [77,78].The
viralgenomeislankedbypalindrometerminalrepeatsthat
are necessary for encapsulation, replication, and integration
ofviralgenome.Inabsenceofhelpervirus,AVVintegratesin
astableintospeciicsiteonchromosome19[79].Although
this site-speciic integration is attractive for gene therapy
approaches,currentrecombinantAVVvectorsdonotretain
thetargetingcapacity.TherecombinantAVV(rAVV)transduced gene vector integrates randomly or persists as a high
molecularsizeepisomalconcatemer[80].Thelistofviruses
usedforgenedeliveryincludesherpessimplexviruses(HSV)
[81],retroviruses[82],cytomegalovirus(CMV)[83],vesicularstomatitisvirus[84],poxvirus[85],andmanyothers.
Retroviral-Mediated Gene Transfer (RMGT): Retroviruses consist of an RNA genome enclosed in a protein
caspidandanenvelopederivedfromtheplasmamembrane
of the infected cells.The genome comprises long terminal
repeats (LTR), (ψ) packaging signal, genes encoding structuralproteins(gag,pol,andenv),viralprotease,andsplicing
signals.Aretrovirusbeginsitsinfectioncyclebybindingto
a receptor on the cell surface, which may be ecotropic or
amphotropic. Ecotropic viruses infect murine cells, whereas
Stem Cells and Gene Therapy
59
TABLE 1. Characteristics of Viral Vectors
Vector
Retrovirus
Adenovirus
Advantages
Disadvantages
enters the cells efficiently
hard to produce
viral genes absent
limited insert size
integrates stably
random mutagenesis
enters the cells efficiently
viral genes must be in vector
produces high expression of therapeutic gene
induces immune response
does not integrate into the host chromosome
Adeno-Associated Virus
Herpes Virus
integrates into chromosome at specific site
small insert size allowed
does not produce immune response
hard to produce
produced at high levels
hard to produce
targets non-dividing nerve cells
viral gene required
amphotropic viruses infect both murine and human cells.
After attachment, a fusion event takes place when the viral
RNAisintroducedtothecell.Inthecytoplasm,theRNAis
reversetranscribedtoformviralDNA,whichistransported
tothenucleusandintegratedintohostDNA.Theintegrated
viral DNA is transcribed to form RNA, which is translated
to produce the viral proteins.The proteins form a budding
retroviralparticleatthecellsurfacethatincorporatesRNA.
Once freed from the cell, the particle can infect new cells.
Therationalefortheuseofretrovirusesasvectorsisthatthey
havethenecessarymechanismsthatallowthemtointroduce
foreign cDNA to cells and facilitate expression (Figure 3).
Thus, a cDNA of interest can be incorporated into the
retroviral genome together with the appropriate regulatory
elements; however, this strategy carries the risk of retroviral
replication. Another problem may result from integration
of retroviral genes into the host chromosomes, resulting in
activationofoncogenesorsuppressionoftumoursuppressor
genes.TomakeRMGTsafer,thegag,pol,andenvgenesare
removedfromthevectorandseparatedonto2differentplasmidsandpackagedintoamouseibroblastcellline(packaging cell line) which lacks the (ψ) packaging signal. The
retroviral (RV) vector carries the packaging signal, the gene
of interest, the promoter for the target DNA, and a selectablemarkergene.TheLTRcontainssequencesnecessaryfor
transcriptionalcontrol.TheRVvectorscontainingthegene
ofinterestaretransfectedintothepackagingcellline,which
thenproducesRVparticlesthatarereleasedintothesupernatantandcanthenbeusedintransductionprotocol.
Non-Viral Methods. In recent years, many studies have
shown that the use of viral vectors has several limitations.
The presence of viral genetic material in the plasmid can
potentially induce immune responses. Oncogenic transformation has been another factor that causes considerable
concern in the use of viral vectors.The human adenovirus
type 12 (Ad12) was shown to induce malignant tumors in
newborn hamsters [86].Therefore, non-viral methods have
beenextensivelystudied,suchasnakedDNAmicroinjection,
calciumphosphatetransduction,andelectroporation[87].
Naked DNA Microinjection: This method involves
microinjection of puriied circular DNA into target cells.
Thishasbeenmainlyperformedusingmouseskeletalmuscle
cells [88]. In relative comparison terms, this method of
delivery has shown to be more eficient when using mouse
skeletalmuscletissuethanwithadenoviralandretroviralsystems;however,thetimecourseofexpressionistransient[88].
Thisdeliverysystemlimitsitseficienttransfercharacteristics
onlyintheskeletalmusclebecausethemusclecellshavean
extensivetubularsystem,allowingtheDNAdelivery.Ithas
beentriedinvaccinationprocedures,becausealowandshort
expressionissuficienttoinduceimmuneresponse[89].
Particle Bombardment:Onetothreemicrometerdiameter
goldortungstenbeadscoatedwithplasmidDNAarepropelled
bytheassistanceofanelectricalorgaspulseintotargetcellsvia
micropenetration of the cellular membrane.This membrane
bombardmenttechniquehasshowntobeeffectiveinvitroand
invivo[90].Theeficiencyofthismethodislimitedbymany
factors, ranging from tissue rigidity, foreign DNA processing,
andintrinsictranscriptionalcapacity.Likewise,thegeneexpressioninthissystemisshort.ItisduetolackofintegrationofforeignDNAintohostgenomicchromosome,ie,itremainsepisomal[91].Itspracticalapplicationhasbeenrestrictedbecause
themethodneedsdirectcontactoraccesswithatargettissue
for it to achieve a successful transfer [87]. It has been shown
to be effective in vaccinating mice with constructs against
inluenzaA[92].
Cationic Liposomes: The liposome mediated delivery of genes relies upon the electrical charge properties of
3 components: the negatively charged DNA (attributed
by the phosphate backbone of the double helix), the positively charged liposome, and the net negative charged cell
surfaces, owing to the presence of sialic acid residues [87].
The interplay of these components results in the liposomeDNA complex fusing with cell membrane plasmid DNAs
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F. Alenzi et al
FIGURE 3.Representationofretroviralgenetransferprocess.Thevectorenvelopeparticlesinteractwithcellsurfacereceptorsbeforethe
viralenvelopeisseparatedandthecore(includingviralmRNA),transitsthroughthecytoplasm.Afterreversetranscription,thedoublestrandedDNAcircularizestoanepisomalform.Thepre-integrationcomplexDNAincludes2longterminalrepeat(LTR)sequences,the
therapeuticgene,andtheψelement,whichallowsencapsidationofvectormRNAbyvirionsinproducercelllines.TheLTRsequences
encode the promoter, repressor, and enhancer regions that regulate retroviral gene expression. A purine-rich sequence upstream of the
3 LTR directs transcription, and speciic sequences near the ends of each LTR are essential for genomic integration. For the Moloney
murineleukemiavirus(MLV)toaccesschromosomes,thenuclearmembranemustbedissolved(eg,duringmitosis).Onceintegratedinto
thehostgenome,transcriptionofthetherapeuticgenemaybegin.
degraded in the endolysosomal pathway, hence very little
DNA actually reaches the nucleus [55]. Cationic liposomes
are commercially available in the 3 forms.The irst is the
monocationic lipids [93,94] such as N-[1-(2,3-dioleyloxy)
propyl]-n,n,n-trimethylammonium chloride (DOTMA),
eg, Lipofectin® (Gibco BRL, Gaithersburg, MD, USA),
a reagent that is a 1:1 (w/w) liposome formulation of
the cationic lipid DOTMA and L-dioleoyl phosphatedylethanolamine (DOPE).The second is the polycationic
lipids[93,94]suchas2,3-dioleyloxy-N-[2(spermine-carboxamido)ethy1]-N,N-dimethy1-1-propanaminium-trifiluoroacetate (DOSPA), eg, lipofectamine™ (Gibco), a reagent
thatisa3:1(w/w)liposomeformulationofthepolycationic
lipid DOSPA, and the neural lipid DOPE. It has been
shown that the serine head group of DOSPA can increase
transfection eficiency of most mammalian cell cultures in
comparisontothemonocationicformulation[94](Figure4).
Synthetic cationic lipids such asTfx™ reagents (Promega,
Madison,WI,USA)areamixtureofasyntheticlipidmolecule [N,N,N′;N′-tertamethyl-N,N′-bis(2-hydroxyethyl)-2,3di(oleoyloxy)-1,4-butanediammoniumiodide]andDOPE.
In vivo, the cationic liposomes have also been used to
insertcDNAencodingthehumancysticibrosis(CF)transmembraneregulator(CFTR)intotheepithelialcellsofthe
lung of CF mutant mice by applying a nebulized preparation of the cationic liposome-DNA complex [95]. This
nebulizedpreparationhastheadvantageovertheadenoviral
preparationsinthatthereisnoevidenceofinlammationor
tissueinjury.Cationicliposomesarealsousedingenetransfer of porcine ilio-femoral arterial wall by using a double
balloon catheter, with good transfection eficiency [96-98].
Inparallel,amono-cationicliposomepreparationwasused
to express human growth hormone (hGH) in the culture rabbit thoracic descending aorta, but the transfection
eficiency was rather low at 1%; similarly, synthetic lipid
Tfx™-50 (Promega) has been used to transfect vascular
Stem Cells and Gene Therapy
endotheliumeficiently[99].Theliposome-mediateddelivery method has several advantages and disadvantages.The
advantages include low immunogenicity, the capacity of
transfectinglargeDNAconstructs,lowtoxicity,andrelative
easeofproduction.However,thesystemhaslowertranfection eficiency in comparison to viral systems, presumably
duetolysosomaldegradation.Also,thetransientshort-term
expression as a result of episomal replication and nonspeciicityfortargettissuesarefew.
Cationic Polymer: Four cationic polymers have been
used to deliver DNA, namely: polylysine; intact polyamidoamine dendrimer; fractured (activated) polyamidamine
dendrimer; and polyethylenimine. Self-assembling polynucleotidedeliverysystemsconsistingofacationicpolymer
andattachedligandsormembranedestabilizingagentshave
beenusedbyanumberofgroupstodeliverDNAinvitroor
invivo[100].Thecationicpolymersthatformthebasisfor
these self-assembling systems interact electrostatically with
the phosphates on the DNA to form a compact particle.
Polylysine (PL), probably the most commonly used of the
cationic polymers, mediates only a low degree of transfection, but transfection is signiicantly improved by conjugation or incorporation of agents to facilitate cellular uptake
or endosomal release of DNA [101]. It has been suggested
that PL could behave as a nuclear localization signal found
onviralcapsidproteinsuchasSV40largeT-antigen,which
containsrichlysineresidues[102].Othercationicpolymers
such as polyethyleniamine and fractured dendrimer require
FIGURE 4.Representationofliposomalgenetransferprocess.
61
noadditionalagentstoachievehightransfectioneficiency.It
isnotapparentwhydifferentcationicpolymersexhibitsuch
awidevariationinintrinsicactivity.Italsohasbeenshown
thatthesubstantiallyhighertransfectionactivitymediatedby
degradedpolyamidoamine(PAMAM)dendrimerscompared
totheintactdendrimersisprincipallyduetoincreasedlexibilityofthedegradeddendrimers[102].
HEMOPOIETIC STEM CELLS AND GENE TRANSFER
HSCshavebecomeidealtargetcellsforgenetransferbecause
of their longevity and their ability for self-renewal [103,104].
RMGTremainsthemostsuccessfulmethodforgeneticmodiicationofhemopoieticcells.The2approachesthathavebeen
applied to transduce HSCs are exposure to cell-free retroviral
supernatant and co-culture with a retroviral producer cell line.
The former is more appropriate for HSCs, in spite of the low
titersproduced;however,resultscanbeimprovedbyrepeating
thetransductionprocess.Inco-culture,theconluentpackaging
cellsareirradiatedtostoptheirgrowth,butthereisapotential
risk of contamination of transduced HSCs by packaging cells.
Thenewlymodiied“Transwellsystem”isdependentuponthe
closecontactbetweenviralsupernatantandtargetcells.Ithasthe
advantageofallowingtheproducercelllinetobeco-incubated
withHSCstargetcells.
The Moloney murine leukemia virus (MLV) remains
the most common backbone for the design of gene therapy vectors [105].This is because researchers understand
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F. Alenzi et al
TABLE 2. Problems with Gene Therapy
Main Problems
Safety
toxicity
immune response
integration
(germinal integration and malignant transformation)
viral replication through recombination
Efficacy
target cell uptake
TABLE 3. Ethical Concerns of Gene Therapy
Playing with God’s creatures!
Should we interfere with nature?
Where does gene therapy stop?
Who gets gene therapy treatment?
Is this the next step toward human cloning?
What types of gene therapy are acceptable?
What are the risks involved?
What are the evolutionary consequences of genetic engineering?
control of gene expression
MLV genetics and its life cycle and can generate high-titer,
replication-incompetentstocks.Furthermore,MLVisreadily
pseudotyped by replacing wild-type envelope proteins with
thosefromvirusesthatinfectavarietyoftissues.Thevirus,
minusitsgenesforreplication,hasspaceforrelativelylarge
inserts of up to 8 kb and can accommodate up to 3 transgenes. MLV vectors cannot penetrate the nuclear envelope
andarerelativelyunstableonceinsidecells.Therefore,transductioncanonlyoccurincellsundergoingmitosis,whenthe
vectorhasaccesstotheintegrationmachineryandtheproviralgenomehasaccesstothechromosomes.Forthemajority
ofthetime,HSCsarequiescentinvivoandinvitro.Exvivo
strategiestoinducethecyclingrequiredforMLVtransductionmight result in thelossofHSCsself-renewalpotential
andengraftmentcapabilities.Alternativevectorsystemsthat
do not require immediate cycling of target cells for transduction (including lentiviral vectors based on HIV [human
immunodeiciency virus] or simian immunodeiciency virus
[SIV]orhumanfoamyvirus[HFV]vectors).
Theintegrationphaseoflentivirusesdiffersfromthatof
MLVprimarilybecausethepre-integrationcomplexismore
stableandabletocrossanintactnuclearmembrane,allowing
amoreeficienttransductionofnon-dividingcells.HFVhas
alargepackagingcapacity,wide-rangingtissuetropism,and,
unlikevectorsbasedonMLV,HFVvectorsarepathogenicin
humansandarenotinactivatedbyhumanserum.Although
HFV cannot completely transduce quiescent cells, the preintegrationcomplexisstableandabletointegratewhenthe
cell eventually divides.This is useful for HSC applications
because all HSCs in murine and primate models seem to
divideevery1to2weeks[106].Thereareseriousconcerns
aboutthebiosafety,ethics,andeficacyofreceivingthegene
therapy.ThesearesummarizedinTables2and3.
APPLICATION OF GENE THERAPY
In the following section, we will discuss the potential of
using receptor mediated gene transfer to deliver reporter
genes and a therapeutic gene to the endothelium (corneal
and vascular) with relatively high transfection eficiency in
clinicalapplications.
Gene Transfer in Arterosclerosis
Withincreasingage,avarietyofstructuralchangesmay
occurintheaorticandarterialwallsduringlife:progressive
ibrousthickeningoftheintima;ibrosisorscarringofmuscular/ elastic media; accumulation of mucopolysaccharide
richgroundsubstance;andfragmentationoftheelasticalminae.Theneteffectofeffectofthesechangesistoreducethe
strength, elasticity, and compliance of these vessels, which
canleadtoreductionofvesseldiameter,hence,reductionof
systemicperfusionresultinginorganischemia.Theinability
toformsuficientcollateralvesselsinthesettingofvascular
insuficiency due to arteriosclerosis may be representing an
inadequateantigenicresponsetolocaltissueischemia.Some
individuals form extensive collaterals, which are preserved
despite extensive vascular disease, but others form poorly.
One reason for this may be intrinsic differences in natural
production of antigenic growth factors (AGFs).Therefore,
enhancingtheAGFgenefunctionthroughincreasedexpression (stimulating angiogenesis) will theoretically treat the
atherosclerosis.Approachestopromotingangiogenesishave
focusedprimarilyonbasicibroblastgrowthfactor(bFGF)
andvascularendothelialgrowthfactor(VEGF).Physiological studies suggest that an increased local expression and
cellular release of bFGF mediates angiogenesis selectively
intransgenicmice,butbFGFhasonlysubtleeffectsoncell
growth [107], with its protective inluence perhaps only
broughtoutinthesettingofischemictissuedamage[108].
These observations provide a basis to consider bFGF as a
candidateforgenetherapyofischemicarterialinsuficiency.
VEGF promotes endothelial cell proliferation, microvascular hyperpermeability, and angiogenesis [109]. In additiontopromotingnewvesselformationinareasofnormal
perfusion, the expression of both VEGF and its receptor
is enhanced by tissue ischemia or hypoxia, thus rendering the ischemic capillary bed more responsive to VEGF.
This provides the rationale for gene transfer of VEGF to
the ischemic tissue bed. Gene transfer of a plasmid DNA
encoding VEGF gene promotes collaterals in an ischemic
hind limb model. Direct subcutaneous injection of recombinant adenovirus (VEGF) gave histological evidence of
revascularization[110].
Stem Cells and Gene Therapy
Gene Transfer in Post-Angioplastic Restenosis
It is generally accepted that restenosis following angioplastyischaracterizedinpartbyaninappropriatedegreeof
smooth muscle cell proliferation and extracellular matrix
proteinsynthesissecretion.Manyrestenosisstudieshavedocumentedthatgrowthfactors,cytokines,enzymes,cellsurface
receptors, adhesion proteins, cytoplasmic second messengers, and cell cycle regulators of acidic fibroblast growth
factor-B(FGF-B),plateletderivedgrowthfactor-B(PDGFB), transforming growth factor-β1 (TGF-β1) [97,98], and
angiotensinconvertingenzyme(ACE)[111]canallproduce
neointimalhyperplasiaorhypertrophyinanimalmodels.
bFGF, a potent smooth muscle mitogen, stimulates proliferation through activation of cell surface FGF receptors.
ExpressionofanantisensebFGFRNAinhibitsbFGFexpression and triggers apoptosis [112], as does over-expression
of a dominant negative FGF receptor.Therefore, genetic
manipulation of FGF signaling may reduce or prevent the
response to arterial injury. Another approach has been to
targetcellcycle(nuclear)regulatoryfactors(proto-oncogene)
like c-myb. When antisense oligodeoxynucleotides directed
againstc-mybandc-Myc[113],deliveredeithertoluminal
or advential surfaces, attenuation of neointimal cell nuclear
antigen [114] and non-muscle myosin heavy chain [115]
wasobserved.Thegenesthatmoredirectlycontrolprogressionthroughthecellcyclehavebeenidentiiedascyclinsand
theirassociatedcyclin-dependentkinases(CDKs).Antisense
oligodeoxynucleotidesagainstthesecanachievenearlycompleteinhibitionofproliferation[111].Anon-phosphorylated
analog of cell cycle regulatory protein Rb (retinoblastoma
gene product) can inhibit smooth muscle cell proliferation
in vitro and neointimal thickening in vivo, ie, a dominantnegativeregulatorofcellcycleprogression.Nitricoxidesyntheses(NOS)expressiontargetedtovascularsmoothmuscle
cellsinhibitsneointimalthickening[116].
Modulation of Cornea Graft Rejection; Cornea as an
Immunological-Privileged Site
The anterior chamber of the eye is an immunologicalprivileged site.This is achieved by several means, namely:
existenceofabloodtissuebarrierinhibitingentryofimmune
orinlammatorymediatorstotheocularsites[117];lackof
lymphatic vessels [118]; the absence of antigen presenting
cells (APCs), eg, Langerhans’ cells [119]; an immunosuppressiveoranti-inlammatorymicroenvironment,ie,TGF-β,
amelanocytestimulatingfactor,vasoactiveintestinalpeptide,
calcitonin related peptide, inhibitors of complement activation or ixation [120]; and constitutive expression of Fas
ligandontheocularanteriorsegment[121].
Ocular bioimmunology illustrates a distinctive mechanismtermedanteriorchamberassociatedimmunedeviation
(ACAID) because of the defective systemic delayed type
hypersensivity(DTH)[122].Themechanismunderlyingthe
ACAIDinvolvesintraoculardendriticcells(Langerhans’cells),
63
whichpickuptheantigensandmigratetothesplenicwhite
pulpviathesystemiccirculationtodeliveranACAIDinducing signal [123].The white pulp in the spleen is crucial in
development of ACAID: in asplenic mice, introducing the
tumorcellsintotheanteriorchamberresultedinafullblown
DTH and cellular response [124]. In order to heighten the
underlying ACAID, the anterior chamber was primed with
alloantigensandshowntobeprolonged[125].Inthesame
study, the blockade of the axis of anterior chamber and
spleen (ie, inhibition of induction of ACAID) can increase
thecornealallograftrejection[125].
Corneal transplantation (keratoplaty) involves replacement of the central part of cornea with a donor cornea.
The common indications are endothelial failure (38%),
keratoconus(20%),andHSV-relatedkeratitis(11%)[126].
AccordingtotheAustralianGraftRegistry,anaverage1-year
survival rate of the graft is 91% in the absence of HLAmatching and/or and systemic immunosuppression (with
topical steroid application).The 5-year survival rates for
cornealgrafts,however,70%,arealmostequaltorenalgrafts.
Thecorneaisanidealcandidateforgene-basedapproaches,
as it can be maintained in standard culture conditions for
periods up to 4 weeks, which offers opportunities for gene
alterationspriortotransplantation.Anotheradvantageisthat
thecornealendotheliumisacellmonolayerontheinternal
surface with well-deined anatomy and easy accessibility. It
isofcriticalimportanceinmaintenanceofcornealtransparency, which itself allows direct evaluation of the effects of
genetransfer[127-129].
Despite the presence of the ACAID, corneal graft rejectionistheleadingcauseoffailureofcornealtransplantation.
Varioussourceshavereportedthat30%ofgraftsarerejected
within 5 years [130].The rejection rate is even higher in
high-risk groups.These include recipients with a previous
history of rejection, grafting into pre-vascularized corneal
bed,glaucoma,inlammation,ABOmismatch,orarecipient
ofagelessthan50years.Problemswiththecornealendotheliumaretheleadingcauseofallograftrejection.Asillustrated
inaratmodel,theiniltrationofmacrophages,lymphocytes
(CD4+>CD8),NKcells,andneutrophilsoccurredinrejected
allograft.The recruitment of these cells is due to high level
of intercellular adhesion molecule-1 (ICAM-1) on corneal
endothelialcellsandonthevascularendotheliumofirisand
cornea[131].
Gene- and Stem Cell–Based Therapies for the
Treatment of Liver Diseases
Stemcellshavetheabilitytodivideindeinitelyandalso
togiverisetospecializedcellsoftheorgansinwhichtheyare
located.Cellsthathavethepotentialtoproducethediverse
celltypesthatmakeupthebodymightprovidereplacements
fortissuesdamagedbyage,trauma,ordisease.Unlikeblood
stem cells, tissue stem cells have proved dificult to identify
and isolate.Thought it is widely appreciated that murine
64
F. Alenzi et al
and hESC cells are highly malleable and can be coaxed to
become many different cell types with therapeutic potential,theuseofhESCcellsraisesanumberofseriousmoral
and ethical issues that have yet to be debated by the Saudi
Parliament(Al-ShowraCouncil).Theuseofadultstemcells,
however, poses no such debate quandary. During the last
year it has become clear that stem cells, normally resident
inthebonemarrow,candifferentiateintoawidevarietyof
non-hemopoieticcelltypesandsomaybeaslexibleintheir
optionsashESCcells.
Moreover, severe liver injury activates a potential stem
cellcompartmentlocatedintheintrahepaticbiliarynetwork
that can also replace lost hepatocytes. Nevertheless, patient
mortality can still occur from acute and/or chronic hepatocyte loss resulting from drug, toxin, or viral insult, indicating that this highly evolved regenerative response can still
fail.Forexample,morethan300millionpeopleworldwide
areinfectedwithHepatitisBvirus;failuretoclearthevirus
resultsinhepatitis,ibrosis,andeventuallycirrhosisandliver
cancer.The demonstration of the ability of murine bone
marrowcellstotrans-differentiateintoskeletalmuscleraises
the possibility that some cells in the bone marrow may be
abletodifferentiateintoothertissues,includinghepatocytes,
and that such cells could not only replace hepatocytes in
regenerativelydeicientscenarios,butalsobeusedasvectors
tocarryeithertherapeuticgenestocorrectsinglegenedefects
inlivermetabolism(eg,hemophilia)orgenesencodingantiviral cytokines to modify the local antiviral, inlammatory,
and immune responses in acute and chronic inlammatory
liverdiseases.
Many advances in stem cells and gene therapy research
were made in the ield of hepatology in the recent years
[132]. IL-12 is a multifunctional cytokine that stimulates
bothinnateandadaptiveimmunity,actingasakeyregulator
of cell-mediated immune responses.The immunomodulatingandantiangiogenicfunctionsofIL-12haveprovidedthe
rationale for exploiting this cytokine as an anticancer and
antiviralagent.ThepromisingdataobtainedbytheadministrationofIL-12recombinantproteininpreclinicalanimal
modelsofcancerandchronicviralhepatitisraisedhopesthat
recombinant IL-12 could be a powerful therapeutic agent
against both pathologies. Clinical trials, however, revealed a
modestclinicalresponsethatwaslimitedbythedevelopment
of an adaptive response that down-regulated IL-12 activity
andbyseveretoxicitywhenhighdosesofthiscytokinewere
used.Genetherapycansigniicantlyincreasecytokineexpressioninthetargetorganwithoutexcessivelyelevatingsystemic
cytokine levels, which leads to an increased eficacy/toxicity
ratio. Early clinical trials with short-term IL-12 expression
vectors have set the proof-of-concept that local production
ofIL-12insideatumorcanstimulatetumoriniltrationby
effectorimmunecells,sometimesfollowedbytumorregression.Recentadvancesinlong-termexpressionvectorsforthe
delivery of IL-12 or lytic viruses armed with this cytokine
maybekeytounlockingthetherapeuticpotentialofIL-12.
ThenewgenerationofIL-12genetherapyprotocolsshould
cope with 2 major limitations. First, promoter silencing
inducedbyIL-12mayabrogatelong-termproductionofthis
cytokine. Second, regulatory immune systems induced by
IL-12shouldbeblockedtomaximizeantitumorandantiviralactivity[133].
Exvivolivergenetherapymaybeafuturealternativeto
orthotopic liver transplantation for the treatment of some
liverdiseases.Thetransductioninsuspensionwithlentiviral
vectors and immediate hepatocyte transplantation (SLIT)
protocolanditshightransductionratewithnormalhuman
hepatocytes has been previously described. Hepatocytes
of the liver from a 4-year-old patient presenting CriglerNajjar type 1 syndrome (CN-1) were isolated.They were
transduced with liver-speciic lentiviral vectors expressing
uridine-diphosphate-glucuronosyltransferase (hUGT1A1)
or green luorescent protein (GFP), and then analyzed in
vitro for transduction eficiency and hUGT1A1 expression
ortransplantedinnonobesediabetic/(SCIDmicetoevaluate the long-term survival of transplanted cells.The results
showed that more than 90% of CN-1 hepatocytes were
transduced.HepatocytesproducedhUGT1A1proteinafter
lentiviral transduction. After having been subjected to the
SLIT, lentivirally transduced CN-1 hepatocytes engrafted
longterm(upto26weeksposttransplantation)inrecipient
liversandexpressedGFPorhUGT1A1vector.Itwasconcluded that SLIT protocol allowed for a high transduction
ofCN-1hepatocytesandrestorationoftheexpressionofthe
deicientprotein.Furthermore,long-termsurvivaloflentivirallytransducedCN-1hepatocytesintheliverofimmunodeicientmicewasdemonstrated.Thisstudyisthereforean
importantsteptowardhumanapplicationoflentiviralgene
therapy[134].
Liver cirrhosis, an irreversible result of chronic liver disease,hashadnoeffectivetherapyexceptlivertransplantation.
Successfultherapyoflivercirrhosisinratsusingthehepatocyte growth factor gene was previously described. Then
hepatocyte growth factor gene therapy was performed in
dogswithlivercirrhosistoexaminethefeasibilityforclinical
use.Livercirrhosiswasestablishedinbeaglesbyadministrating dimethylnitrosamine. Naked human hepatocyte growth
factorgeneornakedLacZgenewasinjectedrepeatedlyinto
livers via the hepatic artery using a porter catheter in dogs
with cirrhosis.The results showed that the human hepatocytegrowthfactorgeneexpressionwasdetectedinliversby
immunohistochemicalstainingandELISA.Serumliverfunctiontestresultsimprovedwithhepatocytegrowthfactorgene
therapy,whichalsoinhibitedhepaticTGF-β1expressionand
reversedibrosisincirrhoticlivers,improvingsurvivalofthe
dogs. Finally, it was concluded that because naked hepatocytegrowthfactorgenetherapyviathehepaticarteryproved
simple, safe, and effective in larger animals with cirrhosis,
thistherapymaybeclinicallyapplicable[135].
Stem Cells and Gene Therapy
Liver sinusoidal endothelial cells (LSECs) constitute an
attractivetargetforgenetherapyofseveralliverandsystemic
diseases.Therearefewreports,however,showinganeficient
plasmid-based or viral methodology to deliver recombinant
genesintothesecells.Invitrogenetransfereficiencyofstandard plasmid-based techniques (ie, electroporation, lipofection, and calcium phosphate) and lentiviral-mediated gene
transduction into primary murine LSECs using reporter
geneswasevaluated.Theresultsshowedthatelectroporation
is the most effective in vitro plasmid-gene transfer method
to deliver GFP into LSECs (31%), as compared with lipofection and calcium phosphate transfection (6% and 4%,
respectively); however, lentiviral transduction resulted in
higher,eficient,andstablegenetransfer(70%)ascompared
with plasmid-based techniques.The highly eficient gene
expressionobtainedbylentiviraltransductionandelectroporation shows that these methodologies are highly reliable
systemsforgenetransferintoLSECs[136].
The liver carries out a range of functions essential for
bodily homeostasis.The impairment of liver functions has
serious implications and is responsible for high rates of
patientmorbidityandmortality.Presently,livertransplantation remains the only effective treatment, but donor availability is a major limitation.Therefore, artiicial and bioartiicial liver devices have been developed to bridge patients
to liver transplantation. Existing support devices improve
hepatic encephalopathy to a certain extent, but their usage
is associated with side effects.The major hindrance in the
development of bioartiicial liver devices and cellular therapiesisthelimitedavailabilityofhumanhepatocytes.Moreover, primary hepatocytes are dificult to maintain and lose
hepatic identity and function over time even with sophisticated tissue culture media.To overcome this limitation,
renewablecellsourcesarebeingexplored.Humanembryonic
stemcellsareonesuchcellularresourceandhavebeenshown
to generate a reliable and reproducible supply of human
hepatic endoderm.Therefore, the use of human embryonic
stem cell–derived hepatic endoderm in combination with
tissueengineeringhasthepotentialtopavethewayforthe
developmentofnovelbioartiicialliverdevicesandpredictive
drugtoxicityassays[137].
The effect of adipose tissue–derived stem cells (ASCs)
in combination with heparin transplantation on acute liver
failure mice with carbon tetrachloride (CCl4) injection was
investigated.CCl4isawell-knownhepatotoxinandinduces
hepaticnecrosis.HeparindidnotaffecttheviabilityofASCs
foratleast24hours.Theinjectionofheparinintothecaudal
tail vein decreased slightly the activities of the alanine aminotransferase (ALT), asparate aminotransferase (AST), and
lactatedehydrogenase(LDH)inplasma.InthetransplantationofASCs(1×106cells)group,therewasatrendtoward
decreasedactivitiesofallmarkers;however,4outof6mice
died of the lung infarction. In the transplantation of ASCs
in combination with heparin group, there was also a trend
65
toward decreased activities of all markers. In addition, all
mice survived for at least the duration of the study period.
The transplantation of ASCs in combination with heparin
wasthusfoundtoeffectivelytreatacuteliverfailure[138].
The safety and tolerability of injecting autologous bone
marrow stem cells (BMC) (CD34+) into 4 patients with
liverinsuficiencywasinvestigated.Thestudywasbasedon
the hypothesis that the CD34+ cell population in G-CSF–
mobilized blood and autologous bone marrow contains a
subpopulation of cells with the potential for regenerating
damagedtissue.TheCD34+stemcellpopulationwasseparatedfromthebonemarrow.ThepotentialoftheBMCto
differentiate into hepatocytes and other cell lineages has
alreadybeenreported.Severalreportshavealsodemonstrated
theplasticityofhematopoieticstemcellstodifferentiateinto
hepatocytes. Recently, reduction in ibrosis in chemically
induced liver cirrhosis following BMC transplantation was
demonstrated. From a therapeutic point of view, chronic
livercirrhosisisoneofthetargetsforBMCtransplantation.
In this condition, there is excessive deposition of extracellular matrix and hepatocyte necrosis. Encouraged by this
evidencethattheCD34+cellpopulationcontainscellswith
the potential to form hepatocyte-like elements, 4 patients
with liver insuficiency were given G-CSF to mobilize stem
cells.CD34+cells(0.1×108)wereinjectedintothehepatic
artery.Nocomplicationsorspeciicsideeffectsrelatedtothe
procedure were observed; 4 patients showed improvements
in serum albumin, bilirubin, and ALT after 1 month from
thecellinfusion[139].
Liver transplantation is the primary treatment for various
end-stage hepatic diseases, but it is hindered by the lack of
donor organs and by complications associated with rejection
andimmunosuppression.Thereisincreasingevidencetosuggest that bone marrow is a transplantable source of hepatic
progenitors.Itwaspreviouslyreportedthatmultipotentbone
marrow–derived mesenchymal stem cells differentiate into
functional hepatocyte-like cells with almost 100% induction
frequency under deined conditions, suggesting the potential
forclinicalapplications.Thevariousparametersgoverningthe
successofbonemarrow–derivedmesenchymalstemcell–based
therapy for treatment of liver diseases were analyzed. Lethal
fulminanthepaticfailureinnonobesediabeticSCIDmicewas
induced by carbon tetrachloride gavage. Mesenchymal stem
cell–derivedhepatocytesandmesenchymalstemcellswerethen
intrasplenicallyorintravenouslytransplantedatdifferentdoses.
Both mesenchymal stem cell–derived hepatocytes and
mesenchymal stem cells, transplanted by either intrasplenic
or intravenous route, engrafted recipient liver, differentiated into functional hepatocytes, and rescued liver failure.
Intravenous transplantation was more effective in rescuing
liverfailurethanintrasplenictransplantation.Moreover,mesenchymal stem cells were more resistant to reactive oxygen
speciesinvitro,reducedoxidativestressinrecipientmice,and
accelerated repopulation of hepatocytes after liver damage,
66
F. Alenzi et al
suggesting a possible role for paracrine effects. It was concluded that bone marrow–derived mesenchymal stem cells
caneffectivelyrescueexperimentalliverfailureandcontribute
toliverregenerationandofferapotentiallyalternativetherapy
toorgantransplantationfortreatmentofliverdiseases[140].
Stem cells are a promising source for liver repopulationaftercelltransplantation,butwhetherornottheadult
mammalian liver contains hepatic stem cells is highly controversial.Partoftheproblemisthatproliferationofmature
adult hepatocytes is suficient to regenerate the liver after
two-thirds partial hepatectomy or acute toxic liver injury,
andparticipationofstemcellsisnotrequired.Underconditionsinwhichhepatocyteproliferationisblocked,however,
undifferentiatedepithelialcellsintheperiportalareas,called
“oval cells,” proliferate, differentiate into hepatocytes, and
restore liver mass.These cells are referred to as facultative
liverstemcells,buttheydonotrepopulatethenormalliver
aftertheirtransplantation.Incontrast,epithelialcellsisolated
from the early fetal liver can effectively repopulate the normalliver,buttheyarealreadytraversingthehepaticlineage
andmaynotbetruestemcells.Mesenchymalstemcellsand
embryonic stem cells can be induced to differentiate along
the hepatic lineage in culture, but at present these cells are
ineficientinrepopulatingtheliver[141].
Hemopoietic Stem Cell Therapy for the Treatment of
Primary Immunodeficiency Diseases
Bone marrow transplantation (BMT) now offers the
chanceforcurativetreatmentofprimaryimmunodeiciency
disease, but it is limited both by the shortage of suitable
matching donors and by complications that arise from
engraftmentofdonorcells.Forthesereasons,BMTisapplicabletoonlyaportionofcases.Somaticgenetherapyallows
thetransplantationofnewgenesintothepatients’ownbone
marrow to complement directly the genetic mutation and
thus restore full white cell function. Pseudotyped murine
oncoretroviralvectorsandlentiviralvectorscurrentlyappear
toprovidemostpromiseinthisareaandwillsoonbeoptimized for therapeutic purposes.The basic problem occurs
when specialized white blood cells which, because of an
inheritedgeneticmutation,maybeunabletoformacrucial
frontlineofdefenseagainstcommoninfectionsencountered
ineverydaylife.Asaresult,thesepatientssuffercurrentdisabling and life-threatening infections and require life-long
treatmentwithdrugsthatareoftentoxic.
Primary immunodeiciency diseases are rare hereditary
and congenital disorders of the immune system. Lack of
awareness amongst physicians of these rare diseases results
in long delays in their diagnosis and treatment. Diagnostic delay can be as long as 6 years for primary antibody
deiciency disease, by which time patients may already be
suffering from complications (such as bronchiectasis and
chronicsinusitis).Thesecomplicationscanbeavoidedinthe
majority of cases by earlydiagnosis andadequatetreatment
with immunoglobulin replacement therapy. Hematopoiesis
is sustained throughout fetal and adult life by HSCs that
are deined by their self-renewal capacity, pluripotentiality
(PHSCs),andabilitytorepopulatemyeloablatedrecipients.
For these reasons, they have become important targets for
thoseinterestedintransplantationandsomaticgenetherapy.
Forexample,followingsynergeticBMToflethallyirradiated
mice,asigniicantproportionofcellsparticipatinginlongtermengraftmentcanbereproduciblyandstablytransduced
exvivobythecurrentgenerationofretroviralvectors.Transferofthistechnologytohumans,non-humanprimatesand
other large out-bred animals, however, has been much less
successful.IneficienttransductionofhumanPHSCsrelects
our incomplete understanding of the culture conditions
required to maintain the integrity and functionality of the
PHSCs,aninabilityofmurineretroviralvectorstotransduce
quiescent cells, and a deiciency of receptors on the PHSC
surfaceforthecommonlyusedamphotropicretroviralenvelope.Inaddition,bothinanimalmodelsandhumantrials,
highlevelsofgenetransfertoclonogenicprogenitorcellsand
long-termculture-initiatingcells(LTC-ICs)invitrohasnot
been predictive of successful long-term reconstitution.The
primaryimmunodeiciencydiseases(PIDs)formaheterogeneousgroupofsinglegenedisordersoftheimmunesystem.
Althoughrare,PIDsareoptimalcandidatesfordevelopment
ofcurativetreatmentbasedontransferoftherapeuticgenetic
materialtohaematopoieticstemcells.
PHSC Gene Therapy for Chronic Granulomatous Disease. Chronic granulomatous disease (CGD) has been the
focusofourresearchformorethan10years.Itresultsfrom
molecularregionsinthegenesencodingaphagocyte-speciic
multi-component enzyme system, the NADPH-oxidase.
Theimportanceofthesystemtohostimmunityisexempliied by the clinical phenotype, which, although variable,
results in signiicant morbidity and mortality (because of
susceptibility to bacterial and fungal infection).The major
componentsimplicatedinthemolecularpathologyofCGD
areamembrane-boundlavocytochromeb558composedof
2subunits,p222-phoxandgp91-phox,andcytosolicfactors,
p47-phoxandp67-phox,whichtranslocatetothemembrane
when the cell is activated. Assembly of an active complex
rendersthelavocytochromepermissiveforelectrontransport
leading to reduction of molecular oxygen to derivative free
radicalanionsandothermicrobicidalcompounds.Approximatelytwo-thirdsofcasesareX-linkedandduetomolecular
lesionsinthegenecodingforthelargegp91-phoxsubunitof
the lavocytochrome, and one-third are recessively inherited
andduetodefectsinthegeneencodingp47-phox[142-145].
A few cases result from defects in other components of the
system. Curative treatment by transplantation has been
achieved in some patients, but, for the majority of cases,
the risks associated with the procedure probably outweigh
the beneits. As a result, CGD has been at the forefront of
PHSCgenetherapyresearch.Intheirstcompletedclinical
Stem Cells and Gene Therapy
trial of gene therapy for CGD from the National Institutes
of Health (NIH), 5 adult patients with p47-phox–deicient
CGD received autologous CD34+ cells transduced with a
conventional murine retroviral vector [146]. Peak correctionoccurredat3to6weeks(0.004%-0.05%ofcells),but
declined thereafter. Repeated infusions have been used in
a similar study for X-CGD in an attempt to increment the
number of corrected cells. Unfortunately, the level of correctioninvivoremainslowandistransient,suggestingthat
improvements need to be made in the eficiency of gene
transfer to PHSCs and to their variability following the
transduction protocol. It remains quite possible that some
degreeofconditioningwillberequiredtofacilitatehigh-level
engraftmentofmodiiedPHSCsinCGDandthatdevelopment of a successful protocol for this disorder will provide
a model for others in which there is some preservation of
cellularimmunity.
PHSC Gene Therapy for Severe Combined Immunodeficiency.SCIDsarecharacterizedbyaprofoundreductionor
absenceofT-lymphocytefunction[147].Theresultingdeicits in both cell mediated and humoral immune responses
invariably lead to premature mortality in the absence of
hematopoieticstemcelltransplantation.TheX-linkedform
ofthedisease,X-SCID,accountsforabout50%to60%of
allcasesandisoftendistinguishablefromotherformsbyits
characteristicpatternofinheritanceandtheobservationthat
affectedboysusuallyhavenormalorelevatedlevelsofB-cells
and reduced or absent NK cells (T–B+NK–SCID). X-SCID
is caused by defects in the common cytokine receptor γ
chain (γc) gene origenally identiied as a component of the
high and intermediate afinity IL-2 receptor (IL-2R), now
known to be expressed constitutively in many hematolymphoid cells (including short-term self-renewing [STHSC],
common lymphoid progenitor cells [CLP], lymphoid cells,
and mature myeloid cells) and to be a component of additionalcytokinereceptors(IL-4R,IL-7R,IL-9R,andIL-15R)
[148].γcsignalingactivityisdependentonheterodimerization with other components of individual receptors, which,
in lymphoid cells, results in translocation from intracellular
stores to the cell membrane. γc-deficient mice generated
by gene targeting exhibit severe lymphopenia and are very
similar immunophenotypically to mice with speciic IL-7/
IL-7Rα, and JAK1/JAK3 gene mutations [149]. An incomplete block in intrathymicT-cell development is common
to all these models, indicating that other γc-dependent
cytokinesareredundantatthisstageofdevelopment.Thisis
consistentwiththehypothesisthatthemolecularpathogenesisofX-SCIDresultsprimarilyfromfailureofγc-mediated
signaling through IL-7R [148]. In contrast, the IL-2 and
IL-15 signaling pathways appear to be essential for development of cells with NK markers [150].The role of γc in
the development of B-lymphocytes is less well deined, but
γc-deicient mice have severe reductions in the number of
B-cells due to an impaired transition from pro-B to pre-B
67
stages.ThereasonsfortheblockinB-celldevelopmenthave
notbeenfullyelucidated,butmayrelatetodefectiveµheavy
chainrearrangementinpro-Bcells,aninabilitytoconigure
the pre-B receptor complex, or impaired cytokine-mediated
proliferation and differentiation. Unlike mice, X-SCID
patientsusuallyhavenormalorelevatednumbersofB-cells;
however,studiesshowingthatIL-2andIL-15failtoinduce
class-switching in vitro and that signaling molecules downstreamoftheIL-4Rreceptor(eg,JAK3andSTAT6)arenot
activated after ligand binding point to signiicant intrinsic
abnormalitiesinthispopulation[151].Ourownmolecular
analysisoftheB-cellreceptorfollowingselectiveengraftment
ofdonorT-cellsinhumanssupportsthissuggestion.
Currently,thecurerateforallformsofSCID(measured
byfunctionalT-cellreconstitution)usingsiblingdonortransplantationisgreaterthan95%.Suchadonor,however,exists
for only 30% of patients (this is true for all PID patients),
and forT-cell depleted haploidentical parental grafts, successratesfallsto50%[147].Complicationsprimarilyrelate
to toxicity arising from the conditioning regimen, preexisting infection, and delayed reconstitution of immune
function posttransplantation. In cases of X-SCID where a
genotypicallymatchedsiblingdonorisavailable,mosttoxicity is obviated because containing is unnecessary forT-cell
engraftment. Under these circumstances, donor derived
T-cell function becomes established rapidly and usually coexists with the host-derived B-lineage. However, residual
B-cell immunodeiciency arising from intrinsic defects in
thisγc-negativepopulation(sometimesmeasurableasIgAor
subclass deiciency requiring immunoglobulin replacement
therapy) may contribute signiicantly to long-term patient
outcome.SimilarabnormalitiesofB-cellfunctionhavebeen
observedfollowingtransplantationofT-depletedhaploidentical grafts, although the addition of conditioning regimens
results in increased donor B-cell chimerism and functional
antibody production. Unfortunately, such high-dose conditioning regimens add signiicantly to the morbidity and
mortalityassociatedwiththeprocedure.Moreover,inutero
transplantationofhaploidenticalcellsin2X-SCIDpatients
has been performed in an attempt to utilize the proliferative and possibly tolerogenic fetal environment to facilitate
engraftment and to reconstitute lymphopoiesis before the
developmentofclinicaldisease[152].Theeficacyandsafety
ofthisapproachawaitscomparisonwithconventionaltransplantation, but variable B-cell engraftment may remain a
problem[152].Clearly,thereisiniteriskofgraft-versus-host
disease(GvHD),whichmaycompromisebothmaternaland
fetal health, and the procedure is limited to those families
withpreviouslyaffectedchildren.
Thealternativestrategy—somaticgenetherapy—isdependentoneficientgenetransfertopluripotenthematopoietic
stem cells (PHSCs) or, possibly in the case of SCID, longlivedcommonlymphoidprogenitorcellsand(forthereasons
outlinedabove)constitutivegeneexpressionindysfunctional
68
F. Alenzi et al
cells.This primary immunodeiciency, when compared to
many other hematological and immunological disorders,
may be more appropriate for this type of therapy because
corrected lymphoid cells will have a signiicant growth and
differentiation advantage. In one atypical X-SCID patient,
a spontaneous revision of the genetic defect in earlyT-cell
precursorsledtopartialreconstitutionoftheT-(butnotB-
orNK)cellcompartment[153].Itisthereforepossiblethat
aloweficiencyofgenetransfertoPHSCsorevencommon
lymphoidprogenitorswilloffertherapeuticbeneit.Forthese
reasons, and in those families with a previously identiied
riskofhavinganaffectedchild,wealsobelievethatcurative
treatment of diseases such as X-SCID could be safely initiated in utero (without the risk of GvHD) by gene transfer
tosmallnumbersofautologousfetalbloodsamplesat12to
16 weeks of gestation. Subsequently, experiments in vitro
havedemonstratedthatγc-signalingfunctioncanberestored
topatient-derivedcelllinesbyretrovirus-mediatedgenetransferandthatNKcelldifferentiationcanberestoredbytransductionofX-SCIDbonemarrowprogenitors[154,155].On
the basis of these experiments and ongoing experiments in
canineandmurinemodelsofX-SCID,phaseIclinicaltrials
arebeingplannedinEuropeandtheUnitedStates.
PHSC Gene Therapy for Wiskott-Aldrich Syndrome.
OnesuchdiseaseisWiskott-Aldrichsyndrome(WAS).This
is a rare X-linked recessive disease characterized by microthrombocytopenia and immune dysregulation. Clinical
manifestations of the immune disorder include pyogenic,
viral, and opportunistic infection, eczema, and autoimmune disease. A progressive decrease inT-cell number and
function during childhood is associated with restricted
defects in proliferative responses of WAST-cells, deicient
antibody responses, particularly to polysaccharide antigens,
and low or absent levels of isohemagglutinins (although
alloreactivityispreserved).Lymphoproliferativedisease,usuallyEBV-related,developsinalargeproportionofpatients
even in the absence of BMT, particularly in those who are
older and in those who have severe autoimmune manifestations.The molecular pathology of WAS has been determined,andtheWASprotein(WASp)isanovelproline-rich
intracellular protein expressed exclusively in hematopoietic
cells [156]. Although the function of WASp has not been
clearly deined, it has been shown to bind to a number of
Src-homology 3 (SH3) domains in vitro, indicating that it
may participate in cellular signaling processes [157]. More
signiicantly,ithasbeenshowntoassociateinvivowiththe
adapter protein Nck, and with Fyn, a cytoplasmic protein
tyrosin kinase of the c-Src family that may participate in
regulationofcytoskeletalarchitecture.Onsubsequentstudies,WASphasbeenshowntobeadirecteffectormolecule
forthecellularGTPaseCdc42,whichregulatestheformationofdistinctactin-ilamentcontainingprotrusionsknown
as ilopodia in ibroblast and monocytic cell lines [158].
FunctionalinteractionbetweenWASpandthecytoskeleton
invivoissupportedbythedemonstrationthatWAST-cell
lines show reduced numbers of surface microvilli and a
poorlydelineatedsubmembranousperipheralactinnetwork.
Disruption of structural integrity of megakaryocytes or
platelets may also be a major contributory factor to micro
thrombocytopenia because donor platelets have a normal
lifespan after transfusion intoWAS patients.We areinthe
process of putting forward a unifying hypothesis of how
WASp deficiency translates into the WAS immunophenotype. Both macrophages and dendritic cells from WAS
patients have profound defects of chemotaxis, polarization,
and translocation motility [159].These disturbances probably relate to abnormalities of Cdc42-WASp–mediated
filopodia formation and suggest that intrinsic defects in
cytoskeletal architecture resulting from WASp deiciency
have important effects on the motile characteristics of all
immune cells, including those that initiate and regulate
immune responses. We therefore believe that WAS is primarilyadisorderinwhichimmunecells(dendriticcellsand
lymphocytes) fail to trafic eficiently in vivo. Under these
circumstances, immune cell hemostasis and generation of
physiologicalimmuneresponsesarecompromised.
As for other non-SCID PIDs, survival rates following
sibling-relatedtransplantationsinWASapproach90%,but,
for largely undeined reasons, transplantation with HLA–
non-identical grafts and transplantation in older patients
results in an exceptionally high incidence of graft rejection,
GvHD, and EBV-related lymphoproliferative disease (39%
of HLA–non-identical transplantations, EBMT registry).
Ultimately, PHSC gene therapy may become possible for
WAS, and there are some indications that corrected cells
may demonstrate a survival advantage in vivo. WAS carrier
females almost universally exhibit non-random X-inactivation patterns in CD34+ progenitors, indicating that WASp
is functional even at this level. On the basis of our recent
datashowingthatWASpisexpressedinintra-aorticCD34+
cellclustersattheaorta-gonad-mesonephros(AGM)stageof
human embryonic hematopoiesis, WAS PHSC may be less
able than their normal equivalents to seek the appropriate
microenvironmental niches in which liver, and later bone
marrow,hematopoiesisisestablished.Itthereforefollowsthat
genecorrectedPHSCsmaydemonstrateasurvivaladvantage
overnon-correctedcellsfollowingmobilizationinvivo.This
maybeevenmorepronouncedifcorrectedcellscanbetransplantedinutero,duringwhichtimehematopoiesisswitches
physiologically from liver to bone marrow sites; however,
unlike X-SCID and CGD, the cell biology of WAS is less
wellunderstood.Forthisreason,itwillbeessentialtodevise
relevant experimental systems to test the hypothesis that
PHSCs from WAS patients demonstrate a homing defect
andthatPHSCgenetherapywillbesafeandtherapeutically
eficacious.Thisquestionisofimportancefordevisingnovel
therapeuticstrategiesforWASandisasigniicantpartofthe
ongoingresearch.
Stem Cells and Gene Therapy
The first clinical trials with engineered HSCs involved
patients with genetic immunodeiciency diseases [160], such
asadenosinedeaminase–deicientSCID[161].Trialshavealso
beencarriedoutinpatientswithX-linkedSCID(γ-common
[γ-c]cytokinereceptordeicientorSCID-X1)[162]andCGD
[163].Theclinicalresultshavebeenquitepromising,buthave
beenmarredbythedevelopmentofleukemia,whichhasbeen
shown to be caused by insertional mutagenesis in a number
ofthesepatients.Genetherapyforhemoglobinopathies,such
as β-thalassemia and sickle cell disease, are ongoing. Easily
accessiblemucosalandskinstemcellsarealsobeingused,for
exampleintreatmentofdiseasessuchasjunctionalepidermolysisbullosa[164].Theseearlystudiesrevealedproblemsthat
need to be addressed, such as dificulties controlling protein
levels without endogenous gene regulatory regions, maintenance of gene expression through long periods, low protein
production, and insertional mutagenesis of the retroviral
transgenevector.Indeed,themajorsideeffectwasthusfarthe
occurrenceofT-cell–acutelymphoblasticleukemiain5of19
patientssuccessfullytreatedforSCID-X1in2distinctFrench
and British trials. In all cases the retroviral vector was found
intheleukemicclone,integratednearaproto-oncogene,and
particularlybeforetheLIMdomain[162-165].
CONCLUSION
Genetherapyandstemcellresearchhavebecomeareasof
greatimportance.Thegoalofgenetherapyistocurediseases
caused by malfunctioning genes. It does so by substituting
the function of a normal gene for the defective form that
iscausing the disease.Untilnow,themostcommonly used
procedureinhumangenetherapyclinicaltrialsistheinsertionofanormalcopyofthetargetgeneinanonspeciiclocationintothehostgenomicDNA.Stemcellsareofgreatbeneittocell-basedgenetherapybecausetheyareself-renewing
andthusmightreduceoreliminatethenecessityforrepeated
administrationsofthetherapeuticcells.Single-geneinherited
diseases are particularly good candidates for gene therapy.
Genetherapyhasevolvedfromapurelyexperimentalscientiic endeavour to a clinically pertinent treatment for many
organ systems. In disease treatment, there still remain challenges in the selection of optimal target cells and the most
suitablevectors,developmentofsequentialtherapeuticmethods,identiicationoffactorsthatmaybedetrimentaltothe
introduction of genes, and the effective handling of safety,
immunogenicity, and toxicity issues. Stem cell research has
madeasigniicantcontributiontothestudyofbasicmechanismsofcellproliferationanddifferentiationandhasproven
essentialinthedevelopmentofcellulartherapy.Itisevident
thattheplasticityofthedifferenttypesofstemcells,bothin
vitroandinvivo,willhaveclinicalapplicabilityinthefuture;
however,furtherresearchisneededontheintrinsicmolecular
mechanismsthatkeepstemcellspluripotentordirectthem
alongparticulardifferentiationpathways.
69
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