Gene Therapy for Skin Diseases - CSHL Pperspectivesinmedicine.cshlp.org/content/4/4/a015149.full.pdf · Either delivery system can be applied forex vivo or in vivo correction routes

Gene Therapy for Skin Diseases

Emily Gorell, Ngon Nguyen, Alfred Lane, and Zurab Siprashvili

Department of Dermatology, Stanford School of Medicine, Palo Alto, California 94305

Correspondence: [email protected]

The skin possesses qualities that make it desirable for gene therapy, and studies have focusedon gene therapy for multiple cutaneous diseases. Gene therapy uses a vector to introducegenetic material into cells to alter gene expression, negating a pathological process. This canbe accomplished with a variety of viral vectors or nonviral administrations. Although resultsare promising, there are several potential pitfalls that must be addressed to improve the safetyprofile to make gene therapy widely available clinically.

Gene-based therapeutics are broadly definedas using a vector to introduce nucleic acids

into cells with the intention of altering geneexpression to prevent, halt, or reverse a patho-logical process (Kay 2011).

The skin is an attractive tissue for gene ther-apy for several reasons. It is an easily accessibleorgan associated with monogenetic diseasesas well as chronic wounds, which may benefitfrom gene therapy approaches (Khavari et al.2002). Although the idea of gene transfer is rel-atively straightforward and initial attemptsshowed promising results, the future of genetherapeutic trials was tempered by the subse-quent development of T-cell leukemia second-ary to vector insertions near the LMO2 proto-oncogene in patients with severe combinedimmunodeficiency and Wiskott-Aldrich syn-drome (Cavazzana-Calvo et al. 2000; Hacein-Bey-Abina et al. 2002, 2008; Howe et al. 2008;Boztug et al. 2010). Although this made re-searchers reevaluate the initial simplicity ofsuch an approach and focus on challenges relat-ed to insertional mutagenesis, immunogenicity,

and vector stability in the host (Kay 2011), clearevidence of clinical benefits lead to increasedgene therapy clinical studies, resulting in 1800gene transfer trials around the world (Ginn et al.2013).

GENE TRANSFER TECHNIQUES

As a superficial organ, easy to manipulate andobserve, the epidermis was one of the first tar-gets for cell isolation (Rheinwald and Green1975), in vitro tissue engineering (Green et al.1979; Bell et al. 1981; Burke et al. 1981), and invivo experimental gene transfer (Williams et al.1991). In gene therapy techniques, genetic ma-terial is usually transferred using modified vec-tors, either directly into a subject’s epidermaltissue (in vivo), or indirectly (ex vivo) in whichcells are removed from the host, subjected togenetic manipulation, then reconstituted intothe subject’s skin (Fig. 1).

Independent of the route used in geneticmaterial transfer, there are two fundamentallydifferent gene delivery systems: viral and nonvi-

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Copyright # 2014 Cold Spring Harbor Laboratory Press; all rights reserved; doi: 10.1101/cshperspect.a015149

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ral. Either delivery system can be applied for exvivo or in vivo correction routes.

Gene Transfer with Viral Vectors

Viral vectors are the most effective vehicles ofgene transfer because of their inherent ability toefficiently infect cells (Table 1). Numerous vi-ruses are under investigation for gene delivery,but the most commonly used viruses to targetcutaneous tissue are retroviruses, adenoviruses(AdV), and adeno-associated viruses (AAV). Al-though they provide superior efficacy in genetransfer, the main drawbacks are virus-associ-ated toxicity as well as costly and complicatedmanufacturing.

Retrovirus

Retroviruses have the longest history of use ingene therapy and are still the most frequentlyused vectors for cutaneous gene transfer (Edel-stein et al. 2007). Retroviral vectors use reversetranscriptase to back-transcribe their RNA intoDNA and use integrase to integrate into in-fected host genome. They are capable of highlevel transduction efficiency in culture (nearly100%) and providing acoding capacityof 8–9 kb.

Vectors derived from oncoviral and lenti-viral retroviruses are commonly used in gene

therapy. The best example of an oncoretro-virus is the Moloney murine leukemia virus(MMLV), whereas lentiviruses originate fromhuman immunodeficiency virus (HIV). HIV-based viruses have a pronounced advantageover oncoretroviruses, namely the ability to in-fect nondividing cells owing to their ability todeliver the viral preintegration complex (PIC)across the nuclear membrane. Because epider-mal stem cell populations have a low rate ofmitotic activity, lentiviral vectors are more at-tractive for in vivo therapy.

On the other hand, oncoretroviruses areefficient for ex vivo gene delivery allowing trans-duction of epidermal stem cells, which is es-sential for long-term transgene expression inreconstituted skin tissue. Both oncoretrovirusesand lentivectors delivered ex vivo provide thecapacity for therapeutic gene expression inskin regenerated from transduced keratinocytes(KC) for several epidermal turnover cycles prov-ing successful targeting of epidermal progenitorcells (Chen et al. 2002; Larcher et al. 2007; Si-prashvili et al. 2010).

General design of retroviral vectors mini-mizes the potential to form a replication com-petent retrovirus (RCR). Vector constructionshould retain several elements that are impor-tant for the viral life cycle, such as RNA pack-

Administer correctedcells to patient

(grafting, injection, etc.)

Geneticcorrection

Culturepatient’s

cells

Obtainpatient’s

cells via skinbiopsy

Directlyadministervector topatient

Ex vivo genetherapy

In vivo genetherapy

Figure 1. Gene therapy delivery methods.

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aging signals and cis-acting viral sequences,such as 50- and 30- long terminal repeats (LTR).Other viral sequences are removed and replacedwith the gene of interest. To generate the thera-peutic vector, viral proteins are supplied in transtogether with the transgene construct withinpackaging cells derived from established celllines (Miller 1992).

Additional genetic engineering of the tar-geting construct is directed to create self-inacti-vating (SIN) vectors including generation of afusion 50 LTR promoter, to control therapeuticgene expression, and introduction of a deletion

within the U3 region of the 30 LTR. This strategyis applicable for both lentiviral and MMLV-de-rived vectors.

Lentiviral vectors are more complex becauseof accessory proteins and sequences that allownuclear import of viral PIC. For safety reasons,lentiviral vectors are usually produced by tran-sient transfection methods in packaging celllines. Pseudotyping lentiviral vectors with thesurface glycoprotein from vesicular stomatitisvirus G protein increases stability and titer,and broadens the tropism (Dull et al. 1998;Cooray et al. 2012).

Table 1. Gene delivery vector characterization

Gene vector Advantages Disadvantages Therapeutic use

Retrovirus(oncoretrovirus)

High transduction efficiency in vitroSustained gene expressionAcceptable coding capacity of ,9 kbNo immunogenicity

Risk of insertionalmutagenesis

Potential for silencingHigh production costsDoes not transduce

nondividing cellsProne for recombination

Inherited geneticdisorders

Systemic diseasesReplacement gene

therapyTrans-splicing RNA

repairLentivirus High transduction efficiency in vitro

Acceptable transduction efficiency invivo

Sustained gene expressionAcceptable coding capacity of ,9 kbNo immunogenicity


Potential for silencingHigh production costsProne to recombination


Systemic diseasesReplacement gene

therapy

Adenovirus High transduction efficiency in vivoHigh coding capacity of ,38 kbDoes not integrate into host genomeStableHigh titer

ImmunogenicTransient gene expressionHigh production costs

Antitumor therapyChronic wounds

Adeno-associatedvirus

Transduction in vivo and ex vivoSustained gene expressionStableHigh titerLow immunogenicity

Low coding capacity,5 kb


Immunogenic afterrepeated administration

High production costs


Systemic diseasesHomology-directed

mutation repair

Nonviral (plasmidDNA)

Low production costsHigh coding capacityDoes not integrate into host genomeIn vivo administration inexpensive

and well toleratedNo immunogenicityStable

Low efficiencyTransient expressionNo selectivityRisk of off-target genome

editing using TALEN,CRISPR systems

VaccinationChronic woundsAntitumor therapyInherited genetic

disordersGenome editing

and homology-directed

Mutation repair

Cutaneous Gene Therapy

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Adenovirus (AdV)

The most commonly used vector today is AdV,used in 23.3% of all viral gene therapy trialsworldwide (Ginn et al. 2013). They can carry alarger DNA load than retroviruses, are able toachieve high transduction efficiency in a varietyof cell types including nondividing cells, andproduce high albeit transient levels of gene ex-pression because of the episomal nonintegratedstate of the viral genome. These double-strand-ed DNAviruses can be rendered replication de-fective by substitution of the essential E1 genewithout an apparent effect on viral growth.“Gutted” helper-dependent adenovirus is gen-erated by stripping the majority of viral protein-encoding genes leaving essentially inverted ter-minal repeats and the packaging sequence at the50-end of the viral genome (Dormond and Ka-men 2011).

Expression of AdV in the skin is brief, last-ing only about 2 wk presumably attributable tolack of genomic integration and possibly delayedcytotoxic effects (Hirsch et al. 2006). Other thantissue repair, adenovirus-based immunother-apy has emerged as a potential direction, butconclusive statements on the usefulness of cuta-neous therapeutic strategies will have to awaitfurther studies (de Gruijl and van de Ven 2012).

Adeno-Associated Virus (AAV)

The nonpathogenic AAV-2 subtype of adeno-associated viruses, a member of the parvovirusfamily, is a common gene therapy vector. It ischaracterized by stability of the viral capsid, lowimmunogenicity, the ability to transduce bothdividing and nondividing cells, the potential tointegrate site specifically and to achieve long-term gene expression even in vivo. AAV vectorshave an insert capacity of �4.5 kb. Proliferationdepends on the presence of a helper virus suchas AdV or herpes virus.

Studies have shown that cutaneous trans-duction using AAV is possible both ex vivoand in vivo, although strong evidence for effi-cacy, duration, and vector integration is lacking.For ex vivo use, one study showed up to 70%transduced KC, whereas another showed short-lived gene expression (,4 d) only in immor-

talized KC, with lack of infection in primarycells (Braun-Falco et al. 1999; Gagnoux-Pala-cios et al. 2005). Interestingly, packaging of anAAV vector with capsid serotype 6 increased KCtransduction frequency 5 logs compared withthe same vector packaged with capsid sero-type 2 (Petek et al. 2010). In vivo gene transferinto porcine skin has been successfully shownafter intradermal injection of recombinant AAVparticles, which led to transgene expression inepidermal KCs for more than 6 wk, althoughwith diminished transgene expression and aninflammatory response after readministrationattempts (Hengge and Mirmohammadsadegh2000). Therefore, recent improvements madein AAV vector design and production highlightsthe therapeutic potential of this vector in cuta-neous gene therapy.

Nonviral Gene Therapy

Nonviral gene transfer techniques possess sev-eral advantages including cost-effective pro-duction of large amounts of vector, low toxicity,low immunogenicity, and preferential safetycompared with viral vectors (Table 1), as thereis no risk of RCR. Furthermore, nonviral genetransfer is usually characterized by transientgene expression and low transfection efficiency.Short-term gene expression may be desirable forwound healing or bone regeneration. Long-term gene expression can be achieved by select-ing stable clones ex vivo. Targeting loss of func-tion mutations is achieved by introducing aplasmid DNA (pDNA) or RNA encoding thegene of interest. Conversely, for gain-of-func-tion mutations, therapies that reduce geneexpression such as RNA interference and micro-RNA can be used. Moreover, recent develop-ments in engineered nucleases to create breaksin the genome following repair based on homol-ogous recombination using exogenous donortemplates makes nonviral gene therapy vectorseven more desirable to target monogenic diseas-es (Porteus and Baltimore 2003). These breakscan be generated by several methods: zinc fin-ger nucleases (Pabo et al. 2001), clustered reg-ularly interspaced short palindromic repeats(CRISPR) (Mali et al. 2013; Wang et al. 2013),

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or transcription activator-like effector nucleases(TALEN) (Osborn et al. 2013).

Plasmid DNA Design

The simplest and most straightforward gene de-livery vehicle is pDNA. Plasmids are propagatedin bacteria, therefore they contain a bacterialreplication origin and a selection marker—agene conferring antibiotic resistance. Tissue-specific promoters, enhancers, splicing introns,and other regulatory elements of mammalianmaintenance devices such as a locus control re-gion, ensure that the therapeutic gene is ade-quately expressed in target human tissue. Inclu-sion of insulating elements on each side of theexpression cassette ensure limited influence onother genes and flanking sequence with trans-poson elements that allow chromosomal inte-gration of the entire transcription unit (Tol-machov 2011). To further improve the safetyprofile of pDNA, minicircle DNA lacking thebacterial backbone sequence, an antibiotic re-sistance gene, and an origin of replication weredeveloped with greatly increased efficiency oftransgene expression in vitro and in vivo (Dar-quet et al. 1997; Chen 2003).

Plasmid DNA Administration

The most desirable method of cutaneous DNAdelivery is topical application, yet the stratumcorneum (SC) prevents DNA transport acrossthe phospholipid-rich layer. Topically appliednaked pDNA in aqueous solution can reachthe epidermis via hair follicles. Although theefficiency of transgene delivery with this routeis low, for hepatitis B surface antigen (HBsAg),specific antibody and cellular responses wereinduced in the same order of magnitude asthose produced by intramuscular injection ofthe commercially available recombinant HBsAgpolypeptide vaccine (Fan et al. 1999).

Several methods were developed to crossthe SC barrier, albeit more invasive than topicalapplication. Direct injection of interleukin-8pDNA into porcine skin resulted in DNA up-take by KC and the appropriate biological re-sponse of neutrophil recruitment (Hengge et al.

1995). Hypodermic needle use often causespain and inflammation at the injection site;therefore, there is a need to develop needle-free gene delivery strategies. One of the methodsthat increases skin permeability is based on aballistic pDNA projectile across the cutaneousbarrier. The first account of successful needle-free pDNA delivery was reported in 1991 using agene gun. pDNA covered gold particles 2–5 mmin diameter were shot into the skin driven byhelium gas without evidence of skin injuryand 10%–20% delivery efficiency (Williams etal. 1991). Today other high-pressure flow meth-ods are used, mainly for immunization purpos-es, such as liquid jet injection (Mohammed et al.2010), which directs a pressurized liquid to makea pathway into the skin and epidermal powderimmunization, which accelerates dried-powdervaccine particles into the skin at supersonicspeed (Dean and Chen 2004).

The other methods of physical/mechanicalgene delivery are sonoporation (ultrasound-mediated gene transfer), electroporation, andmagneto-permeabilization. Sonoporation re-fers to transient porosities in the cell mem-branes induced by ultrasound (cyclic soundpressure with frequency range .20 kHz) anduptake of DNA or drug microbubbles into thecells (Miller et al. 2002). Electroporation hasbeen used for transdermal drug delivery by in-creasing skin permeability by applying an elec-tric field, which surpasses the electrical capacityof the cell membrane. A combination of long,low voltage pulses is used for DNA transfer. Thefirst successful in vivo pDNA electrotransfer wasachieved in 1991 using newborn mice (Tito-mirov et al. 1991). To avoid unwanted electrodecontact with the subject during electropora-tion, magnetic fields were generated using ahand-held flat magnet applicator to delivergreen fluorescent protein-encoded pDNA toguinea pig skin in vivo (Kardos and Rabussay2012). Magneto-permeabilization provides sev-eral advantages over electroporation: there is noneed for invasive electrodes, it is more cost ef-fective, and there is greater tissue penetration bythe magnetic field.

Microneedles (MN) have emerged as a po-tential new approach for minimally invasive de-



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livery of epidermal gene transfer (Pearton et al.2012). The dimensions of MN are within themicron range and consequently their penetra-tion, on topical application, is restricted to themost superficial layers of skin (i.e., the viableepidermis and papillary dermis). Such devicesare widely used for vaccination and fall into atleast four design categories: hollow, solid, coat-ed, and dissolving (Kim and Prausnitz 2011).Coated and dissolving MNs incorporate drugwithin the body of the needle, providing simul-taneous skin puncture and delivery. DissolvingMN can be manufactured as biodegradable ar-ray needles with time-dependent release (Kom-mareddy et al. 2012).

To further enhance topical, dermal, ortransdermal gene delivery efficacy, many cation-ic polymers have been studied both in vitro andin vivo. However, in recent years there has beena focus on nanoparticle (NP) biodegradablecarrier systems (Ditto et al. 2009). NPs vary insize from 1.5 to 1000 nm and are readily grafta-ble with cationic polymers, nuclear localizationsignals, peptides, and polyethylene glycol toprovide the ability to escape endosomes, navi-gate to the nucleus, target the site, and evade theimmune system (Raghavachari and Fahl 2002).NPs are attractive delivery vehicles for genesilencing purposes as well. Spherical nucleicacid NP conjugates (gold cores surrounded bya dense shell of highly oriented, covalently im-mobilized siRNA) freely penetrate almost 100%of keratinocytes in vitro in mouse skin and hu-man epidermis within hours after application(Zheng et al. 2012).

Ultimately, the fact that nucleic acid in ei-ther naked or formulated form displays lowtransfection efficiency in vivo compared withthat of viral vectors limits its usefulness forgene therapy.

REVERTANT MOSAICISM

Revertant mosaicism, also called “natural genetherapy,” has received attention recently in thedermatological community. Revertant mosai-cism refers to the presence of two geneticallyheterogeneous populations of cells resultingfrom spontaneous genetic correction during

mitosis (Almaani et al. 2010). Cutaneous rever-tant mosaicism manifests as areas of pheno-typically normal skin unaffected by disease. Inpatients with the bullous genodermatoses epi-dermolysis bullosa (EB), revertant areas haveepidermal homeostasis indistinguishable fromnormal skin. Cases of revertant mosaicismhave been described in all subtypes of EB, en-compassing several genes, with multiple mech-anisms of correction (Al Aboud et al. 2003;Smith et al. 2004; De Luca et al. 2009; Almaaniet al. 2010; Pasmooij et al. 2010, 2012; Lai-Cheong et al. 2011; Pasmooij and Jonkman2012; van den Akker et al. 2012). Reversion hasalso been described as a mechanism for the phe-notype observed in patients with ichthyosis withconfetti, as revertant stem cells are positively se-lected or there is an increased rate of mitoticrecombination (Choate et al. 2010).

SKIN DISEASES TREATED WITH GENETRANSFER

Epidermolysis Bullosa (EB)

EB is a family of inherited genetic blistering skindisorders associated with gene defects affectinggene expression of the basal epidermis. Fifteengenes and 13 proteins have been characterizedand are responsible for the specific subtypes ofthis disease (Has et al. 2012; Marinkovich 2012).Molecular alterations in a number of specificgenes responsible for EB have been increasinglywell characterized over the past 15 years (Ma-rinkovich 1993; Uitto et al. 1994; Korge andKrieg 1996; Paller 1996; Uitto and Pulkkinen1996; Dang and Murrell 2008). Malfunction inany of their corresponding proteins mediatingepidermal adhesion results in skin fragility andblistering.

There are three main types of EB:EB simplex(EBS), junctional EB (JEB), and dystrophic EB(DEB), each affecting different levels of the epi-dermis (Fig. 2). EBS is most often caused bydominant mutations in the genes encoding forkeratin 5 or keratin 14, and is usually a milderphenotype than the other two forms of EB, withblisters mainly on areas of major trauma. JEBis caused by recessive mutations in the genes for

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collagen XVII, integrin a6b4, or laminin 332.Herlitz JEB is usually lethal within the first2 years of life. Non-Herlitz JEB is characterizedby chronic skin blistering, dental anomalies,and alopecia. DEB is attributable to mutationsin the gene (COL7A1) encoding type VII colla-gen (C7), and can be recessive (RDEB) or dom-inant. The more severe RDEB results in chronicblistering and scarring, esophageal strictures,mitten deformity of the hands and feet, andearly death from malnutrition, sepsis, or aggres-sive squamous cell carcinoma (Fine et al. 2000;Uitto and Richard 2005; Marinkovich 2012).

Recessive Dystrophic EpidermolysisBullosa (RDEB)

Many different groups throughout the worldhave attempted to correct RDEB. Our group

has focused on RDEB gene therapy for morethan 20 years, exploring nonviral methodssuch as the use ofFC31 bacteriophage integrasealong with a plasmid expressing COL7A1 cDNA,to target chromosomal integration of the trans-gene into keratinocytes (Ortiz-Urda et al. 2002)or fibroblasts (Ortiz-Urda et al. 2003). In 2010,using a retrovirus-based therapy, RDEB KCswere corrected with COL7A1 cDNA and long-term durable expression of C7 seen when graft-ing human skin onto an immunodeficientmouse model (Siprashvili et al. 2010). Aphase-1 clinical trial of ex vivo gene trans-fer in human subjects with RDEB using thisretrovirus has been approved by the Food andDrug Administration (FDA) and is currently inprogress.

Similarly, another group attempting ex vivocorrection of DEB also created transplantable

EB simplexKeratin 5/14

Junctional EB

Dystrophic EBCollagen VII

Collagen XVII

Laminin-332/311

Cell membrane

a6b4Integrin

BP230

Plectin

Hemidesmosome

Laminadensa

Interstitialcollagen fibrils

Figure 2. Proteins affected in epidermolysis bullosa. (From Marinkovich 2012; modified, with permission, fromMcGraw-Hill.)



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autologous skin equivalents using a retroviralvector to transduce C7 into primary humanKCs, which they grafted onto immunocompro-mised mice. These grafts showed characteristicsof normal human epidermis, including AF for-mation (Gache et al. 2004). Correction of spon-taneous homozygous mutations was thenshown in two canines with DEB. Neither dogexperienced adverse immunological events. Al-though one dog showed continued expressionof C7, the other did not have as effective a trans-duction, which resulted in blistering and graftloss after 5 mo (Gache et al. 2011).

An alternative approach to grafting is genetransfer via injection. Woodley et al. injected alentiviral vector expressing C7 into DEB skingrafted onto immunocompromised mice, pro-ducing C7 expression and AF formation. Eachinjection provided C7 at the basement mem-brane for 3 mo (Woodley et al. 2004b). Alsousing a lentiviral vector, they showed that cor-rected fibroblasts can be injected intradermallyto restore C7 expression at the basement mem-brane (Woodley et al. 2003).

Several groups have attempted to bypasspathogenic mutations to reverse the DEB phe-notype. Using antisense oligoribonucleotide(AON) therapy, Goto et al. induced “skipping”of a specific exon whose mutation creates a pre-mature termination codon. Skipping this exonresulted in the restoration of the open readingframe. After synthesizing the AON, it was inject-ed into DEB keratinocytes with exon 70 muta-tions. A small number of these keratinocytesproduced C7 and formed anchoring fibrils. In-jection into rats grafted with DEB KCs and fi-broblasts induced low amounts of C7 expres-sion. Unfortunately, AON is degraded easily incells, so therapeutic effects would be short lived(Goto et al. 2006b).

Another approach for RDEB keratinocytecorrection is to use trans-splicing to reduce thesize of the exogenous COL7A1 transgene. Trans-splicing uses the cell’s spliceosome to recombineendogenous target pre-mRNA and an exoge-nous RNA molecule, which replaces part of thetarget pre-mRNA. However, this approach ismutation specific. Murauer et al. (2011) per-formed retroviral transduction of RDEB kerati-

nocytes with a 30 pre-trans-splicing molecule,which resulted in expression of C7 at the base-ment membrane as well as the formation ofstructures similar to AFs in vitro.

Although it is thought that keratinocytesare responsible for the majority of C7 produc-tion (Regauer et al. 1990), there is debate asto whether keratinocytes or fibroblasts are thebest target for DEB gene therapy. After trans-ducing both RDEB keratinocytes and fibroblasts(Chen et al. 2002), Woodley et al. showed thatlentiviral-corrected RDEB fibroblasts couldbe used to create a skin equivalent to normalC7 expression (Woodley et al. 2004a). In anoth-er study, retroviral gene transfer was performed,creating skin grafts from DEB fibroblasts andkeratinocytes. Transduced fibroblasts had moreC7 in the dermal–epidermal junction thantransduced keratinocytes (Goto et al. 2006a).

Recently, Osborn et al. (2013) have had suc-cess using a novel genome editing tool, TALENs,to correct a specific mutation in RDEB fibro-blasts. These particular corrected fibroblastswere then reprogrammed into inducible pluri-potent stem cells that showed normal C7 expres-sion and deposition in a teratoma mouse mod-el. In addition to on-target correction of themutations, three off-target editing events weredocumented highlighting the necessity of estab-lishing a safety profile of such an in situ ap-proach.

Junctional Epidermolysis Bullosa (JEB)

In 2006, Mavilio et al. (2006) published a reportof successful ex vivo correction of LAMB3 geneusing autologous skin grafts for a subject withnonlethal JEB using MLV retrovirus. Nineepidermal sheets were grafted onto wounded ar-eas of the patient’s lower extremities. Normallevels of laminin-332 (previously known as lam-inin 5) were produced in the grafted areas for upto 1 year. Bands specific to the vector size wereseen in each of the follow-up biopsies, indicatingthe proliferation of transduced keratinocytes.No blisters, infection, immune response, or in-flammation were observed (Mavilio et al. 2006).

Using a model of lethal Herlitz JEB micewith homozygous LAMB3 mutations, Endo

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et al. (2012) attempted to perform in uterogene transfer. A lentiviral vector encoding forLAMB3 was injected into the amniotic space.Laminin-332 was incorporated into the base-ment membrane of the skin and the oral muco-sa. Although none of the mice survived morethan 48 h, harvested transduced skin showedcorrect expression of laminin-332 for the 6 moduration of the experiment (Endo et al. 2012).

Similar to the approach used by Murauer etal. (2011) for RDEB, Dallinger et al. (2003) useda system to splice out specific mutant exons(spliceosome-mediated RNA trans-splicing,SMarT) to show correction of a particular col-lagen 17 mutation in nonlethal JEB keratino-cytes in vitro.

Epidermolysis Bullosa Simplex (EBS)

An AAV gene-targeting vector with promotertrap design targeting was used to correct theKRT14 gene in EBS KCs. Fully functional epi-dermis was seen for 20 wk postgrafting ontoSCID mice (Petek et al. 2010).

As most patients with EBS have heterozy-gous dominant negative mutations, the defec-tive protein must first be removed before thecorrect protein can be restored. As such, shortinhibitory RNAs (siRNAs) and SMarTare beinginvestigated as methods for disease correctionfor EBS (Wally et al. 2010; Bowden 2011; Cham-cheu et al. 2012).

Pachyonychia Congenita

Pachyonychia congenita is another dominantnegative disease stemming from a keratin muta-tion resulting in painful plantar keratoderma. In2010, a double-blindphase-1bstudyusing siRNAwas performedononesubject for17 wk.Onefootwas treated with siRNA injection, the other with avehicle injection. Regression of the callus anddecreased tenderness were seen on the siRNAtreated foot, but not the vehicle foot. The chang-es seen in the treated foot began to revert to theirbaseline characteristics after the last dose. Un-fortunately, the subject experienced intense painat the injection site. No other adverse eventswere reported (Leachman et al. 2010).

Melanoma

There are currently multiple clinical trials ofgene therapy for melanoma. One study treatedmelanoma patients with autologous geneticallymodified lymphocytes expressing the cancergerm line gene MAGE-A3. Three out of 10 pa-tients showed an increase in circulating anti-MAGE-A3 T cells, indicating a possible clinicalbenefit. No toxicity or adverse side effects wereobserved (Fontana et al. 2009).

A phase-I/-II study of an interleukin-2 (IL-2) intralesional injection mediated by adenovi-rus shows promise as a treatment for metastaticmelanoma and other advanced solid tumors.IL-2 stimulates T-cell proliferation and inducesmetastatic tumor regression. Two subjects (n ¼17) showed complete response, and six showedlocal responses. The most common side effectswere flu-like symptoms and reactions at the in-jection site (Dummer et al. 2008).

There have also been several clinical trialsusing genetically engineered autologous T cellsthat express T-cell receptors against specific tu-mor antigens after retroviral transduction. In astudy using the NY-ESO-1 antigen to treat me-tastatic melanoma as well as metastatic synovialcell sarcoma, clinical responses were seen in 5/11 melanoma patients and 4/6 synovial sar-coma patients. Notably, two melanoma patientshad complete regression for at least 1 year (Rob-bins et al. 2011). Other metastatic melanomatrials used T-cell receptors to a different antigen,MART-1, and showed tumor regression in 2/13patients (Morgan et al. 2006) and 6/20 patients(Johnson et al. 2009). Engineered T-cell recep-tors to gp100 antigen showed a clinical responsein 3/16 patients (Johnson et al. 2009).

Ichthyosis

Lamellar Ichthyosis (LI)

Patients with lamellar ichthyosis (LI) have a de-fective barrier and abnormal differentiation ofthe epidermis because of a transglutaminase 1deficiency. In an effort to correct the diseasephenotype, LI patient keratinocytes were trans-duced with a retroviral vector engineered to ex-press transglutaminase 1. Corrected keratino-



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cytes were grafted on to immunodeficient mice,displaying normal phenotypes (Choate et al.1996; Khavari et al. 2002).

Harlequin Ichthyosis (HI)

The gene ABCA12 is important for lipid secre-tion from lamellar granules; mutations in thisgene result in harlequin ichthyosis (HI), whichis often lethal. Corrective gene transfer wasperformed on keratinocytes from HI patientsusing a cytomegalovirus-based vector in vitro,which restored lamellar granule lipid secretion(Akiyama et al. 2005).

Sjogren-Larsson Syndrome (SLS)

Sjogren-Larsson syndrome (SLS) is a disordercaused by a mutation in the gene ALDH3A2,which codes for fatty aldehyde dehydrogenase(FALDH). This enzyme catalyzes the oxidationof fatty alcohols into fatty acids. Mutationsin ALDH3A2 result in ichthyosis as well as men-tal retardation and spasticity (which can leadto quadraplegia). Using a recombinant AAV2vector, FALDH was transduced into SLS kera-tinocytes. Corrected keratinocytes appearedphenotypically normal with normal FALDH ex-pression (Haug and Braun Falco 2005).

Xeroderma Pigmentosum (XP)

Xeroderma pigmentosum (XP) results from adefective DNA repair mechanism involving nu-cleotide excision repair (NER). Cells without afunctioning NER develop increased UV-in-duced damage, increasing mutagenesis and skincancer development. Current therapy is limitedto surgical tumor resection and recommenda-tions to avoid sunlight (Cleaver et al. 2009).

Researchers used a MLV-derived retrovirusto correct the NER mechanism in keratinocytesfor one XP subtype. When exposed to UV irra-diation, the corrected keratinocytes continuedto correctly repair DNA with UV exposed cellsurvival comparable to wild-type keratinocytes.Corrected keratinocyte holoclones also ap-peared to convey long-term repair (140 popula-tion doublings in this study) while retaining

their DNA-repairing abilities (Warrick et al.2012). Previous attempts at gene therapy forXP were aimed at adenovirus-mediated fibro-blast transduction (Marchetto et al. 2004).

Wound Healing

Wound healing is a complex mechanism char-acterized by the sequence of inflammation (me-diated in part by epidermal growth factor[EGF], platelet derived growth factor [PDGF],and transforming growth factor b [TGF-b])followed by cell proliferation (assisted by PDGFand TGF-b) and angiogenesis (controlled byvascular endothelial growth factor [VEGF]and fibroblast growth factor [FGF]) and thenre-epithelialization (via EGF, TGF-b, PDGF,and FGF) (Barrientos et al. 2008). The mecha-nism of impaired wound healing is often mul-tifactorial: decreased levels of growth factors orgrowth factor receptors, defective function ofdermal fibroblasts, or damaged nitric oxidesynthetase (Khavari et al. 2002).

Gene therapy for wound healing is designedto boost factors that are known to assist withthe wound-healing process. Topically appliedgrowth factors are easily degraded and thereforehave transient effects. It is also difficult forgrowth factors to properly penetrate the woundbed and they may be unable to reach their tar-get cells (Galeano et al. 2003; Margolis et al.2009).

Diabetic mice who received an AAVexpress-ing VEGF-A had increased VEGF-A expressionand subsequently improved wound healing, viaincreased angiogenesis, re-epithelialization, aswell as synthesis and maturation of the extracel-lular matrix, when compared with mice whoreceived an AAV-LacZ control (Galeano et al.2003). Diabetic mice receiving AAV-VEGF alsohad increased collagen deposits and a fastertime to healing (Brem et al. 2009). Using a non-viral method, VEGF was encoded in minicircleplasmid DNA in combination with a cationicdendrimer then injected subcutaneously intomurine diabetic wounds, resulting in high levelsof VEGF expression and complete wound heal-ing within 6 days (Kwon et al. 2012). Using abiodegradable cationic polymer, Sonic Hedge-

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hog gene (which activates the angiogenic signal-ing pathway, including VEGF) was delivered in-tradermally in murine full-thickness wounds.Expression of VEGF and the chemokine stromalcell derived factor 1a were significantly in-creased, resulting in accelerated wound closure(Park et al. 2012). Administration of minicircle-VEGF165 followed by insonication at 1 MHzshowed reasonable wound closure and restoreddiabetic wound microarchitectures to their nor-mal state (Yoon et al. 2009).

Ko et al. (2011) compared EGF and VEGFgene therapy techniques in a diabetic mousemodel using a minigene to deliver VEGF anda plasmid to deliver EGF. Both groups had ac-celerated wound healing when compared withcontrol diabetic mice. Better blood flow wasseen in the VEGF group; however, wound-heal-ing rates were increased in the EGF group, al-though the results were not statistically signifi-cant.

A phase-1 clinical study of periwound in-jection of an adenovirus encoding PDGFBshowed a decrease in the size of chronic venousleg ulcers within 1 month in 14/15 subjects(mean change ¼ 45.2% decrease).

No adverse events were considered related tothe study treatment (Margolis et al. 2009).

Studies have focused on other growth factorsas well. Keratinocytes treated with a plasmid en-coding for EGF showed increased wound heal-ing when compared with nontreated keratino-cytes in a porcine model (Vranckx et al. 2007).When diabetic mice were intradermally injectedwith a plasmid encoding TGF-b1, they experi-enced an increased rate of cell proliferation, amore organized extracellular matrix, and fasterwound closure compared with control groups(Chesnoy et al. 2003). Nonviral gene transferof insulin-like growth factor-1 (IGF-1), a growthfactor that is reduced in diabetic ulcers, intothe wounds of diabetic pigs resulted in signifi-cantly improved wound healing (Hirsch et al.2008).

Netherton Syndrome

Netherton syndrome is a genetic skin disorder inwhich mutations of the SPINK5 gene result in

loss of a serine protease inhibitor LEKTI. Thisresults in premature corneodesmosome degra-dation and defective keratinization. Di et al. cre-atedaSINlentiviralvectorencoding for SPINK5.Transduced keratinocytes showed correction ofLEKTI expression in vitro as well as in a murine/human skin graft model (Di et al. 2011). Anothergroup used an AAV2-mediated viral vector torestore LEKTI in NS keratinocytes. Expressionof SPINK5 after transduction was increased toapproximately 75% of wild-type KCs (Roedlet al. 2011).

CONCLUDING REMARKS

Future optimization of both vector design andadministration strategies should help to makegene therapy available clinically. Challenges in-clude increasing the effectiveness and durabilityof cutaneous nonviral vectors and improvementof viral vehicle safety, mainly associated withgenomic integration and immune response. Asit can be easily monitored for adverse effects, theskin represents a promising organ to explorethese possibilities.

The process of commencing a human genetherapy trial is extremely detailed and specific.As with any clinical trial in the United States, allactivities must be approved by the FDA as wellas an Institutional Review Board (Gorell et al.2011). In addition to FDA review, human genetransfer research proposals must also be review-ed and approved by the Recombinant DNA Ad-visory Committee (O’Reilly et al. 2012). Bio-safety safeguards must also be followed, whichare usually supervised by an Institutional Bio-safety Committee in an academic environment.Despite these difficulties, new gene transfer tri-als are constantly being developed and currentlythere is a gene therapy drug approved by theEuropean Medicines Agency (the European reg-ulatory agency) for treating lipoprotein lipasedeficiency (Moran 2012).

ACKNOWLEDGMENTS

The authors thank the National Institute of Ar-thritis and Musculoskeletal and Skin Diseasesand the National Institute of Health (Grant



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No. R01 AR055914), the California Institute forRegenerative Medicine (Grant No. DR1-01454),and the Epidermolysis Bullosa Medical Re-search Foundation for funding. The authorsalso thank Mark Yamaguma for assistancewith image formatting.

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2014; doi: 10.1101/cshperspect.a015149Cold Spring Harb Perspect Med Emily Gorell, Ngon Nguyen, Alfred Lane and Zurab Siprashvili Gene Therapy for Skin Diseases

Subject Collection The Skin and Its Diseases

InsightsMelanoma: Clinical Features and Genomic

Elena B. Hawryluk and Hensin TsaoDevelopment in the MouseModeling Cutaneous Squamous Carcinoma

Phillips Y. Huang and Allan BalmainWound Healing and Skin Regeneration

Makoto Takeo, Wendy Lee and Mayumi ItoNatural and Sun-Induced Aging of Human Skin

Laure Rittié and Gary J. Fisher

Development and Regeneration of the Hair FollicleEpithelial Stem and Progenitor Cells in The Dermal Papilla: An Instructive Niche for

Bruce A. Morgan

Advanced Treatment for Basal Cell Carcinomas

OroScott X. Atwood, Ramon J. Whitson and Anthony E.

Immunology and Skin in Health and DiseaseJillian M. Richmond and John E. Harris

Epidermal Polarity Genes in Health and Disease

M. NiessenFrederik Tellkamp, Susanne Vorhagen and Carien

and Adhesion in Epidermal Health and DiseaseDesmosomes: Regulators of Cellular Signaling

GreenJodi L. Johnson, Nicole A. Najor and Kathleen J.

Potentials, Advances, and LimitationsInduced Pluripotent Stem Cells in Dermatology:

Ganna Bilousova and Dennis R. Roop

in Adult Mammalian SkinMarkers of Epidermal Stem Cell Subpopulations

Kai Kretzschmar and Fiona M. Watt

The Genetics of Human Skin DiseaseGina M. DeStefano and Angela M. Christiano

Psoriasis

NestlePaola Di Meglio, Federica Villanova and Frank O. Disease

p53/p63/p73 in the Epidermis in Health and

Vladimir A. Botchkarev and Elsa R. FloresCell Therapy in Dermatology

McGrathGabriela Petrof, Alya Abdul-Wahab and John A. Receptors in Skin

Diversification and Specialization of Touch

David M. Owens and Ellen A. Lumpkin

http://perspectivesinmedicine.cshlp.org/cgi/collection/ For additional articles in this collection, see

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Gene Therapy for Skin Diseases - CSHL Pperspectivesinmedicine.cshlp.org/content/4/4/a015149.full.pdf · Either delivery system can be applied forex vivo or in vivo correction routes