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Cutaneous gene delivery
Journal of Dermatological Science (2008) 50, 87—98
www.intl.elsevierhealth.com/journals/jods
INVITED REVIEW ARTICLE
Cutaneous gene delivery Yasushi Kikuchi, Katsuto Tamai *, Yasufumi Kaneda Division of Gene Therapy Science, Graduate School of Medicine, Osaka University, 2-2 Yamada-oka, Suita, Osaka 565-0871, Japan Received 6 June 2007; received in revised form 10 July 2007; accepted 20 July 2007
KEYWORDS Gene therapy; Barrier function; Genodermatosis
Summary Over the past decade, many approaches to transferring genes into the skin have been investigated. However, most such approaches have been specifically aimed against genodermatosis, and have not produced sufficient results. The goal of such research is to develop a method in which genes are transferred easily, efficiently and stably into keratinocytes, especially into keratinocyte stem cells, and in which the transgene expression persists without a reaction from the host immune response. Although accidental development of cancer has occurred in trials of gene therapy for X-linked severe combined immunodeficiency (X-SCID), resulting in slowing of the progress of this research, the lessons of these setbacks have been applied to further research. Moreover, combined with the techniques acquired from tissue engineering, recent developments in our knowledge about stem cells will lead to new treatments for genodermatoses. The present review summarizes the methods by which therapeutic genes can be transferred into keratinocytes, with discussion of how gene transfer efficiency can be improved, with particular emphasis on disruption of the skin barrier function. It concludes with discussion of the challenges and prospects of keratinocyte gene therapy, in terms of achieving efficient and long-lasting therapeutic effects. # 2007 Published by Elsevier Ireland Ltd on behalf of Japanese Society for Investigative Dermatology.
Contents 1. 2. 3.
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Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Structural barrier against gene delivery into the skin . . . . . . . Approaches to gene transfer into the skin . . . . . . . . . . . . . . 3.1. Ex vivo transfer of genes into the skin . . . . . . . . . . . . 3.2. In vivo transfer of genes into the skin . . . . . . . . . . . . . Methods for overcoming the skin barrier against gene delivery . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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* Corresponding author. Tel.: +81 6 6879 3901; fax: +81 6 6879 3909. E-mail address: [email protected] (K. Tamai). 0923-1811/$30.00 # 2007 Published by Elsevier Ireland Ltd on behalf of Japanese Society for Investigative Dermatology. doi:10.1016/j.jdermsci.2007.07.006
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1. Introduction Efficient cutaneous gene delivery in vivo requires precise knowledge of the mechanisms of the barrier function of the skin. The skin is the most superficial part of the body, and is an attractive target for therapeutic gene transfer due to its easy accessibility, ease of observation and easy removal of genetically modified areas if necessary. However, the skin functions as a barrier against invasion by chemicals and pathogens, and it can be difficult for gene transfer methods to overcome this barrier. Many approaches have been tried to improve the efficiency of skin gene transfer, and some of them have been applied to the treatment of diseases by using the skin as a bioreactor or vaccination site. However, such approaches are not effective for clinical treatment of inherited skin diseases. In the present review, we summarize the current state of methods for delivering genes into the skin.
2. Structural barrier against gene delivery into the skin Human skin is composed of four different layers: basal, squamous, granular and horny layers. Keratinocytes are derived from epithelial stem cells that have the ability to proliferate and differentiate into all of these cell types. Keratinocyte stem cells are thought to be located in the basal layer of the epidermis and in the hair follicle bulge. When a defect appears in the skin, keratinocyte stem cells give rise to daughter keratinocytes that migrate to the skin defect [1]. To efficiently transfer genes into keratinocytes, we must overcome the tissue barrier and the cellular barrier. The tissue barrier physically prevents invasion by foreign bodies from the environment, and the cellular barrier impedes intracellular trafficking. Due to these barriers, only molecules smaller than 500 Da can pass through the skin [2]. The tissue barrier function of the skin is mainly due to three structures of the skin. The first is the horny layer, which is the outermost layer of the skin. It contains dead cells that have differentiated from granular keratinocytes, interspersed within a lipidrich matrix, and functions as a cutaneous barrier preventing water loss and invasion by chemicals and pathogens. The horny layer is selectively permeable, and allows only relatively lipophilic compounds to diffuse into the lower layers. The second structure is tight junctions. To reach the underlying epidermis or dermis, molecules passing through the paracellular space must pass
Y. Kikuchi et al. specialized junctions. Tight junctions create a primary barrier to diffusion of solutes via the paracellular route. Recently, claudin was identified as a component of tight junctions [3]. In one study, claudin-1-deficient mice died within 1 day of birth, and exhibited severe defects in the epidermal permeability barrier [4]. The third structure is the desmosome, which mediates cell—cell adhesion and these are located in the intercellular space between adjacent keratinocytes. In desmosomes, intermediate filaments are connected to the transmembrane proteins desmoglein and desmocollin, which belong to the cadherin family. The importance of desmosome junctions is demonstrated in the skin disease pemphigus, in which severe bullous lesions caused by production of an anti-desmoglein autoantibodies lead to leakage of body fluids. Desmosomal cadherins not only mediate cell—cell adhesion but also affect epidermal barrier function. For example, transgenic mice that expressed desmoglein 3 throughout the entire epidermis died within the first 10 days of life; presumably, the structure of their horny layer was altered, leading to changes in the permeability barrier [5].
3. Approaches to gene transfer into the skin The aim of cutaneous gene therapy is to improve skin phenotype via the effects of proteins derived from genes delivered into skin cells. There are two general methods of gene transfer, transient and stable gene transfer, which are selected depending on the basis of the target disease. Transient transgene expression is used for tissue repair, vaccination and anticancer treatment. Stable transgene expression, which involves integration of the transgene into the cell genome, is used for life-long correction of inherited or acquired conditions. In diseases involving recessive loss-of-function mutations such as those involved in many genodermatoses, simple re-introduction of the wild-type gene via viral or non-viral insertion may be sufficient for correction of the phenotype. The two basic approaches to delivery of therapeutic genes into the skin are ex vivo and in vivo gene delivery.
3.1. Ex vivo transfer of genes into the skin In ex vivo gene transfer, skin obtained from the patient is expanded in vitro. A gene is introduced into the expanded skin cells, which are then regrafted onto the patient. An advantage of this approach is the ease with which keratinocytes can
Summary of cutaneous gene transfer protocols for genetic skin disorders
Disease
Gene
Protein
Target cell
Transfer principle
Method
Reference
Year
Junctional epidermolysis bullosa (JEB)
LAMB3
Laminin subunit beta-3
KC
Ex vivo
Retrovirus
[53]
1998
LAMB3 COL17A1 LAMB3 ITGB4 LAMB3
Laminin subunit beta-3 Alpha 1 type XVII collagen Laminin subunit beta-3 Integrin beta-4 Laminin subunit beta-3
KC KC KC KC KC
Ex Ex Ex Ex Ex
[54] [55] [56] [57] [20]
1998 1999 2001 2001 2003
LAMB3 LAMA3 LAMB3
Laminin subunit beta-3 Laminin subunit alpha-3 Laminin subunit beta-3
KC KC KC
[58] [59] [60]
2003 2006 2006
*
LAMB3
Laminin subunit beta-3
KC
Ex vivo Ex vivo In vivo (injection into amniotic cavity) Ex vivo
Retrovirus Retrovirus Retrovirus Retrovirus Sleeping beauty transposase phiC31 integrase Retrovirus Adenovirus + AAV
Retrovirus
[9]
2006
COL7A1 (mini gene)
Alpha 1 type VII collagen
KC
Ex vivo
Retrovirus
[61]
2000
COL7A1 COL7A1 COL7A1 COL7A1 COL7A1 COL7A1
Alpha Alpha Alpha Alpha Alpha Alpha
KC KC/fibroblast Fibroblast KC Fibroblast Fibroblast/ endothelial cells
phiC31 integrase Lentivirus phiC31 integrase Retrovirus Lentivirus Lentivirus
[19] [62] [63] [64] [65] [31]
2002 2002 2003 2003 2003 2004
COL7A1 COL7A1 COL7A1
Alpha 1 type VII collagen Alpha 1 type VII collagen Alpha 1 type VII collagen
KC KC/fibroblast Skin
Ex vivo Ex vivo Ex vivo Ex vivo Ex vivo In vivo (intradermal injection) Ex vivo Ex vivo In vivo (intradermal injection)
Retrovirus Retrovirus Antisense oligoribonucleotide
[66] [67] [68]
2004 2006 2006
Lammelar ichthyosis (LI) Sjogren—Larsson syndrome
TGM1 ALDH3A2
Transglutaminase 1 Fatty aldehyde dehydrogenase
KC KC
Ex vivo Ex vivo
Retrovirus AAV
[69] [70]
1996 2006
X-linked ichthyosis (XLI)
STS STS
Steroid sulfatase Steroid sulfatase
KC KC
Ex vivo Ex vivo
EB virus Retrovirus
[71] [72]
1993 1997
Xeroderma pigmentosus (XP)
XPD
Xeroderma pigmentosum, complementation group D
Fibroblast
Ex vivo
Retrovirus
[73]
1995
Recessive dystrophic epidermolysis bullosa (RDEB)
1 1 1 1 1 1
type type type type type type
VII VII VII VII VII VII
collagen collagen collagen collagen collagen collagen
vivo vivo vivo vivo vivo
Cutaneous gene delivery
Table 1
89
2006 [75] Lentivirus
2004 [32] Adenovirus
Fibroblast
In vivo (subcutaneous injection) Ex vivo XPA
XPA, C, D KC: keratinocyte. * Clinical trial.
KC/fibroblast
[74] Retrovirus Ex vivo
Xeroderma pigmentosum, complementation groups A, B, C Xeroderma pigmentosum, complementation group A Xeroderma pigmentosum, complementation groups A, C, D XPA ,B, C
Fibroblast
Reference Method Transfer principle Target cell Protein Gene Disease
Table 1 (Continued )
1997
Y. Kikuchi et al. Year
90
be genetically manipulated in vitro and a large amount of skin can be obtained from a tiny piece of biopsied skin using well-established techniques. Also, this approach reduces the risk of uncontrolled systemic spread of the introduced viral or non-viral vectors. For efficient transfer of genes into keratinocytes in vitro, viral vectors are mainly used. Several types of viral vector have been developed, including retrovirus, adenovirus, adeno-associated virus (AAV) and lentivirus. Moloney murine-leukemia virus (MMLV)-derived retroviral vectors have been used to transfer genes into keratinocytes for long-term transgene expression. These vectors randomly integrate into genomic DNA, especially in dividing cells, so they are particularly suitable for gene transfer into rapidly proliferating and differentiating epidermal cells. Retroviral vectors have been used for in vitro keratinocyte delivery of the wild-type allele of the defective underlying gene involved in a particular genodermatosis, resulting in expression of the wildtype recombinant protein and phenotypic reversion of the diseased cells (Table 1). In a 1993 study by Gerrard et al. [6], a skin graft was transduced in vitro with the gene for factor IX using a retroviral vector. After that skin was grafted onto the subject, it secreted factor IX into the bloodstream. Thus, genetically engineered keratinocytes have been successfully used as bioreactors for the production of locally and systemically active therapeutic polypeptides, and thus could theoretically be used for the treatment of hormonal and metabolic disorders affecting other organs (Table 2). However, in studies of gene delivery using retroviral vectors (which are the most extensively studied of all the viral vectors), researchers have observed inactivation of gene expression by the host immune response [7]. Also, in certain patients with X-linked severe combined immunodeficiency (X-SCID) who were treated with a retroviral vector, leukemia developed as a result of integration of the therapeutic gene upstream of an oncogene [8]. Thus, there are serious potential limitations to the use of retroviral vectors. However, a recent report by Mavilio et al. [9] demonstrates the great potential benefits of gene therapy using retroviral vectors. They obtained epidermal stem cells from an adult patient with laminin beta-3 chain (laminin 332, formerly laminin 5)-deficient junctional epidermolysis bullosa, and then transduced those stem cells with laminin 332 beta-3 chain cDNA via a retroviral vector. Those genetically corrected stem cells were then cultured into epidermal grafts that produced a functional correction in vivo with no evidence of clonal expansion or selection of specific integration events.
Cutaneous gene delivery
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Table 2 Summary of cutaneous gene transfer protocols for systemic delivery Protein
Target cell
Transfer principle
Method
Reference
Year
Factor IX Erythropoietin Adenosine deaminase Factor IX Ornithine-delta-aminotransferase Ornithine-delta-aminotransferase IL-10
KC KC KC KC KC KC Skin
Retrovirus AAV Retrovirus Retrovirus Retrovirus Adenovirus Naked DNA
[6] [76] [77] [78] [79] [80] [29]
1993 1996 1997 1997 1997 1997 1998
Factor IX Phenylalanine hydroxylase + GTP cyclohydrolase 1 Leptin Erythropoietin
KC KC
Ex vivo Ex vivo Ex vivo Ex vivo Ex vivo Ex vivo In vivo (intradermal injection) Ex vivo Ex vivo
Retrovirus Retrovirus
[81] [82]
1998 2000
Retrovirus Lentivirus
[83] [84]
2001 2001
Retrovirus
[85]
2002
Naked DNA
[86]
2002
Retrovirus AAV Retrovirus Retrovirus
[87] [88] [89] [90]
2003 2004 2006 2007
KC Skin Fibroblast
Phenylalanine hydroxylase + GTP cyclohydrolase 1 IL-4, 6, 10, TGF-beta
Skin
Growth hormone GM-CSF Growth hormone Proinsulin
KC KC KC KC
Ex vivo In vivo (intradermal injection) Ex vivo In vivo (intradermal injection) Ex vivo Ex vivo Ex vivo Ex vivo
KC: keratinocyte.
Adenoviral vectors can easily be obtained at high titers, and can then be efficiently used to transiently transduce both quiescent and actively dividing cells [10], but they are of limited use for longterm gene correction. Adenoviral vectors can elicit a strong immune response in hosts, as in the case of an ornithine transcarbamylase-deficient patient who died of a systemic inflammatory response syndrome (SIRS) after right hepatic artery infusion of an adenoviral vector [11]. In addition, the efficiency with which adenoviral vectors are transfected into epithelial cells is relatively low, due to the low levels of Coxsackie-adenovirus receptor (CAR; the primary adenoviral cellular receptor) on fibroblasts [12] and keratinocytes [13]. Also, studies indicate that the negative charge of membrane glycoproteins reduces the efficiency of adenovirus-mediated gene transfer [14]. For these reasons, researchers have had little success using adenoviral vectors to transfer genes into keratinocytes. To enhance adenovirus-mediated gene delivery into keratinocytes, researchers have studied the use of polycationic molecules and genetically modified adenoviruses with CAR-independent tropism [14,15]. The advantages of AAV vectors are their ability to transduce both dividing and non-dividing cells, their ability to site-specifically integrate and produce long-term gene expression (even in vivo), and their broad tropism allowing efficient
transduction into diverse organs including the skin [16]. Also, AAV vectors have been found to replicate at high levels in a normal skin model [17]. These findings suggest that AAV vectors are skintropic, and are thus useful vectors for skin-directed gene therapy. Lentiviral vectors are suitable for introducing genes into both dividing and non-dividing cells, and for integrating genes into the genome [18]. This indicates that lentiviral vectors are useful for transferring genes into keratinocyte stem cells, which divide very slowly. It also indicates that lentiviral vectors can be used in place of retroviruses for transfection of stem cells, as retrovirus-mediated transfection of stem cells is inefficient. Non-viral vectors have been used in ex vivo gene therapy for several skin diseases. In a study by OrtizUrda et al. [19], integrase phiC31 derived from a Streptomyces phage stably integrated COL7A1 cDNA into the genome of primary epidermal progenitor cells from four unrelated patients with recessive dystrophic epidermolysis bullosa (RDEB). Sleeping Beauty, a member of the TC1/mariner superfamily transposons mobile in vertebrate genome, has also been used in non-viral ex vivo gene therapy [20]. These non-viral agents have failed to successfully integrate into desired sites where they would not disrupt the native function of unrelated genes. However, combined with skin culture technology and stem cell biology, these agents show promise
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Y. Kikuchi et al.
in contributing to new methods of gene therapy and may improve the long-term expression of genes used to treat various diseases.
3.2. In vivo transfer of genes into the skin Many in vivo gene transfer systems have been developed, using both viral and non-viral vectors. These systems, which deliver genes directly into the skin, include topical DNA application, direct DNA injection, electroporation, iontophoresis, sonophoresis and gene guns. Topical application of plasmid DNA is an attractive approach for gene delivery. If successfully developed, it would be useful for cutaneous gene therapy, because it is painless and can easily be applied to large body surface areas. Li and Hoffman [21] reported successful selective introduction of the LacZ reporter gene into hair follicles of mice via topical application of liposomes containing the gene. Hemagglutinating virus of Japan (HVJ)-liposomes have been constructed by fusing DNA-loaded liposomes with UV-inactivated HVJ [22]. Administration of LacZ plasmid DNA with HVJ-liposome into the amniotic fluid of fetal rats has resulted in transformation of entire areas of fetal skin [23]. In a study using an adenoviral vector containing the human carcinoembryonic antigen (CEA) gene, topical application of the adenoviral vector evoked an immune response against the transgene product [24]. Topical application of naked plasmid DNA encoding the hepatitis B surface antigen has also been found to induce antigen-specific immune responses [25]. Recently, an NF-kB decoy, which is synthetic double-stranded DNA that corresponds to NF-kB ciselements and can bind to transcriptional factor as a ‘‘decoy’’ to alter gene transcription, has been developed and found to be a useful tool as a new class of anti-gene agents [26] (Fig. 1). Research
Fig. 1
indicates that application of ointment containing NF-kB decoy oligonucleotides onto atopic dermatitis lesions of NC/Nga mice is an effective means of gene therapy [27]. Tamai et al. used such an NF-kB decoy ointment to treat human patients with atopic dermatitis (unpublished data), and found that it very effectively improved lesions with impaired barrier function, especially facial eczematous, exudatative lesions with acute inflammation. Direct injection of naked DNA into the skin is an efficient way to transfer genes into keratinocytes in vivo [28]. Although it is unknown why intradermally injected plasmid DNA is taken up and expressed by keratinocytes, the resultant transgene products not only affected the local skin lesion, but were also released into the circulation and exerted effects on other target organs [29]. Although this method is relatively efficient, safe and simple, it does not result in long-term constitutive expression of genes, and it cannot be used to quickly introduce genes into a large area of the skin as possible by topical application. When COL7A1 cDNA spanning about 9 kb was injected into the skin of hairless rats, the cDNA was expressed in keratinocytes [30]. This result indicates that naked DNA injection might be useful for the clinical treatment of RDEB. In recent studies using animal models of RDEB and xeroderma pigmentosum (XP), intradermal injection of genetically modified viral vectors resulted in the synthesis of functional protein [31,32]. Electroporation is one of the most popular methods of introducing genes into cells in vitro. The earliest reported study of in vivo electroporation to deliver genes was conducted by Titomirov et al. [33]. They subcutaneously injected plasmid DNA encoding the neomycin resistance gene, followed by high-voltage pulses, and then found neo-resistant colonies in resultant primary skin cell cultures. Recently, the combination of electroporation and
The mechanism of the therapeutic effect of NF-kB decoy oligodeoxynucleotide (ODN).
Cutaneous gene delivery injection of a plasmid encoding keratinocyte growth factor-1 (KGF-1) has been successfully used to promote wound healing [34]. Drabick et al. [35] found that this method can increase transgene expression up to an average of 83-fold, relative to naked DNA injection. They found that transfected cells were principally located in the dermis, and also observed transfected cells in lymph nodes draining electropermeabilized sites, suggesting that directly transfected (epi-)dermal dendritic cells had migrated from the treated site. These findings suggest that this method is broadly applicable to nucleic acid vaccination. The combination of electroporation and topical application of plasmid DNA onto partially stripped skin has been successfully used to deliver DNA into keratinocytes [36]. Iontophoresis, which is the application of an electrical potential that maintains a constant
93 electric current across the skin, enhances the delivery of both ionized and non-ionized moieties. Although there have been no reports of the use of this method for cutaneous gene delivery, it has been found to be an effective way to deliver antisense oligonucleotides into keratinocytes. Sakamoto et al. [37] studied iontophoretic topical delivery of mouse interleukin-10 (IL-10) antisense oligonucleotides, and found that it had a therapeutic effect in atopic dermatitis skin lesions of NC/Nga mice. Ultrasound improves the efficiency of plasmid delivery in vitro by causing the formation of shortlived pores in the plasma membrane (a method know as sonoporation), leading to holes with diameters of up to 100 nm, via acoustic cavitation. Recently, ultrasound-assisted plasmid delivery has also been successfully achieved in vivo by
Fig. 2 Gene expression in the skin 48 h after intra-amniotic injection with microbubble-enhanced ultrasuound. (a) Macroscopic appearance of fetal mouse. (b and c) Fluorescence macroscopic image of GFP-gene transduced skin. (d) Confocal microscopic image of GFP-gene transduced skin. (e and f) X-Gal staining of LacZ gene transduced skin. Magnifications: 40 (e) and 100 (f).
94 enhancing the effects of ultrasound with microbubbles. In a study by Endoh et al. [38], intraamniotic injection of naked DNA with microbubbleenhanced ultrasound resulted in high levels of expression of the DNA in fetal mouse skin (Fig. 2). A gene gun is a needle-free device that can introduce DNA-coated gold particles into target cells or tissues, including the skin [39]. Due to the short-term and inefficient expression of the resulting gene products in vivo, gene guns have demonstrated only limited potential in gene therapy for genetic skin diseases. However, gene guns have been successfully used in immunomodulation, in which cytokine genes are introduced into tumorbearing animals and the resulting gene products secreted into the blood stream [40]. Furthermore, gene guns have been successfully used in DNA vaccination against infectious diseases [41]. Gene gun-mediated DNA vaccination has been shown to induce greater antibody and/or CD8+ T-cell responses, with substantially lower doses (100— 1000-fold) of DNA, than routine needle-based approaches [41].
4. Methods for overcoming the skin barrier against gene delivery To achieve more efficient cutaneous gene delivery, removal of the horny layer is thought to be the best way to disrupt the barrier of the skin. As described above, the most important component of the skin barrier function is the horny layer, which prevents penetration of foreign molecules greater than 500 Da [2]. Tape-stripping using adhesive tape removes the horny layer, but this is not suitable for all-over topical transfer of genes into skin. Without successful penetration of this effective barrier, exogenously applied genes cannot enter keratinocytes. Drug delivery via the skin is an important pharmacological goal, and the horny layer is thus an important structural target in the improvement of drug delivery systems. Several technological advances have been made in the past few decades in overcoming this barrier: electroporation, sonophoresis, iontophoresis and chemical penetration enhancers (CPEs). Electroporation with high voltage and short pulses creates transient aqueous pores in intercellular lipid bilayers, which may account for the increase in skin permeability [42]. Lombry et al. [43] demonstrated that transdermal and topical delivery of macromolecules of at least 40 kDa can be achieved by skin electroporation.
Y. Kikuchi et al. Iontophoresis has been shown to increase the permeability of skin to macromolecules. Kalia et al. [44] reported the successful iontophoretic delivery of functional peptides of 4 kDa (calcitonin and hPTH(1—34)) into keratinocytes. Several studies indicate that iontophoresis can cause skin irritation. However, combining iontophoresis with permeability-enhancing techniques can produce therapeutically effective delivery levels with lower levels of current, thus dramatically reducing the risk of skin discomfort and irritation [45]. Ultrasound is used to increase penetration by pharmacologically active drugs through the skin. This technique is known as sono- or phonophoresis. Recently, ultrasound at frequencies ranging from 20 kHz to 16 MHz has been used to permeabilize skin to allow transdermal drug administration. Sonophoresis is particularly effective at lower frequencies ( f < 100 kHz; low-frequency sonophoresis). The level of transdermal transport produced by low-frequency ultrasound is up to 1000-fold higher than that produced by therapeutic high-frequency ultrasound [46]. There have been several reports of the successful use of this method for delivery of macromolecules; e.g., mannitol, insulin, low-molecular-weight heparin (LMWH) and antisense oligonucleotides (reviewed in [46]). CPEs are chemical compounds that reduce the resistance of the skin barrier to drug diffusion. Many CPEs have been studied, including various surfactants, fatty acids and fatty esters. The most potent CPEs enhance skin permeability by altering the structure of the horny layer [47]. Using in vitro skin impedance guided high-throughput (INSIGHT) screening, Karande et al. [48] discovered mixtures of penetration enhancers that increase the permeability of skin to macromolecules (1—10 kDa) by up to 100-fold without inducing skin irritation. Microneedles, which create micrometer-scale openings, have been created to painlessly pierce the horny layer and thereby transport drugs into the body. Microneedles can transport macromolecules such as insulin, desmopressin, antisense oligonucleotides and plasmid DNA (reviewed in [49]). Also, vaccine delivery using microneedles can induce an immune response to hepatitis B surface antigen encoded by plasmid DNA [50].
5. Conclusion In the past few decades, many methods using viral or non-viral vectors have been developed for skin genes transfer. Although several of these methods appear promising for clinical application (e.g., vaccination and bioreactors), the gene delivery effi-
Cutaneous gene delivery ciency is generally still insufficient for effective treatment of patients with inherited skin diseases affecting the whole body. For treatment of acquired skin disorders, the use of oligonucleotide decoys for key transcriptional regulators such as NF-kB appears to be a potentially powerful tool because of the ease with which it penetrates the skin, compared to plasmid or viral DNA. Improvement of the efficiency of gene delivery requires the development of noninvasive methods that can rapidly overcome the barrier function of the horny cell layer over large areas of skin. In addition, attaining an efficient and continuous therapeutic effect after gene therapy requires (1) targeting of stem cells, (2) site-specific DNA incorporation and (3) suppression of the host immune response against the transgene product. The properties of keratinocyte stem cells have been well characterized, and isolation of keratinocyte stem cells should allow efficient gene transfer into them. The risks that gene therapy entails are clearly illustrated by the accidental development of cancer in X-SCID patients as a result of gene therapy. To reduce the risk of such accidents, we must develop site-specific DNA correction that only affects the disease-related target gene. Transient gene transduction methods have a lower risk of such problems, but they have a narrow range of applications for treatment of inherited skin diseases involving the entire body. Finally, in patients with null mutations in the target gene, gene therapy can result in immune responses to the transduced cells and clearance of them by CD8+ T cells and/or antibodies against the transgene product. Gross et al. [51] found that adoptive transfer of antigen-specific CD4+CD25+ regulatory T cells, which are implicated in peripheral self-tolerance, can down-regulate both B- and T-cell responses, resulting in tolerance of the transgene product. Other studies indicate that molecular bone marrow chimerism resulting from transduction of bone marrow cells with retroviral vectors has resulted in prolonged acceptance of skin grafts [52]. Thus, cutaneous gene therapy is an attractive approach, but is still insufficient in its effectiveness. However, recent findings suggest that major breakthroughs in this field are imminent.
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Cutaneous gene delivery
[59]
[60]
[61]
[62]
[63]
[64]
[65]
[66]
[67]
[68]
[69]
[70]
[71]
[72]
[73]
[74]
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Y. Kikuchi et al. Katsuto Tamai received his MD and PhD degrees from Hirosaki University, Aomori, Japan, in 1986 and 1990, respectively. He became an assistant professor in the Department of Dermatology, Hirosaki University in 1990. He worked under Dr. Jouni Uitto as a Research Fellow in the Department of Dermatology and Cutaneous Biology, Thomas Jefferson University, Philadelphia, USA, from 1991 to
1993. He was associate professor at Hirosaki University from 1999 to 2003. In 2003, he moved to Osaka University Graduate School of Medicine and is currently an associate professor in the Division of Gene Therapy Science (Prof. Y. Kaneda).
www.intl.elsevierhealth.com/journals/jods
INVITED REVIEW ARTICLE
Cutaneous gene delivery Yasushi Kikuchi, Katsuto Tamai *, Yasufumi Kaneda Division of Gene Therapy Science, Graduate School of Medicine, Osaka University, 2-2 Yamada-oka, Suita, Osaka 565-0871, Japan Received 6 June 2007; received in revised form 10 July 2007; accepted 20 July 2007
KEYWORDS Gene therapy; Barrier function; Genodermatosis
Summary Over the past decade, many approaches to transferring genes into the skin have been investigated. However, most such approaches have been specifically aimed against genodermatosis, and have not produced sufficient results. The goal of such research is to develop a method in which genes are transferred easily, efficiently and stably into keratinocytes, especially into keratinocyte stem cells, and in which the transgene expression persists without a reaction from the host immune response. Although accidental development of cancer has occurred in trials of gene therapy for X-linked severe combined immunodeficiency (X-SCID), resulting in slowing of the progress of this research, the lessons of these setbacks have been applied to further research. Moreover, combined with the techniques acquired from tissue engineering, recent developments in our knowledge about stem cells will lead to new treatments for genodermatoses. The present review summarizes the methods by which therapeutic genes can be transferred into keratinocytes, with discussion of how gene transfer efficiency can be improved, with particular emphasis on disruption of the skin barrier function. It concludes with discussion of the challenges and prospects of keratinocyte gene therapy, in terms of achieving efficient and long-lasting therapeutic effects. # 2007 Published by Elsevier Ireland Ltd on behalf of Japanese Society for Investigative Dermatology.
Contents 1. 2. 3.
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Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Structural barrier against gene delivery into the skin . . . . . . . Approaches to gene transfer into the skin . . . . . . . . . . . . . . 3.1. Ex vivo transfer of genes into the skin . . . . . . . . . . . . 3.2. In vivo transfer of genes into the skin . . . . . . . . . . . . . Methods for overcoming the skin barrier against gene delivery . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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* Corresponding author. Tel.: +81 6 6879 3901; fax: +81 6 6879 3909. E-mail address: [email protected] (K. Tamai). 0923-1811/$30.00 # 2007 Published by Elsevier Ireland Ltd on behalf of Japanese Society for Investigative Dermatology. doi:10.1016/j.jdermsci.2007.07.006
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1. Introduction Efficient cutaneous gene delivery in vivo requires precise knowledge of the mechanisms of the barrier function of the skin. The skin is the most superficial part of the body, and is an attractive target for therapeutic gene transfer due to its easy accessibility, ease of observation and easy removal of genetically modified areas if necessary. However, the skin functions as a barrier against invasion by chemicals and pathogens, and it can be difficult for gene transfer methods to overcome this barrier. Many approaches have been tried to improve the efficiency of skin gene transfer, and some of them have been applied to the treatment of diseases by using the skin as a bioreactor or vaccination site. However, such approaches are not effective for clinical treatment of inherited skin diseases. In the present review, we summarize the current state of methods for delivering genes into the skin.
2. Structural barrier against gene delivery into the skin Human skin is composed of four different layers: basal, squamous, granular and horny layers. Keratinocytes are derived from epithelial stem cells that have the ability to proliferate and differentiate into all of these cell types. Keratinocyte stem cells are thought to be located in the basal layer of the epidermis and in the hair follicle bulge. When a defect appears in the skin, keratinocyte stem cells give rise to daughter keratinocytes that migrate to the skin defect [1]. To efficiently transfer genes into keratinocytes, we must overcome the tissue barrier and the cellular barrier. The tissue barrier physically prevents invasion by foreign bodies from the environment, and the cellular barrier impedes intracellular trafficking. Due to these barriers, only molecules smaller than 500 Da can pass through the skin [2]. The tissue barrier function of the skin is mainly due to three structures of the skin. The first is the horny layer, which is the outermost layer of the skin. It contains dead cells that have differentiated from granular keratinocytes, interspersed within a lipidrich matrix, and functions as a cutaneous barrier preventing water loss and invasion by chemicals and pathogens. The horny layer is selectively permeable, and allows only relatively lipophilic compounds to diffuse into the lower layers. The second structure is tight junctions. To reach the underlying epidermis or dermis, molecules passing through the paracellular space must pass
Y. Kikuchi et al. specialized junctions. Tight junctions create a primary barrier to diffusion of solutes via the paracellular route. Recently, claudin was identified as a component of tight junctions [3]. In one study, claudin-1-deficient mice died within 1 day of birth, and exhibited severe defects in the epidermal permeability barrier [4]. The third structure is the desmosome, which mediates cell—cell adhesion and these are located in the intercellular space between adjacent keratinocytes. In desmosomes, intermediate filaments are connected to the transmembrane proteins desmoglein and desmocollin, which belong to the cadherin family. The importance of desmosome junctions is demonstrated in the skin disease pemphigus, in which severe bullous lesions caused by production of an anti-desmoglein autoantibodies lead to leakage of body fluids. Desmosomal cadherins not only mediate cell—cell adhesion but also affect epidermal barrier function. For example, transgenic mice that expressed desmoglein 3 throughout the entire epidermis died within the first 10 days of life; presumably, the structure of their horny layer was altered, leading to changes in the permeability barrier [5].
3. Approaches to gene transfer into the skin The aim of cutaneous gene therapy is to improve skin phenotype via the effects of proteins derived from genes delivered into skin cells. There are two general methods of gene transfer, transient and stable gene transfer, which are selected depending on the basis of the target disease. Transient transgene expression is used for tissue repair, vaccination and anticancer treatment. Stable transgene expression, which involves integration of the transgene into the cell genome, is used for life-long correction of inherited or acquired conditions. In diseases involving recessive loss-of-function mutations such as those involved in many genodermatoses, simple re-introduction of the wild-type gene via viral or non-viral insertion may be sufficient for correction of the phenotype. The two basic approaches to delivery of therapeutic genes into the skin are ex vivo and in vivo gene delivery.
3.1. Ex vivo transfer of genes into the skin In ex vivo gene transfer, skin obtained from the patient is expanded in vitro. A gene is introduced into the expanded skin cells, which are then regrafted onto the patient. An advantage of this approach is the ease with which keratinocytes can
Summary of cutaneous gene transfer protocols for genetic skin disorders
Disease
Gene
Protein
Target cell
Transfer principle
Method
Reference
Year
Junctional epidermolysis bullosa (JEB)
LAMB3
Laminin subunit beta-3
KC
Ex vivo
Retrovirus
[53]
1998
LAMB3 COL17A1 LAMB3 ITGB4 LAMB3
Laminin subunit beta-3 Alpha 1 type XVII collagen Laminin subunit beta-3 Integrin beta-4 Laminin subunit beta-3
KC KC KC KC KC
Ex Ex Ex Ex Ex
[54] [55] [56] [57] [20]
1998 1999 2001 2001 2003
LAMB3 LAMA3 LAMB3
Laminin subunit beta-3 Laminin subunit alpha-3 Laminin subunit beta-3
KC KC KC
[58] [59] [60]
2003 2006 2006
*
LAMB3
Laminin subunit beta-3
KC
Ex vivo Ex vivo In vivo (injection into amniotic cavity) Ex vivo
Retrovirus Retrovirus Retrovirus Retrovirus Sleeping beauty transposase phiC31 integrase Retrovirus Adenovirus + AAV
Retrovirus
[9]
2006
COL7A1 (mini gene)
Alpha 1 type VII collagen
KC
Ex vivo
Retrovirus
[61]
2000
COL7A1 COL7A1 COL7A1 COL7A1 COL7A1 COL7A1
Alpha Alpha Alpha Alpha Alpha Alpha
KC KC/fibroblast Fibroblast KC Fibroblast Fibroblast/ endothelial cells
phiC31 integrase Lentivirus phiC31 integrase Retrovirus Lentivirus Lentivirus
[19] [62] [63] [64] [65] [31]
2002 2002 2003 2003 2003 2004
COL7A1 COL7A1 COL7A1
Alpha 1 type VII collagen Alpha 1 type VII collagen Alpha 1 type VII collagen
KC KC/fibroblast Skin
Ex vivo Ex vivo Ex vivo Ex vivo Ex vivo In vivo (intradermal injection) Ex vivo Ex vivo In vivo (intradermal injection)
Retrovirus Retrovirus Antisense oligoribonucleotide
[66] [67] [68]
2004 2006 2006
Lammelar ichthyosis (LI) Sjogren—Larsson syndrome
TGM1 ALDH3A2
Transglutaminase 1 Fatty aldehyde dehydrogenase
KC KC
Ex vivo Ex vivo
Retrovirus AAV
[69] [70]
1996 2006
X-linked ichthyosis (XLI)
STS STS
Steroid sulfatase Steroid sulfatase
KC KC
Ex vivo Ex vivo
EB virus Retrovirus
[71] [72]
1993 1997
Xeroderma pigmentosus (XP)
XPD
Xeroderma pigmentosum, complementation group D
Fibroblast
Ex vivo
Retrovirus
[73]
1995
Recessive dystrophic epidermolysis bullosa (RDEB)
1 1 1 1 1 1
type type type type type type
VII VII VII VII VII VII
collagen collagen collagen collagen collagen collagen
vivo vivo vivo vivo vivo
Cutaneous gene delivery
Table 1
89
2006 [75] Lentivirus
2004 [32] Adenovirus
Fibroblast
In vivo (subcutaneous injection) Ex vivo XPA
XPA, C, D KC: keratinocyte. * Clinical trial.
KC/fibroblast
[74] Retrovirus Ex vivo
Xeroderma pigmentosum, complementation groups A, B, C Xeroderma pigmentosum, complementation group A Xeroderma pigmentosum, complementation groups A, C, D XPA ,B, C
Fibroblast
Reference Method Transfer principle Target cell Protein Gene Disease
Table 1 (Continued )
1997
Y. Kikuchi et al. Year
90
be genetically manipulated in vitro and a large amount of skin can be obtained from a tiny piece of biopsied skin using well-established techniques. Also, this approach reduces the risk of uncontrolled systemic spread of the introduced viral or non-viral vectors. For efficient transfer of genes into keratinocytes in vitro, viral vectors are mainly used. Several types of viral vector have been developed, including retrovirus, adenovirus, adeno-associated virus (AAV) and lentivirus. Moloney murine-leukemia virus (MMLV)-derived retroviral vectors have been used to transfer genes into keratinocytes for long-term transgene expression. These vectors randomly integrate into genomic DNA, especially in dividing cells, so they are particularly suitable for gene transfer into rapidly proliferating and differentiating epidermal cells. Retroviral vectors have been used for in vitro keratinocyte delivery of the wild-type allele of the defective underlying gene involved in a particular genodermatosis, resulting in expression of the wildtype recombinant protein and phenotypic reversion of the diseased cells (Table 1). In a 1993 study by Gerrard et al. [6], a skin graft was transduced in vitro with the gene for factor IX using a retroviral vector. After that skin was grafted onto the subject, it secreted factor IX into the bloodstream. Thus, genetically engineered keratinocytes have been successfully used as bioreactors for the production of locally and systemically active therapeutic polypeptides, and thus could theoretically be used for the treatment of hormonal and metabolic disorders affecting other organs (Table 2). However, in studies of gene delivery using retroviral vectors (which are the most extensively studied of all the viral vectors), researchers have observed inactivation of gene expression by the host immune response [7]. Also, in certain patients with X-linked severe combined immunodeficiency (X-SCID) who were treated with a retroviral vector, leukemia developed as a result of integration of the therapeutic gene upstream of an oncogene [8]. Thus, there are serious potential limitations to the use of retroviral vectors. However, a recent report by Mavilio et al. [9] demonstrates the great potential benefits of gene therapy using retroviral vectors. They obtained epidermal stem cells from an adult patient with laminin beta-3 chain (laminin 332, formerly laminin 5)-deficient junctional epidermolysis bullosa, and then transduced those stem cells with laminin 332 beta-3 chain cDNA via a retroviral vector. Those genetically corrected stem cells were then cultured into epidermal grafts that produced a functional correction in vivo with no evidence of clonal expansion or selection of specific integration events.
Cutaneous gene delivery
91
Table 2 Summary of cutaneous gene transfer protocols for systemic delivery Protein
Target cell
Transfer principle
Method
Reference
Year
Factor IX Erythropoietin Adenosine deaminase Factor IX Ornithine-delta-aminotransferase Ornithine-delta-aminotransferase IL-10
KC KC KC KC KC KC Skin
Retrovirus AAV Retrovirus Retrovirus Retrovirus Adenovirus Naked DNA
[6] [76] [77] [78] [79] [80] [29]
1993 1996 1997 1997 1997 1997 1998
Factor IX Phenylalanine hydroxylase + GTP cyclohydrolase 1 Leptin Erythropoietin
KC KC
Ex vivo Ex vivo Ex vivo Ex vivo Ex vivo Ex vivo In vivo (intradermal injection) Ex vivo Ex vivo
Retrovirus Retrovirus
[81] [82]
1998 2000
Retrovirus Lentivirus
[83] [84]
2001 2001
Retrovirus
[85]
2002
Naked DNA
[86]
2002
Retrovirus AAV Retrovirus Retrovirus
[87] [88] [89] [90]
2003 2004 2006 2007
KC Skin Fibroblast
Phenylalanine hydroxylase + GTP cyclohydrolase 1 IL-4, 6, 10, TGF-beta
Skin
Growth hormone GM-CSF Growth hormone Proinsulin
KC KC KC KC
Ex vivo In vivo (intradermal injection) Ex vivo In vivo (intradermal injection) Ex vivo Ex vivo Ex vivo Ex vivo
KC: keratinocyte.
Adenoviral vectors can easily be obtained at high titers, and can then be efficiently used to transiently transduce both quiescent and actively dividing cells [10], but they are of limited use for longterm gene correction. Adenoviral vectors can elicit a strong immune response in hosts, as in the case of an ornithine transcarbamylase-deficient patient who died of a systemic inflammatory response syndrome (SIRS) after right hepatic artery infusion of an adenoviral vector [11]. In addition, the efficiency with which adenoviral vectors are transfected into epithelial cells is relatively low, due to the low levels of Coxsackie-adenovirus receptor (CAR; the primary adenoviral cellular receptor) on fibroblasts [12] and keratinocytes [13]. Also, studies indicate that the negative charge of membrane glycoproteins reduces the efficiency of adenovirus-mediated gene transfer [14]. For these reasons, researchers have had little success using adenoviral vectors to transfer genes into keratinocytes. To enhance adenovirus-mediated gene delivery into keratinocytes, researchers have studied the use of polycationic molecules and genetically modified adenoviruses with CAR-independent tropism [14,15]. The advantages of AAV vectors are their ability to transduce both dividing and non-dividing cells, their ability to site-specifically integrate and produce long-term gene expression (even in vivo), and their broad tropism allowing efficient
transduction into diverse organs including the skin [16]. Also, AAV vectors have been found to replicate at high levels in a normal skin model [17]. These findings suggest that AAV vectors are skintropic, and are thus useful vectors for skin-directed gene therapy. Lentiviral vectors are suitable for introducing genes into both dividing and non-dividing cells, and for integrating genes into the genome [18]. This indicates that lentiviral vectors are useful for transferring genes into keratinocyte stem cells, which divide very slowly. It also indicates that lentiviral vectors can be used in place of retroviruses for transfection of stem cells, as retrovirus-mediated transfection of stem cells is inefficient. Non-viral vectors have been used in ex vivo gene therapy for several skin diseases. In a study by OrtizUrda et al. [19], integrase phiC31 derived from a Streptomyces phage stably integrated COL7A1 cDNA into the genome of primary epidermal progenitor cells from four unrelated patients with recessive dystrophic epidermolysis bullosa (RDEB). Sleeping Beauty, a member of the TC1/mariner superfamily transposons mobile in vertebrate genome, has also been used in non-viral ex vivo gene therapy [20]. These non-viral agents have failed to successfully integrate into desired sites where they would not disrupt the native function of unrelated genes. However, combined with skin culture technology and stem cell biology, these agents show promise
92
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in contributing to new methods of gene therapy and may improve the long-term expression of genes used to treat various diseases.
3.2. In vivo transfer of genes into the skin Many in vivo gene transfer systems have been developed, using both viral and non-viral vectors. These systems, which deliver genes directly into the skin, include topical DNA application, direct DNA injection, electroporation, iontophoresis, sonophoresis and gene guns. Topical application of plasmid DNA is an attractive approach for gene delivery. If successfully developed, it would be useful for cutaneous gene therapy, because it is painless and can easily be applied to large body surface areas. Li and Hoffman [21] reported successful selective introduction of the LacZ reporter gene into hair follicles of mice via topical application of liposomes containing the gene. Hemagglutinating virus of Japan (HVJ)-liposomes have been constructed by fusing DNA-loaded liposomes with UV-inactivated HVJ [22]. Administration of LacZ plasmid DNA with HVJ-liposome into the amniotic fluid of fetal rats has resulted in transformation of entire areas of fetal skin [23]. In a study using an adenoviral vector containing the human carcinoembryonic antigen (CEA) gene, topical application of the adenoviral vector evoked an immune response against the transgene product [24]. Topical application of naked plasmid DNA encoding the hepatitis B surface antigen has also been found to induce antigen-specific immune responses [25]. Recently, an NF-kB decoy, which is synthetic double-stranded DNA that corresponds to NF-kB ciselements and can bind to transcriptional factor as a ‘‘decoy’’ to alter gene transcription, has been developed and found to be a useful tool as a new class of anti-gene agents [26] (Fig. 1). Research
Fig. 1
indicates that application of ointment containing NF-kB decoy oligonucleotides onto atopic dermatitis lesions of NC/Nga mice is an effective means of gene therapy [27]. Tamai et al. used such an NF-kB decoy ointment to treat human patients with atopic dermatitis (unpublished data), and found that it very effectively improved lesions with impaired barrier function, especially facial eczematous, exudatative lesions with acute inflammation. Direct injection of naked DNA into the skin is an efficient way to transfer genes into keratinocytes in vivo [28]. Although it is unknown why intradermally injected plasmid DNA is taken up and expressed by keratinocytes, the resultant transgene products not only affected the local skin lesion, but were also released into the circulation and exerted effects on other target organs [29]. Although this method is relatively efficient, safe and simple, it does not result in long-term constitutive expression of genes, and it cannot be used to quickly introduce genes into a large area of the skin as possible by topical application. When COL7A1 cDNA spanning about 9 kb was injected into the skin of hairless rats, the cDNA was expressed in keratinocytes [30]. This result indicates that naked DNA injection might be useful for the clinical treatment of RDEB. In recent studies using animal models of RDEB and xeroderma pigmentosum (XP), intradermal injection of genetically modified viral vectors resulted in the synthesis of functional protein [31,32]. Electroporation is one of the most popular methods of introducing genes into cells in vitro. The earliest reported study of in vivo electroporation to deliver genes was conducted by Titomirov et al. [33]. They subcutaneously injected plasmid DNA encoding the neomycin resistance gene, followed by high-voltage pulses, and then found neo-resistant colonies in resultant primary skin cell cultures. Recently, the combination of electroporation and
The mechanism of the therapeutic effect of NF-kB decoy oligodeoxynucleotide (ODN).
Cutaneous gene delivery injection of a plasmid encoding keratinocyte growth factor-1 (KGF-1) has been successfully used to promote wound healing [34]. Drabick et al. [35] found that this method can increase transgene expression up to an average of 83-fold, relative to naked DNA injection. They found that transfected cells were principally located in the dermis, and also observed transfected cells in lymph nodes draining electropermeabilized sites, suggesting that directly transfected (epi-)dermal dendritic cells had migrated from the treated site. These findings suggest that this method is broadly applicable to nucleic acid vaccination. The combination of electroporation and topical application of plasmid DNA onto partially stripped skin has been successfully used to deliver DNA into keratinocytes [36]. Iontophoresis, which is the application of an electrical potential that maintains a constant
93 electric current across the skin, enhances the delivery of both ionized and non-ionized moieties. Although there have been no reports of the use of this method for cutaneous gene delivery, it has been found to be an effective way to deliver antisense oligonucleotides into keratinocytes. Sakamoto et al. [37] studied iontophoretic topical delivery of mouse interleukin-10 (IL-10) antisense oligonucleotides, and found that it had a therapeutic effect in atopic dermatitis skin lesions of NC/Nga mice. Ultrasound improves the efficiency of plasmid delivery in vitro by causing the formation of shortlived pores in the plasma membrane (a method know as sonoporation), leading to holes with diameters of up to 100 nm, via acoustic cavitation. Recently, ultrasound-assisted plasmid delivery has also been successfully achieved in vivo by
Fig. 2 Gene expression in the skin 48 h after intra-amniotic injection with microbubble-enhanced ultrasuound. (a) Macroscopic appearance of fetal mouse. (b and c) Fluorescence macroscopic image of GFP-gene transduced skin. (d) Confocal microscopic image of GFP-gene transduced skin. (e and f) X-Gal staining of LacZ gene transduced skin. Magnifications: 40 (e) and 100 (f).
94 enhancing the effects of ultrasound with microbubbles. In a study by Endoh et al. [38], intraamniotic injection of naked DNA with microbubbleenhanced ultrasound resulted in high levels of expression of the DNA in fetal mouse skin (Fig. 2). A gene gun is a needle-free device that can introduce DNA-coated gold particles into target cells or tissues, including the skin [39]. Due to the short-term and inefficient expression of the resulting gene products in vivo, gene guns have demonstrated only limited potential in gene therapy for genetic skin diseases. However, gene guns have been successfully used in immunomodulation, in which cytokine genes are introduced into tumorbearing animals and the resulting gene products secreted into the blood stream [40]. Furthermore, gene guns have been successfully used in DNA vaccination against infectious diseases [41]. Gene gun-mediated DNA vaccination has been shown to induce greater antibody and/or CD8+ T-cell responses, with substantially lower doses (100— 1000-fold) of DNA, than routine needle-based approaches [41].
4. Methods for overcoming the skin barrier against gene delivery To achieve more efficient cutaneous gene delivery, removal of the horny layer is thought to be the best way to disrupt the barrier of the skin. As described above, the most important component of the skin barrier function is the horny layer, which prevents penetration of foreign molecules greater than 500 Da [2]. Tape-stripping using adhesive tape removes the horny layer, but this is not suitable for all-over topical transfer of genes into skin. Without successful penetration of this effective barrier, exogenously applied genes cannot enter keratinocytes. Drug delivery via the skin is an important pharmacological goal, and the horny layer is thus an important structural target in the improvement of drug delivery systems. Several technological advances have been made in the past few decades in overcoming this barrier: electroporation, sonophoresis, iontophoresis and chemical penetration enhancers (CPEs). Electroporation with high voltage and short pulses creates transient aqueous pores in intercellular lipid bilayers, which may account for the increase in skin permeability [42]. Lombry et al. [43] demonstrated that transdermal and topical delivery of macromolecules of at least 40 kDa can be achieved by skin electroporation.
Y. Kikuchi et al. Iontophoresis has been shown to increase the permeability of skin to macromolecules. Kalia et al. [44] reported the successful iontophoretic delivery of functional peptides of 4 kDa (calcitonin and hPTH(1—34)) into keratinocytes. Several studies indicate that iontophoresis can cause skin irritation. However, combining iontophoresis with permeability-enhancing techniques can produce therapeutically effective delivery levels with lower levels of current, thus dramatically reducing the risk of skin discomfort and irritation [45]. Ultrasound is used to increase penetration by pharmacologically active drugs through the skin. This technique is known as sono- or phonophoresis. Recently, ultrasound at frequencies ranging from 20 kHz to 16 MHz has been used to permeabilize skin to allow transdermal drug administration. Sonophoresis is particularly effective at lower frequencies ( f < 100 kHz; low-frequency sonophoresis). The level of transdermal transport produced by low-frequency ultrasound is up to 1000-fold higher than that produced by therapeutic high-frequency ultrasound [46]. There have been several reports of the successful use of this method for delivery of macromolecules; e.g., mannitol, insulin, low-molecular-weight heparin (LMWH) and antisense oligonucleotides (reviewed in [46]). CPEs are chemical compounds that reduce the resistance of the skin barrier to drug diffusion. Many CPEs have been studied, including various surfactants, fatty acids and fatty esters. The most potent CPEs enhance skin permeability by altering the structure of the horny layer [47]. Using in vitro skin impedance guided high-throughput (INSIGHT) screening, Karande et al. [48] discovered mixtures of penetration enhancers that increase the permeability of skin to macromolecules (1—10 kDa) by up to 100-fold without inducing skin irritation. Microneedles, which create micrometer-scale openings, have been created to painlessly pierce the horny layer and thereby transport drugs into the body. Microneedles can transport macromolecules such as insulin, desmopressin, antisense oligonucleotides and plasmid DNA (reviewed in [49]). Also, vaccine delivery using microneedles can induce an immune response to hepatitis B surface antigen encoded by plasmid DNA [50].
5. Conclusion In the past few decades, many methods using viral or non-viral vectors have been developed for skin genes transfer. Although several of these methods appear promising for clinical application (e.g., vaccination and bioreactors), the gene delivery effi-
Cutaneous gene delivery ciency is generally still insufficient for effective treatment of patients with inherited skin diseases affecting the whole body. For treatment of acquired skin disorders, the use of oligonucleotide decoys for key transcriptional regulators such as NF-kB appears to be a potentially powerful tool because of the ease with which it penetrates the skin, compared to plasmid or viral DNA. Improvement of the efficiency of gene delivery requires the development of noninvasive methods that can rapidly overcome the barrier function of the horny cell layer over large areas of skin. In addition, attaining an efficient and continuous therapeutic effect after gene therapy requires (1) targeting of stem cells, (2) site-specific DNA incorporation and (3) suppression of the host immune response against the transgene product. The properties of keratinocyte stem cells have been well characterized, and isolation of keratinocyte stem cells should allow efficient gene transfer into them. The risks that gene therapy entails are clearly illustrated by the accidental development of cancer in X-SCID patients as a result of gene therapy. To reduce the risk of such accidents, we must develop site-specific DNA correction that only affects the disease-related target gene. Transient gene transduction methods have a lower risk of such problems, but they have a narrow range of applications for treatment of inherited skin diseases involving the entire body. Finally, in patients with null mutations in the target gene, gene therapy can result in immune responses to the transduced cells and clearance of them by CD8+ T cells and/or antibodies against the transgene product. Gross et al. [51] found that adoptive transfer of antigen-specific CD4+CD25+ regulatory T cells, which are implicated in peripheral self-tolerance, can down-regulate both B- and T-cell responses, resulting in tolerance of the transgene product. Other studies indicate that molecular bone marrow chimerism resulting from transduction of bone marrow cells with retroviral vectors has resulted in prolonged acceptance of skin grafts [52]. Thus, cutaneous gene therapy is an attractive approach, but is still insufficient in its effectiveness. However, recent findings suggest that major breakthroughs in this field are imminent.
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Cutaneous gene delivery
[59]
[60]
[61]
[62]
[63]
[64]
[65]
[66]
[67]
[68]
[69]
[70]
[71]
[72]
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Y. Kikuchi et al. Katsuto Tamai received his MD and PhD degrees from Hirosaki University, Aomori, Japan, in 1986 and 1990, respectively. He became an assistant professor in the Department of Dermatology, Hirosaki University in 1990. He worked under Dr. Jouni Uitto as a Research Fellow in the Department of Dermatology and Cutaneous Biology, Thomas Jefferson University, Philadelphia, USA, from 1991 to
1993. He was associate professor at Hirosaki University from 1999 to 2003. In 2003, he moved to Osaka University Graduate School of Medicine and is currently an associate professor in the Division of Gene Therapy Science (Prof. Y. Kaneda).