View metadata, citation and similar papers at core.ac.uk brought to you by CORE

provided by Elsevier - Publisher Connector

Gene Therapy for Genetic Skin Disease

Paul A. Khavari Dermatology Service, VA Palo Alto Health Care System, Palo Alto, California, U.S.A.; and Department of Dermatology, Stanford University School of Medicine Stanford, California, U.S.A.

Progress in the molecular characterization of monogenic skin disorders disorders, especially those involving mutations in large structural has been a hallmark of the past decade in investigative dermatology proteins such as type VII collagen in dystrophic epidermolysis and has, for the first time, brought the possibility of corrective genetic bullosa (Christiano et al, 1993, 1994; Hilal et al, 1993), are likely therapies within reach. In addition to identification of disease genes, to be intractable to small molecule pharmacotherapy and may be successful corrective gene delivery to the skin requires an understanding only effectively approached by the return of normal protein of disease pathogenesis, gene expression characteristics, stem cell expression. biology, effective gene delivery, and immune modulation (Vogel, 1993; An understanding of the molecular basis of disease pathogenesis Greenhalgh et al, 1994; Krueger et al, 1994; Taichman, 1994; Khavari in these disorders is important in the design of rational genetic and Krueger, 1997; Khavari, 1997). Because of its accessibility, the therapies. Correction of recessive disorders, in principal, requires skin offers an attractive opportunity to refine gene transfer capabilities only re-expression of the normal protein product corresponding to relative to current efforts underway in visceral tissues (Davis et al, 1996; the previously absent or defective genes. This has been recently Hess, 1996; Sokol and Gewirtz, 1996; Blau and Khavari, 1997). achieved in model efforts, as noted below (Choate et al, 1996a, b; Progress in cutaneous gene therapy, however, has lagged behind other Freiberg et al, 1997). A greater challenge exists in the dominant tissues and future advances depend on rigorous studies in well-defined disorders due to dominant-negative mutant proteins that effectively skin disease model systems. In this review, I focus on the current ‘‘poison’’ any wild type protein present, as in the case of keratin 1 progress in therapeutic gene transfer for genetic skin disease and outline and 10 mutants in epidermolytic hyperkeratosis (Cheng et al, 1992; future directions in the field. Rothnagel et al, 1992). Effective genetic correction in these latter instances requires circumventing the negative effects of such mutants SELECTED CANDIDATE GENETIC SKIN DISEASES in an effort to restore the function of the normal protein. Because The genetic basis for a number of hereditary skin diseases has been of the considerable additional technical challenges associated with elucidated (Table I), with remaining uncharacterized genodermatoses gene delivery to human somatic tissue, the recessive genetic skin currently the focus of intensive investigation. Human genetic lesions disorders have been the focus of many current efforts to date. leading to skin blistering, abnormal cutaneous cornification, and SPECIFIC REQUIREMENTS OF GENODERMATOSES cancer predisposition have been well characterized. In the epidermis, COMPARED WITH OTHER CUTANEOUS GENE these mutations affect genes expressed selectively in either basal or suprabasal layers, as well as those expressed in both compartments. TRANSFER APPLICATIONS The site of such genetic defects within the epidermis can produce A recurrent theme in gene therapy efforts in all tissues is the need to characteristic corresponding phenotypic abnormalities. Mutations in tailor gene transfer approaches to specific therapeutic applications (Blau important basal layer genes commonly lead to skin fragility and and Khavari, 1997). In addition to genetic skin diseases, the skin is a blistering, such as is seen with laminin 5 chain gene defects in a potential tissue site for a number of distinct therapeutic gene transfer subset of junctional epidermolysis bullosa patients (Aberdam et al, efforts and these differ considerably in their gene transfer requirements. 1994; Eady and Dunnill, 1994; Pulkkinen et al, 1994; Uitto and Such potential efforts include cutaneous gene transfer for immunization Pulkkinen, 1996). Suprabasal defects lead to abnormal terminal (Raz et al, 1994) and for production of polypeptides for delivery to epidermal differentiation often manifested as hyperkeratosis, as seen the systemic circulation (Fenjves et al, 1989; Fenjves et al, 1994). Each in the case of transglutaminase 1 gene mutations in lamellar ichthyosis of these three applications has distinctive requirements regarding (Russell et al, 1994, 1995; Huber et al, 1995a). A number of these efficiency of gene transfer, durability, regulation of expression of the introduced gene (the ‘‘transgene’’), and transgene immunogenicity. Reprint requests to: Dr. Paul A. Khavari, Department of Dermatology, P204, High efficiency gene transfer that restores normal gene expression MSLS, Stanford University School of Medicine, Stanford, CA 94305. to all cells of a diseased tissue has been among the most challenging

0022-202X/98/$10.50 · Copyright © 1998 by The Society for Investigative Dermatology, Inc. 462 VOL. 110, NO. 4 APRIL 1998 GENE THERAPY FOR GENETIC SKIN DISEASE 463

Table I. Selected candidate genodermatoses

Disordera Selected gene(s) References

Lamellar ichthyosis subset Transglutaminase 1 Huber et al, 1995a; Russell et al, 1995 X-linked ichthyosis Steroid sulfatase Yen et al, 1987 EB simplex Keratins 5 and 14 Bonifas et al, 1991; Coulombe et al, 1991 Pachyonychia congenita type 1/2 Keratins 6a and 16/17 Bowden et al, 1995; McLean et al, 1995 Epidermolytic hyperkeratosis Keratins 1 and 10 Cheng et al, 1992; Rothnagel et al, 1992 Epidermolytic PPK Keratin 9 Reis et al, 1994; Torchard et al, 1994 Non-epidermolytic PPK Keratin 16 Shamsher et al, 1995 Vohwinkel’s syndrome Loricrin Maestrini et al, 1996 Ichthyosis bullosa of Siemens keratin 2e Rothnagel et al, 1994 Junctional EB Laminin 5 α3, β3 and γ2 Aberdam et al, 1994; Pulkkinen et al, 1994 BPAG2 [BP180] McGrath et al, 1995 β4 integrin Vidal et al, 1995 Plectin Smith et al, 1996 Dystrophic EB Type VII collagen Christiano et al, 1993; Hilal et al, 1993 Xeroderma pigmentosum XP group genes Li et al, 1993; Kraemer, 1996 Basal cell nevus syndrome Patched Hahn et al, 1996; Johnson et al, 1996

aEB, epidermolysis bullosa; PPK, palmoplantar keratoderma; XP, xeroderma pigmentosum. goals in many tissues; however, not all applications require it. Immuniza- for monogenic skin disease. Surprisingly, such unwanted transgene tion via gene transfer to the skin, for example, depends on immune specific immune responses have not been widely reported in current elements in skin and does not require uniform transgene delivery to gene therapy trials in other tissues, in stark contrast to inflammatory all cells in a given tissue compartment. This may also be the case in responses seen in humans with viral gene delivery vectors such as systemic delivery of proteins expressed in skin, where the absolute adenovirus (Knowles et al, 1995). Because current pre-clinical models magnitude of total transgene product delivered from the skin impacts of therapeutic cutaneous gene transfer lack an intact human immune the therapeutic outcome more that the percentage of cells in the skin system, the extent to which unwanted immune reactions will pose a that express it. Lasting correction of genetic skin disease, in contrast, challenge may only become clear in human clinical trials. Central may require high efficiency gene transfer, as could be predicted in the requirements for successful genetic correction of genetic skin disease, case of cancer prevention in patients with xeroderma pigmentosum then, may include high efficiency genetic correction, durabilty, consist- and in the correction of cell-intrinsic structural defects such as keratin ent transgene expression levels, and a lack of unwanted immune 5/14 mutations in epidermolysis bullosa simplex (Bonifas et al, 1991; clearing of the therapeutic gene. Coulombe et al, 1991). In such cases, a focal lack of corrective gene expression may be sufficient to perpetuate major elements of disease PROGRESS IN SPECIFIC GENODERMATOSES pathology (i.e., neoplastic transformation in xeroderma pigmentosum and skin fragility in epidermolysis bullosa). High efficiency genetic Recessive ichthyoses The ichthyotic disorders are a heterogeneous correction therefore may be of importance in gene therapy of genoder- family of diseases characterized by abnormal epidermal cornification matoses. (Williams and Elias, 1987). Recently the genetic basis for several of Gene transfer efforts in human visceral tissues have been unsuccessful these disorders has been characterized (Bale and Doyle, 1994) and in reliably sustaining therapeutic gene expression (Davis et al, 1996; found to involve key enzymatic or structural proteins involved in Hess, 1996; Sokol and Gewirtz, 1996; Blau and Khavari, 1997), and epithelial maturation and cutaneous barrier function. Among the the ability to sustain therapeutic gene delivery for prolonged periods recessive ichthysoses are lamellar ichthyosis and X-linked recessive is another capability important in gene therapy for genetic skin disease. ichthyosis. Consistent with the nature of their gene defects in the Whereas the need for such long-term gene delivery may be variable suprabasal epidermal gene expression program, these disorders are to nonexistent in both genetic immunization and protein delivery to characterized by clinical hyperkeratosis. the circulation from the skin, curative efforts in genodermatoses depend Lamellar ichthyosis has been associated with the TGM1 gene (Russell on lasting restoration of normal gene expression. Because this requires et al, 1994, 1995; Huber et al, 1995a) in a subset of patients by genetic durable gene delivery vectors, gene targeting to long-lived skin stem linkage and mutation analysis, and has proven to be a valuable prototype cells, and avoidance of immune clearing, achieving such sustainability disorder for gene therapy (Choate et al, 1996a, b) due to its dramatic has proven to be a formidable challenge. Whereas periodic read- clinical and biochemical phenotype. The TGM1 gene encodes ker- minstration of therapeutic genes in the form of grafting with genetically atinocytes transglutaminase (TGase1) (Thacher et al, 1985; Phillips et al, engineered cells or by direct transfer of vectors to intact human skin 1990), a membrane linked enzyme active in forming the cornified tissue may be envisioned in certain lethal genetic skin disorders, envelope in differentiating keratinocytes. TGase1 catalyzes the forma- achievement of sustainable cutaneous gene delivery is a major require- tion of isodipeptide cross-links between cornified envelope precursor ment for ultimate success. molecules (Greenberg et al, 1991; Kim et al, 1995) and is believed The skin, by virtue of its accessibility to target gene regulation by necessary for normal terminal differentiation and barrier formation in topically applied agents, is an attractive tissue for regulated therapeutic the outer epidermis. Although not present in all lamellar ichthyosis gene production. Such regulated delivery may be of importance in the patients (Bale et al, 1994; Huber et al, 1995b; Parmentier et al, 1995), delivery of proteins to the systemic circulation, such as the hemato- TGM1 gene mutations in affected patients may produce enzymatically poietic growth factor erythropoeitin (Bohl et al, 1997). Many of the inactive TGase1 protein (Huber et al, 1995a). Restoration of functional genes affected in genetic skin disease, however, are expressed at constant TGase1 enzymatic activity to skin from TGase1-negative patients levels within a given tissue compartment, as in the case of laminin 5 represents a possible means of correcting this disorder. and keratin genes within the basal and suprabasal layers, respectively. In order to achieve this, recent work has developed an approach to Precise regulation of the magnitude of therapeutic gene expression in achieving high efficiency transfer of the normal TGase1 gene to skin genetic skin disease may therefore be unneeded in most cases, provided from patients characterized as TGase1-negative (Choate et al, 1996a). gene expression may be restored to levels within a physiologic range. This approach relies on amphotropic retroviral gene delivery vectors The ability of a therapeutic transgene to induce a specific immune for the TGase1 gene (Choate et al, 1996a). Following high efficiency response, although central for the success of intradermal genetic gene transfer into primary patient keratinocytes grown in vitro, restora- immunization, represents a feared complication in gene transfer efforts tion of full length TGase1 protein expression and transglutaminase 464 KHAVARI THE JOURNAL OF INVESTIGATIVE DERMATOLOGY enyzmatic activity was verified. These keratinocytes were then grafted subtypes of epidermolysis bullosa, a key hope centers on correction of to immune deficient mice to regenerate human skin in vivo in an specific underlying genetic defects. approach that recapitulates the histologic, gene expression, clinical, and Molecular alterations in a number of specific genes responsible for functional features of human skin in vivo (Choate et al, 1996b; Medalie epidermolysis bullosa have been increasingly well characterized over et al, 1996; Choate and Khavari, 1997a), with the exception of the past 5 y (Marinkovich, 1993; Uitto et al, 1994; Korge and Krieg, immune function. Lameller ichthyosis patient keratinocytes genetically 1996; Paller, 1996; Uitto and Pulkkinen, 1996). Malfunction in any engineered with the TGase1 vector regenerated skin displaying restored of their corresponding proteins mediating epidermal adhesion, which TGase1 protein expression in vivo and was normalized at the levels of could be envisioned to function as a series of connected links in a histology, clinical surface appearance, and barrier function; patient cells chain, results in skin fragility and blistering. The association of these that received a control vector produced skin with the hyperkeratosis genes with specific clinical phenotypes has shed additional light on their and defective skin barrier function characteristic of the disease (Choate pathogenesis, as, for example, in the case of compound heterozygous et al, 1996b). These findings indicated that successful phenotypic mutations identified in the type VII collagen gene in dystrophic correction of human genetic skin disease tissue can be achieved via epidermolysis bullosa (Tamai et al, 1997), as well as BPAG2 (McGrath this gene transfer approach; however, this correction failed to extend et al, 1996b) and laminin 5 chains (Christiano et al, 1996; McGrath beyond the 1 month timepoint after which transgene expression is et al, 1996a) in junctional epidermolysis bullosa. The majority of genes commonly lost in many cutaneous gene transfer models (Gerrard et al, implicated in epidermolysis bullosa are effectively produced within 1993; Taichman, 1994; Choate et al, 1996b; Fenjves et al, 1996; Choate keratinocytes of the basal epidermal layer (Marinkovich et al, 1993). and Khavari, 1997a; Freiberg et al, 1997). Such loss may be due to a This, combined with the fact that some of these genes are expressed number of potential factors; however, in this approach it appears to be in internal epithelia in addition to skin, underscores the fact that due to silencing of vector promoter elements (Choate and Khavari, attempts at genetic correction in these disorders must be correctly 1997a). Correction of lamellar ichthyosis patient skin tissue, then, targeted. In this regard, it is unknown whether restored expression of could be achieved but not sustained. a basement membrane protein throughout the epidermis may result in X-linked ichthyosis is a genodermatosis that is generally much milder disordered epithelial polarity, therefore it is formally possible that than lamellar ichthyosis (Williams and Elias, 1987; Paige et al, 1994). proteins such as laminin 5 and BPAG2 require expression limited to Due to a loss of functional Arylsulfatase C (Yen et al, 1987; Shapiro the basal epidermal layer. This is in contrast to corrective efforts with et al, 1989; Alperin and Shapiro, 1994; Ballabio and Shapiro, 1995), a TGase1 and steroid sulfatase where mRNA expression throughout the steroid sulfatase believed necessary for a normal desquamation (Epstein epidermis via vector promoters active in all layers failed to impair their et al, 1981), X-linked ichthyosis constitutes another prototype recessive corrective impact (Choate et al, 1996b; Freiberg et al, 1997). The fact disorder for refinement of cutaneous gene transfer. High efficiency that genetically corrected epidermolysis bullosa patient keratinocytes retroviral gene transfer followed by grafting of genetically engineered may have a selective adhesive advantage over uncorrected cells, cells has also recently been used to correct X-linked ichthyosis patient however, may mitigate against these and other unanticipated poten- skin at the level of tissue architecture, gene expression, clinical tial pitfalls. appearance, and function (Freiberg et al, 1997). Although also not Although at an earlier stage than the ichthyoses, efforts at genetic sustained for periods significantly longer than 1 month, this corrective correction of epidermolysis bullosa in model systems have focused on gene transfer disease model offers another opportunity to address the laminin 5 (Gagnoux-Palacios et al, 1996) and BPAG2 in junctional challenge of durable therapeutic gene delivery to the skin. disease and type VII collagen in the dystrophic subtypes. It is interesting Among the other inherited disorders of cornification are dominant to note that revertant mosaicism in the BPAG2 gene – where a disorders due to keratin mutations, such as epidermolytic hyperkeratosis defective gene reverts to normal in a mosaic pattern in the skin – has (K1/K10) (Cheng et al, 1992; Rothnagel et al, 1992), palmoplantar been described in generalized benign atrophic epidermolysis bullosa, keratoderma subtypes (epidermolytic, K9; nonepidermolytic, K16) and has been noted to resemble a natural form of cutaneous gene (Reis et al, 1994; Torchard et al, 1994; Shamsher et al, 1995), and therapy (Jonkman et al, 1997). In the case of laminin 5, defects in one pachyonychia congenita subtypes (K6a/16/17) (Bowden et al, 1995; of the three chains of the laminin 5 trimer (α3, β3, or γ2) are associated McLean et al, 1995). In principal, these diseases represent much greater with junctional epidermolysis bullosa of a severity that extends to challenges to even achieving short-term correction than the recessive include the severe Herlitz subtype (Aberdam et al, 1994; Pulkkinen disorders because of the presence of proposed dominant-negative et al, 1994; Uitto et al, 1994). Laminin 5 γ2 chain gene transfer via mutant keratin molecules. Additional wild type gene expression in this both modified adenoviral and retroviral based approaches has been setting may be ineffective because such mutants can disrupt the function described in the immortalized L5 V5 keratinocyte line (Gagnoux- of normal keratin subunits in forming the intermediate filaments that Palacios et al, 1996). Such gene transfer led to restoration of normal give structural integrity to the cell (Freedberg, 1993; Fuchs, 1995). A features of laminin 5 trimer expression in a basal polarized fashion and number of strategies to overcoming the challenge of dominant-negative this finding was confirmed in cysts of immortalized cells formed in vivo mutants via genetic approaches are being studied, including in situ after subcutaneous injection into nude mice (Gagnoux-Palacios et al, gene repair by oligonucleotide recombination and homologous gene 1996). A next phase of work in genetic correction of epidermolysis recombination; however, none have yet been shown successful in bullosa will involve nontransformed tissue from patients in spatially achieving functional correction in a genodermatosis model. intact epidermis.

Epidermolysis bullosa The array of inherited blistering skin dis- Xeroderma pigmentosum Xeroderma pigmentosum has been orders known as epidermolysis bullosa represent prototypes of gene associated with defects in a number of DNA repair genes and is defects in affecting the basal epidermal program of gene expression characterized by specific inadequacies in DNA repair following ultravi- (Eady and Dunnill, 1994; Uitto et al, 1994; Korge and Krieg, 1996; olet injury (Li et al, 1993; Kraemer, 1996). From the standpoint of Paller, 1996; Uitto and Pulkkinen, 1996). The recent characterization gene targeting, xeroderma pigmentosum represents a pan-epidermal of the genes responsible for specific subtypes of this disease have prototype disorder affecting both basal and suprabasal compartments, revealed a corresponding heterogeneity in molecular defects (Table I). including melanocytes. The disease is characterized by photosensitivity A common theme emerging from these studies has been that these with early onset of cutaneous neoplasias, including basal cell carcinoma, defects affect structural proteins, forming the vital link from the squamous cell carcinoma, and melanoma (Kraemer et al, 1990). Much cytoplasm of basal keratinocytes through basement membrane zone progress has been made in identifying the genes corresponding to the components and on down into the uppermost dermis (Uitto and separate genetic complementation groups of xeroderma pigmentosum Pulkkinen, 1996). Because of the structural nature of these proteins and its variants. This progress has been followed by attempts at genetic and the fact that many have multiple functional domains and can correction in xeroderma pigmentosum cells grown in tissue culture. polymerize to form higher order structures, purely medicinal therapy The XPA and XPD genes have been among those best studied in this has proven of little benefit. Especially in the case of more severe regard and their restoration to cells lacking these genes has been shown VOL. 110, NO. 4 APRIL 1998 GENE THERAPY FOR GENETIC SKIN DISEASE 465

Figure 1. Paradigm for development of gene therapy for genetic skin disease. to return parameters of DNA repair and cell survival after ultraviolet down-modulation by blockade of costimulatory molecules such as injury back toward normal (Marionnet et al, 1993; Myrand et al, CD28 and CD40 (Larsen et al, 1996) shows some promise in this 1996). While corrective gene delivery to all layers of the epithelium regard. A lack of intrinsic vector stamina, however, must be addressed is readily achieved to keratinocytes by regeneration of skin using as well and the characterization of genetic elements conferring durable genetically engineered cells (Choate et al, 1996b; Freiberg et al, 1997), expression on therapeutic vector sequences is an important effort in the need to target melanocytes represents an additional formidable achieving lasting genetic correction. challenge in xerodermal pigmentosum that has not yet been systematic- Correction of abnormal gene expression in genodermatoses may be ally approached. Xeroderma pigmentosum, then, represents another approached by avenues distinct from transfer of the normal gene. serious genetic disorder in which efforts in models of genetic correction Among such methods are oligonucleotide based repair of mutated have been initiated yet in which major technical challenges remain. genes by genetic recombination, direct application of recombinant proteins to affected skin, and heterologous cell therapy that circumvents REQUIRED NEW CAPABILITIES AND ALTERNATIVE immune rejection. These approaches, along with other emerging APPROACHES innovative strategies, may offer attractive future ways to avoid the Widespread successful application of genetic therapies in the treatment traumatic and complicated process of grafting genetically engineered of genetic skin disease is dependent upon the advancement of our cells, which represents a current focus of models of gene therapy of understanding of fundamental biologic processes in the skin as well as genetic disease. the acquisition of new gene delivery capabilities (Fig 1). Important biologic questions in therapeutic gene transfer involve the characteriza- SUMMARY AND CONCLUSIONS tion of cutaneous stem cells and an increased understanding of cutaneous gene expression. Although impressive advances have recently been With identification of the genes involved in the pathogenesis of a made in our understanding of the characteristics of epidermal stem number of inherited cutaneous disorders, the previously bleak prospects cells (Jones and Watt, 1993; Rochat et al, 1994; Bata-Csorgo et al, for patients afflicted by an array of genodermatoses may be changing 1995; Jones et al, 1995; Mathor et al, 1996), no markers solely specific for the better; however, realistic assessment of the formidable challenges for these cells have yet been identified. Furthermore, our understanding remaining to routinely achieving corrective gene delivery in the skin, of the factors controlling stem cell division and the genetic programs indicate that significant additional progress is required before this unique to these tissue progenitors is still in the early stages. In addition technology becomes widely applicable to these patients. Nevertheless, to targeting genetic elements to stem cells, long-term corrective gene advances in new models of in vivo genetic correction highlight the delivery requires sustained transgene expression. Such expression is potential promise for future successful application to patients afflicted dependent on the gene regulatory milieu within a given tissue and, as with genetic skin disease. noted above, this has not supported the achievement of long-term uniform gene expression throughout genetically engineered skin tissue REFERENCES by current approaches, even when vector sequences persist (Gerrard Aberdam D, Galliano MF, Vailly J, et al: Herlitz’s junctional epidermolysis bullosa is linked et al, 1993; Taichman, 1994; Choate et al, 1996b; Fenjves et al, 1996; to mutations in the gene (LAMC2) for the gamma 2 subunit of nicein/kalinin Choate and Khavari, 1997a; Freiberg et al, 1997). The factors causing (LAMININ-5). Nat Genet 6:299–304, 1994 this loss of transgene expression may involve mechanisms fundamental Alperin ES, Shapiro LJ: Characterization of point mutations in patients with X-linked to eukaryotic gene regulation, such as promoter methylation and ichthyosis. Am J Hum Genet 55:209, 1994 Bale SJ, Doyle SZ: The genetics of ichthyosis: a primer for epidemiologists. J Invest heterochromatin formation (Hoeben et al, 1991; Wolffe, 1992). Dermatol 102:49S–50S, 1994 Recently, however, new vectors have been developed that show Bale SJ, Russell LJ, Lee ML, Compton JG, DiGiovanna JJ: Congenital recessive ichthyosis promise in achieving longer term gene expression in human epidermis unlinked to loci for epidermal transglutaminases. J Invest Dermatol 107:808–811 1994 (Deng et al, 1997). An understanding of fundamental relevant biologic Ballabio A, Shapiro LJ: Steroid sulfatase deficiency and X-linked ichthyosis. In: Scriver CR, Beaudet AL, Sly WS, Valle D (eds).The Metabolic and Molecular Bases of Inherited processes will then be important to future improvements in cutaneous Disease. McGraw-Hill, Health Professions Division, New York, 1995, p. 2999 gene transfer. Bata-Csorgo Z, Hammerberg C, Voorhees JJ, Cooper KD: Kinetics and regulation of Parallel to these advances in fundamental skin biology, new technical human keratinocyte stem cell growth in short-term primary ex vivo culture. capabilities in gene delivery are required for sustained genetic correction Cooperative growth factors from psoriatic lesional T lymphocytes stimulate of the genodermatoses. Among these are development of highly proliferation among psoriatic uninvolved, but not normal, stem keratinocytes. J Clin Invest 95:317–327, 1995 efficient vectors that can be directly administered to intact skin without Blau HM, Khavari PA:. Gene therapy. Progress, problems and prospects. Nat Med 3:13– unwanted immune hypersensitivity reactions. Current viral and nonviral 14, 1997 approaches for direct gene transfer fail to meet this criteria (Hengge Bohl D, Naffakh N, Heard JM: Long-term control of erythropoietin secretion by et al, 1995; Lu et al, 1997). An unsuccessful attempt to correct human doxycycline in mice transplanted with engineered primary myoblasts [see comments]. lamellar ichthyosis patient skin tissue by direct TGase1 expression Nat Med 3:299–305, 1997 Bonifas JM, Rothman AL, Epstein EH: Jr Epidermolysis bullosa simplex: evidence in two plasmid injection highlights some of the current challenges facing this families for keratin gene abnormalities [see comments]. Science 254:1202–1205, 1991 approach to gene therapy for genodermatoses (Choate and Khavari, Bowden PE, Haley JL, Kansky A, Rothnagel JA, Jones DO, Turner RJ: Mutation of a 1997b). The development of new vectors and the use of immune type II keratin gene (K6a) in pachyonychia congenita. Nat Genet 10:363–365, 1995 466 KHAVARI THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

Cheng J, Syder AJ, Yu QC, Letai A, Paller AS, Fuchs E: The genetic basis of epidermolytic mediated gene transfer in the nasal epithelium of patients with cystic fibrosis [see hyperkeratosis: a disorder of differentiation-specific epidermal keratin genes. Cell comments]. N Engl J Med 333:823–831, 1995 70:811–819, 1992 Korge BP, Krieg T: The molecular basis for inherited bullous diseases. J Mol Med 74:59– Choate KA, Kinsella TM, Williams ML, Nolan GP, Khavari PA: Transglutaminase 1 70, 1996 delivery to lamellar ichthyosis keratinocytes. Hum Gene Ther 7:2247–2253, 1996a Kraemer KH: Xeroderma pigmentosum knockouts. Lancet 347:278–279, 1996 Choate KA, Medalie DA, Morgan JR, Khavari PA: Corrective gene transfer in the human Kraemer KH, Seetharam S, Seidman MM, et al: Defective DNA repair in humans: clinical skin disorder lamellar ichthyosis. Nat Med 2:1263–1267, 1996b and molecular studies of xeroderma pigmentosum. Basic Life Science 53:95–104, 1990 Choate KA, Khavari PA: Sustainability of keratinocyte gene transfer and cell survival Krueger GG, Morgan JR, Jorgensen C, et al: Genetically modified skin to treat disease: in vivo. Hum Gene Ther 8:895–901, 1997a potential and limitations. J Invest Dermatol 103:76S–84S, 1994 Choate KA, Khavari PA: Direct cutaneous gene delivery in a human genetic skin disease. Larsen CP, Elwood ET, Alexander DZ, et al: Long-term acceptance of skin and cardiac Hum Gene Ther 8:1671–1677, 1997b allografts after blocking CD40 and CD28 pathways. Nature 381:434–438, 1996 Christiano AM, Greenspan DS, Hoffman GG, et al: A missense mutation in type VII Li L, Bales ES, Peterson CA, Legerski RJ: Characterization of molecular defects in collagen in two affected siblings with recessive dystrophic epidermolysis bullosa. Nat xeroderma pigmentosum group C. Nat Genet 5:413–417, 1993 Genet 4:62–66, 1993 Lu B, Federoff HJ, Wang Y, Goldsmith LA, Scott G: Topical application of viral vectors Christiano AM, Anhalt G, Gibbons S, Bauer EA, Uitto J: Premature termination codons for epidermal gene transfer. J Invest Dermatol 108:803–808, 1997 in the type VII collagen gene (COL7A1) underlie severe, mutilating recessive Maestrini E, Monaco AP, McGrath JA, et al: A molecular defect in loricrin, the major dystrophic epidermolysis bullosa. Genomics 21:160–168, 1994 component of the cornified cell envelope, underlies Vohwinkel’s syndrome. Nat Christiano AM, Pulkkinen L, Eady RA, Uitto J: Compound heterozygosity for nonsense Genet 13:70–77, 1996 and missense mutations in the LAMB3 gene in nonlethal junctional epidermolysis Marinkovich MP: The molecular genetics of basement membrane diseases. Arch Dermatol bullosa. J Invest Dermatol 106:775–777, 1996 129:1557–1565, 1993 Coulombe PA, Hutton ME, Letai A, Hebert A, Paller AS, Fuchs E: Point mutations in Marinkovich MP, Keene DR, Rimberg CS, Burgeson RE: Cellular origin of the dermal- human keratin 14 genes of epidermolysis bullosa simplex patients: genetic and epidermal basement membrane. Dev Dyn 197:255–267, 1993 functional analyses. Cell 66:1301–1311, 1991 Marionnet C, Quilliet X, Benoit A, Armier J, Sarasin A, Stary A: Recovery of normal Davis BM, Koc ON, Lee K, Gerson SL: Current progress in the gene therapy of cancer. DNA repair and mutagenesis in trichothiodystrophy cells after transduction of the Curr Opin Oncol 8:499–508, 1996 XPD human gene. Cancer Res 56:5450–5456, 1993 Deng H, Lin Q, Khavari PA: Sustainable cutaneous gene delivery. Nature Biotech 15:1–4, 1997 Mathor MB, Ferrari G, Dellambra E, Cilli M, Mavilio F, Cancedda R, De Luca M: Clonal Eady RA, Dunnill MG: Epidermolysis bullosa: hereditary skin fragility diseases as paradigms analysis of stably transduced human epidermal stem cells in culture. Proc Natl Acad in cell biology. Arch Dermatol Res 287:2–9, 1994 Sci USA 93:10371–10376, 1996 Epstein EH Jr, Williams ML, Elias PM: Steroid sulfatase, X-linked ichthyosis, and stratum McGrath JA, Gatalica B, Christiano AM, et al: Mutations in the 180-kD bullous pemphigoid corneum cell cohesion. Arch Dermatol 117:761–763, 1981 antigen (BPAG2), a hemidesmosomal transmembrane collagen (COL17A1) in Fenjves ES, Gordon DA, Pershing LK, Williams DL, Taichman LB: Systemic distribution generalized atrophic benign epidermolysis bullosa. Nat Genet 11:83–86, 1995 of apolipoprotein E secreted by grafts of epidermal keratinocytes: implications for McGrath JA, Christiano AM, Pulkkinen L, Eady RA, Uitto J: Compound heterozygosity epidermal function and gene therapy. Proc Natl Acad Sci USA 86:8803–8807, 1989 for nonsense ans missense mutations in the LAMB3 gene in nonlethal junctional Fenjves ES, Smith J, Zaradic S, Taichman LB: Systemic delivery of secreted protein by epidermolysis bullosa. J Invest Dermatol 106:1157–1159, 1996a grafts of epidermal keratinocytes: prospects for keratinocyte gene therapy. Hum Gene McGrath JA, Gatalica B, Li K, et al: Compound heterozygosity for a dominant glycine Ther 5:1241–1248, 1994 substitution and a recessive internal duplication mutation in the type XVII collagen Fenjves ES, Yao SN, Kurachi K, Taichman LB: Loss of expression of a retrovirus- gene results in junctional epidermolysis bullosa and abnormal dentition. Am J Pathol transduced gene in human keratinocytes. J Invest Dermatol 106:576–578, 1996 148:1787–1796, 1996b Freedberg IM: Keratin: a journey of three decades. J Dermatol 20:321–328, 1993 McLean WH, Rugg EL, Lunny DP, et al: Keratin 16 and keratin 17 mutations cause Freiberg RA, Choate KA, Deng H, Alperin ES, Shapiro LJ, Khavari PA: A model of pachyonychia congenita. Nat Genet 9:273–278, 1995 corrective gene transfer in X-linked ichthyosis. Human Mol Genet 6:937–933, 1997 Medalie DA, Eming SA, Tompkins RG, Yarmush ML, Krueger GG, Morgan JR: Evaluation Fuchs E: Keratins and the skin. Ann Rev Cell Dev Biol 11:123–153, 1995 of human skin reconstituted from composite grafts of cultured keratinocytes and Gagnoux-Palacios L, Vailly J, Durand-Clement M, Wagner E, Ortonne JP, Meneguzzi human acellular dermis transplanted to athymic mice. J Invest Dermatol 107:121– G: Functional Re-expression of laminin-5 in laminin-gamma2-deficient human 127, 1996 keratinocytes modifies cell morphology, motility, and adhesion. J Biol Chem Myrand SP, Topping RS, States JC: Stable transformation of xeroderma pigmentosum 271:18437–18444, 1996 group A cells with an XPA minigene restores normal DNA repair and mutagenesis Gerrard AJ, Hudson DL, Brownlee GG, Watt FM: Towards gene therapy for haemophilia of UV-treated plasmids. Carcinogenesis 17:1909–1917, 1996 B using primary human keratinocytes. Nat Genet 3:180–183, 1993 Paige DG, Emilion GG, Bouloux PM, Harper JI: A clinical and genetic study of X-linked Greenberg CS, Birckbichler PJ, Rice RH: Transglutaminases: multifunctional cross-linking recessive ichthyosis and contiguous gene defects. Br J Dermatol 131:622–629, 1994 enzymes that stabilize tissues. Faseb J 5:3071–3077, 1991 Paller AS: The genetic basis of hereditary blistering disorders. Curr Opin Pediatr 8:367– Greenhalgh DA, Rothnagel JA, Roop DR: Epidermis: an attractive target tissue for gene 371, 1996 therapy. J Invest Dermatol 103:63S–69S, 1994 Parmentier L, Blanchet-Bardon C, Nguyen S, Prud’homme JF, Dubertret L, Weissenbach Hahn H, Wicking C, Zaphiropoulous PG, et al: Mutations of the human homolog of J: Autosomal recessive lamellar ichthyosis: identification of a new mutation in Drosophila patched in the nevoid basal cell carcinoma syndrome. Cell 85:841– transglutaminase 1 and evidence for genetic heterogeneity. Hum Mol Genet 4:1391– 851, 1996 1395, 1995 Hengge UR, Chan EF, Foster RA, Walker PS, Vogel JC: Cytokine gene expression in Phillips MA, Stewart BE, Qin Q, Chakravarty R, Floyd EE, Jetten AM, Rice RH: epidermis with biological effects following injection of naked DNA. Nat Genet Primary structure of keratinocyte transglutaminase. Proc Natl Acad Sci USA 87:9333– 10:161–166, 1995 9337, 1990 Hess P: Gene therapy: a brief review. Clin Lab Med 16:197–211, 1996 Pulkkinen L, Christiano AM, Airenne T, Haakana H, Tryggvason K, Uitto J: Mutations Hilal L, Rochat A, Duquesnoy P, et al: A homozygous insertion-deletion in the type VII in the gamma 2 chain gene (LAMC2) of kalinin/laminin 5 in the junctional forms collagen gene (COL7A1) in Hallopeau-Siemens dystrophic epidermolysis bullosa. of epidermolysis bullosa. Nat Genet 6:293–297, 1994 Nat Genet 5:287–293, 1993 Raz E, Carson DA, Parker SE, et al: Intradermal gene immunization: the possible role of Hoeben RC, Migchielsen AA, van der Jagt RC, van Ormondt H, van der Eb AJ: DNA uptake in the induction of cellular immunity to viruses. Proc Natl Acad Sci Inactivation of the Moloney murine leukemia virus long terminal repeat in murine USA 91:9519–9523, 1994 fibroblast cell lines is associated with methylation and dependent on its chromosomal Reis A, Hennies HC, Langbein, et al: Keratin 9 gene mutations in epidermolytic position. J Virol 65:904–912, 1991 palmoplantar keratoderma (EPPK). Nat Genet 6:174–179, 1994 Huber M, Rettler I, Bernasconi K, et al: Mutations of keratinocyte transglutaminase in Rochat A, Kobayashi K, Barrandon Y: Location of stem cells of human hair follicles by lamellar ichthyosis. Science 267:525–528, 1995a clonal analysis. Cell 76:1063–1073, 1994 Huber M, Rettler I, Bernasconi K, Wyss M, Hohl D: Lamellar ichthyosis is genetically Rothnagel JA, Dominey AM, Dempsey LD, et al: Mutations in the rod domains of keratins heterogeneous – cases with normal keratinocyte transglutaminase. J Invest Dermatol 1 and 10 in epidermolytic hyperkeratosis. Science 257:1128–1130, 1992 105:653–654, 1995b Rothnagel JA, Traupe H, Wojcik S, et al: Mutations in the rod domain of keratin 2e in Johnson RL, Rothman AL, Xie J, et al: Human homolog of patched, a candidate gene patients with ichthyosis bullosa of Siemens. Nat Genet 7:485–490, 1994 for the basal cell nevus syndrome. Science 272:1668–1671, 1996 Russell LJ, DiGiovanna JJ, Hashem N, Compton JG, Bale SJ: Linkage of autosomal Jones PH, Watt FM: Separation of human epidermal stem cells from transit amplifying recessive lamellar ichthyosis to chromosome 14q. Am J Hum Genet 55:1146– cells on the basis of differences in integrin function and expression. Cell 73:713– 1152, 1994 724, 1993 Russell LJ, DiGiovanna JJ, Rogers GR, Steinert PM, Hashem N, Compton JG, Bale SJ: Jones PH, Harper S, Watt FM: Stem cell patterning and fate in human epidermis. Cell Mutations in the gene for transglutaminase 1 in autosomal recessive lamellar 80:83–93, 1995 ichthyosis. Nat Genet 9:279–283, 1995 Jonkman MF, Scheffer H, Stulp R, et al: Revertant mosaicism in epidermolysis bullosa Shamsher MK, Navsaria HA, Stevens HP, et al: Novel mutations in keratin 16 gene caused by mitotic gene conversion. Cell 88:543–551, 1997 underly focal non-epidermolytic palmoplantar keratoderma (NEPPK) in two families. Khavari PA. Therapeutic gene delivery to the skin. Mol Med Today 3:533–538, 1997 Hum Mol Genet 4:1875–1881, 1995 Khavari PA, Krueger GG: Cutaneous gene therapy. Dermatologic Clinics 15:27–35, 1997 Shapiro LJ, Yen P, Pomerantz D, Martin E, Rolewic L, Mohandas T: Molecular studies Kim SY, Chung SI, Steinert PM: Highly active soluble processed forms of the of deletions at the human steroid sulfatase locus. Proc Natl Acad Sci USA 86:8477– transglutaminase 1 enzyme in epidermal keratinocytes. J Biol Chem 270:18026– 8481, 1989 18035, 1995 Smith FJ, Eady RA, Leigh IM, et al: Plectin deficiency results in muscular dystrophy with Knowles MR, Hohneker KW, Zhou Z, et al: A controlled study of adenoviral-vector- epidermolysis bullosa. Nat Genet 13:450–457, 1996 VOL. 110, NO. 4 APRIL 1998 GENE THERAPY FOR GENETIC SKIN DISEASE 467

Sokol DL, Gewirtz AM: Gene therapy: basic concepts and recent advances. Crit Rev Uitto J, Pulkkinen L: Molecular complexity of the cutaneous basement membrane zone. Eukaryot Gene Expr 6:29–57, 1996 Mol Biol Rep 23:35–46, 1996 Taichman LB: Epithelial gene therapy. In: Leigh I, Lane B, Watt F (eds). The Keratinocyte Uitto J, Pulkkinen L, Christiano AM: Molecular basis of the dystrophic and junctional Handbook. Cambridge University Press, Cambridge, 1994, p. 543 forms of epidermolysis bullosa: mutations in the type VII collagen and kalinin Tamai K, Ishida-Yamamoto A, Matsuo S, et al: Compound heterozygosity for a nonsense (laminin 5) genes. J Invest Dermatol 103:39S–46S, 1994 mutation and a splice site mutation in the type VII collagen gene (COL7A1) in Vidal F, Aberdam D, Miquel C, et al: Integrin beta 4 mutations associated with junctional recessive dystrophic epidermolysis bullosa. Lab Invest 76:209–217, 1997 epidermolysis bullosa with pyloric atresia. Nat Genet 10:229–234, 1995 Vogel JC: Keratinocyte gene therapy. Arch Dermatol 129:1478–1483, 1993 Thacher SM, Rice RH, Greenberg CS, Birckbichler PJ, Rice RH: Keratinocyte-specific Williams ML, Elias PM: Genetically transmitted, generalized disorders of cornification. transglutaminase of cultured human epidermal cells: relation to cross-linked envelope The Ichthyoses Dermatol Clin 5:155–178, 1987 formation and terminal differentiation. Transglutaminases: multifunctional cross- Wolffe AP: New insights into chromatin function in transcriptional control. Faseb J 6:3354– linking enzymes that stabilize tissues. Cell 40:685–695, 1985 3361, 1992 Torchard D, Blanchet-Bardon C, Serova O, et al: Epidermolytic palmoplantar keratoderma Yen PH, Allen E, Marsh B, Mohandas T, Wang N, Taggart RT, Shapiro LJ: Cloning and cosegregates with a keratin 9 mutation in a pedigree with breast and ovarian cancer. expression of steroid sulfatase cDNA and the frequent occurrence of deletions in Nat Genet 6:106–110, 1994 STS deficiency: implications for X-Y interchange. Cell 49:443–454, 1987