- Email: [email protected]
Gene Delivery to the Hair Follicle
ORIGINAL ARTICLE
Gene Delivery to the Hair Follicle Manabu Ohyama, Jonathan C. Vogel Dermatology Branch, National Cancer Institute, National Institutes of Health
Skin and appendages such as hair follicles are attractive candidates for gene therapy targets because they are easily accessible and can be removed and genetically manipulated in culture. Hair follicles are of special interest because our understanding of hair follicle biology and pathophysiology has progressed signi¢cantly in recent years, and we now have a much better understanding of how genes, encoding transcription factors, growth factors, and cytokines regulate both hair follicle development and the cycles of hair follicle growth (anagen, catagen, and telogen) (Cotsarelis and Millar, 2001; Millar, 2002). Also important is the characterization of an increasing number of genetic mutations that a¡ect hair
growth and can result in hair loss (Cotsarelis and Millar, 2001). Gene therapy could be used to introduce genes that manipulate hair follicle growth and cycling or that replace the mutated defective gene with a normal wildtype gene. As our understanding of the polygenic basis for a number of alopecias improves, gene-based therapies might also be designed to provide more promising treatments than current palliative therapies for hair loss. This review will describe some of the recent progress in gene delivery to hair follicles and discuss examples of how gene delivery can cause phenotypic changes in hair follicles. Key words: hair follicle/gene therapy/ hair cycle/ anagen. JID Symposium Proceedings 8:204 ^206, 2003
THERAPEUTIC USES OF GENE DELIVERY
£ammatory processes (alopecia areata, AA), although long-term expression of the therapeutic gene may not always be necessary to correct hair loss in these situations. In general, treatment of polygenic hair follicle disorders will require the identi¢cation of genes able to initiate anagen or maintain normal hair follicle cycling. The possibility that genetic therapies may someday treat polygenic alopecias is based on understanding the molecular basis of hair follicle formation during development and on understanding how growth factors, cytokines, and transcription factors initiate and regulate the hair cycle. The hair follicle cycle is controlled by ‘‘signals’’ between the dermal components, such as the dermal papilla, and follicular keratinocytes. These signals are precisely regulated in terms of anatomical location (where expressed) and timing (when expressed).We are learning which genes may be able to initiate anagen onset or in£uence early anagen, including WNT, Sonic Hedgehog (SHH), and STAT3, as well as which genes can prolong or maintain the anagen phase, including FGF7 and WNT (Van Steensel et al, 2000; Cotsarelis and Millar, 2001). Genetic therapies that use these genes to initiate and maintain anagen could be useful in treating hair loss of di¡erent etiologies. Additionally, genes that could increase hair follicle size, such as SHH, or inhibiting genes controlling anagen-catagen transformation, such as FGF5, may be useful in treatment of androgenetic alopecias. Finally, if there is complete loss of hair follicles, gene therapies may be bene¢cial in initiating new hair follicle formation similar to the hair follicle formation that occurs during embryonic development (WNT, b-catenin, and others) (Van Steensel et al, 2000; Cotsarelis and Millar, 2001).
T
here are two major reasons for introducing genes into hair follicles for therapeutic purposes. The ¢rst is to treat single-gene mutations that a¡ect hair shaft growth. The second is to treat polygenic disorders of the hair follicle cycle that result in hair loss. For single-gene mutations, signi¢cant progress has been made in recent years in identifying the genes responsible for abnormal hair phenotypes in di¡erent genodermatoses. The genetic basis for an increasing number of conditions, including generalized atrichias, ectodermal dysplasias, monilethrix, Netherton’s syndrome, and Menkes disease, has been de¢ned (Cotsarelis and Millar, 2001). E¡ective phenotypic correction of hair follicle disorders due to single-gene defects will require not only long-term expression of the necessary gene but also gene expression in a high percentage of the hair follicles. Additionally, restoring a normal hair phenotype will require normal gene expression in a high percentage of the keratinocytes in each hair follicle. Achieving these goals demands e⁄cient and stable transduction of desired genes into the keratinocyte stem cells that reside in the bulge area of hair follicles and that provide progenitor cells for the di¡erent cell lineages that form the hair follicle (Taylor et al, 2000). Because the bulge stem cells also potentially give rise to epidermis and sebaceous glands (Taylor et al, 2000; Oshima et al, 2001), stable gene expression in those cells might enable us to manipulate phenotypes in those tissues. The same requirements for e⁄cient gene delivery are also important for the treatment of alopecias that have a polygenic basis (androgenetic, telogen e¥uvium, or chemotherapy-induced) and for the treatment of alopecias due to in-
GENE DELIVERY TO THE HAIR FOLLICLE
Manuscript received December 30, 2002; revised March 26, 2003; accepted for publication April 10, 2003 Reprint requests to: Jonathan C. Vogel, Dermatology Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892-1908, USA. Email: [email protected]
Once a gene(s) has been chosen to achieve a therapeutic e¡ect, it has to be e¡ectively delivered to hair follicle keratinocytes either by a direct in vivo approach or during tissue culture by an ex vivo
1087-0024/03/$15.00 . Copyright r 2003 by The Society for Investigative Dermatology, Inc. 204
VOL. 8, NO. 2 OCTOBER 2003
approach. In an in vivo approach, plasmid or viral vectors containing the gene of interest are directly delivered into follicular keratinocytes by methods such as topical application of lipoplexed DNA or a liposome mixture containing the vectors; direct intradermal injection of vectors; or gene gun introduction of vectors into the hair follicle (Vogel, 2000). Although in vivo approaches are simple and direct, usually only transient expression is achievable by them. In contrast, in ex vivo approaches, desired genes are introduced during ex vivo culture. For gene therapy of hair follicles, ex vivo approaches would involve gene introduction into ex vivo organ cultures of hair follicles or into keratinocytes that would be reconstituted and bioengineered to form hair follicles during tissue culture prior to engraftment. Although such ex vivo approaches are more technically demanding, they should be superior in achieving long-term gene expression because keratinocyte stem cells or progenitor cells can be manipulated and e⁄ciently targeted for gene introduction. USES OF HAIR FOLLICLE GENE THERAPYPRESENT AND FUTURE
Hair follicle gene therapy is a relatively new ¢eld of endeavor, but several relevant examples were recently described, beginning with a demonstration that DNA coated with cationic lipid mixtures can be topically applied and taken up by mouse hair follicles (Li and Ho¡man, 1995). Subsequent studies further characterized gene uptake by both mouse and human hair follicles following topical application, and demonstrated how this uptake could be optimized (Domashenko et al, 2000). After ¢rst determining the optimal cationic lipid-to-DNA ratio in the topically applied lipoplexed plasmid DNA expressing a b-galactosidase indicator gene, researchers demonstrated that optimal transfection of matrix keratinocytes occurred when the lipoplexed DNA was applied within three days of anagen onset and that up to 73% of hair follicles could be transfected (Domashenko et al, 2000). These results suggest that the timing of hair follicle gene therapy to anagen onset is an important consideration and that matrix keratinocytes in the leading edge of epithelial migration are preferentially transfected. Although occasional keratinocytes in the bulge stem cell area were transfected, transient gene expression would be anticipated with this in vivo approach. Another possible in vivo approach for gene delivery to hair follicles is by intradermal injection. Either viral vectors or plasmid DNA vectors could be introduced into super¢cial dermis of hairbearing skin in this way. In one study using this approach, adenoviral vectors expressing an SHH gene were injected into mouse skin during the telogen (resting) phase (Sato et al, 1999). As noted above, SHH plays an important role in both hair follicle development and normal hair follicle cycling and is normally expressed in matirix keratinocytes during anagen onset. The researchers wanted to determine if the introduced SHH gene could induce early anagen onset in telogen hair follicles. This study is important because it represents the ¢rst use of hair follicle gene therapy to exert a biological e¡ect and alter the phenotypic behavior of hair follicles. Mice injected intradermally with the SHH-adenoviral vector expressed SHH in diverse cell types (kerationcytes, ¢broblasts, mesenchymal cells) and showed characteristics of early anagen onset with early melanogenesis, increased hair follicle size, and accelerated hair growth in the treated area. In a subsequent study, adenoviral delivery of SHH also induced hair regrowth in a mouse model of chemotherapy-induced alopecia (Sato et al, 2001). These ¢ndings not only indicate that SHH may function as a ‘‘switch’’ for initiating hair growth in alopecia but also suggest the possibility that the hair cycle could be modulated with introduced genes. In vivo gene therapy approaches might someday be used to correct single-point mutations in genes that result in phenotypic hair abnormalities. One example of this approach utilized RNADNA oligonucleotides designed to correct a point mutation in
GENE DELIVERY TO THE HAIR FOLLICLE
205
the tyrosinase gene, introducing them either topically or intradermally into the skin of albino mice during anagen onset (Alexeev et al, 2000). The goal was for these oligonucleotides to be taken up by melanocytes and to correct one copy of the mutant tyrosinase gene allele. Although the mechanism of repair remains to be de¢ned, the oligonucleotide containing the wild-type tyrosinase sequence presumably hybridizes to the mutant tyrosinase gene, and the resulting mismatch at the point mutation is recognized by the cell and repaired with the correct base, yielding the normal tyrosinase allele. Hair follicles that contained a su⁄cient number of melanocytes with corrected tyrosinase genes demonstrated tyrosinase activity and pigmented hair shafts. Corrected hair follicles were demonstrated to stably maintain their phenotype for up to ¢ve months. E⁄ciency of phenotypic correction was low, however, and the future feasibility of this approach will require improved e⁄ciency of gene targeting. Recently, one of the ¢rst examples of ex vivo gene delivery to hair follicles using GFP-expressing adenoviral vectors was reported (Saito et al, 2002). In this study, the GFP gene was delivered to mouse hair follicles by adenoviral vectors during ex vivo organ culture. After collagenase treatment to obtain better penetration of the GFP-adenoviral vectors into root sheath keratinocytes of the hair follicle, a high percentage of hair follicles (up to 79%) were successfully transduced with GFP genes. These GFP-transduced hair follicles were then grafted back onto mice, and in vivo GFP expression was observed in hair matrix keratinocytes and in the shaft of the grafted hair follicles. Although this ex vivo approach holds great promise to e⁄ciently deliver genes to keratinocyte progenitor cells and to achieve long-term expression, the adenoviral vectors used in it are expected to result in transient expression. Gene therapy of the hair follicle is at an early stage of investigation, but these studies clearly demonstrate that genes could be delivered to hair follicles directly in vivo and during ex vivo culture, providing a theoretical basis for hair follicle gene therapy.We also now know how topical gene delivery is optimally performed during early anagen onset because of optimal access to follicular (matrix) keratinocytes. Importantly, these investigations indicate that manipulating the hair cycle to induce anagen is possible with genes expressing transcription factors, growth factors, or cytokines. In spite of the many challenging hurdles that remain to be overcome before genes can be e⁄ciently delivered to hair follicles for therapeutic purposes, the studies described above indicate that progress is being made. Successful gene therapy of hair follicles not only will be useful for treating hair diseases but will also help provide a understanding of hair follicle biology. MO receives ¢nancial support from the Uehara Memorial Foundation.
REFERENCES Alexeev V, Igoucheva O, Domashenko A, Cotsarelis G, Yoon K: Localized in vivo genotypic and phenotypic correction of the albino mutation in skin by RNA-DNA oligonucleotide. Nat Biotechnol 18:43^47, 2000 Cotsarelis G, Millar SE: Towards a molecular understanding of hair loss and its treatment. Trends Mol Med 7:293^301, 2001 Domashenko A, Gupta S, Cotsarelis G: E⁄cient delivery of transgenes to human hair follicle progenitor cells using topical lipoplex. Nat Biotechnol 18:420^423, 2000 Li L, Ho¡man RM: The feasibility of targeted selective gene therapy of the hair follicle. Nat Med 1:705^706, 1995 Millar SE: Molecular mechanisms regulating hair follicle development. J Invest Dermatol 118:216^225, 2002 Oshima H, Rochat A, Kedzia C, Kobayashi K, Barrandon Y: Morphogenesis and renewal of hair follicles from adult multipotent stem cells. Cell 104:233^245, 2001 Saito N, Zhao M, Li L, et al: High e⁄ciency genetic modi¢cation of hair follicles and growing hair shafts. Proc Natl Acad Sci USA 99:13120^13124, 2002
206
OHYAMA AND VOGEL
Sato N, Leopold PL, Crystal RG: E¡ect of adenovirus-mediated expression of Sonic hedgehog gene on hair regrowth in mice with chemotherapy-induced alopecia. J Natl Cancer Inst 93:1858^64, 2001 Sato N, Leopold PL, Crystal RG: Induction of the hair growth phase in postnatal mice by localized transient expression of Sonic hedgehog. J Clin Invest 104:855^864, 1999
JID SYMPOSIUM PROCEEDINGS
Taylor G, Lehrer MS, Jensen PJ, Sun TT, Lavker RM: Involvement of follicular stem cells in forming not only the follicle but also the epidermis. Cell 102:451^461, 2000 Van Steensel MA, Happle R, Steijlen PM: Molecular genetics of the hair follicle: The state of the art. Proc Soc Exp Biol Med 223:1^7, 2000 Vogel JC: Nonviral skin gene therapy. Hum GeneTher 11:2253^2259, 2000
Gene Delivery to the Hair Follicle Manabu Ohyama, Jonathan C. Vogel Dermatology Branch, National Cancer Institute, National Institutes of Health
Skin and appendages such as hair follicles are attractive candidates for gene therapy targets because they are easily accessible and can be removed and genetically manipulated in culture. Hair follicles are of special interest because our understanding of hair follicle biology and pathophysiology has progressed signi¢cantly in recent years, and we now have a much better understanding of how genes, encoding transcription factors, growth factors, and cytokines regulate both hair follicle development and the cycles of hair follicle growth (anagen, catagen, and telogen) (Cotsarelis and Millar, 2001; Millar, 2002). Also important is the characterization of an increasing number of genetic mutations that a¡ect hair
growth and can result in hair loss (Cotsarelis and Millar, 2001). Gene therapy could be used to introduce genes that manipulate hair follicle growth and cycling or that replace the mutated defective gene with a normal wildtype gene. As our understanding of the polygenic basis for a number of alopecias improves, gene-based therapies might also be designed to provide more promising treatments than current palliative therapies for hair loss. This review will describe some of the recent progress in gene delivery to hair follicles and discuss examples of how gene delivery can cause phenotypic changes in hair follicles. Key words: hair follicle/gene therapy/ hair cycle/ anagen. JID Symposium Proceedings 8:204 ^206, 2003
THERAPEUTIC USES OF GENE DELIVERY
£ammatory processes (alopecia areata, AA), although long-term expression of the therapeutic gene may not always be necessary to correct hair loss in these situations. In general, treatment of polygenic hair follicle disorders will require the identi¢cation of genes able to initiate anagen or maintain normal hair follicle cycling. The possibility that genetic therapies may someday treat polygenic alopecias is based on understanding the molecular basis of hair follicle formation during development and on understanding how growth factors, cytokines, and transcription factors initiate and regulate the hair cycle. The hair follicle cycle is controlled by ‘‘signals’’ between the dermal components, such as the dermal papilla, and follicular keratinocytes. These signals are precisely regulated in terms of anatomical location (where expressed) and timing (when expressed).We are learning which genes may be able to initiate anagen onset or in£uence early anagen, including WNT, Sonic Hedgehog (SHH), and STAT3, as well as which genes can prolong or maintain the anagen phase, including FGF7 and WNT (Van Steensel et al, 2000; Cotsarelis and Millar, 2001). Genetic therapies that use these genes to initiate and maintain anagen could be useful in treating hair loss of di¡erent etiologies. Additionally, genes that could increase hair follicle size, such as SHH, or inhibiting genes controlling anagen-catagen transformation, such as FGF5, may be useful in treatment of androgenetic alopecias. Finally, if there is complete loss of hair follicles, gene therapies may be bene¢cial in initiating new hair follicle formation similar to the hair follicle formation that occurs during embryonic development (WNT, b-catenin, and others) (Van Steensel et al, 2000; Cotsarelis and Millar, 2001).
T
here are two major reasons for introducing genes into hair follicles for therapeutic purposes. The ¢rst is to treat single-gene mutations that a¡ect hair shaft growth. The second is to treat polygenic disorders of the hair follicle cycle that result in hair loss. For single-gene mutations, signi¢cant progress has been made in recent years in identifying the genes responsible for abnormal hair phenotypes in di¡erent genodermatoses. The genetic basis for an increasing number of conditions, including generalized atrichias, ectodermal dysplasias, monilethrix, Netherton’s syndrome, and Menkes disease, has been de¢ned (Cotsarelis and Millar, 2001). E¡ective phenotypic correction of hair follicle disorders due to single-gene defects will require not only long-term expression of the necessary gene but also gene expression in a high percentage of the hair follicles. Additionally, restoring a normal hair phenotype will require normal gene expression in a high percentage of the keratinocytes in each hair follicle. Achieving these goals demands e⁄cient and stable transduction of desired genes into the keratinocyte stem cells that reside in the bulge area of hair follicles and that provide progenitor cells for the di¡erent cell lineages that form the hair follicle (Taylor et al, 2000). Because the bulge stem cells also potentially give rise to epidermis and sebaceous glands (Taylor et al, 2000; Oshima et al, 2001), stable gene expression in those cells might enable us to manipulate phenotypes in those tissues. The same requirements for e⁄cient gene delivery are also important for the treatment of alopecias that have a polygenic basis (androgenetic, telogen e¥uvium, or chemotherapy-induced) and for the treatment of alopecias due to in-
GENE DELIVERY TO THE HAIR FOLLICLE
Manuscript received December 30, 2002; revised March 26, 2003; accepted for publication April 10, 2003 Reprint requests to: Jonathan C. Vogel, Dermatology Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892-1908, USA. Email: [email protected]
Once a gene(s) has been chosen to achieve a therapeutic e¡ect, it has to be e¡ectively delivered to hair follicle keratinocytes either by a direct in vivo approach or during tissue culture by an ex vivo
1087-0024/03/$15.00 . Copyright r 2003 by The Society for Investigative Dermatology, Inc. 204
VOL. 8, NO. 2 OCTOBER 2003
approach. In an in vivo approach, plasmid or viral vectors containing the gene of interest are directly delivered into follicular keratinocytes by methods such as topical application of lipoplexed DNA or a liposome mixture containing the vectors; direct intradermal injection of vectors; or gene gun introduction of vectors into the hair follicle (Vogel, 2000). Although in vivo approaches are simple and direct, usually only transient expression is achievable by them. In contrast, in ex vivo approaches, desired genes are introduced during ex vivo culture. For gene therapy of hair follicles, ex vivo approaches would involve gene introduction into ex vivo organ cultures of hair follicles or into keratinocytes that would be reconstituted and bioengineered to form hair follicles during tissue culture prior to engraftment. Although such ex vivo approaches are more technically demanding, they should be superior in achieving long-term gene expression because keratinocyte stem cells or progenitor cells can be manipulated and e⁄ciently targeted for gene introduction. USES OF HAIR FOLLICLE GENE THERAPYPRESENT AND FUTURE
Hair follicle gene therapy is a relatively new ¢eld of endeavor, but several relevant examples were recently described, beginning with a demonstration that DNA coated with cationic lipid mixtures can be topically applied and taken up by mouse hair follicles (Li and Ho¡man, 1995). Subsequent studies further characterized gene uptake by both mouse and human hair follicles following topical application, and demonstrated how this uptake could be optimized (Domashenko et al, 2000). After ¢rst determining the optimal cationic lipid-to-DNA ratio in the topically applied lipoplexed plasmid DNA expressing a b-galactosidase indicator gene, researchers demonstrated that optimal transfection of matrix keratinocytes occurred when the lipoplexed DNA was applied within three days of anagen onset and that up to 73% of hair follicles could be transfected (Domashenko et al, 2000). These results suggest that the timing of hair follicle gene therapy to anagen onset is an important consideration and that matrix keratinocytes in the leading edge of epithelial migration are preferentially transfected. Although occasional keratinocytes in the bulge stem cell area were transfected, transient gene expression would be anticipated with this in vivo approach. Another possible in vivo approach for gene delivery to hair follicles is by intradermal injection. Either viral vectors or plasmid DNA vectors could be introduced into super¢cial dermis of hairbearing skin in this way. In one study using this approach, adenoviral vectors expressing an SHH gene were injected into mouse skin during the telogen (resting) phase (Sato et al, 1999). As noted above, SHH plays an important role in both hair follicle development and normal hair follicle cycling and is normally expressed in matirix keratinocytes during anagen onset. The researchers wanted to determine if the introduced SHH gene could induce early anagen onset in telogen hair follicles. This study is important because it represents the ¢rst use of hair follicle gene therapy to exert a biological e¡ect and alter the phenotypic behavior of hair follicles. Mice injected intradermally with the SHH-adenoviral vector expressed SHH in diverse cell types (kerationcytes, ¢broblasts, mesenchymal cells) and showed characteristics of early anagen onset with early melanogenesis, increased hair follicle size, and accelerated hair growth in the treated area. In a subsequent study, adenoviral delivery of SHH also induced hair regrowth in a mouse model of chemotherapy-induced alopecia (Sato et al, 2001). These ¢ndings not only indicate that SHH may function as a ‘‘switch’’ for initiating hair growth in alopecia but also suggest the possibility that the hair cycle could be modulated with introduced genes. In vivo gene therapy approaches might someday be used to correct single-point mutations in genes that result in phenotypic hair abnormalities. One example of this approach utilized RNADNA oligonucleotides designed to correct a point mutation in
GENE DELIVERY TO THE HAIR FOLLICLE
205
the tyrosinase gene, introducing them either topically or intradermally into the skin of albino mice during anagen onset (Alexeev et al, 2000). The goal was for these oligonucleotides to be taken up by melanocytes and to correct one copy of the mutant tyrosinase gene allele. Although the mechanism of repair remains to be de¢ned, the oligonucleotide containing the wild-type tyrosinase sequence presumably hybridizes to the mutant tyrosinase gene, and the resulting mismatch at the point mutation is recognized by the cell and repaired with the correct base, yielding the normal tyrosinase allele. Hair follicles that contained a su⁄cient number of melanocytes with corrected tyrosinase genes demonstrated tyrosinase activity and pigmented hair shafts. Corrected hair follicles were demonstrated to stably maintain their phenotype for up to ¢ve months. E⁄ciency of phenotypic correction was low, however, and the future feasibility of this approach will require improved e⁄ciency of gene targeting. Recently, one of the ¢rst examples of ex vivo gene delivery to hair follicles using GFP-expressing adenoviral vectors was reported (Saito et al, 2002). In this study, the GFP gene was delivered to mouse hair follicles by adenoviral vectors during ex vivo organ culture. After collagenase treatment to obtain better penetration of the GFP-adenoviral vectors into root sheath keratinocytes of the hair follicle, a high percentage of hair follicles (up to 79%) were successfully transduced with GFP genes. These GFP-transduced hair follicles were then grafted back onto mice, and in vivo GFP expression was observed in hair matrix keratinocytes and in the shaft of the grafted hair follicles. Although this ex vivo approach holds great promise to e⁄ciently deliver genes to keratinocyte progenitor cells and to achieve long-term expression, the adenoviral vectors used in it are expected to result in transient expression. Gene therapy of the hair follicle is at an early stage of investigation, but these studies clearly demonstrate that genes could be delivered to hair follicles directly in vivo and during ex vivo culture, providing a theoretical basis for hair follicle gene therapy.We also now know how topical gene delivery is optimally performed during early anagen onset because of optimal access to follicular (matrix) keratinocytes. Importantly, these investigations indicate that manipulating the hair cycle to induce anagen is possible with genes expressing transcription factors, growth factors, or cytokines. In spite of the many challenging hurdles that remain to be overcome before genes can be e⁄ciently delivered to hair follicles for therapeutic purposes, the studies described above indicate that progress is being made. Successful gene therapy of hair follicles not only will be useful for treating hair diseases but will also help provide a understanding of hair follicle biology. MO receives ¢nancial support from the Uehara Memorial Foundation.
REFERENCES Alexeev V, Igoucheva O, Domashenko A, Cotsarelis G, Yoon K: Localized in vivo genotypic and phenotypic correction of the albino mutation in skin by RNA-DNA oligonucleotide. Nat Biotechnol 18:43^47, 2000 Cotsarelis G, Millar SE: Towards a molecular understanding of hair loss and its treatment. Trends Mol Med 7:293^301, 2001 Domashenko A, Gupta S, Cotsarelis G: E⁄cient delivery of transgenes to human hair follicle progenitor cells using topical lipoplex. Nat Biotechnol 18:420^423, 2000 Li L, Ho¡man RM: The feasibility of targeted selective gene therapy of the hair follicle. Nat Med 1:705^706, 1995 Millar SE: Molecular mechanisms regulating hair follicle development. J Invest Dermatol 118:216^225, 2002 Oshima H, Rochat A, Kedzia C, Kobayashi K, Barrandon Y: Morphogenesis and renewal of hair follicles from adult multipotent stem cells. Cell 104:233^245, 2001 Saito N, Zhao M, Li L, et al: High e⁄ciency genetic modi¢cation of hair follicles and growing hair shafts. Proc Natl Acad Sci USA 99:13120^13124, 2002
206
OHYAMA AND VOGEL
Sato N, Leopold PL, Crystal RG: E¡ect of adenovirus-mediated expression of Sonic hedgehog gene on hair regrowth in mice with chemotherapy-induced alopecia. J Natl Cancer Inst 93:1858^64, 2001 Sato N, Leopold PL, Crystal RG: Induction of the hair growth phase in postnatal mice by localized transient expression of Sonic hedgehog. J Clin Invest 104:855^864, 1999
JID SYMPOSIUM PROCEEDINGS
Taylor G, Lehrer MS, Jensen PJ, Sun TT, Lavker RM: Involvement of follicular stem cells in forming not only the follicle but also the epidermis. Cell 102:451^461, 2000 Van Steensel MA, Happle R, Steijlen PM: Molecular genetics of the hair follicle: The state of the art. Proc Soc Exp Biol Med 223:1^7, 2000 Vogel JC: Nonviral skin gene therapy. Hum GeneTher 11:2253^2259, 2000