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• W2001569366 abstract "Inherited skin blistering conditions collectively named epidermolysis bullosa (EB) cause significant morbidity and mortality due to the compromise of the skin's barrier function, the pain of blisters, inflammation, and in some cases scaring and cancer. The simplex form of EB is usually caused by dominantly inherited mutations in KRT5 or KRT14. These mutations result in the production of proteins with dominant-negative activity that disrupt polymerization of intermediate filaments in the basal keratinocyte layer and result in a weak epidermal–dermal junction. The genome of adeno-associated virus (AAV) vectors can recombine with chromosomal sequence so that mutations can be corrected, or production of proteins with dominant-negative activity can be disrupted. We demonstrate a clinically feasible strategy for efficient targeting of the KRT14 gene in normal and EB-affected human keratinocytes. Using a gene-targeting vector with promoter trap design, targeted alteration of one allele of KRT14 occurred in 100% of transduced cells and transduction frequencies ranged from 0.1 to 0.6% of total cells. EBS patient keratinocytes with precise modifications of the mutant allele are preferentially recovered from targeted cell populations. Single epidermal stem cell clones produced histologically normal skin grafts after transplantation to athymic mice and could generate a sufficient number of cells to transplant the entire skin surface of an individual. Inherited skin blistering conditions collectively named epidermolysis bullosa (EB) cause significant morbidity and mortality due to the compromise of the skin's barrier function, the pain of blisters, inflammation, and in some cases scaring and cancer. The simplex form of EB is usually caused by dominantly inherited mutations in KRT5 or KRT14. These mutations result in the production of proteins with dominant-negative activity that disrupt polymerization of intermediate filaments in the basal keratinocyte layer and result in a weak epidermal–dermal junction. The genome of adeno-associated virus (AAV) vectors can recombine with chromosomal sequence so that mutations can be corrected, or production of proteins with dominant-negative activity can be disrupted. We demonstrate a clinically feasible strategy for efficient targeting of the KRT14 gene in normal and EB-affected human keratinocytes. Using a gene-targeting vector with promoter trap design, targeted alteration of one allele of KRT14 occurred in 100% of transduced cells and transduction frequencies ranged from 0.1 to 0.6% of total cells. EBS patient keratinocytes with precise modifications of the mutant allele are preferentially recovered from targeted cell populations. Single epidermal stem cell clones produced histologically normal skin grafts after transplantation to athymic mice and could generate a sufficient number of cells to transplant the entire skin surface of an individual. Epidermolysis bullosa (EB) is the term used to describe a group of inherited skin diseases that exhibit frequent blistering as the primary phenotype.1Uitto J Richard G Progress in epidermolysis bullosa: genetic classification and clinical implications.Am J Med Genet C Semin Med Genet. 2004; 131C: 61-74Crossref PubMed Scopus (78) Google Scholar,2Fine JD Eady RA Bauer EA Bauer JW Bruckner-Tuderman L Heagerty A et al.The classification of inherited epidermolysis bullosa (EB): Report of the Third International Consensus Meeting on Diagnosis and Classification of EB.J Am Acad Dermatol. 2008; 58: 931-950Abstract Full Text Full Text PDF PubMed Scopus (697) Google Scholar The group is further divided into dystrophic, junctional, hemidesmosomal, and simplex subtypes based on the cleavage plane of the blister and the affected gene. With the exception of the simplex form, most EB is inherited in an autosomal recessive pattern. EB simplex (EBS) is caused by KRT5 and KRT14 mutations that usually result in proteins with dominant-negative activity3Bonifas JM Rothman AL Epstein Jr EH Epidermolysis bullosa simplex: evidence in two families for keratin gene abnormalities.Science. 1991; 254: 1202-1205Crossref PubMed Scopus (342) Google Scholar,4Coulombe PA Hutton ME Letai A Hebert A Paller AS Fuchs E Point mutations in human keratin 14 genes of epidermolysis bullosa simplex patients: genetic and functional analyses.Cell. 1991; 66: 1301-1311Abstract Full Text PDF PubMed Scopus (524) Google Scholar and cause abnormal polymerization of intermediate filaments within the basal keratinocyte layer.5Livingston RJ Sybert VP Smith LT Dale BA Presland RB Stephens K Expression of a truncated keratin 5 may contribute to severe palmar–plantar hyperkeratosis in epidermolysis bullosa simplex patients.J Invest Dermatol. 2001; 116: 970-974Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar Mutational hotspots exist in both KRT5 and KRT14 such that 70% of affected individuals have mutations in one of five locations.6Ehrlich P Sybert VP Spencer A Stephens K A common keratin 5 gene mutation in epidermolysis bullosa simplex–Weber-Cockayne.J Invest Dermatol. 1995; 104: 877-879Abstract Full Text PDF PubMed Scopus (28) Google Scholar,7Stephens K Sybert VP Wijsman EM Ehrlich P Spencer A A keratin 14 mutational hot spot for epidermolysis bullosa simplex, Dowling-Meara: implications for diagnosis.J Invest Dermatol. 1993; 101: 240-243Crossref PubMed Scopus (61) Google Scholar EBS symptoms usually manifest at birth with erythema, widespread blistering, and areas of denuded skin.8Pfender EG Bruckner AL Epidermolysis Bullosa Simplex. In: GeneReviews at Gene Tests: Medical Genetics Information Resource (database online). Copyright, University of Washington, Seattle2008: 1997-2010Google Scholar Secondary complications arise as a result of recurrent blistering and include skin infections, sepsis, nail dystrophy, and pigmentary changes. Current treatment strategies are limited to the use of shoes and clothing that minimize blister formation, lancing of blisters, and prompt treatment of cellulitis with antibiotics.8Pfender EG Bruckner AL Epidermolysis Bullosa Simplex. In: GeneReviews at Gene Tests: Medical Genetics Information Resource (database online). Copyright, University of Washington, Seattle2008: 1997-2010Google Scholar The EBs are a promising category of disease targets for gene therapy strategies because epidermal stem cells reside abundantly in the skin, can be cultured ex vivo, and autologous transplants of modified cells can be performed. Skin tissue contains two types of stem cells: follicular stem cells that reside in the lateral bulge of the hair follicle and contribute to the hair follicle cycle and wound repair,9Ito M Liu Y Yang Z Nguyen J Liang F Morris RJ et al.Stem cells in the hair follicle bulge contribute to wound repair but not to homeostasis of the epidermis.Nat Med. 2005; 11: 1351-1354Crossref PubMed Scopus (975) Google Scholar and interfollicular stem cells that are located in the basal layer of the epidermis. Interfollicular stem cells contribute mainly to homeostasis, renewing the epidermis as layers are continually sloughed.10Jones PH Watt FM Separation of human epidermal stem cells from transit amplifying cells on the basis of differences in integrin function and expression.Cell. 1993; 73: 713-724Abstract Full Text PDF PubMed Scopus (986) Google Scholar In the mid 1970s, conditions were established to grow interfollicular stem cells in culture allowing them to be studied directly, and used therapeutically for the treatment of full thickness burns.11Gallico 3rd GG O'Connor NE Compton CC Kehinde O Green H Permanent coverage of large burn wounds with autologous cultured human epithelium.N Engl J Med. 1984; 311: 448-451Crossref PubMed Scopus (1095) Google Scholar,12Rheinwald JG Green H Serial cultivation of strains of human epidermal keratinocytes: the formation of keratinizing colonies from single cells.Cell. 1975; 6: 331-343Abstract Full Text PDF PubMed Scopus (3835) Google Scholar In an elegant set of experiments, the same group defined three colony phenotypes that arise from single cells in culture, and described them by colony morphology and size.13Barrandon Y Green H Three clonal types of keratinocyte with different capacities for multiplication.Proc Natl Acad Sci USA. 1987; 84: 2302-2306Crossref PubMed Scopus (1076) Google Scholar Cells that give rise to colonies with a “holoclone” phenotype come from the interfollicular stem cell niche and contain small undifferentiated and highly proliferative cells that can be grown almost indefinitely.14Mathor MB Ferrari G Dellambra E Cilli M Mavilio F Cancedda R et al.Clonal analysis of stably transduced human epidermal stem cells in culture.Proc Natl Acad Sci USA. 1996; 93: 10371-10376Crossref PubMed Scopus (151) Google Scholar In a recent successful gene therapy clinical trial for the treatment of recessively inherited junctional EB, cells with a holoclone phenotype were found in the palms and a few unaffected areas of the 36-year-old patient and used to generate skin equivalents for disease treatment.15Mavilio F Pellegrini G Ferrari S Di Nunzio F Di Iorio E Recchia A et al.Correction of junctional epidermolysis bullosa by transplantation of genetically modified epidermal stem cells.Nat Med. 2006; 12: 1397-1402Crossref PubMed Scopus (496) Google Scholar Gene therapy for recessively inherited conditions can be accomplished by adding another copy of the mutated gene; however, genetic disorders such as EBS are resistant to this approach because of the dominant-negative activity of proteins produced by the mutated gene. Therefore, a gene-targeting approach was used to model treatment strategies for EBS. This approach has not been applied therapeutically due to limitations of efficiency, and the inability to generate large numbers of modified cells for transplantation. Adeno-associated virus (AAV)–mediated gene targeting is an efficient method of gene alteration used in primary human cell culture, and offers a safe, effective, and permanent treatment for individuals with EBS. The observation of mutation hot spots in KRT5 and KRT14 suggests that the construction of a few gene-targeting vectors could treat the cells of multiple individuals from different families, simplifying the therapeutic approach in this patient group. Techniques for keratinocyte culture, stratification on artificial matrices, and successful transplantation of skin equivalents to human recipients have been established.16Nanchahal J Dover R Otto WR Allogeneic skin substitutes applied to burns patients.Burns. 2002; 28: 254-257Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar Modification of cells by AAV-mediated gene targeting before transplantation represents the final challenge for affecting a gene therapy strategy to treat this dominantly inherited condition and would allow modified cells to be incorporated into existing autologous transplantation protocols. We demonstrate efficient targeting of KRT14, the ability to recover KRT14-targeted stem cells with a holoclone phenotype, and transplantation of skin equivalents made from either targeted holoclones, or polyclonal populations of modified cells. Using this clinically feasible approach, human skin equivalents functioned normally after transplantation by providing a barrier to water loss and infection and had a histologically normal appearance. This is the first report that demonstrates both efficient transduction of human keratinocytes by AAV vectors, and efficient purification and functional characterization of keratinocyte populations containing targeted genetic alterations. The number of keratinocytes can be expanded in culture so that clinically significant skin grafts can be generated from modified cells. These results form the basis of future studies that should allow clinical application of this strategy for the treatment of EBS or other EB types where no effective treatments have been found. Gene-targeting approaches require efficient infection of cells so that sufficient numbers of independent transduction events due to homologous recombination can be collected for analysis and ultimately for therapeutic use. Human keratinocytes are resistant to transduction by AAV vectors pseudotyped with type 2 capsid serotypes.17Braun-Falco M Eisenried A Büning H Ring J Recombinant adeno-associated virus type 2-mediated gene transfer into human keratinocytes is influenced by both the ubiquitin/proteasome pathway and epidermal growth factor receptor tyrosine kinase.Arch Dermatol Res. 2005; 296: 528-535Crossref PubMed Scopus (15) Google Scholar,18Gagnoux-Palacios L Hervouet C Spirito F Roques S Mezzina M Danos O et al.Assessment of optimal transduction of primary human skin keratinocytes by viral vectors.J Gene Med. 2005; 7: 1178-1186Crossref PubMed Scopus (26) Google Scholar We generated AAV vector preparations using a plasmid containing the human placental alkaline phosphatase reporter and packaging plasmids containing capsid genes from serotypes 2 and 6. Transduction efficiencies were compared in human keratinocytes, or human HT1080 cells to determine whether a type 6 capsid serotype allows efficient transduction of normal human keratinocytes. Vectors pseudotyped with capsids from serotype 2 isolates transduced human keratinocytes poorly when transduction efficiencies in keratinocytes were compared with efficiencies in HT1080 cells. However, transduction frequencies increased by 5 logs when the same vector was packaged with capsids from a serotype 6 isolate(Figure 1). Permanent transduction of replicating cells by AAV vectors occurs by integration of vector genomes at sites of double-strand break repair,19Miller DG Petek LM Russell DW Adeno-associated virus vectors integrate at chromosome breakage sites.Nat Genet. 2004; 36: 767-773Crossref PubMed Scopus (172) Google Scholar or by homologous recombination of vector and chromosomal sequences.20Russell DW Hirata RK Human gene targeting by viral vectors.Nat Genet. 1998; 18: 325-330Crossref PubMed Scopus (262) Google Scholar Because vector integration at random genomic locations occurs in ~3–10% of cells at high infection multiplicities,21Hirata R Chamberlain J Dong R Russell DW Targeted transgene insertion into human chromosomes by adeno-associated virus vectors.Nat Biotechnol. 2002; 20: 735-738Crossref PubMed Scopus (141) Google Scholar homologous recombination usually represents a fraction of total transduction events. A number of strategies have been developed to enhance detection of transduction events that occur by homologous recombination while ignoring transduction that occurs as a result of integration at random genomic locations. Vector designs that require promoter trapping for expression of marker genes can shift the balance of detection toward recombinants because most integration at random locations does not trap the activity of an active promoter.22Gossler A Joyner AL Rossant J Skarnes WC Mouse embryonic stem cells and reporter constructs to detect developmentally regulated genes.Science. 1989; 244: 463-465Crossref PubMed Scopus (393) Google Scholar A promoterless gene expression cassette containing an internal ribosomal entry site was designed to result in the disruption of KRT14 transcription by insertion into exon 3 of KRT14. GFP expression results from the activity of the KRT14 promoter allowing detection of cells containing targeted insertions. GFP expression resulting from integration at random locations requires the relatively rare event of insertion of the cassette into an exon of an actively transcribed gene (Figure 2). The KRT14 gene-targeting vector (Figure 2) was packaged with capsid proteins from a serotype 6 isolate23Gregorevic P Blankinship MJ Allen JM Crawford RW Meuse L Miller DG et al.Systemic delivery of genes to striated muscles using adeno-associated viral vectors.Nat Med. 2004; 10: 828-834Crossref PubMed Scopus (524) Google Scholar and the percentage of GFP expressing normal human keratinocytes was determined by flow cytometry 7 days after infection of human keratinocytes. Transduction frequencies were typical of other targeting vectors with promoter trap design,21Hirata R Chamberlain J Dong R Russell DW Targeted transgene insertion into human chromosomes by adeno-associated virus vectors.Nat Biotechnol. 2002; 20: 735-738Crossref PubMed Scopus (141) Google Scholar ranged from 0.1 to 0.6% of total cells, and increased with increasing multiplicity of infection (Figure 3). GFP positive cells transduced with the AAVK14e3IFsA vector were collected using fluorescence-activated cell sorting and placed in cell culture dishes at limiting dilution. Keratinocyte colonies generated from single cells were ring cloned and expanded for analysis by genomic Southern, a colony forming efficiency assay, and transplantation of skin equivalents to athymic mice. Out of 30 GFP positive clones, 25 grew sufficiently for DNA analysis and all 25 clones contained a targeted insertion of the vector within exon 3 of the KRT14 gene as evidenced by a shift in size of the BamHI fragment from 10.9 to 5.9 kb on Southern blots hybridized with the KRT14 probe (Figure 4a,c). Analysis of the KRT14 target site in three clones revealed a 9.1-kb vector–chromosome junction fragment (Figure 4a, clones 7, 16, and 18). The same blot was stripped of the KRT14 probe and hybridized with a DNA fragment from the GFP gene. A common size fragment was detected in genomic DNA from each of the targeted clones with the exception of clones 7, 16, and 18 where two GFP-hybridizing fragments of 4.6 and 9.1 kb molecular weight were observed (Figure 4b). DNA from two additional clones showed hybridization to an additional GFP-containing fragment (Figure 4b, clones 8 and 21). To determine whether the additional signals result from vector integration at random locations within the genome, genomic DNA from clones 7, 8, 16, 18, and 21 was digested with the EcoRV enzyme that does not cut within the targeting vector sequence. Vectors present at random genomic locations should have unique fragment lengths depending on their proximity to EcoRV sites in the genome. Hybridization of the GFP probe to these samples revealed a single DNA fragment demonstrating that multiple vector copies integrated at the same locus (Figure 4d). These data demonstrate the efficiency of the promoter trap strategy, and the accuracy of gene targeting in keratinocytes using these vectors. Clones 8 and 21 contain a tandem head to tail insertion of two vectors such that a target site vector:chromosome junction fragment and a unit length vector fragment are generated when DNA is digested with an enzyme that cuts once within vector sequence (BamHI) and probed with a DNA fragment from GFP. Clones 7, 16, and 18 also contain two tandem vector insertions at the KRT14 locus with an additional 3.2 kb of vector sequence added to the right junction producing a 9.1 kb vector:chromosome junction fragment. Concatomerization of AAV vectors has been previously described24Russell DW Hirata RK Human gene targeting favors insertions over deletions.Hum Gene Ther. 2008; 19: 907-914Crossref PubMed Scopus (20) Google Scholar,25McLaughlin SK Collis P Hermonat PL Muzyczka N Adeno-associated virus general transduction vectors: analysis of proviral structures.J Virol. 1988; 62: 1963-1973Crossref PubMed Google Scholar and vector genomes in nondividing cells are present as large predominantly head to tail concatomers where gene expression occurs predominantly from episomal forms.26Nakai H Storm TA Kay MA Recruitment of single-stranded recombinant adeno-associated virus vector genomes and intermolecular recombination are responsible for stable transduction of liver in vivo.J Virol. 2000; 74: 9451-9463Crossref PubMed Scopus (155) Google Scholar The number of clones resulting from independent KRT14-targeting events in the sorted cell populations is calculated to be >1,000 based on the number of cells used to initiate targeting experiments and a transduction frequency of 0.5%. However, as multiple keratinocyte colonies are being pooled before sorting, the possibility exists that the same clone could be isolated more than once. To determine whether similar gene-targeting frequencies, and similar frequencies of integration at random genomic locations are observed if cells are cloned immediately after application of the gene-targeting vector, the GFP expression cassette was replaced with a neomycin phosphotransferase gene and G418 selection applied 1 day after infection. Southern blot analysis of genomic DNA from 14 G418 resistant colonies isolated by ring cloning demonstrated that all clones contained an insertion of the gene expression cassette in exon 3 of the KRT14 locus. None of the 14 clones contained vectors integrated at random genomic locations as evidenced by probing the same blot with a DNA fragment homologous to the neo gene supporting the results obtained by sorting and cloning GFP positive cells (Supplementary Figure S1). Colonies generated from sorted GFP positive cells were similar in size, and showed only subtle morphologic differences when chosen for expansion and characterization. A colony forming efficiency assay based on colony size and morphology has been previously described13Barrandon Y Green H Three clonal types of keratinocyte with different capacities for multiplication.Proc Natl Acad Sci USA. 1987; 84: 2302-2306Crossref PubMed Scopus (1076) Google Scholar and colonies having a holoclone phenotype have been shown to have an almost unlimited capacity for expansion, capability for transplantation and differentiation,14Mathor MB Ferrari G Dellambra E Cilli M Mavilio F Cancedda R et al.Clonal analysis of stably transduced human epidermal stem cells in culture.Proc Natl Acad Sci USA. 1996; 93: 10371-10376Crossref PubMed Scopus (151) Google Scholar,27Larcher F Del Rio M Serrano F Segovia JC Ramírez A Meana A et al.A cutaneous gene therapy approach to human leptin deficiencies: correction of the murine ob/ob phenotype using leptin-targeted keratinocyte grafts.FASEB J. 2001; 15: 1529-1538Crossref PubMed Scopus (63) Google Scholar,28Larcher F Dellambra E Rico L Bondanza S Murillas R Cattoglio C et al.Long-term engraftment of single genetically modified human epidermal holoclones enables safety pre-assessment of cutaneous gene therapy.Mol Ther. 2007; 15: 1670-1676Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar and contain cell surface antigens also present on stem cells defined by other criteria.29Senoo M Pinto F Crum CP McKeon F p63 Is essential for the proliferative potential of stem cells in stratified epithelia.Cell. 2007; 129: 523-536Abstract Full Text Full Text PDF PubMed Scopus (689) Google Scholar The colony forming efficiency assay was performed on cells from all 30 GFP positive clones. One quarter of the cells from a single colony were seeded to dishes containing irradiated mouse feeders, allowed to proliferate for 12 days, and fixed and stained with rhodamine B. Colonies were counted and categorized as greater than or less than 4 mm in diameter. Colonies of keratinocyte clones that contained cells that generate large (>4 mm diameter) daughter colonies 95% of the time are classified as holoclones (Figure 4e, clones 4, 9, 14, and 19 outlined in red). Colonies of cells that give rise to a mixture of large daughter colonies and small abortive colonies are classified as meroclones (Figure 4e, clones outlined in blue), and those with cells that only form abortive daughter colonies (<4 mm in diameter) are classified as paraclones (Figure 4e, clones 3, 5, 15, 20, and 23 outlined in yellow).13Barrandon Y Green H Three clonal types of keratinocyte with different capacities for multiplication.Proc Natl Acad Sci USA. 1987; 84: 2302-2306Crossref PubMed Scopus (1076) Google Scholar Of the 30 clones analyzed, 13% were classified as holoclones, 70% as meroclones, and 16% as paraclones (Figure 4e). These frequencies are similar to those observed in unmodified cells from skin biopsies13Barrandon Y Green H Three clonal types of keratinocyte with different capacities for multiplication.Proc Natl Acad Sci USA. 1987; 84: 2302-2306Crossref PubMed Scopus (1076) Google Scholar and demonstrate the feasibility of a gene-targeting approach to skin blistering conditions. Keratinocytes isolated from the skin of a patient with EBS (Dowling-Meara subtype), and containing a common mutation that results in a change of amino acids at position 125 of the protein sequence (R125C) were transduced with the AAVK14e3IFsA vector. Ten GFP positive clones and a polyclonal GFP positive cell population were collected for Southern blot analysis and sequencing. Five clones grew sufficiently for DNA analysis performed by probing genomic DNA with a KRT14 gene fragment (Figure 5a, top) and a GFP gene fragment (Figure 5a, middle). Each clone and the polyclonal population showed targeted insertion of the vector within exon 3 of the KRT14 gene again demonstrated by a shift in the size from 10.9 to 5.9 kb of the KRT14 hybridizing BamHI fragment. However, in contrast to clones obtained by targeting normal cells, every clone, and the majority of the polyclonal cell population, contained a minimum of two vector copies at the KRT14 locus demonstrated by a KRT14–GFP junction fragment (5.9 kb) and an additional GFP-hybridizing fragment of 4.6 kb similar to clones 8 and 21 isolated from transduction of normal cells (Figures 4b and 5a, middle). The location of both GFP-hybridizing fragments at the KRT14 locus was confirmed by digestion of DNA from the same cells with EcoRV and hybridization with a DNA fragment from the GFP gene (Figure 5a, bottom). Again hybridization to a single DNA fragment with the same size as a concatomer of two vector copies within exon 3 of the KRT14 gene was observed. A faint GFP-hybridizing fragment of ~13.8 kb size is seen in the polyclonal population indicating that a small percentage of cells have as many as three vector copies at the KRT14 locus (Figure 5a, lanes middle labeled pc). Vector:chromosome junction fragments were amplified from the targeted allele of each clone, and from the polyclonal cell population to determine whether cells targeted at the mutant allele are present more frequently after growth in culture. All five clones and the vast majority of cells in the polyclonal population contained targeted insertions of the GFP expression cassette at the mutant allele (Figure 5b) suggesting that cells without the mutant protein are at a growth advantage in culture, and that a tandem insertion of at least two vector copies more effectively eliminated transcription of the mutant allele. To determine whether KRT14-targeted keratinocytes retain the ability to form a normally functioning stratified epidermal cell layer in vivo, skin equivalents were constructed from each KRT14-targeted normal keratinocyte clone having a holoclone colony phenotype (Figure 4e, clones 4, 9, 14, 19) and from an EBS-affected individual (Figure 5, unmodified, clone 6, and polyclonal). Full thickness skin grafts were transplanted to the dorsal surface of nude mice and bandaged by suturing devitalized full thickness skin from the graft site over the human graft. The devitalized mouse skin was sloughed by day 8 in each case, and a mature skin appearance was present 8–10 days after surgery (Figure 6a). Mice were killed 13–20 weeks later for histological analysis of the engrafted tissues. The gross appearance of the grafts was normal and the engrafted human skin functioned effectively by physically protecting underlying tissues, preventing infection, and limiting water loss (Figure 6a,c). Hematoxylin and eosin staining of paraffin-embedded tissue sections revealed an organized, stratified epidermal layer with cornification and keratinization in the upper epidermal layers (Figure 6b). The engrafted skin generally contained more cell layers than the adjacent mouse skin and could be distinguished microscopically by the lack of dermal structures in the engrafted tissue, as well as specific staining for human involucrin in the apical layer of keratinocytes with a antibody specific for the human protein (Figure 6b,c). Normal skin architecture and organization is demonstrated by KRT14 immunostaining in the basal layer (Figure 6b,c), and KRT10 immunostaining in the apical layers of the graft (Figure 6b,c). The KRT14-targeted patient clone 6 maintained a normal karyotype after expansion to over 1013 cells demonstrating the proliferative capacity of single clones, and the ability to generate grafts that could cover large skin surfaces (Supplementary Figure S2). These data demonstrate that normal and diseased human keratinocytes can be modified by gene targeting at a specific genomic locus, expanded in culture, and engrafted to form normal functioning skin surfaces. We also show that clonal (Figure 6a–c) and polyclonal populations of KRT14-targeted keratinocytes (Figure 6c, polyclonal) form skin grafts that are histologically indistinguishable from those formed by unmodified cells (Figure 6c, unmodified). Finally, we demonstrate for the first time a growth advantage of phenotypically corrected human keratinocytes from EBS patients. Epidermal stem cells are attractive cell targets for gene modification given their growth characteristics, their ability to be studied in xenotransplant models,30Barrandon Y Li V Green H New techniques for the grafting of cultured human epidermal cells onto athymic animals.J Invest Dermatol. 1988; 91: 315-318Crossref PubMed Scopus (86) Google Scholar and their history of successful use for autologous transplantation when life threatening full thickness burns have occurred.11Gallico 3rd GG O'Connor NE Compton CC Kehinde O Green H Permanent coverage of large burn wounds with autologous cultured human epithelium.N Engl J Med. 1984; 311: 448-451Crossref PubMed Scopus (1095) Google Scholar,31Ronfard V Rives JM Neveux" @default.
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• W2001569366 title "Efficient KRT14 Targeting and Functional Characterization of Transplanted Human Keratinocytes for the Treatment of Epidermolysis Bullosa Simplex" @default.
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• W2001569366 hasConceptScore W2001569366C2777459323 @default.
• W2001569366 hasConceptScore W2001569366C2778266373 @default.
• W2001569366 hasConceptScore W2001569366C2778405274 @default.
• W2001569366 hasConceptScore W2001569366C2781207856 @default.
• W2001569366 hasConceptScore W2001569366C54355233 @default.
• W2001569366 hasConceptScore W2001569366C71924100 @default.

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