Lentiviral Vector-Mediated Gene Transfer to Human Hair Follicles Penkanok Sriwiriyanont, Akira Hachiya, William L. Pickens, Shigeru Moriwaki, Atsushi Ohuchi, Takashi Kitahara, Yoshinori Takema, William J. Kitzmiller, Marty O. Visscher, Alexander Bello, Ryoji Tsuboi, Gary P. Kobinger  Journal of Investigative Dermatology  Volume 129, Issue 9, Pages 2296-2299 (September 2009) DOI: 10.1038/jid.2009.33 Copyright © 2009 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 VSV-G pseudotyped lentiviral vector targeting in human hair cells. (a) The efficacy of VSV-G pseudotyped lentivirus in transducing cultured human HFs was evaluated by X-gal staining 14 days (tri-panel) and 28 days (single panel) after single-transduction or double-transduction protocols. Transduction efficacy was based on the level of β-galactosidase expression throughout the entire length of the HF. After 14 days, a marked increase in X-gal staining was observed in the double-transduced follicles. By 28 days, the transduced cells had migrated and coalesced to one side of the follicle; presumptively, to the bulge region. The inset in the day 28 panel shows a close-up of the X-gal stained region. Control follicles were incubated in DMEM in parallel with transduced follicles. Scale bars=0.5mm. (b) Triple labeling of HFs with β-galactosidase (red), pmel17/gp100 (green) and the nuclear stain, Draq-5 (blue) was used to investigate the capacity of VSV-G pseudotyped lentiviral vector to target follicular melanocytes following double-transduction. Immunofluorescence analyses of HFs harvested throughout the course of the 21-day organ culture period revealed migration of transduced melanocytes from the hair bulb (1 day after transduction) to the lower portion of the HF (14 days after transduction), and finally, completely out of the hair bulb region (21 days after transduction). The images in the vertical column on the right are the center column images shown at higher magnification. (c) Viral targeting of ORS and IRS cells was assessed in cultured HFs 14 days after transduction using antisera against keratin 17 (green) and β-galactosidase (red), then counterstained with Draq-5 (blue). VSV-G pseudotyped lentivirus transduced major differentiated ORS cells, as shown by the co-localization of keratin 17 and β-galactosidase. The lower two panels depict the boxed regions in the upper two panels at higher magnification. (d) Cross-sectional analysis of a representative transduced HF sectioned longitudinally indicates enhancement in β-galactosidase expression in IRS and ORS cells as the distance from the bulb increases. The depicted follicle was stained 14 days after double-transduction, but similar results were observed at 21 days. Scale bars in panels b through d represent 50μm. Journal of Investigative Dermatology 2009 129, 2296-2299DOI: (10.1038/jid.2009.33) Copyright © 2009 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 The persistency of transgene expression in human hair xenografts. (a, b) Here, 3 months after grafting, regenerated hair shafts were documented. The human hairs are well pigmented and exhibit architecture that corresponds to the hair shape of the donor. Hair shafts from straight hair (a) and curly hair (b) donors retain their original characteristic curvatures. Likewise, the original shape of the hair bulbs was also preserved (a, b; insets). Taken together, the preserved nature of the grafted hair indicates the validity of the grafting procedure. Immunofluorescence analysis using β-galactosidase (red) and pmel17/gp100 antibodies (green) revealed long-term transgene expression in bulb melanocytes and matrix cells in both straight (c) and curly (d) human HFs. No transgene expression was observed in control (DMEM-incubated) follicles (e). For procedural comparison, concentrated viral stock was intradermally injected parallel to grafted HFs that had not been previously incubated with virus. Immunofluorescence of β-galactosidase antibody (red) was conducted 6 weeks after injection (f, g). Low expression of β-galactosidase was noted in the IRS keratinocytes (f) and along the injection site (g, arrows). Scale bars in panels a–b and c–g represent 200 and 100μm, respectively. Journal of Investigative Dermatology 2009 129, 2296-2299DOI: (10.1038/jid.2009.33) Copyright © 2009 The Society for Investigative Dermatology, Inc Terms and Conditions