Abstract: Objective To investigate the possibility of gene correction in keratinocytes. Methods Using intradermal injection of a chimeric RNA-DNA oligonucleotide (RDO) or a single stranded oligonucleotide (ssODN) into murine skin, we attempted to make a dominant mutation (R94P) in the conserved α-helical domain of K17. Both K17A-RDO and K17A-ssODN contained a single base mismatch (CGC to CCC) to alter the normal K17 sequence to cause an amino acid substitution. The complexes consisting of oligonucleotides and cationic liposomes were injected into C57BL/6 murine skin on day 2 and day 5 after birth. Skin biopsies were taken on day 8 after birth. Results Histological examination of skin biopsies on day 8 from mice (5/7 for K17A-RDO and 5/5 for K17A-ssODN) showed consistent twisted hair shafts or broken hair follicles at the sebaceous gland level and an occasional rupture of hair bulb and epidermal cyst-like changes. In the injected area, the number of full anagen hair follicles decreased 50%. Injection of the control oligonucleotide, identical to K17A-RDO but containing no mismatch to the normal sequence, did not result in any detectable abnormality (n=5). The genomic DNA isolated by dissection of hair follicles from slides, PCR amplified and subjected to Aci I digestion, showed that the target gene was mutant from CGC to CCC. Conclusions Our results indicate that intradermal injection of the K17A-RDO or K17A-ssODN affected the hair growth and morphology.

Key words: Hair follicle, Animal testing alternatives, Keratin, Base pair mismatch, Point mutation