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Localized in vivo genotypic and phenotypic correction of the albino mutation in skin by RNA-DNA oligonucleotide

Nature Biotechnology volume 18, pages 43–47 (2000)Cite this article

Abstract

We recently demonstrated that an RNA-DNA oligonucleotide corrected a point mutation in the mouse tyrosinase gene, resulting in permanent and inheritable restoration of tyrosinase enzymatic activity, melanin synthesis, and pigmentation changes in cultured melanocytes. In this study, we extended gene correction of melanocytes from tissue culture to live animals, using a chimeric oligonucleotide designed to correct a point mutation in the tyrosinase gene. Both topical application and intradermal injection of this oligonucleotide to albino BALB/c mouse skin resulted in dark pigmentation of several hairs in a localized area. The restored tyrosinase enzymatic activity was detected by dihydroxyphenylacetic acid (DOPA) staining of hair follicles in the treated skin. Tyrosinase gene correction was also confirmed by restriction fragment length polymorphism analysis and DNA sequencing from skin that was positive for DOPA staining and melanin synthesis. Localized gene correction was maintained three months after the last application of the chimeric oligonucleotides. These results demonstrated correction of the tyrosinase gene point mutation by chimeric oligonucleotides in vivo.

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Figure 1: Sequences of the Tyr-A chimeric oligonucleotide and targeted sequences in the tyrosinase gene.
Figure 2: Delivery of the chimeric oligonucleotide in the mouse skin.
Figure 3: Localization of dark pigmented hairs in albino BALB/c mouse.
Figure 4: Detection of tyrosinase activity and melanin in skin sections after intradermal injection of the Tyr-A and Tyr-B chimeric oligonucleotide.
Figure 5: Restriction fragment length polymorphism (RFLP) and DNA sequencing analysis of genomic DNA isolated from DOPA-positive skin biopsies and controls.

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Acknowledgements

We thank Drs. J. Uitto and V.J. Hearing for discussion and critical reading of manuscript. This work was supported in part by grants from the NIH (AR44350; to K.Y.), and the National Alopecia Areata Foundation (to G.C.). V.A. is a recipient of a Dermatology Foundation Fellowship.

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Authors and Affiliations

  1. Department of Dermatology and Cutaneous Biology, Jefferson Institute of Molecular Medicine, Thomas Jefferson University and Jefferson Medical College, Philadelphia, 19107, PA

    Vitali Alexeev, Olga Igoucheva & Kyonggeun Yoon

  2. Department of Biochemistry and Molecular Pharmacology Jefferson Institute of Molecular Medicine, Thomas Jefferson University and Jefferson Medical College, Philadelphia, 19107, PA

    Kyonggeun Yoon

  3. Department of Dermatology, University of Pennsylvania School of Medicine, Philadelphia, PA

    Alla Domashenko & George Cotsarelis

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  1. Vitali Alexeev
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Correspondence to Kyonggeun Yoon.

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Alexeev, V., Igoucheva, O., Domashenko, A. et al. Localized in vivo genotypic and phenotypic correction of the albino mutation in skin by RNA-DNA oligonucleotide. Nat Biotechnol 18, 43–47 (2000). https://doi.org/10.1038/71901

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