Tropism-modified AAV vectors overcome barriers to successful cutaneous therapy.
Autologous human keratinocytes (HK) forming sheet grafts are approved as skin substitutes. Genetic engineering of HK represents a promising technique to improve engraftment and survival of transplants. Although efficacious in keratinocyte-directed gene transfer, retro-/lentiviral vectors may raise safety concerns when applied in regenerative medicine. We therefore optimized adeno-associated viral (AAV) vectors of the serotype 2, characterized by an excellent safety profile, but lacking natural tropism for HK, through capsid engineering. Peptides, selected by AAV peptide display, engaged novel receptors that increased cell entry efficiency by up to 2,500-fold. The novel targeting vectors transduced HK with high efficiency and a remarkable specificity even in mixed cultures of HK and feeder cells. Moreover, differentiated keratinocytes in organotypic airlifted three-dimensional cultures were transduced following topical vector application. By exploiting comparative gene analysis we further succeeded in identifying αvβ8 integrin as a target receptor thus solving a major challenge of directed evolution approaches and describing a promising candidate receptor for cutaneous gene therapy.
1] Center for Molecular Medicine Cologne (CMMC), University of Cologne, Cologne, Germany [2] Department I for Internal Medicine, University Hospital Cologne, Cologne, Germany.
Jessica
J
Sallach
Molecular Physiology and Therapeutics Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, Maryland, USA.
Giovanni
G
Di Pasquale
Centro de Investigacion Energetica Medioambiental y Tecnologica (CIEMAT) Madrid, Center for Biomedical Research on Rare Diseases (CIBERER-U714) and Department of Bioengineering. Universidad Carlos III de Madrid (UC3M), Madrid, Spain.
Fernando
F
Larcher
1] Center for Molecular Medicine Cologne (CMMC), University of Cologne, Cologne, Germany [2] Department of Dermatology, University Hospital of Cologne, Cologne, Germany.
Nadine
N
Niehoff
1] Center for Molecular Medicine Cologne (CMMC), University of Cologne, Cologne, Germany [2] Department of Dermatology, University Hospital of Cologne, Cologne, Germany.
Matthias
M
Rübsam
1] Center for Molecular Medicine Cologne (CMMC), University of Cologne, Cologne, Germany [2] Department I for Internal Medicine, University Hospital Cologne, Cologne, Germany.
Anke
A
Huber
Molecular Physiology and Therapeutics Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, Maryland, USA.
Jay
J
Chiorini
1] Centro de Investigacion Energetica Medioambiental y Tecnologica (CIEMAT) Madrid, Center for Biomedical Research on Rare Diseases (CIBERER-U714) and Department of Bioengineering. Universidad Carlos III de Madrid (UC3M), Madrid, Spain [2] Current address: Molecular Immunology, UCL Institute of Child Health, London, UK.
David
D
Almarza
1] Center for Molecular Medicine Cologne (CMMC), University of Cologne, Cologne, Germany [2] Department of Dermatology, University Hospital of Cologne, Cologne, Germany [3] Cologne Excellence Center on Cellular Stress Responses in Aging-Associated Diseases (CECAD), Cologne, Germany.
Sabine A
SA
Eming
Children's Medical Surgery, Cologne, Germany.
Hikmet
H
Ulus
Department of Pathology & Laboratory Medicine, Anatomic Pathology, UCSF, San Francisco, CA, USA.
Stephen
S
Nishimura
University Cancer Center Leipzig (UCCL), Leipzig, Germany.
Ulrich T
UT
Hacker
1] Center for Molecular Medicine Cologne (CMMC), University of Cologne, Cologne, Germany [2] Department I for Internal Medicine, University Hospital Cologne, Cologne, Germany.
Michael
M
Hallek
1] Center for Molecular Medicine Cologne (CMMC), University of Cologne, Cologne, Germany [2] Department of Dermatology, University Hospital of Cologne, Cologne, Germany [3] Cologne Excellence Center on Cellular Stress Responses in Aging-Associated Diseases (CECAD), Cologne, Germany.
Carien M
CM
Niessen
1] Center for Molecular Medicine Cologne (CMMC), University of Cologne, Cologne, Germany [2] Department I for Internal Medicine, University Hospital Cologne, Cologne, Germany.
Hildegard
H
Büning
D036022:Capsid Proteins / Q000235:genetics; D000229:Dependovirus / Q000235:genetics; D005818:Genetic Engineering; D015316:Genetic Therapy; D005822:Genetic Vectors; D006801:Humans; D039482:Integrin alpha5 / Q000235:genetics; D015603:Keratinocytes / Q000378:metabolism / Q000473:pathology; D010455:Peptides / Q000235:genetics* / Q000627:therapeutic use; D012868:Skin Abnormalities / Q000235:genetics / Q000473:pathology / Q000628:therapy*; D014161:Transduction, Genetic; D018523:Tropism
D036022:Capsid Proteins; D039482:Integrin alpha5; D010455:Peptides