title: Pseudotyped adeno-associated viral vectors for gene transfer in dermal fibroblasts: implications for wound-healing applications.
abstract: BACKGROUND
  Cell-specific gene transfer and sustained transgene expression are goals of cutaneous gene therapy. Pseudotyping strategy with adeno-associated viral (AAV) vectors has the potential to confer unique cellular tropism and transduction efficiency. We hypothesize that pseudotyped AAV vectors have differential tropism and transduction efficiency under normal and wound conditions in dermal fibroblasts.
  
  
  METHODS
  We packaged AAV2 genome with green fluorescent protein reporter in capsids of other serotypes, AAV5, AAV7, and AAV8, producing pseudotyped vectors AAV2/5, AAV2/7, and AAV2/8, respectively. Murine and human dermal fibroblasts were transduced by the different pseudotypes for 24 h at multiplicities of infection 10(2), 10(3), 10(4), and 10(5). We assessed transduction efficiency at days 3 and 7. Experiments were repeated in a simulated wound environment by adding 10 ng/mL platelet-derived growth factor-B to culture media.
  
  
  RESULTS
  Transduction efficiency of the pseudotyped AAV vectors was dose dependent. Multiplicity of infection 10(5) resulted in significantly higher gene transfer. Under normal culture conditions, the pseudotyping strategy conferred differential transduction of dermal fibroblasts, with significantly enhanced transduction of murine cells by AAV2/5 and AAV2/8 compared with AAV2/2. Adeno-associated virus 2/8 was more efficacious in transducing human cells. Under wound conditions, transduction efficiency of AAV2/2, 2/5, and 2/8 was significantly lower in murine fibroblasts. At day 3 under wound conditions, all vectors demonstrated similar transduction efficiency, but by day 7, the three pseudotyped vectors transduced significantly more murine cells compared with AAV2/2. However, in human cells, there was no significant difference in the transduction efficiency of each pseudotype between normal and wound conditions at both 3 and 7 d.
  
  
  CONCLUSIONS
  The AAV pseudotyping strategy represents a gene transfer technology that can result in differential transduction of dermal fibroblasts. The differences in transduction efficiency in murine and human dermal fibroblasts in both the normal and wound environment highlight issues with translatability of gene transfer techniques. These data provide a template for using pseudotyped AAV vectors in cutaneous applications.
authors.affiliation: Division of Pediatric, General, Thoracic, and Fetal Surgery, Cincinnati Children's Hospital Medical Center and the University of Cincinnati College of Medicine, Cincinnati, Ohio 45229-3039, USA.
authors.forename: Swathi
authors.initials: S
authors.lastname: Balaji
authors.forename: Alice
authors.initials: A
authors.lastname: King
authors.forename: Yashu
authors.initials: Y
authors.lastname: Dhamija
authors.forename: Louis D
authors.initials: LD
authors.lastname: Le
authors.forename: Aimen F
authors.initials: AF
authors.lastname: Shaaban
authors.forename: Timothy M
authors.initials: TM
authors.lastname: Crombleholme
authors.forename: Sundeep G
authors.initials: SG
authors.lastname: Keswani
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D000229:Dependovirus / Q000145:classification / Q000235:genetics*; D005347:Fibroblasts / Q000166:cytology / Q000502:physiology*; D018014:Gene Transfer Techniques; D005822:Genetic Vectors / Q000235:genetics*; D049452:Green Fluorescent Proteins / Q000235:genetics; D006801:Humans; D051379:Mice; D008810:Mice, Inbred C57BL; D014161:Transduction, Genetic / Q000379:methods; D014945:Wound Healing / Q000235:genetics*
chemical_list: D049452:Green Fluorescent Proteins
keywords: Adeno-associated virus; Gene therapy; Pseudotyping; Regenerative medicine; Wound healing