title: Genetic incorporation of the protein transduction domain of Tat into Ad5 fiber enhances gene transfer efficacy.
abstract: BACKGROUND
  Human adenovirus serotype 5 (Ad5) has been widely explored as a gene delivery vector for a variety of diseases. Many target cells, however, express low levels of Ad5 native receptor, the Coxsackie-Adenovirus Receptor (CAR), and thus are resistant to Ad5 infection. The Protein Transduction Domain of the HIV Tat protein, namely PTD tat, has been shown to mediate protein transduction in a wide range of cells. We hypothesize that re-targeting Ad5 vector via the PTD tat motif would improve the efficacy of Ad5-mediated gene delivery.
  
  
  RESULTS
  In this study, we genetically incorporated the PTD tat motif into the knob domain of Ad5 fiber, and rescued the resultant viral vector, Ad5.PTD tat. Our data showed the modification did not interfere with Ad5 binding to its native receptor CAR, suggesting Ad5 infection via the CAR pathway is retained. In addition, we found that Ad5.PTD tat exhibited enhanced gene transfer efficacy in all of the cell lines that we have tested, which included both low-CAR and high-CAR decorated cells. Competitive inhibition assays suggested the enhanced infectivity of Ad5.PTD tat was mediated by binding of the positively charged PTD tat peptide to the negatively charged epitopes on the cells' surface. Furthermore, we investigated in vivo gene delivery efficacy of Ad5.PTD tat using subcutaneous tumor models established with U118MG glioma cells, and found that Ad5.PTD tat exhibited enhanced gene transfer efficacy compared to unmodified Ad5 vector as analyzed by a non-invasive fluorescence imaging technique.
  
  
  CONCLUSIONS
  Genetic incorporation of the PTD tat motif into Ad5 fiber allowed Ad5 vectors to infect cells via an alternative PTD tat targeting motif while retaining the native CAR-mediated infection pathway. The enhanced infectivity was demonstrated in both cultured cells and in in vivo tumor models. Taken together, our study identifies a novel tropism expanded Ad5 vector that may be useful for clinical gene therapy applications.
authors.affiliation: Division of Human Gene Therapy, Department of Medicine, University of Alabama at Birmingham, Birmingham, USA. than@uab.edu
authors.forename: Tie
authors.initials: T
authors.lastname: Han
authors.forename: Yizhe
authors.initials: Y
authors.lastname: Tang
authors.forename: Hideyo
authors.initials: H
authors.lastname: Ugai
authors.forename: Leslie E
authors.initials: LE
authors.lastname: Perry
authors.forename: Gene P
authors.initials: GP
authors.lastname: Siegal
authors.forename: Juan L
authors.initials: JL
authors.lastname: Contreras
authors.forename: Hongju
authors.initials: H
authors.lastname: Wu
mesh_terms: D000260:Adenoviruses, Human / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D020816:Amino Acid Motifs; D000818:Animals; D036022:Capsid Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D062727:Coxsackie and Adenovirus Receptor-Like Membrane Protein; D015696:Gene Products, tat / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D015316:Genetic Therapy / Q000379:methods; D005910:Glioma / Q000235:genetics / Q000378:metabolism; D006801:Humans; D051379:Mice; D008819:Mice, Nude; D009368:Neoplasm Transplantation; D009928:Organ Specificity; D017434:Protein Structure, Tertiary; D011991:Receptors, Virus / Q000378:metabolism; D014161:Transduction, Genetic / Q000379:methods*
chemical_list: C568771:CLMP protein, human; C568772:CLMP protein, mouse; D036022:Capsid Proteins; D062727:Coxsackie and Adenovirus Receptor-Like Membrane Protein; D015696:Gene Products, tat; D011991:Receptors, Virus; C018315:hexon capsid protein, Adenovirus