title: Transduction of pancreatic islets with pseudotyped adeno-associated virus: effect of viral capsid and genome conversion.
abstract: BACKGROUND
  Recombinant adeno-associated viral (rAAV) vectors currently show promise for islet gene therapy. In the presence of complementing AAV2 Rep proteins, AAV2 genomes can be packaged with other serotype capsids to assemble infectious virions. During transduction, the ssDNA to dsDNA conversion is one of the major rate-limiting steps that contribute to the slow onset of transgene expression.
  
  
  METHODS
  Using pseudotyping strategy, we produced double-stranded (dsAAV) and single-stranded (ssAAV) rAAV2 genomes carrying the GFP reporter gene packaged into AAV1, AAV2, and AAV5 capsids. The ability of cross-packaged AAV1, AAV2, and AAV5 at the same genome containing particle (gcp) concentration to transduce murine and human pancreatic islets was evaluated by GFP positive cell percentage. Transgenic expression was also determined by transplant transduced human islet into SCID mice.
  
  
  RESULTS
  Pseudotyped rAAV2/1 based vectors transduced murine islets at greater efficiency than either rAAV2/2 or rAAV2/5 vectors. For human islets transduction, the rAAV2/2 vector was more efficient than rAAV2/1 or rAAV2/5 vectors. rAAV2/2 transduced human islets more efficiently than murine islets, while rAAV2/1 transducted murine islets more efficiently than human islets. dsAAV, which do not require second strand synthesis and thus are potentially more efficient, evidenced 5 fold higher transduction ability than ssAAV vectors. Pseudotyped rAAV transduced islet grafts maintained normal function, expressed transgenic product persistently in vivo, and reversed diabetes.
  
  
  CONCLUSIONS
  The transduction efficiency of rAAV vectors was dependent on the cross-packaged capsid. The vector capsids permit species-specific transduction. For human islets, dsAAV2/2 vectors may be the most efficient vector for clinical development.
authors.affiliation: Recanati-Miller Transplantation Institute, Mount Sinai School of Medicine, New York, NY 10029, USA.
authors.forename: Nan
authors.initials: N
authors.lastname: Zhang
authors.forename: Nathalie
authors.initials: N
authors.lastname: Clément
authors.forename: Dongmei
authors.initials: D
authors.lastname: Chen
authors.forename: Shuang
authors.initials: S
authors.lastname: Fu
authors.forename: Haojiang
authors.initials: H
authors.lastname: Zhang
authors.forename: Patricia
authors.initials: P
authors.lastname: Rebollo
authors.forename: R Michael
authors.initials: RM
authors.lastname: Linden
authors.forename: Jonathan S
authors.initials: JS
authors.lastname: Bromberg
mesh_terms: D000256:Adenoviridae / Q000235:genetics*; D000818:Animals; D002213:Capsid; D004247:DNA; D004277:DNA, Single-Stranded; D003921:Diabetes Mellitus, Experimental / Q000601:surgery*; D017930:Genes, Reporter; D015316:Genetic Therapy / Q000379:methods*; D016679:Genome, Viral; D049452:Green Fluorescent Proteins / Q000235:genetics; D006801:Humans; D007515:Islets of Langerhans; D016381:Islets of Langerhans Transplantation; D051379:Mice; D008807:Mice, Inbred BALB C; D016513:Mice, SCID; D014161:Transduction, Genetic / Q000379:methods*; D019076:Transgenes / Q000235:genetics; D014183:Transplantation, Heterologous
chemical_list: D004277:DNA, Single-Stranded; D049452:Green Fluorescent Proteins; D004247:DNA