Successful expansion but not complete restriction of tropism of adeno-associated virus by in vivo biopanning of random virus display peptide libraries.
Targeting viral vectors to certain tissues in vivo has been a major challenge in gene therapy. Cell type-directed vector capsids can be selected from random peptide libraries displayed on viral capsids in vitro but so far this system could not easily be translated to in vivo applications. Using a novel, PCR-based amplification protocol for peptide libraries displayed on adeno-associated virus (AAV), we selected vectors for optimized transduction of primary tumor cells in vitro. However, these vectors were not suitable for transduction of the same target cells under in vivo conditions. We therefore performed selections of AAV peptide libraries in vivo in living animals after intravenous administration using tumor and lung tissue as prototype targets. Analysis of peptide sequences of AAV clones after several rounds of selection yielded distinct sequence motifs for both tissues. The selected clones indeed conferred gene expression in the target tissue while gene expression was undetectable in animals injected with control vectors. However, all of the vectors selected for tumor transduction also transduced heart tissue and the vectors selected for lung transduction also transduced a number of other tissues, particularly and invariably the heart. This suggests that modification of the heparin binding motif by target-binding peptide insertion is necessary but not sufficient to achieve tissue-specific transgene expression. While the approach presented here does not yield vectors whose expression is confined to one target tissue, it is a useful tool for in vivo tissue transduction when expression in tissues other than the primary target is uncritical.
Department of Hematology and Oncology, University of Freiburg Medical Center, Freiburg, Germany.
Stefan
S
Michelfelder
Johannes
J
Kohlschütter
Alexandra
A
Skorupa
Sabrina
S
Pfennings
Oliver
O
Müller
Jürgen A
JA
Kleinschmidt
Martin
M
Trepel
D000595:Amino Acid Sequence; D000818:Animals; D001943:Breast Neoplasms; D002213:Capsid / Q000378:metabolism; D000229:Dependovirus / Q000235:genetics / Q000378:metabolism; D005260:Female; D018014:Gene Transfer Techniques; D015316:Genetic Therapy / Q000379:methods; D005822:Genetic Vectors / Q000235:genetics / Q000378:metabolism; D006801:Humans; D051379:Mice; D008822:Mice, Transgenic; D008969:Molecular Sequence Data; D019151:Peptide Library; D016133:Polymerase Chain Reaction / Q000379:methods; D014161:Transduction, Genetic; D018523:Tropism / Q000235:genetics; D014407:Tumor Cells, Cultured
D019151:Peptide Library