Limits...
Successful expansion but not complete restriction of tropism of adeno-associated virus by in vivo biopanning of random virus display peptide libraries.

Michelfelder S, Kohlschütter J, Skorupa A, Pfennings S, Müller O, Kleinschmidt JA, Trepel M - PLoS ONE (2009)

Bottom Line: Analysis of peptide sequences of AAV clones after several rounds of selection yielded distinct sequence motifs for both tissues.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Hematology and Oncology, University of Freiburg Medical Center, Freiburg, Germany.

ABSTRACT
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.

Show MeSH

Related in: MedlinePlus

Kinetics of circulating AAV peptide library particles is similar to wild-type AAV.A random X7 peptide library or wild-type AAV-2 viruses were injected intravenously at 1×1010 vg per mouse. Blood samples were collected after indicated time points and the amount of circulating viral particles in the serum was determined by real-time PCR. Data represent mean values from n = 3 mice per group, analyzed in triplicates ± SD.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2664470&req=5

pone-0005122-g003: Kinetics of circulating AAV peptide library particles is similar to wild-type AAV.A random X7 peptide library or wild-type AAV-2 viruses were injected intravenously at 1×1010 vg per mouse. Blood samples were collected after indicated time points and the amount of circulating viral particles in the serum was determined by real-time PCR. Data represent mean values from n = 3 mice per group, analyzed in triplicates ± SD.

Mentions: Based on the negative finding above, we hypothesized that selection under in vivo conditions is needed to enrich library clones that are able to bind cellular receptors in tumors, penetrate the tumor tissue and are internalized into tumor cells under physiological circulation conditions after intravenous administration. But we suspected that our novel PCR-based selection of AAV libraries may not be able to distinguish between library particles successfully internalized into target cells, and non-homing particles present in the circulation if the tissue is harvested too early after injection. To minimize the amount of circulating AAV library particles in our tissue samples at the time point of harvest, we analyzed the kinetics of circulating AAV library particles. Therefore, AAV were injected intravenously at 1×1010 vg per mouse, blood samples were collected at various time points, and the amount of circulating particles in the serum was quantified by real-time PCR. Clearance rates were comparable in AAV library particles and wild-type viruses (Figure 3). The amount of circulating genomes decreased in a straight proportional manner. We therefore decided to harvest tissues in AAV library selections 48 hours after virus administration.


Successful expansion but not complete restriction of tropism of adeno-associated virus by in vivo biopanning of random virus display peptide libraries.

Michelfelder S, Kohlschütter J, Skorupa A, Pfennings S, Müller O, Kleinschmidt JA, Trepel M - PLoS ONE (2009)

Kinetics of circulating AAV peptide library particles is similar to wild-type AAV.A random X7 peptide library or wild-type AAV-2 viruses were injected intravenously at 1×1010 vg per mouse. Blood samples were collected after indicated time points and the amount of circulating viral particles in the serum was determined by real-time PCR. Data represent mean values from n = 3 mice per group, analyzed in triplicates ± SD.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2664470&req=5

pone-0005122-g003: Kinetics of circulating AAV peptide library particles is similar to wild-type AAV.A random X7 peptide library or wild-type AAV-2 viruses were injected intravenously at 1×1010 vg per mouse. Blood samples were collected after indicated time points and the amount of circulating viral particles in the serum was determined by real-time PCR. Data represent mean values from n = 3 mice per group, analyzed in triplicates ± SD.
Mentions: Based on the negative finding above, we hypothesized that selection under in vivo conditions is needed to enrich library clones that are able to bind cellular receptors in tumors, penetrate the tumor tissue and are internalized into tumor cells under physiological circulation conditions after intravenous administration. But we suspected that our novel PCR-based selection of AAV libraries may not be able to distinguish between library particles successfully internalized into target cells, and non-homing particles present in the circulation if the tissue is harvested too early after injection. To minimize the amount of circulating AAV library particles in our tissue samples at the time point of harvest, we analyzed the kinetics of circulating AAV library particles. Therefore, AAV were injected intravenously at 1×1010 vg per mouse, blood samples were collected at various time points, and the amount of circulating particles in the serum was quantified by real-time PCR. Clearance rates were comparable in AAV library particles and wild-type viruses (Figure 3). The amount of circulating genomes decreased in a straight proportional manner. We therefore decided to harvest tissues in AAV library selections 48 hours after virus administration.

Bottom Line: Analysis of peptide sequences of AAV clones after several rounds of selection yielded distinct sequence motifs for both tissues.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Hematology and Oncology, University of Freiburg Medical Center, Freiburg, Germany.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus