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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.

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Gene delivery by AAV capsid mutants selected for breast cancer transduction in vivo.AAV luciferase vectors displaying selected peptides or controls (wild-type or VRRPRFW) were injected intravenously into female PymT tumor-bearing mice. After 8 d, representative tissues were harvested and luciferase activities were determined in individual tissues as relative light units (RLU) per mg protein. A: In vivo transduction of tumor tissue in PymT transgenic FVB mice by selected AAV mutants. Bars indicate the median, n = 5 mice per group. * p<0.05 targeted vectors vs. wild-type. # p<0.05 targeted vectors vs. random insert control. B: In vivo transduction of various non-cancerous tissues in PymT transgenic FVB mice by tumor-selected AAV mutants. The dotted line indicates the threshold beyond which luciferase expression data could be reliably delineated from background signal. Data represent mean values ± SEM, n = 5 mice per group. * p<0.05; ** p<0.01 targeted vectors vs. wild-type AAV-2. # p<0.05; ## p<0.01 targeted vectors vs. random insert control.
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pone-0005122-g004: Gene delivery by AAV capsid mutants selected for breast cancer transduction in vivo.AAV luciferase vectors displaying selected peptides or controls (wild-type or VRRPRFW) were injected intravenously into female PymT tumor-bearing mice. After 8 d, representative tissues were harvested and luciferase activities were determined in individual tissues as relative light units (RLU) per mg protein. A: In vivo transduction of tumor tissue in PymT transgenic FVB mice by selected AAV mutants. Bars indicate the median, n = 5 mice per group. * p<0.05 targeted vectors vs. wild-type. # p<0.05 targeted vectors vs. random insert control. B: In vivo transduction of various non-cancerous tissues in PymT transgenic FVB mice by tumor-selected AAV mutants. The dotted line indicates the threshold beyond which luciferase expression data could be reliably delineated from background signal. Data represent mean values ± SEM, n = 5 mice per group. * p<0.05; ** p<0.01 targeted vectors vs. wild-type AAV-2. # p<0.05; ## p<0.01 targeted vectors vs. random insert control.

Mentions: To assess whether the in vivo-selected AAV-2 vectors mediate gene expression in tumor tissue in vivo, we produced luciferase reporter vectors displaying the selected peptide sequences. All vectors titers ranged between 8×1010 to 7×1011 vg/ml (data not shown). Luciferase gene expression in the breast tumor tissue was evaluated 8 days after intravenous injection in tumor-bearing PymT mice in a screening experiment to assess which clones should be investigated in detail. Five of the clones (GEARISA, SGNSGAA, ESGLSQS, EYRDSSG, and DLGSARA) showed an increased transduction of the breast tumor tissue compared to wild-type AAV vectors, whereas unselected control vectors did not mediate any gene expression (data not shown). We chose the most promising vectors for further experiments in a larger group of animals (n = 5 mice per clone). Following intravenous injection, the selected clones transduced tumor tissue up to ∼275-fold more efficient compared to wild-type AAV vectors (Figure 4A). To further investigate the specificity of selected AAV capsid mutants, luciferase expression in several control organs was evaluated (Figure 4B). Moderate de-targeting from the liver by clone ESGLSQS and the unselected control was observed, whereas clones GEARISA and EYRDSSG transduced the liver in a manner comparable to wild-type AAV. DLGSARA gene transduction in liver tissue was significantly increased compared to the unselected control vector. Further, we found a strongly enhanced cardiac luciferase expression for all clones, being statistically significant for GEARISA, EYRDSSG and DLGSARA, and a weakly enhanced cardiac transduction of the unselected control vector compared to wild-type AAV. In regard to tissue specificity, the ESGLSQS clone had the most favorable profile as it transduced tumor tissue but not the liver. However, cardiac gene transduction was seen for this as for almost all the other clones as well. Reproduction of in vivo gene transduction with independent vector preparations for DLGSARA and ESGLSQS precisely confirmed our results (data not shown).


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)

Gene delivery by AAV capsid mutants selected for breast cancer transduction in vivo.AAV luciferase vectors displaying selected peptides or controls (wild-type or VRRPRFW) were injected intravenously into female PymT tumor-bearing mice. After 8 d, representative tissues were harvested and luciferase activities were determined in individual tissues as relative light units (RLU) per mg protein. A: In vivo transduction of tumor tissue in PymT transgenic FVB mice by selected AAV mutants. Bars indicate the median, n = 5 mice per group. * p<0.05 targeted vectors vs. wild-type. # p<0.05 targeted vectors vs. random insert control. B: In vivo transduction of various non-cancerous tissues in PymT transgenic FVB mice by tumor-selected AAV mutants. The dotted line indicates the threshold beyond which luciferase expression data could be reliably delineated from background signal. Data represent mean values ± SEM, n = 5 mice per group. * p<0.05; ** p<0.01 targeted vectors vs. wild-type AAV-2. # p<0.05; ## p<0.01 targeted vectors vs. random insert control.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2664470&req=5

pone-0005122-g004: Gene delivery by AAV capsid mutants selected for breast cancer transduction in vivo.AAV luciferase vectors displaying selected peptides or controls (wild-type or VRRPRFW) were injected intravenously into female PymT tumor-bearing mice. After 8 d, representative tissues were harvested and luciferase activities were determined in individual tissues as relative light units (RLU) per mg protein. A: In vivo transduction of tumor tissue in PymT transgenic FVB mice by selected AAV mutants. Bars indicate the median, n = 5 mice per group. * p<0.05 targeted vectors vs. wild-type. # p<0.05 targeted vectors vs. random insert control. B: In vivo transduction of various non-cancerous tissues in PymT transgenic FVB mice by tumor-selected AAV mutants. The dotted line indicates the threshold beyond which luciferase expression data could be reliably delineated from background signal. Data represent mean values ± SEM, n = 5 mice per group. * p<0.05; ** p<0.01 targeted vectors vs. wild-type AAV-2. # p<0.05; ## p<0.01 targeted vectors vs. random insert control.
Mentions: To assess whether the in vivo-selected AAV-2 vectors mediate gene expression in tumor tissue in vivo, we produced luciferase reporter vectors displaying the selected peptide sequences. All vectors titers ranged between 8×1010 to 7×1011 vg/ml (data not shown). Luciferase gene expression in the breast tumor tissue was evaluated 8 days after intravenous injection in tumor-bearing PymT mice in a screening experiment to assess which clones should be investigated in detail. Five of the clones (GEARISA, SGNSGAA, ESGLSQS, EYRDSSG, and DLGSARA) showed an increased transduction of the breast tumor tissue compared to wild-type AAV vectors, whereas unselected control vectors did not mediate any gene expression (data not shown). We chose the most promising vectors for further experiments in a larger group of animals (n = 5 mice per clone). Following intravenous injection, the selected clones transduced tumor tissue up to ∼275-fold more efficient compared to wild-type AAV vectors (Figure 4A). To further investigate the specificity of selected AAV capsid mutants, luciferase expression in several control organs was evaluated (Figure 4B). Moderate de-targeting from the liver by clone ESGLSQS and the unselected control was observed, whereas clones GEARISA and EYRDSSG transduced the liver in a manner comparable to wild-type AAV. DLGSARA gene transduction in liver tissue was significantly increased compared to the unselected control vector. Further, we found a strongly enhanced cardiac luciferase expression for all clones, being statistically significant for GEARISA, EYRDSSG and DLGSARA, and a weakly enhanced cardiac transduction of the unselected control vector compared to wild-type AAV. In regard to tissue specificity, the ESGLSQS clone had the most favorable profile as it transduced tumor tissue but not the liver. However, cardiac gene transduction was seen for this as for almost all the other clones as well. Reproduction of in vivo gene transduction with independent vector preparations for DLGSARA and ESGLSQS precisely confirmed our results (data not shown).

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