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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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Related in: MedlinePlus

Pathways used for selection of targeted viral capsids by screening random AAV display peptide libraries.For all selection pathways, genomic DNA containing cap gene fragments from internalized library viruses was extracted from the target cells or tissue. Library inserts were amplified by nested PCR and cloned back into the AAV library backbone plasmid pMT-202-6. The resulting pre-selected plasmid library was used to produce a secondary AAV library by transfection into 293T cells and subsequent superinfection with Ad5. Pre-selected AAV libraries were re-subjected to selection on the target cells in vitro or the target tissue in vivo. Preceding the amplification step, the library selection was done according to one of the following three pathways: Pathway A, in vitro selection: A random AAV display peptide library was incubated on primary breast cancer dissociation cultures derived from female tumor-bearing PymT mice. Non-internalized AAV library particles were removed by extensive washing followed by trypsin digestion prior to DNA extraction and AAV insert amplification. Pathway B, in vivo/ex vivo selection: A random AAV display peptide library was injected intravenously into female tumor-bearing PymT mice. After 24 hours, primary tumor cells of the injected mouse were prepared as in pathway A and grown ex vivo for 96 hours prior to DNA extraction and AAV insert amplification. Pathway C, in vivo selection: A random AAV display peptide library was injected as in pathway B in tumor-bearing mice (for selection of tumor-homing AAV) or wild-type mice (for selection of lung homing AAV), respectively. After 48 hours, the target tissue (tumor or lung, respectively) was removed and lysed, and DNA was extracted for AAV insert amplification.
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pone-0005122-g001: Pathways used for selection of targeted viral capsids by screening random AAV display peptide libraries.For all selection pathways, genomic DNA containing cap gene fragments from internalized library viruses was extracted from the target cells or tissue. Library inserts were amplified by nested PCR and cloned back into the AAV library backbone plasmid pMT-202-6. The resulting pre-selected plasmid library was used to produce a secondary AAV library by transfection into 293T cells and subsequent superinfection with Ad5. Pre-selected AAV libraries were re-subjected to selection on the target cells in vitro or the target tissue in vivo. Preceding the amplification step, the library selection was done according to one of the following three pathways: Pathway A, in vitro selection: A random AAV display peptide library was incubated on primary breast cancer dissociation cultures derived from female tumor-bearing PymT mice. Non-internalized AAV library particles were removed by extensive washing followed by trypsin digestion prior to DNA extraction and AAV insert amplification. Pathway B, in vivo/ex vivo selection: A random AAV display peptide library was injected intravenously into female tumor-bearing PymT mice. After 24 hours, primary tumor cells of the injected mouse were prepared as in pathway A and grown ex vivo for 96 hours prior to DNA extraction and AAV insert amplification. Pathway C, in vivo selection: A random AAV display peptide library was injected as in pathway B in tumor-bearing mice (for selection of tumor-homing AAV) or wild-type mice (for selection of lung homing AAV), respectively. After 48 hours, the target tissue (tumor or lung, respectively) was removed and lysed, and DNA was extracted for AAV insert amplification.

Mentions: A random X7 AAV display peptide library (random insert introduced at position R588 VP1 capsid protein numbering) with a diversity of 2×108 random clones (determined at the cloned plasmid level) was produced using a three-step protocol as described previously [14], [35]. For in vitro biopanning (Figure 1, pathway A), 2×106 primary PymT breast cancer cells were incubated with the AAV library at a multiplicity of infection (MOI) of 1.000 vector genomes (vg)/cell in selection round 1, 500 vg/cell in round 2, and 100 vg/cell in round 3. After 96 hours, unbound AAV library particles were removed by 3 washing steps in PBS. Surface-bound library viruses were detached by trypsin digestion for 20 minutes and subsequent washing. Previous work had shown that this additional trypsin digest is essential to enrich internalizing clones for improved transduction of the target cells (M.T., unpublished observation). Whole cellular DNA was extracted using the QIAamp Tissue Kit (Qiagen, Hilden, Germany). The random oligonucleotides contained in AAV library particles internalized into tumor cells were amplified by PCR using the primers 5′-GGTTCTCATCTTTGGGAAGCAAG-3′ and 5′-TGATGAGAATCTGTGGAGGAG-3′. For in vivo/ex vivo biopanning of AAV peptide libraries (Figure 1, pathway B), 1×1010 vg of an AAV library for selection round 1, or 2×108 to 2×109 vg per animal for round 2–4 were injected into the tail vein of female PymT transgenic mice bearing palpable breast tumors. After 24 hours, primary breast cancer cells were prepared as described above and grown in vitro for 96 hours. Oligonucleotide inserts of targeted AAV library particles were amplified by nested PCR using whole cellular DNA as template. Primers were 5′-ATGGCAAGCCACAAGGACGATG-3′ and 5′- CGTGGAGTACTGTGTGATGAAG-3′ for the first PCR and 5′-GGTTCTCATCTTTGGGAAGCAAG-3′ as well as 5′-TGATGAGAATCTGTGGAGGAG-3′ for the second PCR. Pure in vivo library biopanning (Figure 1, pathway C) was performed along the same lines, except that the circulation time was 48 hours and that DNA extraction from the tumor tissue was done without prior ex vivo growth of the cells. To select for lung homing AAV, libraries were injected into the tail vein of 6-week-old female PycB/FVB wild-type mice (n = 2 animals per selection round) as described for tumor selections (Figure 1, pathway C). DNA of whole lung tissue extracts from two animals was extracted, pooled and used as template to amplify the random oligonucleotide of lung-homing AAV. We varied the time of AAV blood circulation before lung harvest in 2 alternative selection approaches (5 minutes followed by a perfusion step, 48 hours in round 1, 48 hours or 6 days for round 2, and 6 days for round 3 to 4). For all selections, PCR products were analyzed by agarose gel electrophoresis to verify correct size, digested with BglI and cloned into the SfiI-digested pMT-202-6 library backbone plasmid [14], [35]. Cloned AAV library plasmids were transformed into electrocompetent E. coli DH5-α (Invitrogen) using the Gene Pulser (Bio-Rad, Hercules, CA). Randomly assigned clones were sequenced using the reverse primer 5′-CAGATGGGCCCCTGAAGGTA-3′. For production of pre-selected AAV peptide libraries, 2×108 293T cells were transfected with the library plasmids at a ratio of 25 plasmids/cell using Qiagen's PolyFect reagent. pUC18 (Invitrogen) served as carrier DNA. Two hours after transfection, 293T cells were superinfected with wild-type adenovirus type 5 (Ad5, supplied by the Laboratoire de Thérapie Génique, Nantes, France) at an MOI of 5 infectious particles/cell for library particle amplification. After 48 h, or when cell lysis became apparent, cells were detached from the culture dish in PBS-MK (140 mM NaCl, 5.5 mM KCl, 8 mM Na2HPO4, 1.5 mM KH2PO4, 1 mM MgCl2) and pooled with supernatants. AAV library particles were harvested by cell lysis via three freeze-thaw cycles. Cellular DNA was removed by incubation with benzonase (Sigma) at 50 U/ml lysate at 37°C for 30 min, followed by Ad5 inactivation at 55°C for 30 min. Viral library preparations were purified using the iodixanol gradient centrifugation method as previously described [42], [43]. The 40% iodixanol fraction containing the purified AAV viruses was stored at −80°C until further use.


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)

Pathways used for selection of targeted viral capsids by screening random AAV display peptide libraries.For all selection pathways, genomic DNA containing cap gene fragments from internalized library viruses was extracted from the target cells or tissue. Library inserts were amplified by nested PCR and cloned back into the AAV library backbone plasmid pMT-202-6. The resulting pre-selected plasmid library was used to produce a secondary AAV library by transfection into 293T cells and subsequent superinfection with Ad5. Pre-selected AAV libraries were re-subjected to selection on the target cells in vitro or the target tissue in vivo. Preceding the amplification step, the library selection was done according to one of the following three pathways: Pathway A, in vitro selection: A random AAV display peptide library was incubated on primary breast cancer dissociation cultures derived from female tumor-bearing PymT mice. Non-internalized AAV library particles were removed by extensive washing followed by trypsin digestion prior to DNA extraction and AAV insert amplification. Pathway B, in vivo/ex vivo selection: A random AAV display peptide library was injected intravenously into female tumor-bearing PymT mice. After 24 hours, primary tumor cells of the injected mouse were prepared as in pathway A and grown ex vivo for 96 hours prior to DNA extraction and AAV insert amplification. Pathway C, in vivo selection: A random AAV display peptide library was injected as in pathway B in tumor-bearing mice (for selection of tumor-homing AAV) or wild-type mice (for selection of lung homing AAV), respectively. After 48 hours, the target tissue (tumor or lung, respectively) was removed and lysed, and DNA was extracted for AAV insert amplification.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0005122-g001: Pathways used for selection of targeted viral capsids by screening random AAV display peptide libraries.For all selection pathways, genomic DNA containing cap gene fragments from internalized library viruses was extracted from the target cells or tissue. Library inserts were amplified by nested PCR and cloned back into the AAV library backbone plasmid pMT-202-6. The resulting pre-selected plasmid library was used to produce a secondary AAV library by transfection into 293T cells and subsequent superinfection with Ad5. Pre-selected AAV libraries were re-subjected to selection on the target cells in vitro or the target tissue in vivo. Preceding the amplification step, the library selection was done according to one of the following three pathways: Pathway A, in vitro selection: A random AAV display peptide library was incubated on primary breast cancer dissociation cultures derived from female tumor-bearing PymT mice. Non-internalized AAV library particles were removed by extensive washing followed by trypsin digestion prior to DNA extraction and AAV insert amplification. Pathway B, in vivo/ex vivo selection: A random AAV display peptide library was injected intravenously into female tumor-bearing PymT mice. After 24 hours, primary tumor cells of the injected mouse were prepared as in pathway A and grown ex vivo for 96 hours prior to DNA extraction and AAV insert amplification. Pathway C, in vivo selection: A random AAV display peptide library was injected as in pathway B in tumor-bearing mice (for selection of tumor-homing AAV) or wild-type mice (for selection of lung homing AAV), respectively. After 48 hours, the target tissue (tumor or lung, respectively) was removed and lysed, and DNA was extracted for AAV insert amplification.
Mentions: A random X7 AAV display peptide library (random insert introduced at position R588 VP1 capsid protein numbering) with a diversity of 2×108 random clones (determined at the cloned plasmid level) was produced using a three-step protocol as described previously [14], [35]. For in vitro biopanning (Figure 1, pathway A), 2×106 primary PymT breast cancer cells were incubated with the AAV library at a multiplicity of infection (MOI) of 1.000 vector genomes (vg)/cell in selection round 1, 500 vg/cell in round 2, and 100 vg/cell in round 3. After 96 hours, unbound AAV library particles were removed by 3 washing steps in PBS. Surface-bound library viruses were detached by trypsin digestion for 20 minutes and subsequent washing. Previous work had shown that this additional trypsin digest is essential to enrich internalizing clones for improved transduction of the target cells (M.T., unpublished observation). Whole cellular DNA was extracted using the QIAamp Tissue Kit (Qiagen, Hilden, Germany). The random oligonucleotides contained in AAV library particles internalized into tumor cells were amplified by PCR using the primers 5′-GGTTCTCATCTTTGGGAAGCAAG-3′ and 5′-TGATGAGAATCTGTGGAGGAG-3′. For in vivo/ex vivo biopanning of AAV peptide libraries (Figure 1, pathway B), 1×1010 vg of an AAV library for selection round 1, or 2×108 to 2×109 vg per animal for round 2–4 were injected into the tail vein of female PymT transgenic mice bearing palpable breast tumors. After 24 hours, primary breast cancer cells were prepared as described above and grown in vitro for 96 hours. Oligonucleotide inserts of targeted AAV library particles were amplified by nested PCR using whole cellular DNA as template. Primers were 5′-ATGGCAAGCCACAAGGACGATG-3′ and 5′- CGTGGAGTACTGTGTGATGAAG-3′ for the first PCR and 5′-GGTTCTCATCTTTGGGAAGCAAG-3′ as well as 5′-TGATGAGAATCTGTGGAGGAG-3′ for the second PCR. Pure in vivo library biopanning (Figure 1, pathway C) was performed along the same lines, except that the circulation time was 48 hours and that DNA extraction from the tumor tissue was done without prior ex vivo growth of the cells. To select for lung homing AAV, libraries were injected into the tail vein of 6-week-old female PycB/FVB wild-type mice (n = 2 animals per selection round) as described for tumor selections (Figure 1, pathway C). DNA of whole lung tissue extracts from two animals was extracted, pooled and used as template to amplify the random oligonucleotide of lung-homing AAV. We varied the time of AAV blood circulation before lung harvest in 2 alternative selection approaches (5 minutes followed by a perfusion step, 48 hours in round 1, 48 hours or 6 days for round 2, and 6 days for round 3 to 4). For all selections, PCR products were analyzed by agarose gel electrophoresis to verify correct size, digested with BglI and cloned into the SfiI-digested pMT-202-6 library backbone plasmid [14], [35]. Cloned AAV library plasmids were transformed into electrocompetent E. coli DH5-α (Invitrogen) using the Gene Pulser (Bio-Rad, Hercules, CA). Randomly assigned clones were sequenced using the reverse primer 5′-CAGATGGGCCCCTGAAGGTA-3′. For production of pre-selected AAV peptide libraries, 2×108 293T cells were transfected with the library plasmids at a ratio of 25 plasmids/cell using Qiagen's PolyFect reagent. pUC18 (Invitrogen) served as carrier DNA. Two hours after transfection, 293T cells were superinfected with wild-type adenovirus type 5 (Ad5, supplied by the Laboratoire de Thérapie Génique, Nantes, France) at an MOI of 5 infectious particles/cell for library particle amplification. After 48 h, or when cell lysis became apparent, cells were detached from the culture dish in PBS-MK (140 mM NaCl, 5.5 mM KCl, 8 mM Na2HPO4, 1.5 mM KH2PO4, 1 mM MgCl2) and pooled with supernatants. AAV library particles were harvested by cell lysis via three freeze-thaw cycles. Cellular DNA was removed by incubation with benzonase (Sigma) at 50 U/ml lysate at 37°C for 30 min, followed by Ad5 inactivation at 55°C for 30 min. Viral library preparations were purified using the iodixanol gradient centrifugation method as previously described [42], [43]. The 40% iodixanol fraction containing the purified AAV viruses was stored at −80°C until further use.

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