Team:Freiburg Bioware/Project/insertion of motifs into surface-exposed loops
From 2010.igem.org
(6 intermediate revisions not shown) | |||
Line 1: | Line 1: | ||
- | |||
{{:Team:Freiburg_Bioware/Head}} | {{:Team:Freiburg_Bioware/Head}} | ||
{{:Team:Freiburg_Bioware/jquery}} | {{:Team:Freiburg_Bioware/jquery}} | ||
{{:Team:Freiburg_Bioware/menu_home}} | {{:Team:Freiburg_Bioware/menu_home}} | ||
- | |||
<html> | <html> | ||
- | <div align=center> | + | <head> |
- | + | </head> | |
- | <table class=MsoTableGrid | + | <body> |
- | + | <h1>Loop Insertion</h1> | |
- | + | <h2>Modification of the Viral Capsid of the AAV2 using Viral Bricks</h2> | |
- | + | <p style="text-align: justify; margin-top: 10px;"> | |
- | + | For therapeutical applications in human gene transfer, the broad tropism for heparan sulfate proteoglycan (HSPG) has to be knocked-out and a novel tropism has to be inserted. | |
- | + | This retargeting can be achieved either by insertion of functional motifs into the two major surface exposed loops or by fusion of these motifs to the N-terminus of the viral coat proteins. | |
- | + | The graphic on the right shows parts of the three-dimensional structure of a viral coat protein. The parts of the loop regions that are coded in the ViralBricks are shown in purple for the 453 loop and in blue for the 587 loop. | |
- | + | </p> | |
- | + | <img style="border: 0px solid ; width: 571px; height: 269px;" alt="" id="Picture 15" | |
- | + | src="https://static.igem.org/mediawiki/2010/8/8f/Freiburg10_loop_insertion_sites.png"> | |
- | + | <p style="font-size: 11px;">Three-dimensional representation of the AAV2 showing the amino acids of the 453 and 587 loops that are coded by the corresponding Viral Bricks.</p> | |
- | + | <h2>Cloning of Viral Bricks into capsid coding parts</h2> | |
- | + | <p>In order to make loop insertions more convenient the following restriction sites were inserted into all capsid coding parts and already existing restriction sites were removed from the constructs. The choice of these restriction sites was reasoned by enzyme performance, buffer compatibilities and the number of existing restriction sites that had to be removed at other positions. | |
- | + | All restriction endonucleases were purchased from NEB. | |
- | + | </p> | |
- | + | <img src="https://static.igem.org/mediawiki/2010/7/73/Freiburg_10_Bioware_Table.png" style="margin-bottom: 30px;"/> | |
- | + | <h1>His-Affinity tag</h1> | |
- | + | <div align="center"> | |
+ | <table class="MsoTableGrid" | ||
+ | style="border: medium none ; margin-left: 180.8pt; border-collapse: collapse;" | ||
+ | border="0" cellpadding="0" cellspacing="0"> | ||
+ | <tbody> | ||
+ | <tr style="height: 142.35pt;"> | ||
+ | <td style="padding: 0cm 5.4pt; width: 300.3pt; height: 142.35pt;" | ||
+ | valign="top" width="400"> | ||
+ | <p class="MsoNormal" style="text-align: left; text-indent: 0cm;" | ||
+ | align="left"><img style="width: 395px; height: 198px;" | ||
+ | src="https://static.igem.org/mediawiki/2010/4/44/Freiburg10_His-tag.png" | ||
+ | alt="" align="left" hspace="12"></p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr style="height: 22.6pt;"> | ||
+ | <td style="padding: 0cm 5.4pt; width: 300.3pt; height: 22.6pt;" | ||
+ | valign="top" width="400"> | ||
+ | <p style="text-align: justify;">Schematic | ||
+ | figure of His-affinity Tag insertion into the viral capsid</p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | </tbody> | ||
</table> | </table> | ||
- | |||
</div> | </div> | ||
- | + | <p style="text-align: justify;">The insertion of a His-Affinity Tag | |
- | <p | + | into the exposed major surface |
- | + | ||
- | + | ||
- | + | ||
- | + | ||
loops of the viral vectors allows their specific affinity purification | loops of the viral vectors allows their specific affinity purification | ||
- | employing e.g. Ni-NTA affinity chromatography. This purification method was | + | employing e.g. Ni-NTA affinity chromatography. This purification method |
- | tested using the cell culture lysate of transfected AAV-293 cells that were | + | was |
- | either grown in DMEM supplemented with 10% FCS or in the serum-free GIBCO® | + | tested using the cell culture lysate of transfected AAV-293 cells that |
- | FreeStyle™ 293 Expression Medium (Invitrogen). The usage of serum-free media is | + | were |
- | a technological modification meant to facilitate the production of pure viral | + | either grown in DMEM supplemented with 10% FCS or in the serum-free |
- | vectors. Purified viral vectors are important for several applications such as | + | GIBCO® |
- | animal models and biophysical characterizations. On the other hand, the elution | + | FreeStyle™ 293 Expression Medium (Invitrogen). The usage of serum-free |
- | of the His-tagged viral vectors allows also enrichment of transgene viral | + | media is |
- | vectors. In order to answer the question if and to what degree viral vectors | + | a technological modification meant to facilitate the production of pure |
- | are transferred into the media, the cells were centrifugated, then divided into | + | viral |
- | the pellet fraction and the supernatant. Physical cell lysis was performed for | + | vectors. Purified viral vectors are important for several applications |
- | both fractions of the two produced batches (serum-free and FCS media) by | + | such as |
- | performing four cycles of freeze and thaw.< | + | animal models and biophysical characterizations. On the other hand, the |
- | + | elution | |
- | < | + | of the His-tagged viral vectors allows also enrichment of transgene |
- | + | viral | |
- | + | vectors. In order to answer the question if and to what degree viral | |
- | + | vectors | |
- | + | are transferred into the media, the cells were centrifugated, then | |
- | + | divided into | |
- | + | the pellet fraction and the supernatant. Physical cell lysis was | |
- | + | performed for | |
- | + | both fractions of the two produced batches (serum-free and FCS media) | |
- | + | by | |
- | + | performing four cycles of freeze and thaw.<br> | |
- | + | </p> | |
- | + | <table class="MsoTableGrid" | |
- | + | style="border: medium none ; border-collapse: collapse; text-align: left; margin-left: auto; margin-right: auto;" | |
- | + | border="0" cellpadding="0" cellspacing="0"> | |
- | + | <tbody> | |
- | + | <tr> | |
- | + | <td | |
- | + | style="padding: 0cm 5.4pt; vertical-align: top; width: 477.3pt;"> | |
- | + | <p class="MsoNormal" style="text-align: left; text-indent: 0cm;" | |
- | + | align="left"><img style="width: 627px; height: 567px;" alt="" | |
- | + | id="Bild 1" | |
+ | src="https://static.igem.org/mediawiki/2010/0/0b/Freiburg10_His-experiment.png"></p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td style="padding: 0cm 5.4pt; width: 477.3pt;" valign="top" | ||
+ | width="636"> | ||
+ | <p class="MsoNormal" style="text-align: left; text-indent: 0cm;" | ||
+ | align="left"><span lang="EN-US"> | ||
+ | <p style="text-align: justify;">Experimental setup for the | ||
+ | purification of His-tag presenting viral particles produced by AAV-293 | ||
+ | cells in DMEM or the protein- and animal-free Freestyle medium. | ||
+ | Purified viral particles can be detected by ELISA using an | ||
+ | anti-poly-Histidine antibody or qPCR for encapsidated vector plasmids, | ||
+ | respectively</p> | ||
+ | </span></p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | </tbody> | ||
</table> | </table> | ||
- | + | <p class="MsoNormal" style="text-align: left; text-indent: 0cm;" | |
- | <p class=MsoNormal | + | align="left"><span lang="EN-US"> </span></p> |
- | lang=EN-US> </span></p> | + | <p class="MsoNormal" style="text-align: left; text-indent: 0cm;" |
- | + | align="left"><span lang="EN-US"> | |
- | <p class=MsoNormal | + | <h2>Material and Methods:</h2> |
- | lang=EN-US>Material and Methods:</span></p> | + | </span></p> |
- | + | <p class="MsoNormal" style="text-align: justify; text-indent: 0cm;" | |
- | <p class=MsoNormal | + | align="left">Transfection of the AAV-293 producer |
- | + | cells was performed in five 10 cm | |
- | petri dishes with 3.6x10^6 cells, resulting in a confluency of about 70-80% | + | petri dishes with 3.6x10^6 cells, resulting in a confluency of about |
- | according to the standard protocol either with cells grown in GIBCO® FreeStyle™ | + | 70-80% |
- | 293 Expression Medium (Invitrogen, protein- and animal-origin free) or in DMEM | + | according to the standard protocol either with cells grown in GIBCO® |
+ | FreeStyle™ | ||
+ | 293 Expression Medium (Invitrogen, protein- and animal-origin free) or | ||
+ | in DMEM | ||
supplemented with 10% FCS (PAA). For transfection, the composite parts | supplemented with 10% FCS (PAA). For transfection, the composite parts | ||
- | pCMV_VP123(587-His) and RepVP123(587-KO)_p5-TATA-less were used in an 1:1 ratio | + | pCMV_VP123(587-His) and RepVP123(587-KO)_p5-TATA-less were used in an |
- | together with | + | 1:1 ratio |
- | and | + | together with pHelper |
- | + | and [AAV2]-left-ITR_pCMV_betaglobin_mVenus_hGH_[AAV2]-right-ITR. Cells | |
- | were divided into the pellet and the supernatant fractions. Physical cell lysis | + | were spun down at 200 x g for five minutes and the |
- | was performed by four cycles of freeze and thaw for all four samples. The cell | + | samples |
- | lysate / supernatant fractions were incubated with 800 µl of His-Affinity Gel | + | were divided into the pellet and the supernatant fractions. Physical |
- | (kindly provided by Zymo | + | cell lysis |
- | agitation. The beads were then collected in 5 ml gravity-flow columns and | + | was performed by four cycles of freeze and thaw for all four samples. |
- | washed five times with one column volume of PBS each. The His-affinity gel was subsequently | + | The cell |
- | washed with PBS, 25 mM Imidazole to remove unspecifically bound proteins. | + | lysate / supernatant fractions were incubated with 800 µl of |
- | Elution was performed in an second step with PBS, 500 mM Imidazole to elute the | + | His-Affinity Gel |
- | His-tagged viral vectors. The genomic titer of the purified viral vectors was | + | (kindly provided by Zymo Resear ch, USA) at 4 °C for |
- | detected via q-PCR. In an ELISA, viral vectors were captured employing the | + | 18 hours with 200 |
- | monoclonal antibody A20 (kindly provided by PD Dr. J. Kleinschmidt, DKFZ, Heidelberg) | + | rpm constant |
+ | agitation. The beads were then collected in 5 ml gravity-flow columns | ||
+ | and | ||
+ | washed five times with one column volume of PBS each. The His-affinity | ||
+ | gel was subsequently | ||
+ | washed with PBS, 25 mM Imidazole to remove unspecifically bound | ||
+ | proteins. | ||
+ | Elution was performed in an second step with PBS, 500 mM Imidazole to | ||
+ | elute the | ||
+ | His-tagged viral vectors. The genomic titer of the purified viral | ||
+ | vectors was | ||
+ | detected via q-PCR. In an ELISA, viral vectors were captured employing | ||
+ | the | ||
+ | monoclonal antibody A20 (kindly provided by PD Dr. J. Kleinschmidt, | ||
+ | DKFZ, Heidelberg) | ||
that exclusively recognizes assembled AAV capsids. His-Tags present in | that exclusively recognizes assembled AAV capsids. His-Tags present in | ||
- | assembled viral capsids were subsequently detected with an HRP-tagged secondary | + | assembled viral capsids were subsequently detected with an HRP-tagged |
- | anti-His-Tag antibody (1:2000 diluted, A7058, Sigma). | + | secondary |
- | + | anti-His-Tag antibody (1:2000 diluted, A7058, Sigma). HRP presence | |
- | peroxidase substrate ABTS. Generation of blue-green color (absorption at 405 | + | was detected using the |
- | nm) was measured in a Tecan Sunrise plate reader. Sample data were blanked with | + | peroxidase substrate ABTS. Generation of blue-green color (absorption |
- | the average of the non-template controls (NTC).< | + | at 405 |
- | + | nm) was measured in a Tecan Sunrise plate reader. Sample data were | |
- | <p class=MsoNormal | + | blanked with |
- | + | the average of the non-template controls (NTC). <br> | |
- | + | </p> | |
- | + | <p class="MsoNormal" style="text-align: left; text-indent: 0cm;" | |
- | lang=EN-US>Results and Discussion | + | align="left"><span lang="EN-US"> |
- | + | <h2>Results and Discussion</h2> | |
- | + | </span></p> | |
- | + | <p class="MsoNormal" | |
- | + | style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><span | |
- | + | lang="EN-US"> | |
- | + | <p style="text-align: justify;">Presence of the His-affinity tag in | |
- | + | the viral capsid was detected and the ELISA enabled quantification of | |
- | + | the | |
- | + | purification procedure efficiency. The absorbance measured for the | |
- | + | elution | |
- | + | fractions of the 1/10 diluted samples sums up to 2.3 for the DMEM- and | |
- | + | 0.5 for | |
- | <p | + | the Free Style 293-grown cells, assigning the DMEM-grown cells a five |
- | + | times | |
- | the viral capsid was detected and the ELISA enabled quantification of the | + | higher production efficiency. Comparison between the cell pellet and |
- | purification procedure efficiency. The absorbance measured for the elution | + | the |
- | fractions of the 1/10 diluted samples sums up to 2.3 for the DMEM- and 0.5 for | + | supernatant fractions revealed that 70 - 80% of the viral particles can |
- | the Free Style 293-grown cells, assigning the DMEM-grown cells a five times | + | be |
- | higher production efficiency. Comparison between the cell pellet and the | + | found inside the producer cells.</p> |
- | supernatant fractions revealed that 70 - 80% of the viral particles can be | + | </span></p> |
- | found inside the producer cells.</span></p> | + | <p class="MsoNormal" |
- | + | style="margin-bottom: 10pt; text-align: left; text-indent: 0cm; line-height: 115%;" | |
- | <p class=MsoNormal style= | + | align="left"><span lang="EN-US"> |
- | 0cm;line-height: | + | <p style="text-align: justify;">According to these results, |
- | + | producer cells should be grown in complex media for <i>in vitro</i> | |
- | <p | + | and cell |
- | + | culture experiments. Use of serum-free produced viral vectors is | |
- | producer cells should be grown in complex media for <i>in vitro</i> and cell | + | recommended |
- | culture experiments. Use of serum-free produced viral vectors is recommended | + | for mouse or other animal experiments and possible therapeutical |
- | for mouse or other animal experiments and possible therapeutical applications | + | applications |
where even the presence of traces amounts of fetal calf serum should be | where even the presence of traces amounts of fetal calf serum should be | ||
avoided. Combination with different purification approaches such as gel | avoided. Combination with different purification approaches such as gel | ||
- | filtration chromatography using i.e. Superdex 200 columns (GE Healthcare) | + | filtration chromatography using i.e. Superdex 200 columns (GE |
- | enables the production of highly purified viral vectors for several different | + | Healthcare) |
- | applications.<br | + | enables the production of highly purified viral vectors for several |
+ | different | ||
+ | applications.</p> | ||
+ | <br style="page-break-before: always;" clear="all"> | ||
</span></p> | </span></p> | ||
- | + | <table class="MsoTableGrid" | |
- | <table class=MsoTableGrid | + | style="border: medium none ; border-collapse: collapse; text-align: left; margin-left: auto; margin-right: auto;" |
- | + | border="0" cellpadding="0" cellspacing="0"> | |
- | + | <tbody> | |
- | + | <tr> | |
- | + | <td style="padding: 0cm 5.4pt; width: 477.3pt;" valign="top" | |
- | + | width="636"> | |
- | + | <p class="MsoNormal" | |
- | + | style="margin-bottom: 10pt; text-align: left; text-indent: 0cm; line-height: 115%;" | |
- | + | align="left"><img style="width: 451px; height: 796px;" alt="" | |
- | + | src="https://static.igem.org/mediawiki/2010/0/0d/Freiburg10_His-data.png" | |
- | + | align="left" hspace="12"></p> | |
- | + | </td> | |
- | + | </tr> | |
- | + | <tr> | |
- | + | <td style="padding: 0cm 5.4pt; width: 477.3pt;" valign="top" | |
- | + | width="636"> | |
- | + | <p class="MsoNormal" | |
- | + | style="margin-bottom: 10pt; text-align: left; text-indent: 0cm; line-height: 115%;" | |
- | + | align="left"><span lang="EN-US">A: Schematic overview of the sandwich | |
- | + | ELISA for the detection of His-tagged viral particles</span></p> | |
- | + | <p class="MsoNormal" | |
- | + | style="margin-bottom: 10pt; text-align: left; text-indent: 0cm; line-height: 115%;" | |
- | + | align="left"><span lang="EN-US">B: ELISA from viral particles produced | |
- | + | by AAV-293 cells in DMEM or Free Style medium, divided into cell pellet | |
- | + | and cell culture supernatant samples. The particles were purified using | |
- | + | Ni-NTA affinity chromatography with Imidazole in PBS as washing and | |
- | + | elution agent</span></p> | |
- | + | <p class="MsoNormal" | |
- | + | style="margin-bottom: 10pt; text-align: left; text-indent: 0cm; line-height: 115%;" | |
+ | align="left"><span lang="EN-US">C: Absorption measurements from plate | ||
+ | shown in B. Undiluted Äkta fractions converted ABTS peroxidase | ||
+ | substrate at 405 nm</span></p> | ||
+ | <p class="MsoNormal" | ||
+ | style="margin-bottom: 10pt; text-align: left; text-indent: 0cm; line-height: 115%;" | ||
+ | align="left"><span lang="EN-US">D: As C, whereas Äkta fractions were | ||
+ | 10-fold diluted</span></p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | </tbody> | ||
</table> | </table> | ||
- | + | <br> | |
- | + | <p class="MsoNormal" style="text-align: left; text-indent: 0cm;" | |
- | + | align="left"> | |
- | + | <h1>Biotinylation Acceptor | |
- | + | Peptide (BAP)</h1> | |
- | <p class=MsoNormal | + | </p> |
- | text-indent:0cm; | + | <div align="center"> |
- | + | <table class="MsoTableGrid" | |
- | + | style="border: medium none ; border-collapse: collapse;" border="0" | |
- | + | cellpadding="0" cellspacing="0"> | |
- | + | <tbody> | |
- | < | + | <tr style="height: 187.45pt;"> |
- | + | <td style="padding: 0cm 5.4pt; width: 327.6pt; height: 187.45pt;" | |
- | Peptide (BAP)</ | + | valign="top" width="437"> |
- | + | <p class="MsoNormal" style="text-indent: 0cm;"><img | |
- | <div align=center> | + | style="width: 419px; height: 244px;" |
- | + | src="https://static.igem.org/mediawiki/2010/e/ea/Freiburg10_BAP-tag.png" | |
- | <table class=MsoTableGrid | + | alt="http://partsregistry.org/wiki/images/a/a9/Freiburg10_ViralBrick_motif_BAP.png" |
- | + | align="left" hspace="12"></p> | |
- | + | </td> | |
- | + | </tr> | |
- | + | <tr style="height: 39.75pt;"> | |
- | + | <td style="padding: 0cm 5.4pt; width: 327.6pt; height: 39.75pt;" | |
- | + | valign="top" width="437"> | |
- | + | <p class="MsoNormal" style="text-indent: 0cm;"><span | |
- | + | style="line-height: 100%;" lang="EN-US"> | |
- | + | <p style="text-align: justify;">Schematic overview of BAP | |
- | + | insertion into AAV-2 viral capsids, followed by biotinylation and | |
- | + | binding of streptavidin-coupled molecules</p> | |
- | + | </span></p> | |
- | + | </td> | |
- | + | </tr> | |
- | + | </tbody> | |
- | + | ||
- | + | ||
</table> | </table> | ||
- | |||
</div> | </div> | ||
- | + | <p style="text-align: justify;">The | |
- | <p | + | BAP |
- | (Biotinylation Acceptor Peptide) included in the Virus Construction Kit is a 15 | + | (Biotinylation Acceptor Peptide) included in the Virus Construction Kit |
- | amino acid peptide identified by Schatz et al. (1993) in a library screening | + | is a 15 |
- | approach and published under the number #85. This peptide with the sequence | + | amino acid peptide identified by Schatz et al. (1993) in a library |
- | GLNDIFEAQKIEWHE contains a central lysine residue that can be specifically | + | screening |
- | biotinylated by the prokaryotic holoenzyme biotin synthetase, encoded by the | + | approach and published under the number #85. This peptide with the |
- | BirA gene of <i>E. coli</i>. Specific biotinylation of this peptide sequence | + | sequence |
- | can be performed in vivo by cotransfecting a plasmid with the BirA gene as | + | GLNDIFEAQKIEWHE contains a central lysine residue that can be |
- | described for the AAV by Arnold et al. (2006) or by an in vitro coupling | + | specifically |
- | approach using the purified Escherichia coli enzyme biotin ligase (BirA). | + | biotinylated by the prokaryotic holoenzyme biotin synthetase, encoded |
- | + | by the | |
- | <p class=MsoNormal style= | + | BirA gene of <i>E. coli</i>. Specific biotinylation of this peptide |
- | specifically coupled to the viral loops can either be used to attach Streptavidin-coupled | + | sequence |
- | molecules to the viral capsid or to chemically couple molecules with a reactive | + | can be performed in vivo by cotransfecting a plasmid with the BirA gene |
- | group to biotin. Consequently, the inserted motif can enable the visualization | + | as |
+ | described for the AAV by Arnold et al. (2006) or by an in vitro | ||
+ | coupling | ||
+ | approach using the purified Escherichia coli enzyme biotin ligase | ||
+ | (BirA). <span lang="EN-US"><br> | ||
+ | </span></p> | ||
+ | <p class="MsoNormal" style="text-indent: 0cm;"><span lang="EN-US"> | ||
+ | <p class="MsoNormal" style="text-indent: 0cm;"><span lang="EN-US"> | ||
+ | <p style="text-align: justify;">Biotin | ||
+ | molecules | ||
+ | specifically coupled to the viral loops can either be used to attach | ||
+ | Streptavidin-coupled | ||
+ | molecules to the viral capsid or to chemically couple molecules with a | ||
+ | reactive | ||
+ | group to biotin. Consequently, the inserted motif can enable the | ||
+ | visualization | ||
of single virus particles by coupling fluorophores to the virus capsid | of single virus particles by coupling fluorophores to the virus capsid | ||
empowering further uses of the Virus Construction Kit in fundamental | empowering further uses of the Virus Construction Kit in fundamental | ||
virological research. In addition, targeting molecules such as | virological research. In addition, targeting molecules such as | ||
Streptavidin-coupled affinity molecules (i.e. Antibodies, Nanobodies or | Streptavidin-coupled affinity molecules (i.e. Antibodies, Nanobodies or | ||
- | Affibodies) can be coupled for manifold targeting approaches.</span></p> | + | Affibodies) can be coupled for manifold targeting approaches.</p> |
- | + | </span></p> | |
- | <p class=MsoNormal style= | + | </span></p> |
- | + | <p class="MsoNormal" style="text-indent: 0cm;"><span lang="EN-US"> | |
- | <span lang=EN-US | + | <h2>Materials |
- | + | and Methods:<span lang="EN-US"> </span></h2> | |
- | < | + | </span></p> |
- | + | <p class="MsoNormal" style="text-indent: 0cm;"><span lang="EN-US"> | |
- | + | <p style="text-align: justify;">Transfection | |
- | + | of the | |
- | + | AAV-293 producer cells was performed in five 10 cm petri dishes with | |
- | + | 3.4x10^6 | |
- | + | cells (grown in DMEM supplemented with 10% FCS) resulting in a | |
- | <p class=MsoNormal style= | + | confluence of |
- | + | 70-80% according to the standard protocol. Producer cells were | |
- | <p | + | harvested and |
- | AAV-293 producer cells was performed in five 10 cm petri dishes with 3.4x10^6 | + | together with the culture supernatant subjected to four cycles of |
- | cells (grown in DMEM supplemented with 10% FCS) resulting in a confluence of | + | freeze and |
- | 70-80% according to the standard protocol. Producer cells were harvested and | + | thaw cell lysis. The cell lysate was centrifuged at 4000 rpm for 15 |
- | together with the culture supernatant subjected to four cycles of freeze and | + | minutes and |
- | thaw cell lysis. The cell lysate was centrifuged at 4000 rpm for 15 minutes and | + | concentrated in a VivaSpin VS2002 column with 10 kDa molecular weight |
- | concentrated in a VivaSpin VS2002 column with 10 kDa molecular weight cut-off to | + | cut-off to |
- | yield two milliliters. Genomic DNA attached to the virus particles was degraded | + | yield two milliliters. Genomic DNA attached to the virus particles was |
- | using 250 units of Benzonase (EC 3.1.30.2, Sigma-Aldrich) at 37 °C for one | + | degraded |
- | hour. The concentrated cell lysate was washed three times with 4 ml 20 mM | + | using 250 units of Benzonase (EC 3.1.30.2, Sigma-Aldrich) at 37 °C for |
- | bis-Tris buffer pH 6.0, 100 mM NaCl. From this concentrated sample, 500 µl were | + | one |
- | loaded on the ÄKTA purifier (GE Healthcare) equipped with a Superdex 200 gel | + | hour. The concentrated cell lysate was washed three times with 4 ml 20 |
- | filtration column (GE Healthcare). This purification is also described by <span | + | mM |
- | class=apple-converted-space><span style= | + | bis-Tris buffer pH 6.0, 100 mM NaCl. From this concentrated sample, 500 |
- | around the void volume giving a UV absorbance peak were pooled and applied to a | + | µl were |
- | Amicon Ultra column (Millipore, size limit 100 kDa, 2 ml loading volume) and | + | loaded on the ÄKTA purifier (GE Healthcare) equipped with a Superdex |
- | concentrated to 500 µl. This purified virus sample was washed four times with 10 | + | 200 gel |
- | mM Tris-buffer pH 8.0 buffer which is recommended for the BirA biotin ligase | + | filtration column (GE Healthcare). This purification is also described |
- | that was kindly provided by Avidity LCC (Colorado, USA).</span></span></span></p> | + | by <span class="apple-converted-space"><span style="color: black;">Smith |
- | + | et al. (2003). Fractions | |
- | <p class=MsoNormal style= | + | around the void volume giving a UV absorbance peak were pooled and |
- | lang=EN-US style= | + | applied to a |
- | was mixed according to the manufacturer protocol with each 72 µl Biomix A, | + | Amicon Ultra column (Millipore, size limit 100 kDa, 2 ml loading |
+ | volume) and | ||
+ | concentrated to 500 µl. This purified virus sample was washed four | ||
+ | times with 10 | ||
+ | mM Tris-buffer pH 8.0 buffer which is recommended for the BirA biotin | ||
+ | ligase | ||
+ | that was kindly provided by Avidity LCC (Colorado, USA).</span></span></p> | ||
+ | </span></p> | ||
+ | <p class="MsoNormal" style="text-indent: 0cm;"><span | ||
+ | style="color: black;" lang="EN-US"> | ||
+ | <p style="text-align: justify;">The | ||
+ | BAP-containing viral vector sample (500 µl) | ||
+ | was mixed according to the manufacturer protocol with each 72 µl Biomix | ||
+ | A, | ||
Biomix B and Biotin. To reach maximal biotinylation, a volume of 5 µl | Biomix B and Biotin. To reach maximal biotinylation, a volume of 5 µl | ||
- | containing 25000 units of the biotin ligase BirA was added to the reaction | + | containing 25000 units of the biotin ligase BirA was added to the |
- | mixture and incubated for 6 h at 30 °C. In order to remove unbound biotin, the | + | reaction |
- | biotinylated viral vectors were washed five times with 10 mM Tris buffer pH 8.0. | + | mixture and incubated for 6 h at 30 °C. In order to remove unbound |
- | </ | + | biotin, the |
- | + | biotinylated viral vectors were washed five times with 10 mM Tris | |
- | <p class=MsoNormal style= | + | buffer pH 8.0. |
- | + | </p> | |
- | + | </span></p> | |
- | <p | + | <p class="MsoNormal" style="text-indent: 0cm;"><span |
- | + | style="color: black;" lang="EN-US"> | |
- | depicted in figure A. For the detection of successfully biotinylated viral | + | <p style="text-align: justify;">Biotinylation |
- | vectors, MaxiSorp 96-well plates (Nunc) were coated with 200 ng monoclonal A20 | + | was verified with an ELISA as |
- | antibody per well for eight hours at 4°C, and blocked over night with PBST + | + | depicted in figure A. For the detection of successfully biotinylated |
- | 0.5 % BSA. This plate was incubated for 1 h at room temperature with 100 µl of | + | viral |
+ | vectors, MaxiSorp 96-well plates (Nunc) were coated with 200 ng | ||
+ | monoclonal A20 | ||
+ | antibody per well for eight hours at 4°C, and blocked over night with | ||
+ | PBST + | ||
+ | 0.5 % BSA. This plate was incubated for 1 h at room temperature with | ||
+ | 100 µl of | ||
serial diluted viral vectors. After washing the plates three times, the | serial diluted viral vectors. After washing the plates three times, the | ||
- | recommended amount of Streptavidin-HRP conjugate (Sigma-Aldrich) was added to | + | recommended amount of Streptavidin-HRP conjugate (Sigma-Aldrich) was |
- | each well for 1 h at room temperature. Again, the plates were washed three | + | added to |
- | times followed by detection of absorbance caused by converted ABTS substrate at | + | each well for 1 h at room temperature. Again, the plates were washed |
- | 405 nm. Additionally, the genomic AAV-2 titer was determined by qPCR.</ | + | three |
- | + | times followed by detection of absorbance caused by converted ABTS | |
- | <span class=apple-converted-space><span | + | substrate at |
- | line-height:115%;font-family: | + | 405 nm. Additionally, the genomic AAV-2 titer was determined by qPCR.</p> |
- | + | </span></p> | |
- | </span></span | + | <span class="apple-converted-space"><span |
- | + | style="font-size: 11pt; line-height: 115%; font-family: "Calibri","sans-serif"; color: black;" | |
- | + | lang="EN-US"></span></span><span class="apple-converted-space"><span | |
- | + | style="color: black;" lang="EN-US"> </span></span> | |
- | + | <table class="MsoTableGrid" | |
- | + | style="border: medium none ; border-collapse: collapse; text-align: left; margin-left: auto; margin-right: auto;" | |
- | + | border="0" cellpadding="0" cellspacing="0"> | |
- | lang=EN-US | + | <tbody> |
- | + | <tr> | |
- | <table class=MsoTableGrid | + | <td style="padding: 0cm 5.4pt; width: 477.3pt;" valign="top" |
- | + | width="636"> | |
- | + | <p class="MsoNormal" style="text-indent: 0cm;"><img | |
- | + | style="width: 587px; height: 539px;" alt="" | |
- | + | src="https://static.igem.org/mediawiki/2010/2/2c/Freiburg10_BAP-data.png" | |
- | + | align="left" hspace="12"></p> | |
- | + | </td> | |
- | + | </tr> | |
- | + | <tr> | |
- | + | <td style="padding: 0cm 5.4pt; width: 477.3pt;" valign="top" | |
- | + | width="636"> | |
- | + | <p class="MsoNormal" style="text-indent: 0cm;"><span | |
- | + | class="apple-converted-space"><span style="color: black;" lang="EN-US">A: | |
- | + | Schematic overview of the detection of BAP-presenting viral particles | |
- | + | immobilized by A20 capture antibodies. Biotinylated capsids can be | |
- | + | detected by streptavidin-coupled HRP</span></span></p> | |
- | + | <p class="MsoNormal" style="text-indent: 0cm;"><span | |
- | + | class="apple-converted-space"><span style="color: black;" lang="EN-US">B: | |
- | + | ELISA AAV-2 particles carrying a BAP insertion in loop 587 which can be | |
+ | specifically biotinylated in vitro</span></span></p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | </tbody> | ||
</table> | </table> | ||
- | + | <span class="apple-converted-space"><span | |
- | + | style="font-size: 11pt; line-height: 115%; font-family: "Calibri","sans-serif"; color: black;" | |
- | + | lang="EN-US"> | |
- | + | ||
- | + | ||
- | line-height:115%;font-family: | + | |
- | + | ||
</span></span> | </span></span> | ||
- | + | <p class="MsoNormal" style="text-indent: 0cm;"><span | |
- | <p class=MsoNormal | + | class="apple-converted-space"><span style="color: black;" lang="EN-US"> |
- | text-indent:0cm; | + | <h2>Results |
- | + | and Discussion:</h2> | |
- | + | </span></span><span lang="EN-US"> </span></p> | |
- | + | <p class="MsoNormal" style="text-indent: 0cm;"><span lang="EN-US"> | |
- | lang=EN-US | + | <p style="text-align: justify;">As |
- | lang=EN-US> </span></p> | + | can be seen in |
- | + | figure B, biotinylation of assembled AAV particles was achieved with | |
- | <p class=MsoNormal style= | + | the |
- | figure B, biotinylation of assembled AAV particles was achieved with the | + | dilution series ranging from 2- to 128-fold. Correlating this assay |
- | dilution series ranging from 2- to 128-fold. Correlating this assay with the | + | with the |
- | qPCR experiment for the detection of encapsidated vector plasmid (genomic titer | + | qPCR experiment for the detection of encapsidated vector plasmid |
+ | (genomic titer | ||
using CMV-promoter primers) yields a value of 4.70E+07 DNase-resistant | using CMV-promoter primers) yields a value of 4.70E+07 DNase-resistant | ||
- | particles (DRP). Consequently, the presence of approximately 3.6x10^5 biotinylated | + | particles (DRP). Consequently, the presence of approximately 3.6x10^5 |
- | DRPs can be detected using the described ELISA. Compared to the virus particle | + | biotinylated |
- | detection ELISA emploing A20 as capture- and detection antibody, the detection | + | DRPs can be detected using the described ELISA. Compared to the virus |
- | limit of the biotinylated viral capsids is about 10fold more sensitive. This | + | particle |
- | may be explained by the higher affinity of strepavidin towards biotin relative | + | detection ELISA emploing A20 as capture- and detection antibody, the |
- | to the A20 antibody affinity or multiple biotinylation of a single virus | + | detection |
- | particle.</span></p> | + | limit of the biotinylated viral capsids is about 10fold more sensitive. |
- | + | This | |
- | <span | + | may be explained by the higher affinity of strepavidin towards biotin |
- | + | relative | |
- | </span> | + | to the A20 antibody affinity or multiple biotinylation of a single |
- | + | virus | |
- | < | + | particle.</p> |
- | + | <p style="text-align: justify;"><br> | |
- | + | </p> | |
- | + | </span></p> | |
- | <p class=MsoNormal | + | <span |
- | + | style="font-size: 20pt; line-height: 115%; font-family: "Calibri","sans-serif";" | |
- | binding domain (Z34C)</ | + | lang="EN-US"></span><span style="font-size: 20pt; line-height: 115%;" |
- | + | lang="EN-US"></span> | |
- | <table class=MsoTableGrid | + | <p class="MsoNormal" style="text-align: left; text-indent: 0cm;" |
- | + | align="left"> | |
- | + | <h1>Miniaturized antibody | |
- | + | binding domain (Z34C)</h1> | |
- | + | </p> | |
- | + | <table class="MsoTableGrid" | |
- | + | style="border: medium none ; border-collapse: collapse;" border="0" | |
- | + | cellpadding="0" cellspacing="0"> | |
- | + | <tbody> | |
- | + | <tr style="height: 184.6pt;"> | |
- | + | <td style="padding: 0cm 5.4pt; width: 477.3pt; height: 184.6pt;" | |
- | + | valign="top" width="636"> | |
- | + | <p class="MsoNormal" style="text-indent: 0cm;"><img | |
- | + | style="width: 451px; height: 225px;" | |
- | + | src="https://static.igem.org/mediawiki/2010/6/63/Freiburg10_Z34C-tag.png" | |
- | + | alt="" align="left" hspace="12"></p> | |
- | + | </td> | |
- | + | </tr> | |
+ | <tr style="height: 23.7pt;"> | ||
+ | <td style="padding: 0cm 5.4pt; width: 477.3pt; height: 23.7pt;" | ||
+ | valign="top" width="636"> | ||
+ | <p style="text-align: justify;">Schematic overview of Z34C | ||
+ | insertion into AAV-2 viral capsids enabling antibody arming for therapy</p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | </tbody> | ||
</table> | </table> | ||
- | + | <p class="MsoNormal" style="text-indent: 0cm;"><span lang="EN-US"> | |
- | <p class=MsoNormal style= | + | <p style="text-align: justify;">The |
- | targeting approach is to utilize a minimized fragment of the | + | idea of this |
- | href="http://www.ncbi.nlm.nih.gov/protein/153106"><span | + | targeting approach is to utilize a minimized fragment of the <a |
- | style= | + | href="http://www.ncbi.nlm.nih.gov/protein/153106"><span |
- | lang=EN-US> that was first described in Staphylococcus aureus. These | + | style="color: windowtext; text-decoration: none;" lang="EN-US">Staphylococcal |
- | gram-positive bacteria have evolved the 508 amino acid long protein A that has | + | Protein A</span></a><span lang="EN-US"> that was first described |
- | a high affinity for the Fc-domain of antibodies to protect itself from the | + | in Staphylococcus aureus. These |
- | immune system. Binding to the constant region of the antibodies is accomplished | + | gram-positive bacteria have evolved the 508 amino acid long protein A |
+ | that has | ||
+ | a high affinity for the Fc-domain of antibodies to protect itself from | ||
+ | the | ||
+ | immune system. Binding to the constant region of the antibodies is | ||
+ | accomplished | ||
by the </span><a href="http://www.ncbi.nlm.nih.gov/protein/1Q2NA"><span | by the </span><a href="http://www.ncbi.nlm.nih.gov/protein/1Q2NA"><span | ||
- | + | style="color: windowtext; text-decoration: none;" lang="EN-US">Z-Domain</span></a><span | |
- | lang=EN-US> of Protein A that is 58-59 amino acids long, has alone a high | + | lang="EN-US"> of Protein A that is 58-59 amino acids long, has |
- | affinity (Kd= 14,9 nM) for the antibodies and a three-helix bundle structure. | + | alone a high |
+ | affinity (Kd= 14,9 nM) for the antibodies and a three-helix bundle | ||
+ | structure. | ||
In </span><a href="http://www.ncbi.nlm.nih.gov/pubmed/8650153"><span | In </span><a href="http://www.ncbi.nlm.nih.gov/pubmed/8650153"><span | ||
- | + | style="color: windowtext; text-decoration: none;" lang="EN-US">[Braisted | |
- | 1996]</span></a><span lang=EN-US> the authors reduced the secundary | + | & Wells; |
- | structure to an two-helix bundle. This size reduction has lead to an drastic | + | 1996]</span></a><span lang="EN-US"> the authors reduced the |
- | reduction of the affinity for IgG (>10^5 fold) which could be recovered by | + | secundary |
- | 13 amino acid exchanges resulting in a 38 amino acid long peptide with an | + | structure to an two-helix bundle. This size reduction has lead to an |
+ | drastic | ||
+ | reduction of the affinity for IgG (>10^5 fold) which could be | ||
+ | recovered by | ||
+ | 13 amino acid exchanges resulting in a 38 amino acid long peptide with | ||
+ | an | ||
satisfying affinity for IgG (Kd = 185 nM) termed </span><a | satisfying affinity for IgG (Kd = 185 nM) termed </span><a | ||
- | href="http://www.ncbi.nlm.nih.gov/protein/1ZDAA"><span | + | href="http://www.ncbi.nlm.nih.gov/protein/1ZDAA"><span |
- | style= | + | style="color: windowtext; text-decoration: none;" lang="EN-US">Z38</span></a><span |
+ | lang="EN-US">. | ||
This binding domain was subsequently improved in </span><a | This binding domain was subsequently improved in </span><a | ||
- | href="http://www.ncbi.nlm.nih.gov/pubmed/9294166"><span | + | href="http://www.ncbi.nlm.nih.gov/pubmed/9294166"><span |
- | style= | + | style="color: windowtext; text-decoration: none;" lang="EN-US">[Starovasnik |
- | lang=EN-US> by the insertion of a disulfide bridge connecting the ends of | + | et al.; 1997]</span></a><span lang="EN-US"> by the insertion of a |
+ | disulfide bridge connecting the ends of | ||
the helices leading to the binding domain </span><a | the helices leading to the binding domain </span><a | ||
- | href="http://www.ncbi.nlm.nih.gov/protein/1ZDCA"><span | + | href="http://www.ncbi.nlm.nih.gov/protein/1ZDCA"><span |
- | style= | + | style="color: windowtext; text-decoration: none;" lang="EN-US">Z34C</span></a><span |
+ | lang="EN-US"> which | ||
shows an increased affinity for IgG (Kd = 20 nM).</span></p> | shows an increased affinity for IgG (Kd = 20 nM).</span></p> | ||
- | + | </span></p> | |
- | <p | + | <span |
- | font-family: | + | style="font-size: 10pt; line-height: 200%; font-family: "Arial","sans-serif"; color: black;" |
- | + | lang="EN-US"> </span> | |
- | <table class=MsoTableGrid | + | <table class="MsoTableGrid" |
- | + | style="border: medium none ; border-collapse: collapse; text-align: left; margin-left: auto; margin-right: auto;" | |
- | + | border="0" cellpadding="0" cellspacing="0"> | |
- | + | <tbody> | |
- | + | <tr> | |
- | + | <td | |
- | + | style="padding: 0cm 5.4pt; vertical-align: top; width: 477.3pt;"> | |
- | + | <p class="MsoNormal" style="text-indent: 0cm;"><span | |
- | + | style="font-size: 10pt; line-height: 200%; font-family: "Arial","sans-serif"; color: black;"><img | |
- | + | style="border: 0px solid ; width: 524px; height: 309px;" id="Grafik 30" | |
- | + | src="https://static.igem.org/mediawiki/2010/c/cd/Freiburg10_Z34C-history.png" | |
- | + | alt=""></span></p> | |
- | + | </td> | |
- | + | </tr> | |
- | + | <tr> | |
- | + | <td style="padding: 0cm 5.4pt; width: 477.3pt;" valign="top" | |
- | + | width="636"> | |
- | + | <p class="MsoNormal" style="text-indent: 0cm;"><span | |
+ | style="font-size: 10pt; line-height: 200%; font-family: "Arial","sans-serif"; color: black;" | ||
+ | lang="EN-US">Development of the Z34C motif, a miniaturized antibody | ||
+ | binding motif</span></p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | </tbody> | ||
</table> | </table> | ||
- | + | <span | |
- | + | style="font-size: 10pt; line-height: 200%; font-family: "Arial","sans-serif"; color: black;" | |
- | font-family: | + | lang="EN-US"><br> |
- | </span><span lang=EN-US>This engineered antibody binding domain of 34 amino | + | </span><span lang="EN-US"> |
- | acids was then inserted into capsids of different viral vectors amongst others | + | <p style="text-align: justify;">This engineered antibody binding domain |
- | also the AAV. In | + | of 34 |
- | href="http://www.ncbi.nlm.nih.gov/pmc/articles/PMC155067"><span | + | amino |
- | style= | + | acids was then inserted into capsids of different viral vectors amongst |
- | lang=EN-US> the </span><a | + | others |
- | href="http://www.ncbi.nlm.nih.gov/protein/1ZDCA"><span | + | also the AAV. In <a |
- | style= | + | href="http://www.ncbi.nlm.nih.gov/pmc/articles/PMC155067"><span |
- | was inserted at position 587 into the capsid of the AAV resulting in viral | + | style="color: windowtext; text-decoration: none;" lang="EN-US">[Ried |
+ | et al.; 2002]</span></a><span lang="EN-US"> the </span><a | ||
+ | href="http://www.ncbi.nlm.nih.gov/protein/1ZDCA"><span | ||
+ | style="color: windowtext; text-decoration: none;" lang="EN-US">Z34C</span></a><span | ||
+ | lang="EN-US"> domain | ||
+ | was inserted at position 587 into the capsid of the AAV resulting in | ||
+ | viral | ||
vector that can be targeted to different target cells without genetic | vector that can be targeted to different target cells without genetic | ||
engineering. This targeting approach was then improved in </span><a | engineering. This targeting approach was then improved in </span><a | ||
- | href="http://www.ncbi.nlm.nih.gov/pubmed/15922956"><span | + | href="http://www.ncbi.nlm.nih.gov/pubmed/15922956"><span |
- | style= | + | style="color: windowtext; text-decoration: none;" lang="EN-US">[Gigout |
- | + | et al.; 2005]</span></a> by the creation of | |
- | recombinant VP-Proteins what resulted in 4 to 5 orders of magnitude more | + | mosaic vectors that contain only ~25% of |
- | infectiosity compared to all-mutant viruses | + | recombinant VP-Proteins what resulted in 4 to 5 orders of magnitude |
- | + | more | |
- | < | + | infectiosity compared to all-mutant viruses</p> |
- | + | </span><span style="font-size: 12pt; line-height: 200%;" lang="EN-US"></span> | |
- | + | <p class="MsoNormal" style="text-align: left; text-indent: 0cm;" | |
- | <p class=MsoNormal | + | align="left"> |
- | + | <h2>Material and Methods:</h2> | |
- | + | </p> | |
- | <p class=MsoNormal | + | <p class="MsoNormal" style="text-align: justify; text-indent: 0cm;" |
- | + | align="left">Transfection of the | |
- | AAV-293 producer cells was performed for three different loop insertions of | + | AAV-293 producer cells was performed for three different loop |
- | Z34C in each three 10 cm petri dishes with 3.4x10^6 cells resulting in a | + | insertions of |
- | confluence of about 70-80%. For the transfection, either the composite parts pCMV_VP123(453-Z34C), | + | Z34C in each three 10 cm petri dishes with 3.4x10^6 cells resulting in |
- | pCMV_VP123(587KO- Z34C) or pCMV_VP123(587Ko- Z34C-Spacer) were cotransfected | + | a |
- | with pHelper, | + | confluence of about 70-80%. For the transfection, either the composite |
- | and | + | parts pCMV_VP123(453-Z34C), |
- | culture supernatant and subjected to four cycles of freeze and thaw cell lysis. | + | pCMV_VP123(587KO- Z34C) or pCMV_VP123(587Ko- Z34C-Spacer) were |
- | The cell lysate was centrifuged at 4000 rpm for 15 minutes and concentrated in | + | cotransfected |
- | a VivaSpin VS2002 (Sartorius Stedem) with a 10 kDa molecular weight cut-off to | + | with pHelper, |
- | two milliliters and washed three to five times with 20 mM Bis-Tris buffer, pH 6. | + | [AAV2]-left-ITR_pCMV_betaglobin_mVenus_hGH_[AAV2]-right-ITR |
- | The cell lysates were incubated with 250 units of Benzonase (Sigma-Aldrich) to | + | and RepVP123(587KO). The producer cells were harvested |
- | remove contaminant genomic DNA and loaded on an ÄKTA purifier (GE Healthcare) equipped | + | with the |
- | with a Superdex 200 gel filtration column (GE Healthcare). Fractions (500 µl) | + | culture supernatant and subjected to four cycles of freeze and thaw |
- | around the void volume containing viral particles were collected and used in a | + | cell lysis. |
- | sandwich ELISA. 96 well plates were coated with 200 ng Cetuximab (Imclone/Merck/Bristol-Myers | + | The cell lysate was centrifuged at 4000 rpm for 15 minutes and |
- | Squibb). Detection was performed using the monoclonal antibody A20 (kindly | + | concentrated in |
- | provided by PD Dr. J. Kleinschmidt, DKFZ Heidelberg) that was biotinylated | + | a VivaSpin VS2002 (Sartorius Stedem) with a 10 kDa molecular weight |
- | using a Biotinylation kit (Dojindo, Japan) and Streptavidin-HRP (Sigma-Aldrich). | + | cut-off to |
- | The HRP presence was detected by the conversion of the substrate ABTS at 405 | + | two milliliters and washed three to five times with 20 mM Bis-Tris |
- | nm. The average of the non-template controls (NTC) was subtracted from the | + | buffer, pH 6. |
- | sample data.< | + | The cell lysates were incubated with 250 units of Benzonase |
- | + | (Sigma-Aldrich) to | |
- | + | remove contaminant genomic DNA and loaded on an ÄKTA purifier (GE | |
- | + | Healthcare) equipped | |
- | + | with a Superdex 200 gel filtration column (GE Healthcare). Fractions | |
- | <span lang=EN-US | + | (500 µl) |
- | + | around the void volume containing viral particles were collected and | |
- | </span> | + | used in a |
- | + | sandwich ELISA. 96 well plates were coated with 200 ng Cetuximab | |
- | <p class=MsoNormal | + | (Imclone/Merck/Bristol-Myers |
- | text-indent:0cm; | + | Squibb). Detection was performed using the monoclonal antibody A20 |
- | + | (kindly | |
- | + | provided by PD Dr. J. Kleinschmidt, DKFZ Heidelberg) that was | |
- | lang=EN-US>Results and Discussion:</span></p> | + | biotinylated |
- | + | using a Biotinylation kit (Dojindo, Japan) and Streptavidin-HRP | |
- | <div align=center> | + | (Sigma-Aldrich). |
- | + | The HRP presence was detected by the conversion of the substrate ABTS | |
- | <table class=MsoTableGrid | + | at 405 |
- | + | nm. The average of the non-template controls (NTC) was subtracted from | |
- | + | the | |
- | + | sample data.<br> | |
- | + | <span style="font-size: 12pt; line-height: 200%;" lang="EN-US"></span> <br> | |
- | + | </p> | |
- | + | <span lang="EN-US"></span> | |
- | + | <p class="MsoNormal" style="text-align: left; text-indent: 0cm;" | |
- | + | align="left"><span lang="EN-US"> | |
- | + | <h2>Results and Discussion:</h2> | |
- | + | </span></p> | |
- | + | <div align="center"> | |
- | + | <table class="MsoTableGrid" | |
- | + | style="border: medium none ; width: 384.6pt; border-collapse: collapse;" | |
- | + | border="0" cellpadding="0" cellspacing="0" width="513"> | |
- | + | <tbody> | |
- | + | <tr style="height: 305.8pt;"> | |
- | + | <td style="padding: 0cm 5.4pt; width: 384.6pt; height: 305.8pt;" | |
- | + | valign="top" width="513"> | |
- | + | <p class="MsoNormal" style="text-align: center; text-indent: 0cm;" | |
- | + | align="center"><img style="width: 421px; height: 722px;" alt="" | |
- | + | src="https://static.igem.org/mediawiki/2010/2/23/Freiburg10_Z34C_affinity_data.png" | |
- | + | align="left" hspace="12"></p> | |
- | + | </td> | |
- | + | </tr> | |
- | + | <tr style="height: 15.45pt;"> | |
- | + | <td style="padding: 0cm 5.4pt; width: 384.6pt; height: 15.45pt;" | |
- | + | valign="top" width="513"> | |
- | + | <p class="MsoNormal" style="text-align: left; text-indent: 0cm;" | |
- | + | align="left"><span lang="EN-US">A: Sandwich-ELISA scheme for the | |
+ | detection of Z34C-presenting viral particles. Immobilization is | ||
+ | achieved by binding a IgG molecule. Intact viral capsids can be | ||
+ | specifically bound by the biotinylated A20 antibody which can be | ||
+ | detected by Strepavidin-HRP</span></p> | ||
+ | <p class="MsoNormal" style="text-align: left; text-indent: 0cm;" | ||
+ | align="left"><span lang="EN-US">B: ELISA using the principle described | ||
+ | in A for loop insertion samples obtained by gel filtration | ||
+ | chromatography of 453- and 587-Z34C particles. Undiluted and 10-fold | ||
+ | diluted samples were employed</span></p> | ||
+ | <p class="MsoNormal" style="text-align: left; text-indent: 0cm;" | ||
+ | align="left"><span lang="EN-US">C: ELISA signals obtained by | ||
+ | absorbance measurements from B at 405 nm, undiluted samples</span></p> | ||
+ | <p class="MsoNormal" style="text-align: left; text-indent: 0cm;" | ||
+ | align="left"><span lang="EN-US">D: ELISA signals obtained by | ||
+ | absorbance measurements from B at 405 nm, diluted samples</span></p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | </tbody> | ||
</table> | </table> | ||
- | |||
</div> | </div> | ||
- | + | <p class="MsoNormal" style="text-align: left; text-indent: 0cm;" | |
- | <p class=MsoNormal | + | align="left"><span lang="EN-US"> |
- | lang=EN-US | + | <p style="text-align: justify;">Three samples of different viral |
- | + | particles with insertions of the | |
- | <p | + | antibody-binding motif Z34C were successfully purified by the gel |
- | + | filtration | |
- | antibody-binding motif Z34C were successfully purified by the gel filtration | + | chromatography. The fractions around the void volume were subsequently |
- | chromatography. The fractions around the void volume were subsequently used in | + | used in |
- | two different sandwich ELISAs. The first one aims at the detection of the | + | two different sandwich ELISAs. The first one aims at the detection of |
- | particle’s ability to bind IgG-antibodies. The therapeutical antibody Cetuximab | + | the |
- | was employed to test the affinity of the virus particles. As displayed in | + | particle’s ability to bind IgG-antibodies. The therapeutical antibody |
- | figure 1A, only Z34C-presenting particles should be immobilized and only | + | Cetuximab |
- | assembled viral capsid will be detected due to the affinity of the A20 antibody | + | was employed to test the affinity of the virus particles. As displayed |
- | (as described above). Figure 1B shows that absorbance signal were obtained in | + | in |
- | case of the 587KO-Z34C and 587KO-Z34C-spacer insertions. Loop 453-Z34C did not | + | figure 1A, only Z34C-presenting particles should be immobilized and |
- | yield any significant absorbance signals. In addition, the assay was performed | + | only |
- | with undiluted samples and those which were 10-fold diluted in PBST + 0.5 % | + | assembled viral capsid will be detected due to the affinity of the A20 |
- | BSA. Signal strengths in the undiluted ELISA samples indicated that the assay | + | antibody |
- | was saturated, therefore the diluted sample data were used for evaluation. The | + | (as described above). Figure 1B shows that absorbance signal were |
+ | obtained in | ||
+ | case of the 587KO-Z34C and 587KO-Z34C-spacer insertions. Loop 453-Z34C | ||
+ | did not | ||
+ | yield any significant absorbance signals. In addition, the assay was | ||
+ | performed | ||
+ | with undiluted samples and those which were 10-fold diluted in PBST + | ||
+ | 0.5 % | ||
+ | BSA. Signal strengths in the undiluted ELISA samples indicated that the | ||
+ | assay | ||
+ | was saturated, therefore the diluted sample data were used for | ||
+ | evaluation. The | ||
ABTS conversion indicates that highest amount of Z34C-presenting viral | ABTS conversion indicates that highest amount of Z34C-presenting viral | ||
particles is present in the Äkta fractions 7-9 of the two different 587 | particles is present in the Äkta fractions 7-9 of the two different 587 | ||
- | insertions. No viral particles with a binding affinity for the IgG antibody | + | insertions. No viral particles with a binding affinity for the IgG |
- | were present in the 453 samples.</span></p> | + | antibody |
- | + | were present in the 453 samples.</p> | |
- | <div align=center> | + | </span></p> |
- | + | <div align="center"> | |
- | <table class=MsoTableGrid | + | <table class="MsoTableGrid" |
- | + | style="border: medium none ; border-collapse: collapse;" border="0" | |
- | + | cellpadding="0" cellspacing="0"> | |
- | + | <tbody> | |
- | + | <tr style="height: 593.75pt;"> | |
- | + | <td style="padding: 0cm 5.4pt; width: 318.25pt; height: 593.75pt;" | |
- | + | valign="top" width="424"> | |
- | + | <p class="MsoNormal" style="text-align: left; text-indent: 0cm;" | |
- | + | align="left"><img style="width: 410px; height: 793px;" alt="" | |
- | + | src="https://static.igem.org/mediawiki/2010/0/0b/Freiburg10_Z34C_titration_data.png" | |
- | + | align="left" hspace="12"></p> | |
- | + | </td> | |
- | + | </tr> | |
- | + | <tr style="height: 39.25pt;"> | |
- | + | <td style="padding: 0cm 5.4pt; width: 318.25pt; height: 39.25pt;" | |
- | + | valign="top" width="424"> | |
- | + | <p class="MsoNormal" style="text-align: left; text-indent: 0cm;" | |
- | + | align="left"><span lang="EN-US">A: Sandwich-ELISA scheme for the | |
- | + | detection of AAV-2 particles. Immobilization is achieved by binding to | |
- | + | the A20 capture molecule which also acts as the biotinylated detection | |
- | + | antibody after washing. Intact viral capsids can be specifically | |
- | + | detected by Strepavidin-HRP</span></p> | |
- | + | <p class="MsoNormal" style="text-align: left; text-indent: 0cm;" | |
- | + | align="left"><span lang="EN-US">B: ELISA using the principle described | |
- | + | in A for loop insertion samples obtained by gel filtration | |
- | + | chromatography of 453- and 587-Z34C particles. Undiluted and 10-fold | |
- | + | diluted samples were employed</span></p> | |
- | + | <p class="MsoNormal" style="text-align: left; text-indent: 0cm;" | |
- | + | align="left"><span lang="EN-US">C: qPCR data for the | |
- | + | CMV-promoter-based DRP-titer determination. Samples as in B</span></p> | |
+ | <p class="MsoNormal" style="text-align: left; text-indent: 0cm;" | ||
+ | align="left"><span lang="EN-US">D: ELISA signals obtained by | ||
+ | absorbance measurements from B at 405 nm, diluted samples</span></p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | </tbody> | ||
</table> | </table> | ||
- | |||
</div> | </div> | ||
- | + | <p class="MsoNormal" style="text-align: justify; text-indent: 0cm;" | |
- | <p class=MsoNormal | + | align="left">A second approach was conducted to |
- | + | reveal the reason for the absence | |
- | + | of Z34C-presenting particles in case of the 453 insertion. The sandwich | |
- | + | ELISA, | |
- | + | ||
- | of Z34C-presenting particles in case of the 453 insertion. The sandwich ELISA, | + | |
as shown in figure 2A, uses the A20 antibody to capture and detect all | as shown in figure 2A, uses the A20 antibody to capture and detect all | ||
- | assembled viral capsids independent of the presence of a Z34C motif giving the | + | assembled viral capsids independent of the presence of a Z34C motif |
- | so-called physical AAV titer (see figure 2D). To correlate these measurements, | + | giving the |
- | we additionally conducted qPCR measurements to determine the amount of encapsidated | + | so-called physical AAV titer (see figure 2D). To correlate these |
- | vector plasmids, the so-called DRP (DNAse-resistant particle) titer (see figure | + | measurements, |
- | 2C). Comparison reveals that both assays yielded a peak signal around the Äkta | + | we additionally conducted qPCR measurements to determine the amount of |
- | fraction 8. The qPCR data indicated that all samples contain approximately 2 x | + | encapsidated |
- | 10^9 copies of the vector plasmid per milliliter. Assuming equal packaging | + | vector plasmids, the so-called DRP (DNAse-resistant particle) titer |
- | efficiencies for the vector plasmids, all three loop insertion approaches | + | (see figure |
- | contain comparable amounts of viral capsids. The sandwich-ELISA yielded a peak | + | 2C). Comparison reveals that both assays yielded a peak signal around |
- | around fraction eight for all three loop-insertion approaches. Since the signal | + | the Äkta |
- | strength for the 587KO-Z34C samples is significantly higher, it can be assumed that | + | fraction 8. The qPCR data indicated that all samples contain |
- | additionally to the affinity of the A20 antibody for assembled viral capsids, | + | approximately 2 x |
- | the Z34C-containing viral particles add a high affinity for the Fc-part of the | + | 10^9 copies of the vector plasmid per milliliter. Assuming equal |
- | detection antibody. Hence the qPCR-titration only depends on the number of | + | packaging |
- | encapsidated AAV-vector plasmids and not on the viral capsid and its degree of | + | efficiencies for the vector plasmids, all three loop insertion |
- | modification, it is the more quantitative method of particle titration which | + | approaches |
- | proved equal for all three loop insertion approaches.< | + | contain comparable amounts of viral capsids. The sandwich-ELISA yielded |
- | + | a peak | |
- | + | around fraction eight for all three loop-insertion approaches. Since | |
- | lang=EN-US | + | the signal |
- | + | strength for the 587KO-Z34C samples is significantly higher, it can be | |
- | < | + | assumed that |
- | + | additionally to the affinity of the A20 antibody for assembled viral | |
- | + | capsids, | |
- | <p class=MsoNormal | + | the Z34C-containing viral particles add a high affinity for the Fc-part |
- | page-break-after:avoid | + | of the |
- | amino acid functional motif into the two most promising integration sites of | + | detection antibody. Hence the qPCR-titration only depends on the number |
- | the AAV-2 capsid (see figure 3) revealed no integration of modified viral | + | of |
- | capsid proteins (VP1-3) into assembled viral capsids for the 453 integration | + | encapsidated AAV-vector plasmids and not on the viral capsid and its |
- | site. As also seen by the data obtained for the linker-containing insertion, | + | degree of |
- | the 587 region tolerates insertions of at least 39 amino acids. For the 453 | + | modification, it is the more quantitative method of particle titration |
- | integration site, either the inserted Z34C motif loses its ability to bind IgG | + | which |
- | molecules or the influence of the inserted motif causes strong structural | + | proved equal for all three loop insertion approaches.<span lang="EN-US"> |
- | changes that disturb the ability of the modified VP proteins to be integrated | + | <br> |
- | into the virus capsid.</span></p> | + | </span></p> |
- | + | <p class="MsoNormal" | |
- | + | style="text-align: left; text-indent: 0cm; page-break-after: avoid;" | |
- | + | align="left"><span lang="EN-US"> | |
- | <table class=MsoTableGrid | + | <p style="text-align: justify;">Summarizing, the insertion of a 34/39 |
- | + | amino acid functional motif into the two most promising integration | |
- | + | sites of | |
- | + | the AAV-2 capsid (see figure 3) revealed no integration of modified | |
- | + | viral | |
- | + | capsid proteins (VP1-3) into assembled viral capsids for the 453 | |
- | + | integration | |
- | + | site. As also seen by the data obtained for the linker-containing | |
- | + | insertion, | |
- | + | the 587 region tolerates insertions of at least 39 amino acids. For the | |
- | + | 453 | |
- | + | integration site, either the inserted Z34C motif loses its ability to | |
- | + | bind IgG | |
- | + | molecules or the influence of the inserted motif causes strong | |
- | + | structural | |
- | + | changes that disturb the ability of the modified VP proteins to be | |
- | + | integrated | |
+ | into the virus capsid.</p> | ||
+ | </span></p> | ||
+ | <span lang="EN-US"> </span> | ||
+ | <table class="MsoTableGrid" | ||
+ | style="border: medium none ; border-collapse: collapse; text-align: left; margin-left: auto; margin-right: auto;" | ||
+ | border="0" cellpadding="0" cellspacing="0"> | ||
+ | <tbody> | ||
+ | <tr style="height: 209.35pt;"> | ||
+ | <td | ||
+ | style="padding: 0cm 5.4pt; vertical-align: top; width: 477.3pt; height: 209.35pt;"> | ||
+ | <p class="MsoNormal" style="text-align: left; text-indent: 0cm;" | ||
+ | align="left"><img | ||
+ | style="border: 0px solid ; width: 571px; height: 269px;" alt="" | ||
+ | id="Picture 15" | ||
+ | src="https://static.igem.org/mediawiki/2010/8/8f/Freiburg10_loop_insertion_sites.png"></p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td style="padding: 0cm 5.4pt; width: 477.3pt;" valign="top" | ||
+ | width="636"> | ||
+ | <p class="MsoNormal" style="text-align: left; text-indent: 0cm;" | ||
+ | align="left"><span lang="EN-US">Loop insertion sites visualized in the | ||
+ | 3D structure of a single viral coat protein (VP3)</span></p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | </tbody> | ||
</table> | </table> | ||
+ | <p class="MsoNormal" style="text-align: left; text-indent: 0cm;" | ||
+ | align="left"><span lang="EN-US"> </span></p> | ||
+ | </body> | ||
+ | </html> | ||
- | |||
- | |||
- | |||
- | |||
- | |||
{{:Team:Freiburg_Bioware/Footer}} | {{:Team:Freiburg_Bioware/Footer}} |
Latest revision as of 03:36, 28 October 2010
Loop Insertion
Modification of the Viral Capsid of the AAV2 using Viral Bricks
For therapeutical applications in human gene transfer, the broad tropism for heparan sulfate proteoglycan (HSPG) has to be knocked-out and a novel tropism has to be inserted. This retargeting can be achieved either by insertion of functional motifs into the two major surface exposed loops or by fusion of these motifs to the N-terminus of the viral coat proteins. The graphic on the right shows parts of the three-dimensional structure of a viral coat protein. The parts of the loop regions that are coded in the ViralBricks are shown in purple for the 453 loop and in blue for the 587 loop.
Three-dimensional representation of the AAV2 showing the amino acids of the 453 and 587 loops that are coded by the corresponding Viral Bricks.
Cloning of Viral Bricks into capsid coding parts
In order to make loop insertions more convenient the following restriction sites were inserted into all capsid coding parts and already existing restriction sites were removed from the constructs. The choice of these restriction sites was reasoned by enzyme performance, buffer compatibilities and the number of existing restriction sites that had to be removed at other positions. All restriction endonucleases were purchased from NEB.
His-Affinity tag
Schematic figure of His-affinity Tag insertion into the viral capsid |
The insertion of a His-Affinity Tag
into the exposed major surface
loops of the viral vectors allows their specific affinity purification
employing e.g. Ni-NTA affinity chromatography. This purification method
was
tested using the cell culture lysate of transfected AAV-293 cells that
were
either grown in DMEM supplemented with 10% FCS or in the serum-free
GIBCO®
FreeStyle™ 293 Expression Medium (Invitrogen). The usage of serum-free
media is
a technological modification meant to facilitate the production of pure
viral
vectors. Purified viral vectors are important for several applications
such as
animal models and biophysical characterizations. On the other hand, the
elution
of the His-tagged viral vectors allows also enrichment of transgene
viral
vectors. In order to answer the question if and to what degree viral
vectors
are transferred into the media, the cells were centrifugated, then
divided into
the pellet fraction and the supernatant. Physical cell lysis was
performed for
both fractions of the two produced batches (serum-free and FCS media)
by
performing four cycles of freeze and thaw.
Experimental setup for the
purification of His-tag presenting viral particles produced by AAV-293
cells in DMEM or the protein- and animal-free Freestyle medium.
Purified viral particles can be detected by ELISA using an
anti-poly-Histidine antibody or qPCR for encapsidated vector plasmids,
respectively |
Material and Methods:
Transfection of the AAV-293 producer
cells was performed in five 10 cm
petri dishes with 3.6x10^6 cells, resulting in a confluency of about
70-80%
according to the standard protocol either with cells grown in GIBCO®
FreeStyle™
293 Expression Medium (Invitrogen, protein- and animal-origin free) or
in DMEM
supplemented with 10% FCS (PAA). For transfection, the composite parts
pCMV_VP123(587-His) and RepVP123(587-KO)_p5-TATA-less were used in an
1:1 ratio
together with pHelper
and [AAV2]-left-ITR_pCMV_betaglobin_mVenus_hGH_[AAV2]-right-ITR. Cells
were spun down at 200 x g for five minutes and the
samples
were divided into the pellet and the supernatant fractions. Physical
cell lysis
was performed by four cycles of freeze and thaw for all four samples.
The cell
lysate / supernatant fractions were incubated with 800 µl of
His-Affinity Gel
(kindly provided by Zymo Resear ch, USA) at 4 °C for
18 hours with 200
rpm constant
agitation. The beads were then collected in 5 ml gravity-flow columns
and
washed five times with one column volume of PBS each. The His-affinity
gel was subsequently
washed with PBS, 25 mM Imidazole to remove unspecifically bound
proteins.
Elution was performed in an second step with PBS, 500 mM Imidazole to
elute the
His-tagged viral vectors. The genomic titer of the purified viral
vectors was
detected via q-PCR. In an ELISA, viral vectors were captured employing
the
monoclonal antibody A20 (kindly provided by PD Dr. J. Kleinschmidt,
DKFZ, Heidelberg)
that exclusively recognizes assembled AAV capsids. His-Tags present in
assembled viral capsids were subsequently detected with an HRP-tagged
secondary
anti-His-Tag antibody (1:2000 diluted, A7058, Sigma). HRP presence
was detected using the
peroxidase substrate ABTS. Generation of blue-green color (absorption
at 405
nm) was measured in a Tecan Sunrise plate reader. Sample data were
blanked with
the average of the non-template controls (NTC).
Results and Discussion
Presence of the His-affinity tag in
the viral capsid was detected and the ELISA enabled quantification of
the
purification procedure efficiency. The absorbance measured for the
elution
fractions of the 1/10 diluted samples sums up to 2.3 for the DMEM- and
0.5 for
the Free Style 293-grown cells, assigning the DMEM-grown cells a five
times
higher production efficiency. Comparison between the cell pellet and
the
supernatant fractions revealed that 70 - 80% of the viral particles can
be
found inside the producer cells.
According to these results,
producer cells should be grown in complex media for in vitro
and cell
culture experiments. Use of serum-free produced viral vectors is
recommended
for mouse or other animal experiments and possible therapeutical
applications
where even the presence of traces amounts of fetal calf serum should be
avoided. Combination with different purification approaches such as gel
filtration chromatography using i.e. Superdex 200 columns (GE
Healthcare)
enables the production of highly purified viral vectors for several
different
applications.
A: Schematic overview of the sandwich ELISA for the detection of His-tagged viral particles B: ELISA from viral particles produced by AAV-293 cells in DMEM or Free Style medium, divided into cell pellet and cell culture supernatant samples. The particles were purified using Ni-NTA affinity chromatography with Imidazole in PBS as washing and elution agent C: Absorption measurements from plate shown in B. Undiluted Äkta fractions converted ABTS peroxidase substrate at 405 nm D: As C, whereas Äkta fractions were 10-fold diluted |
Biotinylation Acceptor Peptide (BAP)
Schematic overview of BAP
insertion into AAV-2 viral capsids, followed by biotinylation and
binding of streptavidin-coupled molecules |
The
BAP
(Biotinylation Acceptor Peptide) included in the Virus Construction Kit
is a 15
amino acid peptide identified by Schatz et al. (1993) in a library
screening
approach and published under the number #85. This peptide with the
sequence
GLNDIFEAQKIEWHE contains a central lysine residue that can be
specifically
biotinylated by the prokaryotic holoenzyme biotin synthetase, encoded
by the
BirA gene of E. coli. Specific biotinylation of this peptide
sequence
can be performed in vivo by cotransfecting a plasmid with the BirA gene
as
described for the AAV by Arnold et al. (2006) or by an in vitro
coupling
approach using the purified Escherichia coli enzyme biotin ligase
(BirA).
Biotin
molecules
specifically coupled to the viral loops can either be used to attach
Streptavidin-coupled
molecules to the viral capsid or to chemically couple molecules with a
reactive
group to biotin. Consequently, the inserted motif can enable the
visualization
of single virus particles by coupling fluorophores to the virus capsid
empowering further uses of the Virus Construction Kit in fundamental
virological research. In addition, targeting molecules such as
Streptavidin-coupled affinity molecules (i.e. Antibodies, Nanobodies or
Affibodies) can be coupled for manifold targeting approaches.
Materials
and Methods:
Transfection
of the
AAV-293 producer cells was performed in five 10 cm petri dishes with
3.4x10^6
cells (grown in DMEM supplemented with 10% FCS) resulting in a
confluence of
70-80% according to the standard protocol. Producer cells were
harvested and
together with the culture supernatant subjected to four cycles of
freeze and
thaw cell lysis. The cell lysate was centrifuged at 4000 rpm for 15
minutes and
concentrated in a VivaSpin VS2002 column with 10 kDa molecular weight
cut-off to
yield two milliliters. Genomic DNA attached to the virus particles was
degraded
using 250 units of Benzonase (EC 3.1.30.2, Sigma-Aldrich) at 37 °C for
one
hour. The concentrated cell lysate was washed three times with 4 ml 20
mM
bis-Tris buffer pH 6.0, 100 mM NaCl. From this concentrated sample, 500
µl were
loaded on the ÄKTA purifier (GE Healthcare) equipped with a Superdex
200 gel
filtration column (GE Healthcare). This purification is also described
by Smith
et al. (2003). Fractions
around the void volume giving a UV absorbance peak were pooled and
applied to a
Amicon Ultra column (Millipore, size limit 100 kDa, 2 ml loading
volume) and
concentrated to 500 µl. This purified virus sample was washed four
times with 10
mM Tris-buffer pH 8.0 buffer which is recommended for the BirA biotin
ligase
that was kindly provided by Avidity LCC (Colorado, USA).
The
BAP-containing viral vector sample (500 µl)
was mixed according to the manufacturer protocol with each 72 µl Biomix
A,
Biomix B and Biotin. To reach maximal biotinylation, a volume of 5 µl
containing 25000 units of the biotin ligase BirA was added to the
reaction
mixture and incubated for 6 h at 30 °C. In order to remove unbound
biotin, the
biotinylated viral vectors were washed five times with 10 mM Tris
buffer pH 8.0.
Biotinylation
was verified with an ELISA as
depicted in figure A. For the detection of successfully biotinylated
viral
vectors, MaxiSorp 96-well plates (Nunc) were coated with 200 ng
monoclonal A20
antibody per well for eight hours at 4°C, and blocked over night with
PBST +
0.5 % BSA. This plate was incubated for 1 h at room temperature with
100 µl of
serial diluted viral vectors. After washing the plates three times, the
recommended amount of Streptavidin-HRP conjugate (Sigma-Aldrich) was
added to
each well for 1 h at room temperature. Again, the plates were washed
three
times followed by detection of absorbance caused by converted ABTS
substrate at
405 nm. Additionally, the genomic AAV-2 titer was determined by qPCR.
A: Schematic overview of the detection of BAP-presenting viral particles immobilized by A20 capture antibodies. Biotinylated capsids can be detected by streptavidin-coupled HRP B: ELISA AAV-2 particles carrying a BAP insertion in loop 587 which can be specifically biotinylated in vitro |
Results
and Discussion:
As
can be seen in
figure B, biotinylation of assembled AAV particles was achieved with
the
dilution series ranging from 2- to 128-fold. Correlating this assay
with the
qPCR experiment for the detection of encapsidated vector plasmid
(genomic titer
using CMV-promoter primers) yields a value of 4.70E+07 DNase-resistant
particles (DRP). Consequently, the presence of approximately 3.6x10^5
biotinylated
DRPs can be detected using the described ELISA. Compared to the virus
particle
detection ELISA emploing A20 as capture- and detection antibody, the
detection
limit of the biotinylated viral capsids is about 10fold more sensitive.
This
may be explained by the higher affinity of strepavidin towards biotin
relative
to the A20 antibody affinity or multiple biotinylation of a single
virus
particle.
Miniaturized antibody binding domain (Z34C)
Schematic overview of Z34C insertion into AAV-2 viral capsids enabling antibody arming for therapy |
The
idea of this
targeting approach is to utilize a minimized fragment of the Staphylococcal
Protein A that was first described
in Staphylococcus aureus. These
gram-positive bacteria have evolved the 508 amino acid long protein A
that has
a high affinity for the Fc-domain of antibodies to protect itself from
the
immune system. Binding to the constant region of the antibodies is
accomplished
by the Z-Domain of Protein A that is 58-59 amino acids long, has
alone a high
affinity (Kd= 14,9 nM) for the antibodies and a three-helix bundle
structure.
In [Braisted
& Wells;
1996] the authors reduced the
secundary
structure to an two-helix bundle. This size reduction has lead to an
drastic
reduction of the affinity for IgG (>10^5 fold) which could be
recovered by
13 amino acid exchanges resulting in a 38 amino acid long peptide with
an
satisfying affinity for IgG (Kd = 185 nM) termed Z38.
This binding domain was subsequently improved in [Starovasnik
et al.; 1997] by the insertion of a
disulfide bridge connecting the ends of
the helices leading to the binding domain Z34C which
shows an increased affinity for IgG (Kd = 20 nM).
|
Development of the Z34C motif, a miniaturized antibody binding motif |
This engineered antibody binding domain of 34 amino acids was then inserted into capsids of different viral vectors amongst others also the AAV. In [Ried et al.; 2002] the Z34C domain was inserted at position 587 into the capsid of the AAV resulting in viral vector that can be targeted to different target cells without genetic engineering. This targeting approach was then improved in [Gigout et al.; 2005] by the creation of mosaic vectors that contain only ~25% of recombinant VP-Proteins what resulted in 4 to 5 orders of magnitude more infectiosity compared to all-mutant viruses
Material and Methods:
Transfection of the
AAV-293 producer cells was performed for three different loop
insertions of
Z34C in each three 10 cm petri dishes with 3.4x10^6 cells resulting in
a
confluence of about 70-80%. For the transfection, either the composite
parts pCMV_VP123(453-Z34C),
pCMV_VP123(587KO- Z34C) or pCMV_VP123(587Ko- Z34C-Spacer) were
cotransfected
with pHelper,
[AAV2]-left-ITR_pCMV_betaglobin_mVenus_hGH_[AAV2]-right-ITR
and RepVP123(587KO). The producer cells were harvested
with the
culture supernatant and subjected to four cycles of freeze and thaw
cell lysis.
The cell lysate was centrifuged at 4000 rpm for 15 minutes and
concentrated in
a VivaSpin VS2002 (Sartorius Stedem) with a 10 kDa molecular weight
cut-off to
two milliliters and washed three to five times with 20 mM Bis-Tris
buffer, pH 6.
The cell lysates were incubated with 250 units of Benzonase
(Sigma-Aldrich) to
remove contaminant genomic DNA and loaded on an ÄKTA purifier (GE
Healthcare) equipped
with a Superdex 200 gel filtration column (GE Healthcare). Fractions
(500 µl)
around the void volume containing viral particles were collected and
used in a
sandwich ELISA. 96 well plates were coated with 200 ng Cetuximab
(Imclone/Merck/Bristol-Myers
Squibb). Detection was performed using the monoclonal antibody A20
(kindly
provided by PD Dr. J. Kleinschmidt, DKFZ Heidelberg) that was
biotinylated
using a Biotinylation kit (Dojindo, Japan) and Streptavidin-HRP
(Sigma-Aldrich).
The HRP presence was detected by the conversion of the substrate ABTS
at 405
nm. The average of the non-template controls (NTC) was subtracted from
the
sample data.
Results and Discussion:
A: Sandwich-ELISA scheme for the detection of Z34C-presenting viral particles. Immobilization is achieved by binding a IgG molecule. Intact viral capsids can be specifically bound by the biotinylated A20 antibody which can be detected by Strepavidin-HRP B: ELISA using the principle described in A for loop insertion samples obtained by gel filtration chromatography of 453- and 587-Z34C particles. Undiluted and 10-fold diluted samples were employed C: ELISA signals obtained by absorbance measurements from B at 405 nm, undiluted samples D: ELISA signals obtained by absorbance measurements from B at 405 nm, diluted samples |
Three samples of different viral
particles with insertions of the
antibody-binding motif Z34C were successfully purified by the gel
filtration
chromatography. The fractions around the void volume were subsequently
used in
two different sandwich ELISAs. The first one aims at the detection of
the
particle’s ability to bind IgG-antibodies. The therapeutical antibody
Cetuximab
was employed to test the affinity of the virus particles. As displayed
in
figure 1A, only Z34C-presenting particles should be immobilized and
only
assembled viral capsid will be detected due to the affinity of the A20
antibody
(as described above). Figure 1B shows that absorbance signal were
obtained in
case of the 587KO-Z34C and 587KO-Z34C-spacer insertions. Loop 453-Z34C
did not
yield any significant absorbance signals. In addition, the assay was
performed
with undiluted samples and those which were 10-fold diluted in PBST +
0.5 %
BSA. Signal strengths in the undiluted ELISA samples indicated that the
assay
was saturated, therefore the diluted sample data were used for
evaluation. The
ABTS conversion indicates that highest amount of Z34C-presenting viral
particles is present in the Äkta fractions 7-9 of the two different 587
insertions. No viral particles with a binding affinity for the IgG
antibody
were present in the 453 samples.
A: Sandwich-ELISA scheme for the detection of AAV-2 particles. Immobilization is achieved by binding to the A20 capture molecule which also acts as the biotinylated detection antibody after washing. Intact viral capsids can be specifically detected by Strepavidin-HRP B: ELISA using the principle described in A for loop insertion samples obtained by gel filtration chromatography of 453- and 587-Z34C particles. Undiluted and 10-fold diluted samples were employed C: qPCR data for the CMV-promoter-based DRP-titer determination. Samples as in B D: ELISA signals obtained by absorbance measurements from B at 405 nm, diluted samples |
A second approach was conducted to
reveal the reason for the absence
of Z34C-presenting particles in case of the 453 insertion. The sandwich
ELISA,
as shown in figure 2A, uses the A20 antibody to capture and detect all
assembled viral capsids independent of the presence of a Z34C motif
giving the
so-called physical AAV titer (see figure 2D). To correlate these
measurements,
we additionally conducted qPCR measurements to determine the amount of
encapsidated
vector plasmids, the so-called DRP (DNAse-resistant particle) titer
(see figure
2C). Comparison reveals that both assays yielded a peak signal around
the Äkta
fraction 8. The qPCR data indicated that all samples contain
approximately 2 x
10^9 copies of the vector plasmid per milliliter. Assuming equal
packaging
efficiencies for the vector plasmids, all three loop insertion
approaches
contain comparable amounts of viral capsids. The sandwich-ELISA yielded
a peak
around fraction eight for all three loop-insertion approaches. Since
the signal
strength for the 587KO-Z34C samples is significantly higher, it can be
assumed that
additionally to the affinity of the A20 antibody for assembled viral
capsids,
the Z34C-containing viral particles add a high affinity for the Fc-part
of the
detection antibody. Hence the qPCR-titration only depends on the number
of
encapsidated AAV-vector plasmids and not on the viral capsid and its
degree of
modification, it is the more quantitative method of particle titration
which
proved equal for all three loop insertion approaches.
Summarizing, the insertion of a 34/39
amino acid functional motif into the two most promising integration
sites of
the AAV-2 capsid (see figure 3) revealed no integration of modified
viral
capsid proteins (VP1-3) into assembled viral capsids for the 453
integration
site. As also seen by the data obtained for the linker-containing
insertion,
the 587 region tolerates insertions of at least 39 amino acids. For the
453
integration site, either the inserted Z34C motif loses its ability to
bind IgG
molecules or the influence of the inserted motif causes strong
structural
changes that disturb the ability of the modified VP proteins to be
integrated
into the virus capsid.
Loop insertion sites visualized in the 3D structure of a single viral coat protein (VP3) |