Difference between revisions of "Part:BBa K404003"

Revision as of 01:59, 27 October 2010

[AAV2]-Rep-VP123(ViralBrick-587KO-empty)_p5-TATAless

In our terminology the term “RepVP123” encompasses the whole AAV2 genome excluding the ITRs. The rep locus comprises four proteins related to genome replication while the cap locus codes for the proteins VP1, VP2, VP3 and the assembly-associated protein (AAP), which are required for viral capsid assembly. Source of the RepVP123 BioBrick supplied within iGEM team Freiburg_Bioware 2010 Virus Construction Kit is the wild-type AAV2 RepVP123, as provided e. g. in the pAAV vector from Stratagene. In order to introduce iGEM standard and additionally enabling the possibility to modify the viral capsid via integration of certain motives within the viral loops 453 and 587 a total of twelve mutations within RepVP123 (see Figure 1) and additionally two mutations within the pSB1C3 backbone were performed either by Site-Directed Mutagenesis (SDM) or by ordering and cloning of specifically designed gene sequences matching the required demands. Modifying the pSB1C3 led to iGEM team Freiburg_Bioware’s variant of this backbone, pSB1C3_001.

$Description: \\132.230.232.133\x\users\FreiGem\iGEM2010\Stefan\Pictures_Results\RepCap_complete_modifications_arrows.jpg$

Figure 1 Mutations implemented into RepVP123 in order to establish both iGEM standard and loop insertion capability. Green arrows indicate integrated restriction sites, red red arrows indicate deleted restriction sites. KpnI was deleted first and reinstated afterwards. (see text).

Plasmid name:	Functionality (determinded in cell culture via transduction and flow cytometry ):	4x mutations (PstI (310), BamHI (859), SalI (1239), PstI (4073))	inserted rep fragment	inserted cap fragment	reinstated KpnI	pAAV	pSB1C3_001	HSPG-ko
pAAV_RC (wild-type)	yes					x
pAAV_RC_4x mutant	yes	x				x
pAAV_RC_inserts	no	x	x	x		x
pAAV_RC_Cap	yes	x		x		x
pAAV_RC_RepVP123	yes	x	x	x	x	x
pSB1C3_RepVP123_ p5TATAless	yes	x	x	x	x		x
pSB1C3_RepVP123_ HSPG-ko_p5TATAless	yes	x	x	x	x		x	x

Figure 2 Table contains complete overview about all plasmids containing RepVP123 which were used by iGEM team Freiburg_Bioware 2010.

Modularization: Removing iGEM restriction sites and establishing loop insertion capability

Modifications in Rep

$Description: \\132.230.232.133\x\users\FreiGem\iGEM2010\Stefan\Pictures_Results\Rep_synthesis_marked.jpg$

Figure 3 Restriction sites within the wild-type rep gene sequence, which were removed via cloning of synthetized rep gene fragment into the plasmid. The red box indicates the region spanned by the synthetic sequence.

Making the RepVP123 wild-type compatible with the iGEM standards required the removal of five restriction sites (see Figure 1). This was achieved using site-directed mutagenesis for PstI (position 310) and PstI (4073). The remaining three iGEM restriction sites EcoRI (1578), PstI (1773) and EcoRI (1796) were replaced by a synthetic gene fragment, since the rep ORF contained these restriction sites in close proximity to each other plus an additional KpnI restriction site which was also not desired (see Figure 2). This gene fragment was cloned into the rep gene using HindIII and SwaI, which are single-cutting restriction enzymes adjacent to the target area. Additionally, BamHI (859) and SalI (1239) were removed, because these enzymes were required for genetically inserting the loop modifications in VP123.

Modifications in VP123

In order to implement the restriction sites necessary for targeting via loop insertions, the gene coding for the VP proteins was modified as well. The introduction of these restriction required up to four base mutations in a row, hence it was decided to synthesize this gene fragment and replace the wild-type sequence in RepVP123 as well.

$Description: \\132.230.232.133\x\users\FreiGem\iGEM2010\Stefan\Pictures_Results\Cap_synthesis_marked.jpg$

Figure 4 Restriction sites within cap sequence showing introduced loop insertion restriction sites into cap to enable cloning of targeting or purification motifs into both 453 and 587 loops. Again, the red box indicates gene sequence which was synthetized.

Figure 5 Results for transduction efficiency measured by flow cytometry. Fluorescence is measured in surviving cells. The tested RepVP123 containing four point mutation to delete iGEM and loop insertion restriction sites does not show any difference within mVenus expression compared to the wild-type and therefore can be verified working.

Alongside to creating an iGEM compatible plasmid, infectivity of the modified construct was tested in cell culture via flow cytometry. Then experiments confirmed that single cloning steps did not interfere with natural viral infectivity at first (see above). But cloning of the synthesized rep gene fragment into the plasmid dramatically reduced transduction efficiency as detected by flow cytometry. Scrutinizing each mutation and its potential impact, suggested that abolished transduction was related to the mutation, which removed the KpnI (1721) site. This site is located within a splice site, which is crucial for the Rep proteins, and thus even silent mutations may interfere with virus production (see extra topic “Rep proteins”).

xxx

Description: Fluorescence of HT1080 cells transduced with FRAGE AN ADRIAN:WELCHES GOI? and P325 and P326 both containing RepCap in which the synthetized gene sequences were inserted. As control,pAAV_RC (P50) containing wtRepCap was used. mVenus expression within cells clearly state that tested constructs does not meet expectations.

Figure 8 AAV-293 cells were transfected with three plasmids pHelper, pSB1C3_[AAV2]-left-ITR_pCMV_betaglobin_mVenus _hGH_[AAV2]-right-ITR and pAAV_RC_inserts (see above) or pAAV_RC (see below) providing essential genes and proteins for producing viral particles. After 48 hour post transfection, viral particles were harvested by freeze-thaw lysis and centrifugation followed by HT1080 transduction. mVenus expression of viral genomes was determined by flow cytomery 24 hours post infection. Fluorescence is measured in surviving cells. Results show that insertion of both rep and cap syntheses disrupts viral infectivity.

Therefore, additional constructs were designed containing only the synthetic cap sequence or both, the synthesized sequences plus a re-mutation of KpnI (1721), in order to re-establish the wild-type splice site within the rep ORF. Results from cell culture obtained via FACS revealed that in fact the poor results were related to the KpnI restriction site deletion. Both constructs showed a transduction efficiency corresponding to the unmodified wild-type RepVP123’s transduction efficiency.

Figure 9 Fluorescence of cells transduced with mVenus carrying rAAV measured by flow cytometry. AAV-293 cells were transfected with three plasmids pHelper, pSB1C3_[AAV2]-left-ITR_pCMV_betaglobin_mVenus_hGH_[AAV2]-right-ITR and RepVP123 constructs providing essential genes and proteins for producing viral particles. 48 hours post transfection, viral particles were harvested by freeze-thaw lysis and centrifugation followed by HT1080 transduction. mVenus expression of viral genomes was determined by flow cytometry 24 hours post infection. Results show that cap integration does not influence infectivity. Fluorescence is measured in surviving cells. Recreation of KpnI within rep splice site recovers transduction efficiency.

Modularization: Adapting pSB1C3 to loop insertions – pSB1C3_001

To fulfill iGEM requirements all plasmids need to be submitted in pSB1C3, therefore primers were ordered for amplifying RepVP123 containing all modifications done so far by PCR and cloning the into pSB1C3. Still, pSB1C3 contains two restriction sites for SspI and PvuII restriction enzymes in its CAT marker. Since these are necessary for cloning ViralBricks in this vector, the iGEM Team Freiburg_Bioware 2010 decided in agreement with iGEM Headquarters to implement a new standard for the pSB1C3 backbone which was named pSB1C3_001. Both restriction sites interfering with ViralBrick insertions were mutated to make SspI and PvuII single-cutters (see method development).

Figure 10 Comparison of pSB1C3 (upper row) and pSB1C3_001 (lower row). Deletions of SspI and PvuII are marked by red boxes.

RepVP123 containing both rep and cap synthetic gene fragments including the re-mutation of KpnI and the downstream p5TATA-less promotor was cloned into the newly constructed pSB1C3_001. Testing this newly assembled plasmid in cell culture revealed unexpected data. Not only did the newly assembled plasmid work (see Figure 10), but in comparison to pAAV containing the same RepVP123 construct, pSB1C3_001 showed an about 3 times higher transduction efficiency. Although exact reasons are still unknown, these results are probably related to the reduced length of pSB1C3_001 compared to the original pAAV plasmid of approximately 1000 base pairs.

Figure 11 AAV-293 cells were transfected with three plasmids pHelper, pSB1C3_001_[AAV2]-Rep-VP123_p5-TATAless or pAAV_RC_IRCK and pSB1C3_[AAV2]-left-ITR_pCMV_beta-globin_mVenus_hGH_[AAV2]-right-ITR providing essential genes and proteins for producing viral particles. After 48 hours post transfection, viral particles were harvested by freeze-thaw lysis and centrifugation followed by HT1080 transduction. mVenus expression of viral genomes was determined by flow cytomery after 24 hours post infection. Fluorescence is measured in surviving cells. Results showed functionality of RepVP123 within pSB1C3_001 vector and additionally increased transduction efficiency.

Turning-off natural tropism: HSPG-knock-out

Shutting-down the natural viral tropism is essential for targeting specifically tumor cells and not infecting healthy cells. Therefore, the iGEM team Freiburg_Bioware 2010 decided to knock-out the viral natural tropism delivered by the heperan sulfate proteoglycan-(HSPG) binding site within the viruses 587 loop. The knock-out was cloned by designing primers containing the required base exchanges and performing a SDM. Like performed before, this RepVP123 variant was tested in cell culture as well and evaluated by flow cytometry. Results show that mutation of HSPG-binding motif has severe impact on transduction efficiency thus enabling a viral particle carrying this knock-out and additional targeting motifs, e.g. within the loops or presented via N-terminal fusion to bind target cells’ receptors and therefore infecting target cells at a much higher rate compared to unspecific infection of other cell types within an organism (see Figure 12).

To quantify differences in infectivity, the infectious titer of viral particles built-up of RepVP123 with and without HSPG binding motif was determined by qPCR (see Figure 14) for different cell lines. Results show that the implemented HSPG-knock-out verifies results obtained from flow cytometry, infectious titers severely compared to RepVP123 with intact HSPG binding motif.

$Description: \\132.230.232.133\x\users\FreiGem\iGEM2010\Stefan\Pictures_Results\HSPG-ko_modified.jpg$

Figure 12 Alignment of 587 loop within viral VP123: The upper sequence shows a strand containing the HSPG binding motif (AGA, in red boxes), the lower sequence contains the HSPG-ko introduced by the iGEM team Freiburg_Bioware 2010 (GCT and GCC, blue boxes).

Figure 13 Transduction efficiency of HT1080 cells measured by flow cytometry. Fluorescence is measured in surviving cells. Knock-out of HSPG binding motif greatly reduces transduction efficiency compared to RepVP123 containing the motiv.

Figure 14 Infectious titers of RepVP123 with and without natural HSPG binding motif tested in different cell lines via qPCR. Shutting-down the HSPG binding motif reduces infectious titer in both HT1080 and HeLa cell lines. For A431 cells, no infectious titer could be detected via qPCR, which is probably related to poor transduction efficiency of A431 cells.

Overview of RepVP123 plasmid

Modularization: Overview

In our terminology the term “RepVP123” encompasses the whole AAV2 genome excluding the ITRs. The rep locus comprises four proteins related to genome replication while the cap locus codes for the proteins VP1, VP2, VP3 and the assembly-associated protein (AAP), which are required for viral capsid assembly. Source of the RepVP123 BioBrick supplied within iGEM team Freiburg_Bioware 2010 Virus Construction Kit is the wild-type AAV2 RepVP123, as provided e. g. in the pAAV vector from Stratagene. In order to introduce iGEM standard and additionally enabling the possibility to modify the viral capsid via integration of certain motives within the viral loops 453 and 587 a total of twelve mutations within RepVP123 (see Figure 1) and additionally two mutations within the pSB1C3 backbone were performed either by Site-Directed Mutagenesis (SDM) or by ordering and cloning of specifically designed gene sequences matching the required demands. Modifying the pSB1C3 led to iGEM team Freiburg_Bioware’s variant of this backbone, pSB1C3_001.

$Description: \\132.230.232.133\x\users\FreiGem\iGEM2010\Stefan\Pictures_Results\RepCap_complete_modifications_arrows.jpg$

Figure 1 Mutations implemented into RepVP123 in order to establish both iGEM standard and loop insertion capability. Green arrows indicate integrated restriction sites, red red arrows indicate deleted restriction sites. KpnI was deleted first and reinstated afterwards. (see text).

Plasmid name:	Functionality (determinded in cell culture via transduction and flow cytometry ):	4x mutations (PstI (310), BamHI (859), SalI (1239), PstI (4073))	inserted rep fragment	inserted cap fragment	reinstated KpnI	pAAV	pSB1C3_001	HSPG-ko
pAAV_RC (wild-type)	yes					x
pAAV_RC_4x mutant	yes	x				x
pAAV_RC_inserts	no	x	x	x		x
pAAV_RC_Cap	yes	x		x		x
pAAV_RC_RepVP123	yes	x	x	x	x	x
pSB1C3_RepVP123_ p5TATAless	yes	x	x	x	x		x
pSB1C3_RepVP123_ HSPG-ko_p5TATAless	yes	x	x	x	x		x	x

Figure 2 Table contains complete overview about all plasmids containing RepVP123 which were used by iGEM team Freiburg_Bioware 2010.

Modularization: Removing iGEM restriction sites and establishing loop insertion capability

Modifications in Rep

$Description: \\132.230.232.133\x\users\FreiGem\iGEM2010\Stefan\Pictures_Results\Rep_synthesis_marked.jpg$

Figure 3 Restriction sites within the wild-type rep gene sequence, which were removed via cloning of synthetized rep gene fragment into the plasmid. The red box indicates the region spanned by the synthetic sequence.

Making the RepVP123 wild-type compatible with the iGEM standards required the removal of five restriction sites (see Figure 1). This was achieved using site-directed mutagenesis for PstI (position 310) and PstI (4073). The remaining three iGEM restriction sites EcoRI (1578), PstI (1773) and EcoRI (1796) were replaced by a synthetic gene fragment, since the rep ORF contained these restriction sites in close proximity to each other plus an additional KpnI restriction site which was also not desired (see Figure 2). This gene fragment was cloned into the rep gene using HindIII and SwaI, which are single-cutting restriction enzymes adjacent to the target area. Additionally, BamHI (859) and SalI (1239) were removed, because these enzymes were required for genetically inserting the loop modifications in VP123.

Modifications in VP123

In order to implement the restriction sites necessary for targeting via loop insertions, the gene coding for the VP proteins was modified as well. The introduction of these restriction required up to four base mutations in a row, hence it was decided to synthesize this gene fragment and replace the wild-type sequence in RepVP123 as well.

$Description: \\132.230.232.133\x\users\FreiGem\iGEM2010\Stefan\Pictures_Results\Cap_synthesis_marked.jpg$

Figure 4 Restriction sites within cap sequence showing introduced loop insertion restriction sites into cap to enable cloning of targeting or purification motifs into both 453 and 587 loops. Again, the red box indicates gene sequence which was synthetized.

Modularization: Adapting pSB1C3 to loop insertions – pSB1C3_001

To fulfill iGEM requirements all plasmids need to be submitted in pSB1C3, therefore primers were ordered for amplifying RepVP123 containing all modifications done so far by PCR and cloning the into pSB1C3. Still, pSB1C3 contains two restriction sites for SspI and PvuII restriction enzymes in its CAT marker. Since these are necessary for cloning ViralBricks in this vector, the iGEM Team Freiburg_Bioware 2010 decided in agreement with iGEM Headquarters to implement a new standard for the pSB1C3 backbone which was named pSB1C3_001. Both restriction sites interfering with ViralBrick insertions were mutated to make SspI and PvuII single-cutters (see method development).

Figure 10 Comparison of pSB1C3 (upper row) and pSB1C3_001 (lower row). Deletions of SspI and PvuII are marked by red boxes.

RepVP123 containing both rep and cap synthetic gene fragments including the re-mutation of KpnI and the downstream p5TATA-less promotor was cloned into the newly constructed pSB1C3_001. Testing this newly assembled plasmid in cell culture revealed unexpected data. Not only did the newly assembled plasmid work (see Figure 10), but in comparison to pAAV containing the same RepVP123 construct, pSB1C3_001 showed an about 3 times higher transduction efficiency. Although exact reasons are still unknown, these results are probably related to the reduced length of pSB1C3_001 compared to the original pAAV plasmid of approximately 1000 base pairs.

Figure 11 AAV-293 cells were transfected with three plasmids pHelper, pSB1C3_001_[AAV2]-Rep-VP123_p5-TATAless or pAAV_RC_IRCK and pSB1C3_[AAV2]-left-ITR_pCMV_beta-globin_mVenus_hGH_[AAV2]-right-ITR providing essential genes and proteins for producing viral particles. After 48 hours post transfection, viral particles were harvested by freeze-thaw lysis and centrifugation followed by HT1080 transduction. mVenus expression of viral genomes was determined by flow cytomery after 24 hours post infection. Fluorescence is measured in surviving cells. Results showed functionality of RepVP123 within pSB1C3_001 vector and additionally increased transduction efficiency.

Turning-off natural tropism: HSPG-knock-out

Shutting-down the natural viral tropism is essential for targeting specifically tumor cells and not infecting healthy cells. Therefore, the iGEM team Freiburg_Bioware 2010 decided to knock-out the viral natural tropism delivered by the heperan sulfate proteoglycan-(HSPG) binding site within the viruses 587 loop. The knock-out was cloned by designing primers containing the required base exchanges and performing a SDM. Like performed before, this RepVP123 variant was tested in cell culture as well and evaluated by flow cytometry. Results show that mutation of HSPG-binding motif has severe impact on transduction efficiency thus enabling a viral particle carrying this knock-out and additional targeting motifs, e.g. within the loops or presented via N-terminal fusion to bind target cells’ receptors and therefore infecting target cells at a much higher rate compared to unspecific infection of other cell types within an organism (see Figure 12).

To quantify differences in infectivity, the infectious titer of viral particles built-up of RepVP123 with and without HSPG binding motif was determined by qPCR (see Figure 14) for different cell lines. Results show that the implemented HSPG-knock-out verifies results obtained from flow cytometry, infectious titers severely compared to RepVP123 with intact HSPG binding motif.

$Description: \\132.230.232.133\x\users\FreiGem\iGEM2010\Stefan\Pictures_Results\HSPG-ko_modified.jpg$

Figure 12 Alignment of 587 loop within viral VP123: The upper sequence shows a strand containing the HSPG binding motif (AGA, in red boxes), the lower sequence contains the HSPG-ko introduced by the iGEM team Freiburg_Bioware 2010 (GCT and GCC, blue boxes).

Figure 13 Transduction efficiency of HT1080 cells measured by flow cytometry. Fluorescence is measured in surviving cells. Knock-out of HSPG binding motif greatly reduces transduction efficiency compared to RepVP123 containing the motiv.

Figure 14 Infectious titers of RepVP123 with and without natural HSPG binding motif tested in different cell lines via qPCR. Shutting-down the HSPG binding motif reduces infectious titer in both HT1080 and HeLa cell lines. For A431 cells, no infectious titer could be detected via qPCR, which is probably related to poor transduction efficiency of A431 cells.

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
INCOMPATIBLE WITH RFC[21]
Illegal BamHI site found at 3611
Illegal XhoI site found at 1913
Illegal XhoI site found at 2099
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
INCOMPATIBLE WITH RFC[1000]
Illegal BsaI site found at 4137
Illegal BsaI site found at 4319
Illegal BsaI site found at 4356
Illegal SapI site found at 3048

@@ Line 1: / Line 1: @@
 __NOTOC__
 <partinfo>BBa_K404003 short</partinfo>
+<html>
+<head>
+  <h2 style="margin-left: 0cm; text-indent: 0cm;"><a
+ name="_Toc275885921"></a><a name="_Toc275817880"><span
+ lang="EN-US">Overview of RepVP123 plasmid</span></a></h2>
+  <h3 style="margin-left: 0cm; text-indent: 0cm;"><a
+ name="_Toc275885922"></a><a name="_Toc275817881"><span
+ lang="EN-US">Modularization: Overview</span></a></h3>
+  <p class="MsoNormal"
+ style="text-indent: 0cm; line-height: 150%; page-break-after: avoid;"><span
+ lang="EN-US">In our terminology the term “RepVP123”
+encompasses the
+whole AAV2 genome excluding the ITRs. The <i>rep</i> locus
+comprises four
+proteins related to genome replication while the <i>cap</i>
+locus codes for the
+proteins VP1, VP2, VP3 and the assembly-associated protein (AAP), which
+are
+required for viral capsid assembly. Source of the RepVP123 BioBrick
+supplied
+within iGEM team Freiburg_Bioware 2010 Virus Construction Kit is the
+wild-type
+AAV2 RepVP123, as provided e. g. in the pAAV vector from Stratagene. In
+order
+to introduce iGEM standard and additionally enabling the possibility to
+modify
+the viral capsid via integration of certain motives within the viral
+loops 453
+and 587 a total of twelve mutations within RepVP123 (see </span><span
+ lang="EN-US">Figure 1</span><span lang="EN-US">)
+and additionally two mutations
+within the pSB1C3 backbone were performed either by Site-Directed
+Mutagenesis
+(SDM) or by ordering and cloning of specifically designed gene
+sequences matching
+the required demands. Modifying the pSB1C3 led to iGEM team
+Freiburg_Bioware’s
+variant of this backbone, pSB1C3_001.</span></p>
+  <table class="MsoTableGrid"
+ style="border: medium none ; border-collapse: collapse; width: 636px; height: 523px; text-align: left; margin-left: auto; margin-right: auto;"
+ border="0" cellpadding="0" cellspacing="0">
+    <tbody>
+      <tr>
+        <td
+ style="border: 1pt solid windowtext; padding: 0cm 5.4pt; vertical-align: top; width: 462.5pt;">
+        <p class="MsoNormal"
+ style="text-align: center; text-indent: 0cm; line-height: 150%; page-break-after: avoid;"
+ align="center"><img style="width: 605px; height: 451px;"
+ id="Picture 17"
+ src="https://static.igem.org/mediawiki/2010/0/0b/Freiburg10_pAAV_complete_mutations.png"
+ alt="Description: \\132.230.232.133\x\users\FreiGem\iGEM2010\Stefan\Pictures_Results\RepCap_complete_modifications_arrows.jpg"></p>
+        <p class="MsoCaption" style="text-indent: 0cm;"><a
+ name="_Ref275820820"><span lang="EN-US">Figure </span></a><span
+ lang="EN-US">1</span> <span
+ style="font-size: 8pt; color: windowtext; font-weight: normal;"
+ lang="EN-US">Mutations implemented into <i>RepVP123</i>
+in order to establish both iGEM standard and loop insertion capability.
+Green arrows indicate integrated restriction sites, red red arrows
+indicate deleted restriction sites. KpnI was deleted first and
+reinstated afterwards. (see text).</span></p>
+        </td>
+      </tr>
+    </tbody>
+  </table>
+  <span
+ style="font-size: 8pt; line-height: 150%; font-family: &quot;Calibri&quot;,&quot;sans-serif&quot;;"
+ lang="EN-US"><br style="page-break-before: always;"
+ clear="all">
+  </span>
+  <p class="MsoNormal"
+ style="text-indent: 0cm; line-height: 150%;"><span
+ style="font-size: 8pt; line-height: 150%;" lang="EN-US">&nbsp;</span></p>
+  <table class="MsoNormalTable"
+ style="border-collapse: collapse; width: 651px; height: 261px; text-align: left; margin-left: auto; margin-right: auto;"
+ border="0" cellpadding="0" cellspacing="0">
+    <tbody>
+      <tr style="height: 27pt;">
+        <td
+ style="border: 1pt solid windowtext; padding: 0.75pt 0.75pt 0cm; width: 86.45pt; height: 27pt;">
+        <p class="MsoNormal"
+ style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><span
+ style="font-size: 10pt; color: black;" lang="EN-US">Plasmid
+name:</span></p>
+        </td>
+        <td
+ style="border-style: solid solid solid none; border-color: windowtext windowtext windowtext -moz-use-text-color; border-width: 1pt 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 77.35pt; height: 27pt;"
+ width="103">
+        <p class="MsoNormal"
+ style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><span
+ style="font-size: 10pt; color: black;" lang="EN-US">Functionality
+(determinded in cell culture via transduction and flow cytometry ):</span></p>
+        </td>
+        <td
+ style="border-style: solid solid solid none; border-color: windowtext windowtext windowtext -moz-use-text-color; border-width: 1pt 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 77.95pt; height: 27pt;"
+ width="104">
+        <p class="MsoNormal"
+ style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><span
+ style="font-size: 10pt; color: black;" lang="EN-US">4x
+mutations (PstI (310), BamHI (859), SalI (1239), PstI (4073))</span></p>
+        </td>
+        <td
+ style="border-style: solid solid solid none; border-color: windowtext windowtext windowtext -moz-use-text-color; border-width: 1pt 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 42.5pt; height: 27pt;"
+ width="57">
+        <p class="MsoNormal"
+ style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><span
+ style="font-size: 10pt; color: black;">inserted <i>rep</i>
+fragment</span></p>
+        </td>
+        <td
+ style="border-style: solid solid solid none; border-color: windowtext windowtext windowtext -moz-use-text-color; border-width: 1pt 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 49.65pt; height: 27pt;"
+ width="66">
+        <p class="MsoNormal"
+ style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><span
+ style="font-size: 10pt; color: black;">inserted<i> cap</i>
+fragment</span></p>
+        </td>
+        <td
+ style="border-style: solid solid solid none; border-color: windowtext windowtext windowtext -moz-use-text-color; border-width: 1pt 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 50.2pt; height: 27pt;"
+ width="67">
+        <p class="MsoNormal"
+ style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><span
+ style="font-size: 10pt; color: black;">reinstated KpnI</span></p>
+        </td>
+        <td
+ style="border-style: solid solid solid none; border-color: windowtext windowtext windowtext -moz-use-text-color; border-width: 1pt 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 24pt; height: 27pt;"
+ width="32">
+        <p class="MsoNormal"
+ style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><span
+ style="font-size: 10pt; color: black;">pAAV</span></p>
+        </td>
+        <td
+ style="border-style: solid solid solid none; border-color: windowtext windowtext windowtext -moz-use-text-color; border-width: 1pt 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 52.45pt; height: 27pt;"
+ width="70">
+        <p class="MsoNormal"
+ style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><span
+ style="font-size: 10pt; color: black;">pSB1C3_001</span></p>
+        </td>
+        <td
+ style="border-style: solid solid solid none; border-color: windowtext windowtext windowtext -moz-use-text-color; border-width: 1pt 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 27.45pt; height: 27pt;"
+ width="37">
+        <p class="MsoNormal"
+ style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><span
+ style="font-size: 10pt; color: black;">HSPG-ko</span></p>
+        </td>
+      </tr>
+      <tr style="height: 15pt;">
+        <td
+ style="border-style: none solid solid; border-color: -moz-use-text-color windowtext windowtext; border-width: medium 1pt 1pt; padding: 0.75pt 0.75pt 0cm; width: 86.45pt; height: 15pt;"
+ width="115">
+        <p class="MsoNormal"
+ style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><span
+ style="font-size: 10pt; color: black;" lang="EN-US">pAAV_RC
+(wild-type)</span></p>
+        </td>
+        <td
+ style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 77.35pt; height: 15pt;"
+ width="103">
+        <p class="MsoNormal"
+ style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><span
+ style="font-size: 10pt; color: black;" lang="EN-US">yes</span></p>
+        </td>
+        <td
+ style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 77.95pt; height: 15pt;"
+ width="104">
+        <p class="MsoNormal"
+ style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><span
+ style="font-size: 10pt; color: black;" lang="EN-US">&nbsp;</span></p>
+        </td>
+        <td
+ style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 42.5pt; height: 15pt;"
+ nowrap="nowrap" valign="bottom" width="57">
+        <p class="MsoNormal"
+ style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;"
+ align="left"><span style="font-size: 10pt; color: black;">&nbsp;</span></p>
+        </td>
+        <td
+ style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 49.65pt; height: 15pt;"
+ nowrap="nowrap" valign="bottom" width="66">
+        <p class="MsoNormal"
+ style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;"
+ align="left"><span style="font-size: 10pt; color: black;">&nbsp;</span></p>
+        </td>
+        <td
+ style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 50.2pt; height: 15pt;"
+ nowrap="nowrap" valign="bottom" width="67">
+        <p class="MsoNormal"
+ style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;"
+ align="left"><span style="font-size: 10pt; color: black;">&nbsp;</span></p>
+        </td>
+        <td
+ style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 24pt; height: 15pt;"
+ nowrap="nowrap" valign="bottom" width="32">
+        <p class="MsoNormal"
+ style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;"
+ align="left"><span style="font-size: 10pt; color: black;">x</span></p>
+        </td>
+        <td
+ style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 52.45pt; height: 15pt;"
+ nowrap="nowrap" valign="bottom" width="70">
+        <p class="MsoNormal"
+ style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;"
+ align="left"><span style="font-size: 10pt; color: black;">&nbsp;</span></p>
+        </td>
+        <td
+ style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 27.45pt; height: 15pt;"
+ nowrap="nowrap" valign="bottom" width="37">
+        <p class="MsoNormal"
+ style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;"
+ align="left"><span style="font-size: 10pt; color: black;">&nbsp;</span></p>
+        </td>
+      </tr>
+      <tr style="height: 15pt;">
+        <td
+ style="border-style: none solid solid; border-color: -moz-use-text-color windowtext windowtext; border-width: medium 1pt 1pt; padding: 0.75pt 0.75pt 0cm; width: 86.45pt; height: 15pt;"
+ width="115">
+        <p class="MsoNormal"
+ style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><span
+ style="font-size: 10pt; color: black;" lang="EN-US">pAAV_RC_4x
+mutant</span></p>
+        </td>
+        <td
+ style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 77.35pt; height: 15pt;"
+ width="103">
+        <p class="MsoNormal"
+ style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><span
+ style="font-size: 10pt; color: black;" lang="EN-US">yes</span></p>
+        </td>
+        <td
+ style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 77.95pt; height: 15pt;"
+ width="104">
+        <p class="MsoNormal"
+ style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><span
+ style="font-size: 10pt; color: black;" lang="EN-US">x</span></p>
+        </td>
+        <td
+ style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 42.5pt; height: 15pt;"
+ nowrap="nowrap" valign="bottom" width="57">
+        <p class="MsoNormal"
+ style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;"
+ align="left"><span style="font-size: 10pt; color: black;">&nbsp;</span></p>
+        </td>
+        <td
+ style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 49.65pt; height: 15pt;"
+ nowrap="nowrap" valign="bottom" width="66">
+        <p class="MsoNormal"
+ style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;"
+ align="left"><span style="font-size: 10pt; color: black;">&nbsp;</span></p>
+        </td>
+        <td
+ style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 50.2pt; height: 15pt;"
+ nowrap="nowrap" valign="bottom" width="67">
+        <p class="MsoNormal"
+ style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;"
+ align="left"><span style="font-size: 10pt; color: black;">&nbsp;</span></p>
+        </td>
+        <td
+ style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 24pt; height: 15pt;"
+ nowrap="nowrap" valign="bottom" width="32">
+        <p class="MsoNormal"
+ style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;"
+ align="left"><span style="font-size: 10pt; color: black;">x</span></p>
+        </td>
+        <td
+ style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 52.45pt; height: 15pt;"
+ nowrap="nowrap" valign="bottom" width="70">
+        <p class="MsoNormal"
+ style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;"
+ align="left"><span style="font-size: 10pt; color: black;">&nbsp;</span></p>
+        </td>
+        <td
+ style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 27.45pt; height: 15pt;"
+ nowrap="nowrap" valign="bottom" width="37">
+        <p class="MsoNormal"
+ style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;"
+ align="left"><span style="font-size: 10pt; color: black;">&nbsp;</span></p>
+        </td>
+      </tr>
+      <tr style="height: 15pt;">
+        <td
+ style="border-style: none solid solid; border-color: -moz-use-text-color windowtext windowtext; border-width: medium 1pt 1pt; padding: 0.75pt 0.75pt 0cm; width: 86.45pt; height: 15pt;"
+ width="115">
+        <p class="MsoNormal"
+ style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><span
+ style="font-size: 10pt; color: black;" lang="EN-US">pAAV_RC_inserts
+        </span></p>
+        </td>
+        <td
+ style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 77.35pt; height: 15pt;"
+ width="103">
+        <p class="MsoNormal"
+ style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><span
+ style="font-size: 10pt; color: black;" lang="EN-US">no</span></p>
+        </td>
+        <td
+ style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 77.95pt; height: 15pt;"
+ width="104">
+        <p class="MsoNormal"
+ style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><span
+ style="font-size: 10pt; color: black;" lang="EN-US">x</span></p>
+        </td>
+        <td
+ style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 42.5pt; height: 15pt;"
+ nowrap="nowrap" valign="bottom" width="57">
+        <p class="MsoNormal"
+ style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;"
+ align="left"><span style="font-size: 10pt; color: black;">x</span></p>
+        </td>
+        <td
+ style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 49.65pt; height: 15pt;"
+ nowrap="nowrap" valign="bottom" width="66">
+        <p class="MsoNormal"
+ style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;"
+ align="left"><span style="font-size: 10pt; color: black;">x</span></p>
+        </td>
+        <td
+ style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 50.2pt; height: 15pt;"
+ nowrap="nowrap" valign="bottom" width="67">
+        <p class="MsoNormal"
+ style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;"
+ align="left"><span style="font-size: 10pt; color: black;">&nbsp;</span></p>
+        </td>
+        <td
+ style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 24pt; height: 15pt;"
+ nowrap="nowrap" valign="bottom" width="32">
+        <p class="MsoNormal"
+ style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;"
+ align="left"><span style="font-size: 10pt; color: black;">x</span></p>
+        </td>
+        <td
+ style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 52.45pt; height: 15pt;"
+ nowrap="nowrap" valign="bottom" width="70">
+        <p class="MsoNormal"
+ style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;"
+ align="left"><span style="font-size: 10pt; color: black;">&nbsp;</span></p>
+        </td>
+        <td
+ style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 27.45pt; height: 15pt;"
+ nowrap="nowrap" valign="bottom" width="37">
+        <p class="MsoNormal"
+ style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;"
+ align="left"><span style="font-size: 10pt; color: black;">&nbsp;</span></p>
+        </td>
+      </tr>
+      <tr style="height: 15pt;">
+        <td
+ style="border-style: none solid solid; border-color: -moz-use-text-color windowtext windowtext; border-width: medium 1pt 1pt; padding: 0.75pt 0.75pt 0cm; width: 86.45pt; height: 15pt;"
+ width="115">
+        <p class="MsoNormal"
+ style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><span
+ style="font-size: 10pt; color: black;" lang="EN-US">pAAV_RC_Cap</span></p>
+        </td>
+        <td
+ style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 77.35pt; height: 15pt;"
+ width="103">
+        <p class="MsoNormal"
+ style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><span
+ style="font-size: 10pt; color: black;" lang="EN-US">yes</span></p>
+        </td>
+        <td
+ style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 77.95pt; height: 15pt;"
+ width="104">
+        <p class="MsoNormal"
+ style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><span
+ style="font-size: 10pt; color: black;" lang="EN-US">x</span></p>
+        </td>
+        <td
+ style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 42.5pt; height: 15pt;"
+ nowrap="nowrap" valign="bottom" width="57">
+        <p class="MsoNormal"
+ style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;"
+ align="left"><span style="font-size: 10pt; color: black;">&nbsp;</span></p>
+        </td>
+        <td
+ style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 49.65pt; height: 15pt;"
+ nowrap="nowrap" valign="bottom" width="66">
+        <p class="MsoNormal"
+ style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;"
+ align="left"><span style="font-size: 10pt; color: black;">x</span></p>
+        </td>
+        <td
+ style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 50.2pt; height: 15pt;"
+ nowrap="nowrap" valign="bottom" width="67">
+        <p class="MsoNormal"
+ style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;"
+ align="left"><span style="font-size: 10pt; color: black;">&nbsp;</span></p>
+        </td>
+        <td
+ style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 24pt; height: 15pt;"
+ nowrap="nowrap" valign="bottom" width="32">
+        <p class="MsoNormal"
+ style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;"
+ align="left"><span style="font-size: 10pt; color: black;">x</span></p>
+        </td>
+        <td
+ style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 52.45pt; height: 15pt;"
+ nowrap="nowrap" valign="bottom" width="70">
+        <p class="MsoNormal"
+ style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;"
+ align="left"><span style="font-size: 10pt; color: black;">&nbsp;</span></p>
+        </td>
+        <td
+ style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 27.45pt; height: 15pt;"
+ nowrap="nowrap" valign="bottom" width="37">
+        <p class="MsoNormal"
+ style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;"
+ align="left"><span style="font-size: 10pt; color: black;">&nbsp;</span></p>
+        </td>
+      </tr>
+      <tr style="height: 15pt;">
+        <td
+ style="border-style: none solid solid; border-color: -moz-use-text-color windowtext windowtext; border-width: medium 1pt 1pt; padding: 0.75pt 0.75pt 0cm; width: 86.45pt; height: 15pt;"
+ width="115">
+        <p class="MsoNormal"
+ style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><span
+ style="font-size: 10pt; color: black;" lang="EN-US">pAAV_RC_RepVP123</span></p>
+        </td>
+        <td
+ style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 77.35pt; height: 15pt;"
+ width="103">
+        <p class="MsoNormal"
+ style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><span
+ style="font-size: 10pt; color: black;" lang="EN-US">yes</span></p>
+        </td>
+        <td
+ style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 77.95pt; height: 15pt;"
+ width="104">
+        <p class="MsoNormal"
+ style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><span
+ style="font-size: 10pt; color: black;" lang="EN-US">x</span></p>
+        </td>
+        <td
+ style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 42.5pt; height: 15pt;"
+ nowrap="nowrap" valign="bottom" width="57">
+        <p class="MsoNormal"
+ style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;"
+ align="left"><span style="font-size: 10pt; color: black;">x</span></p>
+        </td>
+        <td
+ style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 49.65pt; height: 15pt;"
+ nowrap="nowrap" valign="bottom" width="66">
+        <p class="MsoNormal"
+ style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;"
+ align="left"><span style="font-size: 10pt; color: black;">x</span></p>
+        </td>
+        <td
+ style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 50.2pt; height: 15pt;"
+ nowrap="nowrap" valign="bottom" width="67">
+        <p class="MsoNormal"
+ style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;"
+ align="left"><span style="font-size: 10pt; color: black;">x</span></p>
+        </td>
+        <td
+ style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 24pt; height: 15pt;"
+ nowrap="nowrap" valign="bottom" width="32">
+        <p class="MsoNormal"
+ style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;"
+ align="left"><span style="font-size: 10pt; color: black;">x</span></p>
+        </td>
+        <td
+ style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 52.45pt; height: 15pt;"
+ nowrap="nowrap" valign="bottom" width="70">
+        <p class="MsoNormal"
+ style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;"
+ align="left"><span style="font-size: 10pt; color: black;">&nbsp;</span></p>
+        </td>
+        <td
+ style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 27.45pt; height: 15pt;"
+ nowrap="nowrap" valign="bottom" width="37">
+        <p class="MsoNormal"
+ style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;"
+ align="left"><span style="font-size: 10pt; color: black;">&nbsp;</span></p>
+        </td>
+      </tr>
+      <tr style="height: 18pt;">
+        <td
+ style="border-style: none solid solid; border-color: -moz-use-text-color windowtext windowtext; border-width: medium 1pt 1pt; padding: 0.75pt 0.75pt 0cm; width: 86.45pt; height: 18pt;"
+ width="115">
+        <p class="MsoNormal"
+ style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><span
+ style="font-size: 10pt; color: black;" lang="EN-US">pSB1C3_RepVP123_
+p5TATAless</span></p>
+        </td>
+        <td
+ style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 77.35pt; height: 18pt;"
+ width="103">
+        <p class="MsoNormal"
+ style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><span
+ style="font-size: 10pt; color: black;" lang="EN-US">yes</span></p>
+        </td>
+        <td
+ style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 77.95pt; height: 18pt;"
+ width="104">
+        <p class="MsoNormal"
+ style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><span
+ style="font-size: 10pt; color: black;" lang="EN-US">x</span></p>
+        </td>
+        <td
+ style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 42.5pt; height: 18pt;"
+ nowrap="nowrap" valign="bottom" width="57">
+        <p class="MsoNormal"
+ style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;"
+ align="left"><span style="font-size: 10pt; color: black;">x</span></p>
+        </td>
+        <td
+ style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 49.65pt; height: 18pt;"
+ nowrap="nowrap" valign="bottom" width="66">
+        <p class="MsoNormal"
+ style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;"
+ align="left"><span style="font-size: 10pt; color: black;">x</span></p>
+        </td>
+        <td
+ style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 50.2pt; height: 18pt;"
+ nowrap="nowrap" valign="bottom" width="67">
+        <p class="MsoNormal"
+ style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;"
+ align="left"><span style="font-size: 10pt; color: black;">x</span></p>
+        </td>
+        <td
+ style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 24pt; height: 18pt;"
+ nowrap="nowrap" valign="bottom" width="32">
+        <p class="MsoNormal"
+ style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;"
+ align="left"><span style="font-size: 10pt; color: black;">&nbsp;</span></p>
+        </td>
+        <td
+ style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 52.45pt; height: 18pt;"
+ nowrap="nowrap" valign="bottom" width="70">
+        <p class="MsoNormal"
+ style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;"
+ align="left"><span style="font-size: 10pt; color: black;">x</span></p>
+        </td>
+        <td
+ style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 27.45pt; height: 18pt;"
+ nowrap="nowrap" valign="bottom" width="37">
+        <p class="MsoNormal"
+ style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;"
+ align="left"><span style="font-size: 10pt; color: black;">&nbsp;</span></p>
+        </td>
+      </tr>
+      <tr style="height: 27pt;">
+        <td
+ style="border-style: none solid solid; border-color: -moz-use-text-color windowtext windowtext; border-width: medium 1pt 1pt; padding: 0.75pt 0.75pt 0cm; width: 86.45pt; height: 27pt;"
+ width="115">
+        <p class="MsoNormal"
+ style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><span
+ style="font-size: 10pt; color: black;" lang="EN-US">pSB1C3_RepVP123_
+HSPG-ko_p5TATAless</span></p>
+        </td>
+        <td
+ style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 77.35pt; height: 27pt;"
+ width="103">
+        <p class="MsoNormal"
+ style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><span
+ style="font-size: 10pt; color: black;" lang="EN-US">yes</span></p>
+        </td>
+        <td
+ style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 77.95pt; height: 27pt;"
+ width="104">
+        <p class="MsoNormal"
+ style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><span
+ style="font-size: 10pt; color: black;" lang="EN-US">x</span></p>
+        </td>
+        <td
+ style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 42.5pt; height: 27pt;"
+ nowrap="nowrap" valign="bottom" width="57">
+        <p class="MsoNormal"
+ style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;"
+ align="left"><span style="font-size: 10pt; color: black;">x</span></p>
+        </td>
+        <td
+ style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 49.65pt; height: 27pt;"
+ nowrap="nowrap" valign="bottom" width="66">
+        <p class="MsoNormal"
+ style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;"
+ align="left"><span style="font-size: 10pt; color: black;">x</span></p>
+        </td>
+        <td
+ style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 50.2pt; height: 27pt;"
+ nowrap="nowrap" valign="bottom" width="67">
+        <p class="MsoNormal"
+ style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;"
+ align="left"><span style="font-size: 10pt; color: black;">x</span></p>
+        </td>
+        <td
+ style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 24pt; height: 27pt;"
+ nowrap="nowrap" valign="bottom" width="32">
+        <p class="MsoNormal"
+ style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;"
+ align="left"><span style="font-size: 10pt; color: black;">&nbsp;</span></p>
+        </td>
+        <td
+ style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 52.45pt; height: 27pt;"
+ nowrap="nowrap" valign="bottom" width="70">
+        <p class="MsoNormal"
+ style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;"
+ align="left"><span style="font-size: 10pt; color: black;">x</span></p>
+        </td>
+        <td
+ style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 27.45pt; height: 27pt;"
+ nowrap="nowrap" valign="bottom" width="37">
+        <p class="MsoNormal"
+ style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal; page-break-after: avoid;"
+ align="left"><span style="font-size: 10pt; color: black;">x</span></p>
+        </td>
+      </tr>
+    </tbody>
+  </table>
+  <p class="MsoCaption" style="text-align: left;"
+ align="left"><span lang="EN-US">Figure </span><span
+ lang="EN-US">2</span> <span
+ style="color: windowtext; font-weight: normal;" lang="EN-US">Table
+contains complete overview about all plasmids containing <i>RepVP123</i>
+which
+were used by iGEM team Freiburg_Bioware 2010.</span></p>
+  <h3 style="margin-left: 0cm; text-indent: 0cm;"><a
+ name="_Toc275885923"></a><a name="_Toc275817882"><span
+ lang="EN-US">Modularization: Removing iGEM
+restriction
+sites and establishing loop insertion capability</span></a></h3>
+  <h4 style="margin-left: 0cm; text-indent: 0cm;"><a
+ name="_Toc275885924"></a><a name="_Toc275817883"><span
+ lang="EN-US">Modifications in Rep</span></a></h4>
+  <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="border: 1pt solid windowtext; padding: 0cm 5.4pt; vertical-align: top; width: 460.6pt;">
+        <p class="MsoNormal"
+ style="text-align: center; text-indent: 0cm; page-break-after: avoid;"
+ align="center"><img style="width: 576px; height: 191px;"
+ id="Picture 4"
+ src="https://static.igem.org/mediawiki/2010/b/bc/Freiburg10_rep_synthetic_gene_fragment.png"
+ alt="Description: \\132.230.232.133\x\users\FreiGem\iGEM2010\Stefan\Pictures_Results\Rep_synthesis_marked.jpg"></p>
+        <p class="MsoCaption"><a name="_Ref275818017"></a><a
+ name="_Ref275818042"><span lang="EN-US">Figure </span></a><span
+ lang="EN-US">3</span> <span
+ style="font-size: 8pt; color: windowtext; font-weight: normal;"
+ lang="EN-US">Restriction sites within the wild-type <i>rep</i>
+gene sequence, which were removed via cloning of synthetized <i>rep
+        </i>gene
+fragment into the plasmid. The red box indicates
+the region spanned by the synthetic sequence.</span></p>
+        </td>
+      </tr>
+    </tbody>
+  </table>
+  <p class="MsoNormal" style="text-indent: 0cm;"><span
+ lang="EN-US">Making
+the <i>RepVP123</i>
+wild-type compatible with the iGEM standards required the removal of
+five
+restriction sites (see </span><span lang="EN-US">Figure
+</span><span lang="EN-US">).
+This was achieved using site-directed mutagenesis for PstI (position
+) and
+PstI (4073). The remaining three iGEM restriction sites EcoRI (1578),
+PstI
+(1773) and EcoRI (1796) were replaced by a synthetic gene fragment,
+since the <i>rep
+  </i>ORF contained these restriction sites in close proximity to
+each other plus
+an additional KpnI restriction site which was also not desired (see </span><span
+ lang="EN-US">Figure 2</span><span lang="EN-US">).
+This gene fragment was cloned into
+the <i>rep</i> gene using HindIII and SwaI, which are
+single-cutting
+restriction enzymes adjacent to the target area. Additionally, BamHI
+(859) and
+SalI (1239) were removed, because these enzymes were required for
+genetically
+inserting the loop modifications in VP123.</span></p>
+  <h4 style="margin-left: 0cm; text-indent: 0cm;"><a
+ name="_Toc275885925"></a><a name="_Toc275817884"><span
+ lang="EN-US">Modifications in VP123</span></a></h4>
+  <p class="MsoNormal" style="text-indent: 0cm;"><span
+ lang="EN-US">In
+order to
+implement the restriction sites necessary for targeting via loop
+insertions,
+the gene coding for the VP proteins was modified as well. The
+introduction of
+these restriction required up to four base mutations in a row, hence it
+was
+decided to synthesize this gene fragment and replace the wild-type
+sequence in <i>RepVP123</i>
+as well.</span></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="border: 1pt solid windowtext; padding: 0cm 5.4pt; vertical-align: top; width: 460.6pt;">
+        <p class="MsoNormal"
+ style="text-align: center; text-indent: 0cm; page-break-after: avoid;"
+ align="center"><img style="width: 574px; height: 218px;"
+ id="Picture 5"
+ src="https://static.igem.org/mediawiki/2010/c/cd/Freiburg10_cap_synthetic_gene_fragment.png"
+ alt="Description: \\132.230.232.133\x\users\FreiGem\iGEM2010\Stefan\Pictures_Results\Cap_synthesis_marked.jpg"></p>
+        <p class="MsoCaption"><span lang="EN-US">Figure
+        </span><span lang="EN-US">4</span> <span
+ style="font-size: 8pt; color: windowtext; font-weight: normal;"
+ lang="EN-US">Restriction sites within <i>cap</i>
+sequence showing introduced loop insertion restriction sites into <i>cap</i>
+to enable cloning of targeting or purification motifs into both 453 and
+loops. Again, the red box indicates gene sequence which was
+synthetized.</span></p>
+        </td>
+      </tr>
+    </tbody>
+  </table>
+  <p class="MsoNormal" style="text-indent: 0cm;"><span
+ lang="EN-US">&nbsp;</span></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="border: 1pt solid windowtext; padding: 0cm 3.5pt; vertical-align: top; width: 460.6pt;">
+        <p class="MsoNormal"
+ style="text-align: center; text-indent: 0cm; page-break-after: avoid;"
+ align="center"><img style="width: 511px; height: 302px;"
+ alt="" id="Chart 7"
+ src="https://static.igem.org/mediawiki/2010/6/6b/Freiburg10_wt_vs_4xmutant.png"></p>
+        <p class="MsoCaption"><span lang="EN-US">Figure
+        </span><span lang="EN-US">5</span> <span
+ style="color: windowtext; font-weight: normal;" lang="EN-US">Results
+for transduction efficiency measured by flow cytometry. Fluorescence is
+measured in surviving cells. The tested <i>RepVP123</i>
+containing four point mutation to delete iGEM and loop insertion
+restriction sites does not show any difference within mVenus expression
+compared to the wild-type and therefore can be verified working.</span></p>
+        </td>
+      </tr>
+    </tbody>
+  </table>
+  <p class="MsoNormal" style="text-indent: 0cm;"><span
+ lang="EN-US">Alongside
+to
+creating an iGEM compatible plasmid, infectivity of the modified
+construct was
+tested in cell culture via flow cytometry. Then experiments confirmed
+that
+single cloning steps did not interfere with natural viral infectivity
+at first
+(see above). But cloning of the synthesized <i>rep</i>
+gene fragment into the
+plasmid dramatically reduced transduction efficiency as detected by
+flow
+cytometry. Scrutinizing each mutation and its potential impact,
+suggested that
+abolished transduction was related to the mutation, which removed the
+KpnI
+(1721) site. This site is located within a splice site, which is
+crucial for
+the Rep proteins, and thus even silent mutations may interfere with
+virus
+production (see extra topic “Rep proteins”).</span></p>
+  <span
+ style="font-size: 11pt; line-height: 200%; font-family: &quot;Calibri&quot;,&quot;sans-serif&quot;;"
+ lang="EN-US"><br style="page-break-before: always;"
+ clear="all">
+  </span>
+  <p class="MsoNormal"
+ style="text-indent: 0cm; text-align: center;"><span
+ lang="EN-US">&nbsp;<img alt="xxx"
+ src="https://static.igem.org/mediawiki/2010/f/fd/Freiburg10_Freiburg10_facs_table1.png"><br>
+  </span></p>
+  <br>
+  <p style="text-align: center;" class="MsoNormal">&nbsp;<img
+ style="width: 636px; height: 396px;" alt=""
+ src="https://static.igem.org/mediawiki/2010/3/3a/Freiburg10_Freiburg10_facs_table2.png"></p>
+  <br>
+  <p class="MsoNormal"
+ style="text-indent: 0cm; line-height: 150%;"><span
+ lang="EN-US"></span></p>
+  <p class="MsoNormal"
+ style="text-indent: 0cm; line-height: 150%;"><span
+ lang="EN-US"></span></p>
+  <table
+ style="width: 474px; text-align: left; margin-left: auto; margin-right: auto;"
+ border="0" cellpadding="0" cellspacing="0">
+    <tbody>
+      <tr>
+        <td style="width: 464px;">
+        <p class="MsoNormal"
+ style="margin-bottom: 10pt; text-align: center; text-indent: 0cm; line-height: normal; page-break-after: avoid;"
+ align="center"><span style="font-size: 10pt;"><img
+ style="width: 395px; height: 215px;" id="Chart 18"
+ src="https://static.igem.org/mediawiki/2010/7/73/Freiburg10_KpnI_deleted.png"
+ alt="Description: Fluorescence of HT1080 cells transduced with FRAGE AN ADRIAN:WELCHES GOI? and P325 and P326 both containing RepCap in which the synthetized gene sequences were inserted. As control,pAAV_RC (P50) containing wtRepCap was used. mVenus expression within cells clearly state that tested constructs does not meet expectations."></span></p>
+        <p style="text-align: justify;" class="MsoCaption"><span
+ lang="EN-US">Figure </span><span lang="EN-US">8</span>
+        <span style="color: windowtext; font-weight: normal;"
+ lang="EN-US">AAV-293
+cells were transfected with three plasmids pHelper,
+pSB1C3_[AAV2]-left-ITR_pCMV_betaglobin_mVenus _hGH_[AAV2]-right-ITR and
+pAAV_RC_inserts (see above) or pAAV_RC (see below) providing essential
+genes and proteins for producing viral particles. After 48 hour post
+transfection, viral particles were harvested by freeze-thaw lysis and
+centrifugation followed by HT1080 transduction. mVenus expression of
+viral genomes was determined by flow cytomery 24 hours post
+infection. </span><span
+ style="color: windowtext; font-weight: normal;" lang="EN-US">Fluorescence
+is measured in surviving cells</span><span
+ style="color: windowtext; font-weight: normal;" lang="EN-US">.
+Results show that insertion of both <i>rep</i>
+and <i>cap</i> syntheses disrupts viral infectivity.</span></p>
+        </td>
+      </tr>
+    </tbody>
+  </table>
+  <p class="MsoNormal"
+ style="text-indent: 0cm; line-height: 150%;"><span
+ lang="EN-US"></span></p>
+  <p class="MsoNormal"
+ style="text-indent: 0cm; line-height: 150%;"><span
+ lang="EN-US">Therefore,
+additional constructs were designed containing only the synthetic <i>cap</i>
+sequence or both, the synthesized sequences plus a re-mutation of KpnI
+(1721), in
+order to re-establish the wild-type splice site within the <i>rep</i>
+ORF. Results
+from cell culture obtained via FACS revealed that in fact the poor
+results were
+related to the KpnI restriction site deletion. Both constructs showed a
+transduction efficiency corresponding to the unmodified wild-type <i>RepVP123</i>’s
+transduction efficiency.</span></p>
+  <div align="center">
+  <table class="MsoTableGrid"
+ style="border: medium none ; border-collapse: collapse;"
+ border="0" cellpadding="0" cellspacing="0">
+    <tbody>
+      <tr style="height: 177.6pt;">
+        <td
+ style="border: 1pt solid windowtext; padding: 0cm 5.4pt; vertical-align: top; width: 464.4pt; height: 177.6pt;">
+        <p class="MsoNormal"
+ style="margin-bottom: 10pt; text-align: center; text-indent: 0cm; line-height: 115%; page-break-after: avoid;"
+ align="center"><img style="width: 625px; height: 233px;"
+ alt="" id="Chart 3"
+ src="https://static.igem.org/mediawiki/2010/6/62/Freiburg10_KpnI_reinstated.png"></p>
+        <p class="MsoCaption"><span lang="EN-US">Figure
+        </span><span lang="EN-US">9</span> <span
+ lang="EN-US">F</span><span
+ style="color: windowtext; font-weight: normal;" lang="EN-US">luorescence
+of cells transduced with mVenus carrying rAAV measured by flow
+cytometry. AAV-293 cells were transfected with three plasmids pHelper,
+pSB1C3_[AAV2]-left-ITR_pCMV_betaglobin_mVenus_hGH_[AAV2]-right-ITR and <i>RepVP123</i>
+constructs providing essential genes and proteins for producing viral
+particles. 48 hours post transfection, viral particles were
+harvested by freeze-thaw lysis and centrifugation followed by HT1080
+transduction. mVenus expression of viral genomes was determined by flow
+cytometry 24 hours post infection. Results show that <i>cap </i>integration
+does not influence infectivity. </span><span
+ style="color: windowtext; font-weight: normal;" lang="EN-US">Fluorescence
+is measured in surviving cells.</span><span
+ style="color: windowtext; font-weight: normal;" lang="EN-US">
+Recreation
+of KpnI within <i>rep</i> splice site recovers
+transduction efficiency.</span></p>
+        </td>
+      </tr>
+    </tbody>
+  </table>
+  </div>
+  <p class="MsoNormal"
+ style="text-indent: 0cm; line-height: 150%;"><span
+ lang="EN-US">&nbsp;</span></p>
+  <h3 style="margin-left: 0cm; text-indent: 0cm;"><a
+ name="_Toc275885926"></a><a name="_Toc275817885"><span
+ lang="EN-US">Modularization: Adapting
+pSB1C3 to loop
+insertions – pSB1C3_001</span></a></h3>
+  <p class="MsoNormal"
+ style="text-indent: 0cm; line-height: 150%;"><span
+ lang="EN-US">To
+fulfill iGEM requirements all plasmids need to be submitted in pSB1C3,
+therefore primers were ordered for amplifying <i>RepVP123</i>
+containing all
+modifications done so far by PCR and cloning the into pSB1C3. Still,
+pSB1C3
+contains two restriction sites for SspI and PvuII restriction enzymes
+in its
+CAT marker. Since these are necessary for cloning ViralBricks in this
+vector,
+the iGEM Team Freiburg_Bioware 2010 decided in agreement with iGEM
+Headquarters
+to implement a new standard for the pSB1C3 backbone which was named
+pSB1C3_001.
+Both restriction sites interfering with ViralBrick insertions were
+mutated to
+make SspI and PvuII single-cutters (see method development).</span></p>
+  <table class="MsoTableGrid"
+ style="border: medium none ; margin-left: auto; border-collapse: collapse; text-align: left; margin-right: auto;"
+ border="0" cellpadding="0" cellspacing="0">
+    <tbody>
+      <tr>
+        <td
+ style="border: 1pt solid windowtext; padding: 0cm 5.4pt; vertical-align: top; width: 460.6pt;">
+        <p class="MsoNormal"
+ style="text-align: center; text-indent: 0cm; page-break-after: avoid;"
+ align="center"><img style="width: 584px; height: 278px;"
+ id="Picture 16"
+ src="https://static.igem.org/mediawiki/2010/d/d1/Freiburg10_pSB1C3_001_mutations.png"
+ alt=""></p>
+        <p class="MsoCaption"><span lang="EN-US">Figure
+        </span><span lang="EN-US">10</span> <span
+ style="color: windowtext; font-weight: normal;" lang="EN-US">Comparison
+of pSB1C3 (upper row) and pSB1C3_001 (lower row). Deletions of SspI and
+PvuII are marked by red boxes.</span></p>
+        </td>
+      </tr>
+    </tbody>
+  </table>
+  <p class="MsoNormal"><i><span lang="EN-US">RepVP123</span></i><span
+ lang="EN-US">
+containing both <i>rep</i> and <i>cap</i>
+synthetic gene fragments including
+the re-mutation of KpnI and the downstream p5TATA-less promotor was
+cloned into
+the newly constructed pSB1C3_001. Testing this newly assembled plasmid
+in cell
+culture revealed unexpected data. Not only did the newly assembled
+plasmid work
+(see Figure 10), but in comparison to pAAV containing the same <i>RepVP123</i>
+construct, pSB1C3_001 showed an about 3 times higher transduction
+efficiency.
+Although exact reasons are still unknown, these results are probably
+related to
+the reduced length of pSB1C3_001 compared to the original pAAV plasmid
+of
+approximately 1000 base pairs.</span></p>
+  <span
+ style="font-size: 11pt; line-height: 200%; font-family: &quot;Calibri&quot;,&quot;sans-serif&quot;;"
+ lang="EN-US"></span><span lang="EN-US">&nbsp;</span>
+  <table
+ style="border: medium none ; width: 466px; border-collapse: collapse; text-align: left; margin-left: auto; margin-right: auto;"
+ class="MsoTableGrid" border="0" cellpadding="0"
+ cellspacing="0">
+    <tbody>
+      <tr style="height: 209.85pt;">
+        <td
+ style="border: 1pt solid windowtext; padding: 0cm 5.4pt; vertical-align: top; width: 4661px; height: 209.85pt;">
+        <p class="MsoNormal"
+ style="text-align: center; text-indent: 0cm; page-break-after: avoid;"
+ align="center"><span
+ style="font-size: 10pt; line-height: 200%;"><img
+ style="width: 516px; height: 258px;" alt="" id="Chart 15"
+ src="https://static.igem.org/mediawiki/2010/2/2b/Freiburg10_pAAV_pSB1C3_001.png"></span></p>
+        <p class="MsoCaption"><span lang="EN-US">Figure
+        </span><span lang="EN-US">11</span> <span
+ style="color: windowtext; font-weight: normal;" lang="EN-US">AAV-293
+cells were transfected with three plasmids pHelper,
+pSB1C3_001_[AAV2]-Rep-VP123_p5-TATAless or pAAV_RC_IRCK and
+pSB1C3_[AAV2]-left-ITR_pCMV_beta-globin_mVenus_hGH_[AAV2]-right-ITR
+providing essential genes and proteins for producing viral particles.
+After 48 hours post transfection, viral particles were harvested by
+freeze-thaw lysis and centrifugation followed by HT1080 transduction.
+mVenus expression of viral genomes was determined by flow cytomery
+after 24 hours post infection. </span><span
+ style="color: windowtext; font-weight: normal;" lang="EN-US">Fluorescence
+is measured in surviving cells.</span><span
+ style="color: windowtext; font-weight: normal;" lang="EN-US">&nbsp;Results
+showed functionality of <i>RepVP123</i>
+within pSB1C3_001 vector and additionally increased transduction
+efficiency.</span></p>
+        </td>
+      </tr>
+    </tbody>
+  </table>
+  <h4 style="margin-left: 0cm; text-indent: 0cm;"><a
+ name="_Toc275885927"></a><a name="_Toc275817886"><span
+ lang="EN-US">Turning-off natural tropism:
+HSPG-knock-out</span></a></h4>
+  <p class="MsoNormal" style="text-indent: 0cm;"><span
+ lang="EN-US">Shutting-down
+the
+natural viral tropism is essential for targeting specifically tumor
+cells and
+not infecting healthy cells. Therefore, the iGEM team Freiburg_Bioware
+decided to knock-out the viral natural tropism delivered by the heperan
+sulfate
+proteoglycan-(HSPG) binding site within the viruses 587 loop. The
+knock-out was
+cloned by designing primers containing the required base exchanges and
+performing a SDM. Like performed before, this <i>RepVP123</i>
+variant was tested
+in cell culture as well and evaluated by flow cytometry. Results show
+that
+mutation of HSPG-binding motif has severe impact on transduction
+efficiency
+thus enabling a viral particle carrying this knock-out and additional
+targeting
+motifs, e.g. within the loops or presented via N-terminal fusion to
+bind target
+cells’ receptors and therefore infecting target cells at a much higher
+rate
+compared to unspecific infection of other cell types within an organism
+(see </span><span lang="EN-US">Figure 12</span><span
+ lang="EN-US">).</span></p>
+  <p class="MsoNormal" style="text-indent: 0cm;"><span
+ lang="EN-US">To
+quantify
+differences in infectivity, the infectious titer of viral particles
+built-up of
+  <i>RepVP123</i> with and without HSPG binding motif was
+determined by qPCR (see
+  </span><span lang="EN-US">Figure 14</span><span
+ lang="EN-US">) for
+different cell
+lines. Results show that the implemented HSPG-knock-out verifies
+results obtained
+from flow cytometry, infectious titers severely compared to <i>RepVP123</i>
+with intact HSPG binding motif.</span></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 style="height: 258.85pt;">
+        <td
+ style="border: 1pt solid windowtext; padding: 0cm 5.4pt; vertical-align: top; width: 459.85pt; height: 258.85pt;">
+        <p class="MsoNormal"
+ style="text-indent: 0cm; page-break-after: avoid;"><img
+ style="width: 595px; height: 379px;" id="Picture 2"
+ src="https://static.igem.org/mediawiki/2010/9/9d/Freiburg10_HSPG-ko_sequence.png"
+ alt="Description: \\132.230.232.133\x\users\FreiGem\iGEM2010\Stefan\Pictures_Results\HSPG-ko_modified.jpg"></p>
+        <p class="MsoCaption" style="text-align: left;"
+ align="left"><span lang="EN-US">Figure </span><span
+ lang="EN-US">12</span> <span
+ style="color: windowtext; font-weight: normal;" lang="EN-US">Alignment
+of 587 loop within viral VP123: The upper sequence shows a strand
+containing the HSPG binding motif (AGA, in red boxes), the lower
+sequence contains the HSPG-ko introduced by the iGEM team
+Freiburg_Bioware 2010 (GCT and GCC, blue boxes).</span></p>
+        </td>
+      </tr>
+    </tbody>
+  </table>
+  <p class="MsoNormal" style="text-indent: 0cm;"><span
+ lang="EN-US">&nbsp;</span></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="border: 1pt solid windowtext; padding: 0cm 3.5pt; vertical-align: top; width: 460.6pt;">
+        <p class="MsoNormal"
+ style="text-align: center; text-indent: 0cm; page-break-after: avoid;"
+ align="center"><img style="width: 467px; height: 229px;"
+ alt="" id="Chart 9"
+ src="https://static.igem.org/mediawiki/2010/0/01/Freiburg10_columns_HSPG_HSPG_ko.png"></p>
+        <p class="MsoCaption"><a name="_Ref275882130"><span
+ lang="EN-US">Figure
+        </span></a><span lang="EN-US">13</span>
+        <span style="color: windowtext; font-weight: normal;"
+ lang="EN-US">Transduction
+efficiency of HT1080 cells measured by flow cytometry.&nbsp;</span><span
+ style="color: windowtext; font-weight: normal;" lang="EN-US">Fluorescence
+is measured in surviving cells.</span><span
+ style="color: windowtext; font-weight: normal;" lang="EN-US">
+Knock-out of
+HSPG binding motif greatly reduces transduction efficiency compared to <i>RepVP123</i>
+containing the motiv.</span></p>
+        </td>
+      </tr>
+    </tbody>
+  </table>
+  <p class="MsoNormal" style="text-indent: 0cm;"><span
+ lang="EN-US">&nbsp;</span></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="border: 1pt solid windowtext; padding: 0cm 3.5pt; vertical-align: top; width: 460.6pt;">
+        <p class="MsoCaption"
+ style="text-align: center; page-break-after: avoid;"
+ align="center"><img style="width: 535px; height: 456px;"
+ alt="" id="Chart 1"
+ src="https://static.igem.org/mediawiki/2010/b/bc/Freiburg10_qPCR_HSPG_HSPG_ko.png"></p>
+        <p class="MsoCaption" style="text-align: center;"
+ align="center"><a name="_Ref275883877"><span
+ lang="EN-US">Figure </span></a><span
+ lang="EN-US">14</span> <span
+ style="color: windowtext; font-weight: normal;" lang="EN-US">Infectious
+titers of <i>RepVP123</i> with and without natural HSPG
+binding motif tested in different cell lines via qPCR. Shutting-down
+the HSPG binding motif reduces infectious titer in both HT1080 and HeLa
+cell lines. For A431 cells, no infectious titer could be detected via
+qPCR, which is probably related to poor transduction efficiency of A431
+cells.</span></p>
+        </td>
+      </tr>
+    </tbody>
+  </table>
+  <br>
+  <br>
+  <br>
+</head>
+<body>
+<h2 style="margin-left: 0cm; text-indent: 0cm;"><a
+ name="_Toc275885921"></a><a name="_Toc275817880"><span
+ lang="EN-US">Overview of RepVP123 plasmid</span></a></h2>
+<h3 style="margin-left: 0cm; text-indent: 0cm;"><a
+ name="_Toc275885922"></a><a name="_Toc275817881"><span
+ lang="EN-US">Modularization: Overview</span></a></h3>
+<p class="MsoNormal"
+ style="text-indent: 0cm; line-height: 150%; page-break-after: avoid;"><span
+ lang="EN-US">In our terminology the term “RepVP123”
+encompasses the
+whole AAV2 genome excluding the ITRs. The <i>rep</i> locus
+comprises four
+proteins related to genome replication while the <i>cap</i>
+locus codes for the
+proteins VP1, VP2, VP3 and the assembly-associated protein (AAP), which
+are
+required for viral capsid assembly. Source of the RepVP123 BioBrick
+supplied
+within iGEM team Freiburg_Bioware 2010 Virus Construction Kit is the
+wild-type
+AAV2 RepVP123, as provided e. g. in the pAAV vector from Stratagene. In
+order
+to introduce iGEM standard and additionally enabling the possibility to
+modify
+the viral capsid via integration of certain motives within the viral
+loops 453
+and 587 a total of twelve mutations within RepVP123 (see </span><span
+ lang="EN-US">Figure 1</span><span lang="EN-US">)
+and additionally two mutations
+within the pSB1C3 backbone were performed either by Site-Directed
+Mutagenesis
+(SDM) or by ordering and cloning of specifically designed gene
+sequences matching
+the required demands. Modifying the pSB1C3 led to iGEM team
+Freiburg_Bioware’s
+variant of this backbone, pSB1C3_001.</span></p>
+<table class="MsoTableGrid"
+ style="border: medium none ; border-collapse: collapse; width: 636px; height: 523px; text-align: left; margin-left: auto; margin-right: auto;"
+ border="0" cellpadding="0" cellspacing="0">
+  <tbody>
+    <tr>
+      <td
+ style="border: 1pt solid windowtext; padding: 0cm 5.4pt; vertical-align: top; width: 462.5pt;">
+      <p class="MsoNormal"
+ style="text-align: center; text-indent: 0cm; line-height: 150%; page-break-after: avoid;"
+ align="center"><img style="width: 605px; height: 451px;"
+ id="Picture 17"
+ src="https://static.igem.org/mediawiki/2010/0/0b/Freiburg10_pAAV_complete_mutations.png"
+ alt="Description: \\132.230.232.133\x\users\FreiGem\iGEM2010\Stefan\Pictures_Results\RepCap_complete_modifications_arrows.jpg"></p>
+      <p class="MsoCaption" style="text-indent: 0cm;"><a
+ name="_Ref275820820"><span lang="EN-US">Figure </span></a><span
+ lang="EN-US">1</span> <span
+ style="font-size: 8pt; color: windowtext; font-weight: normal;"
+ lang="EN-US">Mutations implemented into <i>RepVP123</i>
+in order to establish both iGEM standard and loop insertion capability.
+Green arrows indicate integrated restriction sites, red red arrows
+indicate deleted restriction sites. KpnI was deleted first and
+reinstated afterwards. (see text).</span></p>
+      </td>
+    </tr>
+  </tbody>
+</table>
+<span
+ style="font-size: 8pt; line-height: 150%; font-family: &quot;Calibri&quot;,&quot;sans-serif&quot;;"
+ lang="EN-US"><br style="page-break-before: always;"
+ clear="all">
+</span>
+<p class="MsoNormal"
+ style="text-indent: 0cm; line-height: 150%;"><span
+ style="font-size: 8pt; line-height: 150%;" lang="EN-US">&nbsp;</span></p>
+<table class="MsoNormalTable"
+ style="border-collapse: collapse; width: 651px; height: 261px; text-align: left; margin-left: auto; margin-right: auto;"
+ border="0" cellpadding="0" cellspacing="0">
+  <tbody>
+    <tr style="height: 27pt;">
+      <td
+ style="border: 1pt solid windowtext; padding: 0.75pt 0.75pt 0cm; width: 86.45pt; height: 27pt;">
+      <p class="MsoNormal"
+ style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><span
+ style="font-size: 10pt; color: black;" lang="EN-US">Plasmid
+name:</span></p>
+      </td>
+      <td
+ style="border-style: solid solid solid none; border-color: windowtext windowtext windowtext -moz-use-text-color; border-width: 1pt 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 77.35pt; height: 27pt;"
+ width="103">
+      <p class="MsoNormal"
+ style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><span
+ style="font-size: 10pt; color: black;" lang="EN-US">Functionality
+(determinded in cell culture via transduction and flow cytometry ):</span></p>
+      </td>
+      <td
+ style="border-style: solid solid solid none; border-color: windowtext windowtext windowtext -moz-use-text-color; border-width: 1pt 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 77.95pt; height: 27pt;"
+ width="104">
+      <p class="MsoNormal"
+ style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><span
+ style="font-size: 10pt; color: black;" lang="EN-US">4x
+mutations (PstI (310), BamHI (859), SalI (1239), PstI (4073))</span></p>
+      </td>
+      <td
+ style="border-style: solid solid solid none; border-color: windowtext windowtext windowtext -moz-use-text-color; border-width: 1pt 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 42.5pt; height: 27pt;"
+ width="57">
+      <p class="MsoNormal"
+ style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><span
+ style="font-size: 10pt; color: black;">inserted <i>rep</i>
+fragment</span></p>
+      </td>
+      <td
+ style="border-style: solid solid solid none; border-color: windowtext windowtext windowtext -moz-use-text-color; border-width: 1pt 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 49.65pt; height: 27pt;"
+ width="66">
+      <p class="MsoNormal"
+ style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><span
+ style="font-size: 10pt; color: black;">inserted<i> cap</i>
+fragment</span></p>
+      </td>
+      <td
+ style="border-style: solid solid solid none; border-color: windowtext windowtext windowtext -moz-use-text-color; border-width: 1pt 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 50.2pt; height: 27pt;"
+ width="67">
+      <p class="MsoNormal"
+ style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><span
+ style="font-size: 10pt; color: black;">reinstated KpnI</span></p>
+      </td>
+      <td
+ style="border-style: solid solid solid none; border-color: windowtext windowtext windowtext -moz-use-text-color; border-width: 1pt 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 24pt; height: 27pt;"
+ width="32">
+      <p class="MsoNormal"
+ style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><span
+ style="font-size: 10pt; color: black;">pAAV</span></p>
+      </td>
+      <td
+ style="border-style: solid solid solid none; border-color: windowtext windowtext windowtext -moz-use-text-color; border-width: 1pt 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 52.45pt; height: 27pt;"
+ width="70">
+      <p class="MsoNormal"
+ style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><span
+ style="font-size: 10pt; color: black;">pSB1C3_001</span></p>
+      </td>
+      <td
+ style="border-style: solid solid solid none; border-color: windowtext windowtext windowtext -moz-use-text-color; border-width: 1pt 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 27.45pt; height: 27pt;"
+ width="37">
+      <p class="MsoNormal"
+ style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><span
+ style="font-size: 10pt; color: black;">HSPG-ko</span></p>
+      </td>
+    </tr>
+    <tr style="height: 15pt;">
+      <td
+ style="border-style: none solid solid; border-color: -moz-use-text-color windowtext windowtext; border-width: medium 1pt 1pt; padding: 0.75pt 0.75pt 0cm; width: 86.45pt; height: 15pt;"
+ width="115">
+      <p class="MsoNormal"
+ style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><span
+ style="font-size: 10pt; color: black;" lang="EN-US">pAAV_RC
+(wild-type)</span></p>
+      </td>
+      <td
+ style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 77.35pt; height: 15pt;"
+ width="103">
+      <p class="MsoNormal"
+ style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><span
+ style="font-size: 10pt; color: black;" lang="EN-US">yes</span></p>
+      </td>
+      <td
+ style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 77.95pt; height: 15pt;"
+ width="104">
+      <p class="MsoNormal"
+ style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><span
+ style="font-size: 10pt; color: black;" lang="EN-US">&nbsp;</span></p>
+      </td>
+      <td
+ style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 42.5pt; height: 15pt;"
+ nowrap="nowrap" valign="bottom" width="57">
+      <p class="MsoNormal"
+ style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;"
+ align="left"><span style="font-size: 10pt; color: black;">&nbsp;</span></p>
+      </td>
+      <td
+ style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 49.65pt; height: 15pt;"
+ nowrap="nowrap" valign="bottom" width="66">
+      <p class="MsoNormal"
+ style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;"
+ align="left"><span style="font-size: 10pt; color: black;">&nbsp;</span></p>
+      </td>
+      <td
+ style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 50.2pt; height: 15pt;"
+ nowrap="nowrap" valign="bottom" width="67">
+      <p class="MsoNormal"
+ style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;"
+ align="left"><span style="font-size: 10pt; color: black;">&nbsp;</span></p>
+      </td>
+      <td
+ style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 24pt; height: 15pt;"
+ nowrap="nowrap" valign="bottom" width="32">
+      <p class="MsoNormal"
+ style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;"
+ align="left"><span style="font-size: 10pt; color: black;">x</span></p>
+      </td>
+      <td
+ style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 52.45pt; height: 15pt;"
+ nowrap="nowrap" valign="bottom" width="70">
+      <p class="MsoNormal"
+ style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;"
+ align="left"><span style="font-size: 10pt; color: black;">&nbsp;</span></p>
+      </td>
+      <td
+ style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 27.45pt; height: 15pt;"
+ nowrap="nowrap" valign="bottom" width="37">
+      <p class="MsoNormal"
+ style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;"
+ align="left"><span style="font-size: 10pt; color: black;">&nbsp;</span></p>
+      </td>
+    </tr>
+    <tr style="height: 15pt;">
+      <td
+ style="border-style: none solid solid; border-color: -moz-use-text-color windowtext windowtext; border-width: medium 1pt 1pt; padding: 0.75pt 0.75pt 0cm; width: 86.45pt; height: 15pt;"
+ width="115">
+      <p class="MsoNormal"
+ style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><span
+ style="font-size: 10pt; color: black;" lang="EN-US">pAAV_RC_4x
+mutant</span></p>
+      </td>
+      <td
+ style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 77.35pt; height: 15pt;"
+ width="103">
+      <p class="MsoNormal"
+ style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><span
+ style="font-size: 10pt; color: black;" lang="EN-US">yes</span></p>
+      </td>
+      <td
+ style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 77.95pt; height: 15pt;"
+ width="104">
+      <p class="MsoNormal"
+ style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><span
+ style="font-size: 10pt; color: black;" lang="EN-US">x</span></p>
+      </td>
+      <td
+ style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 42.5pt; height: 15pt;"
+ nowrap="nowrap" valign="bottom" width="57">
+      <p class="MsoNormal"
+ style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;"
+ align="left"><span style="font-size: 10pt; color: black;">&nbsp;</span></p>
+      </td>
+      <td
+ style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 49.65pt; height: 15pt;"
+ nowrap="nowrap" valign="bottom" width="66">
+      <p class="MsoNormal"
+ style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;"
+ align="left"><span style="font-size: 10pt; color: black;">&nbsp;</span></p>
+      </td>
+      <td
+ style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 50.2pt; height: 15pt;"
+ nowrap="nowrap" valign="bottom" width="67">
+      <p class="MsoNormal"
+ style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;"
+ align="left"><span style="font-size: 10pt; color: black;">&nbsp;</span></p>
+      </td>
+      <td
+ style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 24pt; height: 15pt;"
+ nowrap="nowrap" valign="bottom" width="32">
+      <p class="MsoNormal"
+ style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;"
+ align="left"><span style="font-size: 10pt; color: black;">x</span></p>
+      </td>
+      <td
+ style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 52.45pt; height: 15pt;"
+ nowrap="nowrap" valign="bottom" width="70">
+      <p class="MsoNormal"
+ style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;"
+ align="left"><span style="font-size: 10pt; color: black;">&nbsp;</span></p>
+      </td>
+      <td
+ style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 27.45pt; height: 15pt;"
+ nowrap="nowrap" valign="bottom" width="37">
+      <p class="MsoNormal"
+ style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;"
+ align="left"><span style="font-size: 10pt; color: black;">&nbsp;</span></p>
+      </td>
+    </tr>
+    <tr style="height: 15pt;">
+      <td
+ style="border-style: none solid solid; border-color: -moz-use-text-color windowtext windowtext; border-width: medium 1pt 1pt; padding: 0.75pt 0.75pt 0cm; width: 86.45pt; height: 15pt;"
+ width="115">
+      <p class="MsoNormal"
+ style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><span
+ style="font-size: 10pt; color: black;" lang="EN-US">pAAV_RC_inserts
+      </span></p>
+      </td>
+      <td
+ style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 77.35pt; height: 15pt;"
+ width="103">
+      <p class="MsoNormal"
+ style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><span
+ style="font-size: 10pt; color: black;" lang="EN-US">no</span></p>
+      </td>
+      <td
+ style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 77.95pt; height: 15pt;"
+ width="104">
+      <p class="MsoNormal"
+ style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><span
+ style="font-size: 10pt; color: black;" lang="EN-US">x</span></p>
+      </td>
+      <td
+ style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 42.5pt; height: 15pt;"
+ nowrap="nowrap" valign="bottom" width="57">
+      <p class="MsoNormal"
+ style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;"
+ align="left"><span style="font-size: 10pt; color: black;">x</span></p>
+      </td>
+      <td
+ style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 49.65pt; height: 15pt;"
+ nowrap="nowrap" valign="bottom" width="66">
+      <p class="MsoNormal"
+ style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;"
+ align="left"><span style="font-size: 10pt; color: black;">x</span></p>
+      </td>
+      <td
+ style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 50.2pt; height: 15pt;"
+ nowrap="nowrap" valign="bottom" width="67">
+      <p class="MsoNormal"
+ style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;"
+ align="left"><span style="font-size: 10pt; color: black;">&nbsp;</span></p>
+      </td>
+      <td
+ style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 24pt; height: 15pt;"
+ nowrap="nowrap" valign="bottom" width="32">
+      <p class="MsoNormal"
+ style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;"
+ align="left"><span style="font-size: 10pt; color: black;">x</span></p>
+      </td>
+      <td
+ style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 52.45pt; height: 15pt;"
+ nowrap="nowrap" valign="bottom" width="70">
+      <p class="MsoNormal"
+ style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;"
+ align="left"><span style="font-size: 10pt; color: black;">&nbsp;</span></p>
+      </td>
+      <td
+ style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 27.45pt; height: 15pt;"
+ nowrap="nowrap" valign="bottom" width="37">
+      <p class="MsoNormal"
+ style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;"
+ align="left"><span style="font-size: 10pt; color: black;">&nbsp;</span></p>
+      </td>
+    </tr>
+    <tr style="height: 15pt;">
+      <td
+ style="border-style: none solid solid; border-color: -moz-use-text-color windowtext windowtext; border-width: medium 1pt 1pt; padding: 0.75pt 0.75pt 0cm; width: 86.45pt; height: 15pt;"
+ width="115">
+      <p class="MsoNormal"
+ style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><span
+ style="font-size: 10pt; color: black;" lang="EN-US">pAAV_RC_Cap</span></p>
+      </td>
+      <td
+ style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 77.35pt; height: 15pt;"
+ width="103">
+      <p class="MsoNormal"
+ style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><span
+ style="font-size: 10pt; color: black;" lang="EN-US">yes</span></p>
+      </td>
+      <td
+ style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 77.95pt; height: 15pt;"
+ width="104">
+      <p class="MsoNormal"
+ style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><span
+ style="font-size: 10pt; color: black;" lang="EN-US">x</span></p>
+      </td>
+      <td
+ style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 42.5pt; height: 15pt;"
+ nowrap="nowrap" valign="bottom" width="57">
+      <p class="MsoNormal"
+ style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;"
+ align="left"><span style="font-size: 10pt; color: black;">&nbsp;</span></p>
+      </td>
+      <td
+ style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 49.65pt; height: 15pt;"
+ nowrap="nowrap" valign="bottom" width="66">
+      <p class="MsoNormal"
+ style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;"
+ align="left"><span style="font-size: 10pt; color: black;">x</span></p>
+      </td>
+      <td
+ style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 50.2pt; height: 15pt;"
+ nowrap="nowrap" valign="bottom" width="67">
+      <p class="MsoNormal"
+ style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;"
+ align="left"><span style="font-size: 10pt; color: black;">&nbsp;</span></p>
+      </td>
+      <td
+ style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 24pt; height: 15pt;"
+ nowrap="nowrap" valign="bottom" width="32">
+      <p class="MsoNormal"
+ style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;"
+ align="left"><span style="font-size: 10pt; color: black;">x</span></p>
+      </td>
+      <td
+ style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 52.45pt; height: 15pt;"
+ nowrap="nowrap" valign="bottom" width="70">
+      <p class="MsoNormal"
+ style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;"
+ align="left"><span style="font-size: 10pt; color: black;">&nbsp;</span></p>
+      </td>
+      <td
+ style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 27.45pt; height: 15pt;"
+ nowrap="nowrap" valign="bottom" width="37">
+      <p class="MsoNormal"
+ style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;"
+ align="left"><span style="font-size: 10pt; color: black;">&nbsp;</span></p>
+      </td>
+    </tr>
+    <tr style="height: 15pt;">
+      <td
+ style="border-style: none solid solid; border-color: -moz-use-text-color windowtext windowtext; border-width: medium 1pt 1pt; padding: 0.75pt 0.75pt 0cm; width: 86.45pt; height: 15pt;"
+ width="115">
+      <p class="MsoNormal"
+ style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><span
+ style="font-size: 10pt; color: black;" lang="EN-US">pAAV_RC_RepVP123</span></p>
+      </td>
+      <td
+ style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 77.35pt; height: 15pt;"
+ width="103">
+      <p class="MsoNormal"
+ style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><span
+ style="font-size: 10pt; color: black;" lang="EN-US">yes</span></p>
+      </td>
+      <td
+ style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 77.95pt; height: 15pt;"
+ width="104">
+      <p class="MsoNormal"
+ style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><span
+ style="font-size: 10pt; color: black;" lang="EN-US">x</span></p>
+      </td>
+      <td
+ style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 42.5pt; height: 15pt;"
+ nowrap="nowrap" valign="bottom" width="57">
+      <p class="MsoNormal"
+ style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;"
+ align="left"><span style="font-size: 10pt; color: black;">x</span></p>
+      </td>
+      <td
+ style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 49.65pt; height: 15pt;"
+ nowrap="nowrap" valign="bottom" width="66">
+      <p class="MsoNormal"
+ style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;"
+ align="left"><span style="font-size: 10pt; color: black;">x</span></p>
+      </td>
+      <td
+ style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 50.2pt; height: 15pt;"
+ nowrap="nowrap" valign="bottom" width="67">
+      <p class="MsoNormal"
+ style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;"
+ align="left"><span style="font-size: 10pt; color: black;">x</span></p>
+      </td>
+      <td
+ style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 24pt; height: 15pt;"
+ nowrap="nowrap" valign="bottom" width="32">
+      <p class="MsoNormal"
+ style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;"
+ align="left"><span style="font-size: 10pt; color: black;">x</span></p>
+      </td>
+      <td
+ style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 52.45pt; height: 15pt;"
+ nowrap="nowrap" valign="bottom" width="70">
+      <p class="MsoNormal"
+ style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;"
+ align="left"><span style="font-size: 10pt; color: black;">&nbsp;</span></p>
+      </td>
+      <td
+ style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 27.45pt; height: 15pt;"
+ nowrap="nowrap" valign="bottom" width="37">
+      <p class="MsoNormal"
+ style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;"
+ align="left"><span style="font-size: 10pt; color: black;">&nbsp;</span></p>
+      </td>
+    </tr>
+    <tr style="height: 18pt;">
+      <td
+ style="border-style: none solid solid; border-color: -moz-use-text-color windowtext windowtext; border-width: medium 1pt 1pt; padding: 0.75pt 0.75pt 0cm; width: 86.45pt; height: 18pt;"
+ width="115">
+      <p class="MsoNormal"
+ style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><span
+ style="font-size: 10pt; color: black;" lang="EN-US">pSB1C3_RepVP123_
+p5TATAless</span></p>
+      </td>
+      <td
+ style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 77.35pt; height: 18pt;"
+ width="103">
+      <p class="MsoNormal"
+ style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><span
+ style="font-size: 10pt; color: black;" lang="EN-US">yes</span></p>
+      </td>
+      <td
+ style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 77.95pt; height: 18pt;"
+ width="104">
+      <p class="MsoNormal"
+ style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><span
+ style="font-size: 10pt; color: black;" lang="EN-US">x</span></p>
+      </td>
+      <td
+ style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 42.5pt; height: 18pt;"
+ nowrap="nowrap" valign="bottom" width="57">
+      <p class="MsoNormal"
+ style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;"
+ align="left"><span style="font-size: 10pt; color: black;">x</span></p>
+      </td>
+      <td
+ style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 49.65pt; height: 18pt;"
+ nowrap="nowrap" valign="bottom" width="66">
+      <p class="MsoNormal"
+ style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;"
+ align="left"><span style="font-size: 10pt; color: black;">x</span></p>
+      </td>
+      <td
+ style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 50.2pt; height: 18pt;"
+ nowrap="nowrap" valign="bottom" width="67">
+      <p class="MsoNormal"
+ style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;"
+ align="left"><span style="font-size: 10pt; color: black;">x</span></p>
+      </td>
+      <td
+ style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 24pt; height: 18pt;"
+ nowrap="nowrap" valign="bottom" width="32">
+      <p class="MsoNormal"
+ style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;"
+ align="left"><span style="font-size: 10pt; color: black;">&nbsp;</span></p>
+      </td>
+      <td
+ style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 52.45pt; height: 18pt;"
+ nowrap="nowrap" valign="bottom" width="70">
+      <p class="MsoNormal"
+ style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;"
+ align="left"><span style="font-size: 10pt; color: black;">x</span></p>
+      </td>
+      <td
+ style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 27.45pt; height: 18pt;"
+ nowrap="nowrap" valign="bottom" width="37">
+      <p class="MsoNormal"
+ style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;"
+ align="left"><span style="font-size: 10pt; color: black;">&nbsp;</span></p>
+      </td>
+    </tr>
+    <tr style="height: 27pt;">
+      <td
+ style="border-style: none solid solid; border-color: -moz-use-text-color windowtext windowtext; border-width: medium 1pt 1pt; padding: 0.75pt 0.75pt 0cm; width: 86.45pt; height: 27pt;"
+ width="115">
+      <p class="MsoNormal"
+ style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><span
+ style="font-size: 10pt; color: black;" lang="EN-US">pSB1C3_RepVP123_
+HSPG-ko_p5TATAless</span></p>
+      </td>
+      <td
+ style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 77.35pt; height: 27pt;"
+ width="103">
+      <p class="MsoNormal"
+ style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><span
+ style="font-size: 10pt; color: black;" lang="EN-US">yes</span></p>
+      </td>
+      <td
+ style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 77.95pt; height: 27pt;"
+ width="104">
+      <p class="MsoNormal"
+ style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><span
+ style="font-size: 10pt; color: black;" lang="EN-US">x</span></p>
+      </td>
+      <td
+ style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 42.5pt; height: 27pt;"
+ nowrap="nowrap" valign="bottom" width="57">
+      <p class="MsoNormal"
+ style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;"
+ align="left"><span style="font-size: 10pt; color: black;">x</span></p>
+      </td>
+      <td
+ style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 49.65pt; height: 27pt;"
+ nowrap="nowrap" valign="bottom" width="66">
+      <p class="MsoNormal"
+ style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;"
+ align="left"><span style="font-size: 10pt; color: black;">x</span></p>
+      </td>
+      <td
+ style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 50.2pt; height: 27pt;"
+ nowrap="nowrap" valign="bottom" width="67">
+      <p class="MsoNormal"
+ style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;"
+ align="left"><span style="font-size: 10pt; color: black;">x</span></p>
+      </td>
+      <td
+ style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 24pt; height: 27pt;"
+ nowrap="nowrap" valign="bottom" width="32">
+      <p class="MsoNormal"
+ style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;"
+ align="left"><span style="font-size: 10pt; color: black;">&nbsp;</span></p>
+      </td>
+      <td
+ style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 52.45pt; height: 27pt;"
+ nowrap="nowrap" valign="bottom" width="70">
+      <p class="MsoNormal"
+ style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;"
+ align="left"><span style="font-size: 10pt; color: black;">x</span></p>
+      </td>
+      <td
+ style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 0.75pt 0.75pt 0cm; width: 27.45pt; height: 27pt;"
+ nowrap="nowrap" valign="bottom" width="37">
+      <p class="MsoNormal"
+ style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal; page-break-after: avoid;"
+ align="left"><span style="font-size: 10pt; color: black;">x</span></p>
+      </td>
+    </tr>
+  </tbody>
+</table>
+<p class="MsoCaption" style="text-align: left;"
+ align="left"><span lang="EN-US">Figure </span><span
+ lang="EN-US">2</span> <span
+ style="color: windowtext; font-weight: normal;" lang="EN-US">Table
+contains complete overview about all plasmids containing <i>RepVP123</i>
+which
+were used by iGEM team Freiburg_Bioware 2010.</span></p>
+<h3 style="margin-left: 0cm; text-indent: 0cm;"><a
+ name="_Toc275885923"></a><a name="_Toc275817882"><span
+ lang="EN-US">Modularization: Removing iGEM
+restriction
+sites and establishing loop insertion capability</span></a></h3>
+<h4 style="margin-left: 0cm; text-indent: 0cm;"><a
+ name="_Toc275885924"></a><a name="_Toc275817883"><span
+ lang="EN-US">Modifications in Rep</span></a></h4>
+<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="border: 1pt solid windowtext; padding: 0cm 5.4pt; vertical-align: top; width: 460.6pt;">
+      <p class="MsoNormal"
+ style="text-align: center; text-indent: 0cm; page-break-after: avoid;"
+ align="center"><img style="width: 576px; height: 191px;"
+ id="Picture 4"
+ src="https://static.igem.org/mediawiki/2010/b/bc/Freiburg10_rep_synthetic_gene_fragment.png"
+ alt="Description: \\132.230.232.133\x\users\FreiGem\iGEM2010\Stefan\Pictures_Results\Rep_synthesis_marked.jpg"></p>
+      <p class="MsoCaption"><a name="_Ref275818017"></a><a
+ name="_Ref275818042"><span lang="EN-US">Figure </span></a><span
+ lang="EN-US">3</span> <span
+ style="font-size: 8pt; color: windowtext; font-weight: normal;"
+ lang="EN-US">Restriction sites within the wild-type <i>rep</i>
+gene sequence, which were removed via cloning of synthetized <i>rep
+      </i>gene
+fragment into the plasmid. The red box indicates
+the region spanned by the synthetic sequence.</span></p>
+      </td>
+    </tr>
+  </tbody>
+</table>
+<p class="MsoNormal" style="text-indent: 0cm;"><span
+ lang="EN-US">Making
+the <i>RepVP123</i>
+wild-type compatible with the iGEM standards required the removal of
+five
+restriction sites (see </span><span lang="EN-US">Figure
+</span><span lang="EN-US">).
+This was achieved using site-directed mutagenesis for PstI (position
+) and
+PstI (4073). The remaining three iGEM restriction sites EcoRI (1578),
+PstI
+(1773) and EcoRI (1796) were replaced by a synthetic gene fragment,
+since the <i>rep
+</i>ORF contained these restriction sites in close proximity to
+each other plus
+an additional KpnI restriction site which was also not desired (see </span><span
+ lang="EN-US">Figure 2</span><span lang="EN-US">).
+This gene fragment was cloned into
+the <i>rep</i> gene using HindIII and SwaI, which are
+single-cutting
+restriction enzymes adjacent to the target area. Additionally, BamHI
+(859) and
+SalI (1239) were removed, because these enzymes were required for
+genetically
+inserting the loop modifications in VP123.</span></p>
+<h4 style="margin-left: 0cm; text-indent: 0cm;"><a
+ name="_Toc275885925"></a><a name="_Toc275817884"><span
+ lang="EN-US">Modifications in VP123</span></a></h4>
+<p class="MsoNormal" style="text-indent: 0cm;"><span
+ lang="EN-US">In
+order to
+implement the restriction sites necessary for targeting via loop
+insertions,
+the gene coding for the VP proteins was modified as well. The
+introduction of
+these restriction required up to four base mutations in a row, hence it
+was
+decided to synthesize this gene fragment and replace the wild-type
+sequence in <i>RepVP123</i>
+as well.</span></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="border: 1pt solid windowtext; padding: 0cm 5.4pt; vertical-align: top; width: 460.6pt;">
+      <p class="MsoNormal"
+ style="text-align: center; text-indent: 0cm; page-break-after: avoid;"
+ align="center"><img style="width: 574px; height: 218px;"
+ id="Picture 5"
+ src="https://static.igem.org/mediawiki/2010/c/cd/Freiburg10_cap_synthetic_gene_fragment.png"
+ alt="Description: \\132.230.232.133\x\users\FreiGem\iGEM2010\Stefan\Pictures_Results\Cap_synthesis_marked.jpg"></p>
+      <p class="MsoCaption"><span lang="EN-US">Figure
+      </span><span lang="EN-US">4</span> <span
+ style="font-size: 8pt; color: windowtext; font-weight: normal;"
+ lang="EN-US">Restriction sites within <i>cap</i>
+sequence showing introduced loop insertion restriction sites into <i>cap</i>
+to enable cloning of targeting or purification motifs into both 453 and
+loops. Again, the red box indicates gene sequence which was
+synthetized.</span></p>
+      </td>
+    </tr>
+  </tbody>
+</table>
+<p class="MsoNormal" style="text-indent: 0cm;"><span
+ lang="EN-US">&nbsp;</span><span lang="EN-US"></span>&nbsp;</p>
+<h3 style="margin-left: 0cm; text-indent: 0cm;"><a
+ name="_Toc275885926"></a><a name="_Toc275817885"><span
+ lang="EN-US">Modularization: Adapting
+pSB1C3 to loop
+insertions – pSB1C3_001</span></a></h3>
+<p class="MsoNormal"
+ style="text-indent: 0cm; line-height: 150%;"><span
+ lang="EN-US">To
+fulfill iGEM requirements all plasmids need to be submitted in pSB1C3,
+therefore primers were ordered for amplifying <i>RepVP123</i>
+containing all
+modifications done so far by PCR and cloning the into pSB1C3. Still,
+pSB1C3
+contains two restriction sites for SspI and PvuII restriction enzymes
+in its
+CAT marker. Since these are necessary for cloning ViralBricks in this
+vector,
+the iGEM Team Freiburg_Bioware 2010 decided in agreement with iGEM
+Headquarters
+to implement a new standard for the pSB1C3 backbone which was named
+pSB1C3_001.
+Both restriction sites interfering with ViralBrick insertions were
+mutated to
+make SspI and PvuII single-cutters (see method development).</span></p>
+<table class="MsoTableGrid"
+ style="border: medium none ; margin-left: auto; border-collapse: collapse; text-align: left; margin-right: auto;"
+ border="0" cellpadding="0" cellspacing="0">
+  <tbody>
+    <tr>
+      <td
+ style="border: 1pt solid windowtext; padding: 0cm 5.4pt; vertical-align: top; width: 460.6pt;">
+      <p class="MsoNormal"
+ style="text-align: center; text-indent: 0cm; page-break-after: avoid;"
+ align="center"><img style="width: 584px; height: 278px;"
+ id="Picture 16"
+ src="https://static.igem.org/mediawiki/2010/d/d1/Freiburg10_pSB1C3_001_mutations.png"
+ alt=""></p>
+      <p class="MsoCaption"><span lang="EN-US">Figure
+      </span><span lang="EN-US">10</span> <span
+ style="color: windowtext; font-weight: normal;" lang="EN-US">Comparison
+of pSB1C3 (upper row) and pSB1C3_001 (lower row). Deletions of SspI and
+PvuII are marked by red boxes.</span></p>
+      </td>
+    </tr>
+  </tbody>
+</table>
+<p class="MsoNormal"><i><span lang="EN-US">RepVP123</span></i><span
+ lang="EN-US">
+containing both <i>rep</i> and <i>cap</i>
+synthetic gene fragments including
+the re-mutation of KpnI and the downstream p5TATA-less promotor was
+cloned into
+the newly constructed pSB1C3_001. Testing this newly assembled plasmid
+in cell
+culture revealed unexpected data. Not only did the newly assembled
+plasmid work
+(see Figure 10), but in comparison to pAAV containing the same <i>RepVP123</i>
+construct, pSB1C3_001 showed an about 3 times higher transduction
+efficiency.
+Although exact reasons are still unknown, these results are probably
+related to
+the reduced length of pSB1C3_001 compared to the original pAAV plasmid
+of
+approximately 1000 base pairs.</span></p>
+<span
+ style="font-size: 11pt; line-height: 200%; font-family: &quot;Calibri&quot;,&quot;sans-serif&quot;;"
+ lang="EN-US"></span><span lang="EN-US">&nbsp;</span>
+<table
+ style="border: medium none ; width: 466px; border-collapse: collapse; text-align: left; margin-left: auto; margin-right: auto;"
+ class="MsoTableGrid" border="0" cellpadding="0"
+ cellspacing="0">
+  <tbody>
+    <tr style="height: 209.85pt;">
+      <td
+ style="border: 1pt solid windowtext; padding: 0cm 5.4pt; vertical-align: top; width: 4661px; height: 209.85pt;">
+      <p class="MsoNormal"
+ style="text-align: center; text-indent: 0cm; page-break-after: avoid;"
+ align="center"><span
+ style="font-size: 10pt; line-height: 200%;"><img
+ style="width: 516px; height: 258px;" alt="" id="Chart 15"
+ src="https://static.igem.org/mediawiki/2010/2/2b/Freiburg10_pAAV_pSB1C3_001.png"></span></p>
+      <p class="MsoCaption"><span lang="EN-US">Figure
+      </span><span lang="EN-US">11</span> <span
+ style="color: windowtext; font-weight: normal;" lang="EN-US">AAV-293
+cells were transfected with three plasmids pHelper,
+pSB1C3_001_[AAV2]-Rep-VP123_p5-TATAless or pAAV_RC_IRCK and
+pSB1C3_[AAV2]-left-ITR_pCMV_beta-globin_mVenus_hGH_[AAV2]-right-ITR
+providing essential genes and proteins for producing viral particles.
+After 48 hours post transfection, viral particles were harvested by
+freeze-thaw lysis and centrifugation followed by HT1080 transduction.
+mVenus expression of viral genomes was determined by flow cytomery
+after 24 hours post infection. </span><span
+ style="color: windowtext; font-weight: normal;" lang="EN-US">Fluorescence
+is measured in surviving cells.</span><span
+ style="color: windowtext; font-weight: normal;" lang="EN-US">&nbsp;Results
+showed functionality of <i>RepVP123</i>
+within pSB1C3_001 vector and additionally increased transduction
+efficiency.</span></p>
+      </td>
+    </tr>
+  </tbody>
+</table>
+<h4 style="margin-left: 0cm; text-indent: 0cm;"><a
+ name="_Toc275885927"></a><a name="_Toc275817886"><span
+ lang="EN-US">Turning-off natural tropism:
+HSPG-knock-out</span></a></h4>
+<p class="MsoNormal" style="text-indent: 0cm;"><span
+ lang="EN-US">Shutting-down
+the
+natural viral tropism is essential for targeting specifically tumor
+cells and
+not infecting healthy cells. Therefore, the iGEM team Freiburg_Bioware
+decided to knock-out the viral natural tropism delivered by the heperan
+sulfate
+proteoglycan-(HSPG) binding site within the viruses 587 loop. The
+knock-out was
+cloned by designing primers containing the required base exchanges and
+performing a SDM. Like performed before, this <i>RepVP123</i>
+variant was tested
+in cell culture as well and evaluated by flow cytometry. Results show
+that
+mutation of HSPG-binding motif has severe impact on transduction
+efficiency
+thus enabling a viral particle carrying this knock-out and additional
+targeting
+motifs, e.g. within the loops or presented via N-terminal fusion to
+bind target
+cells’ receptors and therefore infecting target cells at a much higher
+rate
+compared to unspecific infection of other cell types within an organism
+(see </span><span lang="EN-US">Figure 12</span><span
+ lang="EN-US">).</span></p>
+<p class="MsoNormal" style="text-indent: 0cm;"><span
+ lang="EN-US">To
+quantify
+differences in infectivity, the infectious titer of viral particles
+built-up of
+<i>RepVP123</i> with and without HSPG binding motif was
+determined by qPCR (see
+</span><span lang="EN-US">Figure 14</span><span
+ lang="EN-US">) for
+different cell
+lines. Results show that the implemented HSPG-knock-out verifies
+results obtained
+from flow cytometry, infectious titers severely compared to <i>RepVP123</i>
+with intact HSPG binding motif.</span></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 style="height: 258.85pt;">
+      <td
+ style="border: 1pt solid windowtext; padding: 0cm 5.4pt; vertical-align: top; width: 459.85pt; height: 258.85pt;">
+      <p class="MsoNormal"
+ style="text-indent: 0cm; page-break-after: avoid;"><img
+ style="width: 595px; height: 379px;" id="Picture 2"
+ src="https://static.igem.org/mediawiki/2010/9/9d/Freiburg10_HSPG-ko_sequence.png"
+ alt="Description: \\132.230.232.133\x\users\FreiGem\iGEM2010\Stefan\Pictures_Results\HSPG-ko_modified.jpg"></p>
+      <p class="MsoCaption" style="text-align: left;"
+ align="left"><span lang="EN-US">Figure </span><span
+ lang="EN-US">12</span> <span
+ style="color: windowtext; font-weight: normal;" lang="EN-US">Alignment
+of 587 loop within viral VP123: The upper sequence shows a strand
+containing the HSPG binding motif (AGA, in red boxes), the lower
+sequence contains the HSPG-ko introduced by the iGEM team
+Freiburg_Bioware 2010 (GCT and GCC, blue boxes).</span></p>
+      </td>
+    </tr>
+  </tbody>
+</table>
+<p class="MsoNormal" style="text-indent: 0cm;"><span
+ lang="EN-US">&nbsp;</span></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="border: 1pt solid windowtext; padding: 0cm 3.5pt; vertical-align: top; width: 460.6pt;">
+      <p class="MsoNormal"
+ style="text-align: center; text-indent: 0cm; page-break-after: avoid;"
+ align="center"><img style="width: 467px; height: 229px;"
+ alt="" id="Chart 9"
+ src="https://static.igem.org/mediawiki/2010/0/01/Freiburg10_columns_HSPG_HSPG_ko.png"></p>
+      <p class="MsoCaption"><a name="_Ref275882130"><span
+ lang="EN-US">Figure
+      </span></a><span lang="EN-US">13</span>
+      <span style="color: windowtext; font-weight: normal;"
+ lang="EN-US">Transduction
+efficiency of HT1080 cells measured by flow cytometry.&nbsp;</span><span
+ style="color: windowtext; font-weight: normal;" lang="EN-US">Fluorescence
+is measured in surviving cells.</span><span
+ style="color: windowtext; font-weight: normal;" lang="EN-US">
+Knock-out of
+HSPG binding motif greatly reduces transduction efficiency compared to <i>RepVP123</i>
+containing the motiv.</span></p>
+      </td>
+    </tr>
+  </tbody>
+</table>
+<p class="MsoNormal" style="text-indent: 0cm;"><span
+ lang="EN-US">&nbsp;</span></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="border: 1pt solid windowtext; padding: 0cm 3.5pt; vertical-align: top; width: 460.6pt;">
+      <p class="MsoCaption"
+ style="text-align: center; page-break-after: avoid;"
+ align="center"><img style="width: 535px; height: 456px;"
+ alt="" id="Chart 1"
+ src="https://static.igem.org/mediawiki/2010/b/bc/Freiburg10_qPCR_HSPG_HSPG_ko.png"></p>
+      <p class="MsoCaption" style="text-align: center;"
+ align="center"><a name="_Ref275883877"><span
+ lang="EN-US">Figure </span></a><span
+ lang="EN-US">14</span> <span
+ style="color: windowtext; font-weight: normal;" lang="EN-US">Infectious
+titers of <i>RepVP123</i> with and without natural HSPG
+binding motif tested in different cell lines via qPCR. Shutting-down
+the HSPG binding motif reduces infectious titer in both HT1080 and HeLa
+cell lines. For A431 cells, no infectious titer could be detected via
+qPCR, which is probably related to poor transduction efficiency of A431
+cells.</span></p>
+      </td>
+    </tr>
+  </tbody>
+</table>
+<br>
+<br>
+<br>
+</body>
+</html>