Part:BBa_K404212

ViralBrick-587KO-RGD

ViralBrick-587KO-RGD
BioBrick Nr.	BBa_K404212
RFC standard	RFC 25
Requirement	pSB1C3
Source
Submitted by	[http://2010.igem.org/Team:Freiburg_Bioware FreiGEM 2010]

The RGD integrin binding motif including a knockout of the natural HSPG tropism, ready for insertion into the 587 loop

Usage and Biology

Integrins are transmembrane proteins that, among other functions, mediate cell attachment to surrounding tissues. They bind to a motif consisting of the amino acids arginine, glycine and aspartic acid (RGD in one-letter code). Because Integrin is highly expressed in many tumor cell lines (Albelda et al., 1990), (Damjanovich, Albelda, Mette, & Buck, 1992), (Lessey et al., 1995), (Smythe, LeBel, Bavaria, Kaiser, & Albelda, 1995), (Gladson & Cheresh, 1991), AAV particles displaying the RGD motif on various positions in their capsid proteins have been created by (Shi et al., 2003). Particles displaying RGD at amino acid positions 584 & 588 as well as 453 or 587 (Boucas et al., 2009) showed transduction efficiencies similar to wt AAV, even when the cells’ HSPG receptors were blocked by heparin sulfate or when the natural HSPG binding motif on the capsid surface was knocked out. To further broaden the area of therapeutic application, we created a ViralBrick containing the RGD motive to specifically target cells with low HSPG-/high Integrin expression.

Restriction sites

Restriction site	Enzyme	Recognition sequence	Activity	Temp.
upstream 587	[http://www.neb.com/nebecomm/products/productR3136.asp BamHI-HF]		100;50;10;100	37°C
downstream 587	[http://www.neb.com/nebecomm/products/productR3151.asp PvuII-HF]		0;25;0;100	37°C

Results

We could show a differential targeting towards tumor cells overexpressing integrin on their surface using AAV particles containing the RGD loop insertion with knocked out HSPG tropism. Titers were determined via qPCR (for details, see [http://2010.igem.org/Team:Freiburg_Bioware/Project/Results#targeting team wiki])

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 10
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]