Team:Freiburg Bioware/BioBricks/capsid coding


Capsid Coding


Basic Capsidcoding Constructs

The category of basic capsid coding constructs contains different BioBricks that can be used to produce different kinds of viral therapy vectors. All of these constructs are compatible to the ViralBrick standard and can therefor be modifyed in a single cloning step to contain functional peptide in the major surface exposed loops.

The Basic Capsid Coding Parts

  • [AAV-2]-Rep-VP123_p5-TATA-less is the main capsid coding plasmid.
  • pCMV_[AAV-2]-VP123 is ment for co-transfections aiming to produce mosaic viruses.
  • pCMV_[AAV-2]-VP13 and pCMV_[AAV-2]-VP13 are ment for co-transfections when using N-terminal fusion.
  • pCMV_[AAV-2]-VP1, pCMV_[AAV-2]-VP2 and pCMV_[AAV-2]-VP3 are constructs code for only one of the three viral coat proteins and can be used to produce the viral coat portein, to purify them and to perform InVitro assembly of the viral shell.

ViralBricks for Loop replacement

ViralBricks are short nucleotide sequences derived from the gene of the viral coat proteins VP1-3 where they encode the two major surface exposed loops around the positions 453 and 587. Functional peptides are integrated in the loop tip which will be present on the surface of viral vectros that have been modified with the corresponding ViralBrick.

Different ViralBrick versions:

Different functional motifs:

Versions of the Rep-Vp123_p5-TATA-less

The construct [AAV-2]-RepVP123_p5-TATA-less is the construct that is cloned from an AAV2 Helper-free System which is derived from the genome of the Adeno-associated Virus in which the ITRs were removed. The expression of the eight gene products is regulated by three promoters. The p40 regulates the expression of the viral coat proteins and the AAP whereas the promoters p5 and p19 regulate the expression of the Rep proteins that are important for the production of the vector plasmid and the packaging. The usage of a TATA-less p5 promoter that is located downstream of the open reading frames favoures the production of the short Rep variants over the long ones which is conductive for the production of viral vectors.

Versions of the pCMV_Vp123

The different versions of pCMV_VP123 are ment for the production of mosaic viruses by transfecting this plamid together with [AAV2]-Rep-VP123_P5-TATA-less. The composite part pCMV_VP123 contains the ORF of the Viral Coat Proteins VP123 under the control of the control of the Cytomegalovirus promoter. By providing modified and unmodified viral coat proteins the resulting viral vector will be a chimera. The ratio of modified to unmodified viral coat proteins will depend on the number of plasmids a cell was transfected with and has impect on the functionality of the viral vector.