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User:VolkerMorath
From 2010.igem.org
=> Vector Plasmid
=> Capsid Coding Parts
=> Miscellaneous System parts
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=> Safety
References:
Shielding the Viral Vector
Layers of Specificity
Employment of viral vectors for means of therapy is idea in the context of personalized medicie that gets more and more interest. In such applications the reduction of side effects and the safety of the patient in general is of the highest priority.
In order to satisfy this requirement we designed our Therapy Vector with several layers of Specificity:
The targeting of the viral vector towards the desired target cell (e.g. tumor cells) is the basic idea behind the emplyment of viral vectors for therapeutical means. There for the natural tropismn has to be knocked down and a desired tropism has to be introduced that allows differential targeting of pathological but not of off-target cells. To fulfill this mission our Virus Construction Kit offers you different solutions.
Off-target cells that were transduced by mistake can be preserved from an undesired therapy effect when the therapeutic gene is controley by a tissue specific promoter. For this mean a promoter has to be used that is as specific for the pathological tissue as possible. We included the human telomerase promoter (phTERT) which is often activated in tumor cells and is there for able to allow differential experssion of a therapeutic geneproduct in pathological cells.
For reasons of safety Therapeutic vector do not directly trigger appoptosis in the successfully targeted cells. To include one further layer of specificity and safety we decided to arm our therapy vector with different prodrug convertases. Neither the single application of the harmless prodrug nor the single expression of the convertase has a noteworthy effect of the transduced cell. Only in cells that express the prodrug convertase and have a sufficient cytoplasmatic concentration of the belonging prodrug apoptosis is triggered. This dependency of the therapy on a prodrug can be employed to protect tissues or other persons that could come in contact with the therapeutical vector. This aspect was specially inportant for the development of a viral vector that is able to infect humans in the context of a undergraduate project for the iGEM competition. Therefor this approach gained our preference over other possibly equivalent arming possibilities described in the tumor therapy with viral vectors.
In order to satisfy this requirement we designed our Therapy Vector with several layers of Specificity:
ViralBrick compatible version of the capsid coding sequence
The insertion of sequences for functional peptides into the viral capsid is a well established and characterized possibility to retarget and modify the viral vectors. Over the last decade many different locations in the viral sequence have been evaluated for their quality to insert small peptides. We decided to use the well etablished 587 and the newly identifyed 453 integration site for our Virus Construction Kit.
For reasony of flexibility and usability we decided not to use single cutting restriction sites in stead of PCR-technology. This approach provids the advantage that all capsid coding constructs can be modifyed using ViralBricks in a single cloning step but also beared the risk to harm the functionality of the viral gene sequence. The functionality of the modified version with the introduced single cutting restriction sites was tested and the functionality was determined as comparable with the unmodifyed sequence.
In order to have these single cutting restriction sites two restriction sites had to be remooved from the Rep-protein coding region of pAAV-RepCap and the restriction sites had to be introduced into the Capsid coding region as show in the following figure.
For reasony of flexibility and usability we decided not to use single cutting restriction sites in stead of PCR-technology. This approach provids the advantage that all capsid coding constructs can be modifyed using ViralBricks in a single cloning step but also beared the risk to harm the functionality of the viral gene sequence. The functionality of the modified version with the introduced single cutting restriction sites was tested and the functionality was determined as comparable with the unmodifyed sequence.
In order to have these single cutting restriction sites two restriction sites had to be remooved from the Rep-protein coding region of pAAV-RepCap and the restriction sites had to be introduced into the Capsid coding region as show in the following figure.
