Ljubljana

Synthetic biology provides the possibility to extend our defense against disease by employing our intelligence. In the spirit of synthetic biology we can combine different functional parts with known properties to assemble new cellular functions which do not yet exist in nature.

Abstract

HIV-1 virus is one of the most difficult targets for therapy because it hijacks our immune system and particularly because the virus mutates rapidly. This allows the selection of mutated variant strains which bypass the inhibitors that bind to specific residues on their targets. Currently the most effective therapy consists of an inhibitor cocktail that decreases the probability of HIV to overcome all target sequences at the same time.

We propose a different strategy, where we target a specific FUNCTION of virus, rather than any particular sequence. This viral function triggers a cellular response which can either employ antiviral defense or lead to a destruction of infected cells to prevent spread of the infection.

For implementation of this idea we have selected two functions: viral attachment to the cellular co-receptors CD4 and CCR5 and viral proprotein processing by its own protease. Binding of virus to T-cells leads to the formation of CD4-CCR5 heterodimer. In our device, formation of this heterodimer triggers reconstitution of a split protein (split ubiquitin or TEVP) which activates the specific proteolytic activity. This releases the T7 RNA polymerase from the membrane anchor and leads to transcription of the effector gene, which prevents the spread of viral infection.
The second implementation of this idea was to utilize the HIV-protease, which is required for viral maturation and cleaves a specific amino acid sequence. This target sequence was engineered between the membrane anchor and T7 polymerase. Activation of viral protease similarly as above releases the T7 polymerase and starts the defense program.

Important points of both approaches are to use the amplification of the signal, achieved by the use of T7 polymerase and the system's versatility, as we can select any number of different genes to be activated by the detection of viral function. The crucial aspect of our approach is that this system is not sensitive to viral mutations and is not activated only in case where the mutation leads to the loss of the viral function, which however also renders virus harmless at the same time.

We have tested many different constructs, contributing more than 70 new BioBricks and successfully demonstrated on mammalian cell culture activation of response gene by infection with HIV-1 pseudovirus.