Project Description

Disrupting the flow of biological information at the level of mRNA is a safer alternative to conventional gene therapy, wherein insertional mutagenesis can occur through integrating vectors. In addition, the ability to regulate the level of expression of a gene using such vectors proves difficult. Therefore, we aim to create a modular, self-replicating RNA system using Hepatitis C Virus (HCV) derived RNA dependent RNA polymerase (RdRp). This drives production of siRNA directed against the enzyme dipeptidyl peptidase-IV (DPP-IV) which is elevated in type 2 diabetes and is the target of major drug studies. The replicon contains control modules, exhibiting a negative feedback mechanism provided by: an MS2 domain linked to RdRp, thereby controlling RdRp translation and therefore controlling replication, and an aptazyme switch to regulate expression levels of our siRNA. Validation of our system and the testing of modules will be performed in human (Huh 7.5) and E. coli cells.

Our project is to use replicons, self-replicating RNA systems as a novel method of gene silencing to help treat diseases such as type 2 diabetes, alzheimer's, cancer and many more. In this project we are focusing on treating type 2 diabetes, but in theory our biobrick can be used to help treat a vast number of diseases and can be used for many more purposes than just gene silencing. We are utilizing the well characterized Hepatitis C virus subtype 1b RNA dependent RNA polymerase (RdRp), a protein that effectively copies a strand of mRNA, a polymerase for RNA rather than DNA.

The main advantages of this method of gene silencing as opposed to conventional gene silencing are that this method is cheaper, it can be performed in situe, and perhaps most importantly is safer, as there is no incorporation into the genome meaning that genes which are not the target of the therapy are not affected. In addition, the gene silencing machinery existing as RNA as opposed to in DNA makes it easily accessible, and the addition of extra RNA (or alternatively certain biobricks bolted on to our biobrick) could be used to create a feedback mechanism, where the expression of the target genes is regulated, as opposed to just reduced by a constant amount.

\begin{eqnarray} \label{system1} \frac{dm}{dt} &=& \alpha_m - \beta_m m - k_s ms \\ \label{system2} \frac{ds}{dt} &=& \alpha_s - \beta_s s - p_s k_s ms - k_r sr \\ \label{system3} \frac{dr}{dt} &=& \alpha_r - \beta_r r - p_r k_r sr \end{eqnarray}

\begin{eqnarray} \label{system1} \frac{dm}{dt} &=& \alpha_m - \beta_m m - k_s ms \\ \label{system2} \frac{ds}{dt} &=& \alpha_s - \beta_s s - p_s k_s ms - k_r sr \\ \label{system3} \frac{dr}{dt} &=& \alpha_r - \beta_r r - p_r k_r sr \end{eqnarray}

To test our biobrick, we decided to focus on using it to inhibit the gene DPP4. Dipeptidyl peptidase-4 is a gene responsible for the inhibition of insulin, among other functions. DPP4 inhibitors are already used as treatment for type-2 diabetes, but pills containing them must be taken regularly. Our replicon system should in theory exist permanently in human cells, and continuously inhibit DPP4, removing the need for type-2 diabetes sufferers to continuously take medication.

To achieve this holy grail of treatment, we wish to develop a system whereby small interfering RNAs (siRNAs) are produced intermittently; without having the need to systematically introduce siRNAs through injectable delivery into patients. SiRNAs are small RNA molecules that bind to certain mRNA sequences (which can be chosen nearly arbitrarily), and cause the cell's machinery to cleave the mRNA. As we circumvent the biological flow of information at the level of mRNA, our system overcomes the limitation of gene therapeutic methods which utilize vectors that routinely integrate into the host genome, and can down regulate or activate genes.