Question

You’ve identified an antigenic protein unique to multidrug resistant Staphylococcus aureus (MRSA). You’d like to create a MRSA vaccine based on an in vitro transcribed mRNA. Several answers will be acceptable, since there are several technical approaches to carry out the same tasks. Specifically:

(1 pt) What molecular method could you use to clone the gene from S. aureus? (Name a specific approach from earlier in the course).

(1 pt) What will be the components of your IVT mRNA itself?

(1 pt) What steps could you take to reduce the immunogenicity of the IVT mRNA?

(1 pt) What is one way you might deliver the IVT mRNA into a patient?

(1 pt) You find that the S. aureus protein is poorly expressed in human cells. How could you change your

         mRNA sequence to boost ex

Answer 1

To clone  the gene from S. aureus, PCR based cloning would be a good strategy. Using gene specific primers with appropriate restriction sites the gene encoding the anigenic protein can be cloned into plasmid vector compatible with in vitro translation methods.

Since the IVT mRNA will be used for translation into the mammalian system and its origin is bacterial, it should contains eukaryotic translation compatible elements which ensure the translation of IVT mRNA. These include 5' UTR, ribosome binding site, translation start site, stop site followed by 3' UTR .

To decrease the immunogenicity of IVT mRNA it should resemeble with eukaryotic mRNA, for that 5' cap and poly A tail will be good option. Also eukaryotic cell's cytoplasm contains RNAses as defence against viral RNAs. To avoid the IVT mRNA from degradation in cytoplasm best method will be to choose delivary methods which will deliver IVT mRNA directly into the nucleus.

Site specific delivary methods like lentiviral vectors can be used to deliver the IVT mRNA to a patient.

To enhance the expression of S. aureus protein into human cells few strategies can be used like:

1. Genetic code optimisation.

2. Addition of poly A tail and 5' cap.