In today’s culture negativity is usually frowned upon; maintaining a positive outlook is almost essential to a successful scientific career, but an innovative team of researchers have put some negativity to good use in improving transfection of genome editing complexes using negatively charged proteins and nucleic acid transfection reagents.
Conventional protein-based therapeutics usually focus on extracellular targets, due to the inability of proteins to spontaneously penetrate mammalian cellular barriers. Research has shown that nucleic acids can be protected via liposomal complexation and effectively delivered to cells.
Feeling a little adventurous, John A. Zuris and colleagues at Harvard University decided to venture into the possibility of delivering therapeutic protein cargo directly to cells.
To effectively deliver their desired cargo to cells of interests, the team smartly designed protein complexes that allow for endocytosis and protect against enzymatic degradation by endogenous proteases.
Specifically, Zuris and colleagues engineered fuse proteins with supernegatively charged GFP with the complex protected by cationic lipids and deliver the complex into cultured mammalian cells.
Here is a summary of their findings:
- The technique works well at low nanomolar protein concentraions, requiring less protein to achieve the same delivery compared to other conventional methods.
- CRSPR-cas9 complexed with polyanionic single guide RNA (sgRNA) can be efficiently delivered into mammalian cells.
- The delivered Cas9-sgRNA complex induces high genome editing specificity by an average of tenfold when compared to plasmid transfection.
- Cationic lipid guide protein complex delivery works in vivo as ascertained by the delivery of functional Cre recombinase and functional Cas9-sgRNA complexes to hair cells in the inner ear of live mice.
The team deduced that “intracellular delivery of polyanionic proteins and protein-nucleic acid complexes protected by cationic lipids” holds great promises for research and protein-based therapeutics.
To learn more about this promising masterpiece visit Nature Biotechnology, October , 2014.
Additionally visit the Harvard Gazette to watch videos describing this important work and technical details of the cationic lipid protection of therapeutic protein agents in depth.