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Cationic lipid-mediated delivery of proteins enables efficient protein-based genome editing in vitro and in vivo.

Zuris JA, Thompson DB, Shu Y, Guilinger JP, Bessen JL, Hu JH, Maeder ML, Joung JK, Chen ZY, Liu DR - Nat. Biotechnol. (2014)

Bottom Line: Current methods of protein delivery commonly suffer from low tolerance for serum, poor endosomal escape and limited in vivo efficacy.This approach mediates the potent delivery of nM concentrations of Cre recombinase, TALE- and Cas9-based transcription activators, and Cas9:sgRNA nuclease complexes into cultured human cells in media containing 10% serum.Delivery of unmodified Cas9:sgRNA complexes resulted in up to 80% genome modification with substantially higher specificity compared to DNA transfection.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Chemistry &Chemical Biology, Harvard University, Cambridge, Massachusetts, USA. [2] Howard Hughes Medical Institute, Harvard University, Cambridge, Massachusetts, USA.

ABSTRACT
Efficient intracellular delivery of proteins is needed to fully realize the potential of protein therapeutics. Current methods of protein delivery commonly suffer from low tolerance for serum, poor endosomal escape and limited in vivo efficacy. Here we report that common cationic lipid nucleic acid transfection reagents can potently deliver proteins that are fused to negatively supercharged proteins, that contain natural anionic domains or that natively bind to anionic nucleic acids. This approach mediates the potent delivery of nM concentrations of Cre recombinase, TALE- and Cas9-based transcription activators, and Cas9:sgRNA nuclease complexes into cultured human cells in media containing 10% serum. Delivery of unmodified Cas9:sgRNA complexes resulted in up to 80% genome modification with substantially higher specificity compared to DNA transfection. This approach also mediated efficient delivery of Cre recombinase and Cas9:sgRNA complexes into the mouse inner ear in vivo, achieving 90% Cre-mediated recombination and 20% Cas9-mediated genome modification in hair cells.

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Strategy for delivering proteins into mammalian cells by fusion or non-covalent complexation with polyanionic macromolecules and complexation with cationic lipids. (a) Recombinases, transcriptional-activator-like effector (TALE) proteins, and Cas9 endonucleases bind nucleic acids and are natively cationic (net theoretical charges are shown in black) and are not efficiently complexed with cationic lipids. These proteins can be rendered highly anionic, however, by fusion to either a supernegatively charged protein such as (−30)GFP, or by complexation with polyanionic nucleic acids. (b) We envisioned that cationic lipids commonly used to transfect DNA and RNA would complex with the resulting highly anionic proteins or protein:nucleic acid complexes, mediating their delivery into mammalian cells.
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Figure 1: Strategy for delivering proteins into mammalian cells by fusion or non-covalent complexation with polyanionic macromolecules and complexation with cationic lipids. (a) Recombinases, transcriptional-activator-like effector (TALE) proteins, and Cas9 endonucleases bind nucleic acids and are natively cationic (net theoretical charges are shown in black) and are not efficiently complexed with cationic lipids. These proteins can be rendered highly anionic, however, by fusion to either a supernegatively charged protein such as (−30)GFP, or by complexation with polyanionic nucleic acids. (b) We envisioned that cationic lipids commonly used to transfect DNA and RNA would complex with the resulting highly anionic proteins or protein:nucleic acid complexes, mediating their delivery into mammalian cells.

Mentions: We hypothesized that proteins that are highly anionic could be delivered by the same electrostatics-driven complexation used by cationic liposomal reagents for nucleic acid delivery (Fig. 1a). Although few proteins natively possess the highly anionic character of nucleic acids, we speculated that translational fusion or non-covalent complexation with a polyanionic molecule may render the resulting protein or protein complex sufficiently anionic to be efficiently delivered by common cationic lipid reagents. We demonstrate that fusion of proteins with an engineered supernegatively charged GFP28 enables efficient complexation and delivery of proteins into cultured mammalian cells by cationic lipids. Our approach is effective even at low nanomolar protein concentrations and in the presence of serum, requiring 1,000-fold less protein to achieve similar functional protein delivery levels than methods that use fusion to cationic peptides or proteins.15 We further show that Cas9 nuclease protein complexed with polyanionic single guide RNA (sgRNA) can be efficiently delivered in functional form into mammalian cells using cationic lipid formulations. Delivery of Cas9:sgRNA complexes is highly efficient (up to 80% modification of cultured human cells from a single treatment) and also induces higher genome modification specificity (typically ~10-fold) compared with plasmid transfection. Finally, we demonstrate that this protein delivery approach can be effective in vivo by delivering functional Cre recombinase and functional Cas9:sgRNA complexes to hair cells in the inner ear of live mice. These findings suggest that the intracellular delivery of polyanionic proteins and protein:nucleic acid complexes by cationic lipids may significantly expand the scope of research and therapeutic applications of proteins.


Cationic lipid-mediated delivery of proteins enables efficient protein-based genome editing in vitro and in vivo.

Zuris JA, Thompson DB, Shu Y, Guilinger JP, Bessen JL, Hu JH, Maeder ML, Joung JK, Chen ZY, Liu DR - Nat. Biotechnol. (2014)

Strategy for delivering proteins into mammalian cells by fusion or non-covalent complexation with polyanionic macromolecules and complexation with cationic lipids. (a) Recombinases, transcriptional-activator-like effector (TALE) proteins, and Cas9 endonucleases bind nucleic acids and are natively cationic (net theoretical charges are shown in black) and are not efficiently complexed with cationic lipids. These proteins can be rendered highly anionic, however, by fusion to either a supernegatively charged protein such as (−30)GFP, or by complexation with polyanionic nucleic acids. (b) We envisioned that cationic lipids commonly used to transfect DNA and RNA would complex with the resulting highly anionic proteins or protein:nucleic acid complexes, mediating their delivery into mammalian cells.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4289409&req=5

Figure 1: Strategy for delivering proteins into mammalian cells by fusion or non-covalent complexation with polyanionic macromolecules and complexation with cationic lipids. (a) Recombinases, transcriptional-activator-like effector (TALE) proteins, and Cas9 endonucleases bind nucleic acids and are natively cationic (net theoretical charges are shown in black) and are not efficiently complexed with cationic lipids. These proteins can be rendered highly anionic, however, by fusion to either a supernegatively charged protein such as (−30)GFP, or by complexation with polyanionic nucleic acids. (b) We envisioned that cationic lipids commonly used to transfect DNA and RNA would complex with the resulting highly anionic proteins or protein:nucleic acid complexes, mediating their delivery into mammalian cells.
Mentions: We hypothesized that proteins that are highly anionic could be delivered by the same electrostatics-driven complexation used by cationic liposomal reagents for nucleic acid delivery (Fig. 1a). Although few proteins natively possess the highly anionic character of nucleic acids, we speculated that translational fusion or non-covalent complexation with a polyanionic molecule may render the resulting protein or protein complex sufficiently anionic to be efficiently delivered by common cationic lipid reagents. We demonstrate that fusion of proteins with an engineered supernegatively charged GFP28 enables efficient complexation and delivery of proteins into cultured mammalian cells by cationic lipids. Our approach is effective even at low nanomolar protein concentrations and in the presence of serum, requiring 1,000-fold less protein to achieve similar functional protein delivery levels than methods that use fusion to cationic peptides or proteins.15 We further show that Cas9 nuclease protein complexed with polyanionic single guide RNA (sgRNA) can be efficiently delivered in functional form into mammalian cells using cationic lipid formulations. Delivery of Cas9:sgRNA complexes is highly efficient (up to 80% modification of cultured human cells from a single treatment) and also induces higher genome modification specificity (typically ~10-fold) compared with plasmid transfection. Finally, we demonstrate that this protein delivery approach can be effective in vivo by delivering functional Cre recombinase and functional Cas9:sgRNA complexes to hair cells in the inner ear of live mice. These findings suggest that the intracellular delivery of polyanionic proteins and protein:nucleic acid complexes by cationic lipids may significantly expand the scope of research and therapeutic applications of proteins.

Bottom Line: Current methods of protein delivery commonly suffer from low tolerance for serum, poor endosomal escape and limited in vivo efficacy.This approach mediates the potent delivery of nM concentrations of Cre recombinase, TALE- and Cas9-based transcription activators, and Cas9:sgRNA nuclease complexes into cultured human cells in media containing 10% serum.Delivery of unmodified Cas9:sgRNA complexes resulted in up to 80% genome modification with substantially higher specificity compared to DNA transfection.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Chemistry &Chemical Biology, Harvard University, Cambridge, Massachusetts, USA. [2] Howard Hughes Medical Institute, Harvard University, Cambridge, Massachusetts, USA.

ABSTRACT
Efficient intracellular delivery of proteins is needed to fully realize the potential of protein therapeutics. Current methods of protein delivery commonly suffer from low tolerance for serum, poor endosomal escape and limited in vivo efficacy. Here we report that common cationic lipid nucleic acid transfection reagents can potently deliver proteins that are fused to negatively supercharged proteins, that contain natural anionic domains or that natively bind to anionic nucleic acids. This approach mediates the potent delivery of nM concentrations of Cre recombinase, TALE- and Cas9-based transcription activators, and Cas9:sgRNA nuclease complexes into cultured human cells in media containing 10% serum. Delivery of unmodified Cas9:sgRNA complexes resulted in up to 80% genome modification with substantially higher specificity compared to DNA transfection. This approach also mediated efficient delivery of Cre recombinase and Cas9:sgRNA complexes into the mouse inner ear in vivo, achieving 90% Cre-mediated recombination and 20% Cas9-mediated genome modification in hair cells.

Show MeSH