METHOD AND COMPOSITION FOR A PROTEIN TRANSDUCTION TECHNOLOGY AND ITS APPLICATIONS

• US 20090298111A1
• Filed: 05/28/2008
• Published: 12/03/2009
• Est. Priority Date: 05/28/2008
• Status: Active Grant

• Current Assignee: Wayne State University
• Original Assignee: Wayne State University
• Inventors: Wang, Jianjun, Li, Qianqian
• Granted Patent: US 8,722,348 B2
• Time in Patent Office: Days
• US Class Current: 435/29
• CPC Class Codes: A61K 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• CPC Class Codes: A61K 47/543 Lipids, e.g. triglycerides; Polyamines, e.g. spermine or spermidine
• CPC Class Codes: A61K 47/58 obtained by reactions only involving carbon-to-carbon unsaturated bonds, e.g. poly[meth]acrylate, polyacrylamide, polystyrene, polyvinylpyrrolidone, polyvinylalcohol or polystyrene sulfonic acid resin
• CPC Class Codes: C12N 15/88 using microencapsulation, e.g. using amphiphile liposome vesicle
• CPC Class Codes: C12P 21/00 Preparation of peptides or proteins single cell protein C12N1/00
• CPC Class Codes: G01N 2458/15 Non-radioactive isotope labels, e.g. for detection by mass spectrometry
• CPC Class Codes: G01N 33/5023 on expression patterns
• CPC Class Codes: G01N 33/58 involving labelled substances G01N33/53 takes precedence
• CPC Class Codes: G01N 33/68 involving proteins, peptides or amino acids involving lipoproteins G01N33/92

Abstract

A protein transduction method for efficiently delivery of exogenous proteins into mammalian cells is invented, which has the capability of targeting different cellular compartments and protection from degradation of the delivered proteins from cellular proteases. A composition for treat proteins has cation reagents, lipids and enhancers in a carrier. The method can be used in a number of ways including: production of large quantities of properly folded, post-translationally modified proteins using mammalian cell machinery, a in-cell fluorescence spectroscopy and imaging using small molecule fluorophores and a in-cell NMR spectroscopy using living mammalian cells. The method permits cell biology at atomic resolution that is physiologically and pathological relevant and permits protein therapy to treat human diseases. The method can also be used to deliver exogenous protein inside mammalian cells, wherein the exogenous proteins follow a similar secretion pathway as that of the endogenous protein.

Claims

1. A method of protein transduction into mammalian cells, the method comprising the steps of:

modifying a protein with a reagent that enables the protein to be efficiently delivered into mammalian cells; and

delivering the protein into mammalian cells.

2. The method according to claim 1, further including the step of expressing the protein in bacteria prior to said modifying step.

3. The method according to claim 2, further including the step of in vivo labeling the proteins with the NMR-active isotopes during bacterial expression.

4. The method according to claim 1, further including the step of in vitro labeling the protein with small molecule fluorescence probes prior to modifying the protein.

5. The method according to claim 3, further including monitoring protein activity.

20. The use of the method according to claim 1 for studying cellular structural biology.

22. The use according to claim 20, wherein said therapy includes delivering biologically active materials into target cellular compartment.

6. A composition for treating proteins, said composition comprising:

cation reagents;

lipids; and

enhancers in a carrier.

7. The composition according to claim 6, wherein said cation reagent is polyethylenimine.

8. The composition according to claim 6, wherein said lipid is selected from the group consisting essentially of DOTAP, DOPE, POPC, and DMPE.

9. The composition according to claim 6, wherein said enhancer is an enhancer that significantly enhances cell loading of cationized proteins.

10. The composition according to claim 9, wherein said enhancer selected from the group consisting essentially of DMSO, MG132, CaCl₂ and growth factor.

11. The composition according to claim 6, further including at least one selected from the group consisting essentially of cell membrane surfactants, stabilizers, and other inert carriers.

12. The use of a composition according to claim 6 for enabling in-cell NMR spectroscopy using living mammalian cells.

13. The use of a composition according to claim 6 for enabling in-cell fluorescence spectroscopy and imaging using small fluorophores and living mammalian cells.

15. A method of treating a protein to enable proper in cell folding by exposing the protein to the composition of claim 6 for enabling utilization of cell folding machinery of the mammalian cells.

16. A method of treating a protein to enable proper in cell post-translational modification by exposing the protein to the composition of claim 6 for enabling the utilization of cell post-translational modification machinery within mammalian cells.

17. A method of analyzing secretion pathways of exogenous proteins after being transduced into mammalian cells using fluorescence imaging by:

exposing the protein to the composition of claim 6.

delivering the protein into mammalian cells; and

analyzing the secretion pathway of the protein using the method of claim 13.

18. The use of a composition according to claim 6 for producing large quantities of properly folded protein.

19. The use of the composition according to claim 6 for producing large quantities of properly post-translationally modified proteins.

21. The use of the composition according to claim 6 for creating proteins for use in protein therapy.

23. The use according to claim 21, wherein said therapy includes delivering biologically active material selected from the group consisting essentially of proteins, siRNA and genes.

14. A method of specifically delivery of exogenous protein into a target sub-cellular compartment.