Supercharged proteins for cell penetration

A supercharged, protein technology, applied in the field of supercharged proteins for cell penetration, can solve the problems of easy preparation, stability or universality of cytotoxicity

Inactive Publication Date: 2011-05-18
PRESIDENT & FELLOWS OF HARVARD COLLEGE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Each of these delivery systems is beneficial for specific applications; however, in most cases, questions remain regarding cytotoxicity, ease of preparation, stability, or generalizability

Method used

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  • Supercharged proteins for cell penetration
  • Supercharged proteins for cell penetration
  • Supercharged proteins for cell penetration

Examples

Experimental program
Comparison scheme
Effect test

example 1

[0475] Example 1: Supercharging a protein confers extraordinary resilience

[0476] Materials and methods

[0477] Design programs and supercharged protein sequences

[0478] Solvent exposed residues of AvNAPSA Inside). A charged or highly polar solvent exposed residue (DERKNQ) was mutated to Asp or Glu for supernegative charge or to Lys or Arg for superpositive charge. Other surface-exposed positions to be mutated in green fluorescent protein (GFP) variants were selected based on the sequence variability of said positions among GFP homologs.

[0479] Protein Expression and Purification

[0480] Synthetic genes optimized for codon usage in E. coli were purchased from DNA 2.0, cloned into pET expression vector (Novegen) and overexpressed in E. coli BL21(DE3)pLysS at 15°C for 5-10 hours . Cells were harvested by centrifugation and lysed by sonication. Proteins were purified by Ni-NTA agarose chromatography (Qiagen), buffer exchanged to 100 mM NaCl, 50 mM potassium phosph...

example 2

[0516] Example 2: Supercharged proteins can be used to efficiently deliver nucleic acids into cells

[0517] Figure 5 showed that supercharged GFP can non-specifically and reversibly associate with oppositely charged macromolecules ("protein Velcro"). The interaction can lead to the formation of a precipitate. Unlike aggregates of denatured protein, these pellets contained folded fluorescent GFP and were soluble in 1M salt. Shown: +36GFP alone; +36GFP mixed with -30GFP; +36GFP mixed with tRNA; +36GFP mixed with tRNA in 1M NaCl; superfolded GFP ("sfGFP"; -7GFP); 30GFP mixed with sfGFP.

[0518] Image 6 shows that superpositively charged GFP binds siRNA. The binding stoichiometry between the two components was determined by mixing +36GFP with siRNA in different ratios (over 30 minutes at 25°C) and running the mixture on a 3% agarose gel (Kumar) et al., 2007, Nature, 449:39; which is incorporated herein by reference). The +36GFP:siRNA ratios tested were 0:1, 1:1, 1:2, 1:...

example 3

[0530] Example 3: Mammalian Cell Penetration, siRNA Transfection, and DNA Transfection of Supercharged Green Fluorescent Protein

[0531] Industry literature has recently described the resurfacing of proteins by extensive mutagenesis of non-conserved solvent-exposed residues without abolishing their structure or function (Lawrence MS, Phillips KJ, Liu DR (2007) , Supercharging proteins canimpart unusual resilience (Supercharging proteins canimpart unusual resilience), Journal of the American Chemical Society (J.Am.Chem.Soc.) 129: 10110-10112; applied on June 1, 2007 and served as WO 2007 / 143574 International PCT Patent Application PCT / US07 / 70254 published on December 13, 2007; U.S. Provisional Patent Application U.S.S.N. 60 / 810,364 filed on June 2, 2006 and Applied U.S.S.N. 60 / 836,607; each of which is incorporated herein by reference). When the replacement residues are all positively or all negatively charged, the resulting "supercharged" protein retains its activity while a...

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Abstract

Compositions, systems and related methods for delivering a supercharged protein or a complex of a supercharged protein and therapeutic agent (e g, nucleic acid, peptide, small molecule) to cells are disclosed. Superpositively charged proteins may be associated with nucleic acids (which typically have a net negative charge) via electrostatic interactions. The systems and methods may involve altering the primary sequence of a protein in order to ''supercharge'' the protein (e g, to generate a superpositively-charged protein). The compositions may be used to treat proliferative diseases, infectious diseases, cardiovascular diseases, inborn errors in metabolism, genetic diseases, etc.

Description

[0001] Related Application Cross Reference [0002] This application claims priority under 35 U.S.C. §119(e) to U.S. Provisional Patent Applications USSN 61 / 048,370, filed April 28, 2008, and USSN 61 / 105,287, filed October 14, 2008; applications are incorporated herein by reference. [0003] governmental support [0004] This invention was made with US Government support under Contract No. R01 GM065400 awarded to National Institutes of Health / NIGMS. The US Government has certain rights in this invention. Background technique [0005] The effectiveness of an agent intended for therapeutic diagnostic or other applications is often highly dependent on its ability to penetrate cell membranes or tissues to induce a desired change in biological activity. Although many therapeutic, diagnostic, or other product candidates (whether proteins, nucleic acids, small organic molecules, or small inorganic molecules) exhibit promising biological activity in vitro, many fail to reach or ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/00A61K47/42
CPCA61K38/17A61P31/00A61P35/00
Inventor 大卫·R·刘布赖恩·R·麦克诺顿詹姆斯·约瑟夫·克罗尼坎戴维·B·汤普森
Owner PRESIDENT & FELLOWS OF HARVARD COLLEGE
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