Compositions and methods for delivering nucleic acid to a cell

a nucleic acid and cell technology, applied in the field of compositions and methods for delivering nucleic acid to cells, can solve the problems of poor in vivo transfection efficiency, insufficient stability of liposome-nucleic acid complexes, and inability to commercialize therapeutic nucleic acid containing liposomes

Inactive Publication Date: 2014-03-06
MERRIMACK PHARMACEUTICALS INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0033]In certain embodiments of the compositions described herein, the composition comprises no more than about 5 mol % or 1 mol % DOPE. In certain embodiments, the composition is essentially free of DOPE (e.g., has less than 0.1% DOPE by weight). In certain embodiments, transfection of the nucleic acid into the cell is at least about 10% more efficient (or at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% more efficient) when DOPE is absent or, if present, is present at a concentration of no more than 5 mol. % of total lipid, compared to transfection of the nucleic acid into the cell with a composition identical except for the presence of cationic lipid at a concentration of more than 5 mol. % of total lipid; in certain embodiments, the transf...

Problems solved by technology

Liposome technology has been developed and commercialized for the delivery of conventional pharmaceutical agents, but to date therapeutic nucleic acid containing liposomes have not been commercialized.
To date many publications demonstrate that liposome-plasmid DNA complexes can mediate efficient transient expression of a gene in cultured cells but poor in vivo transfection efficiencies.
Unlike viral...

Method used

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  • Compositions and methods for delivering nucleic acid to a cell
  • Compositions and methods for delivering nucleic acid to a cell
  • Compositions and methods for delivering nucleic acid to a cell

Examples

Experimental program
Comparison scheme
Effect test

example 1

Lipid Formulation with Added Polyamine

Abbreviations

[0085]GFP: Green fluorescent protein[0086]MES: 2-(N-morpholino)ethanesulfonic acid[0087]TE buffer: Tris / EDTA buffer[0088]HBS: HEPES buffer[0089]PEI: polyethyleneimine[0090]Hank's' BSS: Hank's' balanced salt solution[0091]DOTAP: dioleoyl trimethylammonium propane[0092]DOPC: 1,2-dioleoyl-sn-glycero-3-phosphocholine[0093]Chol: cholesterol[0094]DOPE: 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine[0095]CHEMS: cholesteryl hemisuccinate[0096]PEG: polyethylene glycol[0097]PEG-DSG: PEG-distearoylglycerol[0098]DOSPA: N-(1-(2,3-dioleyloxy)propyl)-N-(2-(sperminecarboxamido)ethyl)-N,N-dimethyl-ammonium trifluoroacetate[0099]CHIM: cholesterol imidazole derivative[0100]DiI(3)-DS: a cationic lipid dye:

Liposome Preparation

[0101]The polyamine was mixed with GFP plasmid DNA in 50% ethanol / 50% 20 mM MES, pH 5.1. The lipid mixtures were dissolved in 50% ethanol / 50% 2 mM TE buffer, pH 8.0. The polyamine mixture and the lipid mixture were heated at 60° C. ...

example 2

Method

[0110]DNA (100 μg) was prepared in a 50% ethanol solution as described above. From a stock solution of PEI 600, aliquots were added to in such volumes to give N / P=0, 0.67, 1.33 and 4. Separately, a solution of 2 mM TE buffer, pH 8.0 and ethanol (50% v / v) were prepared. The solution were heated at 60° C. for 2 min and mixed. The samples were cooled and dialysed as above. DNA concentration and dye accessibility was measured as above.

Results

[0111]

%plate1plate2DyeSample[dna]ng / mlstdev[dna]ng / mlstdevAccesstdev[DNA]ug / mlstdevnakedDNA729.116.8779.1217.893.62.3123.370.53N / P 0.67695.121.76771.9617.9590.02.1123.160.54N / P 1.33505.753.72527.7919.2395.83.5615.830.58N / P 4.0467.522.63506.8517.892.23.2815.210.53

Conclusion

[0112]Addition of PEI to DNA does not inhibit the binding of Picogreen®. Therefore any protection afforded to DNA during the liposome encapsulation method even with PEI included within the formulation must come from lipid encapsulation.

example 3

[0113]The following experiment was conducted to investigate whether cationic lipid can be removed from the liposome composition.

Method

[0114]Lipid Formulation=DOTAP / DOPC / Chol / PEG-DSG / DiI(3)-DS 30 / 1500 / 1000 / 30 / 3 nmol per μg DNA

Formulation 1: Lipid as above mixed with DNA (plus PEI N / P=0.9)

Formulation 2: DNA plus PEI N / P=0.9

Formulation 3: Lipid as above mixed with DNA

Formulation 4: Lipid as above (except no DOTAP) mixed with DNA (plus PEI N / P=0.9)

Results

[0115]

% DyeFormulationAccessStdevSize nm121.50.9183.5 ± 60293.22.4N.D331.21248.8 ± 67.2424.00.7289.6 ± 112.5

Conclusion

[0116]Using the DNA pre-contacted with a polyamine or polymeric amine, liposomal particles can be made without using any cationic lipid, that entrap >75% of the DNA.

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Abstract

Provided are novel compositions useful for delivering nucleic acids to cells. Also provided are methods for making and using such compositions.

Description

RELATED APPLICATIONS[0001]This application is a continuation of PCT International Application No. PCT / US2012 / 025324, filed Feb. 15, 2012, which claims the benefit of U.S. Provisional Patent Application No. 61 / 443,246, filed Feb. 15, 2011. The contents of each of the foregoing applications are incorporated herein by reference in their entirety.BACKGROUND OF THE INVENTION[0002]Introducing nucleic acids into living cells is an important process in modern biological research, industry, and medicine. Efficient delivery of a functional nucleic acid into a living cell is an indispensable component of genetic engineering, recombinant protein production, and medical technologies known as gene therapy.[0003]For example, gene therapy involves the transfer of normal, functional genetic material into specific cells to correct an abnormality due to a deficient or defective gene product. A variety of methods have been developed to facilitate both in vivo, in vitro, or ex vivo gene transfer.[0004]N...

Claims

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Application Information

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IPC IPC(8): A61K9/127A61K47/48A61K48/00
CPCA61K9/1271A61K48/0091A61K47/48823A61K48/0008C12N15/88A61K47/6913A61K48/0033C12N15/113C12N2310/14C12N2310/531C12N2320/32
Inventor HAYES, MARK E.KIRPOTIN, DIMITRI B.
Owner MERRIMACK PHARMACEUTICALS INC
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