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Lipid encapsulated interfering RNA

a technology of interfering rna and lipids, which is applied in the direction of aerosol delivery, drug compositions, metabolic disorders, etc., can solve the problems of immune response, safety concerns, and potential undesired immune responses

Inactive Publication Date: 2005-03-24
PROTIVA BIOTHERAPEUTICS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

A further embodiment of the present invention provides a method of treating a disease or disorder in a mammalian subject. A therapeutically effective amount of a nucleic acid-lipid particle comprising a cationic lipid, a non-cationic lipid, a conjugated lipid that inhibits aggregation of particles, and siRNA is administered to the mammalian subject (e.g., a rodent such as a mouse, a primate such as a human or a monkey) with the disease or disorder. In some embodiments, the disease or disorder is associated with expression and / or overexpression of a gene and expression or overexpression of the gene is silenced by the siRNA. In some embodiments, the disease is a viral disease such as, for example, hepatitis (e.g., Hepatitis A, Hepatitis B, Hepatitis C, Hepatitis D, Hepatitis E, Hepatitis G, or a combination thereof). In some embodiment, the disease or disorder is a liver disease or disorder, such as, for example, dyslipidemia.

Problems solved by technology

Viral vectors are relatively efficient gene delivery systems, but suffer from a variety of safety concerns, such as potential for undesired immune responses.
In addition, these systems induce immune responses that compromise delivery with subsequent injections.
Cationic liposome complexes, however, are large, poorly defined systems that are not suited for systemic applications and can elicit considerable toxic side effects (Harrison, et al., Biotechniques 19:816 (1995); Li, et al., The Gene 4:891 (1997); Tam, et al, Gene Ther.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

example 1

SNALP Formulations Encapsulating siRNA

This example demonstrates encapsulating siRNA in SNALP formulated with either short- or long-chain PEG-DAG and produced by continuously mixing organic lipid and aqueous buffer solutions. PEG-DAG lipids employed were PEG-dimyristylglycerol (C14) (PEG-DMG) and PEG-distearylglycerol (C18) (PEG-DSG). Anti-α-galactosidase (β-gal) siRNA encapsulated in DSPC:Cholesterol:DODMA:PEG-DMG / PEG-DSG SNALP by this method resulted in ≧90% encapsulation (Ribogreen Assay) and ˜120 nm particle size (Malvern sizer). The preparations had the following characteristics:

4 ml prep: anti-B-gal siRNA in DSPC:Chol:DODMA:PEG-DMG liposomes

Initial mix=94% encapsulation Post dilution mix=98% encapsulation Post incubation mix=97% encapsulation Post overnight dialysis=96% encapsulation Particle size=109.7 nm Polydispersity=0.14

8 ml prep: anti-B-gal siRNA in DSPC:Chol:DODMA:PEG-DMG liposomes Post dilution & incubated mix=89% Post overnight dialysis=91% Particle siz...

example 2

Downregulation of Intracellular Expression in Cells by Delivering In Vitro an SNALP Formulation Encapsulating siRNA

This example demonstrates downregulation of β-Gal expression in CT26.CL25 cells delivered in vitro DSPC:Cholesterol:DODMA:PEG-DMG liposomes encapsulating anti-β-Gal siRNA. The results are depicted in FIG. 1.

In vitro delivery of 0.2 μg Oligofectamine-encapsulated anti-β-Gal siRNA decreased β-Gal activity by about 60% in comparison to unexposed control cells. Encapsulating 1.5 μg anti-β-Gal siRNA in DSPC:Cholesterol:DODMA:PEG-DMG liposomes decreased β-Gal activity by about 30% in comparison to unexposed control cells.

example 3

Assays for Serum Stability

Lipid / therapeutic nucleic acid particles formulated according to the above noted techniques can be assayed for serum stability by a variety of methods.

For instance, in a typical DNase 1 digestion, 1 μg of DNA encapsulated in the particle of interest is incubated in a total volume of 100 μL of 5 mM HEPES, 150 mM NaCl, 10.0 mM MgCl2 pH 7.4. DNase treated samples are treated with either 100 or 10 U of DNase I (Gibco-BRL). 1.0% Triton X-100 can be added in control experiments to ensure that lipid formulations are not directly inactivating the enzyme. Samples are incubated at 37° C. for 30 min after which time the DNA is isolated by addition of 500 μL of DNAZOL followed by 1.0 mL of ethanol. The samples are centrifuged for 30 min at 15,000 rpm in a tabletop microfuge. The supernatant is decanted and the resulting DNA pellet is washed twice with 80% ethanol and dried. This DNA is resuspended in 30 μL of TE buffer. 20 μL of this sample is loaded on a 1.0% agar...

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Abstract

The present invention provides compositions and methods for silencing gene expression by delivering nucleic acid-lipid particles comprising a siRNA molecule to a cell.

Description

FIELD OF THE INVENTION The present invention relates to compositions and methods for the therapeutic delivery of a nucleic acid by delivering a serum-stable lipid delivery vehicle encapsulating the nucleic acid to provide efficient RNA interference (RNAi) in a cell or mammal. More particularly, the present invention is directed to using a small interfering RNA (siRNA) encapsulated in a serum-stable lipid particle having a small diameter suitable for systemic delivery. BACKGROUND OF THE INVENTION RNA interference (RNAi) is an evolutionarily conserved, sequence specific mechanism triggered by double stranded RNA (dsRNA) that induces degradation of complementary target single stranded mRNA and “silencing” of the corresponding translated sequences (McManus and Sharp, Nature Rev. Genet. 3:737 (2002)). RNAi functions by enzymatic cleavage of longer dsRNA strands into biologically active “short-interfering RNA” (siRNA) sequences of about 21-23 nucleotides in length (Elbashir, et al., Gen...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A61K9/127A61K48/00C12N15/113C12N15/88
CPCA61K9/1272A61P1/16A61P31/14A61P31/20A61P3/06A61K47/50A61K9/127A61K48/00
Inventor MACLACHLAN, IANAMBEGIA, ELLEN GRACEHEYES, JAMES
Owner PROTIVA BIOTHERAPEUTICS
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