Inex Pharmaceuticals Corporation

The University of British Columbia

Peter Scherrer

Pieter Rutter Cullis

Michael J Hope

Steven Michial Ansell

Sean C Semple

Yuan-Peng Zhang

Sandra K Klimuk

Troy Harasym

Dan Debeyer

Marcel B Bally

Jeffery J Wheeler

Dorothy L Reimer

Thomas D Madden

Kim F Wong

Norbert Maurer

Jeffery Jerome Wheeler

Phalgun Joshi

Ljiljana Kojic

Tao Chen

Peter Cullis

David Tirrell

Jung Soo Kim

Deirdre McIntosh

Nancy Dos Santos

Olga Ludkovski

Edward George Saravolac

Yuehua He

Methods for the preparation of a lipid-nucleic acid composition are provided. According to the methods, a mixture of lipids containing a protonatable or deprotonatable lipid, for example an amino lipid and a lipid such as a PEG- or Polyamide oligomer-modified lipid is combined with a buffered aqueous solution of a charged therapeutic agent, for example polyanionic nucleic acids, to produce particles in which the therapeutic agent is encapsulated in a lipid vesicle. Surface charges on the lipid particles are at least partially neutralized to provide surface-neutralized lipid-encapsulated compositions of the therapeutic agents. The method permits the preparation of compositions with high ratios of therapeutic agent to lipid and with encapsulation efficiencies in excess of 50%.

Lipid-encapsulated polyanionic nucleic acid

Novel lipid-nucleic acid particulate complexes which are useful for in vitro or in vivo gene transfer are described. The particles can be formed using either detergent dialysis methods or methods which utilize organic solvents. Upon removal of a solubilizing component (i.e., detergent or an organic solvent) the lipid-nucleic acid complexes form particles wherein the nucleic acid is serum-stable and is protected from degradation. The particles thus formed have access to extravascular sites and target cell populations and are suitable for the therapeutic delivery of nucleic acids.

Lipid-nucleic acid particles prepared via a hydrophobic lipid-nucleic acid complex intermediate and use for gene transfer

The present invention relates generally to the amphiphilic polyelectrolyte, poly(2-ethylacrylic acid) and covalently bonded lipids to generate Lipo-PEAA. These Lipo-PEAA are then used to make pH-sensitive liposomes which become unstable, permeable or fusogenic with certain pH changes. In addition, this invention generally describes methods for delivering therapeutic compounds and drugs to target cells by administering to a host the pH-sensitive liposomes of the present invention.

Polyanionic polymers which enhance fusogenicity

Lipidic compositions with superior characteristics for in vivo delivery of oligodeoxynucleotides (ODN) can easily and efficiently be made in the form of small multilamellar vesicles. The compositions contain a population of nucleic acid-containing lipid vesicles in a liquid carrier, and at least a portion of the lipid vesicles are small multilamellar vesicles. The small multilamellar vesicles are made from a lipid component including 20-30 mol % of an ionizable amino lipid such as DODAP, and a steric barrier lipid such as PEG-CerC ; and an oligodeoxynucleotide contained in the lumen or interlamellar spaces of the small multilamellar vesicles. The ODN and lipid components are preferably present in the small multilamellar vesicles in a mole ratio of from 0.15 to 0.25. The compositions of the invention can be made by preparing two solutions: a lipid mixture with 20-30 mol % of the ionizable amino lipid, the steric barrier lipid and additional lipid components selected from among neutral lipids and sterols in an ethanolic solvent; and a solution of oligodeoxynucleotide in an aqueous solvent having a pH at which the ionizable amino lipid is positively charged. The lipid mixture is added to the solution of oligodeoxynucleotide to form a mixture containing lipid vesicles which is then massed through a filter to produce sized lipid vesicles in a solution containing ethanol. The ethanol is then removed, for example by dialysis. Then, the pH of the solution surrounding the sized lipid vesicles is increased to reduce the net positive charge on the exterior of the sized lipid vesicles.

Composition containing small multilamellar oligodeoxynucleotide-containing lipid vesicles

The present invention relates to lipid-based formulations for nucleic acid delivery to cells, methods for the preparation of such formulations and, in particular, to lipid encapsulated plasmids. The compositions are safe and practical for clinical use. In addition, the present invention provides methods for introducing nucleic acids into cells and for inhibiting tumor growth in cells using such lipid-nucleic acid formulations.

Methods for encapsulating nucleic acids in lipid bilayers

Lipid-therapeutic agent particles are prepared containing a charged therapeutic agent encapsulated in lipid portion containing at least two lipid components including a protonatable or deprotonatable lipid such as an amino lipid and a lipid that prevents particle aggregation during lipid-therapeutic agent particle formation such as a PEG-modified or polyamide oligomer-modified lipid. Other lipid components may also be present and these include a neutral lipid such as DSPC, POPC, DOPE or SM, and a sterol such as Chol. The therapeutic agent is encapsulated by combining a mixture of the lipids with a buffered aqueous solution of a charged therapeutic agent to form an intermediate mixture containing lipid-encapsulated therapeutic agent particles, and changing the pH of the intermediate mixture to neutralize at least some surface charges on the particles. The method permits high ratios of therapeutic agent to lipid and encapsulation efficiencies in excess of 50%. The method is particularly useful for preparing lipid-encapsulated nucleic acids such as an antisense polyanionic nucleic acid having exclusively phosphodiester linkages. The encapsulated nucleic acid can be contacted with a cell to introduce the nucleic acid into the cell such as for treatment or prevention of a disease characterized by aberrant expression of a gene. A pharmaceutical composition may be prepared containing the lipid-encapsulated therapeutic agent particles and a carrier.

Charged therapeutic agents encapsulated in lipid particles containing four lipid components

This invention relates to a novel Solvent Extraction and Direct Hydration (SEDH) method for preparing lipid-nucleic acid particles which are useful for the introduction of nucleic acids (e.g., plasmid DNA, antisense molecules, ribozymes, etc.) into cells. The lipid-nucleic acid particles prepared using the methods of the present invention have enhanced circulation characteristics and serum stability and, thus, they are extremely effective as nucleic acid delivery vehicles.

Preparation of lipid-nucleic acid particles using a solvent extraction

Lipid-nucleic acid particles prepared via a hydrophobic lipid-nucleic

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Peter Scherrer