Gene expression and delivery systems and uses
OwnerJeff Nordstrom, Bruce Freimark +1inventors
InventorsJeff Nordstrom, Bruce Freimark, Deepa Deshpande
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Abstract
Plasmid expression systems for delivery of DNA coding sequences to a mammal are described which provide expression of multiple coding sequences from a single plasmid. Also described are particular lipid/DNA delivery systems having advantageous characteristics of size, charge ratio, and proportion of supercoiled DNA, and methods of preparing and using such delivery systems for treatment or as immunization adjuvants.
Claims44
A plasmid for expression of recombinant eucaryotic genes comprising:
- a first transcription unit comprising a first transcriptional control sequence transcriptionally linked with a first 5′-untranslated region, a first synthetic intron, a first coding sequence, and a first synthetic 3′-untranslated region/poly(A) signal, wherein said first synthetic intron is between said control sequence and said first coding sequence;
- and
- a second transcription unit comprising a second transcriptional control sequence transcriptionally linked with a second 5′-untranslated region, a second synthetic intron, a second coding sequence, and a second synthetic 3′-untranslated region/poly(A) signal, wherein said second synthetic intron is between said control sequence and said second coding sequence.
A plasmid for expression of recombinant eucaryotic genes, comprising an intron having variable splicing, a first coding sequence, and a second coding sequence.
A plasmid for expression of recombinant eucaryotic genes comprising:
- a transcriptional control sequence transcriptionally linked with a first coding sequence, an IRES sequence, a second coding sequence, and a 3′-untranslated region/poly(A) signal, wherein said IRES sequence is between said first coding sequence and said second coding sequence;
- and
- an intron between said promoter and said first coding sequence.
A DNA sequence coding for human IL-12 subunit, comprising a synthetic nucleotide sequence having less than 50% identity to a natural human IL-12 subunit coding sequence.
A composition for delivery of a DNA molecule in a mammal, comprising
- a cationic lipid with a neutral co-lipid, prepared as a liposome having an extrusion size of about 800 nanometers;
- and
- a quantity of DNA comprising a coding sequence.
A composition for delivery of a DNA molecule in a mammal, comprising
- a cationic lipid with a neutral co-lipid;
- and
- a quantity of DNA comprising a coding sequence,
- wherein said cationic lipid and said DNA are present in a negative to positive charge ratio of about 1:3.
A method for preparing a composition for delivery of a DNA to a mammal, comprising the steps of:
- a. preparing a DNA comprising a coding sequence;
- b. preparing liposomes having an extrusion size of about 800 nm, wherein said liposomes comprise a cationic lipid and a neutral co-lipid;
- and
- c. combining said liposomes with said DNA in amounts such that said cationic lipid and said DNA are present in a negative to positive charge ratio of about 1:3.
A method of treatment of a mammalian condition or disease, comprising administering to a mammal suffering from said condition or disease an amount of a composition for delivery of a DNA molecule in a mammal,
- wherein said DNA comprises a coding sequence encoding a therapeutic molecule or a subunit thereof, and
- wherein said composition comprises a cationic lipid, a neutral co-lipid, and said DNA, and has a negative to positive charge ratio of about 1:3 for said cationic lipid and said DNA.
A vaccine adjuvant comprising a cationic lipid, a neutral co-lipid, and DNA,
- wherein said DNA comprises a sequence encoding the p40 subunit of IL-12 and the p35 subunit of IL-12, and
- wherein said cationic lipid and said DNA are present in a negative to positive charge ratio of about 1:3.
A method of enhancing the response of a mammal to a vaccine, comprising the step of administering to said mammal a vaccine and an adjuvant,
- wherein said adjuvant comprises a cationic lipid, a neutral co-lipid, and DNA, said DNA comprising a sequence encoding the p40 subunit of IL-12 and the p35 subunit of IL-12, and
- wherein said cationic lipid and said DNA are present in a negative to positive charge ratio of about 1:3.
Dependencies are inferred from the claim text.