Sonochemical induction of ABCA1 expression and compositions therefor

• US 9,393,328 B2
• Filed: 06/30/2015
• Issued: 07/19/2016
• Est. Priority Date: 03/14/2013
• Status: Active Grant

• Current Assignee: SonoThera, Inc.
• Original Assignee: Sonogene, LLC
• Inventors: Coles, Eric, Davidson, Michael
• Primary Examiner(s): Long, Scott
• Application Number:
  US14/788,259
• Publication Number:
  US 20150343098A1
• Time in Patent Office: 385 Days
• US Class Current: 1/1
• CPC Class Codes: A61K 31/7088 Compounds having three or more nucleosides or nucleotides
• CPC Class Codes: A61K 31/7105 Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
• CPC Class Codes: A61K 31/713 Double-stranded nucleic acids or oligonucleotides
• CPC Class Codes: A61K 41/0028 Disruption, e.g. by heat or ultrasounds, sonophysical or sonochemical activation, e.g. thermosensitive or heat-sensitive liposomes, disruption of calculi with a medicinal preparation and ultrasounds
• CPC Class Codes: A61K 45/06 Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
• CPC Class Codes: A61K 48/00 Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
• CPC Class Codes: A61K 48/0033 the non-active part being non-polymeric
• CPC Class Codes: A61K 49/223 Microbubbles, hollow microspheres, free gas bubbles, gas microspheres
• CPC Class Codes: A61M 37/0092 using ultrasonic, sonic or infrasonic vibrations, e.g. phonophoresis
• CPC Class Codes: A61P 3/06 Antihyperlipidemics
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: C12N 15/00 Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor mutants or genetically engineered microorganisms, per se C12N1/00, C12N5/00, C12N7/00; new plants per se A01H; plant reproduction by tissue culture techniques A01H4/00; new animals per se A01K67/00; use of medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases, gene therapy A61K48/00
• CPC Class Codes: C12N 15/85 for animal cells

Abstract

The present invention provides methods for transfecting cells (e.g., liver cells) to express ABCA1 and increase HDL in the blood. The compositions described herein comprise a pharmaceutically acceptable aqueous carrier containing sonochemically-active microspheres together with a plasmid DNA construct encoding an active form of ABCA1 and at least one promoter for the expression thereof. Preferably, the sonochemically-active microspheres comprise, consist essentially of, or consist of gas bubbles (e.g., a fluorocarbon gas, such as octafluoropropane) encapsulated within protein-containing or lipid-containing shells (e.g., human serum albumin shells). The microspheres are disruptable by exposure to ultrasonic acoustic energy to release the encapsulated gas.

Claims

1. A method for increasing HDL-cholesterol levels in the blood of a subject, the method comprising the steps of:

(a) intravenously co-administering to the subject a plasmid vector encoding the active form of ATP-binding cassette transporter A1 (ABCA1), and sonochemically-active microspheres;

wherein the vector comprises an expressible open reading frame encoding the active form of ABCA1 and at least one sequence adapted to promote expression of the open reading frame in a mammalian cell; and

wherein the sonochemically-active microspheres comprise gas bubbles encapsulated within shells comprising a protein, a lipid, or a combination thereof, the microspheres being disruptable upon exposure to ultrasonic acoustic energy to release the encapsulated gas bubbles;

(b) ultrasonically imaging a tissue of the subject to be transfected by the plasmid while the plasmid and microspheres are circulating through the vasculature of the tissue and thereby detecting the presence of the microspheres in the vasculature of the tissue; and

(c) while the microspheres are present in the tissue, applying pulses of ultrasonic energy to the tissue at an acoustical energy level higher than a level required for imaging and at a sufficient energy level to disrupt the microspheres and release the gas bubbles therefrom, the pulses of ultrasonic energy and release of gas bubbles thereby temporarily increasing the porosity of cells in the tissue to facilitate entry of the plasmid into the cells;

wherein the plasmid transfects the cells and elicits production of an increased level of HDL-cholesterol in the blood of the subject compared to the HDL-cholesterol level in the blood of the subject prior to administering the plasmid.

2. The method of claim 1, wherein the microspheres have an average particle size in the range of about 0.5 to about 20 micrometers.

3. The method of claim 1, wherein the gas bubbles comprise a fluorocarbon gas.

4. The method of claim 1, wherein the shells comprise human serum albumin.

5. The method of claim 1, wherein the active form of ABCA1 has the amino acid sequence of SEQ ID NO:

1.

6. The method of claim 1, wherein the open reading frame has the nucleotide sequence of SEQ ID NO:

2.

7. The method of claim 1, wherein the at least one sequence adapted to promote expression of the open reading frame comprises a cytomegalovirus promoter.

8. The method of claim 1, wherein the plasmid is administered in an aqueous carrier at a concentration in the range of about 0.5 to about 50 milligrams per milliliter.

9. The method of claim 1, wherein the microspheres are administered in an aqueous carrier at a concentration in the range of about 10⁸ to about 10⁹ microspheres per milliliter.

10. The method of claim 1, wherein the plasmid and the microspheres are administered as a mixture in one aqueous carrier.

11. The method of claim 1, wherein the plasmid and the microspheres are administered in separate aqueous carriers.

12. The method of claim 1, wherein the plasmid and microspheres are administered in an aqueous carrier comprising physiological saline, optionally buffered at physiological pH.

13. The method of claim 1, wherein the plasmid encoding ABCA1 also encodes at least one material selected from the group consisting of (a) a protein other than ABCA1 involved in lipid metabolism of transport, and (b) an siRNA that targets a protein involved in lipid metabolism or transport.

14. The method of claim 1, wherein an additional biologically active agent is co-administered along with the plasmid and microspheres.

15. The method of claim 14, wherein the additional biologically active agent comprises at least one material selected from the group consisting of (a) a drug for treating a condition relating to lipid metabolism or transport, (b) a plasmid encoding a protein other than ABCA1 involved in lipid metabolism of transport, and (c) a plasmid encoding an siRNA that targets a protein involved in lipid metabolism or transport.

16. The method of claim 1, wherein the tissue comprises liver tissue, intestinal parenchymal tissue, or a combination thereof.

17. A method for increasing HDL-cholesterol levels in the blood of a subject, the method comprising the steps of:

(a) intravenously administering to the subject a composition comprising a mixture of a nucleic acid encoding an expressible open reading frame and sonochemically-active microspheres in a pharmaceutically acceptable aqueous carrier;

wherein the nucleic acid of the mixture consists of a plasmid vector, the expressible open reading frame consists of an expressible open reading frame encoding the active form of ATP-binding cassette transporter A1 (ABCA1), and the plasmid vector also includes at least one sequence adapted to promote expression of the open reading frame in a mammalian cell; and

wherein the sonochemically-active microspheres comprise gas bubbles encapsulated within shells comprising a protein, a lipid, or a combination thereof, the microspheres being disruptable upon exposure to ultrasonic acoustic energy to release the encapsulated gas bubbles;

(b) ultrasonically imaging a tissue of the subject to be transfected by the plasmid vector while the composition is circulating through the vasculature of the tissue and thereby detecting the presence of the microspheres in the vasculature of the tissue; and

(c) while the microspheres are present in the tissue, applying pulses of ultrasonic energy to the tissue at an acoustical energy level higher than a level required for imaging and at a sufficient energy level to disrupt the microspheres and release the gas bubbles therefrom, the pulses of ultrasonic energy and release of gas bubbles thereby temporarily increasing the porosity of cells in the tissue to facilitate entry of the plasmid into the cells;

wherein the tissue comprises liver tissue, intestinal parenchymal tissue, or a combination thereof;

the plasmid transfects the cells and elicits production of an increased level of HDL-cholesterol in the blood of the subject compared to the HDL-cholesterol level in the blood of the subject prior to administering the composition; and

the pulses are applied at a rate of about 6 to 8 pulses per minute, with pulse durations in the range of about 500 to about 2000 milliseconds per pulse, and at an acoustic energy mechanical index (MI) of about 1 to about 2 MI.

18. The method of claim 17, wherein the microspheres have an average particle size in the range of about 0.5 to about 20 micrometers.

19. The method of claim 17, wherein the gas bubbles comprise a fluorocarbon gas, and the shells comprise human serum albumin.

20. The method of claim 17, wherein the open reading frame encoding the active form of ABCA1 has the nucleotide sequence of SEQ ID NO:

2.

21. The method of claim 17, wherein the at least one sequence adapted to promote expression of the open reading frame comprises a cytomegalovirus promoter.

22. The method of claim 17, wherein the plasmid is administered in an aqueous carrier at a concentration in the range of about 0.5 to about 50 milligrams per milliliter, and the microspheres are administered in an aqueous carrier at a concentration in the range of about 10⁸ to about 10⁹ microspheres per milliliter.

23. The method of claim 17, wherein an additional biologically active agent is co-administered along with the plasmid and microspheres.

24. The method of claim 23, wherein the additional biologically active agent comprises at least one material selected from the group consisting of (a) a drug for treating a condition relating to lipid metabolism or transport, (b) a plasmid encoding a protein other than ABCA1 involved in lipid metabolism of transport, and (c) a plasmid encoding an siRNA that targets a protein involved in lipid metabolism or transport.