WO2014160196A2 - Sonochemical induction of abca1 expression and compositions therefor - Google Patents
Sonochemical induction of abca1 expression and compositions therefor Download PDFInfo
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- WO2014160196A2 WO2014160196A2 PCT/US2014/026018 US2014026018W WO2014160196A2 WO 2014160196 A2 WO2014160196 A2 WO 2014160196A2 US 2014026018 W US2014026018 W US 2014026018W WO 2014160196 A2 WO2014160196 A2 WO 2014160196A2
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Classifications
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- A61K48/0008—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition
- A61K48/0025—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid
- A61K48/0033—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid the non-active part being non-polymeric
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- C—CHEMISTRY; METALLURGY
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
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- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
Definitions
- This invention relates to compositions comprising sonochemically-active microspheres and a plasmid encoding ATP-binding cassette transporter Al, and methods of inducing ATP- binding cassette transporter Al expression in vivo.
- High-density lipoprotein is the largest of the five major groups of lipoproteins.
- lipoproteins include chylomicrons, very low-density lipoprotein (VLDL), intermediate- density lipoprotein (IDL), and low-density lipoprotein (LDL).
- VLDL very low-density lipoprotein
- IDL intermediate- density lipoprotein
- LDL low-density lipoprotein
- HDL and other lipoproteins enable cholesterol and other lipids (e.g., triglycerides) to be transported within the bloodstream, despite their hydrophobic nature. About thirty percent of blood cholesterol is carried by HDL in healthy adults.
- ATP-binding cassette transporter Al (ABCA1) is an integral cell membrane protein that exports excess cholesterol from cells in conjunction with phospholipid that is necessary for the initial lipidation of ApoAl to form nascent high density lipoprotein cholesterol (HDL-c).
- LXR liver X factor
- HDL-c is formed in the liver and the intestines by the lipidation of apolipoprotein Al (apoAl) mediated by the ABCA1 transporter protein.
- apoAl apolipoprotein Al
- Numerous studies of cultured cells, human HDL deficiencies, and animal models have shown that ABCA1 is a major determinant of plasma HDL levels and a potent atheroprotective factor.
- the role of ABCA1 in the liver for the formation of plasma HDL is well established, but it has been unclear whether stimulation of ABCA1 production will enhance lipidation and secretion of nascent HDL thereby resulting in an increase in plasma HDL-c levels.
- HDL The metabolism of HDL is complex and several factors contribute to the transport of cholesterol from arteries to the liver for excretion or re-use (reverse cholesterol transport).
- Some key players in reverse cholesterol transport include ABCA1, ABCG1, apoAl, apoE, LXR, Niemann Pick proteins 1 and 2 (NPC1 and NPC2) sterol regulating element binding protein (SREBP), CD36, acyl cholesterol acyl transferase (ACAT) and scavenger receptor Al (SRA1).
- SREBP sterol regulating element binding protein
- ACAT acyl cholesterol acyl transferase
- SRA1 scavenger receptor Al
- Many of these proteins have been considered as drug targets for enhancing blood HDL-c levels; however, the effects of targeting, enhancing the activity of, or otherwise interfering with the normal activity and expression of any of these individual proteins is unpredictable.
- High cholesterol also commonly is treated by inhibiting production of cholesterol in the liver (e.g., by inhibiting HMG-CoA reduct
- Plasma HDL-c and LDL-c levels are routinely measured as indicators of systemic atherosclerosis resulting in arterial blockage.
- HDL can remove cholesterol from within arteries and transport the cholesterol back to the liver for excretion or re -utilization.
- Individuals with higher levels of HDL-c have a reduced tendency for cardiovascular diseases.
- Low HDL-c cholesterol levels (less than about 40 mg/dL or about 1 mmol/L) are associated with increased risk of heart disease.
- Tangier disease also known as "familial alpha-lipoprotein deficiency”
- a rare inherited disorder, mutations in chromosome 9q31 lead to an inactive form of ABCA1.
- the inactive ABCA1 leads to severely depressed levels of HDL in the blood.
- compositions useful for transfecting cells e.g., liver cells
- 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.
- the sonochemically-active microspheres comprise, consist essentially of, or consist of gas bubbles (e.g., a fluorocarbon gas, such as octafluoropropane or perfluorohexane) encapsulated within protein-containing or lipid-containing shells (e.g., human serum albumin shells).
- gas bubbles e.g., a fluorocarbon gas, such as octafluoropropane or perfluorohexane
- the microspheres are disruptable by exposure to ultrasonic energy (sonication) to release the encapsulated gas.
- Embodiment 1 comprises a composition useful for transfecting cells comprising a mixture of a plasmid vector encoding an active form of ATP-binding cassette transporter Al (ABCA1) and sonochemically-active microspheres in a pharmaceutically acceptable aqueous carrier.
- 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.
- 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.
- Embodiment 2 comprises the composition of embodiment 1 wherein the microspheres have an average particle size in the range of about 0.5 to about 20 micrometers.
- Embodiment 3 comprises the composition of embodiment 1 or embodiment 2 wherein the gas comprises a fluorocarbon gas.
- Embodiment 4 comprises the composition of any one of embodiments 1 to 3 wherein the shells comprise human serum albumin.
- Embodiment 5 comprises the composition of any one of embodiments 1 to 4 wherein the active form of ABCA1 has the amino acid sequence of SEQ ID NO: 1.
- Embodiment 6 comprises the composition of any one of embodiments 1 to 5 wherein the open reading frame has the nucleotide sequence of SEQ ID NO: 2.
- Embodiment 7 comprises the composition of any one of embodiments 1 to 6 wherein the at least one sequence adapted to promote expression of the open reading frame comprises a cytomegalovirus promoter.
- Embodiment 8 comprises the composition of any one of embodiments 1 to 7 wherein the plasmid is present in the composition at a concentration in the range of about 0.5 to about 50 mg/mL.
- Embodiment 9 comprises the composition of any one of embodiments 1 to 8 wherein the microspheres are present in the composition at a concentration in the range of about 10 8 to about K) microspheres per milliliter.
- Embodiment 10 comprises the composition of any one of embodiments 1 to 9 wherein the aqueous carrier comprises physiological saline, optionally buffered at physiological pH.
- Embodiment 11 comprises the composition of any one of embodiments 1 to 10 further comprising 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 ABCAl involved in lipid metabolism of transport, and (c) a plasmid encoding an siR A that targets a protein involved in lipid metabolism or transport.
- Embodiment 12 comprises the composition of any one of embodiments 1 to 11 wherein the plasmid encoding ABCAl also encodes at least one material selected from the group consisting of (a) a protein other than ABCAl involved in lipid metabolism of transport, and (b) an siRNA that targets a protein involved in lipid metabolism or transport.
- Embodiment 13 comprises a composition useful for transfecting cells comprising a mixture of about 0.5 to about 50 mg/mL of a plasmid vector encoding an active form of ATP- binding cassette transporter Al (ABCAl) and about 10 8 to about 10 & microspheres per milliliter of sonochemically-active microspheres in a pharmaceutically acceptable aqueous carrier;
- a composition useful for transfecting cells comprising a mixture of about 0.5 to about 50 mg/mL of a plasmid vector encoding an active form of ATP- binding cassette transporter Al (ABCAl) and about 10 8 to about 10 & microspheres per milliliter of sonochemically-active microspheres in a pharmaceutically acceptable aqueous carrier;
- vector comprises an expressible open reading frame encoding the active form of
- the sonochemically-active microspheres comprise
- Embodiment 14 comprises a method of transfecting a tissue in vivo to express an active form of ABCA1 in cells of the tissue, the method comprising the steps of (a) intravenously coadministering to a subject a plasmid vector encoding the active form of ATP-binding cassette transporter Al (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 a
- Embodiment 15 comprises the method of embodiment 14 wherein the microspheres have an average particle size in the range of about 0.5 to about 20 micrometers.
- Embodiment 16 comprises the method of embodiment 14 or embodiment 15 wherein the gas comprises a fluorocarbon gas.
- Embodiment 17 comprises the method of any one of embodiments 14 to 16 wherein the shells comprise human serum albumin.
- Embodiment 18 comprises the method of any one of embodiments 14 to 17 wherein the active form of ABCA1 has the amino acid sequence of SEQ ID NO: 1.
- Embodiment 19 comprises the method of any one of embodiments 14 to 18 wherein the at least one sequence adapted to promote expression of the open reading frame comprises a cytomegalovirus promoter.
- Embodiment 20 comprises the method of any one of embodiments 14 to 19 wherein the plasmid is administered in an aqueous carrier at a concentration in the range of about 0.5 to about 50 mg/mL.
- Embodiment 21 comprises the method of any one of embodiments 14 to 20 wherein the microspheres are administered in an aqueous carrier at a concentration in the range of about 10 8 to about 10 9 microspheres per milliliter.
- Embodiment 22 comprises the method of any one of embodiments 14 to 21 wherein the plasmid and the microspheres are administered as a mixture in one aqueous carrier.
- Embodiment 23 comprises the method of any one of embodiments 14 to 21 wherein the plasmid and the microspheres are administered in separate aqueous carriers.
- Embodiment 24 comprises the method of any one of embodiments 14 to 23 wherein an additional biologically active agent is co-administered along with the plasmid and microspheres.
- Embodiment 25 comprises the method of embodiment 24 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 siR A that targets a protein involved in lipid metabolism or transport.
- 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 siR A that targets a protein involved in lipid metabolism or transport.
- Embodiment 26 comprises the method of any one of embodiments 14 to 25 wherein the tissue comprises liver tissue, intestinal parenchymal tissue, or a combination thereof.
- Embodiment 27 comprises use of a composition of any one of embodiments 1 to 13 to enhance high density lipoprotein cholesterol in the blood of a subject (e.g., to treat
- Embodiment 28 comprises use of a composition of any one of embodiments 1 to 13 for the preparation of a medicament to enhance high density lipoprotein cholesterol in the blood of a subject (e.g., to treat atherosclerosis).
- FIG. 1 provides the amino acid sequence of human ABCAl (SEQ ID NO: 1) utilized in the examples herein.
- FIG. 2 provides the open reading frame nucleotide sequence of human ABCAl (SEQ ID NO: 1) utilized in the examples herein.
- FIG. 3 illustrates the nucleotide sequence of the plasmid (SEQ ID NO: 3) utilized in the examples herein.
- FIG. 4 provides a schematic illustration of the ABCAl plasmid utilized in the examples described herein.
- FIG. 5 provides a graph of blood HDL-c in rats following sonochemical treatment with compositions comprising OPTISON microspheres and plasmids encoding ABCAl, apoAl, or a combination thereof.
- compositions useful for transfecting cells to express ABCAl comprise a pharmaceutically acceptable aqueous carrier containing sonochemically-active microspheres together with a plasmid DNA construct encoding an active form of ABCAl and at least one promoter for the expression thereof.
- the sonochemically- active microspheres comprise, consist essentially of, or consist of gas bubbles encapsulated within protein-containing or lipid-containing shells. The microspheres are disruptable by sonication (i.e., exposure to ultrasonic acoustic energy), releasing the encapsulated gas.
- the present invention provides a method of inducing ABCAl expression in cells in vivo, and a method enhancing high density lipoprotein cholesterol in the blood.
- These methods comprise intravenously co-administering an ABCAl plasmid and sonochemically active microspheres as described herein to a subject, ultrasonically imaging the a target tissue (e.g., the liver or intestinal parenchymal tissue) of the subject to detect when the microspheres are circulating through the vasculature of the tissue.
- the imaging typically is performed by external application of ultrasonic acoustic energy at a mechanical index (MI; which is defined as the peak negative acoustic pressure divided by the square of the imaginh frequancy) less than about 0.4 MI.
- MI mechanical index
- pulses of ultrasonic acoustic energy are applied to the tissue at an acoustic energy level higher than the energy level needed for imaging (typically greater than 1 MI, preferably greater than 1.3 MI, and up to about 2 MI), and of sufficiently high acoustic energy to disrupt the shells of the microspheres and release the gas bubbles encapsulated by the shells.
- the pulses preferably are applied at an acoustic frequency of about 1 to about 7 MHz.
- the acoustic energy of the pulses and the release of gas bubbles in the tissue temporarily increases the porosity of the tissue cell membranes (a process referred to herein as "sonoporation").
- Sonoporation of a tissue such as the liver comprises supplying ultrasonic acoustic energy pulses to the tissue with the ultrasound imaging probe.
- the pulses are applied while ultrasonically imaging the tissue so that the pulses are applied primarily when the microspheres (and thus also the co-administered plasmid) are present.
- the pulses typically are applied at a rate of about 6 to about 8 pulses per minute for a total of about 5 to about 20 pulses.
- the pulse duration typically is in the range of about 500 to about 2000 milliseconds per pulse.
- the acoustic energy pulses disrupt the microspheres and release the encapsulated gas.
- co-administration refers to administering two or more materials to the same individual during the same therapeutic session. Such co-administration can involve administering material separately, or together within the same composition. The co-administration can be simultaneous or can be temporally separated. In addition, sites of co-administration can be in the same location or different locations.
- plasmid and grammatical variations thereof refers to small circular DNA that is physically separate from, and can replicate independently of, chromosomal DNA within a cell (i.e., in an episome), and which commonly are found as small (e.g., about one thousand to about one million base pairs) circular, double-stranded DNA molecules in bacteria. Artificial plasmids are used as vectors in molecular cloning, serving to drive the replication of recombinant DNA sequences within episomes in the host cells without altering the chromosomal DNA of the host cells.
- episomally transfected and grammatical variations thereof refer to non-insertional (non-integrating) transfection with exogenous episomal nucleic acid, such as DNA, siRNA, RNA, or mRNA (e.g. a plasmid or other episomal vector) to produce a cell with unaltered chromosomal DNA, in which the a polypeptide encoded by the episomal DNA is expressed within the target cells (e.g., liver cells) without genomic integration of the exogenous DNA.
- exogenous episomal nucleic acid such as DNA, siRNA, RNA, or mRNA (e.g. a plasmid or other episomal vector) to produce a cell with unaltered chromosomal DNA, in which the a polypeptide encoded by the episomal DNA is expressed within the target cells (e.g., liver cells) without genomic integration of the exogenous DNA.
- target cells e.g., liver cells
- the term "episome” and grammatical variations thereof refers to closed circular DNA molecules that are replicated in the nucleus, and is intended to encompass exogenous plasmids introduced into host cells such as liver cells.
- the plasmid encodes the active form of ABCA1 and also encodes regulatory elements (e.g., a promoter) to facilitate episomal expression of the ABCA1 protein.
- active form of ABCA1 refers to the ABCA1 protein of SEQ ID NO: 1 and variations thereof comprising conservative substitutions in SEQ ID NO: 1 and sharing at least about 95 percent sequence identity (e.g., at least about 95, 96, 97, 98, or 99 % sequence identity) with SEQ ID NO: 1 and retaining the lysine residues at positions 939 and 1952 of SEQ ID NO: 1.
- Percentage values stated herein are on a weight- weight basis (i.e., "weight percent” or “percent by weight”) when referring to a concentration, and on a number basis when referring to a quantity or countable number of items, as the context will make evident and unless otherwise specified.
- a "therapeutically effective dosage” is an amount (e.g., a total of about 0.5 to about 10 mL of a single composition or co-administered compositions comprising about 0.5 to about 50 mg/mL of the ABCA1 plasmid and about 10 8 to about 10 9 of the microspheres per milliliter) such that when administered in conjunction with sonication of the liver, the plasmids transfect cells of the tissue to express the nucleic acid, subsequently resulting in enhanced HDL-c level in the blood, or other effects targeted by the therapy.
- the dosage and number of doses e.g.
- plasmids administered to a subject will vary depending upon a variety of factors, including the route of administration, patient conditions and characteristics (sex, age, body weight, health, size), extent of symptoms, concurrent treatments, frequency of treatment and the effect desired, the concentration of plasmids in the composition, and the like.
- the dosage does not exceed about 5 mL of composition over a 10 minute period.
- Suitable safe dosages of ultrasonic contrast agents provide a useful guideline for use in the methods described herein. Such safe dosages are well known in the art and are documented in literature from ultrasonic contrast agent manufacturers.
- Sonochemicaiiy-active microspheres suitable for use in the compositions and methods described herein include any protein-based or lipid-based gas-filled microspheres (also known as “mierobubbles”) that can be used as ultrasonic imaging contrast agents.
- the microspheres comprise a physiologically acceptable gas (e.g., a non-toxic gas such as nitrogen, air, oxygen, argon, or a fluorocarbon such as octafluoropropane (also known as "perflutren”) or
- the shel l of the microspheres can comprise a protein (e.g., human serum albumin), a lipid material (e.g., phospholipids, phosphocholine lipids, and polyethoxylates thereof), or a combination thereof. Some lipid-based microspheres may also contain galactose in the shell. Protein-containing microspheres can include relatively small amounts (e.g., less than about 1 percent) of fatty acids (e.g., caprylic acid), amino acids or amino acid derivatives (e.g., N-acyltryptophan), or other formulation aids.
- the microspheres typically have a mean particle size (i.e., effective mean diameter) in the range of about 1 to about 10 micrometers (preferably about 1 to about 5 micrometers). Preferably, at least about 95 % of the
- microspheres have a diameter of less than about 10 micrometers.
- the microspheres preferably are present in the composition at a concentration in the range of about 10 s to about K) of the microspheres per milliliter.
- the compositions described herein preferably are prepared by simple mixing of a microsphere suspension (e.g., as supplied by the manufacturer) with a solution of the plasmid or plasmids that are to be co-administered.
- the pharmaceutically active aqueous carrier of the compositions comprises water (e.g., deionized, pyrogen-free water), and preferably includes one or more salts (e.g., sodium chloride, phosphate, citrate, and the like).
- the aqueous carrier comprises physiological saline (about 0.9 percent NaCl), phosphate buffered saline, and the like.
- the carrier optionally can include other soluble materials (e.g., dextrose), preservatives, and the like, to provide a solution that is generally sterile, safe, isotonic and compatible with blood.
- the compositions have a physiological pH (e.g., about pH 6.4 to 7.5).
- microsphere formulations useful in the compositions and methods described herein include the OPTSON brand microspheres (available from GE Healthcare),
- compositions comprise human serum albumen encapsulated octafluoropropane microspheres, such as
- OPTISON microspheres According the manufacturer, OPTISON microsphere suspensions from GE Healthcare comprise about 5 x 10 8 to about 8 x 10 8 microspheres per mL of suspension.
- the microspheres comprise perflutren (octafluoropropane) gas bubbles encapsulated within shells of human serum albumin.
- the microspheres have a mean particle diameter of about 3 to about 4,5 ⁇ with about 95% of the microspheres having a diameter of less than about 10 ⁇ .
- the microspheres are suspended in a physiological saline solution (about 0,9 percent by weight NaCl in water).
- the compositions also can include less than about 1 percent eaprylic acid, and less than about 1 percent N-acyltryptophan.
- Each milliliter of OPTISON microspheres reportedly comprises about 10 mg of human serum albumin, about 0.2 to 0.3 mg of perflutren, about 0.2 mg N- acetyltryptophan, and about 0.12 mg eaprylic acid in 0.9% aqueous sodium chloride at a pH of about 6.4-7.4.
- the headspace of the vial containing the suspension is filled w r ith perflutren gas.
- the manufacturer recommends that the injection rate should not exceed about 1 mL per second (maximum total dose should not exceed about 5 mL in any 10 minute period, and maximum total dose should not exceed about 8.7 mL in any one patient study).
- IMA VIST is an injectable suspension developed by Alliance Pharmaceutical.
- microspheres reportedly comprise perflexane (perfluorohexane) microbubbles encapsulated in a lipid-based shell comprising l,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), hydroxyethyl starch and poloxomer (a nonionic triblock copolymer composed of a central hydrophobic chain of polyoxypropylene flanked by two hydrophilic chains of polyoxy ethylene).
- DMPC l,2-dimyristoyl-sn-glycero-3-phosphocholine
- poloxomer a nonionic triblock copolymer composed of a central hydrophobic chain of polyoxypropylene flanked by two hydrophilic chains of polyoxy ethylene.
- DEFINITY perflutren lipid microspheres are provided as an injectable suspension.
- the DEFINITY material is supplied as components that upon activation yield perflutren lipid microspheres.
- the material is supplied in a vial containing a clear, colorless, sterile, non-pyrogenic, hypertonic liquid, which upon activation with the aid of a VIALMIX brand activator, provides a homogeneous, opaque, milky white injectable suspension of perflutren lipid microspheres.
- the suspension of activated DEFINITY microspheres is administered by intravenous injection.
- the perflutren lipid microspheres are composed of octafluoropropane encapsulated in an outer lipid shell consisting of (R) ⁇ hexadecanoic acid- 1 -[(phosphonoxy)methyl]- 1 ,2-ethanediy 1 ester, monosodium salt (abbreviated DPPA); (R)-4-hydroxy-N,N,N-trimethyllO-oxo-7-[(l-oxohexadecyl)oxy]-3,4,9- trioxa-4-phosphapentacosan-l-aminium- 4-oxide inner salt (abbreviated DPPC); and (R)-a-[6- hydroxy-6-oxido-9-[(l -oxohexadecyl)oxy]5,7, 11 -trioxa-2-aza-6-phosphahexacos- 1 -ylj- ⁇ - methoxypoly(ox-l ,2-e
- DPPA has a molecular weight of 670, empirical formula of CssHesOsPNa, and following structural formula:
- DPPC has a molecular weight of 734, empirical formula of C4oH 8 oN0 8 P, and following structural formula:
- MPEG5000 DPPE has an approximate molecular weight of 5750 represented by empirical formula C26 5 H 527 NOi 23 PNa, and the following structural formula:
- the DEFINITY component vial Prior to VIALMIX activation, the DEFINITY component vial reportedly contains 6.52 mg/mL octafluoropropane in the headspace. Each mL of the clear liquid reportedly contains 0.75 mg lipid blend (consisting of 0.045 mg DPPA, 0.401 mg DPPC, and 0.304 mg MPEG5000 DPPE), 103.5 mg propylene glycol, 126.2 mg glycerin, 2.34 mg sodium phosphate monobasic monohydrate, 2.16 mg sodium phosphate dibasic heptahydrate, and 4.87 mg sodium chloride in water (pH is 6.2-6.8).
- each mL of the milky white suspension reportedly contains a maximum of 1.2 x 10 10 perflutren lipid microspheres, and about 150 ⁇ . (1.1 mg/mL) octafluoropropane.
- the microsphere mean particle size is about 1.1 ⁇ to 3.3 ⁇ , with 98% of the microspheres having a diameter of less than 10 ⁇ .
- Plasmid design Plasmids generally comprise a strong viral promoter to drive the in vivo transcription and translation of the encoded gene (or complementary DNA, RNA, siRNA, or niDNA) of interest (which is present as an open reading frame). Intron A may can be included to improve m NA stability and hence increase protein expression. Plasmids also typically include a strong polyadenylation/transcriptional termination signal, such as bovine growth hormone or rabbit beta-globulin polyadenylation sequences.
- the plasmid provided the genetic material from which the protein of interest is expressed, optimizing vector design for maximal protein expression is desirable. For example, the codon usage can be adjusted to better conform to eukaryotic cells. Another factor to consider is the choice of promoter. Examples of promoters include the simian virus 40 (SV40) promoter, the Rous Sarcoma Vims (RSV) promoter, and the cytomegalovirus (CMV) promoter. In addition, expression rates can sometimes be improved by inclusion of enhancer sequences, adenovirus tripartite leader (TPL) sequences, or modifications to the polyadenylation and transcriptional termination sequences.
- SV40 simian virus 40
- RSV Rous Sarcoma Vims
- CMV cytomegalovirus
- expression rates can sometimes be improved by inclusion of enhancer sequences, adenovirus tripartite leader (TPL) sequences, or modifications to the polyadenylation and transcriptional termination sequences.
- Non-limiting examples of episomal plasmid vectors suitable for use as vectors for transfection of liver cells include SV40-based vectors, Epstein- Barr virus-based vectors, papilloma virus-based vectors, BK virus-based vectors, and the like, which are well known in the molecular genetics art.
- Non limiting examples of episomal vectors suitable for use as non-integrating vectors for transfection of eukaryotic cells include simian virus 40-based vectors, Epstein-Barr virus-based vectors, papilloma virus-based vectors, BK virus-based vectors, and the like, which are well known in the molecular genetics art.
- an additional biologically active agent is co-administered along with the plasmid and microspheres.
- additional biologically active agents include, for examples, a drug for treating a condition relating to lipid metabolism or transport, a plasmid encoding a protein other than ABCA1 involved in lipid metabolism of transport, and a plasmid encoding an siRNA that targets a protein involved in lipid metabolism or transport.
- the plasmid encoding the active ABCA1 can also encode an a protein other than ABCA1 involved in lipid metabolism of transport, or a siRNA that targets a protein involved in lipid metabolism or transport.
- Non-limiting examples of drugs for treating a condition relating to lipid metabolism or transport include HMG-CoA inhibitors, such as statins (e.g., atorvastatin, cerivastatin, fluvastatin, lovastatin, mevastatin, pitavastatin, pravastatin, rosuvastatin, simvastatin, and combinations thereof); bile acid inhibitors (e.g., cholestyramine, colestipol, colesevalam, and combinations thereof); fibric acid derivatives (e.g., clofibrate, gemfibrozil, fenofibrate) which may lower LDL-c and raise HDL-c; and niacin.
- statins e.g., atorvastatin, cerivastatin, fluvastatin, lovastatin, mevastatin, pitavastatin, pravastatin, rosuvastatin, simvastatin, and combinations thereof
- bile acid inhibitors
- proteins involved in lipid metabolism or transport include, e.g., ABCA1, ABCGl, apoAl, apoA2, apoE, LXR, NPCl, NPC2, SREBP, CD36, ACAT, SRAl, and HMG- CoA.
- Such enzymes or siRNA targeting such proteins may be encoded by a plasmid coadministered with the ABCA1 plasmid, or in some cases can be encoded the same plasmid as the ABCAl .
- Co-administration of a drug or a plasmid encoding a lipid metabolism/transport protein or siRNA can have the advantage of tailoring treatment to the specific condition suffered by the patient, by invoking multiple action sites, multiple metabolic targets, or both.
- the materials to be co-administered can be selected e.g., to HDL-c and lower LDL-c in patients where both types of cholesterol are outside the recommended levels, or to raise HDL-c and lower total triglycerides, etc..
- different tissues may be transfected in a given subject, e.g., to target optimal tissues that may be involved in the particular lipid metabolic pathways of interest. For example, one tissue may be transfected to express one protein or siRNA, while a different tissue of the same subject may be transfected to express another protein of siRNA.
- An apoAl expression plasmid was constructed by subcloning an 804 bp human apoAl
- PCR cDNA product into expression vector pMIR0125 (Minis Bio Corp, Madison, Wisconsin) containing human apoE hepatic control region (HCR), human ubiquitin C promoter and first intron.
- HCR hepatic control region
- pMIR0332-HCRUbC- h apoAl was sequenced and the resulting clone matched the reported human apoAl sequence.
- An ABCAl expression vector was purchased from Origene (ABCAl (NM 005502) Human cDNA ORF Clone, Cat. No.
- FIG. 1 provides the amino acid sequence of ABCAl (SEQ ID NO: 1) encoded in the open reading frame of the vector.
- FIG. 2 provides the nucleotide sequence of the ABCAl open reading frame (SEQ ID NO: 2) of the vector, while FIG. 3 provides the nucleotide sequence of the entire vector (SEQ ID NO: 3).
- FIG. 4 provides a schematic map of the ABCAl plasmid.
- the plasmids i.e., apoAl plasmid, ABCAl plasmid, or a combination of apoAl and ABCAl plasmids
- apoAl plasmid i.e., ABCAl plasmid, or a combination of apoAl and ABCAl plasmids
- Anesthetic Inhalable gas mixture of oxygen and 1.5-3.0% isoflurane.
- a warming bed maintained rat body temperature at 37°C.
- Tail vein injections were performed using a 26 GA 3/4-inch catheter.
- OPTISON microspheres About 1 mL of OPTISON microspheres was mixed with (1) apoAl DNA plasmid (approximately 8 mg in 1 mL), or (2) ABCAl plasmid (approximately 7.3 mg in 1 mL), or (3) a combination of apoAl and ABCAl plasmids at the same concentrations per mL as used for the individual plasmids. Plasmid volume and concentration ofDNA. All plasmids alone or in combination were mixed with OPTISON microspheres for about 15 to about 30 seconds prior to injection with a 3 mL injection syringe and then co-administered or co-infused. The infusion rate was manually performed at a timed rate of about 2 to 3 mL/minute and the total infusion duration was about 50 to about 70 seconds.
- a VIVID I brand imaging system (GE Healthcare Systems, Milwaukee, Wisconsin) equipped with a 3S ultrasound probe was utilized for all liver sonications. All acoustic energy settings remained within FDA guidelines outlined for diagnostic use (ALARA principle).
- the rat liver was continually visualized using external ultrasound to verify appearance of OPTISON mixture within the liver vasculature and parenchyma.
- Low mechanical index (MI) ultrasound acoustic energy was used for imaging (e.g., ⁇ 0.4 MI) whereas, higher MI (about 1.3 MI or greater) was used for therapy. No surgical abdominal incision was ever performed; external ultrasound was used to visualize the liver. The abdomen was shaved to eliminate surface hair.
- the ultrasound parameters consisted of an continuous low mechanical index (MI ⁇ 0.4) ultrasound exposure for "imaging” followed by a two-second “pulse” for therapeutic effect for a total of 10 “pulses.”
- a pulse was defined as a relatively higher (MI >1.3) burst of an ultrasound pulse.
- the "pulse” length was about 2 seconds with a pulse interval of about 8 seconds for a total of 10 pulses.
- FIG. 5 provides a graph of blood HDL-c levels of the treated rats, including the baseline level and levels for three days post-treatment.
- the selective hepatic transduction of ABCAl through sonoporation described herein resulted in enhanced blood HDL-c relative to baseline HDL-c concentration.
- a single sonochemical treatment with an ABCAl plasmid and sonochemically active microsphere composition in conjunction with sonication of the liver resulted in a 15% increase in HDL-c.
- a surprising finding in this study was that the ABCAl plasmid therapy resulted in superior increases in HDL-c relative to treatment with an apoAl plasmid or the combination of ABCAl and apoAl plasmids under the same sonication conditions.
Abstract
Description
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AU2014244014A AU2014244014B2 (en) | 2013-03-14 | 2014-03-13 | Sonochemical induction of ABCA1 expression and compositions therefor |
CA2902047A CA2902047A1 (en) | 2013-03-14 | 2014-03-13 | Sonochemical induction of abca1 expression and compositions therefor |
JP2016502027A JP6550039B2 (en) | 2013-03-14 | 2014-03-13 | Sonochemical induction of ABCA1 expression and composition therefor |
EP14772576.6A EP2970991B1 (en) | 2013-03-14 | 2014-03-13 | Sonochemical induction of abca1 expression and compositions therefor |
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US9393328B2 (en) | 2016-07-19 |
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