US20130142760A1 - Atherosclerosis inhibition via modulation of monocyte-macrophage phenotype using apo a-i milano gene transfer - Google Patents

Atherosclerosis inhibition via modulation of monocyte-macrophage phenotype using apo a-i milano gene transfer Download PDF

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US20130142760A1
US20130142760A1 US13/816,381 US201113816381A US2013142760A1 US 20130142760 A1 US20130142760 A1 US 20130142760A1 US 201113816381 A US201113816381 A US 201113816381A US 2013142760 A1 US2013142760 A1 US 2013142760A1
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macrophage
phenotype
milano
plasmid
inflammatory
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Prediman K. Shah
Behrooz Sharifi
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Cedars Sinai Medical Center
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—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 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/775—Apolipopeptides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]
    • 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
    • C12N2799/00—Uses of viruses
    • C12N2799/02—Uses of viruses as vector
    • C12N2799/021—Uses of viruses as vector for the expression of a heterologous nucleic acid
    • C12N2799/025—Uses of viruses as vector for the expression of a heterologous nucleic acid where the vector is derived from a parvovirus

Definitions

  • This invention relates to the treatment of vascular diseases, including atherosclerosis; and to monitoring the treatment of vascular diseases, including monitoring surface markers on macrophages.
  • Atherosclerosis is just one of several types of arteriosclerosis, which is characterized by thickening and hardening of artery walls (e.g., coronary arteries, carotid arteries, aorta, and ileofemoral arteries). Over time, this material thickens, hardens and may eventually block or severely narrow the arteries. More than 61 million Americans suffer from some form of cardiovascular disease, including high blood pressure, coronary heart disease, stroke, congestive heart failure, and other conditions. More than 2,600 Americans die every day because of cardiovascular diseases.
  • Various embodiments of the present invention provide for a method, comprising: providing a composition comprising a recombinant adeno-associated virus (rAAV) vector comprising an exogenous gene encoding ApoA-I Milano, or fragment thereof and administering the composition to a mammal in need of changing the phenotype of a monocyte or macrophage from a proinflammatory M1 phenotype to an anti-inflammatory M2 phenotype to change the phenotype of the monocyte or macrophage from the proinflammatory M1 phenotype to the anti-inflammatory M2 phenotype.
  • rAAV recombinant adeno-associated virus
  • the method can further comprise monitoring monocyte or macrophage phenotypic switching in the mammal. In various embodiments, the method can further comprise assessing the efficacy of a treatment for atherosclerosis in the mammal.
  • the rAAV vector can be a rAAV8 vector comprising an exogenous gene encoding ApoA-I Milano or a fragment thereof.
  • the rAAV8 can be produced by co-transfecting a host cell with a first plasmid and a second plasmid, wherein the first plasmid genome for the rAAV8 is derived from AAV serotype 2 and the second plasmid is derived from AAV serotypes 2 and 8 (Rep2Cap8).
  • the rAAV vector can be a rAAV2 vector comprising an exogenous gene encoding ApoA-I Milano or a fragment thereof.
  • the rAAV2 can be produced by co-transfecting a host cell with a first plasmid and a second plasmid, wherein the first plasmid genome for the rAAV2 is derived from AAV serotype 2 and the second plasmid is derived from AAV serotype 2 (Rep2Cap2).
  • the monocyte or macrophage can be a circulating monocyte or macrophage. In various embodiments, the monocyte or macrophage can be a peritoneal monocyte or macrophage.
  • changing the phenotype of the monocyte or macrophage from the proinflammatory M1 phenotype to the anti-inflammatory M2 phenotype can inhibit or treat atherosclerosis. In various embodiments, changing the phenotype of the monocyte or macrophage from the proinflammatory M1 phenotype to the anti-inflammatory M2 phenotype can reduce atherosclerosis. In certain embodiments, the atherosclerosis can be reduced in a whole aorta, an aortic sinus, an inominate artery or combinations thereof.
  • changing the phenotype of the monocyte or macrophage from the proinflammatory M1 phenotype to the anti-inflammatory M2 phenotype can reduce plaque lipid content in an aortic sinus, an innominate artery, or both. In various embodiments, changing the phenotype of the monocyte or macrophage from the proinflammatory M1 phenotype to the anti-inflammatory M2 phenotype can reduce plaque macrophage content in an aortic sinus, an innominate artery or both.
  • the rAAV vector can be produced by providing a first plasmid comprising ApoA-I Milano or a fragment thereof; providing a second plasmid complimentary to the first plasmid and which comprises components for rescue and packaging; co-transfecting the first plasmid and the second plasmid into a host cell; and generating a quantity of the rAAV vector from the co-transfected host cell.
  • the pair of the first plasmid and the second plasmid can be selected such that the rAAV vector is targeted for delivery to a specific tissue type.
  • the second plasmid can further comprise AAV rescue and packaging components derived from an AAV serotype selected from the group consisting of AAV1, AAV2, AAV5, AAV7, AAV8, AAV9, AAV10 and combinations thereof.
  • Embodiments of the present invention provide for a method, comprising: measuring the expression level of one or more markers selected from the group consisting of MCP-1, IL-6, TNF- ⁇ , Arg-1, Ym-1, and CD206 to monitor monocyte or macrophage phenotypic switching in a mammal in need thereof; and determining the presence of a phenotypic switch from a proinflammatory M1 macrophage to an anti-inflammatory M2 macrophage when MCP-1, IL-6 and/or TNF- ⁇ is down-regulated, and/or Arg-1, Ym-1 and/or CD206 is up-regulated, or determining the absence of a phenotypic switch from the proinflammatory M1 macrophage to the anti-inflammatory M2 macrophage when MCP-1, IL-6 and/or TNF-a is not down-regulated, and/or Arg-1, Ym-1 and/or CD206 is not up-regulated, or determining the presence of a phenotypic switch from an anti-inflammatory M2
  • Embodiments of the present invention provide for a method for assessing the efficacy of a treatment for atherosclerosis in a mammalian subject in need thereof, comprising: determining the phenotype of the macrophages in the mammalian subject; and making a determination that a treatment is providing beneficial results to a mammalian subject if a phenotypic switch from proinflammatory M1 macrophage to anti-inflammatory M2 macrophage is detected, or making a determination that a treatment is not providing beneficial results to the mammalian subject if a phenotypic switch from anti-inflammatory M2 macrophage to proinflammatory M1 macrophage is detected.
  • the treatment for atherosclerosis is treatment with ApoA-1 Milano.
  • FIG. 1 depicts expression of apo A1 Milano in the mice with rAAV in accordance with various embodiments of the present invention.
  • Serum Levels of apo A1 Milano at 4, 12, 24 weeks after injection virus and a group of mice with the empty vector is used as a control.
  • the data represent mean ⁇ SD from 12 mice per group. These data, analyzed by Student's unpaired t-test, show there was statistically significant difference among AAV8 apo A1 Milano and another three groups.
  • FIG. 2 depicts the comparison of serum levels of apo A1 Milano between mice with rAAV8 and rAAV2 at 4, 12, 24 weeks after IV injection virus in accordance with various embodiments of the present invention.
  • the data represent mean ⁇ SD from 12 mice per group. These data show there was statistically significant difference between rAAV8apo A1 Milano and rAAV2 apo A1 Milano.
  • FIG. 3 depicts the expression of apo A1 Milano in the mice with rAAV in accordance with various embodiments of the present invention. Serum levels of apo A1 Milano at 4, 12, 24 weeks after IM injection virus are shown. The data represent mean ⁇ SD from 10 mice per group. These data show there was no statistically significant difference between rAAV2 apo A1 Milano and rAAV2 vector control.
  • FIG. 4 depicts serum levels of apo A1 Milano in mice from IM and IV delivery with rAAV2 in accordance with various embodiments of the present invention. These data show there was statistically significant difference among AAV2apo A1 Milano IM and AAV2apo A1 Milano IV groups in 4 w, 12 w as well as 20 w (p ⁇ 0.001).
  • FIG. 5 depicts real-time PCR Quantitative analysis of apoA1 Milano mRNA expression in mice tissues in accordance with various embodiments of the present invention. Values were normalized against GAPDH mRNA. Data showed a significantly higher level of apo A1 Milano.
  • FIG. 6B depicts the extent of the atherosclerotic lesions in the total aorta were quantified after Oil Red 0 staining in accordance with various embodiments of the present invention.
  • FIG. 8 depicts the average percent lesion areas in accordance with various embodiments of the present invention in innominate artery (A) and aortic sinus (B) in rAAV8 vector control, rAAV8 apoA1 Milano and rAAV2 vector control; rAAV2 apo A1 Milano are shown.
  • FIG. 9 depicts the lipid content determined in innominate artery and aortic sinus atheromatous plaque by Oil-Red 0 staining in accordance with various embodiments of the present invention.
  • the lipid content was significantly lower in mice treated with rAAV8 apo A1 Milano compared with vector control or rAAV2 apo A1 Milano.
  • FIG. 10 depicts quantitative data of macrophage immunoreactivity in the innominate artery lesions (A) and aotic sinus lesions (B) in accordance with various embodiments of the present invention.
  • the macrophage immunoreactive area was significantly lower in mice treated with rAAV8 apo A1 Milano compared with vector control or rAAV2 apo A1 Milano.
  • C To assess macrophage infiltration into atherosclerotic plaques, immunostaining is used.
  • “Beneficial results” may include, but are in no way limited to, lessening or alleviating the severity of the disease condition, preventing the disease condition from worsening, reducing the likelihood of the disease condition worsening, curing the disease condition and prolonging a patient's life or life expectancy.
  • Gene transfer or “gene delivery” refers to methods or systems for reliably inserting foreign DNA into host cells. Such methods can result in transient expression of non-integrated transferred DNA, extrachromosomal replication and expression of transferred replicons (e.g., episomes), or integration of transferred genetic material into the genomic DNA of host cells. Gene transfer provides a unique approach for the treatment of acquired and inherited diseases. A number of systems have been developed for gene transfer into mammalian cells. See, e.g., U.S. Pat. No. 5,399,346.
  • Vector refers to any genetic element, such as a plasmid, phage, transposon, cosmid, chromosome, virus, virion, etc., which is capable of replication when associated with the proper control elements and which can transfer gene sequences between cells.
  • the term includes cloning and expression vehicles, as well as viral vectors.
  • AAV vector refers to any vector derived from any adeno-associated virus serotype, including, without limitation, AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-7, AAV-8, AAV-9, and AAV-10 and the like.
  • AAV vectors can have one or more of the AAV wild-type genes deleted in whole or in part, preferably the Rep and/or Cap genes, but retain functional flanking ITR sequences. Functional ITR sequences are generally necessary for the rescue, replication, packaging and potential chromosomal integration of the AAV genome.
  • an AAV vector is defined herein to include at least those sequences required in cis for replication and packaging (e.g., functional ITRs) of the virus.
  • the ITRs need not be the wild-type nucleotide sequences, and may be altered (e.g., by the insertion, deletion or substitution of nucleotides) so long as the sequences provide for functional rescue, replication and packaging.
  • Recombinant virus refers to a virus that has been genetically altered (e.g., by the addition or insertion of a heterologous nucleic acid construct into the particle).
  • AAV virion refers to a complete virus particle, such as a wild-type (“wt”) AAV virus particle (i.e., including a linear, single-stranded AAV nucleic acid genome associated with an AAV capsid protein coat).
  • wt wild-type
  • AAV virus particle i.e., including a linear, single-stranded AAV nucleic acid genome associated with an AAV capsid protein coat.
  • single-stranded AAV nucleic acid molecules of either complementary sense i.e., “sense” or “antisense” strands
  • the AAV capsid protein coat can be from any of the various AAV serotypes depending on the target of the AAV virion.
  • a “recombinant AAV virion” or “rAAV virion” is defined herein as an infectious, replication-defective virus composed of an AAV protein shell, encapsidating a heterologous DNA molecule of interest (e.g., genes encoding ApoA-I Milano) which is flanked on both sides by AAV ITRs.
  • a rAAV virion may be produced in a suitable host cell which has had an AAV vector, AAV Rep and Cap functions and helper virus functions introduced therein.
  • the host cell is rendered capable of producing AAV replication and capsid proteins that are required for replicating and packaging the AAV vector (i.e., containing a recombinant nucleotide sequence of interest) into recombinant virion particles for subsequent gene delivery.
  • the complete transgene may consist of a promoter, the coding sequences, usually a cDNA and a polyadenylation signal.
  • a transgene may also include regulatory sequences and intron regions. Promoters that would regulate transgene expression may include constitutive, inducible and tissue-specific promoters.
  • transfection is used herein to refer to the uptake of foreign DNA by a cell.
  • a cell has been “transfected” when exogenous DNA has been introduced inside the cell membrane.
  • transfection techniques are generally known in the art. See, e.g., Graham et al. (1973) Virology, 52:456, Sambrook et al. (1989) Molecular Cloning, a laboratory manual, Cold Spring Harbor Laboratories, New York, Davis et al. (1986) Basic Methods in Molecular Biology, Elsevier, and Chu et al. (1981) Gene 13:197.
  • Such techniques can be used to introduce one or more exogenous DNA moieties, such as a plasmid vector and other nucleic acid molecules, into suitable host cells.
  • the term refers to both stable and transient uptake of the genetic material.
  • transduction denotes the delivery of a DNA molecule to a recipient cell either in vivo or in vitro, via any method of gene delivery, including replication-defective viral vectors, such as via a rAAV.
  • heterologous as it relates to nucleic acid sequences such as gene sequences and control sequences, denotes sequences that are not normally joined together and/or are not normally associated with a particular virus. Allelic variation or naturally occurring mutational events do not give rise to heterologous DNA, as used herein.
  • DNA is meant to refer to a polymeric form of deoxyribonucleotides (i.e., adenine, guanine, thymine and cytosine) in double-stranded or single-stranded form, either relaxed or supercoiled. This term refers only to the primary and secondary structure of the molecule, and does not limit it to any particular tertiary forms. Thus, this term includes single- and double-stranded DNA found, inter alia, in linear DNA molecules (e.g., restriction fragments), viruses, plasmids, and chromosomes.
  • linear DNA molecules e.g., restriction fragments
  • viruses e.g., plasmids, and chromosomes.
  • sequences may be described herein according to the normal convention of giving only the sequence in the 5′ to 3′ direction along the non-transcribed strand of DNA (i.e., the strand having the sequence homologous to the mRNA).
  • the term captures molecules that include the four bases adenine, guanine, thymine and cytosine, as well as molecules that include base analogues which are known in the art.
  • a “gene” or “coding sequence” or a sequence which “encodes” a particular protein is a nucleic acid molecule that is transcribed (in the case of DNA) and translated (in the case of mRNA) into a polypeptide in vitro or in vivo when placed under the control of appropriate regulatory sequences; although one of skill in the art will readily appreciate that various polynucleotides do not operate in this fashion (e.g., antisense RNA, siRNA, ribozymes, wherein the RNA transcript is the product).
  • the boundaries of the coding sequence are determined by a start codon at the 5′ (i.e., amino) terminus and a translation stop codon at the 3′ (i.e., carboxy) terminus.
  • a gene can include, but is not limited to, cDNA from prokaryotic or eukaryotic mRNA, genomic DNA sequences from prokaryotic or eukaryotic DNA, and even synthetic DNA sequences.
  • a transcription termination sequence will usually be located 3′ to the gene sequence.
  • a “gene” starts with a promoter region containing multiple regulatory elements, possibly including enhancers, for directing transcription of the coding region sequences; (ii) includes coding sequences, which start at the transcriptional start site that is located upstream of the translational start site and ends at the transcriptional stop site, which may be quite a bit downstream of the stop codon (a polyadenylation signal is usually associated with the transcriptional stop site and is located upstream of the transcriptional stop); and (iii) may contain introns and other regulatory sequences to modulate expression and improve stability of the RNA transcript.
  • control elements refers collectively to promoter regions, polyadenylation signals, transcription termination sequences, upstream regulatory domains, origins of replication, internal ribosome entry sites (“IRES”), enhancers, and the like, which collectively provide for the replication, transcription and translation of a coding sequence in a recipient cell. Not all of these control elements need always be present, so long as the selected coding sequence is capable of being replicated, transcribed and translated in an appropriate host cell.
  • promoter region is used herein in its ordinary sense to refer to a nucleotide region including a DNA regulatory sequence, wherein the regulatory sequence is derived from a gene which is capable of binding RNA polymerase and initiating transcription of a downstream (3′-direction) coding sequence.
  • “Operably linked” refers to an arrangement of elements wherein the components so described are configured so as to perform their usual function.
  • control elements operably linked to a coding sequence are capable of effecting the expression of the coding sequence.
  • the control elements need not be contiguous with the coding sequence, so long as they function to direct the expression thereof.
  • intervening untranslated yet transcribed sequences can be present between a promoter sequence and the coding sequence and the promoter sequence can still be considered “operably linked” to the coding sequence.
  • nucleotide sequences in a particular nucleic acid molecule For the purpose of describing the relative position of nucleotide sequences in a particular nucleic acid molecule throughout the instant application, such as when a particular nucleotide sequence is described as being situated “upstream,” “downstream,” “5′,” or “3′” relative to another sequence, it is to be understood that it is the position of the sequences in the non-transcribed strand of a DNA molecule that is being referred to as is conventional in the art.
  • “Homology” and “homologous” as used herein refer to the percent of identity between two polynucleotide or two polypeptide moieties. The correspondence between the sequences from one moiety to another can be determined by techniques known in the art. For example, homology can be determined by a direct comparison of the sequence information between two polypeptide molecules by aligning the sequence information and using readily available computer programs. Alternatively, homology can be determined by hybridization of polynucleotides under conditions which form stable duplexes between homologous regions, followed by digestion with single-stranded-specific nuclease(s), and size determination of the digested fragments.
  • Two DNA or two polypeptide sequences are “substantially homologous” to each other when at least about 80%, preferably at least about 90%, and most preferably at least about 95% of the nucleotides or amino acids, respectively, match over a defined length of the molecules, as determined using the methods above.
  • isolated refers to the fact that the indicated molecule is present in the substantial absence of other biological macromolecules of the same type.
  • an “isolated nucleic acid molecule which encodes a particular polypeptide” refers to a nucleic acid molecule that is substantially free of other nucleic acid molecules that do not encode the subject polypeptide.
  • an “isolated vector” refers to a vector that is substantially free of other vectors that differ from the subject vector.
  • the subject molecule or vector may include some additional bases or moieties that do not deleteriously affect the basic characteristics of the composition.
  • a “purified” as used herein when referring to a vector refers to a quantity of the indicated vector that is present in the substantial absence of other biological macromolecules.
  • a “purified vector” refers to a composition that includes at least 80% subject vector, preferably at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% subject vector with respect to other components of the composition
  • “Mammal” as used herein refers to any member of the class Mammalia, including, without limitation, humans and nonhuman primates such as chimpanzees and other apes and monkey species; farm animals such as cattle, sheep, pigs, goats and horses; domestic mammals such as dogs and cats; laboratory animals including rodents such as mice, rats and guinea pigs, and the like.
  • the term does not denote a particular age or sex. Thus, adult and newborn subjects, as well as fetuses, whether male or female, are intended to be included within the scope of this term.
  • the inventors evaluated the effects of intravenously administered rAAV8 encoding Apo A-I Milano on aortic and innominate artery atherosclerosis, plaque composition and phenotype of circulating mononuclear cells in Apo E-/-Apo A1-/-mice.
  • rAAV8 mediated Apo A-I Milano gene transfer reduces all plaques; for example, plaque in whole aorta, aortic sinuses and innominate arteries. The plaque that remains is more table and less likely to rupture and trigger a blood clot. Further, the inventors evaluated the effects of rAAV2 mediated Apo A-I Milano gene transfer using a single intramuscular injection, on murine atherosclerosis and macrophage phenotype.
  • rAAV2-Milano significantly reduces aortic atherosclerosis despite absence of detectable levels of circulating transgene; these effects are associated with adoption of an anti-inflammatory phenotype in macrophages.
  • various embodiments of the present invention are based, at least in part, upon these findings.
  • Various embodiments of the present invention provide for a method of changing the phenotype of monocytes and macrophages from a proinflammatory M1 phenotype to an anti-inflammatory M2 phenotype.
  • the method comprises providing a composition comprising a recombinant adeno-associated virus (rAAV) vector comprising an exogenous gene encoding ApoA-I Milano or a fragment thereof; and administering the composition to a mammal in need thereof to change the phenotype of monocytes or macrophages from a proinflammatory M1 phenotype to an anti-inflammatory M2 phenotype.
  • rAAV recombinant adeno-associated virus
  • the method further comprises monitoring monocyte or macrophage phenotypic switching in the mammal. In other embodiments, the method further comprises assessing the efficacy of a treatment for atherosclerosis in the mammal.
  • the rAAV vector is produced by the process of: (i) providing a first plasmid that comprises ApoA-I Milano or a fragment thereof, (ii) providing a second plasmid that is complementary to the first plasmid and which comprises components for rescue and packaging, (iii) co-transfecting the first and second plasmids into a host cell, and (iv) generating a quantity of said rAAV vector from said co-transfected host cell, wherein the pair of said first and second plasmids is selected such that said rAAV vector is targeted for delivery to a specific tissue type.
  • the second plasmid further comprises AAV rescue and packaging components derived from an AAV serotype selected from the group consisting of AAV1, AAV2, AAV5, AAV7, AAV8, AAV9, AAV10 and combinations thereof.
  • the recombinant adeno-associated virus (rAAV) vector comprising an exogenous gene encoding ApoA-I Milano or a fragment thereof is rAAV8 vector comprising an exogenous gene encoding ApoA-I Milano or a fragment thereof.
  • the recombinant adeno-associated virus (rAAV) vector comprising an exogenous gene encoding ApoA-I Milano or a fragment thereof is rAAV2 vector comprising an exogenous gene encoding ApoA-I Milano or a fragment thereof.
  • the monocyte or macrophage is a circulating monocyte or macrophage. In other embodiments, the monocyte or macrophage is a peritoneal monocyte or macrophage. In various embodiments, very low levels of circulating transgene product are present. In various embodiments, changing monocyte or macrophage phenotype inhibits atherosclerosis.
  • changing the phenotype of monocytes or macrophages from a proinflammatory M1 phenotype to an anti-inflammatory M2 phenotype reduces atherosclerosis.
  • changing the phenotype of monocytes or macrophages from a proinflammatory M1 phenotype to an anti-inflammatory M2 phenotype reduces atherosclerosis in whole aorta.
  • changing the phenotype of monocytes or macrophages from a proinflammatory M1 phenotype to an anti-inflammatory M2 phenotype reduces atherosclerosis in aortic sinuses.
  • changing the phenotype of monocytes or macrophages from a proinflammatory M1 phenotype to an anti-inflammatory M2 phenotype reduces atherosclerosis in the innominate artery.
  • changing the phenotype of monocytes or macrophages from a proinflammatory M1 phenotype to an anti-inflammatory M2 phenotype reduces plaque.
  • changing the phenotype of monocytes or macrophages from a proinflammatory M1 phenotype to an anti-inflammatory M2 phenotype reduces plaque lipid content in aortic sinuses. In various embodiments, changing the phenotype of monocytes or macrophages from a proinflammatory M1 phenotype to an anti-inflammatory M2 phenotype reduces plaque lipid content in the innominate artery. In various embodiments, changing the phenotype of monocytes or macrophages from a proinflammatory M1 phenotype to an anti-inflammatory M2 phenotype reduces plaque macrophage content in aortic sinuses. In various embodiments, changing the phenotype of monocytes or macrophages from a proinflammatory M1 phenotype to an anti-inflammatory M2 phenotype reduces plaque macrophage content in innominate arteries.
  • Various embodiments of the present invention provide for monitoring macrophage phenotypic switching in a mammalian subject in need thereof.
  • the phenotypic switch is between a proinflammatory M1 macrophage and an anti-inflammatory M2 macrophage.
  • the mammalian subject has atherosclerosis.
  • the mammalian subject is or has been treated with apo A1 Milano.
  • the method comprises measuring the expression level of one or more markers selected from the group consisting of MCP-1, IL-6, TNF-a, Arg-1, Ym-1 and CD206; and (a) determining the presence of a phenotypic switch from proinflammatory M1 macrophage to anti-inflammatory M2 macrophage when MCP-1, IL-6 and/or TNF-a is down-regulated, and/or Arg-1, Ym-1 and/or CD206 is up-regulated, or (b) determining the absence of a phenotypic switch from proinflammatory M1 macrophage to anti-inflammatory M2 macrophage when MCP-1, IL-6 and/or TNF-a is not down-regulated, and/or Arg-1, Ym-1 and/or CD206 is not up-regulated, or (c) determining the presence of a phenotypic switch from anti-inflammatory M2 macrophage to proinflammatory M1 macrophage when MCP-1, IL-6 and/or TNF is up-regulated, and/
  • the treatment of atherosclerosis is treatment with apo A1 Milano.
  • the method comprises: determining the phenotype of the macrophages in the mammalian subject; and (a) making a determination that the treatment is providing beneficial results to the mammalian subject if a phenotypic switch from proinflammatory M1 macrophage to anti-inflammatory M2 macrophage is detected, or (b) making a determination that the treatment is not providing beneficial results to the mammalian subject if a phenotypic switch from anti-inflammatory M2 macrophage to proinflammatory M1 macrophage is detected.
  • the treatment with apo A1 Milano may be provided in variety of ways known in the art. Examples include, but are not limited to IM or IV rAAV gene transfer (e.g., rAAV serotype 2, rAAV serotype 8), administration of a composition comprising apo A1 Milano protein, administration of a composition comprising apo A1 Milano protein via a drug eluting stent.
  • the vectors of the present invention are based on the vector described in U.S. Pat. No. 5,474,935, with the transgene being ApoA-I Milano or a fragment thereof, for changing the phenotype of monocytes and macrophages from a proinflammatory M1 phenotype to an anti-inflammatory M2 phenotype and the treatment of atherosclerosis.
  • Preparation of rAAV vectors can be as described in Chatterjee, S. & K. K. Wong, Adeno-associated virus vectors for the delivery of ribozymes.
  • rAAV-5 vector used in the present invention incorporates a CBA promoter.
  • the construction of the vectors of the present invention can be completed by widely recognized means for manufacturing AAV virions, which entails co-transfection of a host cell with two different, yet complementing plasmids.
  • One of these contains the therapeutic or reporter transgene sandwiched between the two cis acting AAV ITRs.
  • the AAV components that are needed for rescue and subsequent packaging of progeny recombinant genomes are provided in trans by a second plasmid encoding the viral open reading frames for rep and cap proteins.
  • any number of other techniques for construction of the vectors of the present invention may be used as will be recognized by one of skill in the art. See, e.g. Gao, G.
  • U.S. Pat. No. 5,658,776 refers to packaging systems and processes for packaging AAV vectors that replace the AAV P5 promoter with a heterologous promoter.
  • 5,622,856 refers to constructs and methods for AAV vector production, which provide constructs formed by moving the homologous P5 promoter to a position 3′ to the rep genes, and optionally flanking the rep-cap and repositioned P5 promoter with FRT sequences.
  • the ITRs and portions of the genome of the first plasmid and the rep and cap proteins of the second plasmid can be derived from any serotype of AW vector.
  • the rAAV virions of the present invention can be specifically tailored to target a subject tissue with greater specificity. It is well known in the art that AAV serotype has a significant impact on tissue-specific gene expression (Hauck, B. et al., “Generation and characterization of chimeric recombinant AAV vectors,” Mol Ther, Vol. 7, pp. 419-25 (2003); Chao, H.
  • the DNA element of the first plasmid may be derived from one AAV serotype
  • the rep proteins may be derived from another AAV serotype
  • the cap proteins may be derived from still another AAV serotype.
  • the AAV vector genome can be pseudotyped by packaging with capsids from different AAV serotypes, which has been effective in directing rAAV gene therapies to specific tissues (Weitzman, M. et al., “Breaking the barriers to global gene delivery,” Nature Biotechnology, Vol.
  • capsids derived from AAV serotypes 1, 8, 9 and 10 may be particularly effective in intramuscular injections. Further, capsids derived from AAV serotypes 1, 7 and 8 may be particularly effective for hematopoietic stem cell transduction. Still further, capsids derived from AAV serotype 8 may be particularly effective targeting the liver.
  • Various embodiments of the present invention also relate to the treatment, prevention, inhibition, stabilization and/or induction of regression of atherosclerosis, as well as the treatment, prevention, inhibition and/or stabilization of any disease or physiological condition in which atherosclerosis (or atherogenesis) plays a role.
  • the methods of the present invention may be particularly useful in treating atherosclerosis when caused by invasive techniques, such as percutaneous transluminal coronary angioplasty (“PTCA”); insertion of a bypass graft or stent insertion; treatment of restenosis following stent placement; or as a result of bypass graft insertion.
  • PTCA percutaneous transluminal coronary angioplasty
  • insertion of a bypass graft or stent insertion treatment of restenosis following stent placement; or as a result of bypass graft insertion.
  • rAAV virions including heterologous DNA corresponding to an ApoA-I Milano coding sequence are generated by any conventional technique known in the art.
  • the recombinant AAV virions of the present invention can be produced by a standard methodology that generally involves the steps of: (1) introducing an AAV vector plasmid into a host cell; (2) introducing an AAV helper construct into the host cell, where the helper construct includes AAV coding regions capable of being expressed in the host cell to complement AAV helper functions missing from the AAV vector; (3) introducing one or more helper viruses and/or accessory function vectors into the host cell, wherein the helper virus and/or accessory function vectors provide accessory functions capable of supporting efficient rAAV virion production in the host cell; and (4) culturing the host cell to produce rAAV virions.
  • the AAV vector, AAV helper construct and the helper virus or accessory function vector(s) can be introduced into the host cell either simultaneously or serially, using standard transfection techniques. Any number of other approaches may also be used, as will be readily recognized by one of skill in the art.
  • AAV vectors are constructed using known techniques to at least provide, as operatively linked components in the direction of transcription, (a) control elements including a transcriptional initiation region, (b) the ApoA-I Milano DNA of interest and (c) a transcriptional termination region. Moreover, any coding sequence sufficiently homologous to the ApoA-I Milano coding sequence so as to exhibit functional properties substantially similar to the ApoA-I Milano coding sequence may be used in connection with alternate embodiments of the present invention.
  • the control elements are selected to be functional in the targeted cell(s).
  • the resulting construct, which contains the operatively linked components may be bounded (5′ and 3′) with functional AAV ITR sequences.
  • the nucleotide sequences of AAV ITR regions are known.
  • AAV ITRs used in the vectors of the invention need not have a wild-type nucleotide sequence, and may be altered (e.g., by the insertion, deletion or substitution of nucleotides).
  • AAV ITRs may be derived from any of several AAV serotypes, including, without limitation, AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-7, AAV-8, AAV-9, AAV-10 and the like. See, e.g. Gao et al., J. Virol. 2004 June; 78(12):6381-8; Weitzman, M. et al. (2005); Wang, Z. et al. (2005); and Wang, L. et al. (2005).
  • 5′ and 3′ ITRs that flank a selected nucleotide sequence in an AAV expression vector need not necessarily be identical or derived from the same AAV serotype or isolate, so long as they function as intended (i.e., to allow for excision and replication of the bounded ApoA-I Milano nucleotide sequence of interest).
  • the rAAV genome encoding the ApoA-I Milano transgenes within AAV ITRs may be packaged in virion capsids derived from any AAV serotype including AAV-1, AAV-2, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10 and the like. See, e.g. Gao et al. (2004); Weitzman, M. et al. (2005); Wang, Z. et al. (2005); and Wang, L. et al. (2005).
  • the virions described above are useful for changing the phenotype of monocytes and macrophages from a proinflammatory M1 phenotype to an anti-inflammatory M2 phenotype, for treating atherosclerosis, for reducing plaque, as well as for preventing and treating coronary heart disease and/or vascular disease and thus are useful for the manufacture of pharmaceutical compositions which contain an effective amount of rAAV-ApoA-I Milano vectors in admixture with inorganic or organic, solid or liquid, pharmaceutically acceptable carriers.
  • another aspect of this invention is a composition for changing the phenotype of monocytes and macrophages from a proinflammatory M1 phenotype to an anti-inflammatory M2 phenotype, for treating atherosclerosis, for reducing plaque, as well as for preventing and treating coronary heart disease and/or vascular disease described herein in combination with a pharmaceutically acceptable excipient.
  • compositions according to the invention are those which are suitable for oral, transdermal, topical, or parenteral, such as intramuscular or intravenous, administration to humans, and which contain the pharmacologically active rAAV transfected vectors together with a pharmaceutically acceptable carrier.
  • the dosage depends on various factors such as the age, weight, severity of vascular condition, and other factors a doctor might identify.
  • the therapeutic compositions are administered via suppository, or in tablet or capsule formulations for oral delivery.
  • Oral formulations usually include such normally employed additives such as binders, fillers, carriers, preservatives, stabilizing agents, emulsifiers, buffers and excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, and the like.
  • These compositions take the form of solutions, suspensions, tablets, pills, capsules, enterics, sustained release formulations, powders, and the like.
  • Oral formulations for gene therapy are known in the art. See, e.g. Chen, J. et al. (2004) World J. Gastroenterol 10(1):112-116. Further, other oral formulations are contemplated for use in the present invention as will be recognized by one of skill in the art.
  • transdermal and topical administration include salves, tinctures, creams, lotions, transdermal patches, transplanted skin, genetically engineered skin, stent coatings and suppositories.
  • traditional binders, carriers and excipients may include, for example, polyalkylene glycols or triglycerides.
  • a transdermal patch may be used for delivering therapeutics. See, e.g. U.S. Pat. No. 4,638,043.
  • Transdermal and topical formulations for gene therapy are known in the art. See, e.g. Jensen, T G (2004) Expert Opin Biol Ther. 4(5):677-82. Further, other transdermal and topical formulations are contemplated for use in the present invention as will be recognized by one of skill in the art.
  • Particularly suitable dosage forms for parenteral administration are sterile aqueous solutions of the pharmacologically active rAAV transfected vectors in water-soluble form, for example, a water-soluble salt, or sterile aqueous injection suspensions which contain substances increasing the viscosity, for example, sodium, carboxymethyl cellulose, sorbitol and/or dextran, and optionally stabilizers.
  • the pharmacologically active rAAV transfected vectors, with or without adjuvants can also be in lyophilized form and brought into solution prior to parenteral administration by the addition of suitable solvents.
  • an injectable composition of the invention may be a solution that is ready for injection, or a dry soluble composition that is ready to be combined with a solvent just prior to use, or a liquid concentrate ready for dilution prior to administration.
  • a composition for injection strict attention must be paid to tonicity adjustment to avoid irritation.
  • the vehicle normally has no therapeutic activity and is nontoxic, but presents the pharmacologically active rAAV transfected vectors to the body tissues or circulation in a form appropriate for absorption. Absorption normally will occur most rapidly and completely when the pharmacologically active rAAV transfected vectors is presented as an aqueous solution. However, modification of the vehicle with water-miscible liquids or substitution with water-immiscible liquids can affect the rate of absorption. In preparing the compositions which are suitable for subcutaneous injection, one can use aqueous vehicles, water-miscible vehicles, and nonaqueous vehicles. Certain aqueous vehicles are recognized officially because of their valid use in parenterals generally.
  • Water-miscible vehicles are also useful in the formulation of the parenteral composition of this invention. These solvents are used primarily to affect the solubility of the pharmacologically active rAAV transfected vectors. These solvents may include, for example, ethyl alcohol, polyethylene glycol and propylene glycol.
  • compositions of this invention may include an appropriate solubilizer, substances to make a solution isotonic, substances to act as antioxidants, and substances that act as a preservative to prevent the growth of microorganisms. These substances will be present in an amount that is appropriate for their function, but will not adversely affect the action of the composition as a treatment for disease conditions as contemplated herein.
  • the sterile, parenterally injectable composition of this invention and other therapeutic formulations suitable for delivery to a mammal in accordance with various embodiments of the present invention can be readily prepared by routine experimentation by the skilled artisan.
  • Guidance as to suitable pharmaceutical formulations is provided by Remington: The Science and Practice of Pharmacy 19 th Ed.
  • the rAAV virions encoding ApoA-I Milano are delivered to a mammal in a sufficient quantity and by a sufficient delivery route so as to effect gene transfer.
  • this provides an effective way for changing the phenotype of monocytes and macrophages from a proinflammatory M1 phenotype to an anti-inflammatory M2 phenotype, for treating atherosclerosis, for reducing plaque, and an effective treatment for atherosclerosis in mammals.
  • a sufficient and therapeutic quantity may be from about 1 ⁇ 10 10 vector genome/kg to about 1 ⁇ 10 14 vector genome/kg of rAAV-ApoA-I Milano vectors in vivo.
  • the ApoA-I Milano vector may be delivered to a subject by first transducing multipotent stem cells (e.g., bone marrow cells, blood stem cells, stromal cells, mesenchymal stem cells, etc.) with a quantity of the rAAV-ApoA-I Milano vector, and then transplanting these cells into a mammal.
  • multipotent stem cells e.g., bone marrow cells, blood stem cells, stromal cells, mesenchymal stem cells, etc.
  • the rAAV-ApoA-I Milano vector may be introduced into a mammal by direct intramuscular or intravenous injection, or directly into the artery at the site of PTCA or stent placement by any conventional methodology, as will be readily appreciated by one of skill in the art.
  • the rAAV virions of the present invention can be delivered as a single administration or as a treatment regimen, e.g., daily, weekly, or at any other suitable time interval, as will be readily recognized by one of skill in the art.
  • one serotype of rAAV virion can be delivered as a single administration followed by delivery of a different serotype of rAAV virion.
  • the present invention is also directed to a kit for changing the phenotype of monocytes and macrophages from a proinflammatory M1 phenotype to an anti-inflammatory M2 phenotype, for treating atherosclerosis, for reducing plaque, and for the treatment of atherosclerosis and related disease conditions in a subject.
  • the kit is an assemblage of materials or components, including at least one means of effecting gene transfer of ApoA-I Milano in a subject in accordance with various embodiments of the present invention. The exact nature of the components configured in the inventive kit depends on its intended purpose and on the particular methodology that is employed.
  • kits are configured for changing the phenotype of monocytes and macrophages from a proinflammatory M1 phenotype to an anti-inflammatory M2 phenotype.
  • Other embodiments of the kit are configured for the purpose of treating atherosclerosis and/or related disease conditions in a mammalian subject.
  • Still other embodiments are configured for the purpose of preventing the onset of atherosclerosis that is the result of another therapy, e.g., angioplasty or stent placement.
  • the kit is configured particularly for the purpose of treating human subjects.
  • Instructions for use may be included with the kit.
  • “Instructions for use” typically include a tangible expression describing the preparation of virions and/or at least one method parameter, such as the relative amounts of rAAV-ApoA-I Milano vector genome, dosage requirements and administration instructions, and the like, typically for an intended purpose.
  • the kit also contains other useful components, such as, diluents, buffers, pharmaceutically acceptable carriers, specimen containers, syringes, stents, catheters, and pipetting or measuring tools.
  • the materials or components assembled in the kit can be provided to the practitioner stored in any convenient and suitable ways that preserve their operability and utility.
  • the components can be in dissolved, dehydrated, or lyophilized form; they can be provided at room, refrigerated, or frozen temperatures.
  • the components are typically contained in suitable packaging material(s).
  • packaging material refers to one or more physical structures used to house the contents of the kit.
  • the packaging material is constructed by well known methods, preferably to provide a sterile, contaminant-free environment.
  • the packaging materials employed in the kit are those customarily utilized in the field.
  • a package refers to a suitable solid matrix or material such as glass, plastic, paper, foil, and the like, capable of holding the individual kit components.
  • a package can be a glass vial used to contain suitable quantities of a composition containing a volume of rAAV-ApoA-I Milano vector.
  • the packaging material generally has an external label which indicates the contents and/or purpose of the kit and/or its components.
  • the tools, kits, and methods of the present invention may be implemented in connection with a gene therapeutic approach for treating atherosclerosis and related disease conditions, or in connection with monitoring phenotypic switches.
  • the various embodiments of the present invention may therefore provide a means for prevention or reduction of the likelihood of the aforementioned disease conditions.
  • the embodiments of the invention are also suitable for use in connection with monitoring the success of ongoing or completed therapeutic intervention. For instance, a subject's serum may be tested prior to treatment to screen for circulating levels of ApoA-I Milano; during the course of treatment (e.g., to enhance a physician's ability to implement an effective treatment regimen); and/or following the completion of an intervention to determine a level of success (e.g., lifestyle changes, angioplasty and bypass surgery).
  • mice received one intravenous injection of 1.2 ⁇ 10 12 vector genome copies of rAAV8-Milano or rAAV8-Control (12 mice per group). Four weeks after injection, mice were placed on high fat diet. Twenty weeks later mice were euthanized and the extent of atherosclerosis in the en reze aorta, aortic sinuses and innominate artery was measured. Oil-red 0 staining and Moma-2 staining were used to measure lipid content and macrophage content of the plaques respectively. Quantitative PCR (qPCR) was used to analyze phenotype of macrophages.
  • qPCR Quantitative PCR
  • ApoE-/-ApoA-/-double knockout received one intramuscular injection of 1.2 ⁇ 10 12 vector genome copies of rAAV2-Milano or rAAV2-Control (10 mice per group).
  • mice Four weeks after intramuscular injection, mice were placed on high fat diet. Twenty weeks later, mice were euthanized and the expression of Apo A-I Milano mRNA in liver was measured by quantitative PCR (qPCR).
  • qPCR quantitative PCR
  • the phenotype of macrophages in the liver, peritoneal macrophages, and peripheral blood mononuclear cells (PBMC) were determined by qPCR.
  • the quantitative extent of total aorta plaques was evaluated by oil-red O staining ELISA analysis was performed to measure serum level of the Apo A-I Milano.
  • qPCR analysis of PBMC revealed that the relative expression level of MCP1, IL-6, and TNF- ⁇ mRNAs were lower and the expression level of Arg-1 as well as YM1 mRNA were higher in the rAVV2-Milano group compared to the Control group.
  • MCP-1, IL-6, and TNF- ⁇ mRNA were lower and YM1 was higher in the liver of rAAV-2 Milano group.
  • the expression of MCP-1, IL-6, and TNF- ⁇ mRNAs was lower and those of YM1 and Arg-1 were higher in the peritoneal macrophages of mice injected with rAAV2-Milano compared to the Control group.
  • rAAV vectors of the present invention are completed by co-transfecting a host cell with two different plasmids.
  • rAAV virions are prepared with the plasmids derived from various AAV serotypes.
  • ApoA-I Milano is sandwiched between the two cis acting AAV ITRs.
  • the AAV rep and cap proteins are provided in trans by a second plasmid encoding the viral open reading frames for rep and cap proteins of AAV.
  • the first plasmid genome is derived from AAV serotype 2
  • the second plasmid is derived from AAV serotype 2 (Rep2Cap2).
  • rAAV5 the first plasmid genome is derived from AAV serotype 5 and the second plasmid is derived from AAV serotype 5 (Rep5Cap5).
  • rAAV1 the first plasmid genome is derived from AAV serotype 2 and the second plasmid is derived from AAV serotypes 2 and 1 (Rep2Cap1).
  • rAAV7 the first plasmid genome is derived from AAV serotype 2 and the second plasmid is derived from AAV serotypes 2 and 7 (Rep2Cap7).
  • rAAV8 the first plasmid genome is derived from AAV serotype 2 and the second plasmid is derived from AAV serotypes 2 and 8 (Rep2Cap8).
  • rAAV9 the first plasmid genome is derived from AAV serotype 2 and the second plasmid is derived from AAV serotypes 2 and 9 (Rep2Cap9).
  • Other virions may be readily implemented as part of the present invention, as will be recognized by one of skill in the art.
  • a rAAV viral vector plasmid was constructed based on vectors previously constructed and utilized in the inventors' laboratory for the purpose of apo A1 Milano expression.
  • the specific rAAV vector serotypes used in this study contain each AAV serotype 2 and 8 viral capsid and a single-stranded DNA containing AAV2 inverted terminal repeat and encoding the human apo A1 Milano gene cDNA driven by a cytomegalovirus (CMV) immediate-early promoter/enhancer.
  • CMV cytomegalovirus
  • EGFP enhanced green fluorescent protein
  • NautCellsTM (Microbix Biosystems Inc. Canada), a reliable and traceable 293 human embryo kidney (HEK) cell clone producing a high titre of rAAV vectors, were grown and maintained in high glucose DMEM (Invitrogen) culture medium containing 10% fetal bovine serum, 100 units/ml-100 mg/ml penicillin-streptomycin in 5% CO 2 at 37° C.
  • DMEM Invitrogen
  • Subcultured actively growing NautCellsTM were placed in 15 cm culture dishes with high glucose DMEM and incubated in 5% CO 2 at 37° C. overnight. The medium was changed the next day and used for transfection 2-4 h.
  • a plasmid mixture consisting of 4 ug of rAAV vector (individual constructs), 4 ug of AAV packging plasmid XX2(AAV rep2 and cap2) or p5E18-VD287(AAV rep2 and cap8), and 12 mg of adenovirus helper plasmid XX6-80 were mixed with EC buffer (Qiagen Inc., Valencia, Calif.) to a final volume of 700 ul.
  • Enhancer 120 ul; Qiagen Inc. was added to each tube and vortexed immediately for 10 s. The tubes were placed at room temperature for 10 min. Fresh DMEM culture medium (4 ml) was added to each individual tube and mixed by pipetting up and down three times. The medium was then laid drop-wise onto NautCellTM while the dish was gently swirled. Transfected NautCellsTM were scraped with a cell lifter at 66-72 h post-transfection in the presence of medium. The cells from five dishes were combined in a 50 ml disposable centrifuge tube, collected by spinning in Sorvall TC centrifuge at 1,000 rpm for 8 min at RT. The media are discarded, and the cell pellets were stored at ⁇ 80° C. for later use.
  • the cell pellets were resuspended in 1.5 ml of 150 mM NaCl, 50 mM Tris-HCl, pH 8.5.
  • the cells subjected to five cycles of freezing (dry ice-ethanol bath) and thawing (37° C. water bath) with vortexing for 30 s after each thawing.
  • the lysed cells were incubated with 0.5% deoxycholate (Fluka) in the presence of 50 u/ml Benzonase (Sigma) at 37° C. for 30 min.
  • the lysate was clarified and recovered by centrifugation at 4500 g at 4° C. for 20 min.
  • rAAV particles Purification of rAAV particles was accomplished by discontinuous iodixanol density gradient centrifugation method as previously described by Muzyczka et al. [2]. The virus was concentrated and desalted by centrifugation through the Amicon ultre-15 centrifugal filter devices (Millipore 100K NMWL device).
  • rAAV vector genome titers were determined by dot-blot assay using RNA Detector Northern Blotting Kit (KPL) according to the manufacturer's instructions, and the titers were rAAV2/2 6.2 ⁇ 10e12 genome copies/ml, rAAV2/8 5.6 ⁇ 19e12 genome copies/ml.
  • Viral transducing units were measured by transduction of 293 cells in the presence of adenovirus helper with MOI followed by FACS.
  • mice on C57BL/6 background were kindly provided by Dr. Linda Curtiss (Department of Immunology, The Scripps Research Institute, La Jolla, Calif.) and maintained as a breeding colony. Mice 6-8 weeks old weighing 15-21 g were used for this study and males and females were equally represented. The animals were randomly divided into 6 groups (Table 1) and injected virus at a doses of 1.2 ⁇ 10e12 genome copies in 320 ul PBS (IV) 150 ul PBS (IM) per mouse, tail vein injection and intramuscular injection into the hind leg.
  • IV 150 ul PBS
  • rAAV vector administration into apoE/apoA1 double KO mice Serotypes 8 and 2 expressing EGFP-apo A1 Milano and EGFP (vector control) cDNA were evaluated in mice. Details of the total number of mice tested, routes of vector administration are listed.
  • RNA was extracted from brain, lung, heart, liver, spleen, kidney, intestine and muscle with TRIzol reagent (Invitrogen) according to the manufacturer's protocol.
  • First-strand cDNA was synthesized from 2 ug of total RNA in a final volume 20 ul using a Omniscript Reverse Transcription Kit (Qiagen, Valencia, Calif., USA). The cDNA was used. Real-time quantitative PCR analysis using an iO5 Real-Time PCR Detection System (Bio-Rad Laboratories, Hercules, Calif., USA).
  • Serum levels of human apo A1-Milano in the mice with rAAVs were determined by ELISA.
  • ELISA plates were coated with an antihuman apo A1 monoclonal antibody (Calbiochem) at a concentration of 4 ug/ml and incubated at 4° C. overnight. The wells were washed, blocked with 1% fetal bovine serum PBS, and diluted standards (Calbiochem) or serum samples were added to the wells and incubated at 4° C. overnight. After washing, rabbit antihuman apo A1 antibody was added and incubated at room temperature for 2 hours.
  • the extent of the atherosclerotic lesions in the total aorta were quantified after oil red O staining, as described previously [4, 5]. Briefly, the thoracic cavity was opened; the aortic arch as well as the aorta were exposed, and were photographed under a dissecting microscope. After capturing the images, most of the vascular tree was dissected intact. The luminal surface was exposed, and then the aortic tissue was fixed with Histo-choice tissue fixative (Amresco, Solon, Ohio). After tissue fixation, the adventitial tissue was removed and aortic tissue was stained for lipids with Oil-Red O.
  • Histo-choice tissue fixative Amresco, Solon, Ohio
  • the aortas were pinned to a dark surface, and images were captured by using a Spot digital camera (Diagnostic Instruments, Sterling Heights, Mich.). The extent of the atherosclerotic lesions was quantified using Image-Pro Plus (version 4, Media Cybernetics, Silver Spring, Md.). Lesions were reported as percentage of the total aortic area consisting of thoracic aorta (ending at the final intercostals artery), and abdominal aorta ending at the iliac difurcation.
  • aortic arch was fixed in 4% paraformaldehyde overnight and then embedded in Optimal Cutting Temperature (OCT).
  • OCT Optimal Cutting Temperature
  • the frozen tissues were serially sectioned into 8-um sections from the aortic arch using a Leica cryostat and kept in a ⁇ 80° C. freezer for ⁇ 1 month before use. Tissue was stained with Oil-Red O.
  • the lesion areas were quantified by Image-Pro Plus using the hematoxylin-stained sections (mean of 6 sections per mouse; 5 mice per group).
  • macrophage immunoreactivity was detected by immunostaining of frozen sections of innominate and aortic sinus with a 1:100 dilution of rat anti-mouse monocyte/macrophage (MOMA-2) monoclonal antibody (Seiotec). After washing, the sections were incubated with the goat anti-Rat secondary antibodies (Jacksin Immuno Research Lab), and then to apply LSAB streptavidin-HRP 25 min followed by color development. The sections were counterstained with hematoxyli. Photomicroscopy was performed using an Olympus microscope. The percentage of plaque area occupied by MOMA-2 staining, reflecting macrophage immunoreactivity, was calculated from the captured images using the Image-Pro Plus software.
  • MOMA-2 rat anti-mouse monocyte/macrophage
  • PBMC peripheral blood mononuclear cell
  • peritoneal macrophages 3% Brewer thioglycollate medium activity
  • liver using TRIzol.
  • the sequences of the primer pairs used in this analysis are indicated in Table 2.
  • the reaction conditions consisted of one 3 min cycle at 94° C., followed by 40 cycles of 94° C. for 10 s, 60° C. for 10 s, and 72° C. for 10 s, which concluded by the melting curve analysis process and run 1.8% agarose gel.
  • Plasma samples of KO apoE/apoA1 mice IV or IM injected with rAAV-apo a1 Milano were tested for the presence of antibodies against apo A1 Milano using an ELISA.
  • 96 wells plates were coated with 1 ug/ml human apoA1 in 0.1M NaHCO 3 pH 9.4, 100 ul/well and then incubated at 4° C. for 12-24 hours. Plates were washed three times with PBS-0.05% Tween-20, 5 min/time.
  • Plates were blocked with 1% BSA in PBS 200 ul/well at R/T 2 h or 4° C., O/N. Plates were washed three times with PBS-Tween-20, 5 min/time.
  • Dilute plasma samples (1:16) were applied in duplicate.
  • Dilute Anti-human apo A1 Milano cAb to final concentration of 1 ug/ml as positive control.
  • Negative controls included noninjected and rAAV EGFP injected mice. Plates were incubated 4° C. for 12-24 hours or RT 2 h. Plates were washed three times with PBS-Tween, 5 min/time.
  • Detection Ab anti-mouse IgG-HRP was added in a dilution of 1:2000. Plates were incubated for 1 hour at RT. Plates were washed 6 times with PBS-0.05% Tween-20, 5 min/time. Absorbance was measured at 450 nm on a microplate reader. Serum antibody against human apo A1-Milano levels were calculated by comparison with the standard curve.
  • rAAV transducer efficacies were possible in several organs in two serotypes.
  • a single mouse was killed for each rAAV vector group and total RNA was extracted from brain, lung, heart, liver, spleen, kidney and muscle.
  • Atherosclerosis progression in the total aorta was determined by Oil-Red 0 staining
  • Mean lesion areas were highly correlated with plasma levels of human apo A1 Milano in mice which were treated with rAAV8.
  • the lipid content was determined by Oil-Red 0 staining.
  • the reduction in lesion size was associated with reduced macrophage immunoreactivity.
  • the inventors subjected monocyte, macrophage as well as liver from mice treated with rAAV apo A1 Milano to real time PCR analysis using primer pairs (Table 2) for molecules that have been used classify monocytes/macrophages into the M1 and M2 subtypes [6, 7].
  • Mouse GAPDH was measured as a housekeeping gene for normalization purposes.

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