WO2009124055A1 - Human apoa-i mutants - Google Patents

Abstract

A polypeptide based on human apoA-I, comprising a polypeptide sequence containing amino acids 25-267 of human apoA-I, or containing an amino acid sequence at least 90% identical to amino acids 25-267 of human apoA-I. The polypeptide sequence is substituted at amino acids corresponding to amino acids 208-214 of human apoA-I with an amino acid sequence comprising a proline residue, and preferentially associates with an HDL subclass. Polynucleotides encoding the polypeptide, compositions containing the polypeptide, and methods of using the polypeptide are provided.

Description

DESCRIPTION HUMAN APOA-I MUTANTS

The present application claims the benefit of U.S. Provisional Patent

Application Serial No. 60/041,236, filed March 31, 2008, and U.S. Provisional Patent Application Serial No. 61/085,755, filed August 1, 2008, the entire disclosures of which are specifically incorporated herein by reference.

This invention was made with government support under NIH Cardiovascular

Pathophysiology and Biochemistry Training Program Award Number 5T32 HL07237, NIH ROl Award Number HL68661, and NIH ROl Award Number HL057334, all awarded by the National Institutes of Health (NIH). The government has certain rights in the invention.

BACKGROUND

I. Field of Invention

This invention generally relates to medicine. More specifically it concerns methods and compositions involving apolipoprotein A-I, such as polypeptides containing apolipoprotein A-I sequences and their use in treating vascular disease.

II. Related Art

Reverse cholesterol transport is a process in which excess cholesterol in peripheral tissues is transported to the liver by high density lipoprotein (HDL) particles. Epidemiological studies indicate an inverse relationship between HDL-cholesterol levels and coronary artery disease (Miller, 1987; Castelli et al, 1986). This cardioprotective role of HDL is related to the ability of apolipoprotein A-I (apoA-I), which is the major protein component of HDL, to promote reverse cholesterol transport (Assman and Gotto, 2004; Singh et al, 2007; Lewis, 2006; Rader, 2006) involving ABC transporters and SR-BI [Assman and Gotto, 2004; Singh et al, 2007; Lewis, 2006; Rader, 2006; Wang et al, 2001; Kozarsky et al, 1997; Rigotti et al, 1997, and to activate lecithin-cholesterol acyltransferase (LCAT), an enzyme involved in cholesterol esterification (Sorci-Thomas et al, 1998). In addition, HDL has anti-inflammatory and anti-oxidant properties (Assman and Gotto, 2004, Barter, 1997; Barter et al, 2004), and can stimulate nitric oxide production by endothelial cells [Assman and Gotto, 2004, Mineo et al, 2006). Most species, including mice, generate monodisperse HDL particles, while primates and humans make several HDL subclasses. The two major subclasses are HDL₂ and HDL3 (Reschly et al., 2002). HDL₂ is larger, more buoyant and has a higher ratio of apoA-I to apolipoprotein A-II (apo A-II) than HDL₃. In addition, apolipoprotein E is found almost exclusively on HDL₂. Distinct functional capacities of these HDL subclasses have been identified [Mowri et al, 1987; Kontush et al, 2007; Asztalos, 2004). Further, epidemiological data suggests that HDL₂ is more atheroprotective than HDL₃ under certain conditions (Miller, 1987). Studies in apoA-I transgenic animals (Rubin et al, 1991), adenoviral gene transfer in mice (Reardon et al, 2001), and transfected hepatocytes (Thurberg et al, 1996) revealed that the ability to form distinct HDL subclasses is inherent in the sequence of human apo A-I.

The primary structure of apoA-I contains 267 amino acid residues arranged as an 18 residue signal peptide, a 6 residue pro-peptide, and a 243 residue mature polypeptide. Variants of apoA-I have been identified in the human population, including a variant identified in an Italian cohort with low rates of coronary heart disease [US 5,876,968, herein incorporated by reference]. This apoA-I Milano variant contains a substitution of cysteine for arginine at residue 173 of the mature apoA-I amino acid sequence. Experiments indicate that the apoA-I Milano variant increases reverse cholesterol transport, activates LCAT, and has positive effects on atherosclerotic lesions in mice models. The identification of additional apoA-I variants and mutants effecting the level of HDL or HDL subclasses will increase the arsenal of pharmacological agents for combating cardiovascular disease.

SUMMARY

In one aspect, the present invention provides an isolated polypeptide containing a polypeptide sequence based on the 267 amino acid sequence of human apoA-I (SEQ ID NO:1). The isolated polypeptide associates with HDL, and in various embodiments, preferentially associates an HDL subclass, such as HDL₂ or HD L3. In some embodiments, the isolated polypeptide includes amino acids 25-267 of the human apoA-I amino acid sequence. In these embodiments, at amino acids corresponding to amino acids 208-214 of the human apoA-I amino acid sequence, the isolated polypeptide is substituted with an amino acid sequence containing a proline residue. In other embodiments, the isolated polypeptide includes an amino acid sequence at least 90% identical to amino acids 25-267 of the human apoA-I amino acid sequence, and is substituted at amino acids corresponding to amino acids 208-214 of the human apoA-I amino acid sequence with an amino acid sequence containing a proline residue. In various embodiments, the isolated polypeptide can also include amino acids 19-24 of the human apoA-I amino acid sequence, which produces the pro-form of apoA- I.

In certain embodiments, the proline-containing substituting amino acid sequence in the isolated polypeptide can be represented as X₁X₂XSPX₄XSXO, where each of X₁ - X₆ is independently any amino acid. In particular embodiments, the substituting amino acid sequence is PCS2 (SEQ ID NO: 2) or PCS3 (SEQ ID NO:3).

The isolated polypeptide can be further substituted with a cysteine residue at an amino acid position corresponding to amino acid 27, 30, 33, 39, 45, 49, 54, 58, 61, 96, 123, 131, 151, 173, 215, or 238 of the apoA-I amino acid sequence. As a result, the cysteine-substituted polypeptide can dimerise via the cysteine residue. In addition, the isolated polypeptide can contain a trimerizing module, which mediates the formation of trimers of the isolated polypeptide.

In certain embodiments, the isolated polypeptide includes the altered apoA-I amino acid sequence set forth in SEQ ID NO:4 or SEQ ID NO:5. In other embodiments, the isolated polypeptide includes amino acids 25-267 of SEQ ID NO:4 or SEQ ID NO:5.

The present invention also provides polynucleotides encoding any version of the isolated polypeptide. In some embodiments, the polynucleotide includes the altered apoA-I nucleotide sequence set forth in SEQ ID NO: 7 or SEQ ID NO:8. In other embodiments, the polynucleotide includes nucleotides 73-801 of SEQ ID NO:7 or SEQ ID NO:8.

In another aspect of the present invention, a pharmaceutical composition is provided that includes any version of the isolated polypeptide or multimer of the isolated polypeptide, along with a pharmaceutically acceptable carrier. The pharmaceutical composition can in addition contain a lipid bound to the isolated polypeptide or multimer. In some embodiments, the lipid is phosphatidyl choline, phosphatidyl serine, phosphatidyl ethanolamine, phosphatidal choline, phosphatidal ethanolamine, alkyl acyl glycerophosphoryl choline or alkyl acyl glycerophosphoryl ethanolamine, or any combination of these lipids. In various embodiments, the pharmaceutical composition can further include cholesterol, cholesteryl ester, sphingomyelin, or any combination of these lipids. In further aspects of the present invention, an expression vector is provided for an apo- lipoprotein expression in a cell. The expression vector includes a promoter active in the cell and operably linked to a DNA segment encoding any version of the isolated polypeptide. The cell for expression can be a liver cell, macrophage (including a Kupffer cell), lymphocyte, enterocyte, smooth muscle cell or endothelial cell. In some embodiments, the expression vector includes a signal peptide encoding DNA segment linked to the DNA segment encoding the isolated polypeptide such that the isolated polypeptide is secreted from the cell. In certain embodiments, the signal peptide can be the apoA-I signal peptide set forth in amino acids 1- 18 of SEQ ID NO:l.

A method of modulating HDL in a subject in need of such modulation is also provided. The method includes administering to a subject any version of the isolated polypeptide or multimer of the isolated polypeptide in an amount sufficient to modulate HDL. As used herein, "modulate" means to alter the distribution, composition, and/or level of an HDL subclass, such as HDL₂ or HD L3.

The isolated polypeptide or multimer can be administered as part of a pharmaceutical composition, and can contain bound lipid. Alternatively, the isolated polypeptide or multimer can be provided by an expression vector delivered to the subject. In some embodiments, the expression vector is a viral vector, which can be a vaccinia virus, adenovirus, retrovirus, herpes virus, cytomegalovirus, lentivirus or adenovirus-associated virus vector.

In another aspect, a method of treating a vascular disease in a subject in need of such treatment is provided. The method includes administering to the subject any version of the isolated polypeptide or multimer of the isolated polypeptide in an amount sufficient to increase the level of a beneficial HDL subclass. In certain embodiments, the HDL subclass is HDL₂ or HDL3. The isolated polypeptide or multimer can be administered as part of a pharmaceutical composition, and can contain bound lipid. Alternatively, the isolated polypeptide or multimer can be provided by an expression vector delivered to the subject.

Any embodiment discussed with respect to one aspect of the invention applies to other aspects of the invention as well.

The embodiments in the Example section are understood to be embodiments of the invention that are applicable to all aspects of the invention. The use of the term "or" in the claims is used to mean "and/or" unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and "and/or." Moreover, with any listing of items using the term "or", it is contemplated that in some embodiments one or more of those items may be excluded as an embodiment of the invention.

Following long-standing patent law, the words "a" and "an," when used in conjunction with the word "comprising" in the claims or specification, denotes one or more, unless specifically noted.

The novel features which are believed to be characteristic of the invention, both as to its organization and method of operation, together with further objects and advantages will be better understood from the following description when considered in connection with the accompanying figures. It is to be expressly understood, however, that each of the figures is provided for the purpose of illustration and description only and is not intended as a definition of the limits of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG 1. Strategy for generating human apoA-I proline containing sequence mutants. The illustration is an example of how each PCS mutant was generated.

FIG. 2. Recombinant apoA-I distributions to HDL₂ or HDL₃. Wild-type or the indicated PCS mutants were incubated with HDL (HDL₂ + HDL₃), the lipoproteins separated on equilibrium density gradients and the fractions probed for the T7-tagged human apoA-I. The recombinant apoA-I distributions to HDL₂ and HDL₃ were normalized by dividing by endogenous apoA-I distribution to the HDL subclasses. These values for HDL₂ (open bars) and HDL₃ (black bars) were expressed as percentages, n = 6, ^aHDL₂, p<0.05, ^bHDL₂, p<0.05, ΗDL₃, p<0.05, ^dHDL₃, p<0.05 compared to T7-HuA-I.

FIG. 3. Self-association behavior of lipid-free recombinant apoA-I proteins. Lipid- free apoA-I (1 mg/mL) was cross-linked with BS₃ and electrophoresed on a 6-12% Tricine gel. Lanes: Lane M, high molecular weight standard, Lane 1, Non-cross-linked T7-HuA-I, Lane 2, cross-linked T7-HuA-I (NPCS→PCS3), Lane 3, cross-linked T7-HuA-I (NPCS→PCS2), Lane 4, cross-linked T7-HuA-I. FIG. 4. DMPC clearance (lipid binding affinity) of recombinant apoA-I proteins. DMPC vesicles were incubated (1 :2.5 w/w) apoA-I:DMPC at 24°C in the absence of protein (solid black diamond) and presence of T7-HuA-I (NPCS→PCS3) (open circle), T7-HuA-I (NPCS→PCS2) (solid black triangle), or T7-HuA-I (solid black square).

FIG. 5. Electrophoresis of rHDL assembled with recombinant apoA-I proteins. The rHDL particles were prepared using the sodium cholate dialysis method with POPC, wild- type/mutant apoA-I, and sodium cholate in a molar ratio of 80:1 :108, run on a 6-20% nondenaturing polyacryl amide gradient gel, and stained with Coomassie G-250. Lanes: M, high molecular weight standards with their corresponding diameters to the left; Lane 1, T7- HuA-I (NPCS→PCS3); Lane 2, T7-HuA-I (NPCS→PCS2); Lane 3, T7-HuA-I.

FIGs. 6A-6B. Lipid-free and lipid-associated (isothermal) guanidine HCl denaturation stability. Lipid-free (FIG. 6A), and lipid-associated (FIG. 6B) wild-type and recombinant apoA-I proteins. The results from three separate experiments are plotted. The line represents the best fit curve, which includes both a sigmoid and hyperbola.

FIGs. 7A-7F. Scatchard analysis of association of (FIGs. 7 A and 7B) T7-HuA-I,

(FIGs. 7C-7D) T7-HuA-I (NPCS→PCS2), and (FIGs. 7E and 7F) T7-HuA-I (NPCS→PCS3) with HDL₂ or HDL₃. Increasing amounts of recombinant apoA-I proteins were incubated with 200 μg HDL₂ or HDL₃. HDL bound apoA-I was separated from lipid-free apoA-I by agarose gel electrophoresis. Bound and free recombinant apoA-I were detected by immunoblotting with HRP-conjugated T7 antibody. The results from three separate experiments are plotted. The line represents the best fit curve.

FIGs. 8A-8B. (FIG. 8A) Substituting PCS2 for NPCS may increase hydrophobic face exposure leading to preferential association with HDL₂, (FIG. 8B) Substituting PCS3 for NPCS may decrease hydrophobic face exposure leading to preferential association with HDL₃. Note the amino acids that constitute the sequence between putative helices 7 and 8 are outlined in bold lines and the potential size (black half-sphere) of the hydrophobic face is noted above the wheels.

FIGs. 9A-9C. Model for wild-type human apoA-I (FIG. 9A), HuA-I (NPCS→PCS2) mutant (FIG. 9B), HuA-I (NPCS→PCS3) mutant (FIG. 9C) association with spherical HDL₂ and HDL₃. The 10 amphipathic α-helices of apoA-I are represented by cylinders. Note the sequence between putative helices 7 and 8 is in bold. FIG. 10. Illustration of the location and amino acid sequence for the PCS between the putative helices 3 and 4 and 4 and 5 and the NPCS between the putative helices 7 and 8 in wild-type human apoA-I and the two PCS mutants of human apoA-I. Note that the mutants contain two copies of the PCS; one at its native position and one between helices 7 and 8.

FIGs. 1 IA-11C. Secretion of apoproteins by non-transfected and stably transfected

McA7⁴ cells. Non-transfected and stably transfected Mc A7⁴ cells in T75 flasks were incubated with 7mL of DMEM/high glucose phenol red free media, plus LPDS [0.25mg/mL] for 24hrs. The total medium was concentrated down to 2mL and 150μl was subject to western blotting using a rabbit anti-human species-specific (FIG. HA), rabbit anti-rat species-specific antibody (FIG. HB), or rabbit anti-rat apoE antibody (FIG. HC) and goat anti-rabbit HRP conjugated antibody.

FIGs. 12A-12B. Human apoA-I stably transfected McA7⁴ cells bias the density of secreted HDL. Non-transfected McA7⁴ cells or McA7⁴ cells transfected with wild-type human apoA-I (FIG. 12A), or HuA-I NPCS→PCS2 or HuA-I NPCS→PCS3 (FIG. 12B) in T 150 flasks were incubated with 14mL of DMEM/high glucose phenol red free media, plus LPDS for 24 hrs. The medium was concentrated down to 2mL and fractionated on a 10-20% NaBr density gradient. 150μl of each fraction was subject to western blotting using a rabbit anti-human apoA-I for stably transfected McA7⁴ cells or anti-rat apoA-I species specific antibody for non-transfected McA7⁴ cells and goat anti-rabbit HRP conjugated antibody. After exposure by chemiluminescence, the relative amount of human or rat apoA-I was determined using Alphalmager Software. Note, indicated at the base of the X-axis is the density range of human HDL subclasses and free protein on this density gradient.

FIGs. 13A-13C. Stably transfected human apoA-Is bias the size of α-HDL particles secreted from stably transfected McA7⁴ cells. McA7⁴ cells in T 150 flasks were incubated with 14mL DMEM/high glucose phenol red free media, plus LPDS for 24hrs. The media underwent sequential flotation centrifugation to isolate HDL within a density range of 1.063- 1.21g/mL and 150μl/lane was loaded directly onto a 2-36% non-denaturing gradient gel, Lanes: Lane 1, Wild-type human apoA-I, Lane 2, HuA-I NPCS→PCS2, Lane 3, HuA-I NPCS→PCS3 (FIG. 13A); or 75μl/lane was loaded onto a 0.7% agarose gel, Lanes: Lane 1, Non-transfected, Lane 2, Wild-type human apoA-I, Lane 3, HuA-I NPCS→PCS2, Lane 4, HuA-I NPCS→PCS3, Lane 5, Isolated Human HDL, Lane 6, Discoidal HDL (FIG. 13B); Lanes, Lane 1, Non-transfected, Lane 2, Isolated Rat HDL, Lane 3, Isolated Human HDL, Lane 4, Discoidal HDL (FIG. 13C). The proteins were electrotransferred to Immobilon P and probed with anti-human apoA-I (FIG. 13A-13B) or anti-rat species specific antibody (FIG. 13C) and goat anti-rabbit HRP conjugated antibody.

FIGs. 14A-14B. Endogenous rat apoA-I follows the distribution of human apoA-I in stably transfected wild-type (FIG. 14A) or PCS mutant human apoA-Is (FIG. 14B). The density gradient fractions were probed with a rabbit anti-rat species-specific antibody and goat anti-rabbit HRP conjugated antibody. After exposure by chemiluminescence, the relative amount of rat apoA-I was determined using Alphalmager Software. Note, indicated at the base of the X-axis is the density range of human HDL subclasses and free protein on this density gradient.

FIG. 15. Endogenous rat apoA-I largely cohabitates with HDL that mimic native rat HDL. 400μl of each HDL density peak generated McA7⁴ cells was added to 40μl of 50% slurry of protein A-sepharose beads. The fraction and beads were centrifuged and the supernatant was transferred to a new tube. 1.5μl of species-specific human apoA-I antibody was added to the supernatant and then rotated head to bottom at 4°C overnight. 50μl of 50% slurry of protein A-sepharose beads was added and rotated head to bottom at 4°C for 2hr. The beads were precipitated by centrifugation and the supernatant carefully removed. The beads were washed 3X with IXPBS and 20μl of 6X SDS-sample buffer plus 80μl of dH₂O were added to the beads and heated at 95°C for 5min, then put back on ice for 2min. The mixture was centrifuged and transferred to a fresh tube, where it was then run on a 4-20% SDS-PAGE, transferred to immobilon P and immunodecorated with species-specific rat apoA-I antibody.

FIG. 16. Wild-type and PCS mutant human apoA-I's have an equal capacity for cholesterol efflux from J774 macrophages at high protein concentrations. J774 macrophages were loaded with cholesterol by incubation with [³H] cholesterol and 25μg/mL Ac-LDL in the presence of an ACAT inhibitor as described in methods. Following the labeling period, the cells were washed and incubated with CPT-cAMP to specifically up-regulate ABCA-I. Lipid efflux was initiated with the application of ImL total volume/well DMEM in the presence or absence of wild-type/mutant human apoA-I protein (0-16μg). After 4hrs, the media were removed and the radioactivity was measured comparing counts of media over total counts (cells + media) x 100. DETAILED DESCRIPTION I. ApoA-1

The amino acid sequence of the 243 residue mature human apoA-I polypeptide contains ten amino acid repeats of 11 or 22 amino acids that are organized as amphipathic α- helices (Frank and Marcel, 2000; Ajees et al., 2006). The overall structure of human apoA-I is illustrated schematically in FIG. 1, which shows the ten amphipathic α-helices separated by proline containing sequences ("PCS") or the non-proline containing sequence ("NPCS") between helices 7 and 8. Structure/function relationships have been identified for specific helical repeats within human apoA-I. Helices 1, 9, and 10 are essential for initial lipid binding (Davidson et al., 1996; Saito et al., 2004) and helices 6 and partly 7 are critical for maximal activation of LCAT (Sorci-Thomas et al., 1998). Two crystal structures of lipid- free human apoA-I have been reported. The crystal structure forms a horseshoe shape. The crystal structure of the whole protein forms a four helix bundle N-terminus and a smaller C-terminal domain (Ajees et al., 2006). Experiments indicate that the N-terminus of lipid-free apoA-I forms a stable four-helix bundle, while the C-terminus forms a hydrophobic random coil.

An in vitro binding assay can be used to detect the association of mouse and human apoA-I with mature human HDL subclasses (Reschly et al., 2002). In the assay, murine apoA-I associates preferentially with HDL₂ while the human apoA-I associates equally well with HDL₂ and HD L₃. The affinity for HDL subclasses parallels the size and density of HDL in human and mouse plasma, with humans forming distinct HDL subclasses and mouse forming monodisperse HDL particles. When the human sequence from residues 166-209 (helices 7 and 8) are substituted for the homologous sequence in murine apoA-I, a chimeric molecule is produced that shows affinity for both HDL₂ and HD L3 in the in vitro assay, thus resembling human apoA-I in lipoprotein association properties (Reschly et al., 2002).

In some embodiments of the present invention, the isolated polypeptide comprises amino acids 25-267 of the human apoA-I amino acid sequence (SEQ ID NO:1), with a substitution at amino acids corresponding to amino acids 208-214 of the human apoA-I amino acid sequence. Amino acids 25-267 represents the mature apoA-I polypeptide. The substitution occurs in the interhelical region between helices 7 and 8 of human apoA-I. In human apoA-I, this interhelical region lacks a proline residue. In the polypeptide embodiments, an amino acid sequence comprising a proline residue is substituted in this interhelical region. In other embodiments, the isolated polypeptide comprises a polypeptide sequence at least 90% identical, and more particularly at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical, to the mature apoA-I amino acid sequence, with a substitution at amino acids corresponding to amino acids 208-214 of the human apoA-I amino acid sequence. The substitution comprises an amino acid sequence containing a proline residue. Any such polypeptide sequence is contemplated as long as the isolated polypeptide containing the polypeptide sequence preferentially associates with an HDL subclass.

Percent identity can be calculated using any published algorithm. For example, alignment of sequences for comparison may be conducted by the local homology algorithm of Smith and Waterman (1981), by the homology alignment algorithm of Needleman and Wunsch (1970), by the search for similarity method of Pearson and Lipman (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), the Best Fit sequence program described by Devereux et al. (1984), preferably using the default settings, or by inspection. Preferably, percent identity is calculated by using alignment tools known to and readily ascertainable to those of skill in the art.

In certain embodiments, the isolated polypeptide comprises a sequence about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85% or greater than 85% identical, to the mature apoA-I amino acid sequence, with a substitution at amino acids corresponding to amino acids 208-214 of the human apoA-I amino acid sequence.

An apoA-I sequence included herein, whether based on the human apoA-I sequence of SEQ ID NO:1 or on a sequence at least 90% or more identical to SEQ ID NO:1, can be referred to as an "altered" apoA-I sequence. In this context, the term "altered" refers to the substitution of a proline-containing sequence at amino acids corresponding to amino acids 208-214 of SEQ ID NO:1. The proline-containing sequence can be any sequence containing at least one proline residue so long as the isolated polypeptide incorporating the proline- containing sequence preferentially associates with an HDL subclass. For example, in embodiments where amino acids 208-214 of SEQ ID NO:1 are substituted with a proline- containing sequence of seven amino acids, one of the seven amino acids is proline, each of the six remaining positions can be any one of the twenty naturally encoded amino acids, and any combination of amino acids in the six remaining positions is provided as long as the isolated polypeptide preferentially associates with an HDL subclass. For instance, in particular embodiments where the proline-containing sequence in the isolated polypeptide is represented as X₁X₂XsPX₄XsXe, each of residues X₁ - X₆ can be any one of the twenty naturally encoded amino acids, and the proline-containing sequence can include any combination of X₁ - X₆ amino acids as long as the isolated polypeptide preferentially associates with an HDL subclass.

In certain embodiments, the isolated polypeptide can be 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, about 300, about 325, about 350, about 375, about 400, about 425, about 450, about 475, about 500, about 525, about 550, about 575, about 600, about 625, about 650, about 675, about 700, about 725, about 750, about 775, about 800, about 825, about 850, about 875, about 900, about 925, about 950, about 975, about 1000, about 1100, about 1200, about 1300, about 1400, about 1500, about 1750, about 2000, about 2250, about 2500 or greater amino acid residues, or any range derivable therein. In other embodiments, the isolated polypeptide can be about 25, about 50, about 75, about 100, about 125, about 150, about 175, about 200, about 225 or greater amino acid residues, or any range derivable therein.

As used herein, the term "preferentially associates" refers to the preferential association of a polypeptide of the present invention with an HDL subclass in vitro and/or in vivo. Using various methods of separation, such as zonal and analytical centrifugation, differential precipitation, gradient gel electrophoresis, or adsorption chromatography, different subclasses of HDL can be obtained. A polypeptide preferentially associates with an HDL subclass when the affinity of the polypeptide for one HDL subclass is greater than the affinity of the polypeptide for other HDL subclasses (within experimental error).

II. Variants Polypeptides containing altered apoA-I sequences can be based on a sequence at least

90% identical to amino acids 25-267 of SEQ ID NO:1. Such polypeptides can be considered as sequence variants of SEQ ID NO:1. Any sequence variant of amino acids 25-267 of SEQ ID NO:1 is included in the present invention as long as the isolated polypeptide containing the sequence variant preferentially associates with an HDL subclass. Amino acid sequence variants of the polypeptides of the present invention can be substitutional, insertional or deletion variants. Deletion variants lack one or more residues of the native protein that are not essential for function, and are exemplified by variants lacking secretory signal sequences or signal sequences directing a protein to bind to a particular part of a cell. Insertional mutants typically involve the addition of material at a non-terminal point in the polypeptide. This may include the insertion of an immunoreactive epitope or simply a single residue. Terminal additions, called fusion proteins, are discussed below.

Substitutional variants typically contain the exchange of one amino acid for another at one or more sites within the protein, and may be designed to modulate one or more properties of the polypeptide, such as stability against proteolytic cleavage, without the loss of other functions or properties. Substitutions of this kind preferably are conservative, that is, one amino acid is replaced with one of similar shape and charge. Conservative substitutions are well known in the art and include, for example, the changes of: alanine to serine; arginine to lysine; asparagine to glutamine or histidine; aspartate to glutamate; cysteine to serine; glutamine to asparagine; glutamate to aspartate; glycine to proline; histidine to asparagine or glutamine; isoleucine to leucine or valine; leucine to valine or isoleucine; lysine to arginine; methionine to leucine or isoleucine; phenylalanine to tyrosine, leucine or methionine; serine to threonine; threonine to serine; tryptophan to tyrosine; tyrosine to tryptophan or phenylalanine; and valine to isoleucine or leucine.

In various embodiments, a sequence variant of amino acids 25-267 SEQ ID NO:1 can be 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280 or greater amino acid residues, or any range derivable therein. In other embodiments, a sequence variant can be about 25, about 50, about 75, about 100, about 125, about 150, about 175, about 200, about 225 or greater amino acid residues, or any range derivable therein.

In certain embodiments, a sequence variant can be about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85% or greater than 85% identical to amino acids 25-267 SEQ ID NO: 1.

The foregoing discussion concerning sequence variants of amino acids 25-267 of SEQ

ID NO:1 can also be applied more narrowly to the amino acid sequence corresponding to amino acids 208-214 of SEQ ID NO:1. That is, the pro line-containing sequence substituted for amino acids 208-214 can be considered a sequence variant of amino acids 208-214 of SEQ

ID NO:1. Viewed in this way, in some embodiments, the pro line-containing sequence can contain an amino acid substitution, or a biologically functionally equivalent amino acid substitution, for one or more of the seven positions corresponding to amino acids 208-214 of SEQ ID NO:1. Also, in some embodiments, the pro line-containing sequence can be an insertional or deletion variant of amino acids 208-214 of SEQ ID NO:1. Any substitutional, insertional or deletion variant of amino acids 208-214 of SEQ ID NO:1 is contemplated so long as the variant sequence contains a proline residue and the isolated polypeptide incorporating the variant sequence can associate preferentially with an HDL subclass. Thus, the variant sequence can be, but is not limited to, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or greater amino acid residues, or any range derivable therein.

In certain embodiments, the proline-containing variant sequence is about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95% or greater than 95% identical to amino acids 208-214 of SEQ ID NO:1.

In some embodiments, functionally equivalent amino acid substitutions are preferred. The term "biologically functional equivalent" is well understood in the art and signifies amino acids with similar chemical and biological properties. For example, certain amino acids may be substituted for other amino acids in a protein structure without appreciable loss of interactive binding capacity with structures such as, for example, binding sites on substrate molecules. Since it is the interactive capacity and nature of a protein that defines that protein's biological functional activity, certain amino acid substitutions can be made in a protein sequence and nevertheless produce a protein with like properties. It is thus contemplated by the inventors that various changes can be made in a protein sequence without appreciable loss of its biological utility or activity.

It also will be understood that amino acid sequences can include additional residues, such as additional N- or C-terminal amino acids, and yet still be essentially as set forth in one of the sequences disclosed herein, so long as the sequence meets the criteria set forth above, including the maintenance of biological protein activity where protein expression is concerned.

In making such changes, the hydropathic index of amino acids may be considered. The importance of the hydropathic amino acid index in conferring interactive biologic function on a protein is generally understood in the art (Kyte & Doolittle, 1982). It is accepted that the relative hydropathic character of the amino acid contributes to the secondary structure of the resultant protein, which in turn defines the interaction of the protein with other molecules, for example, enzymes, substrates, receptors, DNA, antibodies, antigens, and the like.

It also is understood in the art that the substitution of like amino acids can be made effectively on the basis of hydrophilicity. U.S. Patent 4,554,101, incorporated herein by reference, states that the greatest local average hydrophilicity of a protein, as governed by the hydrophilicity of its adjacent amino acids, correlates with a biological property of the protein.

As detailed in U.S. Patent 4,554,101, the following hydrophilicity values have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0 ± 1); glutamate (+3.0 ± 1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-

0.5 ± 1); alanine (-0.5); histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4).

It is understood that an amino acid can be substituted for another having a similar hydrophilicity value and still produce a biologically equivalent and immunologically equivalent protein. In such changes, the substitution of amino acids whose hydrophilicity values are within ±2 is preferred, those that are within ±1 are particularly preferred, and those within ±0.5 are even more particularly preferred.

As outlined above, amino acid substitutions generally are based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like. Exemplary substitutions that take into consideration the various foregoing characteristics are well known to those of skill in the art and include: arginine and lysine; glutamate and aspartate; serine and threonine; glutamine and asparagine; and valine, leucine and isoleucine.

III. Fusion Proteins A specialized kind of insertional variant is the fusion protein. This molecule generally has all or a substantial portion of the native molecule, linked at the N- or C-terminus, to all or a portion of a second polypeptide. For example, fusions typically employ leader sequences from other species to permit the recombinant expression of a protein in a heterologous host. Another useful fusion includes the addition of an immunologically active domain, such as an antibody epitope, to facilitate purification of the fusion protein. Inclusion of a cleavage site at or near the fusion junction will facilitate removal of the extraneous polypeptide after purification. Other useful fusions include linking of functional domains, such as active sites from enzymes such as a hydrolase, glycosylation domains, cellular targeting signals, multimerization domains, or transmembrane regions.

A. Multimeric Forms U.S. patent publication No. 20030181372, hereby incorporated by reference, describes naturally occurring variants and artificially generated mutants of human apoA-I that have one or more cysteine substitutions at various amino acid positions in the protein. As described, cysteine substitutions can occur at residues 7, 10, 13, 17, 20, 22, 27, 30, 33, 39, 45, 49, 54, 58, 61, 96, 123, 131, 215 and 238 of human apoA-I. The cysteine substitutions can mediate dimer formation of the apoA-I polypeptide via the creation of disulphide bonds. Similarly, in certain embodiments of the present invention, the altered apoA-I sequence can comprise one or more cysteine substitutions at amino acids corresponding to amino acids 27, 30, 33, 39, 45, 49, 54, 58, 61, 96, 123, 131, 215 and 238 of SEQ ID NO:1. Each cysteine substitution can mediate the formation of dimers of the isolated polypeptide.

In some embodiments, the addition of a trimerising module mediates the formation of trimers of the isolated polypeptide. Various examples of trimerising modules are described in U.S. Patent No. 6,897,039, hereby incorporated by reference. One example of a trimerising module is the tetranectin trimerising structural element, which is also described in detail in WO 98/56906, hereby incorporated by reference. The trimerising activity of the tetranectin trimerising structural element is due to a coiled coil structure that can interact with the coiled coil structure of two other tetranectin trimerising structural elements to form a stable trimer. Another example, which is also described in WO 95/31540, hereby incorporated by reference, is the neck region of collectin polypeptides. In general, a trimerising module can be based on any trimerising domain found in proteins that naturally form trimers. In some embodiments of the present invention, the altered apoA-I sequence can comprise a trimerising module, and thus these embodiments can form trimers. In particular embodiments, the trimerising module is the tetranectin trimerising structural element or the neck region of a collectin polypeptide.

B. Protein Purification

Protein purification or isolation techniques are well known to those of skill in the art. These techniques involve, at one level, the crude fractionation of the cellular milieu to polypeptide and non-polypeptide fractions. Having separated the polypeptide from other proteins, the polypeptide of interest may be further purified using chromatographic and electrophoretic techniques to achieve partial or complete purification (or purification to homogeneity). Analytical methods particularly suited to the preparation of a pure peptide are ion-exchange chromatography, exclusion chromatography, polyacrylamide gel electrophoresis, and isoelectric focusing. A particularly efficient method of purifying peptides is fast protein liquid chromatography or even HPLC.

Certain aspects of the present invention concern the purification, and in particular embodiments, the substantial purification, of a polypeptide or protein. As used herein, the term "isolated" or "purified" in relation to polypeptides or proteins refers to a composition, isolatable from other components, wherein the protein or peptide is purified to any degree relative to its naturally-obtainable state. An "isolated" or "purified" polypeptide or protein therefore also refers to a polypeptide or protein, free from the environment in which it may naturally occur.

Generally, "isolated" or "purified" will refer to a polypeptide or protein composition that has been subjected to fractionation to remove various other components, and which composition substantially retains its biological activity. Where the term "substantially isolated" or "substantially purified" is used, this designation will refer to a composition in which the polypeptide or protein forms the major component of the composition, such as constituting about 50%, about 60%, about 70%, about 80%, about 90%, about 95% or more of the proteins in the composition.

Various methods for quantifying the degree of purification of the polypeptide or protein will be known to those of skill in the art in light of the present disclosure. These include, for example, determining the specific activity of an active fraction, or assessing the amount of polypeptides within a fraction by SDS/PAGE analysis. A preferred method for assessing the purity of a fraction is to calculate the specific activity of the fraction, to compare it to the specific activity of the initial extract, and to thus calculate the degree of purity, herein assessed by a "-fold purification number." The actual units used to represent the amount of activity will, of course, be dependent upon the particular assay technique chosen to follow the purification and whether or not the polypeptide or protein exhibits a detectable activity.

Various techniques suitable for use in protein purification will be well known to those of skill in the art. These include, for example, precipitation with ammonium sulfate, PEG, antibodies and the like or by heat denaturation, followed by centrifugation; chromatography steps such as ion exchange, gel filtration, reverse phase, hydroxylapatite and affinity chromatography; isoelectric focusing; gel electrophoresis; and combinations of such and other techniques. As is generally known in the art, it is believed that the order of conducting the various purification steps may be changed, or that certain steps may be omitted, and still result in a suitable method for the preparation of a substantially purified polypeptide or protein.

There is no general requirement that the polypeptide or protein always be provided in their most purified state. Indeed, it is contemplated that less substantially purified products will have utility in certain embodiments. Partial purification may be accomplished by using fewer purification steps in combination, or by utilizing different forms of the same general purification scheme. For example, it is appreciated that a cation-exchange column chromatography performed utilizing an HPLC apparatus will generally result in a greater "- fold" purification than the same technique utilizing a low pressure chromatography system. Methods exhibiting a lower degree of relative purification may have advantages in total recovery of protein product, or in maintaining the activity of an expressed protein.

IV. Lipids

A neutral fat may comprise a glycerol and/or a fatty acid. A typical glycerol is a three carbon alcohol. A fatty acid generally is a molecule comprising a carbon chain with an acidic moiety (e.g., carboxylic acid) at an end of the chain. The carbon chain of a fatty acid may be of any length, however, it is preferred that the length of the carbon chain be of from at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, to 30 or more carbon atoms, and any range derivable therein. An example of a range is from about 8 to about 16 carbon atoms in the chain portion of the fatty acid. In certain embodiments the fatty acid carbon chain may comprise an odd number of carbon atoms, however, an even number of carbon atoms in the chain may be preferred in certain embodiments. A fatty acid comprising only single bonds in its carbon chain is called saturated, while a fatty acid comprising at least one double bond in its chain is called unsaturated. The fatty acid may be branched, though in embodiments of the present invention, it is unbranched.

Specific fatty acids include, but are not limited to, linoleic acid, oleic acid, palmitic acid, linolenic acid, stearic acid, lauric acid, myristic acid, arachidic acid, palmitoleic acid, arachidonic acid, ricinoleic acid, tuberculosteric acid, lactobacillic acid. An acidic group of one or more fatty acids is covalently bonded to one or more hydroxyl groups of a glycerol. Thus, a monoglyceride comprises a glycerol and one fatty acid, a diglyceride comprises a glycerol and two fatty acids, and a triglyceride comprises a glycerol and three fatty acids.

A phospholipid generally comprises either glycerol or a sphingosine moiety, an ionic phosphate group to produce an amphipathic compound, and one or more fatty acids. Types of phospholipids include, for example, phosphoglycerides, wherein a phosphate group is linked to the first carbon of glycerol of a diglyceride, and sphingophospholipids (e.g., sphingomyelin), wherein a phosphate group is esterified to a sphingosine amino alcohol. Another example of a sphingophospholipid is a sulfatide, which comprises an ionic sulfate group that makes the molecule amphipathic. A phospholipid may, of course, comprise further chemical groups, such as for example, an alcohol attached to the phosphate group. Examples of such alcohol groups include serine, ethanolamine, choline, glycerol and inositol. Thus, specific phosphoglycerides include a phosphatidyl serine, a phosphatidyl ethanolamine, a phosphatidyl choline, a phosphatidyl glycerol or a phosphatidyl inositol. Other phospholipids include a phosphatidic acid or a diacetyl phosphate. In one aspect, a phosphatidylcholine comprises a dioleoylphosphatidylcholine (a.k.a. cardiolipin), an egg phosphatidylcholine, a dipalmitoyl phosphalidycholine, a monomyristoyl phosphatidylcholine, a monopalmitoyl phosphatidylcholine, a monostearoyl phosphatidylcholine, a monooleoyl phosphatidylcholine, a dibutyroyl phosphatidylcholine, a divaleroyl phosphatidylcholine, a dicaproyl phosphatidylcholine, a diheptanoyl phosphatidylcholine, a dicapryloyl phosphatidyl choline, a dilinoleoyl phosphatidyl choline, or a distearoyl phosphatidyl choline.

A steroid is a four-membered ring system derivative of a phenanthrene. Steroids often possess regulatory functions in cells, tissues and organisms, and include, for example, hormones and related compounds in the progestagen (e.g., progesterone), glucocor^ticoid (e.g., Cortisol), mineralocorticoid (e.g., aldosterone), androgen (e.g., testosterone) and estrogen (e.g., estrone) families. Cholesterol is another example of a steroid, and generally serves primarily structural functions though regulatory functions may also be involved. Vitamin D is another example of a sterol, and is involved in calcium absorption from the intestine.

In some embodiments, the polypeptide comprising an altered apoA-I sequence, or multimers of the polypeptide, can be associated with one or more lipids. In certain embodiments, the lipid is phosphatidyl choline, phosphatidyl serine, phosphatidyl ethanolamine, phosphatidal choline, phosphatidal ethanolamine, alkyl acyl glycerophosphoryl choline or alkyl acyl glycerophosphoryl ethanolamine, cholesterol, cholesteryl ester, or sphingomyelin, or any combination thereof.

V. Polynucleotides Polynucleotides encoding any version of the polypeptides of the present invention are provided. For example, any polynucleotide encoding amino acids 25-267 of SEQ ID NO:1, or an amino acid sequence at least 90% identical to amino acids 25-267 of SEQ ID NO:1, is provided. Due to the degeneracy of the genetic code, the same polypeptide embodiment can be encoded by different nucleotide sequences, and any polynucleotide containing any nucleotide sequence encoding a polypeptide embodiment of the present invention is contemplated. A polynucleotide can be a DNA molecule, an RNA molecule, or any combination of the two, and can be single or double stranded. Besides encoding altered apoA-I sequences, a polynucleotide can contain non-coding regions and can encode additional amino acid sequences.

The polynucleotide sequence (cDNA) of the human apoA-I coding region is set forth in SEQ ID NO:6. In various embodiments, a polynucleotide includes the nucleotide sequence (cDNA) of 801 bases set forth in SEQ ID NO:7 or SEQ ID NO:8, each encoding an altered apoA-I polypeptide containing PCS2 (SEQ ID NO:7) or PCS3 (SEQ ID NO:8). In other embodiments, a polynucleotide includes nucleotides 73-801 of SEQ ID NO:7 or SEQ ID NO:8, which encode amino acids corresponding to amino acids 25-267 of human apoA-I. In various embodiments, any polynucleotide encoding amino acids 25-267 of SEQ ID NOs: 1, 4 and 5 is provided.

In certain embodiments, a polynucleotide encoding a polypeptide of the present invention can contain a nucleic acid sequence of 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, 1000, 1010, 1020, 1030, 1040, 1050, 1060, 1070, 1080, 1090, 1095, 1100, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 9000, 10000, or more nucleotides, nucleosides, or base pairs. In other embodiments, a polynucleotide encoding a polypeptide of the present invention can be about 75, about 100, about 150, about 200, about 250, about 300, about 350, about 400, about 450, about 500, about 550, about 600, about 650, about 700 or more nucleotides, nucleosides, or base pairs. DNA segments encoding polypeptides containing altered apoA-I sequences can be prepared using standard molecular techniques. One of skill in the art would be well equipped to manipulate DNA using standard recombinant techniques, which are described in Sambrook et al, (1989) and Ausubel et al, 1996, both incorporated herein by reference. Such methods can also be used to prepare DNA segments encoding polypeptides at least 90% identical to amino acids 25-267 of SEQ ID NO: 1.

A. Expression Vectors

Expression vectors for apo-lipoprotein expression in a cell are included within the scope of the present invention. In various embodiments, the expression vector includes a promoter active in a cell and operably linked to a DNA segment encoding a polypeptide containing an altered apoA-I sequence. The cell for expression is preferably a liver cell, macrophage (including a Kupffer cell), lymphocyte, enterocyte, smooth muscle cell or endothelial cell. In some embodiments, the expression vector includes a signal peptide encoding DNA segment linked to the DNA segment encoding the isolated polypeptide such that the isolated polypeptide is secreted from the cell. In certain embodiments, the signal peptide can be the apoA-I signal peptide set forth in amino acids 1-18 of SEQ ID NO:1. The expression vector can be used for various purposes, including gene therapy in a patient, or the production of polypeptide in cell cultures.

1. Vectors Native and modified polypeptides may be encoded by a nucleic acid molecule comprised in a vector. The term "vector" is used to refer to a carrier nucleic acid molecule into which a nucleic acid sequence can be inserted for introduction into a cell where it can be replicated. A nucleic acid sequence can be "exogenous," which means that it is foreign to the cell into which the vector is being introduced or that the sequence is homologous to a sequence in the cell but in a position within the host cell nucleic acid in which the sequence is ordinarily not found. Vectors include plasmids, cosmids, viruses (bacteriophage, animal viruses, and plant viruses), and artificial chromosomes {e.g., YACs). One of skill in the art would be well equipped to construct a vector through standard recombinant techniques, which are described in Sambrook et al (1989) and Ausubel et al. (1996), both incorporated herein by reference. In addition to encoding a modified polypeptide such as variant apo-lipoprotein, a vector may encode non-modified polypeptide sequences such as a tag or targetting molecule. Useful vectors encoding such fusion proteins include pIN vectors (Inouye et al, 1985), vectors encoding a stretch of histidines, and pGEX vectors, for use in generating glutathione S-transferase (GST) soluble fusion proteins for later purification and separation or cleavage. A targetting molecule is one that directs the expressed polypeptide to a particular organ, tissue, cell, or other location in a subject's body.

The term "expression vector" refers to a vector containing a nucleic acid sequence coding for at least part of a gene product capable of being transcribed. In some cases, RNA molecules are then translated into a protein, polypeptide, or peptide. In other cases, these sequences are not translated, for example, in the production of antisense molecules or ribozymes. Expression vectors can contain a variety of "control sequences," which refer to nucleic acid sequences necessary for the transcription and possibly translation of an operably linked coding sequence in a particular host organism. In addition to control sequences that govern transcription and translation, vectors and expression vectors may contain nucleic acid sequences that serve other functions as well and are described infra.

a. Promoters and Enhancers A "promoter" is a control sequence that is a region of a nucleic acid sequence at which initiation and rate of transcription are controlled. It may contain genetic elements at which regulatory proteins and molecules may bind such as RNA polymerase and other transcription factors. The phrases "operatively positioned," "operatively linked," "under control," and "under transcriptional control" mean that a promoter is in a correct functional location and/or orientation in relation to a nucleic acid sequence to control transcriptional initiation and/or expression of that sequence. A promoter may or may not be used in conjunction with an "enhancer," which refers to a cis-acting regulatory sequence involved in the transcriptional activation of a nucleic acid sequence.

A promoter may be one naturally associated with a gene or sequence, as may be obtained by isolating the 5' non-coding sequences located upstream of the coding segment and/or exon. Such a promoter can be referred to as "endogenous." Similarly, an enhancer may be one naturally associated with a nucleic acid sequence, located either downstream or upstream of that sequence. Alternatively, certain advantages will be gained by positioning the coding nucleic acid segment under the control of a recombinant or heterologous promoter, which refers to a promoter that is not normally associated with a nucleic acid sequence in its natural environment. A recombinant or heterologous enhancer refers also to an enhancer not normally associated with a nucleic acid sequence in its natural environment. Such promoters or enhancers may include promoters or enhancers of other genes, and promoters or enhancers isolated from any other prokaryotic, viral, or eukaryotic cell, and promoters or enhancers not "naturally occurring," i.e., containing different elements of different transcriptional regulatory regions, and/or mutations that alter expression. In addition to producing nucleic acid sequences of promoters and enhancers synthetically, sequences may be produced using recombinant cloning and/or nucleic acid amplification technology, including PCR™, in connection with the compositions disclosed herein (see U.S. Patent 4,683,202, U.S. Patent 5,928,906, each incorporated herein by reference). Furthermore, it is contemplated the control sequences that direct transcription and/or expression of sequences within non-nuclear organelles such as mitochondria, chloroplasts, and the like, can be employed as well.

Naturally, it may be important to employ a promoter and/or enhancer that effectively directs the expression of the DNA segment in the cell type, organelle, and organism chosen for expression. Those of skill in the art of molecular biology generally know the use of promoters, enhancers, and cell type combinations for protein expression, for example, see Sambrook et al. (1989), incorporated herein by reference. The promoters employed may be constitutive, tissue-specific, inducible, and/or useful under the appropriate conditions to direct high level expression of the introduced DNA segment, such as is advantageous in the large- scale production of recombinant proteins and/or peptides. The promoter may be heterologous or endogenous.

Table 1 lists several elements/promoters that may be employed, in the context of the present invention, to regulate the expression of a gene. This list is not intended to be exhaustive of all the possible elements involved in the promotion of expression but, merely, to be exemplary thereof. Table 2 provides examples of inducible elements, which are regions of a nucleic acid sequence that can be activated in response to a specific stimulus.

The identity of tissue-specific promoters or elements, as well as assays to characterize their activity, is well known to those of skill in the art. Examples of such regions include the human LIMK2 gene (Nomoto et al. 1999), the somatostatin receptor 2 gene (Kraus et al., 1998), murine epididymal retinoic acid-binding gene (Lareyre et al., 1999), human CD4 (Zhao-Emonet et al., 1998), mouse alpha2 (XI) collagen (Tsumaki, et al., 1998), DlA dopamine receptor gene (Lee, et al., 1997), insulin- like growth factor II (Wu et al., 1997), human platelet endothelial cell adhesion molecule- 1 (Almendro et al., 1996), and the SM22α promoter.

Also contemplated as useful in the present invention are the dectin-1 and dectin-2 promoters. Additional viral promoters, cellular promoters/enhancers and inducible promoters/enhancers that could be used in combination with the present invention are listed in Tables 1 and 2. Additionally any promoter/enhancer combination (as per the Eukaryotic Promoter Data Base EPDB) could also be used to drive expression of structural genes encoding oligosaccharide processing enzymes, protein folding accessory proteins, selectable marker proteins or a heterologous protein of interest. Alternatively, a tissue-specific promoter (Tables 3 and Table 4) may be employed with the nucleic acid molecules of the present invention.

TABLE 3: Candidate Tissue-S ecific Promoters

TABLE 4: Candidate Promoters

b. Initiation Signals and Internal Ribosome Binding Sites

A specific initiation signal also may be required for efficient translation of coding sequences. These signals include the ATG initiation codon or adjacent sequences.

Exogenous translational control signals, including the ATG initiation codon, may need to be provided. One of ordinary skill in the art would readily be capable of determining this and providing the necessary signals. It is well known that the initiation codon must be "in-frame" with the reading frame of the desired coding sequence to ensure translation of the entire insert.

The exogenous translational control signals and initiation codons can be either natural or synthetic. The efficiency of expression may be enhanced by the inclusion of appropriate transcription enhancer elements.

In certain embodiments of the invention, the use of internal ribosome entry sites (IRES) elements are used to create multigene, or polycistronic, messages. IRES elements are able to bypass the ribosome scanning model of 5'- methylated Cap dependent translation and begin translation at internal sites (Pelletier and Sonenberg, 1988). IRES elements from two members of the picornavirus family (polio and encephalomyo carditis) have been described (Pelletier and Sonenberg, 1988), as well an IRES from a mammalian message (Macejak and Sarnow, 1991). IRES elements can be linked to heterologous open reading frames. Multiple open reading frames can be transcribed together, each separated by an IRES, creating polycistronic messages. By virtue of the IRES element, each open reading frame is accessible to ribosomes for efficient translation. Multiple genes can be efficiently expressed using a single promoter/enhancer to transcribe a single message (see U.S. Patents 5,925,565 and 5,935,819, herein incorporated by reference).

c. Multiple Cloning Sites

Vectors can include a multiple cloning site (MCS), which is a nucleic acid region that contains multiple restriction enzyme sites, any of which can be used in conjunction with standard recombinant technology to digest the vector. (See Carbonelli et ah, 1999, Levenson et al, 1998, and Cocea, 1997, incorporated herein by reference.) "Restriction enzyme digestion" refers to catalytic cleavage of a nucleic acid molecule with an enzyme that functions only at specific locations in a nucleic acid molecule. Many of these restriction enzymes are commercially available. Use of such enzymes is widely understood by those of skill in the art. Frequently, a vector is linearized or fragmented using a restriction enzyme that cuts within the MCS to enable exogenous sequences to be ligated to the vector. "Ligation" refers to the process of forming phosphodiester bonds between two nucleic acid fragments, which may or may not be contiguous with each other. Techniques involving restriction enzymes and ligation reactions are well known to those of skill in the art of recombinant technology.

d. Splicing Sites

Most transcribed eukaryotic RNA molecules will undergo RNA splicing to remove introns from the primary transcripts. Vectors containing genomic eukaryotic sequences may require donor and/or acceptor splicing sites to ensure proper processing of the transcript for protein expression. (See Chandler et al, 1997, incorporated herein by reference.)

e. Termination Signals

The vectors or constructs of the present invention will generally comprise at least one termination signal. A "termination signal" or "terminator" is comprised of the DNA sequences involved in specific termination of an RNA transcript by an RNA polymerase. Thus, in certain embodiments a termination signal that ends the production of an RNA transcript is contemplated. A terminator may be necessary in vivo to achieve desirable message levels.

In eukaryotic systems, the terminator region may also comprise specific DNA sequences that permit site-specific cleavage of the new transcript so as to expose a polyadenylation site. This signals a specialized endogenous polymerase to add a stretch of about 200 A residues (polyA) to the 3 ' end of the transcript. RNA molecules modified with this polyA tail appear to be more stable and are translated more efficiently. Thus, in other embodiments involving eukaryotes, it is preferred that the terminator comprises a signal for the cleavage of the RNA, and it is more preferred that the terminator signal promotes polyadenylation of the message. The terminator and/or polyadenylation site elements can serve to enhance message levels and/or to minimize read through from the cassette into other sequences.

Terminators contemplated for use in the invention include any known terminator of transcription described herein or known to one of ordinary skill in the art, including but not limited to, for example, the termination sequences of genes, such as for example the bovine growth hormone terminator or viral termination sequences, such as for example the SV40 terminator. In certain embodiments, the termination signal may be a lack of transcribable or translatable sequence, such as due to a sequence truncation.

f. Polyadenylation Signals

In expression, particularly eukaryotic expression, one will typically include a polyadenylation signal to effect proper polyadenylation of the transcript. The nature of the polyadenylation signal is not believed to be crucial to the successful practice of the invention, and/or any such sequence may be employed. Preferred embodiments include the SV40 polyadenylation signal and/or the bovine growth hormone polyadenylation signal, which are convenient and/or known to function well in various target cells. Polyadenylation may increase the stability of the transcript or may facilitate cytoplasmic transport.

g. Origins of Replication

In order to propagate a vector in a host cell, it may contain one or more origin of replication sites (often termed "ori"), which is a specific nucleic acid sequence at which replication is initiated. Alternatively an autonomously replicating sequence (ARS) can be employed if the host cell is yeast.

h. Selectable and Screenable Markers

In certain embodiments of the invention, cells containing a nucleic acid construct of the present invention may be identified in vitro or in vivo by including a marker in the expression vector. Such markers would confer an identifiable change to the cell permitting easy identification of cells containing the expression vector. Generally, a selectable marker is one that confers a property that allows for selection. A positive selectable marker is one in which the presence of the marker allows for its selection, while a negative selectable marker is one in which its presence prevents its selection. An example of a positive selectable marker is a drug resistance marker.

Usually the inclusion of a drug selection marker aids in the cloning and identification of transformants, for example, genes that confer resistance to neomycin, puromycin, hygromycin, DHFR, GPT, zeocin and histidinol are useful selectable markers. In addition to markers conferring a phenotype that allows for the discrimination of transformants based on the implementation of conditions, other types of markers including screenable markers such as GFP, whose basis is colorimetric analysis, are also contemplated. Alternatively, screenable enzymes such as herpes simplex virus thymidine kinase (tk) or chloramphenicol acetyltransferase (CAT) may be utilized. One of skill in the art would also know how to employ immunologic markers, possibly in conjunction with FACS analysis. The marker used is not believed to be important, so long as it is capable of being expressed simultaneously with the nucleic acid encoding a gene product. Further examples of selectable and screenable markers are well known to one of skill in the art.

2. Host Cells

As used herein, the terms "cell," "cell line," and "cell culture" may be used interchangeably. All of these terms also include their progeny, which is any and all subsequent generations. It is understood that all progeny may not be identical due to deliberate or inadvertent mutations. In the context of expressing a heterologous nucleic acid sequence, "host cell" refers to a prokaryotic or eukaryotic cell, and it includes any transformable organism that is capable of replicating a vector and/or expressing a heterologous gene encoded by a vector. A host cell can, and has been, used as a recipient for vectors. A host cell may be "transfected" or "transformed," which refers to a process by which exogenous nucleic acid, such as a modified protein-encoding sequence, is transferred or introduced into the host cell. A transformed cell includes the primary subject cell and its progeny.

Host cells may be derived from prokaryotes or eukaryotes, including yeast cells, insect cells, and mammalian cells, depending upon whether the desired result is replication of the vector or expression of part or all of the vector-encoded nucleic acid sequences. Numerous cell lines and cultures are available for use as a host cell, and they can be obtained through the American Type Culture Collection (ATCC), which is an organization that serves as an archive for living cultures and genetic materials (on the world wide web at atcc.org). An appropriate host can be determined by one of skill in the art based on the vector backbone and the desired result. A plasmid or cosmid, for example, can be introduced into a prokaryote host cell for replication of many vectors. Bacterial cells used as host cells for vector replication and/or expression include DH5α, JM 109, and KC8, as well as a number of commercially available bacterial hosts such as SURE^® Competent Cells and Solopack™ Gold Cells (Stratagene^®, La Jolla, CA). Alternatively, bacterial cells such as E. coli LE392 could be used as host cells for phage viruses. Appropriate yeast cells include Saccharomyces cerevisiae, Saccharomyces pombe, and Pichia pastoris. Examples of eukaryotic host cells for replication and/or expression of a vector include

HeLa, NIH3T3, Jurkat, 293, Cos, CHO, Saos, and PC 12. Many host cells from various cell types and organisms are available and would be known to one of skill in the art. Similarly, a viral vector may be used in conjunction with either a eukaryotic or prokaryotic host cell, particularly one that is permissive for replication or expression of the vector.

Some vectors may employ control sequences that allow it to be replicated and/or expressed in both prokaryotic and eukaryotic cells. One of skill in the art would further understand the conditions under which to incubate all of the above described host cells to maintain them and to permit replication of a vector. Also understood and known are techniques and conditions that would allow large-scale production of vectors, as well as production of the nucleic acids encoded by vectors and their cognate polypeptides, proteins, or peptides.

3. Expression Systems

Numerous expression systems exist that comprise at least a part or all of the compositions discussed above. Prokaryote- and/or eukaryote -based systems can be employed for use with the present invention to produce nucleic acid sequences, or their cognate polypeptides, proteins and peptides. Many such systems are commercially and widely available.

The insect cell/baculovirus system can produce a high level of protein expression froma heterologous nucleic acid segment, such as described in U.S. Patent No. 5,871,986,

4,879,236, both herein incorporated by reference, and which can be bought, for example, under the name MaxBac^® 2.0 from Invitrogen^® and BacPack™ Baculovirus Expression

System From Clontech^®.

In addition to the disclosed expression systems of the invention, other examples of expression systems include Stratagene^®'s Complete Control™ Inducible Mammalian

Expression System, which involves a synthetic ecdysone -inducible receptor, or its pET

Expression System, an E. coli expression system. Another example of an inducible expression system is available from Invitrogen^®, which carries the T-Rex™ (tetracycline- regulated expression) System, an inducible mammalian expression system that uses the full- length CMV promoter. Invitrogen^® also provides a yeast expression system called the Pichia methanolica Expression System, which is designed for high-level production of recombinant proteins in the methylotrophic yeast Pichia methanolica. One of skill in the art would know how to express a vector, such as an expression construct, to produce a nucleic acid sequence or its cognate polypeptide, protein, or peptide.

4. Viral Vectors There are a number of ways in which expression vectors may be introduced into cells.

In certain embodiments of the invention, the expression vector comprises a virus or engineered vector derived from a viral genome. The ability of certain viruses to enter cells via receptor-mediated endocytosis, to integrate into host cell genome and express viral genes stably and efficiently have made them attractive candidates for the transfer of foreign genes into mammalian cells (Ridgeway, 1988; Nicolas and Rubenstein, 1988; Baichwal and Sugden, 1986; Temin, 1986). The first viruses used as gene vectors were DNA viruses including the papovaviruses (simian virus 40, bovine papilloma virus, and polyoma) (Ridgeway, 1988; Baichwal and Sugden, 1986) and adenoviruses (Ridgeway, 1988; Baichwal and Sugden, 1986). These have a relatively low capacity for foreign DNA sequences and have a restricted host spectrum. Furthermore, their oncogenic potential and cytopathic effects in permissive cells raise safety concerns. They can accommodate only up to 8 kb of foreign genetic material but can be readily introduced in a variety of cell lines and laboratory animals (Nicolas and Rubenstein, 1988; Temin, 1986).

The retroviruses are a group of single-stranded RNA viruses characterized by an ability to convert their RNA to double-stranded DNA in infected cells; they can also be used as vectors. It is also contemplated that lentiviral vectors can be used as expression vectors.

Lentiviruses belong to the retrovirus family but differ from simple retroviruses in that they are able to transducer and productively infect non-dividing cells, such as resting T-cells, dendritic cells, macrophages, skeletal or cardiac muscles, neural cells, hepatocytes and various cells of the hematopoietic system (Zufferey et al , 1997; Zufferey et al , 1998; Pfeifer, 2004; Dodart et al, 2005; Mautino et al, 2002; Miyoshi et al, 1998; Naldini et al, 1996; Naldini, 1998; and

US patent 6,013,516, all incorporated by reference herein). Other viral vectors may be employed as expression constructs in the present invention. Vectors derived from viruses such as vaccinia virus (Ridgeway, 1988; Baichwal and Sugden, 1986; Coupar et al, 1988) adeno-associated virus (AAV) (Ridgeway, 1988; Baichwal and Sugden, 1986; Hermonat and

Muzycska, 1984) and herpesviruses may be employed. They offer several attractive features for various mammalian cells (Friedmann, 1989; Ridgeway, 1988; Baichwal and Sugden, 1986; Coiφar et ai, 1988; Horwich et al, 1990).

VI. Pharmaceutical Formulations, Delivery, and Treatment Regimens

In another aspect of the present invention, pharmaceutical compositions are provided that include a polypeptide of the present invention, or multimer of the polypeptide, along with a pharmaceutically acceptable carrier. In various embodiments, the pharmaceutical composition can in addition contain a lipid bound to the isolated polypeptide or multimer. In some embodiments, the lipid is phosphatidyl choline, phospatidyl serine, phosphatidyl ethanolamine, phosphatidal choline, phosphatidal ethanolamine, alkyl acyl glycerophosphoryl choline or alkyl acyl glycerophosphoryl ethanolamine, or any combination of these lipids. In various embodiments, the pharmaceutical composition can further include cholesterol, cholesteryl ester, sphingomyelin, or any combination of these lipids.

Methods of modulating HDL in a subject in need of such modulation are also provided. In various embodiments, the method includes administering to a subject a polypeptide of the present invention, or multimer of the polypeptide, in an amount sufficient to modulate HDL. The administration can result in modulating HDL by altering the distribution, composition or level of an HDL subclass, such as HDL₂ or HD L₃. The composition of an HDL subclass can be altered by changing the amount and/or type of lipid in an HDL subclass, by changing the amount and/or type of protein in an HDL subclass, or by changing both lipid and protein composition in an HDL subclass. For example, the amount of apoE associated with HDL₂ may be altered when a particular polypeptide containing an altered apoA-I sequence is administered. Modulation of HDL can also include increasing the ability of an HDL subclass to package lipid and protein into an HDL particle, which can lead to enhanced absorption of cholesterol from the gut. The level of an HDL subclass can be altered by increasing or decreasing the amount of the subclass in the subject, where the amount of a subclass can be expressed as milligrams per deciliter of blood, for example. In some embodiments, the method includes the step of identifying a subject in need of modulation before administration of the polypeptide or multimer. In some embodiments, the method includes determining the composition, level and/or distribution of an HDL subclass in a subject before and/or after administration.

The polypeptide or multimer can be administered as part of a pharmaceutical composition, and can contain bound lipid. Alternatively, the isolated polypeptide or multimer can be provided by an expression vector delivered to the subject. In some embodiments, the expression vector is a viral vector, which can be a vaccinia virus, adenovirus, retrovirus, herpes virus cytomegalovirus, lentivirus or adenovirus-associated virus vector.

In another aspect, methods of treating a vascular disease in a subject in need of such treatment are provided. In various embodiments, the method includes administering to a subject a polypeptide of the present invention, or multimer of the polypeptide, in an amount sufficient to increase the level of a beneficial HDL subclass. In certain embodiments, the HDL subclass is HDL₂ or HD L₃. The polypeptide or multimer can be administered as part of a pharmaceutical composition, and can contain bound lipid. Alternatively, the isolated polypeptide or multimer can be provided by an expression vector delivered to the subject. The vascular disease can be atherosclerosis, coronary heart disease, peripheral vascular disease, stroke, or mesenteric ischemia. In some embodiments, the method includes the step of identifying a subject in need of treatment before administration of the polypeptide or multimer. In some embodiments, the method includes determining the composition, level and/or distribution of an HDL subclass in a subject before and/or after treatment.

In certain embodiments, the subject has atherosclerotic disease, such as angina, transient ischemic attacks, dislipidemia, or metabolic syndrome. In certain embodiments, the subject has an abnormal apoA-I level and/or function, an abnormal HDL level and/or function, or both abnormal apoA-I and HDL levels and/or functions. In certain embodiments, the subject has a cardiac problem with an abnormal apoA-I level and/or function, an abnormal HDL level and/or function, or both abnormal apoA-I and HDL levels and/or functions.

The term "beneficial HDL subclass" refers to any HDL subclass that promotes or enhances the well-being of the subject with respect to the medical treatment of his or her condition, which includes treatment of vascular diseases. A list of nonexhaustive examples of promoting or enhancing the well-being includes extension of the subject's life by any period of time, a decrease or delay in the progression of the disease, a decrease in pain to the subject that can be attributed to the subject's condition, a decrease in the severity of the disease, a decrease in the risk of establishing the disease, an increase in the therapeutic effect of a therapeutic agent, an improvement in the prognosis of the condition or disease, a decrease in the amount or frequency of administration of a therapeutic agent, an alteration in the treatment regimen of the subject that reduces invasiveness of treatment, and a decrease in the severity or frequency of side effects from a therapeutic agent. A. Administration

To modulate HDL or treat vascular disease in accordance with the present invention, one would generally contact a cell with a polypeptide containing an altered apoA-I sequence, or introduce such a polypeptide into the circulation. This can be accomplished either by administering a composition containing the polypeptide or containing an expression construct encoding the polypeptide. The route of administration will vary, naturally, with the particular circumstances of the patient and the composition, and include, e.g., intradermal, transdermal, parenteral, intravenous, intramuscular, intranasal, subcutaneous, percutaneous, intratracheal, intraperitoneal, intratumoral, perfusion, lavage, direct injection, and oral administration and formulation. In particular embodiments, the route of administration is intravenous. In particular embodiments, for example with a formulation, the route of administration is a subcutaneous depot injection.

The composition can be given by single or multiple injection, or by continuous administration. Generally, the dose of the therapeutic composition via continuous administration will be equivalent to that given by a single or multiple injections, adjusted over a period of time during which the continuous administration occurs. Treatment regimens can vary as well, and can depend on such circumstances as the health and age of the patient, the severity of the disease, and the type of composition. Obviously, certain therapeutic situations will require more aggressive treatment, while at the same time, certain patients cannot tolerate more taxing protocols. The clinician will be best suited to make such decisions based on the efficacy and toxicity (if any) of the therapeutic formulations.

The treatments may include various "unit doses." Unit dose is defined as containing a predetermined-quantity of the therapeutic composition. The quantity to be administered, and the particular route and formulation, are within the skill of those in the clinical arts. A unit dose need not be administered as a single injection but may comprise continuous infusion over a set period of time. Unit dose of the present invention may conveniently be described in terms of plaque forming units (pfu) for a viral construct. Unit doses range from 10³, 10⁴, 10⁵, 10⁶, 10⁷, 10⁸, 10⁹, 10¹⁰, 10¹¹, 10¹², 10¹³ pfu and higher. Alternatively, depending on the kind of virus and the titer attainable, one will deliver 1 to 100, 10 to 50, 100-1000, or up to about 1 x 10⁴, 1 x 10⁵, 1 x 10⁶, 1 x 10⁷, 1 x 10⁸, 1 x 10⁹, 1 x 10¹⁰, 1 x 10¹¹, 1 x 10¹², 1 x 10¹³, 1 x 10¹⁴, or 1 x 10¹⁵ or higher infectious viral particles (vp) to the patient or to the patient's cells. B. Injectable Compositions and Formulations

An expression construct encoding a polypeptide containing an altered apoA-I sequence can be administered parenterally, intravenously, intradermally, intramuscularly, transdermally or even intraperitoneally as described in U.S. Patents 5,543,158, 5,641,515 and 5,399,363 (each specifically incorporated herein by reference in its entirety).

Injection of nucleic acid constructs may be delivered by syringe or any other method used for injection of a solution, as long as the expression construct can pass through the particular gauge of needle required for injection. For example, a needleless injection system has been described (U.S. Patent 5,846,233, incorporated by reference herein) having a nozzle defining an ampule chamber for holding the solution and an energy device for pushing the solution out of the nozzle to the site of delivery. A syringe system has also been described for use in gene therapy that permits multiple injections of predetermined quantities of a solution precisely at any depth (U.S. Patent 5,846,225, incorporated by reference herein).

Solutions of active substances, whether polypeptides or expression constructs, may be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions may also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions (U.S. Patent 5,466,468, specifically incorporated herein by reference in its entirety). In all cases the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and/or vegetable oils. Proper fluidity may be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous, intratumoral and intraperitoneal administration. In this connection, sterile aqueous media that can be employed will be known to those of skill in the art in light of the present disclosure. For example, one dosage may be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion, (see for example, "Remington's Pharmaceutical Sciences" 15th Edition, pages 1035-1038 and 1570-1580). Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject. Moreover, for human administration, preparations should meet sterility, pyrogenicity, general safety and purity standards as required by FDA Office of Biologies standards.

Sterile injectable solutions are prepared by incorporating the active substances in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

The compositions disclosed herein may be formulated in a neutral or salt form.

Pharmaceutically-acceptable salts, include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like. Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms such as injectable solutions, drug release capsules and the like.

As used herein, "carrier" includes any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, and the like. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions.

The phrase "pharmaceutically-acceptable" or "pharmacologically-acceptable" refers to molecular entities and compositions that do not produce an allergic or similar untoward reaction when administered to a human. The preparation of an aqueous composition that contains a protein as an active ingredient is well understood in the art. Typically, such compositions are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid prior to injection can also be prepared.

C. Combination Therapies

In order to increase the effectiveness of a given treatment or therapy, it may be desirable to combine the compositions and methods of the invention with an agent effective in the treatment of vascular or cardiovascular disease or disorder. For example, a polypeptide of the present invention can be used in combination with an agent that lowers plasma lipids (e.g., a statin), or in combination with an agent that increases plasma HDL (e.g., niacin, fibrates, or cholesterol absorption inhibitors). In some embodiments, it is contemplated that a conventional therapy or agent, including but not limited to, a pharmacological therapeutic agent, a surgical therapeutic agent (e.g., a surgical procedure) or a combination thereof, may be combined with treatment directed to altering levels of beneficial HDL subclasses. Thus, in certain embodiments, a therapeutic method of the present invention may comprise altering the level of a beneficial HDL subclass in combination with another therapeutic agent.

This process may involve contacting the cell(s) with an agent(s) and a polypeptide of the present invention, or a multimer of the polypeptide, at the same time or within a period of time wherein separate administration of the polypeptide or multimer and an agent to a cell, tissue or organism produces a desired therapeutic benefit. The terms "contacted" and "exposed," when applied to a cell, tissue or organism, are used herein to describe the process by which the polypeptide or multimer and/or therapeutic agent is delivered to a target cell, tissue or organism or is placed in direct juxtaposition with the target cell, tissue or organism. The cell, tissue or organism may be contacted (e.g., by administration) with a single composition or pharmacological formulation that includes both the polypeptide or multimer and one or more agents, or by contacting the cell with two or more distinct compositions or formulations, wherein one composition includes a polypeptide or multimer and the other includes one or more agents.

The polypeptide or multimer may precede, be co-current with and/or follow the other agent(s) by intervals ranging from minutes to weeks. In embodiments where the polypeptide or multimer and other agent(s) are applied separately to a cell, tissue or organism, one would generally ensure that a significant period of time did not expire between the time of each delivery, such that the polypeptide or multimer and agent(s) would still be able to exert an advantageously combined effect on the cell, tissue or organism. For example, in such instances, it is contemplated that one may contact the cell, tissue or organism with two, three, four or more modalities substantially simultaneously (i.e., within less than about a minute) as the polypeptide or multimer. In other aspects, one or more agents may be administered within of from substantially simultaneously, about 1 minute, about 5 minutes, about 10 minutes, about 20 minutes about 30 minutes, about 45 minutes, about 60 minutes, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours about 8 hours, about 12 hours, about 18 hours, about 24 hours, about 36 hours, about 48 hours, about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 14 days, about 21 days, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, or about 12 months, and any range derivable therein, prior to and/or after administering the polypeptide or multimer.

Various combination regimens of the polypeptide or multimer and one or more agents may be employed. Non-limiting examples of such combinations are shown below, wherein a composition polypeptide or multimer is "A" and the other agent is "B":

A/B/A B/A/B B/B/A A/A/B A/B/B B/A/A A/B/B/B B/A/B/B B/B/B/A B/B/A/B A/A/B/B A/B/A/B A/B/B/A B/B/A/A

B/A/B/A B/A/A/B A/A/A/B B/A/A/A A/B/A/A A/A/B/A

Administration of the polypeptide or multimer to a cell, tissue or organism may follow general protocols for the administration of vascular or cardiovascular therapeutics, taking into account the toxicity, if any. It is expected that the treatment cycles would be repeated as necessary. In particular embodiments, it is contemplated that various additional agents may be applied in any combination with the present invention.

1. Pharmacological Therapeutic Agents

Pharmacological therapeutic agents and methods of administration, dosages, etc., are well known to those of skill in the art (see for example, the "Physicians Desk Reference",

Goodman & Gilman's "The Pharmacological Basis of Therapeutics", "Remington's

Pharmaceutical Sciences", and "The Merck Index, Eleventh Edition", incorporated herein by reference in relevant parts), and may be combined with the invention in light of the disclosures herein. Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject, and such individual determinations are within the skill of those of ordinary skill in the art.

Non-limiting examples of a pharmacological therapeutic agent that may be used in the present invention include an antihyperlipoproteinemic agent, an antiarteriosclerotic agent, an antithrombotic/fibrinolytic agent, a blood coagulant, an antiarrhythmic agent, an antihypertensive agent, a vasopressor, a treatment agent for congestive heart failure, an antianginal agent, or any combination thereof.

a. Antihyperlipoproteinemics

In certain embodiments, administration of an agent that lowers the concentration of one of more blood lipids and/or lipoproteins, known herein as an "antihyperlipoproteinemic," may be combined with a therapy according to the present invention, particularly in treatment of atherosclerosis and thickenings or blockages of vascular tissues. In certain aspects, an antihyperlipoproteinemic agent may comprise an aryloxyalkanoic/fibric acid derivative, a resin/bile acid sequesterant, a HMG CoA reductase inhibitor, a nicotinic acid derivative, a thyroid hormone or thyroid hormone analog, a miscellaneous agent or a combination thereof. (i) Aryloxyalkanoic Acid/Fibric Acid Derivatives

Non-limiting examples of aryloxyalkanoic/fibric acid derivatives include beclobrate, enzafϊbrate, binifibrate, ciprofibrate, clinofibrate, clofibrate (atromide-S), clofϊbric acid, etofibrate, fenofϊbrate, gemfibrozil (lobid), nicofibrate, pirifibrate, ronifibrate, simfibrate and theofibrate.

(ii) Resins/Bile Acid Sequesterants

Non-limiting examples of resins/bile acid sequesterants include cholestyramine (cholybar, questran), colestipol (colestid) and polidexide.

(iii) HMG CoA Reductase Inhibitors Non- limiting examples of HMG CoA reductase inhibitors include lovastatin

(mevacor), pravastatin (pravochol) or simvastatin (zocor).

(iv) Nicotinic Acid Derivatives

Non-limiting examples of nicotinic acid derivatives include nicotinate, acepimox, niceritrol, nicoclonate, nicomol and oxiniacic acid.

(v) Thryroid Hormones and Analogs

Non-limiting examples of thyroid hormones and analogs thereof include etoroxate, thyropropic acid and thyroxine.

(vi) Miscellaneous Antihyperlipoproteinemics

Non-limiting examples of miscellaneous antihyperlipoproteinemics include acifran, azacosterol, benfluorex, β-benzalbutyramide, carnitine, chondroitin sulfate, clomestrone, detaxtran, dextran sulfate sodium, 5, 8, 11, 14, 17-eicosapentaenoic acid, eritadenine, furazabol, meglutol, melinamide, mytatrienediol, ornithine, γ-oryzanol, pantethine, pentaerythritol tetraacetate, α-phenylbutyramide, pirozadil, probucol (lorelco), β-sitosterol, sultosilic acid-piperazine salt, tiadenol, triparanol and xenbucin.

b. Antiarteriosclerotics

Non-limiting examples of antiarteriosclerotics include pyridinol carbamate and liver X receptor (LXR) antagonists. c. Antithrombotic/Fibrinolytic Agents

In certain embodiments, administration of an agent that aids in the removal or prevention of blood clots may be combined with administration of a polypeptide or multimer of the present invention, particularly in treatment of athersclerosis and vasculature (e.g., arterial) blockages. Non-limiting examples of antithrombotic and/or fibrinolytic agents include anticoagulants, anticoagulant antagonists, antiplatelet agents, thrombolytic agents, thrombolytic agent antagonists or combinations thereof.

In certain aspects, antithrombotic agents that can be administered orally, such as, for example, aspirin and wafarin (Coumadin), are preferred.

(i) Anticoagulants

Non-limiting examples of anticoagulants include acenocoumarol, ancrod, anisindione, bromindione, clorindione, coumetarol, cyclocumarol, dextran sulfate sodium, dicumarol, diphenadione, ethyl biscoumacetate, ethylidene dicoumarol, fluindione, heparin, hirudin, lyapolate sodium, oxazidione, pentosan polysulfate, phenindione, phenprocoumon, phosvitin, picotamide, tioclomarol and warfarin.

(ii) Antiplatelet Agents

Non-limiting examples of antiplatelet agents include aspirin, a dextran, dipyridamole (persantin), heparin, sulfmpyranone (anturane) and ticlopidine (tic lid).

(iii) Thrombolytic Agents Non-limiting examples of thrombolytic agents include tissue plaminogen activator

(activase), plasmin, pro-urokinase, urokinase (abbokinase) streptokinase (streptase), and anistreplase/ APSAC (eminase).

d. Blood Coagulants

In certain embodiments wherein a patient is suffering from a hemmorage or an increased likelyhood of hemmoraging, an agent that may enhance blood coagulation may be used. Non-limiting examples of a blood coagulation promoting agent include thrombolytic agent antagonists and anticoagulant antagonists. (i) Anticoagulant Antagonists

Non-limiting examples of anticoagulant antagonists include protamine and vitamin Kl.

(ii) Thrombolytic Agent Antagonists and Antithrombotics Non-limiting examples of thrombolytic agent antagonists include amiocaproic acid

(amicar) and tranexamic acid (amstat). Non- limiting examples of antithrombotics include anagrelide, argatroban, cilstazol, daltroban, defibrotide, enoxaparin, fraxiparine, indobufen, lamoparan, ozagrel, picotamide, plafibride, tedelparin, ticlopidine and triflusal.

e. Antiarrhythmic Agents Non- limiting examples of antiarrhythmic agents include Class I antiarrythmic agents

(sodium channel blockers), Class II antiarrythmic agents (beta-adrenergic blockers), Class II antiarrythmic agents (repolarization prolonging drugs), Class IV antiarrhythmic agents (calcium channel blockers) and miscellaneous antiarrythmic agents.

(i) Sodium Channel Blockers Non- limiting examples of sodium channel blockers include Class IA, Class IB and

Class IC antiarrhythmic agents. Non-limiting examples of Class IA antiarrhythmic agents include dispyramide (norpace), procainamide (pronestyl) and quinidine (quinidex). Non- limiting examples of Class IB antiarrhythmic agents include lidocaine (xylocaine), tocainide (tonocard) and mexiletine (mexitil). Non-limiting examples of Class IC antiarrhythmic agents include encainide (enkaid) and flecainide (tambocor).

(ii) Beta Blockers

Non-limiting examples of beta blockers, otherwise known as β-adrenergic blockers, β- adrenergic antagonists or Class II antiarrhythmic agents, include acebutolol (sectral), alprenolol, amosulalol, arotinolol, atenolol, befunolol, betaxolol, bevantolol, bisoprolol, bopindolol, bucumolol, bufetolol, bufuralol, bunitrolol, bupranolol, butidrine hydrochloride, butofϊlolol, carazolol, carteolol, carvedilol, celiprolol, cetamolol, cloranolol, dilevalol, epanolol, esmolol (brevibloc), indenolol, labetalol, levobunolol, mepindolol, metipranolol, metoprolol, moprolol, nadolol, nadoxolol, nifenalol, nipradilol, oxprenolol, penbutolol, pindolol, practolol, pronethalol, propanolol (inderal), sotalol (betapace), sulfϊnalol, talinolol, tertatolol, timolol, toliprolol and xibinolol. In certain aspects, the beta blocker comprises an aryloxypropanolamine derivative. Non-limiting examples of aryloxypropanolamine derivatives include acebutolol, alprenolol, arotinolol, atenolol, betaxolol, bevantolol, bisoprolol, bopindolol, bunitrolol, butofϊlolol, carazolol, carteolol, carvedilol, celiprolol, cetamolol, epanolol, indenolol, mepindolol, metipranolol, metoprolol, moprolol, nadolol, nipradilol, oxprenolol, penbutolol, pindolol, propanolol, talinolol, tertatolol, timolol and toliprolol.

(iii) Repolarization Prolonging Agents

Non-limiting examples of agents that prolong repolarization, also known as Class III antiarrhythmic agents, include amiodarone (cordarone) and sotalol (betapace).

(iv) Calcium Channel Blockers/ Antagonist

Non-limiting examples of a calcium channel blocker, otherwise known as a Class IV antiarrythmic agent, include an arylalkylamine (e.g., bepridile, diltiazem, fendiline, gallopamil, prenylamine, terodiline, verapamil), a dihydropyridine derivative (felodipine, isradipine, nicardipine, nifedipine, nimodipine, nisoldipine, nitrendipine) a piperazinde derivative (e.g., cinnarizine, flunarizine, lidoflazine) or a micellaneous calcium channel blocker such as bencyclane, etafenone, magnesium, mibefradil or perhexiline. In certain embodiments a calcium channel blocker comprises a long-acting dihydropyridine (nifedipine- type) calcium antagonist.

(v) Miscellaneous Antiarrhythmic Agents Non-limiting examples of miscellaneous antiarrhymic agents include adenosine

(adenocard), digoxin (lanoxin), acecainide, ajmaline, amoproxan, aprindine, bretylium tosylate, bunaftine, butobendine, capobenic acid, cifenline, disopyranide, hydroquinidine, indecainide, ipatropium bromide, lidocaine, lorajmine, lorcainide, meobentine, moricizine, pirmenol, prajmaline, propafenone, pyrinoline, quinidine polygalacturonate, quinidine sulfate and viquidil.

f. Antihypertensive Agents

Non-limiting examples of antihypertensive agents include sympatholytic, alpha/beta blockers, alpha blockers, anti-angiotensin II agents, beta blockers, calcium channel blockers, vasodilators and miscellaneous antihypertensives. (i) Alpha Blockers

Non-limiting examples of alpha blockers, also known as α-adrenergic blockers or α- adrenergic antagonists, include amosulalol, arotinolol, dapiprazole, doxazosin, ergoloid mesylates, fenspiride, indoramin, labetalol, nicergoline, prazosin, terazosin, tolazoline, trimazosin and yohimbine. In certain embodiments, an alpha blocker may comprise a quinazoline derivative. Non-limiting examples of quinazoline derivatives include alfuzosin, bunazosin, doxazosin, prazosin, terazosin and trimazosin.

(ii) Alpha/Beta Blockers

In certain embodiments, an antihypertensive agent is both an alpha and beta adrenergic antagonist. A non- limiting examples of an alpha/beta blocker includes labetalol (normodyne, trandate).

(iii) Anti-Angiotension II Agents

Non-limiting examples of anti-angiotension II agents include angiotensin converting enzyme inhibitors and angiotension II receptor antagonists. Non- limiting examples of angiotension converting enzyme inhibitors (ACE inhibitors) include alacepril, enalapril (vasotec), captopril, cilazapril, delapril, enalaprilat, fosinopril, lisinopril, moveltopril, perindopril, quinapril and ramipril.. Non- limiting examples of an angiotensin II receptor blocker, also known as an angiotension II receptor antagonist, an ANG receptor blocker or an ANG-II type-1 receptor blocker (ARBS), include angiocandesartan, eprosartan, irbesartan, losartan and valsartan.

(iv) Sympatholytics

Non-limiting examples of a sympatholytic include a centrally acting sympatholytic or a peripherially acting sympatholytic. Non-limiting examples of centrally acting sympatholytics, also known as an central nervous system (CNS) sympatholytics, include clonidine (catapres), guanabenz (wytensin) guanfacine (tenex) and methyldopa (aldomet). Non-limiting examples of peripherally acting sympatholytics include a ganglion blocking agent, an adrenergic neuron blocking agent, a β-adrenergic blocking agent or a alpha 1- adrenergic blocking agent. Non-limiting examples of ganglion blocking agents include mecamylamine (inversine) and trimethaphan (arfonad). Non-limiting examples of adrenergic neuron blocking agents include guanethidine (ismelin) and reserpine (serpasil). Non-limiting examples of β-adrenergic blockers include acenitolol (sectral), atenolol (tenormin), betaxolol (kerlone), carteolol (cartrol), labetalol (normodyne, trandate), metoprolol (lopressor), nadanol (corgard), penbutolol (levatol), pindolol (visken), propranolol (inderal) and timolol (blocadren). Non-limiting examples of alpha 1 -adrenergic blockers include prazosin (minipress), doxazocin (cardura) and terazosin (hytrin).

(v) Vasodilators

In certain embodiments a cardiovasculator therapeutic agent may comprise a vasodilator (e.g., a cerebral vasodilator, a coronary vasodilator or a peripheral vasodilator). In certain preferred embodiments, a vasodilator comprises a coronary vasodilator. Non-limiting examples of coronary vasodilators include amotriphene, bendazol, benfurodil hemisuccinate, benziodarone, chloracizine, chromonar, clobenfurol, clonitrate, dilazep, dipyridamole, droprenilamine, efloxate, erythrityl tetranitrane, etafenone, fendiline, floredil, ganglefene, herestrol bis(β -diethyl aminoethyl ether), hexobendine, itramin tosylate, khellin, lidoflanine, mannitol hexanitrane, medibazine, nicorglycerin, pentaerythritol tetranitrate, pentrinitrol, perhexiline, pimefylline, trapidil, tricromyl, trimetazidine, trolnitrate phosphate and visnadine.

In certain aspects, a vasodilator may comprise a chronic therapy vasodilator or a hypertensive emergency vasodilator. Non-limiting examples of chronic therapy vasodilators include hydralazine (apresoline) and minoxidil (loniten). Non-limiting examples of hypertensive emergency vasodilators include nitroprusside (nipride), diazoxide (hyperstat IV), hydralazine (apresoline), minoxidil (loniten) and verapamil.

(vi) Miscellaneous Antihypertensives

Non-limiting examples of miscellaneous antihypertensives include ajmaline, γ- aminobutyric acid, bufeniode, cicletainine, ciclosidomine, cryptenamine tannate, fenoldopam, flosequinan, ketanserin, mebutamate, mecamylamine, methyldopa, methyl 4-pyridyl ketone thiosemicarbazone, muzolimine, pargyline, pempidine, pinacidil, piperoxan, primaperone, a protoveratrine, raubasine, rescimetol, rilmenidene, saralasin, sodium nitrorusside, ticrynafen, trimethaphan camsylate, tyrosinase and urapidil.

In certain aspects, an antihypertensive may comprise an arylethanolamine derivative, a benzothiadiazine derivative, a JV-carboxyalkyl(peptide/lactam) derivative, a dihydropyridine derivative, a guanidine derivative, a hydrazines/phthalazine, an imidazole derivative, a quanternary ammonium compound, a reserpine derivative or a suflonamide derivative. Arylethanolamine Derivatives. Non-limiting examples of arylethanolamine derivatives include amosulalol, bufuralol, dilevalol, labetalol, pronethalol, sotalol and sulfinalol.

Benzothiadiazine Derivatives. Non-limiting examples of benzothiadiazine derivatives include althizide, bendroflumethiazide, benzthiazide, benzylhydrochlorothiazide, buthiazide, chlorothiazide, chlorthalidone, cyclopenthiazide, cyclothiazide, diazoxide, epithiazide, ethiazide, fenquizone, hydrochlorothizide, hydroflumethizide, methyclothiazide, meticrane, metolazone, paraflutizide, polythizide, tetrachlormethiazide and trichlormethiazide.

7V-carboxyalkyl(peptide/lactam) Derivatives. Non-limiting examples of N- carboxyalkyl(peptide/lactam) derivatives include alacepril, captopril, cilazapril, delapril, enalapril, enalaprilat, fosinopril, lisinopril, moveltipril, perindopril, quinapril and ramipril.

Dihydropyridine Derivatives. Non-limiting examples of dihydropyridine derivatives include amlodipine, felodipine, isradipine, nicardipine, nifedipine, nilvadipine, nisoldipine and nitrendipine.

Guanidine Derivatives. Non- limiting examples of guanidine derivatives include bethanidine, debrisoquin, guanabenz, guanacline, guanadrel, guanazodine, guanethidine, guanfacine, guanochlor, guanoxabenz and guanoxan.

Hydrazines/Phthalazines. Non- limiting examples of hydrazines/phthalazines include budralazine, cadralazine, dihydralazine, endralazine, hydracarbazine, hydralazine, pheniprazine, pildralazine and todralazine.

Imidazole Derivatives. Non-limiting examples of imidazole derivatives include clonidine, lofexidine, phentolamine, tiamenidine and tolonidine.

Quanternary Ammonium Compounds. Non-limiting examples of quanternary ammonium compounds include azamethonium bromide, chlorisondamine chloride, hexamethonium, pentacynium bis(methylsulfate), pentamethonium bromide, pentolinium tartrate, phenactropinium chloride and trimethidinium methosulfate.

Reserpine Derivatives. Non-limiting examples of reserpine derivatives include bietaserpine, deserpidine, rescinnamine, reserpine and syrosingopine. Suflonamide Derivatives. Non-limiting examples of sulfonamide derivatives include ambuside, clopamide, furosemide, indapamide, quinethazone, tripamide and xipamide.

g. Vasopressors

Vasopressors generally are used to increase blood pressure during shock, which may occur during a surgical procedure. Non-limiting examples of vasopressors, also known as antihypotensives, include amezinium methyl sulfate, angiotensin amide, dimetofrine, dopamine, etifelmin, etilefrin, gepefrine, metaraminol, midodrine, norepinephrine, pholedrine and synephrine.

h. Treatment Agents for Congestive Heart Failure Non- limiting examples of agents for the treatment of congestive heart failure include anti-angiotension II agents, afterload-preload reduction treatment, diuretics and inotropic agents.

(i) Afterload-Preload Reduction

In certain embodiments, a patient that cannot tolerate an angiotension antagonist may be treated with a combination therapy. Such therapy may combine adminstration of hydralazine (apresoline) and isosorbide dinitrate (isordil, sorbitrate).

(ii) Diuretics

Non-limiting examples of a diuretic include a thiazide or benzothiadiazine derivative (e.g., althiazide, bendroflumethazide, benzthiazide, benzylhydrochlorothiazide, buthiazide, chlorothiazide, chlorothiazide, chlorthalidone, cyclopenthiazide, epithiazide, ethiazide, ethiazide, fenquizone, hydrochlorothiazide, hydroflumethiazide, methyclothiazide, meticrane, metolazone, paraflutizide, polythizide, tetrachloromethiazide, trichlormethiazide), an organomercurial (e.g., chlormerodrin, meralluride, mercamphamide, mercaptomerin sodium, mercumallylic acid, mercumatilin dodium, mercurous chloride, mersalyl), a pteridine (e.g., furterene, triamterene), purines (e.g., acefylline, 7-morpholinomethyltheophylline, pamobrom, protheobromine, theobromine), a steroid including an aldosterone antagonist (e.g., canrenone, oleandrin, spironolactone), a sulfonamide derivative (e.g., acetazolamide, ambuside, azosemide, bumetanide, butazolamide, chloraminophenamide, clofenamide, clopamide, clorexolone, diphenylmethane-4,4'-disulfonamide, disulfamide, ethoxzolamide, furosemide, indapamide, mefruside, methazolamide, piretanide, quinethazone, torasemide, tripamide, xipamide), a uracil (e.g., aminometradine, amisometradine), a potassium sparing antagonist (e.g., amiloride, triamterene)or a miscellaneous diuretic such as aminozine, arbutin, chlorazanil, ethacrynic acid, etozolin, hydracarbazine, isosorbide, mannitol, metochalcone, muzolimine, perhexiline, ticrnafen and urea.

(iii) Inotropic Agents

Non-limiting examples of positive inotropic agents, also known as cardiotonics, include acefylline, an acetyldigitoxin, 2-amino-4-picoline, amrinone, benfurodil hemisuccinate, bucladesine, cerberosine, camphotamide, convallatoxin, cymarin, denopamine, deslanoside, digitalin, digitalis, digitoxin, digoxin, dobutamine, dopamine, dopexamine, enoximone, erythrophleine, fenalcomine, gitalin, gitoxin, glycocyamine, heptaminol, hydrastinine, ibopamine, a lanatoside, metamivam, milrinone, nerifolin, oleandrin, ouabain, oxyfedrine, prenalterol, proscillaridine, resibufogenin, scillaren, scillarenin, strphanthin, sulmazole, theobromine and xamoterol.

In particular aspects, an intropic agent is a cardiac glycoside, a beta-adrenergic agonist or a phosphodiesterase inhibitor. Non- limiting examples of cardiac glycosides includes digoxin (lanoxin) and digitoxin (crystodigin). Non-limiting examples of β-adrenergic agonists include albuterol, bambuterol, bitolterol, carbuterol, clenbuterol, clorprenaline, denopamine, dioxethedrine, dobutamine (dobutrex), dopamine (intropin), dopexamine, ephedrine, etafedrine, ethylnorepinephrine, fenoterol, formoterol, hexoprenaline, ibopamine, isoetharine, isoproterenol, mabuterol, metaproterenol, methoxyphenamine, oxyfedrine, pirbuterol, procaterol, protokylol, reproterol, rimiterol, ritodrine, soterenol, terbutaline, tretoquinol, tulobuterol and xamoterol. A non-limiting example of a phosphodiesterase inhibitor include amrinone (inocor).

i. Antianginal Agents Antianginal agents may comprise organonitrates, calcium channel blockers, beta blockers and combinations thereof.

Non-limiting examples of organonitrates, also known as nitrovasodilators, include nitroglycerin (nitro-bid, nitrostat), isosorbide dinitrate (isordil, sorbitrate) and amyl nitrate (aspirol, vaporole). 2. Surgical Therapeutic Agents

In certain aspects, the secondary therapeutic agent may comprise a surgery of some type, which includes, for example, preventative, diagnostic or staging, curative and palliative surgery. Surgery, and in particular a curative surgery, may be used in conjunction with other therapies, such as the present invention and one or more other agents.

Such surgical therapeutic agents for vascular and cardiovascular diseases and disorders are well known to those of skill in the art, and may comprise, but are not limited to, performing surgery on an organism, providing a cardiovascular mechanical prostheses, angioplasty, coronary artery reperfusion, catheter ablation, providing an implantable cardioverter defibrillator to the subject, mechanical circulatory support or a combination thereof. Non-limiting examples of a mechanical circulatory support that may be used in the present invention comprise an intra-aortic balloon counterpulsation, left ventricular assist device or combination thereof.

The present invention may be better understood by referring to the accompanying examples, which are intended for illustration purposes only and should not in any sense be construed as limiting the scope of the invention as defined in the claims appended hereto.

EXAMPLES

In the following examples, the locations of peptide sequences (i.e. residues 184-190) are given relative to the 243 amino acid sequence of mature human apoA-I. The mature polypeptide corresponds to residues 25-267 of SEQ ID NO:1. Also, the sequence of apoA-I set forth in SEQ ID NO:1 is referred to as "wild type" human apoA-I, while polypeptides containing altered apoA-I sequences are referred to as "mutant" apoA-I.

The abbreviations used are: CAD, Coronary Artery Disease; HDL, high density lipoprotein; apo, apolipoprotein, PCS, proline containing sequence; NPCS, Non-proline containing sequence; LCAT, lecithin-cholesterol acyl transferase; ABC, ATP Binding Cassette; SR-BI, Scavenger Receptor B Class I; NTD, N-terminal domain; CTD, C-terminal domain; NDGGE, nondenaturing gradient gel electrophoresis; rHDL, reconstituted HDL; GdnHCl, Guanidine Hydrochloride; BS₃, bis(sulfosuccinimidyl)suberate; POPC, sn-\- palmitoyl-5/?-2-oleoyl-phosphatidylcholine; DMPC, dimyristoylphosphatidylcholine; McA7⁴, McArdle-7777 Rat Hepatoma; LDL, Low density lipoprotein; VLDL, Very low density lipoprotein; HRP, Horse Radish Peroxidase; FBS, Fetal bovine serum; HS, Horse serum; DMEM, Dulbecco's modified eagle medium; ACAT, Acyl CoA transferase; BSA, Bovine serum albumin; NaBr, Sodium bromide; CPT-cAMP, 8-(4-Chlorophenylthio)-adenosine-3',5'- cyclic monophosphate, sodium salt; LPDS, lipoprotein deficient serum.

EXAMPLE 1 MATERIALS AND METHODS

The rat hepatoma cell line McA-RH7777 (ATCC CRL 1601) and the J774 macrophages (ATCC TIB-67) were obtained from American Type Culture Collection (Manassas, VA). [1,2-³H] cholesterol (lmCi/mL) was obtained from Perkin Elmer Life Sciences, Inc (Boston, MA). 8-(4-chlorophenylthio)-cAMP, and Sandoz acyl- CoAxholesterol acyltransferase (ACAT) inhibitor were obtained from Sigma (St. Louis, MO). Biogel A-5m chromatography beads were obtained from Bio-Rad (Hercules, CA). The HRP -T7 antibody was purchased from Novagen (Gibbstown, NJ). Immobilon P transfer membranes and Centriprep YM-30 were obtained from Millipore Corp. (Bedford, MA.). Enhanced chemilluminescence (ECL) western blotting kit was obtained from GE Biosciences Inc (Piscataway, NJ). Superscript Reverse Transcriptase II, Lipofectamine 2000, Geneticin (G418), and the pcDNA3.1 (-) vector were purchased from Invitrogen (Carlsbad, CA).

Elements common to all recombinant apoA-I cDNAs: The restriction enzymes, Vent polymerase and T4 DNA ligase were purchased from New England Biolabs. The QuickChange site-directed mutagenesis kits and Pfu DNA polymerase were from the Stratagene, Inc. The oligonucleotide primers were made from the Integrated DNA Technologies (IDT), Inc. The pET28 vector from Novagen was used for bacterial expression of apoA-I.

Generation of recombinant apoA-I: The wild-type human apoA-I cDNA was subcloned into the bacterial expression vector pET28c as described (Reschly et ah, 2002). Briefly, reverse transcription-PCR of total human liver RNA was used to obtain a human apoA-I cDNA. A 5' oligonucleotide (5 '-get egg cat ttc tgg ate caa gat gaa ccc-3') (SEQ ID NO: 9) contained a BamHI site and a 3' oligonucleotide (5'-tgc aag ctt tea ctg ggt gtt gag ctt ct tag-3') (SEQ ID NO:10) contained a HindIII site, allowing subcloning of the human apoA-I cDNA into the corresponding regions of the pET28c vector. The recombinant protein contains a poly-His sequence and a T7 tag at the N-terminus of the protein. The PCR-based QuickChange site directed mutagenesis kit was used to replace the natural NPCS "NGGARLA" (SEQ ID NO: 11) between putative helices 7 and 8 of human apoA-I (residues 184-190) with the PCS present between other helices of the human protein (i.e. PCSl- QLGPVTQ (SEQ ID NO: 12), residues 63-69; PCS2-KVQPYLD (SEQ ID NO:2), residues 96-102; PCS3-KVEPLRA (SEQ ID NO:3), residues 118-124; PCS4-KLSPLGE (SEQ ID NO: 13), residues 140-146; PCS5-HLAPYSD (SEQ ID NO: 14), residues 162-168; PCS6- KAKPALE (SEQ ID NO: 15), residues 206-212; and PCS7-GLLPVLE (SEQ ID NO: 16), residues 217-224). Each plasmid was transformed into protease-minus BL21(DE3)pLys E. coli strain (Novagen) and protein purification was carried out as described (Reschly et al., 2002). The concentration of recombinant human apoA-I was determined by Bradford protein assay (BIORAD).

Cross-linking experiments: Lipid-free or lipid-bound wild-type/mutant apoA-I (1 mg/mL) in 50 mM sodium phosphate and 50 mM NaCl at pH 7.2 were incubated for 30 min with BS₃ (Pierce, final concentration 0.5 mM). The reaction mixture was quenched by adding 1 M Tris-HCl and 5x SDS-PAGE sample buffer and then loaded on a 6-12% Tricine/PAGE gel. The populations of different oligomers were estimated using the band intensities of the oligomers as quantified using the software of Kodak Gel Logic 100 Imaging System (Ren et a/., 2005).

Circular dichroism spectroscopy: Circular Dichroism (CD) measurements of lipid- free and lipid-bound wild-type/mutant apoA-I were carried out on an AVIV model 62DS CD spectrometer (AVIV Instruments, Inc., Lakewood NJ) with a variable temperature capability under computer control within ± 0.2⁰C. Lyophilized protein samples were suspended in 50 mM sodium phosphate and 50 mM NaCl at pH 7.2, and were adjusted to a concentration of 0.1 mg/mL using the same buffer. Measurements were made at room temperature using a 0.1- cm quartz cell. Five scans between 200 and 250 nm were acquired and averaged. A base-line scan was subtracted to produce the final average scan. The percentage of α-helix content was calculated from the molar ellipticity at 222 nm, as described (Ren et al., 2005).

Preparation and characterization of apoA-I:rHDL particles: The rHDL particles were prepared using the sodium cholate dialysis method with phosphatidylcholine (POPC) (Avanti Polar Lipids), wild-type/mutant apoA-I, and sodium cholate in a molar ratio of 80:1 :108 (Ren et al., 2005). POPC was dissolved in CHCl₃ and dried under nitrogen, then resuspended in 50 mM sodium phosphate and 50 mM NaCl, pH 7.2. After being vortexed thoroughly, sodium cholate was added into the mixture followed by vortexing for another 3 min. The solution was incubated at 37°C and vortexed every 15 min until completely clear. Human apoA-I protein was added, and the protein/lipid mixture was incubated for 1 h at 37°C. Sodium cholate was removed by the bio-bead method (Ren et al., 2005). Size was examined by non-denaturing gradient gel electrophoresis (NDGGE) as described (Reschly et al, 2002).

Lipid-associated (isothermal) guanidine HCl (GdnHCl) denaturation studies:

GdnHCl denaturation was performed as described (Ren et al., 2005) and monitored by CD at 222 nm in a 0.1 -cm path length cuvette at 20⁰C.

DMPC (lipid binding) clearance assay: Ten milligrams of DMPC (Avanti Polar Lipids Inc., AL) were dissolved in a mixture of chloroform and methanol (3:1 v/v), dried using N₂, and placed under vacuum for at least 1O h. 1 mL of pre-warmed buffer was added (10 mM Tris-HCl, pH 7.2, 150 mM NaCl, and 0.5 mM EDTA) for a final lipid concentration of 10 mg/mL and vortexed several times, 30 seconds each. Using a 200 nm filter, unilamellar vesicles (-200 nm in diameter) were prepared by extrusion. The protein-induced transformation of DMPC vesicles into protein/DMPC discoidal complexes were monitored as a function of time (Ren et al., 2005). Two different ratios of apoA-I and DMPC (1 :1 and 1 :2.5, w/w) were used in this assay. ApoA-I/DMPC vesicles in buffer were added into a 1 mL thermostat cuvette and mixed for 5-10 seconds at 24°C. The clearance of solution was monitored using a Perkin-Elmer spectrophotometer (model Lambda 3B) at 490 nm. All solutions were pre -incubated at 24°C before being used in the reaction.

Isolation of human HDL₂ and HDL₃: HDL₂ (p = 1.063-1.098 g/mL) and HDL₃ (p =

1.13-1.21 g/mL) were isolated from human plasma obtained from female donors by sequential flotation as described (Reschly et al., 2002).

ApoA-I quantitation by immunoturbidometry: The endogenous apoA-I in the isolated HDL was determined by immunoturbidometry using kits from Roche and Molecular Biochemicals or Boehringer Mannheim (Reschly et al., 2002). The human apoA-I standards were purchased from Sigma or Northwest Lipid Laboratory (Seattle, WA).

In vitro association of recombinant human apoA-I with isolated human HDL:

The recombinant human apoA-I proteins were incubated with different proportions of HDL₂ and HDL₃ (i.e., 1 :1, 2:1 or 1 :2) according to their endogenous apoA-I content. In a typical 545 μl assay, 10 μg of wild-type/mutant recombinant human apoA-I was incubated with an

HDL preparation containing 625 μg of total endogenous apoA-I for 30 min at 4°C. Following incubation, the final volume of the assay mixture was adjusted to 2.0 mL with Tris Sodium Chloride EDTA (TSE) buffer (10 mM Tris, 140 mM NaCl, 0.25 mM EDTA and 0.15 mM sodium azide), pH 7.4 and subjected to a 10-20% equilibrium density gradient separation (Reschly et ah, 2002). 0.4 mL fractions were collected using an ISCO gradient collector. The gradient fractions corresponding to HDL₂ and HD L₃ were determined from the A₂go tracings.

To detect the recombinant and endogenous apoA-I in these fractions, alternate fractions were electrophoresed on 4-20% SDS-PAGE gels, proteins transferred to Immobilon- P, and immunodecorated with mouse monoclonal anti-T7 antibody HRP conjugate (1 :5000 dilution, Novagen) to detect recombinant apoA-I or with rabbit anti-huA-I antibodies (1 :25000 dilution)/goat anti-rabbit-IgG-HRP secondary antibody (1 :10,000 dilution) for endogenous and recombinant huA-I detection. The immuno-decorated proteins were visualized with enhanced chemiluminescence (ECL; Amersham/Pharmacia/Molecular Dynamics). The protein bands were quantitated by Alpha-Imager (ImageQuant, Genomic Solutions Inc). In order to compare data between experiments the recombinant apoA-I distributions were normalized to the HDL₂:HDL3 proportions by dividing them by the corresponding endogenous apoA-I distributions. Endogenous apoA-I is an appropriate surrogate for starting HDL proportions.

Concentration-dependent binding of recombinant apoA-I to HDL subclasses: In order to determine the binding curves of wild-type/mutant human apoA-I proteins for HDL subclasses, increasing amounts of recombinant apoA-I (1.25-51 μg) were added to isolated HDL₂ or HDL₃ (200 μg total protein) in 545 μl of TSE buffer. The reaction was incubated at 4°C for 30 min. An aliquot from each assay was electrophoresed on 0.7% agarose gels to separate HDL bound from unbound protein. Following transfer to Immobilon-P, specifically probing with the mouse monoclonal anti-T7 antibody, as described above, recombinant human apoA-I bound to HDL and unbound were calculated and used to determine the IQ of association. The blots were also immunoblotted for apoE and apoA-IV to assess remodeling of the HDL during the incubation. Previous studies have demonstrated little remodeling at the concentrations of HDL and recombinant proteins used in these studies.

Stable cell lines: McA-RH7777 (McA7⁴) cells were stably transfected with the recombinant apoA-I - pcDNA3.1 expression vectors using Lipofectamine 2000. Stable transfectants were selected using the neomycin analogue G418 (400μg/mL), and the media of the individual colonies were screened for the presence of the wild-type or mutant human apoA-I proteins by immunob lotting using species-specific antibodies [20]. Stable cell lines were maintained in DMEM supplemented with 4500mg/L D-glucose, 10% horse serum, 5% fetal bovine serum, 1% penicillin/streptomycin, 1% glutamine and 400μg/mL of G418. All cell lines were maintained for no more than 3 months, during which time no changes in human apoprotein expression levels were observed.

Preparation of lipoprotein-defϊcient serum (LPDS): Lipoproteins were removed from fetal bovine serum by centrifugation in NaBr, p = 1.25g/mL, twice. The bottom two- thirds of the serum was removed and dialyzed extensively against phosphate-buffered saline. Protein concentration was determined by Lo wry assay (Lo wry et al, 1951) using bovine serum albumin as a standard. LPDS was used in the media at a final concentration of 0.25mg/mL.

Secretion of lipoproteins from McA7⁴ cells: Cells were seeded at 9 x 10⁶ in T 150 flasks. After 24hrs, the medium was removed, and the cells were washed 3 times with sterile phosphate-buffered saline (PBS) and incubated with 14mL of DMEM/high glucose, LPDS (0.25mg/mL), 1% penicillin/streptomycin, and 1% glutamine. After a 24hr incubation, the LPDS-containing cell medium was removed, spun at 3000rpm in a table top centrifuge to remove cell debris, and supplemented with 21μg/mL aprotinin, O.lmM phenylmethysulfonyl fluoride, 0.1% EDTA, 0.02% azide, and ImM BHT. The media underwent sequential flotation centrifugation to isolate HDL within a density range of 1.063-1.21g/mL to be analyzed on non-denaturing gradient and agarose gels or it was concentrated to 2mL using a YM-30 centricon and immediately analyzed by NaBr gradient centrifugation.

J774 macrophage HDL generation protocol: J774 macrophages were seeded and grown up to 80% confluency in 12 well plates, and then cholesterol loaded by incubating with ImL of DMEM supplemented with 1%FBS, 2μg/mL ACAT inhibitor, and 25μg/mL Ac-LDL for 48hrs. Following the loading period, the cells were incubated for 12hrs with 0.2% (w/v) BSA and 2μg/mL ACAT inhibitor in DMEM with 0.3mM CPT-cAMP for 12hrs to specifically up-regulate ABCA-I. Lipid efflux was initiated with the application of ImL total volume/well made up of DMEM containing 25 μg of recombinant wild-type or mutant human apoA-I protein in this case carrying a T7-tag. After 24hrs, the media was removed, and 150μl was run on a 2-36% NDGG or adjusted to 2mL with IXPBS and fractionated on a 10-20% NaBr density gradient. Following both fractionations samples were subjected to western blotting and probed with an HRP conjugated T7 antibody for the T7 tag on recombinant proteins.

Gradient centrifugal separation of lipoproteins from McA7⁴ cell or macrophage medium: 2mL of the medium from McA7⁴ cells or J774 macrophages was layered at the interface of a 10-20% NaBr gradient and centrifuged in a Beckman SW41Ti rotor at

38,000rpm for 66hrs at 15°C (Reschly et al, 2002). The densities of individual fractions were determined by refractometry of a treatment medium blank included in the run. 31, 0.4mL fractions were collected using an ISCO gradient collector with UV monitor (Instrument

Specialties Co., Lincoln, NE), dialyzed against Tris-buffered saline (1OmM Tris, 15OmM NaCl, 0.01% EDTA, 2OmM NaN₃, pH 7.4), and used for the apoprotein analysis via western blotting using T7 or species-specific antibodies.

Non-denaturing gradient gels: The LPDS-containing medium with HDL secreted from non-transfected and stably transfected McA7⁴ cells or J774 macrophages was loaded (150 μl/lane) directly onto 2-36% non-denaturing gradient gels. The standards (high molecular weight electrophoresis calibration kit, Amersham Biosciences Inc.) had the following radii: thyro globulin, 8.5nm; ferritin, 6.1nm; catalase 4.6nm; lactate dehydrogenase, 4.1nm; albumin, 3.55nm. Gels were electrophoretically separated in 9OmM Tris, 8OmM boric acid, 2.5mM sodium azide and EDTA, pH 8.3, for 24hrs at 360mA/gel. The next day, the proteins were electrotransferred to Immobilon P membrane overnight and then probed with T7 or species-specific antibodies followed by ECL detection.

Agarose electrophoresis: To separate preβ and α HDL, LPDS-containing cell medium with HDL secreted from non-transfected and stably transfected McA7⁴ cells was loaded (75 μl/lane) directly onto 0.7% agarose gels prepared and run in 25mM tricine, 3mM calcium lactate, and 0.05% sodium azide, pH 8.6. Following the transfer to Immobilon-P, the immunoblots were decorated with species-specific human or rat apoA-I followed by ECL detection.

Antibody production: Rabbit anti-human or anti-rat apoA-I antibodies were prepared in rabbits as described previously (Hay and Getz, 1979). To prepare antibodies that distinguish between rat and human apoA-I, polyclonal antibodies to human apoA-I were passed through a column containing rat HDL. The antibodies to the human apoproteins that cross-react with the rat apoproteins remained on the column, and the species-specific antibodies were eluted. Antibodies to the rat apoproteins were purified in a similar fashion. The specificity of the antibodies was confirmed by western blotting against human and rat apoA-I (Thurberg et al, 1996).

Species-specific immunoprecipitation of McA7⁴ cell peak density fractions: 400μl of each peak NaBr density fraction was added to 40μl of 50% slurry of protein A-sepharose beads (Sigma, P3391) and incubated at 4°C rotating head to bottom for lhr. The fraction and beads were centrifuged at 1200rpm for 2min at 4°C and the supernatant was transferred to a new tube. 1.5μl of species-specific human apoA-I antibody was added to the supernatant and then rotated head to bottom at 4°C overnight. 50μl of 50% slurry of protein A-sepharose beads was added and rotated head to bottom at 4°C for 2hr. The beads were precipitated by centrifugation at 1500rpm for 2min at 4°C. The supernatant was carefully removed and the beads were washed 3X with IXPBS. 20μl of 6X SDS-sample buffer and 80μl of dH₂O were added to the beads and heated at 95°C for 5min, then put back on ice for 2min. The mixture was centrifuged at 2400rpm for 2min at room temperature and transferred to a fresh tube, where it was then run on a 4-20% SDS-PAGE, transferred to immobilon P and immunodecorated with species-specific rat apoA-I antibody.

Preparation and characterization of apoA-I:rHDL particles: The rHDL substrate for the LCAT assays was prepared at a molar ratio of 80:4:1 sn-l-palmitoyl-sn-2- phosphatidylcholine (POPC): cholesterol :apo A-I with a trace amount of [³H] cholesterol for the LCAT assays as described (Sorci-Thomas et al, 1998). The rHDLs were purified by passage through a Superose 12 (Amersham Biosciences and Molecular Dynamics) column (55 x 1.8cm) in 1OmM Tris, 14OmM NaCl, 0.25mM EDTA and 0.15mM sodium azide, pH 7.4. The final molar composition of the rHDL and size based on non-denaturing gradient gel electrophoresis (Sorci-Thomas et al, 1998) were determined.

Exogenous LCAT assay: LCAT activation was performed as described (Sorci-

Thomas et al., 1998) using rHDL as a substrate containing POPC: cholesterol :apo A-I and [ H] cholesterol. The reaction was carried out at 37°C and the conversion of ³H cholesterol to cholesteryl ester was determined by thin layer chromatography (TLC). To quantitate radiolabeled cholesterol and cholesteryl ester, these regions were cut from the TLC silica and counted using a scintillation counter. The fractional esterification rate was multiplied by the nanomoles of the substrate cholesterol in the assay, corrected for the background, and converted to the nanomoles of the cholesteryl ester formed/hour/mL of LCAT. Cholesterol efflux: J774 murine macrophages were seeded and grown up to 80% confluency in 12 well plates, and then cholesterol loaded/labeled by incubating with DMEM supplemented with 1% FBS, 2μg/mL ACAT inhibitor, 3μCi/mL [1,2-³H] cholesterol, and 25μg/mL acetylated LDL for 48hrs [36]. Following the labeling period, the cells were washed and incubated for 12hrs with 0.2% (w/v) BSA and 2μg/mL ACAT inhibitor in DMEM with 0.3mM CPT-cAMP to specifically up-regulate ABCA-I. Lipid efflux was initiated with the application of ImL total volume/well DMEM in the presence or absence of wild-type/mutant human apoA-I protein (0-16μg). After 4hrs, the media was removed and the lipids from the media and cells were extracted by the Bligh and Dyer procedure (Bligh and Dyer, 1959). The % cholesterol efflux was measured comparing radioactivity counts of media over total counts (cells + media) x 100.

Statistical Analysis: Results are expressed as means ± S.D. Group differences were tested by analysis of variance and student's f-tests. The significance level was set at p < 0.05.

RESULTS

ApoA-I recombinant protein HDL subclass association has been shown to parallel

HDL generation in vivo (Reschly et al., 2002). Therefore, the capacity of mutant and wild- type human apoA-I proteins to associate with HDL subclasses were examined. The mutant proteins are designated by the specific PCS replacing the NPCS between residues 184-190. Thus, when the PCS between helices 3 and 4 (i.e. PCS2) is inserted in place of the NPCS at residues 184-190 of human apoA-I, the mutant is designated NPCS→PCS2, as illustrated in figure 1.

HDL preferential association assay: Recombinant apoA-I constructs were synthesized, each containing N-terminal tags consisting of a His tag for purification and a T7 tag for selective identification. It has been shown by comparing recombinant wild-type apoA- I (T7-HuA-I) protein with purified plasma apoA-I that the presence of the N-terminal tag did not significantly affect the properties of the protein (Reschly et al, 2002). The recombinant proteins displayed the expected molecular weights determined by SDS-PAGE. From densitometric scanning of the gel, the purity of the recombinant proteins was > 95%. The ability of the various recombinant proteins to associate with mature HDL subclasses was examined. In this association assay, lipid- free recombinant apoA-I was incubated with different ratios of human HDL₂ and HD L3 (i.e. 1 :1, 1 :2, and 2:1) at an endogenous to recombinant apoA-I weight ratio of 62.5:1. At this ratio the added apoA-I "tags" the HDL without significantly altering the HDL size or density. Assuming two to four molecules of apoA-I per HDL, there are about 20 HDL particles (HDL₂ + HDL₃) per recombinant apoA-I molecule in the association assays. With this excess of HDL, the recombinant apoA-I binds to the HDL subclass for which it has the highest affinity.

The HDL subclasses after incubation with the recombinant apoA-I were separated on NaBr equilibrium density gradients, and the fractions were analyzed for the presence of endogenous apoA-I and recombinant proteins by immunoblotting for the T7 tag. In order to compare data among experiments, the recombinant apoA-I distributions were normalized to the starting HDL₂:HDL₃ proportions by dividing the recombinant apoA-I distributions by the corresponding endogenous apoA-I distribution. Endogenous apoA-I was found to be an appropriate surrogate for starting HDL subclass proportions, as demonstrated by the similar HDL subclass ratio calculated from A₂go tracings and from the densitometric scanning of the western blot for endogenous apoA-I.

T7-HuA-I distributed to HDL subclasses in a pattern that followed closely that of endogenous apoA-I (FIG. 2). On average about 50% of T7-HuA-I distributed to HDL₂ and 50% to HD L₃, showing that T7-HuA-I bound with approximately equal affinity to HDL₂ and HDL₃. The human apoA-I mutants in which PCS2 replaced NPCS (i.e. T7-HuA-I (NPCS→PCS2)) preferentially associated in a statistically significant manner with HDL₂ (75- 80%), while only 20-25% associated with HDL₃. On the other hand, the human apoA-I mutant in which PCS3 replaced NPCS (i.e. T7-HuA-I (NPCS→PCS3)) preferentially associated in a statistically significant manner with HDL₃ (70-75%), with only 25-30% associating with HDL₂. The substitution of the other PCS of human apoA-I for NPCS had no statistically significant effect on the HDL subclass distribution of recombinant proteins. These data indicate that the nature of the sequence between putative helices 7 and 8 influences the binding specificity of human apoA-I for HDL subclasses. The physical properties of the two mutants with different HDL subclass distribution then examined.

Self-association of lipid-free apoA-I proteins: The self-association properties of the recombinant apoA-I proteins in the lipid-free state were examined by cross-linking with BS₃. It has been shown that T7-HuA-I self-association properties were similar to those of isolated human plasma apoA-I (Reschly et ah, 2002). T7-HuA-I (NPCS→PCS3) and T7-HuA-I

(NPCS→PCS2) had differing self-association properties (FIG. 3). T7-HuA-I and the T7- HuA-I (NPCS→PCS3) mutant generated a higher percentage of trimer, tetramer, and pentamers than the T7-HuA-I (NPCS→PCS2) mutant. These findings indicate that the specific sequence within the segment between helices 7 and 8 affects the self-association properties of the lipid- free protein.

DMPC (lipid binding) clearance assay: The lipid affinity of the recombinant mutant apoA-I proteins were measured using the DMPC clearance assay. Figure 4 illustrates that wild-type and mutant recombinant apoA-I proteins display a similar, but not identical ability to clear DMPC vesicles. This trend was observed when using either a ratio of apoA-I:DMPC (w/w) of 1 :1 (data not shown) or 1 :2.5. The clearance by T7-HuA-I and T7-HuA-I (NPCS→PCS3) mutant were essentially identical while the clearance by the T7-HuA-I (NPCS→PCS2) mutant was somewhat slower though reaching the same maximum.

Physicochemical properties of rHDL particles: One of the primary functions of apoA-I is to assemble HDL particles. A surrogate for this is represented by the ability to generate HDL-like particles with a physiologically relevant lipid. Recombinant apoA-I was reconstituted in particles containing POPC and cholate in a molar ratio of 80:1 :108 of POPC:apoA-I:cholate. Recombinant wild-type apoA-I:rHDL had a hydrodynamic diameter of 10.3 nm (FIG. 5 and Table 5) and contained 2 apoA-I molecules per rHDL. This is not different from plasma derived apoA-I:rHDL (Reschly et ah, 2002). The size and composition of rHDL particles containing each of the two mutants were very close to one another and not different from the wild-type protein (Table 5).

Recombinant apoA-I secondary structure: To ascertain the secondary structure, the α-helical content of lipid-free and lipid-bound recombinant apoA-I proteins was examined by circular dichroism. The α-helical contents for lipid-free T7-HuA-I and T7-HuA-I (NPCS→PCS3) mutant were similar, while the T7-HuA-I (NPCS→PCS2) mutant appeared to exhibit a higher degree of lipid-free α-helicity (Table 5). The lipid-associated α-helicity of the two mutant proteins closely resembled that of T7-HuA-I.

Lipid-free/lipid-associated isothermal stability of recombinant apoA-I proteins:

Investigations in several laboratories have shown that the denaturation of lipid-free apoA-I is a reversible process which has a midpoint of denaturation (Dy₂) close to 1.0 M GdnHCl (Saito et ah, 2004). Furthermore, the effectiveness of GdnHCl in unfolding apoA-I is significantly decreased when apoA-I is complexed with DMPC. The Dy₂ of lipid-free T7-HuA-I was similar to published values using plasma human apoA-I (Saito et al, 2004). The denaturation profiles for lipid-free wild-type and mutant recombinant apoA-I proteins were similar (FIG. 6A and Table 6). While the Dy₂ for lipid-associated T7-HuA-I was similar to reported values for lipid-associated plasma human apoA-I, both mutants when lipid-associated appeared to be more stable than T7-HuA-I to guanidine denaturation (FIG. 6B and Table 7).

Binding curves of recombinant apoA-I to HDL: In the association assays presented in Figure 2, a small amount of recombinant apoA-I was incubated with a mixture OfHDL₂ and HDL₃ so that these two HDL subclasses competed for the added recombinant apoA-I. The results from these experiments suggested that the affinity of some of these recombinants differ for the two HDL subclasses. Having confirmed that overall there are only modest differences, if any, in the physico chemical properties between the two PCS mutants exhibiting preferential binding to HDL subclasses and wild-type human apoA-I, the affinity of recombinant apoA-I proteins for each HDL subclass were examined. Since added apoA-I did not cause remodeling of HDL or significant desorption of endogenous apoA-I (see below), the binding curves of apoA-I for HDL subclasses could be measured directly using Scatchard analysis. For this analysis non-denaturing agarose electrophoresis was employed to separate free and bound recombinant apoA-I. The Scatchard analysis was based on reversible binding of apoA- I to a site on an HDL particle as shown in equation 1,

apoA-I + site <→ complex Equation 1 : K_d = (apoA-I)_free (site)_free/C_x

Since (Site)_free = (Sites)_totai - (Sites)_occupied = (Sites)_totai - (apoA-I)_boUnd and C_x = (ApoA-I)_boUnd, the value of K_<j and (Sites)_totai could be assessed as parameters using non- linear least square analysis.

For determination of these binding curves, between 1.25-51 μg of recombinant apoA-I was incubated with 200 μg of isolated HDL₂ or HDL₃, corresponding to 130 or 150 μg of endogenous apoA-I. Scatchard analysis (Figure 7) demonstrated that T7-HuA-I binding to HDL₂ (IQ=3.2μM) and HDL₃ (K₄=I.6μM) are similar. The HuA-I (NPCS→PCS2) mutant displayed higher affinity for HDL₂ (K_d=5.3μM) than HDL₃ (K_d>lM), consistent with the association assay (Figure 2). On the other hand, the HuA-I (NPCS→PCS3) mutant displayed a higher affinity for HDL₃ (K_d=6.7μM) versus HDL₂ (K_d>lM). Thus the two assays used for determining the association of recombinant mutant apoA-I proteins with HDL revealed similar preferences for HDL subclasses. HDL remodeling in binding assays: To ensure that wild-type or mutant apoA-I affinity for an HDL subclass was being measured rather than an artifact of HDL remodeling, the HDL size following the addition of the highest concentration (51 μg) of apoA-I used in the binding assays was examined. When 51 μg of recombinant apoA-I was incubated with either HDL subclass in the binding assays, the Stokes diameter did not change, nor was apoE or apoA-I released from the particle as examined by western blotting, indicating that by this parameter major HDL remodeling did not occur.

Cell lines expressing human apoA-I: Having identified human apoA-I PCS mutants that preferentially associate with individual HDL subclasses in vitro and retain the physical properties of the wild-type human apoA-I protein, next examined was whether these particular mutants could preferentially generate HDL particles when stably transfected in McA7⁴ cells. McA7⁴ cells are a rat hepatoma cell line that secretes VLDL- and HDL-like spherical particles (Thurberg et ah, 1996). Previously, it was shown that HDL association preferences parallel the HDL profile in vivo (Reschly et ah, 2002).

Secretion of apoproteins by non-transfected and stably transfected McA7⁴ cells:

Since the HDL profiles produced by endogenous rat apoA-I are compared with those stably transfected with human apoA-I cDNAs, it is necessary to work with clones that secrete similar levels of apoA-I. Using species-specific antibodies, the amount of human and rat apoA-I in the non-transfected and stably transfected cell lines was measured. FIG. 1 IA demonstrates that in the clones chosen for analysis, wild-type human apoA-I and the PCS mutant apoA-I proteins are secreted at similar levels. Compared to non-transfected McA7⁴ cells, the level of endogenous rat apoA-I secreted into the media is decreased 4-fold in the cells expressing the human proteins (FIG. HB). Furthermore, as a control to confirm that the transfection of DNA was not disturbing protein production, the amount of secreted rat apoE between non- transfected and transfected cells was examined and no differences were observed (FIG. HC).

Human apoA-I stably transfected McA7⁴ cells bias the density of secreted HDL:

Having established that stably transfected McA7⁴ cells were secreting similar levels of apoA- I, an equilibrium NaBr density gradient was employed to examine the density of HDL particles secreted by the non-transfected and stable transfected McA7⁴ cells into LPDS- containing medium. The distributions of endogenous rat apoA-I and transfected human apoA- I proteins in the gradient fractions were determined by western blotting using species-specific antibodies. The distribution of endogenous rat apoA-I on the HDL particles secreted by non- transfected McA7⁴ cells is shown in FIG. 12A. The peak density of the rat apoA-I containing HDL particles is 1.1037g/mL. The second peak of rat apoA-I likely represents poorly lipidated or lipid-free rat apoA-I. The relative monodispersity of the HDL particles secreted from non-transfected cells is consistent with the presence of one major subclass of HDL in rat plasma. However in contrast, when the cells stably transfected with wild-type human apoA-I were examined, two major HDL particles were observed, with peaks at densities at 1.0901g/mL and 1.1557g/mL (FIG. 12A). These densities fall within the density range corresponding to HDL₂ and HDL₃ found in human plasma, respectively.

The distribution of the two PCS mutants was next examined. HuA-I (NPCS→PCS2), which preferentially associated with HDL₂ in vitro, generates one major HDL with a peak density between 1.1037-1.1191g/mL (FIG. 12B). In contrast, the peak of the HDL generated by the HuA-I (NPCS→PCS3) mutant, which preferentially associated with HDL₃ in vitro, is at 1.1362g/mL, but also appears to have a shoulder at a lower density (FIG. 12B). These studies have been confirmed by examining two independent stable transfected cell lines in duplicate for each apoA-I protein.

Stably transfected human apoA-Is bias the size of α-HDL particles secreted from stably transfected Mc A7⁴ cells: To determine the size of the particles secreted from the stable transfected cells, lipoproteins secreted from each stable cell line into LPD S -containing medium were isolated by ultracentrifugation sequential flotation between the density range of 1.063-1.2 lg/mL and separated on a 2-36% non-denaturing gradient gel that was probed with species-specific antibodies. The rat HDL particles from non-transfected cells had a diameter of 11.9nm. Two discrete sized HDL particles were observed for wild-type human apoA-I at 10.6 and 8.6nm in diameter, which is within the range for the HDL subclasses typically seen in human plasma. In contrast, the HuA-I (NPCS→PCS2) mutant generated a single size HDL particle with a diameter of 10.6nm. However, the HuA-I (NPCS→PCS3) mutant generated two different sized HDL particles, 10.6 and 8.6nm, with 80% of the particles generated being the smaller 8.6nm particle (FIG. 13A). These studies have been confirmed by examining two independent stable transfected cell lines in duplicate for each apoA-I protein.

To further examine the properties of the HDLs secreted by the non-transfected and stably transfected McA7⁴ cells, their electrophoretic mobility was analyzed. Discoidal and spherical HDL have distinct electrophoretic mobility based on charge, with discoidal HDL migrating to preβ and spherical HDL migrating to α. The majority (95-100%) of the HDL particles secreted from McA7⁴ cells have been shown to be spherical (Y ao et al, 1993). As seen in Figure 13B, the HDL secreted from both non-transfected and stably transfected McA7⁴ cells are α in mobility.

Endogenous rat apoA-I follows the distribution of human apoA-I in stably transfected Mc A7⁴ cells: Having determined the distribution of rat apoA-I in non-transfected cells and human apoA-I on HDL in the transfected cells, next investigated was whether the expression of the human protein influenced the distribution of endogenous rat apoA-I (FIG. 14). Comparison of the data in FIGs. 12A-B and 14 illustrates that in the presence of the human proteins, rat apoA-I is redistributed to follow in some measure the human protein, and in turn, its distribution is distinct for each of the stable transfected cells. One possible explanation for our results is that the human apoA-I proteins are remodeling the rat HDL extracellularly. However, this is not believed to be the explanation since the incubation of non-transfected McA7⁴ cells with exogenous recombinant human apoA-I had no effect on the distribution of rat apoA-I on the HDL secreted from the cells and did not generate human apoA-I particles observed in FIG. 12 (data not shown).

Endogenous rat apoA-I largely cohabitates with HDL that mimic native rat

HDL: Since rat apoA-I was observed following the distribution of human apoA-I in transfected McA7⁴ cells, next examined was whether rat and human apoA-I could be found on the same HDL particle. To address this question, from the three different human apoA-I transfected cell lines, the peak fractions described in the NaBr gradient fractionation in FIG. 12 were obtained. The HDL in these peak fractions were immunoprecipitated using species- specific human apoA-I antibody and then probed with species-specific rat apoA-I. Conversely using fractions from non-transfected cells, density fractions that corresponded to the peak fractions identified in wild-type human apoA-I transfected cells were examined. While there was rat apoA-I on human apoA-I associated HDL for the denser wild-type human apoA-I generated HDL, as well as the particle generated by HuA-I (NPCS→PCS3), rat apoA- I was found preferentially cohabitated with human apoA-I on HDL particles that most closely resembled the density of native rat HDL (FIG. 15).

The recombinant human apoA-Is yield no major differences in two major functions of apoA-I: To confirm that the PCS mutant human apoA-I proteins were able to promote cholesterol efflux, the wild-type and PCS mutant human apoA-I proteins were incubated with [³H] cholesterol labeled, acetylated LDL loaded J774 macrophages. There was no difference in the efflux capacity of the proteins in the absence of ABCA-I induction. In the presence of ABCA-I induction, low concentrations of the mutant human apoA-I proteins were somewhat less effective in promoting cholesterol efflux than wild-type human apoA-I. However, there was no difference in the extent of efflux promoted by the 3 human proteins at the highest concentration of protein added (FIG. 16). This indicates that overall, the PCS mutants are capable of promoting cholesterol efflux from loaded macrophages just as well as wild-type human apoA-I. Since HDL are also formed by the interaction of lipid-free apoA-I with ABCA-I, the ability of wild-type and PCS mutant human apoA-I proteins to generate HDL was examined. No difference in HDL density or size was observed (data not shown).

As a second assessment of apoA-I function, the ability of the recombinant apoA-I proteins to activate LCAT was examined. LCAT activation analysis was performed by examining the conversion of [³H] cholesterol, as a component of apoA-I containing reconstituted HDL to cholesteryl ester. The conversion of [³H] cholesterol to [³H]cholesteryl ester was determined by lipid extraction of the incubation mixture followed by thin layer chromatography. The fractional esterifϊcation rate was kept below 15% to maintain first order kinetics. No major differences in the ability of the recombinant mutant human apoA-I proteins ability to activate human LCAT versus recombinant wild-type or plasma human apoA-I was observed (data not shown).

EXAMPLE 2

FIG. 8 illustrates the helical wheel projections of the original putative helices 7 and 8 and the sequences formed by the exchange of the NPCS for PCS2 (FIG. 8A) or PCS3 (FIG. 8B). These projections highlight the size of the hydrophobic face of the resultant helices. This face is more hydrophobic in the case of the PCS2 mutant than the PCS3 mutant and may more tightly associate with the surface of HDL₂ having a lower radius of curvature and thus a larger lipid surface (FIG. 9B). In contrast, the HuA-I (NPCS→PCS3) mutant may have a smaller hydrophobic face, thus favorably binding to a smaller, more curved lipid surface (FIG. 9C).

The generation of a mutant human apoA-I containing three copies of either PCS2 or

PCS3, one at its native location, one substituted for NPCS between putative helices 7 and 8, and another PCS elsewhere in the human protein does not further alter the association properties of the resultant mutant protein.

Table 5. Characterization of recombinant apoA-I proteins in rHDL and lipid- free forms.

a Particle diameters were determined from native polyacrylamide gel electrophoresis using reference globular proteins (Fig. 5). n=2. b The number of proteins per particle was determined by SDS-PAGE of rHDL cross-linked with BS₃. n=2 c The percent α-helical content of lipid-free recombinant and plasma-derived proteins were estimated from molar ellipticity at 222 nm. n = 3.

The percent α-helical content of recombinant and plasma apoA-LrHDL. n = 2.

Table 6. Guanidine HCl denaturation of lipid-free recombinant apoA-I proteins.

aThe y- intercept of the denaturation curve, represented in calories. «=3. b The slope of the denaturation curve, represented in calories. «=3 c The midpoint concentration of Gdn-HCl where half of lipid-free recombinant is folded and unfolded, n = 3; ^ p<0.05, compared to T7-HuA-I.

Table 7. Guanidine HCl denaturation of lipid-associated recombinant apoA-I proteins.

aThe y- intercept of the denaturation curve, represented in calories. «=3. b The slope of the denaturation curve, represented in calories. «=3 c The midpoint concentration of Gdn-HCl where half of lipid-associated recombinant is folded and unfolded. « = 3, Vθ-0001, compared to T7-HuA-I; ^¥p<λ0001, compared to T7-HuA-I (NPCS→PCS3). REFERENCES

The following references, whose reference numbers are noted in brackets throughout this application, are incorporated by reference herein:

U.S. Patent 4,554,101

U.S. Patent 4,683,202

U.S. Patent 4,879,236 U.S. Patent 5,339,363

U.S. Patent 5,466,468

U.S. Patent 5,543,158

U.S. Patent 5,641,515

U.S. Patent 5,846,225 U.S. Patent 5,846,233

U.S. Patent 5,871,986

U.S. Patent 5,876,968

U.S. Patent 5,925,565

U.S. Patent 5,928,906 U.S. Patent 5,935,819

U.S. Patent 6,013,516

U.S. Patent 6,897,039

US 20030181372

WO 95/31540 WO 98/56906

Ajees et al, (2006) Proc Natl Acad Sci USA 103, 2126-2131

Almendro et al, J Immunol, 157:5411-5421, 1996.

Angel et al, Cell, 49:729, 1987b.

Angel et al, MoI. Cell Biol, 7:2256, 1987a. Assmann G and Gotto AM Circulation 109, 1118-14, 2004

Asztalos BF Curr Opin Cardiol 19, 385-391, 2004

Atchison and Perry, Cell, 46:253, 1986.

Atchison and Perry, Cell, 48:121, 1987.

Ausubel, ed., Current protocols in molecular biology, New York, John Wiley & Sons, 1996. Baichwal and Sugden, In: Gene Transfer, Kucherlapati R, ed., New York, Plenum Press, pp. 117-148, 1986.

Banerji et al, Cell, 27:299, 1981.

Banerji et al, Cell, 35:729, 1983. Barter Clin Exp Pharmacol Physiol 24, 286-287, 1997

Barter et al, Circ Res 95, 764-772, 2004

Berkhout et al, Cell, 59:273, 1989.

Blanav et al, EMBO J., 8:1139, 1989.

Bligh and Dyer Can J Biochem Physiol 37, 911-917, 1959 Boshavt et al, Cell, 41 :521, 1985.

Bosze et al, EMBO J., 5: 1615, 1986.

Braddock et al, Cell, 58:269, 1989.

Bulla and Siddiqui, J. Virol, 62:1437, 1986.

Campbell and Villarreal, MoI Cell Biol, 8:1993, 1988. Campere and Tilghman, Genes and Dev., 3:537, 1989.

Campo et al, Nature, 303:77, 1983.

Carbonelli et al FEMS Microbiol Lett. 177(1): 75-82, 1999.

Castelli et al, JAMA 256, 2835-2838, 1986

Celander and Haseltine, J. Virology, 61 :269, 1987. Celander et al, J. Virology, 62:1314, 1988.

Chandler et al, Cell, 33:489, 1983.

Chandler et al, Proc Natl Acad Sci USA. 94(8):3596-3601, 1997.

Chatterjee et al, Proc. N at' I Acad. Sci. USA., 86:9114, 1989.

ClonTech. Yeast Protocols Handbook 1997. Cocea, Biotechniques. 23(5):814-816, 1997.

Cohen et al, J. Cell. Physiol, 5:75, 1987.

Costa et al, MoI Cell. Biol, 8:81, 1988.

Coupar et al, Gene, 68:1-10, 1988.

Cripe et al, EMBO J., 6:3745, 1987. Dandolo et al, J. Virology, 47:55, 1983.

Davidson et al, Proc Natl Acad Sci USA 93, 13605-13610, 1996

De Villiers et al, Nature, 312:242, 1984.

Deschamps et al, Science, 230:1174, 1985.

Devereux et al, Nucl. Acid Res., 12:387-395, 1984. Dodart et al, Proc Natl Acad Sci USA 102, 1211-1216, 2005

Edbrooke et al., Mol. Cell. Biol., 9:1908, 1989.

Feng and Holland, Nature, 334:6178, 1988.

Firak and Subramanian, MoI. Cell. Biol, 6:3667, 1986. Foecking and Hofstetter, Gene, 45(1): 101-5, 1986.

Frank and Marcel J Lipid Res 41, 853-872, 2000

Friedmann, Science, 244:1275-1281, 1989.

Fujita et al, Cell, 49:357, 1987.

Gittcs et al., Cell, 33:717, 1983. Gloss et al, EMBO J, 6:3735, 1987.

Godbout et al, Mol Cell. Biol, 8:1169, 1988.

Goodbourn and Maniatis, Proc. Nat 'I Acad. Sci. USA, 85:1447, 1988.

Goodbourn et al, Cell, 45:601, 1986.

Greene et al, Immunology Today, 10:272, 1989. Grosschedl and Baltimore, Cell, 41 :885, 1985.

Haslinger and Karin, Proc. Nat'l Acad. Sci. USA., 82:8572, 1985.

Hauber and Cullen, J. Virology, 62:673, 1988.

Hay and Getz, J Lipid Res 20, 334-348, 1979

Hen et al, Nature, 321 :249, 1986. Hensel et al, Lymphokine Res., 8:347, 1989.

Hermonat and Muzyczka, Proc. Nat'l. Acad. Sci. USA, 81 :6466-6470, 1984.

Herr and Clarke, Cell, 45:461, 1986.

Hirochika ^ α/., J. Virol, 61 :2599, 1987.

Holbrook e^/., Virology, 157:211, 1987. Horlick and Benfϊeld, MoI Cell. Biol, 9:2396, 1989.

Horwich et al J. Virol, 64:642-650, 1990.

Huang et al, Cell, 27:245, 1981.

Hug et al, MoI Cell Biol, 8:3065, 1988.

Hwang et al, MoI Cell. Biol, 10:585, 1990. Imagawa et al, Cell, 51 :251, 1987.

Imbra and Karin, Nature, 323:555, 1986.

Imler et al, MoI Cell. Biol, 7:2558, 1987.

Imperiale and Nevins, MoI Cell. Biol, 4:875, 1984.

Inouye et al , Nucleic Acids Res. , 13:3101-3109, 1985. Jakobovits et al., Mol. Cell. Biol., 8:2555, 1988.

Jameel and Siddiqui, MoI. Cell. Biol., 6:710, 1986.

Jaynes et al., Mol. Cell. Biol, 8:62, 1988.

Johnson et al, MoI. Cell. Biol, 9:3393, 1989. Kadesch and Berg, MoI Cell. Biol, 6:2593, 1986.

Karin et al, MoI Cell. Biol, 7:606, 1987.

Katinka et al, Cell, 20:393, 1980.

Kawamoto et al, MoI Cell. Biol, 8:267, 1988.

Kiledjian et al, MoI Cell. Biol, 8:145, 1988. Klamut et al, Mol Cell. Biol, 10:193, 1990.

Koch et al, MoI Cell. Biol, 9:303, 1989.

Kontush et al, Arterioscler Throm Vase Biol 27, 1843-1849, 2007

Kozarsky et al, Nature 387, 414-417, 1997

Kraus et al. FEBS Lett., 428(3): 165-170, 1998. Rriegler and Botchan, In: Eukaryotic Viral Vectors, Y. Gluzman, ed., Cold Spring Harbor: Cold Spring Harbor Laboratory, NY, 1982.

Rriegler and Botchan, MoI Cell. Biol, 3:325, 1983.

Rriegler et al, Cell, 38:483, 1984a.

Rriegler et al, Cell, 53:45, 1988. Rriegler et al, In: Cancer Cells 2 /Oncogenes and Viral Genes, Van de Woude et al eds, Cold Spring Harbor, Cold Spring Harbor Laboratory, 1984b.

Rriegler et al, In: Gene Expression, D. Hamer and M. Rosenberg, eds., New York: Alan R. Liss, 1983.

Ruhl et al, Cell, 50:1057, 1987. Runz et al, Nucl Acids Res., 17:1121, 1989.

Ryte and Doolittle, J. MoI Biol, 157(l):105-132, 1982.

Lareyre et al, J Biol Chem., 274(12):8282-8290, 1999.

Laspia et al, Cell, 59:283, 1989.

Latimer et al, MoI Cell. Biol, 10:760, 1990. Lee et al, J Auton Nerv Syst. 74(2-3):86-90, 1997.

Lee et al, Nature, 294:228, 1981.

Lee et al, Nucleic Acids Res., 12:4191-206, 1984.

Levenson et al, Hum Gene Ther. 20;9(8):1233-1236, 1998.

Levinson et al, Nature, 295:79, 1982. Lewis Curr Opin Cardiol 21, 345-352, 2006

Un et al, MoI Cell. Biol, 10:850, 1990.

Lowry et al, (1951) J Biol Chem 193, 265-275

Luria et al, EMBO J., 6:3307, 1987. Lusky and Botchan, Proc. Nat 7 Acad. Sci. USA., 83 :3609, 1986.

Lusky et al, MoI Cell. Biol, 3:1108, 1983.

Macejak and Sarnow, Nature, 353:90-94, 1991.

Majors and Varmus, Proc. Nat'l Acad. Sci. USA., 80:5866, 1983.

Mautino and Morgan, AIDS Patient Care STDS. 2002 Jan;16(l):l 1-26. Review. McNeall et al, Gene, 76:81, 1989.

Miksicek et al, Cell, 46:203, 1986.

MiXlQX ΗE Am Heart J i n, 589-597, 1987

Mineo et al, Circ Res 98, 1352-1364, 2006

Miyoshi et al, Development of a self-inactivating lentivirus vector. J. Virol. 72, 8150-8157, 1998

Mordacq and Linzer, Genes and Dev., 3:760, 1989.

Moreau et al, Nucl Acids Res., 9:6047, 1981.

Mowri et al, Cell, 48:691, 1987.

Naldini L Lentiviruses as gene transfer agents for delivery to non-dividing cells. Curr Opin Biotechnol 9, 457-463, 1998

Naldini L et al, In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector. Science 272, 263-267, 1996

Needleman and Wunsch, J. MoI Biol. 48: 443, 1970.

Ng et al, Nuc. Acids Res., 17:601, 1989. Nicolas and Rubenstein, In: Vectors: A survey of molecular cloning vectors and their uses, Rodriguez and Denhardt (eds.), Stoneham: Butterworth, pp. 493-513, 1988.

Nomoto et al, Gene, 236(2):259-271, 1999.

Ondek et al, EMBO J., 6:1017, 1987.

Palmiter et al, Nature, 300:611, 1982. Pearson and Lipman, Proc. Natl. Acad. Sci. U.S.A. 85: 2444, 1988

Pech et al, MoI Cell. Biol, 9:396, 1989.

Pelletier and Sonenberg, Nature, 334:320-325, 1988.

Pfϊefer A Lentivial transgenesis. Transgenic Res. 13(6):513-522, 2004

Picard and Schaffner, Nature, 307:83, 1984. Pinkert et al, Genes and Dev., 1 :268, 1987.

Ponta et al, Proc. Nat 'I Acad. Sci. USA., 82:1020, 1985.

Forton et al, MoI Cell. Biol., 10:1076, 1990.

Queen and Baltimore, Cell, 35:741, 1983. Quinn et al., Mol. Cell. Biol., 9:4713, 1989.

Rader DJ J Clin Invest 116, 3090-3100, 2006

Reardon et al, Biochemistry 40, 13670-13680, 2001

Redondo et al, Science, 247:1225, 1990.

Reisman and Rotter, MoI Cell. Biol, 9:3571, 1989. Remington's Pharmaceutical Sciences, 15^th ed., pages 1035-1038 and 1570-1580, Mack Publishing Company, Easton, PA, 1980.

Ren et al, Biochemistry 44, 14907-14919, 2005

Reschly et al, JBiol Chem 277, 9645-9654, 2002

Resendez Jr. et al, MoI Cell. Biol, 8:4579, 1988. Ridgeway, In: Vectors: A survey of molecular cloning vectors and their uses, Rodriguez RL, Denhardt DT, ed., Stoneham:Butterworth, pp. 467-492, 1988.

Rigotti et al, Proc Natl Acad Sci USA 94, 12610-12615, 1997

Kittling et al., Nucl. Acids Res., 17:1619, 1989.

Rosen et al, Cell, 41 :813, 1988. Rubin EM, Ishida BY, Clift SM and Krauss RM Proc Natl Acad Sci USA 88, 434-438, 1991

Saito et al, Prog Lipid Res 43, 350-380, 2004

Sakai et al, Genes and Dev., 2:1144, 1988.

Sambrook et al, In: Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor

Press, Cold Spring Harbor, NY, 1989. Satake et al, J. Virology, 62:970, 1988.

Schaffher et al, J. MoI Biol, 201 :81, 1988.

Searle et al, MoI Cell. Biol, 5:1480, 1985.

Sharp and Marciniak, Cell, 59:229, 1989.

Shaul and Ben-Levy, EMBO J., 6:1913, 1987. Sherman et al, MoI Cell. Biol, 9:50, 1989.

Singh IM, Shishehbor MH, and Ansell BJ, JAMA 298, 786-798, 2007

Sleigh and Lockett, J EMBO, 4:3831, 1985.

Smith and Waterman, Adv. Appl. Math. 2: 482, 1981

Sorci-Thomas et al, JBiol Chem 273, 11776-11782, 1998 Spalholz et al, Cell, 42:183, 1985.

Spandau and Lee, J. Virology, 62:421, 1988.

Spandidos and Wilkie, EMBO J, 2: 1193, 1983.

Stephens and Hentschel, Biochem. J, 248:1, 1987. Stuart et al, Nature, 317:828, 1985.

Sullivan and Peterlin, MoI. Cell. Biol, 7:3315, 1987.

Swartzendruber and Lehman, J. Cell. Physiology, 85:179, 1975.

Takebe et al, MoI. Cell. Biol, 8:466, 1988.

Tavernier et al, Nature, 301 :634, 1983. Taylor and Kingston, MoI Cell. Biol. , 10 : 165 , 1990a.

Taylor and Kingston, MoI Cell. Biol, 10:176, 1990b.

Taylor et al, J. Biol. Chem., 264:15160, 1989.

Temin, In: Gene Transfer, Kucherlapati (ed.), New York: Plenum Press, pp. 149-188, 1986.

Thiesen et α/., J. Virology, 62:614, 1988. Thurberg et al, J Biol Chem 271, 6062-6070, 1996

Tranche et al, MoI Biol. Med., 7:173, 1990.

Tranche et al, MoI Cell. Biol, 9:4759, 1989.

Tsumaki et al, JBiol Chem. 273(36):22861-22864, 1998.

Tyndall et al, Nuc. Acids. Res., 9:6231, 1981. Vannice and Levinson, J. Virology, 62: 1305, 1988.

Vasseur et al, Proc. Nat 'I Acad. Sci. USA., 77:1068, 1980.

Wang and Calame, Cell, 47:241, 1986.

Wang N, Silver DL, Thiele C, and Tall AR, J Biol Chem 276, 23742-23747, 2001

Weber et al, Cell, 36:983, 1984. Weinberger et al. MoI Cell. Biol. , 8:988, 1984.

Winoto and Baltimore, Cell, 59:649, 1989.

Wu et al, Biochem. Biophys. Res. Commun., 233(l):221-6, 1997.

Yao Z, Lauer SJ, Sanan DA, and Fazio S, Arterioscler Thomb Vase Biol 13, 1476-1486, 1993

Yutzey et al. MoI Cell. Biol, 9:1397, 1989. Zhao-Emonet et al, Biochem. Biophys. Acta., 1442(2-3): 109-19, 1998.

Zufferey et al, J Virol 72, 9873-9880, 1998

Zufferey et al, Multiply attenuated lentiviral vector achieves efficient gene delivery in vivo. Nat Biotechnol 12, 871-875, 1997

Claims

1. An isolated polypeptide comprising a polypeptide sequence selected from the group consisting of:

(a) amino acids 25-267 of SEQ ID NO:1, in which amino acids 208-214 of SEQ ID NO: 1 are substituted with an amino acid sequence comprising a proline residue; and

(b) an amino acid sequence at least 90% identical to amino acids 25-267 of SEQ ID NO: 1, in which amino acids 208-214 of SEQ ID NO:1 are substituted with an amino acid sequence comprising a proline residue; wherein the isolated polypeptide preferentially associates with an HDL subclass.

2. The isolated polypeptide of claim 1, wherein the polypeptide sequence is the sequence of l(a).

3. The isolated polypeptide of claim 1, wherein the substituting amino acid sequence in l(a) or l(b) is X₁X₂X₃PX₄XsXe, wherein each OfX₁ - X₆ is independently any amino acid.

4. The isolated polypeptide of claim 3 , wherein the substituting sequence in 1 (a) or 1 (b) is the amino acid sequence set forth in SEQ ID NO:2 or SEQ ID NO:3.

5. The isolated polypeptide of claim 1, further comprising amino acids 19-24 of SEQ ID NO:1.

6. The isolated polypeptide of claim 1, wherein the HDL subclass is subclass 2 or 3.

7. The isolated polypeptide of claim 1 , wherein the polypeptide sequence is further substituted with a cysteine residue at an amino acid position corresponding to amino acid 27, 30, 33, 39, 45, 49, 54, 58, 61, 96, 123, 131, 151, 173, 215, or 238 of SEQ ID NO:1.

8. A dimer of the isolated polypeptide of claim 7.

9. The isolated polypeptide of claim 1, further comprising a trimerising module.

10. A trimer of the isolated polypeptide of claim 9.

11. The isolated polypeptide of claim 1 , wherein the polypeptide sequence is the sequence of l(a) and the substituting amino acid sequence is X₁X₂X₃PX₄XsXe, wherein each OfX₁ - X₆ is independently any amino acid.

12. The isolated polypeptide of claim 11, wherein the substituting sequence is the amino acid sequence set forth in SEQ ID NO:2 or SEQ ID NO:3.

13. The isolated polypeptide of claim 1 comprising the sequence set forth in SEQ ID NO:4 or SEQ ID NO:5.

14. The isolated polypeptide of claim 1 comprising amino acids 25-267 of SEQ ID NO:4 or SEQ ID NO:5.

15. A polynucleotide encoding the isolated polypeptide of any one of claims 1-7, 9 and 11-14.

16. A pharmaceutical composition comprising the isolated polypeptide or multimer of any one of claims 1-14 and a pharmaceutically acceptable carrier.

17. The pharmaceutical composition of claim 16, further comprising lipid bound to the isolated polypeptide or multimer.

18. The pharmaceutical composition of claim 17, wherein the lipid is phosphatidyl choline, phosphatidyl serine, phosphatidyl ethanolamine, phosphatidal choline, phosphatidal ethanolamine, alkyl acyl glycerophosphoryl choline or alkyl acyl glycerophosphoryl ethanolamine, or any combination thereof.

19. The pharmaceutical composition of claim 18, further comprising cholesterol, cholesteryl ester or sphingomyelin, or any combination thereof.

20. An expression vector for apo-lipoprotein expression in a cell, comprising a promoter active in the cell and operably linked to a DNA segment encoding the isolated polypeptide of any one of claims 1-7, 9 and 11-14.

21. The expression vector of claim 20, wherein the cell is a liver cell, macrophage, Kupffer cell, lymphocyte, enterocyte, smooth muscle cell or endothelial cell.

22. The expression vector of claim 20, wherein the vector comprises a DNA segment encoding a signal peptide linked to the DNA segment encoding the isolated polypeptide, whereby the isolated polypeptide is secreted from the cell.

23. The expression vector of claim 22, wherein the signal peptide has the amino acid sequence set forth in amino acids 1-18 of SEQ ID NO:1.

24. A method of modulating HDL subclass in a subject in need of such modulation, comprising administering the isolated polypeptide or multimer of any one of claims 1-14 to the subject in an amount sufficient to modulate HDL.

25. The method of claim 24, wherein administration of the isolated polypeptide or multimer alters HDL subclass 2 or 3 distribution.

26. The method of claim 24, wherein administration of the isolated polypeptide or multimer alters HDL subclass 2 composition.

27. The method of claim 24, wherein administration of the isolated polypeptide or multimer alters the level of HDL subclass 3 composition.

28. The method of claim 24, wherein the isolated polypeptide or multimer is provided to the subject in a pharmaceutical composition.

29. The method of claim 28, wherein the polypeptide or multimer contains bound lipid.

30. The method of claim 24, wherein the isolated polypeptide or multimer is provided by an expression vector delivered to the subject, the expression vector comprising a promoter operably linked to a DNA segment encoding the isolated polypeptide.

31. The method of claim 30, wherein the expression vector comprises a viral vector.

32. The method of claim 31 , wherein the viral vector is a vaccinia virus, adenovirus, retrovirus, herpesvirus, cytomegalovirus, lentivirus or adeno-associated virus.

33. A method of treating a vascular disease in a subject in need of such treatment, comprising administering the isolated polypeptide or multimer of any one of claims 1 - 14 to the subject in an amount sufficient to increase the level of a beneficial HDL subclass.

34. The method of claim 33, wherein the vascular disease is atherosclerosis.

35. The method of claim 33, wherein the vascular disease is one of coronary heart disease, peripheral vascular disease, stroke, or mesenteric ischemia.

36. The method of claim 33, wherein the HDL subclass is subclass 2.

37. The method of claim 33, wherein the HDL subclass is subclass 3.

38. The method of claim 33, wherein the isolated polypeptide or multimer is provided to the subject in a pharmaceutical composition.

39. The method of claim 38, wherein the polypeptide or multimer contains bound lipid.

40. The method of claim 33, wherein the isolated polypeptide is provided by an expression vector delivered to the subject, the expression vector comprising a promoter operably linked to a DNA segment encoding the isolated polypeptide,

41. The method of claim 40, wherein the expression vector comprises a viral vector.

42. The method of claim 41 , wherein the viral vector is a vaccinia virus, adenovirus, retrovirus, herpesvirus, cytomegalovirus, lentivirus or adeno-associated virus.

43. The method of claim 24, further comprising identifying a subject in need of modulation before administration of the isolated polypeptide or multimer.

44. The method of claim 33, further comprising identifying a subject in need of treatment before administration of the isolated polypeptide or multimer.

45. The method of claim 24, further comprising determining the composition, level and/or distribution of an HDL subclass in the subject before and/or after modulation.

46. The method of claim 33, further comprising determining the composition, level and/or distribution of an HDL subclass in the subject before and/or after treatment.

47. The method of claim 33, further comprising administering an agent effective in the treatment of vascular disease.