Soluble truncated apoM proteins and medical uses thereof

Field of the invention

The present invention relates to soluble truncated apoM proteins and derivatives thereof, capable of raising the apoA1 and HDL concentration in a mammal upon administration into the blood circulation of said mammal, corresponding nucleic acids as well as vectors and host cells containing these. In addition, the present invention is directed to the use of said soluble truncated apoM proteins and derivatives thereof for manufacturing medicaments, in particular medicaments for raising the HDL concentration, for the prophylaxis and/or treatment of atherosclerosis, and also for increasing alveolar surfactants, in particular for the prophylaxis and/or treatment of Respiratory Distress Syndrome (RDS) and Acute Respiratory Distress Syndrome (ARDS).

Background of the invention

An important function of high-density lipoproteins (HDL) is reverse cholesterol transport (RCT), a process in which cholesterol from the extra-hepatic tissue is taken up by HDL and delivered to the liver for excretion into the bile. In addition, HDL has also been shown to have antioxidant, anti-inflammatory and anti-thrombotic properties. These properties contribute to HDL anti-atherogenicity and HDL concentrations are inversely correlated to cardiovascular disease. HDL is heterogeneous in density, size, lipid composition and apolipoprotein (apo) content, which might explain the diverse functions of the particle. Several methods are available to distinguish between different subclasses of HDL, i.e. ultracentrifugation for density, non-denaturing gel for size and native agarose gel for electrophoretic mobility. The small discoidal nascent HDL particles have a pre-β migrating property on an agarose gel while the larger spherical mature HDL migrates as β-particles.

The liver is the primary origin of plasma HDL and the liver specific deficiency of the ATP binding cassette A1 (AbcA1), that controls the rate-limiting step in HDL particle assembly, leads to an 80% reduction of plasma HDL. Nascent immature pre-β migrating particles can also be formed in extra hepatic tissue during the initial steps of RCT. In addition, there is data showing that pre-β migrating particles can be generated when

larger HDL particles are metabolized. Catabolism of HDL particles includes remodelling, selective cholesterol uptake and whole particle uptake by the liver or kidney. It has been demonstrated in vivo that the size of the particle determines the fate of HDL, mature HDL particles being catabolised mainly by the liver, whereas pre-β migrating particles are catabolised in the kidney.

In addition to the size and density of the HDL, the apolipoprotein content modulates the particle ^' s function and catabolism. Known apolipoproteins that are capable of associating with HDL are, for example, apoAI, apoAII, apoE, apoAIV, apoCs and apoM. The most abundant HDL apolipoprotein is apoAI and it is important in RCT, since lipid free apoAI has been shown both in vivo and in vitro to associate with lipids forming nascent pre-β migrating particles. In addition, apoAI plays a role in the maturation of the nascent particles since it is required for the normal function of lecithin cholesterol ester transferase (LCAT), an enzyme that converts free cholesterol to cholesterol esters. Therefore, apoAI also plays a central role in determining the lipid composition of the HDL particles.

Due to its protective effect on atherosclerosis much research has been carried out on HDL, apolipoprotein function and RCT, the main goal being to increase plasma HDL levels. However, it is increasingly recognized that the function of HDL, in addition to plasma HDL concentrations, determines the particle ^' s anti-atherogenic properties. For example, overexpressing either apoAI or apoAII increases HDL cholesterol. However, only the overexpression of apoAI is atheroprotective, while HDL from apoAII overexpressing transgenic mice is pro-inflammatory.

One apolipoprotein that recently had been shown to modulate HDL function in a beneficial manner is apoM, which was discovered in 1θ99 (Xu, N., and Dahlback, B. (1999). A novel human apolipoprotein (apoM). J. Biol. Chem. 274, 31286-31290) by cloning an unrecognized 26 kDa protein in triglyceride-rich lipoproteins. Even though it has been shown that apoM can be associated with other lipoprotein particles, it is primarily present on HDL. Hence, the plasma concentration of apoM highly correlates with HDL in healthy humans (Axler, O., Ahnstrom, J., and Dahlback, B. (2007). An ELISA for apolipoprotein M reveals a strong correlation to total cholesterol in human plasma. J. Lipid Res. 48, 1772-1780.). Mice lacking apoM expression due to genetic deletion of the transcription factor Hnf-iαTcrT ^7" or gene silencing of apoM by RNAi have an atypical lipoprotein profile with abnormally large HDL. In addition, these mice lack pre-β migrating

particles. Conversely, over-expression of apoM by recombinant adenovirus in mice increases the amount of pre-β migrating particles and total HDL levels in plasma (Wolfrum, C, Poy, M. N., and Stoffel, M. (2005). Apolipoprotein M is required for prebeta- HDL formation and cholesterol efflux to HDL and protects against atherosclerosis. Nat. Med. 11 , 418-422.). Furthermore, isolating apoM-containing HDL particles from human plasma by immunoaffinity chromatography leads to an additional peak in the lipoprotein profile that corresponds to the size of pre-β migrating particles (Christoffersen, C, Nielsen, L. B., Axler, O., Andersson, A., Johnsen, A.H., and Dahlback, B. (2006). Isolation and characterization of human apolipoprotein M-containing lipoproteins. J Lipid Res 47, 1833-1843.). These results strongly argue in favour of an important role of apoM in HDL metabolism. Further, assessing cholesterol efflux from lipid-laden macrophages, apoM- containing pre-β migrating particles are better acceptors for cholesterol compared to apoM-deficient pre-β migrating particles, indicating a role for apoM in RCT, which would imply that the protein is anti-atherogenic (Christoffersen et al., Wolfrum et al., see above). Indeed, over-expression of apoM by adenovirus in atherogenic-prone LDL receptor null mice (LdIr ^' ' ^' ) for only 3 weeks lead to a reduction in atherosclerosis, showing that the apoM protein has beneficial effects on atherogenesis (Wolfrum et al., 2005, see above).

The apoM gene is only expressed in the liver and kidney and encodes 188 amino acids. Due to a glycosylation site in humans the protein is 26 kDa compared to 22 kDa in mouse. Structural computer modelling of apoM suggests that the protein belongs to the lipocalin family and in vitro findings indicate that apoM has the capability of binding to retinols, all-trans retinoic acid and 9-cis retinoic acid with its lipid binding pocket. However, the in vivo ligand of apoM remains to be determined. ApoM retains its 20 amino acids long signal peptide, like the HDL associated protein paraoxonase-1. It has been speculated that the signal peptide, which contains an α-helix, is incorporated into the phosholipid layer of the lipoprotein particle. This could explain why apoM in the circulation can only be detected when attached to lipoprotein particles and not in a free form.

Atherosclerosis ("hardening of the artery) is associated with the deposition of cholesterol, cholesterol-esters, lipoproteins, collagen, calcification and sometimes even ossification of arteries leading to an increase in wall thickness, a corresponding decrease in the elasticity of the arterial wall and often partial or complete occlusion of the artery, all of which can eventually result in coronary thrombosis or infarction.

Respiratory Distress Syndrome (RDS) is a breathing disorder of premature newborns in which the air sacs (alveoli) in a newborn ^' s lung do not remain open because the production of surfactant is absent or insufficient. Normally, the lungs produce a mixture of lipids (fats) and proteins called surfactant, which acts as a wetting agent and lines the surface of the air sacs, where it lowers the surface tension and allows the air sac to remain open throughout the respiratory cycle. Usually, production of surfactant begins after about 34 weeks of pregnancy. The more premature the newborn, the greater the likelihood that RDS will develop after birth. Next to the avoidance of premature delivery RDS strategies include injecting the mother with a corticosteroid drug, typically betamethasone or dexamethason, that crosses the placenta and accelerates the production of surfactant, as well as oxygen supplementation, oxygen intubation of the newborn and the use of surfactant preparations to reduce complications such as pneumothorax.

The Acute Respiratory Distress Syndrome (ARDS) is defined by noncardiogenic pulmonary edema and respiratory failure in the seriously ill patient. The cardinal feature of ARDS, refractory hypoxemia, is caused by formation of protein-rich alveolar edema after damage to the integrity of the lung ^' s alveolar-capillary barrier. Alveolar-capillary damage in ARDS can be initiated by physical or chemical injury or by extensive activation of innate inflammatory responses. Such damage causes the lung ^' s edema safety factor to decrease by about half and edema develops at low capillary pressures. Widespread alveolar flooding in ARDS impairs alveolar ventilation, excludes oxygen and inactivates surfactant; this in turn decreases lung compliance, increases dispersion of ventilation and perfusion and produces intrapulmonary shunt.

Hence, the surfactant concentration in the alveoli plays a critical role not only in the normal breathing mechanism but is also an important factor involved in ARDS and RDS pathology, its absence or reduction (e.g. by inactivation in ARDS) contributing to alveolar dysfunction and edema formation.

It is the object of the present invention to provide new compounds useful for manufacturing medicaments, in particular medicaments for the therapeutic and/or prophylactic treatment of atherosclerosis and related diseases, in particular ischemic cardiovascular disease, stroke, etc., as well as ARDS and/or RDS.

When further elucidating the mechanism of natural apoM in HDL metabolism it was surprisingly found that soluble apoM derivatives are useful for preparing medicaments that increase HDL concentration as well as for preparing medicaments that increase the concentration of alveolar surfactant.

In a first aspect, the present invention relates to a soluble derivative of the apoM protein.

In particular, this aspect of the present invention relates to a polypeptide comprising an amino acid sequence having at least 50 or at least 60 %, preferably at least 70 %, more preferably at least 80 %, even more preferably at least 90 %, most preferably at least 95 % amino acid sequence identity to the polypeptide of SEQ ID NO: 1 or 7, wherein the polypeptide and/or a fragment thereof comprising said amino acid sequence i) is soluble under physiological conditions, ii) reduces the renal excretion of ApoAI in a mammal, preferably human, iii) raises the ApoAI and HDL concentration in a mammal, preferably human, upon intravenous administration of a pharmacologically effective amount thereof.

SEQ ID NO. 1 : QLTTLGVDGKEFPEVHLGQWYFIAGAAPTKEELATFDPVDNIVFNMAAGSAPMQLH LRATIRMKDGLCVPRKWIYHLTEGSTDLRTEGRPDMKTELFSSSCPGGIMLNETGQG YQRFLLYNRSPHPPEKCVEEFKSLTSCLDSKAFLLTPRNQEACELSNN

SEQ ID NO. 7:

IYQCPEHSQLTTLGVDGKEFPEVHLGQWYFIAGAAPTKEELATFDPVDNIVFNMAAG SAPMQLHLRATIRMKDGLCVPRKWIYHLTEGSTDLRTEGRPDMKTELFSSSCPGGIM LNETGQGYQRFLL YNRSPHPPEKCVEEFKSLTSCLDSKAFLLTPRNQEACELSNN

SEQ ID NO: 1 represents the amino acid sequence of human apoM lacking the N- terminal 27 amino acids. SEQ ID NO: 7 represents the amino acid sequence of human apoM lacking the N-terminal 19 amino acids. The term "amino acid sequence identity", as used herein, is meant to indicate the percentage of identical amino acids in said amino acid sequence, comprised by the polypeptide of the invention, relative to the amino acid sequence in SEQ ID NO: 1 or 7. For determining the identity among amino acid sequences, the skilled person can revert to a number of standard algorithms known to those of skill in the art.

Preferably, the BLAST programs at http://www.expasy.org/tools/blast/ and http://www.ncbi.nlm. nih.gov/BLAST/Blast.cgi?CMD=Web&LAYOUT=Two- Windows&AUTO_FORMAT=Semiauto&ALIGNMENTS=250&ALIG NMENT_VIEW=Pairwi se&CDD_SEARCH=on&CLIENT=web&DATABASE=nr&DESC RIPTIONS=500&ENTRE Z_QUERY=%28none%29&EXPECT=10&FILTER=L&FORMAT_OBJ ECT=Alignment&F ORMAT_TYPE=HTML&l_THRESH=0.005&MATRIX_NAME=BLOSUM62& amp;NCBI_GI=on &PAGE=Proteins&PROGRAM=blastp&SERVICE=plain& SET_DEFAULTS.x=41&SET_D EFAULTS.y=5&SHOW_OVERVIEW=on&END_OF_HTTPGET=Yes& SHOW_LINKOUT= yes&GET_SEQUENCE=yes, more preferably with the default settings, are used for identifying the amino acid sequence identity of said amino acid sequence comprised by a polypeptide or functional derivative of the present invention relative to SEQ ID NO 1 or 7.

The polypeptide of the present invention may consist of only 50 up to 100 % of the amino acids of SEQ ID NO: 1 or 7 as long as the required functional properties for practicing the present invention are given. The polypeptide of the present invention may also comprise more than 50 to 100 % of the amino acids of SEQ ID NO: 1 or 7, for example, additional signal peptides, purification tags (e.g. poly-his-tags) for ease of identification and purification, preferably polypeptide components that are enzymatically cleavable by naturally occurring enzymes, more preferably enzymes present in recombinant cells for producing polypeptides according to the invention, most preferably enzymes that are present in liver, kidney and/or blood.

The term "polypeptide according to the present invention" also encompasses functional derivatives of the polypeptide of the invention having the properties (functions) identified above. A functional derivative of the polypeptide of the present invention is meant to encompass any amino acid sequence and/or chemical derivative thereof (consisting of natural and/or non-natural amino acid equivalents, e.g. enzymatically or chemically derivatised at one or more functional group, e.g. by glycosylate, PEGylation (PEG= poly(ethylene glycol) or poly(ethyleneoxide) derivatisation, polyamide-PEG derivatisation, alkanoylation, carbamoylation, urea formation, including dimeric peptides (e.g. dimerisation via cycteine bonds), etc.), that has substantially sufficient accessible amino acid residues or non-natural equivalents to establish the requirements i) to iii) identified above.

In summary, the polypeptides according to the invention are defined by a structural requirement in combination with a functional limitation.

Requirement i), i.e. that the polypeptide and/or a fragment thereof, comprising said amino acid sequence having at least 50 to 100 % amino acid sequence identity to SEQ ID NO 1 or 7, is soluble under physiological conditions, means that said polypeptide and/or fragment does not precipitate substantially, preferably less than 10 %, more preferably less than 5 %, most preferably less than 1 %, within 1 h, preferably 12 h, more preferably 24 h, most preferably 48 h, at room temperature, i.e. approximately 20 ⁰ C, in physiological saline, preferably in blood serum.

Requirement ii), i.e. that the polypeptide and/or a fragment thereof, comprising said amino acid sequence having at least 50 to 100 % amino acid sequence identity to SEQ ID NO 1 or 7, reduces the renal excretion of ApoAI in a mammal, preferably human, means that that upon preferably intravenous administration of said polypeptide in a pharmacologically effective amount, renal excretion of ApoAI into the urine of a mammal, preferably selected from rodents, preferably rat or mouse, and humans, is reduced by at least 10 %, preferably at least 20 %, more preferably at least 50 %, most preferably at least 80 % and very most preferred at least 95 %.

Requirement iii), i.e. that the polypeptide and/or a fragment thereof, comprising said amino acid sequence having at least 50 to 100 % amino acid sequence identity to SEQ ID NO 1 or 7, raises the ApoAI and HDL concentration in a mammal, preferably human, upon intravenous administration of a pharmacologically effective amount thereof, means that the intravenous administration of a pharmacologically effective amount of the polypeptide must result in an increase in ApoAI and HDL, preferably plasma HDL, by at least 1 %, preferably at least 5 %, more preferably at least 10 %, most preferably at least 20 % relative to the injection of saline vehicle alone (statistical significance provided), preferably plasma ApoAI, by at least 10 %, preferably at least 20 %, more preferably at least 50 %, most preferably at least 80 %, and very most preferred at least 95 % relative to the injection of saline vehicle alone (statistical significance provided) . Most preferably said requirement is tested in mice, rats or humans, e.g. by intravenously injecting polypeptides according to the invention and measuring apoAI content in the urine and/or serum. As a result of injecting polypeptides according to the invention apoAI concentrations in the urine will decrease by at least 10 %, preferably at least 20 %, more preferably at least 40 %, most preferably at least 80 % relative to the injection of saline vehicle alone. At the same time, ApoAI levels in the serum will increase at least 10 %,

preferably at least 20 %, more preferably at least 50 %, most preferably at least 80 % relative to the injection of saline vehicle alone.

The present invention has been experimentally investigated using chimeric polypeptides comprising the amino acids of SEQ ID NO: 1 or 7 and the N-terminal 26 or 25 amino acids of albumin or apoAI, respectively (Alb/apoM and apoA1/apoM, respectively), both N-terminal sequences including a peptide cleavage site (which is missing in apoM). The amino acid sequences of these polypeptides are listed in SEQ ID NO 2 and 3 (Alb/apoM and apoA1/apoM, respectively).

SEQ ID NO. 2:

MKWVTFISLLFLFSSA YSRGVFRREA/roCP£HrøLTTLGVDGKEFPEVHLGO WYFIAGA APTKEELATFDPVDNIVFNMAAGSAPMQLHLRATIRMKDGLCVPRKWIYHLTEGSTDLR TEGRPDMKTELFSSSCPGGIMLNETGQGYQRFLL YNRSPHPPEKCVEEFKSLTSCLDSKAF LLTPRNQEACELSNN

SEQ ID NO: 3:

MKAAVLTLAVLFLTGSOARHFWOOD/rOCf^HSOLTTLGVDGKEFPEVHLGO WYFIAGA APTKEELATFDPVDNIVFNMAAGSAPMQLHLRATIRMKDGLCVPRKWIYHLTEGSTDLR TEGRPDMKTELFSSSCPGGIMLNETGQGYQRFLLYNRSPHPPEKCVEEFKSLTSCLDSKA F LLTPRNQEACELSNN

In a preferred embodiment, the present invention is directed to polypeptides according to the invention having an amino acid sequence set forth in SEQ ID NO: 2 and 3. In a more preferred embodiment the present invention is directed to polypeptides according to the invention having an amino acid sequence set forth in SEQ ID NO: 2 and 3, wherein the sequence IYQCPEHS is deleted (marked in italic letters in the above sequences).

Investigating the above embodiments of the present invention it was demonstrated that the polypeptides of the present invention have one or more, more preferably all of the following biological effects.

Polypeptides of the present invention located in the blood circulation of mammals being soluble themselves or comprising soluble fragments thereof will prevent loss of apoAI in the kidney. As a result, apoAI concentrations in the blood increase, and, apoAI being a major determinant of HDL concentration, this consequently leads to increased HDL formation.

In summary, pharmacologically effective amounts of soluble polypeptides of the present invention in the blood of mammals will raise HDL concentration by inhibiting renal clearance of apoAI without affecting HDL formation in the liver.

Therefore and as a preferred embodiment, polypeptides of the present invention will significantly raise HDL concentration, preferably by at least 1 %, more preferably by at least 5 %, even more preferably by at least 10 %, most preferably by at least 20 %, by significantly inhibiting, preferably by at least 10 %, more preferably at least 15 %, even more preferably by at least 20 %, most preferably by at least 50 %, the renal clearance of apoAI without substantially affecting HDL formation in the liver when introduced into the blood of a mammal, preferably human, in a pharmacologically effective amount. The term "significant" as used herein in the context of raising the HDL concentration and inhibiting renal clearance of apoAI is understood from a statistical standpoint, a significant change being statistically relevant when compared to a reference point, e.g. vehicle administration.

In another aspect the present invention relates to an isolated and purified nucleic acid, comprising a nucleic acid selected from the group consisting of:

(i) the nucleic acid of SEQ ID NO 4, 5, 6 or 8; (ii) a nucleic acid having a sequence of at least 60 % identity, preferably at least 70 % identity, more preferred at least 80 % identity, even more preferably 90 %, most preferred at least 95 % identity with the nucleic acid sequence of SEQ ID NO 4, 5, 6 or 8;

(iii) a nucleic acid that hybridizes to a nucleic acid of (i) or (ii); (iv) a nucleic acid, wherein said nucleic acid is derivable by substitution, addition and/or deletion of one of the nucleic acids of (i), (ii) oder (iii); (v) a fragment of any of the nucleic acids of (i) to (iv), that hybridizes to a nucleic acid of (i), coding for a polypeptide according to the invention. (The above hybridization is preferably performed under stringent conditions (see Maniatis & Sambrook)).

SEQ ID NO: 4 is the nucleic acid sequence coding for SEQ ID NO: 1

SEQ ID NO. 4: CAACTGACAACTCTGGGCGTGGATGGGAAGGAGTTCCCAGAGGTCCACTTGGGCCAGTG GTACTTTATCGCAGGGGCAGCTCCCACCAAGGAGGAGTTGGCAACTTTTGACCCTGTGGA CAACATTGTCTTCAATATGGCTGCTGGCTCTGCCCCGATGCAGCTCCACCTTCGTGCTAC C ATCCGCATGAAAGATGGGCTCTGTGTGCCCCGGAAATGGATCTACCACCTGACTGAAGGG

AGCACAGATCTCAGAACTGAAGGCCGCCCTGACATGAAGACTGAGCTCTTTTCCAGC TCA TGCCCAGGTGGAATCATGCTGAATGAGACAGGCCAGGGTTACCAGCGCTTTCTCCTCTAC AATCGCTCACCACATCCTCCCGAAAAGTGTGTGGAGGAATTCAAGTCCCTGACTTCCTGC CTGGACTCCAAAGCCTTCTTATTGACTCCTAGGAATCAAGAGGCCTGTGAGCTGTCCAAT AACTGA

SEQ ID NO: 8 is the nucleic acid sequence coding for SEQ ID NO: 7

SEQ ID NO. 8: ATCTACCAGTGCCCTGAGCACAGTCAACTGACAACTCTGGGCGTGGATGGGAAGGAGTTC CCAGAGGTCCACTTGGGCCAGTGGTACTTTATCGCAGGGGCAGCTCCCACCAAGGAGGA GTTGGCAACTTTTGACCCTGTGGACAACATTGTCTTCAATATGGCTGCTGGCTCTGCCCC G ATGCAGCTCCACCTTCGTGCTACCATCCGCATGAAAGATGGGCTCTGTGTGCCCCGGAAA TGGATCTACCACCTGACTGAAGGGAGCACAGATCTCAGAACTGAAGGCCGCCCTGACAT GAAGACTGAGCTCTTTTCCAGCTCATGCCCAGGTGGAATCATGCTGAATGAGACAGGCCA GGGTTACCAGCGCTTTCTCCTCTACAATCGCTCACCACATCCTCCCGAAAAGTGTGTGGA GGAATTCAAGTCCCTGACTTCCTGCCTGGACTCCAAAGCCTTCTTATTGACTCCTAGGAA T CAAGAGGCCTGTGAGCTGTCCAATAAC In a preferred embodiment the nucleic acid sequence of the present invention is SEQ ID NO: 5 or 6, which code for the amino acid sequences in SEQ ID NO: 2 and 3, respectively.

SEQ ID NO. 5: ATGAAGTGGGTAACCTTTCTCCTCCTCCTCTTCGTCTCCGGCTCTGCTTTTTCCAGGGGT GT GTTTCGCCGAGAAGCAATCTACCAGTGCCCTGAGCACAGTCAACTGACAACTCTGGGCGT GGATGGGAAGGAGTTCCCAGAGGTCCACTTGGGCCAGTGGTACTTTATCGCAGGGGCAG CTCCCACCAAGGAGGAGTTGGCAACTTTTGACCCTGTGGACAACATTGTCTTCAATATGG CTGCTGGCTCTGCCCCGATGCAGCTCCACCTTCGTGCTACCATCCGCATGAAAGATGGGC TCTGTGTGCCCCGGAAATGGATCTACCACCTGACTGAAGGGAGCACAGATCTCAGAACTG AAGGCCGCCCTGACATGAAGACTGAGCTCTTTTCCAGCTCATGCCCAGGTGGAATCATGC TGAATGAGACAGGCCAGGGTTACCAGCGCTTTCTCCTCTACAATCGCTCACCACATCCTC CCGAAAAGTGTGTGGAGGAATTCAAGTCCCTGACTTCCTGCCTGGACTCCAAAGCCTTCT TATTGACTCCTAGGAATCAAGAGGCCTGTGAGCTGTCCAATAAC

SEQ ID NO. 6:

ATGAAAGCTGCGGTGCTGACCTTGGCCGTGCTCTTCCTGACGGGGAGCCAGGCTCGG CAT

TTCTGGCAGCAAGATATCTACCAGTGCCCTGAGCACAGTCAACTGACAACTCTGGGC GTG GATGGGAAGGAGTTCCCAGAGGTCCACTTGGGCCAGTGGTACTTTATCGCAGGGGCAGCT CCCACCAAGGAGGAGTTGGCAACTTTTGACCCTGTGGACAACATTGTCTTCAATATGGCT GCTGGCTCTGCCCCGATGCAGCTCCACCTTCGTGCTACCATCCGCATGAAAGATGGGCTC TGTGTGCCCCGGAAATGGATCTACCACCTGACTGAAGGGAGCACAGATCTCAGAACTGA AGGCCGCCCTGACATGAAGACTGAGCTCTTTTCCAGCTCATGCCCAGGTGGAATCATGCT GAATGAGACAGGCCAGGGTTACCAGCGCTTTCTCCTCTACAATCGCTCACCACATCCTCC CGAAAAGTGTGTGGAGGAATTCAAGTCCCTGACTTCCTGCCTGGACTCCAAAGCCTTCTT ATTGACTCCTAGGAATCAAGAGGCCTGTGAGCTGTCCAATAAC

The term "% (percent) identity" as known to the skilled artisan and used herein indicates the degree of relatedness among 2 or more nucleic acid molecules that is determined by agreement among the sequences. The percentage of "identity" is the result of the

percentage of identical regions in 2 or more sequences while taking into consideration the gaps and other sequence peculiarities.

The identity of related nucleic acid molecules can be determined with the assistance of known methods. In general, special computer programs are employed that use algorithms adapted to accommodate the specific needs of this task. Preferred methods for determining identity begin with the generation of the largest degree of identity among the sequences to be compared. Computer programs for determining the identity among two sequences comprise, but are not limited to, the GCG-program package, including GAP (Devereux et al., Nucleic Acids Research 12 (12):387 (1984); Genetics Computer Group University of Wisconsin, Madison, (Wl)); BLASTP, BLASTN, and FASTA (Altschul et al., J. Molec. Biol 215:403/410 (1990)). The BLAST X program can be obtained from the National Center for Biotechnology Information (NCBI) and from other sources (BLAST handbook, Altschul et al., NCB NLM NIH Bethesda, MD 20894). Also, the well- known Smith-Waterman algorithm can be used for determining identity.

Preferred parameters for sequence comparison comprise the following:

The gap program is also suited to be used with the above-mentioned parameters. The above-mentioned parameters are standard parameters (default) for nucleic acid comparisons.

Further exemplary algorithms, gap opening penalties, gap extension penalties, comparison matrix, including those in the program handbook, Wisconsin-package, version 9, September 1997, can also be used. The selection depends on the comparison to be done and further, whether a comparison among sequence pairs, for which GAP or Best Fit is preferred, or whether a comparison among a sequence and a large sequence databank, for which FASTA or BLAST is preferred, is desired.

The nucleic acid molecules according to the invention may be prepared synthetically by methods well-known to the skilled person, but also may be isolated from suitable DNA

libraries and other publicly-available sources of nucleic acids and subsequently may optionally be mutated. The preparation of such libraries or mutations is well-known to the person skilled in the art.

In a preferred embodiment, the nucleic acid molecules of the invention are cDNA, genomic DNA, synthetic DNA, RNA or PNA, either double-stranded or single-stranded (i.e. either a sense or an antisense strand). Fragments of these molecules, which are encompassed within the scope of the invention, may be produced by, for example, the polymerase chain reaction (PCR) or generated synthetically using DNA synthesis or by reverse transcription using mRNA from liver or kidney.

In some instances the present invention also provides novel polynucleotides encoding the polypeptides of the present invention characterized in that they have the ability to hybridize to a specifically referenced nucleic acid sequence, preferably under stringent conditions. Next to common and/or standard protocols in the prior art for determining the ability to hybridize to a specifically referenced nucleic acid sequence under stringent conditions, it is preferred to analyse and determine the ability to hybridize to a specifically referenced nucleic acid sequence under stringent conditions by comparing the nucleotide sequences of the two proteins, which may be found in gene databases (e.g. http://www.ncbi. nlm.nih.gov/entrez/query.fcgi?db=nucleotide) with alignment tools (e.g., http://www.ncbi.nlm. nih.gov/blast/Blast.cgi?CMD=Web&LAYOUT=TwoWindows&A UTO_FORMAT=Semiauto&PAGE=Nucleotides&NCBI_GI=yes& ;FILTER=L&HITLIST_SI ZE=IOOaSHOW-OVERVIEW=VeSSIAUTO-FORIvIAT=VeSaSHOW-LINKOUT=VeS ).

The nucleic acid of the present invention is preferably operably linked to a promoter that governs expression in suitable vectors and/or host cells producing the polypeptides of the present invention in vitro or in vivo.

In a preferred embodiment the nucleic acid of the present invention is one that is operably linked to a promoter selected from the group consisting of the MCK promoter, the RSV promoter, the CMV promoter, a tetracycline-regulatable promoter, a doxycycline-regulatable promoter, and a promoter capable of being recognized by RNA- dependent RNA polymerase.

Preferably, the isolated and purified nucleic acid is in the form of a recombinant vector, such as a viral vector. The selection of a suitable vector and expression control

sequences as well as vector construction are within the ordinary skill in the art. Preferably, the viral vector is selected from the group consisting of an adenovirus, an adeno-associated viral vector, a retroviral vector, a Herpes simplex viral vector, a lentiviral vector, a Sindbis viral vector, or a Semliki forest viral vector. Preferably, the isolated and purified nucleic acid encoding and expressing the protein or polypeptide is operably linked to a promoter selected from the group consisting of the MCK promoter, the CMV promoter, a tetracycline-regulatable promoter, and a doxycycline-regulatable promoter.

Suitable vectors are reviewed in Kay et al., Nature Medicine 7: 33-40 (2001); Somia et al., Nature Reviews 1 : 91-99 (2000); and van Deutekom et al., Neuromuscular Disorders 8: 135-148 (1998). Preferably, the viral vector is an adenovirus (preferred examples are described in Acsadi et al., Hum. Gene Then 7(2): 129-140 (1996); Quantin et al., PNAS USA 89(7): 2581-2584 (1992); and Ragot et al., Nature 361 (6413): 647-650 (1993)), an adeno-associated viral vector (preferred examples are described in Rabinowitz et al., Curr. Opin. Biotechnol. 9(5): 470-475 (1998)), a retroviral vector (preferred examples are described in Federico, Curr. Opin. Biotechnol. 10(5): 448-453 (1999)), a Herpes simplex viral vector (see, e.g., Latchman, Gene 264(1): 1-9 (2001)), a lentiviral vector, a Sindbis viral vector, or a Semliki forest viral vector. Suitable promoters for operable linkage to the isolated and purified nucleic acid are known in the art. Preferably, the isolated and purified nucleic acid encoding the protein is operably linked to a promoter selected from the group consisting of the muscle creatine kinase (MCK) promoter (Jaynes et al., MoI. Cell Biol. 6: 2855-2864 (1986)), the cytomegalovirus (CMV) promoter, a tetracycline- regulatable promoter (Gossen et al., PNAS USA 89: 5547-5551 (1992)), and a doxycycline-regulatable promoter (Gossen et al. (1992), supra). Vector construction, including the operable linkage of a coding sequence with a promoter and other expression control sequences, is within the ordinary skill in the art.

Hence and in a further aspect, the present invention relates to a recombinant vector, comprising a nucleic acid according to the invention, preferably said recombinant vector being capable of producing a polypeptide according to the invention.

Preferably, the recombinant vector according to the invention is one, wherein the vector is a viral vector selected from the group consisting of an adenoviral vector, an adeno- associated viral vector, a retroviral vector, a Herpes simplex viral vector, a lentiviral vector, a Sindbis viral vector, and a Semliki forest viral vector.

A further aspect of the present invention is directed to a host cell comprising a nucleic acid according to the invention and/or a vector according to the invention, preferably being capable of producing polypeptides according to the present invention.

Furthermore, the present invention encompasses an antibody that specifically binds a polypeptide according to the invention but not to natural mammalian apoM. The antibodies may be polyclonal or monoclonal antibodies. As used herein, the term "antibody" refers not only to whole antibody molecules, but also to antigen-binding fragments, e.g., Fab, F(ab') ₂ , Fv, and single chain Fv fragments. Also included are chimeric antibodies, preferably humanized antibodies. Such antibodies are useful as research tools for distinguishing between natural apoM and polypeptides according to the invention. A further aspect relates to a hybridoma cell line, expressing a monoclonal antibody according to the invention.

First experiments in vitro and in vivo in mammals have demonstrated the efficacy of the polypeptides, nucleic acids and vectors to inhibit renal clearance of apoA1 without affecting HDL formation in the liver, thus, increasing HDL formation and decreasing the risk of atherosclerotic lesions. These results demonstrate the potential utility of these compounds to be formulated into therapeutically and/or prophylactically effective pharmaceutical formulations.

In a preferred embodiment the present invention provides for a pharmaceutical composition compromising a polypeptide, a nucleic acid and/or a recombinant vector according to the invention as well as a pharmaceutically acceptable carrier (or excipient).

Suitable carriers or excipients are well-known in the art. A carrier or excipient may be a solid, semi-solid or liquid material which may serve as a vehicle or medium for the active ingredient. One of ordinary skill in the art in the field of preparing compositions can readily select the proper form and mode of administration depending upon the particular characteristics of the product selected, the disease or condition to be treated, the stage of the disease or condition, and other relevant circumstances (Remington ^' s Pharmaceutical Sciences, Mack Publishing Co. (1990)). The proportion and nature of the pharmaceutically acceptable carrier or excipient are determined by the solubility and chemical properties of the pharmaceutically active compound selected, the chosen route of administration, and standard pharmaceutical practice. The pharmaceutical preparation

may be adapted for oral, parenteral intravenous, subcutaneous, intramuscular or inhalative administration and may be administered to the patient in the form of tablets, capsules, suppositories, solution, suspensions or the like. The pharmaceutically active compounds of the present invention, while effective themselves, can be formulated and administered in the form of their pharmaceutically acceptable salts, such as acid addition salts or base addition salts, for purposes of stability, convenience of crystallization, increased solubility, and the like.

The polypeptide or polypeptides can be formulated and administered in the form of their pharmaceutically acceptable salts, such as acid addition salts or base addition salts, for purposes of stability, solubility and activity. Nucleotides and/or vectors in form or adeno- adeno associated, Sindbis or Semlili Forest virus vectors may be administered intravenously.

For preparing pharmaceutical compositions of the invention, it is preferred that the polypeptide, nucleic acid and/or vector is formulated in such a manner that they provide for a pharmaceutically effective amount of the soluble and active polypeptide and/or a soluble and active fragment (after enzymatic and/or hydrolytic cleavage) of the polypeptide of the invention in the blood circulation, and in particular in the kidney, of the treated mammal, preferably human, to prevent loss of apoA1 in the kidney without affecting pre-β-HDL formation in the liver.

Another aspect of the present invention is directed to the use of at least one of the following, a polypeptide, a nucleic acid and/or a recombinant vector according to the present invention, for preparing a medicament.

With respect to the vectors of the present invention and to ensure effective transfer of the vectors of the present invention, it is preferred that about 1 to about 5,000 copies of the vector according to the invention be employed per cell to be contacted, based on an approximate number of cells to be contacted in view of the given route of administration, and it is even more preferred that at least about 3 to about 300 pfu enter each cell. However, this is merely a general guideline, which by no means precludes use of a higher or lower amount, as might be warranted in a particular application, either in vitro or in vivo. The actual dose and schedule can vary depending on whether the composition is administered in combination with other compositions, e.g. pharmaceutical compositions, or depending on interindividual differences in pharmacokinetics, drug

disposition, and metabolism. Similarly, amounts can vary in in vitro applications depending on the particular type of cell or the means by which the vector is transferred. One skilled in the art easily can make any necessary adjustments in accordance with the necessities of the particular situation.

The compounds of the present invention have demonstrated their utility for raising the HDL concentration. Hence and in a preferred embodiment, the present invention relates to the use of a polypeptide, a nucleic acid and/or a recombinant vector according to the invention for the preparation of a medicament for raising the HDL concentration.

High HDL blood contents have been shown to be of importance for preventing and/or treating atheroscleosis. Hence and in a preferred embodiment, the present invention relates to the use of a polypeptide, a nucleic acid and/or a recombinant vector according to the invention for the preparation of a medicament for the prophylaxis and/or therapy of atherosclerosis.

Next to HDL formation ApoA1 is also directly involved in the transport of phospholipids into alveoli for surfactant action. Hence and in a preferred embodiment, the present invention relates to the use of a polypeptide, a nucleic acid and/or a recombinant vector according to the invention for the preparation of a medicament for increasing the content of alveolar surfactants.

An increase of alveolar surfactants is an important measure for preventing and/or treating Respiratory Distress Syndrome (RDS) in newborns and also for preventing and/or treating Acute Respiratory Distress Syndrome by preventing and/or treating edema formation. Hence and in a preferred embodiment, the present invention relates to the use of a polypeptide, a nucleic acid and/or a recombinant vector according to the invention for the preparation of a medicament for the prophylaxis and/or therapy of Respiratory Distress Syndrome.

Furthermore, another aspect of the present invention concerns a method of treatment, wherein a pharmacologically effective amount of the above pharmaceutical composition is administered to a patient in need thereof, preferably a patient suffering from atheroscleorsis, ARDS or RDS.

In effecting treatment of a subject suffering from said diseases, at least one compound of the present invention can be administered in any form or mode which makes the therapeutic polypeptide or therapeutic fragment thereof bioavailable in an effective amount, including oral or parenteral routes. For example, compositions of the present invention can be administered subcutaneously, intramuscularly, intravenously, by inhalation and the like. One skilled in the art in the field of preparing formulations can readily select the proper form and mode of administration depending upon the particular characteristics of the product selected, the disease or condition to be treated, the stage of the disease or condition and other relevant circumstances (see. e.g. Remington ^' s Pharmaceutical Sciences, Mack Publishing Co. (1990)).

Another aspect of the present invention is directed to a method of raising the HDL concentration in a mammal, which method comprises administering to the mammal a therapeutically effective amount of a polypeptide, a nucleic acid and/or a recombinant vector according to the invention.

In a preferred embodiment, the present invention relates to a method of treating and/or preventing atherosclerosis in a mammal, which method comprises administering to the mammal a therapeutically effective amount of a polypeptide, a nucleic acid and/or a recombinant vector according to the invention.

In a further embodiment, the present invention relates to a method of treating and/or preventing Respiratory Distress Syndrome and/or Acute Respiratory Distress Syndrome in a mammal, which method comprises administering to the mammal a therapeutically effective amount of a polypeptide, a nucleic acid and/or a recombinant vector according to the invention.

In said methods, the vector is preferably a recombinant viral vector selected from the group consisting of an adenovirus, an adeno-associated viral vector, or a lentiviral vector.

In said methods, the polypeptide, nucleic acid and/or recombinant vector is/are preferably administered by intravenous or local application, preferably into the kidney.

Moreover, the present invention encompasses a) methods of producing a polypeptide, wherein a host cell according to the invention is cultured and said polypeptide is purified; b) methods of producing a nucleic acid according to the invention, wherein a host cell

according to the invention is cultured and said nucleic acid is purified; c) methods of producing a vector according to the invention, wherein a host cell according to the invention is cultured and said vector is purified; d) methods of producing an antibody according to the invention, wherein a hybridoma cell according to the invention is cultured and said antibody is purified.

Figures

Fig. 1 Characterization of flag-tagged apoM

Fig. 1A) is a schematic drawing of the surface probability plot of apoM calculated according to a formula of Emini et al. (J. Virol., 55:836-839 (1985). The 8 amino acid flag epitope was inserted at indicated amino acids. The total length of apoM with flag epitope is 196 amino acids.

Fig. 1 B) shows the over-expression of flag constructs, apoM and GFP in HepG2 cells. Cell lysates and medium were analyzed by SDS-PAGE and immunoblotted using anti-apoM antibodies and anti-flag antibodies.

Fig. 1C) One-dimensional native agarose gel electrophoresis and immunoblotting of medium from HepG2 cells transfected with apoM117, apoM or GFP expression vectors. The blotted 1 D native agarose gel was probed with either anti-apoM antibodies or anti-flag antibodies.

Fig. 1 D) FPLC analysis of plasma from TcfT' ^~ mice infected with either Ad-apoM117 or Ad-GFP. Cholesterol was measured by a colorimetric assay and presented as absorbance at 505 nm.

Fig. 2 Cleavage of the signal peptide of apoM inhibits pre-β migrating particle formation and affects full-length apoM in a dominant negative fashion

Fig. 2 A) HepG2 cells were transfected with expression vectors for apoM, hybrids Al/M, alb/M and GFP and cell extracts were analyzed by PAGE and immunoblotting. Media were analyzed by SDS-PAGE and immunoblotted with anti-apoM antibodies.

Fig. 2 B) 1 D native agarose gel of medium from HepG2 cells transfected with apoM, hybrid Al/M, hybrid alb/M or GFP. The blotted agarose gel was probed with anti- apoAI antibodies. Fig. 2 C) Effects of truncated apoM on expression of wildtype apoM in co-transfected HEK293 cells. Cells were transfected with 10ug GFP or co-transfected with

constant amount (4 μg) of apoM117 flag and increasing amount (1-6μg) of AI/M117 HA. Cell lysates and medium were subjected to SDS-PAGE and immunoblotted using either anti-HA antibodies or anti-flag antibodies. For cell lysate loading control, anti-TATA binding protein (Tbp) antibodies were used. Endogenous secreted apoM was detected by using anti-apoM antibodies.

Fig. 3. Dominant negative activity mediated by heterodimerization between wild-type and truncated apoM

Fig. 3. A) HepG2 cell lysates, transfected with apoM or alb/M expression vectors, were incubated with either PBS, or cross-linking reagents dimethyl 3, 3'- dithiobispropionimate (DTBP) and bismaleimidohexane (BMH). SDS-PAGE under non-reducing conditions and immunoblotting using anti-apoM antibodies showed a band twice the molecular weight of apoM. Under reducing conditions only the monomer was detected in PBS and DTBP-treated cell lysate. In contrast, the dimer persisted in BMH-treated samples as BMH cross-linking could not be reversed.

Fig. 3. B) HEK293 cells were co-transfected with the following combinations of expression vectors: apoM117 HA and AI/M117 flag, apoM117 flag and AI/M117 HA, ApoM117 flag and ApoM117 HA or AI/M117 flag and AI/M117 HA. The cell lysates were immunoprecipitated with anti-M2 flag agarose. The immunoprecipitates were subjected to SDS-PAGE and immunoblotted using anti-

HA antibodies.

Fig. 3. C) Truncated apoM affects formation of pre-b migrating particles in vivo. C57BL/6 mice injected with 1 x 10 ⁹ pfu adenovirus expressing GFP, apoM, AI/M117 or

Al/M. 7 days post-infection, plasma containing 0.1 μM EDTA and 1.5 mM DTNB were analyzed on a 1-D native agarose gel and immunoblotted using anti-apoAI antibodies.

Fig. 3. D) Liver homogenates from C57BL/6 mice injected with 1 x 10 ⁹ pfu adenovirus expressing GFP, apoM117 or AI/M117, were incubated with either PBS, or cross- linking reagent bismaleimidohexane (BMH). SDS-PAGE under non-reducing conditions and immunoblottning using anti-apoM antibodies showed a band twice the molecular weight of apoM. Under reducing conditions only the monomer was detected in PBS liver homogenates. In contrast, the dimer persisted in BMH- treated samples as BMH cross-linking could not be reversed.

Fig. 4. Effect of hepatic expression of truncated apoM on lipoprotein metabolism

Fig. 4A) FPLC analysis of plasma from Ad-GFP, Ad-apoM, Ad-AI/M and Ad-AI/M infected mice. Cholesterol was measured by colorimetric assay and presented as absorbance at 505 - 650 nm.

Fig. 4B) Expression of plasma apolipoprotein in Ad-GFP, Ad-apoM, Ad-AI/M and Ad- Al/M infected mice. Plasma was analyzed 7 days postinjection by SDS-PAGE and immunoblotting.

Fig. 5 Truncated apoM affects apoAI metabolism Fig. 5A) shows apoAI plasma clearance. I ¹²⁵ -labeled apoAI was injected into mice treated with Ad-GFP, Ad-apoM, Ad-AI/M and Ad-AI/M and clearance was measured over 24 h (n=4 per group).

Fig. 5D) shows ¹²⁵ l-apoAI levels in urine during a 24 hrs collection (n=4 per group).

Fig. 5C) shows ¹²⁵ l-apoAI accumulation in different liver, intestine, kidney and heart 24 hrs after injection of mice that were treated with Ad-GFP, Ad-ApoM, Ad-AI/M and

Ad-AI/M infected mice adenovirus (n=4 per group). Fig. 5D) shows ApoAI expression in liver and urine. Mice were injected with Ad-GFP, Ad- apoM, Ad-AI/M and Ad-AI/M and specimens were taken after 6 days. Urine samples were collected during a 24 h interval. Liver cell lysates (20μg protein) and urine (15 μL) were analyzed by SDS-PAGE and immunoblotting. Fig. 6 HDL and apoAI metabolism in TTR-al/M transgenic mice Fig. 6A) is a Western blot analysis of urine from TTR-AI/M transgenic mice and wildtype littermates using specific anti-apoAI antibodies. Fig. 6B) is an FPLC analysis of plasma from TTR-AI/M transgenic mice and wildtype littermates. Cholesterol was measured by colorimetric assay and presented as absorbance at 505 - 650 nm. Fig. 6B) shows an immunoblot of plasma from transgenic mice and wildtype littermates using anti-apoAI and apoM antibodies. Fig. 6C) shows a 1-D native agarose gel electrophoresis and immunoblotting of plasma from TTR-AI/M transgenic mice and wildtype littermates using anti-apoA1 antibodies. Fig. 6D) is a Western blot analysis of urine from TTR-AI/M transgenic mice and wildtype littermates using specific anti-apoAI antibodies.

Fig. 7 Truncated apoM exacerbates the formation of atherosclerotic lesions Figs. 7A-C relate to C57BI/6 mice fed 0.02% cholesterol diet for 11 weeks, followed by injection of Ad-GFP, Ad-apoM and Ad-AI/M. Plasma cholesterol and lesion areas were studied 3 weeks post-infection.

Figs. 7D-E relate to LdIr-/- mice fed 0.02% cholesterol diet for 16 weeks, followed by injection of Ad-GFP ₁ Ad-apoM and Ad-AI/M. Plasma cholesterol and lesion areas were studied 3 weeks post-infection.

Figs. 7A & D) show plasma cholesterol levels (n=8 per group). Figs. 7B & E) show the area oil red o staining in the attic root (n=8 per group). Figs. 7C & F) are representative pictures of oil red o stainings of aortic roots.

7C No lesions can be recognized in Ad-GFP and Ad-apoM treated mice, whereas

Ad-AI/M treated mice show small lesions.

7F Fewer lesions in Ad-apoM and Ad-apoAI treated mice compared to AdGFP. Markedly enhanced lesion formation in Ad-AI/M treated mice.

In the following the subject-matter of the invention will be described in more detail referring to specific embodiments which are not intended to be construed as limiting to the scope of the invention.

Examples

Experimental procedures

Animals

All animal models were maintained on a 12 hours light/dark cycle in a pathogen-free animal facility. All mutant animals used in this study were crossed to a C57BI/6 background. Animals were fed a high fat diet (Harland Teklad) containing 50% fat for 12 weeks. The modified AIN76A semisynthetic diet (0.02% cholesterol) was purchased from Harlan Teklad (Blackthorn, Bicester, UK).

Generation of transgenic TTR-Alb./apoM mice

Adenoviruses

The cDNAs of Flag-tagged apoM and hybrid apoM were cloned into plasmid AdδCMV K- NpA (Viraquest) and recombinant adenoviruses were generated using standard procedures. For in vivo experiments, mice were injected through the tail vein with 1x10 ⁹ pfu of adenovirus. Empty virus expressing only GFP served as control (Ad-GFP).

Expression vectors

The apoM flag-constructs were generated by inserting Flag sequences into the respective positions in the apoM cDNA using PCR. The hybrids Al/M and Alb/M were generated by PCR by replacing the sequence of amino acids 1-27 of the murine apoM cDNA with the N-terminal 18 amino acid residue sequence of apoAI and the N-terminal 24 amino acid sequence of albumin, to generate hybrids Al/M and alb/M, respectively. All sequences were confirmed by dideoxynucleotide sequencing.

Cell culture and transient transfection

HEK293 and HepG2 cells were cultured with DMEM medium containing 25 mM glucose 10% FCS. Transfections were performed using Fugene reagent (Roche Diagnostics, Switzerland) according to the manufacture's instructions.

Cross linking of protein in cells

HepG2 cells were split into 10 cm Petri dishes and grown to 80% confluency before infection with 1 x 10 ⁷ pfu adenovirus. Two days after the cells were harvest and respunded in reaction buffer. Cross linking experiments were performed as previously described (Akpinar et al, Cell Metabolism 2: 385-397, 2005). Cross linking reagents and buffers: bismaleimidohexane (BMH) (Sigma Aldrich) was dissolved in DMSO and incubated in PBS, and dimethyl-3,3'-dithiobispropionimidate (DTBP) (Sigma Aldrich) was dissolved in water and incubated in 0.2M tris-ethanolamine (pH 8.0).

lmmunoprecipitation of Flag epitope

Transfected HEK293 cells were washed twice with PBS and the cells were lysed in RIPA buffer in the presence of protease inhibitors for 20 min at 4 ⁰ C. Cell lysates were centrifuged for 5 min at 10,000 x g at 4 ⁰ C and the supernatant were incubated with M2 flag agarose (Sigma Aldrich) at 4°C for 16 h. The samples were then washed 3 times with ice cold PBS and eluted with sample buffer, according to the manufacture's protocol. The eluate was subject for SDS-PAGE.

lmmunoblotting

Plasma or whole cell extracts were separated by SDS-PAGE and transferred onto a nitrocellulose membrane (Schleicher & Schuell, Germany) by electroblotting. ApoM was detected with anti-apoM antiserum (ref) (1 :1000), anti-flag antibodies (Sigma Aldrich) or anti-HA antibodies (Convance, USA). Other apolipoproteins were detected using affinity

purified antibodies (BioDesign, USA) (1 :1000). Primary antibodies were incubated at 4 ⁰ C over night.

Plasma lipids and lipoprotein analysis Blood samples were treated with 0.1 μM EDTA and 1.5mM DTNB (5,5-dithiobis-2-nitro- benzoic acid) to inhibit LCAT activity. Cholesterol and triglyceride levels were determined using colorimetric assay (Roche, Switzerland). Lipoprotein separation (described previously ref), by fast performance liquid chromatography (FPLC), was performed by loading 0.2 μl plasma onto two serial connected Superose-6 FPLC columns (HR10/30) and run at 0.4 ml/min.

Detection of lipoproteins by native gel electrophoresis

Lipoproteins were separated by native agarose gel electrophoresis (0.8% agarose in 10 mM Tris, pH 8.6). The agarose gel was blotted by capillary transfer onto a nitrocellulose membrane in ddH ₂ 0 and apolipoproteins were detected using specific antibodies.

Laboratory measurements

Blood samples were taken from mice using heparinized capillary tubes. An aliquot of each sample was treated with 1.5 mM DTNB (5,5-Dithiobis-2-nitro-benzoic acid) to inhibit LCAT activity. Plasma cholesterol, triglycerides and phospholipids levels were determined using a colorimetric assay system (Roche, Wako, USA).

Radiolabeling of apoA1

Human apoA1 was labeled according to McFarlan et al. using freshly prepared [ ¹²⁵ I]CI solution. Unbound free iodine or iodine labelled tracer was separated from HDL by PD- 10 columns (Pharmacia). The integrity of apoAI was checked by FPLC separation. Mice were injected with 100 nCi of radiolabeled HDL. Blood was taken at different times after injection and counted using a gamma-counter.

Quantification of atherosclerosis C57BI/6 mice were fed a diet containing 0.02% cholesterol for 8 weeks, after this period the animals were injected with either Ad-GFP, Ad-ApoM or Ad-AI/M. Ldlr' ^" mice were fed the same diet for 16 weeks and then were injected with either Ad-GFP, Ad-ApoAI, Ad-

ApoM or Ad-AI/M. Mice were sacrificed 3 weeks post injection and atherosclerosis was quantified as reported previously (Wolfrum et al., Nature Medicine 11418-422, 2005).

Statistical Analysis Results are given as mean ± SD. Statistical analyses were performed by using a Student's Mest, and the null hypothesis was rejected at the 0.05 level.

Results

Characterization of flag-tagged apoM To generate recombinant apoM that can be recognized by antibodies in the native form Flag epitopes were inserted in regions of the apoM protein where the surface probability is the highest (amino acid 57, 117, 152 and 180) (Fig. 1A). All constructs were expressed in HepG2 cells and expression was assessed by immunoblotting. ApoM 152 could not be detected by the apoM antibody used (raised against a peptide containing the amino acids 140 to 159 of apoM) (Richter et al, Diabetes 52:2989-2995, 2003), since the insertion of the flag is interrupting the apoM antibody epitope, but could be visualized using anti-flag antibodies. Interestingly, only apoM117 was secreted from cells, showing that insertion of the Flag epitope at positions 57, 152 and 180 affects apoM secretion (Fig. 1 B). Previous studies have shown that apoM is involved in the formation of pre-β migrating particles (Wolfrum et al, Nature Medicine 11418-422, 2005). To test whether apoM117 was functional, formation of pre-β migrating particles in HepG2 cells was examined. Similar to wild type apoM, apoM117 induced formation of pre-β migrating particles that could be detected by anti-Flag antibodies (Fig. 1C). Lastly, it was assessed whether apoM117 was functional in vivo by injecting apoM117 adenovirus into TcfT' ^~ mice. These mice have an abnormal lipoprotein profile that can be rescued by overexpressing wildtype apoM (Wolfrum et al, Nature Medicine 11418-422, 2005). ApoM117 was indeed able to restore the lipoprotein profile of Tcfr^ (Fig. 1 D). Together, these results demonstrate that an insertion of a Flag epitope at amino acid 117 in apoM is compatible with normal apoM function.

Cleavage of the signal peptide of apoM inhibits pre-β migrating particle formation and affects full length apoM in a dominant negative fashion

To elucidate the biological role of the retained signal peptide in apoM, apoM hybrids were generated, in which amino acid residues 1-19 of apoM were replaced either by the N-terminal 25 amino acid residues of apoAI (to generate hybrid Al/M), or amino acids 1- 26 of albumin (to generate hybrid alb/M), thereby fusing the signal peptides of apoAI and

albumin, including their signal cleavage recognition sequences, with apoM lacking its hydrophobic N-terminus. These constructs were predicted to express a truncated form of apoM that is unable to associate with HDL. Vectors expressing the hybrids (Al/M, alb/M), full-length (wildtype) apoM or GFP (control) were transfected in HepG2 cells and apoM secretion into the media was measured. As shown in figure 2A both hybrid constructs were able to express mutant apoM proteins in cells and that could also be secreted into the medium (Fig. 2A). Therefore, the ability of the mutant apoM hybrids to assemble and secret pre-β HDL migrating particles from transfected HepG2 cells was examined. Figure 2B shows that apoM hybrids, in contrast to wildtype apoM, were unable to induce formation of pre-β migrating particles. This result demonstrates that the signal peptide of apoM is important for the formation of pre-β migrating particles in vitro.

Next, it was investigated whether the truncated form of apoM had an effect on the expression of full-length apoM. Constant amounts of Flag-tagged wildtype apoM (p- apoM117Flag) were cotransfected with increasing amounts of HA-tagged alb/M (p- AI/M117HA) in HepG2 and the expression of hybrid and wildtype apoMs was analyzed in cell lysates and medium by immunoblotting (Fig. 2C). Increasing expression of truncated apoM (AI/M117HA) led to a dose-dependent decrease of full-length (apoM117Flag) in cell lysates and medium. In addition to inhibiting full-length apoM117Flag, AI/M117HA also inhibited the secretion of human endogenous apoM into the media (Fig. 2C). Together, these data indicated that truncated apoM acts as a dominant negative regulator of apoM expression/secretion.

Homo-and heterodimerization of apoM and truncated apoM To further elucidate the apparent dominant-negative effect of truncated apoM on wildtype apoM, it was hypothesized that the truncated form interacts with the wildtype form and thereby prevents its secretion. To test this, HepG2 cells were transfected with full length and truncated apoM expression vectors and cells were treated with the cross linkers DTBP or BMH. DTBP and BMH stabilize existing dimers, however, DTBP is cleavable under reducing condition. Under non-reducing conditions several bands could be detected in PBS, DTBP and BMH treated cells for both the full-length apoM and truncated apoM (Fig. 3A). When treating the samples with β-mercaptoethanol, only the monomeric form of apoM could be detected in the PBS and DTBP treated cells. On the contrary, BMH treated cells under reducing condition revealed marked bands. These data indicate that both full-length and truncated (Figure 3A) apoM can form dimers in vitro.

The most intense immunoreactive band in the cross linking experiments corresponded to a protein of about 50 kDa, leading to speculation that apoM might form homodimers and that truncated apoM can form heterodimers with the full length apoM. To test this hypothesis HEK293 cells were co-transfected with either apoM11-HA and AI/M117-flag, apoM117-flag and AI/M117-HA, apoM117-flag and apoM117-HA or AI/M117-flag and AI/M117-HA. lmmunoprecipitations were performed from cell lysates using anti-flag antibodies, followed by a SDS-PAGE and immuno-blotting with anti-HA antibodies. Figure 3B shows that full length (lane 3) and truncated (lane 4) apoM can interact to form homodimers. Interestingly, the truncated apoM also interacted with the full-length apoM to form heterodimers (lane 1 and 2), a finding that offers a mechanistic explanation for the dominant-negative activity of truncated apoM.

Truncated apoM inhibits formation of pre-β migrating particles in vivo To test whether the in vitro effects of the truncated apoM also occurred in vivo, C57BI/6 mice were injected with either recombinant adenoviruses expressing GFP, apoM, AI/M117 or Al/M (Ad-GFP, Ad-apoM, Ad-AI/M117 or Ad-AI/M, respectively) and plasma and livers were analyzed 6 days post injection. As demonstrated previously, over- expression of apoM led to an increase in plasma levels of pre-β migrating HDL. In contrast, over-expression of the hybrids profoundly reduced the occurrence of pre-β migrating particles. Furthermore, injection of Ad-ApoM, Ad-AI/M117 and Ad-AI/M decreased β-HDL levels compared to Ad-GFP injected mice (Fig. 3C). When analyzing liver homogenates from mice infected with Ad-apoM117 or Ad-AI/M117 the apoM dimers and heterodimers were detected (Fig. 3D). These results demonstrate that the truncated form of apoM acts as a dominant negative mutant and inhibits pre-β migrating particle formation in vivo.

Over-expression of truncated apoM in vivo has minor effects on plasma lipoprotein metabolism Pre-β migrating particles are precursors for mature HDL in the circulation. It was therefore of interest to examine whether the truncated apoM had any effect on plasma lipoprotein metabolism. Mice were injected with recombinant adenoviruses Ad-GFP, Ad- ApoM, Ad-AI/M117 and Ad-AI/M and total plasma cholesterol and triglycerides were measured after 7 days. No significant changes in the plasma levels were detected in Ad- GFP ₁ Ad-AI/M117 and Ad-AI/M injected animals, however, there was an about 2-fold

increase in plasma cholesterol and an about 3-fold reduction of plasma triglycerides in mice that received Ad-ApoM (Table 1).

Table 1. Total plasma cholesterol and triglycerides in C57BI/6 mice, measured 7 days after injection of either 1x109 pfu AD-GFP or Ad-AI/M117 (*P<0,02)

Analyzing the plasma lipoprotein profile by FPLC from infected mice revealed that Ad- AI/M117 and Ad-AI/M did not influence the lipoprotein profile when pared to Ad-GFP. Overexpression of apoM in vivo increased the HDL peak, as shown previously (Fig. 4A). Because the expression of truncated apoM did not disturb the size and quantity of lipoprotein particles, it was examined whether the apolipoprotein levels in plasma were changed. The apoM levels were increased in Ad-ApoM compared to Ad-GFP and, consistent with the in vitro data, apoM was lower in plasma from mice injected with either Ad-AI/M117 or Ad-AI/M, confirming that truncated apoM inhibits endogenous apoM secretion. Plasma apoAI was slightly increased in mice expressing apoM, AI/M117 and Al/M. ApoE was also affected, where Ad-ApoM and Ad-AI/M increased the expression compared to Ad-GFP. However, injection of Ad-AI/M117 did not seem to alter apoE levels. Furthermore, injection of Ad-ApoM, Ad-AI/M117 and Ad-AI/M increased apoC levels in plasma, with the strongest effect in mice over-expressing apoM. The levels of apoB48, a very low density and low density lipoprotein associated protein were also determined. All three apoM adenoviruses increased apoB48 in plasma, indicating that apoM affects the other lipoprotein classes. Finally, the flag epitope of Ad-AI/M117 was detected in plasma, demonstrating that the truncated apoM is secreted from the liver into the circulation (Fig. 4B). The altered apolipoprotein content in plasma most likely reflects changed apolipoprotein content on the particle, which might affect the function of the lipoproteins and or compensate for decreased apoM expression, even if the total plasma cholesterol and triglyceride levels are not altered.

Truncated apoM affects apoAI metabolism

ApoAI is the primary apolipoprotein attached to HDL (approximately 70% of all apolipoprotein) and it is thought to play an important role for pre-β migrating particle formation, either by acquired lipids from cells or during lipoprotein remodeling (Assmann G et al. Annu. Rev. Med. 54, 321-341 , 2003). Since truncated apoM decreases apoAI containing pre-β migrating particles, studies were performed to elucidate whether other components of apoAI metabolism were affected. Mice treated with Ad-GFP, Ad-ApoM, Ad-AI/M117 or Ad-AI/M were injected after 6 days with radio-labeled ¹²⁵ I-ApOAI. Blood was collected over a 24 h period to measure the clearance of ¹²⁵ I-ApOAI from the circulation. As seen in figure 5A, ¹²⁵ l-apoAI rapidly cleared from the blood in all four groups. Interestingly, the clearance of apoAI after 30 min was much greater in Ad-GFP and Ad-ApoM mice compared to Ad-AI/M117 and Ad-AI/M treated animals (56.6 ± 0.82% and 50.1 ± 2.09% vs. 29.2 ± 2.55% and 42.2 ± 1.11%, respectively). The difference in clearance was maintained throughout the experiment and at 24 hours, mice over- expressing AI/M117 or Al/M, had 2-fold more radiolabeled apoAI in plasma when compared to Ad-GFP and Ad-ApoM treated mice (Fig. 5C). It was then investigated whether there was an altered catabolism of apoAI in the kidney, since it is well known that lipid-free apoAI is rapidly catabolized through the kidney (Assmann G et al. Annu. Rev. Med. 54, 321-341 , 2003). Therefore, an increase in ApoAI plasma clearance should also be reflected in increased excretion of ¹²⁵ I-ApOAI in the urine. Indeed, the urine of Ad-GFP and Ad-ApoM injected mice exhibited a «2-fold increase in ¹²⁵ l-apoAI compared to Ad-AI/M117 and Ad-AI/M mice during a 24 h urine collection (Fig. 5B). Consistent with this finding, it was found that truncated apoM also affected the uptake of apoAI in the kidney. Measurements of ¹²⁵ lodine in the liver, kidney, intestine and heart 24 h postinjection revealed no differences in accumulation of apoAI in liver, intestine and heart between the four groups. However, an about 2-fold greater accumulation of apoAI in the in kidneys of Ad-ApoM, Ad-AI/M117 and Ad-AI/M treated mice was measured compared to Ad-GFP injected mice (Fig. 5C).

To corroborate the above kinetic findings, it was assessed whether the free apoM also affected catabolism of endogenous apoAI. Mice were injected with Ad-GFP, Ad-ApoM,

Ad-AI/M117 and Ad-AI/M and ApoAI secretion was measured in the urine by western blotting. As early as 4 days after recombinant adenovirus injection, apoAI could not be detected in the urine of mice over-expressing AI/M117 and Al/M (Fig. 5D), whereas mice infected with Ad-GFP or Ad-apoM secreted apoAI in the urine. To assess whether truncated apoM also affected apoAI expression in the liver, homogenates from the adenovirus-injected mice were analyzed on SDS-PAGE and by immunoblotting. The

expression of apoAI varied within the groups, however, no marked effect on hepatic expression of ApoAI could be detected (Fig. 5E).

Liver-specific transgenic mice overexpressing Ad-AI/M117 To study the metabolic effects of long-term overexpression of truncated apoM transgenic mice expressing AI/M117 under the control of the liver-specific transthyretin (TTR) promoter were generated. Two independent lines (TTR-AI/M117 #1 and #2) were generated. Western blot analysis of liver cell lysates revealed that AI/M117 was expressed in both lines (data not shown). Plasma apolipoprotein levels were analyzed by immunoblotting and lipoprotein composition by fast protein liquid chromatography (FPLC) fractioning. Plasma apoM levels were drastically reduced in TTR-AI/M117 mice compared to control littermates, while plasma apoAI levels were «2-fold increased (Fig. 6A). The expression of other lipoproteins including apoA2, apoCIII and apoE did not change significantly (data not shown). FPLC analysis of plasma from TTR-AI/M117 mice led to a decreased HDL peak compared to controls (Fig. 6B). Native gel electrophoresis analysis of plasma from TTR-AI/M117 and wildtype littermates revealed decreased preβ- migrating HDL levels and increased β-HDL concentrations, respectively (Fig. 6C). lmmunoblot analysis of apoAI in the urine revealed a complete absence of apoAI in TTR-AI/M117 transgenic mice, whereas apoAI was readily detectable in the urine of wildtype littermates (Fig. 6D). Together, these results show that expression of free apoM not only affects the concentration of pre-β migrating particles but also have profound effects on apoAI catabolism, by retaining ApoAI in the circulation and increasing apoAI accumulation in the kidney. This subsequently leads to reduced elimination of apoAI through urinary excretion.

Truncated apoM exacerbated atherosclerotic lesion formation

Finally, it was investigated, whether truncated apoM is pro-atherogenic, since it decreases plasma pre-β migrating particles, which are know to be anti-atherogenic due to the involvement in RCT. Mice (C57BI/6) were fed an atherogenic diet, containing 0.02% cholesterol, for eight weeks followed by injection of either Ad-GFP, Ad-ApoM or Ad-AI/M. Three weeks post-injection plasma cholesterol levels were increased in mice expressing apoM compared to GFP (242 ± 13.2 mg/dL vs. 177 ± 13.9 mg/dL), consistent with previous studies (Wolfrum et al., Nature Medicine 11418-422, 2005). Interestingly, over-expression of the truncated form of apoM led to a decrease in plasma cholesterol (133 ± 9.8 mg/dL) (Fig. 7A). Quantitative measurements of atherosclerotic lesion size in the aortic root showed no visible lesions in mice that received either Ad-GFP or Ad-

ApoM. Strikingly, all mice over-expressing Al/M had small fatty streak (299 ± 226 μm ² ), demonstrating that hepatic expression of truncated apoM is pro-atherogenic (Fig. 7B ₁ C).

It was then elucidated whether truncated apoM is pro-atherogenic in a mouse model that carries its main cholesterol in the LDL fraction. LdIr ^1' mice were fed a semisynthetic 0.02% cholesterol diet for 16 weeks followed by injection of Ad-GFP, Ad-ApoAI, Ad- ApoM and Ad-AI/M. The plasma cholesterol levels three week post-injection showed a similar trend as the C57BI/6 atherosclerotic study, an increase of cholesterol in mice expressing apoM or apoAI compared to GFP (717 ±15.1 mg/dL and 800 ± 26.1 mg/dL vs. 614 ± 56.0 mg/dL, respectively) and a decrease in mice expressing Al/M (505 ± 36.6 mg/dL) (Fig. 7D). Figure 7E shows the lesion area for the four different groups. Similar to the C57BI/6 atherosclerotic study, over-expression of apoAI or apoM in the liver led to a reduction of atherosclerosis compared to GFP (116,379 ± 26,476 μm ² , 77,884 ± 14,274 μm ² vs. 179,684 ± 32,871 μm ² , respectively). In contrast, over-expression of truncated apoM in Lό\f' ^~ mice increased the formation of atherosclerotic lesions (287,260 ± 37,019 μm ² ). This demonstrates that hepatic expression of apoM lacking its signal peptide is also pro-atherogenic in atherosclerotic models in which the majority of cholesterol is carried in the LDL lipoprotein fraction.