WO2024236336A1 - Lipc variant in the treatment of hypercholesterolemia and atherosclerotic cardiovascular disease - Google Patents

Abstract

Inventors have performed a pioneer study (Figure 5) by overexpressing control eGFP, human WT and E97G LIPC in full Ldlr"/" females fed with a pro-atherogenic diet for 9 weeks, using AAV8 viruses as described before. Their results revealed that the E97G LIPC expression 1) strongly reduces plasma cholesterol levels in Ldlr/" females; 2) potently decreases the size of aortic atherosclerotic lesions. These preliminary data support the hypothesis of a beneficial role of LIPC E97G towards the development of atherosclerosis and reinforce the need for further studies. Interestingly, they also open the prospect of new therapeutic solutions in familial hypercholesterolemia (EH) patients with partial/total EDER deficiency. The present invention relates to a method for treating hypercholesterolemia, including homozygous and heterozygous EH, and related ASCVD in a subject in need thereof comprising administering a therapeutically effective amount of a LIPC variant to said subject in need thereof.

Description

LIPC VARIANT IN THE TREATMENT OF HYPERCHOLESTEROLEMIA AND ATHEROSCLEROTIC CARDIOVASCULAR DISEASE

FIELD OF THE INVENTION:

The invention is in the field of atherosclerotic cardiovascular disease (ASCVD) and hypercholesterolemia.

BACKGROUND OF THE INVENTION:

The development of atherosclerotic cardiovascular disease (ASCVD), the main cause of mortality worldwide, is strongly influenced by circulating lipoprotein levels.^1,2 Elevated low- density lipoprotein (LDL) cholesterol (LDL-C) is a well-established causal risk factor for ASCVD, whereas the role of high-density lipoprotein cholesterol (HDL-C) in ASCVD development remains to be further characterized.^1,2 Plasma LDL-C concentrations are strongly affected by genetics, with heritability explaining ~40% to 60% of plasma levels.³ Genetically modulated LDL-C concentrations typically have a polygenic origin but can also be caused by a codominant disease attributable to biallelic or monoallelic pathogenic variants (with for instance familial hypercholesterolemia - FH - which is the most frequent monogenic disorder worldwide with a prevalence of 1/310).^4,5 So far, however, only a few genes with a strong causal relationship to plasma LDL-C levels have been identified. Even fewer monogenic causes of low LDL-C, familial hypobetalipoproteinemia (FHBL), have been identified. These monogenic disorders were recently classified into 2 classes based on molecular mechanisms: FHBL attributable to lipoprotein secretion defects (FHBL-SD1, -SD2, and -SD3 for MTTP, APOB, and S AR IB-related FHBL) or to enhanced catabolism of apolipoprotein B (apoB)-containing lipoprotein (FHBL-EC1 and -EC2 for ANGPTL3- and PCSK9-related FHBL).⁶ Furthermore, pathogenic variants in the ANGPTL3 gene have been identified in cases of familial combined hypolipidemia, a condition characterized by very low concentrations of circulating very-low- density lipoprotein (VLDL), LDL, and HDL.⁷ Despite the suspected polygenic origins of unexplained cases of extremely elevated or reduced LDL-C concentrations, polygenic risk scores have so far yielded lower effect sizes compared with the effect of monogenic variants.^8,9 This suggests that additional monogenic variants that cause very low plasma LDL-C concentrations or combined hypolipidemia remain to be characterized.

Knowledge of the molecular mechanisms and genes implicated in extreme lipoprotein phenotypes is of direct clinical relevance. Accordingly, many of the recent therapeutics developed to lower plasma LDL-C levels and that provided breakthroughs in ASCVD risk reduction involved the products of genes implicated in FHBL and familial combined hypolipidemia such as PCSK9 and ANGPTL3.¹ Despite the effectiveness of these new therapies in lowering LDL-C, a need to identify additional mechanisms to effectively lower plasma LDL-C remains.

SUMMARY OF THE INVENTION:

The invention relates to a method for treating hypercholesterolemia and atherosclerotic cardiovascular disease (ASCVD) in a subject in need thereof comprising administering a therapeutically effective amount of a LIPC variant to said subject in need thereof In particular, the invention is defined by claims.

DETAILED DESCRIPTION OF THE INVENTION:

Using next-generation sequencing, inventors have identified a novel dominant rare variant in the LIPC gene, encoding for hepatic lipase, which cosegregates with the phenotype. They characterized the impact of this LIPC-E97G variant on circulating lipid and lipoprotein levels in family members using nuclear magnetic resonance-based lipoprotein profiling and lipidomics. To uncover the mechanisms underlying the combined hypocholesterolemia, they used protein homology modeling, measured triglyceride lipase and phospholipase activities in cell culture, and studied the phenotype of APOE*3. Leiden. CETP mice after LIPC-E97G overexpression.

Inventors have performed a pioneer study (Figure 5) by overexpressing control eGFP, human WT and E97G LIPC in full Ldlr ' males and females fed with a pro-atherogenic diet for 14 weeks, using AAV8 viruses as described before [1], Their results revealed that the E97G LIPC expression 1) strongly reduces plasma cholesterol levels in Ldlr ^ males and females; 2) potently decreases the extent of aortic atherosclerotic lesions. These preliminary data support the hypothesis of a beneficial role of LIPC E97G towards the development of atherosclerosis and reinforce the need for further studies. Interestingly, they also open the prospect of new therapeutic solutions in FH patients with partial/total LDLR deficiency.

Accordingly, in a first aspect, the invention relates to a method for treating hypercholesterolemia and atherosclerotic cardiovascular disease (ASCVD) in a subject in need thereof comprising administering a therapeutically effective amount of a LIPC variant to said subject in need thereof.

As used herein, the term "treatment" or "treat" refer to both prophylactic or preventive treatment as well as curative or disease modifying treatment, including treatment of patient at risk of contracting the disease or suspected to have contracted the disease as well as patients who are ill or have been diagnosed as suffering from a disease or medical condition, and includes suppression of clinical relapse. The treatment may be administered to a subject having a medical disorder or who ultimately may acquire the disorder, in order to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of a disorder or recurring disorder, or in order to prolong the survival of a subject beyond that expected in the absence of such treatment. By "therapeutic regimen" is meant the pattern of treatment of an illness, e.g., the pattern of dosing used during therapy. A therapeutic regimen may include an induction regimen and a maintenance regimen. The phrase "induction regimen" or "induction period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the initial treatment of a disease. The general goal of an induction regimen is to provide a high level of drug to a patient during the initial period of a treatment regimen. An induction regimen may employ (in part or in whole) a "loading regimen", which may include administering a greater dose of the drug than a physician would employ during a maintenance regimen, administering a drug more frequently than a physician would administer the drug during a maintenance regimen, or both. The phrase "maintenance regimen" or "maintenance period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the maintenance of a patient during treatment of an illness, e.g., to keep the patient in remission for long periods of time (months or years). A maintenance regimen may employ continuous therapy (e.g., administering a drug at a regular interval, e.g., weekly, monthly, yearly, etc.) or intermittent therapy (e.g., interrupted treatment, intermittent treatment, treatment at relapse, or treatment upon achievement of a particular predetermined criteria [e.g., disease manifestation, etc.]).

As used herein, the term "subject", "individual," or "patient" is used interchangeably and refers to any subject for whom diagnosis, treatment, or therapy is desired, particularly humans. Other subjects may include cattle, dogs, cats, guinea pigs, rabbits, rats, mice, horses, and the like. In some embodiments the subject is a human.

As used herein, the term “atherosclerotic cardiovascular disease” (ASCVD) refers to cardiovascular diseases (i.e. coronary artery disease including myocardial infarction ; ischemic stroke and peripheral artery disease) linked to atherosclerosis development.

In a particular embodiment, the atherosclerotic cardiovascular disease is selected from the group consisting of but not limited to: dyslipidemia and atherosclerotic cardiovascular disease (ASCVD), low density lipoprotein (LDL)-driven ASCVD, triglyceride-driven ASCVD, remnants-driven ASCVD lipoprotein a Lp(a)-driven ASCVD, chronic inflammatory disease- driven ASCVD, inflammatory ASCVD, hypercholesterolemia, familial hypercholesterolemia including homozygous familial hypercholesterolemia, chronic kidney disease, diabetes mellitus, blood pressure or human immunodeficiency virus infection

As used herein, the term “hypercholesterolemia” refers to an increase in low-density lipoprotein (LDL) cholesterol (LDL-C) levels which constitutes a major risk for the development of atherosclerosis and ASCVD.

In a particular embodiment, the hypercholesterolemia is Familial hypercholesterolemia (FH).

As used herein, the term “Familial hypercholesterolemia” (FH) refers to an inherited disorder of lipid metabolism that predisposes a person to premature severe cardiovascular disease (CVD) (Kolansky et al., (2008), Am J Cardiology, 102(11): 1438- 1443). FH can be either an autosomal dominant or an autosomal recessive disease that results from mutations in the low density lipoprotein receptor (LDLR), or in at least 3 different genes that code for proteins involved in hepatic clearance of LDL-C can cause FH. Examples of such defects include mutations in the gene coding for the LDL receptor (LDLR) that removes LDL-C from the circulation, and in the gene for apolipoprotein (Apo) B, which is the major protein of the LDL particle. In all cases, FH is characterized by an accumulation of LDL-C in the plasma from birth and subsequent development of tendon xanthomas, xanthelasmas, atherosclerosis, and ACCVD. FH can be classified as either heterozygous FH (heFH) or homozygous FH (hoFH) depending on whether the individual has a genetic defect in one (heterozygous) or both (homozygous) copies of the implicated gene.

In a particular embodiment, the hypercholesterolemia is homozygous Familial hypercholesterolemia (HoFH).

In a particular embodiment, the hypercholesterolemia is heterozygous Familial hypercholesterolemia (HeFH).

As used herein, the term “LIPC” also called as Hepatic lipase (HL), or hepatic triglyceride lipase (HTGL) refers to lipase C, hepatic type. LIPC is a form of lipase, catalyzing the hydrolysis of triacylglyceride. Hepatic lipase is coded by chromosome 15. hepatic lipase is to convert intermediate-density lipoprotein (IDL) to low-density lipoprotein (LDL). LIPC plays an important role in triglyceride level regulation in the blood by maintaining steady levels of IDL, HDL and LDL.

The naturally occurring human LIPC gene has a nucleotide sequence as shown in Genbank Accession number NM 000236 and the naturally occurring human LIPC protein has an aminoacid sequence as shown in Genbank Accession number NP 000227. The murine nucleotide and amino acid sequences have also been described (Genbank Accession numbers NM_008280, NM_001324472, NM_001324473 and NP 001311401, NP 001311402, NP-032306).

The sequence of said human protein can be found under the Uniprot accession number Pl 1150. The amino acid sequence is represented by SEQ ID NO: 1 :

MDTSPLCFSI LLVLCIFIQSSALGQSLKPE PFGRRAQAVE TNKTLHEMKT RFLLFGETNQGCQIRINHPD TLQECGFNSS LPLVMIIHGW SVDGVLENWI WQMVAALKSQ PAQPVNVGLV DWITLAHDHY TIAVRNTRLV GKEVAALLRW LEESVQLSRS HVHLIGYSLG AHVSGFAGSSIGGTHKIGRI TGLDAAGPLF EGSAPSNRLS PDDANFVDAI HTFTREHMGL SVGIKQPIGH YDFYPNGGSF QPGCHFLELY RHIAQHGFNA ITQTIKCSHE RSVHLFIDSL LHAGTQSMAYPCGDMNSFSQ GLCLSCKKGR CNTLGYHVRQ EPRSKSKRLF LVTRAQSPFK VYHYQFKIQFINQTETPIQT TFTMSLLGTK EKMQKIPITL GKGIASNKTY SFLITLDVDI GELIMIKFKW ENSAVWANVW DTVQTIIPWS TGPRHSGLVL KTIRVKAGET QQRMTFCSEN TDDLLLRPTQEKIFVKCEIK SKTSKRKIR.

The sequence of said human nucleotide sequence can be found under the Uniprot accession number NM 000236. The human nucleotide sequence is represented by SEQ ID NO: 2:

1 agaaattacc aagaaagcct ggaccccggg tgaaacggag aaatggacac aagtcccctg

61 tgtttctcca ttctgttggt tttatgcatc tttatccaat caagtgccct tggacaaagc

121 ctgaaaccag agccatttgg aagaagagct caagctgttg aaacaaacaa aacgctgcat

181 gagatgaaga ccagattcct gctctttgga gaaaccaatc agggctgtca gattcgaatc

241 aatcatccgg acacgttaca ggagtgcggc ttcaactcct ccctgcctct ggtgatgata

301 atccacgggt ggtcggtgga cggcgtgcta gaaaactgga tctggcagat ggtggccgcg

361 ctgaagtctc agccggccca gccagtgaac gtggggctgg tggactggat caccctggcc

421 cacgaccact acaccatcgc cgtccgcaac acccgccttg tgggcaagga ggtcgcggct

481 cttctccggt ggctggagga atctgtgcaa ctctctcgaa gccatgttca cctaattggg

541 tacagcctgg gtgcacacgt gtcaggattt gccggcagtt ccatcggtgg aacgcacaag

601 attgggagaa tcacagggct ggatgccgcg ggacctttgt ttgagggaag tgcccccagc

661 aatcgtcttt ctccagatga tgccaatttt gtggatgcca ttcatacctt tacccgggag

721 cacatgggcc tgagcgtggg catcaaacag cccataggac actatgactt ctatcccaac

781 gggggctcct tccagcctgg ctgccacttc ctagagctct acagacatat tgcccagcac

841 ggcttcaatg ccatcaccca gaccataaaa tgctcccacg agcgatcggt gcaccttttc 901 atcgactcct tgctgcacgc cggcacgcag agcatggcct acccgtgtgg tgacatgaac

961 agcttcagcc agggcctgtg cctgagctgc aagaagggcc gctgcaacac gctgggctac

1021 cacgtccgcc aggagccgcg gagcaagagc aagaggctct tcctcgtaac gcgagcccag

1081 tcccccttca aagtttatca ttaccagttc aagatccagt tcatcaacca aactgagaca

1141 ccaatacaaa caacttttac catgtcacta ctcggaacaa aagagaaaat gcagaaaatt

1201 cccatcactc tgggcaaagg aattgctagt aataaaacgt attcctttct tatcacgctg

1261 gatgtggata tcggcgagct gatcatgatc aagttcaagt gggaaaacag tgcagtgtgg

1321 gccaatgtct gggacacggt ccagaccatc atcccatgga gcacagggcc gcgccactca

1381 ggcctcgttc tgaagacgat cagagtcaaa gcaggagaaa cccagcaaag aatgacattt

1441 tgttcagaaa acacagatga cctactactt cgcccaaccc aggaaaaaat cttcgtgaaa

1501 tgtgaaataa agtctaaaac atcaaagcga aagatcagat gagatttaat gaagacccag

1561 tgtaaagaat aaatgaatct tactccttat ctggaatggc tgccttattt agaagccaaa

1621 attacataaa gaatctcaca caaagcttaa ataaagttta gatttaaggg gggtatgttt

1681 cactctcata aactgagctt ttaaaaacga tatgatataa ctattttcca gtatcagtac

1741 aaccaaaaat gtcccaattt tatctctggt taaatgttaa tatatgcaaa taagtacaaa

1801 tgcaagagcc attttagatc tgttgttcta atttacaatg tcttttataa ttaagtttcc

1861 cacatgtcct tgctagttta ttatgaccca tttttgaacc atgctagaaa gatacttttt

1921 tattaggtaa ctagtgcttc aataaagcaa gattctaatt aatcaccact agttgtgtat

1981 accaaataca catcctttaa gacaatacag ttcttaaaag aagaaaaatt ataattattc

2041 taatatttat acaaatcata taacgatttc acagaaccaa acacttacaa gtagaattct

2101 actaaaacta cttgtgataa cagtgaaagc tagacatgtg gtcattttaa taatggaatt

2161 acaaattaaa taaacataat tattatatct tactaatgct tgcaacaaaa gctaatttta

2221 aaaacttgga atgtctcaaa gcatttgcta tggactgaat gtttgagtca ccccaaaatt

2281 catatgctga aatctaatcc ccaatgtgat ggtgtttgga ggtggggcct ttgggaggtg

2341 atcaggtcat gagggtggag ccctcataag tgggattagt gcccttatag gggataaagg

2401 atcagaactc tctgccatgt gcggatatga gaaggcaact atatgcaaac caggaagttg

2461 accctcacct ggaagtagac taccagatct gctggcacct ttatcttgga cttctcagct

2521 tccagaactg tgagaaataa atgtttcttg tttaagcca

As used herein, the term "LIPC variant" is, with respect to nucleic acids, polypeptide or protein, to be understood as a polynucleotide which differs in comparison to the nucleic acid from which it is derived by one or more changes in the nucleotide sequence. The nucleic acid from which variant is derived is also known as the parent nucleic acid. Typically, a variant is constructed artificially, preferably by gene-technological means. Typically, the parent nucleic acid is a wild-type nucleic acid or part thereof. The variants usable in the present invention may also be derived from homologs, orthologs, or paralogs of the parent nucleic acid. The changes in the nucleotide sequence may be exchanges, insertions, deletions, 5' truncations, or 3' truncations, or any combination of these changes, which may occur at one or several sites. In particular embodiment, a variant usable in the present invention exhibits a total number of up to 600 (up to 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 1 00, 1 1 0, 120, 130, 140, 150, 160, 1 70, 180, 190, 200, 300, 400, 500 or 600) changes in the nucleotide sequence. The nucleotide exchanges may be led to non-conservative and/or preferably conservative amino acid exchanges as set out below with respect to polypeptide variants. Alternatively, or additionally, a "variant" as used herein can be characterized by a certain degree of sequence identity to the parent nucleic acid from which it is derived. More precisely, a nucleic acid variant in the context of the present invention exhibits at least 80% sequence identity to its parent nucleic acid.

Particularly, the sequence identity of nucleic acid variants is over a continuous stretch of 20, 30, 40, 45, 50, 60, 70, 80, 90, 1 00, 150, 200, 250, 300, 400, 500, 600 or more amino acids, more preferably over the entire length of the reference nucleic acid (the parent nucleic acid). The term "at least 80% sequence identity" is used throughout the specification also with regard to nucleic acid sequence comparisons. This term preferably refers to a sequence identity of at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% to the respective reference nucleic acid or to the respective reference nucleic acid. Particularly, the nucleic acid in question and the reference nucleic acid or exhibit the indicated sequence identity over a continuous stretch as specified above.

The terms "polypeptide" and "protein" are used interchangeably and refer to a polymeric form of amino acids of any length, which can include coded and non-coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having modified peptide backbones. The term includes fusion proteins, including, but not limited to, fusion proteins with a heterologous amino acid sequence, fusions with heterologous and homologous signal sequences, with or without N-terminal methionine residues; immunologically tagged proteins; and the like.

In the context of the invention, the LIPC variant is LIPC-E97G.

Typically, such LIPC variant allows to strongly reduce plasma cholesterol levels in Ldlr ^/_ males and females; and potently decrease the size of aortic atherosclerotic lesions. Accordingly, in a particular embodiment, the LIPC variant is a variant that promotes the activity of LIPC.

In some embodiments, the variant that promotes the activity of LIPC is a polypeptide having at least having at least 70% of identity with the sequence of SEQ ID NO: 1 or a fragment thereof. According to the invention a first amino acid sequence having at least 70% of identity with a second amino acid sequence means that the first sequence has 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90; 91; 92; 93; 94; 95; 96; 97; 98; 99 or 100% of identity with the second amino acid sequence. Sequence identity is frequently measured in terms of percentage identity (or similarity or homology); the higher the percentage, the more similar are the two sequences. Methods of alignment of sequences for comparison are well known in the art. Various programs and alignment algorithms are described in: Smith and Waterman, Adv. Appl. Math., 2:482, 1981; Needleman and Wunsch, J. Mol. Biol, 48:443, 1970; Pearson and Lipman, Proc. Natl. Acad. Sci. U.S.A., 85:2444, 1988; Higgins and Sharp, Gene, 73:237-244, 1988; Higgins and Sharp, CABIOS, 5: 151-153, 1989; Corpet et al. Nuc. Acids Res., 16: 10881-10890, 1988; Huang et al, Comp. Appls Biosci., 8: 155-165, 1992; and Pearson et al, Meth. Mol. Biol, 24:307-31, 1994). Altschul et al, Nat. Genet., 6: 119-129, 1994, presents a detailed consideration of sequence alignment methods and homology calculations. By way of example, the alignment tools ALIGN (Myers and Miller, CABIOS 4: 11-17, 1989) or LFASTA (Pearson and Lipman, 1988) may be used to perform sequence comparisons (Internet Program® 1996, W. R. Pearson and the University of Virginia, fasta20u63 version 2.0u63, release date December 1996). ALIGN compares entire sequences against one another, while LFASTA compares regions of local similarity. These alignment tools and their respective tutorials are available on the Internet at the NCSA Website, for instance. Alternatively, for comparisons of amino acid sequences of greater than about 30 amino acids, the Blast 2 sequences function can be employed using the default BEOSUM62 matrix set to default parameters, (gap existence cost of 11, and a per residue gap cost of 1). When aligning short peptides (fewer than around 30 amino acids), the alignment should be performed using the Blast 2 sequences function, employing the PAM30 matrix set to default parameters (open gap 9, extension gap 1 penalties). The BEAST sequence comparison system is available, for instance, from the NCBI web site; see also Altschul et al, J. Mol. Biol, 215:403-410, 1990; Gish. & States, Nature Genet., 3:266- 272, 1993; Madden et al. Meth. EnzymoE, 266: 131-141, 1996; Altschul et al, Nucleic Acids Res., 25:3389-3402, 1997; and Zhang & Madden, Genome Res., 7:649-656, 1997. As used herein, the term "fragment" refers to a physically contiguous portion of the primary structure of the polypeptide (i.e. SEQ ID NO: 1). In some embodiments, the fragment comprises at least 8 consecutive amino acids of SEQ ID NO: 1. In some embodiments, the fragment comprises 8; 9; 10; 11; 12; 13; 14; 15; 16; 17; 18; 19; 20; 21; 22; 23; 24; 25; 26; 27; 28; 29; 30; 31; 32; 33; 34; 35; 36; 37; 38; 39; 40; 41; 42; 43; 44; 45; 46; 47; 48; 49; 50; 51; 52; 53; 54; 55; 56; 57; 58; 59; 60; 61; 62; 63; 64; 65; 66; 67; 68; 69; 70; 71; 72 ; 73 ; 74 ; 75 ; 76 ; 77 ; 78 ; 79 ; 80 ; 81 : 82; 83; 84; 85; or 86 consecutive amino acids.

The polypeptides of the present invention are produced by any technique known per se in the art, such as, without limitation, any chemical, biological, genetic or enzymatic technique, either alone or in combination. For instance, knowing the amino acid sequence of the desired sequence, one skilled in the art can readily produce said polypeptides, by standard techniques for production of amino acid sequences. For instance, they can be synthesized using well- known solid phase method, preferably using a commercially available peptide synthesis apparatus (such as that made by Applied Biosystems, Foster City, California) and following the manufacturer's instructions. Alternatively, the polypeptides of the present invention can be synthesized by recombinant DNA techniques as is now well-known in the art. For example, these fragments can be obtained as DNA expression products after incorporation of DNA sequences encoding the desired (poly)peptide into expression vectors and introduction of such vectors into suitable eukaryotic or prokaryotic hosts that will express the desired polypeptide, from which they can be later isolated using well-known techniques.

In some embodiments, it is contemplated that the polypeptide of the invention used in the therapeutic methods of the present invention may be modified in order to improve their therapeutic efficacy. Such modification of therapeutic compounds may be used to decrease toxicity, increase circulatory time, or modify biodistribution. For example, the toxicity of potentially important therapeutic compounds can be decreased significantly by combination with a variety of drug carrier vehicles that modify biodistribution. A strategy for improving drug viability is the utilization of water-soluble polymers. Various water-soluble polymers have been shown to modify biodistribution, improve the mode of cellular uptake, change the permeability through physiological barriers; and modify the rate of clearance from the body. To achieve either a targeting or sustained-release effect, water-soluble polymers have been synthesized that contain drug moieties as terminal groups, as part of the backbone, or as pendent groups on the polymer chain. Polyethylene glycol (PEG) has been widely used as a drug carrier, given its high degree of biocompatibility and ease of modification. Attachment to various drugs, proteins, and liposomes has been shown to improve residence time and decrease toxicity. PEG can be coupled to active agents through the hydroxyl groups at the ends of the chain and via other chemical methods; however, PEG itself is limited to at most two active agents per molecule. In a different approach, copolymers of PEG and amino acids were explored as novel biomaterials which would retain the biocompatibility properties of PEG, but which would have the added advantage of numerous attachment points per molecule (providing greater drug loading), and which could be synthetically designed to suit a variety of applications.

In some embodiments, the variant that promotes the expression of LIPC is a nucleic acid molecule that encodes for the polypeptide as described above. As used herein, the term "nucleic acid molecule" has its general meaning in the art and refers to a DNA or R A molecule. However, the term captures sequences that include any of the known base analogues of DNA and RNA such as, but not limited to 4-acetylcytosine, 8-hydroxy-N6-methyladenosine, aziridinylcytosine, pseudoisocytosine, 5-(carboxyhydroxylmethyl) uracil, 5 -fluorouracil, 5- bromouracil, 5- carboxymethylaminomethyl-2 -thiouracil, 5 -carboxymethylaminomethyluracil, dihydrouracil, inosine, N6-isopentenyladenine, 1 -methyladenine, 1 - methylpseudouracil, 1-methylguanine, 1- methylinosine, 2,2-dimethylguanine, 2- methyladenine, 2-methylguanine, 3-methylcytosine, 5- methylcytosine, N6-methyladenine, 7- methylguanine, 5 -methylaminomethyluracil, 5- methoxyamino-methyl-2 -thiouracil, beta-D- mannosylqueosine, 5'- methoxycarbonylmethyluracil, 5 -methoxyuracil, 2-methylthio-N6- isopentenyladenine, uracil- 5 -oxy acetic acid methylester, uracil-5 -oxy acetic acid, oxybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2 -thiouracil, 2-thiouracil, 4- thiouracil, 5 -methyluracil, -uracil-5- oxyacetic acid methylester, uracil-5 -oxyacetic acid, pseudouracil, queosine, 2-thiocytosine, and 2,6-diaminopurine.

In some embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least 50% with SEQ ID NO: 2. According to the invention a first nucleic acid sequence having at least 50% of identity with a second nucleic acid sequence means that the first sequence has 50; 51; 52; 53; 54; 55; 56; 57; 58; 59; 60; 61; 62; 63; 64; 65; 66; 67; 68; 69; 70; 71; 72; 73; 74; 75; 76; 77; 78; 79; 80; 81; 82; 83; 84; 85; 86; 87; 88; 89; 90; 91; 92; 93; 94; 95; 96; 97; 98; 99; or 100 % of identity with the second nucleic acid sequence.

In a particular embodiment, the invention relates to a nucleic acid encoding human LIPC protein comprising a sequence having at least 70% sequence identity with the sequence SEQ ID NO 1.

In a particular embodiment, the nucleic acid (such as DNA, RNA, mRNA) or the amino acid related to LIPC variant (as a protein) is comprised in a vector. Thus, in particular embodiment, the present invention relates also to a method for treating hypercholesterolemia and atherosclerotic cardiovascular disease (ASCVD) in a subject in need thereof, comprising administering said subject a vector comprising LIPC variant.

In particular embodiment, the present invention relates to a method for treating hypercholesterolemia and atherosclerotic cardiovascular disease (ASCVD) in a subject in need thereof, comprising administering said subject a vector comprising DNA or RNA encoding a protein of interest.

In particular embodiment, the present invention relates to a method for treating a hypercholesterolemia and atherosclerotic cardiovascular disease (ASCVD) in a subject in need thereof, comprising administering said subject a vector comprising LIPC variant.

In particular embodiment, the present invention relates to a method for treating hypercholesterolemia and atherosclerotic cardiovascular disease (ASCVD) in a subject in need thereof, comprising administering said subject a vector comprising LIPC-E97G.

As used herein, the term “vector” has its general meaning in the art and refers to the vehicle or agents by which a nucleic acid molecule can be introduced into cells, so as to transform/transfect the cell and promote expression (e.g. transcription and/or translation) of the introduced sequence.

According to the invention, vectors include viral vectors or non- viral vectors.

In some embodiments, the method according to the invention, wherein the vector is a viral or non-viral vector.

Non-viral vectors mainly comprise chemical systems that are not of viral origin and generally include chemical methods such as cationic liposomes and polymers. Non-viral vectors useful in the practice of the present invention has very well known in the art. According to the invention non-viral vector includes transfection agents as defined above.

According to the invention, non-viral vectors include but are not limited to liposomes such as cationic liposomes (including neutral and cationic lipids), electroporation, direct injection, cell fusion, particle bombardment, biopolymer nanoparticle, calcium phosphate coprecipitation, DEAE-dextran-mediated transfer and viral vector-mediated transfer, solid-lipid nanoparticles (SLNs or LNPs) such as [(4-hydroxybutyl)azanediyl]di(hexane-6,l-diyl) bis(2- hexyldecanoate)-based nanoparticles; niosomes; polymers such as cationic polymers; polymers-based nanoparticles such polyethylenimine(PEI)-based nanoparticles; lipopeptides- based nanoparticles such as lipid 1 ,2-dilinoleyloxy-3 -dimethylaminopropane (DLin-DMA)- based nanoparticles, dilinoleylmethyl-4-dimethylaminobutyrate (DLin-MC3-DMA)-based nanoparticles, ALC-0315 -based nanoparticles, ALC-0159-based nanoparticles SM-102-based nanonparticles and ; and chitosans as described in Toualbi L, et al. International Journal of Molecular Sciences, Maier.M et al. Molecular Therapy (2013), Shriane D et al.Biol Pharrn Bull (2018). Non- viral vectors according to the invention include also the non- viral vectors described in patent WO2017049245, W02018081480 and W02021016430.

In some embodiments, the method according to the invention, wherein the non-viral vector is cationic a polymers-based nanoparticle, and more particularly is a polyethylenimine(PEI)-based nanoparticle.

In some embodiments, the method according to the invention, wherein the non-viral vector is in vzvo-jetRNA® or in vzvo-jetPEI®.

In some embodiments, the method according to the invention, wherein the vector is a non-viral vector comprising ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) encoding a protein of interest. In some embodiments, the method according to the invention, wherein the vector is a non-viract vector when the acid nucleic encodes for LIPC or LIPC variant.

In some embodiments, the method according to the invention, wherein the vector is a non-viral vector comprising messenger ribonucleic acid (mRNA) encoding LIPC or LIPC variant.

Thus, in a particular embodiment, the present invention relates to a method for treating hypercholesterolemia and atherosclerotic cardiovascular disease (ASCVD) in a subject in need thereof, comprising administering said subject a non-viral vector comprising a messenger ribonucleic acid (mRNA) encoding a protein of interest, and in particular mRNA encoding LIPC or LIPC variant.

In some embodiments, the method according to the invention, the vector is a viral vector. Viral vectors useful in the practice of the present invention can be constructed utilizing methodologies well known in the art of molecular biology. Typically, viral vectors carrying nucleic acid encoding the protein of interest, suitable regulatory elements and elements necessary for production of viral proteins which mediate cell transduction. Examples of viral vector include but are not limited to retrovirus, adenovirus, adeno-associated virus (AAV), herpes virus, pox virus, human foamy virus (HEV), and lentivirus.

In some embodiments, the method according to the invention, wherein the viral vector is a lentivirus (LV) vector, adenovirus vector or an adeno-associated virus (AAV) vector.

As used herein, the term “lentivirus” refers to enveloped RNA particles measuring approximately 120 nm in size are efficient drug delivery tools and more particularly gene delivery tools. The LV binds to, and enters into target cells through its envelope proteins which confer its pseudotype. Once the LV has entered into the cells, it releases its capsid components and undergoes reverse transcription of the lentiviral RNA before integrating the proviral DNA into the genome of target cells. Non-integrative lentiviral vectors have been generated by modifying the properties of the vector integration machinery and can be used for transient gene expression. Virus-like particles lacking a provirus have also been generated and can be used to deliver proteins or messenger RNA. LV can be used for example, for gene addition, RNA interference, exon skipping or gene editing. All of these approaches can be facilitated by tissue or cell targeting of the LV via its pseudotype.

Lentivirus-like particles are described for example in (Aoki et al., 2011; Kaczmarczyk et al., 2011; McBumey et al., 2006; Muratori et al., 2010). Examples of lentivirus-like particles are VLPs generated by co-expressing in producer cells, a syncytin protein with a gag fusion protein (Gag fused with the gene of interest). The drug and/or syncytin may be, either displayed on the surface of the particles, or enclosed (packaged) into the particles. The syncytin protein is advantageously displayed on the surface of the particles, such as coupled to the particles or incorporated into the envelope of (enveloped) virus particles or virus-like particles to form pseudotyped enveloped virus particles or virus-like particles. The drug is coupled to the particles or packaged into the particles. For example, the drug is coupled to viral capsids or packaged into viral capsids, wherein said viral capsids may further comprise an envelope, preferably pseudotyped with syncytin. In some preferred embodiments, the drug is packaged into the particles pseudotyped with syncytin protein. The drug which is packaged into particles is advantageously a heterologous gene of interest which is packaged into viral vector particles, preferably retroviral vector particles, more preferably lentiviral vector particles.

As used herein, the term “adenovirus” refers to medium-sized (90-100 nm), nonenveloped (without an outer lipid bilayer) viruses with an icosahedral nucleocapsid containing a double stranded DNA genome.

In a particular embodiment, the method according to the invention, wherein the viral vector is an adeno-associated virus (AAV) vector.

As used herein, the term “AAV vector” refers to a vector derived from an adeno- associated virus serotype, including without limitation AAV1, AAV2, AAV3, AAV4, AA5, AAV6, AAV7, AAV8, AAV9, AAVrhlO or any other serotypes of AAV that can infect humans, monkeys or other species. AAV vectors can have one or more of the AAV wild-type genes deleted in whole or part, preferably the rep and/or cap genes, but retain functional flanking ITR sequences. Functional ITR sequences are necessary for the rescue, replication and packaging of the AAV virion. Thus, an AAV vector is defined herein to include at least those sequences required in cis for replication and packaging (e. g., functional ITRs) of the virus. The ITRs need not be the wild-type nucleotide sequences, and may be altered, e. g by the insertion, deletion or substitution of nucleotides, so long as the sequences provide for functional rescue, replication and packaging. AAV expression vectors are constructed using known techniques to at least provide as operatively linked components in the direction of transcription, control elements including a transcriptional initiation region, the nucleic acid molecule of the present invention and a transcriptional termination region. The control elements are selected to be functional in a mammalian cell. The resulting construct which contains the operatively linked components is bounded (5' and 3’) with functional AAV ITR sequences. By "adeno-associated virus inverted terminal repeats " or "AAV ITRs" is meant the art-recognized regions found at each end of the AAV genome which function together in cis as origins of DNA replication and as packaging signals for the virus. AAV ITRs, together with the AAV rep coding region, provide for the efficient excision and rescue from, and integration of a nucleotide sequence interposed between two flanking ITRs into a mammalian cell genome. The nucleotide sequences of AAV ITR regions are known. See, e.g., Kotin, 1994; Berns, KI "Parvoviridae and their Replication" in Fundamental Virology, 2nd Edition, (B. N. Fields and D. M. Knipe, eds.) for the AAV-2 sequence. As used herein, an "AAV ITR" does not necessarily comprise the wild-type nucleotide sequence, but may be altered, e.g., by the insertion, deletion or substitution of nucleotides. Additionally, the AAV ITR may be derived from any of several AAV serotypes, including without limitation, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8 or AAV9. Furthermore, 5’ and 3’ ITRs which flank a selected nucleotide sequence in an AAV vector need not necessarily be identical or derived from the same AAV serotype or isolate, so long as they function as intended, i.e., to allow for excision and rescue of the sequence of interest from a host cell genome or vector, and to allow integration of the heterologous sequence into the recipient cell genome when AAV Rep gene products are present in the cell.

In some embodiments, the AAV vector of the present invention is a double-stranded, self-complementary AAV (scAAV) vector. Alternatively, to the use of single-stranded AAV vector, self-complementary vectors can be used. The efficiency of AAV vector in terms of the number of genome-containing particles required for transduction, is hindered by the need to convert the single-stranded DNA (ssDNA) genome into double-stranded DNA (dsDNA) prior to expression. This step can be circumvented through the use of self-complementary vectors, which package an inverted repeat genome that can fold into dsDNA without the requirement for DNA synthesis or base-pairing between multiple vector genomes. Resulting self- complementary AAV (scAAV) vectors have increased resulting expression of the transgene. For an overview of AAV biology, ITR function, and scAAV constructs, see McCarty D M. Self-complementary AAV vectors; advances and applications. Mol. Ther. 2008 October; 16 (10): at pages 1648-51, first full paragraph, incorporated herein by reference for disclosure of AAV and scAAV constructs, ITR function, and role of ATRS ITR in scAAV constructs. A rAAV vector comprising a ATRS ITR cannot correctly be nicked during the replication cycle and, accordingly, produces a self-complementary, double-stranded AAV (scAAV) genome, which can efficiently be packaged into infectious AAV particles. Various rAAV, ssAAV, and scAAV vectors, as well as the advantages and drawbacks of each class of vector for specific applications and methods of using such vectors in gene transfer applications are well known to those of skill in the art (see, for example, Choi V W, Samulski R J, McCarty D M. Effects of adeno-associated virus DNA hairpin structure on recombination. J. Virol. 2005 June; 79(11):6801-7; McCarty D M, Young S M Jr, Samulski R J. Integration of adeno-associated virus (AAV) and recombinant AAV vectors. Annu Rev Genet. 2004; 38:819-45; McCarty D M, Monahan P E, Samulski R J. Self-complementary recombinant adeno-associated virus (scAAV) vectors promote efficient transduction independently of DNA synthesis. Gene Ther. 2001 August; 8(16): 1248-54; and McCarty D M. Self-complementary AAV vectors; advances and applications. Mol. Ther. 2008 October; 16(10): 1648-56; all references cited in this application are incorporated herein by reference for disclosure of AAV, rAAV, and scAAV vectors).

In some embodiments, the AAV vector is an AAV8 vector.

In a particular embodiment, the LIPC variant in the context of the invention is obtained by genome editing.

As used herein, the term "gene," refers to a DNA region encoding a gene product, as well as all DNA regions which regulate the production of the gene product, whether or not such regulatory sequences are adjacent to coding and/or transcribed sequences. A gene includes, but is not limited to, promoter sequences, enhancers, silencers, insulators, boundary elements, terminators, polyadenylation sequences, post-transcription response elements, translational regulatory sequences such as ribosome binding sites and internal ribosome entry sites, replication origins, matrix attachment sites, and locus control regions.

As used herein, the term "gene expression" refers to the conversion of the information, contained in a gene, into a gene product. A gene product can be the direct transcriptional product of a gene (e.g., mRNA, tRNA, rRNA, antisense RNA, ribozyme, structural RNA or any other type of RNA) or a protein produced by translation of an mRNA. Gene products also include RNAs which are modified, by processes such as capping, polyadenylation, methylation, and editing, and proteins modified by, for example, methylation, acetylation, phosphorylation, ubiquitination, ADP-ribosylation, myristilation, and glycosylation.

As used herein, the term "genome editing" refers to the substitution, deletion, and/or introduction of genetic material at a target site in the cell's genome, which restores, corrects, disrupts, and/or modifies expression of a gene or gene product. Genome editing contemplated in particular embodiments comprises introducing one or more nuclease variants into a cell to generate DNA lesions at or proximal to a target site in the cell' s genome, optionally in the presence of a donor repair template.

As used herein, the term "genetically engineered" or "genetically modified" refers to the chromosomal or extrachromosomal addition of extra genetic material in the form of DNA or RNA to the total genetic material in a cell. Genetic modifications may be targeted or nontargeted to a particular site in a cell's genome. In one embodiment, genetic modification is site specific. In one embodiment, genetic modification is not site specific.

As used herein, the term "gene therapy" refers to the introduction of extra genetic material into the total genetic material in a cell that restores, corrects, or modifies expression of a gene or gene product, or for the purpose of expressing a therapeutic polypeptide. In particular embodiments, introduction of genetic material into the cell's genome by genome editing that restores, corrects, disrupts, or modifies expression of a gene or gene product, or for the purpose of expressing a therapeutic polypeptide is considered gene therapy.

Genome editing compositions and methods contemplated in various embodiments comprise nuclease variants, designed to bind and cleave a target site in a LIPC gene. In particular compositions genome editing compositions contemplated herein comprise a polynucleotide encoding a nuclease variant or megaTAL that binds and cleaves a target site in LIPC gene. The nuclease variants contemplated in particular embodiments, can be used to introduce a double-strand break in a target.

Polynucleotide sequence, which may be repaired by non-homologous end joining (NHEJ) in the absence of a polynucleotide template, e.g., a donor repair template, or by homology directed repair (HDR), i.e., homologous recombination, in the presence of a donor repair template. Nuclease variants contemplated in certain embodiments, can also be designed as nickases, which generate single-stranded DNA breaks that can be repaired using the cell's base-excision-repair (BER) machinery or homologous recombination in the presence of a donor repair template. NHEJ is an error-prone process that frequently results in the formation of small insertions and deletions that disrupt gene function. Homologous recombination requires homologous DNA as a template for repair and can be leveraged to create a limitless variety of modifications specified by the introduction of donor DNA containing the desired sequence at the target site, flanked on either side by sequences bearing homology to regions flanking the target site.

In a particular embodiment, the genome editing compositions contemplated herein comprise a polynucleotide encoding a homing endonuclease variant or megaTAL that targets a LIPC gene.

In a particular embodiment, the genome editing compositions contemplated herein comprise one or more polynucleotides encoding a homing endonuclease variant or megaTAL and an end-processing enzyme, e.g., Trex2.

Techniques for recombinant (i.e., engineered) DNA, peptide and oligonucleotide synthesis, immunoassays, tissue culture, transformation (e.g., electroporation, lipofection), enzymatic reactions, purification and related techniques and procedures may be generally performed as described in various general and more specific references in microbiology, molecular biology, biochemistry, molecular genetics, cell biology, virology and immunology as cited and discussed throughout the present specification. See, e.g., Sambrook et al, Molecular Cloning: A Laboratory Manual, 3d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y.; Current Protocols in Molecular Biology (John Wiley and Sons, updated July 2008); Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience; Glover, DNA Cloning: A Practical Approach, vol. I & H (TRL Press, Oxford Univ. Press USA, 1985); Current Protocols in Immunology (Edited by: John E. Coligan, Ada M. Kruisbeek, David H. Margulies, Ethan M. Shevach, Warren Strober 2001 John Wiley & Sons, NY, NY); Real-Time PCR: Current Technology and Applications, Edited by Julie Logan, Kirstin Edwards and Nick Saunders, 2009, Caister Academic Press, Norfolk, UK; Anand, Techniques for the Analysis of Complex Genomes, (Academic Press, New York, 1992); Guthrie and Fink, Guide to Yeast Genetics and Molecular Biology (Academic Press, New York, 1991); Oligonucleotide Synthesis (N. Gait, Ed., 1984); Nucleic Acid The Hybridization (B. Hames & S. Higgins, Eds., 1985); Transcription and Translation (B. Hames & S. Higgins, Eds., 1984); Animal Cell Culture (R. Freshney, Ed., 1986); Perbal, A Practical Guide to Molecular Cloning (1984); Next- Generation Genome Sequencing (Janitz, 2008 Wiley- VCH); PCR Protocols Methods in Molecular Biology) (Park, Ed., 3rd Edition, 2010 Humana Press); Immobilized Cells And Enzymes (IRL Press, 1986); the treatise, Methods In Enzymology (Academic Press, Inc., N. Y.); Gene Transfer Vectors For Mammalian Cells (J. H. Miller and M. P. Calos eds., 1987, Cold Spring Harbor Laboratory); Harlow and Lane, Antibodies, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1998); Immunochemical Methods In Cell And Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987); Handbook Of Experimental Immunology, Volumes I-IV (D. M. Weir andCC Blackwell, eds., 1986); Roitt, Essential Immunology, 6th Edition, (Blackwell Scientific Publications, Oxford, 1988); Current Protocols in Immunology (Q. E. Coligan, A. M. Kruisbeek, D. H. Margulies, E. M. Shevach and W. Strober, eds., 1991); Annual Review of Immunology; as well as monographs in journals such as Advances in Immunology.

As used herein, the term "Recombination" refers to a process of exchange of genetic information between two polynucleotides, including but not limited to, donor capture by non- homologous end j oining (NHEJ) and homologous recombination. For the purposes of this disclosure, "homologous recombination (HR)" refers to the specialized form of such exchange that takes place, for example, during repair of double-strand breaks in cells via homology- directed repair (HDR) mechanisms. This process requires nucleotide sequence homology, uses a "donor" molecule as a template to repair a "target" molecule (i.e., the one that experienced the double-strand break), and is variously known as "non-crossover gene conversion" or "short tract gene conversion," because it leads to the transfer of genetic information from the donor to the target. Without wishing to be bound by any particular theory, such transfer can involve mismatch correction of heteroduplex DNA that forms between the broken target and the donor, and/or "synthesis- dependent strand annealing," in which the donor is used to resynthesize genetic information that will become part of the target, and/or related processes. Such specialized HR often results in an alteration of the sequence of the target molecule such that part or all of the sequence of the donor polynucleotide is incorporated into the target polynucleotide.

As used herein, the term "NHEJ" or "non-homo logons end joining" refers to the resolution of a double-strand break in the absence of a donor repair template or homologous sequence. NHEJ can result in insertions and deletions at the site of the break. NHEJ is mediated by several sub-pathways, each of which has distinct mutational consequences. The classical NHEJ pathway (cNHEJ) requires the KU/DNA-PKcs/Lig4/XRCC4 complex, ligates ends back together with minimal processing and often leads to precise repair of the break. Alternative NHEJ pathways (altNHEJ) also are active in resolving dsDNA breaks, but these pathways are considerably more mutagenic and often result in imprecise repair of the break marked by insertions and deletions. While not wishing to be bound to any particular theory, it is contemplated that modification of dsDNA breaks by end-processing enzymes, such as, for example, exonucl eases, e.g., Trex2, may increase the likelihood of imprecise repair. As used herein, the term "Cleavage" refers to the breakage of the covalent backbone of a DNA molecule. Cleavage can be initiated by a variety of methods including, but not limited to, enzymatic or chemical hydrolysis of a phosphodiester bond. Both single-stranded cleavage and double- stranded cleavage are possible. Double-stranded cleavage can occur as a result of two distinct single-stranded cleavage events. DNA cleavage can result in the production of either blunt ends or staggered ends. In certain embodiments, polypeptides and nuclease variants, e.g., homing endonuclease variants, megaTALs, etc. contemplated herein are used for targeted double-stranded DNA cleavage. Endonuclease cleavage recognition sites may be on either DNA strand.

As used herein, the term "exogenous" molecule is a molecule that is not normally present in a cell, but that is introduced into a cell by one or more genetic, biochemical or other methods. Exemplary exogenous molecules include, but are not limited to small organic molecules, protein, nucleic acid, carbohydrate, lipid, glycoprotein, lipoprotein, polysaccharide, any modified derivative of the above molecules, or any complex comprising one or more of the above molecules.

As used herein, the term "endogenous" molecule is one that is normally present in a particular cell at a particular developmental stage under particular environmental conditions. Additional endogenous molecules can include proteins.

In some embodiments, the LIPC variant in the contexte of the invention comprises an endonuclease.

As used herein, the term “endonuclease” refers to enzymes that cleave the phosphodiester bond within a polynucleotide chain. Some, such as Deoxyribonuclease I, cut DNA relatively nonspecifically (without regard to sequence), while many, typically called restriction endonucleases or restriction enzymes, and cleave only at very specific nucleotide sequences. The mechanism behind endonuclease-based genome inactivating generally requires a first step of DNA single or double strand break, which can then trigger two distinct cellular mechanisms for DNA repair, which can be exploited for DNA inactivating: the error prone nonhomologous end-joining (NHEJ) and the high-fidelity homology-directed repair (HDR).

In a particular embodiment, the endonuclease is CRISPR-cas.

As used herein, the term “CRISPR-Cas” has its general meaning in the art and refers to clustered regularly interspaced short palindromic repeats associated which are the segments of prokaryotic DNA containing short repetitions of base sequences. In some embodiment, the endonuclease is CRISPR-Cas9 which is from Streptococcus pyogenes. The CRISPR/Cas9 system has been described in US 8697359 Bl and US 2014/0068797. In some embodiments, the endonuclease is CRISPR-Cpfl which is the more recently characterized CRISPR from Provotella and Francisella 1 (Cpfl) in (Zetsche et al., 2015).

As used herein the terms "administering" or "administration" refer to the act of injecting or otherwise physically delivering a substance as it exists outside the body (e.g., a LIPC variant) into the subject, such as by oral, mucosal, intradermal, intravenous, subcutaneous, intramuscular, intraperitoneal delivery and/or any other method of physical delivery described herein or known in the art. When a disease, or a symptom thereof, is being treated, administration of the substance typically occurs after the onset of the disease or symptoms thereof. When a disease or symptoms thereof, are being prevented, administration of the substance typically occurs before the onset of the disease or symptoms thereof. In a particular embodiment, the LIPC variant is administered by oral, intravenous or subcutaneous route.

By a "therapeutically effective amount" is meant a sufficient amount of the agent of the present invention for reaching a therapeutic effect. It will be understood, however, that the total daily usage of the compounds and compositions of the present invention will be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular subject will depend upon a variety of factors including the disorder being treated and the severity of the disorder; activity of the specific compound employed; the specific composition employed, the age, body weight, general health, sex and diet of the subject; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific compound employed; and like factors well known in the medical arts. For example, it is well within the skill of the art to start doses of the compound at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. However, the daily dosage of the products may be varied over a wide range from 0.01 to 4,000 mg per adult per day. Typically, the compositions contain 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 250, 500 and 1000 mg of the active ingredient for the symptomatic adjustment of the dosage to the subject to be treated. A medicament typically contains from about 0.01 mg to about 1000 mg of the active ingredient. An effective amount of the drug is ordinarily supplied at a dosage level from 0.0002 mg/kg to about 50 mg/kg of body weight per day, especially from about 0.001 mg/kg to 10 mg/kg of body weight per day. In another aspect, the LIPC variant according to the invention is combined with a classical treatment of hypercholesterolemia and atherosclerotic cardiovascular disease (ASCVD).

Accordingly, in a second aspect, the invention relates to the LIPC variant for use according to the invention, and a classical treatment as a combined preparation for simultaneous, separate or sequential use in the treatment of hypercholesterolemia and atherosclerotic cardiovascular disease (ASCVD) in a subject in need thereof

As used herein, the terms “combined treatment”, “combined therapy” or “therapy combination” refer to a treatment that uses more than one medication. The combined therapy may be dual therapy or bi-therapy.

As used herein, the term “administration simultaneously” refers to administration of at least 2 or 3 active ingredients by the same route and at the same time or at substantially the same time. The term “administration separately” refers to an administration of at least 2 or 3 active ingredients at the same time or at substantially the same time by different routes. The term “administration sequentially” refers to an administration of at least 2 or 3 active ingredients at different times, the administration route being identical or different.

As used herein, the term “classical treatment” refers to treatments well known in the art and used to treat hypercholesterolemia and atherosclerotic cardiovascular disease (ASCVD). Typically, such classical treatment refers to compounds targetting LDLR pathway such as statins and/or PCSK9 inhibitors.

In a particular embodiment, the classical treatment is statins.

As used herein, the term “statins” also known as HMG-CoA reductase inhibitors refers to a class of lipid-lowering medications that reduce illness and mortality in those who are at high risk of cardiovascular disease. These drugs block a substance the liver needs to make cholesterol. Statins are selected from the group consiting of but not limited to : atorvastatin (Lipitor), fluvastatin (Lescol XL), lovastatin (Altoprev), pitavastatin (Livalo), pravastatin, rosuvastatin (Crestor) and simvastatin (Zocor).

In a particular embodiment, the classical treatment PCSK9 inhibitor.

As used herein, the term “PCSK9 inhibitor” refers to a natural or synthetic compound that directly or indirectly decreases the PCSK9 activity that has a biological effect to inhibit or significantly reduce the activity or expression PCSK9. It thus refers to any compound able to directly or indirectly decrease the transcription, translation, post-translational modification or activity of PCSK9. It includes intracellular as well as extracellular PCSK9 inhibitors. The activator or inhibitor of PCSK9 activity is a small organic molecule, an aptamer an antibody or a polypeptide.

As used herein the term “small organic molecule” refers to a molecule of a size comparable to those organic molecules generally used in pharmaceuticals. The term excludes biological macro molecules (e. g. proteins, nucleic acids, etc.). Typically, small organic molecules range in size up to about 5000 Da, more preferably up to 2000 Da, and most preferably up to about 1000 Da.

As used herein the term “aptamers” refers to a class of molecule that represents an alternative to antibodies in term of molecular recognition. Aptamers are oligonucleotide or oligopeptide sequences with the capacity to recognize virtually any class of target molecules with high affinity and specificity.

As used herein the term "antibody" refers to any antibody-like molecule that has an antigen binding region, and this term includes antibody fragments that comprise an antigen binding domain such as Fab', Fab, F(ab')2, single domain antibodies (DABs or VHH), TandAbs dimer, Fv, scFv (single chain Fv), dsFv, ds-scFv, Fd, linear antibodies, minibodies, diabodies, bispecific antibody fragments, bibody, tribody (scFv-Fab fusions, bispecific or trispecific, respectively); sc-diabody; kappa(lamda) bodies (scFv-CL fusions); DVD-Ig (dual variable domain antibody, bispecific format); SIP (small immunoprotein, a kind of minibody); SMIP ("small modular immunopharmaceutical" scFv-Fc dimer; DART (ds-stabilized diabody "Dual Affinity ReTargeting"); small antibody mimetics comprising one or more CDRs and the like. The techniques for preparing and using various antibody-based constructs and fragments are well known in the art. Particularly, in the context of the invention, the antibody is a single domain antibody. The term “single domain antibody” has its general meaning in the art and refers to the single heavy chain variable domain of antibodies of the type that can be found in Camelid mammals which are naturally devoid of light chains. Such single domain antibody are also called VHH or “nanobody®”. For a general description of (single) domain antibodies, reference is also made to the prior art cited above, as well as to EP 0 368 684, Ward et al. (Nature 1989 Oct 12; 341 (6242): 544-6), Holt et al., Trends Biotechnol., 2003, 21(11):484- 490; and WO 06/030220, WO 06/003388. In the context of the invention, the amino acid residues of the single domain antibody are numbered according to the general numbering for VH domains given by the International ImMunoGeneTics information system aminoacid numbering (http://imgt.cines.fr/). Particularly, in the context of the invention, the antibody is a single chain variable fragment. The term "single chain variable fragment" or "scFv fragment" refers to a single folded polypeptide comprising the VH and VL domains of an antibody linked through a linker molecule. In such a scFv fragment, the VH and VL domains can be either in the VH - linker - VL or VL - linker - VH order. In addition to facilitate its production, a scFv fragment may contain a tag molecule linked to the scFv via a spacer. A scFv fragment thus comprises the VH and VL domains implicated into antigen recognizing but not the immunogenic constant domains of corresponding antibody. In a particular embodiment, the inhibitor of PCSK9 activity is an intrabody having specificity for PCSK9. As used herein, the term "intrabody" generally refers to an intracellular antibody or antibody fragment. Antibodies, in particular single chain variable antibody fragments (scFv), can be modified for intracellular localization. Such modification may entail for example, the fusion to a stable intracellular protein, such as, e.g., maltose binding protein, or the addition of intracellular trafficking/localization peptide sequences, such as, e.g., the endoplasmic reticulum retention. In some embodiments, the intrabody is a single domain antibody. In a particular embodiment, the inhibitor of PCSK9 activity is Evolocumab commercialized as Repatha® (or AMG 145) by Amgen and has the following formula in the art:C_1B«H_S6«N₎«₄₈0iwS5_<. In a particular embodiment, the antibody is Alirocumab commercialized as Praluent (REGN727 or SAR2365553) by Sanofi and Regeneron Pharmaceuticals and has the following formula in the art:

As used herein, the term “polypeptide” refers to a polypeptide that specifically bind to PCSK9, can be used as a PCSK9 activator or inhibitor that bind to and activate or sequester the PCSK9 protein, thereby stimulating or preventing it from signaling. Polypeptide refers both short peptides with a length of at least two amino acid residues and at most 10 amino acid residues, oligopeptides (11-100 amino acid residues), and longer peptides (the usual interpretation of "polypeptide", i.e. more than 100 amino acid residues in length) as well as proteins (the functional entity comprising at least one peptide, oligopeptide, or polypeptide which may be chemically modified by being glycosylated, by being lipidated, or by comprising prosthetic groups). The definition of polypeptides also comprises native forms of peptides/proteins in mycobacteria as well as recombinant proteins or peptides in any type of expression vectors transforming any kind of host, and also chemically synthesized peptides. In a particular, the PCSK9 activity inhibitor is an intracellular peptide. Typically, intracellular peptide disturbs transmission of signals of PCSK9 mainly in the cytosol, mitochondria, and/or nucleus. In a particular embodiment, the polypeptide against PCSK9 activity is BMS-962476 as characterized by the amino acid sequences disclosed in WO 2011130354. This polypeptide is also described in Mitchell et al 2010 (J Pharmacol Exp Ther. 2014 Aug;350(2):412-24. doi: 10.1124/jpet.114.214221. Epub 2014 Jun 10.) In a particular embodiment, the PCSK9 inhibitor is an inhibitor of PCSK9 expression. An "inhibitor of PCSK9 expression" refers to a natural or synthetic compound that has a biological effect to inhibit or significantly reduce the expression of the gene encoding for PCSK9. Typically, the inhibitor of PCSK9 expression has a biological effect on one or more of the following events: (1) production of an RNA template from a DNA sequence (e.g., by transcription); (2) processing of an RNA transcript (e.g., by splicing, editing, 5' cap formation, and/or 3' end formation); (3) translation of an RNA into a polypeptide or protein; and/or (4) post-translational modification of a polypeptide or protein.

In some embodiments, the inhibitor of PCSK9 expression is an antisense oligonucleotide. Anti-sense oligonucleotides, including anti-sense RNA molecules and antisense DNA molecules, would act to directly block the translation of PCSK9 mRNA by binding thereto and thus preventing protein translation or increasing mRNA degradation, thus decreasing the level of PCSK9 proteins, and thus activity, in a cell. For example, antisense oligonucleotides of at least about 15 bases and complementary to unique regions of the mRNA transcript sequence encoding PCSK9 can be synthesized, e.g., by conventional phosphodiester techniques and administered by e.g., intravenous injection or infusion. Methods for using antisense techniques for specifically alleviating gene expression of genes whose sequence is known are well known in the art (e.g. see U.S. Pat. Nos. 6,566,135; 6,566,131; 6,365,354; 6,410,323; 6,107,091; 6,046,321; and 5,981,732).

In a particular embodiment, the inhibitor of PCSK9 expression is a shRNA. shRNA is generally expressed using a vector introduced into cells, wherein the vector utilizes the U6 promoter to ensure that the shRNA is always expressed. This vector is usually passed on to daughter cells, allowing the gene silencing to be inherited. The shRNA hairpin structure is cleaved by the cellular machinery into siRNA, which is then bound to the RNA-induced silencing complex (RISC). This complex binds to and cleaves mRNAs that match the siRNA to which it is bound.

In some embodiments, the inhibitor of PCSK9 expression is a small inhibitory RNAs (siRNAs). PCSK9 expression can be reduced by contacting the subject or cell with a small double stranded RNA (dsRNA), or a vector or construct causing the production of a small double stranded RNA, such that PCSK9 expression is specifically inhibited (i.e. RNA interference or RNAi). Methods for selecting an appropriate dsRNA or dsRNA-encoding vector are well known in the art for genes whose sequence is known (e.g. see Tuschl, T. et al. (1999); Elbashir, S. M. et al. (2001); Hannon, GJ. (2002); McManus, MT. et al. (2002); Brummelkamp, TR. et al. (2002); U.S. Pat. Nos. 6,573,099 and 6,506,559; and International Patent Publication Nos. WO 01/36646, WO 99/32619, and WO 01/68836). In a particular embodiment, the siRNA is ALN-PCS02 developed and commercialized by Novartis (as Inclisirian).

In some embodiments, inhibitor of PCSK9 expression is a ribozyme. Ribozymes are enzymatic RNA molecules capable of catalyzing the specific cleavage of RNA. The mechanism of ribozyme action involves sequence specific hybridization of the ribozyme molecule to complementary target RNA, followed by endonucleolytic cleavage. Engineered hairpin or hammerhead motif ribozyme molecules that specifically and efficiently catalyze endonucleolytic cleavage of PCSK9 mRNA sequences are thereby useful within the scope of the present invention. Specific ribozyme cleavage sites within any potential RNA target are initially identified by scanning the target molecule for ribozyme cleavage sites, which typically include the following sequences, GUA, GUU, and GUC. Once identified, short RNA sequences of between about 15 and 20 ribonucleotides corresponding to the region of the target gene containing the cleavage site can be evaluated for predicted structural features, such as secondary structure, that can render the oligonucleotide sequence unsuitable. The suitability of candidate targets can also be evaluated by testing their accessibility to hybridization with complementary oligonucleotides, using, e.g., ribonuclease protection assays.

In some embodiments, the inhibitor of PCSK9 expression is an endonuclease. The term “endonuclease” refers to enzymes that cleave the phosphodiester bond within a polynucleotide chain. Some, such as Deoxyribonuclease I, cut DNA relatively nonspecifically (without regard to sequence), while many, typically called restriction endonucleases or restriction enzymes, and cleave only at very specific nucleotide sequences. The mechanism behind endonuclease-based genome inactivating generally requires a first step of DNA single or double strand break, which can then trigger two distinct cellular mechanisms for DNA repair, which can be exploited for DNA inactivating: the errorprone nonhomologous end-joining (NHEJ) and the high-fidelity homology-directed repair (HDR). In a particular embodiment, the endonuclease is CRISPR- cas. As used herein, the term “CRISPR-cas” has its general meaning in the art and refers to clustered regularly interspaced short palindromic repeats associated which are the segments of prokaryotic DNA containing short repetitions of base sequences. In some embodiment, the endonuclease is CRISPR-cas9 which is from Streptococcus pyogenes. The CRISPR/Cas9 system has been described in US 8697359 Bl and US 2014/0068797. In some embodiment, the endonuclease is CRISPR-Cpfl which is the more recently characterized CRISPR from Provotella and Francisella 1 (Cpfl) in Zetsche et al. (“Cpfl is a Single RNA-guided Endonuclease of a Class 2 CRISPR-Cas System (2015); Cell; 163, 1-13). In a further embodiment, the PCSK9 inhibitor is selected from the group consisting of but not limited to: alirocumab (Sanofi); inclisirian (Novartis); tafolecimab (Innovent Biologies Inc); ebronucimab (Akeso Inc); lerodalcibep (LIB Therapeutics LLC); SHR-1209 (Jiangsu Hengrui Medicine Co Ltd); cepadacursen sodium (Civi Biopharma Inc); DC- 371739(Guangzhou Jiayue Pharmaceutical Technology Co Ltd); MK-0616 (Merck & Co Inc); NN-6434 (Novo Nordisk AS); AZD-0780 (AstraZeneca Pic); B-1655 (Tasly Pharmaceutical Group Co Ltd); RBD-7022 (Suzhou Ribo Life Sciences Co Ltd); CiVi-008 (Civi Biopharma Inc); KFPH-020 (Jiangsu Carephar Pharmaceutical Co Ltd); NYX-330 (Nyrada Inc); NYXPCSK-9i212 (Nyrada Inc); PBGENE-PCSK9 (Precision Biosciences Inc); STP-135G (Simaomics Ltd); SX-PCK9 (Alexion Pharmaceuticals Inc); CTX-330 (CRISPR Therapeutics AG); ALD-306 (Lundbeck Seattle BioPharmaceuticals Inc).

Another object of the present invention is a pharmaceutical composition comprising a nucleic acid and/or a vector and/or a host cell of the present invention. The nucleic acid in the pharmaceutical composition may be any nucleic acid of the invention as defined above.

More particularly, the invention relates to a pharmaceutical composition comprising a LIPC variant.

In a particular embodiment, the invention relates to a pharmaceutical composition comprising LIPC- E97G.

In a particular embodiment, the invention relates to a pharmaceutical composition comprising a vector comprising the nucleic acid (such as DNA, RNA, mRNA) or the amino acid related to LIPC variant (as a protein).

The host cell in the pharmaceutical composition may be any host cell of the invention as defined above.

The pharmaceutical composition may be in any form that can be administered to a human or an animal.

The pharmaceutical composition according to the invention for use in the treatment of hypercholesterolemia and atherosclerotic cardiovascular disease in a subject in need thereof.

Administration may be carried out directly, i.e. pure or substantially pure, or after mixing of the nucleic acid and/or a vector and/or a host cell of the present invention with a pharmaceutically acceptable carrier and/or medium.

In a particular embodiment, the invention relates to a pharmaceutical composition comprising the LIPC variant for use according to the invention, and a classical treatment as a combined preparation for simultaneous, separate or sequential use in the treatment of hypercholesterolemia and atherosclerotic cardiovascular disease (ASCVD) in a subject in need thereof.

As used herein, the term “classical treatment” refers to treatments well known in the art and used to treat hypercholesterolemia and atherosclerotic cardiovascular disease (ASCVD). Typically, such classical treatment refers to compounds targetting LDLR pathway such as statins and/or PCSK9 inhibitors as described above.

As used herein, the terms "Pharmaceutically" or "pharmaceutically acceptable" refer to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to a mammal, especially a human, as appropriate. A pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. The pharmaceutical compositions of the present invention for oral, sublingual, subcutaneous, intramuscular, intravenous, transdermal, local or rectal administration, the active principle, alone or in combination with another active principle, can be administered in a unit administration form, as a mixture with conventional pharmaceutical supports, to animals and human beings. Suitable unit administration forms comprise oral-route forms such as tablets, gel capsules, powders, granules and oral suspensions or solutions, sublingual and buccal administration forms, aerosols, implants, subcutaneous, transdermal, topical, intraperitoneal, intramuscular, intravenous, subdermal, transdermal, intrathecal and intranasal administration forms and rectal administration forms. Typically, the pharmaceutical compositions contain vehicles which are pharmaceutically acceptable for a formulation capable of being injected. These may be in particular isotonic, sterile, saline solutions (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride and the like or mixtures of such salts), or dry, especially freeze-dried compositions which upon addition, depending on the case, of sterilized water or physiological saline, permit the constitution of injectable solutions. The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. 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. Solutions comprising compounds of the invention as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can 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 polypeptide (or nucleic acid encoding thereof) can be formulated into a composition 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. The carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetables oils. The proper fluidity can 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, aluminium monostearate and gelatin. Sterile injectable solutions are prepared by incorporating the active polypeptides in the required amount in the appropriate solvent with several 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 vacuumdrying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. 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 the type of injectable solutions described above, but drug release capsules and the like can also be employed. 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 and intraperitoneal administration. In this connection, sterile aqueous media which can be employed will be known to those of skill in the art in light of the present disclosure. For example, one dosage could 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. 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.

The invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention.

FIGURES:

Figure 1. A novel variant in LIPC cosegregates with familial combined hypocholesterolemia. Pedigree of family with familial combined hypocholesterolemia. Squares indicate male family members; circles, female family members. Slashes indicate deceased individuals. Roman numerals to the left of the pedigree indicate the generation; numerals to the upper left of each symbol indicate the individual family member. Basic lipid parameters of the recruited family members are indicated in the table below the pedigree. Values of total cholesterol, triglyceride, and low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol (HDL-C) levels below the fifth percentile for age and sex are in bold.

Figure 2. E97G alters hepatic lipase substrate specificity. A and B, Triglyceride (TG) lipase activity and phospholipase Al (PLA1) activity in medium of heparin-treated immortalized human hepatocytes with overexpression of wild-type LIPC (LIPC-WT), LIPC- E97G, or LIPC-S168G. C and D, Triglyceride lipase activity and PLA1 activity in medium of heparin-treated immortalized human hepatocytes with a wild-type allele, a heterozygous presence of the E97G variant, or a corrected wild-type allele. Each enzymatic activity was corrected for the amount of released hepatic lipase (HL). E, Lipidomics data of plasma of control individuals (n=5) or E97G carriers (n=3). Values are depicted in nanomoles per liter (phosphatidylethanolamine [PE], lysophosphatidylethanolamine [LysoPE], phosphatidylinositol [PI], ceramides [Cer]) or micromoles per liter (fatty acids [FAs], phosphatidylcholine [PC], lysophosphatidylcholine [LysoPC], sphingomyelin [SM]). F, Ratios of plasma FA levels between control individuals (n=5) and E97G carriers (n=3). G, Phospholipid (PL) ratios of lysophospholipids/phospholipids in control individuals (n=5) or family members (n=3). Cell culture data are of 3 independent experiments with a technical duplicate. Statistical significance determined by Mann-Whitney tests, ns Indicates not significant; and RFU, relative fluorescence units. *P<0.05; **P<0.01; ***P<0.001.

Figure 3. Overexpression of the LIPC-E97G variant markedly lowers LDL-C and HDL-C in APOE*3.Leiden.CETP mice. A, Experimental setup of mice study overexpressing AAV-TBG-eGFP, AAV-TBG-LIPC, and AAV-TBG-LIPC-E97G in APOE* 3. Leiden. CETP mice. B, Human hepatic lipase (HL) levels as determined by ELISA in preheparin and postheparin plasma of mice overexpressing low or high doses of AAV-TBG-eGFP, AAV-TBG- LIPC, and AAV-TBG-LIPC-E97G. C and D, Triglyceride (TG) lipase activity and phospholipase Al (PLA1) activity in plasma of APOE *3. Leiden. CETP mice overexpressing low or high doses of AAV-TBG-eGFP, AAV-TBG-LIPC, and AAV-TBG-LIPC-E97G and after heparin injection. E, Triglycerides (TG), phospholipids, and cholesterol concentrations in plasma of APOE*3.Leiden.CETP mice overexpressing low or high doses of AAV-TBG-eGFP, AAV-TBG-LIPC, and AAV-TBG-LIPC-E97G. F, Cholesterol and triglyceride (TG) concentrations in fast protein liquid chromatography (FPLC)-separated pooled plasma of APOE *3. Leiden. CETP mice overexpressing high doses of AAV-TBG-eGFP, AAVTBG- LIPC, and AAV-TBG-LIPC-E97G. G through I, Cholesterol (CHOL) and D7-cholesterol (D7- CHOL) levels extracted from liver (G), feces (H), and plasma (I) of APOE*3. Leiden. CETP mice overexpressing high doses of AAV-TBG-eGFP, AAV-TBG-LIPC, and AAV-TBG-LIPC- E97G and injected with D7-cholesterol 3 days before death. n=5 or 6 per group. A 1-way ANOVA with Tukey correction for multiple comparisons was used for statistical analysis, with a P value cutoff at P<0.05. eGFP indicates enhanced green fluorescent protein; and LIPC, lipase C, hepatic type. *P<0.05; **P<0.01; ***P<0.001.

Figure 4. Overexpression of the LIPC-E97G variant promotes peripheral cholesterol uptake. A and C, Decay of plasma 3H activity (glycerol tri[3H]oleate, hydrolysable; A) and plasma 14C activity ([14C]cholesteryl oleate, nonhydro lysable) levels (C) in APOE*3. Leiden. CETP mice overexpressing AAV-TBG-eGFP, AAV-TBG-LIPC, and AAV-TBG-LIPC-E97G injected with very-lowdensity lipoprotein (VLDL)-like particles. B and D, Decay of plasma 3H activity (glycerol tri[3H]oleate, hydrolysable; B) and plasma 14C activity ([14C]cholesteryl oleate, nonhydrolysable) levels (D) in APOE*3. Leiden. CETP micel4 overexpressing AAV-TBG-eGFP, AAV-TBG-LIPC, and AAV3-TBG-LIPC-E97G injected with radiolabeled murine VLDL. E, Liver 14C activity (cholesteryl ester, nonhydrolysable; left) and liver 3H activity (triolein, hydrolysable) levels (right) in APOE *3. Leiden. CETP mice overexpressing AAV-TBG-eGFP, AAV-TBG-LIPC, and AAV- TBG-LIPC-E97G injected with VLDL-like particles. F, Liver 14C activity (cholesteryl ester, nonhydrolysable; left) and liver 3H activity (triolein, hydrolysable levels (right) in APOE* 3. Leiden. CETP mice overexpressing AAV-TBG-eGFP, AAV-TBG-LIPC, and AAV- TBG-LIPC-E97G injected with radiolabeled murine VLDL. G and I, Tissue 14C activity (cholesteryl ester, nonhydrolysable; G) and 3H activity (triolein, hydrolysable; I) levels in APOE *3. Leiden. CETP mice overexpressing AAV-TBG-eGFP, AAV-TBG-LIPC, and AAV- TBG-LIPC-E97G injected with VLDL-like particles. H and J, Tissue 14C activity (cholesteryl ester, nonhydrolysable; H) and 3H activity (triolein, hydrolysable; J) levels in APOE *3. Leiden. CETP mice overexpressing AAV-TBG-eGFP, AAV-TBG-LIPC, and AAV- TBG-LIPC-E97G injected with radiolabeled murine VLDL. n=8 per group. A 1-way ANOVA with Tukey correction for multiple comparisons was used for statistical analysis, with a P value cutoff at P<0.05. gWAT indicates gonadal white adipose tissue; iBAT, interscapular brown adipose tissue; sBAT, subscapular brown adipose tissue; and sWAT, subcutaneous white adipose tissue; and TGRL, triglyceride-rich lipoprotein. *P<0.05, human wild-type (LIPC-WT) vs LIPC-E97G. $P<0.05, enhanced green fluorescent protein (eGFP) vs LIPC-WT. #P<0.05, eGFP vs LIPC-E97G.

Figure 5. Effect of LIPC E97G expression on plasma cholesterol levels & aortic atherosclerosis progression in Ldlr-/- mice.

EXAMPLES:

EXAMPLE 1:

Material & Methods

Family Studies

The proband and his relatives were recruited in Lyon to investigate the genetic cause of a familial combined hypolipidemia (GENELIP study [From Known to New Genes in Dyslipidemia]; ClinicalTrials.com registration No. NCT03939039). GENELIP aims to decipher the mechanisms involved in the occurrence or modulation of dyslipidemia in patients referred for primary dyslipidemia. After the identification of 1 potential candidate gene and a confirmative cosegregation analysis, the members of the pedigree were recruited to the HYPOCHOL study (A Genetically-Based Strategy to Identify New Targets in Cholesterol Metabolism; ClinicalTrials.com registration No. NCT02354079) of the CHOPIN program for functional studies. Written informed consent was obtained from all participants in accordance with the principles of the Declaration of Helsinki and the French bioethics law. GENELIP and HYPOCHOL protocols were approved by the University Hospital Center of Lyon (France) and Nantes (CHU Nantes, France), respectively.

The GENELIP study also obtained the agreement of the ethical committee of the Commission Nationale de ITnformatique et des Libertes (No. 920434).

Targeted Next-Generation Sequencing

After genomic DNA extraction, DNA from the proband and patient IV.2 was sequenced and analyzed as previously detailed with the DysliSEQ custom design.10 This panel includes coding exons and intron/exon junctions of 311 genes selected from published literature: (1) genes identified in monogenic dyslipidemia, (2) genes identified in genome-wide association studies in lipid metabolism through direct or indirect effects, (3) genes associated with dyslipidemia in mice, and (4) single-nucleotide polymorphisms already described in familial hypercholesterolemia genetic risk scores and in genome-wide association studies (with P>5.10- 8). Among these genes, a first intention panel was defined for FHBL (APOB, PCSK9, ANGPTL3), abetalipoproteinemia (ABL, OMIM 200100; MTTP), and chylomicron retention disease (CMRD, OMIM No. 246700; SAR1B). In the absence of a variant in the first intention panel, relevant variants were selected as previously describedl 0 (Table S 1.3). The only relevant variant common to the proband and his granddaughter and that cosegregated with the combined hypocholesterolemia observed in the family after Sanger sequencing was the E97G variant in the LIPC gene. In addition, the patients exhibited a combined hypolipidemia and a decrease of phospholipids concentration, as observed in mice after LIPC overexpression.¹¹

Mouse Studies

All mouse studies were performed with APOE*3. Leiden. CETP mice that have a humanized plasma lipid profile¹² and that were bred at Leiden University Medical Centre, Leiden, the Netherlands. All mice experiments were approved by the ethics committee of Pays de la Loire (France, 006) and the Ministere de 1’enseignement superieur de la recherche et de 1’innovation (France; APAFIS 26862) or the Central Committee on Animal Experimentation of the Netherlands (AVD 11600202010187) and Animal Welfare Body of the Leiden University Medical Center and conducted in accordance with institutional guidelines. At ~10 weeks of age, male mice were placed on a diet containing 0.5% cholesterol and 15% cocoa butter (Ssniff, No. S8854-E035 EF 4021-04T) for 3 to 5 weeks. Mice were injected intravenously with 3x1010 or 3x1011 genome copies of adeno-associated viruses (AAV8) under the thyroxin-binding globulin (TBG) promoter and containing enhanced green fluorescent protein (eGFP), wild-type lipase C, hepatic type (LIPC), or LIPC-E97G (Vector Biolabs). Mice were monitored weekly for changes in plasma cholesterol and triglyceride levels and body weight. Four weeks after injection, experiments to pheno typically characterize the mice were started as described in the Supplemental Material, each with at least a 1-week interval.

Statistical Analyses

For mice studies, a 1-way ANOVA with Tukey correction for multiple comparisons was used, with a 2-sided P value cutoff set at P<0.05. For cell culture studies, a nonparametric Mann- Whitney test was used with a 2-sided P value cutoff set at P<0.05.

Results

A Novel Variant in LIPC Cosegregates With Familial Combined Hypocholesterolemia A 61 -year-old patient was admitted to the lipid clinic of the University Hospital in Lyon (France) to explore a combined hypocholesterolemia inherited as a dominant phenotype over 4 generations. He was referred for low LDL-C before statin treatment while presenting with coronary artery disease requiring a first coronary stenting. He needed subsequent iterative coronary stentings at 62 and 73 years of age. In addition, he had evolving silent carotid artery plaques, which progressed from 7% (NASCET [North American Symptomatic Carotid Endarterectomy Trial]) with an increased intimamedia thickness (1.23 mm) when he was 61 years of age to 75% when he was 73 years of age. He was a former heavy smoker between 18 and 36 years of age and had well-controlled type 2 diabetes since 53 years of age. He had a body mass index of 25.3 kg/m2 and android adiposity. A phenotypic characterization of the patient (patient II.8, Figure 1) revealed extremely low values of circulating cholesterol, LDL- C, HDL-C, phospholipids, and apolipoprotein Al levels (below the fifth percentile for age and sex; Figure 1 and data not shown). In contrast, circulating triglycerides and apoB levels were within the low but normal range. Liver transaminases were mildly increased (aspartate aminotransferase, 54 UI/L; alanine aminotransferase, 54 UI/L when he was 71 years of age), but the fibrosis-4 indexes (a liver fibrosis score) were within the normal interval range (fibrosis- 4 index, 1.5 and 1.0 when he was 62 and 74 years of age, respectively), suggesting the absence of liver fibrosis. To clarify the origin of the hypocholesterolemic phenotype, family members of the patient were recruited for plasma and DNA collection. Plasma lipid analyses revealed that a combined hypocholesterolemia below the fifth percentile for age and sex was present in multiple family members and followed an autosomal dominant mode of inheritance (Figure 1 and data not shown). More detailed nuclear magnetic resonance lipoprotein profiling of 3 affected family members (III.9, IV.4, IV.5) revealed profound changes in lipoprotein composition and sizes compared with age- and sexmatched individuals (data not shown). Whereas VLDL particle numbers of affected family members were within the normal range, VLDL particle size was reduced compared with control subjects (data not shown). In contrast, LDL particle size was normal, but LDL particle numbers were low in affected family members, concordant with circulating apoB levels, which were within the lower range in these individuals (data not shown). Furthermore, we found an elevated number of large HDL particles and a near absence of small and medium HDL particles in affected family members, resulting in an elevated average HDL particle size (data not shown). These large HDL particles are relatively triglyceride enriched, as indicated by the low HDL-C in these individuals and the elevated HDL-triglycerides/HDL-C ratio (data not shown). In terms of ASCVD, only the proband (II.8) had ASCVD, with no knowledge of clinical ASCVD in the other family members. To elucidate the genetic origin of the hypocholesterolemia in the family, DNA from the proband was sequenced with the DysliSEQ custom next-generation sequencing panel design based on ontology of lipid disorders.10 No rare single nucleotide or copy number variation was found in coding exons of genes involved in FHBL (ANGPTL3, APOB, MTTP, PCSK9, SAR1B), and the polygenic risk score (0.91, 49th percentile) did not support a polygenic hypobetalipoproteinemia. Our rare variant filtering criteria (data not shown) enabled the detection of a rare missense variant, P.(Glu97Gly) or E97G, in the LIPC gene. The LIPC gene encodes for HL, a protein known to be involved in lipid metabolism. The LIPC-E97G variant is not reported in any large genetic data set (gnomAD and >300 000- individuals of the UK Biobank database) and cosegregated with the combined hypocholesterolemia in the family (Figure 1). To further confirm the absence of another candidate gene, we performed a wholegenome sequencing analysis of all recruited family members, leading to the identification of 3 variants that cosegregate with the hypocholesterolemic phenotype (data not shown). Of these variants, the LIPC-E97G variant was predicted to be the most conserved (Genomic Evolutionary Rate Profiling score, 4.6) and the most deleterious by 9 prediction algorithms (data not shown). This result confirmed that the combined hypocholesterolemia might be related to the LIPC-E97G variant.

LIPC-E97G Modifies the Structural Conformation of HL

HL is part of a family of glycerol-sn-1 -fatty acid hydrolases that includes lipoprotein lipase (LPL) and endothelial lipase (EL).¹³ HL has intermediate triglyceride lipase and phospholipase activities and hydrolyses triglycerides and phospholipids on circulating lipoproteins such as HDL, intermediate-density lipoprotein, and chylomicron remnants.¹⁴ Besides its lipolytic actions, HL also functions as a ligand to facilitate the uptake of lipoproteins by heparan sulfate proteoglycans or other cell surface receptors.¹⁵ To determine how the E97G variant might affect HL functionality, we performed an in silico analysis using homology modeling. A homology model for HL was created from a recently established crystal structure of LPL (6OB0,16 data not shown). Using this model, we observed that E97 is not part of the catalytic triad (SI 68, DI 94, and H279) but lies in relatively close proximity to the triad owing to protein folding (data not shown). Furthermore, we observed the existence of a salt bridge between E97 and K276, an amino acid present in the lid region of HL (residues 244-277; data not shown). This lid region has been shown to determine substrate access to the HL catalytic site.¹⁷ The conformation of the lateral chain of K276 was found to be similar to the corresponding lateral chain in LPL (Lys265, lid region from residues 233-266), indicating a conservation of this particular motif between the 2 lipases (data not shown). The importance of this structural motif is underlined by the observation that K265 is hydrogen bonded to the inhibitor present in the catalytic site of an experimental structure of LPL (data not shown).

We next virtually mutated E97G in the HL homology model and optimized the geometry/energy of the resulting structure. The E97G variant significantly changes the conformation of K276 (data not shown). Instead of with E97, K276 is now involved in a salt bridge with the carboxylate group of D280 [d(Hz3...OEl)=1.80 A] in the mutant model. These results show that E97 and K276 might be important for structural features of the catalytic site. By significantly affecting the conformation of K276, E97G alters the HL lid region and might consequently modify the substrate access to the HL catalytic site.

LIPC-E97G Alters HL Substrate Specificity

We next set out to determine the impact of the E97G variant on the HL triglyceride lipase and phospholipase activities. We overexpressed wild-type human LIPC (LIPC-WT), LIPC-E97G, or LIPC-S168G, a catalytically inactive mutant, in immortalized human hepatocytes and determined triglyceride lipase and phospholipase activities in the medium after treatment with heparin, which releases HL from cell surface heparan sulfate proteoglycan binding sites (data not shown). Triglyceride lipase and phospholipase activities of the released HL were identical between mock-treated and LIPC-WT-overexpressing cells when corrected for the amount of released HL (Figure 2A and 2B) but were nearly absent in the LIPC-S168G- o verexpressing cells. A striking finding was that cells overexpressing LIPC-E97G had a moderately reduced triglyceride lipase activity level but a 4-fold increased phospholipase activity level (P<0.01; Figure 2A and 2B). To investigate whether the heterozygous presence of E97G, as found in our family, similarly affects triglyceride lipase and phospholipase activities, we introduced the E97G variant into immortalized human hepatocytes using CRISPR-Cas9 and recorrected the variant to the wild-type allele (data not shown). After heparin treatment, the heterozygous presence of E97G did not affect triglyceride lipase activity in the medium compared with wild-type cells (Figure 2C and 2D). However, we observed a 7-fold increase in HL phospholipase activity (P<0.01) with heterozygous E97G presence (Figure 2Cand 2D). This effect was completely reversed when E97G was corrected to the wild-type allele (Figure 2C and 2D). These data suggest that the E97G variant specifically increases HL phospholipase activity without increasing triglyceride lipase activity. To confirm a change in HL substrate specificity in carriers of the E97G variant, we carried out detailed lipidomics on plasma of family members (III.9, IV.4, IV.5) and compared these data with data from normo Epidemic individuals (data not shown). HL has phospholipase Al activity and hydrolyzes phospholipids at the SN-1 position to form a fatty acid and a lysophospho lipid. In agreement with increased HL phospholipase activity in carriers of the E97G variant, plasma lipidomics data of the E97G carriers showed a clear reduction in overall fatty acids compared with control subjects, with a moderate reduction in fatty acid/apoB ratios (Figure 2E and data not shown). Polyunsaturated fatty acids were significantly more reduced than monounsaturated fatty acids, in agreement with the literaturel8 on in vitro HL substrate preference (Figure 2F). A more striking finding was that phospholipid concentrations were significantly reduced in plasma of E97G carriers, and a concomitant 3- to 5 -fold increase in lysophospholipids/phospholipids ratios was observed (Figure 2E and 2G). Furthermore, an increased ratio of lysophospholipids and a decreased ratio of phospholipids versus circulating apoB levels were found in E97G carriers compared with control subjects, suggesting significant changes in the composition of circulating apoB containing lipoprotein particles (data not shown). Together, these data suggest that the E97G variant alters substrate specificity by significantly increasing HL phospholipase activity levels.

Overexpression of the Human LIPC-E97G Variant Markedly Lowers LDL-C and HDL-C in APOE*3.Leiden.CETP Mice

To determine how the increased phospholipase activity of the E97G variant causes combined hypocholesterolemia, we overexpressed LIPC-WT, LIPC-E97G, and an eGFP control in hepatocytes of the humanized APOE*3. Leiden. CETP mouse model. To do so, we used 2 different doses of adeno-associated virus (AAV8; 3x1010 genome copies [low] and 3x1011 genome copies [high]; Figure 3A). On a Western diet, APOE*3. Leiden. CETP mice have a humanized lipoprotein profile, with cholesterol being carried principally on VLDL and LDL.12 To confirm the successful dose-dependent overexpression of human HL, we performed ELISA on preheparin and postheparin plasma and found similar levels of HL in mice overexpressing LIPC-WT and LIPC-E97G (Figure 3B). The large increase in circulating HL after heparin injection also confirms the localization of human HL on endothelial heparan sulfate proteoglycan (Figure 3B). Confirming our cell culture and lipidomics studies, the overexpression of E97G in mice strongly increased plasma phospholipase activity, but not triglyceride lipase activity levels, in postheparin plasma compared with eGFP or LIPC-WT overexpression (Figure 3C and 3D).

Overexpression of LIPC-WT had only a modest impact on plasma lipid levels (Figure 3E and data not shown). In contrast, LIPC-E97G overexpression did strongly affect plasma lipoprotein metabolism, with significant dose-dependent reductions in plasma cholesterol, HDL-C, non-HDL-C, phospholipid, and triglyceride levels (Figure 3E and data not shown). In line with the phenotype observed in familial carriers of the LIPC-E97G, cholesterol levels in VLDL, LDL, and HDL fractions were markedly lower in mice overexpressing LIPC-E97G compared with LIPC- and eGFP-overexpressing mice (Figure 3F). In contrast to family data, triglyceride levels in VLDL and LDL fractions were also markedly lower in mice overexpressing LIPCE97G (Figure 3F). Furthermore, a mild enrichment of triglycerides in HDL was observed in mice overexpressing LIPC-E97G, as was seen in our familial LIPC-E97G carriers (Figure 3F and data not shown). We confirmed the profound lowering of plasma lipid levels on overexpression of LIPC-E97G during different metabolic challenges, including postprandial lipemia and fasting/ refeeding experiments (data not shown). Overall, these data show that overexpression of LIPC-E97G in mice results in increased hepatic phospholipase activity and leads to a profound reduction in circulating plasma lipid levels.

Functional Consequences of the Overexpression of the LIPC-E97G Variant

We first hypothesized that the reduced LDL-C and HDLC levels associated with the LIPC-E97G variant were caused by an increased hepatic cholesterol clearance such as an increased hepatic cholesteryl ester uptake or increased hepatic receptor-mediated endocytosis of circulating lipoproteins as a result of hydrolysis of phospholipids on the lipoprotein surface.^19,20 A moderate reduction in the hepatic expression of 3-hydroxy- 3-methylglutaryl- CoA reductase Hmgcr, the rate-limiting enzyme in cholesterol synthesis (data not shown), was found. However, no significant enrichment of either hepatic triglycerides or free or esterified cholesterol was observed in the livers of mice overexpressing the E97G variant (data not shown). Consistently, after an intravenous injection of D7-labeled cholesterol 3 days before death, we found no increase in hepatic D7-cholesterol and fecal D7-cholesterol excretion in LIPC-E97G-high- overexpressing mice (Figure 3G and 3H). Given that D7-cholesterol labels were cleared more rapidly from the blood of LIPC-E97G-high-o verexpressing mice, this suggests that hepatic catabolism and reverse cholesterol transport are unlikely to be the main cause of the reduced plasma cholesterol levels (Figure 31). In addition, no impact of the overexpression of LIPC-E97G on VLDL secretion rates compared with overexpression of LIPCWT or eGFP was found (data not shown).

To more directly investigate the impact of LIPC-E97G on lipoprotein kinetics, we overexpressed high doses of eGFP, LIPC-WT, or LIPC-E97G in APOE*3.Leiden.CETP mice and injected these mice with VLDL-like particles or murine VLDL particles labeled with hydrolysable glycerol tri[3H]oleate ([3H]triolein) and [14C] cholesteryl oleate (CO), which is not hydrolysable by HL and is a tracer of triglyceride-rich lipoprotein (TGRL) core remnant particle uptake. Plasma decay of radiolabeled VLDL-like particles was followed, and mice were killed 10 minutes (VLDLlike particles) or 15 minutes (murine VLDL) after injection to determine tissue distribution of the radiolabels (Figure 4A and 4B). No major impact of LIPCWT overexpression was found on [3H]triolein and [14C]CO decay, whereas [3H]triolein and [14C]CO plasma levels were cleared moderately faster in LIPC-E97G- compared with LIPC- WT-o verexpressing mice when injected with VLDL-like particles but not when injected with murine VLDL (Figure 4A-4D). More remarkable differences were observed with a detailed analysis of tissue distributions. These data indicated that [3H]triolein uptake was significantly and [14C]CO uptake was moderately reduced in the liver after clearance of both types of particles (Figure 4E and 4F). Instead, [3H]triolein and [14C] CO labels were increasingly taken up by different adipose tissue depots and oxidative tissues (Figure 4G-4I). These data suggest that the LIPC-E97G variant is responsible for the combined hypocholesterolemic phenotype observed in our family by promoting the uptake of cholesterol-containing remnants by extrahepatic tissues. In support, a nonsignificant increase in cholesterol levels was observed in the muscle and gonadal white adipose tissue of mice overexpressing LIPC-E97G data not shown). It should be noted that these effects are likely not mediated through an apoB-mediated pathway because no significant differences in the tissue clearance profile were observed between the clearance of VLDL-like particles (without apoB) and endogenous murine VLDL (with apoB).

EXAMPLE 2:

The E97G LIPC variant was initially identified in a proband who was admitted to the lipid clinic of the University Hospital in Lyon to investigate the origin of a combined hypocholesterolemial. Despite very low plasma cholesterol levels, the proband shows coronary stenosis and evolutive carotid atherosclerosis 1 raising the question about the putative pro- atherogenic impact of the E97G LIPC variant. To address this critical question for the clinical relevance of E97G LIPC manipulation in human, we performed a pioneer study by overexpressing control eGFP, human WT and E97G LIPC in full Ldlr-/- females fed with a proatherogenic diet for 9 weeks, using AAV8 viruses as described before¹. Our results (Figure 5) revealed that the E97G LIPC expression 1) strongly reduces plasma cholesterol levels in Ldlr-/- females; 2) potently decreases the size of aortic atherosclerotic lesions. These preliminary data fuel the debate about the pro- or anti-atherogenic role of the E97G LIPC variant and reinforce the need for further studies. Interestingly, it also opens the prospect of new therapeutic solutions in FH patients with part i a 1/total LDLR deficiency.

REFERENCES: Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure.

Claims

CLAIMS:

1. A method for treating hypercholesterolemia and atherosclerotic cardiovascular disease (ASCVD) in a subject in need thereof comprising administering a therapeutically effective amount of a LIPC variant to said subject in need thereof.

2. The method according to claim 1, wherein, the LIPC variant is LIPC-E97G.

3. The method according to claim 1, wherein, the LIPC variant is obtained by genome editing.

4. The method according to claim 1, wherein, the atherosclerotic cardiovascular disease is selected from the groupe consisting of but not limited to: dyslipidemia and atherosclerotic cardiovascular disease (ASCVD), low density lipoprotein (LDL)-driven ASCVD, triglyceride-driven ASCVD, remnants-driven ASCVD lipoprotein a Lp(a)- driven ASCVD, chronic inflammatory disease-driven ASCVD, inflammatory ASCVD, hypercholesterolemia, familial hypercholesterolemia including homozygous familial hypercholesterolemia, chronic kidney disease, diabetes mellitus, blood pressure or human immunodeficiency virus infection.

5. The method according to claim 1, wherein the hypercholesterolemia is Familial hypercholesterolemia (homozygous or heterozygous FH).

6. A nucleic acid encoding human LIPC protein comprising a sequence having at least 70% sequence identity with the sequence SEQ ID NO 1.

7. A recombinant vector comprising the nucleic acid according to claim 6.

8. A vector according to claim 7 characterized in that it is an adenovirus, a plasmid, a YAC (Yeast Artificial Chromosomes) or a BAC (Bacterial Artificial Chromosome).

9. The vector according to claims 7 to 8, wherein the vector is an AAV vector.

10. The vector according to claim 9, wherein the vector is AAV8.

I L A host cell comprising a nucleic acid according to claim 6 and/or a vector according to claim 9.

12. A pharmaceutical composition comprising a LIPC variant.

13. The pharmaceutical composition according to claim 12 comprising a vector comprising the nucleic acid (such as DNA, RNA, mRNA) or the amino acid related to LIPC variant (as a protein).

14. The pharmaceutical composition according to claims 12 to 13 for use in the treatment of hypercholesterolemia and atherosclerotic cardiovascular disease in a subject in need thereof.