WO2010134806A1 - Use of lipoprotein lipase (lpl) in therapy - Google Patents

Abstract

The present invention relates to a lipoprotein lipase (LPL) therapeutic for use in: reducing the number and/or size of chylomicrons in a subject; redistributing triglycerides in chylomicrons to very low-density lipoprotein (VLDL) in a subject; or increasing clearance of chylomicron remnants in a subject. Further, the invention relates to An LPL therapeutic suitable for use in the invention may be selected from the group consisting of: a) an LPL protein with an amino acid sequence as shown in SEQ ID NO: 3 or SEQ ID NO: 4, or a derivative of either thereof; b) an LPL protein with an amino acid sequence which comprises a contiguous segment having at least 90% sequence identity to SEQ ID NO: 3 or 4 when optimally aligned which has equal or greater LPL activity than a protein under a), or a derivative thereof; or c) a nucleic acid encoding a) or b), or a derivative thereof.

Description

USE OF LIPOPROTEIN LIPASE (LPL) IN THERAPY

Field of the invention

The present invention relates to compositions for use in reducing the number and/or size of chylomicrons or chylomicron remnants in a subject, redistributing triglycerides in chylomicrons to very low-density lipoprotein (VLDL) and/or to VLDL- containing lipid/liprotein fractions in a subject or increasing clearance of chylomicron remnants in a subject. The invention also relates to a method for the use of the compositions in such applications. The compositions may be used in the manufacture of a medicament for use in such applications.

Background to the invention

Dietary fats (from food) are absorbed through the gut where they are assembled into chylomicrons, microscopic, minute fat particles formed during fat digestion and assimilation that directly enter the lymphatic system. Intestinal triglycerides, or chylomicrons, containing approximately 85 percent triglycerides, are then delivered through the bloodstream mainly to the liver, where they are processed (the normal half- life of chylomicrons is about 10 minutes).

Chylomicrons may aberrantly accumulate in patients with one of a number of conditions. There is a need for a general treatment for conditions characterized by excess levels of chylomicrons and/or triglycerides.

Summary of the invention

It is demonstrated herein that patients suffering from elevated levels of chylomicrons may effectively be treated with an adeno-associated virus based gene therapy vector which expresses a truncated mutant of the lipoprotein lipase (LPL) protein. In these patients, increases in VLDL was observed. That is to say, there is an apparent shift from chylomicrons to VLDL over time. Triglycerides which are present in the chylomicron fraction appear to be shifted, i.e. redistributed, into the VLDL fraction (and/or to VLDL-containing lipid/lipoprotein fraction). As a consequence, the level of chylomicrons in the patients may be reduced such that symptoms associated with elevated levels of chylomicrons are ameliorated.

Accordingly, the invention relates to the use of an LPL therapeutic for use in resolving chylomicrons. That is to say, an LPL therapeutic may be used to reduce the amount of, for example number of and/or size of, chylomicrons in a subject. The size, for example total volume, of the chylomicron fraction (or chylomicron remnant fraction) may be reduced. Thus, the term "size" in relation to the chylomicron fraction may refer to the total volume of that fraction or to the average size (such as diameter) of chylomicrons in a subject.

A reduction of the number and/or size of chylomicrons refers to the fact that that the number and/or size are reduced after administration of an LPL therapeutic as compared to the number and/or size prior to administration.

In particular, an LPL therapeutic may be used in the invention such that chylomicrons are resolved to VLDLs, such as trigylcerides in chylomicrons are resolved to very low-density lipoprotein (VLDL). That is to say, the use of an LPL therapeutic may result in an apparent shift in lipid/lipoprotein profiles with an apparent shift from chylomicrons to VLDL, in particular triglycerides present in chylomicrons may shift to the VLDL fraction. This may result in a reduction in the amount, for example in terms of the number and/or volume, of chylomicrons in a subject.

According to the invention, there is thus provided a lipoprotein lipase (LPL) therapeutic for use in reducing the number and/or size, for example volume, of chylomicrons in a subject.

The invention also provides an LPL therapeutic for use in redistributing triglycerides in chylomicrons to very low-density lipoprotein (VLDL) or to a VLDL- containing lipid/lipoprotein fraction in a subject.

The invention also concerns an LPL therapeutic of the invention may be used in increasing clearance of chylomicron remnants in a subject.

An LPL therapeutic of the invention may be used in the treatment of a condition which is characterized by: (i) an elevated number and/or size of chylomicrons and/or chylomicron remnants; and/or (ii) reduced or inadequate processing of chylomicrons and/or chylomicron remnants. An LPL therapeutic of the invention may be used in the treatment of a condition characterized or accompanied by elevated triglyceride levels.

An LPL therapeutic of the invention may be used in the treatment of a subject having reduced levels of lipoprotein lipase (LPL) and/or a relative LPL deficiency. An LPL therapeutic according to any one of the preceding claims, wherein the

LPL therapeutic may be a member selected from the group consisting of: a) an LPL protein with an amino acid sequence as shown in SEQ ID NO: 3 or 4, or a derivative of either thereof; b) an LPL protein with an amino acid sequence which comprises a contiguous segment having at least 90% sequence identity to SEQ ID NO: 3 or 4 when optimally aligned which has equal or greater LPL activity than a protein under a), or a derivative thereof; or c) a nucleic acid encoding a) or b), or a derivative thereof.

An LPL therapeutic of the invention may comprise a nucleic acid which comprises a DNA coding sequence encoding an RNA having at least 90% sequence identity to nucleotides 452 through 1795 of SEQ ID NO: 1 or nucleotides 452 through 1789 of SEQ ID NO: 1.

An LPL therapeutic of the invention may be a nucleic acid which comprises a DNA coding sequence that hybridizes under stringent conditions to the reverse complement of nucleotides 452 through 1795 of SEQ ID NO: 1 or nucleotides 452 through 1789 of SEQ ID NO: 1.

An LPL therapeutic of the invention may be an LPL protein with a contiguous segment of at least 95% sequence identity to SEQ ID NO: 3 or 4.

An LPL therapeutic of the invention may be administered to the subject in a gene therapy vector. The gene therapy vector may be a viral vector, such as an adeno- associated virus (AAV).

An LPL therapeutic of the invention may be administered parenterally. A pharmaceutical composition comprising an LPL therapeutic as defined herein and a pharmaceutically acceptable carrier is also provided by the invention. The invention also provides: a method for use in reducing the number and/or size of chylomicrons or chylomicron remnants in a subject, which method comprises administering to a subject in need thereof an effective amount of an LPL therapeutic; a method for use in redistributing triglycerides in chylomicrons to very low- density lipoprotein (VLDL) in a subject, which method comprises administering to a subject in need thereof an effective amount of an LPL therapeutic; a method for use in increasing clearance of chylomicron remnants in a subject, which method comprises administering to a subject in need thereof an effective amount of an LPL therapeutic; use of an LPL therapeutic in the manufacture of a medicament for use in reducing the number and/or size of chylomicrons and/or chylomicron remnants in a subject; - use of an LPL therapeutic in the manufacture of a medicament for use in redistributing triglycerides in chylomicrons to very low-density lipoprotein (VLDL) in a subject; and use of an LPL therapeutic in the manufacture of a medicament for use in increasing clearance of chylomicron remnants in a subject.

Any LPL therapeutic as described herein may be used in such a method or use.

Brief description of the figures

Fig. 1 shows results for four patients (A, B, C and E) one year post-treatment with AAV1-LPL^S447X. The light coloured bars show the VLDL-triglycerides and the dark coloured bars show the chylomicron-triglycerides.

Fig. 2 shows distribution of triglycerides and cholesterol over the Sf >400 (chylomicrons) and Sf 20-400 (VLDL) fractions in plasma from LPLD patients, before and after treatment with AAV1-LPL^S447X. Fasting plasma was collected from LPLD patients before (week -3) and after (week 52) IM administration of AAV1-LPL^S447X. Plasma samples were further fractionated by ultracentrifugation, and Sf >400 (corresponding to CM) and Sf 20-400 fractions (corresponding to VLDL) were collected. A decrease in triglyceride and cholesterol content of the Sf >400 fraction was observed, and a concomitant increase of triglycerides and cholesterol in the Sf 20-400 fraction, after treatment. Individual results are shown, as well as the mean ± SEM.

Fig. 3 shows plasma apoB levels in LPLD patients, before and after treatment with AAV1-LPL^S447X. Fasting plasma was collected from LPLD patients before (week -3) and after (week 12 and week 52) IM administration of AAV1-LPL^S447X. Plasma samples were further fractionated by ultracentrifugation, and total apoB levels were determined in the Sf 20-400 fraction (ie containing particles of a density corresponding to VLDL) by nephelometry. An increase in apoB was observed at week 12 and 52, in particular in the high dose cohort (IxIO¹² gc/kg), indicating an increased number of particles in the Sf 20-400 fraction. Individual results are shown, as well as the mean ± SEM.

Brief description of the sequence listing

SEQ ID NO: 1 sets out the mRNA sequence of the human LPL (NCBI GenBank accession number: NM 000237), in which a signal peptide is encoded by nucleotides 371 through 451, and the mature peptide is encoded by nucleotides 452 through 1795 (SEQ ID NO: 2). See also Wion, et al., Science 235 (4796), 1638-1641 (1987); Sparkes et al, Genomics 1 (2), 138-144 (1987); Mattei et al., Cytogenet. Cell Genet. 63 (1), 4546 (1993); Zechner, Curr. Opin. Lipidol. 8 (2), 77-88 (1997); Fisher et al., Atherosclerosis 135 (2), 145-159 (1997); and Beisiegel, Eur. Heart J. 19, A20-A23 (1998). SEQ ID NO: 2 sets out the amino acid sequence of a pre-LPL peptide (NCBI

GenBank accession number: NP 000228), showing a protein having a signal peptide at amino acids 1 through 27, prior to the mature LPL peptide sequence (SEQ ID NO: 3). SEQ ID NO: 3 sets out the amino acid sequence of the mature wild type LDL polypeptide showing amino acids designated 1 through 448 herein. SEQ ID NO: 4 sets out the amino acid sequence of the mature LDL^S447X polypeptide, showing amino acids designated 1 through 446 herein (SEQ ID NO: 1). See also Wion, et al., Science 235 (4796), 1638-1641 (1987); Sparkes et al., Genomics 1 (2), 138-144 (1987); Mattei et al., Cytogenet. Cell Genet. 63 (1), 45-46 (1993); Zechner, Curr. Opin. Lipidol. 8 (2), 77-88 (1997); Fisher et al., Atherosclerosis 135 (2), 145-159 (1997); and Beisiegel, Eur. Heart J. 19, A20 A23 (1998); Groenemeijer et al., Circulation 1997,95: 2628-2635; Gagne et al., Clin. Genet. 1999,55 (6): 450-454). Detailed description of the invention

The present invention relates to a method of treating conditions characterized by elevated levels of chylomicrons and/or triglycerides. It is demonstrated herein that patients suffering from elevated levels of chylomicrons may effectively be treated with an adeno-associated virus based gene therapy vector which expresses a truncated mutant of the lipoprotein lipase (LPL) protein. In these patients, there is a modification in lipid/lipoprotein profiles with an apparent shift from chylomicrons to VLDL. Metabolism in these patients is such that the size of the amount and/or concentration of triglycerides in the chylomicron fraction is reduced. This is accompanied by a rise in the amount and/or concentration of triglycerides in other lipoprotein fractions, in particular the VLDL fraction (and/or a VLDL-containing lipid/lipoprotein fraction). That is to say, there is a shift in triglyceride amount and/or concentration from the chylomicron fraction to the VLDL fraction (and/or to VLDL-containing lipid/lipoprotein fractions). This is what is meant by a redistribution of triglycerides from chylomicrons to other lipoprotein fractions such as VLDL in particular.

The Sf >400 fraction (see Figs. 2 and 3) contains the (large) CM, and the Sf 20- 400 fraction is likely a mix of smaller CM or CM remnants, and VLDL. What is thus seen is a shift from most of the TG and cholesterol in a very buoyant fraction (large CM) to a less buoyant fraction, ie a change in size and/or composition of the CM particle. This less buoyant fraction corresponds to what is normally termed 'VLDL'.

Without wishing to be bound by any particular theory, it is believed that elevating LPL levels (typically in patients with relative LPL deficiency) results in a breakdown of chylomicrons, for example by decreasing the size and/or amount of chylomicrons, i.e. more efficiently processing chylomicrons. This results in the increased production of chylomicron remnants which may then be transported to other tissues, such as the liver, and/or other changes in lipid/lipoproteins and/or carbohydrate metabolism. These remnants may then stimulate the liver, for example, to de novo produce triglycerides and then export them, for example in the VLDL fraction. Thus, excess carbohydrates or lipids/lipoproteins in the liver, for example, may be redistributed as triglycerides into VLDL and exported from the liver. According to the invention, there is thus provided a lipoprotein lipase (LPL) therapeutic for use in reducing the number and/or size, for example volume, of chylomicrons in a subject.

The reduction in such number and/or size of chylomicrons may be at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90% or more when the pre-treatment number and/or size is compared with the post-treatment (with an LPL therapeutic) number and/or size.

Accordingly, LPL therapeutics provide a general therapy for conditions characterized by elevated levels of chylomicrons and/or triglycerides. Thus, an LPL therapeutic may be administered to a subject with elevated levels or chylomicrons and/or triglycerides. Elevated in this context refers to an increase in chylomicrons and/or triglycerides in comparison with a normal subject who exhibits no chylomicron/triglyceride related symptoms. Such a subject may be homozygous for the wild type LPL gene.

Extremely elevated levels of triglycerides encompass subjects demonstrating concentrations of triglycerides of at least about lOmmol/1 TG. However, it will also be possible to treat according to the invention, subjects demonstrating a level of triglycerides of at least about 4.5mmol/l TG or those demonstrating a level of trigylcerides of at least about 2.3mmol/l TG. These concentrations are set out as blood or plasma concentrations.

A may be homozygous for the wild type lipoprotein lipase (LPL) gene. A subject suitable for treatment according to the invention may have an LPL deficiency, such as a relative LPL deficiency. That is to say, a subject where the ratio of LPL activity to triglyceride concentration is reduced as compared to a subject having a homozygous wild type LPL genotype. Accordingly, a subject suitable for treatment according to the invention may be homozygous for a mutation in the LPL gene or may be heterozygous for a mutation in the LPL gene. Any decrease in the level of LPL (as compared to a wild type homozygote) may be treatable according to the invention. The method comprises administering to a subject an effective amount of a lipoprotein lipase (LPL) therapeutic. As used herein, LPL therapeutic encompasses any substance capable of providing LPL activity, such as a protein having LPL activity or a nucleic acid encoding such a protein. For the purposes of this invention LPL activity may be defined with reference to EC 3.1.1.34, for example the ability to catalyze a reaction of the form triacylglycerol + H₂O = diacylglycerol + a carboxylate.

Thus, an LPL therapeutic suitable for use in the invention may have the amino acid sequence as set out in: NCBI Accession Number NM_000228 (SEQ ID NO: 3 herein and Fig. 2/SEQ ID NO: 3 of WOO 1/00220).

In one embodiment, the LPL therapeutic may be an LPL protein with an amino acid sequence as shown in SEQ ID NO: 4, herein referred to as LPL^S447X proteins or peptides (see also Fig. 1/SEQ ID NO: 1 of WO01/00220). In general, these LPL^S447X proteins are shorter than well-known wild type LPL, which has 448 amino acids. An LPL therapeutic according to the invention may comprise a compound such as a peptide fragment, a modified peptide fragment, an analogue or a pharmacologically acceptable salt of LPL (full-length wild type mature protein) or LPL having amino acids 447-448 truncated from the carboxy terminal of a wild-type LPL. Such compounds are collectively referred to herein as LPL peptides, proteins or polypeptides, although LPL^S447X peptides (or polypeptide or proteins) are also referred to herein. LPL and LPL^S447X peptides may include homo logs of the wild-type mature LPL sequence or from amino acids 1 through 446 in the case of LPL^S447X peptides, including homo logs from species other than Homo sapiens (which may have veterinary applications). LPL and LPL^S447X peptides may include derivatives and naturally occurring iso forms or genetic variants of wild type LPL. The use of derivatives and variants of the LPL and LPL^S447X proteins is also encompassed within the invention.

Derivatives of LPL and LPL^S447X polypeptides include, in particular, polypeptides with LPL activity which have the same amino acid sequence as the LPL or LPL^S447X polypeptides, but in which some N- or O-glycosylation sites have been modified or eliminated. Derivatives also include C-terminal hydroxymethyl derivatives, O-modified derivatives (e. g., C-terminal hydroxymethyl benzyl ether), and N-terminally modified derivatives including substituted amides such as alkylamides and hydrazides.

Within an LPL therapeutic of the invention, a peptidic structure maybe coupled directly or indirectly to a modifying group. The term "modifying group" is intended to include structures that are directly attached to the peptidic structure (e. g., by covalent coupling), as well as those that are indirectly attached to the peptidic structure (e. g., by a stable non-covalent association or by covalent coupling to additional amino acid residues, or mimetics, analogues or derivatives thereof, which may flank the MCP-3 core peptidic structure). For example, the modifying group can be coupled to the amino -terminus or carboxy-terminus of an LPL therapeutic structure, or to a peptidic or peptidomimetic region flanking the core domain. Alternatively, the modifying group can be coupled to a side chain of an amino acid residue of the LPL therapeutic, or to a peptidic or peptido-mimetic region flanking the core domain (e. g., through the epsilon amino group of a lysyl residue (s), the carboxyl group of an aspartic acid residue (s) a glutamic acid residue (s), a hydroxy group of a tyrosyl residue (s), serine residue (s) a threonine residue (s) other suitable reactive group on an amino acid side chain). Modifying groups covalently coupled to the peptidic structure can be attached by means and using methods well known in the art for linking chemical structures, including, for example, amide, alkylamino, carbamate or urea bonds.

In some embodiments, the modifying group may comprise a cyclic, heterocyclic or polycyclic group. The term "cyclic group", as used herein, includes cyclic saturated or unsaturated (i. e., aromatic) group having from 3 to 10, 4 to 8, or 5 to 7 carbon atoms.

Exemplary cyclic groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cyclooctyl. Cyclic groups may be unsubstituted or substituted at one or more ring positions. A cyclic group may for example be substituted with halogens, alkyls, cycloalkyls, alkenyls, alkynyls, aryls, heterocycles, hydroxyls, aminos, nitros, thiols amines, imines, amides, phosphonates, phosphines, carbonyls, carboxyls, silyls, ethers, thioethers, sulfonyls, sulfonates, selenoethers, ketones, aldehydes, esters,-CF3, - CN.

The term "heterocyclic group" includes cyclic saturated, unsaturated and aromatic groups having from 3 to 10, 4 to 8, or 5 to 7 carbon atoms, wherein the ring structure includes about one or more heteroatoms. Heterocyclic groups include pyrrolidine, oxolane, thiolane, imidazole, oxazole, piperidine, piperazine, morpholine. The heterocyclic ring may be substituted at one or more positions with such substituents as, for example, halogens, alkyls, cycloalkyls, alkenyls, alkynyls, aryls, other heterocycles, hydroxyl, amino, nitro, thiol, amines, imines, amides, phosphonates, phosphines, carbonyls, carboxyls, silyls, ethers, thioethers, sulfonyls, selenoethers, ketones, aldehydes, esters,-CF3, -CN. Heterocycles may also be bridged or fused to other cyclic groups as described below. The term "polycyclic group" as used herein is intended to refer to two or more saturated, unsaturated or aromatic cyclic rings in which two or more carbons are common to two adjoining rings, so that the rings are "fused rings". Rings that are joined through non- adjacent atoms are termed "bridged" rings. Each of the rings of the polycyclic group may be substituted with such substituents as described above, as for example, halogens, alkyls, cycloalkyls, alkenyls, alkynyls, hydroxyl, amino, nitro, thiol, amines, imines, amides, phosphonates, phosphines, carbonyls, carboxyls, silyls, ethers, thioethers, sulfonyls, selenoethers, ketones, aldehydes, esters,-CF3, or-CN.

The term "alkyl" refers to the radical of saturated aliphatic groups, including straight chain alkyl groups, branched-chain alkyl groups, cycloalkyl (alicyclic) groups, alkyl substituted cycloalkyl groups, and cycloalkyl substituted alkyl groups. In some embodiments, a straight chain or branched chain alkyl has 20 or fewer carbon atoms in its backbone (C₁-C₂₀ for straight chain, C3-C20 for branched chain), or 10 or fewer carbon atoms . In some embodiments, cycloalkyls may have from 4-10 carbon atoms in their ring structure, such as 5, 6 or 7 carbon rings. Unless the number of carbons is otherwise specified, "lower alkyl" as used herein means an alkyl group, as defined above, having from one to ten carbon atoms in its backbone structure. Likewise, "lower alkenyl" and "lower alkynyl" have chain lengths often or less carbons.

The term "alkyl" (or "lower alkyl") as used throughout the specification and claims is intended to include both "unsubstituted alkyls" and "substituted alkyls", the latter of which refers to alkyl moieties having substituents replacing a hydrogen on one or more carbons of the hydrocarbon backbone. Such substituents can include, for example, halogen, hydroxyl, carbonyl (such as carboxyl, ketones (including alkylcarbonyl and arylcarbonyl groups), and esters (including alkyloxycarbonyl and aryloxycarbonyl groups) ), thiocarbonyl, acyloxy, alkoxyl, phosphoryl, phosphonate, phosphinate, amino, acylamino, amido, amidine, imino, cyano, nitro, azido, sulfhydryl, alkylthio, sulfate, sulfonate, sulfamoyl, sulfonamido, heterocyclyl, aralkyl, or an aromatic or hetero aromatic moiety. The moieties substituted on the hydrocarbon chain can themselves be substituted, if appropriate. For instance, the substituents of a substituted alkyl may include substituted and unsubstituted forms of aminos, azidos, iminos, amidos, phosphoryls (including phosphonates and phosphinates), sulfonyls (including sulfates, sulfonamidos, sulfamoyls and sulfonates), and silyl groups, as well as ethers, alkylthio s, carbonyls (including ketones, aldehydes, carboxylates, and esters), -CF₃, - CN and the like. Exemplary substituted alkyls are described below. Cycloalkyls can be further substituted with alkyls, alkenyls, alkoxys, alkylthios, aminoalkyls, carbonyl-substituted alkyls,-CF3, -CN, and the like.

The terms "alkenyl" and "alkynyl" refer to unsaturated aliphatic groups analogous in length and possible substitution to the alkyls described above, but that contain at least one double or triple bond respectively.

The term "aralkyl", as used herein, refers to an alkyl or alkylenyl group substituted with at least one aryl group. Exemplary aralkyls include benzyl (i. e., phenylmethyl), 2- naphthylethyl, 2- (2-pyridyl)propyl, and the like. The term "alkylcarbonyl", as used herein, refers to-C(O)-alkyl. Similarly, the term "arylcarbonyl" refers to-C(O)-aryl. The term "alkyloxycarbonyl", as used herein, refers to the group-C(O)-O-alkyl, and the term "aryloxycarbonyl" refers to-C(O)-O- aryl. The term "acyloxy" refers to -0-C(O)-R₇, in which R₇ is alkyl, alkenyl, alkynyl, aryl, aralkyl or heterocyclyl. The term "amino", as used herein, refers to-N(Rα)(Rp), in which R_α and Rp; are each independently hydrogen, alkyl, alkyenyl, alkynyl, aralkyl, aryl, or in which R₀- and Rp; together with the nitrogen atom to which they are attached form a ring having 4-8 atoms. Thus, the term "amino", as used herein, includes unsubstituted, monosubstituted (e. g., mono alky lamino or monoarylamino), and disubstituted (e. g., dialkylamino or alkylarylamino) amino groups.

The term "amido" refers to -C(O)-N(Rs)(Rg), in which Rs and R9 are as defined above. The term "acylamino" refers to-N(R's)C(O)-R₇, in which R₇ is as defined above and R^T8 is alkyl.

As used herein, the term "nitro" means -NO₂ ; the term "halogen" designates-F, - Cl, - Br or-I; the term "sulfhydryl" means-SH; and the term "hydroxyl" means-OH.

The term "aryl" as used herein includes 5-, 6- and 7-membered aromatic groups that may include from zero to four heteroatoms in the ring, for example, phenyl, pyrrolyl, furyl, thiophenyl, imidazolyl, oxazole, thiazolyl, triazolyl, pyrazolyl, pyridyl, pyrazinyl, pyridazinyl and pyrimidinyl, and the like. Those aryl groups having heteroatoms in the ring structure may also be referred to as "aryl heterocycles" or

"heteroaromatics". The aromatic ring can be substituted at one or more ring positions with such substituents as described above, as for example, halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, amino, nitro, sulfhydryl, imino, amido, phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl, sulfonamido, ketone, aldehyde, ester, a heterocyclyl, an aromatic or hetero aromatic moiety,-CF₃, -CN, or the like. Aryl groups can also be part of a polycyclic group. For example, aryl groups include fused aromatic moieties such as naphthyl, anthracenyl, quinolyl, indolyl, and the like.

Modifying groups may include groups comprising biotinyl structures, fluorescein- containing groups, a diethylene-triaminepentaacetyl group, a (-) - menthoxyacetyl group, a N- acetylneuraminyl group, a cholyl structure or an iminiobiotinyl group. An LPL therapeutic may be modified at its carboxy terminus with a cholyl group according to methods known in the art (for example see: Wess, G. et al. (1993) Tetrahedron Letters, 34: 817-822; cholyl derivatives and analogues may also be used as modifying groups, such as Aic (3-(O- aminoethyl-iso) -cholyl), which has a free amino group that can be used to further modify the LPL therapeutic. A modifying group may be a "biotinyl structure", which includes biotinyl groups and analogues and derivatives thereof (such as a 2-iminobiotinyl group). In another embodiment, the modifying group may comprise a "fluorescein-containing group", such as a group derived from reacting an LPL therapeutic peptide with 5 -(and 6-)-carboxyfluorescein, succinimidyl ester or fluorescein isothiocyanate. In various other embodiments, the modifying group (s) may comprise an N-acetylneuraminyl group, a trans-4- cotininecarboxyl group, a 2- imino-1-imidazolidineacetyl group, an (S) -(-)-indoline-2- carboxyl group, a (-)- menthoxyacetyl group, a 2-norbomaneacetyl group, a gamma- oxo-5-acenaphthenebutyryl, a (-) -2-oxo-4-thiazolidinecarboxyl group, a tetrahydro-3- furoyl group, a 2-iminobiotinyl group, a diethylenetriaminepentaacetyl group, a 4- morpholinecarbonyl group, a 2-thiopheneacetyl group or a 2-thiophenesulfonyl group. Variants and derivatives of LPL and LPL^S447X polypeptides also include polypeptides having substantial sequence similarity to the LPL (SEQ ID NO: 2) polypeptide or to the LPL^S447X (SEQ ID NO: 3) polypeptide, such as at least about 90%, at least about 95%, at least about 98% or at least about 99% sequence identity to a corresponding portion of those sequences, the corresponding portion being any contiguous sequence of any length, such as at least about 10, at least about 20, at least about 30, at least about 40 or at least about 50 or more amino acids. Such variant polypeptides typically have LPL activity, or another LPL-like property, preferably equal to or greater than an LPL or LPL^S447X polypeptide. In some embodiments, one or more chemically similar amino acids may be substituted for amino acids in the LPL or LPL^S447X polypeptide sequence (so as to provide conservative amino acid substitutions). Amino acid substitutions that reduce LPL activity, of which more than 50 have been disclosed, such as the substitution of a Ser residue for Asn at position 291 (Asn291 Ser), the substitution of Asn for Asp at position 9 (Asp9Asn), the substitution of GIu for GIy at position 188 (Glyl88GIu, see Monsalve et al,. J Clin. Invest. 1990, 86(3):728-734) or Asp250Asn (Ma et al. Genomics. 1992, 13:649-653) should typically be avoided in preferred embodiments.

In yet another embodiment, an LPL therapeutic suitable for use in the invention is a nucleic acid encoding a wild type LPL polypeptide or encoding an LPL^S447X polypeptide or a derivative of such a polypeptide as described above. In an alternative embodiment, the nucleic acid may comprise a DNA coding sequence encoding an RNA having at least about 90% sequence identity to nucleotides 452 through 1795 of SEQ ID NO: 1, which stretch of nucleotides encodes the mature wild type LPL peptide or to nucleotides 452 to 1789 of SEQ ID NO: 1, which stretch of nucleotides encodes the _LpLs447x _polypeptide

Yet alternatively, an LPL therapeutic suitable for use in the invention may comprise a nucleic acid which comprises a DNA coding sequence that hybridizes, for example under stringent conditions, to the reverse complement of nucleotides 452 through 1795 of SEQ ID NO : 1.

Herein, two nucleic acid or protein sequences may be considered substantially identical (or "essentially similar") if, when optimally aligned, they share at least about 70% sequence identity. In alternative embodiments, substantial sequence identity may imply sequence identity of, for example, at least 75%, at least 90% or at least 95%, at least 98% or at least 99%.

Optimal alignment of sequences for comparisons of identity may be conducted using a variety of algorithms, such as the local homology algorithm of Smith and Waterman, 1981, Adv. Appl. Math 2: 482, the homology alignment algorithm of Needleman and Wunsch, 1970, J MoI. Biol. 48: 443, the search for similarity method of Pearson and Lipman, 1988, Proc. Natl. Acad. Sci. USA 85: 2444, and the computerised implementations of these algorithms (such as GAP, BESTFIT, FASTA and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, Madison, WI, U.S.A.). Sequence alignment may also be carried out using the BLAST algorithm, described in Altschul et al, 1990, J MoI. Biol. 215: 403-10 (using the published default settings). Software for performing BLAST analysis may be available through the National Center for Biotechnology Information (through the internet at http://www.ncbi.nlm.nih.gov/). The BLAST algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence that either match or satisfy some positive -valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighbourhood word score threshold. Initial neighbourhood word hits act as seeds for initiating searches to find longer HSPs. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Extension of the word hits in each direction is halted when the following parameters are met: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T and X determine the sensitivity and speed of the alignment. The BLAST programs may use as defaults a word length (W) of 11, the BLOSUM62 scoring matrix (Henikoff and Henikoff, 1992, Proc. Natl. Acad. Sci. USA 89: 10915-10919) alignments (B) of 50, expectation (E) of 10 (which may be changed in alternative embodiments to 1 or 0.1 or 0.01 or 0.001 or 0.0001; although E values much higher than 0.1 may not identify functionally similar sequences, it is useful to examine hits with lower significance, E values between 0.1 and 10, for short regions of similarity), M=5, N=4, for nucleic acids a comparison of both strands. For protein comparisons, BLASTP may be used with defaults as follows: G=I 1 (cost to open a gap) ; E=I (cost to extend a gap); E=IO (expectation value, at this setting, 10 hits with scores equal to or better than the defined alignment score, S, are expected to occur by chance in a database of the same size as the one being searched; the E value can be increased or decreased to alter the stringency of the search. ); and W=3 (word size, default is 11 for BLASTN, 3 for other blast programs).

The BLOSUM matrix assigns a probability score for each position in an alignment that is based on the frequency with which that substitution is known to occur among consensus blocks within related proteins. The BLOSUM62 (gap existence cost = 11; per residue gap cost = 1; lambda ratio = 0.85) substitution matrix is used by default in BLAST 2.0. A variety of other matrices may be used as alternatives to BLOSUM62, including: PAM30 (9,1,0.87); PAM70 (10,1,0.87) BLOSUM80 (10,1,0.87) ; BLOSUM62 (11,1,0.82) and BLOSUM45 (14,2,0.87). One measure of the statistical similarity between two sequences using the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. In alternative embodiments of the invention, nucleotide or amino acid sequences are considered substantially identical if the smallest sum probability in a comparison of the test sequences is less than about 1, preferably less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001. It is well known in the art that, a number of modifications and changes can be made in the structure of a polypeptide without substantially altering the biological function of that peptide, to obtain a biologically equivalent polypeptide. In one aspect of the invention, LPL therapeutics may include peptides that differ from a portion of the wild-type LPL or LPL^S447X sequence by conservative amino acid substitutions. As used herein, the term "conserved amino acid substitutions" refers to the substitution of one amino acid for another at a given location in the peptide, where the substitution can be made without loss of function. In making such changes, substitutions of like amino acid residues can be made, for example, on the basis of relative similarity of side- chain substituents, for example, their size, charge, hydrophobicity, hydrophilicity, and the like, and such substitutions may be assayed for their effect on the function of the peptide by routine testing.

In some embodiments, conserved amino acid substitutions may be made where an amino acid residue is substituted for another having a similar hydrophilicity value (e. g., within a value of plus or minus 2.0), where the following hydrophilicity values are assigned to amino acid residues (as detailed in United States Patent No. 4,554,101, incorporated herein by reference): Arg (+3.0); Lys (+3.0); Asp (+3.0); GIu (+3.0); Ser (+0.3); Asn (+0.2); GIn (+0.2); GIy (0); Pro (-0.5); Thr (-0.4); Ala (-0.5); His (-0.5); Cys (-1.0); Met (-1.3); VaI (-1.5); Leu (-1.8); lie (-1.8); Tyr (-2.3); Phe (-2.5); and Trp (-3.4). In alternative embodiments, conserved amino acid substitutions may be made where an amino acid residue is substituted for another having a similar hydropathic index (e. g., within a value of plus or minus 2.0). In such embodiments, each amino acid residue may be assigned a hydropathic index on the basis of its hydrophobicity and charge characteristics, as follows: lie (+4.5); VaI (+4.2); Leu (+3.8); Phe (+2.8); Cys (+2.5); Met (+1.9); Ala (+1.8); GIy (-0.4); Thr (-0.7); Ser (-0.8); Trp (-0.9); Tyr (-1.3); Pro (-1.6); His (-3.2); GIu (-3.5); GIn (-3.5); Asp (- 3.5); Asn (-3.5); Lys (-3.9); and Arg (-4.5). In alternative embodiments, conserved amino acid substitutions may be made where an amino acid residue is substituted for another in the same class, where the amino acids are divided into non-polar, acidic, basic and neutral classes, as follows: non-polar: Ala, VaI, Leu, He, Phe, Trp, Pro, Met; acidic: Asp, GIu; basic: Lys, Arg, His; neutral: GIy, Ser, Thr, Cys, Asn, GIn, Tyr. In a preferred embodiment of the invention, the LPL therapeutic is administered to a subject in a gene therapy vector (or, to put it another way, a LPL therapeutic of the invention may be a gene therapy vector which comprises a nucleic acid encoding an LPL or LPL^S447X peptide or a derivative of either thereof as described above). Any such gene therapy vector will typically be one that is suitable for gene therapy of mammals, preferably gene therapy o f humans

A preferred nucleic acid construct according to the invention is a viral gene therapy vector. Viral gene therapy vectors are well known in the art. Vectors may be prepared from one of a number of different type of viruses, including adenoviruses, parvoviruses such as adeno-associated viruses (AAV), herpes viruses (HSV), lentiviruses and retroviruses.

Particularly preferred gene therapy vectors in the context of the present invention are parvoviral vectors. Thus, in this preferred aspect the invention relates the use of animal parvoviruses, in particular dependoviruses such as infectious human or simian AAV, and the components thereof (e.g., an animal parvovirus genome) for use as vectors for introduction and/or expression of the nucleotide sequences encoding an LPL or LPL^S447X peptide or a derivative of either in, for example, mammalian cells.

Viruses of the Parvoviridae family are small DNA animal viruses. The family Parvoviridae may be divided between two subfamilies: the Parvovirinae, which infect vertebrates, and the Densovirinae, which infect insects. Members of the subfamily Parvovirinae are herein referred to as the parvoviruses and include the genus Dependovirus and are especially useful for use in the invention. As may be deduced from the name of their genus, members of the Dependovirus are unique in that they usually require coinfection with a helper virus such as adenovirus or herpes virus for productive infection in cell culture. The genus Dependovirus includes AAV, which normally infects humans (e.g., serotypes 2, 3A, 3B, 5, and 6) or primates (e.g., serotypes 1 and 4, which are thought to have been originated from monkeys, but also infect humans), and related viruses that infect other warm-blooded animals (e.g., bovine, canine, equine, and ovine adeno-associated viruses). Further information on AAV serotypes and on strategies for engineering hybrid AAV vectors derived from AAV serotypes is described in Wu et al. (2006, Molecular Therapy 1_4:316-327). For convenience the present invention is further exemplified and described herein by reference to AAV. It is however understood that the invention is not limited to AAV but may equally be applied to hybrid AAV vectors derived from two or more different AAV serotypes, to modified AAVs (i.e. artificially modified AAVs) and to other parvoviruses and hybrids thereof.

Preferred adenoviral vectors are modified to reduce the host response as reviewed by Russell (2000, J. Gen. Virol. 81: 2573-2604), or as described in US20080008690 and by Zaldumbide and Hoeben (Gene Therapy 2008:239-246).

AAV is able to infect a number of mammalian cells. See, e.g., Tratschin et al. (1985, MoI. Cell Biol. 5:3251-3260) and Grimm et al. (1999, Hum. Gene Ther. Jj):2445-2450). However, AAV transduction of human synovial fibroblasts is significantly more efficient than in similar murine cells, Jennings et al., Arthritis Res, 3:1 (2001), and the cellular tropicity of AAV differs among serotypes. See, e.g., Davidson et al. (2000, Proc. Natl. Acad. Sci. USA, 97:3428-3432), who discuss differences among AA V2, AAV4, and AAV5 with respect to mammalian CNS cell tropism and transduction efficiency. In a preferred embodiment, a host cell of the invention is any mammalian cell that may be infected by a parvoviral virion, for example, but not limited to, a muscle cell, a liver cell, a nerve cell, a glial cell and an epithelial cell. In a preferred embodiment a host cell of the invention is a human cell.

The genomic organization of all known AAV serotypes is very similar. The genome of AAV is a linear, single- stranded DNA molecule that is less than about 5,000 nucleotides (nt) in length. Inverted terminal repeats (ITRs) flank the unique coding nucleotide sequences for the non- structural replication (Rep) proteins and the structural (VP) proteins. The VP proteins (VPl, -2 and -3) form the capsid. The terminal 145 nt are self-complementary and are organized so that an energetically stable intramolecular duplex forming a T-shaped hairpin may be formed. These hairpin structures function as an origin for viral DNA replication, serving as primers for the cellular DNA polymerase complex. Following wtAAV infection in mammalian cells the Rep genes (i.e. Rep78 and Rep52) are expressed from the P5 promoter and the P19 promoter, respectively and both Rep proteins have a function in the replication of the viral genome. A splicing event in the Rep ORF results in the expression of actually four Rep proteins (i.e. Rep78, Rep68, Rep52 and Rep40). However, it has been shown that the unspliced mRNA, encoding Rep78 and Rep52 proteins, in mammalian cells are sufficient for AAV vector production. Also in insect cells, the Rep78 and Rep52 proteins suffice for AAV vector production. An LPL therapeutic of thee invention may be a recombinant parvo viral or AAV vector suitable for use in gene therapy. A "recombinant parvoviral or AAV vector" (or "rAAV vector") herein refers to a vector comprising a nucleic acid sequence encoding an LPL protein as defined herein that is flanked by at least one parvoviral or AAV inverted terminal repeat sequences (ITRs). Such rAAV vectors can be replicated and packaged into infectious viral particles when present in a host cell, such as an insect cell, that is expressing AAV rep and cap gene products (i.e. AAV Rep and Cap proteins). Typically then, an LPL therapeutic of the invention in the form of an AAV vector does not comprise nucleic acid sequences encoding Rep or Cap proteins.

When an rAAV vector is incorporated into a larger nucleic acid construct (e.g. in a chromosome or in another vector such as a plasmid or baculovirus used for cloning or transfection), then the rAAV vector is typically referred to as a "pro-vector" which can be "rescued" by replication and encapsidation in the presence of AAV packaging functions and necessary helper functions.

In the recombinant parvoviral (rAAV) vectors of the invention the at least one nucleotide sequence(s) encoding a gene product of interest for expression in a mammalian cell, preferably is/are operably linked to at least one mammalian cell- compatible expression control sequence, e.g., a promoter. Many such promoters are known in the art (see Sambrook and Russel, 2001, supra). Constitutive promoters that are broadly expressed in many cell-types, such as the CMV promoter may be used. However, more preferred will be promoters that are inducible, tissue-specific, cell-type- specific, or cell cycle-specific.

For example, muscle-specific over-expression of an LPL protein may advantageously be induced by adeno-associated virus (AAV) -mediated transduction of muscle cells. Muscle is amenable to AAV-mediated transduction, and different serotypes may be used (AAVl, AAV6, AAV7, AAV8). Transduction of muscle is accomplished by intramuscular injection of AAV-LPL in multiple sites. Multiple sites, keeping the local viral dose low, will help to prevent LPL- induced myopathy or vector-induced immune responses. This has been an effective method for long-term transduction of muscle using serotype 1, however intravenous administration using other serotypes may also be applicable (AAV6, AAVB8).

Alternatively, for liver-specific expression a promoter may be selected from an αl-anti-trypsin (AAT) promoter, a thyroid hormone-binding globulin promoter, an albumin promoter, a LPS (thyroxine-binding globlin) promoter, an HCR-ApoCII hybrid promoter, an HCR-hAAT hybrid promoter, an AAT promoter combined with the mouse albumin gene enhancer (EaIb) element and an apo lipoprotein E promoter. Other examples include the E2F promoter for tumour-selective, and, in particular, neurological cell tumour-selective expression (Parr et al, 1997, Nat. Med. 3:1145-9) or the IL-2 promoter for use in mononuclear blood cells (Hagenbaugh et al., 1997, J Exp Med; 185: 2101-10).

In the context of the invention "at least one parvoviral inverted terminal repeat nucleotide sequence" is understood to mean a palindromic sequence, comprising mostly complementary, symmetrically arranged sequences also referred to as "A," "B," and "C" regions. The ITR functions as an origin of replication, a site having a "cis" role in replication, i.e., being a recognition site for trans acting replication proteins such as e.g. Rep 78 (or Rep68) which recognize the palindrome and specific sequences internal to the palindrome. One exception to the symmetry of the ITR sequence is the "D" region of the ITR. It is unique (not having a complement within one ITR). Nicking of single-stranded DNA occurs at the junction between the A and D regions. It is the region where new DNA synthesis initiates. The D region normally sits to one side of the palindrome and provides directionality to the nucleic acid replication step. A parvovirus replicating in a mammalian cell typically has two ITR sequences. It is, however, possible to engineer an ITR so that binding sites are on both strands of the A regions and D regions are located symmetrically, one on each side of the palindrome. On a double-stranded circular DNA template (e.g., a plasmid), the Rep78- or Rep68- assisted nucleic acid replication then proceeds in both directions and a single ITR suffices for parvoviral replication of a circular vector. Thus, one ITR nucleotide sequence can be used in the context of the present invention. Preferably, however, two or another even number of regular ITRs are used. Most preferably, two ITR sequences are used. A preferred parvoviral ITR is an AAV ITR. For safety reasons it may be desirable to construct a recombinant parvoviral (rAAV) vector that is unable to further propagate after initial introduction into a cell in the presence of a second AAV. Such a safety mechanism for limiting undesirable vector propagation in a recipient may be provided by using rAAV with a chimeric ITR as described in US2003148506.

The term "flanked" with respect to a sequence that is flanked by another element(s) herein indicates the presence of one or more of the flanking elements upstream and/or downstream, i.e., 5' and/or 3', relative to the sequence. The term "flanked" is not intended to indicate that the sequences are necessarily contiguous. For example, there may be intervening sequences between the nucleic acid encoding the transgene and a flanking element. A sequence that is "flanked" by two other elements (e.g. ITRs), indicates that one element is located 5' to the sequence and the other is located 3' to the sequence; however, there may be intervening sequences therebetween. In a preferred embodiment a nucleotide sequence of (i) is flanked on either side by parvoviral inverted terminal repeat nucleotide sequences.

Thus, in an LPL therapeutic of the invention the nucleic acid encoding an LPL protein is also comprises at least one parvoviral or AAV ITR. Preferably, the nucleotide sequence encoding the LPL protein is flanked by a parvoviral or an AAV ITR (of a mixture thereof) on either side. Any parvoviral or AAV ITR may be used in the constructs of the invention, including ITRs from AAVl, AAV2, AA V4, and/or AAV5. ITRs of AAV2 are most preferred.

Methods for the preparation of recombinant parvoviral (rAAV) virions (comprising a recombinant parvoviral (rAAV) vector as defined above) are well known to those skilled in the art. For example, rAAV virions may be prepared in an insect cell. Preferably, the method comprises the steps of: (a) culturing an insect cell as defined in herein above under conditions such that recombinant parvoviral (rAAV) vector is produced; and, (b) recovery of the recombinant parvoviral (rAAV) vector. It is understood here that the recombinant parvoviral (rAAV) vector produced in the method preferably is an infectious parvoviral or AAV virion that comprise the recombinant parvoviral (rAAV) vector nucleic acids. Growing conditions for insect cells in culture, and production of heterologous products in insect cells in culture are well-known in the art and described e.g. in the above cited references on molecular engineering of insects cells. Preferred methods and constructs for the production of rAAV virions of the invention are disclosed in e.g. WO2007/046703 and WO2007/148971.

The insect cell may be any cell that is suitable for the production of heterologous proteins. Preferably the insect cell allows for replication of baculoviral vectors and can be maintained in culture. More preferably the insect cell also allows for replication of recombinant parvoviral vectors, including rAAV vectors. For example, the cell line used can be from Spodoptera frugiperda, Drosophila cell lines, or mosquito cell lines, e.g., Aedes albopictus derived cell lines. Preferred insect cells or cell lines are cells from the insect species which are susceptible to baculovirus infection, including e.g. S2 (CRL-1963, ATCC), Se301, SeIZD2109, SeUCRl, Sf9, Sf900+, Sf21, BTI-TN-5B1-4, MG-I, Tn368, HzAmI, Ha2302, Hz2E5, High Five (Invitrogen, CA, USA) and expresSF+^® (US 6,103,526; Protein Sciences Corp., CT, USA). A preferred insect cell according to the invention is an insect cell for production of recombinant parvoviral vectors.

AAV sequences that may be used in the present invention for the production of a recombinant AAV virion, for example by the use of insect cells, can be derived from the genome of any AAV serotype. Generally, the AAV serotypes have genomic sequences of significant homology at the amino acid and the nucleic acid levels, provide an identical set of genetic functions, produce virions which are essentially physically and functionally equivalent, and replicate and assemble by practically identical mechanisms. For the genomic sequence of the various AAV serotypes and an overview of the genomic similarities see e.g. GenBank Accession number U89790; GenBank Accession number JO 1901; GenBank Accession number AF043303; GenBank Accession number AF085716; Chlorini et al. (1997, J. Vir. 71 : 6823-33); Srivastava et al. (1983, J. Vir. 45:555-64); Chlorini et al. (1999, J. Vir. 73:1309-1319); Rutledge et al. (1998, J. Vir. 72:309-319); and Wu et al. (2000, J. Vir. 74: 8635-47). AAV serotypes 1, 2, 3, 4 and 5 are preferred source of AAV nucleotide sequences for use in the context of the present invention. Preferably the AAV ITR sequences for use in the context of the present invention are derived from AAVl, AA V2, and/or AA V4. Likewise, the Rep (Rep78/68 and Rep52/40) coding sequences are preferably derived from AAVl, AAV2, and/or AAV4. The sequences coding for the VPl, VP2, and VP3 capsid proteins for use in the context of the present invention may however be taken from any of the known 42 serotypes, more preferably from AAVl , AA V2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8 or AAV9 or newly developed AAV-like particles obtained by e.g. capsid shuffling techniques and AAV capsid libraries, or from newly designed, developed or evolved ITR' s. It is within the technical skills of the skilled person to select the most appropriate virus or virus subtype. Some subtypes may be more appropiate than others for a certain type of tissue.

Preferably, the nucleotide sequences encoding a parvoviral Rep protein and/or a parvoviral capsid protein is operably linked to expression control sequences for expression in an insect cell. These expression control sequences will at least include a promoter that is active in insect cells. Techniques known to one skilled in the art for expressing foreign genes in insect host cells can be used to practice the invention. Methodology for molecular engineering and expression of polypeptides in insect cells is described, for example, in Summers and Smith. 1986. A Manual of Methods for Baculo virus Vectors and Insect Culture Procedures, Texas Agricultural Experimental Station Bull. No. 7555, College Station, Tex.; Luckow. 1991. In Prokop et al, Cloning and Expression of Heterologous Genes in Insect Cells with Baculovirus Vectors' Recombinant DNA Technology and Applications, 97-152; King, L. A. and R. D. Possee, 1992, The baculovirus expression system, Chapman and Hall, United Kingdom; O'Reilly, D. R., L. K. Miller, V. A. Luckow, 1992, Baculovirus Expression Vectors: A Laboratory Manual, New York; W. H. Freeman and Richardson, C. D., 1995, Baculovirus Expression Protocols, Methods in Molecular Biology, volume 39; US 4,745,051; US2003148506; and WO 03/074714. Suitable promoters for transcription of the nucleotide sequences comprised in the first and the second construct of the invention include e.g. the polyhedron (PoH), plO, p35, IE-I or ΔIE-1 promoters and further promoters described in the above references.

AAV Rep and ITR sequences are particularly conserved among most serotypes. The Rep78 proteins of various AAV serotypes are e.g. more than 89% identical and the total nucleotide sequence identity at the genome level between AAV2, AAV3A, AAV3B, and AAV6 is around 82% (Bantel-Schaal et al., 1999, J. Virol, 73(2):939- 947). Moreover, the Rep sequences and ITRs of many AAV serotypes are known to efficiently cross-complement (i.e., functionally substitute) corresponding sequences from other serotypes in production of AAV particles in mammalian cells. US2003148506 reports that AAV Rep and ITR sequences also efficiently cross- complement other AAV Rep and ITR sequences in insect cells.

The AAV VP proteins are known to determine the cellular tropism of the AAV virion. The VP protein-encoding sequences are significantly less conserved than Rep proteins and genes among different AAV serotypes. The ability of Rep and ITR sequences to cross-complement corresponding sequences of other serotypes allows for the production of pseudotyped rAAV particles comprising the capsid proteins of one serotype (e.g., AAV5) and the Rep and/or ITR sequences of another AAV serotype (e.g., AAV2). Such pseudotyped rAAV particles are a part of the present invention. Modified "AAV" sequences also can be used in the context of the present invention, e.g. for the production of rAAV vectors in insect cells. Such modified sequences e.g. include sequences having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or more nucleotide and/or amino acid sequence identity (e.g., a sequence having about 75-99% nucleotide sequence identity) to an AAVl , AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8 or AAV9 ITR, Rep, or VP can be used in place of wild-type AAV ITR, Rep, or VP sequences.

Although similar to other AAV serotypes in many respects, AAV5 differs from other human and simian AAV serotypes more than other known human and simian serotypes. In view thereof, the production of rAAV 5 can differ from production of other serotypes in insect cells. Where methods of the invention are employed to produce rAAV5, it is preferred that one or more constructs comprising, collectively in the case of more than one construct, a nucleotide sequence comprising an AAV5 ITR, a nucleotide sequence comprises an AAV5 Rep coding sequence (i.e. a nucleotide sequence comprises an AAV5 Rep78). Such ITR and Rep sequences can be modified as desired to obtain efficient production of rAAV5 or pseudotyped rAAV5 vectors in insect cells. E.g., the start codon of the Rep sequences can be modified, VP splice sites can be modified or eliminated, and/or the VPl start codon and nearby nucleotides can be modified to improve the production of rAAV5 vectors in the insect cell. Preferably the methods for producing recombinant parvoviral virions further comprises the step of affinity-purification of the (virions comprising the) recombinant parvoviral (rAAV) vector using an anti-AAV antibody, preferably an immobilised antibody. The anti-AAV antibody preferably is an monoclonal antibody. A particularly suitable antibody is a single chain camelid antibody or a fragment thereof as e.g. obtainable from camels or llamas (see e.g. Muyldermans, 2001, Biotechnol. 74: 277- 302). The antibody for affinity-purification of rAAV preferably is an antibody that specifically binds an epitope on a AAV capsid protein, whereby preferably the epitope is an epitope that is present on capsid protein of more than one AAV serotype. E.g. the antibody may be raised or selected on the basis of specific binding to AAV2 capsid but at the same time also it may also specifically bind to AAVl, AAV3 and AAV5 capsids.

General methods for gene therapy are known in the art. See for example, U. S. Pat. No. 5,399,346 by Anderson et al. (incorporated herein by reference). A biocompatible capsule for delivering genetic material is described in PCT Publication WO 95/05452 by Baetge et al. Methods of gene transfer into hematopoietic cells have also previously been reported (see Clapp, D. W. , et al. , Blood 78: 1132-1139 (1991) ; Anderson, Science 288: 627-9 (2000) ; and , Cavazzana-Calvo et al., Science 288: 669- 72 (2000), all of which are incorporated herein by reference). In the treatment according to the invention, the number and/or size of chylomicrons or chylomicron remnants may be reduced by administering to a subject an effective amount of an LPL therapeutic as defined herein. That is to say, the total size (or volume) of the chylomicron fraction or the chylomicron remnant fraction may be reduced by administering to a subject an effective amount of an LPL therapeutic as defined herein. Also, treatment according to the invention may result in an increase in clearance of chylomicron remnants. This is intended to indicate that more chylomicron remnants may be cleared in a specified period of time as would the case in the subject if not treated according to the invention.

Treatment according to the invention may results in redistribution of triglycerides in chylomicrons to other lipoprotein fractions, in particular very low-density lipoprotein (VLDL) in a subject. This is not intended to imply a direct relationship between a reduction of concentration of triglycerides in chylomicrons and VLDL. However, treatment according to the invention may result in a reduction in triglyceride amount and/or concentration in chylomicrons accompanied by a rise in amount and/or concentration of triglycerides in other lipoprotein fractions such as VLDL in particular (or a lipid/lipoprotein fraction containing VLDL).

An LPL therapeutic as described herein may be used in the treatment of a condition which is characterized by: (i) an elevated number and/or size of chylomicrons and/or chylomicron remnants; and/or (ii) reduced or inadequate processing of chylomicrons and/or chylomicron remnants.

An LPL therapeutic as described herein may be used in the treatment of a condition characterized or accompanied by elevated triglyceride levels. Such levels of triglycerides may be evident in the blood or in some other tissue, for example the lymph, adipose tissue, the liver or the spleen.

Such conditions are known to those skilled in the art and such conditions may accordingly be treated. For example, the treatment may be used in the treatment of partial LPL deficiency, metabolic syndrome, hyperuricemia, dyslipidemia, neuropathy, such as peripheral/central neuropathy, insulin resistance, difficult to treat diabetes, the long-term consequences of diabetes, memory impairment, depression, cardiovascular indications such as deep vein thrombosis and in the prophylaxis of pancreatitis. Also, in view of the ability of an LPL therapeutic to stimulate triglyceride export from the liver, it is also use an LPL therapeutic according to the invention in the treatment of conditions associated with a fatty liver, alcoholic steatotic hepatitis and hepatocellular carcinoma and in the prophylaxis of cirrhosis.

The invention also pertains to a pharmaceutical composition comprising an LPL therapeutic as herein defined optionally in combination with a pharmaceutical carrier, diluent and/or adjuvant. Any suitable pharmaceutically acceptable diluent, adjuvant, carrier or excipient can be used in the present compositions (See e.g., Remington: The Science and Practice of Pharmacy, Alfonso R. Gennaro (Editor) Mack Publishing Company, April 1997). Preferred pharmaceutical forms would be in combination with sterile saline, dextrose solution, or buffered solution, or other pharmaceutically acceptable sterile fluids. Alternatively, a solid carrier, may be used such as, for example, microcarrier beads. Such compositions include the LPL therapeutic in an effective amount, sufficient to provide a desired therapeutic or prophylactic effect, and a pharmaceutically acceptable carrier or excipient. An "effective amount" includes a therapeutically effective amount or a prophylactically effective amount.

A "therapeutically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result, such as a reduction of chylomicron numbers and/or size, i.e. the volume of the chylomicron fraction may be reduced. A therapeutically effective amount of an LPL therapeutic may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the LPL therapeutic to elicit a desired response in the individual. Dosage regimens may be adjusted to provide the optimum therapeutic response. A therapeutically effective amount is also typically one in which any toxic or detrimental effects of the LPL therapeutic are outweighed by the therapeutically beneficial effects.

In particular embodiments, a range for therapeutically or prophylactically effective amounts of LPL therapeutic may be 0.01 nM-0.1M, 0.1 nM-0.1M, 0.1 nM- 0.05M, 0.05 nM-15 M or 0.01 nM-10 M. It is to be noted that dosage values may vary with the severity of the condition to be alleviated. For any particular subject, specific dosage regimens may be adjusted over time according to the individual need and the professional judgement of the person administering or supervising the administration of the compositions. Dosage ranges set forth herein are exemplary only and do not limit the dosage ranges that may be selected by medical practitioners.

For gene therapy vectors, the dosage to be administered may depend to a large extent on the condition and size of the subject being treated as well as the therapeutic formulation, frequency of treatment and the route of administration. Regimens for continuing therapy, including dose, formulation, and frequency may be guided by the initial response and clinical judgment. The parenteral route of injection into the interstitial space of tissue may be preferred, although other parenteral routes, such as inhalation of an aerosol formulation, may be required in specific administration. In some protocols, a formulation comprising the gene and gene delivery system in an aqueous carrier is injected into tissue in appropriate amounts.

The tissue target may be specific, for example the muscle or liver tissue, or it may be a combination of several tissues, for example the muscle and liver tissues. Exemplary tissue targets may include liver, skeletal muscle, heart muscle, adipose deposits, kidney, lung, vascular endothelium, epithelial and/or hematopoietic cells.

In one embodiment, the effective dose range for small animals (mice), following intramuscular injection, is between IxIO¹⁰ and IxIO¹⁵ genome copy (gc) /kg, and for larger animals (cats) and possibly human subjects, between IxIO¹¹ and IxIO¹² gc/kg. Again in mice, levels of transgene expression, as measured by LPL in post-heparin plasma, reach up to 300 ng/ml (measured using a commercial ELISA from DaiMppon), and expression is long-term (>1 year) following a one-time administration. The amount of active compound in the compositions of the invention may vary according to factors such as the disease state, age, sex, and weight of the individual. Dosage regimens may be adjusted to provide the optimum therapeutic response. For example, a single dose may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation.

It may be advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. "Dosage unit form" as used herein refers to physically discrete units suited as unitary dosages for subjects to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the invention may be dictated by the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and by the limitations inherent in the art of compounding such an active compound for the treatment of a condition in individuals.

As used herein "pharmaceutically acceptable carrier" or "exipient" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. In one embodiment, the carrier is suitable for parenteral administration, which includes intravenous, intraperitoneal or intramuscular administration. Alternatively, the carrier may be suitable for sublingual or oral administration. Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the pharmaceutical compositions of the invention is contemplated.

Supplementary active compounds can also be incorporated into the pharmaceutical compositions of the invention. Guidance on co-administration of additional therapeutics may for example be found in the Compendium of

Pharmaceutical and Specialties (CPS) of the Canadian Pharmacists Association.

Pharmaceutical compositions are typically sterile and stable under the conditions of manufacture and storage. Pharmaceutical compositions may be formulated as a solution, microemulsion, liposome, or other ordered structure suitable to accommodate high drug concentration. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. 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. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, monostearate salts and gelatin. The LPL therapeutic may be administered in a time or controlled release formulation, for example in a composition which includes a slow release polymer or other carriers that will protect the compound against rapid release, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers may for example be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, polylactic acid and polylactic, polyglycolic copolymers (PLG).

Many methods for the preparation of such formulations are patented or generally known to those skilled in the art.

Examples

LPLD, also known as familial chylomicronemia, is caused by a loss of function mutation in the LPL gene with consequent loss of LPL activity. This results in severe hypertriglyceridemia and chylomicronemia as triglycerides (TG) in chylomicrons (CM) and TG rich- lipoproteins are no longer hydrolyzed. Recurrent pancreatitis is the most frequent complication. In this study, safety and efficacy of gene therapy in adult LPLD patients with LPL^S447X, a naturally occurring gain of function mutation, delivered in a non-replicating, non-integrating adeno associated virus (Ross et al. Hum. Gene Therapy 15(9). 906-919, 2004).

A total of 14 subjects were enrolled in two dose groups. Subjects in group 1 (n=6) received 3x10¹¹ gc/kg while subjects in group 2 (n=8) received 1x10¹² gc/kg of AAV1-LPL^S447X (alipogene tiparvovec), via a single series of intramuscular injections in the lower extremities. Safety and efficacy was evaluated over a period of 12 weeks with long term follow-up planned for 15 years. According to the study design, all subjects, except two in group 1, were also immunosuppressed with cyclosporine and mycophenolate mofetil for the initial 12 weeks. All 14 subjects safely completed the study and are currently being longitudinally followed. An important reduction (>40%) in fasting TG levels was observed in the majority of subjects beginning 2 weeks post-injection and was maintained for the duration of the study (12 weeks). From week 2 post injection (when the decrease in plasma TG levels is first observed) to the end of year 1, several clinical, biochemical and hematological features of LPLD significantly improved. Approximately 4 months post injection, fasting TG levels tended to drift back towards pretreatment values but this increase in TG levels was associated with important changes in the composition and characteristics of the CM and other lipoproteins compared to baseline. In particular, ultracentrifugation data available at weeks 26, 39 and 52 suggest a shift of TG concentration from the CM to the VLDL fraction (see Figs 1 to 3). The Sf >400 fraction (see Figs. 2 and 3) contains the (large) CM, and the Sf 20-400 fraction is likely a mix of smaller CM or CM remnants, and VLDL. What is thus seen is a shift from most of the TG and cholesterol in a very buoyant fraction (large CM) to a less buoyant fraction, ie a change in size and/or composition of the CM particle. This less buoyant fraction corresponds to what is normally termed 'VLDL'.

These data demonstrate that gene therapy with LPL^S447X is safe and well tolerated and suggest that a single dose is associated with significant clinical improvement and changes in lipoprotein characteristics and composition for at least 1 year post dosing.

Claims

1. A lipoprotein lipase (LPL) therapeutic for use in reducing the number and/or size of chylomicrons and/or chylomicron remnants in a subject.

2. An LPL therapeutic for use in redistributing triglycerides in chylomicrons to very low-density lipoprotein (VLDL) in a subject.

3. An LPL therapeutic for use in increasing clearance of chylomicron remnants in a subject.

4. An LPL therapeutic according to any one of the preceding claims for use in the treatment of a condition which is characterized by: (i) an elevated number and/or size of chylomicrons and/or chylomicron remnants; and/or (ii) reduced or inadequate processing of chylomicrons and/or chylomicron remnants.

5. An LPL therapeutic according to any one of claims 1 to 3 for use in the treatment of a condition characterized or accompanied by elevated triglyceride levels.

6. An LPL therapeutic according to any one of the preceding claims for use in the treatment of a subject having reduced levels of lipoprotein lipase (LPL) and/or a relative LPL deficiency.

7. An LPL therapeutic according to any one of the preceding claims, wherein the LPL therapeutic is a member selected from the group consisting of: a) an LPL protein with an amino acid sequence as shown in SEQ ID NO: 3 or SEQ ID NO: 4, or a derivative of either thereof; b) an LPL protein with an amino acid sequence which comprises a contiguous segment having at least 90% sequence identity to SEQ ID NO: 3 or 4 when optimally aligned which has equal or greater LPL activity than a protein under a), or a derivative thereof; or c) a nucleic acid encoding a) or b), or a derivative thereof.

8. An LPL therapeutic according to any one of the preceding claims, wherein the LPL therapeutic comprises a nucleic acid which comprises a DNA coding sequence encoding an RNA having at least 90% sequence identity to nucleotides 452 through 1795 of SEQ ID NO: 1 or nucleotides 452 through 1789 of SEQ ID NO: 1.

9. An LPL therapeutic according to any one of the preceding claims, wherein the LPL therapeutic is a nucleic acid which comprises a DNA coding sequence that hybridizes under stringent conditions to the reverse complement of nucleotides 256 through 1795 of SEQ ID NO: 1 or nucleotides 452 through 1789 of SEQ ID NO: 1.

10. An LPL therapeutic according to any one of claims 7 to 9, wherein the LPL therapeutic is an LPL protein with a contiguous segment of at least 95% sequence identity to SEQ ID NO: 3 or 4 respectively.

11. An LPL therapeutic according to any one of the preceding claims, wherein the LPL therapeutic is administered to the subject in a gene therapy vector.

12. An LPL therapeutic use according to claim 11, wherein the gene therapy vector is a viral vector.

13. An LPL therapeutic according to claim 12, wherein the viral vector comprises adeno-associated virus (AAV).

14. An LPL therapeutic according to any one of the preceding claims, wherein the LPL therapeutic is administered parenterally.

15. A pharmaceutical composition comprising an LPL therapeutic as defined in any one of the preceding claims and a pharmaceutically acceptable carrier.

16. A method for use in reducing the number and/or size of chylomicrons and/or chylomicron remnants in a subject, which method comprises administering to a subject in need thereof an effective amount of an LPL therapeutic.

17. A method for use in redistributing triglycerides in chylomicrons to very low- density lipoprotein (VLDL) in a subject, which method comprises administering to a subject in need thereof an effective amount of an LPL therapeutic.

18. A method for use in increasing clearance of chylomicron remnants in a subject, which method comprises administering to a subject in need thereof an effective amount of an LPL therapeutic.

19. Use of an LPL therapeutic in the manufacture of a medicament for use in reducing the number and/or size of chylomicrons and/or chylomicron remnants in a subject.

20. Use of an LPL therapeutic in the manufacture of a medicament for use in redistributing triglycerides in chylomicrons to very low-density lipoprotein (VLDL) in a subject.

21. Use of an LPL therapeutic in the manufacture of a medicament for use in increasing clearance of chylomicron remnants in a subject.

22. A method according to any one of claims 16 to 18 or use according to any one of claims 19 to 21, wherein the LPL therapeutic is as defined in any one of claims 7 to 14.