Lipoprotein lipase deficiency and methods for assessing lipoprotein lipase bioavailability

Abstract

Described herein are methods and kits for the use of ANGPTL3, ANGPTL4, ANGPTL8, and / or ANGPTL3 / 8 inhibitors in the field of severe hypertriglyceridemia, particularly for distinguishing complete or partial lipoprotein lipase (LPL) deficiency and assessing LPL bioavailability in subjects. As described, these inhibitors can be used to distinguish subjects with significant basal LPL activity from controls with no or little LPL activity. The inhibitors can also be used to distinguish recurrent from persistent chylomicronemia in subjects with severe hypertriglyceridemia or chylomicronemia. Administration of the inhibitors can also replace post-heparin LPL activity measurement or genotyping in identifying disorders of triglyceride and HDL-cholesterol metabolism.

Description

[Technical Field]

[0001] This International PCT application was filed concurrently with U.S. Provisional Patent Application No. 63 / 587,192, entitled "Assessment of Chylomicronemia with Vitamin E," filed October 2, 2023, the entire contents of which are incorporated herein by reference.

[0002] The present invention relates to the field of lipoprotein lipase (LPL) deficiency, and in particular to distinguishing between complete and partial lipoprotein lipase (LPL) deficiency and assessing the bioavailability of LPL in a subject. [Background technology]

[0003] Hypertriglyceridemia is characterized by the accumulation of triglyceride (TG)-rich lipoproteins in the blood, derived from exogenous (chylomicrons and remnants) or endogenous (VLDL and remnants) metabolic pathways. Hypertriglyceridemia exists in several forms, and the most severe form, hyperchylomicronemia, is defined by the accumulation of chylomicrons, lipoproteins that transport dietary fats, in the blood after a meal. Thus, chylomicronemia is a postprandial metabolic disorder characterized by triglyceridemia exceeding 10 mmol / L. The prevalence of chylomicronemia (all causes) is estimated at 1 in 600 individuals in Western countries. Chylomicronemia can be persistent or recurrent, and several genetic, physiological, environmental, or health factors may contribute to the occurrence of this condition. Chylomicronemia is considered persistent when triglyceridemia is almost always greater than 10 mmol / L and is resistant to conventional lipid-lowering treatment.

[0004] Complete lipoprotein lipase (LPL) deficiency is a well-known and most severe form of persistent chylomicronemia. It is associated with familial hyperchylomicronemia syndrome (FCS), a rare genetic disorder affecting approximately 1–3 individuals per million. Loss-of-function mutations in the LPL gene or genes regulating LPL activity, such as APOCII, APOAV, LMF1, and GPIHBP1, have been identified as the cause of this disorder. However, mutations or high polygenic scores in other genes involved in the triglyceride metabolism pathway can also cause severe hypertriglyceridemia or persistent chylomicronemia. Patients with persistent chylomicronemia (not limited to FCS) are at significant risk for recurrent acute pancreatitis and other conditions.

[0005] With the expansion of personalized medicine, access to innovative therapies for severe hypertriglyceridemia, chylomicronemia, or LPL deficiency is expected to become increasingly limited to patients with genetically confirmed hyperchylomicronemia, such as those with two loss-of-function (null) mutations that cause loss of LPL function or those in whom post-heparin LPL activity assays confirm the absence of bioavailable LPL protein. However, existing assays for measuring post-heparin circulating LPL activity are either invasive or have limitations in reproducibility, validity, antibody sensitivity, substrate stability, or environmental friendliness.

[0006] Another challenge with LPL is that genotypic confirmation does not necessarily correspond to the phenotypic manifestation of the disease. Indeed, several factors, including unidentified and / or unreported mutations in the LPL gene or genes in the same biological pathway; epigenetic modifications that prevent proper transcription of the LPL gene; circulating antibodies against LPL, secondary factors, or a combination of primary and secondary factors, can inhibit LPL activity and cause persistent chylomicronemia or severe hypertriglyceridemia. Therefore, patients with persistent chylomicronemia or severe hypertriglyceridemia who do not have a loss-of-function (null) FCS-causing mutation may not have reasonable access to appropriate treatment, even though they have the same phenotypic and clinical profile as FCS patients.

[0007] Angiopoietin-like proteins (ANGPTLs) are a family of proteins. ANGPTL3 is one of the most well-described ANGPTLs and is a key regulator of lipid metabolism due to its inhibition of LPL activity. Over the past decade, ANGPTL3 has emerged as a novel therapeutic target for lowering blood LDL-C and triglycerides. Many ANGPTL3 inhibitors, including monoclonal antibodies, antisense oligonucleotides, and small interfering ribonucleic acid (siRNA), are currently available or in clinical trials and / or have shown promising results for the treatment of a wide range of lipid-related diseases. ANGPTL3 inhibitors are LPL-dependent, and therefore the presence of LPL is essential for their efficacy (Ahmad Z, et al. J Am Coll Cardiol. 2021 Jul 13;78(2):193-195). After a meal, ANGPTL3 interacts with ANGPTL8, and their interaction exerts a profound effect on LPL. Due to this LPL dependency, the efficacy of dual inhibition of ANGPTL3 and ANGPTL8 also depends on the presence of LPL, and this cooperation increases lipolysis via the LPL pathway by up to 100-fold.

[0008] Angiopoietin-like protein 4 (ANGPTL4) is also known to inhibit LPL. However, unlike ANGPTL3 and ANGPTL8, which are effective in the postprandial state, ANGPTL4 may play an important role in the fasting state. Its paracrine role is almost exclusively limited to adipose tissue, and a phase I clinical trial was initiated in June 2022.

[0009] Although new ANGPTL inhibitor treatments appear to be very promising for lowering LDL cholesterol, remnants, and / or triglycerides, the use and development of ANGPTL3 inhibitors, ANGPTL4 inhibitors, or ANGPTL3 and / or ANGPTL8 inhibitors is solely for therapeutic purposes. These inhibitors have not previously been proposed for diagnostic purposes, to assess LPL bioavailability, or as a clinical and qualitative replacement for post-heparin LPL measurement, or to distinguish between complete and partial LPL deficiency.

[0010] Some existing or novel treatments for severe hypertriglyceridemia, such as ANGPTL3, ANGPTL4, or ANGPTL3 / 8 inhibitors, require the presence of LPL for efficacy, whereas others (e.g., APOC3 inhibitors) are LPL-independent. Given the chronic nature of hypertriglyceridemia susceptibility, the half-life, and cost of treatment, assessing LPL bioavailability is essential to establish the likelihood of response and evaluate the expected TG-lowering effect of therapeutics.

[0011] For example, AAV-based LPL gene replacement therapy for patients with persistent chylomicronemia and LPL deficiency is only available to patients with complete LPL deficiency and is only available once in a lifetime. Access to treatment requires demonstrating not only genetic background but also the lack of LPL bioavailability. In contrast, genome editing strategies (e.g., CRISPR-Cas and others) targeting ANGPTL3 inhibition are likely to be implemented every decade or once in a lifetime, and LPL bioavailability is essential to establishing access to treatment.

[0012] Therefore, there is a need for new methods and assays for assessing lipoprotein lipase (LPL) deficiency or for assessing LPL bioavailability that are non-invasive, sensitive, stable, and / or user-friendly, and that, from a clinical perspective, can replace post-heparin LPL measurement or other unvalidated and poorly reproducible approaches.

[0013] There is also a need for new methods and assays that will make personalized medicine treatment of hypertriglyceridemia, chylomicronemia, or LPL deficiency accessible to more than just patients with genotype-confirmed disease.

[0014] In particular, there is a need for methods to distinguish between complete and partial lipoprotein lipase (LPL) deficiency in a subject, e.g., between patients with complete LPL deficiency and patients with residual activity of the protein.

[0015] There is also a need for methods of diagnosing multifactorial chylomicronemia and / or familial hyperchylomicronemia syndrome (FCS) in a subject.

[0016] There is also a need for methods to assess the bioavailability of PLs in patients with persistent chylomicronemia and / or familial hyperchylomicronemia syndrome (FCS).

[0017] There is also a need for improved methods for treating subjects with complete or partial lipoprotein lipase (LPL) deficiency, or subjects lacking bioavailable LPL.

[0018] The present invention addresses these needs and other needs as will become apparent from a review of the following disclosure and description of the features of the invention. Summary of the Invention

[0019] In certain aspects, the present invention relates to a method for distinguishing between complete and partial lipoprotein lipase (LPL) deficiency in a subject, the method comprising: (i) administering to the subject at least one inhibitor of ANGPTL3, ANGPTL4, ANGPTL8, and / or ANGPTL3 / 8; and (ii) measuring the subject's response to the inhibitor, wherein the response identifies the subject as having complete or partial LPL deficiency.

[0020] In another particular aspect, the present invention relates to a method for assessing the bioavailability of lipoprotein lipase (LPL) in patients with persistent chylomicronemia and / or familial hyperchylomicronemia syndrome (FCS), comprising: (i) administering to the patient at least one inhibitor of ANGPTL3, ANGPTL4, ANGPTL8, and / or ANGPTL3 / 8; and (ii) measuring the patient's response to the inhibitor, wherein a reduction in blood triglycerides in the patient is indicative of LPL bioavailability, and wherein a lack of a reduction in blood triglycerides in the patient is indicative of a lack or rarity of bioavailable LPL.

[0021] In another specific aspect, the present invention relates to a method of distinguishing between recurrent and persistent chylomicronemia in a subject with severe hypertriglyceridemia or chylomicronemia, comprising the steps of: (i) administering to a subject at least one inhibitor of ANGPTL3, ANGPTL4, ANGPTL8, and / or ANGPTL3 / 8; and (ii) measuring the subject's response to the inhibitor.

[0022] In another specific aspect, the present invention relates to a method for replacing post-heparin LPL activity measurement in identifying triglyceride and HDL-cholesterol metabolism disorders, comprising replacing the post-heparin LPL activity measurement with administration of at least one inhibitor of ANGPTL3, ANGPTL4, ANGPTL8, and / or ANGPTL3 / 8 to a subject.

[0023] In another particular embodiment, the present invention relates to a kit for distinguishing between complete and partial lipoprotein lipase (LPL) deficiency in a subject.

[0024] In another particular aspect, the present invention provides a method for producing a (i) To distinguish between complete and partial lipoprotein lipase (LPL) deficiency in subjects; (ii) identifying the subject as having recurrent chylomicronemia; (iii) identifying the subject as having persistent chylomicronemia; and (iv) identifying the subject as having hyperchylomicronemia syndrome (FCS); The present invention relates to the use of at least one inhibitor of ANGPTL3, ANGPTL4, ANGPTL8, and / or ANGPTL3 / 8 for at least one of the above.

[0025] In a further particular aspect, the present invention provides a method for producing a composition comprising: (i) To identify genetic mutations responsible for complete LPL deficiency in human subjects; (ii) to identify human subjects likely to respond to LPL-dependent drugs; (iii) to identify human subjects who are likely to be unresponsive to LPL-dependent drugs; (iv) identifying human subjects eligible for ANGPTL3 inhibition genome editing strategies; (v) identifying human subjects eligible for LPL gene replacement therapy or LPL genome editing; and / or (iv) replacing post-heparin LPL activity assays in identifying disorders of triglyceride and HDL-cholesterol metabolism; The present invention relates to the use of at least one inhibitor of ANGPTL3, ANGPTL4, ANGPTL8, and / or ANGPTL3 / 8 for at least one of the above.

[0026] In a further specific aspect, the present invention relates to a method for treating a subject with complete or partial lipoprotein lipase (LPL) deficiency, which comprises: (i) administering to the subject at least one inhibitor of ANGPTL3, ANGPTL4, ANGPTL8, and / or ANGPTL3 / 8; (ii) assessing the subject's response to the inhibitor by measuring the subject's triglyceridemia or the level of chylomicrons in the subject's blood, wherein the response identifies whether the subject has bioavailable LPL; and (iii) selecting a treatment plan taking the response into consideration.

[0027] Further aspects, advantages and features of the present invention will become more apparent upon reading the following non-limiting description of preferred embodiments, which are illustrative and should not be construed as limiting the scope of the invention. [Brief explanation of the drawings]

[0028] In order that the present invention may be readily understood, embodiments of the invention are illustrated by the following figures.

[0029] [Figure 1] Figure 1 is a scatter plot showing the linear relationship between FCS diagnostic scores and post-heparin plasma LPL activity* using scoring systems Model A (circles) and Model B (triangles). LPL: lipoprotein lipase; FCS and arrows indicate the cutoff values ​​for the familial chylomicronemia syndrome diagnostic score in Model A (left) and Model B (right). *The mean value was used for subjects with LPL activity obtained by both methods.

[0030] Further details and advantages of the present invention will become apparent from the detailed description that follows. DETAILED DESCRIPTION OF THE INVENTION

[0031] In the following description of the embodiments, reference is made to the accompanying drawings, which illustrate examples in which the invention may be practiced. It will be understood that other embodiments may be practiced without departing from the scope of the invention. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0032] The present invention relates to the field of lipoprotein lipase (LPL) deficiency and is scientifically based on the fact that ANGPTL3, ANGPTL4, ANGPTL8, and / or ANGPTL3 / 8 inhibitors can upregulate lipoprotein lipase (LPL).Accordingly, according to the present invention, these inhibitors can be used to distinguish subjects with significant basal LPL activity from subjects with no or little LPL activity.

[0033] In one particular aspect, the present invention relates to a method that allows distinguishing between complete and partial lipoprotein lipase (LPL) deficiency. In another aspect, the present invention relates to a method for assessing lipoprotein lipase (LPL) bioavailability in patients with persistent chylomicronemia and / or familial hyperchylomicronemia syndrome (FCS).

[0034] One aspect of the present invention relates to a method for distinguishing between complete and partial lipoprotein lipase (LPL) deficiency in a subject. In one embodiment, the method comprises (i) administering to a subject at least one inhibitor of ANGPTL3, ANGPTL4, ANGPTL8, and / or ANGPTL3 / 8, and (ii) measuring the subject's response to the inhibitor. According to the method, the response identifies the subject as having complete or partial LPL deficiency (i.e., the presence of some LPL that can be upregulated by the inhibitor).

[0035] Another aspect of the present invention relates to a method for diagnosing partial LPL deficiency, multifactorial chylomicronemia, and / or complete LPL deficiency in a subject. In one embodiment, the method comprises (i) administering to a subject at least one inhibitor of ANGPTL3, ANGPTL4, ANGPTL8, and / or ANGPTL3 / 8, and (ii) measuring the subject's response to the inhibitor. According to the method, the subject's response identifies the subject as having partial LPL deficiency, multifactorial chylomicronemia, or complete LPL deficiency.

[0036] Another aspect of the present invention relates to a method for assessing the bioavailability of lipoprotein lipase (LPL) in a patient with persistent chylomicronemia and / or familial hyperchylomicronemia syndrome (FCS). In one embodiment, the method comprises (i) administering to the patient at least one inhibitor of ANGPTL3, ANGPTL4, ANGPTL8, and / or ANGPTL3 / 8, and (ii) measuring the patient's response to the inhibitor. According to the method, a decrease in the patient's blood triglycerides confirms the bioavailability of LPL, and a lack of a decrease in the patient's blood triglycerides indicates a lack or rarity of bioavailable LPL.

[0037] Another aspect of the present invention relates to a method for distinguishing between recurrent chylomicronemia and persistent chylomicronemia in a subject with severe hypertriglyceridemia or chylomicronemia. In one embodiment, the method comprises: (i) administering to the subject at least one inhibitor of ANGPTL3, ANGPTL4, ANGPTL8, and / or ANGPTL3 / 8; and (ii) measuring the subject's response to the inhibitor. According to the method, the measured response identifies the subject as having recurrent chylomicronemia, persistent chylomicronemia, complete LPL deficiency, or familial chylomicronemia syndrome (FCS). To obtain a more definitive conclusion, steps (i) and (ii) are preferably repeated at different intervals (e.g., at least twice, at least three times, or more, at intervals of one week, several weeks, one month, several months, or more).

[0038] Another aspect of the present invention relates to a method for replacing post-heparin LPL activity measurement in identifying triglyceride and HDL-cholesterol metabolism disorders, the method comprising clinically replacing the post-heparin LPL activity measurement with administration of at least one inhibitor of ANGPTL3, ANGPTL4, ANGPTL8, and / or ANGPTL3 / 8 to a subject.

[0039] As used herein, the term "subject" includes humans, and more preferably includes a human patient in need of treatment. Even more preferably, the subject is a human patient diagnosed with or suspected of having severe hypertriglyceridemia (including, but not limited to, persistent chylomicronemia).

[0040] The present invention refers to the measurement of chylomicrons, vitamin E, and / or triglycerides, etc., in a subject's blood. In the context of the present invention, the term "blood" broadly encompasses not only whole blood, but also blood components such as plasma and serum. It is within the capabilities of one skilled in the art to determine whether measurements should be made in whole blood, serum, and / or plasma.

[0041] As used herein, the term "ANGPTL3" or "angiopoietin-like protein 3" refers to the protein encoded by the ANGPTL3 gene (Gene ID: 27329) in humans, which is also known as ANL3, ANG-5, FHBL2, and ANGPT5.

[0042] As used herein, the term "ANGPTL4" or "angiopoietin-like protein 4" refers to the protein encoded by the ANGPTL4 gene (Gene ID: 51129) in humans, which is also known as NL2, ARP4, FIAF, HARP, PGAR, HFARP, TGQTL, UNQ171, and pp1158.

[0043] As used herein, the term "ANGPTL8" or "angiopoietin-like protein 8" refers to the protein encoded by the ANGPTL8 gene (Gene ID: 55908) in humans, which is also known as RIFL, TD26, PRO1185, PVPA599, and C19orf80.

[0044] As used herein, the term "ANGPTL3 / 8" refers to the complex formed by ANGPTL8 and ANGPTL3.

[0045] The present invention encompasses the use of any suitable ANGPTL3, ANGPTL4, ANGPTL8, and / or ANGPTL3 / 8 inhibitor that can upregulate LPL and thereby distinguish subjects with basal LPL activity from subjects with little or no LPL activity. The inhibitor can be effective against one or more of the ANGPTL3, ANGPTL4, ANGPTL8 proteins, or the ANGPTL3 / 8 complex. Suitable inhibitors include, but are not limited to, antibodies (e.g., monoclonal antibodies); immunogens; gene silencers such as antisense oligonucleotides, small interfering ribonucleic acids (siRNAs), microRNAs, and small chemical molecules (e.g., oral drugs). ANGPTL3 antibodies include, but are not limited to, evinacumab (Evkeeza™) and the ANGPTL3 antibodies described in U.S. Patent Application Publication No. 2020 / 0061189, U.S. Patent Application Publication No. 2022 / 0127348, or U.S. Patent Application Publication No. 2022 / 0153825 (Regeneron Pharmaceuticals). Antibodies against ANGPTL8 include, but are not limited to, those described in WO 2017 / 177181. Antibodies against the ANGPTL3 / 8 complex include, but are not limited to, those described in U.S. Patent Application Publication No. 2022 / 0110537. Antisense oligonucleotides that inhibit ANGPTL3 protein synthesis include, but are not limited to, bupanorthene (Ionis Pharmaceuticals) and those described in International Publication No. 2020 / 099525 (Lipigon Pharmaceuticals AB). siRNAs against ANGPTL3 messenger RNA include, but are not limited to, ARO-ANG3 (Arrowhead Pharmaceuticals). Further examples include microRNA inhibitors against ANGPTL3 and / or ANGPTL8, such as those described in U.S. Patent Nos. 11,058,710 and 10,844,383 (Dasman Diabetes Institute).Also contemplated are CRISPR-based gene editing techniques (e.g., in vivo-based ANGPTL3, ANGPTL4, and / or ANGPTL8 editing via liposomes or adenoviral vectors), or any other techniques that inhibit ANGPTL3, ANGPTL4, ANGPTL8, and / or ANGPTL3 / 8. Preferably, an ANGPTL8 inhibitor is used in combination with an ANGPTL3 inhibitor, rather than used alone.

[0046] In one embodiment, LPL deficiency (e.g., complete LPL deficiency) is assessed by administering an ANGPTL3 inhibitor, an ANGPTL4 inhibitor, an ANGPTL8 inhibitor, and / or an ANGPTL3 / 8 inhibitor to a subject for a short period of time (e.g., less than 3 months, less than 2 months, or less than 1 month).

[0047] Any suitable method or route may be used to administer an inhibitor of ANGPTL3, ANGPTL4, ANGPTL8, and / or ANGPTL3 / 8 to a subject. These routes include oral, sublingual, rectal, topical, injection (e.g., intravenous, intramuscular, subcutaneous), etc. One skilled in the art can readily identify the optimal route of administration depending on various factors, such as the recommended or medically approved route for administering the inhibitor, potential side effects, and the expected rapidity of response.

[0048] Many suitable methods, techniques, or assays can be used to measure a subject's response to an inhibitor. In some embodiments, the response to an inhibitor is assessed by measuring the subject's triglyceridemia, i.e., the level of triglycerides in the subject's blood, or by measuring the subject's glycerolemia, i.e., the level of glycerol in the subject's blood. Furthermore, as described in U.S. Provisional Patent Application No. 63 / 587,192, entitled "Assessing Chylomicronemia Using Vitamin E," filed October 2, 2023, and incorporated herein by reference, vitamin E is absorbed and transported by chylomicrons. Therefore, according to the present invention, vitamin E can be used as a valuable surrogate marker for the level of chylomicrons and chylomicron remnants. In some embodiments of the methods of the invention disclosed herein, the response to an inhibitor is measured by assessing the subject's blood vitamin E level (i.e., the level of vitamin E in the subject's blood). In some embodiments, the subject's response to an inhibitor is measured both before and after administration of the inhibitor. Measurement of triglycerides, free glycerol, and / or vitamin E in a subject's blood can be performed using any suitable medical test.

[0049] In one embodiment, assessing lipoprotein lipase (LPL) bioavailability involves administering an ANGPTL3, ANGPTL4, ANGPTL8, and / or ANGPTL3 / 8 inhibitor for a short period of time (e.g., less than 3 months, less than 2 months, or less than 1 month) and measuring the subject's triglyceridemia (e.g., blood triglyceride levels) before and after administration of the inhibitor.

[0050] In some embodiments, the measured response identifies the subject as having a complete or partial LPL deficiency. In some embodiments, the measured response determines whether LPL is bioavailable. Furthermore, the measured response also provides information about health risks to LPL.

[0051] In some embodiments, the response to the inhibitor is measured by assessing the subject's triglyceridemia. In some embodiments, a low reduction in blood triglycerides after administration of the inhibitor to a subject indicates low LPL activity. In one particular embodiment, a reduction in blood triglycerides of 20% or more (e.g., ≥20%, ≥25%, ≥30%, ≥35%, ≥40%, ≥45%, or more) after administration of the inhibitor indicates the presence of LPL activity and LPL bioavailability, thereby ruling out complete LPL deficiency. In other embodiments, a reduction in blood triglycerides of less than 20% (e.g., <20%, <15%, <10%, <5%, or less) suggests a lack of LPL bioavailability and / or persistent chylomicronemia. Furthermore, no response or a reduction in blood triglycerides of less than 5% suggests complete LPL deficiency.

[0052] For greater certainty, if blood triglyceride reduction is about 20% and / or less than 20%, it is preferable to verify the diagnosis, e.g., to confirm the condition of persistent chylomicronemia or FCS. In one embodiment, the method further comprises using an LPL deficiency diagnostic scoring system to confirm the appropriate diagnosis of complete or partial LPL deficiency. In one embodiment, the method further comprises using vitamin E levels and / or lipolysis markers and / or assessing circulating lipoproteins in conjunction with the diagnostic scoring system.

[0053] Those skilled in the art can easily determine the amount and / or type of inhibitor to administer to a subject. These may depend on various parameters, such as the age, health status of the subject, fasting conditions, the nature of the inhibitor, and the use of a single or multiple inhibitors. For example, without wishing to be bound by theory, because chylomicronemia is a postprandial phenotype, administering two inhibitors (e.g., an ANGPTL3 inhibitor and an ANGPTL8 inhibitor), or administering a compound that inhibits both ANGPTL3 and ANGPTL8, may provide a more pronounced effect on postprandial LPL upregulation than using an ANGPTL3 inhibitor alone, making it a more sensitive diagnostic test or test to evaluate LPL bioavailability. However, in most cases, using a single inhibitor (e.g., an ANGPTL3 inhibitor) is believed to be sufficient. Selecting the appropriate dosage, administration schedule, administration method, etc. is within the capabilities of those skilled in the art. In certain embodiments, these parameters are in accordance with the accepted monography for the therapeutic use of inhibitors.

[0054] Advantageously, knowing a subject's status (e.g., whether they have complete or partial LPL deficiency) allows healthcare professionals to make more informed decisions regarding the patient's treatment plan. Thus, in some embodiments, the method further includes selecting a treatment plan taking into account the subject's response to the inhibitor. In some embodiments, the treatment plan aims to reduce the patient's triglyceridemia and / or chylomicronemia. For example, if a patient is diagnosed with complete LPL deficiency or familial chylomicronemia syndrome (FCS), the treatment plan may include using an LPL-independent agent, such as an ApoC3 inhibitor, using LPL gene replacement therapy, and / or using a genome editing strategy, preferably in combination with an adapted very-low-fat diet plus medium-chain fatty acids (MCTs). On the other hand, if a patient is diagnosed with partial LPL deficiency or multifactorial chylomicronemia, depending on the clinical phenotype and comorbidities, the treatment plan may include the use of LPL-dependent drugs such as ANGPTL3 inhibitors and / or ANGPTL3 / 8 inhibitors, or LPL-independent drugs alone or in combination with an adapted diet.

[0055] Another aspect of the present invention relates to a kit for distinguishing between complete and partial lipoprotein lipase (LPL) deficiency in a subject. In one embodiment, the kit comprises (i) an inhibitor of ANGPTL3, ANGPTL4, ANGPTL8, and / or ANGPTL3 / 8, and (ii) components for measuring triglycerides, vitamin E, and / or free glycerol blood levels in a subject.

[0056] As will be appreciated, the present invention provides numerous advantages and benefits. For example, the present invention allows for the diagnosis of individuals with multifactorial chylomicronemia or other causes of persistent chylomicronemia, or for the diagnosis of individuals with residual LPL bioavailability from individuals with complete LPL deficiency or familial hyperchylomicronemia syndrome (FCS). Advantageously, this can be done without relying on the typical post-heparin LPL activity assay, which is known to be invasive and provide variable results between laboratories. The present invention therefore provides a non-invasive and reliable alternative to the common post-heparin LPL activity assay. Therefore, the use of inhibitors of ANGPTL3, ANGPTL4, ANGPTL8, and / or ANGPTL3 / 8 may be useful for replacing the post-heparin LPL activity assay in identifying disorders of triglyceride and HDL-cholesterol metabolism.

[0057] The use of inhibitors of ANGPTL3, ANGPTL4, ANGPTL8, and / or ANGPTL3 / 8 may also be useful to replace, confirm, and / or refute a presumptive genetic diagnosis of persistent chylomicronemia or FCS in individuals in need of such a diagnosis. Because not all mutations and factors causing complete LPL deficiency have been identified or reported, the present invention may enable the identification of some unidentified cases of persistent chylomicronemia, complete LPL deficiency, and / or FCS. Thus, the present invention may be used to confirm whether a novel genetic mutation is responsible for complete LPL deficiency. Similarly, the present invention may be used to prove that a newly identified mutation is not loss-of-function (null). For example, if a novel mutation is discovered and the individual responds to an ANGPTL inhibitor, this may indicate that the mutation is non-null and may increase the severity of the disease rather than causing it.

[0058] Furthermore, the present invention facilitates patient treatment based on the bioavailability of LPL.

[0059] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, numerous equivalents to the specific procedures, embodiments, claims, and examples described herein. Such equivalents are considered to be within the scope of this invention and covered by the claims appended hereto. The present invention is further illustrated by the following examples, which should not be construed as further or specifically limiting. [Example]

[0060] Example 1: Correlation between chylomicronemia diagnostic score and post-heparin lipoprotein lipase activity A study was conducted to correlate the FCS diagnostic scoring system with postheparin activity.

[0061] Briefly, postheparin plasma LPL activity was measured using a colorimetric assay in 29 subjects with persistent chylomicronemia (20 with FCS and 9 with MCS). Correlation analyses were performed to estimate the linear relationship between LPL activity and two diagnostic scoring systems that integrated (Model A) or did not (Model B) apolipoprotein B and free glycerol, a surrogate marker of triglyceride hydrolysis.

[0062] As shown in Figure 1, a significant difference (p<0.001) was observed in post-heparin LPL activity between FCS and MCS. Both scoring systems were significantly correlated with post-heparin LPL activity (Model A:r s =-0.64, p=0.001; Model B: r s =-0.54, p=0.002).

[0063] Overall, when postheparin LPL activity levels from both assays were combined, the association between LPL activity and Model A score tended to be slightly stronger than the association with Model B score (rs =-0.64; p<0.001 vs. r s =-0.54; p=0.002) (see Figure 1). The sensitivity and specificity for predicting LPL activity <20% were similar for both scoring systems (see Table 4). [Table 1]

[0064] These results suggest that the chylomicronemia diagnostic scoring system correlates with LPL activity and contributes appropriately to distinguishing FCS from MCS.

[0065] Therefore, this study supports the idea that inhibitors of ANGPTL3, ANGPTL4, ANGPTL8, and / or ANGPTL3 / 8 can be used as an alternative to measuring LPL activity after heparin administration in identifying disorders of triglyceride and HDL cholesterol metabolism. For example, if the response to circulating triglyceride reduction with ANGPTL3, ANGPTL3 / 8, or ANGPTL4 inhibitors is less than 20%, the presence or absence of bioavailable LPL can be confirmed using the FCS diagnostic scoring system.

[0066] Therefore, the present invention provides a non-invasive and reliable alternative to the common post-heparin LPL activity assay, and therefore may serve as a clinical substitute for post-heparin LPL activity assays for decision-making purposes.

[0067] Example 2: Identification of a novel, unidentified LPL gene mutation responsible for FCS Evinacumab is an angiopoietin-like protein 3 (ANGPTL3) monoclonal antibody developed for the treatment of a wide range of lipid disorders. As mentioned herein, the effectiveness of evinacumab in lowering blood triglycerides depends on the bioavailability of LPL.

[0068] We evaluated LPL genotype and drug response in patients with persistent chylomicronemia treated with evinacumab.

[0069] In brief, a 20-week phase II clinical trial of evinacumab was conducted in patients with severe hypertriglyceridemia and chylomicronemia to evaluate its safety and efficacy. The study included three cohorts, one of which included patients with persistent chylomicronemia and LPL deficiency (LPLD). LPL genotype was assessed in all patients. Plasma triglyceride (TG) levels were measured at baseline and every 2 weeks for 20 weeks. Genotype-specific responses to evinacumab were assessed by comparing TG levels before and after treatment.

[0070] In this phase II study, one patient was identified as homozygous for a previously unreported mutation (E282X) in the LPL gene, and this patient did not respond to evinacumab (no TG reduction after 20 weeks), consistent with the lack of LPL bioavailability. Therefore, this mutation is suspected to be the cause of LPLD (OMIM:609708).

[0071] Thus, this study supports that inhibitors of ANGPTL3, ANGPTL4, ANGPTL8, and / or ANGPTL3 / 8 may also be used to identify genetic mutations responsible for complete LPL deficiency.

[0072] Headings are used herein for reference and to aid in locating particular sections. These headings are not intended to limit the scope of the concepts described therein, and these concepts may be applicable to other sections throughout the specification. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

[0073] The singular forms "a," "an," and "the" also include the corresponding plural forms unless the context clearly dictates otherwise. For example, reference to "an inhibitor" includes one or more of such inhibitors, and reference to "the method" includes reference to equivalent steps and methods known to those skilled in the art that may modify or substitute for the methods described herein.

[0074] Unless otherwise noted, all numerical values ​​expressing ingredient quantities, reaction conditions, concentrations, properties, and the like used in the specification and claims should be understood to be modified by the term "about." At the very least, each numerical parameter should be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Accordingly, unless otherwise indicated, the numerical parameters set forth in the specification and appended claims are approximations that may vary depending upon the properties sought. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the embodiments are approximations, the numerical values ​​set forth in the specific examples are reported as precisely as possible. However, any numerical value inherently contains certain errors resulting from variations in experiments, testing measurements, statistical analyses, and the like.

[0075] It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications and changes in light thereof will be suggested to those skilled in the art and are within the scope of the present invention and the appended claims.

Claims

1. 1. A method of distinguishing between complete and partial lipoprotein lipase (LPL) deficiency in a subject, comprising: (i) administering to a subject at least one inhibitor of ANGPTL3, ANGPTL4, ANGPTL8, and / or ANGPTL3 / 8; (ii) measuring the subject's response to the inhibitor; and wherein the response identifies the subject as having complete or partial LPL deficiency.

2. 10. The method of claim 1, wherein measuring the subject's response comprises measuring the subject's triglyceridemia.

3. 3. The method of claim 1 or 2, wherein measuring the subject's response comprises measuring the subject's glycerolemia.

4. 3. The method of claim 1 or 2, wherein measuring the subject's response comprises measuring the level of chylomicrons in the subject's blood.

5. 5. The method of claim 4, wherein the subject's chylomicron levels are measured by assessing the level of vitamin E in the blood.

6. 6. The method of any one of claims 1 to 5, wherein a decrease of 20% or more in blood triglycerides after administration of the at least one inhibitor indicates the presence of bioavailable LPL or partial LPL deficiency.

7. The method of any one of claims 1 to 5, wherein a decrease in blood triglycerides of less than 20% after administration of the at least one inhibitor indicates a lack of bioavailability of LPL.

8. The method of any one of claims 1 to 5, wherein a lack of response or a decrease in blood triglycerides of less than 5% after administration of the at least one inhibitor indicates complete LPL deficiency.

9. 9. The method of any one of claims 1 to 8, further comprising using an LPL deficiency clinical diagnostic scoring system to confirm an appropriate diagnosis of complete or partial LPL deficiency.

10. 10. The method of claim 9, further comprising using a marker of lipolysis in conjunction with the diagnostic scoring system or further comprising assessing circulating lipoproteins.

11. The method of any one of claims 1 to 10, wherein the response further provides information about the subject's health risk of having an LPL deficiency.

12. The method according to any one of claims 1 to 11, wherein the LPL deficiency indicates at least one of multifactorial chylomicronemia, persistent chylomicronemia, complete LPL deficiency, or familial chylomicronemia syndrome (FCS).

13. The method of any one of claims 1 to 12, further comprising selecting a treatment regimen in consideration of said response.

14. 14. The method of claim 13, wherein the treatment regimen is adapted for the treatment of multifactorial chylomicronemia or the treatment of hyperchylomicronemia syndrome (FCS).

15. 14. The method of claim 13, wherein the treatment plan comprises reducing triglyceridemia and / or glycerolemia in the subject.

16. the subject has complete LPL deficiency, the treatment regimen includes using an LPL-independent drug such as an ApoC3 inhibitor; The method of claim 13.

17. the subject has partial LPL deficiency, the treatment regimen comprises administering to the subject an ANGPTL3 inhibitor and / or an ANGPTL3 / 8 inhibitor; The method of claim 13.

18. 18. The method of any one of claims 1 to 17, wherein the ANGPTL3, ANGPTL4, ANGPTL8, and / or ANGPTL3 / 8 inhibitor is selected from the group consisting of antibodies; immunogens; antisense oligonucleotides; small interfering ribonucleic acid (siRNA); microRNA; and small molecule compounds that inhibit the expression and / or biological activity of at least one of ANGPTL3, ANGPTL4, ANGPTL8, and ANGPTL3 / 8.

19. 19. The method of claim 18, wherein the ANGPTL3 antibody is evinacumab.

20. 19. The method of claim 18, wherein the antisense oligonucleotide is bupanorthene.

21. 19. The method of claim 18, wherein the siRNA is ARO-ANG3.

22. (i) administering to the patient at least one inhibitor of ANGPTL3, ANGPTL4, ANGPTL8, and / or ANGPTL3 / 8; (ii) measuring the patient's response to the inhibitor; and Including, The reduction in blood triglycerides in the patient confirms the bioavailability of LPL; The lack of reduction in blood triglycerides in the patient indicates a lack or scarcity of bioavailable LPL. A method for assessing the bioavailability of lipoprotein lipase (LPL) in patients with persistent chylomicronemia and / or familial hyperchylomicronemia syndrome (FCS).

23. 23. The method of claim 22, wherein measuring the patient's response comprises measuring the patient's triglyceridemia.

24. 24. The method of claim 22 or 23, wherein measuring the patient's response comprises measuring the patient's glycerolemia.

25. 24. The method of claim 22 or 23, wherein measuring the patient's response comprises measuring the level of chylomicrons in the patient's blood.

26. 26. The method of claim 25, wherein the patient's chylomicron levels are measured by assessing the level of vitamin E in the blood.

27. The method of any one of claims 22 to 26, further comprising selecting a treatment regimen taking into account the bioavailability of the LPL.

28. The LPL is absent or rare, the treatment regimen includes using an LPL-independent drug such as an ApoC3 inhibitor; 28. The method of claim 27.

29. the LPL is bioavailable; the treatment regimen includes using an LPL-dependent drug, such as an ANGPTL3 inhibitor; 28. The method of claim 27.

30. 30. The method of any one of claims 22 to 29, wherein the ANGPTL3, ANGPTL4, ANGPTL8, and / or ANGPTL3 / 8 inhibitor is selected from the group consisting of antibodies; immunogens; antisense oligonucleotides; small interfering ribonucleic acid (siRNA); microRNA; and small molecule compounds that inhibit the expression and / or biological activity of at least one of ANGPTL3, ANGPTL4, ANGPTL8, and ANGPTL3 / 8.

31. 19. The method of claim 18, wherein the ANGPTL3 antibody is evinacumab.

32. (i) administering to a subject at least one inhibitor of ANGPTL3, ANGPTL4, ANGPTL8, and / or ANGPTL3 / 8; (ii) measuring the subject's response to the inhibitor; 1. A method for distinguishing between recurrent and persistent chylomicronemia in a subject with severe hypertriglyceridemia or chylomicronemia, comprising:

33. 33. The method of claim 32, wherein measuring the subject's response comprises measuring the subject's triglyceridemia.

34. 34. The method of claim 32 or 33, wherein measuring the subject's response comprises measuring the subject's glycerolemia.

35. 34. The method of claim 32 or 33, wherein measuring the subject's response comprises measuring the level of chylomicrons in the subject's blood.

36. The method of any one of claims 32 to 35, wherein steps (i) and (ii) are repeated at different intervals.

37. 36. The method of any one of claims 32 to 35, wherein steps (i) and (ii) are repeated after one or several weeks.

38. 1. A method for replacing LPL activity measurement after heparin administration in identifying disorders of triglyceride and HDL-cholesterol metabolism, comprising: The method comprises replacing the measurement of LPL activity after heparin administration with administration of at least one inhibitor of ANGPTL3, ANGPTL4, ANGPTL8, and / or ANGPTL3 / 8 to the subject.

39. (i) an inhibitor of ANGPTL3, ANGPTL4, ANGPTL8, and / or ANGPTL3 / 8; (ii) a component for measuring blood levels of triglycerides and / or glycerol in a subject; 1. A kit for distinguishing between complete and partial lipoprotein lipase (LPL) deficiency in a subject, comprising:

40. (i) distinguishing between complete and partial lipoprotein lipase (LPL) deficiency in a subject; (ii) identifying the subject as having recurrent chylomicronemia; (iii) identifying the subject as having persistent chylomicronemia; and (iv) identifying the subject as having hyperchylomicronemia syndrome (FCS); Use of at least one inhibitor of ANGPTL3, ANGPTL4, ANGPTL8, and / or ANGPTL3 / 8 for at least one of the following:

41. Use of at least one inhibitor of ANGPTL3, ANGPTL4, ANGPTL8, and / or ANGPTL3 / 8 to identify genetic mutations responsible for complete LPL deficiency in a human subject.

42. Use of at least one inhibitor of ANGPTL3, ANGPTL4, ANGPTL8, and / or ANGPTL3 / 8 to identify human subjects susceptible to response to LPL-dependent drugs.

43. Use of at least one inhibitor of ANGPTL3, ANGPTL4, ANGPTL8, and / or ANGPTL3 / 8 to identify human subjects who are likely to be unresponsive to LPL-dependent drugs.

44. Use of at least one inhibitor of ANGPTL3, ANGPTL4, ANGPTL8, and / or ANGPTL3 / 8 to identify human subjects eligible for an ANGPTL3 inhibition genome editing strategy.

45. Use of at least one inhibitor of ANGPTL3, ANGPTL4, ANGPTL8, and / or ANGPTL3 / 8 to identify human subjects eligible for LPL gene replacement therapy or LPL genome editing.

46. Use of at least one inhibitor of ANGPTL3, ANGPTL4, ANGPTL8, and / or ANGPTL3 / 8 to replace the LPL activity measurement test after heparin administration in identifying disorders of triglyceride and HDL-cholesterol metabolism.

47. (i) administering to a subject at least one inhibitor of ANGPTL3, ANGPTL4, ANGPTL8, and / or ANGPTL3 / 8; (ii) assessing the subject's response to the inhibitor by measuring the subject's triglyceridemia or the level of chylomicrons in the subject's blood, wherein the response identifies the subject as having complete or partial LPL deficiency; (iii) selecting a treatment regimen taking into account said response; 1. A method of treating a subject having complete or partial lipoprotein lipase (LPL) deficiency, comprising:

48. 48. The method of claim 47, wherein the treatment plan comprises reducing triglyceridemia in the subject.

49. the response identifies the subject as having complete LPL deficiency; the treatment regimen comprises administering to the subject at least one LPL-independent agent; 48. The method of claim 47.

50. 50. The method of claim 49, wherein the at least one LPL-independent agent comprises an ApoC3 inhibitor.

51. the response identifies the subject as having partial LPL deficiency; the treatment regimen comprises administering to the subject an ANGPTL3 inhibitor and / or an ANGPTL3 / 8 inhibitor; 48. The method of claim 47.

52. 52. The method of any one of claims 47 to 51, wherein the subject has chylomicronemia and / or familial chylomicronemia syndrome (FCS).

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