Cyclopropane-tagging method for tracking fatty acid transport between cells and tissues in vivo and in cell culture

Abstract

The invention relates to methods of tracking fatty acids and / or fatty acyl chains between an origin cell and one or more destination cells or locations in a multicellular structure, methods of diagnosis and screening related thereto, and nucleic acids encoding cyclopropane fatty acid synthase for use therein.

Description

[0001] Cyclopropane-tagging method fortracking fatty acid transport between cells and tissues in vivo and in cell culture

[0002] Background

[0003] Fatty acids (FAs) are major components of most lipids including glycerophospholipids, sphingolipids, diacylglycerols and triacylglycerols (Chandel, 2021). Fatty acid (FA) transport between different cell-types and tissues in the body is mediated by a diverse array of lipoproteins and transporters (Getz, 2018; Thomas and Tampe, 2020). It is essential for health and its disruption can be a causal factor in neurodegeneration, cancer and metabolic diseases (Currie et al., 2013; Fukushima and Lopaschuk, 2016; Islimye et al., 2022; Snaebjornsson et al., 2020; Yoon et al., 2021). Inter-organ FA transport, for example, between the gut, adipose tissue, liver, heart and muscle is mediated via route- selective lipoprotein particles and healthy crosstalk is disrupted in many metabolic diseases including obesity and type 2 diabetes (Denimal et al., 2023; Heeren and Scheja, 2021). Local intercellular transport of FAs between cells within organs is also emerging as a key contributor to health and disease. For example, metabolically stressed neurons secrete potentially toxic FAs that are stored and metabolized in astrocytes and microglia during oxidative stress and neurodegenerative disease (Islimye et al., 2022; Mallick et al., 2024; Ralhan et al., 2021 ; Yen and Yu, 2023). In the context of cancer, many tumours uptake large quantities of FAs from cells in the tumour microenvironment (TME) and beyond, such as cancer-associated fibroblasts (CAFs) and adipocytes (Baenke et al., 2013; Broadfield et al., 2021).

[0004] Stable and radioactive isotopes are widely used to track FA transport and metabolism (Faubert et al., 2021; Fernandez-Garcia et al., 2020). These isotope labelled FAs are administered from an exogenous source, such as the medium for cells cultured in vitro, or intravenous or dietary boluses for living animals and humans (Bartman et al., 2023; DeBerardinis and Keshari, 2022; Fernandez-Garcia et al., 2020). Other types of FA tracers, also administered via an exogenous source, are chemically synthesized to contain fluorophores like BODIPY or clickable alkyne tags covalently linked to the alkyl chain (Boldyrev et al., 2007; Gao and Hannoush, 2018; Johnson et al., 1991; Thiele et al., 2012). A major limitation of current isotopic and chemically modified FA tracers is that it is challenging to deliver them selectively to a defined cell-type within a living animal or a multicellular in vitro model such as an organoid. This substantially restricts the ability to monitor intercellular and inter-organ FA transport and metabolism. To overcome this barrier, it is necessary to develop genetically encoded methods that allow conditional and bioorthogonal tagging of FAs in vivo in a manner that preserves their bioactivity. A bacterial enzyme, cyclopropane fatty acyl phospholipid synthase (CFAse), converts a C=C double bond in the long-chain fatty acyl chains of phospholipids to a cyclopropane group (John Peter et al., 2022) (Figure 1A). Cyclopropane FA chains have similar biophysical properties to their unsaturated counterparts but they can be distinguished via mass spectrometry due to a molecular weight increase of 14 Daltons (Cronan and Luk, 2022; Grogan and Cronan, 1997; John Peter et al., 2022; Zhang and Rock, 2008). CFAse has previously been used for tracing intracellular phospholipid trafficking between organelles within a cell grown in vitro (John Peter and Kornmann, 2024; John Peter et al., 2022). We now develop genetically encoded methods enabling CFAse to be used to trace quantitatively, and in vivo, the extracellular transport and metabolism of free fatty acids, neutral lipids and phospholipids between different cell-types within a living animal. Our novel methods also enable CFAse to be used for quantitative tracing of FAs incorporated into neutral lipids, as well as phospholipids. We name these methods conditional cyclopropane fatty acid tagging (eFAT). As a proof-of-principle, eFAT is implemented in the genetic model organism Drosophila, first validating the technology in cell culture and then applying it in vivo to the study of intra-organ and inter-organ lipid transport between cells.

[0005] Summary of the invention

[0006] Provided herein is a method of tracking fatty acids and / or fatty acyl chains between an origin cell and one or more destination cells or locations in a multicellular structure, the method comprising:

[0007] (a) expressing cyclopropane fatty acid synthase (CFAse) under a conditional and / or cell-type specific expression system in the origin cell;

[0008] (b) catalysing the addition of a mass-tag to one or more fatty acids and / or fatty acyl chains in the origin cell with CFAse by introducing a methylene group from S-adenosyl methionine (SAM) at double bonds in the fatty acids and / or fatty acyl chains to form a cyclopropane ring, producing a mass-tagged cyclopropane derivative;

[0009] (c) detecting and / or quantifying the mass-tagged cyclopropane derivative in the one or more destination cells or locations.

[0010] The origin cell and destination cell may be different cell-types or the same cell-type. The mass-tagged cyclopropane derivative may be detected and / or quantified sequentially or simultaneously in two or more different cell-types. Alternatively, the destination location may be an extracellular location. For example, the destination location may be an extracellular fluids and / or secreted or excreted body fluid or substance. Examples include blood, plasma, hemolymph, lymph, cerebrospinal fluid, perspiration, urine or faeces. The mass-tagged cyclopropane derivative may be detected and / or quantified sequentially or simultaneously in both cellular and extracellular locations.

[0011] The origin cell and destination cell may be different cell-types or tissues in a living organism, organoid, or cell culture system.

[0012] The fatty acids may be monounsaturated fatty acids. The fatty acyl chains may be phospholipid or neutral lipid fatty acyl chains. The monounsaturated fatty acids may be palmitoleic acid and / or oleic acid. The fatty acyl chains may be part of phosphatidylethanolamines (PEs), phosphatidylcholines (PCs), phosphatidylserines (PSs), phosphatidylinositols (Pls), phosphatidyl glycerols (PGs), ether lipids (ELs), phosphatidic acids (PAs), lysophospholipids (LPs), acylcarnitines (AcCar), diacylglycerols (DGs) and / or triacylglycerols (TGs). The CFAse may convert the fatty acid chain C16:1 into the cyclopropane fatty acid chain C17:0c and / or the fatty acid chain C18:1 into the cyclopropane fatty acid chain C19:0c.

[0013] The mass-tagged cyclopropane derivative may have equivalent bioactivity to a corresponding non-tagged fatty acid and / or molecule comprising a fatty acyl chain. For example, the mass-tagged cyclopropane derivative may retain more than 50%, more than 60%, more than 70%, more than 75%, more than 80%, more than 85%, more than 90%, more than 95%, more than 98%, more than 99%, or 100% of a bioactivity. The bioactivity may be growth, viability, rate of development, rate of hatching, survival, or body weight.

[0014] The CFAse may be expressed under a conditional promotor or a constitutive promotor. The CFAse may be expressed under the control of a conditional and / or cell-type specific expression system. The cell-type specific expression system may be specific for the origin cell. The CFAse may be expressed in all of the origin cells in the multicellular structure, or in a subset of the origin cells in the multicellular structure. The conditional or cell-type specific expression system may be selected from GAL4 / UAS, QF / QUAS, Flp / FRT, LexA / LexAop, Cre / Lox, Dre / Rox, Vika / Vox, FLEx, Tet-On / Tet-Off, conditional degron tags such as auxin-inducible degron (AID) or optogenetic methods such as ShineGAL4 / UAS. The CFAse may be heterologous to the organism or cell-type in which it is expressed. The CFAse may be expressed transiently. The CFAse may be introduced into the origin cell by transfection or transduction of DNA encoding CFAse or delivery of mRNA encoding CFAse. The CFAse may be introduced into the origin cell by a viral vector, for example a lentiviral vector. The CFAse may be introduced by CRISPR gene editing. The CFAse may be expressed from an integrated gene. Alternatively, the CFAse may be expressed from a nonintegrating vector or plasmid. The conditional expression system may be an optogenetic expression system.

[0015] The CFAse may be a bacterial CFAse or plant CFAse. The bacterial CFAse may be an Escherichia coli CFAse or CFAsea Pseudomonas fluorescens CFAse, the plant CFAse may be a Gossypium hirsutum CFAse, (Genbank: AAT74600) or a Sterculia foetida CFAse (GenBank: AAM33848.1). The CFAse may have an amino acid sequence comprising SEQ ID NO: 1 or SEQ ID NO: 5. The CFAse may have an amino acid sequence having at least 70%, at least 80%, at least 90% at least 95% or at least 99% identity or 100% identity to SEQ ID NO: 1 or 5. The CFAse may be encoded by a wild-type sequence or a codon optimised sequence. The CFAse sequence may be codon optimised for expression in a mammalian cell. For example, the codon optimised sequence may be optimised for expression in a human cell or a mouse cell. Alternatively, the codon optimised sequence may be optimised for expression in an insect cell. The CFAse may be encoded by a DNA sequence of SEQ ID NO: 2, 3, 4 or 6.

[0016] The CFAse may be fused to a targeting moiety targeting it to an organelle. The organelle may be the plasma membrane, the nuclear membrane, the endoplasmic reticulum, the Golgi apparatus, a mitochondrion, or a lipid vesicle. The lipid vesicle may be a lipid droplet, a peroxisome or a lysosome. The CFAse may be fused to a label. The label may be fluorescent protein (e.g. GFP).

[0017] The targeting moiety targeting the endoplasmic reticulum may be from the Ubc6 or Sec61beta proteins. The targeting moiety targeting the plasma membrane may be a myristylation signal, which may be from a Src protein. The targeting moiety targeting the nuclear membrane may be from the Lamin B receptor (LBR) or other nuclear membrane protein. The targeting moiety targeting the Golgi apparatus may be from Golgin-245 or Golgin-97 or the alternative open reading frame (altORF) within the mRNA of the centromere protein CENP-R. The targeting moiety targeting mitochondria may be from the Tom70 protein. The targeting moiety targeting the peroxisome may be from a peroxisomal membrane protein (PMP), which may be Pmp34 or Pex26. The targeting moiety targeting the lysosome may be from Lampl , Lamp2, or Ard1. The targeting moiety targeting the lipid droplet may be from CideA or a Peripilin protein such as Lsd-2 or Plinl . The targeting moiety targeting lipid vesicles may be from VAMP2 or another secretory vesicle membrane protein, Rab5 or another Rab GTPase or other endosomal membrane proteins, or from synaptophysin, synapsin, synaptobrevin (Syb or nSyb) or other synaptic or intracellular vesicle proteins. The multicellular structure may be a living organism, an organoid or a cell culture system. The living organism may be a mouse, a rat, a zebrafish, a Drosophila or a C.elegans. The organoid may be a patient-derived organoid. The cell culture system may be a cell coculture system or a multi-cell-type culture system. The multicellular structure may be an in vitro or ex vivo structure. The organoid or cell culture system may comprise pluripotent stem cells (iPSCs), or differentiated cells derived therefrom.

[0018] The multicellular structure may further comprise one or more extracellular fluids and / or secreted or excreted body fluid or substances. Examples include plasma, hemolymph, lymph, cerebrospinal fluid, perspiration, urine or faeces.

[0019] In some embodiments, the multicellular structure is in vitro. In some embodiments, the multicellular structure is ex vivo. In some embodiments, the multicellular structure is in vivo. According, in some embodiments, the method is an in vitro method. In some embodiments, the method is an ex vivo method. In some embodiments, the method is an in vivo method.

[0020] The iPSCs may be obtained from a patient or animal having or suspected of having a disease or disorder associated with lipid transport and / or metabolism. The disease or disorder may be an inborn error of lipid metabolism, a cancer or a neurodegenerative disease. The inborn error of lipid metabolism may be Very long-chain acyl-coenzyme A dehydrogenase deficiency, Malonyl-CoA decarboxylase deficiency, Primary carnitine deficiency, Niemann-Pick disease, Wolman disease, Cholesteryl ester storage disease, Fabry disease, Farber's disease, Gangliosidoses, Gaucher disease, Krabbe disease, Metachromatic leukodystrophy, Mitochondrial trifunctional protein deficiency, Tangier disease or Wolman disease. The cancer may be carcinoma, gastric cancer, pancreatic cancer, colon cancer, colorectal cancer, lung cancer, prostate cancer, testicular cancer, liver cancer, neuroblastoma or glioma. The neurodegenerative disease may be Alzheimer’s disease, Parkinson's disease, Amyotrophic lateral sclerosis, Friedreich ataxia, Huntington's disease, Lewy body disease, primary lateral sclerosis, progressive muscular atrophy, or Spinal muscular atrophy.

[0021] The origin cell may be selected from a neuron, a glia, a hepatocyte, an adipocyte, an enterocyte, a muscle cell, an epidermal cell, a kidney cell, a stem cell, a hematopoietic cell or a blood cell. The destination cell may be selected from a neuron, a glia, a hepatocyte, an adipocyte, an enterocyte, a muscle cell, an epidermal cell, a kidney cell, a stem cell, a hematopoietic cell or a blood cell. Alternatively, the origin cell and / or destination cell may be a cancer or tumour cell.

[0022] The origin cell may be a neuron and a destination cell may be a glial cell or the destination cell may be a glial cell and the origin cell may be a neuron. The origin cell may be an adipocyte and a destination cell may be an adipocyte or the destination cell may be a hepatocyte and the origin cell may be an adipocyte. The origin cell may be a cancer cell or tumour cell and the destination cell may be a non-cancerous cell. The destination cell may be a cancer cell or a tumour cell and the origin cell may be a non-cancerous cell. The non- cancerous cell may be a cell in the tumour microenvironment. Alternatively, the origin and destination cells may both be cancer cells or tumour cells.

[0023] The detection and / or quantification may be carried out by any suitable detection or quantification method, i.e. a method suitable for detecting the mass-tagged cyclopropane derivative in the multicellular structure. The detection and / or quantification may be carried out by mass spectrometry (MS), Mass spectrometry imaging (MSI) or nuclear magnetic resonance (NMR).

[0024] The mass-tagged cyclopropane derivative may comprise one or more rare isotopes. The rare isotope may be a stable isotope or a radioactive isotope. For example, the stable isotope may be13Carbon,15Nitrogen or2Hydrogen (deuterium)17Oxygen or18Oxygen or the radioactive isotope may be11Carbon,14Carbon or3Hydrogen (tritium).

[0025] The fatty acids and / or fatty acyl chains may be tagged with a rare isotope to be incorporated into the mass-tagged cyclopropane derivative. Alternatively, the methylene group from S-adenosyl methionine (SAM) may be tagged with a rare isotope to be incorporated into the mass-tagged cyclopropane derivative.

[0026] The isotope labelled fatty acids and / or fatty acyl chains or S-adenosyl methionine may be administered from an exogenous source. The exogenous source may be a cell culture medium, an intravenous or intraperitoneal injection, gavage, a dietary supplement, dietary bolus, or supplementation of the drinking water.

[0027] The mass-tag may be between 13 and 20 daltons. For example, the mass tag may be 14 daltons. Further provided is a method of diagnosing a disease or disorder associated with lipid transport and / or lipid metabolism, the method comprising:

[0028] (a) expressing CFAse under a conditional and / or cell-type specific expression system in a first cell-type, wherein the first cell-type contains one or more molecules comprising one or more mono-unsaturated fatty acyl chains;

[0029] (b) culturing an organoid, multi-cell-type culture system or cell co-culture system comprising one or more cells of the first cell-type and one or more cells of a second celltype, wherein the first cell-type is a cell obtained from a patient suspected of having a disease or disorder associated with lipid transport and / or lipid metabolism, and wherein the second cell-type is a cell-type which accepts or is suspected to accept molecules containing mono-unsaturated fatty acyl chains from the first cell-type under physiological conditions;

[0030] (c) producing a mass-tagged cyclopropane derivative by adding a mass-tag to one or more molecules comprising fatty acyl chains in the first cell-type, wherein the mass-tag is added by catalysing the introduction of a methylene group from SAM at double bonds within the fatty acyl chains with CFAse to form a cyclopropane ring;

[0031] (d) quantifying the amount or concentration of mass-tagged cyclopropane derivative in the second cell-type;

[0032] (e) comparing the amount or concentration of mass-tagged cyclopropane derivative in the second cell-type to a control value; wherein a change in the amount or concentration of mass tagged cyclopropane derivative relative to the control value is predictive of a disease or disorder associated with lipid transport and / or metabolism.

[0033] Further provided is a method of stratifying patients having a disease or disorder associated with lipid transport and / or metabolism, the method comprising:

[0034] (a) expressing CFAse under a conditional and / or cell-type specific expression system in a first cell-type, wherein the first cell-type contains one or more molecules comprising one or more mono-unsaturated fatty acyl chains;

[0035] (b) culturing an organoid, multi-cell-type culture system or cell co-culture system comprising one or more cells of the first cell-type and one or more cells of a second celltype, wherein the first cell-type is a cell obtained from a patient suspected of having a disease or disorder associated with lipid transport and / or lipid metabolism, and wherein the second cell-type is a cell-type which accepts or is suspected to accept molecules comprising mono-unsaturated fatty acyl chains from the first cell-type under physiological conditions; (c) producing a mass-tagged cyclopropane derivative by adding a mass-tag to one or more molecules comprising fatty acyl chains in the first cell-type, wherein the mass-tag is added by catalysing the introduction of a methylene group from S-Adenosyl methionine (SAM) at double bonds within the fatty acyl chains with CFAse to form a cyclopropane ring;

[0036] (d) quantifying the amount or concentration of mass-tagged cyclopropane derivative in the second cell-type;

[0037] (e) comparing the amount or concentration of mass-tagged cyclopropane derivative in the second cell-type to a control value;

[0038] (f) stratifying the patient into a risk category based on the difference between the amount or concentration of mass-tagged cyclopropane derivative and the control value.

[0039] The control value may be determined by carrying out steps (a) to (d) of the method of diagnosing a disease or disorder associated with lipid transport and / or metabolism, wherein the first cell-type is replaced with a wild-type cell of the same type, or a cell of the same type obtained from a healthy donor.

[0040] Alternatively, the control value may be a predicted value or expected value.

[0041] Further provided is a method of identifying the destination of a molecule comprising monounsaturated fatty acyl chains in a multicellular structure, the method comprising:

[0042] (a) expressing cyclopropane fatty acid synthase (CFAse) under a conditional and / or cell-type specific expression system in a first cell-type, wherein the first cell-type contains molecules comprising one or more mono-unsaturated fatty acyl chains;

[0043] (b) culturing an organoid, multi-cell-type culture system or cell co-culture system comprising one or more cells of the first cell-type, and one or more cells of at least one further cell-type, wherein the further cell-type is a cell-type that accepts or is suspected to accept molecules containing fatty acyl chains from the first cell-type;

[0044] (c) producing a mass-tagged cyclopropane derivative by adding a mass-tag to one or more molecules comprising fatty acyl chains in the first cell-type, wherein the mass-tag is added by catalysing the addition of a methylene group from SAM at double bonds within the fatty acyl chains with CFAse to form a cyclopropane ring;

[0045] (d) detecting the presence of the mass-tagged cyclopropane derivative in at least one further cell-type; wherein detection of the mass-tagged cyclopropane derivative in the further cell-type identifies the further cell-type as a destination of the molecule comprising monounsaturated fatty acyl chains. The first cell-type may be obtained from a patient suspected of having a disease or disorder associated with lipid transport and / or metabolism.

[0046] In the methods described herein, instead of (or in addition to) detecting or quantifying the mass-tagged cyclopropane derivative in a destination cell, second cell-type or further celltype, the mass-tagged cyclopropane derivative may be detected in an extracellular location. Suitable extracellular locations include extracellular fluids and / or secreted or excreted body fluid or substances. Examples include plasma, hemolymph, lymph, cerebrospinal fluid, perspiration, urine or faeces.

[0047] The organoid, multi-cell-type culture system or cell co-culture system may comprise at least two different cell-types. For example, the organoid, multi-cell-type culture system or cell co-culture system may comprise more than three, more than five, more than ten, or more than twenty different cell-types.

[0048] Detection and / or quantification of the mass-tagged cyclopropane derivative may be carried out in one or more further cell-types simultaneously or sequentially.

[0049] Further provided is a method of screening therapeutic agents for use in the correction of defects in lipid transport and / or metabolism, the method comprising:

[0050] (a) providing a multicellular structure comprising one or more cells of a first cell-type having, or suspected of having a defect in lipid transport and / or metabolism, and one or more cells of at least one further cell-type, wherein the first cell-type contains molecules comprising one or more fatty acyl chains and wherein the further cell-type is a cell-type that accepts or is suspected to accept molecules comprising fatty acyl chains from the first cell-type;

[0051] (b) expressing cyclopropane fatty acid synthase (CFAse) under a conditional and / or cell-type specific expression system in the first cell-type,

[0052] (c) producing a mass-tagged cyclopropane derivative by adding a mass-tag to one or more molecules comprising fatty acyl chains in the first cell-type, wherein the mass-tag is added by catalysing the addition of a methylene group from SAM at double bonds within the fatty acyl chains with CFAse to form a cyclopropane ring;

[0053] (d) quantifying the mass-tagged cyclopropane derivative in the second cell-type to establish the baseline level of fatty acid transport;

[0054] (e) administering a therapeutic agent for use in the correction of defective lipid transport and / or metabolism to the multicellular structure; (f) quantifying mass-tagged cyclopropane derivative in the second cell-type after administration;

[0055] (g) comparing the levels of the mass-tagged cyclopropane derivative in the second celltype after administering a therapeutic agent to the baseline level or to a control value.

[0056] The control value may be a predicted value or expected value.

[0057] The first cell-type may be obtained from a patient suspected of having a disease or disorder associated with lipid transport and / or metabolism. Alternatively, the first cell-type may obtained from a healthy donor. The first cell-type may obtained from a vertebrate or invertebrate model organism. The vertebrate or invertebrate model organism may comprise one or more genetic mutations associated with, or suspected to be associated with, a disease or disorder of lipid transport and / or metabolism in humans. The first eukaryotic cell-type may be a pluripotent stem cell. The first eukaryotic cell-type may be a cell line, for example, an immortalised human or animal cell line.

[0058] The first eukaryotic cell-type may have been modified or conditioned to replicate the symptoms of a disease or disorder associated with lipid transport and / or metabolism. The first cell-type may have been modified to comprise one or more genetic mutations associated with, or suspected to be associated with, defects in lipid transport and / or metabolism. The first cell-type may have been exposed to or one or more environmental conditions associated with, or suspected to be associated with, defects in lipid transport and / or metabolism. For example, the first cell-type may have been exposed to thermal stress, metabolic stress, oxidative stress, mechanical stress, pH stress, pathogens, hormones, growth factors, inflammatory mediators, heavy metals, solvents, toxins or radiation.

[0059] The multicellular structure may further comprise one or more cells of a third, fourth or further cell-type. The multicellular structure may be an organoid or cell culture system comprising some or all of the cell-types found in a corresponding organ or tissue. For example, the organoid or cell culture system may comprise some or all of the cell-types found in the liver, kidneys, intestines, heart, stomach, pancreas, lungs, skin or brain.

[0060] The at least one further cell-type may be wild-type cells, or cells obtained from a healthy donor. The cells of the second cell-type may have the same disease or disorder, or have undergone the same modification or conditioning as the first cell-type. The baseline level or control value of step (f) may be a value indicative of an abnormal or pathological state in the first cell-type. The baseline level or control value may be higher than if the cells of the first cell-type were wild-type or unconditioned cells of the same type, or cells of the same type obtained from a healthy donor. Alternatively, the baseline level or control value may be lower than if the cells of the first cell-type were wild-type or unconditioned cells of the same type, or cells of the same type obtained from a healthy donor.

[0061] A change in the levels of the mass-tagged cyclopropane derivative in the second cell-type relative to baseline levels may indicate the therapeutic agent is capable of modifying lipid transport and / or metabolism. Therapeutic agents capable of modifying lipid transport and / or metabolism may be identified as candidate therapeutic agents for use in the treatment of defects in lipid transport and / or metabolism. A change in the levels of the mass-tagged cyclopropane derivative in the second cell-type after administering a therapeutic agent in comparison to the baseline or control value may represent a partial or complete reversal of the defect in lipid transport and / or metabolism.

[0062] The levels of the mass-tagged cyclopropane derivative may be determined by the amount or concentration of mass-tagged cyclopropane derivative.

[0063] Further provided is a nucleic acid having a sequence of SEQ ID NO: 2, 3, 4, 6, 7, 8, 9, 10, 11 or 12.

[0064] Further provided is a recombinant expression vector comprising the sequence of SEQ ID NO: 2, 3, 4, 6, 7, 8, 9, 10, 11 or 12.

[0065] Further provided is a recombinant expression vector, wherein the nucleic acid sequence is under the control of a conditional and / or cell-type specific expression system.

[0066] The recombinant expression vector of the invention may be under the control of the expression system GAL4 / UAS, QF / QUAS, Flp / FRT, LexA / LexAop, Cre / Lox, Dre / Rox, Vika / Vox, FLEx, Tet-On / Tet-Off, conditional degron tags such as auxin-inducible degron (AID) or optogenetic methods such as ShineGAL4 / UAS.

[0067] Further provided is a host cell comprising a recombinant expression vector of the invention.

[0068] The host cell may be an iPSC. Further provided is a genetically modified organism expressing the CFAse of SEQ ID NO: 1 or 5 under the control of a conditional and / or cell-type specific expression system.

[0069] The organism may have the expression system GAL4 / UAS, QF / QUAS, Flp / FRT, LexA / LexAop, Cre / Lox, Dre / Rox, Vika / Vox, FLEx, Tet-On / Tet-Off, conditional degron tags such as auxin-inducible degron (AID) or optogenetic methods such as ShineGAL4 / UAS.

[0070] Brief description of the figures

[0071] Figure 1 : CFAse cyclo-tags lipids in Drosophila S2 cells

[0072] A. Scheme representing the CFAse reaction. Briefly CFAse uses SAM as a Carbon donor to transform C16:1 fatty acid chain contained within a phospholipid (PL) such as phosphatidylethanolamine into a cyclopropane fatty acid C17:0c.

[0073] B. Schemes representing the CFAse constructs used in the study. The coding sequence of Escherichia coli or Pseudomonas fluorescens CFAse was codon optimized for insect cell expression and cloned in frame with a V5 tag and a sequence targeting one of the following subcellular compartments: ER (with Ubc6 or Sec61 beta), plasma membrane (myristylation site), mitochondria (Tom70). The diagram below represents the orientation of CFAse and its organelle targeting sequence in a membrane lipid bilayer.

[0074] C. Western blot depicting the expression levels of the different CFAse constructs in Drosophila S2 cell. S2 Cells were transfected with actin-Gal4, actin-GFP, and UAS-CFAse. Anti-V5 was used to detect the CFAse constructs, GFP-HA acts as a transfection control.

[0075] □. Representative GCMS-FAME chromatogram depicting the retention time of different S2 cell fatty acids: C16:1 , C16:0, C18:1 and C18:0 and their cyclo-tag counterpart C17:0c and C19:0c. Control cells without CFAse (top, grey) do not contain detectable level of cyclotagged fatty acids while CFAse expressing cells contain C17:0c and C19:0c.

[0076] E. Dot plot showing the relative abundance of the principal fatty acids in S2 cells measured by GCMS-FAME. Cells are transfected with control vector (grey) or CFAse targeted to the ER (orange), to mitochondria (purple) or to the plasma membrane (blue).

[0077] F. Dot plot showing the % of cyclo-conversion of C16:1 and C18:1 in these cells.

[0078] G. Phospholipid class identified by LCMS analysis in S2 lipid extract. S2 cell contain more phospholipids such as phosphatidylethanolamine (PE), phosphatidylcholine (PC) and phosphatidylserine (PS) than neutral lipids.

[0079] H. Plot showing the percentage conversion of cyclo-tagged lipid species of the principal lipid classes in S2 cells. Control cells are depicted with grey dots and CFAse expressing cells with orange dots (UAS-CFAse. ER. 7). Phosphatidylethanolamines (PEs), phosphatidylcholines (PCs), phosphatidylinositols (Pls), phosphatidylglycerols (PGs), lysophospholipids (LPs), acylcarnitines (AcCar) and triacylglycerols (TGs) are shown.

[0080] I. Heatmap representing the square root transformed relative abundance of the principal lipid species identified for the selected lipid classes PC, PE, and PI. Cells expressing GFP on the left (- CFAse), and cells expressing CFAse.ER.1 on the right (+). Lipid species containing cyclo-tagged fatty acids are indicated in red and were found only in CFAse expressing cells.

[0081] Figure 2: CFAse is not toxic in larvae, and cyclopropane tags lipids in vivo

[0082] A. Western blot depicting the expression levels of the different CFAse constructs in larvae using a ubiquitous driver (tubuline-GAL4). Anti-V5 was used to detect CFAse constructs, loading control is Histone H3.

[0083] B. Time to pupariation for indicated genotypes.

[0084] C. % of adult emerging for the depicted genotypes.

[0085] D. Adult female weight for indicated genotypes.

[0086] E. Dot plot showing the relative abundance of principal fatty acids in L3 larvae measured by G CMS- FA ME. Larvae are expressing ubiquitously GFP (grey) or CFAse targeted to the ER (orange), to mitochondria (purple) or plasma membrane (blue). Genotypes shown on the graph from left to right are: GFP, CFAse::ER.1 , CFAse::ER.2, Mito::CFAse, PM::CFAse and PM::PfCFAse.

[0087] F. Dot plot showing the % of cyclo-conversion of C16:1 and C18:1 in larvae of the indicated genotype. Genotypes shown on the graph from left to right are: CFAse:: ER.1 , CFAse::ER.2, Mito::CFAse, PM::CFAse and PM::PfCFAse.

[0088] □. Relative abundance of neutral lipids and phospholipids in larvae lipid extract (left). Relative abundance of each phospholipid class (right).

[0089] H. Plot depicting the percentage of lipid species cyclo-tagged in the principal lipid classes measured by LCMS. Larvae expressing GFP are in depicted with grey dots (tub>GFP) and larvae expressing CFAse (tub> CFAse. ER. 7) with orange dots.

[0090] I. Heatmap representing the square root transformed relative abundance of the principal lipid species identified for the selected phospholipid classes PC and PE. Larvae expressing GFP on the left (- CFAse), and larvae expressing CFAse.ER.1 on the right (+). Lipid species containing cyclopropane fatty acids are indicated in red and were found only in larvae expressing CFAse.

[0091] Figure 3: eFAT can be used to track lipid transfer within the CNS. A. Scheme of the Drosophila larval central nervous system (CNS) in ventral view (left) or in cross section (right). The cortex (Cx, grey), the region containing the neural stem cells and most neuronal and glial cell nuclei, and the neuropil (Np, dark grey), the region dense in lipid-rich neuronal processes, are depicted. Anterior-posterior (A-P) and dorso-ventral (D-V) axes are indicated.

[0092] B. Confocal images of CNS cross sections showing nuclei (stained with DAPI, left panel) and membrane GFP expression under a the control of a neuronal driver (nSyb- Gal4, right panel). Nuclei are restricted to the cortex, whereas neuronal processes visualised with membrane GFP are present in both cortex and neuropil.

[0093] C. OrbiSIMS images of CNS cross-sections showing the spatial distributions of the fatty acids C16:0, C16:1 and C17:0c, as well as the C17:0c / C16:1 ratio from control larvae expressing GFP (top row) or experimental larvae expressing CFAse.ER. 1 (bottom row) under the control of a neuronal driver (nSyb-Gal4). Expression of CFAse induces C17:0c accumulation strongly in the neuropil and more weakly in a subregion of the cortex.

[0094] D. OrbiSIMS images of CNS cross-sections showing the spatial distributions of the fatty acids C18:0, C18:1 and C19:0c, as well as the C19:0c / C18:1 ratio from control larvae expressing GFP (top row) or experimental larvae expressing CFAse (bottom row) under the control of a neuronal driver (nSyb-Gal4). Expression of CFAse induces C19:0c accumulation strongly in the neuropil and more weakly in a subregion of the cortex.

[0095] Figure 4: eFAT can be used to track lipid transfer between organs.

[0096] A. Scheme depicting a simplified cross section of a Drosophila larvae showing the internal organisation of tissues secreting lipids in the hemolymph and the CNS. The fat body, analogue to the mammalian liver / adipose tissue (orange), and the gut (light blue) can both secrete lipoproteins. Skeletal muscles are also depicted as a large tissue that is not known for lipid secretion activity. The Central Nervous System (CNS) is depicted as a tissue receiving lipids.

[0097] B. Dot plot showing the % of cyclo-tagging of C16:1 in selected larval tissue: Fat Body, Gut Carcass and CNS when CFAse is expressed within these tissues. Control without CFAse (GFP, grey), and CFAse expressing tissue (UAS-CFAse.ER.1). Tissue-specific drivers were used for each tissue: Lpp-Gal4 for the fat body, Mef2-Gal4 for the skeletal muscles, Mex-Gal4 for the gut and nsyb-Gal4 and repo-Gal4 for mature neurons, and glia respectively.

[0098] C. Heatmap representing the square root transformed relative abundance of the principal fatty acids circulating in the hemolymph when CFAse is expressed in the indicated tissues. Larvae expressing GFP on the left (- CFAse), and larvae expressing CFAse. ER.1 on the right (+) in the fat body (Lpp-Gal4), gut (Mex-Gal4) or Muscles (Mef2-Gal4). Cyclo-tagged fatty acids are indicated in red.

[0099] D. Plot showing the percentage of cyclo-tagged lipids in circulating hemolymph of larvae expressing CFAse specifically in fat body (Lpp-Gal4), gut (Mex-Gal4) or Muscles (Mef2- Gal4).

[0100] E. Heatmap representing the square root transformed relative abundance of the principal fatty acids found in the larval CNS when CFAse is expressed in the indicated tissues. Larvae expressing GFP are on the left (- CFAse), and larvae expressing CFAse (CFAse. ER.1) on the right (+) in the fat body (Lpp-Gal4), gut (Mex-Gal4) or Muscles (Mef2-Gal4). Cyclo- tagged fatty acids are indicated in red.

[0101] F. Plot showing the percentage of cyclo-tagged lipids in the CNS of larvae expressing CFAse specifically in fat body (Lpp-Gal4), gut (Mex-Gal4) or Muscles (Mef2-Gal4).

[0102] G.OrbiSIMS images of CNS cross-sections showing the spatial distributions of the fatty acids C16:0, C16:1 and C17:0c, as well as the C17:0c / C16:1 ratio from control larvae expressing GFP (top row) or experimental larvae expressing CFAse (bottom row) under the control of a fat body-specific driver (Lpp-Gal4).

[0103] H. Quantifications of the C16:0, C16:1 and C17:0c signal intensities per pixel for the indicated genotypes in panel G. Expression of CFAse in the fat body leads to remote accumulation of C17:0c in the CNS.

[0104] Figure 5: Protein sequence from Escherichia coli CFAse (Taxon: 562, NCBI entry:

[0105] WP_ 139961088.1)

[0106] Figure 6: DNA sequence from Escherichia coli CFAse optimized for insect cell expression

[0107] Figure 7: DNA sequence from Escherichia coli CFAse optimized for mice expression

[0108] Figure 8: DNA sequence from Escherichia coli CFAse optimized for human cell expression

[0109] Figure 9: Protein sequence from Pseudomonas fluorescens CFAse (Taxon: 294, NCBI entry: KAF1008382.1)

[0110] Figure 10: DNA sequence from Pseudomonas fluorescens CFAse optimized for insect cell expression

[0111] Figure 11 : DNA sequence 2xV5::CFAse[Ecol]::Sec61 B. Escherichia coli CFAse sequence codon optimized for insect expression is indicated in grey.

[0112] Figure 12: DNA sequence 2xV5::CFAse[Ecol]::llbc6.

[0113] Escherichia coli CFAse sequence codon optimized for insect expression is indicated in grey.

[0114] Figure 13: DNA sequence Tom70::CFAse[Ecol]:: 2xV5.

[0115] Escherichia coli CFAse sequence codon optimized for insect expression is indicated in grey.

[0116] Figure 14: DNA sequence myr::CFAse[Ecol]::2xV5.

[0117] Escherichia coli CFAse sequence codon optimized for insect expression is indicated in grey.

[0118] Figure 15: DNA sequence myr::CFAse[Pflu]::2xV5.

[0119] Pseudomonas fluorescens CFAse sequence codon optimized for insect expression is indicated in grey.

[0120] Figure 16: DNA sequence 2xV5::CFAse[Ecol]::Sec61B.

[0121] Escherichia coli CFAse sequence codon-optimized for murine expression is indicated in grey.

[0122] Detailed description

[0123] Fatty acids (FAs) are exchanged between cells and tissues in health and disease. To understand the in vivo functions of FAs, it is important to be able to track their extracellular transport from one defined cell or tissue type to another in a quantitative manner. With existing stable isotope and chemically modified FA tracers, this is very challenging in any multicellular context such as a living animal or an in vitro organoid. Here we develop conditional cyclopropane fatty acid tagging (eFAT) to overcome this limitation. eFAT is a genetically encoded method enabling phospholipids with monounsaturated FA chains to be tagged in vivo, in a defined cell-type and in a conditional manner. The resultant cell-type specific FA tracer can then be used for quantitative tracing of metabolism and extracellular transport to other cells. As a proof-of-concept, we validate eFAT in the genetic model organism Drosophila, first in cell culture and then in live animals. We demonstrate that eFAT can be used to trace inter-organ FA transport in vivo from adipose tissue or enterocytes into the circulation and then on to remote tissues such as the central nervous system. In combination with mass spectrometry imaging, we show that eFAT also be used to monitor extracellular FA transport between cell-types within a tissue, such as the CNS. eFAT is a powerful method for tracing lipid exchange with many potential applications in biomedical research, drug screening and diagnostics.

[0124] We used Drosophila as a model to develop conditional cyclopropane fatty acid tagging (eFAT). eFAT is a bio-orthogonal and conditional method for the in vivo generation of a FA tracer in a cell-type specific manner. Using this approach, palmitoleate (C16:1) and oleate (C18:1) FA chains can be mass-tagged in a wide range of phospholipids in living animals without compromising growth, development or survival. Proof-of-principle was provided showing how eFAT can be implemented in vivo in a quantitative manner to trace both intraorgan and inter-organ extracellular FA transport in a living animal. These first applications of eFAT revealed new biology, showing that the developing CNS uptakes more of the two major monounsaturated FAs from the fat body than from the gut. This suggests a greater CNS contribution of FAs from de novo synthesis and / or desaturation in the fat body than from the diet.

[0125] CFAse can be expressed in a cell-type specific manner in any animal model, including mice, or in organoid or cell co-culture using a variety of conditional expression systems such as GAL4 / UAS, LexA / LexAop, Flp / FRT, Cre / Lox and FLEx as well as optogenetic variants of these that allow precise temporal control. Alternatively, CFAse can be delivered via plasmids or lentiviruses into cell co-cultures or organoids formed from induced pluripotent stem cells (iPSCs). We detected the 14 dalton FA mass tag added by CFAse using conventional mass spectrometry methods such as GC-MS and LC-MS. This provides sufficient resolution to distinguish between tissues that can be isolated via dissection. We also provided evidence that other analytical methods with greater spatial resolution can be used to detect cyclopropanated lipids and their routes of transport within a tissue, for example the CNS, with single-cell resolution. Hence, we combined eFAT with high resolution mass spectrometry imaging using OrbiSIMS. Using this powerful imaging approach, we quantified the enrichment of the C16:1 pool with cyclopropanated C17:1 (C17:0c / C16:1 ratio) in both the neuropil and cortex of the developing CNS. This revealed new biology, namely that neurons make a greater contribution to the total C16:1 of the neuropil than the cortex, whereas adipocytes contribute more to the cortex than to the neuropil. These findings illustrate that, in principle, eFAT can be used to determine the FA source cells / tissues for any destination cell-type of interest. eFAT has multiple applications in mechanistic lipid metabolism research, the clinical stratification of patients and drug screening. The key advance over other technologies is that the invention allows the movement of fatty acids from one cell-type to another to be monitored and quantified in 2D cell co-culture or in more complex 3D cell culture systems such as organoids (Whitehouse et al., 2023; Yang et al., 2023). Organoids show great promise in many therapeutic areas including cancer (Driehuis et al., 2020; Polak et al., 2024; Wensink et al., 2021) and neurodegeneration (Eichmuller and Knoblich, 2022; Jusop et al., 2023; Venkataraman et al., 2022). These may be derived from patients in good health or from those with diseases of inborn errors of lipid metabolism, or in more common diseases with an underlying genetic basis where altered lipid transport and / or metabolism have been implicated, such as cancer and neurodegeneration. Patient derived organoids and co-cultures expressing CFAse in a cell-type specific manner have several applications. For example, they may provide a diagnostic tool for the detection and stratification of defects in lipid transport / metabolism. In another example, they may be used to screen for drugs that correct defective lipid transport in organoid models of pancreatic cancer, or in organoid or neuronal-glial co-culture models of neurodegenerative disease.

[0126] Cyclopropane fatty acid synthase, also known as cyclopropane fatty acyl phospholipid synthase, (CFAse) is an enzyme belonging to the family of transferases. The CFAse enzyme converts mono-unsaturated fatty acids such as palmitoleic and oleic acid into their cyclopropane derivatives. This adds a 14 dalton mass tag but largely preserves their geometry and function in cell and organelle membranes.

[0127] The terms “mass-tagged”, “cyclo-tagged” and “cyclopropane-tagged” are used interchangeably and refer to a cyclopropane derivative of a fatty acid or fatty acyl chain which has an increased mass relative to the native fatty acid or fatty acyl chain, allowing the tagged molecule to be detected by assays such as mass spectrometry, mass spectrometry imaging or NMR.

[0128] The term cell-type refers to a group of cells sharing a common morphology, phenotype or gene expression programme. Examples of cell-types include neurons, glia, adipocytes, hepatocytes and cancer cells.

[0129] The term conditional expression system refers to a gene expression system comprising one or more regulatory elements which allow for spatial or temporal control of gene expression at the RNA or protein level. Such systems allow the expression of a particular gene to be restricted to specific cell-types or tissues, to specific developmental timeframes, or to specific environmental conditions.

[0130] The term cell-type specific expression system refers to a gene expression system comprising one or more regulatory elements which restrict the expression of the gene to one or more particular cell-types or tissues.

[0131] Conditional expression systems include cell-type specific expression systems, such as cell-type specific promoters, which are only able to initiate transcription of a gene in specific cell-types, inducible systems, which allow RNA or protein expression to be controlled by external factors such as the presence of a drug, light, temperature or another environmental condition, or recombinase systems, which activate or delete a gene in a conditional manner via site-specific recombination. Recombinase systems may be cellspecific or ubiquitous. Examples of such systems include GAL4 / UAS, QF / QUAS, Flp / FRT, LexA / LexAop, Cre / Lox, Dre / Rox, Vika / Vox, FLEx, Tet-On / Tet-Off, conditional degron tags such as auxin-inducible degron (AID) or optogenetic methods such as ShineGAL4 / UAS.

[0132] In embodiments employing a conditional expression system, the method may comprise a step of exposing the cell, cell culture-system or multicellular structure to the relevant condition or conditions necessary to trigger expression. As used herein, the term conditional expression system also encompasses inducible expressions systems; expression is induced by exposing the cell, cell culture-system or multicellular structure to the relevant condition or conditions necessary to trigger expression.

[0133] The term patient as used herein refers to a human subject who has, or is suspected of having, a disease or disorder, or is receiving, has received, or is intended to receive a medical treatment, diagnostic procedure, or prophylactic intervention in relation to a disease or disorder.

[0134] The patient may be a patient suspected of having a disease or disorder associated with lipid transport and / or metabolism, for example a disease or disorder associated with defects in lipid transport and / or metabolism. The disease or disorder may be an inborn error of lipid metabolism, a cancer or a neurodegenerative disease. The inborn error of lipid metabolism may be Very long-chain acyl-coenzyme A dehydrogenase deficiency, Malonyl-CoA decarboxylase deficiency, Primary carnitine deficiency, Niemann-Pick disease, Wolman disease, Cholesteryl ester storage disease, Fabry disease, Farber's disease, Gangliosidoses, Gaucher disease, Krabbe disease, Metachromatic leukodystrophy, Mitochondrial trifunctional protein deficiency, Tangier disease or Wolman disease. The cancer may be carcinoma, gastric cancer, pancreatic cancer, colon cancer, colorectal cancer, lung cancer, prostate cancer, testicular cancer, liver cancer, neuroblastoma or glioma. The neurodegenerative disease may be Alzheimer’s disease, Parkinson's disease, Amyotrophic lateral sclerosis, Friedreich ataxia, Huntington's disease, Lewy body disease, primary lateral sclerosis, progressive muscular atrophy, or Spinal muscular atrophy.

[0135] Examples Methods

[0136] UAS-CFAse constructs

[0137] Escherichia coli CFAse coding sequence was optimized for insect cell expression using the Genewiz codon optimization algorithm (Azenta Life Sciences) and fused in frame with 2x V5 tag on one end (C-term for plasma membrane and mitochondrial outer membrane, N-term for ER targeting), and an organelle targeting sequence on the opposite end. CFAse was targeted to the ER outer membrane using full length Sec61B / CG10130 (Summerville et al., 2016), or Ubc6 / CG5830 (aa255-281); to the plasma membrane using the myristoylation target sequence of Src64b / CG7524 (aa1-90) (Melom et al., 2013); and to the mitochondria outer membrane using aa1-33 from Tom70 / CG6756. The construct was then synthesized by Azenta Life Sciences and cloned into the UAS expression vector pW100 (created in this study based on pWALIUM10-roe, DGRC Stock #1471). The five constructs generated were named respectively: 2xUAS-loxP-6xUAS-loxP-2xV5::EcCFAse::Sec61 > 2xUAS-loxP-6xUAS-loxP-2xV5::EcCFAse::Ubc6 2xUA S-loxP-6xUA S-loxP-myr::EcCFAse::2x V5 2xUAS-loxP-6xUAS-loxP-myr::PfCFAse::2xV5 2xUAS-loxP-6xUAS-loxP-Tom70::.EcCFAse::2xV5.

[0138] All CFAse constructs were also cloned in a LexAop2 expression vector pJFRC13 (Addgene, #26224) (Pfeiffer et al., 2010). Table 1 lists all the new stocks generated in this study.

[0139] Fly transgenics

[0140] Transgenic flies were generated by cpC31 mediated insertion. Briefly, embryos of the background w1118; nos-cpC31; attP40 were injected with the p8xUAS-CFAse plasmid (~500ng / pL) to obtain F0 crossed to itself. Candidate recombinant flies were selected in the following generation (F1) based on presence of the mini-w+ transgene (orange eyes) and balanced over TM6B. 2xllAS CFAse lines were obtained by removal of loxP-6xL / AS- loxP by Cre-mediated recombination and verified by PCR.

[0141] Fly stocks and husbandry

[0142] Flies were kept at 25°C in a humidity-controlled chamber and raised on standard yeastbased diet. The majority of stocks used in this study were obtained from Bloomington Drosophila Stock Centre including: nSyb-Gal4 (#51635), Mef2-Gal4 (#27390), Mex1-Gal4 (#91368), repo-Gal4 (#7415), Cyp4g15-Gal4 (#39103), moody-Gal4 (#90883); (Padiath et al., 2006): 16951681), alarm-Gal4 (#Q7032), UA S-GFP (#35786). tubulin-Gal4 (II) was obtained from J.P. Vincent+ and Lpp-Gal4 (III) was kindly gifted by S. Eaton (Brankatschk and Eaton, 2010).

[0143] S2 cell culture and transfections

[0144] Drosophila S2 cells were kept in Schneider’s medium (Gibco, 21720-04) supplemented with Penicillin-Streptomycin (10000 U / rnL, Gibco) and 10% FBS (Sigma-Aldrich) at 25°C. For transfection, cells were pelleted 5 min at 5000 rpm and resuspended in fresh medium, the number of cells in the suspension was measured and cells were seeded in 6-well plates at a density of 3x106cells per well. After 20min, the transfection mix was prepared according to the Effectene manufacturer instruction (QIAGEN, #301427) with 0.3 pg of the following plasmids pWA-GAL4 (Usui et al., 1999), pAct5C-GFP-3xHA (Banerjee et al., 2017) and the pUAS-CFAse plasmid of interest. For negative control cells, only the two plasmids pWA-GAL.4 and pAct5C-GFP-3xHA were used. The transfection mix was then mixed with fresh medium and added drop by drop to the well to a final volume of 2.5mL. Transfected cells were then kept for 48 to 72 hrs in the incubator at 25°C before protein and lipid extraction.

[0145] Lipid extraction

[0146] For S2 cells, transfected cells were pelleted and rinsed once with PBS 1X. After removal of the PBS, 400 pL of methanol / chloroform (2:1, v / v) was added. Cells were then sonicated to resuspend the pellet and kept at -80°C until processed.

[0147] For larvae and individual tissues, late-stage larvae (L3) were floated in 30% glycerol rinsed three times with PBS, weighed and dissected in insect Ringer (Jarosz, 1988). Samples were then homogenized Precellys® metal tubes (Bertin Technologies, P002290-LYSK0- A.0) containing 1.4 mm ceramic beads (Bertin Technologies) and 500pL of methanol / chloroform (2:1, v / v). For each replicate, we used respectively 5 whole-larvae, 5 fat bodies, 5 guts, 5 carcasses (for muscles) or 10 brains. Samples were kept at -80°C until lipid extraction. Hemolymph extraction was modified form Fernando and colleagues’ protocol (Fernando et al., 2019). Briefly, 10 late-stage larvae (L3) were bled in a drop of Ringer (20pL) on a small plastic petri dish. The hemolymph extracted was then filtered through a 0.22-pm filter unit, collected in 200pL of ultrapure water and transferred to a glass vial containing 500pL of methanol / chloroform (2:1, v / v).

[0148] Lipids were extracted using an adapted Folch extraction method (Lubojemska et al., 2021). For the two-phase extraction 166pL of chloroform containing heneicosanoic acid as internal standard (Sigma-Aldrich, H-5149) and 300pL ultrapure water (100pL for hemolymph) were added to each sample for a final solvent ratio of 2:2: 1.8 methanol / chloroform / water. Samples were then vortexed, and centrifugated for 10min at 3000 rpm, before collection of the apolar phase in a glass vial. For analysis, a volume of lipid material corresponding to 1 / 8 whole larvae, 1 / 2 gut, 1 / 2 fat body, 1 / 2 carcass, 10 brain or 10 hemolymph was transferred to a glass insert (Agilent). The organic phase was then dried under vacuum and washed twice with methanol.

[0149] GC-MS based detection of cyclopropane-tagged fatty acids

[0150] The dry apolar samples, and dry FA standards were redissolved in 20 pL of methanol / chloroform (2:1, v / v) and 5 pL of the derivatization agent, trimethylphenylammonium hydroxide solution (Sigma-Aldrich, #79266). 1-palmitoyl-2-cis- 9,10-methylenehexadecanoyl-sn-glycero-3-phosphocholine was used as standard for C17:0c (Avanti Polar Lipids, 857501 P-1 MG) and cis-9,10-Methyleneoctadecanoic acid for C19:0c (Cayman Chemical, #24824). The analysis of derivatized apolar metabolites was performed using an Agilent 7890B-7000C GC-QqQ system (Agilent Technologies) operating in electron ionisation (El) mode as described previously (Lubojemska et al., 2021). A sample of 1 pl was injected into a deactivated splitless liner (270°C) using helium as the carrier gas (0.9 ml / min) and transferred to a 30 m x 0.25 mm DB-5MS + 10 m DuraGuard capillary column. The initial oven temperature was 70°C (1 minute), then ramped up to 230°C (15°C per minute), then to 325°C (at 25°C per minute, then a 3- minute hold). Data analysis was performed using Agilent Masshunter Quantitative Analysis software (Agilent Technologies, v10.1). FA methyl-esters (FAMEs) were identified by comparison to authentic standards. Raw ion counts FAMEs were normalized to the internal standard and to larval group wet weights.

[0151] LC-MS based detection of cyclopropane-tagged lipids

[0152] For LCMS analysis, 10pL of SPLASH® II LI PI DOM IX® (Avanti Polar Lipids, 330709W- 1EA) was added to each organic phase sample before drying under vacuum. Lipids were re-suspended in butanol / methanol (1 :1, v / v) containing 5 pM ammonium formate. The LC- MS method was adapted from Amiar et al. (32). Lipids were separated by injecting 5 pL aliquots onto a 2.1 x 100 mm, 1.8 pm C18 Zorbax Elipse plus column (Agilent) using a Dionex UltiMate 3000 LC system (ThermoScientific). A 20 min elution gradient of 45% to 100% Solvent B was used, followed by a 5 min wash of 100% Solvent B and 3 min reequilibration, where Solvent A was 10 mM ammonium formate in water (Optima HPLC grade, Fisher Chemical) and Solvent B was water / acetonitrile / isopropanol (5:20:75, v / v / v) with 10 mM ammonium formate (Optima HPLC grade, Fisher Chemical). Other parameters were as follows: flow rate 600 pL / min; column temperature 60°C; autosampler temperature 10°C. MS was performed with positive / negative polarity switching using a Exploris 240 Orbitrap (Thermo Scientific) equipped with a OptaMax™ NG ion source.

[0153] Mass calibration was performed before analysis using 1 -Point Mass Calibration. MS parameters were as follows: spray voltage 3.5 kV and 2.5 kV for positive and negative modes, respectively; ion transfer tube was set at 350°C and vaporizer at 400°C; sheath and auxiliary gases were 60 and 15 arbitrary units, respectively. The MS1 scans were acquired at 60,000 resolution (Full Width Half Maximum) over a range of m / z 150-2000. Default charge state was 1 and the expected peak width was 15 s. The RF lens was 70%, the AGC target was standard, the maximum injection time was auto, and microscan was 1. Advanced peak determination and internal mass calibration using EASY-ICTM were enabled. AcquireX Deep Scan was used to create a background ion exclusion list from the matrix blank and an ion inclusion list from a pooled quality control (PBQC) sample. Default Deep Scan settings were used except that [M+H]+1 , [M-NH3+H]+1 and [M-H]-1 were preferred ions and isotopes of fragmented precursors were excluded. Then, three injections of the PBQC were performed by AcquireX to generate MS2 spectra.

[0154] Monoisotopic precursor selection was enabled, the minimum intensity was 20000, dynamic exclusion was set at 1 time and 5s, and targeted mass and targeted mass exclusions had 5 ppm mass windows. Ten precursor ions per scan were selected within a 0.4 Da isolation window and were fragmented by high energy collision-induced dissociation (10%, 20%, 45% normalized stepped collision energy), and their MS2 fragment ions were resolved in the Orbitrap at 15,000 resolution (FWHM) with standard AGC target, maximum injection time of 100 ms, and 1 microscan. After each ID injection, the m / z for resolved ions were automatically appended to the exclusion list for the subsequent injection. This entire procedure was repeated using the same samples but in negative ion mode. Data was recorded using Xcalibur 4.6.67.17 software (Thermo Scientific). Qualitative and quantitative analyses were performed using Free Style 1.8 (Thermo Scientific), Progenesis (Nonlinear Dynamics). Lipid annotation was performed using the R tool LipidMatch v.4.2 (Koelmel et al., 2017) using customized in-silico fragment libraries for cyclo-tagged lipid identification. Data visualization was performed using R (v. 4.3.2). Detection of cyclopropane-tagged lipids using mass spectrometry imaging

[0155] Larval CNS were dissected in PBS and fixed in 4% PFA / PBS for 30min at RT. After fixation, samples were washed briefly with PBS and stained with 13pM Toluidine Blue O for 30min. Stained CNSs were washed with PBS, then with ammonium formate (150mM) before embedding in 4% carboxymethyl cellulose in ammonium formate (150mM) and flash frozen in a bath of dry ice and 2-methylbutane. 10pm cross sections were then made using a Leica CM3050 S Cryostat (Leica Microsystems, Wetzlar, Germany) and mounted on ITO slides (Sigma-Aldrich, #703176) cleaned beforehand with acetone / water (70 / 30), chloroform / methanol (2 / 1) and then hexane. Sections on slides were then vacuum packed and stored at -80°C until analysis. Sections were analysed at 25°C with an OrbiSIMS (HybridSIMS, IONTOF GMbH) instrument (Newell et al., 2020; Passarelli et al., 2017). OrbiSIMS images were acquired using a 20 keV Ar3500+ quasi-continuous GCIB analysis beam with a spot size of ~3 pm using a sawtooth raster mode, a current of ~13 pA, a duty cycle of 10-15 %, a cycle length of 200 ps and surface potential of approximately -30 V. The total ion dose for each image was between 1.91 * 109 and 2.17 x 1011 ions cm-2 with 1 shot per pixel. Image areas are between 150 pm x 150 pm and 250 pm x 250 pm, with a 5 pm pixel size. The Orbitrap mass detector was operated in negative-ion polarity using a sawtooth raster mode with a mass resolution of 240,000 @ 200 m / z and an injection time of 2961 ms, with the automatic gain control switched off. Mass spectral information was acquired for the mass range m / z 100 - 1 ,000. Putative peak annotations are based on exact mass and isotope distribution analysis, and the assignments are: palmitic acid (016:0), m / z 255.2329 [C16H31O2]- (mass deviation -0.3 ppm), adduct [M-H]-; palmitoleic acid (016:1), m / z 253.217 [C16H29O2]- (mass deviation -0.3 ppm), adduct [M-H]-; cis-9,10-methylene- hexadecanoic acid (017:0c), m / z 267.233 [C17H31O2]- (mass deviation -0.3 ppm), adduct [M-H]-; stearic acid (018:0), m / z 283.2641 [C18H35O2]- (mass deviation -0.5 ppm), adduct [M-H]-; oleic acid (018:1), m / z 281.2485 [C18H33O2]- (mass deviation -0.4 ppm), adduct [M-H]-; cis-9,10-methyleneoctadecanoic acid / dihydrosterculic acid (019:0c), m / z 295.264 [C19H35O2]- (mass deviation -0.3 ppm), adduct [M-H]-. The OrbiSIMS instrument was controlled using SurfaceLab software (IONTOF GMbH), integrating an application programming interface provided by ThermoFisher Scientific. Image and spectral analyses were performed using SurfaceLab and R softwares. Images shown correspond to ROI selections of the CNS (outer dotted line) that exclude the surrounding embedding medium. For quantifications, pixel values of zero denote that the intensity is below the limit of detection. All images and graphs were obtained using centroid data.

[0156] Immunostainings 10pm CNS transversal sections were made as described for mass spectrometry imaging but were mounted on Superfrost Plus Adhesion Microscope slides (epredia, J1800AMNZ). Sections were rinsed with PBS, and incubated in blocking solution (PBS 1X / FBS 10% / Triton X-100 0.25%) for 45min at RT. Slides were then incubated in Rabbit anti-GFP primary antibody (Invitrogen, A11122) overnight at 4°C, briefly washed with PBS-Tween 0.1%, and incubated in secondary antibody goat anti-rabbit Alexa-Fluor 647 1 :400 (Invitrogen, A21245) 2hr at room temperature. After a wash in PBS-Tween 0.1%, slides were incubated with DAPI (1 :10000 in PBS) and mounted in anti-fade mounting medium (Vectashield, #1-1-1000). Images were acquired with a STELLARIS 8 inverted confocal microscope (Leica) using a 40x objective (HC PL APO CS2 40x / 1.30 oil, Leica) and images were analyzed using Fiji / lmageJ (v1.54i).

[0157] Western Blots

[0158] S2 cells were pelleted, rinsed once with PBS 1X and resuspended in RIPA buffer (ThermoFisher, #89900) supplemented with phosphatase and protease inhibitors. For Larvae, 10 late-stage L3 larvae were homogenized in 150pL of RIPA using the Precellys® soft tissue kit (Bertin Technologies, P000912-LYSK0). For both types of samples, protein concentration was measured using Pierce BSA protein assay in microplate (ThermoFisher, #23227). 10pg of protein was then diluted in LDS sample buffer (1 / 1 , v / v) (Invitrogen, #NP0007), boiled for 3min, and loaded on pre-cast gel (Invitrogen, #NW04127). Proteins were then transferred onto PVDF membrane by semi-dry transfer, blocked and incubated with the following primary antibodies: anti-V5 tag (Rabbit, 1 :1000, Novus Biologicals #NB600-381), anti-HA tag (Rabbit, 1 :2000, Cell Signalling Technology #3724), Anti-H3 (Rabbit, Cell Signalling Technology #9715). Membranes were washed briefly and incubated with the appropriate horseradish peroxidase -coupled secondary antibody, antiRabbit (Sigma-Aldrich, #A6667) or anti-Mouse (Sigma-Aldrich, #A9917). Finally, western blots were revealed using ECL Prime Western Blotting Detection Reagents (Cytiva, #RPN2232) and imaged using an Amersham Imagequant 800.

[0159] Time to pupariation, eclosion rate and weight measurement Briefly, cages were set with 60-70 female to 30 males and let lay egg on grape juice-agar plate for 3 hours. 24 hours later, 25 first instar (L1) larvae were collected for each replicate and transferred to vials containing standard yeast food. The number of puparia was then quantified two times a day. For eclosion rate, the number of empty puparia was quantified one week after the beginning of adult eclosion. After eclosion adults were transferred to a new vial containing standard yeast diet, and their weight was measured one-week posteclosion. Data Analysis

[0160] All statistical analysis were performed using R version 4.3.2 (October 31 , 2023) (R Core Team, 2018). Most plots were generated using ggplot2 (v3.4.4). The percentage conversion to cyclo was calculated by dividing the amount of cyclo-tagged species of a given precursor lipid or FA by the sum of the precursor and the cyclo-tagged species amounts.

[0161] Example 1

[0162] To implement cFAS as an intercellular and inter-organ tracing technology, we designed versions of CFAse from Escherichia coli and Pseudomonas fluorescens that are codon optimised for expression in Drosophila, mouse or humans (Figures 5 to 11). Drosophila- optimised CFAses from E.coli and P. fluorescens were developed further, with a modification at either the N or C terminus to add a V5 epitope tag. V5-tagged CFAse was targeted either to the endoplasmic reticulum (ER), plasma membrane (PM) or outer mitochondrial membrane (Mito). Well characterised localization sequences were chosen from Drosophila proteins, Sec61b and Ubc6 for the ER, Src64b for the PM and Tom70 for Mito (Figures 12 to 16). In each case, open reading frames were designed such that the CFAse catalytic site is proximal to the cytoplasmic leaflet of each of these membranes (Figure 1B). ER, PM and Mito localized versions of V5-tagged CFAse were cloned into Drosophila UAS and LexAop vectors for expression via the GAL4 / UAS system or the orthogonal LexA / LexAop system respectively (Brand and Perrimon, 1993; Pfeiffer et al., 2010) (Table 1).

[0163] ER, PM and Mito localized UAS versions of V5-tagged CFAse were first expressed in vitro in Drosophila S2 cells under the control of GAL4 expressed from a ubiquitous promoter / enhancer (Figure 1C). The CFAse enzyme converts mono-unsaturated fatty acids such as palmitoleic and oleic acid into their cyclopropane derivatives, adding a 14 dalton mass tag (Figure 1A). GC-MS spectra indicate that UAS-CFAse that is V5-tagged and targeted to the ER is capable of converting palmitoleic (C16:1) and oleic C18:1) acids into their cyclopropanated counterparts C17:0c and C19:0c (Figure 1D). Percentage conversions for ER, PM and Mito localized CFAse variants were in the ranges of 10-30% for CI 6:1 and 2-10% for CI 8:1 and, for C16:1. This detectably decreased the relative abundance of untagged C16:1 (Figure 1E-1F). For the two ER-localised CFAse versions, the percentage conversion to the cyclopropanated derivative was approximately twice as high for the Sec61beta (CFAse::ER.1) version than for the Ubc6 (CFAse::ER.1) version (Figure 1F). This demonstrates that the CFAse fusion with Sec61beta (CFAse::ER.1) substantially improves the efficiency of cyclopropane tagging in the ER membrane. Using LC-MS, we observed that the expression of CFAse::ER.1 in S2 cells does not substantially alter the relative proportions of phospholipids (LPC, LPE, PC, PE, PG, PI, PS or TGs) (Figure 1G). ER localized CFAse was, however, able to cyclopropanate a wide range of phospholipids with conversion rates that, depending upon their fatty acyl composition, could approach 15-35% for PE, PC, PI, PG, LPC, AcCar and TG (Figure 1H). Heatmaps indicate that cyclopropanation of phospholipids is reproducible between biological replicates, depends upon CFAse and in its absence is negligible (Figure 11).

[0164] Example 2

[0165] To implement eFAT in vivo, we expressed ER, PM and Mito localized versions of V5- tagged UAS-CFAse throughout the Drosophila body using the ubiquitous GAL4 driver (tubulin-GAL4) (Figure 2A). Quantitations of the time taken to complete larval development, the adult eclosion (hatching) rate and the adult weight indicate that ER, PM and Mito localized versions of CFAse do not interfere with Drosophila development or viability (Figure 2B to 2D). One exception was ubiquitous expression of Sec61b version of ER localized CFAse, which decreased adult eclosion by -25% and adult weight by -30% (Figure 2C-2D). Nevertheless, viability and body weight were restored by decreasing the number of UAS sites from 8 to 2, which is expected to decrease the expression level (Figure 2C-2D). Consistent with the findings in S2 cells, GC-MS analysis indicates that ubiquitous expression of ER, PM or Mito localized versions of CFAse throughout the larval body does not interfere with the relative abundances of the major fatty acids (Figure 2E). Importantly, ubiquitous ER, PM or Mito CFAse converted 3-9% of total C16:1 and lower amounts of total C18:1 into their cyclopropanated counterparts and, for Sec61b CFAse, decreasing the number of UAS sites from 8 to 2 did give a small decrease in conversion (Figure 2F). The lower enrichment of cyclopropanated lipids in larvae than in S2 cells likely reflects the very high abundance of neutral rather than phospholipids in whole larvae (Figure 2G). This corresponds to the large quantities of TAG known to be stored in the fat body of larvae. Consistent with this, cyclopropanation of FAs specifically within the PE and PC pools approached 30-40% for some phospholipid species, similar if not higher than in S2 cells (Figure 2H-2I).

[0166] Example 3

[0167] We used the eFAT technology to track FA transport and metabolism from cell-to-cell within a tissue. We selected the CNS as this is a small and complex tissue containing many different cell-types and subtypes that would be challenging to dissociate and analyse individually using conventional mass spectrometry. The developing CNS consists of two major anatomical compartments, the cortex and the neuropil (Figure 3A). The cortex contains the nuclei of almost all neurons and glia, whereas the neuropil contains the lipid rich processes of neurons (Figure 3B). To map the spatial distributions of cyclopropanetagged FAs in the cortex and neuropil of the CNS, we used OrbiSIMS, a high chemical and spatial resolution mass spectrometry imaging instrument (Newell et al., 2020; Passarelli et al., 2017). The Orbitrap analyzer on this hybrid secondary ion mass spectrometry imaging (SIMS) instrument provides high mass resolution (>240000 at mlz 200) with <2 ppm mass accuracy, enabling peaks to be assigned with high confidence. Using OrbiSIMS with a 5 pm pixel size, we tracked the distribution of FAs that were cyclopropane-tagged specifically in the ER of neurons using nSyb-GAL4 to drive UAS-CFAse::ER. 1 expression (Figure 3B-3D). This revealed CFAse-dependent signals for both of the major cyclopropanated FAs, C17:0c and C19:0c, in both the cortex and neuropil (Figure 3C-3D). Signals from cyclopropanated FAs were detected at higher levels in the neuropil than in the cortex, similar to the signals from endogenous untagged FAs (Figure 3C-3D). The C17:0c / C16:1 and C19:0c / C18:1 ratios were also greater in the neuropil than in the cortex, with most pixel values above 0.7, indicating that neuron-derived FAs make a substantial contribution to the overall FA content of the neuropil. Careful comparison of the nSyb- GAL4 expression pattern and the C17:0c / C16:1 ratio suggests that cyclopropane-tagged FAs extend closer to the surface of the cortex than GFP (Figures 3B and 3C). This suggests that FAs derived from the ER phospholipids of neurons may be transported to other cell-types that are restricted to the superficial cortex such as neural stem cells.

[0168] Example 4

[0169] We next used the eFAT technology to track FA transport between cell and tissue types in Drosophila larvae. An ER localized and V5-tagged version of CFAse (UAS-CFAse::ER. 7) was expressed specifically in either the fat body, gut enterocytes, muscles, neurons or glia using Lpp-GAL4, Mex-GAL4, Mef2-GAL4, nSyb-GAL4 or repo-GAL4 respectively (Figure 4A-4B). FA analysis by mass spectrometry revealed that, within each tissue, total cyclopropane FA enrichments of 15%-35% were detected for gut, muscles, neurons and glia but only ~5% in the fat body, lower than in the other tissues as expected (Figure 4B). Given the lower cyclopropane enrichment of the fat body, it is striking that this "source" tissue contributes a higher cyclopropane enrichment to the hemolymph (blood) than the muscles or the gut (Figure 4C-4D). The fat body also contributes more than the gut to the cyclopropane enrichment of an important "destination" tissue, the central nervous system (CNS) (Figure 4E-4F). Mass spectrometry imaging of cross sections of the developing CNS using the OrbiSIMS platform revealed that the signal from adipocyte-derived cyclopropane-tagged FA (C17:0c) is substantially enriched within the CNS of CFAse expressing but not control genotype animals (Figure 4G-4H). The signals from endogenous untagged FAs and from adipocyte-derived 017:0c were similarly distributed, with both higher in the neuropil than in the cortex (Figure 4G). This indicates that cyclopropane-tagged FAs can cross the blood-brain-barrier and accumulate in both of the major anatomical compartments of the CNS. Interestingly, we also observed that the adipocyte-derived C17:0c / C16:1 ratio was higher in the cortex (up to 0.4) than the neuropil (less than 0.15), the reverse of the neuron-derived C17:0c / C16:1 ratio (Figure 4G, compare with Figure 3C). This is consistent with membranes in the cortex receiving a greater proportional contribution of FAs from adipocytes than those in the neuropil.

[0170] Together these eFAT inter-organ transport experiments provide in vivo evidence that the developing CNS obtained C16:1 and C18:1 from the circulation, derived mostly from the phospholipids of adipocytes rather than gut enterocytes.

[0171] Table 1

[0172] Stiort / name Full Genotype

[0173]

[0174]

[0175]

[0176]

[0177]

[0178]

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Claims

Claims1. A method of tracking fatty acids and / or fatty acyl chains between an origin cell and one or more destination cells or locations in a multicellular structure, the method comprising:(a) expressing cyclopropane fatty acid synthase (CFAse) under a conditional and / or cell-type specific expression system in the origin cell;(b) catalysing the addition of a mass-tag to one or more fatty acids and / or fatty acyl chains in the origin cell with CFAse by introducing a methylene group from S-adenosyl methionine (SAM) at double bonds in the fatty acids and / or fatty acyl chains to form a cyclopropane ring, producing a mass-tagged cyclopropane derivative;(c) detecting the mass-tagged cyclopropane derivative in the one or more destination cells or locations.

2. The method of claim 1 wherein the origin cell and destination cell are different celltypes or the same cell-type.

3. The method of claim 1 wherein the destination location is an extracellular fluid and / or secreted or excreted body fluid or substance.

4. The method of any one of claims 1 to 3, wherein the fatty acids are monounsaturated fatty acids and / or the fatty acyl chains are phospholipid or neutral lipid fatty acyl chains.

5. The method of claim 4 wherein monounsaturated fatty acids are palmitoleic acid and / or oleic acid and / or the fatty acyl chains may be part of phosphatidylethanolamines (PEs), phosphatidylcholines (PCs), phosphatidylserines (PSs), phosphatidylinositols (Pls), phosphatidyl glycerols (PGs), ether lipids (ELs), phosphatidic acids (PAs), lysophospholipids (LPs), acylcarnitines (AcCar), diacylglycerols (DGs) and / or triacylglycerols (TGs).

6. The method of any one of claims 1 to 5 wherein the cell-type specific expression system is specific for the origin cell.

7. The method of any one of claims 1 to 6 wherein CFAse is heterologous to the organism or cell-type in which it is expressed.

458. The method of claim 7 wherein the CFAse is expressed from an integrated gene or from a nonintegrating vector or plasmid.

9. The method of any one of claims 1 to 8 wherein the CFAse has an amino acid sequence a sequence having at least 70%, at least 80%, at least 90% at least 95% or at least 99% identity or 100% to SEQ ID NO: 1 or 5.

10. The method of any one of claims 1 to 9 wherein the CFAse is encoded by a DNA sequence having at least 70%, at least 80%, at least 90% at least 95% or at least 99% identity or 100% to SEQ ID NO: 2, 3, 4 or 6.

11. The method of any one of claims 1 to 10 wherein CFAse is fused to a targeting moiety targeting it to the plasma membrane, the nuclear membrane, the endoplasmic reticulum, the Golgi apparatus, a mitochondrion, or a lipid vesicle, optionally wherein the lipid vesicle is a lipid droplet, a peroxisome or a lysosome.

12. The method of claim 11, wherein the CFAse is fused to a targeting moiety targeting it to the endoplasmic reticulum.

13. The method of claim 12, wherein the targeting moiety targeting the endoplasmic reticulum is Sec61beta.

14. The method of any one of claims 1 to 13 wherein the multicellular structure is living organism, an organoid or a cell culture system.

15. The method of claim 14, wherein organoid or cell culture system comprises pluripotent stem cells (iPSCs), or differentiated cells derived therefrom.

16. The method of claim 15 wherein the iPSCs are obtained from a patient or animal having, or suspected of having, a disease or disorder associated with lipid transport and / or metabolism, optionally wherein the disease or disorder is an inborn error of lipid metabolism, a cancer or a neurodegenerative disease.

17. The method of claim 14 wherein the living organism is a mouse, a rat, a zebrafish, Drosophila melanogaster or C.elegans.

18. The method of any one of claims 1 to 17 wherein the detection is carried out by mass spectrometry (MS), mass spectrometry imaging (MSI) or nuclear magnetic resonance (NMR).

19. The method of any one of claims 1 to 18 wherein the mass-tagged cyclopropane derivative comprises a rare isotope, and wherein rare isotope is:(a) a stable isotope selected from13Carbon,15Nitrogen or2Hydrogen (deuterium)17Oxygen or18Oxygen; or(b) a radioactive isotope selected from11Carbon,14Carbon or3Hydrogen (tritium).

20. A method of diagnosing a disease or disorder associated with lipid transport and / or metabolism, the method comprising:(a) expressing CFAse under a conditional and / or cell-type specific expression system in a first cell-type, wherein the first cell-type contains one or more molecules comprising one or more mono-unsaturated fatty acyl chains;(b) culturing an organoid, multi-cell-type culture system or cell co-culture system comprising one or more cells of the first cell-type and one or more cells of a second celltype, wherein the first cell-type is a cell obtained from a patient suspected of having a disease or disorder associated with lipid transport and / or lipid metabolism, and wherein the second cell-type is a cell-type which accepts or is suspected to accept molecules comprising mono-unsaturated fatty acyl chains from the first cell-type under physiological conditions;(c) producing a mass-tagged cyclopropane derivative by adding a mass-tag to one or more molecules comprising fatty acyl chains in the first cell-type, wherein the mass-tag is added by catalysing the introduction of a methylene group from S-Adenosyl methionine (SAM) at double bonds within the fatty acyl chains with CFAse to form a cyclopropane ring;(d) quantifying the amount or concentration of mass-tagged cyclopropane derivative in the second cell-type;(e) comparing the amount or concentration of mass-tagged cyclopropane derivative in the second cell-type to a control value; wherein a change in the amount or concentration of mass-tagged cyclopropane derivative relative to the control value is predictive of a disease or disorder associated with lipid transport and / or metabolism.

21. The method of claim 20, wherein the control value is determined by carrying out steps (a) to (d) of claim 20 wherein the first cell-type is replaced with a wild-type cell of the same type, or a cell of the same type obtained from a healthy donor.

22. The method of claim 20, wherein the control value is a predicted value or an expected value.

23. A method of identifying the destination of a molecule comprising mono-unsaturated fatty acyl chains in a multicellular structure, the method comprising:(a) expressing cyclopropane fatty acid synthase (CFAse) under a conditional and / or cell-type specific expression system in a first cell-type, wherein the first cell-type comprises molecules comprising one or more mono-unsaturated fatty acyl chains;(b) culturing an organoid, multi-cell-type culture system or cell co-culture system comprising one or more cells of the first cell-type, and one or more cells of at least one further cell-type, wherein the further cell-type is a cell-type that accepts, or is suspected to accept, molecules comprising fatty acyl chains from the first cell-type;(c) producing a mass-tagged cyclopropane derivative by adding a mass-tag to one or more molecules comprising fatty acyl chains in the first cell-type, wherein the mass-tag is added by catalysing the addition of a methylene group from SAM at double bonds within the fatty acyl chains with CFAse to form a cyclopropane ring;(d) detecting the presence of the mass-tagged cyclopropane derivative in at least one further cell-type; wherein detection of the mass-tagged cyclopropane derivative in a further cell-type identifies the further cell-type as a destination of the molecule comprising monounsaturated fatty acyl chains.

24. A method of screening therapeutic agents for use in the correction of defect in lipid transport and / or metabolism, the method comprising:(a) providing a multicellular structure comprising one or more cells of a first cell-type having a defect in lipid transport and / or metabolism, and one or more cells of at least one further cell-type, wherein the first cell-type contains molecules comprising one or more fatty acyl chains and wherein the further cell-type is a cell-type that accepts or is suspected to accept molecules comprising fatty acyl chains from the first cell-type;(b) expressing cyclopropane fatty acid synthase (CFAse) under a conditional and / or cell-type specific expression system in the first cell-type;(c) producing a mass-tagged cyclopropane derivative by adding a mass-tag to one or more molecules comprising fatty acyl chains in the first cell-type, wherein the mass-tag isadded by catalysing the addition of a methylene group from SAM at double bonds within the fatty acyl chains with CFAse to form a cyclopropane ring;(d) quantifying the mass-tagged cyclopropane derivative in the second cell-type to establish the baseline level of fatty acid transport;(e) administering a therapeutic agent proposed for use in the correction of defective lipid transport and / or metabolism to the multicellular structure;(f) quantifying the mass-tagged cyclopropane derivative in the second cell-type after administration;(g) comparing the levels of the mass-tagged cyclopropane derivative in the second celltype after administering the therapeutic agent to the baseline or to a control value.

25. A nucleic acid encoding a CFAse having a sequence of SEQ ID NO: 2, 3, 4 or 6.

26. A recombinant expression vector comprising the sequence of SEQ ID NO: 2, 3, 4, 6, 7, 8, 9, 10, 11 or 12.

27. A host cell comprising the recombinant expression vector of claim 26.

28. A genetically modified organism expressing the CFAse of SEQ ID NO: 1 or 5 under the control of a conditional and / or cell-type specific expression system.

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