Stereoinversion method for cell-type specific labelling of amino-acids

Abstract

The invention relates to methods of tracking the movement of an amino acid of interest or a metabolite thereof in a eukaryotic multicellular structure, methods of diagnosis and screening related thereto, and nucleic acids encoding amino-acid stereo- isomerase for use therein.

Description

[0001] Stereoinversion method for cell-type specific labelling of amino-acids and their metabolites

[0002] Background

[0003] Amino acids are important for growth and metabolism in health and disease (Felig, 1975; Ling et al., 2023; Soeters et al., 2004). Inadequate intake of dietary amino acids is a global health problem that can lead to severe childhood malnutrition, stunting and kwashiorkor (Hegsted, 1972; Semba, 2016). Disruptions in amino acid metabolism are involved in many diseases including cancer (Endicott et al., 2021) and there are numerous inborn errors such as phenylketonuria, homocystinuria, tyrosinemia and maple syrup urine disease (Ziegler et al., 2023). In maple syrup urine disease, toxic levels of essential branched chain amino acids (BCAAs) such as leucine, as well as their metabolites, accumulate due to inherited mutations in the catabolic enzymes that would normally break them down (Dimou et al., 2022; Du et al., 2022). The metabolism of BCAAs is also implicated in many cancers, suggesting that it might be utilized in therapeutic interventions (Peng et al., 2020; Silva et al., 2017; Xu et al., 2023a; Xu et al., 2023b). Importantly, healthy physiology necessitates that BCAAs and other amino acids are transported between different cell-types and different tissues within the body (J. Ryan et al., 2021). A well-known example of this is starvation, during which amino acids are transported from "wasting" muscle into the circulation, then taken up by the liver and catabolized into nutrients that are released back into circulation to supply other starving tissues such as the brain (Cahill, 1970). The redistribution of amino acids away from muscles also occurs in cancer cachexia and sarcopenia during ageing (Beaudry and Law, 2022; He et al., 2024; Layman, 2024; Siqueira et al., 2024). Other examples of amino acid mobilisations from one tissue or cell-type to another include transport across the blood-brain-barrier and shuttling between neurons and glia, both of which are disrupted in neurodegenerative diseases (Adla et al., 2024; Aldana et al., 2020; Sidoryk-Wegrzynowicz et al., 2024; Sperringer et al., 2017).

[0004] For proteins, tracing their intercellular transport in live animals is feasible using established genetically encoded methods such as BirA*G3 and iSLET, which tag the entire secretome with biotin (Droujinine et al., 2021 ; Kim et al., 2021 ; Yang et al., 2022). In addition, in cell co-culture, CTAP is an alternative method for cell-selective tagging of the protein secretome via lysine racemase (Gauthier et al., 2013; Tape et al., 2014). For free amino acids and their metabolites however, it is currently very challenging to trace their in vivo movements from one specific organelle, cell, or tissue to another. Hence, although amino acids labelled with stable or radioactive isotopes or other tags are extensively used, they do have to be administered broadly from an exogenous source, such as the culture medium for cells in vitro, or an intravenous injection or dietary bolus for living animals and humans (Bartman et al., 2023; DeBerardinis and Keshari, 2022; Fernandez-Garcia et al., 2020). To overcome this limitation, we have invented conditional activation of amino acids by stereoinversion (CAAS), a genetically-encoded method that can generate, in living animals, cell-type specific sources of labelled or unlabelled amino acids. CAAS utilizes the finding that most metabolic enzymes in animals are stereoselective for L rather than D amino acids (Riehl, 2010). For this reason, animals and their cells in culture require substantial amounts of the L-stereoisomer of most, if not all, essential amino acids, including BCAAs. CAAS is a genetically encoded method that utilizes microbial amino-acid racemases / epimerases to convert specific D-amino acids into their bioactive L-steroisomers and metabolites within specific organelles, cells or tissues in a bio-orthogonal manner. CAAS can be utilized in either bulk or tracing modes depending upon the primary objective. In tracing mode, it generates a defined cellular source of an isotope labelled L-amino acid for studies of inter-organelle, inter-cellular or inter-organ transport. In bulk mode, it produces a localized source of an unlabelled L-amino acid for growth, survival and metabolism research.

[0005] Brief description of the invention

[0006] Provided herein is a method of tracking the movement of an amino acid of interest or a metabolite thereof in a eukaryotic multicellular structure, from or in a first location of the eukaryotic multicellular structure, the method comprising:

[0007] (a) expressing an exogenous stereo-isomerase in the first location of the eukaryotic multicellular structure, for example using an conditional and / or cell-type specific expression system;

[0008] (b) introducing an isomer counterpart of the amino acid of interest into the nutritional supply of the eukaryotic multicellular structure, wherein the isomer counterpart is labelled at a chiral centre with a stable isotope or radioactive isotope of hydrogen and optionally further labelled with at least one additional stable isotope or radioactive isotope;

[0009] (c) catalysing the interconversion of the isomer counterpart of the amino acid of interest with the amino-acid stereo-isomerase to form a converted amino acid in the first location of the eukaryotic multicellular structure; detecting or quantifying the converted amino acid or a metabolite thereof in the first location and / or in one or more further locations of the multicellular structure.

[0010] The method may further comprise a step of providing the eukaryotic cell or tissue with a nutritional supply. The isomer counterpart of the amino acid of interest is labelled at a chiral centre with a stable isotope of hydrogen or a radioactive isotope of hydrogen (i.e. deuterium or tritium) that is acted upon by the amino-acid stereo-isomerase. Hence the isomer counterpart of the amino acid of interest loses at least one mass unit when a first interconversion from one isomer to another (for example from D to L or from L to D) is catalysed by the action of the amino-acid stereo- isomerase. This loss of mass is maintained during subsequent interconversions, and no further change in mass occurs. In some embodiments, the isomer counterpart is additionally labelled with at least one additional rare isotope (e.g. of hydrogen, sulphur, carbon, oxygen or nitrogen). The optional additional label is not lost during interconversions of the amino acid from one isomer to another. Hence, the additional label allows converted amino acids to be distinguished from other amino acids in the eukaryotic multicellular structure or from the nutritional supply thereof.

[0011] In some embodiments, the isomer counterpart of the amino acid of interest is labelled at the alpha-carbon of the amino acid with deuterium or tritium. The deuterium or tritium is lost during a first interconversion of the amino acid from one isomer to another (for example from D to L or from L to D). The isomer counterpart of the amino acid of interest may be additionally labelled with a rare isotope.

[0012] In some embodiments, the optional additional label is not at the chiral centre of the amino acid labelled with a stable isotope of hydrogen or a radioactive isotope of hydrogen (i.e. deuterium or tritium).

[0013] In some embodiments, the optional additional label is not at any chiral centre of the amino acid.

[0014] In preferred embodiments, the optional additional label is not lost from the amino acid during any interconversion of the amino acid from one isomer to another.

[0015] In some embodiments, the optional additional label is any additional rare isotope label (e.g. a rare isotope of hydrogen, sulphur, carbon, oxygen or nitrogen) at any location of the amino acid, with the exception that the additional label is not a rare isotope of hydrogen (i.e. deuterium or tritium) on a chiral centre, for example a chiral centre that may be acted on by the stereo- isomerase. In some embodiments, the amino acid is labelled with deuterium or tritium at the alpha carbon of the amino acid, and the amino acid is optionally labelled with an additional rare isotope label (e.g. a rare isotope of hydrogen, sulphur, carbon, oxygen or nitrogen) at a further location, with the exception that the additional label is not a deuterium or tritium on a chiral centre acted on by the stereo-isomerase.

[0016] In embodiments where the first label is deuterium, the amino acid loses one mass unit when it is interconverted. In embodiments where the first label is tritium, the amino acid loses two mass units when it is interconverted.

[0017] The alpha carbon of the amino acid is the carbon immediately adjacent to the carboxyl group and comprises the amine group of the amino acid.

[0018] The “additional label” described herein may alternatively be referred to as a “second label”.

[0019] The first location may be a eukaryotic cell-type. The one or more further locations may be the same eukaryotic cell-type and / or a different eukaryotic cell-type. Alternatively, the one or more further locations may be an extracellular fluid and / or secreted or excreted body fluid.

[0020] The multicellular structure may comprise at least two different cell-types. For example, the multicellular structure may comprise more than three, more than five, more than ten, or more than twenty different cell-types. The eukaryotic cell-type may be a neuron, a glial cell, a hepatocyte, an adipocyte, an enterocyte, a muscle cell, an epidermal cell, a stem cell, a hematopoietic cell, a blood cell, or a cancer cell. The tissue may be a tissue comprising neurons, glia, hepatocytes, adipocytes, enterocytes, muscle cells, epidermal cells, stem cells hematopoietic cells, blood cells or cancer cells. The multicellular structure may further comprise one or more extracellular fluids and / or secreted or excreted body fluid or substances. Examples of such fluids include blood, plasma, hemolymph, lymph, cerebrospinal fluid, perspiration, urine or faeces.

[0021] The method may be for tracking the movement of an amino acid of interest or a metabolite thereof from an origin cell to a destination cell in a multicellular structure, or to a destination extracellular fluid and / or secreted or excreted body fluid. For example, the first location may be an origin cell and the one or more further locations may be different eukaryotic cell-types. Alternatively, the origin cell and destination cell may both be cells of the first cell-type. Alternatively, the first location may be an origin cell and the one or more further locations may be a destination extracellular fluid and / or secreted or excreted body fluid or substance. The one or more further locations may include both destination cells and extracellular fluids and / or secreted or excreted body fluids or substances.

[0022] Alternatively, the method may be for tracking the movement of an amino acid of interest or a metabolite thereof between one or more organelles within a single cell.

[0023] The converted-amino acid or a metabolite thereof may be detected sequentially or simultaneously in more than one different cell-type, tissue, extracellular fluid, or secreted or excreted body fluid.

[0024] The origin cell may be selected from a neuron, a glia, a hepatocyte, an adipocyte, an enterocyte, a muscle cell, an epidermal 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 stem cell, a hematopoietic cell or a blood cell. Alternatively, the origin cell and / or destination cell may be a cancer or tumour cell. 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 healthy cell and a destination cell may be a non-cancerous cell or the destination cell may be a cancer cell and the origin cell may be a non-cancerous cell. Alternatively, the origin and destination cells may both be cancer or tumour cells.

[0025] Provided herein is a method of determining the function of an L-amino acid of interest in a eukaryotic cell or tissue, the method comprising:

[0026] (a) providing the eukaryotic cell or tissue with a nutritional supply comprising the L-amino acid of interest (unconverted L-amino);

[0027] (b) measuring one or more biological activities of the cell or tissue suspected of being modulated by the L-amino acid;

[0028] (c) expressing an amino-acid stereo-isomerase in the cell or tissue, for example using an conditional and / or cell-type specific expression system;

[0029] (d) catalysing the conversion of an amino acid of interest from an L-amino acid to a D-amino acid with the amino-acid stereo-isomerase, resulting in a reduction in the amount or concentration of L-amino acid in the eukaryotic cell or tissue;

[0030] (e) detecting a change in one or more biological activities of the cell-type or tissue following the reduction in the amount or concentration of the L-amino acid. Provided herein is a method of determining the function of an L-amino acid of interest in a eukaryotic cell or tissue, the method comprising:

[0031] (a) providing a eukaryotic cell or tissue with a nutritional supply comprising the L-amino acid of interest;

[0032] (b) measuring one or more biological activities of the cell or tissue suspected of being modulated by the L-amino acid;

[0033] (c) expressing the amino-acid stereo-isomerase in the cell or tissue, for example using an conditional and / or cell-type specific expression system;

[0034] (d) partially or completely replacing the L-amino acid in the nutritional supply of the cell with its D-amino acid counterpart (unconverted D-amino acid);

[0035] (e) catalysing the conversion of the D-amino acid obtained from the nutritional supply to its L- amino acid counterpart with the amino-acid stereo-isomerase, resulting in an increase in the amount or concentration of L-amino acid in the eukaryotic cell or tissue;

[0036] (f) detecting a change in one or more biological activities of the cell or tissue following the increase in the amount or concentration of the L-amino acid.

[0037] The unconverted D-amino acid or unconverted L-amino acid may comprise one or more rare isotopes. The rare isotope may be a stable isotope or a radioactive isotope.

[0038] The unconverted D-amino acid or unconverted L-amino acid may be labelled at a chiral centre with a rare isotope. The rare isotope may be2Hydrogen (deuterium) or3Hydrogen (tritium).

[0039] The unconverted D-amino acid or unconverted L-amino acid may be further labelled (for example outside of a chiral centre) with a rare isotope. The rare isotope may be an isotope of Carbon, Nitrogen, Hydrogen, Oxygen or Sulphur. The rare isotope may be a stable isotope or a radioactive isotope. The stable isotope may be13Carbon,15Nitrogen or2Hydrogen (deuterium)17Oxygen or18Oxygen. The radioactive isotope may be11Carbon,14Carbon or3Hydrogen (tritium).

[0040] If a radioactive isotope of sulphur is used, this may be,27Sulfur,28Sulfur29Sulfur,30Sulfur,31Sulfur,35Sulfur,37Sulfur,38Sulfur,39Sulfur,40Sulfur,41Sulfur,42Sulfur,43Sulfur,44Sulfur,45Sulfur,46Sulfur,47Sulfur,48Sulfur or49Sulfur.

[0041] If a stable isotope of sulphur is used, this may be32sulphur,33sulphur,34sulphur or36sulphur. The additional label may be retained following conversion or metabolism of the unconverted D-amino acid or unconverted L-amino acid amino acid. Metabolites of the amino acid of interest may therefore also be labelled with the additional label.

[0042] The unconverted D-amino acid or unconverted L-amino acid may be present in the nutritional supply in a fixed molar ratio of D- to L isomers of the amino acid. Alternatively, the D-amino acid may be the sole isomer of the amino acid in the nutritional supply.

[0043] An unconverted amino acid is an amino acid which has not been acted on by an amino-acid stereo-isomerase.

[0044] The isotope labelled D-amino acid or L-amino acid may be administered from an exogenous source. The exogenous source may be a cell culture medium, or an intravenous or dietary bolus.

[0045] The biological activity may be metabolism, cell growth or cell survival. The biological activity may be linked to an amino acid sensing or amino acid regulated pathway.

[0046] The L-amino acid of interest may be an amino acid which is present in eukaryotic cells as an L stereoisomer. The D-amino acid may be an amino acid which is not present in Eukaryotic cells as a D stereoisomer. Alternatively, the D-amino acid may be an amino acid which is present in Eukaryotic cells predominantly but not exclusively as the L stereoisomer.

[0047] The L amino acid of interest may be L-alanine, L-arginine, L-asparagine, L-aspartic acid, L- cysteine, L-glutamic acid, L-glutamine, L-histidine, L-isoleucine, L-leucine, L-lysine, L- methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L- valine, L-cystine, L-selenocysteine, L-alpha-alanine, L-norleucine or L-homoserine. The L- amino acid of interest may be a branched chain amino acid. The L amino acid of interest may be Leucine or Isoleucine. The D-amino acid counterpart may be D-Leucine or and D-allo- Isoleucine.

[0048] The nutritional supply may be a form of cell culture medium, an intravenous or intraperitoneal injection, gavage, a dietary supplement or supplementation of the drinking water.

[0049] The amino-acid stereo-isomerase may be expressed under a conditional promotor or a constitutive promotor. The amino-acid stereo-isomerase may be expressed under the control of a conditional or cell-type specific expression system. The cell-type specific expression system may be specific for the origin cell. The amino-acid stereo-isomerase 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 and / 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 amino-acid stereo-isomerase may be heterologous to the organism or cell-type in which it is expressed. The amino-acid stereo- isomerase may be expressed transiently. The amino-acid stereo-isomerase may be introduced into the origin cell by transfection or transduction of DNA encoding the amino-acid stereo- isomerase or delivery of mRNA encoding the amino-acid stereo-isomerase. The amino-acid stereo-isomerase may be introduced into the origin cell by a viral vector, for example a lentiviral vector. The amino-acid stereo-isomerase may be introduced by CRISPR gene editing. The amino-acid stereo-isomerase may be expressed from an integrated gene. Alternatively, aminoacid stereo-isomerase may be expressed from a nonintegrating vector or plasmid. The conditional expression system may be an optogenetic expression system.

[0050] The amino-acid stereo-isomerase may be expressed as a cytosolic protein. The amino-acid stereo-isomerase may be fused to a localisation sequence targeting it to a sub-cellular compartment or 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.

[0051] The amino-acid stereo-isomerase may be a racemase or epimerase. The racemase or epimerase may be a pyridoxal-5-phosphate dependent racemase or epimerase or a pyridoxal- 5-phosphate independent racemase or epimerase.

[0052] The amino-acid stereo-isomerase may be a wild-type amino-acid stereo-isomerase or a codon optimised amino-acid stereo-isomerase. The amino-acid stereo-isomerase 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 amino-acid stereo-isomerase may be a broad spectrum amino-acid stereo-isomerase. For example, the amino-acid stereo-isomerase may be a broad spectrum racemase or epimerase.

[0053] The racemase or epimerase may be from Lactobacillus fermentum. For example, the racemase or epimerase may be isoleucine-2-epimerase (Lafer ILEP). The racemase or epimerase may be from Vibrio cholerae (UniProt: Q9KSE5.3). The racemase or epimerase may have an amino acid sequence of SEQ ID NO: 1. The racemase or epimerase 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.

[0054] The racemase or epimerase may be encoded by a nucleic 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: 2 or 3. The racemase or epimerase may be encoded by a DNA sequence of SEQ ID NO: 2 or 3.

[0055] The racemase or epimerase may be fused to a targeting moiety targeting it to an organelle.

[0056] The racemase or epimerase may be fused to a label. The label may be a fluorescent protein (e.g. GFP, mNeonGreen, RFP, dsRed, mCherry or mScarlet) or another tag (e.g HA-tag, FLAG-tag, His-tag, V5-tag, Myc-tag, Sun-tag, Halo-tag, Clip-tag).

[0057] The multicellular structure may be a living organism, and organoid or a cell culture system. The living organism may be a vertebrate or invertebrate model organism. The living organism may be a mouse, a rat, a zebrafish, a Drosophila or a C.elegans. The cell culture system may be an organoid or cell co-culture system.

[0058] The organoid or cell culture system may comprise human cells, and / or a cells from a nonhuman animal. The non-human animal may be a vertebrate or invertebrate model organism. The cells may be human, mouse, rat, zebrafish, Drosophila or C.elegans cells, or a combination thereof. The human cells may be patient-derived cells, for example, patient- derived induced pluripotent stem cells (iPSCs) or patient-derived somatic cells. Alternatively, the human cells may be from a healthy subject. The human cells may be cancerous or non- cancerous cells. The human cells may be derived from an immortalised human cell line. The human cells may contain one or more genetic mutations associated, or suspected to be associated with, neurodegeneration, cancer, metabolic disease, or another disease or disorder associated with amino acid transport and / or metabolism.

[0059] The organoid or cell culture system may comprise pluripotent stem cells, for example iPSCs, or differentiated cells derived therefrom. The organoid or cell culture system may comprise embryonic stem cells. The embryonic stem cells may be non-human embryonic stem cells. The multicellular structure may be an in vitro or ex vivo structure. The organoid may be a patient-derived organoid or another human-derived organoid, for example, an organoid derived from a healthy human subject. The organoid may be derived from a vertebrate or invertebrate model organism. The cell co-culture system may comprise CHO, CACO-2, HEK293, HeLa, ID8, MEF, MCF-7, S2, Sf9, U2OS, 3T3, U87, or U-251 cells.

[0060] The iPSCs may be obtained from a patient or animal suspected to have a disease or disorder associated with amino acid transport and / or metabolism. The disease or disorder may be a metabolic disease or an inborn error of amino acid or protein metabolism, cancer or neurodegeneration. The metabolic disease or inborn error of amino acid or protein metabolism may be phenylketonuria, homocystinuria, tyrosinemia or maple syrup urine disease, Isovaleric acidemia, 3-Methylcrotonyl-CoA carboxylase deficiency, 3-Methylglutaconic aciduria types I- IV, 3-Hydroxy-3- methylglutaryl-CoA lyase deficiency, 2-methyl-3-hydroxyisobutyric aciduria, Methylbutyryl-CoA dehydrogenase deficiency, Mitochondrial acetoacetyl-CoA thiolase deficiency, 3-Hydroxyisobutyryl- CoA deacylase deficiency, 3-Hydroxyisobutyric aciduria, Propionic acidemia, Methylmalonic acidemia, type-2 diabetes, atherosclerosis, hypertension, heart failure, obesity or kidney disease. The cancer may be carcinoma, gastric cancer, pancreatic cancer, breast 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.

[0061] The vertebrate or invertebrate model organism may be a genetically modified model organism, a transgenic organism, knock-out organism, knock-in organism, or disease model, including but not limited to cancer models, neurodegeneration models, metabolic disease models, cardiovascular disease models, or inflammatory disease models. The vertebrate or invertebrate model organism may comprise one or more genetic mutations associated, or suspected to be associated with, neurodegeneration, cancer, metabolic disease, or another disease or disorder associated with amino acid transport and / or metabolism in humans.

[0062] The detection and / or quantification may be carried out by any suitable detection or quantification method, i.e. a method suitable for detecting and / or quantifying the L-amino acid of interest or a metabolite thereof in the multicellular structure. For example, in some embodiments the detection may be mass spectrometry (MS), mass spectrometry imaging (MSI) or nuclear magnetic resonance (NMR), scintillation counting, autoradiography, gamma counting or imaging using a fluorescent or other reporter.

[0063] Further provided is a nucleic acid of SEQ ID NO: 2 or 3. Further provided is a recombinant expression vector comprising a nucleic acid of SEQ ID NO: 2 or 3.

[0064] Further provided is an expression vector of the invention, wherein the nucleic acid sequence is under the control of a conditional and / or cell-type specific expression system. The 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 auxininducible degron (AID) or optogenetic methods such as ShineGAL4 / UAS. The cell-type specific expression system may be specific for the origin cell.

[0065] Further provided is a host cell comprising the recombinant expression vector of the invention.

[0066] The host cell may be a CHO cell, CACO-2 cell , HEK293 cell, HeLa cell, ID8 cell, MEF cell, MCF-7 cell, S2 cell, Sf9 cell, U2OS cell, 3T3 cell or an iPSC.

[0067] Further provided is a genetically modified organism expressing the racemase or epimerase of SEQ ID NO: 1 under the control of a conditional and / or cell-type specific expression system.

[0068] The expression system may be 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.

[0069] Further provided is a method of diagnosing a disease or disorder associated with amino acid transport and / or metabolism, the method comprising:

[0070] (a) expressing an amino-acid stereo-isomerase under a conditional and / or cell-type specific expression system in a first eukaryotic cell-type obtained from a patient suspected of having a disease or disorder associated with amino acid transport and / or metabolism;

[0071] (b) culturing an organoid or cell culture system comprising one or more cells of the first celltype and optionally further comprising one or more cells of a second eukaryotic cell-type;

[0072] (c) providing the organoid or cell culture system with a nutritional supply;

[0073] (d) introducing a labelled L-amino acid of interest or labelled D-amino acid counterpart of the L-amino acid of interest into the nutritional supply of the organoid or cell culture system, wherein the labelled L-amino acid or D-amino acid counterpart is labelled at a chiral centre with a stable isotope or a radioactive isotope of hydrogen;

[0074] (e) catalysing the interconversion of the D-amino acid and the L-amino acid of interest with the amino-acid stereo-isomerase; (f) quantifying the amount or concentration of the labelled and / or converted L-amino acid or a metabolite thereof in the first cell-type and / or second cell-type;

[0075] (g) comparing the amount or concentration of the labelled and / or converted L-amino acid or a metabolite thereof in the first cell-type and / or second cell-type to a control value; wherein a change in the amount of the L-amino acid or metabolite thereof in the first cell-type and / or second cell-type relative to the control value is predictive of a disease or disorder associated with amino acid transport and / or metabolism.

[0076] Further provided herein is a method of stratifying patients having a disease or disorder associated with amino acid transport and / or metabolism, the method comprising:

[0077] (a) expressing an amino-acid stereo-isomerase under a conditional and / or cell-type specific expression system in a first eukaryotic cell-type obtained from a patient having, or suspected of having, a disease or disorder associated with amino acid transport and / or metabolism;

[0078] (b) culturing an organoid or cell culture system comprising one or more cells of the first celltype and optionally further comprising one or more cells of a second eukaryotic cell-type;

[0079] (c) providing the organoid or cell culture system with a nutritional supply;

[0080] (d) introducing a labelled L-amino acid of interest or labelled D-amino acid counterpart of the L-amino acid of interest into the nutritional supply of the organoid or cell culture system, wherein the L-amino acid or D-amino acid counterpart is labelled with a stable isotope or a radioactive isotope of hydrogen at a chiral centre;

[0081] (e) catalysing the interconversion of the D-amino acid and L-amino acid of interest with the amino-acid stereo-isomerase;

[0082] (f) quantifying the amount or concentration of the labelled and / or converted L-amino acid or a metabolite thereof in the first cell-type and / or second cell-type;

[0083] (g) comparing the amount or concentration of the labelled and / or converted L-amino acid or a metabolite thereof in the first cell-type and / or second cell-type to a control value;

[0084] (h) stratifying the patient into a risk category based on the difference between the amount or concentration of the labelled and / or converted L-amino acid or a metabolite thereof and the control value.

[0085] The control value may be determined by carrying out steps (a) to (f) of the method of diagnosing a disease or disorder associated with amino acid 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. Alternatively, the control value may be a predicted or expected value. Further provided is a method of screening therapeutic agents for use in the treatment of a disease or disorder associated with amino acid transport and / or metabolism, the method comprising:

[0086] (a) expressing an amino-acid stereo-isomerase under a conditional and / or cell-type specific expression system in a first eukaryotic cell-type;

[0087] (b) culturing an organoid or cell culture system comprising one or more cells of the first eukaryotic cell-type and optionally further comprising one or more cells of a second eukaryotic cell-type;

[0088] (c) providing the organoid or cell culture system with a nutritional supply;

[0089] (d) introducing a labelled L-amino acid of interest or a labelled D-amino acid counterpart of the L-amino acid of interest into the nutritional supply of the organoid or cell culture system, wherein the L-amino acid or D-amino acid counterpart is labelled at a chiral centre with a stable isotope or a radioactive isotope of hydrogen;

[0090] (e) catalysing the interconversion of the D-amino acid and L-amino acid of interest with the amino-acid stereo-isomerase;

[0091] (f) quantifying the amount or concentration of the labelled and / or converted L-amino acid in the first and / or second cell-type to establish the baseline level;

[0092] (g) administering a pharmaceutically active amount of the therapeutic agent to the organoid or cell culture system;

[0093] (h) quantifying the amount or concentration of the labelled and / or converted L-amino acid or a metabolite thereof in the first cell-type and / or second cell-type following the administration of the therapeutic agent;

[0094] (i) comparing the amount or concentration of the labelled and / or converted L-amino acid or metabolite thereof in the first cell-type and / or second cell-type after administration of the therapeutic agent to the baseline level of step (f).

[0095] The first eukaryotic cell-type may be obtained from a patient suspected of having a disease or disorder associated with amino acid transport and / or metabolism. Alternatively, the first eukaryotic cell-type may be obtained from a healthy donor. The first eukaryotic cell-type may be obtained from a vertebrate or invertebrate model organism. The vertebrate or invertebrate model organism may comprise one or more genetic mutations associated, or suspected to be associated with, neurodegeneration, cancer, metabolic disease, or another disease or disorder of amino acid 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. The first eukaryotic cell-type may have been modified or conditioned to replicate the symptoms of a disease or disorder associated with amino acid transport and / or metabolism. The first eukaryotic cell-type may have been modified to comprise one or more genetic mutations associated, or suspected to be associated with, neurodegeneration, cancer, metabolic disease, or another disease or disorder associated with amino acid transport and / or metabolism in humans. The first eukaryotic cell-type may have been exposed to one or more environmental conditions associated with, or suspected to be associated with, disruption to amino acid transport and / or metabolism. For example, the first eukaryotic 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.

[0096] The organoid or cell culture system may further comprise one or more cells of a third, fourth or further eukaryotic cell-type. The organoid or cell culture system may comprise 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.

[0097] The cells of the second eukaryotic cell-type may be wild-type cells or cells obtained from a healthy donor. The cells of the second eukaryotic cell-type may have the same disease or disorder, or have undergone the same modification or conditioning as the first eukaryotic celltype.

[0098] The baseline level of step (f) may be indicative of an abnormal or pathological state in the first and / or second cell-type. The baseline level of the labelled and / or converted L-amino acid may be higher than a wild-type or unconditioned cell of the same type, or a cell of the same type obtained from a healthy donor. The baseline level of the labelled and / or converted L-amino acid may be lower than in a wild-type or unconditioned cell of the same type, or a cell of the same type obtained from a healthy donor

[0099] The change in the amount or concentration of the labelled and / or converted L-amino acid or metabolite thereof in the first cell-type and / or second cell-type relative to baseline levels may indicate that the therapeutic agent is capable of modifying amino acid transport and / or metabolism. Therapeutic agents capable of modifying amino acid transport and / or metabolism may be identified as candidate therapeutic agents for use in the treatment of a disease or disorder associated with disruption to amino acid transport and / or metabolism. The change in the amount or concentration of the of the L-amino acid or metabolite thereof in the first cell-type and / or second cell-type relative to baseline levels may represent a partial or complete reversal of abnormal levels of a disease or disorder associated with disruption to amino acid transport and / or metabolism.

[0100] The amount or concentration of the of the L-amino acid or metabolite thereof in the first celltype and / or second cell-type following the administration of the therapeutic agent may be functionally similar or equivalent to the levels in a wild-type cell of the same type, or a cell of the same type obtained from a healthy donor.

[0101] The disease or disorder may be a metabolic disease or an inborn error of protein or amino acid metabolism, cancer or neurodegeneration. The metabolic disease or inborn error of protein or amino acid metabolism may be phenylketonuria, homocystinuria, tyrosinemia or maple syrup urine disease, Isovaleric acidemia, 3-Methylcrotonyl-CoA carboxylase deficiency, 3- Methylglutaconic aciduria types l-IV, 3-Hydroxy-3- methylglutaryl-CoA lyase deficiency, 2- methyl-3-hydroxyisobutyric aciduria, Methylbutyryl-CoA dehydrogenase deficiency, Mitochondrial acetoacetyl-CoA thiolase deficiency, 3-Hydroxyisobutyryl- CoA deacylase deficiency, 3-Hydroxyisobutyric aciduria, Propionic acidemia or Methylmalonic acidemia, type- 2 diabetes, atherosclerosis, hypertension, heart failure, obesity or kidney disease. The cancer may be carcinoma, gastric cancer, pancreatic cancer, breast 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.

[0102] The stable isotope or radioactive isotope of hydrogen may be2H or3H. The stable isotope or radioactive isotope may be lost on conversion of the D-amino acid to the L-amino acid, resulting in a mass change. The D-amino acid may be labelled with additional rare isotopes, for example outside of the chiral centre.

[0103] Brief description of the figures

[0104] Figure 1. Racemase / epimerase-dependent mass tagging of L-amino acids and their metabolites.

[0105] (A) D-to-L interconversion strategy. Diagram shows a deuterium atom (d) at a chiral centre of a generic isotope labelled D-amino acid substrate (left). This is exchanged for a hydrogen atom (H) during the racemase reaction with a hydrogen from water to produce the L-amino acid (centre). The loss of one mass unit is maintained during subsequent racemization (centre, right). Incorporation of stable or unstable isotopes (red asterisk) in the rest of the molecule (R) of the D-amino acid substrate allows the racemase-dependent L-amino acid and its metabolites to be distinguished by mass spectrometry from those in the racemase- independent pool.

[0106] (B) L-to-D-to-L interconversion strategy. Diagram shows a deuterium atom (d) at a chiral centre of a generic isotope labelled L-amino acid substrate (left). This is exchanged for a hydrogen atom (H) during the racemase reaction with a hydrogen from water to produce the D-amino acid (centre). The loss of one mass unit is maintained during subsequent racemization (centre, right). Incorporation of stable or unstable isotopes (red asterisk) in the rest of the molecule (R) of the L-amino acid substrate allows the racemase-dependent L-amino acid and its metabolites to be distinguished by mass spectrometry from those in the racemase-independent pool.

[0107] Figure 2. Diagram of reactions catalyzed by Lafer ILEP, isoleucine-2-epimerase from Lactobacillus fermentum Lactobacillus fermentum was recently renamed as Limosilactobacillus fermentum: Taxid: 334390. NCBI entry WP_012391664.

[0108] Figure 3. Protein sequence of Lafer ILEP, isoleucine-2-epimerase from Lactobacillus fermentum Lactobacillus fermentum was recently renamed as Limosilactobacillus fermentum: Taxid: 334390. NCBI entry WP_012391664.

[0109] Figure 4. DNA sequence encoding Lafer ILEP, codon-optimized for Drosophila melanogaster

[0110] Figure 5. DNA sequence encoding Lafer ILEP, codon-optimized for Mus musculus

[0111] Figure 6. Schematics of in vitro and in vivo constructs

[0112] Diagrams are depicted for constructs and transgenes expressing Lafer ILEP-HA in cultured human cells in vitro or in Drosophila or mice in vivo. Figure 7. Lafer ILEP-HA is active in human HEK293 cells. Graph depicts racemisation as the ratio of 13C6-L-Leucine to 13C6-D-Leucine in extracts from either control genotype HEK293 cells (open circles) or HEK293 cells transiently transfected with a plasmid expressing Lafer ILEP-HA (filled squares). Prior to the experiment, HEK293 cells were cultured in regular DMEM containing unlabelled L-Leucine. For the experiment, cell cultures were incubated for 90 minutes in DMEM containing dialysed fetal calf serum and 0.4mM each of unlabelled L- Leucine and 13C6-D-Leucine before the cells were harvested for chiral GC mass spectrometry. Lafer ILEP-HA transfected but not control cells efficiently generate 13C6 L- Leucine from 13C6 D-Leucine. This indicates that Lafer ILEP-HA has racemase activity in human cells.

[0113] Figure 8. Lafer ILEP-HA is active in Drosophila melanogaster. Graph depicts levels of L and D branched-chain amino acids, normalized to their L-stereoisomers, in larval hemolymph (insect blood) of control animals (Ctrl 1 and 2, light and dark grey bars) and animals expressing Lafer ILEP-HA specifically in gut enterocytes (J\IP1>LaferlLEP-HA, black bars), raised on a complete diet. In control animals, only the natural L-amino acids (L-Leucine and L-lsoleucine) as well as trace amounts of D-Leucine are detected. In Lafer ILEP expressing animals, D-allo- Isoleucine and D-Leucine are detected at similar or about half the level of their L-amino acid counterparts respectively. Metabolites were quantified by GC-MS using a chiral column.

[0114] Figure 9: Lafer ILEP-dependent mass tagging of L-leucine in vivo in D. melanogaster Relative quantities of the three major deuterated Leucine isotopomers (d9, d10 and d11) in the hemolymph (blood) of Drosophila melanogaster larvae, following 2 hour exposure to diet containing d10-D-Leucine. Note that the "pure" standard of commercial d10-D-leucine (left triplet of histograms) used in this experiment contains -10% d9 and d11 isotopomers. In genetic control animals (Lpp>GFP, central triplet of histograms) the isotopomer ratios are unchanged compared to the d10-D-leucine standard. In LaferILEP-HA expressing animals (Lpp>LaferlLEP-HA, right triplet of histograms), the proportion of d9 and d10 isotopomers respectively increases and decreases. This is consistent with conversion of d10 to d9 leucine by stereoinversion (Figure 1 and Figure 2). Metabolites were quantified by GC-MS.

[0115] Figure 10. Lafer ILEP-HA expression in Drosophila melanogaster does not disrupt development. 100% of control genotype animals (grey triangles) and experimental genotype animals expressing Lafer ILEP-HA specifically in gut enterocytes (black circles) complete larval development at a similar time (hours) on a complete diet containing the essential amino acid L-Leucine. Figure 11. Lafer ILEP expression in Drosophila melanogaster confers the ability to develop on a D-leucine diet. 0% of control genotype animals (grey triangles) complete larval development on a diet in which L-Leucine is replaced with D-Leucine. In contrast, 100% of animals expressing Lafer ILEP-HA specifically in gut enterocytes (black circles) complete larval development on schedule on this D-leucine diet.

[0116] Figure 12: Lafer ILEP expression in mice does not disrupt juvenile growth. On a complete diet containing the essential amino acid L-Leucine, body weight gain (in grams) increases similarly over time for P21 to P35 (0-14 days post weaning) juvenile female mice, Mus musculus, of a control genotype (black circles) or an experimental genotype expressing Lafer ILEP-HA specifically in liver hepatocytes (grey triangles).

[0117] Figure 13: Lafer ILEP-HA expression in mice confers the ability to grow on a D-leucine diet. On a diet in which 75% of L-Leucine is replaced with D-Leucine (D-leucine diet), body weight gain (in grams) from P21 to P35 (0-14 days post weaning) in juvenile female mice, Mus musculus, is much lower for the control genotype (black circles) than the experimental genotype expressing Lafer ILEP specifically in liver hepatocytes (grey triangles). Note that hepatocyte expression of Lafer ILEP confers a body growth rate on the D-leucine diet that is similar to that of control genotype animals on complete diet (Figure 9).

[0118] Figure 14: Lafer ILEP-HA expression in human HEK293 cells confers the ability to grow in D-leucine medium.

[0119] Graph depicts confluence (area of dish occupied by cells) over time of human HEK293 cells as measured by an Incucyte cell culture monitor. Control cells (open circles) or cells transfected with a LaferILEP-HA expressing plasmid (filled squares) were seeded at identical density and incubated with DMEM containing dialysed fetal calf serum and 0.8mM D-Leucine (no L- Leucine). Control cells fail to grow in the absence of L-Leucine and cannot utilize D-Leucine, whereas LaferILEP-HA expressing cells grow at a near normal rate.

[0120] Detailed description of the invention

[0121] To understand the in vivo functions of amino acids and their metabolites, it is important to be able to track their mobilization and transport from one organelle, cell or tissue to another. Currently, however, this is challenging in any multicellular context such as an organoid culture or a living animal. Here we invent conditional activation of amino acids by stereoinversion (CAAS) to overcome this limitation. CAAS is a genetically encoded technology that utilizes microbial amino-acid stereo-isomerases (racemases or epimerases) to convert, in a conditional and bio-orthogonal manner, D-amino acids into their bioactive L-amino acid counterparts within specific organelles, cells or tissues. As a proof-of-principle, we generated transgenic Drosophila and mouse CAAS models for two essential branched chain amino acids (BCAAs), leucine and isoleucine. We use these models to demonstrate that CAAS enables the in vivo addition of an isotope tag to L-BCAAs in a cell-type specific manner. The localized generation of these stable isotope tracers allows transport of BCAAs and their metabolites, from one specific cell or tissue type to another, to be monitored in live animals. CAAS can also be adapted to bulk mode, to produce substantial cell-type specific sources of unlabelled L- BCAAs that can modulate growth and other processes. For example, gut-specific (in Drosophila) or liver-specific (in mouse) CAAS is sufficient to confer growth and survival of developing animals on a D-leucine holidic diet deficient for L-leucine. CAAS can be adapted to other amino acids and may have wide applications in biomedical research, drug screening and diagnostics.

[0122] This study introduces conditional activation of amino acids by stereoinversion (CAAS) as a new bio-orthogonal genetically encoded method for organelle, cell or tissue specific production of L-amino acids. The key advance over other technologies is that the CAAS invention allows the movement of amino acids from one organelle / cell / tissue type to another to be monitored and quantified. Exemplars for leucine and isoleucine in both Drosophila and mouse, using the LaferILEP racemase, demonstrate that CAAS has wide applicability and can be used either in tracing or bulk modes. In other words, CAAS can generate a cell-type specific source of unlabelled or labelled L-amino acids and it also enables cell-type specific isotope tracing of L- amino acid transport and metabolism.

[0123] CAAS can be readily adapted to bulk or tracing modes by adjustment of the concentration of the D-amino acid substrate provided for the amino-acid stereo-isomerase reaction. In general, the concentrations suitable for tracing mode will be considerably lower than those optimal for bulk mode. Further refinements are possible by modifying genetic elements that regulate the expression level of the amino-acid stereo-isomerase. For the tracing mode, we provided an example of how deuterium labelling of the chiral centre of an amino acid can be used to generate in vivo an amino acid isotope tracer in a cell-type specific manner. This also has the technical advantage that amino-acid stereo-isomerase -dependent L-amino acids can be detected via a mass change with conventional mass spectrometry, without the need for a specialized chiral column to separate L and D amino acids. To distinguish the L-amino acid generated by deuterium loss at the chiral centre from endogenous L-amino acids, we also used deuterium to label other atoms in the molecule but would also be feasible to use stable or radioactive isotopes of carbon or nitrogen for this purpose. To gain detailed spatial information about amino acid transport and metabolism between individual cells within complex tissues, we envisage combining CAAS with high resolution mass spectrometry imaging instruments such as OrbiSIMS (Newell et al., 2020; Passarelli et al., 2017).

[0124] We used conditional gene expression systems to activate LaferILEP epimerase in the cytoplasm of specific cell-types within live animals. LaferILEP is very specific for leucine and isoleucine but the CAAS method could also be used with broader specificity racemases or epimerases as specificity can be applied by the choice of the D-amino acid substrate that is supplied. Any pyridoxal-5-phosphate dependent or independent racemases or epimerase suitable for CAAS could also be delivered to specific cell-types using other methods including transfection / electroporation of DNA or viral transduction. These and other methods are suitable for implementation of the CAAS method in cell co-culture, embryo culture, organ culture, or other 3D in vitro models such as organoids (Whitehouse et al., 2023; Yang et al., 2023). Moreover, CAAS racemases or epimerases could be fused to other coding sequences such as those conferring localization to organelles including the ER, Golgi, peroxisome, plasma membrane or mitochondria. These modifications may enable CAAS to be used to study amino acid trafficking and metabolism at very high spatial resolution within a cell.

[0125] CAAS has multiple applications in mechanistic research into amino acid metabolism. It can also be combined with genetic manipulations of genes of interest to help elucidate interorganelle, intercellular and inter-organ pathways of amino acid transport and metabolism. In addition, CAAS may be useful for studying amino acid sensing and signalling pathways, such as those involving mTOR or GCN2 (Ben-Sahra and Manning, 2017; Costa-Mattioli and Walter, 2020; Gonzalez and Hall, 2017; Ryoo, 2024).

[0126] CAAS also has potential uses in medical diagnosis and drug screening. In this context, CAAS could be implemented in cell co-culture or in organoids generated from induced pluripotent stem cells (iPSCs) derived from healthy or diseased subjects (Driehuis et al., 2020; Wensink et al., 2021). In one example, this may aid the diagnosis and stratification of patients with diseases that have an underlying genetic basis such as cancer, neurodegeneration and inborn errors of amino-acid metabolism (Eichmuller and Knoblich, 2022; Ling et al., 2023; Manoli and Venditti, 2016; Venkataraman et al., 2022). In other examples, CAAS may provide a useful drug-screening platform in the contexts of co-cultures of tumour cells with cancer associated fibroblasts (CAFs), or organoid models of colorectal cancer, gastric cancer, glioblastoma, liver cancer, or pancreatic cancer, (Liu et al., 2021 ; Liu et al., 2023; Polak et al., 2024; Silva et al., 2017; Strating et al., 2023; Xu et al., 2023b; Zhao et al., 2024) The term cell-type refers to a group of cells sharing a common genotype, phenotype or genetic lineage. Examples of cell-types include neurons, glia, adipocytes or hepatocytes.

[0127] 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.

[0128] 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.

[0129] 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.

[0130] 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.

[0131] The disease or disorder may be a metabolic disease or an inborn error of amino acid or protein metabolism, cancer or neurodegeneration. The metabolic disease or inborn error of amino acid or protein metabolism may be phenylketonuria, homocystinuria, tyrosinemia or maple syrup urine disease, Isovaleric acidemia, 3-Methylcrotonyl-CoA carboxylase deficiency, 3- Methylglutaconic aciduria types l-IV, 3-Hydroxy-3- methylglutaryl-CoA lyase deficiency, 2- methyl-3-hydroxyisobutyric aciduria, Methylbutyryl-CoA dehydrogenase deficiency, Mitochondrial acetoacetyl-CoA thiolase deficiency, 3-Hydroxyisobutyryl- CoA deacylase deficiency, 3-Hydroxyisobutyric aciduria, Propionic acidemia, Methylmalonic acidemia, type-2 diabetes, atherosclerosis, hypertension, heart failure, obesity or kidney disease. The cancer may be carcinoma, gastric cancer, pancreatic cancer, breast 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.

[0132] The enzymes used in the methods of the invention act on amino acids. The enzymes catalyse the conversion of a first isomer or epimer of an amino acid into another isomer or epimer (for example the D to L form, or vice versa). The enzymes are stereo-isomerases that act on amino acids, otherwise known as amino-acid stereo-isomerases. The stereo-isomerase may be a racemase (i.e. an amino acid racemase) or a epimerase (i.e. an amino acid epimerase). “Amino acid racemase” as used herein refers to a racemase enzyme that acts on amino acids. “Amino acid epimerase” as used herein refers to an epimerase enzyme that acts on amino acids.

[0133] In some embodiments, there is provided a method of tracking the movement of an amino acid of interest or a metabolite thereof in a eukaryotic multicellular structure, from or within a first location of the eukaryotic multicellular structure, the method comprising:

[0134] (a) expressing an exogenous amino-acid stereo-isomerase in the first location of the eukaryotic multicellular structure, for example using an conditional and / or cell-type specific expression system;

[0135] (b) introducing a D-isomer counterpart of the amino acid of interest into the nutritional supply of the multicellular structure, wherein the D-isomer counterpart is labelled at a chiral centre (for example the alpha carbon) with deuterium or tritium and comprises a second label, wherein second label is at least one additional stable isotope or radioactive isotope;

[0136] (c) catalysing the interconversion of the D-isomer counterpart of the amino acid of interest with the amino-acid stereo-isomerase to from the L-isomer of the amino acid of interest in the first location of the eukaryotic multicellular structure;

[0137] (d) detecting or quantifying the converted L-isomer of the amino acid of interest comprising the second label, or a metabolite thereof, in the first location and / or in one or more further locations of the eukaryotic multicellular structure;

[0138] (e) wherein the second label is not lost from the amino acid during any interconversion of the amino acid from one isomer to another. In some embodiments, there is provided a method of tracking the movement of an amino acid of interest or a metabolite thereof in a eukaryotic multicellular structure, from or within a first location of the eukaryotic multicellular structure, the method comprising:

[0139] (a) expressing an exogenous amino-acid stereo-isomerase in the first location of the eukaryotic multicellular structure, for example using an conditional and / or cell-type specific expression system;

[0140] (b) introducing an L-isomer counterpart of the amino acid of interest into the nutritional supply of the multicellular structure, wherein the L-isomer counterpart is labelled at a chiral centre (for example the alpha carbon) with deuterium or tritium and comprises a second label, wherein second label is at least one additional stable isotope or radioactive isotope;

[0141] (c) catalysing the interconversion of the L-isomer counterpart of the amino acid of interest with the amino-acid stereo-isomerase to form the D-isomer of the amino acid of interest in the first location of the eukaryotic multicellular structure;

[0142] (d) detecting or quantifying the converted D-isomer of the amino acid of interest comprising the second label, or a metabolite thereof, in the first location and / or in one or more further locations of the eukaryotic multicellular structure, or detecting or quantifying an L-isomer of the amino acid of interest comprising the second label that has undergone at least two interconversions, or a metabolite thereof;

[0143] (e) wherein the second label is not lost from the amino acid during any interconversion of the amino acid from one isomer to another.

[0144] In embodiments where an L-isomer of the amino acid of interest that is labelled with deuterium or tritium is used, the step of detection or quantification may comprise detection or quantification of the amino acid of interest after it has undergone at least one interconversion from L to D, or after it has undergone at least two interconversions, i.e. from L to D and then from D back to L. Generally the detected or quantified L or D amino acid comprises the additional or second label. However, the detected or quantified L or D amino acid may not comprise the deuterium or tritium label on the alpha carbon.

[0145] Examples

[0146] Methods

[0147] LaferILEP cloning and constructs

[0148] Isoleucine epimerase (ILEP) from Lactobacillus fermentum* (abbreviated LaferILEP) was expressed as a genomic DNA construct in cells, tissues or whole, animals. It could also be delivered as mRNA or via RNA- or DNA-viruses. Lafer protein sequence from the NCBI database (entry WP_012391664) was retrieved and a linker-HA-linker-HA epitope tag inserted instead of the natural stop codon (at the C-terminus of the protein). This synthetic nucleotide sequence encoding LaferILEP-linker-HA-linker-HA protein was then codon optimised for expression in Drosophila melanogaster using the Gene Optimiser algorithm (ThermoFisher Scientific) and manual corrections of the poorest codons in the Drosophila genome. The codon optimised LaferILEP sequence was then synthesised by Genewiz (Azenta) and subcloned into the UAS fly expression vector pWALIUM10-roe from the TRiP project at Harvard. T ransgenic UAS- LaferILEP- HA D. melanogaster were created by injection of this plasmid DNA construct (pW10_Lafer) into embryos and subsequent selection of transgenic F1 offspring based on marker expression.

[0149] Transgenic mice were created under contract by Ozgene (Perth, Australia) by homologybased recombination at the Rosa26 locus in mouse ES cells of a C57BL / 6 genetic background. The Lafer-linker-HA-linker-HA sequence created for fly expression was used as the basis for developing the mouse / mammalian sequence. In addition, a tdTomato-NLS was fused to the C-terminus after a self-cleaving P2A peptide so that both Lafer-linker-HA-linker- HA and tdTomato-NLS proteins could be translated from the same open reading frame. The resulting bicistronic protein sequence was codon-optimized for mouse (Mus musculus) using the Gene Optimiser algorithm (ThermoFisher Scientific). The final DNA sequence was used to design a ROSA26 knock-in construct for Cre-mediated Lafer-linker-HA-linker-HA expression via the flip-excision (FLEx) system (Schnutgen et al., 2003).

[0150] In vivo bioassays in Drosophila melanogaster

[0151] Expression in Drosophila was achieved using the GAL4 / UAS method (Brand and Perrimon, 1993). UAS-LaferILEP-HA flies were crossed to flies carrying an enterocyte-specific Gal4 driver (NP1-Gal4) in order to drive LaferILEP-HA expression specifically in the gut of offspring. Alternatively, UAS-LaferILEP-HA flies were crossed to flies carrying a fat body GAL4 driver (Lpp-Gal4) to drive LaferILEP-HA expression specifically in the adipose tissue of offspring. Offspring embryos from these crosses were sterilized with sodium hypochlorite and transferred under sterile conditions to vials of a chemically-defined diet (HolFast) containing either L-Leucine (control) or D-Leucine (experimental). HolFast was optimised from a previous holidic diet and supports much faster growth and development (Froldi et al., 2019; Sorge et al., 2025). Growth and developmental progression were monitored and pupariation scored as evidence of completion of the larval phase of development.

[0152] To detect D- and L-Leucine in fly samples, we collected hemolymph (blood) from larvae using an established NMR protocol (Fernando et al., 2019). LaferILEP-HA was expressed in adipose tissue of the larva (using Lpp-Gal4) and compared to two control crosses containing either the driver Lpp-Gal4 or UAS-LaferILEP-HA. Larvae were raised conventionally on a standard diet containing L-Leucine and L-lsoleucine.

[0153] Proof of concept of the CAAS mass-tagging method used d10-D-Leucine (CDN Isotopes, D- 5607), where all carbon-bound hydrogen (1H) atoms are substituted for deuterium (2H). Larvae of control (Lpp-Gal4>UAS-GFP) or experimental genotype (Lpp-Gal4> UAS-LaferILEP-HA) were raised on a standard diet, starved for 1.5h and then refed for 2h with only d10-D-Leucine and L-glutamate (the latter for its orexigenic effect) in agarose containing blue food dye, which is used to assess whether larvae have ingested food. Groups of larvae were then bled as mentioned above to extract hemolymph for analysis of circulating Leucine. As the compound (d10-D-Leucine) does contain some d9-Leucine, the graph shows measurements of the pure compound and after larvae of the two genotypes had consumed the compound. Statistical analysis used two-way ANOVA with Tukey’s multiple comparison test.

[0154] In vivo bioassays in Mus musculus

[0155] All studies were performed under a UK Home office approved project license and in accordance with institutional welfare guidelines and local ethical committees. All results are reported in line with ARRIVE 2.0 guidelines (Percie du Serf et al., 2020). Mice carrying a FLExLaferILEP-linker-HA-linker-HA construct were crossed to Albumin-Cre animals in order to drive LaferILEP-HA expression specifically in liver hepatocytes. Juvenile offspring from this cross were separated from their mothers at weaning age (P21 ± 2days) and transferred to custom designed chemically-defined diets containing either D-Leucine or L-Leucine (Envigo / lnotiv diets 220646 and 220644 respectively). Weights were recorded daily for 14 days of juvenile growth after weaning at 21 days old.

[0156] Chiral GC-MS detection of D- and L-Leucine

[0157] Gas chromatography-based discrimination of D- and L-enantiomers of amino acids by mass spectrometry (GC-MS, Agilent 7890A-7000C) used a chiral column (Agilent Chirasil L-Val) and a two-step derivatisation procedure with acetyl chloride in isopropanol and pentafluoropropionic anhydride in ethyl acetate based on a previously described method (Lorenzo et al., 2015). The dry polar extracts of fly or mouse origin were derivatised by first adding 50pl of a 1 :4 (v:v) mix of acetyl chloride and isopropanol, followed by 2 minutes of vortexing and 5 minutes of incubation in an ultrasonic bath. The mixture was then incubated at 80°C for one hour and afterwards dried under a stream of nitrogen gas. The dry residue was then again dissolved in 25pl of pentrafluoropropionic anhydride and 25pl of ethyl acetate, vortexed for 3-5 minutes and then derivatised at 65°C for 30 minutes. The mix was then dried again under nitrogen and the residue redissolved in 50pl dichloromethane. 1 l was injected in splitless mode with an inlet temperature of 250°C and a flow rate of 1 ml / min. The oven was set to 75°C for 3 minutes, followed by a ramp of 8°C / min to 120°C, which was held for 2 minutes and a further ramp of 10°C / min to 160°C. The mass-spectrometer was operating in scan mode in the m / z range of 50-350. Separation of stereoisomers was confirmed with pure standards and detected levels were normalised with an internal standard spiked into samples at the earliest possible timepoint.

[0158] To detect racemisation by loss of a deuterium atom at the chiral centre (see Figure 1 for explanation), conventional gas chromatography with mass spectrometry was employed. Polar extracts were derivatised with methoxyamine in pyridine overnight, followed by a silylation reaction with BSTFA and 1 % TMS. Derivatised extracts were then separated by GC-MS (Agilent 7890A-7000C) on an Agilent DB-5ms column.

[0159] ★Lactobacillus fermentum was recently renamed as Limosilactobacillus fermentum, taxid: 334390.

[0160] Example 1

[0161] CAAS in tracing mode provides specific cells or tissues with primary access to a labelled L- amino acid. The racemase or epimerase will convert D-amino acids labelled with stable isotopes such as deuterium,13C or15N, into their labelled L-amino acid counterparts only in specific cells or tissues. In order to circumvent the need for chiral chromatography or chiral derivatization to detect the conversion of D to L amino acids, we have devised a racemase or epimerase-dependent mass tagging strategy. Racemase or epimerase-dependent mass tagging exploits a reaction mechanism shared between amino-acid racemases or epimerases of the pyridoxal-5-phosphate (PLP) dependent and independent classes, which involves hydrogen exchange with water at the chiral centre of the amino acid (Yoshimura and Esak, 2003). In this way, an exogenously supplied D-amino acid substrate labelled with deuterium at the chiral centre will lose one mass unit during conversion into its L-amino acid stereoisomer, which can then be detected via mass spectrometry. Any subsequent racemase or epimerasedependent interconversions of L and D-leucine will nevertheless maintain this mass loss (Figure 1A). Additional deuterium or other isotope labelled atoms incorporated into the initial D-amino acid substrate allow the L-amino acid and its metabolites generated by conditional expression of the racemase or epimerase to be distinguished via mass spectrometry from the pools generated from other reactions (Figure 1A).

[0162] As a proof-of-principle, we have applied the CAAS invention to BCAAs, which are essential for animal growth, development and survival. We tested several amino acid racemases or epimerases and chose isoleucine-2-epimerase from Lactobacillus fermentum* (Lafer I LEP) as it is known to be specific for two branched-chain amino acid substrates, leucine and isoleucine (Mutaguchi et al., 2018). Lafer I LEP interconverts D-leucine and L-leucine, as well as D-allo- isoleucine and L-isoleucine (Figure 2 and Figure 3). The GAL4 / UAS system (Brand and Perrimon, 1993) in fruit flies (Drosophila melanogaster, hereafter Drosophila) and the FLEx system (Schnutgen et al., 2003) in mice (Mus musculus) were used to generate DNA vectors and transgenic animals that express, in a conditional and tissue-specific manner, codon- optimized and HA epitope tagged variants of LaferILEP (Figure 4, Figure 5 and Figure 6). Vectors were also created for transient or stable transfection of cultured cells with LaferILEP expressed from a strong, constitutive promoter (Figure 6).

[0163] In transiently transfected human HEK293 cells, LaferILEP-expressing cells are able to racemise a significant proportion of 13C6-labelled D-Leucine after 90 minutes of incubation (Figure 7). In Drosophila, using a chiral GC-MS assay, we show that codon-optimised LaferILEP-HA expressed in the gut (NP1-GAL4:UAS-LaferlLEP-HA, abbreviated as NP1> LaferILEP- HA) of animals raised on a complete L-amino acid diet generate substantial quantities of D-leucine and D-allo-isoleucine, which are largely absent in genetic controls (Figure 8). This indicates that bacterial LaferILEP-HA is an effective leucine racemase and isoleucine epimerase not only in the context of human cell culture but also in a living animal. Importantly, gut enterocyte-specific expression of LaferILEP-HA produces D-leucine and D- allo-isoleucine in the insect blood (called hemolymph, Figure 8). This demonstrates that LaferILEP-HA can be used to produce a source of unlabelled amino acids that are transported from one tissue to another, in this case from the gut to the hemolymph.

[0164] To use codon-optimised LaferILEP-HA in tracing mode (Figure 1), we also developed an isotopic labelling strategy. We reasoned that d10-D-Leucine, which has deuterium (2Hydrogen) at the chiral centre and at other atomic positions, would enable monitoring of the D to L transition by conversion of a d10-D-Leucine substrate into a d9-L-leucine product, with the loss of 1 dalton. Drosophila larvae of two control genotypes or those expressing LaferILEP-HA specifically in adipose tissue called fat body (Lpp> LaferILEP-HA) were exposed to a diet containing d10-D-Leucine. Following only 2 hr exposure to this diet, significant enrichment of d9-leucine could be detected in the hemolymph in a LaferILEP-HA dependent manner (Figure 9). This finding provides an experimental validation that LaferILEP-HA can be used in tracing mode to generate a defined cellular source of labelled L-amino acid that is distinguishable from its labelled D-amino acid substrate as well as from endogenous unlabelled amino acids. It also indicates that LaferILEP-HA in stable isotope tracing mode can be used to detect the transport of leucine from one tissue to another, in this case from the adipose tissue to the hemolymph.

[0165] Example 2

[0166] To use codon-optimised LaferILEP-HA in bulk mode, we developed in vivo bioassays in Drosophila and mice (Mus musculus). We asked whether LaferILEP-HA activity is sufficient to produce enough L-leucine from D-leucine to support normal a rate of growth and development, a biological context with a higher demand for essential amino acids than adulthood. In Drosophila raised on a standard complete diet, conditional expression of LaferILEP-HA in gut enterocytes (NP1> LaferILEP-HA) did not alter the rate of larval development or impact upon survival, strongly suggesting that it is not toxic (Figure 10). Importantly, on a diet with L-leucine entirely replaced by D-leucine, 0% of animals of a control genotype survive, whereas 100% of enterocyte LaferILEP-HA expressing animals were able to complete normal development on time (Figure 11). This striking result demonstrates that gut LaferILEP-HA activity in Drosophila can provide sufficient amounts of L-leucine to bypass the dietary requirement for this essential amino acid. In mice, we assessed the ability of liver-specific expression of LaferILEP-HA to confer a normal rate of juvenile growth (mass gain) on diets supplemented with D-leucine but containing decreased levels of L-Leucine. We chose to reduce L-Leucine levels to 25% of the standard amount, based on a previous study (Xiao and Guo, 2022) and our own observations that less than 25% of the normal amount of dietary L-leucine causes substantial weight loss beyond that ethical or permissible in our animal licence. We first raised juvenile mice with conditional expression of LaferILEP-HA in liver hepatocytes (Albumin-Cre x FLEx::Lafer ILEP) on a complete diet containing 100% L-leucine. This did not compromise the survival of juvenile mice or significantly alter their growth rate, compared to the control genotype, over two week period (Figure 12). This shows that hepatocyte LaferILEP-HA expression is not harmful for juvenile development and therefore unlikely to be toxic (Figure 12). Next, we raised juvenile animals on a diet in which 75% of L-Leucine is replaced with D-Leucine. On this diet, there was negligible juvenile body weight gain for the control genotype, but animals expressing hepatocyte LaferILEP-HA gained mass at the normal rate (Figure 13). Hence, hepatocyte LaferILEP-HA activity in Drosophila can provide sufficient amounts of L-leucine to bypass the dietary requirement for this essential amino acid.

[0167] In human cell culture, LaferILEP-HA can also confer cell proliferation and growth in the presence of D-Leucine and the absence of L-Leucine. Control (untransfected) or LaferILEP- HA expressing H EK-293 cells were cultured in DM EM devoid of L-Leucine but containing D- Leucine and their confluence measured over time as a proxy for cell growth and proliferation. Under these conditions, control cells are unable to grow but Lafer ILEP-HA expression confers the ability to utilise D-Leucine for growth (Figure 14). Together, the bulk-mode experiments provide examples in human cells, Drosophila and mice that show that LaferILEP has racemase / epimerase activity that can generate sufficient amounts of L-leucine from D-leucine to support growth and cell proliferation in many different biological contexts.

[0168] Sequences

[0169] References

[0170] Adla, S.K., Virtanen, H., Thongsodsaeng, T., Huttunen, K.M., 2024. Amino acid transporters in neurological disorders and neuroprotective effects of cysteine derivatives. Neurochem I nt 177, 105771.

[0171] Aldana, B.I., Zhang, Y, Jensen, P, Chandrasekaran, A., Christensen, S.K., Nielsen, T.T., Nielsen, J.E., Hyttel, P, Larsen, M.R., Waagepetersen, H.S., Freude, K.K., 2020. Glutamateglutamine homeostasis is perturbed in neurons and astrocytes derived from patient iPSC models of frontotemporal dementia. Mol Brain 13, 125.

[0172] Bartman, C.R., Faubert, B., Rabinowitz, J.D., DeBerardinis, R.J., 2023. Metabolic pathway analysis using stable isotopes in patients with cancer. Nat Rev Cancer 23, 863-878.

[0173] Beaudry, A. G., Law, M.L., 2022. Leucine Supplementation in Cancer Cachexia: Mechanisms and a Review of the Pre-Clinical Literature. Nutrients 14.

[0174] Ben-Sahra, I., Manning, B.D., 2017. mTORCI signaling and the metabolic control of cell growth. Curr Opin Cell Biol 45, 72-82.

[0175] Brand, A.H., Perrimon, N., 1993. Targeted gene expression as a means of altering cell fates and generating dominant phenotypes. Development 118, 401-415.

[0176] Cahill, G.F., Jr., 1970. Starvation in man. N Engl J Med 282, 668-675.

[0177] Costa-Mattioli, M., Walter, P, 2020. The integrated stress response: From mechanism to disease. Science 368.

[0178] DeBerardinis, R.J., Keshari, K.R., 2022. Metabolic analysis as a driver for discovery, diagnosis, and therapy. Cell 185, 2678-2689.

[0179] Dimou, A., Tsimihodimos, V., Bairaktari, E., 2022. The Critical Role of the Branched Chain Amino Acids (BCAAs) Catabolism-Regulating Enzymes, Branched-Chain Aminotransferase (BCAT) and Branched-Chain alpha-Keto Acid Dehydrogenase (BCKD), in Human Pathophysiology. I nt J Mol Sci 23.

[0180] Driehuis, E., Kretzschmar, K., Clevers, H., 2020. Establishment of patient-derived cancer organoids for drug-screening applications. Nat Protoc 15, 3380-3409.

[0181] Droujinine, I. A., Meyer, A.S., Wang, D., Udeshi, N.D., Hu, Y, Rocco, D., McMahon, J. A., Yang, R., Guo, J., Mu, L., Carey, D.K., Svinkina, T, Zeng, R., Branon, T., Tabatabai, A., Bosch, J. A., Asara, J.M., Ting, A.Y, Carr, S.A., McMahon, A.P., Perrimon, N., 2021. Proteomics of protein trafficking by in vivo tissue-specific labeling. Nat Commun 12, 2382.

[0182] Du, C., Liu, WJ., Yang, J., Zhao, S.S., Liu, H.X., 2022. The Role of Branched-Chain Amino Acids and Branched-Chain alpha-Keto Acid Dehydrogenase Kinase in Metabolic Disorders. Front Nutr 9, 932670.

[0183] Eichmuller, O.L., Knoblich, J. A., 2022. Human cerebral organoids - a new tool for clinical neurology research. Nat Rev Neurol 18, 661-680.

[0184] Endicott, M., Jones, M., Hull, J., 2021. Amino acid metabolism as a therapeutic target in cancer: a review. Amino Acids 53, 1169-1179. Felig, P, 1975. Amino acid metabolism in man. Annu Rev Biochem 44, 933-955.

[0185] Fernandez-Garcia, J., Altea-Manzano, P., Pranzini, E., Fendt, S.M., 2020. Stable Isotopes for Tracing Mammalian-Cell Metabolism In Vivo. Trends Biochem Sci 45, 185-201.

[0186] Fernando, T, Sawala, A., Bailey, A.P, Gould, A.P., Driscoll, PC., 2019. An Improved Method for Measuring Absolute Metabolite Concentrations in Small Biofluid or Tissue Samples. J Proteome Res 18, 1503-1512.

[0187] Froldi, F., Pachnis, P, Szuperak, M., Costas, O., Fernando, T., Gould, A.P, Cheng, L.Y., 2019. Histidine is selectively required for the growth of Myc-dependent dedifferentiation tumours in the DrosophilaCNS. EMBO J. 38, e99895 - 99812.

[0188] Gauthier, N.P, Soufi, B., Walkowicz, W.E., Pedicord, V.A., Mavrakis, K.J., Macek, B., Gin, D.Y., Sander, C., Miller, M.L., 2013. Cell-selective labeling using amino acid precursors for proteomic studies of multicellular environments. Nat Methods 10, 768-773.

[0189] Gonzalez, A., Hall, M.N., 2017. Nutrient sensing and TOR signaling in yeast and mammals. EMBO J 36, 397-408.

[0190] He, W, Connolly, E.D., Cross, H.R., Wu, G., 2024. Dietary protein and amino acid intakes for mitigating sarcopenia in humans. Crit Rev Food Sci Nutr, 1-24.

[0191] Hegsted, D.M., 1972. Deprivation syndrome or protein-calorie malnutrition. Nutr Rev 30, SI- 54.

[0192] J. Ryan, P, Riechman, S.E., Fluckey, J.D., Wu, G., 2021. Interorgan Metabolism of Amino Acids in Human Health and Disease, in: Wu, G. (Ed.), Amino Acids in Nutrition and Health: Amino Acids in Gene Expression, Metabolic Regulation, and Exercising Performance. Springer International Publishing, Cham, pp. 129-149.

[0193] Kim, K.E., Park, I., Kim, J., Kang, M.G., Choi, WG., Shin, H., Kim, J.S., Rhee, H.W, Suh, J.M., 2021. Dynamic tracking and identification of tissue-specific secretory proteins in the circulation of live mice. Nat Commun 12, 5204.

[0194] Layman, D.K., 2024. Impacts of protein quantity and distribution on body composition. Front Nutr 11 , 1388986.

[0195] Ling, Z.N., Jiang, Y.F., Ru, J.N., Lu, J.H., Ding, B., Wu, J., 2023. Amino acid metabolism in health and disease. Signal Transduct Target Ther 8, 345.

[0196] Liu, J., Li, P, Wang, L., Li, M., Ge, Z., Noordam, L., Lieshout, R., Verstegen, M.M.A., Ma, B., Su, J., Yang, Q., Zhang, R., Zhou, G., Carrascosa, L.C., Sprengers, D., JNM, I. J., Smits, R., Kwekkeboom, J., van der Laan, L.J.W, Peppelenbosch, M.P., Pan, Q., Cao, W, 2021. Cancer- Associated Fibroblasts Provide a Stromal Niche for Liver Cancer Organoids That Confers Trophic Effects and Therapy Resistance. Cell Mol Gastroenterol Hepatol 11 , 407-431.

[0197] Liu, Y, Li, N., Zhu, Y, 2023. Pancreatic Organoids: A Frontier Method for Investigating Pancreatic- Related Diseases. Int J Mol Sci 24.

[0198] Lorenzo, M.P, Dudzik, D., Varas, E., Gibellini, M., Skotnicki, M., Zorawski, M., Zarzycki, W, Pellati, F., Garcia, A., 2015. Optimization and validation of a chiral GC-MS method for the determination of free D-amino acids ratio in human urine: application to a gestational diabetes mellitus study. J Pharm Biomed Anal 107, 480-487. Manoli, I., Venditti, C.P., 2016. Disorders of branched chain amino acid metabolism. Transl Sci Rare Dis 1 , 91-110.

[0199] Mutaguchi, Y, Kasuga, K., Kojima, I., 2018. Production of d-Branched-Chain Amino Acids by Lactic Acid Bacteria Carrying Homologs to Isoleucine 2-Epimerase of Lactobacillus buchneri. Front Microbiol 9, 1540.

[0200] Newell, C.L., Vorng, J.L., MacRae, J. I., Gilmore, I.S., Gould, A.P., 2020. Cryogenic OrbiSIMS Localizes Semi-Volatile Molecules in Biological Tissues. Angew Chem Int Ed Engl 59, 18194- 18200.

[0201] Passarelli, M.K., Pirkl, A., Moellers, R., Grinfeld, D., Kollmer, F., Havelund, R., Newman, C.F., Marshall, PS., Arlinghaus, H., Alexander, M.R., West, A., Horning, S., Niehuis, E., Makarov, A., Dollery, C.T., Gilmore, I.S., 2017. The 3D OrbiSI MS-label-free metabolic imaging with subcellular lateral resolution and high mass-resolving power. Nat Methods 14, 1175-1183.

[0202] Peng, H., Wang, Y, Luo, W, 2020. Multifaceted role of branched-chain amino acid metabolism in cancer. Oncogene 39, 6747-6756.

[0203] Percie du Serf, N., Hurst, V., Ahluwalia, A., Alam, S., Avey, M.T., Baker, M., Browne, WJ., Clark, A., Cuthill, I.C., Dirnagl, U., Emerson, M., Garner, P, Holgate, S.T, Howells, D.W, Karp, N.A., Lazic, S.E., Lidster, K., MacCallum, C.J., Macleod, M., Pearl, E.J., Petersen, O.H., Rawle, F., Reynolds, P, Rooney, K., Sena, E.S., Silberberg, S.D., Steckler, T., Wurbel, H., 2020. The ARRIVE guidelines 2.0: Updated guidelines for reporting animal research. J Cereb Blood Flow Metab 40, 1769-1777.

[0204] Polak, R., Zhang, E.T., Kuo, C.J., 2024. Cancer organoids 2.0: modelling the complexity of the tumour immune microenvironment. Nat Rev Cancer 24, 523-539.

[0205] Riehl, J.P., 2010. Mirror-image asymmetry: An introduction to the origin and consequences of chirality. John Wiley & Sons.

[0206] Ryoo, H.D., 2024. The integrated stress response in metabolic adaptation. J Biol Chem 300, 107151.

[0207] Schnutgen, F., Doerflinger, N., Calleja, C., Wendling, O., Chambon, P, Ghyselinck, N.B., 2003. A directional strategy for monitoring Cre-mediated recombination at the cellular level in the mouse. Nat Biotechnol 21 , 562-565.

[0208] Semba, R.D., 2016. The Rise and Fall of Protein Malnutrition in Global Health. Ann Nutr Metab 69, 79-88.

[0209] Sidoryk-Wegrzynowicz, M., Adamiak, K., Struzynska, L., 2024. Astrocyte-Neuron Interaction via the Glutamate-Glutamine Cycle and Its Dysfunction in Tau-Dependent Neurodegeneration. Int J Mol Sci 25.

[0210] Silva, L.S., Poschet, G., Nonnenmacher, Y, Becker, H.M., Sapcariu, S., Gaupel, A.C., Schlotter, M., Wu, Y, Kneisel, N., Seiffert, M., Hell, R., Hiller, K., Lichter, P, Radlwimmer, B., 2017. Branched-chain ketoacids secreted by glioblastoma cells via MCT1 modulate macrophage phenotype. EMBO Rep 18, 2172-2185.

[0211] Siqueira, J.M., Vega, M., Pimentel, G.D., 2024. Amino acids and cancer: potential for therapies? Curr Opin Clin Nutr Metab Care 27, 47-54.

[0212] Soeters, P.B., van de Poll, M.C., van Gemert, WG., Dejong, C.H., 2004. Amino acid adequacy in pathophysiological states. J Nutr 134, 1575S-1582S. Sorge, S., Girard, V., Lampe, L., Tixier, V., Weaver, A., Higgins, T., Gould, A.P., 2025. A Drosophila holidic diet optimized for growth and development. Dev Cell 60, 1636- 1648. e 1634.

[0213] Sperringer, J. E., Addington, A., Hutson, S.M., 2017. Branched-Chain Amino Acids and Brain Metabolism. Neurochem Res 42, 1697-1709.

[0214] Strating, E., Verhagen, M.P., Wensink, E., Dunnebach, E., Wijler, L., Aranguren, I., De la Cruz, A.S., Peters, N.A., Hageman, J.H., van der Net, M.M.C., van Schelven, S., Laoukili, J., Fodde, R., Roodhart, J., Nierkens, S., Snipped, H., Gloerich, M., Rinkes, I.B., Elias, S.G., Kranenburg, O., 2023. Co-cultures of colon cancer cells and cancer-associated fibroblasts recapitulate the aggressive features of mesenchymal-like colon cancer. Front Immunol 14, 1053920.

[0215] Tape, C.J., Norrie, I.C., Worboys, J.D., Lim, L., Lauffenburger, D.A., Jorgensen, C., 2014. Cellspecific labeling enzymes for analysis of cell-cell communication in continuous co-culture. Mol Cell Proteomics 13, 1866-1876.

[0216] Venkataraman, L., Fair, S.R., McElroy, C.A., Hester, M.E., Fu, H., 2022. Modeling neurodegenerative diseases with cerebral organoids and other three-dimensional culture systems: focus on Alzheimer's disease. Stem Cell Rev Rep 18, 696-717.

[0217] Wensink, G.E., Elias, S.G., Mullenders, J., Koopman, M., Boj, S.F., Kranenburg, O.W, Roodhart, J.M.L., 2021. Patient-derived organoids as a predictive biomarker for treatment response in cancer patients. NPJ Precis Oncol 5, 30.

[0218] Whitehouse, C., Corbett, N., Brownlees, J., 2023. 3D models of neurodegeneration: implementation in drug discovery. Trends Pharmacol Sci 44, 208-221.

[0219] Xiao, F, Guo, F, 2022. Impacts of essential amino acids on energy balance. Mol Metab 57, 101393.

[0220] Xu, E., Ji, B., Jin, K., Chen, Y, 2023a. Branched-chain amino acids catabolism and cancer progression: focus on therapeutic interventions. Front Oncol 13, 1220638.

[0221] Xu, F., Jiang, H.L., Feng, WW, Fu, C., Zhou, J.C., 2023b. Characteristics of amino acid metabolism in colorectal cancer. World J Clin Cases 11 , 6318-6326.

[0222] Yang, R., Meyer, A. S., Droujinine, I. A., lldeshi, N.D., Hu, Y, Guo, J., McMahon, J. A., Carey, D.K., Xu, C., Fang, Q., Sha, J., Qin, S., Rocco, D., Wohlschlegel, J., Ting, A.Y, Carr, S.A., Perrimon, N., McMahon, A.P, 2022. A genetic model for in vivo proximity labelling of the mammalian secretome. Open Biol 12, 220149.

[0223] Yang, S., Hu, H., Kung, H., Zou, R., Dai, Y, Hu, Y, Wang, T., Lv, T., Yu, J., Li, F., 2023. Organoids: The current status and biomedical applications. MedComm (2020) 4, e274.

[0224] Yoshimura, T., Esak, N., 2003. Amino acid racemases: functions and mechanisms. J Biosci Bioeng 96, 103-109.

[0225] Zhao, Y, Li, S., Zhu, L., Huang, M., Xie, Y, Song, X., Chen, Z., Lau, H.C., Sung, J. J., Xu, L., Yu, J., Li, X., 2024. Personalized drug screening using patient-derived organoid and its clinical relevance in gastric cancer. Cell Rep Med 5, 101627.

[0226] Ziegler, S.G., Kim, J., Ehmsen, J.T., Vernon, H.J., 2023. Inborn errors of amino acid metabolism - from underlying pathophysiology to therapeutic advances. Dis Model Meeh 16.

Claims

Claims1. A method of tracking the movement of an amino acid of interest or a metabolite thereof in a eukaryotic multicellular structure, from or within a first location of the eukaryotic multicellular structure, the method comprising:(a) expressing an exogenous amino-acid stereo-isomerase in the first location of the eukaryotic multicellular structure, for example using an conditional and / or cell-type specific expression system;(b) introducing an isomer counterpart of the amino acid of interest into the nutritional supply of the multicellular structure, wherein the isomer counterpart is labelled at a chiral centre with a stable isotope or radioactive isotope of hydrogen, and optionally further labelled with at least one additional stable isotope or radioactive isotope;(c) catalysing the interconversion of the isomer counterpart of the amino acid of interest with the amino-acid stereo-isomerase to form a converted amino acid in the first location of the eukaryotic multicellular structure;(d) detecting or quantifying the converted amino acid or a metabolite thereof in the first location and / or in one or more further locations of the eukaryotic multicellular structure.

2. The method of claim 1 , wherein:(a) the isomer counterpart is an L-amino acid and the converted amino acid is a D- amino acid; or(b) the isomer counterpart is a D-amino acid and the converted amino acid is an L- amino acid.

3. The method of claim 1 or 2, wherein the first location is an origin cell and the one or more further locations is a destination cell, extracellular fluid and / or a secreted or excreted body fluid or substance.

4. The method of claim 3, wherein the cell-type specific expression system is specific for the origin cell.

5. The method of claim 1 , wherein the first location is an organelle within a single cell of the eukaryotic multicellular structure and the one or more further locations are(i) different organelles within the same cell; or(ii) different cells.

366. A method of determining the function of an L-amino acid of interest in a eukaryotic cell or tissue, the method comprising:(a) providing the eukaryotic cell or tissue with a nutritional supply comprising the L- amino acid of interest (non-converted L-amino acid);(b) measuring one or more biological activities of the cell or tissue suspected of being modulated by the L-amino acid;(c) expressing an amino-acid stereo-isomerase in the cell or tissue, for example using an conditional and / or cell-type specific expression system;(d) catalysing the conversion of an amino acid of interest from an L-amino acid to a D- amino acid with the amino-acid stereo-isomerase, resulting in a reduction in the amount or concentration of L-amino acid in the eukaryotic cell or tissue;(e) detecting a change in one or more biological activities of the cell-type or tissue following the reduction in the amount or concentration of the L-amino acid.

7. A method of determining the function of an L-amino acid of interest in a eukaryotic cell or tissue, the method comprising:(a) providing a eukaryotic cell or tissue with a nutritional supply comprising the L-amino acid of interest;(b) measuring one or more biological activities of the cell or tissue suspected of being modulated by the L-amino acid;(c) expressing an amino-acid stereo-isomerase in the cell or tissue, for example using an conditional and / or cell-type specific expression system;(d) partially or completely replacing the L-amino acid in the nutritional supply of the cell with its D-amino acid counterpart (non-converted D-amino acid);(e) catalysing the conversion of the D-amino acid obtained from the nutritional supply to its L-amino acid counterpart with the amino-acid stereo-isomerase, resulting in an increase in the amount or concentration of L-amino acid in the eukaryotic cell or tissue;(f) detecting a change in one or more biological activities of the cell or tissue following the increase in the amount or concentration of the L-amino acid.

8. The method of any one of claims 1 to 7, wherein the unconverted D-amino acid or unconverted L-amino acid is labelled at a chiral centre with a rare isotope, optionally wherein the rare isotope is2Hydrogen (deuterium) or3Hydrogen (tritium).

9. The method of any one of claims 1 to 8, wherein the unconverted D-amino acid or unconverted L-amino acid is further labelled, optionally wherein the rare isotope is37selected from13Carbon,15Nitrogen or2Hydrogen (deuterium)17Oxygen or18Oxygen, 11Carbon,14Carbon or3Hydrogen (tritium).

10. The method of any one of claims 1 to 9 wherein the nutritional supply is in the form of cell culture medium, intravenous or intraperitoneal injection, gavage, a dietary supplement or supplementation of the drinking water.

11. The method of any one of claims 1 to 10 wherein the conditional and / or cell-type specific expression system is 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.

12. The method of any one of claims 1 to 11 wherein the L-amino acid of interest is Leucine or Isoleucine and the D-amino acid counterpart is D-Leucine or D-allo-lsoleucine.

13. The method of any one of claims 1 to 12, wherein the amino-acid stereo-isomerase is a racemase or epimerase.

14. The method of claim 13, wherein the racemase or epimerase is from Lactobacillus fermentum, optionally wherein the racemase or epimerase is isoleucine-2-epimerase (Lafer ILEP).

15. The method of any one of claims 1 to 14 wherein the racemase or epimerase has 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.

16. The method of any one of claims 1 to 15 wherein the racemase or epimerase is encoded by a nucleic 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: 2 or 3.

17. The method of any one of claims 1 to 16 wherein the multicellular structure is a living organism, an organoid or a cell culture system.

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

19. The method of claim 17, wherein the cell culture system comprises pluripotent stem cells or differentiated cells derived therefrom, optionally wherein the pluripotent stem cells are induced pluripotent stem cells.

20. The method of claim 17, wherein the cell culture system comprises human, mouse, rat, zebrafish, Drosophila or C.elegans cells.

21. The method of claim 17, wherein the organoid is a mouse-derived organoid, a rat- derived organoid, a primate-derived organoid, a patient-derived organoid or other human- derived organoid.

22. The method of any one of claims 1 to 21 , wherein the detection or quantification is carried out by mass spectrometry (MS), mass spectrometry imaging (MSI), nuclear magnetic resonance (NMR), scintillation counting, autoradiography, gamma counting or imaging using a fluorescent or other reporter.

23. A nucleic acid comprising a sequence of SEQ ID NO: 2 or 3.

24. A recombinant expression vector comprising the nucleic acid sequence of claim 23.

25. A host cell comprising the recombinant expression vector of claim 24.

26. A genetically modified organism expressing the nucleic acid of claim 23 under the control of a conditional and / or cell-type specific expression system.

27. A method of diagnosing a disease or disorder associated with amino acid transport and / or metabolism, the method comprising:(a) expressing an amino-acid stereo-isomerase under a conditional and / or cell-type specific expression system in a first eukaryotic cell-type obtained from a patient suspected of having a disease or disorder associated with amino acid transport and / or metabolism;(b) culturing an organoid or cell culture system comprising one or more cells of the first cell-type and optionally further comprising one or more cells of a second eukaryotic cell-type;(c) providing the organoid or cell culture system with a nutritional supply;(d) introducing a labelled L-amino acid of interest or labelled D-amino acid counterpart of the L-amino acid of interest into the nutritional supply of the organoid or cellculture system, wherein the L-amino acid or D-amino acid counterpart is labelled with a stable isotope or a radioactive isotope of hydrogen at a chiral centre;(e) catalysing the interconversion of the D-amino acid and L-amino acid of interest with the amino-acid stereo-isomerase;(f) quantifying the amount or concentration of the labelled and / or converted L-amino acid or a metabolite thereof in the first cell-type and / or second cell-type;(g) comparing the amount or concentration of the labelled and / or converted L-amino acid or a metabolite thereof in the first cell-type and / or second cell-type to a control value; wherein a change in the amount of the labelled and / or converted L-amino acid or metabolite thereof in the first cell-type and / or second cell-type relative to the control value is predictive of a disease or disorder associated with amino acid transport and / or metabolism.

28. The method of claim 27, wherein:(a) the control value is determined by carrying out steps (a) to (f) of claim 27, 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; or(b) the control value is a predicted or expected value.

29. A method of screening therapeutic agents for use in the treatment of a disease or disorder associated with amino acid transport and / or metabolism, the method comprising:(a) expressing an amino-acid stereo-isomerase under a conditional and / or cell-type specific expression system in a first eukaryotic cell-type having a disease or disorder associated with amino acid transport and / or metabolism;(b) culturing an organoid or cell culture system comprising one or more cells of the first eukaryotic cell-type and optionally further comprising one or more cells of a second eukaryotic cell-type;(c) providing the organoid or cell culture system with a nutritional supply;(d) introducing a labelled L-amino acid of interest or labelled D-amino acid counterpart of the L-amino acid of interest into the nutritional supply of the organoid or cell culture system, wherein the L-amino acid or D-amino acid counterpart is labelled with a stable isotope or a radioactive isotope of hydrogen at a chiral centre;(e) catalysing the interconversion of the D-amino acid and L-amino acid of interest with the amino-acid stereo-isomerase;(f) quantifying the amount or concentration of the converted L-amino acid in the first and / or second cell-type to establish the baseline level;(g) administering a pharmaceutically active amount of the therapeutic agent to the organoid or cell culture system;(h) quantifying the amount or concentration of the labelled and / or converted L-amino acid or a metabolite thereof in the first cell-type and / or second cell-type following the administration of the therapeutic agent;(i) comparing the amount or concentration of the labelled and / or converted L-amino acid or metabolite thereof in the first cell-type and / or second cell-type cell after administration of the therapeutic agent to the baseline level of step (f).

30. The method of claim 29, wherein the first eukaryotic cell-type is obtained from a patient suspected of having a disease or disorder associated with amino acid transport and / or metabolism.

31. The method of claim 29 or claim 30, wherein a change in the amount or concentration of the labelled and / or converted L-amino acid or metabolite thereof in the first cell-type and / or second cell-type relative to baseline levels indicates that the therapeutic agent is capable of modifying amino acid transport and / or metabolism.

32. The method of any one of claims 29 to 31 , wherein the disease or disorder is a metabolic disease or an inborn error of amino acid metabolism, cancer or neurodegeneration.

33. The method of claim 32, wherein:(i) the metabolic disease or inborn error of protein or amino acid metabolism is phenylketonuria, homocystinuria, tyrosinemia or maple syrup urine disease, Isovaleric acidemia, 3-Methylcrotonyl-CoA carboxylase deficiency, 3-Methylglutaconic aciduria types l-IV, 3-Hydroxy-3- methylglutaryl-CoA lyase deficiency, 2-methyl-3- hydroxyisobutyric aciduria, M ethyl butyryl-CoA dehydrogenase deficiency, Mitochondrial acetoacetyl-CoA thiolase deficiency, 3-Hydroxyisobutyryl- CoA deacylase deficiency, 3-Hydroxyisobutyric aciduria, Propionic acidemia or Methylmalonic acidemia, Type-2 diabetes, atherosclerosis, hypertension, heart failure, obesity or kidney disease;(ii) the cancer is carcinoma, gastric cancer, pancreatic cancer, breast cancer, colon cancer, colorectal cancer, lung cancer, prostate cancer, testicular cancer, liver cancer, neuroblastoma or glioma; or(iii) the neurodegenerative disease is 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.42