Methods of treating neurological diseases

Abstract

This disclosure relates to methods of inhibiting transport of choline and choline- containing compounds in a cell, such as a vascular cell in the central nervous system, using inhibitors of MFSD7c or MFSD2a. Also disclosed are methods of treating CNS diseases and conditions in a subject using the inhibitors.

Description

[0001] Methods of Treating Neurological Diseases

[0002] Technical field

[0003] The invention relates generally to neurology. In particular, the invention relates to methods of inhibiting transport of choline or choline-containing compounds in a cell, such as a vascular cell in the central nervous system, by inhibiting MFSD proteins.

[0004] Background

[0005] Acetylcholine is an important neurotransmitter that is involved in many cognitive functions of the brain, including memory, attention and learning. The deficit of cholinergic activity (i.e. the activity of acetylcholine) is associated with several neurological diseases and conditions. These include Alzheimer's disease, which is a disease characterized by impaired cognitive functions such as memory, thinking and behaviour. This is thought to be due to the accumulation of abnormal protein structures, such as P-amyloid plaques, in the brain, which disrupt normal communication and degeneration of brain cells. The decline in cholinergic activity is primarily due to the loss of cholinergic neurons. In Alzheimer's disease, cholinesterase inhibitors, which inhibit the degradation of acetylcholine, are often prescribed for mild to moderate symptoms, with the aim to stimulate cholinergic activity. However, the benefits of this therapy for Alzheimer’s patients arc modest at best.

[0006] Elevated or excessive cholinergic activity, on the other hand, is associated with other types of neurological diseases and conditions. Parkinson's disease, for example, is a neurological disease that affects movement control. The symptoms of Parkinson's disease include involuntary shaking, muscle rigidity and difficulty with balance and coordination. These symptoms often develop gradually and worsen over time. Furthermore, patients often experience other non-motor symptoms such as sleep disturbances, chance in speech and swallowing as well as depression. There is no cure for Parkinson's disease, although there are various treatment options that are available to manage its symptoms and improve the quality of life for affected individuals. These treatments include, for example, medications that aim to increase dopamine levels in the brain, such as levodopa, dopamine agonists or Mao-B inhibitors. In Parkinson's disease, there is an imbalance between dopamine and acetylcholine in certain parts of the brain. Specifically, there is an overactivity of the cholinergic system in relation to the dopaminergic system. This imbalance is thought to contribute to the motor symptoms of the disease. Epilepsy, on the other hand, is a neurological disorder that is characterized by recurrent and unprovoked seizures. These seizures arc due to abnormal electrical activity in the brain and can involve elevated levels of neurotransmitters such as glutamate, gamma-aminobutyric acid (GABA) and acetylcholine.

[0007] There is a need to develop more effective treatments for neurological diseases and conditions such as Alzheimer's disease, Parkinson's disease and epilepsy. There is also need to develop new ways to slow down or halt the progression of these diseases.

[0008] It would be desirable to overcome or ameliorate at least one of the above-described problems, or at least to provide a useful alternative.

[0009] Summary

[0010] Disclosed herein is a method of promoting synthesis and / or accumulation of acetylcholine in a brain parenchymal cell or tissue in a subject, the method comprising administering an effective amount of an inhibitor of MFSD7c to the subject to promote synthesis and / or accumulation of acetylcholine in the brain parenchymal cell or tissue of the subject.

[0011] Disclosed herein is a method of inhibiting choline transport in a vascular cell in the central nervous system (CNS), the method comprising contacting the vascular cell with an effective amount of an inhibitor of MFSD7c for a sufficient time and under conditions to inhibit choline transport in the vascular cell.

[0012] Disclosed herein is a method of treating a CNS disease or condition in a subject, the method comprising administering an effective amount of an inhibitor of MFSD7c to the subject to treat the CNS disease or condition in the subject. Disclosed herein is a method of preventing or delaying the progression of a CNS disease or condition in a subject, the method comprising administering an effective amount of an inhibitor of MFSD7c to the subject to prevent or delay progression of the CNS disease or condition in the subject.

[0013] Disclosed herein is an inhibitor of MFSD7c, for use in treating or preventing a CNS disease or condition in a subject, wherein the MFSD7c inhibitor is to be administered to the subject.

[0014] Disclosed herein is the use of an inhibitor of MFSD7c in the manufacture of a medicament for treating or preventing a CNS disease or condition in a subject, wherein the MFSD7c inhibitor is to be administered to the subject..

[0015] Disclosed herein is a mammalian cell comprising an exogenous nucleic acid encoding choline kinase A (CHKA) and MFSD7c.

[0016] Disclosed herein is a kit comprising a mammalian cell as defined herein, and an isotopically-labelled choline or an analogue thereof.

[0017] Disclosed herein is the use of a mammalian cell or a kit as defined herein for screening for an inhibitor of MFSD7c.

[0018] Disclosed herein is a lysophosphatidylcholinc (LPC) composition comprising at least one LPC selected from the group consisting of LPC-16:0, LPC-16: 1, LPC-18:0, LPC- 18:1, LPC-18:2, LPC-18:3, LPC-20:4, LPC-20:5, and LPC-22:6.

[0019] Disclosed herein is the use of an LPC composition as defined herein for promoting synthesis and / or accumulation of acetylcholine in a brain parenchymal cell or tissue in a subject.

[0020] Brief description of the drawings Embodiments of the present invention are hereafter described, by way of non-limiting example only, with reference to the accompanying drawings in which:

[0021] Figure 1. Comprehensive metabolomics identifies changes in the levels of choline and choline metabolites in the brain of Major Facilitator Superfamily Domain Containing 7C (Mfsd7c) knockout. A) Increased levels of glycolytic metabolite lactate and decreased levels of mitochondrial metabolites in Mfsd7c knockout brain. B) Heatmap of metabolites with differential changes in the brains of Mfsd7c knockout compared to that of WT embryos. C) Increased levels of acylcamitines in the brains of Mfsd7c knockout embryos. D) Increased levels of choline and decreased levels of CDP- choline and L-carnitine in the brains of Mfsd7c knockout embryos. E) Levels of L- carnitine, choline, and CDP-choline were unchanged in the fetal livers of WT and Mfsd7c knockout embryos. F) Heatmap of neutral lipid species that are differentially changed in the fetal livers of Mfsd7c knockouts compared to that of WT embryos. The results showed the accumulation of neutral lipids in the fetal livers of the Mfsd7c knockout embryos. The brains and livers were collected at E14.5. N=8 per genotype. ***p<Q.001, **P<0.01, *P<0.05; non-parametric t-test (Mann Whitney test); ns. not significant. Data are mean and SD; each dot represents one mouse.

[0022] Figure 2. Expression of MFSD7c increases choline uptake. A) Overexpression of mouse Mfsd7c (mMfsd7c) and human Mfsd7c (hMfsd7c) increases choline uptake in HEK293 cells. CHT1 (SLC5a7), a neuronal choline transporter, was included as a positive control. B) Dose curve for choline uptake from mock, hMfsd7c, and S203Y mutant. C) Time course for choline uptake from mock, hMfsd7c, and S203Y mutant. Experiments were performed at least twice in triplicates. D) Transport activity of mock, medaka Mfsd7c (MeMfsd7c), zebrafish Mfsd7c isoform a (DaMfsd7c_a), hMfsd7c, zebrafish Mfsd7c isoform b (DaMfsd7c_b), and frog Mfsd7c (XeMfsd7c). e) Complementation assays of hMfsd7c with the knockout of yeast S. cerevisiae choline transporter HNM1. Transport activity of wild-type S. cerevisiae strain and HNM1 mutant strain with and without overexpression of hMfsd7c. The results show that expression of hMfsd7c overcomes the loss of choline transport activity in HNM1 mutant yeast cells. Three different overexpression yeast clones were used. F) Choline transport activity by CHT1 and hMfsd7c in the presence of hemicholinium-3 (HC-3), a competitive inhibitor of CHT1. G) Structure of choline, betaine, L-carnitine, and ethanolamine. H-K) Uptake assays of hMfsd7c. S203Y, and / or CHT1 with betaine, L- carnitine and acetylcamitine, and ethanolamine. Each symbol represents one biological replicate. All of these experiments were repeated at least twice in triplicate. Data arc expressed as mean ± SD. ****P<0.0001, ***P<0.001, **P<0.01, ns: not significant. Oneway ANOVA for A, D, E, F, H, and K; Two-way ANOVA for b and c; t-test for I and J.

[0023] Figure 3. MFSD7c exhibits properties of a facilitative transporter for choline. A) Transport activity of hMfsd7c is not sodium-dependent. B) Import of choline by hMfsd7c is strongly increased by co-expression of human choline kinase A (hChKA). C) Time course of choline uptake by hMfsd7c when co-expressed with hChKA. D) Dose curve of choline uptake by hMfsd7c and S203Y mutant when co-expressed with hChKA. Data are expressed as mean ± SD. These experiments were repeated at least twice in triplicate. *”P< 0.0001, **P< 0.01. *P< 0.05. One-way ANOVA: a and b. Two- way ANOVA: c and d. E) Import of choline by hMfsd7c is also significantly increased with co-cxprcssion of choline acyltransfcrasc (CHAT). F) Co-cxprcssion of hMfsd7c with ethanolamine kinase 1 (ETNK1) increases uptake of ethanolamine. G) Coexpression of hMfsd7c with hChKA slightly increased uptake of L-camitine. H) hMfsd7c or mMfsd7c mediates the release of choline from cells. Before release: the levels of choline in the cells; After release: the remaining levels of choline in the cells after 30 mins incubation with plain DMEM. There was a reduction of intracellular choline due to the release into the medium. Data are expressed as mean ± SD. These experiments were repeated at least twice in triplicate. ****P< 0.0001, ***P< 0.001, t-test: c-g; One-way ANOVA: h. I) Uptake of choline by hMfsd7c increases membrane potential in HEK293 cells. Representative exemplar traces of HEK293 cells cotransfected with empty pIRES2-eGFP plasmid and hChKA plasmid (top), hChKA with hMfsd7c (middle), and hChKA with S203Y (bottom). The effect of 200 pM choline on the resting membrane potential changes was induced by active import of choline by hMfsd7c, but not mutant S203Y. J) Quantification of resting membrane potential changes induced with 100 and 200 pM choline. Data are expressed as mean ± SD. ****p< 0.0001; ns, not significant; One-way ANOVA. Figure 4. MFSD7c regulates choline levels in the brain. A) Western blot analysis of the micro-vessels in the brains. Mfsd7c band was reduced in EcMfsd7c-KO compared to controls (Mfsd7cf / f). Mock: 15|_ig proteins from mock transfected HEK239 cells; OV : 1.5 g protein lysates from overexpression of hMfsd7c in HEK293 cells used for interpretation of Mfsd7c band. B) Radioactive signals in the blood at 5 mins postinjection of radioactive choline in EcMfsd7c-K0 (Ec-KO) and control (f / f) mice. C-G) The radioactive signals in the brain (C), livers (D), kidneys (E), lungs (F), and hearts (G) of EcMfsd7c-KO and control mice after 30 mins of choline injection. There was a significant reduction of radioactive signals in the brain of EcMfsd7c-KO mice compared to controls. Each symbol represents one biological replicate. **P< 0.01; t-test. H) Illustration of maternal deuterated choline delivery to the embryos. Choline -d9 is used for the synthesis of LPC-d9 by the maternal liver which is then delivered to the embryos. I) Levels of endogenous choline and choline-d9 in the brains and livers of WT / HET and KO embryos. Endogenous choline and choline-d9 were significantly increased in the brains but not in the liver of KO embryos. Each symbol represents one mouse. **P< 0.01 ; 'P< 0.05, t-test. J) Illustration of deuterated LPC-d49 tracing experiment in adult mice. Plasma LPC-d49 is taken up to the brain by Mfsd2a for PC synthesis. Cholinedi 3 released in the brains after the remodelling of LPC / PC-d49 is imported back to the endothelial cells by Mfsd7c. K) Levels of LPC-d49, LPC-d31 , and LPC-dl 3 the plasma of controls and EcMfsd7c-KO mice at 2hr post-injection of LPC-d49. L) Levels of PC- d49, PC-d31, and PC-dl3 in the brains of controls and EcMfsd7c-KO mice at 2hr postinjection of LPC-d49. M) Levels of endogenous choline and choline-dl3 in the brains of controls and EcMfsd7c-KO mice at 2hr post-injection of LPC-d49. Each symbol represents one mouse. **P< 0.01, ***P< 0.001; t-test and two-way ANOVA (in k and 1).

[0024] Figure 5. Defective choline export in the brain might confer to the phenotypes in the mice and the pathogenesis of Fowler syndrome. A) Illustration of treatment regimen for the mice with choline deficient diet (CDD). B) Maternal choline deficiency affects embryonic development. Arrow shows failed development of eyes in a WT embryo. C) Quantification of body length of WT, HET, and KO E14.5 embryos from normal chow diet (ND) and CDD diet. Each symbol represents one mouse. D) Depletion of choline ameliorated the dilation of the CNS blood vessels in the cortical regions but not in the ganglionic eminences (GE) of E14.5 KO embryos. E) Diameters of CNS blood vessels in the neocortex of KO embryos were decreased after CDD treatment. Note that there was no improvement in the phenotypes of the blood vessels in the ganglionic eminence (GE) regions. Data are expressed as mean ± SD. Each symbol represents the averaged diameters of blood vessels from one image. n=3 embryos per genotype. ****P< 0.0001. One-way ANOVA. F) Western blot analysis of protein expression for indicated missense mutations. G) Immunofluorescent analysis of the localization of the indicated mutant proteins in HEK293 cells. H) Missense mutations of Mfsd7c affects choline import activity. Percentage of choline transport activity of the indicated mutants compared to wild-type (WT) Mfsd7c. Data are expressed as mean ± SD. Each symbol represents one replicate. 0.0001, **P< 0.01; One-way ANOVA. Mean percentage of transport activity for each mutant was shown on top of each bar. I) Pedigree of the patient’s families. J) Sanger sequencing results confirm the mutation P276S from the mother and L483R from the father in a non-consanguinity family. K) MRI images of the patient taken at 4 months of age. Arrowhead shows signs of calcification. Arrows show delayed development of cerebellum and ncocortcx.

[0025] Figure 6. Choline transport properties of Mfsd7c, related to Figure 3. A) Mfsd7c activity is not sodium dependent. In these experiments, Mfsd7c was co-expressed with choline kinase A (hChKA). Transport assays were performed in transport buffer with lithium (Li+) in replacement of sodium (Na+). ****P<0.0001. One-way ANOVA was used. B-C) Dose -curve of choline transport activity by hMfsd7c and S203Y mutant. Experiments were stopped after 30 mins of choline incubation. Experiments were performed in triplicate. D) Competition assays of choline with indicated compounds. A 5-fold excess (250pM) of cold choline and ethanolamine, but not other indicated compounds reduced the import of radioactive choline [3H]-choline. Experiments were repeated twice in triplicate. Data are expressed as mean ± SD. E) Representative images of co-expression of CHKA in RFP plasmid with hMfsd7c in GFP plasmid for single cell patch clamp. Cells with co-cxprcssion of the two fluorescent proteins were selected for patch clamp. Note that Mfsd7c (magenta) is localized in the plasma membrane. Figure 7. Increased levels of choline and acetylcholine in the brain of adult EcMfsd7c-KO mice. A) choline and acetylcholine levels in the brain, liver, and plasma of EcMfsd7c-KO and control mice under normal chow diet. B) choline and acetylcholine levels in the brain, liver, and plasma of EcMfsd7c-KO and control mice after feeding for 1-1.5months with choline deficient diet. Each symbol represents one mouse. Data arc expressed as mean ± SD. **P<0.001, *P<0.05; t-test was used.

[0026] Figure 8. Missense mutations of Mfsd7c affect choline transport activity. A) Transport activity of the missense mutations of Mfsd7c that were reported previously in the literature. Transport activity of these missense mutations was expressed as percentage of wildtype (WT) protein. Each symbol represents one replicate. Experiments were repeated at least twice. B) Fluorescent microscope analysis of the localization of the missense mutations in HEK293 cells. Localization of these missense mutations to the plasma membrane as similar to the wild-type protein was not affected.

[0027] Figure 9. Evaluation of plasticity in hippocampal slices of EcMfsd7c-KO mice. A) Induction of long-term potentiation (late-LTP) by strong tetanization protocol (STET) resulted in stable late LTP in both control and EcMfsd7c- knockout mice (n=6-10 hippocampal slices with 3 mice per genotype). B) Induction of early LTP by weak tetanization protocol (WTET) resulted in stable early-LTP EcMfsd7c- knockout, but not control mice (n=3). Late-LTP and early-LTP are widely studied models of learning and memory at a cellular level. The results show that the memory of EcMfsd7c- knockout mice might be maintained during ageing. A triplet of arrows represents the point of induction of Late-LTP by STET (100Hz, 100 pulses at 0.2 ms, 3 tunes with a 10-minute interval), while a single arrow represents the point of induction of early-LTP by 100 Hz, 21 pulses applied only once. Closed circles represent the potentiated pathway, while open circles represent control inputs from the same neuronal population. In all cases, synaptic plasticity, represented by changes in excitatory field potentials (fEPSP), was recorded for up to 180 minutes.

[0028] Figure 10. A) Experimental procedure for co-injection of deuterated CPC and LPC to control (Mfsd7cf / r) and cKO (EcMfsd7-cKO) mice in=4 per genotype). B) Endogenous choline levels in control and cKO mice. C) The level of labelled choline from LPC (choline-dl3) and GPC (choline-d9) in the brain of control and cKO mice. These results show that with co-injection with the same molar concentration of LPC and GPC in blood, LPC pathway results in a higher level of choline in the brain. D) Hypothesized pathways of LPC and GPC transport at the BBB and their subsequent metabolism.

[0029] Figure 11. Circulation of original forms of the injected LPC and GPC in periphery. A) In the plasma, 300 pM of GPC is mostly diminished after 2h of injection whereas about 1-2% of the injected LPC is still detected. B) GPC is absorbed more by the liver rather than LPC whereas C) in the muscle, LPC is more abundantly detected.

[0030] Figure 12. In periphery, choline-containing metabolites derived from LPC are also more abundant than those from GPC. A) in plasma; B) in liver; C) in muscle.

[0031] Figure 13. Choline-containing phospholipids (PLs) derived from LPC are more abundant than those from GPC in the brain as well as some peripheral tissues. A) In the brain, PLs products from LPC is much higher than those from GPC. B) PLs products from LPC in the plasma is slightly higher than GPC-derived PLs. C) In the liver, LPC- derived PLs is slightly higher than those from the injected GPC. D) Tn the muscle, only metabolized LPCs from the injected LPC is detected but not the ones from GPC.

[0032] Detailed description

[0033] The present invention is predicated on the discovery that Major Facilitator Superfamily Domain Containing 7C (MFSD7c) is a choline transporter that helps to regulate choline levels in the brain. Using Mfsd7c knockout animal models and specially developed cellbased assays, the inventors have found that MFSD7c mediates bidirectional choline transport in vascular cells of the brain and blood-brain bander (BBB), and may play a role in reducing brain parenchymal choline levels through choline export at the BBB. Choline is required for acetylcholine synthesis in neurons. Inhibiting MFSD7c is expected to increase the levels of choline available for acetylcholine synthesis in the brain, which is potentially beneficial for managing CNS disorders associated with reduced cholinergic signaling, such as Alzheimer’s disease. This disclosure is further predicated on the discovery that lysophosphatidylcholine (LPC) delivers choline to the brain via the MFSD2a transporter, and is a preferred source of choline in the brain compared to other choline-containing phospholipids such as glyccrophosphatidylcholinc. MFSD2a activity at the BBB is thus expected to affect choline and acetylcholine levels in the brain. Tn particular, inhibiting MFSD2a is expected to reduce choline and acetylcholine levels in the brain and can potentially benefit patients with disorders arising from abnormally high acetylcholine signaling, such as patients with Parkinson’s disease and epilepsy.

[0034] Without being bound by theory, the inventors used Mfsd7c knockout (KO) mice (MfscT c1") and cell-based assays to reveal that MFSD7c is a conserved choline transporter. Comprehensive metabolomics was performed and it was found that choline- related metabolites were altered in the brains but not in the livers of Mfsd7c'1' embryos. The inventors tested the choline transport function of MFSD7c by expressing human MFSD7c in mammalian cells and showed that it increased choline uptake into the cells. Furthermore, single-cell patch-clamp experiments showed that import of choline by MFSD7c is electrogenic. Missense mutations of Mfsd7c have been described in patients with Fowler syndrome. Employing in vitro cell-based transport assays, the inventors showed that engineered MFSD7c containing missense mutations found in Fowler patients had abolished or reduced choline transport activity. Furthermore, conditional knockout mice lacking Mfsd7c in CNS endothelial cells had reduced import of exogenous choline to the brain. Tracer study suggests that MFSD7c is also required for choline export from the brain. Collectively, the work identifies MFSD7c as a long- sought choline transporter at the blood-brain barrier (BBB). The identification of MFSD7c as a choline transporter suggests that defective transport of choline may be the cause of Fowler syndrome.

[0035] Interestingly, the inventors found that choline and acetylcholine levels were significantly increased in the brains of Mfsd7c KO mice. It was found that MFSD7c in CNS endothelial cells is involved in choline export from brain parenchyma to the bloodstream, which reduces brain choline levels for acetylcholine synthesis. Thus, inhibiting MFSD7c (which is specifically expressed at the BBB) is expected raise the levels of brain choline for acetylcholine synthesis, which can benefit patients with diminished cholinergic signaling in the CNS.

[0036] The inventors have further developed an in vitro assay which can be used to screen for drugs targeting MFSD7c. It was found that, surprisingly, choline uptake through MFSD7c is increased in the presence of cholinc-mctabolising enzymes such as choline kinase A (CHKA). The assay uses mammalian cells such as HEK293 which have been engineered to co-express human choline kinase A (CHKA) and human MFSD7c. The engineered cells can be used together with a labelled choline molecule (e.g., radiolabeled choline) to investigate the effect of potential drug candidates on choline transport through MFSD7c.

[0037] The inventors also discovered that circulating lysophosphatidylcholines (LPC) is a preferred source of choline in the brain and in some peripheral tissues compared to alpha-glycerophosphatidylcholine (GPC), which is widely used as a choline supplement. It was found that choline from LPC accumulates to a higher level than choline from GPC in the brain; choline-containing phospholipids (PLs) derived from LPC are more abundant than those from GPC in the brain as well as in some peripheral tissues; and choline-containing metabolites derived from LPC are also more abundant than those from GPC in certain peripheral tissues. Using Mfsd7c conditional knockout mice, it was shown that reducing MFSD7c activity led to the accumulation of much higher levels of LPC-derived choline metabolites in the brain than GPC-derived choline metabolites. Taken together, the results suggest that LPC is the preferred carrier of choline to the brain, and LPC supplementation can potentially be used alone or in combination with MFSD7c inhibitors to boost brain choline and acetylcholine levels in patients with reduced cholinergic signalling.

[0038] Accordingly, disclosed herein is a method of promoting synthesis and / or accumulation of acetylcholine in a brain parenchymal cell or tissue in a subject, the method comprising administering an effective amount of an inhibitor of MFSD7c to the subject to promote synthesis and / or accumulation of acetylcholine in the brain parenchymal cell or tissue of the subject. Also disclosed herein is a method of inhibiting synthesis and / or accumulation of acetylcholine in a brain parenchymal cell or tissue in a subject, the method comprising administering an effective amount of an inhibitor of MFSD2a to the subject to inhibit synthesis and / or accumulation of acetylcholine in the brain parenchymal cell or tissue of the subject.

[0039] Disclosed herein is a method of treating, preventing or delaying the progression of a CNS disease or condition in a subject, the method comprising administering an effective amount of an inhibitor of MFSD7c to the subject to treat, prevent or delay the progression of the CNS disease or condition in the subject.

[0040] Disclosed herein is a mammalian cell comprising an exogenous nucleic acid encoding choline kinase A (CHKA) and MFSD7c.

[0041] Disclosed herein is a lysophosphatidylcholine (LPC) composition comprising at least one LPC selected from the group consisting of LPC-16:0, LPC-16: 1, LPC-18:0, LPC- 18:1, LPC-18:2, LPC-18:3, LPC-20:4, LPC-20:5, and LPC-22:6.

[0042] The term “inhibit” is used consistently with its use in the art, i.e., meaning to cause or facilitate a qualitative or quantitative decrease in choline transport in the cell. The inhibition may be a decrease in choline transport by at least 5% as compared to a reference level (e.g., the level of choline transport in a cell not treated with the inhibitor). The decrease may be, for example, a decrease of at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%. at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90% or up to and including a 100% decrease or any decrease between 5 to 100% as compared to a reference level.

[0043] As used herein, the terms “ isrupt” and “disruption” refer to any modification that decreases or eliminates expression and / or functional activity of a nucleic acid or an expression product thereof. For example, disruption of a gene includes within its scope any genetic modification that decreases or eliminates expression of the gene and / or functional activity of a corresponding mRNA product (e.g., mRNA and / or protein). Genetic modifications include complete or partial inactivation, suppression, deletion, interruption, blockage, or down-regulation of a nucleic acid (e.g., a gene). Illustrative genetic modifications include, but arc not limited to, gene knockout, gene knockdown, inactivation, mutation (e.g., insertion, deletion, point, or frameshift mutations that disrupt the expression or activity of the gene product), or use of inhibitory nucleic acids (e.g., inhibitory RNAs such as sense or antisense RNAs, molecules that mediate RNA interference such as siRNA, shRNA, miRNA; etc.), or any other molecule that inhibits the activity of the gene or level or functional activity of an expression product of the gene.

[0044] As used herein, “Major Facilitator Superfamily Domain Containing 7C (MFSD7c)” is a protein expressed in vascular cells of the blood-brain barrier (BBB). It is also known as Feline Leukemia Virus subgroup C Cellular Receptor family, member 2 (FLVCR2). In one embodiment, MFSD7c is a polypeptide that is identified by UniProtKB Q9UPI3.

[0045] As used herein, “Major Facilitator Superfamily Domain Containing 2A (MFSD2a)” is a protein expressed in vascular cells of the blood-brain barrier (BBB). In one embodiment, MFSD2a is a polypeptide that is identified by UniProtKB Q8NA29.

[0046] As used herein, the terms “nucleic acid”, “nucleic acid molecule”, “nucleic acid sequence”, “polynucleotide”, or “oligonucleotide” can comprise a polymeric form of nucleotides of any length, can comprise DNA and / or RNA, and can be single-stranded, double-stranded, or multiple stranded. One strand of a nucleic acid also refers to its complement.

[0047] The terms “polypeptide”, “proteinaceous molecule”, “peptide” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues and to variants and synthetic analogues of the same. Thus, these terms apply to amino acid polymers in which one or more amino acid residues is a synthetic non-naturally-occurring amino acid, such as a chemical analogue of a corresponding naturally-occurring amino acid, as well as to naturally-occurring amino acid polymers. These terms do not exclude modifications, for example, glycosylations, acetylations, phosphorylations and the like. Soluble forms of the subject proteinaceous molecules are particularly useful. Included within the definition arc, for example, polypeptides containing one or more analogues of an amino acid including, for example, unnatural amino acids or polypeptides with substituted linkages.

[0048] As used herein “sequence identity” refers to the number (or fraction expressed as a percentage %) of identical or similar amino acids or nucleotide bases in a comparison between a test and a reference polypeptide or polynucleotide. Sequence identity can be determined by sequence alignment of nucleic acid or protein sequences to identify regions of similarity or identity. For purposes herein, sequence identity is generally determined by alignment to identify identical residues. Alignment can be local or global, but for purposes herein alignment is generally a global alignment where the full-length of each sequence is compared. Matches, mismatches and gaps can be identified between compared sequences. Gaps are null amino acids or nucleotides inserted between the residues of aligned sequences so that identical or similar characters are aligned. Generally, there can be internal and terminal gaps. Sequence identity can be determined by taking into account gaps as the number of identical residues / length of the shortest sequencexlOO. When using gap penalties, sequence identity can be determined with no penalty for end gaps (e.g., terminal gaps are not penalised). Alternatively, sequence identity can he determined without taking into account gaps as the number of identical positions / lcngth of the total aligned sequencexlOO.

[0049] Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are known to those skilled in the art, for instance, using publicly available computer software available on internet web sites such as http: / / blast.ncbi.nlm.nih.gov / or http: / / www.ebi.ac.uk / Tools / emboss / ). Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. As used herein “sequence variation” generally refers to differences in residues (such as nucleotide or amino acid residues) between two or more nucleic acid or polypeptide sequences under comparison. Such differences may be expressed as a fraction or a percentage of the number of residues in the compared sequences. Sequence variations herein include nucleotide and amino acid substitutions, insertions, deletions and sequence inversions, and may be naturally-occurring or engineered. Contemplated sequence variations are those which do not substantially affect any biological activity of a nucleic acid or polypeptide. For example, in the case of polypeptides, the sequence variations may be conservative amino acid substitutions and / or are located in segments of the polypeptide which do not contain the following: an active site, an allosteric site, a chelating site, a site for protein modification (e.g., a phosphorylation, acetylation, glycosylation or cleavage site), a site for intramolecular interaction (e.g., a site of a disulphide or other covalent or non-covalent bond), a binding site for a receptor, ligand, antigen, nucleic acid, protein, lipid, ion or metabolite, a site for an intermolecular covalent or non-covalent interaction, or a multimerisation site (including a dimerisation site). A skilled person can identify appropriate segments and sites which minimally affect a biological activity of a polypeptide using, for example, structural or homology data available for the polypeptide.

[0050] As used herein, the phrase “consisting essentially of’ in the context of a recited subunit sequence (e.g., amino acid sequence) indicates that the sequence may comprise at least one additional upstream subunit (e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more upstream subunits; e.g., amino acids) and / or at least one additional downstream subunit (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more upstream subunits; e.g., amino acids), wherein the number of upstream subunits and the number of downstream subunits are independently selectable.

[0051] As used herein, a “phosphatidylcholine” refers to a phospholipid comprising a choline moiety and two fatty acid chains attached to a glycerol backbone; a “lysophosphatidylcholine” refers to a phospholipid comprising a choline moiety and one fatty acid chain attached to a glycerol backbone; “glycerophosphatidylcholine” or “alpha-glycerophosphocholine” refers to a phospholipid comprising a choline moiety and no fatty acid chains attached to a glycerol backbone. Fatty acids in a phosphatidylcholine or lysophosphatidylcholinc may be of any length, and may be saturated or unsaturated (mono- or polyunsaturated).

[0052] As used herein, the term “subject” includes any animal, including mammals. Mammals include, but are not limited to, farm animals (such as, for example, horse, cow, pig), companion animals (such as, for example, dog, cat), laboratory animals (such as, for example, mouse, rat, rabbits), and non-human primates (such as, for example, apes and monkeys). In some embodiments, the subject is a human. In some embodiments, the subject is a patient under the care of a physician.

[0053] The terms “treating”, “treatment” and the like include relieving, reducing, alleviating, ameliorating or otherwise inhibiting the effects of the metabolic disease for at least a period of time. It is also to be understood that terms “treating”, “treatment” and the like do not imply that the disease, or a symptom thereof, is permanently relieved, reduced, alleviated, ameliorated or otherwise inhibited and therefore also encompasses the temporary relief, reduction, alleviation, amelioration or otherwise inhibition of the disease, or of a symptom thereof.

[0054] As used herein a “therapeutically effective amount” or “effective amount” is an amount that is non-toxic to the subject and sufficient to effect desired outcomes in a subject (i.c., achieve therapeutic efficacy). For purposes of this disclosure, a therapeutically effective amount of an inhibitor or composition is an amount that is sufficient to palliate, ameliorate, stabilise, reverse, prevent, slow or delay the progression of a disease state. A therapeutically effective amount can be administered in one or more administrations.

[0055] A “CNS disease or condition” herein is any disease or condition that affects the central nervous system. Exemplary CNS diseases and disorders include but are not limited to Huntingtons's Disease (HD), Parkinson's Disease (PD), Amyotropic Lateral Sclerosis (ALS). Alzheimer's Disease, Lewy body dementia, Multiple System Atrophy, spinal and bulbar muscular atrophy (Kennedy's disease), Tourette Syndrome, Autosomal dominant spinocerebellar ataxia (SCA) (e.g., Type 1 SCA1, Type 2 SCA2, Type 3 (Machado-Joseph disease) SCA3 / MJD, Type 6 SCA6, Type 7 SCA7, Type 8 SCA8. Friedreich's Ataxia and Dentatorubral pallidoluysian atrophy DRPLA / Haw-River syndrome), epilepsy, stroke, schizophrenia, age associated memory impairment, autism, attention-deficit disorder, bipolar disorder, and depression. Particularly contemplated in this disclosure arc CNS diseases and conditions associated with aberrant cholinergic signalling, such as abnormally elevated or diminished cholinergic signalling.

[0056] MFSD7c and MFSD2a inhibitors

[0057] This disclosure provides inhibitors for inhibiting MFSD7c or MFSD2a.

[0058] In some embodiments, the inhibitor of MFSD7c or MFSD2a is an inhibitory nucleic acid molecule, a site-specific nuclease (SSN) system, a polypeptide or a small molecule.

[0059] In some embodiments, the MFSD7c or MFSD2a inhibitor is capable of disrupting a gene, or an RNA product thereof, encoding MFSD7c or MFSD2a. In one embodiment, the MFSD7c or MFSD2a inhibitor is capable of modifying a gene encoding a MFSD7c or MFSD2a to reduce its expression. The MFSD7c or MFSD2a inhibitor may comprise a site-specific nuclease (SSN) targeting a gene encoding MFSD7c or MFSD2a, or an RNA product of the gene.

[0060] In some embodiments, the MFSD7c inhibitor comprises an inhibitory nucleic acid molecule. Examples of inhibitory nucleic acid molecules include, but are not limited to, antisense nucleic acid molecules, small interfering RNAs (siRNAs), and short hairpin RNAs (shRNAs). Such inhibitory nucleic acid molecules can be designed to target any region of a MFSD7c-encoding nucleic acid molecule, such as an mRNA molecule encoding MFSD7c. In some embodiments, the antisense RNA, siRNA, or shRNA hybridizes to a sequence within a genomic nucleic acid molecule or mRNA molecule encoding MFSD7c and decreases expression of the MFSD7c polypeptide in a cell in the subject. In some embodiments, the MFSD7c inhibitor comprises an antisense RNA molecule comprising a nucleic acid sequence that hybridizes to a complementary sequence in a genomic nucleic acid molecule or mRNA molecule encoding MFSD7c, and decreases expression of the MFSD7c polypeptide in a cell in the subject. In some embodiments, the MFSD7c inhibitor comprises an siRNA molecule comprising a nucleic acid sequence that hybridizes to a complementary sequence in a genomic nucleic acid molecule or mRNA molecule encoding MFSD7c, and decreases expression of the MFSD7c polypeptide in a cell in the subject. In some embodiments, the MFSD7c inhibitor comprises an shR A molecule comprising a nucleic acid sequence that hybridizes to a complementary sequence in a genomic nucleic acid molecule or mRNA molecule encoding MFSD7c, and decreases expression of the MFSD7c polypeptide in a cell in the subject.

[0061] In some embodiments, the MFSD2a inhibitor comprises an inhibitory nucleic acid molecule. Examples of inhibitory nucleic acid molecules include, but are not limited to, antisense nucleic acid molecules, small interfering RNAs (siRNAs), and short hairpin RNAs (shRNAs). Such inhibitory nucleic acid molecules can be designed to target any region of a nucleic acid molecule encoding MFSD2a, such as an mRNA molecule encoding MFSD2a. In some embodiments, the antisense RNA, siRNA, or shRNA hybridizes to a sequence within a genomic nucleic acid molecule or mRNA molecule encoding MFSD2a and decreases expression of the MFSD2a polypeptide in a cell in the subject. In some embodiments, the MFSD2a inhibitor comprises an antisense RNA molecule comprising a nucleic acid sequence that hybridizes to a complementary sequence in a genomic nucleic acid molecule or mRNA molecule encoding MFSD2a, and decreases expression of the MFSD2a polypeptide in a cell in the subject. In some embodiments, the MFSD2a inhibitor comprises an siRNA molecule comprising a nucleic acid sequence that hybridizes to a complementary sequence in a genomic nucleic acid molecule or mRNA molecule encoding MFSD2a and decreases expression of the MFSD2a, polypeptide in a cell in the subject. In some embodiments, the inhibitor comprises an shRNA molecule comprising a nucleic acid sequence that hybridizes to a complementary sequence in a genomic nucleic acid molecule or mRNA molecule encoding MFSD2a, and decreases expression of the MFSD2a polypeptide in a cell in the subject.

[0062] Nucleic acid inhibitors can be advantageous for the methods herein in several ways. For example, they can be designed to target specific genes or RNA sequences with a high degree of specificity, thus minimising the risk of undesirable off-target or side effects; they can be highly potent and effective at low doses; and they can cross the blood-brain barrier when provided in a suitable carrier, such as an extracellular vesicle or a lipid nanoparticlc.

[0063] Nucleic acid inhibitors according to the present disclosure may comprise or consist of DNA and / or RNA. Nucleic acid inhibitors may be single-stranded (e.g. in the case of antisense oligonucleotides (e.g. gapmers)). Nucleic acid inhibitors may be doublestranded or may comprise double-stranded region(s) (e.g. in the case of siRNA, shRNA, etc.). Inhibitory nucleic acids may comprise both double-stranded and single stranded regions (e.g. in the case of shRNA and pre-miRNA molecules, which are doublestranded in the stem region of the hairpin structure, and single-stranded in the loop region of the hairpin structure).

[0064] The gene, mRNA or cDNA sequence of Mfsd7c or Mfsd2a may be used to design a nucleic acid inhibitor according to methods known in the art. Such sequences may be accessed on known databases of genomic information, for example, the GenBank and RefSeq databases maintained by the National Center for Biotechnology Information (NCBI), USA. Exemplary amino acid and mRNA sequences of human MFSD7c and MFSD2a are provided as SEQ ID NO: 1-4 herein. Mfsd7c or Mfsd2a mRNA or cDNA sequence can be used to design a nucleic acid inhibitor or a silencing RNAi modulator or antisense molecule which inhibits MFSD7c or MFSD2a for use in the methods disclosed here hi.

[0065] Human MFSD7c amino acid sequence

[0066] MVNEGPNQEESDDTPVPESALQADPSVSVHPSVSVHPSVSINPSVSVHPSSSAH PSALAQPSGLAHPSSSGPEDLSVIKVSRRRWAVVLVFSCYSMCNSFQWIQYGSI NNIFMHFYGVSAFAIDWLSMCYMLTYIPLLLPVAWLLEKFGLRTIALTGSALN CLGAWVKLGSLKPHLFPVTVVGQLICSVAQVFILGMPSRIASVWFGANEVSTA CSVAVFGNQLG1A1GFLVPPVLVPN1EDRDELAYH1S1MFY11GGVATLLL1LV11V FKEKPKYPPSRAQSLSYALTSPDASYLGSTARLFKNLNFVLLVITYGLNAGAFY ALSTLLNRMV1WHYPGEEVNAGR1GLT1V1AGMLGAV1SG1WLDRSKTYKETT LVVYIMTLVGMVVYTFTLNLGHLWVVFITAGTMGFFMTGYLPLGFEFAVELT

[0067] YPESEGISSGLLNISAQVFGIIFTISQGQIIDNYGTKPGNIFLCVFLTLGAALTAFI

[0068] KADLRRQKANKETLENKLQEEEEESNTSKVPTAVSEDHL

[0069] (SEQ ID NO: 1)

[0070] Human MFSD7c mRNA sequence

[0071] ATGGTGAATGAAGGTCCCAACCAGGAAGAGAGCGATGACACCCCTGTGCC

[0072] GGAGTCCGCACTCCAAGCGGACCCCAGCGTCTCGGTCCATCCCAGCGTCTC

[0073] GGTCCATCCCAGCGTCTCCATCAACCCCAGCGTCTCTGTCCACCCCAGCAG

[0074] TTCGGCCCACCCCAGTGCCTTAGCCCAACCCAGTGGCTTGGCTCACCCCAG

[0075] TAGCTCGGGCCCTGAGGACCTCAGCGTGATCAAGGTGAGCAGGCGCCGTT

[0076] GGGCCGTGGTCCTGGTGTTTAGCTGCTACTCCATGTGCAACTCCTTTCAGT

[0077] GGATCCAGTACGGCTCCATCAATAACATCTTCATGCACTTCTACGGTGTCA

[0078] GTGCCTTTGCCATTGACTGGCTGTCCATGTGCTACATGCTGACTTACATCCC

[0079] TCTGCTCCTGCCAGTGGCTTGGCTGCTGGAGAAGTTCGGCCTGCGCACCAT

[0080] TGCTCTCACTGGCTCGGCTCTCAACTGCCTGGGGGCCTGGGTGAAGCTGGG

[0081] CAGCCTGAAGCCGCATCTCTTTCCGGTCACCGTGGTGGGCCAGCTCATCTG

[0082] CTCTGTGGCCCAGGTTTTCATCCTGGGCATGCCCTCCCGCATCGCTTCCGTC

[0083] TGGTTCGGGGCTAATGAGGTTTCAACAGCCTGCTCCGTGGCTGTCTTTGGC

[0084] AATCAGCTTGGAATTGCGATTGGGTTCTTGGTCCCTCCTGTTTTGGTACCCA

[0085] ACATTGAAGACCGGGACGAGCTTGCCTACCACATCAGCATCATGTTCTATA

[0086] TAATAGGAGGTGTGGCCACTCTCCTCCTCATCCTTGTCATCATTGTGTTCAA

[0087] GGAGAAACCTAAATATCCCCCCAGCAGGGCCCAATCCCTGAGCTATGCCTT

[0088] GACCTCTCCTGATGCCTCATACTTAGGTTCCATCGCCCGGCTCTTCAAAAA

[0089] TCTCAACTTTGTGCTGCTTGTCATCACCTATGGTCTGAATGCTGGTGCTTTT

[0090] TATGCCTTGTCCACTCTTCTGAATCGCATGGTGATCTGGCACTACCCGGGG

[0091] GAAGAAGTGAATGCTGGAAGAATTGGCCTGACGATCGTCATTGCAGGAAT

[0092] GCTTGGGGCTGTGATCTCAGGAATCTGGCTGGATAGGTCCAAAACCTACA

[0093] AAGAGACAACCCTGGTAGTCTATATCATGACACTGGTGGGCATGGTGGTG

[0094] TACACGTTTACCTTGAACCTGGGACACCTGTGGGTAGTGTTCATCACTGCT

[0095] GGCACAATGGGCTTCTTTATGACTGGCTATCTCCCACTGGGATTTGAGTTT

[0096] GCTGTGGAGCTCATGTACCCAGAATCAGAAGGCATCTCCTCCGGCCTCCTC AACATATCTGCACAGGTATTTGGGATCATCTTTACCATCTCCCAGGGCCAG

[0097] ATTATTGACAACTATGGAACCAAGCCTGGGAACATCTTCCTGTGTGTGTTC

[0098] CTTACTCTTGGAGCAGCCCTCACTGCATTCATTAAGGCAGATCTCCGGAGA

[0099] CAGAAAGCAAACAAAGAAACTCTTGAGAACAAACTCCAAGAGGAGGAGG

[0100] AGCiACiAGCAACACCAGCAAAGTGCCCACTGCTGTGTCAGAGGATCATCTC

[0101] TGA

[0102] (SEQ ID NO: 2)

[0103] Human MFSD2a amino acid sequence

[0104] MAKGEGAESGSAAGLLPTSILQSTERPAQVKKEPKKKKQQLSVCNKLCYALG

[0105] GAPYQVTGCALGFFLQIYLLDVAQVGPFSASIILFVGRAWDAITDPLVGLCISK

[0106] SPWTCLGRLMPWIIFSTPLAVIAYFLIWFVPDFPHGQTYWYLLFYCLFETMVTC

[0107] FHVPYSALTMFISTEQTERDSATAYRMTVEVLGTVLGTAIQGQIVGQADTPCF

[0108] QDLNSSTVASQSANHTHGTTSHRETQKAYLLAAGVIVCIYIICAVILILGVREQ

[0109] REPYEAQQSEPIAYFRGLRLVMSHGPYIKLITGFLFTSLAFMLVEGNFVLFCTY

[0110] TLGFRNEFQNLLLAIMLSATLTIPIWQWFLTRFGKKTAVYVGISSAVPFLILVAL

[0111] MESNLITTYAVAVAAGTSVAAAFLLPWSMLPDVTDDFHLKQPHFHGTEPTFFSF

[0112] YVFFTKFASGVSLGISTLSLDFAGYQTRGCSQPERVKFTLNMLVTMAPIVLILL

[0113] GLLLFKMYPIDEERRRQNKKALQALRDEASSSGCSETDSTELASIL

[0114] (SEQ ID NO: 3)

[0115] Human MFSD2a mRNA sequence

[0116] ATGGCCAAAGGAGAAGGCGCCGAGAGCGGCTCCGCGGCGGGGCTGCTACC

[0117] CACCAGCATCCTCCAAAGCACTGAACGCCCGGCCCAGGTGAAGAAAGAAC

[0118] CGAAAAAGAAGAAACAACAGTTGTCTGTTTGCAACAAGCTTTGCTATGCA

[0119] CTTGGGGGAGCCCCCTACCAGGTGACGGGCTGTGCCCTGGGTTTCTTCCTT

[0120] CAGATCTACCTATTGGATGTGGCTCAGGTGGGCCCTTTCTCTGCCTCCATC

[0121] ATCCTGTTTGTGGGCCGAGCCTGGGATGCCATCACAGACCCCCTGGTGGGC

[0122] CTCTGCATCAGCAAATCCCCCTGGACCTGCCTGGGTCGCCTTATGCCCTGG

[0123] ATCATCTTCTCCACGCCCCTGGCCGTCATTGCCTACTTCCTCATCTGGTTCG

[0124] TGCCCGACTTCCCACACGGCCAGACCTATTGGTACCTGCTTTTCTATTGCCT CTTTGAAACAATGGTCACGTGTTTCCATGTTCCCTACTCGGCTCTCACCATG TTCATCAGCACCGAGCAGACTGAGCGGGATTCTGCCACCGCCTATCGGATG ACTGTGGAAGTGCTGGGCACAGTGCTGGGCACGGCGATCCAGGGACAAAT CGTGGGCCAAGCAGACACGCCTTGTTTCCAGGACCTCAATAGCTCTACAGT

[0125] AGCTTCACAAAGTGCCAACCATACACATGGCACCACCTCACACAGGGAAA

[0126] CGCAAAAGGCATACCTGCTGGCAGCGGGGGTCATTGTCTGTATCTATATAA TCTGTGCTGTCATCCTGATCCTGGGCGTGCGGGAGCAGAGAGAACCCTATG AAGCCCAGCAGTCTGAGCCAATCGCCTACTTCCGGGGCCTACGGCTGGTCA TGAGCCACGGCCCATACATCAAACTTATTACTGGCTTCCTCTTCACCTCCTT GGCTTTCATGCTGGTGGAGGGGAACTTTGTCTTGTTTTGCACCTACACCTTG GGCTTCCGCAATGAATTCCAGAATCTACTCCTGGCCATCATGCTCTCGGCC ACTTTAACCATTCCCATCTGGCAGTGGTTCTTGACCCGGTTTGGCAAGAAG ACAGCTGTATATGTTGGGATCTCATCAGCAGTGCCATTTCTCATCTTGGTG GCCCTCATGGAGAGTAACCTCATCATTACATATGCGGTAGCTGTGGCAGCT GGCATCAGTGTGGCAGCTGCCTTCTTACTACCCTGGTCCATGCTGCCTGAT GTCATTGACGACTTCCATCTGAAGCAGCCCCACTTCCATGGAACCGAGCCC ATCTTCTTCTCCTTCTATGTCTTCTTCACCAAGTTTGCCTCTGGAGTGTCACT

[0127] GGGCATTTCTACCCTCAGTCTGGACTTTGCAGGGTACCAGACCCGTGGCTG

[0128] CTCGCAGCCGGAACGTGTCAAGTTTACACTGAACATGCTCGTGACCATGGC TCCCATAGTTCTCATCCTGCTGGGCCTGCTGCTCTTCAAAATGTACCCCATT GATGAGGAGAGGCGGCGGCAGAATAAGAAGGCCCTGCAGGCACTGAGGG ACGAGGCCAGCAGCTCTGGCTGCTCAGAAACAGACTCCACAGAGCTGGCT

[0129] AGCATCCTCTAG

[0130] (SEQ ID NO: 4)

[0131] In some embodiments, the nucleic acid inhibitor according to the present disclosure is an antisense nucleic acid as described herein. In some embodiments, the nucleic acid inhibitor comprises an antisense nucleic acid as described herein. In some embodiments, the nucleic acid inhibitor encodes an antisense nucleic acid as described herein.

[0132] As used herein, an “antisense nucleic acid” refers to a nucleic acid molecule (e.g., DNA or RNA) which is complementary to at least a portion of a target nucleotide sequence (e.g., of RNA encoding a target gene described herein). Antisense nucleic acids according to the present disclosure are preferably single-stranded nucleic acids, and bind via complementary Watson-Crick base-pairing to a target nucleotide sequence. Complementary base-pairing may involve hydrogen bonding between complementary base pairs. Antisense nucleic acids may be provided as single-stranded molecules, as for example in the case of antisense oligonucleotides, or may be comprised in doublestranded molecular species, as for example in the case of siRNA, shRNA and pre- miRNA molecules.

[0133] Complementary base-pairing between the antisense nucleic acid and its target nucleotide sequence may be complete. In such embodiments the antisense nucleic acid comprises, or consists of, the reverse complement of its target nucleotide sequence, and complementary base-pairing occurs between each nucleotide of the target nucleotide sequence and complementary nucleotides in the antisense nucleic acid. Alternatively, complementary base-pairing between the antisense nucleic acid and its target nucleotide sequence may be incomplete / partial. In such embodiments complementary base-pairing occurs between some, but not all, nucleotides of the target nucleotide sequence and complementary nucleotides in the antisense nucleic acid.

[0134] Such binding between nucleic acids through complementary base pairing may be referred to as “hybridisation”. Through binding to its target nucleotide sequence, an antisense nucleic acid may form a nucleic acid complex comprising (i) the antisense nucleic acid and (ii) a target nucleic acid comprising the target nucleotide sequence.

[0135] The nucleotide sequence of an antisense nucleic acid is sufficiently complementary to its target nucleotide sequence such that it binds or hybridises to the target nucleotide sequence. It will be appreciated that an antisense nucleic acid preferably has a high degree of sequence identity to the reverse complement of its target nucleotide sequence. In some embodiments, the antisense nucleic acid comprises or consists of a nucleotide sequence having at least 75% sequence identity (e.g., one of at least 75%, 76%, 77%, 78%. 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%. 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater sequence identity) to the reverse complement of its target nucleotide sequence. In some embodiments, the nucleic acid inhibitor is an antisense oligonucleotide (ASO). ASOs are single-stranded nucleic acid molecules comprising or consisting of an antisense nucleic acid to a target nucleotide sequence. An antisense oligonucleotide according to the present disclosure may comprise or consist of an antisense nucleic acid as described herein.

[0136] ASOs can modify expression of RNA molecules comprising their target nucleotide sequence by altering splicing, or by recruiting RNase H to degrade RNA comprising the target nucleotide sequence. RNase H recognises nucleic acid complex molecules formed when the ASO binds to RNA comprising its target nucleotide sequence. ASOs according to the present disclosure may comprise or consist of an antisense nucleic acid according to the present disclosure. ASOs may comprise 10 to 40 (e.g., 17 to 30, 20 to 27, 21 to 23) nucleotides in length. Many ASOs are designed as chimeras, comprising a mix of bases with different chemistries, or as gapmers, comprising a central DNA portion surrounded by “wings” of modified nucleotides. ASOs sometimes comprise alterations to the sugar-phosphate backbone in order to increase their stability and / or reduce / prevent RNAse H degradation, such as phosphorothioate linkages, phosphorodiamidate linkages such as phosphorodiamidate morpholino linkages (PMOs), and may comprise, e.g., peptide nucleic acids (PNAs), locked nucleic acids (LNAs), methoxyethyl nucleotide modifications, e.g. 2’-O-methyl (2’-0Me) and 2’-O- methoxyethyl (MOE) ribose modifications and / or 5 ’-methylcytosine modifications.

[0137] The nucleic acid inhibitor may be an RNA interference (RNAi) agent (e.g., siRNA, shRNA or miRNA-based shRNA or gRNA for CRISR / CAS9 knockout) or a nucleic acid encoding an RNAi agent that reduces expression of a gcnc / mRNA.

[0138] The term “RNAi agent” or “RNAi” as used interchangeably herein, refers to an agent that contains RNA, and which mediates the targeted cleavage of an RNA transcript via an RNA-induced silencing complex (RISC) pathway. The RNAi agent directs the sequence-specific degradation of mRNA through a process known as RNA interference (RNAi). The RNAi agent modulates, e.g., inhibits, the expression of a gene in a cell, e.g., a cell within a subject, such as a mammalian subject. The term “RNAi agent” includes both shRNAs, or precursor RNAs that are processed by RISC into siRNAs, as well as the siRNAs themselves that inhibits the expression of an endogenous gene.

[0139] In some embodiments, each strand of the RNAi agent may range from 12-30 nucleotides in length. For example, each strand may be between 14—30 nucleotides in length, 17—30 nucleotides in length, 25—30 nucleotides in length, 27—30 nucleotides in length, 17—23 nucleotides in length, 17—21 nucleotides in length. 17—19 nucleotides in length, 19-25 nucleotides in length, 19-23 nucleotides in length, 19-21 nucleotides in length, 21-25 nucleotides in length, or 21-23 nucleotides in length.

[0140] In some embodiments, the inhibitory nucleic acid comprises an antisense nucleic acid described herein, e.g., as part of a larger nucleic acid species. For example, in some embodiments, an inhibitory nucleic acid may be an siRNA, dsiRNA, miRNA, shRNA, pri-miRNA, pre-miRNA, saRNA or snoRNA comprising an antisense nucleic acid described herein.

[0141] In some embodiments, the inhibitory nucleic acid is a small interfering RNA (siRNA). As used herein, “siRNA” refers to a double-stranded RNA molecule having a length between 17 to 30 base pairs, which is capable of engaging the RNA interference (RNAi) pathway for the targeted degradation of target RNA. Double-stranded siRNA molecules may be formed as a nucleic acid complex of RNA strands having a high degree of complementarity. The strand of the double-stranded siRNA molecule having complementarity to a target nucleotide sequence may be referred to as the antisense strand or the guide strand, and the other strand may be referred to as the sense strand or passenger strand.

[0142] In some embodiments, the guide or antisense strand of an siRNA according to the present disclosure may comprise or consist of an antisense nucleic acid as described herein.

[0143] The sense strand and antisense strand typically form a duplex double stranded RNA (“dsRNA”). The duplex region of an RNAi agent may be 12-30 nucleotide pairs in length. For example, the duplex region can be between 14-30 nucleotide pairs in length, 17-30 nucleotide pairs in length, 27-30 nucleotide pairs in length. 17-23 nucleotide pairs in length, 17-21 nucleotide pairs in length, 17-19 nucleotide pairs in length, 19-25 nucleotide pairs in length, 19-23 nucleotide pairs in length, 19-21 nucleotide pairs in length, 21 -25 nucleotide pairs in length, or 21 -23 nucleotide pairs in length. In another example, the duplex region is selected from 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, and 27 nucleotides in length.

[0144] The RNAi agent may contain one or more overhang regions and / or capping groups at the 3’-cnd, 5’-cnd, or both ends of one or both strands e.g., comprising one or two or three nucleotides (e.g. a UU 3’ overhang, a TT 3’ overhang, or a CCA 5’ overhang). The overhang can be 1—6 nucleotides in length, for instance 2—6 nucleotides in length, 1—5 nucleotides in length, 2—5 nucleotides in length, 1—4 nucleotides in length, 2—4 nucleotides in length, 1—3 nucleotides in length, 2—3 nucleotides in length, or 1—2 nucleotides in length. The overhangs can be the result of one strand being longer than the other, or the result of two strands of the same length being staggered. The overhang can form a mismatch with the target mRNA or it can be complementary to the gene sequences being targeted or can be another sequence. The first and second strands can also be joined, e.g., by additional bases to form a hairpin, or by other non-base linkers.

[0145] In some embodiments, a passenger strand of an siRNA according to the present disclosure may comprise a ‘CCA’ modification at the 5’ end, i.e., the addition of nucleotides ‘CCA’. In some embodiments, a passenger strand of an siRNA according to the present disclosure may comprise a ‘TT’ modification at the 3’ end, e.g., replacing the 3’ two nucleotides.

[0146] In some embodiments an siRNA according to the present disclosure may be contained within a longer shRNA sequence that undergoes processing to form the siRNA.

[0147] In some embodiments, the inhibitory nucleic acid is a dicer small interfering RNA (dsiRNA). As used herein, “dsiRNA” refers to a double-stranded RNA molecule having a length of about 27 base pairs, which is processed by Dicer to siRNA for RNAi- mediated degradation of target RNA. DsiRNAs are described e.g. in Raja et al., Asian J Pharm Sci. (2019) 14(5): 497-510, which is hereby incorporated by reference in their entirety.

[0148] DsiRN As arc optimised for Dicer processing and may have increased potency compared with 21 -mer siRNAs (see e.g. Kim et al., Nat Biotechnol. (2005) 23(2):222-226), which may be related to the link between Dicer-mediated nuclease activity and RISC loading.

[0149] In some embodiments, the inhibitory nucleic acid is a micro RNA (miRNA), or a precursor thereof (e.g., a primiRNA or a pre-miRNA). miRNA molecules have a similar structure to siRNA molecules, but arc encoded endogenously, and derived from processing of short hairpin RNA molecules. They are initially expressed as long primary transcripts (pri-miRNAs), which are processed within the nucleus into 60 to 70 nucleotide hairpins (pre-miRNAs), which are further processed in the cytoplasm into smaller species that interact with RISC and target mRNA. miRNAs comprise “seed sequences” that are essential for binding to target mRNA. Seed sequences usually comprise six nucleotides and are situated at positions 2 to 7 at the miRNA 5’ end.

[0150] In some embodiments, the inhibitory nucleic acid is a short hairpin RNA (shRNA). shRNA molecules comprise sequences of nucleotides having a high degree of complementarity that associate with one another through complementary base pairing to form the stem region of the hairpin. The sequences of nucleotides having a high degree of complementarity may be linked by one or more nucleotides that form the loop region of the hairpin. shRNA molecules may be processed (e.g., via catalytic cleavage by DICER) to form siRNA or miRNA molecules. shRNA molecules may have a length of between 35—100 (e.g., 40—70) nucleotides. The stem region of the hairpin may have a length between 17—30 (e.g., 20-27) base pairs. The stem region may comprise G:U pairings to stabilise the hairpin structure. An shRNA sequence described herein may comprise sequences that will be subsequently processed into shorter siRNA strand(s). siRNA, dsiRNA, miRNAs and shRNAs for the targeted inhibition of gene and / or protein expression of one or more of MFSD7c or MFSD2a may be identified / designed in accordance with principles and / or using tools well known to the skilled person. Parameters and tools for designing siRNA and shRNA molecules are described e.g. in Fakhr et al., Cancer Gene Therapy (2016) 23:73-82 (hereby incorporated by reference in its entirety). Software that may be used by the skilled person for the design of such molecules is summarised in Table 1 of Fakhr et al., Cancer Gene Therapy (2016) 23:73- 82, and includes c.g. siRNA Wizard (InvivoGcn). Details for making such molecules can be found in the websites of commercial vendors such as Ambion, Dharmacon, GcnScript, Invitrogcn and OligoEnginc.

[0151] In some embodiments, the inhibitor of MFSD7c is a site-specific nuclease (SSN) system. SSN systems will generally comprise a sequence-specific nuclease that recognises a target nucleic acid sequence. The sequence- specific nuclease may be, for example, a wild-type, engineered or chimeric nuclease. The SSN system may target a gene, or RNA product thereof, encoding a MFSD7c protein. The SSN system may further comprise a donor template nucleic acid molecule for introducing specific sequence modifications at or adjacent to the target nucleic acid sequence.

[0152] In some embodiments, the inhibitor of MFSD2a is a site-specific nuclease (SSN) system. SSN systems will generally comprise a sequence-specific nuclease that recognises a target nucleic acid sequence. The sequence- specific nuclease may be, for example, a wild-type, engineered or chimeric nuclease. The SSN system may target a gene, or RNA product thereof, encoding a MFSD2a protein. The SSN system may further comprise a donor template nucleic acid molecule for introducing specific sequence modifications at or adjacent to the target nucleic acid sequence.

[0153] The term “donor template nucleic acid”, as used herein, refers to a nucleic acid molecule that can be used by one or more cellular proteins to modify the sequence of a target nucleic acid after a sequence-specific nuclease described herein has altered the target nucleic acid. In some embodiments, the donor template nucleic acid is a double-stranded nucleic acid molecule. In some embodiments, the donor template nucleic acid is a single-stranded nucleic acid molecule. In some embodiments, the donor template nucleic acid is linear molecule. In some embodiments, the donor template nucleic acid is circular (e.g., a plasmid). In some embodiments, the donor template nucleic acid is an exogenous nucleic acid molecule. In some embodiments, the donor template nucleic acid is an endogenous nucleic acid molecule (e.g., a chromosome). In some embodiments the donor template is a DNA molecule. In some embodiments, the donor template is an RNA molecule.

[0154] In some embodiments the SSN system is capable of disrupting a gene encoding MFSD7c or MFSD2a by modifying the nucleic acid sequence of the gene. Such modifications may include inserting, deleting or substituting one or more nucleic acid residues in the gene. The SSN system may comprise a donor template nucleic acid for introducing the nucleotide modification. In some embodiments, the SSN system is capable of disrupting a gene encoding MFSD7c or MFSD2a by nucleotide insertion or deletion.

[0155] SSNs capable of being engineered to generate target nucleic acid sequence-specific double- or single-strand breaks include zinc-fmger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced palindromic repeats / CRISPR-associated nuclease (CRISPR / Cas) systems.

[0156] In some embodiments the SSN system is a ZFN system, a TALEN system, a CRTSPR / Cas9 system, a CRISPR / Cas12 system or a CRISPR / Cas13 system.

[0157] In some embodiments, the SSN system is a CRISPR / Cas system. The CRISPR / Cas system may include a Cas nuclease and a guide RNA (gR A) with a guide sequence complementary to a target nucleic acid sequence (e.g., a gene sequence encoding MFSD7c or MFSD2a). The CRISPR / Cas system may additionally comprise a donor template nucleic acid for introducing specific sequence modifications at or adjacent to the target nucleic acid sequence. The CRISPR / Cas system may comprise a plurality of gRNAs and / or donor templates.

[0158] In one embodiment the SSN system is a CRISPR / Cas9 system. The CRISPR / Cas9 system may comprise a gRNA, and the gRNA may comprise a CRISPR RNA (crRNA) containing the guide sequence. The gRNA may also comprise a trans-activating crRNA (tracrRNA) for processing the crRNA to its mature form. Alternatively, the gRNA may be an engineered construct comprising both the crRNA and tracRNA in a single nucleic acid molecule (i.e., a single guide RNA or sgRNA). In one embodiment the SSN system is a CRISPR / Casl2 system, such as a CRISPR / Casl2a (also known as CRISPR / Cpfll) or CRISPR / Casl2b (also known as CR1SPR / C2cl) system. In such an embodiment, the CRISPR / Cas system may comprise a single gRNA molecule containing the guide sequence.

[0159] The inventors have found that the following gRNAs can be used with CRISPR / Cas9 systems to knockout MFSD7c in cell lines.

[0160] Flvcr2-oligol-gRNA3-THPl

[0161] 5’-CACCGGTCCACTCTTCTGAATCGCA-3’ (SEQ ID NO: 5)

[0162] Hvcr2-oligo2-gRNA3-THPl

[0163] 5’-AAACTGCGATTCAGAAGAGTGGACC-3’ (SEQ ID NO: 6)

[0164] In some embodiments, the inhibitor of MFSD7c is a nucleic acid inhibitor, wherein the nucleic acid inhibitor comprises, encodes or binds to a nucleic acid sequence having at least 70% sequence identity to a nucleic acid sequence of SEQ ID NO: 5 or SEQ ID NO: 6, or a complementary sequence thereof. The nucleic acid inhibitor may comprise, encode or bind to a nucleic acid sequence having at least 70% sequence identity, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to a nucleic acid sequence of SEQ ID NO: 5 or SEQ ID NO: 6, or a complementary sequence thereof

[0165] In some embodiments, the inhibitor of MFSD7c is a nucleic acid inhibitor, wherein the nucleic acid inhibitor comprises, encodes or binds to a nucleic acid sequence of SEQ ID NO: 5 or SEQ ID NO: 6, or a complementary sequence thereof.

[0166] In one embodiment, the MFSD7c or MFSD2a inhibitor is a small molecule. Small molecule inhibitors that bind to target mRNA / proteins described herein, or their binding partners, can be identified by screening of small molecule libraries. As used herein, a “small molecule” refers to an organic compound with low molecular weight, typically less than 1000 daltons, such as between 200 and 800 daltons.

[0167] In one embodiment, the MFSD7c or MFSD2a inhibitor is a peptide or polypeptide.

[0168] Suitable peptides and polypeptides include but arc not limited to peptide aptamers, monobodies, antibodies, anticalins, Kunitz domains, avimers, knottins, fynomers, atrimers, DARPins, affibodies, nanobodies (i.e., single-domain antibodies (sdAbs)), affilins, armadillo repeat proteins (ArmRPs), OBodies and fibronectin - reviewed e.g., in Rcvcrdatto ct al., Curr Top Med Chcm. 2015; 15(12): 1082-1101, which is hereby incorporated by reference in its entirety (see also e.g., Boersma et al., J Biol Chem (2011) 286:41273-85 and Emanuel et al., Mabs (2011) 3:38-48). Inhibitors include peptides / polypeptides that can be identified by screening of libraries of the relevant peptide s / polypeptides. The peptide / polypeptide inhibitors may be referred to as inhibitory peptides / polypeptides.

[0169] Pharmaceutical compositions

[0170] This disclosure also provides pharmaceutical compositions comprising the MFSD7c or MFSD2a inhibitor as described herein and a pharmaceutically acceptable carrier.

[0171] Representative pharmaceutically acceptable earners include any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, stabilisers, gels, binders, excipients, disintegration agents, lubricants, sweetening agents, flavouring agents, dyes, such like materials and combinations thereof, as would be known to a skilled person (see, e.g., Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, pp. 1289-1329, incorporated herein by reference). Except insofar as any conventional carrier is incompatible with the an inhibitor of this disclosure, its use in the pharmaceutical compositions is contemplated.

[0172] The pharmaceutical compositions may be in a variety of forms. These include, for example, liquid, semi-solid and solid dosage forms, such as liquid solutions (e.g., injectable and infusible solutions), suspensions or emulsions, gels, powders and depositories. The preferred form depends on the intended mode of administration and therapeutic application. Suitable pharmaceutical compositions can be administered intravascularly, intravenously, subcutaneously, intramuscularly, intraosseously, intracerebroventricularly, via any mucosal surface (e.g., orally, sublingually, buccally, intranasally, rcctally or via the pulmonary route), or by surgical intervention. A preferred mode of administration is parenteral (e.g., intravenous, intramuscular, intranasal, intracerebroventricular). In some embodiments, the compositions are in the form of injectable or infusible solutions. In some embodiments, the compositions are in the form of intranasal preparations, such as nasal drops, nasal sprays or nasal rinses. In some embodiments, the pharmaceutical composition is administered by intravenous infusion or injection. In other embodiments, the pharmaceutical composition is administered by intranasal inhalation, spray or irrigation.

[0173] Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic / aqueous solutions, emulsions or suspensions, including saline and buffered media. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers, such as those based on Ringer’s dextrose, and the like. Preservatives and other additives can also be present such as for example, antimicrobials, antioxidants, chelating agents, and inert gases and the like.

[0174] In embodiments where the inhibitor is an inhibitory nucleic acid molecule or a sitc- specific nuclease (SSN) system, the carrier may include compounds capable of complexing, encapsulating or otherwise interacting with the inhibitory nucleic acid, one or more components of the SSN system, or with a vector encoding components of the SSN system, to form a stable composite for administration without affecting the activity of the nucleic acid inhibitor or SSN system. Non-limiting examples of such carriers include viral vectors, polymer-based carriers, protein-based carriers, lipid-based carriers and extracellular vesicles. In some embodiments, the pharmaceutical composition comprises a carrier selected from a lipid nanoparticle (LNP), a polymeric nanoparticle, an extracellular vesicle and a viral vector. These carriers are capable of penetrating the blood-brain barrier and being taken up by vascular cells of the CNS.

[0175] In some embodiments, the pharmaceutical composition is a lipid nanoparticlc (LNP) composition. The LNP composition may comprise an inhibitory nucleic acid molecule, one or more nucleic acid or polypeptide components of an SSN system, or a nucleic acid vector encoding one or more nucleic acid or polypeptide components of an SSN system. As used herein, the term “lipid nanoparticlc” refers to a nanoparticlc assembled from lipids, in particular one or more lipids selected from a cationic lipid, an ionisable lipid, a non-cationic lipid, a conjugated lipid and cholesterol, wherein the nucleic acid is fully encapsulated within the lipid. LNPs may contain multiple lipid layers as well as microdomains of lipids and nucleic acids and may thus be distinguished from lipoplexes (in which the nucleic acid is not encapsulated), micelles (which contain a lipid monolayer) and liposomes (which only contain a lipid bilayer).

[0176] The LNPs may contain one or more lipid conjugates. The term “lipid conjugate” refers to a conjugated lipid that inhibits aggregation of lipid particles. Such lipid conjugates include, but are not limited to, polyamide oligomers (e.g., ATTA-lipid conjugates), PEG-lipid conjugates (such as PEG coupled to dialkyloxypropyls, PEG coupled to di acyl glycerols, PEG coupled to cholesterol, PEG coupled to phosphatidylcthanolamincs), PEG conjugated to ceramides, cationic PEG lipids, and mixtures thereof. PEG can be conjugated directly to the lipid or may be linked to the lipid via a linker moiety. Any linker moiety suitable for coupling the PEG to a lipid can be used including, e.g., non-ester containing linker moieties and ester-containing linker moieties. In preferred embodiments, non-ester containing linker moieties are used.

[0177] The LNPs may contain one or more non-cationic lipids, which may be amphipathic or neutral lipids. The term “amphipathic lipid” refers, in part, to any suitable material wherein the hydrophobic portion of the lipid material orients into a hydrophobic phase, while the hydrophilic portion orients toward the aqueous phase. Hydrophilic characteristics derive from the presence of polar or charged groups such as carbohydrates, phosphate, carboxylic, thio, amino, sulphydryl, nitro, hydroxyl, and other like groups. Hydrophobicity can be conferred by the inclusion of apolar groups that include, but are not limited to, long-chain saturated and unsaturated aliphatic hydrocarbon groups and such groups substituted by one or more aromatic, cycloaliphatic, or heterocyclic group(s). Examples of amphipathic lipids include, but are not limited to, phospholipids, aminolipids, and sphingolipids.

[0178] Representative examples of phospholipids include, but are not limited to, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoylolcoyl phosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine, dipalmitoylphosphatidylcholine, dioleoylphosphatidylcholine, distearoylphosphatidylcholine, and dilinoleoylphosphatidylcholine. Other compounds lacking in phosphorus, such as sphingolipid, glycosphingolipid families, diacylglycerols, and P-acyloxyacids, are also within the group designated as amphipathic lipids. Additionally, the amphipathic lipids described above can be mixed with other lipids including triglycerides and sterols.

[0179] The term “neutral lipid” refers to any of a number of lipid species that exist either in an uncharged or neutral zwitterionic form at a selected pH. At physiological pH, such lipids include, for example, diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, sphingomyelin, cephalin, cholesterol, cerebrosides, and diacylglycerols.

[0180] The term “non-cationic lipid” refers to any amphipathic lipid as well as any other neutral lipid or anionic lipid.

[0181] The term “anionic lipid” refers to any lipid that is negatively charged at physiological pH. These lipids include, but are not limited to, phosphatidylglycerols, cardiolipins, diacylphosphatidylserines, diacylphosphatidic acids, N-dodecanoyl phosphatidylethanolamines, N-succinyl phosphatidylethanolamines, N- glutarylphosphatidylethanolamines, lysylphosphatidylglycerols, palmitoylol eyolphosphatidyl glycerol (POPG), and other anionic modifying groups joined to neutral lipids. LNPs may contain one or more cationic lipids. The term “cationic lipid” refers to any of a number of lipid species that carry a net positive charge at a selected pH, such as physiological pH (c.g., pH of about 7.0). It has been surprisingly found that cationic lipids comprising alkyl chains with multiple sites of unsaturation, e.g., at least two or three sites of unsaturation, arc particularly useful for forming lipid particles with increased membrane fluidity. Non-limiting examples of cationic lipids include N,N- dioleyl-N,N-dimethylammonium chloride (DODAC), l,2-dioleyloxy-N,N- dimethylaminopropane (DODMA), l,2-distearyloxy-N,N-dimethylaminopropane (DSDMA), N-(l-(2,3-diolcyloxy)propyl)-N,N,N-trimcthylammonium chloride (DOTMA), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(l-(2,3- dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP), 3-(N-(N’,N’- dimethylaminoethane)-carbamoyl)cholesterol (DC-Chol), N-(l ,2-dimyristyloxyprop-3- yl)-N,N-dimethyl-N-hydroxyethyl ammonium bromide (DMIR1E), 2,3-dioleyloxy-N- [2(spermine-carboxamido)ethyl]-N,N-dimethyl-l-propanaminium trifluoroacetate (DOSPA), dioctadecylamidoglycyl spermine (DOGS), 3-dimethylamino-2-(cholest-5- en-3-beta-oxybutan-4-oxy)-l-(cis,cis-9,12-octadecadienoxy)propane (CLinDMA), 2- [5’-(cholest-5-en-3.beta.-oxy)-3’oxapentoxy)-3-dimethy-1 -(cis,cis-9’, 1 -2’- octadecadienoxy)propane (CpLinDMA), N,N-dimethyl-3,4-dioleyloxybenzylamine (DMOBA), 1,2-N,N’ -diole ylcarbamyl-3-dimethylaminopropane (DOcarbDAP), 1,2- N ,N ’ -Dilinoleylcarbamyl-3-dimethylaminopropane (DLincarbDAP), 1 ,2-

[0182] Dilinoleoylcarbamyl-3-dimethylaminopropane (DLinCDAP), and mixtures thereof. Tn some cases, the cationic lipids comprise a protonatablc tertiary amine (c.g., pH titratable) head group, C18 alkyl chains, ether linkages between the head group and alkyl chains, and 0 to 3 double bonds. Such lipids include, c.g., DSDMA, DLinDMA, DLenDMA, and DODMA.

[0183] The term “hydrophobic lipid” refers to compounds having apolar groups that include, but are not limited to, long-chain saturated and unsaturated aliphatic hydrocarbon groups and such groups optionally substituted by one or more aromatic, cycloaliphatic, or heterocyclic group(s). Suitable examples include, but are not limited to, diacylglycerol, dialkylglycerol, N-N-dialkylamino, 1,2 -diacyloxy- 3 -aminopropane, and 1 ,2-dialkyl-3-aminopropane. In one embodiment, the LNP composition comprises a lysophosphatidylcholine (LPC) or phosphatidylcholine (PC). The LPC may be, for example, one or more LPCs selected from LPC-16:0, LPC-16:1, LPC-18:0, LPC-18:!, LPC-18:2, LPC-18:3, LPC-20:4, LPC-20:5, and LPC-22:6. The PC may be, for example, one or more PCs selected from PC-16:0, PC-16:1, PC-18:0, PC-18:1, PC-18:2, PC-18:3, PC-20:4, PC-20:5, and PC- 22:6.

[0184] In some embodiments, the pharmaceutical composition is a polymeric nanoparticle. The polymeric nanoparticlc composition may comprise an inhibitory nucleic acid molecule, one or more nucleic acid or polypeptide components of an SSN system, or a nucleic acid vector encoding one or more nucleic acid or polypeptide components of an SSN system. The polymeric nanoparticle may comprise a polycationic polymer such as, for example, polyethyleneimine (PEI), poly (P-amino esters), poly(amidoamine) (PAMAM), cationic polyacrylates and cationic poly(amino acids).

[0185] In some embodiments, the pharmaceutical composition is an extracellular vesicle composition. The extracellular vesicle composition may comprise an inhibitory nucleic acid molecule, one or more nucleic acid or polypeptide components of an SSN system, or a nucleic acid vector encoding one or more nucleic acid or polypeptide components of an SSN system. As used herein, an “extracellular vesicle” refers to a lipid bilayer- delimited nano- or micro-sized non -replicative particle secreted or shed from a eukaryotic cell. Extracellular vesicles may range in size from about 10 nm to about 10 pm. Non-limiting examples of extracellular vesicles include ectosomes (released from the plasma membrane), cxosomcs (derived from endosomes), apoptotic bodies (release by cells undergoing apoptosis), oncosomes (released by cancer cells) and migrasomes (released by migrating cells).

[0186] In some embodiments, the extracellular vesicle is an exosome. The term “exosome” refers to an extracellular vesicle of endocytic origin with a diameter of about 30 nm to about 200 nm. Exosomes may be suitably identified by detecting known protein markers such as CD9, CD63, CD81, HSP70. HSP90, Alix, TsglOl, Annexin (such as Annexin I, Annexin II or Annexin V), Flotillin (such as Flotillin- 1 or Flotillin-2), and others. The exosomes or extracellular vesicles may be derived from any cell. In some embodiments, the extracellular vesicles are derived from a cell from the nervous system, such as from neural cells (e.g., glial cells, astrocytes, neurons, oligodendrocytes, spindle neurons, microglia, neural stem cells, etc.) or vascular cells (e.g., endothelial cells, mural cells, pericytes, capillary-associated microglia, etc.). The cells can be in the form of a cell culture, a tissue sample, or parts thereof. The cells may be autologous with respect to the subject to be treated, although allogeneic or even xenogeneic cells may be used.

[0187] Exosomes and extracellular vesicles produced or released by cells may be isolated and / or purified using techniques well-known in the art. These include but are not limited to filtration (such as ultrafiltration), centrifugation (such as density gradient centrifugation), and chromatography (such as ion-exchange or affinity chromatography), either alone or in combination.

[0188] The exosomes or extracellular vesicles may be loaded with the nucleic acid inhibitor using methods known in art, such electroporation, lipofection and heat shock.

[0189] In some embodiments, the pharmaceutical composition is a viral vector composition. The viral vector composition may comprise an inhibitory nucleic acid molecule or a nucleic acid vector encoding one or more nucleic acid or polypeptide components of an SSN system. The viral vector is preferably an recombinant virus. Both replication- competent and replication-defective viruses may be used. Exemplary viruses include, but arc not limited to retroviruses such as Moloney murine leukemia virus (MMLV) and lentiviruses; adenoviruses and adeno-associated viruses; and alphaviruses (e.g., Semliki Forest virus (SFV)). In some embodiments, the viral vector is an adenovirus, an adeno- associated virus (AAV) or a lentivirus.

[0190] Engineered cells, kits

[0191] This disclosure provides engineered cells, kits and methods to screen for compounds (e.g., choline or choline-containing compounds) that are transported by MFSD7c or MFSD2a, and to screen for drugs that modulate the transport activity of MFSD7c or MFSD2a.

[0192] Ceils may be engineered to express exogenous MFSD7c or MFSD2a through the introduction of a suitable vector or expression construct. Surprisingly, the inventors found that co-cxprcssion of a cholinc-mctabolising enzyme (c.g., choline kinase A) can increase MFSD-mediated transport. Engineered cells herein are preferably of mammalian origin.

[0193] Disclosed herein is a mammalian cell comprising an exogenous nucleic acid encoding choline kinase A (CHKA) and MFSD7c. Also disclosed herein is a mammalian cell comprising an exogenous nucleic acid encoding MFSD2a.

[0194] The exogenous nucleic acid may be a vector or expression cassette.

[0195] By “vector” or “expression vector” is meant a nucleic acid molecule, preferably a DNA molecule derived, for example, from a plasmid, bacteriophage or virus, into which a nucleic acid sequence may be inserted or cloned. A vector preferably contains one or more unique restriction sites and may be capable of autonomous replication in a defined host cell including a target cell or a progenitor cell thereof, or be integrable with the genome of the defined host such that the cloned sequence is reproducible. Accordingly, the vector may be an autonomously replicating vector, i.e., a vector that exists as an cxtrachromosomal entity, the replication of which is independent of chromosomal replication, e.g., a linear or closed circular plasmid, an cxtrachromosomal element, a minichromosomc, or an artificial chromosome. The vector may contain any means for assuring self-replication. Alternatively, the vector may be one which, when introduced into the host cell, is integrated into the genome and replicated together with the chromosome(s) into which it has been integrated. A vector system may comprise a single vector or plasmid, two or more vectors or plasmids, which together contain the total DNA to be introduced into the genome of the host cell, or a transposon. The choice of the vector will typically depend on the compatibility of the vector with the host cell into which the vector is to be introduced. The vector may also include a selection marker such as an antibiotic resistance gene that can be used for selection of suitable transformants. Examples of such resistance genes are well known to those of skill in the art.

[0196] The term “expression cassette’’ or “expression construct” generally includes at least a control sequence operahly linked to a nucleotide sequence of interest. In this manner, for example, promoters in operable connection with the nucleotide sequences to be expressed are provided in expression constructs for expression in a host cell. Conventional compositions and methods for preparing and using constructs and host cells are well known to one skilled in the art, see for example, Molecular- Cloning: A Laboratory Manual, 3rdedition Volumes 1, 2, and 3. J. F. Sambrook, D. W. Russell, and N. Irwin, Cold Spring Harbor Laboratory Press, 2000.

[0197] By “control element” or “control sequence” is meant nucleic acid sequences (e.g., DNA) necessary for expression of an operably linked coding sequence in a particular host cell. The control sequences that are suitable for eukaryotic cells include transcriptional control sequences such as promoters, polyadenylation signals, transcriptional enhancers, translational control sequences such as translational enhancers and internal ribosome binding sites (IRES), nucleic acid sequences that modulate mRNA stability, as well as targeting sequences that target a product encoded by a transcribed polynucleotide to an intracellular compartment within a cell or to the extracellular environment. Such elements may comprise mutant, truncated, and hybrid sequences, and may be obtained from genes encoding polypeptides either homologous or hctcrologous to the host cell.

[0198] The term “operably connected” or “operably linked” as used herein refers to a juxtaposition wherein the components so described are in a relationship permitting them to function in their intended manner. For example, a regulatory sequence (e.g., a promoter) “operably linked” to a nucleotide sequence of interest (e.g., a coding and / or non-coding sequence) refers to positioning and / or orientation of the control sequence relative to the nucleotide sequence of interest to permit expression of that sequence under conditions compatible with the control sequence. The control sequences need not be contiguous with the nucleotide sequence of interest, so long as they function to direct its expression. Thus, for example, intervening non-coding sequences (e.g., untranslated, yet transcribed, sequences) can be present between a promoter and a coding sequence, and the promoter sequence can still be considered “operably linked” to the coding sequence.

[0199] The codon usage in an expression construct or cassette of this disclosure may be adapted according to the host cell in which the polypeptide will be expressed. These steps may be carried out according to methods well known to one skilled in the art.

[0200] The nucleic acid may be introduced into the host cell by any method known by the skilled person, such as electroporation, viral transduction, competent cell transformation, protoplast transformation, protoplast fusion, “gene gun” transformation, PEG-mediated transformation, lipid-assisted transformation or transfection, chemically mediated transfection, lithium acetate-mediated transformation, liposome-mediated transformation, etc. More than one copy of a nucleic acid may be inserted into a host cell to increase production of a polypeptide disclosed herein.

[0201] A number of mammalian cell lines are known in the art and include immortalised cell lines available from the American Type Culture Collection (ATCC), such as, but not limited to. Chinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), Madin-Darby bovine kidney (“MDBK”) cells, Human Embryonic Kidney (HEK293) cells, as well as others.

[0202] Methods known in the art may be used to trace the movement of choline or choline- containing or choline precursor compounds such as LPC in cells or tissues. Suitable methods include but are not limited to high-performance liquid chromatography (HPLC), liquid chromatography-mass spectrometry (LC-MS), tandem mass spectrometry (such as MS / MS or LC-MS / MS), enzymatic assays and radiochemical assays. Enzymatic assays may rely on the use of specific enzymes to catalyse the conversion of choline or LPC into a detectable product. For example, to detect choline, the enzyme choline oxidase can be used to produce hydrogen peroxide, which can be detected colorimetrically or fluorimetrically. In another example, the action of phospholipase A2 and choline oxidase may be coupled to produce a chromogenic or fluorescent signal from LPC. Radiochemical assays using isotopically labelled choline and LPC molecules are particularly useful for tracing the transport, distribution and metabolic fate of choline and LPC in cells and tissues. For example, |51 l / dcutcrium |- or L14CJ-labcllcd choline or LPC may be used in combination with scintillation counting or autoradiography to detect choline / LPC and their metabolic products in cells or tissues.

[0203] Disclosed herein is a kit comprising a mammalian cell as defined herein. The mammalian cell may comprise a nucleic acid molecule encoding exogenous MFSD2a and / or MFSD7c, and optionally further encoding choline kinase A (CHKA). The kit may further comprise an isotopically-labcllcd choline or an analogue thereof. Alternatively or additionally, the kit may comprise an isotopically-labelled LPC or an analogue thereof.

[0204] As used herein, a choline or LPC analogue is a compound which is capable of interacting with and being transported by MFSD7c and / or MFSD2a and which may thus be used as a replacement for choline or LPC in the cell-based methods and assays herein.

[0205] Disclosed herein is the use of a mammalian cell as defined herein or a kit as defined herein for screening for a compound capable of being transported by MFSD7c or MFSD2a.

[0206] Disclosed herein is the use of a mammalian cell as defined herein or a kit as defined herein for screening for an inhibitor of MFSD7c or MFSD2a.

[0207] Cells engineered to express MFSD7c or MFSD2a may be incubated with labelled choline or LPC, optionally in the presence of a candidate drug or inhibitor, for a suitable length of time (such as 30 min to 2 hr) to allow MFSD2a- or MFSD7c -mediated transport. Cell pellets may then be harvested to quantify the uptake of choline or LPC. The cell-based assay may be performed in a high-throughput manner, such as multi-well plates.

[0208] Methods of treatment The disclosure provides methods of regulating choline and / or acetylcholine levels in cells or tissues by modulating transport through MFSD7c or MFSD2a. In some embodiments, the methods comprise inhibiting MFSD7c to inhibit MFSD7c -mediated choline transport into and / or out of a cell. In some embodiments, the methods comprise inhibiting MFSD7c to promote the synthesis and / or accumulation of choline and / or acetylcholine in a cell or tissue. In alternative embodiments, the methods comprise inhibiting MFSD2a to inhibit MFSD2a-mediated LPC transport into and / or out of a cell. In some embodiments, the methods comprise inhibiting MFSD2a to inhibit the synthesis and / or accumulation of choline and / or acetylcholine in a cell or tissue.

[0209] In some embodiments, methods herein comprise inhibiting MFSD7c or MFSD2a in a vascular cell of the central nervous system (CNS), such as a vascular cell of the bloodbrain barrier (BBB), to regulate choline and / or acetylcholine levels in tissues of the CNS, e.g., in brain parenchyma. MFSD7c and MFSD2a are highly expressed in vascular cells of the CNS. Advantageously, inhibiting MFSD7c- or MFSD2a-mediated transport can be used to treat CNS diseases associated with dysfunctional cholinergic signalling, such as abnormally high or abnormally low levels of acetylcholine signalling.

[0210] Inhibitors as defined in this disclosure may be used to inhibit MFSD2a or MFSD7c in the methods herein. In some embodiments, the inhibitor is an inhibitory nucleic acid molecule, a site-specific nuclease (SSN) system, a polypeptide or a small molecule. In some embodiments, the MFSD2a or MFSD7c inhibitor is an inhibitory nucleic acid molecule that hybridises to a MFSD2a or MFSD7c mRNA molecule, and the inhibitory nucleic acid molecule is selected from an antisense nucleic acid molecule, a small interfering RNA (siRNA), and a short hairpin RNA (shRNA). In some embodiments, the MFSD2a or MFSD7c inhibitor is a site-specific nuclease (SSN) system. In some embodiments, the MFSD2a or MFSD7c inhibitor is a CRISPR / Cas9 system. In some embodiments, the inhibitor of MFSD7c is a nucleic acid inhibitor, wherein the nucleic acid inhibitor comprises, encodes or binds to a nucleic acid sequence having at least 70% sequence identity to a nucleic acid sequence of SEQ ID NO: 5 or SEQ ID NO: 6, or a complementary sequence thereof. In some embodiments, the inhibitor of MFSD7c is a nucleic acid inhibitor, wherein the nucleic acid inhibitor comprises, encodes or binds to a nucleic acid sequence of SEQ ID NO: 5 or SEQ ID NO: 6, or a complementary sequence thereof.

[0211] Disclosed herein is a method of promoting synthesis and / or accumulation of acetylcholine in a brain parenchymal cell or tissue in a subject, the method comprising administering an effective amount of an inhibitor of MFSD7c to the subject to promote synthesis and / or accumulation of acetylcholine in the brain parenchymal cell or tissue of the subject.

[0212] Disclosed herein is a method of inhibiting choline transport in a vascular cell in the central nervous system (CNS), the method comprising contacting the vascular cell with an effective amount of an inhibitor of MFSD7c for a sufficient time and under conditions to inhibit choline transport in the vascular cell.

[0213] In some embodiments, the vascular cell in the methods herein is an endothelial cell or a mural cell (e.g., a pericyte) of the CNS. The vascular cell may be present in a subject suffering from a CNS disease or condition associated with diminished cholinergic signalling. In one embodiment, the MFSD7c inhibitor inhibits parenchymal choline import into the vascular cell and / or choline export into the blood stream from the vascular cell.

[0214] Disclosed herein is a method of treating a CNS disease or condition in a subject, the method comprising administering an effective amount of an inhibitor of MFSD7c to the subject to treat the CNS disease or condition in the subject. In one embodiment, the CNS disease or condition is associated with diminished cholinergic signaling. The diminished cholinergic signaling may be diminished as compared to a reference (e.g., cholinergic signaling in a healthy subject).

[0215] Disclosed herein is a method of preventing or delaying the progression of a CNS disease or condition in a subject, the method comprising administering an effective amount of an inhibitor of MFSD7c to the subject to prevent or delay progression of the CNS disease or condition in the subject. In one embodiment, the CNS disease or condition is associated with diminished cholinergic signaling. Disclosed herein is an inhibitor of MFSD7c, for use in treating or preventing a CNS disease or condition in a subject, wherein the MFSD7c inhibitor is to be administered to the subject, fn some embodiments, the CNS disease or condition is associated with diminished cholinergic signalling in a subject. The MFSD7c inhibitor may be an inhibitor as defined in this disclosure.

[0216] Disclosed herein is the use of an inhibitor of MFSD7c in the manufacture of a medicament for treating or preventing a CNS disease or condition in a subject, wherein the MFSD7c inhibitor is to be administered to the subject. In some embodiments, the CNS disease or condition is associated with diminished cholinergic signalling in a subject. The MFSD7c inhibitor may be an inhibitor as defined in this disclosure.

[0217] In some embodiments, the CNS disease or condition is a neurological, cerebrovascular, or psychiatric disease or condition. Non-limiting examples of CNS diseases and conditions that may be treated with MFSD7c inhibitors include neurodegenerative diseases and neurocognitive disorders such as dementia and Alzheimer’s disease, cerebrovascular diseases such as stroke, and psychiatric or psychological conditions such as depression and schizophrenia. In some embodiments, the CNS disease or condition is Alzheimer’s disease, stroke, depression or schizophrenia.

[0218] Disclosed herein is a method of inhibiting synthesis and / or accumulation of acetylcholine in a brain parenchymal cell or tissue in a subject, the method comprising administering an effective amount of an inhibitor of MFSD2a to the subject to inhibit synthesis and / or accumulation of acetylcholine in the brain parenchymal cell or tissue of the subject.

[0219] Disclosed herein is a method of inhibiting lysophosphatidylcholine (LPC) transport in a vascular cell in the central nervous system (CNS), the method comprising contacting the vascular cell with an effective amount of an inhibitor of MFSD2a for a sufficient time and under conditions to inhibit LPC transport in the vascular cell. In some embodiments, the vascular cell in the methods herein is an endothelial cell or a mural cell (e.g., a pericyte) of the CNS. The vascular cell may be present in a subject suffering from a CNS disease or condition associated with elevated or excessive cholinergic signalling. In one embodiment, the MFSD2a inhibitor inhibits LPC import from the blood stream into the vascular cell and / or parenchymal LPC export from the vascular cell.

[0220] Disclosed herein is a method of treating a CNS disease or condition in a subject, the method comprising administering an effective amount of an inhibitor of MFSD2a to the subject to treat the CNS disease or condition in the subject. In one embodiment, the CNS disease or condition is associated with elevated or excessive cholinergic signaling. The elevated or excessive cholinergic signaling may be elevated or may be excessive as compared to a reference (e.g., cholinergic signaling in a healthy subject).

[0221] Disclosed herein is a method of preventing or delaying the progression of a CNS disease or condition in a subject, the method comprising administering an effective amount of an inhibitor of MFSD2a to the subject to prevent or delay progression of the CNS disease or condition in the subject. In one embodiment, the CNS disease or condition is associated with elevated or excessive cholinergic signaling.

[0222] Disclosed herein is an inhibitor of MFSD2a, for use in treating or preventing a CNS disease or condition in a subject, wherein the MFSD2a inhibitor is to be administered to the subject. In some embodiments, the CNS disease or condition is associated with elevated or excessive cholinergic signalling. The MFSD2a inhibitor may be an inhibitor as defined in this disclosure.

[0223] Disclosed herein is the use of an inhibitor of MFSD2a in the manufacture of a medicament for treating or preventing a CNS disease or condition in a subject, wherein the MFSD2a inhibitor is to be administered to the subject. In some embodiments, the CNS disease or condition is associated with elevated or excessive cholinergic signalling. The MFSD2a inhibitor may be an inhibitor as defined in this disclosure. In some embodiments, the CNS disease or condition is a neurological, cerebrovascular, or psychiatric disease or condition. Non-limiting examples of CNS diseases and conditions that may be treated with MFSD2a inhibitors include Parkinson’s disease, epilepsy and vascular Fowler syndrome. In some embodiments, the CNS disease or condition is Parkinson’s disease, epilepsy or vascular Fowler syndrome.

[0224] Inhibitor therapies herein may be accompanied by suitable adjunct therapies to increase the uptake of the inhibitor in vivo. By way of non-limiting example, hyperosmotic solutions, focused ultrasound and / or electromagnetic fields can be used to increase the entry of the inhibitor into vascular cells of the CNS.

[0225] Dosage regimens may be adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. An inhibitor or composition of the present disclosure can be administered on multiple occasions. Intervals between single dosages can be daily, weekly, monthly or yearly. Intervals can also be irregular as indicated by clinical assessment in the patient. Alternatively, the inhibitor or composition can be administered as a sustained release formulation, in which case less frequent administration is required. Dosage and frequency vary depending on the half-life of the inhibitor or active agent in the patient.

[0226] It is to be noted that dosage values may vary with the type and severity of the disease or condition to be alleviated. It is to be further understood that for any particular subject, specific dosage regimens may be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the inhibitor or composition, and that dosage ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed composition.

[0227] LPC compositions, combination therapy This disclosure further provides lysophosphatidylcholine (LPC) compositions which can be used to deliver choline to the brain of a subject through MFSD2a-mediated transport. The compositions may also be used to deliver fatty acids to the brain, in particular arachidonic acid and docosahcxacnoic acid (DHA). LPC compositions herein contain higher concentrations of LPCs than are typically found in human plasma and arc expected to supplement endogenous sources of choline in a subject.

[0228] Disclosed herein is a lysophosphotidylcholine (LPC) composition comprising at least one LPC selected from the group consisting of LPC-16:0, LPC-16: 1, LPC-18:0, LPC- 18:1, LPC-18:2, LPC-18:3, LPC-20:4, LPC-20:5, and LPC-22:6. The LPC composition may comprise at least one, at least two, at least three, at least four, at least five, at least six, at least seven, or at least eight LPCs selected from the group consisting of LPC- 16:0, LPC-16:!, LPC-18:0, LPC-18:!, LPC-18:2, LPC-18:3, LPC-20:4, LPC-20:5, and LPC-22:6. The LPC composition may comprise one, two, three, four, five, six, seven, eight or all nine LPCs selected from the group consisting of LPC-16:0, LPC-16:1, LPC- 18:0, LPC-18:1, LPC-18:2, LPC-18:3, LPC-20:4, LPC-20:5, and LPC-22:6.

[0229] Fatty acids in LPC compositions herein are identified by an X:Y notation, wherein X is the number of carbon atoms in the fatty acid chain, and Y is the number of double bonds in the fatty acid chain. For example, the saturated 16-carbon fatty acid palmitic acid is represented by 16:0, the polyunsaturated 18-carbon fatty acid linoleic acid is represented by 18:2, the polyunsaturated fatty acid arachidonic acid is represented by 20:4, and the polyunsaturated fatty acid DHA is represented by 22:6.

[0230] In some embodiments, the LPC composition comprises LPC-20:4 and LPC-22:6. Advantageously, LPC compositions containing LPC-20:4 and LPC-22:6 are able to supply arachidonic acid and DHA to supplement endogenous levels of the fatty acids. This can be beneficial for managing neurodegenerative diseases or neurocognitive disorders such as Alzheimer’s disease and dementia.

[0231] In some embodiments, each LPC in the LPC composition is present at a concentration of at least 50 pM. In some embodiments, each LPC is present at a concentration of about 50 jiM to about 500 pM. such as about 50 p.M. about 60 pM, about 70 pM, about 80 pM, about 90 M. about 100 pM, about 110 pM, about 120 pM, about 130 pM, about 140 pM, about 150 pM, about 160 pM. about 170 pM, about 180 pM, about 190 pM. about 200 pM, about 210 pM, about 220 pM, about 230 pM, about 240 pM, about 250 pM, about 260 pM, about 270 pM, about 280 pM, about 290 pM, about 300 pM, about 310 pM, about 320 pM, about 330 pM, about 340 pM, about 350 pM, about 360 pM, about 370 pM, about 380 pM, about 390 pM, about 400 pM, about 410 pM, about 420 pM, about 430 pM, about 440 pM, about 450 pM, about 460 pM, about 470 pM, about 480 pM, about 490 pM, or about 500 pM.

[0232] In some embodiments, the LPC composition is a nutritional composition.

[0233] Nutritional compositions herein can be produced by conventional formulation techniques and food production techniques. The nutritional composition can be formulated as a liquid preparation such as an elixir, suspension, syrup, emulsion, ampoule; or a solid preparation such as gel, gum, drop, powder, granule, pill, tablet (including sugar-coated tablet, film-coated tablet), capsule, package agent, powder, and the like.

[0234] The nutritional composition may be given as a food or beverage, added to a food beverage, or directly administered to the stomach or bowels of a subject. When the nutritional composition is provided as food or beverage, such products can include liquid products such as drinks, milk products such as milk, milk beverage, yogurt, jelly products such as jelly drinks, jelly, gum products, powder products, granular products, sheet products, capsule products, tablet products, solid products such as snack bar, cookie, and the like.

[0235] The LPC composition may further comprise food-safe additives, including but not limited to a sweetener, colorant, preservative, thickening stabiliser, antioxidant, fungicide, gum base, bittering agent, enzyme, gloss agent, acidulant, seasoning, emulsifier, flavour enhancement agent, and the like.

[0236] The amount of the LPC composition to be administered can be individually determined based on the age, gender, body weight, meal condition, and the like, of the subject. In embodiments, LPC compositions herein may be used to boost choline and / or acetylcholine levels in the brain of a subject. In further embodiments, LPC compositions may be used in combination with the MFSD7c inhibitors of this disclosure to promote synthesis and / or accumulation of acetylcholine in the brain, and / or to treat, prevent or delay the progression of CNS diseases associated with diminished cholinergic signalling.

[0237] The administration of the combination of the inhibitor and LPC composition may result not only in a beneficial effect, c.g., an additive or synergistic therapeutic effect, for instance, with regard to promoting synthesis and / or accumulation of acetylcholine in the brain parenchyma, or with regard to treating, preventing or delaying the progression of a CNS disease or condition associated with diminished cholinergic signalling, but also in further surprising beneficial effects. Such other effects may include fewer side effects, an improved quality of life or a decreased morbidity, compared with a monotherapy applying only the inhibitor or only the LPC composition.

[0238] Disclosed herein is the use of an LPC composition as defined herein for promoting synthesis and / or accumulation of acetylcholine in a brain parenchymal cell or tissue in a subject. In some embodiments, the subject is suffering from a CNS disease or condition associated with diminished cholinergic signalling, such as Alzheimer’s disease, stroke, depression or schizophrenia. In some embodiments, the LPC composition is administered or is to be administered to the subject in combination with an inhibitor of MFSD7c.

[0239] Disclosed herein is a method of promoting synthesis and / or accumulation of acetylcholine in a brain parenchymal cell or tissue in a subject, the method comprising administering an effective amount of an inhibitor of MFSD7c in combination with a LPC composition as defined herein to the subject to promote synthesis and / or accumulation of acetylcholine in the brain parenchymal cell or tissue of the subject.

[0240] Disclosed herein is a method of treating a CNS disease or condition in a subject, the method comprising administering an effective amount of an inhibitor of MFSD7c in combination with a LPC composition as defined herein to the subject to treat the CNS disease or condition in the subject. In some embodiments, the CNS disease or condition is associated with diminished cholinergic signalling.

[0241] Disclosed herein is a method of preventing or delaying the progression of a CNS disease or condition in a subject, the method comprising administering an effective amount of an inhibitor of MFSD7c in combination with a LPC composition as defined herein to the subject to prevent or delay the progression of the CNS disease or condition in the subject. In some embodiments, the CNS disease or condition is associated with diminished cholinergic signalling.

[0242] Disclosed herein is an inhibitor of MFSD7c, for use in treating or preventing a CNS disease or condition in a subject, wherein the inhibitor is to be administered to the subject in combination with an LPC composition as defined herein. In some embodiments, the CNS disease or condition is associated with diminished cholinergic signalling.

[0243] Disclosed herein is the use of an inhibitor of MFSD7c in the manufacture of a medicament for treating or preventing a CNS disease or condition in a subject, wherein the inhibitor is to be administered to the subject in combination with an LPC composition as defined herein. In some embodiments, the CNS disease or condition is associated with diminished cholinergic signalling.

[0244] A therapeutically effective amount of each of the inhibitor and LPC composition (i.e., the combination partners) may be administered simultaneously or sequentially in any order, and the components may be administered separately or as a fixed formulation.

[0245] The effective dosage of each of the combination partners may vary depending on the particular inhibitor or LPC composition employed, the mode of administration, the disease or condition being treated, and the severity of the condition being treated. Thus, the dosage regimen of the combination therapy is selected in accordance with a variety of factors including the route of administration and clinical condition of the subject. A person of ordinary skill can readily determine the effective amount of the combination partners required to alleviate, counter or arrest the progress of the CNS disease or condition.

[0246] As used herein, “and / or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative (or).

[0247] As used in this application, the singular form “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “an agent” includes a plurality of agents, including mixtures thereof.

[0248] Throughout this specification and the claims which follow, unless the context requires otherwise, the word “comprise”, and variations such as “comprises” and “comprising”, will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

[0249] The reference in this specification to any prior publication (or information derived from it), or to any matter which is known, is not, and should not be taken as an acknowledgment or admission or any form of suggestion that that prior publication (or information derived from it) or known matter forms part of the common general knowledge in the field of endeavour to which this specification relates.

[0250] Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications which fall within the spirit and scope. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any two or more of said steps or features.

[0251] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this invention belongs. Certain embodiments of the invention will now be described with reference to the following examples which are intended for the purpose of illustration only and are not intended to limit the scope of the generality hereinbefore described.

[0252] EXAMPLES

[0253] Materials and methods

[0254] Mice

[0255] Global deletion of Mfsd7c has been described previously. To generate the postnatal deletion of Mfsd7c in the blood brain ban er, Mfsd7cmwas crossed with Cdh5-CreERT mice to generate Mfsd7cm, Cdh5-CreERT2 mice (EcAf / ir / 7c-KO). At postnatal day 15 (P15) the pups were injected daily with three doses of 100 pg tamoxifen prepared in olive oil via intraperitoneal injection. Male and female mice with age >7-8 weeks old were used for experiments. Mice were maintained at a constant temperature of 20°C with 12-hour light / 12-hour dark cycle on normal chow diets. All experimental protocols and procedures in the protocol R19-0567 were approved by IACUC committees under National University of Singapore.

[0256] Plasmids

[0257] All plasmids were constructed in pcDNA3.1 vector for overexpression unless stated otherwise. hChKA (D10704.1), ETNK1, CHAT (NM_020986.4) gene were synthesized from GenScript and inserted into pcD A3.1. pcDNA3.1hMfsd7c, pcDNA3. lhMfsd7cT430M and S203 Y were generated previously. The other missense mutations including hMfsd7cT430A, P276S. L483R, P340L, and T430R were generated by mutagenesis and confirmed by Sanger sequencing. Zebrafish (DaMfsd7c_a, XM_688497.7; DaMfsd7c_b: XM_003200755.5), medaka fish (MeMfsd7c, XM_004082328.4), and frog Mfsd7c (XeMfsd7c, NM_001016982.2) coding sequences were synthesized by GenScript and cloned into pcDNA3.1 plasmid for overexpression. The pESC-HIS-hMfsd7c plasmid (Genscript) was used for overexpression on the yeast S. cerevisiae.

[0258] Untargeted metabolite analysis Untargeted metabolomics analysis was performed at the Genome Analysis Center, Research Unit Molecular Endocrinology and Metabolism. Helmholtz Center Munich. Frozen wildtype and Mfsd7c knockout embryonic brain and liver samples at E14.5 were weighed and placed into pre-cooled (dry ice) 2 ml homogenization tubes containing ceramic beads with a diameter of 1.4 mm (Precellys® Keramik-Kit 1 .4 mm). Pre-cooled water with a ratio of 5 ul / mg tissue was added into the tubes. The samples were then homogenized in Precellys 24 homogenizer (PEQLAB Biotechnology GmbH, Germany) equipped with an integrated cooling unit for 3 times 20 s at 5,500 rpm, with 30 second intervals (to ensure freezing temperatures in sample vials) between the homogenization steps. A hundred pl of the brain and liver homogenates were loaded onto two separate 2 ml 96-deep well plate, one plate for the brain sample set and the other plate for the liver samples. Three types of quality control samples were analyzed in concert with the experimental samples in each sample set: samples generated from a pool of human plasma, samples generated from a small portion of each experimental sample from the respective sample set served as technical replicate throughout the data set; and extracted water samples served as process blanks. Experimental samples and controls were randomized across the metabolomics analysis.

[0259] The homogenates in each well of the 2 ml 96-deep well plate were extracted with 500 pl methanol, containing four recovery standards to monitor the extraction efficiency. After centrifugation, the supernatant was split into aliquots in two 96-well microplates. The first 2 aliquots of each sample set were used for ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS / MS) analysis in positive electrospray ionization modes (i.e., early and late eluting compounds), 1 aliquot of each sample set was used for UPLC-MS / MS analysis in negative ionization mode, and the rest were kept as reserve for backup. The extract aliquots were dried on a TurboVap 96 (Zymark).

[0260] Prior to UPLC-MS / MS analysis, the dried extract samples were reconstituted in acidic or basic LC-compatible solvents, each of which contained 8 or more standard compounds at fixed concentrations to ensure injection and chromatographic consistency. The UPLC-MS / MS platform utilized a Waters Acquity UPLC with Waters UPLC BEH 08-2.1x100 mm, 1.7 pm columns and a Thermo Scientific Q-Exactive high re solution / accurate mass spectrometer interfaced with a heated electrospray ionization (HESI-II) source and Orbitrap mass analyzer operated at 35,000 mass resolution. Extracts reconstituted in acidic conditions were gradient eluted using water and methanol containing 0.1% formic acid, while the basic extracts, which also used water / methanol, contained 6.5mM ammonium bicarbonate. The MS analysis alternated between MS and data-dependent MS2 scans using dynamic exclusion, and the scan range was from 80-1000 m / z.

[0261] Metabolites were identified by automated comparison of the ion features in the experimental samples to a reference library of chemical standard entries that included retention time, molecular weight (m / z), preferred adducts, and in-source fragments as well as associated MS spectra and curated by visual inspection for quality control using software developed at Metabolon. Chromatographic peaks were quantified using area- under-the-curve.

[0262] In vivo transport of radiolabelled choline

[0263] To study the uptake of choline into the brain, control (Mfsd7ca!) and EcM fsd7c KO mice aged 4-6 months was intravenously injected 2mM radiolabelled choline (stock: 333mM choline with 0.05pCi / pl). After 5 mins of injection, 50pl blood was collected and the radioactive levels were quantified to monitor the initial radiolabelled levels in the circulation. After 30 mins of injection, mice were perfused with 20ml of PBS. Then, brains, lungs, hearts, kidneys, and livers were collected for quantification of radioactive signals. Similar amount of tissues was homogenized in RIPA buffer and the radioactive signals were quantified by liquid scintillation counter. The radioactive signals from tissue from each mouse were normalized to radioactive signals from blood collected at 5 mins post-injection.

[0264] Choline-d9 and LPC-d49 experiments

[0265] For stable isotopic tracing with choline-d9 (containing 9 deuterium atoms) injection, pregnant Mfsd7c+I- mice at 12.5 days of gestation were injected daily by IV route with an amount of 2mM choline-d9 / day until gestation day 14.5 (total of 3 doses of choline- d9 from E12.5-14.5). The embryos were collected at E15.5 after the last dose at E14.5 and genotyped. The brains and livers of embryos were harvested for lipid extraction for lipidomic analysis. Briefly, equal amount of brain and liver lysates from WT, HET and KO embryos were extraeted with l-butanol:methanol (ratio 1:1) method for choline and lipid analyses by LC-MS / MS.

[0266] For stable isotopic LPC-d49 (Sigma: 870308P-5MG; containing choline-d! 3, glycerol- d5, and palmitatc-d31) injection, EcMfsdl7c-KO and control mice aged 5-8 months were injected with a dose of 200pM LPC-d49 in 6% BSA. After 2 hours of injection, the mice were perfused with cold PBS to remove blood and the brains and livers were collected. Brains and livers were homogenized in PBS and the same amount of protein lysates was used for lipid extraction with chloroforrmmcthanol method. Organic phase was used for lipid analysis and aqueous phase (upper phase) was used for choline analysis by mass- spectrometry.

[0267] Lipid and choline quantification by LC / MSMS

[0268] Samples were randomized before extraction and analysis. Blank samples (10 pL MilliQ water), matrix blanks (samples extracted without spiking internal standards, see below), and pooled QC samples were used to assess method performance. In addition, diluted pooled QC samples were used to assess response linearity. Blanks, blank extracts, QC, and diluted QC were interspersed with study samples throughout the analytical run. Phospholipids and choline were separated using HILIC column (Kinetex 2.6 pm HILIC 100 A, 150 x 2.1 mm, Phenomenex, Torrance, CA, USA) on an Agilent 6495 A2 and 6495C2 (Agilent Technologies). Mobile phases A (50% acetonitrile (LC-MS grade, Thermo Fisher Scientific Inc.) 50% 25 mM ammonium formate (Sigma-Aldrich) pH = 3.5) and B (95% acetonitrile and 5% of 25 mM ammonium formate, pH = 4.6) were mixed at the following gradient: 0-6 min, 99-75% B; 6-7 min, 75-10% B; 7-7.1 min, 10-99% B; 7.1-10.1 min, 99% B. The flow rate was 0.6 mL / min, and the sample injection volume was 1 pL. MS parameters were as follows: electrospray ionization, gas temperature 200°C, gas flow 12 L / minute, sheath gas flow 12 L / minute, and capillary voltage 3,500 V. Phospholipids were quantified at the sum composition level using multiple reaction monitoring (MRM) using transition to phosphocholine headgroup (m / z 184 for endogenous lipids and adjusted to appropriate m / z for deuterated lipids). Choline was quantified using multiple reaction monitoring (MRM) using transition 104 to 60 (adjusted to appropriate m / z for deuterated choline). These internal standards (IS) were used for lipidomics and choline analysis by LC MS / MS. Internal standards solutions were prepared in butanol / methanol (BuMe) (1:1, v / v) or chloroform / mcthanol (1:2) containing phospholipid IS SPLASH Mix (Avanti, 330707) (containing 5.3 pM 15:0-18:1 (d7) PC, 0.19 pM 15:0-18: 1 (d7) PE, 0.13 pM 15 :0-l 8: l(d7) PS (Na Salt), 0.93 pM 15:0-18: l(d7) PG (Na Salt), 0.27pM 15:0-18:l(d7) PI (NH4 Salt), 0.27 pM 15:0-18:l(d7) PA (Na Salt), 1.20 pM 18:l(d7) Lyso PC, 0.27 pM18:l(d7) Lyso PE, 13.32 pM18:l(d7) dl8:l-18:l(d9) SM. Choline-d9 was used as IS in choline analysis.

[0269] Transport assays in HEK293 cells

[0270] Human (hMfsd7c) or mouse (mMfsd7c) Mfsd7c cDNA constructed in pcDNA3.1 was transfected to HEK293 cells using lipofectamine 2000. Mock was treated with empty plasmid. For experiments in which human choline kinase A (hChKA) was used, 1-1.5pg pcDNA3.1 hChKA plasmid was co-transfected with 2-2.5pg plasmid of hMfsd7c or mutant plasmids. Cells transfected with 1-1.5pg pcDNA3.1 hChKA plasmid was used as control. After 18-30 hours post-transfection, cells were used for transport assays with 100pM [3H] choline (ARC: ART 0197) in DMEM containing 10%FBS. The transport assays were stopped after Ihour incubation at 37°C by washing once with cold plain DMEM medium. Cell pellets were lyzed in RIPA buffer and transferred to scintillation vials for quantification of radioactive signal using Tricarb liquid scintillation counter. Transport activity of MFSD7c was expressed as DMP for [3H] isotopes and CPM [14C] isotopes. For dose dependent transport activity, HEK293 cells were similarly transfected with hMfsd7c or hMfsd7cS203Y mutant plasmid alone or co-transfected with hChKA. Transport activity was conducted with 20, 45, 90, and 250 pM [3H] choline in DMEM containing 10% FBS for 1 hr at 37°C. For time-dependent assay, hMfsd7c was transfected or co-transfected with hChKA. Transfected HEK293 cells were incubated with lOOpM [3H] choline in DMEM containing 10%FBS for 15, 30, 60, and 120 mins at 37°C. For transport assay with [,4C] ethanolamine, [3H] L-carnitine, and [3H] acetylcarnitine, [3H] palmitoyl-carnitine. [14C] octanoyl-carnitine, HEK293 cells were cotransfected with MFSD7c and hChKA as described above. Transport assays were performed with I OOLI [14C] ethanolamine, ImM [3H] L-carnitine, ImM [3H] acetylcarnitine, lOpM [3H] palmitoyl-carnitine, or lOOpM [14C] octanoyl-carnitine in DMEM with 10%FBS for 1 hour. Cells were washed once with cold plain DMEM and lyzed with RIPA buffer for radioactive quantification. For import assays with the missense mutants, these plasmids were co-transfected with hChKA to HEK293 cells. Import assays were performed with lOOpM [3HJ choline in DMEM containing 10% FBS for 60 mins. Radioactive signals from cells were quantified and converted to percentage of WT activity.

[0271] For export assays, HEK293 cells were transfected with empty, hMfsd7c, or mMfsd7c plasmids. After 24 hours of transfection, cells were loaded with 500pM [3H] choline for 1 hour. The cells were then washed to remove remaining [3H] choline in the medium and incubated with DMEM medium to stimulate the release. The cells were harvested at 0 (before release) and Ihour (after release) after incubation with DMEM for radioactive quantification as described above.

[0272] Sodium and pH-dependent transport assays

[0273] For sodium dependent assays, sodium is replaced with lithium in transport buffer (NaCl buffer: 150mM NaCl, 5mM KC1, lOmM HEPES-Na, pH 7.4; LiCl buffer: 150mM LiCl. 5mM KC1, 10mM HEPES, pH 7.4 with HO). For pH dependent assays, NaCl buffer was adjusted to pH 6.5 and pH 8.5. For transport assay conditions, overnight hMfsd7c and hChKA co-transfected HEK293 cells were washed once with the same buffer before incubated with lOOpM [3HJ choline for 15 minutes at 37°C. Cells were lysed in RIPA buffer and mixed with scintillation fluid for radioactive quantification.

[0274] Electrophysiology

[0275] For single cell recording of membrane potential alteration during transport of choline, HEK293FT cells were cultured in DMEM medium supplemented with 10% fetal bovine serum and 1% penicillin and streptomycin and maintained in 5% CO2 incubator at 37°C. For transfection, cells were seeded on the Petri dishes and grown overnight. Subsequently, 2 pg of empty pIRES2-EGFP (empty plasmid), pIRES2-EGFP hMfsd7c or mutant pIRES2-EGFP S203Y plasmid was co-transfected with 2 pg of pIRES2- DsRed hChKA plasmid using lipofectamine 2000. The transfected cells were incubated for 24 h in 5% CO2 incubator at 37°C. At 48 hours post-transfection, the cells were split and seeded on poly-D lysine coated cover slips for one more day before recording. Whole-cell patch-clamp recordings and data analysis

[0276] To record the changes in resting membrane potential, the internal solution (pipette solution) contained 130mM K-gluconatc, lOnM KC1, 5mM EGTA, lOmM HEPES, I mM MgCE, 0.5mM Na iGTP, 4mM Mg-ATP, 10mM Na-phoshocreatine, pH 7.4 (adjusted with KOH) was filled in the pipette tip. The external solution contained: lOmM Glucose, 125mM NaCl, 25mM NaHC03, 1.25mM NaH2PO4.2H20, 2.5mM KC1, 1.8mM CaC12, ImM MgC12, pH 7.4 (300-310 mOsm). Single cells with both EGFP (empty, hMfsd7c, S203Y plasmid) and DsRed (hChKA) fluorescence was visualized under fluorescent microscope for patching. Resting membrane potential was recorded under current clamp with an Axopatch200B or multiclamp 200B amplifier (Molecular Device) with 0 pA current injection. Subsequently, 200 uM choline was perfused to the cells after 2 minutes of stable base line recording. After 5 min of recording, the ligand was washed away. Approximately, 10 individual cells were recorded for each condition. The data were analyzed using Graphpad Prism V software (San Diego, CA) and Microsoft (Seattle, WA) Excel. Data are expressed as mean values ± SEM.

[0277] Expression of human Mfsd7c in 5. cerevisiae Hnml mutants

[0278] Saccharomyces cerevisiae acquires choline via HNMl. The S. cerevisiae Hnml mutants were acquired from Dharmacons. Wildtype and Hnml mutant yeasts were grown in Yeast extract-Peptone-Dextrose (YPD) medium at 30°C overnight before transformation. The pESC-HIS-hMfsd7c plasmid was transformed into wildtypc and Hnml mutant yeasts following a previously described protocol. After heat shock at 42UC for 30 minutes and recovery in YPD for 2 hours, the yeasts were spread on 2% agar plates prepared in yeast nitrogen base (YNB) medium (Sigma Aldrich) supplemented with 2% glucose and yeast synthetic drop-out medium supplements without histidine (Sigma Aldrich). Positive colonies were picked up randomly and further confirmed by transport assay and western blot.

[0279] Transport assay for yeast cells

[0280] Wild type, Hnml mutant and transformed yeast cells were grown in YNB medium with 2% glucose and drop-out medium supplements without histidine at 30°C overnight followed by induction of hMfsd7c expression in YNB medium with 2% galactose for at least 5 hours at 30°C. An 800μl of yeast cells at OD600= 1 was centrifuged, washed twice with distilled water, then incubated in PBS containing 100 pM [3H] choline for 10 mins. The transport assay was stopped by washing twice with PBS containing 100 μM choline. Yeast cells were lysed with lysis buffer containing 2% Triton-X, 1 % SDS, 100mM NaCl, lOmM Tris-HCl (pH 8.0), ImM EDTA (pH8.0) at 95°C for 5 mins for radioactive quantification by liquid scintillation counter.

[0281] Western blot analysis

[0282] For protein extraction, transfected HEK293 cells with hMfsd7c, mMfsd7c, hMfsd7c mutants were lysed with RIPA buffer. For validation of deletion of Mfsd7c in mice, micro-vessels from PBS perfused brains of WT and EcMfsd7c-KO mice were isolated, homogenized and lysed with RIPA buffer. For Western blot analysis of hMfsd7c expression in yeasts, 1ml of yeast culture at OD600=1 from WT, Hnml mutant yeasts or transformed yeasts was used for total protein extraction. BCA assay was used for total protein quantification. These antibodies were used: VDAC (CellSignaling CS4866), CoxIV (Invitrogen, MA5-15686), OPA1 (BD biosciences, 612602), MRPS35 (Protein biotech, 16457), CHKA (CellSignaling, CS13422S), HM0X1 (Proteintech, 10701-1- AP), NDUFS1 (Proteintech, 12444-1-AP). Western hlot analysis was conducted as previously described.

[0283] Fowler patient recruitment

[0284] A five-month-old infant born at 39wld with multifocal epilepsy, ventriculomegaly, developmental delay, and lisscnccphaly was identified via GcncMatchcr collaboration. She was born to a 38 years old mother. Pregnancy was complicated by gestational diabetes and maternal hypertension. There was concern for lisscnccphaly on prenatal ultrasound and MRI. Prenatal CMA and karyotype were normal. She was delivered vaginally complicated by shoulder dystocia. She required brief positive pressure ventilation and was admitted to the NICU on NIPPV. Apgars at 1 and 5 minutes were 4 and 9 respectively. Birth parameters include length of 49 cm (47th percentile WHO), weight of 2.86 kg (20th percentile WHO) and head circumference of 32 cm (6th percentile WHO). Her whole exome sequencing at birth showed two variants of uncertain significance in FLVCR2, C.826OT (Maternal) and c,1448T>G (Paternal). The missense mutations were confirmed by Sanger sequencing. There was no family history of a similar presentation. This was the first pregnancy between this patient’s parents. Mother had a previous miscarriage and a healthy older daughter. Parents report no consanguinity.

[0285] RNA sequencing and analysis

[0286] For bulk RNA sequencing of whole brains from El 4.5 wildtype and KO embryos from normal chow diet (ND), this has been deposited in GEO database with the accession number: GSE148854. For RNA sequencing of isolated endothelial cells from heterozygous (HET) and KO embryos at E14.5, the dataset GSE129838 was reanalyzed. For RNA sequencing of whole brains from E14.5 wildtype embryos from choline deficient diet (CDD), pregnant wildtype mice were fed with CDD diet from the breeding until embryo collection. The whole brain of E14.5 embryos was collected for bulk RNA sequencing. This dataset has been deposited in the GEO database with the following accession number: GSE239589.

[0287] Choline and acetylcholine assays

[0288] Choline and acetylcholine from plasma and brain samples were measured by colorimetric assay (Abeam: ab65345). Tissues were homogenized in choline buffer. Choline and acetylcholine were assayed according to the manufacturer’s instructions.

[0289] Transmission electron microscopy

[0290] Wild-type (n=3) and global knockout embryos of Mfsd7c (n=3) at E14.5 were collected from the same pregnant mice and fixed in 2.5% glutaraldehyde for 24 hours at room temperature. Brain sections were prepared and were post- fixed with 1% osmium tetroxide for 1 hour and dehydrated using a serial ethanol concentration. Images were taken using a Fei Quanta650 transmission electron microscope.

[0291] Statistical analysis

[0292] Data was analyzed using GraphPrism9 software. Statistical significance was calculated using r-test. One or Two-way ANOVA as indicated in the figure legends. P value < 0.05 was considered as statistically significant. Example 1 : MFSD7c facilitates choline transport via the plasma membrane at the blood-brain barrier

[0293] Misscnsc mutations of MFSD7c (also known as FLVCR2) have been described in Fowler patients. The major clinical symptoms reported in these patients are the severe dilation of cerebral blood vessels and mild microcephaly. Most severe cases result in embryonic death, but some patients have survived after birth. Although the cause of Fowler syndrome is unclear, it is linked to the loss of functions of MFSD7c, an orphan transporter. To gain better understanding about the disease mechanisms, the molecular role of MFSD7c must be elucidated. It was recently reported that mice with a global deletion of Mfsd7c had late gestation lethality with severe dilation of central nervous system (CNS) vasculature. Specific deletion of Mfsd7c in endothelial cells also resulted in similar phenotypes, indicating its critical roles in the blood vessels. Mfsd7c knockout embryos exhibited multiple neurological phenotypes that are probably linked to primary defects of the CNS vasculature. These phenotypes resemble the glomeruloid structures in the CNS blood vessels of patients with Fowler syndrome.

[0294] MFSD7c belongs to the Major Facilitator Superfamily (MFS) of transporters which facilitate the movement of small molecules through cell membranes. MFSD7c was reported to exert heme import activity, but this observation has not been confirmed. Recently, it was shown that MFSD7c is involved in thermogenesis in response to heme. Besides its localization in the mitochondria, it was shown that MFSD7c is expressed in the plasma membrane. Furthermore, it was shown that MFSD7c is expressed in endothelial cells of the CNS vasculature.

[0295] Utilizing mouse knockout models coupled with comprehensive metabolomics, it was revealed the specific changes in metabolites was due to the loss of Mfsd7c. These results demonstrate that MFSD7c is a transporter for choline. It was shown that MFSD7c facilitates choline transport via the plasma membrane in a concentration-dependent manner. Unexpectedly, it was found that lack of Mfsd7c at the BBB causes choline accumulation in the brain. Stable isotopic tracing study reveals that lysophosphatidylcholine (LPC) and Mfsd2a pathway delivers choline to the brain. The excessive amount of choline is then released to be exported out of the brain via Mfsd7c. The current work identifies MFSD7c as a choline transporter and provides a foundation that facilitates the understanding of the disease mechanisms of Fowler syndrome. Additionally, the identification of MFSD7c as a choline transporter at the BBB lays the groundwork for future research to reveal the metabolic fates of choline in the brain.

[0296] Lack of Mfsd7c affects choline levels in the brain

[0297] Deletion of Mfsd7c resulted in late gestation lethality in mice. Because MFSD7c is predicted to be a membrane transporter, its deletion is anticipated to cause insufficient transport of essential nutrients to the brain. To dc-orphanizc the physiological ligands for MFSD7c, a comprehensive analysis of metabolites in the brains of wildtype (WT) and global Mfsd7c knockout (Mfsd7c '~) embryos was performed at gestation day 14.5 (E14.5), a time point when knockout embryos already exhibited vascular growth defects. This analysis encompassed more than 520 metabolites from metabolic pathways for carbohydrates, lipids, amino acids, and cofactors. First, it was found that the levels of glycolytic metabolites such as lactate were significantly increased, whereas the levels of 3 -phosphoglycerate (3 -PGA) and phosphoenolpyruvate (PEP) were reduced (Figure la). These metabolic changes are congruent with the increased glycolysis and possibly reduced mitochondrial activity as a consequence of hypoxia that occurs in the brain of Mfsd7c '~ embryos. MFSD7c was shown to regulate heme transport. However, heme and bilirubin levels in brains were comparable between Mfsd7c~'~ and WT embryos (results not shown). Second, increased levels of several long chain acylcarnitines were detected (Figure lb, c; black arrowheads), whereas the levels of L-carnitinc were significantly reduced in the brain of Mfsd7c '~ embryos (Figure Id). Nevertheless, it was ruled out that MFSD7c transports these acylcarnitincs by performing the import assay with palmitoylcarnitine (C16:0 carnitine) and octanoylcamitine (C8:0 carnitine) (results not shown). Again, these changes might reflect the defects of mitochondrial activity due to hypoxia in the knockout brain. Indeed, mitochondrial morphology and the expression level of several mitochondrial markers was reduced in the brain of Mfsd7c '~ embryos (results not shown). Interestingly, it was noted the increased levels of choline, palmitoylcholine, and oleoylcholine in the brains of MfsdTc1" embryos relative to WT embryos (Figure lb; red arrowheads). In contrast, the levels of CDP-choline were reduced in Mfsd7c '~ embryos (Figure Id). These changes in choline metabolites were specific to the brain as their levels were unaltered in the livers from Mfsd7c '~ embryos (Figure le). Instead, a striking increase in the levels of several neutral lipids such as monoacylglycerols and diacylglycerols was detected in the livers of Mfsd7c '~ embryos (Figure If, arrowheads). Deficiency of choline has been linked to mitochondrial defects. These differential changes in the levels of choline and choline metabolites in the brain and the accumulation of neutral lipids in the livers of Mfsd7c' embryos suggest that MFSD7c may be involved in choline metabolism.

[0298] MFSD7c mediates transport of choline

[0299] The accumulation of choline and decrease in CDP-choline levels in the brains of Mfsd7c embryos suggests that MFSD7c is important for regulation of choline levels. Thus, the inventors tested whether MFSD7c regulates choline levels by a transport mechanism. Human (hMFSD7c) or mouse Mfsd7c (mMfsd7c) was expressed in HEK293 cells and utilized radioactive [3H] -choline for transport assays. Interestingly, overexpression of human or mouse MFSD7c led to 1.5-2 fold increase of intracellular levels of radioactive signals, implicating that MFSD7c mediates the import of choline (Figure 2a). The choline transport assay was validated by including neuronal choline transporter CHT1 (also known as SLC5a7) as a positive control (Figure 2a). The import of choline mediated by hMFSD7c was increased with the increased levels of choline in medium and was slightly increased over time (Figure 2b-c). To reaffirm that MFSD7c mediates choline import to the cells, the missense mutation, S203Y, was included in the transport assays. In comparison to wild-type MFSD7c, S203Y mutant exhibited abolished choline transport activity when incubated with different concentrations of choline (Figure 2b- c). The reduced import of choline of the wildtypc and mutant was not due to reduced protein expression levels nor defective localization in the plasma membrane (results not shown). MFSD7c is conserved from fish to man, but not in yeasts (results not shown). Choline import function for several MFSD7c orthologs including the ones from zebrafish (DaMfsd7c_a and DaMfsd7c_b isoforms), medaka fish (MeMfsd7c), and frog (XeMfsd7c) were tested. Consistently, these MFSD7c orthologs exhibited equal choline transport activity compared to hMFSD7c (Figure 2d). In the yeast S. cerevisiae, HNM1 was shown to import choline as well as L-carnitine. Complementation assays of hMFSD7c in Hnml S. cerevisiae knockout were performed. First, it was shown that Hnml knockout cells exhibited reduced choline uptake compared to wild-type yeast cells (Figure 2e). Second, when wild-type yeast cells and Hnml mutant yeast cells were overexpressed with human MFSD7c, they both exhibited significantly increased choline uptake compared to parental cells (Figure 2e, three different clones were used). These results indicate that hMFSD7c can rescue the loss of yeast Hnml and reaffirm that MFSD7c is a choline transporter.

[0300] Choline transport by neuronal choline transporter CHT1 is inhibited by hemicholinium- 3 (HC3), a competitive inhibitor of CHT1. Hemicholinium-3 inhibited CHT1, but not MFSD7c, suggesting that these two choline transporters exert different transport properties (Figure 2f). SLC44al and SLC44a2 has been reported to be choline transporters. However, the inventors were unable to show choline import activity for these transporters under the tested conditions (results not shown). MFSD7c prefers choline as a substrate as it did not transport betaine, a choline metabolite that is also found in the brain (Figure 2g-h). Similarly, Mfsd7c did not transport L-carnitine, acetylcarnitine, and ethanolamine, and acetylcholine (Figure 2i-k). Together, the results reveal that MFSD7c is a conserved transporter for choline in vertebrates.

[0301] MFSD7c functions as a facilitative transporter for choline

[0302] It was noted that the transport activity of MFSD7c was slightly enhanced over time (Figure 2c), suggesting that intracellular levels of choline might affect the influx of choline. Thus, its transport properties were characterized by testing whether MFSD7c utilizes cations for transport activity. Replacement of sodium with lithium did not affect choline import by MFSD7c (Figure 3a; Figure 6a). Next, it was tested whether lowering intracellular choline levels could increase the influx of choline via MFSD7c. To test this hypothesis, hMfsd7c was co-expressed with human choline kinase A (hChKA), which phosphorylates choline to phosphorylcholine, thus lowering the intracellular concentration of choline. Remarkably, co-expression of hMfsd7c with hChKA increased choline uptake by approximately 9-fold (Figure 3b), whereas sole expression of hMfsd7c increased choline uptake by 1.5-2 fold (Figure 2a). In the presence of hChKA, the import of choline was significantly increased over time (Figure 3c). Co-expression of hChKA with hMfsd7c also greatly enhanced choline uptake in a dose-dependent manner (Figure 3d; Figure 6b). The kinetics (Km) and Vmax for choline import by human Mfsd7c under this condition was lOOpM and 0.117 pmol / well / 60 mins, respectively. Choline uptake by S203Y mutant in the absence of hChKA was completely inhibited (Figure 2b-c). Interestingly, it was noted that there was a slight increase in choline import in S203Y mutant when co-expressed with hChKA, indicating that the transport activity of S203Y is severely delayed, but not abolished (Figure 3d; Figure 6c). The kinetics (Km) and Vmax for importing choline by S203Y mutant under this condition was 1197 M and 0.039 pmol / well / 60 mins, respectively.

[0303] It was also examined whether expression of choline acetyltransferase (CHAT), a neuronal enzyme for acetylcholine synthesis, would increase choline uptake by converting choline into acetylcholine. Indeed, co-expression of CHAT and hMfsd7c greatly increased choline uptake (Figure 3e). In the absence of ETNK1, an ethanolamine kinase, MFSD7c did not increase uptake of ethanolamine (above Figure 2k). However, transport of ethanolamine by MFSD7c was increased to a significant level when ETNK1 was co-expressed (Figure 3f). Interestingly, co-expression of hMfsd7c with hChKA slightly increased L-carnitine uptake (Figure 3g). However, L- carnitine is unable to compete for choline import, suggesting that L-camitine is a weak ligand (Figure 6d).

[0304] Furthermore, it was tested whether the transport of choline by MFSD7c is bidirectional. Indeed, the results showed that expression of hMfsd7c is required for the release of intracellular choline (Figure 3h). Choline is a positively charged molecule. Import of choline would increase the membrane potential. To this end, whole-cell patch clamp of HEK293 cells co-expressing hMfsd7c or S203Y mutant with hChKA was performed. Cells with overexpression of hChKA alone were used as a control (Figure 6e). Incubation of control cells or cells expressing S203Y mutant with I OOIJ M or 200LI M choline did not significantly increase membrane potential (Figure 3i). In contrast, expression of hMfsd7c significantly increased membrane potential when I OOLI M or 200, u M choline was included in the bath solution (Figure 3i, j). Interestingly, removal of choline reversed the increased membrane potential, suggesting that the influx of choline generates the membrane potential. These results show that the import of choline by MFSD7c is electrogenic. Collectively, the results show that MFSD7c facilitates the transport of choline and that it behaves like a channel for the movement of choline via the plasma membrane.

[0305] Lack of Mfsd7c results in elevated levels of choline in the brain

[0306] Choline uptake at the BBB has been documented in previous studies. However, the identity of the long-sought choline transporter was unknown. To test if MFSD7c play a role for choline transport in vivo, conditional knockout mice for Mfsd7c in the endothelial cells using Cdh5-CrcERT was generated (Figure 4a). Next, the inventors intravenously injected controls (MfsdTcf / f) and MfsdTcf / fCdh5-CreERT (hereafter, EcMfsdl7c-KO) mice with a dose (2mM in blood) of radioactive choline and measured the distribution of radioactive signals in the brains, lungs, livers, hearts, and kidneys after 30 minutes. This supraphysiologic al dose was chosen as the half-life of choline in plasma is within 3 minutes. The radioactive choline levels in blood were indifferent between the two genotypes after 5 mins choline injection, indicating that choline was equally delivered to the blood of the mice (Figure 4b). Importantly, it was found that radioactive signals were significantly reduced in the brains of EcMfsdl7c-KO mice compared to control mice (Figure 4c). However, it was noted that the majority of injected choline was present in peripheral organs such as livers and kidneys; and the radioactivity in the organs was comparable between the genotypes, indicating that reduced choline import to the brain was specific in EcMfsdl7c-KO mice (Figure 4d-g). These results indicate that Mfsd7c can mediate the uptake of exogenous choline at the blood-brain barrier, a result which is consistent with the reported findings in the literature. Nevertheless, the injected amount of choline was well above physiological concentrations of choline in blood. Perhaps, Mfsd7c mediates the import of a small amount of choline to the brain at this supraphysiologic al dose.

[0307] Metabolomic analysis showed that the levels of choline in the brain of Mfsd7c- / -embryos were elevated (Figure Id). As Mfsd7c is expressed on both the brain-facing and bloodfacing sides of CNS endothelial cells, this suggests that Mfsd7c is also required for choline import from the brain parenchyma. To gain insights into the mechanism by which choline level is elevated in the brain of Mfsd7c- / -embryos, deuterated choline (choline-d9) was injected to the circulation of Mfsdl7c pregnant dams and Mfsd7c- / -embryos were harvested for lipidomic analysis of choline-containing lipids and choline (Figure 4h). It was found that the total levels of endogenous phospholipids such as lysophosphatidylcholinc (LPC), phosphatidylcholine (PC), and sphingomyelin (SM) as well as the deuterated phospholipids containing choline-d9 in the brains and livers of Mfsd7c knockout and control littermates were comparable (results not shown). Interestingly, it was found that the levels of endogenous choline and choline-d9 were significantly elevated in the brains but not livers of Mfsd7c KO embryos (Figure 4i). Free choline import to the brain is expected to be suppressed in the KO embryos. These results suggest that choline imported to the brains of Mfsd7c KO embryos must be via other routes. It was suspected that lysophosphatidylcholine (LPC) delivers choline to the brain of Mfsd7c- / -' embryos (Figure 4h). These above results suggest a possibility that MFSD7c is also necessary for the export of choline from the brain parenchyma.

[0308] Lysophosphatidylcholine is one of the most abundant lipids in the plasma, and is imported to the brain by MFSD2a via the BBB for phospholipid synthesis. To test the hypothesis that choline from LPC is accumulated in the brain of Mfsd7c knockout mice, a dose (300 M in blood) of deuterated LPC-d49 which consists of choline-d13, palmitate-d31, and glycerol-d5 was injected into the circulation of EcMfsdl7c-KO and control mice and mass spectrometry was employed for detection of choline-dl3 and deuterated LPC and PC (Figure 4j). In the plasma, a part of LPC-d49 was remodelled to produce LPC-d31 and LPC-d13. However, LPC-d49 was still the major deuterated LPC in the blood of the mice at 2 hours post-injection (Figure 4k). In the brain, deuterated PC species were detected, which were present with similar amounts in the phospholipid pool of EcMfsdl7c-KO and control mice. These results indicate that plasma LPC-d49 was equally imported to the brains for PC synthesis (Figure 41). Remarkably, it was found that most of the deuterated PC species in the brains of the mice contained palmitate-d31 (PC-d31), instead of the intact form of LPC-d49 (PC-d49) (Figure 41). This indicates that there was a dramatic remodelling of plasma derived LPC in the brain. As a result, the levels of choline-dl3 in the brains of EcMfsdl7c-KO mice were significantly increased compared to that of control mice (Figure 4m). Consistently, the levels of endogenous choline were confirmed to be elevated in EcMfsd7c-KQ in the same analysis (Figure 4m; Figure 7a-b), whereas the levels of choline-dl3 in the plasma and livers of EcMfsdl7c-KO and control mice were comparable, highlighting a specific elevation of choline in the brain of EcMfsdl7c-KO mice (results not shown). Furthermore, the levels of acetylcholine were increased in the brains of EcMfsdl7c-KO adult mice, especially from mice fed with choline deficient diet (CDD) (Figure 7a-b). Since free choline import to the brain is likely suppressed or reduced in EcMfsd7c-K0 mice (Scc Figure 4c), these results reveal an unexpected finding that excessive choline from LPC / PC metabolism is transported out of the brain and this mechanism is mediated by the activity of MFSD7c at the BBB.

[0309] Defective choline export in the brain may be a causal factor and the missense mutations of MFSD7c in Fowler patients result in reduced choline transport activity.

[0310] At this point, it is unclear whether deficiency of choline is a causal factor for the phenotypes of Mfsd7c- / -embryos or it is due to choline accumulation in the brain. If choline is utilized for phospholipid synthesis, it is expected that the brain phospholipid profile of Mfsd7c knockout mice must be changed. Lipidomic analysis of the brains from Mfsd7c knockouts and controls was performed. However, there was no significant changes in the levels of major phospholipids containing choline such as LPC, PC, and SM due to Mfsd7c knockout embryos and mice (results not shown). Additionally, the brain transcriptomes of Mfsd7c- / -embryos was compared to that of wild-type embryos obtained from dietary choline deficient mice as it was argued that deficiency of choline would recapitulate the deletion of Mfsd7c (results not shown). Nevertheless, no significant change in gene expression from wild-type embryos obtained from dams fed with choline deficient diet compared to that of Mfsd7c- / -embryos was observed. These results indicate that deficiency of choline is unlikely to be the cause for the phenotypes in the brain of Mfsd7c- / -embryos. Next, it was tested whether depletion of choline from diets would improve the phenotypes in Mfsd7c- / -embryos. Choline was depleted from the diets for pregnant Mfsd7c mice and the CNS blood vessels of Mfsd7c- / -embryos were examined. Interestingly, it was found that depletion of choline significantly reduced the dilation of blood vessels in the cortices of E14.5 Mfsd7c- / -embryos (Figure 5a-e). Nonetheless, maternal choline deficiency did not prevent the migratory defects of the blood vessels to the ganglionic eminence (GE) regions of these Mfsd7c- / -embryos (Figure 5d), probably due to the dominant source of choline from LPC. Although the dietary intervention approach did not completely reverse the phenotypes of Mfsd7c';' embryos, these results suggest that accumulation of choline might be detrimental to the developing blood vessels. Together, the results point to a critical role of MFSD7c for the regulation of choline levels in the brain.

[0311] Missense mutations of MFSD7c have been reported in patients with Fowler syndrome. However, it remains unknown whether these mutations cause a loss or gain of choline function of MFSD7c. Thus, mutagenesis was performed to generate several homozygous and compound heterozygous missense mutations including S203Y, T430R, and T430M and their transport activity was tested. These homozygous missense mutations S203Y, T430R, and T430M were chosen as the first two former mutations caused lethal Fowler syndrome, whereas the latter mutation caused non-lethal Fowler syndrome. The activity of missense mutations P340L and T430A that were previously reported in a non-lethal Fowler patient was also tested. First, it was confirmed that these missense mutations did not affect the expression and localization of MFSD7c (Figure 5f-g). Second, the choline transport activity was measured and it was found that most of these missense mutations of MFSD7c exhibited significantly reduced or abolished choline transport activity (Figure 5h). A patient homozygous for S203Y was aborted at 20 weeks during gestation. Choline transport activity of this missense mutation was abolished (Figure 5h). The T430M mutation was associated with non-lethal Fowler syndrome, whereas homozygous T430R mutation was previously reported to be lethal. It was shown that T430M had a reduction of approximately 62% activity, whereas T430R is an inactive mutant (Figure 5h). Previously, compound heterozygous mutants (P340L and T430A) were found in a 2 year-old patient, who is still living. His brain MRI showed mild microcephaly. It was found that both alleles had partially decreased choline transport activity (Figure 5h). The current study also identified a patient with phenotypes associated with Fowler syndrome. Whole exome sequencing identified two missense mutations of MFSD7c coding sequence that lead to a substitution of proline at position 276 to serine (P276S) inherited from the mother and leucine at position 483 to arginine (L483R) from the father (Figure 5i-k). The patient exhibits severe microcephaly with neurological disorders (Figure 5k). Transport activity of these missense mutants showed that P276S retained approximately 50% choline transport activity, whereas L483R is likely unstable, leading to a severe reduction of activity in MFSD7c (Figure 5f, h). Expending these results, the missense mutations that have been reported in the literature were also tested. It was found that most of lethal mutations caused a significant reduction of choline transport activity (Figure 8). Nevertheless, there were several missense mutations which led to partial loss of choline transport activity, but the patients carrying these mutations exhibited severe clinical features. It is unclear whether other factors also contributed to the pathological conditions in these cases. Together, these results suggest that reduced choline transport activity of MFSD7c may be associated with the pathogenesis of Fowler syndrome. The discovery of the molecular function of MFSD7c as a choline transporter is an important step to facilitate with the understanding of the disease mechanisms in the patients with Fowler syndrome.

[0312] Inhibition of MFSD7c leads to increased cognitive functions in mouse ageing models.

[0313] The plasticity of hippocampal slices was evaluated in wild- type (Control) and EcMfsd7c conditional KO mice (cKO). The results suggest that the memory of cKO mice might be maintained during ageing (Figure 9).

[0314] Example 2; Circulating LPC, not GPC, is the major source of choline for the brain

[0315] To identify the major source of choline in the brain, the same amount of lysophosphatidylcholinc (LPC) and glyccrophosphatidylcholinc (GPC) were coinjected into wild-type (Control) and EcMfsd7c conditional knockout mice (cKO) (Figure lOd). It was found that, in cKO mice, inhibiting MFSD7c expression led to the accumulation of significantly higher levels LPC-derived choline compared to GPC- derived choline in the brain (Figure 10c). Choline-derived phospholipids (PLs) originating from LPC were also found to be more abundant in the brain and peripheral tissues than choline-derived PLs originating from GPC (Fig. 13). Differences were also observed in the level of choline-containing metabolites derived from LPC and GPC in peripheral tissue (Fig. 12), with LPC-derived metabolites more abundant in plasma, liver and muscle than GPC-derived metabolites. The results suggests that LPC is the preferred carrier of choline to the brain. Thus, supplements containing LPC may be a better vehicle to deliver choline to the brain and to boost acetylcholine levels in the brain compared to supplements containing GPC.

[0316] It will be appreciated that many further modifications and permutations of various aspects of the described embodiments are possible. Accordingly, the described aspects arc intended to embrace all such alterations, modifications, and variations that fall within the spirit and scope of the appended claims.

Claims

CLAIMS1. A method of promoting synthesis and / or accumulation of acetylcholine in a brain parenchymal cell or tissue in a subject, the method comprising administering an effective amount of an inhibitor of MFSD7c to the subject to promote synthesis and / or accumulation of acetylcholine in the brain parenchymal cell or tissue of the subject.

2. The method of claim 1 , wherein the inhibitor is an inhibitory nucleic acid molecule, a site-specific nuclease (SSN) system, a polypeptide or a small molecule.

3. The method of claim 2, wherein the inhibitory nucleic acid molecule is an antisense nucleic acid molecule, a small interfering RNA (siRNA), or a short hairpin RNA (shRNA) that hybridizes to a MFSD7c mRNA.

4. The method of claim 2, wherein the SSN system is a CRISPR / Cas system.

5. The method of any one of claims 1 to 4, wherein the inhibitor inhibits parenchymal choline import into a vascular cell in the central nervous system (CNS) and / or choline export into the blood stream from the vascular cell.

6. The method of any one of claims 1 to 5, wherein the vascular cell is an endothelial cell or a mural cell.

7. The method of any one of claims 1 to 6, wherein the subject is suffering from a CNS disease or condition associated with diminished cholinergic signalling.

8. The method of claim 7, wherein the CNS disease or condition is a neurological, cerebrovascular, or psychiatric disease or condition.

9. The method of claim 8, wherein the CNS disease or condition is Alzheimer’s disease, stroke, depression or schizophrenia.

10. The method of any one of claims 1 to 9, further comprising administering to the subject a lysophosphatidylcholine (LPC) composition comprising at least one LPCselected from the group consisting of LPC-16:0, LPC-16:1, LPC-18:0, LPC-18:1, LPC-18:2, LPC-18:3, LPC-20:4, LPC-20:5, and LPC-22:6.

11. The method of claim 10, wherein the LPC composition comprises LPC-20:4 and LPC-22:6.

12. The method of claim 10 or 11, wherein each LPC in the LPC composition is present at a concentration of about 50 pM to about 500 LI\L13. A method of inhibiting choline transport in a vascular cell in the central nervous system (CNS), the method comprising contacting the vascular cell with an effective amount of an inhibitor of MFSD7c for a sufficient time and under conditions to inhibit choline transport in the vascular cell.

14. The method of claim 13, wherein the vascular cell is present in a subject suffering from a CNS disease or condition associated with diminished cholinergic signalling.

15. A method of treating a CNS disease or condition in a subject, the method comprising administering an effective amount of an inhibitor of MFSD7c to the subject to treat the CNS disease or condition in the subject.

16. A method of preventing or delaying the progression of a CNS disease or condition in a subject, the method comprising administering an effective amount of an inhibitor of MFSD7c to the subject to prevent or delay progression of the CNS disease or condition in the subject.

17. The method of claim 15 or 16, wherein the CNS disease or condition is associated with diminished cholinergic signalling.

18. The method of any one of claims 15 to 17, further comprising administering to the subject a lysophosphatidylcholine (LPC) composition comprising at least one LPC selected from the group consisting of LPC-16:0, LPC-16:1 , LPC-18:0, LPC-18:1 , LPC-18:2, LPC-18:3, LPC-20:4, LPC-20:5, and LPC-22:6.

19. An inhibitor of MFSD7c, for use in treating or preventing a CNS disease or condition in a subject, wherein the inhibitor is to be administered to the subject.

20. Use of an inhibitor of MFSD7c in the manufacture of a medicament for treating or preventing a CNS disease or condition in a subject, wherein the MFSD7c inhibitor is to be administered to the subject.

21. The inhibitor for use of claim 19 or the use of claim 20, wherein the inhibitor is an inhibitory nucleic acid molecule, a site-specific nuclease (SSN) system, a polypeptide or a small molecule.

22. The inhibitor for use of claim 19 or 21 or the use of claim 20 or 21, wherein the CNS disease or condition is associated with diminished cholinergic signalling.

23. The inhibitor for use of any one of claims 19, 21 or 22 or the use of any one of claims 20 to 22, wherein the inhibitor is to be administered to the subject in combination with a lysophosphatidylcholinc (LPC) composition comprising at least one LPC selected from the group consisting of LPC-16:0, LPC-16:1, LPC-18:0, LPC-18:1, LPC-18:2, LPC-18:3, LPC-20:4, LPC-20:5, and LPC-22:6.

24. A mammalian cell comprising an exogenous nucleic acid encoding choline kinase A (CHKA) and MFSD7c.

25. A kit comprising a mammalian cell of claim 24, and an isotopically-labelled choline or an analogue thereof.

26. Use of a mammalian cell of claim 24 or a kit of claim 25 for screening for an inhibitor of MFSD7c.

27. A lysophosphatidylcholinc (LPC) composition comprising at least one LPC selected from the group consisting of LPC-16:0, LPC-16:1, LPC-18:0, LPC-18:

1. LPC-18:2, LPC-18:3, LPC-20:4, LPC-20:5, and LPC-22:6.

28. The LPC composition of claim 27, wherein the LPC composition comprises LPC- 20:4 and LPC-22:6.

29. The LPC composition of claim 27 or 28, wherein each LPC is present at a concentration of about 50 pM to about 500 pM.

30. The LPC composition of any one of claims 27 to 29, wherein the LPC composition is a nutritional composition.

31. Use of an LPC composition of any one of claims 27 to 30 for promoting synthesis and / or accumulation of acetylcholine in a brain parenchymal cell or tissue in a subject.

32. The use of claim 31, wherein the subject is suffering from a CNS disease or condition associated with diminished cholinergic signalling.

33. The use of claim 31 or 32, wherein the LPC composition is administered to the subject in combination with an inhibitor of MFSD7c.

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