Compositions and methods for delivery of peptides or proteins to the central nervous system

Abstract

The present disclosure provides lipid nanoparticles for treating, preventing, or diagnosing diseases, disorders, trauma, or injuries of the central nervous system. The lipid nanoparticles comprise a non-cationic helper lipid, a sterol, a hydrophilic polymer-lipid conjugate, an ionizable amino lipid with a pKa of 5.0 to 7.0, and an mRNA having a nucleic acid sequence encoding a secreted polypeptide for treating, preventing, or diagnosing diseases, disorders, trauma, or injuries of the central nervous system. The secreted polypeptide is capable of being secreted from cells of the central nervous system into the interstitial fluid and / or cerebrospinal fluid of a subject. Additionally, methods for administering lipid nanoparticles and uses of such lipid nanoparticles for treating, preventing, or diagnosing diseases, disorders, trauma, or injuries of the central nervous system are provided.

Description

[Technical Field]

[0001] (CROSS-REFERENCE TO RELATED APPLICATIONS) This application claims the benefit of U.S. Provisional Patent Application No. US 63 / 430,740, filed December 7, 2022.

[0002] The present disclosure relates to compositions and methods for treating, preventing, or diagnosing diseases or disorders of the central nervous system. In particular, the compositions include lipid nanoparticles for delivery of mRNA (LNP:LNP-mRNA). [Background technology]

[0003] Neurological disorders are often chronic and impose a significant burden on patients, their families, and the healthcare system. [4] Despite decades of extensive research into potential treatments, the clinical success rate of central nervous system (CNS)-targeted therapies is low compared to other organs. [6] Beyond the complexity of neurotransmission, it is widely believed that a major hurdle in brain therapy is delivering therapeutic agents to the target site. [1] Targeting the brain is particularly challenging, primarily due to the presence of the blood-brain barrier (BBB), which shields the brain parenchyma from the periphery and prevents the passage of most small molecules and almost all large molecules. [7, 11]

[0004] Although progress toward clinical application of research has been slow, progress is being made in understanding the molecular basis of many neurological disorders and diseases. For example, the pathophysiology of very common diseases, such as stroke, traumatic brain injury (TBI), neurodegenerative diseases, and glioblastoma, is now well understood, and several specific signaling pathways are known to hold therapeutic promise. A wealth of knowledge exists about how altered expression of specific proteins influences, and sometimes directly causes, disease progression and maintenance. In virtually every central nervous system disorder, the absence or downregulation of one or many endogenous proteins is known to contribute to the disease pathology. Therefore, protein replacement or supplementation represents an attractive therapeutic avenue [3]. However, pharmacological control of these disease mechanisms remains challenging.

[0005] Despite advances in recombinant protein synthesis, direct supplementation has several drawbacks, limiting its potential application in CNS therapy. The main challenges are the inability of proteins to cross the BBB and reach the brain parenchyma, as well as their short half-life, limiting the time course of therapeutic effects. The LNP strategy has been used to deliver small interfering RNAs (siRNAs) to neurons in vivo to inhibit the expression of SLC26A11, which was determined to be important and necessary for neuronal swelling [12, 13]. Similarly, mRNA encapsulated in SS-cleavable proton-activated lipid-like material (ssPalm) nanoparticles has been shown to deliver exogenous protein-encoding mRNA to neurons and astrocytes via intracerebroventricular (ICV) administration

[14] . Intrathecal injection of LNP-mRNA was found to target mostly the dorsal root ganglia, as measured by intracellular protein production. Similarly, delivery of an LNP-mRNA construct encoding human IL-10 to neurons, astrocytes, and microglia / macrophages at the site of spinal cord injury promoted neuroprotection and functional recovery in a spinal cord injury model [5, 10]. Thus, LNP-mRNA may offer therapeutic potential as an alternative to conventional approaches for treating, preventing, and diagnosing central nervous system diseases, disorders, trauma, or injuries. Summary of the Invention

[0006] The present disclosure seeks to address one or more problems in the art for treating, preventing, or diagnosing diseases, disorders, trauma, or injuries of the central nervous system.

[0007] The present invention is based, in part, on the serendipitous discovery that LNP-mRNA formulations can deliver mRNA to brain cells (e.g., glial cells) when injected into the brain via intracerebroventricular (ICV) or lumbar intrathecal injection. Furthermore, certain LNP-mRNA compositions, when injected into cerebrospinal fluid (CSF), preferentially target glial cells, causing them to express the peptide or protein (herein "polypeptide") encoded by the mRNA and subsequently secrete therapeutically effective amounts of the peptide or protein into the CSF. In some embodiments, the LNP-encapsulated mRNA causes glial cells in the brain to produce and secrete the desired peptide or protein into the extracellular environment of the brain parenchyma. Specifically, a therapeutically effective amount of the secreted peptide or protein acts extracellularly or is taken up by the target cell. Alternatively, such a protein or peptide may have diagnostic value in the CSF. In particular, the glial cells are oligodendrocytes or astrocytes. In this way, the desired peptide or protein encoded by the mRNA can achieve therapeutically effective amounts in the CSF when measured distal to the injection site.

[0008] The method presented here involves delivering genetic material (mRNA) to a subset of brain cells via direct injection (e.g., intracerebroventricular or intrathecal injection), instructing these cells to translate the mRNA into a desired protein, which is then secreted into the extracellular space. The desired protein then diffuses throughout the brain compartment and can be measured at significant concentrations in cerebrospinal fluid (CSF) collected distal to the initial injection site. Importantly, these cells continue to translate the mRNA into protein for an extended period related to the half-life of the mRNA, thereby achieving a sustained supply of protein to the brain extracellular environment and CSF.

[0009] According to one aspect of the present disclosure, there is provided a method for treating, preventing, or diagnosing a disease, disorder, trauma, or injury of the central nervous system, comprising contacting lipid nanoparticles (LNPs) with cells of the central nervous system of a subject. The lipid nanoparticles are (a) Ionizable aminocationic lipids with pKa between 5.0 and 7.0; (b) noncationic helper lipids; (c) sterols; (d) a hydrophilic polymer-lipid conjugate; and (e) mRNA encoding a secreted polypeptide for treating, preventing, or diagnosing a disease, disorder, trauma, or injury of the central nervous system, wherein the secreted polypeptide is capable of being secreted from cells of the central nervous system into the interstitial fluid and / or cerebrospinal fluid of a subject, and upon contact of the LNP with the cell, the mRNA enters the cell and is translated within the cell, thereby producing the polypeptide.

[0010] Following production of the polypeptide, the polypeptide is secreted from the cells into the interstitial fluid and / or cerebrospinal fluid (CSF) of the subject, and the secreted polypeptide is present in the CSF of the subject at a first concentration at a first time point and at a second concentration at a subsequent second time point. The first time point is 3 hours after contacting the LNP with the cells, and the second time point is 48 hours after contacting the LNP with the cells. The second concentration is at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of the first concentration. The contacting step can be performed in vitro or in vivo.

[0011] In one embodiment, the cells of the central nervous system are selected from one or more of: (a) glial cells and (b) ependymal cells.

[0012] In another embodiment, the glial cells are selected from astrocytes or oligodendrocytes and the ependymal cells are selected from the ventricles or choroid plexus.

[0013] According to further embodiments, the secreted polypeptide is endogenous to cells of the central nervous system or is modified to enhance activity in the brain.

[0014] According to another embodiment, the secreted polypeptide is diffusible within the interstitial fluid and / or cerebrospinal fluid of the subject.

[0015] According to another embodiment, the non-cationic helper lipid is a generally cylindrical lipid.

[0016] In another embodiment, the non-cationic helper lipid is distearoylphosphatidylcholine (DSPC) or dioleoylphosphatidylglycerol (DOPG).

[0017] In another embodiment, the non-cationic helper lipid has a phosphatidylethanolamine content of less than 2 mol %.

[0018] According to a further embodiment, the sterol is cholesterol.

[0019] In a further embodiment, the polymer lipid conjugate is PEG-DMG.

[0020] In another embodiment, the molar ratio of ionizable amino cationic lipid / non-cationic helper lipid / sterol / polymer lipid conjugate is 50 / 10 / 38-39 / 1-2.

[0021] According to further embodiments, the method comprises injection into the subject's central nervous system via an intracerebroventricular (ICV) or lumbar intrathecal route, a cisternal route, or via a catheter.

[0022] According to a further aspect, there is provided a use of a pharmaceutical formulation comprising lipid nanoparticles (LNPs) for contacting cells of the central nervous system of a subject to treat, prevent, or diagnose a disease, disorder, trauma, or injury of the central nervous system. (a) Ionizable aminocationic lipids with pKa between 5.0 and 7.0; (b) noncationic helper lipids; (c) sterols; (d) a hydrophilic polymer-lipid conjugate; and (e) mRNA encoding a secreted polypeptide for treating, preventing, or diagnosing a disease, disorder, trauma, or injury of the central nervous system, wherein the secreted polypeptide is capable of being secreted from cells of the central nervous system into the interstitial fluid and / or cerebrospinal fluid of a subject after administration of the lipid nanoparticles.

[0023] Upon contact of the LNP with a cell, the mRNA enters the cell and is translated within the cell to produce a polypeptide.

[0024] After production of the polypeptide, the polypeptide is secreted from the cells into the interstitial fluid and / or cerebrospinal fluid (CSF) of the subject.

[0025] The secreted polypeptide is present in the subject's CSF at a first concentration at a first time point, and at a second concentration at a subsequent second time point.

[0026] The first time point is 3 hours after contacting the LNPs with the cells, and the second time point is 48 hours after contacting the LNPs with the cells.

[0027] The second concentration is at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of the first concentration.

[0028] The contacting can be effected in vitro or in vivo.

[0029] According to another aspect, there is provided a use of lipid nanoparticles for contacting cells of the central nervous system of a subject for the manufacture of a medicament for treating, preventing or diagnosing a disease, disorder, trauma or injury of the central nervous system. (a) Ionizable aminocationic lipids with pKa between 5.0 and 7.0; (b) noncationic helper lipids; (c) sterols; (d) a hydrophilic polymer-lipid conjugate; and (e) The lipid nanoparticles comprise mRNA having a nucleic acid sequence encoding a secretory polypeptide for treating, preventing, or diagnosing a disease, disorder, trauma, or injury of the central nervous system. The secretory polypeptide can be secreted from cells of the central nervous system into the interstitial fluid and / or cerebrospinal fluid of a subject after administration of the lipid nanoparticles. Upon contact of the LNPs with the cells, the mRNA enters the cells and is translated within the cells, thereby producing the polypeptide. Following production of the polypeptide, the polypeptide is secreted from the cells into the interstitial fluid and / or cerebrospinal fluid (CSF) of the subject, and the secreted polypeptide is present in the CSF of the subject at a first concentration at a first time point and at a second concentration thereafter at a second time point. The first time point is 3 hours after contacting the LNPs with the cells, and the second time point is 48 hours after contacting the LNPs with the cells. The second concentration is at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of the first concentration, and the contacting is performed in vitro or in vivo.

[0030] In another embodiment, the cell of the central nervous system is a glial cell selected from an astrocyte or an oligodendrocyte, or an ependymal cell of the ventricle or choroid plexus.

[0031] In a further embodiment of the aforementioned use, the non-cationic lipid is a generally cylindrical lipid.

[0032] In another embodiment of the aforementioned use, the non-cationic lipid is distearoylphosphatidylcholine (DSPC) or dioleoylphosphatidylglycerol (DOPG).

[0033] In another embodiment of the aforementioned use, the content of phosphatidylethanolamine is less than 2 mol %.

[0034] In another embodiment of the aforementioned use, the sterol is cholesterol.

[0035] In a further embodiment of the aforementioned use, the polymer lipid conjugate is PEG-DMG.

[0036] In another embodiment of the aforementioned use, the molar ratio of ionizable amino cationic lipid / non-cationic helper lipid / sterol / polymer lipid conjugate is 50 / 10 / 38-39 / 1-2.

[0037] In another embodiment of the aforementioned uses, the therapeutic polypeptide or peptide is endogenous to cells of the central nervous system or is modified to enhance activity in the brain.

[0038] In another embodiment of the aforementioned use, the therapeutic polypeptide or peptide is diffusible in the interstitial fluid and / or cerebrospinal fluid.

[0039] According to another aspect of the present disclosure, there is provided a lipid nanoparticle for contacting cells of the central nervous system of a subject to treat, prevent, or diagnose a disease, disorder, trauma, or injury of the central nervous system, the lipid nanoparticle comprising: (a) Ionizable aminocationic lipids with pKa between 5.0 and 7.0; (b) noncationic helper lipids; (c) sterols; (d) a hydrophilic polymer-lipid conjugate; and (e) mRNAs having a nucleic acid sequence encoding a secretable polypeptide for treating, preventing, or diagnosing a disease, disorder, trauma, or injury of the central nervous system, wherein the secretable polypeptide is capable of being secreted from cells of the central nervous system into the interstitial fluid and / or cerebrospinal fluid of a subject.

[0040] Upon contact of the LNP with a cell, the mRNA enters the cell and is translated within the cell to produce a polypeptide.

[0041] After production of the polypeptide, the polypeptide is secreted from the cells into the interstitial fluid and / or cerebrospinal fluid (CSF) of the subject.

[0042] The secreted polypeptide is present in the subject's CSF at a first concentration at a first time point and at a second concentration at a later second time point.

[0043] The first time point is 3 hours after contacting the LNPs with the cells, and the second time point is 48 hours after contacting the LNPs with the cells.

[0044] The second concentration is at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of the first concentration.

[0045] The contacting can be in vitro or in vivo.

[0046] According to another aspect of the present disclosure, there is provided a method for treating, preventing or diagnosing diseases, disorders, trauma or injury of the central nervous system.The method comprises administering lipid nanoparticles to the central nervous system of a subject.The lipid nanoparticles comprise a neutral lipid, a sterol, a hydrophilic polymer-lipid conjugate, an ionizable amino lipid with a pKa of less than 7.0; and an mRNA encoding a secretory protein for treating, preventing or diagnosing diseases, disorders, trauma or injury of the central nervous system.The secretory protein can be secreted from cells of the central nervous system into the cerebrospinal fluid of the subject.

[0047] In one embodiment of the foregoing aspect, the cell of the central nervous system is a glial cell selected from an astrocyte or an oligodendrocyte, or an ependymal cell of the ventricle or choroid plexus.

[0048] In another embodiment of the foregoing aspect, the lipid nanoparticles are part of a pharmaceutical formulation.

[0049] In another embodiment of the foregoing aspect, the secreted polypeptide is endogenous to cells of the central nervous system or is modified to enhance activity in the brain.

[0050] In further embodiments of the foregoing aspects, the secreted polypeptide is diffusible within the interstitial fluid and / or cerebrospinal fluid of the subject.

[0051] In further embodiments of the foregoing aspects, the non-cationic helper lipid is a generally cylindrical lipid.

[0052] According to further embodiments of the aforementioned aspect, the non-cationic lipid is distearoylphosphatidylcholine (DSPC) or dioleoylphosphatidylglycerol (DOPG).

[0053] According to further embodiments of the aforementioned aspect, the non-cationic helper lipid has a phosphatidylethanolamine content of less than 2 mol %.

[0054] According to further embodiments of the aforementioned aspect, the sterol is cholesterol.

[0055] According to a further embodiment of the foregoing aspect, the polymer lipid conjugate is PEG-DMG.

[0056] According to a further embodiment of the aforementioned aspect, the molar ratio of ionizable amino cationic lipid / non-cationic helper lipid / sterol / polymer lipid conjugate is 50 / 10 / 38-39 / 1-2.

[0057] Alternatively, the ionizable amino cationic lipid having a pKa of 5.0 to 7.0 may be nor-MC3 and / or compound 22.

[0058] In further examples of any of the foregoing aspects or embodiments of the present disclosure, administration may comprise injection into the subject's central nervous system intracerebroventricularly (ICV), via a lumbar intrathecal route, via a cisternal route, or via a catheter.

[0059] In a further example of any of the foregoing aspects or embodiments of the present disclosure, the lipid nanoparticles may be part of a pharmaceutical formulation that is injectable into a subject's central nervous system via an intracerebroventricular (ICV) route, a lumbar intrathecal route, a cisternal route, or via a catheter. [Brief explanation of the drawings]

[0060] [Figure 1] FIG. 1 is a diagram illustrating a proposed mechanism for delivering secreted proteins to the central nervous system using LNP-mRNA as described herein. [Figure 2] Figure 2 is a schematic diagram of intracerebroventricular (ICV) injection of LNP-mRNA encoding the fluorescent reporter protein mCherry® into the mouse brain (injected into both sides of the mouse brain (bottom right)). The image on the left shows mCherry® expression in a brain section of the corpus callosum white matter tract 2 days after injection. mCherry® was used as a reporter to visualize cells that translate mRNA cargo into mCherry® protein, but are not secreting mCherry® (arrow indicates the orientation of the slice taken from posterior to anterior of the corpus callosum). Cells instructed to express mCherry® are widely distributed throughout the brain, but the most prominent mCherry® protein is found in the corpus callosum. [Figure 3A] Figure 3A shows brain images showing uptake of LNP-mRNA in oligodendrocytes (olig2+) after ICV injection. Uptake of LNP-mRNA encoding mCherry® induced mCherry® expression, visualized in immunostained oligodendrocytes in mouse white matter tracts. The dashed box in the top image (100 μm scale) represents the magnified area in the corresponding bottom image (20 μm scale). [Figure 3B]Figure 3B shows brain images of LNP-mRNA uptake in astrocytes (GFAP+) after ICV injection. Uptake of LNP-mRNA encoding mCherry® induced mCherry® expression, visualized in immunostained astrocytes in the white matter tracts of mice. The dashed box in the top image (100 μm scale) represents the magnified area in the corresponding bottom image (20 μm scale). [Figure 4] Figure 4 shows brain images of mice after intracerebroventricular (ICV) injection of LNP-mRNA encoding mCherry®. Images show immunostaining of mCherry®-expressing cells in the choroid plexus and ventricular wall of the mice at various magnifications. [Figure 5] Figure 5 shows spinal cord images of LNP-mRNA uptake. Mice were injected intrathecally with LNPs encapsulating mRNA encoding mCherry®. Animals were perfused, and fixed tissue was harvested 2 days after LNP-mRNA injection. The images show immunostaining of spinal cord cells expressing mCherry®. [Figure 6] Figure 6 is a schematic diagram of the protocol used to demonstrate that LNP-mRNA can induce the production and secretion of desired proteins into the CSF. The concentration of FGF21 protein in cerebrospinal fluid (CSF) was measured after bilateral ICV injection of mice with two different formulations of LNP-mRNA encoding FGF-21 (i.e., LNP(1)-FGF-21 (LNP-G FGF-21) and LNP(2)-FGF-21 (LNP-H FGF-21)) (graph on the right side of the figure). The concentration was compared to a CSF control and an LNP-mRNA EPO-negative control (i.e., EPO-encoding mRNA does not result in an increase in FGF-21). This demonstrates the specificity of the assay, but importantly, the protein production and secretion are specific to the mRNA-encoded protein. After LNP-mRNA FGF-21 injection, CSF was collected and FGF-21 was measured using the U-PLEX FGF21 assay. [Figure 7]Figure 7 shows the change in CSF FGF-21 concentration at 3 and 48 hours after injection of varying doses of LNP-mRNA encoding FGF-21 (LNP(2)-FGF-21) injected intravenously. CSF FGF-21 protein was measured using a quantitative U-PLEX assay. The dashed line corresponds to the CSF FGF-21 concentration at 3 hours after injection for a 1 mg / mL mRNA dose. [Figure 8] Figure 8 shows the concentration of EPO protein in the CSF after ICV injection of mice with LNP-mRNA (LNP-EPO) encoding the secreted factor EPO (graph on the right side of the figure). After injection, CSF was collected and EPO was measured using the U-PLEX EPO assay described herein (shown on the left side of the figure). Control CSF is a negative control from uninjected animals, and LNP-FGF-21 is a negative injection control demonstrating that LNP-mRNA FGF-21 production does not alter EPO concentrations in the CSF. [Figure 9] Figure 9 shows the change in CSF EPO concentration at 3 and 48 hours after ICV injection of varying doses of LNP-mRNA encoding EPO (LNP-EPO). CSF EPO protein was measured using a quantitative U-PLEX assay. The dashed line corresponds to CSF ​​[EPO] 3 hours after injection of 1 mg / mL of mRNA. [Figure 10] Figure 10 shows coronal and sagittal views of a mouse brain with intracerebroventricular (ICV) injection sites (grey triangle "ICV injection" - coronal and sagittal views), lateral ventricle (LV) injection sites (black), cerebrospinal fluid (CSF) collection sites (grey triangle "CSF collection" - sagittal view only), and the approximate distance between injection and collection sites (i.e., 8 mm - sagittal view only). Detailed Description

[0061] The following detailed description will be better understood when read in conjunction with the accompanying drawings. For the purpose of illustrating the invention, the drawings show embodiments of the invention. However, the invention is not limited to the precise arrangements, embodiments, and apparatus shown.

[0062] The present disclosure provides compositions for treating, preventing, or diagnosing central nervous system conditions, diseases, disorders, trauma, or injuries using lipid nanoparticles containing mRNA encoding a secretable protein, also referred to herein as "LNP-mRNA." By delivering LNP-mRNA to glial cells, the glial cells can be converted into "CNS protein bioreactors" that secrete the protein or peptide encoded by the mRNA. Without being bound by theory, an example of the delivery method of the present invention is shown in FIG. 1. Referring to the example shown in FIG. 1, lipid nanoparticles containing mRNA encoding a secretable therapeutic protein are injected into the CNS of a subject (i.e., intracerebroventricular (ICV) injection; lumbar intrathecal injection (IT); or cisterna magna injection). While a mouse is shown as an example in the figure, the subject includes all mammals, including humans. The LNP-mRNA is taken up by brain cells (e.g., glial cells) by endocytosis, and the mRNA is translated into protein in the cell's cytoplasm. The secreted protein is then secreted from the cells into the interstitial fluid and / or cerebrospinal fluid (CSF), allowing the protein to diffuse to target sites and exert a therapeutic, diagnostic, or prophylactic effect in one or more regions of the central nervous system. In another embodiment, the protein is a diagnostic agent. As illustrated, in some advantageous embodiments of the present disclosure, proteins secreted by glial cells are diffusible, thereby providing brain-wide delivery within a subject. A variety of secreted proteins, peptides, combinations of two or more proteins, combinations of two or more peptides, or combinations of proteins and peptides can be delivered to the central nervous system. The mRNA can encode proteins or peptides, including, but not limited to, antibodies, growth factors, and other therapeutic, diagnostic, or prophylactic proteins described herein.

[0063] In some embodiments, the lipid nanoparticles comprise four lipid components, as described herein. In some embodiments, this comprises an ionizable lipid, a helper lipid, a hydrophilic polymer-lipid conjugate (e.g., PEG-lipid), and cholesterol or other sterol, as described herein. Such LNP compositions have been shown herein to be particularly effective in delivering mRNA to the cytoplasm of cells in the central nervous system, where it is translated into a secretory protein and ultimately secreted into interstitial fluid and / or cerebrospinal fluid.

[0064] Previous studies have demonstrated that LNP-mRNA uptake in CNS neurons is possible via direct ICV or IT injection. These studies used LNP-mRNA to produce intracellular proteins, either as a reporter marker to confirm LNP uptake

[14] or to express biologically active proteins [6, 14]. Previous studies have relied on direct LNP uptake in diseased / dysregulated cell types to treat neurological disorders by altering the local microenvironment via mRNA-produced proteins. Instead, in this disclosure, we use a consistent subset of neurons, namely glial cells, as a CNS protein bioreactor, specifically to produce and secrete proteins for biodistribution throughout the brain and at therapeutically sufficient concentrations.

[0065] secreted proteins Proteins or peptides encoded by the mRNA of lipid nanoparticles are collectively referred to herein as "secreted proteins," meaning that the proteins can cross central nervous system cell membranes and exert extracellular therapeutic or prophylactic effects within one or more regions of the central nervous system. Secreted proteins may contain a secretory signal that facilitates their secretion. Proteins may already possess a secretory signal sequence (e.g., growth factors and cytokines) or may be genetically modified to contain a secretory signal sequence. Without limitation, secretory signals are located at the amino terminus of secreted proteins and trigger translocation of newly synthesized proteins to the plasma membrane via the endoplasmic reticulum, the Golgi apparatus, and the plasma membrane for secretion into interstitial fluid and / or cerebrospinal fluid. Alternatively, in one example of the present disclosure, secreted proteins are secreted via a "non-classical" pathway that does not rely on the inclusion of a secretory signal. Furthermore, secreted proteins may also be naturally released by central nervous system cells, such as astrocytes, oligodendrocytes, and ependymal cells of the ventricles and choroid plexus, under certain environmental conditions.

[0066] The ability of a protein or peptide to be secreted from central nervous system cells can be assessed in vitro by the method described in Example 3 herein. Briefly, in this assay, brain glial cells (astrocytes and oligodendrocytes) are cultured and the cell culture is treated with LNP-mRNA encoding a candidate protein. Protein secretion is assayed by collecting the cell supernatant and quantifying the protein amount, for example, by immunoblotting, ELISA, or U-PLEX assay. Supernatants are collected at various time points to estimate the peak time of production / secretion. Typically, in vitro assays are followed by in vivo tests using rodents to confirm protein secretion. Such in vivo tests are described in the Examples section herein.

[0067] For proteins for which intracellular localization after secretion is desired, a sequence encoding a cell-penetrating peptide (CPP), such as the TAT peptide, can be included. Thus, the engineered mRNA sequence can contain both a signal sequence and a CPP sequence to direct secretion and promote intracellular uptake of the protein by target cells, respectively.

[0068] Secreted proteins can treat or prevent diseases, disorders, trauma, or injuries of the central nervous system. Typically, the proteins are secreted from cells of the central nervous system into the cerebrospinal fluid of a subject and diffuse to the target site.

[0069] LNP-mRNA encoding secreted proteins can be used for purposes other than the treatment and / or prevention of disease, disorders, trauma, or injury. LNP-mRNA can be used to treat symptoms of aging, preventative medicine, and / or as part of a personalized medicine regimen. In a further embodiment, LNPs can be used for diagnostic purposes.

[0070] Non-limiting examples of diseases, disorders, trauma or injuries and secreted proteins that can be used to treat them are shown in Table 1 below. JPEG2025540306000001.jpg193159

[0071] It should be understood that two or more secreted proteins can be used in combination to treat or prevent a disease, disorder, trauma, or injury of the central nervous system. Alternatively, or additionally, one or more mutations can be introduced into a secreted protein to increase its half-life in the central nervous system. Furthermore, a particular secreted protein can be used to treat or prevent one or more disease indications.

[0072] The proteins may also be diagnostic agents for evaluating patients for diseases, disorders, trauma, or injuries of the central nervous system, hi another embodiment, the proteins may be used prophylactically to prevent diseases, disorders, trauma, or injuries of the central nervous system.

[0073] Furthermore, the secreted protein may be a protein fragment (i.e., a peptide), a protein domain, or a peptide sequence. The protein may also be post-translationally modified.

[0074] mRNA As used herein, the term "messenger RNA" or "mRNA" refers to a polynucleotide that encodes and expresses secreted proteins.

[0075] As used herein, the term "encapsulation" refers to the incorporation of mRNA within a lipid nanoparticle and refers to any association of the mRNA with a lipid component or compartment of the lipid nanoparticle. In one example of the present disclosure, the mRNA is present in the core of the LNP.

[0076] As used herein, mRNA encompasses both modified and unmodified mRNA. In one embodiment, mRNA comprises one or more coding and non-coding regions. mRNA can be purified from natural sources, produced using recombinant expression systems and optionally purified, or chemically synthesized. Modifications to mRNA can improve the immunogenicity, stability, translation efficiency and fidelity of the mRNA, and increase the amount of protein produced from the mRNA.

[0077] In those embodiments in which the mRNA is chemically synthesized, the mRNA can include nucleoside analogs, such as analogs with chemically modified bases or sugars, and / or backbone modifications. In some embodiments, the mRNA can include natural nucleosides (e.g., adenosine, guanosine, cytidine, uridine); nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolopyrimidine, 3-methyladenosine, 5-methylcytidine, C-5-propynyl-cytidine, C-5-propynyl-uridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodour ... The base may be or contain: C5-propynyluridine, C5-propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanine, 2-thiocytidine, pseudouridine, 5-methylcytidine; chemically modified bases; biologically modified bases (e.g., methylated bases); intercalated bases; modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose); and / or modified phosphate groups (e.g., phosphorothioate and 5'-N-phosphoramidite linkages).

[0078] The mRNA of the present disclosure can be synthesized according to any of various known methods.For example, in certain embodiments, the mRNA can be synthesized through in vitro transcription (IVT).Briefly, IVT is typically carried out using a linear or circular DNA template, which contains a promoter, a pool of ribonucleotide triphosphates, a buffer system that may contain DTT and magnesium ions, and a suitable RNA polymerase (for example, T3, T7 or SP6 RNA polymerase), DNAse I, pyrophosphatase, and / or RNAse inhibitor.

[0079] In some embodiments, in vitro synthesized mRNA may be purified prior to encapsulation into LNPs to remove unwanted impurities, including various enzymes and other reagents used during mRNA synthesis.

[0080] The present disclosure can be used to formulate and encapsulate mRNAs of various lengths. In some embodiments, the present disclosure can be used to formulate and encapsulate in vitro synthesized mRNAs of lengths of about 0.1 to 20 kb, about 1 to 20 kb, about 1 to 15 kb, about 1 to 10 kb, about 5 to 20 kb, about 5 to 15 kb, about 5 to 12 kb, about 5 to 10 kb, about 8 to 20 kb, or about 8 to 15 kb. Alternatively, peptides or proteins produced from mRNAs can be between about 500 Da and about 200 kDa.

[0081] Typically, mRNA synthesis involves the addition of a "cap" at the 5' end and a "tail" at the 3' end. The presence of the cap is important for conferring resistance to nucleases found in most eukaryotic cells, while the presence of the tail serves to protect the mRNA from degradation by exonucleases.

[0082] In some embodiments, the mRNA comprises a 5' and / or 3' untranslated region. In some embodiments, the 5' untranslated region comprises one or more elements that affect mRNA stability or translation, such as an iron-responsive element. In some embodiments, the 5' untranslated region may be between about 50 and 500 nucleotides in length.

[0083] In some embodiments, the 3' untranslated region comprises one or more of a polyadenylation signal, a binding site for a protein that affects the stability of the mRNA's location within a cell, or one or more binding sites for an miRNA. In some embodiments, the 3' untranslated region may be 50 to 500 nucleotides or more in length.

[0084] While mRNA provided from an in vitro transcription reaction may be desirable in certain embodiments, other mRNA sources are contemplated, such as mRNA produced from bacteria, fungi, plants, and / or animals.

[0085] Lipid nanoparticle formulations The lipid nanoparticles (LNPs) described herein induce the uptake of messenger RNA into the cytoplasm of cells in the central nervous system. Such uptake is facilitated by the inclusion of ionizable lipids. Additionally, the LNPs contain a non-cationic helper lipid component, a sterol, and a hydrophilic polymer-lipid conjugate.

[0086] Ionizable lipids The ionizable lipid described herein may be an ionizable cationic amino lipid. The ionizable cationic amino lipid may have a pKa such that it is positively charged at low pH and near-neutral at physiological pH. This allows electrostatic interactions between the lipid and negatively charged mRNA during initial formulation. Because the ionizable cationic amino lipid is near-neutral at physiological pH, toxicity is reduced. Without being limited by theory, after endocytosis, the acidic environment of the endosome increases the positive charge of the ionizable cationic amino lipid, promoting fusion with the anionic lipid in the endosomal membrane, followed by membrane destabilization and release of the mRNA into the cytoplasm of CNS cells for translation into secreted proteins. As previously described, the translated protein is then transported to the plasma membrane via the endoplasmic reticulum and Golgi apparatus network and secreted into the interstitial fluid and / or cerebrospinal fluid. Alternatively, the ionizable cationic amino lipid may be an ionizable amino cationic lipid as described in WO2022 / 246571 [2]. Alternatively, the ionizable cationic amino lipid may have a pKa of less than 7.0.

[0087] The neutral form of the ionizable amino cationic lipid has a calculated logarithm of the partition coefficient between water and 1-octanol (i.e., cLogP) greater than 8. In some embodiments, the ionizable cationic amino lipid has a pKa of 5.0 to 7.0, more typically 6.0 to 6.8.

[0088] Non-cationic helper lipids As used herein, "non-cationic helper lipid" refers to a neutral or anionic structural lipid that can be incorporated into lipid nanoparticles.

[0089] As used herein, an "anionic lipid" refers to a lipid that is negatively charged at physiological pH. Non-limiting examples include phosphatidylglycerol lipids such as 1,2-dioleoyl-sn-glycero-3-phosphoglycerol (DOPG).

[0090] As used herein, a "neutral lipid" is a structured lipid that is neutral (including net neutral) at physiological pH and typically comprises a lipid selected from sphingomyelin, phosphatidylcholine lipids, or mixtures thereof. The term "neutral lipid" includes zwitterionic lipids.

[0091] In some embodiments, the neutral lipid is selected from sphingomyelin, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC), and dipalmitoyl-phosphatidylcholine (DPPC). In certain embodiments, the neutral lipid is DOPC, DSPC, or sphingomyelin. In one embodiment, the neutral lipid is DOPC. The neutral lipid component may comprise a mixture of two or more different neutral lipids. Alternatively, the "neutral lipid" may be a cylindrical lipid. The "neutral lipid" may be distearoylphosphatidylcholine (DSPC). The "neutral lipid" may have a phosphatidylethanolamine content of less than 2 mol%. The "neutral lipid" may be a sphingomyelin, a phosphatidylcholine lipid, or a mixture thereof. The "neutral lipid" may be selected from sphingomyelin, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC) and dipalmitoyl-phosphatidylcholine (DPPC); or a mixture of two or more different neutral lipids.

[0092] sterols LNPs, in some embodiments, further comprise a sterol. The term "sterol" refers to a naturally occurring or synthetic compound having a gonane skeleton and a hydroxyl moiety attached to one of its rings, typically the A ring.

[0093] Examples of sterols include cholesterol, or cholesterol derivatives, the latter of which refers to a cholesterol molecule with a gonane structure and one or more additional functional groups.

[0094] Cholesterol derivatives include β-sitosterol, 3-sitosterol, campesterol, stigmasterol, fucosterol, or stigmastanol, dihydrocholesterol, ent-cholesterol, epicholesterol, desmosterol, cholestanol, cholestanone, cholestenone, cholesteryl-2′-hydroxyethyl ether, cholesteryl-4′-hydroxybutyl ether, 3β[N-(N′N′-dimethylaminoethyl)carbamoylcholesterol (DC-Chol)], 24(S)-hydroxycholesterol, 25-hydroxycholesterol, 25(R)-27-hydroxycholesterol, 22-oxacholesterol, 23-oxacholesterol, 24-oxacholesterol, cycloartenol, 22-ketosterol, 20-hydroxysterol, 7-hydroxycholesterol, The hydroxycholesterol may be selected from one or more of 19-hydroxycholesterol, 22-hydroxycholesterol, 25-hydroxycholesterol, 7-dehydrocholesterol, 5α-cholest-7-en-3β-ol, 3,6,9-trioxaoctan-1-ol-cholesteryl-3e-ol, dehydroergosterol, dehydroepiandrosterone, lanosterol, dihydrolanosterol, lanostenol, lumisterol, cytocalciferol, calcipotriol, coprostanol, cholecalciferol, lupeol, ergocalciferol, 22-dihydroegocalciferol, ergosterol, brassicasterol, tomatidine, tomatine, ursolic acid, cholic acid, chenodeoxycholic acid, zymosterol, diosgenin, fucosterol, fecosterol, or salts or esters thereof.

[0095] Hydrophilic polymer lipid conjugates In one embodiment, the lipid nanoparticle comprises a hydrophilic polymer-lipid conjugate that can be incorporated into the LNP. The conjugate comprises a lipid covalently attached (optionally via a linker group) to a hydrophilic polymer chain. Examples of hydrophilic polymers include polyethylene glycol (PEG), polyvinylpyrrolidone, polyvinylmethylether, polyhydroxypropylmethacrylate, polyhydroxypropylmethacrylamide, polyhydroxyethylacrylate, polymethacrylamide, polydimethylacrylamide, polymethyloxazoline, polyethyloxazoline, polyhydroxyethyloxazoline, polyhydroxypropyloxazoline, polysarcosine, and polyaspartamide. In one embodiment, the hydrophilic polymer-lipid conjugate is a PEG-lipid conjugate. The hydrophilic polymer-lipid conjugate may also be a naturally occurring or synthetic oligosaccharide-containing molecule, such as monosialoganglioside (GM1). The ability of a given hydrophilic polymer-lipid conjugate to improve the circulation life of the LNP herein can be easily determined by those skilled in the art using known methodologies. The PEG-lipid conjugate may be 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol (DMG-PEG).

[0096] Administration route In some embodiments, the lipid nanoparticles containing mRNA are part of a pharmaceutical composition. The pharmaceutical composition may provide prophylactic (preventive), ameliorative, or therapeutic effects. The pharmaceutical composition is administered in any suitable dosage. The types of administration used to introduce the LNP-mRNA include any route that delivers the LNP-mRNA to a central nervous system (CNS) site where the extracellular space contains interstitial fluid and / or cerebrospinal fluid. This site includes the brain and / or spinal cord. Administration methods include, but are not limited to, intracerebroventricular (ICV) injection, lumbar intrathecal injection, cisternal injection, and the use of a catheter (e.g., an intrathecal catheter) to introduce the LNP-mRNA into the brain or spinal cord (see Figure 10).

[0097] The LNP-mRNA compositions described herein can be administered to a subject. As used herein, a "subject" can be a human, non-human primate, rat, mouse, cow, horse, pig, sheep, goat, dog, cat, etc. The subject may be suspected of having or at risk of having a central nervous system disease, disorder, trauma, or injury, as known to those skilled in the art. Some examples are shown in Table 1.

[0098] Methods and Materials ICV microinjection All experimental protocols were approved by the University of British Columbia Committee on Animal Care and conducted in accordance with the guidelines of the Canadian Council of Animal Care. Adult C57Bl6 mice (P40-P60) were anesthetized with 1-2% isoflurane and immobilized in a stereotaxic frame. A small hole (1 mm diameter) was drilled in the skull to allow access to the brain (-0.5 mm anterior-posterior (AP), ±1.0 mm medial-lateral (ML), -1.8 mm dorsal-ventral (DV) from bregma - see Figure 10). A glass micropipette (40 μm tip diameter) was connected to a Hamilton® syringe, and LNP-mRNA was injected at a rate of 200 nl / min using an infusion pump (Harvard Apparatus®, Holliston, MA). The total volume of LNP-mRNA injected was 1 μL (0.05–1 mg mRNA / ml in sterile PBS). The LNP-mRNA formulation was injected sequentially into both lateral ventricles. After withdrawing the needle, the skin on the skull was sutured, and the mice were housed singly. For mCherry® imaging experiments, the mice were housed for 2 days, then transcardially perfused with PBS and 4% paraformaldehyde, and the brains were harvested. For FGF-21 measurement in cerebrospinal fluid (CSF), the mice were housed for 3 or 48 hours after injection, after which CSF was collected by cisternal puncture (see Figure 10).

[0099] IT injection All experimental protocols were approved by the University of British Columbia Animal Care Committee and conducted in accordance with the guidelines of the Canadian Committee on Animal Care. Adult C57Bl6 mice (P40-P60) were anesthetized with 1-2% isoflurane and the hair at the lower spine was removed with an electric shaver. Mice were continuously administered 1-2% isoflurane via a nose cone with a 15 mL conical tube placed under their abdomen. LNP-mRNA (5 μL) was injected between the L5 and L6 lobes using a Hamilton syringe (10 μL).

[0100] LNP formulation LNPs were prepared by dissolving mRNA in 25 mM sodium acetate (pH 4.0) and dissolving a given mole percent of lipid components in absolute ethanol. Lipids in ethanol and mRNA in buffer were mixed at a volume ratio of 1:3 using a T-junction with a dual syringe pump. These solutions were pumped through the T-junction at a total flow rate of 20 mL / min (5 mL / min for the lipid-containing syringe and 15 mL / min for the mRNA-containing syringe). The mixture was then dialyzed overnight against at least 100 volumes of 1x phosphate-buffered saline (pH 7.4) using a Spectro / Por dialysis membrane (molecular weight cutoff 12,000-14,000 Da). If necessary, LNPs were concentrated using Amicon Ultra® 10,000 MWCO (molecular weight cutoff) regenerated cellulose concentrators.

[0101] Lipid nanoparticles with different compositions, as listed in Table 2 below, were assayed. LNPs consist of four lipid components: (1) an ionizable lipid (e.g., nor-MC3 and Compound 22); (2) a noncationic helper lipid (e.g., DSPC or DOPG); (3) a sterol (e.g., cholesterol); and (4) a polymer-lipid conjugate (e.g., PEG-DMG), as shown in Table 2 below. LNPs were prepared by loading them with mRNA encoding the fluorescent reporter protein mCherry® or fibroblast growth factor 21 (FGF21) NM_020013.4 or erythropoietin (EPO) NM_000799. The fluorescent lipid dye DiO was included in some LNP formulations to enable tracking of LNP intracerebral diffusion independently of reporter protein expression. In some experiments (LNPs: A, B, C, D), the lipophilic dye DiO (DiOC), a green fluorescent lipophilic carbocyanine dye, was incorporated into the LNPs. 18 (3)) was used. The charge ratio of nitrogen to phosphate was 6. JPEG2025540306000002.jpg82159

[0102] The ionizable amino cationic lipid nMC3 described in Table 2 above is the lipid designated nMC3 (nor-MC3) described in WO2022 / 246571[2] on page 8. The ionizable amino cationic lipid is compound 22 of PCT / CA2023 / 051274, entitled "Amino Acid-Containing Ionizable Lipids for Therapeutic Agent Delivery," with an international filing date of September 27, 2023.

[0103] Imaging Brains were sliced ​​into 300 μm-thick sections before immunostaining and imaging under a confocal microscope (Zeiss®). Individual cell types were labeled with common cell markers (rabbit anti-Olig2 (1:200) for oligodendrocytes and rat anti-GFAP (1:500) for astrocytes and ependymal cells). Alexa-647® dye-conjugated goat anti-rabbit (or anti-rat) secondary antibodies were imaged using a 622 nm laser line. The lipophilic dye DiO (incorporated into LNPs) was imaged using a 488 nm laser line. mCherry® fluorescent protein was imaged using a 561 nm laser line.

[0104] CSF collection CSF was collected as previously described [8]. Briefly, a glass capillary tube was pulled and trimmed to an inner diameter of approximately 0.5 mm. Mice were anesthetized with a three-component anesthetic (fentanyl® 0.05 mg / kg, midazolam 5 mg / kg, and dexmedetomidine 0.5 mg / kg). Ophthalmic ointment was applied, and the animal's neck and skull were shaved. The mouse was fixed in a stereotaxic apparatus at an angle of approximately 135° from the body, and a sagittal incision was made below the occipital region. The subcutaneous tissue was dissected to expose the dura mater of the cisterna magna (see Figure 10). CSF (2–10 μL) was collected by piercing the dura mater with a capillary tube. The collected CSF was then transferred to a clean tube and stored at -80°C for future analysis. o Immediately frozen at C.

[0105] Measurement of FGF-21 and EPO CSF was collected from mice 3 or 48 hours after injection of LNP-mRNA (FGF-21) or LNP-mRNA (EPO). The presence of FGF-21 or EPO was quantified using the U-PLEX FGF-21 assay or the U-PLEX EPO assay (Meso Scale Discovery®, USA).

[0106] Example Example 1: LNP-mRNA induces widespread expression of a reporter protein in the brain and spinal cord.

[0107] To verify the feasibility of using brain cells to induce the diffusion of secreted proteins throughout the brain, we first examined the distribution patterns of LNP-mRNA and downstream protein expression after intracerebroventricular (ICV) injection. LNPs containing the lipophilic dye DiO and carrying mRNA encoding mCherry® were injected bilaterally into the lateral ventricles of adult (p40-60) mice. After 2 days, brains were harvested and imaged for mCherry® protein expression and accumulation.

[0108] The distribution pattern of the DiO dye contained in the LNP formulation indicated that LNPs were distributed throughout the corpus callosum, a major white matter tract in the brain. Importantly, robust expression of the mCherry fluorescent protein reporter was readily observed along the corpus callosum, as well as along the ventricular walls and the choroid plexus within the ventricles. The reporter protein was expressed over a range of more than 3 mm anterior-posteriorly and more than 4 mm laterally (Figure 2). Further examination of the cell types producing mCherry® within the white matter revealed expression in two glial cell types: oligodendrocytes, the Olig2+ cells responsible for myelination of neuronal axons, and astrocytes immunostained for GFAP (Figure 3). Furthermore, ependymal cells lining the ventricular walls and choroid plexus ependymal cells expressed mCherry® (Figure 4). This demonstrates that LNP-mRNA injection can be used to widely produce exogenous proteins within cells of the CNS.

[0109] In another experiment, LNP-mRNA encoding mCherry® was injected intrathecally into the lumbar region between L5 and L6. Robust expression of mCherry® was observed throughout the entire length of the spinal cord ( FIG. 5 ). This demonstrates that LNP-mRNA injection can be used to achieve widespread production of exogenous proteins within cells of the CNS via multiple delivery routes.

[0110] Example 2: LNP-mRNA expression of secreted proteins results in widespread diffusion within the brain.

[0111] The wide distribution of LNP-mRNA throughout the CNS, both in the structures responsible for CSF production and throughout the white matter tracts, suggests the potential for the use of LNP-mRNA in the production of secreted proteins that diffuse throughout the CNS. We tested this "bioreactor" approach by injecting LNP(1) [or G]-mRNA-FGF-21 and LNP(2) [or H]-mRNA-FGF-21, shown in Table 2 (obtained from NanoVation Therapeutics Inc.), encoding fibroblast growth factor 21 (FGF-21), a secreted growth factor primarily produced by hepatocytes and therefore typically absent in the brain. Three or 48 hours after bilateral intracerebroventricular injection, CSF was collected from the cisterna magna (the largest CSF reservoir in the brain) and the presence of FGF-21 was quantified using the U-PLEX assay. CSF FGF-21 concentrations were significantly higher for LNP(1) [or G]-mRNA-FGF-21 (2,790.3 pg / mL) and LNP(2) [or H]-mRNA-FGF-21 (23,765 pg / mL) than for uninjected controls (control: 164.3 pg / mL) and injected controls (LNP-mRNA-EPO: 28.6 pg / mL) (Figure 6). FGF21 levels in CSF were elevated for at least 48 hours (Figure 7; LNP-mRNA-FGF-21, CSF collection, 3 hours post-injection: 30,740 pg / mL, 48 hours: 27,719 pg / mL). Furthermore, FGF-21 levels in the CSF varied dose-dependently depending on the injected LNP-mRNA concentration (Figure 7; LNP-mRNA-FGF-21 at 1 mg / mL: 30,740 pg / mL, 0.5 mg / mL: 30,576 pg / mL, 0.2 mg / mL: 31,604 pg / mL, 0.05 mg / mL: 1,213 pg / mL). In another experiment, LNP-mRNA encoding erythropoietin (EPO) was intracerebroventricularly injected. At 3 or 48 hours after bilateral intracerebroventricular injection, CSF samples were collected from the cisterna magna, and the presence of EPO was quantified using the U-PLEX assay. High levels of EPO were found in the CSF (FIG. 8; uninjected control: 8.5 pg / ml, injected control LNP-FGF-21: 15.0 pg / ml, LNP-mRNA-EPO: 396,899 pg / ml).EPO levels in the CSF were elevated for at least 48 hours (Figure 9; LNP-mRNA-EPO 3 hours: 396,899 pg / mL, 48 hours: 144,286 pg / mL). Furthermore, EPO levels in the CSF varied in a dose-dependent manner depending on the injected LNP-mRNA concentration (Figure 9; LNP-mRNA-EPO 1 mg / mL: 396,899 pg / mL, 0.5 mg / mL: 218,629 pg / mL). This data indicates that secreted proteins, such as growth factors and erythropoietin, can diffuse throughout the brain and reach sites of action distal to the actual site of LNP-mRNA uptake and expression. Remarkably, this data demonstrates that protein production, secretion, and diffusion throughout the brain are consistent over at least 48 hours.

[0112] Example 3: In vitro method to assess whether the protein encoded by the mRNA can be secreted into the cerebrospinal fluid from cells of the central nervous system

[0113] An in vitro primary culture assay is used to test the ability of central nervous system cells (e.g., astrocytes and oligodendrocytes (OLs)) to secrete proteins from LNP-mRNA. In parallel experiments, primary astrocytes and primary OLs are cultured from perinatal mixed glial cultures using a standard method (commonly known as the McCarthy and deVelis method). Briefly, brains are harvested from P0-P3 mice, the cerebral cortex is dissected, and the cells are mechanically dissociated by trituration. After successive washing steps, the cells are cultured at 37°C and 5-8% CO2 for 3-6 days. This results in a "mixed glial culture" consisting of a sublayer of astrocytes, an overlying layer of oligodendrocyte progenitor cells (OPCs), and loosely adherent microglia. Shaking the plate detaches the microglia, leaving astrocytes and OPCs behind. Further differential shaking steps detach the OPCs. These OPCs are then cultured separately and differentiated into mature OLs using OL-specific medium. In parallel, the remaining layer of astrocytes is passaged several times to obtain astrocyte-enriched cultures. The purity of the astrocyte and OL cultures can be confirmed using GFAP and O4 / MBP immunostaining, respectively.

[0114] Astrocyte and OL cultures are used to assay protein secretion. Primary cells (astrocytes or OLs) are treated with LNP-mRNA encoding the protein. Protein secretion is assayed by collecting cell supernatants and quantifying protein levels by immunoblotting, ELISA, or U-PLEX. Supernatants are collected at various time points to estimate peak production / secretion. While the purpose of this assay is to focus on whether the protein is successfully secreted, it can also serve as a quantifiable assay for optimal LNP-mRNA formulations and concentrations and to test the production / secretion efficiency of various mRNA constructs.

[0115] The detailed description and examples are intended to illustrate the preparation and properties of particular lipid nanoparticle mRNA formulations, but are not intended to limit the scope of the invention in any way.

[0116] While various embodiments of the present invention are disclosed herein, many adaptations and modifications may be made within the scope of the present invention in accordance with the common general knowledge of those skilled in the art. Such modifications include the substitution of known equivalents for any aspect of the invention to achieve the same result in substantially the same way. Numerical ranges include the numbers defining the range. As used herein, the term "including" is used as an open-ended term substantially equivalent to the phrase "including, but not limited to," and the word "comprises" has a corresponding meaning. As used herein, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, reference to "one" includes plurals of such. The citation of a reference herein does not constitute an admission that such reference is prior art to embodiments of the present invention. The present invention includes all embodiments and variations substantially as described herein with reference to the examples and drawings.

[0117] References (1) AKHTAR, A. et al., "Neurodegenerative diseases and effective drug delivery: A review of challenges and novel therapies," J. Control Release (2021) 10:330:1152-1167 (2) CIUFOLINI, MA et al. WO2022 / 246571 (3) DIMITROV, DS “Therapeutic proteins” Methods Mol Biol. (2012) 899:1-26 (4) FEIGIN, VL et al., “The Global Burden of Neurological Disorders: Translating Evidence into Policy,” Lancet Neurol. (2020) 19(3): 255-265 (5) GAL, L. et al. "Recovery of motor function after spinal cord injury by delayed intraspinal delivery of nucleoside-modified mRNA encoding human IL-10." Research (2023) 6:0056 (6) GRIBKOFF, VK and KACZMAREK, LK. “The Need for New Approaches in CNS Drug Discovery: Why Drugs Fail and What Can Be Done to Improve Outcomes.” Neuropharmacology (2017) 120:11-19 (7) LIEBNER, S. et al., “Functional Morphology of the Blood-Brain Barrier in Health and Disease,” Acta Neuropathol. (March 2018) 135(3):311-336 (8) LIU, L. and DUFF, KA. "A technique for continuous collection of cerebrospinal fluid from the cisterna magna of mice." J Vis Exp. (2008) 21:960 (9) NABHAN, JF et al., "Intrathecal delivery of lipid nanoparticle-encapsulated frataxin mRNA to the dorsal root ganglion: a potential treatment for Friedreich's ataxia." Sci Rep. (2016) 17:6:20019 (10) PARDI, N. et al. WO2023 / 086830 (11) PARDRIDGE, "The Blood-Brain Barrier: A Bottleneck in Brain Drug Development," NeuroRx. (2005) 2(1): 3-14 (12) RUNGTA, RL et al. "Suppression of neuronal gene expression in the brain by delivery of siRNA via lipid nanoparticles" Molecular Therapy Nucleic Acids (2013) 2, e136 (13) RUNGTA, RL et al., "Cellular mechanisms underlying neuronal swelling in cytotoxic edema," Cell (2015) 161: 610-621 (14) TANAKA, H. et al., "In vivo delivery of mRNA encapsulated in lipid nanoparticles to brain neurons and astrocytes by intracerebroventricular administration," Mol. Pharmaceuticals (2018) 15, 2060-2067

Claims

1. 1. A method for treating, preventing, or diagnosing a disease, disorder, trauma, or injury of the central nervous system, said method comprising: contacting lipid nanoparticles (LNPs) with cells of the central nervous system of a subject, wherein the lipid nanoparticles comprise: (a) an ionizable amino cationic lipid having a pKa of 5.0 to 7.0; (b) a non-cationic helper lipid; (c) sterols; (d) a hydrophilic polymer-lipid conjugate; and (e) comprising mRNA encoding a secreted polypeptide for treating, preventing, or diagnosing a disease, disorder, trauma, or injury of the central nervous system, wherein the secreted polypeptide is capable of being secreted from the cells of the central nervous system into the interstitial fluid and / or cerebrospinal fluid of the subject; Upon contact of the LNP with the cell, the mRNA enters the cell and is translated within the cell to produce the polypeptide; after production of the polypeptide, the polypeptide is secreted from the cells into the interstitial fluid and / or the cerebrospinal fluid (CSF) of the subject; the secreted polypeptide is present in the CSF of the subject at a first concentration at a first time point and at a second concentration at a subsequent second time point; The first time point is 3 hours after contacting the LNP with the cells, and the second time point is 48 hours after contacting the LNP with the cells; the second concentration is at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of the first concentration; The method, wherein the contacting step is carried out in vitro or in vivo.

2. The cells of the central nervous system (a) glial cells; and (b) Ependymal cells The method of claim 1 , wherein the ion exchange is selected from one or more of the following:

3. 3. The method of claim 2, wherein the glial cells are selected from astrocytes or oligodendrocytes, and the ependymal cells are selected from the ventricles or choroid plexus.

4. 4. The method of any one of claims 1 to 3, wherein the secreted polypeptide is endogenous to the cells of the central nervous system or is modified to enhance activity in the brain.

5. The method of any one of claims 1 to 4, wherein the secreted polypeptide is diffusible within the interstitial fluid and / or the cerebrospinal fluid of the subject.

6. 6. The method of any one of claims 1 to 5, wherein the non-cationic helper lipid is a generally cylindrical lipid.

7. The method according to any one of claims 1 to 6, wherein the non-cationic helper lipid is distearoylphosphatidylcholine (DSPC) or dioleoylphosphatidylglycerol (DOPG).

8. 8. The method of claim 1, wherein the non-cationic helper lipid has a phosphatidylethanolamine content of less than 2 mol %.

9. The method according to any one of claims 1 to 8, wherein the sterol is cholesterol.

10. The method of any one of claims 1 to 9, wherein the polymer lipid conjugate is PEG-DMG.

11. 11. The method of any one of claims 1 to 10, wherein the molar ratio of the ionizable amino cationic lipid / the non-cationic helper lipid / the sterol / the polymer lipid conjugate is 50 / 10 / 38-39 / 1-2.

12. 12. The method of any one of claims 1 to 11, wherein the administration comprises injection into the central nervous system of the subject via an intracerebroventricular (ICV) or lumbar intrathecal route, a cisternal route, or a catheter.

13. 1. Use of a pharmaceutical formulation comprising lipid nanoparticles (LNPs) for contacting cells of the central nervous system of a subject to treat, prevent, or diagnose a disease, disorder, trauma, or injury of the central nervous system, wherein the lipid nanoparticles comprise: (a) an ionizable amino cationic lipid having a pKa of 5.0 to 7.0; (b) a non-cationic helper lipid; (c) sterols; (d) a hydrophilic polymer-lipid conjugate; and (e) comprising mRNA encoding a secreted polypeptide for treating, preventing, or diagnosing a disease, disorder, trauma, or injury of the central nervous system, wherein the secreted polypeptide is capable of being secreted from the cells of the central nervous system into the interstitial fluid and / or cerebrospinal fluid of the subject after administration of the lipid nanoparticle; Upon contact of the LNP with the cell, the mRNA enters the cell and is translated within the cell to produce the polypeptide; after production of the polypeptide, the polypeptide is secreted from the cells into the interstitial fluid and / or the cerebrospinal fluid (CSF) of the subject; the secreted polypeptide is present in the CSF of the subject at a first concentration at a first time point and at a second concentration at a subsequent second time point; The first time point is 3 hours after contacting the LNP with the cells, and the second time point is 48 hours after contacting the LNP with the cells; the second concentration is at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of the first concentration; The use, wherein the contacting is carried out in vitro or in vivo.

14. 1. Use of lipid nanoparticles for contacting cells of the central nervous system of a subject for the manufacture of a medicament for treating, preventing, or diagnosing a disease, disorder, trauma, or injury of the central nervous system, wherein the lipid nanoparticles comprise: (a) an ionizable amino cationic lipid having a pKa of 5.0 to 7.0; (b) a non-cationic helper lipid; (c) sterols; (d) a hydrophilic polymer-lipid conjugate; and (e) comprising mRNA having a nucleic acid sequence encoding a secreted polypeptide for treating, preventing, or diagnosing a disease, disorder, trauma, or injury of the central nervous system, wherein the secreted polypeptide is capable of being secreted from the cells of the central nervous system into the interstitial fluid and / or cerebrospinal fluid of the subject after administration of the lipid nanoparticle; Upon contact of the LNP with the cell, the mRNA enters the cell and is translated within the cell to produce the polypeptide; after production of the polypeptide, the polypeptide is secreted from the cells into the interstitial fluid and / or the cerebrospinal fluid (CSF) of the subject; the secreted polypeptide is present in the CSF of the subject at a first concentration at a first time point and at a second concentration at a subsequent second time point; The first time point is 3 hours after contacting the LNP with the cells, and the second time point is 48 hours after contacting the LNP with the cells; the second concentration is at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of the first concentration; The use, wherein the contacting is carried out in vitro or in vivo.

15. 15. The use according to claim 13 or 14, wherein the cells of the central nervous system are glial cells selected from astrocytes or oligodendrocytes, or ependymal cells of the ventricles or choroid plexus.

16. The use according to any one of claims 13 to 15, wherein the non-cationic lipid is a generally cylindrical lipid.

17. The use according to any one of claims 13 to 16, wherein the non-cationic lipid is distearoylphosphatidylcholine (DSPC) or dioleoylphosphatidylglycerol (DOPG).

18. The use according to any one of claims 13 to 17, wherein the phosphatidylethanolamine content is less than 2 mol%.

19. The use according to any one of claims 13 to 18, wherein the sterol is cholesterol.

20. The use according to any one of claims 13 to 19, wherein the polymer lipid conjugate is PEG-DMG.

21. 21. The use according to any one of claims 13 to 20, wherein the molar ratio of the ionizable amino cationic lipid / the non-cationic helper lipid / the sterol / the polymer lipid conjugate is 50 / 10 / 38-39 / 1-2.

22. 22. The use of any one of claims 13 to 21, wherein the therapeutic polypeptide or peptide is endogenous to the cells of the central nervous system or has been modified to enhance activity in the brain.

23. The use according to any one of claims 13 to 22, wherein the therapeutic polypeptide or peptide is diffusible in the interstitial fluid and / or the cerebrospinal fluid.

24. 1. A lipid nanoparticle for contacting cells of the central nervous system of a subject to treat, prevent, or diagnose a disease, disorder, trauma, or injury of the central nervous system, said lipid nanoparticle comprising: (a) an ionizable amino cationic lipid having a pKa of 5.0 to 7.0; (b) a non-cationic helper lipid; (c) sterols; (d) a hydrophilic polymer-lipid conjugate; and (e) comprising an mRNA having a nucleic acid sequence encoding a secreted polypeptide for treating, preventing, or diagnosing a disease, disorder, trauma, or injury of the central nervous system, wherein the secreted polypeptide is capable of being secreted from the cells of the central nervous system into the interstitial fluid and / or cerebrospinal fluid of the subject; Upon contact of the LNP with the cell, the mRNA enters the cell and is translated within the cell to produce the polypeptide; after production of the polypeptide, the polypeptide is secreted from the cells into the interstitial fluid and / or the cerebrospinal fluid (CSF) of the subject; the secreted polypeptide is present in the CSF of the subject at a first concentration at a first time point and at a second concentration at a subsequent second time point; The first time point is 3 hours after contacting the LNP with the cells, and the second time point is 48 hours after contacting the LNP with the cells; the second concentration is at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of the first concentration; The lipid nanoparticles are characterized in that the contact is carried out in vitro or in vivo.

25. 25. The lipid nanoparticle of claim 24, wherein the cell of the central nervous system is a glial cell selected from an astrocyte or an oligodendrocyte, or an ependymal cell of the ventricle or choroid plexus.

26. The lipid nanoparticle of claim 24 or 25, wherein the lipid nanoparticle is part of a pharmaceutical formulation.

27. The lipid nanoparticle of any one of claims 24 to 26, wherein the secreted polypeptide is endogenous to the cells of the central nervous system or has been modified to enhance activity in the brain.

28. The lipid nanoparticle of any one of claims 24 to 27, wherein the secreted polypeptide is diffusible within the interstitial fluid and / or the cerebrospinal fluid of the subject.

29. The lipid nanoparticle of any one of claims 24 to 28, wherein the non-cationic helper lipid is a generally cylindrical lipid.

30. The lipid nanoparticle of any one of claims 26 to 31, wherein the non-cationic lipid is distearoylphosphatidylcholine (DSPC) or dioleoylphosphatidylglycerol (DOPG).

31. The lipid nanoparticle of any one of claims 26 to 32, wherein the non-cationic helper lipid has a phosphatidylethanolamine content of less than 2 mol%.

32. The lipid nanoparticle of any one of claims 24 to 31, wherein the sterol is cholesterol.

33. The lipid nanoparticle of any one of claims 24 to 32, wherein the polymer lipid conjugate is PEG-DMG.

34. The lipid nanoparticle according to any one of claims 24 to 33, wherein the molar ratio of the ionizable amino cationic lipid / the non-cationic helper lipid / the sterol / the polymer lipid conjugate is 50 / 10 / 38-39 / 1-2.

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