WO2023165582A1

WO2023165582A1 - Targeted delivery system and method

Info

Publication number: WO2023165582A1
Application number: PCT/CN2023/079438
Authority: WO
Inventors: 孙怡迪; 周昌阳; 毛少帅; 彭文博
Original assignee: 益杰立科(上海)生物科技有限公司
Priority date: 2022-03-04
Filing date: 2023-03-03
Publication date: 2023-09-07

Abstract

Provided is use of a lipid nanoparticle (LNP) delivery system in preparing a drug for the prevention and/or treatment of a central nervous system disease, wherein the LNP comprises the following components on the basis of molar percentage: about 45%-50% ionizable lipid, 37.5%-45% cholesterol-based lipid, 9%-10% structural lipid, and 1.5%-2.5% PEGylated lipid.

Description

Targeted delivery systems and methods

technical field

This application relates to the field of biomedicine, in particular to a lipid delivery carrier and delivery method for the central nervous system.

Background technique

Currently, there remains a need for effective therapies for the treatment of CNS diseases, such as those caused directly or indirectly by the loss, aberrant expression or dysregulation of neuronal cellular proteins. Several obstacles exist in implementing effective therapeutic regimens for CNS diseases, primarily due to the separation and isolation of CNS tissue by the impermeable blood-brain barrier (BBB) and the uniquely complex membrane composition of neurons.

Significant differences exist between the CSF environment and the IV environment

1. Comparison of characteristics and components of cerebrospinal fluid and human blood

2. Differences in flow characteristics

Fluid circulation in cerebrospinal fluid: cerebrospinal fluid moves unidirectionally from the ventricles outward, but moves multidirectionally in the subarachnoid space. Eventually drains into the venous system, diluting macromolecules, lipids, and insoluble molecules into the blood.

The blood circulation is a non-Newtonian fluid while the cerebrospinal fluid circulation is a Newtonian fluid mainly affected by Brownian motion. Fluctuation dynamics follow the laws of the fluctuation-dissipation theorem (unlike blood). Its translation and rational motion in CSF are based on the Stokes-Einstein and Stokes-Einstein-Debye relational equations.

The degrees of freedom alone follow a Gaussian distribution Hydrodynamics Wall effects mainly affect the movement of nanoparticles rather than thermal effects

(The hydrodynamic mean value of CSV conductance is 8-24 mm ³ k.Pa-1.S-1 depending on age). Therefore, the diffusivity of nanoparticles in CSF can be based on

references:

Uma,B.,et al."Modeling of a Nanoparticle Motion in a Newtonian Fluid:A Comparison Between Fluctuating Hydrodynamics and Generalized Langevin Procedures."International Conference on Micro/Nanoscale Heat Transfer.Vol.54778.American Society of Mechanical Engineers, 2012 .

Ekstedt, J.A.N."CSF hydrodynamic studies in man.2.Normal hydrodynamic variables related to CSF pressure and flow."Journal of Neurology, Neurosurgery&Psychiatry 41.4(1978):345-353.

Cells in the Nervous System Deliver LNP Differently from Cells in Human Tissues

Major lipid components in non-CNS cell membranes: phospholipids, cholesterol, glycoproteins, carbohydrate groups. In the central nervous system, sphingolipids, glycosphingolipids, and sialic acid are mainly found in gangliosides, which play an important role in regulating neuronal plasticity in glycosides compared with other types of cells. role. Specifically, sialic acid acts as a cellular receptor (with a specific protein: GPI-nexin) attached to the outer leaflet of the plasma membrane to facilitate signal transduction, neurotransmission, interaction with neuroregulatory proteins, and cell-cell recognition and proliferation.

cell signal

Cellular medicines in the central nervous system are regulated by lipoproteins, especially apoE4.

Neuronal uptake of lipid nanoparticles

In contrast to glial cells, neurons are non-phagocytic (cannot actively endocytose LNP particles). Neuronal uptake of LNP is facilitated by the attachment of astrocyte-secreted apolipoprotein E to nanoparticles, which can be recognized by low-density lipoprotein (LDL) receptors and subsequent neuronal endocytosis in vivo.

These properties above indicate that the endocytic performance of LNP in the CNS and the way of cell transport are significantly different from those injected with serum or intramuscularly. Currently, gene therapy has become an effective treatment for a variety of diseases, and while promising for non-neuronal diseases, liposomes are impermeable to the BBB, and the unique and complex membrane composition of neuronal cells is critical for delivering nucleic acid drugs to the interior of neuronal cells. presents unique challenges (Svennerhol et al., Biochimica et Biophysica Acta, 1992, 1128: 1-7).

Contents of the invention

The present application provides a method for delivering nucleic acids to cells in the central nervous system using lipid nanoparticles, which can remain stable under specific pH and protein concentration environments such as cerebrospinal fluid; the lipid nanoparticles are based on The construction of nerve cell membrane components is conducive to its fusion with nerve cell membranes, mediating entry into cells, and releasing embedded nucleic acids such as mRNA.

The present invention is based in part on the surprising discovery that lipid- or polymer-based nanoparticles loaded with nucleic acid molecules can be administered directly into the CNS space (e.g., by intrathecal administration) and efficiently penetrate neuronal cell membranes, resulting in nucleic acid molecules in the CNS space. Intracellular delivery in neurons in the brain and/or spinal cord. Surprisingly, the lipid- or polymer-based nanoparticles described in this application can effectively deliver nucleic acid molecules into the cells of the central nervous system, even neurons located deep in the center of the brain and spine and those difficult to treat. motor neuron. Thus, the present invention provides an improved and efficient method for CNS delivery of nucleic acid drugs and holds promise as an effective therapy for the treatment of a variety of CNS diseases.

In one aspect, the application provides the use of a lipid nanoparticle (LNP) delivery system in the preparation of a drug for the prevention and/or treatment of the central nervous system, wherein the LNP comprises the following components in terms of molar percentage: ionizable lipid Cholesterol-based lipids 37.5%-45%, structured lipids 9-10%, pegylated lipids 1.5%-2.5%.

In certain embodiments, wherein said LNP comprises the following components: about 45-50% ionizable lipids, 37.5%-38.5% cholesterol-based lipids, 9-10% structured lipids, pegylated lipids Quality 1.5%-2.5%.

In certain embodiments, wherein said LNP comprises the following components: about 50% ionizable lipids, 37.5%-38.5% cholesterol-based lipids, 9-10% structured lipids, 1.5% pegylated lipids %-2.5%.

In certain embodiments, wherein said LNP comprises the following components: about 50% ionizable lipids, 37.5%-38.5% cholesterol-based lipids, 10% structured lipids, 1.5%-pegylated lipids 2.5%.

In certain embodiments, wherein said LNP comprises the following components: about 45-50% ionizable lipids, 38.5%-45% cholesterol-based lipids, 9-10% structured lipids, PEGylated lipids Quality 1.5%-2.5%.

In certain embodiments, wherein said LNP comprises the following components: about 45% ionizable lipids, 38.5-45% cholesterol-based lipids, 9-10% structured lipids, 1.5% pegylated lipids -2.5%.

In certain embodiments, wherein said LNP comprises the following components: about 45% ionizable lipids, 44-45% cholesterol-based lipids, 9-10% structured lipids, 1.5% pegylated lipids -2.5%.

In certain embodiments, wherein said LNP comprises the following components: about 45% ionizable lipids, 44-45% cholesterol-based lipids, 9% structured lipids, 1.5%-2.5% pegylated lipids %.

In certain embodiments, wherein the ionizable lipid is selected from MC-3, LPO1 and combinations thereof.

In certain embodiments, wherein the structural lipid is selected from DPPC, DSPC, DOPE and combinations thereof.

In certain embodiments, wherein the PEGylated lipid is selected from DAG-PEG, DAA-PEG, DMG-PEG, DSPE-PEG, C8-PEG, DOG-PEG, ceramide PEG, and combinations thereof.

In certain embodiments, wherein the cholesterol-based lipid comprises cholesterol or PEGylated cholesterol.

In certain embodiments, wherein said liposome comprises a combination selected from:

MC3, cholesterol, DSPC, DMG-PEG;

LP01, cholesterol, DOPE, DMG-PEG;

LP01, cholesterol, DSPC, DMG-PEG; and

MC3/LP01, cholesterol, DSPC, DMG-PEG.

In another aspect, the present application provides a method of delivering a therapeutic agent to the central nervous system (CNS), comprising: intrathecally administering to a subject in need of delivery a combination comprising a therapeutic agent encapsulated within a lipid nanoparticle wherein the lipid nanoparticle comprises the following components based on molar percentages: about 45-50% ionizable lipids, 37.5%-45% cholesterol-based lipids, 9-10% structured lipids, polyethylene Diolated lipids 1.5%-2.5%.

In certain embodiments, wherein the cells in the brain and/or spinal cord are selected from motor neurons, oligodendrocytes, oligodendrocytes, astrocytes, glial cells, Anterior horn cells and dorsal root ganglia and combinations thereof.

In certain embodiments, wherein the therapeutic agent comprises a nucleic acid.

In certain embodiments, wherein the nucleic acid is selected from siRNA, miRNA, pri-miRNA, messenger RNA (mRNA), clustered regularly interspaced short palindromic repeat (CRISPR)-related nucleic acid, single guide RNA (sgRNA) ), CRISPR-RNA (crRNA), trans-activating crRNA (tracrRNA), plasmid DNA (pDNA), transfer RNA (tRNA), antisense oligonucleotide (ASO), guide RNA, double-stranded DNA (dsDNA), single One or more of stranded DNA (ssDNA), single stranded RNA (ssRNA) and double stranded RNA (dsRNA).

In certain embodiments, wherein the therapeutic agent comprises mRNA.

In certain embodiments, wherein said protein encoded by said mRNA normally functions in said neurons in said brain and/or spinal cord.

In certain embodiments, wherein intracellular delivery of said mRNA results in intracellular expression of said protein encoded by said mRNA within the cytosol of said neuron.

In certain embodiments, wherein the intracellular delivery of the mRNA results in the expression of the protein encoded by the mRNA and, after expression, secretion from the neuron to the outside of the cell.

MC3, cholesterol, DSPC, DMG-PEG;

LP01, cholesterol, DOPE, DMG-PEG;

LP01, cholesterol, DSPC, DMG-PEG; and

MC3/LP01, cholesterol, DSPC, DMG-PEG.

In another aspect, the present application provides a composition for treating central nervous system diseases or disorders, which comprises lipid nanoparticles encapsulated with therapeutic agents, wherein the lipid nanoparticles comprise the following Components: ionizable lipids approximately 45-50%, cholesterol-based lipids 37.5%-45%, structured lipids 9-10%, pegylated lipids 1.5%-2.5%.

In certain embodiments, it is formulated as a liquid suitable for administration in cerebrospinal fluid.

In certain embodiments, it is formulated for administration by intrathecal injection.

In some embodiments, the intrathecal injection comprises injection into the hippocampal region of the brain parenchyma, intracerebroventricular injection and/or spinal orthotopic injection.

In certain embodiments, the disease or disorder of the central nervous system comprises Parkinson's syndrome, Angelman syndrome or spinal cord injury disease.

In another aspect, the present application provides a method for treating a central nervous system disease or disorder, comprising administering lipid nanoparticles comprising a therapeutic agent to the cerebrospinal fluid of the subject, wherein the lipid The nanoparticles comprise the following components: ionizable lipids approximately 45-50%, cholesterol-based lipids 37.5%-45%, structured lipids 9-10%, and pegylated lipids 1.5%-2.5%.

In certain embodiments, said administering comprises intrathecal injection.

Those skilled in the art can easily perceive other aspects and advantages of the present application from the following detailed description. In the following detailed description, only exemplary embodiments of the present application are shown and described. As those skilled in the art will appreciate, the content of the present application enables those skilled in the art to make changes to the specific embodiments which are disclosed without departing from the spirit and scope of the invention to which this application relates. Correspondingly, the drawings and descriptions in the specification of the present application are only exemplary rather than restrictive.

Description of drawings

The particular features of the invention to which this application relates are set forth in the appended claims. The features and advantages of the invention to which this application relates can be better understood with reference to the exemplary embodiments described in detail hereinafter and the accompanying drawings. A brief description of the accompanying drawings is as follows:

Figure 1 shows the results of transfection of the lipid nanoparticles described in this application into the striatum of Ai9 transgenic mice.

Figure 2 shows the evaluation of transfection effect by spinal injection in mice.

Figure 3 shows the transfection evaluation of spinal injection in adult mice.

What Fig. 4 shows is that the lipid nanoparticle transfection Ai9 transgenic mouse brain result map described in the application

Detailed ways

The implementation of the invention of the present application will be described in the following specific examples, and those skilled in the art can easily understand other advantages and effects of the invention of the present application from the content disclosed in this specification.

Example

Example 1

Example 1.1 Preparation of Lipid Nanoparticles

By fully mixing the organic phase and the aqueous phase, LNP nanoparticles are prepared to achieve effective embedding of gene editing tools or other nucleic acid drugs.

Wherein, the organic phase: contains at least one ionizable lipid, at least one supporting lipid, at least one amphiphilic block copolymer and cholesterol, and is dissolved by an organic solvent that is miscible with water. The organic solvent is preferably selected from ethanol, acetonitrile, acetone and the like.

Aqueous phase: an aqueous solution of gene editing tools, wherein the content of nucleic acid substances (such as mRNA) is 0.5-50% (w/v), and the pH is 3.0-7.0. The aqueous salt solution is selected from: citrate buffer, phosphate buffer, Tris-HCl buffer system.

The mixing of organic phase and aqueous phase can be achieved by microfluidic and impinging flow reactors. The embedding efficiency of gene editing tool RNA can be optimized by adjusting the N/P ratio of the system, and the N/P ratio is 1:1 to 9:1.

The optimization of embodiment 1.2 embedding rate

LNP was prepared by the method of Example 1.1, and LNP was prepared by regulating the molar ratio of ionizable lipid and cationic lipid, and its embedding rate for more than 2000bp mRNA was characterized. (RNA is derived from RNA extracted from yeast, purchased from McLean, Cat. No. R822593)

Characterization method of embedding rate: The embedding rate of prepared LNP is determined by Quant-iTTM RNA Reagent and Kit assay.

Specific detection method:

Solution configuration: Dilute an appropriate amount of 20×TE buffer solution to 1× with ultrapure water, which is enough for the day’s experiment. Dilute the concentrated dye solution with 1× TE (large range 25-1000ng/ml RNA) at a ratio of 1:200, (small range 1-50ng/ml RNA) at a ratio of 1:2000. Wrap it in gold foil or store it in a dark place away from light. Dilute TritonX-100 to 5% concentration with 1×TE buffer for use.

Sample processing: set ultrapure water with RNA concentration of 0 as the blank background, set TE buffer instead of LNP with 5% TritonX-100 as the free RNA assay sample, and set LNP with 5% TritonX-100 as the total RNA assay Sample. Take the LNP sample to be determined and add an equal volume of 5% TritonX-100, 1×TE solution to the blank, and incubate at 50-60°C for 5-10 minutes.

The incubated LNP samples were diluted to 10-400 times with 1×TE buffer solution (appropriately adjusted according to the RNA concentration of the sample group). After dilution, 100 μl was added to a 96-well plate, and then 100 μl of the diluted concentrated dye solution was added in the dark, and the fluorescence absorbance was measured within 5 minutes. The measurement conditions are an excitation wavelength of 480 nm and an emission wavelength of 520 nm.

Calculation: Embedding rate=(absorbance of total RNA-absorbance of free RNA)/absorbance of total RNA×100%

The results are shown in Table 1. The formulas (#1-#7) of the present invention ensure a relatively high embedding rate of large fragments of mRNA (above 93%).

Table 1 Preparation of LNP and determination of embedding rate

Example 2 Evaluation of transfection effect of striatal cells injected into lateral ventricle

Lipid nanoparticles were prepared by formula #2 in Table 1, and Cre mRNA was embedded (the Cre mRNA sequence is GenBank: AAL31698.1, and the Cre mRNA sequences used in subsequent examples are all of this sequence). Stereotaxic injections of Ai9 transgenic mice were performed using a standard stereotaxic apparatus (Model 68528, RWD Life Sciences) under isoflurane anesthesia (4% induction and 2% maintenance) or 5% chloral hydrate. The mouse skull was exposed and cleaned, a small craniotomy was performed, and a pulled glass micropipette manufactured by a micropipette stretcher (Shutter Instrument Co.) was placed in the striatum (experimental group: A/P+1.2 mm; M/L: 2.0mm; D/V 3.0mm). Subsequently, we injected 1uL of LNP sample (100ng/ul mRNA-embedded LNP at a speed of 120nL/min) through a pressure microinjector (KDS-310-plus, KD Scientific). Leave the injection needle in place for 5 min before slowly withdrawing to allow the LNP to diffuse. Five days after the injection, the mice were sacrificed humanely, and the brain slices were taken, and observed by laser confocal, the results are shown in Figure 1. LNP can effectively transfect the striatum of the injected area, showing obvious red fluorescence. And compared to neuron cells (neuron), LNP transfected more glial cells (Glial cells), showing that it can be efficiently delivered to the central nervous system (brain tissue) to treat central nervous diseases such as Parkinson's.

Example 3 P0 mouse spinal injection transfection effect evaluation

Prepare lipid nanoparticles using recipe #2 in Table 1, and simultaneously embed Cre mRNA samples. Fix the P0 mouse of the Ai9 transgenic mouse with the index finger and middle finger, put the sample into the Hamilton injection needle, insert the needle at 30° obliquely below the spine, insert the needle at a depth of 1mm, and then insert the needle parallel to the spine, push the needle for injection, and stay for 1min Withdraw the injection slowly. The LNP sample was mixed with Fastgreen dye at a ratio of 10:1 to trace the position of the injected sample in the spinal cord, and each mouse was injected with 2ul sample (concentration 100ng/uL). The mouse spine was taken 5 days after injection for confocal laser, and the results showed that LNP can effectively transfect the P0 mouse spine (Figure 2), proving that it has the potential of efficient delivery to the central nervous system (spinal cord) for Angelman syndrome Gene therapy for rare diseases.

Example 4 Evaluation of Transfection Effect of Adult Ai9 Transgenic Mice Spinal Injection

Prepare lipid nanoparticles using recipe #2 in Table 1, and embed Cre mRNA samples at the same time. Mice were anesthetized with 1% sodium pentobarbital at a ratio of 0.16 mL/24 g. After the mouse was anesthetized, the hair around the injured part of the back of the mouse was shaved with a shaver; a small incision was made at the exposed epidermis to fully expose the subcutaneous tissue; Stretch the tissue to expose the spine, use micro forceps to break the lamina along the intervertebral space or use micro scissors to cut the lamina along the intervertebral space to expose the spinal cord, inject #2 formula LNP at the T8-T10 position , each mouse was injected with 2uL sample (concentration 100ng/uL). Five days after the injection, the mouse spine was taken for laser confocal, and the results showed that LNP can effectively transfer Stained adult mouse spine (Figure 3), it was proved that it has the potential of efficient delivery to the central nervous system (spinal cord), so as to be applied to the repair and treatment of various spinal cord injury diseases.

Example 5 Evaluation of transfection effect of adult Ai9 transgenic mouse brain parenchyma injected into hippocampus region

Lipid nanoparticles were prepared using recipe #7 in Table 1, and samples of Cre mRNA were embedded at the same time. Stereotaxic injections were performed on Ai9 transgenic adult mice using a standard stereotaxic apparatus (Model 68528, RWD Life Sciences) under isoflurane anesthesia (4% induction and 2% maintenance) or 1.25% tribromoethanol. The mouse skull was exposed and cleaned, and a cranial drill was used to perforate the skull, and a glass micropipette made by a micropipette stretcher (Shutter Instrument Co.) was placed in the hippocampus (experimental group, control group: AP- 2.00mm; ML+-1.5mm; DV-1.5mm). 2 uL of LNP samples were injected (at a rate of 300 nL/min) via a pressure microsyringe (Nanoject III, Drummond). Leave the injection needle in place for 5 minutes before slowly withdrawing it, allowing the LNP to seep down the needle. Four days after the injection, the mice were sacrificed under the conditions of animal welfare, and the brain slices were taken, and observed through a laser section rapid scanning microscope (VS200, Olympus). The results showed that LNP can effectively transfect parts of the CA3 and DG regions in the hippocampal structure Granulosa cells and glial cells (Figure 4). It has been shown to be effectively delivered to the central nervous system (brain tissue) for the treatment of related central system diseases.

Claims

The use of a lipid nanoparticle (LNP) delivery system in the preparation of a drug for the prevention and/or treatment of the central nervous system, wherein the LNP comprises the following components in terms of molar percentage: about 45-50% of ionizable lipids, Cholesterol-based lipids 37.5%-45%, structured lipids 9-10%, pegylated lipids 1.5%-2.5%.
The use according to claim 1, wherein said LNP comprises the following components: about 45-50% ionizable lipids, 37.5%-38.5% cholesterol-based lipids, 9-10% structured lipids, polyethylene glycol Alcoholated lipid 1.5%-2.5%.
The use according to any one of claims 1-2, wherein the LNP comprises the following components: about 50% ionizable lipids, 37.5%-38.5% cholesterol-based lipids, and 9-10% structured lipids , Pegylated lipids 1.5%-2.5%.
The use according to any one of claims 1-3, wherein the LNP comprises the following components: about 50% ionizable lipids, 37.5%-38.5% cholesterol-based lipids, 10% structured lipids, poly Glycolated lipids 1.5%-2.5%.
The use according to claim 1, wherein said LNP comprises the following components: about 45-50% of ionizable lipids, 38.5%-45% of lipids based on cholesterol, 9-10% of structured lipids, polyethylene glycol Alcoholated lipid 1.5%-2.5%.
The use according to any one of claims 1 and 5, wherein the LNP comprises the following components: about 45% ionizable lipids, 38.5-45% cholesterol-based lipids, 9-10% structured lipids, Pegylated lipids 1.5%-2.5%.
Use according to any one of claims 1 and 5-6, wherein the LNP comprises the following components: about 45% ionizable lipids, 44-45% cholesterol-based lipids, 9-10% structured lipids %, pegylated lipids 1.5%-2.5%.
Use according to any one of claims 1 and 5-7, wherein the LNP comprises the following components: about 45% ionizable lipids, 44-45% cholesterol-based lipids, 9% structured lipids, Pegylated lipids 1.5%-2.5%.
The use according to any one of claims 1-8, wherein the ionizable lipid is selected from MC-3, LPO1 and combinations thereof.
The use according to any one of claims 1-9, wherein the structural lipid is selected from DPPC, DSPC, DOPE and combinations thereof.
The use according to any one of claims 1-10, wherein the pegylated lipid is selected from the group consisting of DAG-PEG, DAA-PEG, DMG-PEG, DSPE-PEG, C8-PEG, DOG-PEG, Ceramide PEG and combinations thereof.
The use according to any one of claims 1-11, wherein the cholesterol-based lipid comprises cholesterol or PEGylated cholesterol.
Use according to any one of claims 1-12, wherein said liposome comprises a combination selected from the group consisting of:

MC3, cholesterol, DSPC, DMG-PEG;

LP01, cholesterol, DOPE, DMG-PEG;

LP01, cholesterol, DSPC, DMG-PEG; and

MC3/LP01, cholesterol, DSPC, DMG-PEG.
A method of delivering a therapeutic agent to the central nervous system (CNS), comprising: intrathecally administering a composition comprising a therapeutic agent encapsulated in a lipid nanoparticle to a subject in need of delivery; wherein, calculated by molar percentage, The lipid nanoparticles comprise the following components: about 45-50% ionizable lipids, 37.5%-45% cholesterol-based lipids, 9-10% structured lipids, 1.5%-2.5% pegylated lipids %.
The method according to claim 14, wherein said LNP comprises the following components: ionizable lipids about 45-50%, cholesterol-based lipids 37.5%-38.5%, structured lipids 9-10%, polyethylene glycol Alcoholated lipid 1.5%-2.5%.
The method according to any one of claims 14-15, wherein the LNP comprises the following components: ionizable lipids about 50%, cholesterol-based lipids 37.5%-38.5%, structured lipids 9-10% , Pegylated lipids 1.5%-2.5%.
The method according to any one of claims 14-16, wherein the LNP comprises the following components: about 50% ionizable lipids, 37.5%-38.5% cholesterol-based lipids, 10% structured lipids, poly Glycolated lipids 1.5%-2.5%.
The method according to claim 14, wherein said LNP comprises the following components: about 45-50% ionizable lipids, 38.5%-45% cholesterol-based lipids, 9-10% structured lipids, polyethylene glycol Alcoholated lipid 1.5%-2.5%.
The method according to any one of claims 14 and 18, wherein the LNP comprises the following components: about 45% ionizable lipids, 38.5-45% cholesterol-based lipids, 9-10% structured lipids, Pegylated lipids 1.5%-2.5%.
The method according to any one of claims 14 and 18-19, wherein the LNP comprises the following components: ionizable lipids about 45%, cholesterol-based lipids 44-45%, structured lipids 9-10% %, pegylated lipids 1.5%-2.5%.
The method according to any one of claims 14 and 18-20, wherein the LNP comprises the following components: about 45% ionizable lipids, 44-45% cholesterol-based lipids, 9% structured lipids, Pegylated lipids 1.5%-2.5%.
The method according to any one of claims 14-21, wherein the cells in the brain and/or spinal cord are selected from motor neurons, oligodendrocytes, oligodendrocytes, astrocytes in the spinal cord Glial cells, glial cells, anterior horn cells, and dorsal root ganglia, and combinations thereof.
The method of any one of claims 14-22, wherein the therapeutic agent comprises a nucleic acid.
The method according to claim 23, wherein the nucleic acid is selected from siRNA, miRNA, pri-miRNA, messenger RNA (mRNA), clustered regularly interspaced short palindromic repeat (CRISPR) related nucleic acid, single guide RNA (sgRNA), CRISPR-RNA (crRNA), trans-activating crRNA (tracrRNA), plasmid DNA (pDNA), transfer RNA (tRNA), antisense oligonucleotide (ASO), guide RNA, double-stranded DNA (dsDNA) , one or more of single-stranded DNA (ssDNA), single-stranded RNA (ssRNA) and double-stranded RNA (dsRNA).
The method of any one of claims 14-24, wherein the therapeutic agent comprises mRNA.
The method of claim 25, wherein said protein encoded by said mRNA normally functions in said neurons in said brain and/or spinal cord.
The method according to any one of claims 25-26, wherein the intracellular delivery of the mRNA results in intracellular expression of the protein encoded by the mRNA within the cytosol of the neuron.
The method according to any one of claims 25-27, wherein the intracellular delivery of the mRNA results in the expression of the protein encoded by the mRNA and the extracellular secretion from the neuron after expression.
The method according to any one of claims 14-28, wherein the ionizable lipid is selected from MC-3, LPO1, and combinations thereof.
The method according to any one of claims 14-29, wherein the structural lipid is selected from DPPC, DSPC, DOPE and combinations thereof.
The method according to any one of claims 14-30, wherein the pegylated lipid is selected from DAG-PEG, DAA-PEG, DMG-PEG, DSPE-PEG, C8-PEG, DOG-PEG, Ceramide PEG and combinations thereof.
The method of any one of claims 14-31, wherein the cholesterol-based lipid comprises cholesterol or PEGylated cholesterol.
The method according to any one of claims 14-32, wherein said liposome comprises a combination selected from the group consisting of:

MC-3, cholesterol, DSPC, DMG-PEG;

LP01, cholesterol, DOPE, DMG-PEG;

LP01, cholesterol, DSPC, DMG-PEG; and

MC3/LP01, cholesterol, DSPC, DMG-PEG.
A composition for treating diseases or disorders of the central nervous system, comprising lipid nanoparticles encapsulated with a therapeutic agent, wherein calculated by molar percentage, the lipid nanoparticles comprise the following components: ionizable lipids about 45-50%, cholesterol-based lipids 37.5%-45%, structured lipids 9-10%, pegylated lipids 1.5%-2.5%.
The composition according to claim 34, wherein said LNP comprises the following components: ionizable lipids about 45-50%, cholesterol-based lipids 37.5%-38.5%, structured lipids 9-10%, polyethylene glycol Diolated lipids 1.5%-2.5%.
The composition according to any one of claims 34-35, wherein the LNP comprises the following components: ionizable lipids about 50%, cholesterol-based lipids 37.5%-38.5%, structured lipids 9-10% %, pegylated lipids 1.5%-2.5%.
The composition according to any one of claims 34-36, wherein the LNP comprises the following components: about 50% ionizable lipids, 37.5%-38.5% cholesterol-based lipids, 10% structured lipids, Pegylated lipids 1.5%-2.5%.
The composition according to claim 34, wherein said LNP comprises the following components: ionizable lipids about 45-50%, cholesterol-based lipids 38.5%-45%, structured lipids 9-10%, polyethylene glycol Diolated lipids 1.5%-2.5%.
The composition according to any one of claims 34 and 38, wherein the LNP comprises the following components: about 45% ionizable lipids, 38.5-45% cholesterol-based lipids, 9-10% structured lipids , Pegylated lipids 1.5%-2.5%.
The composition according to any one of claims 34 and 38-39, wherein the LNP comprises the following components: ionizable lipids about 45%, cholesterol-based lipids 44-45%, structured lipids 9- 10%, pegylated lipids 1.5%-2.5%.
The composition according to any one of claims 34 and 38-40, wherein the LNP comprises the following components: about 45% ionizable lipids, 44-45% cholesterol-based lipids, 9% structured lipids , Pegylated lipids 1.5%-2.5%.
The composition according to any one of claims 34-41, formulated as a liquid suitable for administration in cerebrospinal fluid.
The composition according to any one of claims 34-42, formulated for administration by intrathecal injection.
The composition according to any one of claims 34-43, the intrathecal injection comprises brain parenchymal hippocampus region injection, lateral ventricle injection and/or spinal orthotopic injection.
The composition according to any one of claims 34-44, wherein the central nervous system disease or disorder comprises Parkinson's syndrome, Angelman syndrome or spinal cord injury disease.
A method for treating a central nervous system disease or disorder, comprising administering lipid nanoparticles comprising a therapeutic agent to the cerebrospinal fluid of the subject, wherein the lipid nanoparticles comprise the following components in terms of molar percentage: About 45-50% ionized lipids, 37.5%-45% cholesterol-based lipids, 9-10% structured lipids, 1.5%-2.5% pegylated lipids.
The method of claim 46, wherein the LNP comprises the following components: about 45-50% ionizable lipids, 37.5%-38.5% cholesterol-based lipids, 9-10% structured lipids, polyethylene glycol Alcoholated lipid 1.5%-2.5%.
The method according to any one of claims 46-47, wherein the LNP comprises the following components: ionizable lipids about 50%, cholesterol-based lipids 37.5%-38.5%, structured lipids 9-10% , Pegylated lipids 1.5%-2.5%.
The method according to any one of claims 46-48, wherein the LNP comprises the following components: about 50% ionizable lipids, 37.5%-38.5% cholesterol-based lipids, 10% structured lipids, poly Glycolated lipids 1.5%-2.5%.
The method of claim 46, wherein the LNP comprises the following components: about 45-50% ionizable lipids, 38.5%-45% cholesterol-based lipids, 9-10% structured lipids, polyethylene glycol Alcoholated lipid 1.5%-2.5%.
The method according to any one of claims 46 and 50, wherein the LNP comprises the following components: about 45% ionizable lipids, 38.5-45% cholesterol-based lipids, 9-10% structured lipids, Pegylated lipids 1.5%-2.5%.
The method according to any one of claims 46 and 50-51, wherein the LNP comprises the following components: ionizable lipids about 45%, cholesterol-based lipids 44-45%, structured lipids 9-10% %, pegylated lipids 1.5%-2.5%.
The method according to any one of claims 46 and 50-52, wherein the LNP comprises the following components: about 45% ionizable lipids, 44-45% cholesterol-based lipids, 9% structured lipids, Pegylated Lipid 1.5%-2.5%
The method according to any one of claims 46-53, formulated as a liquid suitable for administration in cerebrospinal fluid.
The method according to any one of claims 46-54, said administering comprising intrathecal injection.
The method according to any one of claims 46-55, wherein the intrathecal injection includes injection into the hippocampal region of the brain parenchyma, intracerebroventricular injection and/or spinal orthotopic injection.
The method according to any one of claims 46-56, wherein the central nervous system disease or disorder comprises Parkinson's syndrome, Angelman syndrome or spinal cord injury disease.