Kidney-targeted mrna-LNP delivery technology and disease therapy method

Abstract

Disclosed is a method for treating nephropathy, which comprises targeted delivery of a nucleic acid composition by means of topical administration. Renal tubular cells are transfected with neutral LNPs by means of ureteral retrograde administration, or renal glomerular cells are transfected with positive and neutral LNPs by means of abdominal aorta administration, thereby better targeting renal lesion cells. The mRNA-LNP technology can be applied to the targeted delivery of a kidney-specific cell population by means of the selection of a drug delivery way, thereby achieving therapy for kidney genetic diseases.

Description

A kidney-targeted mRNA-LNP delivery technology and disease treatment method Technical Field

[0001] The present invention belongs to the field of pharmaceuticals, and in particular relates to a kidney-targeted mRNA-LNP delivery technology and a disease treatment method. Background Art

[0002] There are over 160 types of inherited kidney diseases, most of which lack effective treatments. Based on the site of onset, inherited kidney diseases are primarily divided into two categories: tubular and glomerular diseases, typified by autosomal dominant polycystic kidney disease (ADPKD) and Alport syndrome (AS). ADPKD is the most common monogenic inherited kidney disease, with an incidence of 1 in 400 to 1 in 1000, affecting approximately 12 million people worldwide. The causative genes for ADPKD are Pkd1 and Pkd2, encoding PC1 and PC2 proteins, respectively, accounting for 85% and 15% of cases. Loss of PC1 protein function alters cell polarity, increasing cell proliferation and leading to the formation of cysts. These cysts continuously secrete cystic fluid, further exacerbating their expansion. Vesicles initially develop in the proximal tubules and collecting ducts, eventually spreading throughout the kidney, leading to loss of renal function. ADPKD patients generally do not develop symptoms before the age of 30. After the age of 45, the disease enters a rapidly progressive phase, with approximately half of patients progressing to end-stage renal disease (ESRD) by the age of 60. Currently, there is a lack of effective treatments for ADPKD. Tolvaptan is the only approved drug for the treatment of ADPKD. However, this drug is severely hepatotoxic and is only indicated for the rapidly progressive stage of the disease, severely limiting its application. Therefore, new treatment strategies are urgently needed. AS was the first hereditary glomerular disease to be listed on the National List of Rare Diseases. AS is the most common hereditary progressive nephritis in children. It is primarily caused by genetic defects in type IV collagen α3, α4, and α5, which constitute the glomerular basement membrane. X-linked dominant AS (XLAS), caused by mutations in the Col4α5 gene, accounts for approximately 85% of all AS patients. AS is characterized by proteinuria, progressive renal function decline, ocular abnormalities, and sensorineural hearing loss. XLAS patients often develop ESRD during adolescence. However, clinical treatment options for this disease are limited, and current treatment options are mainly symptomatic conservative treatment.

[0003] Gene therapy is a fundamental treatment for genetic diseases. Advances in gene therapy technology have led to breakthroughs in numerous areas, including the liver, eyes, and muscles. Many gene therapy / gene editing interventions are currently being or have already been clinically implemented, bringing hope to patients with genetic diseases. By designing the sequence of messenger RNA (mRNA), it is theoretically possible to express any protein, such as antigens, therapeutic proteins, gene editing elements, and so on. Therefore, it can be used in a wide range of fields, including vaccine development, disease treatment, and gene editing. Furthermore, due to its simple production process, low cost, and short lead time, mRNA is also highly favored in clinical translation. At the end of 2020, the successful development of an mRNA COVID-19 vaccine demonstrated its strong potential for application. However, because mRNA carries a negative charge and is easily degraded by enzymes, a safe and efficient delivery vehicle is crucial for its effectiveness in target cells. Lipid nanoparticles (LNPs) are considered one of the best delivery vehicle options, with over 90% of mRNA drug projects in clinical development relying on LNPs.

[0004] LNPs are typically composed of ionizable cationic lipids, auxiliary phospholipids, cholesterol, and polyethylene glycol lipids. Among them, ionizable cationic lipids are key to achieving efficient mRNA loading and lysosomal escape, and are also the focus of various research institutions and R&D companies. Ionizable cationic lipid molecules can be protonated in low pH environments (such as pH 4.0), thereby binding to mRNA to complete its encapsulation. In physiological environments (such as pH 7.4), they are deprotonated and uncharged. Therefore, this type of LNP is actually a neutral LNP during use. This type of neutral LNP has been the most widely studied and has the most mature application in vaccine development and liver mRNA targeted delivery. Recent studies have shown that lung-targeted mRNA delivery can be achieved by introducing positively charged cationic lipids (such as the quaternary ammonium lipid DOTAP) into LNPs, while spleen-targeted mRNA delivery can be achieved by introducing electronegative anionic lipids (such as the phosphate lipid 18:1PA).

[0005] However, there is currently a lack of effective mRNA-LNP delivery technology and treatment strategies for the kidneys, and basic and clinical research on gene therapy for hereditary kidney diseases is currently almost blank. Based on the above background, in the present invention, by constructing various types of LNPs: including traditional neutral LNPs, positive LNPs (containing permanent cationic lipid molecules), and negative LNPs (containing electronegative anionic lipid molecules), precise delivery to kidney organs and specific cells is achieved through local administration. On the one hand, it effectively solves the practical problems faced by kidney diseases and brings new breakthroughs to the treatment of the entire field of kidney diseases. On the other hand, it greatly broadens the depth and breadth of application of mRNA-LNP drugs.

[0006] SUMMARY OF THE INVENTION

[0007] The present invention provides a method for treating kidney disease, comprising targeted delivery of a nucleic acid composition containing lipid nanoparticles (LNPs) and nucleic acid to a patient in need thereof through local administration.

[0008] According to the present invention, the kidney disease is selected from hereditary kidney disease, chronic kidney disease (such as renal vascular disease, infectious kidney damage, diabetic nephropathy, etc.), acute kidney injury, nephrotic syndrome (such as minimal change nephropathy, membranous nephropathy, focal segmental glomerulosclerosis, etc.), nephritis (such as acute glomerulonephritis, chronic glomerulonephritis, acute interstitial nephritis, chronic interstitial nephritis, hepatitis B virus-related nephritis, idiopathic acute tubulointerstitial nephritis, etc.), kidney stones, renal failure, kidney transplantation, kidney tumors (such as renal cell carcinoma, transitional cell carcinoma, Wilms' carcinoma, renal sarcoma, etc.).

[0009] According to the present invention, the kidney disease is selected from diseases related to the glomerulus, for example, congenital steroid-resistant nephrotic syndrome, Dennis-Drasher syndrome, Fraser syndrome, WAGR syndrome (Wilms tumor, aniridia, genitourinary system abnormalities, mental retardation and developmental delay), Pierson syndrome, nail-patellar syndrome, Schimke immune-osteodystrophy, mitochondrial disease with steroid-resistant nephrotic syndrome, Fabry disease, Alport syndrome, benign familial hematuria (thin basement membrane), Fechtner syndrome (Alport syndrome with macrothrombocytopenia), Alport syndrome with leiomyomatosis, Familial amyloidosis; and diseases related to the renal tubules, such as proximal tubular diseases: renal glycosuria, disaccharidemia, lysine protein intolerance, proximal renal tubular acidosis, hypophosphatemic rickets, nephrotic cystinosis, primary renal Fanconi syndrome, Fanconi-Bickel syndrome (hepatorenal glycogenosis), Low syndrome, Dunn's disease type 1 and type 2, hereditary renal hypouricemia, cystinuria type 1-3, X-linked hypophosphatemic rickets, Hartnap disease, iminoglycosuria, disaccharidemia, large limb ascending and distal tubular diseases: Bartter syndrome, familial hypocalcemic hypercalcemia type 1-4, severe neonatal hyperparathyroidism, autosomal dominant hypocalcemia, Gitelman syndrome, pseudohyperaldosteronism type 2 (Gordon syndrome), SeSAME syndrome (EAST syndrome), hypomagnesemia, familial juvenile hyperuricemia nephropathy type 1-6, autosomal dominant hypocalcemia, Kenny-Caffey syndrome type 2, HNF1B-related nephropathy, collecting duct diseases: Liddle syndrome, distal renal tubular acidosis, pseudohypoaldosteronism type 1, nephrogenic diabetes insipidus types 1 and 2, inappropriate antidiuretic nephrogenic syndrome, significant mineralocorticoid excess, congenital adrenal hyperplasia types 1-5, etc.

[0010] According to the present invention, the nucleic acid is siRNA, miRNA, primary-miRNA, messenger RNA (mRNA), clustered regularly interspaced short palindromic repeats (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-stranded DNA (ssDNA), single-stranded RNA (ssRNA) and double-stranded RNA (dsRNA), such as protein replacement therapy mRNA, base repair mRNA / sgRNA, gene repair mRNA / sgRNA / HDR, etc.

[0011] According to the present invention, the mass ratio of total lipids to nucleic acids in the nucleic acid composition is 1-100:1, preferably 10-80:1, more preferably 20-60:1 (e.g., 20:1, 30:1, 40:1, 50:1, 60:1), or even 40:1.

[0012] According to the present invention, the nucleic acid composition is a three-component lipid nanoparticle comprising, in addition to the nucleic acid, an ionizable cationic lipid and a helper lipid, wherein the helper lipid is selected from steroids, steroid derivatives, PEG lipids or phospholipids.

[0013] According to the present invention, the nucleic acid composition comprises four components. In addition to the nucleic acid, the lipid nanoparticles include ionizable cationic lipids, permanent cationic lipids and phospholipids.

[0014] According to the present invention, the nucleic acid composition is a five-component lipid nanoparticle comprising, in addition to the nucleic acid, an ionizable cationic lipid, a phospholipid, a steroid and a PEG lipid, and optionally an electropositive cationic lipid and / or an electronegative anionic lipid.

[0015] According to the present invention, the lipid nanoparticles comprise, based on molar percentages, 10-65% of ionizable cationic lipids (e.g., 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%), 0-50% of electropositive cationic lipids and / or electronegative anionic lipids (e.g., 0%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%). %, 45%, 50%), 5-30% phospholipids (e.g., 5%, 10%, 15%, 20%, 25%, 30%), 15-50% steroids (e.g., 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%), and 0.5-5% PEG lipids (e.g., 0.5%, 1.0%, 1.5%, 2.0%, 2.5%, 3.0%, 3.5%, 4.0%, 4.5%, 5.0%).

[0016] For example, based on molar percentages, the lipid nanoparticles contain 50% ionizable cationic lipids (e.g., SM102, MC3), 10% phospholipids (e.g., DSPC), 38.5% steroids (e.g., cholesterol), and 1.5% PEG lipids (e.g., DMG-PEG2000).

[0017] For another example, based on molar percentage, the lipid nanoparticles contain 35% ionizable cationic lipids (e.g., C12-200), 16% phospholipids (e.g., DOPE), 46.5% steroids (e.g., cholesterol) and 2.5% PEG lipids (e.g., DMG-PEG2000).

[0018] For another example, based on molar percentage, the lipid nanoparticles contain 45% ionizable cationic lipids (e.g., SM102), 9% phospholipids (e.g., DSPC), 34.65% steroids (e.g., cholesterol), 1.35% PEG lipids (e.g., DMG-PEG2000), and 10% electropositive cationic lipids (e.g., DOTAP) or electronegative anionic lipids (e.g., 18PA).

[0019] For another example, based on molar percentage, the lipid nanoparticles contain 35% ionizable cationic lipids (e.g., SM102), 7% phospholipids (e.g., DSPC), 26.95% steroids (e.g., cholesterol), 1.05% PEG lipids (e.g., DMG-PEG2000), and 30% electropositive cationic lipids (e.g., DOTAP) or electronegative anionic lipids (e.g., 18PA).

[0020] According to the present invention, the ratio of ionizable cationic lipid to nucleic acid is about 100:1 to about 1:5 (mass ratio), the ratio of steroid to ionizable cationic lipid is about 10:1 to about 1:20 (molar ratio), the ratio of phospholipid to ionizable cationic lipid is about 5:1 to about 1:50 (molar ratio), and the ratio of PEG lipid to ionizable cationic lipid is about 3:1 to about 1:300 (molar ratio).

[0021] According to the present invention, ionizable cationic lipids include SM102 (heptadecan-9-yl-8-((2-hydroxyethyl)(6-oxo-6-((decyloxy)hexyl)amino)octanoate), 5A2-SC8, C12-200, or DLin-MC3-DMA (dilinoleylmethyl 4-(N,N-dimethylamino)butanoate). 5A2-SC8 (Zhou et al., Proc. Natl. Acad. Sci., 113(3):520-525, 2016), DLin-MC3-DMA (Jayaraman et al., Angew. Chem. Int. Ed., 51:8529-8533, 2012), and C12-200 (Love et al., Proc. Natl. Acad. Sci. USA, 107:1864-1869, 2010) were synthesized and purified by following published protocols.

[0022] According to the present invention, the ionizable cationic lipid may also include the dendrimers shown in CN108271360A.

[0023] According to the present invention, the phospholipid is an amphoteric phospholipid, including DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine), DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine), and DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine).

[0024] According to the present invention, the steroid or its derivative is a sterol, cholestane or its derivative, such as cholesterol.

[0025] According to the present invention, the PEG lipid is shown in the following structural formula:

[0026] in:

[0027] R 12 and R 13 Each is independently alkyl (C≤24), alkenyl (C≤24), or a substituted form of any of these groups;

[0028] Re is hydrogen, alkyl (C≤8) or substituted alkyl (C≤8); and

[0029] x is 1-250.

[0030] According to the present invention, the PEG lipid is represented by the following formula:

[0031] in:

[0032] n1 is 5-250; and

[0033] n2 and n3 are each independently 2-25.

[0034] According to the present invention, the PEG lipid is 1,2-dimyristoyl-sn-glycero-methoxy(polyethylene glycol) MW 2000 (DMG-PEG2000).

[0035] According to the present invention, the electropositive cationic lipids (i.e., permanent cationic lipids, the two are used interchangeably in the present invention) contain positively charged groups regardless of pH, such as quaternary ammonium groups, including DOTAP (1,2-dioleoyl-3-trimethylammonium-propane), DOTAP-Cl (1,2-dioleoyl-3-trimethylammonium-propane (chloride)), dimethyldioctadecyl ammonium (DDAB), 1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (EPC):

[0036] According to the present invention, the electropositive cationic lipid is not limited thereto, as long as it contains a quaternary ammonium head and a hydrophobic carbon chain tail.

[0037] According to the present invention, the electronegative anionic lipids contain a phosphate group, including PS lipids, such as dioleoyl-sn-glycerophosphatidylserine (sodium salt) (DOPS), PG lipids, such as 1,2-dioleoyl-sn-glycero-phosphatidylglycerol (DOPG), PA lipids, such as 1,2-dioleoyl-sn-glycero-3-phosphate (sodium salt) (18PA), 1,2-dimyristoyl-sn-glycero-3-phosphate (sodium salt) (14PA), sn-(3-oleoyl-2-hydroxy)-glycero-1-phospho-sn-3'-(1',2'-dioleoyl)-glycerol (ammonium salt) (18:1Hemi BMP, 18BMP), preferably 18PA, 14PA and 18BMP.

[0038] According to the present invention, the electronegative anionic lipid is not limited thereto, as long as it contains a phosphoric acid, a carboxylic acid head and a hydrophobic carbon chain tail.

[0039] The lipid nanoparticle size is 10-500 nm (e.g., 20 nm, 50 nm, 100 nm, 150 nm, 200 nm, 250 nm, 300 nm, 350 nm, 400 nm, 450 nm), preferably 100-150 nm.

[0040] According to the present invention, the local administration is to retrogradely administer the neutral LNP through the ureter to transfect the renal tubular cells.

[0041] According to the present invention, the local administration is to administer positive and neutral LNPs through the abdominal aorta to transfect glomerular cells (targeting kidney podocytes). Neutral and positive LNPs have similar efficiency, but neutral LNPs are preferred.

[0042] The present invention also provides a use of neutral LNP in preparing a drug for targeted delivery to renal lesion cells, wherein the drug is retrogradely administered through the ureter to transfect renal tubular cells.

[0043] The present invention also provides use of positive or neutral LNP (preferably neutral LNP) in preparing a drug for targeted delivery to renal lesion cells, wherein the drug is administered through the abdominal aorta to transfect glomerular cells.

[0044] The present invention also provides a kit comprising a nucleic acid composition containing neutral LNP and nucleic acid, an instrument or device for retrograde drug administration through the ureter, such as an anesthetic, a syringe (e.g., 100 μL, containing 50 μL of drug) and its needle (e.g., 34G), a renal arteriovenous clamp, and optional instructions for use.

[0045] The present invention also provides a kit comprising a nucleic acid composition containing a positive or neutral LNP (preferably a neutral LNP) and a nucleic acid, an instrument or device for administering the drug through the abdominal aorta, such as an anesthetic, a syringe (e.g., 1 mL, containing 250 μL of drug) and a needle thereof (e.g., 34G), an abdominal artery clamp, a renal vein clamp, and optional instructions for use.

[0046] According to the present invention, the neutral LNP is selected from SM102 LNP, 5A2-SC8 LNP, MC3 LNP and C12-200 LNP.

[0047] According to the present invention, LNP containing permanent cationic lipids is positive LNP, LNP containing neither permanent cationic lipids nor electronegative anionic lipids is neutral LNP, and LNP containing electronegative anionic lipids is negative LNP. Beneficial effects

[0048] The inventors have developed a method for kidney-targeted gene drug delivery and treatment using mRNA-LNPs, primarily delivering drugs to renal lesion cells through a localized drug delivery strategy. The main innovations and key points are as follows:

[0049] LNPs with different properties (positive, negative, and neutral) were prepared, and mRNA-LNPs were retrogradely administered through the ureter. It was found that the nanomedicine mainly transfected renal tubular cells, and that LNPs with different properties corresponded to different types of tubular cells. Among them, neutral LNP had the best transfection effect and specificity, while negative LNP and positive LNP were poor (the former was slightly better than the latter).

[0050] Neutral LNPs have better transfection efficiency in renal tubular cells via retrograde administration through the ureter and are broad-spectrum, that is, they are applicable to any type of neutral LNP (such as the well-known SM102 LNP, MC3 LNP, C12-200 LNP, etc.).

[0051] By administering LNPs through the abdominal aorta, the inventors found that LNPs primarily transfected glomerular cells, and that different LNPs (positive, negative, and neutral) had different abilities to transfect glomerular cells. Both positive and neutral LNPs were better able to target renal podocytes.

[0052] Based on the above conclusions, the inventors can apply mRNA-LNP technology to the targeted delivery of specific kidney cell populations by selecting the route of administration, thereby achieving the treatment of specific kidney genetic diseases.

[0053] The inventors conducted experiments in an autosomal dominant polycystic kidney disease mouse model with a mutation at the R3277C site of polycystin 1 (PC1) encoded by the Pkd1 gene (the main lesions were tubular cells). By retrograde administration through the ureter, they demonstrated that this site could be corrected by an mRNA-LNP-dependent adenine base editor (ABE) and a therapeutic effect was achieved.

[0054] The inventors have developed a broad-spectrum, universal mRNA-LNP kidney-targeted delivery technology that can efficiently and specifically transfect tubular cells and glomerular cells, providing a new gene therapy approach for the treatment of hereditary kidney diseases.

[0055] Currently, targeted delivery of mRNA is difficult, especially for organ and cell-specific delivery outside the liver. In this application, the inventors successfully achieved precise targeted delivery of renal tubular cells, renal vascular endothelial cells, renal glomerular cells, interstitial cells, etc. through strategies such as LNP screening, ureteral retrograde / renal pelvic antegrade administration, and abdominal aorta administration. This effect has important reference significance and value for the treatment of diseases such as hereditary kidney diseases, chronic kidney disease, acute kidney injury, nephrotic syndrome, nephritis, kidney stones, renal failure, kidney transplantation, and kidney tumors; it also provides important reference significance for expanding the application scenarios of mRNA drugs.

[0056] BRIEF DESCRIPTION OF THE DRAWINGS

[0057] To more clearly describe the technical solution of the present invention, a brief introduction will be given below in conjunction with the accompanying drawings. Obviously, these drawings are only some specific embodiments described in this application. The present invention includes but is not limited to these drawings.

[0058] Figure 1-1 shows that the neutral LNP (LNP1) screened by ureteral retrograde drug delivery has the strongest affinity for mouse kidneys;

[0059] Figure 1-2 shows the safety of ureteral retrograde surgery and the safety verification of LNP on mouse kidneys;

[0060] Figure 2 shows that ABE-LNP can effectively edit the kidneys of reporter mice;

[0061] Figure 3 shows the identification of LNP-targeted renal tubular cell types by single-cell sequencing;

[0062] Figure 4-1 shows that the positive and neutral LNPs screened by abdominal aorta administration have the highest efficiency in targeting glomeruli;

[0063] Figure 4-2 shows the safety of abdominal aorta surgery and the safety of LNP on mouse kidneys;

[0064] Figure 5 shows that targeting Pkd1 R3277C sgRNA in vitro screening and treatment results showed that neutral ABE+sgPkd1-LNP can effectively treat ADPKD mouse disease; and

[0065] Figure 6 shows targeting of Col4α5 R373X sgRNA screening and verification of editing efficiency at the cell level. DETAILED DESCRIPTION

[0066] In order to further understand the present invention, the preferred embodiments of the present invention will be described below in conjunction with the embodiments. These descriptions are only examples to illustrate the features and advantages of the technical solutions of the present invention, and are not intended to limit the scope of protection of the present invention.

[0067] Example 1

[0068] Different LNPs have varying affinities for organs or tissues. To identify LNPs with high affinity for the kidneys, the inventors screened multiple LNPs in vivo. Based on the SM102 LNP formulation of the FDA-approved COVID-19 mRNA vaccine (Spikevax), and leveraging their Selective Organ Delivery Technology (SORT) and related research experience, the inventors synthesized positive, neutral, and negative LNPs.

[0069] First, the inventors transcribed Luciferase mRNA (SEQ ID NO: 1) in vitro and encapsulated it into LNPs with different properties using microfluidic technology to create a complete expression system (Figure 1-1A). The specific formula of the LNPs is shown in Table 1-1 below, with a total lipid to RNA mass ratio of 40:1.

[0070] Table 1-1 (corresponding to Figure 1-1A)

[0071] Table 1-2 (corresponding to Figures 1-1B and C) Note: Values ​​are Mean±SD

[0072] Table 1-3 (corresponding to Figure 1-2D)

[0073] Refer to Tables 1-2 and 1-3 above, and refer to Figures 1-1 and 1-2 for a detailed description of this embodiment. Particle size testing revealed that the LNPs had a particle size of approximately 100 nm to 150 nm, with good uniformity (Figure 1-1B). Testing the encapsulation efficiency revealed that most LNPs had a high mRNA encapsulation efficiency, exceeding 80% (Figure 1-1C). Under anesthesia, surgical instruments were used to make an approximately 1 cm incision in the mouse's abdomen / dorsum at the kidney site to expose the kidney. A vascular clamp was used to clamp the renal artery and vein. Using a microsyringe with an extremely fine needle, 50 μL of Luciferase-LNP was injected into the mouse kidney via retrograde ureteral / antegrade renal pelvis injection surgery, clamping the proximal mid-section of the ureter (Figure 1-1A). After 15 minutes, the renal artery and vein clamps were removed to restore renal blood supply; after 30 minutes, the ureteral clamp was removed to restore urine flow. The mouse wound was sutured and the mouse was raised normally after recovery. After 6 hours, the signal intensity of LNPs in different groups was evaluated by in vivo imaging of small animals and imaging of major organs. The imaging results in Figure 1-1D and the quantitative statistical results of the total fluorescence value of the kidney in Figure 1-1E showed that compared with positive LNP (LNP2, LNP3) and negative LNP (LNP4, LNP5), neutral LNP (LNP1) was expressed at the highest level in the kidney, indicating that neutral LNP (LNP1) has the strongest affinity for kidney cells. In addition, under this surgical method and injection volume, no obvious abnormalities were found in the renal function and cell morphology of mice (Figure 1-2), indicating that this operation method and LNP are safe. Analysis of the total fluorescence of different organs showed that compared with negative LNP, the expression of neutral LNP (LNP1) in the kidney has higher specificity, and there is almost no leakage of extrarenal organs (Figure 1-1F). Therefore, by rationally designing positive, neutral, and negative LNPs and combining them with a retrograde ureteral / anterograde renal pelvis injection method in mice to screen for high-affinity LNPs, the inventors screened for a neutral LNP (LNP1) that exhibited the strongest expression and highest specificity in the kidney. Based on these results, other neutral LNPs (LNP6 and LNP7) were tested and similarly demonstrated kidney-specific delivery (Figures 1-1D-F).

[0074] Example 2

[0075] To further explore whether the gene editor encapsulated by the high-affinity LNP screened by the inventors can efficiently edit the kidneys, the inventors constructed membrane-tagged green florescent protein (mGFP) (SEQ ID NO: 2) reporter mice suitable for adenine base editors (ABE): a membrane localization signal was inserted before the GFP sequence, and the CAG base at the front position was replaced with a TAG base (stop codon), causing premature translation termination and no complete functional mGFP protein to be produced. The inventors designed a suitable guide RNA (sgmGFP) (SEQ ID NO: 5) for this site and used the ABE editor to convert the A opposite to the T to a G. The original TAG base became a CAG base, the stop codon disappeared, and mGFP could be translated normally, emitting green fluorescence on the cell membrane. To verify the feasibility of this reporter system, the inventors stably introduced the above-mentioned mGFP with a stop codon (TAG) into HEK 293T cells, mixed ABE and sgmGFP at an appropriate ratio, and transiently transfected the reporter cells. After 24 hours, most cells produced mGFP protein (Figure 2A), proving that the system designed by the inventors can well report whether ABE can work properly.

[0076] To further verify whether the screened neutral LNPs can efficiently play an editing role in vivo after encapsulating ABE, the inventors stabilized the above-mentioned reporter system into the mouse ROSA26 site to construct mGFP-reporter mice. Neutral LNPs encapsulating ABE mRNA (SEQ ID NO: 3) and sgmGFP (mass ratio of 2: 1) were injected into the kidneys of 1-month-old mice using ureteral retrograde / renal pelvic anterograde surgery. Three days later, the mouse kidneys were harvested for protein immunoblotting (WB) and frozen sections to observe protein expression (Figure 2B). The inventors found that when the total injection volume was controlled at 50 μL, as the ABE+sgmGFP-LNP content increased, WB showed an increase in mGFP protein expression, showing a significant dose-dependent relationship (Figure 2C and Table 2-1). We then performed frozen sections on the kidneys, and mGFP immunofluorescence staining found that as the dose increased, the luminous area of ​​the kidneys gradually increased. When the total amount of nucleic acid was increased to 4 mg / kg, half of the kidney area was illuminated (Figure 2D and Table 2-2). This result suggests that the LNP-encapsulated gene editor identified by the inventors can effectively edit mouse kidneys after surgical administration, with the editing efficiency showing a dose-dependent relationship. Zoomed-in results showed that the edited regions were primarily concentrated in the medulla, with some areas of the cortex also being edited (Figure 2E).

[0077] Table 2-1 (corresponding to Figure 2C)

[0078] Table 2-2 (corresponding to Figure 2D)

[0079] Example 3

[0080] Next, the inventors analyzed which renal intrinsic cell types were transfected and edited by the neutral ABE-LNP. The kidneys of mice after surgical administration were digested, and macrophages were removed by flow cytometry after obtaining single-cell samples, and GFP-positive cells were subjected to single-cell sequencing (scRAN-seq) to identify their cell types (Figure 3A). As shown in Figures 3B-C and Table 3, the inventors obtained the gene expression profile of GFP-positive cells. The main cell groups were collecting duct cells, proximal tubule cells, Henle's ring cells, distal tubule cells, mesenchymal cells and vascular endothelial cells, and the proportions of the above-mentioned various cell types were 27.7%, 17.0%, 16.4%, 15.5%, 13.8% and 9.6% respectively (Figure 3D). To further evaluate whether the above cells were edited, the inventors performed fluorescent staining, and the results showed that collecting duct cells, distal tubule cells, loop cells of Henle, proximal tubule cells, mesenchymal cells, and vascular endothelial cells all co-localized with mGFP. Statistics showed that the number of edited cells accounted for 45.5%, 33.0%, 21.6%, 7.2%, 12.1%, and 11.5% of the total number of their respective cell types (Figure 3E). This shows that ABE-LNP based on this administration method can efficiently transfect and edit a variety of renal cells, among which tubular cells have the highest probability of being transfected and edited.

[0081] Table 3 (corresponding to Figures 3B and C)

[0082] Example 4

[0083] Furthermore, the inventors compared the transfection of LNPs to the kidneys after different administration methods. LNPs with different properties (neutral, positive, negative) were injected through the abdominal aorta (same as Figure 1-1LNP1, LNP2, LNP4). Cre-LNPs encapsulating Cre mRNA (SEQ ID NO: 4) were injected into Ai14 reporter mice (CAG-loxP-stop-loxP-tdTomato). Cre can cut the loxP site to allow target cells to express tdTomato fluorescent protein (Figure 4-1A). The inventors performed organ imaging 48 hours after surgery, and neutral and positive LNPs were able to better target the kidneys (Figure 4-1B). The inventors further observed through section staining that neutral and positive LNPs had better targeting and specificity for podocytes (Figures 4-1C-E and Table 4-1). Therefore, the inventors used abdominal aorta injection of different LNPs to screen out positive and neutral LNPs with the best effect on targeting kidney podocytes. Furthermore, under this surgical procedure and injection volume, observation of renal morphology (Figure 4-2A) showed no significant abnormalities. Measurements of serum creatinine (Scr) levels (Figure 4-2B and Table 4-2) and blood urea nitrogen (BUN) levels (Figure 4-2C and Table 4-3) indicated normal renal function in the mice. PAS staining (Figure 4-2D and Table 4-4) and Masson staining (Figure 4-2E and Table 4-5) revealed no renal tubular damage and no renal fibrosis. These results indicate that no significant abnormalities in renal function or structure were observed in the mice, suggesting that this surgical procedure and LNP are safe.

[0084] Table 4-1 (corresponding to Figure 4-1E)

[0085] Table 4-2 (corresponding to Figure 4-2B)

[0086] Table 4-3 (corresponding to Figure 4-2C)

[0087] Table 4-4 (corresponding to Figure 4-2D)

[0088] Table 4-5 (corresponding to Figure 4-2E)

[0089] Example 5

[0090] ADPKD is a typical tubular renal disease, with the collecting duct and proximal tubule being the first sites for cyst formation. Given that ABE-LNPs, based on ureteral retrograde / renal pelvic antegrade injection, can efficiently infect renal tubular cells, the inventors believe they can be used for gene therapy of ADPKD. RC / RCThe mouse is recognized in the field as the model mouse that best simulates the clinical patient's disease situation, and it is a single base site mutation. The inventors selected this mouse as a model mouse and tried to perform gene editing therapy. In order to verify whether the R3277C site can be corrected by ABE and encode normal amino acids, the inventors stabilized the approximately 350bp bases before and after the above site into 293T cells, and based on the base characteristics near R3277C, combined with the ABE editing window, designed 3 sgRNAs (Figure 5A) (SEQ ID NO: 6-8). The ABE plasmid and the 3 sgRNA plasmids were transfected into the above cells respectively, and the editing efficiency was measured after DNA extraction. The results showed that compared with the second and third sgRNAs, the first sgRNA (A5) can effectively edit the mutation site, with an editing efficiency of nearly 80% (Figure 5B and Table 5-1). Sequence alignment analysis found that A5 corrected the site to a normal amino acid without causing changes in the amino acid sequence of nearby sites, suggesting that A5 efficiently and specifically completes gene editing of the target site. To further verify whether A5 can correct endogenous Pkd1 gene mutations, the inventors isolated Pkd1 RC / RC Mouse embryo fibroblasts (MEF) were transfected with ABE mRNA and A5 sgRNA using the above-mentioned neutral LNP. After 72 hours, DNA was extracted and the editing efficiency was measured. It was found that ABE+A5 sgRNA-LNP could effectively edit endogenous Pkd1. RC / RC The mutation was mutated, and the editing efficiency was about 30% (Figure 5C and Table 5-2). At the same time, the inventors collected the membrane proteins of MEF cells before and after editing. WB results showed that the PC1 protein expression of MEF cells after editing was effectively restored (Figure 5D). The above results suggest that the inventors can use the above surgery and editing delivery system to try to treat ADPKD disease mice. The inventors inserted Pkd1 F / F ;Cre / Esr1+ and Pkd1 RC / RC Crossbreeding of mice to obtain Pkd1 F / RCCre / Esr1+ mice. The inventors performed interventions on these mice as shown in Figure 5E: Tamoxifen was used to knock out one normal Pkd1 allele at P9 (where P represents postnatal day, the same below), inducing disease in the mice. ABE+A5 sgPkd1-LNPs were surgically administered via ureteral retrograde / renal pelvic antegrade injection into the left and right kidneys at P30 and P33, respectively. Mice were harvested at P120 and the therapeutic effects were observed. Results showed that compared with the ABE+sgCtrl-LNP (sgCtrl: SEQ ID NO: 12) group, the kidneys in the ABE+A5 sgRNA-LNP group were significantly smaller (Figure 5F), with a lower kidney-to-weight ratio (Figure 5G and Table 5-3), fewer renal cysts (Figures 5H-I and Table 5-3), and renal function was effectively restored (Figure 5J and Table 5-3). After collecting the mouse kidneys and extracting DNA, the editing efficiency was measured and it was found that the proportion of mouse kidney cells edited ranged from 0.31% to 1.96% (Figure 5K and Table 5-4). By analyzing the correlation between editing efficiency and cyst coefficient, it was found that editing efficiency was significantly positively correlated with therapeutic effect, that is, the higher the editing efficiency, the better the therapeutic effect, and the greater the reduction in cyst coefficient (Figure 5L). The above results suggest that the LNP encapsulated ABE+A5sgRNA screened by the inventors can be delivered to the kidneys through ureteral retrograde / renal pelvic antegrade injection surgery to effectively complete Pkd1 F / RC Gene editing can effectively treat ADPKD.

[0091] Table 5-1 (corresponding to Figure 5B)

[0092] Table 5-2 (corresponding to Figure 5C)

[0093] Table 5-3 (corresponding to Figures 5G, I, and J)

[0094] Table 5-4 (corresponding to Figure 5K)

[0095] Example 6

[0096] AS is a typical glomerular disease, and the main site of disease is the podocytes of the glomeruli. R373X After screening sgRNA (Figure 6A), the inventors confirmed by Sanger sequencing that sgRNA2 (SEQ ID NO: 10) had a stronger editing efficiency (about 80%) for the mutation site (Figure 6B). The inventors further used sgRNA2 to modify the mutant Col4α5 R373XCell-level repair was performed, and both WB and immunofluorescence results confirmed that the ABE system could efficiently restore Col4α5 protein expression ( Figure 6C-D ).

[0097] Table 6 (corresponding to Figure 6B)

[0098] The above description of the specific embodiments is only intended to help understand the core concept of the present invention. It should be noted that, for those skilled in the art, without departing from the principles of the present invention, several improvements and modifications may be made to the technical solution of the present invention, and such improvements and modifications also fall within the scope of protection claimed in the claims of the present invention.

[0099] mRNA sequence information

[0100] sgRNA sequence information

Claims

1. A method for treating kidney disease, comprising delivering a nucleic acid composition comprising lipid nanoparticles (LNPs) and nucleic acid to a patient in need thereof by local targeted administration.

2. The method of claim 1, wherein the kidney disease is selected from the group consisting of hereditary kidney disease, chronic kidney disease, acute kidney injury, nephrotic syndrome, nephritis, kidney stones, renal failure, kidney transplantation, and kidney tumors.

3. The method according to claim 1, wherein the renal disease is selected from diseases associated with the glomerulus, for example, congenital steroid-resistant nephrotic syndrome, Dennis-Draser syndrome, Fraser syndrome, WAGR syndrome (Wilms tumor, aniridia, genitourinary anomalies, mental retardation), Pierson syndrome, nail-patellar syndrome, Schimke immune-osteodystrophy, mitochondrial disease with steroid-resistant nephrotic syndrome, Fabry disease, Alport syndrome, benign familial hematuria (thin basement membrane), Fechtner syndrome (Alport syndrome with macrothrombocytopenia), Alport syndrome with leiomyomatosis, familial amyloidosis; and diseases associated with the renal tubules, for example, proximal tubule diseases: renal glycosuria, disaccharidemia, lysine protein intolerance, proximal renal tubular acidosis, hypophosphatemic rickets, nephrotic cystinosis, primary renal Fanconi syndrome, Fanconi syndrome. Nie-Bickel syndrome (hepatorenal glycogenosis), Lowe's syndrome, Dunn's disease types 1 and 2, hereditary renal hypouricemia, cystinuria types 1-3, X-linked hypophosphatemic rickets, Hartnap disease, iminoglycosuria, disaccharidemia, large limb ascending and distal tubule diseases: Bartter syndrome, familial hypocalcemic hypercalcemia types 1-4, severe neonatal hyperparathyroidism, autosomal dominant hypocalcemia, Gitelman syndrome, pseudohyperaldosteronism type 2 (Gordon syndrome), syndrome), SeSAME syndrome (EAST syndrome), hypomagnesemia, familial juvenile hyperuricemic nephropathy types 1-6, autosomal dominant hypocalcemia, Kenneth-Caffey syndrome type 2, HNF1B-related nephropathy, collecting duct diseases: Liddle syndrome, distal renal tubular acidosis, pseudohypoaldosteronism type 1, nephrogenic diabetes insipidus types 1 and 2, inappropriate antidiuretic nephrogenic syndrome, overt mineralocorticoid excess, and congenital adrenal hyperplasia types 1-5.

4. The method of claim 1, wherein the nucleic acid is selected from siRNA, miRNA, primary-miRNA, messenger RNA (mRNA), clustered regularly interspaced short palindromic repeats (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-stranded DNA (ssDNA), single-stranded RNA (ssRNA) and double-stranded RNA (dsRNA), preferably mRNA (protein replacement therapy), mRNA / sgRNA (base repair), mRNA / sgRNA / HDR (gene repair).

5. The method of claim 1, wherein the LNP comprises an ionizable cationic lipid and a helper lipid selected from a steroid, a steroid derivative, a PEG lipid, or a phospholipid.

6. The method of claim 1, wherein the LNP comprises an ionizable cationic lipid, a permanent cationic lipid (electropositive cationic lipid), and a phospholipid.

7. The method of claim 1, wherein the LNP comprises ionizable cationic lipids, phospholipids, steroids and PEG lipids, and optionally electropositive cationic lipids and / or electronegative anionic lipids.

8. The method according to any one of the preceding claims, wherein the mass ratio of total lipids to nucleic acids in the nucleic acid composition is 1-100:

1.

9. The method of claim 7, wherein the LNP comprises, on a mole percentage basis: 10-65% ionizable cationic lipids, 0-50% electropositive cationic lipids and / or electronegative anionic lipids, 5-30% phospholipids, 15-50% steroids and 0.5-5% PEG lipids.

10. The method of claim 7, wherein the ratio of ionizable cationic lipid to nucleic acid is about 100:1 to about 1:5 (mass ratio), the ratio of steroid to ionizable cationic lipid is about 10:1 to about 1:20 (molar ratio), the ratio of phospholipid to ionizable cationic lipid is about 5:1 to about 1:50 (molar ratio), and the ratio of PEG lipid to ionizable cationic lipid is about 3:1 to about 1:300 (molar ratio).

11. The method according to any one of claims 5 to 7, wherein the ionizable cationic lipid is selected from SM102 (heptadecan-9-yl-8-((2-hydroxyethyl)(6-oxo-6-((decyloxy)hexyl)amino)octanoate), 5A2-SC8, C12-200 or DLin-MC3-DMA (4-(N,N-dimethylamino)butanoic acid (dicinoleyl)methyl ester).

12. The method according to any one of claims 5 to 7, wherein the phospholipid is selected from DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine), DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine), and DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine).

13. The method according to claim 5 or 7, wherein the steroid is selected from sterols and cholestane, preferably cholesterol.

14. The method of claim 5 or 7, wherein the PEG lipid is represented by the formula: in: R 12 and R 13 Each is independently alkyl (C≤24), alkenyl (C≤24), or a substituted form of any of these groups; Re is hydrogen, alkyl (C≤8) or substituted alkyl (C≤8); and x is 1-250.

15. The method of claim 5 or 7, wherein the PEG lipid is represented by the formula: in: n1 is 5-250; and n2 and n3 are each independently 2-25.

16. The method of claim 5 or 7, wherein the PEG lipid is 1,2-dimyristoyl-sn-glycero-methoxy(polyethylene glycol) (MW 2000) (DMG-PEG2000).

17. The method according to claim 6 or 7, wherein the electropositive cationic lipid contains a quaternary ammonium head and a hydrophobic carbon chain tail, and is preferably selected from DOTAP (1,2-dioleoyl-3-trimethylammonium-propane), DOTAP-Cl (1,2-dioleoyl-3-trimethylammonium-propane (chloride)), dimethyldioctadecyl ammonium (DDAB), and 1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (EPC).

18. The method according to claim 7, wherein the electronegative anionic lipid contains a phosphoric acid or carboxylic acid head and a hydrophobic carbon chain tail, including PS lipids, such as dioleoyl-sn-glycerophosphatidylserine (sodium salt) (DOPS), PG lipids, such as 1,2-dioleoyl-sn-glycero-phosphatidylglycerol (DOPG), PA lipids, such as 1,2-dioleoyl-sn-glycero-3-phosphate (sodium salt) (18PA), 1,2-dimyristoyl-sn-glycero-3-phosphate (sodium salt) (14PA), sn-(3-oleoyl-2-hydroxy)-glycero-1-phospho-sn-3'-(1',2'-dioleoyl)-glycerol (ammonium salt) (18:1HemiBMP, 18BMP).

19. The method according to any one of the preceding claims, wherein the LNP has a nanoparticle size of 10-500 nm, preferably 100-150 nm.

20. The method of claim 1, wherein the local administration is retrograde administration of neutral LNPs through the ureter to transfect renal tubular cells.

21. The method according to claim 1, wherein the local administration is to administer positive or neutral LNPs through the abdominal artery to transfect glomerular cells (targeting kidney podocytes), preferably neutral LNPs.

22. The method of claim 20 or 21, wherein the neutral LNP is selected from SM102 LNP, 5A2-SC8 LNP, MC3 LNP, and C12-200 LNP.

23. Use of neutral LNP in preparing a drug for targeted delivery to renal lesion cells, wherein the drug is retrogradely administered through the ureter to transfect renal tubular cells.

24. Use of positive or neutral LNP (preferably neutral LNP) in the preparation of a drug for targeted delivery to renal lesion cells, wherein the drug is administered via abdominal artery to transfect glomerular cells.

25. Use according to claim 23 or 24, wherein the neutral LNP is selected from SM102 LNP, 5A2-SC8 LNP, MC3 LNP and C12-200 LNP.

26. A kit comprising a nucleic acid composition comprising neutral LNPs and a nucleic acid, a device or apparatus for retrograde administration through the ureter, and optionally instructions for use.

27. A kit comprising a nucleic acid composition comprising a positive or neutral LNP (preferably a neutral LNP) and a nucleic acid, a device or apparatus for administration via the abdominal artery, and optionally instructions for use.

28. The kit according to claim 26 or 27, wherein the neutral LNP is selected from SM 102 LNP, 5A2-SC8 LNP, MC3 LNP and C12-200 LNP.

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