A liver-targeted gene therapy for lald

Abstract

The present application relates to gene therapy, in particular to liver-targeted gene therapy for LALD, and specifically to a gene therapy vector (such as an AAV vector) containing a LAL-encoding gene, which achieves efficient and specific expression of a therapeutic gene in the liver. Experiments show that the LAL gene delivered by the vector can efficiently express LAL protein in a liver-specific, stable and dose-dependent manner, and can significantly repair the ultrastructural damage of hepatocytes in the model, and can not only inhibit the development of liver fibrosis in LALD mice with severe disease, but more importantly, can to some extent make the fibrosis reversible, providing a basis for future clinical treatment of LALD patients in the middle and late stages.

Description

Technical Field

[0001] This invention relates to gene therapy, and more particularly to a gene therapy for liver-targeted treatment of LALD. Background Technology

[0002] Lysosomal acid lipase deficiency (LALD) is a rare autosomal recessive metabolic disorder caused by a mutation in the lysosomal acid lipase gene (LIPA) located on chromosome 10q23.31. This mutation results in a deficiency or loss of the encoded lysosomal acid lipase (LAL), leading to impaired hydrolysis of cholesterol esters and triglycerides within cells, which accumulate in various tissues and cells, causing liver and cardiovascular diseases and their complications. Based on the degree of enzyme deficiency and clinical presentation, LALD is classified into two clinical phenotypes: Wolman disease (WD) and cholesterol ester storage disease (CESD). WD is characterized by hepatomegaly with liver dysfunction, dyslipidemia, hepatosplenomegaly, pulmonary fibrosis, adrenal calcification, and adrenal insufficiency. The disease typically manifests in infants within the first month of life, and they fail to thrive, likely due to liver disease and the inability to absorb nutrients through the intestinal wall. The median lifespan of untreated WD infants is 3.7 months. Partial loss-of-function LIPA mutations, typically with 1-12% of normal activity, cause CESD, a later-onset, less severe form of the disease. Although CESD does not necessarily lead to premature death, it is associated with significant morbidity, including liver fibrosis and cirrhosis (as well as liver failure).

[0003] Current treatment for LALD includes symptomatic supportive care (lipid-lowering therapy) and etiological treatment targeting the enzyme deficiency. WD clinical treatment primarily focuses on symptomatic support, including low-fat or fat-free formula feeding, parenteral nutrition, fat-soluble vitamin supplementation, and glucocorticoid and mineralocorticoid replacement therapy. Early hematopoietic stem cell transplantation can prevent liver failure and death. In 2000, the first successful bone marrow transplant in a WD child was reported in the United States. In 2007, Israeli researchers reported a successful umbilical cord blood transplant in a WD child. Some reports indicate that a few children can survive up to 11 years, but the transplant success rate is low. CESD patients generally receive lipid-lowering drugs such as statins and cholestyramine; some patients experience a decrease in low-density lipoprotein (LDL) levels, but these are ineffective against liver lesions.

[0004] In December 2015, the FDA approved Sebelipase alfa as the first orphan drug for the treatment of late-onset LALD. Sebelipase alfa is a recombinant human LAL extracted from the egg white of eggs produced by genetically modified chickens. This recombinant human LAL is likely taken up by mannose and M6P receptors, but its detailed histological biodistribution has not been reported. In patients with late-onset LALD, the most significant effects are the normalization of liver transaminase (AST) levels and a significant decrease in LDL-C, lasting for at least 5 years. However, biopsies in individual cases showed liver fibrosis and worsening lobular inflammation. Patients with early-onset LALD have high survival rates, with improvements primarily in gastrointestinal symptoms, growth and development, and AST levels.

[0005] Adeno-associated virus (AAV) was first discovered in the 1960s. AAV cannot self-replicate, and wild-type AAV has never been found to cause disease in humans. Recombinant AAV (rAAV) removes 96% of the wild-type AAV genome, further ensuring its safety. AAV has a broad host range, capable of transducing both dividing and quiescent cells. AAV exhibits strong targeting, allowing for selective transfection of specific organs. Its physical properties are stable, and rAAV can stably express exogenous genes long-term. These characteristics make AAV a vector for breakthrough gene therapy, used to treat genetically modified diseases. AAV gene therapy covers various disease types, including ophthalmic diseases, neurological diseases, muscle diseases, and cardiovascular diseases. Currently, 21 AAV projects in development globally involve lysosomal-related metabolic diseases, mostly focusing on carbohydrate metabolism disorders; there are currently no projects in development targeting LALD (Lactosomal Deficiency Disease).

[0006] In 2022, P. Lam et al. used the recombinant adeno-associated virus vector rscAAVrh74.mCMV.LIPA to deliver the functional LIPA gene to LALD mice, achieving high expression levels and sustained enzyme activity, thereby correcting defects and reducing lipid accumulation. In this study, a dose of 2.1E13 vg / kg was required for improvement in hepatosplenomegaly in LALD mice, far exceeding the conventional doses for AAV in mouse experiments. This patent uses a broad-spectrum CMV promoter to initiate LIPA expression in various tissues throughout the body. Because cell division rates differ across tissues—for example, liver cells divide rapidly while eye cells divide slowly—the viral vector persists for varying durations in different tissues, hindering secondary administration. To address this issue, this class of AAV drugs, in addition to employing a hepatotropic rAAV capsid, typically uses a liver-specific promoter, ensuring that the AAV drug targets the liver and is specifically expressed only in the liver. Furthermore, the main symptoms of this disease are hepatosplenomegaly.

[0007] Therefore, there is an urgent need to design a liver-specific, high-efficiency rAAV particle that expresses LAL protein, so that it can effectively improve the symptoms of hepatosplenomegaly in LALD patients at the lowest possible dosage, while reducing the production of AAV neutralizing antibodies and hepatotoxicity. Summary of the Invention

[0008] In view of the shortcomings of the prior art described above, the purpose of this invention is to provide a gene therapy for liver-targeted treatment of LALD, in order to solve the problems in the prior art.

[0009] To achieve the above and other related objectives, the present invention first provides a recombinant AAV (rAAV) vector, wherein the rAAV vector comprises a gene expression cassette and AAV ITR sequences located at the 5' and 3' ends of the gene expression cassette; the gene expression cassette comprises:

[0010] A) The polynucleotide sequence encoding LAL;

[0011] B) A liver-specific transcriptional regulatory region, wherein the liver-specific transcriptional regulatory region is operatively linked to the polynucleotide sequence encoding LAL;

[0012] C) Polynucleotide signal, wherein the polynucleotide signal is located at the 3' end of the gene expression cassette;

[0013] The polynucleotide sequence encodes an amino acid sequence as shown in SEQ ID NO.2.

[0014] The present invention also provides an rAAV vector system, which includes a packaging plasmid, an expression plasmid, and an auxiliary plasmid, wherein the expression plasmid is the above-mentioned rAAV vector.

[0015] The present invention also provides an rAAV particle, wherein the rAAV particle comprises the above-described rAAV carrier and AAV capsid, or is obtained by viral packaging via the above-described rAAV carrier system.

[0016] The present invention also provides a pharmaceutical composition comprising the above-described rAAV carrier, or the above-described rAAV carrier system, or the above-described rAAV particles, and a pharmaceutically acceptable carrier, diluent, or excipient.

[0017] The present invention also provides the use of the above-described rAAV carrier, or the above-described rAAV carrier system, or the above-described rAAV particles, or the above-described pharmaceutical composition in the preparation of a medicament for treating LALD.

[0018] The present invention also provides a method for preparing rAAV vector particles, the method comprising culturing cells transfected with any of the above-described rAAV vectors and recovering rAAV particles from the supernatant of the transfected cells.

[0019] The present invention also provides a method for treating LALD, the method comprising administering to a subject a therapeutically effective amount of the above-described rAAV carrier or the above-described pharmaceutical composition.

[0020] As described above, the gene therapy for liver-targeted treatment of LALD of the present invention has the following beneficial effects: the obtained rAAV particles that specifically and efficiently express LAL protein in the liver are screened, which can effectively improve the symptoms of hepatosplenomegaly in LALD patients at the lowest possible dosage, while reducing the production of AAV neutralizing antibodies and hepatotoxicity.

[0021] 1) In young mice: The gene therapy vector encoding LAL delivered LAL stably and dose-dependently resulted in efficient expression of the LAL protein, significantly restoring serum and hepatic LAL enzyme activity in mice. At the experimental endpoint, Lipa-KO mice showed increased body weight, and the volume and weight of the liver and spleen, as well as the weight of other related organs, returned to near-normal levels. Furthermore, serum transaminase (ALT, AST) levels were significantly reduced, indicating effective relief of liver injury. In addition, lipid metabolism abnormalities were also restored, especially with high-dose drug (6E12 vg / kg), which significantly repaired the ultrastructural damage of hepatocytes in the model.

[0022] 2) For older Lipa-KO mice with more severe fibrosis: The gene therapy vector encoding LAL was used to deliver LAL. The presence of rAAV ITR DNA, transcribed LIPA mRNA and expressed LAL protein was detected in Lipa-KO mice. The stability and dose-dependency of LAL protein expression activity were determined by LAL enzyme activity assay in serum, liver and spleen tissues.

[0023] Furthermore, 13 weeks after injection, the volume and weight of the liver and spleen, as well as the weight of other related organs, in mice were significantly reduced, approaching the tissue weight and morphology of normal mice. Serum liver function indicators (ALT, AST) showed significant improvement, and blood lipid indicators (TG, CHO, LDL-C, HDL-C, NEFA, and FC) also recovered to some extent. Blood lipid indicators in the liver and spleen tissues of mice were also significantly improved. In summary, through effective gene delivery and expression, the function of LAL was restored, and not only was the development of liver fibrosis inhibited, but more importantly, fibrosis could be made reversible to a certain extent. The therapeutic and salvage efficacy in severely diseased LALD mice provides a basis for the mid-to-late stage clinical treatment of LALD patients in the future. Attached Figure Description

[0024] Figure 1The diagram shows a candidate molecular expression vector constructed in Example 1 of this invention. “p6-7K(p6-10)” is an ssAAV genome; “p6-7K(p6-13)” is a scAAV genome; and “p6-7K(p6-14)” is a scAAV genome.

[0025] Figure 2 The diagram shows a comparison of the yields of the three types of viral stock solutions prepared in Example 2 of this invention.

[0026] Figure 3 The figures shown are schematic diagrams comparing candidate molecules in in vitro cells in Example 3 of this invention. Figure A shows a Western blotting diagram of LAL protein expression and secretion after infection with different MOI rAAV-Huh7 cells; Figure B shows a schematic diagram of LAL activity in the supernatant after infection with different MOI rAAV-Huh7 cells; Figure C shows a schematic diagram of LAL activity in cells after infection with different MOI rAAV-Huh7 cells; Figure D shows a Western blotting diagram of N-glycosylation modification analysis of LAL protein; Figure E shows a schematic diagram of the M6P modification level of LAL verified by the mannose-6-phosphate (M6P) receptor binding experiment in K562 cells.

[0027] Figure 4 The figures shown are comparative schematic diagrams of the candidate molecules in in vivo experiments in Example 4 of this invention. Figure A shows a timeline diagram of the mouse experiment endpoint; Figure B shows a WB diagram of LAL expression in the liver; Figure C shows a WB diagram of LAL glycosylation modification in the liver as determined by enzyme digestion; Figure D shows a schematic diagram of the number of AAV vector genomes in the liver; Figure E shows a schematic diagram of LIPA mRNA levels in the liver; Figure F shows a schematic diagram of LAL activity in the liver; Figure G shows a schematic diagram of the number of AAV vector genomes in the spleen; Figure H shows a schematic diagram of LIPA mRNA levels in the spleen; Figure I shows a schematic diagram of LAL activity in the spleen.

[0028] Figure 5 The figures show schematic diagrams of drug expression in young mice in Example 5 of this invention. Figure A shows a schematic diagram of rAAV ITR genome number detection in the liver using ITR as the amplification target; Figure B shows a schematic diagram of LIPA mRNA level in the liver; Figure C shows a WB diagram of LAL expression in the liver; Figure D shows a schematic diagram of LAL activity in serum; Figures EF show schematic diagrams of LAL activity in the liver and spleen.

[0029] Figure 6The figures shown are schematic diagrams illustrating the changes in body weight and organ weight of mice in each group during Example 5 of this invention. Figure A shows a timeline diagram of the mouse experiment endpoint; Figure B shows a schematic diagram of the changes in body weight of mice in each group; Figure C shows photographs of the liver and spleen of mice in each group; Figure DJ shows a comparison of the relative body weight percentages of the liver, spleen, ileum, duodenum, jejunum, bilateral inguinal fat, and back brown fat of mice in each group.

[0030] Figure 7 The figures show the changes in lipid metabolism indicators and tissue staining in mice in each group in Example 5 of this invention. Figures AB show the changes in liver function indicators (ALT, AST); Figure CH shows the changes in blood lipid metabolism indicators (TG, CHO, NEFA, FC, HDL-C, LDL-C); Figure IL shows the changes in TG and TC levels in the liver and spleen; Figure MN shows the Oil Red O staining images of liver and spleen tissues.

[0031] Figure 8 The figures shown are pathological changes in the liver and spleen of mice in each group in Example 5 of this invention. Figure A shows the HE staining of liver tissue; Figure B shows the Masson staining of liver tissue; Figure C shows the electron microscopy results of the liver; Figure D shows the mRNA expression levels of related genes in the liver; Figure E shows the immunohistochemical staining score of CD68 protein expression in the liver; Figure F shows the WB diagram of autophagy-related genes; Figure G shows the HE staining of spleen tissue; Figure H shows the immunohistochemical staining score of CD68 protein expression in the spleen.

[0032] Figure 9 The figures show schematic diagrams of drug expression in aged mice in Example 6 of this invention. Figure A shows a schematic diagram of rAAV ITR genome number detection in the liver using ITR as the amplification target; Figure B shows a schematic diagram of LIPA mRNA level in the liver; Figure C shows a WB diagram of LAL expression in the liver; Figure D shows a schematic diagram of LAL activity in serum; Figures EF show schematic diagrams of LAL activity in the liver and spleen.

[0033] Figure 10 The figures shown are schematic diagrams illustrating the changes in body weight and organ weight of mice in each group during Example 6 of this invention. Figure A shows a timeline diagram of the mouse experiment endpoint; Figure B shows a schematic diagram of the changes in body weight of mice in each group; Figure C shows photographs of the liver and spleen of mice in each group; Figure DJ shows a comparison of the relative body weight percentages of the liver, spleen, ileum, duodenum, jejunum, bilateral inguinal fat, and back brown fat of mice in each group.

[0034] Figure 11The figures show the changes in lipid metabolism indicators and tissue staining in mice in each group in Example 6 of this invention. Figures AB show the changes in liver function indicators (ALT, AST); Figure CH shows the changes in blood lipid metabolism indicators (TG, CHO, NEFA, FC, HDL-C, LDL-C); Figure IL shows the changes in TG and TC levels in the liver and spleen; Figure MN shows the Oil Red O staining images of liver and spleen tissues.

[0035] Figure 12 The figures shown are pathological changes in the liver and spleen of mice in each group in Example 6 of this invention. Figure A shows a liver tissue HE staining image; Figure B shows a liver tissue Masson staining image; Figure C shows a WB image of autophagy-related genes; Figure D shows a spleen tissue HE staining image. Detailed Implementation

[0036] This invention first provides a recombinant AAV (rAAV) vector, wherein the rAAV vector comprises a gene expression cassette and AAV ITR sequences located at the 5' and 3' ends of the gene expression cassette; the gene expression cassette comprises:

[0037] A) The polynucleotide sequence encoding LAL;

[0038] B) A liver-specific transcriptional regulatory region, wherein the liver-specific transcriptional regulatory region is operatively linked to the polynucleotide sequence encoding LAL;

[0039] C) Polynucleotide signal, wherein the polynucleotide signal is located at the 3' end of the gene expression cassette;

[0040] The polynucleotide sequence encodes an amino acid sequence as shown in SEQ ID NO.2.

[0041] In some embodiments of the present invention, the liver-specific transcriptional regulatory region contains a liver-specific promoter.

[0042] In a preferred embodiment of the present invention, the liver-specific promoter is selected from the TBG promoter, the Alb promoter, or the ApoEHCR-hAAT promoter. Specifically, the nucleotide sequence of the TBG promoter is shown in SEQ ID NO.7.

[0043] In some embodiments of the present invention, the liver-specific transcriptional regulatory region further comprises an enhancer. For example, the enhancer is an Alpha mic / bik element. Specifically, the nucleotide sequence of the Alpha mic / bik element is shown in SEQ ID NO. 3.

[0044] In some embodiments of the present invention, the polynucleotide signal (PA) is selected from simian virus 40 polyadenylation signal (SV40PA), bovine growth hormone polyadenylation signal (BGHPA), human growth hormone polyadenylation signal (HGHpA), artificially synthesized polyadenylation signal 48 (PA48), or fragments thereof.

[0045] In a preferred embodiment of the present invention, the nucleotide sequence of the SV40PA is as shown in SEQ ID NO.5.

[0046] In a preferred embodiment of the present invention, the nucleotide sequence of the BGHPA is as shown in SEQ ID NO. 6.

[0047] In some embodiments of the present invention, in order to enhance the expression efficiency of the target protein, the expression cassette further includes one or more expression enhancement elements; for example, the expression cassette includes a WPRE3 element, which is used to promote the stability and translation efficiency of LIPAmRNA. Specifically, the nucleotide sequence of the WPRE3 element is shown in SEQ ID NO.4.

[0048] In some embodiments of the present invention, the polynucleotide encoding LAL is codon-optimized.

[0049] In some embodiments of the invention, the polynucleotide sequence encoding LAL comprises, or is substantially composed of, a sequence that is at least 65%, at least 70%, at least 75%, at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89%, more typically 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher of the nucleotide sequence shown in SEQ ID NO.1, and encodes a protein that retains LAL activity.

[0050] In some embodiments of the invention, the rAAV vector described herein comprises, or is substantially composed of, a polynucleotide sequence encoding LAL, which has, for example, at least 65%, at least 70%, at least 75%, at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89%, more typically 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity with the amino acid sequence shown in SEQ ID NO. 2, and the protein retains LAL activity.

[0051] In some embodiments of the present invention, the rAAV vector is a self-complementary AAV (scAAV) vector, wherein the scAAV vector contains a self-complementary AAV vector genome.

[0052] Specifically, the nucleotide sequence of the 5' ITR sequence is shown in SEQ ID NO.15; and / or, the nucleotide sequence of the 3' ITR sequence is shown in SEQ ID NO.16.

[0053] In some embodiments of the present invention, the rAAV vector is a single-stranded AAV (ssAAV) vector. The ssAAV vector contains a single-stranded AAV vector genome.

[0054] Specifically, the nucleotide sequence of the 5' ITR sequence is shown in SEQ ID NO.17; and / or, the nucleotide sequence of the 3' ITR sequence is shown in SEQ ID NO.18.

[0055] In some embodiments of the present invention, the rAAV carrier comprises any of the following structures:

[0056] 1) 5'-ITR-Alpha mic / bik-TBG-encoding LAL polynucleotide sequence-WPRE3-SV40PA-3';

[0057] 2) 5'-ITR-Alpha mic / bik-TBG-encoding LAL polynucleotide sequence-BGHPA-ITR-3';

[0058] 3) 5'-ITR-TBG-encoding LAL polynucleotide sequence-WPRE3-SV40PA ITR-3'.

[0059] In some embodiments of the present invention, the rAAV vector shown in structure 1) is an ssAAV vector, the nucleotide sequence of its 5' ITR sequence is shown in SEQ ID NO. 17; and / or, the nucleotide sequence of its 3' ITR sequence is shown in SEQ ID NO. 18.

[0060] In some embodiments of the present invention, the rAAV vector shown in structure 2) is an scAAV vector, the nucleotide sequence of its 5' ITR sequence is as shown in SEQ ID NO. 15; and / or, the nucleotide sequence of its 3' ITR sequence is as shown in SEQ ID NO. 16.

[0061] In some embodiments of the present invention, the rAAV vector shown in structure 3) is an scAAV vector, the nucleotide sequence of its 5' ITR sequence is shown in SEQ ID NO. 15; and / or, the nucleotide sequence of its 3' ITR sequence is shown in SEQ ID NO. 16.

[0062] In some embodiments of the present invention, the rAAV vector comprises any of the following nucleotide sequences:

[0063] 1) The nucleotide sequence shown in SEQ ID NO. 8;

[0064] 2) The nucleotide sequence shown in SEQ ID NO. 9;

[0065] 3) The nucleotide sequence shown in SEQ ID NO.10.

[0066] The present invention also provides an rAAV vector system, which includes a packaging plasmid, an expression plasmid, and an auxiliary plasmid, wherein the expression plasmid is the above-mentioned rAAV vector.

[0067] After the packaging plasmid, expression plasmid, and helper virus plasmid are transferred into cells, all of their nucleic acid sequences are integrated into the cells to produce adeno-associated virus.

[0068] The present invention also provides an rAAV particle, wherein the rAAV particle comprises the above-described rAAV carrier and AAV capsid, or is obtained by viral packaging via the above-described rAAV carrier system.

[0069] In some embodiments of the present invention, the serotype of the AAV capsid is selected from AAV1, AAV2, AAV5, AAV6, AAV8, AAV9 or AAV-LK03.

[0070] Furthermore, the serotype of the AAV capsid is selected from AAV2, AAV8, AAV9 or AAV-LK03; the AAV capsid of the above serotypes is an AAV capsid targeting the liver.

[0071] Furthermore, the serotype of the AAV capsid is AAV8, and its nucleotide sequence is shown in SEQ ID NO.11.

[0072] The present invention also provides a pharmaceutical composition comprising the above-described rAAV carrier, or the above-described rAAV carrier system, or the above-described rAAV particles, and a pharmaceutically acceptable carrier, diluent, or excipient.

[0073] In the pharmaceutical composition provided by the present invention, the rAAV carrier or rAAV particles are a single active ingredient, or they can be combined with one or more other active ingredients useful for the treatment of LALD disease to form a combined formulation.

[0074] The content of the active ingredient in the composition is a safe and effective amount, which should be adjustable by those skilled in the art. For example, the dosage of the active ingredient in the rAAV carrier and pharmaceutical composition depends on the subject / patient's weight, the type of application, and the condition and severity of the disease.

[0075] The term "pharmaceutically acceptable" means that a compound, material, composition, and / or dosage form is suitable for use in contact with human and animal tissues, within reasonable medical judgment, without excessive toxicity, irritation, allergic reactions, or other problems or complications, and has a reasonable benefit / risk ratio.

[0076] The term "pharmaceutically acceptable carrier, diluent, or excipient" includes, but is not limited to, any adjuvant, carrier, excipient, gliding agent, sweetener, diluent, preservative, dye / coloring agent, flavor enhancer, surfactant, wetting agent, dispersant, suspending agent stabilizer, isotonic agent, solvent, surfactant, or emulsifier approved by the U.S. Food and Drug Administration for use in humans and / or livestock.

[0077] The present invention also provides the use of the above-described rAAV carrier, or the above-described rAAV carrier system, or the above-described rAAV particles, or the above-described pharmaceutical composition in the preparation of a medicament for treating LALD.

[0078] The present invention also provides a method for preparing rAAV vector particles, the method comprising culturing cells transfected with any of the above-described rAAV vectors and recovering rAAV particles from the supernatant of the transfected cells.

[0079] The present invention also provides a method for treating LALD, the method comprising administering to a subject a therapeutically effective amount of the above-described rAAV carrier, or the above-described carrier system, or the above-described pharmaceutical composition. In the present invention, LALD disease in a subject can be treated by administering to a subject a therapeutically effective amount of the above-described rAAV carrier, or the above-described carrier system, or the above-described pharmaceutical composition.

[0080] The term "subject" includes animals, such as mammals. In some embodiments, the mammal is a primate. In some embodiments, the mammal is a human. In some embodiments, the subject is livestock, such as cattle, sheep, goats, dairy cows, pigs, etc.; or domesticated animals, such as dogs and cats. In some embodiments (e.g., particularly in a research context), the subject is rodents (e.g., mice, rats, hamsters), rabbits, primates, or pigs (e.g., inbred pigs), etc. The term "subject" may be used interchangeably with "subject" or "object of action."

[0081] The term "treatment" refers to the delivery of a drug or composition to a subject to affect physiological outcomes. In some embodiments, treatment refers to the treatment of a disease in a subject (e.g., a human), including (a) suppressing the disease, such as halting the development of the disease or preventing its progression; (b) alleviating the disease, such as causing the remission of the disease state; (c) curing the disease; and (d) preventing the onset of the disease.

[0082] The term "application" refers to the introduction of a drug or composition into a subject.

[0083] In some embodiments of the invention, the routes of administration for applying the above-described carrier or a composition comprising the above-described carrier include conventional and pharmaceutically acceptable routes of administration, including but not limited to: direct delivery to the desired organ (e.g., the liver (optionally via the hepatic artery) or the lung), oral, inhalation, intranasal, intratracheal, intra-arterial, intraocular, intravenous, intramuscular, subcutaneous, intradermal, and other routes of administration. Routes of administration may be combined if desired.

[0084] The following specific examples illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention.

[0085] Before further describing specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terminology used in the embodiments of the present invention is for describing specific embodiments and not for limiting the scope of protection of the present invention; in the specification and claims of the present invention, unless otherwise expressly stated in the text, the singular forms "a", "an" and "this" include the plural forms.

[0086] When numerical ranges are given in the embodiments, it should be understood that, unless otherwise stated in the present invention, both endpoints of each numerical range and any value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art. In addition to the specific methods, apparatus, and materials used in the embodiments, based on the knowledge of the prior art possessed by one of ordinary skill in the art and the description of this invention, any prior art methods, apparatus, and materials similar to or equivalent to those described, apparatus, and materials in the embodiments of this invention may be used to implement the present invention.

[0087] This invention relates to the administration of an rAAV vector containing a polynucleotide expressing LAL to inhibit or reverse liver fibrosis in individuals with LALD. As demonstrated in the “Examples” section, administration of the rAAV vector described herein leads to the restoration of LAL protein expression in gene knockout mice. Administration of the rAAV vector described herein will result in the inhibition and reversal of liver fibrosis, including bringing the volume and weight of the liver and spleen, the weight of other related organs closer to normal mouse tissues and morphology, significant improvement in serum liver function indicators (ALT, AST), and some degree of recovery in lipid indicators (TG, CHO, LDL-C, HDL-C, NEFA, and FC).

[0088] Example 1: Design and Construction of Candidate Molecular Expression Vectors

[0089] The theoretical upper limit of AAV genome loading capacity is approximately 4.7 kb (including both ITRs), while the actual space available for inserting foreign genes is about 4.4-4.5 kb. When the length of the foreign gene exceeds the loading capacity of AAV, the packaging process becomes unstable, which may lead to the inability of viral particles to assemble properly or to produce an incomplete genome. Wild-type AAV is a single-stranded DNA virus (ssAAV), which requires host-mediated second-strand synthesis to initiate the transcription process, resulting in slow onset of action and high dosage. Self-complementary AAV (scAAV) forces the formation of head-to-head inverted repeats by mutating one side of the ITR, resulting in double stranding upon nuclear insertion, which can initiate gene transcription, thus achieving faster and higher expression levels of foreign genes; however, due to the deletion or mutation of one of the ITRs, its loading capacity is only 2.3 kb.

[0090] This invention combines different elements (Alphamic / bik, WPRE, SV40PA, BGHPA, TBG) (SEQ 3-7) to ensure that the AAV loading capacity is not exceeded. Simultaneously, codon optimization of the LIPA gene sequence is performed (SEQ 1-2). The aim is to screen for a liver-specific, highly efficient rAAV particle expressing LAL protein, ensuring that it effectively improves hepatosplenomegaly symptoms in LALD patients at the lowest possible dosage, while reducing the production of AAV neutralizing antibodies and hepatotoxicity. Specific candidate molecular expression vectors are as follows: Figure 1 As shown in (SEQ 8-10), p6-7k is the plasmid name of AAV8; p6-10 / p6-13 / p6-14 are the names of three candidate molecular target gene expression vectors.

[0091] Example 2 Virus Packaging

[0092] 1) Plasmid preparation: Engineered bacteria containing pAAV8, pHelper and the three rAAV target plasmids constructed in Example 1 were cultured in a fermenter. After alkaline lysis of the bacterial cells, filtration clarification, anion chromatography and affinity chromatography purification, and ultrafiltration concentration, five high-purity plasmids were obtained, and their sequences are shown in SEQ 8-12 respectively.

[0093] 2) Virus Packaging and Purification: pAAV8, pHelper, and the target plasmid were transfected into HEK293S cells via PEI at a mass ratio of 6:1:3. After 72 hours, the cell supernatant was collected, filtered through a 0.45 μm membrane, concentrated by ultrafiltration, subjected to affinity chromatography, and then subjected to ion exchange chromatography. After ultrafiltration and concentration, the AAV virus stock solution was obtained. The yields of the three virus stock solutions were as follows: Figure 2 As shown, there is no significant difference in the total amount of virus harvested at the end.

[0094] 3) Virus quality control testing: Testing was commissioned to Guangdong Sansheng Pharmaceutical Co., Ltd., as detailed in Table 1.

[0095] Table 1 Quality Control Test Table for Candidate Molecular Virus Stock Solution

[0096]

[0097]

[0098] Example 3: Comparison of effects in in vitro cells

[0099] The candidate molecules from Example 2 were used to infect the hepatocyte line Huh.7. Seven days after infection, the expression levels and activity of LAL protein in different groups were observed. Candidate molecules were screened based on their ability to express LAL in the Huh.7 cell line.

[0100] In this embodiment, p6-7k (p6-11) was used as a negative control. Its nucleotide sequence is shown in SEQ ID NO.13. This plasmid only expresses luciferase, does not carry the LIPA gene, and does not express LAL protein.

[0101] 3.1 Infection of Huh.7 cells

[0102] 1) One day before infection, Huh.7 cells were re-rotated in complete medium of DMEM + 10% FBS + 1% PS and evenly seeded into 6-well plates at 4E5 cells / well, and cultured overnight at 37°C with 5% CO2.

[0103] 2) Discard the culture medium in the 6-well plate and wash the cells with 1 mL / well PBS. Infect Huh.7 cells with each virus in a serum-free state, with infection MOIs of 1E5 vg / cell, 5E5 vg / cell, and 1E6 vg / cell.

[0104] 3) Replace the medium with 5% FBS 24 hours after infection, and supplement with 5% FBS medium on the 4th day after infection. Culture for a total of 7 days under 5% CO2 and 37℃ conditions.

[0105] 4) Aspirate the supernatant from each well and centrifuge at 13000 rpm for 5 min at 4℃; collect the supernatant after centrifugation;

[0106] 5) Wash the cells in the 6-well plate with pre-cooled PBS and discard the PBS. Add 200 μL to each well. IP LysisBuffer (containing protease inhibitor) and mix by pipetting; place on ice for 5 min; centrifuge at 13000g at 4℃ for 10 min; collect the supernatant;

[0107] 6) The concentration of proteins in cell supernatant and cell lysate was detected using the BCA assay kit.

[0108] 3.2 Western blot analysis of LAL expression in supernatant and cells

[0109] 1) Mix Huh.7 cell supernatant and cell lysis buffer with 5x Loading buffer at a ratio of 4:1, i.e., add 40 μL of sample to 10 μL of 5× Loading buffer, place the mixed sample on a metal bath and heat at 100℃ for 10 min.

[0110] 2) Perform SDS-PAGE gel electrophoresis on the prepared samples. The sample loading volume is 30 μg, and the protein marker loading volume is 5 μL. Set the voltage to 100V, and stop the electrophoresis when the loading buffer moves to the bottom of the gel.

[0111] 3) Thoroughly wet the PVDF membrane with anhydrous methanol for about 1 minute. Thoroughly wet the filter paper, PVDF membrane, and pre-prepared gel with transfer solution. Turn on the transfer equipment and place the membranes on the bottom plate in the following order from bottom to top: 2 layers of filter paper, PVDF membrane, protein gel, and 2 layers of filter paper. Then cover the top plate and place the membrane in the transfer apparatus. Set the parameters to 25V, 1.3A, and 10min for the transfer.

[0112] 4) After the transfer is complete, wash once with PBST solution, then place in blocking solution containing 5% skim milk powder PBST on a shaker and block at room temperature for 2 hours.

[0113] 5) Discard the blocking solution, wash 3 times with PBST solution, add rabbit anti-LAL protein antibody, and incubate overnight at 4°C on a shaker;

[0114] 6) Discard the above solution, wash 3 times with PBST solution, add goat anti-rabbit antibody, and incubate on a shaker at room temperature for 1 hour;

[0115] 7) Discard the above solution, wash three times with PBST solution, then incubate with chromogenic solution for 1 min. Expose and image using a protein imager and analyze the results;

[0116] 8) Add an appropriate amount of PBST solution and place on a shaker for 10 min, then discard the PBST solution. Repeat this process 3 times, then add the blot stripping solution and incubate on a shaker at 37°C for 15 min.

[0117] 9) Discard the stripping solution, wash 3 times with PBST solution, add anti-Tubulin antibody, and incubate on a shaker at room temperature for 1 hour;

[0118] 10) Discard the above solution, wash three times with PBST solution, then add chromogenic solution and incubate for 1 min. Expose and image using a protein imager and analyze the results. Figure 3 A).

[0119] 3.3 Examination of LAL activity in supernatant and cells

[0120] 1) Preparation of standards: Dilute 10mM 4-MU solution with PBS to final concentrations of 3.33μM, 1.11μM, 0.37μM, 0.1233μM, 0.04111μM, 0.013704μM, 0.0045679μM, and 0μM, for a total of 8 concentrations;

[0121] 2) Cell lysis buffer The protein samples were diluted to the appropriate factor using IP Lysis Buffer; the cell supernatant was diluted to the appropriate factor using PBS containing 0.5% CA-630.

[0122] 3) Prepare a substrate containing 1% Triton X-100, 0.345 mM, 0.0325% 4-MUP solution, and 90.9 mM sodium acetate solution.

[0123] 4) Dilute 30 mM Lalistat2 with PBS to 30 μM Lalistat2;

[0124] 5) Add the standard to the 96-well black plate, with a volume of 100 μL per well. Perform 3 replicates for each gradient. Add the standard to each plate.

[0125] 6) Sample loading: Each sample requires 6 wells: 3 non-inhibition wells (50 μL substrate, 10 μL PBS, 40 μL sample per well); 3 inhibition wells (50 μL substrate, 10 μL 30 μM Lalistat2, 40 μL sample per well).

[0126] 7) Seal the membrane with an ELISA membrane and centrifuge at 2000g for 1 min;

[0127] 8) The reaction was incubated at 37 (±1)℃ in a 0% CO2 incubator for 21 (±0.5) h;

[0128] 9) Add 200 μL of 150 mM EDTA (pH 11.5) to each well to terminate the reaction;

[0129] 10) Before termination, set the program in the microplate reader and plate the plate; shake and mix for 7 seconds in the microplate reader, read the fluorescence, excitation wavelength 365nm, emission wavelength 460nm. Figure 3 B-3C).

[0130] 3.4 Enzyme digestion test for LAL glycosylation modification

[0131] 1) Sample denaturation: Add 10 μL of 10×Glycoprotein Denaturing Buffer to 90 μL of cell supernatant and mix thoroughly; incubate at 100℃ for 10 min;

[0132] 2) Prepare a 40 μL Endo H digestion system: 20 μL 4.1 denatured sample, 4 μL 10×Gly Buffer 3, 4 μL Endo H (ddH2O for control group), 12 μL ddH2O;

[0133] 3) Prepare a 40 μL pNGase F digestion system: 20 μL 4.1 denatured sample, 4 μL 10×Gly Buffer 2, 4 μL 10% NP-40, 2 μL PNGase F (control group is ddH2O), 10 μL ddH2O;

[0134] 4) The prepared Endo H digestion system and PNGase F digestion system were digested at 37℃ for 1 hour;

[0135] 5) After enzyme digestion, LAL was detected by Western blotting (WB). Figure 3 D).

[0136] 3.5K562 cell assay for LAL protein M6P modification

[0137] 1) Viral infection and harvesting of supernatant;

[0138] 2) K562 cells were incubated for cell counting, and the cells were diluted with complete culture medium to 1E6 cells / mL.

[0139] 3) Add 200 μL of K562 cells at a density of 1E6 cells / mL to each well of a 24-well plate.

[0140] 4) Each sample has 6 wells, with 3 replicates containing 50 μL of 100 mM M6P and 3 replicates containing 50 μL of culture medium.

[0141] 5) Add 250 μL of cell supernatant to each well.

[0142] 6) After mixing thoroughly, incubate overnight in a 37°C, 5% CO2 incubator.

[0143] 7) Aspirate the supernatant, wash the cells with pre-cooled PBS and discard the supernatant; then add PBS to each well. IP LysisBuffer (containing protease inhibitor), mix well by pipetting, place on ice for 5 min, centrifuge at 13000g at 4℃ for 10 min, and collect the supernatant.

[0144] 8) The protein concentration of the collected supernatant was detected using a BCA kit, and LAL activity was detected according to the method in step 3. Figure 3 E).

[0145] Depend on Figure 3 It can be seen that Huh.7 cells exhibit dose-dependent responses to all three candidate molecules. Figure 3 The expression levels and activities of LAL in the secretory supernatant and lysate of Huh.7 cells infected with A-3C and P6-7K (P6-13) were significantly higher than those of other candidate molecules. Figure 3 AC); the candidate molecule P6-7K (P6-13) infected Huh.7 cells and secreted into the cell supernatant. LAL is glycosylated and can be taken up by K562 cells. Figure 3 D, 3E).

[0146] Example 4: In vivo effect comparison

[0147] 4.1 Drug administration to mice

[0148] The experimental design in this embodiment is shown in Table 2.

[0149] Table 2 Experimental Design Table

[0150]

[0151] 4.2 Liver and spleen tissue lysis and protein concentration detection

[0152] 1) Preparation of tissue lysis buffer: 0.1M sodium phosphate (pH 6.8), 1mM EDTA, 10mM DTT, 0.5% CA-630, 1× protease inhibitor;

[0153] 2) Add 0.7 mL of tissue lysis buffer to approximately 20 mg of tissue, place it on ice and then place it in an ultrasonic homogenizer for homogenization. Set the homogenization parameters as follows: power 100 W, working time 1 min, ultrasonic on time 2 s, ultrasonic off time 3 s, and alarm temperature 50 ℃.

[0154] 3) After crushing, centrifuge at 10000g for 15 minutes, and collect the supernatant; repeat 3 times.

[0155] 4) The concentration of liver samples was detected using a BCA detection kit;

[0156] 5) Store in a refrigerator at -70℃ for later use.

[0157] 4.3 Western blot analysis of LAL expression in liver

[0158] 1) The prepared liver sample and 5x Loading buffer were mixed at a ratio of 4:1, that is, 40 μL of sample was added to 10 μL of 5× Loading buffer. The mixed sample was placed on a metal bath and heated at 100℃ for 10 min.

[0159] 2) Perform SDS-PAGE gel electrophoresis on the prepared samples. The sample loading volume is 20 μg, and the protein marker loading volume is 5 μL. Set the voltage to 100V, and stop the electrophoresis when the loading buffer moves to the bottom of the gel.

[0160] 3) Thoroughly wet the PVDF membrane with anhydrous methanol for about 1 minute. Thoroughly wet the filter paper, PVDF membrane, and pre-formed gel with transfer solution. Turn on the transfer apparatus and place the membrane on the bottom plate in the following order from bottom to top: 2 layers of filter paper, PVDF membrane, protein gel, and then 2 layers of filter paper. Then cover with the top plate and place the apparatus in the transfer chamber. Set the parameters to 25V, 1.3A, and 10min for the transfer process.

[0161] 4) After the transfer is complete, wash once with PBST solution, then place in blocking solution containing 5% skim milk powder PBST on a shaker and block at room temperature for 2 hours.

[0162] 5) Discard the blocking solution, wash 3 times with PBST solution, add rabbit anti-LAL protein antibody, and incubate overnight at 4°C on a shaker;

[0163] 6) Discard the above solution, wash 3 times with PBST solution, add goat anti-rabbit antibody, and incubate on a shaker at room temperature for 1 hour;

[0164] 7) Discard the above solution, wash three times with PBST solution, then incubate with chromogenic solution for 1 min. Expose and image using a protein imager and analyze the results;

[0165] 8) Add an appropriate amount of PBST solution and place on a shaker for 10 min, then discard the PBST solution. Repeat this process 3 times, then add the blot stripping solution and incubate on a shaker at 37°C for 15 min.

[0166] 9) Discard the stripping solution, wash three times with PBST solution, add rabbit anti-HSP90 antibody, and incubate on a shaker at room temperature for 1.5 h;

[0167] 10) Discard the above solution, wash 3 times with PBST solution, add goat anti-rabbit antibody, and incubate on a shaker at room temperature for 1 hour;

[0168] 11) Discard the above solution, wash three times with PBST solution, and then incubate with chromogenic solution for 1 min. Expose and image the solution using a protein imaging system and analyze the results. Figure 4 B).

[0169] 4.4 Enzyme digestion assay for LAL glycosylation modification in liver

[0170] 1) Sample denaturation: Add 8 μL of 10×Glycoprotein Denaturing Buffer to 160 μg of liver sample, then add ddH2O to make up the volume to 80 μL and mix thoroughly; incubate at 100℃ for 10 min;

[0171] 2) Prepare a 40 μL Endo H digestion system: 20 μL of 1) moderately denatured sample, 4 μL of 10×Gly Buffer 3, 4 μL Endo H (ddH2O for control group), and 12 μL of ddH2O;

[0172] 3) Prepare a 40 μL pNGase F digestion system: 20 μL 1) moderately denatured sample, 4 μL 10×Gly Buffer 2, 4 μL 10% NP-40, 2 μL PNGase F (control group is ddH2O), 10 μL ddH2O;

[0173] 4) The prepared Endo H digestion system and PNGase F digestion system were digested at 37℃ for 1 hour;

[0174] 5) After enzyme digestion, LAL was detected by Western blotting (WB). Figure 4 C).

[0175] 4.5 Examination of the number of AAV vectors in the liver and spleen

[0176] 4.5.1 DNA extraction from liver and spleen tissues

[0177] 1) Add 600 μL of lysis buffer RLA to the tissue and repeatedly grind it with an RNase-Free tissue grinding stick until the tissue is completely homogenized. After homogenization, the liquid will become significantly more viscous.

[0178] 2) Centrifuge at 13000g for 5 min, take 200 μL of supernatant and add 200 μL of GB to it, then pipette and mix repeatedly.

[0179] 3) Then add 200 μL of anhydrous ethanol and mix thoroughly by repeatedly pipetting.

[0180] 4) Transfer the above solution and precipitate together into the adsorption column CB3, centrifuge at 13000g for 1min, and discard the waste liquid;

[0181] 5) Add 500 μL of buffer GD to the adsorption column CB3, centrifuge at 13000g for 1 min, and discard the waste liquid;

[0182] 6) Add 600 μL of wash buffer PW to the RNase-Free adsorption column CR4, centrifuge at 13000g for 1 min, discard the waste liquid; repeat once.

[0183] 7) Centrifuge at 13000g for 2 minutes, then discard the waste liquid. Place the RNase-Free adsorption column CR4 at room temperature for 2 minutes.

[0184] 8) Transfer the RNase-Free adsorption column CR4 into a new RNase-Free centrifuge tube, add 100 μL of ddH2O to the middle of the adsorption membrane, incubate at room temperature for 2 min, centrifuge at 13000g for 2 min to obtain the DNA solution;

[0185] 9) Detect the concentration using a nanodrop and store in a refrigerator at -70°C.

[0186] 4.5.2 Detection of the number of AAV vectors in liver and spleen

[0187] 1) Preparation of standard: The p6-7K (p6-13) plasmid was digested with ScaI enzyme, and the linearization of the plasmid was confirmed by nucleic acid gel detection. Then, it was recovered using a gel recovery kit, and the concentration was detected and converted into copy number.

[0188] 2) The standard was first diluted with nuclease-free water to 2.5E7 copies / μL, and then serially diluted to obtain 8 gradients: 2.5E6 copies / μL, 2.5E5 copies / μL, 2.5E4 copies / μL, 2.5E3 copies / μL, 2.5E2 copies / μL, 2.5E1 copies / μL, and 0 copies / μL.

[0189] 3) Sample dilution: Dilute the sample with nuclease-free water to 90 ng / μL;

[0190] 4) Prepare the reaction solution according to the table below.

[0191] reagents Usage per well (μL) TB GreenPremix Ex Taq II(Tli RNaseH Plus)(2×) 10 LAL PCR Forward Primer 0.8 LAL PCR Reverse Primer 0.8 ROX Reference Dye or Dye II(50×) 0.4 Samples or standards 2 Sterilized water 6 Total 20

[0192] Real-time PCR reaction settings: 95℃ for 30s, 1 cycle; 95℃ for 5s, 60℃ for 34s, 40 cycles. Results are as follows: Figure 4 D、 Figure 4 As shown in G.

[0193] 4.6 Examination of LIPA mRNA levels in liver and spleen

[0194] 4.6.1 RNA extraction from liver and spleen tissues

[0195] 1) Add 600 μL of lysis buffer RLA to the tissue and repeatedly grind it with an RNase-Free tissue grinding stick until the tissue is completely homogenized. After homogenization, the liquid will become significantly more viscous.

[0196] 2) Centrifuge at 13000g for 5 min, and add 200 μL of supernatant to the genomic DNA removal column;

[0197] 3) Add to the genomic DNA removal column, centrifuge at 13000g for 1 min, take out the filtrate, add 200μL of 70% ethanol, and mix thoroughly by pipetting repeatedly.

[0198] 4) Transfer all the obtained solutions together into an RNase-Free adsorption column CR4, centrifuge at 13000g for 1 min, and discard the waste liquid;

[0199] 5) Add 700 μL of protein removal solution RW3 to the RNase-Free adsorption column CR4, centrifuge at 13000g for 1 min, and discard the waste liquid;

[0200] 6) Add 500 μL of wash buffer RW to the RNase-Free adsorption column CR4, centrifuge at 13000g for 1 min, discard the waste liquid; repeat once.

[0201] 7) Centrifuge at 13000g for 2 minutes and discard the waste liquid. Place the RNase-Free adsorption column CR4 at room temperature for 2 minutes to thoroughly dry any residual washing liquid in the adsorption material;

[0202] 8) Transfer the RNase-Free adsorption column CR4 into a new RNase-Free centrifuge tube, add 50 μL of RNase-Free ddH2O to the middle of the adsorption membrane, incubate at room temperature for 2 min, centrifuge at 13000g for 2 min to obtain the RNA solution;

[0203] 9) Detect the concentration using a nanodrop and store in a refrigerator at -70°C.

[0204] 4.6.2 Reverse transcription

[0205] Dissolve 800 ng of RNA solution in water to prepare a 20 μL volume. Add 4 μL of 5×PrimeScript RTMaster Mix to each tube. Set the PCR program to 37℃ for 15 min; 85℃ for 5 sec; 4℃ to ∞ to obtain cDNA.

[0206] 4.6.3 Quantitative Real-Time PCR

[0207] Add 5 μL of cDNA sample to 95 μL of nuclease-free water and mix thoroughly, i.e., dilute 20-fold. Then prepare the real-time PCR reaction solution according to the table below. Real-Time PCR reaction settings: 95℃ for 30 s, 1 cycle; 95℃ for 5 s, 60℃ for 34 s, 40 cycles. Results are as follows: Figure 4 E, Figure 4 As shown in H.

[0208] reagents Usage per well (μL) TB GreenPremix Ex Taq II(Tli RNaseH Plus)(2×) 10 LIPAF(18S rRNAF) 0.8 LIPAR(18s rRNAR) 0.8 ROX Reference Dye or Dye II(50×) 0.4 cDNA 2 Sterilized water 6 Total 20

[0209] 4.7 Examination of LAL activity in liver and spleen

[0210] 1) Preparation of standards: Dilute 10mM 4-MU solution with PBS to final concentrations of 3.33μM, 1.11μM, 0.37μM, 0.1233μM, 0.04111μM, 0.013704μM, 0.0045679μM, and 0μM, for a total of 8 concentrations;

[0211] 2) Dilute the processed tissue samples to an appropriate ratio using tissue lysis buffer;

[0212] 3) Prepare a substrate containing 1% Triton X-100, 0.345 mM, 0.0325% 4-MUP solution, and 90.9 mM sodium acetate solution;

[0213] 4) Dilute 30 mM Lalistat2 with PBS to 30 μM Lalistat2;

[0214] 5) Add the standard to the 96-well black plate, with a volume of 100 μL per well. Perform 3 replicates for each gradient. Add the standard to each plate.

[0215] 6) Sample loading: Each sample requires 6 wells. 3 non-inhibition wells: 50 μL substrate, 10 μL PBS, and 40 μL sample per well; 3 inhibition wells: 50 μL substrate, 10 μL 30 μM Lalistat2, and 40 μL sample per well.

[0216] 7) Seal the membrane with an ELISA membrane and centrifuge at 2000g for 1 min;

[0217] 8) The reaction was incubated at 37 (±1)℃ in a 0% CO2 incubator for 21 (±0.5) h;

[0218] 9) Add 200 μL of 150 mM EDTA (pH 11.5) to each well to terminate the reaction;

[0219] 10) Before termination, set the program in the microplate reader, plate the plate, and mix thoroughly with the microplate reader for 7 seconds. Read the fluorescence; the excitation wavelength is 365 nm, and the emission wavelength is 460 nm. Results are as follows: Figure 4 F, Figure 4 As shown in Figure I.

[0220] Depend on Figure 4 It can be seen that there is no significant difference in the genomic level of rAAV vectors in the liver and spleen among the three candidate molecules. Figure 4 (D and 4G); Liver LIPA mRNA levels: In the medium- and high-dose groups, the LIPA mRNA levels of the candidate molecule p6-7K (p6-13) were significantly higher than in other groups. However, there was no significant difference in LIPA mRNA levels in the spleen. This indicates that the three candidate molecules can only be expressed in the liver, and cannot be expressed in the spleen, demonstrating that all three candidate molecules exhibit liver-specific expression. Figure 4 E and 4H); Liver LAL expression levels: p6-7K(p6-13)>p6-7K(p6-14)>p6-7K(p6-10), and all were dose-dependent, consistent with the in vitro evaluation results in Case 3. Figure 4B); LAL enzyme activity in liver and spleen: p6-7K(p6-13)>p6-7K(p6-14)>p6-7K(p6-10); Glycosylation modification: all three candidate molecules have glycosylation modification ( Figure 4 C). Although the three candidate molecules can only be expressed in the liver ( Figure 4 E), expression cannot be initiated within the spleen ( Figure 4 H), but LAL enzyme activity can be detected in the spleen ( Figure 4 I) This indicates that the LAL protein expressed by the candidate molecule has the correct M6P modification and can be delivered to distal tissues expressing M6PR, and... Figure 4 Conclusion C is consistent.

[0221] Example 5: Non-clinical efficacy trial 1 (young mice)

[0222] 5.1 Drug administration to mice

[0223] The principle of this experimental design is to use lysosomal acid lipase deficiency (Lipa-KO) mice to simulate the occurrence and development of lysosomal acid lipase deficiency disease and to evaluate the efficacy of the test product p6-7K (p6-13) in Lipa-KO mice.

[0224] Nine-week-old C57BL / 6J and Lipa-KO mice that passed quarantine were weighed before grouping. Animals that were too large or too small were excluded based on weight, and then randomly paired into groups according to weight. This experiment consisted of five groups: normal control group (G1), model control group (G2), treatment group 1 (G3, low-dose treatment group), treatment group 2 (G4, medium-dose treatment group), and treatment group 3 (G5, high-dose treatment group). The normal control group consisted of nine-week-old wild-type C57BL / 6J mice, while the other groups consisted of model mice: Lipa-KO mice.

[0225] The day of drug administration was defined as D0 in this experiment. The experiment consisted of 5 groups, with 8 animals in each group, half male and half female. The detailed grouping and drug dosage are shown in Table 3 below:

[0226] Table 3. Pharmacodynamic design of young mice

[0227]

[0228] In this embodiment, p6-7k (p6-5) was used as a negative control, and its nucleotide sequence is shown in SEQ ID NO.14. This plasmid expresses only GFP fluorescent protein, does not carry the LIPA gene, and does not express LAL protein.

[0229] 5.2 Tissue Sample Collection

[0230] 1) Trial endpoint: D96 (fasting the day before euthanasia);

[0231] 2) Method of euthanasia: After CO2 euthanasia, blood is collected, left to stand for 30-60 minutes, then centrifuged at 4°C for 10-15 minutes at 5000 rpm. The serum is then aliquoted into pre-labeled EP tubes and stored in an ultra-low temperature freezer.

[0232] 3) Sample collection process: Mice were euthanized with CO2 on the same day, and tissue samples were collected, including liver, spleen and other tissues. The weight of the liver and spleen was measured and photographed.

[0233] 4) Sample segmentation requirements: Samples should be placed on ice packs during segmentation;

[0234] 5) Tissue collection, aliquoting, and preservation: ① Liver: Divided into 7 portions, 5 portions were quick-frozen with dry ice and then stored in an ultra-low temperature freezer; 2 portions were preserved with PFA; ② Spleen: Divided into 5 portions, 3 portions were quick-frozen with dry ice and then stored in an ultra-low temperature freezer; 2 portions were preserved with PFA.

[0235] Note: The PFA-preserved tissues of the liver and spleen in the above samples were used for pathological analysis and immunohistochemistry.

[0236] 5.3 Expression of the target gene

[0237] 5.3.1 For details on the method for detecting the genome number of AAV vectors in the liver, please refer to Example 4. Figure 5 A);

[0238] 5.3.2 For details on the method for detecting LIPA mRNA expression levels in the liver, please refer to Example 4. Figure 5 B);

[0239] 5.3.3 Method for detecting LAL enzyme activity in serum

[0240] 1) Separate serum: After standing at room temperature for 15 minutes, centrifuge at 3500 rpm for 15 minutes and collect the supernatant;

[0241] 2) Preparation of standards: Dilute 10mM 4-MU solution with PBS to final concentrations of 3.33μM, 1.11μM, 0.37μM, 0.1233μM, 0.04111μM, 0.013704μM, 0.0045679μM, and 0μM, for a total of 8 concentrations;

[0242] 3) Dilute the serum with PBS to the appropriate concentration;

[0243] 4) Prepare a substrate containing 1% Triton X-100, 0.345 mM, 0.0325% 4-MUP solution, and 90.9 mM sodium acetate solution;

[0244] 5) Dilute 30 mM Lalistat2 with PBS to 30 μM Lalistat2;

[0245] 6) Add the standard to the 96-well black plate, with a volume of 100 μL per well. Perform 3 replicates for each gradient. Add the standard to each plate.

[0246] 7) Sample loading: Each sample requires 6 wells: 3 non-inhibition wells (50 μL substrate, 10 μL PBS, 40 μL sample per well); 3 inhibition wells (50 μL substrate, 10 μL 30 μM Lalistat2, 40 μL sample per well).

[0247] 8) Seal the membrane with an ELISA membrane and centrifuge at 2000g for 1 min;

[0248] 9) The reaction was incubated at 37 (±1)℃ in a 0% CO2 incubator for 21 (±0.5) h;

[0249] 10) Add 200 μL of 150 mM EDTA (pH 11.5) to each well to terminate the reaction;

[0250] 11) Before termination, set the program in the microplate reader and plate the plate; shake and mix for 7 seconds in the microplate reader, read the fluorescence, excitation wavelength 365nm, emission wavelength 460nm. Figure 5 D).

[0251] 5.3.4 For details on the method for detecting LAL enzyme activity in the liver and spleen, please refer to Example 4. Figure 5 E, Figure 5 F);

[0252] 5.3.5 For details of the LAL WB test method in the liver, please refer to Example 4 ( Figure 5 C).

[0253] Depend on Figure 5 The experimental results are as follows: 1) The genomic level of rAAV ITR in the liver shows a significant dose-dependent effect. Figure 5 A); 2) LIPA mRNA levels in the liver: LIPA mRNA transcribed from the vector in the liver increased significantly with increasing dose, also showing a dose-dependent effect; the levels of ITR DNA and LIPA mRNA in liver tissue indicate that the presence and expression of the therapeutic vector p6-7K (p6-13) in vivo are dose-dependent. Figure 5 B); 3) Liver LAL expression level: The LAL protein level in treatment groups G3, G4, and G5 was significantly higher than that in G1 and G2, and showed an increasing trend with increasing dose. Figure 5C); 4) Serum LAL enzyme activity level: The serum LAL enzyme activity levels in the treatment groups G3, G4, and G5 were significantly higher than those in G1 and G2, and showed an increasing trend with increasing dose, exhibiting a clear dose-dependent effect. Figure 5 D); 5) LAL activity in liver and spleen: Control group (G1, G2): LAL enzyme activity in liver and spleen remained at a low level (because the vector control did not express LAL protein). Treatment group (G3-G5): LAL enzyme activity in liver and spleen was significantly increased compared with the control group, and in a dose-dependent manner (G5>G4>G3), indicating that the LAL protein expressed by p6-7K (p6-13) has high enzyme activity and correct M6P modification, and can be transferred to the distal tissue (spleen) expressing M6PR. Figure 5 E and 5F).

[0254] 5.4 Changes in mouse body weight and the weight of various organs

[0255] Depend on Figure 6 The experimental results are as follows: 1) Body weight: Compared with the G2 group Lipa-KO mice, after treatment with p6-7K (p6-13), the body weight of mice in the G3-G5 groups, i.e., the three treatment groups, increased significantly. Figure 6 B); 2) Macroscopic photographs of the liver and spleen: At the experimental endpoint, the livers of group G2 showed a significantly lighter color, appearing pale yellow, in stark contrast to group G1, suggesting possible fatty degeneration. The livers of the remaining treatment groups were uniformly dark brown, with a dense texture, consistent with the color and morphological characteristics of normal livers, similar to G1. There were no significant differences in color and morphological characteristics among the treatment groups. Macroscopic photographs of the spleen showed that, compared to the spleens of group G1 mice, the spleens of group G2 mice were significantly larger, and the spleen volume in each treatment group recovered to a similar level to the normal group. Figure 6 C); 3) Liver weight: Compared with the G2 group, after p6-7K (p6-13) treatment, the liver weight of mice in each treatment group was significantly reduced and returned to normal levels. Figure 6 D); 4) Spleen weight: Compared with the G2 group, after p6-7K (p6-13) treatment, the spleen weight of mice in each treatment group was significantly reduced and returned to normal levels. Figure 6 E); 5) Jejunum, ileum, and duodenum weights: Compared with the G2 group, after p6-7K (p6-13) treatment, the weights of the jejunum, ileum, and duodenum in mice in each treatment group were significantly reduced, returning to normal levels. Figure 6 F, 6G, 6H); 6) Bilateral inguinal fat: Compared with the G2 group, after p6-7K (p6-13) treatment, the fat weight of mice in each treatment group was significantly increased (F ... Figure 6 I); 7) Brown fat on the back: There was no significant difference between the treatment groups and the G2 group. Figure 6 J).

[0256] 5.5 Changes related to oils and fats

[0257] 5.5.1 ALT, AST, TG, CHO, HDL-C and LDL-C in the blood were detected using a biochemical analyzer;

[0258] 5.5.2 NEFA and FC in blood were detected using a kit, and the steps are as follows:

[0259] 1) Separate serum: After standing at room temperature for 15 minutes, centrifuge at 3500 rpm for 15 minutes and collect the supernatant;

[0260] 2) Nanjing Jiancheng Free Fatty Acid (NEFA) Assay Kit for Detecting NEFA in Serum

[0261] ① Add 200 μL of reagent 1 to each well of a 96-well plate, then add 4 μL of ultrapure water, standard, and serum sample respectively, and perform 3 replicates for each. After mixing, incubate at 37°C for 5 min and measure the OD value using a microplate reader at a wavelength of 600 nm.

[0262] ② Add 50 μL of reagent II to each well, mix well, incubate at 37℃ for 5 min, and measure the OD value using a microplate reader at a wavelength of 546 nm.

[0263] 3) Free cholesterol (FC) colorimetric test kit FC

[0264] Add 5 μL of ultrapure water to the corresponding well of a 96-well plate, add 5 μL of standard to the corresponding well of a 96-well plate, and add 5 μL of the sample to be tested to the corresponding well of a 96-well plate. Then add 250 μL of reagent one to each well, mix well, incubate at 37°C for 10 min, and measure the OD value using a microplate reader at a wavelength of 510 nm.

[0265] 5.5.3 TG and TC in liver and spleen were detected using a kit.

[0266] 1) Extract lipids from the liver and spleen using the Lipid Extraction Kit.

[0267] ① Add 500 μL of Lipid Extraction Buffer to approximately 20 mg of tissue (record the actual weight of the tissue) and vortex for 1-2 minutes;

[0268] ② Homogenize the sample using a handheld homogenizer. After homogenization, centrifuge at 10000g for 5 minutes at 4℃ and collect the supernatant.

[0269] ③ Mix by rotating at room temperature for 15-20 minutes, then centrifuge at 10000g for 5 minutes and collect the supernatant;

[0270] ④ Dry in a drying incubator at 37°C until all liquid has evaporated;

[0271] ⑤ Dissolve lipids with 200 μL Lysis Buffer II / Suspension Buffer;

[0272] ⑥ Use an ultrasonic homogenizer for homogenization. The homogenization parameters are: power 100W, ultrasonic on time 5s, ultrasonic off time 5s, alarm temperature 50℃, and homogenization until the sample is clear.

[0273] 2) Nanjing Jiancheng Total Cholesterol (TC) Assay Kit: This kit detects TC levels in the liver and spleen.

[0274] ① Experimental preparation: Remove the TC assay kit from the 4℃ refrigerator and allow it to equilibrate to room temperature. Turn on the microplate reader and set the detection program.

[0275] ② Add 250 μL of working solution to each well of a 96-well plate, then add 2.5 μL of ultrapure water, standard, and serum sample respectively, and make two replicates for each. Centrifuge at 300g for 1 min to mix, incubate at 37℃ for 10 min, and measure the OD value using a microplate reader at a wavelength of 500 nm.

[0276] 3) Nanjing Jiancheng Triglyceride (TG) Assay Kit for detecting TG in liver and spleen

[0277] ① Experimental preparation: Remove the TG assay kit from the 4℃ refrigerator and allow it to equilibrate to room temperature. Turn on the microplate reader and set the detection program.

[0278] ② Add 250 μL of working solution to each well of a 96-well plate, then add 2.5 μL of ultrapure water, standard, and serum sample respectively, and make two replicates for each. Centrifuge at 300g for 1 min to mix, incubate at 37℃ for 10 min, and measure the OD value using a microplate reader at a wavelength of 500 nm.

[0279] 5.5.4 The liver and spleen were prepared into frozen sections and then sent to Wuhan Saiweier Biotechnology Co., Ltd. for further testing and analysis.

[0280] Depend on Figure 7 The experimental results are as follows: 1) Liver function indicators (ALT, AST): G2 group: ALT and AST levels were significantly higher than those in the normal control group, indicating severe hepatocellular damage; G3-G5 treatment groups: ALT and AST levels were significantly lower than those in the G2 group and returned to normal mouse levels; the drug had a protective effect on liver function, and there was no difference among the treatment groups. Figure 7 A and 7B).

[0281] 2) Lipid metabolism indicators: Triglycerides (TG): The levels in group G2 were significantly higher than those in group G1, while the levels in treatment groups G3-G5 were significantly lower than those in group G2, reaching normal levels. Figure 7 C); Cholesterol (CHO): No significant difference among groups ( Figure 7D); Non-esterified fatty acids (NEFA): No significant differences among groups ( Figure 7 E); Free cholesterol (FC): The level was significantly higher in group G2 than in group G1, and significantly lower in the G3-G5 treatment groups than in group G2, reaching normal levels. Figure 7 F); High-density lipoprotein cholesterol (HDL-C): The G2 group showed a decreasing trend compared to the G1 group, while the G3-G5 treatment groups were higher than the G2 group to normal levels. Figure 7 G); Low-density lipoprotein cholesterol (LDL-C): The G2 group was significantly higher than the G1 group, and the G3-G5 treatment groups were significantly lower than the G2 group to normal levels. Figure 7 H).

[0282] 3) Liver and spleen lipid concentrations: There was no significant difference in liver and spleen triglyceride (TG) levels between the G3-G5 treatment group and the G2 group. Figure 7 I and 7J); the TC levels in the liver and spleen of group G2 were significantly higher than those of group G1, and the levels in treatment groups G3-G5 were significantly lower than those in group G2, reaching normal levels. Figure 7 K and 7L).

[0283] 4) Oil Red O staining of the liver: In group G1, hepatocytes appeared light red under Oil Red O staining. In group G2, hepatocytes appeared bright red, and macrophages were distributed in patches, also appearing bright red. Compared to group G2, hepatocytes in treatment groups G3-G5 appeared light red, while macrophages were distributed in focal patches, also appearing bright red. AI calculations showed that the Oil Red O area in treatment groups G3-G5 was significantly lower than that in group G2, reaching normal levels. Figure 7 M).

[0284] 5) Oil Red O staining of spleen: In group G1, the tissue appeared light blue under Oil Red O staining. Compared with group G1, macrophages in group G2 showed a small focal distribution and appeared bright red. Compared with group G2, macrophages in the spleen of treatment groups G3-G5 showed a clustered distribution and appeared bright red. AI calculations showed that the Oil Red area in treatment groups G3-G5 was significantly lower than that in group G2. Figure 7 N).

[0285] The above results indicate that p6-7K (p6-13) can significantly increase LAL enzyme activity in the liver and spleen (in a dose-dependent manner); reduce serum TG levels, increase HDL-C, and decrease LDL-C; improve liver function and reduce TC content and lipid distribution area in the liver and spleen.

[0286] 5.6 Pathological changes in the liver and spleen

[0287] For liver and spleen HE, IHC, and Masson assays, liver tissue was prepared into paraffin sections and sent to Wuhan Saiweier Biotechnology Co., Ltd. for subsequent testing and analysis. The segmented tissue was preserved in electron microscopy solution and transferred to a third-party company for sectioning, photographing, and electron microscopy analysis, and an analysis report was issued. The methods for detecting the expression levels of inflammation and fibrosis-related gene mRNAs in the liver are detailed in Example 4; the methods for the Western blot (WB) test of autophagy-related proteins in the liver are also detailed in Example 4.

[0288] Depend on Figure 8 The experimental results are as follows:

[0289] 1) Liver pathological examination: In group G1, the liver tissue structure was clear, and all samples showed mild hepatocellular steatosis without obvious ballooning degeneration, fibrosis, or inflammatory cell infiltration. Compared with group G1, group G2 samples showed increased severity and extent of hepatocellular steatosis, with many samples showing ballooning degeneration. All samples showed moderate fibrosis and severe inflammatory cell infiltration. Under Masson staining, abundant collagen fiber proliferation was observed around macrophages and in the perisinusoidal region, appearing blue. Compared with group G2, group G3 samples showed a decrease in the number and extent of ballooning degeneration, and a reduction in the severity and extent of hepatocellular steatosis, fibrosis, and inflammatory cell infiltration. Under Masson staining, abundant collagen fiber proliferation was observed around macrophages and in the perisinusoidal region, appearing blue. Compared to group G2, group G4 samples showed reduced and smaller extent of hepatocyte steatosis, fibrosis, and inflammatory cell infiltration, with no obvious ballooning degeneration. Masson staining revealed a small amount of collagen fiber proliferation around macrophages and in the perisinusoidal region, appearing blue. Compared to group G2, group G5 showed reduced and smaller extent of hepatocyte steatosis, fibrosis, and inflammatory cell infiltration, with no obvious ballooning degeneration. Masson staining revealed a small amount of collagen fiber proliferation around macrophages, appearing blue. Figure 8 A and 8B).

[0290] 2) Results of liver electron microscopy:

[0291] Compared with group G1, group G2 exhibited progressive pathological features: ① Nucleus and cytoplasm structure: Significantly indented and deformed nuclear membranes (from mild to severe), suggesting abnormal nuclear membrane tension or cellular stress. Mild cytoplasmic swelling and a reduced number of organelles reflected impaired metabolic function. ② Mitochondrial dysfunction: A significant reduction in the number of mitochondria, accompanied by moderate to severe swelling, cristae-like lesions, and matrix degradation, suggesting mitochondrial energy metabolism failure and oxidative damage. Abnormal lipid droplet accumulation in some mitochondria may interfere with respiratory chain function. ③ Endoplasmic reticulum stress (ERS) and mitochondrial-associated endoplasmic reticulum membrane (MAM) disruption: Endoplasmic reticulum dilatation and vesicle formation, accompanied by degranulation, consistent with ERS characteristics. Complete disappearance of MAM structure indicates a break in the mitochondrial-endoplasmic reticulum functional coupling, leading to abnormal regulation of lipid metabolism, calcium homeostasis, and apoptosis signaling. ④ Lipid metabolism disorder: Accumulation of large amounts of lipid droplets (large in size and quantity) and cholesterol crystals, suggesting severe lipotoxic damage and bile acid metabolism imbalance. The complete disappearance of glycogen granules reflects the depletion of energy reserves and is directly related to mitochondrial dysfunction. ⑤ Autophagy inhibition: No autophagy was observed in any samples, possibly due to inhibition of the autophagy-lysosomal pathway or excessive lipotoxicity leading to loss of autophagy clearance capacity.

[0292] Compared to group G2, the key differences in group G5 were: ① Structural repair: The ultrastructure of hepatocytes (nuclear membrane, mitochondria, endoplasmic reticulum) in group G5 was basically restored to normal levels (G1), indicating that high-dose drugs had a significant reversal effect on model-induced organelle damage. ② Improved lipid metabolism: The number / volume of lipid droplets decreased and there was no cholesterol crystallization, suggesting that the drug alleviated lipotoxicity by inhibiting lipogenesis (such as downregulating SREBP-1c) or promoting fatty acid oxidation (such as activating PPARα). ③ Glycogen distribution: The number of granules was significantly restored, but the distribution was uneven (one case of abnormal aggregation), suggesting that glycogen synthase (GS) activity was partially restored, but the regulatory mechanism may not have been fully normalized. Two possible reasons are speculated: First, regional differences in GS activity: The drug may have partially restored GS activity by activating the AKT / GSK3β pathway, but residual local GS phosphorylation (inactivated form) led to uneven synthesis. Second, insulin signaling was not fully restored: insulin resistance (such as increased phosphorylation of IRS-1Ser307) in the G2 group was not completely reversed, and the regulation of glucose uptake by hepatocytes still showed heterogeneity. ④ Autophagy activation: autophagy levels were significantly higher than in the G1 and G2 groups, indicating that the drug may restore metabolic homeostasis by regulating the autophagy-lysosomal pathway, clearing damaged mitochondria and lipid droplets.

[0293] Group G2 presented with typical non-alcoholic steatohepatitis (NASH). High-dose drug (G5) significantly repaired the ultrastructural damage of hepatocytes in the model, with its mechanism of action involving autophagy activation, MAM functional reconstruction, and regulation of lipid metabolism homeostasis. Despite the minor issue of uneven glycogen distribution, the core efficacy endpoint met the criteria for clinical translation. These results provide important morphological evidence for subsequent preclinical studies. Figure 8 C).

[0294] 3) Liver-related protein detection: Real-time PCR was used to detect inflammation and fibrosis-related genes. The results showed that the expression of related genes in group G2 was significantly higher than that in group G1, while the expression in group G5 (high dose) was significantly lower than that in group G2, with some genes returning to normal levels (see [link to relevant data]). Figure 8 D), which is related to HE staining ( Figure 8 A) and Masson staining ( Figure 8 B) The conclusions are consistent.

[0295] IHC was used to detect the macrophage surface marker CD68 ( Figure 8 E), the experimental results showed that CD68 expression in the G2 group was significantly higher than that in the G1 group, and that in the G3-G5 treatment groups it was significantly lower than that in the G2 group. This is consistent with Masson staining ( Figure 8 B) and CD68 qPCR (see B) Figure 8 D) The test results are consistent.

[0296] Western blot was used to test for autophagy-related genes. Figure 8 F), the experimental results showed that compared with group G1, group G2 had significantly lower P62 protein and significantly higher LC3Ⅱ protein, indicating that the autophagy pathway was blocked. In group G5 (high dose), P62 protein and LC3Ⅱ protein basically returned to normal levels. Figure 8 F) indicates that p6-7K (p6-13) can restore the autophagy signaling pathway, which is consistent with the electron microscopy results. Figure 8 C).

[0297] 4) Pathological examination of the spleen: In group G1, a very small number of spleen tissue samples showed mild white pulp injury, with no obvious inflammatory cell infiltration. Compared with group G1, group G2 showed an increased number, severity, and extent of white pulp injury, with all samples showing severe inflammatory cell infiltration and extramedullary hematopoietic cell aggregation. Compared with group G2, group G3 showed a decreased number, severity, and extent of white pulp injury, with reduced inflammatory cell infiltration, and no obvious extramedullary hematopoietic cell aggregation. Compared with group G2, group G4 showed reduced inflammatory cell infiltration, with no obvious white pulp injury or extramedullary hematopoietic cell aggregation. Compared with group G2, group G5 showed reduced inflammatory cell infiltration, with no obvious white pulp injury or extramedullary hematopoietic cell aggregation. Figure 8 G).

[0298] IHC was used to detect the macrophage surface marker CD68 ( Figure 8 The experimental results showed that CD68 expression in the G2 group was significantly higher than that in the G1 group, and that the expression in the G3-G5 treatment groups was significantly lower than that in the G2 group, which was consistent with the results of HE staining (Figure G).

[0299] Example 6: Non-clinical efficacy trial two (older rats)

[0300] 6.1 Drug administration to mice

[0301] This study used older Lipa-KO mice with a longer history of the disease, whose liver tissue had reached a level of fibrosis. The main objective was to test the therapeutic and rescue effects of p6-7K (p6-13) on severely diseased mice with lysosomal acid lipase deficiency. Currently, there are no studies on the reversibility of fibrosis in older mice; it has only been shown to inhibit fibrosis in younger mice. This study aimed to investigate whether p6-7K (p6-13) could make fibrosis reversible.

[0302] C57BL / 6J and Lipa-KO mice that passed quarantine were weighed before grouping. Animals that were too large or too small were excluded based on weight, and then randomly paired into groups according to weight. This experiment consisted of four groups: normal control group (G1), model control group (G2), treatment group 1 (G4), and treatment group 2 (G5). Group G1 consisted of wild-type C57BL / 6J mice, while the other groups consisted of model mice: Lipa-KO mice.

[0303] The day of administration in this study was defined as D0 (Day 0). The detailed grouping and dosage of the study are shown in Table 4.

[0304] Table 4. Pharmacodynamic Design of Older Mice

[0305]

[0306] In this embodiment, p6-7k (p6-5) was used as a negative control, and its nucleotide sequence is shown in SEQ ID NO.14. This plasmid expresses only GFP fluorescent protein, does not carry the LIPA gene, and does not express LAL protein.

[0307] 6.2 Tissue Sample Collection

[0308] 1) Trial endpoint: D91 (fasting the day before euthanasia);

[0309] 2) Method of euthanasia: Same as in Example 5.2.

[0310] 3) Material sourcing process: Same as Example 5.2.

[0311] 4) Organization segmentation requirements: Same as Example 5.2.

[0312] 5) Organize, collect, package, and preserve: Same as in Example 5.2.

[0313] Note: The PFA-preserved tissues of the liver and spleen in the above samples were used for pathological analysis and immunohistochemistry.

[0314] 6.3 Target gene expression status

[0315] 6.3.1 The method for detecting the genome number of AAV vectors in the liver is detailed in Example 4, and the results are as follows: Figure 9 As shown in Figure A.

[0316] 6.3.2 The method for detecting LIPA mRNA expression levels in the liver is detailed in Example 4, and the results are as follows: Figure 9 As shown in B.

[0317] 6.3.3 The method for detecting LAL enzyme activity in serum is detailed in Example 5.3.3, and the results are as follows: Figure 9 As shown in D.

[0318] 6.3.4 The method for detecting LAL enzyme activity in the liver and spleen is detailed in Example 4, and the results are as follows: Figure 9 E, Figure 9 As shown in F.

[0319] 6.3.5 The method for LAL WB testing in the liver is detailed in Example 4, and the results are as follows: Figure 9 As shown in C.

[0320] Depend on Figure 9 The experimental results are as follows: 1) The genomic level of rAAV ITR in the liver shows a significant dose-dependent effect. Figure 9 A); 2) LIPA mRNA levels in the liver: LIPA mRNA transcribed from the vector in the liver increased significantly with increasing dose, also showing a dose-dependent effect; the levels of ITR DNA and LIPA mRNA in liver tissue indicated that the presence and expression of the therapeutic vector p6-7K (p6-13) in vivo were dose-dependent. Figure 9 B); 3) Liver LAL expression level: The LAL protein level in the treatment groups (G4, G5) was significantly higher than that in the G1 and G2 groups, and showed an increasing trend with increasing dose. Figure 9 C); 4) Serum LAL enzyme activity level: The serum LAL enzyme activity level in the treatment groups (G4, G5) was significantly higher than that in G1 and G2, and showed an increasing trend with increasing dose, exhibiting a clear dose-dependent effect. Figure 9D); 5) LAL activity in liver and spleen: Control group (G1, G2): LAL enzyme activity in liver and spleen remained at a low level (because the vector control did not express LAL protein). Treatment group (G4-G5): LAL enzyme activity in liver and spleen was significantly increased compared with the control group, and in a dose-dependent manner (G5>G4), indicating that the LAL protein expressed by p6-7K (p6-13) has high enzyme activity. Figure 9 E and 9F).

[0321] 6.4 Changes in mouse body weight and organ weight

[0322] Depend on Figure 10 The experimental results are as follows: 1) Compared with the G2 group Lipa-KO mice, after treatment with p6-7K (p6-13), the body weight of the treatment groups (G4, G5) mice showed an increasing trend, but there was no statistically significant difference. Figure 10 B); 2) Gross photographs of the liver and spleen: Compared to the liver color and morphology of the G1 group mice, the liver color of the G2 group mice was significantly lighter, appearing pale yellow, showing obvious fatty lesions. Compared to the liver and spleen tissue volume of the G1 group mice, the liver and spleen tissue volume of the G2 group mice was significantly enlarged. After 13 weeks of treatment, compared to the G2 group, the livers of the treatment groups (G4, G5) were dark brown, approaching the color and morphology of normal livers, and the volume of the liver and spleen was significantly reduced. Figure 10 C); 3) Liver and spleen weight: Compared with the G2 group, the liver and spleen weights of mice in the G4 and G5 groups were significantly reduced after p6-7K (p6-13) treatment. Figure 10 D and Figure 10 E); 4) Ileal weight analysis: There was no significant difference in ileal weight among the groups. Figure 10 F); Duodenum and jejunum weight: The weight of group G2 was significantly higher than that of group G1. Compared with group G2, the weight of group G5 was significantly lower. Figure 10 G and 10H); 5) Bilateral inguinal fat: The weight of bilateral inguinal fat in group G2 was significantly lower than that in group G1. Compared with group G2, the weight in group G5 was significantly higher ( Figure 10 I); 6) Back brown fat: The weight of back brown fat in group G2 was significantly lower than that in group G1. There was no significant difference between the treatment groups (G4, G5) and group G2. Figure 10 J).

[0323] 6.5 Changes related to oils and fats

[0324] 6.5.1 ALT, AST, TG, CHO, HDL-C and LDL-C in the blood were detected using a biochemical analyzer;

[0325] 6.5.2 For details on the detection of NEFA, FC in blood, TG and TC in liver and spleen using kits, please refer to Example 5;

[0326] 6.5.3 The liver and spleen were prepared into frozen sections and then sent to Wuhan Saiweier Biotechnology Co., Ltd. for further testing and analysis.

[0327] Depend on Figure 11 The experimental results are as follows: 1) Liver function indicators (ALT, AST): In group G2, ALT and AST levels were significantly higher than in group G1. In the treatment groups (G4, G5), ALT and AST levels were significantly lower than in group G2. This indicates that the drug has a certain effect on the recovery of liver function and can effectively repair liver damage, providing a potential target for the treatment of related diseases. Figure 11 A and Figure 11 B).

[0328] 2) Lipid metabolism indicators: There were no statistically significant differences in serum TG and CHO levels among the groups. Figure 11 C and Figure 11 D); NEFA: Compared with group G2, serum NEFA levels in groups G4 and G5 were significantly increased after administration ( Figure 11 E); FC: Compared with group G2, serum FC levels in groups G4 and G5 showed a decreasing trend after drug administration, but the difference was not statistically significant. Figure 11 F); HDL-C: Compared with group G2, serum HDL-C levels in groups G4 and G5 were significantly increased after drug administration (F); Figure 11 G); LDL-C: There were no statistically significant differences in serum LDL-C levels among the groups. Figure 11 H).

[0329] 3) Liver and spleen lipid concentrations: Compared with the G2 group, the TG levels in the liver tissue of mice in the G4 and G5 groups were significantly decreased. Figure 11 I). There was no significant difference in TG levels in the spleen tissue of mice among the different groups, and the difference was statistically insignificant. Figure 11 J); The levels of TC in the liver and spleen of group G2 were significantly higher than those of group G1, while the levels of TC in treatment groups G4 and G5 were significantly lower than those in group G2, reaching normal levels. Figure 11 K and 11L).

[0330] 4) Oil Red O staining of the liver: In group G1, hepatocytes appeared light red under Oil Red O staining. Compared to group G1, in group G2, hepatocytes appeared bright red under Oil Red O staining, and macrophages were distributed in patches, also appearing bright red. Compared to group G2, in the treatment groups (G4, G5), hepatocytes appeared light red or pale blue under Oil Red O staining, and macrophages were focally distributed, with some areas coalescing into patches, appearing bright red. AI calculations showed that the Oil Red O area in the treatment groups (G4, G5) was significantly lower than that in group G2, reaching normal levels. Figure 11 M).

[0331] 5) Oil Red O staining of spleen: In group G1, the spleen tissue appeared light blue under Oil Red O staining. Compared with group G1, in group G2, macrophages were focally distributed and appeared bright red under Oil Red O staining. Compared with group G2, macrophages in the spleen of the treatment groups (G4, G5) were focally distributed and appeared bright red. AI calculations showed that the Oil Red area in the treatment groups (G4, G5) was significantly lower than that in group G2. Figure 11 N).

[0332] The above results indicate that administration of different doses of p6-7K (p6-13) significantly increased the LAL protease activity levels in the liver and spleen of mice in a dose-dependent manner. Furthermore, p6-7K (p6-13) effectively reduced the total cholesterol (TC) content in the liver and spleen, significantly downregulated TG levels in the liver of mice, and effectively reduced the distribution area of ​​lipids in the liver and spleen.

[0333] 6.6 Pathological changes in the liver and spleen

[0334] For the HE and Masson assays of liver and spleen, liver tissue was prepared into paraffin sections and sent to Wuhan Saiweier Biotechnology Co., Ltd. for subsequent testing and analysis. The Western blot (WB) method for detecting autophagy-related proteins in the liver is detailed in Example 4.

[0335] Depend on Figure 12 The experimental results are as follows:

[0336] 1) Liver pathological examination: In group G1, the liver tissue structure was clear, and all samples showed mild hepatocellular steatosis without obvious ballooning degeneration, fibrosis, or inflammatory cell infiltration. Compared with group G1, the degree and extent of hepatocellular steatosis in group G2 samples were more severe, and all samples showed hepatocellular ballooning degeneration, moderate fibrosis, and severe inflammatory cell infiltration; under Masson staining, a large amount of collagen fiber proliferation was observed around macrophages and in the sinusoids, appearing blue. Compared with group G2, the degree and extent of hepatocellular steatosis, fibrosis, and inflammatory cell infiltration in group G4 samples were reduced, and no obvious ballooning degeneration was observed; under Masson staining, a large amount of collagen fiber proliferation was observed around macrophages and in the sinusoids, appearing blue. Compared with group G2, the degree and extent of hepatocellular steatosis, fibrosis, and inflammatory cell infiltration in group G5 samples were reduced, and no obvious ballooning degeneration was observed; under Masson staining, a small amount of collagen fiber proliferation was observed around macrophages, appearing blue. Figure 12 A and Figure 12 B).

[0337] All model mice exhibited moderate to severe fibrosis at enrollment (week 0). Masson staining revealed abundant collagen fiber proliferation around macrophages and in the perisinusoidal region, appearing blue. Compared to the model mice at enrollment, the G2 group showed increased fibrosis, with Masson staining again revealing abundant collagen fiber proliferation around macrophages and in the perisinusoidal region, appearing blue. Compared to the model mice at enrollment, the G4 group showed decreased fibrosis, with Masson staining revealing a relatively large amount of collagen fiber proliferation around macrophages and in the perisinusoidal region, appearing blue. Compared to the model mice at enrollment, the G5 group showed decreased fibrosis, with Masson staining revealing a small amount of collagen fiber proliferation around macrophages and in the perisinusoidal region, appearing blue. This indicates that the p6-7K (p6-13) test sample not only inhibits the development of liver fibrosis but, more importantly, can reversibly induce fibrosis to a certain extent. Figure 12 B).

[0338] 2) Examination of relevant proteins in the liver: Western blotting was used to examine autophagy-related genes ( Figure 12 C) The experimental results showed that compared with the G1 group, the P62 protein level in the G2 group was significantly lower and the LC3Ⅱ protein level was significantly higher, indicating that the autophagy pathway was blocked. In the G5 group, the P62 protein and LC3Ⅱ protein levels were basically restored to normal levels (see Figure C), indicating that p6-7K (p6-13) can restore the autophagy signaling pathway, which is consistent with the conclusions of the efficacy test in young mice.

[0339] 3) Spleen pathological examination: Pathological examination showed that the spleen tissue structure in group G1 was clear, with no obvious white pulp damage or inflammatory cell infiltration. Compared with group G1, samples in group G2 all showed white pulp damage, severe inflammatory cell infiltration, and extramedullary hematopoietic cell aggregation. Compared with group G2, the treatment groups (G4, G5) showed reduced degree and extent of white pulp damage and inflammatory cell infiltration, a smaller number of extramedullary hematopoietic cell samples, and a smaller aggregation range. Figure 12 D).

[0340] The sequences involved in this application are as follows:

[0341] LIPA gene (SEQ ID NO.1)

[0342]

[0343]

[0344]

[0345]

[0346]

[0347]

[0348]

[0349]

[0350]

[0351]

[0352]

[0353]

[0354] The primer sequences involved are as follows:

[0355]

[0356]

[0357] The above embodiments are for illustrating the implementation schemes disclosed in this invention and should not be construed as limiting the invention. Furthermore, various modifications and variations of the methods listed herein will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been specifically described in conjunction with various specific preferred embodiments, it should be understood that the invention should not be limited to these specific embodiments. In fact, various modifications as described above that are obvious to those skilled in the art to obtain the invention should be included within the scope of this invention.

Claims

1. An rAAV carrier, characterized in that, The rAAV vector comprises a gene expression cassette and AAV ITR sequences located at the 5' and 3' ends of the gene expression cassette; the gene expression cassette comprises: A) The polynucleotide sequence encoding LAL; B) A liver-specific transcriptional regulatory region, wherein the liver-specific transcriptional regulatory region is operatively linked to the polynucleotide sequence encoding LAL; C) Polynucleotide signal, wherein the polynucleotide signal is located at the 3' end of the gene expression cassette; The polynucleotide sequence encodes an amino acid sequence as shown in SEQ ID NO.

2.

2. The rAAV carrier according to claim 1, characterized in that, The liver-specific transcriptional regulatory region contains a liver-specific promoter; preferably, the liver-specific promoter is selected from the TBG promoter, the Alb promoter, or the ApoEHCR-hAAT promoter; more preferably, the nucleotide sequence of the TBG promoter is shown in SEQ ID NO.7; And / or, the organ-specific transcriptional regulatory region further includes an enhancer; preferably, the enhancer is an Alpha mic / bik element; more preferably, the nucleotide sequence of the Alpha mic / bik element is as shown in SEQ ID NO.3; And / or, the polynucleotide signal is selected from simian virus 40 polyadenylation signal, bovine growth hormone polyadenylation signal, human growth hormone polyadenylation signal, synthetic polyadenylation signal 48 or fragments thereof; preferably, the nucleotide series of the simian virus 40 polyadenylation signal is as shown in SEQ ID NO.5; preferably, the nucleotide series of the bovine growth hormone polyadenylation signal is as shown in SEQ ID NO.

6.

3. The rAAV carrier according to claim 1, characterized in that, The rAAV vector further includes one or more expression enhancement elements; the expression enhancement elements are located upstream of the polynucleotide signal; preferably, the expression enhancement element is WPRE3; more preferably, the nucleotide sequence of WPRE3 is shown in SEQ ID NO.

4.

4. The rAAV carrier according to any one of claims 1 to 3, characterized in that, The polynucleotide sequence encoding LAL has been codon-optimized; preferably, the polynucleotide encoding LAL comprises the nucleotide sequence shown in SEQ ID NO.

1.

5. The rAAV carrier according to claim 1, characterized in that, The rAAV vector is an scAAV vector; preferably, the nucleotide sequence of the 5' ITR sequence of the scAAV vector is as shown in SEQ ID NO.15; and / or, the nucleotide sequence of the 3' ITR sequence is as shown in SEQ ID NO.16; Alternatively, the rAAV vector may be an ssAAV vector; preferably, the nucleotide sequence of the 5' ITR sequence of the ssAAV vector is shown in SEQ ID NO.17; and / or, the nucleotide sequence of the 3' ITR sequence is shown in SEQ ID NO.

18.

6. The rAAV carrier according to claim 1, characterized in that, The rAAV carrier comprises any of the following structures: 1) 5'-ITR-Alpha mic / bik-TBG-encoding LAL polynucleotide sequence-WPRE3-SV40PA-ITR-3', wherein the rAAV vector is an ssAAV vector; 2) 5'-ITR-Alpha mic / bik–TBG-encoding LAL polynucleotide sequence-BGHPA-ITR-3', wherein the rAAV vector is an scAAV vector; 3) 5'-ITR-TBG-encoding LAL polynucleotide sequence-WPRE3-SV40PA–ITR-3', wherein the rAAV vector is an scAAV vector.

7. The rAAV carrier according to claim 1, characterized in that, The rAAV vector contains any of the following nucleotide sequences: 1) The nucleotide sequence shown in SEQ ID NO. 8; 2) The nucleotide sequence shown in SEQ ID NO. 9; 3) The nucleotide sequence shown in SEQ ID NO.

10.

8. An rAAV carrier system, characterized in that, The rAAV vector system comprises a packaging plasmid, an expression plasmid, and an auxiliary plasmid, wherein the expression plasmid is any one of the rAAV vectors described in claims 1 to 7.

9. An rAAV particle, characterized in that, The rAAV particles comprise the rAAV vector and AAV capsid as described in any one of claims 1 to 7, or are obtained by viral packaging using the rAAV vector system as described in claim 8.

10. The rAAV particles according to claim 9, characterized in that, The serotype of the AAV capsid is selected from AAV1, AAV2, AAV5, AAV6, AAV8, AAV9 or AAV-LK03; preferably, the serotype of the AAV capsid is selected from AAV2, AAV8, AAV9 or AAV-LK03.

11. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the rAAV carrier as described in any one of claims 1 to 7, or the rAAV carrier system as described in claim 8, or the rAAV particles as described in claim 9 or 10, and a pharmaceutically acceptable carrier, diluent, or excipient.

12. Use of the rAAV carrier of any one of claims 1 to 7, or the rAAV carrier system of claim 8, or the rAAV particles of claim 9 or 10, or the pharmaceutical composition of claim 11 in the preparation of a medicament for treating LALD.

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