COL4A5 truncated mutant and application thereof

Abstract

The invention provides a COL4A5 truncated mutant, related nucleic acid and a drug product thereof, and application of the COL4A5 truncated mutant in preparation of a drug for treating Alport syndrome. According to the present invention, the COL4A5 truncated mutant can be assembled with COL4A3 and COL4A4 collagen in cells to form a trimer, and the trimer is secreted out of the cells, such that the COL4A5 gene defect of the Alport syndrome patient can be rescued; animal experiments prove that the COL4A5 truncated mutant disclosed by the invention can obviously improve the renal function of a mouse with the Alport syndrome and has a certain treatment effect on abnormal retina structure of the mouse, so that the symptom of a disease is improved to a certain extent, and the progress of the disease is relieved. Therefore, the invention provides a new treatment choice for gene therapy of the Alport syndrome, and has great clinical application prospect and potential value.

Description

[0001] Cross-references

[0002] This application claims priority to Chinese Patent Application No. 202410535332.3, filed on April 30, 2024, entitled "COL4A5 Truncated Mutant and Its Application", the entire contents of which are incorporated herein by reference. Technical Field

[0003] This invention relates to the field of biomedical technology, specifically to a COL4A5 truncated mutant, its related nucleic acids and pharmaceutical products, and their application in the preparation of drugs for the treatment of Alport syndrome. Background Technology

[0004] Alport syndrome is a hereditary kidney disease characterized by progressive kidney dysfunction, eye abnormalities, and sensorineural hearing loss. It is caused by mutations in the COL4A3, COL4A4, and COL4A5 genes encoding the type IV collagen α3 / 4 / 5 chains. In normal individuals, the proteins COL4A3, COL4A4, and COL4A5, encoded by COL4A3 / 4 / 5, form collagen trimers and are secreted extracellularly to exert their functional activity. However, in Alport syndrome patients, mutations in these genes result in the loss of their ability to assemble into collagen trimers and / or be secreted extracellularly, leading to the disease.

[0005] Based on different inheritance patterns, Alport syndrome can be divided into autosomal dominant Alport syndrome, autosomal recessive Alport syndrome, and X-linked Alport syndrome. Clinically, the most common type is X-linked Alport syndrome with the COL4A5 mutation, accounting for approximately 85% of cases. The progression of Alport syndrome varies depending on the mutated gene and mutation type, but the vast majority of Alport syndrome patients will develop end-stage kidney disease (ESKD), making Alport syndrome one of the leading hereditary kidney diseases causing ESKD. Currently, the main treatment goal for Alport syndrome is to prolong the disease progression time. Angiotensin-converting enzyme inhibitors are commonly used clinically to delay the initiation of renal replacement therapy and prolong patient life. However, patients need to take medication for a long time, and the therapeutic effect is quite limited, especially for patients in the later stages of the disease. The main renal replacement therapies for patients who develop end-stage renal disease are dialysis and kidney transplantation. However, dialysis severely impacts patients' quality of life and also causes complications. The main problem with kidney transplantation is the shortage of kidney donors. Therefore, new treatment strategies for Alport syndrome urgently need to be developed.

[0006] Collagen deficiency is the primary cause of Alport syndrome, and gene therapy to re-express collagen holds potential therapeutic value. Currently, the main gene therapy options for Alport syndrome are homologous recombination repair based on CRISPR / Cas9 and exon skipping mediated by antisense oligonucleotides. CRISPR / Cas9 homologous recombination for COL4A5 repair remains at the cellular level, and its efficiency, efficacy, and safety in vivo have not been proven. Furthermore, this method requires the design of different CRISPR / Cas9 systems for patients with different mutations, significantly increasing drug development costs (Daga, Sergio, et al. "New frontiers to cure Alport syndrome: COL4A3 and COL4A5 gene editing in podocyte-lineage cells." European Journal of Human Genetics 28.4(2020):480-490). Antisense oligonucleotide-mediated exon skipping (Yamamura, Tomohiko, et al. "Development of an exon skipping therapy for X-linked Alport syndrome with truncating variants in COL4A5." Nature Communications 11.1(2020):2777) is currently in phase I clinical trials. However, not all mutation types meet the treatment criteria for antisense oligonucleotides, and antisense oligonucleotide drugs are expensive and require long-term administration.

[0007] Adeno-associated virus (AAV) vectors have become the most commonly used gene replacement therapy vectors in clinical practice due to their safety, low immunogenicity, and long-term sustained expression. However, AAV vectors have limited packaging capacity, only 4.7 kb. Within this 4.7 kb, promoters and transcription terminators must be considered, and the length of the inserted transgene must be within 4 kb. Considering that some specific promoters or highly efficient promoters are longer, the transgene length may need to be even shorter. The COL4A3, COL4A4, and COL4A5 genes are around 5.1 kb in length, far exceeding the packaging capacity of AAV. Therefore, developing a functional truncated peptide of type IV collagen that can retain its original functional activity while meeting the delivery requirements of AAV vectors has been a key research focus in the treatment of Alport syndrome. Summary of the Invention

[0008] Purpose of the invention

[0009] The purpose of this invention is to provide a COL4A5 truncated mutant that can retain the collagen trimer assembly and secretion activity while meeting the delivery requirements of AAV vectors, its related nucleic acid and drug products, and its application in the preparation of drugs for the treatment of Alport syndrome.

[0010] Solution

[0011] To achieve the above objectives, the present invention adopts the following technical solution:

[0012] In a first aspect, the present invention provides a COL4A5 truncated mutant, wherein the COL4A5 truncated mutant is a polypeptide selected from the following:

[0013] (1) A polypeptide having an amino acid sequence as shown in SEQ ID NO:1 or 2; or

[0014] (2) A polypeptide derived from (1) having one or more amino acids substituted, deleted or added in the amino acid sequence shown in SEQ ID NO:1 or 2 and having the activity of forming collagen trimers with COL4A3 and COL4A4 and being secreted outside the cell.

[0015] Preferably, the COL4A5 truncated mutant is a polypeptide with an amino acid sequence as shown in SEQ ID NO:1 or 2; more preferably, the COL4A5 truncated mutant is a polypeptide with an amino acid sequence as shown in SEQ ID NO:2;

[0016] Optionally, the COL4A5 truncated mutant further includes a peptide tag and / or a signal peptide;

[0017] Preferably, the peptide tag is selected from: Flag, HA, His, and Myc tags;

[0018] Preferably, the peptide tag is located at the C-terminus of the functional truncated peptide;

[0019] Preferably, the signal peptide is located at the N-terminus of a functional truncated peptide.

[0020] In a second aspect, the present invention provides a polynucleotide that encodes a truncated COL4A5 mutant as described in the first aspect above.

[0021] In a feasible implementation, the polynucleotide is DNA or mRNA.

[0022] Preferably, the polynucleotide is DNA containing the sequence shown in SEQ ID NO:3 or 4, or mRNA corresponding to the DNA, which respectively encodes the amino acid sequence shown in SEQ ID NO:1 or 2.

[0023] Thirdly, the present invention provides a nucleic acid construct comprising a polynucleotide as described in the second aspect above, and optionally, at least one expression regulatory element operatively linked to the polynucleotide.

[0024] Fourthly, the present invention provides a recombinant expression vector comprising the polynucleotides as described in the second aspect above, or comprising the nucleic acid constructs as described in the third aspect above.

[0025] Preferably, the recombinant expression vector is a recombinant viral vector, more preferably a recombinant AAV vector, and even more preferably, the recombinant AAV vector contains a podocyte-specific promoter, under which the polynucleotides described in the second aspect above are expressed under the control of the podocyte-specific promoter.

[0026] Fifthly, the present invention provides the use of the COL4A5 truncated mutant as described in the first aspect above, the polynucleotide as described in the second aspect above, the nucleic acid construct as described in the third aspect above, and / or the recombinant expression vector as described in the fourth aspect above in the preparation of a medicament for the treatment of Alport syndrome.

[0027] In a feasible implementation, the treatment of Alport syndrome includes any one or more of the following:

[0028] (1) To reduce, alleviate, improve or reverse the kidney symptoms or condition of Alport syndrome;

[0029] (2) To alleviate, relieve, improve or reverse the ocular symptoms or conditions of Alport syndrome;

[0030] (3) To alleviate, relieve, improve or reverse ear symptoms or conditions of Alport syndrome.

[0031] In a feasible implementation, the drug is a gene therapy drug.

[0032] In the most preferred embodiment, the active ingredient of the drug is a recombinant AAV expression vector containing a polynucleotide as described in the second aspect above or a nucleic acid construct as described in the third aspect above, which is suitable for delivery by injection to the kidney, eye and / or ear of a patient with Alport syndrome, preferably the kidney and / or eye.

[0033] In a sixth aspect, the present invention provides a pharmaceutical composition comprising the COL4A5 truncated mutant as described in the first aspect above, the polynucleotide as described in the second aspect above, the nucleic acid construct as described in the third aspect above, and / or the recombinant expression vector as described in the fourth aspect above.

[0034] Preferably, the pharmaceutical composition is an injection, preferably a sustained-release injection; in a further preferred embodiment, the pharmaceutical composition is in the form of the above-mentioned recombinant AAV carrier loaded on a hydrogel.

[0035] In a preferred embodiment, the pharmaceutical composition comprises the recombinant expression vector as described in the fourth aspect above, preferably the recombinant AAV expression vector, as the active ingredient, and is prepared in the form of an injectable preparation for delivery by injection to the kidney, eye, and / or ear of a patient with Alport syndrome, preferably the kidney and / or eye.

[0036] In a seventh aspect, the present invention provides a method for treating Alport syndrome, the method comprising: administering to a subject in need a therapeutically effective amount of a COL4A5 truncated mutant as described in the first aspect above, a polynucleotide as described in the second aspect above, a nucleic acid construct as described in the third aspect above, a recombinant expression vector as described in the fourth aspect above, and / or a pharmaceutical composition as described in the sixth aspect above.

[0037] Preferably, the treatment is gene therapy; more preferably, the gene therapy is achieved by administering a therapeutically effective amount of a recombinant AAV vector containing nucleotides encoding the truncated recombinant COL4A5 peptide described in the first aspect to a subject in need (preferably to their kidneys, eyes, and / or ears).

[0038] The "therapeutic effective dose" can vary depending on the recipient, the organ involved, the symptoms, the method of administration, etc. It can be determined based on the doctor's judgment, taking into account factors such as the type of dosage form, the method of administration, the patient's age and weight, and the patient's symptoms.

[0039] Beneficial effects

[0040] Through scientific design and extensive screening, this invention has yielded a truncated COL4A5 mutant that retains the activity of natural COL4A5 collagen in assembling into a trimer with COL4A3 and COL4A4 collagen and being secreted extracellularly, while its size is suitable for AAV vector delivery. The inventors have demonstrated through in vitro and in vivo experiments that the truncated COL4A5 mutant can assemble into a trimer with COL4A3 and COL4A4 collagen and be secreted extracellularly. Furthermore, when delivered to Alport syndrome mouse models, it significantly improves renal function and has a certain therapeutic effect on retinal structural abnormalities, thereby alleviating disease symptoms and slowing disease progression to some extent. These results indicate that the truncated COL4A5 mutant of this invention has great clinical application potential in the treatment of Alport syndrome. In particular, recombinant AAV vectors containing its encoding nucleic acid can be used for gene therapy of Alport syndrome, thus possessing significant potential clinical application value. Attached Figure Description

[0041] One or more embodiments are illustrated by way of example with reference to the accompanying drawings, and these illustrative examples are not intended to limit the embodiments. The term "illustrative" as used herein means "serving as an example, embodiment, or illustration." Any embodiment illustrated herein as "illustrative" is not necessarily to be construed as superior to or better than other embodiments.

[0042] Figure 1 The design and functional validation of the Col4α5 truncated gene are shown. Figure A shows a schematic diagram of the design principle and functional validation method of the Col4α5 truncated gene; Figure B shows the functional validation results of the formation and secretion of the Col4α5 truncated gene trimer; Figure C shows the detection results of the intracellular interaction of the Col4α5 truncated gene trimer; Figure D shows the detection results of the secretion and extracellular interaction of the Col4α5 truncated gene trimer; and Figure E shows the validation results of the packaging and expression of AAV-CMV-mini-Col4α5(Je) and AAV-CMV-mini-Col4α5(K) viruses.

[0043] Figure 2 The plasmid maps of the recombinant vectors AAV-CMV-mini-Col4α5 (Je, Figure A) and AAV-CMV-mini-Col4α5 (K, Figure B) are shown.

[0044] Figure 3The diagram shows the construction and phenotypic validation of the XLAS disease model mouse. Figure A shows the design and sequencing results of gene mutation sites in XLAS mice, Figure B shows the collagen staining results of the kidneys of XLAS mice, Figure C shows the structural changes of the glomerular basement membrane and podocytes in XLAS mice, and Figure D shows the progressive decline of kidney function in XLAS mice.

[0045] Figure 4 This study shows the differences in renal function between mini Col4α5 transgenic model mice and XLAS disease model mice under the XLAS background. Figure A shows the construction process and design principle of the R373X mini Col4α5 model mouse. Figure B shows collagen trimer staining after mini Col4α5 expression in XLAS mice. Figure C shows the changes in urinary protein / creatinine ratio after mini Col4α5 expression in XLAS mice. Figure D shows the changes in blood urea nitrogen after mini Col4α5 expression in XLAS mice. Figure E shows the changes in serum creatinine after mini Col4α5 expression in XLAS mice.

[0046] Figure 5 This study shows the differences in renal pathology between mini Col4α5 transgenic model mice and XLAS disease model mice under XLAS background. Figure A shows the improvement in tubular damage after mini Col4α5 expression in XLAS mice; Figure B shows the statistical results of different treatment groups in Figure A; Figure C shows the improvement in renal fibrosis after mini Col4α5 expression in XLAS mice; Figure D shows the statistical results of different treatment groups in Figure C; Figure E shows the glomerular structure after mini Col4α5 expression in XLAS mice; and Figure F shows the statistical results of podocyte number after mini Col4α5 expression in XLAS mice.

[0047] Figure 6 The phenotypic validation of AAV-CMV-mini Col4α5(K) in vivo delivery mice is shown. Figure A shows a schematic diagram of subretinal injection of AAV2-CMV-mini Col4α5(K), Figure B shows the re-expression of Col4α3 after subretinal injection of AAV2-CMV-mini Col4α5(K), Figure C shows the re-expression of Col4α4 after subretinal injection of AAV2-CMV-mini Col4α5(K), Figure D shows the expression of mini Col4α5(K) after subretinal injection of AAV2-CMV-mini Col4α5(K), and Figure E shows the improvement of fundus abnormalities in XLAS mice after subretinal injection of AAV2-CMV-mini Col4α5(K). Detailed Implementation

[0048] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below in conjunction with embodiments. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0049] Furthermore, to better illustrate the present invention, numerous specific details are provided in the following detailed embodiments. Those skilled in the art should understand that the present invention can be practiced without certain specific details. In some embodiments, materials, elements, methods, and means well known to those skilled in the art are not described in detail in order to highlight the spirit of the invention.

[0050] Unless otherwise expressly stated, throughout the specification and claims, the term "comprising" or its variations such as "including" or "comprises" shall be understood to include the stated elements or components without excluding other elements or other components.

[0051] The present invention will be further described in detail below through examples.

[0052] Example 1: Col4α5 protein structure and Col4α5 truncated gene design

[0053] In this study, truncated functional genes were designed and optimized based on the amino acid sequence of mouse Col4α5. Col4α5 truncated mutants were designed using structural analysis and alignment. The N-terminal 7S domain and C-terminal NC1 domain are mainly responsible for trimer assembly and secretion, while the middle Gly-XY repeat domain mediates the formation of the trimer helical structure. Besides the Gly-XY repeat domain, the middle region contains multiple glycosylation sites affecting collagen stability and cysteine ​​residues that form disulfide bonds. Simply deleting the middle Gly-XY repeat domain indiscriminately may disrupt the stability of the trimer structure. Furthermore, the Gly-XY repeat domain contains multiple interacting protein sites, such as binding sites for HSP47, integrins, and heparin. HSP47 is a core protein responsible for transferring collagen from the endoplasmic reticulum to the Golgi apparatus for glycosylation and other modifications; the absence of HSP47 binding sites affects collagen stability. Integrins mediate the adhesion of type IV collagen in cells and the matrix, which is crucial for the stability of the glomerular basement membrane. Heparin and heparan sulfate proteoglycan (HSPG) binding sites are responsible for supporting the interaction between cells and type IV collagen by binding to HSPGs on the cell surface. Based on the above distribution of the main functional regions of Col4α5, we constructed a series of Col4α5 truncated mutants (hereinafter referred to as mini-Col4α5). The structural schematic diagram of the designed Col4α5 truncated mutants is shown in [Figure number missing]. Figure 1 A contains mutants that delete the N-terminal 7S domain, mutants that delete the C-terminal NC1 domain, mutants that delete Gly-XY amino acids from positions 61 to 153, mutants that delete amino acids from positions 542 to 655 (including cysteine ​​residues and multiple Gly-XY repeats), mutants that delete Gly-XY amino acids from positions 672 to 780, mutants that delete Gly-XY amino acids from positions 1143 to 1254, and mutants that delete a portion of the multiple interruptions present in the Gly-XY repeat domain (amino acids 245-250, 346-355, 444-453, 481-487, 597-599, and 659-662). (852-856 amino terminus) and combinations of amino acids with different segments deleted, such as: F (deleting amino acids at positions 542-655 and 672-780), Fe (deleting amino acids at positions 542-655, 672-780 and 1143-1254), J (deleting amino acids at positions 542-655, 672-919, 1107-1130 and 1268-1273), Je (deleting amino acids at positions 542-655, 672-919, 1107-1130, 1143-1254 and 1268-1273), K (deleting amino acids at positions 542-655, 672-919, 967-1139 and 1268-1273).

[0054] Example 2: Expression of Col4α5 truncated mutant

[0055] In a series of designed Col4α5 truncated mutants, we constructed different Col4α5 truncated mutants into the pcDNA5 / FRT vector, which was obtained from Invitrogen (catalog number V601020), using polymerase chain reaction (PCR).

[0056] To verify the ability of each Col4α5 truncated mutant to form trimers with wild-type Col4α3 / 4 and their secretion, we designed a trimer formation and secretion experiment based on (Omachi, Kohei, et al. "A split-luciferase-based trimer formation assay as a high-throughput screening platform for therapeutics in Alportsyndrome." Cell Chemical Biology 25.5 (2018):634-643). Specifically, we ligated a GS linker-LgBiT fragment (sequence shown in SEQ ID NO:5) to the C-terminus of each Col4α5 truncated mutant and a GS linker-SmBiT fragment (sequence shown in SEQ ID NO:6) to the C-terminus of natural Col4α3.

[0057] SEQ ID NO:5 is shown below:

[0058] GSSGGGGSGGGGSSGVFTLEDFVGDWEQTAAYNLDQVLEQGGVSSLLQNLAVSV

[0059] TPIQRIVRSGENALKIDIHVIIPYEGLSADQMAQIEEVFKVVYPVDDHHFKVILPYG

[0060] TLVIDGVTPNMLNYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLITPDGSMLFRVTINS;

[0061] SEQ ID NO:6 is shown below:

[0062] GSSGGGGSGGGGSSGVTGYRLFEEIL.

[0063] LgBiT and SmBiT proteins bind tightly in space to form a functional protein, which emits light upon the addition of an appropriate substrate. The intensity of the luminescence signal is used to determine the formation of the trimer. Both the substrate and buffer are derived from... The Live Cell Assay System kit was purchased from Promega (catalog number N2011).

[0064] Specifically, the expression plasmid of the fusion protein of the Col4α5 truncated mutant and LgBiT was first transfected into HEK293T cells. After 6 hours of PEI transfection, the medium was changed, and the cells were harvested and the protein was extracted after 48 hours. The expression of the Col4α5 truncated mutant fused with LgBiT was detected by Western blotting. The results showed that the fusion proteins of each Col4α5 truncated mutant and LgBiT could be expressed normally in the cells.

[0065] Similarly, the fusion protein of Col4α3 and SmBiT, as well as Col4α4, were also verified and expressed.

[0066] Example 3: Trimer formation and secretory activity detection of Col4α5 truncated mutant

[0067] 1. Trimer formation and secretion detection

[0068] Trimer formation and secretion assays were performed with reference to Omachi, Kohei, et al. "A split-luciferase-based trimer formation assay as a high-throughput screening platform for therapy in Alport syndrome." Cell Chemical Biology 25.5(2018):634-643.

[0069] Specifically, the fusion proteins of each Col4α5 truncated mutant and LgBiT, along with Col4α3-SmBiT and Col4α4, were co-transfected into HEK293T cells. 48 hours after transfection, substrates were added, and the luminescence signal values ​​in the cell culture medium and cells were measured. The assembly ability of each Col4α5 truncated mutant with Col4α3 and Col4α4 and its ability to be secreted extracellularly were determined based on the strength of the luminescence signal value. A higher luminescence signal value indicates a stronger collagen assembly and secretion ability.

[0070] The results of the trimer formation and secretion experiments are as follows: Figure 1As shown in B.

[0071] Figure 1 B shows that deletion of both the N-terminal 7S domain and the C-terminal NC1 domain affects trimer formation and secretion. Deletion of Gly-XY amino acids from positions 61 to 153 severely affects trimer secretion. Deletion of some of the multiple interruptions in the Gly-XY repeat domain (amino acids 245-250, 346-355, 444-453, 481-487, 597-599, 659-662, and 852-856) also significantly reduces trimer secretion. These results suggest that multiple interruptions in the Gly-XY repeat domain play an important role in trimer secretion, and the Gly-XY repeat domain and non-collagen domains cannot be deleted arbitrarily.

[0072] according to Figure 1 Based on the results of the trimer formation and secretion experiments of B, we finally identified two mini Col4α5 truncated functional mutants that meet the AAV packaging requirements: mutant Je (3564bp) (deleting amino acids at positions 542-655, 672-919, 1107-1130, 1143-1254, and 1268-1273) and mutant K (3453bp) (deleting amino acids at positions 542-655, 672-919, 967-1139, and 1268-1273). Their amino acid sequences are shown in SEQ ID NO:1 and 2, respectively. In the following text, these two mutants can also be referred to as Col4α5-Je and Col4α5-K, respectively. Figure 1 B shows that both mutants can form trimers with Col4α3 and Col4α4 in vitro and be secreted extracellularly. In particular, mutant K shows superior secretion ability compared to wild-type Col4α5. This suggests that the roles of different regions of the Gly-XY repeat domain in trimer formation and secretion are not entirely the same, and deletion of some regions of the Gly-XY repeat domain may even help trimer secretion.

[0073] 2. Immunoprecipitation (Co-IP) assay to verify trimer formation and secretion.

[0074] We further verified using co-immunoprecipitation (Co-IP) experiments that the Col4α5 truncated mutants Je and K can form trimers with Col4α3 / 4 in cells, and the presence of trimers can also be detected in the culture supernatant.

[0075] To perform co-immunoprecipitation (Co-IP) experiments, we first constructed pcDNA5-Col4α5-Flag, pcDNA5-Col4α5-del C-Flag, pcDNA5-Col4α5-Je-Flag, pcDNA5-Col4α5-K-Flag, pcDNA5-Col4α3, and pcDNA5-Col4α4 plasmids. Then, using PEI, we transiently transfected HEK293T cells with the above Col4α5 or truncated mutant expression plasmids along with Col4α3 and Col4α4 expression plasmids. After 48 hours, we collected the supernatant and added a protease inhibitor (Roche 04693132001) and ethyelenediaminetetraacetic acid (EDTA) to the supernatant. Cells were lysed with NETN buffer, which consisted of 50 mmol / L Tris-HCl (pH 7.8), 150 mmol / L NaCl, and 1 mmol / L... Cells were lysed with EDTA, 1% NP-40, 1 mmol / L dithiothreitol (DTT), and a protease inhibitor (Roche 04693132001). After centrifugation, the supernatant was collected, and Flag beads were added to the supernatant. Flag beads can bind to proteins tagged with Flag (pcDNA5-Col4α5-Flag, pcDNA5-Col4α5-del C-Flag, pcDNA5-Col4α5-Je-Flag, pcDNA5-Col4α5-K-Flag). The presence of Flag, Col4α3, and Col4α4 was then detected by Western blotting.

[0076] See results Figure 1 C and 1D. By Figure 1 From C and 1D we can see:

[0077] (1) pcDNA5-Col4α5-Flag interacts with pcDNA5-Col4α3 and pcDNA5-Col4α4, and serves as a positive control.

[0078] (2) pcDNA5-Col4α5-del C-Flag does not interact with pcDNA5-Col4α3 and pcDNA5-Col4α4 and cannot be secreted into the culture medium, so it serves as a negative control.

[0079] (3) pcDNA5-Col4α5-Je / K-Flag interacts with both pcDNA5-Col4α3 and pcDNA5-Col4α4 and can be secreted into the culture medium, proving that Col4α5-Je / K forms a trimer with Col4α3 and Col4α4 and is secreted into the extracellular space.

[0080] The Flag antibody used in this experimental procedure was Abcam Ab205606 reagent, the Col4α3 antibody was Chondrex 7076 reagent, the Col4α4 antibody was Chondrex 7073 reagent, and the Col4α5 antibody was Cosmo Bio SGE-C453 reagent. The Co-IP experiment on collagen trimer interactions is referenced (Kobayashi, Takehiro, and Makoto Uchiyama. "Characterization of assembly of recombinant type IV collagen α3, α4, and α5 chains in transfected cell strains." Kidney International 64.6 (2003): 1986-1996).

[0081] Example 4: Construction of AAV plasmid for mini Col4α5 and viral expression

[0082] Based on the cellular level experiments in Example 3, we demonstrated that the Col4α5 truncated mutants Je and K possess collagen formation and secretion functions. Furthermore, we commissioned Paizhen Biotechnology to construct the recombinant AAV vectors AAV-CMV-mini-Col4α5(Je) and AAV-CMV-mini-Col4α5(K) for the Col4α5 truncated mutants Je and K (their plasmid maps are shown below). Figure 2 A and 2B, such as Figure 2 As shown in Figures A and 2B, a Flag tag was introduced at the C-terminus of mini-Col4α5(Je) and mini-Col4α5(K) (to facilitate subsequent protein detection and identification), and the virus was packaged. Ultimately, we obtained HEK293T cells infected with AAV-CMV-mini-Col4α5(Je) or AAV-CMV-mini-Col4α5(K) virus. Western blotting results showed that the virus could be well expressed in HEK293T cells (see Appendix). Figure 1 E). This proves that we have successfully designed and packaged the AAV-CMV-mini-Col4α5-Je / K virus.

[0083] Example 5: Construction and Phenotypic Detection of XLAS Disease Model Mouse

[0084] 1. Querying COL4A5 clinical mutation sites and constructing a mouse disease model

[0085] Based on the COL4A5 clinical mutation database provided by ARUP Laboratories, we confirmed the COL4A5 mutation in X-linked Alport syndrome (XLAS), specifically the COL4A5 exon 19 c.1117C>T mutation, with the amino acid sequence COL4A5R373X. This mutation is highly conserved in this region in humans and mice. Based on this mutation information, we commissioned the Nanjing Institute of Model Animals to construct Col4α5R373X mutant mice as a mouse model for X-linked Alport syndrome. The institute used CRISPR / Cas9 technology, employing homologous recombination repair to introduce the target mutation site. The brief procedure is as follows: Cas9 mRNA and sgRNA were obtained through in vitro transcription; donor DNA with the mutated sequence was synthesized; and Cas9 mRNA, sgRNA, and donor DNA were microinjected into C57BL / 6J mouse zygotes. F0 generation mice were obtained by selecting embryos. PCR-positive and F0 generation mice were crossed with wild-type C57BL / 6J mice to obtain positive F1 generation mice, which are the Col4α5R373X mutant mice. DNA was extracted from the toes of the constructed mice and sequenced. Sanger sequencing results showed that a C>T mutation was indeed present at exon 19 of Col4α5 (see...). Figure 3 A) indicates that the Col4α5R373X mutant mouse was successfully constructed.

[0086] 2. Phenotypic validation of the XLAS disease model mouse

[0087] We confirmed the deletion of Col4α3 / 4 / 5 in the glomerular basement membrane of Col4α5R373X mutant mice using immunofluorescence staining. The results are shown in [Figure number missing]. Figure 3 B.

[0088] We observed mouse glomeruli using transmission electron microscopy, and the results are attached. Figure 3 As shown in C; Figure 3 C indicates that glomerular podocytes in the disease model mice showed fusion and abnormalities in the glomerular basement membrane structure.

[0089] In addition, kidney function in the disease model mice was examined. Specifically, mice of different ages were placed in metabolic cages, and urine was collected at different time points after 24 hours. Albumin levels in the urine were detected using the BethylMouse Albumin ELISA Kit (catalog number E99-134), and creatinine levels in the urine were detected using the sarcosine oxidase method (kit purchased from Nanjing Jiancheng Bioengineering Institute (catalog number C011-2-1)). Results are as follows: Figure 3 As shown in D, Figure 3 D shows that Col4α5R373X mutant mice exhibit an increasing ratio of urinary protein to urinary creatinine with age, suggesting a continuous decline in renal function in Col4α5R373X mutant mice.

[0090] The above results indicate that the Col4α5R373X mutant mice exhibit typical XLAS disease symptoms and can be used as an XLAS disease model. Therefore, they will be referred to as XLAS disease model mice below.

[0091] Example 6: Establishment of the mini Col4α5 mouse transgenic model and detection of kidney function

[0092] 1. Construction of mini-Col4α5 transgenic mice

[0093] To verify that the restoration of mini-Col4α5 expression in podocytes of the constructed XLAS disease model mice can promote the re-secretion and deposition of trimers, thereby alleviating disease progression, we first designed mini-Col4α5-K transgenic mice, Nphs1 promoter-rtTA-3G mice, and TRE3G-Col4α5-K mice. In these mice, the Nphs1 promoter drives the specific expression of rtTA-3G in mouse podocytes. Upon addition of doxycycline, doxycycline binds to free rtTA-3G and then to the TRE3G promoter, driving the expression of the target gene (Col4α5-K) following the TRE3G promoter (the mechanism is described in [link to mechanism description]). Figure 4A). The construction method, induction time, and concentration of this mouse were all based on the reference (Lin, Xiaobo, et al. "Feasibility of repairing glomerular basement membrane defects in Alportsyndrome." Journal of the American Society of Nephrology: JASN 25.4(2014):687). In that reference, the researchers designed two types of mice: Nphs1 promoter-rtTA-3G mice and (tetO)7 / CMV-Col4α3cDNA mice, and then hybridized the two types of mice to obtain Nphs1 promoter-rtTA-3G;(tetO)7 / CMV-Col4α3cDNA mice. In our study, we used a single-vector method to construct Nphs1 promoter-rtTA-3G;TRE3G-Col4α5-K mice. Nphs1 promoter-rtTA-3G;TRE3G-Col4α5-K mice can be obtained with just one construction, without the need for further hybridization. This method references the following literature: (Katigbak, Alexandra, et al. "Inducible genome editing with conditional CRISPR / Cas9 mice." G3: Genes, Genomes, Genetics 8.5 (2018): 1627-1635) (Ye, Lin, et al. "Generation of induced pluripotent stem cells using site-specific integration with phage integrase." Proceedings of the National Academy of Sciences 107.45 (2010): 19467-19472). Based on the above design, we commissioned Cyagen Biosciences to construct this mouse. The brief construction process is as follows: First, we constructed a single vector, Nphs1 promoter-rtTA-3G;TRE3G-Col4α5-K, which has a homologous arm and can be directionally inserted into the genomic safe site Hipp11 (H11). Simultaneously, we obtained Cas9 mRNA and sgRNA through in vitro transcription. Cas9 mRNA, sgRNA, and the single vector with the homologous arm were microinjected into C57BL / 6J mouse zygotes, and F0 generation mice were obtained by selecting embryos.PCR-sequencing positive and F0 generation mice were crossed with wild-type C57BL / 6J mice to obtain positive F1 generation mice, resulting in the final Nphs1promoter-rtTA-3G;TRE3G-Col4α5-K mice. Genotyping results showed that Nphs1promoter-rtTA-3G;TRE3G-Col4α5-K was effectively inserted into the mouse genome.

[0094] Furthermore, we crossed the constructed Nphs1 promoter-rtTA-3G;TRE3G-Col4α5-K mice with the Col4α5R373X mutant mice constructed in Example 5 to obtain Col4α5R373X;Nphs1 promoter-rtTA-3G;TRE3G-Col4α5-K mice, which were used for subsequent experiments. We can control the expression time and level of mini-Col4α5-K in Col4α5R373X disease model mice by adjusting the induction time and concentration of Doxycycline, thereby observing the therapeutic effect of mini-Col4α5-K on the XLAS disease model.

[0095] 2. Phenotypic validation of mini-Col4α5 transgenic mice

[0096] For the Col4α5R373X;Nphs1 promoter-rtTA-3G;TRE3G-Col4α5-K mice constructed above, mice were fed a diet containing 0.15% Doxycycline on day 21 (P21). After 6 weeks of treatment, kidney section staining revealed the expression of mini-Col4α5-K in podocytes, and the re-expression of Col4α3 / 4 was also observed (see...). Figure 4 B). The above experiments demonstrate that after podocyte-specific expression of mini-Col4α5-K, the re-expression of Col4α3 / 4 can be restored (this may be because after re-expression of mini-Col4α5-K, the expressed mini-Col4α5-K can form a trimer with Col4α3 / 4, thereby making the protein level of Col4α3 / 4 more stable), suggesting that the Col4α5 truncated mutant Col4α5-K has the function of collagen trimer assembly and secretion.

[0097] After 16 weeks of Dox feeding, we collected urine samples from mice to detect urinary protein and creatinine. The results showed that after overexpression of mini-Col4α5-K in podocytes, the urinary protein-to-creatinine ratio (U-ACR) in mouse urine was lower (see [link to study]). Figure 4C) suggests that overexpression of mini-Col4α5-K in podocytes can not only restore the re-expression of Col4α3 / 4, but also alleviate the disease progression to some extent.

[0098] Meanwhile, we measured the levels of blood urea nitrogen and creatinine in mouse blood samples. The results showed that overexpression of mini-Col4α5-K in podocytes could reduce blood urea nitrogen and serum creatinine levels and slow disease progression (see...). Figure 4 (D and 4E).

[0099] The reagents used for detecting mouse kidney function in this part of the experiment are as follows: Urinary creatinine was measured using the sarcosine oxidase method; the kit was purchased from Nanjing Jiancheng Bioengineering Institute (catalog number C011-2-1). Urinary protein was measured using the MouseAlbumin ELISA Kit (catalog number E99-134) from Bethyl. Mouse plasma urea nitrogen was measured using the diacetyl oxime colorimetric method; the kit was purchased from Nanjing Jiancheng Bioengineering Institute (catalog number C013-1-1). Mouse plasma and urinary creatinine were measured using the sarcosine oxidase method; the kit was purchased from Nanjing Jiancheng Bioengineering Institute (catalog number C011-2-1).

[0100] Example 7: Histopathological examination of kidney tissue in a mini Col4α5 mouse transgenic model

[0101] The kidneys of the mini-Col4α5 transgenic mice constructed in Example 6 were sectioned and stained. The glomerular structure was observed by transmission electron microscopy, renal fibrosis was observed by Masson staining, and renal tubular damage was observed by PAS staining.

[0102] Experimental results are as follows Figure 5 As shown in the figure, Figure A shows the improvement of tubular damage after expressing mini Col4α5 in XLAS mice; Figure B shows the statistical results of different treatment groups in Figure A; Figure C shows the improvement of renal fibrosis after expressing mini Col4α5 in XLAS mice; Figure D shows the statistical results of different treatment groups in Figure C; Figure E shows the glomerular structure after expressing mini Col4α5 in XLAS mice; and Figure F shows the statistical results of podocyte number after expressing mini Col4α5 in XLAS mice. Figure 5 The results showed that the mini-Col4α5 transgenic mice exhibited more intact glomerular podocyte foot processes and more normal glomerular basement membranes, while also showing lower levels of fibrosis and tubular damage. These experiments indicate that the mini-Col4α5 transgenic mice exhibit better kidney function.

[0103] The sample preparation process for electron microscopy was as follows: Under a dissecting microscope, the renal cortex was cut into small pieces containing glomeruli (1-4 mm in diameter), and the samples were placed in 2.5% glutaraldehyde fixative. After sample processing, the samples were sectioned and prepared by the Institute of Hematology and Blood Diseases Hospital, Chinese Academy of Medical Sciences. The Masson staining kit was purchased from Solarbio (catalog number G1340), and the glycogen PAS staining solution kit (periodic acid-Schiff staining solution kit) was purchased from Solarbio (catalog number G1281).

[0104] Example 8: Phenotypic Validation of AAV-CMV-mini Col4α5 In Vivo Delivery

[0105] In addition to severe kidney damage, patients with X-linked Alport syndrome have a certain probability of experiencing visual abnormalities, which significantly impacts their quality of life. A common clinical ocular abnormality is punctate retinopathy; we observed the absence of Col4α3 / 4 / 5 and punctate lesions in the retinas of Col4α5R373X mice.

[0106] We packaged AAV2 adeno-associated virus using the AAV-CMV-miniCol4α5(K) viral vector constructed in Example 4 to obtain AAV2-CMV-miniCol4α5(K) virus. Then, the AAV2-CMV-miniCol4α5(K) virus was injected subretinally into RPE cells of Col4α5R373X mutant mice constructed as in Example 5. Figure 6 A) The subretinal injection procedure is as follows (Huang, Peirong, et al. "Subretinal injection in mice to study retinal physiology and disease." Nature Protocols 17.6(2022):1468-1485). The injection volume per eye is 1 μL, and the viral load is 1*102. 10 vg. Three weeks later, sections of the eyeball were stained, and the results were as follows. Figure 6 As shown in BD; Figure 6 BD analysis showed that the AAV2-CMV-miniCol4α5(K) injection group exhibited collagen re-expression.

[0107] In addition, we used the MicroIV small animal retinal imaging system manufactured by Phoenix Research Labs to observe the fundus phenotype of mice, and the results are as follows: Figure 6 As shown in E; Figure 6E showed that mice in the AAV2-CMV-miniCol4α5(K) injection group had fewer punctate lesions in their fundus, suggesting that AAV2-CMV-miniCol4α5(K) has a certain therapeutic effect on retinal structural abnormalities.

[0108] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A truncated COL4A5 mutant, characterized in that, The COL4A5 truncated mutant is a polypeptide selected from the following: (1) A polypeptide having an amino acid sequence as shown in SEQ ID NO:1 or 2; or (2) A polypeptide derived from (1) having one or more amino acids substituted, deleted or added in the amino acid sequence shown in SEQ ID NO:1 or 2 and having the activity of forming collagen trimers with COL4A3 and COL4A4 and being secreted outside the cell.

2. The COL4A5 truncated mutant according to claim 1, characterized in that, The COL4A5 truncated mutant is a polypeptide with an amino acid sequence as shown in SEQ ID NO:1 or 2, preferably a polypeptide with an amino acid sequence as shown in SEQ ID NO:2; Optionally, the COL4A5 truncated mutant further includes a peptide tag and / or a signal peptide; Preferably, the peptide tag is selected from: Flag, HA, His, and Myc tags; Preferably, the peptide tag is located at the C-terminus of the functional truncated peptide; Preferably, the signal peptide is located at the N-terminus of a functional truncated peptide.

3. A polynucleotide encoding a truncated COL4A5 mutant as described in claim 1 or 2.

4. The polynucleotide according to claim 3, characterized in that, The polynucleotide is DNA or mRNA; Preferably, the polynucleotide is DNA containing the sequence shown in SEQ ID NO:3 or 4, or mRNA corresponding to the DNA.

5. A nucleic acid construct comprising a polynucleotide as described in claim 3 or 4, and optionally, at least one expression regulatory element operatively linked to said polynucleotide.

6. A recombinant expression vector comprising the polynucleotide as described in claim 3 or 4, or comprising the nucleic acid construct as described in claim 5.

7. The recombinant expression vector according to claim 6, characterized in that, The recombinant expression vector is a recombinant viral vector, preferably a recombinant AAV vector, and more preferably, the recombinant AAV vector contains a promoter selected from the following: a podocyte-specific promoter, an inner ear cell-specific promoter, and a retinal cell-specific promoter, and the polynucleotide as described in claim 3 or 4 is expressed under the control of the promoter.

8. Use of the COL4A5 truncated mutant as described in claim 1 or 2, the polynucleotide as described in claim 3 or 4, the nucleic acid construct as described in claim 5, and / or the recombinant expression vector as described in claim 6 or 7 in the preparation of a medicament for the treatment of Alport syndrome.

9. The use according to claim 8, characterized in that, The treatment of Alport syndrome includes any one or more of the following: (1) To reduce, alleviate, improve or reverse the kidney symptoms or condition of Alport syndrome; (2) To alleviate, relieve, improve or reverse the ocular symptoms or conditions of Alport syndrome; (3) To alleviate, relieve, improve or reverse ear symptoms or conditions of Alport syndrome; And / or, the drug is a gene therapy drug.

10. A pharmaceutical composition comprising the COL4A5 truncated mutant as claimed in claim 1 or 2, the polynucleotide as claimed in claim 3 or 4, the nucleic acid construct as claimed in claim 5, and / or the recombinant expression vector as claimed in claim 6 or 7, and optionally a pharmaceutically acceptable vector or excipient; Preferably, the pharmaceutical composition is an injection, more preferably a sustained-release injection, and even more preferably the form of the above-mentioned recombinant AAV carrier loaded on a hydrogel.

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