Polynucleotide sequence for simultaneously reducing blood lipid and blood pressure and application thereof

Abstract

The application discloses a polynucleotide sequence for simultaneously reducing blood fat and blood pressure and application thereof. Cell and mouse experiments show that the application can significantly inhibit the expression levels of multiple blood fat and blood pressure molecular targets such as proprotein convertase subtilisin / kexin type 9 (PCSK9), angiotensinogen (AGT) and beta-hydroxy-beta-methylglutaryl-coenzyme a reductase (HMGCR), thereby reducing the blood fat and blood pressure levels of a related disease model mouse. The application is expected to be applied to treating related cardiovascular and cerebrovascular diseases complicated with high blood fat and high blood pressure symptoms.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and provides a polynucleotide sequence that simultaneously lowers blood lipids and blood pressure and its applications. Background Technology

[0002] Pathological cardiovascular and cerebrovascular diseases mainly include coronary artery disease, cerebrovascular disease, and peripheral artery disease, affecting more than 500 million people worldwide and causing more than 18 million deaths, accounting for about one-third of global deaths. In China, 21-26% of people aged 20 and above are diagnosed with hyperlipidemia-related lesions such as increased intima-media thickness, vascular wall inflammation, and lipid plaque deposition, which worsen with age. The incidence of these symptoms can exceed half in people over 60 years of age. Regarding hypertension, more than 800 million adults worldwide have a systolic blood pressure greater than 140 mmHg, and in China, the proportion of people aged 35 and above with hypertension is over 40%. These two conditions have a remarkably high complication rate among patients with cardiovascular and cerebrovascular diseases, represented by stroke, atherosclerosis, and coronary heart disease, even reaching over 75% in some populations. This demonstrates that hyperlipidemia and hypertension are crucial driving forces in the occurrence and progression of cardiovascular and cerebrovascular diseases.

[0003] While current lipid-lowering and blood pressure-lowering drugs are widely used and have a rapid onset of action, they have single targets, short duration of action, difficulty in eradicating relapse, and require multiple medications, thus increasing the risk of accumulating adverse reactions. For example, first-line statins are frequently reported to cause side effects such as muscle pain, rhabdomyolysis, neurological damage (depression, cognitive and memory impairment, peripheral neuropathy), and liver and kidney toxicity. This is closely related to their systemic non-specific inhibition of HMG-CoA reductase and steroid synthesis after oral administration. On the other hand, frequent use of antihypertensive drugs also carries the risk of accumulating various adverse reactions. Among them, adrenergic receptor blockers can cause cardiovascular side effects such as congestive heart failure due to non-specific inhibition of α and β adrenergic receptors in the heart; diuretics may cause polyuria, dehydration, and hyponatremia, leading to various physiological dysfunctions related to osmotic pressure abnormalities; and commonly used drugs of the renin-angiotensin-aldosterone system, angiotensin-converting enzyme (ACE) inhibitors, can cause persistent dry cough due to the simultaneous inhibition of ACE-regulated bradykinin and substance P degradation, which can activate the cough reflex. In addition, ACE inhibitors can also inhibit the proliferation of hematopoietic stem cells, causing anemia, and can cause adverse reactions such as eczema when applied to the skin.

[0004] In summary, current lipid-lowering and blood pressure-lowering drugs have the following problems: their pharmacological mechanisms are relatively simple, making it difficult to simultaneously address multiple targets and pathways such as lowering blood lipids, lowering blood pressure, and suppressing immune inflammation; in addition, their effects are short-lived, so they often need to be used in combination and taken frequently, which not only causes inconvenience to patients but also increases the risk of the cumulative effect of non-specific adverse reactions of each drug.

[0005] To address the aforementioned issues, several long-acting RNAi drugs have been developed both domestically and internationally in recent years, such as Inclisiran targeting hepatic PCSK9 and Zilebesiran targeting hepatic AGT. While these drugs, through GalNAc glycosylation modification of siRNA molecules, can achieve long-term efficacy of 3-6 months, mitigating the need for frequent medication, these new drugs still act on a single target, and their long-term efficacy and safety require further systematic evaluation with larger patient samples. AAV (autologous adenovirus) vectors have been commonly used in gene therapy for the past decade, with key advantages including extremely low pathogenicity, efficient multi-tissue delivery, and long-term stable (over 2 years) gene expression. Furthermore, AAVs can carry multiple nucleic acid or protein drugs. These advantages make AAV vectors potentially capable of simultaneously lowering blood lipids, lowering blood pressure, and controlling blood sugar with a single dose, with efficacy lasting for 5 years or even longer, thus providing a superior therapeutic tool for solving the aforementioned problems. Building upon previous work, this invention further modifies and optimizes novel AAV vectors, resulting in the novel AAV vector T68, which exhibits higher transduction efficiency in the liver and pancreas compared to wild-type AAV5, 8, and 9. Furthermore, the applicant has utilized neural network learning-guided design to develop an expression cassette system that simultaneously targets multiple lipid-lowering and blood pressure-lowering molecules. This system targets molecules in the liver and pancreas that regulate lipid metabolism and blood pressure, such as PCSK9, AGT, and HMGCR, aiming to achieve both lipid-lowering and blood pressure-lowering effects with a single drug. Ultimately, this will enable long-lasting (2 years or more) lipid-lowering and blood pressure-lowering effects with a single intravenous injection, providing an efficient and convenient tool for the prevention and treatment of cardiovascular and cerebrovascular diseases. Summary of the Invention

[0006] In view of this, the purpose of the present invention is to provide a polynucleotide sequence that simultaneously lowers blood lipids and blood pressure and its applications.

[0007] To achieve the above objectives, the present invention provides the following technical solution: The present invention provides a polynucleotide sequence that simultaneously lowers blood lipids and blood pressure, selected from any one or more of shHMG1, shHMG2, shHMG3, shPCSK2, shAGT1, shAGT2, shAGT3, shPALL1, shPALL2, shHALL1, and shHALL2, and their nucleotide sequences are shown in SEQ ID NO.1 to 11, respectively.

[0008] As one of the preferred technical solutions, the polynucleotide sequence is shHMG2, shPCSK2, shAGT1, shPALL1, shPALL2, shHALL1, shHALL2.

[0009] As a further preferred technical solution, the polynucleotide sequence is shAGT1, shPALL1, shPALL2, shHALL1, shHALL2.

[0010] As a further preferred technical solution, the polynucleotide sequence is shAGT1, shPALL1, shPALL2.

[0011] The present invention also provides a transgenic expression cassette comprising the aforementioned polynucleotide sequence.

[0012] As one of the preferred technical solutions, the transgenic expression cassette also includes a promoter required to drive expression, and is more preferably the U6 promoter with a nucleotide sequence as shown in SEQ ID NO.12.

[0013] As one of the preferred technical solutions, the transgenic expression cassette also includes a CB promoter (SEQ ID NO. 25) to drive the expression of green fluorescent protein GFP to trace the viral transduction efficiency (amino acid sequence as shown in SEQ ID NO. 14, coding gene sequence as shown in SEQ ID NO. 13), flanked by a normal ITR and a mutated ITR, so as to package the lipid-lowering and blood pressure-lowering expression cassette as a self-complementary AAV vector into AAV particles (Wang Z et al., Gene Ther (2003). 10(26): 2105-11; Douglas M McCarty, Mol Ther (2008). 16(10): 1648-56).

[0014] The present invention also provides a gene delivery system comprising AAV capsid protein and the aforementioned transgene expression cassette.

[0015] As one of the preferred technical solutions, the AAV capsid protein is selected from: AAV5, AAV8, AAV9, AAVT02, and AAVT68, and their amino acid sequences are shown in SEQ ID NO.16, SEQ ID NO.18, SEQ ID NO.20, SEQ ID NO.22, and SEQ ID NO.24, respectively.

[0016] As one of the further preferred technical solutions, the nucleotide sequences of the encoding genes of each AAV capsid protein are shown in SEQ ID NO.15, SEQ ID NO.17, SEQ ID NO.19, SEQ ID NO.21, and SEQ ID NO.23, respectively.

[0017] As one of the further preferred technical solutions, the AAV capsid protein is AAVT02 or AAVT68. AAVT02 is an engineered serotype used in the newly launched Pepidacoq injection in 2025 (patent WO2019 / 241324A1), which has been shown to have good liver targeting in clinical trials. AAVT68 is a novel AAV serotype vector that has been modified and optimized by introducing three phosphorylation site mutations (S424G, S456G and S468G) on the basis of T02.

[0018] As a further preferred technical solution, the AAV capsid protein is AAVT68.

[0019] As one of the preferred technical solutions, the method for preparing the AAV capsid protein includes: three plasmid transfection, cell lysis, removal of nucleic acids and organic impurities, affinity chromatography, ion exchange chromatography, and gradient ultracentrifugation with cesium chloride and iodixanol. (Xiao X et al., J Virol (1998). 72(3): 2224-32; Grieger JC et al., Mol Ther (2016). 24(2): 287-97; Blessing D et al., Mol Ther Methods Clin Dev (2018). 13: 14-26) As one of the further preferred technical solutions, triple plasmid transfection includes: Plasmid 1, cis-element plasmids with ITRs, such as expression cassettes shAGT1, shPALL1 and shPALL2, SEQ ID NO. 5, 8, 9; Plasmid 2, AAV Rep / Cap plasmid, has a capsid coding sequence, such as AAV5, AAV8, AAV9, AAVT02, AAVT68; Plasmid 3, an auxiliary plasmid containing adenovirus components, can promote the replication, assembly, and packaging of AAV viruses.

[0020] The AAV particles produced by HEK293 cells were then purified by affinity chromatography and iodixanol density gradient ultracentrifugation.

[0021] As one of the preferred technical solutions, the gene delivery system consists of AAVT68 and a transgenic expression cassette containing a polynucleotide sequence, wherein the polynucleotide sequence is shAGT1, shPALL1 or shPALL2, as shown in SEQ ID NO.5, SEQ ID NO.8 and SEQ ID NO.9 respectively.

[0022] The present invention also provides the application of the aforementioned polynucleotide sequences, transgenic expression cassettes, or gene delivery systems in the preparation of drugs that simultaneously lower blood lipids and blood pressure.

[0023] As one of the preferred technical solutions, this drug inhibits multiple targets, including PCSK9, HMGCR, and AGT.

[0024] As one of the preferred technical solutions, the types of diseases treated by this drug include hyperlipidemia, hypercholesterolemia, hypertension, and other cardiovascular and cerebrovascular diseases complicated with hyperlipidemia and hypertension.

[0025] As one of the further preferred technical solutions, hyperlipidemia and hypercholesterolemia are caused by mutations in the PCSK9-related gene.

[0026] As one of the further preferred technical solutions, the cardiovascular and cerebrovascular diseases refer to diseases caused by hyperlipidemia and hypertension, or high-risk factors, including but not limited to atherosclerosis, stroke, coronary heart disease, and arrhythmia.

[0027] The present invention also provides a drug that simultaneously lowers blood lipids and blood pressure, comprising the aforementioned polynucleotide sequence, transgenic expression cassette or gene delivery system.

[0028] The beneficial effects of this invention are: This invention discloses a multinucleotide sequence that simultaneously lowers blood lipids and blood pressure, and its applications. Cell and mouse experiments show that this invention can significantly inhibit the expression levels of multiple lipid-lowering and blood pressure-lowering molecular targets, such as subtilisin convertase (PCSK9), angiotensinogen (AGT), and β-hydroxy-β-methylglutaryl-CoA reductase (HMGCR), thereby reducing blood lipid and blood pressure levels in mice with related diseases. This invention holds promise for the treatment of cardiovascular and cerebrovascular diseases complicated by hyperlipidemia and hypertension.

[0029] Other advantages, objectives, and features of the invention will be set forth in part in the description which follows, and in part will be apparent to those skilled in the art from the following examination, or may be learned from practice of the invention. The objectives and other advantages of the invention can be realized and obtained through the following description. Attached Figure Description

[0030] To make the objectives, technical solutions, and advantages of the present invention clearer, the preferred embodiments of the present invention will be described in detail below with reference to the accompanying drawings, wherein: Figure 1The inhibitory effects of multipurpose polynucleotides on Flag-tagged hyperlipidemia and hypertension target molecules transfected into HEK293 cells. A shows the inhibitory effect of shHMG1-3 on hHMGCR-Flag protein transfected into HEK293 cells. B shows the inhibitory effect of shPCSK2 on hPCSK9-Flag protein transfected into HEK293 cells, with Inclisiran (30 mM) added as a positive control. C shows the inhibitory effect of shAGT1-3 on hAGT-Flag protein transfected into HEK293 cells. D is a schematic diagram of the multipurpose lipid-lowering and blood pressure-lowering polynucleotide sequences shPALL1-2 and shHALL1-2 obtained by stem-loop and linker tandem. E shows the dual inhibitory effect of shAGT1 and shPALL1-2 on hPCSK9-Flag and hAGT-Flag proteins transfected into HEK293 cells, with Inclisiran (30 mM) added as a positive control. F represents the dual inhibitory effect of shHALL1-2 on hHMGCR-Flag and hAGT-Flag proteins transfected into HEK293 cells. n = 3, *p < 0.05, **p < 0.01, ***p < 0.001.

[0031] Figure 2 The AAVT68 serotype exhibits excellent transduction efficiency in liver and pancreatic cells. In the diagram, A shows AAVT68 (YC5) and its template AAVT02 (843), with T68 containing three phosphorylation site mutations: S424G, S456G, and S468G. B represents AAV8, 9, T02, and T68 packaged with GFP at an MOI of 3 × 10⁻⁶. 4 Human liver cells (Huh7) were transduced using vg / cell, and GFP fluorescence intensity was statistically analyzed. CE was used to package GFP with AAV5, 8, T02, and T68 at a concentration of 3 × 10⁻⁶. 12 Human PCSK9 transgenic mice were fed a high-fat diet at a dose of vg / kg for 8 weeks. Fourteen days later, GFP fluorescence intensity and protein expression were measured in liver and pancreatic tissues, corresponding to the transduction efficiency of the respective AAV serotypes. n = 5, *p < 0.05, **p < 0.01, ***p < 0.001.

[0032] Figure 3The AAVT68-packaged lipid-lowering and blood pressure-lowering polynucleotide sequences inhibit multiple endogenous target molecules in human liver cells. A shows the structural diagrams of T68-AGT1, T68-PALL1, and T68-PALL2, including the AAVT68 capsid, shAGT1, shPALL1, and shPALL2 polynucleotide sequences, and the CB-GFP expression element used to trace transduction efficiency. B shows the three AAVs at an MOI of 2 × 10⁻⁶. 4 Huh7 cells were transduced using vg / cell, and GFP expression showed high transduction efficiency. CD1s (T68-AGT1, T68-PALL1, and T68-PALL2) were used at an MOI of 2 × 10⁻⁶. 4 Huh7 cells were transduced with vg / cell for 48 hours, and the expression of endogenous PCSK9 and AGT proteins was detected by Western blot. EF was T68-AGT1 and T68-PALL2 at MOI = 2 × 10⁻⁶. 4 Huh7 cells were transduced with vg / cell for 48 hours. Endogenous PCSK9 and AGT proteins secreted in the culture supernatant were detected by Western blot. Inclisiran (30 mM) was added as a positive control. n = 4-5, ***p < 0.001 Figure 4 The effect of T68-PALL2 on reducing blood lipids and body fat in hyperlipidemic and hypertension model mice. In this study, A was a humanized PCSK9 transgenic mouse model of hyperlipidemia and hypertension induced by a high-fat diet and L-NAME water intake for 5 weeks, followed by tail vein injection of T68-PALL2 at a dose of 3 × 10⁻⁶. 12 Vg / kg, lipid-related indicators were measured at 2 and 4 weeks after injection. B shows the expression of PCSK9 and AGT in the liver of hyperlipidemic and hypertensive model mice 4 weeks after T68-PALL2 injection, and the protein levels were statistically analyzed. CD shows the detection and statistical analysis of four lipid parameters (LDL, T-CHO, TG, and HDL) 2 and 4 weeks after T68-PALL2 injection. E shows the body fat distribution of mice treated with T68-PALL2 for 4 weeks. FG shows the aorta and liver oil red of model mice treated with T68-PALL2 for 4 weeks, and the fat deposition was statistically analyzed. H shows the fluorescent staining of SMA (green) and CD68 (red) on aortic sections of model mice treated with T68-PALL2 for 4 weeks to show macrophage infiltration. n = 6-7. ** p < 0.01, *** p < 0.001.

[0033] Figure 5The effect of T68-PALL2 on blood pressure reduction in hyperlipidemic and hypertension model mice. Specifically, AB mice were fed a high-fat diet and L-NAME water for 5 weeks to induce a hyperlipidemic and hypertension model, and then T68-PALL2 was injected via tail vein at a dose of 3 × 10⁻⁶. 12 Vg / kg was used to measure pressure-related parameters at 2 and 4 weeks post-injection, and mean systolic blood pressure (SBP) and diastolic blood pressure (DBP) were statistically analyzed. C represents the statistical values ​​of mouse body weight and heart rate. n = 6-7. *p < 0.05, **p < 0.01, ***p < 0.001. Detailed Implementation

[0034] The present invention will be further described below with reference to specific embodiments. However, the present invention should not be limited to the embodiments described herein, but rather these embodiments are intended to illustrate and define the scope of the invention to those skilled in the art.

[0035] Unless otherwise stated, the nucleic acid or polynucleotide sequences listed in this article are in single-stranded form only, oriented from 5' to 3', from left to right. Nucleic acids and amino acids provided in this article follow the format recommended by the IUPACIUB Biochemical Nomenclature Committee, or (for amino acids) use single-letter or three-letter codes.

[0036] Unless otherwise defined, all technical and scientific terms used herein, including all parameters, dimensions, materials, and constructions, have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein is intended to describe particular embodiments and not to impede the invention.

[0037] Unless otherwise stated, the different items described in this invention can be used in any combination. Furthermore, it is contemplated that in some embodiments, any item or combination of items may be excluded or omitted.

[0038] Unless otherwise stated, standard methods known to those skilled in the art can be used to produce recombinant and synthetic peptides or proteins thereof, design nucleic acid sequences, produce transformed cells, construct recombinant AAV mutants, modify capsid proteins, packaging vectors (expressing AAV Rep and / or Cap sequences), and transiently or stably transfected packaging cells. These techniques are known to those skilled in the art. See, for example, Molecular Cloning: A Laboratory Manual, Second Edition (Cold Spring Harbor, NY, 1989); Recent Protocols in Molecular Biology, et al. (Green Publishing Associates, Inc. and John Wiley & Sons, Inc., NY).

[0039] All publications, patent applications, patents, nucleic acid and amino acid sequences, and other references mentioned in this invention are incorporated herein by reference in their entirety.

[0040] It should be understood that for any method described herein that includes more than one step, the order of the steps is not necessarily limited to the order described in those embodiments.

[0041] definition A "vector" refers to one or more macromolecules that encapsulate polynucleotides, facilitating their delivery to target cells in vitro or in vivo. Types of vectors include, but are not limited to, plasmids, viral vectors, liposomes, and other gene delivery vectors. The polynucleotide to be delivered is sometimes referred to as an "expression cassette" or "transgenic cassette," which may contain, but is not limited to, coding sequences for certain proteins or synthetic polypeptides that can enhance, inhibit, weaken, protect, trigger, or prevent certain biological and physiological functions; coding sequences of interest in vaccine development (e.g., polynucleotides expressing proteins, polypeptides, or peptides suitable for evoking an immune response in mammals); coding sequences for RNAi materials (e.g., shRNA, siRNA, antisense oligonucleotides); or optional biomarkers.

[0042] “Transduction,” “transfection,” “conversion,” or the terms used herein refer to the process of delivering exogenous nucleic acids into host cells, followed by transcription and translation of the polynucleotide product, which includes the introduction of exogenous polynucleotides into host cells using recombinant viruses.

[0043] "Gene delivery" refers to the introduction of exogenous polynucleotides into cells for gene transfer, and can include targeting, binding, uptake, transport, replicon integration, and expression.

[0044] "Gene expression" or "expression" refers to the process of gene transcription, translation, and post-translational modification to produce the RNA or protein product of the gene.

[0045] Unless otherwise stated, "polynucleotide" generally refers to a polymeric form of nucleotides of any length, including deoxyribonucleotides or ribonucleotides, mixed sequences thereof, or the like. Polynucleotides can include unmodified and modified nucleotides, such as methylated or restricted nucleotides and nucleotide analogs. As used herein, the term polynucleotide is interchangeable with double-stranded and single-stranded molecules. Unless otherwise stated, the polynucleotides described herein in any embodiment include double-stranded forms and each of two complementary single-stranded forms known or predicted to constitute the double-stranded form.

[0046] The terms "expression cassette" and "transgenic expression cassette" are synonymous. A "transgenic cassette" refers to a polynucleotide fragment encoding a specific protein, polypeptide, or RNAi element that can be cloned into a plasmid vector. In some embodiments, the "cassette" may also be packaged into AAV particles and used as a viral genome to deliver the transgenic product to target cells. Other regulatory elements may also be included, such as specific promoters / enhancers, polyA, and regulatory introns, to enhance or degrade the expression of the transgenic product.

[0047] The term “adeno-associated virus (AAV)” as used herein encompasses natural AAVs (types 1–11, avian AAVs, bovine AAVs, canine AAVs, equine AAVs, and sheep AAVs) and any other known artificially engineered AAVs, or those subsequently discovered and invented. See, for example, Bernard N. Fields et al., VIROLOGY. Vol. 2, Chapter 69 (4th edition, Lippincott-Raven Publishers, e.g., Gao G et al., J. Virol (2004) 78: 6381–6388). Genomic sequences of various AAV serotypes, as well as sequences of the ITR, Rep, and Cap proteins, are known in the art. Such sequences can be found in the literature or in public databases such as GenBank®. See, for example, GenBank login numbers NC 002077, NC001401, NC 001729, NC 001863, NC 001829, NC 001862, NC 000883, NC 001701, NC 001510, AF063497, U89790, AF043303, AF028705, AF028704, J02275, JO1901; J02275, XO1457, AF288061, AHO09962, AY028226, AY028223, NC 001358, NC 001540, AF513851, AF513852, AY530579, AY631965, AY631966; their entire contents are incorporated herein by reference. See also, for example, Srivistava et al., J.Virol (1983). 45:555; Chiorini et al., J.Virol (1998). 71:6823; Chiorini et al., J.Virol (1999). 73:1309; Bantel-Schaal et al., J.Virol (1999). 73:939; Xiao et al., J.Virol (1999). 73:3994; J.Virol (1999). 73:3994. Muramatsu et al., Virology (1996). 221:208. International patent publications WO 00 / 28061, WO 99 / 61601, WO 98 / 11244: US Patent No. 6,156.303.

[0048] The term "and / or" means that the relevant items can be interpreted separately with or without any combination of (and) or (or).

[0049] In this article, “treatment” refers to any agent, chemical substance or substance that at least in some way helps to improve the course and prognosis of a disease, or to inhibit or delay the onset of a disease.

[0050] The term "recombinant" in polynucleotides refers to polynucleotides that are synthetic products resulting from numerous cloning steps, producing constructs different from natural polynucleotides, as is known to those skilled in the art. Recombinant viruses are viral particles containing recombinant polynucleotides.

[0051] The term “approximately” refers to a measurable value with an acceptable deviation of 10% or less.

[0052] The term "subject" in most cases refers to a human patient or healthy person. However, in some embodiments, it also refers to non-human primates (such as rhesus monkeys) or sometimes even non-primate mammals (such as mice, rats, rabbits, pigs, dogs, and cattle).

[0053] AAV-based multipurpose lipid-lowering and blood pressure-lowering agents and their uses Developing novel delivery systems to ensure the long-term stable expression of anti-angiogenic proteins and peptides is essential, allowing patients to use the drugs less frequently and making it far more convenient than traditional therapies. Due to its low pathogenicity, broad tissue / organ affinity in vivo, and ability to stably express proteins over a long period, AAV is widely considered a promising gene vector.

[0054] The basic building blocks of functional AAV particles are capsid proteins, which include VP1, VP2, and VP3. Those skilled in the art will understand that VP2 and VP3 proteins undergo transcription and translation at the initiation site within the VP1 protein. That is, the VP1 nucleic acid and amino acid sequence includes the sequences of VP2 and VP3. Capsid proteins not only regulate AAV assembly during replication but also promote viral interaction with receptors on the plasma membrane and entry into target cells (Venkatakrishnan B et al., J Virol (2013). 87(9): 4974-84; Walters RW et al., J Virol (2004). 78(7): 3361-71; Kronenberg Set al., J Virol (2005). 79(9): 5296-303; Uhrig S et al., Gene Ther (2012). 19(2): 210-8; Wang D et al., Nat Rev Drug Discov (2019). 18(5): 358-78; Zhang R et al., Nat Commun (2019). 10(1): 3760). Overall, capsid proteins determine the tissue / organ affinity or transduction efficiency of AAV serotypes.

[0055] The capsid proteins used in this paper are novel capsid proteins designed and synthesized by the applicant that are distinct from wild-type serotypes (e.g., AAV5, 8, 9, T02, T68), as previously published by the applicant (Wang Y et al., Mol Ther NucleicAcids (2022). 21(28): 293-306; Luo L et al., Biomaterials (2024). 304:122403). The applicant has now discovered that these AAV serotypes exhibit superior retinal transduction efficiency compared to wild-type and have potential applications in ocular gene delivery.

[0056] Unless otherwise stated, cis-elements in this invention refer to transgenic cassettes packaged in AAV particles and expressed in target cells to produce polynucleotide or protein products with therapeutic effects. Indications for transgenic cassettes include, but are not limited to, hypertension, hyperlipidemia, hypercholesterolemia, and other cardiovascular and cerebrovascular diseases complicated with hypertension and hyperlipidemia, including but not limited to stroke, atherosclerosis, and arrhythmia.

[0057] In addition to the sequence encoding the protein product, the transgenic cassette contains numerous regulatory elements to enable the transgene to be packaged into the virus, such as a normal 145 bp ITR, a shortened ITR of approximately 100 bp, located lateral to the protein-coding sequence, and in the promoter region. The transgenic cassette also contains polynucleotide elements for controlling protein product expression, such as origin of replication, polyadenylation signals, internal ribosome entry sites (IRES) or 2A signals (e.g., P2A, T2A, F2A), promoters, and enhancers (e.g., CMV promoters with vertebrate β-actin, β-globulin, or β-globulin regulatory elements, or other hybrid CMV promoters, referred to as CB and CAG promoters, EF1 promoters, hypoxia-responsive elements, ubiquitin promoters, T7 promoters, SV40 promoters, VP16, or VP64 promoters). Promoters and enhancers can be activated by chemicals or hormones (e.g., doxycycline or tamoxifen) to ensure gene expression at specific time points. Furthermore, promoters and enhancers can be natural, artificial, or chimeric sequences, i.e., prokaryotic or eukaryotic sequences.

[0058] In some embodiments, the inducible regulatory element for gene expression may be a tissue or organ-specific promoter or enhancer, including but not limited to promoters specific to various types of retinal cells, such as: broad-spectrum RNA transcription promoters (e.g., the U6 promoter), ganglion cell-specific promoters (e.g., the Tuj1 promoter), astrocyte and Müller cell-specific promoters (e.g., the GFAP or vimentin promoter), and retinal pigment epithelium-specific promoters (e.g., the RPE65 or VMD2 promoter), as well as liver, pancreas, spleen, and lung cancer cell-specific or preferred promoters.

[0059] In some embodiments, the gene delivery system of the present invention is used to assist in cell transplantation therapy. Specifically, transgenic AAV particles can be used to transduce various cell types in vitro to produce stable cell lines expressing protein products, which can then be introduced in vivo for therapeutic purposes. Cell types include, but are not limited to, liver cells, pancreatic A cells, pancreatic B cells, etc.

[0060] In certain embodiments, the potential cell transplantation is autologous to the subject, allowing for in vitro culture. The principles and techniques for introducing or transplanting cells into the subject are known to those skilled in the art.

[0061] In some embodiments, anti-angiogenic AAV particles are produced by transfecting HEK293 cells with a tri-plasmid: a cis-element plasmid (plasmid 1), an AAVRep / Cap plasmid (plasmid 2), and a helper plasmid (plasmid 3). AAV particles are harvested from the culture medium and lysates of HEK293 cells. Purification methods are represented by affinity chromatography, ion-exchange chromatography, and gradient ultracentrifugation with cesium chloride and iodixanol. Chemicals or reagents associated with AAV production and purification include, but are not limited to, chemicals or reagents used for cell culture (e.g., cell culture medium components include bovine, equine, goat, chicken, or other vertebrate serum, glutamine, glucose, sucrose, sodium pyruvate, phenol red, penicillin, kanamycin, streptomycin, tetracycline, and other antibiotics). Used for cell lysis, polynucleotide precipitation, or ultracentrifugation (e.g., Triton X-100, NP-40, sodium deoxycholate, sodium dodecyl sulfate, domiphen bromide, sodium dodecyl salicylate, sodium chloride, magnesium chloride, calcium chloride, barium chloride, nitrates, potassium chloride, ammonium chloride, ammonium persulfate, ammonium sulfate, PEG-20, PEG-40, PEG-400, PEG-2000, PEG-6000, PEG-8000, PEG-20000, Tris-HCl, Tris-acetate, manganese chloride, phosphates, bicarbonates, cesium chloride, methanol, ethanol, glycerol, iodixanol, isopropanol, butanol, benzoinase, DNase). I, RNase), affinity column materials (e.g., AAVX affinity resin, heparin sulfate proteoglycan and mucin resin, other materials associated with AAV-specific antibodies), ion exchange chromatography materials and wash buffers (e.g., hydrochloric acid, sulfuric acid, acetic acid, formic acid, nitric acid, urea, acetone, chloroform, acetonitrile, trifluoroacetic acid, sodium hydroxide, potassium hydroxide, barium hydroxide, ammonium hydroxide, Tris base or other organic amines, poloxamer 188, Tween 20, Tween 40, Tween 80, guanidine hydrochloride).

[0062] In some embodiments, the protein product encoded by the transgenic cassette is attached to an oligopeptide tag (e.g., Flag, 6×His, 2×HA, Myc), which facilitates the purification of these proteins. Those skilled in the art will understand the techniques and procedures associated with protein purification. In short, the transgenic plasmid is transfected into eukaryotic cells (e.g., HEK293 and CHO cells), and the target protein is then collected by affinity chromatography. For example, Flag-M2 resin beads are commonly used to specifically attract Flag-tagged proteins, followed by elution with 3×Flag-soluble oligopeptides. Alternatively, nickel hyponitrotriacetate (Ni-NTA) columns can be used for reversible binding, followed by specific purification of 6×His-tagged proteins.

[0063] Example 1: In vitro screening of polynucleotides targeting molecules that lower blood lipids and blood pressure This invention further improves upon published literature by using a genetic neural network (GNN) model to design highly efficient lipid-lowering and blood pressure-targeting shRNAs (Knott, Simon RV, et al., Mol Cell (2014). 56: 796-807; Pelossof, R., et al., Nat Biotechnol (2017). 35: 350-353; La Rosa M, et al., Int J MolSci (2022): 23, 14211; Lee M, et al., Front Genet (2023). It comprehensively learns from multiple relevant shRNA databases and combines 3D structural and thermodynamic analyses of the siRNA-mRNA binding of the target molecule PCSK9, while also analyzing off-target effects. This study analyzed the potential thermodynamics and 3D structure of microRNA binding (non-100% complementary base pairing), and analyzed polynucleotide molecules that could simultaneously inhibit multiple targets such as PCSK9, HMGCR, and AGT. Polynucleotide molecules with high target scores and low off-target effects were selected for cell testing. It was found that shHMG2 (SEQ ID NO. 2), shPCSK2 (SEQ ID NO. 4), and shAGT1 (SEQ ID NO. 5) achieved inhibition efficiencies of over 80% for flag-tagged target molecules transfected in HEK293 cells. Figure 1 Next, this invention connects multiple lipid-lowering and blood pressure-lowering polynucleotide sequences using special linker sequences and stem-loop structures to obtain shPALL1 (SEQ ID NO. 8), shPALL2 (SEQ ID NO. 9), shHALL1 (SEQ ID NO. 10), and shHALL2 (SEQ ID NO. 11). Figure 1 Cellular data showed that shPALL1 and shPALL2 inhibited PCSK9-Flag protein transfected in HEK293 cells by 83% and 86%, respectively, and AGT-Flag protein by 78% and 76%, respectively. Surprisingly, shAGT1 not only inhibited AGT-Flag protein levels by 82%, but also inhibited PCSK9-Flag protein levels by 79%, demonstrating the simultaneous inhibitory effect of polynucleotide sequences on both PCSK9 and AGT targets. Figure 1(E); In addition, shHALL1 and shHALL2 can achieve an inhibitory effect of more than 75% on the levels of HMGCR and AGT protein, which are the targets for lowering blood lipids and blood pressure. Figure 1 (F) further demonstrates the effectiveness of multi-purpose sequences.

[0064] Example 2: Excellent transduction efficiency of AAVT68 serotype in liver and pancreatic cells To screen the most suitable AAV vectors for delivering lipid-lowering and blood pressure-lowering expression cassettes, this invention conducted in vitro and in vivo transduction tests on several commonly used liver-targeting AAVs (AAV5, AAV8, AAV9, T02, and T68, SEQ ID NO. 15-24). Since the liver and pancreas are the two most critical organs for regulating blood lipids, blood glucose, and blood pressure, the transduction efficiency in the liver and pancreas was the primary focus. In vitro experiments showed that the transduction efficiency of T02 and T68 serotypes in human liver cells Huh7 was significantly higher than that of AAV8 and 9, with overall GFP fluorescence intensity reaching 7-9 times the transduction efficiency of AAV8 and 9. Figure 2 Due to potential differences in AAV transduction efficiency between in vivo and in vitro, this project selected human hPCSK9 transgenic mice (Biocytok catalog number 112751) that had been modeled on a high-fat diet for 8 weeks and injected them with 3×10⁻⁶ AAV via the tail vein. 12 Various AAV serotypes packaged with GFP at vg / kg doses were tested. The T68 serotype showed transduction efficiency in the liver of hyperlipidemic mouse models that was 16, 5.8, and 1.6 times higher than that of AAV5, T02, and AAV8, respectively; and transduction efficiency in the pancreas of hyperlipidemic mouse models that was 10.7, 8.9, and 2.1 times higher than that of AAV5, T02, and AAV8, respectively. Figure 2 The presence of CE indicates that T68 is a serotype with excellent delivery efficiency of the aforementioned expression cassette.

[0065] Example 3: Inhibitory effect of AAV formulations packaged with the above sequence on endogenous targets. To test whether the AAV reagent containing the aforementioned lipid-lowering and blood pressure-lowering sequences could inhibit endogenous molecular targets in human liver cells, the applicant initiated the production process of anti-angiogenic AAV particles by transfecting with three plasmids: cis-element plasmids (shAGT1, shPALL1, and shPALL2, SEQ ID NO. 5, 8, 9), AAVT68 capsid plasmid, and adenovirus helper plasmid (Xiao Lab, XX680), along with 1 mg / ml PEI (polyethyleneimine, Thermo CAS 9002-98-6). The process was terminated using one-quarter of the volume of 20 g / L CDM4 solution in Hyclone cell culture medium (Cytiva SH.31035). HEK293 cells (ATCC, CRL-1573) were lysed 40-80 hours after transfection with a solution of 0.1% Triton X-100, 0.1% NP40, 0.3% sodium deoxycholate, or 0.005% sodium dodecyl sulfate. Polynucleotides were then removed by repeated freezing and thawing, sonication, and treatment with 20 mM sodium sulfate added. The solution was then used to lyse HEK293 cells (ATCC, CRL-1573) with one or any combination of the following: benzonase (50 U / ml), DNase I, or RNase; 0.5 mM doxofene; isopropanol; isopropanol; 50 mM ammonium sulfate; or 300 mM sodium chloride. The cells were then centrifuged at 12000 rpm for 15-45 minutes. The supernatant containing AAV particles was loaded into an AAVX affinity column and then eluted sequentially with buffer A (50 mM Tris-acetate or Tris-HCl, 500 mM sodium chloride, 2 mM magnesium chloride, 0.05% poloxamer 188, pH = 6.5-7.5), buffer B (1 M acetic acid, 500 mM sodium chloride, 0.05% poloxamer 188, pH = 2-5), and 3 M urea solution. The eluted AAV particles were then concentrated to a specific volume for use by gradient ultracentrifugation with iodixanol (consisting of iodixanol solutions at concentrations of 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, and 80%). The packaged AAV reagents are abbreviated as T68AGT1, T68PALL1, and T68PALL2. Data shows that the three AAV reagents can effectively transduce the human liver cell line Huh7 (JCRB cell bank No. 0403). Figure 3 The drug (AB) showed an inhibitory effect on PCSK9 protein levels in human liver cells exceeding 85%, higher than the 79% inhibition rate of Inclisiran (Leqvio, Novartis); it also reduced AGT protein levels in the culture supernatant by 77% and 84%, respectively. Figure 3 This is an effect that Inclisiran does not have (CF).

[0066] Example 4: The effect of a multi-purpose AAV formulation on reducing blood lipids and blood pressure in model mice. This invention first induced a hyperlipidemia and hypertension model in human PCSK9 overexpressing mice by subjecting them to a high-fat diet and L-NAME water intake for up to 5 weeks, followed by injection of AAV reagent (Model group, Figure 4 (A). Western blot results showed that T68PALL2 achieved 86% and 77% inhibition of PCSK9 and AGT proteins in the liver of model mice, respectively. Figure 4 Regarding blood lipids, injections of T68PALL2 for 4 weeks reduced serum low-density lipoprotein (LDL) from 6.43 to 0.44 mmol / L, serum total cholesterol (T-CHO) from 10.4 to 2.7 mmol / L, and serum triglycerides (TG) from 1.86 to 0.734 mmol / L, essentially restoring them to normal levels. Figure 4 (CD); In addition, T68PALL2 significantly improved body fat levels, aortic lipoplasty, and hepatic steatosis in hyperlipidemic and hypertensive model mice. Figure 4 Correspondingly, macrophage infiltration in the arterial plaque was also significantly alleviated (in the context of EG); Figure 4 (H). The above results demonstrate the significant ameliorative effect of the multipurpose AAV reagent of this invention on blood lipid levels, atherosclerosis, and hepatic fat deposition in model mice.

[0067] Blood pressure monitoring showed that 2 to 4 weeks after T68PALL2 injection, the mean systolic blood pressure (SBP) of the hyperlipidemic hypertension model mice decreased from 142.6-146.9 mmHg to 115.2-118.0 mmHg; the mean diastolic blood pressure (DBP) decreased from 90.0-96.1 mmHg to 71.4-73.0 mmHg. Figure 5 In the middle AB group, average weight also decreased by about 6 g, but heart rate did not change significantly. Figure 5 (C) This further illustrates the lipid-lowering and blood pressure-lowering functions of this AAV reagent in model mice.

[0068] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A polynucleotide sequence that simultaneously lowers blood lipids and blood pressure, characterized in that, Selected from any one or more of shHMG1, shHMG2, shHMG3, shPCSK2, shAGT1, shAGT2, shAGT3, shPALL1, shPALL2, shHALL1, and shHALL2, with their nucleotide sequences shown in SEQ ID NO.1 to 11, respectively.

2. A transgenic expression cassette, characterized in that, It comprises the polynucleotide sequence of claim 1.

3. A gene delivery system, characterized in that, It contains AAV capsid protein and the transgenic expression cassette of claim 2.

4. The gene delivery system according to claim 3, characterized in that, The AAV capsid protein is selected from AAV5, AAV8, AAV9, AAVT02, and AAVT68, and their amino acid sequences are shown in SEQ ID NO.16, SEQ ID NO.18, SEQ ID NO.20, SEQ ID NO.22, and SEQ ID NO.24, respectively.

5. The gene delivery system according to claim 4, characterized in that, The nucleotide sequences encoding the genes of each AAV capsid protein are shown in SEQ ID NO.15, SEQ ID NO.17, SEQ ID NO.19, SEQ ID NO.21, and SEQ ID NO.23, respectively.

6. The gene delivery system according to claim 4, characterized in that, The AAV capsid protein is AAVT02 or AAVT68.

7. The gene delivery system according to claim 4, characterized in that, The method for preparing the AAV capsid protein includes: three plasmid transfection, cell lysis, removal of nucleic acids and organic impurities, affinity chromatography, ion exchange chromatography, and gradient ultracentrifugation with cesium chloride and iodixanol.

8. The use of the polynucleotide sequence of claim 1, the transgenic expression cassette of claim 2, or the gene delivery system of claim 3 in the preparation of a drug that simultaneously lowers blood lipids and lowers blood pressure.

9. The application according to claim 8, characterized in that, This drug inhibits multiple targets, including PCSK9, HMGCR, and AGT.

10. A drug that simultaneously lowers blood lipids and blood pressure, characterized in that, It comprises the polynucleotide sequence of claim 1, the transgenic expression cassette of claim 2, or the gene delivery system of claim 3.

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