Gene delivery vector, gene delivery system and preparation method and application thereof

Abstract

The invention relates to a gene delivery vector, a gene delivery system and a preparation method and application thereof. The gene delivery system provided by the invention comprises the gene delivery carrier provided by the invention and lipid nanoparticles; the lipid nanoparticles encapsulate the gene delivery vector. In the prior art, related researches on treatment of metabolic disorder diseases or diseases (such as OTCD) based on lipid nanoparticle LNP delivery are not found temporarily, so that the carrier containing the OTC-circRNA drug gene based on LNP delivery is constructed for the first time, and the carrier can be used for treating the metabolic disorder diseases or diseases. The effectiveness and safety of treatment are researched in vivo and in vitro, a new scheme is provided for clinical treatment of patients with gene defects, and the application value is important. The OTC-circRNA-encapsulated lipid nanoparticles disclosed by the invention are good in stability in vivo, good in safety and long in curative effect lasting time.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to a gene delivery vector, a gene delivery system, its preparation method, and its application. Background Technology

[0002] Metabolic disorders include lipid metabolism disorders and protein metabolism disorders, especially ornithine transcarbamate deficiency (OTCD). OTC is an X-linked recessive genetic disorder, the most common type of UCD caused by mutations in the OTC gene. The OTC gene is located on chromosome Xp21.1, and mutation types include duplication, deletion, missense, and nonsense mutations. Clinically, it is characterized by elevated blood ammonia, elevated blood citrulline, decreased glutamine, and elevated urinary orotic acid levels. The clinical manifestations and severity of OTCD are mainly influenced by OTC enzyme activity and blood ammonia levels. Based on clinical presentation, it can be divided into early-onset and late-onset types. Early-onset OTCD primarily affects male infants, typically presenting in the neonatal period with rapid onset, severe illness, and a high risk of developing into inherited metabolic encephalopathy, often resulting in death within the first week of life. Late-onset hyperammonemia occurs in infancy and adulthood, with relatively mild clinical symptoms, but it is often life-threatening due to acute hyperammonemia induced by stress factors such as high-protein diets and medications.

[0003] Currently, there is no specific cure for over-the-counter (OTCD). Clinically, the treatment goal and principle is to reduce blood ammonia levels as much as possible to avoid hyperammonemia and its resulting neurological damage. Current treatment methods mainly include dietary control, medication, hemodialysis, and liver transplantation. Medications include sodium benzoate or sodium phenylbutyrate, arginine or citrulline, and L-carnitine. For severe hyperammonemia (blood ammonia concentration consistently above 500 μmol / L) or when blood ammonia levels fail to decrease after antiammonia treatment, hemodialysis is necessary, which is currently the fastest and most effective method for lowering blood ammonia. In addition, liver transplantation can significantly reduce blood ammonia levels, improve dietary and medication treatments, and significantly improve the patient's quality of life. However, due to factors such as the scarcity of donor livers, high surgical risks leading to high mortality, the occurrence of immune rejection, the inability to maintain the therapeutic effect for life, and the inability to reverse existing neurological damage through liver transplantation, the clinical application of liver transplantation in the treatment of OTCD is greatly limited. Therefore, the development of new treatment methods for OTCD is of great significance for its clinical treatment.

[0004] The goal of gene therapy is to address acute hyperammonemia by transiently expressing OTC enzymes and to prevent hyperammonemia through stable long-term repair of OTC genes. Currently, gene therapy for OTCD has made some progress at the cellular level and in mouse models. For example, Prieve et al. developed a hybrid mRNA technology delivery system (HMT) and validated it in an OTCD mouse model. Moscioni et al. used a recombinant AAV vector construct to verify its effectiveness in correcting liver metabolic defects in two OTCD mouse models. Yang et al. used a dual AAV system to deliver CRISPR / Cas9 nucleases to neonatal OTC-defective mice. However, due to the high immunogenicity and toxicity of the delivery vector, its small packaging capacity, and rapid mRNA decay, it requires long-term repeated administration, limiting its clinical application.

[0005] Liposome delivery systems represent the most advanced technology in novel drug delivery systems. They possess nanoscale cell-like membrane structures, exhibiting high biocompatibility and low immunogenicity. Simultaneously, they can protect the biological activity or function of delivered drugs or active groups, prolong drug half-life, and reduce toxicity. Furthermore, by modifying the structure or surface of lipid molecules, liposomes can be endowed with specific biological effects, thus expanding their applications in the biomedical field. Circular RNA (circRNA) is a class of endogenous non-coding RNA molecules widely found in eukaryotic cells, playing a crucial role in organismal development. circRNA molecules have a closed circular structure, are unaffected by RNA exonucleases, exhibit more stable expression, and are less prone to degradation. With the development of biotechnology and molecular medicine, artificial circRNAs have been designed as a new type of vaccine for the treatment and prevention of diseases. With continuous advancements in circRNA design and in vitro and in vivo synthesis technologies, circRNAs are expected to achieve rapid development in areas such as infectious disease vaccines, cancer immunotherapy, and alternative therapies for rare diseases.

[0006] In summary, liposome delivery systems offer high biocompatibility and low immunogenicity, while also protecting the biological activity or function of the delivered drug or active group, prolonging the drug's half-life, and reducing its toxicity. Meanwhile, circRNAs can achieve cap-independent protein translation, thus becoming a stable platform for the delivery of immune and therapeutic proteins. Engineered circRNAs show great potential in the fields of therapy and gene regulation. Currently, there are no studies on the use of LNP-based delivery of OTC gene circRNAs for the treatment of metabolic disorders or conditions (especially OTCD). Therefore, this study aims to investigate the construction of LNP-based circRNAs. OTC- The efficacy and safety of circRNA in treating metabolic disorders or conditions (especially OTCD). Summary of the Invention

[0007] The purpose of this invention is to provide an encapsulation OTC -Lipid nanoparticles (LNPs) of circRNA are used as drug delivery devices for the treatment of metabolic disorders or conditions. To achieve the above objectives, the technical solution adopted in this invention is as follows: In a first aspect, the present invention provides a gene delivery vector comprising an OTC gene and a nucleic acid encoding circRNA, wherein the OTC gene and the nucleic acid encoding circRNA are operatively linked, and the nucleic acid encoding circRNA comprises a PIE element and an IRES sequence, wherein... The OTC gene contains any one of (a1)-(a3): (a1) The nucleotide sequence shown in SEQ ID NO: 1; (a2) A nucleotide sequence that has more than 90% identity with the nucleotide sequence shown in SEQ ID NO: 1 and has the same or similar biological activity or function; (a3) A nucleotide sequence consisting of the nucleotide sequence shown in (a1) or (a2) by substitution and / or deletion and / or addition of one or more nucleotides, and having the same or similar biological activity or function. The PIE element comprises an E1 sequence and an E2 sequence. The nucleic acid of the E1 sequence includes any one of (b1)-(b3): (b1) The nucleotide sequence shown in SEQ ID NO: 2; (b2) A nucleotide sequence that has more than 90% identity with the nucleotide sequence shown in SEQ ID NO: 2 and has the same or similar biological activity or function; (b3) A nucleotide sequence consisting of the nucleotide sequence shown in (b1) or (b2) by substitution and / or deletion and / or addition of one or more nucleotides, and having the same or similar biological activity or function. The nucleic acid of the E2 sequence includes any one of (c1)-(c3): (c1) The nucleotide sequence shown in SEQ ID NO: 3; (c2) A nucleotide sequence that has more than 90% identity with the nucleotide sequence shown in SEQ ID NO: 3 and has the same or similar biological activity or function; (c3) A nucleotide sequence consisting of the nucleotide sequence shown in (c1) or (c2) by substitution and / or deletion and / or addition of one or more nucleotides, and having the same or similar biological activity or function. The nucleic acid in the IRES sequence includes any one of (d1)-(d3): (d1) The nucleotide sequence shown in SEQ ID NO: 4; (d2) A nucleotide sequence that has more than 90% identity with the nucleotide sequence shown in SEQ ID NO: 4 and has the same or similar biological activity or function; (d3) A nucleotide sequence consisting of the nucleotide sequence shown in (d1) or (d2) by substitution and / or deletion and / or addition of one or more nucleotides, and having the same or similar biological activity or function.

[0008] In some preferred embodiments, the OTC gene is the sequence shown in SEQ ID NO: 1.

[0009] In some preferred embodiments, the E1 sequence is the sequence shown in SEQ ID NO: 2.

[0010] In some preferred embodiments, the E2 sequence is the sequence shown in SEQ ID NO: 3.

[0011] In some preferred embodiments, the IRES sequence is the IRES sequence of CVB3 shown in SEQ ID NO: 4.

[0012] In some implementations, a CMV promoter sequence is also included, wherein the nucleic acid of the CMV promoter sequence comprises any one of (e1)-(e3): (e1) The nucleotide sequence shown in SEQ ID NO: 5; (e2) A nucleotide sequence that has more than 90% identity with the nucleotide sequence shown in SEQ ID NO: 5 and has the same or similar biological activity or function; (e3) A nucleotide sequence consisting of the nucleotide sequence shown in (e1) or (e2) by substitution and / or deletion and / or addition of one or more nucleotides, and having the same or similar biological activity or function.

[0013] In some preferred embodiments, the CMV promoter sequence is the sequence shown in SEQ ID NO: 5.

[0014] In some implementations, an intron sequence is also included, wherein the nucleic acid of the intron sequence comprises any one of (f1)-(f3): (f1) The nucleotide sequence shown in SEQ ID NO: 6; (f2) A nucleotide sequence that has more than 90% identity with the nucleotide sequence shown in SEQ ID NO: 6 and has the same or similar biological activity or function; (f3) A nucleotide sequence consisting of the nucleotide sequence shown in (f1) or (f2) by substitution and / or deletion and / or addition of one or more nucleotides, and having the same or similar biological activity or function.

[0015] In some embodiments, an SV40 terminator sequence is also included. In some more preferred embodiments, the SV40 terminator sequence is the sequence shown in SEQ ID NO: 7.

[0016] In a second aspect, the present invention provides a gene delivery system comprising a gene delivery vector and lipid nanoparticles according to claim 1; wherein the lipid nanoparticles encapsulate the gene delivery vector.

[0017] In some preferred embodiments, the gene delivery vector is a plasmid. In some more preferred embodiments, the plasmid is any one of pUC57, pUC19, pET-32a(+), and pET-28a(+). In some further preferred embodiments, the plasmid is pUC57. In some further more preferred embodiments, the sequence of the pUC57 plasmid is the sequence shown in SEQ ID NO: 10.

[0018] In some preferred embodiments, the raw materials for lipid nanoparticles (LNPs) comprise, by molar percentage: 40-50% ionizable cationic lipid (SM-102); 10-15% 1,2-distearate-sn-glycerol-3-phosphorylcholine (DSPC); 35-40% cholesterol; and 1-3% 1,2-dimyristoyl-sn-glycerol-3-methoxy polyethylene glycol-2000 (DMG-PEG 2000).

[0019] In some preferred embodiments, the lipids in the lipid nanoparticles comprise, by molar percentage: 50% ionizable cationic lipid (SM-102); 10% 1,2-distearate-sn-glycerol-3-phosphorylcholine (DSPC); 38.5% cholesterol; and 1.5% 1,2-dimyristoyl-sn-glycerol-3-methoxy polyethylene glycol-2000 (DMG-PEG 2000).

[0020] In some preferred embodiments, the N:P ratio in the raw materials of the lipid nanoparticles ranges from (4-6):1.

[0021] In some preferred embodiments, the dispersion index (PDI) of the lipid nanoparticles encapsulating the gene delivery vector is 0.1-0.2.

[0022] In some preferred embodiments, the encapsulation efficiency of the lipid nanoparticles is above 90%.

[0023] In some preferred embodiments, at the end of lipid nanoparticle encapsulation, the free circRNA (unencapsulated circRNA) is less than 10%.

[0024] In some preferred embodiments, the total concentration of CircRNA is 110-120 µg / mL at the start of lipid nanoparticle encapsulation.

[0025] In some preferred embodiments, the concentration of free circRNA (unencapsulated circRNA) is 8-10 µg / mL at the end of lipid nanoparticle encapsulation.

[0026] In some preferred embodiments, the zeta potential of the lipid nanoparticles encapsulating the gene delivery vector is -2 to -4 mV.

[0027] In some preferred embodiments, the lipid nanoparticles encapsulating the gene delivery vector have a particle size of 80-120 nm.

[0028] Thirdly, the present invention provides a method for preparing a gene delivery system, comprising the following steps: (1) Preparation of gene delivery vectors; (2) The gene delivery vector was transcribed in vitro to obtain purified linear RNA products; (3) Circularizing the purified linear RNA product, including: ①Circularize using T4 RNA ligase at 15-18℃ for 1.5-3 hours; ② At 35-38℃, use ribonuclease R for 25-35 minutes to degrade the uncircularized linear RNA to obtain circRNA; (4) Purify the circRNA obtained in step (3); (5) Then, microfluidic technology is used to encapsulate the purified circRNA obtained in step (4).

[0029] Fourthly, the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of the gene delivery system described above or the gene delivery system prepared by the method described above.

[0030] In this invention, "treatment" should be interpreted in the broadest sense, encompassing the meaning of "adjunctive treatment," which refers to treatments used to enhance the effectiveness of the treatment.

[0031] In this invention, "treatment" may also include the meaning of "prevention".

[0032] In this invention, the term "prevention" includes reducing the likelihood of a patient developing or worsening a disease or symptom.

[0033] In this invention, the term "treatment" and other similar synonyms include the following meanings: (i) To prevent the occurrence of disease or condition in mammals, especially when such mammals are susceptible to the disease or condition but have not yet been diagnosed with it; (ii) To suppress a disease or symptom, that is, to curb its development; (iii) To alleviate a disease or symptom, that is, to cause the condition of the disease or symptom to subside; or (iv) To alleviate the symptoms caused by the disease or condition.

[0034] As used herein, the terms "effective amount," "therapeutic effective amount," or "pharmaceutical effective amount" refer to an amount of at least one drug or compound that, when taken orally, is sufficient to alleviate, to some extent, one or more symptoms of the disease or condition being treated. The result may be a reduction and / or relief of signs, symptoms, or causes, or any other desired change in the biological system. For example, an "effective amount" for treatment is the amount of a composition comprising the compounds disclosed in this invention required to provide significant symptom relief in a clinical setting. Effective amounts suitable for any individual case can be determined using techniques such as dose escalation testing.

[0035] As used in this invention, the terms "administration," "application," and "dosage" refer to methods that deliver a compound or composition to the desired site for biological action. These methods include, but are not limited to, parenteral injection (including intravenous, subcutaneous, intraperitoneal, intramuscular, and intra-arterial injection or infusion), local administration, and enteral administration. Those skilled in the art are familiar with the administration techniques that can be used with the compounds and methods described in this invention.

[0036] In this invention, "pharmaceutical composition" refers to a formulation of the compounds of this invention with a medium generally accepted in the art for delivering biologically active or functional compounds to mammals (such as humans). This medium includes pharmaceutically acceptable carriers. The purpose of the pharmaceutical composition is to facilitate administration to the organism, thereby promoting the absorption of the active ingredient and enabling it to exert its biological activity or function.

[0037] When using the pharmaceutical composition, a safe and effective amount of the compound of the present invention is applied to the subject requiring treatment (the subject includes mammals, such as humans). Of course, the specific dosage should also take into account factors such as the route of administration and the health status of the subject requiring prevention or treatment, which are all within the scope of the skills of a skilled physician.

[0038] In some preferred embodiments, the dosage of LNP-hOTC-circRNA is 0.5~3 mg / kg; the dosing cycle is once every 5-8 weeks.

[0039] In a sixth aspect, the present invention provides the use of the gene delivery vector, gene delivery system, gene delivery vector prepared by the method described above, gene delivery system prepared by the method described above, and pharmaceutical composition described above in the preparation of a medicament for treating metabolic disorders or conditions; wherein the metabolic disorders or conditions include lipid metabolism disorders and protein metabolism disorders.

[0040] In some preferred embodiments, the metabolic disorder or condition includes ornithine transcarbamylase deficiency (OTCD).

[0041] Otolaryngitis-related disease (OTCD) is the most common urea cycle disorder. Early-onset patients typically present with severe hyperammonemia in the neonatal period, leading to serious complications and a very high early mortality rate. Existing treatments (including ammonia-lowering drugs and liver transplantation) are palliative or high-risk. Adeno-associated virus (AAV)-based gene therapy has shown promise but faces significant limitations, including high immunogenicity, loss of cell-free DNA, and the risk of viral integration. Liposome delivery systems offer high biocompatibility and low immunogenicity, while also protecting the biological activity or function of the delivered drug or active group, prolonging drug half-life, and reducing toxicity. circRNAs enable cap-independent protein translation, thus serving as a stable platform for the delivery of immune and therapeutic proteins. Engineered circRNAs show great potential in therapeutic and gene regulation fields.

[0042] The beneficial effects of the present invention include at least the following: Existing technologies have not yet yielded any research on the use of lipid nanoparticles (LNPs) to deliver OTC-circRNA genes for the treatment of metabolic disorders or conditions (such as OTCD). Therefore, this invention is the first to construct a vector containing OTC-circRNA drug genes delivered via LNPs, which can be used to treat metabolic disorders or conditions (especially ornithine transcarbamate deficiency). The efficacy and safety of this treatment will be studied in vitro and in vivo, providing a new approach for the clinical treatment of patients with such gene defects and has significant application value.

[0043] This invention rationally designs nucleic acids encoding circRNA containing PIE elements and IRES sequences, and optimizes the cyclization reaction conditions, achieving a cyclization efficiency of over 95%, even approaching 100%. The lipid nanoparticles encapsulating OTC-circRNA of this invention exhibit good in vivo stability, good safety, and long-lasting therapeutic effects.

[0044] Other features and advantages of the present invention will be described in detail in the following detailed description section. Attached Figure Description

[0045] Figure 1 The results of hOTC-circRNA quality control and LNP embedding are shown. Figure (a) shows that the circular RNA is resistant to RNase R cleavage; Figure (b) shows the circularization interface verification (hOTC-circRNA): the prepared circular RNA was circularized in the specified circularization region; Figure (c) shows that the Agilent 2100 quality control results indicate that the in vitro prepared circular RNA has high integrity, no degradation, and the fragment size is as expected, with no other extraneous bands; Figure (d) shows the particle size distribution of the LNP-hOTC-circRNA sample.

[0046] Figure 2 The data show the transfection of Hep2.2.15 cell lines with LNP-hOTC-circRNA and LNP-EGFP-circRNA. Figure (a) shows a photograph taken 24 hours after transfection; Figure (b) shows Western blotting to verify OTC protein expression after LNP-hOTC-circRNA transfection; Figure (c) shows a statistical graph of OTC protein grayscale values ​​(with GADPH as an internal reference). All data are expressed as mean ± standard deviation. Compared with the control group, **** P < 0.0001, *** P < 0.001, ** P < 0.01, * P < 0.05, ns P ≥ 0.05.

[0047] Figure 3 The data show the transfection status of Huh1 cell lines with LNP-hOTC-circRNA and LNP-EGFP-circRNA. Figure (a) shows a photograph taken 24 hours after transfection; Figure (b) shows Western blotting to verify OTC protein expression after LNP-hOTC-circRNA transfection; Figure (c) shows a statistical graph of OTC protein grayscale values ​​(with GADPH as an internal reference). All data are expressed as mean ± standard deviation. Compared with the control group, **** P < 0.0001, *** P < 0.001, ** P < 0.01, * P < 0.05, nsP ≥ 0.05.

[0048] Figure 4 The data show the transfection status of BRL cell lines with LNP-hOTC-circRNA and LNP-EGFP-circRNA. Figure (a) shows a photograph of BRL cells 24 hours after transfection; Figure (b) shows Western blotting to verify OTC protein expression after LNP-hOTC-circRNA transfection; Figure (c) shows a statistical graph of OTC protein grayscale values ​​(with GADPH as an internal reference). All data are expressed as mean ± standard deviation. Compared with the control group, **** P < 0.0001, *** P < 0.001, ** P < 0.01, * P < 0.05, ns P ≥ 0.05.

[0049] Figure 5 The figures show the main targeting of LNP-ECGF-circRNA / PBS after administration, specifically the distribution of the fluorescently labeled EGFP delivered by the LNP SM102 formulation via tail vein injection. Figure (a) shows the labeling fluorescence mainly distributed in mouse liver cells; Figure (b) shows the distribution of the labeling fluorescence in lung tissue; Figure (c) shows the distribution of the labeling fluorescence in brain tissue; Figure (d) shows the distribution of the labeling fluorescence in spleen tissue; Figure (e) shows the distribution of the labeling fluorescence in kidney tissue; Figure (f) shows the distribution of the labeling fluorescence in the duodenum; and Figure (g) shows the distribution of the labeling fluorescence in the heart.

[0050] Figure 6 The figures show the general condition of mice after LNP-hOTC-circRNA intervention. Figure (a) shows the clinical phenotypic changes in the intervention group and the non-intervention group of the OTCD-P225L male mouse model. Figure (b) shows the changes in body weight of mice in each group during the 8-week observation period (N=6-8 mice / group).

[0051] Figure 7 The survival curves and blood ammonia levels at different time points in mice after LNP-hOTC-circRNA intervention are shown in Figure (a). Figure (a) shows the changes in survival curves of male mice in the LNP-hOTC-circRNA intervention group, the unintervention group, and the WT group. Figure (b) shows the changes in blood ammonia levels at different time points after LNP-hOTC-circRNA intervention. All data are expressed as mean ± standard deviation. Compared with the WT group, **** P < 0.0001, *** P < 0.001, ** P < 0.01, * P < 0.05, and ns P ≥ 0.05.

[0052] Figure 8The table shows the expression of OTC protein in mice after LNP-hOTC-circRNA intervention. Figures (a) and (c) show the expression of OTC protein in liver tissue at different time points (1d, 3d, 7d, 2w, 4w, and 8w) after LNP-hOTC-circRNA intervention (N=5 mice / group). Figures (b) and (d) show the statistical analysis results of OTC protein grayscale values ​​using GADPH as an internal control (N=5 mice / group). All data are expressed as mean ± standard deviation. Compared with the WT group, **** P < 0.0001, *** P < 0.001, ** P < 0.01, * P < 0.05, ns P ≥ 0.05.

[0053] Figure 9 This shows the immunohistochemical characteristics of liver tissue in mice at different time points after LNP-hOTC-circRNA intervention.

[0054] Figure 10 The data show the ATP and NAD+ / NADH levels in mice after LNP-hOTC-circRNA intervention (N=4~5 / group). All data are expressed as mean ± standard deviation. Compared with the WT group, **** P < 0.0001, *** P < 0.001, ** P < 0.01, * P < 0.05, ns P ≥ 0.05.

[0055] Figure 11 This paper shows the changes in biochemical indicators at different time points in mice after LNP-hOTC-circRNA intervention. The results indicate the liver function-related indicators (N=4~8 / group) in OTCD-P225L hemizygous male mice and WT male mice after LNP-hOTC-circRNA intervention: ALT (Alanine Aminotransferase); AST (Aspartate Transaminase); ALB (Albumin); TBIL (Total Bilirubin); CHO (Total Cholesterol); TG (Triacylglycerol); HDL (High Density Lipoprotein); LDL (Low Density Lipoprotein). All data are expressed as mean ± standard deviation. Compared with the WT group, **** P < 0.0001, *** P < 0.001, ** P < 0.01, * P < 0.05, ns P ≥ 0.05.

[0056] Figure 12 This paper shows the changes in serum amino acid and carnitine profiles in mice at different time points after LNP-hOTC-circRNA intervention. MS / MS analysis was used to analyze serum metabolite levels at different time points before and after LNP-hOTC-circRNA intervention (N=4~5 / group). Cit: citrulline; Gln: glutamine; Arg: arginine; Orn: ornithine; C0: free carnitine; C2: acetylcarnitine. All data are expressed as mean ± standard deviation. Compared with the WT group, **** P < 0.0001, *** P < 0.001, ** P < 0.01, * P < 0.05, ns P ≥ 0.05.

[0057] Figure 13 This shows the liver hepatic atherosclerosis (HE) in mice at different time points after LNP-hOTC-circRNA intervention.

[0058] Figure 14 This displays the plasmid map constructed in Example 1. Detailed Implementation

[0059] To make the objectives, technical solutions, and beneficial effects of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. Examples of the embodiments are shown in the accompanying drawings. It should be understood that the specific embodiments described in the following embodiments of the invention are merely illustrative examples of specific implementations of the invention and are intended to explain the invention, but do not constitute a limitation thereof.

[0060] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein. In the description of this application, unless otherwise stated, terms such as "multiple / a variety" mean two / a kind or more.

[0061] In the description of this invention, it should be noted that unless specific conditions are specified in the examples, conventional conditions are followed. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0062] Example 1

[0063] 1. Construction and quality control of hOTC-cirRNA plasmid ① Design a DNA template containing the CMV promoter sequence (as shown in SEQ ID NO: 5), the ORF sequence of the target gene hOTC (human OTC) (as shown in SEQ ID NO: 1), a PIE element, an IRES sequence (as shown in SEQ ID NO: 4), an intron arm sequence (5'T4 intron arm, as shown in SEQ ID NO: 6), and an SV40 terminator sequence (as shown in SEQ ID NO: 7). The PIE element includes an inverse complementary sequence (containing E1 sequence (SEQ ID NO: 2) and E2 sequence (SEQ ID NO: 3)) to promote circularization.

[0064] ② Obtain a linear DNA template through PCR amplification or gene synthesis, and clone it into the pUC57 plasmid vector (as shown in SEQ ID NO: 10). The constructed plasmid map is shown below. Figure 14 As shown.

[0065] ③ Restriction enzyme digestion was used to verify the correctness of the vector, and Sanger sequencing was used to confirm the PIE element and splice site (see results). Figure 1 a).

[0066] 2. In vitro transcription ① In vitro transcription using the MEGAscript kit: Reaction system: containing 1 μg linear DNA template, T7 RNA polymerase, NTPs (1 mM each), and transcription buffer (37℃, 6 hours).

[0067] DNase treatment: Add DNase I (37°C, 30 minutes) to remove DNA template.

[0068] ② The linear RNA product was purified using the RNA Clean & Concentrator Kit.

[0069] 3. RNA circularization ① Optimize cyclization reaction conditions: Use T4 RNA ligase (containing ATP and PEG 8000, incubated at 16°C for 3 hours).

[0070] ② The uncircularized linear RNA was degraded by treatment with ribonuclease R at 37°C for 30 minutes to obtain circularized RNA (circRNA). The RNA circularization efficiency in this example reached 99.6%.

[0071] 1) RNase R digestion method to verify hOTC-circRNA Equal volumes of linear precursor RNA and circular RNA were digested with RNase R (RNase R / RNA = 2 U / μg) at 37 °C for 15 min, then recovered by lithium chloride precipitation, and detected by 1.5% ordinary agarose gel electrophoresis (150 V, 15 min).

[0072] 2) Ring interface verification The purified circular RNA was further reverse transcribed into cDNA using an N6 random primer. PCR primers were then designed and sequencing was performed to detect the correctness of the circularization interface (primers are shown in Table 1, results are available in Table 1). Figure 1 b).

[0073] Table 1. Primers for PCR sequencing verification of hOTC-circRNA circularization interface

[0074] 4. Purification and Quantification ① The circRNA was purified by HPLC using an Agilent 1260 Infinity system, and a C18 column was used to separate the circRNA from impurities.

[0075] ②Nanodrop was used to quantify RNA concentration, and integrity was assessed using an Agilent 2100 Bioanalyzer.

[0076] Following the Agilent 2100 chip operating procedure, the gel-dye mixture, marker, ladder, and sample were added sequentially to the corresponding chip wells, and then placed into the Agilent 2100 bioanalyzer for analysis (results can be found in [link to results]). Figure 1 c).

[0077] 5. LNP encapsulation The encapsulation uses lipid nanoparticles containing ionizable cationic lipid SM102: by molar percentage, it contains 50% ionizable cationic lipid (SM-102); 1,2-distearate-sn-glycerol-3-phosphorylcholine (DSPC); 38.5% cholesterol; 1.5% 1,2-dimyristoyl-sn-glycerol-3-methoxy polyethylene glycol-2000 (DMG-PEG 2000); (see particle size) Figure 1 d) As a comparative example, lipid nanoparticles containing the ionizable cationic lipid ALC-0315 were used: by molar percentage, containing ionizable cationic lipid (ALC-0315): 50%; 1,2-distearate-sn-glycerol-3-phosphorylcholine (DSPC): 10%; cholesterol: 38.5%; 1,2-dimyristoyl-sn-glycerol-3-methoxy polyethylene glycol-2000 (DMG-PEG2000): 1.5%; LNPs are encapsulated using microfluidic technology, including the following steps: Step 1) Dissolve the purified hOTC-circRNA in a sodium citrate solution with a pH of 4-4.5 to form an aqueous phase, with the N / P ratio controlled at 6:1; Step 2) Dissolve the lipid nanoparticle raw materials of this embodiment and the comparative example in ethanol to form an oil phase; Step 3) By controlling the precise mixing and self-assembly of the oil and water phases with a microfluidic pump and a microfluidic chip, and using microfluidic technology to prepare purified hOTC-circRNA encapsulated in lipid nanoparticles at a flow rate ratio of 1:3, the process is carried out.

[0078] The quality control of the synthesized hOTC-circRNA plasmid and LNP encapsulation in this embodiment was performed, and the results are detailed in [link to documentation]. Figure 1 And Table 2.

[0079]

[0080] The results show that the LNP-encapsulated in this embodiment... hOTC - The circRNA has a particle size of 111.9 nm, which is at the nanoscale, making it suitable for endocytosis through the cell membrane; and the encapsulation efficiency can reach over 90%, indicating that more than 90% of the circRNA is effectively encapsulated in LNPs, reducing the risk of circRNA degradation in the in vitro environment. Meanwhile, LNP- hOTC The PDI of circRNA indicates its uniform distribution, which helps improve the distribution of LNP in vivo and liver targeting, thereby improving gene delivery efficiency. The ratio of free circRNA concentration to total circRNA concentration is less than 10%, indicating that most of the circRNA is effectively encapsulated in LNP with a low leakage rate, which helps to increase the effective concentration of circRNA in target cells, thereby improving gene delivery efficiency. The zeta potential is a suitable negative charge, indicating that the lipid nanoparticles have high stability in the dispersion system, which is beneficial for maintaining the long-term stability and effectiveness of the gene delivery vector in vivo.

[0081] In the comparative example, when the ionizable cationic lipid was ALC-0315, the zeta potential of the lipid nanoparticles was 0.26 mV, which is less stable than the lipid nanoparticles in this embodiment, making it unfavorable for maintaining the long-term stability and effectiveness of the gene delivery vector in vivo.

[0082] Example 2: Cellular experiments confirm LNP- hOTC -circRNA can successfully express OTC protein

[0083] ①HepG2, Hep2.2.15, Huh1 and mouse liver cells BRL were purchased from Baidi Company and identified by STR. After receiving the cells, they were placed in a 5% CO2, 37℃ cell culture incubator for 24 h according to the instructions.

[0084] ② Wipe the biosafety cabinet with 75% UV sterilization for half an hour. Under aseptic conditions, discard the original culture medium in the biosafety cabinet, wash the cells with PBS, use an appropriate amount of 0.25% trypsin, and place the cells in a 37°C incubator to digest the cells for 2-3 minutes (observe the cells become rounded under a regular optical microscope).

[0085] ③ Add DMEM medium containing 10% fetal bovine serum and 1% penicillin antibiotics at a concentration of more than 0.25% trypsin to terminate digestion, and further mix the cells by pipetting. Transfer the cells to 15 mL EP tubes and centrifuge (1000 rpm, room temperature, 5 min). Discard the supernatant and resuspend the cells. Passage the cells at a ratio of 1:2 or 1:3.

[0086] ① Cell plating: Plating is performed the day before transfection (using 6-well plates) to ensure that the confluence of cells in each well reaches 30% to 40%, and to ensure that the confluence of cells reaches 70% to 80% the next day.

[0087] ② Cell transfection: Before transfection, transfection can be performed when the cell confluence reaches 70%–80% as observed under a microscope; after removing the old culture medium, replace 1.5 mL of fresh culture medium in each well. Add 2 μg LNP- according to the grouping and requirements to each well. hOTC -circRNA / LNP- EGFP - circRNA or LNP- hOTC -mRNA / LNP- EGFP -mRNA, gently shake the cell culture plate back and forth, the final concentration of circRNA or mRNA in each well is approximately 1 μg / mL, incubate at room temperature for 5-10 minutes. Incubate the cells at 37°C in a 5% CO2 incubator for 24 hours.

[0088] Within 24 hours after transfection, the 6-well plate was photographed under a fluorescence microscope, and images were taken at different magnifications according to the groups.

[0089] ① Collect the corresponding cells, centrifuge at 1000 rpm for 5 min, discard the supernatant, add 100-200 μL of lysis buffer (with added protease / phosphatase inhibitor), add small steel balls, place in a cryogenic ball mill for grinding, and then sonicate in an ultrasonic homogenizer (sonicate for 10 s, pause for 10 s, sonicate for 10 min, power at 80 Hz).

[0090] ② After complete lysis on ice, centrifuge (13000 rpm, 4℃, 15 min) and collect the supernatant, which is the total protein extract.

[0091] ③ BCA assay: 96-well plate: Dilute 2 μL of original total protein extract with 18 μL of PBS, and make 2-3 replicates per sample. BCA working solution preparation: Prepare the working solution according to the total number of samples and the instructions.

[0092] ④ Protein denaturation: To ensure consistent protein concentration, the samples were first diluted with 1×PBS buffer, followed by the addition of 5×SDS protein loading buffer. The mixed samples were then boiled at 100°C to denature the proteins. Finally, the denatured samples were stored at -20°C for use in subsequent experiments.

[0093] 5. Western Blot ① Sample preparation: See section 4.7, Tissue protein extraction, for specific steps.

[0094] ② Gel preparation: Prepare separating gels and stacking gels of different concentrations according to the different molecular weights of the target proteins.

[0095] ③ Sample Loading and Gel Running: After the gel solidifies, begin assembly. Pour freshly prepared 1× electrophoresis buffer into the inner tank and load the sample. Then, add an appropriate amount of 1× electrophoresis buffer to the outer tank until the marker line is reached. Next, set the relevant electrophoresis parameters, including voltage and time. First, set the voltage to 80V and run for 30 minutes; then, increase the voltage to 120V and continue running for 1 hour to complete the entire electrophoresis process.

[0096] ④ Semi-dry transfer membrane: Activate the PVDF membrane with methanol beforehand, and sandwich the filter paper, protein gel, and PVDF membrane together. Be sure to remove air bubbles. Transfer the membrane at a constant current of 200 mA for 2 hours.

[0097] ⑤ Sealing: Place the transferred PVDF membrane into the sealing solution (TBST solution containing 5% skim milk powder), place it on a shaker, and incubate at room temperature for 1-2 hours.

[0098] ⑥ Primary antibody incubation: Prepare the required primary antibody, incubate overnight on a shaker at 4°C.

[0099] ⑦ Secondary antibody incubation: Wash 3 times with TBST solution for 10 min each time, add the prepared secondary antibody dilution solution, incubate at room temperature for 1-2 h, then wash 3 times with TBST solution for 10 min each time.

[0100] ⑨ Data Analysis: Grayscale values ​​were measured using ImageJ software, and statistical analysis was performed using GraphPad Prism.

[0101] Because hepatocellular carcinoma cell lines (Hep2.2.15 and Huh1, etc.) exhibit defective OTC protein expression, these cell lines were used to verify LNP- at the cellular level. hOTC -circRNA effect, and WB verification of OTC protein expression.

[0102] The results showed that the raw material formulation containing SM102 encapsulated LNP- hOTC -circRNA can successfully enter the cell and express EGFP fluorescence and OTC proteins (such as...). Figure 2 , Figure 3 and Figure 4 (As shown).

[0103] Example 3: Validation of LNP- in the OTCD-P225L mouse model hOTC - circRNA efficacy and safety assessment This embodiment validates LNP- in the OTCD-P225L mouse model. hOTC -Efficacy and safety assessment of circRNA tail vein injection.

[0104] 1. Animal condition and grouping Mice were randomly divided into three groups (n=6-8 per group) based on age and genotype: WT group, OTCD group, and OTCD- hOTC -circRNA intervention group (i.e., using LNP-) hOTC - circRNA intervention in OTCD group). Age: 3 weeks. Diet: 70% protein diet (Teklad custom diet, Madison WI), feed changed once a week.

[0105] 2. Before intervention, verify the targeting of LNP SM102 formulation: WT mice were injected with LNP-EGFP and PBS via the tail vein, respectively. After 24 hours, various organs of the mice were collected, frozen and embedded, sectioned, and photographed under a microscope.

[0106] 3. Intervention: Mice were injected intravenously via the tail vein at 3 weeks of age with a dose of 1 mg / kg of LNP- hOTC -circRNA or dosed PBS. Dosage cycle: once every 6 weeks.

[0107] 4. Observation indicators: blood metabolism, urine metabolism, blood ammonia, biochemistry (ALT, AST, ALB, TBA, CHO, TG, HDL, LDL), OTC protein levels, weight changes, changes in survival curve, etc. at different time points before and after intervention.

[0108] ① Record changes in mouse weight, survival rate, mortality rate, phenotype, etc. after intervention. The recorder enters the animal room twice a day to record the changes.

[0109] ② Mouse tail gene extraction: A 0.5 cm long tail was cut from a 1-day-old or deceased mouse and transferred to a 1.5 mL centrifuge tube, which was then stored in an ice box. 50 µL of a prepared 50 mM NaOH solution (0.1 g NaOH + 50 mL ddH2O) was added, and the mixture was heated in a 95°C metal bath for 30 min, followed by standing at 4°C for 2–3 min. An equal volume of pH 7.4, 1 M Tris-HCl (6.055 g Tris + 50 mL hydrochloric acid) was added, and the mixture was thoroughly mixed. The supernatant was collected by centrifugation. The mixture was then centrifuged at 13,000 rpm for 3 min at room temperature. 80 μL of the supernatant was transferred to a new centrifuge tube and stored at -20°C.

[0110] ③ Detection of mouse blood metabolites: Blood samples were collected via the orbital sinus using dried blood smears, dried in a cool, dry place, and stored at -20℃ or -80℃. Suitable samples were selected, and blood smears of approximately 1 mm in size were applied to qualified blood spots and placed in 96-well plates. The samples were then sent to the Genetic Metabolism Laboratory of the Sixth Affiliated Hospital of Sun Yat-sen University for further analysis of blood amino acid and carnitine profiles.

[0111] ④ Plasma collection: Based on the animal's weight, 1% sodium pentobarbital was injected intraperitoneally into the mother and newborn mice. When the eyelid reflex disappeared, blood was collected from the orbital venous sinus. After blood collection, the anticoagulant EP tubes were placed on ice for 1 hour and then centrifuged (3000 rpm × 15 min). The supernatant was then transferred to a -80℃ freezer for storage.

[0112] ⑤ Obtaining Liver and Other Tissues from Mice: After euthanizing the mice, the abdominal skin was first disinfected by spraying with 75% alcohol. Then, a small section of the abdominal skin was gently grasped with forceps. Next, the skin was carefully cut open with curved scissors to allow for further manipulation. After cutting the skin, the abdominal tissues were exposed. Next, the stomach, located below the liver, was located, and the connection between the stomach and esophagus was carefully severed. The stomach was grasped with forceps, and then the connective tissue behind the peritoneum was cut with curved scissors until the rectum was reached. Care must be taken during this process to ensure the integrity of vital tissues such as the gastrointestinal tract. The entire liver, gastrointestinal tract, and other tissues were carefully removed and thoroughly rinsed in pre-cooled PBS (phosphate-buffered saline). Next, the ribs were cut, and the heart and lung tissues were separated and obtained. After removing the mouse's head, the top of the skull was carefully removed to obtain complete brain tissue. Tissues from the liver, duodenum, kidney, heart, lungs, and brain were rapidly flash-frozen in liquid nitrogen to maintain freshness, and then stored at -80°C. These tissues will be used for subsequent Western blot (WB) analysis. Simultaneously, a portion of liver tissue was collected, flash-frozen in liquid nitrogen, and then stored at -80°C; this portion will be used for transcriptomics analysis. Finally, portions of liver, duodenum, and kidney tissue were fixed in 4% paraformaldehyde for paraffin embedding and sectioning, followed by HE staining and immunohistochemical analysis.

[0113] ⑥ Plasma ALT, AST, albumin, and total bilirubin detection: Remove the plasma sample to be tested from the -80℃ freezer, thaw it on ice, and mix well. Use the corresponding indicator kits to detect the above-mentioned indicators respectively. Preparation of working reagents: For the single-reagent method, use directly; for the two-reagent method, apply R1 and R2 separately. After setting the corresponding parameters on the fully automated biochemical analyzer, load the sample and automatically measure the results.

[0114] ⑦ Tissue protein extraction: Same as cell protein extraction steps.

[0115] ⑧Western Blot: The cell procedure is the same.

[0116] The LNP SM102 formulation showed the most significant expression in mouse liver cells (e.g., Figure 5 (as shown in a), but because the drug administration method was changed to tail vein administration, it can be seen that it can also reach other organs of the mouse.

[0117] ②LNP- hOTC -circRNA can improve the clinical phenotype and reduce mortality in the OTCD-P225L mouse model. LNP- hOTCFollowing circRNA intervention, the skin wrinkling and hair thinning in the OTCD-P225L mouse model were significantly improved compared to the untreated group during the 8-week observation period (e.g., Figure 6 As shown in a); their body weight also improved compared to the untreated group, although it has not yet reached the level of the WT group mice (e.g., Figure 6 (As shown in b). Survival curves revealed that LNP- hOTC -circRNA can significantly reduce the mortality rate of OTCD-P225L mouse models 20-30 days after birth (e.g., circRNA can significantly reduce the mortality rate of mice in the OTCD-P225L mouse model 20-30 days after birth). Figure 7 As shown in a), and blood ammonia levels also improved significantly (as shown in a). Figure 7 (As shown in b). Further examination of the expression level of OTC protein in mouse liver revealed that the expression in the intervention group was significantly higher than that in the non-intervention group (nearly 60%~80%), and it maintained this level for a longer period of time (e.g., as shown in b). Figure 8 (As shown in a and 8b). Simultaneously, immunohistochemistry was also used to assess LNP- hOTC - LNP-related OTC protein expression in mouse liver tissue was observed after circRNA intervention. hOTC -circRNA can significantly improve the expression of its OTC protein (e.g. Figure 9 (As shown). Finally, the energy metabolism-related indicators ATP and NAD in mice were further examined after 3 days of intervention. + / NADPH-related indicators revealed LNP- hOTC -mRNA can improve its expression (e.g. Figure 10 (As shown).

[0118] ②LNP- hOTC -circRNA can improve lipid metabolism and increase albumin levels, and does not cause abnormal liver function during a short observation period. To further evaluate LNP- hOTC The efficacy and safety of circRNA were further investigated, including blood liver function and lipid metabolism-related indicators at different time points, revealing that LNP- hOTC -circRNA treatment can improve lipid metabolism and increase albumin levels (e.g., ... Figure 11 (as shown in the figure), and will not cause significant abnormalities in liver function.

[0119] ③LNP- hOTC -circRNA can improve metabolic disorders in an OTCD-P225L mouse model At the same time, the inventor discovered LNP- hOTC -circRNA intervention can also significantly improve metabolic disorders in the body, including increasing the levels of citrulline, arginine, and C2, and significantly decreasing the levels of glutamine and ornithine, which are maintained at relatively normal levels for a longer period of time (e.g. Figure 12 (As shown).

[0120] ④LNP- hOTC -circRNA did not cause pathological changes during the short observation period. Finally, HE staining was used to assess the pathological changes in mouse liver tissue after LNP-hOTC-circRNA intervention. The results showed that LNP-hOTC-circRNA intervention did not cause significant pathological damage to the liver over a prolonged period (e.g., ...). Figure 13 (As shown).

[0121] Based on the above data, we can see how LNP- can be observed over a short period of time. hOTC -circRNA intervention can effectively improve the clinical performance of the OTCD-P225L mouse model and the therapeutic effect can last for 6-8 weeks.

[0122] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and do not constitute a limitation on the content of the present invention. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solutions of the present invention, including combining various technical features in any other suitable manner. These simple modifications and combinations should also be regarded as the content disclosed in the present invention and all fall within the protection scope of the present invention.

Claims

1. A gene delivery vector, characterized in that, The gene delivery vector comprises an OTC gene and a nucleic acid encoding circRNA, the OTC gene and the nucleic acid encoding circRNA being operatively linked, the nucleic acid encoding circRNA comprising a PIE element and an IRES sequence, wherein... The OTC gene contains any one of (a1)-(a3): (a1) The nucleotide sequence shown in SEQ ID NO: 1; (a2) A nucleotide sequence that has more than 90% identity with the nucleotide sequence shown in SEQ ID NO: 1 and has the same or similar biological activity or function; (a3) A nucleotide sequence consisting of the nucleotide sequence shown in (a1) or (a2) by substitution and / or deletion and / or addition of one or more nucleotides, and having the same or similar biological activity or function. The PIE element comprises an E1 sequence and an E2 sequence. The nucleic acid of the E1 sequence includes any one of (b1)-(b3): (b1) The nucleotide sequence shown in SEQ ID NO: 2; (b2) A nucleotide sequence that has more than 90% identity with the nucleotide sequence shown in SEQ ID NO: 2 and has the same or similar biological activity or function; (b3) A nucleotide sequence consisting of the nucleotide sequence shown in (b1) or (b2) by substitution and / or deletion and / or addition of one or more nucleotides, and having the same or similar biological activity or function. The nucleic acid of the E2 sequence includes any one of (c1)-(c3): (c1) The nucleotide sequence shown in SEQ ID NO: 3; (c2) A nucleotide sequence that has more than 90% identity with the nucleotide sequence shown in SEQ ID NO: 3 and has the same or similar biological activity or function; (c3) A nucleotide sequence consisting of the nucleotide sequence shown in (c1) or (c2) by substitution and / or deletion and / or addition of one or more nucleotides, and having the same or similar biological activity or function. The nucleic acid in the IRES sequence includes any one of (d1)-(d3): (d1) The nucleotide sequence shown in SEQ ID NO: 4; (d2) A nucleotide sequence that has more than 90% identity with the nucleotide sequence shown in SEQ ID NO: 4 and has the same or similar biological activity or function; (d3) A nucleotide sequence consisting of the nucleotide sequence shown in (d1) or (d2) by substitution and / or deletion and / or addition of one or more nucleotides, and having the same or similar biological activity or function.

2. A gene delivery system, characterized in that, The gene delivery system comprises the gene delivery vector and lipid nanoparticles according to claim 1; the lipid nanoparticles encapsulate the gene delivery vector.

3. The gene delivery system according to claim 2, characterized in that, The gene delivery vector is a plasmid.

4. The gene delivery system according to claim 2, characterized in that, The raw materials of the lipid nanoparticles, by molar percentage, comprise: ionizable cationic lipid (SM-102): 40-50%; 1,2-distearate-sn-glycerol-3-phosphorylcholine (DSPC): 10-15%; cholesterol: 35-40%; 1,2-dimyristoyl-sn-glycerol-3-methoxy polyethylene glycol-2000 (DMG-PEG 2000): 1-3%.

5. The gene delivery system according to claim 2 or 4, characterized in that, The N:P ratio in the raw material of the lipid nanoparticles is in the range of (4-6):1; and / or The lipid nanoparticles encapsulating the gene delivery vector have a dispersion index (PDI) of 0.1-0.2; and / or The encapsulation efficiency of lipid nanoparticles is over 90%; and / or At the end of lipid nanoparticle encapsulation, free circRNA was less than 10%; and / or At the start of lipid nanoparticle encapsulation, the total concentration of CircRNA was 110-120 µg / mL; and / or At the end of lipid nanoparticle encapsulation, the concentration of free circRNA was 8-10 µg / mL; and / or The zeta potential of the lipid nanoparticles encapsulating the gene delivery vector is -2 to -4 mV.

6. The gene delivery system according to claim 5, characterized in that, The lipid nanoparticles encapsulating the gene delivery vector have a particle size of 80-120 nm.

7. A method for preparing a gene delivery system, characterized in that, Includes the following steps: (1) Preparation of gene delivery vectors; (2) The gene delivery vector was transcribed in vitro to obtain purified linear RNA products; (3) Circularizing the purified linear RNA product, including: ①Circularize using T4 RNA ligase at 15-18℃ for 1.5-3 hours; ② At 35-38℃, use ribonuclease R for 25-35 minutes to degrade the uncircularized linear RNA to obtain circRNA; (4) Purify the circRNA obtained in step (3); (5) Then, microfluidic technology is used to encapsulate the purified circRNA obtained in step (4).

8. A pharmaceutical composition, characterized in that, Containing a therapeutically effective amount of the gene delivery vector according to claim 1, or the gene delivery system according to any one of claims 2-5, or the gene delivery vector prepared according to the method of claim 6, or The gene delivery system prepared according to the method of claim 7.

9. The use of the gene delivery vector according to claim 1, the gene delivery system according to any one of claims 2-5, the gene delivery system prepared by the method according to claim 6, the gene delivery system prepared by the method according to claim 7, or the pharmaceutical composition according to claim 8 in the preparation of a medicament for treating metabolic disorders or conditions; wherein the metabolic disorders or conditions include lipid metabolism disorders and protein metabolism disorders.

10. The application according to claim 9, wherein the metabolic disorder or condition comprises ornithine transcarbamylase deficiency.

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