Recombinant human granulocyte colony stimulating factor encoded by a small molecule regulated AAV expression vector

Abstract

The present application provides a kind of AAV small molecule regulatory expression vector encoded recombinant human granulocyte colony stimulating factor.It is specifically provided that a kind of construct for inducing and regulating the expression of recombinant human granulocyte colony stimulating factor (rhG-CSF), the vector comprising the construct, the corresponding host cell, gene delivery system, pharmaceutical composition, and its use and administration method.The AAV small molecule regulatory expression vector constructed by the present application can open the expression of rhG-CSF by taking small molecules when white blood cells need to be increased, and the drug can be precisely regulated, so as to achieve the purpose of continuous and controllable endogenous rhG-CSF production.In addition, the AAV small molecule regulatory expression vector constructed by the present application has very low leakage expression, and can induce very high level of target gene expression when very low small molecule level exists, thereby effectively improving the safety of gene therapy drugs.

Description

Technical Field

[0001] This invention belongs to the field of gene therapy, and more specifically relates to a recombinant human granulocyte colony-stimulating factor encoded by an AAV small molecule regulatory expression vector. Background Technology

[0002] In recent years, with the deepening of basic research on tumors, the methods and drug choices for tumor treatment have been constantly updated. Tumor treatment has gradually evolved into a comprehensive treatment plan that includes surgery, radiotherapy, chemotherapy, targeted drugs, and immunotherapy drugs. However, chemotherapy remains the foundation of tumor treatment, especially for patients with advanced tumors, where it is still one of the main treatment options. However, chemotherapy causes many toxic side effects, among which myelosuppressive toxicity is relatively common. Chemotherapy-induced neutropenia (CIN) and febrile neutropenia (FN) are the main adverse events caused by myelosuppressive chemotherapy drugs, and are the most serious hematological toxicities and clinical complications of myelosuppressive chemotherapy. Because granulocytes have the shortest average survival time, approximately 6-8 hours, myelosuppression first manifests as a decrease in white blood cells. Preventing severe neutropenia during chemotherapy is crucial, as this can increase medical costs, antibiotic use, or prolong hospitalization, and may even lead to a reduction or delay in the use of chemotherapy drugs. Therefore, preventing or treating neutropenia is fundamental to ensuring adequate doses or intensive chemotherapy.

[0003] White blood cell boosting agents are the main drugs used to increase the white blood cell count in the body, targeting the toxic side effects caused by chemotherapy and radiotherapy for tumors. Based on their composition, white blood cell boosting agents are mainly divided into three categories: the first category is oral chemical preparations; the second category is traditional Chinese medicine preparations, including oral and injectable formulations; and the third category is biological injectable agents—granulocyte colony-stimulating factor (G-CSF), which is the most commonly used drug for treating severe myelosuppression and is the first-line treatment for chemotherapy-associated neutropenia in domestic and international clinical guidelines. It includes both short-acting and long-acting types. Although there are currently three generations of recombinant human G-CSF (rhG-CSF), even the third generation, with a much longer half-life than the first two generations, still has a half-life of only 43.9–62.8 hours in the human body.

[0004] Therefore, there is an urgent need in the field to develop a long-acting recombinant rhG-CSF and its derivatives with in vivo modulotropic expression levels to avoid frequent dosing, thereby treating or preventing severe neutropenia and other leukopenia- and granulocytopenia-related conditions throughout the chemotherapy cycle. Summary of the Invention

[0005] The purpose of this invention is to address the technical deficiencies in existing technologies by developing a novel, long-acting white blood cell-boosting drug with adjustable expression levels and its derivatives—AAV-tet on inducible-hG-CSF transgenic vectors—for gene therapy of diseases related to leukopenia and granulocytopenia, such as neutropenia. After transferring a functional copy of hG-CSF to the patient, it provides continuous and controllable endogenous production of hG-CSF.

[0006] Another object of the present invention is to provide the use of the AAV-tet on inducible-hG-CSF transgenic vector in the preparation of a medicament for treating neutropenia and other conditions related to leukopenia and granulocytopenia.

[0007] In a first aspect of the invention, a construct for inducing and regulating the expression of recombinant human granulocyte colony-stimulating factor (rhG-CSF) is provided, the construct comprising the structure shown in formula (I) from the 5'-3' end:

[0008] Equation (I) is Z1-Z2-Z3-Z4-Z5-Z6-Z7-Z8-Z9-Z10.

[0009] Z1 is a tetracycline reaction element-regulated promoter;

[0010] Z2 is a Kozak sequence;

[0011] Z3 is the signal peptide sequence;

[0012] Z4 is the granulocyte colony-stimulating factor (hG-CSF) gene;

[0013] Z5 is a polyA element 1;

[0014] Z6 is a polyA element 2;

[0015] Z7 is an rTetR sequence;

[0016] Z8 is a 2A peptide sequence;

[0017] Z9 is a tTS sequence;

[0018] Z10 is the chicken β-actin promoter;

[0019] In the formula, each "-" represents an independent bond or nucleotide linkage sequence;

[0020] Z4 is selected from the following group:

[0021] (i) A sequence as shown in SEQ ID NO:1;

[0022] (ii) A sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the sequence shown in SEQ ID NO:1;

[0023] (iii) A sequence obtained by optionally adding, deleting and / or replacing at least one nucleotide based on the sequence shown in SEQ ID NO:1.

[0024] In another preferred embodiment, Z1 is selected from the group consisting of: TRE promoter, CMV promoter, CAG promoter, CBA promoter, EFS promoter, EF1a promoter, SFFV promoter, hPGK promoter, mPGK promoter, SV40 promoter, CBh promoter, MSCV promoter, UBC promoter, or combinations thereof; preferably, Z1 is selected from the group consisting of: TRE promoter, CMV promoter, EF1a promoter, EFS promoter, SFFV promoter, hPGK promoter, or combinations thereof.

[0025] In another preferred embodiment, Z1 is a TRE promoter with the sequence shown in SEQ ID NO:4.

[0026] In another preferred embodiment, the sequence of Z2 is as shown in SEQ ID NO:3.

[0027] In another preferred embodiment, the sequence of Z3 is as shown in SEQ ID NO:2.

[0028] In another preferred embodiment, Z5 is selected from the group consisting of bGH polyA, SV40 polyA, Rabbit gbpA, hGHpolyA, HSV TK poly(A), or combinations thereof.

[0029] In another preferred embodiment, Z5 is SV40 polyA.

[0030] In another preferred embodiment, Z6 is selected from the group consisting of bGH polyA, SV40 polyA, Rabbit gbpA, hGHpolyA, HSV TK poly(A), or combinations thereof.

[0031] In another preferred embodiment, Z6 is bGH polyA.

[0032] In another preferred embodiment, the sequence of Z7 is as shown in SEQ ID NO:5.

[0033] In another preferred embodiment, Z8 is selected from the group consisting of T2A sequences, P2A sequences, E2A sequences, F2A sequences, or combinations thereof; preferably, Z8 is a T2A sequence.

[0034] In another preferred embodiment, the sequence of Z9 is as shown in SEQ ID NO:6.

[0035] In another preferred embodiment, the sequence of Z10 is as shown in SEQ ID NO:7.

[0036] In a second aspect of the invention, a carrier is provided, the carrier comprising the constructs as described in the first aspect of the invention.

[0037] In another preferred embodiment, the vector is selected from the group consisting of DNA, RNA, viral vectors, plasmids, transposons, other gene transfer systems, or combinations thereof.

[0038] In another preferred embodiment, the vector is a plasmid.

[0039] In another preferred embodiment, the viral vector includes: lentivirus, adenovirus, AAV virus, retrovirus, or a combination thereof.

[0040] In another preferred embodiment, the carrier is an AAV carrier.

[0041] In another preferred embodiment, the serotypes of the AAV vector include, but are not limited to, the following groups: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, and AAV-DJ.

[0042] In another preferred embodiment, the serotype of the AAV vector is AAV8.

[0043] In another preferred embodiment, the AAV carrier is AAV8.

[0044] In another preferred embodiment, the AAV carrier is AAV2 / 8, which contains an ITR element of AAV2 and a capsid protein of AAV8.

[0045] In another preferred embodiment, the vector further includes elements selected from the group consisting of promoters, enhancers, transcriptional enhancer elements (WPREs), long terminal repeat sequences (LTRs), etc.

[0046] In another preferred embodiment, the vector comprises one or more promoters operatively linked to a construct, enhancer, intron, transcription termination signal, polyadenylation sequence, origin of replication, selectivity marker, nucleic acid restriction site, and / or homologous recombination site as described in the first aspect of the invention.

[0047] In another preferred embodiment, the enhancer is a CMV enhancer.

[0048] In another preferred embodiment, the selective label is an ampicillin resistance (AmpR) label.

[0049] In another preferred embodiment, the nucleic acid restriction site is the SmaI site.

[0050] In another preferred embodiment, the carrier includes the structure shown in Formula II from the 5'-3' end:

[0051] A1-A2-A3(II)

[0052] In the formula, each "-" represents an independent bond or nucleotide linkage sequence;

[0053] A1 is an ITR-L sequence;

[0054] A2 is a construct as described in the first aspect of the present invention; and

[0055] A3 is an ITR-R sequence.

[0056] In another preferred embodiment, A1 is an ITR-L sequence of AAV2.

[0057] In another preferred embodiment, A3 is an ITR-R sequence of AAV2.

[0058] In another preferred embodiment, the vector comprises a sequence as shown in SEQ ID NO:8.

[0059] In a third aspect of the invention, a host cell is provided, the host cell comprising a vector as described in the second aspect of the invention, or having a construct as described in the first aspect of the invention integrated into its genome.

[0060] In another preferred embodiment, the host cell includes a prokaryotic cell or a eukaryotic cell.

[0061] In another preferred embodiment, the host cell is selected from the group consisting of Escherichia coli, yeast cells, and mammalian cells.

[0062] In another preferred embodiment, the host cell is a HEK293F cell.

[0063] In a fourth aspect of the invention, a gene delivery system is provided, comprising: a construct as described in the first aspect of the invention or a vector as described in the second aspect of the invention, and an AAV capsid protein.

[0064] In another preferred embodiment, the AAV capsid protein is a natural AAV capsid protein or an artificially modified AAV capsid protein.

[0065] In another preferred embodiment, the AAV includes, but is not limited to, the following group: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, and AAV-DJ.

[0066] In another preferred embodiment, the AAV capsid protein is the capsid protein of AAV8.

[0067] In a fifth aspect of the invention, a pharmaceutical composition is provided, comprising:

[0068] (i) the constructs as described in the first aspect of the invention, the vectors as described in the second aspect of the invention, the host cells as described in the third aspect of the invention, the gene delivery systems as described in the fourth aspect of the invention, or combinations thereof; and

[0069] (ii) Pharmaceutically acceptable carriers, diluents or excipients.

[0070] In another preferred embodiment, component (i) accounts for 0.1-99.9 wt% of the total weight of the pharmaceutical composition, more preferably 10-99.9 wt%, and even more preferably 70-99 wt%.

[0071] In another preferred embodiment, the dosage form of the pharmaceutical composition is selected from the group consisting of: lyophilized dosage form and liquid dosage form.

[0072] In another preferred embodiment, the dosage form of the pharmaceutical composition is an injection.

[0073] In another preferred embodiment, the pharmaceutical composition is administered via intravenous injection, subcutaneous injection, or intramuscular injection.

[0074] In another preferred embodiment, the pharmaceutical composition is an injectable dosage form intended for intravenous, subcutaneous, or intramuscular injection.

[0075] In another preferred embodiment, the pharmaceutically acceptable carrier includes, but is not limited to, solvents, dispersion media, coatings, antibacterial or antifungal agents, isotonic agents, and absorption delay agents.

[0076] In another preferred embodiment, the pharmaceutically acceptable carrier is an injectable carrier. Preferably, the pharmaceutically acceptable carrier includes saline solutions, including but not limited to: buffered saline, physiological saline, phosphate buffer, citrate buffer, acetate buffer, bicarbonate buffer, sucrose solution, salt solution, polysorbate solution, or combinations thereof.

[0077] In another preferred embodiment, the pharmaceutically acceptable carrier may further comprise additives, including but not limited to: stabilizers, preservatives, transfection promoters that facilitate cell uptake, or combinations thereof.

[0078] In another preferred embodiment, the pharmaceutical composition may be used alone or in combination.

[0079] In another preferred embodiment, the combined use includes: combined use with other therapeutic agents used to treat conditions associated with leukopenia and granulocytopenia.

[0080] In a sixth aspect of the invention, a kit is provided, the kit comprising:

[0081] (Z1) A first container, and a construct as described in the first aspect of the invention, a carrier as described in the second aspect of the invention, a host cell as described in the third aspect of the invention, a gene delivery system as described in the fourth aspect of the invention, a pharmaceutical composition as described in the fifth aspect of the invention, or a combination thereof, located within the first container; and

[0082] (Z2) The second container, and the doxycycline located within the second container.

[0083] In another preferred embodiment, the concentration of doxycycline is 0.001 to 1000 ng / μL, more preferably 0.01 to 100 ng / μL, and even more preferably 0.1 to 10 ng / μL, such as 0.1 ng / μL, 0.5 ng / μL, 1 ng / μL, 5 ng / μL, 10 ng / μL, etc.

[0084] In a seventh aspect of the invention, there is provided the use of a construct as described in the first aspect of the invention, a vector as described in the second aspect of the invention, a host cell as described in the third aspect of the invention, a gene delivery system as described in the fourth aspect of the invention, a pharmaceutical composition as described in the fifth aspect of the invention, a kit as described in the sixth aspect of the invention, or a combination thereof, for the preparation of a formulation or composition.

[0085] In another preferred embodiment, the formulation or composition further includes doxycycline.

[0086] In another preferred embodiment, the concentration of doxycycline is 0.001 to 1000 ng / μL, more preferably 0.01 to 100 ng / μL, and even more preferably 0.1 to 10 ng / μL, such as 0.1 ng / μL, 0.5 ng / μL, 1 ng / μL, 5 ng / μL, 10 ng / μL, etc.

[0087] In another preferred embodiment, the formulation or composition is a medicine for treating conditions associated with leukopenia and granulocytopenia in subjects in need.

[0088] In another preferred embodiment, the conditions associated with leukopenia and granulocytopenia include, but are not limited to: ① chemotherapy-induced neutropenia (CIN) and fever with granulocytopenia (FN); ② neutropenia caused by infections such as bacteria, viruses, protozoa, and rickettsia, such as Gram-negative bacillus infections, such as typhoid and paratyphoid bacillus infections, such as influenza, viral hepatitis, chickenpox, rubella, cytomegalovirus infections, such as malaria, leishmaniasis, and other protozoan infections; ③ neutropenia caused by some hematologic disorders or autoimmune diseases; or combinations thereof.

[0089] In another preferred embodiment, the condition associated with leukopenia and granulocytopenia is caused by tumor radiotherapy or chemotherapy.

[0090] In another preferred embodiment, the subject is a human or a non-human mammal (such as a mouse, rat, rabbit, etc.).

[0091] In another preferred embodiment, the subject is an untreated patient.

[0092] In another preferred embodiment, the subject is a patient who has been treated with recombinant human G-CSF.

[0093] In another preferred embodiment, the subject is a patient who has been treated with recombinant human G-CSF but has not been cured or has relapsed.

[0094] In another preferred embodiment, the recombinant human G-CSF includes: short-acting recombinant human G-CSF and / or long-acting recombinant human G-CSF.

[0095] In another preferred embodiment, the recombinant human G-CSF includes: Filgrastim, Lenograstim, Pegfilgrastim, Benefilgrastim (F627), or a generic version thereof, or a combination thereof.

[0096] In another preferred embodiment, the formulation or composition has effects selected from the group consisting of:

[0097] (a) Increase white blood cell count or white blood cell level;

[0098] (b) Increase granulocyte count or granulocyte level; and / or

[0099] (c) Increase hG-CSF protein expression or hG-CSF protein level.

[0100] In another preferred embodiment, the formulation or composition is applied to cells.

[0101] In another preferred embodiment, the formulation or composition also has effects selected from the group consisting of:

[0102] (i) Improve hematopoietic function;

[0103] (ii) Improves anemia symptoms;

[0104] (iii) Stem cell mobilization;

[0105] (iv) Enhance the function of monocytes, granulocytes, eosinophils and / or macrophages; and / or

[0106] (v) Prevent infection complications or reduce the risk of infection.

[0107] In another preferred embodiment, the improvement of hematopoietic function includes improving bone marrow hematopoietic function.

[0108] In another preferred embodiment, the improvement of bone marrow hematopoietic function includes stimulating the proliferation, differentiation, and activation of hematopoietic stem cells in the bone marrow.

[0109] In another preferred embodiment, the anemia includes aplastic anemia.

[0110] In another preferred embodiment, the infectious complications or risk of infection are caused by a decrease in white blood cells or granulocytes.

[0111] In another preferred embodiment, the formulation or composition is also used to treat hematologic disorders.

[0112] In another preferred embodiment, the preparation or composition is also used to treat anemia.

[0113] In another preferred embodiment, the formulation or composition is also used to promote peripheral blood stem cell transplantation.

[0114] In another preferred embodiment, the formulation or composition is also used to enhance the body's immunity.

[0115] In another preferred embodiment, the formulation or composition is also used as an adjunct treatment for infectious diseases.

[0116] In an eighth aspect of the invention, a method for in vitro enhancement of blood parameters is provided, the method comprising the steps of: (S1) administering to a desired object a construct as described in the first aspect of the invention, a vector as described in the second aspect of the invention, a host cell as described in the third aspect of the invention, a gene delivery system as described in the fourth aspect of the invention, a pharmaceutical composition as described in the fifth aspect of the invention, a kit as described in the sixth aspect of the invention, or a combination thereof.

[0117] In another preferred embodiment, the method is for non-disease treatment purposes or non-disease diagnosis purposes.

[0118] In another preferred embodiment, the object is a cell.

[0119] In another preferred embodiment, the blood parameters are selected from the group consisting of:

[0120] (a) White blood cell count or white blood cell level; and / or

[0121] (b) Granulocyte count or granulocyte level.

[0122] In another preferred embodiment, the method further includes the step of: (S2) applying doxycycline to the desired object.

[0123] In another preferred embodiment, steps (S1) and (S2) are performed simultaneously or sequentially.

[0124] In another preferred embodiment, the concentration of doxycycline is 0.001 to 1000 ng / μL, more preferably 0.01 to 100 ng / μL, and even more preferably 0.1 to 10 ng / μL, such as 0.1 ng / μL, 0.5 ng / μL, 1 ng / μL, 5 ng / μL, 10 ng / μL, etc.

[0125] In a ninth aspect of the present invention, a method for increasing the expression level or hG-CSF protein level in vitro is provided, the method comprising the steps of: (S1) administering to a desired object a construct as described in the first aspect of the present invention, a vector as described in the second aspect of the present invention, a host cell as described in the third aspect of the present invention, a gene delivery system as described in the fourth aspect of the present invention, a pharmaceutical composition as described in the fifth aspect of the present invention, a kit as described in the sixth aspect of the present invention, or a combination thereof.

[0126] In another preferred embodiment, the method is for non-disease treatment purposes or non-disease diagnosis purposes.

[0127] In another preferred embodiment, the object is a cell.

[0128] In another preferred embodiment, the method further includes the step of: (S2) applying doxycycline to the desired object.

[0129] In another preferred embodiment, steps (S1) and (S2) are performed simultaneously or sequentially.

[0130] In another preferred embodiment, the concentration of doxycycline is 0.001 to 1000 ng / μL, more preferably 0.01 to 100 ng / μL, and even more preferably 0.1 to 10 ng / μL, such as 0.1 ng / μL, 0.5 ng / μL, 1 ng / μL, 5 ng / μL, 10 ng / μL, etc.

[0131] In a tenth aspect of the invention, a treatment method for conditions related to leukopenia and granulocytopenia is provided, the method comprising the steps of:

[0132] (S1) Administer to the desired subject the construct as described in the first aspect of the invention, the vector as described in the second aspect of the invention, the host cell as described in the third aspect of the invention, the gene delivery system as described in the fourth aspect of the invention, the pharmaceutical composition as described in the fifth aspect of the invention, the kit as described in the sixth aspect of the invention, or a combination thereof.

[0133] In another preferred embodiment, the administration method is intravenous injection or subcutaneous injection.

[0134] In another preferred embodiment, the subject is a human or a non-human mammal (such as a mouse, rat, rabbit, etc.).

[0135] In another preferred embodiment, the subject is an untreated patient.

[0136] In another preferred embodiment, the subject is a patient who has been treated with recombinant human G-CSF.

[0137] In another preferred embodiment, the subject is a patient who has been treated with recombinant human G-CSF but has not been cured or has relapsed.

[0138] In another preferred embodiment, the recombinant human G-CSF includes: short-acting recombinant human G-CSF and / or long-acting recombinant human G-CSF.

[0139] In another preferred embodiment, the recombinant human G-CSF includes: Filgrastim, Lenograstim, Pegfilgrastim, Benefilgrastim (F627), or a generic version thereof, or a combination thereof.

[0140] In another preferred embodiment, the dosage of the gene delivery system is 8E9vg / mouse-2E11vg / mouse, more preferably 8E9vg / mouse-4E10vg / mouse, and even more preferably 8E9vg / mouse.

[0141] In another preferred embodiment, the method further includes step (S2): administering doxycycline to the subject in need.

[0142] In another preferred embodiment, steps (S1) and (S2) are performed simultaneously or sequentially.

[0143] In another preferred embodiment, the concentration of doxycycline is 0.001 to 1000 ng / μL, more preferably 0.01 to 100 ng / μL, and even more preferably 0.1 to 10 ng / μL, such as 0.1 ng / μL, 0.5 ng / μL, 1 ng / μL, 5 ng / μL, 10 ng / μL, etc.

[0144] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0145] Figure 1 The gene map of the AAV-tet on inducible-hG-CSF plasmid is shown.

[0146] Figure 2 The results of qPCR testing of relative mRNA expression in 293 cells after transfection with the pAAV-CMV-hG-CSF plasmid vector are shown.

[0147] Figure 3 The results of ELISA testing of protein expression in 293 cells after transfection with the pAAV-CMV-hG-CSF plasmid vector are shown.

[0148] Figure 4 The effect of the AAV-CMV-hG-CSF viral vector on leukocytes of wild-type C57BL / 6J mice was demonstrated.

[0149] Figure 5 The effects of the AAV-CMV-hG-CSF viral vector on granulocytes of wild-type C57BL / 6J mice were demonstrated.

[0150] Figure 6 The effects of AAV8-hG-CSF transgenic vectors with different promoters on leukocytes and granulocytes in wild-type C57BL / 6J mice were demonstrated.

[0151] Figure 7 The effects of different administration routes of the AAV8-hG-CSF transgenic vector on leukocytes and granulocytes in wild-type C57BL / 6J mice were demonstrated.

[0152] Figure 8 The results show the hG-CSF protein expression level 24 hours after transfection of 293 cells with the AAV-tet on inducible-hG-CSF plasmid vector using ELISA.

[0153] Figure 9 The effects of AAV8-tet on inducible-hG-CSF viral vector on leukocytes in wild-type C57BL / 6J mice were demonstrated, both with and without drug administration. Detailed Implementation

[0154] Through extensive and in-depth research and numerous screenings, the inventors have, for the first time, creatively constructed an all-in-one doxycycline small molecule inducible regulatory AAV expression vector, enabling the packaging and delivery of all elements using a single AAV. This doxycycline small molecule inducible regulatory expression vector exhibits extremely low leakage expression, allowing for more precise regulation of target gene expression. Furthermore, the doxycycline small molecule inducible regulatory expression vector constructed in this invention achieves high levels of target gene expression even in the presence of extremely low levels of doxycycline inducible molecules, which helps reduce the toxic side effects of gene therapy drugs and improves their safety. This invention was completed based on these findings.

[0155] the term

[0156] To facilitate a clearer understanding of this disclosure, certain terms are first defined. As used herein, unless otherwise expressly specified herein, each of the following terms shall have the meaning given below.

[0157] The term “about” can refer to a value or composition within an acceptable range of error for a particular value or composition as determined by a person skilled in the art, which will depend in part on how the value or composition is measured or determined.

[0158] The term “administration” means the physical introduction of the product of the present invention into a subject using any of the various methods and delivery systems known to those skilled in the art, including intravenous, intratumoral, intramuscular, subcutaneous, intraperitoneal, spinal, or other parenteral routes of administration, such as by injection or infusion.

[0159] As used herein, the term "expression construct" refers to a single-stranded or double-stranded polynucleotide isolated from a naturally occurring gene or modified to contain a non-naturally occurring nucleic acid fragment. Expression constructs may contain control sequences required to express the coding sequences of this invention.

[0160] As used herein, the term “recombinant” refers to a nucleic acid, vector, polypeptide, or protein produced by DNA recombination (cloning) methods and distinguishable from natural or wild-type nucleic acids, vectors, polypeptides, or proteins.

[0161] As used herein, the term "host cell" refers to, for example, microbial, yeast, insect, and mammalian cells that can or have been used as rAAV vector recipients. This term includes the progeny of the transduced original cell. Therefore, as used herein, "host cell" generally refers to a cell that has been transduced with a foreign DNA sequence. It should be understood that due to natural, accidental, or artificial mutations, the progeny of a single parent cell may not necessarily be morphologically identical or complementary to the original parent in terms of genome or total DNA.

[0162] As used herein, the term "transfection" refers to the introduction of genetic material into a cell for the purpose of genetic modification. Transfection can be achieved by a variety of methods known in the art, such as transduction or electroporation.

[0163] As used in this article, "vector" refers to a recombinant plasmid or virus containing polynucleotides delivered to host cells in vitro or in vivo. "Recombinant" means different from what is normally found in nature.

[0164] As used herein, the term "gene therapy" refers to the insertion of a nucleic acid sequence (e.g., a nucleic acid containing a promoter operatively linked to a polynucleotide encoding a transgene (e.g., human granulocyte colony-stimulating factor)) into the cells and / or tissues of an individual to treat a disease or condition. Such transgenes may be exogenous. Exogenous molecules or sequences should be understood as molecules or sequences that are not normally present in the cells, tissues, and / or individual to be treated.

[0165] Unless otherwise specified, nucleotide sequences in this article are shown only as single strands, in a 5' to 3' orientation, from left to right. Nucleotides and amino acids in this article are represented in the manner recommended by the IUPAC IUB Committee on Biochemistry Nomenclature, or (for amino acids) in the form of single-letter or three-letter codes as per 37 CFR §1.822 and established usage.

[0166] As used herein, the term "coding sequence" or the sequence "coding" refers to a region of DNA or RNA (the transcribed region) that "encodes" a specific protein. When under the control of appropriate regulatory regions (e.g., promoters), coding sequences are transcribed (DNA) and translated (RNA) into polypeptides in vitro or in vivo. The boundaries of a coding sequence are defined by a start codon at the 5' (amino) end and a translation stop codon at the 3' (carboxyl) end. Coding sequences can include, but are not limited to, prokaryotic or eukaryotic cDNA, prokaryotic or eukaryotic genomic DNA, and synthetic DNA sequences. The transcription stop sequence may be located on the 3' side of the coding sequence.

[0167] As used herein, the term "promoter" refers to a nucleic acid sequence or segment that functions to control the transcription of one or more genes (or coding sequences), is located upstream of the transcription start site in the transcriptional direction, and is structurally identified as containing a DNA-dependent RNA polymerase binding site, a transcription start site, and any other DNA sequence, including but not limited to transcription factor binding sites, repressor and activator protein binding sites, and any other nucleotide sequence known to those skilled in the art to directly or indirectly regulate the amount of transcription from a promoter. A "constitutive" promoter is a promoter that is active under most physiological and developmental conditions. An "inducible" promoter is a promoter regulated by physiological or developmental conditions. A "tissue-specific" promoter is a promoter that is preferentially active in a specific type of differentiated cell / tissue.

[0168] As used in this article, the term "enhancer" is a cis-acting element that stimulates or represses transcription of adjacent genes. Enhancers that repress transcription are also called "silencers." Enhancers can exert their effects (e.g., they can be associated with coding sequences) at distances of up to thousands of base pairs (kb) downstream of coding sequences and transcribed regions in either direction.

[0169] As used herein, the terms "AAV viral particle," "AAV viral unit," or "AAV vector particle" refer to a viral particle composed of at least one AAV capsid protein and an AAV vector encapsulating polynucleotides. If the particle contains heterologous polynucleotides (i.e., polynucleotides other than the wild-type AAV genome, such as transgenes delivered to mammalian cells), it is generally referred to as an "AAV vector particle" or simply "AAV vector." Therefore, the production of AAV vector particles necessarily includes the production of AAV vectors. Thus, the vector is contained within the AAV vector particle.

[0170] As used herein, the terms "cell culture medium" and "culture medium" refer to a solution containing nutrients that provide nourishment for the continuous growth of cultured eukaryotic cells. Typically, these solutions provide essential and non-essential amino acids, vitamins, energy sources, lipids, and trace elements required for minimum cell growth and / or survival. The solution may also contain components that enhance growth and / or survival, including hormones and growth factors. The pH and salt concentration of the solution are formulated to be optimal for cell survival and proliferation.

[0171] As used in this article, “cell viability” refers to the ability of cultured cells to survive under a given set of culture conditions or experimental variations. The term also refers to the proportion of cells that survive at a specific time relative to the total number of live and dead cells in the culture at that time.

[0172] The term “cell density” as used in this article refers to the number of cells present in a given volume of culture medium.

[0173] As used in this article, the term "inoculation" refers to the process of providing cell cultures to a bioreactor dish. On the one hand, the cells have previously been propagated in the bioreactor dish. On the other hand, the cells have been immediately frozen and thawed before being provided to the bioreactor dish. The term refers to any number of cells, including single cells.

[0174] Chemotherapy's myelosuppressive toxicity

[0175] Chemotherapy is a cytotoxic drug that inhibits rapidly dividing cells, with myelosuppressive toxicity being a common effect. Bone marrow is the body's hematopoietic tissue, where cells proliferate rapidly; therefore, chemotherapy interferes with bone marrow's hematopoietic function, leading to myelosuppression. Severe myelosuppression not only affects a patient's tolerance and sensitivity to cancer treatment but also leads to adverse consequences such as reduced dosage of anti-tumor drugs, prolonged treatment cycles, or interruption of treatment. This reduces treatment effectiveness, promotes disease progression, lowers the patient's quality of life, increases the patient's financial burden, and negatively impacts prognosis.

[0176] Chemotherapy-induced neutropenia (CIN) and febrile neutropenia (FN) are major adverse events caused by myelosuppressive chemotherapy drugs, representing the most serious hematological toxicities and clinical complications of myelosuppressive chemotherapy. Because granulocytes have the shortest average survival time, approximately 6-8 hours, myelosuppression initially manifests as a decrease in white blood cells. Preventing severe neutropenia during chemotherapy is crucial, as it can increase medical costs, antibiotic use, or prolong hospital stays, and may even lead to a reduction or delay in chemotherapy drug use. Multiple studies have demonstrated that the incidence of CIN can be extremely high, potentially leading to life-threatening complications such as septic shock and sepsis; conversely, the risk is significantly reduced if CIN is avoided. Therefore, preventing or treating CIN is fundamental to ensuring adequate or intensive chemotherapy doses.

[0177] Chemotherapy-induced neutropenia (CIN)

[0178] CIN refers to an absolute neutrophil count (ANC) in peripheral blood induced by the use of myelosuppressive chemotherapy drugs, specifically an ANC < 2.0 × 10⁻⁶ in a complete blood count. 9 / L. According to the National Cancer Institute's Common Terminology Standard for Adverse Events (CTS) version 5.0, neutropenia is classified into four grades; Grade 1: ANC (1.5 to <2.0) × 10⁻⁶. 9 / L; Grade 2: ANC (1.0~<1.5)×109 / L; Grade 3: ANC (0.5~<1.0)×10 9 / L; Grade 4: ANC < 0.5 × 10 9 / L.

[0179] Chemotherapy-induced fever with neutropenia (FN)

[0180] FN refers to severe neutropenia accompanied by fever. Severe neutropenia is defined as ANC < 0.5 × 10⁻⁶. 9 / L (level 4) or ANC is (0.5~<1.0)×10 9 / L (Level 3), but is expected to decrease to <0.5×10 in the following 48 hours. 9 / L; Fever is defined as a single oral temperature measurement ≥38.3℃ or ≥38.0℃ lasting for more than 1 hour.

[0181] Leukocyte-stimulating agents

[0182] White blood cell growth regulators are mainly classified into three categories according to the nature of their ingredients.

[0183] The first category consists of oral chemical preparations, mainly including leucogen, vitamin B4, squalene, inosine, and lithium carbonate. Leucogen is one of the more effective oral Western medicines, with an efficacy rate of 60%-70%, but it may cause allergic dermatitis. Vitamin B4, squalene, inosine, and lithium carbonate all have the disadvantages of slow action, low efficacy, unstable effects, and long treatment courses. Squalene has been reported to cause pharyngitis after taking it; inosine can occasionally cause allergic reactions due to gastric discomfort; lithium carbonate's clinical use is limited because it can cause changes in blood lithium concentration, and there are currently no methods to detect lithium concentration; vitamin B4, being a nucleic acid precursor, has the potential to promote tumor development, which remains to be investigated.

[0184] The second category is traditional Chinese medicine (TCM) preparations, including oral and injectable formulations. Oral TCM preparations include Diyu Shengbai tablets, Fufan Zaofan pills, Shengxuebao mixture, Shengbai mixture, and Yixuesheng capsules, among others. Injectable TCM preparations include Shengbai injection, Shengmai injection, and Huangqi injection. Among TCM preparations for increasing white blood cell count, most adjuvant TCM preparations lack evidence-based medicine support and suffer from drawbacks such as unclear mechanisms of action and effective components, difficulty in quantifying their onset of action, and challenges in using quantitative indicators for efficacy.

[0185] The third category is biological injectable agents—granulocyte colony-stimulating factor (G-CSF), a glycoprotein synthesized by vascular endothelial cells, monocytes, and fibroblasts. It binds to G-CSF receptors on the cell membrane and promotes the proliferation and differentiation of hematopoietic stem cells into neutrophils by activating downstream signaling pathways such as serine / threonine kinase AKT. At the same time, it mobilizes mature neutrophils from the bone marrow into the peripheral blood, enhancing the functions of neutrophils such as adhesion, migration, phagocytosis, and bactericidal activity. As the most commonly used drug for treating severe myelosuppression and the first choice in domestic and international clinical guidelines for the treatment of radiotherapy and chemotherapy-related neutropenia, it includes both short-acting and long-acting types.

[0186] Recombinant human granulocyte colony-stimulating factor (rhG-CSF)

[0187] First-generation recombinant human G-CSFs are all short-acting, requiring daily or multiple weekly injections, with white blood cell counts every 2-3 days. These include Filgrastim, developed by Amgen (USA), and Lenograstim, developed by Chugai Pharmaceutical (Japan). Filgrastim is a recombinant protein expressed in *E. coli*, containing 175 amino acids with a molecular weight of 19 kDa. Its unglycosylated form has a half-life of 3.5 hours in humans. Lenograstim is a recombinant protein produced by mammalian CHO cells, containing 174 amino acids and exhibiting glycosylation; its half-life in humans is also only 3 hours.

[0188] The second-generation rhG-CSF, Pegfilgrastim, also developed by Amgen, is a once-injectable chemotherapy drug. It involves PEGylation (20kD-PEG) at the N-terminus of the Filgrastim protein molecule. This modification approximately doubles the molecular weight of Pegfilgrastim, reducing renal excretion and increasing its half-life from 3.5 hours to 15-80 hours. However, the PEGylation reduces the affinity of G-CSF for its receptor, thus extending the half-life at the cost of decreased biological activity. This is because PEG's steric shielding effect weakens the interaction between the protein drug and its receptor, reducing its biological activity; the larger the molecular weight, the greater the reduction in activity. Furthermore, high-molecular-weight PEG is not easily metabolized in vivo and tends to accumulate in the liver and other sites, causing macromolecular syndrome and potentially damaging the liver with long-term use. In addition, PEG conjugated to protein drugs may induce anti-PEG antibodies, reducing efficacy and safety.

[0189] Although there have been reports in recent years of anti-PEG monoclonal antibodies being used to develop analytical tools for PEGylation therapy or drug release, related research is still in the preclinical stage. Currently, most of the long-acting recombinant rhG-CSFs approved for marketing in China, as well as those under investigation, are generic versions of Pegfilgrastim.

[0190] The third generation is Benefilgrastim (F627): developed by Yifan Pharmaceuticals, it is an rhG-CSF dimer (rhG-CSF / IgG2-Fc fusion protein) expressed by CHO cells based on Fc fusion protein technology. Although its half-life in the human body is much longer than the previous two generations, it is only 43.9 to 62.8 hours.

[0191] After long-term research, the inventors of this application have developed a long-acting recombinant rhG-CSF and its derivatives based on the star gene therapy vector AAV. In normal mice and myelosuppressed mouse models, its ability to increase white blood cell count lasts for several months. This means that in humans, it may be possible to prevent or treat severe neutropenia and other conditions related to leukopenia and granulocytopenia that occur throughout the entire chemotherapy cycle with only one dose. This has significant application prospects.

[0192] In addition, rhG-CSF can also be used in the treatment of hematological diseases, as an adjunct to tumor treatment, and in the treatment of infectious diseases.

[0193] For example, rhG-CSF can stimulate the proliferation, differentiation and activation of hematopoietic stem cells in the bone marrow, thereby improving bone marrow hematopoietic function and treating bone marrow hematopoietic dysfunction; rhG-CSF can be used to treat myelodysplastic syndromes and help improve the patient's hematopoietic function; rhG-CSF can improve anemia symptoms (such as aplastic anemia) by stimulating bone marrow hematopoiesis and increasing the patient's blood cell levels.

[0194] rhG-CSF can also be used for stem cell mobilization before peripheral blood hematopoietic stem cell transplantation, improving the success rate of transplantation. rhG-CSF can also enhance the function of monocytes, granulocytes, eosinophils and macrophages, thereby improving the body's anti-tumor and anti-infection immunity. Although it is not directly used to treat infectious diseases, it can assist in the treatment of certain infectious diseases by enhancing the body's immunity.

[0195] Main advantages of the invention

[0196] 1. Due to the short lifespan of human granulocytes, existing recombinant human granulocyte colony-stimulating factor (rhG-CSF) injections, although capable of promoting leukocyte production, have a half-life of only 2-3 days in vivo. Therefore, repeated injections are required to maintain their efficacy, causing significant inconvenience and discomfort for patients. Subcutaneous delivery of recombinant human granulocyte colony-stimulating factor via an AAV carrier can achieve sustained endogenous rhG-CSF production, thus resolving the problem with a single injection.

[0197] 2. This invention constructs a doxycycline small molecule inducible regulatory expression vector to regulate the expression of rhG-CSF. Therefore, when it is necessary to increase white blood cell count, the expression of rhG-CSF can be activated by taking the small molecule, allowing for precise drug regulation.

[0198] 3. The all-in-one doxycycline small molecule inducible regulatory expression vector constructed in this invention is relatively small, and all elements can be packaged and delivered using a single AAV.

[0199] 4. The doxycycline small molecule inducible regulatory expression vector constructed in this invention has extremely low leakage expression through the optimization of regulatory elements, that is, the expression of the target gene is extremely low in the absence of doxycycline, thus the regulation is more precise.

[0200] 5. The doxycycline small molecule inducible regulatory expression vector constructed in this invention has a high level of target gene induction expression even in the presence of extremely low levels of doxycycline inducible molecules. Therefore, oral small molecule inducible drugs have practical clinical application value in achieving very low dosage of small molecules. Moreover, the high level of small molecule induction expression means that the amount of virus to be injected can also be controlled at a low level to reduce the toxic side effects of gene therapy drugs and improve the safety of gene therapy drugs.

[0201] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or under conditions recommended by the manufacturer, or according to methods listed in laboratory manuals such as *Molecular Cloning: A Laboratory Manual*, *Cellular Laboratory Manual*, and CFDA experimental guidelines familiar to those skilled in the art. Unless otherwise stated, percentages and parts are weight percentages and parts by weight. All reagents and raw materials used are commercially available and can be purchased through public channels.

[0202] Sequence information

[0203]

[0204]

[0205]

[0206]

[0207]

[0208]

[0209] Example 1: Preparation of AAV2 / 8-hG-CSF or AAV8-hG-CSF transgenic vectors

[0210] In this embodiment, an AAV2 / 8-hG-CSF or AAV8-hG-CSF transgenic vector is provided, which is prepared by the following method:

[0211] ① Enzyme digestion and recovery of the vector backbone:

[0212] Digest the existing laboratory vector backbone with EcoRl restriction endonuclease (see backbone structure). Figure 1 The medium skeleton was then recovered by agarose gel electrophoresis and stored at 4°C.

[0213] ② Amplification and recovery of the target gene:

[0214] Design primers, amplify the hG-CSF target fragment by PCR, recover it by agarose gel electrophoresis, and store it at 4°C.

[0215] ③ Homologous recombination:

[0216] The target plasmid was constructed using the homologous recombinase of Vazyme (2x CloneExpress Mix, C115-02-AA).

[0217] ④ Chemical transformation:

[0218] The homologous recombination product obtained in step ③ was added to DH5α competent cells (Solabio, C1100), placed on ice for 30 min, then heat-shocked at 45℃ for 45 s, then placed on ice for 3 min, and then added to antibiotic-free LB medium. The cells were activated at 37℃ and shaken at 220 rpm for 1 h, then plated and incubated at 37℃ overnight.

[0219] ⑤ Select single clones, perform bacterial culture PCR identification, and then select positive clones;

[0220] ⑥ Sanger sequencing (GENEWIZ);

[0221] ⑦ Plasmid extraction (NucleoBond Xtra Midi kit for transfection-grade plasmid DNA, MCHEREY-NAGEL, 740410.5), plasmid map as shown. Figure 1 As shown.

[0222] Example 2: In vitro validation experiment—detection of AAV2 / 8-hG-CSF or AAV8-hG-CSF transgenic cells at the cellular level Due to the expression of the vector hG-CSF

[0223] ① Resuscitation and passage of adherent HEK293 cells;

[0224] ② Cell plating:

[0225] 18–24 hours before transfection, cells are seeded into 24-well plates, and an appropriate number of cells are added to the culture medium to ensure the formation of a monolayer with 60–80% confluence before transfection.

[0226] ③ Transfection:

[0227] A. Preparation of premixed system: Take 150 μL of serum-free DMEM medium, add 4.5 μg of plasmid, mix thoroughly, add 13.5 μL of PEI, mix well and let stand for 10 min, add to well plate, mix gently, and incubate in a 37℃ carbon dioxide incubator.

[0228] B. 72 hours after transfection, the cell supernatant was removed, the cell pellet was collected, and the relative expression of the hG-CSF gene was detected by qPCR.

[0229] C. 72 hours after transfection, cell supernatant was collected, and the expression of hG-CSF protein was detected using the Human G-CSF ELISA Kit (Beyotime, PG340);

[0230] like Figure 2 As shown, the qPCR test results indicate successful cell transfection and detection of relative expression (mRNA expression) of the hG-CSF gene. Furthermore, high levels of relative expression (mRNA expression) of the hG-CSF gene were still detected at durations of 72 hours and 96 hours.

[0231] like Figure 3 As shown, the ELISA test results indicate that cell transfection was successful and hG-CSF protein expression was detected.

[0232] Example 3: In vivo validation experiment – ​​In vivo validation of AAV2 / 8-hG-CSF or AAV8-hG-CSF in wild-type mice Efficacy trials of gene therapy drugs

[0233] ① Virus dilution:

[0234] High-dose group: 2E12 vg / mL (pre-diluted)

[0235] Medium-dose group: 2E11 vg / mL (pre-diluted)

[0236] Medium-dose group: 2E10 vg / mL (pre-diluted)

[0237] ② Administration method: subcutaneous injection

[0238] ③ Mice: 6-8 week old C57 mice (mice of the same sex from the same batch)

[0239] ④ Experimental grouping and drug dosage:

[0240]

[0241]

[0242] ⑤ Testing indicators: Complete blood count:

[0243] Twelve mice were randomly divided into three groups. Before administration, blood samples were taken to measure the levels of white blood cells and granulocytes in the mice's blood. Then, the mice were subcutaneously inoculated according to the table above. After administration, blood samples were taken periodically to measure the levels of white blood cells and granulocytes in the mice.

[0244] like Figure 4 and Figure 5 As shown, each mouse in the experimental group was subcutaneously injected with 2×10 9 vg, 2×10 10 vg, 2×10 11 The results for the AAV2 / 8-hG-CSF or AAV8-hG-CSF virus in vg are as follows:

[0245] (1) As can be seen from the figure, there was no significant difference in white blood cells and granulocytes among the mice in each group before injection;

[0246] (2) Before day 22 of injection, 2×10 11 The rate of increase in white blood cell and granulocyte counts in the vg group mice was >2×10 10 VG group mice >2×10 9 vg group mice;

[0247] (3) 2×10 before 85 days after injection 11 The white blood cell and granulocyte counts of mice in the vg group were higher than 2 × 10⁻⁶. 10 The vg group mice, but after 85 days, there was no difference in the number of white blood cells and granulocytes between the two groups of mice;

[0248] (4) In addition, 2×10 11 vg group and 2×10 10 In the vg group mice, the white blood cell and granulocyte counts remained above 2 × 10⁻⁶ after injection.9 VG group mice, 2×10 11 vg group and 2×10 10 The white blood cell and granulocyte counts of mice in the vg group fluctuated significantly, but the count was 2×10⁻⁶. 9 After the white blood cell and granulocyte counts of the vg group mice increased slowly, they remained at a relatively stable level.

[0249] (5) Overall, subcutaneous inoculation with AAV2 / 8-hG-CSF or AAV8-hG-CSF virus can sustainably stimulate an increase in white blood cells and granulocytes. To date (218 days until the experimental endpoint), white blood cell and granulocyte levels remain at a high level, consistently higher than the control group. These results indicate that the AAV-hG-CSF transgenic vector of this invention possesses a significant and long-lasting effect in increasing white blood cells and granulocytes, and can be used to prevent and treat diseases related to leukopenia and granulocytopenia.

[0250] In addition, we further optimized the transgenic vector and attempted subcutaneous inoculation of AAV-hG-CSF transgenic vector viruses with different promoters, such as... Figure 6 As shown, the dosage was 2×10 10 In addition to the CMV promoter, AAV-hG-CSF transgenic vector viruses corresponding to the EF1α, EFS, SFFV, and hPGK promoters also significantly increase white blood cell and granulocyte counts; for example... Figure 7 As shown, the administration method was further optimized by subcutaneous injection, intramuscular injection, and intravenous injection of the AAV-hG-CSF transgenic vector virus corresponding to the CMV promoter. The results showed that, up to the experimental endpoint of 100 days, all three different administration methods could significantly induce an increase in white blood cells and granulocytes in mice. The results of different promoters and different administration methods further corroborated the therapeutic effect of the drug and improved the broadness of its effects.

[0251] Example 4: In vitro validation experiment—validation of pAAV-tet oninducible-hG-CSF vector at the cellular level Effect of inducing and regulating the expression of hG-CSF protein

[0252] To precisely regulate the expression levels of target gene therapeutic drugs, we constructed a doxycycline small molecule inducible regulatory AAV expression vector, pAAV-tet on inducible-hG-CSF. This expression vector is an optimized vector obtained after extensive screening and validation of its structure and elements. Compared with expression vectors designed using other elements or structures (e.g., those containing only a single polyA element, or those with different elements and their order), it achieves superior induction of the target protein expression.

[0253] To verify the effect of doxycycline on the induction of the target protein, we transfected pAAV-tet on inducible-hG-CSF plasmid into 293 cells. After induction for 24 hours with doxycycline (without or with different doses, 0.1 ng / µl, 0.5 ng / µl, 1 ng / µl, 5 ng / µl, and 10 ng / µl), the supernatant was collected, and the hG-CSF protein level in the supernatant was detected by ELISA. Figure 8 As shown, the basal leakage expression was very low without doxycycline, indicating high safety and facilitating precise regulation. Furthermore, the addition of 0.1 ng / ul doxycycline resulted in the induction of 10 ng / ml hG-CSF protein expression, demonstrating that this small-molecule inducible expression vector can achieve relatively high target protein expression even in the presence of low-dose small-molecule inducers, thus possessing practical clinical application value.

[0254] Example 5: In vivo validation experiment—In vivo validation of AAV8-tet on inducible-hG- in wild-type mice CSF doxycycline small molecule inducible gene therapy drug's induction and regulation effect on leukocytes

[0255] To further verify the regulatory role of this small molecule inducible expression vector in vivo, we packaged AAV8-tet on inducible-hG-CSF (as an example) and then subcutaneously injected this small molecule-regulated expression recombinant human granulocyte colony-stimulating factor gene therapy drug at a dose of 8E9 vg / mouse. Afterwards, the white blood cell levels in the two groups of mice were measured, either without medication or with medication at 625 mg / kg doxycycline.

[0256] like Figure 9 As shown, the results indicated that the white blood cell count in mice not fed the small molecule inducer increased only slightly, with little significant change, while the white blood cell count in mice fed 625 mg / kg doxycycline increased continuously to 60-fold. This demonstrates the in vivo regulatory effect of the small molecule inducible expression vector on human granulocyte colony-stimulating factor and on white blood cell increase. These results also prove that the AAV-tet on inducible-hG-CSF viral vector exhibits virtually no leakage expression, demonstrating high safety and facilitating precise regulation.

[0257] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A construct for inducing and regulating the expression of recombinant human granulocyte colony-stimulating factor (rhG-CSF), characterized in that, The construct includes the structure shown in formula (I) from the 5'-3' end: Equation (I) is Z1-Z2-Z3-Z4-Z5-Z6-Z7-Z8-Z9-Z10. Z1 is a tetracycline reaction element-regulated promoter; Z2 is a Kozak sequence; Z3 is the signal peptide sequence; Z4 is the granulocyte colony-stimulating factor (hG-CSF) gene; Z5 is a polyA element 1; Z6 is a polyA element 2; Z7 is an rTetR sequence; Z8 is a 2A peptide sequence; Z9 is a tTS sequence; Z10 is the chicken β-actin promoter; In the formula, each "-" represents an independent bond or nucleotide linkage sequence; Z4 is selected from the following group: (i) A sequence as shown in SEQ ID NO:1; (ii) A sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the sequence shown in SEQ ID NO:1; (iii) A sequence obtained by optionally adding, deleting and / or replacing at least one nucleotide based on the sequence shown in SEQ ID NO:

1.

2. The construct as claimed in claim 1, characterized in that, The Z1 is selected from the group consisting of: TRE promoter, CMV promoter, CAG promoter, CBA promoter, EFS promoter, EF1a promoter, SFFV promoter, hPGK promoter, mPGK promoter, SV40 promoter, CBh promoter, MSCV promoter, UBC promoter, or a combination thereof; preferably, the Z1 is selected from the group consisting of: TRE promoter, CMV promoter, EF1a promoter, EFS promoter, SFFV promoter, hPGK promoter, or a combination thereof.

3. The construct as claimed in claim 1, characterized in that, Z1 is a TRE promoter, the sequence of which is shown in SEQ ID NO:

4.

4. The construct as claimed in claim 1, characterized in that, The Z5 is selected from the group consisting of bGH polyA, SV40 polyA, Rabbit gbpA, hGH polyA, HSV TK poly(A), or combinations thereof; preferably, the Z5 is SV40 polyA.

5. A carrier, characterized in that, The carrier includes the construct as described in any one of claims 1-4.

6. A host cell, characterized in that, The host cell includes the vector as described in claim 5, or the genome of which is integrated with a construct as described in any one of claims 1-4.

7. A gene delivery system, characterized in that, include: The construct as described in any one of claims 1-4 or the vector as described in claim 5, and the AAV capsid protein.

8. A pharmaceutical composition, characterized in that, include: (i) the construct as described in any one of claims 1-4, the vector as described in claim 5, the host cell as described in claim 6, the gene delivery system as described in claim 7, or a combination thereof; and (ii) Pharmaceutically acceptable carriers, diluents or excipients.

9. A reagent kit, characterized in that, The kit includes: (Z1) A first container, and a construct as described in any one of claims 1-4, a vector as described in claim 5, a host cell as described in claim 6, a gene delivery system as described in claim 7, a pharmaceutical composition as described in claim 8, or a combination thereof, located within the first container; and (Z2) The second container, and the doxycycline located within the second container.

10. Use of a construct as described in any one of claims 1-4, a vector as described in claim 5, a host cell as described in claim 6, a gene delivery system as described in claim 7, a pharmaceutical composition as described in claim 8, a kit as described in claim 9, or a combination thereof, characterized in that, This is used to prepare a formulation or composition for the treatment of conditions associated with leukopenia or granulocytopenia in subjects in need.

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