AAV vector for retina infection, adalimumab and application of AAV vector and adalimumab

Abstract

The invention relates to a retina-infected AAV vector, an adalimumab and application thereof. Specifically, the invention provides an easily available AAV vector capable of efficiently infecting the retina and application thereof, and also provides a high-expression adalimumab coding sequence and application thereof. According to the AAV vector and the adalimumab coding sequence, on one hand, the side effect can be reduced through local administration and relatively low dosage, and on the other hand, the lifelong benefit effect of one-time administration can be realized by utilizing the long-term expression ability.

Description

AAV vector for infecting retina, adalimumab and use thereof Technical Field

[0001] The present invention belongs to the field of biotechnology and specifically relates to an AAV vector for infecting the retina, adalimumab, and applications thereof. Background Art

[0002] Uveitis, also known as uveitis, is a general term for inflammation of the iris, ciliary body, and choroid. Uveitis is a chronic disease that often occurs in young adults. It has numerous forms and complex etiologies. Improper treatment can lead to blindness, making it a significant cause of blindness and a global concern.

[0003] Since its pathogenesis and recurrence mechanism are not yet fully understood, its prevention is difficult to achieve and the therapeutic effect is also very unsatisfactory. Therefore, seeking a reasonable and effective therapeutic drug has become a problem urgently needed to be solved in the field of ophthalmology.

[0004] Currently, the first-line treatments for uveitis are steroids (such as dexamethasone) and TNFα inhibitors. However, TNFα inhibitors require systemic administration, which can lead to significant side effects, and the monthly dosing can lead to poor compliance.

[0005] Adeno-associated virus (AAV) has broad application prospects in the field of human gene therapy. Due to its long-term gene expression ability and non-pathogenicity, it has been widely used in various studies in tissues such as liver, muscle, heart, brain, eye, and kidney.

[0006] However, there are still few AAV vectors that can efficiently infect the retina, and their infection efficiency is low, so a higher titer is required to achieve a certain effective infection rate.

[0007] Adalimumab is a humanized monoclonal antibody against human tumor necrosis factor (TNF). It is a disulfide-bonded dimer of the heavy and light chains of the human monoclonal D2E7. Adalimumab specifically binds to TNFα and blocks its interaction with p55 and p75 cell surface TNF receptors, controlling inflammation and reducing immune responses, potentially ameliorating autoimmune diseases. However, current adalimumab is relatively expensive and its expression level is insufficient. A higher-expression adalimumab is needed to reduce industrial costs, thereby lowering medication costs and benefiting patients.

[0008] Therefore, there is an urgent need in this field to develop an easily accessible AAV vector that can efficiently infect the retina and a high expression level of adalimumab. On the one hand, local administration and relatively low dosage can reduce side effects. On the other hand, the long-term expression ability can achieve a lifelong benefit effect of a single dose.

[0009] Summary of the Invention

[0010] The purpose of the present invention is to provide an AAV vector that is easy to obtain and can efficiently infect the retina.

[0011] Another object of the present invention is to provide a pharmaceutical composition comprising the AAV vector provided by the present invention.

[0012] Another object of the present invention is to provide a use of the AAV vector of the present invention for preparing a preparation or pharmaceutical composition for treating uveitis.

[0013] Another object of the present invention is to provide a high expression level adalimumab coding sequence and its application.

[0014] In a first aspect of the present invention, a polynucleotide encoding adalimumab is provided, the polynucleotide comprising:

[0015] (1) the heavy chain coding sequence shown in SEQ ID NO: 5 and the light chain coding sequence shown in SEQ ID NO: 10; or

[0016] (2) A conservative derivative sequence obtained by adding, deleting, modifying and / or replacing at least one nucleotide on the basis of the heavy chain coding sequence or the light chain coding sequence in (1), without changing the amino acid sequence encoded thereby.

[0017] In another preferred example, the conserved derived sequence has at least 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence shown in SEQ ID NO: 5 or SEQ ID NO: 10.

[0018] In the second aspect of the present invention, an expression cassette is provided, wherein the expression cassette comprises the polynucleotide according to the first aspect of the present invention.

[0019] In another preferred embodiment, the expression cassette comprises one or more promoters, which are operably linked to the polynucleotide or a fragment thereof, an enhancer, an intron, a transcription termination signal, a polyadenylation sequence, an origin of replication, a selective marker, a nucleic acid restriction site, and / or a homologous recombination site.

[0020] In another preferred embodiment, the expression cassette includes a plasmid vector or a viral vector.

[0021] In another preferred embodiment, the viral vector is selected from the group consisting of a lentiviral vector, an adenoviral vector, an adeno-associated viral vector (AAV), or a combination thereof. Preferably, the expression cassette is an AAV vector.

[0022] In another preferred example, the expression cassette is selected from the following group: expression cassette L93, expression cassette L5, expression cassette Y817 or expression cassette Y981, wherein the expression cassette L93 has the nucleotide sequence shown in SEQ ID NO: 12; the expression cassette L5 has the nucleotide sequence shown in SEQ ID NO: 13; the expression cassette Y817 has the nucleotide sequence shown in SEQ ID NO: 14; the expression cassette Y981 has the nucleotide sequence shown in SEQ ID NO: 24.

[0023] In another preferred embodiment, the expression cassette has a nucleotide sequence selected from the group consisting of:

[0024] (a) the nucleotide sequence shown in SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 24;

[0025] (b) a nucleotide sequence that is ≥95% (preferably ≥98%) identical to the sequence shown in SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 24; or

[0026] (c) a sequence having 1-30 (preferably 1-15, more preferably 1-9) nucleotides truncated or added to the 5' and / or 3' ends of the sequences shown in SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 24.

[0027] In another preferred embodiment, the expression cassette has a structure shown in Formula A or Formula B from the 5'-3' end: X0-X1-X2-X3-X4-X5 (A) X0-X1-X2-X5-X4-X3 (B)

[0028] In the formula, each "-" is independently a bond or a nucleotide linking sequence;

[0029] X0 is the heavy chain regulatory element;

[0030] X1 is the heavy chain coding sequence SEQ ID NO: 5;

[0031] X2 is poly(A) signal sequence 1;

[0032] X3 is the light chain regulatory element;

[0033] X4 is the light chain coding sequence SEQ ID NO: 10; and

[0034] X5 is the poly(A) signal sequence 2.

[0035] In another preferred example, the heavy chain regulatory element or the light chain regulatory element is each selected from the following group: an enhancer element, a promoter element, an intron element, or a combination thereof.

[0036] In another preferred embodiment, the heavy chain regulatory element and the light chain regulatory element may be the same or different.

[0037] In another preferred embodiment, the enhancer element is the CMV enhancer, and its sequence is shown in SEQ ID NO: 1.

[0038] In another preferred embodiment, the promoter element is selected from the following group: CMV promoter, CBA promoter, CAG promoter, Cbh promoter, EF-1α promoter, or a combination thereof.

[0039] In another preferred embodiment, the heavy chain regulatory element includes a CMV promoter or a CBA promoter, and the sequences thereof are shown in SEQ ID NO: 2 or SEQ ID NO: 3, respectively.

[0040] In another preferred embodiment, the light chain regulatory element includes EF-1α promoter 1 or EF-1α promoter 2, and the sequences thereof are shown in SEQ ID NO: 7 or SEQ ID NO: 8, respectively.

[0041] In another preferred embodiment, the heavy chain regulatory element includes: SV40 intron, chimeric intron, or a combination thereof.

[0042] In another preferred embodiment, the heavy chain regulatory element includes an SV40 intron, and its sequence is shown in SEQ ID NO:4.

[0043] In another preferred embodiment, the light chain regulatory element includes a TPL intron, the sequence of which is shown in SEQ ID NO:9.

[0044] In another preferred example, the poly(A) signal sequence 1 and the poly(A) signal sequence 2 may be the same or different.

[0045] In another preferred embodiment, the poly(A) signal sequence 1 is a bGH poly(A) signal sequence, and its sequence is shown in SEQ ID NO: 6.

[0046] In another preferred embodiment, the poly(A) signal sequence 2 is the SV40 poly(A) signal sequence, and its sequence is shown in SEQ ID NO:11.

[0047] In another preferred embodiment, the expression cassette has a structure shown in Formula I from the 5'-3' end: Z0-Z1-Z2-Z3-Z4-Z5-Z6-Z7-Z8-Z9-Z10-Z11-Z12-Z13 (I)

[0048] In the formula, each "-" is independently a bond or a nucleotide linking sequence;

[0049] Z0 is the ITR-L sequence;

[0050] Z1 is an enhancer element;

[0051] Z2 is promoter element A;

[0052] Z3 is absent or contains intron element B;

[0053] Z4 is SEQ ID NO: 5;

[0054] Z5 is none or poly(A) signal sequence 1;

[0055] Z6 is absent or promoter element C;

[0056] Z7 is absent or contains intron element D;

[0057] Z8 is no or self-cleaved peptide;

[0058] Z9 is SEQ ID NO: 10;

[0059] Z10 is poly(A) signal sequence 2;

[0060] Z11 is the ITR-R sequence;

[0061] Z12 is absent or promoter element E; and

[0062] Z13 is none or an optional tag sequence.

[0063] In another preferred embodiment, the sequence of Z0 is shown in SEQ ID NO: 19.

[0064] In another preferred embodiment, the Z1 is a CMV enhancer, and its sequence is shown in SEQ ID NO: 1.

[0065] In another preferred embodiment, the Z2 is selected from the following group: CMV promoter, CAG promoter, Cbh promoter, CAG promoter, EF-1α promoter, CBA promoter, or a combination thereof.

[0066] In another preferred embodiment, the Z2 is a CMV promoter or a CBA promoter, and the sequences thereof are shown in SEQ ID NO: 2 or SEQ ID NO: 3, respectively.

[0067] In another preferred embodiment, the Z3 includes: an SV40 intron, a chimeric intron, or a combination thereof.

[0068] In another preferred embodiment, the Z3 is an SV40 intron, and its sequence is shown in SEQ ID NO:4.

[0069] In another preferred embodiment, the Z3 is a chimeric intron.

[0070] In another preferred embodiment, the Z5 is a bGH poly (A) signal sequence, the sequence of which is shown in SEQ ID NO: 6.

[0071] In another preferred embodiment, the Z6 is selected from the following group: CMV promoter, CBA promoter, Cbh promoter, CAG promoter, EF-1α promoter.

[0072] In another preferred embodiment, the Z6 is EF-1α promoter 1 or EF-1α promoter 2, and the sequences thereof are shown in SEQ ID NO: 7 or SEQ ID NO: 8, respectively.

[0073] In another preferred embodiment, the Z7 is a TPL intron, and its sequence is shown in SEQ ID NO:9.

[0074] In another preferred embodiment, the Z8 is selected from T2A, P2A, F2A, E2A, or a combination thereof.

[0075] In another preferred embodiment, the Z10 is the SV40 poly (A) signal sequence, the sequence of which is shown in SEQ ID NO:11.

[0076] In another preferred embodiment, the sequence of Z11 is shown in SEQ ID NO: 20.

[0077] In another preferred embodiment, the Z12 is the ampicillin resistance gene (AmpR) promoter.

[0078] In another preferred embodiment, the label includes a resistance marker and / or a fluorescent marker.

[0079] In another preferred embodiment, the Z13 is a kanamycin resistance gene (kanR).

[0080] In another preferred embodiment, the length of each nucleotide linker sequence is 0-30 nt, preferably 1-15 nt.

[0081] In another preferred embodiment, the expression cassette has a structure shown in Formula Ia from the 5'-3' end: Z0-Z1-Z2-Z3-Z4-Z5-Z6-Z7-Z9-Z10-Z11-Z12-Z13 (Ia)

[0082] In the formula, each "-" is independently a bond or a nucleotide linking sequence;

[0083] Z0-Z4 have the definitions as above;

[0084] Z5 is poly(A) signal sequence 1;

[0085] Z6 is promoter element C;

[0086] Z7, Z9-Z13 have the same definitions as above.

[0087] In another preferred embodiment, the expression cassette has a structure shown in Formula Ib from the 5'-3' end: Z0-Z1-Z2-Z3-Z4-Z8-Z9-Z10-Z11-Z12-Z13 (Ib)

[0088] In the formula, each "-" is independently a bond or a nucleotide linking sequence;

[0089] Z0-Z4 have the definitions as above;

[0090] Z8 is a self-cleaving peptide;

[0091] Z9-Z13 have the same definitions as above.

[0092] In another preferred embodiment, the expression cassette has a structure shown in Formula Ic from the 5'-3' end: Z0-Z1-Z2-Z3-Z4-Z5-Z10-Z9-Z8-Z7-Z6-Z11-Z12-Z13 (Ic)

[0093] In the formula, each "-" is independently a bond or a nucleotide linking sequence;

[0094] Z0-Z13 have the definitions described above.

[0095] In the third aspect of the present invention, there is provided a use of the polynucleotide according to the first aspect of the present invention or the expression cassette according to the second aspect of the present invention for preparing a formulation or composition for treating indications for which adalimumab is applicable.

[0096] In another preferred embodiment, the indications for the adalimumab include but are not limited to: retinal diseases and autoimmune diseases.

[0097] In another preferred embodiment, the retina-related diseases include but are not limited to uveitis.

[0098] In another preferred embodiment, the autoimmune disease includes but is not limited to ankylosing spondylitis, rheumatoid arthritis, polyarticular juvenile idiopathic arthritis, psoriasis, psoriatic arthritis, plaque psoriasis, ulcerative colitis, Crohn's disease (including childhood Crohn's disease), and hidradenitis suppurativa.

[0099] In a fourth aspect of the present invention, an engineered AAV capsid protein is provided, wherein the AAV capsid protein has an amino acid sequence selected from the group consisting of:

[0100] (a) the amino acid sequence shown in SEQ ID NO: 16, SEQ ID NO: 17 or SEQ ID NO: 18; and

[0101] (b) an amino acid sequence that is at least 95% (preferably at least 98%, most preferably at least 99%) identical to any one of SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 18.

[0102] In the fifth aspect of the present invention, a nucleic acid molecule is provided, which encodes the AAV capsid protein as described in the fourth aspect of the present invention.

[0103] In another preferred embodiment, the nucleic acid molecule has a nucleotide sequence selected from the group consisting of:

[0104] (a) the nucleotide sequence shown in SEQ ID NO: 21, 22 or 23; and

[0105] (b) a nucleotide sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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% identical to any one of SEQ ID NO:21, SEQ ID NO:22, and SEQ ID NO:23.

[0106] In a sixth aspect of the present invention, there is provided a use of the AAV capsid protein according to the fourth aspect of the present invention, for:

[0107] (i) preparing a medicament for preventing and / or treating a retina-related disease in a subject in need thereof; and / or

[0108] (ii) delivering the target gene packaged in the AAV capsid protein to the retina of the subject for expression.

[0109] In another preferred embodiment, the subject includes a human or a non-human mammal.

[0110] In another preferred embodiment, the non-human mammals include but are not limited to non-human primates, sheep, dogs, cats, horses, cows, chickens, rats, mice, etc.

[0111] In another preferred embodiment, the retina-related disease is selected from the following group: uveitis, retinal vein occlusion, macular edema after retinal vein occlusion, wet (neovascular, exudative) age-related macular degeneration, myopic macular degeneration, diabetic macular edema, diabetic retinopathy, retinopathy of prematurity, ischemic retinopathy, retinitis pigmentosa, retinal capillary dilation, retinal and / or choroidal neovascularization, neovascular glaucoma, corneal and / or iris neovascularization secondary to retinal disease, or a combination thereof.

[0112] In another preferred embodiment, the retinal vein occlusion includes: branch retinal vein occlusion, hemi-retinal vein occlusion, and central retinal vein occlusion.

[0113] In another preferred embodiment, the retinal and / or choroidal neovascularization includes retinal and / or choroidal neovascularization secondary to retinal diseases, retinal and / or choroidal neovascularization caused by trauma, and idiopathic retinal and / or choroidal neovascularization.

[0114] In another preferred embodiment, the retinal and / or choroidal neovascularization secondary to retinal diseases is secondary to retinal diseases including but not limited to those selected from the group consisting of uveitis, retinal vein occlusion, retinal degenerative disorders, hereditary retinal and / or choroidal diseases, and eye tumors.

[0115] In another preferred embodiment, the retina-related disease is a retina-related disease with high expression of TNFα, wherein the high expression of TNFα refers to the ratio of the TNFα expression level E1 in inflammatory tissues or inflammatory cells to the TNFα expression level E0 in normal tissues or normal cells (i.e., E1 / E0) ≥1.5, preferably ≥2.0, and more preferably ≥3.0.

[0116] In another preferred embodiment, the inflamed tissue includes but is not limited to: iris, ciliary body, choroid, retina, lens, and cornea.

[0117] In another preferred embodiment, the inflammatory cells include but are not limited to: rod cells, cone cells, bipolar cells, horizontal cells, ganglion cells, amacrine cells, interreticular cells, Muller cells, pigment epithelial cells, and ganglion cells.

[0118] In the seventh aspect of the present invention, a recombinant AAV vector is provided, wherein the recombinant AAV vector has: the AAV capsid protein as described in the fourth aspect of the present invention, and an exogenous expression cassette encoding a target gene packaged in the AAV capsid protein.

[0119] In another preferred embodiment, the capsid protein of the recombinant AAV vector is encoded by the nucleic acid molecule as described in the fifth aspect of the present invention.

[0120] In another preferred embodiment, the rep gene of the recombinant AAV vector can be derived from any AAV serotype.

[0121] In another preferred embodiment, the AAV serotype includes but is not limited to: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, and AAVrh10.

[0122] In another preferred embodiment, the rep gene of the recombinant AAV vector is derived from AAV2.

[0123] In another preferred embodiment, the recombinant AAV vector is AAV-Y659, and its capsid protein has the amino acid sequence shown in SEQ ID NO:16.

[0124] In another preferred embodiment, the recombinant AAV vector is AAV-Y660, and its capsid protein has the amino acid sequence shown in SEQ ID NO:17.

[0125] In another preferred example, the recombinant AAV vector is AAV-Y689, and its capsid protein has the amino acid sequence shown in SEQ ID NO:18.

[0126] In another preferred embodiment, the exogenous expression cassette has a structure shown in Formula II from the 5'-3' end:

[0127] A1-(A2-A3-A4-A5-A6)n-A7-A8-A9(II)

[0128] In the formula, each "-" is independently a bond or a nucleotide linking sequence;

[0129] A1 is the ITR-L sequence;

[0130] A2 is absent or enhancer element;

[0131] A3 is absent or promoter element i;

[0132] A4 is absent or contains intronic elements;

[0133] A5 is the target gene;

[0134] A6 is the poly(A) signal sequence;

[0135] A7 is the ITR-R sequence;

[0136] A8 is none or promoter element ii;

[0137] A9 is no or optional tag sequence; and

[0138] n is a positive integer, preferably 1≤n≤4.

[0139] In another preferred embodiment, the sequence of A1 is shown as SEQ ID NO: 19.

[0140] In another preferred embodiment, the A2 is a CMV enhancer, and its sequence is shown in SEQ ID NO: 1.

[0141] In another preferred embodiment, the A3 is selected from the following group: CMV promoter, CAG promoter, Cbh promoter, CAG promoter, EF-1α promoter.

[0142] In another preferred embodiment, the A3 is a CMV promoter or a CAG promoter, and the sequences thereof are shown in SEQ ID NO: 2 or SEQ ID NO: 3, respectively.

[0143] In another preferred embodiment, the A3 is EF-1α promoter 1 or EF-1α promoter 2, and the sequences thereof are shown in SEQ ID NO: 7 or SEQ ID NO: 8, respectively.

[0144] In another preferred embodiment, the A4 is an SV40 intron or a TPL intron, and the sequences thereof are shown in SEQ ID NO: 4 or SEQ ID NO: 9, respectively.

[0145] In another preferred embodiment, the A5 is used to treat retina-related diseases or autoimmune diseases.

[0146] In another preferred embodiment, the A5 is an adalimumab coding sequence.

[0147] In another preferred embodiment, the A5 is a reporter gene.

[0148] In another preferred embodiment, the A5 is a firefly luciferase and green fluorescent protein (FLuc:GFP) fusion protein gene.

[0149] In another preferred embodiment, the A5 may be a plurality of target genes in series.

[0150] In another preferred embodiment, the A6 is a bGH poly (A) signal sequence or a SV40 poly (A) signal sequence, and the sequences thereof are shown in SEQ ID NO: 6 or SEQ ID NO: 11, respectively.

[0151] In another preferred embodiment, the sequence of A7 is shown in SEQ ID NO: 20.

[0152] In another preferred embodiment, the A8 is the promoter of the ampicillin resistance gene (AmpR).

[0153] In another preferred embodiment, the label includes a resistance marker and / or a fluorescent marker.

[0154] In another preferred embodiment, the A9 is a kanamycin resistance gene (kanR).

[0155] In another preferred embodiment, the promoter element i and promoter element ii may be the same or different promoter elements.

[0156] In another preferred example, when n>1, in one group (A2-A3-A4-A5-A6)n, compared with other groups (A2-A3-A4-A5-A6), the respective A2, A3, A4, A5, and A6 may be the same or different.

[0157] In another preferred embodiment, the exogenous expression cassette is the expression cassette described in the second aspect of the present invention.

[0158] In another preferred example, the exogenous expression cassette comprises the sequence shown in SEQ ID NO: 15.

[0159] In an eighth aspect of the present invention, a genetically engineered cell for producing the recombinant AAV vector according to the seventh aspect of the present invention is provided, wherein the genetically engineered cell contains:

[0160] (i) a first nucleic acid construct, wherein the first nucleic acid construct contains an exogenous target gene; and

[0161] (ii) a second nucleic acid construct comprising rep and cap genes, wherein the cap gene encodes the AAV capsid protein as described in the fourth aspect of the present invention.

[0162] In another preferred embodiment, the genetically engineered cells are eukaryotic cells.

[0163] In another preferred embodiment, the genetically engineered cells are selected from the group consisting of 293T cells, HEK293 cells, Sf9 cells, and BHK cells.

[0164] In another preferred embodiment, the genetically engineered cells further contain:

[0165] (iii) a third nucleic acid construct, wherein the third nucleic acid construct is a helper plasmid, which has the function of assisting virus packaging to form virus particles.

[0166] In another preferred embodiment, the helper plasmid is derived from adenovirus (Ad), herpes simplex virus (HSV), or other helper plasmids with helper functions.

[0167] In another preferred embodiment, the first nucleic acid construct may further contain an exogenous reporter gene.

[0168] In another preferred embodiment, the exogenous reporter gene is selected from the following group: GFP, hGH, SEAP, LacZ, CAT, luciferase, etc., or a combination thereof.

[0169] In another preferred embodiment, the first nucleic acid construct, the second nucleic acid construct and / or the third nucleic acid construct may be temporarily present in the genetically engineered cell, or may be stably integrated into the genome of the genetically engineered cell.

[0170] In another preferred embodiment, the recombinant AAV vector as described in the seventh aspect of the present invention can be packaged inside the genetically engineered cells.

[0171] In another preferred embodiment, the exogenous target gene is an adalimumab coding sequence.

[0172] In another preferred example, the first nucleic acid construct is expression cassette L93, expression cassette L5, expression cassette Y817 or expression cassette Y981, wherein the expression cassette L93 has the nucleotide sequence shown in SEQ ID NO: 12; the expression cassette L5 has the nucleotide sequence shown in SEQ ID NO: 13; the expression cassette Y817 has the nucleotide sequence shown in SEQ ID NO: 14; and the expression cassette Y981 has the nucleotide sequence shown in SEQ ID NO: 24.

[0173] In another preferred embodiment, the rep gene in the second nucleic acid construct is derived from an optional AAV serotype.

[0174] In another preferred embodiment, the rep gene in the second nucleic acid construct is the rep2 gene, ie, the rep gene derived from AAV2.

[0175] In another preferred embodiment, the second nucleic acid construct contains the nucleic acid molecule as described in the fifth aspect of the present invention.

[0176] In the ninth aspect of the present invention, a host cell is provided, which contains the recombinant AAV vector as described in the seventh aspect of the present invention, or an exogenous expression cassette as described in the second aspect of the present invention or a polynucleotide as described in the first aspect of the present invention integrated into its chromosome.

[0177] In another preferred embodiment, the host cell is a mammalian cell, and the mammal includes human and non-human mammals.

[0178] In another preferred embodiment, the host cell is selected from the group consisting of HEK cells, rod cells, cone cells, bipolar cells, horizontal cells, ganglion cells, amacrine cells, interreticular cells, Muller cells, pigment epithelial cells, and ganglion cells.

[0179] In a tenth aspect of the present invention, there is provided a pharmaceutical composition comprising:

[0180] (i) the polynucleotide according to the first aspect of the present invention, the expression cassette according to the second aspect of the present invention, the recombinant AAV vector according to the seventh aspect of the present invention, or the host cell according to the ninth aspect of the present invention, as an active ingredient; and

[0181] (ii) a pharmaceutically acceptable carrier, diluent or excipient.

[0182] In another preferred embodiment, the component (i) accounts for 0.1-99.9 wt %, preferably 10-99.9 wt %, and more preferably 70-99 wt % of the total weight of the pharmaceutical composition.

[0183] In another preferred embodiment, the dosage form of the pharmaceutical composition is selected from the following group: lyophilized dosage form, liquid dosage form.

[0184] In another preferred embodiment, the pharmaceutical composition is in the form of an injection.

[0185] In another preferred embodiment, the administration site of the pharmaceutical composition includes periocular, intravitreal, suprachoroidal or subretinal.

[0186] In another preferred embodiment, the pharmaceutical composition is in the form of an injection for subretinal injection or intravitreal injection.

[0187] In another preferred embodiment, the pharmaceutically acceptable carrier includes but is not limited to: a solvent, a dispersion medium, a coating, an antibacterial or antifungal agent, an isotonic agent, and an absorption delaying agent.

[0188] In another preferred embodiment, the pharmaceutically acceptable carrier is an injection carrier. Preferably, the pharmaceutically acceptable carrier includes saline, and the saline includes but is not limited to: buffered saline, physiological saline, phosphate buffer, citrate buffer, acetate buffer, bicarbonate buffer, sucrose solution, saline solution, polysorbate solution, or a combination thereof.

[0189] In another preferred embodiment, the pharmaceutically acceptable carrier may further contain additives, including but not limited to: stabilizers, preservatives, transfection promoters that facilitate cell uptake, or combinations thereof.

[0190] In another preferred embodiment, the pharmaceutical composition can be used alone or in combination for the treatment of retinal diseases or autoimmune diseases.

[0191] In another preferred embodiment, the combined use includes: combined use with other drugs for treating retinal-related diseases or autoimmune diseases.

[0192] In another preferred embodiment, the other drugs for treating retinal diseases include: glucocorticoids (such as prednisone, dexamethasone, etc.), atropine, epinephrine, cyclophosphamide, chlorambucil, methotrexate, azathioprine, cyclosporine, or a combination thereof.

[0193] In another preferred embodiment, the other drugs for treating autoimmune diseases include: etanercept, infliximab, golimumab, and certolizumab pegol.

[0194] In the eleventh aspect of the present invention, there is provided a use of the recombinant AAV vector as described in the seventh aspect of the present invention for preparing a preparation or composition for treating retinal-related diseases.

[0195] In another preferred embodiment, the administration site of the preparation or composition includes periocular, intravitreal, suprachoroidal or subretinal.

[0196] In another preferred embodiment, the preparation or composition is administered by intravitreal injection.

[0197] In the twelfth aspect of the present invention, a method for treating retinal-related diseases is provided, the method comprising the steps of administering to a subject in need thereof an expression cassette as described in the second aspect of the present invention, a recombinant AAV vector as described in the seventh aspect of the present invention, a host cell as described in the ninth aspect of the present invention, or a pharmaceutical composition as described in the tenth aspect of the present invention.

[0198] In another preferred embodiment, the administration site includes periocular, intravitreal, suprachoroidal or subretinal.

[0199] In another preferred embodiment, the administration method is intravitreal injection.

[0200] In another preferred embodiment, the subject in need includes humans and non-human mammals.

[0201] In another preferred embodiment, in the recombinant AAV vector, the dosage of AAV-Y659 is 2E+9-1E+13 vg, preferably 5E+10-2E+12 vg, and more preferably 1E+11-2E+11 vg.

[0202] In another preferred embodiment, in the recombinant AAV vector, the dosage of AAV-Y660 is 2E+9-1E+13 vg, preferably 5E+10-2E+12 vg, and more preferably 1E+11-2E+11 vg.

[0203] In the thirteenth aspect of the present invention, a method for transfecting the retina in vitro is provided, comprising the steps of: transfecting the retina with the recombinant AAV vector described in the seventh aspect of the present invention.

[0204] In the fourteenth aspect of the present invention, a chimeric antigen receptor CAR is provided, wherein the heavy chain coding sequence of the antigen binding region scFv of the CAR is shown in SEQ ID NO: 5; the light chain coding sequence of the scFv is shown in SEQ ID NO: 10.

[0205] In the fifteenth aspect of the present invention, an engineered immune cell is provided, wherein the engineered immune cell expresses an exogenous CAR as described in the fourteenth aspect of the present invention.

[0206] In another preferred embodiment, the engineered immune cells are selected from the following group:

[0207] (i) Chimeric antigen receptor αβ T cells (CAR-T cells);

[0208] (ii) chimeric antigen receptor γδ T cells (CAR-T cells);

[0209] (iii) chimeric antigen receptor NKT cells (CAR-NKT cells);

[0210] (iv) Chimeric antigen receptor NK cells (CAR-NK cells).

[0211] In another preferred embodiment, the engineered immune cells include autologous or allogeneic αβT cells, γδT cells, NKT cells, NK cells, or a combination thereof.

[0212] In another preferred embodiment, the engineered immune cells are CAR-T cells.

[0213] In a sixteenth aspect of the present invention, a method for producing adalimumab or an antigen-binding fragment thereof is provided, comprising the steps of:

[0214] (a) culturing the host cell according to the ninth aspect of the present invention under suitable conditions to obtain a culture containing adalimumab or an antigen-binding fragment thereof;

[0215] (b) isolating and / or recovering the adalimumab or antigen-binding fragment thereof from the culture; and

[0216] (c) Optionally, purifying and / or modifying the adalimumab or antigen-binding fragment thereof obtained in step (b).

[0217] In the seventeenth aspect of the present invention, an immunoconjugate is provided, comprising:

[0218] (a) an antibody portion, which is encoded by a polynucleotide as described in the first aspect of the invention; and (b) a conjugate portion selected from the group consisting of a detectable label, a drug, an enzyme, a gold nanoparticle or nanorod, a nanomagnetic particle, a viral coat protein or a VLP, or a combination thereof.

[0219] In another preferred embodiment, the (a) part and the coupling part are coupled via a chemical bond or a linker.

[0220] In another preferred embodiment, the coupling portion is selected from the following group: fluorescent or luminescent markers, radioactive markers, MRI (magnetic resonance imaging) or CT (computer tomography) contrast agents, or enzymes capable of producing detectable products, radionuclides, biotoxins, cytokines (such as IL-2, etc.), antibodies, antibody Fc fragments, antibody scFv fragments, gold nanoparticles / nanorods, viral particles, liposomes, nanomagnetic particles, prodrug-activating enzymes (for example, DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)) or any form of nanoparticles.

[0221] In another preferred embodiment, the radioactive nuclides include:

[0222] (i) a diagnostic isotope selected from the group consisting of Tc-99m, Ga-68, F-18, I-123, I-125, I-131, In-111, Ga-67, Cu-64, Zr-89, C-11, Lu-177, Re-188, or a combination thereof; and / or

[0223] (ii) therapeutic isotopes, wherein the therapeutic isotopes are selected from the group consisting of Lu-177, Y-90, Ac-225, As-211, Bi-212, Bi-213, Cs-137, Cr-51, Co-60, Dy-165, Er-169, Fm-255, Au-198, Ho-166, I-125, I-131, Ir-192, Fe-59, Pb-212, Mo-99, Pd-103, P-32, K-42, Re-186, Re-188, Sm-153, Ra223, Ru-106, Na24, Sr89, Tb-149, Th-227, Xe-133, Yb-169, Yb-177, or a combination thereof.

[0224] In another preferred embodiment, the drug is a drug for the targeted treatment of retinal-related diseases or autoimmune diseases.

[0225] In another preferred embodiment, the immunoconjugate contains: a multivalent (eg, bivalent) antibody portion.

[0226] In the eighteenth aspect of the present invention, a method for preventing and / or treating an autoimmune disease is provided, the method comprising administering to a subject in need thereof an antibody encoded by the polynucleotide as described in the first aspect of the present invention, an expression cassette as described in the second aspect of the present invention, a host cell as described in the ninth aspect of the present invention, an engineered immune cell as described in the fifteenth aspect of the present invention, or an immunoconjugate as described in the seventeenth aspect of the present invention, or a combination thereof.

[0227] In another preferred embodiment, the subject includes mammals, such as humans.

[0228] In another preferred embodiment, the engineered immune cells or the CAR immune cells contained in the active ingredient combination are cells derived from the subject (autologous cells).

[0229] In another preferred embodiment, the engineered immune cells or CAR immune cells contained in the active ingredient combination are cells derived from healthy individuals (allogeneic cells).

[0230] In another preferred embodiment, the method can be used in combination with other treatment methods.

[0231] In a nineteenth aspect of the present invention, a method for diagnosing an autoimmune disease is provided, comprising the steps of:

[0232] (i) obtaining a sample from a diagnostic subject, and contacting the sample with the antibody encoded by the polynucleotide according to the first aspect of the present invention, or the immunoconjugate according to the seventeenth aspect of the present invention, or a combination thereof; and

[0233] (ii) detecting whether an antigen-antibody complex is formed, wherein the formation of the complex indicates that the subject is a confirmed patient of an autoimmune disease.

[0234] In another preferred embodiment, the sample is a blood sample or a throat swab sample, or a sample from other tissues and organs.

[0235] In a twentieth aspect of the present invention, a method for increasing the titer of AAV virus particles is provided, the method comprising the steps of:

[0236] (i) providing the expression cassette as described in the second aspect of the present invention as the expression cassette to be modified, and changing the sequential connection order of the light chain regulatory element, the light chain coding sequence and the poly(A) signal sequence in the expression cassette to be modified to the reversed connection order of the poly(A) signal sequence, the light chain coding sequence, and the light chain regulatory element, thereby obtaining a new expression cassette with reversed element order; and

[0237] (ii) The new expression cassette obtained in (i) is packaged into an AAV vector and expressed, thereby obtaining AAV virus particles with increased titer.

[0238] In another preferred embodiment, the expression cassette to be modified is expression cassette L93, having the nucleotide sequence shown in SEQ ID NO:12.

[0239] In another preferred embodiment, the new expression cassette with the elements sequence reversed is expression cassette Y981, having the nucleotide sequence shown in SEQ ID NO:24.

[0240] In another preferred embodiment, the expression cassette to be modified has a structure shown in formula A from the 5'-3' end, and the new expression cassette with the elements reversed in sequence has a structure shown in formula B from the 5'-3' end: X0-X1-X2-X3-X4-X5 (A) X0-X1-X2-X5-X4-X3 (B)

[0241] In the formula, each "-" is independently a bond or a nucleotide linking sequence;

[0242] X0 is the heavy chain regulatory element;

[0243] X1 is the heavy chain coding sequence SEQ ID NO: 5;

[0244] X2 is poly(A) signal sequence 1;

[0245] X3 is the light chain regulatory element;

[0246] X4 is the light chain coding sequence SEQ ID NO: 10; and

[0247] X5 is the poly(A) signal sequence 2.

[0248] In the twenty-first aspect of the present invention, a packaging plasmid is provided, comprising: the nucleic acid molecule according to the fifth aspect of the present invention and the rep gene.

[0249] In another preferred embodiment, the rep gene may be derived from any AAV serotype.

[0250] In a twenty-second aspect of the present invention, a plasmid system is provided, comprising:

[0251] (Z1) a first plasmid containing an exogenous gene of interest;

[0252] (Z2) a second plasmid, which is the packaging plasmid according to the twenty-first aspect of the present invention; and

[0253] (Z3) A third plasmid, which is a helper plasmid that assists virus packaging to form virus particles.

[0254] In another preferred embodiment, the helper plasmid is derived from adenovirus (Ad), herpes simplex virus (HSV), or other helper plasmids with helper functions.

[0255] In another preferred embodiment, the exogenous target gene is an adalimumab coding sequence.

[0256] In the twenty-third aspect of the present invention, a kit is provided, comprising: a first plasmid, a second plasmid and a third plasmid in the plasmid system as described in the twenty-second aspect of the present invention, and a first container for packaging the first plasmid, a second container for packaging the second plasmid and a third container for packaging the third plasmid.

[0257] In another preferred embodiment, the kit further contains instructions, which describe a method for using the kit to prepare the recombinant AAV vector as described in the seventh aspect of the present invention, or the genetically engineered cell as described in the eighth aspect of the present invention.

[0258] In another preferred embodiment, any two of the first container, the second container, and the third container may be the same or different containers.

[0259] In the twenty-fourth aspect of the present invention, there is provided the use of the packaging plasmid as described in the twenty-first aspect of the present invention, the plasmid system as described in the twenty-second aspect of the present invention, or the kit as described in the twenty-third aspect of the present invention for preparing the recombinant AAV vector as described in the seventh aspect of the present invention, or the genetically engineered cell as described in the eighth aspect of the present invention.

[0260] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features described in detail 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 listed here one by one. BRIEF DESCRIPTION OF THE DRAWINGS

[0261] Figure 1 shows a map of expression cassettes L93, L5 and Y817.

[0262] Figure 2 shows immunoblotting of HEK293T cells infected with Y660-L5 / L93 / Y817 virus.

[0263] Figure 3 shows immunoblotting of mice injected intravitreally with Y606, Y609, Y659, Y660, and Y689-L5 viruses.

[0264] Figure 4 shows the luciferase activity detection of mice injected intravitreally with Y606, Y609, Y659, Y660, and Y689-Y770 viruses.

[0265] Figure 5 shows the staining of eye sections of mice injected intravitreally with Y606, Y609, Y659, Y660, and Y689-Y770 viruses.

[0266] FIG6 shows the luciferase activity detection of rats injected intravitreally with Y659, Y660-Y770 viruses.

[0267] FIG7 shows that in an in vitro experiment, the L93 expression cassette containing the optimized adalimumab coding sequence of the present invention has significantly better intracellular and extracellular Ada expression levels than other adalimumab coding sequences.

[0268] FIG8 shows that in an in vivo experiment, the L93 expression cassette comprising the optimized adalimumab coding sequence of the present invention has significantly better intracellular and extracellular Ada expression levels than other adalimumab coding sequences.

[0269] FIG9 shows that in an in vivo experiment, the Ada expression levels packaged by Y659 and Y660 of the present invention were significantly better than those of Y606 and Y609.

[0270] FIG10 shows that the binding ability of Ada expressed by using the AAV vector Y659 to encapsulate the L93 expression cassette to soluble TNF-α is significantly better than that of the standard.

[0271] FIG11 shows that the protective effect of Ada expressed by L93 expression cassette packaged by AAV vector Y659 on TNF-α-induced apoptosis of L-929 mouse fibroblasts is also better than that of the standard product.

[0272] FIG12 shows that the expression cassette Y981 after preparation process optimization has a better expression level of Ada both inside and outside the cells than that of the expression cassette L93.

[0273] Figure 13 shows a map of expression cassette Y981.

[0274] [Corrected 01.03.2024 according to Regulation 26] Figure 14 shows the efficacy of viruses obtained by intravitreal administration of AAV vector Y659, packaging expression cassettes Y981 and L93, and intraperitoneal injection of the positive drug adalimumab in a mouse EAU model. Clinical score difference = score 14 days after modeling minus score before modeling. Data are mean ± SEM. Twelve eyes, 6 animals per group; *, p ≤ 0.05; ***, p ≤ 0.001.

[0275] FIG15 shows fundus color photographs of the mice in the intraperitoneal injection of adalimumab, intravitreal injection of vehicle, Y981, and L93 groups 14 days after the establishment of the EAU model.

[0276] [Corrected 01 / 03 / 2024 in accordance with Rule 26] Figure 16 shows the expression of adalimumab in the aqueous humor and vitreous of cynomolgus monkeys 6 weeks after a single intravitreal injection of the Y981 and L93 expression cassettes packaged with the AAV vector Y659. Data are mean ± SD; six eyes were included in each group, including three animals.

[0277] [Corrected 01.03.2024 according to Rule 26] Figure 17 shows the efficacy of viruses obtained using AAV vector Y659 to package the expression cassette Y981 and a control GFP gene expression cassette in the EAU mouse model. Data are presented as mean ± SD; n = 8 for the AAV-GFP group and n = 14 for the AAV-Y981 group.

[0278] FIG18 shows fundus color photographs of the control group (AAV-GFP) and experimental group (AAV-Y981) animals 17 days after the establishment of the mouse EAU model. DETAILED DESCRIPTION

[0279] After extensive and in-depth research and extensive screening, the inventors have developed, for the first time, a recombinant AAV vector capable of efficiently infecting the retina and a high-expression adalimumab coding sequence. On the one hand, the recombinant AAV vector and adalimumab coding sequence of the present invention can be applied in the prevention and / or treatment of retinal diseases. Due to their high expression efficiency, only a relatively small dosage is required to achieve the desired effect, which helps reduce industrial production costs and time costs, thereby reducing medication costs and benefiting patients. On the other hand, the adalimumab coding sequence of the present invention, due to its high expression level, also offers significant advantages in areas such as autoimmune diseases. This is the basis for the completion of the present invention.

[0280] the term

[0281] In order that the present disclosure may be more readily understood, certain terms are first defined. As used in this application, unless otherwise expressly provided herein, each of the following terms shall have the meaning given below.

[0282] As used herein, when used in reference to a specific recited value, the term "about" means that the value may vary by no more than 1% from the recited value. For example, as used herein, the expression "about 100" includes all values ​​between 99 and 101 (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0283] As used herein, the terms "comprising" or "including" may be open, semi-closed, or closed. In other words, the terms also include "consisting essentially of" or "consisting of."

[0284] As used herein, the term "treatment" refers to administering to a patient an internal or external therapeutic agent, comprising a recombinant AAV vector or expression cassette provided herein, and a pharmaceutical composition thereof. The patient has one or more symptoms of a disease for which the therapeutic agent is known to have a therapeutic effect. Typically, the therapeutic agent is administered to the patient in an amount effective to alleviate one or more symptoms of the disease (a therapeutically effective amount).

[0285] As used herein, the terms "optional" or "optionally" mean that the subsequently described event or circumstance may occur but need not.

[0286] Sequence identity is determined by comparing two aligned sequences along a predetermined comparison window (which can be 50%, 60%, 70%, 80%, 90%, 95% or 100% of the length of the reference nucleotide sequence or protein) and determining the number of positions at which identical residues occur. Typically, this is expressed as a percentage. The measurement of sequence identity of nucleotide sequences is a method well known to those skilled in the art.

[0287] As used herein, the term "subject in need thereof" refers to any mammal or non-mammal. Mammals include, but are not limited to, humans, vertebrates such as rodents, non-human primates, cows, horses, dogs, cats, pigs, sheep, and goats.

[0288] As used herein, the terms "AAV vector," "recombinant AAV vector," "recombinant adeno-associated virus vector," "rAAV," "recombinant AAV virus particle," "recombinant virus particle," and "virus particle" are used interchangeably to refer to modified AAV virus particles that can be used for transport, transduction, and specific expression of the contained exogenous gene of interest at a targeted site, preferably the targeted site being the retina.

[0289] Expression cassette

[0290] The present invention provides an expression cassette comprising the polynucleotide according to the first aspect of the present invention. The expression cassette of the present invention further comprises one or more promoters operably linked to the polynucleotide or a fragment thereof, an enhancer, an intron, a transcription termination signal, a polyadenylation sequence, an origin of replication, a selectable marker, a nucleic acid restriction site, and / or a homologous recombination site.

[0291] The expression cassette can be a plasmid vector or a viral vector. Preferably, the expression cassette of the present invention is selected from the following group: expression cassette L93, expression cassette L5, expression cassette Y817, wherein the expression cassette L93 has the nucleotide sequence shown in SEQ ID NO: 12; the expression cassette L5 has the nucleotide sequence shown in SEQ ID NO: 13; the expression cassette Y817 has the nucleotide sequence shown in SEQ ID NO: 14; the expression cassette Y981 has the nucleotide sequence shown in SEQ ID NO: 24. When the expression cassette of the present invention is a viral vector, preferably an AAV vector, a preferred embodiment is that the expression cassette of the present invention is an exogenous expression cassette carried or contained in the recombinant AAV vector as described in the seventh aspect of the present invention, or the recombinant AAV vector as a whole can also be regarded as the expression cassette of the present invention.

[0292] Preparations, compositions, pharmaceutical compositions

[0293] The present invention provides a preparation or composition (preferably a pharmaceutical composition), which contains (a) the polynucleotide as described in the first aspect of the present invention, the expression cassette as described in the second aspect of the present invention, the recombinant AAV vector as described in the seventh aspect of the present invention, or the host cell as described in the ninth aspect of the present invention as an active ingredient; and (b) a pharmaceutically acceptable carrier, diluent or excipient.

[0294] In another preferred embodiment, the preparation or composition is used to treat eye diseases. Preferably, the pharmaceutical composition is used to treat retinal diseases, and more preferably, to treat uveitis.

[0295] To facilitate clinical application, the preparation or composition of the present invention can be contained in an injectable device (such as an injection needle), which can contain a single dose of the pharmaceutical composition. The injectable device can be contained in a medicine box for convenient storage and use. During transportation, the small container containing the drug suspension needs to be placed on dry ice. It should be stored in a freezer at -80°C.

[0296] The preparation or composition of the present invention may also include instructions for use to facilitate those skilled in the art to use it in the correct manner.

[0297] The preparation or composition of the present invention can be administered in a safe and effective amount. "Safe and effective amount" means that the amount of the active ingredient is sufficient to significantly improve the condition or symptoms without causing serious side effects.

[0298] The safe and effective amount of the present invention may vary depending on the mode of administration and the severity of the disease to be treated. The preferred safe and effective amount can be determined by one of ordinary skill in the art based on various factors (e.g., through clinical trials). Such factors include, but are not limited to: pharmacokinetic parameters of the drug, such as drug tissue distribution, bioavailability, metabolism, half-life, etc.; the severity of the disease to be treated, the patient's weight, the patient's immune status, the route of administration, etc. For example, depending on the urgency of the treatment, several divided doses may be administered daily, or the dose may be reduced proportionally.

[0299] "Pharmaceutically acceptable carrier, diluent, or excipient" refers to one or more compatible solid or liquid fillers or gels suitable for human use and possessing sufficient purity and low toxicity. "Compatibility" as used herein refers to the ability of the components of a formulation or composition to intermix with the active ingredient of the present invention without significantly reducing the efficacy of the active ingredient.

[0300] The formulation or composition can be a liquid or solid, such as a powder, gel or paste. Preferably, the composition is a liquid, preferably an injectable liquid. Suitable excipients will be known to those skilled in the art.

[0301] The formulation or composition may comprise a physiologically acceptable sterile aqueous or anhydrous solution, dispersion, suspension or emulsion, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Suitable aqueous and non-aqueous carriers, diluents, solvents or excipients include water, ethanol, polyols and suitable mixtures thereof.

[0302] Preferably, in the present invention, the recombinant AAV vector can be administered to the eye subretinal or intravitreally. In either mode of administration, the recombinant AAV vector is preferably provided as an injectable liquid. Preferably, the injectable liquid is provided as a capsule or syringe.

[0303] Examples of pharmaceutically acceptable carriers include cellulose and its derivatives (such as sodium carboxymethyl cellulose, sodium ethyl cellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (such as stearic acid, magnesium stearate), calcium sulfate, vegetable oils (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (such as propylene glycol, glycerol, mannitol, sorbitol, etc.), emulsifiers (such as ), wetting agents (such as sodium lauryl sulfate), colorants, flavorings, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.

[0304] Adeno-associated virus

[0305] Because adeno-associated virus (AAV) is smaller than other viral vectors, has no pathogenicity, and can transfect both dividing and non-dividing cells, AAV-based gene therapy methods for the eye, especially hereditary retinal degenerative diseases, have received widespread attention.

[0306] Adeno-associated virus (AAV), also known as adeno-associated virus, belongs to the genus Dependinovirus in the family Parvoviridae. It is the simplest single-stranded DNA defective virus discovered to date and requires a helper virus (usually an adenovirus) for replication. AAV can infect a wide variety of cells and can stably integrate into the genome of the cells they infect in a site-specific manner. They can infect a wide range of cells without affecting cell growth, morphology, or differentiation, and do not appear to be involved in human pathology.

[0307] AAV contains an inverted terminal repeat (ITR) region of approximately 145 bases at each end, which serves as the origin of viral replication and is also crucial for viral packaging. The rest of the genome is divided into two important regions with encapsidation functions: the left portion of the genome, which contains the rep gene involved in viral replication and viral gene expression; and the right portion of the genome, which contains the cap gene encoding the viral capsid protein.

[0308] Recombinant adeno-associated virus (rAAV) vectors, derived from the non-pathogenic wild-type adeno-associated virus, are considered one of the most promising gene transfer vectors due to their safety, broad host cell range (dividing and non-dividing cells), low immunogenicity, and prolonged in vivo expression of foreign genes. They are widely used in gene therapy and vaccine research worldwide. After more than a decade of research, the biological properties of rAAV have been thoroughly understood, particularly with extensive data on their efficacy in various cell types, tissues, and in vivo experiments. In medical research, rAAV has been used in gene therapy studies for a variety of diseases (both in vitro and in vivo). As a unique gene transfer vector, it is also widely used in gene function studies, disease model development, and the generation of knockout mice.

[0309] AAV capsid protein determines that AAV has tissue cell specificity. Therefore, since AAV was proven to have clinical application prospects, designing new AAV capsids to obtain new features has always been the goal that researchers have been pursuing. With the advancement of technology, the strategy for developing new capsids has gradually evolved. At present, the capsid protein development strategy is mainly divided into rational design, directed evolution and computer-aided design. Improving the targeting and efficient transduction of target tissue cells is an important strategy to reduce toxic side effects in AAV gene therapy. In a preferred embodiment of the present invention, the capsid protein of the AAV vector has an amino acid sequence as shown in SEQ ID NO: 16, SEQ ID NO: 17 or SEQ ID NO: 18, or its amino acid sequence has at least 95% (preferably at least 98%) identity with any of the sequences shown in SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 18. Preferably, the capsid protein of the AAV vector of the present invention is encoded by a nucleic acid molecule as described in the fifth aspect of the present invention.

[0310] AAV vectors can be prepared using standard methods in the art. Adeno-associated viruses of any serotype are suitable. Methods for purifying vectors can be found in, for example, U.S. Patent Nos. 6,566,118, 6,989,264, and 6,995,006, the disclosures of which are incorporated herein by reference in their entirety. The preparation of hybrid vectors is described, for example, in PCT Application No. PCT / US2005 / 027091, the disclosures of which are incorporated herein by reference in their entirety. The use of vectors derived from AAV for transporting genes in vitro and in vivo has been described (see, for example, International Patent Application Publication Nos. WO91 / 18088 and WO93 / 09239; U.S. Patent Nos. 4,797,368, 6,596,535, and 5,139,941, and European Patent No. 0488528, all of which are incorporated herein by reference in their entirety). These patent publications describe various AAV-derived constructs in which the rep and / or cap genes are deleted and replaced with a gene of interest, and the use of these constructs to deliver genes of interest in vitro (into cultured cells) or in vivo (directly into an organism).

[0311] In a specific embodiment, the production of AAV vectors requires a DNA plasmid comprising ITR-L, a recombinant target genome, and ITR-R, wherein ITR-L and ITR-R are located on either side of the recombinant genome. The above-mentioned DNA plasmid, a plasmid encoding the AAV cap / rep gene (e.g., the packaging plasmid described in aspect 21 of the present invention), and auxiliary genes provided by adenovirus or herpes virus can be simultaneously introduced into suitable host cells using known techniques, such as by transfection, to produce AAV viral vectors. The DNA plasmid can be expressed in the host cell and packaged into viral particles.

[0312] Preferably, the recombinant AAV vector of the present invention is a high-affinity retinal vector. High retinal affinity means that when the AAV vector is administered to a subject in need, the affinity of the recombinant AAV vector of the present invention to the retinal tissue or cells of the subject is 50%, preferably 80%, and optimally 100% higher than that of other AAV vectors. Specifically, high retinal affinity is reflected in two aspects: (1) the recombinant AAV vector of the present invention can express higher amounts of multiple target proteins in retinal tissues or cells, including but not limited to adalimumab; (2) the recombinant AAV vector of the present invention can infect more types of retinal cells, and the high affinity is also reflected in the increase in the number of infected cells.

[0313] Treatment

[0314] The present invention provides a method for treating retinal diseases, which comprises administering to a subject in need thereof an expression cassette as described in the second aspect of the present invention, a recombinant AAV vector as described in the seventh aspect of the present invention, a host cell as described in the ninth aspect of the present invention, or a pharmaceutical composition as described in the tenth aspect of the present invention.

[0315] The present invention also provides a method for preventing and / or treating an autoimmune disease, the method comprising administering to a subject in need thereof an antibody encoded by the polynucleotide of the first aspect of the invention, the expression cassette of the second aspect of the invention, the host cell of the ninth aspect of the invention, the engineered immune cell of the fifteenth aspect of the invention, or the immunoconjugate of the seventeenth aspect of the invention, or a combination thereof.

[0316] The expression cassette as described in the second aspect of the present invention or the recombinant AAV vector as described in the seventh aspect of the present invention may contain a gene encoding a target gene, such as an adalimumab encoding gene.

[0317] The pharmaceutical composition of the present invention can be administered alone or in combination with other therapeutic drugs (eg, formulated in the same pharmaceutical composition).

[0318] As used herein, treating a disease means administering a polynucleotide or recombinant AAV vector as described herein to ameliorate or alleviate one or more symptoms of the disease.

[0319] Preferably, in the therapeutic method of the present invention, the administration of the expression cassette or the recombinant AAV vector is local administration and a single injection.

[0320] Preferably, in the therapeutic method of the present invention, the administration site of the expression cassette or the recombinant AAV vector includes but is not limited to the periocular, intravitreal, suprachoroidal or subretinal.

[0321] The method of the present invention comprises introducing a nucleic acid sequence encoding a gene of interest (therapeutic gene) into the vitreous cavity of the eye. Preferably, the method comprises contacting cells with a vector (preferably a virus, more preferably an adeno-associated virus) comprising the vector encoding the therapeutic gene. Preferably, the cells are retinal cells, preferably rods, cones, bipolar cells, horizontal cells, ganglion cells, amacrine cells, interstitial cells, Muller cells, pigment epithelial cells, and / or ganglion cells.

[0322] Compared with the existing intravenous injection treatment method (1 time / 4 weeks, systemic administration), the treatment method of the present invention has obvious advantages such as one-time administration can provide lifelong benefits, is more targeted, and has fewer side effects.

[0323] The main advantages of the present invention include:

[0324] 1. Using the recombinant AAV vector of the present invention, efficient and sustained expression of the target gene in the retina can be achieved.

[0325] 2. The recombinant AAV vector of the present invention is administered topically, requiring only a single injection. Compared with existing treatments, it can effectively reduce side effects and improve patient compliance.

[0326] 3. The recombinant AAV vector of the present invention has long-term expression capability, thereby achieving the effect of lifelong benefits after a single administration.

[0327] 4. The adalimumab coding sequence of the present invention has a high expression level, which is conducive to reducing industrial production costs and time costs, thereby reducing medication costs and benefiting patients.

[0328] 5. The adalimumab of the present invention can be used to prevent and / or treat autoimmune diseases, changing the drawback of the existing treatment regimen of "one disease, one drug" and achieving the effect of "one drug for multiple uses".

[0329] 6. The present invention also optimizes the AAV preparation process by switching the connection order of some elements in the genome packaged in the capsid protein, ultimately increasing the titer of AAV virus particles.

[0330] 7. The adalimumab of the present invention can be used to treat autoimmune uveitis and can also prolong the early intervention or early treatment window period of the disease, which is beneficial to improving the prognosis.

[0331] 8. Adalimumab (Ada) expressed by the adalimumab coding sequence of the present invention has high secretion expression and good activity.

[0332] 9. The recombinant AAV vector of the present invention can efficiently infect ocular cells, thereby synthesizing and secreting high expression levels of target proteins, such as adalimumab, in the eye.

[0333] 10. The specific expression cassette designed in the present invention can significantly increase the expression level of the target gene (e.g., the coding sequence of adalimumab) in cells and outside cells, and when the recombinant AAV vector of the present invention is used to package and express the above-mentioned specific expression cassette, it can also significantly increase the expression level of the target gene (e.g., the coding sequence of adalimumab) in the vitreous body and aqueous humor.

[0334] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. The experimental methods in the following examples, for which specific conditions are not 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 according to the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are by weight.

[0335] Sequence information

[0336] CMV (human cytomegalovirus early) enhancer sequence (SEQ ID NO: 1):

[0337] CMV (human cytomegalovirus early) promoter sequence (SEQ ID NO: 2):

[0338] CBA (chicken β-actin) promoter sequence (SEQ ID NO: 3):

[0339] SV40 intron sequence (SEQ ID NO: 4):

[0340] Ada-HC codon optimized sequence (SEQ ID NO: 5):

[0341] bGH poly(A) (bovine growth hormone polyadenylation) signal peptide (SEQ ID NO: 6):

[0342] EF-1α (human elongation factor EF-1α strong constitutive) promoter sequence 1 (SEQ ID NO: 7):

[0343] EF-1α (human elongation factor EF-1α strong constitutive) promoter sequence 2 (SEQ ID NO: 8):

[0344] TPL intron sequence (SEQ ID NO: 9):

[0345] Ada-LC codon optimized sequence (SEQ ID NO: 10):

[0346] SV40 poly(A) (SV40 polyadenylation) signal peptide (SEQ ID NO: 11):

[0347] Nucleotide sequence of expression cassette L93 (SEQ ID NO: 12):

[0348] Nucleotide sequence of expression cassette L5 (SEQ ID NO: 13):

[0349] Nucleotide sequence of expression cassette Y817 (SEQ ID NO: 14):

[0350] Nucleotide sequence of expression cassette Y770 (SEQ ID NO: 15):

[0351] AAV-Y659 capsid protein amino acid sequence (SEQ ID NO: 16):

[0352] AAV-Y660 capsid protein amino acid sequence (SEQ ID NO: 17):

[0353] AAV-Y689 capsid protein amino acid sequence (SEQ ID NO: 18):

[0354] ITR-L sequence (SEQ ID NO: 19):

[0355] ITR-R sequence (SEQ ID NO: 20):

[0356] AAV-Y659 cap gene sequence (SEQ ID NO: 21):

[0357] Cap gene sequence of AAV-Y660 (SEQ ID NO: 22):

[0358] AAV-Y689 cap gene sequence (SEQ ID NO: 23):

[0359] Nucleotide sequence of expression cassette Y981 (SEQ ID NO: 24)

[0360] Materials, reagents, and consumables

[0361] Unless otherwise specified, all materials and reagents used can be obtained from commercial sources:

[0362] Example 1 Construction of expression cassettes L93, L5 and Y817

[0363] The adalimumab heavy chain (SEQ ID NO: 5), light chain (SEQ ID NO: 10) and various regulatory elements were assembled with the ITR-containing pUC vector backbone using seamless cloning technology according to the kit instructions, as shown in Figure 1, to construct expression cassettes L93, L5 and Y817, respectively.

[0364] Example 2: Using AAV vector Y660 to package different expression cassettes and detecting their Ada expression levels

[0365] The plasmid containing the AAV rep2 and cap sequences corresponding to Y660 (SEQ ID NO: 22), the adenovirus Helper plasmid containing the adenovirus helper gene, and the three expression cassettes L93 / L5 / Y817 constructed in Example 1 were transiently transfected into HEK293T cells to construct an in vitro expression system for recombinant AAV virus particles.

[0366] The obtained recombinant viral particles were used to infect HEK293T cells at a multiplicity of infection of 1E+5. After 72 hours, the cells were harvested and protein was extracted using a total cell protein extraction kit. Western blotting was performed to detect Ada expression. Specifically, after obtaining the protein sample, 30 μl was added to 10 μl of 4× SDS loading buffer (SDS buffer without the reducing agent DTT). After mixing, 15 μl of the sample was taken for Western blotting.

[0367] As shown in Figure 2, no Ada target band was detected in the blank control sample (BL), while Ada target bands were detected in the L93 / L5 / Y817 group. The protein migrated at the correct molecular weight of approximately 130 KD, as indicated by the arrow in the figure.

[0368] The above results showed that Ada expression could be detected in all three expression cassettes, among which L93 had the highest expression level.

[0369] Example 3: Using different AAV vectors to package the expression cassette L5 and detecting its Ada expression level

[0370] The three plasmid transient transfection methods in Example 2 were used to package the expression cassette L5 encoding adalimumab using AAV vectors Y606, Y609, Y659, Y660, and Y689, respectively, and recombinant AAV virus particles were obtained in HEK293T cells, wherein the AAV rep gene corresponding to Y659 is rep2, and the sequence of the AAV cap gene is shown in SEQ ID NO: 21; the AAV rep gene corresponding to Y689 is rep2, and the sequence of the AAV cap gene is shown in SEQ ID NO: 23.

[0371] The recombinant viral particles were administered intravitreally to 8-week-old C57BL / 6 mice (2E+9 vg / eye). Three weeks later, the mouse retinas were isolated and retinal proteins were extracted using a tissue protein extraction kit. Western blotting was performed to detect Ada expression. Specifically, 10 μl of 4× SDS loading buffer (without the reducing agent DTT) was added to 30 μl of protein sample, mixed thoroughly, and 15 μl of the sample was taken for Western blotting.

[0372] As shown in Figure 3 , obvious Ada target bands were detected in the Y606, Y609, Y659, and Y660 packaging L5 groups, as indicated by the arrows in the figure.

[0373] The above results showed that the Ada expression levels of Y659 and Y660 were significantly higher than those of Y606 and Y609, among which Y606 was wild-type AAV2 and Y609 was AAV2.7m8 vector.

[0374] Example 4: Packaging reporter gene Y770 using different AAV vectors and detecting its luciferase activity

[0375] The three plasmid transient transfection methods in Example 2 were used to package the luciferase-expressing reporter gene vector Y770 (which contains the expression cassette Y770 sequence as shown in SEQ ID NO: 15) using AAV vectors Y606, Y609, Y659, Y660, and Y689, respectively, and recombinant AAV virus particles were obtained in HEK293T cells.

[0376] The above recombinant virus particles were administered into the vitreous cavity of 8-week-old C57BL / 6 mice (the amount of virus used was 2E+9 vg / eye). Three weeks later, the mouse retinas were isolated and proteins were extracted. The luciferase activity was detected using a glow-type firefly luciferase reporter gene detection kit, and the protein concentration of the samples was detected using a BCA protein concentration assay kit. The luciferase activity of each group was normalized to per microgram of protein.

[0377] As shown in FIG4 , the results showed that the luciferase activity of Y659 packaged was significantly higher than that of Y606 and Y609, wherein Y606 was a wild-type AAV2 and Y609 was an AAV2.7m8 vector.

[0378] According to the expression results of the reporter genes, it is shown that the Y659 and Y660 recombinant AAV vectors of the present invention can efficiently express a variety of target proteins, not limited to Ada.

[0379] Example 5 Immunofluorescence staining of reporter gene Y770 using different AAV vectors

[0380] As described in Example 4, AAV vectors Y606, Y609, Y659, Y660, and Y689 were used to package the reporter gene Y770, respectively, and recombinant AAV virus particles were obtained in HEK293T cells.

[0381] The above recombinant virus particles were administered into the vitreous cavity of 8-week-old C57BL / 6 mice (the amount of virus used was 2E+9 vg / eye). Three weeks later, the mouse eyes were peeled off, fixed with 4% paraformaldehyde, infiltrated with 30% sucrose solution, embedded in OCT, and frozen sections were performed for immunofluorescence staining.

[0382] As shown in Figure 5, the green fluorescence shown in the figure is the signal of the anti-GFP antibody. The results show that the fluorescence intensity of the reporter gene packaged by Y659 and Y660 is better than that of Y606 and Y609, where Y606 is wild-type AAV2 and Y609 is AAV2.7m8 vector.

[0383] Based on the above reporter gene expression results, it was further verified that the Y659 and Y660 recombinant AAV vectors of the present invention can efficiently express a variety of target proteins in ocular tissues, not limited to Ada.

[0384] Based on the above immunofluorescence staining method, the affinity of AAV vectors Y606, Y609, Y659, Y660, and Y689 to the retina was further evaluated according to the reporter gene fluorescence signal.

[0385] The results show that Y659 and Y660 of the present invention can infect more types and numbers of retinal cells and exhibit higher affinity to the retina.

[0386] Example 6: Using AAV vectors Y659 and Y660 to package reporter gene Y770 and detect its luciferase activity

[0387] As described in Example 4, AAV vectors Y659 and Y660 were used to package the reporter gene Y770, respectively, and recombinant AAV virus particles were obtained in HEK293T cells.

[0388] The two viral particles were administered intravitreally to 8-week-old SD rats (the dose was 2E+9 vg / μl, with each virus injected in two gradient volumes: 1 μl and 4 μl). Three weeks later, the rat retinas were isolated and protein extracted. Luciferase activity was measured using a glow-type firefly luciferase reporter gene assay kit. Luciferase activity was higher in the group receiving 4 μl of virus than in the group receiving 1 μl of virus.

[0389] As shown in FIG6 , the results showed that Y659 was superior to Y660, and the luciferase activity was dose-dependent.

[0390] Example 7: Using AAV vector Y659 or Y660 to package gene expression cassette L93 and other adalimumab coding sequences, the extracellular and intracellular expression levels of Ada were compared.

[0391] The three plasmid transient transfection methods in Example 2 were adopted, and the AAV vector Y659 was used to package the gene expression cassette L93 and other adalimumab coding sequences (i.e., the heavy chain sequence SEQ ID NO: 26 and the light chain sequence SEQ ID NO: 27 in the WO2022094106A1 patent. Except for the different adalimumab coding sequences, other regulatory elements were the same as the design of the L93 expression cassette), and recombinant AAV virus particles were obtained in HEK293T cells.

[0392] HEK293T cells were infected with the above viral particles and harvested 72 hours later. Protein was extracted using a total cell protein extraction kit, and the extracellular and intracellular expression of Ada was detected using Western blotting and an Ada ELISA kit.

[0393] Furthermore, the above virus particles were administered into the vitreous cavity of 8-week-old C57BL / 6 mice (the amount of virus used was 2E+9 vg / eye). Three weeks later, the mouse retinas were isolated, and retinal proteins were extracted using a tissue protein extraction kit. Protein immunoblotting experiments and Ada ELISA kits were used to detect the extracellular and intracellular expression levels of Ada.

[0394] As shown in Figure 7, in vitro experiments on HEK293T cells showed that the intracellular and extracellular Ada expression levels of the L93 expression cassette of the present invention were significantly better than those of other adalimumab coding sequences, wherein: in terms of extracellular expression, the expression level of the L93 expression cassette of the present invention was as high as 190 ng / ml, which was approximately 21 times the expression level of other adalimumab coding sequences; in terms of intracellular expression, the expression level of the L93 expression cassette of the present invention was as high as 6502 ng / ml, which was approximately 57 times the expression level of other adalimumab coding sequences.

[0395] As shown in Figure 8, in vivo experiments in mice further demonstrated that the intracellular and extracellular Ada expression levels of the L93 expression cassette of the present invention were also significantly superior to those of other adalimumab coding sequences. Specifically, in terms of extracellular expression, the expression level of the L93 expression cassette of the present invention could reach up to 443 ng / mg protein, which was approximately twice the expression level of other adalimumab coding sequences; and in terms of intracellular expression, the expression level of the L93 expression cassette of the present invention could reach up to 78 ng / mg protein, which was approximately twice the expression level of other adalimumab coding sequences.

[0396] The above results show that when using the same regulatory elements and the same AAV vector, the expression level of the L93 expression cassette of the present invention is much higher than that of the expression cassette containing other adalimumab coding sequences. The fundamental reason is that the adalimumab coding sequence of the present invention has the superior property of significantly increasing the expression level.

[0397] Example 8: Using different AAV vectors to package the expression cassette L93 and detecting the Ada content and activity secreted into the extracellular space

[0398] The three plasmid transient transfection methods in Example 2 were used to package the expression cassette L93 encoding adalimumab using AAV vectors Y606, Y609, Y659, and Y660, respectively, and recombinant AAV virus particles were obtained in HEK293T cells.

[0399] HEK293T cells were infected with the above viral particles. 72 hours later, the cells and cell culture medium were collected. Protein was extracted using a total cell protein extraction kit and immunoblotted to determine Ada expression. Ada ELISA kit was used to measure Ada content and activity in the cell culture medium. Two methods were used to detect Ada activity: a) binding activity, using an ELISA assay to measure the binding ability of Ada in the culture medium to soluble TNF-α; and b) cell viability. TNF-α can induce apoptosis in L-929 mouse fibroblasts. Adalimumab specifically binds to TNF-α, blocking its apoptotic effect on target cells and neutralizing its cytotoxicity to L-929 cells. The apoptosis-inhibiting bioactivity of the test sample was calculated using a TNF-α cytotoxicity neutralization assay by comparing the EC50 values ​​of the standard sample with those of the test sample.

[0400] Furthermore, the above virus particles were administered into the vitreous cavity of 8-week-old SD rats (the amount of virus used was 2E+9 vg / eye). After 6 weeks, the aqueous humor of the rats was collected, and the rat retinas were separated. Retinal proteins were extracted using a tissue protein extraction kit, and protein immunoblotting experiments were performed to detect the expression level of Ada. The Ada ELISA kit was also used to detect the Ada content and activity in the aqueous humor of each virus-administered group.

[0401] As shown in FIG9 , in vivo experiments in rats showed that the Ada expression levels of Y659 and Y660 packaged in the present invention were significantly better than those of Y606 and Y609, wherein Y606 is a wild-type AAV2 and Y609 is an AAV2.7m8 vector.

[0402] As shown in FIG10 , the binding ability of Ada expressed by the L93 expression cassette packaged using the AAV vector Y659 to soluble TNF-α was significantly better than that of the standard, and the reference standard used was adalimumab injection (Humira).

[0403] As shown in Figure 11, Ada expressed by the L93 expression cassette packaged using the AAV vector Y659 also has a better protective effect on TNF-α-induced apoptosis of L-929 mouse fibroblasts than the standard product, and the reference standard used is adalimumab injection (Humira).

[0404] Example 9: Dose-effect experiment using AAV vector Y659 or Y660 to package gene expression cassette L93

[0405] The three plasmid transient transfection methods in Example 2 were used to package the expression cassette L93 encoding adalimumab using AAV vector Y659 or Y660, respectively, and recombinant AAV virus particles were obtained in HEK293T cells.

[0406] The above viral particles were administered intravitreally to 8-week-old SD rats (using three injection gradients: 1E+9 vg / eye, 2E+10 vg / eye, and 5E+11 vg / eye). Six weeks later, the rats' aqueous humor was collected, and the retinas were isolated. Retinal proteins were extracted using a tissue protein extraction kit and immunoblotted to measure Ada expression. Ada ELISA kit was also used to measure Ada content and activity in the aqueous humor of each virus-treated group.

[0407] The results showed that the expression level and activity of Ada were dose-dependent.

[0408] Example 10: Efficacy experiment using AAV vector Y659 to package gene expression cassette L93 and control group gene expression cassette

[0409] The three plasmid transient transfection methods in Example 2 were used to package the expression cassette L93 encoding adalimumab, other vectors encoding adalimumab sequences, and a control vector expressing GFP, respectively, using the AAV vector Y659, and recombinant AAV virus particles were obtained in HEK293T cells.

[0410] The above viral particles were administered intravitreally to 8-week-old C57BL / 6 mice (using a viral dose of 2E+9 vg / eye). Three weeks later, ophthalmoscopic examination of both eyes revealed no abnormalities, and an experimental autoimmune uveitis (EAU) model was established in mice. The protective effects of the experimental and control viruses in EAU mice were evaluated. The day of model establishment was designated Day 0.

[0411] Fundus color photographs were taken with a fundus camera on D0, D11, D14, D25, D39, and D53 after modeling, and clinical scores (0-4 points) were calculated based on the images. The scoring criteria are shown in Table 1. Before each examination, the animals were anesthetized with Zotai (25-50 mg / kg, ip) and xylazine hydrochloride (5 mg / kg, ip) injections.

[0412] Table 1

[0413] At the end of the in vivo experiment (Day 53), animals were euthanized by cervical dislocation after CO2 inhalation. Intact eyeballs were removed, fixed, paraffin-embedded, and stained with H&E. Central sections were selected for H&E staining. Histological changes in the eyeballs were observed under a light microscope and scored according to histopathology, as shown in Table 2.

[0414] Table 2

[0415] The TNF-α content in the eyes of mice in the experimental group and the control group was detected at three time points: D14, D25 and D53 after modeling.

[0416] The results showed that the experimental group injected with Y659-encapsulated L93 virus encoding the expression cassette of adalimumab had a significant protective effect on mice with experimental autoimmune uveitis (EAU), and the protective effect was improved compared with the group injected with Y659-encapsulated virus encoding other adalimumab sequences.

[0417] Example 11 Exploring the protective effect of the adalimumab coding sequence of the present invention on other autoimmune disease animal models

[0418] In this example, the inventors further verified the protective effect of the adalimumab coding sequence of the present invention on other autoimmune disease animal models.

[0419] The results show that administration of an AAV vector packaging the virus encoding the adalimumab sequence of the present invention can effectively protect one or more autoimmune disease animal models (e.g., rheumatoid arthritis, ankylosing spondylitis, psoriatic arthritis, plaque psoriasis, ulcerative colitis, Crohn's disease).

[0420] Example 12 Preparation process optimization

[0421] To further increase the titer of recombinant AAV virus particles, the inventors optimized the preparation process:

[0422] For example, process optimization was performed by changing the orientation of the light chain regulatory elements and CDS sequences in expression cassette L93. Specifically, the structure of expression cassette L93, as shown in Formula A, was changed to the structure of expression cassette B. More specifically, the modified expression cassette had the following pattern: Z0-Z1-Z2-Z3-Z4-Z5-Z10-Z9-Z8-Z7-Z6-Z11-Z12-Z13 (referring to the changes made to the structure of Formula I). ​​The modified expression cassette was named expression cassette Y981, and its nucleotide sequence is shown in SEQ ID NO: 24.

[0423] As shown in Figure 12, in vitro experiments on HEK293T cells showed that after the preparation process was optimized, the expression level of Ada in the expression cassette Y981 was better than that of the expression cassette L93 both inside and outside the cells.

[0424] As shown in FIG13 , a map of the above-mentioned expression cassette Y981 is shown.

[0425] Example 13: Comparative Efficacy Experiment Using AAV Vector Y659 to Package Gene Expression Cassette Y981 and L93 Gene Expression Cassette

[0426] In this example, the method of Example 10 was basically used to conduct the comparative efficacy experiment, except that a different expression cassette was used and the subsequent administration of the virus was changed to 1E+9 vg / eye. Specifically:

[0427] The three plasmid transient transfection methods in Example 2 were used to package the expression cassettes Y981 and L93 encoding adalimumab using the AAV vector Y659, and recombinant AAV virus particles were obtained in HEK293T cells.

[0428] The above viral particles (1E+9 vg / eye) and vehicle were administered intravitreally to 8-week-old C57BL / 6 mice. Three weeks later, fundus imaging and examination of both eyes revealed no abnormalities, and an experimental autoimmune uveitis (EAU) model was established in mice. The positive control group received an intraperitoneal injection of adalimumab (3 mg / kg) 1 day prior to modeling, followed by administration every 3 days until the end of the experiment. The day of modeling was designated Day 0.

[0429] On the 14th day after modeling, fundus color photographs were taken again with a fundus camera, and clinical scores (0-4 points) were calculated based on the images. The scoring criteria are shown in Table 1. Before each examination, the animals were anesthetized with Zotai (25-50 mg / kg, ip) and xylazine hydrochloride (5 mg / kg, ip) injections.

[0430] The clinical scoring criteria are shown in Table 1.

[0431] As shown in Figures 14 and 15, compared to the vehicle group, intraperitoneal injection of the positive drug adalimumab, Y981, and L93 all produced protective effects on the mouse retina. In particular, viral vector-encapsulated Y981 significantly improved retinal damage in mice, with an effect superior to that of L93 and the positive drug adalimumab.

[0432] Example 14: Single intravitreal injection of AAV vector Y659 packaging gene expression cassettes Y981 and L93 in cynomolgus monkeys and detection of adalimumab expression in aqueous humor and vitreous

[0433] Six healthy male cynomolgus macaques aged 3-8 years were enrolled. Before dosing, 30-50 μl of aqueous humor and vitreous fluid were collected to determine baseline adalimumab levels. The animals were randomly divided into two groups of three each and received a single intravitreal injection of the viral vectors Y981 and L93 at a viral dose of 5E+11 vg / eye. The day of administration was designated Day 0. To alleviate local ocular inflammation, anti-inflammatory and immunosuppressive interventions were administered within one week after intravitreal administration: Diclofenac eye ointment twice daily; Dimethoprim eye drops and Dimethoprim eye ointment twice daily; and atropine eye ointment (Dishan) twice daily for one week after intravitreal administration. Prednisone 1 mg / kg was then administered intramuscularly once daily. Six weeks later, 30-50 μl of aqueous humor and vitreous fluid were collected. Adalimumab levels in the aqueous humor and vitreous were determined by ELISA before and six weeks after dosing.

[0434] As shown in Figure 16, adalimumab was detected in both the aqueous humor and vitreous of the animals six weeks after a single intravitreal injection of the Y981 and L93 viruses. The Y981 expression cassette mediated adalimumab expression in the aqueous humor at a level four times higher than that of the L93 expression cassette, and the Y981 expression cassette mediated adalimumab expression in the vitreous was also significantly higher than that of the L93 expression cassette. Similar to Example 13, these experimental results also demonstrated that the Y981 expression cassette was superior to the L93 expression cassette.

[0435] Example 15: Efficacy experiment using AAV vector Y659 to package gene expression cassette Y981 and control group GFP gene expression cassette

[0436] In this example, the method in Example 10 was basically used to conduct the drug efficacy comparison experiment, except that a different expression cassette was used. Specifically:

[0437] The three plasmid transient transfection methods in Example 2 were used to package the expression cassette Y981 encoding adalimumab and the control vector expressing GFP, respectively, using the AAV vector Y659, and recombinant AAV virus particles were obtained in HEK293T cells.

[0438] The above viral particles were administered intravitreally to 8-week-old C57BL / 6 mice (1E+8 vg / eye). Three weeks later, ophthalmoscopic examination revealed no abnormalities in both eyes, and an experimental autoimmune uveitis (EAU) model was established in mice. The protective effects of the experimental and control viruses in EAU mice were evaluated. The day of model establishment was designated Day 0.

[0439] Fundus color photographs were taken with a fundus camera on D0, D11, D14, D25, D39, and D53 after modeling, and clinical scores (0-4 points) were calculated based on the images. The scoring criteria are shown in Table 1. Before each examination, the animals were anesthetized with Zotai (25-50 mg / kg, ip) and xylazine hydrochloride (5 mg / kg, ip) injections.

[0440] The clinical scoring criteria are shown in Table 1.

[0441] As shown in Figure 17, ocular symptoms gradually intensified after modeling and peaked at 17 days. Animals treated with AAV-Y981 showed significantly improved ocular symptoms compared to the control group. Furthermore, the AAV-GFP mice in the control group already showed clinical symptoms at day 11, while the AAV-Y981 group showed almost no disease changes. This suggests that the AAV-Y981 group also delayed the onset of EAU in mice, thereby extending the window for early intervention or treatment of EAU and improving the prognosis of EAU.

[0442] As shown in Figure 18, fundus images were taken 17 days after modeling in both the control (AAV-GFP) and experimental (AAV-Y981) groups. These fundus images revealed multiple focal lesions and more than five linear retinal lesions in the control group, along with mild vasculitis and old lesions. In contrast, the experimental group had fewer focal and linear lesions (<5).

[0443] The results showed that the experimental group injected with Y659 virus encapsulating the expression cassette Y981 encoding adalimumab had a significant protective effect on mice with experimental autoimmune uveitis (EAU).

[0444] All documents mentioned in this application are incorporated herein by reference, just as if each document were incorporated herein by reference individually. It should also be understood that after reading the above teachings of the present invention, those skilled in the art may make various changes or modifications to the present invention, and that such equivalents also fall within the scope of the claims appended hereto.

Claims

1. A polynucleotide encoding adalimumab, characterized in that The polynucleotide comprises: (1) the heavy chain coding sequence shown in SEQ ID NO: 5 and the light chain coding sequence shown in SEQ ID NO: 10; or (2) A conservative derivative sequence obtained by adding, deleting, modifying and / or replacing at least one nucleotide on the basis of the heavy chain coding sequence or the light chain coding sequence in (1), without changing the amino acid sequence encoded thereby.

2. An expression cassette, characterized in that The expression cassette comprises the polynucleotide of claim 1.

3. Use of the polynucleotide according to claim 1 or the expression cassette according to claim 2 for preparing a preparation or composition, characterized in that: The preparation or composition is used to treat indications for which adalimumab is applicable.

4. An engineered AAV capsid protein, characterized in that The AAV capsid protein has an amino acid sequence selected from the group consisting of: (a) the amino acid sequence shown in SEQ ID NO: 16, SEQ ID NO: 17 or SEQ ID NO: 18; and (b) an amino acid sequence that is at least 95% (preferably at least 98%, most preferably at least 99%) identical to any one of SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO:

18.

5. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the AAV capsid protein according to claim 4.

6. A use of the AAV capsid protein according to claim 4, characterized in that: Used for: (i) preparing a medicament for preventing and / or treating a retina-related disease in a subject in need thereof; and / or (ii) delivering the target gene packaged in the AAV capsid protein to the retina of the subject for expression.

7. A recombinant AAV vector, characterized in that The recombinant AAV vector comprises: the AAV capsid protein according to claim 4, and an exogenous expression cassette encoding a target gene packaged in the AAV capsid protein.

8. A genetically engineered cell for producing the recombinant AAV vector according to claim 7, characterized in that: The genetically engineered cells contain: (i) a first nucleic acid construct, wherein the first nucleic acid construct contains an exogenous target gene; and (ii) a second nucleic acid construct comprising rep and cap genes, wherein the cap gene encodes the AAV capsid protein according to claim 4.

9. A host cell, characterized in that The host cell contains the recombinant AAV vector according to claim 7, or an exogenous expression cassette according to claim 2 or a polynucleotide according to claim 1 is integrated into its chromosome.

10. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises: (i) the polynucleotide of claim 1, the expression cassette of claim 2, the recombinant AAV vector of claim 7, or the host cell of claim 9; and (ii) a pharmaceutically acceptable carrier, diluent or excipient.

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