Vector comprising sequence encoding anti-TNF antibody and inflammation-inducing promoter

Abstract

The present invention provides a vector comprising a nucleotide sequence encoding an anti-TNF antibody or fragment thereof wherein the nucleotide sequence encoding the anti-TNF antibody or fragment thereof is operably linked to an inflammation inducible promoter.

Description

Field of the Invention

[0001] The present invention relates to a vector for preventing or treating inflammatory eye diseases. Background of the Invention

[0003] Chronic inflammation of the eye can lead to cumulative damage, ultimately leading to severe visual loss. Chronic non-infectious uveitis is a vision-threatening intraocular inflammation that accounts for 10% of blindness in working-age people and has a disproportionately large economic burden (see, for example, Joltikov, KA and Lobo-Chan, AM, 2021. Frontiers in Medicine, 8: 695904). Uveitis can include intraocular inflammation that affects the uvea and adjacent structures (such as the cornea, vitreous humor, retina, and optic nerve). Most commonly, uveitis is idiopathic, but can be associated with infection, malignancy, or underlying inflammatory conditions, such as spondyloarthritis, sarcoidosis, juvenile idiopathic arthritis (JIA), inflammatory bowel disease, rheumatoid arthritis, tubulointerstitial nephritis, and other autoinflammatory diseases (see, for example, Rosenbaum, JT, et al., 2019. Seminars in Arthritis and Rheumatism, 49 (3), pp. 438-445).

[0004] At present, the first-line treatment for non-infectious uveitis is corticosteroids, which can be applied superficially, periocularly, intraocularly or systemically. However, some problems are associated with this treatment option. Systemic administration of corticosteroids has many well-known side effects that can cause adverse events, and although topical administration of corticosteroids can reduce the required concentration, because the ocular concentration of the drug decreases over time, repeated injections are required. Due to the recurrence of the condition, patients may need to maintain continuous systemic corticosteroid treatment, which can cause many adverse effects (see, for example, Valenzuela, RA, et al., 2020. Frontiers in Pharmacology, 11: 655). Local treatment options, including intraocular steroid implants and intravitreal injections, do not significantly improve the clinical situation. Although recurrence is effectively reduced in milder diseases, these local treatment options are associated with significant adverse events (such as cataracts and glaucoma).

[0005] Immunosuppressant therapy (IMT) is an alternative to corticosteroid therapy, and immunosuppressant therapy includes antimetabolites, calcineurin inhibitors and alkylating agents. When conventional corticosteroids and IMT fail, biological agents and biologics, such as TNF inhibitors, IL-1 blockers and anti-CD20, can be used. However, these medicaments are associated with adverse events. For example, the adverse effects associated with TNF inhibitors include the development of autoimmune diseases, increased infection risk, injection site reactions, increased risk of malignant tumors and demyelination disorders (see, for example, Valenzuela, RA, et al., 2020.Frontiers in Pharmacology, 11: 655).

[0006] Therefore, new methods for treating or preventing inflammatory eye diseases such as uveitis are needed. Summary of the invention

[0007] The present inventors have developed a gene therapy for the treatment or prevention of inflammatory eye diseases such as uveitis, in which an anti-inflammatory TNF inhibitor is delivered to the eye.

[0008] The inventors have surprisingly demonstrated that vectors encoding TNF inhibitors under the control of an inflammation-inducible promoter can allow inflammation-inducible expression of TNF inhibitors in the eye. When the expression of TNF inhibitors is coupled to an inflammation-inducible promoter, gene therapy can therefore provide adaptable and responsive dosage levels to prevent or treat inflammatory eye diseases. Such gene therapy can prevent the recurrence of inflammation and / or maintain inflammation at subclinical levels, thereby preventing cumulative damage while reducing the occurrence of adverse events.

[0009] In one aspect, the present invention provides a vector comprising a nucleotide sequence encoding a TNF inhibitor.

[0010] In preferred embodiments, the TNF inhibitor is an anti-TNF antibody or a fragment thereof. Any suitable anti-TNF antibody or fragment thereof may be used. In some embodiments, the TNF inhibitor is any one of: adalimumab or a fragment thereof, infliximab or a fragment thereof, golimumab or a fragment thereof, or certolizumab pegol or a fragment thereof. In some embodiments, the TNF inhibitor is adalimumab or a fragment thereof or infliximab or a fragment thereof.

[0011] In some embodiments, the TNF inhibitor is an anti-TNF antibody fragment. Any suitable anti-TNF antibody fragment can be used. In some embodiments, the anti-TNF antibody fragment is an antigen binding fragment (Fab), a fragment antibody (F(ab')2), a single-chain antibody (scFv) or a single domain antibody (sdAb). In some embodiments, the anti-TNF antibody fragment is an antigen binding fragment (Fab).

[0012] In some embodiments, the TNF inhibitor is adalimumab or a fragment thereof. In some embodiments, the TNF inhibitor is an antigen-binding fragment (Fab) of adalimumab. In some embodiments, the TNF inhibitor is an anti-TNF antibody or a fragment thereof comprising one or more CDR regions selected from SEQ ID NOs: 1 to 6 or derivatives thereof comprising one amino acid substitution. In some embodiments, the TNF inhibitor is an anti-TNF antibody or a fragment thereof comprising CDR regions HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3, or a fragment thereof, wherein the CDR regions HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3 respectively comprise or consist of: SEQ ID NOs: 1, 2, 3, 4, 5, and 6 or derivatives thereof comprising one amino acid substitution. In some embodiments, the TNF inhibitor is an anti-TNF antibody or a fragment thereof comprising a heavy chain and / or a light chain, wherein the heavy chain comprises or consists of a sequence having at least 70% identity to SEQ ID NO: 7, and the light chain comprises or consists of a sequence having at least 70% identity to SEQ ID NO: 8.

[0013] In some embodiments, the heavy chain is encoded by a nucleotide sequence having at least 70% identity to SEQ ID NO: 47, and / or the light chain is encoded by a nucleotide sequence having at least 70% identity to SEQ ID NO: 48. The nucleotide sequence encoding the heavy chain and the nucleotide sequence encoding the light chain can be connected via a linker sequence. Suitably, the linker sequence encodes a 2A self-cleaving peptide and / or an enzymatically cleavable peptide motif. In some embodiments, the linker sequence encodes a 2A self-cleaving peptide having at least 70% sequence identity to any one of SEQ ID NO: 55-58. The nucleotide sequence encoding the heavy chain and / or the nucleotide sequence encoding the light chain can each be operably connected to a signal sequence. In some embodiments, the signal sequence encodes a signal peptide selected from any one of the following: human growth hormone (HGH) signal peptide, interleukin-2 (IL-2) signal peptide, CD5 signal peptide, immunoglobulin kappa light chain signal peptide, trypsinogen signal peptide, serum albumin signal peptide and prolactin signal peptide.

[0014] In some embodiments, the nucleotide sequence encoding a TNF inhibitor encodes an anti-TNF antibody or fragment comprising or consisting of a heavy chain having a sequence at least 70% identical to SEQ ID NO: 7, optionally a 2A self-cleaving peptide having at least 70% sequence identity to any one of SEQ ID NOs: 55-58, and a light chain comprising or consisting of a sequence at least 70% identical to SEQ ID NO: 8. In some embodiments, the nucleotide sequence encoding a TNF inhibitor encodes an anti-TNF antibody or fragment comprising or consisting of an amino acid sequence at least 70% identical to SEQ ID NO: 65.

[0015] In some embodiments, the nucleotide sequence encoding the TNF inhibitor comprises or consists of a nucleotide sequence having at least 70% identity to SEQ ID NO: 47, a nucleotide sequence having at least 70% sequence identity to SEQ ID NO: 59 or 60, and a nucleotide sequence having at least 70% identity to SEQ ID NO: 48. In some embodiments, the nucleotide sequence encoding the TNF inhibitor comprises or consists of a nucleotide sequence having at least 70% identity to SEQ ID NO: 66.

[0016] In a preferred embodiment, the nucleotide sequence encoding the TNF inhibitor is operably linked to an inflammation-inducible promoter. Any suitable inflammation-inducible promoter may be used. Suitably, the inflammation-inducible promoter comprises one or more inflammation-inducible transcription factor binding motifs selected from the group consisting of: an AP-1 transcription factor binding motif; an NF-κB transcription factor binding motif; an IRF transcription factor binding motif; a STAT transcription factor binding motif; and a NFAT transcription factor binding motif or any combination thereof.

[0017] In some embodiments, the inflammation-inducible promoter comprises one or more AP-1 binding motifs and / or one or more NF-κB binding motifs. In some embodiments, the inflammation-inducible promoter comprises two or more AP-1 binding motifs and / or two or more NF-κB binding motifs, three or more AP-1 binding motifs and / or three or more NF-κB binding motifs, four or more AP-1 binding motifs and / or four or more NF-κB binding motifs, or five or more AP-1 binding motifs and / or five or more NF-κB binding motifs. In some embodiments, the inflammation-inducible promoter comprises at least one AP-1 binding motif coupled to at least one NF-κB binding motif. In some embodiments, the inflammation-inducible promoter comprises five AP-1 binding motifs coupled to five NF-κB binding motifs. Suitably, the AP-1 binding motif comprises or consists of SEQ ID NO: 70, or comprises or consists of any one of SEQ ID NO: 71-73 or a derivative thereof comprising one nucleotide substitution. Suitably, the NF-κB binding motif comprises or consists of SEQ ID NO:74, or comprises or consists of SEQ ID NO:75 or a derivative thereof comprising two or fewer nucleotide substitutions. In some embodiments, the inflammation-inducible promoter comprises or consists of a nucleotide sequence having at least 70% identity to SEQ ID NO:76.

[0018] In some embodiments, the vector comprises a nucleotide sequence that is at least 70% identical to SEQ ID NO:77.

[0019] The vector may comprise any other suitable vector elements. The nucleotide sequence encoding the TNF inhibitor may be operably linked to a polyadenylation sequence. Suitably, the polyadenylation sequence is selected from any one of the following: bovine growth hormone (bGH) polyadenylation sequence, SV40 polyadenylation sequence, and rabbit β-globin polyadenylation sequence. In some embodiments, the polyadenylation sequence comprises or consists of a nucleotide sequence having at least 70% identity to SEQ ID NO: 78. The nucleotide sequence encoding the TNF inhibitor may be operably linked to a woodchuck hepatitis post-transcriptional regulatory element (WPRE). In some embodiments, the WPRE comprises or consists of a nucleotide sequence having at least 70% identity to SEQ ID NO: 79. The nucleotide sequence encoding the TNF inhibitor may be operably linked to an intron. Suitably, the intron is selected from a β-globin intron or an SV40 intron. In some embodiments, the intron comprises or consists of a nucleotide sequence having at least 70% identity to SEQ ID NO: 80.

[0020] In a preferred embodiment, the vector is a viral vector. Any suitable viral vector may be used. Suitably, the viral vector is any of the following: a parvoviral vector, preferably an adeno-associated virus (AAV) vector, an adenoviral vector, a herpes simplex virus vector, an anellovirus vector, a retroviral vector or a lentiviral vector.

[0021] In a preferred embodiment, the vector is an adeno-associated virus (AAV) vector. In a preferred embodiment, the vector is an AAV vector particle. The AAV vector particle can be pseudotyped to confer ocular tissue tropism. Suitably, the AAV vector particle comprises an AAV2 capsid protein or an AAV2 capsid variant protein, optionally wherein the AAV2 capsid variant is selected from any one of the following: AAV2.tYF, AAV2.7m8, R100, AAV2.GL and AAV2.NN. The vector may comprise one or more inverted terminal repeats (ITRs).

[0022] In some embodiments, the vector comprises or consists of a nucleotide sequence that is at least 70% identical to SEQ ID NO:91.

[0023] In one aspect, the present invention provides a vector comprising or consisting of a nucleotide sequence having at least 70% identity to SEQ ID NO: 91. The vector may be a viral vector. The vector may be an AAV vector.

[0024] In one aspect, the invention provides a cell comprising a vector of the invention. The cell may be an isolated cell.

[0025] In one aspect, the invention provides kits for producing the vectors of the invention.

[0026] In one aspect, the present invention provides a pharmaceutical composition comprising the vector of the present invention or the cell of the present invention. The vector or cell may be combined with a pharmaceutically acceptable carrier, diluent or excipient.

[0027] In one aspect, the invention provides a vector according to the invention, a cell according to the invention and / or a pharmaceutical composition according to the invention for use as a medicament.

[0028] In one aspect, the present invention provides use of a vector according to the present invention, a cell according to the present invention or a pharmaceutical composition according to the present invention for the preparation of a medicament.

[0029] In one aspect, the invention provides a method comprising administering a vector according to the invention, a cell according to the invention or a pharmaceutical composition according to the invention to a subject in need thereof.

[0030] In one aspect, the present invention provides a vector for preventing or treating an inflammatory eye disease, wherein the vector comprises a nucleotide sequence encoding a TNF inhibitor, and wherein the nucleotide sequence encoding a TNF inhibitor is operably linked to an inflammation-inducible promoter.

[0031] In one aspect, the present invention provides use of a vector in the preparation of a medicament for preventing or treating an inflammatory eye disease, wherein the vector comprises a nucleotide sequence encoding a TNF inhibitor, and wherein the nucleotide sequence encoding the TNF inhibitor is operably linked to an inflammation-inducible promoter.

[0032] In one aspect, the invention provides a method for preventing or treating an inflammatory eye disease, wherein the method comprises administering a vector to a subject in need thereof, wherein the vector comprises a nucleotide sequence encoding a TNF inhibitor, and wherein the nucleotide sequence encoding the TNF inhibitor is operably linked to an inflammation-inducible promoter.

[0033] In one aspect, the present invention provides a vector according to the present invention or a pharmaceutical composition according to the present invention for use in preventing or treating an inflammatory eye disease.

[0034] In one aspect, the present invention provides use of a vector according to the present invention or a pharmaceutical composition according to the present invention for preparing a medicament for preventing or treating an inflammatory eye disease.

[0035] In one aspect, the present invention provides a method for preventing or treating an inflammatory eye disease, comprising administering a vector according to the present invention or a pharmaceutical composition according to the present invention to a subject in need thereof.

[0036] The inflammatory eye disease may be any inflammatory eye disease. Suitably, the inflammatory eye disease is uveitis. The vector or pharmaceutical composition may be administered in response to a recurrence of the inflammatory eye disease, in particular, wherein the inflammatory eye disease is uveitis.

[0037] The vector or pharmaceutical composition can be administered by any suitable route. Suitably, the vector or pharmaceutical composition is administered intraocularly. In some embodiments, the vector or pharmaceutical composition is administered via intravitreal injection, subretinal injection, direct retinal injection, subconjunctival injection, sub-Tenon injection, or suprachoroidal injection. In some embodiments, the vector or pharmaceutical composition is administered via intravitreal injection.

[0038] The vector or pharmaceutical composition may be administered in any suitable regimen. Suitably, the vector or pharmaceutical composition is administered as a single dose. Suitably, the vector is administered at a dose of at least about 1E10 vg / mL, at least about 1E11 vg / mL, at least about 1E12 vg / mL, or at least about 5E12 vg / mL. Suitably, the vector is administered at a dose of at least about 1E9 vg / eye, at least about 1E10 vg / eye, or at least about 1E11 vg / eye. Suitably, the vector is administered at a dose of about 1E9 vg / eye to about 5E12 vg / eye. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 A-1C: Topical administration of anti-TNF antibodies inhibits experimental autoimmune uveoretinitis (EAU)

[0041] B10.RIII mice were immunized for experimental autoimmune uveoretinitis (EAU) and eyes were monitored using Topical Endoscopic Fundal Imaging (TEFI) starting on day 10 to select experimental mice that exhibited clinically evident disease. On day 10, each group of mice was injected with 15 μg of infliximab or vehicle control (EAU) via the intravitreal route. Eyes were enucleated (day 14) and retinal infiltrates were characterized. (1A) Representative fundus images, (1B) clinical disease scores, and (1C) flow cytometric analysis of total CD45+ cell counts from a single eye on day 14. **P<0.005; data are expressed as mean + / - SEM and are representative of two independent experiments.

[0042] Figure 2 :Therapeutic vector design

[0043] Schematic diagram showing the vector organization of CMV.Infliximab Fab (expression under the control of the constitutive CMV promoter) and AP1-NFkB.Infliximab Fab (expression under the control of an inflammation-inducible promoter containing 5 repeats of AP1 and NFkB binding sites). The heavy and light chains of the Infliximab Fab are separated by a self-cleaving 2A peptide.

[0044] Figure 3 A-3B: Constitutive Therapeutic Transgene Expression

[0045] (3A) HEK-293T (standard cell line for AAV development) or ARPE-19 (ocular cell line) cells were transduced with AAV2.CMV.Infliximab or AAV.CMV.NULL vectors [MOI 1E5 vg / cell], and culture supernatants were assayed using a clinical IFX ELISA kit. Detectable expression of infliximab Fab in both cell types was evident by 72 hours (approximately 30 ng / ml). ****P<0.0001. Data are expressed as mean + / - SEM. (3B) AAV2.CMV.Infliximab or AAV.CMV.NULL was administered to the eyes of healthy B10.RIII mice at 5E12 vg / ml by intravitreal (IVT) injection. Four weeks after AAV, mice were sacrificed, eyes were dissected, and supernatants (retina and vitreous) were assayed using a clinical IFX ELISA kit. Detectable expression of infliximab Fab in vivo (approximately 1.5 ng / ml) was observed in eyes receiving the 5E12 vg / ml dose. *P<0.05. Data are presented as mean + / - SEM, with each data point representing a single eye.

[0046] Figure 4 A-4B: Inducible transgene expression in vitro

[0047] HEK-293T cells transduced with AAV2.AP1-NFkB.EGFP (reporter vector) or AAV2.AP1-NFkB.Infliximab (therapeutic vector) were stimulated with recIL-1b (2 ng / mL). (4A) Activation resulted in GFP expression visible at 24 h, with an increase in intensity at 72 hr, where no GFP signal was observed with AAV2.AP1-NFkB.NULL (control vector). Images were captured on EVOSFL, 10X magnification. ( Figure 4 B) Stimulation resulted in rapid induction of infliximab Fab expression (8 hr), with accumulation reaching approximately 20 ng / ml at 72 hr. ****P<0.0001; data are expressed as mean + / - SEM.

[0048] Figure 5 A-5B: Inducible transgene expression in vivo

[0049] AAV2.AP1-NFkB.EGFP (reporter) or AAV.AP1-NFkB.NULL (control) at a dose of 5E12 vg / ml was administered to C57BL / 6J mice by intravitreal (IVT) injection. (5A) Four weeks after AAV injection, mice were immunized to induce experimental autoimmune uveoretinitis (EAU) and imaged to monitor the onset of ocular inflammation. Representative fundus and OCT images at day 14 of EAU show clear clinical signs of disease (perivascular sheathing and vitreous infiltration), as well as induction of GFP expression. No clinical signs of disease or expression of the GFP transgene were observed in mice that received AAV alone (no EAU). (5B) Groups of mice were injected with AAV2.AP1-NFkB.Infliximab or AAV2.AP1-NFkB.NULL in the contralateral eye and EAU was induced 4 weeks after AAV. At 3 weeks after EAU, mice were sacrificed, eyes were dissected and supernatants (retina and vitreous) were determined using a clinical IFXELISA kit. Detectable expression of infliximab Fab was observed only in EAU eyes receiving therapeutic vectors and not in controls. *P<0.05. Data are expressed as mean + / - SEM, where each data point represents a single eye.

[0050] Figure 6 A-6C: In vivo evaluation of the efficacy of constitutive therapeutic transgenes

[0051] To demonstrate the efficacy of constitutive therapeutic transgenes, groups of mice were injected with AAV7m8.CMV.Infliximab or AAV7m8.CMV.NULL in the contralateral eye, followed by intravitreal administration of recombinant human TNF (rec_hTNF) 4 weeks after AAV. At 18 hr (peak of inflammatory response to rec_hTNF), representative fundus and OCT images showed increased inflammation (vitreal infiltration) in control (empty) eyes compared with infliximab eyes (6A). In mice receiving only AAV, no clinical symptoms of the disease were observed. Mice were sacrificed at 18 hr, and anatomical eyes were prepared for flow cytometry analysis to determine the absolute number of Ly6C+ monocytes (the main infiltrate in this model) from a single eye. A significant reduction in the number of monocytes in eyes receiving therapeutic vectors rather than controls (6B) was further highlighted by paired (contralateral eye) analysis (6C). **Wilcoxon signed rank test; *P<0.05Wilcoxon matched pair analysis.

[0052] Figure 7 A-7C: Constitutive expression of other anti-TNF biologics in vitro and in vivo

[0053] HEK-293T cells were transfected with huTNFRI-huIgG plasmids, and culture supernatants were assayed using an anti-human TNF antibody ELISA kit. Detectable expression of huTNFRI-huIgG was evident at 48 hr (7A). HEK-293T cells were transduced with AAV7m8.CMV.huTNFRI-huIgG or AAV7m8.CMV.NULL vectors [MOI 1E5 vg / cell], and culture supernatants were assayed using an anti-human TNF antibody ELISA kit. Detectable expression was evident at 72 hr (approximately 30 ng / ml) (7B). ****P<0.0001. Data are expressed as mean + / - SEM. AAV7m8.CMV.huTNFRI-huIgG or AAV7m8.CMV.NULL was administered to the eyes of healthy C57BL / 6J mice at a range of doses [2E8 or 2E9 vg / eye] by intravitreal (IVT) injection. At 4 weeks post-AAV, mice were sacrificed, eyes dissected and supernatants (retina and vitreous) were assayed using an anti-human TNF antibody ELISA kit. Detectable expression of approximately 4 ng / ml and 9 ng / ml of huTNFRI-huIgG was observed in eyes receiving 2E8 and 2E9 vg / eye doses, respectively (7C). ns-not significant; one-way ANOVA; **P<0.05. Data are expressed as mean + / - SEM, with each data point representing a single eye.

[0054] Figure 8 A-8C: In vitro biological activity of a constitutively expressed anti-TNF transgene

[0055] HEK-BLUE TNF reporter cells were transfected with AAV.CMV.huTNFRI-huIgG, AAV.CMV.huTNFRI-msIgG, AAV.CMV.msTNFRI-msIgG, AAV.CMV.ADALIMUMAB plasmids or AAV2.CMV.Infliximab for 48 hr. Cells were then stimulated with recombinant human TNF or mouse TNF (0.5 ng / ml) for another 24 hours and NFkB activation was assessed. All TNFRI antibody-like plasmid constructs inhibited huTNF and msTNF-mediated activation in reporter cell lines compared to NULL or recTNF alone (8A). For monoclonal Fab-based anti-TNF biologics, plasmid expression of adalimumab Fab (8B) and AAV-mediated infliximab Fab expression (8C) both inhibited huTNF activation.

[0056] Fig. 9 A-9B: Expression of an inducible anti-TNF transgene and in vitro evaluation of biological activity

[0057] HEK-293T cells were transduced with AAV7m8.AP1-NFkB.huTNFRI-huIgG or AAV7m8.AP1-NFkB.NULL vectors [MOI 1E5 vg / cell] and stimulated with IL-1b (2 ng / ml). Stimulation resulted in robust induction of huTNFRI-huIgG expression by 24 hr, with accumulation reaching approximately 25 ng / ml at 48 hr (9A). One-way ANOVA; **P<0.0001. Data are expressed as mean + / - SEM. Conditioned media were "spiked" with recombinant human or mouse TNF (final concentration 10 ng / ml) and incubated with HEK-BLUE reporter cells for 24 hr. NFkB activation was robustly induced in reporter cells in response to both huTNF stimulation alone or mTNF stimulation, or with conditioned media from AAV7m8.AP1-NFkB.NULL. When supplemented with conditioned medium from cells transduced with AAV7m8.AP1-NFkB.huTNFRI-huIgG and stimulated with IL-1b, NFkB activation in reporter cells was completely inhibited (9B). One-way ANOVA; **p<0.05 ***p<0.001; Data are expressed as mean + / - SEM.

[0058] Fig.10 A-10B: Expression of an inducible anti-TNF transgene and in vivo evaluation of biological activity

[0059] Mice were injected with AAV7m8.AP1-NFkB.huTNFRI-huIgG and EAU was induced 4 weeks after AAV. On day 19 after EAU, when mild to moderate clinical signs of inflammation were observed (disease peak not yet reached), mice were sacrificed, eyes dissected and supernatants (retina and vitreous) were assayed using anti-human TNF antibody ELISA. Clinical disease drives detectable expression of huTNFRI-huIgG in EAU eyes that received therapeutic vectors but not controls (AAV only) (10A). Mice were injected with AAV7m8.AP1-NFkB.huTNFRI-huIgG and AAV7m8.AP1-NFkB.NULL (contralateral eye control) at 2E9vg / eye. Four weeks after AAV, mice received bilateral administration of recombinant human TNF (rec_hTNF), were sacrificed 18hr later, eyes were dissected and huTNFRI-huIgG expression in ocular supernatants (retina and vitreous) was assayed. Acute activation triggered a significant increase in huTNFRI-huIgG expression compared to controls (10B). ****P<0.0001. Data are presented as mean + / - SEM, where each data point represents a single eye.

[0060] Fig.11A-11B: In vivo evaluation of the efficacy of inducible therapeutic transgenes

[0061] B10.RIII mice were injected with AAV7m8.AP1-NFkB.huTNFRI-IgG or AAV7m8.CMV.NULL [2E9 vg / eye] in the contralateral eye and then immunized for EAU 4 weeks after AAV. On day 11, representative fundus and OCT images showed increased inflammation in eyes receiving control empty vectors (11A). The contralateral eyes of the same three animals receiving inducible therapeutic vectors showed significantly reduced clinical inflammation (both perivascular sheathing and vitreous infiltration). At this time point, mice were sacrificed and dissected eyes were prepared for flow cytometric analysis to determine the absolute number of CD45+ (all leukocytes), CD3+ (lymphocytes), CD4+ (Th T cells) and CD11b+ (macrophages and monocytes) populations from a single eye (11B). We observed a trend towards reduced immune cell infiltrates with AAV7m8.AP1-NFkB.huTNFRI-IgG compared to AAV7m8.CMV.NULL. DETAILED DESCRIPTION OF THE INVENTION

[0063] Various preferred features and embodiments of the invention will now be described by way of non-limiting examples.

[0064] It must be noted that as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0065] As used herein, the terms "comprising, comprises and comprised of" are synonymous with "including and includes" or "containing and contains" and are inclusive or open-ended and do not exclude additional, unrecited members, elements or steps. The terms "comprising, comprises and comprised of" also include the term "consisting of".

[0066] Numerical ranges are inclusive of the numbers defining the range.Unless otherwise indicated, any nucleic acid sequences are written left to right in 5' to 3' orientation and amino acid sequences are written left to right in amino to carboxyl orientation, respectively.

[0067] The disclosures discussed herein are provided only because they were disclosed prior to the filing date of the present application. Nothing herein should be construed as an admission that such disclosures constitute prior art for the claims appended hereto. All disclosures mentioned in the specification are incorporated herein by reference.

[0068] The present disclosure is not limited to the exemplary methods and materials disclosed herein, and any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the embodiments of the present disclosure. Those skilled in the art will appreciate that they can combine all features of the present invention disclosed herein without departing from the scope of the disclosed invention.

[0069] TNF inhibitors

[0070] The vectors of the invention comprise a nucleotide sequence encoding a TNF inhibitor. The inventors have surprisingly shown that such vectors can be used to prevent or treat inflammatory eye diseases.

[0071] As used herein, "TNF inhibitors" can be any protein that inhibits the inflammatory response to TNF. Tumor necrosis factor (TNF) is also called cachexin or cachectin, and may also be referred to as tumor necrosis factor alpha (TNF-α). TNF is synthesized as a transmembrane protein (mTNF) and cut into soluble TNF (sTNF). There are two very closely related types of TNF, TNF-α and TNF-β. The activity of both TNFs is mediated by binding to TNF receptors TNFR1 and TNFR2. TNF binding can activate several signal transduction pathways, including transcription factor activation, proteases, and protein kinases. This signal transduction can lead to the activation of target cells, which leads to inflammation and immune responses by releasing several cytokines and initiating apoptotic pathways (see Gerriets, V., et al., 2021. "Tumor necrosis factor inhibitors". In Stat Pearls).

[0072] Example TNF inhibitors include adalimumab, infliximab, golimumab, certolizumab pegol, etanercept, XPro1595, XENP345, R1antTNF, Atrosab, and Atrosimab (see, e.g., Lis, K., Kuzawińska, O., and E., 2014. AMS, 10 (6), p. 1175; and Fischer, R., et al., 2020. Frontiers in cell and developmental biology, 8, p. 401). Suitably, the TNF inhibitor can inhibit TNF activity by directly binding to TNF. For example, the TNF inhibitor can be an anti-TNF antibody or a fragment thereof (e.g., adalimumab, infliximab, golimumab, certolizumab), or can comprise a TNF binding domain of a TNFR receptor (e.g., etanercept). Alternatively, the TNF inhibitor can inhibit TNF activity by binding to a TNF receptor. For example, the TNF inhibitor can be a TNF mutant protein (e.g., XPro1595, XENP345, R1antTNF) or can be an anti-TNFR antibody or a fragment thereof (e.g., Atrosab and Atrosimab).

[0073] Anti-TNF antibodies or fragments thereof

[0074] In a preferred embodiment, the TNF inhibitor is an anti-TNF antibody or fragment thereof.

[0075] Antibodies are glycoproteins belonging to the immunoglobulin superfamily. Antibodies are usually made of basic structural units, each of which has two heavy chains and two light chains. Antibodies can recognize antigens via fragment antigen binding (Fab) variable regions. The fragment crystallizable region (Fc region) is the tail region of the antibody, which can allow the antibody to activate the immune system. The hinge region is an extension of the heavy chain connecting the Fab and Fc regions.

[0076] "Heavy chain variable region" or "VH" refers to a fragment of an antibody's heavy chain that contains three CDRs inserted between flanking extensions called framework regions, which form a scaffold to support the CDRs. "Light chain variable region" or "VL" refers to a fragment of an antibody's light chain that contains three CDRs inserted between framework regions.

[0077] "Complementarity determining region" or "CDR" with respect to an antibody or antigen binding fragment thereof refers to a highly variable loop in the variable region of the heavy or light chain of an antibody. The CDRs can interact with the antigen conformation and determine, to a large extent, the binding to the antigen. The heavy and light chain variable regions each contain three CDRs (heavy chain CDRs 1, 2, and 3 and light chain CDRs 1, 2, and 3, numbered from amino terminus to carboxyl terminus).

[0078] The CDR of the heavy chain variable region and light chain variable region of antibody can use the prediction software available in the art, such as using Abysis algorithm, or using IMGT / V-QUEST software, predict from the heavy chain variable region sequence and light chain variable region sequence of antibody (see, for example, Lefranc et al., 2009 NAR37: D1006-D1012 and Lefranc 2003, Leukemia17: 260-266). The CDR region identified by any algorithm in the two is considered to be equally suitable for the present invention. The length of CDR can vary (depending on the antibody predicted to have them), and can vary between heavy chain and light chain. Therefore, the three heavy chain CDRs of complete antibody can have different lengths (or can have the same length), and the three light chain CDRs of complete antibody can have different lengths (or can have the same length). For example, the length of CDR can range from 2 or 3 amino acids to 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids. In particular, the length of a CDR may be 3-14 amino acids, such as at least 3 amino acids and less than 15 amino acids.

[0079] Suitable anti-TNF antibodies are known in the art. In addition, anti-TNF antibodies and fragments and / or derivatives thereof can be prepared using methods known to those skilled in the art. Such methods include phage display, methods for generating human antibodies or humanized antibodies, or methods using transgenic animals or plants engineered to produce human antibodies. Phage display libraries of partially or completely synthetic antibodies are available, and the libraries can be screened for antibodies or fragments thereof that can bind to target molecules. Phage display libraries of human antibodies are also available. Once identified, the amino acid sequence or polynucleotide sequence encoding the antibody (or its fragment and / or derivative) can be isolated and / or determined. The sequence of the antibody can be used to design its suitable fragment and / or derivative.

[0080] In a preferred embodiment, the anti-TNF antibody or fragment thereof is an anti-TNF antibody fragment. An "anti-TNF antibody fragment" may be a fragment of an anti-TNF antibody, or a genetically engineered product of one or more fragments of an anti-TNF antibody, which fragment is involved in binding to TNF. Examples include antigen binding fragments (Fab), fragment antibodies (F(ab')2), variable regions (Fv), single chain antibodies (scFv), single domain antibodies (sdAb), and camelid antibodies (VHH). The use of anti-TNF antibody fragments may be advantageous because it may reduce inflammation associated with the Fc region.

[0081] In some embodiments, the anti-TNF antibody fragment is an antigen-binding fragment (Fab), a fragment antibody (F(ab')2), a single-chain antibody (scFv), or a single-domain antibody (sdAb).

[0082] In a preferred embodiment, the anti-TNF antibody fragment is an antigen binding fragment (Fab). "Antigen binding fragment" (Fab) refers to the region of an antibody that binds to an antigen. It is composed of a constant region and a variable region of each of the heavy chain and the light chain.

[0083] In other embodiments, the anti-TNF antibody fragment is a fragment antibody (F(ab')2). "Fragment antibody" (F(ab')2) refers to the region of an antibody that remains after digestion of the Fc region, while leaving some hinge regions intact.

[0084] In other embodiments, the anti-TNF antibody fragment is a single-chain antibody (scFv). "Single-chain antibody" (scFv) refers to an engineered antibody consisting of a light chain variable region and a heavy chain variable region directly connected to each other or connected to each other via a peptide linker sequence. The peptide linker sequence is generally about 10 to 25 amino acids in length, rich in glycine for flexibility, and rich in serine or threonine for solubility. The peptide linker sequence can connect the N-terminus of the heavy chain variable region to the C-terminus of the light chain variable region, or vice versa.

[0085] In other embodiments, the anti-TNF antibody fragment is a single domain antibody (sdAb). "Single domain antibody" (sdAb), also known as nanobody, refers to an antibody fragment consisting of a single monomer variable antibody domain. Therefore, sdAb can be a heavy chain variable region (VH) or a light chain variable region (VL). Examples of single domain antibodies include, but are not limited to, VHH fragments and VNAR fragments. Single domain antibodies can also be produced by splitting the dimeric variable domains from ordinary IgG molecules into monomers.

[0086] TNF inhibitors may comprise at least one CDR (e.g., HCDR3) that can be predicted from an anti-TNF antibody (or a variant of such a predicted CDR, e.g., a variant having one, two, or three amino acid substitutions). It should be understood that molecules containing three or fewer CDR regions (e.g., a single CDR or even a portion thereof) may be able to retain the antigen binding activity of the antibody from which the CDR is derived. Molecules containing two CDR regions have been described in the art as being able to bind to a target antigen, e.g., in the form of minibodies (see, e.g., Vaughan and Sollazzo, 2001, Combinational Chemistry & High Throughput Screening, 4, 417-430). Molecules containing a single CDR have been described that can exhibit strong binding activity to a target (see, e.g., Nicaise et al., 2004, Protein Science, 13: 1882-91).

[0087] The TNF inhibitor may comprise one or more variable heavy chain CDRs, such as one, two or three variable heavy chain CDRs. Alternatively or additionally, the TNF inhibitor may comprise one or more variable light chain CDRs, such as one, two or three variable light chain CDRs. The TNF inhibitor may comprise three heavy chain CDRs and / or three light chain CDRs (and more particularly a heavy chain variable region comprising three CDRs and / or a light chain variable region comprising three CDRs), wherein at least one CDR, preferably all CDRs, may be from an anti-TNF antibody, or may be selected from one of the CDR sequences provided below.

[0088] The TNF inhibitor can comprise any combination of variable heavy chain CDRs and light chain CDRs, such as one variable heavy chain CDR together with one variable light chain CDR, two variable heavy chain CDRs together with one variable light chain CDR, two variable heavy chain CDRs together with two variable light chain CDRs, three variable heavy chain CDRs together with one or two variable light chain CDRs, one variable heavy chain CDR together with two or three variable light chain CDRs, or three variable heavy chain CDRs together with three variable light chain CDRs. Preferably, the TNF inhibitor comprises three variable heavy chain CDRs (HCDR1, HCDR2, and HCDR3) and / or three variable light chain CDRs (LCDR1, LCDR2, and LCDR3).

[0089] One or more CDRs present in a TNF inhibitor may not all be from the same antibody, as long as the domain has the above-mentioned binding activity. Thus, one CDR may be predicted from the heavy chain or light chain of an anti-TNF antibody, while another CDR present may be predicted from a different anti-TNF antibody. In this case, it may be preferred to predict CDR3 from an anti-TNF antibody. However, in particular, if more than one CDR is present in a TNF inhibitor, it is preferred to predict CDRs from an anti-TNF antibody. A combination of CDRs from different antibodies, particularly from antibodies that bind to the same desired region or epitope, may be used.

[0090] In preferred embodiments, the TNF inhibitor comprises three CDRs predicted from the variable heavy chain sequence of an anti-TNF antibody and / or three CDRs predicted from the variable light chain sequence of an anti-TNF antibody.

[0091] The present invention includes "variants" of the following CDR regions. As used herein, the term "variant" is defined in the section "Variants, Derivatives and Fragments" below. It should be understood that one or more amino acid substitutions can be made in a CDR while retaining antigen binding ability. For example, a CDR variant can contain 3 or less amino acid substitutions, such as 3 amino acid substitutions, 2 amino acid substitutions, or 1 amino acid substitution. In particular, in some embodiments, a CDR variant contains one amino acid substitution and retains antigen binding ability. A variant can be a variant having at least 80% or 90% identity to a CDR.

[0092] The examples of antibodies and fragments thereof and / or derivatives that can be used in the present invention are further described below. The TNF inhibitor may comprise or consist of an amino acid sequence comprising the CDRs described herein, and substitution, variation, modification, replacement, deletion and / or addition of one or more amino acid residues may occur in the framework region. The derivatives described herein may retain TNF binding ability. The derivatives may be able to bind to TNF at a level of at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or 100% of the corresponding reference amino acid sequence.

[0093] TNF binding affinity can be determined by the equilibrium binding constant (KD), which can be determined by any suitable assay such as surface plasmon resonance (see, e.g., Shealy, DJ, et al., 2010. mAbs, 2(4), pp. 428-439). Suitably, the anti-TNF antibody or fragment thereof can bind to soluble TNF with a binding affinity of about 1000 pM or less, about 900 pM or less, about 800 pM or less, about 700 pM or less, about 600 pM or less, about 500 pM or less, about 400 pM or less, about 300 pM or less, about 200 pM or less, or about 100 pM or less (e.g., as determined by surface plasmon resonance). Suitably, the anti-TNF antibody or fragment thereof may bind to soluble TNF with a binding affinity (e.g., as determined by surface plasmon resonance) of about 0.1 pM to about 1000 pM, about 1 pM to about 1000 pM, about 10 pM to about 1000 pM, 0.1 pM to about 100 pM, about 1 pM to about 100 pM, or about 10 pM to about 100 pM. Suitably, the anti-TNF antibody or fragment thereof may bind to transmembrane TNF with a binding affinity (e.g., as determined by surface plasmon resonance) of about 10000 pM or less, about 9000 pM or less, about 8000 pM or less, about 7000 pM or less, about 6000 pM or less, about 5000 pM or less, or about 4000 pM or less.

[0094] In some embodiments, the TNF inhibitor is selected from any one of: adalimumab or a fragment and / or derivative thereof; infliximab or a fragment and / or derivative thereof; golimumab or a fragment and / or derivative thereof; and certolizumab pegol or a fragment and / or derivative thereof.

[0095] In some embodiments, the TNF inhibitor is selected from: adalimumab, or a fragment and / or derivative thereof; and infliximab, or a fragment and / or derivative thereof.

[0096] In some embodiments, the TNF inhibitor is selected from any one of the following: an adalimumab fragment or a derivative thereof; an infliximab fragment or a derivative thereof; a golimumab fragment or a derivative thereof; and a certolizumab fragment or a derivative thereof.

[0097] In some embodiments, the TNF inhibitor is selected from any one of: an adalimumab fragment or a derivative thereof; and an infliximab fragment or a derivative thereof.

[0098] Adalimumab

[0099] In preferred embodiments, the TNF inhibitor is adalimumab or a fragment and / or derivative thereof. In some embodiments, the TNF inhibitor is adalimumab Fab, adalimumab F(ab')2, adalimumab scFv, adalimumab sdAb or a derivative thereof. In some embodiments, the TNF inhibitor is adalimumab Fab or a derivative thereof.

[0100] Adalimumab It is a recombinant fully human IgG1 monoclonal antibody that specifically binds to TNF to neutralize the activity of the cytokine. Technicians will be able to use conservative mutations and / or TNF adalimumab inhibition mechanism knowledge to generate adalimumab derivatives (see, for example, Hu, S., et al., 2013. Journal of Biological Chemistry, 288 (38), pp. 27059-27067). For example, it has been shown that the following adalimumab variants have KD values ​​comparable to wild-type adalimumab: L178K, L178N, Q160N, L116N, T118N, A122N, Q179N, L183N and T199N (see, for example, Reslan, M., et al., 2020. International Journal of Biological Macromolecules, 158, pp. 189-196).

[0101] In some embodiments, the TNF inhibitor is a fragment of adalimumab or a derivative thereof. In some embodiments, the TNF inhibitor is adalimumab Fab, adalimumab F(ab')2, adalimumab scFv or adalimumab sdAb or a derivative thereof. In some embodiments, the TNF inhibitor is adalimumab Fab or a derivative thereof.

[0102] Suitable adalimumab Fab derivatives include those described in Yoshikawa, M., et al., 2022. The Journal of Biochemistry, mvac040; and Nakamura, H., et al., 2020. Biological and Pharmaceutical Bulletin, 43 (3), pp. 418-423. Suitably, the adalimumab Fab derivatives may be selected from one or more of the following: H: K137C-L: I117C, H: K137C-L: F209C, H: S138C-L: F116C, H: S140C-L: S114C, and H: V177C-L: Q160C.

[0103] In some embodiments, the TNF inhibitor is or is derived from an anti-TNF antibody (e.g., a Fab, F(ab')2, scFv, or sdAb), wherein the antibody comprises one or more CDR regions selected from SEQ ID NO: 1-6 or variants thereof. In other words, in some embodiments, the TNF inhibitor comprises one or more CDR regions selected from SEQ ID NO: 1-6 or variants thereof.

[0104] In some embodiments, the TNF inhibitor comprises:

[0105] (i) a HCDR1 having an amino acid sequence of SEQ ID NO: 1 or a variant thereof, a HCDR2 having an amino acid sequence of SEQ ID NO: 2 or a variant thereof, and / or a HCDR3 having an amino acid sequence of SEQ ID NO: 3 or a variant thereof; and / or

[0106] (ii) LCDR1 having the amino acid sequence of SEQ ID NO: 4 or a variant thereof, LCDR2 having the amino acid sequence of SEQ ID NO: 5 or a variant thereof, and / or LCDR3 having the amino acid sequence of SEQ ID NO: 6 or a variant thereof.

[0107] In some embodiments, the TNF inhibitor comprises a HCDR2 having an amino acid sequence of SEQ ID NO: 2 or a variant thereof and / or a LCDR2 having an amino acid sequence of SEQ ID NO: 5 or a variant thereof. For adalimumab, CDRs L2 and H2 contribute to most of the interactions with the antigen (see Hu, S., et al., 2013. Journal of Biological Chemistry, 288 (38), pp. 27059-27067).

[0108] In some embodiments, the TNF inhibitor comprises:

[0109] (i) a HCDR1 having an amino acid sequence of SEQ ID NO: 1 or a variant thereof, a HCDR2 having an amino acid sequence of SEQ ID NO: 2 or a variant thereof, and a HCDR3 having an amino acid sequence of SEQ ID NO: 3 or a variant thereof; and / or

[0110] (ii) LCDR1 having the amino acid sequence of SEQ ID NO: 4 or a variant thereof, LCDR2 having the amino acid sequence of SEQ ID NO: 5 or a variant thereof, and LCDR3 having the amino acid sequence of SEQ ID NO: 6 or a variant thereof.

[0111] In some embodiments, the TNF inhibitor comprises or consists of a heavy chain, wherein the heavy chain comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical, or 100% identical to SEQ ID NO:7.

[0112] In some embodiments, the TNF inhibitor comprises or consists of a light chain, wherein the light chain comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical, or 100% identical to SEQ ID NO:8.

[0113] In some embodiments, the TNF inhibitor comprises or consists of a heavy chain and a light chain, wherein the heavy chain comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical, or 100% identical to SEQ ID NO:7, and the light chain comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 96%, at least 97%, at least 98%, at least 99% identical, or 100% identical to SEQ ID NO:8.

[0114] In some embodiments, the TNF inhibitor comprises or consists of:

[0115] (i) a heavy chain comprising an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:7, wherein the amino acid sequence comprises a HCDR1 having the amino acid sequence of SEQ ID NO:1 or a variant thereof, a HCDR2 having the amino acid sequence of SEQ ID NO:2 or a variant thereof, and a HCDR3 having the amino acid sequence of SEQ ID NO:3 or a variant thereof; and / or

[0116] (ii) a light chain comprising an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:8, wherein the amino acid sequence comprises a LCDR1 having the amino acid sequence of SEQ ID NO:4 or a variant thereof, a LCDR2 having the amino acid sequence of SEQ ID NO:5 or a variant thereof, and a LCDR3 having the amino acid sequence of SEQ ID NO:6 or a variant thereof.

[0117] In some embodiments, the TNF inhibitor comprises or consists of a heavy chain, wherein the heavy chain comprises or consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical or 100% identical to SEQ ID NO:9 or 10.

[0118] In some embodiments, the TNF inhibitor comprises or consists of a light chain, wherein the light chain comprises or consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical, or 100% identical to SEQ ID NO: 11.

[0119] In some embodiments, the TNF inhibitor comprises or consists of a heavy chain and a light chain, wherein the heavy chain comprises or consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical, or 100% identical to SEQ ID NO:9, and the light chain comprises or consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 96%, at least 97%, at least 98%, at least 99% identical, or 100% identical to SEQ ID NO:11.

[0120] In some embodiments, the TNF inhibitor comprises or consists of:

[0121] (i) a heavy chain comprising or consisting of an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:9, wherein the amino acid sequence comprises a HCDR1 having the amino acid sequence of SEQ ID NO:1, a HCDR2 having the amino acid sequence of SEQ ID NO:2, and a HCDR3 having the amino acid sequence of SEQ ID NO:3; and / or

[0122] (ii) a light chain comprising or consisting of an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:11, wherein the amino acid sequence comprises a LCDR1 having the amino acid sequence of SEQ ID NO:4, a LCDR2 having the amino acid sequence of SEQ ID NO:5, and a LCDR3 having the amino acid sequence of SEQ ID NO:6.

[0123]

[0124]

[0125]

[0126] Infliximab

[0127] In some embodiments, the TNF inhibitor is infliximab or a fragment and / or derivative thereof. In some embodiments, the TNF inhibitor is infliximab Fab, infliximab F(ab')2, infliximab scFv, infliximab sdAb or a derivative thereof. In some embodiments, the TNF inhibitor is infliximab Fab or a derivative thereof.

[0128] Infliximab It is a chimeric monoclonal antibody against human TNF. It binds to soluble and transmembrane forms of TNF at picomolar concentrations. The skilled person will be able to generate infliximab derivatives using conservative mutations and / or knowledge of the infliximab inhibition mechanism of TNF (see, e.g., Liang, S., et al., 2013. Journal of Biological Chemistry, 288(19), pp.13799-13807).

[0129] In some embodiments, the TNF inhibitor is a fragment of infliximab or a derivative thereof. In some embodiments, the TNF inhibitor is infliximab Fab, infliximab F(ab')2, infliximab scFv or infliximab sdAb or a derivative thereof. In some embodiments, the TNF inhibitor is infliximab Fab or a derivative thereof.

[0130] In some embodiments, the TNF inhibitor is or is derived from an anti-TNF antibody (e.g., a Fab, F(ab')2, scFv, or sdAb), wherein the antibody comprises one or more CDR regions selected from SEQ ID NOs: 12-17 or variants thereof. In other words, in some embodiments, the TNF inhibitor comprises one or more CDR regions selected from SEQ ID NOs: 12-17 or variants thereof.

[0131] In some embodiments, the TNF inhibitor comprises:

[0132] (i) a HCDR1 having an amino acid sequence of SEQ ID NO: 12 or a variant thereof, a HCDR2 having an amino acid sequence of SEQ ID NO: 13 or a variant thereof, and / or a HCDR3 having an amino acid sequence of SEQ ID NO: 14 or a variant thereof; and / or

[0133] (ii) LCDR1 having the amino acid sequence of SEQ ID NO: 15 or a variant thereof, LCDR2 having the amino acid sequence of SEQ ID NO: 16 or a variant thereof, and / or LCDR3 having the amino acid sequence of SEQ ID NO: 17 or a variant thereof.

[0134] In some embodiments, the TNF inhibitor comprises:

[0135] (i) a HCDR1 having an amino acid sequence of SEQ ID NO: 12 or a variant thereof, a HCDR2 having an amino acid sequence of SEQ ID NO: 13 or a variant thereof, and a HCDR3 having an amino acid sequence of SEQ ID NO: 14 or a variant thereof; and / or

[0136] (ii) LCDR1 having the amino acid sequence of SEQ ID NO: 15 or a variant thereof, LCDR2 having the amino acid sequence of SEQ ID NO: 16 or a variant thereof, and LCDR3 having the amino acid sequence of SEQ ID NO: 17 or a variant thereof.

[0137] In some embodiments, the TNF inhibitor comprises or consists of a heavy chain, wherein the heavy chain comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical, or 100% identical to SEQ ID NO:18.

[0138] In some embodiments, the TNF inhibitor comprises or consists of a light chain, wherein the light chain comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical, or 100% identical to SEQ ID NO:19.

[0139] In some embodiments, the TNF inhibitor comprises or consists of a heavy chain and a light chain, wherein the heavy chain comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical, or 100% identical to SEQ ID NO:18, and the light chain comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical, or 100% identical to SEQ ID NO:19.

[0140] In some embodiments, the TNF inhibitor comprises or consists of:

[0141] (i) a heavy chain comprising an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 18, wherein the amino acid sequence comprises a HCDR1 having the amino acid sequence of SEQ ID NO: 12 or a variant thereof, a HCDR2 having the amino acid sequence of SEQ ID NO: 13 or a variant thereof, and a HCDR3 having the amino acid sequence of SEQ ID NO: 14 or a variant thereof; and / or

[0142] (ii) a light chain comprising an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 19, wherein the amino acid sequence comprises a LCDR1 having the amino acid sequence of SEQ ID NO: 15 or a variant thereof, a LCDR2 having the amino acid sequence of SEQ ID NO: 16 or a variant thereof, and a LCDR3 having the amino acid sequence of SEQ ID NO: 17 or a variant thereof.

[0143] In some embodiments, the TNF inhibitor comprises or consists of a heavy chain, wherein the heavy chain comprises or consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical or 100% identical to SEQ ID NO: 20 or 21.

[0144] In some embodiments, the TNF inhibitor comprises or consists of a light chain, wherein the light chain comprises or consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical, or 100% identical to SEQ ID NO:22.

[0145] In some embodiments, the TNF inhibitor comprises or consists of a heavy chain and a light chain, wherein the heavy chain comprises or consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical, or 100% identical to SEQ ID NO:20, and the light chain comprises or consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical, or 100% identical to SEQ ID NO:22.

[0146] In some embodiments, the TNF inhibitor comprises or consists of:

[0147] (i) a heavy chain comprising or consisting of an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 20, wherein the amino acid sequence comprises a HCDR1 having the amino acid sequence of SEQ ID NO: 12, a HCDR2 having the amino acid sequence of SEQ ID NO: 13, and a HCDR3 having the amino acid sequence of SEQ ID NO: 14; and / or

[0148] (ii) a light chain comprising or consisting of an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:22, wherein the amino acid sequence comprises a LCDR1 having the amino acid sequence of SEQ ID NO:15, a LCDR2 having the amino acid sequence of SEQ ID NO:16, and a LCDR3 having the amino acid sequence of SEQ ID NO:17.

[0149]

[0150]

[0151]

[0152] Golimumab

[0153] In some embodiments, the TNF inhibitor is golimumab or a fragment and / or derivative thereof. In some embodiments, the TNF inhibitor is golimumab Fab, golimumab F(ab')2, golimumab scFv, golimumab sdAb or a derivative thereof. In some embodiments, the TNF inhibitor is golimumab Fab or a derivative thereof.

[0154] Golimumab is a human IgG1 TNF antagonist monoclonal antibody. A skilled person will be able to generate golimumab derivatives using conservative mutations and / or knowledge of the golimumab inhibition mechanism of TNF (see, eg, Shealy, DJ, et al., 2010. MAbs, 2(4), pp. 428-439).

[0155] In some embodiments, the TNF inhibitor is a fragment of golimumab or a derivative thereof. In some embodiments, the TNF inhibitor is golimumab Fab, golimumab F(ab')2, golimumab scFv or golimumab sdAb or a derivative thereof. In some embodiments, the TNF inhibitor is golimumab Fab or a derivative thereof.

[0156] In some embodiments, the TNF inhibitor is or is derived from an anti-TNF antibody (e.g., a Fab, F(ab')2, scFv, or sdAb), wherein the antibody comprises one or more CDR regions selected from SEQ ID NOs: 23-28 or variants thereof. In other words, in some embodiments, the TNF inhibitor comprises one or more CDR regions selected from SEQ ID NOs: 23-28 or variants thereof.

[0157] In some embodiments, the TNF inhibitor comprises:

[0158] (i) a HCDR1 having an amino acid sequence of SEQ ID NO: 23 or a variant thereof, a HCDR2 having an amino acid sequence of SEQ ID NO: 24 or a variant thereof, and / or a HCDR3 having an amino acid sequence of SEQ ID NO: 25 or a variant thereof; and / or

[0159] (ii) LCDR1 having the amino acid sequence of SEQ ID NO: 26 or a variant thereof, LCDR2 having the amino acid sequence of SEQ ID NO: 27 or a variant thereof, and / or LCDR3 having the amino acid sequence of SEQ ID NO: 28 or a variant thereof.

[0160] In some embodiments, the TNF inhibitor comprises:

[0161] (i) a HCDR1 having an amino acid sequence of SEQ ID NO: 23 or a variant thereof, a HCDR2 having an amino acid sequence of SEQ ID NO: 24 or a variant thereof, and a HCDR3 having an amino acid sequence of SEQ ID NO: 25 or a variant thereof; and / or

[0162] (ii) LCDR1 having the amino acid sequence of SEQ ID NO: 26 or a variant thereof, LCDR2 having the amino acid sequence of SEQ ID NO: 27 or a variant thereof, and LCDR3 having the amino acid sequence of SEQ ID NO: 28 or a variant thereof.

[0163] In some embodiments, the TNF inhibitor comprises or consists of a heavy chain, wherein the heavy chain comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical, or 100% identical to SEQ ID NO:29.

[0164] In some embodiments, the TNF inhibitor comprises or consists of a light chain, wherein the light chain comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical, or 100% identical to SEQ ID NO:30.

[0165] In some embodiments, the TNF inhibitor comprises or consists of a heavy chain and a light chain, wherein the heavy chain comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical, or 100% identical to SEQ ID NO:29, and the light chain comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical, or 100% identical to SEQ ID NO:30.

[0166] In some embodiments, the TNF inhibitor comprises or consists of:

[0167] (i) a heavy chain comprising an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 29, wherein the amino acid sequence comprises a HCDR1 having the amino acid sequence of SEQ ID NO: 23 or a variant thereof, a HCDR2 having the amino acid sequence of SEQ ID NO: 24 or a variant thereof, and a HCDR3 having the amino acid sequence of SEQ ID NO: 25 or a variant thereof; and / or

[0168] (ii) a light chain comprising an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:30, wherein the amino acid sequence comprises a LCDR1 having the amino acid sequence of SEQ ID NO:26 or a variant thereof, a LCDR2 having the amino acid sequence of SEQ ID NO:27 or a variant thereof, and a LCDR3 having the amino acid sequence of SEQ ID NO:28 or a variant thereof.

[0169] In some embodiments, the TNF inhibitor comprises or consists of a heavy chain, wherein the heavy chain comprises or consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical or 100% identical to SEQ ID NO:31 or 32.

[0170] In some embodiments, the TNF inhibitor comprises or consists of a light chain, wherein the light chain comprises or consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical, or 100% identical to SEQ ID NO:33.

[0171] In some embodiments, the TNF inhibitor comprises or consists of a heavy chain and a light chain, wherein the heavy chain comprises or consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical, or 100% identical to SEQ ID NO:31, and the light chain comprises or consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 96%, at least 97%, at least 98%, at least 99% identical, or 100% identical to SEQ ID NO:33.

[0172] In some embodiments, the TNF inhibitor comprises or consists of:

[0173] (i) a heavy chain comprising or consisting of an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:31, wherein the amino acid sequence comprises a HCDR1 having the amino acid sequence of SEQ ID NO:23, a HCDR2 having the amino acid sequence of SEQ ID NO:24, and a HCDR3 having the amino acid sequence of SEQ ID NO:25; and / or

[0174] (ii) a light chain comprising or consisting of an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:33, wherein the amino acid sequence comprises a LCDR1 having the amino acid sequence of SEQ ID NO:26, a LCDR2 having the amino acid sequence of SEQ ID NO:27, and a LCDR3 having the amino acid sequence of SEQ ID NO:28.

[0175]

[0176]

[0177]

[0178] Certolizumab

[0179] In some embodiments, the TNF inhibitor is certolizumab pegol or a fragment and / or derivative thereof. In some embodiments, the TNF inhibitor is certolizumab pegol Fab, certolizumab pegol F(ab')2, certolizumab pegol scFv, certolizumab pegol sdAb or a derivative thereof. In some embodiments, the TNF inhibitor is certolizumab pegol Fab or a derivative thereof.

[0180] Certolizumab is a humanized antigen-binding fragment (Fab') of a monoclonal antibody, usually conjugated to polyethylene glycol. Administration. A skilled person will be able to generate certolizumab derivatives using conservative mutations and / or knowledge of the certolizumab inhibition mechanism of TNF (see, for example, Lee, JU, et al., 2017. International journal of molecular sciences, 18(1), p. 228).

[0181] In some embodiments, the TNF inhibitor is a fragment of certolizumab pegol or a derivative thereof. In some embodiments, the TNF inhibitor is certolizumab pegol Fab, certolizumab pegol F(ab')2, certolizumab pegol scFv or certolizumab pegol sdAb or a derivative thereof. In some embodiments, the TNF inhibitor is certolizumab pegol Fab or a derivative thereof.

[0182] In some embodiments, the TNF inhibitor is or is derived from an anti-TNF antibody (e.g., a Fab, F(ab')2, scFv, or sdAb), wherein the antibody comprises one or more CDR regions selected from SEQ ID NOs: 34-39 or variants thereof. In other words, in some embodiments, the TNF inhibitor comprises one or more CDR regions selected from SEQ ID NOs: 34-39 or variants thereof.

[0183] In some embodiments, the TNF inhibitor comprises:

[0184] (i) a HCDR1 having an amino acid sequence of SEQ ID NO: 34 or a variant thereof, a HCDR2 having an amino acid sequence of SEQ ID NO: 35 or a variant thereof, and / or a HCDR3 having an amino acid sequence of SEQ ID NO: 36 or a variant thereof; and / or

[0185] (ii) LCDR1 having the amino acid sequence of SEQ ID NO: 37 or a variant thereof, LCDR2 having the amino acid sequence of SEQ ID NO: 38 or a variant thereof, and / or LCDR3 having the amino acid sequence of SEQ ID NO: 39 or a variant thereof.

[0186] In some embodiments, the TNF inhibitor comprises:

[0187] (i) a HCDR1 having an amino acid sequence of SEQ ID NO: 34 or a variant thereof, a HCDR2 having an amino acid sequence of SEQ ID NO: 35 or a variant thereof, and a HCDR3 having an amino acid sequence of SEQ ID NO: 36 or a variant thereof; and / or

[0188] (ii) LCDR1 having the amino acid sequence of SEQ ID NO: 37 or a variant thereof, LCDR2 having the amino acid sequence of SEQ ID NO: 38 or a variant thereof, and LCDR3 having the amino acid sequence of SEQ ID NO: 39 or a variant thereof.

[0189] In some embodiments, the TNF inhibitor comprises or consists of a heavy chain, wherein the heavy chain comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical, or 100% identical to SEQ ID NO:40.

[0190] In some embodiments, the TNF inhibitor comprises or consists of a light chain, wherein the light chain comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical, or 100% identical to SEQ ID NO:41.

[0191] In some embodiments, the TNF inhibitor comprises or consists of a heavy chain and a light chain, wherein the heavy chain comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical, or 100% identical to SEQ ID NO:40, and the light chain comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical, or 100% identical to SEQ ID NO:41.

[0192] In some embodiments, the TNF inhibitor comprises or consists of:

[0193] (i) a heavy chain comprising an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:40, wherein the amino acid sequence comprises a HCDR1 having the amino acid sequence of SEQ ID NO:34 or a variant thereof, a HCDR2 having the amino acid sequence of SEQ ID NO:35 or a variant thereof, and a HCDR3 having the amino acid sequence of SEQ ID NO:36 or a variant thereof; and / or

[0194] (ii) a light chain comprising an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:41, wherein the amino acid sequence comprises a LCDR1 having the amino acid sequence of SEQ ID NO:37 or a variant thereof, a LCDR2 having the amino acid sequence of SEQ ID NO:38 or a variant thereof, and a LCDR3 having the amino acid sequence of SEQ ID NO:39 or a variant thereof.

[0195] In some embodiments, the TNF inhibitor comprises or consists of a heavy chain, wherein the heavy chain comprises or consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical or 100% identical to SEQ ID NO:42 or 43.

[0196] In some embodiments, the TNF inhibitor comprises or consists of a light chain, wherein the light chain comprises or consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical, or 100% identical to SEQ ID NO:44.

[0197] In some embodiments, the TNF inhibitor comprises or consists of a heavy chain and a light chain, wherein the heavy chain comprises or consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical, or 100% identical to SEQ ID NO:42, and the light chain comprises or consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical, or 100% identical to SEQ ID NO:44.

[0198] In some embodiments, the TNF inhibitor comprises or consists of:

[0199] (i) a heavy chain comprising or consisting of an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:42, wherein the amino acid sequence comprises a HCDR1 having the amino acid sequence of SEQ ID NO:34, a HCDR2 having the amino acid sequence of SEQ ID NO:35, and a HCDR3 having the amino acid sequence of SEQ ID NO:36; and / or

[0200] (ii) a light chain comprising or consisting of an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:44, wherein the amino acid sequence comprises a LCDR1 having the amino acid sequence of SEQ ID NO:34, a LCDR2 having the amino acid sequence of SEQ ID NO:35, and a LCDR3 having the amino acid sequence of SEQ ID NO:36.

[0201]

[0202]

[0203] TNFR domain

[0204] In some embodiments, the TNF inhibitor comprises the TNF binding domain of a TNF receptor (TNFR).

[0205] TNF signals through two receptors (TNFR1 and TNFR2) that share a similar structural arrangement, with an N-terminal extracellular domain (ECD) consisting of four cysteine-rich domains (CRDs), an α-helical transmembrane domain, and a cytoplasmic domain. The two receptors differ most in the cytoplasmic domain, with TNFR1 having a death domain that is absent in TNFR2 (see, e.g., Bodmer, JL, et al. Trends in biochemical sciences, 27(1), pp. 19-26).

[0206] In some embodiments, the TNF inhibitor comprises a soluble form of a TNF receptor (TNFR). In some embodiments, the TNF inhibitor comprises a soluble form of TNFR1 or TNFR2. In some embodiments, the TNF inhibitor comprises or consists of a soluble form of TNFR2.

[0207] The TNF binding domain and / or the soluble form of the TNF receptor may be fused to any suitable domain. Suitably, the TNF binding domain and / or the soluble form of the TNF receptor may be coupled to an Fc domain (eg the Fc portion of human IgG1).

[0208] Etanercept

[0209] In some embodiments, the TNF inhibitor is etanercept or a derivative thereof.

[0210] Etanercept is a fusion protein consisting of the TNFR2 domain coupled to the Fc portion of human IgG1. A skilled person will be able to generate etanercept derivatives using conservative mutations and / or knowledge of the etanercept inhibition mechanism of TNF (see, e.g., Lamanna, WC, et al., 2017. Scientific reports, 7(1), pp. 1-8).

[0211] In some embodiments, the TNF inhibitor comprises or consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical, or 100% identical to SEQ ID NO: 45. In some embodiments, the TNF inhibitor comprises or consists of an amino acid sequence of SEQ ID NO: 45.

[0212] LPAQVAFTPYAPEPGSTCRLREYYDQTAQMCCSKCSPGQHAKVFCTKTSDTVCDSCEDSTYTQLWNWVPECLSCGSRCSSDQVETQACTREQNRICTCRPGWYCALSKQEGCRLCA PLRKCRPGFGVARPGTETSDVVCKPCAPGTFSNTTSSTDICRPHQICNVVAIPGNASMDAVCTSTSPTRSMAPGAVHLPQPVSTRSQHTQPTPEPSTAPSTSFLLPMGPSPPAEGST GDEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPA PIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0213] Example Etanercept sequence (SEQ ID NO: 45)

[0214] Other TNF inhibitors

[0215] In some embodiments, the TNF inhibitor is a fusion protein consisting of the extracellular domain of TNFR1 coupled to the Fc portion of human IgG1.

[0216] In some embodiments, the TNF inhibitor comprises or consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical, or 100% identical to SEQ ID NO: 93. In some embodiments, the TNF inhibitor comprises or consists of an amino acid sequence of SEQ ID NO: 93.

[0217] MGLSTVPDLLLPLVLLELLVGIYPSGVIGLVPHLGDREKRDSVCPQGKYIHPQNNSICCTKCHKGTYLYNDCPGPGQDTDCRECESGSFTASENHLRHCLSCSKCRKEM GQVEISSCTVDRDTVCGCRKNQYRHYWSENLFQCFNCSLCLNGTVHLSCQEKQNTVCTCHAGFFLRENECVSCSNCKKSLECTKLCLPQIENVKGTEDSGTTDKTHTCPP CPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0218] Example fusion protein sequence (SEQ ID NO:93)

[0219] Signal peptide

[0220] The TNF inhibitor may be operably linked to one or more signal peptides.

[0221] "Signal peptide" may refer to a short peptide that directs protein insertion into the membrane of the endoplasmic reticulum. Signal peptides are typically N-terminal extensions of newly synthesized secretory and membrane proteins, with a length of 16 to 30 amino acid residues, and are composed of a hydrophilic, usually positively charged N-terminal region, a central hydrophobic domain, and a C-terminal region with a cleavage site for a signal peptidase. In addition to these common features, signal peptides do not share sequence similarity, and some signal peptides are more than 50 amino acid residues in length (see, e.g., Kapp, K., et al., 2009. Protein transport into the endoplasmicreticulum, pp. 1-16).

[0222] TNF inhibitors can be operably linked to any suitable signal peptide. The SPdb signal peptide database is a repository of experimentally determined and computationally predicted signal peptides (see, e.g., Choo, KH, et al., 2005. BMC bioinformatics, 6 (1), pp. 1-8). Suitable signal peptides include human growth hormone (HGH) signal peptide, interleukin-2 (IL-2) signal peptide, CD5 signal peptide, immunoglobulin kappa light chain signal peptide, trypsinogen signal peptide, serum albumin signal peptide or prolactin signal peptide.

[0223] In some embodiments, the TNF inhibitor is operably linked to one or more signal peptides selected from any one of the following: human growth hormone (HGH) signal peptide, interleukin-2 (IL-2) signal peptide, CD5 signal peptide, immunoglobulin kappa light chain signal peptide, trypsinogen signal peptide, serum albumin signal peptide and prolactin signal peptide. In some embodiments, the TNF inhibitor is operably linked to one or more human growth hormone (HGH) signal peptides.

[0224] In some embodiments, the TNF inhibitor is operably linked to one or more signal peptides comprising or consisting of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical, or 100% identical to SEQ ID NO: 46. In some embodiments, the TNF inhibitor is operably linked to one or more signal peptides comprising or consisting of the amino acid sequence of SEQ ID NO: 46.

[0225] MATGSRTSLLLAFGLLCLPWLQEGSA

[0226] Example HGH signal peptide (SEQ ID NO: 46)

[0227] Example Nucleotide Sequence

[0228] The TNF inhibitor may be encoded by any suitable nucleotide sequence.

[0229] In some embodiments, the nucleotide sequence is codon optimized, for example, codon optimized for expression in humans. Different cells differ in the selection of their specific codons. This codon bias corresponds to the bias in the relative abundance of a specific tRNA in a cell type. By changing the codons in the sequence, it is customized to match the relative abundance of the corresponding tRNA, and expression may be increased. For the same reason, expression may be reduced by deliberately selecting codons known to be rare in a specific cell type for the corresponding tRNA. Therefore, an additional degree of translational control may be obtained. For mammalian cells (e.g., humans) and for a variety of other organisms, codon selection tables are known in the art.

[0230] In a preferred embodiment, the TNF inhibitor is an anti-TNF antigen binding fragment (Fab). Suitably, the nucleotide sequence encoding the anti-TNF Fab may comprise, from 5' to 3': a first signal sequence; nucleotides encoding the heavy chain; a linker sequence; a second signal sequence; and nucleotides encoding the light chain. Suitably, the nucleotide sequence encoding the anti-TNF Fab may comprise, from 5' to 3': a first signal sequence; nucleotides encoding the light chain; a linker sequence; a second signal sequence; and nucleotides encoding the heavy chain.

[0231] Heavy and light chains

[0232] The nucleotides encoding the heavy chain and the nucleotides encoding the light chain can encode any heavy chain and light chain combination described herein. For example, the nucleotide sequence encoding the anti-TNF Fab can include from 5' to 3':

[0233] (a) a first signal sequence; a nucleotide encoding an adalimumab heavy chain or a derivative thereof; a linker sequence; a second signal sequence; and a nucleotide encoding an adalimumab light chain or a derivative thereof;

[0234] (b) a first signal sequence; a nucleotide encoding an infliximab heavy chain or a derivative thereof; a linker sequence; a second signal sequence; and a nucleotide encoding an infliximab light chain or a derivative thereof;

[0235] (c) a first signal sequence; nucleotides encoding a golimumab heavy chain or a derivative thereof; a linker sequence; a second signal sequence; and nucleotides encoding a golimumab light chain or a derivative thereof;

[0236] (d) a first signal sequence; a nucleotide encoding a certolizumab pegol heavy chain or a derivative thereof; a linker sequence; a second signal sequence; and a nucleotide encoding a certolizumab pegol light chain or a derivative thereof;

[0237] (e) a first signal sequence; nucleotides encoding an adalimumab light chain or a derivative thereof; a linker sequence; a second signal sequence; and nucleotides encoding an adalimumab heavy chain or a derivative thereof;

[0238] (f) a first signal sequence; nucleotides encoding an infliximab light chain or a derivative thereof; a linker sequence; a second signal sequence; and nucleotides encoding an infliximab heavy chain or a derivative thereof;

[0239] (g) a first signal sequence; nucleotides encoding a golimumab light chain or a derivative thereof; a linker sequence; a second signal sequence; and nucleotides encoding a golimumab heavy chain or a derivative thereof; or

[0240] (h) a first signal sequence; nucleotides encoding a certolizumab pegol light chain or a derivative thereof; a linker sequence; a second signal sequence; and nucleotides encoding a certolizumab pegol heavy chain or a derivative thereof.

[0241] In some embodiments, the nucleotide sequence encoding anti-TNF Fab may include from 5' to 3': a first signal sequence; nucleotides encoding adalimumab heavy chain or its derivatives; a linker sequence; a second signal sequence; and nucleotides encoding adalimumab light chain or its derivatives. In some embodiments, the nucleotide sequence encoding anti-TNF Fab may include from 5' to 3': a first signal sequence; nucleotides encoding adalimumab light chain or its derivatives; a linker sequence; a second signal sequence; and nucleotides encoding adalimumab heavy chain or its derivatives.

[0242] In some embodiments, the nucleotide sequence encoding the heavy chain of adalimumab comprises or consists of a nucleotide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical, or 100% identical to SEQ ID NO: 47. In some embodiments, the nucleotide sequence encoding the heavy chain of adalimumab comprises or consists of SEQ ID NO: 47.

[0243] gaggtgcaactggtggaaagcggcggcggcctggtccagcctggaaggtccctgagactgagctgtgccgccagcggctttaccttcgacgactacgccatgcactgggtgcgccaggcccctggcaagggcctggaatgggtctccgccatcacctggaatag cggccacatcgactacgccgatagcgtggaaggcagattcaccatcagccgggacaacgccaagaactctctgtatctgcaaatgaacagcctgcgggctgaagatacagccgtgtactattgcgccaaagtgagctacctctccaccgccagcagcctggact attggggacagggcaccctggtgaccgtgtctagcgcctccacaaagggcccttctgtgtttccactggctccaagctccaaaagcacatctggaggaaccgctgccctgggctgcctggttaaggactacttccccgagcctgtgaccgtgagctggaacagc ggcgccctgacatctggtgttcataccttccctgccgttctgcaatcttctggactctacagcctgtcttctgtggtgaccgtgcccagcagcagccttggaacacagacctacatctgcaatgtgaaccacaagcctagcaacaccaaggtggacaagaaggtg

[0244] Example nucleotide sequence encoding adalimumab heavy chain (SEQ ID NO: 47)

[0245] In some embodiments, the nucleotide sequence encoding the adalimumab light chain comprises or consists of a nucleotide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical or 100% identical to SEQ ID NO: 48. In some embodiments, the nucleotide sequence encoding the adalimumab light chain comprises or consists of SEQ ID NO: 48.

[0246] gacatccagatgacccagagcccttcctcactgagcgccagcgtgggcgacagagtgactattacatgcagagccagccaaggcatccggaactacctggcctggtatcagcagaagcccggcaaagcccctaagctgctgatctacgccgccagcaccc tgcaaagcggcgtgcctagcagattcagcggctcaggctctggcactgatttcaccctgaccatctcctctctgcaacctgaggacgtggccacatactactgccagagatacaacagagccccatacacctttggccagggcacaaaagtggaaatcaag agaaccgtggccgctcccagtgtgttcatcttcccccccagtgatgagcagctgaagtccggcacagcctctgtcgtgtgcctgctgaacaacttctaccccagagaggccaaggtgcagtggaaggtggataatgccctgcaaagcggcaacagccagg agagcgtgacagagcaggacagcaaggacagcacctacagcctctctagcacactgaccctgagcaaggccgactacgagaagcacaaggtgtacgcatgcgaggtgacccaccagggcctgagcagtcctgtgaccaagagcttcaaccggggcgagtgt

[0247] Example nucleotide sequence encoding adalimumab light chain (SEQ ID NO: 48)

[0248] In some embodiments, the nucleotide sequence encoding anti-TNF Fab may include from 5' to 3': a first signal sequence; nucleotides encoding the heavy chain of infliximab or its derivatives; a linker sequence; a second signal sequence; and nucleotides encoding the light chain of infliximab or its derivatives. In some embodiments, the nucleotide sequence encoding anti-TNF Fab may include from 5' to 3': a first signal sequence; nucleotides encoding the light chain of infliximab or its derivatives; a linker sequence; a second signal sequence; and nucleotides encoding the heavy chain of infliximab or its derivatives.

[0249] In some embodiments, the nucleotide sequence encoding the heavy chain of infliximab comprises or consists of a nucleotide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical, or 100% identical to SEQ ID NO: 49. In some embodiments, the nucleotide sequence encoding the light chain of infliximab comprises or consists of SEQ ID NO: 49.

[0250] gaggtgaaactggaggaaagtggaggaggcctcgttcaaccaggcggctccatgaagctgtcgtgtgtggcatctggcttcatcttcagcaatcactggatgaactgggtcaggcaatctcctgagaaggggctagagtgggtggcggagatccgctcaaaat caatcaattccgccacacattatgcagagtcagtaaaagggcggttcaccatttctagagatgacagcaaaagcgccgtgtacctccagatgaccgacctgcgaacagaggacactggggtctactactgctcccggaactactatggctccacctatgactac tggggccaagggaccacattgacagtatcctcagcctccactaaaggtccttcagtgtttccgctggctccctcctccaaaagtacgtcaggcggcaccgctgctctgggctgtctggtgaaggattacttccctgaacctgtgactgtttcctggaacagtg gagccttgacttcaggagtccacacatttccggcagtgctccagagcagtggtctctattccctaagcagtgtagtgaccgtgccctctagcagcctcggaacccagacatacatctgcaatgtcaatcacaagccaagcaatacaaaagtggacaagaaggtt

[0251] Example nucleotide sequence encoding the heavy chain of infliximab (SEQ ID NO: 49)

[0252] In some embodiments, the nucleotide sequence encoding the heavy chain of infliximab comprises or consists of a nucleotide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical, or 100% identical to SEQ ID NO: 50. In some embodiments, the nucleotide sequence encoding the light chain of infliximab comprises or consists of SEQ ID NO: 50.

[0253] gacattctcctgacccagtcccctgctatcttgtctgtctcccccggagagcgcgtctccttctcttgcagagcttcccagtttgtgggcagcagcattcactggtatcagcagagaacaaatggatcaccaaggcttttgatcaagtatgcttcagaaa gcatgagtgggataccatccaggtttagtggaagtggctctggtactgacttcactctctctctataaacacggtggaaagcgaagatattgctgactattactgtcagcaaagccatagctggccatttacttttggatcagggaccaacctggaagtcaag agaactgtggccgcgccttcggtttttattttccccccatctgatgaacagctgaagagcggtacagccagtgtagtgtgcctgctcaacaacttctaccctagagaagccaaggtgcagtggaaggtcgacaatgcattacagagcgggaacagccagg aaagtgttactgagcaggatagcaaggacagcacctactctctgtctagcacactcactttgtctaaagcagattatgagaaacataaagtttatgcctgtgaagttacccaccagggcctgagcagtcccgtcaccaagtctttcaaccgcggggagtgc

[0254] Example nucleotide sequence encoding the light chain of infliximab (SEQ ID NO: 50)

[0255] signal sequence

[0256] The first signal sequence and the second signal sequence can encode the same signal peptide or different signal peptides. In some embodiments, the first signal sequence and the second signal sequence encode the same signal peptide. The first signal sequence and the second signal sequence can be any signal sequence described herein.

[0257] In some embodiments, the first signal sequence and / or the second signal sequence encodes any one of the following: a human growth hormone (HGH) signal peptide, an interleukin-2 (IL-2) signal peptide, a CD5 signal peptide, an immunoglobulin kappa light chain signal peptide, a trypsinogen signal peptide, a serum albumin signal peptide, and a prolactin signal peptide.

[0258] In some embodiments, the first signal sequence and / or the second signal sequence encodes a human growth hormone (HGH) signal peptide. In some embodiments, the first signal sequence and the second signal sequence encode a human growth hormone (HGH) signal peptide.

[0259] In some embodiments, the first signal sequence and / or the second signal sequence comprises or consists of a nucleotide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical or 100% identical to any one of SEQ ID NOs: 51-54. In some embodiments, the first signal sequence and / or the second signal sequence comprises or consists of a nucleotide sequence of any one of SEQ ID NOs: 51-54.

[0260] atggcaacaggtagtcgaaccagcctattactggccttcggtctcctgtgtctgccctggcttcaagagggctct gc

[0261] Example HGH signal sequence (SEQ ID NO: 51)

[0262] atggccacgggaagtcggacttccttactactcgcctttggtcttctttgcttgccatggctccaggagggtagtg ca

[0263] Example HGH signal sequence (SEQ ID NO: 52)

[0264] atggccacaggctctcggaccagcctgctgctggccttcggcctgctgtgtctgccttggctgcaagagggca gcgcc

[0265] Example HGH signal sequence (SEQ ID NO: 53)

[0266] atggctaccggcagcagaaccagcctgctgctggcattcggccttctgtgcctgccttggctgcaagagggct ctgcc

[0267] Example HGH signal sequence (SEQ ID NO: 54)

[0268] Adapter sequence

[0269] The joint sequence can comprise one or more cleavage sites. As used herein, "cleavage site" can include a nucleotide sequence encoding a specific peptide sequence and a nucleotide sequence encoding a self-cleaving peptide, at which a site-specific protease can cleave or cut (cut) peptides (also referred to as enzymatically cleavable peptide motifs).

[0270] In preferred embodiments, the linker sequence encodes a cleavage site. In some embodiments, the linker sequence encodes a self-cleaving peptide and / or an enzymatically cleavable peptide motif.

[0271] In some embodiments, the linker sequence encodes a 2A self-cleaving peptide. A 2A self-cleaving peptide is a class of 18-22 amino acid long peptides that can induce ribosomal skipping during protein translation in cells. Suitable 2A self-cleaving peptides include T2A, P2A, E2A and F2A or derivatives thereof.

[0272] In some embodiments, the linker sequence encodes a 2A self-cleaving peptide having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity, or 100% identity to any one of SEQ ID NOs: 55-58. In some embodiments, the linker sequence encodes a 2A self-cleaving peptide comprising or consisting of any one of SEQ ID NOs: 55-58.

[0273] In some embodiments, the linker sequence encodes a 2A self-cleaving peptide that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical, or 100% identical to SEQ ID NO: 55. In some embodiments, the linker sequence encodes a 2A self-cleaving peptide comprising or consisting of SEQ ID NO: 55.

[0274] ATNFSLLKQAGDVEENPGP

[0275] Example P2A peptide sequence (SEQ ID NO:55)

[0276] EGRGSLLTCGDVEENPGP

[0277] Example T2A peptide sequence (SEQ ID NO:56)

[0278] QCTNYALLKLAGDVESNPGP

[0279] Example E2A peptide sequence (SEQ ID NO:57)

[0280] VKQTLNFDLLKLAGDVESNPGP

[0281] Example F2A peptide sequence (SEQ ID NO:58)

[0282] In some embodiments, the linker sequence comprises a nucleotide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical, or 100% identical to any one of SEQ ID NOs: 59-60. In some embodiments, the linker sequence comprises any one of SEQ ID NOs: 59-60.

[0283] gctacgaatttttcattgctcaagcaagcggggagatgtggaggagaaccctggcccc

[0284] Example P2A nucleotide sequence (SEQ ID NO: 59)

[0285] gctaccaacttcagcctcctgaaacaggccggcgatgtggaggaaaaccctggacct

[0286] Example P2A nucleotide sequence (SEQ ID NO: 60)

[0287] The linker sequence may comprise any other suitable nucleotide sequence, such as a nucleotide sequence that facilitates expression of the anti-TNF Fab. Suitably, the linker sequence may comprise a furin site and / or a fusion protein linker sequence.

[0288] In some embodiments, the linker sequence comprises a furin site. Furin is a protease that can cut at the conserved polybasic RNRR site. Suitably, the furin site encodes RKRR. In some embodiments, the linker sequence comprises a fusion protein linker sequence. The fusion protein linker sequence can join two protein domains together. Suitably, the fusion protein linker sequence encodes SGSG.

[0289] In some embodiments, the linker sequence encodes from 5' to 3': a furin site, a fusion protein linker sequence, and a 2A self-cleaving peptide.

[0290] In some embodiments, the linker sequence encodes an amino acid that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical, or 100% identical to SEQ ID NO: 61. In some embodiments, the linker sequence encodes an amino acid that comprises or consists of SEQ ID NO: 61.

[0291] RKRRSGSGATNFSLLKQAGDVEENPGP

[0292] Example linker peptide sequence (SEQ ID NO:61)

[0293] In some embodiments, the linker sequence comprises or consists of a nucleotide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical or 100% identical to any one of SEQ ID NOs: 62-63. In some embodiments, the linker sequence comprises or consists of any one of SEQ ID NOs: 62-63.

[0294] aggaagcggcgttccgggagcggggctacgaatttttcattgctcaagcaagcggggagatgtggaggagaa ccctggcccc

[0295] Example linker sequence (SEQ ID NO: 62)

[0296] agaaagcggagaagcggaagcggtgctaccaacttcagcctcctgaaacaggccggcgatgtggaggaaa accctggacct

[0297] Example linker sequence (SEQ ID NO: 63)

[0298] Other sequences

[0299] The nucleotide sequence encoding the anti-TNF Fab may comprise any other suitable sequence. For example, the anti-TNF Fab may comprise an HA tag for detection. Suitably, the HA tag may comprise SEQ ID NO: 64 or consist of SEQ ID NO: 64.

[0300] YPYDVPDYA

[0301] Example HA tag (SEQ ID NO: 64)

[0302] Example anti-TNF Fab sequence

[0303] In some embodiments, the nucleotide sequence encoding the anti-TNF Fab encodes an amino acid sequence from 5' to 3' comprising or consisting of: an amino acid sequence that has at least 70% identity to SEQ ID NO:46, an amino acid sequence that has at least 70% identity to SEQ ID NO:7, an amino acid sequence that has at least 70% sequence identity to any one of SEQ ID NOs:55-58, an amino acid sequence that has at least 70% identity to SEQ ID NO:46, and an amino acid sequence that has at least 70% identity to SEQ ID NO:8.

[0304] In some embodiments, the nucleotide sequence encoding the anti-TNF Fab encodes an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical, or 100% identical to SEQ ID NO: 65. In some embodiments, the nucleotide sequence encoding the anti-TNF Fab encodes the amino acid sequence of SEQ ID NO: 65.

[0305] MATGSRTSLLLAFGLLCLPWLQEGSAEVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSAITWNSGHIDYADSVEGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAKVSYL STASSLDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVRKRRSGSGATN FSLLKQAGDVEENPGPMATGSRTSLLLAFGLLCLPWLQEGSADIQMTQSPSSSLSASVGDRVTITCRASQGIRNYLAWYQQKPGKAPKLLIYAASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDVATY YCQRYNRAPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0306] Example Adalimumab Fab amino acid sequence (SEQ ID NO: 65)

[0307] In some embodiments, the nucleotide sequence encoding the anti-TNF Fab comprises or consists of, from 5' to 3', a nucleotide sequence having at least 70% identity to any one of SEQ ID NOs: 51-54, a nucleotide sequence having at least 70% identity to SEQ ID NO: 47, a nucleotide sequence having at least 70% sequence identity to SEQ ID NOs: 59 or 60, a nucleotide sequence having at least 70% identity to any one of SEQ ID NOs: 51-54, and a nucleotide sequence having at least 70% identity to SEQ ID NO: 48.

[0308] In some embodiments, the nucleotide sequence encoding the anti-TNF Fab comprises or consists of a nucleotide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical, or 100% identical to SEQ ID NO: 66. In some embodiments, the nucleotide sequence encoding the anti-TNF Fab comprises or consists of SEQ ID NO: 66.

[0309]

[0310] Example Adalimumab Fab nucleotide sequence (SEQ ID NO: 66)

[0311] In some embodiments, the nucleotide sequence encoding the anti-TNF Fab encodes an amino acid sequence from 5' to 3' comprising or consisting of: an amino acid sequence that has at least 70% identity to SEQ ID NO:46, an amino acid sequence that has at least 70% identity to SEQ ID NO:18, an amino acid sequence that has at least 70% sequence identity to any one of SEQ ID NOs:55-58, an amino acid sequence that has at least 70% identity to SEQ ID NO:46, and an amino acid sequence that has at least 70% identity to SEQ ID NO:19.

[0312] In some embodiments, the nucleotide sequence encoding the anti-TNF Fab encodes an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical, or 100% identical to SEQ ID NO: 67. In some embodiments, the nucleotide sequence encoding the anti-TNF Fab encodes the amino acid sequence of SEQ ID NO: 67.

[0313] MATGSRTSLLLAFGLLCLPWLQEGSAEVKLEESGGGLVQPGGSMKLSCVASGFIFSNHWMNWVRQSPEKGLEWVAEIRSKSINSATHYAESVKGRFTISRDDSKSAVYLQMTDLRTEDTGVYYCSRN YYGSTYDYWGQGTTLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVRKRRSGSGATN FSLLKQAGDVEENPGPMATGSRTSLLLAFGLLCLPWLQEGSADILLTQSPAILSVSPGERVSFSCRASQFVGSSIHWYQQRTNGSPRLLIKYASESMSGIPSRFSGSGSGTDFTLSINTVESEDIADY YCQQSHSWPFTFGSGTNLEVKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0314] Example Infliximab Fab amino acid sequence (SEQ ID NO: 67)

[0315] In some embodiments, the nucleotide sequence encoding the anti-TNF Fab comprises or consists of, from 5' to 3', a nucleotide sequence having at least 70% identity to any one of SEQ ID NOs: 51-54, a nucleotide sequence having at least 70% identity to SEQ ID NO:49, a nucleotide sequence having at least 70% sequence identity to SEQ ID NOs: 59 or 60, a nucleotide sequence having at least 70% identity to any one of SEQ ID NOs: 51-54, and a nucleotide sequence having at least 70% identity to SEQ ID NO:50.

[0316] In some embodiments, the nucleotide sequence encoding the anti-TNF Fab comprises or consists of a nucleotide sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical, or 100% identical to SEQ ID NO: 68. In some embodiments, the nucleotide sequence encoding the anti-TNF Fab comprises or consists of SEQ ID NO: 68.

[0317]

[0318] Example Infliximab Fab nucleotide sequence (SEQ ID NO: 68)

[0319] Promoter

[0320] The vector of the present invention may comprise a promoter, preferably an inflammation-inducible promoter. Suitably, the promoter may be operably linked to a nucleotide sequence encoding a TNF inhibitor (e.g., an anti-TNF antibody fragment). The term "operably linked" may mean that the components are in a relationship that allows them to function in their intended manner.

[0321] "Promoter" may refer to a region of DNA that causes the initiation of transcription of a gene. The promoter is usually located near the transcription start site of the gene, upstream of the DNA (towards the 5' region of the sense strand). Any suitable promoter may be used, and the technician may easily make this selection.

[0322] The promoter generally comprises a "core" region and a "proximal" region. A "core promoter region" may comprise promoter elements, such as a transcription start site, an RNA polymerase binding site, and general transcription factor binding sites (e.g., a TATA box, a B recognition element). A "proximal promoter region" may comprise, for example, major regulatory elements and specific transcription factor binding sites required for promoting effective and controllable transcription. The size and composition of both the core promoter region and the proximal promoter region generally vary in a gene-specific manner.

[0323] In some embodiments, the promoter is an ocular tissue-specific promoter. As used herein, an "ocular tissue-specific promoter" is a promoter that preferentially promotes gene expression in ocular cells (eg, photoreceptors, RPE cells, retinal ganglion cells, etc.).

[0324] In other embodiments, the promoter is a constitutive promoter. As used herein, a "constitutive promoter" is a promoter that is always active. Exemplary constitutive promoters include the chicken beta-actin (CBA) promoter or a variant or fragment thereof. The promoter may comprise or consist of the CAG promoter or a variant or fragment thereof.

[0325] Inflammation-inducible promoter

[0326] The vector of the present invention preferably comprises an inflammation-inducible promoter. Suitably, the nucleotide sequence encoding the TNF inhibitor (e.g., anti-TNF antibody fragment) is operably linked to the inflammation-inducible promoter. The promoter can promote the expression of the TNF inhibitor in response to inflammation.

[0327] Use of an inflammation-inducible promoter is advantageous because TNF inhibitors can be expressed when inflammation occurs at subclinical levels and thereby prevent damage caused by flare-ups before symptoms occur.

[0328] As used herein, "inflammation-inducible promoter" may refer to a promoter that preferentially promotes the expression of an operably linked transgene in response to inflammation. At the tissue level, inflammation may be characterized by redness, swelling, fever, pain, and / or loss of tissue function (see, e.g., Chen, L., et al., 2018. Oncotarget, 9(6), p.7204). At the cellular level, inflammation may be characterized by elevated levels of proinflammatory cytokines, activated immune cells, or acute phase proteins. Inflammation may be considered acute, i.e., an immediate response to a noxious stimulus, or chronic, i.e., long-term inflammation.

[0329] Suitably, an inflammation-inducible promoter can promote higher expression of an operably linked transgene in response to higher levels of inflammation. Higher expression can be measured, for example, by measuring the expression of a transgene (e.g., green fluorescent protein (GFP)) operably linked to a promoter, wherein the expression of the transgene is related to the ability of the promoter to promote gene expression. The level of inflammation can be determined by suitable methods known in the art (see, for example, Menzel et al., 2021, Antioxidants, 10 (3), p.414). For example, the measurement of the level of inflammatory markers can be taken from an appropriate medium, such as a body fluid. Suitable body fluids may include blood, urine, or vitreous humor.

[0330] Suitably, inflammation-inducible promoters can promote higher expression of transgenic responses in ocular cells (e.g., photoreceptors, RPE cells, retinal ganglion cells, etc.) that are operably connected to higher levels of inflammation in ocular cells. The level of inflammation in the eye can be determined by suitable non-invasive methods known in the art. For example, by grading ocular inflammation (see, e.g., McNeil, R., 2016. Eye news, 22 (5), pp. 1-4-19) or by laser flare photometry (laser flarephotometry) (see, e.g., Tugal-Tutkun, I. and Herbort, CP, 2010. International ophthalmology, 30 (5), pp. 453-464).

[0331] Inflammation-inducible promoters can be (or can be derived from) promoters associated with genes with increased expression in response to inflammation. Suitably, inflammation-inducible promoters can be (or can be derived from) promoters associated with genes with increased expression in response to inflammation (e.g., uveitis) in ocular cells. For example, genes with increased expression in uveitis can include CCL16, IL15, CCL7, CYSLTR1, IL17, IL4, CCL8, IL15RA, CCR1, CDC25A, LAT, MEF2B, CSF3, MAP2K7, IL20, TYK2, MAF, COL1A1, IKBKG, MGC27165, IL10, CIITA, NFATC2IP, MEF2D, GSK3A, TH1L, IL3, IL10RB, SMA D9, C19orf10, IL13RA2, TGFB2, LEP, HAVCR2, SOCS5, IRF1, TXLNA, RFX2, FADD, TGIF, CCR4, RFX1, MIF, IL10RA, CXCL5, LTA, IGFBP3, NFKBIB, CXCL13, IKBKE, MEF2A, LAG3, CAM1, AMHR2, CCL15 and PDGFB (see, e.g., Li, Z., et al., 2008. The Journal of Immunology, 181(7), pp. 5147-5157). Methods for identifying promoters associated with genes are known to those skilled in the art.

[0332] In some embodiments, the inflammation-inducible promoter comprises an IFN-β minimal promoter or a fragment or derivative thereof. In some embodiments, the inflammation-inducible promoter comprises a nucleotide sequence at least 70% identical to SEQ ID NO:69 or a fragment thereof. In some embodiments, the inflammation-inducible promoter comprises a nucleotide sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO:69 or a fragment thereof. In some embodiments, the inflammation-inducible promoter comprises SEQ ID NO:69.

[0333] agcttgaataaaatgaatattagaagctgttagaataagagaaaatgacagaggaaaactgaaagggagaactgaaagtgggaaattcctctgaggcagaaaggaccatcccttataaatagcacaggccatgaaggaagatcattctcactgcagcctttgacagcctttgcctcatcttg

[0334] Example IFN-β minimal promoter (SEQ ID NO: 69)

[0335] The inflammation-inducible promoter may contain one or more inflammation-associated transcription factor binding motifs. "Inflammation-associated transcription factor binding motifs" or "inflammation-associated transcription factor binding sites" may refer to nucleotide sequences to which inflammation-associated transcription factors bind. Exemplary inflammation-associated transcription factors include, but are not limited to, AP-1, NF-κB, IRF, STAT, and NFAT (see, e.g., Smale ST. Cell. 2010 Mar 19; 140 (6): 833-44., Platanitis E, Decker T. Front Immunol. 2018 Nov 13; 9: 2542., Pessler F, Dai L, Cron RQ, Schumacher HR. Autoimmun Rev. 2006 Feb; 5 (2): 106-10.). Exemplary inflammation-associated transcription factor binding motifs may include, but are not limited to, AP-1 binding motifs, NF-κB binding motifs (κB sites), interferon stimulation response elements (ISREs), gamma interferon activation sequences (GASs), and NFAT binding motifs.

[0336] In some embodiments, the inflammation-inducible promoter comprises at least one inflammation-associated transcription factor binding motif, which can be selected from an AP-1 transcription factor binding motif; an NF-κB transcription factor binding motif; an IRF transcription factor binding motif; a STAT transcription factor binding motif; and a NFAT transcription factor binding motif; or any combination thereof.

[0337] In some embodiments, the inflammation-inducible promoter comprises two or more inflammation-associated transcription factor binding motifs. In some embodiments, the inflammation-inducible promoter comprises three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more inflammation-associated transcription factor binding motifs.

[0338] AP-1 binding motif

[0339] In some embodiments, the inflammation-inducible promoter comprises one or more AP-1 binding motifs.

[0340] Activator protein 1 (AP-1) is a transcription factor that regulates gene expression in response to a variety of stimuli, including cytokines, growth factors, stress, and bacterial and viral infections. "AP-1 binding motif", also known as "AP-1 transcription factor binding motif" or "AP-1 promoter site", is a DNA sequence to which the AP-1 transcription factor can bind (see, e.g., Kim, H., et al., 1997. Biochemical Journal, 324 (2), pp. 547-553).

[0341] In some embodiments, the inflammation-inducible promoter comprises two or more AP-1 binding motifs. In some embodiments, the inflammation-inducible promoter comprises three or more AP-1 binding motifs. In some embodiments, the inflammation-inducible promoter comprises four or more AP-1 binding motifs. In some embodiments, the inflammation-inducible promoter comprises five or more AP-1 binding motifs. In some embodiments, the inflammation-inducible promoter comprises five AP-1 binding motifs.

[0342] Exemplary AP-1 binding motifs are shown as SEQ ID NO: 70 (wherein "s" is g or c and "m" is a or c), SEQ ID NO: 71, SEQ ID NO: 72, and SEQ ID NO: 73. Any other variants or derivatives of AP-1 binding can be used in the present invention.

[0343] In some embodiments, the inflammation-inducible promoter comprises the nucleotide sequence of SEQ ID NO:70.

[0344] tgastma

[0345] Example AP-1 binding consensus motif (SEQ ID NO: 70)

[0346] In some embodiments, the inflammation-inducible promoter comprises the nucleotide sequence of SEQ ID NO: 71 or a derivative thereof having one or two nucleotide substitutions.

[0347] tgagtca

[0348] Example AP-1 binding motif 1 (SEQ ID NO: 71)

[0349] In some embodiments, the inflammation-inducible promoter comprises the nucleotide sequence of SEQ ID NO: 72 or a derivative thereof having one or two nucleotide substitutions.

[0350] tgactca

[0351] Example AP-1 binding motif 2 (SEQ ID NO: 72)

[0352] In some embodiments, the inflammation-inducible promoter comprises the nucleotide sequence of SEQ ID NO: 73 or a derivative thereof having one or two nucleotide substitutions.

[0353] tgactaa

[0354] Example AP-1 binding motif 3 (SEQ ID NO: 73)

[0355] NF-kB binding motif

[0356] In some embodiments, the inflammation-inducible promoter comprises one or more NF-kB binding motifs.

[0357] Nuclear factor kappa-light-chain enhancer of activated B cells (NF-kB) is a protein complex that controls DNA transcription in response to stimuli such as stress, cytokines, free radicals, heavy metals, ultraviolet radiation, oxidized LDL, and bacterial or viral antigens. "NF-kB binding motif", also known as "NF-kB transcription factor binding motif" or "NF-kB promoter site", is a DNA sequence to which the NF-kB transcription factor can bind (see, e.g., Natoli, G., 2006. FEBS letters, 580 (12), pp. 2843-2849.).

[0358] In some embodiments, the inflammation-inducible promoter comprises two or more NF-kB binding motifs. In some embodiments, the inflammation-inducible promoter comprises three or more NF-kB binding motifs. In some embodiments, the inflammation-inducible promoter comprises four or more NF-kB binding motifs. In some embodiments, the inflammation-inducible promoter comprises five or more NF-kB binding motifs. In some embodiments, the inflammation-inducible promoter comprises five NF-kB binding motifs.

[0359] Exemplary NF-kB binding motifs are shown as SEQ ID NO: 74 (wherein "r" is a purine, "y" is a pyrimidine and "n" is any nucleotide) and SEQ ID NO: 75. Any other variants or derivatives of NF-kB binding can be used in the present invention.

[0360] In some embodiments, the inflammation-inducible promoter comprises the nucleotide sequence of SEQ ID NO:74.

[0361] gggrnyyycc

[0362] Example NF-κB binding consensus motif (SEQ ID NO: 74)

[0363] In some embodiments, the inflammation-inducible promoter comprises the nucleotide sequence of SEQ ID NO: 75 or a derivative thereof having one or two nucleotide substitutions.

[0364] ggggactttccact

[0365] Example NF-κB binding motif (SEQ ID NO: 75)

[0366] Other inflammation-associated transcription factor binding motifs

[0367] In some embodiments, the inflammation-inducible promoter comprises one or more, two or more, three or more, four or more, or five or more interferon-stimulated response elements (ISREs). ISREs (also known as IRF transcription factor binding motifs) are DNA sequences to which IRF transcription factors can bind. All IRF family members have an N-terminal DNA binding domain that recognizes ISREs, which can be characterized by a consensus sequence AANNGAAA (see, e.g., Yanai, H., et al., 2012. Oncoimmunology, 1 (8), pp. 1376-1386).

[0368] In some embodiments, the inflammation-inducible promoter comprises one or more, two or more, three or more, four or more, or five or more interferon gamma activation sequences (GAS). GAS (also known as "STAT transcription factor binding motif") is a DNA sequence to which STAT transcription factors can bind. Several members of the STAT protein family, particularly STAT1, STAT2, STAT3, STAT4, and STAT6, act as transcription factors in regulating inflammatory responses. STAT transcription factors bind to similar sequences and may have a palindromic core motif TTCN. 2-4 GAA (see, eg, Ehret, GB, et al., 2001. Journal of Biological Chemistry, 276(9), pp. 6675-6688).

[0369] In some embodiments, the inflammation-inducible promoter comprises one or more, two or more, three or more, four or more, or five or more NFAT binding motifs. The NFAT transcription factor binding motif is a DNA sequence to which the NFAT transcription factor can bind. The NFAT family synergizes with the AP-1 protein on the DNA element containing adjacent NFAT and AP-1 binding sites to regulate the expression of the inducible gene. The NFAT binding motif can be characterized by the consensus sequence TGGAAA (see, e.g., Macian, F., et al., 2001. Oncogene, 20 (19), pp. 2476-2489).

[0370] Example Inflammation-Inducible Promoter

[0371] In some embodiments, the inflammation-inducible promoter comprises a combination of two or more different inflammation-associated transcription factor binding motifs. For example, the inflammation-inducible promoter may comprise any combination of: one or more AP-1 binding motifs, one or more NF-κB binding motifs, one or more gamma interferon activation sequences (GAS), one or more interferon-stimulated response elements (ISRE), and one or more NFAT binding motifs.

[0372] In some embodiments, the inflammation-inducible promoter comprises one or more AP-1 binding motifs and / or one or more NF-κB binding motifs. In some embodiments, the inflammation-inducible promoter comprises two or more AP-1 binding motifs and / or two or more NF-κB binding motifs. In some embodiments, the inflammation-inducible promoter comprises three or more AP-1 binding motifs and / or three or more NF-κB binding motifs. In some embodiments, the inflammation-inducible promoter comprises four or more AP-1 binding motifs and / or four or more NF-κB binding motifs. In some embodiments, the inflammation-inducible promoter comprises five or more AP-1 binding motifs and / or five or more NF-κB binding motifs.

[0373] The transcription factor binding motifs associated with inflammation can be coupled. The term "coupling" can mean that the transcription factor binding motifs associated with inflammation are in a relationship that allows them to function in their intended manner (e.g., in conjunction with the transcription factor associated with inflammation). Suitably, the transcription factor binding motifs associated with inflammation can be connected by short nucleotide sequences or can be directly connected or any combination thereof. Suitably, the transcription factor binding motifs associated with inflammation are connected by a linker sequence of 1 to about 20 nucleotides or 1 to about 10 nucleotides. Suitably, the transcription factor binding motifs associated with inflammation are directly connected (i.e., without a linker sequence in between).

[0374] Suitably, the inflammation-inducible promoter may comprise one or more inflammation-associated transcription factor binding sites and a minimal promoter (e.g., IFN-β minimal promoter). Suitably, the inflammation-inducible promoter may comprise a proximal promoter region comprising one or more inflammation-associated transcription factor binding sites. Suitably, the inflammation-inducible promoter may comprise a proximal promoter region and a core promoter region comprising one or more inflammation-associated transcription factor binding sites, the core promoter region being derived from a gene having selective expression in response to inflammation.

[0375] In some embodiments, the inflammation-inducible promoter comprises or consists of a nucleotide sequence that is at least 70% identical to SEQ ID NO: 76 or a fragment thereof. Suitably, the inflammation-inducible promoter comprises or consists of a nucleotide sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 76 or a fragment thereof. In some embodiments, the inflammation-inducible promoter comprises or consists of the sequence SEQ ID NO: 76 or a fragment thereof.

[0376] tgagtcactgactcagtgagtcactgactcagtgagtaaggaattctggggactttccactggggactttccactggggactttccactggggactttccactggggactttccactcctgcaggagcttgaataaaatgaatattagaagct gttagaataagagaaaatgacagaggaaaactgaaagggagaactgaaagtgggaaattcctctgaggcagaaaggaccatcccttataaatagcacaggccatgaaggaagatcattctcactgcagcctttgacagcctttgcctcatcttg

[0377] Example Inflammation-Inducible Promoter (SEQ ID NO: 76)

[0378] In some embodiments, the nucleotide sequence encoding the TNF inhibitor operably linked to the inflammation-inducible promoter comprises or consists of a nucleotide sequence at least 70% identical to SEQ ID NO: 77 or a fragment thereof. Suitably, the nucleotide sequence encoding the TNF inhibitor operably linked to the inflammation-inducible promoter comprises or consists of a nucleotide sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 77 or a fragment thereof. In some embodiments, the nucleotide sequence encoding the TNF inhibitor operably linked to the inflammation-inducible promoter has a nucleotide sequence comprising or consisting of the sequence SEQ ID NO: 77 or a fragment thereof.

[0379]

[0380] Example Inflammation-Inducible Promoter and Adalimumab Fab Nucleotide Sequence (SEQ ID NO: 77)

[0381] Other regulatory elements

[0382] The vector of the present invention may contain one or more additional regulatory elements, which may act before or after transcription. Suitably, the nucleotide sequence encoding the TNF inhibitor is operably linked to one or more additional regulatory elements, which may act before or after transcription.

[0383] As used herein, "regulatory element" may refer to any nucleotide sequence that promotes polypeptide expression, such as acting to increase transcript expression or enhance mRNA stability. Suitable regulatory elements include, for example, enhancer elements, post-transcriptional regulatory elements, introns, polyadenylation sites, and Kozak sequences.

[0384] Enhancer

[0385] The vector of the present invention may include an enhancer. Suitably, the nucleotide sequence encoding the TNF inhibitor is operably linked to the enhancer. The enhancer can promote the expression of the TNF inhibitor in ocular cells (e.g., retinal ganglion cells, RPE cells, photoreceptors, glial cells).

[0386] An "enhancer" or "enhancer element" may refer to a region of DNA that can be bound by a protein (activator) to increase the likelihood that transcription of a particular gene will occur. Enhancers are cis-acting. They can be located as far as 1 M bp (1,000,000 bp) from a gene, either upstream or downstream of the start site.

[0387] The vector of the present invention may comprise an ocular tissue-specific enhancer. Suitably, the enhancer may be operably linked to the nucleotide sequence encoding the TNF inhibitor.

[0388] As used herein, a "tissue-specific enhancer" is an enhancer that preferentially promotes the expression of a gene in a specific cell or tissue. Suitably, a tissue-specific enhancer can promote the higher expression of a gene in a specific cell type compared to other cell types. Higher expression can be measured, for example, by measuring the expression of a transgenic (e.g., green fluorescent protein (GFP)) operably connected to an enhancer, wherein the expression of the transgenic is related to the ability of the enhancer to promote gene expression. Suitable tissue-specific enhancers will be known to those skilled in the art. The enhancer can be a retina-specific enhancer, preferably a retinal ganglion-specific enhancer. Suitably, the enhancer can be (or can be derived from) an enhancer associated with a gene having selective expression in human retinal cells. The method of identifying the enhancer region associated with a gene will be known to those skilled in the art.

[0389] polyadenylation sequence

[0390] The vector of the present invention may comprise a polyadenylation sequence. Suitably, the nucleotide sequence encoding the TNF inhibitor is operably linked to the polyadenylation sequence. The polyadenylation sequence may be inserted after the nucleotide sequence to improve transgenic expression.

[0391] The polyadenylation sequence usually comprises a polyadenylation signal, a polyadenylation site and a downstream element: the polyadenylation signal comprises a sequence motif recognized by the RNA cleavage complex; the polyadenylation site is the site of cleavage at which the poly-A tail is added to the mRNA; the downstream element is a GT-rich region, which is usually located just downstream of the polyadenylation site and is important for efficient processing.

[0392] Suitable polyadenylation sequences will be known to those skilled in the art (see, e.g., Schambach, A., et al., 2007. Molecular Therapy, 15(6), pp. 1167-1173; and Choi, JH et al., 2014. Molecular brain, 7(1), pp. 1-10). Example polyadenylation sequences include bovine growth hormone (bGH) polyadenylation sequence, SV40 polyadenylation sequence, and rabbit β-globin polyadenylation sequence.

[0393] In some embodiments, the polyadenylation sequence comprises or consists of a nucleotide sequence that is at least 70% identical to SEQ ID NO: 78 or a fragment thereof. Suitably, the polyadenylation sequence comprises or consists of a nucleotide sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 78 or a fragment thereof.

[0394] In some embodiments, the polyadenylation sequence comprises or consists of the nucleotide sequence of SEQ ID NO: 78 or a fragment thereof.

[0395] gcctcgactgtgccttctagttgccagccatctgttgtttgcccctcccccgtgccttccttgaccctggaaggtgccactcccactgtcctttcctaataaaatga ggaaattgcatcgcattgtctgagtaggtgtcattctattctggggggtggggtggggcaggacagcaagggggaggattgggaagacaatagcaggcatgctgggga

[0396] Example bGH polyadenylation sequence (SEQ ID NO: 78)

[0397] Post-transcriptional regulatory elements

[0398] The vector of the present invention may comprise a post-transcriptional regulatory element. Suitably, the nucleotide sequence encoding the TNF inhibitor is operably linked to a post-transcriptional regulatory element.

[0399] The vector of the invention may comprise a woodchuck hepatitis post-transcriptional regulatory element (WPRE). Suitably, the nucleotide sequence encoding the TNF inhibitor is operably linked to the WPRE.

[0400] Suitable WPRE sequences are known to those skilled in the art (see, for example, Zufferey, R., et al., 1999. Journal of virology, 73 (4), pp. 2886-2892; and Zanta-Boussif, MA et al., 2009. Gene therapy, 16 (5), pp. 605-619). Suitably, WPRE is a wild-type WPRE or a mutant WPRE. For example, WPRE can be mutated to eliminate translation of woodchuck hepatitis virus X protein (WHX), for example by mutating the WHX ORF translation start codon.

[0401] In some embodiments, the WPRE comprises or consists of an amino acid sequence at least 70% identical to SEQ ID NO: 79, or a fragment thereof. Suitably, the WPRE comprises or consists of a nucleotide sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 79, or a fragment thereof.

[0402] In some embodiments, the WPRE comprises or consists of the nucleotide sequence of SEQ ID NO: 79, or a fragment thereof.

[0403] aatcaacctctggattacaaaatttgtgaaagattgactggtattcttaactatgttgctccttttacgctatgtggatacgctgctttaatgcctttgtatcatgctattgcttcccgtatggctttcattttc tcctccttgtataaatcctggttgctgtctctttatgaggagttgtggcccgttgtcaggcaacgtggcgtggtgtgcactgtgtttgctgacgcaacccccactggttggggcattgccaccacctgtcagctcc tttccgggactttcgctttccccctccctattgccacggcggaactcatcgccgcctgccttgcccgctgctggacaggggctcggctgttgggcactgacaattccgtggtgttgtcggggaaatcatcgtcct ttccttggctgctcgcctgtgttgccacctggattctgcgcgggacgtccttctgctacgtcccttcggccctcaatccagcggaccttccttcccgcggcctgctgccggctctgcggcctcttccgcgtcttcg

[0404] Example WPRE sequence (SEQ ID NO: 79)

[0405] Intron

[0406] The vector of the present invention may contain an intron. Suitably, the nucleotide sequence encoding the TNF inhibitor is operably linked to the intron. An intron may be inserted between the promoter and the nucleotide sequence encoding the TNF inhibitor to increase expression.

[0407] Suitable introns are known to those skilled in the art (see, e.g., Powell, SK, et al., 2015. Discovery medicine, 19(102), p.49), and may include MVM introns, F.IX truncated intron 1, chimeric β-globin / immunoglobulin heavy chain introns, chimeric adenovirus / immunoglobulin introns, and SV40 introns.

[0408] In some embodiments, the intron is the SV40 intron.

[0409] In some embodiments, the intron comprises or consists of a nucleotide sequence that is at least 70% identical to SEQ ID NO: 80 or a fragment thereof. Suitably, the intron comprises or consists of a nucleotide sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 80 or a fragment thereof.

[0410] In some embodiments, the intron comprises or consists of the nucleotide sequence of SEQ ID NO: 80 or a fragment thereof.

[0411] gtaagtatcaaggttacaagacaggtttaaggagaccaatagaaactgggcttgtcgagacagagaagactct tgcgtttctgataggcacctattggtcttactgacatccactttgcctttctctccacag

[0412] Example SV40 intron (SEQ ID NO: 80)

[0413] Kozak sequence

[0414] The vector of the present invention may comprise a Kozak sequence. Suitably, the nucleotide sequence encoding the TNF inhibitor is operably linked to the Kozak sequence. The Kozak sequence may be inserted before the start codon to improve the initiation of translation.

[0415] Suitable Kozak sequences will be known to those skilled in the art (see, for example, Kozak, M., 1987. Nucleic acids research, 15(20), pp. 8125-8148). The consensus Kozak sequence in vertebrates may have the sequence of SEQ ID NO:95 or SEQ ID NO:96.

[0416] Suitably, the Kozak sequence may comprise or consist of the nucleotide sequence of SEQ ID NO: 95 or 96, or a variant thereof having five or fewer deletions, substitutions or insertions. Suitably, the variant may have four or fewer, three or fewer, two or fewer or one deletion, substitution or insertion. Suitably, the variant may have three or fewer, two or fewer or one deletion and / or three or fewer, two or fewer or one substitution. Suitably, the variant may have three or fewer, two or fewer or one deletion and / or three or fewer, two or fewer or one substitution. Suitably, the variant may have one deletion and / or one substitution. Suitably, the variant may have one deletion and one substitution.

[0417] gccrccatgg

[0418] Example consensus Kozak sequence 1 (SEQ ID NO: 95)

[0419] gccgccrccatgg

[0420] Example consensus Kozak sequence 2 (SEQ ID NO: 96)

[0421] Inflammation suppressing oligonucleotides

[0422] The vector of the present invention may comprise a sequence encoding an inflammation suppressing oligonucleotide. Suitably, the nucleotide sequence encoding a TNF inhibitor is operably linked to the sequence encoding an inflammation suppressing oligonucleotide.

[0423] Inserting the sequence encoding the inflammation suppressive oligonucleotide into the vector can reduce innate immunity and T cell response and enhance gene expression by "cloaking" the vector from inducing unwanted immune responses. Suitable sequences are known to those skilled in the art (see, for example, Chan, YK, et al., 2021. Science translational medicine, 13 (580), p. eabd3438).

[0424] The sequence encoding inflammation suppressive oligonucleotide can antagonize TLR9 activation. In some embodiments, the inflammation suppressive oligonucleotide can be a TLR9 suppressive oligonucleotide. In some embodiments, the sequence encoding inflammation suppressive oligonucleotide includes one or more TLR9i sequences, such as one or more, two or more, three or more TLR9i sequences. Suitable TLR9i sequences are known in the art, and suitable TLR9i sequences are shown in SEQ ID NO:81. In some embodiments, the TLR9i sequence comprises or consists of the following: a nucleotide sequence that is at least 92%, at least 96% or 100% identical to SEQ ID NO:81. The TLR9i sequence can be separated by any suitable linker (e.g., AAAAA linker).

[0425] tttagggttagggttagggttaggg

[0426] Example TLR9i sequence (SEQ ID NO: 81)

[0427] In some embodiments, the sequence encoding the inflammation suppressive oligonucleotide comprises or consists of a nucleotide sequence that is at least 70% identical to SEQ ID NO: 82 or a fragment thereof. Suitably, the sequence encoding the inflammation suppressive oligonucleotide comprises or consists of a nucleotide sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 82 or a fragment thereof.

[0428] In some embodiments, the sequence encoding the inflammation suppressive oligonucleotide comprises or consists of the nucleotide sequence SEQ ID NO: 82 or a fragment thereof.

[0429] tttagggttagggttagggttagggaaaaatttagggttagggttagggttagggaaaaatttagggttagggttagggttagggaaaaatgcagcggtaagttcccatccaggttttttgcagcggtaagttcccatccaggttttttgcagcggtaagttcccatccaggttttt

[0430] Example io2 sequence (SEQ ID NO: 82)

[0431] Carrier

[0432] The vectors of the invention may be capable of transducing ocular cells (eg, retinal ganglion cells, RPE cells, photoreceptors, glial cells). In some embodiments, the vectors of the invention are capable of specifically transducing ocular cells.

[0433] In some embodiments, the vector of the present invention can transduce retinal cells. In some embodiments, the vector of the present invention can specifically transduce retinal cells. The retina is a multilayer membrane that is arranged in the inner posterior chamber of the eye and perceives images of the visual world, which are transmitted to the brain via the optic nerve. In order from the inside of the eye to the outside, the retina includes a neurosensory retinal layer and a retinal pigment epithelium, wherein the choroid is located outside the retinal pigment epithelium.

[0434] In some embodiments, the vectors of the present invention are capable of transducing retinal ganglion cells. In some embodiments, the vectors of the present invention are capable of specifically transducing retinal ganglion cells. Retinal ganglion cells are a type of neuron located near the inner surface of the retina of the eye.

[0435] Suitable vectors for transducing ocular cells include viral vectors such as parvoviral vectors (e.g., AAV vectors), lentiviral vectors, adenoviral vectors, and also non-viral delivery systems (see, e.g., Rodrigues, GA, et al., 2019. Pharmaceutical research, 36(2), pp. 1-20).

[0436] The vector of the present invention can be a viral vector. The viral vector of the present invention is preferably an adeno-associated virus (AAV), although it is contemplated that other viral vectors can be used. In some embodiments, the viral vector is any one of the following: a parvoviral vector, an adenoviral vector, a herpes simplex virus vector, an anellovirus vector, a retroviral vector, or a lentiviral vector.

[0437] The vector of the present invention can be in the form of a viral vector particle. In some embodiments, the viral vector is any one of the following: a parvovirus vector particle, an adenovirus vector particle, a herpes simplex virus vector particle, an anellovirus vector particle, a retrovirus vector particle, or a lentivirus vector particle. Preferably, the viral vector of the present invention is in the form of an AAV vector particle.

[0438] Methods for preparing and modifying viral vectors and viral vector particles (e.g., those derived from AAV) are known in the art. Suitable methods are described in Ayuso, E. et al., 2010. Current gene therapy, 10 (6), pp. 423-436, Merten, OW et al., 2016. Molecular Therapy-Methods & Clinical Development, 3, p. 16017; and Nadeau, I. and Kamen, A., 2003. Biotechnology advances, 20 (7-8), pp. 475-489.

[0439] Parvovirus vector

[0440] The vector of the present invention may be a parvoviral vector. The vector of the present invention may be in the form of a parvoviral vector particle.

[0441] Parvovirus, and especially adeno-associated virus (AAV), provides a universal platform for the rational design of human gene therapy vectors. Typically, all parvoviruses are composed of small, non-enveloped capsids containing a single-stranded DNA genome. Suitably, parvovirus vectors are from the subfamily Parvovirinae, which includes Dependoparvovirus, Protoparvovirus, and Bocaparvovirus. The vector of the present invention can be a hybrid gene therapy vector based on parvovirus (see, for example, Fakhiri, J. and Grimm, D., 2021. Molecular Therapy, 29 (12), pp. 3359-3382).

[0442] In some embodiments, the vector of the present invention is a parvoviral vector. In some embodiments, the vector of the present invention is in the form of a parvoviral vector. Some parvoviral vectors are also called adeno-associated viruses because they cannot replicate efficiently in host cells without co-infection of the host cells with a helper virus (such as an adenovirus).

[0443] In a preferred embodiment, the vector of the present invention is an adeno-associated virus (AAV) vector. In a preferred embodiment, the vector of the present invention is in the form of an AAV vector particle.

[0444] AAV genome

[0445] The AAV vector or AAV vector particle may comprise an AAV genome or a fragment or derivative thereof. The AAV genome is a polynucleotide sequence that can encode the functions required to produce AAV particles. These functions include functions that play a role in the replication and packaging cycle of AAV in host cells, including encapsidation of the AAV genome into AAV particles. Naturally occurring AAV is replication-defective and relies on the provision of trans-helper functions to complete the replication and packaging cycle. Therefore, the AAV genome of the AAV vector of the present invention is typically replication-defective.

[0446] The AAV genome can be in single-stranded form (positive or negative sense), or alternatively in double-stranded form. The use of the double-stranded form allows bypassing the DNA replication step in the target cell, thus accelerating transgene expression.

[0447] Naturally occurring AAV can be classified according to different biological systems. The AAV genome can be from any naturally derived AAV serotype, isolate or clade.

[0448] AAV can refer to its serotype. Serotype corresponds to a variant subspecies of AAV, which has a unique reactivity that can be used to distinguish it from other variant subspecies due to the expression spectrum of its capsid surface antigens. Typically, AAV vector particles with a specific AAV serotype do not efficiently cross-react with neutralizing antibodies specific to any other AAV serotype. AAV serotypes include AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, and AAV11. In some embodiments, the AAV vector of the present invention is an AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, or AAV9 serotype or a variant thereof. In some embodiments, the AAV vector of the present invention is an AAV2 serotype or a variant thereof.

[0449] The AAV genome may also contain packaging genes, such as rep and / or cap genes that encode the packaging function of the AAV particles. The rep gene encodes one or more of the proteins Rep78, Rep68, Rep52, and Rep40 or their variants. The cap gene encodes one or more capsid proteins, such as VP1, VP2, and VP3 or their variants. These proteins constitute the capsid of the AAV particles that determine the AAV serotype.

[0450] The AAV genome can be the complete genome of a naturally occurring AAV. For example, a vector comprising a complete AAV genome can be used to prepare an AAV vector or vector particle.

[0451] Preferably, the AAV genome is derivatized for the purpose of administration to a patient. Such derivatization is standard in the art, and the present invention encompasses the use of any known derivative of the AAV genome, as well as any derivative that can be generated by applying techniques known in the art. The AAV genome can be a derivative of any naturally occurring AAV. Suitably, the AAV genome is a derivative of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10 or AAV11. Suitably, the AAV genome is a derivative of AAV2.

[0452] Derivatives of the AAV genome include any truncated or modified forms of the AAV genome that allow the expression of transgenes in vivo from the AAV vector of the present invention. Generally, the AAV genome may be significantly truncated to include minimal viral sequences, but still retain the above functions. For safety reasons, this is preferred to reduce the risk of vector recombination with wild-type viruses, and also to avoid triggering cellular immune responses due to the presence of viral gene proteins in target cells.

[0453] Typically, the derivative will contain at least one inverted terminal repeat (ITR), preferably more than one ITR, such as two or more ITRs. One or more ITRs may be derived from an AAV genome having a different serotype, or may be a chimeric or mutated ITR. Preferred mutant ITRs have mutant ITRs with a trs (terminal resolution site) deletion. This deletion allows the genome to continue to replicate to generate a single-stranded genome containing both the coding sequence and the complementary sequence, i.e., a self-complementary AAV genome. This allows bypassing DNA replication in the target cell, and thus enables accelerated transgene expression.

[0454] The AAV genome may comprise one or more ITR sequences from any naturally derived serotype, isolate or clade of AAV or variants thereof. The AAV genome may comprise at least one (such as two) AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10 or AAV11 ITRs or variants thereof.

[0455] Suitably, the AAV genome may comprise at least one, such as two, AAV2 ITRs or variants thereof. In some embodiments, the AAV genome comprises an AAV2 5'ITR and / or an AAV2 3'ITR.

[0456] In some embodiments, the AAV genome comprises a 5' ITR that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 83. In some embodiments, the AAV genome comprises a 5' ITR that comprises or consists of SEQ ID NO: 83.

[0457] ctgcgcgctcgctcgctcactgaggccgcccgggcaaagcccgggcgtcgggcgacctttggtcgcccgg cctcagtgagcgagcgagcgcgcagagaggggagtggccaactccatcactaggggttcct

[0458] Example AAV2 5' ITR (SEQ ID NO: 83)

[0459] In some embodiments, the AAV genome comprises a 3' ITR that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 84. In some embodiments, the AAV genome comprises a 3' ITR comprising or consisting of SEQ ID NO: 84.

[0460] aggaacccctagtgatggagttggccactccctctctgcgcgctcgctcgctcactgaggccgggcgaccaa aggtcgcccgacgcccgggctttgcccgggcggcctcagtgagcgagcgagcgcgcag

[0461] Example AAV2 3'ITR (SEQ ID NO: 84)

[0462] One or more ITRs may flank the nucleotide sequence encoding the TNF inhibitor at either end. Preferably, one or more ITRs are included to aid in the formation of concatemers of the AAV vector in the host cell nucleus, for example after the single-stranded vector DNA is converted to double-stranded DNA by the action of a host cell DNA polymerase. The formation of such episomal concatemers protects the AAV vector during the life of the host cell, thereby allowing for prolonged expression of the transgene in vivo.

[0463] Suitably, the AAV genome may comprise one or more AAV2 ITR sequences flanking the nucleotide sequence encoding the TNF inhibitor. Suitably, the AAV genome may comprise two AAV2 ITR sequences flanking either side of the nucleotide sequence encoding the TNF inhibitor.

[0464] Suitably, the ITR element will be the only sequence retained from the natural AAV genome in the derivative. The derivative will preferably not contain the rep gene and / or cap gene of the natural genome and any other sequence of the natural genome. Due to the above reasons, and in order to reduce the possibility of vector integration into the host cell genome, this is preferred. In addition, reducing the size of the AAV genome allows other sequence elements (such as regulatory elements) to be incorporated into the vector in addition to the transgene to increase flexibility.

[0465] Therefore, the following parts can be removed in the derivatives of the present invention: an inverted terminal repeat (ITR) sequence, a replication (rep) gene, and a capsid (cap) gene. However, the derivatives may additionally include one or more rep genes and / or cap genes or other viral sequences of the AAV genome. Naturally occurring AAV integrates at a high frequency at a specific site on human chromosome 19 and exhibits a negligible frequency of random integration, making it possible to tolerate the retention of integration capacity in AAV vectors in a therapeutic setting.

[0466] The present invention also encompasses providing AAV genome sequences in an order and configuration different from the native AAV genome sequence. The present invention also encompasses replacing one or more AAV sequences or genes with sequences from another virus or with chimeric genes composed of sequences from more than one virus. Such chimeric genes may be composed of sequences of two or more related viral proteins from different viral species.

[0467] AAV capsid protein

[0468] AAV vector particles can be encapsidated by capsid proteins. The serotype can promote transduction of ocular cells (e.g., retinal ganglion cells, RPE cells, photoreceptors, glial cells), such as specific transduction of ocular cells. AAV vector particles can be ocular tissue-specific vector particles. AAV vector particles can be encapsidated by ocular tissue-specific capsids. AAV vector particles can contain ocular tissue-specific capsid proteins.

[0469] In some embodiments, the AAV vector particle is a retina-specific vector particle. In some embodiments, the AAV vector particle is encapsidated by a retina-specific capsid. In some embodiments, the AAV vector particle comprises a retina-specific capsid protein.

[0470] In some embodiments, the AAV vector particles are retinal ganglion-specific vector particles. In some embodiments, the AAV vector particles are encapsidated with retinal ganglion-specific capsids. In some embodiments, the AAV vector particles comprise retinal ganglion-specific capsid proteins.

[0471] Suitably, the AAV vector particles can be in a transcapsidated form, in which an AAV genome or derivative having ITRs of one serotype is packaged in a capsid of a different serotype. AAV vector particles also include chimeric forms, in which a mixture of unmodified capsid proteins from two or more different serotypes constitutes the viral capsid. AAV vector particles also include chemically modified forms with ligands adsorbed to the capsid surface. For example, such ligands can include antibodies for targeting specific cell surface receptors.

[0472] When the derivative comprises capsid proteins, i.e., VP1, VP2, and / or VP3, the derivative may be a chimeric, shuffled, or capsid-modified derivative of one or more naturally occurring AAVs. Specifically, the present invention encompasses providing capsid protein sequences from different serotypes, clades, clones, or isolates of AAV in the same vector (i.e., pseudotyped vector). The AAV vector may be in the form of a pseudotyped AAV vector particle.

[0473] Derivatives that are chimeric, reorganized or capsid modified are usually selected to provide one or more desired functions for AAV vectors. Therefore, compared with AAV vectors containing naturally occurring AAV genomes, these derivatives can show increased gene delivery efficiency, reduced immunogenicity (humoral or cell), changed tropism range and / or improved ocular cell targeting. The increased gene delivery efficiency can be affected by improved receptor or co-receptor binding on the cell surface, improved internalization, improved intracellular and intranuclear transport, improved viral particle uncoating, and improved conversion of single-stranded genomes to double-stranded forms. The increased efficiency can also be related to the changed tropism range or targeting of ocular cells, so that the vector dose will not be diluted by administration to unwanted tissues.

[0474] Chimeric capsid proteins include those generated by recombination between capsid coding sequences of two or more naturally occurring AAV serotypes. This can be done, for example, by a marker rescue method, in which a non-infectious capsid sequence of one serotype is co-transfected with a capsid sequence of a different serotype, and directed selection is used to select a capsid sequence with desired properties. The capsid sequences of different serotypes can be altered by homologous recombination in cells, thereby generating new chimeric capsid proteins.

[0475] For example, directed evolution methods have been used to generate novel AAV vectors that can more effectively cross biological barriers and target specific cell types. An example is the identification of AAV.7m8 variants, which can effectively deliver genes to all retinal layers of both mice and primates after intravitreal injection. Similarly, SH10 (an AAV6 variant) has an increased tropism for glial cells after intravitreal delivery, and has been shown to rescue retinal function in rat models of RP (see, e.g., Rodrigues, GA, et al., 2019. Pharmaceutical research, 36 (2), pp. 1-20).

[0476] Chimeric capsid proteins also include those generated by engineering the capsid protein sequence to transfer specific capsid protein domains, surface loops, or specific amino acid residues between two or more capsid proteins, such as between two or more capsid proteins of different serotypes.

[0477] Shuffled or chimeric capsid proteins can also be generated by DNA shuffling or by error-prone PCR. Hybrid AAV capsid genes can be created by randomly fragmenting the sequences of related AAV genes (e.g., those encoding capsid proteins of multiple different serotypes), and then reassembling these fragments in a self-starting polymerase reaction, which may also result in crossovers in regions with sequence homology. Libraries of hybrid AAV genes created in this way by shuffling capsid genes of several serotypes can be screened to identify viral clones with desired functions. Similarly, error-prone PCR can be used to randomly mutate AAV capsid genes to create a diverse library of variants, which can then be selected to obtain desired properties.

[0478] The sequence of the capsid gene can also be genetically modified to introduce specific deletions, substitutions or insertions relative to the natural wild-type sequence. In particular, the capsid gene can be modified by inserting an unrelated protein or peptide sequence in the open reading frame of the capsid coding sequence or at the N-terminus and / or C-terminus of the capsid coding sequence. The unrelated protein or peptide can advantageously be a protein or peptide as a part of a specific cell type, thereby imparting improved binding to the target cell or improving the specificity of the vector targeting a specific cell population. The unrelated protein can also be a protein, i.e., an epitope or affinity tag, that assists in the purification of viral particles as a part of the production process. Usually the insertion site is selected so as not to interfere with other functions of the viral particles, such as internalization and transportation of the viral particles.

[0479] For example, AAV variants have been generated by site-directed mutagenesis of surface-exposed tyrosine residues, which prevent capsid phosphorylation and subsequent ubiquitination and proteasome-mediated degradation. AAV2, AAV8, and AAV9 carrying these mutations have been shown to have increased transduction efficiency both in vitro and in vivo (see, e.g., Petrs-Silva, H., et al., 2009. Molecular therapy, 17(3), pp. 463-471).

[0480] The capsid protein can be an artificial capsid protein. As used herein, the term "artificial capsid" refers to a capsid particle comprising an amino acid sequence that does not exist in nature or an amino acid sequence that has been engineered (e.g., modified) from a naturally occurring capsid amino acid sequence. In other words, when the artificial capsid amino acid sequence and the parent capsid amino acid sequence are aligned, the artificial capsid protein comprises a mutation or change in the amino acid sequence compared to the sequence of the parent capsid from which it is derived.

[0481] The capsid protein can comprise a mutation or modification relative to a wild-type capsid protein that improves the ability to transduce ocular cells relative to unmodified or wild-type viral particles. The improved ability to transduce ocular cells can be measured, for example, by measuring the expression of a transgene carried by the AAV vector particles, such as GFP, wherein the expression of the transgene in the ocular cells correlates with the ability of the AAV vector particles to transduce ocular cells.

[0482] Suitably, the AAV vector particles of the invention are AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8 or AAV9 vector particles or variants thereof. AAV vector particles having these serotypes can transduce ocular cells.

[0483] The AAV vector particles of the present invention may comprise AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8 or AAV9 capsid proteins or variants thereof. Suitably, the AAV vector particles may comprise AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8 or AAV9 capsid proteins VP1, VP2 and VP3 or variants thereof.

[0484] In one embodiment, the AAV vector particles comprise one or more AAV2 ITR sequences flanking a nucleotide sequence encoding a TNF inhibitor and an AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8 or AAV9 capsid protein or a variant thereof. In one embodiment, the AAV vector particles comprise an AAV2 genome and an AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8 or AAV9 capsid protein or a variant thereof.

[0485] AAV2 vectors and their variants

[0486] In some embodiments, the AAV vector particle is an AAV2 vector particle or a variant thereof. In some embodiments, the AAV vector particle comprises an AAV2 capsid protein or a variant thereof. Suitably, the AAV vector particle may comprise AAV2 capsid proteins VP1, VP2 and VP3 or a variant thereof.

[0487] AAV2 variants include AAV2.tYF, AAV2.7m8, R100, AAV2.GL, AAV2.NN, LSV1, R195-003 and dyno-86m. In some embodiments, the AAV vector particles are AAV2 vector particles, AAV2.tYF vector particles, AAV2.7m8 vector particles, R100 vector particles, AAV2.GL vector particles, AAV2.NN vector particles, LSV1 vector particles, R195-003 vector particles or dyno-86m vector particles. In some embodiments, the AAV vector particles comprise AAV2 capsid protein, AAV2.tYF capsid protein, AAV2.7m8 capsid protein, R100 capsid protein, AAV2.GL capsid protein, AAV2.NN capsid protein, LSV1 capsid protein, R195-003 capsid protein or dyno-86m capsid protein. Suitably, the AAV vector particles may comprise AAV2 capsid proteins VP1, VP2 and VP3, AAV2.tYF capsid proteins VP1, VP2 and VP3, AAV2.7m8 capsid proteins VP1, VP2 and VP3, R100 capsid proteins VP1, VP2 and VP3, AAV2.GL capsid proteins VP1, VP2 and VP3, AAV2.NN capsid proteins VP1, VP2 and VP3, LSV1 capsid proteins VP1, VP2 and VP3, R195-003 capsid proteins VP1, VP2 and VP3, or dyno-86m capsid proteins VP1, VP2 and VP3.

[0488] In some embodiments, the AAV vector particles are AAV2 vector particles, AAV2.tYF vector particles, AAV2.7m8 vector particles, R100 vector particles, AAV2.GL vector particles or AAV2.NN vector particles. In some embodiments, the AAV vector particles comprise AAV2 capsid proteins, AAV2.tYF capsid proteins, AAV2.7m8 capsid proteins, R100 capsid proteins, AAV2.GL capsid proteins or AAV2.NN capsid proteins. Suitably, the AAV vector particles may comprise AAV2 capsid proteins VP1, VP2 and VP3, AAV2.tYF capsid proteins VP1, VP2 and VP3, AAV2.7m8 capsid proteins VP1, VP2 and VP3, R100 capsid proteins VP1, VP2 and VP3, AAV2.GL capsid proteins VP1, VP2 and VP3, or AAV2.NN capsid proteins VP1, VP2 and VP3.

[0489] In some embodiments, the AAV vector particle is an AAV2 vector particle. In some embodiments, the AAV vector particle comprises AAV2 capsid proteins. In some embodiments, the AAV vector particle comprises AAV2 capsid proteins VP1, VP2, and VP3.

[0490] Suitably, the AAV2 VP1 capsid protein may comprise or consist of the amino acid sequence SEQ ID NO: 85 or a variant that is at least 90% identical to SEQ ID NO: 85. Suitably, the variant may be at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 85. Suitably, the AAV2 VP2 and VP3 capsid proteins may be N-terminal truncations of SEQ ID NO: 85, or N-terminal truncations of variants that are at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 85.

[0491] MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPGYKYLGPFNGLDKGEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRVLEPLGLVEEPVKTAPGKKRPVEHSPVEPDSSSGTGKAGQQPARKRLNFGQTGDADSVPDPQPLGQPPAAPSGLGTNTMATGSGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVITTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTNTPSGTTTQSRLQFSQAGASDIRDQSRNWLPGPCYRQQRVSKTSADNNNSEYSWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQGSEKTNVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRGNRQAATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPSTTFSAAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRNL

[0492] Example AAV2 VP1 capsid protein (SEQ ID NO:85)

[0493] In some embodiments, the AAV vector particle is an AAV2.tYF vector particle. In some embodiments, the AAV vector particle comprises an AAV2.tYF capsid protein. In some embodiments, the AAV vector particle comprises an AAV2.tYF capsid protein VP1, VP2, and VP3. A single phenylalanine (F) substitution for tyrosine (Y) has increased efficacy of AAV2 after intraocular injection (see, e.g., Petrs-Silva, H., et al., 2009. Molecular therapy, 17 (3), pp. 463-471).

[0494] Suitably, the AAV2.tYF VP1 capsid protein may comprise or consist of the amino acid sequence SEQ ID NO: 86 or a variant that is at least 90% identical to SEQ ID NO: 86. Suitably, the variant may be at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 86. Suitably, the AAV2.tYF VP2 and VP3 capsid proteins may be N-terminal truncations of SEQ ID NO: 86, or N-terminal truncations of variants that are at least 90% identical, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 86.

[0495] MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPGYKYLGPFNGLDKGEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADAEFQERLKEDTSF GGNLGRAVFQAKKRVLEPLGLVEEPVKTAPGKKRPVEHSPVEPDSSSSGTGKAGQQPARKRLNFGQTGDADSVPDPQPLGQPPAAPSGLGTNTMATGSGAPMADNNEGADGV GNSSGNWHCDSTWMGDRVITTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTQNDGTTTI ANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLY F LSRTNTPSGTTTQSRLQFSQAGASDIRDQSRNWLPGPCYRQQRVSKTSADNNNSEF SWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQGSEKTNVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRGNRQAATADVNTQGVLPGMVWQDRDVYLQ GPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPSTTFSAAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTR F LTRNL

[0496] Example AAV2.tYF VP1 capsid protein (SEQ ID NO: 86)

[0497] In some embodiments, the AAV vector particle is an AAV2.7m8 vector particle. In some embodiments, the AAV vector particle comprises an AAV2.7m8 capsid protein. In some embodiments, the AAV vector particle comprises an AAV2.7m8 capsid protein VP1, VP2, and VP3. AAV2.7m8 is an engineered capsid with 10 amino acids inserted in the variable region VIII (VR-VIII) of the adeno-associated virus (AAV) surface, which results in the ability to effectively transduce retinal cells after intravitreal administration (see, e.g., Bennett, A., et al., 2020. Journal of structural biology, 209 (2), p. 107433).

[0498] Suitably, the AAV2.7m8 VP1 capsid protein may comprise or consist of the amino acid sequence SEQ ID NO: 87 or a variant that is at least 90% identical to SEQ ID NO: 87. Suitably, the variant may be at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 87. Suitably, the AAV2.7m8 VP2 and VP3 capsid proteins may be N-terminal truncations of SEQ ID NO: 87, or N-terminal truncations of variants that are at least 90% identical, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 87.

[0499] MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPGYKYLGPFNGLDKGEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRVLEPLGLVEEPVKTAPGKKRPVEHSPVEPDSSSGTGKAGQQPARKRLNFGQTGDADSVPDPQPLGQPPAAPSGLGTNTMATGSGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVITTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTNTPSGTTTQSRLQFSQAGASDIRDQSRNWLPGPCYRQQRVSKTSADNNNSEYSWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQGSEKTNVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRGN LALGETTRPA RQAATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPSTTFSAAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRNL

[0500] Example AAV2.7m8 VP1 capsid protein (SEQ ID NO:87)

[0501] In some embodiments, the AAV vector particle is an R100 vector particle. In some embodiments, the AAV vector particle comprises an R100 capsid protein. In some embodiments, the AAV vector particle comprises R100 capsid proteins VP1, VP2, and VP3. Compared to wild-type AAV, R100 demonstrates excellent transduction of human retinal cells (see, e.g., Kotterman, M., et al., 2021.bioRxiv2021.06.24.449775).

[0502] Suitably, the R100 VP1 capsid protein may comprise or consist of the amino acid sequence SEQ ID NO: 88 or a variant that is at least 90% identical to SEQ ID NO: 88. Suitably, the variant may be at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 88. Suitably, the R100 and VP3 capsid proteins may be N-terminal truncations of SEQ ID NO: 88, or N-terminal truncations of variants that are at least 90% identical, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 88.

[0503] MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPGYKYLGPFNGLDKGEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRVLEPLGLVEEPVKTAPGKKRPVEHSPVEPDSSSGTGKAGQQPARKRLNFGQTGDADSVPDPQPLGQPPAAPSGLGTNTMATGSGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVITTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTNTPSGTTTQSRLQFSQAGASDIRDQSRNWLPGPCYRQQRVSKTSADNNNSEYSWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQGSEKTNVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRGN LAISDQT RQAATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPSTTFSAAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRNL

[0504] Example R100 VP1 capsid protein (SEQ ID NO:88)

[0505] In some embodiments, the AAV vector particles are AAV2.GL vector particles. In some embodiments, the AAV vector particles comprise AAV2.GL capsid protein. In some embodiments, the AAV vector particles comprise AAV2.GL capsid protein VP1, VP2 and VP3. In some embodiments, the AAV vector particles are AAV2.NN vector particles. In some embodiments, the AAV vector particles comprise AAV2.NN capsid protein. In some embodiments, the AAV vector particles comprise AAV2.NN capsid protein VP1, VP2 and VP3. In mice, dogs and non-human primates, after intravitreal injection, AAV2.GL and AAV2.NN mediate extensive and high levels of retinal transduction (see, e.g., Pavlou, M., et al., 2021. EMBO molecular medicine, 13 (4), p.e13392).

[0506] Suitably, the AAV2.GL VP1 capsid protein may comprise or consist of the amino acid sequence SEQ ID NO: 89 or a variant that is at least 90% identical to SEQ ID NO: 89. Suitably, the variant may be at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 89. Suitably, the AAV2.GL VP2 and VP3 capsid proteins may be N-terminal truncations of SEQ ID NO: 89 or N-terminal truncations of variants that are at least 90% identical, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 89.

[0507] MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPGYKYLGPFNGLDKGEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRVLEPLGLVEEPVKTAPGKKRPVEHSPVEPDSSSGTGKAGQQPARKRLNFGQTGDADSVPDPQPLGQPPAAPSGLGTNTMATGSGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVITTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTNTPSGTTTQSRLQFSQAGASDIRDQSRNWLPGPCYRQQRVSKTSADNNNSEYSWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQGSEKTNVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRGN AAAGLSPPTRAA RQAATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPSTTFSAAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRNL Instance AAV2.GL VP1 capsid protein (SEQ ID NO:89)

[0508] Suitably, the AAV2.NN VP1 capsid protein may comprise or consist of the amino acid sequence SEQ ID NO: 90 or a variant that is at least 90% identical to SEQ ID NO: 90. Suitably, the variant may be at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 90. Suitably, the AAV2.NN VP2 and VP3 capsid proteins may be N-terminal truncations of SEQ ID NO: 90 or N-terminal truncations of variants that are at least 90% identical, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 90.

[0509] MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPGYKYLGPFNGLDKGEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRVLEPLGLVEEPVKTAPGKKRPV EHSPVEPDSSSSGTGKAGQQPARKRLNFGQTGDADSVPDPQPLGQPPAAPSGLGTNTMATGSGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVITTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSP RDWQRLINNNWGFRPKRLNFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQ YLYYLSRTNTPSGTTTQSRLQFSQAGASDIRDQSRNWLPGPCYRQQRVSKTSADNNNSEYSWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQGSEKTNVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRGN AAANNPTPSRAARQAATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPSTTFSAAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRNL Example AAV2.NN VP1 capsid protein (SEQ ID NO: 90)

[0510] In some embodiments, the AAV vector particle is an LSV1 vector particle. In some embodiments, the AAV vector particle comprises an LSV1 capsid protein. In some embodiments, the AAV vector particle comprises LSV1 capsid proteins VP1, VP2, and VP3. Loop exchange variant 1 (LSV1) transduces the retina and retinal pigment epithelium (RPE) from the vitreous body and is based on AAV2.5T (substitution from aa 571-579) (see, e.g., Baker, CK, et al., 2022, Molecular Therapy, 30 (4), p. 575).

[0511] Suitably, the LSV1 VP1 capsid protein may comprise or consist of the amino acid sequence SEQ ID NO: 94 or a variant that is at least 90% identical to SEQ ID NO: 94. Suitably, the variant may be at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 94. Suitably, the LSV1 VP2 and VP3 capsid proteins may be N-terminal truncations of SEQ ID NO: 94 or N-terminal truncations of variants that are at least 90% identical, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 94.

[0512] MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPGYKYLGPFNGLDKGEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRVLEPFGLVEEGAKTAPTGKRIDDHFPKRKKARTEEDSKPSTSSDAEAGPSGSQQLQI PAQPASSLGADTMSAGGGGPLGDNNQGADGVGNASGDWHCDSTWMGDRVVTKSTRTWVLPSYNNHQYREIKSGSVDGSNANAYFGYSTPWGYFDFNRFHSHWSPRDWQRLINNYWGFRPRSLRVKIFNIQVKEVTVQDSTTTIANNLTSTVQVFTDDDYQLPYVVGNGTEGCLPAFPPQVF TLPQYGYATLNRDNTENPTERSSFCLEYFPSKMLRTGNNFEFTYNFEEVPFHSSFAPSQNLFKLANPLVDQYLYRFVSTNNTGGVQFNKNLAGRYANTYKNWFPGPMGRTQGWNLGSGVNRASVSAFATTNRMELEGASYQVPPQPNGMTNNLQGSNTYALENTMIFNSQPANPGTTATY LEGNMLITSESETQPVNRVAYNVGGQMLAHKFKSGDAPTTGTYNLQEIVPGSVWMERDVYLQGPIWAKIPETGAHFHPSPAMGGFGLKHPPPMMLIKNTPVPGNITSFSDVPVSSFITQYSTGQVTVEMEWELKKENSKRWNPEIQYTNNYNDPQFVDFAPDSTGEYRTTRPIGTRYLTRPL

[0513] Example LSV1 capsid protein (SEQ ID NO: 94)

[0514] In some embodiments, the AAV vector particle is an R195-003 vector particle. In some embodiments, the AAV vector particle comprises an R195-003 capsid protein. In some embodiments, the AAV vector particle comprises an R195-003 capsid protein VP1, VP2, and VP3 (see, e.g., Human Gene Therapy Methods 2022; 33(23-24): A27-A28).

[0515] In some embodiments, the AAV vector particle is a dyno-86m vector particle. In some embodiments, the AAV vector particle comprises a dyno-86m capsid protein. In some embodiments, the AAV vector particle comprises a dyno-86m capsid protein VP1, VP2, and VP3 (see, e.g., Molecular Therapy 2023; 31(4), S1, p.1284).

[0516] In some embodiments, the AAV vector particles comprise one or more AAV2 ITR sequences flanking a nucleotide sequence encoding a TNF inhibitor and an AAV2 capsid protein or a variant thereof. In some embodiments, the AAV vector particles comprise an AAV2 genome and an AAV2 capsid protein, or a variant thereof.

[0517] Other parvovirus vectors

[0518] In some embodiments, the vector of the present invention is a proto-parvovirus vector. In some embodiments, the vector form of the present invention is a proto-parvovirus vector. Proto-parvoviruses have been widely studied and used as vectors, including minute virus of mice (MVM), rat parvovirus H1, and LuIII virus. Recently discovered human variants include bufavirus (BuV), tusavirus (TuV), and cutavirus (CuV) (see, e.g., Fakhiri, J. and Grimm, D., 2021. Molecular Therapy, 29 (12), pp. 3359-3382).

[0519] In some embodiments, the vector of the present invention is a bocavirus vector. In some embodiments, the vector of the present invention is in the form of a bocavirus vector. The use of human bocavirus 1 (HBoV1) as a parvovirus vector for gene delivery has been described (see, e.g., Shao, L., et al., 2021. Frontiers in Microbiology, 12, p. 1463).

[0520] Other viral vectors

[0521] Retroviral and lentiviral vectors

[0522] The vector of the present invention may be a retroviral vector or a lentiviral vector. The vector of the present invention may be a retroviral vector particle or a lentiviral vector particle.

[0523] Retroviral vectors can be derived from or derivable from any suitable retrovirus. A large number of different retroviruses have been identified. Examples include murine leukemia virus (MLV), human T-cell leukemia virus (HTLV), mouse mammary tumor virus (MMTV), Rous sarcoma virus (RSV), Fujinami sarcoma virus (FuSV), Moloney murine leukemia virus (Mo-MLV), FBR murine osteosarcoma virus (FBR MSV), Moloney murine sarcoma virus (Mo-MSV), Abelson murine leukemia virus (A-MLV), avian myelocytoma virus-29 (MC29) and avian erythroblastosis virus (AEV).

[0524] Retroviruses can be broadly divided into two categories, ‘simple’ and ‘complex’. Retroviruses can be even further divided into seven groups. Five of these groups represent retroviruses with oncogenic potential. The remaining two groups are lentiviruses and foamy viruses.

[0525] The basic structure of retroviral and lentiviral genomes share many common features, such as 5'LTR and 3'LTR. Positioned between or within them are packaging signals that enable the genome to be packaged, primer binding sites, integration sites that enable integration into the host cell genome, and gag, pol, and env genes that encode packaging components (these are polypeptides required to assemble viral particles). Lentiviruses have additional features, such as rev and RRE sequences in HIV, which enable efficient export of RNA transcripts of the integrated provirus from the nucleus of infected target cells to the cytoplasm.

[0526] In the provirus, these genes are flanked at both ends by regions called long terminal repeats (LTRs). LTRs are responsible for proviral integration and transcription. LTRs also act as enhancer-promoter sequences and can control the expression of viral genes.

[0527] The LTR itself is an identical sequence that can be divided into three elements: U3, R, and U5. U3 is derived from a sequence unique to the 3' end of the RNA. R is derived from a sequence that is repeated at both ends of the RNA. U5 is derived from a sequence unique to the 5' end of the RNA. The sizes of the three elements can vary greatly in different retroviruses.

[0528] In a defective retroviral vector genome, gag, pol and env may be absent or not functional.

[0529] In a typical retroviral vector, at least a portion of one or more protein coding regions necessary for replication can be removed from the virus. This makes the viral vector replication defective. Portions of the viral genome can also be replaced by a library encoding candidate regulatory parts, which are operably connected to the regulatory control region and the reporter part in the vector genome to generate a vector comprising the candidate regulatory parts, which can transduce the target host cell and / or integrate its genome into the host genome.

[0530] Lentivirus vectors are part of a larger group of retroviral vectors. In short, lentiviruses can be divided into primate and non-primate groups. Examples of primate lentiviruses include, but are not limited to, human immunodeficiency virus (HIV), the pathogen of human acquired immunodeficiency syndrome (AIDS); and simian immunodeficiency virus (SIV). Examples of non-primate lentiviruses include the prototype "slow virus" Vesna / Medi virus (VMV), as well as the related caprine arthritis encephalitis virus (CAEV), equine infectious anemia virus (EIAV), and the recently described feline immunodeficiency virus (FIV) and bovine immunodeficiency virus (BIV).

[0531] The lentivirus family differs from retroviruses in that lentiviruses have the ability to infect both dividing and non-dividing cells. In contrast, other retroviruses, such as MLV, are unable to infect non-dividing cells or slowly dividing cells, such as cells that make up, for example, muscle, brain, lung, and liver tissue.

[0532] As used herein, a lentiviral vector is a vector comprising at least one component that can be derived from a lentivirus. Preferably, the component is involved in the biological machinery of the vector to infect cells, express genes or replicate.

[0533] The lentiviral vector may be a "primate" vector. The lentiviral vector may be a "non-primate" vector (i.e., derived from a virus that does not primarily infect primates, especially humans). Examples of non-primate lentiviruses may be any member of the Lentiviridae family that does not naturally infect primates.

[0534] As examples of lentivirus-based vectors, HIV-1 and HIV-2-based vectors are described below.

[0535] HIV-1 vectors contain cis-acting elements also found in simple retroviruses. It has been shown that sequences extending into the gag open reading frame are important for packaging HIV-1. Therefore, HIV-1 vectors usually contain relevant parts of gag in which the translation start codon has been mutated. In addition, most HIV-1 vectors also contain a part of the env gene including RRE. Rev binds to RRE, and RRE allows full-length or single-spliced ​​mRNA to be transported from the nucleus to the cytoplasm. In the absence of Rev and / or RRE, full-length HIV-1 RNA accumulates in the nucleus. Alternatively, constitutive transport elements from certain simple retroviruses such as Mason-Pfizer monkey virus can be used to alleviate the need for Rev and RRE. The efficient transcription of the HIV-1 LTR promoter requires the viral protein Tat.

[0536] Most HIV-2-based vectors are structurally very similar to HIV-1 vectors. Similar to HIV-1-based vectors, HIV-2 vectors also require RRE for efficient transport of full-length or singly spliced ​​viral RNA.

[0537] Preferably, the viral vector used in the present invention has a minimal viral genome. The so-called "minimal viral genome" should be understood as having been manipulated to remove non-essential elements and retain essential elements to provide the functions required for infection, transduction and delivery of the nucleotide sequence of interest to the target host cell. Further details of this strategy can be found in WO1998 / 017815.

[0538] Preferably, the plasmid vector used to produce the viral genome in the host cell / packaging cell will have sufficient lentiviral genetic information to allow the RNA genome to be packaged into viral particles capable of infecting target cells but unable to replicate independently in the presence of packaging components to produce infectious viral particles in the final target cell. Preferably, the vector lacks functional gag-pol and / or env genes and / or other genes essential for replication.

[0539] However, the plasmid vector used to produce the viral genome in the host cell / packaging cell will also contain transcriptional regulatory control sequences operably linked to the lentiviral genome to direct the transcription of the genome in the host cell / packaging cell. These regulatory sequences can be natural sequences associated with the transcribed viral sequence (i.e., the 5' U3 region), or they can be a heterologous promoter, such as another viral promoter (e.g., CMV promoter).

[0540] The vector can be a self-inactivating (SIN) vector in which the viral enhancer and promoter sequences have been deleted. SIN vectors can be generated in vivo and transduce non-dividing cells with efficacy similar to that of wild-type vectors. Transcriptional inactivation of the long terminal repeats (LTRs) in the SIN provirus will prevent mobilization by replication-competent viruses. This will also enable regulation of gene expression from internal promoters by eliminating any cis-acting effects of the LTRs.

[0541] The vector may be integration-defective. For example, an integration-defective lentiviral vector (IDLV) may be produced by packaging the vector with a catalytically inactive integrase (such as an HIV integrase with a D64V mutation in the catalytic site), or by modifying or deleting essential att sequences from the vector LTR, or by a combination of the above.

[0542] Adenoviral vector

[0543] The vector of the present invention may be an adenovirus vector. The vector of the present invention may be an adenovirus vector particle.

[0544] Adenoviruses are double-stranded linear DNA viruses that do not pass through an RNA intermediate. There are more than 50 different human adenovirus serotypes, divided into 6 subgroups based on gene sequence homology. The natural targets of adenoviruses are respiratory and gastrointestinal epithelia, usually causing only mild symptoms. Serotypes 2 and 5 (with 95% sequence homology) are most commonly used in adenovirus vector systems and are often associated with upper respiratory tract infections in young people.

[0545] Adenoviruses have been used as vectors for gene therapy and heterologous gene expression. The large (36 kb) genome can accommodate up to 8 kb of foreign insert DNA and can replicate efficiently in complementing cell lines to produce up to 10 12 Therefore, adenovirus is one of the best systems for studying gene expression in primary non-replicating cells.

[0546] The expression of viral genes or exogenous genes from the adenoviral genome does not require replicative cells. Adenoviral vectors enter cells through receptor-mediated endocytosis. Once inside the cell, adenoviral vectors rarely integrate into the host chromosome. Instead, they function as an episome (independent of the host genome) as a linear genome in the host cell nucleus. Therefore, the use of recombinant adenoviruses alleviates the problems associated with random integration into the host genome.

[0547] Herpes simplex virus vector

[0548] The vector of the present invention may be a herpes simplex virus vector. The vector of the present invention may be a herpes simplex virus vector particle.

[0549] Herpes simplex virus (HSV) is a neurotropic DNA virus with favorable properties as a gene delivery vector. HSV is highly infectious, so HSV vectors are effective vehicles for delivering exogenous genetic material to cells. Viral replication is easily destroyed by empty mutations in immediate early genes, which can be trans-complemented in vitro, which enables the direct production of high-titer pure preparations of non-pathogenic vectors. The genome is very large (152Kb), and many viral genes are dispensable for in vitro replication, which allows them to be replaced with large transgenes or multiple transgenes. Latent infection of wild-type viruses leads to the persistence of episomal viruses in sensory nerve nuclei during the host's lifetime. The vector is non-pathogenic, cannot be reactivated and persists for a long time. Latent active promoter complexes can be used for vector design to achieve long-term stable transgenic expression in the nervous system. Due to the wide expression pattern of cell receptors recognized by the virus, HSV vectors transduce a wide range of tissues. The increasing understanding of the process involving cell entry has allowed the targeting of the tropism of HSV vectors.

[0550] Vaccinia virus vector

[0551] The vector of the present invention may be a vaccinia virus vector. The vector of the present invention may be a vaccinia virus vector particle.

[0552] Vaccinia virus is a large enveloped virus with a linear double-stranded DNA genome of approximately 190 kb. Vaccinia virus can accommodate up to about 25 kb of foreign DNA, which also makes it useful for the delivery of large genes. Many attenuated vaccinia virus strains suitable for gene therapy applications are known in the art, such as MVA and NYVAC strains.

[0553] Anellovirus vector

[0554] The vector of the present invention may be an anacycloviral vector. The vector of the present invention may be an anacycloviral vector particle.

[0555] Anelloviruses are small, single-stranded, circular DNA viruses. They are extremely diverse and have not been associated with any disease to date. Anelloviruses can infect entire populations, and there is no evidence of disease association or clearance of the virus from infected individuals (see, e.g., Venkataraman, T., et al., 2022.bioRxiv 2022.03.28.486145).

[0556] Non-viral delivery systems

[0557] In some embodiments, the vector is a non-viral vector. Suitable non-viral delivery systems are known to the skilled person (see, for example, Zulliger, R., et al., 2015. Journal of Controlled Release, 219, pp. 471-487; and Oliveira, AV, et al., 2017. Materials Science and Engineering: C, 77, pp. 1275-1289.).

[0558] In some embodiments, the vector is a plasmid. In some embodiments, the plasmid is modified to promote cellular and / or nuclear uptake. In some embodiments, the plasmid is contained in a non-viral particle (e.g., a lipoplex particle or a polyplex particle).

[0559] Non-viral delivery systems include, but are not limited to, transfection methods. Here, transfection includes the process of delivering genes to target cells using non-viral vectors. Typical transfection methods include electroporation, DNA gene guns, lipid-mediated transfection, compacted DNA-mediated transfection, liposomes, immunoliposomes, lipofectin, cationic agent-mediated transfection, cationic surface amphiphiles (CFAs), and combinations thereof.

[0560] Instance carrier

[0561] The vector of the present invention may comprise, from 5' to 3', a promoter (eg, an inflammation-inducible promoter) and a nucleotide sequence encoding a TNF inhibitor.

[0562] The carrier of the present invention may further comprise any other suitable element, such as any other element described herein or one or more spacer sequences. The spacer sequence may comprise, for example, at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10), at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine or at least ten nucleotide bases. The spacer sequence may comprise a restriction site to enable insertion of one or more additional elements.

[0563] In a preferred embodiment, the vector of the present invention is an AAV vector. In some embodiments, the AAV genome comprises from 5' to 3': 5'ITR; inflammation-inducible promoter; nucleotide sequence encoding a TNF inhibitor; and 3'ITR. In some embodiments, the AAV genome comprises from 5' to 3': 5'ITR; inflammation-inducible promoter; nucleotide sequence encoding a TNF inhibitor; polyadenylation sequence; and 3'ITR. In some embodiments, the AAV genome comprises from 5' to 3': 5'ITR; inflammation-inducible promoter; nucleotide sequence encoding a TNF inhibitor; WPRE; polyadenylation sequence and 3'ITR. In some embodiments, the AAV genome comprises from 5' to 3': 5'ITR; inflammation-inducible promoter; intron; nucleotide sequence encoding a TNF inhibitor; WPRE; polyadenylation sequence; and 3'ITR. In some embodiments, the AAV genome comprises from 5' to 3': a 5' ITR; an inflammation-inducible promoter; an intron; a nucleotide sequence encoding a TNF inhibitor; a WPRE; a polyadenylation sequence; a sequence encoding an inflammation-suppressing oligonucleotide; and a 3' ITR.

[0564] In one aspect, the invention provides a vector comprising or consisting of a nucleotide sequence at least 70% identical to SEQ ID NO: 91 or a fragment thereof. Suitably, the vector comprises or consists of a nucleotide sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 91 or a fragment thereof. In some embodiments, the vector comprises or consists of the nucleotide sequence SEQ ID NO: 91 or a fragment thereof.

[0565] In some embodiments, the AAV genome comprises or consists of an amino acid sequence that is at least 70% identical to SEQ ID NO: 91 or a fragment thereof. Suitably, the AAV genome comprises or consists of a nucleotide sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 91 or a fragment thereof. In some embodiments, the AAV genome comprises or consists of the nucleotide sequence SEQ ID NO: 91 or a fragment thereof.

[0566]

[0567] Example AAV vector encoding adalimumab Fab (SEQ ID NO: 91)

[0568] In one aspect, the invention provides a vector comprising or consisting of a nucleotide sequence at least 70% identical to SEQ ID NO: 92 or a fragment thereof. Suitably, the vector comprises or consists of a nucleotide sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 92 or a fragment thereof. In some embodiments, the vector comprises or consists of the nucleotide sequence SEQ ID NO: 92 or a fragment thereof.

[0569] In some embodiments, the AAV genome comprises or consists of an amino acid sequence that is at least 70% identical to SEQ ID NO: 92 or a fragment thereof. Suitably, the AAV genome comprises or consists of a nucleotide sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 92 or a fragment thereof. In some embodiments, the vector comprises or consists of the nucleotide sequence SEQ ID NO: 92 or a fragment thereof.

[0570]

[0571] Example AAV vector encoding infliximab Fab (SEQ ID NO: 92)

[0572] Variants, derivatives, analogs and fragments

[0573] In addition to the specific polypeptides and polynucleotides mentioned herein, the present invention also encompasses variants, derivatives and fragments thereof.

[0574] In the context of the present invention, a "variant" of any given sequence refers to a sequence in which a particular sequence of residues (whether amino acids or nucleic acid residues) has been modified in such a way that the polypeptide or polynucleotide in question retains at least one or all of its endogenous functions. Variant sequences can be obtained by adding, deleting, substituting, modifying, replacing and / or mutating at least one residue present in a naturally occurring polypeptide or polynucleotide.

[0575] As used herein, the term "derivative" in relation to a protein or polypeptide of the invention includes any substitution, variation, modification, replacement, deletion and / or addition from or to one (or more) amino acid residues in the sequence, provided that the resulting protein or polypeptide retains at least one or all of its endogenous functions.

[0576] Generally, amino acid substitutions (eg, from 1, 2 or 3 to 10 or 20 substitutions) can be made, provided that the modified sequence retains the desired activity or ability.Amino acid substitutions may include the use of non-naturally occurring analogs.

[0577] The polypeptides used in the present invention may also have deletions, insertions or substitutions of amino acid residues that produce silent changes and result in functionally equivalent polypeptides. Deliberate amino acid substitutions may be made based on similarities in the polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathic properties of the residues, as long as the endogenous function is retained. For example, negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; and amino acids with uncharged polar head groups with similar hydrophilic values ​​include asparagine, glutamine, serine, threonine, and tyrosine.

[0578] Conservative substitutions can be made, for example according to the following table. Amino acids in the same block in the second column and amino acids in the same row in the third column can be substituted for each other:

[0579]

[0580] The effects of addition, deletion, substitution, modification, replacement and / or variation can be predicted using any suitable prediction tool such as SIFT (Vaser, R., et al., 2016. Nature protocols, 11 (1), pp. 1-9), PolyPhen-2 (Adzhubei, I., et al., 2013. Current protocols in human genetics, 76 (1), pp. 7-20), CADD (Rentzsch, P., et al., 2021. Genome medicine, 13 (1), pp. 1-12), REVEL (Ioannidis, NM, et al., 2016. The American Journal of Human Genetics, 99 (4), pp. 877-885), MetaLR (Dong, C., et al., 2015. Human molecular genetics, 24 (8), pp. 2125-2137) and / or MutationAssessor (Reva, B., et al., 2011. Nucleic acids Research, 39 (17), pp. e118-e118) or predicted based on clinical data such as ClinVar (Landrum, MJ, et al., 2016. Nucleic acids research, 44 (D1), pp. D862-D868). Suitable additions, deletions, substitutions, modifications, replacements and / or variations can be considered tolerable, benign and / or likely to be benign.

[0581] Generally, a variant may have a certain identity with a wild-type amino acid sequence or a wild-type nucleotide sequence.

[0582] In the present context, variant sequences are considered to include amino acid sequences that may be at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85% or at least 90% identical, suitably at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the subject sequence. Although variants may also be considered in terms of similarity (i.e., amino acid residues having similar chemical properties / functions), in the context of the present invention, it is preferred to express in terms of sequence identity.

[0583] In this context, variant sequences are considered to include nucleotide sequences that may be at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85% or at least 90% identical to the subject sequence, suitably at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical. Although variants may also be considered with respect to similarity, in the context of the present invention, it is preferred to express variants with respect to sequence identity.

[0584] Suitably, reference to a sequence having a percentage identity to any one of the SEQ ID NOs detailed herein refers to the sequence having that percentage identity over the entire length of the referenced SEQ ID NO.

[0585] Sequence identity comparisons can be performed by eye, or more usually, with the aid of available sequence comparison programs. These commercially available computer programs can calculate the percent identity between two or more sequences.

[0586] Percent identity can be calculated over contiguous sequences by aligning one sequence with the other and comparing each amino acid or nucleotide in one sequence directly to the corresponding amino acid or nucleotide in the other sequence, one residue at a time. This is called an "ungapped" alignment. Typically, such ungapped alignments are performed only over relatively short residue numbers.

[0587] While this is a very simple and consistent approach, it does not take into account that, for example, an insertion or deletion in an amino acid or nucleotide sequence in an otherwise identical pair of sequences may result in the subsequent residue or codon being placed outside the alignment, thus potentially resulting in a substantial reduction in percent identity when a global alignment is performed. Therefore, most sequence comparison methods are designed to produce an optimal alignment, taking into account possible insertions and deletions without overly penalizing the overall identity score. This is achieved by inserting "gaps" in the sequence alignment in an attempt to maximize local identity.

[0588] In some embodiments, the present invention relates to the comparison of the sequence alignment of amino acid sequence with the same number of gaps. However, these more complex methods assign "gap penalties" to each gap present in the comparison, so that for the same number of identical amino acids or nucleotides, a sequence alignment with as few gaps as possible (reflecting the higher correlation between the two compared sequences) will achieve a higher score than a sequence alignment with many gaps. Usually an "affine gap cost" is used, which charges a relatively high cost to the existence of a gap, and charges a smaller penalty to each subsequent residue in the gap. This is the most commonly used gap scoring system. High gap penalties will produce an optimized comparison with less gaps. Most alignment programs allow modification of gap penalties. However, when using this type of software to compare sequences, it is preferred to use default values. For example, when using the GCG Wisconsin Bestfit package, the default gap penalty for amino acid sequences is gap-12 and each extension-4.

[0589] Therefore, calculation of the maximum percent identity first requires the production of an optimal alignment, which takes into account gap penalties. A suitable computer program for performing such an alignment is the GCG Wisconsin Bestfit package (see, for example, Devereux, J., et al., 1984. Nucleic acids research, 12(1), pp. 387-395). Examples of other software that can perform sequence comparisons include, but are not limited to, BLAST packages (see, e.g., Altschul, SF, et al., 1990. Journal of molecular biology, 215 (3), pp. 403-410), BLAST 2 (see, e.g., Tatusova, TA and Madden, TL, 1999. FEMS microbiology letters, 174 (2), pp. 247-250), FASTA (see, e.g., Pearson, WR and Lipman, DJ, 1988. PNAS, 85 (8), pp. 2444-2448.), EMBOSS Needle (Madeira, F., et al., 2019. Nucleic acids research, 47 (W1), pp. W636-W641) and the GENEWORKS alignment tool suite. For some applications, it is preferred to use EMBOSS Needle.

[0590] Although the final percent identity can be measured, the alignment process itself is usually not based on an all-or-nothing pairwise comparison. Instead, a scaled similarity score matrix is ​​often used, assigning scores to each pairwise comparison based on chemical similarity or evolutionary distance. An example of such a matrix that is commonly used is the BLOSUM62 matrix.

[0591] Once the software has produced the optimal alignment, it is possible to calculate the percentage of sequence identity. The software typically performs this as part of a sequence comparison and generates a numerical result. The percentage of sequence identity can be calculated as the number of identical residues as a percentage of the total residues in the SEQ ID NO referred to.

[0592] "Fragment" is also a variant, and the term generally refers to a selected region of a polypeptide or polynucleotide that is of interest functionally or in, for example, an assay. Thus, a "fragment" refers to an amino acid or nucleic acid sequence that is a portion of a full-length polypeptide or polynucleotide.

[0593] Such variants, derivatives and fragments can be prepared using standard recombinant DNA techniques such as site-directed mutagenesis. Where insertion is required, synthetic DNA encoding the insert can be prepared, together with 5' and 3' flanking regions corresponding to the naturally occurring sequences on both sides of the insertion site. The flanking regions will contain convenient restriction sites corresponding to the sites in the naturally occurring sequence, so that the sequence can be cut with an appropriate enzyme and the synthetic DNA can be connected to the nick. The DNA is then expressed according to the present invention to prepare the encoded polypeptide. These methods only illustrate many standard techniques known in the art for manipulating DNA sequences, and other known techniques can also be used.

[0594] Vectors, kits and systems

[0595] In one aspect where the vector is a viral vector, the invention provides a vector encoding a viral genome of the invention. The vector can be a transfer vector as described herein. For example, the vector can be a plasmid and / or the viral genome can be operably linked to a promoter (e.g., a viral promoter, such as a CMV promoter).

[0596] In one aspect, the invention provides a kit or system for producing a vector (e.g., a viral vector) of the invention. The kit or system may be a viral packaging kit or system or a viral production kit or system. As used herein, a "viral packaging kit or system" may include one or more components for packaging a viral vector of the invention, and optionally instructions. As used herein, a "viral production kit or system" may include one or more components for producing a viral vector of the invention, and optionally instructions.

[0597] The kit or system may comprise a transfer vector encoding the viral genome of the present invention, and optionally one or more auxiliary vectors. The kit or system may further comprise a host cell (e.g., a packaging cell or a production cell) and / or other reagents (e.g., a transfection reagent, a culture medium, etc.). The kit or system may further comprise any other suitable components for packaging and / or producing the viral vector of the present invention, and optionally instructions.

[0598] cell

[0599] In one aspect, the invention provides a cell comprising a vector of the invention (e.g., a viral vector). The cell may be an isolated cell. Suitably, the cell is a mammalian cell, such as a human cell. The cell may be an isolated human cell.

[0600] Suitably, the cell can be a producer cell. The term "producer cell" includes cells that produce viral particles after transient transfection, stable transfection or vector transduction of all elements necessary for producing viral particles, or any cell that is engineered to stably contain elements necessary for producing viral particles. In some embodiments, the producer cell is an AAV producer cell. Suitable producer cells are known to those skilled in the art (see, for example, Martin, J., et al. 2013. Human gene therapy methods, 24 (4), pp. 253-269), and may include HEK293, COS-1, COS-7, CV-1, HeLa, CHO and A549 cell lines. In some embodiments, the producer cell is a HEK293 cell or a derivative thereof (e.g., HEK293T cell).

[0601] Suitably, the cell can be a packaging cell. The term "packaging cell" includes cells containing some or all of the elements necessary for packaging a recombinant viral genome. Typically, such packaging cells contain one or more vectors capable of expressing viral structural proteins (e.g., AAV rep and cap genes) and / or one or more genes encoding viral structural proteins have been integrated into the genome of the packaging cell. After subsequent steps of transient transfection, transduction, or stable integration of each additional required element, cells containing only some of the elements required to produce enveloped viral particles can be used as intermediate reagents for generating viral particle producer cell lines. The term "packaging cell" encompasses these intermediate reagents. In some embodiments, the packaging cell is an AAV packaging cell. Suitable packaging cells are known to those skilled in the art (see, e.g., Martin, J., et al. 2013. Human gene therapy methods, 24 (4), pp. 253-269).

[0602] Pharmaceutical composition

[0603] In one aspect, the invention provides a pharmaceutical composition comprising a vector or a cell of the invention. In a preferred embodiment, the pharmaceutical composition comprises a vector of the invention in the form of a viral vector particle.

[0604] A pharmaceutical composition is a composition comprising or consisting of a therapeutically effective amount of a pharmaceutically active agent (eg, a carrier). The pharmaceutical composition preferably comprises a pharmaceutically acceptable carrier, diluent, or excipient (including combinations thereof).

[0605] The term "pharmaceutically acceptable" includes formulations that are sterile and pyrogen-free. The carrier, diluent and / or excipient must be "acceptable" in the sense of being compatible with the carrier and not harmful to the recipient thereof. Typically, the carrier, diluent and excipient will be saline or an infusion medium that is sterile and pyrogen-free, however, other acceptable carriers, diluents and excipients may be used.

[0606] Acceptable carriers, diluents and excipients for therapeutic use are well known in the pharmaceutical field. The choice of pharmaceutical carrier, excipient or diluent can be selected with respect to the intended route of administration and standard pharmaceutical practice. The pharmaceutical composition may include any suitable binder, lubricant, suspending agent, coating agent or solubilizing agent as a carrier, excipient or diluent (or in addition to a carrier, excipient or diluent).

[0607] Examples of pharmaceutically acceptable carriers include, for example, water, saline solutions, alcohol, silicone, wax, petroleum vaseline, vegetable oils, polyethylene glycol, propylene glycol, liposomes, sugars, gelatin, lactose, amylose, magnesium stearate, talc, surfactants, silicic acid, viscous paraffin, perfume oils, fatty acid mono- and diglycerides, petroleum ether fatty acid esters, hydroxymethylcellulose, polyvinyl pyrrolidone, and the like.

[0608] The vector, cell or pharmaceutical composition according to the present invention can be administered in a manner suitable for treating and / or preventing the diseases described herein. Suitable routes of administration are known to those skilled in the art. The amount and frequency of administration can be determined by a technician, for example depending on factors such as the subject's condition and the type and severity of the subject's disease. The pharmaceutical composition can be formulated accordingly.

[0609] The vector, cell or pharmaceutical composition according to the present invention can be topically administered to the eye. Suitably, the vector, cell or pharmaceutical composition according to the present invention is administered by subretinal injection, direct retinal injection, subconjunctival injection, sub-Tenon injection, periocular injection, suprachoroidal injection or intravitreal injection. The pharmaceutical composition can be formulated accordingly.

[0610] In some embodiments, the vector, cell or pharmaceutical composition of the present invention is administered intraocularly. The term "intraocular" may refer to the interior of the eye, so intraocular administration may involve administration to the interior of the subject's eye. In some embodiments, the vector, cell or pharmaceutical composition is administered to the subject's eye by subretinal injection, direct retinal injection, suprachoroidal injection or intravitreal injection. Technicians will be familiar with and can well perform individual subretinal injections, direct retinal injections, suprachoroidal injections or intravitreal injections (see, e.g., Hartman, RR and Kompella, UB, 2018. Journal of Ocular Pharmacology and Therapeutics, 34 (1-2), pp. 141-153).

[0611] In some embodiments, the vector, cell or pharmaceutical composition is administered to the subject's eye by subretinal injection, suprachoroidal injection or intravitreal injection. In a preferred embodiment, the vector, cell or pharmaceutical composition of the invention is administered by intravitreal injection.

[0612] The pharmaceutical composition may comprise the vector or cell of the present invention in an infusion medium (eg, a sterile isotonic solution). The pharmaceutical composition may be packaged in an ampoule, a disposable syringe or a multiple dose vial made of glass or plastic.

[0613] The vector, cell or pharmaceutical composition can be administered in a single dose or multiple doses. Suitably, the vector, cell or pharmaceutical composition can be administered in a single, disposable dose. The pharmaceutical composition can be formulated accordingly.

[0614] Vectors, cells or pharmaceutical compositions can be administered at different doses (e.g. measured by viral genome (vg) / mL). In any case, a physician can determine the actual dose that will be most suitable for any individual subject, and the dose may vary, for example, with the age, weight and response of a particular subject.

[0615] Suitably, the carrier of the present invention is present in an amount of at least about 10 10 vg / mL, at least about 10 11 vg / mL, at least about 10 12 vg / mL or at least about 5x10 12 vg / mL. Suitably, the carrier of the present invention is administered at a dose of about 10 13 vg / mL or less, about 10 12 vg / mL or less or about 10 11 vg / mL or less. Suitably, the vector of the present invention is administered at a dose of about 10 10 to about 10 13 vg / mL, or about 1011 to about 10 13 vg / mL. Suitably, the carrier of the present invention is administered at a dose of about 10 10 to about 10 12 vg / mL. Suitably, the carrier of the present invention is administered at a dose of about 10 11 to about 10 13 vg / mL. Suitably, the carrier of the present invention is administered at a dose of about 10 12 to about 10 13 vg / mL. Suitably, the carrier of the present invention is administered at a dose of about 10 12 About 5x10 12 The pharmaceutical composition can be formulated accordingly.

[0616] Suitably, the carrier of the present invention is present in an amount of at least about 10 9 vg / eye, at least about 2x10 9 vg / eye, at least about 5x10 9 vg / eye, at least about 10 10 vg / eye, at least about 2x10 10 vg / eye, at least about 5x10 10 vg / eye or at least about 10 11 vg / eye. Suitably, the carrier of the present invention is administered at a dose of about 10 13 vg / eye or smaller or about 5x10 12 vg / eye or less. Suitably, the carrier of the present invention is administered at a dose of about 10 9 vg / eye is about 5x10 12 vg / eye, about 10 10 vg / eye is about 5x10 12 vg / eye, about 10 10 vg / eye to about 10 12 vg / eye or about 10 10 vg / eye is about 5x10 11 The dose was administered at vg / eye.

[0617] Suitably, the vector of the present invention can be administered in combination with one or more other therapeutic agents. One or more other therapeutic agents can be administered separately, simultaneously or sequentially. The pharmaceutical composition may further comprise one or more other therapeutic agents. For example, the vector of the present invention can be administered in combination with one or more immunosuppressants (e.g., antimetabolites, calcineurin inhibitors, and alkylating agents).

[0618] The vectors of the invention can reduce the need for immunosuppressant therapy. In some embodiments, the vectors of the invention are administered in the absence of immunosuppressant therapy (ie, the subject is not undergoing immunosuppressant therapy).

[0619] The invention further includes a kit comprising the vector, cell and / or pharmaceutical composition of the invention. Preferably, the kit is used in the method as described herein and is used as described herein, e.g., a therapeutic method as described herein. Preferably, the kit comprises instructions for using the kit components.

[0620] Methods for treating and / or preventing disease

[0621] In one aspect, the invention provides a vector, a cell and / or a pharmaceutical composition according to the invention for use as a medicament.

[0622] In one aspect, the present invention provides use of a vector, a cell or a pharmaceutical composition according to the present invention in the preparation of a medicament.

[0623] In one aspect, the invention provides a method of administering a therapeutically effective amount of a vector, cell or pharmaceutical composition according to the invention to a subject in need thereof.

[0624] The vector, cell or pharmaceutical composition can be administered to any subject in need thereof. The subject can be a mammal (e.g., a human). The vector, cell or pharmaceutical composition according to the present invention can be administered to a subject suffering from an inflammatory eye disease or at risk of an inflammatory eye disease.

[0625] Inflammatory eye disease

[0626] The vector, cell or pharmaceutical composition according to the present invention can be used to prevent and / or treat inflammatory eye diseases.

[0627] In one aspect, the present invention provides a vector, a cell or a pharmaceutical composition according to the present invention for use in preventing and / or treating an inflammatory eye disease.

[0628] In one aspect, the present invention provides use of a vector, a cell or a pharmaceutical composition according to the present invention in the preparation of a medicament for preventing or treating an inflammatory eye disease.

[0629] In one aspect, the present invention provides a method for preventing or treating an inflammatory eye disease, the method comprising administering to a subject in need thereof a therapeutically effective amount of a vector, cell or pharmaceutical composition according to the present invention.

[0630] As used herein, "inflammatory eye disease" may refer to any condition associated with inflammation of the eye, including uveitis, scleritis, keratitis, conjunctivitis, iritis, chorioretinitis, choroiditis, retinitis, and retinochoroiditis.

[0631] After administering the vector, cell and / or pharmaceutical composition according to the present invention, one or more symptoms of inflammatory eye diseases can be prevented and / or treated in a subject. Any suitable method for determining the severity of inflammatory eye diseases can be used (see, e.g., McNeil, R., 2016. Eye news, 22(5), pp. 1-4).

[0632] The vector, cell and / or pharmaceutical composition according to the present invention can prevent and / or reduce intraocular inflammation. Any suitable method can be used to determine intraocular inflammation. Suitable methods for quantifying intraocular inflammation include laser flash photometry (see, e.g., Tugal-Tutkun, I. and Herbort, CP, 2010. International ophthalmology, 30(5), pp.453-464).

[0633] The vector, cell and / or pharmaceutical composition according to the present invention can reduce the recurrence rate of inflammatory eye diseases. Any suitable method can be used to determine the recurrence rate. For example, a recurrence (or flare) of uveitis is usually defined as anterior chamber cell and / or vitreous opacity graded ≥2+ using the SUN grading system (see, e.g., McNeil, R., 2016. Eye news, 22(5), pp. 1-4).

[0634] The vector, cell and / or pharmaceutical composition according to the present invention can prevent and / or reduce vision loss. Inflammatory eye diseases such as uveitis are the main causes of visual morbidity (see, e.g., Durrani, OM, et al., 2004. British Journal of Ophthalmology, 88(9), pp.1159-1162). The vector, cell and / or pharmaceutical composition according to the present invention can maintain or improve vision.

[0635] Uveitis

[0636] In a preferred embodiment, the inflammatory eye disease is uveitis.

[0637] In one aspect, the present invention provides a vector, a cell or a pharmaceutical composition according to the present invention for use in preventing and / or treating uveitis.

[0638] In one aspect, the present invention provides use of a vector, a cell or a pharmaceutical composition according to the present invention in the preparation of a medicament for preventing or treating uveitis.

[0639] In one aspect, the present invention provides a method for preventing or treating uveitis, the method comprising administering a therapeutically effective amount of a vector, a cell or a pharmaceutical composition according to the present invention to a subject in need thereof.

[0640] Uveitis refers to a group of intraocular inflammatory diseases of the uvea (i.e., iris, ciliary body, and choroid) and adjacent structures (including cornea, vitreous humor, retina, and optic nerve). Uveitis can be classified based on the primary anatomical site of inflammation (i.e., anterior, middle, and posterior) and / or etiological origin (including infectious, non-infectious, or masquerade) (see, e.g., Rosenbaum, JT, et al., 2019. Seminars in Arthritis and Rheumatism, 49(3), pp.438-445; and Standardization of Uveitis Nomenclature (SUN) Working Group, 2005. American journal of ophthalmology, 140(3), pp.509-516).

[0641] Uveitis can be anterior uveitis, intermediate uveitis, posterior uveitis or panuveitis. Uveitis can be infectious, non-infectious or masquerading. In a preferred embodiment, uveitis is non-infectious uveitis.

[0642] In some embodiments, the uveitis is selected from one or more of the following: sympathetic ophthalmia, birdshot chorioretinopathy, sarcoid uveitis, intermediate uveitis, Vogt Koyanaga Harada syndrome, JIA-associated uveitis, idiopathic retinal vasculitis, HLA-B27-associated non-anterior uveitis.

[0643] In some embodiments, the uveitis is sympathetic ophthalmia. Sympathetic ophthalmia is a rare bilateral granulomatous uveitis that occurs following surgery or accidental trauma to one eye (see, e.g., Damico, FM, et al., 2005. Seminars in ophthalmology, 20(3), pp. 191-197).

[0644] In some embodiments, the uveitis is birdshot chorioretinopathy. Birdshot chorioretinopathy is a rare, chronic, bilateral posterior uveitis (see, e.g., Levinson, RD, et al., 2006. American journal of ophthalmology, 141(1), pp. 185-187).

[0645] In some embodiments, the uveitis is sarcoid uveitis. Sarcoid uveitis may also be referred to as sarcoid-associated uveitis. Sarcoidosis is a disease that causes noncaseating granulomatous inflammation in one or more organs. The most common ocular manifestations are uveitis, dry eyes, and conjunctival nodules (see, e.g., Jamilloux, Y., et al., 2014. Autoimmunity reviews, 13 (8), pp. 840-849).

[0646] In some embodiments, the uveitis is intermediate uveitis.Intermediate uveitis can refer to inflammation in the anterior vitreous, ciliary body, and peripheral retina (see, eg, Babu, BM and Rathinam, SR, 2010. Indian journal of ophthalmology, 58(1), p. 21).

[0647] In some embodiments, the uveitis is Vogt Koyanaga Harada syndrome. Vogt-Koyanagi-Harada syndrome is a bilateral chronic diffuse granulomatous panuveitis, often associated with neurological, auditory, and cutaneous manifestations (see, e.g., Fang, W. and Yang, P., 2008. Current eye research, 33(7), pp. 517-523).

[0648] In some embodiments, the uveitis is JIA-associated uveitis. JIA is the most common rheumatic disease in childhood, of which JIA-associated uveitis is its most common extra-articular manifestation (see, e.g., Clarke, SL, et al., 2016. Pediatric Rheumatology, 14(1), pp. 1-11).

[0649] In some embodiments, uveitis is idiopathic retinal vasculitis. Retinal vasculitis is a vision-threatening inflammatory eye condition involving retinal blood vessels. Based on the cause, retinal vasculitis can be classified as idiopathic or secondary to infection, neoplasia, or systemic inflammatory disease (see, e.g., Talat, L., et al., 2014. Journal of ophthalmology, 197675).

[0650] In some embodiments, the uveitis is HLA-B27 associated non-anterior uveitis. Human leukocyte antigen (HLA)-B27 associated uveitis is the most commonly diagnosed cause of acute anterior uveitis (see, e.g., Loh, AR and Acharya, NR, 2010. American journal of ophthalmology, 150(4), pp. 534-542). Example

[0651] The present invention will now be further described by way of examples, which are intended to help those of ordinary skill in the art to practice the present invention and are not intended to limit the scope of the present invention in any way.

[0652] Example 1 - In vivo topical administration of anti-TNF antibody fragments inhibits experimental autoimmune uveoretinitis (EAU)

[0653] B10.RIII mice were immunized for experimental autoimmune uveoretinitis (EAU), and eyes were monitored using Topical Endoscopic Fundal Imaging (TEFI) starting on day 10 to select experimental mice that exhibited clinically evident disease. On day 10, each group of mice was injected with 15 μg of infliximab or vehicle control (EAU) via the intravitreal route. Eyes were enucleated (day 14) and retinal infiltrates were characterized. Representative fundus images ( Figure 1 A) Clinical disease scores ( Figure 1 B) and flow cytometric analysis of total CD45+ cell numbers from individual eyes on day 14 ( Figure 1 C), Demonstrates the efficacy of intravitreal infliximab-derived Fab molecules in inhibiting inflammation and retinal infiltration in the B10.RIII EAU model. **P<0.005; data are expressed as mean + / - SEM and are representative of two independent experiments.

[0654] Example 2 - Therapeutic vector design

[0655] Figure 2 : is a schematic diagram showing the vector organization of CMV.Infliximab Fab (constitutive CMV promoter) and AP1-NFkB.Infliximab Fab (inflammation-inducible promoter containing 5 repeats of AP1 and NFkB binding sites). The heavy and light chains of Infliximab Fab are separated by a self-cleaving 2A peptide to generate two separate peptides, and then Fab is formed in situ. After transduction of cells, AAV persists as episomal DNA in the nucleus.

[0656] Example 3 - In vitro and in vivo evaluation of constitutive transgene expression

[0657] To evaluate constitutive infliximab Fab transgene expression, HEK-293T (a standard cell line for AAV development) or ARPE-19 (an ocular cell line) cells were transduced with AAV2.CMV.Infliximab or AAV.CMV.NULL vectors [MOI 1E5 vg / cell] and culture supernatants were assayed for expression using a clinical IFX ELISA kit ( Figure 3 A). Detectable expression of Infliximab Fab from both cell types was evident by 72 hours (approximately 30 ng / ml). ****P<0.0001. Data are expressed as mean + / - SEM.

[0658] To evaluate transgene expression in vivo, AAV2.CMV.Infliximab or AAV.CMV.NULL was administered by intravitreal (IVT) injection into the eyes of healthy B10.RIII mice at 5E12 vg / ml. Four weeks after AAV, mice were sacrificed, eyes were dissected and supernatants (retina and vitreous) were assayed using a clinical IFX ELISA kit ( Figure 3 B). Detectable expression of infliximab Fab in vivo (approximately 1.5 ng / ml) was observed in eyes receiving the 5E12 vg / ml dose. *P<0.05. Data are presented as mean + / - SEM, where each data point represents a single eye.

[0659] These data demonstrate that AAV can mediate constitutive expression of anti-TNF Fab.

[0660] Example 4 - Evaluation of in vitro and in vivo clinical models of inducible transgene expression

[0661] To demonstrate the inducibility of the inflammatory responsive promoter design, HEK-293T cells transduced with AAV2.AP1-NFkB.EGFP (reporter vector) or AAV2.AP1-NFkB.Infliximab (therapeutic vector) were stimulated with recIL-1b (2 ng / mL). Activation resulted in GFP expression visible at 24 h, increasing in intensity by 72 hr, where no GFP signal was observed with AAV2.AP1-NFkB.NULL (control vector) ( Figure 4 A). Higher GFP signals were observed with the constitutive AAV.CMV.EGFP vector. Stimulation resulted in a rapid induction of infliximab Fab expression (8 hr), with accumulation reaching approximately 20 ng / ml at 72 hr ( Figure 4 B). Images captured on EVOS FL, 10X magnification. ****P<0.0001; data are expressed as mean + / - SEM.

[0662] To demonstrate the in vivo inducibility of the inflammatory responsive promoter design, AAV2.AP1-NFkB.EGFP (reporter) or AAV.AP1-NFkB.NULL (control) at a dose of 5E12 vg / ml was administered to C57BL / 6J mice by intravitreal (IVT) injection. Four weeks after AAV injection, mice were immunized to induce experimental autoimmune uveoretinitis (EAU) and imaged to monitor the onset of ocular inflammation. Representative fundus and OCT images at day 14 of EAU show clear clinical signs of disease (perivascular sheathing and vitreous infiltration), as well as induction of GFP expression ( Figure 5 A) In mice that received AAV alone (without EAU), no clinical signs of disease or expression of the GFP transgene were observed.

[0663] To demonstrate the inducibility of the therapeutic transgene in vivo, mice in each group were injected with AAV2.AP1-NFkB.Infliximab or AAV2.AP1-NFkB.NULL in the contralateral eye and EAU was induced 4 weeks after AAV. At 3 weeks after EAU, mice were sacrificed, eyes were dissected and supernatants (retina and vitreous) were assayed using a clinical IFX ELISA kit ( Figure 5 B). Detectable expression of infliximab Fab was observed only in EAU eyes receiving therapeutic vehicle and not in controls. *P<0.05. Data are presented as mean + / - SEM, with each data point representing a single eye.

[0664] These data demonstrate the in vitro and in vivo inducibility of inflammatory responsive promoters designed to express either a reporter gene (eGFP) or a therapeutic (anti-TNFFab) transgene.

[0665] Example 5 - In vivo evaluation of the efficacy of a constitutive therapeutic transgene

[0666] To demonstrate the efficacy of a constitutive therapeutic transgene, groups of mice were injected with AAV7m8.CMV.Infliximab or AAV7m8.CMV.NULL in the contralateral eye, followed by intravitreal administration of recombinant human TNF (rec_hTNF) 4 weeks after AAV. At 18 hr (peak of the inflammatory response to rec_hTNF), representative fundus and OCT images show increased inflammation (vitreal infiltration) in control (null) eyes compared to infliximab eyes ( Figure 6A). In mice that received AAV alone, no clinical signs of disease were observed. Mice were sacrificed at 18 hr and eyes were dissected for flow cytometric analysis to determine the absolute number of Ly6C+ monocytes (the predominant infiltrate in this model) from individual eyes. A significant reduction in monocyte numbers was observed in eyes that received the therapeutic vector but not the control ( Figure 6 B), this effect was further highlighted by paired (contralateral eye) analysis ( Figure 6 C). **Wilcoxon signed-rank test; *P<0.05Wilcoxon matched pairs analysis.

[0667] These data demonstrate the efficacy of a constitutive promoter design encoding a therapeutic (infliximab Fab) transgene to inhibit human TNF-mediated inflammation in vivo.

[0668] Example 6 - In vitro and in vivo constitutive expression of other anti-TNF biologics

[0669] To evaluate constitutive expression of additional anti-TNF biologics, HEK-293T cells were transfected with huTNFRI-huIgG plasmid and culture supernatants were assayed using an anti-human TNF antibody ELISA kit ( Figure 7 A). Detectable expression of huTNFRI-huIgG was evident by 48 hr. To evaluate AAV-mediated transgene expression, HEK-293T cells were transduced with AAV7m8.CMV.huTNFRI-huIgG or AAV7m8.CMV.NULL vectors [MOI 1E5 vg / cell] and culture supernatants were assayed using an anti-human TNF antibody ELISA kit ( Figure 7 B) Detectable expression of huTNFRI-IgG was evident by 72 hr (approximately 30 ng / ml).

[0670] To evaluate constitutive transgenic expression of huTNFRI-huIgG in vivo, AAV7m8.CMV.huTNFRI-huIgG or AAV7m8.CMV.NULL was administered into the eyes of healthy C57BL / 6J mice at a range of doses [2E8 or 2E9 vg / eye] by intravitreal (IVT) injection. At 4 weeks post-AAV, mice were sacrificed, eyes dissected, and supernatants (retina and vitreous) were assayed using an anti-human TNF antibody ELISA kit ( Figure 7 C). In vivo detectable expression of approximately 4 ng / ml and 9 ng / ml of huTNFRI-huIgG was observed in eyes receiving doses of 2E8 vg / eye and 2E9 vg / eye, respectively.

[0671] These data provide additional evidence that gene therapy vectors can mediate constitutive expression of multiple clinically relevant anti-TNF biologics.

[0672] Example 7 - In vitro biological activity of a constitutively expressed anti-TNF transgene

[0673] To determine whether the transgenic product is bioactive and functional, we evaluated AAV plasmids encoding adalimumab Fab, huTNFRI-huIgG, and two modified constructs engineered to express the huTNFRI extracellular domain fused to mouse Fc (huTNFRI-msIgG) and the murine version (msTNFRI-msIgG). We also evaluated the bioactivity of infliximab Fab using AAV2.CMV.Infliximab viral vectors.

[0674] HEK-BLUE TNF reporter cells were first transfected with AAV.CMV.huTNFRI-huIgG, AAV.CMV.huTNFRI-msIgG, AAV.CMV.msTNFRI-msIgG, AAV.CMV.ADALIMUMAB plasmids or AAV2.CMV.Infliximab for 48 hr. Cells were stimulated with recombinant human TNF or mouse TNF (0.5 ng / ml) for another 24 hr and NFkB activation was assessed. All TNFRI antibody-like plasmid constructs were biologically active, inhibiting huTNF- and msTNF-mediated activation in the reporter cell lines compared to empty or recTNF alone ( Figure 8 A). For monoclonal Fab-based anti-TNF biologics (all human-specific), plasmid expression of adalimumab Fab ( Figure 8 B) or AAV-mediated infliximab Fab expression ( Figure 8 C) Both inhibited the activation of huTNF.

[0675] These data demonstrate that vectored anti-TNF biologics, including huTNFRI-huIgG and adalimumab, are biologically active and neutralize TNF-mediated signaling and NFkB activation in vitro.

[0676] Example 8 - Expression and evaluation of biological activity of inducible anti-TNF transgenes in vitro and in vivo clinical disease models

[0677] To further demonstrate the inducibility of anti-TNF biologics via inflammatory responsive promoters, HEK-293T cells were transduced with AAV7m8.AP1-NFkB.huTNFRI-huIgG or AAV7m8.AP1-NFkB.NULL vectors [MOI 1E5 vg / cell] and stimulated with IL-1b (2 ng / ml). We selected this construct for further characterization because huTNFRI-huIgG can effectively neutralize both human and mouse TNF, which facilitates in vivo efficacy testing in mice. Stimulation resulted in robust induction of huTNFRI-huIgG expression by 24 hr, with accumulation reaching approximately 25 ng / ml (48 hr) at 48 hr. Fig. 9 A).

[0678] To determine the biological activity of the induced huTNFRI-huIgG protein, conditioned media (cell supernatants collected at 48 hr from expression assays) were "spike" with recombinant human or mouse TNF (final concentration 10 ng / ml) and incubated with HEK-BLUE reporter cells for 24 hr. NFkB activation was robustly induced in reporter cells in response to both huTNF stimulation or mTNF stimulation, or using conditioned media from AAV7m8.AP1-NFkB.NULL. NFkB activation in reporter cells was completely inhibited when supplemented with conditioned media from cells transduced with AAV7m8.AP1-NFkB.huTNFRI-huIgG and stimulated with IL-1b, indicating strong biological activity of the induced transgene ( Fig. 9 B).

[0679] To demonstrate in vivo inducibility of the therapeutic transgene, mice were injected with AAV7m8.AP1-NFkB.huTNFRI-huIgG and EAU was induced 4 weeks after AAV. At day 19 after EAU, when mild to moderate clinical signs of inflammation were observed (disease peak had not yet been reached), mice were sacrificed, eyes were dissected and supernatants (retina and vitreous) were assayed using anti-human TNF antibody ELISA. We found that clinical disease drove detectable expression of huTNFRI-huIgG in EAU eyes that received the therapeutic vector but not the control (AAV only) ( Fig.10 A).

[0680] Using the recombinant human TNF model, we also evaluated the inducibility of the huTNFRI-huIgG transgene in response to acute inflammatory stimuli. Mice were injected with AAV7m8.AP1-NFkB.huTNFRI-huIgG and AAV7m8.AP1-NFkB.NULL (contralateral eye control) at 2E9vg / eye. Four weeks after AAV, mice received bilateral administration of recombinant human TNF (rec_hTNF), were sacrificed 18hr later, eyes were dissected and huTNFRI-huIgG expression in ocular supernatants (retina and vitreous) was determined. Acute activation triggered a significant increase in huTNFRI-huIgG expression compared to controls ( Fig.10 B).

[0681] Using two different disease models (one with chronic low-grade inflammation and the other with acute ocular inflammation), these data demonstrate the in vitro and in vivo inducibility and sensitivity of an inflammation-responsive promoter designed to express a bioactive therapeutic (anti-TNF biologic) transgene.

[0682] Example 9 - In vivo evaluation of the efficacy of inducible therapeutic transgenes

[0683] To demonstrate the efficacy of the inducible therapeutic transgene, B10.RIII mice were injected with AAV7m8.AP1-NFkB.huTNFRI-huIgG or AAV7m8.CMV.NULL [2E9 vg / eye] in the contralateral eye and then immunized 4 weeks after AAV for EAU. Representative fundus and OCT images at day 11 show increased inflammation (perivascular sheathing and vitreous infiltration) in eyes receiving the control empty vector ( Fig.11 A). The contralateral eyes of the same four animals that received the inducible therapeutic vector showed significantly reduced clinical inflammation (both perivascular sheathing and vitreous infiltration). Mice were sacrificed at this time point and dissected eyes were prepared for flow cytometric analysis to determine the absolute numbers of CD45+ (all leukocytes), CD3+ (lymphocytes), CD4+ (Th T cells), and CD11b+ (macrophages and monocytes) populations from individual eyes ( Fig.11 B). We observed a trend towards reduced immune cell infiltrates with AAV7m8.AP1-NFkB.huTNFRI-huIgG compared to AAV7m8.CMV.NULL.

[0684] These data demonstrate the in vivo efficacy of inducible anti-TNF in topically suppressing ocular inflammation in a mouse model of human uveitis.

[0685] Example 10 - Method

[0686] Viral vectors: Plasmid design, cloning, and sequencing performed in-house. Ultrapure AAV2 preparations generated, QC, and quantified by Vector Builder. Ultrapure AAV7m8 preparations generated, QC, and quantified by Vector BioLabs.

[0687]

[0688]

[0689] Cell lines: HEK 293T or ARPE-19 cells cultured using standard complete medium. Plasmid transduction of cells was performed using liposomes at a concentration of 0.25 μg / ml. AAV transduction of cells was performed at a range of multiplicity of infection (MOI; 1E4-1E5 vg / cell).

[0690] Anti-TNF constructs: Infliximab Fab is expressed via the heavy and light chains encoding infliximab Fab, separated by a self-cleaving 2A peptide to generate two separate peptides, which then form Fab in situ. Adalimumab Fab is similarly expressed, but encodes the heavy and light chains of adalimumab Fab. For both Fabs, a human growth hormone (HGH) signal peptide is added to the N-terminus of both the heavy and light chains to allow secretion.

[0691] For antibody-like molecules, huTNFRI-huIgG is expressed by encoding the extracellular domain of human tumor necrosis factor receptor type 1 (TNFRI, also known as p55 receptor) (N-terminal 211 amino acid residues, which include the natural signal peptide), followed by the Fc region of human IgG1. Similarly, huTNFRI-msIgG is expressed, but the Fc region of human IgG1 is switched to the Fc region of mouse IgG1. Similarly, msTNFRI-msIgG is expressed, but the extracellular domain of huTNFRI (N-terminal 211 amino acid residues, which include the natural signal peptide) is additionally switched to the extracellular domain of msTNFRI (N-terminal 212 amino acid residues, which include the natural signal peptide). These soluble fusion proteins bind to and neutralize TNF.

[0692] Infliximab Fab and huTNFRI-huIgG characterization: The levels of secreted Fab or IgG (cell culture or ex vivo retinal supernatant) were determined using the clinical IFX ELISA kit (R-Biopharm) or the anti-human TNF antibody ELISA kit (LS Bio; LS-F55832). The biological activity of infliximab Fab or huTNFRI-huIgG (i.e., the ability to neutralize human / mouse TNF) was evaluated using TNF-αReporter HEK 293 cells (Invivogen), which allows monitoring of NF-kB pathway activation.

[0693] Therapeutic interventions (AAV or biologics): Intravitreal injections were performed under ketamine-based recovery anesthesia using a 33G Hamilton syringe under an operating microscope to inject volumes up to 2 μl.

[0694] Uveitis model: Experimental autoimmune uveoretinitis (EAU) is an established preclinical model of human non-infectious uveitis (Khalili, H., et al., Sci Rep, 2016.6: p.36905). Using a standard immunization protocol, retinal peptides (RBP-3) and additional adjuvants (CFA and pertussis toxin) are induced by appropriate systemic routes (e.g., subcutaneous or intraperitoneal injections). EAU susceptibility is mouse strain dependent, with different RBP-3 epitopes triggering acute, severe inflammation (B10.RIII) or persistent disease with reduced disease severity (C57BL / 6J). Initial therapeutic AAV2.Infliximab vector testing used the B10.RIII strain, and subsequent therapeutic AAV7m8.huTNFRI-huIgG vectors used two strains.

[0695] Recombinant human TNF model: To evaluate the efficacy of therapeutic anti-TNF vectors against human proteins, acute inflammation can be induced by intravitreal injection of recombinant human TNF (20 ng / eye). Susceptibility is not mouse strain dependent, with similar inflammatory kinetics in B10.RIII or C57BL / 6J.

[0696] Clinical Assessments: The Micron IV system allows for repeated in vivo ocular assessments of disease severity (fundus, fluorescence, OCT and ERG). Single retinas are processed ex vivo for routine 15-color flow cytometry (FACS) to immuno-phenotype and quantify CD45+ infiltrating cell populations.

[0697] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of chemistry, biochemistry, molecular biology, microbiology and immunology, which are within the capabilities of one of ordinary skill in the art. Such techniques are explained in the literature. See, for example: Skoog, DA, et al. (2013) Fundamentals of Analytical Chemistry, 9th edition, Cengage learning; Walker JM (2009) The Protein Protocols Handbook, 3rd edition, Springer Nature; Green, MR and Sambrook, J. (2012) Molecular Cloning: A Laboratory Manual, 4th edition, Cold Spring Harbor Laboratory Press; Ausubel, FM, et al. (2003) Current Protocols in Molecular Biology, John Wiley & Sons; Hill, AJ (2013) DNA Sequencing Protocols, Humana Press; Nielsen, BS and Jones, J. (2021) In Situ Hybridization Protocols, Springer US; Herdewijn, P. (2010) Oligonucleotide Synthesis: Methods and Applications, Humana Press; and Luo, Y. (2019) CRISPR Gene Editing: Methods and Protocols, Springer New York. Each of these general texts is incorporated herein by reference.

Claims

1. A vector comprising a nucleotide sequence encoding an anti-TNF antibody or a fragment thereof, wherein the nucleotide sequence encoding the anti-TNF antibody or a fragment thereof is operably linked to an inflammation-inducible promoter.

2. The vector according to claim 1, wherein the anti-TNF antibody or fragment thereof is an antibody fragment, preferably wherein the antibody fragment is an antigen-binding fragment (Fab), a fragment antibody (F(ab')2), a single-chain antibody (scFv) or a single-domain antibody (sdAb).

3. A vector comprising a nucleotide sequence encoding an anti-TNF antibody fragment selected from the group consisting of an antigen-binding fragment (Fab), a fragment antibody (F(ab')2), a single-chain antibody (scFv) and a single-domain antibody (sdAb).

4. The vector according to claim 3, wherein the nucleotide sequence encoding the anti-TNF antibody fragment is operably linked to an inflammation-inducible promoter.

5. The vector according to any one of the preceding claims, wherein the anti-TNF antibody or fragment thereof is any one of the following: adalimumab or a fragment thereof, infliximab or a fragment thereof, golimumab or a fragment thereof, or certolizumab pegol or a fragment thereof.

6. The vector according to any one of the preceding claims, wherein the anti-TNF antibody or fragment thereof is adalimumab or a fragment thereof or infliximab or a fragment thereof.

7. The vector according to any one of the preceding claims, wherein the anti-TNF antibody or fragment thereof is adalimumab or a fragment thereof.

8. The vector according to any one of the preceding claims, wherein the anti-TNF antibody or fragment thereof is an antigen binding fragment (Fab).

9. The vector according to any one of the preceding claims, wherein the anti-TNF antibody or fragment thereof is an antigen-binding fragment (Fab) of adalimumab.

10. The vector according to any one of the preceding claims, wherein the anti-TNF antibody or fragment thereof comprises one or more CDR regions selected from SEQ ID NO: 1 to 6 or a derivative thereof comprising one amino acid substitution.

11. The vector according to any one of the preceding claims, wherein the anti-TNF antibody or fragment thereof comprises CDR regions HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3, and the CDR regions HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3 respectively comprise or consist of: SEQ ID NO: 1, 2, 3, 4, 5 and 6, or a derivative thereof comprising one amino acid substitution.

12. The vector according to any of the preceding claims, wherein the anti-TNF antibody or fragment thereof comprises a heavy chain comprising or consisting of a sequence having at least 70% identity to SEQ ID NO:7 and / or a light chain comprising or consisting of a sequence having at least 70% identity to SEQ ID NO:

8.

13. The vector according to claim 12, wherein the heavy chain is encoded by a nucleotide sequence having at least 70% identity to SEQ ID NO:47, and / or the light chain is encoded by a nucleotide sequence having at least 70% identity to SEQ ID NO:

48. The vector according to claim 13 , wherein the nucleotide sequence encoding the heavy chain and the nucleotide sequence encoding the light chain are connected via a linker sequence.

15. The vector according to claim 14, wherein the linker sequence encodes a 2A self-cleaving peptide and / or an enzymatically cleavable peptide motif, preferably wherein the linker sequence encodes a 2A self-cleaving peptide having at least 70% sequence identity to any one of SEQ ID NOs: 55-58.

16. The vector according to any one of claims 13 to 15, wherein the nucleotide sequence encoding the heavy chain and / or the nucleotide sequence encoding the light chain is operably linked to a signal sequence, optionally wherein the signal sequence encodes a signal peptide selected from any one of the following: human growth hormone (HGH) signal peptide, interleukin-2 (IL-2) signal peptide, CD5 signal peptide, immunoglobulin kappa light chain signal peptide, trypsinogen signal peptide, serum albumin signal peptide and prolactin signal peptide.

17. The vector according to any of the preceding claims, wherein the nucleotide sequence encoding the anti-TNF antibody or fragment encodes a heavy chain comprising or consisting of a sequence having at least 70% identity to SEQ ID NO:7, a 2A self-cleaving peptide having at least 70% sequence identity to any one of SEQ ID NOs:55-58, and a light chain comprising or consisting of a sequence having at least 70% identity to SEQ ID NO:

8.

18. The vector according to any one of the preceding claims, wherein the nucleotide sequence encoding the anti-TNF antibody or fragment thereof comprises or consists of: A nucleotide sequence having at least 70% identity to SEQ ID NO:47, a nucleotide sequence having at least 70% sequence identity to SEQ ID NO:59 or 60, and a nucleotide sequence having at least 70% identity to SEQ ID NO:

48.

19. The vector according to any one of the preceding claims, wherein the nucleotide sequence encoding the anti-TNF antibody or fragment thereof comprises or consists of: A nucleotide sequence that is at least 70% identical to SEQ ID NO:

66.

20. The vector according to any one of claims 1, 2 or 4-19, wherein the inflammation-inducible promoter comprises one or more inflammation-inducible transcription factor binding motifs selected from the group consisting of: AP-1 transcription factor binding motif; NF-κB transcription factor binding motif; IRF transcription factor binding motif; STAT transcription factor binding motif; and NFAT transcription factor binding motif or any combination thereof.

21. The vector of any one of claims 1, 2, or 4-20, wherein the inflammation-inducible promoter comprises one or more AP-1 binding motifs and / or one or more NF-κB binding motifs.

22. The vector of any one of claims 1, 2 or 4-21, wherein the inflammation-inducible promoter comprises two or more AP-1 binding motifs and / or two or more NF-κB binding motifs, three or more AP-1 binding motifs and / or three or more NF-κB binding motifs, four or more AP-1 binding motifs and / or four or more NF-κB binding motifs, or five or more AP-1 binding motifs and / or five or more NF-κB binding motifs.

23. The vector of any one of claims 1, 2, or 4-22, wherein the inflammation-inducible promoter comprises at least one AP-1 binding motif coupled to at least one NF-κB binding motif.

24. The vector of any one of claims 1, 2, or 4-23, wherein the inflammation-inducible promoter comprises five AP-1 binding motifs coupled to five NF-κB binding motifs.

25. The vector of any one of claims 20-24, wherein the AP-1 binding motif comprises or consists of SEQ ID NO: 70, or wherein the AP-1 binding motif comprises or consists of: Any one of SEQ ID NOs: 71-73 or a derivative thereof comprising a nucleotide substitution.

26. The vector according to any one of claims 20-25, wherein the NF-κB binding motif comprises or consists of SEQ ID NO: 74, or wherein the NF-κB binding motif comprises or consists of: SEQ ID NO: 75 or a derivative thereof comprising two or fewer nucleotide substitutions.

27. The vector of any one of claims 1, 2, or 4-26, wherein the inflammation-inducible promoter comprises or consists of a nucleotide sequence that is at least 70% identical to SEQ ID NO:

76.

28. The vector of any one of the preceding claims, wherein the vector comprises a nucleotide sequence that is at least 70% identical to SEQ ID NO:

77.

29. The vector according to any one of the preceding claims, wherein the nucleotide sequence encoding the anti-TNF antibody or fragment thereof is operably linked to a polyadenylation sequence, preferably wherein the polyadenylation sequence is selected from any one of the following: bovine growth hormone (bGH) polyadenylation sequence, SV40 polyadenylation sequence and rabbit β-globin polyadenylation sequence.

30. The vector of claim 29, wherein the polyadenylation sequence comprises or consists of a nucleotide sequence that is at least 70% identical to SEQ ID NO:

78.

31. The vector of any one of the preceding claims, wherein the nucleotide sequence encoding the anti-TNF antibody or fragment thereof is operably linked to a Woodchuck Hepatitis Posttranscriptional Regulatory Element (WPRE).

32. The vector of claim 31, wherein the WPRE comprises or consists of a nucleotide sequence having at least 70% identity to SEQ ID NO:

79.

33. The vector according to any one of the preceding claims, wherein the nucleotide sequence encoding the anti-TNF antibody or fragment thereof is operably linked to an intron, optionally wherein the intron is selected from a β-globin intron or an SV40 intron.

34. The vector of claim 33, wherein the intron comprises or consists of a nucleotide sequence having at least 70% identity to SEQ ID NO:

80.

35. The vector of any one of the preceding claims, wherein the vector is a viral vector.

36. The vector according to any of the preceding claims, wherein the vector is any one of a parvoviral vector, an adenoviral vector, a herpes simplex virus vector, an anelloviral vector, a retroviral vector or a lentiviral vector, preferably wherein the vector is an adeno-associated virus (AAV) vector.

37. The vector of any of the preceding claims, wherein the vector is an AAV vector particle.

38. The vector of claim 37, wherein the AAV vector particles are pseudotyped to confer ocular tissue tropism.

39. The vector of claim 37 or 38, wherein the AAV vector particle comprises an AAV2 capsid protein or an AAV2 capsid variant protein, optionally wherein the AAV2 capsid variant is selected from any one of the following: AAV2.tYF, AAV2.7m8, R100, AAV2.GL and AAV2.NN.

40. The vector of any one of the preceding claims, wherein the vector comprises one or more inverted terminal repeats (ITRs).

41. The vector of any one of the preceding claims, wherein the vector comprises a nucleotide sequence that is at least 70% identical to SEQ ID NO:

91.

42. A vector comprising a nucleotide sequence having at least 70% identity to SEQ ID NO:

91.

43. An isolated cell comprising the vector of any one of claims 1-42.

44. A kit for producing the vector of any one of claims 1-42.

45. A pharmaceutical composition comprising the vector according to any one of claims 1 to 42 or the isolated cell according to claim 43, in combination with a pharmaceutically acceptable carrier, diluent or excipient.

46. ​​The vector according to any one of claims 1 to 42, the isolated cell according to claim 43 and / or the pharmaceutical composition according to claim 45 for use as a medicament.

47. Use of a vector according to any one of claims 1 to 42, an isolated cell according to claim 43 or a pharmaceutical composition according to claim 45 in the preparation of a medicament.

48. A method comprising administering to a subject in need thereof a vector according to any one of claims 1 to 42, an isolated cell according to claim 43, or a pharmaceutical composition according to claim 45.

49. A vector for preventing or treating an inflammatory eye disease, wherein the vector comprises a nucleotide sequence encoding a TNF inhibitor, and wherein the nucleotide sequence encoding the TNF inhibitor is operably linked to an inflammation-inducible promoter.

50. Use of a vector in the preparation of a medicament for preventing or treating an inflammatory eye disease, wherein the vector comprises a nucleotide sequence encoding a TNF inhibitor, and wherein the nucleotide sequence encoding the TNF inhibitor is operably linked to an inflammation-inducible promoter.

51. A method for preventing or treating an inflammatory eye disease, wherein the method comprises administering a vector to a subject in need thereof, wherein the vector comprises a nucleotide sequence encoding a TNF inhibitor, and wherein the nucleotide sequence encoding the TNF inhibitor is operably linked to an inflammation-inducible promoter.

52. The vector according to any one of claims 1 to 42 or the pharmaceutical composition according to claim 45 for use in preventing or treating inflammatory eye diseases.

53. Use of a carrier according to any one of claims 1 to 42 or a pharmaceutical composition according to claim 45 in the preparation of a medicament for preventing or treating inflammatory eye diseases.

54. A method for preventing or treating an inflammatory eye disease, the method comprising administering the vector according to any one of claims 1 to 42 or the pharmaceutical composition according to claim 45 to a subject in need thereof.

55. The vector or pharmaceutical composition of claim 49 or 52, the use of claim 50 or 53, or the method of claim 51 or 54, wherein the inflammatory eye disease is uveitis.

56. The vector or pharmaceutical composition according to any one of claims 49, 52 or 55, the use according to any one of claims 50, 53 or 55, or the method according to any one of claims 51 or 54-55, wherein the vector or pharmaceutical composition is administered intraocularly.

57. The vector or pharmaceutical composition of any one of claims 49, 52 or 55-56, the use of any one of claims 50, 53 or 55-56, or the method of any one of claims 51 or 54-56, wherein the vector or pharmaceutical composition is administered via intravitreal injection, subretinal injection, direct retinal injection, subconjunctival injection, sub-Tenon injection or suprachoroidal injection.

58. The vector or pharmaceutical composition according to any one of claims 49, 52 or 55-57, the use according to any one of claims 50, 53 or 55-57, or the method according to any one of claims 51 or 54-57, wherein the vector or pharmaceutical composition is administered via intravitreal injection.

59. The vector or pharmaceutical composition according to any one of claims 49, 52 or 55-58, the use according to any one of claims 50, 53 or 55-58, or the method according to any one of claims 51 or 54-58, wherein the vector or pharmaceutical composition is administered as a single dose.

60. The vector or pharmaceutical composition of any one of claims 49, 52 or 55-59, the use of any one of claims 50, 53 or 55-59, or the method of any one of claims 51 or 54-59, wherein the vector is administered at a dose of at least about 1E10 vg / mL, at least about 1E11 vg / mL, at least about 1E12 vg / mL or at least about 5E12 vg / mL.

61. The vector or pharmaceutical composition of any one of claims 49, 52 or 55-60, the use of any one of claims 50, 53 or 55-60, or the method of any one of claims 51 or 54-60, wherein the vector is administered at a dose of at least about 1E9 vg / eye, at least about 1E10 vg / eye or at least about 1E11 vg / eye, preferably wherein the vector is administered at a dose of about 1E9 vg / eye to about 5E12 vg / eye.

62. The vector or pharmaceutical composition of any one of claims 49, 52 or 55-61, the use of any one of claims 50, 53 or 55-61, or the method of any one of claims 51 or 54-61, wherein the vector or pharmaceutical composition is administered in response to a recurrence of an inflammatory eye disease, preferably wherein the inflammatory eye disease is uveitis.

Images