Pharmaceutical composition and application thereof

Abstract

Relates to pharmaceutical compositions and uses thereof. In particular to a pharmaceutical composition containing rAAV particles and application thereof.

Description

Pharmaceutical composition and use thereof

[0001] This disclosure claims priority to the following patent applications: Chinese patent application filed on August 23, 2023, with application number CN202311073344.0; Chinese patent application filed on August 24, 2023, with application number CN202311077883.1; the entire contents of the foregoing patent applications are incorporated by reference into this disclosure. Technical Field

[0002] The present disclosure belongs to the field of biomedicine, and particularly relates to a pharmaceutical composition comprising recombinant adeno-associated virus (rAAV) particles, and its use as a medicine. Background Art

[0003] Adeno-associated virus (AAV) belongs to the genus Dependovirus of the family Parvoviridae. The AAV genome consists of a single-stranded DNA molecule containing approximately 4.7 kilobases (kb) and composed of two major open reading frames encoding the nonstructural Rep (replication) and structural Cap (capsid) proteins. The AAV coding region is flanked by two cis-acting inverted terminal repeats (ITRs) of approximately 145 nucleotides in length, with interrupted palindromes that fold into a hairpin structure that acts as a primer in the initiation of DNA replication.

[0004] As a vector, AAV shows great potential for the treatment of genetic diseases and gene diseases and can be used for gene supplementation therapy (also known as gene enhancement therapy). It restores the loss or disorder of gene function caused by mutation by complementing the missing gene function, thereby restoring the biological function of the target cell to a normal physiological state. Studies have shown that the performance of AAV-based vectors in preclinical disease models and human clinical trials shows the prospect of application in the treatment of several diseases. For example, it can achieve efficient gene transfer into retinal cells and long-term continuous expression (Boye et al. Mol Ther. 2013 Mar; 21(3): 509-19. Trapani et al. Prog Retin Eye Res. 2014 Nov; 43: 108-28.). The AAV vectors currently used in clinical practice have been genetically engineered to be latent in the absence of helper virus. Their safety and long-term transgene expression have been extensively tested in rodent models, non-human primates, and multiple human trials (MacLaren et al. Lancet. 2014 Mar 29; 383(9923): 1129-37.; Maguire et al. N Engl J Med. 2008 May 22; 358(21): 2240-8; Simonelli et al. Mol Ther. 2010 Mar; 18(3): 643-50; Nathwani et al. N Engl J Med. 2014 Nov 20; 371(21): 1994-2004).

[0005] Recombinant adeno-associated virus (rAAV) is derived from a non-pathogenic wild-type adeno-associated virus and has the advantages of good safety, a wide range of host cells (dividing and non-dividing cells), low immunogenicity, and long-term expression of foreign genes in vivo. It is regarded as one of the most promising gene delivery vectors and has been widely used in gene therapy and vaccine research worldwide (Snyder 1999; Xiao, Lentz, and Samulski 2012).

[0006] In order to make AAV safe for clinical use, AAV has been genetically modified at several locations within its genome. For example, the Rep gene required for viral replication and the elements required for site-specific integration have been eliminated from the AAV genome in many viral vectors. This recombinant AAV (rAAV) exists in an extrachromosomal state and has a very low integration efficiency for genomic DNA. If not completely eliminated, the possibility of rAAV inducing random mutagenesis in host cells is therefore reduced. Due to these characteristics and the lack of pathogenicity, rAAV has shown great prospects as a gene therapy vector in many aspects of preclinical and clinical applications. New serotype vectors are also the focus of current clinical development.

[0007] AAV research also focuses on the development of AAV preparations for human administration. How to develop AAV preparations that maintain long-term stability and strong titer of AAV and maintain infection efficiency and biological activity after being subjected to various conditions and pressures is an important issue that needs to be addressed.

[0008] Summary of the Invention

[0009] The present disclosure provides a pharmaceutical composition comprising adeno-associated virus (AAV) particles, a method for preparing the pharmaceutical composition, and a method for treating or preventing a disease or related pharmaceutical uses thereof.

[0010] The present disclosure provides a pharmaceutical composition comprising recombinant adeno-associated virus (rAAV) particles and an adjuvant. In some embodiments, the adjuvant comprises Hepes and / or BTP. Exemplarily, the adjuvant comprises Hepes, BTP, or a combination of Hepes and BTP.

[0011] In some embodiments, the pharmaceutical composition comprises Hepes. Hepes is also known as N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid. Hepes acts as a buffer and stabilizer in the pharmaceutical composition, allowing the pharmaceutical composition to maintain stability and high biological potency over a longer period of time (e.g., 1 week, 2 weeks, 3 weeks, 1 microliter, 2 microliter, 3 microliter, etc.).

[0012] In some embodiments, the pharmaceutical composition comprises BTP. BTP, also known as Bis-tris propane, can act as a buffer, stabilizer, etc. In some embodiments, the pharmaceutical composition comprises Hepes and BTP.

[0013] In some embodiments, the concentration of Hepes or BTP is from about 1 mM to about 100 mM. For example, from about 1 mM to about 50 mM, from about 1 mM to about 55 mM, from about 1 mM to about 60 mM, from about 1 mM to about 70 mM, from about 1 mM to about 80 mM, from about 5 mM to about 100 mM, from about 1 mM to about 80 mM, from about 5 mM to about 70 mM, from about 5 mM to about 50 mM, from about 5 mM to about 45 mM, from about 5 mM to about 40 mM, from about 5 mM to about 35 mM, from about 5 mM to about 30 mM, from about 5 mM to about 25 mM, from about 5 mM to about 20 mM In some embodiments, the concentration of Hepes or BTP is about 5mM to about 50mM. In some embodiments, the concentration of Hepes or BTP is about 5mM to about 25mM. In some embodiments, the concentration of Hepes or BTP is about 10mM to about 50mM. In some embodiments, the concentration of Hepes or BTP is about 5mM to about 25mM. In some embodiments, the concentration of Hepes or BTP is about 10mM.

[0014] In some embodiments, the concentration of Hepes or BTP is about 1 mM, about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM, about 11 mM, about 12 mM, about 13 mM, about 14 mM, about 15 mM, about 16 mM, about 17 mM, about 18 mM, about 19 mM, about 20 mM, about 22 mM, about 25 mM, about 26 mM, about 28 mM, about 30 mM, about 35mM, about 40mM, about 43mM, about 45mM, about 48mM, about 50mM, about 55mM, about 57mM, about 58mM, about 60mM, about 62mM, about 65mM, about 68mM, about 70mM, about 72mM, about 75mM, about 78mM, about 80mM, about 82mM, about 85mM, about 88mM, about 90mM, about 92mM, about 95mM, about 100mM.

[0015] In some embodiments, the pH of the pharmaceutical composition is from about 4.0 to about 10.0, for example, from about 5.0 to about 10.0, about 6.0 to about 10.0, about 5.0 to about 9.0, about 5.5 to about 9.0, about 5.5 to about 8.5, about 6.5 to about 8.5, about 5.5 to about 8.0, about 5.5 to about 7.5, about 5.5 to about 7.0, about 6.0 to about 7.0, about 6.0 to about 7.5, about 6.0 to about 8.0, about 6.0 to about 8.5, about 6.0 to about 9.0, about 4.0 to about 6.0, about 4.0 to about 5.5, about 4.0 to about 5.0, about 4.0 to about 4.8, 4.0 to about 4.5, about 4.8 to about 5.0, about 5.0 to about 5.5, about 4.5 to about 4.8, or any range therebetween. In some embodiments, the pH of the pharmaceutical composition is about 6.0 to about 8.0. In some embodiments, the pH of the buffer or the pharmaceutical composition is about 6.0 to about 7.5.

[0016] In some embodiments, the pH of the buffer or the pharmaceutical composition is about 4.0, about 4.1, about 4.2, about 4.3, about 4.4, about 4.5, about 4.6, about 4.7, about 4.8, about 4.9, about 5.0, about 5.1, about 5.2, about 5.3, about 5.4, about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5, about 6.6, about 6.7 , about 6.8, about 6.9, about 7.0, about 7.1, about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7, about 7.8, about 7.9, about 8.0, about 8.1, about 8.2, about 8.3, about 8.4, about 8.5, about 8.6, about 8.7, about 8.8, about 8.9, about 9.0, about 9.1, about 9.2, about 9.3, about 9.4, about 9.5, about 9.6, about 9.7, about 9.8, about 9.9, about 10.0.

[0017] Typically, the pH of the pharmaceutical composition obtained by replacing the buffer is substantially consistent with the pH of the buffer. It is also well known to those skilled in the art that pH drift may sometimes occur during the pharmaceutical preparation process, but the pH drift of the pharmaceutical preparation is generally small (e.g., within a range of ±0.8). In some embodiments, the pH drift of the pharmaceutical preparation is within a range of ±0.5.

[0018] In some embodiments, the pharmaceutical composition as described in any one of the above, it comprises a surfactant. In some embodiments, the surfactant is a nonionic surfactant. In some embodiments, the surfactant is selected from poloxamer (e.g., poloxamer 188), polysorbate (e.g., polysorbate 20 (i.e., PS20), polysorbate 80 (i.e., PS80)), polyhydroxyalkyl, Triton, sodium dodecyl sulfate, sodium lauryl sulfate, sodium octyl glucoside, lauryl-sulfobetaine, myristyl-sulfobetaine, linoleyl-sulfobetaine, stearyl-sulfobetaine, lauryl-sarcosine, myristyl-sarcosine, linoleyl-sarcosine, stearyl-sarcosine, linoleyl-sweet In some embodiments, the surfactant is a copolymer of sodium lauryl sulfate (SDS) or polysorbate. In some embodiments, the surfactant is poloxamer 188 and / or polysorbate 80. In some embodiments, the surfactant is poloxamer 188. In some embodiments, the surfactant is a polysorbate 80. In some embodiments, the surfactant is a polysorbate 80.

[0019] In some embodiments, the concentration of the surfactant is from about 0.001% (w / v) to about 1.0% (w / v), for example, from about 0.001% (w / v) to about 0.8% (w / v), from about 0.001% (w / v) to about 0.5% (w / v), from about 0.001% (w / v) to about 0.2% (w / v), from about 0.001% (w / v) to about 0.1% (w / v), from about 0.001% (w / v) to about 0.08% (w / v), from about 0.001% (w / v) to about 0.05% (w / v), from about 0.001% (w / v) to about 0.03% (w / v), from about 0.001% (w / v) to about 0.02% (w / v), from about 0.0 01% (w / v) to about 0.01% (w / v), about 0.005% (w / v) to about 0.08% (w / v), about 0.005% (w / v) to about 0.05% (w / v), about 0.005% (w / v) to about 0.03% (w / v), about 0.005% (w / v) to about 0.02% (w / v), about 0.005% (w / v) to about 0.01% (w / v), about 0.005% (w / v) to about 0.1% (w / v), about 0.005% (w / v) to about 0.15% (w / v), about 0.005% (w / v) to about 0.18% (w / v), about 0.008% (w / v) to about 0.18% (w / v), about 0. 008% (w / v) to about 0.15% (w / v), about 0.005% (w / v) to about 0.2% (w / v), about 0.005% (w / v) to about 0.3% (w / v), about 0.01% (w / v) to about 1.0% (w / v), about 0.01% (w / v) to about 0.8% (w / v), about 0.01% (w / v) to about 0.5% (w / v), about 0.01% (w / v) to about 0.2% (w / v), about 0.01% (w / v) to about 0.1% (w / v), about 0.01% (w / v) to about 0.18% (w / v), about 0.01% (w / v) to about 0.18% (w / v), about 0.01% (w / v) to about 0. 15% (w / v), about 0.01% (w / v) to about 0.1% (w / v), about 0.01% (w / v) to about 0.08% (w / v), about 0.01% (w / v) to about 0.06% (w / v), about 0.01% (w / v) to about 0.04% (w / v), about 0.02% (w / v) to about 0.2% (w / v), about 0.02% (w / v) to about 0.15% (w / v), about 0.02% (w / v) to about 0.1% (w / v), about 0.02% (w / v) to about 0.08% (w / v), about 0.02% (w / v) to about 0.04% (w / v), about 0.04% (w / v) to about 0.2% (w / v), about 0.In some embodiments, the concentration of the surfactant is from about 0.04% (w / v) to about 0.1% (w / v), from about 0.04% (w / v) to about 0.08% (w / v), and any range therebetween. In some embodiments, the concentration of the surfactant is from about 0.001% (w / v) to about 0.05% (w / v). In some embodiments, the concentration of the surfactant is from about 0.01% (w / v) to about 0.02% (w / v).

[0020] In some embodiments, the concentration of the surfactant is about 0.001% (w / v), 0.002% (w / v), 0.003% (w / v), 0.004% (w / v), 0.005% (w / v), 0.006% (w / v), 0.007% (w / v), 0.008% (w / v), 0.009% (w / v), 0.01% (w / v), 0.011% (w / v), 0.012% (w / v), 0.013% (w / v), 0.015% (w / v), 0.018% (w / v), 0.019% (w / v), 0.02% (w / v), about 0.01% (w / v), about 0.02% (w / v), about 0.03% (w / v), about 0.04% (w / v), about 0.05% (w / v), about 0.06% (w / v), about 0.07% (w / v), about 0.08% (w / v), about 0.09% (w / v), about 0.1% (w / v), about 0.11% (w / v), about 0.12% (w / v), about 0.13% (w / v), About 0.14% (w / v), about 0.15% (w / v), about 0.16% (w / v), about 0.17% (w / v), about 0.18% (w / v), about 0.19% (w / v), about 0.2% (w / v)), about 0.3% (w / v)), about 0.4% (w / v)), about 0.5% (w / v)), about 0.6% (w / v)), about 0.7% (w / v)), about 0.8% (w / v).

[0021] In some embodiments, the pharmaceutical composition as described in any one of the above items comprises one or more of saccharides, alcohols, and sugar alcohols. "One or more" in the present disclosure includes one, two, or more than two.

[0022] Any sugar may be used in the present disclosure, such as a monosaccharide, disaccharide, or polysaccharide, or a water-soluble glucan, including, for example, fructose, glucose, mannose, sorbose, xylose, maltose, lactose, sucrose, dextran, trehalose, pullulan, dextrin, cyclodextrin, soluble starch, hydroxyethyl starch, and carboxymethyl cellulose. In particular embodiments, the sugar may be sucrose, glucose, trehalose, or a combination thereof. In certain embodiments, the trehalose may be trehalose dihydrate. A sugar alcohol is defined as a hydrocarbon having between about 4 and about 8 carbon atoms and a hydroxyl group. Non-limiting examples of alcohols or sugar alcohols in the present disclosure include mannitol, sorbitol, inositol, galactitol, tretol, xylitol, and arabitol. In certain embodiments, mannitol may be used as a sugar alcohol additive. In certain embodiments, the buffer formulation contains both a sugar and a sugar alcohol additive. In certain embodiments, the sugar may be sucrose. In certain embodiments, the sugar is trehalose. In certain embodiments, the alcohol is glycerol. Sugars, alcohols and sugar alcohols can be used alone or in combination. In some embodiments, the pharmaceutical composition comprises sugars and alcohols. In some embodiments, the pharmaceutical composition comprises sucrose and glycerol.

[0023] In some embodiments, the concentration of the carbohydrate is from about 0.1% (w / v) to about 10% (w / v), for example, from about 0.1% (w / v) to about 9% (w / v), from about 0.1% (w / v) to about 8% (w / v), from about 0.1% (w / v) to about 7% (w / v), from about 0.1% (w / v) to about 6% (w / v), from about 0.1% (w / v) to about 5% (w / v), from about 0.1% (w / v) to about 3% (w / v), from about 1% (w / v) to about 9% (w / v), from about 1% (w / v) to about 8% (w / v), from about 1% (w / v) to about / v) to about 7% (w / v), about 1% (w / v) to about 6% (w / v), about 1% (w / v) to about 5% (w / v), about 1% (w / v) to about 3% (w / v), about 1.5% (w / v) to about 9% (w / v), about 1.5% (w / v) to about 8% (w / v), about 1.5% (w / v) to about 7% (w / v), about 1.5% (w / v) to about 6% (w / v), about 1.5% (w / v) to about 5% (w / v), about 1.5% (w / v) to about 3% (w / v), and any range between these points.

[0024] In some embodiments, the concentration of the carbohydrate is about 0.1% (w / v), about 0.2% (w / v), about 0.5% (w / v), about 0.6% (w / v), about 0.7% (w / v), about 0.8% (w / v), about 0.9% (w / v), about 1.0% (w / v), about 1.1% (w / v), about 1.2% (w / v), about 1.3% (w / v), about 1.4% (w / v), about 1.5% (w / v), about 1.6% (w / v), about 1.7% (w / v), about 1.8% (w / v), about 1.9% (w / v), about 2 .1% (w / v), about 2.2% (w / v), about 2.3% (w / v), about 2.4% (w / v), about 2.5% (w / v), about 2.6% (w / v), about 2.7% (w / v), about 2.8% (w / v), about 2.9% (w / v), about 3.0% (w / v), about 3.1% (w / v), about 3.2% (w / v), about 3.3% (w / v), about 3.4% (w / v), about 3.5% (w / v), about 3.6% (w / v), about 3.7% (w / v), about 3.8% (w / v), about 3.9% (w / v) ), about 4.0% (w / v), about 4.1% (w / v), about 4.2% (w / v), about 4.3% (w / v), about 4.4% (w / v), about 4.5% (w / v), about 4.6% (w / v), about 4.7% (w / v), about 4.8% (w / v), about 4.9% (w / v), about 5.0% (w / v), about 5.1% (w / v), about 5.2% (w / v), about 5.3% (w / v), about 5.5% (w / v), about 5.5% (w / v), about 5.6% (w / v), about 5.7% (w / v), about 5.8% (w / v), about 5.9% (w / v), about 6.0% (w / v), about 6.1% (w / v), about 6.3% (w / v), about 6.5% (w / v), about 6.7% (w / v), about 6.8% (w / v), about 7.1% (w / v), about 7.3% (w / v), about 7.5% (w / v), about 7.7% (w / v), about 7.8% (w / v), about 7.9% (w / v), about 8.0% (w / v), about 8.5% (w / v), about 9.0% (w / v), about 9.5% (w / v), about 10.0% (w / v).

[0025] In some embodiments, the concentration of the alcohol is from about 0.01% (w / v) to about 10% (w / v), for example, from about 0.01% (w / v) to about 8% (w / v), from about 0.01% (w / v) to about 9% (w / v), from about 0.01% (w / v) to about 8% (w / v), from about 0.01% (w / v) to about 7% (w / v), from about 0.01% (w / v) to about 5% (w / v), from about 0.01% (w / v) to about 4% (w / v), from about 0.01% (w / v) to about 3% (w / v), from about 0.01% (w / v) to about 1% (w / v), from 0.05% (w / v) to about 1% (w / v), from 0.05% (w / v) to about 1.5% (w / v), or from 0.05% (w / v) to about 1.5% (w / v). to about 2% (w / v), 0.05% (w / v) to about 3.5% (w / v), 0.05% (w / v) to about 5.0% (w / v), 0.05% (w / v) to about 6.0% (w / v), 0.05% (w / v) to about 7.0% (w / v), about 0.1% (w / v) to about 1% (w / v), about 0.1% (w / v) to about 0.8% (w / v), about 0.1% ( w / v) to about 0.7% (w / v), about 0.1% (w / v) to about 0.6% (w / v), about 0.1% (w / v) to about 0.5% (w / v), about 0.1% (w / v) to about 0.4% (w / v), about 0.2% (w / v) to about 0.8% (w / v), about 0.1% (w / v) to about 0.3% (w / v), about 0.2% (w / v) to about 2% (w / v).

[0026] In some embodiments, the concentration of the alcohol is about 0.01% (w / v), about 0.05% (w / v), about 0.015% (w / v), about 0.02% (w / v), about 0.01% (w / v), about 0.08% (w / v), about 0.1% (w / v), about 0.15% (w / v), about 0.18% (w / v), about 0.2% (w / v), about 0.22% (w / v), about 0.25% (w / v), about 0.28% (w / v), about 0.3% (w / v), about 0.35% (w / v), about 0.4% (w / v), about 0.45% (w / v), about 0.5% (w / v), about 0.55% (w / v), about 0.6% (w / v), about 0.65% (w / v), about 0.7% (w / v), about 0.8% (w / v), about 0.9% (w / v), about 1% (w / v), about 2% (w / v), about 3% (w / v), about 4% (w / v), about 5% (w / v), about 6% (w / v), about 7% (w / v), about 8% (w / v), about 9% (w / v), about 10.0% (w / v).

[0027] In some embodiments, the pharmaceutical composition of any of the above items comprises an amino acid. Amino acids include, but are not limited to, arginine, glycine, cysteine, histidine, and the like. In some embodiments, the amino acid is histidine. For example, the amino acid is L-histidine.

[0028] In some embodiments, the concentration of the amino acid is from about 1 mM to about 50 mM. For example, from about 1 mM to about 40 mM, from about 1 mM to about 35 mM, from about 1 mM to about 30 mM, from about 1 mM to about 25 mM, from about 1 mM to about 15 mM, from about 1 mM to about 20 mM, from about 1 mM to about 10 mM, from about 1 mM to about 5 mM, from about 2 mM to about 40 mM, from about 2 mM to about 35 mM, from about 2 mM to about 30 mM, from about 2 mM to about 25 mM, from about 2 mM to about 25 mM, from about 2 mM to about 25 mM. To about 20mM, about 2mM to about 20mM, about 2mM to about 5mM, about 3mM to about 40mM, about 3mM to about 35mM, about 3mM to about 30mM, about 3mM to about 35mM, about 3mM to about 35mM, about 3mM to about 30mM, about 3mM to about 30mM, about 3mM to about 5mM, about 2mM to about 8mM, about 3mM to about 10mM, and any ranges between these points.

[0029] In some embodiments, the concentration of the amino acid is about 1 mM, about 2 mM, about 2.5 mM, about 3 mM, about 3.5 mM, about 4 mM, about 4.5 mM, about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM, about 20 mM, about 30 mM, about 40 mM, about 50 mM.

[0030] In some embodiments, the pharmaceutical composition as described in any one of the above comprises an inorganic salt. Exemplary, the inorganic salt includes but is not limited to sodium salt and / or magnesium salt. In some embodiments, the sodium salt is sodium chloride. In some embodiments, the magnesium salt is magnesium chloride. "Magnesium chloride" can be anhydrous magnesium chloride, or a solvate of magnesium chloride formed with a solvent (e.g., magnesium chloride hexahydrate). In some specific embodiments, "magnesium chloride" described in this disclosure refers to magnesium chloride hexahydrate.

[0031] In some embodiments, the concentration of the sodium salt is from about 10 mM to about 500 mM, for example, from about 20 mM to about 500 mM, from about 30 mM to about 500 mM, from about 50 mM to about 500 mM, from about 60 mM to about 500 mM, from about 70 mM to about 500 mM, from about 80 mM to about 500 mM; from about 20 mM to about 400 mM, from about 30 mM to about 400 mM, from about 50 mM to about 400 mM, from about 60 mM to about 500 mM, from about 70 mM to about 500 mM, from about 80 mM to about 500 mM; 0mM to about 400mM, about 70mM to about 400mM, about 80mM to about 400mM; about 20mM to about 350mM, about 30mM to about 350mM, about 50mM to about 350mM, about 60mM to about 350mM, about 70mM to about 350mM, about 80mM to about 350mM; about 20mM to about 250mM, about 30mM to about 250mM, about 50mM to about 250mM M, about 60mM to about 250mM, about 70mM to about 250mM, about 80mM to about 250mM; about 20mM to about 200mM, about 30mM to about 200mM, about 50mM to about 200mM, about 60mM to about 200mM, about 70mM to about 200mM, about 80mM to about 200mM; about 20mM to about 180mM, about 30mM to about 180mM, about 50mM to about 180mM, about 60mM to about 180mM, about 70mM to about 180mM, about 80mM to about 180mM; about 20mM to about 150mM, about 30mM to about 150mM, about 50mM to about 150mM, about 60mM to about 150mM, about 70mM to about 150mM, about 80mM to about 150mM; about 100mM to about 150mM; or any range therebetween.

[0032] In some embodiments, the concentration of the sodium salt is about 10 mM, about 15 mM, about 20 mM, about 25 mM, about 30 mM, about 35 mM, about 40 mM, about 45 mM, about 50 mM, about 55 mM, about 60 mM, about 65 mM, about 70 mM, about 75 mM, about 80 mM, about 85 mM, about 90 mM, about 95 mM, about 100 mM, about 110 mM, about 120 mM, about 125 mM, about 130 mM, about 135 mM, about 140 mM, about 145 mM. M, about 150mM, about 155mM, about 160mM, about 165mM, about 170mM, about 175mM, about 180mM, about 185mM, about 190mM, about 195mM, about 200mM, about 210mM, about 220mM, about 250mM, about 260mM, about 270mM, about 280mM, about 290mM, about 300mM, about 350mM, about 400mM, about 450mM, about 500mM, or any range therebetween, or any range therebetween.

[0033] In some embodiments, the concentration of the magnesium salt is from about 0.1 mM to about 50 mM, for example, from about 0.5 mM to about 50 mM, from about 0.5 mM to about 45 mM, from about 0.5 mM to about 40 mM, from about 0.5 mM to about 35 mM, from about 0.5 mM to about 30 mM, from about 0.5 mM to about 25 mM, from about 0.5 mM to about 20 mM, from about 0.5 mM to about 15 mM, from about 0.5 mM to about 10 mM, from about 0.1 mM to about 15 mM. M to about 15mM, about 0.1mM to about 10mM, about 0.5mM to about 8mM, about 0.5mM to about 7mM, about 0.5mM to about 6mM, about 0.5mM to about 5mM, about 1mM to about 5mM, about 1mM to about 2mM, about 1mM to about 3mM, about 1mM to about 4mM, about 2mM to about 5mM, about 2mM to about 4mM, about 2mM to about 3mM, or any range therebetween.

[0034] In some embodiments, the concentration of the magnesium salt is about 0.1 mM, about 0.2 mM, about 0.3 mM, about 0.4 mM, about 0.5 mM, about 0.6 mM, about 0.7 mM, about 0.8 mM, about 0.9 mM, about 1.0 mM, about 1.1 mM, about 1.2 mM, about 1.3 mM, about 1.4 mM, about 1.5 mM, about 1.6 mM, about 1.7 mM, about 1.8 mM, about 1.9 mM, about 2.0 mM, about 2.1 mM, about 2.2 mM, about 2.3 mM, about 2.4 mM, about 2.5 mM, about 2.6 mM, about 2.7 mM, about 2.8 mM, about 2.9 mM, about 3.0 mM , about 3.2mM, about 3.5mM, about 3.7mM, about 4.0mM, about 4.5mM, about 4.8mM, about 5.0mM, about 5.5mM, about 6.0mM, about 6.5mM, about 7.0mM, about 7.5mM, about 8.0mM, about 8.5mM, about 9.0mM, about 9.5mM, about 10.0mM, about 12mM, about 14mM, about 15mM, about 16mM, about 18mM, about 19mM, about 20mM, about 22mM, about 25mM, about 28mM, about 30mM, about 35mM, about 40mM, about 45mM, about 50mM, or any range between any of the foregoing points.

[0035] In some embodiments, in any of the above pharmaceutical compositions, the titer of recombinant adeno-associated virus (rAAV) particles is about 1×10 8 Vg / mL~about 1×10 14 Vg / mL, for example, about 1×10 9 Vg / mL~about 1×10 14 Vg / mL, about 1×10 10Vg / mL~about 1×10 14 Vg / mL, about 1×10 11 Vg / mL~about 1×10 14 Vg / mL, about 1×10 11 Vg / mL~about 9×10 13 Vg / mL, about 1×10 12 Vg / mL~about 9×10 13 Vg / mL, about 1×10 12 Vg / mL~about 8×10 13 Vg / mL, about 1×10 12 Vg / mL~about 7×10 13 Vg / mL, about 1×10 12 Vg / mL~about 6×10 13 Vg / mL, about 1×10 12 Vg / mL~about 5×10 13 Vg / mL, about 1×10 12 Vg / mL~about 2×10 13 Vg / mL, and any range between these points.

[0036] In certain embodiments, the sugars, alcohols, surfactants, and amino acids used in the present disclosure can further improve the physical stability, chemical stability, and biological activity of the pharmaceutical composition, thereby maintaining high titer of the rAAV particles and making them suitable for long-term stable storage and use.

[0037] In some embodiments, the pharmaceutical composition comprises recombinant adeno-associated virus (rAAV) particles, and an excipient; wherein the excipient can be any of the following:

[0038] (a) about 1 mM to about 100 mM Hepes,

[0039] about 10 mM to about 500 mM of the sodium salt, about 1 mM to about 50 mM of the magnesium salt,

[0040] 0.1% (w / v) to about 10% (w / v) sugars,

[0041] about 0.01% (w / v) to about 10% (w / v) of alcohol,

[0042] 1 mM to about 50 mM of an amino acid, and,

[0043] about 0.001% (w / v) to about 0.05% (w / v) of a surfactant; the surfactant is selected from poloxamer 188 and / or polysorbate 80; the pH of the pharmaceutical composition is about 4.0 to about 10.0;

[0044] (b) about 1 mM to about 100 mM BTP,

[0045] about 10 mM to about 500 mM of the sodium salt, about 1 mM to about 50 mM of the magnesium salt,

[0046] 0.1% (w / v) to about 10% (w / v) sugars,

[0047] about 0.01% (w / v) to about 10% (w / v) of alcohol,

[0048] 1 mM to about 50 mM of an amino acid, and,

[0049] about 0.001% (w / v) to about 0.05% (w / v) of a surfactant; the surfactant is selected from poloxamer 188 and / or polysorbate 80; the pH of the pharmaceutical composition is about 4.0 to about 10.0;

[0050] (c) about 5 mM to about 50 mM Hepes,

[0051] about 10 mM to about 200 mM sodium chloride, and about 1 mM to about 10 mM magnesium chloride hexahydrate,

[0052] about 1% (w / v) to about 10% (w / v) of a sugar selected from sucrose and / or trehalose;

[0053] about 0.05% (w / v) to about 5% (w / v) glycerol,

[0054] about 1 mM to about 10 mM histidine,

[0055] about 0.001% (w / v) to about 0.05% (w / v) of a surfactant; the surfactant is selected from poloxamer 188 and / or polysorbate 80; the pH of the pharmaceutical composition is about 4.0 to about 10.0, for example, about 6.0 to about 8.0;

[0056] (d) about 5 mM to about 50 mM BTP,

[0057] about 10 mM to about 200 mM sodium chloride, and about 1 mM to about 10 mM magnesium chloride hexahydrate,

[0058] about 1% (w / v) to about 10% (w / v) of a sugar selected from sucrose and / or trehalose;

[0059] about 0.05% (w / v) to about 5% (w / v) glycerol,

[0060] about 1 mM to about 10 mM histidine,

[0061] about 0.001% (w / v) to about 0.05% (w / v) of a surfactant; the surfactant is selected from poloxamer 188 and / or polysorbate 80; the pH of the pharmaceutical composition is about 4.0 to about 10.0, for example, about 6.0 to about 8.0;

[0062] (e) about 10 mM to about 30 mM Hepes,

[0063] about 50 mM to about 200 mM sodium chloride, and about 1 mM to about 5 mM magnesium chloride hexahydrate,

[0064] about 1% (w / v) to about 5% (w / v) sucrose,

[0065] about 0.1% (w / v) to about 0.5% (w / v) glycerol,

[0066] about 1 mM to about 5 mM histidine,

[0067] about 0.001% (w / v) to about 0.02% (w / v) of poloxamer 188; the pH of the pharmaceutical composition is about 6.0 to about 8.0, for example, about 6.0 to about 7.5;

[0068] (f) about 10 mM to about 30 mM BTP,

[0069] about 50 mM to about 200 mM sodium chloride, and about 1 mM to about 5 mM magnesium chloride hexahydrate,

[0070] about 1% (w / v) to about 5% (w / v) sucrose,

[0071] about 0.1% (w / v) to about 0.5% (w / v) glycerol,

[0072] about 1 mM to about 5 mM histidine,

[0073] about 0.001% (w / v) to about 0.02% (w / v) of poloxamer 188; the pH of the pharmaceutical composition is about 6.0 to about 8.0, for example, about 6.0 to about 7.5;

[0074] (g) about 5 mM to about 20 mM Hepes,

[0075] about 100 mM to about 150 mM sodium chloride, and about 1 mM to about 5 mM magnesium chloride hexahydrate,

[0076] about 1% (w / v) to about 5% (w / v) sucrose,

[0077] about 0.1% (w / v) to about 0.5% (w / v) glycerol,

[0078] about 1 mM to about 5 mM histidine,

[0079] about 0.01% (w / v) to about 0.02% (w / v) of poloxamer 188; the pH of the pharmaceutical composition is about 6.0 to about 8.0, for example, about 6.0 to about 7.5;

[0080] (h) about 5 mM to about 20 mM Hepes,

[0081] about 80 mM to about 120 mM sodium chloride, and about 1 mM to about 5 mM magnesium chloride hexahydrate,

[0082] about 1% (w / v) to about 5% (w / v) sucrose,

[0083] about 0.1% (w / v) to about 0.5% (w / v) glycerol,

[0084] about 1 mM to about 5 mM histidine,

[0085] about 0.01% (w / v) to about 0.02% (w / v) of poloxamer 188; the pH of the pharmaceutical composition is about 6.0 to about 8.0, for example, about 6.0 to about 7.5;

[0086] (i) about 5 mM to about 20 mM Hepes,

[0087] about 120 mM to about 180 mM sodium chloride, and about 1 mM to about 5 mM magnesium chloride hexahydrate,

[0088] about 1% (w / v) to about 5% (w / v) sucrose,

[0089] about 0.1% (w / v) to about 0.5% (w / v) glycerol,

[0090] about 1 mM to about 5 mM histidine,

[0091] about 0.01% (w / v) to about 0.02% (w / v) of poloxamer 188; the pH of the pharmaceutical composition is about 6.0 to about 8.0, for example, about 6.0 to about 7.5;

[0092] In some embodiments, in any of the above pharmaceutical compositions, the titer of recombinant adeno-associated virus (rAAV) particles is about 1×10 8 Vg / mL~about 1×10 14 Vg / mL, for example, about 1×10 9 Vg / mL~about 1×10 14 Vg / mL, about 1×10 10 Vg / mL~about 1×10 14 Vg / mL, about 1×1011 Vg / mL~about 1×10 14 Vg / mL, about 1×10 11 Vg / mL~about 9×10 13 Vg / mL, about 1×10 12 Vg / mL~about 9×10 13 Vg / mL, about 1×10 12 Vg / mL~about 8×10 13 Vg / mL, about 1×10 12 Vg / mL~about 7×10 13 Vg / mL, about 1×10 12 Vg / mL~about 6×10 13 Vg / mL, about 1×10 12 Vg / mL~about 5×10 13 Vg / mL, about 1×10 12 Vg / mL~about 2×10 13 Vg / mL, and any range between these points.

[0093] In some embodiments, the pharmaceutical composition comprises any of the following:

[0094] (1) Approximately 1×10 11 Vg / mL~about 9×10 13 Vg / mL of recombinant adeno-associated virus (rAAV) particles

[0095] About 10mM Hepes,

[0096] About 100 mM sodium chloride, and about 2 mM magnesium chloride hexahydrate,

[0097] About 2.5% (w / v) sucrose,

[0098] About 0.25% (w / v) glycerol,

[0099] About 5mM histidine,

[0100] about 0.01% (w / v) of poloxamer 188; the pH of the pharmaceutical composition is about 6.5 to about 8.0, for example about 7.5;

[0101] (2) Approximately 1×10 11 Vg / mL~about 9×10 13 Vg / mL of recombinant adeno-associated virus (rAAV) particles

[0102] About 10mM BTP,

[0103] About 100 mM sodium chloride, and about 2 mM magnesium chloride hexahydrate,

[0104] About 2.5% (w / v) sucrose,

[0105] About 0.25% (w / v) glycerol,

[0106] About 5mM histidine,

[0107] about 0.01% (w / v) of poloxamer 188; the pH of the pharmaceutical composition is about 6.5 to about 8.0, for example about 7.5;

[0108] (3) Approximately 1×10 11 Vg / mL~about 9×10 13 Vg / mL of recombinant adeno-associated virus (rAAV) particles

[0109] About 10mM Hepes,

[0110] About 100 mM sodium chloride, and about 2 mM magnesium chloride hexahydrate,

[0111] About 2.5% (w / v) sucrose,

[0112] About 0.25% (w / v) glycerol,

[0113] About 5mM histidine,

[0114] about 0.01% (w / v) polysorbate 80; the pH of the pharmaceutical composition is about 6.5 to about 8.0, for example about 7.5;

[0115] (4) About 1×10 11 Vg / mL~about 9×10 13 Vg / mL of recombinant adeno-associated virus (rAAV) particles

[0116] About 10mM Hepes,

[0117] About 150 mM sodium chloride, and about 2 mM magnesium chloride hexahydrate,

[0118] About 2.5% (w / v) sucrose,

[0119] About 0.25% (w / v) glycerol,

[0120] About 5mM histidine,

[0121] about 0.01% (w / v) poloxamer 188; the pH of the pharmaceutical composition is about 5.0 to about 7.0, for example about 6.0;

[0122] (5) About 1×10 11 Vg / mL~about 9×10 13Vg / mL of recombinant adeno-associated virus (rAAV) particles

[0123] About 10mM BTP,

[0124] About 150 mM sodium chloride, and about 2 mM magnesium chloride hexahydrate,

[0125] About 2.5% (w / v) sucrose,

[0126] About 0.25% (w / v) glycerol,

[0127] About 5mM histidine,

[0128] about 0.01% (w / v) poloxamer 188; the pH of the pharmaceutical composition is about 5.0 to about 7.0, for example about 6.0;

[0129] (6) About 1×10 11 Vg / mL~about 9×10 13 Vg / mL of recombinant adeno-associated virus (rAAV) particles

[0130] About 10mM Hepes,

[0131] About 150 mM sodium chloride, and about 2 mM magnesium chloride hexahydrate,

[0132] About 2.5% (w / v) sucrose,

[0133] About 0.25% (w / v) glycerol,

[0134] About 5mM histidine,

[0135] about 0.01% (w / v) polysorbate 80; the pH of the pharmaceutical composition is about 5.0 to about 7.0, for example about 6.0.

[0136] In some embodiments, in any one of the pharmaceutical compositions of (1) to (3), the pH is about 6.5 to about 8.0, for example, about 7.0 to about 8.0, about 7.0 to about 7.5, or about 7.5 to about 8.0. Exemplarily, the pH is about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, about 7.0, about 7.1, about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7, about 7.8, about 7.9, or about 8.0.

[0137] In some embodiments, in the pharmaceutical composition of any one of (1)-(3), the pH is about 7.5.

[0138] In some embodiments, in any of the pharmaceutical compositions of (4) to (6), the pH is about 5.0 to about 7.0, for example, about 5.5 to about 7.0, about 5.5 to about 6.5, about 5.5 to about 6.0, or about 6.0 to about 6.5. Exemplarily, the pH is about 5.0, about 5.1, about 5.2, about 5.3, about 5.4, about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, or about 7.0.

[0139] In some embodiments, in the pharmaceutical composition of any one of (4)-(6), the pH is about 6.0.

[0140] In some embodiments, the recombinant adeno-associated virus (rAAV) particle comprises a nucleic acid molecule and an AAV capsid.

[0141] AAV capsids and AAV particles

[0142] The AAV capsid comprises or is derived from any natural or recombinant AAV serotype, including but not limited to: PHP.B, PHP.A, AAV1, AAV2, AAV2G9, AAV3, AAV3a, AAV3b, AAV3-3, AAV4, AAV4-4, AAV5, AAV6, AAV6.1, AAV6.2, AAV6.1.2, AAV7, AAV7.2, AAV8, AAV9, AAV9.11, AAV9.13, AAV9.16, AAV9.24, AAV9.45, AAV9.47, AAV9.61, AAV9.68, AAV9.84, AAV9.9, AAV10, AAV11, AAV12, AAV16.3, AAV24.1, AAV27.3, AAV42.12, AAV42-1b, AAV42-2, AAV42-3a, AAV42-3b, AAV42-4, AAV42-5a, AAV42-5b, AAV42-6b, AAV42-8, AAV42-10, AAV42-11, AAV42-12, AAV42-13, AAV42-15, AAV42-aa, AAV43-1, AAV43-12, AAV43-20, AAV43-21, AAV43-23, AAV43-25, AAV43-5, AAV44.1, AAV44.2, AAV44.5, AAV223.1, AAV223.2, AAV223.4, AAV223.5, AAV223.6, AAV223.7, AAV1-7 / rh.48, AAV1-8 / rh.49, AAV2-15 / rh.62, AAV2-3 / rh.61, AAV2-4 / rh.50, AAV2-5 / rh.51, AAV3.1 / hu.6, AAV3.1 / hu.9, AAV3-9 / rh.52, AAV3-11 / rh.53, AAV4-8 / r11.64, AAV4-9 / rh.54, AAV4-19 / rh.55, AAV5-3 / rh.57, AAV5-22 / rh.58, AAV7.3 / hu.7, AAV16.8 / hu.10, AAV16.12 / hu.11, AAV29.3 / bb.1, AAV29.5 / bb.2, AAV106.1 / hu.37, AAV114.3 / hu.40, AAV127.2 / hu.41, AAV127.5 / hu.42, AAV128.3 / hu.44, AAV130.4 / hu.48, AAV145.1 / hu.53, AAV145.5 / hu.54, AAV145.6 / hu.55, AAV161.10 / hu.60, AAV161.6 / hu.61, AAV33.12 / hu.17,AAV33.4 / hu.15、AAV33.8 / hu.16、AAV52 / hu.19、AAV52.1 / hu.20、AAV58.2 / hu.25、AAVA3.3、AAVA3.4、AAVA3.5、AAVA3.7、AAVC1、AAVC2、AAVC5、AAV-DJ 、AAV-DJ8、AAVF3、AAVF5、AAVH2、AAVrh.72、AAVhu.8、AAVrh.68、AAVrh.70、AAVpi.1、AAVpi.3、AAVpi.2、AAVrh.60、AAVrh.44、AAVrh.65、AAVrh.55、AAV rh.47, AAVrh.69, AAVrh.45, AAVrh.59, AAVhu.12, AAVH6, AAVLK03, AAVH-1 / hu.1, AAVH-5 / hu.3, AAVLG-10 / rh.40, AAVLG-4 / rh.38, AAVLG-9 / hu.39, AAVN721-8 / rh.43, AAVCh.5, AAVCh.5R1, AAVcy.2, AAVcy.3, AAVcy.4, AAVcy.5, AAVCy.5R1, AAVCy.5R2, AAVCy.5R3, AAVCy.5R4, AAVcy.6, AAVhu.1, AAVh u.2、AAVhu.3、AAVhu.4、AAVhu.5、AAVhu.6、AAVhu.7、AAVhu.9、AAVhu.10、AAVhu.11、AAVhu.13、AAVhu.15、AAVhu.16、AAVhu.17、AAVhu.18、AAVhu.20、 AAVhu.21、AAVhu.22、AAVhu.23.2、AAVhu.24、AAVhu.25、AAVhu.27、AAVhu.28、AAVhu.29、AAVhu.29R、AAVhu.31、AAVhu.32、AAVhu.34、AAVhu.35、AAVhu. .37、AAVhu.39、AAVhu.40、AAVhu.41、AAVhu.42、AAVhu.43、AAVhu.44、AAVhu.44R1、AAVhu.44R2、AAVhu.44R3、AAVhu.45、AAVhu.46、AAVhu.47、AAVhu.44 8、AAVhu.48R1、AAVhu.48R2、AAVhu.48R3、AAVhu.49、AAVhu.51、AAVhu.52、AAVhu.54、AAVhu.55、AAVhu.56、AAVhu.57、AAVhu.58、AAVhu.60、AAVhu.61、AAVhu.63, AAVhu.64, AAVhu.66, AAVhu.67, AAVhu.68, AAVhu.14 / 9, AAVhu.t19, AAVrh.2, AAVrh.2R, AAVrh.8, AAVrh.8R, AAVrh.10, AAVrh.12, AAVrh.13, AAVrh.13R, AAVrh.14, AAVrh.17, AAVrh.18, AAVrh.19, AAVrh.20, AAVrh.21, AAVrh.22, AAVrh.23, AAVrh.24, AAVrh.25, AAVrh.31, AAVrh.32, AAVrh.33, AAVrh.34, AAVrh.35, AAVrh.36, AAVrh.37, AAVrh.37R2, AAVrh.38, AAVrh.39, AAVrh.40, AAVrh.46, AAVrh.48, AAVrh.48.1, AAVrh.48.1.2, AAVrh.48.2, AAVrh.49, AAVrh.51, AAVrh.52, AAVrh.53, AAVrh.54, AAVrh.56, AAVrh.57, AAVrh.58, AAVrh.61, AAVrh.64, AAVrh.64R1, AAVrh.64R2, AAVrh.67, AAVrh.73, AAVrh.74, AAVrh8R, AAVrh8R A586R mutant, AAVrh8R R533A mutant, AAAV, BAAV, goat AAV, bovine AAV, sheep AAV, AAVhE1.1, AAVhEr1.5, AAVhER1.14, AAVhEr1.8, AAVhEr1.16, AAVhEr1.18, AAVhEr1.35, AAVhEr1.7, AAVhEr1.36, AAVhEr2.29, AAVhEr2.4, AAVhEr2.16, AAVhEr2.30, AAVhEr2.31, AAVhEr2.36, AAVhER1.23, AAVhEr3.1, AAV2.5T, AAV-PAEC, AAV-LK01, AAV-LK02, AAV-LK03, AAV-LK04, AAV-LK05, AAV-LK06, AAV-LK07, AAV-LK08, AAV-LK09, AAV-LK10, AAV-LK11, AAV-LK12, AAV-LK13, AAV-LK14, AAV-LK15, AAV-LK16, AAV-LK17, AAV-LK18, AAV-LK19, AAV-PAEC2, AAV-PAEC4, AAV-PAEC6, AAV-PAEC7AAV-PAEC8, AAV-PAEC11, AAV-PAEC12, AAV-2-pre-miRNA-101, AAV-8h, AAV-8b, AAV-h, AAV-b, AAV SM 10-2, AAVShuffle 100-1, AAV Shuffle 100-3, AAV Shuffle 100-7, AAV Shuffle 10-2, AAVShuffle 10-6, AAV Shuffle 10-8, AAV Shuffle 100-2, AAV SM 10-1, AAV SM 10-8, AAVSM 100-3, AAV SM 100-10, BNP61 AAV, BNP62 AAV, BNP63 AAV, AAVrh.50, AAVrh.43, AAVrh.62, AAVrh.48, AAVhu.19, AAVhu.11, AAVhu.53, AAV4-8 / rh.64, AAVLG-9 / hu.39, AAV54.5 / hu.23, AAV54.2 / hu.22, AAV54.7 / hu.24, AAV54.1 / hu.21, AAV54.4R / hu.27, AAV46.2 / hu.28, AAV46.6 / hu.29, AAV128.1 / hu.43, true type AAV (ttAAV), UPENNAAV 10, Japanese AAV 10 serotype, AAV CBr-7.1, AAV CBr-7.10, AAV CBr-7.2, AAV CBr-7.3, AAVCBr-7.4, AAV CBr-7.5, AAV CBr-7.7, AAV CBr-7.8, AAV CBr-B7.3, AAV CBr-B7.4, AAVCBr-E1, AAV CBr-E2, AAV CBr-E3, AAV CBr-E4, AAV CBr-E5, AAV CBr-e5, AAV CBr-E6, AAVCBr-E7, AAV CBr-E8, AAV CHt-1, AAV CHt-2, AAV CHt-3, AAV CHt-6.1, AAV CHt-6.10, AAVCHt-6.5, AAV CHt-6.6, AAV CHt-6.7, AAV CHt-6.8, AAV CHt-P1, AAV CHt-P2, AAV CHt-P5, AAV CHt-P6, AAV CHt-P8, AAV CHt-P9, AAV CKd-1, AAV CKd-10, AAV CKd-2, AAV CKd-3, AAVCKd-4, AAV CKd-6, AAV CKd-7,AAV CKd-8、AAV CKd-B1、AAV CKd-B2、AAV CKd-B3、AAV CKd-B4、AAV CKd-B5、AAV CKd-B6、AAV CKd-B7、AAV CKd-B8、AAV CKd-H1、AAV CKd-H2、AAV CKd-H3、AAV CKd-H4、AAV CKd-H5、AAV CKd-H6、AAV CKd-N3、AAV CKd-N4、AAV CKd-N9、AAV CLg-F1、AAV CLg-F2、AAV CLg-F3、AAV CLg-F4、AAV CLg-F5、AAV CLg-F6、AAV CLg-F7、AAV CLg-F8、AAV CLv-1、AAV CLv1-1、AAV Clv1-10、AAV CLv1-2、AAV CLv-12、AAV CLv1-3、AAV CLv-13、AAV CLv1-4、AAV Clv1-7、AAV Clv1-8、AAV Clv1-9、AAV CLv-2、AAV CLv-3、AAV CLv-4、AAV CLv-6、AAV CLv-8、AAV CLv-D1、AAV CLv-D2、AAV CLv-D3、AAV CLv-D4、AAV CLv-D5、AAV CLv-D6、AAV CLv-D7、AAV CLv-D8、AAV CLv-E1、AAV CLv-K1、AAV CLv-K3、AAV CLv-K6、AAV CLv-L4、AAV CLv-L5、AAV CLv-L6、AAV CLv-M1、AAV CLv-M11、AAV CLv-M2、AAV CLv-M5、AAV CLv-M6、AAV CLv-M7、AAV CLv-M8、AAV CLv-M9、AAV CLv-R1、AAV CLv-R2、AAV CLv-R3、AAV CLv-R4、AAV CLv-R5、AAV CLv-R6、AAV CLv-R7、AAV CLv-R8、AAV CLv-R9、AAV CSp-1、AAV CSp-10、AAV CSp-11、AAV CSp-2、AAV CSp-3、AAV CSp-4、AAV CSp-6、AAV CSp-7、AAVCSp-8、AAV CSp-8.10、AAV CSp-8.2、AAV CSp-8.4、AAV CSp-8.5、AAV CSp-8.6、AAV CSp-8.7、AAV CSp-8.8、AAV CSp-8.9、AAV CSp-9, AAV.hu.48R3, AAV.VR-355, AAV3B, AAV4, AAV5, AAVF1 / HSC1, AAVF11 / HSC11, AAVF12 / HSC12, AAVF13 / HSC13, AAVF14 / HSC14, AAVF15 / HSC15, AAVF16 / HSC16, AAVF17 / HSC17, AAVF2 / HSC2, AAVF3 / HSC3, AAVF4 / HSC4, AAVF5 / HSC5, AAVF6 / HSC6, AAVF7 / HSC7, AAVF8 / HSC8, AAVF9 / HSC9, PHP.B (AAV-PHP.B), PHP.A (AAV.PHP.A), G2B-26, G2B-13, TH1.1-32, TH1.1-35, AAVPHP.B2, AAVPHP.B3, AAVPHP.N / PHP.B-DGT, AAVPHP.B-EST, AAVPHP.B-GGT, AAVPHP.B-ATP, AAVPHP.B-ATT-T, AAVPHP.B-DGT-T, AAVPHP.B-GGT-T, AAVPHP.B-SGS, AAVPHP.B-AQP, AAVPHP.B-QQP, AAVPHP.B-SNP(3), AAVPHP.B-SNP, AAVPHP.B-QGT, AAVPHP.B-NQT, AAVPHP.B-EGS, AAVPHP.B-SGN, AAVPHP.B-EGT, AAVPHP.B-DST, AAVPHP.B-DST, AAVPHP.B-STP, AAVPHP.B-PQP, AAVPHP.B-SQP, AAVPHP.B-QLP, AAVPHP.B-TMP, AAVPHP.B-TTP, AAVPHP.S / G2A12, AAVG2A15 / G2A3, AAVG2B4, AAVG2B5, AAVDJ8 and their variants.,

[0143] In some embodiments, the AAV capsid can be modified or mutated, for example, containing one or more mutations of Y252F, Y272F, Y444F, Y500F, Y700F, Y704F, Y730F, Y275F, Y281F, Y508F, Y576F, Y612G, Y673F, and Y720F; for example, containing one or more mutations of F129L, D418E, K531E, L584F, V598A, and H642N; for example At least one of the Tyr residues at positions 252, 272, 444, 500, 700, 704 and 730 of AAV2 is substituted with, for example, a Phe residue; for example, the AAV2 capsid contains an N587A, E548A or N708A mutation; for example, the AAV9 capsid contains a T446F mutation; for example, the AAV capsid contains a V708K mutation; for example, the AAV capsid is generated via an AAV9 capsid library having mutations in amino acids 390-627 (VP1 numbering).

[0144] This disclosure incorporates in its entirety WO2022247917A, WO2018232055 (e.g., Table 1 thereof), WO2005033321, WO2015168666, WO2015121501, WO2015038958, WO2016065001, WO2016130589, WO2016049230, WO2016134375, WO2017100671, WO2017083722, WO2017015102, WO2017058892, WO2017066764, US9546112, US7198951, US9233131, US6156303, US9624274, US9475845, US8734809, US20130224836, US20140359799, US20150315612, US20150376240, US20150159173, US20150376607, US20150238550, US20160369298, US20160361439, US20170145405, and AAV capsids in NPulicherla et al. (Molecular Therapy 19(6):1070-1078(2011)). In some specific embodiments, the AAV capsid used in the present disclosure is the AAV2 and AAV9 capsids in the above-mentioned prior art.

[0145] In some embodiments, the AAV capsid is engineered, for example, a hybrid AAV capsid derived from two or more parental serotypes. For example, the AAV capsid can be AAV2G9, which comprises sequences from AAV2 and AAV9. The sequence of AAV2G9 in US20160017005 is incorporated herein in its entirety.

[0146] In some embodiments, the AAV capsid is an AAV2 or AAV9 capsid, for example comprising the amino acid sequence shown in SEQ ID NO:41 or 42.

[0147] In some embodiments, in addition to the encoded heterologous payload, the AAV particle can comprise all or part of the vector genome of any naturally occurring and / or recombinant AAV capsid nucleotide sequence or variant.

[0148] In some embodiments, the AAV particles can be replication-defective (e.g., they lack sequences encoding functional Rep and Cap proteins in the vector genome). In some specific embodiments, the replication-defective AAV particles can lack most or all parental coding sequences and essentially carry only one or two AAV ITR sequences and a polynucleotide of interest for delivery to a cell, tissue, organ, or organism.

[0149] In some embodiments, the AAV particle can be a recombinant AAV (rAAV) particle.

[0150] In some embodiments, the AAV particles can be selected from single-stranded AAV particles (e.g., ssAAV) and self-complementary AAV particles (e.g., scAAV). By skipping second-strand synthesis, scAAV achieves rapid expression in cells.

[0151] In some embodiments, the pharmaceutical composition comprises recombinant adeno-associated virus (rAAV) particles and excipients; the recombinant adeno-associated virus (rAAV) particles comprise a nucleic acid molecule and an AAV9 capsid, and the excipients comprise any one of (a)-(f) and (h).

[0152] In some embodiments, the pharmaceutical composition comprises recombinant adeno-associated virus (rAAV) particles and an excipient; the recombinant adeno-associated virus (rAAV) particles comprise a nucleic acid molecule and an AAV9 capsid, and the excipient comprises any one of (1)-(3).

[0153] In some embodiments, the pharmaceutical composition comprises recombinant adeno-associated virus (rAAV) particles and excipients; the recombinant adeno-associated virus (rAAV) particles comprise a nucleic acid molecule and an AAV2 capsid, and the excipients comprise any one of (a)-(f) and (i).

[0154] In some embodiments, the pharmaceutical composition comprises recombinant adeno-associated virus (rAAV) particles and an excipient; the recombinant adeno-associated virus (rAAV) particles comprise a nucleic acid molecule and an AAV2 capsid, and the excipient comprises any one of (4)-(6).

[0155] In some embodiments, the nucleic acid molecule or AAV particle is derived from any of the nucleic acid molecules or AAV particles of WO2023093905A, which are incorporated herein by reference. In some embodiments, the nucleic acid molecule or AAV particle is derived from any of the VEGF-binding molecules or AAV particles of WO2023155918A, which are incorporated herein by reference.

[0156] Nucleic Acid Molecule I

[0157] The present disclosure provides a nucleic acid molecule comprising a polynucleotide encoding an AADC protein (the amino acid sequence encoding the AADC protein is, for example, as shown in SEQ ID NO: 1), wherein the polynucleotide is at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, or at least 95% identical to SEQ ID NO: 3 (e.g., at least 80.83%, at least 81.21%, at least 79.18%, at least 79.63%, or more identical), or at least 95% identical to any one of SEQ ID NOs: 4-7. In some embodiments, the polynucleotide encoding the AADC protein comprises a polynucleotide sequence as shown in any one of SEQ ID NOs: 3-7.

[0158] The present disclosure provides a nucleic acid molecule comprising a polynucleotide encoding a GDNF protein (the amino acid sequence encoding the AADC protein is, for example, as shown in SEQ ID NO: 2), wherein the polynucleotide is at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% identical to SEQ ID NO: 8 (e.g., at least 75.13%, at least 73.99%, at least 77.52%, at least 77.83%, or more identical), or at least 95% identical to any one of SEQ ID NOs: 9-12. In some embodiments, the polynucleotide encoding the GDNF protein comprises a polynucleotide sequence as shown in any one of SEQ ID NOs: 8-12.

[0159] In some embodiments of the present disclosure, AADC protein and GDNF protein encompass analogs thereof.

[0160] The present disclosure provides a nucleic acid molecule comprising a first polynucleotide and a second polynucleotide, wherein the first polynucleotide comprises a polynucleotide encoding an AADC protein (the amino acid sequence encoding the AADC protein is, for example, as shown in SEQ ID NO: 1), and the second polynucleotide comprises a polynucleotide encoding a GDNF protein (the amino acid sequence encoding the AADC protein is, for example, as shown in SEQ ID NO: 2). In some embodiments, the first polynucleotide and the second polynucleotide are operably linked, and the first polynucleotide and the second polynucleotide can be located in the same polynucleotide, the same plasmid, or the same expression cassette. In other embodiments, the first polynucleotide and the second polynucleotide are independently present in two mutually independent polynucleotides, or two mutually independent different plasmids, or two different expression cassettes.

[0161] In some embodiments, the amino acid sequence of the AADC protein comprises or is shown as SEQ ID NO: 1, and the amino acid sequence of the GDNF protein comprises or is shown as SEQ ID NO: 2.

[0162] In some embodiments, the first polynucleotide sequence encoding the AADC protein and / or the second polynucleotide sequence encoding the GDNF protein are codon-optimized. For example, the codon-optimized polynucleotide sequence encoding the AADC protein is at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, or at least 95% identical to SEQ ID NO: 3; and the codon-optimized polynucleotide sequence encoding the GDNF protein is at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% identical to SEQ ID NO: 8.

[0163] In some embodiments, the polynucleotide sequence encoding the AADC protein comprises a sequence having at least 95% identity to any one of SEQ ID NOs: 4-7, and / or the polynucleotide sequence encoding the GDNF protein comprises a sequence having at least 95% identity to any one of SEQ ID NOs: 9-12. In some specific embodiments, the polynucleotide sequence encoding AADC comprises or is set forth in any one of SEQ ID NOs: 3-7; the polynucleotide sequence encoding GDNF comprises or is set forth in any one of SEQ ID NOs: 8-12.

[0164] In this disclosure, "at least 95% identity" is meant to encompass at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more identity.

[0165] The codon-optimized polynucleotide expressing an AADC protein (e.g., any one of SEQ ID NOs: 4-7) increases the expression of the AADC protein compared to a wild-type or non-codon-optimized polynucleotide (e.g., SEQ ID NO: 3). The codon-optimized polynucleotide expressing a GDNF protein (e.g., any one of SEQ ID NOs: 9-12) increases the expression of the GDNF protein compared to a wild-type or non-codon-optimized polynucleotide (e.g., SEQ ID NO: 8). The increase is an increase in protein expression of at least 10%, at least 20%, at least 30%, at least 50%, at least 75%, at least 100%, or at least several times (including 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) compared to a wild-type or non-codon-optimized polynucleotide.

[0166] In some embodiments, the nucleic acid molecules provided herein further comprise the same expression control sequence operably linked to the first polynucleotide and the second polynucleotide, or two identical or different expression control sequences operably linked to the first polynucleotide and the second polynucleotide, respectively, wherein the expression control sequence comprises (c) a promoter and / or (d) an enhancer.

[0167] In some embodiments, the nucleic acid molecules provided by the present disclosure further comprise any one or any combination of the following:

[0168] (a) 5' inverted terminal repeat (5'ITR);

[0169] (b) 3' inverted terminal repeat (3'ITR);

[0170] (e) intron;

[0171] (f) post-transcriptional regulatory elements;

[0172] (g) polyadenylation signal (polyA);

[0173] (h) Multiple cloning site (MCS).

[0174] In some specific embodiments, any combination of (a)-(h) can satisfy the function of expressing the target gene (AADC and / or GDNF) (eg, expression in the brain of a subject (eg, substantia nigra, striatum)).

[0175] In some specific embodiments, the polynucleotide of any one of (a) to (h) or any combination thereof is operably linked to the polynucleotide encoding the AADC protein and / or the polynucleotide encoding the GDNF protein.

[0176] In some specific embodiments, the 5' ITR and / or 3' ITR are derived from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV9.47, AAV9(hu14), AAV10, AAV11, AAV12, AAVrh8, AAVrh10, AAV-DJ or AAV-DJ8; for example, from AAV2, AAV9.

[0177] In some specific embodiments, the promoter is selected from CMV, CAG, CBh, EFS, EF1 (eg, EF-1α), PGK, SV40, Ubi, RSV, or any combination thereof.

[0178] In some specific embodiments, the enhancer is selected from Ubi, CMV, RSV enhancer or any combination thereof.

[0179] In some embodiments, the intron is selected from MVM, SV40, βGlobin, EF1 (eg, EF-1α), a hybrid intron, or any combination thereof.

[0180] In some specific embodiments, the polyA is selected from PA75 polyA, SV40 polyA, hGH polyA, BGH polyA, rbGlob polyA, or any combination thereof.

[0181] In some embodiments, the post-transcriptional regulatory element is selected from WPRE, HPRE, or a combination thereof.

[0182] In some embodiments, the nucleic acid molecule comprises a polynucleotide encoding an AADC protein, a CMV enhancer (e.g., CMV enhancer 1), and a CBA promoter. Optionally, the first polynucleotide further comprises a hybrid intron and polyA, wherein the polyA is selected from SV40 polyA and hGH polyA; and / or the nucleic acid molecule comprises a polynucleotide encoding a GDNF protein, a CMV enhancer (e.g., CMV enhancer 2), and a CMV promoter. Optionally, the second polynucleotide further comprises a β-globin intron and polyA, wherein the polyA is selected from PA75 polyA and SV40 polyA; wherein the CMV enhancer 1 and CMV enhancer 2 are both CMV enhancers, and the two may be the same or different.

[0183] In some embodiments, AADC and GDNF in the polynucleotide can be expressed in different expression frameworks or connected via a linker (eg, a P2A sequence) to share a common expression framework.

[0184] The first polynucleotide and the second polynucleotide are operably linked to express the AADC protein and the GDNF protein at the same target administration site in the subject. This can prevent the therapeutic efficacy of the combined use of the two proteins from being affected by inconsistent administration sites and reduce side effects caused by leakage of one protein to another site. Referring to the Examples of the present disclosure, the first polynucleotide and the second polynucleotide were operably linked and administered to disease model mice, demonstrating significant therapeutic effects.

[0185] In some embodiments, the first polynucleotide and the second polynucleotide are connected by a third polynucleotide, for example, the third polynucleotide encodes an amino acid sequence having a linker function. The connection order can be from the 5' end to the 3' end: first polynucleotide-third polynucleotide-second polynucleotide, or from the 5' end to the 3' end: second polynucleotide-third polynucleotide-first polynucleotide. In some specific embodiments, the amino acid sequence encoded by the third polynucleotide is as shown in any one of SEQ ID NOs: 43-46, for example, the sequence of the third polynucleotide is as shown in any one of SEQ ID NOs: 47-50 or has at least 80%, at least 90%, at least 95%, or at least 98% identity thereto.

[0186] In some embodiments, the polynucleotide encoding the AADC protein and the polynucleotide encoding the GDNF protein are expressed by the same regulatory element, or by two different regulatory elements, including but not limited to 5'ITR, 3'ITR, promoter, enhancer, intron, polyA, post-transcriptional regulatory elements, etc.

[0187] In some embodiments, the nucleic acid molecule comprises, from the 5' end to the 3' end:

[0188] a) CMV enhancer, CBA promoter, a first polynucleotide encoding AADC protein, a second polynucleotide encoding GDNF protein, and polyA;

[0189] In some optional embodiments, a hybrid intron is further included between the CBA promoter and the first polynucleotide encoding the AADC protein;

[0190] In some optional embodiments, a WPRE sequence is further included between the second polynucleotide encoding the GDNF protein and the polyA.

[0191] In some specific embodiments, the nucleic acid molecule comprises: a CMV enhancer, a CBA promoter, a hybrid intron, a first polynucleotide encoding an AADC protein, a second polynucleotide encoding a GDNF protein, a WPRE sequence, and polyA.

[0192] b) CMV enhancer, CBA promoter, a second polynucleotide encoding a GDNF protein, a first polynucleotide encoding an AADC protein, and polyA;

[0193] In some optional embodiments, a hybrid intron is further included between the CBA promoter and the second polynucleotide encoding the GDNF protein;

[0194] In some optional embodiments, a WPRE sequence is further included between the first polynucleotide encoding the AADC protein and the polyA.

[0195] In some specific embodiments, the nucleic acid molecule comprises: a CMV enhancer, a CBA promoter, a hybrid intron, a second polynucleotide encoding a GDNF protein, a first polynucleotide encoding an AADC protein, a WPRE sequence, and polyA.

[0196] c) CMV enhancer, CBA promoter, a first polynucleotide encoding an AADC protein, polyA, CMV enhancer, CMV promoter, a second polynucleotide encoding a GDNF protein, and polyA;

[0197] In some optional embodiments, a hybrid intron is further included between the CBA promoter and the first polynucleotide encoding the AADC protein;

[0198] In some optional embodiments, a beta globin intron is further included between the CMV promoter and the second polynucleotide encoding the GDNF protein;

[0199] In some optional embodiments, a WPRE sequence is further included between the second polynucleotide encoding the GDNF protein and the polyA.

[0200] In some specific embodiments, the nucleic acid molecule comprises:

[0201] CMV enhancer 1, CBA promoter, hybrid intron, a first polynucleotide encoding AADC protein, polyA, CMV enhancer 2, CMV promoter, a second polynucleotide encoding GDNF protein, and polyA; or,

[0202] CMV enhancer 1, CBA promoter, hybrid intron, a first polynucleotide encoding an AADC protein, polyA, CMV enhancer 2, CMV promoter, a second polynucleotide encoding a GDNF protein, a WPRE sequence, and polyA; or,

[0203] CMV enhancer 1, CBA promoter, hybrid intron, first polynucleotide encoding AADC protein, polyA, CMV enhancer 2, CMV promoter, beta globin intron, second polynucleotide encoding GDNF protein and polyA.

[0204] d) CMV enhancer, CMV promoter, a second polynucleotide encoding a GDNF protein, polyA, CMV enhancer, CBA promoter, a first polynucleotide encoding an AADC protein, and polyA;

[0205] In some optional embodiments, a beta globin intron or a MVM intron is further included between the CMV promoter and the second polynucleotide encoding the GDNF protein;

[0206] In some optional embodiments, a hybrid intron is further included between the CBA promoter and the first polynucleotide encoding the AADC protein;

[0207] In some optional embodiments, a WPRE sequence is further included between the first polynucleotide encoding the AADC protein and the polyA;

[0208] In some specific embodiments, the nucleic acid molecule comprises:

[0209] CMV enhancer 2, CMV promoter, beta globin intron, a second polynucleotide encoding a GDNF protein, polyA, CMV enhancer 1, CBA promoter, a hybrid intron, a first polynucleotide encoding an AADC protein, and polyA;

[0210] CMV enhancer 2, CMV promoter, a second polynucleotide encoding a GDNF protein, polyA, CMV enhancer 1, CBA promoter, a hybrid intron, a first polynucleotide encoding an AADC protein, a WPRE sequence, and polyA; or,

[0211] CMV enhancer 2, CMV promoter, MVM intron, a second polynucleotide encoding a GDNF protein, polyA, CMV enhancer 1, CBA promoter, a hybrid intron, a first polynucleotide encoding an AADC protein, a WPRE sequence, and polyA.

[0212] e) CMV enhancer, CBA promoter, a first polynucleotide encoding an AADC protein, a third polynucleotide, a second polynucleotide encoding a GDNF protein, and polyA;

[0213] In some optional embodiments, a hybrid intron is further included between the CBA promoter and the first polynucleotide encoding the AADC protein;

[0214] In some optional embodiments, a WPRE sequence is further included between the second polynucleotide encoding the GDNF protein and the polyA;

[0215] In some specific embodiments, the nucleic acid molecule comprises: a CMV enhancer, a CBA promoter, a hybrid intron, a first polynucleotide encoding an AADC protein, a third polynucleotide, a second polynucleotide encoding a GDNF protein, a WPRE sequence, and polyA.

[0216] f) CMV enhancer, CBA promoter, the second polynucleotide encoding GDNF protein, the third polynucleotide, the first polynucleotide encoding AADC protein, and polyA;

[0217] In some optional embodiments, a hybrid intron is further included between the CBA promoter and the second polynucleotide encoding the GDNF protein;

[0218] In some optional embodiments, a WPRE sequence is further included between the first polynucleotide encoding the AADC protein and the polyA;

[0219] In some specific embodiments, the nucleic acid molecule comprises: a CMV enhancer, a CBA promoter, a hybrid intron, a second polynucleotide encoding a GDNF protein, a third polynucleotide, a first polynucleotide encoding an AADC protein, a WPRE sequence, and polyA.

[0220] g) CMV enhancer, CBA promoter, the first polynucleotide or the second polynucleotide, and polyA;

[0221] In some optional embodiments, a hybrid intron is further included between the CBA promoter and the first polynucleotide, or a hybrid intron is further included between the CBA and the second polynucleotide;

[0222] In some optional embodiments, a WPRE sequence is further included between the first polynucleotide and polyA, or a WPRE sequence is further included between the second polynucleotide and polyA;

[0223] In some specific embodiments, the nucleic acid molecule comprises: a CMV enhancer, a CBA promoter, a hybrid intron, a first polynucleotide, and polyA;

[0224] CMV enhancer, CBA promoter, hybrid intron, second polynucleotide and polyA;

[0225] CMV enhancer, CBA promoter, hybrid intron, first polynucleotide, WPRE sequence and polyA; or, CMV enhancer, CBA promoter, hybrid intron, second polynucleotide, WPRE sequence and polyA.

[0226] In some embodiments, the polyA is selected from hGH polyA, PA75 polyA, or SV40 polyA.

[0227] In some embodiments, a polynucleotide is provided, wherein the nucleic acid molecule comprises, from the 5' end to the 3' end:

[0228] (1) CMV enhancer-CBA promoter-hybrid intron-first polynucleotide encoding AADC-third polynucleotide-second polynucleotide encoding GDNF-WPRE-SV40 polyA;

[0229] (2) CMV enhancer-CBA promoter-hybrid intron-second polynucleotide encoding GDNF-third polynucleotide-first polynucleotide encoding AADC-WPRE-SV40 polyA;

[0230] (3) CMV enhancer-CBA promoter-hybrid intron-first polynucleotide encoding AADC-SV40 polyA-CMV enhancer-CMV promoter-second polynucleotide encoding GDNF-PA75 polyA;

[0231] (4) CMV enhancer-CBA promoter-hybrid intron-first polynucleotide encoding AADC-SV40 polyA-CMV enhancer-CMV promoter-second polynucleotide encoding GDNF-WPRE-PA75 polyA;

[0232] (5) CMV enhancer-CBA promoter-hybrid intron-first polynucleotide encoding AADC-SV40 polyA-CMV enhancer-CMV promoter-β-globin intron-second polynucleotide encoding GDNF-PA75 polyA;

[0233] (6) CMV enhancer-CMV promoter-β-globin intron-second polynucleotide encoding GDNF-PA75 poly A-CMV enhancer-CBA promoter-hybrid intron-first polynucleotide encoding AADC-SV40 poly A;

[0234] (7) CMV enhancer-CMV promoter-second polynucleotide encoding GDNF-PA75 poly A-CMV enhancer-CBA promoter-hybrid intron-first polynucleotide encoding AADC-WPRE-SV40 poly A; or

[0235] (8) CMV enhancer-CMV promoter-MVM intron-second polynucleotide encoding GDNF-PA75 poly A-CMV enhancer-CBA promoter-hybrid intron-first polynucleotide encoding AADC-WPRE-SV40 poly A.

[0236] In some embodiments, a polynucleotide is provided, which comprises, from 5' to 3' direction, the following 1)-13) or any combination thereof:

[0237] 1) 5'ITR, CMV enhancer 1, CBA promoter, hybrid intron, AADC encoding polynucleotide or GDNF encoding polynucleotide, hGH polyA, 3'ITR;

[0238] 2) 5'ITR, CMV enhancer 1, CBA promoter, hybrid intron, AADC encoding polynucleotide or GDNF encoding polynucleotide, SV40 polyA, 3'ITR;

[0239] 3) 5'ITR, CMV enhancer 1, CBA promoter, hybrid intron, AADC encoding polynucleotide or GDNF encoding polynucleotide, WPRE, SV40 polyA, 3'ITR;

[0240] 4) 5'ITR, CMV enhancer 1, CBA promoter, hybrid intron, AADC encoding polynucleotide, P2A, GDNF encoding polynucleotide, WPRE, SV40 polyA, 3'ITR;

[0241] 5) 5'ITR, CMV enhancer 1, CBA promoter, hybrid intron, GDNF encoding polynucleotide, P2A, AADC encoding polynucleotide, WPRE, SV40 polyA, 3'ITR;

[0242] 6) 5'ITR, MCS, CMV enhancer 1, CBA promoter, hybrid intron, AADC encoding polynucleotide, SV40 polyA, CMV enhancer 2, CMV promoter, GDNF encoding polynucleotide, PA75 polyA, 3'ITR;

[0243] 7) 5'ITR, MCS, CMV enhancer 1, CBA promoter, hybrid intron, AADC encoding polynucleotide, SV40 polyA, CMV enhancer 2, CMV promoter, GDNF encoding polynucleotide, WPRE, PA75 polyA, 3'ITR;

[0244] 8) 5'ITR, MCS, CMV enhancer 1, CBA promoter, hybrid intron, AADC encoding polynucleotide, SV40 polyA, CMV enhancer 2, CMV promoter, β-globin intron, GDNF encoding polynucleotide, PA75 polyA, 3'ITR;

[0245] 9) 5'ITR, CMV enhancer 2, CMV promoter, β-globin intron, GDNF encoding polynucleotide, PA75 polyA, CMV enhancer 1, CBA promoter, hybrid intron, AADC encoding polynucleotide, SV40 polyA, 3'ITR;

[0246] 10) 5'ITR, CMV enhancer 2, CMV promoter, GDNF encoding polynucleotide, PA75 polyA, CMV enhancer 1, CBA promoter, hybrid intron, AADC encoding polynucleotide, WPRE, SV40 polyA, 3'ITR;

[0247] 11) 5'ITR, CMV enhancer 2, CMV promoter, MVM intron, GDNF encoding polynucleotide, PA75 polyA, CMV enhancer 1, CBA promoter, hybrid intron, AADC encoding polynucleotide, WPRE, SV40 polyA, 3'ITR;

[0248] 12) 5'ITR, CMV enhancer 1, CBA promoter, hybrid intron, AADC encoding polynucleotide, P2A, GDNF encoding polynucleotide, WPRE, SV40 polyA, 3'ITR;

[0249] 13) 5'ITR, CMV enhancer 2, CMV promoter, β-globin intron, GDNF encoding polynucleotide, PA75 polyA, CMV enhancer 1, CBA promoter, hybrid intron, AADC encoding polynucleotide, SV40 polyA, 3'ITR.

[0250] In some specific embodiments,

[0251] The CMV enhancer 1 comprises or is represented by the sequence shown in SEQ ID NO: 27;

[0252] The CMV enhancer 2 comprises or is represented by the sequence shown in SEQ ID NO: 37;

[0253] The CBA promoter comprises or is as shown in SEQ ID NO: 28;

[0254] The CMV promoter comprises or is as shown in SEQ ID NO:35;

[0255] The beta globin intron comprises or is represented by the sequence shown in SEQ ID NO: 38;

[0256] The hybrid intron comprises or is as shown in SEQ ID NO: 29;

[0257] The MVM intron comprises or is as shown in SEQ ID NO: 39;

[0258] The PA75 polyA comprises or is as shown in SEQ ID NO: 36;

[0259] The SV40 polyA comprises or is as shown in SEQ ID NO: 32;

[0260] The hGH polyA comprises or is as shown in SEQ ID NO: 30;

[0261] The HPRE comprises or is as shown in SEQ ID NO: 33;

[0262] The WPRE comprises or is as shown in SEQ ID NO: 34;

[0263] The 5'ITR comprises or or the sequence shown in SEQ ID NO: 26;

[0264] The 3'ITR comprises or is as shown in SEQ ID NO:31;

[0265] The MCS comprises or is as shown in SEQ ID NO:40; and / or

[0266] The P2A comprises or has a sequence as shown in SEQ ID NO:33.

[0267] In some embodiments, a nucleic acid molecule is provided, comprising a polynucleotide having at least 95% identity to a sequence set forth in any one of SEQ ID NOs: 13-25, 140-141. In some embodiments, the polynucleotide comprises a sequence set forth in any one of SEQ ID NOs: 13-25, 140-141.

[0268] In some embodiments, the AADC protein (e.g., the AADC protein shown in SEQ ID NO: 1) comprises a variant thereof (the variant is, for example, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or more identical to SEQ ID NO: 1, NP_000781.2, NP_001076440.2, NP_001229815.2, NP_001229816.2, NP_001229817.2, NP_001229818.2, NP_001229819.2) or a fragment thereof, wherein the variant or fragment has the same or similar biological activity or function as the AADC protein; the GDNF protein (e.g., the GDNF protein shown in SEQ ID NO: 2) comprises a variant thereof (the variant is, for example, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or more identical to SEQ ID NO: 1, NP_000781.2, NP_001076440.2, NP_001229815.2, NP_001229816.2, NP_001229817.2, NP_001229818.2, NP_001229819.2) or a fragment thereof. NO:2, NP_001177397.1, NP_001177398.1, NP_001265027.1, NP_954701.1 have at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or more identity) or a fragment thereof, wherein the variant or fragment has the same or similar biological activity or function as the GDNF protein.

[0269] In some embodiments, the polynucleotide comprises a precursor molecule that is processed inside the cell.AADC and / or GDNF polynucleotides or processed forms thereof can be encoded in a plasmid, vector, genome, or other polynucleotide expression vehicle for delivery to the cell.

[0270] In some embodiments, the vector comprises an intact replicon, such that it can replicate in the cell, for example, after transfection, infection, or transformation of the cell. In some embodiments, the vector is or is derived from a retrovirus, an adenovirus, a herpes virus, a baculovirus, a papillomavirus, or a modified or reconstructed version thereof. In some embodiments, methods of delivering the vector include, but are not limited to, direct delivery of naked DNA or delivery via cationic or anionic liposomes, via complexation with a cationic polymer, or via complexation with a protein or polypeptide.

[0271] In some embodiments, the polynucleotide is designed as a component of an AAV vector genome (or viral genome) and packaged into rAAV particles, which are processed intracellularly to produce AADC and / or GDNF proteins, which may be wild-type proteins or variants thereof.

[0272] In some embodiments, the polynucleotide can be the payload of the rAAV particle. The AAV capsid and AAV particle are any of the above.

[0273] In some embodiments, the AAV capsid is an AAV2 or AAV9 capsid, for example comprising the amino acid sequence shown in SEQ ID NO:41 or 42.

[0274] In some embodiments, in addition to the encoded heterologous payload, the AAV particle can comprise all or part of the vector genome of any naturally occurring and / or recombinant AAV capsid nucleotide sequence or variant.

[0275] In some embodiments, the AAV particles can be replication-defective (e.g., they lack sequences encoding functional Rep and Cap proteins in the vector genome). In some specific embodiments, the replication-defective AAV particles can lack most or all parental coding sequences and essentially carry only one or two AAV ITR sequences and a polynucleotide of interest for delivery to a cell, tissue, organ, or organism.

[0276] In some embodiments, the AAV particle can be a recombinant AAV (rAAV) particle.

[0277] In some embodiments, the AAV particles can be selected from single-stranded AAV particles (e.g., ssAAV) and self-complementary AAV particles (e.g., scAAV). By skipping second-strand synthesis, scAAV achieves rapid expression in cells.

[0278] In some embodiments, the AAV particle encapsulates a polynucleotide that is at least 95% identical to the sequence set forth in any one of SEQ ID NOs: 13-25, 140-141.

[0279] In some embodiments, the AAV particle encapsulates a polynucleotide comprising a sequence shown in any one of SEQ ID NOs: 13-25, 140-141.

[0280] Regulatory elements

[0281] The vector genome (or viral genome) of any of the aforementioned polynucleotides or AAV particles of the present disclosure comprises at least one regulatory element, which enables the replication, transcription and translation of the target gene (eg, AADC, GDNF) in the payload.

[0282] In some embodiments, the regulatory elements include, but are not limited to, sequences for transcription initiation and / or termination, promoter and / or enhancer sequences, efficient RNA processing signals (e.g., splicing and polyadenylation signals), sequences that stabilize cytoplasmic mRNA, sequences that enhance translation efficiency (e.g., Kozak consensus sequences), sequences that enhance protein stability, and / or sequences that enhance protein processing and / or secretion. Exemplary regulatory elements include, but are not limited to, promoters, enhancers, introns, endogenous miRNAs, post-transcriptional regulatory elements (PREs), polyadenylation (PolyA) signal sequences, and upstream enhancers (USEs).

[0283] In some embodiments, the regulatory elements (e.g., promoters) described below drive expression of the payload in a target tissue, e.g., the brain (e.g., striatum, substantia nigra) of a subject, for a period of time, e.g., 1, 2, 3, 4, 5, 6, 7 days, 1, 2, 3, 4 weeks, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60 years or more. For another example, 1-2 weeks, 1-3 weeks, 1-4 weeks, 1-2 months, 1-4 months, 1-6 months, 2-6 months, 3-6 months, 3-9 months, 4-8 months, 6-12 months, 1-2 years, 1-5 years, 2-5 years, 3-6 years, 3-8 years, 4-8 years, 5-10 years, 10-20 years, 10-30 years, 20-40 years, 20-50 years, or the lifetime of the subject.

[0284] Inverted terminal repeats (ITRs)

[0285] The vector genome (or viral genome) of the polynucleotide or AAV particle of any of the aforementioned disclosures comprises at least one ITR, for example, has two ITRs, flanking the payload at the 5' and 3' ends, respectively.

[0286] In some embodiments, the ITR functions as an origin of replication.

[0287] In some embodiments, the ITRs comprise sequence regions that are complementary and symmetrically arranged.

[0288] In some embodiments, the ITRs may consist of naturally occurring polynucleotide sequences or recombinantly derived polynucleotide sequences.

[0289] In some embodiments, the ITR can be derived from the same or different serotype as the capsid. In some embodiments, the 5' ITR and 3' ITR can be derived from the same serotype or from different serotypes. For example, the 5' ITR and 3' ITR are both derived from AAV2, or both are derived from AAV9. For another example, the 5' ITR comprises the sequence shown in SEQ ID NO: 26, and / or the 3' ITR comprises the nucleotide sequence shown in SEQ ID NO: 31.

[0290] In some embodiments, the length of each ITR can be about 100 to about 150 nucleotides, for example, 100-105 nucleotides, 106-110 nucleotides, 111-115 nucleotides, 116-120 nucleotides, 121-125 nucleotides, 126-130 nucleotides, 131-135 nucleotides, 136-140 nucleotides, 141-145 nucleotides, or 146-150 nucleotides. In one embodiment, the length of the ITR is 140-142 nucleotides, for example, 141 nucleotides. Non-limiting examples of ITR lengths are 102, 140, 141, 142, 145 nucleotides in length, and nucleotides that are at least 95% identical thereto.

[0291] promoter

[0292] The vector genome (or viral genome) of any of the aforementioned polynucleotides or AAV particles of the present disclosure comprises at least one promoter, including but not limited to species-specific, inducible, tissue-specific or cell cycle-specific promoters.

[0293] In some embodiments, the promoter drives expression of a protein or polypeptide (eg, AADC and / or GDNF) encoded in the payload of the vector genome of the AAV particle.

[0294] In some embodiments, the promoter is specific or tropic for the target tissue, for example, a promoter capable of expressing the payload in neural tissue.

[0295] In some embodiments, the promoter can be a viral promoter, a plant promoter, a mammalian promoter, or a human promoter.

[0296] In some embodiments, the promoter includes but is not limited to CMV, CBA (including derivatives CAG, CBh, etc.), EF-1α, PGK, UBC, RSV, EFS, EF1, GUSB (hGBp), UCOE (promoter of HNRPA2B1-CBX3), NSE, Synapsin, MeCP2, MeP418, MeP426, VMD2, MRHO, TRE, Ac5, Polyhedrin, CaMKIIa, Gall, TEF1, GDS, ADH1, Ubi, GFAP or PKG promoter, and can be selected from neurofilament light (NFL) promoter, neurofilament heavy (NFH) promoter, SCN8A promoter, frataxin (FXN) promoter (or FRDA promoter), H1 promoter, RNA pol III promoter (e.g., U6 or H1), small nuclear RNA (ULB or ULA) promoter. In some embodiments, the promoter is a liver promoter, such as human alpha-1-antitrypsin (hAAT) and thyroxine-binding globulin (TBG), or a skeletal muscle promoter, such as desmin, MCK, or synthetic C5-12.

[0297] In some embodiments, the AAV vector genome of the present disclosure comprises two promoters, for example, CMV and CBA promoters, whose sequences are shown in SEQ ID NOs: 35 and 28, respectively; another example is EF1α promoter and CMV promoter, or any combination of any two of the aforementioned promoters.

[0298] In some embodiments, the promoter is a tissue-specific expression element that can restrict expression to certain cell types, including but not limited to a muscle-specific promoter, a B cell promoter, a monocyte promoter, a leukocyte promoter, a macrophage promoter, a pancreatic acinar cell promoter, an endothelial cell promoter, a lung tissue promoter, an astrocyte promoter, or a nervous system promoter, which can be used to restrict expression to neurons, astrocytes, or oligodendrocytes.

[0299] In some embodiments, the promoter is a neuronal tissue-specific expression element, including but not limited to neuron-specific enolase (NSE), platelet-derived growth factor (PDGF), platelet-derived growth factor B chain (PDGF-β), synaptophysin (Syn), methyl CpG binding protein 2 (MeCP2), Ca 2+ / Calmodulin-dependent protein kinase II (CaMKII), metabotropic glutamate receptor 2 (mGluR2), neurofilament light (NFL) or heavy (NFH), β-globin minigene nβ2, preproenkephalin (PPE), enkephalin (Enk) and excitatory amino acid transporter 2 (EAAT2) promoters. Non-limiting examples of tissue-specific expression elements for astrocytes include glial fibrillary acidic protein (GFAP) and EAAT2 promoters. Non-limiting examples of tissue-specific expression elements for oligodendrocytes include myelin basic protein (MBP) promoter.

[0300] In some embodiments, the promoter is a truncated or variant of the aforementioned promoter, and is less than 1 kb in length, for example, 200-300, 200-400, 300-400, 200-500, 200-600 or more in length.

[0301] In some embodiments, the promoter can be a combination of two or more components of the same or different starting or parental promoters, such as CMV and CBA.

[0302] enhancer

[0303] The vector genome (or viral genome) of any of the aforementioned polynucleotides or AAV particles of the present disclosure comprises at least one enhancer, including but not limited to species-specific, inducible, tissue-specific or cell cycle-specific enhancers.

[0304] In some embodiments, the enhancer is specific or tropic for the target tissue, for example, an enhancer that regulates expression of the payload in neural tissue.

[0305] In some embodiments, the enhancer can be or be derived from a viral enhancer, a plant enhancer, a mammalian enhancer, or a human enhancer.

[0306] In some embodiments, the enhancer can be located upstream or downstream of the promoter and operably linked to the promoter. When the enhancer is present, the expression of the target gene (e.g., AADC and / or GDNF) is enhanced by at least 20%, at least 50%, at least 80%, at least 100%, or at least several times (including 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 times or more) compared to when the enhancer is not present.

[0307] In some embodiments, the enhancer includes but is not limited to EF-1α, Ubc, human b-actin, CAG, TRE, Ac5, Polyhedrin, CaMKIIa, Gall, TEF1, GDS, ADH1, Ubi and α-1-antitrypsin (hAAT), or variants or fragments thereof. In some embodiments, the enhancer is selected from IRBP, RSV or CMV enhancers, or variants or fragments thereof.

[0308] In some embodiments, the enhancer comprises or is the enhancer shown in SEQ ID NO: 27 or 37, or a variant or fragment thereof.

[0309] introns

[0310] The vector genome (or viral genome) of any of the aforementioned polynucleotides or AAV particles of the present disclosure may contain an intron or a portion thereof.

[0311] In some embodiments, the introns include but are not limited to MVM (67-97 bp), F.IX truncated intron 1 (about 300 bp), β-globin SD / immunoglobulin heavy chain splice acceptor (about 250 bp), adenovirus splice donor / immunoglobulin splice acceptor (about 500 bp), SV40 late splice donor / splice acceptor (19S / 16S) (180 bp) and hybrid adenovirus splice donor / IgG splice acceptor (about 230 bp), hemoglobin intron, hybrid intron, β-globin intron, or variants or fragments of the above introns.

[0312] In some embodiments, the intron has a length of 100-800 nucleotides, such as about 100, about 200, about 300, about 400, about 500, about 600 nucleotides, or 200-250, 200-300, 100-200, 300-500, 500-600, 400-600, or 100-200 nucleotides.

[0313] In some embodiments, the intron in the technical solution of the present disclosure is selected from hybrid intron and β-globin intron, and their sequences are shown in SEQ ID NO: 29 and 38, respectively.

[0314] Untranslated region (UTR)

[0315] The polynucleotide of any of the foregoing or the vector genome (or viral genome) of the AAV particles of the present disclosure may include an untranslated region (UTR), and the UTR is selected from 5'UTR and / or 3'UTR to regulate (e.g., increase, decrease or reduce) polynucleotide stability and protein production. Typically, the 5'UTR starts at the transcription start site and ends at the start codon, and the 3'UTR starts immediately after the stop codon and ends at the transcription termination signal. The UTR may be a wild type, a variant thereof or an artificial UTR.

[0316] In some embodiments, the 5'UTR includes a Kozak sequence. In other embodiments, the 5'UTR does not include a Kozak sequence.

[0317] In some embodiments, the 3'UTR is AU-rich. In some embodiments, the 3'UTR is selected from: Class I AREs, such as but not limited to c-Myc and MyoD, which contain several dispersed copies of the AUUUA motif within the U-rich region; Class II AREs, such as but not limited to GM-CSF and TNF-a, which have two or more overlapping UUAUUUA(U / A)(U / A) nonamers; Class III ARES, such as but not limited to c-Jun and myogenin

[0318] In some embodiments, the 3'UTR may include an oligo(dT) sequence for templated addition of a polyadenylation sequence (PolyA).

[0319] Polyadenylation sequence (PolyA)

[0320] The polynucleotide of any of the aforementioned items of the present disclosure or the vector genome (or viral genome) of the AAV particle may contain a sequence encoding PolyA, and the PolyA may be a wild type, a variant thereof, or a modified PolyA for protein translation.

[0321] In some embodiments, the sequence encoding Poly A is located between the 3' end of the payload encoding sequence and the 5' end of the 3' ITR.

[0322] In some embodiments, the sequence encoding Poly A is 0-500 nucleotides in length, for example, about 75, about 100, about 110, about 120, about 150, about 160, about 200, or about 300 nucleotides, or for example, 50-100, 50-150, 50-160, 50-200, 60-100, 60-150, 60-160, 60-200, 70-100, 70-150, 70-160, 70-200, 80-100, 80-150, 80-160, 80-200, 90-100, 90-150, 90-160, or 90-200.

[0323] In some embodiments, the poly A sequence may encode a continuous poly A or a discontinuous poly A. When encoding a discontinuous poly A, the coding sequence may be interrupted or separated by other nucleotides. For example, the poly A sequence may have at least two segments of 60 adenylate residues, and the at least two segments of 60 adenylate residues are separated by a sequence comprising 10-90 nucleotides.

[0324] In some embodiments, the sequence encoding PolyA or polyA in the technical solutions of the present disclosure is selected from β-globin polyA, SV40 polyA, bGH polyA, PA75 polyA, MeCP2 polyA, RDH1 polyA, BGH polyA, SPA49polyA, sNRP-TK65 polyA, sNRP polyA, TK65 polyA, or variants or fragments of the above PolyA.

[0325] In some embodiments, the polyA or variants or fragments thereof are shown in SEQ ID NO: 32 and 36 in the technical solutions of the present disclosure.

[0326] The polyA or coding sequences thereof in WO2016005324, WO2016005004, WO2016091391, WO2019036513, and WO2020074642 are incorporated herein in their entirety, and can all be used in the technical solutions disclosed herein.

[0327] Filling sequence

[0328] The vector genome (or viral genome) of any of the polynucleotides or AAV particles disclosed herein may comprise a stuffer sequence such that the length of the vector genome is the optimal size for packaging, for example, such that the length of the vector genome is about 2.3 kb, about 4.6 kb, about 4.7 kb, or about 5.1 kb.

[0329] In some embodiments, the vector genome is a single-stranded or double-stranded genome, and the packaged AAV particles are ssAAV or scAAV.

[0330] In some embodiments, a vector genome may contain one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) stuffer sequences.

[0331] In some embodiments, the stuffer sequence can be located between or within multiple regulatory elements, such as between two introns, 3' to the 5'ITR sequence, 5' to the 5'ITR sequence, 5' to the 3'ITR sequence, 3' to the 3'ITR sequence, and before or after regions such as promoters, introns, enhancers, polyA, multiple cloning site (MCS) regions, exons, etc.

[0332] Methods for producing or preparing rAAV particles

[0333] The present disclosure provides methods for producing, preparing and / or modifying rAAV particles. The rAAV particle production, preparation and / or modification methods in WO200028004, WO200123001, WO2004112727, WO 2005005610, WO2005072364, WO2013123503, WO2015191508 and US20130195801 are introduced herein in full. The rAAV particles can have the characteristics of enhancing delivery efficiency, can be effectively packaged, and can successfully infect target cells (e.g., mammalian or human cells) with high frequency and minimal toxicity.

[0334] In some embodiments, a method for producing rAAV particles is provided, comprising packaging any polynucleotide or vector genome (or viral genome) or vector disclosed herein into an AAV capsid. In some specific embodiments, the method comprises the following steps: 1) co-transfecting competent bacterial cells with a baculovirus vector and a viral construct vector and / or an AAV payload construct vector, 2) isolating the resulting viral construct expression vector and the AAV payload construct expression vector and transfecting viral replicating cells separately, 3) isolating and purifying the resulting payload and viral construct particles comprising the viral construct expression vector or the AAV payload construct expression vector, 4) co-infecting viral replicating cells with both the AAV payload and the viral construct particles comprising the viral construct expression vector or the AAV payload construct expression vector, and 5) harvesting and purifying the AAV particles comprising the viral genome.

[0335] In some embodiments, a method for producing rAAV particles is provided, comprising the steps of:

[0336] 1) co-transfecting mammalian cells (e.g., HEK293 cells) with any polynucleotide or vector genome (or viral genome) or vector disclosed herein, and constructs expressing Rep and Cap genes and helper constructs (to achieve helper functions);

[0337] 2) Harvest and purify rAAV particles containing the viral genome.

[0338] In some embodiments, the viral genome of the aforementioned rAAV particles optionally encodes a selection marker. The selection marker can include a cell surface marker, such as any protein expressed on the cell surface, including but not limited to a receptor CD marker, a lectin, an integrin, or a truncated form thereof.

[0339] In some embodiments, an AAV production system is provided for producing rAAV particles of the present disclosure, wherein the production system comprises:

[0340] 1) A polynucleotide sequence encoding the AAV capsid;

[0341] 2) any polynucleotide or vector genome (or viral genome) or vector disclosed herein; and

[0342] 3) Sufficient AAV rep function and helper functions to allow packaging of the polynucleotide or vector genome (or viral genome) or vector in (b) into AAV capsids.

[0343] In the aforementioned embodiment, the AAV capsid is selected from any of the AAV capsids disclosed herein, such as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV9.47, AAV9(hu14), AAV10, AAV11, AAV12, AAVrh8, AAVrh10, AAV-DJ or AAV-DJ8.

[0344] In some embodiments, sufficient AAV rep and helper functions are provided by packaging cells, which may contain three plasmids: pHelper, pRC9, and pGOI.

[0345] In some embodiments, the Rep gene encodes a nonstructural protein that regulates functions, such as replication of the AAV genome, and can be selected from Rep78, Rep68, Rep52, and Rep40. Rep78 and Rep68 are typically transcribed from the p5 promoter, while Rep52 and Rep40 are typically transcribed from the p19 promoter. The Cap gene encodes the structural proteins VP1, VP2, and / or VP3 that assemble to form the viral capsid. The Cap gene is typically transcribed from the p40 promoter.

[0346] In some embodiments, rAAV particles produced by the above-described AAV production system are provided.

[0347] Nucleic Acid Molecule II

[0348] The present disclosure provides a VEGF-binding molecule, which comprises the immunoglobulin-like domain 2 (R2D2) of VEGFR2 and / or the immunoglobulin-like domain 3 (R2D3) of VEGFR2; or, the VEGF-binding molecule consists of R2D2 and / or R2D3.

[0349] In some embodiments, a VEGF-binding molecule is provided, comprising: a first VEGF-binding domain and a second VEGF-binding domain, wherein the first VEGF-binding domain comprises the immunoglobulin-like domain 2 (R2D2) of VEGFR2 and the immunoglobulin-like domain 3 (R2D3) of VEGFR2, or consists of R2D2-R2D3; and the second VEGF-binding domain comprises VEGF Trap or an anti-VEGF antibody or an antigen-binding fragment thereof, or consists of VEGF Trap or an anti-VEGF antibody or an antigen-binding fragment thereof.

[0350] In some embodiments, the VEGF Trap in the second VEGF binding domain is the immunoglobulin-like domain 2 (R1D2) of VEGFR1 and / or the immunoglobulin-like domain 3 (R2D3) of VEGFR2.

[0351] In some embodiments, the anti-VEGF antibody in the second VEGF binding domain is bevacizumab or ranibizumab.

[0352] In some embodiments, a VEGF-binding molecule is provided, comprising:

[0353] a first VEGF-binding domain and a second VEGF-binding domain, wherein the first VEGF-binding domain comprises or consists of R2D2-R2D3;

[0354] The second VEGF binding domain comprises R1D2-R2D3 or consists of R1D2-R2D3, or comprises the heavy chain variable region (VH) and light chain variable region (VL) of an anti-VEGF antibody. Exemplarily, the anti-VEGF antibody is bevacizumab or ranibizumab.

[0355] As a non-limiting illustration, R2D2-R2D3 represents a VEGF binding domain formed by R2D2 linked to R2D3; wherein R2D2 and R2D3 may be directly linked or indirectly linked (eg, linked via a linker).

[0356] In some embodiments, a VEGF-binding molecule is provided, comprising one or more selected from R1D2, R2D2, and R2D3. For example, the molecule comprises one R1D2, one R2D2, and two R2D3s, or consists of one R1D2, one R2D2, and two R2D3s. For example, the molecule comprises one R2D2 and one R2D3, or consists of one R2D2 and one R2D3.

[0357] In some embodiments, the VEGF-binding molecule comprises amino acid mutations that improve its stability.

[0358] In some embodiments, the VEGF-binding molecule comprises amino acid mutations that improve its affinity for the ligand.

[0359] In some embodiments, the VEGF-binding molecule comprises amino acid mutations that improve its affinity for the ligands VEGF-A and / or VEGF-C.

[0360] In some embodiments, the VEGF-binding molecule comprises both amino acid mutations that improve its stability and amino acid mutations that improve its affinity for the ligands VEGF-A and / or VEGF-C.

[0361] In a first aspect, in some embodiments, R2D2 and / or R2D3 of the VEGF binding molecule comprises an amino acid mutation at any one or more positions selected from the group consisting of: position 162, position 221, position 222, position 274, position 276, position 277, position 280, position 284, position 288, position 289, and position 313.

[0362] The sites of the above-mentioned amino acid mutations are sites in the natural sequence relative to the amino acid sequence shown in SEQ ID NO: 51.

[0363] In some embodiments, the R2D2 and / or R2D3 comprise amino acid mutations at positions selected from any one or more of the following groups:

[0364] -162nd;

[0365] - 162nd and 221st;

[0366] - 162nd and 222nd;

[0367] - 162nd and 274th;

[0368] - 162nd and 276th;

[0369] - 162nd and 277th;

[0370] - 162nd and 289th;

[0371] - 162nd and 313th;

[0372] - 221st and 222nd;

[0373] - 277th and 289th;

[0374] - 162nd, 221st and 222nd;

[0375] - 162nd, 277th and 289th;

[0376] -162nd, 221st, 222nd and 274th.

[0377] In some embodiments, the R2D2 and / or R2D3 comprises an amino acid mutation at a position selected from any one of the following groups:

[0378] -162nd;

[0379] -162nd, 221st, 222nd and 274th.

[0380] In some embodiments, the R2D2 and / or R2D3 comprise any one or more groups of amino acid mutations selected from the following:

[0381] -162A, 162V, 162S, 162I, or 162L;

[0382] -221S or 221N;

[0383] -222K;

[0384] -274F or 274I;

[0385] -276Q;

[0386] -277R;

[0387] -280E;

[0388] -284H;

[0389] -288Y;

[0390] -289Y;

[0391] -313R.

[0392] In some embodiments, the R2D2 and / or R2D3 comprise any one or more groups of amino acid mutations selected from the following:

[0393] -C162A, C162V, C162S, C162I, or C162L;

[0394] -Y221S or Y221N;

[0395] -R222K;

[0396] -N274F or N274I;

[0397] -D276Q;

[0398] -L277R;

[0399] -Q280E;

[0400] -E284H;

[0401] -F288Y;

[0402] -L289Y;

[0403] -L313R.

[0404] In some embodiments, the R2D2 and / or R2D3 comprise any one or more groups of amino acid mutations selected from the following:

[0405] -162V or 162I;

[0406] -162V / 274F; 162V / 274I; 162I / 274F; or 162I / 274I;

[0407] -221S / 222K;

[0408] -277R / 289Y;

[0409] -162V / 221S / 222K;

[0410] -162V / 276Q;

[0411] -162V / 289Y;

[0412] -162V / 277R / 289Y;

[0413] -162V / 313R;

[0414] -162V / 221S / 222K / 274F.

[0415] In some embodiments, the R2D2 and / or R2D3 comprise any one or more groups of amino acid mutations selected from the following:

[0416] -C162V or C162I;

[0417] -C162V / N274F; C162V / N274I; C162I / N274F; or C162I / N274I;

[0418] -Y221S / R222K;

[0419] -L277R / L289Y;

[0420] -C162V / Y221S / R222K;

[0421] -C162V / D276Q;

[0422] -C162V / L289Y;

[0423] -C162V / L277R / L289Y;

[0424] -C162V / L313R;

[0425] -C162V / Y221S / R222K / N274F.

[0426] In some embodiments, the R2D2 and / or R2D3 comprise any one or more groups of amino acid mutations selected from the following:

[0427] -162V;

[0428] -162V / 221S / 222K / 274F.

[0429] In some embodiments, the R2D2 and / or R2D3 comprise any one or more groups of amino acid mutations selected from the following:

[0430] -C162V;

[0431] -C162V / Y221S / R222K / N274F.

[0432] In some embodiments, the VEGF-binding molecules comprising the aforementioned amino acid mutations have improved stability relative to a wild-type molecule of the same molecular form.

[0433] In a second aspect, in some embodiments, R2D2 and / or R2D3 of the VEGF binding molecule comprises an amino acid mutation at one or more positions selected from the group consisting of: 135, 197, 199, 213, 215, 219, 257, 275, 277, 278, 283, 289, 313, and 314.

[0434] The sites of the above-mentioned amino acid mutations are sites in the natural sequence relative to the amino acid sequence shown in SEQ ID NO: 51.

[0435] In some embodiments, the R2D2 and / or R2D3 comprises an amino acid mutation at position 283.

[0436] In some embodiments, the R2D2 and / or R2D3 comprise any one or more groups of amino acid mutations selected from the following:

[0437] -135Y;

[0438] -197Y;

[0439] -199Q or 199T;

[0440] -213N;

[0441] -215F or 215H;

[0442] -219I;

[0443] -257Q or 257T;

[0444] -275L or 275E;

[0445] -277R;

[0446] -278D, 278S, or 278E;

[0447] -283T;

[0448] -289Y;

[0449] -313Y;

[0450] -314S or 314Q.

[0451] In some embodiments, the R2D2 and / or R2D3 comprise any one or more groups of amino acid mutations selected from the following:

[0452] -V135Y;

[0453] -M197Y;

[0454] -F199Q or F199T;

[0455] -M213N;

[0456] -I215F or I215H;

[0457] -V219I;

[0458] -D257Q or D257T;

[0459] -R275L or R275E;

[0460] -L277R;

[0461] -K278D, K278S, or K278E;

[0462] -S283T;

[0463] -L289Y;

[0464] -L313Y;

[0465] -M314S or M314Q.

[0466] In some embodiments, the R2D2 and / or R2D3 comprises 283T.

[0467] In some embodiments, the R2D2 and / or R2D3 comprises the amino acid mutation S283T.

[0468] In some embodiments, the VEGF-binding molecules comprising the aforementioned amino acid mutations have improved ligand binding activity relative to the wild-type of the same molecular form.

[0469] In a third aspect, in some embodiments, R2D2 and / or R2D3 of the VEGF-binding molecule comprises an amino acid mutation at any one or more positions selected from the group consisting of: position 162, position 221, position 222, position 274, position 276, position 277, position 280, position 284, position 288, position 289, position 313; and

[0470] The invention also comprises an amino acid mutation at any one or more positions selected from the group consisting of: position 135, position 197, position 199, position 213, position 215, position 219, position 257, position 275, position 277, position 278, position 283, position 289, position 313, and position 314.

[0471] In some embodiments, R2D2 and / or R2D3 of the VEGF binding molecule comprises an amino acid mutation at any position selected from the group consisting of:

[0472] - 162nd and 275th;

[0473] - 162nd and 283rd;

[0474] - 162nd and 313th;

[0475] - 162nd, 274th and 283rd;

[0476] - 162nd, 221st, 222nd and 283rd;

[0477] -162nd, 221st, 222nd, 274th and 283rd.

[0478] In some embodiments, the R2D2 and / or R2D3 comprises an amino acid mutation at any position selected from the group consisting of:

[0479] - 162nd and 283rd;

[0480] -162nd, 221st, 222nd, 274th and 283rd.

[0481] In some embodiments, the R2D2 and / or R2D3 comprises:

[0482] (i) any one or more groups of amino acid mutations selected from the following:

[0483] -162A, 162V, 162S, 162I, or 162L;

[0484] -221S;

[0485] -222K;

[0486] -274F or 274I;

[0487] -276Q;

[0488] -277R;

[0489] -280E;

[0490] -284H;

[0491] -288Y;

[0492] -289Y;

[0493] -313R; and

[0494] (ii) any one or more groups of amino acid mutations selected from the following:

[0495] -135Y;

[0496] -197Y;

[0497] -199Q or 199T;

[0498] -213N;

[0499] -215F or 215H;

[0500] -219I;

[0501] -257Q or 257T;

[0502] -275L or 275E;

[0503] -277R;

[0504] -278D, 278S, or 278E;

[0505] -283T;

[0506] -289Y;

[0507] -313Y;

[0508] -314S or 314Q.

[0509] In some embodiments, the R2D2 and / or R2D3 comprises:

[0510] (i) any one or more groups of amino acid mutations selected from the following:

[0511] -C162A, C162V, C162S, C162I, or C162L;

[0512] -Y221S;

[0513] -R222K;

[0514] -N274F or N274I;

[0515] -D276Q;

[0516] -L277R;

[0517] -Q280E;

[0518] -E284H;

[0519] -F288Y;

[0520] -L289Y;

[0521] -L313R; and

[0522] (ii) any one or more groups of amino acid mutations selected from the following:

[0523] -V135Y;

[0524] -M197Y;

[0525] -F199Q or F199T;

[0526] -M213N;

[0527] -I215F or I215H;

[0528] -V219I;

[0529] -D257Q or D257T;

[0530] -R275L or R275E;

[0531] -L277R;

[0532] -K278D, K278S, or K278E;

[0533] -S283T;

[0534] -L289Y;

[0535] -L313Y;

[0536] -M314S or M314Q.

[0537] In some embodiments, the R2D2 and / or R2D3 comprise amino acid mutations selected from any one or more of the following groups:

[0538] -162V / 275L;

[0539] -162V / 283T;

[0540] -162V / 313Y;

[0541] -162V / 274F / 283T;

[0542] -162V / 221S / 222K / 283T;

[0543] -162V / 221S / 222K / 274F / 283T.

[0544] In some embodiments, the R2D2 and / or R2D3 comprise amino acid mutations selected from any one or more of the following groups:

[0545] -C162V / R275L;

[0546] -C162V / S283T;

[0547] -C162V / L313Y;

[0548] -C162V / N274F / S283T;

[0549] -C162V / Y221S / R222K / S283T;

[0550] -C162V / Y221S / R222K / N274F / S283T.

[0551] In some embodiments, the R2D2 and / or R2D3 comprise amino acid mutations selected from any one or more of the following groups:

[0552] -162V / 283T;

[0553] -162V / 221S / 222K / 274F / 283T.

[0554] In some embodiments, the R2D2 and / or R2D3 comprise amino acid mutations selected from any one or more of the following groups:

[0555] -C162V / S283T;

[0556] -C162V / Y221S / R222K / N274F / S283T.

[0557] In the context of the present disclosure, “ / ” indicates that the amino acid mutations exist simultaneously in the same VEGF-binding molecule.

[0558] In the context of this disclosure, ordinal numbers such as "first" and "second" cannot be understood as limiting the quantity, level or order of technical features; they are only used to distinguish different technical features, steps, and elements.

[0559] In the above schemes, the mutations in R2D2 and / or R2D3 of the VEGF-binding molecule may contain or consist solely of the above mutations.

[0560] In some embodiments, R2D2 and / or R2D3 of VEGFR2 are wild-type or functional variants having 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% sequence identity to wild-type.

[0561] In some embodiments, the immunoglobulin-like domain 2 and the immunoglobulin-like domain 3 of wild-type VEGFR2 have the same amino acid sequence as R2D2 and R2D3 in the sequence shown in SEQ ID NO:51.

[0562] In some embodiments, the immunoglobulin-like domain 2 (R2D2) and the immunoglobulin-like domain 3 (R2D3) of VEGFR2 contain one or more amino acid differences compared to the wild type (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid differences); the amino acid differences include amino acid substitutions, insertions or deletions.

[0563] In some embodiments, the amino acid mutations are relative to the immunoglobulin-like domain 2 and immunoglobulin-like domain 3 of wild-type VEGFR2, wherein the amino acid sequence of wild-type VEGFR2 is shown in SEQ ID NO: 51.

[0564] In some embodiments, the immunoglobulin-like domain 2 and immunoglobulin-like domain 3 of wild-type VEGFR2 have the amino acid sequence shown in positions 123-327 of SEQ ID NO: 51.

[0565] In some embodiments, R2D2-R2D3 has the amino acid sequence shown in positions 123-327 of SEQ ID NO:51.

[0566] In some embodiments, the immunoglobulin-like domain 2 (R2D2) of wild-type VEGFR2 has the amino acid sequence shown in SEQ ID NO: 51, positions 123-225 (shown as single underlines), and the immunoglobulin-like domain 3 (R2D3) of wild-type VEGFR2 has the amino acid sequence shown in SEQ ID NO: 51, positions 226-327 (shown as double underlines).

[0567] In the context of the present disclosure, the amino acid position numbers associated with R2D2 and R2D3 all use the natural numbering of the amino acid sequence shown in SEQ ID NO: 51, i.e., counting starts from methionine (M) at position 1 of SEQ ID NO: 51. The amino acid sequence of wild-type VEGFR2 is:

[0568] In some embodiments, the first VEGF binding domain consists of R2D2-R2D3.

[0569] In some embodiments, the first VEGF binding domain has VEGF-C binding ability. In some embodiments, the first VEGF binding domain has VEGF-C binding ability, and VEGF-A binding ability.

[0570] In some embodiments, the VEGF-binding molecule has increased stability and / or ligand binding activity.

[0571] In some embodiments, the second VEGF binding domain has the ability to bind VEGF-A.

[0572] In some embodiments, the second VEGF-binding domain has VEGF-A binding ability and has no or weak VEGF-C binding ability.

[0573] In some embodiments, the second VEGF binding domain comprises or consists of VEGF Trap.

[0574] In some embodiments, the VEGF Trap comprises the immunoglobulin-like domain 2 of VEGFR1 (R1D2) and the immunoglobulin-like domain 3 of VEGFR2 (R2D3).

[0575] In some embodiments, the immunoglobulin-like domain 2 (R1D2) of VEGFR1 is wild-type or a functional variant of R1D2 having 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% sequence identity to the wild-type.

[0576] In some embodiments, wild-type R1D2 has the same amino acid sequence as the immunoglobulin-like domain 2 in wild-type VEGFR1 as shown in SEQ ID NO:52.

[0577] In some embodiments, the wild-type R1D2 has the amino acid sequence shown in positions 129-231 of SEQ ID NO:52.

[0578] In some embodiments, the R1D2 functional variant comprises one or more amino acid differences compared to the wild type (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid differences); the amino acid differences include amino acid substitutions, insertions or deletions.

[0579] In the context of the present disclosure, the amino acid position numbers related to R1D2 all adopt the natural numbering of the amino acid sequence shown in SEQ ID NO: 52, that is, counting starts from methionine (M) at position 1 of SEQ ID NO: 52.

[0580] Wild-type VEGFR1 amino acid sequence:

[0581] In some embodiments, the VEGF Trap is aflibercept or conbercept. In some embodiments, the VEGF Trap is the VEGFR portion of aflibercept or the VEGFR portion of conbercept.

[0582] In some embodiments, the anti-VEGF-A antibody or antigen-binding fragment thereof is a scFv, Fv, Fab, or Fab' fragment.

[0583] In some embodiments, the anti-VEGF-A antibody is bevacizumab or ranibizumab. In some embodiments, the anti-VEGF-A antibody is a Fab of bevacizumab.

[0584] The present disclosure also provides a VEGF-binding molecule comprising an immunoglobulin-like domain 2 (R2D2) of VEGFR2 and an immunoglobulin-like domain 3 (R2D3) of VEGFR2, wherein the immunoglobulin-like domain 2 (R2D2) of VEGFR2 and / or the immunoglobulin-like domain 3 (R2D3) of VEGFR2 comprise an amino acid mutation at any one or more positions selected from the group consisting of position 162, position 221, position 222, position 274, position 276, position 277, position 280, position 284, position 288, position 289, and position 313. The positions of the amino acid mutations are positions in the natural sequence relative to the amino acid sequence shown in SEQ ID NO: 51.

[0585] In some embodiments, the R2D2 and / or R2D3 comprise amino acid mutations at any one or more positions selected from the group consisting of:

[0586] -162nd;

[0587] - 162nd and 221st;

[0588] - 162nd and 222nd;

[0589] - 162nd and 274th;

[0590] - 162nd and 276th;

[0591] - 162nd and 277th;

[0592] - 162nd and 289th;

[0593] - 162nd and 313th;

[0594] - 221st and 222nd;

[0595] - 277th and 289th;

[0596] - 162nd, 221st and 222nd;

[0597] - 162nd, 277th and 289th;

[0598] -162nd, 221st, 222nd and 274th.

[0599] In some embodiments, the R2D2 and / or R2D3 comprises an amino acid mutation at any position selected from the group consisting of:

[0600] -162nd;

[0601] -162nd, 221st, 222nd and 274th.

[0602] In some embodiments, the R2D2 and / or R2D3 comprise any one or more groups of amino acid mutations selected from the following:

[0603] -162A, 162V, 162S, 162I, or 162L;

[0604] -221S or 221N;

[0605] -222K;

[0606] -274F or 274I;

[0607] -276Q;

[0608] -277R;

[0609] -280E;

[0610] -284H;

[0611] -288Y;

[0612] -289Y;

[0613] -313R.

[0614] In some embodiments, the R2D2 and / or R2D3 comprise any one or more groups of amino acid mutations selected from the following:

[0615] -C162A, C162V, C162S, C162I, or C162L;

[0616] -Y221S;

[0617] -R222K;

[0618] -N274F or N274I;

[0619] -D276Q;

[0620] -L277R;

[0621] -Q280E;

[0622] -E284H;

[0623] -F288Y;

[0624] -L289Y;

[0625] -L313R.

[0626] In some embodiments, the R2D2 and / or R2D3 comprise any one or more groups of amino acid mutations selected from the following:

[0627] -162V or 162I;

[0628] -162V / 274F; 162V / 274I; 162I / 274F; or 162I / 274I;

[0629] -221S / 222K;

[0630] -277R / 289Y;

[0631] -162V / 221S / 222K;

[0632] -162V / 276Q;

[0633] -162V / 289Y;

[0634] -162V / 277 / 289Y;

[0635] -162V / 313R;

[0636] -162V / 221S / 222K / 274F.

[0637] In some embodiments, the R2D2 and / or R2D3 comprise any one or more groups of amino acid mutations selected from the following:

[0638] -C162V or C162I;

[0639] -C162V / N274F; C162V / N274I; C162I / N274F; or C162I / N274I;

[0640] -Y221S / R222K;

[0641] -L277R / L289Y;

[0642] -C162V / Y221S / R222K;

[0643] -C162V / D276Q;

[0644] -C162V / L289Y;

[0645] -C162V / L277R / L289Y;

[0646] -C162V / L313R;

[0647] -C162V / Y221S / R222K / N274F.

[0648] In some embodiments, the R2D2 and / or R2D3 comprise any one or more groups of amino acid mutations selected from the following:

[0649] -162V;

[0650] -162V / 221S / 222K / 274F.

[0651] In some embodiments, the R2D2 and / or R2D3 comprise any one or more groups of amino acid mutations selected from the following:

[0652] -C162V;

[0653] -C162V / Y221S / R222K / N274F.

[0654] In some embodiments, the R2D2 and / or R2D3 comprises an amino acid mutation at any one or more positions selected from the group consisting of: position 135, position 197, position 199, position 213, position 215, position 219, position 257, position 275, position 277, position 278, position 283, position 289, position 313, and position 314. The positions of the amino acid mutations are positions in the natural sequence relative to the amino acid sequence of SEQ ID NO: 51.

[0655] In some embodiments, the R2D2 and / or R2D3 comprises an amino acid mutation at position 283.

[0656] In some embodiments, the R2D2 and / or R2D3 comprise any one or more groups of amino acid mutations selected from the following:

[0657] -135Y;

[0658] -197Y;

[0659] -199Q or 199T;

[0660] -213N;

[0661] -215F or 215H;

[0662] -219I;

[0663] -257Q or 257T;

[0664] -275L or 275E;

[0665] -277R;

[0666] -278D, 278S, or 278E;

[0667] -283T;

[0668] -289Y;

[0669] -313Y;

[0670] -314S or 314Q.

[0671] In some embodiments, the R2D2 and / or R2D3 comprise any one or more groups of amino acid mutations selected from the following:

[0672] -V135Y;

[0673] -M197Y;

[0674] -F199Q or F199T;

[0675] -M213N;

[0676] -I215F or I215H;

[0677] -V219I;

[0678] -D257Q or D257T;

[0679] -R275L or R275E;

[0680] -L277R;

[0681] -K278D, K278S, or K278E;

[0682] -S283T;

[0683] -L289Y;

[0684] -L313Y;

[0685] -M314S or M314Q.

[0686] In some embodiments, the R2D2 and / or R2D3 comprises the amino acid mutation 283T.

[0687] In some embodiments, the R2D2 and / or R2D3 comprises the amino acid mutation S283T.

[0688] In some embodiments, the R2D2 and / or R2D3 comprises an amino acid mutation at any one or more positions selected from the group consisting of: 162, 221, 222, 274, 276, 277, 280, 284, 288, 289, 313; and

[0689] The invention also comprises an amino acid mutation at any one or more positions selected from the group consisting of: position 135, position 197, position 199, position 213, position 215, position 219, position 257, position 275, position 277, position 278, position 283, position 289, position 313, and position 314.

[0690] In some embodiments, the R2D2 and / or R2D3 comprise amino acid mutations at any one or more positions selected from the group consisting of:

[0691] - 162nd and 275th;

[0692] - 162nd and 283rd;

[0693] - 162nd and 313th;

[0694] - 162nd, 274th and 283rd;

[0695] - 162nd, 221st, 222nd and 283rd;

[0696] -162nd, 221st, 222nd, 274th and 283rd.

[0697] In some embodiments, the R2D2 and / or R2D3 comprise amino acid mutations at any one or more positions selected from the group consisting of:

[0698] - 162nd and 283rd;

[0699] -162nd, 221st, 222nd, 274th and 283rd.

[0700] In some embodiments, the R2D2 and / or R2D3 comprises:

[0701] (i) any one or more groups of amino acid mutations selected from the following:

[0702] -162A, 162V, 162S, 162I, or 162L;

[0703] -221S;

[0704] -222K;

[0705] -274F or 274I;

[0706] -276Q;

[0707] -277R;

[0708] -280E;

[0709] -284H;

[0710] -288Y;

[0711] -289Y;

[0712] -313R; and

[0713] (ii) any one or more groups of amino acid mutations selected from the following:

[0714] -135Y;

[0715] -197Y;

[0716] -199Q or 199T;

[0717] -213N;

[0718] -215F or 215H;

[0719] -219I;

[0720] -257Q or 257T;

[0721] -275L or 275E;

[0722] -277R;

[0723] -278D, 278S, or 278E;

[0724] -283T;

[0725] -289Y;

[0726] -313Y;

[0727] -314S or 314Q.

[0728] In some embodiments, the R2D2 and / or R2D3 comprises:

[0729] (i) any one or more groups of amino acid mutations selected from the following:

[0730] -C162A, C162V, C162S, C162I, or C162L;

[0731] -Y221S;

[0732] -R222K;

[0733] -N274F or N274I;

[0734] -D276Q;

[0735] -L277R;

[0736] -Q280E;

[0737] -E284H;

[0738] -F288Y;

[0739] -L289Y;

[0740] -L313R; and

[0741] (ii) any one or more groups of amino acid mutations selected from the following:

[0742] -V135Y;

[0743] -M197Y;

[0744] -F199Q or F199T;

[0745] -M213N;

[0746] -I215F or I215H;

[0747] -V219I;

[0748] -D257Q or D257T;

[0749] -R275L or R275E;

[0750] -L277R;

[0751] -K278D, K278S, or K278E;

[0752] -S283T;

[0753] -L289Y;

[0754] -L313Y;

[0755] -M314S or M314Q.

[0756] In some embodiments, the R2D2 and / or R2D3 comprise any one or more groups of amino acid mutations selected from the following:

[0757] -162V / 275L;

[0758] -162V / 283T;

[0759] -162V / 313Y;

[0760] -162V / 274F / 283T;

[0761] -162V / 221S / 222K / 283T;

[0762] -162V / 221S / 222K / 274F / 283T.

[0763] In some embodiments, the R2D2 and / or R2D3 comprise any one or more groups of amino acid mutations selected from the following:

[0764] -C162V / R275L;

[0765] -C162V / S283T;

[0766] -C162V / L313Y;

[0767] -C162V / N274F / S283T;

[0768] -C162V / Y221S / R222K / S283T;

[0769] -C162V / Y221S / R222K / N274F / S283T.

[0770] In some embodiments, the R2D2 and / or R2D3 comprise any one group of amino acid mutations selected from the group consisting of:

[0771] -162V / 283T;

[0772] -162V / 221S / 222K / 274F / 283T.

[0773] In some embodiments, the R2D2 and / or R2D3 comprise any one group of amino acid mutations selected from the group consisting of:

[0774] -C162V / S283T;

[0775] -C162V / Y221S / R222K / N274F / S283T.

[0776] In some embodiments, the VEGF binding molecule consists of R2D2-R2D3.

[0777] In some embodiments, the VEGF-binding molecule comprises the amino acid sequence set forth in any one of SEQ ID NOs: 55-56, 112-115, or an amino acid sequence having at least 80% sequence identity thereto.

[0778] In some embodiments, the VEGF-binding molecule consists of the amino acid sequence set forth in any one of SEQ ID NOs: 55-56, 112-115, or an amino acid sequence having at least 80% sequence identity thereto.

[0779] In some embodiments, the VEGF-binding molecule has VEGF-C binding ability.

[0780] In some embodiments, the VEGF-binding molecule has increased stability and / or ligand binding activity.

[0781] In some embodiments, a VEGF-binding molecule comprising an amino acid mutation has increased stability and / or ligand binding activity relative to wild-type.

[0782] In some embodiments, any of the aforementioned VEGF-binding molecules optionally further comprises: a first VEGF-binding domain, a second VEGF-binding domain, and a multimeric component. In some embodiments, the multimeric component includes but is not limited to immunoglobulin Fc or a variant thereof.

[0783] In some embodiments, the Fc comprises any one of a hinge region (e.g., an upper, middle, and / or lower hinge region), a CH2, or a CH3 domain, or any combination thereof. In some embodiments, the Fc comprises at least a CH2 and a CH3 domain. In some embodiments, the Fc comprises a hinge region, a CH2, and a CH3 domain.

[0784] In some embodiments, Fc is derived from immunoglobulins of any species. In some embodiments, Fc is derived from human immunoglobulins. In some embodiments, Fc is derived from immunoglobulins of other mammalian species, including but not limited to rodents (e.g., mice, rats, rabbits, guinea pigs) or non-human primates (e.g., chimpanzees, macaques) species.

[0785] In some embodiments, the Fc is derived from any subtype of immunoglobulin, including but not limited to human IgG1, IgG2, IgG3, IgG4, IgD, IgA, IgE, or IgM antibodies. In some embodiments, the Fc is from human IgG1.

[0786] In some embodiments, the multimeric component is an Fc variant having an amino acid sequence that differs from a naturally occurring immunoglobulin Fc by no more than 20 amino acids, e.g., no more than 15, no more than 10, or no more than 5 amino acids. In some embodiments, the amino acid differences comprise amino acid substitutions, insertions, or deletions. In some embodiments, the Fc variant has reduced effector function (e.g., FcγR binding).

[0787] In some embodiments, the multimeric component enables the VEGF-binding molecule to form a dimer, trimer, tetramer, pentamer, hexamer, heptamer, octamer, nonamer, or decamer.

[0788] In some embodiments, the VEGF-binding molecule is a dimer (including homodimers, heterodimers).

[0789] In some embodiments, the Fc comprises an IgG1 hinge region, CH2, and CH3 domains.

[0790] In some embodiments, the VEGF binding domain is fused with a multimeric component (such as Fc of human immunoglobulin), and the resulting fusion protein has extremely excellent VEGF binding effect, good stability, long half-life, and can multivalently bind to VEGF molecules.

[0791] In some embodiments, the VEGF-binding molecule comprises, from N-terminus to C-terminus, any one selected from the group consisting of:

[0792] R2D2-R2D3-Fc;

[0793] R1D2-R2D3-R2D2-R2D3-Fc;

[0794] R1D2-R2D3-Linker-R2D2-R2D3-Fc;

[0795] R2D2-R2D3-R1D2-R2D3-Fc;

[0796] R2D2-R2D3-Linker-R1D2-R2D3-Fc.

[0797] In some embodiments, the VEGF-binding molecule comprises a heavy chain and a light chain, wherein the heavy chain comprises, from N-terminus to C-terminus, R2D2-R2D3-Fc-linker-VH-CH1, and the light chain comprises, from N-terminus to C-terminus, VL-CL.

[0798] The “-” indicates that the two domains are directly or indirectly connected, but the relative position from the N-terminus to the C-terminus remains unchanged.

[0799] In some embodiments, any two adjacent domains in the VEGF-binding molecules of the present disclosure can be linked directly or through a linker.

[0800] In some embodiments, the linker is a peptide linker.

[0801] In some embodiments, the linker is represented by the following formula: (GS) a (GGS) b (GGGS) c (GGGGS) d (GGGGG) e , a, b, c, d, e are independently integers greater than or equal to 0; or the linker is selected from: (EAAAK)3, (EAAAR)3, (EGGGK)3, (EGGGR)3, (DAAAR)3, (DAAAK)3, (DGGGR)3 or (DGGGK)3; or the linker is (G x S) y , wherein x is selected from an integer of 1-5, and y is selected from an integer of 1-6.

[0802] In some embodiments, the linker is GGGGS. In some embodiments, in some embodiments, the VEGF binding molecule is selected from the group consisting of:

[0803] R2D2-R2D3-Fc;

[0804] R1D2-R2D3-R2D2-R2D3-Fc;

[0805] R1D2-R2D3-Linker-R2D2-R2D3-Fc;

[0806] R2D2-R2D3-R1D2-R2D3-Fc;

[0807] R2D2-R2D3-Linker-R1D2-R2D3-Fc.

[0808] In some embodiments, the VEGF-binding molecule comprises a heavy chain and a light chain, wherein the heavy chain is sequentially arranged from N-terminus to C-terminus: R2D2-R2D3-Fc-linker-VH-CH1, and the light chain is sequentially arranged from N-terminus to C-terminus: VL-CL.

[0809] In some embodiments, the VEGF-binding molecule comprises the amino acid sequence set forth in any one of SEQ ID NOs: 53-54, SEQ ID NOs: 57-99, SEQ ID NOs: 102-111, SEQ ID NO: 116, or an amino acid sequence having at least 80% sequence identity thereto.

[0810] In some embodiments, the VEGF binding molecule consists of the amino acid sequence set forth in any one of SEQ ID NOs: 53-54, SEQ ID NOs: 57-99, SEQ ID NOs: 102-111, SEQ ID NO: 116, or an amino acid sequence having at least 80% sequence identity thereto.

[0811] In some embodiments, the heavy chain of the VEGF-binding molecule comprises the amino acid sequence of SEQ ID NO: 100, or an amino acid sequence having at least 80% sequence identity thereto; and the heavy chain of the VEGF-binding molecule comprises the light chain of SEQ ID NO: 101, or an amino acid sequence having at least 80% sequence identity thereto.

[0812] In some embodiments, the heavy chain of the VEGF-binding molecule comprises the amino acid sequence of SEQ ID NO: 117, or an amino acid sequence having at least 80% sequence identity thereto; and the light chain of the VEGF-binding molecule comprises the amino acid sequence of SEQ ID NO: 118, or an amino acid sequence having at least 80% sequence identity thereto.

[0813] In some embodiments, the VEGF-binding molecule comprises or consists of the amino acid sequence of SEQ ID NO: 94, or an amino acid sequence having at least 80% sequence identity thereto.

[0814] In some embodiments, the VEGF-binding molecule comprises or consists of the amino acid sequence of SEQ ID NO: 111, or an amino acid sequence having at least 80% sequence identity thereto.

[0815] In some embodiments, the VEGF-binding molecules of the present disclosure include the full-length or partial protein of any of the aforementioned VEGF-binding molecules, or mutant proteins, functional derivatives, functional fragments, biologically active peptides, fusion proteins, isoforms, or salts thereof obtained by further mutation on the basis of any of the aforementioned VEGF-binding molecules. For example, fusion proteins comprising VEGF-binding molecules, monomers, dimers, trimers, or multimers of the VEGF-binding molecules, various modified forms of the VEGF-binding molecules (such as PEGylation, polyethylene glycolation, glycosylation, albumin conjugation or fusion, albumin antibody conjugation or fusion, hydroxyethylation, removal of O-glycosylation, etc.), and homologs of the VEGF-binding molecules in various species. Modification of the VEGF-binding molecules does not adversely affect the activity of the therapeutically relevant protein.

[0816] In some embodiments, the VEGF-binding molecule has increased stability and / or ligand binding activity.

[0817] In some embodiments, a VEGF-binding molecule comprising an amino acid mutation has increased stability and / or ligand binding activity relative to wild-type.

[0818] In some embodiments, the VEGF-binding molecules of the disclosure have an HPLC purity of at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% at 40°C, 75% humidity for two weeks.

[0819] In some embodiments, the VEGF-binding molecules of the disclosure have an HPLC purity of at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% at 40°C, 75% humidity for four weeks.

[0820] In some embodiments, the VEGF-binding molecules of the disclosure have an HPLC purity of at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% at room temperature, 75% humidity for two weeks.

[0821] In some embodiments, the VEGF-binding molecules of the disclosure have an HPLC purity of at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% at room temperature and 75% humidity for four weeks.

[0822] In some embodiments, HPLC purity testing can be performed using the method described in the Examples.

[0823] In some embodiments, the VEGF-binding molecules of the present disclosure have a binding activity for VEGF-A of IC50 ≤ 10 nM, ≤ 5 nM, or ≤ 2 nM, such as an IC50 of 0.01 nM-10 nM, 0.05 nM-5 nM, 0.05 nM-2 nM, 0.05 nM-1.5 nM, 0.05 nM-1.2 nM, or 0.05 nM-1 nM; and / or

[0824] The VEGF-binding molecules disclosed herein have a binding activity IC50 for VEGF-C of ≤10 nM, ≤5 nM, or ≤2 nM. For example, the VEGF-binding molecules disclosed herein have a binding activity IC50 for VEGF-C of 0.01 nM-10 nM, 0.05 nM-5 nM, 0.05 nM-2 nM, 0.05 nM-1.5 nM, 0.05 nM-1.2 nM, or 0.05 nM-1 nM.

[0825] In some embodiments, the binding kinetics K of the VEGF-binding molecules of the present disclosure to VEGF-A is D ≤1×10 -9 , for example, K D ≤1×10 -10 , K D ≤9×10 -11 , K D ≤8×10 -11 , K D ≤7×10 -11 , K D ≤6×10 -11 , K D ≤5×10 -11 , K D ≤4×10-11 ;

[0826] The binding kinetics of the VEGF-binding molecules of the present disclosure to VEGF-B D ≤1×10 -9 , for example, K D ≤4×10 -10 , K D ≤3×10 -10 , K D ≤2×10 -10 , K D ≤1×10 -10 , K D ≤9×10 -11 , K D ≤8×10 -11 , K D ≤7×10 -11 , K D ≤6×10 -11 , K D ≤5×10 -11 , K D ≤4×10 -11 , K D ≤3×10 -11 , K D ≤2×10 -11 and / or

[0827] The binding kinetics of the VEGF-binding molecules of the present disclosure to VEGF-C are D ≤1×10 -9 , for example, K D ≤1×10 -10 , K D ≤9×10 -11 , K D ≤8×10 -11 , K D ≤7×10 -11 , K D ≤6×10 -11 , K D ≤5×10 -11 , K D ≤4×10 -11 , K D ≤3×10 -11 , K D ≤2×10 -11 .

[0828] In some embodiments, the binding activity of the VEGF-binding molecules of the present disclosure to a ligand can be determined using the methods described in Example 3 or Example 5.

[0829] Nucleic acids and vectors

[0830] The present disclosure also provides a nucleic acid molecule encoding the VEGF-binding molecule of the present disclosure. In some embodiments, the nucleic acid of the present disclosure can be RNA, DNA or cDNA. According to some embodiments of the present disclosure, the nucleic acid of the present disclosure is an isolated nucleic acid.

[0831] The present disclosure also provides a DNA molecule encoding any of the aforementioned VEGF-binding molecules of the present disclosure.

[0832] The nucleic acids of the present disclosure may also be in the form of, present in, and / or be part of a vector, such as a plasmid, cosmid, YAC, or viral vector. The vector may in particular be an expression vector, i.e., a vector that provides for expression of the VEGF-binding molecule in vitro and / or in vivo (i.e., in a suitable host cell, host organism, and / or expression system). The expression vector typically comprises at least one nucleic acid of the present disclosure, operably linked to one or more suitable expression control elements (e.g., promoters, enhancers, terminators, etc.). The selection of such elements and their sequences for expression in a particular host is within the skill of the art. Regulatory and other elements useful or necessary for expression of the VEGF-binding molecules of the present disclosure are, for example, promoters, enhancers, terminators, integration factors, selection markers, leader sequences, and reporter genes.

[0833] The nucleic acids of the present disclosure can be prepared or obtained by known means (eg, by automated DNA synthesis and / or recombinant DNA technology) based on the information of the amino acid sequence of the polypeptides of the present disclosure, and / or can be isolated from suitable natural sources.

[0834] In some embodiments, the nucleic acid molecule comprises a transgene or polynucleotide encoding any of the VEGF-binding molecules of the disclosure.

[0835] In some embodiments, the transgene comprises a nucleic acid sequence that is at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 99%, or 100% identical to the nucleic acid sequence of any one of SEQ ID NOs: 128-136, or is a codon-optimized variant of the nucleic acid sequence of SEQ ID NOs: 128-136.

[0836] In some embodiments, the transgenic nucleic acid sequence is operably connected to a constitutive promoter. In other embodiments, the transgenic nucleic acid sequence is operably connected to an inducible promoter. In some cases, the transgenic nucleic acid sequence is operably connected to a tissue-specific or cell type-specific regulatory element.

[0837] In the context of the present disclosure, transgene refers to the gene of interest, as well as the nucleic acid encoding any of the VEGF-binding molecules of the present disclosure.

[0838] In some embodiments, the isolated nucleic acid further comprises a promoter operably linked to the transgene.

[0839] In some embodiments, the promoter is selected from the group consisting of a cytomegalovirus (CMV) promoter, a Rous sarcoma virus (RSV) promoter, an MMT promoter, an EF-1alpha promoter, an UB6 promoter, a chicken beta-actin promoter, a CAG promoter, an RPE65 promoter, and an opsin promoter, such as a CMV promoter.

[0840] In some embodiments, the isolated nucleic acid further comprises an enhancer, eg, the enhancer is a CMV enhancer.

[0841] In some embodiments, the isolated nucleic acid further comprises an intron.

[0842] In some embodiments, the isolated nucleic acid further comprises a Kozak sequence. In some embodiments, the Kozak sequence is located between an intron and a transgene encoding a VEGF-binding molecule.

[0843] In some embodiments, the isolated nucleic acid further comprises a 5'-UTR and a 3'-UTR.

[0844] In some embodiments, the isolated nucleic acid further comprises a WPRE sequence.

[0845] In some embodiments, the isolated nucleic acid further comprises a polyA sequence, such as an sv40 polyA sequence.

[0846] The present disclosure also provides a vector comprising any of the above-mentioned isolated nucleic acids. In some embodiments, the vector is a plasmid or a viral vector. In some embodiments, the virus is selected from adeno-associated virus (AAV), helper-dependent adenovirus, retrovirus, herpes simplex virus, lentivirus, adenovirus, adeno-associated virus vector, poxvirus, Japanese hemagglutinating virus-liposome (HVJ) complex, Moloney murine leukemia virus, and HIV-based viruses.

[0847] In some embodiments, the vector is a recombinant adeno-associated viral vector.

[0848] The present disclosure also provides a recombinant adeno-associated virus vector comprising:

[0849] (i) a transgene encoding a VEGF-binding molecule as defined in any one of the preceding claims;

[0850] (ii) enhancers (e.g., CMV enhancer);

[0851] (iii) a promoter (e.g., CMV promoter);

[0852] (iv) 5′-UTR and 3′-UTR;

[0853] (v) post-transcriptional regulatory elements (e.g., WPRE); and

[0854] (vi) polyadenylation signal (polyA, e.g., SV40 polyA);

[0855] Optionally, containing (vii) a Kozak sequence, and / or, (viii) an intron;

[0856] Any one or any combination of (ii) to (viii) is operably linked to (i).

[0857] In some embodiments, the recombinant adeno-associated viral vector comprises a 5' ITR and / or a 3' ITR. The 5' ITR and / or the 3' ITR are derived from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV9.47, AAV9(hu14), AAV10, AAV11, AAV12, AAVrh8, AAVrh10, AAV-DJ, or AAV-DJ8; for example, derived from AAV2 or AAV9.

[0858] In some embodiments, the promoter is selected from CMV, CAG, CBh, EFS, EF1 (eg, EF-1α), PGK, SV40, Ubi, RSV, or any combination thereof.

[0859] In some embodiments, the enhancer is selected from Ubi, CMV, RSV enhancer, or any combination thereof.

[0860] In some embodiments, the intron is selected from MVM, SV40, βGlobin, EF1 (eg, EF-1α), a hybrid intron, or any combination thereof.

[0861] In some specific embodiments, the polyA is selected from PA75 polyA, SV40 polyA, hGH polyA, BGH polyA, rbGlob polyA, or any combination thereof.

[0862] In some embodiments, the post-transcriptional regulatory element is selected from WPRE, HPRE, or a combination thereof.

[0863] In some embodiments, a recombinant adeno-associated viral vector comprises any of the aforementioned isolated nucleic acids and a capsid protein.

[0864] In some embodiments, the capsid protein is selected from the following serotypes: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or variants thereof.

[0865] In some embodiments, the capsid protein has an amino acid sequence comprising the amino acid sequence shown in SEQ ID NO: 127, or an amino acid sequence that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 127.

[0866] The novel VEGF-binding molecules provided herein serve as novel VEGF inhibitors that can be administered in either protein or nucleic acid drug forms. Functionally, they can simultaneously bind to both VEGF-A and VEGF-C, resulting in higher VEGF inhibition efficiency. In terms of efficacy, these VEGF-binding molecules exhibit enhanced stability. In particular, the nucleic acid drug form can effectively reduce dosing frequency, making them promising drugs for clinical development.

[0867] AAV capsids and AAV particles

[0868] The polynucleotides of any of the aforementioned items of the present disclosure can be encapsidated with the following AAV capsids to form AAV particles. The AAV capsids are as shown in any of the aforementioned items. In some specific embodiments, the AAV capsids used in the present disclosure are the AAV2 and AAV9 capsids described in the prior art.

[0869] In some embodiments, the AAV capsid is engineered, for example, a hybrid AAV capsid derived from two or more parental serotypes. For example, the AAV capsid can be AAV2G9, which comprises sequences from AAV2 and AAV9. The sequence of AAV2G9 in US20160017005 is incorporated herein in its entirety.

[0870] In some embodiments, the AAV capsid is an AAV2 or AAV9 capsid.

[0871] In some embodiments, in addition to the encoded heterologous payload, the AAV particle can comprise all or part of the vector genome of any naturally occurring and / or recombinant AAV capsid nucleotide sequence or variant.

[0872] In some embodiments, the AAV particles can be replication-defective (e.g., they lack sequences encoding functional Rep and Cap proteins in the vector genome). In some specific embodiments, the replication-defective AAV particles can lack most or all parental coding sequences and essentially carry only one or two AAV ITR sequences and a nucleic acid of interest for delivery to a cell, tissue, organ, or organism.

[0873] In some embodiments, the AAV particle can be a recombinant AAV (rAAV) particle.

[0874] In some embodiments, the AAV particles can be selected from single-stranded AAV particles (e.g., ssAAV) and self-complementary AAV particles (e.g., scAAV). By skipping second-strand synthesis, scAAV achieves rapid expression in cells.

[0875] In some embodiments, the AAV particle is encapsulated with a nucleic acid encoding a VEGF-binding molecule comprising the amino acid sequence of any one of SEQ ID NOs: 53-54, 57-99, 102-61, and 116; the VEGF-binding molecule comprises the heavy chain of SEQ ID NO: 100 and the light chain of SEQ ID NO: 101; or the VEGF-binding molecule comprises the heavy chain of SEQ ID NO: 117 and the light chain of SEQ ID NO: 118.

[0876] In some embodiments, the AAV particle encapsidates a nucleic acid that is at least 95% identical to any one of SEQ ID NOs: 128-136.

[0877] In some embodiments, the AAV particle encapsidates a nucleic acid comprising a sequence set forth in any one of SEQ ID NOs: 128-136.

[0878] In some embodiments, the capsid protein of the AAV particle has an amino acid sequence comprising the amino acid sequence set forth in SEQ ID NO: 127, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 127.

[0879] Regulatory elements

[0880] The vector genome (or viral genome) of the nucleic acid or AAV particle of any of the foregoing disclosures comprises at least one regulatory element that enables replication, transcription and translation of the gene of interest (eg, VEGF-binding molecule) in the payload.

[0881] In some embodiments, the regulatory elements include, but are not limited to, sequences for transcription initiation and / or termination, promoter and / or enhancer sequences, efficient RNA processing signals (e.g., splicing and polyadenylation signals), sequences that stabilize cytoplasmic mRNA, sequences that enhance translation efficiency (e.g., Kozak consensus sequences), sequences that enhance protein stability, and / or sequences that enhance protein processing and / or secretion. Exemplary regulatory elements include, but are not limited to, promoters, enhancers, introns, endogenous miRNAs, post-transcriptional regulatory elements (PREs), polyadenylation (PolyA) signal sequences, and upstream enhancers (USEs).

[0882] In some embodiments, the regulatory elements (e.g., promoters) described below drive expression of the payload in a target tissue (e.g., an eye of a subject) for a period of time, for example, 1, 2, 3, 4, 5, 6, 7 days, 1, 2, 3, 4 weeks, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60 years or more. For another example, 1-2 weeks, 1-3 weeks, 1-4 weeks, 1-2 months, 1-4 months, 1-6 months, 2-6 months, 3-6 months, 3-9 months, 4-8 months, 6-12 months, 1-2 years, 1-5 years, 2-5 years, 3-6 years, 3-8 years, 4-8 years, 5-10 years, 10-20 years, 10-30 years, 20-40 years, 20-50 years, or the lifetime of the subject.

[0883] Inverted terminal repeats (ITRs)

[0884] The vector genome (or viral genome) of the polynucleotide or AAV particle of any of the aforementioned disclosures comprises at least one ITR, for example, has two ITRs, flanking the payload at the 5' and 3' ends, respectively.

[0885] In some embodiments, the ITR functions as an origin of replication.

[0886] In some embodiments, the ITRs comprise sequence regions that are complementary and symmetrically arranged.

[0887] In some embodiments, the ITRs may consist of naturally occurring polynucleotide sequences or recombinantly derived polynucleotide sequences.

[0888] In some embodiments, the ITR can be derived from the same or a different serotype as the capsid. In some embodiments, the 5' ITR and 3' ITR can be derived from the same serotype or from different serotypes. For example, both the 5' ITR and the 3' ITR are derived from AAV2, or both are derived from AAV9. In another example, the 5' ITR comprises the sequence set forth in SEQ ID NO: 126, and / or the 3' ITR comprises the nucleotide sequence set forth in SEQ ID NO: 137.

[0889] In some embodiments, the length of each ITR can be about 100 to about 150 nucleotides, for example, 100-105 nucleotides, 106-110 nucleotides, 111-115 nucleotides, 116-120 nucleotides, 121-125 nucleotides, 126-130 nucleotides, 131-135 nucleotides, 136-140 nucleotides, 141-145 nucleotides, or 146-150 nucleotides. In one embodiment, the length of the ITR is 140-142 nucleotides, for example, 141 nucleotides. Non-limiting examples of ITR lengths are 102, 140, 141, 142, 145 nucleotides in length, and nucleotides that are at least 95% identical thereto.

[0890] promoter

[0891] The vector genome (or viral genome) of any of the aforementioned polynucleotides or AAV particles of the present disclosure comprises at least one promoter, including but not limited to species-specific, inducible, tissue-specific or cell cycle-specific promoters.

[0892] In some embodiments, the promoter drives expression of a protein or polypeptide (eg, a VEGF-binding molecule) encoded in the payload of the vector genome of the AAV particle.

[0893] In some embodiments, the promoter is specific or tropic for the target tissue, for example, a promoter that expresses the payload in ocular tissue.

[0894] In some embodiments, the promoter can be a viral promoter, a plant promoter, a mammalian promoter, or a human promoter.

[0895] In some embodiments, the promoter includes but is not limited to CMV, CBA (including derivatives CAG, CBh, etc.), EF-1α, PGK, UBC, RSV, EFS, EF1, GUSB (hGBp), UCOE (promoter of HNRPA2B1-CBX3), NSE, Synapsin, MeCP2, MeP418, MeP426, VMD2, MRHO, TRE, Ac5, Polyhedrin, CaMKIIa, Gall, TEF1, GDS, ADH1, Ubi, GFAP or PKG promoter, and can be selected from neurofilament light (NFL) promoter, neurofilament heavy (NFH) promoter, SCN8A promoter, frataxin (FXN) promoter (or FRDA promoter), H1 promoter, RNA pol III promoter (e.g., U6 or H1), small nuclear RNA (ULB or ULA) promoter. In some embodiments, the promoter is a liver promoter, such as human alpha-1-antitrypsin (hAAT) and thyroxine-binding globulin (TBG), or a skeletal muscle promoter, such as desmin, MCK, or synthetic C5-12.

[0896] In some embodiments, the AAV vector genome of the present disclosure comprises two promoters, such as CMV and CBA promoters, the CMV sequence is shown in SEQ ID NO: 125; another example is EF1α promoter and CMV promoter, or any combination of any two of the aforementioned promoters.

[0897] In some embodiments, the promoter is a tissue-specific expression element that can restrict expression to certain cell types, including but not limited to a muscle-specific promoter, a B cell promoter, a monocyte promoter, a leukocyte promoter, a macrophage promoter, a pancreatic acinar cell promoter, an endothelial cell promoter, a lung tissue promoter, an astrocyte promoter, or a nervous system promoter, which can be used to restrict expression to neurons, astrocytes, or oligodendrocytes.

[0898] In some embodiments, the promoter is a truncated or variant of the aforementioned promoter, and is less than 1 kb in length, for example, 200-300, 200-400, 300-400, 200-500, 200-600 or more in length.

[0899] In some embodiments, the promoter can be a combination of two or more components of the same or different starting or parental promoters, such as CMV and CBA.

[0900] enhancer

[0901] The vector genome (or viral genome) of any of the aforementioned polynucleotides or AAV particles of the present disclosure comprises at least one enhancer, including but not limited to species-specific, inducible, tissue-specific or cell cycle-specific enhancers.

[0902] In some embodiments, the enhancer is specific or tropic for the target tissue, for example, an enhancer that regulates expression of the payload in neural tissue.

[0903] In some embodiments, the enhancer can be or be derived from a viral enhancer, a plant enhancer, a mammalian enhancer, or a human enhancer.

[0904] In some embodiments, the enhancer can be located upstream or downstream of the promoter and operably linked to the promoter. When the enhancer is present, the expression of the target gene (e.g., VEGF-binding molecule) is enhanced by at least 20%, at least 50%, at least 80%, at least 100%, or at least several times (including 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 times or more) compared to when the enhancer is not present.

[0905] In some embodiments, the enhancer includes but is not limited to EF-1α, Ubc, human b-actin, CAG, TRE, Ac5, Polyhedrin, CaMKIIa, Gall, TEF1, GDS, ADH1, Ubi and α-1-antitrypsin (hAAT), or variants or fragments thereof. In some embodiments, the enhancer is selected from IRBP, RSV or CMV enhancers, or variants or fragments thereof.

[0906] In some embodiments, the enhancer comprises or is the enhancer shown in SEQ ID NO: 124 or a variant or fragment thereof.

[0907] introns

[0908] The vector genome (or viral genome) of any of the aforementioned polynucleotides or AAV particles of the present disclosure may contain an intron or a portion thereof.

[0909] In some embodiments, the introns include but are not limited to MVM (67-97 bp), F.IX truncated intron 1 (about 300 bp), β-globin SD / immunoglobulin heavy chain splice acceptor (about 250 bp), adenovirus splice donor / immunoglobulin splice acceptor (about 500 bp), SV40 late splice donor / splice acceptor (19S / 16S) (180 bp) and hybrid adenovirus splice donor / IgG splice acceptor (about 230 bp), hemoglobin intron, hybrid intron, β-globin intron, or variants or fragments of the above introns.

[0910] In some embodiments, the intron is 100-800 nucleotides in length, such as about 100, about 200, about 300, about 400, about 500, about 600 nucleotides, or 200-250, 200-300, 100-200, 300-500, 500-600, 400-600, or 100-200 nucleotides.

[0911] In some embodiments, the intron in the technical solution of the present disclosure is selected from hybrid introns and β-globin introns.

[0912] Untranslated region (UTR)

[0913] The polynucleotide of any of the foregoing or the vector genome (or viral genome) of the AAV particles of the present disclosure may include an untranslated region (UTR), and the UTR is selected from 5'UTR and / or 3'UTR to regulate (e.g., increase, decrease or reduce) polynucleotide stability and protein production. Typically, the 5'UTR starts at the transcription start site and ends at the start codon, and the 3'UTR starts immediately after the stop codon and ends at the transcription termination signal. The UTR may be a wild type, a variant thereof or an artificial UTR.

[0914] In some embodiments, the 5'UTR includes a Kozak sequence. In other embodiments, the 5'UTR does not include a Kozak sequence.

[0915] In some embodiments, the 3'UTR is AU-rich. In some embodiments, the 3'UTR is selected from: class I AREs, such as but not limited to c-Myc and MyoD, which contain several dispersed copies of the AUUUA motif within the U-rich region; class II AREs, such as but not limited to GM-CSF and TNF-α, which possess two or more overlapping UUAUUUA(U / A)(U / A) nonamers; and class III ARES, such as but not limited to c-Jun and myogenin.

[0916] In some embodiments, the 3'UTR may include an oligo(dT) sequence for templated addition of a polyadenylation sequence (PolyA).

[0917] Polyadenylation sequence (PolyA)

[0918] The polynucleotide of any of the aforementioned items of the present disclosure or the vector genome (or viral genome) of the AAV particle may contain a sequence encoding PolyA, and the PolyA may be a wild type, a variant thereof, or a modified PolyA for protein translation.

[0919] In some embodiments, the sequence encoding Poly A is located between the 3' end of the payload encoding sequence and the 5' end of the 3' ITR.

[0920] In some embodiments, the sequence encoding Poly A is 0-500 nucleotides in length, for example, about 75, about 100, about 110, about 120, about 150, about 160, about 200, or about 300 nucleotides, or for example, 50-100, 50-150, 50-160, 50-200, 60-100, 60-150, 60-160, 60-200, 70-100, 70-150, 70-160, 70-200, 80-100, 80-150, 80-160, 80-200, 90-100, 90-150, 90-160, or 90-200.

[0921] In some embodiments, the poly A can encode continuous poly A or discontinuous poly A. When encoding discontinuous poly A, the coding sequence can be interrupted or separated by other nucleotides. For example, the poly A has at least two segments of 60 adenylic acids, and the at least two segments of 60 adenylic acids are separated by a sequence comprising 10-90 nucleotides.

[0922] In some embodiments, the sequence encoding PolyA or polyA in the technical solutions of the present disclosure is selected from β-globin polyA, SV40 polyA, bGH polyA, PA75 polyA, MeCP2 polyA, RDH1 polyA, BGH polyA, SPA49polyA, sNRP-TK65 polyA, sNRP polyA, TK65 polyA, or variants or fragments of the above PolyA.

[0923] In some embodiments, the polyA shown in SEQ ID NO: 139 or a variant or fragment thereof in the technical solution of the present disclosure.

[0924] The polyA or coding sequences thereof in WO2016005324, WO2016005004, WO2016091391, WO2019036513, and WO2020074642 are fully introduced herein, and can all be used in the technical solutions disclosed herein.

[0925] Filling sequence

[0926] The vector genome (or viral genome) of any of the polynucleotides or AAV particles disclosed herein may comprise a stuffer sequence such that the length of the vector genome is the optimal size for packaging, for example, such that the length of the vector genome is about 2.3 kb, about 4.6 kb, about 4.7 kb, or about 5.1 kb.

[0927] In some embodiments, the vector genome is a single-stranded or double-stranded genome, and the packaged AAV particles are ssAAV or scAAV.

[0928] In some embodiments, a vector genome may contain one or more (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) stuffer sequences.

[0929] In some embodiments, the stuffer sequence can be located between or within multiple regulatory elements, such as between two introns, 3' to the 5'ITR sequence, 5' to the 5'ITR sequence, 5' to the 3'ITR sequence, 3' to the 3'ITR sequence, and before or after regions such as promoters, introns, enhancers, polyA, multiple cloning site (MCS) regions, exons, etc.

[0930] Method for producing or preparing recombinant adeno-associated virus (rAAV) particles

[0931] The present disclosure provides methods for producing, preparing and / or modifying rAAV particles. The rAAV particle production, preparation and / or modification methods in WO200028004, WO200123001, WO2004112727, WO 2005005610, WO2005072364, WO2013123503, WO2015191508 and US20130195801 are introduced herein in full. The rAAV particles can have the characteristics of enhancing delivery efficiency, can be effectively packaged, and can successfully infect target cells (e.g., mammalian or human cells) with high frequency and minimal toxicity.

[0932] In some embodiments, a method for producing rAAV particles is provided, comprising packaging any nucleic acid or vector genome (or viral genome) or vector disclosed herein into an AAV capsid. In some specific embodiments, the method comprises the following steps:

[0933] 1) co-transfecting competent bacterial cells with the baculovirus vector and the viral construct vector and / or the AAV payload construct vector,

[0934] 2) separating the obtained viral construct expression vector and AAV payload construct expression vector and transfecting them into virus-replicating cells respectively,

[0935] 3) isolating and purifying the resulting payload and viral construct particles comprising the viral construct expression vector or the AAV payload construct expression vector,

[0936] 4) co-infecting virally replicating cells with both the AAV payload and the viral construct particles comprising the viral construct expression vector or the AAV payload construct expression vector, and

[0937] 5) Harvest and purify AAV particles containing the viral genome.

[0938] In some embodiments, a method for producing rAAV particles is provided, comprising the steps of:

[0939] 1) co-transfecting mammalian cells (e.g., HEK293 cells) with any nucleic acid or vector genome (or viral genome) or vector disclosed herein, and constructs expressing Rep and Cap genes and helper constructs (to achieve helper functions);

[0940] 2) Harvest and purify rAAV particles containing the viral genome.

[0941] In some embodiments, the viral genome of the aforementioned rAAV particles optionally encodes a selection marker. The selection marker can include a cell surface marker, such as any protein expressed on the cell surface, including but not limited to a receptor CD marker, a lectin, an integrin, or a truncated form thereof.

[0942] In some embodiments, an AAV production system is provided for producing rAAV particles of the present disclosure, wherein the production system comprises:

[0943] 1) Nucleic acid sequence encoding AAV capsid;

[0944] 2) any nucleic acid or vector genome (or viral genome) or vector disclosed herein; and

[0945] 3) auxiliary packaging elements having sufficient AAV rep functions and helper functions to allow packaging of the nucleic acid or vector genome (or viral genome) or vector in (b) into AAV capsids.

[0946] The nucleic acid or vector genome (or viral genome) or vector comprises any nucleic acid encoding a VEGF-binding molecule disclosed above.

[0947] The AAV capsid is selected from any of the aforementioned AAV capsids disclosed herein, such as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV9.47, AAV9(hu14), AAV10, AAV11, AAV12, AAVrh8, AAVrh10, AAV-DJ, AAV-DJ8, or a variant of any of the aforementioned.

[0948] In some embodiments, the helper packaging plasmid, including elements sufficient for AAV rep function and helper function, is provided by packaging cells, which may contain any one or more of the three plasmids: pHelper, pRC9, and pGOI.

[0949] Exemplarily, "helper function" or "helper virus function" refers to a function encoded in the helper virus genome that allows AAV replication and packaging (in conjunction with other requirements for replication and packaging as described herein). In the present disclosure, "helper virus function" can be provided in many ways, including by providing a helper virus or providing, for example, a polynucleotide sequence encoding the necessary function to the producer cell in transit.

[0950] Exemplarily, "AAV rep function" refers to the function provided by the Rep gene and / or the Cap gene.

[0951] In some embodiments, the Rep gene encodes a nonstructural protein that regulates functions, such as replication of the AAV genome, and can be selected from Rep78, Rep68, Rep52, and Rep40. Rep78 and Rep68 are typically transcribed from the p5 promoter, while Rep52 and Rep40 are typically transcribed from the p19 promoter. The Cap gene encodes the structural proteins VP1, VP2, and / or VP3 that assemble to form the viral capsid. The Cap gene is typically transcribed from the p40 promoter.

[0952] In some embodiments, rAAV particles produced by the above-described AAV production system are provided.

[0953] In some embodiments, the pharmaceutical composition comprises the nucleic acid molecule described in any one of the preceding claims.

[0954] In some embodiments, the pharmaceutical composition comprises recombinant adeno-associated virus (rAAV) particles and excipients; the recombinant adeno-associated virus (rAAV) particles comprise nucleic acid molecule I and AAV9 capsid, and the excipients comprise any one of (a)-(f) and (h).

[0955] In some embodiments, the pharmaceutical composition comprises recombinant adeno-associated virus (rAAV) particles and excipients; the recombinant adeno-associated virus (rAAV) particles comprise nucleic acid molecule I and AAV9 capsid, and the excipients comprise any one of (1)-(3).

[0956] In some embodiments, the pharmaceutical composition comprises recombinant adeno-associated virus (rAAV) particles and excipients; the recombinant adeno-associated virus (rAAV) particles comprise nucleic acid molecule II and AAV9 capsid, and the excipients comprise any one of (a)-(f) and (i).

[0957] In some embodiments, the pharmaceutical composition comprises recombinant adeno-associated virus (rAAV) particles and excipients; the recombinant adeno-associated virus (rAAV) particles comprise nucleic acid molecule II and AAV9 capsid, and the excipients comprise any one of (4)-(6).

[0958] The pharmaceutical composition of the present disclosure further comprises a solvent. The solvent in the pharmaceutical composition is selected from, but not limited to, non-toxic physiologically acceptable liquid carriers, such as physiological saline, water for injection, glucose solution (such as 5% glucose injection, glucose sodium chloride injection), etc.

[0959] The present disclosure also provides a lyophilized preparation comprising a pharmaceutical composition, wherein the lyophilized preparation is obtained by freeze-drying any of the aforementioned pharmaceutical compositions.

[0960] The present invention adopts the method of dilute freezing and concentrated dissolving to prepare a reconstituted solution with increased protein concentration, and the reconstituted solution has a good appearance and good stability.

[0961] In some embodiments, the composition is substantially stable over an extended period of time. "Stable" or "storage-stable" or "substantially stable" means that the composition does not significantly degrade and / or lose activity over an extended period of time. For example, a storage-stable composition does not have significant impurities due to degradation of the composition over an extended period of time, for example, less than 10% impurities, or less than 9%, or less than 8%, or less than 7%, or less than 6%, or less than 5%, or less than 4%, or less than 3%, or less than 2%, or less than 1% degradation products over an extended period of time. In some cases, a storage-stable composition has less than 5% impurities over an extended period of time. In some cases, a storage-stable composition substantially retains its activity over an extended period of time, for example, the composition retains 100%, or greater than 99%, or greater than 98%, or greater than 97%, or greater than 96%, or greater than 95%, or greater than 94%, or greater than 93%, or greater than 92%, or greater than 91%, or greater than 90%, or greater than 85%, or greater than 80%, or greater than 75% of its activity over an extended period of time. For example, a storage-stable composition may retain greater than 90% of its activity over an extended period of time. In some cases, a storage-stable composition retains greater than 95% of its activity over an extended period of time. A longer period of time is a period of time, such as more than 1 week, or more than 2 weeks, or more than 3 weeks, or more than 1 month, or more than 2 months, or more than 3 months, or more than 4 months, or more than 6 months, or more than 9 months, or more than 1 year, or more than 1.5 years (e.g., 18 months), or more than 2 years, or more than 2.5 years (e.g., 30 months), or more than 3 years, or more than 3.5 years (e.g., 42 months), or more than 4 years, or more than 4.5 years (e.g., 54 months), or more than 5 years. For example, a longer period of time can be more than 6 months. In some cases, a longer period of time is more than 9 months. In some cases, a longer period of time is more than 1 year (e.g., 12 months). In some cases, a longer period of time is more than 1.5 years (e.g., 18 months). In some cases, a longer period of time is more than 2 years (e.g., 24 months). In some embodiments, the storage-stable composition is substantially stable over an extended period of time at ambient temperature, for example, a temperature of 20-40° C., or 25-35° C., or 25-30° C. In some cases, the storage-stable composition is substantially stable over an extended period of time at a temperature below ambient temperature, for example, a temperature of 0-20° C., or 0-15° C., or 0-10° C., or 2 to 8° C.

[0962] In certain embodiments, the rAAV particles in the pharmaceutical composition have stability or substantially stability (e.g., stable Vg, purity, IU, and / or biological activity) when stored at a temperature range of about -80°C to about 40°C for at least 6 months. In certain embodiments, the viral particles have stability when stored at a temperature range of about -80°C to about 25°C for at least 6 months. In certain embodiments, the viral vector has stability when stored at a temperature range of about -20°C to about 18°C ​​for at least 6 months. In certain embodiments, the viral vector has stability when stored at a temperature range of about -20°C to about 4°C for at least 6 months. In certain embodiments, the viral vector has stability when stored at a temperature range of about 4°C to about 40°C for at least 6 months. In certain embodiments, the viral vector has stability when stored at a temperature range of about 4°C to about 25°C for at least 6 months. In certain embodiments, the viral vector has stability when stored at a temperature range of about 4°C to about 18°C ​​for at least 6 months.

[0963] The present disclosure further provides a product, comprising a container containing the pharmaceutical composition, liquid preparation, lyophilized preparation or reconstituted solution as described above. In some embodiments, the container can be, but is not limited to, a neutral borosilicate glass tube injection bottle.

[0964] The present disclosure also provides use of the pharmaceutical composition, liquid preparation, lyophilized preparation, reconstituted solution or product described in any of the above items in preparing a drug for treating a disease.

[0965] The present disclosure also provides a method for treating or preventing a disease, comprising administering to a subject in need thereof an effective amount of the pharmaceutical composition, liquid preparation, lyophilized preparation, reconstituted solution, or product described above.

[0966] The present disclosure also provides the pharmaceutical composition as described in any one of the above items, the liquid preparation as described in any one of the above items, the lyophilized preparation as described in any one of the above items, the reconstituted solution as described in any one of the above items, or the product as described in any one of the above items, for use in treating a disease.

[0967] In some embodiments, the disease is selected from a disease associated with abnormal angiogenesis, a central nervous system disease or condition.

[0968] In some embodiments, the disease is a disease associated with abnormal angiogenesis; in some embodiments, the disease associated with abnormal angiogenesis is an vascular eye disease or cancer.

[0969] In some embodiments, the disease is a central nervous system disease or symptom; preferably, the central nervous system disease or symptom is a neurodegenerative disease or symptom, more preferably a movement disorder, a sleep disorder, and most preferably Parkinson's disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0970] FIG1A shows the amount of AADC protein expressed by each AAV plasmid after AADC codon optimization; FIG1B shows the amount of GDNF protein expressed by each AAV plasmid after GDNF codon optimization.

[0971] FIG2A shows the immunofluorescence signal of AADC in the striatum of mice after AAV2 and AAV9 serotype transfection; FIG2B shows the immunofluorescence coverage area of ​​AADC in the striatum of mice after AAV2 and AAV9 serotype transfection.

[0972] FIG3A shows the expression level of AADC in the striatum after injection of AAV2-02A and AAV9-02A; FIG3B shows the expression level of GDNF in the striatum after injection of AAV2-02A and AAV9-02A.

[0973] Figure 4A shows the response of mice to L-DOPA after injection of HRPDAAV02-AI (i.e., 02AI, the same below) and HRPDAAV03-DI (i.e., 03DI, the same below) (counterclockwise rotation); Figure 4B shows the response of mice to L-DOPA (contralateral forelimb utilization) after injection of HRPDAAV02-AI and HRPDAAV03-DI.

[0974] Figure 5A shows the response of mice to L-DOPA after injection of HRPDAAV02-AI and HRPDAAV03-DI drugs (DOPA concentration on the non-administration side); Figure 5B shows the response of mice to L-DOPA after injection of HRPDAAV02-AI and HRPDAAV03-DI drugs (DOPA concentration on the administration side); Figure 5C shows the response of mice to L-DOPA after injection of HRPDAAV02-AI and HRPDAAV03-DI drugs (DOPA concentration in the blood); Figure 5D shows the protection of dopamine neurons in mice after injection of HRPDAAV02-AI and HRPDAAV03-DI drugs, and the immunofluorescence signal density of dopamine nerve terminals was statistically analyzed.

[0975] Figure 6A shows the response of each group of animals to apomorphine after 6-OHDA modeling; Figure 6B shows the response of rats to L-DOPA after injection of HRPDAAV02-AI and HRPDAAV03-DI (rotation test); Figure 6C shows the test results of the rat model established in the cylinder test experiment; Figure 6D shows the response of rats to L-DOPA after injection of HRPDAAV02-IA and HRPDAAV03-DI (contralateral forelimb utilization); 6E shows the results of the test of the metabolic capacity of the striatum to L-DOPA after administration of HRPDAAV02-IA and HRPDAAV03-DI (DOPA ratio of the affected side / healthy side); 6F shows the protective effect of HRPDAAV-03DI on DA neurons.

[0976] In the present disclosure, **** represents p<0.0001, *** represents p between 0.0001 and 0.001, both of which indicate extremely significant statistical differences; ** represents p between 0.001 and 0.01, indicating a very significant statistical difference; * represents p between 0.01 and 0.05, indicating a significant statistical difference.

[0977] Figure 7: Schematic representation of the molecular format of VEGF binding molecules.

[0978] FIG8 : Schematic diagram of the vector structure containing the nucleic acid molecule expressing the VEGF binding molecule.

[0979] Figure 9: Schematic diagram of AAV packaging plasmids.

[0980] Figure 10: Results of stability test of VEGF-binding molecules in rabbit double vitreous.

[0981] FIG11A shows the results of fundus fluorescein angiography (FFA) 7 days after rat CNV modeling and 14 days after drug administration.

[0982] FIG11B : The results of the inhibition test of fluorescein sodium leakage in choroidal neoformation in rat CNV model 14 days after drug administration.

[0983] Figure 12: Expression detection results of AAV-1174 and ADVM-022 in rabbit aqueous humor, vitreous, retina and choroid.

[0984] FIG13 : The results of the test on the inhibition of choroidal neoplastic fluorescein sodium leakage by administering AAV-1174 and ADVM-022 to the mouse CNV model.

[0985] Figure 14: Results of investigation on the effects of different formulations on virus stability.

[0986] Figure 15: Results of investigation on the effect of temperature on the pH of the buffer solution (pH 7.5, 27.2°C).

[0987] Figure 16: Results of investigating the effect of temperature on the pH of buffer solutions with different pH values.

[0988] Figure 17: Results of investigation on pH sensitivity to temperature of different buffer systems. DETAILED DESCRIPTION

[0989] the term

[0990] In order to make the present disclosure more easily understood, certain technical and scientific terms are specifically defined below. Unless otherwise explicitly defined herein, all other technical and scientific terms used herein have the meanings commonly understood by those skilled in the art to which the present disclosure belongs.

[0991] "Buffer" refers to a buffer that tolerates changes in pH through the action of its acid-base conjugate components. Examples of buffers that control pH within an appropriate range include acetate, succinate, gluconate, histidine, oxalate, lactate, phosphate, citrate, tartrate, fumarate, glycylglycine, and other organic acid buffers.

[0992] A "histidine buffer" is a buffer containing histidine ions. Examples of histidine buffers include acetate-histidine, succinate-histidine, histidine-histidine hydrochloride, hydrochloride-histidine, and sulfate-histidine. For example, a histidine-hydrochloride buffer is a mixture of histidine and hydrochloric acid, or histidine and histidine hydrochloride.

[0993] A "pharmaceutical composition" refers to a mixture containing one or more compounds described herein, or their physiologically / pharmaceutically acceptable salts or prodrugs, together with other chemical components, such as physiologically / pharmaceutically acceptable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration to an organism, facilitating absorption of the active ingredient and thereby exerting its biological activity. As used herein, the terms "pharmaceutical composition" and "preparation" are not mutually exclusive.

[0994] Unless otherwise specified, the solvent in the solution form of the pharmaceutical composition described in the present disclosure is water.

[0995] As used herein, the term "about" refers to a value that is within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which value depends in part on how the value is measured or determined (i.e., the limitations of the measurement system). For example, "about" can mean within 1 or more than 1 standard deviation per practice in the art. Alternatively, "about" or "substantially comprising" can mean a range of up to 20%. Furthermore, particularly with respect to biological systems or processes, the term can mean up to an order of magnitude or up to 5 times the value. Unless otherwise indicated, when a specific value appears in the application and claims, the meaning of "about" or "substantially comprising" should be assumed to be within an acceptable error range for that specific value.

[0996] The pharmaceutical compositions disclosed herein can achieve a stable effect: the antibodies therein substantially retain their physical stability and / or chemical stability and / or biological activity after storage. Preferably, the pharmaceutical compositions substantially retain their physical and chemical stability as well as their biological activity after storage. The storage period is generally selected based on the intended shelf life of the pharmaceutical composition. Currently, there are various analytical techniques for measuring protein stability after storage at a selected temperature for a selected period of time.

[0997] A stable pharmaceutical antibody formulation is one in which no significant change is observed after storage at refrigerated temperatures (2-8°C) for at least 3 months, preferably 6 months, more preferably 1 year, and even more preferably up to 2 years. Additionally, stable liquid formulations include those that exhibit the desired characteristics after storage at 25°C for a period of time, including 1 month, 3 months, 6 months, or 40°C for 1 month. Typical acceptable standards for stability are as follows: typically no more than about 10%, preferably no more than about 5%, of the antibody monomers degrade as measured by SEC-HPLC. By visual analysis, the pharmaceutical antibody formulation is colorless or yellow, clear to slightly opalescent. The concentration, pH, and weight-gram molecular osmotic pressure concentration of the formulation vary by no more than ±10%. Typically, no more than about 10%, preferably no more than about 5%, of truncation is observed, and typically no more than about 10%, preferably no more than about 5%, of aggregation is formed.

[0998] An antibody "retains its physical stability" in a pharmaceutical formulation if it shows no significant increase in aggregation, precipitation, and / or denaturation as measured by visual inspection of color and / or clarity, or by UV light scattering, size exclusion chromatography (SEC), and dynamic light scattering (DLS). Changes in protein conformation can be assessed by fluorescence spectroscopy (which determines protein tertiary structure) and by FTIR spectroscopy (which determines protein secondary structure).

[0999] An antibody "retains its chemical stability" in a pharmaceutical formulation if it shows no significant chemical changes. Chemical stability can be assessed by detecting and quantifying chemically altered forms of the protein. Degradation processes that often change the chemical structure of a protein include hydrolysis or truncation (assessed by methods such as size exclusion chromatography and SDS-PAGE), oxidation (assessed by methods such as peptide mapping in combination with mass spectrometry or MALDI / TOF / MS), deamidation (assessed by methods such as ion exchange chromatography, capillary isoelectric focusing, peptide mapping, isoaspartate measurement), and isomerization (assessed by measuring isoaspartate content, peptide mapping, etc.).

[1000] "AAV" is an abbreviation for adeno-associated virus and can be used to refer to the virus itself or its derivatives. Unless otherwise specified, it encompasses all AAV subtypes and naturally occurring and recombinant forms. "AAV" includes, but is not limited to, AAV type 1 (AAV1), AAV type 2 (AAV2), AAV type 3 (AAV3), AAV type 4 (AAV4), AAV type 5 (AAV5), AAV type 6 (AAV6), AAV type 7 (AAV7), AAV type 8 (AAV8), AAV type 9 (AAV9), AAV type 10 (AAV10), AAVrh10 (AAVrh10), and different species of avian AAV, bovine AAV, canine AAV, equine AAV, primate AAV, non-primate AAV, and ovine AAV.

[1001] AAV is a non-pathogenic parvovirus composed of a 4.7 kb single-stranded DNA genome within a non-enveloped icosahedral capsid. The genome contains three open reading frames (ORFs) flanked by inverted terminal repeats (ITRs) that serve as viral replication origins and packaging signals. The Rep ORF encodes four non-structural proteins that play a role in viral replication, transcriptional regulation, site-specific integration, and virion assembly. The Cap ORF encodes three structural proteins (VP1-3) that assemble to form the 60-mer viral capsid. Finally, the ORF, which exists as an alternative reading frame within the cap gene, produces assembly activation protein (AAP), a viral protein that localizes the AAV capsid protein in the nucleus and plays a role in the capsid assembly process.

[1002] The genomic sequences of various serotypes of AAV, as well as natural terminal repeat (ITR) sequences, Rep protein sequences, and capsid subunit sequences are known in the art. Such sequences can be found in the literature or in public databases such as GenBank. See, for example, GenBank accession numbers NC_002077.1 (AAV1), AF063497.1 (AAV1), NC_001401.2 (AAV2), AF043303.1 (AAV2), J01901.1 (AAV2), U48704.1 (AAV3), NC_001729.1 (AAV3), NC_001829.1 (AAV4), U8 9790.1 (AAV4), NC_006152.1 (AAV5), AF085716.1 (AAV5), AF028704.1 (AAV6), NC_006260.1 (AAV7), AF513851.1 (AAV7), AF513852.1 (AAV8) NC_006261.1 (AAV8), and AY530579.1 (AAV9). Also, Srivistava et al. (1983) J. Virology 45:555; Chiorini et al. (1998) J. Virology 71:6823; Chiorini et al. (1999) J. Virology 73:1309; Bantel-Schaal et al. (1999) J. Virology 73:939; Xiao et al. (1999) J. Virology 73:3994; Muramatsu et al. (1996) Virology y221:208; Shade et al., (1986) J. Virol. 58:921; Gao et al. (2002) Proc. Nat. Acad. Sci. USA 99:11854; Moris et al. (2004) Virology 33:375-383; and WO00 / 28061, WO99 / 61601, WO98 / 11244, US6156303, WO2012145601A, WO2017197355A, WO2018022905A. The above are incorporated into the present disclosure in their entirety.

[1003] For the GH loop or loop IV of the AAV capsid, see, eg, van Vliet et al. (2006) Mol. Ther. 14:809; Padron et al. (2005) J. Virol. 79:5047; and Shen et al. (2007) Mol. Ther. 15:1955.

[1004] "AAV viral particle" or "AAV virion" refers to a viral particle composed of at least one AAV capsid protein and an encapsidated AAV polynucleotide.

[1005] "rAAV" refers to recombinant adeno-associated virus. "Recombinant" as applied to polynucleotides means that the polynucleotide is the product of various combinations of cloning, restriction, or ligation steps, as well as other processes that result in a construct that is different from a polynucleotide found in nature. Recombinant viruses are viral particles that include a recombinant polynucleotide.

[1006] If an "AAV virion" includes a heterologous polynucleotide (i.e., a polynucleotide other than the wild-type AAV genome, such as a gene product to be delivered to the target cell (e.g., a transgene, RNAi, etc.), it is often referred to as a "recombinant AAV (rAAV) virion" or "rAAV viral particle" or "rAAV vector virus." Typically, the heterologous polynucleotide is flanked by at least one, and usually two, AAV inverted terminal repeats (ITRs).

[1007] "rAAV vector" encompasses rAAV virions comprising rAAV polynucleotides; and also encompasses polynucleotides encoding rAAV (e.g., single-stranded polynucleotides encoding rAAV (ss-rAAV); double-stranded polynucleotides encoding rAAV (ds-rAAV), such as plasmids encoding rAAV; etc.).

[1008] "AAV variant," "AAV mutant," or "capsid-variant rAAV" refers to a viral particle composed of: (a) a variant AAV capsid protein, wherein the variant AAV capsid protein comprises at least one amino acid difference (e.g., an amino acid substitution, insertion, or deletion) relative to a corresponding parental AAV capsid protein, wherein the AAV capsid protein is different from or does not correspond to the amino acid sequence of a naturally occurring AAV capsid protein; and optionally, (b) comprises a heterologous polynucleotide encoding a heterologous gene product, wherein the variant AAV capsid protein confers increased binding to a heparanoid or heparan sulfate proteoglycan compared to the binding of an AAV viral particle comprising the corresponding parental AAV capsid protein.

[1009] "Packaging" refers to the series of intracellular events that lead to the assembly and encapsidation of AAV particles.

[1010] The "rep" and "cap" genes refer to polynucleotide sequences that encode the replication and encapsidation proteins of adeno-associated virus. AAV rep and cap are referred to herein as AAV "packaging genes."

[1011] "Helper virus" refers to a virus that allows AAV (e.g., wild-type AAV) to be replicated and packaged by mammalian cells. Various helper viruses for AAV are known in the art and include adenoviruses, herpes viruses, and pox viruses such as cowpox. Adenoviruses encompass many different subgroups, but the most commonly used is adenovirus type 5 in subgroup C; viruses of the herpes family include, for example, herpes simplex virus (HSV) and Epstein-Barr virus (EBV), as well as cytomegalovirus (CMV) and pseudorabies virus (PRV). Many adenoviruses, herpes viruses, and the like of human, non-human mammalian, and avian origin are known and available from institutions such as ATCC.

[1012] "Helper virus function" or "helper function" refers to a function encoded in the helper virus genome that allows AAV replication and packaging (in conjunction with other requirements for replication and packaging as described herein). In the present disclosure, "helper virus function" can be provided in many ways, including by providing a helper virus or providing, for example, a polynucleotide sequence encoding the necessary function to the producer cell in transit.

[1013] An "infectious" virus or viral particle is one that includes a suitably assembled viral capsid and is capable of delivering a polynucleotide component to a cell for which the virus species is tropistic, without necessarily implying that the virus has any ability to replicate. Assays for counting infectious viral particles are well known in the art. Viral infectivity can be expressed as a ratio of infectious viral particles to total viral particles. Methods for determining the ratio of infectious viral particles to total viral particles are known in the art. See, e.g., Grainger et al. (2005) Mol. Ther. 11:S337 (describing TCID 50 Infectious titer determination); Zolotukhin et al. (1999) Gene Ther. 6:973.

[1014] "Tropism" or "specificity" refers to the preferential targeting of viruses (e.g., AAV) to cells of a particular host species or specific cell types within a host species. For example, viruses that can infect heart, lung, liver, and muscle cells have a wider (i.e., increased) tropism relative to viruses that may only infect lung and muscle cells. Tropism can also include the dependence of viruses on cell surface molecules of a particular type of host. For example, some viruses may only infect cells with surface glycosaminoglycans, while other viruses may only infect cells with sialic acid (this dependence can be tested as potential host cells for viral infection using various cell lines lacking specific class molecules). In some cases, the tropism of a virus describes the relative preference of the virus. For example, a first virus may be able to infect all cell types, but is more successful in infecting these cells with surface glycosaminoglycans. If a second virus also prefers the same characteristics (e.g., the second virus is also more successful in infecting these cells with surface glycosaminoglycans), it can be considered that the second virus has a similar (or consistent) tropism to the first virus, even if the absolute transduction efficiency is not similar. For example, a second virus may be more effective than a first virus at infecting each given cell type tested, but if the relative preferences are similar (or consistent), the second virus can still be considered to have a tropism similar (or consistent) to that of the first virus. In some embodiments, the tropism of virions comprising variant AAV capsid proteins of the present disclosure is not altered relative to naturally occurring virions. In some embodiments, the tropism of virions comprising variant AAV capsid proteins of the present disclosure is expanded (i.e., broadened) relative to naturally occurring virions. In some embodiments, the tropism of virions comprising variant AAV capsid proteins of the present disclosure is reduced relative to naturally occurring virions.

[1015] "Polynucleotide" refers to a polymeric form of nucleotides of any length, or their analogs, including deoxyribonucleotides or ribonucleotides. A polynucleotide may include modified nucleotides, such as methylated nucleotides and nucleotide analogs, and may be interrupted by non-nucleotide components. Modifications to the nucleotide structure may be made before or after assembly of the polymer. Polynucleotides refer interchangeably to double-stranded molecules and single-stranded molecules, and unless otherwise indicated, the polynucleotides of the present disclosure encompass both the double-stranded form and the two complementary single-stranded forms that comprise the double-stranded form.

[1016] "Homology" or "identity" refers to the sequence similarity between two polynucleotide sequences or between two polypeptides. When a position in the two compared sequences is occupied by the same nucleotide or amino acid monomer subunit, for example, if every position in two DNA molecules is occupied by the same nucleotide, then the molecules are homologous at that position. The percentage homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared × 100%. For example, if 6 out of 10 positions in the two sequences match or are homologous when the sequences are optimally aligned, then the two sequences are 60% homologous. Sequence similarity can be measured in many different ways. To determine sequence identity, sequences can be aligned using methods and computer programs, including BLAST, which is available at the Internet address ncbi.nlm.nih.gov / BLAST / . Another alignment algorithm is FASTA, which is available in the Genetics Computing Group (GCG) package of Madison, Wisconsin, USA, a wholly owned subsidiary of Oxford Molecular Group, Inc. Other alignment techniques are described in Enzymology, Vol. 266: Computer Methods for Macromolecular Sequence Analysis (1996), ed. Doolittle, Academic Press, Inc., an adaptation of the methods of Harcourt Brace & Co., San Diego, California, USA. Of particular interest are alignment programs that tolerate gaps in sequences. Smith-Waterman is a type of algorithm that tolerates gaps in sequence alignments. See Meth. Mol. Biol. 70: 173-187 (1997). The GAP program, which uses the Needleman and Wunsch alignment method, can also be used to align sequences. See J. Mol. Biol. 48: 443-453 (1970).

[1017] "Gene" refers to a polynucleotide containing at least one open reading frame that can encode a specific gene product after transcription and sometimes translation. "Gene" or "coding sequence" refers to the in vitro or in vivo nucleotide sequence that encodes the gene product. In some cases, a gene consists of or essentially consists of the coding sequence, which is the sequence that encodes the gene product. In other cases, a gene includes additional non-coding sequences. For example, a gene may or may not include regions before and after the coding region (e.g., 5'UTR, 3'UTR, and intervening sequences (introns) between the individual coding segments (exons)).

[1018] "Gene product" is a molecule produced by the expression of a specific gene, such as a polypeptide, aptamer, interfering RNA, mRNA, etc. In some embodiments, the "gene product" is a polypeptide, peptide, protein or interfering RNA, including short interfering RNA (siRNA), miRNA or small hairpin RNA (shRNA). In some specific embodiments, the gene product is a therapeutic gene product, such as a therapeutic polypeptide. The therapeutic gene product confers a beneficial effect on the cell, tissue or mammal in which it is located, and the beneficial effect includes improving the signs or symptoms of a condition or disease, preventing or inhibiting a condition or disease, or conferring a desired characteristic. When a gene encodes a polypeptide, "gene product" and "product of expression of the target gene" can be used interchangeably.

[1019] "Polypeptide," "peptide," and "protein" refer to amino acid polymers of any length and include amino acid polymers that have been modified, for example, by disulfide bond formation, glycosylation, lipidation, phosphorylation, or conjugated to a labeling component.

[1020] "Regulatory elements" or "regulatory sequences" are nucleotide sequences that participate in molecular interactions that contribute to the functional regulation of a polynucleotide, including replication, duplication, transcription, splicing, translation, or degradation of the polynucleotide. Regulation can affect the frequency, speed, or specificity of a process and can be enhancing or inhibitory in nature. Known control elements include, for example, transcriptional regulatory sequences such as promoters and enhancers. A promoter is a region of DNA that is capable of binding RNA polymerase under certain conditions and initiating transcription of a coding region that is typically located downstream (in the 3' direction) of the promoter.

[1021] An "expression vector" is a vector comprising a region encoding a target gene product, and is used to achieve expression of the gene product in the intended target cell, the vector comprising a polynucleotide encoding the gene product of interest. The expression vector also includes control elements operatively linked to the coding region to promote expression of the gene product in the target. A combination of control elements such as promoters, enhancers, UTRs, miRNA targeting sequences, and the like, and one or more genes operatively linked thereto for expression is sometimes referred to as an "expression cassette." Many expression cassettes are known and available in the art, or can be easily constructed from components available in the art.

[1022] "Operably linked" or "operably linked" refers to a juxtaposition of genetic elements wherein the elements are in a relationship permitting them to operate in their intended manner. For example, a promoter is operably linked to a coding polynucleotide sequence if the promoter helps initiate transcription of the coding polynucleotide sequence; there may be intervening nucleic acid residues between the promoter and the coding polynucleotide sequence as long as this functional relationship is maintained.

[1023] "Administering" or "introducing" refers to delivering a vector for expression of a recombinant gene or protein to a cell, a cell and / or an organ of a subject, or a subject. Such administration or introduction can occur in vivo, in vitro, or ex vivo. A vector for expression of a gene product can be introduced into a cell by:

[1024] In cells: transfection, which generally refers to the insertion of heterologous DNA into cells by physical means (e.g., calcium phosphate transfection, electroporation, microinjection, or lipofection); infection, which generally refers to the introduction of an infectious agent, i.e., a virus; or transduction, which generally refers to the stable infection of a cell with a cell or the transfer of genetic material from one microorganism to another microorganism by a viral agent (e.g., a bacteriophage). "Transformation" is generally used to refer to bacteria that include heterologous DNA or cells that express oncogenes and have been converted to a continuous growth mode, such as tumor cells. The vector used to "transform" the cell can be a plasmid, a virus, or other medium. Depending on the method used to administer, introduce, or insert the heterologous DNA (i.e., the vector) into the cell, the cell is generally referred to as being "transduced," "infected," "transfected," or "transformed." "Transduction," "transfection," and "transformation" can be used interchangeably herein without regard to the method of introduction of the heterologous DNA.

[1025] "Host cell" refers to a cell that has been transduced, infected, transfected, or transformed with a vector, and encompasses the initially transduced, infected, transfected, or transformed cell and its progeny. The vector can be a plasmid, a viral particle, a bacteriophage, or the like. Culture conditions such as temperature and pH will be apparent to those skilled in the art.

[1026] "Treatment" is generally used to mean obtaining a desired pharmacological and / or physiological effect. The effect may be prophylactic in terms of completely or partially preventing a disease or its symptoms, e.g., reducing the likelihood of the disease or its symptoms occurring in a subject, and / or may be therapeutic in terms of partially or completely curing the disease and / or adverse reactions caused by the disease. "Treatment" covers any treatment of a disease in a mammal and includes: (a) preventing the disease from occurring in a subject who may be predisposed to the disease but has not yet been diagnosed as having the disease; (b) inhibiting or halting the development of the disease; or (c) alleviating the disease (or the symptoms it causes) or causing the disease to regress. The therapeutic agent may be administered before, during, or after the onset of the disease or injury. In particular, treatment of an ongoing disease wherein the treatment stabilizes or reduces the patient's undesirable clinical symptoms. Some preferred embodiments are to perform such treatment before the function of the affected tissue is completely lost. Some preferred embodiments are to administer the disclosed treatment during, and in some cases after, the symptomatic phase of the disease.

[1027] An "effective amount" encompasses an amount sufficient to ameliorate or prevent the symptoms or signs of a medical condition. An effective amount also means an amount sufficient to permit or facilitate diagnosis. The effective amount for a particular subject may vary depending on factors such as the condition to be treated, the subject's overall health, the route and dosage of administration, and the severity of side effects. An effective amount can be the maximum dose or dosage regimen that avoids significant side effects or toxic effects.

[1028] "Retinal cell" in this disclosure may refer to any of the cell types that comprise the retina, such as retinal ganglion cells; amacrine cells; horizontal cells; bipolar cells; photoreceptor cells including rods and cones; Müller glial cells; astrocytes (e.g., retinal astrocytes); and retinal pigment epithelial cells.

[1029] "Individual," "subject," and "patient" are used interchangeably herein and include, but are not limited to, humans and non-human primates, such as monkeys, humans and other mammals (e.g., horses, sheep, goats, dogs, cats, and rodents (e.g., mice, rats, etc.)), preferably humans.

[1030] In this disclosure, "polypeptide" and "protein" are used interchangeably.

[1031] Example

[1032] The present disclosure is further described below with reference to the following examples, but these examples are not intended to limit the scope of this disclosure. Experimental methods in the examples herein where specific conditions are not specified generally follow conventional conditions, such as those in the Cold Spring Harbor Laboratory Manual of Antibody Techniques and the Molecular Cloning Manual, or the conditions recommended by the raw material or product manufacturer. Reagents where the specific source is not specified are commercially available.

[1033] Example 1. AADC and GDNF polynucleotide structure design

[1034] In this embodiment, the coding polynucleotides of AADC and GDNF are sequenced and engineered for transduction of cells or for in vivo transduction of humans and animals. The sequences can be inserted into plasmids, lentiviruses, adenoviruses, mRNA-LNPs, and adeno-associated virus (AAV) vectors, for example, by recombinant adeno-associated virus (rAAV) to achieve in vivo and in vitro transduction. The specific sequences between the ITRs of the rAAV plasmid structure are shown in Table 1. The enhancers used may include, for example, the CMV enhancer, and the promoters may include, for example, the CMV promoter and the CBA promoter. The sequences in Table 2 are all codon-unoptimized.

[1035] Table 1. Modified AADC / GDNF polynucleotide sequences (Note: AADC / GDNF includes both AADC and GDNF, the same below)

[1036] Example 2. Codon optimization of AADC and GDNF

[1037] 1. In this example, four codon-optimized polynucleotide sequences were designed for AADC and GDNF, respectively. The wild-type polynucleotide sequence of AADC is shown in SEQ ID NO:3, and the codon-optimized sequences AADC01, AADC02, AADC03, and AADC04 are shown in SEQ ID NOs:4-7, respectively. The sequence identities of SEQ ID NOs:4-7 to SEQ ID NO:3 are 80.83%, 81.21%, 79.18%, and 79.63%, respectively. The wild-type polynucleotide sequence of GDNF is shown in SEQ ID NO:8, and the codon-optimized sequences GDNF01, GDNF02, GDNF03, and GDNF04 are shown in SEQ ID NOs:9-12, respectively. The sequence identities of SEQ ID NOs:9-12 to SEQ ID NO:8 are 75.13%, 73.99%, 77.52%, and 77.83%, respectively.

[1038] AADC01, AADC02, AADC03, and AADC04 were substituted for the AADC sequence in HRPDAAV02-A, respectively, to obtain HRPDAAV02-A(AADC01), HRPDAAV02-A(AADC02), HRPDAAV02-A(AADC03), and HRPDAAV02-A(AADC04). GDNF 01, GDNF 02, GDNF 03, and GDNF 04 were substituted for the GDNF sequence in HRPDAAV02-A, respectively, to obtain HRPDAAV02-A(GDNF01), HRPDAAV02-A(GDNF02), HRPDAAV02-A(GDNF03), and HRPDAAV02-A(GDNF04). Screening was then performed to obtain the codon-optimized AADC and GDNF sequences (AADC01 and GDNF04) with the highest expression and function. The results are shown in Figures 1A and 1B.

[1039] After replacing AADC and GDNF in HRPDAAV02-A and HRPDAAV03-D with AADC01 (SEQ ID NO: 3) and GDNF04 (SEQ ID NO: 12), HRPDAAV02-AI (SEQ ID NO: 24) and HRPDAAV03-DI (SEQ ID NO: 25) were obtained. HRPDAAV02-AI and HRPDAAV03-DI correspond to 02AI and 03DI, respectively. The structures of the two are shown in Table 2. The codon-optimized sequence of HRPDAAV02-AI can also be SEQ ID NO: 140, and the codon-optimized sequence of HRPDAAV03-DI can also be SEQ ID NO: 141.

[1040] Table 2. Sequence structures of HRPDAAV02-AI and HRPDAAV03-DI

[1041] 2. HRPDAAV-02A was packaged and purified using AAV2 serotype (amino acid sequence see SEQ ID NO: 41) and AAV9 serotype (amino acid sequence see SEQ ID NO: 42) to obtain AAV2-02A and AAV9-02A.

[1042] Male Sprague-Dawley rats were anesthetized with 50 mg / kg of Zotalitol (ip) and placed in a stereotaxic apparatus. A midsagittal incision was made along the skull to expose the bregma. Position 1 was located in the right striatum: AP -0.0 mm, ML -2.6 mm, DV -4.7 mm; position 2: AP 0.0 mm, ML -2.6 mm, DV -4.7 mm. A dental drill was used to open the skull. A microsyringe was inserted and 2 μL of the test drug was automatically injected into each site using a microperistaltic pump (0.2 μL / min). After injection, the needle was left in place for 10 minutes, then slowly withdrawn and the wound sutured. Each rat was subcutaneously injected with 0.3 mL of levofloxacin to prevent infection. The rats were housed for 8 weeks after surgery.

[1043] 1) Rats were perfused with 4% paraformaldehyde and brains were removed. Postfixed for at least 24 hours, the specimens were transferred to a 30% sucrose solution and thoroughly dehydrated. Coronal sections of the striatum were sliced ​​using a freezing microtome at a thickness of 30 μm, placed in cryopreservative solution, and stored at 4°C. Brain sections were washed three times in PBS, treated with 0.3% Triton X100 for 10 minutes, and blocked in PBS containing 10% goat serum for 1 hour. The primary antibody was rabbit anti-AADC (Absin, abs110350) (1:1000) and incubated overnight at 4°C. The next day, sections were washed three times with PBS for 5 minutes each, incubated with donkey anti-rabbit IgG Alexa Fluor 488 antibody (1:1500) and the nuclear dye DAPI (1:15000) for 1 hour at room temperature, washed three times with PBS, and mounted with Dako anti-fluorescence quenching mounting medium. Observation and photography were performed under an Olympus fluorescence microscope. As shown in Figures 2A and 2B , the staining results showed that the fluorescent area of ​​AADC was significantly larger after injection of AAV9-02A than that of AAV2-02A (P<0.01, AAV2-02A vs AAV9-02A), demonstrating that AAV9 has a higher expression efficiency in the striatum than AAV2.

[1044] 2) Rats were raised for 4 or 8 weeks after brain localization surgery and then decapitated for extraction of striatal tissue on the injected side. Tissue was weighed and stored at -80°C. Pre-chilled PBS (1 mg tissue: 10 μL PBS) was added to the tissue for lysis. The sample was thoroughly homogenized using a pre-chilled tissue grinder. 100 μL of the homogenate was used for HPLC experiments, 40 μL for tissue DNA extraction and qPCR experiments, and the remaining volume was used for ELISA experiments.

[1045] AADC and GDNF concentrations were measured by ELISA. The ELISA experiment was performed strictly according to the instructions of the kit (RayBiotech, ELH-DDC). The specific experimental steps are as follows:

[1046] The method for detecting AADCs is as follows: 100 μL of dissolved standard or sample was added to each well of the kit (RayBiotech, ELH-DDC), incubated at room temperature for 2.5 hours, dried, and washed; 100 μL of biotin antibody was added to each well, incubated at room temperature for 1 hour, dried, and washed; 100 μL of Streptavidin solution was added to each well, incubated at room temperature for 45 minutes, dried, and washed; 100 μL of TMB was added to each well, incubated at room temperature for 30 minutes, dried, and washed. 50 μL of stop solution was added to each well. Spectrometers were immediately read at 450 nm.

[1047] The method for detecting GDNF is as follows: 100 μL of dissolved standard or sample was added to each well of the kit (R&D, DY212), incubated at room temperature for 2 hours, dried, and washed; 100 μL of detection antibody was added to each well, incubated at room temperature for 2 hours, dried, and washed; 100 μL of Streptavidin-HRP solution was added to each well, incubated at room temperature for 20 minutes, dried, and washed; 100 μL of substrate solution was added to each well, incubated at room temperature for 20 minutes. 50 μL of stop solution was added to each well. Spectrometers were immediately read at 450 nm.

[1048] As shown in Figures 3A and 3B , the results of target gene expression level detection showed that the expression levels of AADC and GDNF in the striatum of the AAV9-02A group were significantly higher than those of the AAV2-02A group, demonstrating that AAV9 has a higher transduction efficiency in the striatum than AAV2.

[1049] Example 3. Pharmacodynamic Verification of AAV Delivery and Expression of Target Protein

[1050] 1. Validation of drug efficacy in MPTP-induced mouse PD model

[1051] In this example, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) was used to establish a mouse model of Parkinson's disease. MPTP is highly lipid-soluble and easily crosses the blood-cerebrospinal fluid barrier. Once inside the brain, it is converted to methyl-phenylpyridinium ion (MPP+) by glial cell monoamine oxidase B. MPP+ is actively taken up by DA transporters into the mitochondria of dopamine (DA) neurons, leading to DA neuronal degeneration and death. This process closely mimics the symptoms and pathogenesis of Parkinson's disease. Intraperitoneal injection of MPTP for five consecutive days in adult male mice can induce DA neuronal apoptosis and cause Parkinson's-like symptoms in the mice.

[1052] Mice were divided into four groups: a sham-operated control group (normal control), a model control group (MPTP model control, the same below), an 02AI group (HRPDAAV-02AI), and an 03DI group (HRPDAAV-03DI). Eight-week-old male mice were injected unilaterally with AAV9 capsids into the striatum. The sham-operated and model control groups underwent sham surgery. C57BL / 6N male mice were anesthetized with 1% sodium pentobarbital (35-50 mg / kg) intraperitoneally. After confirming anesthesia, the mice were placed in a prone position on a stereotaxic apparatus (Rayward, Model 68018). After routine disinfection of the instruments, the scalp was incised, and the soft tissue and periosteum were bluntly dissected to expose the right parietal bone. A bone window was drilled 0.8 mm anterior to the bregma (AP = +0.8 mm) and 1.8 mm lateral to the midline (ML = 1.8 mm). AAV was injected using a microsyringe (1 μL, outer diameter: 0.55 mm). The needle was advanced at a speed of 1 mm / min to 3 mm below the dura mater (DV = -3 mm) and withdrawn to 2.8 mm (DV = -2.8 mm). The liquid was then injected at a constant rate of 0.2 μL / min. After the injection, the needle was retained for 3 min and withdrawn at a speed of 1 mm / min.

[1053] Twenty-eight days after the striatal injection, mice in the model control and treatment groups were intraperitoneally injected with 30 mg / kg of MPTP (10 mL / kg). Mice in the sham-operated group were intraperitoneally injected with 10 mL / kg of normal saline. Five days after modeling, mice rested for three days before behavioral testing.

[1054] 1) Rotation test

[1055] To verify the response of AADC delivered by HRPDAAV-02AI and HRPDAAV-03DI to L-DOPA following unilateral striatal expression, a rotational test was performed. Each group of mice was gavaged with 20 mg / kg of L-DOPA and 6 mg / kg of benserazide. Following L-DOPA administration, the mice were placed in the test environment for acclimatization. The number of rotations was recorded 45 minutes after administration, and the number of rotations on the unaffected side (left-sided rotations) was measured within 30 minutes.

[1056] The results are shown in Figure 4A and Table 3. Compared with the sham control group and the model control group, the HRPDAAV-02AI and HRPDAAV-03DI groups had significantly increased rotation times after L-DOPA administration ("02AI group vs. model control group", p = 0.07; "03DI group vs. model control group", p = 0.01), demonstrating that the AADC expressed by AAV delivery has a good response effect to L-DOPA.

[1057] Table 3. Results of mouse rotation test

[1058] 2) Cylinder test

[1059] To examine the forelimb preference of mice under baseline conditions, a cylinder test was performed. Four days after the last MPTP injection, mice were gavage-administered L-DOPA (10 mg / kg) plus benserazide (3 mg / kg). The mice were then placed in a transparent cylinder (10 cm diameter, 45 cm height). The number of times the left, right, or bilateral forelimbs touched the wall was recorded over a 5-minute period. Each mouse, using its hind limbs as a support, touched the wall with one or both forelimbs before returning to the bottom of the cylinder, and was counted as a single forelimb touch. (A bilateral forelimb touch was counted if one limb touched the wall first, followed by the other limb.) After each experiment, the cylinder was wiped with alcohol. Left forelimb usage was analyzed as follows: Left forelimb usage (%) = (left + 0.5 bilateral) / (right + left + bilateral) × 100.

[1060] The results, shown in Figure 4B and Table 4, show that after L-DOPA administration, contralateral forelimb utilization increased in the HRPDAAV-02AI and HRPDAAV-03DI groups compared with the model control group, with the 02AI group increasing by 20.4% and the 03DI group increasing by 8.2%. This further demonstrates the L-DOPA response of AADCs delivered by AAV.

[1061] Table 4. Mouse cylinder test results

[1062] 3) Dopamine detection in striatum and blood

[1063] After the behavioral experiment, mice in each group were gavage-administered L-DOPA (20 mg / kg, 5 mL / kg) and benserazide (5 mg / kg, 5 mL / kg). Mice were sacrificed 1 hour after the administration. Peripheral blood was collected before sacrifice for dopamine analysis. After sacrifice, the striatum of the healthy and affected sides was obtained. DOPA was detected using UPLC-MS / MS.

[1064] As shown in Figure 5A and Table 5-1, DOPA detection in the left striatum (non-drug administration side) revealed that DOPA levels in both the model control group and the drug administration group were significantly lower than those in the sham operation control group, demonstrating the success of MPTP modeling.

[1065] Table 6-1. DOPA detection results of the left striatum of mice (non-drug administration side)

[1066] As shown in Figure 5B and Table 5-2, DOPA analysis in the right striatum (administered side) revealed a significant increase in DOPA in both the HRPDAAV-02AI and HRPDAAV-03DI groups compared to the model control group. DOPA levels returned to those in the sham-operated control group.

[1067] Table 5-2. DOPA detection results of the right striatum of mice (drug administration side)

[1068] Referring to FIG5C and Table 6-3, plasma DOPA detection revealed little difference among the groups, demonstrating that the effects of HRPDAAV-02AI and HRPDAAV-03DI were mainly confined to the striatum of the brain, with no significant leakage.

[1069] Table 5-3. Mouse plasma DOPA test results

[1070] 4) Immunohistochemistry of brain tissue

[1071] Each group of mice was perfused through the heart to obtain brain tissue for fluorescent immunohistochemistry detection. The specific method was as follows: after gross observation of the experimental mice, the heart tissue in the chest cavity was freed, an intravenous infusion needle was inserted from the left ventricle and fixed, the right atrial appendage was cut open, and pre-cooled PBS was used for perfusion until transparent liquid flowed out of the right atrial appendage. Then 4% paraformaldehyde was perfused through the heart, the brain tissue was cut, and placed in a fixative (4% paraformaldehyde solution). The fixed tissue was sliced ​​using paraffin for tyrosine dehydrogenase TH staining.

[1072] As shown in Figure 5D and Table 6, fluorescence immunohistochemistry analysis revealed that the TH fluorescence intensity was comparable between the sham and model control groups, while the TH fluorescence intensity in the right striatum (dose side) of the HRPDAAV-02AI and HRPDAAV-03DI groups was significantly increased compared to the left striatum (non-dose side), demonstrating that HRPDAAV-02AI and HRPDAAV-03DI drugs have a strong protective effect on dopamine neurons in mice.

[1073] Table 6. Results of immunohistochemistry of mouse brain tissue

[1074] 2. Validation of drug efficacy in 6-OHDA-induced PD rat model

[1075] In this example, 6-OHDA was used to establish a rat PD model. 6-OHDA is a neurotoxin. Due to its structure being similar to dopamine (DA), it is often mistakenly taken up into DA neurons as a neurotransmitter. It selectively causes DA neuron death by forming hydroxyl free radicals, inhibiting mitochondrial oxidative respiratory chain complexes, and interfering with ATP synthesis. The DA content in the damaged striatum decreases, causing rats to develop symptoms similar to human PD. The model is reliable, stable, and irreversible, and its behavioral deficits can be quantitatively analyzed intuitively. It is widely used to evaluate anti-Parkinson's drugs, especially those that are effective against DA and its receptors. Therefore, this example uses a unilateral MFB injection of 6-OHDA to induce a Parkinson's model to evaluate the efficacy of HRPDAAV-02AI and HRPDAAV-03DI in treating Parkinson's disease.

[1076] The method is as follows: SD rats were divided into 6 experimental groups, namely sham operation control group (i.e. normal control group), model control group, 02AI high-dose group (1E+10vg / dose), 02AI low-dose group (2E+9vg / dose), 03DI high-dose group (1E+10vg / dose), and 03DI low-dose group (2E+9vg / dose), all using AAV9 capsid. Rats were anesthetized by intraperitoneal injection of 3% sodium pentobarbital (at a dose of 50-60 mg / kg) and fixed in a prone position. After routine disinfection of the instruments, the scalp was incised, the soft tissue and periosteum were bluntly peeled off, the right parietal bone was exposed, the connective tissue near the midline was removed, the sagittal suture, anterior fontanel and lambdoid point were determined, and a microsyringe (specification: 10 μL, outer diameter: 0.5 mm) containing the modeling reagent was fixed on a stereotaxic instrument for coordinate positioning. The striatum was positioned on the right side of the rat with the anterior fontanel as the positioning center point, and a bone window was drilled 0.0 mm in front of the anterior fontanel (AP=+0.0 mm) and 3 mm lateral to the midline (ML=3 mm). The needle was inserted at a speed of 1 mm / min, advanced 6 mm below the dura mater (DV = -6 mm), withdrawn to 5.8 mm (DV = -5.8 mm), and 2 μL of liquid was injected at a constant speed of 0.6 μL / min. After the injection, the needle was withdrawn to 3.8 mm (DV = -3.8 mm), and 2 μL of liquid was injected at a constant speed of 0.6 μL / min. After the two-point injection, the needle was retained for 3 minutes, and the needle withdrawal speed was 2 mm / min. The scalp was sutured and disinfected, and placed on a 37°C heating pad until the rat woke up. Penicillin sodium (80,000 units / day) and ibuprofen oral solution were injected for three consecutive days after the operation.

[1077] Modeling was performed 3 weeks after the striatum was injected with drugs. 3% sodium pentobarbital was used for intraperitoneal anesthesia. The injection dose was 60 mg / kg and the injection volume was 2 mL / kg. After anesthesia induction, the rats were transferred to the operating table and fixed in a prone position on a brain stereotaxic instrument to ensure that the brain was on a horizontal plane. The rats' eyelid reflex and pain response were observed. The surgery could be started only after the eyelid reflex reaction and the pain response of the limbs and tail disappeared. Use the smallest drill bit to drill vertically, taking care not to deviate and injure the brain tissue. Position the rat on the right side (drug administration side) and drill a bone window 2.0 mm (AP = -2.0 mm) posterior to the anterior fontanelle and 2 mm (ML = 2 mm) beside the midline. The needle was advanced at a speed of 6 mm / min to 8.8 mm below the dura mater (DV = -8.8 mm) and withdrawn to 8.5 mm (-8.5 mm). 6-OHDA was injected at a constant rate of 0.6 μL / min, with each rat receiving 4 μL of 6-OHDA (5 μg / μL). After the injection, the needle was retained for 3 minutes and withdrawn at a speed of 2 mm / min. The scalp was sutured and disinfected, and the rat was placed on a 37°C heating pad until awake. For three consecutive days postoperatively, penicillin sodium (80,000 units / day) and ibuprofen oral solution were administered orally.

[1078] Three weeks after modeling, the rats were tested for their response to apomorphine. Apomorphine (0.5 mg / kg) was administered intraperitoneally to the rats. Ten minutes later, the rats were placed in the test environment and the number of rotations within 30 minutes was recorded.

[1079] As shown in FIG6A , the 6-OHDA model rats showed a significantly increased number of rotations compared to the sham-operated control group, demonstrating that the modeling experiment was successful.

[1080] 1) Rotation test

[1081] To examine the response of exogenously expressed AADC in the striatum to exogenous L-DOPA, we measured left-sided rotations in rats following oral administration of L-DOPA. Rats were given L-DOPA (20 mg / kg, 5 mL / kg) plus benserazide (5 mg / kg, 5 mL / kg) by oral gavage. 45 minutes later, the rats were placed in the test environment, and the number of rotations was recorded over a 30-minute period.

[1082] As shown in Figure 6B, the model control group did not show a significant increase in the number of rotations compared with the sham-operated control group, while the high- and low-dose administration groups of HRPDAAV-02AI and HRPDAAV-03DI showed a significant increase compared with the model control group, indicating that the AADC expressed by AAV delivered to the striatum has a good response to exogenous L-DOPA drugs.

[1083] 2) Cylinder test

[1084] To examine rats' forelimb preference under baseline conditions, a cylinder test was conducted. Each group of rats was placed in a 20-cm-diameter transparent cylinder. The number of times the rats' left, right, and bilateral forelimbs touched the wall within 10 minutes was observed and recorded. Each time the rats, using their hind limbs as a fulcrum, touched the wall with one or both forelimbs and then returned to the bottom of the cylinder, a single touch was counted (a bilateral touch was counted if one limb touched the wall first, followed by the other limb). The percentage of left forelimb usage was calculated as: Left forelimb usage (%) = (left + 0.5 bilateral) / (right + left + bilateral) × 100.

[1085] As shown in Figure 6C, the left forelimb utilization rate of the model mice and the drug-treated group decreased compared with the blank control group, which once again verified the effectiveness of the model.

[1086] To detect the utilization of L-DOPA in AAV drug-treated rats, L-DOPA (5 mg / kg, 5 mL / kg) and benserazide (2.5 mg / kg, 5 mL / kg) were orally administered to the rats. Thirty minutes later, the rats were placed in a transparent cylinder with a diameter of 20 cm. The number of times the left and right forelimbs and bilateral forelimbs touched the wall within 10 minutes was observed and recorded.

[1087] As shown in Figure 8D , the model control group showed no significant improvement in left forelimb utilization after L-DOPA supplementation, whereas the HRPDAAV-02AI and HRPDAAV-03DI groups showed significant improvement in left forelimb utilization compared to the model control group, demonstrating that L-DOPA utilization in rats was significantly enhanced after administration. This further demonstrates that AADC expressed in the striatum by AAV delivery has a strong response to exogenous L-DOPA.

[1088] 3) Detection of the striatal metabolic capacity of L-DOPA after administration of HRPDAAV-02AI and HRPDAAV-03DI

[1089] After the behavioral experiment, rats in each group were gavage-administered L-DOPA (20 mg / kg, 5 mL / kg) and benserazide (5 mg / kg, 5 mL / kg). Rats were sacrificed 1 hour after the administration. The striatum of the healthy and affected sides was obtained. DOPA was detected using UPLC-MS / MS.

[1090] Referring to Figure 6E and Table 7, the DOPA ratio of the affected side to the healthy side was detected, and it was found that the DOPA level in the affected side of the model control group was significantly decreased compared with the drug-treated group, demonstrating the success of the 6-OHDA model.

[1091] Table 7. DOPA test results of bilateral striatum of rats

[1092] DOPA analysis of the striatum (affected side) on the treated side revealed significant increases in DOPA levels in both the HRPDAAV-02AI and HRPDAAV-03DI groups compared to the model control group. The DOPA ratio between the affected side and the healthy side in each treatment group was significantly elevated compared to the model control group. This demonstrates that the striatum's ability to metabolize L-DOPA is significantly enhanced after administration of HRPDAAV-02AI and HRPDAAV-03DI.

[1093] 4) Detection of the protective effect of HRPDAAV-03DI on DA neurons

[1094] The rats in each group were perfused through the heart to obtain brain tissue for fluorescent immunohistochemistry detection. The specific method was as follows: after the experimental rats were grossly observed, the heart tissue in the chest cavity was freed, an intravenous infusion needle was inserted from the left ventricle and fixed, the right atrial appendage was cut open, and pre-cooled PBS was used for perfusion until transparent liquid flowed out of the right atrial appendage. Then 4% paraformaldehyde was used for transcardial perfusion, the brain tissue was cut, and placed in a fixative (4% paraformaldehyde solution). The fixed tissue was sliced ​​using paraffin for tyrosine dehydrogenase TH staining.

[1095] As shown in Figure 6F, the ratio of TH-positive cells on the affected side to the contralateral side was analyzed by fluorescence immunohistochemistry. It was found that the survival rate of TH cells on the affected side was less than 15% in the experimental model control group compared with the sham operation group, indicating that the 6-OHDA model was successfully established.

[1096] The TH fluorescence intensity ratio of the affected side to the healthy side in the low-dose HRPDAAV-03DI group was significantly increased compared with the model control group, demonstrating that HRPDAAV-03DI has a good protective effect on dopamine neurons in rats and improves the survival rate of TH cells.

[1097] Example 4: VEGF binding molecules and point mutation design

[1098] This example designs three molecular forms of VEGF-binding molecules ( FIG. 7 ), which are as follows from N-terminus to C-terminus:

[1099] Molecular form 1: VEGFR1 D2 + VEGFR2 D3 + VEGFR2 D2D3 + hinge region and CH2CH3 constant region of human IgG1;

[1100] Molecular form 2: VEGFR2 D2D3 + VEGFR1 D2 + VEGFR2 D3 + hinge region and CH2CH3 constant region of human IgG1;

[1101] Molecular format 3: Heavy chain: VEGFR2 D2D3 + hinge region and CH2CH3 constant region of human IgG1 + bevacizumab VH + CH1; Light chain: bevacizumab VL + CL.

[1102] To improve the stability and ligand affinity of the fusion protein, a series of point mutations were designed in the VEGF trap backbone molecule (i.e., the VEGFR2 D2D3 domain, i.e., the underlined portion above) in the VEGF binding molecule:

[1103] 1. Based on the VEGFR2 D2D3 sequence (SEQ ID NO: 69), five mutants were designed, and the cysteine ​​at position 162 (C162) was mutated to A / V / S / I / L, respectively.

[1104] 2. Based on the VEGFR2 D2D3 sequence (SEQ ID NO: 69), 6 single point mutations and 2 combination mutations were designed (Table 1).

[1105] 3. Based on the VEGFR2 D2D3 sequence (SEQ ID NO: 69), single point mutations were designed in the D2 or D3 domain (Tables 2 and 3).

[1106] Table 8: VEGF trap backbone molecule VEGFR2 D3 mutations

[1107] Table 9: VEGF trap backbone molecule VEGFR2 D2 mutations

[1108] Table 10: VEGF trap backbone molecules VEGFR2 D3 mutations

[1109] Example 5: Construction of plasmids expressing VEGF binding molecules, protein expression and purification

[1110] The VEGF-binding molecules designed in Example 6 were synthesized by whole gene synthesis, wherein the VEGF-C antagonistic activity was mainly provided by VEGFR2 D2D3, and the VEGF-A antagonistic activity was mainly provided by VEGFR1D2-VEGFR2D3 (Aflibercept) or Bevacizumab Fab.

[1111] After optimizing the expression host for amino acids, the genes encoding the above nucleotide sequences were inserted into the pTT5 vector, and the expression vectors were designed and synthesized according to the above sequences, and sequence verification was performed.

[1112] VEGF binding molecule expression and purification: The qualified plasmid was transfected into human 293F cells with PEI and cultured continuously. 293F cells were cultured with serum-free culture medium (Shanghai Aopuma Biotechnology, OPM-293CD03) to the logarithmic growth phase for cell transfection. 45μ plasmid was dissolved in 10mL I Reduced Serum Medium (GIBCO, 31985-070) was mixed, followed by the addition of 200 μg of PEI, mixing, incubation at room temperature for 15 minutes, and addition to 50 mL of cells. Cell culture conditions were: 5% CO2, 37°C, 125 rpm / min. During the culture period, feed was added on the first and third days until the cell viability was less than 70%. The cell supernatant was collected and centrifuged. The cell culture fluid after centrifugation was loaded onto a protein A column, washed with phosphate buffer, eluted with glycine hydrochloride buffer (pH 2.7 0.1M Gly-HCl), neutralized with 1M Tris hydrochloride, pH 9.0, and dialyzed against phosphate buffer to obtain the purified protein. SDS-PAGE and SEC-HPLC were used to confirm the acquisition of the target fusion protein.

[1113] Example 6: Determination of Binding Activity of VEGF Binding Molecules to VEGF-A and VEGF-C Ligands

[1114] The inhibitory effect of VEGF-binding molecules on KDR signaling activation was evaluated by detecting the expression level of NFAT-RE Luc2P KDR cell reporter gene induced by VEGF-A. The experimental steps are as follows:

[1115] 3A.1 Culture medium preparation: Complete medium: high glucose DMEM (90%) + FBS (10%); Complete medium: high glucose DMEM (90%) + FBS (10%), 100 μg / mL hygromycin B, 250 μg / mL G-418. Assay medium: high glucose DMEM (99%) + FBS (1%).

[1116] 3A.2 VEGF Binding Molecule Sample Dilution: Serially dilute the sample to be tested to a maximum concentration of 300 nM, followed by a 3-fold serial dilution. VEGFA Dilution: The final VEGFA concentration is 250 ng / mL.

[1117] 3A.3 Preparation of reporter gene cell line: After digestion of Glo-ResponseTM NFAT-RE-luc2P / KDR HEK293 cells, wash the cells with 5 mL of PBS and resuspend the cells in assay medium to a density of 8 × 10 5 / mL, 50 μl was added to each well of a 96-well cell culture plate, i.e. the number of cells was 4×10 4 / hole.

[1118] 3A.4 VEGF-binding molecule treatment: Add VEGF-binding molecules of varying concentrations to the cells at 50 μl / well and incubate at 37°C, 5% CO2 for 6 h. Repeat three times for each concentration. Also, establish control groups with or without VEGF-A.

[1119] 3A.5 Color Development: Equilibrate Bright-Glo Color Development Solution to room temperature. Add 50 μL of Bright-Glo Color Development Solution to the 96-well plate after incubation. Vortex and mix at room temperature for 10 minutes, then let stand for 2 minutes. Place the plate on a multi-function microplate reader and read the chemiluminescence value.

[1120] 3A.6 Data Processing:

[1121] Top value = fluorescence value of the group with VEGF-A + VEGF binding molecule concentration of 0 nM

[1122] Bottom value = fluorescence value of the group without VEGF-A

[1123] Inhibition rate = (fluorescence value - lowest value) / (highest value - lowest value) * 100%

[1124] Graphpad: The results were analyzed using a four-parameter fitting formula to obtain a dose-response curve. The four-parameter fitting formula is: Y = (AD) / (1+(X / C)^B)+D

[1125] A: Estimated asymptote under the curve; B: Slope of the curve; C: IC 50 ; D: asymptote estimate on the curve.

[1126] To evaluate the binding activity of VEGF-binding molecules to VEGF-C, this example uses a BAF3-VEGFR2 / R3 cell proliferation assay. BAF3-VEGFR2 / R3 is a BA-F3 cell line that overexpresses human VEGFR2 / R3. Cell growth is dependent on exogenously added growth factor VEGF-C. The basic experimental steps are as follows:

[1127] 3B.1 Cell culture: The above cell lines were cultured in RPMI1640 containing 10% FBS (supplemented with 10 ng / mL mouse IL-3, 1 μg / mL puromycin, and 250 μg / mL G418) at a concentration of 1*105 to 1.5*10 5 Place in a 37°C, 5% CO2 incubator with a cell density not exceeding 1*10 6 pieces / mL.

[1128] 3B.2 Cell proliferation assay: Cells in the logarithmic growth phase were washed three times with PBS, centrifuged at 800 rpm for 3 min, and then the cell density was adjusted with RPMI1640 (FBS: 2%, 143 ng / mL VEFG-C). 4 50 μL of cell titer-glo was added to each well of a 96-well plate. Next, 50 μL of serially diluted VEGF-binding molecules (maximum concentration 300 nM, 3-fold dilution) was added. Control groups without VEGF-C and with VEGF-C alone were also established. After 3 days of culture, 30 μL of cell titer-glo was added, mixed, and the cells were counted.

[1129] 3B.3 Data Processing:

[1130] Maximum value = fluorescence value of VEGF-C + VEGF binding molecule concentration 0 group

[1131] Lowest value = fluorescence value of the group without VEGF-C

[1132] Inhibition rate = (fluorescence value - lowest value) / (highest value - lowest value) * 100%

[1133] Graphpad: The results were analyzed using a four-parameter fitting formula to obtain a dose-response curve. The four-parameter fitting formula is: Y = (AD) / (1+(X / C)^B)+D

[1134] Where A: the estimated asymptote under the curve; B: the slope of the curve; C: IC 50 ; D: asymptote estimate on the curve.

[1135] In this example, the binding activities of VEGF-A, VEGF-C and VEGF binding molecules were evaluated by the above two in vitro experiments. The specific data are shown in Table 11:

[1136] Table 11: Molecular activity test data after single point mutation based on molecular form 1

[1137] The in vitro activities of the VEGF-binding molecules of the present disclosure against VEGF-A and VEGF-C are comparable to those of the positive controls Aflibercept and OPT-302.

[1138] Table 12: Activity data of proteins produced in the CHO-K1 system after combining some single-point mutations with C162V “-” indicates that the activity was not detected.

[1139] Table 13: Activity data of proteins produced by CHO-K1 system after combined mutation

[1140] As shown in Tables 12 and 13, PR1160 is a VEGFR2 D2D3 (C162V)-Fc fusion protein. Although it has some VEGF-A inhibitory activity in vitro, its activity is weaker than that of the positive control, Aflibercept. The inhibitory activities of the other fusion proteins against VEGF-A and VEGF-C are comparable to those of the two positive control molecules, respectively. As shown in Table 14, the inhibitory activities of the VEGF-binding molecules PR1145, PR1174, PR1163, and PR1186 against VEGF-A and VEGF-C are comparable to those of the two positive control molecules (Aflibercept and OPT-302), respectively, and are stronger than the inhibitory activities of PR1010 and Bevazicumab against VEGF-A.

[1141] Example 8: Thermal stability analysis of different VEGF binding molecules

[1142] The thermal stability of fusion proteins is an important criterion for evaluating the quality of molecules. Developing a fusion protein molecule with better thermal stability has positive implications for process production, drug delivery, etc. In this example, an accelerated thermal stability evaluation was performed on the candidate molecules.

[1143] The specific experimental steps are as follows: dilute the candidate molecule to 1 mg / mL and divide it into 3 1.5mL EP tubes, 100μL / tube; take one of the tubes as the 0 time point sample and freeze it. The remaining samples are placed in a constant temperature and humidity incubator at 40°C and 75% humidity, and one tube of sample is taken at two weeks and four weeks for freezing. After collecting the samples of the three time points (0, 2, and 4 weeks), place them at 4°C to melt, centrifuge at 12000rpm / min for 10 minutes to remove the precipitate, and take the supernatant for HPLC detection. The HPLC used in this example is the ACQUITY Arc type produced by Waters, and the column is XBridge BEH SEC 3.5μm, flow rate 0.5mL / min, 30μL of sample was drawn, and the final HPLC chromatogram of the sample was shown in the following table.

[1144] Although the parent molecule PR1010 of Form 1 initially achieved 90% purity, after two weeks at 40°C, its purity dropped below 30%, suggesting that its stability needed to be improved. The purity of the PR1094 molecule, which introduced the VEGFR2 C162V mutation into PR1010, was further increased to over 95% in HEK 293F cells, and after four weeks at 40°C, the SEC purity remained around 90%.

[1145] According to the HPLC results, after the C162V mutation, the thermal stability of the molecule was significantly improved, both in the VEGFR2 D2D3-Fc fusion protein (PR1160 compared to PR1088) and in molecular form 1 (PR1094 compared to RP1010).

[1146] In addition, point mutations such as N274F (PR1113), N274I (PR1114), D276Q (PR1118), Q280E (PR1123), E284H (PR1125), Y221S / R222K (PR1135), and L313R (PR1136) can also significantly improve the stability of the fusion protein by stabilizing the VEGFR2 D2D3 domain.

[1147] To further study the effects of combined mutations based on C162V / I on protein expression and purification, we subsequently used CHO-K1 to express the combined mutant molecules: in most cases, combined mutations can further improve the stability of the molecules (such as PR1145 / PR1149 compared to PR1094).

[1148] The results are shown in Tables 14 and 15, wherein PR1088 to PR1136 (Table 8-1) were obtained by HEK 293F expression, and PR1094 to RP1179 (Table 8-2) were obtained by CHO-K1 expression.

[1149] Table 14: Thermal stability analysis results of different VEGF binding molecules

[1150] Table 15: Thermal stability analysis results of different VEGF binding molecules

[1151] Example 9: Determination of the affinity between VEGF binding molecules and different ligands using SPR technology

[1152] Surface plasmon resonance (SPR) was used to detect the affinity between different VEGF-binding molecules and VEGF-A / B / C proteins (VEGF-binding molecules were captured on a Protein A chip. The coupling level was set at 100 RU, and detection was performed using a Biacore instrument (T200, GE Healthcare, BIAC-B20-03). The running buffer was HBS-EP+ (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.05% surfactant P20). Diluted antibodies (6.25, 12.5, 25, 50, 100 nM) were flowed through the experimental channel and the control channel (100 nM VEGF-A / B / C) at a flow rate of 30 μl / min for 3 minutes, dissociated for 5 minutes, and then washed with regeneration buffer 10 mM glycine pH 1.5 (GE The cells were run at a flow rate of 30 μl / min for 30 seconds (Biacore Healthcare, BR-1003-54). Data were analyzed using Biacore 8K evaluation software.

[1153] The affinity constants of the VEGF binding molecules for human VEGF-A, VEGF-B, and VEGF-C proteins were calculated using the Multi-Cycle method. The calculated kinetic parameters Ka, Kd, ​​and KD values ​​are shown in the table below.

[1154] Table 16: Affinity results of VEGF binding molecules with different ligands

[1155] Example 10: In vivo stability evaluation of VEGF binding molecules

[1156] To determine the stability of VEGF binding molecules in vivo, the changes in the molecular concentration in ocular tissue after intravitreal injection of the molecules were detected. The specific experiments are as follows:

[1157] Fourteen New Zealand rabbits, half male and half female, weighing 1.5-2.5 kg, were purchased and divided into four groups: a high-dose PR1145 group, a low-dose PR1145 group, a high-dose PR1163 group, and a blank control group. All procedures were performed on both eyes. Vitreous and aqueous humor samples were collected from both eyes of the 12 treated groups 3 hours, 3 days, 7 days, 14 days, and 28 days after intravitreal administration. The target protein content in the samples was determined using an enzyme-linked immunosorbent assay. The experiments were performed by Chengdu Hetuo Chuangzhan Biotechnology Co., Ltd. The dosages administered are shown in Table 17.

[1158] Table 17: Dosing regimen for in vivo stability evaluation of VEGF binding molecules

[1159] Figure 10 shows the levels of VEGF-binding molecules in the rabbits measured at 3 hours, 3 days, 7 days, 14 days, and 28 days after administration to the rabbit vitreous. The results showed that the PR1145 high-dose group, the PR1145 low-dose group, and the PR1163 high-dose group reached maximum drug concentrations in the animals 3 days after injection, and PR1145 and PR1163 were detectable in the animals for 14 days, indicating that the VEGF-binding molecules have good in vivo stability.

[1160] Example 11: In vivo efficacy evaluation of VEGF binding molecules

[1161] To evaluate the inhibitory effect of VEGF-binding molecules on choroidal neovascularization, a laser-induced choroidal neovascularization (CNV) model was used for efficacy validation. This model is currently widely used internationally to validate anti-choroidal neovascularization drugs. The specific experiments are as follows:

[1162] Forty-two rats, half male and half female, aged 8-10 weeks, were purchased and divided into seven groups: a high-dose PR1145 group, a low-dose PR1145 group, a control group 1 (Aflibercept), a control group 2 (OPT302), and a negative control group (PBS). The dosing schedule is shown in Table 11. Six animals per group underwent bilateral CNV laser treatment. One week later, 4 μL of the protein was injected into both eyes. Fundus angiography was performed two weeks after administration to assess drug efficacy. PR1145 (3.75 mg / mL) and Aflibercept (2.5 mg / mL) were administered at equimolar doses, with a molar ratio of Aflibercept (2.5 mg / mL) to OPT302 (10 mg / mL) of 1:4.

[1163] Table 18: Dosage regimen for in vivo efficacy evaluation of VEGF binding molecules

[1164] Fundus photography of rats was performed before modeling, and no abnormalities were observed in the fundus of any rat. As shown in Figure 11A, significant laser-induced sodium fluorescein leakage was observed in the PBS group 7 days after modeling, indicating successful model establishment. All rats were dosed 7 days after CNV modeling, and fluorescein fundus angiography (FFA) was performed 14 days after drug administration. Increased sodium fluorescein leakage was observed in the PBS group, indicating that the extent of laser-induced choroidal neovascularization continued to progress and showed a trend of aggravation within 21 days after modeling. The results of FFA assay 14 days after drug administration to investigate the inhibition of choroidal neovascularization by VEGF-binding molecules are shown in Figure 11B. Compared with the PBS group, the high-dose PR1145 group, the low-dose PR1145 group, Aflibercept, and OPT302 all significantly improved laser-induced sodium fluorescein leakage in the choroidal neovascularization, with the improvement rate of PR1145 (3.75 mg / mL) being 15.8%, higher than the improvement rate of Aflibercept (2.5 mg / mL) (12.5%). It has comparable improvement effects to PR1145 (3.75 mg / mL) and OPT302 (10 mg / mL), but the dosage of PR1145 is significantly lower.

[1165] Example 12: Structure of AAV-delivered gene expression cassettes

[1166] The expression vector provided in this embodiment contains a nucleic acid molecule for expressing a VEGF-binding molecule, the structure of which is shown in FIG8 . From the 5' end to the 3' end, the expression vector contains: a CMV enhancer, a promoter, a 5'UTR, a Kozak sequence (gccacc), a VEGFR-binding molecule coding sequence, a 3'UTR, a WPRE, and an SV40 polyA. The nucleic acid sequence of the CMV enhancer is shown in SEQ ID NO: 124, the nucleic acid sequence of the CMV promoter is shown in SEQ ID NO: 125, the 5'UTR is shown in SEQ ID NO: 126, and the Kozak sequence (gccacc) is shown. The coding sequence of the VEGF-binding molecule is shown in any one of SEQ ID NOs: 128-136 (wherein SEQ ID NOs: 128-130 correspond to the codon-optimized sequences 1, 2, and 3 of PR1145; SEQ ID NOs: 131-133 correspond to the codon-optimized sequences 1, 2, and 3 of PR1174; SEQ ID NOs: 134-135 correspond to the codon-optimized sequences 1, 2, and 3 of PR1175; and SEQ ID NOs: 136-137 correspond to the codon-optimized sequences 1, 2, and 3 of PR1176. NO: 134-136 correspond to codon-optimized sequences 1, 2, and 3 of PR1163), the 3'UTR sequence is shown in SEQ ID NO: 137, the WPRE sequence is shown in SEQ ID NO: 138, and the SV40 polyA sequence is shown in SEQ ID NO: 139.

[1167] Example 13: Construction of expression vector

[1168] An expression cassette was constructed, and an AAV packaging plasmid expressing the VEGF binding molecule protein gene was constructed through routine molecular biological operations such as enzyme digestion, ligation, transformation, and cloning screening and identification (see Figure 9). The AAV packaging plasmid includes: CMV enhancer, promoter, 5'UTR, Kozak sequence (gccacc), VEGFR-binding molecule, 3'UTR, WPRE and SV40polyA. The expression cassette is flanked by inverted terminal repeat sequences (ITRs), and EcoRV and BSMI are enzyme digestion sites.

[1169] Example 14: AAV virus preparation and purification

[1170] The amino acid sequence of the AAV capsid used to deliver the gene expression cassette in this example is shown in SEQ ID NO: 127. Referring to the method for packaging and purifying recombinant AAV virus reported by Martin Lock et al., the AAV Rep and Cap protein expression plasmids, a helper plasmid, and the expression vector of Example 13 were co-transfected into HEK293 cells using PEI to package and prepare recombinant adeno-associated virus. 48 hours after transfection, the cells and culture supernatant were harvested and the AAV virus was purified using iodixanol ultracentrifugation gradient (see Martin Lock, et al. Rapid, Simple, and Versatile Manufacturing of Recombinant Adeno-Associated Viral Vectors at Scale. HUMAN GENE THERAPY, 2010. 21: 1259-1271) to obtain the recombinant adeno-associated virus vector, and the viral titer was measured by real-time fluorescence quantitative PCR.

[1171] Example 15: Intraocular expression experiment

[1172] To determine the expression and changes of the pharmacodynamic protein of AAV-1174 (expressing PR1174 (nucleic acid sequence: SEQ ID NO: 131)) and ADVM-022 (see rAAV2.7m8-aflibercept in WO2017218974) in the vitreous, aqueous humor, retina, and choroid of the rabbit eyes after injection into the rabbit eyes, the following experiment was conducted. 32 New Zealand rabbits, aged 2 to 5 months, were purchased and divided into 4 groups of 8 rabbits each. The vitreous of both eyes were injected according to the doses in Table 12 (each sample was injected with two high and low doses). On day 58 after injection, the vitreous, aqueous humor, retina, and choroid of both eyes were collected. At each time point, the number of samples collected for each dosing group was 2 rabbits and 4 eyes. The samples were sent back to our laboratory and the pharmacodynamic protein content was determined by enzyme-linked immunosorbent assay.

[1173] Table 19: Dose groups for intraocular expression experiments

[1174] Figure 12 shows the intraocular expression results. At D58, both the AAV-1174 high-dose group and the AAV-1174 low-dose group stably expressed PR1174 protein in the rabbit aqueous humor, vitreous, retina, and choroid, and the protein expression level was higher than that of ADVM-022.

[1175] Example 12: In vivo efficacy experiment

[1176] The laser-induced choroidal neovascularization model is currently a classic model widely used internationally to verify anti-choroidal neovascularization drugs. In order to verify the inhibitory effect of AAV-1174 on choroidal neovascularization, this model was used for efficacy verification.

[1177] Thirty-six rats, half male and half female, aged 8-10 weeks, were purchased and divided into 6 groups, 6 rats in each group. Both eyes were administered with intravitreal injection of AAV-1174 (expressing PR1174 (nucleic acid sequence: SEQ ID NO: 131) pharmacodynamic protein) according to the dosage in Table 13. Three weeks after administration, both eyes were laser-modeled, and fundus angiography was performed one week after modeling to detect the drug efficacy.

[1178] Table 20: Dose groups for in vivo efficacy experiments

[1179] Table 21: Results of inhibition of choroidal neovascularization

[1180] Fundus photography was performed on the rats before modeling, and no abnormalities were found in the fundus of all rats. All rats underwent CNV modeling 21 days after administration and FFA detection 28 days after administration. 7 days after modeling, obvious laser spot sodium fluorescein leakage was observed in the PBS group, indicating that the model was successfully established; 28 days after administration, FFA detection of fluorescent spot grading scores was performed, and the ratio of grade 3 leakage spots was detected. The results are shown in Table 21 and Figure 13. There was a statistically significant difference between the AAV-1174 (high dose) group and the PBS group. AAV-1174 had a significant inhibitory effect on laser-induced choroidal neoplasia sodium leakage.

[1181] Screening and stability evaluation of formulation ingredients

[1182] In this example, a pharmaceutical composition comprising viral (recombinant adeno-associated virus, rAAV) particles was prepared, and the exemplary steps were as follows:

[1183] 1. Prepare a preparation stock solution containing rAAV particles and perform sterile filtration on the preparation stock solution: a) Filter using a 0.2 μm PES filter membrane (syringe filter), filter membrane model KM2EKVS.

[1184] 2. Preparation of stock solution: After sterilization, the virus suspension is ultrafiltered using the sterile preparation mother liquor to complete the solvent replacement: a) Before ultrafiltration, first retain a sample of the virus suspension and test the physical and chemical properties as the initial value; b) Use purified water to perform the initial centrifugation wash of the ultrafiltration centrifuge tube, and the centrifugation parameter is 1500g*2min; c) Use the sterile preparation mother liquor to perform the initial centrifugation wash of the ultrafiltration centrifuge tube twice, and the centrifugation parameter is 1500g*2min; d) Use the sterile preparation mother liquor to centrifuge the virus suspension sample four times to replace the liquid. For each liquid replacement, the volume of the virus concentrate should be 1 / 6 of the sterile preparation mother liquor added, and the centrifugation parameter is 1500g*1min (about 2 to 3 centrifugations).

[1185] 3. Sterile filter the ultrafiltered virus stock solution.

[1186] 4. Prepare virus solution (preparation): Perform Vg test on the ultrafiltered virus solution and dilute the virus solution according to the test results.

[1187] 5. Test the virus solution for thermal stability and freeze-thaw stability after adjusting the concentration, and pay special attention to several indicators such as virus titer, IU, appearance, and virus biological activity.

[1188] The rAAV particles in the following Test Examples 1 to 6 contain nucleic acid molecule I, for example, the rAAV particles are HRPDAAV-02AI or HRPDAAV-03DI; the rAAV particles in Test Examples 7 to 11 contain nucleic acid molecule I, for example, the rAAV particles are AAV-1145.

[1189] Test Example 1: Component Screening of Preparation Formula

[1190] 1) Prepare a preparation stock solution and sterilize and filter the preparation stock solution. Refer to Table 22 for the excipients in the preparation stock solution. 2) After sterilization, use the sterile preparation stock solution to ultrafilter the virus suspension to complete solvent exchange. 3) Sterilize and filter the ultrafiltered virus stock solution. 4) Perform a Vg test on the ultrafiltered virus solution and dilute the virus solution to 8.5E12 vg / ml (8.5×10 12 vg / ml).

[1191] V. Test the heat stability and freeze-thaw stability of the adjusted viral solution. Assess the genome titer (Vg) using qPCR, and examine several other parameters, including log(IU), appearance, and viral bioactivity. After storage at 25°C for 1-6 months, 5°C for 1-18 months, and repeated freeze-thaw cycles, examine the appearance, genomic titer (Vg) using qPCR, and infectious titer (IU) using TCID50 / qPCR.

[1192] The stability results are shown in Table 23 and Figure 14. Under accelerated (25°C) and long-term (5°C) conditions, the viral Vg counts of the three formulations fluctuated, but overall did not show a downward trend. Under long-term conditions, the IU of the F2 and F3 formulations under accelerated 6M conditions decreased significantly, retaining the histidine in the formulations.

[1193] Table 22. Screening prescription

[1194] Table 23. Stability screening results “N / A” means not tested, “W” means week, and “M” means month.

[1195] Test Example 2: Component Screening of Preparation Formula

[1196] A viral solution containing rAAV particles was prepared. For excipient information, see Table 24. Stability was assessed at 25°C, 5°C, and ≤-60°C. The results are shown in Table 25. VG showed consistent trends across storage conditions. At 25°C, IU showed a downward trend, while the trends were essentially the same under the other three conditions. The formulation had comparable quality when stored at 2-8°C and below -60°C.

[1197] Table 24. Screening prescription

[1198] Table 25. Stability test results

[1199] Test Example 3: pH Screening of Formulations

[1200] 1) Prepare a formulation stock solution according to the following formula: 5 mM L-histidine, 100 mM sodium chloride, 2 mM magnesium chloride hexahydrate, 0.25% (w / v) glycerol, 10 mM BTP, 2.50% (w / v) sucrose, and 0.01% (w / v) poloxamer 188; and adjust the pH of the formulation stock solution to 6.0, 6.5, 7.0, 7.5, or 8.0 at 25°C using sodium hydroxide or hydrochloric acid solution. 2) Sterilize and filter the adjusted pH of the formulation stock solution. 3) After sterilization, ultrafilter the virus suspension using the sterile formulation stock solution to complete solvent exchange. 4) Sterilize and filter the ultrafiltered virus stock solution. 5) Perform a vg test on the ultrafiltered virus solution and dilute the virus solution to (6-7)E12 vg / ml based on the test results.

[1201] The virus solution after concentration adjustment was tested for thermal stability and freeze-thaw stability, with particular attention paid to several indicators, including virus titer, IU, appearance, and viral bioactivity. The results are shown in Table 27. Within the pH range of 6-8, after 3M accelerated elution, no significant decrease in indicators such as VG and IU was observed.

[1202] Table 26. Test conditions

[1203] Table 27. Investigation of the effect of different pH on virus stability “N / A” means not tested.

[1204] Test Example 4. Effect of other buffer systems in the preparation on virus stability

[1205] 1) Prepare a formulation stock solution according to the information for Formulation F10 shown in Table 28, and adjust the pH of the formulation stock solution to 7.5 at 25°C. 2) Sterilize and filter the formulation stock solution. 3) After sterilization, ultrafilter the virus suspension using the sterile formulation stock solution to complete solvent exchange. 4) Sterilize and filter the ultrafiltered virus stock solution. 5) Perform a Vg test on the ultrafiltered virus solution and, based on the test results, dilute the virus solution to 9.6E12 vg / ml. After adjusting the concentration, test the virus solution for thermal stability and freeze-thaw stability, focusing on several indicators such as virus titer, IU, appearance, and viral bioactivity. Viral bioactivity is determined by infecting cells with the virus and detecting protein expression by SDS-PAGE.

[1206] The results are shown in Table 29. Viral genome titer, AAV capsid protein purity, infectivity titer, biological activity, and pH all met quality standards under long-term 12M and accelerated 3M conditions, with no significant changes. Biological activity was assessed by measuring AADC expression (pg / μg). AADC expression was 5.7 pg / μg at T0, with no significant changes after 3M of long-term and accelerated treatment. The AAV viral vector empty ratio met quality standards under long-term 6M and accelerated 3M conditions, with no significant changes.

[1207] Table 28. Excipients in Formulation F10

[1208] Table 29. Effects of other buffer systems in the formulation on virus stability “N / A” means not tested.

[1209] Test Example 5. Investigation of the Effect of Temperature on the pH of the Formulation

[1210] 1. Prepare a stock solution of the formulation with a pH of 7.5 at 27.2°C according to the formulation in F10.

[1211] 2. Take about 3 ml of the preparation mother solution and put it into a centrifuge tube. First, use a pH meter to measure the pH value in an ice-water bath when the temperature drops from room temperature to about 2°C, and record it; then record the pH change when the solution is heated from 2°C to 30°C.

[1212] The results are shown in Figure 15. Temperature has a certain effect on the pH of the mother liquor. The pH increases as the temperature decreases, reaching 8.08 at 2°C. The pH decreases as the temperature increases, with a pH change of approximately 0.24 for every 10°C change in temperature. The pH decreases to 7.42 at 30°C.

[1213] 3. Using sodium hydroxide or hydrochloric acid solution, adjust the preparation stock solution to five different pH values ​​at 25°C: 6.0, 6.5, 7.0, 7.5, and 8.0, and adjust it to pH 7.5 at 5°C. Record the pH change of the solution from 2°C to 30°C.

[1214] The results are shown in Figure 16. At 25°C, all five mother solutions with different pH values ​​experience changes in pH as a result of temperature changes. The pH decreases with increasing temperature, and increases with decreasing temperature. Within the pH range of 6.0-8.0, a 10°C change in solution temperature results in a pH change of approximately 0.2.

[1215] Test Example 6. Investigation of pH Temperature Sensitivity of Different Buffers

[1216] 1. Prepare buffer stock solutions using different buffer solutions. The buffer range is shown in Table 30.

[1217] 2. Take about 3 ml of different buffer solutions into a centrifuge tube. First, use a pH meter to measure the pH value in an ice-water bath while the temperature drops from 30°C to about 2°C, and record it. Then, record the pH change when the solution is heated from 2°C to 30°C.

[1218] Table 30. Buffering range of different buffers

[1219] The results, as shown in Figure 17, show that the pH values ​​of all three buffer systems, BTP, Tris, and HEPES, decrease with increasing temperature. For BTP and Tris, the pH value decreases by approximately 0.024 with a 1°C increase in temperature. For HEPES, the pH value decreases by approximately 0.001 with a 1°C increase in temperature. The HEPES buffer system exhibits high stability.

[1220] Test Example 7. Investigating the Effect of Different pH Ranges on Stability

[1221] 1) Prepare the formulation stock solution according to the recipe shown in Test Example 3, and adjust the formulation stock solution to 6.5, 7.0, 7.5, 8.0, and 8.5 (F11-F15) at 25°C using sodium hydroxide or hydrochloric acid solution.

[1222] 2) After sterilization, the virus suspension is ultrafiltered using the sterile preparation stock solution to complete solvent replacement.

[1223] 3) The ultrafiltered virus stock solution is sterilized and filtered.

[1224] 4) The ultrafiltration virus solution was tested for Vg, and the virus solution was diluted to (1.1-1.4)E13 vg / ml based on the test results. The virus solution after concentration adjustment was tested for viral thermal stability (placed at 25°C), and several indicators such as virus particle size, titer, IU, appearance, viral bioactivity, and MFI were observed. The virus particle size was observed by dynamic light scattering (DLS), and the degradation of virus particles was investigated by microfluidic imaging particle analysis (MFI). IP (representing virus particles containing incomplete genes) and FP (representing virus particles with complete gene sequences) were detected by AUC (analytical ultracentrifuge). The results are shown in Table 32. After placing at 1M-2M, the particle size of each pH gradient sample was stable, and the purity decreased to a certain extent.

[1225] Table 31. Prescriptions of the tested stock solutions and preparations

[1226] Table 32. Storage stability test at 25°C

[1227] Test Example 8. Effects of Temperature and Formulation pH Range on Virus Stability

[1228] 1. A high-temperature forced degradation experiment was conducted on the product stock solution to examine the effect of temperature on product quality. The experimental conditions and results are shown in Table 33. The results showed that different high-temperature degradation conditions had no significant effect on the purity of the product stock solution.

[1229] Table 33. High temperature forced degradation experimental conditions and purity test results

[1230] 2. Investigate the effects of different acid and alkali conditions on product stability.

[1231] 1) According to the formulation of F10, adjust the sodium chloride to 150 mM, prepare a formulation stock solution, and filter the formulation stock solution to remove bacteria. Adjust the pH of the formulation stock solution to 5, 9, or 6.8 (corresponding to F16-F18, respectively) at 25°C using sodium hydroxide or hydrochloric acid solution.

[1232] 2) Filter using a 0.2 μm PES filter membrane (syringe filter), the filter membrane model is KM2EKVS.

[1233] 3) After sterilization, the virus suspension is ultrafiltered using the sterile preparation stock solution to complete the solvent replacement.

[1234] 4) Sterile filter the ultrafiltered virus stock solution. Perform a vg test on the ultrafiltered virus solution and dilute the virus solution to (1.1-1.2)E13 vg / ml based on the test results.

[1235] Stability testing of formulations at different pH levels was performed according to the conditions in Table 26, focusing on viral titer and purity. The results are shown in Table 34. At 5°C, virus purity did not significantly decrease across the three pH conditions. Compared to 5°C, purity decreased at pH 9.0 after 1M acceleration at 25°C, indicating that high pH conditions had an impact on purity. At 40°C, both VG and purity decreased, with a greater decrease in VG at pH 5.0.

[1236] Table 34. Effect of different pH values ​​of the formulation on virus stability

[1237] Test Example 9. Effect of Preparation pH Range on Virus Stability

[1238] Prepare a stock solution according to the formulation in Table 35 and sterilize and filter the stock solution. After sterilization, ultrafilter the virus suspension using the sterile stock solution to complete solvent exchange. Sterile filter the ultrafiltered virus stock solution. Test the ultrafiltered virus solution for Vg and, based on the test results, dilute the virus solution to a value of 9.8E12-1.3E13 Vg / ml.

[1239] The virus solution after concentration adjustment was tested for thermal stability and freeze-thaw stability, with particular attention paid to several indicators, including virus titer and purity. The results, shown in Table 36, show that the product's VG and purity remained stable after 3 months under long-term storage conditions. Under accelerated conditions at 25°C, the product's VG remained stable, with a slight decrease in purity at 3M, but still meeting quality standards.

[1240] Table 35. Formulations of preparations to be screened

[1241] Table 36. Effect of formulation pH range on virus stability “--” means not detected

[1242] Test Example 10. Effect of Preparation pH on Virus Tm Value

[1243] According to the formulation in Table 37, a preparation stock solution was prepared and sterilized and filtered. After sterilization, the sterile preparation stock solution was used to ultrafilter the virus suspension to complete solvent exchange. The ultrafiltered virus stock solution was sterilized and filtered. The Tm value of the ultrafiltered virus solution was tested. The results are shown in Table 38. Different pH conditions of the preparation had a significant effect on the virus Tm value.

[1244] Table 37. Test formulations

[1245] Table 38. Effect of pH of the preparation on the Tm value of the virus

[1246] Test Example 11. Effect of formulation on virus stability

[1247] According to the formulation in Table 39, a preparation stock solution was prepared and sterilized and filtered. After sterilization, the virus suspension was ultrafiltered using the sterile preparation stock solution to complete solvent exchange. The ultrafiltered virus stock solution was sterilized and filtered. The ultrafiltered virus solution was tested for Vg and diluted to (1.2-1.4) E13 vg / ml based on the test results.

[1248] The virus solution after adjusting the concentration was tested for thermal stability and freeze-thaw stability, and several indicators such as virus titer, purity, potency, IU, appearance, and viral bioactivity were observed. The results are shown in Table 40. After 1 month, 2 months, 3 months, and 6 months under long-term conditions, the product VG, purity, in vitro biological activity, and infectivity titer remained stable.

[1249] Table 39. Test formulations

[1250] Table 40. Effect of the test formulation on virus stability

[1251] The present disclosure also uses the following sequence:

Claims

1. A pharmaceutical composition comprising recombinant adeno-associated virus (rAAV) particles and excipients; wherein: The auxiliary material comprises Hepes; preferably, the concentration of Hepes is about 1 mM to about 100 mM, preferably about 5 mM to about 50 mM, preferably about 5 mM to about 25 mM.

2. The pharmaceutical composition according to claim 1, wherein The rAAV comprises a nucleic acid molecule, wherein the nucleic acid molecule comprises a polynucleotide encoding a VEGF binding molecule, wherein the VEGF binding molecule comprises a first VEGF binding domain and a second VEGF binding domain; wherein The first VEGF binding domain comprises immunoglobulin-like domain 2 (R2D2) of VEGFR2 and immunoglobulin-like domain 3 (R2D3) of VEGFR2; The second VEGF binding domain comprises a VEGF Trap, or comprises an anti-VEGF antibody or an antigen-binding fragment thereof; Preferably, the VEGF Trap comprises the immunoglobulin-like domain 2 (R1D2) of VEGFR1 and / or the immunoglobulin-like domain 3 (R2D3) of VEGFR2.

3. The pharmaceutical composition according to claim 2, wherein: The first VEGF binding domain comprises an amino acid mutation at any one or more positions selected from the group consisting of: position 162, position 221, position 222, position 274, position 276, position 277, position 280, position 284, position 288, position 289 and position 313; the positions of the amino acid mutations are positions in the natural sequence relative to the amino acid sequence of SEQ ID NO: 51; or, The first VEGF binding domain comprises an amino acid mutation at any one or more positions selected from the group consisting of: position 135, position 197, position 199, position 213, position 215, position 219, position 257, position 275, position 277, position 278, position 283, position 289, position 313 and position 314; the positions of the amino acid mutations are positions in the natural sequence relative to the amino acid sequence of SEQ ID NO: 51; preferably, the first VEGF binding domain comprises an amino acid mutation at position 283; Preferably, the first VEGF binding domain comprises any one or more groups of amino acid mutations selected from the following: -162V or 162I; -162V / 274F; 162V / 274I; 162I / 274F; or 162I / 274I; -221S / 222K; -277R / 289Y; -162V / 221S / 222K; -162V / 276Q; -162V / 289Y; -162V / 277R / 289Y; -162V / 313R; -162V / 221S / 222K / 274F; More preferably, it comprises any one or more groups of amino acid mutations selected from the following: -162V； -162V / 221S / 222K / 274F; or, Preferably, the first VEGF binding domain comprises any one or more groups of amino acid mutations selected from the following: -135Y； -197Y； -199Q or 199T; -213N； -215F or 215H; -219I； -257Q or 257T; -275L or 275E; -277R； -278D, 278S or 278E; -283T； -289Y； -313Y； -314S or 314Q; More preferably, the first VEGF binding domain comprises the amino acid mutation 283T.

4. The VEGF binding molecule according to any one of claims 2 to 3, wherein: The first VEGF binding domain comprises an amino acid mutation at any one or more positions selected from the group consisting of: position 162, position 221, position 222, position 274, position 276, position 277, position 280, position 284, position 288, position 289, position 313; and comprising an amino acid mutation at any one or more positions selected from the group consisting of: 135, 197, 199, 213, 215, 219, 257, 275, 277, 278, 283, 289, 313, and 314; Preferably, the first VEGF binding domain comprises: (i) any one or more groups of amino acid mutations selected from the following: -162A, 162V, 162S, 162I or 162L; -221S； -222K； -274F or 274I; -276Q； -277R； -280E； -284H； -288Y； -289Y； -313R; and (ii) any one or more groups of amino acid mutations selected from the following: -135Y； -197Y； -199Q or 199T; -213N； -215F or 215H; -219I； -257Q or 257T; -275L or 275E; -277R； -278D, 278S or 278E; -283T； -289Y； -313Y； -314S or 314Q; Preferably, the first VEGF binding domain comprises amino acid mutations selected from any one or more of the following groups: -162V / 275L; -162V / 283T; -162V / 313Y; -162V / 274F / 283T; -162V / 221S / 222K / 283T; -162V / 221S / 222K / 274F / 283T; More preferably, the first VEGF binding domain comprises amino acid mutations selected from any one or more of the following groups: -162V / 283T; -162V / 221S / 222K / 274F / 283T.

5. The pharmaceutical composition according to any one of claims 2 to 4, wherein: The VEGF binding molecule further comprises Fc; preferably, the Fc is the Fc of human IgG1, human IgG2, human IgG3 or human IgG4; preferably, The VEGF binding molecule comprises any one selected from the following from N-terminus to C-terminus: R2D2-R2D3-Fc; R1D2-R2D3-R2D2-R2D3-Fc; R1D2-R2D3-Linker-R2D2-R2D3-Fc; R2D2-R2D3-R1D2-R2D3-Fc; R2D2-R2D3-Linker-R1D2-R2D3-Fc; Alternatively, the VEGF binding molecule comprises a heavy chain and a light chain, wherein the heavy chain comprises from N-terminus to C-terminus: R2D2-R2D3-Fc-Linker-VH-CH1, and the light chain comprises from N-terminus to C-terminus: VL-CL; Preferably, the VH and VL are the VH and VL of bevacizumab or the VH and VL of ranibizumab; Preferably, the linker is a peptide linker; More preferably, the linker is selected from: (GS)a(GGS)b(GGGS)c(GGGGS)d(GGGGG)e, wherein a, b, c, d, e are independently integers greater than or equal to 0; or The linker is selected from: (EAAAK)3, (EAAAR)3, (EGGGK)3, (EGGGR)3, (DAAAR)3, (DAAAK)3, (DGGGR)3 or (DGGGK)3; or The linker is (G x S) y , wherein x is selected from an integer of 1-5, and y is selected from an integer of 1-6.

6. A pharmaceutical composition according to any one of claims 2 to 5, wherein: The VEGF binding molecule comprises an amino acid sequence as shown in any one of SEQ ID NOs: 55-56, SEQ ID NOs: 112-115, or an amino acid sequence having at least 90% sequence identity thereto; or The VEGF binding molecule consists of an amino acid sequence shown in any one of SEQ ID NOs: 55-56, SEQ ID NOs: 112-115, or an amino acid sequence having at least 90% sequence identity thereto; Preferably, 1) The VEGF binding molecule comprises an amino acid sequence as shown in any one of SEQ ID NOs: 53-54, SEQ ID NOs: 57-99, SEQ ID NOs: 102-111, SEQ ID NO: 116, or an amino acid sequence having at least 90% sequence identity thereto; 2) the heavy chain of the VEGF-binding molecule comprises the amino acid sequence of SEQ ID NO: 100, or an amino acid sequence having at least 90% sequence identity thereto, and the light chain of the VEGF-binding molecule comprises the amino acid sequence of SEQ ID NO: 101, or an amino acid sequence having at least 90% sequence identity thereto; or 3) The heavy chain of the VEGF-binding molecule comprises the amino acid sequence shown in SEQ ID NO: 117, or an amino acid sequence having at least 90% sequence identity thereto, and the light chain of the VEGF-binding molecule comprises the amino acid sequence shown in SEQ ID NO: 118, or an amino acid sequence having at least 90% sequence identity thereto.

7. The pharmaceutical composition according to any one of claims 2 to 6, wherein The nucleic acid molecule comprises a nucleic acid sequence having at least 70% identity to the nucleic acid sequence shown in any one of SEQ ID NOs: 128-136; Preferably, the nucleic acid molecule further comprises any one or any combination of the following: Kozak sequence, 5'-UTR and 3'-UTR, WPRE sequence, polyA sequence, intron; the Kozak sequence is preferably located between the intron and the polynucleotide encoding the VEGF binding molecule, and the polyA sequence is preferably the sv40 polyA sequence.

8. The pharmaceutical composition according to claim 1, wherein The rAAV comprises a nucleic acid molecule comprising a first polynucleotide and a second polynucleotide, wherein the first polynucleotide encodes an aromatic L-amino acid decarboxylase (AADC) protein and the second polynucleotide encodes a glial cell line-derived neurotrophic factor (GDNF) protein; Preferably, the AADC protein comprises the amino acid sequence shown in SEQ ID NO: 1, and / or the GDNF protein comprises the amino acid sequence shown in SEQ ID NO: 2; Preferably, the first polynucleotide comprises a sequence that is at least 75% identical to SEQ ID NO:3 or a sequence that is at least 95% identical to any one of SEQ ID NOs:4-7, and / or the second polynucleotide comprises a sequence that is at least 70% identical to SEQ ID NO:8 or a sequence that is at least 95% identical to any one of SEQ ID NOs:9-12.

9. The pharmaceutical composition according to claim 8, wherein The nucleic acid molecule further comprises any one or any combination of a 5' inverted terminal repeat (5'ITR), a 3' inverted terminal repeat (3'ITR), an intron, a post-transcriptional regulatory element, a polyadenylation signal (polyA) and a multiple cloning site (MCS); The 5'ITR and / or 3'ITR are derived from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV9.47, AAV9 (hu14), AAV10, AAV11, AAV12, AAV13, AAVrh8, AAVrh10, AAV-DJ or AAV-DJ8; preferably derived from AAV2 or AAV9; The promoter is selected from CMV, CAG, CBA, CBh, EFS, EF1, PGK, SV40, Ubi, RSV promoter or any combination thereof; The enhancer is selected from Ubi, CMV, RSV enhancer or any combination thereof; the intron is selected from MVM, SV40, β-globin, EF-1α, hybrid intron or any combination thereof; The polyA is selected from PA75 polyA, SV40 polyA, hGH polyA, BGH polyA, rbGlob polyA or Any combination thereof; and / or the post-transcriptional regulatory element is selected from WPRE, HPRE or a combination thereof; Preferably, the nucleic acid molecule comprises from the 5' end to the 3' end: (1) CMV enhancer-CBA promoter-hybrid intron-first polynucleotide-third polynucleotide-second polynucleotide encoding GDNF-WPRE-SV40 polyA; (2) CMV enhancer-CBA promoter-hybrid intron-second polynucleotide encoding GDNF-third polynucleotide-first polynucleotide-WPRE-SV40 polyA; (3) CMV enhancer-CBA promoter-hybrid intron-first polynucleotide-SV40 polyA-CMV enhancer-CMV promoter-second polynucleotide encoding GDNF-PA75 polyA; (4) CMV enhancer-CBA promoter-hybrid intron-first polynucleotide-SV40 polyA-CMV enhancer-CMV promoter-second polynucleotide encoding GDNF-WPRE-PA75 polyA; (5) CMV enhancer-CBA promoter-hybrid intron-first polynucleotide-SV40 polyA-CMV enhancer-CMV promoter-β-globin intron-second polynucleotide encoding GDNF-PA75 polyA; (6) CMV enhancer-CMV promoter-β-globin intron-second polynucleotide encoding GDNF-PA75 poly A-CMV enhancer-CBA promoter-hybrid intron-first polynucleotide-SV40 polyA; (7) CMV enhancer-CMV promoter-second polynucleotide encoding GDNF-PA75 poly A-CMV enhancer-CBA promoter-hybrid intron-first polynucleotide-WPRE-SV40 poly A; or (8) CMV enhancer-CMV promoter-MVM intron-second polynucleotide encoding GDNF-PA75 poly A-CMV enhancer-CBA promoter-hybrid intron-first polynucleotide-WPRE-SV40 polyA.

10. The pharmaceutical composition according to claim 8 or 9, wherein The nucleic acid molecule comprises a sequence as shown in any one of SEQ ID NOs: 13-25, 140-141 or a sequence having at least 95% sequence identity with any one of SEQ ID NOs: 13-25, 140-141.

11. The pharmaceutical composition according to any one of claims 1 to 10, further comprising an inorganic salt, wherein the inorganic salt is preferably a sodium salt and / or a magnesium salt; the sodium salt is preferably sodium chloride, and the magnesium salt is preferably magnesium chloride, more preferably magnesium chloride hexahydrate.

12. The pharmaceutical composition according to any one of claims 1 to 11, wherein The concentration of the sodium salt is about 10 mM to about 500 mM, preferably about 10 mM to about 200 mM, more preferably about 100 mM to about 150 mM; and / or, The concentration of the magnesium salt is about 0.1 mM to about 50 mM, preferably about 1 mM to about 10 mM, and more preferably about 1 mM to about 5 mM.

13. The pharmaceutical composition according to any one of claims 1 to 12, further comprising one or more of sugars, alcohols, and sugar alcohols; Preferably, the sugar is selected from sucrose, mannose and / or trehalose, more preferably sucrose; Preferably, the alcohol is glycerol; Preferably, the pharmaceutical composition comprises sugars and alcohols, wherein: The concentration of the saccharide is about 0.1% (w / v) to about 10% (w / v), preferably about 1% (w / v) to about 10% (w / v), preferably about 1% (w / v) to about 5% (w / v); and / or, The concentration of the alcohol is about 0.01% (w / v) to about 10% (w / v), preferably about 0.05% (w / v) to about 5% (w / v), preferably about 0.1% (w / v) to about 0.5% (w / v).

14. The pharmaceutical composition according to any one of claims 1 to 13, further comprising a surfactant; Preferably, the surfactant is poloxamer 188 and / or polysorbate 80, more preferably poloxamer 188; Preferably, the concentration of the surfactant is about 0.001% (w / v) to about 1% (w / v), preferably about 0.001% (w / v) to about 0.05% (w / v), preferably about 0.01% (w / v) to about 0.02% (w / v).

15. The pharmaceutical composition according to any one of claims 1 to 14, further comprising an amino acid, wherein the amino acid is preferably histidine; Preferably, the concentration of the amino acid is from about 1 mM to about 50 mM, preferably from about 1 mM to about 10 mM, more preferably from about 1 mM to about 5 mM.

16. The pharmaceutical composition according to any one of claims 1 to 15, wherein The pH of the pharmaceutical composition is about 4.0 to about 10, preferably about 6.0 to about 7.

5.

17. The pharmaceutical composition according to any one of claims 1 to 16, wherein The titer of the recombinant adeno-associated virus (rAAV) particles was about 1×10 8 Vg / mL~about 1×10 14 Vg / mL, preferably about 1×10 11 Vg / mL~about 9×10 13 Vg / mL, preferably about 1×10 12 Vg / mL~about 9×10 13 Vg / mL.

18. A pharmaceutical composition, wherein The pharmaceutical composition comprises recombinant adeno-associated virus (rAAV) particles, and: (a) about 1 mM to about 100 mM Hepes, about 10 mM to about 500 mM of the sodium salt, about 1 mM to about 50 mM of the magnesium salt, 0.1% (w / v) to about 10% (w / v) sugars, about 0.01% (w / v) to about 10% (w / v) of alcohol, 1 mM to about 50 mM of an amino acid, and, about 0.001% (w / v) to about 0.05% (w / v) of a surfactant; the surfactant is selected from poloxamer 188 and / or polysorbate 80; the pH of the pharmaceutical composition is about 4.0 to about 10.0; (c) about 5 mM to about 50 mM Hepes, about 10 mM to about 200 mM sodium chloride, and about 1 mM to about 10 mM magnesium chloride hexahydrate, about 1% (w / v) to about 10% (w / v) of a sugar selected from sucrose and / or trehalose; about 0.05% (w / v) to about 5% (w / v) glycerol, About 1 mM to about 10 mM histidine, About 0.001% (w / v) to about 0.05% (w / v) of a surfactant; the surfactant is selected from poloxamer 188 and / or polysorbate 80; the pH of the pharmaceutical composition is about 4.0 to about 10.0, preferably about 6.0 to about 8.0; (e) about 5 mM to about 30 mM Hepes, about 50 mM to about 200 mM sodium chloride, and about 1 mM to about 5 mM magnesium chloride hexahydrate, about 1% (w / v) to about 5% (w / v) sucrose, about 0.1% (w / v) to about 0.5% (w / v) glycerol, About 1 mM to about 5 mM histidine, about 0.001% (w / v) to about 0.02% (w / v) of poloxamer 188; the pH of the pharmaceutical composition is about 6.0 to about 8.0, preferably about 6.0 to about 7.5; (g) about 5 mM to about 20 mM Hepes, about 100 mM to about 150 mM sodium chloride, and about 1 mM to about 5 mM magnesium chloride hexahydrate, about 1% (w / v) to about 5% (w / v) sucrose, about 0.1% (w / v) to about 0.5% (w / v) glycerol, About 1 mM to about 5 mM histidine, about 0.01% (w / v) to about 0.02% (w / v) of poloxamer 188; the pH of the pharmaceutical composition is about 6.0 to about 8.0, preferably about 6.0 to about 7.5; Preferably, the titer of the recombinant adeno-associated virus (rAAV) particles is about 1×10 8 Vg / mL~about 1×10 14 Vg / mL, preferably about 1×10 11 Vg / mL~about 9×10 13 Vg / mL, preferably about 1×10 12 Vg / mL~about 9×10 13 Vg / mL; Preferably, the pharmaceutical composition comprises: (1) Approximately 1×10 11 Vg / mL~about 9×10 13 Vg / mL of recombinant adeno-associated virus (rAAV) particles About 10mM Hepes, about 100 mM sodium chloride, and about 2 mM magnesium chloride hexahydrate, About 2.5% (w / v) sucrose, About 0.25% (w / v) glycerol, About 5mM histidine, about 0.01% (w / v) of poloxamer 188; the pH of the pharmaceutical composition is about 6.5 to about 8.0, preferably about 7.5; (2) Approximately 1×10 11 Vg / mL~about 9×10 13 Vg / mL of recombinant adeno-associated virus (rAAV) particles About 10mM Hepes, about 100 mM sodium chloride, and about 2 mM magnesium chloride hexahydrate, About 2.5% (w / v) sucrose, About 0.25% (w / v) glycerol, About 5mM histidine, About 0.01% (w / v) polysorbate 80; the pH of the pharmaceutical composition is about 6.5 to about 8.0, preferably about 7.5; (3) Approximately 1×10 11 Vg / mL~about 9×10 13 Vg / mL of recombinant adeno-associated virus (rAAV) particles About 10mM Hepes, about 150 mM sodium chloride, and about 2 mM magnesium chloride hexahydrate, About 2.5% (w / v) sucrose, About 0.25% (w / v) glycerol, About 5mM histidine, about 0.01% (w / v) of poloxamer 188; the pH of the pharmaceutical composition is about 5.0 to about 7.0, preferably about 6.0; (4) Approximately 1×10 11 Vg / mL~about 9×10 13 Vg / mL of recombinant adeno-associated virus (rAAV) particles About 10mM Hepes, about 150 mM sodium chloride, and about 2 mM magnesium chloride hexahydrate, About 2.5% (w / v) sucrose, About 0.25% (w / v) glycerol, About 5mM histidine, About 0.01% (w / v) polysorbate 80; the pH of the pharmaceutical composition is about 5.0 to about 7.0, preferably about 6.

0.

19. The pharmaceutical composition according to any one of claims 1 to 18, wherein The recombinant adeno-associated virus (rAAV) particle comprises a nucleic acid molecule and an AAV capsid; Preferably, the AAV capsid is selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV9.47, AAV9(hu14), AAV10, AAV11, AAV12, AAV13, AAVPHP.B, AAVrh74, AAVrh8, AAVrh10, AAV-DJ or AAV-DJ8, more preferably AAV2 or AAV9.

20. A pharmaceutical composition comprising recombinant adeno-associated virus (rAAV) particles and excipients; the recombinant adeno-associated virus (rAAV) particles comprising a nucleic acid molecule and an AAV9 capsid, and the excipients comprising any one of the following: (a) about 1 mM to about 100 mM Hepes, about 10 mM to about 500 mM of the sodium salt, about 1 mM to about 50 mM of the magnesium salt, 0.1% (w / v) to about 10% (w / v) sugars, about 0.01% (w / v) to about 10% (w / v) of alcohol, 1 mM to about 50 mM of an amino acid, and, about 0.001% (w / v) to about 0.05% (w / v) of a surfactant; the surfactant is selected from poloxamer 188 and / or polysorbate 80; the pH of the pharmaceutical composition is about 4.0 to about 10.0; (c) about 5 mM to about 50 mM Hepes, about 10 mM to about 200 mM sodium chloride, and about 1 mM to about 10 mM magnesium chloride, about 1% (w / v) to about 10% (w / v) of a sugar selected from sucrose and / or trehalose; about 0.05% (w / v) to about 5% (w / v) glycerol, About 1 mM to about 10 mM histidine, About 0.001% (w / v) to about 0.05% (w / v) of a surfactant; the surfactant is selected from poloxamer 188 and / or polysorbate 80; the pH of the pharmaceutical composition is about 4.0 to about 10.0, preferably about 6.0 to about 8.0; (e) about 10 mM to about 30 mM Hepes, about 50 mM to about 200 mM sodium chloride, and about 1 mM to about 5 mM magnesium chloride hexahydrate, about 1% (w / v) to about 5% (w / v) sucrose, about 0.1% (w / v) to about 0.5% (w / v) glycerol, About 1 mM to about 5 mM histidine, about 0.001% (w / v) to about 0.02% (w / v) of poloxamer 188; the pH of the pharmaceutical composition is about 6.0 to about 8.0, preferably about 6.0 to about 7.5; (h) about 5 mM to about 20 mM Hepes, about 80 mM to about 120 mM sodium chloride, and about 1 mM to about 5 mM magnesium chloride hexahydrate, about 1% (w / v) to about 5% (w / v) sucrose, about 0.1% (w / v) to about 0.5% (w / v) glycerol, About 1 mM to about 5 mM histidine, about 0.01% (w / v) to about 0.02% (w / v) of poloxamer 188; the pH of the pharmaceutical composition is about 6.0 to about 8.0, preferably about 6.0 to about 7.5; Preferably, the titer of the recombinant adeno-associated virus (rAAV) particles is about 1×10 8 Vg / mL~about 1×10 14 Vg / mL, preferably about 1×10 11 Vg / mL~about 9×10 13 Vg / mL, preferably about 1×10 12 Vg / mL~about 9×10 13 Vg / mL; Preferably, the auxiliary material comprises any one of the following: (1) Approximately 1×10 11 Vg / mL~about 9×10 13 Vg / mL of recombinant adeno-associated virus (rAAV) particles About 10mM Hepes, about 100 mM sodium chloride, and about 2 mM magnesium chloride hexahydrate, About 2.5% (w / v) sucrose, About 0.25% (w / v) glycerol, About 5mM histidine, about 0.01% (w / v) of poloxamer 188; the pH of the pharmaceutical composition is about 6.5 to about 8.0, preferably about 7.5; (3) Approximately 1×10 11 Vg / mL~about 9×10 13 Vg / mL of recombinant adeno-associated virus (rAAV) particles About 10mM Hepes, about 100 mM sodium chloride, and about 2 mM magnesium chloride hexahydrate, About 2.5% (w / v) sucrose, About 0.25% (w / v) glycerol, About 5mM histidine, About 0.01% (w / v) polysorbate 80; the pH of the pharmaceutical composition is about 6.5 to about 8.0, preferably about 7.5; Preferably, the pharmaceutical composition comprises the nucleic acid molecule according to any one of claims 2 to 10; Alternatively, the pharmaceutical composition comprises recombinant adeno-associated virus (rAAV) particles and excipients; the recombinant adeno-associated virus (rAAV) particles comprise nucleic acid molecules and AAV2 capsids, and the excipients comprise any one of the following: (a) about 1 mM to about 100 mM Hepes, about 10 mM to about 500 mM of the sodium salt, about 1 mM to about 50 mM of the magnesium salt, 0.1% (w / v) to about 10% (w / v) sugars, about 0.01% (w / v) to about 10% (w / v) of alcohol, 1 mM to about 50 mM of an amino acid, and, about 0.001% (w / v) to about 0.05% (w / v) of a surfactant; the surfactant is selected from poloxamer 188 and / or polysorbate 80; the pH of the pharmaceutical composition is about 4.0 to about 10.0; (c) about 5 mM to about 50 mM Hepes, about 10 mM to about 200 mM sodium chloride, and about 1 mM to about 10 mM magnesium chloride, about 1% (w / v) to about 10% (w / v) of a sugar selected from sucrose and / or trehalose; about 0.05% (w / v) to about 5% (w / v) glycerol, About 1 mM to about 10 mM histidine, About 0.001% (w / v) to about 0.05% (w / v) of a surfactant; the surfactant is selected from poloxamer 188 and / or polysorbate 80; the pH of the pharmaceutical composition is about 4.0 to about 10.0, preferably about 6.0 to about 8.0; (e) about 10 mM to about 30 mM Hepes, about 50 mM to about 200 mM sodium chloride, and about 1 mM to about 5 mM magnesium chloride hexahydrate, about 1% (w / v) to about 5% (w / v) sucrose, about 0.1% (w / v) to about 0.5% (w / v) glycerol, About 1 mM to about 5 mM histidine, about 0.001% (w / v) to about 0.02% (w / v) of poloxamer 188; the pH of the pharmaceutical composition is about 6.0 to about 8.0, preferably about 6.0 to about 7.5; (i) about 5 mM to about 20 mM Hepes, about 120 mM to about 180 mM sodium chloride, and about 1 mM to about 5 mM magnesium chloride hexahydrate, about 1% (w / v) to about 5% (w / v) sucrose, about 0.1% (w / v) to about 0.5% (w / v) glycerol, About 1 mM to about 5 mM histidine, about 0.01% (w / v) to about 0.02% (w / v) of poloxamer 188; the pH of the pharmaceutical composition is about 6.0 to about 8.0, preferably about 6.0 to about 7.5; Preferably, the titer of the recombinant adeno-associated virus (rAAV) particles is about 1×10 8 Vg / mL~about 1×10 14 Vg / mL, preferably about 1×10 11 Vg / mL~about 9×10 13 Vg / mL, preferably about 1×10 12 Vg / mL~about 9×10 13 Vg / mL; Preferably, the auxiliary material comprises any one of the following: (4) Approximately 1×10 11 Vg / mL~about 9×10 13 Vg / mL of recombinant adeno-associated virus (rAAV) particles About 10mM Hepes, about 150 mM sodium chloride, and about 2 mM magnesium chloride hexahydrate, About 2.5% (w / v) sucrose, About 0.25% (w / v) glycerol, About 5mM histidine, about 0.01% (w / v) of poloxamer 188; the pH of the pharmaceutical composition is about 5.0 to about 7.0, preferably about 6.0; (6) Approximately 1×10 11 Vg / mL~about 9×10 13 Vg / mL of recombinant adeno-associated virus (rAAV) particles About 10mM Hepes, about 150 mM sodium chloride, and about 2 mM magnesium chloride hexahydrate, About 2.5% (w / v) sucrose, About 0.25% (w / v) glycerol, About 5mM histidine, About 0.01% (w / v) polysorbate 80; the pH of the pharmaceutical composition is about 5.0 to about 7.0, preferably about 6.

0.

21. A lyophilized preparation, which is obtained by freeze-drying the pharmaceutical composition according to any one of claims 1 to 20, or which can form the pharmaceutical composition according to any one of claims 1 to 20 after being reconstituted.

22. A reconstituted solution, which is prepared by reconstituted the lyophilized preparation according to claim 21.

23. A product comprising a container containing the pharmaceutical composition of any one of claims 1 to 20, the lyophilized preparation of claim 27, or the reconstituted solution of claim 22.

24. A method for treating or preventing a disease, comprising administering to a subject in need thereof a therapeutically or preventively effective amount of the pharmaceutical composition of any one of claims 1 to 20, the lyophilized preparation of claim 21, the reconstituted solution of claim 22, or the preparation of claim 23; preferably, the disease is selected from a disease associated with abnormal angiogenesis, a central nervous system disease or symptom.

25. The method according to claim 24, wherein: The disease is a disease related to abnormal angiogenesis; Preferably, the disease associated with abnormal angiogenesis is an vascular eye disease or cancer.

26. The method according to claim 24, wherein: The disease is a central nervous system disease or symptom; preferably, the central nervous system disease or symptom is a neurodegenerative disease or symptom, more preferably a movement disorder, a sleep disorder, and most preferably Parkinson's disease.

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