WO2025113432A1 - Gene-based drug for treating x-linked retinoschisis - Google Patents

Abstract

A gene-based drug for treating X-linked retinoschisis. The drug comprises a gene expression cassette for expressing retinoschisin (RS1 protein), can achieve high-level expression of RS1 protein, and delivers RS1 protein to target cells by means of constructing a new AAV virus vector, which can effectively compensate for X-linked retinoschisis and related eye diseases caused by RS1 gene mutation, and can fundamentally treat X-linked retinoschisis and complications thereof, and improve vision.

Description

A gene drug for the treatment of X-linked retinoschisis

The present invention claims priority to international application PCT/CN2023/134413 filed on November 27, 2023, entitled “A Genetic Medicine for Treating X-linked Retinoschisis”, the entire contents of which, including the appendix, are incorporated herein by reference.

Technical Field

The present invention relates to a gene medicine for treating X-chromosome linked retinoschisis, in particular to a recombinant adeno-associated virus vector carrying a human RS1 gene expression frame, which is a gene medicine for treating X-chromosome linked retinoschisis and belongs to the field of biotechnology.

Background Art

Congenital retinoschisis, also known as X-linked retinoschisis (XLRS), is a vitreoretinal degenerative disease. The first description of radial cystic changes in the macular area of two brothers was by German doctor Hass J in 1898. It was reported as X-linked in 1913, and the name X-linked retinoschisis was first used by Jager GM in 1935. The main clinical feature of XLRS is split lesions, which mainly accumulate on both sides of the retina, causing separation between the retinal nerve fiber layer and the retinal ganglion cell layer. XLRS patients mainly have mild to severe progressive loss of central vision, radial streaks caused by foveal fissures, OCT showing inner retinal splitting, and ERG showing negative waveforms (severe decrease in b-wave amplitude and mild decrease in a-wave amplitude). At the same time, the natural course of the disease in XLRS patients has certain variations. Some patients have obvious progression before the age of 10, while others are relatively stable in childhood. Most patients develop retinal atrophy in adulthood, leading to further deterioration of visual function. There is currently no treatment that can effectively reverse the progression of the disease, and the blindness rate of adult patients with XLRS exceeds 60%. XLRS is inherited in an X-linked recessive manner and mainly occurs in males. Most cases are familial. Cases of the disease have been reported all over the world. Literature reports that the prevalence of the disease in men is 1/5000-1/25000. There is currently no exact prevalence in my country. It is preliminarily estimated that there are at least 100,000 people with the disease in my country, and there are new cases every year. At the same time, there are reports of about 35,000 patients in the United States and the European Union. The natural course of congenital retinoschisis has certain variations. Most patients develop the disease within 10 years of age and have severe visual impairment; while others have relatively stable conditions during childhood. Most patients develop retinal atrophy in adulthood, leading to further deterioration of visual function. There is currently no treatment that can effectively reverse the progression of the disease, and the blindness rate of adult patients with XLRS exceeds 60%.

XLRS is mainly caused by mutations in the RS1 gene of patients. More than 400 mutations have been found so far (www.hgmd.org), including missense mutations, nonsense mutations, splicing mutations, small deletions/insertions, large deletions/insertions, and complex structural variations; missense mutations account for about 50% and are the most common type of variation. Except for complex structural variations, other mutation types have been found in our Chinese patients. The distribution of the above mutation types is basically the same, and no high-incidence mutation types have been found. Nonsense mutations, frameshift mutations caused by deletions/insertions, and large gene deletions usually lead to premature termination of truncated proteins that cannot function effectively. Since missense mutations only involve base changes at a certain site, it has been found that: 1) If located in the discoid structure region, it often causes protein folding errors, resulting in changes in spatial conformation, so that the protein cannot be secreted and is retained in the endoplasmic reticulum. 2) If base mutations occur in the sequence encoding the flanking part of the discoid structure, the RS1 subunit cannot assemble an oligomeric complex with adhesion function connected by disulfide bonds. 3) If a mutation occurs in the signal peptide region, it is found that the synthesis or secretion of RS1 protein is affected, and it is further rapidly degraded by proteases and cannot perform normal functions. 4) In addition, there are a few missense mutations that neither interfere with protein folding nor affect the normal secretion of cells, but disrupt the assembly of RS1 protein into an octamer oligomeric complex. Most mutations are missense mutations in exons 4-6 encoding the DS discoid domain. In summary, the main pathogenic mechanism of XLRS is RS1 gene mutation, which leads to RS1 protein secretion disorders, inability to octamerize, and reduced function of secreted octamer protein.

Currently, there is no drug that can effectively treat XLRS. Carbonic anhydrase inhibitors can be used to treat macular cystic lesions caused by XLRS, but the treatment effect is not great and is unstable. In addition, the treatment of complications caused by XLRS is also a focus of clinical research. For example, vitreous hemorrhage and retinal detachment caused by XLRS. The incidence of these two complications is high, and both complications may cause blindness in patients. RS1 gene mutations can cause vitreous hemorrhage in 3%-21% of patients, which is mainly caused by retinal splitting or vitreous traction on retinal blood vessels or extraretinal neovascularization. Mild symptoms are generally gradually absorbed and eventually disappear; severe symptoms are treated surgically. Nearly 20% of patients will experience retinal detachment, with holes caused by the split outer layer, the surrounding of the split cavity, or vitreous traction. Fluid can enter the retina through the holes, and severe retinal detachment can cause full-thickness retinal tearing. In addition, XLRS can also cause complications such as refractive error, strabismus, and neovascular glaucoma optic disc atrophy. Refractive error and strabismus can usually be treated through optical correction. The treatment of neovascular glaucoma optic disc atrophy first requires the elimination of new blood vessels and surgical treatment. However, even if these complications are effectively treated, the patient's vision is still in a poor state. Therefore, only gene therapy can make up for the patient's mutated RS1 gene, which is a fundamental way to cure XLRS.

Summary of the invention

Problem that the invention aims to solve

The current drug treatment for XLRS is ineffective and unstable, so there is no drug that can effectively treat XLRS. In addition, XLRS can also cause complications such as vitreous hemorrhage and retinal detachment, and current drugs have no way to fundamentally treat complications and improve vision.

Solutions for solving problems

[1] A gene expression cassette comprising a coding sequence for a retinoschisis protein and, optionally, a promoter, an enhancer and/or an intron;

Preferably, the coding sequence of the retinal splitting protein comprises a sequence as shown in any one of SEQ ID NO: 7 to 9 or a sequence that has at least 85% identity with a sequence as shown in any one of SEQ ID NO: 7 to 9.

[2] The gene expression cassette according to [1], wherein the promoter comprises a CMV promoter and/or a human retinoschisis protein gene promoter;

Preferably, the promoter comprises CMV promoter or human retinoschisis protein gene promoter;

Preferably, the CMV promoter comprises the sequence as described in SEQ ID NO:3 or a sequence having at least 85% identity thereto;

Preferably, the human retinal splitting protein gene promoter comprises a sequence as described in SEQ ID NO:11 or a sequence having at least 85% identity thereto.

[3] The gene expression cassette according to [1] or [2], wherein the enhancer comprises CMV, EF1α and/or IRBP;

Preferably, the enhancer CMV comprises the sequence as described in SEQ ID NO: 10 or a sequence having at least 85% identity thereto;

Preferably, the enhancer EF1α comprises the sequence as described in SEQ ID NO:12 or a sequence having at least 85% identity thereto;

Preferably, the enhancer IRBP comprises a sequence as described in SEQ ID NO:13 or a sequence having at least 85% identity thereto.

[4] The gene expression cassette according to any one of [1] to [3], wherein the intron comprises a CMVc intron, a human retinoschisis protein gene intron and/or an SV40 intron;

Optionally, the human retinoschisis protein gene intron comprises any intron of the human retinoschisis protein gene, preferably the first intron of the human retinoschisis protein gene, which comprises the sequence as described in SEQ ID NO: 15 or a sequence having at least 85% identity thereto;

Preferably, the CMVc intron comprises the sequence as described in SEQ ID NO:14 or a sequence having at least 85% identity thereto;

Preferably, the SV40 intron comprises a sequence as described in SEQ ID NO:16 or a sequence having at least 85% identity thereto.

[5] The gene expression cassette according to any one of [1] to [4], wherein the gene expression cassette further comprises a polyadenylation region;

Optionally, the polyadenylation region is selected from human growth hormone or SV40 polyadenylation region;

Preferably, the polyadenylation region comprises the SV40 polyadenylation region;

More preferably, the SV40 polyadenylation region comprises the sequence shown in SEQ ID NO:5 or a sequence having at least 85% identity thereto.

[6] The gene expression cassette according to any one of [1] to [5], wherein the structure of the gene expression cassette is as follows:

[Promoter]-[coding sequence of retinoschisis protein]-[polyadenylation region];

Preferably, it is [partial sequence of the 5' end of the promoter]-[enhancer]-[partial sequence of the 3' end of the promoter]-[coding sequence of the retinoschisis protein]-[polyadenylation region];

More preferably, it is [partial sequence at the 5' end of the promoter]-[enhancer]-[partial sequence at the 3' end of the promoter]-[partial sequence at the 5' end of the coding sequence of the retinoschisis protein]-[intron]-[partial sequence at the 3' end of the coding sequence of the retinoschisis protein]-[polyadenylation region].

[7]. A gene expression cassette according to any one of [1] to [6], wherein the nucleotide sequence of the gene expression cassette is as shown in SEQ ID NO:32.

[8]. A gene delivery vector comprising the gene expression cassette according to any one of [1] to [7].

[9] The gene delivery vector according to [8], wherein the gene delivery vector is a viral vector derived from a virus;

Preferably, the gene delivery vector is a recombinant adeno-associated virus.

[10] The gene delivery vector according to [9], wherein the recombinant adeno-associated virus comprises a capsid protein, and the gene expression cassette is encapsidated within the capsid protein;

Optionally, the capsid protein is selected from any one of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9 and AAV10 adeno-associated virus serotypes or variants thereof.

[11]. The gene delivery vector according to [10], wherein the capsid protein is AAV2 capsid protein or a variant thereof;

Preferably, the capsid protein is an AAV2 capsid protein variant;

More preferably, the AAV2 capsid protein variant comprises a sequence as shown in SEQ ID NO:24 or SEQ ID NO:25, or a sequence that is at least 85% identical to SEQ ID NO:24 or SEQ ID NO:25.

[12] A pharmaceutical composition comprising the gene expression cassette described in any one of [1] to [7] or the gene delivery vector described in any one of [8] to [11],

and, optionally, a pharmaceutically acceptable carrier.

[13] Use of the gene expression cassette described in any one of [1] to [7] or the gene delivery vector described in any one of [8] to [11] in the preparation of a drug for treating a disease;

Optionally, the disease is an eye disease;

Preferably, the eye disease is an eye disease associated with X-linked retinoschisis of the eye;

More preferably, the eye disease is selected from one or more of vitreous hemorrhage, retinal detachment, refractive error, strabismus and neovascular glaucoma optic disc atrophy.

[14] A method for treating a disease, comprising administering to a subject a therapeutically effective amount of the gene expression cassette described in any one of [1] to [7], the gene delivery vector described in any one of [8] to [11], or the pharmaceutical composition described in [12];

Optionally, the disease is an eye disease;

Preferably, the eye disease is an eye disease associated with X-linked retinoschisis of the eye;

More preferably, the eye disease is selected from one or more of vitreous hemorrhage, retinal detachment, refractive error, strabismus and neovascular glaucoma optic disc atrophy.

Effects of the Invention

In some embodiments, the gene expression cassettes provided by the present disclosure can achieve high-level expression of retinoschisis protein (RS1 protein);

In some embodiments, the gene delivery vector provided by the present disclosure can effectively deliver the retinoschisis protein (RS1 protein) into target cells;

In some embodiments, the pharmaceutical compositions provided by the present disclosure can effectively compensate for ocular diseases associated with X-linked retinoschisis in the eye due to RS1 gene mutations.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a schematic diagram of the pscAAV-CMV-hRS1-SV40 polyA vector structure.

ITR: inverted terminal repeat, a reverse terminal repeat sequence with a length of 145bp; CMV promoter: human CMV promoter sequence; hRS1: human RS1 gene sequence; SV40 polyA: SV40 virus polyA sequence; AvrII, SacI, StuI, SpeI, and Sa1I are all restriction enzyme cutting sites.

Figure 2 is a schematic diagram of the pscAAV-CMV-RS1opt1-SV40 polyA vector structure.

Figure 3 is a schematic diagram of the pscAAV-CMV-RS1opt2-SV40 polyA vector structure.

Figure 4 is a schematic diagram of the pscAAV-CMV-RS1opt3-SV40 polyA vector structure.

Figure 5 is a schematic diagram of the pscAAV-CMV-GFP vector structure.

Figure 6 is a schematic diagram of the pscAAV-scRS-IRBP-GFP vector structure.

Figure 7 is a schematic diagram of the pscAAV-scRS-CMV-hRS1opt2-SV40 polyA vector structure.

Figure 8 is a schematic diagram of the pscAAV-scRS-EF1α-hRS1opt2-SV40 polyA vector structure.

Figure 9 is a schematic diagram of the pscAAV-scRS-IRBP-hRS1opt2-SV40 polyA vector structure.

Figure 10 is a schematic diagram of the pscAAV-scRS-IRBP-hRS1opt2-CMVc Intron-SV40 polyA vector structure.

Figure 11 is a schematic diagram of the pscAAV-scRS-IRBP-hRS1opt2-Intron1-SV40 polyA vector structure.

Figure 12 is a schematic diagram of the pscAAV-scRS-IRBP-hRS1opt2-SV40 Intron-SV40 polyA vector structure.

FIG. 13 shows the fundus fluorescence level of C57BL/6J mice 4 weeks after intravitreal injection of GFP recombinant virus.

The recombinant AAV virus scAAV-CMV-GFP was injected into the left eye of C57BL/6J mice at a dose of 1E+9 vg/mouse (viral genome, vg) through the vitreous cavity, and the scAAV-scRS-IRBP-GFP was injected into the right eye of C57BL/6J mice at a dose of 1E+9 vg/mouse (viral genome, vg) through the vitreous cavity. C57BL/6J mice were sampled 28 days after injection, and frozen sections were used to detect the main expression position of GFP fluorescence in the retina.

FIG. 14 shows the expression level of RS1 protein after Rb1 cells were transfected with plasmids.

First, Rb1 cells were plated at a density of 1.5E+5 cells/well in a 24-well cell culture plate. The cells were cultured overnight at 37°C and 5% _CO2 , and then transfected with the following plasmids: pscAAV-CMV-hRS1 (i.e., pscAAV-CMV-hRS1-SV40 polyA), pscAAV-CMV-RS1opt1 (pscAAV-CMV-RS1opt1-SV40 polyA), pscAAV-CMV-RS1opt2 (i.e., pscAAV-CMV-RS1opt2-SV40 polyA), and pscAAV-CMV-RS1opt3 (i.e., pscAAV-CMV-RS1opt3-SV40 polyA). 1 μg of plasmid DNA was transfected into each well. After 72 hours of transfection, the cells were collected, the cells on the cell culture plate were scraped off with a cell scraper, and 100 μl of RIPA protein lysis buffer (containing 100 mM protease inhibitor) was incubated on ice for 30 min, then transferred to a new 1.5 ml centrifuge tube and centrifuged at 4 °C 12000 rpm for 15 min. The supernatant (protein sample) was collected and the total protein concentration of the sample was detected using the BCA kit. The sample was diluted to the same concentration according to the original protein concentration obtained by the test, and the expression level of RS1 protein in the sample was analyzed using the Jess automatic protein expression analysis system.

FIG. 15 shows the expression level of RS1 protein after ARPE-KO cells were transfected with plasmids.

First, ARPE-KO cells were plated at a density of 1.5E+5 cells/well in a 24-well cell culture plate. The cells were cultured overnight at 37°C and 5% _CO2 , and then transfected with pscAAV-CMV-hRS1 (i.e., pscAAV-CMV-hRS1-SV40 polyA) and pscAAV-CMV-RS1opt2 (i.e., pscAAV-CMV-RS1opt2-SV40 polyA), respectively. 1 μg of plasmid DNA was transfected into each well. Cells were collected 72 hours after transfection, and the cells on the cell culture plate were scraped off with a cell scraper. 100 μl of RIPA protein lysis buffer (containing 100 mM protease inhibitor) was added to each well, and the cells were incubated on ice for 30 minutes. The cells were then transferred to a new 1.5 ml centrifuge tube and centrifuged at 12000 rpm at 4°C for 15 minutes. The supernatant (protein sample) was collected, and the total protein concentration of the sample was detected using a BCA kit. The sample was diluted to the same concentration according to the original protein concentration obtained by the test, and the expression level of RS1 protein in the sample was analyzed using the Jess fully automatic protein expression analysis system.

FIG. 16 shows the expression level of RS1 protein after ARPE-KO cells were transfected with plasmids.

First, ARPE-KO cells were plated at a density of 1.5E+5 cells/well in a 24-well cell culture plate. The cells were cultured overnight at 37°C and 5% _CO2 , and then transfected with the following plasmids: pscAAV-scRS-CMV-hRS1opt2 (i.e., pscAAV-scRS-CMV-hRS1opt2-SV40 polyA), pscAAV-scRS-EF1α-hRS1opt2 (i.e., pscAAV-scRS-EF1α-hRS1opt2-SV40 polyA), and pscAAV-scRS-IRBP-hRS1opt2 (i.e., pscAAV-scRS-IRBP-hRS1opt2-SV40 polyA). 1 μg of plasmid DNA was transfected into each well. After 72 hours of transfection, the cells were collected, the cells on the cell culture plate were scraped off with a cell scraper, and 100 μl of RIPA protein lysis buffer (containing 100 mM protease inhibitor) was incubated on ice for 30 min, then transferred to a new 1.5 ml centrifuge tube and centrifuged at 4 °C 12000 rpm for 15 min. The supernatant (protein sample) was collected and the total protein concentration of the sample was detected using the BCA kit. The sample was diluted to the same concentration according to the original protein concentration obtained by the test, and the expression level of RS1 protein in the sample was analyzed using the Jess automatic protein expression analysis system.

FIG. 17 shows the expression level of RS1 protein after ARPE-KO cells were transfected with plasmids.

First, ARPE-KO cells were plated in a 24-well cell culture plate at a density of 1.5E+5 cells/well. After the cells were cultured overnight at 37°C and 5% CO ₂ , the cells were transfected with the following plasmids: pscAAV-scRS-IRBP-hRS1opt2-CMVc Intron (i.e., pscAAV-scRS-IRBP-hRS1opt2-CMVc Intron-SV40 polyA), pscAAV-scRS-IRBP-hRS1opt2-Intron1 (i.e., pscAAV-scRS-IRBP-hRS1opt2-Intron1-SV40 polyA), and pscAAV-scRS-IRBP-hRS1opt2-SV40Intron (i.e., pscAAV-scRS-IRBP-hRS1opt2-CMVc Intron-SV40 polyA). polyA), transfect 1 μg of plasmid DNA per well, collect cells 72 hours after transfection, scrape cells on the cell culture plate with a cell scraper, add 100 μl RIPA protein lysis buffer (containing 100 mM protease inhibitor) to each well, incubate on ice for 30 minutes, then transfer to a new 1.5 ml centrifuge tube, centrifuge at 4 ° C 12000 rpm for 15 minutes. Collect the supernatant (protein sample), use the BCA kit to detect the total protein concentration of the sample, dilute the sample to the same concentration according to the original protein concentration detected, and use the Jess automatic protein expression analysis system to analyze the expression level of RS1 protein in the sample.

FIG. 18 shows the changes in retinal cyst volume in RS1 ^R213W mice 8 weeks after intravitreal injection of recombinant virus (AAV.IVT15-RS1).

Seventeen 4-week-old RS1 ^R213W mice were randomly selected and divided into 3 groups. One group of 5 mice was injected with 1 μl PBS into the vitreous cavity as the control group G1; one group of 6 mice was injected with 2E+9 vg/eye (viral genome, vg) into the vitreous cavity as the low-dose test group G2; the last group of 6 mice was injected with 1E+10 vg/eye (viral genome, vg) into the vitreous cavity as the high-dose test group G3; OCT fundus photography of RS1 ^R213W mice was performed at different time points (4 weeks and 8 weeks) after intravitreal injection, and 25 photos were taken for each eye. The size of the retinal cyst in each OCT fundus photograph was then scored (scoring criteria: 0 points, no cyst; 1 point, very mild; 2 points, mild; 3 points, moderate; 4 points, severe; 5 points, very severe).

FIG. 19 shows the electrophysiological ERG changes in RS1 ^R213W mice 12 weeks after intravitreal injection of the recombinant virus (AAV.IVT15-RS1).

Seventeen 4-week-old RS1 ^R213W mice were randomly selected and divided into 3 groups. One group of 5 mice was injected with 1 μl PBS into the vitreous cavity as the control group G1; one group of 6 mice was injected with 2E+9 vg/eye (viral genome, vg) into the vitreous cavity as the low-dose test group G2; the last group of 6 mice was injected with 1E+10 vg/eye (viral genome, vg) into the vitreous cavity as the high-dose test group G3; electrophysiological ERG detection was performed on RS1 ^R213W mice 12 weeks after intravitreal injection to observe the changes of a wave and b wave under dark adaptation and light adaptation.

Figure 20 is a schematic diagram of the vector structure of the final vector pscAAV-scRS CMV-hRS1opt2-SV40 intron-SV40 polyA.

FIG21 shows the changes in cyst volume of each group 4 and 8 weeks after treatment.

FIG. 22 shows the ERG baseline level detection results of each group.

FIG. 23 shows the changes in ERG levels in each group 8 weeks after administration.

FIG. 24 shows the changes in ERG levels in each group 16 weeks after administration.

FIG. 25 shows the changes in ERG levels in each group 24 weeks after administration.

FIG. 26 shows the mRNA expression level of RS1 gene in each group 24 weeks after administration.

DETAILED DESCRIPTION

Various exemplary embodiments, features and aspects of the present invention will be described in detail below. The word "exemplary" used here means "used as an example, embodiment or illustrative". Any embodiment described here as "exemplary" is not necessarily interpreted as being superior or better than other embodiments.

In addition, in order to better illustrate the present invention, numerous specific details are provided in the following specific embodiments. It should be understood by those skilled in the art that the present invention can be implemented without certain specific details. In other examples, methods, means, equipment and steps well known to those skilled in the art are not described in detail in order to highlight the subject matter of the present invention.

Unless otherwise stated, the units used in this specification are all international standard units, and the numerical values and numerical ranges appearing in the present invention should be understood to include the inevitable systematic errors in industrial production.

In this specification, the word "may" means both performing a certain process and not performing a certain process.

In this specification, the references to "some specific/preferred embodiments", "other specific/preferred embodiments", "embodiments", etc., mean that the specific elements (e.g., features, structures, properties and/or characteristics) described in connection with the embodiments are included in at least one embodiment described herein, and may or may not exist in other embodiments. In addition, it should be understood that the elements may be combined in various embodiments in any suitable manner.

In this specification, the numerical range expressed using "a numerical value A to a numerical value B" means a range including the endpoints numerical values A and B.

In the present specification, the use of “substantially” or “essentially” means that the standard deviation from a theoretical model or theoretical data is within a range of 5%, preferably 3%, and more preferably 1%.

In this specification, the word "may" means both performing a certain process and not performing a certain process.

In the present specification, "optional" or "optionally" means that the event or situation described below may or may not occur, and the description includes cases where the event occurs and cases where it does not occur.

In this specification, the terms "include" and "have" and any variations thereof are intended to cover non-exclusive inclusions. For example, a process, method, device, product or equipment comprising a series of steps is not limited to the listed steps or modules, but may optionally include steps not listed, or may optionally include other steps inherent to these processes, methods, products or equipment.

In this specification, the term "plurality" refers to two or more than two. "And/or" describes the association relationship of associated objects, indicating that three relationships can exist. For example, A and/or B can mean: A exists alone, A and B exist at the same time, and B exists alone. The character "/" generally indicates that the associated objects before and after are in an "or" relationship.

In this specification, the references to "some specific/preferred embodiments", "other specific/preferred embodiments", "embodiments", etc., mean that the specific elements (e.g., features, structures, properties and/or characteristics) described in connection with the embodiments are included in at least one embodiment described herein, and may or may not exist in other embodiments. In addition, it should be understood that the elements may be combined in various embodiments in any suitable manner.

According to the present disclosure, the terms "polypeptide", "protein" and "peptide" are used interchangeably herein to refer to a polymeric form of amino acids of any length, which may include coded and non-coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides with similar peptide backbones.

According to the present disclosure, the terms "nucleic acid molecule", "polynucleotide", "polynucleic acid", and "nucleic acid" are used interchangeably to refer to a polymeric form of nucleotides of any length, whether deoxyribonucleotides or ribonucleotides, or their analogs.

According to the present disclosure, the terms "upstream" and "downstream" are relative terms that define the linear position of at least two elements located in a nucleic acid molecule (whether single-stranded or double-stranded) oriented in the 5' to 3' direction.

According to the present disclosure, "fusion protein" and "fusion polypeptide" refer to hybrid polypeptides comprising protein domains from at least two different proteins. Any protein provided herein can be produced by any method known in the art. For example, the protein provided herein can be produced via recombinant protein expression and purification, which is particularly suitable for fusion proteins comprising a peptide linker. Methods for recombinant protein expression and purification are well known and include those described in Green and Sambrook, Molecular Cloning: A Laboratory Manual (4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2012)), the entire contents of which are incorporated herein by reference.

According to the present disclosure, the term "amino acid" may include natural amino acids, unnatural amino acids, amino acid analogs and all their D and L stereoisomers. According to the present disclosure, the amino acid three-letter code and one-letter code used are as described in J.biol.chem, 243, p3558 (1968). The amino acids and their abbreviations and English abbreviations in the present disclosure are as follows: histidine (His, H); serine (Ser, S); glutamic acid (Glu, E); glutamine (Gln, Q); glycine (Gly, G); threonine (Thr, T); phenylalanine (Phe, F); aspartic acid (Asp, D); tyrosine (Tyr, Y); leucine (Leu, L); isoleucine (Ile, I); arginine (Arg, R); alanine (Ala, A); valine (Val, V); tryptophan (Trp, W); methionine (Met, M); asparagine (Asn, N); cysteine (Cys, C); lysine (Lys, K); proline (Pro, P).

According to the present disclosure, amino acid "addition" refers to adding amino acids at the C-terminus or N-terminus of an amino acid sequence. According to the present disclosure, amino acid "deletion" refers to deleting 1, 2 or 3 or more amino acids from an amino acid sequence. According to the present disclosure, amino acid "insertion" refers to inserting an amino acid residue at an appropriate position in an amino acid sequence, and the inserted amino acid residues may also be all or partly adjacent to each other, or the inserted amino acids may not be adjacent to each other. According to the present disclosure, amino acid "substitution" refers to replacing an amino acid residue at a certain position in an amino acid sequence with other amino acid residues; wherein, the "substitution" may be a conservative amino acid substitution.

According to the present disclosure, "conservative modification", "conservative substitution" or "conservative replacement" refers to the replacement of an amino acid in a protein with another amino acid having similar characteristics (e.g., charge, side chain size, hydrophobicity/hydrophilicity, main chain conformation and rigidity, etc.), so that changes can be made frequently without changing the biological activity of the protein. Those skilled in the art know that, in general, single amino acid replacements in non-essential regions of a polypeptide do not substantially change the biological activity (see, e.g., Watson et al. (1987) Molecular Biology of the Gene, The Benjamin/Cummings Pub. Co., p. 224, (4th ed.)). In addition, replacement of amino acids with similar structure or function is unlikely to destroy biological activity. Exemplary conservative substitutions are set forth below in "Exemplary Amino Acid Conservative Substitutions".

Exemplary conservative amino acid substitutions

According to the present disclosure, "identity" refers to the sequence similarity between two polynucleotide sequences or between two polypeptides. When the positions in the two compared sequences are occupied by the same base or amino acid monomer subunit, for example, if every position of the two DNA molecules is occupied by adenine, then the molecules are homologous at that position. The percent identity 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, when the sequences are optimally aligned, if 6 out of 10 positions in the two sequences are matched or homologous, then the two sequences are 60% homologous. In general, the comparison is made when the two sequences are aligned to obtain the maximum percent identity.

According to the present disclosure, "administering," "giving," and "treating" as applied to animals, humans, experimental subjects, cells, tissues, organs, or biological fluids, refers to the contact of an exogenous drug, therapeutic agent, diagnostic agent, or composition with an animal, human, subject, cell, tissue, organ, or biological fluid. "Administering," "giving," and "treating" may refer to, for example, treatment, pharmacokinetics, diagnosis, research, and experimental methods. Treatment of cells includes contact of an agent with a cell, and contact of an agent with a fluid, wherein the fluid is in contact with the cell. "Administering," "giving," and "treating" also means in vitro and ex vivo treatment of, for example, a cell by an agent, a diagnostic agent, a binding composition, or by another cell. "Treatment," when applied to humans, veterinary medicine, or research subjects, refers to therapeutic, prophylactic or preventative measures, research, and diagnostic applications.

According to the present disclosure, "treatment" means administering an internal or external therapeutic agent, such as any of the antibodies disclosed herein, to a patient who has one or more symptoms of a disease for which the therapeutic agent is known to have a therapeutic effect. Typically, the therapeutic agent is administered in an amount effective to alleviate one or more symptoms of a disease in the treated patient or population, either by inducing regression of such symptoms or inhibiting the development of such symptoms to any clinically measurable extent. The amount of a therapeutic agent effective to alleviate any specific disease symptom (also referred to as a "therapeutically effective amount") may vary according to a variety of factors, such as the patient's disease state, age, and weight, and the ability of the drug to produce the desired therapeutic effect in the patient. Whether the disease symptom has been alleviated can be evaluated by any clinical test method commonly used by physicians or other health care professionals to evaluate the severity or progression of the symptom.

According to the present disclosure, the term "prevention" refers to the preventive treatment of subjects who do not have a disease now and in the past but are at risk of developing a disease or who have had a disease in the past and do not have a disease now but are at risk of recurrence of the disease. In certain embodiments, the subject has a higher risk of developing a disease or a higher risk of recurrence of the disease compared to the average healthy member of the subject population.

According to the present disclosure, an "effective amount" includes an amount sufficient to improve or prevent the symptoms or symptoms of a medical condition. An effective amount also means an amount sufficient to allow or facilitate diagnosis. The effective amount for a particular patient or veterinary subject may vary depending on factors such as the condition to be treated, the patient's overall health, the method, 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.

According to the present disclosure, a "therapeutically effective amount" is an amount sufficient to provide a therapeutic benefit in the treatment of a condition or sufficient to delay or minimize one or more symptoms associated with a condition. A therapeutically effective amount refers to an amount of a therapeutic agent, alone or in combination with other therapies, that provides a therapeutic benefit in the treatment of a condition. The term "therapeutically effective amount" can include an amount that improves overall therapy; reduces or avoids symptoms, signs, or causes of a condition; and/or enhances the therapeutic efficacy of another therapeutic agent.

According to the present disclosure, a "prophylactically effective amount" is an amount sufficient to prevent a condition or one or more symptoms associated with a condition or to prevent its recurrence. A prophylactically effective amount refers to an amount of a therapeutic agent, alone or in combination with other agents, that provides a prophylactic benefit in preventing a condition. The term "prophylactically effective amount" may include an amount that improves overall prevention or enhances the prophylactic efficacy of another prophylactic agent.

According to the present disclosure, the term "subject" refers to a human (i.e., a male or female of any age, e.g., a pediatric subject (e.g., an infant, child, or adolescent) or an adult subject (e.g., a young, middle-aged, or elderly person)) or a non-human animal. In certain embodiments, the non-human animal is a mammal (e.g., a primate (e.g., a cynomolgus monkey or a rhesus monkey), a commercially relevant mammal (e.g., a cow, pig, horse, sheep, goat, cat, or dog), or a bird. The non-human animal can be male or female at any stage of development. The non-human animal can be a transgenic animal or a genetically engineered animal.

According to the present disclosure, "vector" refers to a macromolecule or an association of macromolecules that includes or is associated with a polynucleotide and can be used to mediate delivery of the polynucleotide to a cell. Illustrative vectors include, for example, plasmids, viral vectors (i.e., viruses such as adeno-associated viruses), liposomes, and other gene delivery vehicles.

According to the present disclosure, "expression vector" encompasses vectors, such as plasmids, minicircles, viral vectors, liposomes, etc., that include a gene expression cassette encoding a gene product of interest and are used to deliver the gene expression cassette to intended target cells.

According to the present disclosure, the term "AAV" is an abbreviation for adeno-associated virus and can be used to refer to the virus itself or its derivatives. The term covers all subtypes and naturally occurring and recombinant forms unless otherwise required. The term "AAV" includes AAV type 1 (AAV-1), AAV type 2 (AAV-2), AAV type 3 (AAV-3), AAV type 4 (AAV-4), AAV type 5 (AAV-5), AAV type 6 (AAV-6), AAV type 7 (AAV-7), AAV type 8 (AAV-8), AAV type 9 (AAV-9), avian AAV, bovine AAV, canine AAV, equine AAV, primate AAV, non-primate AAV and ovine AAV. "Primate AAV" refers to AAV that infects primates, "non-primate AAV" refers to AAV that infects non-primate mammals, "bovine AAV" refers to AAV that infects bovine mammals, etc.

According to the present disclosure, "AAV virus" or "AAV virus particle" or "rAAV vector particle" refers to a virus particle composed of at least one AAV capsid protein (usually composed of all capsid proteins of wild-type AAV) and an encapsidated polynucleotide. If the particle includes a heterologous polynucleotide (i.e., a polynucleotide other than the wild-type AAV genome, such as a transgene to be delivered to a mammalian cell), it is generally referred to as a recombinant AAV vector or rAAV. Typically, the heterologous polynucleotide is flanked by AAV inverted terminal repeats (ITRs).

According to the present disclosure, the term "gene", "coding sequence" or "coding gene" refers to a nucleotide sequence that encodes a gene product in vitro or in vivo. The term "transgene" refers to a coding sequence or gene delivered into a cell by a vector. The coding sequence or gene can encode a peptide or polypeptide molecule.

According to the present disclosure, the term "operably linked" refers to the juxtaposition of genetic elements (e.g., promoters, enhancers, termination signal sequences, polyadenylation sequences, etc.), wherein the elements are in a relationship that permits them to operate in the intended manner. For example, a promoter is operably linked to a coding region if the promoter helps initiate transcription of a coding sequence. Intervening residues may exist between the promoter and the coding region as long as this functional relationship is maintained.

According to the present disclosure, the term "heterologous" refers to an entity that is derived from a different genotype than the rest of the entity to which it is being compared. For example, a polynucleotide introduced into a plasmid or vector derived from a different species by genetic engineering techniques is a heterologous polynucleotide. As another example, a promoter removed from its native coding sequence and operably linked to a coding sequence to which it is not naturally found is a heterologous promoter. Thus, for example, a rAAV comprising a heterologous nucleic acid encoding a heterologous gene product is a rAAV comprising a nucleic acid that is not normally contained in a naturally occurring wild-type AAV, and the encoded heterologous gene product is a gene product that is not normally encoded by a naturally occurring wild-type AAV.

According to the present disclosure, the term "endogenous" with respect to nucleotide molecules or gene products refers to nucleic acid sequences (eg, genes or genetic elements) or gene products (eg, RNA, proteins) that are naturally present in or associated with a host virus or cell.

DETAILED DESCRIPTION OF THE INVENTION

<Gene expression cassette>

According to some embodiments of the present disclosure, there is provided a gene expression cassette comprising a coding sequence of a retinoschisis protein and, optionally, one or more of a promoter, an enhancer and an intron.

According to the present disclosure, XLRS is caused by a mutation in a gene called RS1 on the X chromosome, which encodes a protein called retinoschisin (or RS1 protein). Retinoschisin is a structural protein expressed and secreted by photoreceptors and bipolar cells that strongly and specifically binds to the surface of many cells in the retina. The protein is used as an adhesive to maintain the structural integrity of the layers of the retina. In the absence of normal retinoschisin, the layers of the retina split, intercellular communication is interrupted, and retinal cells and ultimately vision are lost. XLRS patients typically show a weakened b wave in the electroretinogram (ERG) measurement of their retina. Patients with nonsense mutations typically suffer from more severe disease than patients with missense mutations.

Retinoschisis protein (RS1 protein) is expressed throughout the neural retina during retinal development. Following development and into adulthood, it is expressed by photoreceptors. RS1 protein is a secreted protein that is primarily localized to the inner segment (IS) of rod and cone photoreceptors and, to a lesser extent, to the outer plexiform layer. RS1 protein contains a discoidin domain that allows it to form a homo-octameric complex.

In some embodiments of the present disclosure, the retinoschisis protein (RS1 protein) comprises the following amino acid sequence (SEQ ID NO: 1) or a sequence having at least 85% identity with the following amino acid sequence:

In some embodiments of the present disclosure, the coding sequence of the retinal splitting protein comprises a sequence as shown in SEQ ID NO:4 or a sequence having at least 85% identity thereto; further, the sequence as shown in SEQ ID NO:4 may also be codon optimized.

According to the present disclosure, the term "codon optimization" refers to a polynucleotide sequence modified from its native form. Such modification results in a difference in one or more base pairs, with or without changes in its corresponding amino acid sequence, which may enhance or inhibit the expression of a gene and/or the cellular response to the modified polynucleotide sequence. A coding sequence is a part of an mRNA sequence that encodes an amino acid for translation. During the translation process, each of the 61 trinucleotide codons is translated into one of the 20 amino acids, thereby resulting in degeneracy or redundancy in the genetic code. However, different cell types and different animal species utilize tRNAs (each carrying an anticodon) that encode the same amino acid at different frequencies. When a gene sequence contains codons that are not frequently represented by corresponding tRNAs, the ribosome translation mechanism may slow down, thereby hindering effective translation. Expression can be improved by "codon optimization" of a specific species, in which the coding sequence is changed to encode the same protein sequence, while codons (Cid-Arregui et al., 2003; Journal of Virology 77:4928) that are highly represented and/or utilized by highly represented human proteins are utilized.

In some specific embodiments, the codon-optimized coding sequence of the retinal splitting protein comprises a sequence as shown in any one of SEQ ID NO:7 to 9 or a sequence that has at least 85% identity with a sequence as shown in any one of SEQ ID NO:7 to 9.

In some embodiments of the present disclosure, in addition to the sequence encoding the protein product, the gene expression cassette further comprises polynucleotide elements for controlling the expression of the protein product, such as promoters, enhancers, introns, polyadenylation signals, etc. The promoters and enhancers may be natural or artificial or chimeric sequences, i.e., prokaryotic or eukaryotic sequences.

According to the present disclosure, a "promoter" encompasses a DNA sequence that directs RNA polymerase binding and thereby promotes RNA synthesis. Promoters and corresponding protein or polypeptide expression can be ubiquitous (meaning that they are strongly active in a wide range of cells, tissues, and species) or cell type-specific, tissue-specific, or species-specific. Promoters can be "constitutive" (meaning that they are continuously active) or "inducible" (meaning that the promoter can be activated or inactivated by the presence or absence of biological or abiotic factors).

In some optional embodiments, the promoter has a CMV promoter or other hybrid CMV promoter (called CB and CAG promoter) of vertebrate β-actin, β-globulin or β-globulin regulatory elements, EF1 promoter, hypoxia response element, ubiquitin promoter, T7 promoter, SV40 promoter, VP16 or VP64 promoter, or human retinoschisis protein gene promoter; preferably, it is a CMV promoter or a human retinoschisis protein gene promoter, more preferably, the CMV promoter comprises a sequence as described in SEQ ID NO:3 or a sequence having at least 85% identity thereto, and the human retinoschisis protein gene promoter comprises a sequence as described in SEQ ID NO:11 or a sequence having at least 85% identity thereto.

In some further preferred embodiments, the promoter comprises the human retinoschisis protein gene promoter.

According to the present disclosure, "enhancer" encompasses cis-acting elements that stimulate or inhibit transcription of adjacent genes. Enhancers that inhibit transcription are also referred to as "silencers". Enhancers can act in any direction at a distance of several thousand base pairs (kb) from the coding sequence and the position downstream of the transcription region (i.e., can be associated with the coding sequence). Exemplary, an example of a suitable enhancer is the CMV enhancer. Other suitable enhancers include enhancers suitable for the desired target tissue indication.

In some specific embodiments, the enhancer comprises enhancer CMV, enhancer EF1α and/or enhancer IRBP; the enhancer CMV comprises the sequence as described in SEQ ID NO:10 or a sequence having at least 85% identity thereto; the enhancer EF1α comprises the sequence as described in SEQ ID NO:12 or a sequence having at least 85% identity thereto; the enhancer IRBP comprises the sequence as described in SEQ ID NO:13 or a sequence having at least 85% identity thereto.

In some further preferred embodiments, the enhancer comprises enhancer CMV.

In some specific embodiments, the enhancer is located in the sequence of the promoter; exemplarily, the enhancer is inserted into the sequence of the promoter between positions 242 and 243, 243 and 244, 244 and 245, 245 and 246, 246 and 247, 247 and 248, 248 and 249, 249 and 250, or 250 and 251.

According to the present disclosure, "introns" include splice donor/acceptor regions. Introns are DNA polynucleotides that are transcribed into RNA by intron splicing and removed during mRNA processing. The expression of gene expression cassettes containing introns is generally higher than that of those gene expression cassettes that do not have introns.

In some specific embodiments, the introns in the above-mentioned gene expression cassette include one or more of CMVc introns, human retinoschisis protein gene introns, and SV40 introns, and the introns are located in the coding sequence of the retinoschisis protein; illustratively, the introns are inserted between positions 45 and 46, positions 46 and 47, positions 47 and 48, positions 48 and 49, positions 49 and 50, positions 50 and 51, positions 51 and 52, positions 52 and 53, positions 53 and 54, positions 54 and 55, positions 55 and 56, positions 56 and 57, positions 57 and 58, positions 58 and 59, positions 59 and 60, positions 60 and 61, positions 61 and 62, positions 62 and 63, positions 63 and 64, or positions 64 and 65 of the coding sequence of the retinoschisis protein.

In some preferred embodiments, the human retinoschisis protein gene intron can be any one or more introns in the human retinoschisis protein gene, for example, the first, second, third, fourth, or fifth intron, preferably the first intron of the human retinoschisis protein gene, which comprises the sequence as described in SEQ ID NO:15 or a sequence having at least 85% identity thereto.

In other preferred embodiments, the CMVc intron comprises a sequence as described in SEQ ID NO:14 or a sequence having at least 85% identity thereto.

In other preferred embodiments, the SV40 intron comprises a sequence as described in SEQ ID NO:16 or a sequence having at least 85% identity thereto.

In some further preferred embodiments, the intron comprises an SV40 intron.

In some optional embodiments of the present disclosure, the gene expression cassette further comprises a polyadenylation region (or polynucleotide tailing signal or polyadenylation signal).

As understood in the art, RNA polymerase II transcripts are terminated by cleavage and addition of a polyadenylation region, which may also be referred to as a polynucleotide tailing signal, a polyA signal, a polyA region, or a polyA tail. The polyA region contains multiple consecutive adenosine monophosphates, which typically have repeats of the motif AAUAAA. Several effective polyadenylation sites have been identified, including those from SV40, bovine growth hormone, human growth hormone, and rabbit beta-globin. The most effective polyA signal for expressing a transgene in a mammalian cell may depend on the cell type and species of interest, as well as the specific vector used. In some embodiments of the present disclosure, the gene expression cassette includes a polyA region selected from the group consisting of SV40, bovine growth hormone (bGH), human growth hormone (hGH), and beta-globin (beta globin).

In some optional embodiments, the polyA region is a human growth hormone polyA region. In some specific embodiments, the human growth hormone polyA region includes a sequence as shown in SEQ ID NO: 19 or a sequence having at least 85% identity thereto.

In some preferred embodiments, the polyA region is the SV40 polyA region. In some specific embodiments, the SV40 polyA region includes a sequence as shown in SEQ ID NO: 5 or a sequence having at least 85% identity thereto.

In some embodiments of the present disclosure, in addition to the sequence encoding the protein product, the gene expression cassette further comprises a variety of regulatory elements to enable the gene expression cassette to be packaged into a virus.

According to the present disclosure, the term "inverted terminal repeat (ITR)" includes any AAV viral terminal repeat or synthetic sequence that forms a hairpin structure and acts as a cis-element to mediate viral replication, packaging and integration. ITRs herein include, but are not limited to, terminal repeats from types 1-11 AAV (avian AAV, bovine AAV, canine AAV, equine AAV and sheep AAV). In addition, the AAV terminal repeat sequence does not have to have a natural terminal repeat sequence, as long as the terminal repeat sequence can be used for viral replication, packaging and integration.

In some exemplary embodiments, the ITR can be upstream and downstream ITRs from the AAV2 genome, optionally, the upstream ITR comprises a sequence as described in SEQ ID NO:2 or a sequence having at least 85% identity thereto; the downstream ITR intron comprises a sequence as described in SEQ ID NO:6 or a sequence having at least 85% identity thereto.

The gene expression cassette includes from 5' to 3' end:

[Promoter]-[Coding sequence of retinoschisis protein (RS1 protein)]-[Polyadenylation region].

Further, [partial sequence of the 5' end of the promoter] - [enhancer] - [partial sequence of the 3' end of the promoter] - [coding sequence of the retinoschisis protein (RS1 protein)] - [polyadenylation region].

Further, [partial sequence at the 5' end of the promoter] - [enhancer] - [partial sequence at the 3' end of the promoter] - [partial sequence at the 5' end of the coding sequence of the retinoschisis protein (RS1 protein)] - [intron] - [partial sequence at the 3' end of the coding sequence of the retinoschisis protein (RS1 protein)] - [polyadenylation region].

Furthermore, [upstream ITR]-[partial sequence of the 5' end of the promoter]-[enhancer]-[partial sequence of the 3' end of the promoter]-[partial sequence of the 5' end of the coding sequence of the retinoschisis protein (RS1 protein)]-[intron]-[partial sequence of the 3' end of the coding sequence of the retinoschisis protein (RS1 protein)]-[polyadenylation region]-[downstream ITR].

In some specific embodiments, the 5' partial sequence of the promoter includes positions 1 to 241, 1 to 242, 1 to 243, 1 to 244, 1 to 245, 1 to 246, 1 to 247, 1 to 248, 1 to 249 or 1 to 250 of the sequence of the promoter, and the 3' partial sequence of the promoter includes the sequence after position 241, 242, 243, 244, 245, 246, 247, 248, 249, 250 or 251 of the sequence of the promoter, and the 5' partial sequence of the promoter and the 3' partial sequence of the promoter constitute the complete sequence of the promoter. In other specific embodiments, the 5' end portion of the coding sequence of the retinoschisis protein (RS1 protein) comprises positions 1 to 45, 1 to 46, 1 to 47, 1 to 48, 1 to 49, 1 to 50, 1 to 51, 1 to 52, 1 to 53, 1 to 54, 1 to 55, 1 to 56, 1 to 57, 1 to 58, 1 to 59, 1 to 60, 1 to 61, 1 to 62, 1 to 63 or 1 to 64 of the coding sequence of the retinoschisis protein (RS1 protein), wherein The 3' end portion sequence of the coding sequence of the retinoschisis protein (RS1 protein) includes the sequence after position 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63 or 64 of the coding sequence of the retinoschisis protein (RS1 protein), and the 5' end portion sequence of the coding sequence of the retinoschisis protein (RS1 protein) and the 3' end portion sequence of the coding sequence of the retinoschisis protein (RS1 protein) constitute the complete coding sequence of the retinoschisis protein (RS1 protein).

<Gene delivery vector>

In some aspects of the present disclosure, the gene expression cassette of the present disclosure is used to deliver a gene (encoding a fusion polypeptide) to an animal cell, for example, to determine the effect of the gene on cell viability and/or function, to treat a cell disorder, etc. Therefore, in some aspects of the present disclosure, a gene delivery vector is provided, comprising the gene expression cassette of the present disclosure. In some preferred embodiments, the gene delivery vector is used to express a transgene (encoding a fusion polypeptide) in a mammalian cell.

The gene delivery vector of the present disclosure encompasses any convenient gene delivery vector for delivering a polynucleotide sequence to a mammalian cell. For example, the vector can include a single-stranded nucleic acid or a double-stranded nucleic acid, such as a single-stranded DNA or a double-stranded DNA. For example, the gene delivery vector can be a DNA, such as a naked DNA, such as a plasmid or a mini-circle, etc. The vector can include a single-stranded RNA or a double-stranded RNA, including a modified form of RNA. In another example, the gene delivery vector can be an RNA, such as an mRNA or a modified mRNA.

As another example, the gene delivery vector can be a viral vector derived from a virus, such as an adenovirus, an adeno-associated virus (AAV), a lentivirus, a herpes virus, an alpha virus, or a retrovirus, such as Moloney murine leukemia virus (M-MuLV), Moloney murine sarcoma virus (MoMSV), Harvey murine sarcoma virus (HaMuSV), murine mammary tumor virus (MuMTV), gibbon ape leukemia virus (GaLV), feline leukemia virus (FLV), foamy virus, Friend murine leukemia virus, murine stem cell virus (MSCV), and Rous sarcoma virus (RSV) or a lentivirus. Although embodiments covering the use of adeno-associated viruses are described in more detail below, it is expected that the skilled artisan will recognize that similar knowledge and skills in the art can also be applied to non-AAV gene delivery vectors.

In some embodiments, the gene delivery vector is a recombinant adeno-associated virus (rAAV). In this embodiment, the gene expression cassette is flanked by functional AAV inverted terminal repeat (ITR) sequences at the 5' and 3' ends. "Functional AAV ITR sequences" refer to ITR sequences that are used to rescue, replicate, and package AAV virus particles as expected. Therefore, the AAV ITRs used in the gene delivery vectors of the present disclosure do not need to have wild-type nucleotide sequences and can be altered by insertion, deletion, or substitution of nucleotides, or the AAV ITRs can be derived from any of several AAV serotypes, for example, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10. Preferred AAV vectors have all or part of the wild-type Rep gene and Cap gene deleted, but retain functional flanking ITR sequences. In specific embodiments, the AAV viral vector is an AAV variant. In some embodiments, the AAV variant is an AAV viral vector comprising a variant AAV capsid (or referred to as an AAV capsid protein variant).

In some embodiments, the gene expression cassette is encapsidated in an AAV capsid, which can be derived from any adeno-associated virus serotype, including but not limited to AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, etc., any of which can serve as a gene delivery vector. For example, the AAV capsid can be a wild-type capsid or a natural capsid. However, like ITR, the capsid does not need to have a wild-type nucleotide sequence, but as long as the capsid is capable of transducing mammalian cells, it can be changed relative to the wild-type sequence by insertion, deletion or substitution of nucleotides in the VP1, VP2 or VP3 sequence. In other words, the AAV capsid can be a variant AAV capsid, which includes one or more amino acid substitutions, deletions or insertions relative to the parent capsid protein or AAV capsid protein derived from.

The AAV capsid is an icosahedron, composed of 60 VP capsid protein monomers, including 5 VP1 monomers, 5 VP2 monomers and 50 VP3 monomers. VP1, VP2, and VP3 monomers are all transcribed and translated from the Cap gene of AAV. Among them, VP1 is the longest, containing about 735 amino acids. VP2 and VP3 are "truncated versions" of VP1, which do not contain some amino acids at the N-terminus of the VP1 protein. By convention, the capsid protein modification site is named according to the amino acid sequence of the VP1 protein.

The AAV capsid protein variant can be an AAV capsid protein variant containing a targeting peptide that is targeted to a specific tissue. In some specific embodiments, the targeting peptide is a targeting peptide that has good targeting to the eye, and the target peptide that can be selected is shown in SEQ ID NO: 26 or SEQ ID NO: 27.

In some more specific embodiments, the AAV capsid protein variant is an AAV2 capsid protein variant.

In some preferred embodiments, the AAV2 capsid protein variant is AAV2 capsid protein variant IVT13 or IVT15.

The above-mentioned targeting peptides IVT13 and IVT15 are described in PCT International Application No. PCT/CN2023/095837, which is incorporated herein by reference. The targeting peptides IVT13, IVT15, and the sequences of IVT13, IVT15 and the specific construction methods are also described in Example 3 below.

In some preferred embodiments, the AAV2 capsid protein variant is the AAV2 capsid protein variant IVT15, which is obtained by inserting the targeting peptide 15 (AAAGNGRAHAAA (SEQ ID NO: 27)) with good targeting to the eye at the 587th amino acid position of AAV2 VP1. That is, the targeting peptide is located between the 587th amino acid and the 588th amino acid of AAV2 VP1, and the AAV2 capsid protein/AAV2 containing the capsid protein is obtained.

In other preferred embodiments, the AAV2 capsid protein variant is the AAV2 capsid protein variant IVT13, which is obtained by inserting the targeting peptide 13 (AAARGSLAA (SEQ ID NO: 26)) with good targeting to the eye at the 587th amino acid position of AAV2 VP1. That is, the targeting peptide is located between the 587th amino acid and the 588th amino acid of AAV2 VP1, and the AAV2 capsid protein/AAV2 containing the capsid protein is obtained.

Preferably, rAAV is replication-defective, as the AAV vector is unable to independently replicate and package its genome. For example, when cone cells are transduced with rAAV viral particles, genes are expressed in the transduced cone cells, however, due to the fact that the transduced cone cells lack the AAV rep and cap genes and the auxiliary function genes, rAAV cannot replicate.

Standard methods can be used to produce gene delivery vectors (e.g., rAAV virus particles) that encapsidate the gene expression cassette of the present disclosure. For example, in the case of rAAV virus particles, the AAV expression vector according to the present disclosure can be introduced into the production cell, and then the AAV auxiliary construct is introduced, wherein the auxiliary construct comprises an AAV coding region that can be expressed in the production cell and the AAV coding region can supplement the AAV auxiliary function that does not exist in the AAV vector. Then the helper virus and/or another vector are introduced into the production cell, wherein the helper virus and/or another vector provide auxiliary functions that can support effective rAAV virus production. Then, the production cell is cultured to produce rAAV. These steps are performed using standard methods. The replication-defective AAV virus particles that encapsidate the recombinant AAV vector of the present disclosure are prepared using AAV packaging cells and packaging technology by standard techniques known in the art.

Any concentration of viral particles suitable for effective transduction of mammalian cells can be prepared to contact mammalian cells in vitro or in vivo. Similarly, any total number of viral particles suitable for providing appropriate cell transduction to impart the desired effect or treat disease can be administered to a mammal. Any suitable number of vectors can be administered to the eye of a mammal or primate.

The viral vector can be formulated into a pharmaceutical composition comprising any suitable unit dosage of the vector, which can be administered to a subject to produce a change in the subject or to treat a disease in the subject.

In some cases, the multiplicity of infection (MOI) can be used to measure the unit dose of a pharmaceutical composition. MOI refers to the ratio or number of cells to which a vector or viral genome can be delivered to a nucleic acid.

When preparing rAAV compositions, any host cell for producing rAAV viral particles can be used, including but not limited to mammalian cells (e.g., 293 cells), insect cells (e.g., SF9 cells), microorganisms, and yeast. The host cell can also be a packaging cell or a production cell, in which the AAV rep gene and cap gene are stably maintained in the host cell, and the AAV vector genome is stably maintained and packaged in the production cell. Exemplary packaging and production cells are derived from SF-9, 293, A549 or HeLa cells. AAV vectors are purified and formulated using standard techniques known in the art.

<Pharmaceutical Composition>

As disclosed herein, in some aspects of the present disclosure, a pharmaceutical composition is provided, comprising a fusion polypeptide, a polynucleotide, a gene expression cassette or a gene delivery vector provided by the present disclosure, and, optionally, a pharmaceutically acceptable carrier.

In some embodiments, the pharmaceutical composition described in the present disclosure contains the above-mentioned fusion polypeptide, polynucleotide, gene expression cassette or the above-mentioned gene delivery vector as an active ingredient.

In some specific embodiments, the pharmaceutical composition of the present disclosure contains a gene delivery vector as an active ingredient, and the gene delivery vector is a recombinant adeno-associated virus. In these embodiments, the pharmaceutical composition includes about 1×10 ⁸ to about 1×10 ¹⁵ viral genomes (vg), about 1×10 ⁹ to about 1×10 ¹⁴ vg, about 1×10 ⁹ to about 1×10 ¹³ vg, such as 2.0×10 ⁹ vg, 1×10 ¹⁰ vg.

Individual doses are usually not less than the amount required to produce a measurable effect on the subject and can be determined based on the pharmacokinetics and pharmacology of absorption, distribution, metabolism and excretion ("ADME") of the pharmaceutical composition or its byproducts and thus based on the disposition of the composition in the subject. This includes consideration of route of administration and dosage. Effective doses and/or dosage regimens can be readily determined empirically based on preclinical assays, based on safety and escalation and dose range trials, individual clinician-patient relationships, and in vitro and in vivo assays.

As used herein, the term "pharmaceutically acceptable carrier" includes saline, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration.

The pharmaceutical composition is typically formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, such as intravenous, intraarterial, subcutaneous, intraperitoneal, intrathecal, intramuscular, or injection or infusion. Thus, delivery can be systemic or local. For example, for delivery to the retina, subretinal or intravitreal injections can be used (see, e.g., Ochakovski et al., Front Neurosci. 2017; 11: 174; Xue et al., Eye (Lond). 2017 Sep; 31(9): 1308-1316).

In some specific embodiments, the pharmaceutical compositions of the present disclosure are designed, engineered, or adapted for administration to primates (eg, non-human primates and human subjects) by intravitreal or subretinal injection.

Methods for formulating suitable pharmaceutical compositions are known in the art, see, for example, Remington: The Science and Practice of Pharmacy, 21st ed., 2005; and Drugs and the Pharmaceutical Sciences: a Series of Textbooks and Monographs (Dekker, NY).

<Use and method>

In some aspects of the present disclosure, the present disclosure provides a method for treating or preventing a disease (e.g., an ocular disease) in a subject in need thereof, the method comprising administering an effective amount of the fusion polypeptide, polynucleotide, gene expression cassette, gene delivery vector or pharmaceutical composition of the present disclosure to a subject in need thereof.

In other aspects of the present disclosure, the present disclosure also provides use of the fusion polypeptide, polynucleotide, gene expression cassette or gene delivery vector of the present disclosure in the preparation of a medicament for treating a disease (eg, an ocular disease).

In some specific embodiments, the gene expression cassette, gene delivery vector or pharmaceutical composition of the present disclosure can at least partially improve eye diseases associated with X-linked retinoschisis of the eye. In some embodiments, the gene expression cassette, gene delivery vector or pharmaceutical composition (coding sequence of fusion polypeptide) of the present disclosure can be delivered to the eye of a human subject.

In some embodiments, the method as disclosed herein and the gene expression cassette, gene delivery vector or pharmaceutical composition are used to prevent or treat eye diseases or conditions that respond to retinoschisis protein (RS1 protein) in vivo. In some optional embodiments, the eye diseases include vitreous hemorrhage, retinal detachment, refractive error, strabismus, neovascular glaucoma, optic disc atrophy and the like due to RS1 gene mutations.

In some specific embodiments, the gene expression cassette, gene delivery vector or pharmaceutical composition can be administered parenterally by intravenous injection or oral infusion. In some more specific embodiments, the gene expression cassette, gene delivery vector or pharmaceutical composition is applied to the eye by injection, for example, to the retina, lower retina or vitreous. In other more specific embodiments, the gene expression cassette, gene delivery vector or pharmaceutical composition is applied by retinal injection, lower retinal injection or intravitreal injection. In other more specific embodiments, the gene expression cassette, gene delivery vector or pharmaceutical composition is applied locally or directly to the tissue or organ of interest, for example, by injection into the liver. In some embodiments, the method includes a single administration; in other embodiments, when the attending clinician considers it appropriate, multiple administrations can be performed over time.

The subject can be a mammal, including, for example, a human subject in need of treatment for a particular disease (eg, an ocular disease associated with X-linked retinoschisis of the eye).

In some specific embodiments, a gene delivery vector (recombinant adeno-associated virus) is administered to a subject, and the therapeutically effective amount for effecting a change or producing a therapeutic effect in the subject may be about 1×10 ⁸ viral genomes (vg) or more, and in some cases, about 1×10 ⁹ , 1×10 ¹⁰ , 1×10 ¹¹ , 1×10 ¹² , or 1×10 ¹³ viral genomes or more.

Example

The embodiments of the present invention will be described in detail below in conjunction with the examples, but those skilled in the art will appreciate that the following examples are only used to illustrate the present invention and should not be considered to limit the scope of the present invention. If no specific conditions are specified in the examples, they are carried out according to normal conditions or the conditions recommended by the manufacturer. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be obtained commercially.

Example 1: Construction of plasmid vector expressing RS1 gene

In order to construct an AAV vector plasmid (i.e., a gene delivery vector) carrying an RS1 gene expression cassette (gene expression cassette), this example first constructed an expression vector of the RS1 gene and optimized its codons. On this basis, an AAV vector plasmid carrying different promoter sequences, enhancer sequences, and intron sequences was constructed, and then an AAV vector plasmid containing an RS1 gene expression cassette with the optimal expression element was further constructed.

The plasmid in this example was synthesized by Universal Biosystems (Anhui) Co., Ltd.

1. Construction of AAV vector carrying RS1 gene expression cassette

(1) Construction of plasmid pscAAV-CMV-hRS1-SV40 polyA

The pscAAV-CMV-hRS1-SV40 polyA plasmid vector contains:

i) the upstream ITR (left ITR) from the AAV2 genome, the nucleotide sequence of which is shown in SEQ ID NO: 2;

ii) constitutive CMV promoter (CMV promoter), the nucleotide sequence of which is shown in SEQ ID NO: 3;

iii) human RS1 (hRS1) gene, the nucleotide sequence of which is shown in SEQ ID NO:4;

iv) SV40 polyA, the nucleotide sequence of which is shown in SEQ ID NO:5;

v) Downstream ΔITR (ITR/right ITR) from the AAV2 genome, the nucleotide sequence is shown in SEQ ID NO:6.

The constructed pscAAV-CMV-hRS1-SV40 polyA is shown in Figure 1.

Upstream ITR nucleotide sequence (SEQ ID NO: 2):

CMV promoter nucleotide sequence (SEQ ID NO: 3):

Human RS1 gene nucleotide sequence (SEQ ID NO: 4):

SV40 polyA nucleotide sequence (SEQ ID NO:5):

Downstream ΔITR nucleotide sequence (SEQ ID NO: 6):

(2) Construction of plasmid pscAAV-CMV-RS1 opt1-SV40 polyA

The hRS1 gene sequence in the plasmid pscAAV-CMV-hRS1-SV40 polyA was replaced with the RS1opt1 (SEQ ID NO:7), a sequence after the RS1 codon was optimized, to construct pscAAV-CMV-RS1opt1-SV40 polyA (Figure 2).

RS1opt1 nucleotide sequence (SEQ ID NO:7):

(3) Construction of plasmid pscAAV-CMV-RS1opt2-SV40 polyA

The hRS1 gene sequence in the plasmid pscAAV-CMV-hRS1-SV40 polyA was replaced by the RS1opt2 (SEQ ID NO:8), a sequence after the RS1 codon was optimized, to construct pscAAV-CMV-RS1opt2-SV40 polyA (Figure 3).

RS1 opt2 nucleotide sequence (SEQ ID NO:8):

(4) Construction of plasmid pscAAV-CMV-RS1opt3-SV40 polyA

The hRS1 gene sequence in the plasmid pscAAV-CMV-hRS1-SV40 polyA was replaced with the RS1opt3 (SEQ ID NO:9), a sequence after the RS1 codon was optimized, to construct pscAAV-CMV-RS1 opt3-SV40 polyA (Figure 4).

RS1opt3 nucleotide sequence (SEQ ID NO:9):

2. Construction of AAV vector plasmids carrying RS1 gene expression cassettes with different enhancer sequences

(1) Construction of plasmid pscAAV-scRS-CMV-hRS1opt2-SV40 polyA

The pscAAV-scRS-CMV-hRS1 opt2-SV40 polyA plasmid vector contains:

i) ITR from the AAV2 genome, the sequence of which is shown in SEQ ID NO: 2;

ii) positions 1 to 246 of the human RS1 gene promoter (hRS1 promoter, abbreviated as scRS) as shown in SEQ ID NO: 11;

iii) CMV enhancer (CMV enhancer), the sequence of which is shown in SEQ ID NO: 10;

iv) positions 247 to 275 of the promoter of the human RS1 gene as shown in SEQ ID NO: 11;

v) RS1 opt2, the sequence of RS1 gene after codon optimization, as shown in SEQ ID NO: 8;

vi) SV40 polyA, the sequence of which is shown in SEQ ID NO: 5;

vii) ΔITR from the AAV2 genome, the sequence of which is shown in SEQ ID NO:6.

The constructed pscAAV-scRS-CMV-hRS1opt2-SV40 polyA is shown in Figure 7.

CMV enhancer nucleotide sequence (SEQ ID NO: 10):

Human RS1 gene promoter sequence (SEQ ID NO: 11):

(2) Construction of plasmid pscAAV-scRS-EF1α-hRS1opt2-SV40 polyA

Replace the plasmid with EF1α enhancer (EF1αenhancer, SEQ ID NO: 12)

pscAAV-scRS-CMV-hRS1opt2-SV40 CMV enhancer in polyA was constructed

pscAAV-scRS-EF1α-hRS1opt2-SV40 polyA (Figure 8).

EF1α enhancer nucleotide sequence (SEQ ID NO: 12):

(3) Construction of plasmid pscAAV-scRS-IRBP-hRS1 opt2-SV40 polyA

The IRBP enhancer (IRBP enhancer, SEQ ID NO: 13) was used to replace the CMV enhancer in the plasmid pscAAV-scRS-CMV-hRS1 opt2-SV40 polyA to construct pscAAV-scRS-IRBP-hRS1 opt2-SV40 polyA (Figure 9).

IRBP enhancer nucleotide sequence (SEQ ID NO: 13):

3. Construction of AAV vectors carrying different intron sequences, enhancers and RS1 gene expression cassettes

(1) Construction of plasmid pscAAV-scRS-IRBP-hRS1opt2-CMVc Intron-SV40 polyA

pscAAV-scRS-IRBP-hRS1opt2-CMVc Intron-SV40 polyA contains:

i) ITR from the AAV2 genome, the sequence of which is shown in SEQ ID NO: 2;

ii) human RS1 gene promoter, the sequence of which is shown in SEQ ID NO:11;

iii) IRBP enhancer, the sequence of which is shown in SEQ ID NO:13;

iv) positions 1-52 of the codon-optimized sequence RS1_opt2 of the RS1 gene as shown in SEQ ID NO:8;

v) CMVc intron (CMVc Intron), the sequence of which is shown in SEQ ID NO:14;

vi) positions 53-675 of the codon-optimized sequence RS1_opt2 of gene RS1 as shown in SEQ ID NO:8;

vii) SV40 polyA, the sequence of which is shown in SEQ ID NO: 5;

viii) ΔITR from the AAV2 genome, the sequence of which is shown in SEQ ID NO:6.

The constructed pscAAV-scRS-IRBP-hRS1opt2-CMVc Intron-SV40 polyA is shown in Figure 10.

CMVc intron nucleotide sequence (SEQ ID NO: 14):

(2) Construction of plasmid pscAAV-scRS-IRBP-hRS1opt2-Intron1-SV40 polyA

The first intron of RS1 gene (Intron1, SEQ ID NO: 15) replaced the CMVc intron in pscAAV-scRS-IRBP-hRS1 opt2-CMVc Intron-SV40 polyA to construct pscAAV-scRS-IRBP-hRS1 opt2-Intron1-SV40 polyA (Figure 11).

The nucleotide sequence of the first intron (Intron1) of RS1 gene (SEQ ID NO: 15):

(3) Construction of plasmid pscAAV-scRS-IRBP-hRS1opt2-SV40 Intron-SV40 polyA

The sequence of SV40 intron (SV40intron, SEQ ID NO: 16) was used to replace the CMVc intron in pscAAV-scRS-IRBP-hRS1opt2-CMVc Intron-SV40 polyA to construct pscAAV-scRS-IRBP-hRS1opt2-SV40 Intron-SV40 polyA (Figure 12).

SV40 intron nucleotide sequence (SEQ ID NO: 16):

5. Construction of AAV vectors carrying GFP gene expression cassettes with different promoters

(1) Construction of plasmid pscAAV-CMV-GFP

The pscAAV-CMV-GFP plasmid vector contains:

i) upstream ITR from the AAV2 genome, the sequence of which is shown in SEQ ID NO: 2;

ii) constitutive CMV promoter, the sequence of which is shown in SEQ ID NO: 3;

iii) human beta Globin intron, the sequence of which is shown in SEQ ID NO: 17;

iv) GFP fluorescent protein gene (GFP), the sequence of which is shown in SEQ ID NO: 18;

v) human growth hormone polynucleotide tailing signal (hGH polyA signal), the sequence of which is shown in SEQ ID NO:19;

vi) Downstream ΔITR from the AAV2 genome, the sequence is shown in SEQ ID NO:6.

The construction of pscAAV-CMV-GFP is shown in Figure 5.

Human β-globin intron nucleotide sequence (SEQ ID NO: 17):

GFP fluorescent protein gene nucleotide sequence (SEQ ID NO: 18):

The polynucleotide tailing signal nucleotide sequence of human growth hormone (SEQ ID NO: 19):

(2) Construction of plasmid pscAAV-scRS-IRBP-GFP

The pscAAV-scRS-IRBP-GFP plasmid vector contains:

i) upstream ITR from the AAV2 genome, the sequence of which is shown in SEQ ID NO: 2;

ii) IRBP enhancer, the sequence of which is shown in SEQ ID NO:13;

iii) human RS1 gene promoter, the sequence of which is shown in SEQ ID NO:4;

iv) GFP fluorescent protein gene, the sequence of which is shown in SEQ ID NO: 18;

v) SV40 polyA, the sequence of which is shown in SEQ ID NO: 5;

vi) Downstream ΔITR from the AAV2 genome, the sequence is shown in SEQ ID NO:6.

The constructed pscAAV-scRS-IRBP-GFP is shown in FIG6 .

Example 2: Screening of gene expression cassettes

The plasmid vector was obtained from Example 1. First, the plasmid containing pscAAV-CMV-GFP and pscAAV-scRS-IRBP-GFP was packaged with a three-plasmid packaging system (commissioned by Heyuan Biotechnology (Shanghai) Co., Ltd.) with reference to the literature (XiaoX, et al. J Virol. 1998; 72 (3): 2224-2232.). The three plasmids included plasmid pscAAV-CMV-GFP (or pscAAV-scRS-IRBP-GFP), AAV5RC plasmid (capsid & replication plasmid) and Helper plasmid (or Ad Helper plasmid, auxiliary plasmid, providing auxiliary factors required for the generation of AAV). The AAV virus was separated, purified and packaged by iodixanol density gradient centrifugation. The recombinant AAV virus was injected subretinaally into C57BL/6J mice at 1E+9 vg/eye. The eyeballs were collected 4 weeks after the injection, frozen sections were cut and photographed under a fluorescent inverted microscope for detection.

WERI-Rb1 cells (purchased from Wuhan Pronocell Life Science Co., Ltd., catalog number CL-0465) and ARPE-KO cells (ARPE-19 cell line with CYP4V2 gene knockout (Gene ID: 285440) commissioned by Suzhou GeneWeiZhi Biotechnology Co., Ltd., March 1, 2022) were cultured and plated in 24-well cell culture plates at a density of 1.5 × 10 ⁵ /well. The plasmid vectors pscAAV-CMV-hRS1, pscAAV-CMV-RS1opt1-SV40 polyA, pscAAV-CMV-RS1opt2-SV40 polyA, and pscAAV-CMV-RS1opt3-SV40 polyA were transfected into WERI-Rb1 cells, with 1 μg of plasmid DNA per well, and cell samples were collected 72 hours after transfection; the plasmid vectors pscAAV-CMV-hRS1-SV40 polyA, pscAAV-CMV-RS1opt2-SV40 polyA, pscAAV-scRS-CMV-hRS1opt2-SV40 polyA, pscAAV-scRS-EF1α-hRS1opt2-SV40 polyA, pscAAV-scRS-IRBP-hRS1opt2-SV40 polyA, and pscAAV-scRS-IRBP-hRS1opt3-CMVc-Intron-SV40 ARPE-KO cells were transfected with 1 μg of plasmid DNA per well, pscAAV-scRS-IRBP-hRS1opt2-Intron1-SV40 polyA, and pscAAV-scRS-IRBP-hRS1opt2-SV40 Intron-SV40 polyA, respectively. Total cell protein was collected using protein lysis buffer, and RS1 protein level was detected using the Jess fully automated protein expression analysis system.

From the results in Figure 13, it can be seen that compared with mice injected with scAAV-CMV-GFP recombinant AAV virus into the vitreous cavity, the green fluorescence level on the retina of mice injected with scAAV-scRS-IRBP-GFP recombinant AAV virus is higher.

From the results in Figures 14 and 15, it can be seen that among the plasmids pscAAV-CMV-RS1opt1-SV40 polyA, pscAAV-CMV-RS1opt2-SV40 polyA, and pscAAV-CMV-RS1opt3-SV40 polyA, the RS1 protein expression level in WERI-Rb1 cells and ARPE-KO cells transfected with plasmid pscAAV-CMV-RS1opt2-SV40 polyA was the highest, and RS1opt2 (SEQ ID NO: 8) was the optimal codon sequence.

From the results in Figure 16, it can be seen that among the plasmids pscAAV-scRS-CMV-hRS1opt2-SV40 polyA, pscAAV-scRS-EF1α-hRS1opt2-SV40 polyA, and pscAAV-scRS-IRBP-hRS1opt2-SV40 polyA, the RS1 protein expression level of ARPE-KO cells transfected with plasmid pscAAV-scRS-CMV-hRS1opt2-SV40 polyA was the highest, and the CMV enhancer (SEQ ID NO: 10) was the optimal enhancer.

From the results in Figure 17, it can be seen that among the plasmids pscAAV-scRS-IRBP-hRS1opt2-CMVc-Intron-SV40 polyA, pscAAV-scRS-IRBP-hRS1opt2-Intron1-SV40 polyA, and pscAAV-scRS-IRBP-hRS1opt2-SV40 Intron-SV40 polyA, the RS1 protein expression level of ARPE-KO cells transfected with the plasmid pscAAV-scRS-IRBP-hRS1opt2-SV40 Intron-SV40 polyA was the highest, and SV40 Intron (SEQ ID NO: 16) was the optimal intron.

Example 3: Design and construction of AAV capsid protein expression plasmid pAAV-RC2_IVT

1. Construction of intermediate plasmid RC2_IVB-NotI plasmid

In this embodiment, a reverse P5 promoter sequence was added upstream of the Rep sequence on pAAV-RC2 (purchased from CellBiolabs, catalog number: VPK-410-SER2), and a NotI restriction endonuclease site was inserted at 1752bp of the Cap2 sequence. The intermediate plasmid RC2_IVB-NotI plasmid was commissioned to Anhui General Biotechnology for construction. The RC2_IVB-NotI plasmid vector sequence is shown in SEQ ID NO:31.

2. Construction of AAV capsid protein expression plasmid encoding the capsid protein of interest

The AAV capsid protein expression plasmid encoding the capsid protein of interest was constructed using the Gibson assembly method (for specific steps, see Gibson Chemical Transformation Protocol (E2611), Gibson assembly of the PCR fragment and the intermediate plasmid RC2_IVB-NotI plasmid constructed in step 1 of NotI linearization, and then obtain different AAV capsid plasmids encoding capsid proteins of interest. In the process of obtaining the PCR fragment, PCR has no template, and the primers can be paired by themselves for PCR amplification. The primer sequences are as shown in Table 1:

Table 1:

Experimental results: In this example, the plasmid DNA to be constructed was subjected to restriction enzyme digestion and Sanger sequencing, proving that the expression plasmid of the corresponding capsid protein was successfully constructed. The amino acid sequence of the AAV capsid protein expressed in the obtained AAV capsid protein expression plasmid is shown in Table 2 below.

Table 2:

Example 4: Preparation and testing of recombinant AAV virus

1. The optimized expression element combination screened in Example 2 was constructed into the final vector pscAAV-scRS-CMV-hRS1 opt2-SV40 intron-SV40 polyA. The plasmid was synthesized by Universal Biosystems (Anhui) Co., Ltd. and the virus was packaged.

The final vector pscAAV-scRS CMV-hRS1opt2-SV40 intron-SV40 polyA is constructed in the same manner as in Example 1, and comprises:

i) upstream ITR from the AAV2 genome, the nucleotide sequence of which is shown in SEQ ID NO: 2;

ii) CMV enhancer, the sequence of which is shown in SEQ ID NO: 10;

ii) human RS1 gene promoter, the sequence of which is shown in SEQ ID NO:11;

iv) positions 1 to 52 of the codon-optimized sequence RS1opt2 of gene RS1 as shown in SEQ ID NO:8;

v) SV40 intron, the sequence of which is shown in SEQ ID NO: 16;

vi) positions 53 to 675 of the codon-optimized sequence RS1_opt2 of gene RS1 as shown in SEQ ID NO:8;

vii) SV40 polyA, the nucleotide sequence of which is shown in SEQ ID NO:5;

viii) Downstream ΔITR from the AAV2 genome, the sequence is shown in SEQ ID NO:6.

The expression cassette sequence in the final vector is shown in SEQ ID NO:32:

2. The recombinant AAV virus was packaged by the three-plasmid co-transfection method (commissioned to Guangzhou Paizhen Biotechnology Co., Ltd. for preparation). The three plasmids included the plasmid pscAAV-scRS CMV-hRS1 opt2-SV40 intron-SV40 polyA, the RC plasmid (capsid & replication plasmid; containing the nucleotide sequence of the capsid Cap, i.e., the AAV capsid protein expression plasmids RC2_IVT15 and RC2_IVT13 constructed in Example 3) and the Helper plasmid (or Ad Helper plasmid, auxiliary plasmid, providing the auxiliary factors required for the generation of AAV), which were named AAV.IVT15-RS1 and AAV.IVT13-RS1.

3. Use the ddPCR method to determine the genome titer of the prepared AAV virus.

The specific process is as follows:

Primers ITR-F, ITR-R and probe ITR-P were designed in ITR. The 5' end of the probe was labeled with 6-carboxyfluorescein (6-FAM) and the 3' end was labeled with tetramethylrhodamine (TAMRA):

ITR-F: 5’-GGAACCCCTAGTGATGGAGTT-3’ (SEQ ID NO: 28),

ITR-R: 5’-CGGCCTCAGTGAGCGA-3’(SEQ ID NO:29),

ITR-P: 5’-CACTCCCTCTCTGCGCGCTCG-3’ (SEQ ID NO: 30).

ITR-F and ITR-R were used as primers to specifically amplify the ITR fragment with a length of 62 bp. The ddPCR probe method was used, and the 2×ddPCR Mix (probe method) reagent (Sinafo, Suzhou, China) was used. The viral genome titer was detected using a fluorescent ddPCR instrument (model: DQ24, Sinafo). The operation process was referred to the instructions for the 2×ddPCR Mix (probe method) reagent. The titer of AAV.IVT15-RS1 and AAV.IVT13-RS1 viruses was 1E+13 vg/mL.

Example 5: Establishment of RS1 ^R213W mouse model

The RS1 mutation mouse model was prepared by Beijing Innovicon Pharmaceutical Technology Co., Ltd. For the specific construction method, please refer to the patent document with the patent name: A method for preparing an RS1 point mutation mouse model and its use, and patent publication number: CN115968834A.

Example 6: Treatment of X-staining in RS1 ^R213W mice by intravitreal injection of recombinant virus (AAV.IVT15-RS1) Systemic retinoschisis

17 newborn mice were randomly selected from the RS1 ^R213W mouse model obtained in Example 5 and divided into 3 groups. OCT fundus photography and ERG detection were performed when the mice were 3-4 weeks old as the baseline of the experimental mice.

Six mice in group 1 were injected intravitreally with the AAV.IVT15-RS1 recombinant virus prepared in Example 4 at a dose of 2×10 ⁹ vg/eye 4 weeks after birth, serving as the low-dose test group G2.

Six mice in group 1 were injected intravitreally with the AAV.IVT15-RS1 recombinant virus prepared in Example 4 at a dose of 1×10 ¹⁰ vg/eye 4 weeks after birth, serving as the high-dose test group G3.

In one group, five mice were injected with PBS through the vitreous cavity 4 weeks after birth, serving as the control group G1 for injection of therapeutic virus.

At 4 and 8 weeks after administration, OCT fundus photography was performed on the three groups of RS1 ^R213W mice, with 25 photos taken for each eye, and then the size of the retinal cyst in each OCT fundus photograph was scored (scoring criteria: 0 point, no cyst; 1 point, very mild; 2 points, mild; 3 points, moderate; 4 points, severe; 5 points, very severe). Electrophysiological ERG detection was performed at 12 weeks to observe the changes in a wave and b wave under dark adaptation and light adaptation.

From the results in Figure 18, it can be seen that at 4 weeks of drug administration, the size of the retinal cyst cavity among the three groups of mice was slightly different; as the drug administration time passed, at 8 weeks of drug administration, the cyst cavity area of the G1 group of mice increased, while the cyst cavity areas of the G2 and G3 groups were smaller than that of the G1 group, and showed a downward trend compared to 4 weeks of drug administration, showing a therapeutic effect.

As shown in the results of Figure 19, compared with the G1 control group, the G2 group with a dose of 2×10 ⁹ vg/eye of RS1 ^R213W mice administered by intravitreal injection 12 weeks later could significantly improve visual function in both light and dark reactions; the G3 group with a dose of 1×10 ¹⁰ vg/eye could also significantly improve visual function in partial dark reactions.

The results showed that the recombinant virus (AAV.IVT15-RS1) at a dose of 2×10 ⁹ vg/eye injected into the RS1 ^R213W mouse model via the vitreous cavity could effectively inhibit retinal cystic schisis and increase the electrical signal levels of photoreceptor cells and bipolar cells, thereby achieving the purpose of treating X-linked retinoschisis.

Example 7: Treatment of RS1 ^R213W newborn mice with X by intravitreal injection of recombinant virus (AAV.IVT13-RS1) Chromosomal-linked retinoschisis

20 newborn mice were randomly selected from the RS1 ^R213W mouse model obtained in Example 5 and divided into 4 groups. OCT fundus photography and ERG detection were performed on the mice at 3-4 weeks of age as the baseline of the experimental mice.

In one group, five mice were injected with PBS through the vitreous cavity 4 weeks after birth, serving as the control group G1 for injection of therapeutic virus.

Among them, 5 mice in group 1 were injected intravitreally with the AAV.IVT13-RS1 recombinant virus prepared in Example 4 at a dose of 5×10 ⁹ vg/eye 4 weeks after birth, serving as the high-dose group G2.

Among them, 5 mice in group 1 were injected intravitreally with the AAV.IVT13-RS1 recombinant virus prepared in Example 4 at a dose of 2×10 ⁹ vg/eye 4 weeks after birth, serving as the low-dose group G3.

The last group of 5 mice was injected intravitreally with the AAV.IVT13-RS1 recombinant virus prepared in Example 4 at a dose of 8×10 ⁸ vg/eye 4 weeks after birth, serving as the low-dose group G4.

Four weeks and eight weeks after administration, four groups of RS1 ^R213W mice were photographed with OCT fundus. Twenty-five photos were taken for each eye, and the size of the retinal cyst in each OCT fundus photo was scored (scoring criteria: 0, no cyst; 1, very mild; 2, mild; 3, moderate; 4, severe; 5, very severe). Electrophysiological ERG detection was performed at 8, 16, and 24 weeks after administration: the changes of a-wave and b-wave under different light intensities (0.00316 cd·s/m ² , 0.01 cd·s/m ² , 0.1 cd·s/m ² , 1 cd·s/m ² , 10 cd·s/m ² ) under dark adaptation and light adaptation were observed. After 24 weeks of testing, the mice were sampled, RNA was extracted from the mouse retina, and RT-qPCR was performed to detect the expression of the target gene RS1.

From the results in Figure 21, it can be seen that 4 weeks after administration, the degree of cyst splitting in the G2 high-dose group and the G3 medium-dose group was significantly reduced, and the cyst in the G4 low-dose group also showed a trend of reduction, but it has not yet reached a statistical difference; 8 weeks after administration, the cyst in the control group entered the atrophy stage from the splitting stage, and the cyst in the G2-G4 treatment groups all recovered to almost no cyst.

As can be seen from the results of Figure 22, there was no significant difference in the ERG amplitudes of the four groups at baseline. Eight weeks after administration, the b-wave amplitude of the G1 mice in the control group was significantly reduced compared with the baseline, while the dark-adapted b-wave amplitudes of the G2 high-dose group, the G3 medium-dose group, and the G4 low-dose group were significantly higher than those of the G1 control group (Figure 23). 16 weeks after administration, the b-wave amplitude of the mice in the treatment group increased over time, among which the b-wave amplitude of the G2 medium-dose group had recovered to a level that was not statistically different from that of wild-type C57BL/6J mice (Figure 24). 24 weeks after administration, the b-wave amplitude of the mice in each treatment group continued to increase, and the dark-adapted b-wave amplitudes of the G2 high-dose group and the G3 medium-dose group were both restored to a level that was not significantly different from that of C57BL/6J mice, and the therapeutic effect was very significant (Figure 25).

From the results in Figure 26, it can be seen that the drug can be effectively transduced and expressed in mice. The expression level of RS1 gene in the retinal tissue of mice in the treatment group was significantly higher than that in the control group, and showed a certain dose dependence.

The results showed that the recombinant virus AAV.IVT13-RS1 could be effectively expressed in vivo after intravitreal injection into the RS1 ^R213W mouse model, promoting the recovery of the split retinal cyst to near normal levels, while increasing the electrical signal levels of photoreceptor cells and bipolar cells, and the ERG b-wave amplitude could be restored to near normal levels, showing excellent therapeutic effects.

RC2_IVB-NotI plasmid vector sequence (SEQ ID NO:31)

It should be noted that, although the technical solution of the present invention is introduced with specific examples, those skilled in the art will appreciate that the present invention should not be limited thereto.

The embodiments of the present invention have been described above, and the above description is exemplary, not exhaustive, and is not limited to the disclosed embodiments. Many modifications and variations will be apparent to those of ordinary skill in the art without departing from the scope and spirit of the described embodiments. The selection of terms used herein is intended to best explain the principles of the embodiments, practical applications, or technical improvements in the market, or to enable other persons of ordinary skill in the art to understand the embodiments disclosed herein.

Claims (14)

A gene expression cassette comprising a coding sequence for a retinoschisis protein and, optionally, a promoter, an enhancer and/or an intron; Preferably, the coding sequence of the retinal splitting protein comprises a sequence as shown in any one of SEQ ID NO: 7 to 9 or a sequence that has at least 85% identity with a sequence as shown in any one of SEQ ID NO: 7 to 9. The gene expression cassette according to claim 1, wherein the promoter comprises a CMV promoter and/or a human retinoschisis protein gene promoter; Preferably, the promoter comprises CMV promoter or human retinoschisis protein gene promoter; Preferably, the CMV promoter comprises the sequence as described in SEQ ID NO:3 or a sequence having at least 85% identity thereto; Preferably, the human retinal splitting protein gene promoter comprises a sequence as described in SEQ ID NO:11 or a sequence having at least 85% identity thereto. The gene expression cassette according to claim 1 or 2, wherein the enhancer comprises CMV, EF1α and/or IRBP; Preferably, the enhancer CMV comprises the sequence as described in SEQ ID NO: 10 or a sequence having at least 85% identity thereto; Preferably, the enhancer EF1α comprises the sequence as described in SEQ ID NO:12 or a sequence having at least 85% identity thereto; Preferably, the enhancer IRBP comprises a sequence as described in SEQ ID NO:13 or a sequence having at least 85% identity thereto. The gene expression cassette according to any one of claims 1 to 3, wherein the intron comprises a CMVc intron, a human retinoschisis protein gene intron and/or an SV40 intron; Optionally, the human retinoschisis protein gene intron comprises any intron of the human retinoschisis protein gene, preferably the first intron of the human retinoschisis protein gene, which comprises the sequence as described in SEQ ID NO: 15 or a sequence having at least 85% identity thereto; Preferably, the CMVc intron comprises the sequence as described in SEQ ID NO:14 or a sequence having at least 85% identity thereto; Preferably, the SV40 intron comprises a sequence as described in SEQ ID NO:16 or a sequence having at least 85% identity thereto. The gene expression cassette according to any one of claims 1 to 4, wherein the gene expression cassette further comprises a polyadenylation region; Optionally, the polyadenylation region is selected from human growth hormone or SV40 polyadenylation region; Preferably, the polyadenylation region comprises the SV40 polyadenylation region; More preferably, the SV40 polyadenylation region comprises the sequence shown in SEQ ID NO:5 or a sequence having at least 85% identity thereto. The gene expression cassette according to any one of claims 1 to 5, wherein the structure of the gene expression cassette is as follows: [Promoter]-[coding sequence of retinoschisis protein]-[polyadenylation region]; Preferably, it is [partial sequence of the 5' end of the promoter]-[enhancer]-[partial sequence of the 3' end of the promoter]-[coding sequence of the retinoschisis protein]-[polyadenylation region]; More preferably, it is [partial sequence at the 5' end of the promoter]-[enhancer]-[partial sequence at the 3' end of the promoter]-[partial sequence at the 5' end of the coding sequence of the retinoschisis protein]-[intron]-[partial sequence at the 3' end of the coding sequence of the retinoschisis protein]-[polyadenylation region]. The gene expression cassette according to any one of claims 1 to 6, wherein the nucleotide sequence of the gene expression cassette is as shown in SEQ ID NO:32. A gene delivery vector comprising the gene expression cassette according to any one of claims 1 to 7. The gene delivery vector according to claim 8, wherein the gene delivery vector is a viral vector derived from a virus; Preferably, the gene delivery vector is a recombinant adeno-associated virus. The gene delivery vector according to claim 9, wherein the recombinant adeno-associated virus comprises a capsid protein, and the gene expression cassette is encapsidated within the capsid protein; Optionally, the capsid protein is selected from any one of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9 and AAV10 adeno-associated virus serotypes or variants thereof. The gene delivery vector according to claim 10, wherein the capsid protein is AAV2 capsid protein or a variant thereof; Preferably, the capsid protein is an AAV2 capsid protein variant; More preferably, the AAV2 capsid protein variant comprises a sequence as shown in SEQ ID NO:24 or SEQ ID NO:25, or a sequence that is at least 85% identical to SEQ ID NO:24 or SEQ ID NO:25. A pharmaceutical composition comprising the gene expression cassette according to any one of claims 1 to 7 or the gene delivery vector according to any one of claims 8 to 11, and, optionally, a pharmaceutically acceptable carrier. Use of the gene expression cassette according to any one of claims 1 to 7 or the gene delivery vector according to any one of claims 8 to 11 in the preparation of a drug for treating a disease; Optionally, the disease is an eye disease; Preferably, the eye disease is an eye disease associated with X-linked retinoschisis of the eye; More preferably, the eye disease is selected from one or more of vitreous hemorrhage, retinal detachment, refractive error, strabismus and neovascular glaucoma optic disc atrophy. A method for treating a disease, comprising administering to a subject a therapeutically effective amount of the gene expression cassette according to any one of claims 1 to 7, the gene delivery vector according to any one of claims 8 to 11, or the pharmaceutical composition according to claim 12; Optionally, the disease is an eye disease; Preferably, the eye disease is an eye disease associated with X-linked retinoschisis of the eye; More preferably, the eye disease is selected from one or more of vitreous hemorrhage, retinal detachment, refractive error, strabismus and neovascular glaucoma optic disc atrophy.