Retina-targeted AAV capsid protein variants and uses thereof

Abstract

The present invention relates to retina-targeting AAV capsid protein variants, isolated nucleic acids encoding same, vectors and host cells comprising said nucleic acids, and recombinant AAV vectors comprising said AAV capsid protein variants and therapeutic uses thereof.

Description

Technical Field

[0001] The present invention relates to the field of adeno-associated virus (AAV), and specifically to AAV capsid protein variants, isolated nucleic acids encoding the same, vectors and host cells containing the nucleic acids, and recombinant AAV vectors containing the AAV capsid protein variants and their therapeutic uses. Background Art

[0002] Recombinant adeno-associated virus (rAAV) is the most commonly used vector for in vivo gene replacement therapy and gene editing, but selective transduction of specific tissues after in vivo delivery remains a challenge. AAV transduction has multiple steps, including binding to cell surface receptors, intracellular trafficking, transgene expression, etc., all of which may limit the efficacy of the vector. It has been proposed to use AAV capsid protein evolution strategies to improve or expand the capsid targeting of AAV so that genes can be selectively delivered to specific target tissues or cell types, thereby increasing the viral transduction rate.

[0003] CN 112011571 A discloses an AAV9 capsid protein variant AAV-PHP.eB obtained by AAV capsid protein evolution. The amino acid sequence of the variant replaces the original amino acid AQ with the DGTLAVPFK peptide between positions 586 and 589 corresponding to the amino acid sequence of the wild-type AAV9 capsid protein. The author pointed out that the replacement can improve the infection efficiency of the capsid protein to the nervous system; and the application of the AAV viral vector containing the capsid variant in the treatment of spinal muscular atrophy by intrathecal administration was detected through model mice. However, the document does not involve research on targeting retinal tissue.

[0004] In view of the application prospects of AAV in the treatment of inherited retinal diseases, there is still a need in this field to develop new capsid protein variants to improve the specific targeting and transduction efficiency of recombinant adeno-associated viruses and the transgenes they carry to retinal cells. SUMMARY OF THE INVENTION

[0006] In the development of new capsid protein variants, the inventors obtained the capsid protein variants of the present invention by screening capsid protein variants. Compared with the wild-type AAV9 capsid protein, the AAV capsid variant of the present invention has an amino acid sequence substitution of SEQ ID NO.5 between amino acid positions 586 and 589 (amino acid positions are numbered according to SEQ ID NO:6). The capsid protein variant according to the present invention produced by the substitution is referred to as CapX-120 in this article. Through the expression of reporter genes and the comparison of fluorescence intensity, it was demonstrated in animal experiments that the capsid protein variant CapX-120 can enhance the transduction efficiency of adeno-associated virus vectors to retinal tissue. Thus, the use of the capsid protein variant according to the present invention makes it possible to construct an efficient and suitable AAV virus vector for the specific tissue targeting required for the treatment of ophthalmic diseases, improve the therapeutic effect on related diseases, and minimize the toxicity caused by off-target effects.

[0007] Therefore, in a first aspect, the present invention provides an adeno-associated virus (AAV) capsid protein variant, which has the amino acid sequence of SEQ ID NO.5 inserted between amino acid positions 586 and 589 relative to the parent AAV capsid protein to replace residues 587-588 of the parent AAV capsid protein, wherein the amino acid positions are numbered according to SEQ ID NO:6.

[0008] In some embodiments, compared with the parent AAV capsid protein, the capsid protein variant according to the present invention confers increased transduction of the AAV virus particles containing it to retinal cells, preferably, the retinal cells are selected from: photoreceptor cells, RPE layer cells, outer nuclear layer cells, inner nuclear layer cells and ganglion layer cells. In some embodiments, the retinal cells are cone cells and / or rod cells. In some embodiments, relative to other cell types of the retina, the capsid protein variant confers a higher transduction rate to cones and rod cells of the retina.

[0009] In some embodiments, the parent AAV capsid protein is selected from the group consisting of capsid proteins of AAV serotypes of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, and AAV13. In some embodiments, the parent AAV capsid protein has a surface-exposed hypervariable loop VIII that corresponds in structure to an AAV9 capsid protein. In some preferred embodiments, the parent AAV capsid protein is an AAV9 capsid protein.

[0010] In some more preferred embodiments, the parent AAV capsid protein is an AAV9 capsid protein comprising or consisting of the following amino acid sequence:

[0011] (a) an amino acid sequence of amino acids 203 to 736 of SEQ ID NO: 6;

[0012] (b) an amino acid sequence of amino acids 137 to 736 of SEQ ID NO: 6; or

[0013] (c) The amino acid sequence of SEQ ID NO:6.

[0014] In some embodiments, the parent AAV capsid protein is a VP1 capsid protein and comprises the amino acid sequence of SEQ ID NO: 6 or an amino acid sequence having at least 95%, 96%, 97%, 98%, 99%, 99.5% or 100% identity thereto. In some embodiments, the capsid protein variant according to the present invention is a variant of the parent AAV capsid protein, and preferably comprises the amino acid sequence of SEQ ID NO: 4 or a VP1 capsid protein having at least 95%, 96%, 97%, 98%, 99%, 99.5% or 100% identity thereto.

[0015] In some embodiments, the parent AAV capsid protein is a VP2 capsid protein and comprises an amino acid sequence of amino acids 137 to 736 of SEQ ID NO: 6, or an amino acid sequence having at least 95%, 96%, 97%, 98%, 99%, 99.5% or 100% identity thereto. In some embodiments, the capsid protein variant according to the present invention is a variant of the parent AAV capsid protein, and preferably a VP2 capsid protein comprising an N-terminal truncated amino acid sequence starting at amino acid 137 of SEQ ID NO: 4, or an amino acid sequence having at least 95%, 96%, 97%, 98%, 99%, 99.5% or 100% identity thereto.

[0016] In some embodiments, the parent AAV capsid protein is a VP3 capsid protein and comprises an amino acid sequence of amino acids 203 to 736 of SEQ ID NO: 6, or an amino acid sequence having at least 95%, 96%, 97%, 98%, 99%, 99.5% or 100% identity thereto. In some embodiments, the capsid protein variant according to the present invention is a variant of the parent AAV capsid protein, and preferably a VP3 capsid protein comprising an N-terminal truncated amino acid sequence starting at amino acid 203 of SEQ ID NO: 4, or an amino acid sequence having at least 95%, 96%, 97%, 98%, 99%, 99.5% or 100% identity thereto.

[0017] In some embodiments, the capsid protein variant according to the present invention is a VP1, VP2 or VP3 capsid protein comprising the amino acid sequence of SEQ ID NO: 4 or its N-terminal truncated sequence. In other embodiments, the capsid protein variant according to the present invention is a VP1, VP2 or VP3 capsid protein encoded by a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 3.

[0018] In some embodiments, the capsid protein variant according to the present invention is a VP1, VP2 or VP3 capsid protein derived from a transcript encoding the amino acid sequence of SEQ ID NO:4 or comprising the nucleotide sequence of SEQ ID NO:3.

[0019] In a second aspect, the present invention provides an isolated nucleic acid, wherein the nucleic acid comprises a polynucleotide encoding an AAV capsid protein variant according to the present invention.

[0020] In some embodiments, a nucleic acid according to the invention comprises:

[0021] (a) a polynucleotide encoding SEQ ID NO:4, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100% identical thereto; or

[0022] (b) the nucleotide sequence of SEQ ID NO:3, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100% identical thereto.

[0023] In some embodiments, the nucleic acid according to the present invention is capable of transcribing and expressing at least one or any two or all three AAV capsid proteins selected from the following: VP1, VP2 and VP3. In this article, the expression "capable of transcribing and expressing" with respect to nucleic acid means that when the nucleic acid is placed under the control of a suitable promoter, it can be transcribed and expressed in a suitable cell into which the nucleic acid is introduced to produce the gene product mentioned above.

[0024] In a third aspect, the present invention provides a nucleic acid vector comprising a nucleic acid according to the present invention encoding an AAV capsid protein variant according to the present invention. In some embodiments, the nucleic acid vector further comprises a nucleic acid encoding an AAV REP protein. In some embodiments, the nucleic acid vector is a plasmid.

[0025] In a fourth aspect, the present invention provides an isolated host cell comprising a nucleic acid according to the present invention encoding an AAV capsid protein variant according to the present invention. In some embodiments, the host cell is a recombinant adeno-associated viral vector (rAAV) production cell. In some embodiments, the nucleic acid stably transfects the production cell. In other embodiments, the production cell also comprises a nucleic acid encoding an AAV REP protein (especially the REP protein of AAV9 or AAV2). In other embodiments, the production cell also comprises a nucleic acid capable of trans-providing the helper virus functions required for AAV replication and assembly (e.g., E1a and E1b helper genes from adenovirus, and / or E4, E2a and VA helper genes). In other embodiments, the production cell also comprises an rAAV genome carrying a heterologous nucleic acid.

[0026] In a fifth aspect, the present invention provides the use and method of using a nucleic acid according to the present invention, a nucleic acid vector according to the present invention, or a host cell or a production cell according to the present invention encoding an AAV capsid protein variant according to the present invention for preparing a recombinant adeno-associated virus (rAAV) vector. In some embodiments, the use or method comprises:

[0027] (i) providing a production cell according to the invention;

[0028] (ii) culturing the cells under conditions that allow packaging to produce rAAV virions comprising the recombinant AAV genome; and

[0029] (iii) harvesting the cultured host cells or the culture medium to collect the produced recombinant AAV viral vector.

[0030] In a sixth aspect, the present invention provides a recombinant adeno-associated virus (rAAV) vector comprising a capsid protein variant according to the present invention or produced using a production cell of the present invention comprising a nucleic acid according to the present invention.

[0031] In some embodiments, the rAAV vector comprising the capsid protein variant according to the present invention exhibits increased transduction of retinal cells relative to rAAV vectors comprising wild-type AAV capsid proteins or relative to reference rAAV vectors comprising parental capsid proteins, preferably, the retinal cells are selected from the group consisting of: cone and rod layer cells, RPE layer cells, outer nuclear layer cells, inner nuclear layer cells and ganglion layer cells. In some embodiments, the retinal cells are photoreceptor cells, cone cells and / or rod cells.

[0032] In some embodiments, the rAAV vector according to the present invention comprises in its genome:

[0033] a. 5' and 3' AAV inverted terminal repeat (ITR) sequences, and

[0034] b. an expression construct comprising a heterologous nucleic acid located between the 5' and 3' ITRs, wherein the heterologous nucleic acid encodes a target gene product,

[0035] Preferably, the expression construct comprises the following elements functionally linked to each other in the transcriptional direction:

[0036] - promoter,

[0037] - a heterologous nucleic acid,

[0038] - Transcription terminator.

[0039] In some embodiments, the heterologous nucleic acid encodes a target gene product for gene replacement, gene suppression or gene editing. Preferably, the target gene product is a protein or RNA.

[0040] An important and controversial issue in retinal gene therapy is that subretinal injection can cause transient separation of RPE from photoreceptors, which has adverse effects on the diseased retina. Relatively speaking, intravitreal delivery is the least invasive method of retinal delivery and can therefore transduce the retina more safely. However, when injected intravitreally, most viral vectors, including most AAV serotypes, do not transduce photoreceptors and RPE. See Gene therapy of inherited retinal degenerations: prospects and challenges, Hum. Gene Ther. 2015; 26: 193-200; see also https: / / doi.org / 10.1016 / j.molmed.2018.06.006. Therefore, the development of AAV serotypes that can effectively transduce photoreceptors by intravitreal injection is urgently needed in the art.

[0041] As shown in the examples, the capsid protein variants of the present invention exhibited higher transduction properties to retinal cells than the parent capsid protein by intravitreal injection, especially to the main target of inherited retinal degeneration (IRD), photoreceptors (rod and cone layer), showing a nearly saturated transduction level. Therefore, the capsid protein variants of the present invention can be advantageously used in the treatment of inherited retinal-related diseases, including IRD.

[0042] In some preferred embodiments, accordingly, the present invention provides a rAAV vector according to the present invention, which comprises a heterologous nucleic acid for the treatment of inherited retinal diseases in the genome. In some embodiments, the heterologous nucleic acid encodes a target gene product, which can be used for gene replacement, gene suppression or gene editing of genes associated with inherited retinal diseases. Examples of such genes include, for example, but are not limited to, congenital amaurosis-related genes such as RPE65, achromatopsia-related genes such as CNGB3, choroideremia-related genes such as CHM and REP1, Leber hereditary optic neuropathy-related genes such as ND4, macular degeneration-related genes such as VEGF, sFLt1 and CD59, retinitis pigmentosa-related genes such as ChR2, MERTK, RLBP1 and PDE6B, X-linked retinitis pigmentosa-related genes such as RPGR, and X-linked retinoschisis-related genes such as RS1.

[0043] In some embodiments, preferably, in the rAAV vector according to the present invention, the heterologous gene is combined with a retinal tissue or cell type specific promoter, such as a rod and / or cone cell specific promoter. In some cases, this combination will be beneficial to reduce the rAAV administration dose and / or toxicity.

[0044] In a seventh aspect, the present invention provides a pharmaceutical composition comprising a recombinant AAV viral vector according to the present invention and a pharmaceutically acceptable carrier.

[0045] In an eighth aspect, the present invention provides a method for delivering a heterologous nucleic acid to a subject in need thereof, wherein the method comprises administering to the subject a recombinant AAV viral vector according to the present invention or a pharmaceutical composition according to the present invention. Accordingly, the present invention also provides the use of a recombinant AAV viral vector according to the present invention or a pharmaceutical composition according to the present invention in the preparation of a medicament for delivering a heterologous nucleic acid to a subject in need thereof.

[0046] In some embodiments according to this aspect of the invention, the rAAV vector comprises an AAV9 capsid protein variant having an amino acid sequence of SEQ ID NO: 11 between amino acid positions 586 and 589. In some embodiments, the methods according to the invention are used to treat or prevent inherited retinal-related diseases, such as congenital amaurosis, achromatopsia, retinitis (X-linked retinitis pigmentosa), choroideremia, Leber hereditary optic neuropathy, macular degeneration (such as age-related macular degeneration), X-linked retinoschisis, diabetic retinopathy, diabetic macular edema, or choroidal neovascularization.

[0047] In some embodiments, the delivery comprises intravitreal injection or subretinal injection. More preferably, the delivery is performed by intravitreal injection, which is less invasive.

[0048] In the ninth aspect, the present invention provides a method for delivering heterologous nucleic acid to cells cultured in vitro or in vitro, comprising contacting the cells with the rAAV viral vector according to the present invention. Preferably, the cells are retinal cells, such as rod cells and / or cone cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 The schematic diagram shows the pRDAV-CMV-EGFP-Cap9 backbone plasmid (Construct 1). ITR: AAV inverted terminal repeat (ITR); CMV promoter: cytomegalovirus promoter; EGFP: gene encoding enhanced green fluorescent protein; shortpA: short polyadenylation signal; p40: p40 promoter from AAV9; CAP9: gene encoding capsid protein of wild-type AAV9 serotype.

[0050] Figure 2 The pRDAV-CMV-EGFP-Cap9Δ-588 backbone plasmid generated based on construct 1 is schematically shown (construct 2).

[0051] Figure 3 Schematic diagram showing the results of microscopic examination (×40) of mouse retinas infected with CapX-120 capsid variant viruses.

[0052] Figure 4 Schematic diagram showing the results of microscopic examination (×200 magnification) of mouse retinas infected with CapX-120 capsid variant viruses.

[0053] Figure 5The mean fluorescence intensity and fold change of fluorescence intensity detected in the cone and rod layers, RPE layer, outer nuclear layer, inner nuclear layer and ganglion layer of mouse retinal tissue after intravitreal injection of CapX-120 capsid variant virus relative to the control rAAV9 virus are shown.

[0054] Figure 6 Shown are changes in transduction rates (ie, number of EGFP-positive cells) in the outer and inner nuclear layers of the retina resulting from infection with CapX-120 capsid variant viruses relative to control rAAV9 viruses.

[0055] Figure 7 The capsid protein variant of SEQ ID NO: 4 is aligned with the sequence of SEQ ID NO: 6. The sequence alignment shows that the amino acid residues of SEQ ID NO: 4 correspond to the position numbers of SEQ ID NO: 6. As can be seen from the figure, between the amino acid residues 586-589 corresponding to SEQ ID NO: 6, SEQ ID NO: 4 has an inserted amino acid sequence YGNPSQKL. DETAILED DESCRIPTION OF THE INVENTION

[0057] Unless defined otherwise hereinafter, all technical and scientific terms used in this specification have the same meaning as those of ordinary skill in the art to which the present invention belongs. All publications, patent applications, patents and other references mentioned herein are fully incorporated by reference. In addition, the materials, methods and examples described herein are only illustrative and are not intended to be restrictive. Other features, purposes and advantages of the present invention will be apparent from this specification and the accompanying drawings and from the appended claims.

[0058] definition

[0059] In this article, the term "about" when used in conjunction with a numerical value is intended to encompass numerical values ​​within a range having a lower limit that is 5% less than the specified numerical value and an upper limit that is 5% greater than the specified numerical value. The term is also intended to encompass numerical values ​​within the range of ±1%, ±0.5%, or ±0.1% of the specified number.

[0060] Herein, the expression "and / or" is used to indicate any one of the related listed items, or any and all possible combinations of a plurality of the related listed items.

[0061] In this article, the term "comprising" or "including" means including the mentioned elements, integers or steps, or groups of elements, integers or steps, but does not exclude any other elements, integers or steps, or other groups of elements, integers or steps. In this article, when the term "comprising" or "including" is used, unless otherwise indicated, the situation consisting of the mentioned elements, integers or steps is also covered. For example, when referring to a polypeptide / protein "comprising" a specific sequence, it is also intended to cover polypeptides / proteins consisting of the specific sequence.

[0062] As used herein, the term "AAV capsid protein variant" or "AAV variant capsid protein" refers to an AAV capsid protein having at least one modification (including deletion, insertion and / or substitution) in the amino acid sequence relative to the parent AAV capsid protein. The variant AAV capsid protein may have at least 90%, 91%, or 95% identity to the amino acid sequence of the parent capsid protein.

[0063] Herein, "parent AAV capsid protein" or "parent capsid protein" refers to an AAV capsid protein that serves as a template for introducing capsid protein mutations at positions 586-589 according to the present invention. The parent of the variant AAV capsid protein may be a naturally occurring or "wild-type" AAV capsid protein; but it may also be a capsid protein that is not a wild-type capsid protein, for example, a capsid protein in which one or more (e.g., no more than 10 or 5, 4, 3, 2, 1) amino acid modifications are introduced into the natural AAV capsid protein. It should be understood that, in the case where the parent is a non-natural capsid protein, it does not contain the capsid protein mutations at positions 586-589 according to the present invention. In some embodiments, preferably, the parent capsid protein is a natural AAV9 serotype capsid protein, for example, a VP1 protein having an amino acid sequence of SEQ ID NO: 6, or a corresponding VP2 or VP3 protein having an N-terminal truncation.

[0064] In this article, when referring to the amino acid position in the AAV capsid protein or its segment, it refers to the amino acid position numbered according to SEQ ID NO:6. The amino acid position corresponding to the amino acid position of SEQ ID NO:6 in the sequence of the capsid protein can be determined by comparing the amino acid sequence of the AAV capsid protein mentioned with SEQ ID NO:6. For example, when referring to position 586 of the capsid protein, it refers to the 586th amino acid residue of SEQ ID NO:6, or the amino acid residue that appears at the corresponding position of other capsid proteins after comparison. It should be understood that in the present disclosure, when the AAV capsid protein variant involves the insertion or deletion of amino acids in a certain or certain specific amino acid position segment, the alignment of the AAV capsid protein variant with SEQ ID NO:6 in the segment can be visually detected, and if necessary, a gap is introduced in one or both of the two sequences to ensure that after the introduction of the gap, the segment of the AAV capsid protein and the corresponding segment of SEQ ID NO:6 are in the corresponding position region in the alignment and have the corresponding amino acid residue number. For example, for an AAV capsid protein variant (e.g., SEQ ID NO:4) in which the SEQ ID NO:5 sequence (YGNPSQKL) is inserted between positions 586-589, the alignment of the variant with SEQ ID NO:6 in this segment can be visually checked, and if necessary, a gap is introduced before position 587 of SEQ ID NO:6, so that the insertion region of the variant is in the corresponding position region with positions 586-589 of SEQ ID NO:6 after the gap is introduced in the alignment, and has the insertion sequence YGNPSQKL between the amino acid residues corresponding to positions 586-589 of SEQ ID NO:6. See. Figure 7 Sequence alignment for amino acid position determination can be performed using the Basic Local Alignment Search Tool available from https: / / blast.ncbi.nlm.nih.gov / Blast.cgi using default parameters.

[0065] In this article, "AAV virus particle" and "AAV virus particle" are used interchangeably and refer to a complete virus particle comprising an AAV capsid and an AAV nucleic acid genome (including a wild-type AAV genome and a recombinant AAV genome) packaged in the capsid. In this regard, the AAV nucleic acid molecule chain packaged into any one AAV virus particle can be a sense (e.g., "positive") chain or an "antisense" chain, and the two chains have equal infectivity.

[0066] Herein, the terms "recombinant AAV vector" and "recombinant AAV virions" and "recombinant AAV virus particles" are used interchangeably to refer to non-wild-type recombinant AAV virus particles that can serve as a carrier of a target nucleic acid. Typically, the viral vector comprises a capsid and a viral genome packaged therein, and preferably the viral genome comprises a target nucleic acid to be delivered to a target cell or tissue inserted therein. Herein, "recombinant" may be abbreviated as "r", for example, recombinant AAV may be referred to as rAAV. Therefore, herein, the terms "recombinant AAV virions" and "recombinant AAV vectors" may also be used interchangeably with "rAAV virions", "rAAV virus particles" or "rAAV" or "rAAV vectors". Typically, recombinant AAV vectors are infectious but replication-defective.

[0067] Herein, for the purposes of the present disclosure, rAAV capsids may be from or derived from various adeno-associated virus serotypes, including but not limited to AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, and AAV13. For example, the rAAV may have a viral protein capsid composed of AAV capsid proteins and / or variants thereof from the serotypes.

[0068] Herein, for the purposes of this disclosure, the viral genome packaged in the rAAV vector is recombinant, that is, the AAV genomic DNA has genetic modifications relative to the wild-type AAV genomic DNA. In order to produce recombinant AAV virus particles that can deliver the target nucleic acid to tissues or cells, it is generally only necessary to retain the reverse terminal repeat (ITR) cis element in the genome, and the remaining sequences required for viral packaging can be provided in trans. Therefore, typically, rAAV can have one or more AAV wild-type genes that are completely or partially deleted, such as rep and / or cap genes, and are thus replication-defective; but retain the functional flanking ITR sequences required for the rescue, replication and packaging of AAV virus particles. More typically, the recombinant AAV viral genome packaged in the rAAV virus particles can retain only the functional ITR sequence, and preferably contains or consists of one or more exogenous nucleotide sequences located between two AAVITR sequences. It should be understood that, with respect to rAAV, the functional ITR sequence can be, but not necessarily, a wild-type nucleotide sequence, which can be changed, for example, by the insertion, deletion or substitution of nucleotides, as long as it still provides the functions required for rescue, replication and packaging. Therefore, in this article, rAAV vector is a viral vector that at least contains the functional ITR required for viral replication and packaging in cis. It should also be understood that the recombinant AAV viral genome can contain two or more (eg, three) ITR sequences, and these ITR sequences can be the same or different.

[0069] As used herein, the terms AAV "inverted terminal repeat" or "ITR" are used interchangeably to refer to the functional inverted terminal repeat cis-acting elements from the AAV viral genome and encompass wild-type ITR sequences and variant ITR sequences.

[0070] The wild-type ITR of the natural AAV virus contains a Rep protein binding site (RBS) and a terminal resolution site trs (terminal resolution site) in the sequence. It can be bound and recognized by the Rep protein and produce an incision at the trs, and can form a unique "T"-shaped secondary structure, which plays an important role in the life cycle of the AAV virus. The earliest isolated AAV virus, AAV2, has a "reverse terminal repeat sequence" (ITR) with a length of 145bp and a palindromic-hairpin structure located at both ends of the genome. Afterwards, different ITR sequences were found in AAV viruses of various serotypes, but they can all form a hairpin structure and have a Rep binding site. Recombinant AAV viral vectors based on these wild-type ITR sequences are generally single-stranded AAV vectors (ssAAV).

[0071] Relative to the wild-type ITR sequence, the variant ITR can be, for example, a non-natural ITR sequence comprising one or more nucleotide deletions, substitutions, and / or additions, and / or truncations, but still functional, i.e., capable of being used to produce rAAV viral vectors. It has been found that, unlike the above-mentioned ssAAV, by modifying the ITR, deleting the trs sequence and optionally the D sequence in the ITR sequence on one side of the AAV virus, the packaged recombinant AAV viral vector can carry self-complementary genomic DNA, thereby producing a virus called scAAV (self-complementary AAV). The packaging capacity of the scAAV viral vector is half that of the ssAAV viral vector, about 2.2 kb-2.5 kb, but the transduction efficiency is higher after infecting cells. See, for example, Self-complementary AAV Vectors; Advances and Applications, https: / / doi.org / 10.1038 / mt.2008.171. Such variant ITR sequences that can be used to produce scAAV viruses are also referred to herein as ΔITR. The present disclosure contemplates not only ssAAV vectors generated by combining two wild-type ITRs, but also scAAV vectors generated by combining a ΔITR sequence with a wild-type ITR.

[0072] As those skilled in the art will appreciate, the viral capsid protein and viral genome ITR sequence of the recombinant adeno-associated virus can come from the same or different AAV virus serotypes. In this article, therefore, the recombinant adeno-associated virus can be represented by the AAV virus serotype from which the viral capsid is derived, or by the AAV virus serotype from which the viral capsid and viral genome ITR sequence are derived. In the latter case, in this article, the identifier " / " is used for separation, and the identifier " / " is followed by the source serotype of the capsid, and the identifier " / " is preceded by the source serotype of the ITR. For example, the number 9 in the expression recombinant AAV9 indicates that the recombinant adeno-associated virus has a capsid from the AAV9 serotype; and the number before the identifier " / " in the expression recombinant AAV2 / 9 indicates that the recombinant adeno-associated virus has a wild-type or variant ITR sequence from AAV2, and the number after the identifier " / " indicates that the recombinant adeno-associated virus has a capsid protein from AAV9. In some embodiments, for example, the rAAV virus according to the present invention can be an AAV9 virus having a variant capsid protein from an AAV9 serotype, or an AAV2 / 9 virus further comprising ITR sequences from AAV2.

[0073] As used herein, the term "host cell" refers to a cell into which an exogenous polynucleotide has been introduced, including the progeny of such cells. Examples of host cells include, but are not limited to, microorganisms, yeast cells, insect cells, and mammalian cells. In some embodiments, the host cell is a mammalian cell. In some embodiments, the host cell is a retinal cell. Depending on the context in which the term is used, the host cell may be an in vitro, ex vivo, or in vivo cell. In other embodiments, the host cell is a production cell for producing an rAAV vector according to the present invention, for example, a "HEK293 cell" or a "293 cell", or a cell line derived from the cell.

[0074] Herein, in some embodiments, the term "producer cell" or "producer cell line" refers to a cell population that can grow and divide continuously or for a long time in vitro. It is known in the art that during the storage or passage of such a clonal cell population, the karyotype may undergo spontaneous or induced changes, and thus may not be completely identical to the ancestral cell or culture, but still maintain the desired characteristics of the original producer cell. Such producer cells or cell lines are encompassed within the scope of the present invention.

[0075] As used herein, the terms "exogenous" or "heterologous" are used interchangeably to describe a nucleic acid or protein and refer to a nucleic acid or protein that is exogenous or heterologous relative to a virus, host cell, subject, or other organism containing the nucleic acid or protein, or relative to a flanking nucleic acid or polypeptide to which it is attached, i.e., the nucleic acid or protein is present in a non-natural state in the virus, host cell, subject, or organism, or is non-naturally attached to the flanking nucleic acid or polypeptide. For example, a nucleic acid that is introduced into a particular viral genome or host cell or subject by recombinant techniques so as to be attached to a sequence that is not naturally attached thereto or is in a non-natural chromosomal or cellular location or state is heterologous relative to the viral genome or host cell or subject. Thus, a nucleic acid that is inserted into the same host cell as the cell from which it is derived or into the same organism as the organism from which it is derived, but is present in a non-natural state, e.g., in a different copy number or under the control of different regulatory elements, is exogenous or heterologous.

[0076] In this article, "isolated" nucleic acid refers to a nucleic acid molecule that is artificially synthesized or separated from at least some components of the natural environment containing it. For example, an isolated nucleic acid can be part of a larger nucleic acid, or part of a carrier or material composition, or can be contained in a cell and still be "isolated" if the larger nucleic acid, carrier, material composition or specific cell is not the natural environment of the nucleic acid. In some embodiments, the "isolated" nucleic acid is enriched at least about 10 times, 100 times, 1000 times, 10,000 times or more relative to the starting material.

[0077] As used herein, the term "functional connection" is also referred to as "operably linked", meaning that the specified components are in a relationship that allows them to function in an intended manner. For example, a promoter or enhancer sequence is operably linked to a coding sequence if it stimulates or regulates transcription of the coding sequence in a suitable host cell or other expression system. In general, promoters that are operably linked to a transcribable sequence are continuous with the transcribable sequence, i.e., they are cis-acting. However, some transcriptional regulatory sequences (such as enhancers) do not need to be physically adjacent to or in close proximity to the coding sequence whose transcription they enhance.

[0078] In this article, the term sequence "identity" is used to describe the sequence structure similarity between two amino acid sequences or polynucleotide sequences. To determine the identity percentage of two amino acid sequences or two nucleic acid sequences, the sequences can be compared for the best comparison purpose (for example, a room can be introduced in one or both of the first and second amino acid sequences or nucleic acid sequences for the best comparison or a non-homologous sequence can be abandoned for the purpose of comparison). In a preferred embodiment, for the purpose of comparison, the length of the compared reference sequence is at least 30%, preferably at least 40%, more preferably at least 50%, 60%, and even more preferably at least 70%, 80%, 90%, or 100% of the length of the reference sequence. Subsequently, the amino acid residues or nucleotides at the corresponding amino acid positions or nucleotide positions are compared. When the position in the first sequence is occupied by the same amino acid residue or nucleotide at the corresponding position in the second sequence, the molecules are identical at this position.

[0079] Mathematical algorithms can be used to compare sequences and calculate percent identity between two sequences. In a preferred embodiment, the Needlema and Wunsch ((1970) J. Mol. Biol. 48: 444-453) algorithm (available at http: / / www.gcg.com) that has been integrated into the GAP program of the GCG software package is used, using a Blossum 62 matrix or a PAM250 matrix and a gap weight of 16, 14, 12, 10, 8, 6 or 4 and a length weight of 1, 2, 3, 4, 5 or 6 to determine the percent identity between two amino acid sequences. In another preferred embodiment, the GAP program in the GCG software package is used (available at http: / / www.gcg.com) to determine the percent identity between two nucleotide sequences using the NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70 or 80 and a length weight of 1, 2, 3, 4, 5 or 6. A particularly preferred parameter set (and the one that should be used unless otherwise stated) is the Blossum 62 scoring matrix with a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5.

[0080] The percent identity between two amino acid or nucleotide sequences can also be determined using the algorithm of E. Meyers and W. Miller, ((1989) CABIOS, 4: 11-17), which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weighted remainder table, a gap length penalty of 12, a gap penalty of 4).

[0081] As used herein, the term "conservative" amino acid or nucleotide changes refers to neutral or nearly neutral amino acid or nucleotide changes that result in the protein or nucleic acid molecule containing the amino acid or nucleotide changes substantially maintaining the original function. For example, conservative amino acid substitutions are substitutions or replacements of amino acids with side chains having similar biochemical properties (e.g., charge, hydrophobicity, and size). Such conservatively modified variants are additional to polymorphic variants, interspecies homologs, and alleles and do not exclude them. The following 8 groups contain amino acids that are conservative substitutions for each other: 1) alanine (A), glycine (G); 2) aspartic acid (D), glutamic acid (E); 3) asparagine (N), glutamine (Q); 4) arginine (R), lysine (K); 5) isoleucine (I), leucine (L), methionine (M), valine (V); 6) phenylalanine (F), tyrosine (Y), tryptophan (W); 7) serine (S), threonine (T); and 8) cysteine ​​(C), methionine (M) (see, e.g., Creighton, Proteins (1984)). Those skilled in the art can easily detect the conservative nature of amino acid or nucleotide changes in a particular polypeptide sequence or nucleotide sequence by conventional technical means, such as functional assays.

[0082] As used herein, "individual" or "subject" refers to a mammal. Examples of mammals include, but are not limited to, humans, non-human primates (e.g., cynomolgus monkeys, rhesus monkeys), rodents, and other mammals, e.g., cattle, pigs, horses, dogs. As used herein, mammals include individuals at all stages of development (including embryonic and fetal stages).

[0083] As used herein, the term "treatment" refers to a clinical intervention intended to alter the natural course of a disease in the individual being treated. Desired therapeutic effects include, but are not limited to, preventing the onset or recurrence of the disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of progression of the disease, ameliorating or relieving the disease state, and alleviating or improving prognosis. The term "treatment" also encompasses modification or improvement of at least one physical parameter, including physical parameters that may not be discernible by the patient.

[0084] As used herein, the term "prevention" refers to preventing or delaying the onset or development or progression of a disease or condition. As used herein, "prevention" generally refers to hospital intervention performed before at least one symptom of a disease occurs.

[0085] Various aspects of the invention are described below.

[0086] Capsid protein variants

[0087] Adeno-associated virus (AAV) is a non-pathogenic parvovirus consisting of a 4.7 kb single-stranded DNA genome within a non-enveloped capsid. The genome contains three open reading frames (ORFs) flanked by inverted terminal repeats (ITRs) that serve as the origin of viral replication and packaging signals. The rep ORF encodes four nonstructural proteins that play a role in viral replication, transcriptional regulation, site-specific integration, and virion assembly. The cap ORF encodes three structural proteins (VP1-3) that assemble to form the viral capsid.

[0088] The AAV viral capsid is composed of a total of 60 VP monomers from three VP capsid proteins, arranged in an icosahedral structure, wherein the molar ratio of the three capsid proteins VP1:VP2:VP3 is "optimal" at approximately 1:1:10. The three capsid proteins VP1, VP2, and VP3 are all encoded by the cap gene of AAV and are produced from the same transcript and regulated by the p40 promoter. The three VP proteins have a common C-terminal sequence, but different N-terminal start sites; VP2 and VP3 produced by alternative splicing are two truncated forms of VP1. As an example, the capsid protein sequence of AAV9 is shown below, in which the VP1-specific amino acid sequence is shown in black and bold (VP1 unique region). The amino acid sequence shared by VP1 and VP2 ("VP1 / VP2 common region") is underlined and italicized; the entire VP3 is contained in VP1 and VP2, and is an amino acid shared by all three capsid proteins ("common VP3 region"), which is bold and italicized below.

[0089]

[0090] VP1 and VP2 are mainly located in the nucleus, while the monomeric VP3 is distributed in both the nucleus and the cytoplasm. The outer surface of the assembled AAV capsid is composed of the VP3 sequence (including VP3 and the C-termini of VP1 and VP2), and the N-termini of VP1 and VP2 are located inside the capsid.

[0091] The VP has nine protruding loops called variable regions (VRs). VRs vary among AAV serotypes and are responsible for serotype-specific differences in receptor binding. Due to their exposed position and their function in receptor binding, the VRs forming the protruding loops are ideal locations for capsid modifications aimed at redirecting or expanding AAV tropism (i.e., cell surface targeting).

[0092] In order to develop new cell / tissue-targeted AAV capsid protein variants, the inventors screened AAV capsid protein variants. By inserting a random sequence into the capORF at the amino acid position corresponding to the top of the VR-VIII loop (position 586-588), and constructing an AAV transfer plasmid containing a reporter gene and the cap ORF to couple the genotype and phenotype of the AAV capsid variant with reporter gene expression, a plasmid library was generated. Subsequently, through capsid variant screening and tissue distribution detection in animals, AAV capsid protein variants that can more efficiently and / or more specifically transduce retinal cells (especially photoreceptor cells) under vitreous administration were identified. Thus, the inventors established new capsid protein variants of the present invention.

[0093] Therefore, in one aspect, the present invention provides AAV capsid protein variants. In one embodiment, the AAV capsid protein variant according to the present invention is derived from a parent AAV capsid protein, which inserts a peptide (YGNPSQKL) of SEQ ID NO: 11 between amino acid positions 586 and 589 of the parent capsid protein to replace the original 587-588 residues (according to the AAV9 VP1 numbering of SEQ ID NO: 6). The capsid protein modification according to the present invention does not interfere with capsid assembly and genome packaging, and allows rAAV to obtain modified retinal cell targeting.

[0094] In other embodiments, the present invention provides an AAV capsid protein variant, comprising: an inserted peptide located between amino acids 586 and 589 of the parent AAV capsid protein to replace the amino acid residues at the original positions 567-568, and the peptide comprises the amino acid sequence of SEQ ID NO: 5. In some embodiments, the parent AAV capsid protein is a capsid protein of an AAV serotype selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12 and AAV13. In some preferred embodiments, the parent AAV capsid protein is an AAV9 serotype capsid protein. In some embodiments, the parent AAV capsid protein comprises or consists of an AAV9 capsid protein consisting of the following amino acid sequence:

[0095] (a) an amino acid sequence of amino acids 203 to 736 of SEQ ID NO: 6;

[0096] (b) an amino acid sequence of amino acids 137 to 736 of SEQ ID NO: 6;

[0097] (c) the amino acid sequence of SEQ ID NO: 6;

[0098] (d) an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identical to the amino acid sequence of any one of (a) to (c); or

[0099] (e) has one or more (e.g., 1, 2, 3, 4, 5, 7, 9, 9 or 10) amino acid residue substitutions, deletions and / or additions (preferably conservative amino acid substitutions) compared to the amino acid sequence of any one of (a) to (c).

[0100] In other embodiments, the AAV capsid protein variant according to the present invention is derived from a transcript encoding the following amino acid sequence, wherein the amino acid sequence is the amino acid sequence shown in SEQ ID NO:6 having an inserted peptide sequence of SEQ ID NO.11 between amino acid positions 586 and 589. In this article, the expression "derived from" a transcript related to a capsid protein variant means that the transcript is capable of producing the capsid protein variant when it is under conditions suitable for its expression to produce protein, including different forms of capsid protein variants produced from the transcript by using alternative translation start sites and / or alternative splicing, such as VP1, VP2 or VP3 capsid protein variants. In some embodiments, therefore, the AAV capsid protein of the present invention derived from the transcript can be a capsid protein variant selected from VP1, VP2 and VP3. In some preferred embodiments, the AAV capsid protein variant according to the present invention is derived from a transcript encoding the amino acid sequence shown in SEQ ID NO:4. In some preferred embodiments, the AAV capsid protein variant according to the present invention is derived from a transcript comprising the nucleotide sequence shown in SEQ ID NO:3.

[0101] In some embodiments, in addition to the mutations at positions 586-589 according to the present invention, the capsid protein variants according to the present invention may or may not include amino acid substitutions at one or more other amino acid positions, for example, 2 to 5, 5 to 10, or 10 to 15 amino acid substitutions.

[0102] In some embodiments, the AAV capsid protein variants according to the present invention can be further modified, for example, to enhance or extend the delivery of the recombinant vector. Methods for constructing and screening recombinant AAV capsid protein libraries are well known in the art. See, for example, US 2005 / 0053922 and US 2009 / 0202490, the disclosures of which are incorporated herein by reference in their entirety.

[0103] The AAV capsid protein variants disclosed herein are identified by intravitreal injection into animals and retinal tissue detection. In some embodiments, the variant capsid proteins disclosed herein confer increased retinal cell transduction to the rAAV virions containing it compared to transducing retinal cells using rAAV virions containing the corresponding parental AAV capsid protein or wild-type AAV. For example, relative to AAV virions containing parental AAV capsid proteins or wild-type AAV, the variant capsid proteins of the present invention cause retinal cells (especially retinal cells in vivo) to take up more AAV virions. In some implementations, AAV virions containing variant capsid proteins of the present invention preferentially transduce one or more retinal cells (e.g., rod cells; cone cells), for example, compared with other retinal cells, the transduction efficiency of the one or more retinal cells is 2 times or 5 times or more. In some embodiments, the retinal cells are selected from: photoreceptor cells (e.g., cones and rods), retinal pigment epithelial (RPE) layer cells, outer nuclear layer cells, inner nuclear layer cells (e.g., bipolar cells) and optic nerve layer cells. In some embodiments, the retinal cell is a photoreceptor cell (e.g., rod; cone). In some embodiments, the retinal cell is a retinal ganglion cell (RGC). In some embodiments, the retinal cell is a retinal pigment epithelial cell (RPE cell). In some embodiments, the retinal cell is a bipolar cell. A variety of methods for measuring gene expression in the art can be used to assess the transduction efficiency of the AAV virus particles to be tested to retinal cells in vitro or in vivo, such as an increase in transduction efficiency, selection of transduced cell types, transduction priority, etc. For example, an rAAV genome containing a reporter gene (e.g., a fluorescent protein under the control of a constitutive promoter) can be packaged using AAV capsid proteins, and in an in vitro cell-based assay (e.g., after transfection of a target cell or a group of target cells) or in an animal model-based assay, by detecting the expression of the reporter gene (e.g., fluorescence microscopy), transduction efficiency and / or transduced cell type selectivity / priority can be assessed.

[0104] rAAV production

[0105] In some aspects, the present invention provides the use of nucleic acids encoding the capsid protein variants of the present invention, vectors and host cells comprising the same in preparing rAAV vectors.

[0106] The general principles of rAAV production are reviewed in, for example, Carter, 1992, Current Opinions in Biotechnology, 1533-539; and Muzyczka, 1992, CUM Topics in Microbial. and Immunol., 158: 97-129). Various methods are described in the following documents: WO 95 / 13365; WO 95 / 13392; WO 96 / 17947; PCT / US98 / 18600; WO 97 / 09441; WO 97 / 08298; WO 97 / 21825; WO 97 / 06243; WO 99 / 11764; US 5,786,211; US ​​5,871,982; and US 6,258,595. These documents, in particular the parts thereof related to rAAV production, are hereby incorporated by reference in their entirety.

[0107] It has been confirmed that the two inverted terminal repeat sequences (ITR) in the AAV genome are cis-acting elements necessary for AAV integration, replication, rescue and packaging. The two open reading frames located between the ITR sequences in the wild-type AAV genome, called Rep and Cap, encode four Rep proteins (Rep78, Rep68, Rep52 and Rep40) and three Cap proteins (VP1, VP2 and VP3) involved in AAV replication and packaging, as well as one assembly activation protein (AAP). The transcription of Rep78 and Rep68 is controlled by the p5 promoter, the transcription of Rep52 and Rep40 is initiated by the p19 promoter, and the p40 promoter regulates the transcription of Cap proteins VP1, VP2 and VP3.

[0108] Generally, to produce rAAV virions, it is necessary to retain the cis-acting element ITR in the rAAV genome, but the REP and CAP proteins required for replication and packaging can be provided in trans. Therefore, typically, a vector (e.g., a plasmid) carrying an rAAV genome that has deleted all or part of the rep and / or cap genes can be used to transfect a suitable host cell (e.g., a suitable packaging cell) that can provide the deleted AAV function (e.g., rep and / or cap proteins) in trans to produce infectious rAAV virions.

[0109] Herein, the nucleic acid vector carrying the rAAV genome to be transferred into the target cell is also referred to as an "AAV transfer vector", which can be in the form of various nucleic acid vectors suitable for gene transfer, for example, a plasmid. Herein, the AAV functions that are missing from the rAAV genome and need to be provided in trans to assist the rAAV genome in packaging into productive AAV virus particles are also referred to as "AAV auxiliary functions", such as the rep and / or cap genes deleted from the rAAV genome, or nucleic acids encoding AAV rep and / or cap expression products or functional variants thereof. In some embodiments, the AAV auxiliary function can be provided by a vector encoding the auxiliary function, for example, in the form of a plasmid, phage, transposon, cosmid, virus or virus particle. Typically, such a vector provides transient expression of AAV rep and / or cap nucleic acids, but lacks AAV ITR, and therefore can neither replicate nor package itself. In other embodiments, the AAV auxiliary function can be provided by cells or cell lines that stably express the auxiliary function.

[0110] Adeno-associated virus (AAV) is a non-enveloped virus of the Parvoviridae family, belonging to the genus Dependoparvovirus, and requires the presence of a helper virus to replicate and assemble to produce infectious virions. A virus that allows AAV (e.g., wild-type AAV) to be replicated and packaged by mammalian cells is also referred to herein as an "AAV helper virus." Various helper viruses for AAV are known in the art, including adenovirus, herpes virus, and poxvirus. In this article, the functions required for AAV replication and packaging encoded by the helper virus genome are also referred to herein as "helper virus functions," for example, E1a and E1b helper genes from adenovirus, and E4, E2a, and VA helper genes. Helper virus functions can be provided in the form of plasmids or other vectors, and / or in the form of stable cell lines expressing the helper genes. In addition, as will be appreciated by those skilled in the art, AAV helper functions (rep / cap genes) and helper virus functions (e.g., E4, E2a, and VA helper genes) can be provided on a single or separate vector. In some aspects, helper virus functions can also be provided by wild-type adenovirus. For example, a cell line containing and stably expressing the AAV Rep and Cap proteins can be transduced with wild-type adenovirus and an AAV transfer vector, thereby producing infectious rAAV viral particles.

[0111] In some aspects, the technology for producing rAAV particles involves providing the rAAV genome to be packaged, rep and cap genes, and helper virus functions to the cells. Such technology is well known in the art. For example, rAAV production cells (or packaging cells) comprising the following components can be produced: rAAV genome, AAV rep and cap genes separated from the rAAV genome (i.e., not in the rAAV genome), and helper virus functions. Afterwards, the production cells can be used to produce the infectious rAAV particles of interest.

[0112] In some aspects, therefore, the present invention provides a production cell (or packaging cell) that can be used to prepare a recombinant adeno-associated virus (rAAV) vector, which comprises a cap gene encoding a capsid protein variant of the present invention, and optionally further comprises a nucleic acid encoding an AAV rep gene and / or a nucleic acid encoding a helper virus function. In some embodiments, the production cell also comprises an rAAV genome. In some embodiments, the rAAV genome comprises the cap gene and / or the rep gene. In other embodiments, the rAAV genome does not comprise the cap gene and the rep gene.

[0113] In other aspects, the present invention also provides a method for preparing a recombinant adeno-associated virus (rAAV) vector. In some embodiments, the method comprises:

[0114] (i) providing a production cell according to the invention;

[0115] (ii) culturing the cells under conditions that allow packaging to produce rAAV virions comprising the recombinant AAV genome; and

[0116] (iii) harvesting the cultured host cells or the culture medium to collect the produced recombinant AAV viral vector.

[0117] In some embodiments, the production cell comprises a nucleic acid according to the invention encoding an AAV capsid protein variant according to the invention. In some embodiments, the nucleic acid according to the invention comprises:

[0118] (a) a polynucleotide encoding SEQ ID NO:4, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100% identical thereto; or

[0119] (b) the nucleotide sequence of SEQ ID NO:3, or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100% identical thereto.

[0120] In some embodiments, the nucleic acid according to the present invention is capable of transcribing and expressing at least one, or any two, or all three AAV capsid proteins selected from the group consisting of: VP1, VP2, and VP3.

[0121] In some embodiments, the nucleic acid according to the present invention is introduced into the production cell in the form of a vector. In some embodiments, the vector further comprises a nucleic acid encoding an AAV REP protein. In some embodiments, the vector is a plasmid.

[0122] In some embodiments, the nucleic acid according to the present invention is stably transfected and integrated into the genome of the producer cell.

[0123] In other embodiments, the production cell further comprises (e.g., stably integrated) a nucleic acid encoding an AAV REP protein (particularly an AAV9 or AAV2 REP protein). In other embodiments, the production cell further comprises (e.g., stably integrated or introduced via a vector) a nucleic acid capable of providing helper virus functions in trans (e.g., E1a and E1b helper genes, and / or E4, E2a and VA helper genes from adenovirus).

[0124] In some embodiments, step (i) of the method according to the present invention comprises: introducing a recombinant AAV genome comprising a heterologous nucleic acid into a production cell according to any of the above embodiments. In some embodiments, the rAAV genome to be introduced into the host cell lacks AAV rep and cap DNA. In some embodiments, the rAAV genome to be introduced into the host cell comprises cap DNA encoding a capsid protein variant of the present invention, but lacks AAV rep DNA. In some embodiments, the rAAV genome is introduced into the host cell via an AAV transfer vector.

[0125] In some embodiments, the rAAV production method according to the present invention further comprises: a step of generating a production cell (or cell line). In some embodiments, the production cell (or cell line) expresses one or more of the following components necessary for rAAV genome packaging: an rAAV genome lacking AAV rep and cap genes, an AAV rep gene separated from the rAAV genome, and a cap gene encoding a capsid protein variant according to the present invention. Afterwards, the production cell can be infected with a helper virus (such as an adenovirus) to produce infectious rAAV virus particles.

[0126] In some preferred embodiments, the rAAV production method according to the present invention comprises: co-transfection with three plasmids to produce production cells (or cell lines), wherein the three plasmids are: a plasmid (transfer plasmid) providing an rAAV genome containing heterologous nucleic acid and flanking ITRs, a plasmid providing AAV auxiliary functions (cap and / or rep proteins), and a plasmid providing auxiliary virus (such as adenovirus or herpes simplex virus) functions. In other embodiments, the present invention also contemplates the use of adenovirus or baculovirus to replace the above-mentioned plasmids to introduce AAV auxiliary functions and auxiliary virus functions.

[0127] rAAV

[0128] In some aspects, the present invention provides rAAV vectors comprising variant capsid proteins according to the present invention or produced using the rAAV production method according to the present invention. In some embodiments, the rAAV vector according to the present invention has an rAAV genome packaged in a viral capsid composed of a capsid protein variant of the present invention. In some embodiments, the rAAV genome according to the present invention is a ssAAV genome or a scAAV genome.

[0129] In some embodiments, the recombinant AAV vector according to the present invention comprises in its genome:

[0130] a. AAV inverted terminal repeat (ITR) sequences, and

[0131] b. an expression construct comprising a heterologous nucleic acid, wherein the heterologous nucleic acid encodes a gene product of interest,

[0132] Preferably, the expression construct comprises the following elements functionally linked to each other in the transcriptional direction:

[0133] - promoter,

[0134] - a heterologous nucleic acid encoding a gene product of interest,

[0135] - Transcription terminator.

[0136] In some embodiments, the genome lacks AAV rep and cap DNA, ie, no AAV rep or cap DNA is present in the genome.

[0137] In some embodiments, the rAAV genome disclosed herein comprises two AAV ITRs flanking the expression construct. In some embodiments, the ITR sequences flanking the expression construct in the rAAV genome are native, variant or modified AAV ITR sequences. In some embodiments, at least one ITR sequence is a native, variant or modified AAV ITR sequence. In some embodiments, both ITR sequences are native, variant or modified AAV ITR sequences. In some embodiments, one of the two flanking ITRs is a modified AAV ITR sequence that allows the generation of a self-complementary genome, and the other ITR is a native AAV ITR sequence.

[0138] In the rAAV genome according to the present invention, the AAV ITR may be derived from or may be derived from any AAV serotype, including but not limited to AAV serotypes AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10, AAV-11, AAV-12, and AAV-13. The nucleotide sequences of the genomes of the AAV serotypes are known. For example, the complete genome of AAV-1 is provided in GenBank Accession No. NC_002077; the complete genome of AAV-2 is provided in GenBank Accession No. NC001401 and Srivastava et al., Virol. [Virology], 45: 555-564 {1983); the AAV-9 genome is provided in Gao et al., J. Virol. [Journal of Virology], 78: 6381-6388 (2004).

[0139] In some embodiments, the heterologous nucleic acid contained in the rAAV genome is operably linked to transcription control DNA, particularly promoter DNA, enhancer DNA and polyadenylation signal sequence DNA that are functional in the target cell to form an expression construct. The expression construct may also include intron sequences to facilitate the processing of RNA transcripts when expressed in mammalian cells.

[0140] The heterologous nucleic acid contained in the rAAV according to the present invention is not particularly limited. In some embodiments, the heterologous nucleic acid encodes a gene product to be expressed in a target tissue / cell (e.g., one or more retinal cells). In some embodiments, the expression of the gene product in the target cell / tissue will be beneficial to the treatment or prevention of a disease or disorder (e.g., retinal-related lesions) associated with a genetic defect of the tissue. In other embodiments, the heterologous nucleic acid comprises a polynucleotide encoding a marker or reporter protein.

[0141] In some embodiments, the heterologous nucleic acid encodes a target gene product for gene replacement, gene suppression or gene editing. Preferably, the target gene product is a protein or RNA.

[0142] Gene replacement is the most "direct" gene therapy method, which generally involves introducing a nucleic acid encoding a functional protein into a host cell and expressing it in the host cell to compensate for the lack of the functional protein caused by a defective gene (e.g., a mutated retinopathy-related gene) in the host cell. As those skilled in the art will appreciate, depending on the size and properties of the functional protein, a full-length functional protein can be expressed by introducing a nucleic acid molecule encoding the protein into a host cell, or by introducing multiple nucleic acids encoding a portion of the protein into the same host cell. In some embodiments, the rAAV according to the present invention comprises an expression construct, wherein the expression construct comprises at least one heterologous nucleic acid for gene replacement operably linked to a promoter. In some embodiments, the at least one heterologous nucleic acid encodes a therapeutic protein.

[0143] Gene suppression can be achieved by RNA interference (RNAi) means such as antisense nucleic acids, siRNA or microRNA, to inhibit the expression or translation of defective genes in host cells, thereby treating or preventing functional disorders caused by the defective genes. In some embodiments, the rAAV according to the present invention comprises an expression construct, wherein the expression construct comprises at least one heterologous nucleic acid for gene suppression operably linked to a promoter. In some embodiments, the at least one heterologous nucleic acid, after being introduced into the host cell for expression, will result in the production of siRNA or microRNA to inhibit the expression or translation of defective genes in the host cell.

[0144] Gene editing is another gene therapy method for targeted repair of gene defects, including but not limited to ZFN, TALEN and CRISP / Cas editing. In some embodiments, the rAAV according to the present invention comprises an expression construct, wherein the expression construct comprises at least one heterologous nucleic acid for gene editing (especially CRISP / Cas9 editing) operably linked to a promoter. In some embodiments, the expression of the heterologous nucleic acid in the target cell / tissue will target the repair of the gene defect in the cell / tissue.

[0145] The promoter for being connected to heterologous nucleic acid in expression construct is not particularly limited. In some embodiments of the rAAV vector disclosed herein, the heterologous nucleic acid encoding the gene product is operably connected to a constitutive promoter. Suitable constitutive promoters include, for example, cytomegalovirus promoter (CMV), CMV early enhancer / chicken β-actin (CBA) promoter / rabbit β-globin intron (CAG) 7, human elongation factor 1α promoter (EF1α), human phosphoglycerate kinase promoter (PGK), mitochondrial heavy chain promoter, ubiquitin promoter. In other embodiments, the heterologous nucleic acid encoding the gene product is operably connected to an inducible promoter. In some cases, the heterologous nucleic acid encoding the gene product is operably connected to a tissue-specific or cell type-specific regulatory element. For example, in some cases, the heterologous encoding the gene product is operably connected to a photoreceptor-specific regulatory element (e.g., a photoreceptor-specific promoter), which is an element that, for example, gives selective expression of an operably connected gene in a photoreceptor cell. Suitable photoreceptor-specific regulatory elements include, for example, the rhodopsin promoter; the rhodopsin kinase promoter; the beta phosphodiesterase gene promoter; the retinitis pigmentosa gene promoter; the opsin gene promoter; the retinoschisis protein gene promoter.

[0146] In addition to the promoter and the heterologous nucleic acid, other regulatory sequences may be included in the expression construct as needed. The terms "regulatory sequence" and "expression control sequence" are used interchangeably herein and refer to nucleic acid sequences that induce, inhibit or otherwise control the transcription of proteins of the coding nucleic acid sequence that is effectively connected thereto. Regulatory sequences may be, for example, initiation sequences, enhancer sequences, intron sequences, and transcription terminator sequences, etc. In some embodiments, the expression construct further comprises a Kozak sequence located upstream (i.e., 5') of the heterologous nucleic acid.

[0147] Pharmaceutical composition

[0148] In one aspect, the present invention provides a pharmaceutical composition comprising the rAAV vector of the present invention. The rAAV virions according to the present disclosure can be formulated according to known methods after purification to prepare pharmaceutically useful compositions. The compositions of the present disclosure can be formulated using techniques known in the art for administration to mammalian subjects, such as humans.

[0149] When the delivery system is formulated as a solution or suspension, the delivery system is in an acceptable carrier, such as an aqueous carrier. A variety of aqueous carriers can be used, such as water, buffered water, 0.8% saline, 0.3% glycine, hyaluronic acid, etc. These compositions can be sterilized by conventional well-known sterilization techniques, or can be aseptically filtered. The resulting aqueous solution can be packaged for use as is, or lyophilized, and the lyophilized preparation is combined with a sterile solution before administration.

[0150] The pharmaceutical composition according to the present invention may contain pharmaceutically acceptable auxiliary substances to approach physiological conditions, such as pH adjusters and buffers, tonicity adjusters, wetting agents, etc., such as sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, etc. The pharmaceutical composition according to the present invention may or may not contain a preservative.

[0151] The genomic titer of a viral vector, such as the genomic titer of a viral vector in a composition and formulation disclosed herein, can be determined using a variety of standard methods, including, for example, using PCR to determine the genomic titer of a viral vector. The genomic titer of a viral vector is determined using qPCR and ddPCR.

[0152] In a preferred embodiment, the pharmaceutical composition of the present invention is in the form of a pharmaceutical preparation formulated for vitreous delivery.

[0153] Therapeutic applications

[0154] Inherited retinal diseases encompass a large heterogeneous group of genetic disorders that affect approximately 1 in 3000 people (over 2 million worldwide) and are a major source of severe vision loss or blindness. AAV-based gene therapy has been proposed, in which recombinant adeno-associated viruses (rAAV) are used to deliver genes to one or more types of cells in the retina, for example, to replace a missing gene or correct a defective gene.

[0155] The novel capsid protein variants of the present invention improve the targeting and transduction efficiency of AAV to various retinal cells when delivered intravitreally, thus providing a more effective means for gene delivery-based treatment of retinal-related eye diseases.

[0156] Therefore, in one aspect, the present invention provides a method for treating a disease using a recombinant AAV vector of the present invention or a pharmaceutical composition comprising the same. In one embodiment, the method comprises administering any recombinant AAV vector or pharmaceutical composition of the present invention to a subject in need thereof. The recombinant AAV vector or pharmaceutical composition can be administered by any suitable route. In some embodiments, the treatment is therapeutic. In other embodiments, the treatment is preventive. In some embodiments, the subject is a mammal, wherein the mammal is particularly a human, a primate, a dog, a horse, a cow, and particularly a human subject.

[0157] Ocular diseases that can be treated using the variant rAAV vectors or viral particles and / or methods disclosed herein include, but are not limited to, monogenic diseases, complex genetic diseases, acquired diseases, and traumatic injuries. Examples of these diseases include, but are not limited to, congenital amaurosis, achromatopsia, retinitis (X-linked retinitis pigmentosa), choroideremia, Leber hereditary optic neuropathy, macular degeneration (such as age-related macular degeneration), X-linked retinoschisis, diabetic retinopathy, diabetic macular edema, or choroidal neovascularization. Examples of such disease-related genes include, for example, but not limited to, congenital amaurosis-related genes such as RPE65, achromatopsia-related genes such as CNGB3, choroideremia-related genes such as CHM and REP1, Leber hereditaryoptic neuropathy-related genes such as ND4, macular degeneration-related genes such as VEGF, sFLt1 and CD59, retinitis pigmentosa-related genes such as ChR2, MERTK, RLBP1 and PDE6B, X-linked retinitis pigmentosa-related genes such as RPGR, and X-linked retinoschisis-related genes such as RS1. The gene products delivered by the AAV variant capsid according to the present invention can be used to change the level of gene products or gene product activities directly or indirectly associated with retinal diseases and trauma.

[0158] One of ordinary skill in the art will be able to readily determine the amount of rAAV virions effective for a subject and monitor disease treatment by testing changes in one or more functional or anatomical parameters, such as visual acuity, visual field, electrophysiological responses to light and dark, color vision, contrast sensitivity, anatomy, retinal health and vasculature, ocular motility, fixation preference and stability. Some non-limiting methods for assessing retinal function and changes therein include: assessing visual acuity (e.g., best corrected visual acuity [BCVA], movement, navigation, object detection and discrimination); assessing visual fields (e.g., static and dynamic perimetry); performing a clinical examination (e.g., slit lamp examination of the anterior and posterior segments of the eye); assessing electrophysiological responses to all light and dark wavelengths (e.g., all forms of electroretinograms (ERGs) [full-view, multifocal, and pattern], all forms of visual evoked potentials (VEPs), electrooculograms (EOGs), color vision, dark adaptation, and / or contrast sensitivity). Non-limiting methods for assessing anatomy and retinal health and changes therein include optical coherence tomography (OCT), fundus photography, adaptive optics scanning laser ophthalmoscopy (AO-SLO), fluorescence and / or autofluorescence; measuring eye motility and eye movements (e.g., nystagmus, fixation preference and stability), measuring reported outcomes (patient-reported changes in visually and non-visually guided behaviors and activities, patient-reported outcomes [PROs], questionnaire-based quality of life assessments, measures of daily activities and neurological function (e.g., functional magnetic resonance imaging (MRI)).

[0159] In some embodiments, an effective amount of rAAV virions results in a reduction in the rate of loss of retinal function, anatomical integrity, or retinal health in a subject, such as a 2-fold, 3-fold, 4-fold, or 5-fold or more reduction in the rate of loss, and therefore disease progression, such as a 10-fold or more reduction in the rate of loss, and therefore disease progression.

[0160] In some embodiments, rAAV virions are administered to a subject by intraocular injection, for example, by intravitreal injection, by subretinal injection, by suprachoroidal injection, or by any other convenient mode of administration or route that results in the delivery of rAAV virions to the eye. Other convenient modes of administration or routes include, but are not limited to, intravenous, intraarterial, periocular, intracameral, subconjunctival, and subtenon injections and topical and intranasal administration. In some preferred embodiments, rAAV virions are administered by vitreous injection. In some embodiments, when administered by intravitreal injection, rAAV virions are able to pass through the vitreous of the subject and through the inner limiting membrane, and / or move more efficiently through the retinal layers than the ability of AAV virions comprising the corresponding parental AAV capsid protein.

[0161] On the one hand, the present invention also provides the use of the recombinant AAV viral vector of the present invention or the pharmaceutical composition of the present invention in the preparation of a medicament for the above method. In some embodiments, the medicament is used to deliver heterologous nucleic acid to the retina of a subject. In other embodiments, the medicament is used to treat or prevent retinal related diseases, for example, congenital amaurosis, achromatopsia, retinitis (X-linked pigmentary retinitis), choroideremia, Leber hereditary optic neuropathy, macular degeneration (such as age-related macular degeneration), X-linked retinoschisis, diabetic retinopathy, diabetic macular edema, or choroidal neovascularization. In another embodiment, the medicament is in the form of a vitreous injection preparation. Example

[0162] The technical scheme in the embodiment of the present invention is clearly and completely described below. Obviously, the described embodiment is only a part of the embodiment of the present invention, rather than all the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative work are within the scope of protection of the present invention. Unless otherwise specified, the various reaction reagents involved in the embodiments can be purchased through commercial channels.

[0163] Example 1. Construction of a backbone plasmid for an AAV9 capsid protein variant library

[0164] 1. Construction of pRDAV-CMV-EGFP-Cap9 backbone plasmid (construct 1)

[0165] The original plasmid pRDAV-CMV-EGFP (basket gene storage) contains EcoRI and MluI restriction sites after EGFP. Construct 1 (such as Figure 1 Schematically shown).

[0166] Amplification of the complete ORF fragment of Cap9

[0167] Using the complete AAV9 genome as a template, PCR amplified the complete Cap9 ORF fragment and introduced restriction sites, wherein the Cap9 complete ORF fragment refers to an ORF fragment starting from the P40 promoter and ending at the Cap9 termination codon TAA, with a length of 2637 bp, wherein a restriction endonuclease MluI restriction site is introduced at the P40 end, and a restriction endonuclease EcoRI restriction site is introduced at the Cap9 termination codon end.

[0168] The primer sequences used in the PCR amplification are as follows:

[0169] Upstream primer (SEQ ID NO:7): 5'-ACGCGTacgtcaaaaagggtggagc-3'

[0170] Downstream primer (SEQ ID NO: 8): 5'-GAATTCttacagattacgagtcaggtatctg-3'

[0171] The PCR amplification program was as follows: pre-denaturation at 95°C for 2 min, denaturation at 95°C for 15 s, annealing at 56°C for 15 s, extension at 72°C for 30 s, repeated denaturation to extension for 35 cycles, finishing extension at 72°C for 2 min, and termination at 4°C.

[0172] Generation of construct 1

[0173] The PCR product was digested with EcoRI and MluI and recovered by gel electrophoresis to obtain the target fragment. The original plasmid pRDAV-CMV-EGFP was double digested with EcoRI and MluI, and the obtained target fragment was connected to the digested plasmid backbone to obtain the plasmid backbone pRDAV-CMV-EGFP-Cap9, i.e., construct 1 ( Figure 1 The size is 10191 bp and the sequence is shown in SEQ ID NO.1.

[0174] 2. Construction of pRDAV-CMV-EGFP-Cap9Δ-588 backbone plasmid (construct 2)

[0175] The fragment from the 881st base position (c.881) to the 1985th base position (c.1985) on the wild-type Cap9 gene was selected, and the alanine codon corresponding to the amino acid 587 position was mutated to a tyrosine codon (A587Y). At the same time, the GGTAACCGTT AACTT sequence (which contains the BestEII and HpaI restriction sites, BestEII restriction digestion produces sticky ends, and HpaI restriction digestion produces blunt ends, so that the restriction digestion product produced by the BestEII and HpaI double digestion has a sticky end at one end and a blunt end at the other end, thereby preventing self-ligation and facilitating the middle region of the restriction site to be replaced by the randomized library fragment, such as Figure 2The 3 bases encoding the original amino acid residue at position 588 were replaced by 5 bases (as shown in the figure), thereby obtaining the Cap9Δ-588 sequence encoding the mutation at position 587 and the deletion at position 588. The DraIII restriction site was introduced at position c.881 of the Cap9Δ-588 sequence, and the BamHI restriction site was introduced at position c.1985, and the gene synthesis was entrusted to Qingke Biotechnology Co., Ltd. The synthetic product was constructed into construct 1 by enzyme ligation to replace the corresponding fragment between the DraIII and BamHI restriction sites in the original Cap9, and construct 2, i.e., pRDAV-CMV-EGFP-Cap9Δ-588, was obtained. The sequence of construct 2 is shown in SEQ ID NO.2.

[0176] Example 2 Construction of AAV capsid variant virus library

[0177] Linearized backbone vector

[0178] Construct 2 in Example 1 was used as the backbone vector, and double digested with BestEII and HpaI to linearize the backbone vector. The digestion system was: construct 2 (3 μg), CutSmart Buffer (5 μL), restriction endonuclease BestEII (0.5 μL), restriction endonuclease HpaI (0.5 μL), water was added to 50 μL, and incubated at 37°C for 3 to 4 hours. After digestion, the linearized vector was recovered by gel cutting.

[0179] Synthetic Oligo Library

[0180] The Oligo sequence used is as follows:

[0181] Oligo-F:5'-GTAACNNNNNNNNN-3'

[0182] Oligo-R: 5'-NNNNNNNNN-3'

[0183] Oligo was denatured at 95 degrees and slowly annealed at room temperature to obtain the annealing product.

[0184] Library construction and screening

[0185] The oligo annealing product was connected to the linearized backbone (construct 2) using T4 DNA ligase to obtain a recombinant plasmid. The ligation system was: annealing product (2 μL), linearized backbone vector (25 ng), T4 DNA ligase (0.5 μL), ligation buffer (5 μL), water was added to 10 μL, and incubated at 16°C overnight to obtain a recombinant plasmid with oligo inserted.

[0186] The recombinant plasmid was transformed into E. coli HB101 competent cells as follows.

[0187] 1) Add 10 μl of recombinant plasmid to 100 μl of HB101 competent cells in Eppendorf tubes, tap the tube wall several times to mix thoroughly, and place on ice for 30 min;

[0188] 2) Place the Eppendorf tube in a 42°C water bath for 90 seconds;

[0189] 3) Slowly add 0.5 mL of LB medium into the Eppendorf tube and shake at 37°C, 80 rpm for 45 min;

[0190] 4) Spread the bacterial solution on LB plates containing ampicillin (0.1 g / L) and culture at 37°C overnight.

[0191] A single colony was randomly picked and inoculated into a culture tube (LB medium containing 0.1 g / L ampicillin), and cultured overnight at 37°C with shaking at 200 rpm.

[0192] Plasmid extraction was performed as follows.

[0193] 1) Centrifuge the bacterial solution obtained above at 12000 rpm for 1 minute and discard the supernatant medium;

[0194] 2) Add 250 μL of buffer P1 / RNaseA mixture and vortex at high speed to resuspend the bacteria;

[0195] 3) Add 250 μL of buffer P2 and invert 8-10 times;

[0196] 4) Add 350 μL of buffer P3 and immediately invert and mix 8-10 times to completely neutralize the solution;

[0197] 5) Centrifuge at 13000 rpm for 10 minutes and pass the supernatant through the column;

[0198] 6) Centrifuge at 12000 for 1 minute, discard the waste liquid, add 500 μL of PW1, centrifuge at 12000 for 1 minute, discard the waste liquid;

[0199] 7) Add 600 μL of PW2, centrifuge at 12,000 g for 1 minute, and discard the supernatant;

[0200] 8) Repeat washing once: repeat step 7;

[0201] 9) Idle at 12000 rpm for 2 minutes;

[0202] 10) Add 30-50 μL of 55°C preheated eluent, let stand for 2 minutes, and centrifuge at 12,000 rpm for 1 minute.

[0203] The positive plasmids that have been tested for concentration and identified by enzyme digestion were numbered and 10 μL was taken for sequencing. The positive plasmids were stored in a -20°C refrigerator to obtain the AAV capsid library.

[0204] The AAV9 capsid protein (CAP9) VP1 variant numbered CapX-120 was obtained by sequencing, and its corresponding nucleotide sequence is shown in SEQ ID NO.3, and the corresponding amino acid sequence is shown in SEQ ID NO.4. The sequence information obtained by sequencing was compared with the original capsid protein (CAP9) sequence, and the alternative amino acid sequences of the variant at positions 587 and 588 are shown in SEQ ID NO.5.

[0205] Example 3 Packaging and Production of Mutant AAV Viruses and Control AAV Viruses

[0206] Reference (Xiao X, et al. Production of High-Titer Recombinant Adeno-Associated Virus Vectors in the Absence of Helper Adenovirus. J Virol. 1998; 72(3): 2224-2232), the three-plasmid packaging system was used to package and purify the recombinant AAV virus. In brief, the packaging plasmid expressing the Rep gene of AAV, the auxiliary plasmid providing the auxiliary function of AdV, and the transfer plasmid carrying the ITR of AAV and expressing the variant CAP9 and the green fluorescent protein eGFP (inserted with the variant CAP9) were Figure 2 The plasmids shown) or transfer plasmids expressing wild-type CAP9 and green fluorescent protein eGFP ( Figure 1 The plasmid shown in the figure was mixed at a molar ratio of 1:1:1 and co-transfected into HEK293 cells using the calcium phosphate method. 72 hours after transfection, the cells and culture supernatant were collected, and the recombinant AAV virus was purified by PEG8000-NaCl&CH3Cl extraction. The AAV virus product was dissolved with 0.01% Pluronic F68-PBS, and the rAAV titer was determined by dot hybridization. The AAV capsid variant virus numbered CapX-120 and the control rAAV9 virus were obtained.

[0207] Example 4 Biodistribution of AAV capsid variant viruses in mice

[0208] AAV virus injection

[0209] 6-8 weeks, 18-20g male SPF C57 mice were selected, rAAV9 virus encoding fluorescent protein EFGP was used as a control, and the AAV capsid variant virus described in this article was used as an experimental group, with at least 3 mice in each group. The capsid variant virus CapX-120 was injected intravitreally at a dose of 2E10gc / kg. After injection, the mice were killed 28 days later, and frozen sections of each tissue were prepared to observe EGFP fluorescence.

[0210] Mouse tissue sections and immunofluorescence comparison

[0211] A. Preparation of frozen sections

[0212] 1) Tissue fixation: Fix the tissue in fresh tissue fixative for more than 24 hours, remove the tissue from the fixative, and use a scalpel to smooth the tissue at the target site.

[0213] 2) Dehydration: Place the trimmed tissue in a 15% sucrose solution and dehydrate it in a 4°C refrigerator until it sinks to the bottom. Then transfer it to a 30% sucrose solution and dehydrate it in a 4°C refrigerator until it sinks to the bottom.

[0214] 3) OCT embedding: Take out the dehydrated tissue, use filter paper to slightly absorb the surface water, place it on the embedding table with the cut surface facing up, drip OCT embedding agent around the tissue, and place the embedding table on the quick freezing table of the freezing microtome for quick freezing embedding. After the OCT turns white and hardens, it can be sliced. Fresh tissue does not need to be fixed and dehydrated for direct frozen sectioning. Use a scalpel to directly flatten the tissue of the target area, embed it with OCT embedding agent, and slice it.

[0215] 4) Sectioning: Fix the embedding table on the slicer, cut the tissue surface roughly and then start sectioning. The thickness of the slice is 8-10μm. Place a clean slide flat on the cut tissue slice, stick the tissue on the slide, and write a label.

[0216] B. Observe fluorescence

[0217] The frozen sections were protected with anti-fading mounting medium (ProLong, Thermo Fischer), and the EGFP protein was observed and images were collected under a fluorescence microscope using a blue excitation band module (488 nm). The images were statistically analyzed using ImageJ software.

[0218] result

[0219] Capsid protein mutant CapX-120

[0220] The experimental results showed that compared with the control group, after the AAV capsid variant CapX-120 virus infected the mouse retina, the intracellular fluorescence intensity was significantly enhanced, the number of infected cells increased, and the infection rate of cones and rods was almost saturated ( Figure 3 and4 ). According to the fluorescence analysis results of ImageJ software, the average fluorescence intensity caused by CapX-120 virus infection in the cone and rod layer, RPE layer, outer nuclear layer, and inner nuclear layer is higher than that of AAV9 virus, which is 1 to 2 times that of AAV9 virus ( Figure 5 ); For the countable outer nuclear layer and inner nuclear layer, the EGFP-positive cell rate after CapX-120 virus infection was higher than that of AAV9 virus, which was 2 to 3 times that of AAV9 virus ( Figure 6 ). The above results show that the AAV capsid protein mutant CapX-120 can significantly enhance the transduction efficiency of the retina and can be used as a better choice for AAV treatment of ophthalmic diseases.

[0221] Some embodiments of the present invention:

[0222] 1. An adeno-associated virus (AAV) capsid protein variant, which inserts the amino acid sequence of SEQ ID NO.5 between amino acid positions 586 and 589 of the parent AAV capsid protein to replace residues 587-588 of the parent AAV capsid protein, wherein the amino acid positions are numbered according to SEQ ID NO:6.

[0223] 2. The AAV capsid protein variant of embodiment 1, wherein, compared with the parent AAV capsid protein, the capsid protein variant confers increased transduction of the AAV virus particles comprising it to retinal cells, preferably, the retinal cells are selected from: photoreceptor cells, retinal pigment epithelium (RPE) layer cells, outer nuclear layer cells, inner nuclear layer cells and optic ganglion layer cells; more preferably, the retinal cells are rod cells and / or cone cells,

[0224] Still more preferably, the capsid protein variant confers a higher transduction rate to the cones and rods of the retina relative to other cell types of the retina.

[0225] 3. The capsid protein variant of embodiment 1 or 2, wherein the parent AAV capsid protein is an AAV9 capsid protein.

[0226] 4. The capsid protein variant of any one of embodiments 1-3, wherein the parent AAV capsid protein is an AAV9 capsid protein comprising or consisting of the following amino acid sequence:

[0227] (a) an amino acid sequence of amino acids 203 to 736 of SEQ ID NO: 6;

[0228] (b) an amino acid sequence of amino acids 137 to 736 of SEQ ID NO: 6;

[0229] (c) The amino acid sequence of SEQ ID NO:6.

[0230] 5. The capsid protein variant according to any one of embodiments 1 to 4, wherein the capsid protein variant is VP1, VP2 or VP3 capsid protein,

[0231] Preferably, the capsid protein variant:

[0232] (a) comprising an amino acid sequence of SEQ ID NO: 4;

[0233] (b) is encoded by a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 3; or

[0234] (c) derived from a transcript encoding the amino acid sequence of SEQ ID NO:4 or comprising the nucleotide sequence of SEQ ID NO:3.

[0235] 6. An isolated nucleic acid, wherein the nucleic acid comprises a polynucleotide sequence encoding the AAV capsid protein variant of any one of embodiments 1-5.

[0236] 7. The nucleic acid according to embodiment 6, comprising:

[0237] (a) a polynucleotide encoding the amino acid sequence of SEQ ID NO: 4; or

[0238] (b) The nucleotide sequence of SEQ ID NO:3.

[0239] 8. The nucleic acid according to embodiment 6 or 7, wherein the nucleic acid is capable of transcribing and expressing at least one or any two or all three AAV capsid proteins selected from the following: VP1, VP2 and VP3.

[0240] 9. A nucleic acid vector comprising the nucleic acid of any one of embodiments 6-8, optionally further comprising a polynucleotide encoding an AAVREP protein.

[0241] 10. An isolated host cell comprising the nucleic acid of any one of embodiments 6-8, optionally wherein the nucleic acid stably transfects the host cell,

[0242] Preferably, the host cell further comprises nucleic acid encoding AAV REP protein and / or nucleic acid encoding auxiliary viral function.

[0243] 11. Use of the nucleic acid according to any one of embodiments 6 to 8, the nucleic acid vector according to embodiment 9, or the host cell according to embodiment 10 for preparing a recombinant adeno-associated virus (rAAV) vector.

[0244] 12. A recombinant adeno-associated virus (rAAV) vector comprising the capsid protein variant of any one of embodiments 1-5,

[0245] Preferably, the rAAV exhibits increased transduction of retinal cells relative to wild-type AAV, preferably, the retinal cells are selected from the group consisting of photoreceptor cells, RPE layer cells, outer nuclear layer cells, inner nuclear layer cells and ganglion layer cells.

[0246] 13. The rAAV vector according to embodiment 12, wherein the rAAV vector comprises in its genome:

[0247] a. 5' and 3' AAV inverted terminal repeat (ITR) sequences, and

[0248] b. An expression construct comprising a heterologous nucleic acid located between the 5' and 3' ITRs, wherein the heterologous nucleic acid encodes a gene product of interest.

[0249] 14. The rAAV vector according to embodiment 13, wherein the expression construct comprises the following elements functionally linked to each other in the transcriptional direction:

[0250] - a promoter, preferably a retina-specific promoter, such as a rod and / or cone-specific promoter,

[0251] - a heterologous nucleic acid,

[0252] - Transcription terminator.

[0253] 15. An AAV vector according to embodiment 13 or 14, wherein the heterologous nucleic acid encodes a target gene product for gene replacement, gene suppression or gene editing, preferably, the target gene product is a protein or RNA.

[0254] 16. A pharmaceutical composition comprising the recombinant AAV viral vector of any one of embodiments 12-15 and a pharmaceutically acceptable carrier.

[0255] 17. A method of delivering a heterologous nucleic acid to a subject in need thereof, wherein the method comprises administering to the subject the recombinant AAV viral vector of any one of embodiments 12-15 or the pharmaceutical composition of embodiment 16.

[0256] 18. The method of embodiment 17, wherein the method is used to treat or prevent a retina-related disease, such as congenital amaurosis, achromatopsia, retinitis (X-linked retinitis pigmentosa), choroideremia, Leber hereditary optic neuropathy, macular degeneration (such as age-related macular degeneration), X-linked retinoschisis, diabetic retinopathy, diabetic macular edema, or choroidal neovascularization.

[0257] 19. The method of embodiment 17 or 18, wherein the delivering comprises intravitreal injection.

[0258] 20. A method for delivering heterologous nucleic acid to cells cultured in vitro or in vitro, comprising contacting the cells with the rAAV viral vector of embodiments 12-15, preferably, the cells are retinal cells, especially rod cells and / or cone cells.

[0259] 21. Use of the recombinant AAV viral vector according to any one of embodiments 12-15 for preparing a pharmaceutical composition for treating or preventing retina-related diseases, for example, the retina-related diseases are selected from congenital amaurosis, achromatopsia, retinitis (X-linked retinitis pigmentosa), choroideremia, Leber hereditary optic neuropathy, macular degeneration (such as age-related macular degeneration), X-linked retinoschisis, diabetic retinopathy, diabetic macular edema, or choroidal neovascularization, preferably, the pharmaceutical composition is administered by ocular gene delivery (e.g., intravitreal injection).

[0260]

[0261]

[0262]

[0263]

[0264]

[0265]

Claims

1. An adeno-associated virus (AAV) capsid protein variant, which inserts the amino acid sequence of SEQ ID NO.5 between amino acid positions 586 and 589 of the parent AAV capsid protein to replace residues 587-588 of the parent AAV capsid protein, wherein the amino acid positions are numbered according to SEQ ID NO:

6.

2. The AAV capsid protein variant of claim 1, wherein Compared to the parent AAV capsid protein, the capsid protein variant confers increased transduction of the AAV virus particles comprising it to retinal cells, preferably, the retinal cells are selected from: photoreceptor cells, retinal pigment epithelium (RPE) layer cells, outer nuclear layer cells, inner nuclear layer cells and optic ganglion layer cells; more preferably, the retinal cells are rod cells and / or cone cells, Still more preferably, the capsid protein variant confers a higher transduction rate to the cones and rods of the retina relative to other cell types of the retina.

3. The capsid protein variant of claim 1 or 2, wherein the parent AAV capsid protein is an AAV9 capsid protein.

4. An isolated nucleic acid, wherein the nucleic acid comprises a polynucleotide sequence encoding the AAV capsid protein variant of any one of claims 1-3.

5. A nucleic acid vector comprising the nucleic acid of claim 4, optionally further comprising a polynucleotide encoding an AAV REP protein.

6. An isolated host cell comprising the nucleic acid of claim 4, optionally wherein the nucleic acid stably transfects the host cell, Preferably, the host cell further comprises nucleic acid encoding AAV REP protein and / or nucleic acid encoding auxiliary viral function.

7. Use of the nucleic acid according to claim 4, the nucleic acid vector according to claim 5, or the host cell according to claim 6 for preparing a recombinant adeno-associated virus (rAAV) vector.

8. A recombinant adeno-associated virus (rAAV) vector comprising the capsid protein variant of any one of claims 1 to 3, Preferably, the rAAV exhibits increased transduction of retinal cells relative to wild-type AAV, preferably, the retinal cells are selected from the group consisting of photoreceptor cells, RPE layer cells, outer nuclear layer cells, inner nuclear layer cells and ganglion layer cells.

9. A pharmaceutical composition comprising the recombinant AAV viral vector of claim 8 and a pharmaceutically acceptable carrier.

10. A method for delivering a heterologous nucleic acid to a subject in need thereof or to cells cultured ex vivo or in vitro, wherein the method comprises administering the recombinant AAV viral vector of claim 8 or the pharmaceutical composition of claim 9 to the subject or the cells.

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