CN114507692B

CN114507692B - Adeno-associated viral vectors for the treatment of brile disease and uses thereof

Info

Publication number: CN114507692B
Application number: CN202111351703.5A
Authority: CN
Inventors: 张婷婷; 王超; 李捧花
Original assignee: Beijing Solobio Genetechnology Co Ltd; Staidson Beijing Biopharmaceutical Co Ltd
Current assignee: Beijing Solobio Genetechnology Co Ltd; Staidson Beijing Biopharmaceutical Co Ltd
Priority date: 2020-11-16
Filing date: 2021-11-16
Publication date: 2024-07-12
Anticipated expiration: 2041-11-16
Also published as: CN114507692A

Abstract

The invention discloses a recombinant adeno-associated virus (AAV) vector comprising AAVX capsids and a vector genome, wherein the vector genome comprises a nucleic acid sequence encoding a functional alpha-galactosidase A and regulatory sequences that direct the expression of the alpha-galactosidase A sequence in a host cell. The invention also discloses the application of the recombinant adeno-associated virus vector in treating the BuLi disease.

Description

Adeno-associated viral vectors for the treatment of brile disease and uses thereof

Technical Field

The invention relates to the field of gene therapy, in particular to an adeno-associated virus vector for treating Brillouin disease and application thereof.

Background

Fabry disease is a lysosomal storage disorder caused by lysosomal deficiency of α -galactosidase a (α -GalA, GLA) and can lead to systemic accumulation of neutral glycosphingolipids throughout the body, with the accumulation of sphingosine trihexyl (Gb 3) being the predominant cause, mainly in lysosomal endothelial cells, vascular smooth muscle cells, kidney cells, heart muscle cells and dorsal root ganglion cells. The first clinical symptoms that appear are neuralgia, little sweat and tremors associated with the peripheral nervous system and the small nerve fibers of the autonomic nervous system, thereby affecting the quality of life. With age, fatal complications occur mainly in the kidneys, heart and brain. Since fabry disease is an X-chromosome related disease, only male and female heterozygotes develop, the incidence of fabry disease in men is about 1/40000 to 1/117000. The current treatment is mainly enzyme replacement therapy, using CHO cells to express the full-length alpha-galactosidase. Currently, two protein medicines are marketed, one is Fabrazyme of Jianzan corporation, the dosage is 1mg/kg, and the mode of administration is intravenous administration, once every two weeks; another protein drug is replagal from Shire at a dose of 0.2mg/kg by intravenous administration for once every two weeks. The main disadvantages of protein drugs are short half-life in vivo, frequent dosing frequency, poor patient compliance, and heavy economic burden on the patient's home from long-term treatment.

In recent years, gene therapy has shown dramatic efficacy in the areas of rare genetic diseases, cancer and infectious diseases. Gene therapy medicine Luxturna with AAV2 as a vector of Spark Therapeutics is marketed in batches, and brings good news for patients with retina caused by RPE65 gene deficiency. In the case of fabry disease, after transferring the normal α -galactosidase a gene into the affected patient by gene therapy, the potential for therapy is provided by sustained endogenous production of α -galactosidase a, which can be a good solution to patient compliance issues, resulting in better therapeutic results.

Currently, viral vectors are one of the most common delivery methods in the field of gene therapy, and AAV subtypes named serotypes are classified as AAV1-AAV12, mainly in humans and primates, wherein AAV1-6 is isolated from human tissue and has defined antibody reactivity, and thus is more classical and well-established. AAV7 and AAV8 are high-gloss and equal AAV subtypes which are obtained by rescue from heart tissues of macaques through genetic engineering means and are suspected to be destinated in evolution, and AAV9-12 are prepared in tissues of humans and cynomolgus monkeys respectively by using similar technical routes. Although viruses of different AAV serotypes all have a structure of regular icosahedron, their capsid proteins differ in sequence and spatial conformation such that there is a significant difference in their cell surface binding receptors and infectivity (tropism) of cells, e.g., AAV2 has a broad spectrum of infection, especially for neural cells; AAV1 and AAV7 are more efficient in transduction in skeletal muscle; AAV3 readily transduces megakaryocytes; AAV5 and AAV6 have significant advantages in infecting airway epithelial cells, and the like.

However, natural AAV targeting is limited, especially when using AAV vectors for systemic administration, the proportion of target tissue that can be effectively infected varies widely depending on the serotype selection, and other non-targeted tissue cells are potentially at risk for infection. And because natural AAV naturally infects humans and other primates, neutralizing antibodies against natural AAV can be produced in humans and other primates, the half-life of AAV can be greatly reduced, and AAV vector utilization cannot be maximized.

The relative specificity of AAV subtypes for tissue infection and the extent to which neutralizing antibodies are already present or produced in humans or primates are of great significance for the treatment of specific diseases. Thus, the search for optimal vector types in gene therapy for different targeted tissues is critical to the success of treating the relevant diseases.

AAV8 is the currently accepted serotype that is optimal for liver targeting. AAVX is a novel AAV vector obtained using the DNA shuffling technique. Through the in vitro and in vivo targeting study of AAVX vectors and the study of the existence or possibility of generating neutralizing antibodies in vivo and in vitro of primates, a series of studies show that AAVX may be more advantageous than AAV8 in the study of treating liver diseases.

Related studies on the treatment of Fabry diseases using AAV 8-carried GLA are known in the prior art (patent CN107980063 a), but further studies according to the present invention indicate that better therapeutic effects are achieved by using AAVX-carrier-carried GLA.

Disclosure of Invention

The present disclosure also uses adeno-associated viral vector AAVX to mediate transfer and expression of alpha-galactosidase a gene, so that normal alpha-galactosidase a is continuously produced in patient, accumulation of glycosphingolipids is reduced, and the aim of treating the brix disease is achieved, and it has been proved that AAVX vector carrying alpha-galactosidase a has better therapeutic effect in treating the brix disease than currently known AAV8 vector carrying alpha-galactosidase a with best liver targeting.

In one aspect, the present disclosure provides a recombinant adeno-associated virus (AAV) vector comprising a AAVX capsid and a vector genome, wherein the vector genome comprises a nucleic acid sequence encoding a functional α -galactosidase a and an expression control sequence that directs the expression of the α -galactosidase a sequence in a host cell.

In another aspect, the present disclosure provides an isolated host cell comprising the aforementioned recombinant adeno-associated virus (AAV) vector.

In another aspect, the present disclosure provides a pharmaceutical composition comprising a recombinant adeno-associated virus (AAV) vector and/or host cell as described above, and a pharmaceutically acceptable carrier.

In another aspect, the present disclosure provides the use of a recombinant adeno-associated virus (AAV) vector, host cell and/or pharmaceutical composition as described above in the manufacture of a medicament for preventing or treating fabry disease.

In another aspect, the present disclosure provides a method of treating fabry disease in a subject. The method comprises administering to a subject in need thereof a recombinant adeno-associated virus (AAV) vector, host cell, and/or pharmaceutical composition as described previously.

Drawings

FIG. 1 shows a plasmid map associated with the vector construction process.

FIG. 2 shows a comparison of the infection efficiency of AAV2/X and AAV2/8 recombinant viruses in different human hepatocyte lines. Wherein, the left and right graphs are repeated experiments.

FIG. 3 shows a comparison of the infection efficiency of AAV2/X and AAV2/8 recombinant viruses in different human liver cancer primary cells. Wherein, the left side is fluorescence percentage, and the right side is fluorescence intensity.

FIG. 4 shows a fluorescence photograph of different tissues of AAV2/X-CMV-EGFP recombinant virus-dosed animals. Wherein, A, heart, B, lung, C, liver 1, D, liver 2, E, liver 3, F, liver 4, G, liver 5,H, brain, I, testis, J, biceps femoris, K, stomach, L, jejunum, M, kidney, N, spleen.

FIG. 5 shows a fluorescence photograph of different tissues of AAV2/8-CMV-EGFP recombinant virus-dosed animals. Wherein, A, heart, B, lung, C, liver 1, D, liver 2, E, liver 3, F, liver 4, G, liver 5,H, brain, I, testis, J, biceps femoris, K, stomach, L, jejunum, M, kidney, N, spleen.

FIG. 6 shows comparative statistics of AAV2/X and AAV2/8 neutralizing antibodies in 20 human sera.

FIG. 7 shows a block diagram containing various promoter elements.

FIG. 8A shows the results of luciferase activity assays after separate injections of recombinant viruses carrying luciferases with different promoter elements into normal mice.

FIG. 8B shows the results of the detection of GLA enzyme activity after separate injections of GLA-carrying recombinant viruses containing different promoter elements into normal mice.

FIG. 9 shows a structural diagram of a WPRE-containing expression regulatory element.

FIG. 10 shows the results of GLA enzyme activity detection after injection of recombinant viruses containing LP1 promoter-GLA and LP1 promoter-WPRE expression regulatory element-GLA, respectively, into normal mice.

FIG. 11 shows the results of GLA enzyme activity assay after drug candidate ssAAV2/X-LP1-GLA infection of primary hepatocytes in model mice.

FIG. 12A shows the results of GLA enzyme activity assays in different tissues after ssAAV/X-LP 1-GLA and ssAAV/8-LP 1-GLA recombinant viruses were injected into model mice, respectively.

FIG. 12B shows the results of GLA enzyme activity assay in different tissues after candidate drug ssAAV2/X-LP1-GLA was injected into model mice at different doses, respectively.

Detailed Description

I. definition of the definition

The practice of the present invention will employ, unless otherwise indicated, conventional chemical, biochemical, recombinant DNA techniques and immunological methods in the art. Such techniques are well explained in the literature (see, e.g., fundamentalVirology, second edition, vol. I & II (B.N. fields and D.M. Knope.); handbook of ExperimentalImmunology, voIs.I-FV (D.M. Weir and CC. Blackbwell. ,Blackwell ScientificPublications);T.E.Creighton,Proteins:Structures and Molecular Properties(W.H.Freeman and Company, 1993); A.L. Lehninger, biochemistry (Worth Publishers, inc.), sambrook, et al, molecular Cloning: A Laboratory Manual (2 nd edition, 1989); methods In Enzymology (S.Colowick and N.Kaplan. ACADEMIC PRESS, inc.).

In order to facilitate an understanding of the various embodiments of the present disclosure, the following explanation of specific terms is provided:

Adeno-associated virus (AAV): small replication-defective non-enveloped viruses that infect humans and some other primate species. AAV is known not to cause disease and to elicit a very mild immune response. Gene therapy vectors using AAV can infect dividing cells and resting cells, and can remain extrachromosomal without integration into the host cell's genome. These characteristics make AAV an attractive viral vector for gene therapy.

Dosing/administration: an agent, e.g., a therapeutic agent (e.g., recombinant AAV), is provided or administered to a subject by an effective route. Exemplary routes of administration include, but are not limited to, injection (e.g., subcutaneous, intramuscular, intradermal, intraperitoneal, and intravenous), oral, intraductal, sublingual, rectal, transdermal, intranasal, vaginal, and inhalation routes.

Codon optimized: a "codon-optimized" nucleic acid refers to a nucleic acid sequence that has been altered to make the codon optimal for expression in a particular system (e.g., a particular species or group of species). For example, the nucleic acid sequence may be optimized for expression in mammalian cells or in a particular mammalian species (e.g., human cells). Codon optimization does not alter the amino acid sequence of the encoded protein.

Enhancers: a nucleic acid sequence that increases transcription rate by increasing promoter activity.

Introns: the gene contains a piece of DNA which does not contain coding information of protein. Introns are removed prior to translation of the messenger RNA. Hybrid introns: is a combination intron that includes sequences from more than one natural intron.

Inverted Terminal Repeat (ITR): symmetric nucleic acid sequences in the genome of the desired adeno-associated virus are efficiently replicated. ITR sequences are located at each end of the AAV DNA genome. The ITR serves as an origin of replication for viral DNA synthesis and is a cis-element necessary for the production of AAV integrative vectors.

Separating: an "isolated" biological component (e.g., a nucleic acid molecule, protein, virus, or cell) has been substantially isolated or purified from other biological components (e.g., other chromosomal and extra-chromosomal DNA and RNA, proteins, and cells) in the organism or tissues in which the component naturally occurs, or in the organism itself. Nucleic acid molecules and proteins that have been "isolated" include those purified by standard purification methods. The term also includes nucleic acid molecules and proteins prepared by recombinant expression in host cells, as well as chemically synthesized nucleic acid molecules and proteins.

Operatively connected to: the first nucleic acid sequence is operably linked to the second nucleic acid sequence when the first nucleic acid sequence and the second nucleic acid sequence are placed in a functional relationship. For example, a promoter is operably linked to a coding sequence if it affects the transcription or expression of the coding sequence. Typically, operably linked DNA sequences are contiguous and, where necessary to join two protein coding regions, in the same reading frame.

Pharmaceutically acceptable carrier: pharmaceutically acceptable carriers (solvents) that can be used in the present disclosure are conventional. Remington's Pharmaceutical Sciences, by E.W.Martin, mackPublishing co., easton, PA,15th Edition (1975) describes compositions and formulations suitable for drug delivery of one or more therapeutic compounds, molecules or agents.

Generally, the nature of the carrier will depend on the particular mode of administration used. For example, parenteral formulations typically contain injectable fluids which include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol and the like as solvents. For solid compositions (e.g., in the form of powders, pills, tablets, or capsules), conventional non-toxic solid carriers such as pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate may be included. In addition to the biologically neutral carrier, the pharmaceutical composition to be administered may also contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate.

Preventing, treating or ameliorating a disease: "preventing" a disease (e.g., GSD-Ia) refers to inhibiting the overall occurrence of the disease. "treatment" refers to therapeutic intervention that ameliorates signs or symptoms of a disease or pathological condition after onset of the disease. "ameliorating" refers to reducing the number or severity of signs or symptoms of a disease.

Promoter: a DNA region that directs/causes transcription of a nucleic acid (e.g., gene). Promoters include the necessary nucleic acid sequences near the transcription initiation site. Typically, a promoter is located near the gene it transcribes. The promoter region also optionally includes distal enhancer or repressor elements, which may be located thousands of base pairs away from the transcription initiation site.

Recombination: recombinant nucleic acid molecule refers to a nucleic acid molecule having a non-naturally occurring sequence or having a sequence prepared by artificial combination of two sequence fragments (which would otherwise be separate). Such artificial combination may be achieved by chemical synthesis or by artificial manipulation of isolated nucleic acid molecule fragments (e.g., by genetic engineering techniques).

Likewise, a recombinant virus is a virus comprising a sequence that is not naturally occurring or is made by artificial combination of at least two sequences of different origin. The term "recombinant" also includes nucleic acids, proteins and viruses that are altered by the addition, substitution or deletion of only a portion of the native nucleic acid molecule, protein or virus. As used herein, "recombinant AAV" refers to an AAV particle in which a recombinant nucleic acid molecule (e.g., a recombinant nucleic acid molecule encoding G6 Pase-a) is packaged.

Serotypes: a closely related class of microorganisms (e.g., viruses) distinguished by a characteristic set of antigens.

The subject: living multicellular vertebrate organisms, including classes of human and non-human mammals.

And (3) synthesis: the synthetic nucleic acids may be produced in the laboratory by artificial means, for example, they may be chemically synthesized in the laboratory.

Therapeutically effective amount of: an amount of a particular drug or therapeutic agent (e.g., recombinant AAV) sufficient to achieve a desired effect in a subject or cell treated with the agent. The effective amount of the agent depends on a variety of factors including, but not limited to, the subject or cell being treated, and the mode of administration of the therapeutic composition.

And (3) a carrier: vectors are nucleic acid molecules that allow insertion of foreign nucleic acids without disrupting the ability of the vector to replicate and/or integrate in a host cell. A vector may comprise a nucleic acid sequence, such as an origin of replication, that allows it to replicate in a host cell. The vector may also comprise one or more selectable marker genes and other genetic elements. An expression vector is a vector that contains the necessary regulatory sequences to allow transcription and translation of an inserted gene. In some embodiments herein, the vector is an AAV vector.

Sequence identity: identity or similarity between two or more nucleic acid sequences or between two or more amino acid sequences is expressed in terms of identity or similarity between the sequences. Sequence identity can be measured in terms of percent identity; the higher the percentage, the more identical the sequence. Sequence similarity can be measured in terms of percent similarity (taking into account conservative amino acid substitutions); the higher the percentage, the more similar the sequences. Homologs or orthologs of nucleic acid or amino acid sequences have a relatively high degree of sequence identity/similarity when aligned using standard methods. Such homology is more pronounced when the orthologous protein or cDNA is from more closely related species (e.g., human and mouse sequences) than from more closely related species (e.g., human and nematode (C. Elegans) sequences).

The length of the sequence identity comparison may be over the full length of the genome, the full length of the gene coding sequence, or a fragment of at least about 500 to 5000 nucleotides is desirable. Identity in smaller fragments (e.g., having at least about 9 nucleotides, typically at least about 20 to 24 nucleotides, at least about 28 to 32 nucleotides, at least about 36 or more nucleotides) may also be desirable.

The percent identity of amino acid sequences can be readily determined over the full-length protein, polypeptide, about 32 amino acids, about 330 amino acids or peptide fragments thereof, or corresponding nucleic acid sequence coding sequences. Suitable amino acid fragments may be at least about 8 amino acids in length and may be up to about 700 amino acids in length. In general, when referring to "identity", "homology" or "similarity" between two different sequences, the "identity", "homology" or "similarity" is determined with reference to "aligned" sequences. "aligned" sequences or "alignment" refers to a plurality of nucleic acid sequences or protein (amino acid) sequences that generally contain deletions or additional base or amino acid corrections as compared to a reference sequence.

Any public or commercially available multiple sequence alignment program is used for alignment. Sequence alignment programs can be used for amino acid sequences such as the "Clustal X", "MAP", "PIMA", "MSA", "BLOCKMAKER", "MEME" and "Match-Box" programs. Typically, any of these programs is used in default settings, although one skilled in the art can change these settings as desired. Or another algorithm or computer program that provides at least the level of identity or alignment as provided by a reference algorithm or program may be employed by those skilled in the art. See, e.g., J.D. Thomson et al ,Nucl.Acids.Res.,"Acomprehensive comparison of multiple sequence alignments",27(13):2682-2690(1999).

Multiple sequence alignment programs can also be used for nucleic acid sequences. Examples of such programs include "Clustal W", "CAPSequence Assembly", "BLAST", "MAP", and "MEME", which can be accessed through a Web server on the Internet. Other sources of such procedures are known to those skilled in the art. Or also using Vector NTI applications. There are also many algorithms known in the art that can be used to measure nucleotide sequence identity, including those contained in the above-described programs. As another example, it is possible to use(One of the programs in GCG Version 6.1) to compare polynucleotide sequences.An alignment and percent sequence identity of the optimal overlap region between the query and search sequences is provided. For example, one can useThe percent sequence identity between nucleic acid sequences was determined by its default parameters (word length 6, and NOPAM factor for scoring matrices) provided in GCG Version 6.1 (incorporated herein by reference).

In one embodiment, the modified hGLA coding sequence is a codon optimized sequence, optimized for expression in the test species. As used herein, a "subject" is a mammal, e.g., a human, mouse, rat, guinea pig, dog, cat, horse, cow, pig, or non-human primate, such as a monkey, chimpanzee, baboon, or gorilla. In a preferred embodiment, the subject is a human. In one embodiment, the sequence is codon optimized for expression in humans.

The codon optimized coding region can be designed by a variety of different methods. Such optimization may be performed using methods available online (e.g., geneArt), published methods, or companies that provide codon optimization services, such as DNA2.0 (Menlo Park, CA). One method of codon optimization is described, for example, in U.S. International patent publication No. WO 2015/012924, which is incorporated herein by reference in its entirety. See also, for example, U.S. patent publication No. 2014/0032186 and U.S. patent publication No. 2006/0136814. Suitably, the entire length of the Open Reading Frame (ORF) of the product is modified. However, in some embodiments, only a fragment of the ORF may be altered. By using one of these methods, the frequency can be applied to any given polypeptide sequence and nucleic acid fragments encoding the codon-optimized coding region of the polypeptide produced.

Many options are available for making practical changes to codons or for synthesizing codon optimized coding regions designed as described herein. Such modifications or syntheses may be performed using standard and conventional molecular biological procedures well known to those of ordinary skill in the art. In one approach, a series of complementary oligonucleotide pairs, each 80-90 nucleotides in length and spanning the length of the desired sequence, are synthesized by standard methods. These oligonucleotide pairs are synthesized such that they, when annealed, form a double-stranded fragment of 80-90 base pairs containing cohesive ends, e.g., each oligonucleotide in the pair is synthesized to extend 3, 4,5, 6, 7, 8, 9, 10 or more bases beyond the region complementary to the other oligonucleotide in the pair. The single stranded end of each oligonucleotide pair is designed to anneal to the single stranded end of the other oligonucleotide pair. Annealing the oligonucleotide pairs and then annealing about five to six of these double stranded fragments together via cohesive single stranded ends, and then ligating them together and cloning into a standard bacterial cloning vector, such as that available from Invitrogen Corporation, carlsbad, califA carrier. The construct is then sequenced by standard methods. Several of these constructs, consisting of 5 to 6 fragments of 80 to 90 base pairs (i.e., fragments of about 500 base pairs) linked together, were prepared so that the entire desired sequence was displayed as a series of plasmid constructs. The inserts of these plasmids are then cleaved with the appropriate restriction enzymes and ligated together to form the final construct. The final construct was then cloned into a standard bacterial cloning vector and sequenced. Additional methods will be apparent to those skilled in the art. Furthermore, gene synthesis is readily available.

In one embodiment, the modified hGLA genes described herein are genetically engineered into suitable genetic elements (vectors) useful for the production of viral vectors and/or delivery to host cells, such as naked DNA, phage, transposons, cosmids, episomes, and the like, which deliver hGLA sequences carried thereon. The selected vector may be delivered by any suitable method, including transfection, electroporation, liposome delivery, membrane fusion techniques, high speed DNA coated beads, viral infection, and protoplast fusion. Methods for making such constructs are known to those skilled in the art of nucleic acid manipulation and include genetic engineering, recombinant engineering, and synthetic techniques. See, e.g., sambrook et al Molecular Cloning: A Laboratory Manual, coldSpring Harbor Press, cold Spring Harbor, N.Y..

In one aspect, an expression cassette comprising the hGLA nucleic acid sequence is provided. As used herein, an "expression cassette" refers to a nucleic acid molecule comprising a promoter hGLA sequence, and may include other regulatory sequences for this purpose, which can be packaged into the capsid of a viral vector (e.g., a viral particle). Typically, such expression cassettes for generating viral vectors contain hGLA sequences described herein that flank the packaging signal of the viral genome, as well as other expression control sequences, such as those described herein. For example, for AAV viral vectors, the packaging signals are 5 'Inverted Terminal Repeats (ITRs) and 3' ITRs. When packaged into an AAV capsid, the expression cassette and its flanking ITRs are referred to herein as the "recombinant AAV (rAAV) genome" or "vector genome.

Thus, in one aspect, there is provided an adeno-associated viral vector comprising an AAV capsid and at least one expression cassette, wherein the at least one expression cassette comprises a nucleic acid sequence encoding GLA and expression control sequences that direct expression of the GLA sequence in a host cell. The AAV vector further comprises an AAV ITR sequence. In one embodiment, the ITRs are from a different AAV than the one providing the capsid. In a preferred embodiment, the ITR sequence is from AAV9, or a deleted version thereof (Δitr), which may be used for convenience and to speed up regulatory approval (regulatory approval). ITRs from other AAV sources may be selected. When the source of the ITR is from AAV9 and the AAV capsid is from another AAV source, the resulting vector may be referred to as pseudotyped. Typically, the AAV vector genome comprises an AAV 5'itr (hGLA coding sequences and any regulatory sequences) and an AAV 3' itr. Other configurations of these elements may be suitable. Shortened versions of the 5' ITR (termed Δitr) have been described in which the D-sequence and terminal resolution sites (trs) are deleted. In other embodiments, full length AAV 5 'and 3' itrs are used.

Other expression cassettes may be generated using other synthetic hGLA coding sequences described herein and other expression control elements described herein.

The expression cassette typically contains a promoter sequence as part of the expression control sequence, for example, between the selected 5' ITR sequence and the hGLA coding sequence. Illustrative plasmids and vectors described herein use a vector containing a DC172 promoter, a DC190 promoter, or an LP1 promoter.

Other promoters such as constitutive promoters, regulatable promoters [ see, for example, WO 2011/126808 and WO2013/04943], or promoters responsive to physiological signals (cues) may be used in the vectors described herein. The promoter may be selected from different sources, such as the human Cytomegalovirus (CMV) immediate early enhancer/promoter, the SV40 early enhancer/promoter, the JC polymovirus promoter, myelin Basic Protein (MBP) or Glial Fibrillary Acidic Protein (GFAP) promoter, the herpes simplex virus (HSV-1) latency-associated promoter (LAP), the Rous Sarcoma Virus (RSV) Long Terminal Repeat (LTR) promoter, the neuron-specific promoter (NSE), the Platelet Derived Growth Factor (PDGF) promoter, hSYN, the Melanin Concentrating Hormone (MCH) promoter, CBA, the matrix metalloproteinase promoter (MPP) and the chicken beta actin promoter.

In addition to a promoter, the expression cassette and/or vector may contain one or more other suitable transcription initiation, termination, enhancer sequences, effective RNA processing signals such as splice and polyadenylation (polyA) signals; sequences that stabilize cytoplasmic mRNA, such as WPRE; sequences that enhance translation efficiency (i.e., kozak consensus sequences); a sequence that enhances protein stability; and, if desired, sequences which enhance secretion of the encoded product. Examples of suitable polyA sequences include, for example, SV40, SV50, bovine growth hormone (bGH), human growth hormone, and synthetic polyA. One example of a suitable enhancer is the CMV enhancer. Other suitable enhancers include those suitable for liver specificity. In one embodiment, the expression cassette comprises one or more expression enhancers. In one embodiment, the expression cassette contains two or more expression enhancers. These enhancers may be the same, or may be different from each other. For example, the enhancer may include the CMV immediate early enhancer. Such enhancers may be present in two copies located adjacent to each other. Or the two copies of the enhancer may be separated by one or more sequences. In yet another embodiment, the expression cassette further comprises an intron, such as a chicken beta actin intron. Other suitable introns include those known in the art, for example as described in WO 2011/126808. Optionally, one or more sequences may be selected to stabilize the mRNA. An example of such a sequence is a modified WPRE sequence, which can be engineered upstream of the polyA sequence and downstream of the coding sequence [ see, e.g., MA Zanta-Boussif et al, GENE THERAPY (2009) 16:605-619].

These control sequences are "operably linked" to hGLA gene sequences. The term "operably linked" as used herein refers to both expression control sequences that are contiguous with the gene of interest and expression control sequences that function in trans or at a distance to control the gene of interest.

Adeno-associated virus (AAV) viral vectors are AAV DNase resistant particles having an AAV protein capsid into which a nucleic acid sequence is packaged for delivery to a target cell. AAV capsids consist of 60 capsid protein subunits VP1, VP2 and VP3, which are symmetrically arranged in an icosahedral ratio of about 1:1:10 to 1:1:20, depending on the AAV chosen. The AAV capsid may be selected from those known in the art, including variants thereof. In one embodiment, the AAV capsid is selected from those AAV capsids effective to transduce liver cells. In one embodiment, the AAV capsid is selected from AAV1, AAV2, AAV7, AAV8, AAV9, aavrh.10, AAV5, aavhu.11, AAV8DJ, aavhu.32, aavhu.37, aavpi.2, aavrh.8, aavhu.48r3, and variants thereof, see Royo et al, brain Res,2008, month 1, 1190:15-22; Petrosyan et al, GENE THERAPY, month 12 in 2014, 21 (12): 991-1000; holehonnur et al, BMC Neuroscience,2014,15:28; and Cearley et al, mol Ther.2008, month 10, 16 (10): 1710-1718, each of which is incorporated herein by reference. Other AAV capsids useful herein include aavrh.39, aavrh.20, aavrh.25, AAV10, aavbb.1, and AAV bb.2, and variants thereof. As a source of capsids of AAV viral vectors (DNase resistant virions) other AAV serotypes may be selected, including for example AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV6.2, AAV7, AAV8, AAV9, rh10, AAVrh64R1, AAVrh64R2, rh8, rh.10, variants of any known or mentioned AAV or yet to be discovered AAV, see for example US2007/0036760A1; US2009/0197338A1; EP 1310571. See also WO 2003/042397 (AAV 7 and other simian AAV), US patent 7790449 and US patent 7282199 (AAV 8), WO2005/033321 and US 7,906,111 (AAV 9), and WO 2006/110689 and WO 2003/042397 (rh.10). Alternatively, recombinant AAV based on any of the AAV can be used as a source of AAV capsids. These documents also describe that other AAV may be selected for AAV production and are incorporated by reference herein. In some embodiments, an AAV capsid for the viral vector may be produced by mutagenesis (i.e., by insertion, deletion, or substitution) of one of the aforementioned AAV capsids or a nucleic acid encoding the same. In some embodiments, the AAV capsid is chimeric, comprising domains from two or three or four or more of the aforementioned AAV capsid proteins. In some embodiments, the AAV capsid is a chimera of Vpl, vp2, and Vp3 monomers from two or three different AAV or recombinant AAV. As used herein, the term variant, with respect to AAV, refers to any AAV sequence derived from a known AAV sequence, including sharing at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or greater sequence identity over an amino acid or nucleic acid sequence. In another embodiment, the AAV capsid comprises a variant comprising up to about 10% variation from any of the described or known AAV capsid sequences. That is, the AAV capsid shares about 90% to 99.9% identity, about 95% to 99% identity, or about 97% to 98% identity with an AAV capsid provided herein and/or known in the art. In one embodiment, the AAV capsid shares at least 95% identity with the AAV capsid. When determining the percent identity of AAV capsids, the comparison can be made for any variable protein (e.g., vp1, vp2, or vp 3). In one embodiment, the AAV capsid shares at least 95% identity with AAV8 vp 3. In one embodiment, self-complementary AAV is used. In another embodiment, single stranded AAV is used.

In one embodiment, the capsid is an AAV8 capsid or variant thereof. In another embodiment, the capsid is AAVX capsids or variants thereof.

In one embodiment, a self-complementary AAV is provided. The abbreviation "sc" in this context refers to the self-complementary type. "self-complementary AAV" refers to a construct wherein the coding region carried by the recombinant AAV nucleic acid sequence is designed to form an intramolecular double-stranded DNA template. Instead of waiting for cell-mediated synthesis of the second strand upon infection, the two complementary halves of the scAAV will associate to form one double stranded DNA (dsDNA) unit ready for immediate replication and transcription. See, e.g., D M MCCARTY et al, ,"Self-complementary recombinant adeno-associated virus(scAAV)vectorspromote efficient transduction independently of DNA synthesis",Gene Therapy(2001, 8), volume 8, 16, pages 1248-1254. Self-complementary AAV is described, for example, in U.S. patent No. 6,596,535;7,125,717 and 7,456,683, each of which is incorporated herein by reference in its entirety.

Methods of generating and isolating AAV viral vectors suitable for delivery to a subject are known in the art. See, for example, U.S. published patent application No. 2007/0036760 (2 months 15 days 2007); U.S. patent 7790449; U.S. patent 7282199; WO 2003/042397; WO 2005/033321; WO 2006/110689 and US 7588772 B2. In one system, production cell lines are transiently transfected with a construct encoding a transgene flanking the ITR and a construct encoding Rep and Cap. In the second system, packaging cell lines stably providing Rep and Cap were transiently transfected with constructs encoding transgenes flanking the ITRs. In each of these systems, AAV virions are produced in response to infection with a helper or herpes virus, wherein isolation of the rAAV from the contaminating virus is desired. Recently, systems have been developed that do not require infection with helper virus to recover AAV, which also provide the required helper functions in trans (i.e., adenovirus E1, E2a, VA and E4 or herpesvirus UL5, UL8, UL52 and UL29 and herpesvirus polymerase). In these newer systems, the helper functions may be provided by transiently transfecting the cell with a construct encoding the desired helper function, or the cell may be engineered to stably contain a gene encoding the helper function, the expression of which may be controlled at the transcriptional or post-transcriptional level. In yet another system, the ITR-flanked transgenes and rep/cap genes are introduced into insect cells by infection with a baculovirus-based vector. For an overview of these production systems, see, for example, zhang et al ,2009,"Adenovirus-adeno-associated virus hybrid for large-scale recombinant adeno-associated virus production",Human Gene Therapy 20:922-929,, the contents of each of which are incorporated herein by reference in their entirety. Methods of making and using these and other AAV production systems are also described in the following U.S. patents, the respective contents of which are incorporated herein by reference in their entirety ：5,139,941;5,741,683;6,057,152;6,204,059;6,268,213;6,491,907;6,660,514;6,951,753;7,094,604;7,172,893;7,201,898;7,229,823 and 7,439,065.

"Replication defective virus" or "viral vector" refers to a synthetic or artificial viral particle in which an expression cassette containing the relevant gene is packaged in a viral capsid or envelope, wherein any viral genomic sequence also packaged in the viral capsid or envelope is replication defective; i.e., they are unable to produce progeny virions, but retain the ability to infect target cells. In one embodiment, the genome of the viral vector does not include genes encoding enzymes required for replication (the genome may be engineered to be "content-free" —only containing the relevant transgenes flanking the signals required to amplify and package the artificial genome), but these genes may be provided during production. It is therefore considered safe for use in gene therapy because replication and infection by progeny virions do not occur except in the presence of viral enzymes required for replication. Such replication-defective viruses may be adeno-associated viruses (AAV), adenoviruses, lentiviruses (integrated or non-integrated), or another suitable viral source.

Typically, these delivery methods are designed to avoid direct systemic delivery of suspensions containing the AAV compositions described herein. Suitably, this may have the benefit of reduced dose, reduced toxicity and/or reduced unwanted immune response to AAV and/or transgene products compared to systemic administration.

Or other routes of administration (e.g., oral, inhalation, intranasal, intratracheal, intraarterial, intraocular, intravenous, intramuscular, and other parenteral (parental) routes) may be selected.

The hGLA delivery constructs described herein may be delivered in a single composition or in multiple compositions. Optionally, two or more different AAV may be delivered [ see, e.g., WO 2011/126808 and WO 2013/049493]. In another embodiment, such multiple viruses may contain different replication defective viruses (e.g., AAV, adenovirus, and/or lentivirus). Or delivery may be mediated by non-viral constructs, such as "naked DNA," "naked plasmid DNA," RNA, and mRNA; in combination with various delivery compositions and nanoparticles, including, for example, micelles, liposomes, cationic lipid-nucleic acid compositions, polysaccharide (poly-glycan) compositions and other polymers, lipid-based and/or cholesterol-nucleic acid conjugates, and other constructs as described herein. See, for example, X.Su et al, mol.pharmaceuticals, 2011,8 (3), pages 774-787; web publishing, 2011, 3 and 21 days; WO2013/182683, WO 2010/053572, and WO 2012/170930, all of which are incorporated herein by reference, such non-viral hGLA delivery constructs can be administered by the aforementioned routes.

The viral vectors, or non-viral DNA or RNA transfer portions, may be formulated with physiologically acceptable vectors for gene transfer and gene therapy applications. A variety of suitable purification methods may be selected. Examples of purification methods suitable for separating empty capsids from carrier particles are described, for example, in International patent application No. PCT/US16/65976 and its priority document filed on day 2016, 12, 9, U.S. patent application No. 62/322,098 filed on day 4, 13, and U.S. patent application No. 62/266,341 filed on day 2015, 12, 11, and entitled "Scalable Purification Method for AAV" which are incorporated herein by reference. See also the purification methods described in the following documents: international patent application No. PCT/US16/65974 filed on day 2016, 12, 9, and priority documents thereof, U.S. patent application No. 62/322,083 filed on day 2016, 4, 13, and 62/266,351 (AAV 1) filed on day 2015, 12, 11; international patent application No. PCT/US16/66013 filed 12/9/2016, U.S. provisional application No. 62/322,055 filed 4/13/2016 and U.S. provisional application No. 62/266,347 (AAVrh 10) filed 12/11/2015; and International patent application Ser. No. PCT/US16/65970, filed 12/9/2016, and priority applications thereof, U.S. provisional application Ser. Nos. 62/266,357 and 62/266,357 (AAV 9), which are incorporated herein by reference. Briefly, a two-step purification protocol is described that selectively captures and separates genomic containing rAAV vector particles from clarified concentrated supernatant of rAAV-producing cell culture. The method utilizes an affinity capture method performed at high salt concentration followed by an anion exchange resin method performed at high pH to provide rAAV vector particles that are substantially free of rAAV intermediates.

In the case of AAV viral vectors, quantification of viral genome (vg) can be used as a measure of the dose contained in the formulation. The dose of rAAV administered in the methods disclosed herein will vary depending upon, for example, the particular rAAV, mode of administration, therapeutic target, individual, and cell type targeted, and can be determined by standard methods in the art. Dosages may be expressed in units of viral genome (vg) (i.e., 1×10⁷vg、1×10⁸vg、1×10⁹vg、1×10¹⁰vg、1×10¹¹vg、1×10¹²vg、1×10¹³vg、1×l0¹⁴vg、1×10¹⁵vg). doses, respectively, may also be expressed in units of viral genome (vg) per kilogram (kg) of body weight) (i.e., methods for titrating AAV 1×10¹⁰vg/kg、1×10¹¹vg/kg、1×10¹²vg/kg、1×10¹³vg/kg、1×10¹⁴vg/kg、1×10¹⁵vg/kg)., respectively, are described in Clark et al, human gene therapy (hum. Genetherer.)) (1999; 10:1031-1039).

These above-described dosages may be administered in various volumes of carrier, excipient or buffer formulations ranging from about 25 to about 1000 microliters, including all numbers within the range, depending on the size of the area to be treated, the viral titer used, the route of administration, and the desired effect of the method. In one embodiment, the volume of carrier, excipient, or buffer is at least about 25 μl. In one embodiment, the volume is about 50 μl. In another embodiment, the volume is about 75 μl. In another embodiment, the volume is about 100. Mu.L. In another embodiment, the volume is about 125. Mu.L. In another embodiment, the volume is about 150. Mu.L. In another embodiment, the volume is about 175. Mu.L. In yet another embodiment, the volume is about 200 μl. In another embodiment, the volume is about 225. Mu.L. In yet another embodiment, the volume is about 250 μl. In yet another embodiment, the volume is about 275 μl. In yet another embodiment, the volume is about 300 μl. In yet another embodiment, the volume is about 325. Mu.L. In another embodiment, the volume is about 350 μl. In another embodiment, the volume is about 375. Mu.L. In another embodiment, the volume is about 400. Mu.L. In another embodiment, the volume is about 450 μl. In another embodiment, the volume is about 500 μl. In another embodiment, the volume is about 550. Mu.L. In another embodiment, the volume is about 600. Mu.L. In another embodiment, the volume is about 650 μl. In another embodiment, the volume is about 700. Mu.L. In another embodiment, the volume is between about 700 and 1000 μl.

The recombinant vectors described above may be delivered to host cells according to the disclosed methods. The rAAV, preferably suspended in a physiologically compatible carrier, may be administered to a human or non-human mammalian patient. In another embodiment, the composition comprises a carrier, diluent, excipient and/or adjuvant. Suitable vectors can be readily selected by those skilled in the art in view of the indication for which the transfer virus is intended. For example, suitable carriers include saline, which may be formulated with a variety of buffer solutions (e.g., phosphate buffered saline). Other exemplary carriers include sterile saline, lactose, sucrose, calcium phosphate, gelatin, dextran, agar, pectin, peanut oil, sesame oil and water. The buffer/carrier should include components that prevent the rAAV from attaching to the infusion tube but do not interfere with the binding activity in the rAAV.

Optionally, the compositions of the invention may contain other conventional pharmaceutical ingredients, such as preservatives or chemical stabilizers, in addition to the rAAV and carrier. Suitable exemplary preservatives include chlorobutanol, potassium sorbate, sorbic acid, sulfur dioxide, propyl gallate, parabens, ethyl vanillin, glycerol, phenol, and p-chlorophenol. Suitable chemical stabilizers include gelatin and albumin.

The viral vectors described herein can be used to prepare a medicament for delivering hGLA to a subject (e.g., a human patient) in need thereof, providing functional alpha-galactosidase a (GLA) to a subject, and/or treating fabry disease. The course of treatment may optionally include repeated administration of the same viral vector (e.g., AAVX vectors) or different viral vectors (e.g., AAVX and AAV 8). Other combinations may still be selected using the viral vectors and non-viral delivery systems described herein.

The hGLA cDNA sequences described herein may be generated synthetically in vivo using techniques well known in the art. For example, PCR-based accurate synthesis (PAS) using long DNA sequence methods can be used, as described by Xiong et al, PCR-basedaccurate synthesis of long DNA sequences, nature Protocols 1,791-797 (2006). Methods combining double asymmetric PCR and overlap extension PCR methods are described by Young and Dong, two-step totalgene synthesis method, nucleic Acids Res.2004;32 And (7) e59. See also Gordeeva et al ,JMicrobiol Methods.Improved PCR-based gene synthesis method and itsapplication to the Citrobacter freundii phytase gene codon modification.2010, month 5; 81 (2) 147-52.Epub, 3 months 10 days 2010; see also the following patents regarding oligonucleotide synthesis and Gene synthesis, gene seq.2012, month 4; 6 (1) 10-21; US 8008005 and US 7985565. Each of these documents is incorporated herein by reference. In addition, kits and protocols for generating DNA via PCR are commercially available. These include the use of polymerases, including but not limited to Taq polymerase;(New England Biolabs)； High-FIDELITY DNA polymerase (NEW ENGLAND Biolabs); and G2 polymerase (Promega). DNA may also be generated from cells transfected with plasmids containing hGLA sequences described herein. Kits and protocols are known and commercially available and include, but are not limited to, the QIAGEN plasmid kit; Pro Filter plasmid kit (Invitrogen); and Plasmid kit (SIGMA ALDRICH). Other techniques useful herein include sequence-specific isothermal amplification methods, which eliminate the need for thermal cycling. These methods typically use strand displacement DNA polymerases such as Bst DNA polymerase, large fragments (LARGE FRAGMENT) (NEW ENGLAND Biolabs) instead of heat to isolate duplex DNA. DNA can also be generated from RNA molecules by amplification via the use of Reverse Transcriptase (RT), which is an RNA-dependent DNA polymerase. RT polymerizes the DNA strand complementary to the original RNA template and is called cDNA. Such cDNA may then be further amplified by PCR or isothermal methods as described above. Custom DNA can also be generated by commercial sources, including but not limited to GenScript; (Life Technologies) and INTEGRATED DNATECHNOLOGIES.

The term "expression" is used herein in its broadest sense and includes the production of RNA or RNA and proteins. In the case of RNA, the term "expression" or "translation" relates in particular to the production of peptides or proteins. Expression may be transient or may be stable.

The term "translation" in the context of the present invention relates to a process at the ribosome, wherein the mRNA chain controls the assembly of amino acid sequences to produce proteins or peptides.

According to the present invention, a "therapeutically effective amount" as described herein refers to an amount effective to achieve a desired therapeutic result, e.g., to increase the level of functional alpha-galactosidase a in a subject (so as to result in the production of functional alpha-galactosidase a to a level sufficient to ameliorate symptoms of fabry disease) at a dosage and for a necessary period of time.

"Functional hGLA" refers to a gene encoding a native GLA protein, as shown in SEQ ID NO. 3, or another GLA protein that provides at least about 50%, at least about 75%, at least about 80%, at least about 90% or about the same, or more than 100% level of biological activity of the GLA protein that survives naturally, or a naturally occurring variant or polymorph thereof that is not associated with a disease. .

The activity of functional alpha-galactosidase a can be measured relatively easily and methods for determining alpha-galactosidase a activity are well known to those skilled in the art. The activity of alpha-galactosidase can be conveniently measured in blood using the blood spot method as described in Clin. Biochem.45 (15): 1233-8 (2012). The principle of the method is that under the acid pH value, alpha-galactosidase hydrolyzes a substrate 4-methylumbelliferyl-alpha-D-galactopyranoside into 4-methylumbelliferyl and galactose. The addition of an alkaline buffer can terminate the enzymatic reaction and cause 4-methylumbelliferone to fluoresce at a different wavelength than the unhydrolyzed substrate, allowing it to be measured in the presence of a substantial excess of unhydrolyzed substrate. In leukocytes, typically more than 95% of the α -galactosidase activity is α -galactosidase a, whereas in plasma and cultured cells, isozymes, α -galactosidase B can contribute significantly to the total α -galactosidase activity. Alpha-galactosidase a can be measured by utilizing the increased heat load of the a isozymes in the presence of alpha-galactosidase B, and the addition of alpha-galactosidase B in plasma can be inhibited by the addition of a-NAc galactosamine. A key advantage of this method is that only 5ul of dry spots of whole blood on the filter paper are required. This provides the advantage of measuring the α -galactosidase level in real time following vector administration as experiments are performed in fabry Ko mice.

Alpha-galactosidase a activity in plasma can be assessed in end-point hemorrhages in mice following gene transfer. The method is based on the following facts: as described above, the substrate 4-methylumbelliferone-alpha-D-galactopyranoside is hydrolysed by the alpha-galactosidase to 4-methylumbelliferone and galactose at an acidic pH. In addition, standard western blot analysis or standard (ELISA) immunoassays that show antigen levels can also be used to measure alpha-galactosidase.

The level of naturally occurring α -galactosidase a in a subject with fabry disease varies depending on the severity of fabry disease. Patients with severe forms of disease have a level of α -galactosidase a that is less than about 1% of the levels found in normal healthy subjects (referred to herein as "normal levels"). It has been found that when using the methods of treatment of the present invention, the level of functional alpha-galactosidase a can be increased to at least about 1% of normal levels. In some embodiments, the methods of treatment of the present invention result in an increase in the level of functional α -galactosidase a to at least about 2%, at least about 3%, at least about 4%, at least about 10%, about 15%, at least about 20%, or at least about 25% of normal levels. In particular embodiments, the methods of treatment of the invention result in an elevation of the level of functional α -galactosidase a to at least about 30% of normal levels.

In one embodiment, the methods of treatment of the present invention result in an elevation of the level of functional α -galactosidase a to at most normal levels.

In one embodiment, such functional GLA has a sequence with about 95% or greater identity to the native protein or to the full length sequence of the protein encoded by the gene set forth in SEQ ID NO. 3, or at the amino acid level has about 97% or greater, or about 99% or greater identity to the alpha-galactosidase set forth in SEQ ID NO. 4. Such functional GLA proteins may also include natural polymorphs. This identity can be determined by making an alignment of sequences and by using a variety of algorithms and/or computer programs known in the art or commercially available [ e.g., BLAST, exPASy; clustalO; FASTA; using, for example, needleman-Wunsch algorithm, smith-Waterman algorithm ].

It is noted that the terms "a" or "an" mean one or more. Thus, the terms "a" (or "an"), "one or more" and "at least one" can be used interchangeably herein.

The words "comprise" and "comprising" are to be interpreted as inclusive rather than exclusive. The word "composition (consist, consisting)" and variations thereof are to be interpreted as exclusive and not inclusive. Although various embodiments in the specification appear to use the language "comprising," in other instances, related embodiments are also intended to be interpreted and described using the language "consisting of … …" or "consisting essentially of … ….

The term "about" as used herein means 10% (±10%) from a given reference value, unless otherwise specified.

As used herein, "disease," "disorder," and "condition" are used interchangeably to indicate an abnormal condition in a subject.

Unless defined otherwise in the present specification, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art and by reference to the disclosure, this provides one of ordinary skill in the art with a general guidance on many terms used in the present application.

Detailed description of the preferred embodiments

In one aspect, the invention provides a recombinant adeno-associated virus (AAV) vector comprising a AAVX capsid and a vector genome, wherein the vector genome comprises a nucleic acid sequence encoding a functional α -galactosidase and an expression control sequence that directs the expression of the α -galactosidase sequence in a host cell.

Preferably, the AAVX capsid comprises the amino acid sequence shown in SEQ ID NO. 9.

Preferably, the nucleic acid sequence encoding the AAVX capsid comprises the nucleotide sequence shown in SEQ ID NO. 8.

Preferably, the nucleic acid sequence encoding the alpha-galactosidase comprises the sequence of SEQ ID NO. 3 or a nucleotide sequence having at least 80% identity thereto. More preferably, the nucleotide sequence having at least 80% identity to SEQ ID NO. 3 is a codon optimized sequence.

Preferably, the expression control sequence comprises a promoter; preferably, the promoter is an LP1 promoter; more preferably, the LP1 promoter sequence is the sequence set forth in SEQ ID No. 2.

Preferably, the vector further comprises one or more of introns, kozak sequences, polyA, WPRE and post transcriptional regulatory elements.

Preferably, the vector further comprises AAV Inverted Terminal Repeat (ITRs) sequences.

Preferably, the AAV Inverted Terminal Repeats (ITRs) are from AAV of different serotypes, preferably AAV2.

Recombinant adeno-associated virus (AAV) vectors according to the previous aspect, said vectors being selected from the group consisting of adeno-associated virus (AAV) vectors, adenovirus vectors, lentiviral vectors and hybrid virions, preferably said AAV vectors are AAV serotype 2 (AAV 2) vectors, preferably self-complementing adeno-associated virus (scAAV) vectors.

In one aspect, the invention provides an isolated host cell comprising the aforementioned recombinant adeno-associated virus (AAV) vector.

In one aspect, the invention provides a pharmaceutical composition comprising a recombinant adeno-associated virus (AAV) vector and/or host cell as described above, and a pharmaceutically acceptable excipient; preferably, it is formulated for intravenous administration.

In one aspect, the invention provides the use of the aforementioned recombinant adeno-associated virus (AAV) vector, host cell and/or pharmaceutical composition for the preparation of a medicament for preventing or treating fabry disease.

The use according to the previous aspect, wherein the recombinant adeno-associated virus (AAV) vector, host cell and/or pharmaceutical composition may be administered in combination with another therapy.

The use according to the previous aspect, wherein the recombinant adeno-associated virus (AAV) vector, host cell and/or pharmaceutical composition may be administered at a dose of about 1 x 10 ¹⁰ vg/kg to about 1 x 10 ¹⁶ vg/kg; preferably, the recombinant adeno-associated virus (AAV) vector may be administered at a dose of about 5 x 10 ¹³ vg/kg; preferably, the recombinant adeno-associated virus (AAV) vector or composition may be administered at a dose of about 2.5 x 10 ¹² vg/kg; preferably, the recombinant adeno-associated virus (AAV) vector or composition may be administered more than once.

In one aspect, the invention provides a method of treating fabry disease in a subject, the method comprising administering to a subject in need thereof a recombinant adeno-associated virus (AAV) vector, host cell, and/or pharmaceutical composition as described previously. Preferably, the subject is a mammal, more preferably, the subject is a human.

The following examples are illustrative only and are not intended to limit the invention.

EXAMPLE 1 vector construction

1.1 Construction of pSC-DC172-Gluc, pSC-DC190-Gluc and pSC-CMV-Gluc plasmids

DC172 promoter sequence fragment, DC190 promoter sequence fragment, CMV promoter sequence fragment and luciferase Gluc (Gaussia luciferase) sequence fragment with NotI and XbaI cleavage sites at both ends are synthesized respectively. The nucleotide sequence of the DC172 promoter sequence fragment is shown as SEQ ID NO. 1, and the nucleotide sequence of the DC190 promoter sequence fragment is shown as SEQ ID NO. 16.

The pSC-CMV-EGFP plasmid (shown in FIG. 1A) was digested with XhoI and XbaI, and ligated with the XhoI-NotI digested DC172 and the NotI-XbaI digested Gluc fragment to form pSC-DC172-Gluc plasmid (shown in FIG. 1B); ligating the XhoI and NotI double digested DC190 and the NotI and XbaI double digested Gluc fragment to form pSC-DC190-Gluc plasmid (shown in FIG. 1C); the pSC-CMV-EGFP plasmid (shown in FIG. 1A) was digested with XhoI and XbaI, and ligated with the double digested CMV fragment with XhoI and NotI and the double digested Gluc fragment with NotI and XbaI to form the pSC-CMV-Gluc plasmid (shown in FIG. 1D).

The above plasmids were packaged into double-stranded AAV viral vectors (scAAVs) and used for the first round of in-vivo promoter selection.

1.2 Construction of plasmid pSNAV2.0-DC172-GLA, pSNAV2.0-DC172-GLA-wpre, pSNAV2.0-LP1-GLA, and pSNAV2.0-LP1-GLA-wpre

LP1 promoter sequence fragments with XhoI and NotI cleavage sites at two ends are synthesized respectively, alpha-galactosidase A (GLA) nucleotide sequence fragments with NotI and SalI at two ends are contained respectively, and WPRE (wppre) sequence fragments of the post-transcriptional regulatory element of the woodchuck hepatitis virus with SalI at two ends are contained. Wherein the nucleotide sequence of the LP1 promoter sequence is shown as SEQ ID NO. 2; the GLA nucleotide fragment sequence is shown as SEQ ID NO. 3, and the amino acid sequence is shown as SEQ ID NO. 4; the nucleotide sequence of the WPRE sequence fragment is shown in SEQ ID NO. 5.

The pSNAV2.0-EGFP plasmid (shown in FIG. 1E) was digested with XhoI and SalI, and ligated with the XhoI-NotI-digested DC172 promoter sequence fragment and the NotI-SalI-digested GLA sequence fragment to form the pSNAV2.0-DC172-GLA plasmid (shown in FIG. 1F). Wherein, the nucleotide sequence of the DC172-GLA fragment after the DC172 promoter sequence fragment and the GLA sequence fragment are connected is shown in SEQ ID NO. 6.

The pSNAV2.0-DC172-GLA was singly digested with SalI and ligated with the SalI-digested WPRE fragment to form the pSNAV2.0-DC172-GLA-WPRE plasmid (shown in FIG. 1G).

PSNAV2.0-EGFP was digested with XhoI and SalI, and ligated with the fragment of LP1 promoter sequence digested with XhoI and NotI and the fragment of GLA digested with NotI and SalI to form pSNAV2.0-LP1-GLA plasmid (see FIG. 1H). Wherein, the nucleotide sequence of the LP1-GLA fragment after the connection of the LP1 promoter sequence fragment and the GLA sequence fragment is shown as SEQ ID NO. 7.

PSNAV2.0-LP1-GLA was singly digested with SalI and ligated with the SalI digested WPRE fragment to form pSNAV2.0-LP1-GLA-WPRE plasmid (shown in FIG. 1I).

The plasmids are packaged into single-chain AAV virus vectors (ssAAVs) and then are respectively used for screening promoter and expression regulatory elements in a second round.

Example 2 viral packaging and genome titre detection

This example uses HEK293 cells (purchased from ATCC under the designation CRL-1573) as a producer cell line and a conventional three-plasmid packaging system produces recombinant AAV viral vectors. The experimental methods used are all conventional in the art (see Xiao Xiao,Juan Li,and Richard Jude Samulski.Production of high-titer recombinant adeno-associated virus vectors in the absence of helper adenovirus.J.Virol.1998,72(3):2224).

An appropriate amount of purified AAV sample was taken, DNase I digestion reaction mixtures were prepared as described in the following Table (Table 1), incubated at 37℃for 30min, and incubated at 75℃for 10min to inactivate DNase I.

TABLE 1

AAV samples	5μl
		10 XDNase I buffer	5μl
DNase I	1μl
		RNase-free water	39μl
Totalizing	50μl

After the treated AAV purified samples were diluted by an appropriate factor, a Q-PCR reaction system was prepared with reference to the following Table (Table 2), and tested according to the following procedure.

TABLE 2

The primers used therein are described in the following table (Table 3):

TABLE 3 Table 3

Packaging yield results see table below (table 4):

TABLE 4 Table 4

AAV2/X means that the vector genome ITR of the recombinant virus is derived from AAV2 and the packaging shell is derived from AAVX; AAV2/8 means that the vector genome ITR of the recombinant virus is derived from AAV2 and the packaging envelope is derived from AAV8.

Example 3 selection of drug candidates

3.1 Selection of AAV vectors in candidate Agents

Selection of AAV vector targeting

AAV8 is the currently known viral vector with the highest efficiency for liver-targeted transduction. AAVX is a vector obtained by the method of DNA shuffling (DNA shuffling), and this example examined whether AAVX has stronger targeting than AAV8 by in vitro and in vivo liver targeting studies in humans or monkeys.

Comparison of infection efficiency of 3.1.1AAV2/X and AAV2/8 vectors on different human hepatocyte lines

The infection efficiency of the recombinant viruses scAAV2/8-CMV-EGFP and scAAV2/X-CMV-EGFP on 5 human hepatocyte lines (Huh 6, 7402, huh7, hepG2 and 7721, wherein Huh6 was obtained from the upper biosciences biotechnology limited, 7402 and 7721 were obtained from beijing s a derivative, huh7 was obtained from the institute of tumor at the national academy of medical science, and HepG2 was obtained from 301 hospitals) was first examined by flow cytometry, and the evaluation method was adopted to compare the difference in MOI used by a fold with equal infection efficiency of two AAVs on the same cell line. The specific results are shown in FIG. 2. Wherein, the encoding nucleotide sequence of the packaging shell of the AAV2/X related recombinant virus is shown as SEQ ID NO. 8, and the amino acid sequence is shown as SEQ ID NO. 9.

On various human liver cell lines, the infection efficiency of the recombinant virus scAAV2/X-CMV-EGFP is obviously higher than that of the recombinant virus scAAV2/8-CMV-EGFP; when the two viruses are of comparable efficiency, the recombinant virus scAAV2/8-CMV-EGFP infects the human liver Huh-6 cell line with an MOI of about 3 times that of scAAV 2/X-CMV-EGFP; the MOI used by recombinant virus scaV 2/8-CMV-EGFP on human liver 7402 cells was about 10 times that of recombinant virus scaV 2/X-CMV-EGFP; the MOI of the recombinant virus scaV 2/8-CMV-EGFP used when infecting human liver Huh-7 cells is about 100-300 times that of the recombinant virus scaV 2/X-CMV-EGFP; the MOI of the recombinant virus scaV 2/8-CMV-EGFP used when infecting human liver HepG2 cells is about 30-100 times that of the recombinant virus scaV 2/X-CMV-EGFP; the MOI used by the recombinant virus scaV 2/8-CMV-EGFP when infecting human liver 7721 cells was about 30-100 times that of the recombinant virus scaV 2/X-CMV-EGFP. As can be seen, the infection efficiency of the recombinant virus scAAV2/X-CMV-EGFP is far higher than that of the recombinant virus scAAV2/8-CMV-EGFP on various human liver cell lines.

3.1.2 Comparison of infection efficiencies of recombinant viral vectors AAV2/X and AAV2/8 on different human liver primary cells

After demonstrating high transduction efficiency of recombinant viral vector AAV2/X on cell lines, primary hepatocyte transduction experiments of HBV patients were performed. The isolated 5 primary liver cancer cells confirmed by PCR detection are respectively named as follows: HCC307N1, HCC061A2, HCC213F1, HCC893D1, HCC554A4. Cell-corresponding patient information is as shown in table 5:

TABLE 5

HCC indicates liver cancer

And 5, separating and culturing liver cancer primary cells, and respectively infecting the liver cancer primary cells by using recombinant virus scAAV2/8-CMV-EGFP and recombinant virus scAAV 2/X-CMV-EGFP. For HCC307N1 cells, the two recombinant viruses were infected with the same MOI, which was 5000, 15000, 50000,150000, 500000, respectively; for HCC061A2 cells, scAAV2/8-CMV-EGFP adopts MOI of 5000, 15000, 50000,150000, 500000; the MOI of scAAV2/X-CMV-EGFP is 500, 1500, 5000, 15000, 50000; for the remaining 3 cells, 2 viruses were infected with the same MOI of 500, 1500, 5000, 15000, 50000,150000, 500000, respectively, 3 wells were made per MOI. After 48h, the fluorescence microscope was used to take pictures and the GFP positive rate and fluorescence intensity were measured by flow.

As shown in FIG. 3, under the same MOI condition, the positive rate of the recombinant virus scAAV2/X-CMV-EGFP infection is higher than that of the recombinant virus scAAV2/8-CMV-EGFP infection, and the fluorescence is stronger.

By combining the experimental results of the human liver cell line and the experimental results of the primary liver cells, the targeting of the recombinant viral vector AAV2/X to the human liver cells in vitro experiments can be fully proved to have more advantages than the targeting of the recombinant viral vector AAV 2/8.

In vivo targeting comparison of 3.1.3AAV2/X and AAV2/8 vectors in monkeys

6 Cynomolgus monkeys are selected, and the male and female monkeys are divided into 2 groups, namely a scAAV2/8-CMV-EGFP group and a scAAV2/X-CMV-EGFP group. The administration dosage is 1E+12vg/kg, the administration mode is intravenous infusion, and the administration frequency is single administration. All animals were euthanized 7 days after the administration, and tissues of heart, lung, liver, brain, testis, ovary, biceps femoris, stomach, jejunum, kidney, and spleen were collected, and GFP protein expression in each tissue was observed by fluorescence microscopy.

The results of fluorescence microscopy of animal tissues are shown in fig. 4, 5 and table 6, and the results show that GFP protein expression was observed only in liver tissues of each group of animals, but not in the remaining tissues (heart, lung, brain, testis, ovary, biceps femoris, stomach, jejunum, kidney, spleen). Wherein the average GFP positive cell number in liver tissues of 3 animals in the recombinant virus scaAAV 2/8-CMV-EGFP group is 15.00+ -4.47, 8.20+ -2.39, 8.00+ -5.83/visual field, respectively, and the average GFP positive cell number in liver tissues of 3 animals in the recombinant virus scaAAV 2/X-CMV-EGFP group is 123.40 + -8.02, 79.80+ -23.06, 54.40+ -28.01/visual field, respectively. Furthermore, there was no significant difference in GFP protein expression between liver tissues from the liver of the same individual. The result of statistical analysis shows that the number of GFP protein positive cells in liver tissues of the recombinant virus scAAV2/X-CMV-EGFP group animals is obviously higher than that of the recombinant virus scAAV2/8-CMV-EGFP group animals. The in vitro and in vivo experimental results show that AAV2/X has better targeting to liver than AAV2/8 in vitro and in vivo.

TABLE 6 fluorescence microscopy of GFP-positive cells in liver tissue (individual/field of view)

(II) detection of neutralizing antibody level against AAV vector in human or monkey serum

3.1.4 Comparison of AAV2/X and AAV2/8 neutralizing antibody levels in monkey serum

The experiment uses a fixed viral infection index (MOI), the serum is subjected to multiple ratio dilution, and the level of neutralizing antibodies against AAV2/8 and AAV2/X in the cynomolgus monkey serum is compared. In a series of serum dilutions, a dilution was selected in which the efficiency of infection of cells by serum virus mixtures reached 50% of the efficiency of infection of cells by viruses in the absence of serum, the reciprocal of this dilution was used as the amount of neutralizing antibodies, and the level of neutralizing antibodies was evaluated for each viral vector (see Lochrie MA et al, 2006,Virology 353:68-82; mori S et al, 2006,Jpn J Infect Dis 59:285-293 et al). In the experiment, 12 cynomolgus monkey serum is detected, serial dilutions are carried out on the serum, and the levels of neutralizing antibodies against recombinant virus AAV2/8 and neutralizing antibodies against recombinant virus AAV2/X in each sample are respectively judged and compared.

The experimental procedure was as follows: 7402 inoculating 24-well plate, serial gradient diluting of cynomolgus monkey serum sample, mixing serial diluted serum sample with virus liquid 1:1, incubating at 37deg.C for 1 hr, adding mixed liquid of serum sample and virus liquid or virus liquid into cells, and infecting cells, wherein MOI of scaV 2/8-CMV-EGFP and scaV 2/X-CMV-EGFP virus infection is 2000. Cells were harvested 48h later and the infection efficiency was examined by flow cytometry.

The results of the test are shown in Table 7, and the serum sample was diluted in 4 gradients in a range of 5-100 times, and when the amount of neutralizing antibody was less than 5, the neutralizing antibody against the virus was regarded as negative. The 12 serum samples are numbered 1#, 2#, 3#, 4#, 5#, 6#, 7#, 8#, 9#, 10#, 11#, 12#, respectively. Wherein neutralizing antibodies for two virus vectors in the sample # 1, the sample # 2 and the sample # 4 are negative; the neutralizing antibody level of the 3# serum sample and the 12# serum sample against the recombinant virus scAAV2/8 is higher, and the neutralizing antibody against the recombinant virus scAAV2/X is negative; the level of neutralizing antibodies against both recombinant viruses was lower in sample # 7; the neutralizing antibody levels against recombinant virus scAAV2/X in sample # 5, # 6, # 8, # 9, # 10, # 11 were significantly lower than the neutralizing antibody levels against recombinant virus scAAV 2/8.

TABLE 7 detection results of neutralizing antibodies in 12 serum of cynomolgus monkey

Serum numbering	AAV2/X-Nab	AAV2/8-Nab
			1#	＜5	＜5
2#	＜5	＜5
			3#	≤5	＞100
4#	＜5	＜5
			5#	10-50	＞100
6#	10-50	＞100
			7#	10	10
8#	10-50	＞100
			9#	50-100	＞100
10#	50-100	＞100
			11#	50-100	＞100
12#	＜5	10-50

Thus, by comprehensive comparison of AAV2/X and AAV2/8 neutralizing antibody levels in cynomolgus monkey serum, neutralizing antibodies to AAV2/X were significantly lower in the monkey population than neutralizing antibodies to AAV 2/8. Therefore, the recombinant virus AAV2/X is used as a drug delivery carrier, has lower immunogenicity, and is favorable for better playing the therapeutic effect of the drug.

3.1.5 Comparison of neutralizing antibody levels against recombinant viral AAV2/X and recombinant viral AAV2/8 in human serum

The experiment is carried out for 20 human serum detection altogether, serial dilutions are carried out on serum, the cell infection efficiency of serum virus mixture is selected from a series of serum dilutions to reach the dilution of 50% of the cell infection efficiency of virus in no serum, the reciprocal of the dilution is used as the neutralizing antibody amount, and the neutralizing antibody level of recombinant virus AAV2/8 and the neutralizing antibody level of recombinant virus AAV2/X in each sample are respectively evaluated.

The experimental procedure was as follows: human liver 7402 cell line is inoculated into 24 hole plate, human serum sample is serially diluted, two recombinant viruses of scAAV2/8-CMV-EGFP and scAAV2/X-CMV-EGFP adopt virus infection index (MOI) as 2000, serial diluted serum sample is mixed with virus liquid 1:1, after mixing, the mixture of serum sample and virus liquid or virus liquid is added into cells for infecting the cells, and incubation is carried out for 1h at 37 ℃. Cells were harvested 48h later and the infection efficiency was examined by flow cytometry.

The results of the assay are shown in Table 8 and FIG. 6, wherein in 9 samples (2#, 5#, 6#, 7#, 9#, 15#, 16#, 17#, 20#) a higher level of neutralizing antibodies against recombinant virus AAV2/8 than against recombinant virus AAV2/X was observed by assaying 20 healthy human serum for neutralizing antibodies; in 4 samples (4#, 13#, 14#, 18#) the level of neutralizing antibodies against recombinant viral AAV2/X was higher than the level of neutralizing antibodies against recombinant viral AAV 2/8; in 3 samples (1 #, 3#, 8 #) the level of neutralizing antibodies against recombinant viral AAV2/8 was comparable to the level of neutralizing antibodies against recombinant viral AAV 2/X; the 4 samples (10#, 11#, 12#, 19#) were negative for the neutralizing antibody levels of both viruses.

The experiment randomly selects 20 human serum to detect the neutralizing antibody level, and has colony. Experimental results prove that in human population, the neutralizing antibodies against the recombinant virus AAV2/X are fewer, and compared with the recombinant virus AAV2/8, the recombinant virus AAV2/X is more suitable for being used as a drug delivery carrier, so that the reactivity can be reduced, and the efficacy is exerted.

TABLE 8 human serum neutralizing antibody titres of 20 parts

3.2 Selection of promoters in candidate Agents

In order to select a suitable promoter, the present invention uses three different promoters (CMV promoter, DC172 promoter, DC190 promoter) for vector construction in the first stage, the vector main structure is shown in FIG. 7J, and the recombinant virus scaAAV 2/X-CMV-Gluc, recombinant virus scaAAV 2/X-DC172-Gluc, recombinant virus scaAAV 2/X-DC190-Gluc are packaged, and then comparison of expression amounts is performed in mice.

Normal 129 mice were selected as recipients, three dosing groups were set up, one for recombinant virus scAAV2/X-CMV-Gluc, recombinant virus scAAV2/X-DC172-Gluc, and recombinant virus scAAV2/X-DC190-Gluc, respectively, and PBS dosing control groups were set up for a total of 3 experimental animals each. The administration dose of the viral vector is 3E+10vg/patient, and after tail vein injection, tail cutting and blood sampling are respectively carried out at 1,2, 3 and 4 weeks, and the viral vector is used for luciferase activity test, and the specific experimental method refers to the description of a Ganing organism Gaussia Luciferase detection kit.

The results of the first round of in-promoter comparisons are shown in FIG. 8A, with all PBS injected groups showing negative. The comparison result of each detection point shows that the expression level of Gluc genes in the recombinant virus scAAV2/X-DC172-Gluc administration group is highest, the recombinant virus scAAV2/X-DC190-Gluc administration group is next, and the expression level of exogenous genes is started to be lowest, namely, the recombinant virus scAAV2/X-CMV-Gluc administration group.

In the second stage, the DC172 promoter and the LP1 promoter are respectively subjected to vector construction, the main structure of the vector is shown in figure 7K, and the vector is packaged into recombinant viruses ssAAV/X-DC 172-GLA and ssAAV/X-LP 1-GLA, and then a second in vivo comparison experiment is carried out.

Normal mice were selected as recipients, two dosing groups were set up, recombinant virus ssAAV/X-DC 172-GLA and recombinant virus ssAAV/X-LP 1-GLA, respectively, and PBS dosing control groups were set up, with a total of 3 experimental animals per group. The dose of the viral vector is 1E+15vg/patient, and after tail vein injection, tail blood is collected after 2, 3, 4, 5, 6, 7 and 8 weeks, and GLA enzyme activity test is carried out by a substrate fluorescence method. To a 96 Kong Yingguang plate, 10. Mu.l of serum sample to be tested was added, and 40. Mu.l of substrate (5 mM 4-methylumbelliferone-. Alpha. -D-galactoside (ACROS, 337162500) and 100mM N-acetyl-D-galactosamine (Sigma, A2795)) were added and mixed well, and after incubation at 37℃for 1 hour in the absence of light, the reaction was stopped with 0.3 Mglycine-NaOH. Fluorescence readings were performed using different molar concentrations of 4-MU (4-Methylumbelliferone tetramethyl umbelliferone) (Sigma, M1381) standard as quantitative indicators. And drawing a standard curve by using a standard substance, and calculating enzyme activity values of samples at different time points. As a result, as shown in FIG. 8B, the enzyme activity of GLA in recombinant virus ssAAV/X-LP 1-GLA was higher than that of GLA in recombinant virus ssAAV/X-DC 172-GLA at each sampling time point. Thus, the LP1 promoter is selected as the promoter element of the drug candidate.

3.3 Selection of expression regulatory elements in candidate Agents

To confirm the effect of the expression regulatory element, vectors carrying WPRE regulatory elements and vectors not carrying WPRE regulatory elements were constructed and packaged. The main structure of the vector without the WPRE regulatory element is shown in figure 7K, the main structure of the vector with the WPRE regulatory element is shown in figure 9, and the vector is constructed and packaged into recombinant virus ssAAV/X-LP 1-GLA-WPRE.

Normal 129 mice were selected as recipients, 2 dosing groups were set up, ssAAV2/X-LP1-GLA and ssAAV/X-LP 1-GLA-wpre dosing groups, and PBS dosing control groups were set up, for a total of 3 experimental animals per group. The doses of the viral vectors were 3E+10vg/dose, and after tail vein injection, ssAAV2/X-LP1-GLA and ssAAV/X-LP 1-GLA-wpre dosing groups were subjected to tail-clipping blood collection at 2, 3, 4, 5, 6, 7, and 8 weeks for GLA enzyme activity testing.

As shown in FIG. 10, ssAAV2/X-LP1-GLA and ssAAV2/X-LP1-GLA-WPRE experimental groups showed that the expression of foreign genes was improved after WPRE addition, which was only 1.5-2 times that of the viral vector without WPRE.

Example 4 detection of candidate drug in vitro enzymatic Activity

Isolation and culture of murine primary hepatocytes, see literature (Seglen P O.preparation of isolated RAT LIVE CELLS [ J ]. Methods Cell biol.1976, 13:29-83), after adherent culture of isolated murine primary hepatocytes for 16-20h, 1 well Cell count was digested, medium was discarded from each well of cells, and medium was discarded after rinsing the cells 2 times with 2ml of serum-free medium (OPT-MEM). Dilutions of recombinant viruses ssAAV/X-LP 1-GLA virus of different MOI (5, 10, 20, 40 ten thousand respectively) were added to 6-well plates (1 mL of infection volume) while serum-free medium controls without virus were placed and incubated in a 5% CO ₂ 37 ℃incubator. After 5-7h of virus infection, the virus solution was aspirated, cells were washed 1-2 times with 2mL of serum-free medium (OPT-MEM) per well, 2mL of hepatocyte maintenance medium was added per well, and the culture was continued in a 5% CO ₂ incubator at 37 ℃. After 48h of virus infection, cell culture supernatants were collected and cells were collected with a live assay (ph=4.6, 28mM citric acid+44 mM disodium hydrogen phosphate). After three quick-frozen and instant-dissolving of the cells, the protein concentration was detected by BCA kit. The substrate fluorescence method is used for measuring the activity of the intracellular and extracellular alpha-galactosidase A. The results are shown in FIG. 11, where intracellular (FIG. 11A) and extracellular (FIG. 11B) enzyme activities are increasing with the multiplication of the selected infection MOI, and have a pronounced dose-dependent relationship.

Example 5 in vivo pharmacodynamics experiments with candidate drugs

5.1 Comparison of the enzymatic Activity of alpha-galactosidase A in different tissues after administration of recombinant viruses ssAAV2/X-LP1-GLA and ssAAV2/8-LP1-GLA

Animal experiments were performed in a total of two dosing groups and two control groups, each dosing group containing 3 female model mice and 3 male model mice, the model mice being GLA-deficient model mice (Ohshima T et al, proc NATL ACAD SCI U S A (1997). 94 (6): 2540-2544). The two control groups were a wild mouse control group and a blank model mouse (homozygote) control group, respectively. The two doses were injected with 1 E+10vg/dose of recombinant virus ssAAV/X-LP 1-GLA or recombinant virus ssAAV/8-LP 1-GLA, respectively. All groups of animals were sacrificed 7 days after dosing and serum, liver tissue, heart tissue and kidney tissue were extracted. Centrifuging at 4deg.C for 30min after tissue homogenization, centrifuging at 4deg.C for 10min again, centrifuging for the last time, taking out supernatant, and measuring protein concentration by BCA. Serum and tissue alpha-galactosidase A (alpha-Gal A) activity was measured using substrate fluorescence.

The results of GLA enzyme activity detection of serum and different tissues are shown in fig. 12A: with the same dose administration, the level of enzyme activity of α -galactosidase A in the two recombinant viruses ssAAV/X-LP 1-GLA and ssAAV/8-LP 1-GLA administered groups was higher than that of α -galactosidase A in the wild-type control group in serum, liver, kidney, heart tissues, and was found to be higher in the recombinant virus ssAAV/X-LP 1-GLA administered group than in the ssAAV/8-LP 1-GLA administered group. As can be seen from FIG. 12A, after systemic administration, the serum and liver levels of GLA enzyme activity were significantly higher than those of the kidney and heart, indicating that the drug reached less by blood circulation to the kidney and heart, but still higher than that of the GLA enzyme activity in wild-type mice, indicating that recombinant viruses ssAAV/X-LP 1-GLA and ssAAV/8-LP 1-GLA were sufficient to express normal levels of active GLA in the kidney or other affected organs, and that the GLA expression level or enzyme activity level in the recombinant virus ssAAV/X-LP 1-GLA group was significantly higher than that in the recombinant virus sAAV/8-LP 1-GLA group.

5.2 GLA enzymatic Activity analysis of different tissues after administration of recombinant viral AAV2/X vector drug

In vivo animal experiments were tested for the relationship between dose and GLA expression levels, three dose groups were set up, each containing 3 female model mice and 3 male model mice, injected with recombinant virus ssAAV/X-LP 1-GLA at 1E+9vg/alone, 3E+9vg/alone, and 1E+10vg/alone, respectively. Two control groups were set, a wild-type mouse control group and a blank model mouse control group (model mouse), respectively. All groups of animals were sacrificed 7 days after tail vein administration and serum, liver tissue, heart tissue, and kidney tissue were extracted. The tissue homogenization method is described above. After extraction of different histones, BCA measured protein concentration and substrate fluorescence measured serum and tissue α -galactosidase a (α -Gal a) activity.

The results of GLA enzyme activity detection of serum and different tissues are shown in fig. 12B: serum enzyme activity assay results showed that GLA enzyme activity levels increased with increasing doses administered, at low doses, i.e. 1e+9vg, GLA enzyme activity levels were already higher than those of wild-type mice; liver enzyme activity test results show that the GLA enzyme activity level increases with the increase of the administration dosage, and the GLA enzyme activity level is equivalent to that of a wild-type mouse when the low dosage, namely 1E+9vg is used; the kidney enzyme activity test result shows that the GLA enzyme activity level is very low at low and medium doses, and is obviously higher than that of a wild-type mouse at high doses, namely 1E+10vg; the results of the cardiac enzyme activity assay showed that the GLA enzyme activity level increased with increasing doses administered, i.e. 3e+9vg, and that the GLA enzyme activity level was consistent with that of wild-type mice.

Kidney involvement is a prominent feature of fabry disease, caused by the accumulation of neutral glycosphingolipids (mainly ceramide trihexose glycosides) (Gb 3). Thus, the ultrastructure of the mouse kidney parenchyma was evaluated by high resolution electron microscopy. In untreated fabry model mice, podocytes form podophy fusion, gb3 accumulates, filter the split pores to form multiple vesicles and degrade, and the split pore membrane forms a complex. When this occurs, proteinuria and glomerulosclerosis may develop. Following administration of recombinant virus ssAAV/X-LP 1-GLA, a substantial reduction in lipid accumulation throughout the kidney parenchyma was observed in model mice at high doses of 1E+10vg, with the kidney structure tending to normal (not shown). Thus, the ultrastructural findings of fewer, smaller or less dense lysosomes in kidney tissues of all mice in the dosing group showed that both accumulated Gb3 could be consumed and re-accumulation in mice prevented at 1e+10vg dose levels.

Sequence listing

<110> Shu Taishen (Beijing) biopharmaceutical Co., ltd; beijing Sannojiyi biotechnology Limited liability company

<120> Adeno-associated viral vectors for the treatment of brile disease and uses thereof

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115 120 125

Leu Lys Leu Gly Ile Tyr Ala Asp Val Gly Asn Lys Thr Cys Ala Gly

130 135 140

Phe Pro Gly Ser Phe Gly Tyr Tyr Asp Ile Asp Ala Gln Thr Phe Ala

145 150 155 160

Asp Trp Gly Val Asp Leu Leu Lys Phe Asp Gly Cys Tyr Cys Asp Ser

165 170 175

Leu Glu Asn Leu Ala Asp Gly Tyr Lys His Met Ser Leu Ala Leu Asn

180 185 190

Arg Thr Gly Arg Ser Ile Val Tyr Ser Cys Glu Trp Pro Leu Tyr Met

195 200 205

Trp Pro Phe Gln Lys Pro Asn Tyr Thr Glu Ile Arg Gln Tyr Cys Asn

210 215 220

His Trp Arg Asn Phe Ala Asp Ile Asp Asp Ser Trp Lys Ser Ile Lys

225 230 235 240

Ser Ile Leu Asp Trp Thr Ser Phe Asn Gln Glu Arg Ile Val Asp Val

245 250 255

Ala Gly Pro Gly Gly Trp Asn Asp Pro Asp Met Leu Val Ile Gly Asn

260 265 270

Phe Gly Leu Ser Trp Asn Gln Gln Val Thr Gln Met Ala Leu Trp Ala

275 280 285

Ile Met Ala Ala Pro Leu Phe Met Ser Asn Asp Leu Arg His Ile Ser

290 295 300

Pro Gln Ala Lys Ala Leu Leu Gln Asp Lys Asp Val Ile Ala Ile Asn

305 310 315 320

Gln Asp Pro Leu Gly Lys Gln Gly Tyr Gln Leu Arg Gln Gly Asp Asn

325 330 335

Phe Glu Val Trp Glu Arg Pro Leu Ser Gly Leu Ala Trp Ala Val Ala

340 345 350

Met Ile Asn Arg Gln Glu Ile Gly Gly Pro Arg Ser Tyr Thr Ile Ala

355 360 365

Val Ala Ser Leu Gly Lys Gly Val Ala Cys Asn Pro Ala Cys Phe Ile

370 375 380

Thr Gln Leu Leu Pro Val Lys Arg Lys Leu Gly Phe Tyr Glu Trp Thr

385 390 395 400

Ser Arg Leu Arg Ser His Ile Asn Pro Thr Gly Thr Val Leu Leu Gln

405 410 415

Leu Glu Asn Thr Met Gln Met Ser Leu Lys Asp Leu Leu

420 425

<210> 5

<211> 610

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 5

gtcgacaccg gttagtaatg atcgacaatc aacctctgga ttacaaaatt tgtgaaagat 60

tgactggtat tcttaactat gttgctcctt ttacgctatg tggatacgct gctttaatgc 120

ctttgtatca tgctattgct tcccgtatgg ctttcatttt ctcctccttg tataaatcct 180

ggttgctgtc tctttatgag gagttgtggc ccgttgtcag gcaacgtggc gtggtgtgca 240

ctgtgtttgc tgacgcaacc cccactggtt ggggcattgc caccacctgt cagctccttt 300

ccgggacttt cgctttcccc ctccctattg ccacggcgga actcatcgcc gcctgccttg 360

cccgctgctg gacaggggct cggctgttgg gcactgacaa ttccgtggtg ttgtcgggga 420

agctgacgtc ctttccatgg ctgctcgcct gtgttgccac ctggattctg cgcgggacgt 480

ccttctgcta cgtcccttcg gccctcaatc cagcggacct tccttcccgc ggcctgctgc 540

cggctctgcg gcctcttccg cgtcttcgcc ttcgccctca gacgagtcgg atctcccttt 600

gggccgcctc 610

<210> 6

<211> 2595

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 6

gaattggaga tcggtacttc gcgaatgcgt cgagttaatt tttaaaaagc agtcaaaagt 60

ccaagtggcc cttggcagca tttactctct ctgtttgctc tggttaataa tctcaggagc 120

acaaacattc ctggaggcag gagaagaaat caacatcctg gacttatcct ctgggcctct 180

ccccaccccc aggagaggct gtgcaactgt taatttttaa aaagcagtca aaagtccaag 240

tggcccttgg cagcatttac tctctctgtt tgctctggtt aataatctca ggagcacaaa 300

cattcctgga ggcaggagaa gaaatcaaca tcctggactt atcctctggg cctctcccca 360

cccccaggag aggctgtgca actggatcca ggcctgaggc tggtcaaaat tgaacctcct 420

cctgctctga gcagcctggg gggcagacta agcagagggc tgtgcagacc cacataaaga 480

gcctactgtg tgccaggcac ttcacccgag gcacttcaca agcatgcttg ggaatgaaac 540

ttccaactct ttgggatgca ggtgaaacag ttcctggttc agagaggtga agcggcctgc 600

ctgaggcagc acagctcttc tttacagatg tgcttcccca cctctaccct gtctcacggc 660

cccccatgcc agcctgacgg ttgtgtctgc ctcagtcatg ctccattttt ccatcgggac 720

catcaagagg gtgtttgtgt ctaaggctga ctgggtaact ttggatgagc ggtctctccg 780

ctctgagcct gtttcctcat ctgtcaaatg ggctctaacc cactctgatc tcccagggcg 840

gcagtaagtc ttcagcatca ggcattttgg ggtgactcag taaatggtag atcttgctac 900

cagtggaaca gccactaagg attctgcagt gagagcagag ggccagctaa gtggtactct 960

cccagagact gtctgactca cgccaccccc tccaccttgg acacaggacg ctgtggtttc 1020

tgagccaggt acaatgactc ctttcggtaa gtgcagtgga agctgtacac tgcccaggca 1080

aagcgtccgg gcagcgtagg cgggcgactc agatcccagc cagtggactt agcccctgtt 1140

tgctcctccg ataactgggg tgaccttggt taatattcac cagcagcctc ccccgttgcc 1200

cctctggatc cactgcttaa atacggacga ggacagggcc ctgtctcctc agcttcaggc 1260

accaccactg acctgggaca gtgaatcgcg gccgcatatg ccaccatgca gctgaggaac 1320

ccagaactac atctgggctg cgcgcttgcg cttcgcttcc tggccctcgt ttcctgggac 1380

atccctgggg ctagagcact ggacaatgga ttggcaagga cgcctaccat gggctggctg 1440

cactgggagc gcttcatgtg caaccttgac tgccaggaag agccagattc ctgcatcagt 1500

gagaagctct tcatggagat ggcagagctc atggtctcag aaggctggaa ggatgcaggt 1560

tatgagtacc tctgcattga tgactgttgg atggctcccc aaagagattc agaaggcaga 1620

cttcaggcag accctcagcg ctttcctcat gggattcgcc agctagctaa ttatgttcac 1680

agcaaaggac tgaagctagg gatttatgca gatgttggaa ataaaacctg cgcaggcttc 1740

cctgggagtt ttggatacta cgacattgat gcccagacct ttgctgactg gggagtagat 1800

ctgctaaaat ttgatggttg ttactgtgac agtttggaaa atttggcaga tggttataag 1860

cacatgtcct tggccctgaa taggactggc agaagcattg tgtactcctg tgagtggcct 1920

ctttatatgt ggccctttca aaagcccaat tatacagaaa tccgacagta ctgcaatcac 1980

tggcgaaatt ttgctgacat tgatgattcc tggaaaagta taaagagtat cttggactgg 2040

acatctttta accaggagag aattgttgat gttgctggac cagggggttg gaatgaccca 2100

gatatgttag tgattggcaa ctttggcctc agctggaatc agcaagtaac tcagatggcc 2160

ctctgggcta tcatggctgc tcctttattc atgtctaatg acctccgaca catcagccct 2220

caagccaaag ctctccttca ggataaggac gtaattgcca tcaatcagga ccccttgggc 2280

aagcaagggt accagcttag acagggagac aactttgaag tgtgggaacg acctctctca 2340

ggcttagcct gggctgtagc tatgataaac cggcaggaga ttggtggacc tcgctcttat 2400

accatcgcag ttgcttccct gggtaaagga gtggcctgta atcctgcctg cttcatcaca 2460

cagctcctcc ctgtgaaaag gaagctaggg ttctatgaat ggacttcaag gttaagaagt 2520

cacataaatc ccacaggcac tgttttgctt cagctagaaa atacaatgca gatgtcatta 2580

aaagacttac tttaa 2595

<210> 7

<211> 1866

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 7

ccctaaaatg ggcaaacatt gcaagcagca aacagcaaac acacagccct ccctgcctgc 60

tgaccttgga gctggggcag aggtcagaga cctctctggg cccatgccac ctccaacatc 120

cactcgaccc cttggaattt cggtggagag gagcagaggt tgtcctggcg tggtttaggt 180

agtgtgagag gggaatgact cctttcggta agtgcagtgg aagctgtaca ctgcccaggc 240

aaagcgtccg ggcagcgtag gcgggcgact cagatcccag ccagtggact tagcccctgt 300

ttgctcctcc gataactggg gtgaccttgg ttaatattca ccagcagcct cccccgttgc 360

ccctctggat ccactgctta aatacggacg aggacagggc cctgtctcct cagcttcagg 420

caccaccact gacctgggac agtgaatccg gactctaagg taaatataaa atttttaagt 480

gtataatgtg ttaaactact gattctaatt gtttctctct tttagattcc aacctttgga 540

actgaattct agaccaccgc ggccgcatat gccaccatgc agctgaggaa cccagaacta 600

catctgggct gcgcgcttgc gcttcgcttc ctggccctcg tttcctggga catccctggg 660

gctagagcac tggacaatgg attggcaagg acgcctacca tgggctggct gcactgggag 720

cgcttcatgt gcaaccttga ctgccaggaa gagccagatt cctgcatcag tgagaagctc 780

ttcatggaga tggcagagct catggtctca gaaggctgga aggatgcagg ttatgagtac 840

ctctgcattg atgactgttg gatggctccc caaagagatt cagaaggcag acttcaggca 900

gaccctcagc gctttcctca tgggattcgc cagctagcta attatgttca cagcaaagga 960

ctgaagctag ggatttatgc agatgttgga aataaaacct gcgcaggctt ccctgggagt 1020

tttggatact acgacattga tgcccagacc tttgctgact ggggagtaga tctgctaaaa 1080

tttgatggtt gttactgtga cagtttggaa aatttggcag atggttataa gcacatgtcc 1140

ttggccctga ataggactgg cagaagcatt gtgtactcct gtgagtggcc tctttatatg 1200

tggccctttc aaaagcccaa ttatacagaa atccgacagt actgcaatca ctggcgaaat 1260

tttgctgaca ttgatgattc ctggaaaagt ataaagagta tcttggactg gacatctttt 1320

aaccaggaga gaattgttga tgttgctgga ccagggggtt ggaatgaccc agatatgtta 1380

gtgattggca actttggcct cagctggaat cagcaagtaa ctcagatggc cctctgggct 1440

atcatggctg ctcctttatt catgtctaat gacctccgac acatcagccc tcaagccaaa 1500

gctctccttc aggataagga cgtaattgcc atcaatcagg accccttggg caagcaaggg 1560

taccagctta gacagggaga caactttgaa gtgtgggaac gacctctctc aggcttagcc 1620

tgggctgtag ctatgataaa ccggcaggag attggtggac ctcgctctta taccatcgca 1680

gttgcttccc tgggtaaagg agtggcctgt aatcctgcct gcttcatcac acagctcctc 1740

cctgtgaaaa ggaagctagg gttctatgaa tggacttcaa ggttaagaag tcacataaat 1800

cccacaggca ctgttttgct tcagctagaa aatacaatgc agatgtcatt aaaagactta 1860

ctttaa 1866

<210> 8

<211> 2211

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 8

atggctgccg atggttatct tccagattgg ctcgaggaca acctctctga gggcattcgc 60

gagtggtggg acttgaaacc tggagccccg aagcccaaag ccaaccagca aaagcaggac 120

gacggccggg gtctggtgct tcctggctac aagtacctcg gacccttcaa cggactcgac 180

aagggggagc ccgtcaacgc ggcggacgca gcggccctcg agcacgacaa ggcctacgac 240

cagcagctca aagcgggtga caatccgtac ctgcggtata accacgccga cgccgagttt 300

caggagcgtc tgcaagaaga tacgtctttt gggggcaacc tcgggcgagc agtcttccag 360

gccaagaagc gggttctcga acctctcggt ctggttgagg aaggcgctaa gacggctcct 420

ggaaagaaac gtccggtaga gcagtcgcca caagagccag actcctcctc gggcatcggc 480

aagacaggcc agcagcccgc taaaaagaga ctcaattttg gtcagactgg cgactcagag 540

tcagttccag accctcaacc tctcggagaa ccaccagcag cccccacaag tttgggatct 600

aatacaatgg cttcaggcgg tggcgcacca atggcagaca ataacgaagg cgccgacgga 660

gtgggtaatg cctcaggaaa ttggcattgc gattccacat ggctgggcga cagagtcatc 720

accaccagca cccgaacatg ggccttgccc acctataaca accacctcta caagcaaatc 780

tccagtgctt caacgggggc cagcaacgac aaccactact tcggctacag caccccctgg 840

gggtattttg atttcaacag attccactgc catttctcac cacgtgactg gcagcgactc 900

atcaacaaca attggggatt ccggcccaag agactcaact tcaagctctt caacatccaa 960

gtcaaggagg tcacgacgaa tgatggcgtc acgaccatcg ctaataacct taccagcacg 1020

gttcaagtct tctcggactc ggagtaccag ttgccgtacg tcctcggctc tgcgcaccag 1080

ggctgcctcc ctccgttccc ggcggacgtg ttcatgattc cgcaatacgg ctacctgacg 1140

ctcaacaatg gcagccaagc cgtgggacgt tcatcctttt actgcctgga atatttccct 1200

tctcagatgc tgagaacggg caacaacttt accttcagct acacctttga ggaagtgcct 1260

ttccacagca gctacgcgca cagccagagc ctggaccggc tgatgaatcc tctcatcgac 1320

cagtacctgt attacctgaa cagaactcag aatcagtccg gaagtgccca aaacaaggac 1380

ttgctgttta gccgtgggtc tccagctggc atgtctgttc agcccaaaaa ctggctacct 1440

ggaccctgtt accggcagca gcgcgtttct aaaacaaaaa cagacaacaa caacagcaac 1500

tttacctgga ctggtgcttc aaaatataac ctcaatgggc gtgaatccat catcaaccct 1560

ggcactgcta tggcctcaca caaagacgac aaagacaagt tctttcccat gagcggtgtc 1620

atgatttttg gaaaggagag cgccggagct tcaaacactg cattggacaa tgtcatgatc 1680

acagacgaag aggaaatcaa agccactaac cccgtggcca ccgaaagatt tgggactgtg 1740

gcagtcaatc tccagagcag cagcacagac cctgcgaccg gagatgtgca tgttatggga 1800

gccttacctg gaatggtgtg gcaagacaga gacgtatacc tgcagggtcc tatttgggcc 1860

aaaattcctc acacggatgg acactttcac ccgtctcctc tcatgggcgg ctttggactt 1920

aagcacccgc ctcctcagat cctcatcaaa aacacgcctg ttcctgcgaa tcctccggca 1980

gagttttcgg ctacaaagtt tgcttcattc atcacccagt attccacagg acaagtgagc 2040

gtggagattg aatgggagct gcagaaagaa aacagcaaac gctggaatcc cgaagtgcag 2100

tatacatcta actatgcaaa atctgccaac gttgatttta ctgtggacaa caatggactt 2160

tatactgagc ctcgccccat tggcacccgt taccttaccc gtcccctgta a 2211

<210> 9

<211> 736

<212> PRT

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 9

Met Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Asn Leu Ser

1 5 10 15

Glu Gly Ile Arg Glu Trp Trp Asp Leu Lys Pro Gly Ala Pro Lys Pro

20 25 30

Lys Ala Asn Gln Gln Lys Gln Asp Asp Gly Arg Gly Leu Val Leu Pro

35 40 45

Gly Tyr Lys Tyr Leu Gly Pro Phe Asn Gly Leu Asp Lys Gly Glu Pro

50 55 60

Val Asn Ala Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp

65 70 75 80

Gln Gln Leu Lys Ala Gly Asp Asn Pro Tyr Leu Arg Tyr Asn His Ala

85 90 95

Asp Ala Glu Phe Gln Glu Arg Leu Gln Glu Asp Thr Ser Phe Gly Gly

100 105 110

Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Val Leu Glu Pro

115 120 125

Leu Gly Leu Val Glu Glu Gly Ala Lys Thr Ala Pro Gly Lys Lys Arg

130 135 140

Pro Val Glu Gln Ser Pro Gln Glu Pro Asp Ser Ser Ser Gly Ile Gly

145 150 155 160

Lys Thr Gly Gln Gln Pro Ala Lys Lys Arg Leu Asn Phe Gly Gln Thr

165 170 175

Gly Asp Ser Glu Ser Val Pro Asp Pro Gln Pro Leu Gly Glu Pro Pro

180 185 190

Ala Ala Pro Thr Ser Leu Gly Ser Asn Thr Met Ala Ser Gly Gly Gly

195 200 205

Ala Pro Met Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly Asn Ala

210 215 220

Ser Gly Asn Trp His Cys Asp Ser Thr Trp Leu Gly Asp Arg Val Ile

225 230 235 240

Thr Thr Ser Thr Arg Thr Trp Ala Leu Pro Thr Tyr Asn Asn His Leu

245 250 255

Tyr Lys Gln Ile Ser Ser Ala Ser Thr Gly Ala Ser Asn Asp Asn His

260 265 270

Tyr Phe Gly Tyr Ser Thr Pro Trp Gly Tyr Phe Asp Phe Asn Arg Phe

275 280 285

His Cys His Phe Ser Pro Arg Asp Trp Gln Arg Leu Ile Asn Asn Asn

290 295 300

Trp Gly Phe Arg Pro Lys Arg Leu Asn Phe Lys Leu Phe Asn Ile Gln

305 310 315 320

Val Lys Glu Val Thr Thr Asn Asp Gly Val Thr Thr Ile Ala Asn Asn

325 330 335

Leu Thr Ser Thr Val Gln Val Phe Ser Asp Ser Glu Tyr Gln Leu Pro

340 345 350

Tyr Val Leu Gly Ser Ala His Gln Gly Cys Leu Pro Pro Phe Pro Ala

355 360 365

Asp Val Phe Met Ile Pro Gln Tyr Gly Tyr Leu Thr Leu Asn Asn Gly

370 375 380

Ser Gln Ala Val Gly Arg Ser Ser Phe Tyr Cys Leu Glu Tyr Phe Pro

385 390 395 400

Ser Gln Met Leu Arg Thr Gly Asn Asn Phe Thr Phe Ser Tyr Thr Phe

405 410 415

Glu Glu Val Pro Phe His Ser Ser Tyr Ala His Ser Gln Ser Leu Asp

420 425 430

Arg Leu Met Asn Pro Leu Ile Asp Gln Tyr Leu Tyr Tyr Leu Asn Arg

435 440 445

Thr Gln Asn Gln Ser Gly Ser Ala Gln Asn Lys Asp Leu Leu Phe Ser

450 455 460

Arg Gly Ser Pro Ala Gly Met Ser Val Gln Pro Lys Asn Trp Leu Pro

465 470 475 480

Gly Pro Cys Tyr Arg Gln Gln Arg Val Ser Lys Thr Lys Thr Asp Asn

485 490 495

Asn Asn Ser Asn Phe Thr Trp Thr Gly Ala Ser Lys Tyr Asn Leu Asn

500 505 510

Gly Arg Glu Ser Ile Ile Asn Pro Gly Thr Ala Met Ala Ser His Lys

515 520 525

Asp Asp Lys Asp Lys Phe Phe Pro Met Ser Gly Val Met Ile Phe Gly

530 535 540

Lys Glu Ser Ala Gly Ala Ser Asn Thr Ala Leu Asp Asn Val Met Ile

545 550 555 560

Thr Asp Glu Glu Glu Ile Lys Ala Thr Asn Pro Val Ala Thr Glu Arg

565 570 575

Phe Gly Thr Val Ala Val Asn Leu Gln Ser Ser Ser Thr Asp Pro Ala

580 585 590

Thr Gly Asp Val His Val Met Gly Ala Leu Pro Gly Met Val Trp Gln

595 600 605

Asp Arg Asp Val Tyr Leu Gln Gly Pro Ile Trp Ala Lys Ile Pro His

610 615 620

Thr Asp Gly His Phe His Pro Ser Pro Leu Met Gly Gly Phe Gly Leu

625 630 635 640

Lys His Pro Pro Pro Gln Ile Leu Ile Lys Asn Thr Pro Val Pro Ala

645 650 655

Asn Pro Pro Ala Glu Phe Ser Ala Thr Lys Phe Ala Ser Phe Ile Thr

660 665 670

Gln Tyr Ser Thr Gly Gln Val Ser Val Glu Ile Glu Trp Glu Leu Gln

675 680 685

Lys Glu Asn Ser Lys Arg Trp Asn Pro Glu Val Gln Tyr Thr Ser Asn

690 695 700

Tyr Ala Lys Ser Ala Asn Val Asp Phe Thr Val Asp Asn Asn Gly Leu

705 710 715 720

Tyr Thr Glu Pro Arg Pro Ile Gly Thr Arg Tyr Leu Thr Arg Pro Leu

725 730 735

<210> 10

<211> 23

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 10

cgagaacaac gaagacttca aca 23

<210> 11

<211> 18

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 11

cgcggtcagc atcgagat 18

<210> 12

<211> 20

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 12

ccgtggccag caacttcgcg 20

<210> 13

<211> 20

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 13

ctgccaggaa gagccagatt 20

<210> 14

<211> 24

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 14

gtactcataa cctgcatcct tcca 24

<210> 15

<211> 16

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 15

tgcatcagtg agaagc 16

<210> 16

<211> 787

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 16

gtacctcgcg aatgcatcta cgatgcgaga acttgtgcct ccccgtgttc ctgctctttg 60

tccctctgtc ctacttagac taatatttgc cttgggtact gcaaacagga aatgggggag 120

ggattcgaaa cggctttgcg gacactagcg atgcgagaac ttgtgcctcc ccgtgttcct 180

gctctttgtc cctctgtcct acttagacta atatttgcct tgggtactgc aaacaggaaa 240

tgggggaggg attcgaaacg gctttgcgga cactagtgtg tgcatgcgtg agtacttgtg 300

tgtaaatttt tcattatcta taggtaaaag cacacttgga attagcaata gatgcaattt 360

gggacttaac tctttcagta tgtcttattt ctaagcaaag tatttagttt ggttagtaat 420

tactaaacac tgagaactaa attgcaaaca ccaagaacta aaatgttcaa gtgggaaatt 480

acagttaaat accatggtaa tgaataaaag gtacaaatcg tttaaactct tatgtaaaat 540

ttgataagat gttttacaca actttaatac attgacaagg tcttgtggag aaaacagttc 600

cagatggtaa atatacacaa gggatttagt caaacaattt tttggcaaga atattatgaa 660

ttttgtaatc ggttggcagc caatgaaata caaagatgag tctagttaat aatctacaat 720

tattggttaa agaagtatat tagtgctaat ttccctccgt ttgtcctagc ttttctcttc 780

tgtcaac 787

Claims

1. A recombinant adeno-associated viral vector comprising an AAV capsid and a vector genome, wherein the vector genome comprises a nucleotide sequence encoding a functional α -galactosidase a and an expression control sequence that directs the expression of the α -galactosidase a in a host cell, wherein the amino acid sequence of the AAV capsid is shown in SEQ ID No. 9, the expression control sequence comprises a promoter that is an LP1 promoter, and the sequence of the LP1 promoter is shown in SEQ ID No. 2.

2. The recombinant adeno-associated viral vector according to claim 1, wherein the nucleotide sequence encoding the AAV capsid is shown in SEQ ID No. 8.

3. The recombinant adeno-associated virus vector according to claim 1, wherein the nucleotide sequence encoding the α -galactosidase a is shown in SEQ ID No. 3.

4. The recombinant adeno-associated virus vector of any one of claims 1-3, wherein the vector further comprises a post-transcriptional regulatory element.

5. The recombinant adeno-associated viral vector of any one of claims 1-3, wherein the vector further comprises one or more of introns, kozak sequences, polyA, and WPRE.

6. The recombinant adeno-associated viral vector of any one of claims 1-3, wherein the vector further comprises an AAV inverted terminal repeat.

7. The recombinant adeno-associated viral vector of claim 6, wherein the AAV inverted terminal repeat is from an AAV2 serotype.

8. An isolated host cell comprising the recombinant adeno-associated viral vector of any one of claims 1-7.

9. A pharmaceutical composition comprising the recombinant adeno-associated viral vector of any one of claims 1-7 or the host cell of claim 8 and a pharmaceutically acceptable excipient.

10. The pharmaceutical composition of claim 9, wherein the pharmaceutical composition is formulated for intravenous administration.

11. Use of the recombinant adeno-associated viral vector of any one of claims 1-7, the host cell of claim 8 or the pharmaceutical composition of claim 9 or 10 in the manufacture of a medicament for the prevention or treatment of fabry disease.

12. The use of claim 11, wherein the recombinant adeno-associated viral vector, host cell or pharmaceutical composition is administered in combination with another therapy.

13. The use of claim 11 or 12, wherein the recombinant adeno-associated viral vector, host cell or pharmaceutical composition is administered at a dose of 1x10 ¹⁰ vg/kg to 1x10 ¹⁶ vg/kg; the recombinant adeno-associated viral vector or composition is administered more than once.

14. The use of claim 13, wherein the recombinant adeno-associated viral vector is administered at a dose of 5 x 10 ¹³ vg/kg.

15. The use of claim 13, wherein the recombinant adeno-associated viral vector or pharmaceutical composition is administered at a dose of 2.5 x 10 ¹² vg/kg.