Composition for treating congenital gpi deficiency

Abstract

The present disclosure provides an AAV vector including: (a) a promoter including a nucleotide sequence having approximately 90% or more identity to a nucleotide sequence of SEQ ID NO: 3 and having activity greater than or equivalent to that of a promoter comprising the nucleotide sequence of SEQ ID NO: 3; and (b) a polynucleotide encoding PIGO.

Description

Compositions for treating congenital GPI deficiency

[0001] This patent application claims priority to Japanese Patent Application No. 2024-108994, the entire contents of which are incorporated herein by reference. The present disclosure relates to compositions for treating congenital GPI deficiency.

[0002] Glycosylphosphatidylinositol (GPI) anchors GPI-anchored proteins (GPI-APs) to cell membranes (Figure 1). GPI-APs include over 150 proteins, including enzymes, receptors, adhesion molecules, and complement regulatory factors, that play important roles in ontogeny, neurodevelopment, immunity, and fertilization. Thirty genes are known to be involved in the biosynthesis of GPI-APs (Figure 2). Mutations in these genes result in reduced expression and activity of the proteins they encode, leading to reduced expression and structural abnormalities of GPI-APs on the cell membrane surface, resulting in congenital GPI deficiency (IGD). Primary symptoms include intellectual disability, delayed motor development, and epilepsy. Other symptoms include hyperalkaline phosphatasia, abnormalities of the fingers and toes such as shortened distal phalanges and hyponacrosis of the nails, hearing loss, and visceral malformations. To date, 24 genetic mutations causing IGD have been reported. Current treatments for IGD are limited to symptomatic treatments such as controlling convulsions, and there is no fundamental cure.

[0003] Phosphatidylinositol glycan anchor biosynthesis class O (PIGO) is one of the genes involved in the biosynthesis of GPI-AP, and mutations in this gene cause IGD. The inventors previously generated mice with the same mutation as PIGO deficiency patients as a disease model of IGD. These mice closely reflect the symptoms of patients, and many phenotypes were improved by genome editing using the CRISPR / Cas9 system (Non-Patent Document 1). This means that many of the neurological symptoms of IGD are reversible with early treatment, and the development of treatments applicable to humans is needed.

[0004] Kuwayama R, et al., Establishment of mouse model of inherited PIGO deficiency and therapeutic potential of AAV-based gene therapy. Nat Commun. 2022 Jun 3;13(1):3107

[0005] It is an object of the present disclosure to provide methods for treating congenital GPI deficiency.

[0006] The present inventors have conducted extensive research into congenital GPI deficiency, discovered a promoter useful for PIGO gene replacement therapy, and developed an adeno-associated virus (AAV) vector capable of treating congenital GPI deficiency. Accordingly, in one aspect, the present disclosure provides an AAV vector comprising: (a) a promoter comprising a nucleotide sequence having about 90% or more identity to the nucleotide sequence of SEQ ID NO: 3, and having activity equivalent to or greater than that of a promoter consisting of the nucleotide sequence of SEQ ID NO: 3; and (b) a polynucleotide encoding PIGO. In one aspect, the present disclosure provides a composition for treating congenital GPI deficiency due to an abnormality in the PIGO gene, comprising the AAV vector.

[0007] The present disclosure is useful for treating congenital GPI deficiencies.

[0008] Schematic diagram of GPI-anchored protein (GPI-AP). Schematic diagram of GPI-AP biosynthesis and transport. The promoter sequence (SEQ ID NO: 6) upstream of the human PIGO gene is shown. The nucleotide sequence (SEQ ID NO: 8) of pAAVp590hPIGO is shown. Changes in body weight of male congenital GPI deficiency (IGD) model mice that underwent PIGO gene replacement therapy are shown. The results of a hanging test of IGD model mice that underwent PIGO gene replacement therapy are shown. Survival curves of IGD model mice that underwent PIGO gene replacement therapy are shown. Schematic diagram of the promoter region upstream of the PIGO gene. Activity of the endogenous PIGO promoter measured by luciferase assay is shown. CD24 recovery rates in PigoKO Neuro2a cells infected with AAV vectors containing various promoters and the PIGO gene are shown. 1 shows the changes in body weight of IGD model mice intracerebroventricularly administered with AAV vectors containing various promoters and the PIGO gene. 1 shows the results of a hanging test of IGD model mice intracerebroventricularly administered with AAV vectors containing various promoters and the PIGO gene. 1 shows the tremor scores of IGD model mice intracerebroventricularly administered with AAV vectors containing various promoters and the PIGO gene. 1 shows the survival curves of IGD model mice intracerebroventricularly administered with AAV vectors containing various promoters and the PIGO gene.

[0009] Unless otherwise specified, terms used herein have the meanings commonly understood by those skilled in the art of organic chemistry, medicine, pharmacology, molecular biology, microbiology, etc. Definitions of some terms used herein are provided below, but these definitions take precedence over common understandings in this specification.

[0010] As used herein, when a numerical value is accompanied by the term "about," it is intended to encompass a range of ±10% of that value. For example, "about 20" is intended to include "18 to 22." A range of numerical values ​​includes all values ​​between and at the endpoints. "About" in reference to a range applies to both endpoints of the range. Thus, for example, "about 20 to 30" is intended to include "18 to 33."

[0011] Phosphatidylinositol glycan anchor biosynthesis class O (PIGO) is one of the genes involved in the biosynthesis of glycosylphosphatidylinositol (GPI)-anchored proteins (GPI-APs). PIGO, also known as GPI ethanolamine phosphate transferase 3, is a transferase that adds ethanolamine phosphate to the third mannose of GPI, thereby attaching GPI to proteins. Because complete loss of GPI biosynthesis results in embryonic lethality, many patients with congenital GPI deficiency (IGD) have partial defects in GPI biosynthesis, and mutations in the PIGO gene are one of the causes.

[0012] The amino acid sequence of human wild-type PIGO is registered, for example, under GenBank accession number NP_116023.2 (SEQ ID NO: 1). In the present disclosure, PIGO includes naturally occurring allelic products thereof that maintain their function. In some embodiments, PIGO comprises the amino acid sequence of SEQ ID NO: 1. In some embodiments, PIGO consists of the amino acid sequence of SEQ ID NO: 1.

[0013] In the present disclosure, PIGO may consist of the amino acid sequence of SEQ ID NO: 1 with one or several amino acids deleted, substituted, or added, as long as its function is maintained. "Several" preferably means 2 to 7, more preferably 2 to 5, and most preferably 2 to 3 amino acids. Amino acid substitutions are preferably conservative substitutions between similar amino acid residues.

[0014] Furthermore, as long as its function is maintained, PIGO may consist of an amino acid sequence that has at least about 60% or more, preferably about 70% or more, more preferably about 80% or more, even more preferably about 90% or more, particularly preferably about 95% or more, and most preferably about 97%, about 98% or about 99% or more identity with the amino acid sequence of SEQ ID NO: 1 when calculated using BLAST or the like (for example, when using the default, i.e., initial condition, parameters of BLAST).

[0015] With respect to PIGO having a mutation, "PIGO function is maintained" means that cells expressing the mutated PIGO express GPI-AP at a similar level compared to cells expressing wild-type PIGO. Known human PIGO mutations that reduce GPI-AP expression include R119W, T130N, and K1047E. Therefore, the PIGO of the present disclosure does not contain mutations corresponding to R119W, T130N, and K1047E.

[0016] In one embodiment, the polynucleotide encoding PIGO comprises or consists of a nucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 1. Examples of nucleotide sequences that encode the amino acid sequence of SEQ ID NO: 1 include the nucleotide sequence of SEQ ID NO: 2.

[0017] A polynucleotide encoding PIGO may comprise or consist of a nucleotide sequence that has an identity to the nucleotide sequence of SEQ ID NO: 2 of about 60% or more, preferably about 70% or more, more preferably about 80% or more, even more preferably about 90% or more, particularly preferably about 95% or more, and most preferably about 97%, about 98%, or about 99% or more, when calculated using BLAST or the like (e.g., when using the default, i.e., initial, parameters of BLAST).

[0018] A polynucleotide encoding PIGO may have a nucleotide sequence that hybridizes under stringent conditions to a polynucleotide having a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 2. The term "hybridizing under stringent conditions" used herein refers to hybridization that can be performed according to standard methods, such as those described in "Molecular Cloning," T. Maniatis et al., CSH Laboratory (1983). Examples of "stringent conditions" include hybridization in 6x SSC (a solution containing 1.5 M NaCl and 0.15 M trisodium citrate is made into 10x SSC) and 50% formamide at 45°C, followed by washing in 2x SSC at 50°C (Molecular Biology, John Wiley & Sons, NY (1989), 6.3.1-6.3.6), as well as conditions of equivalent stringency.

[0019] In the present disclosure, a promoter is used that contains a nucleotide sequence having about 90% or more identity to the nucleotide sequence of SEQ ID NO: 3 and has activity equivalent to or greater than that of a promoter consisting of the nucleotide sequence of SEQ ID NO: 3. The nucleotide sequence of SEQ ID NO: 3 (referred to as p320 or p320L1) corresponds to the sequence consisting of 320 nucleotides on the 3'-terminal side of the 970-nucleotide nucleotide sequence of the human wild-type PIGO promoter region (SEQ ID NO: 6, referred to as p970 or p970L1). Similarly, the sequence consisting of 590 nucleotides on the 3'-terminal side of p970 (SEQ ID NO: 4) is referred to as p590 or p590L1, and the sequence consisting of 790 nucleotides (SEQ ID NO: 5) is referred to as p790 or p790L1.

[0020] The length of the promoter is not limited as long as it can be introduced into AAV vector.For example, about 4.2 kb of polynucleotide can be introduced into AAV9 vector.When the polynucleotide encoding PIGO is the polynucleotide of SEQ ID NO: 2, which consists of 3270 nucleotides, the upper limit of the number of nucleotides of the promoter that can be introduced into AAV9 vector is about 930.

[0021] In one embodiment, the promoter comprises a nucleotide sequence having about 90% or more identity to the nucleotide sequence of any of SEQ ID NOs: 3-5, e.g., about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% or more identity.

[0022] In one embodiment, the promoter comprises a nucleotide sequence that hybridizes under stringent conditions to a polynucleotide having a nucleotide sequence complementary to any of the nucleotide sequences set forth in SEQ ID NOs: 3-5.

[0023] In some embodiments, the promoter comprises the nucleotide sequence of any of SEQ ID NOs: 3 to 5. In some embodiments, the promoter comprises the nucleotide sequence of any of SEQ ID NOs: 3 to 5 and consists of a portion of the nucleotide sequence of SEQ ID NO: 6. In some embodiments, the promoter consists of the nucleotide sequence of any of SEQ ID NOs: 3 to 5.

[0024] In some embodiments, the promoter comprises a nucleotide sequence having about 90% or greater identity to the nucleotide sequence of SEQ ID NO:4, e.g., about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% or greater identity. In some embodiments, the promoter comprises a nucleotide sequence that hybridizes under stringent conditions to a polynucleotide having a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO:4. In some embodiments, the promoter comprises the nucleotide sequence of SEQ ID NO:4. In some embodiments, the promoter comprises the nucleotide sequence of SEQ ID NO:4 and consists of a portion of the nucleotide sequence of SEQ ID NO:6. In some embodiments, the promoter consists of the nucleotide sequence of SEQ ID NO:4.

[0025] In one embodiment, the promoter comprises a nucleotide sequence having about 90% or more identity, e.g., about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% or more identity, to the nucleotide sequence of any of SEQ ID NOs: 3-5, and comprises the nucleotide sequence of SEQ ID NO: 7. SEQ ID NO: 7 corresponds to the sequence of the 20 nucleotides at the 3' end of the nucleotide sequence of SEQ ID NO: 3.

[0026] Promoter activity can be measured by introducing a nucleic acid construct containing a reporter gene linked downstream of the promoter into nervous system cells and measuring the expression level of the reporter gene. Any reporter gene that directly or indirectly generates a detectable label can be used, including the chloramphenicol acetyltransferase (CAT) gene, green fluorescent protein (GFP) gene, β-glucuronidase (GUS) gene, luciferase gene, and other marker genes. Nervous system cells are not limited as long as they are capable of expressing the reporter gene. Examples include neuronal cells such as Neuro2a cells, SHSY5Y cells, NSC-34 cells, and PC12 cells, and glial cells such as U251 cells and C6 cells. Construction of vectors for introducing nucleic acid constructs, transformation, etc. can be performed using known methods (Molecular Cloning: A Laboratory Manual 2nd edition (1989), Cold Spring Harbor Laboratory Press).

[0027] In some embodiments, the promoter has activity equal to or greater than that of a promoter consisting of the nucleotide sequence of SEQ ID NO: 3. For example, the promoter has about 90% or more, about 95% or more, about 97% or more, about 98% or more, about 99% or more, or about 100% or more of the activity of a promoter consisting of the nucleotide sequence of SEQ ID NO: 3.

[0028] In some embodiments, the promoter has activity equivalent to that of a promoter consisting of the nucleotide sequence of SEQ ID NO: 3. For example, the promoter has about 90% to 110%, about 95% to 105%, about 97% to 103%, about 98% to 102%, about 99% to 101%, or about 100% of the activity of a promoter consisting of the nucleotide sequence of SEQ ID NO: 3.

[0029] In the present disclosure, AAV vectors known in the art can be used. AAV is a non-enveloped virus with a single-stranded DNA genome. The wild-type AAV genome has, between two inverted terminal repeats (ITRs), a Cap gene encoding a capsid protein and a Rep gene encoding a protein with helicase activity necessary for AAV replication. In the genome of an AAV vector, the genomic sequence between the ITRs is usually replaced with a polynucleotide to be introduced into a cell. In the AAV vector of the present disclosure, the genomic sequence between the ITRs is replaced with a sequence containing a promoter and a polynucleotide encoding a PIGO.

[0030] The AAV vector may contain one or more other regulatory sequences, such as enhancers, polyadenylation signals, and post-transcriptional regulatory elements (PREs), as long as they do not reduce the expression of PIGO.

[0031] There are many AAV serotypes, and vectors derived from their native forms or recombinant forms in which capsid proteins, etc., have been modified can be used. For example, AAV vectors derived from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV-rhlO, AAV-DJ, AAVAnc80, or yfAAV9 / 3 can be used. For example, the AAV vectors described in JP 2015-51009 or Iida A, et al., "Systemic delivery of tyrosine-mutant AAV vectors results in robust transduction of neurons in adult mice." Biomed Res Int. 2013;2013:974819 can be used. In one embodiment, the vector is an AAV9 vector.

[0032] AAV has tropism for specific tissues or cells depending on its serotype. The tropism of AAV is determined by the capsid protein on the surface of the virus. The AAV vector in the present disclosure may have a natural capsid protein or an artificially modified capsid protein. In the present disclosure, AAV vectors derived from AAVs having tropism for the nervous system or neural cells, such as AAV1, AAV2, AAV5, AAV8, AAV9, or variants thereof, may be used.

[0033] AAV vectors can be produced, for example, by introducing an AAV vector plasmid containing a promoter and a polynucleotide encoding PIGO, along with inverted terminal repeats (ITRs) at both ends, into packaging cells together with an AAV helper plasmid containing the Rep and Cap genes required for AAV replication and particle formation, and an adenovirus helper plasmid containing adenovirus helper genes required for AAV propagation. Examples of packaging cells include 293T cells, 293 cells, HeLa cells, COS1 cells, and COS7 cells. Viral particles released from packaging cells can be recovered from the packaging cell culture supernatant by purification methods such as centrifugation, filtration, and column purification. An insect cell-baculovirus system may be used to produce large quantities of AAV vectors.

[0034] AAV vectors can be administered together with agents to enhance their transduction efficiency, such as neuraminidase inhibitors, proteasome inhibitors such as MG132, Eeyarestatin I, tritiated thymidine, cisplatin, etoposide, calpain inhibitors, and ubiquitin ligase inhibitors.

[0035] In the Examples described below, when an AAV vector containing a portion of the PIGO promoter region and a polynucleotide encoding PIGO was administered to a mouse model of congenital GPI deficiency (IGD) with the same mutation as that of a PIGO deficiency patient, the symptoms of IGD were improved. Therefore, this vector can be used to treat IGD caused by an abnormality in the PIGO gene.

[0036] There are many known examples of PIGO gene mutations that cause IGD, including I75T, R119W, R119Q, T130N, A137V, F153S, A157T, N162D, G238D, M255I, M280T, M344K, N370S, V424fs, V424Dfs, Q430X, R436W, A452Gfs, A452V, F575V, and R604 Examples of IGD include, but are not limited to, Pfs, T788Hfs, A834Cfs, H871P, G883fs, F903V, L957F, R1035C, V1040I, and K1047E in mice, R119W, T130N, and K1051E in mice (where fs indicates a frameshift and X indicates a stop codon), and other splicing abnormalities and frameshifts. In one embodiment, the PIGO gene mutation is human R119W, T130N, or K1047E, e.g., T130N. Examples of IGD symptoms include mental retardation, intractable epilepsy, hyperalkaline phosphatasia, abnormal facial features, short distal phalanges, nail hypoplasia, Hirschsprung's disease, tetralogy of Fallot, and cleft lip and palate.

[0037] In this disclosure, "treat" or "treatment" means reducing or eliminating the cause of a disease, slowing or halting its progression, and / or reducing, alleviating, ameliorating or eliminating its symptoms in a subject suffering from the disease.

[0038] The subject for treatment of IGD can be of any species, typically a mammal (e.g., human, mouse, rat, hamster, rabbit, cat, dog, cow, sheep, monkey), particularly a human. In some embodiments, the subject is a child, infant, baby, or newborn.

[0039] The AAV vector may be administered systemically or locally, and the administration method is not limited thereto, for example, intravenously, intracerebroventricularly, intracisternally, or intrathecally, preferably intracerebroventricularly, intracisternally, or intrathecally, more preferably intracerebroventricularly.

[0040] A composition comprising an AAV vector may contain, in addition to the AAV vector as an active ingredient, a pharmaceutically acceptable carrier and / or additive. Examples of pharmaceutically acceptable carriers include physiological saline or other physiologically acceptable buffer solutions. Examples of additives include solubilizers, pH adjusters, preservatives, stabilizers, etc. Dosage forms include, but are not limited to, injections, such as liquid injections and solid injections (e.g., lyophilized injections) that are dissolved before use. A composition comprising an AAV vector may be provided as a kit, and the kit may further include a buffer solution for dissolution before use, instructions for use, etc.

[0041] The dosage and frequency of administration of the AAV vector can be appropriately determined by those skilled in the art depending on the animal species, health condition, age, weight, administration route, administration form, etc. of the subject so that an effective amount of AAV vector is administered to the subject. The effective amount in a given situation can be easily determined by routine experimentation and is within the skill and judgment of an ordinary clinician. For example, in the case of mice, about 1 x 10 １０ ~1x10 １２ vg / body, approx. 2x10 １０ ~5x10 １１ vg / body or about 5x10 １０ ~2x10 １１ vg / body, e.g., about 1x10 １１ For example, in humans, about 1 x 10 １２ ~3x10 １５ vg / body or approximately 1 x 10 １３ ~2x10 １５ When administered intravenously to humans, for example, up to about 2 x 10 １４ vg / kg body weight, approximately 1.5x10 １４ vg / kg body weight or approximately 1.1 x 10 １４ For intracerebroventricular, intracisternal, or intrathecal administration to humans, approximately 1 x 10 vg / kg body weight of vector can be administered. １３ ~1x10 １５ vg / body, approx. 2x10 １３ ~5x10 １４ vg / body or about 5x10 １３~2x10 １４ vg / body, e.g., about 1x10 １４ The AAV vector may be administered in a single dose or multiple doses. In some embodiments, the AAV vector is administered in a single dose.

[0042] The timing of AAV vector administration is not limited, but early treatment is preferred.For example, in the case of mice, it can be administered within 4 weeks, 3 weeks, 2 weeks, 1 week or 6 days, 5 days, 4 days, 3 days, 2 days or 1 day after birth.In some embodiments, the AAV vector is administered to mice within 2 days after birth.For example, in the case of humans, it can be administered within 20 years, 15 years, 10 years, 5 years, 3 years, 2 years, 1 year, 6 months, 3 months, 2 months or 6 weeks after birth.

[0043] The AAV vectors of the present disclosure can be used alone or in combination with one or more additional active ingredients, particularly active ingredients for the treatment of IGD. For example, a composition comprising one or more additional active ingredients in addition to the AAV vector can be used.

[0044] "Combined use" of ingredients refers not only to the use of a dosage form containing all ingredients or the use of a combination of dosage forms containing each ingredient separately, but also to the simultaneous administration of each ingredient or the administration of any ingredient with a delay, as long as they are used to treat IGD. Two or more additional active ingredients may also be used in combination. Suitable active ingredients for combination use include, for example, vitamin B1, vitamin B2, vitamin B6, etc.

[0045] In one aspect, there is provided a method for treating congenital GPI deficiency caused by an abnormality in the PIGO gene, comprising administering an effective amount of an AAV vector of the present disclosure to a subject in need of such treatment. In one aspect, there is provided an AAV vector of the present disclosure for treating congenital GPI deficiency caused by an abnormality in the PIGO gene. In one aspect, there is provided a use of an AAV vector of the present disclosure for treating congenital GPI deficiency caused by an abnormality in the PIGO gene. In one aspect, there is provided a use of an AAV vector of the present disclosure in the manufacture of a composition for treating congenital GPI deficiency caused by an abnormality in the PIGO gene.

[0046] For example, the following embodiments are provided: [1] An adeno-associated virus (AAV) vector comprising: (a) a promoter comprising a nucleotide sequence having at least about 90% identity to the nucleotide sequence of SEQ ID NO: 3, and having activity equivalent to or greater than that of a promoter consisting of the nucleotide sequence of SEQ ID NO: 3; and (b) a polynucleotide encoding a phosphatidylinositol glycan anchor biosynthesis class O (PIGO). [2] The vector according to 1 above, wherein the promoter comprises a nucleotide sequence having at least about 90% identity to the nucleotide sequence of any of SEQ ID NOs: 3 to 5. [3] The vector according to 1 or 2 above, wherein the promoter comprises a nucleotide sequence having at least about 90% identity to the nucleotide sequence of SEQ ID NO: 4. [4] The vector according to any of 1 to 3 above, wherein the promoter comprises the nucleotide sequence of SEQ ID NO: 7. [5] The vector according to any of 1 to 4 above, wherein the promoter comprises the nucleotide sequence of any of SEQ ID NOs: 3 to 5. [6] The vector according to any of 1 to 5 above, wherein the promoter comprises the nucleotide sequence of SEQ ID NO: 4. [7] The vector according to any one of 1 to 6, wherein the promoter comprises the nucleotide sequence of any one of SEQ ID NOs: 3 to 5 and consists of a part of the nucleotide sequence of SEQ ID NO: 6. [8] The vector according to any one of 1 to 7, wherein the promoter comprises the nucleotide sequence of SEQ ID NO: 4 and consists of a part of the nucleotide sequence of SEQ ID NO: 6. [9] The vector according to any one of 1 to 8, wherein the promoter consists of the nucleotide sequence of any one of SEQ ID NOs: 3 to 5.

[10] The vector according to any one of 1 to 9, wherein the promoter consists of the nucleotide sequence of SEQ ID NO: 4.

[11] The vector according to any one of 1 to 10, wherein PIGO consists of an amino acid sequence having about 90% or more identity to the amino acid sequence of SEQ ID NO: 1.

[12] The vector according to any one of 1 to 11, wherein PIGO consists of the amino acid sequence of SEQ ID NO: 1.

[13] The vector according to any one of 1 to 12, which is an AAV9 vector.

[14] A composition for treating congenital GPI deficiency caused by an abnormality in the PIGO gene, comprising the vector according to any one of 1 to 13.

[15] The composition according to 14, wherein the abnormality in the PIGO gene is I75T, R119W, R119Q, T130N, A137V, F153S, A157T, N162D, G238D, M255I, M280T, M344K, N370S, V424fs, V424Dfs, Q430X, R436W, A452Gfs, A452V, F575V, R604Pfs, T788Hfs, A834Cfs, H871P, G883fs, F903V, L957F, R1035C, V1040I or K1047E.

[16] The composition according to any one of 14 or 15, wherein the abnormality in the PIGO gene is an R119W, T130N, or K1047E mutation.

[17] The composition according to any one of 14 to 16, wherein the abnormality in the PIGO gene is a T130N mutation.

[18] The composition according to any one of 14 to 17, wherein the composition is administered intravenously, intracerebroventricularly, intracisternally, or intrathecally.

[19] The composition according to any one of 14 to 18, wherein the composition is administered intracerebroventricularly, intracisternally, or intrathecally.

[20] The composition according to any one of 14 to 19, wherein the composition is administered intracerebroventricularly.

[21] The composition according to any one of 14 to 20, wherein the composition is administered in a single dose.

[0047] All documents cited in this specification are incorporated herein by reference. The present invention will be described in more detail in the following examples, but the present invention is not limited to these examples. The above descriptions are all non-limiting, and the present invention is defined in the appended claims, and various modifications are possible within the scope of the technical idea thereof.

[0048] Materials and Methods Construction of Constructs The promoter sequence upstream of the hPIGO gene is shown in Figure 3. In Figure 3, part of hPIGO exon 1 (5'UTR) is shown in capital letters. The region for which primers were designed is underlined. The genome was extracted from human peripheral blood cells, and the following promoter regions were amplified by PCR and inserted into the vectors: A-L1: pAAVp970L1 B-L1: pAAVp790L1 C-L1: pAAVp590L1 D-L1: pAAVp320L1 D-L2: pAAVp320L2

[0049] Preparation of luciferase reporter constructs. Each promoter region was amplified using a forward primer with an EcoRV site at the 5' end and a reverse primer with a HindIII site at the 3' end, followed by restriction enzyme cleavage. The pGL4.11{luc2P} (Promega) vector was cleaved with EcoRV and HindIII, and each promoter region was inserted. The promoter regions of CBA, CMV, and Synapsin I were amplified using existing vectors as templates, and reporter constructs were similarly prepared.

[0050] Construction of pAAVp590hPIGO and pAAVp320hPIGO: 1) p590L1 and p320L1 were amplified using a forward primer with an Xba1 site at the 5' end and a reverse primer with an Age1 site at the 3' end, followed by restriction enzyme digestion. 2) hPIGO cDNA was amplified using a forward primer with an Age1 site at the 5' end and a reverse primer with an EcoRI site at the 3' end, followed by restriction enzyme digestion. 3) pAAV nEFCas9 (Addgene, #87115) was digested with Xba1 and EcoRI, and 1) and 2) were ligated and inserted to obtain pAAVp590hPIGO and pAAVp320hPIGO. The nucleotide sequence of pAAVp590hPIGO is shown in Figure 4. Similarly, promoter portions were amplified using existing vectors containing CBA, CMV, and SynapsinI as templates to obtain pAAVCBAPIGO, pAAVCMVPIGO, and pAAVSynapsin1PIGO.

[0051] Measurement of promoter activity Dual-luciferase activity Dual-luciferase activity was measured using a Promega kit. The above luciferase reporter was introduced into the mouse neuronal cell line Neuro2a by lipofection along with a small amount of internal control plasmid. Two days later, the cells were lysed, and substrate was added, followed by measurement of luminescence using a luminometer. Gene introduction efficiency was corrected using the internal control.

[0052] Production of AAV with AAV9 or AAVGTX serotypes. AAVpro293T cells (24 plates, 15 cm dishes) were lipofected at an equimolar ratio with pAAVPIGO, pAd5 (Helper), and pXR9-pAAV9 (or pGTX), each containing a promoter. The next day, the medium was replaced with serum-free medium and cultured for 5 days. The cells were then harvested and centrifuged at 2000 rpm. PEG (polyethylene glycol) was added to the supernatant, and the mixture was left at 4°C overnight to precipitate the virus. After centrifugation at 2800 g for 15 min at 4°C, the precipitate was solubilized with 5 ml of PBS containing 26 U / ml benzonase (a nucleolytic agent) at 37°C for 1 h and layered on top of an iodixanol gradient (15%, 25%, 40%, 58%). After ultracentrifugation (70Ti rotor, 48,000 rpm, 2 h 10 min), the virus layer (between 40% and 58%) was collected and concentrated on a Vivaspin 20 column. The titer was measured by qPCR using a Taqman probe.

[0053] Infection of Neuro2a As previously reported, PIGO knockout cells of Neuro2a (PigoKO Neuro2a) were generated using the CRISPR / Cas system (Non-Patent Document 1). ５ or 10 ６ The virus was added to the culture medium at 100°C, and the recovery of CD24 expression was measured on days 5 and 8. After harvesting the cells, they were stained with an anti-mouse CD24 antibody (M1 / 69 Biolegend) and a PE-labeled anti-mouse IgG as a secondary antibody, and analyzed using a MACS Quant flow cytometer (Miltenyi Biotec).

[0054] IGD Model Mouse As previously reported, we used an IGD model mouse (KI / KO) in which a mutation (T130N) in the PIGO gene found in human IGD patients was knocked in (Non-Patent Document 1).

[0055] Intraventricular administration: Newborn mice 1-2 days old were anesthetized on ice, and the virus solution was injected into the ventricles 10 cm apart, using the bregma as a guide. １１ vg / mouse.

[0056] Hanging test: A mouse was held onto a wire mesh with a 5 mm or 11 mm grid, and the time it took to fall after being turned upside down was measured for up to 3 minutes.

[0057] Tremor score Two evaluators (one blinded) evaluated the tremor according to the following criteria: Score 1: Occasional tremor Score 2: Continuous slight tremor Score 3: Whole head tremor Score 4: Tremor from the trunk

[0058] Results: Gene replacement therapy was attempted in IGD model mice. AAVPHPeB, a serotype that effectively infects the nervous system in mice, or AAV9, a serotype used in humans, was used to express large amounts of PIGO using the CBA promoter. The virus solution was administered intravenously or intracerebroventricularly on days 1-2 after birth. The results are shown in Figures 5-7. Intravenous administration of AAVPHPeB-PIGO improved many phenotypes, including growth retardation, muscle weakness, and proneness to seizures (Figures 5 and 6), but resulted in a high incidence of hepatocellular carcinoma one year later. Intravenous administration of AAV9CBA-PIGO was less effective than AAVPHPeB-PIGO (Figures 5 and 6), and resulted in a high incidence of hepatocellular carcinoma one year later. Intracerebroventricular administration of AAV9CBA-PIGO was found to be as effective as or even more effective than intravenous administration of AAVPHPeB-PIGO (Figs. 5 to 7), but resulted in a high incidence of liver cancer one year later.

[0059] The promoter region upstream of the PIGO gene was cloned at various lengths, and a luciferase gene was connected downstream of it. These clones were then introduced into Neuro2a cells, a mouse neuronal cell line, and the promoter activity was compared. Figure 8 shows a schematic diagram of the PIGO promoters used. The results are shown in Figure 9. p320L1, p590L1, p790L1, and p970L1 all showed moderate promoter activity, while p320L2 showed lower promoter activity.

[0060] The PIGO gene was linked to the promoter sequence and inserted into an improved version of AAV9, AAVGTX (another name for yfAAV9 / 3, described in Iida A, et al., Systemic delivery of tyrosine-mutant AAV vectors results in robust transduction of neurons in adult mice. Biomed Res Int. 2013;2013:974819). PigoKO Neuro2a cells were infected, and the recovery rates of the GPI-AP CD24 were compared. The results are shown in Figure 10. The endogenous promoters p320L1 and p590L1 showed recovery rates equivalent to those of the strong promoter CBA.

[0061] AAVGTXp590L1PIGO was administered intracerebroventricularly to IGD model mice immediately after birth, and changes in body weight were recorded. A hanging test was performed, and tremor scores were measured. The results are shown in Figures 11 to 13. When AAVGTXp590L1PIGO was administered intracerebroventricularly, it had the same effect as the CBA promoter. Furthermore, survival time was extended (Figure 14).

[0062] The present disclosure provides methods for treating IGD and is useful in the medical field.

Claims

1. An adeno-associated virus (AAV) vector comprising: (a) a promoter comprising a nucleotide sequence having at least about 90% identity to the nucleotide sequence of SEQ ID NO: 3, and having activity equivalent to or greater than that of a promoter consisting of the nucleotide sequence of SEQ ID NO: 3; and (b) a polynucleotide encoding a phosphatidylinositol glycan anchor biosynthesis class O (PIGO).

2. The vector of claim 1, wherein the promoter comprises a nucleotide sequence of any one of SEQ ID NOs: 3 to 5.

3. The vector according to claim 1, wherein the promoter comprises a nucleotide sequence of any one of SEQ ID NOs: 3 to 5 and consists of a part of the nucleotide sequence of SEQ ID NO:

6.

4. The vector according to claim 1, wherein the promoter consists of a nucleotide sequence of any one of SEQ ID NOs: 3 to 5.

5. The vector of claim 1, wherein PIGO consists of an amino acid sequence having approximately 90% or more identity with the amino acid sequence of SEQ ID NO:

1.

6. The vector of claim 1, wherein PIGO consists of the amino acid sequence of SEQ ID NO:

1.

7. The vector of claim 1, which is an AAV9 vector.

8. A composition for treating congenital GPI deficiency caused by a defect in the PIGO gene, comprising the vector according to any one of claims 1 to 7.

9. The composition according to claim 8, wherein the abnormality in the PIGO gene is a T130N mutation.

10. The composition of claim 8, which is administered intravenously, intracerebroventricularly, intracisternally, or intrathecally.

11. The composition of claim 8, which is administered in a single dose.

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