WO2023131345A1 - Médicament et méthode de traitement génique de d'adrénoleucodystrophie liée à l'x - Google Patents

Médicament et méthode de traitement génique de d'adrénoleucodystrophie liée à l'x Download PDF

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WO2023131345A1
WO2023131345A1 PCT/CN2023/071616 CN2023071616W WO2023131345A1 WO 2023131345 A1 WO2023131345 A1 WO 2023131345A1 CN 2023071616 W CN2023071616 W CN 2023071616W WO 2023131345 A1 WO2023131345 A1 WO 2023131345A1
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target
vector
sequence
abcd1
expression
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吴小兵
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北京锦篮基因科技有限公司
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Definitions

  • the present invention relates to the field of gene therapy, and more specifically to nucleic acids, expression constructs, and pharmaceutical compositions comprising the nucleic acid or constructs for treating X-chromosome-linked adrenoleukodystrophy (X-ALD) and for treating X-ALD.
  • ALD method X-chromosome-linked adrenoleukodystrophy
  • X-linked adrenoleukodystrophy is a common metabolic genetic disorder characterized by progressive demyelination of the central nervous system (CNS), adrenal insufficiency, and very long-chain saturated fatty acids accumulation.
  • X-ALD The clinical types of X-ALD include Addison type, cerebral type (childrenal type, adolescent type, and adult type), adrenomyeloneuropathy type (AMN) with or without intracranial demyelination, asymptomatic or presymptomatic type , and the heterozygous type.
  • the disease primarily affects the adrenal cortex and nervous system.
  • the pathological changes of the nervous system are diverse, ranging from inflammatory demyelination to axonal lesions involving the ascending and descending fiber tracts of the spinal cord. (J. Berger, Pathophysiology of X-linked adrenoleukodystrophy, Biochimie 98(2014) 135e142)
  • X-ALD disease is caused by mutations in the ABCD1 gene (ALD).
  • the gene is located at Xq28, consists of 10 exons, and encodes the transmembrane ATP-binding cassette transporter (ATP-binding cassette transporter, ABCD1) on the peroxisome.
  • the ABCD1 protein is responsible for the transport of CoA-activated very long-chain fatty acids (VLCFAs) from the cytoplasm into the peroxisome for degradation. Mutations in the ABCD1 gene will lead to the incapacity of this transmembrane protein, which in turn will cause the accumulation of VLCFA in cells, resulting in neurotoxicity and adrenal toxicity.
  • gene therapy based on recombinant AAV viral vectors is considered to be a safer approach than lentiviruses because it does not depend on the integration of the viral genome in the host cell nucleus.
  • AAV virus also has the advantages of wide host cell range and long expression time in vivo, which make it one of the most promising gene therapy vectors.
  • Cardiotoxicity has been observed in both non-clinical and clinical studies of the marketed AAV gene therapy drug 'Zolgensma'. According to the analysis, this is related to the high-dose use of AAV9 vector and the expression of the target gene in heart tissue.
  • Yi Gong et al. have also pointed out that although the gene construct used ABCD1 could be successfully delivered to CNS cells by IV injection, but significant cardiac tissue toxicity was induced due to the expression level of the transgene in liver and cardiac tissue much higher than that in CNS.
  • the ABCD1 gene is a widely expressed housekeeping gene, and its expression can be detected in most tissues.
  • ABCD1 protein is abundantly expressed in the central nervous system, adrenal gland, testis and other parts, indicating that the metabolism of very long-chain fatty acids in these tissues is relatively strong.
  • the ABCD1 gene is deleted, the ABCD1 protein level in the central nervous system, adrenal gland, testis and other parts decreases, resulting in the blockage of the very long-chain fatty acid metabolic pathway, causing cell oxidative stress and cell death. Therefore, gene therapy for X-ALD needs to take into account both the central and peripheral regions.
  • the inventors have made in-depth research, and by optimizing and combining multiple gene elements, they have proposed a method that can effectively alleviate the main affected organs (central nervous system and adrenal gland) of X-ALD after systemic administration. ) disease burden, and at the same time have a new AAV virus vector with low drug peripheral tissue toxicity, and its use.
  • the expression construct of the present invention realizes high-efficiency expression of ABCD1 through the optimized promoter and ABCD1 encoding nucleic acid; at the same time, by including an optimized combination of miRNA target sequences in the 3'UTR, the toxicity of transgene overexpression to peripheral organs is reduced , especially myocardial tissue toxicity.
  • the invention provides a nucleic acid encoding ABCD1 comprising: SEQ ID No: 1, or at least 90%, 95%, 96%, 97%, 98%, 99%, 99.1% thereof , 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% identical nucleotide sequences.
  • the expression level (preferably, the expression level in mammalian cells) is increased by at least 200% or more, such as at least 300%, 320%, 330%, 340%, 350%, 360% %, 370%, 380%, 390%, or 400%.
  • the ABCD1-encoding nucleic acid according to the invention comprises a Kozak sequence at the 5' end. According to the in vitro transient expression test described in Example 1, it can be determined that the expression efficiency of the encoding nucleic acid is improved compared with the reference nucleic acid.
  • the present invention provides an expression construct comprising the ABCD1 encoding nucleic acid according to the present invention, especially comprising the nucleotide sequence of SEQ ID NO:1.
  • the expression construct according to the present invention further comprises a promoter, such as a constitutive promoter, operably linked to the ABCD1 encoding nucleic acid.
  • a promoter such as a constitutive promoter
  • the promoter comprises a nucleotide sequence selected from SEQ ID No: 2-5, or nucleotides having at least 95%, 96%, 97%, 98%, 99% or 99.5% identity therewith Sequence, especially the nucleotide sequence of SEQ ID No:2.
  • the expression construct according to the present invention further comprises a 3'UTR operably linked to said ABCD1 encoding nucleic acid.
  • the 3'UTR may comprise at least 1 copy (eg, 1-8 copies, such as 2, 3, or 4 copies) of the target sequence of a myocyte-specific miRNA and/or at least 1 copy (eg, 1-8 copies, eg, 1 or 2 copies) of the target sequence of the hepatocyte-specific miRNA.
  • the 3'UTR comprises at least 1 copy (such as 3 copies) of a myocyte-specific miRNA target sequence and at least 1 (such as 2 copies) of a hepatocyte-specific miRNA target sequence.
  • the at least one myocyte-specific miRNA target sequence and the at least one hepatocyte-specific miRNA target sequence may be included in the 3'UTR in various arrangements, but preferably, the myocyte-specific miRNA target sequence is Hepatocyte-specific miRNA target sequences spaced apart.
  • the miRNA target sequences can be connected directly, or can be separated by a few nucleotides, such as 1-5 nucleotides.
  • the muscle cell-specific miRNA target sequence that can be used in the present invention can be selected from miRNA1 target sequence, miRNA206 target sequence and combinations thereof; for example, the miRNA1 target sequence shown in SEQ ID NO:6 and the miRNA1 target sequence shown in SEQ ID NO:7 miRNA206 target sequence.
  • the hepatocyte-specific miRNA target sequence that can be used in the present invention can be a miRNA122 target sequence, such as the miRNA122 target sequence shown in SEQ ID NO:8.
  • the 3'UTR comprises miRNA target sequences arranged as follows: miRNA1 target sequence-miRNA122 target sequence-miRNA1 target sequence-miRNA122 target sequence-miRNA206 target sequence; or miRNA206 target sequence-miRNA122 target sequence- miRNA1 target sequence - miRNA122 target sequence - miRNA122 target sequence - miRNA1 target sequence.
  • the expression construct according to the present invention comprises a 3'UTR operably linked to the ABCD1 encoding nucleic acid, said 3'UTR comprising the nucleotide sequence of SEQ ID NO:12.
  • the invention provides a vector comprising an expression construct according to the invention.
  • the vector is a plasmid or viral vector.
  • the vector is an adeno-associated viral (AAV) vector.
  • the invention also provides a host cell, preferably a mammalian cell, comprising an expression construct or vector according to the invention.
  • the present invention provides a recombinant adeno-associated virus (AAV) vector, wherein said recombinant AAV vector comprises in its genome an expression construct according to the present invention.
  • the recombinant AAV vector comprises in its genome: (a) 5' and 3' AAV inverted terminal repeat (ITR) sequences, and (b) an expression construct located between the 5' and 3' ITR, wherein said expression construct comprises the following elements functionally linked to each other in the direction of transcription: a promoter; a polynucleotide encoding human ABCD1; at least one miRNA target sequence; one or more terminators; and one or more polyA signal sequences .
  • the polyA signal sequence is selected from the group consisting of SV40 late polyA sequence, rabbit ⁇ -globin polyA sequence, and bovine growth hormone polyA sequence, more preferably bovine growth hormone polyA sequence, such as the nucleotide sequence of SEQ ID NO:13.
  • the recombinant AAV viral vector according to the present invention may be a ssAAV vector or a scAAV vector.
  • a recombinant AAV viral vector according to the invention comprises a wild-type AAV2 ITR sequence, or in the case of scAAV vectors, one of said ITRs is a wild-type AAV2 ITR sequence and the other of said ITRs lacks a functional AA2 ⁇ ITR sequence of terminal melting sites (trs) and optionally D sequence.
  • the recombinant AAV viral vector according to the present invention may comprise capsid proteins from any AAV serotype, such as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9 serotypes, especially AAV9 serotypes .
  • the recombinant AAV vector is an AAV2/9 vector having AAV2 ITR sequence and AA9 capsid protein.
  • the present invention also provides a pharmaceutical composition
  • a pharmaceutical composition comprising an expression construct according to the present invention, a vector according to the present invention or a recombinant AAV viral vector according to the present invention and a pharmaceutically acceptable carrier.
  • the pharmaceutical composition may be formulated for different routes of administration, such as intraperitoneal, intramuscular, intraarterial, intravenous, intrathecal or intracerebroventricular administration, as desired.
  • the pharmaceutical composition is an intravenous injection formulation.
  • the formulation comprises a prophylactically or therapeutically effective amount of a recombinant AAV viral vector according to the invention.
  • the invention also relates to the use of an expression construct according to the invention or a vector according to the invention or a recombinant AAV viral vector according to the invention for expressing ABCD1 in a mammalian cell, cell line or cell population, or Use in the preparation of a medicament for expressing ABCD1 in mammalian cells, cell lines or cell groups.
  • the mammalian cells may be in vitro, ex vivo or in vivo.
  • the present invention also provides a method (such as an in vitro, in vivo, or ex vivo method) of expressing ABCD1 in mammalian cells, said method comprising using an expression construct according to the present invention or a method according to the present invention vector to transfect isolated mammalian cells, cell lines or cell populations.
  • the mammalian cell is a cell line, or a cell, such as a fibroblast, isolated from an ABCD1-deficient mammalian subject.
  • the impact of the construct, vector or recombinant AAV virus vector of the present invention on the expression and/or viability of the target protein of the cells can be monitored by testing the expression of the target protein and/or the growth rate of the cell in vitro or in isolated cells.
  • the present invention provides a method for treating or preventing X-chromosome-linked adrenoleukodystrophy (X-ALD) and/or improving symptoms associated with X-ALD, wherein the method comprises administering to a subject in need
  • the method comprises intraperitoneal, intramuscular, intraarterial, intravenous, intrathecal or intracerebroventricular administration, more preferably intravenous injection of a recombinant AAV viral vector according to the invention.
  • the recombinant AAV viral vector is an AAV9 vector.
  • the plasma and tissues of the subject especially the adrenal gland and cerebellum, the biochemical markers of X-ALD disease—very long chain fatty acid VLCFA (especially C26:0 fatty acid)——The content is reduced.
  • VLCFA very long chain fatty acid
  • autophagic activity stimulated by VLCFA accumulation in the subject's spinal cord is alleviated/improved by IV administration of a recombinant AAV viral vector of the invention.
  • the methods of the invention ameliorate/alleviate symptoms of progressive demyelination of the subject's CNS.
  • the present invention also provides an expression construct comprising a 3'UTR operably linked to a gene, wherein said 3'UTR comprises:
  • -miRNA1 target x2 ie, 2 copies of the miRNA1 target sequence
  • -miRNA206 target x1 ie, 1 copy of the miRNA206 target sequence
  • -miRNA122 target x2 ie, 2 copies of the miRNA122 target sequence
  • -miRNA1 target x2 i.e., 2 copies of the miRNA1 target sequence
  • -miRNA122 target x2 i.e., 2 copies of the miRNA122 target sequence
  • -miRNA206 target x1 i.e., 1 copy of the miRNA206 target sequence
  • SEQ ID NO:10 SEQ ID NO:10
  • -miRNA1 target x1 i.e., 1 copy of miRNA1 target sequence
  • -miRNA122 target x1 i.e., 1 copy of miRNA122 target sequence
  • -miRNA1 target x1 i.e., 1 copy of miRNA1 target sequence
  • miRNA122 target x1 i.e., 1 copy of miRNA122 target sequence
  • miRNA206 target x1 i.e., 1 copy of the miRNA206 target sequence
  • the expression construct according to the present invention can be used to selectively reduce the expression of a gene of interest operably linked thereto in muscle cells and liver cells, or to prepare a gene for selectively reducing the expression of a gene of interest in Use in drugs expressed in muscle cells and hepatocytes.
  • Figure 1 shows the schematic diagram of the expression construct used in the present invention ( Figure 1A) and the expression detection results of the optimized ABCD1 encoding nucleic acid ( Figure 1B).
  • ITR ITR sequence of AAV virus
  • CA CA promoter
  • CAR-Mut CAR promoter with mutation
  • hABCD1 natural human ABCD1 encoding nucleic acid
  • coABCD1 optimized ABCD1 encoding nucleic acid
  • EGFP enhanced green Fluorescent protein
  • polyA polyA signal sequence
  • 3'UTR 3' untranslated region.
  • the left panel of Figure 1B shows that the natural human ABCD1-encoding nucleic acid (hABCD1) and the optimized ABCD1-encoding nucleic acid (coABCD1) were transiently expressed in HEK293 cells, the cells were lysed after 48 hours, and the total cellular protein was extracted and separated by electrophoresis on 10% SDS-PAGE Protein bands (the upper left figure shows the ABCD1 protein band, and the lower left figure shows the actin band); the right figure in Figure 1B shows that the fluorescent color results of the bands separated by electrophoresis are relatively gray after grayscale scanning degree value.
  • Figure 2 shows a schematic diagram of the pscAAV-CAR-Gluc plasmid vector.
  • Figure 3 shows that in an assay based on cultured cells in vitro, compared with BHK-21 cells that were not transfected with the plasmid (i.e., blank control), the pscAAV-CAR-Gluc vector and the pscAAV-CAR-MutC-Gluc vector were transfected. Changes in Gluc levels measured in BHK-21 cells with vector, pscAAV-CAR-MutA-Gluc vector and pscAAV-CAR-MutG-Gluc vector. Among them, ** means p ⁇ 0.01.
  • Figure 4 shows that in the EGFP-based expression plasmid vector, the impact of the target sequence embedded in miRNA1, 122, and 206 on the expression of the target gene in muscle cells and liver cells was detected.
  • Figure 5 shows that in the expression plasmid vector based on the Gluc reporter gene, the effect of increasing the number of repetitions of the miRNA target sequence and changing the arrangement and combination of the miRNA target sequence on the expression of the target gene in muscle cells was detected.
  • Figure 6 shows that in the Gluc reporter gene-based expression plasmid vector, the effect of increasing the number of repeats of the miRNA target sequence and changing the arrangement and combination of the miRNA target sequence on the expression of the target gene in hepatocytes was detected.
  • Figure 7 shows a schematic diagram of the basic plasmid pRDAAV-CMV-EGFP used to construct the AAV plasmid vector.
  • Figure 8 shows that using fibroblasts from X-ALD patient 1 and patient 2, the impact of recombinant AAV9-coABCD1 virus transduction on the expression of ABCD1 in cells was determined in the cell slide immunofluorescence assay.
  • the red fluorescence shows the ABCD1 protein in the cytoplasm stained by the dylight549-labeled secondary antibody; the blue fluorescence shows the nuclei stained by dapi.
  • Figure 9 shows the effect of recombinant AAV9-coABCD1 virus transduction on cell growth and proliferation rate.
  • Figures 9A and 9B show the cell densities of patient 2 fibroblasts not inoculated with AAV9-coABCD1 and inoculated with AAV9-coABCD1 at 7 days in culture, respectively.
  • Figure 9C shows the cell density of normal human fibroblasts in culture for 7 days.
  • Figure 10 shows the changes in the content of very long chain fatty acids in various tissues after injection of AA9-coABCD1 or AAV9-coABCD1-miT recombinant virus compared to untreated C57BL6J wild-type mice (WT). Among them, * means p ⁇ 0.05.
  • Figure 11 shows the histopathological changes in the test animals after injection of AA9-coABCD1 or AAV9-coABCD1-miT recombinant virus.
  • Figure 11A shows that inoculation with the optimized recombinant virus rAAV9-coABCD1-miT significantly reduced the pathological changes in the hearts and livers of the tested mice.
  • Black arrows indicate vacuolar degeneration of the heart and nuclear pyknosis and focal necrosis of the liver.
  • Figure 11B shows the autopsy results of a dead mouse in the test animal group inoculated with rAAV9-coABCD1, the histopathological examination results of the heart tissue in the above figure, the black arrow indicates the occurrence of large-area vacuolar degeneration of the myocardium; Red arrows indicate extensive intracardiac thrombus formation; lower panel shows histopathological examination of liver tissue, showing extensive hepatocyte necrosis and nuclear pyknosis.
  • FIG. 12 shows the evaluation of the effect of administering AAV9-coABCD1-miT on the behavior of X-ALD model mice in the rope grabbing experiment. The results showed that intravenous administration of AAV9-coABCD1-miT effectively improved the exercise capacity of model mice.
  • Figure 13 shows that 8 weeks after the optimized drug AAV9-coABCD1-miT was administered to X-ALD model mice, the content of very long chain fatty acids in each tissue was detected, and compared with unadministered X-ALD model mice and wild animals. normal mice for comparison. Among them, * indicates p ⁇ 0.05; NS indicates that the difference is not significant.
  • Figure 14 shows the histopathological results of the adrenal glands of X-ALD model mice treated with AAV9-coABCD1-miT tail vein injection 8 weeks after administration.
  • the red fluorescence shows the ABCD1 protein in the immunofluorescence-labeled cells; the blue fluorescence shows that the nuclei are stained.
  • Figure 15 shows that after AAV9-coABCD1-miT administration, LC3 ⁇ was immunofluorescently labeled on spinal cord tissue sections to evaluate the autophagy and improvement in the nervous system of X-ALD model mice.
  • Figure 16 shows that after intravenous injection of optimized (AAV9-coABCD1-miRT) and unoptimized drugs (AAV9-coABCD1) into normal wild-type mice (2 mice in each group), the Western Blot method was used to determine the ABCD1 protein content.
  • Figure 16A shows the Western Blot detection results, wherein the upper figure shows the ABCD1 band, and the lower figure shows the ⁇ -actin band; wherein, lanes 1 and 2 are mice administered with AAV9-coABCD1 (RD48-1 and RD48 -2); lanes 3-4 are mice administered with AAV9-coABCD1-miRT (RD49-2 and RD49-4); lanes 5 and 6 are normal mouse controls without administration (N1 and N2).
  • Figure 16B shows the quantification results obtained by analyzing the gray value of the Western Blot detection result pictures using the software ImageJ.
  • the invention discloses a gene therapy construct, a pharmaceutical composition and a method for treating X-ALD, especially the construction, preparation and application of a recombinant AAV vector for delivering ABCD1.
  • the expression "and/or”, when used to connect multiple items, means any one of the listed related items, or any and all possible combinations of a plurality or all of the listed related items.
  • recombinant adeno-associated virus can be represented by the AAV virus serotype from which the capsid is derived alone, or by the AAV virus serotype from which the capsid and genomic ITR sequences are derived.
  • the identifier "/" is used for separation, followed by the serotype of origin of the capsid and before the identifier "/" by the serotype of origin of the ITR.
  • the number 9 in the expression recombinant AAV9 indicates that the recombinant adeno-associated virus has a capsid from the AAV9 serotype; while the number before the identifier "/" in the expression recombinant AAV2/9 indicates that the recombinant adeno-associated virus has The wild-type or variant ITR sequence from AAV2, while the number after the identifier "/" indicates that the recombinant adeno-associated virus has a capsid protein from AAV9.
  • sequence identity is used to describe the similarity in sequence structure between two amino acid sequences or polynucleotide sequences.
  • sequences can be aligned for optimal comparison purposes (e.g., the first and second amino acid sequences or the first and second amino acid sequences can be aligned for optimal alignment). Gaps may be introduced into one or both of the first and second nucleic acid sequences or non-homologous sequences may be discarded for comparison purposes).
  • the length of the reference sequence being aligned is at least 30%, preferably at least 40%, more preferably at least 50%, 60% and even more preferably at least 70%, 80%, 90%, 100%.
  • amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide at the corresponding position in the second sequence, then the first and second sequences are identical at that position.
  • the comparison of sequences and the calculation of percent identity between two sequences can be accomplished using a mathematical algorithm.
  • the Needlema and Wunsch ((1970) J. Mol. Biol. 48:444-453) algorithm (available at http://www.gcg.com available), use the Blossum 62 matrix or the PAM250 matrix with gap weights of 16, 14, 12, 10, 8, 6 or 4 and length weights of 1, 2, 3, 4, 5 or 6 to determine the distance between two amino acid sequences. percent identity.
  • using the GAP program in the GCG software package (available at http://www.gcg.com), using the NWSgapdna.CMP matrix and gap weights of 40, 50, 60, 70 or 80 and Length weights of 1, 2, 3, 4, 5 or 6 determine the percent identity between two nucleotide sequences.
  • a particularly preferred parameter set (and one that should be used unless otherwise stated) is the Blossum 62 scoring matrix with a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5.
  • the term "host cell” refers to a cell into which an exogenous polynucleotide has been introduced, including the progeny of such cells.
  • the host cell is any type of cell that can be used to produce a recombinant AAV vector of the invention, for example, mammalian cells (such as HEK 293 cells suitable for production of recombinant AAV by a three-plasmid packaging system) and insect cells ( For example sf9 cells suitable for the production of recombinant AAV by the baculovirus packaging system).
  • regulatory sequence refers to a nucleic acid sequence that induces, represses, or otherwise controls the transcription of a protein of an encoding nucleic acid sequence to which it is operably linked. Regulatory sequences can be, for example, initiation sequences, enhancer sequences, intron sequences, and promoter sequences, among others.
  • exogenous or heterologous are used interchangeably when describing a nucleic acid or protein to mean that the nucleic acid or protein does not naturally exist in the chromosomal or host cell location in which it is found.
  • An exogenous nucleic acid sequence also refers to a sequence that is derived from and inserted into the same host cell or subject but exists in a non-native state, eg, the sequence is present in a different copy number, or is under the control of a different regulatory element.
  • an "isolated" polynucleotide eg, isolated DNA or isolated RNA
  • an "isolated" nucleic acid is enriched at least about 10-fold, 100-fold, 1000-fold, 10,000-fold or more relative to the starting material.
  • an "isolated" polypeptide refers to a polypeptide that is at least partially separated from at least some other components of the native organism or virus in which it is contained. In some embodiments, an “isolated” polypeptide is enriched at least about 10-fold, 100-fold, 1000-fold, 10,000-fold or more relative to the starting material.
  • an “isolated” or “purified” viral vector means that the viral vector has been partially separated from at least some components of the starting material comprising it. In some embodiments, an “isolated” viral vector is enriched at least about 10-fold, 100-fold, 1000-fold, 10,000-fold or more relative to the starting material.
  • viral vector refers to a viral particle (such as an AAV viral particle) capable of serving as a delivery vehicle for a nucleic acid of interest.
  • a viral vector comprises a capsid and a viral genome (for example, viral DNA) packaged therein, and the target nucleic acid to be delivered is inserted into the viral genome.
  • recombinant AAV viral vectors in order to generate recombinant virus particles that can deliver the nucleic acid of interest to tissues or cells, it is usually only necessary to retain the inverted terminal repeat (ITR) cis element in the genome, while the rest required for viral packaging Sequences can be provided in trans.
  • ITR inverted terminal repeat
  • the recombinant AAV viral vectors of the present invention comprise a capsid and a recombinant viral genome packaged therein, wherein the recombinant viral genome comprises or consists of one or more exogenous genes located between two AAV ITR sequences.
  • Source nucleotide sequence composition The two ITR sequences located at the 5' and 3' ends of the recombinant viral genome (i.e., 5'ITR and 3'ITR) may be the same or different.
  • AAV "inverted terminal repeat” refers herein to a cis-acting element from the AAV viral genome that plays an important role in the integration, rescue, replication, and genome packaging of the AAV virus.
  • the ITR sequence of the natural AAV virus contains a Rep protein binding site (Rep binding site, RBS) and a terminal unzipping site trs (terminal resolution site), which can be recognized by the Rep protein and generate a nick at the trs.
  • the ITR sequence can also form a unique "T" letter-shaped secondary structure, which plays an important role in the life cycle of the AAV virus.
  • AAV2 The earliest isolated AAV virus, AAV2, has "inverted terminal repeats" (ITRs) with a palindrome-hairpin structure of 145 bp located at both ends of the genome. Later, different ITR sequences were found in various serotypes of AAV viruses, but they all formed hairpin structures and had Rep binding sites.
  • ITRs inverted terminal repeats
  • Traditional recombinant AAV viral vectors based on these wild-type ITR sequences are generally single-stranded AAV vectors (ssAAV), and the viral genome is packaged in the AAV capsid in a single-stranded form.
  • the genome carried by the recombinant AAV virus vector obtained by packaging can be self-complementary to form a double chain (Wang Z et al., Gene Ther. 2003; 10(26):2105-2111; McCarty DM et al., Gene Ther. 2003; 10(26):2112-2118).
  • the virus thus packaged is a double-stranded AAV virus, that is, scAAV (self-complementary AAV) virus.
  • the packaging capacity of the scAAV viral vector is smaller, only half of the packaging capacity of the ssAAV viral vector, about 2.2kb-2.5kb, but the transduction efficiency after infection of cells is higher.
  • ITR in relation to AAV encompasses wild-type ITRs and variant ITRs.
  • Wild-type ITRs can be from any native AAV virus, such as an AAV2 virus.
  • the wild-type ITR contains a Rep protein binding site (Rep binding site, RBS) and a terminal unzipping site trs (terminal resolution site), which can be recognized by the Rep protein and generate a nick at trs.
  • the wild-type ITR sequence can form a unique "T" letter-shaped secondary structure, which plays an important role in the life cycle of AAV virus.
  • a variant ITR is a non-native ITR sequence which may, for example, be derived from any wild-type AAV ITR sequence and which comprises a deletion, substitution, and/or addition of one or more nucleotides relative to the wild-type ITR, and/ Or truncated, but still functional, ie, can be used to generate ssAAV viral vectors or scAAV viral vectors.
  • a variant ITR is an AAV ITR sequence (also referred to herein as a ⁇ ITR) that has been deleted for a functional trs site and optionally a D region sequence.
  • wild-type ITRs are combined with ⁇ ITRs to generate self-complementary recombinant AAV viral vectors (scAAV).
  • scAAV self-complementary recombinant AAV viral vectors
  • two wild-type ITRs are used in combination to generate single-chain recombinant AAV viral vectors (ssAAV).
  • the AAV proteins VP1, VP2 and VP3 are capsid proteins that interact to form the AAV capsid.
  • Different serotypes of AAV viruses have different tissue infection tropisms, and foreign genes can be transferred to specific organs and tissues by selecting the source serotype of the recombinant AAV virus vector capsid (Wu Z et al., Mol Ther.2006; 14(3):316-327).
  • the recombinant AAV virus vector can have different targeting properties by selecting the source serotype of the capsid.
  • the capsid of the recombinant AAV virus is from an AAV serotype that targets neuronal cells.
  • the recombinant AAV viral vector comprises a capsid from AAV9.
  • the recombinant AAV viral vector comprises a capsid from AAV9 and an ITR from AAV2.
  • treatment refers to medical intervention intended to alter the natural course of a disease in the individual being treated. Desired therapeutic effects include, but are not limited to, preventing the onset or recurrence of the disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, ameliorating or palliation of the disease state, and remission or improved prognosis.
  • the recombinant AAV virus of the present invention after administration to an ABCD1-deficient subject or an X-ALD patient, preferably after systemic administration, reduces the number of affected tissues (especially, the adrenal gland and central nervous system) of the subject. Nervous system) VLCFA content.
  • the recombinant AAV virus of the present invention improves central nervous system damage and/or adrenal damage in a subject after administration to an ABCD1-deficient subject or an X-ALD patient, preferably after systemic administration. In some embodiments, the recombinant AAV virus of the invention improves the exercise capacity of the subject after administration to an ABCD1-deficient subject or an X-ALD patient, preferably after systemic administration.
  • prevention includes the inhibition of the occurrence or development of a disease or symptoms of a particular disease.
  • subjects predisposed to developing X-ALD disease are candidates for prophylactic regimens.
  • prevention refers to medical intervention performed before at least one symptom of a disease occurs. Therefore, in one embodiment, prevention includes administering the gene therapy drug of the present invention in subjects with ABCD1 gene deficiency before the symptoms of X-ALD disease occur, so as to delay the development of the disease or prevent the appearance of the disease.
  • the prevention includes using the gene therapy drug of the present invention to improve the abnormal phagocytosis stimulated by VLCFA in the nervous system, thereby preventing the occurrence of related spinal cord axonal lesions.
  • the gene construct according to the present invention has at least one or more optimized gene elements as follows: (1) optimized ABCD1 encoding nucleic acid (also referred to as coABCD1 for short); (2) optimized constitutive promoter; and (3) optimized combination of miRNA target sequences.
  • the ABCD1-encoding nucleic acid contained in the construct of the present invention may be any polynucleotide capable of encoding functional ABCD1 protein activity. However, in order to facilitate expression in mammalian cells, it is advantageous to optimize the nucleic acid sequence of the nucleic acid encoding the ABCD1 polypeptide.
  • the ABCD1 encoding nucleic acid used in the expression construct of the present invention comprises the polynucleotide sequence of SEQ ID NO: 1, or has at least about 95%, about 96%, about 97%, 98%, Polynucleotide sequences with 99% or greater nucleotide sequence identity.
  • the optimized ABCD1-encoding nucleic acid with respect to a reference nucleic acid (for example, a natural human ABCD1-encoding nucleic acid, such as a nucleic acid having the nucleotide sequence shown in SEQ ID NO: 15), is in an operably linked composition
  • Assays for determining protein expression levels are known in the art. Any such assay can be used by one skilled in the art to determine the degree of optimization achieved in terms of expression efficiency of an optimized ABCD1-encoding nucleic acid compared to a reference nucleic acid.
  • the optimized ABCD1-encoding nucleic acid according to the present invention may comprise a Kozak sequence located upstream of the start codon at the 5' end.
  • the Kozak sequence used in the present invention may be a consensus sequence defined as GCCRCC, wherein R is a purine (ie A or G), and wherein said sequence is located upstream of the start codon.
  • the construct of the invention comprises a Kozak sequence, wherein said Kozak sequence has a 5'-GCCACC-3' sequence. Other different Kozak sequences can also be used in the constructs of the invention.
  • the construct of the present invention may contain any promoter that can be used to promote the expression of an ABCD1-encoding nucleic acid in a mammalian cell of interest.
  • the mutant constitutive promoter CAR-Mut according to the invention is included.
  • the CAR-Mut constitutive promoter of the present invention can efficiently promote the expression of exogenous genes in various tissues, so it is especially suitable for use in the treatment method of the present invention, so as to take into account the peripheral and central therapeutic purposes of X-ALD.
  • the construct of the invention comprises a CAR-Mut promoter comprising a polynucleotide selected from the group consisting of:
  • the polynucleotide has a mutated nucleotide C or G or A at nucleotide 568 of SEQ ID NO:5 or the corresponding position, more preferably T is mutated to C.
  • the mutant promoter of the present invention increases the expression of a gene of interest functionally linked thereto, e.g., makes said gene of interest Gene expression is increased by 1%-70%, eg, at least 5%, 10%, 20%, 30%, 40%, or at least 50%, 60%.
  • the mutant promoter of the present invention increases the expression of the gene of interest functionally linked thereto in mammalian cells or tissues, for example, increases the expression of the gene of interest in mammals relative to the reference promoter. Expression in peripheral tissues and/or central nervous tissues, especially in the central nervous system.
  • the mammal is a human or a non-human mammal, eg, a mouse, a rat and a non-human primate.
  • the promoter comprises a nucleotide sequence selected from any one of SEQ ID NOs: 2 to 4, or differs therefrom by one or several nucleotide substitutions, deletions and/or additions and has Nucleotide sequences with equivalent promoter activity.
  • the promoter comprises or consists of the nucleotide sequence of SEQ ID NO:2.
  • any promoter functional assay known in the art such as the luciferase reporter gene expression assay in Example 1
  • the reference promoter such as SEQ ID NO: 5
  • the promoter to be tested under the same test conditions, compared with the reference promoter (such as SEQ ID NO: 5), if the promoter to be tested has the same or substantially the same activity, for example, the activity of the reference promoter ⁇ 10% , preferably ⁇ 5%, or more preferably ⁇ 1%, then the promoter to be tested can be considered to have equivalent promoter activity.
  • MicroRNA is an endogenous small non-coding RNA that can regulate cellular gene expression in a sequence-specific manner, usually by repressing translation of target mRNAs. Endogenous miRNAs can suppress the expression of transgenes in expression cassettes that contain their perfectly complementary target sequences. The level of repression is related to factors such as the promoter used to express the construct, the corresponding miRNA abundance in the target tissue cells, and the like. According to in-depth research, the inventors found that in the expression construct containing a constitutive promoter, embedding specific types, quantities and arrangements of miRNA target cells in the 3' UTR can increase the safety of gene therapy based on the expression construct sex.
  • the expression constructs of the invention further comprise one or more miRNA target sequences located in the 3'UTR operably linked to the coding nucleic acid sequence of interest.
  • miRNA target sequences located in the 3'UTR operably linked to the coding nucleic acid sequence of interest.
  • inclusion of miRNA target sequences in expression constructs will allow for the modulation (eg, inhibition) of expression of a gene of interest in cells and tissues producing the corresponding miRNA.
  • the expression constructs of the invention comprise one or more miRNA target sequences, so that the expression of ABCD1 can be downregulated in a cell type specific manner.
  • miRNA target sequences that can be used in the present invention include target sequences of muscle cell-specific miRNA and target sequences of hepatocyte-specific miRNA.
  • the muscle cell-specific miRNA target sequence that can be used in the present invention can be selected from miRNA1 target sequence, miRNA206 target sequence and combinations thereof; for example, the miRNA1 target sequence shown in SEQ ID NO:6 and the miRNA1 target sequence shown in SEQ ID NO:7 miRNA206 target sequence.
  • the liver cell-specific miRNA target sequence that can be used in the present invention can be a miRNA122 target sequence, such as the miRNA122 target sequence of SEQ ID NO:8.
  • the liver cell-specific miRNA target sequence contained in the expression construct according to the present invention can be at least one or more, and preferably 2-4, which can be connected in series or arranged at intervals with the muscle cell-specific miRNA target sequence .
  • the muscle cell-specific miRNA target sequence contained in the expression construct according to the present invention can be at least one or more, and preferably 2-4, which can be connected in series or combined with the liver cell-specific miRNA target sequence. Sequence spaced.
  • the miRNA target sequences can be connected directly, or can be separated by a few nucleotides, such as 1-5 nucleotides.
  • an expression construct according to the invention comprises a 3' UTR operably linked to said ABCD1 encoding nucleic acid.
  • the 3'UTR comprises at least 1 copy (eg, 1-8 copies, such as 2, 3, or 4 copies) of the target sequence of a myocyte-specific miRNA and/or at least 1 copy (eg, 1 - 8 copies, eg 1 or 2 copies) of the target sequence of the hepatocyte-specific miRNA.
  • the 3'UTR comprises at least 1 copy (such as 3 copies) of a myocyte-specific miRNA target sequence and at least 1 (such as 2 copies) of a hepatocyte-specific miRNA target sequence.
  • the at least one myocyte-specific miRNA target sequence and the at least one hepatocyte-specific miRNA target sequence may be present in various arrangements in the 3'UTR, but preferably, the myocyte-specific miRNA target sequence Spaced with hepatocyte-specific miRNA target sequences.
  • the 3'UTR comprises miRNA target sequences arranged as follows: miRNA1 target sequence-miRNA122 target sequence-miRNA1 target sequence-miRNA122 target sequence-miRNA206 target sequence; or miRNA206 target sequence-miRNA122 target sequence- miRNA1 target sequence - miRNA122 target sequence - miRNA1 target sequence.
  • the expression construct according to the present invention comprises a 3'UTR operably linked to the ABCD1 encoding nucleic acid, said 3'UTR comprising the nucleotide sequence of SEQ ID NO:12.
  • the invention provides expression constructs.
  • the expression construct of the present invention can be advantageously used for gene therapy of X-ALD disease.
  • the expression construct of the invention comprises the following elements functionally linked to each other in the direction of transcription:
  • -encoding ABCD1 nucleic acid preferably, according to the optimized ABCD1 encoding nucleic acid of the present invention, more preferably comprises the nucleic acid of nucleotide sequence shown in SEQ ID NO:1,
  • the expression construct also includes two ITR sequences.
  • the expression construct may comprise elements arranged as follows: 5'ITR-promoter-ABCD1 coding sequence-miRNA target sequence-polyA-3'ITR.
  • the 5'ITR and 3'ITR are the same.
  • the 5'ITR and the 3'ITR are different and one (preferably the 3'ITR) is a ⁇ ITR lacking a functional trs site.
  • the 5'ITR and 3'ITR in the expression construct are identical and both comprise or consist of the AAV2 ITR sequence.
  • the promoter used in the expression construct of the present invention may be the CAR-Mut promoter described in any of the above embodiments of the present invention.
  • the promoter comprises or consists of the nucleotide sequence of SEQ ID No:2.
  • the promoter comprises or consists of the nucleotide sequence of SEQ ID NO:3.
  • the promoter comprises or consists of the nucleotide sequence of SEQ ID NO:4.
  • the ABCD1-encoding nucleic acid used in the expression construct of the present invention may be the ABCD1-encoding nucleic acid described in any of the above embodiments of the present invention.
  • the encoding nucleic acid comprises or consists of the nucleotide sequence of SEQ ID NO:1.
  • the miRNA target sequence used in the expression construct of the present invention may be the miRNA target sequence described in any of the above embodiments of the present invention, in particular, at least one hepatocyte-specific miRNA target sequence according to the present invention and at least one muscle specific miRNA target sequence according to the present invention.
  • Cell-specific miRNA target sequence preferably, comprises the nucleotide sequence of SEQ ID NO:12.
  • Transcription terminators useful in the present invention include any nucleic acid sequence that can terminate translation of a nucleic acid.
  • the terminator can be "TAG", “TGA” or “TAA”, and the corresponding RNA sequence is “UAG”, “UGA” or “UAA”.
  • the expression construct of the invention further comprises at least one polyA tail located downstream of the ABCD1 encoding nucleic acid and the miRNA target sequence.
  • Any suitable polyA sequence may be used, including but not limited to hGHpolyA, BGHpolyA, SV40 late polyA sequence, rabbit ⁇ -globin polyA sequence, or any variant thereof.
  • polyA is BGHpolyA, such as polyA shown in SEQ ID NO: 13, or has at least 80%, 85%, 90%, 95%, 96%, 97% with SEQ ID NO: 13, A polyA polynucleotide sequence having 98% or 99% nucleotide sequence identity.
  • the invention also provides vectors comprising expression constructs of the invention.
  • the vector is a plasmid (eg, a plasmid used for recombinant viral particle production).
  • the vector is a viral vector, such as a recombinant AAV vector or a baculovirus vector.
  • the genome of the recombinant AAV vector is single-stranded (eg, single-stranded DNA).
  • the genome of the recombinant AAV vector is self-complementary.
  • the present invention also provides host cells, such as mammalian cells or insect cells, comprising the expression construct or vector of the present invention.
  • the cells can be used to produce recombinant AAV viruses.
  • the invention provides recombinant AAV vectors.
  • the recombinant AAV vectors of the present invention are particularly useful for X-ALD disease or ameliorating symptoms associated therewith.
  • the recombinant AAV vector comprises a capsid and nucleic acid located within the capsid, also referred to herein as the "genome of the recombinant AAV vector.”
  • the genome of the recombinant AAV vector contains multiple elements, including but not limited to two inverted terminal repeats (ITRs, i.e., 5'-ITR and 3'-ITR), and other elements located between the two ITRs, including the promoter , a heterologous gene, and a polyA tail.
  • ITRs inverted terminal repeats
  • at least one miRNA target sequence may also be included between the two ITRs.
  • adeno-associated virus includes, but is not limited to, AAV of any serotype, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 AAV, and AAV with AAV with artificially altered capsid proteins.
  • AAV adeno-associated virus
  • the genome sequences of various serotypes and artificial AAVs and their native inverted terminal repeat (ITR) sequences, Rep proteins and capsid cap proteins are known in the art. These sequences can be found in public databases such as GenBank or in the literature.
  • the present invention provides a recombinant AAV viral vector comprising a capsid, wherein the capsid is composed of a capsid protein capable of crossing the blood-brain barrier, such as AAV9, AAVPHP.B, AAVPHP.eB capsid protein.
  • the recombinant AAV vectors of the invention transduce cells of the central nervous system (CNS), including neuronal cells and glial cells, as well as peripheral non-neuronal cells.
  • recombinant AAV vectors are capable of targeting and transducing neuronal cells, astrocytes, and microglia after systemic administration.
  • the recombinant AAV vector is capable of targeting and transducing the peripheral organs and central nervous system of a subject following systemic administration.
  • the recombinant AAV vector is capable of targeting and transducing multiple tissues (e.g., brain, spinal cord, adrenal gland) of a subject following systemic administration, and preferably, is compatible with non-administered recombinant AAV vectors.
  • the recombinant AAV vector resulted in higher expression of the foreign gene of interest (the gene encoding ABCD1 in this application) and/or reduced VLCFA levels in the targeted and transduced tissues compared to control subjects.
  • the therapeutic efficacy of the recombinant AAV vector or gene drug of the present invention can be detected by decreasing the cellular level of saturated linear VLCFA (C24:0 and C26:0, especially C26:0).
  • the recombinant AAV vectors of the invention have a capsid from an AAV9 serotype (also referred to herein as an AAV9 vector); preferably, the recombinant AAV vector has a wild-type or capsid from AAV2 in its genome.
  • AAV9 serotype also referred to herein as an AAV9 vector
  • Variant ITR sequences also referred to herein as AAV2/9 vectors.
  • the two ITR sequences of the recombinant AAV vector of the present invention are full-length ITRs (for example, about 125-145 bp in length, and contain a functional Rep binding site (RBS) and a terminal melting site ( trs)).
  • full-length functional ITRs are used to produce single-chain recombinant AAV vectors (ssAAV).
  • one of the ITRs of the recombinant AAV vector is truncated.
  • truncated ITRs lack functional terminal melting sites trs and are used to produce self-complementary recombinant AAV vectors (scAAV vectors).
  • the present invention provides a recombinant adeno-associated virus (AAV) vector, wherein said recombinant AAV vector comprises in its genome: 5' and 3' AAV inverted terminal repeat (ITR) sequences, and located at Expression construct according to the invention between the 5' and 3'ITR.
  • AAV adeno-associated virus
  • the amount of ABCD1 gene expression or the amount of ABCD1 protein expression in the cells is increased compared with that before the administration , thereby reducing the level of VLCFA in the cell, eg by at least 1-3 fold.
  • the recombinant viral vector of the present invention after being administered to a subject, results in a decrease in VLCFA levels in the adrenal gland and CNS such as cerebellum, brain, spinal cord, for example, at least 0.5 times lower than before administration, for example At least 1-3 times. Determination of this fold reduction can be performed according to standard methods known in the art for the quantification of VLCFAs.
  • AAV vector packaging systems mainly include three-plasmid co-transfection system, adenovirus as helper virus system, Herpes simplex virus type 1 (HSV1) as helper virus packaging system, and baculovirus-based packaging system. system.
  • HSV1 Herpes simplex virus type 1
  • baculovirus-based packaging system baculovirus-based packaging system.
  • Each packaging system has its own characteristics, and those skilled in the art can make appropriate choices according to needs.
  • the three-plasmid co-transfection packaging system is the most widely used AAV vector packaging system because it does not require helper virus and has high safety. It is also the mainstream production system in the world.
  • the slight disadvantage is that the absence of an efficient large-scale transfection method limits the application of the three-plasmid transfection system in the large-scale preparation of AAV vectors.
  • the recombinant AAV viral vectors of the invention can be produced using any suitable method known in the art.
  • the recombinant AAV virus of the present invention is produced using a three-plasmid packaging system.
  • the recombinant AAV virus of the present invention is produced using a baculovirus packaging system.
  • the present invention provides a pharmaceutical composition comprising a recombinant AAV viral vector of the present invention.
  • the pharmaceutical composition of the present invention preferably comprises a pharmaceutically acceptable excipient, diluent or carrier.
  • the pharmaceutical compositions of the present invention may be formulated in any suitable preparation form.
  • Suitable pharmaceutically acceptable excipients, diluents or carriers for formulation are well known in the art and include, for example, phosphate buffered saline, water, emulsions, such as oil/water emulsions, various types of Wet agent, sterile solution, etc.
  • Preparations can be formulated by conventional methods, and administered to subjects in appropriate doses.
  • Administration of a suitably formulated composition can be achieved in different ways, eg. Administration is by intravenous, intraperitoneal, subcutaneous, intramuscular, topical or intradermal. The particular route of administration depends, inter alia, on the type of carrier included in the pharmaceutical composition. The dosage regimen will be determined by the attending physician and other clinical factors.
  • the dosage for any one patient will depend on many factors, including the patient's size, body surface area, age, sex, the particular active agent to be administered, the timing and route used, and the type and phase of the drug used. Infections or diseases, general health conditions, and combinations of other medications.
  • the pharmaceutical compositions of the present invention may include a second active agent.
  • the second active agent is a drug for treating or alleviating X-ALD, or a component capable of reducing side effects when the drug is administered.
  • compositions of the present invention may be administered by any suitable route, including systemic administration and topical administration.
  • the pharmaceutical composition of the present invention is for systemic administration, especially intravenous administration.
  • the present invention provides a pharmaceutical composition comprising a recombinant AAV vector of the present invention, wherein said pharmaceutical composition is an intravenous formulation, or a lyophilized stable formulation suitable for formulation as an intravenous formulation.
  • the pharmaceutical composition of the present invention is suitable for local administration, for example, directly in or near the organ or tissue to be treated in a subject.
  • the present invention provides a pharmaceutical composition comprising a recombinant AAV vector of the present invention, wherein said pharmaceutical composition is a formulation suitable for topical administration.
  • the present invention relates to methods of treating diseases using the recombinant AAV vectors of the present invention or pharmaceutical compositions comprising the same.
  • the disease is X-ALD.
  • the disease is a defect in the ABCD1 gene.
  • the method comprises: administering a recombinant AAV vector or pharmaceutical composition of the invention to a subject in need thereof.
  • the recombinant AAV vector or pharmaceutical composition can be administered by any suitable route, including but not limited to, intramuscular, subcutaneous, intraspinal, intracerebroventricular, intrathecal, intravenous, intradiaphragmatic, intrathoracic, intraperitoneal.
  • the recombinant AAV vector or pharmaceutical composition of the present invention is delivered to a subject by systemic administration, especially intravenous administration.
  • the treatment is therapeutic.
  • the treatment is prophylactic.
  • the subject is a mammal, wherein the mammal is especially a human, primate, dog, horse, cow, especially a human subject.
  • treatment includes any one or more of: (1) arresting or delaying the onset of the disease; (2) lessening the severity of the disease; (3) lessening the severity of the disease; or prevent the onset and/or worsening of at least one symptom of the disease; (4) improve disease-related neurodegeneration and/or behavior of the subject; and (5) prolong the survival of the subject.
  • treatment of X-ALD while encompassing, does not require complete elimination of the disease or symptoms associated therewith.
  • Subjects who can be treated/prevented by the method of the present invention include Addison type, cerebral type (children's brain type, adolescent brain type and adult brain type), adrenomyeloneuropathy type (AMN) with or without intracranial demyelination, Asymptomatic or presymptomatic, and heterozygous.
  • the subject is a patient with cerebral ALD.
  • the subject is an AMN patient.
  • the subject is an asymptomatic patient.
  • the subject is an individual exhibiting symptoms associated with the onset of ALD or AMN, such as accumulation of high levels of VLCFA in plasma.
  • the subject is an individual at risk for ALD or AMN disease due to family history; or an individual whose genetic testing includes one or more mutations associated with ALD or AMN in the ABCD1 gene.
  • the present invention provides a method for treating or preventing X-chromosome-linked adrenoleukodystrophy (X-ALD) and/or improving symptoms associated with X-ALD, wherein the method comprises providing A subject is administered the recombinant AAV viral vector according to the present invention or the pharmaceutical composition according to the present invention.
  • said method comprises intraperitoneal, intramuscular, intraarterial, intravenous, intrathecal or intraventricular administration, more preferably intravenous injection, of said recombinant AAV viral vector.
  • the X-ALD gene therapy drug of the present invention (for example, according to the recombinant AAV virus vector of the present invention or according to the pharmaceutical composition of the present invention) can break through the blood-brain barrier, thereby reducing the extremely long chain in the multi-organ organs of the whole body. fatty acid levels.
  • the adeno-associated virus AAV9 is used as a vector, the drug can have a wider biodistribution, especially to cover the central nervous system, improve the level of very long chain fatty acids in the central nervous system, and prevent white matter lesions.
  • the X-ALD gene therapy drug of the present invention (for example, according to the recombinant AAV viral vector of the present invention or according to the pharmaceutical composition of the present invention) has increased the expression level of the drug in vivo through the optimization of the promoter and the coding gene, and the viral vector Genomic stability is enhanced, resulting in longer-lasting reductions in tissue and blood VLCFA levels.
  • the X-ALD gene therapy drug of the present invention (for example, the recombinant AAV virus vector according to the present invention or the pharmaceutical composition according to the present invention) has reduced toxicity to peripheral tissues and organs.
  • the X-ALD gene therapy drug of the present invention can reduce the toxicity of transgene overexpression to peripheral organs, especially myocardial tissue toxicity.
  • the X-ALD gene therapy drug of the present invention after intravenous injection into the ABCD1 gene-deficient model mice, can Efficient, continuous and stable expression of ABCD1 protein, and the ABCD1 protein produced by expression can participate in the degradation of extremely long-chain fatty acids in cells, reduce its accumulation in cells, and maintain it at a normal level, thereby eliminating cells caused by extremely long-chain fatty acids Various disease symptoms caused by excessive accumulation of fatty acids, to achieve the purpose of treatment.
  • the X-ALD gene therapy medicine of the present invention after intravenous injection and treatment of X-ALD model mice, the extremely long The level of chain fatty acids decreased significantly and returned to normal levels, and the very long chain fatty acids in peripheral tissues and blood also changed significantly.
  • the medicine of the present invention can be used in the treatment of X-ALD by intrathecal injection.
  • the treatment effect of intrathecal injection will be better and more significant for the improvement of abnormal indicators of the central nervous system.
  • the biodistribution of the AAV vector genome will be more concentrated in the central nervous system, and the proportion of vector biodistribution in peripheral organs such as the heart and liver is low (lower exposure), so the safety of the drug will be improved. Further improvement.
  • the present invention realizes the improvement of disease symptoms and simultaneously achieves good drug safety through a uniquely designed and constructed gene expression construct. Below in conjunction with specific embodiment and accompanying drawing, the present invention will be further described:
  • Embodiment 1 ABCD1 gene coding sequence optimization
  • CDS ABCD1 coding sequence
  • the nucleotide sequence of the gene encoding hABCD1 (UniProtKB-P33897) was optimized, and the optimized sequence for improving the expression efficiency of hABCD1 was determined by in vitro expression in HEK293 cell line and detection of ABCD1 protein expression level by WESTERN BLOT (coABCD1).
  • the wild-type ABCD1 coding nucleic acid sequence (from the sequence of NCBI accession number NM_000033.3) and the optimized coABCD1 coding nucleic acid sequence (SEQ ID NO: 1) were synthesized by GenScript Biotechnology Co., Ltd. The synthesized sequence was cloned into the pUC57 simple vector (GenScript Biotechnology, Nanjing). After that, the synthetic wild-type coding sequence and the optimized coding sequence were respectively cloned into the pAAV vector plasmid through KpnI and EcoRI sites, and embedded between the CA promoter and polyA (as shown in Figure 1A). After sequencing and identification, they were frozen and stored in the library and the plasmids were extracted for in vitro transfection experiments.
  • plasmids were miniprepped and HEK293 cell line was transiently transfected using Lipofectamine 2000.
  • the medium was changed 6 hours after transfection, and the cells were collected 48 hours later, and the total protein of the cells was lysed and extracted.
  • the protein bands were separated by 10% SDS-PAGE gel electrophoresis and transferred to PVDF membrane by wet transfer method.
  • Anti-ABCD1 antibody (1:2000, Abcam, ab197013) was used to incubate overnight.
  • ECL-enhanced chemiluminescence reagent (Sanko, C500044) was used for ECL luminescent color development.
  • the color rendering results were processed and analyzed by grayscale scanning.
  • CA promoter composed of the enhancer sequence of human CMV virus and the basal promoter of chicken ⁇ -actin protein
  • the 3' end of the sequence was introduced into the human TATA box binding protein-related factor 1 gene (GenBank: NG_012771.2)
  • the intron sequence from position 62804 to position 62890 was named CAR promoter.
  • CAR-MutC with mutation T568C, the sequence is shown in SEQ ID NO: 2
  • CAR-MutA with mutation T568A, sequence shown in SEQ ID NO: 3
  • CAR-MutG with mutation T568G, sequence shown in SEQ ID NO: 4
  • the pscAAV-CAR-Gluc plasmid vector shown in Figure 2 was constructed, including:
  • Gluc the nucleotide sequence encoding the luciferase reporter gene
  • BGH polyA bovine growth hormone
  • the CAR promoter in the pscAAV vector was replaced with the CAR-Mut promoter sequence to obtain the pscAAV-CAR-Mut-Gluc vector.
  • the well-grown BHK-21 cells were passaged to 24-well plates, and when the density reached 60%, Lipofectamine2000 (Invitrogen, USA) was used to transfect pscAAV-CAR-Gluc, pscAAV-CAR-MutC-Gluc, pscAAV according to the manufacturer's instructions - 3 wells each for CAR-MutA-Gluc and pscAAV-CAR-MutG-Gluc. 48 hours after transfection, 100 ⁇ L of the supernatant was taken from each well, the Gluc level was detected with a Glomax96 microplate luminometer (Promega), and data analysis was performed using the detector software.
  • Lipofectamine2000 Invitrogen, USA
  • the target sequences of miRNA1, 122, and 206 were embedded, and whether the miRNA target sequences were effective in down-regulating the expression of the target gene in cell lines derived from different tissues expected effect.
  • plasmid vectors based on EGFP expression constructs.
  • the CMV promoter drives EGFP gene expression
  • the miRNA1 target sequence miRNA1 Target
  • miRNA206 target sequence miRNA206 Target
  • miRNA122 target sequence miRNA122 Target
  • the plasmids were transiently transfected in C2C12 cell line (myoblasts, preserved by Jinlan Laboratory) and Huh7 cell line (human liver cancer cells, preserved by Jinlan Laboratory). After 48 hours of transfection, the fluorescent signal of the cells was observed under a fluorescent microscope. The effects of different miRNA target sequences located in the 3'UTR on the expression of the target gene EGFP were compared.
  • the combination of miRNA1 and miRNA206 target sequences can significantly reduce the expression level of the target gene in skeletal muscle cells.
  • the miRNA122 target sequence can down-regulate the expression of target genes in hepatocytes.
  • the plasmid vector embedded with the miRNA target sequence had significantly down-regulated expression of the target gene in Huh7 cells and C2C12 cells, which indicated that the 3'UTR containing the miRNA target sequence had specific expression down-regulation ability.
  • the EGFP gene was replaced by the Gluc reporter gene, and the number of repeats of the miRNA target sequence was increased in the 3'UTR and its arrangement and combination were changed, thereby comparing miRNA 1, miRNA 206, and miRNA 122 targets The effect of different combinations of sequences on the expression of the target gene.
  • Transient transfection was performed on the C2C12 cell line (myoblast, preserved by Jinlan Laboratory) and the Huh7 cell line (human liver cancer cell, preserved by Jinlan Laboratory) with the expression plasmid containing the construct. After 48 hours of transfection, the fluorescent signal of the cells was observed.
  • the combination of the three miRNA target sequences also showed a better ability to down-regulate the target gene. Compared with the down-regulation ability of the single-copy miRNA combination, it increased the ability to inhibit the target gene by ⁇ 300%. The expression in hepatocytes was suppressed by more than 90%.
  • Embodiment 4 Construction of recombinant AAV virus
  • An AAV plasmid vector containing an optimized promoter CAR-MutC, an optimized target gene co-ABCD1, and an ABCD1 expression cassette inserted into the 3'UTR miRNA target sequence was constructed.
  • the constructed AAV plasmid vector contains:
  • the coABCD1 nucleic acid sequence was synthesized by GenScript Biotechnology Co., Ltd., and a KpnI restriction site and a Kozak sequence 5'-GCCACC-3' were added upstream of the synthetic sequence, and a taa stop codon and EcoRI enzyme were added downstream cut site.
  • the synthesized sequence was cloned into the pUC57 simple vector (GenScript Biotechnology, Nanjing) to obtain the pUC57-coABCD1 vector.
  • the pUC57-coABCD1 vector and the pRDAAV-CAR-Mut-EGFP vector were digested with KpnI and EcoRI respectively, the coABCD1 fragment and the pRDAAV-CAR-Mut-EGFP vector fragment with the EGFP reporter gene removed were recovered, and the two fragments were ligated and transformed into E.coli DH5 ⁇ competent cells (Qingke Xinye, Beijing) were screened and identified to obtain the pRDAAV-CAR-Mut-coABCD1 vector.
  • the artificially synthesized miRNA target fragment (comprising miRNA122, 206, 1 target sequence, sequence information see SEQ ID No.12) is cloned into between the EcoRI and SalI restriction sites of the pRD.AAV-CAR-Mut-coABCD1 vector
  • the pRD.AAV-CAR-Mut-coABCD1-miT vector was obtained.
  • Example 5 In vitro transduction and functional testing of recombinant AAV9 virus
  • Skin fibroblasts from two X-ALD patients were infected with the prepared recombinant AAV virus at a multiplicity of infection of 10000. Cells were fixed with formalin 72 hours after infection and immunofluorescent staining was performed. Cells were incubated overnight with Anti-ABCD1 antibody (diluted 1:100, Abcam), rinsed, washed with dylight549-labeled secondary antibody for 1 hour, counterstained with dapi, and mounted. Red fluorescence and blue fluorescence were observed under a fluorescence microscope.
  • Skin fibroblasts from X-ALD patients were infected with the prepared recombinant AAV virus at a MOI of 50000. Cell density was observed on day 7 after infection.
  • Example 6 A single tail vein injection of AAV9-coABCD1-miT and AAV9-coABCD1 interferes with C57BL6/J wild-type mice
  • the adeno-associated virus vector containing the ABCD1 expression construct before and after optimization was injected into the tail vein of C57BL6J wild-type mice at a dose of 1.5E+14vg/kg, with 4 mice in each group. Except for one mouse in the unoptimized group that died 3 weeks after administration, the remaining 7 mice were killed 4 weeks after injection, and samples were collected for histopathology and the content of very long chain fatty acids.
  • the histopathological changes in vivo and the changes of very long chain fatty acids before and after 3'UTR optimization are shown in Figures 10 and 11.
  • Necropsy was performed on mice in the non-optimized group that died 3 weeks after administration. Histopathological diagnosis revealed that the heart of the dead mouse had extensive vacuolar degeneration, obvious myocardial fibrosis, and thrombus in the cardiac cavity. Liver pathological examination revealed a large area of hepatocyte necrosis, which spread outwards mainly centered on the central vein. The above pathological changes are considered to be related to the toxicity of the test product. ( Figure 11B)
  • Example 7 AAV9-coABCD1-miT Tail Vein Injection Treats X-ALD Model Mice
  • a B6.129 mouse model in which the ABCD1 gene was knocked out was selected, and the model mouse was purchased from Jackson Lab. Due to the species differences of the mice, the deletion of ABCD1 did not cause serious physiological or behavioral effects in the model mice, but the model mice showed phenotypes such as decreased exercise ability at 10 weeks, and the fibroblasts and The levels of C26:0 in various tissues (adrenal gland, brain, etc.) and blood were significantly higher than those in wild-type mice, indicating that there is pathological accumulation of very long-chain fatty acids in the model mice. This process simulates the pathological process of X-ALD patients.
  • AAV9-coABCD1-miT was delivered by intravenous injection, and the model mice were treated reparatively, and compared with normal wild-type mice and untreated mice.
  • the optimized drug was administered intravenously to 8 X-ALD model mice of the same age at a dose of 5E+13vg/kg. There were 8 wild-type mice and X-ALD model mice of the same age without any intervention.
  • mice The locomotion of the mice was assessed using the grab rope method.
  • the test results can be seen from Figure 12, the average score of normal wild-type mice is 4.5 points; the average score of untreated X-ALD model mice is 0 points, and the mice that were put on the rope fell within 10s. It shows that under the development of the natural history of the diseased mice, the muscles will atrophy and become weak; the average score of the treated diseased mice is 3.7 points, these mice can grasp the rope for more than 30s, and some will try to climb On the rope, some can put the front paw and one hind paw on the rope. Compared with the untreated diseased mice, the treatment effect is very obvious, and the data results of the treatment group are not significantly different from those of the wild type . These results indicate that the optimized drug has a significantly superior therapeutic effect through intravenous administration.
  • the content of very long chain fatty acids was determined by HPLC-MS/MS. First, the experimental mice were dissected and weighed, and then each tissue was ground into powder by liquid nitrogen grinding, and the very long-chain fatty acids were extracted from the tissue through a series of rough extraction and fine extraction using the Matyash extraction method, and used The content of very long chain fatty acids was determined by HPLC-MS/MS, and the detection results are shown in Figure 13.
  • X-ALD model mice were treated by tail vein injection of AAV9-coABCD1-miT, and ABCD1 was immunofluorescently labeled on tissue sections 8 weeks after administration.
  • X-ALD activates autophagy due to the accumulation of very long-chain fatty acids.
  • Autophagy wraps and transports very long-chain fatty acids accumulated in various subcellular organelles to lysosomes. But very long-chain fatty acids must be degraded in peroxisomes, so autophagy does not improve the accumulation of very long-chain fatty acids.
  • the accumulation of very long-chain fatty acids continuously stimulates and activates autophagy, and thus, the autophagic flux is stagnant and ineffective.
  • X-ALD model mice were treated with AAV9-coABCD1-miT tail vein injection. Eight weeks after administration, the spinal cord tissue sections were immunofluorescently labeled with LC3 ⁇ (axonemal dynein light chain 2 ⁇ ). The results are shown in Figure 15. From the results shown in the figure, it can be seen that in the untreated group, the anterior and dorsal horns of the spinal cord showed strong autophagy protein signals of myelin sheath, indicating that autophagy activation was obvious. The autophagy signal in the anterior horn of the spinal cord decreased significantly in the treatment group.
  • the trend of decreasing autophagy signal can also be observed, although the decrease degree is not as good as that in the forefoot area of the spinal cord (this is because AAV9 is more tropistic to the anterior horn cells after intravenous injection), but it can be clearly compared
  • the autophagy activity in the spinal cord decreased after treatment. It shows that after treatment, the problem of autophagy pathway disorder can be improved.
  • Embodiment 8 Evaluation of the safety and optimization degree of drugs
  • the protein content of ABCD1 was determined by Western Blot method to explore the safety and optimization degree of the drug.
  • the optimized (AAV9-coABCD1-miRT) and unoptimized drug (AAV9-coABCD1) were intravenously injected into normal wild-type mice, and compared with untreated normal mice.

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Abstract

La présente invention concerne un acide nucléique et une construction d'expression pour traiter l'adrénoleucodystrophie liée à l'X (X-ALD), une composition pharmaceutique comprenant l'acide nucléique ou la construction, et une méthode de traitement de la X-ALD.
PCT/CN2023/071616 2022-01-10 2023-01-10 Médicament et méthode de traitement génique de d'adrénoleucodystrophie liée à l'x WO2023131345A1 (fr)

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Citations (2)

* Cited by examiner, † Cited by third party
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CN108456697A (zh) * 2018-03-29 2018-08-28 成都优娃生物科技有限公司 应用慢病毒载体EF1α启动子优化表达ABCD1基因治疗肾上腺脑白质营养不良症
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Publication number Priority date Publication date Assignee Title
CN108456697A (zh) * 2018-03-29 2018-08-28 成都优娃生物科技有限公司 应用慢病毒载体EF1α启动子优化表达ABCD1基因治疗肾上腺脑白质营养不良症
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