Recombinant AAV vectors for the treatment of proteinopathy in the central nervous system

Abstract

A recombinant adeno-associated virus (rAAV) vector for the treatment of proteinopathy in the central nervous system, comprising one or two of the following: (a) a nucleotide sequence encoding progranlin (PGRN) and (b) a nucleotide sequence encoding stasmin-2 (STMN2). Also disclosed are codon-optimized coding sequences of PGRN and / or STMN2, and expression cassettes, vectors, and viral particles containing them.

Description

[Technical Field]

[0001] [Cross-reference of related applications] This application claims priority to PCT application number PCT / CN2023 / 096582, filed on 26 May 2023, the disclosures of which are incorporated herein by reference in their entirety.

[0002] [Technical field] This disclosure relates to the technology of gene therapy. In particular, this disclosure provides recombinant adeno-associated virus (rAAV) vectors comprising either one or both of the following: (a) a nucleotide sequence encoding progranlin (PGRN) and (b) a nucleotide sequence encoding stasmin-2 (STMN2), for use in the treatment of neurodegenerative diseases, particularly amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), Parkinson's disease (PD), multiple system atrophy (MSA), Alzheimer's disease (AD), and other proteinopathy. Also provided herein are viral particles comprising the rAAV vector, pharmaceutical compositions comprising the viral particles, and uses thereof.

[0003] [Sequence List] This disclosure includes, as part of its disclosure, a list of sequences. [Background technology]

[0004] Neurodegenerative diseases (NDs) encompass a range of conditions caused by the selective dysfunction and progressive loss of cells in the brain or peripheral nervous system, affecting millions of people. Many NDs are also classified as proteinopathies, as aggregates formed by structurally abnormal proteins are often observed in or around dying nerve cells. These aggregates disrupt the normal function of nerve cells, resulting in either loss of function (LOF) or gain of function (GOF). Examples of these proteinopathies include amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), Huntington's disease (HD), Parkinson's disease (PD), multiple system atrophy (MSA), and Alzheimer's disease (AD).

[0005] Amyotrophic lateral sclerosis (ALS) is a fatal proteinopathy. Motor neurons in ALS patients progressively degenerate, leading to muscle weakness, inability to walk, and chronic impairment of speech and respiratory function, often resulting in early death within 2–5 years of onset. The estimated annual incidence of ALS in Europe is 1.75–3 cases per 100,000 people, with a prevalence of approximately 10–12 cases per 100,000 people. In mainland China, a recent report indicated a prevalence of approximately 1.24 cases per 100,000 people (Zhang J, Liu X, Liang H, Xu S, Wang X, Xu R. Amyotrophic lateral sclerosis in seven provinces of Chinese mainland: A cross-sectional survey from 2015 to 2016. Front Aging Neurosci. 2022 Sep 15; 14:94635).

[0006] Currently, there is no truly effective treatment for ALS, and available treatments primarily focus on symptom relief and prevention of complications. The U.S. Food and Drug Administration (FDA) has approved three small molecule drugs for ALS: riluzole (Sanofi-Aventis), the free radical scavenger edaravone (Radicava®; manufactured by Tanabe Mitsubishi Pharma Corporation), and lilevrio® (sodium phenylbutyrate and taurursodiol; manufactured by Amilik Pharmaceuticals). Riluzole may inhibit glutamatergic neurotransmission and may extend average survival by 3 to 6 months. Both edaravone and lilevrio may slightly reduce the decline in activities of daily living for ALS patients. Adjunctive therapies such as physiotherapy or respiratory care are often used to alleviate symptoms and support patients' daily living. None of the above treatments have been able to reverse disease progression.

[0007] The precise etiology of ALS is largely unknown. The disease is thought to be the result of a complex interaction of genetic and environmental factors. Approximately 10% of ALS cases are inherited in an autosomal dominant pattern and are defined as familial ALS (fALS), while the remaining ALS cases are sporadic (sALS) with no clear pattern, such as a family history. The genetic factors in ALS are diverse. In fALS, genetic association studies have revealed that pathogenic mutations frequently occur in several identified disease-causing genes, such as SOD1, C9ORF72, FUS, and TARDBP (transcriptional activation response DNA-binding protein 43kDa, TDP-43).

[0008] The neuropathological features of ALS include the progressive death of both superior motor neurons (UMNs) and inferior motor neurons (LMNs), with ubiquitin-positive inclusions detected within the dead cells. TDP-43 has been reported to be the main component of these inclusions. TDP-43-positive protein aggregates are widely observed (>95%) in ALS patients, suggesting that the accumulation of TDP-43 aggregates may be a common basis in the pathogenesis of ALS.

[0009] TDP-43 pathology is widely known to contribute to neurodegeneration in either loss-of-function or gain-of-function manners. TDP-43 is a 43 kDa DNA / RNA-binding protein encoded by the TARDBP gene. Physiologically, it plays a crucial role in gene transcription and translation, mRNA transport, and stabilization. TDP-43 is a nuclear protein, and mutant TDP-43 aggregates and loses its physiological location, thereby losing its nuclear function. Through interactions with RNA transcripts of more than 6,000 genes that have important functions in axon formation, synaptic activity, and neuronal cell structure, TDP-43 plays a vital role in regulating neuronal function. For example, under normal conditions, TDP-43 binds to hidden exons (including stop codons) of the statimin-2 (STMN2) gene, repressing its integration and consequently preventing abnormal splicing. In the absence of functional TDP-43, STMN2 protein levels decrease, impairing microtubule stability in the axonal growth cone.

[0010] The gain-of-toxicity (GOF) hypothesis for TDP-43 is supported by observations in animal models, as reported in numerous studies, that overexpression of either wild-type or mutant TDP-43 consistently induces an ALS-like neurodegenerative phenotype. Based on these findings, TDP-43 is a promising target for ALS treatment. However, because the physiological levels of TDP-43 are precisely regulated, directly altering its protein or mRNA levels as a therapeutic approach may not be advantageous.

[0011] Currently being investigated therapies for ALS that target TDP-43 can be classified into three types: removal of TDP-43 aggregates via endogenous mechanisms (e.g., the PGRN pathway), targeted modifiers of TDP-43 toxicity (e.g., Ataxin-2), and genetic manipulation of downstream TDP-43 genes (e.g., STMN2).

[0012] Progranulin (PGRN) is a 593-amino acid secreted protein encoded by the GRN gene. The secreted protein PGRN appears to act extracellularly via the tyrosine kinase ephrin receptor 2 (EphA2) and Notch signaling pathways, and plays a crucial role in neuronal survival and axonal elongation. PGRN also functions as a chaperone within lysosomes to mediate the degradation of misfolded proteins, thus maintaining lysosomal homeostasis. Within lysosomes, PGRN is processed into multiple 6kDa granulin (GRN) peptides.

[0013] PGRN deficiency is causally associated with neuronal ceroid lipofuscinosis type 11 (CLN11), frontotemporal lobar degeneration with TDP-43 aggregation, and GRN-type frontotemporal dementia (FTD-GRN), and is also associated with the progression of other neurodegenerative diseases such as ALS, PD, AD, and autism.

[0014] There is likely a correlation between PGRN heterozygous deficiency and TDP-43 aggregation, possibly mediated by microglia-mediated neuroinflammation. Overexpression of PGRN has been reported to reduce insoluble TDP-43 and restore the ALS phenotype in TDP-43 A315T transgenic mice (Beel, S., Herdewyn, S., Fazal, R. et al. Progranulin reduces insoluble TDP-43 levels, slows down axonal degeneration, and prolongs survival in mutant TDP-43 mice. Mol Neurodegeneration 13, 55 (2018)).

[0015] PR006 from Prevail Therapeutics is a single-dose gene therapy for the treatment of FTD-GRN. A Phase I / II clinical trial drug, PR006 is designed to deliver normal PGRN proteins into the central nervous system (CNS) of FTD patients via an rAAV vector.

[0016] International Publication No. 2021 / 058830 (filed by King's College London) discloses an rAAV vector containing a codon-optimized full-length human programmen coding sequence. The rAAV was evaluated for PGRN protein expression in mice. International Publication No. 2021 / 058830 envisioned the use of expression cassettes and rAAV in the treatment of frontotemporal dementia (FTD), neuronal ceroid lipofuscinosis (CLN11), and amyotrophic lateral sclerosis (ALS), but the application did not provide disease model data.

[0017] As mentioned above, STMN2 mRNA splicing is directly regulated by functional TDP-43. Antisense oligonucleotides (ASOs) targeting hidden exon regions of STMN2 can mimic the function of TDP-43 by suppressing hidden splicing in order to restore full-length STMN2 expression, thereby promoting axonal regeneration in human motor neurons (Michael W. Baughn et al., Mechanism of STMN2 cryptic splice-polyadenylation and its correction for TDP-43 proteinopathies. Science 379,1140-1149 (2023)).

[0018] International Publication No. 2021 / 156832 discloses the miRNA inhibitor miR-485 for the treatment of amyotrophic lateral sclerosis (ALS). In this application, it is explained that the miRNA may increase the expression of a set of genes whose decreased expression is thought to be associated with ALS, one of which is STMN2. However, no data indicating the effect of the miRNA on regulating STMN2 protein expression, or its consequent effect on the progression of ALS, is provided in the application documents.

[0019] International Publication No. 2023 / 018858 discloses a gene-editing statimin-2 (STMN2) system based on a CRISPR-Cas nuclease editing system that may be used for the treatment of ALS. The system comprises a Cas12i polypeptide and a guide RNA that mediates cleavage within the STMN2 gene. However, the published patent application did not provide any experiments or data indicating the therapeutic effect of the system.

[0020] Frontotemporal dementia (FTD) is a clinically diverse neurodegenerative disease and is perhaps the most common form of dementia in younger individuals (45-65 years of age). Mutations in three genes, including C9ORF72, the microtubule-associated protein Tau (MAPT), or GRN, are causally associated with the development of FTD. Previous pathological studies have shown that intracellular deposition of abnormally aggregated proteins is frequently observed in the brain tissue of FTD patients. Major components of these depositions include TDP-43, the microtubule-associated protein Tau, and tumor-associated protein fusion sarcoma (FUS). Therefore, TDP-43 targeted therapies may also be effective in treating FTD patients.

[0021] Neurodegenerative diseases often share similar pathological features. For example, TDP-43-positive inclusion bodies are found not only in ALS and FTD, but also in other neurodegenerative diseases such as primary lateral sclerosis, amyotrophy, Guam-Parkinson-dementia complex, PD, some AD cases, inclusion body myopathy, and some traumatic brain injury.

[0022] Currently researched therapies for proteinopathy typically target a single aspect with a single mechanism of action (MOA), and therefore may not be effective in treating these diseases with complex etiologies. Thus, developing more effective therapies for treating multifaceted proteinopathy remains an unmet medical need. [Overview of the project]

[0023] To develop a more effective treatment for proteinopathies, the inventors have innovatively created a combination therapy that targets both the loss-of-function (LOF) and gain-of-function (GOF) mechanisms of TDP-43, a promising strategy for treating ALS, FTD, and other proteinopathies and a potential game-changer. Furthermore, the inventors modified the nucleotide sequences encoding progranulin (PGRN) and stathmin-2 (STMN2) to optimize their expression when delivered into the human brain via rAAV vectors. Based on these, an rAAV vector expressing the two genes of interest (GOIs) tandemly is provided for use in a therapy that synergistically targets multiple ALS pathologies to achieve a greater therapeutic effect.

[0024] Thus, in a first aspect, the present application provides an isolated nucleic acid molecule comprising a first polynucleotide sequence encoding a first polypeptide and a second polynucleotide sequence encoding a second polypeptide, wherein the first polypeptide is progranulin (PGRN) and the second polypeptide is stathmin-2 (STMN2), or the first polypeptide is stathmin-2 (STMN2) and the second polypeptide is progranulin (PGRN).

[0025] In one embodiment of the first aspect, the first polynucleotide sequence is located upstream on the 5' side of the second nucleotide sequence.

[0026] In one embodiment of the first aspect, progranulin (PGRN) comprises or consists of the polypeptide sequence of SEQ ID NO: 10, or a variant, homolog or ortholog thereof.

[0027] In one embodiment of the first aspect, the polynucleotide sequence encoding PGRN is a wild-type coding sequence, a variant thereof, a homolog or orthomolecule thereof. In a preferred embodiment, the polynucleotide sequence encoding PGRN is a codon-optimized coding sequence. For example, the polynucleotide sequence encoding PGRN is codon-optimized for human expression, to reduce the CpG site, and / or to reduce the CG content. In a particular embodiment, the polynucleotide sequence encoding PGRN is a polynucleotide sequence selected from the group consisting of the polynucleotide sequences shown in any one of SEQ ID NOs: 1 to 8.

[0028] In one embodiment of the first aspect, statimin 2 (STMN2) includes or comprises the polypeptide sequence of sequence number 20, its variants, homologs or orthomolecules thereof.

[0029] In one embodiment of the first aspect, the polynucleotide sequence encoding STMN2 is a wild-type coding sequence, a variant thereof, a homolog or orthomolecule thereof. In a preferred embodiment, the polynucleotide sequence encoding STMN2 is a codon-optimized coding sequence. For example, the polynucleotide sequence encoding STMN2 is codon-optimized for human expression, to reduce the CpG site, and / or to reduce the CG content. In a particular embodiment, the polynucleotide sequence encoding STMN2 is a polynucleotide sequence selected from the group consisting of the polynucleotide sequences shown in any one of sequence numbers 11 to 18.

[0030] In one embodiment of the first aspect, a first polynucleotide sequence and a second polynucleotide sequence are linked in a frame to form a combined construct and functionally linked to a single promoter located 5' upstream of both the first and second nucleotide sequences. For example, the promoter is a constitutive promoter, e.g., an EF1α promoter or an EF1α-derived promoter. The EF1α-derived promoter may be a cleaved form of the EF1α promoter, e.g., an EFS promoter (a shorter version of the EF1α promoter). The EF1α-derived promoter may be a hybrid promoter consisting of an EF1α promoter or an EFS promoter and an additional nucleotide sequence, e.g., an intron sequence. In a particular embodiment, the promoter may be selected from hybrid promoters, such as EFShI1, EFShI2, EFShI3, EFSI4, EFSdI1 and EFSdI2, or their variants, as disclosed in International Publication No. 2023 / 061499. In a particular embodiment, the promoter may be EFShI1 (SEQ ID NO: 27).

[0031] In one embodiment of the first aspect, the isolated nucleic acid molecule further comprises a linker sequence between a first polynucleotide sequence and a second polynucleotide sequence. Preferably, the linker sequence is a self-cleaving peptide or a coding sequence for an intra-sequence ribosome entry site. Preferably, the self-cleaving peptide is a 2A peptide. For example, the 2A peptide is selected from the group consisting of E2A, F2A, T2A, and P2A, and is preferably P2A.

[0032] In one embodiment of the first aspect, the first polynucleotide sequence, linker sequence, and second polynucleotide sequence are codon-optimized as a whole coding region. For example, the entire coding region is codon-optimized for expression in humans. In a particular embodiment, the isolated nucleic acid molecule includes codon-optimized coding regions for both PGRN and STMN2, comprising or consisting of polynucleotide sequences selected from the group consisting of SEQ ID NOs: 21 to 26. In a particular embodiment, the isolated nucleic acid molecule includes codon-optimized coding regions for both PGRN and STMN2, comprising or consisting of polynucleotide sequences, preferably SEQ ID NO: 25, which is SEQ ID NO: 22 or 25.

[0033] In one embodiment of the first aspect, the isolated nucleic acid molecule further comprises a polyadenylation signal. In certain embodiments, the polyadenylation signal is SV40 polyA or human growth hormone (hGH) polyA.

[0034] In one embodiment of the first aspect, the isolated nucleic acid molecule further comprises a post-transcriptional regulator (WPRE) sequence.

[0035] In a second embodiment, the application provides a codon-optimized coding sequence for PGRN. Specifically, the coding sequence for PGRN is codon-optimized for expression in humans and has reduced CpG site and / or CG content. In certain embodiments, the codon-optimized coding sequence for PGRN comprises or consists of the polynucleotide sequences shown in any one of SEQ ID NOs: 1 to 8.

[0036] In a third embodiment, the application provides a codon-optimized coding sequence for STMN2. Specifically, the coding sequence for STMN2 is codon-optimized for expression in humans and has reduced CpG site and / or CG content. In certain embodiments, the codon-optimized coding sequence for STMN2 comprises or consists of the polynucleotide sequences shown in any one of SEQ ID NOs: 11-18.

[0037] In a fourth embodiment, the application provides an expression cassette comprising an isolated nucleic acid molecule of the first embodiment, a codon-optimized coding sequence of PGRN of the second embodiment, or a codon-optimized coding sequence of STMN2 of the third embodiment. Preferably, the expression cassette is suitable for use in recombinant adeno-associated virus (rAAV) vectors.

[0038] In a fifth embodiment, the application provides an rAAV vector comprising an isolated nucleic acid molecule of the first embodiment, a codon-optimized coding sequence of PGRN of the second embodiment, a codon-optimized coding sequence of STMN2 of the third embodiment, or an expression cassette of the fourth embodiment.

[0039] In one embodiment of the fifth aspect, the AAV vector is an AAV vector of the AAV9 serotype or a proprietary ViVec serotype.

[0040] In one embodiment of the fifth aspect, the rAAV vector further comprises two terminal inversion sequences (ITRs). In a preferred embodiment, the ITRs are AAV2 ITRs.

[0041] In a sixth embodiment, the application provides a viral particle comprising an rAAV vector according to the fifth embodiment.

[0042] In a seventh embodiment, the present application provides a composition comprising an rAAV vector according to the fifth embodiment and a pharmaceutically acceptable excipient, such as a pharmaceutical composition.

[0043] In an eighth embodiment, the present application provides a method for treating or preventing a neurodegenerative disease (ND) in a subject requiring such treatment, comprising administering to the subject an rAAV vector according to the fifth embodiment, a viral particle according to the sixth embodiment, or a pharmaceutical composition according to the seventh embodiment. For example, the neurodegenerative disease may be amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), Huntington's disease (HD), Parkinson's disease (PD), multiple system atrophy (MSA), or Alzheimer's disease (AD). In some embodiments, the neurodegenerative disease may be a neuropathy or proteinopathy, in particular a disease associated with TDP-43 aggregation. For example, the neurodegenerative disease may be TDP-43-associated ALS.

[0044] The bicistronic construct and rAAV of this application can simultaneously deliver and enable the expression of two therapeutic polypeptides, namely PGRN and STMN2. The ingenious use of GRN and STMN2 as genes of interest in gene therapy provides a novel strategy for developing potentially more effective therapies that treat neurodegenerative diseases through two synergistic mechanisms. Codon optimization of the coding sequences allows both therapeutic polypeptides to be expressed at desired levels, enabling their intended use in the treatment of specific neurodegenerative diseases. Furthermore, the inventors have surprisingly discovered that the rAAV of this application, through the bicistronic construct identified in this application, can achieve high levels of expression of both genes, which has been difficult with the prior art. [Brief explanation of the drawing]

[0045] [Figure 1] Figures 1A and 1B show schematic diagrams of exemplary combinational constructs containing both PGRN and STMN2 coding sequences in different orders. Figure 1A shows a schematic diagram of a PGRN-P2A-STMN2 combinational construct, and Figure 1B shows a schematic diagram of an STMN2-P2A-PGRN combinational construct. [Figure 2]Figure 2 shows representative images of Western blots illustrating PGRN protein expression in both cell soluble and supernatant samples, as well as STMN2 expression in the cell soluble sample. All samples were obtained from N2A cells transfected with various combination constructs containing codon-optimized coding sequences for STMN2 and PGRN. [Figure 3] Figure 3A shows a schematic diagram of an exemplary construct containing the codon-optimized code sequence for PGRN. Figure 3B shows a schematic diagram of an exemplary construct containing the codon-optimized code sequence for STMN2. [Figure 4] Figure 4 shows representative Western blot images illustrating GRN protein expression in both cell soluble and supernatant samples obtained from SH-SY5Y cells transfected with various constructs containing the GRN codon-optimized coding sequence. [Figure 5] Figure 5 shows representative images of Western blot results for STMN2 protein expression in both cell soluble and supernatant samples taken from SH-SY5Y cells transfected with various constructs containing the STMN2 codon-optimized coding sequence. [Figure 6] Figure 6 shows the Western blot results of GRN and STMN2 protein expression in N2A-AAVR cells transfected with either the AAV9-MC2 or AAV9-MC5 combination construct. GRN protein levels were detected in both the (cell soluble) and extracellular (supernatant) fractions, while only the cellular matrix STMN2 protein level was detected, as STMN2 protein is localized only in the cell matrix. [Figure 7] Figure 7 shows the survival curve of male ALS mice after intrathecal administration of AAV9-MC5. [Figure 8] Figure 8 shows the latency (seconds) in the rotorod test in male ALS mice 8 weeks after intrathecal administration of AAV9-MC5. [Figure 9]Figure 9 shows a statistical analysis of Western blot results of insoluble TDP-43 levels in cortical and spinal cord tissue samples taken from ALS mice 15 weeks after intrathecal administration of AAV9-MC5. L: low dose; H: high dose. [Figure 10] Figure 10 shows a statistical analysis of immunohistochemical staining results for TDP-43 levels in fixed spinal cord tissue samples taken from ALS mice 15 weeks after intrathecal administration of AAV9-MC5. TDP-43 levels are quantified by both the number of TDP-43-positive cells and the total area of ​​TDP-43-positive stained regions. L: low dose; H: high dose. [Figure 11] Figure 11 shows a representative image of TDP-43-positive aggregates in an ALS cell model after gene transfer using AAV9-MC5 (MOI=1E+6), along with a statistical analysis of their number. White arrows indicate TDP-43-positive aggregates. [Modes for carrying out the invention]

[0046] Unless otherwise defined herein, all technical and scientific terms used herein have the meanings that are ordinarily understood by those skilled in the art to which the present invention pertains.

[0047] As used herein, including in the attached claims, singular forms such as “a,” “an,” and “the” include their corresponding plural forms unless the context clearly indicates otherwise.

[0048] In the context of this specification, unless otherwise specified, the term “comprise,” and its variations such as “comprises” and “comprising,” mean the inclusion of the described elements, such as amino acid sequences, nucleotide sequences, properties, processes, or groups thereof, but not the exclusion of other elements, such as amino acid sequences, nucleotide sequences, properties, and processes. Where used herein, “comprise” or its variations may be replaced with “contain,” “include,” or sometimes “have,” or equivalent variations thereof. In some embodiments, the term “comprise” also encompasses the scenario of “consisting of.”

[0049] As used herein, the term “gene” refers to a nucleotide sequence that codes for nucleic acids (such as DNA, e.g., genomic DNA or cDNA) and their corresponding RNA transcripts. As used herein, terms relating to genomic DNA include intervening non-coding and regulatory regions, and may include both the 5' and 3' ends. In some cases, the term includes the transcription sequence, including the 5'-UTR and 3'-UTR, exons, and introns. In some genes, the transcription region will include an “open reading frame” that codes for a polypeptide. In some cases, “gene” includes only the coding sequence necessary to code for a polypeptide (e.g., an “open reading frame” or “coding regions”). In some cases, the term “gene” includes not only the transcription sequence but also non-coding regions, including upstream and downstream regulatory regions, enhancers, and promoters. “Genes” may mean “endogenous genes” or native genes. The term "gene" can mean a "foreign gene" or a "non-natural gene." A non-natural gene can mean a gene that is not normally present in a host organism but has been introduced into the host organism through gene transfer. A non-natural gene can also mean a gene that is not in its original location within the genome of an organism. A non-natural gene can also mean a natural nucleic acid, such as a codon-optimized nucleotide sequence, that contains mutations, insertions, and / or deletions (e.g., non-natural sequences). In the context of this application, unless otherwise stated, "GOI" specifically refers to a CDS region, i.e., a sequence that codes for an amino acid in a protein.

[0050] The terms “polynucleotide,” “oligonucleotide,” and “nucleic acid” are used interchangeably herein and refer to polymers of nucleotides of any length. Polynucleotides may be exogenous or endogenous to cells. Polynucleotides may exist in a cell-free environment. Polynucleotides may be genes or fragments thereof. Polynucleotides may be DNA. Polynucleotides may be RNA. Polynucleotides may have any three-dimensional structure and may perform any known or unknown function. Polynucleotides may contain one or more analogues (e.g., substituted backbone, sugars, or bases).

[0051] The term “isolated nucleic acid” refers to DNA or RNA from which all or part of a polynucleotide that is bound to a polynucleotide found in nature or not bound in nature has been removed. An isolated nucleic acid molecule “comprising” a particular nucleotide sequence may, in addition to the specified sequence, contain manipulably linked regulatory sequences that control the expression of the coding region of the described nucleic acid sequence. Those skilled in the art will understand that, due to codon degeneracy, any particular amino acid sequence can be encoded by multiple different nucleotide sequences.

[0052] In this specification, “expression cassette” refers to a DNA component consisting of one or more, for example, one or two GOIs selected from the GRN and STMN2 genes, which are contained in a vector (e.g., an rAAV vector) and expressed in host cells transfected by the rAAV vector under the control of a regulatory sequence.

[0053] In the context of this application, the term “combination construct” refers to a construct comprising two GOIs, particularly GRN and STMN2. In a preferred embodiment, the combination construct is a bicistronic construct in which two target genes can be transcribed into a single mRNA. For example, the two coding sequences of PGRN and STMN2 are constructed in a frame under the control of the same promoter located 5' upstream of both coding sequences.

[0054] In this specification, “operatively linked” is used to describe two or more components, particularly nucleotide sequences, that are linked in such a way that each component can perform a specified function.

[0055] In this specification, "codon-optimized coding sequence" refers to a nucleotide sequence encoding a protein such as PGRN or STMN2 that has been modified from its wild-type coding sequence to account for codon bias.

[0056] "AAV" stands for adeno-associated virus. "rAAV" stands for recombinant adeno-associated virus.

[0057] "PGRN" refers to the protein progranlin, which is encoded by the gene GRN in humans. "GRN" refers to granulins, a group of secreted peptides produced from progranlin by cleavage within lysosomes.

[0058] "STMN2" stands for Stasmin-2. STMN2 can also be interchangeably referred to as "SCG10".

[0059] A "CpG island" refers to a region within the genome that is rich in CpG sites. A "CpG site" refers to two consecutive nucleotides consisting of cytosine (C) and guanine (G) in the 5' to 3' direction.

[0060] "2A peptides" refer to a group of self-cleaving short-chain (18-22 amino acid) peptides derived from viruses. 2A peptides induce ribosome skipping during translation, resulting in the separation of the 2A sequence ends from downstream proteins.

[0061] "Proteinopathy" refers to neurodegenerative diseases characterized by the accumulation of structurally abnormal proteins, such as TDP-43, which lead to the formation of aggregates or inclusions within the axons of neurons or oligodendrocytes.

[0062] In the context of this application, “subject” means an animal, preferably a mammal, such as a rodent such as a mouse or a rat, or a primate, preferably a higher primate such as a cynomolgus macaque, such as a human. Unless otherwise specified, in the context of this application, the term “subject” is interchangeable with the terms “patient” or “individual.” Expression cassette

[0063] In one embodiment, the expression cassette of the present application is characterized by the expression of a single GOI, for example, a coding sequence of either PGRN or STMN2, preferably a codon-optimized coding sequence of either PGRN or STMN2, particularly those described herein. For example, the codon-optimized coding sequence of PGRN may be selected from any one polynucleotide sequence of SEQ ID NOs: 1 to 8, or from a polynucleotide having at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% sequence identity with any one of SEQ ID NOs: 1 to 8. For example, the codon-optimized coding sequence of STMN2 may be selected from any one polynucleotide sequence of SEQ ID NOs: 11 to 18, or from a polynucleotide having at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% sequence identity with any one of SEQ ID NOs: 11 to 18.

[0064] Codon optimization can be achieved by reducing sequence complexity, adjusting GC content, adjusting codon usage, and / or avoiding rare codons. Codon-optimized coding sequences typically exhibit improved translation efficiency of the target gene (GOI), resulting in higher protein expression. Tools incorporating algorithms for designing codon-optimized coding sequences (e.g., JCat) are readily available to those skilled in the art.

[0065] In a preferred embodiment, the codons of the PGRN and / or STMN2 coding sequences of this application have a codon adaptation index (CAI) of at least 0.75, preferably at least 0.8, and more preferably at least 0.85. CAI is a measure of codon bias. Those skilled in the art will understand that the actual efficiency of any sequence generated by running the algorithm still needs to be verified experimentally.

[0066] In a preferred embodiment, the PGRN and / or STMN2 coding sequences of this application have a reduced number of CpG sites or no CpG sites at all compared to the corresponding wild-type coding sequences. In a preferred embodiment, the PGRN and / or STMN2 coding sequences of this application have a reduced level of CG content, for example, a CG content of 60% or less.

[0067] Codon optimization allows the expression cassettes of this disclosure, after insertion into an AAV vector, to achieve higher and more consistent protein expression or co-expression in neurons in vitro or in vivo. For example, the expression cassettes of this disclosure exhibit superior performance in GOI(s) expression in human cell lines with neuronal characteristics, such as SH-SY5Y cells. Since progranulin is a secreted protein, its expression can be assessed by measuring the protein level in the cell culture supernatant and the total protein amount in both the supernatant and solubilized cell culture medium.

[0068] In a preferred embodiment, the expression cassette of the present application is a bicistronic expression cassette characterized by the co-expression of a PGRN coding sequence and an STMN2 coding sequence spaced by a linker sequence. Either of the coding sequences may be a wild-type coding sequence or a codon-optimized coding sequence. For example, the PGRN coding sequence in the bicistronic expression cassette may be selected from any one of the SEQ ID NOs: 1 to 9 polynucleotide sequences, or from polynucleotides having at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% sequence identity with any one of the SEQ ID NOs: 1 to 9. For example, the STMN2 coding sequence in the bicistronic expression cassette may be selected from any one of the SEQ ID NOs: 11 to 19 polynucleotide sequences, or from polynucleotides having at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% sequence identity with any one of the SEQ ID NOs: 11 to 19.

[0069] In one particular embodiment, the expression cassette of the Disclosure comprises a polynucleotide sequence encoding PGRN and a polynucleotide sequence encoding STMN2, wherein the polynucleotide sequence encoding PGRN is Sequence ID: 1 or Sequence ID: 4. In another particular embodiment, the expression cassette of the Disclosure comprises a polynucleotide sequence encoding PGRN and a polynucleotide sequence encoding STMN2, wherein the polynucleotide sequence encoding STMN2 is Sequence ID: 13 or Sequence ID: 14.

[0070] Since the inventors have found that changing the sequence order has only a limited effect on the relative expression levels and / or transduction levels of the two GOIs, the coding sequences of PGRN and STMN2 can be arranged in any order in the combined construct of this application.

[0071] When the linker sequence of this application is used to link two coding sequences co-expressed within a single rAAV vector, it yields high efficiency and fidelity. As an example of a linker sequence, a sequence encoding a 2A peptide (e.g., P2A, F2A, or E2A) may be used to link two coding sequences of this application. The position of GOI(s) relative to the linker sequence may be adjusted to achieve desired protein expression and function. In a preferred embodiment, a P2A linker sequence is used in the rAAV between two GOIs. In a particular embodiment, the linker sequence of this application comprises or consists of the nucleotide sequence shown in any one of SEQ ID NO: 31, SEQ ID NO: 32, and SEQ ID NO: 33.

[0072] Codon optimization can also be performed on the entire coding region, including both the target gene and the linker sequence. For example, the coding region includes a codon-optimized polynucleotide sequence selected from any one of SEQ ID NOs: 21-26, or a polynucleotide having at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% sequence identity with any one of SEQ ID NOs: 21-26. In a preferred embodiment, the coding region includes a codon-optimized polynucleotide sequence selected from either SEQ ID NOs: 22(MC2) or SEQ ID NOs: 25(MC5). In a more preferred embodiment, the coding region includes a codon-optimized polynucleotide sequence selected from SEQ ID NOs: 25(MC5).

[0073] Apart from the coding sequence or coding region, the expression cassette may further include one or more regulatory sequences. In the context of this application, “regulatory sequence” refers to a nucleotide element that affects the expression of the target gene. Regulatory sequences may be selected from one or more of promoters, enhancers, polyadenylation sequences, and translation termination signals. Certain combinations of regulatory sequences of this disclosure may achieve unexpected effects that improve the expression efficiency of the coding sequence.

[0074] A "promoter" refers to a DNA sequence that enables the initiation of transcription of a downstream gene under its control. Examples of promoters include, but are not limited to, constitutive promoters, cell-type-specific promoters, tissue-specific promoters, and developmental-stage-specific promoters. A tissue-specific promoter may be a brain-specific promoter. Promoter can be a naturally occurring promoter of a gene, a modified version of a naturally occurring promoter, or a synthetic promoter.

[0075] In preferred embodiments, the promoter of this disclosure may be a constitutive promoter. In preferred embodiments, the promoter may be an EF1α-derived promoter (such as an EFS-derived promoter such as the EFShI1 promoter), a CBh promoter, an EF1α promoter, a CAG promoter, an MBP promoter (myelin basic protein promoter), or a promoter derived therefrom. In a particular embodiment, the promoter is an EFShI1 promoter having the nucleotide sequence of SEQ ID NO: 11.

[0076] An "enhancer" is a regulatory DNA sequence that, together with the promoter, can enhance the transcription of GOIs in rAAV. In a preferred embodiment, the expression cassette of this application includes an enhancer. More preferably, the enhancer may be a CMV enhancer, for example, within the CBh promoter.

[0077] In some embodiments, the expression cassette may include intron sequences that function as enhancers. For example, intron sequences derived from the introns or untranslated regions (UTRs) of each GOI may be included in the expression cassette.

[0078] In some cases, a promoter, together with an enhancer and / or intron sequence, is collectively referred to as a “promoter” or “promoter element.” In a preferred embodiment, the promoter is a CBh promoter. In another preferred embodiment, the promoter consists of an EFS promoter and an intron sequence.

[0079] Preferably, the intron sequence has a total length of approximately 200 bp or less, approximately 250 bp or less, approximately 300 bp or less, approximately 350 bp or less, and approximately 400 bp or less.

[0080] For example, the intron sequences of this disclosure are derived from the target gene. For example, the intron sequence consists of one or more fragments derived from one or more intron regions of the target gene.

[0081] In a preferred embodiment, the promoter or promoter / intron element has a length of 1000 bp or less, 900 bp or less, 850 bp or less, 800 bp or less, 700 bp or less, 600 bp or less, 500 bp or less, or 400 bp or less, due to the limited packaging capabilities of the AAV.

[0082] In some cases, if an intron sequence originates from an intron region of the target gene, it may be inserted into the coding sequence (e.g., a codon-optimized coding sequence) at a location corresponding to its position in the native gene, such as between two exons, rather than being located 5' upstream of the coding sequence to constitute a promoter / intron element.

[0083] The Kozak consensus sequence (Kozak sequence), named after the scientist who discovered it, is a nucleic acid sequence motif found in most eukaryotic mRNA transcripts and functions as a protein translation initiation site. The Kozak sequence ensures that proteins are translated accurately and efficiently.

[0084] The expression cassette of this application may include a polyadenylation signal (Poly A). For example, Poly A sequences that may be used in this application include SV40 Poly A, human growth hormone (hGH) Poly A, or bovine growth hormone (bGH) Poly A.

[0085] In one embodiment, the expression cassette of the present disclosure comprises SV40 Poly A having the polynucleotide sequence of SEQ ID NO: 28. In one embodiment, the expression cassette of the present disclosure comprises hGH Poly A having the polynucleotide sequence of SEQ ID NO: 29.

[0086] In some embodiments, the expression cassettes of the present disclosure include a woodchuck hepatitis virus posttranscriptional regulator (WPRE). The WPRE sequence may be located downstream of the GOI and in close proximity to the polyadenylation signal.

[0087] In certain embodiments, the expression cassette of the present disclosure comprises an EFShI1 promoter, a coding sequence for PRGN, a linker sequence encoding the P2A peptide, a coding sequence for STMN2, and an hGH Poly A sequence, as shown in Figures 1A and 1B; or comprises an EFShI1 promoter, a coding sequence for STMN2, a linker sequence encoding the P2A peptide, a coding sequence for PRGN, and an hGH Poly A sequence.

[0088] AAV Vector The expression cassette of this application is suitable for use in rAAV vectors. Therefore, this application provides an rAAV vector containing a coding sequence, or the expression cassette of this application. Unless otherwise specified, the term "rAAV vector" in the context of this application refers to an rAAV vector plasmid.

[0089] The rAAV vector of this application comprises two ITRs and a heterogeneous polynucleotide adjacent to them, e.g., the expression cassette of this application. The ITRs may be of any suitable serotype. Preferably, both ITRs are AAV2 ITRs or their variants. The nucleotide sequences of AAV2 ITRs are known in the art.

[0090] rAAV vectors can be single-stranded AAVs (ssAAVs) or self-complementary AAVs (scAAVs).

[0091] Furthermore, the rAAV virus particles of this application comprise an AAV capsid protein and an rAAV vector encapsulated within the capsid protein. In some embodiments, the rAAV of this application comprises a modified or engineered AAV capsid compared to a wild-type AAV capsid.

[0092] This application envisions the use of any serotype AAV vector. However, rAAV vectors of serotypes exhibiting tissue affinity to the CNS are preferred. For example, the rAAV vectors of this application are preferably AAV9 vectors or ViVec AAV vectors.

[0093] The Vivec AAV vectors are a series of vectors containing an engineered adeno-associated virus (AAV) capsid polypeptide and exhibiting improved CNS affinity compared to the wild-type AAV9 capsid polypeptide. The capsid polypeptide of the ViVec AAV vector contains seven amino acid insertions between amino acid positions Q588 and A589 of the wild-type AAV9 VP1 capsid polypeptide having the amino acid sequence shown in SEQ ID NO: 34. In certain embodiments, the seven amino acid insertion sequences of the ViVec AAV vector are selected from the group of amino acid sequences shown in SEQ ID NOs: 35 to 94, which result in AAV vectors designated ViVec-N001 to ViVec-N060, respectively. In preferred embodiments, ViVec The seven amino acid insertion sequences of the AAV vector were selected from the group of amino acid sequences shown in SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 67, SEQ ID NO: 74 and SEQ ID NO: 77, respectively, and these are ViV Compatible with ec-N002, ViVec-N004, ViVec-N005, ViVec-N006, ViVec-N007, ViVec-N008, ViVec-N021, ViVec-N022, ViVec-N023, ViVec-N024, ViVec-N026, ViVec-N029, ViVec-N030, ViVec-N033, ViVec-N040, and ViVec-N043.

[0094] Pharmaceutical composition The term "pharmaceutical composition" refers to a composition suitable for delivery to a subject. The pharmaceutical compositions of this disclosure comprise isolated nucleic acids, the rAAV vector or viral particles of this disclosure, and pharmaceutically acceptable excipients. Conventional pharmaceutically acceptable excipients are known in the art and may be solid or liquid excipients. In one embodiment, the pharmaceutical composition may be a liquid formulation for injection.

[0095] Delivery method When applied to subjects such as animals, including humans, or to cells, tissues, organs, or biological fluids, the terms “administration,” “administering,” “treating,” and “treatment” as used herein mean the contact of an exogenous drug, therapeutic agent, diagnostic agent, or composition with a subject, cell, tissue, organ, or biological fluid. Cellular treatment includes contact of the reagent with the cell and contact of the reagent with a fluid, where the fluid is in contact with the cell. The terms “administration” and “treatment” also include in vitro and ex vivo treatment of reagents, diagnostic agents, binding compounds, or other cells, such as cells.

[0096] In a preferred embodiment, the rAAV vector of this application may be delivered by intravenous, intraventricular, intrathecal, or intrastriatal administration. In a particular embodiment, the rAAV vector is delivered via an intrathecal route.

[0097] rAAV vectors can be administered as a single dose or in multiple doses. In certain embodiments, rAAV vectors are administered via a single injection.

[0098] The dose of rAAV vector injection may be modified based on the route of administration. The dose may also be modified based on the subject's body weight. Therefore, the dose range is 1.5 × 10⁻⁶. 9 ~1.5×10 14This can be within a wide range covering vg / kg.

[0099] therapeutic use The terms “treat,” “treating,” or “treatment” include treating, or at least alleviating, the symptoms of neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), Huntington’s disease (HD), Parkinson’s disease (PD), multiple system atrophy (MSA), or Alzheimer’s disease (AD), or other proteinopathies, particularly those associated with TDP-43 agglutination. “Associated with” means that the neurodegenerative disease is associated with TDP-43 agglutination, but does not necessarily mean that the neurodegenerative disease is caused by TDP-43 agglutination.

[0100] Subjects suffering from any of these neurodegenerative diseases may be diagnosed by a well-trained neurologist based on clinical diagnostic criteria, including genetic background, medical history, symptoms and signs, as well as the results of neurological and physical examinations.

[0101] In certain embodiments, the subject may be a clinically diagnosed ALS patient. The ALS treated with the rAAV vector of this application may be familial ALS (fALS) or sporadic ALS (sALS).

[0102] Administration of the rAAV vector of this application may alleviate neurodegenerative diseases, such as ALS, as evidenced by suppressed progression of motor neuron death, a reduced number of ubiquitin-positive inclusions, and / or reduced accumulation of TDP-43 aggregates.

[0103] In the treatment of the above-mentioned neurodegenerative diseases, the rAAV vectors of this application, including, for example, combination constructs such as bicistronic rAAV vectors, may be used. Alternatively, two rAAV vectors, each containing one of the PGRN coding sequence and STMN2 coding sequence of this application, may be used. Bicistronic rAAV vectors are preferred. [Examples]

[0104] To facilitate understanding and application of the present invention, the advantages of the invention will be described in more detail with reference to examples and accompanying drawings. However, it should be understood that the following examples are intended to illustrate the invention only and are not intended to limit the scope of the invention. The scope of the invention should be defined by the claims.

[0105] Example 1. Codon-optimized single GOI construct and a combinatorial construct generated by performing codon optimization on the combinatorial construct. To generate combination constructs for expressing both PGRN and STMN2 proteins via rAAV, codon optimization procedures were performed to improve protein expression. The wild-type coding sequences of GRN (G0; SEQ ID NO: 9) and STMN (S0; SEQ ID NO: 19) were ligated by the P2A sequence (P2A-3; SEQ ID NO: 33) to create G0S0 (GpS) and S0G0 (SpG). In G0S0, the progranulin coding sequence was located upstream of the statmin-2 coding sequence, while in S0G0, the order of the two GOIs was reversed. G0S0 and S0G0 were used as reference sequences.

[0106] Subsequently, based on reference sequences G0S0 and S0G0, six manually optimized combination sequences MC1-MC6 (sequence numbers: 21-26) were generated by subjecting the entire coding region, including the coding sequences of both Progranlin and Statomin-2, linked by intervening P2A sequences, to codon optimization. More frequent codons (human-specific) were used to replace wild-type codons in the construct without altering the polypeptide sequence. Hairpin-forming sequences and repetitive sequences were avoided during optimization. These optimized sequences have a reduced CG content while having a codon adaptation index (CAI) greater than 0.75, as calculated by an online tool (https: / / www.genscript.com / tools / rare-codon-analysis).

[0107] MC1, MC2, MC3, and MC6 are four different versions of the codon-optimized construct. In MC1 and MC3, the Programrin code sequence is placed before the Stasmin-2 code sequence, while in MC2 and MC6, the order of the two GOIs is reversed. MC4 and MC5 were generated by reversing the order of the GOIs while maintaining their sequences in M3 and M6, respectively.

[0108] As shown in Figures 1A and 1B, eight different combination sequences (MC1-MC6, GpS, SpG), consisting of the following elements in this order: EFShI1 promoter (SEQ ID NO: 27), first coding sequence, P2A sequence (SEQ ID NO: 31, 32, or 33), second coding sequence, hGH Poly A (SEQ ID NO: 29), and two AAV2 ITRs, were subsequently inserted into the AAV vector backbone plasmid. Detailed information on MC1-MC6 is shown in Table 1.

[0109] [Table 1]

[0110] These six MCs and GpSs were transfected into N2A cells (mouse neuroblastoma cell line, Procell #CL-0168) using jetOPTIMUS® (Polyplus-transfection®, #101000025) according to the instructions provided by the manufacturer. 72 hours after transfection, the supernatant was collected from the culture wells and concentrated to 200 μL (1:10 ratio) using a 30 kDa centrifuge (Amicon® Ultra-4 Centrifugal Filter Unit, Millipore, UFC8030) to measure the amount of secreted progranulin protein.

[0111] Cell lysates were also collected to measure the protein levels of progranlin and statimin-2. Cells in culture wells were washed once with 1×PBS and then 150 μL of RIPA buffer (Beyotime, P0013B or Thermo Scientific) containing a protease inhibitor cocktail (Roche, 11697498001) was added to generate cell lysates. TM The samples were recovered during (#89900). Cell soluble samples were recovered after sonication and centrifugation.

[0112] BCA assay The progranlin protein concentration in both the supernatant and cell soluble samples was measured by BCA assay (Beyotime, P0010S). All samples were boiled at 95°C for 10 minutes after adding reducing loading buffer.

[0113] Western blot Progranlin protein levels in both cell lysates and supernatants were also measured by Western blotting (WB). The entire sample (30 μg loading) was separated on an SDS-polyacrylamide gel (GenScript, M00657) with a colored protein ladder (Thermo Scientific, 26616) and transferred to a nitrocellulose membrane (Pall, 55253088) at 80 V for 2 hours. The membrane was washed in TBST (Solarbio, T1081) and then blocked overnight at 4°C with 5% BSA blocking buffer (Solarbio, SW3015). The membranes were incubated overnight at 4°C with an anti-PGRN antibody (R&D, #AF2420 or Abclonal, #A5124 or Sino Biological, #10826-R007) or an antibody against the housekeeping gene β-actin (Sigma, #A1978), followed by incubation at room temperature for 2 hours with a dye-labeled secondary antibody. The signal was then detected using the ODYSSEY Clx infrared imaging system (LI-COR Biosciences).

[0114] The protein level of statimin-2 in cell soluble samples was measured by Western blotting (WB) using a primary antibody against statimin-2 (Invitrogen, #PA5-21768, Proteintech, #10586-1-AP, or Abcam, #ab185956).

[0115] Sandwich ELISA The protein levels of progranulin in both the cell lysate and supernatant were also measured by sandwich ELISA (Sino Biological, KIT10826) according to the manufacturer's manual.

[0116] result Progranlin levels in the supernatant and cell solubil, as well as statimin-2 levels in the cell solubil, were measured by Western blotting (WB). As shown in Figure 2, among the six candidate strains, MC1 and MC5 expressed more progranlin in both the supernatant and cell solubil, and MC1 surpassed GpS.

[0117] MC1–MC3 similarly expressed high levels of statimin-2. Surprisingly, the expression level of STMN2 in MC3 was higher than that in MC4, meaning that the downstream drop-off (decreased expression) of STMN2, which is usually observed in combined constructs prepared in a single expression cassette driven by a single promoter, did not occur.

[0118] Constructs containing only GRN or STMN2 were also created for use as controls to evaluate combination effects in in vitro functional assays and in vivo studies. Codon-optimized sequences of GRN (G1-G4) or STMN2 (S1-S4) were extracted from MC1-MC6 and inserted into an AAV vector backbone plasmid consisting of the following elements in this order: two AAV2 ITRs adjacent to an EFShI1 promoter, a G or S coding sequence, and an hGH poly A sequence (see Figures 3A and 3B).

[0119] These MC-derived "single GOI" constructs were also transfected into SH-SY5Y cells (human neuroblastoma cell line, Procell, #CL-0208) using the Lipofectamine 3000 transfection reagent (Invitrogen, #L3000150) according to the manufacturer's instructions.

[0120] Cell soluble and supernatant samples were collected as described above to measure the protein expression levels of PGRN or stasmin-2 by WB. As shown in Figures 4 and 5, of the candidate constructs tested, G1 and G4 showed higher protein expression levels of PGRN (Figure 4), while S3 and S4 showed higher expression levels of STMN2 (Figure 5). Interestingly, G1 derived from MC1 and G4 derived from MC5 maintained their expression performance as observed in the combined constructs, whereas, regarding STMN2 expression, only S3 derived from MC3 and S4 derived from MC6 expressed high levels of STMN2, which was not observed in S1 and S2 derived from MC1 and MC2, respectively.

[0121] Example 2. Codon-optimized single GOI constructs and combined constructs generated by performing independent codon optimization for each GOI. Codon optimization was also performed for individual GOIs. Four additional coding sequences containing higher-frequency codons were generated for GRN (named G5-G8, SEQ ID NOs: 5-8) and STMN2 (named S5-S8, SEQ ID NOs: 15-18). All of these sequences were calculated using an online tool (https: / / www.genscript.com / tools / rare-codon-analysis) to have a codon adaptation index (CAI) greater than 0.85, while being free of CpG islands and having a reduced CG content. For reference, wild-type coding sequences for GRN (G0, SEQ ID NO: 9) and STMN2 (S0, SEQ ID NO: 19) were also synthesized.

[0122] Ten candidate constructs having the configurations shown in Figures 3A and 3B were generated using the eight codon-optimized sequences and two wild-type reference sequences described above. Specifically, each of the synthesized PGRN or STMN2 coding sequences was cloned into a vector skeleton having an EFShI1 promoter (SEQ ID NO: 27) and an SV40 Poly A tail (SEQ ID NO: 28) or an hGH Poly A tail (SEQ ID NO: 29), as well as two AAV2 ITRs.

[0123] In addition to MC1-MC6, a second batch of combination constructs was generated containing all 32 possible combinations of the individual codon-optimized sequences of the two GOIs in either G+S or S+G order (G5S5, G5S6, G5S7, G5S8, G6S5, G6S6, G6S7, G6S8, G7S5, G7S6, G7S7, G7S8, G8S5, G8S6, G8S7, G8S8, G1S3, G1S4, and G4S3, as well as the aforementioned constructs in the reverse order), and the protein expression of both GOIs was evaluated as described for the first batch of combination constructs in Example 1.

[0124] Example 3. Evaluation of protein expression in rAAV9 vectors containing combined GOI constructs and single GOI constructs. The combined GOI construct plasmid described in Example 1 was prepared and packaged in AAV9. The rAAV was diluted in culture medium and two different MOIs (1 × 10⁻¹) were used to transduce cells. 5 and 5×10 5 The AAV9-MC5 (G4S4) cells were then added to N2A-AAVR cells (a proprietary N2A cell line overexpressing the AAV receptor). The cells were harvested 72 hours after gene transfer according to the method described above to evaluate protein expression by WB. As shown in Figure 6, AAV9-MC5 (G4S4) expressed higher levels of GRN and STMN2 proteins than AAV9-MC2 (S2G2).

[0125] Example 4. In vivo therapeutic effect of rAAV candidates expressing optimized GRN and STMN2 in the hTDP-43 A315T recombinant ALS mouse model. The therapeutic effect of rAAV, including the selected combination GOI construct MC5, was evaluated in an ALS mouse model (hTDP-43 A315T ALS mouse, a mouse model overexpressing the human TDP-43 A315T transgene to reproduce the mutant TDP-43-induced ALS phenotype).

[0126] AAV9-MC5 was administered to hTDP-43 A315T ALS mice at three different doses (Low dose: 3.00 × 10⁶). 8 vg, mid dose (Mid): 1.00×10 9 vg, high dose (High): 3.00×10 9 The drug was administered via intrathecal injection (vg). Wild-type mice and hTDP-43 A315T ALS mice injected with the vehicle (Veh) alone were included as controls. Median survival time was used to evaluate the therapeutic effect. The results showed a slight improvement in median survival time in the AAV9-MC5 group compared to the vehicle control group (Figure 7).

[0127] Motor function was evaluated using the rotarod test 8 weeks after administration. This test measured the mean latency to fall from the rotating rod, allowing for a comparison of motor coordination ability among treated subjects. The results of the rotarod test are shown in Figure 8. The dotted line in Figure 8 shows the mean fall latency from the rod for the Veh group, which was significantly shorter compared to wild-type healthy mice. Compared to the Veh group, AAV9-MC5-treated mice showed a tendency toward improved motor coordination, as demonstrated by the extended latency to fall in the rotarod test (Figure 8).

[0128] At the 15th week after AAV administration, the surviving mice in the MC5 administration group were euthanized, and brain tissue samples were collected from the cortex and spinal cord. A part of the samples was used for WB analysis, while the remaining samples were fixed with 4% paraformaldehyde (PFA) for subsequent immunostaining experiments. WB analysis revealed a moderate decrease in the insoluble TDP-43 levels in both the cortex and spinal cord after AAV9-MC5 treatment (Figure 9). Furthermore, immunostaining showed a significant decrease in the TDP-43 positive regions, especially in the spinal cord, after MC5 administration (Figure 10).

[0129] Example 5. In vitro therapeutic effects of rAAV candidates expressing optimized GRN and STMN2 in ALS cell models A cell-based system was used to evaluate the effectiveness of the developed rAAV therapy in reducing TDP-43 aggregation. This cell line employed a cell strain engineered using the PiggyBac system to stably express a mutant form of TDP-43 fused to GFP for visualization. The Tet-on promoter controls the expression of TDP-43, enabling the induction of TDP-43 aggregates in cells by doxycycline treatment. As shown in Figure 11, treatment with AAV9-MC5 (MOI = 1×10 6 ) significantly reduced the levels of abnormal TDP-43 aggregates.

[0130] [Sequence Listing] TIFF2026517315000002.tif160162 TIFF2026517315000003.tif240162 TIFF2026517315000004.tif240162 TIFF2026517315000005.tif240162 TIFF2026517315000006.tif240162 TIFF2026517315000007.tif240162 TIFF2026517315000008.tif239162 TIFF2026517315000009.tif240162 TIFF2026517315000010.tif240162 TIFF2026517315000011.tif240162 TIFF2026517315000012.tif240162 TIFF2026517315000013.tif240162 TIFF2026517315000014.tif239162 TIFF2026517315000015.tif245162 TIFF2026517315000016.tif176162

Claims

1. An isolated nucleic acid molecule comprising a first polynucleotide sequence encoding a first polypeptide and a second polynucleotide sequence encoding a second polypeptide, The first polypeptide is progranlin (PGRN) and the second polypeptide is stasmin-2 (STMN2), or the first polypeptide is stasmin-2 (STMN2) and the second polypeptide is progranlin (PGRN), An isolated nucleic acid molecule in which the first polynucleotide sequence is located 5' upstream of the second nucleotide sequence.

2. The isolated nucleic acid molecule according to claim 1, wherein the polypeptide of progranlin (PGRN) includes or is composed of the polypeptide sequence of SEQ ID NO: 10, or its variants, homologs or orthomolecules thereof, and / or the polypeptide of statimin 2 (STMN2) includes or is composed of the polypeptide sequence of SEQ ID NO: 20, or its variants, homologs or orthomolecules thereof.

3. The isolated nucleic acid molecule according to claim 1 or 2, wherein the polynucleotide sequence encoding PGRN is a polynucleotide sequence selected from the group consisting of polynucleotide sequences shown in any one of SEQ ID NOs: 1 to 9, and / or the polynucleotide sequence encoding STMN2 is a polynucleotide sequence selected from the group consisting of polynucleotide sequences shown in any one of SEQ ID NOs: 11 to 19.

4. An isolated nucleic acid molecule according to any one of claims 1 to 3, wherein the first polynucleotide sequence and the second polynucleotide sequence are linked within a frame and functionally linked to a single promoter located upstream of the 5' end of both the first and second nucleotide sequences.

5. The isolated nucleic acid molecule according to claim 4, wherein the promoter is the EF1α promoter, the EFS promoter, or a variant or derivative thereof.

6. The isolated nucleic acid molecule according to claim 5, wherein the promoter is EFShI1 (SEQ ID NO: 27).

7. The isolated nucleic acid molecule according to any one of claims 1 to 6, wherein the isolated nucleic acid molecule further comprises a linker sequence between the first polynucleotide sequence and the second polynucleotide sequence.

8. The isolated nucleic acid molecule according to claim 7, wherein the linker sequence is a coding sequence for a self-cleaving peptide, preferably a coding sequence for a 2A peptide, and more preferably a coding sequence for a P2A peptide.

9. The isolated nucleic acid molecule according to any one of claims 1 to 8, wherein the isolated nucleic acid molecule contains or is composed of a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 21 to 26.

10. The isolated nucleic acid molecule according to claim 9, wherein the isolated nucleic acid molecule comprises or is composed of a polynucleotide sequence which is sequence number 22 or sequence number 25.

11. The isolated nucleic acid molecule according to any one of claims 1 to 10, further comprising one or more regulatory sequences selected from polyadenylation signals, post-transcriptional regulator (WPRE) sequences, and enhancers.

12. The isolated nucleic acid molecule according to claim 11, wherein the polyadenylation signal is human growth hormone (hGH) Poly A.

13. Sequence ID: A codon-optimized coding sequence for a PGRN that contains or is composed of one of the polynucleotide sequences represented by sequences 1 through 8.

14. Sequence ID: A codon-optimized coding sequence for STMN2 containing or composed of one of the polynucleotide sequences shown in sequence IDs 11-18.

15. An isolated nucleic acid molecule according to any one of claims 1 to 12, or an expression cassette comprising a codon-optimized coding sequence according to claim 13 or 14.

16. The expression cassette according to claim 15, which is suitable for use in an adeno-associated virus (rAAV) vector.

17. A recombinant adeno-associated virus (rAAV) vector comprising an isolated nucleic acid molecule according to any one of claims 1 to 12, or a codon-optimized coding sequence according to claim 13 or 14, or an expression cassette according to claim 15 or 16.

18. The rAAV according to claim 17, wherein the rAAV vector is AAV9 serotype or ViVec AAV.

19. The rAAV according to claim 18, wherein the Vivec AAV vector has a capsid polypeptide obtained by inserting seven amino acids between amino acid positions Q588 and A589 of the wild-type AAV9 VP1 capsid protein shown in Sequence ID No. 34, and the Vivec AAV has increased affinity for one or more tissues or cells of the central nervous system (CNS), and / or can produce a higher level of transgene expression in tissues or cells of the central nervous system compared to an rAAV having a capsid of wild-type serotype AAV9.

20. The rAAV according to claim 19, wherein the insertion of the seven amino acids is shown in any one of sequence numbers 35 to 94.

21. The rAAV according to any one of claims 17 to 20, wherein the rAAV further comprises two terminal inversion sequences (ITRs), preferably two AAV2 ITRs.

22. A virus particle containing rAAV as described in any one of claims 17 to 21.

23. A pharmaceutical composition comprising rAAV according to any one of claims 17 to 21, or virus particles according to claim 22, together with a pharmaceutically acceptable excipient.

24. A method for treating or preventing neurodegenerative disease (ND) in a subject requiring such treatment, comprising administering to the subject an rAAV vector according to any one of claims 17 to 21, a viral particle according to claim 22, or a pharmaceutical composition according to claim 23.

25. The method according to claim 24, wherein the neurodegenerative disease is selected from amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), Huntington's disease (HD), Parkinson's disease (PD), multiple system atrophy (MSA), or Alzheimer's disease (AD).

26. The method according to claim 24 or 25, wherein the neurodegenerative disease is related to TDP-43 aggregation.

Images