Treatment of myelin conditions

Abstract

The present disclosure relates to RNA molecules, recombinant nucleic acids and gene therapy vectors for the treatment of diseases and conditions associated with myelination deficiency or dysfunction.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Application No. 63 / 363,529, filed on April 25, 2022. The contents of said application are incorporated herein by reference in their entirety.

[0003] References to sequence listings

[0004] This application incorporates by reference the Sequence Listing submitted in computer readable form as file Sequence_Listing_323429-00201.xml, which was created on April 19, 2023 and contains 258,210 bytes. Technical Field

[0005] The present disclosure relates to compositions and methods for treating inherited and / or acquired myelin disorders. Background Art

[0006] The human central nervous system (CNS), which includes the brain and spinal cord, is made up of two types of cells: neurons and glial cells. Neurons have a cell body that houses the nucleus (where the cell's genes are located), an axon that extends from the cell body (terminating at the axon terminal), and dendrites that branch out from the cell body and connect with the axon terminals of other neurons. Neurons receive information (from other neurons) at the cell body and dendrites, and send this information along their axons to other neurons. Information is transmitted along the length of the axon in the form of an electrical signal called an action potential. At the end of the axon, the action potential triggers the release of neurotransmitters (into the synapse, the small space between cells that the neurotransmitters span). All activities of the central nervous system, namely thinking, processing sensory information, storing memories, and controlling muscles and glands, are carried out by interconnected networks of neurons.

[0007] The glial cells of the CNS support the function of neurons. These cells include astrocytes, microglia and oligodendrocytes. Astrocytes constitute the blood-brain barrier (BBB), provide nutrition to neurons, and maintain extracellular ion balance and neurotransmitter levels. Microglia are phagocytic cells similar to macrophages, which migrate through the CNS and remove damaged or unnecessary substances. Oligodendrocytes are cells that produce myelin in the central nervous system.

[0008] Myelin is a complex substance composed of high levels of saturated long-chain fatty acids, glycosphingolipids, cholesterol and proteins. Oligodendrocytes produce myelin by extending lamellar processes, each of which contacts and tightly wraps a segment of axon multiple times to produce the unique architecture of myelin. On the same axon, adjacent myelin segments belong to different oligodendrocytes, and a single oligodendrocyte can myelinate up to 50 axon segments (Stadelmann et al., 2019). Oligodendrocyte myelin promotes rapid conduction of action potentials and supports the metabolic needs of axons. Oligodendrocytes are also involved in extracellular fluid regulation and provide neuronal nutritional support by producing neurotrophic factors such as glial cell line-derived neurotrophic factor (GDNF), brain-derived neurotrophic factor (BDNF) or insulin-like growth factor-1 (IGF-1) (Bradl and Lassmann, 2010).

[0009] The central nervous system is divided into "grey matter" and "white matter". The grey matter usually contains the cell bodies and dendrite networks of neurons, while the white matter consists of bundles of axons wrapped in myelin produced by oligodendrocytes. The myelin sheath has a high lipid fat content, which is why it appears white. Myelin plays a key role in neuronal communication. Damage to oligodendrocytes can disrupt the integrity of white matter and lead to white matter degeneration (demyelination) and loss of neuronal communication within the brain and spinal cord.

[0010] Inherited or acquired myelin-related disorders affect millions of people, placing a heavy burden on affected individuals and their families. The underlying pathological processes of many of these disorders remain poorly understood, and few disease-modifying therapies exist. Therefore, therapeutic agents for the treatment of these disorders are needed. Summary of the invention

[0011] The present disclosure addresses the above needs from multiple aspects.

[0012] One aspect of the present invention provides a method for reducing the expression of a gene in oligodendrocytes or a method for treating an inherited or acquired myelin disorder, the method comprising providing a composition comprising adeno-associated virus (AAV) particles having a preferential tropism for the surface of oligodendrocytes; wherein the AAV particles encapsidate a polynucleotide; wherein the polynucleotide comprises a 5′ inverted terminal repeat sequence (ITR), a promoter sequence region, a polynucleotide encoding a pri-miRNA or pre-miRNA targeting the gene, an optional post-transcriptional regulatory element (e.g., a woodchuck hepatitis post-transcriptional regulatory element), a polyA signal sequence region, and a 3′ ITR.

[0013] In certain embodiments, the polynucleotide is a pri-miRNA or pre-miRNA scaffold derived from human mir-16-1, miR-21, miR-23a, miRNA-30a, miR-31, miR-122, miR-155 or miR-451, wherein the pri-miRNA or pre-miRNA scaffold does not include the native sequence of the guide strand and the passenger strand of the pre-miRNA; wherein a heterologous guide strand is inserted into the scaffold as a substitute for the native sequence of the guide strand, wherein the heterologous guide strand is complementary to the mRNA of the target gene, wherein after processing of the pri-miRNA or pre-miRNA by a cytoplasmic nuclease, the heterologous guide strand is incorporated into the RISC complex to allow the RISC complex to target the mRNA of the target gene and downregulate the expression of the gene.

[0014] In certain embodiments, the heterologous guide strand is complementary to an mRNA for a protein whose elimination improves the treatment outcome of an inherited or acquired myelin disorder, such as Pelizaeus-Merzbacher disease, multiple system atrophy, or hypomyelination with basal ganglia and cerebellar atrophy.

[0015] In one aspect, the present disclosure provides a method for reducing the expression of a target gene in an oligodendrocyte or a method for treating an inherited or acquired myelin disorder. The method comprises: (1) providing an AAV particle having a preferential tropism for the surface of an oligodendrocyte; wherein the AAV particle encapsulates a nucleic acid with a capsid, and the nucleic acid comprises from 5' to 3': a 5'ITR, a promoter sequence region, a polynucleotide encoding a pri-miRNA or pre-miRNA targeting the target gene, and a 3'ITR; and (2) contacting the AAV particle with the oligodendrocyte. In some embodiments, the nucleic acid may further comprise one or more of a post-transcriptional regulatory element and a polyA signal sequence region between the polynucleotide and the 3'ITR. The pri-miRNA or pre-miRNA may comprise (a) a pri-miRNA or pre-miRNA scaffold, (b) a heterologous guide strand, and (c) a heterologous passenger strand.

[0016] In one embodiment, the pri-miRNA or pre-miRNA scaffold is a human pri-miRNA or pre-miRNA scaffold derived from human microRNA. Examples of the human microRNA include microRNAs derived from human mir-16-1, miR-21, miR-23a, miRNA-30a, miR-31, miR-122, miR-155 or miR-451. Preferably, the human pri-miRNA or pre-miRNA scaffold does not include the natural sequence of the natural guide strand of the human microRNA and the natural sequence of the natural passenger strand. The heterologous guide strand can be inserted into the human pri-miRNA or pre-miRNA scaffold as a substitute for the natural sequence of the natural guide strand. The heterologous guide strand can be complementary to the mRNA of the target gene. After the pri-miRNA or pre-miRNA is processed by a cytoplasmic nuclease, the heterologous guide strand is incorporated into the RISC complex to allow the RISC complex to target the mRNA of the target gene and downregulate the expression of the target gene.

[0017] In the methods described above, the heterologous guide strand can be complementary to the mRNA of a protein, the elimination of which improves the treatment outcome of a hereditary or acquired myelin disorder. In one example, the hereditary or acquired myelin disorder can be Pelizhaus-Merzbacher disease. In the case, the protein can be PLP1. To this end, the heterologous guide strand can comprise a nucleotide sequence that is at least 70%, 80%, 85%, 90% or 95% identical to one of SEQ ID NOs: 42 to 80 (e.g., SEQ ID NO: 54 or SEQ ID NO: 75). In another example, the hereditary or acquired myelin disorder is multiple system atrophy. In the case, the targeted protein can be alpha-synuclein. To this end, the heterologous guide strand can comprise a nucleotide sequence that is at least 70%, 80%, 85%, 90% or 95% identical to one of SEQ ID NOs: 122 to 161. In another example, the inherited or acquired myelin disorder is hypomyelination with basal ganglia and cerebellar atrophy (H-ABC). In such a case, the targeted protein may be the microtubule-associated protein tubulin beta-4a. Thus, the heterologous guide strand may comprise a nucleotide sequence having at least 70%, 80%, 85%, 90% or 95% identity to one of SEQ ID NOs: 204 to 244. Preferably, the nucleotide sequence is 21 to 30 nucleotides in length.

[0018] In another aspect, the present disclosure is characterized in that a RNA molecule comprises a first RNA sequence and a second RNA sequence. The first sequence is substantially complementary to the second RNA sequence, wherein the first RNA sequence has a sequence length of at least 19 nucleotides and is at least 70%, 80%, 85%, 90% or 95% or 100% complementary to a target sequence selected from the group consisting of: SEQ ID NO: 2-40, 82-121 and 163-203. In one embodiment, the target sequence comprises or is SEQ ID NO: 14 or 35. In one embodiment, the RNA molecule may be contained in a pre-miRNA scaffold or a pri-miRNA scaffold, such as a human pri-miRNA or pre-miRNA scaffold derived from a human microRNA. Examples of the human microRNA include human mir-16-1, miR-21, miR-23a, miRNA-30a, miR-31, miR-122, miR-155 and miR-451. In one example, the human microRNA is miRNA-30a. Preferably, the human pri-miRNA or pre-miRNA scaffold does not include the native sequence of the native guide strand of the human microRNA and the native sequence of the native passenger strand. In one example, the first strand is a heterologous guide strand that is inserted into the human pri-miRNA or pre-miRNA scaffold as a substitute for the native sequence of the native guide strand. In one embodiment, the RNA molecule comprises, or consists essentially of, or consists of: Fig.11 Or the sequence of SEQ ID NO:284 or 285 shown in 12.

[0019] The scope of the present disclosure includes: (1) a polynucleotide encoding the RNA molecule described above; (2) an expression cassette or expression vector comprising the polynucleotide; and (3) a host cell comprising the polynucleotide or the expression cassette or expression vector. The expression cassette or expression vector may further comprise one or more of the following from 5' to 3': 5'ITR, a promoter sequence region, a post-transcriptional regulatory element, a polyA signal sequence region, and a 3'ITR. In some embodiments, the expression vector is a viral vector, such as an AAV vector. In one example, the AAV vector has a preferential tropism for oligodendrocytes. Examples of such AAV vectors include AAV / Olig001, AAV / Olig002, and AAV / Olig003. Another example is AAV9 with six glutamic acid residues inserted into the VP2 region, such as described in Powell SK et al., Mol Ther 28(5): 1373-1380.

[0020] The RNA molecules, or polynucleotides, or expression cassettes, or expression vectors, or host cells described above can be used to treat myelin-related disorders. Therefore, the present disclosure further provides a pharmaceutical composition comprising: (a) one or more of the RNA molecules, the polynucleotides, the expression cassettes, the expression vectors, and the host cells; and (b) a pharmaceutically acceptable carrier.

[0021] A method for treating myelin disorders is also provided. The method comprises administering one or more of the RNA molecules, polynucleotides, expression cassettes, expression vectors, and host cells described above to a subject in need thereof. The subject may be a human being. The administration may be performed via injection. In some embodiments, the RNA molecule, or the polynucleotide, or the expression cassette, or the expression vector, or the host cell, or the pharmaceutical composition may be administered to a central nervous system region selected from the group consisting of: brain parenchyma, spinal canal, subarachnoid space, ventricle, cisterna magna, and combinations thereof. The RNA molecule, or the polynucleotide, or the expression cassette, or the expression vector, or the host cell, or the pharmaceutical composition may be administered by a method selected from the group consisting of: intraparenchymal administration, intrathecal administration, intraventricular administration, intracisterna mag ...

[0022] The present disclosure also provides similar RNA molecules, or polynucleotides, or expression cassettes, or expression vectors, or host cells, or pharmaceutical compositions, or methods for treating other myelin disorders in a subject in the same manner as described above. In such cases, the RNA molecule can reduce or inhibit the level or function of a related gene, and the gain of function of the related gene leads to the disorder. Examples of the disorder and related genes include: Alexander disease, which has a gain-of-function mutation in glial fibrillary acid protein (GFAP); Mitchell disease, which has a gain-of-function mutation in acyl-CoA oxidase 1 (ACOX1); autosomal dominant leukodystrophy with autonomic disease (ADLD), which has a genomic duplication of the lamin B1 gene (LMNB1) (or a deletion upstream of the gene), which results in increased expression of the LMNB1 gene; central myelinating leukodystrophy, Waardenburg syndrome, or Hirschsprung's disease. disease, which has a duplication at 22q11.2q13 that includes SOX10, the gene encoding the transcription factor SOX-10; adult polyglucan body disease (APBD), which has mutations that alter the folding of glycogen branching enzyme (GBE1) resulting in a gain of function; hereditary diffuse leukoencephalopathy with spheroid bodies, which has a gain of function mutation in CSF1R, the gene encoding the colony stimulating factor 1 receptor; or Aicardi-Goutieres syndrome, which has a gain of function mutation in IFIH1, the gene encoding the interferon-inducible helicase C domain-containing protein 1.

[0023] Therefore, the present disclosure is characterized in that a kind of RNA molecule reduces or inhibits the level or function of one of the genes described herein. The RNA molecule comprises a first RNA sequence and a second RNA sequence. The first sequence is substantially complementary to the second RNA sequence, wherein the sequence length of the first RNA sequence is at least 19 nucleotides and is at least 90% complementary to the mRNA encoding one of the genes mentioned herein. In one embodiment, the RNA molecule may be contained in a pre-miRNA scaffold or a pri-miRNA scaffold, such as a human pri-miRNA or pre-miRNA scaffold derived from human microRNA. Examples of the human microRNA include human mir-16-1, miR-21, miR-23a, miRNA-30a, miR-31, miR-122, miR-155 and miR-451. Preferably, the human pri-miRNA or pre-miRNA scaffold does not include the natural sequence of the natural guide chain of the human microRNA and the natural sequence of the natural passenger chain. In one example, the first strand is a heterologous guide strand that is inserted into the human pri-miRNA or pre-miRNA scaffold as a replacement for the native sequence of the native guide strand.

[0024] In other embodiments, the disorder is Canavan disease, Krabbedisease, globular cell leukodystrophy, X-linked adrenoleukodystrophy, metachromatic leukodystrophy, hypomyelinating leukodystrophy-2, Niemann-Pick disease type C, 4H leukodystrophy / Pol III-associated leukodystrophy, Zellweger Spectrum Disorder, childhood ataxia with central nervous system hypomyelination, cerebrotendinous xanthomas, SOX10-associated peripheral demyelinating neuropathy, Adult Refsum disease, autism spectrum disorder, Alzheimer's disease, Parkinson's disease, disease, fragile X syndrome, schizophrenia, multiple sclerosis, neuromyelitis optica, progressive multifocal leukoencephalopathy, encephalomyelitis, central pontine myelinolysis, adrenoleukodystrophy, Wallerian Degeneration, optic neuritis, transverse myelitis, amyotrophic lateral sclerosis, Huntington's disease, spinal cord injury, traumatic brain injury, post-radiation injury, neurologic complications of chemotherapy, stroke, acute ischemic optic neuropathy, vitamin E deficiency, isolated vitamin E deficiency syndrome, Bassen-Kornzweig syndrome, Marchiafava-Bignami syndrome, metachromatic leukodystrophy, trigeminal neuralgia, acute disseminated encephalitis, Guillian-Barre syndrome, Marie-Charcot-Tooth disease, or Bell's palsy.

[0025] The details of one or more embodiments of the present disclosure are set forth in the description below. Other features, objects, and advantages of the present disclosure will be apparent from the specification and claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1Shown are the 5' flanking sequence, stem-loop sequence (wherein the guide and passenger strand sequences are depicted in red (bold and underlined) and blue (italic and underlined)), 3' flanking sequence and stem-loop structure of human mir-16-1NR_029486 (mbase accession number MI0000070) (Han et al., 2006), which results in nucleotide bulges due to incomplete complementarity.

[0027] Figure 2 Shown are the 5' flanking sequence, stem-loop sequence (where the guide and passenger strand sequences are depicted in red (bold and underlined) and blue (italic and underlined)), 3' flanking sequence, and stem-loop structure of human miR-21NC_000017.11 (Yue et al., 2010) (mbase accession number MI0000077), which results in nucleotide bulges due to imperfect complementarity.

[0028] Figure 3 Shown are the 5' flanking sequence, stem-loop sequence (where the guide and passenger strand sequences are depicted in red (bold and underlined) and blue (italic and underlined)), 3' flanking sequence, and stem-loop structure of human miR-23a NR_029495 (mbase accession number MI0000079) (van den Berg et al., 2016), which results in nucleotide bulges due to imperfect complementarity.

[0029] Figure 4 Shown are the 5' flanking sequence, stem-loop sequence (where the guide and passenger strand sequences are depicted in red (bold and underlined) and blue (italic and underlined)), 3' flanking sequence and stem-loop structure of human miRNA-30a NR_029504 (mbase accession number MI0000088) (Zeng et al., 2002), which results in nucleotide bulges due to imperfect complementarity.

[0030] Figure 5 Shown are the 5' flanking sequence, stem-loop sequence (where the guide and passenger strand sequences are depicted in red (bold and underlined) and blue (italic and underlined)), 3' flanking sequence, and stem-loop structure of human miR-31NR_029505.1 (mbase accession number MI0000089) (Ely et al., 2008), which results in nucleotide bulges due to imperfect complementarity.

[0031] Figure 6Shown are the 5' flanking sequence, stem-loop sequence (where the guide and passenger strand sequences are depicted in red (bold and underlined) and blue (italic and underlined)), 3' flanking sequence, and stem-loop structure of human miR-122NR_029667 (mbase accession number MI0000442) (Ely et al., 2008), which results in nucleotide bulges due to imperfect complementarity.

[0032] Figure 7 Shown are the 5' flanking sequence, stem-loop sequence (where the guide and passenger strand sequences are depicted in red (bold and underlined) and blue (italic and underlined)), 3' flanking sequence and stem-loop structure of human miR-155NR_030784NC_000021.9 (mbase accession number MI0000681), which results in nucleotide bulges due to imperfect complementarity.

[0033] Figure 8 Shown are the 5' flanking sequence, stem-loop sequence (where the guide strand sequence is depicted in red (bold and underlined)), 3' flanking sequence and stem-loop structure of human miR-451NR_029970 (mbase accession number MI0001729) (Yoda et al., 2013), which results in nucleotide bulges due to imperfect complementarity.

[0034] Fig. 9 The example of target region selection is shown. The target region in the coding sequence of the gene of interest is identified using a non-commercial design tool based on a web-based algorithm. A target region is SEQ ID NO:35, and the sequence is shown in red (bold and underlined). Determine the nucleotide sequence that is completely complementary to the target sequence, and align from 3' to 5' to show complementarity. Below the arrow, SEQ ID NO:75 is shown in blue (italics and underlined) in the 5' to 3' direction.

[0035] Fig.10 The 3' and 5' flanking regions (50-100 nucleotides) and loop regions (shown in black) of human miR-30a used as a backbone are shown. The guide strand of human miR-30a is shown in red (bold and underlined). A sequence of 20 to 22 nucleotides fully complementary to the PLP1 target sequence is substituted after the 5' flanking sequence, followed by a loop sequence from a naturally occurring microRNA, a reverse complement of the guide strand of 20 to 22 nucleotides (modified by deletion positions 10 to 11 to produce a bulge, thereby allowing the guide strand to be preferentially loaded into the RISC complex, depicted in blue (bold and underlined)), followed by a 3' flanking sequence.

[0036] Fig.11Examples of conservative secondary structures of the pre-miRNA polynucleotide of human miR-30a predicted by mfold (SEQ ID NO: 283) and the modified miR-30a-PLP1 polynucleotide of the present disclosure (SEQ ID NO: 284) are shown. The designed pre-miRNA miR-30a-PLP1 has the same secondary structure as the native sequence (with the same framework region, but with different guide sequences and chain sequences), and has a free energy (dG) similar to the native sequence. The resulting hairpin has a conserved loop region and contains the cleavage site required for Dicer to remove the loop and leave the dsRNA duplex. However, the two structures will target different mRNA molecules. In this case, miR-30a-PLP1 targets human PLP1 mRNA. The miR-30a-PLP1 sequence on the right contains the RNA sequence of SEQ ID NO: 75, in which T is replaced by U.

[0037] Fig.12 Another modified miR-30a-PLP1 polynucleotide (SEQ ID NO: 285) of the present disclosure predicted by mfold is shown. The designed pre-miRNA miR-30a-PLP1 has the same secondary structure as the native sequence (with the same framework region, but with different guide sequences and chain sequences), and has a free energy (dG) similar to the native sequence. The resulting hairpin has a conserved loop region and contains a cleavage site required for Dicer to remove the loop and leave a dsRNA duplex. In this case, miR-30a-PLP1 contains an RNA sequence of 21 nucleotides from SEQ ID NO: 14 and SEQ ID 54, and targets human PLP1mRNA. DETAILED DESCRIPTION

[0038] The present disclosure relates to compositions and methods for treating inherited and / or acquired myelin disorders (eg, demyelination).

[0039] 1. Myelin-related diseases

[0040] Myelin-related disorders include any disease or condition associated with demyelination, myelination and insufficient remyelination or dysmyelination of the subject. Such disorders can be hereditary or acquired or both. CNS demyelination may occur due to gene mutation (leukodystrophy), autoimmune disease (e.g., multiple sclerosis) or trauma (e.g., traumatic brain injury, spinal cord injury or ischemic stroke). Furthermore, disturbances in myelin function may play a key role in neurological and psychiatric disorders such as Autism Spectrum Disorder (ASD), Alzheimer's disease (Nasrabady et al., 2018), Multiple System Atrophy (Wenning et al., 2008), Parkinson's disease (Bohnen and Albin, 2011), Fragile X syndrome (Filley, 2016), and schizophrenia (Najjar and Pearlman, 2015).

[0041] Leukodystrophy is a group of rare, mainly inherited neurological disorders that are caused by abnormal production, processing or development of myelin and are the result of genetic defects (mutations). Some forms are present at birth, while others may not show symptoms until a child is in early childhood. A few forms mainly affect adults. Leukodystrophies include Canavan disease (MIM#271900), Pelitzaus-Merzbacher disease (MIM#312080), hypomyelination with basal ganglia and cerebellar atrophy (OMIM#612438), Krabbe disease (globoid cell leukodystrophy, MIM#245200), X-linked adrenoleukodystrophy (MIM#300100), metachromatic leukodystrophy (MLD, MIM#250100), Pelitzaus-Merzbacher disease (or hypomyelination leukodystrophy-2, MIM#608804), Niemann-Pick disease type C (NPC, MIM#257220), autosomal dominant leukodystrophy with autonomic disease (ADLD, MIM#169500), 4H leukodystrophy (Pol III-related leukodystrophy, MIM#607694), Zellweger spectrum disorder (ZSD, MIM#various), childhood ataxia with CNS hypomyelination or CACH (also known as ablative white matter disease or VWMD, MIM#603896), cerebrotendinous xanthomatosis (CTX, MIM#213700), Alexander disease (AXD, MIM#203450), SOX10-related peripheral demyelination Neuropathy, central dysmyelinating leukodystrophy, Waardenburg syndrome and Hirschsprung disease (PCWH, MIM#609136), adult polyglucan body disease (APBD, MIM#263570), hereditary diffuse leukoencephalopathy with spheroids (HDLS, MIM#221820), Acardi-Guterres syndrome (AGS, MIM#many), and adult Refsum disease (MIM#266500).

[0042] A. Pelitzaus-Merzbacher disease

[0043] Pelitzaus-Merzbacher disease (PMD) is a rare, fatal, X-linked, recessive dysmyelinating leukodystrophy that typically presents in the first year of life. Symptoms include hypotonia, nystagmus, and delayed developmental milestones, particularly in motor function. PMD is a progressive disease that causes declines in coordination, motor ability, and intellectual function and leads to early death, often before adulthood. There are approximately 3,500 patients with PMD in the United States (Schmidt et al., 2020), and there are no effective treatments.

[0044] The most common (Elitt et al., 2020) form of PMD is caused by repeated mutations in the gene encoding the myelin protein proteolipid protein 1 (PLP1), one of the most abundant protein components of the myelin sheath. PLP1 facilitates adhesion to the outer membrane surface to produce the unique architecture of the myelin sheath.

[0045] Overexpression of the PLP1 protein in oligodendrocytes triggers dysfunction, impedes normal myelination, and leads to a massive loss of myelin-forming oligodendrocytes in the CNS (Osório and Goldman, 2018). Reducing PLP1 expression to normal levels in patients with gene duplications could be used to restore oligodendrocyte function and improve outcomes.

[0046] Overexpression of PLP1 is an example of "toxic gain of function". In this case, the overexpressed protein causes cytotoxicity. One way to address PLP1 overexpression is to reduce the expression of the PLP1 gene by using RNA interference. This can be achieved through microRNA-based gene therapy. A single administration of an AAV vector that delivers an expression cassette of a therapeutic miRNA precursor targeting PLP1 mRNA can be used to activate endogenous mRNA silencing mechanisms to reduce PLP1 translation in oligodendrocytes. In addition, since PLP1 overexpression is primarily confined to oligodendrocytes, AAV vectors with a higher tropism for oligodendrocytes can be used to improve safety and therapeutic efficacy.

[0047] An antisense oligonucleotide strategy can be used to inhibit overexpression of PLP1 in jimpy mice, which mimic PMD by expressing abnormal PLP and recapitulating cellular, molecular, and neurological features seen in severe PMD. A single dose of an antisense oligonucleotide targeting PPL1 in postnatal jimpy mice restored oligodendrocyte numbers, increased myelination, improved motor performance, normalized respiratory function, and extended lifespan to an endpoint of eight months (Illit et al., 2020).

[0048] The miRNA strategy can also be used to reduce PLP1 overexpression in Plp1-Tg mice (PMD model caused by PLP1 duplication). In mouse PMD models (Plp1 transgenic mice, PLP1 duplication model), direct intraparenchymal injection (into brain tissue, specifically the oligodendrocyte-rich striatum and internal capsule) of AAV vectors with a transgene encoding an artificial miRNA targeting PLP1 mRNA (synthetic miR-155 containing a PLP1-directed short hairpin) under the control of oligodendrocyte-specific (human CNP) prevented oligodendrocyte death, restored myelin, and improved neurological phenotype and survival (Li et al., 2019).

[0049] A PMD therapeutic approach that combines AAV vectors that effectively target oligodendrocytes with a miRNA strategy for reducing PLP1 gene overexpression may be beneficial for the treatment of PMD.

[0050] B. Multiple system atrophy

[0051] Multiple system atrophy (MSA) is a rare, fatal and progressive neurodegenerative disorder characterized by symptoms similar to those of Parkinson's disease, including slow movements, tremors, rigidity (stiffness), ataxia and speech disorders. Symptoms usually appear between the ages of 50 and 60 and progress rapidly. Most MSA patients die from the disease or its complications within 6 to 10 years of symptom onset. There are approximately 15,000 to 50,000 MSA patients in the United States (Multiple System Atrophy Fact Sheet | National Institute of Neurological Disorders and Stroke, n.d.). Although some drugs provide symptom relief, there is no therapy to slow the progression of the disease and there is no cure.

[0052] The pathology of MSA is similar to that of Parkinson's disease, characterized by the accumulation and aggregation of the synaptic protein alpha-synuclein, a protein involved in the release of neurotransmitters. However, unlike Parkinson's disease, in which alpha-synuclein accumulates and aggregates in neurons, MSA is characterized by the abnormal accumulation of alpha-synuclein within oligodendrocytes, known as glial cytoplasmic inclusions. Due to the toxic accumulation of alpha-synuclein in the form of glial cytoplasmic inclusions, the oligodendrocyte-myelin-axon complex is destroyed, causing inflammation, demyelination, and subsequent neuronal loss.

[0053] Accumulation of α-synuclein in oligodendrocytes triggers dysfunction, impedes normal myelination, and leads to secondary neurodegeneration in the CNS (Marmion et al., 2021). Reducing α-synuclein levels in oligodendrocytes of MSA patients is expected to restore inhibition of glial inclusion formation and improve outcomes.

[0054] One way to address α-synuclein overexpression is to use RNA interference to reduce the expression of the α-synuclein gene. This can be achieved through microRNA-based gene therapy. A single administration of an AAV vector that delivers an expression cassette of a therapeutic miRNA precursor targeting α-synuclein mRNA is expected to activate endogenous mRNA silencing mechanisms to reduce α-synuclein translation and subsequent accumulation in oligodendrocytes. In addition, because α-synuclein accumulation in MSA is primarily confined to oligodendrocytes, the use of AAV vectors with a higher tropism for oligodendrocytes is expected to improve safety and therapeutic efficacy.

[0055] Recently, AAV-Olig001, an AAV vector with a unique tropism for oligodendrocytes (Powell et al., 2016), was used to generate a model of MSA in mice. AAV-Olig001 with an α-synuclein transgene produced selective overexpression of α-synuclein in oligodendrocytes, with a tropism of >95% for oligodendrocytes in the dorsal striatum, resulting in demyelination and neuroinflammation similar to human MSA (Williams et al., 2020). Similarly, intrastriatal injection of AAV-Olig001 expressing an α-synuclein transgene in rhesus monkeys resulted in widespread expression of α-synuclein throughout the striatum. Demyelination was observed in the white matter tracts of the corpus callosum and striatum in animals injected with AAV-Olig001-α-synuclein but not in animals injected with AAV-Olig001-GFP, similar to human disease (Mandel et al., 2017).

[0056] Recently, Mavroeidi et al. demonstrated that administration of α-synuclein to cultured mouse oligodendrocytes recruited endogenous oligodendrocyte α-synuclein in toxic aggregates (Mavroeidi et al., 2019). Similarly, endogenous oligodendrocyte α-synuclein was incorporated into pathological aggregates generated by in vivo administration of α-synuclein to the mouse brain (Mavroeidi et al., 2019). Moreover, this was ameliorated in α-synuclein knockout mice. These results suggest that endogenous α-synuclein is required for the formation of intracellular α-synuclein aggregates. Manipulation of α-synuclein expression in oligodendrocytes may provide a rational approach to reduce the accumulation of α-synuclein in glial cytoplasmic inclusions, thereby delaying or preventing the rapid progression of MSA.

[0057] As disclosed herein, it may be advantageous to use novel MSA treatment approaches that combine AAV vectors that effectively target oligodendrocytes with miRNA strategies for reducing α-synuclein gene expression.

[0058] C. Hypomyelination with atrophy of the basal ganglia and cerebellum

[0059] Hypomyelination with basal ganglia and cerebellar atrophy (H-ABC) is a rare, autosomal dominant, dysmyelinating leukodystrophy in children. H-ABC usually presents in early childhood with symptoms such as dystonia, progressive gait disorders, and speech and cognitive deficits. The symptoms and progression of H-ABC are more severe when they occur in the first few months of life, but less severe when symptoms occur later in childhood. Magnetic resonance imaging (MRI) typically shows characteristic hypomyelination and atrophy of the caudate and putamen, as well as cerebellar atrophy (Simons et al., 2013). There are approximately 2,600 patients with H-ABC in the United States (Schmidt et al., 2020), and there is no effective treatment.

[0060] H-ABC is caused by toxic gain-of-function mutations in the TUBB4A gene, which encodes the microtubule-associated protein tubulin β-4a, which heterodimerizes with α-tubulin to form subunits that assemble into microtubules. Microtubules are cables within cells that help support the shape of the cell and move proteins to where they are needed. Tubulin β-4a protein is a microtubule component that is highly expressed in mature oligodendrocytes. Mutations in the Tubb4a gene alter and disrupt microtubule dynamics and ultimately lead to the loss of oligodendrocytes (Curiel et al., 2017; Sase et al., 2020).

[0061] Overexpression of tubulin β-4a protein alters microtubule dynamics in oligodendrocytes and induces dysfunction, impeding normal myelination and leading to a massive loss of myelin-forming oligodendrocytes in the CNS (Currier et al., 2017; Sass et al., 2020). Normalizing tubulin β-4a protein expression in patients could be used to restore oligodendrocyte function and improve outcomes.

[0062] Overexpression of tubulin beta-4a protein is another example of "toxic gain of function". In this case, the overexpressed protein leads to disruption of microtubule dynamics, resulting in cytotoxicity. One way to address overexpression of tubulin beta-4a protein is to reduce expression of the TUBB4A gene by using RNA interference. This can be achieved through microRNA-based gene therapy. A single administration of an AAV vector that delivers an expression cassette of a therapeutic miRNA precursor targeting TUBB4A mRNA can be used to activate endogenous mRNA silencing mechanisms to reduce TUBB4A translation in oligodendrocytes. In addition, since TUBB4A overexpression is primarily confined to oligodendrocytes, AAV vectors with a higher tropism for oligodendrocytes can be used to improve safety and therapeutic efficacy.

[0063] The most common mutation in H-ABC patients is TUBB4A D249N Like all proteins, the tubulin beta-4a protein is made up of amino acids that are linked together like a chain and then folded into a precise configuration. Mutations in the gene that encodes the protein can cause an incorrect amino acid to be incorporated into the chain. In this case, tubulin beta-4a normally has the amino acid aspartic acid at position 249 in the chain. The mutation caused a switch to the amino acid asparagine at this position. This small change can often have a huge effect on the way a protein functions.

[0064] Recently, Sass et al. developed a H-ABC mouse model in which mice have the same mutation in the mouse TUBB4A gene (Sass et al., 2020). These mice (Tubb4a D249N / D249N Mice) showed progressive motor dysfunction, gait abnormalities, poor coordination, and involuntary movements such as twitches and hyporeflexia, similar to those seen in H-ABC patients. D249N / D249N The mice also showed shortened survival compared to controls, loss of myelin staining compared to control mice, altered microtubule behavior and formation, and a significant loss of oligodendrocytes. These results suggest that Tubb4a D249N / D249N H-ABC disease in mice shares many similar symptoms and pathologies with those in humans. Combining AAV vectors that effectively target oligodendrocytes with miRNA and / or gene replacement strategies for normalizing TUBB4A gene expression may be advantageous for H-ABC treatment approaches.

[0065] The present disclosure uses viral vectors (e.g., AAV vectors) to efficiently deliver therapeutic nucleic acids (e.g., siRNA) targeting one or more genes or RNA encoding toxic function gain proteins into cells. In some embodiments, AAV vectors encoding RNAi molecules (e.g., siRNA molecules of the present disclosure) can increase the delivery of active agents to oligodendrocytes. Therapeutic nucleic acids or polynucleotides may be able to significantly inhibit gene expression (e.g., mRNA levels) of toxic function gain proteins in cells; thereby improving defects induced or caused by intracellular proteins, such as protein aggregation and inclusion formation.

[0066] Such inhibitory nucleic acids (e.g., siRNA) can be used to treat various inherited and / or acquired myelin disorders, such as demyelination. According to the present disclosure, methods for treating and / or improving the disorders of patients include administering an effective amount of at least one therapeutic nucleic acid (e.g., a polynucleotide encoding one or more siRNA duplexes) to the cells of the patient and allowing inhibition / silencing of gene expression.

[0067] 2. Nucleic Acids

[0068] Certain aspects of the present disclosure provide one or more inhibitory nucleic acids (e.g., inhibitory RNA molecules), polynucleotides encoding such inhibitory nucleic acids, and transgenes engineered to express such inhibitory nucleic acids. The one or more inhibitory nucleic acids can target the same gene (e.g., hybridize or specifically bind to the same mRNA sequence or different mRNA sequences of the same gene) or different genes (e.g., hybridize or specifically bind to mRNAs of different genes).

[0069] A. Inhibitory Nucleic Acids

[0070] Inhibitory nucleic acids refer to nucleic acids that can bind to a target nucleic acid (e.g., a target RNA) in a cell and reduce or inhibit the level or function of the target nucleic acid in the cell. Examples of inhibitory nucleic acids include antisense oligonucleotides, ribozymes, external guide sequences (EGS) oligonucleotides, small interfering (si) RNA compounds, single-stranded or double-stranded RNA interference (RNAi) compounds, modified bases / locked nucleic acids (LNA), peptide nucleic acids (PNA), and other oligomeric compounds or oligonucleotide mimetics that specifically hybridize with at least a portion of a target nucleic acid and regulate its level or function. In some embodiments, the inhibitory nucleic acid can be antisense RNA, antisense DNA, chimeric antisense oligonucleotides, antisense oligonucleotides comprising modified bonds, interfering RNA (iRNA), short or small interfering RNA (siRNA), microRNA or micro interfering RNA (miRNA), small sequence RNA (stRNA), short hairpin RNA (shRNA), small RNA-induced gene activators (RNAa), small activation RNA (saRNA), or a combination thereof. In some examples, the inhibitory nucleic acid is an inhibitory RNA molecule that mediates RNA interference.

[0071] RNA interference (RNAi) is a process discovered in 1998 (Fire et al., 1998), by which cells regulate gene expression. Double-stranded RNA (dsRNA) in the cytoplasm triggers the RNAi pathway, wherein the double-stranded RNA is processed by the RNAse III-like enzyme DICER into small double-stranded fragments of about 21 to 23 nucleotides in length. These double-stranded fragments are integrated into a multi-subunit protein called the RNA-induced silencing complex (RISC). RISC contains Argonaute proteins, which unravel the double-stranded fragments into passenger strands and guide strands, which are removed from the complex, and the guide strands are complementary to the target sequence in a specific mRNA and guide the RISC complex to cut the specific target mRNA molecule or inhibit its translation (Kotowska-Zimmer et al., 2021). In this way, the gene encoding the mRNA molecule is essentially inactivated or "silenced".

[0072] RNAi technology can use three tools: synthetic siRNA, vector-based shRNA, and artificial miRNA (amiRNA). Synthetic siRNA is an exogenous double-stranded RNA that must be delivered into cells and must overcome stability and pharmacokinetic challenges. shRNA is an artificial RNA molecule with a tight hairpin loop structure that is delivered into cells using a plasmid or viral expression vector. shRNA is usually transcribed by a strong pol III promoter (such as U6 or H1) and enters the RNAi pathway in the form of a hairpin. However, transcription driven by a strong pol III promoter can produce supraphysiological levels of shRNA, thereby saturating the endogenous miRNA biosynthesis machinery and causing toxicity. AmiRNA embeds the target-specific shRNA insert into a scaffold based on natural primary miRNA (pri-miRNA). This ensures correct processing and transport similar to endogenous miRNA, thereby reducing toxicity (Kotoska-Zimmer et al., 2021).

[0073] In some embodiments of the present disclosure, the inhibitory RNA molecule may be siRNA, miRNA (including amiRNA) or shRNA.

[0074] In the art, siRNA is a double-stranded RNA molecule of about 19 to 25 (eg, 19 to 23) base pairs in length that induces RNAi in cells. In some embodiments, siRNA sequences can also be inserted into artificial miRNA scaffolds ("shmiRNA").

[0075] In the art, shRNA is an RNA molecule comprising double-stranded RNA of about 19 to 25 (eg, 19 to 23) base pairs connected by a short loop (eg, about 4 to 11 nucleotides) that induces RNAi in cells.

[0076] In the art, miRNA is an RNA molecule that induces RNAi in a cell, comprising a short (e.g., 19 to 25 base pairs) double-stranded RNA sequence connected by a loop and containing one or more additional double-stranded RNA sequences comprising one or more protrusions (e.g., mismatched or unpaired base pairs). As used herein, the term "miRNA" encompasses endogenous miRNA and exogenous or heterologous miRNA. In certain embodiments, "miRNA" may refer to pri-miRNA or pre-miRNA. During miRNA processing, pri-miRNA transcripts are produced. Pri-miRNA is processed by Drosha-DGCR8 to produce pre-miRNA by excision of one or more sequences, leaving pre-miRNA with 5' flanking regions, guide strands, loop regions, non-guide strands and 3' flanking regions, or pre-miRNA with 5' flanking regions, non-guide strands, loop regions, guide strands and 3' flanking regions. Pre-miRNA is subsequently exported to the cytoplasm and processed by Dicer, producing siRNA with guide strands and non-guide (or passenger) strands. The RISC complex then uses the guide strand to catalyze gene silencing, for example, by identifying a target RNA sequence complementary to the guide strand. Additional descriptions of miRNA can be found in, for example, WO 2008 / 150897. The recognition of the target sequence by miRNA depends primarily on the pairing between the target sequence and the miRNA seed sequence (e.g., nucleotides 1-8 (5' to 3') of the guide strand) (see, for example, Boudreau, RL et al. (2013) Nucleic Acids Res. 41: e9).

[0077] In some embodiments of the present disclosure, the inhibitory RNA molecule forms a hairpin structure. Typically, the RNA forming a hairpin is arranged into a self-complementary "stem-loop" structure, which includes a single nucleic acid encoding a stem portion with a duplex, wherein the duplex includes a sense strand (e.g., a passenger strand) connected to an antisense strand (e.g., a guide strand) by a loop sequence. The passenger strand has complementarity with the guide strand. In some embodiments, the passenger strand has 100% complementarity with the guide strand. In some embodiments, the passenger strand has at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or at least 99% complementarity with the guide strand. The passenger strand and the guide strand may lack complementarity due to base pair mismatches. In some embodiments, the passenger strand and the guide strand of the RNA forming a hairpin may have at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9 or at least 10 mismatches. Typically, the first 2 to 8 nucleotides of the stem (relative to the loop) are referred to as "seed" residues and play an important role in target recognition and binding. The first residue of the stem (relative to the loop) is referred to as the "anchor" residue. In some embodiments, the RNA forming the hairpin has a mismatch at the anchor residue.

[0078] In some embodiments, inhibitory RNA molecules are processed in cells (or subjects) to form "mature miRNAs". Mature miRNAs are the result of a multi-step pathway that is initiated by transcription of primary miRNAs from their miRNA genes or introns by RNA polymerase II or III, which produces initial precursor molecules in biological pathways to produce miRNAs. After transcription, pri-miRNAs (usually more than one thousand nucleotides in length and with a hairpin structure) are processed by the Drosha enzyme, which cuts the pri-miRNA near the junction between the hairpin structure and the ssRNA to produce precursor miRNAs (pre-miRNAs). Pre-miRNAs are exported to the cytoplasm, where they are further reduced by the Dicer enzyme at the pre-miRNA loop in the cytoplasm to produce double-stranded miRNA chains.

[0079] Of the two strands of the miRNA duplex, one arm (the guide strand (miR)) is usually present in higher concentrations and is bound and associated with Argonaute proteins, which are ultimately loaded into the RNA-induced silencing complex. The guide strand miRNA-RISC complex helps regulate gene expression by binding to the complementary sequence of its mRNA (usually located in the 3'UTR of the mRNA). The non-guide strand of the miRNA duplex is called the passenger strand and is usually degraded, but can also persist and function intact or after partial degradation to play a functional role in gene expression.

[0080] In some embodiments, the transgene is engineered to express an inhibitory nucleic acid (e.g., miRNA) with a guide strand targeting a human gene. "Targeting" refers to hybridization or specific binding of an inhibitory nucleic acid to a homologous (e.g., complementary) sequence on a target gene (e.g., an mRNA transcript of a target gene). In some embodiments, the inhibitory nucleic acid of the targeted gene transcript has a complementary region with the target gene of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length. In some embodiments, the length of the complementary region is greater than 30 nucleotides.

[0081] Typically, the guide strand can target a human gene transcript associated with a myelin disease or disorder. Examples include gene transcripts for PLP1 (associated with PMD), SNCA (associated with MSA), or TUBB4A (associated with H-ABC). In some embodiments, the guide strand targeting any of these gene transcripts is encoded by an isolated nucleic acid comprising the following sequence.

[0082] The sequence encoding Homo sapiens proteolipid protein 1 (PLP1) transcript variant 1 mRNA is shown below.

[0083] NCBI reference sequence: PLP1 (NM_000533.5) coding sequence (SEQ ID NO: 1)

[0084] ATGGGCTTGTTAGAGTGCTGTGCATGGCACTGTTCTGTGGCTGTGGACATGAAG

[0085] CCCTCACTGGCACAGAAAAGCTAATTGAGACCTATTTCTCCAAAAACTACCAAGA

[0086] CTATGAGTATCTCATCAATGTGATCCATGCCTTCCAGTATGTCATCTATGGAACT

[0087] GCCTCTTTCTTCTTCCTTTATGGGGCCCTCCTGCTGGCTGAGGGCTTCTACACCA

[0088] CCGGCGCAGTCAGGCAGATCTTTGGCGACTACAAGACCACCATCTGCGGCAAGGG

[0089] CCTGAGCGCAACGGTAACAGGGGGCCAGAAGGGGAGGGGTTCCAGAGGCCAACAT

[0090] CAAGCTCATTCTTTGGAGCGGGTGTGTCATTGTTTGGGAAAATGGCTAGGACATC

[0091] CCGACAAGTTTGTGGGCATCACCTATGCCCTGACCGTTGTGTGGCTCCTGGTGTT

[0092] TGCCTGCTCTGCTGTGCCTGTGTACATTTACTTCAACACCTGGACCACCTGCCAG

[0093] TCTATTGCCTTCCCCAGCAAGACCTCTGCCAGTATAGGCAGTCTCTGTGCTGATG

[0094] CCAGAATGTATGGTGTTCTCCCATGGAATGCTTTCCCTGGCAAGGTTTGTGGCTC

[0095] CAACCTTCTGTCCATCTGCAAAACAGCTGAGTTCCAAATGACCTTCCACCTGTTT

[0096] ATTGCTGCATTGTGGGGGCTGCAGCTACACTGGTTTCCCTGCTCACCTTCATGA

[0097] TTGCTGCCACTTACAACTTTGCCGTCCTTAAACTCATGGGCCGAGGCACCAAGTT

[0098] CTGA

[0099] Exemplary target sequences and guide strands for targeting PLP1 are shown in Table 1 below.

[0100] Table 1. PLP1 target sequences

[0101]

[0102]

[0103] The sequence encoding the Homo sapiens synuclein alpha (SNCA) transcript variant 1 mRNA is shown below.

[0104] NCBI reference sequence: NM_000345.4 coding sequence (SEQ ID NO: 81)

[0105] ATGGATGTATTCATGAAAGGACTTTCAAAGGCCAAGGAGGGAGTTGTGGCTGCTG

[0106] CTGAGAAAACCAAACAGGGTGTGGCAGAAGCAGCAGGAAAGACAAAAGAGGGTGT

[0107] TCTCTATGTAGGCTCCAAAACCAAGGAGGGAGTGGTGCATGGTGTGGCAACAGTG

[0108] GCTGAGAAGACCAAAGAGCAAGTGACAAATGTTGGAGGAGCAGTGGTGACGGGTG

[0109] TGACAGCAGTAGCCCAGAAGACAGTGGAGGGAGCAGGGAGCATTGCAGCAGCCAC

[0110] TGGCTTTGTCAAAAAGGACCAGTTGGGCAAGAATGAAGAAGGAGCCCCACAGGAA

[0111] GGAATTCTGGAAGATATGCCTGTGGATCCTGACAATGAGGCTTATGAAATGCCTT

[0112] CTGAGGAAGGGTATCAAGACTACGAACCTGAAGCCTAA

[0113] Exemplary target sequences and guide strands for targeting human α-synuclein are shown in Table 2 below.

[0114] Table 2 Human α-synuclein target sequences

[0115]

[0116] Shown below is the sequence encoding Homo sapiens tubulin beta 4A class IVa (TUBB4A) transcript variant 1; NCBI Reference Sequence: NM_001289123.2 (SEQ ID NO: 162).

[0117] ATGAGAAGGGGGCTGCGGACCGAGAAACTGAGCGGCTCCCGGGGGCGCAGGGAC

[0118] CGTGCTCCGCCGTCTCCGCCGCATCTTCCACCCTCGCCGCCGCCGCAGCTCCCCG

[0119] CGCTCGTGCCACCGCCGCCGCGTCCACCCTCAGCGCCACCGCCATGCGGGAGATC

[0120] GTGCACCTGCAGGCCGGCCAGTGCGGCAACCAGATCGGGGCCAAGTTTTGGGAGG

[0121] TTATCAGTGACGAACATGGCATCGACCCCACAGGCACATACCATGGGGACAGTGA

[0122] CCTGCAACTGGAGAGGATCAACGTGTACTACAACGAGGCCACAGGAGGAAATTAT

[0123] GTCCCCAGAGCGGTGCTGGTGGACCTGGAACCCGGCACCATGGACTCTGTCCGTT

[0124] CTGGCCCCTTCGGTCAGATCTTTCGGCCGGACAACTTCGTGTTTGGCCAATCCGG

[0125] AGCCGGCAACAACTGGGCAAAGGGGCACTACACGGAGGGCGCAGAGCTGGTGGAC

[0126] GCTGTCCTGGACGTAGTCCGGAAGGAGGCCGAGAGCTGCGACTGCCTTCAGGGCT

[0127] TCCAGCTGACCCACTCGCTGGGGGGTGGCACGGGGTCCGGAATGGGCACGCTGCT

[0128] CATCAGTAAGATCCGCGAGGAGTTCCCAGACCGCATCATGAACACCTTCAGCGTG

[0129] GTGCCCTCGCCCAAAGTGTCAGACACGGTGGTGGAGCCCTACAACGCCACGCTGT

[0130] CTGTGCACCAGCTGGTGGAGAATACGGATGAGACCTACTGCATCGACAACGAGGC

[0131] ACTCTACGACATCTGTTTCCGCACCCTCAAGCTGACCACCCCCACCTACGGGGAC

[0132] CTCAACCACCTGGTGTCGGCCACCATGAGCGGGGTCACCACCTGCCTGCGCTTCC

[0133] CGGGCCAGCTGAACGCCGACCTGCGCAAGCTGGCCGTCAACATGGTTCCCTTTCC

[0134] TCGCCTGCACTTCTTCATGCCCGGCTTCGCACCCCTGACCAGCCGGGGCAGCCAG

[0135] CAGTACCGGGCCCTGACGGTGCCCGAGCTCACCCAGCAGATGTTCGATGCCAAGA

[0136] ACATGATGGCGGCGTGCGACCCGCGCCACGGCCGCTACCTGACCGTGGCCGCCGT

[0137] GTTCCGGGGCCGCATGTCCATGAAGGAGGTGGACGAGCAGATGCTGAGCGTGCAG

[0138] AGCAAGAACAGCAGCTACTTCGTGGAGTGGATCCCCAACAACGTGAAGACGGCCG

[0139] TGTGCGACATCCCGCCCCGCGGCCTGAAGATGGCCGCGACCTTCATCGGCAACAG

[0140] CACGGCCATCCAGGAGCTGTTCAAGCGCATCTCCGAGCAGTTCACGGCCATGTTC

[0141] CGGCGCAAGGCCTTCTTGCACTGGTACACGGGCGAGGGCATGGACGAGATGGAGT

[0142] TCACCGAGGCCGAGAGCAACATGAATGACCTGGTATCTGAGTACCAGCAGTACCA

[0143] GGACGCCACGGCCGAGGAGGGCGAGTTCGAGGAGGAGGCGGAGGAGGAGGTGGCC

[0144] TAG

[0145] Exemplary target sequences and guide strands for targeting human TUBB4A are shown in Table 3 below.

[0146] Table 3. Human TUBB4A target sequences

[0147]

[0148]

[0149] In some embodiments, the inhibitory nucleic acid is 5 to 300 bases in length (e.g., 10 to 30, 15 to 25, 19 to 22, 25 to 50, 40 to 90, 60 to 90, 75 to 100, 90 to 150, 110 to 200, 150 to 250, 200 to 300, etc. nucleotides in length). The inhibitory nucleic acid sequence encoding the pre-miRNA or mature miRNA may be 10 to 50 or 5 to 50 bases in length. In some embodiments, the inhibitory nucleic acid encodes, comprises, consists essentially of, or consists of: Fig.11 Or the sequence of SEQ ID NO:284 or 285 shown in 12.

[0150] B. Bracket

[0151] In certain embodiments, the inhibitory RNA molecule may be encoded in an inhibitory nucleic acid comprising a molecular scaffold. As used herein, a "molecular scaffold" is a framework or starting molecule that forms the sequence or structural basis for designing or making subsequent molecules.

[0152] In some embodiments, the molecular scaffold comprises at least one 5' flanking region or one 3' flanking region or both. As a non-limiting example, the 5' or 3' flanking region may comprise a 5' or 3' flanking sequence, which may have any length and may be derived in whole or in part from a wild-type microRNA sequence, or may be a completely artificial sequence. In some embodiments, one or both of the 5' and 3' flanking sequences may not exist. In some embodiments, the 5' and 3' flanking sequences may have the same or different lengths. In some embodiments, the length of the 5' or 3' flanking sequence may be 1 to 10 nucleotides, 5 to 15 nucleotides, 10 to 30 nucleotides, 20 to 50 nucleotides, greater than 40 nucleotides, greater than 50 nucleotides, greater than 100 nucleotides, or greater than 200 nucleotides.

[0153] In some embodiments, the inhibitory nucleic acid sequence comprising or encoding a pri-miRNA scaffold is at least 200, 250, 260, 270, 280, 290 or 300 bases in length. In some embodiments, the inhibitory nucleic acid comprises or consists of a base sequence that is at least 80% or 90% complementary to a target nucleic acid (e.g., a human mRNA, such as an mRNA of PLP1, SNCA or TUBB4A) (e.g., at least 5, 10, 15, 20, 25 or 30 bases of the target nucleic acid, or at most 30 or 40 bases), or comprises a base sequence having at most 3 mismatches (e.g., at most 1 or at most 2 mismatches) at 10, 15, 20, 25 or 30 bases of the target nucleic acid.

[0154] In some embodiments, the inhibitory nucleic acid is an artificial miRNA (amiRNA). AmiRNA is derived by modifying a natural miRNA to replace the natural targeting region of a pre-mRNA with a targeting region of interest. For example, a naturally occurring, expressed miRNA can be used as a scaffold or framework (e.g., a pri-miRNA scaffold) in which the stem sequence is replaced by the stem sequence of a miRNA targeting a gene of interest. Artificial precursor microRNAs (pre-amiRNAs) are typically processed in a manner that preferentially produces a single stable small RNA.

[0155] The stem forming the stem-loop structure is a minimal inhibitory nucleic acid encoding at least one siRNA, miRNA, shRNA or other RNAi agent described herein. In some embodiments, the siRNA, miRNA, shRNA or other RNAi agent described herein comprises at least one nucleic acid sequence that is partially complementary to or will hybridize with a target sequence. In some embodiments, the 5' arm of the stem-loop structure of the inhibitory nucleic acid comprises a nucleic acid sequence encoding an antisense sequence (i.e., a guide sequence / strand). In some other embodiments, the 3' arm of the stem-loop structure of the inhibitory nucleic acid comprises a nucleic acid sequence encoding the antisense / guide sequence.

[0156] In certain embodiments, the sense sequence and antisense sequence of the stem-loop structure of the inhibitory nucleic acid are separated by a loop sequence (also referred to as a loop motif, linker, or linker motif). The loop sequence may have any length, 4 to 30 nucleotides, 4 to 20 nucleotides, 4 to 15 nucleotides, 5 to 15 nucleotides, 6 to 12 nucleotides, 6 nucleotides, 7 nucleotides, 8 nucleotides, 9 nucleotides, 10 nucleotides, 11 nucleotides, 12 nucleotides, 13 nucleotides, 14 nucleotides, and / or 15 nucleotides.

[0157] Some aspects of the present disclosure relate to a nucleic acid sequence encoding a guide strand targeting a human gene, the guide strand being inserted into a human or non-human (e.g., mouse) pri-miRNA scaffold. In some embodiments, the pri-miRNA scaffold may be selected from mir-16-1, miR-21, miR-23a, miRNA-30a, miR-31, miR-122, miR-155, or miR-451. In some embodiments, the pri-miRNA scaffold is located on either side of an inhibitory nucleic acid targeting a human mRNA (e.g., an mRNA of PLP1, SNCA, or TUBB4A) or its target sequence (e.g., a target sequence encoded by a sequence shown in Tables 1 to 3 above, such as SEQ ID NO: 14 and SEQ ID NO: 35). In some embodiments, the pri-miRNA scaffold is located on either side of an inhibitory nucleic acid comprising or encoding a guide strand (e.g., a guide strand RNA sequence corresponding to a sequence shown in Tables 1 to 3 above or encoded by it, such as SEQ ID NO: 54 and SEQ ID NO: 75). In some embodiments, the pri-miRNA scaffold may be from human miRNA-30a ( Fig.11 , left side). The associated guide strand RNA sequence can be located on the 5' arm or 3' arm of the stem loop. In one example, the guide strand RNA sequence corresponding to SEQ ID NO: 75 or encoded thereby can be located on the 5' arm (see Fig.11, right side). In another example, the guide strand RNA sequence corresponding to SEQ ID NO: 54 or encoded thereby may be located on the 3' arm (see Fig.12 ).

[0158] C. Recombinant Nucleic Acid

[0159] Recombinant nucleic acids disclosed herein include inhibitory nucleic acids described above and plasmids and vector genomes comprising inhibitory nucleic acids. Recombinant nucleic acids, plasmids or vector genomes may include regulatory sequences to regulate proliferation (e.g., proliferation of plasmids) and / or control the expression of transgenic (e.g., inhibitory nucleic acids). Recombinant nucleic acids may also be provided as components of viral vectors (e.g., rAAV vectors). In general, viral vectors include vector genomes comprising recombinant nucleic acids packaged in capsids.

[0160] D. Regulatory elements

[0161] The present disclosure includes recombinant nucleic acids that include transgenes (e.g., transgenes encoding RNA) and various regulatory or control elements (e.g., woodchuck hepatitis post-transcriptional regulatory elements). Generally, regulatory elements are one or more nucleic acid sequences that affect the expression of operably linked polynucleotides. The precise nature of the regulatory elements used for gene expression will vary depending on the organism and cell type, including, for example, promoters, enhancers, introns, etc., with the purpose of promoting correct transcription and / or translation of heterologous polynucleotides. Regulatory control can be affected at the level of transcription, translation, splicing, information stability, etc. Generally, regulatory control elements that regulate transcription are juxtaposed near the 5' end of the transcribed polynucleotide (i.e., upstream). Regulatory control elements can also be located at the 3' end of the transcribed sequence (i.e., downstream) or within the transcript (e.g., in an intron). Regulatory control elements can be located at a certain distance from the transcribed sequence (e.g., 1 to 100, 100 to 500, 500 to 1000, 1000 to 5000, 5000 to 10,000 or more nucleotides). However, due to the length of the vector genome (eg, an AAV vector genome), regulatory control elements are typically within 1 to 1000 nucleotides of the polynucleotide.

[0162] a. Promoter

[0163] As used herein, the term "promoter", such as "eukaryotic promoter", refers to a nucleotide sequence that initiates transcription of a specific gene, or one or more coding sequences in a eukaryotic cell (e.g., oligodendrocyte). Promoters can work together with other regulatory elements or regulatory regions to guide the transcription level of the gene or the one or more coding sequences. These regulatory elements include, for example, transcription binding sites, repressor proteins, and activator protein binding sites, as well as other nucleotide sequences known to act directly or indirectly to regulate the transcription amount of the promoter, including, for example, attenuators, enhancers, and silencers. Promoters are most often located on the same chain and near the transcription start site, i.e., 5' of a gene or coding sequence operably connected thereto. The length of a promoter is generally 100 to 1000 nucleotides. Relative to the expression of the same gene in the absence of a promoter, a promoter typically increases gene expression.

[0164] As used herein, "core promoter" or "minimal promoter" refers to the minimum portion of a promoter sequence required to properly initiate transcription. It may include any of the following: a transcription start site, a binding site for RNA polymerase, and a universal transcription factor binding site. A promoter may also include a proximal promoter sequence (5' of the core promoter) and a distal promoter sequence (3' of the core promoter) containing other major regulatory elements (e.g., enhancers, silencers, boundary elements, insulators).

[0165] Examples of suitable promoters include adenovirus promoters, such as the adenovirus major late promoter; heterologous promoters, such as the cytomegalovirus (CMV) promoter; respiratory syncytial virus promoter; Rous Sarcoma Virus (RSV) promoter; albumin promoter; inducible promoters, such as the mouse mammary tumor virus (Mouse Mammary Tumor Virus) promoter; Virus, MMTV) promoter; metallothionein promoter; heat shock promoter; alpha-1-antitrypsin promoter; hepatitis B surface antigen promoter; transferrin promoter; apolipoprotein A-1 promoter; chicken β-actin (CBA) promoter, elongation factor 1a promoter (EF1a), a hybrid form of the CBA promoter (CBh promoter) and the CAG promoter (cytomegalovirus early enhancer element, and the promoter, first exon and first intron of the chicken β-actin gene, and the splice acceptor of the rabbit β-globin gene) (Alexopoulou et al. (2008) BioMed. Central Cell Biol. 9:2).

[0166] Promoters can be constitutive, tissue-specific or regulated. Constitutive promoters are those that always express an operably linked gene. In some embodiments, constitutive promoters are active in most eukaryotic tissues under most physiological and developmental conditions.

[0167] Regulated promoters are those that can be activated or inactivated. Regulated promoters include inducible promoters, which are normally in an "off" state but can be induced to be "on," and "repressible" promoters, which are normally in an "on" state but can be "off." Many different regulatory factors are known, including temperature, hormones, cytokines, heavy metals, and regulatory proteins. The distinction is not absolute; constitutive promoters can usually be regulated to some extent. In some cases, endogenous pathways can be used to regulate transgene expression, such as using promoters that are naturally downregulated when pathological conditions improve.

[0168] Tissue-specific promoters are promoters that are active only in tissues, cells or organs of a particular type. Typically, tissue-specific promoters are identified by transcriptional activator elements that are specific to a particular tissue, cell and / or organ. For example, a tissue-specific promoter may be more active in one or more (e.g., two, three or four) specific tissues than in other tissues. In certain embodiments, the expression of a gene regulated by a tissue-specific promoter is much higher in the tissue specifically targeted by the promoter than in other tissues. In certain embodiments, a promoter may be very little active or substantially inactive in any tissue except the tissue specifically targeted by the promoter.

[0169] b. Enhancer

[0170] In another aspect, the recombinant nucleic acids described herein may further comprise an enhancer to increase the expression of a transgene (e.g., an RNA molecule disclosed herein). Typically, the enhancer element is located upstream of the promoter element, but may also be located downstream or within another sequence (e.g., a transgene). The enhancer may be located 100 nucleotides, 200 nucleotides, 300 nucleotides, or more upstream or downstream of the modified nucleic acid. An enhancer will typically increase the expression of a transgene (e.g., encoding an inhibitory nucleic acid) beyond the increase in expression provided by the promoter element alone.

[0171] Many enhancers are known in the art, including but not limited to the cytomegalovirus major immediate early enhancer. More specifically, the CMV MIE promoter comprises three regions: a regulator, a unique region, and an enhancer (Isomura and Stinski (2003) Journal of Virology (J.Virol.) 77 (6): 3602-3614). The CMV enhancer region can be combined with another promoter or a portion thereof to form a hybrid promoter to further increase the expression of a nucleic acid operably connected thereto. For example, the CBA promoter or a portion thereof can be combined with the CMV promoter / enhancer or a portion thereof to form a CBA version called a "CBh" promoter, which represents a chicken β-actin hybrid promoter, as described in Gray et al. (2011, Human Gene Therapy (Human Gene Therapy) 22: 1143-1153). Like a promoter, an enhancer can be constitutive, tissue-specific, or regulated.

[0172] c. Filler, spacer and stuffer

[0173] As disclosed herein, recombinant nucleic acids can be used in rAAV vectors. In the case described, recombinant nucleic acids can include additional nucleic acid elements to adjust the length of nucleic acid to a normal size (e.g., about 4.7 to 4.9 kilobases) close to or reaching an acceptable viral genome sequence, so that AAV is packaged into rAAV vectors (Grieger and Samulski (2005) Journal of Virology 79 (15): 9933-9944). Such sequences can be interchangeably referred to as filler sequences, spacer sequences, or stuffing sequences. In certain embodiments, filler DNA is a non-translated (non-protein coding) nucleic acid fragment. In some embodiments, the filler or stuffer polynucleotide sequence is a sequence of about 1-10, 10-20, 20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90-90-100, 100-150, 150-200, 200-250, 250-300, 300-400, 400-500, 500-750, 750-1000, 1000-1500, 1500-2000, 2000-3000, or longer in length.

[0174] AAV vectors generally accept DNA inserts in the range of about 4kb to about 5.2kb or about 4.1 to 4.9kb in size, so that nucleic acid is optimally packaged into AAV capsids. In certain embodiments, rAAV vectors include a total length of about 3.0kb to about 3.5kb, about 3.5kb to about 4.0kb, about 4.0kb to about 4.5kb, about 4.5kb to about 5.0kb, or about 5.0kb to about 5.2kb of vector genome. In certain embodiments, rAAV vectors include a total length of about 4.7kb of vector genome. In certain embodiments, rAAV vectors include self-complementary vector genomes. Although the total length of the self-complementary (sc) vector genome in the rAAV vector is equal to the single-stranded (ss) vector genome (i.e., about 4kb to about 5.2kb), the nucleic acid sequence encoding the sc vector genome (i.e., including transgene, regulatory elements, and ITR) must be only half the length of the nucleic acid sequence encoding the ss vector genome, so that the sc vector genome is packaged in the capsid.

[0175] d. Introns and exons

[0176] In certain embodiments, the recombinant nucleic acid disclosed herein includes, for example, introns, exons and / or a portion thereof. Introns can serve as filling or filling polynucleotide sequences to achieve the appropriate length of the vector genome packaged into the rAAV vector. Compared with the expression without introns and / or exon elements, introns and / or exon sequences can also enhance the expression of transgenic (e.g., RNA disclosed herein) (Kurachi et al. (1995) Journal of Biological Chemistry (J.Biol.Chem.) 270 (10): 576-5281; WO 2017 / 074526). In addition, filling / filling polynucleotide sequences (also referred to as "insulators") are well known in the art, and include but are not limited to those described in WO 2014 / 144486 and WO 2017 / 074526.

[0177] e. Polyadenylation signal sequence (polyA)

[0178] Additional regulatory elements may include stop codons, termination sequences, and polyadenylation (polyA) signal sequences, such as, but not limited to, the bovine growth hormone poly A signal sequence (BHG polyA). The polyA signal sequence drives the efficient addition of a polyadenylic acid "tail" at the 3' end of eukaryotic mRNAs, which directs the termination of gene transcription (see, e.g., Goodwin and Rottman, Journal of Biochemistry (1992) 267(23): 16330-16334). The polyA signal acts as a signal for endonucleolytic cleavage at the 3' end of the newly formed pre-mRNA and for the addition of an RNA segment consisting only of adenine bases to this 3' end. The polyA tail is important for nuclear export, translation, and stability of mRNA. In some embodiments, the poly A can be an SV40 early polyadenylation signal, an SV40 late polyadenylation signal, an HSV thymidine kinase polyadenylation signal, a protamine gene polyadenylation signal, an adenovirus 5E1b polyadenylation signal, a growth hormone polyadenylation signal, a PBGD polyadenylation signal, or a computer-designed polyadenylation signal.

[0179] 3. Expression cassette and expression vector

[0180] The disclosure also provides an expression cassette, which comprises or consists of a recombinant nucleic acid encoding an inhibitory nucleic acid as described above. In the case where this recombinant nucleic acid may not yet comprise a promoter, the expression cassette may additionally comprise a promoter. Therefore, the expression cassette according to the present invention comprises a promoter, a coding sequence and an optional terminator or other elements in a 5' to 3' direction. The expression cassette allows the nucleic acid sequence of interest to be easily transferred to an organism, preferably a cell, and preferably a disease cell.

[0181] The expression cassette of the present disclosure is preferably contained in a vector. Therefore, the vector of the present disclosure allows cells to be transformed with the nucleic acid sequence of interest. Correspondingly, the present disclosure provides a host cell comprising an expression cassette according to the present disclosure or a recombinant nucleic acid according to the present disclosure. The recombinant nucleic acid may also comprise a promoter or an enhancer to allow expression of the nucleic acid sequence of interest.

[0182] Exogenous genetic material (e.g., nucleic acids, expression cassettes, or expression vectors encoding one or more therapeutic or inhibitory RNAs) can be introduced into target cells of interest in vivo by gene transfer methods (e.g., transfection or transduction) to provide genetically modified cells. Various expression vectors (i.e., vehicles for facilitating delivery of exogenous genetic material into target cells) are known to those of ordinary skill in the art. As used herein, "exogenous genetic material" refers to natural or synthetic nucleic acids or oligonucleotides that do not naturally occur in cells; or, even if they naturally occur in cells, they are not transcribed or expressed by cells at biologically significant levels. Therefore, "exogenous genetic material" includes, for example, non-naturally occurring nucleic acids that can be transcribed into RNA.

[0183] As used herein, "cell transfection" refers to the acquisition of new genetic material by cells by incorporating added nucleic acids (DNA, RNA or their hybrids) without the use of a viral delivery vehicle. Therefore, transfection refers to the introduction of nucleic acids into cells using physical or chemical methods. Several transfection techniques are known to those of ordinary skill in the art, including calcium phosphate nucleic acid coprecipitation, strontium phosphate nucleic acid coprecipitation, DEAE-dextran, electroporation, cationic liposome-mediated transfection, and microparticle bombardment promoted by tungsten particles. In contrast, "cell transduction" refers to the process of transferring nucleic acids into cells using DNA or RNA viruses. RNA viruses (e.g., retroviruses) used to transfer nucleic acids into cells are referred to herein as transducing chimeric viruses. The exogenous genetic material contained in the virus can be incorporated into the genome of the transduced cell. Cells transduced with chimeric DNA viruses (e.g., adenoviruses carrying DNA encoding therapeutic agents) may not incorporate exogenous genetic material into their genomes, but may be able to express exogenous genetic material retained outside the chromosomes of the cells.

[0184] Typically, exogenous genetic material can include a heterologous gene (encoding therapeutic RNA or protein) and a promoter that controls the transcription of the new gene. The promoter is characterized by having a specific nucleotide sequence necessary for initiating transcription. Optionally, the exogenous genetic material further includes additional sequences (i.e., enhancers) required for obtaining the desired gene transcription activity. The exogenous genetic material can be introduced into the cell genome immediately downstream of the promoter so that the promoter and the coding sequence are operably connected to allow transcription of the coding sequence. Retroviral expression vectors can include exogenous promoter elements to control the transcription of the inserted exogenous gene. Such exogenous promoters include both constitutive promoters and inducible promoters.

[0185] Naturally occurring constitutive promoters control the expression of basic cellular functions. Therefore, genes under the control of constitutive promoters are expressed under all cell growth conditions. Exemplary constitutive promoters include promoters of the following genes encoding certain constitutive or "housekeeping" functions: hypoxanthine phosphoribosyltransferase, dihydrofolate reductase, adenosine deaminase, phosphoglycerol kinase, pyruvate kinase, phosphoglycerol mutase, actin promoter, ubiquitin, elongation factor-1 and other constitutive promoters known to those of ordinary skill in the art. In addition, many viral promoters function constitutively in eukaryotic cells. These include the early and late promoters of SV40; the long terminal repeats (LTRs) of Moloney Leukemia Virus and other retroviruses; and the thymidine kinase promoter of herpes simplex virus, etc. Therefore, any of the above-mentioned constitutive promoters can be used to control the transcription of heterologous gene inserts.

[0186] Genes controlled by inducible promoters are expressed only in the presence of an inducing agent or are mainly controlled by an inducing agent (e.g., transcription controlled by metallothionein promoters is greatly increased in the presence of certain metal ions). Inducible promoters include response elements (REs) that stimulate transcription when their inducing factors are combined. For example, there are REs for serum factors, steroid hormones, retinoic acid, and cyclic AMP. Promoters containing specific REs can be selected to obtain induction responses, and in some cases, REs themselves can be connected to different promoters, thereby conferring inducibility to recombinant genes. Therefore, by selecting appropriate promoters (constitutive and inducible; strong and weak), both the presence and expression level of therapeutic agents in genetically modified cells can be controlled. If the gene encoding the therapeutic agent is under the control of an inducible promoter, the in situ delivery of the therapeutic agent is triggered by exposing the genetically modified cells in situ to conditions that allow the transcription of the therapeutic agent, such as by injecting a specific inducer of the inducible promoter that controls the transcription of the therapeutic agent. For example, in situ expression of a therapeutic agent encoded by a gene controlled by a metallothionein promoter by a genetically modified cell can be enhanced by contacting the genetically modified cell with a solution containing appropriate (ie, inducing) metal ions in situ.

[0187] Thus, the amount of therapeutic agent delivered in situ is regulated by controlling factors such as: (1) the nature of the promoter used to direct transcription of the inserted gene (i.e., whether the promoter is constitutive or inducible, strong or weak); (2) the number of copies of the exogenous gene inserted into the cell; (3) the number of transduced / transfected cells administered (e.g., implanted) to the patient; (4) the size of the implant (e.g., a graft or encapsulated expression system); (5) the number of implants; (6) the length of time the transduced / transfected cells or implants remain in situ; and (7) the rate at which the genetically modified cells produce the therapeutic agent. Considering the factors disclosed above and the clinical profile of the patient, it is believed that the selection and optimization of these factors to deliver a therapeutically effective dose of a particular therapeutic agent is within the capabilities of one of ordinary skill in the art without undue experimentation.

[0188] In addition to at least one promoter and at least one heterologous nucleic acid encoding a therapeutic agent, the expression vector may also include a selection gene, such as a neomycin resistance gene or a fluorescent protein gene, to facilitate selection of cells that have been transfected or transduced by the expression vector. Alternatively, the cell is transfected with two or more expression vectors, at least one vector containing one or more genes encoding one or more therapeutic agents and another vector containing a selection gene. The selection of appropriate promoters, enhancers, selection genes and / or signal sequences is considered to be within the capabilities of those of ordinary skill in the art without the need for excessive experimentation.

[0189] The coding sequences disclosed herein can be inserted into any type of target or host cell. In the case of an expression vector, the vector can be easily introduced into a host cell, such as a mammal, a bacterium, a yeast, or an insect cell, by any method in the art. For example, the expression vector can be transferred to a host cell by physical, chemical, or biological means.

[0190] Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, liposome transfection, particle bombardment, microinjection, electroporation, etc. Methods for generating cells containing vectors and / or exogenous nucleic acids are well known in the art. See, for example, Sambrook et al. (2012, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York).

[0191] Biological methods for introducing polynucleotides of interest into host cells include the use of DNA and RNA vectors. Viral vectors, and especially retroviral vectors, have become the most widely used methods for inserting genes into mammalian (e.g., human) cells. Other viral vectors can be derived from lentiviruses, poxviruses, herpes simplex virus I, adenoviruses, and adeno-associated viruses, etc. See, e.g., U.S. Patents Nos. 5,350,674 and 5,585,362.

[0192] Chemical means for introducing polynucleotides into host cells include colloidal dispersion systems, such as macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system used as a delivery vehicle in vitro and in vivo is a liposome (e.g., an artificial membrane vesicle).

[0193] As disclosed herein, the RNA molecules described above can be used to treat the disease of a subject. In some embodiments, the polynucleotide encoding the RNA molecule can be inserted into a vector (e.g., a plasmid or a viral vector) or encoded by it. Preferably, the polynucleotide is inserted into a viral vector or encoded by it. The viral vector can be a herpes virus (HSV) vector, a retroviral vector, an adenoviral vector, an AAV vector, a lentiviral vector, etc. In some specific embodiments, the viral vector is an AAV vector. In some embodiments, RNA can be encoded by a retroviral vector (see, e.g., U.S. Patent Nos. 5,399,346, 5,124,263, 4,650,764, and 4,980,289; the contents of each of which are incorporated herein by reference in their entirety).

[0194] Adenoviruses are eukaryotic DNA viruses that can be modified to effectively deliver nucleic acids to various cell types in vivo and have been widely used in gene therapy protocols, including for targeting genes to neural and glial cells. Various replication-defective adenoviruses and minimal adenovirus vectors for nucleic acid therapeutics have been described (see, e.g., PCT Patent Publication Nos. WO199426914, WO 199502697, WO199428152, WO199412649, WO199502697, and WO199622378; the contents of each of which are incorporated by reference in their entirety). Such adenovirus vectors can also be used to deliver the RNA molecules of the present disclosure to cells.

[0195] 4. AAV

[0196] Adeno-associated virus is a widely used gene therapy vector because of its clinical safety record, lack of pathogenicity, ability to infect non-dividing cells (such as neurons), and ability to provide long-term gene expression after a single administration (Hocquemiller et al., 2016). Currently, multiple human and non-human primate AAV serotypes have been identified (Gao et al., 2004). AAV vectors have demonstrated their safety in hundreds of clinical trials worldwide and have demonstrated their clinical efficacy in trials for hemophilia B, spinal muscular atrophy, α1 antitrypsin, and Leber congenital amaurosis (Keeler et al., 2017). Three AAV-based gene therapies have been approved. The first drug, Glybera, was approved by the European Medicines Agency (EMA) in 2012 (but was withdrawn in 2017, primarily due to commercial failure). Luxturna was approved by the FDA in 2017 for the treatment of rare inherited retinal dystrophy, and Zolgensma was approved by the FDA in 2019 for the treatment of spinal muscular atrophy.

[0197] AAVs such as AAV1, AAV2, AAV4, AAV5, AAV6, AAV8, and AAV9 are commonly used gene therapy vectors for CNS applications because they are safe, non-pathogenic, and can infect neurons. However, after direct CNS infusion, these serotypes show a clear neuronal tropism and low expression in oligodendrocytes, especially when gene expression is driven by a constitutive promoter, which limits their potential for the treatment of white matter diseases. AAV1 / 2, AAV2, and AAV8 have been shown to transduce oligodendrocytes, but only when oligodendrocyte-specific promoters are used (Chen et al., 1998; Lawlor et al., 2009; Li et al., 2019). Relying on cell-specific promoters to achieve expression specificity makes it possible to achieve non-selective cellular uptake and leaky transgene expression in non-oligodendrocyte lineage cells through cryptic promoter activity.

[0198] The method described herein to alleviate these problems includes the use of an AAV serotype with high tropism for oligodendrocytes. Recently, using DNA reorganization and directed evolution, a chimeric AAV capsid with strong selectivity for oligodendrocytes, i.e., AAV / Olig001 (Powell et al., 2016), has been described. Subsequently, AAV / Olig001 was confirmed to transduce newborn oligodendrocytes in a Canavan disease mouse model (Francis et al., 2021). Other methods such as random mutagenesis and peptide library insertion can be used to generate a capsid library that can screen the tropism and selectivity of oligodendrocytes.

[0199] As discussed above, the term "adeno-associated virus" and / or "AAV" refers to a parvovirus and its variants having a linear single-stranded DNA genome. Unless otherwise required, the term encompasses all subtypes and both naturally occurring and recombinant forms. Parvoviruses (including AAV) can be used as gene therapy vectors because they can penetrate cells and introduce nucleic acids (e.g., transgenes) into the nucleus. In some embodiments, the introduced nucleic acids (e.g., rAAV vector genomes) form circular concatemers that are retained as episomes in the nucleus of transduced cells. In some embodiments, the transgene is inserted into a specific site in the host cell genome, such as a site on human chromosome 19. In contrast to random integration, site-specific integration is thought to produce predictable long-term expression profiles. The site where AAV is inserted into the human genome is referred to as AAVS1. Once introduced into a cell, the RNA or polypeptide encoded by the nucleic acid can be expressed by the cell. Because AAV is not related to any pathogenic disease in humans, the nucleic acid delivered by AAV can be used to express therapeutic RNA or polypeptides to treat diseases, disorders and / or conditions of human subjects.

[0200] There are multiple AAV serotypes in nature, and at least fifteen wild-type serotypes have been identified from humans to date (i.e., AAV1 to AAV15). Naturally occurring serotypes and variant serotypes are distinguished by having a protein capsid that is serologically distinct from other AAV serotypes. Examples include AAV1, AAV2, AAV, AAV3 (including AAV3A and AAV3B), AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV12, AAVrh10, AAVrh74 (see WO 2016 / 210170), avian AAV, bovine AAV, canine AAV, equine AAV, primate AAV, non-primate AAV, and ovine AAV, as well as recombinantly produced variants (e.g., capsid variants with insertions, deletions, substitutions, etc.), such as variants referred to as AAV2i8, NP4, NP22, NP66, DJ, DJ / 8, DJ / 9, LK3, RHM4-1, and the like. "Primate AAV" refers to AAV that infects primates, "non-primate AAV" refers to AAV that infects non-primate mammals, "bovine AAV" refers to AAV that infects bovine mammals, and so on.

[0201] Serotype uniqueness is determined based on the lack of cross-reactivity between antibodies to one AAV and antibodies to another AAV. Such cross-reactivity differences are due to differences in capsid protein sequences and antigenic determinants (e.g., due to differences in VP1, VP2 and / or VP3 sequences of AAV serotypes). However, some naturally occurring AAVs or artificial AAV mutants (e.g., recombinant AAVs) may not exhibit serological differences from any currently known serotype. These viruses can then be considered as subgroups of the corresponding type, or more simply as variant AAVs. Therefore, as used herein, the term "serotype" refers to both serologically different viruses and viruses that are serologically indistinguishable but may belong to a subgroup or variant of a given serotype.

[0202] Marsic et al. (2014) Molecular Therapy 22(11):1900-1909 provides a comprehensive list and alignment of amino acid sequences of capsids of known AAV serotypes. The genomic sequences of various AAV serotypes as well as the sequences of native ITRs, rep proteins, and capsid subunits are known in the art. Such sequences can be found in the literature or in public databases such as GenBank. See, e.g., GenBank Accession Nos. NC_002077 (AAV1), AF063497 (AAV1), NC_001401 (AAV2), AF043303 (AAV2), NC_001729 (AAV3), NC_001863 (AAV3B), NC_001829 (AAV4), U89790 (AAV4), NC_006152 (AAV5), NC_001862 (AAV6), AF513851 (AAV7), AF513852 (AAV8), and NC_006261 (AAV8); the disclosures of which are incorporated herein by reference. See also, e.g., Srivistava et al. (1983) J. Virology 45:555; Chiorini et al. (1998) J. Virology 71:6823; Chiorini et al. (1999) J. Virology 73:1309; Bantel-Schaal et al. (1999) J. Virology 73:939; Xiao et al. (1999) J. Virology 73:943. 99) Journal of Virology 73:3994; Muramatsu et al. (1996) Virology 221:208; Shade et al. (1986) Journal of Virology 58:921; Gao et al. (2002) Proc. Nat. Acad. Sci. USA 99:11854; Moris et al. (2004) Virology 33:375-383; International Patent Publications WO 00 / 28061, WO 99 / 61601, WO 98 / 11244, WO 2013 / 063379, WO 2014 / 194132, WO 2015 / 121501; and U.S. Pat. No. 6,156,303 and U.S. Pat. No. 7,906,111.

[0203] As discussed herein, "recombinant adeno-associated virus" or "rAAV" is distinguished from wild-type AAV by replacing all or part of the endogenous viral genome with non-native sequences. The incorporation of non-native sequences into the virus defines the viral vector as a "recombinant" vector, and therefore as a "rAAV vector". The rAAV vector may include a heterologous polynucleotide encoding a desired RNA or protein or polypeptide (e.g., an RNA molecule disclosed herein). The recombinant vector sequence may be encapsidated or packaged into an AAV capsid and is referred to as a "rAAV vector", "rAAV vector particle", "rAAV virion", or simply "rAAV".

[0204] For the production of rAAV vectors, the desired VP1:VP2:VP3 ratio may be in the range of about 1:1:1 to about 1:1:100, preferably in the range of about 1:1:2 to about 1:1:50, and more preferably in the range of about 1:1:5 to about 1:1:20. Although the desired ratio of VP1:VP2 may be 1:1, the ratio range of VP1:VP2 may vary from 1:50 to 50:1.

[0205] The present disclosure provides a rAAV vector comprising a polynucleotide sequence from a non-AAV source (e.g., a polynucleotide heterologous to AAV). The flank of the heterologous polynucleotide may be at least one, sometimes two, AAV terminal repeats (e.g., inverted terminal repeats). The heterologous polynucleotide flanked by ITRs (also referred to herein as "vector genomes") typically encodes an RNA or polypeptide of interest or a gene of interest, such as a target for therapeutic treatment. Delivery or administration of the rAAV vector to a subject (e.g., a patient) provides the subject with the encoded RNA / protein / peptide. Therefore, the rAAV vector can be used to transfer / deliver heterologous polynucleotides for expression, such as for the treatment of various diseases, disorders, and conditions.

[0206] The rAAV vector genome usually retains 145 bases of ITR, which is cis-linked to the heterologous nucleic acid sequence that replaces the viral rep and cap genes. Such ITRs can be used to produce recombinant AAV vectors; however, modified AAV ITRs and non-AAV terminal repeats (including partially or completely synthesized sequences) can also be used for this purpose. ITR forms a hairpin structure and acts as a primer for complementary DNA chain synthesis mediated by host cells, for example, after infection. ITR also plays a role in virus packaging, integration, etc. ITR is the only AAV viral element required for cis-carrying AAV genome replication and packaging into rAAV vectors. The rAAV vector genome optionally includes two ITRs, which are generally located at the 5' end and 3' end of the vector genome containing a heterologous sequence (e.g., a transgene encoding a gene of interest, or a nucleic acid sequence of interest, including but not limited to antisense sequences, and siRNA, CRISPR molecules, etc.). 5' and 3' ITRs can both contain the same sequence, or each can contain a different sequence. The AAV ITRs can be from any AAV, including but not limited to serotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11, or any other AAV.

[0207] The rAAV vectors of the present disclosure may comprise ITRs from an AAV serotype (e.g., wild-type AAV2, fragments or variants thereof) that is different from the capsid serotype (e.g., AAV8, Olig001). Such rAAV vectors comprising at least one ITR from one serotype but comprising a capsid from a different serotype may be referred to as hybrid viral vectors (see U.S. Pat. No. 7,172,893). AAV ITRs may include the entire wild-type ITR sequence, or a variant, fragment or modification thereof, but will retain functionality.

[0208] In some embodiments, the rAAV vector genome is linear, single-stranded, and flanked by AAV ITR. Before the heterologous gene is transcribed and translated, the second strand synthesis must be initiated by DNA polymerase (e.g., DNA polymerase in transduced cells) using the free 3'-OH of one of the self-priming ITRs to convert the single-stranded DNA genome of about 4700 nucleotides into a double-stranded form. In some embodiments, the full-length single-stranded vector genome (i.e., sense and antisense) is annealed to produce a full-length double-stranded vector genome. This may occur when multiple rAAV vectors carrying opposite polarity (i.e., sense or antisense) genomes simultaneously transduce the same cell. In any case, once the double-stranded vector genome is formed, the cell can transcribe and translate the double-stranded DNA and express the heterologous gene.

[0209] Since the single-stranded rAAV genome (ssAAV) needs to be converted into double-stranded DNA before expression, the transgenic expression efficiency of rAAV vectors may be hindered. This step can be circumvented by using a self-complementary AAV genome (scAAV), which can package an inverted repeat genome that can be folded into double-stranded DNA without the need for DNA synthesis or base pairing between multiple vector genomes. See, for example, U.S. Patent No. 8,784,799; McCarty (2008) Molec. Therapy 16 (10): 1648-1656; and McCarty et al. (2001) Gene Therapy 8: 1248-1254; McCarty et al. (2003) Gene Therapy 10: 2112-2118.

[0210] The viral capsid of the rAAV vector can be derived from wild-type AAV or variant AAV, such as AAV1, AAV2, AAV3, AAV3A, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVrh10, AAVrh74 (see WO2016 / 210170), AAV12, AAV2i8, AAV1.1, AAV2.5, AAV6.1, AAV6.3.1, AAV9.45, RHM4-1 (SEQ ID NO: WO 2015 / 013313), NO:5), RHM15-1, RHM15-2, RHM15-3 / RHM15-5, RHM15-4, RHM15-6, AAVhu.26, AAV1.1, AAV2.5, AAV6.1, AAV6.3.1, AAV9,45, AAV2i8, AAV29G, AAV2,8G9, AVV-LK03, AAV2-TT, AAV2-TT-S312N, AAV3B-S312N, AAV avian AAV, bovine AAV, canine AAV, equine AAV, primate AAV, non-primate AAV, snake AAV, goat AAV, shrimp AAV, sheep AAV and variants thereof (see, e.g., Fields et al., VIROLOGY, Vol. 2, Chapter 69 (4th ed., Lippincott-Raven Publishers). Capsids can be derived from U.S. Pat. No. 7,906,111, Gao et al. (2004) Journal of Virology 78:6381, Morris et al. (2004) Journal of Virology 33:375, WO 2004005445. 2013 / 063379, WO 2014 / 194132, and including the true AAV (AAV-TT) variants disclosed in WO 2015 / 121501, and RHM4-1, RHM15-1 to RHM15-6 and variants thereof disclosed in WO 2015 / 013313. A full set of AAV cap proteins includes VP1, VP2 and VP3. An ORF comprising a nucleotide sequence encoding an AAV VP capsid protein may contain less than a full set of AAV Cap proteins, or may provide a full set of AAV cap proteins.

[0211] In some embodiments, rAAV vectors comprising capsid proteins encoded by nucleotide sequences derived from more than one AAV serotype (e.g., wild-type AAV serotype, variant AAV serotype) are referred to as "chimeric vectors" or "chimeric capsids" (see U.S. Pat. No. 6,491,907, the entire disclosure of which is incorporated herein by reference). In some embodiments, the chimeric capsid protein is encoded by nucleic acid sequences derived from 2, 3, 4, 5, 6, 7, 8, 9, 10 or more AAV serotypes. In some embodiments, the recombinant AAV vector comprises a capsid sequence derived from, for example, AAV1, AAV2, AAV3, AAV3A, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAVrh74, AAVrh10, AAV2i8, or a variant thereof, resulting in a chimeric capsid protein comprising a combination of amino acids from any of the aforementioned AAV serotypes (see Rabinowitz et al. (2002) Journal of Virology 76(2):791-801). Alternatively, the chimeric capsid may comprise a mixture of VP1 from one serotype, VP2 from a different serotype, VP3 from yet another different serotype, and combinations thereof. For example, a chimeric viral capsid may comprise an AAV1 cap protein or subunit and at least one AAV2 cap protein or subunit. Chimeric capsids may, for example, include AAV capsids with one or more B19 cap subunits, for example, AAV cap proteins or subunits may be replaced by B19 cap proteins or subunits. For example, in one embodiment, the VP3 subunit of the AAV capsid may be replaced by the VP2 subunit of B19. In some embodiments, the chimeric capsid is the Olig001 capsid described in WO2021221995 and WO2014052789, which are incorporated herein by reference.

[0212] In some embodiments, chimeric vectors have been engineered to exhibit altered tropism or tropism for specific tissues or cell types. The term "tropism" refers to the virus preferentially entering certain cells (e.g., oligodendrocytes) or tissue types and / or preferentially interacting with cell surfaces that promote entry into certain cells or tissue types. AAV tropism is generally determined by specific interactions between different viral capsid proteins and their cognate cell receptors (Lincoln (Lykken) et al. (2018) Journal of Neurodevelopmental Disorders (J. Neurodev. Disord.) 10: 16). Preferably, once a virus or viral vector enters a cell, a sequence (e.g., a heterologous sequence, such as a transgene) carried by the vector genome (e.g., rAAV vector genome) will be expressed.

[0213] "Tropism profile" refers to the transduction pattern of one or more target cells in various tissues and / or organs. For example, the tropism profile of a chimeric AAV capsid may be characterized by effective transduction of oligodendrocytes, while only low transduction of neurons, astrocytes, and other CNS cells. See WO2014 / 052789, which is incorporated herein by reference. If such chimeric capsids preferentially transduce oligodendrocytes rather than neurons, astrocytes, and other CNS cell types when directly administered to the CNS, such chimeric capsids may be considered to be "specific for oligodendrocytes", showing a tropism for oligodendrocytes, and are referred to herein as "oligotropism (oligotropism)". In some embodiments, at least about 80% of cells transduced with a capsid specific for oligodendrocytes are oligodendrocytes, for example, at least about 85%, 90%, 95%, 96%, 97%, 98%, 99% or more of the transduced cells are oligodendrocytes.

[0214] In some embodiments, rAAV vectors can be used to treat or prevent "disorders associated with oligodendrocyte dysfunction". As used herein, the term "associated with oligodendrocyte dysfunction" refers to a disease, disorder or condition in which oligodendrocytes are damaged, lost or function abnormally compared to normal oligodendrocytes that are otherwise identical. The term includes diseases, disorders and conditions in which oligodendrocytes are directly affected, as well as diseases, disorders or conditions in which oligodendrocytes are secondary to dysfunction due to damage to other cells. In some embodiments, the disorder associated with oligodendrocyte dysfunction is demyelination.

[0215] In some embodiments, the chimeric AAV capsid with tropism for oligodendrocytes is Olig001 (also referred to as BNP61) or a functional variant of Olig001, comprising sequences from AAV1, AAV2, AAV6, AAV8, and AAV9 (see WO2021221995 and WO 2014 / 052789). The amino acid sequence of the Olig001 capsid protein is shown in the following sequence, wherein VP1 starts from amino acid residue 1 (methionine), VP2 starts from amino acid residue 148 (threonine), and VP3 starts from amino acid residue 203 (methionine).

[0216] Amino acid sequence of Olig001 (BNP61) capsid (SEQ ID NO: 245)

[0217] MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPPKPAERHKDDSRGLVLPGYKYLGP

[0218] FNGLDKGEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADAEFQERLKEDTSF

[0219] GGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSSGIGKTGQQ

[0220] PAKKRLNFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADG

[0221] VGSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNGTSGGATNDNTYF

[0222] GYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLSFKLFNIQVKEVTQNEG

[0223] TKTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMIPQYGYLTLNNG

[0224] SQAVGRSSFYCLEYFPSQMLRTGNNFQFTYTFEDVPFHSSYAHSQSLDRLMNPLI

[0225] DQYLYYLSRTQTTGGTANTQTLGFSQGGPNTMANQAKNWLPGPCYRQQRVSTTTG

[0226] QNNNSNFAWTAGTKYHLNGRNSLANPGIAMATHKDDKERFFPSNGILIFGKQNAA

[0227] RDNADYSDVMLTSEEEIKTTNPVATEEYGIVADNLQQQNTAPQIGTVNSQGALPG

[0228] MVWQNRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGLKHPPPQILIKNTPVPADPP

[0229] TTFNQSKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSTSVDFA

[0230] VNTEGVYSEPHPIGTRYLTRPL

[0231] In some embodiments, the chimeric AAV capsid having tropism for oligodendrocytes is Olig002 (also known as BNP62) or Olig003 (also known as BNP63) (see WO2021221995 and WO 2014 / 052789). In some embodiments, the Olig002 capsid VP1 comprises or consists of the amino acid sequence shown below or a functional variant thereof.

[0232] Amino acid sequence of Olig002 (BNP62) capsid (SEQ ID NO: 246)

[0233] MAADGYLPDWLEDNLSEGIREWWDLKPGAPPKPKANQQKQDDGRGLVLPGYKYLGP

[0234] FNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLRYNHADAEFQERLQEDTSF

[0235] GGNLGRAVFQAKKRVLEPLGLVEEGAKTAPGKKRPVEQSPQEPDSSSSGIGKTGQQ

[0236] PAKKRLNFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADG

[0237] VGSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISSASTGASNDNHYFG

[0238] YSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTDNNGV

[0239] KTIANNLTSTVQVFTDSDYQLPYVLGSAHQGCLPPFPADVFMIPQYGYLTLNNGS

[0240] QAVGRSSFYCLEYFPSQMLRTGNNFQFTYTFEDVPFHSSYAHSQSLDRLMNPLID

[0241] QYLYYLSRTQTTGGTANTQTLGFSQGGPNTMANQAKNWLPGPCYRQQRVSTTTGQ

[0242] NNNSNFAWTAGTKYHLNGRNSLANPGIAMATHKDDKERFFPSNGILIFGKQNAAR

[0243] DNADYSDVMLTSEEEIKTTNPVATEEYGIVADNLQQQNTAPQIGTVNSQGALPGM

[0244] VWQNRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGLKHPPPQILIKNTPVPADPPT

[0245] TFNQSKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSTSVDFAV

[0246] NTEGVYSEPHPIGTRYLTRPL

[0247] In some embodiments, the Olig003 capsid comprises or consists of the amino acid sequence shown below or a functional variant thereof.

[0248] Amino acid sequence of Olig003 (BNP63) capsid (SEQ ID NO: 247)

[0249] MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPPKPAERHKDDSRGLVLPGYKYLGP

[0250] FNGLDKGEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADAEFQERLQGDTSF

[0251] GGNLGRAVFQAKKRVLEPLGLVEEGAKTAPGKKRPVEQSPQEPDSSSGIGETGQQ

[0252] PAKKRLNFGQTGDSESVPDPQPLGEPPATPAAVGPTTMASGGGAPMADNNEGADG

[0253] VGSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISSASTGASNDNHYFG

[0254] YSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLSFKLFNIQVKEVTDNNGV

[0255] KTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMIPQYGYLTLNNGS

[0256] QAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLID

[0257] QYLYYLSRTQTTGGTANTQTLGFSQGGPNTMANQAKNWLPGPCYRQQRVSTTTGQ

[0258] NNNSNFAWTAGTKYHLNGRNSLANPGIAMATHKDDKERFFPSNGILIFGKQNAAR

[0259] DNADYSDVMLTSEEEIKTTNPVATEEYGIVADNLQQQNTAPQIGTVNSQGALPGM

[0260] VWQNRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGLKHPPPQILIKNTPVPADPPT

[0261] TFNQSKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSTSVDFAV

[0262] NTEGVYSEPHPIGTRYLTRPL

[0263] In some embodiments, rAAV vectors comprising chimeric AAV capsids (e.g., Olig001, Olig002, or Olig003) and therapeutic transgenes can be used to treat diseases, disorders, or conditions associated with oligodendrocyte dysfunction. In such diseases, disorders, or conditions, oligodendrocytes are damaged, lost, or malfunction. This may be the result of a direct effect on oligodendrocytes, or the result when oligodendrocytes are secondary to dysfunction due to damage to other cells. In some embodiments, rAAV vectors comprising AAV / Olig001 capsids and polynucleotides encoding RNA molecules described herein are used to treat hereditary and acquired myelin disorders.

[0264] 5. Viral Particles and Production

[0265] Viral vectors (e.g., rAAV vectors) carrying transgenes (e.g., transgenes encoding RNA disclosed herein) can be assembled from polynucleotides encoding transgenes, appropriate regulatory elements, and elements necessary to produce viral proteins that mediate cell transduction. Examples of viral vectors include, but are not limited to, adenoviruses, retroviruses, lentiviruses, herpes viruses, and AAV vectors, and in particular rAAV vectors.

[0266] The vector genome components of the rAAV vector produced according to the method of the present disclosure include at least one transgenic (e.g., a polynucleotide encoding an RNA molecule) and a related expression control sequence for controlling RNA expression. In a preferred embodiment, the vector genome includes a portion of a parvoviral genome, such as an AAV genome that is missing and / or replaced by a transgenic and its related expression control sequence, such as rep and cap. The transgenic is usually inserted near one or two AAV ITR or ITR elements sufficient for viral replication (i.e., the AAV ITR or ITR elements are flanked), replacing the nucleic acid encoding viral rep and cap proteins. Other regulatory sequences suitable for promoting tissue-specific expression of transgenics in target cells (e.g., oligodendrocytes) may also be included.

[0267] A. Packaging cells

[0268] Those skilled in the art will appreciate that rAAV vectors comprising a transgene and lacking viral proteins required for viral replication (e.g., cap and rep) are unable to replicate, as these proteins are required for viral replication and packaging. The cap and rep genes can be provided to a cell (e.g., a host cell, such as a packaging cell) as part of a plasmid that is separate from the plasmid that provides the vector genome with the transgene.

[0269] Packaging cells or production cells refer to cells or cell lines that can be transfected with vectors, plasmids or DNA constructs and provide all missing functions required for complete replication and packaging of viral vectors in trans. The genes required for rAAV vector assembly include vector genomes (e.g., transgenics, regulatory elements and ITRs encoding RNA), AAV rep genes, AAV cap genes and some auxiliary genes from other viruses (e.g., adenoviruses). Ordinary technicians will understand that the genes required for AAV production can be introduced into packaging cells in a variety of ways, including, for example, transfection of one or more plasmids. However, in some embodiments, some genes (e.g., rep, cap, auxiliary genes) may already be present in packaging cells, integrated into the genome or carried on episomes. In certain embodiments, packaging cells express one or more missing viral functions in a constitutive or inducible manner.

[0270] Any suitable packaging cell known in the art can be used to produce packaged viral vectors. Mammalian cells or insect cells are preferred. In the practice of the present disclosure, examples of cells that can be used to produce packaging cells include, for example, human cell lines, such as PER.C6, WI38, MRC5, A549, HEK293 cells (which express functional adenovirus E1 under the control of a constitutive promoter), B-50 or any other HeLa cells (HeLa cell), HepG2, Saos-2, HuH7 and HT1080 cell lines. Suitable non-human mammalian cell lines include, for example, VERO, COS-1, COS-7, MDCK, BHK21-F, HKCC or CHO cells.

[0271] In some embodiments, the packaging cells are capable of growing in suspension culture. In some embodiments, the packaging cells are capable of growing in serum-free medium. For example, HEK293 cells are grown in suspension in serum-free medium. In another embodiment, the packaging cells are HEK293 cells described in U.S. Pat. No. 9,441,206 and deposited as American Type Culture Collection (ATCC) No. PTA 13274. Many rAAV packaging cell lines are known in the art, including but not limited to those disclosed in WO 2002 / 46359.

[0272] Cell lines used as packaging cells include insect cell lines. According to the present disclosure, any insect cell that allows AAV replication and can be maintained in culture can be used. Examples include Spodoptera frugiperda, such as Sf9 or Sf21 cell lines, Drosophila spp. cell lines, or mosquito cell lines, such as cell lines derived from Aedes albopictus. A preferred cell line is the Sf9 cell line of the Spodoptera frugiperda. The following references are incorporated herein for their teachings on the use of insect cells to express heterologous polypeptides, methods for introducing nucleic acids into such cells, and methods for maintaining such cells in culture: Methods in Molecular Biology, Richard (Richard), Humana Press, NJ (1995); O'Reilly et al., Baculovirus Expression Vectors: A Laboratory Manual, Oxford University Press (Oxford Univ. Press) (1994); Samsky et al. (1989) Journal of Virology 63:3822-3828; Kajigaya et al. (1991) Proceedings of the National Academy of Sciences of the United States of America 88:4646-4650; Ruffing et al. (1992) Journal of Virology 66:6922-6930; Kimbauer et al. (1996) Virology 219:37-44; Zhao et al. (2000) Virology 272:382-393; and U.S. Patent No. 6,204,059.

[0273] As another alternative, the viral vectors of the present disclosure can be produced in insect cells using baculovirus vectors to deliver rep / cap genes and rAAV templates, such as described in Urabe et al. (2002) Human Gene Therapy 13: 1935-1943. When using baculovirus to produce AAV, in some embodiments, the vector genome is self-complementary. In some embodiments, the host cell is a baculovirus-infected cell (e.g., an insect cell), which optionally contains additional nucleic acids encoding baculovirus helper functions, thereby promoting the production of viral capsids.

[0274] Packaging cells typically include one or more viral vector functions and auxiliary functions and packaging functions sufficient to cause viral vector replication and packaging. These various functions can be provided to packaging cells together or separately using genetic constructs (e.g., plasmids or amplicons), and they can exist outside the chromosomes in the cell line, or be integrated into the chromosomes of the host cell.

[0275] B. Accessibility

[0276] In the absence of helper virus co-infected cells, AAV cannot replicate in cells. Auxiliary functions include auxiliary virus elements required for establishing active infection of packaging cells, which is required for initiating packaging of viral vectors. Helper viruses generally include adenoviruses or herpes simplex viruses. Adenovirus auxiliary functions generally include adenovirus components adenovirus early region 1A (E1a), E1b, E2a, E4 and virus-related (VA) RNA. Auxiliary functions (such as E1a, E1b, E2a, E4 and VA RNA) can be provided to packaging cells by transfecting cells with one or more nucleic acids encoding various auxiliary elements. Alternatively, host cells (such as packaging cells) may include nucleic acids encoding auxiliary proteins. For example, HEK293 cells are produced by transforming human cells with adenovirus 5 DNA, and now express many adenovirus genes, including but not limited to E1 and E3 (see, for example, Graham et al. (1977) Journal of General Virology (J.Gen.Virol.) 36:59-72). Thus, these helper functions can be provided by the HEK 293 packaging cells without the need to provide them to the cells via, for example, a plasmid encoding them.

[0277] In some embodiments, the packaging cells are transfected with at least: (i) a plasmid comprising a vector genome comprising a transgene and AAV ITRs and further comprising at least one of the following regulatory elements: an enhancer, a promoter, an exon, an intron, and poly A; (ii) a plasmid comprising a rep gene (e.g., AAV2rep) and a cap gene (e.g., Olig001cap); and (iii) a plasmid comprising helper functions.

[0278] Any method for introducing a nucleotide sequence carrying an auxiliary function into a cell host for replication and packaging may be used, including but not limited to electroporation, calcium phosphate precipitation, microinjection, cationic or anionic liposomes, and a combination of liposomes and nuclear localization signals. In some embodiments, the auxiliary function is provided by transfection with a viral vector or by infection with a helper virus, and standard methods for producing viral infections may be used.

[0279] The vector genome may be any suitable recombinant nucleic acid, such as a DNA or RNA construct, and may be single-stranded, double-stranded or duplexed (ie, self-complementary as described in WO 2001 / 92551).

[0280] C. Production of Packaged Viral Vectors

[0281] Viral vectors can be prepared by several methods known to those skilled in the art (see, e.g., WO 2013 / 063379). Preferred methods are described in Grieger et al. (2015) Molecular Therapy 24(2): 287-297, the contents of which are incorporated herein by reference for all purposes. In brief, efficient transfection of HEK293 cells is used as a starting point, where adherent HEK293 cell lines from qualified clinical master cell banks are used to grow in animal component-free suspension conditions in shake flasks and WAVE bioreactors, thereby achieving rapid and scalable rAAV production. Using a triple transfection method (e.g., WO 96 / 40240), HEK293 cell line suspensions harvested 48 hours after transfection can produce more than 1×10 per cell. 5 particles containing vector genomes (vg), or greater than 1×10 per liter of cell culture. 14 More specifically, triple transfection refers to a method of transfecting packaging cells with three plasmids: one plasmid encodes the AAV rep and cap (e.g., Olig001 cap) genes, another plasmid encodes various helper functions (e.g., adenovirus or HSV proteins, such as E1a, E1b, E2a, E4, and VA RNA), and another plasmid encodes a transgene (e.g., the RNA described herein) and various elements that control the expression of the transgene.

[0282] The single-stranded vector genome is packaged into the capsid as a positive strand or a negative strand in approximately equal proportions. In some embodiments of rAAV vectors, the vector genome is in positive strand polarity (i.e., the sense or coding sequence of the DNA strand). In some embodiments of rAAV vectors, the vector is in negative strand polarity (i.e., the antisense or template DNA strand). Given a positive strand nucleotide sequence in the 5' to 3' direction, the negative strand nucleotide sequence in the 5' to 3' direction can be determined as the reverse complement of the positive strand nucleotide sequence.

[0283] To achieve the desired yield, many variables are optimized, such as the selection of a compatible serum-free suspension medium that supports growth and transfection, the choice of transfection reagent, transfection conditions, and cell density.

[0284] rAAV vectors can be purified by standard methods in the art, such as by column chromatography or cesium chloride gradients. Methods for purifying rAAV vectors are known in the art and include methods described in Clark et al. (1999) Human Gene Therapy 10(6):1031-1039; Schenpp and Clark (2002) Methods Mol. Med. 69:427-443; U.S. Pat. No. 6,566,118; and WO 98 / 09657.

[0285] A general purification strategy based on ion exchange chromatography can be used to generate high purity vector preparations of AAV serotypes 1 to 6, 8, 9, and various chimeric capsids (e.g., Olig001). In some embodiments, this process can be completed within one week, produces a high ratio of full capsid to empty capsid (>90% full capsid), provides a post-purification yield suitable for clinical applications (>1×10 13 In some embodiments, such methods are universal for all serotypes and chimeric capsids. Scalable manufacturing technology can be used to manufacture GMP clinical and commercial grade rAAV vectors (e.g., for the treatment of inherited or acquired myelin disorders).

[0286] After the rAAV vectors of the present disclosure are produced and purified, they can be titrated (e.g., the amount of rAAV vector in a sample can be quantified) to prepare a composition for administration to a subject (e.g., a human subject with an inherited or acquired myelin disorder). rAAV vector titration can be accomplished using methods known in the art.

[0287] In some embodiments, the number of viral particles (including particles containing vector genomes and "empty" capsids that do not contain vector genomes) can be determined by electron microscopy, such as transmission electron microscopy (TEM). Such TEM-based methods can provide the number of vector particles (or viral particles in the case of wild-type AAV) in a sample.

[0288] In some embodiments, primers for sequences in the vector genome (e.g., ITR sequences, and / or sequences in transgenic or regulatory elements) can be used to titrate the rAAV vector genome using quantitative PCR (qPCR). By performing qPCR in parallel on dilutions of known concentrations of standards (e.g., plasmids containing vector genome sequences), a standard curve can be generated, allowing the concentration of the rAAV vector to be calculated as the number of vector genomes (vg) per unit volume (e.g., microliters or milliliters). By comparing the number of vector particles measured by, for example, electron microscopy with the number of vector genomes in the sample, the number of empty capsids can be determined. Since the vector genome contains a therapeutic transgene, the vg / kg or vg / ml of the vector sample may be more indicative of the therapeutic amount of the vector that the subject will receive than the number of vector particles, and some vector particles may be empty and do not contain a vector genome. Once the concentration of the rAAV vector genome in the stock solution is determined, it can be diluted into a suitable buffer or dialyzed against a suitable buffer for the preparation of a composition for application to a subject (e.g., a subject with a hereditary or acquired myelin disorder).

[0289] 6. Uses and treatment methods

[0290] The nucleic acids disclosed herein (e.g., RNA molecules or polynucleotides encoding the RNA molecules) can be used for gene therapy treatment and / or prevention of diseases, disorders or conditions. Specifically, they can be used to treat or prevent diseases, disorders or conditions associated with a lack or dysfunction of oligodendrocytes or myelin (e.g., PMD, MSA or H-ABC) by targeting specific target genes, as well as any other conditions and or ailments that can produce therapeutic benefits or improvements by reducing the expression of the relevant target genes, such as diseases, disorders or conditions mediated or associated with an increase in the level or function of the relevant protein (e.g., PLP1, SNCA or TUBB4A) compared to the level or function of the protein in a healthy individual. The vector genomes and / or rAAV vectors described herein can be used for gene therapy treatment and / or prevention of the same diseases, disorders or conditions.

[0291] In some embodiments, the methods of the present disclosure include using a rAAV vector or a pharmaceutical composition thereof to treat a disease, disorder or condition of a subject. In some embodiments, the methods of the present disclosure include using a rAAV vector (e.g., AAV / Olig001) or a pharmaceutical composition thereof to reduce the level of a gene of interest (e.g., PMD, MSA or H-ABC) in a subject in need thereof.

[0292] The nucleic acids, vector genomes and / or rAAV vectors described above can be used to prepare medicaments for treating and / or preventing diseases, disorders or conditions associated with or caused by a deficiency or dysfunction of oligodendrocytes or myelin (e.g., PMD, MSA or H-ABC), as well as any other condition or ailment in which downregulation of one or more related proteins can produce a therapeutic benefit or improvement.

[0293] As used herein, myelin disorders, myelin diseases, myelin-related disorders, myelin-related diseases, myelin disorders and myelin diseases are used interchangeably. It includes any disease, condition (such as the condition caused by traumatic spinal cord injury and cerebral infarction) or disease related to demyelination, myelination and myelination regeneration of the subject. Such diseases can be hereditary or acquired or both. It can be caused by myelination-related conditions or demyelination caused by a variety of neurotoxic injuries. As used herein, "demyelination" refers to the loss of demyelinating behavior or nerve insulating myelin, and is a sign of some neurodegenerative autoimmune diseases, including multiple sclerosis, transverse myelitis, chronic inflammatory demyelinating polyneuropathy and Guillain-Barre syndrome. Leukodystrophy is caused by hereditary enzyme deficiency, which causes abnormal formation, destruction and / or abnormal turnover of myelin in the white matter of CNS. Acquired and inherited myelin disorders have a poor prognosis and can lead to severe disability. Therefore, some embodiments of the present disclosure may include methods for treating neurodegenerative autoimmune diseases in subjects. Oligodendrocytes are required for remyelination of neurons. As used herein, the term "remyelination" refers to regenerating the myelin sheath of a nerve by replacing cells that produce myelin or restoring its function.

[0294] Myelin-related diseases or conditions that can be treated or improved by the methods of the present invention include diseases, disorders or injuries associated with dysmyelination or demyelination in brain cells (e.g., CNS neurons) of a subject. Such diseases include, but are not limited to, diseases and disorders in which the myelin sheath surrounding neurons is missing, incomplete, improperly formed or degenerated. Such diseases include, but are not limited to, multiple sclerosis (MS), neuromyelitis optica (NMO), progressive multifocal leukoencephalopathy (PML), encephalomyelitis (EPL), central pontine myelinolysis (CPM), adrenoleukodystrophy, Alexander disease, Pellishaus-Mertzbacher disease (PMD), Wallerian degeneration, optic neuritis, transverse myelitis, amyotrophic lateral sclerosis (ALS), Huntington's disease, Alzheimer's disease, Parkinson's disease, spinal cord injury, traumatic brain injury, post-radiation injury, neurological complications of chemotherapy, stroke, acute ischemic optic neuropathy, vitamin E deficiency, isolated vitamin E deficiency syndrome, AR, Basen-Kotzweig syndrome, Ma-Bieder syndrome, metachromatic leukodystrophy, trigeminal neuralgia, acute disseminated encephalitis, Guillain-Barré syndrome, Marie-Charcot-Tooth disease, and Bell's palsy.

[0295] Myelin-related diseases or conditions that can be treated or improved by the method of the present invention include diseases or conditions characterized by myelin deficiency. Insufficient myelination in the central nervous system is related to a variety of nervous system disorders. The nervous system disorder includes the following cerebral palsy forms, wherein the congenital defects of the forebrain myelination of children with periventricular leukomalacia can cause nervous system morbidity (Goldman et al., 2008) Goldman, SA, Schanz, S. (Schanz, S.) and Windrem, MS (Windrem, MS) (2008). Stem cell-based strategies for treating pediatric disorders of myelin in children. Human Molecular Genetics (Hum Mol Genet.) 17, R76-83. At the other end of the age spectrum, myelin loss and ineffective repair may contribute to cognitive decline associated with aging (Kohama et al., 2011) Kohama, SG, Rosene, DL and Sherman, LS (2011) Age (Dordr). Age-related changes in human and non-human primate white matter: from myelination disturbances to cognitive decline. Therefore, it is contemplated that effective compositions and methods for enhancing myelination and / or remyelination may have significant therapeutic benefits in preventing disease progression and restoring function in a variety of myelin-related disorders.

[0296] In certain embodiments, the compositions of the present invention may be applied to subjects who do not suffer from and / or are not suspected of suffering from myelin-related disorders, to enhance or promote myelin-dependent processes. In certain embodiments, the compositions described herein may be applied to subjects to promote myelination of CNS neurons, thereby enhancing the cognition of cognitive healthy subjects, and it is known that cognition is a myelin-dependent process. In certain embodiments, the compositions described herein may be administered in combination with cognitive enhancement (mental) agents. Exemplary agents include any drug, supplement or other substance that improves cognitive function, particularly executive function, memory, creativity or motivation in healthy individuals. Non-limiting examples include racetams (e.g., piracetam, oxiracetam, and aniracetam), nutraceuticals (e.g., Bacopa monnieri, Ginseng, Ginkgo biloba, and GABA), stimulants (e.g., amphetamine drugs, methylphenidate, eugeroic, xanthine, and nicotine), L-Theanine, Tolcapone, Levodopa, Atomoxetine, and Desipramine.

[0297] The total dose of the therapeutic agent (e.g., RNA molecule, polynucleotide encoding the RNA molecule, vector genome or vector (e.g., rAAV vector) or cell) will be a therapeutically effective amount, which depends on several factors, including the overall health of the subject, the subject's disease state, the severity of the condition, the observed improvement, and the formulation and route of administration of the selected agent or agents. The determination of a therapeutically effective amount is within the capabilities of those skilled in the art. The exact formulation, route of administration, and dosage can be selected by the individual physician considering the subject's condition.

[0298] In certain embodiments, the cells or nucleotide compositions described herein can be administered in an amount effective to enhance myelin production in the CNS of a subject by increasing the amount of myelin protein (e.g., MBP) by at least 5%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 550%, 600%, 650%, 700%, 750%, 800%, 850%, 900%, 950%, or 1000% as compared to the level of myelin protein in an untreated subject.

[0299] In other embodiments, the cells or nucleotide compositions are administered in an amount effective to promote the survival of CNS neurons in a subject by at least 5%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 550%, 600%, 650%, 700%, 750%, 800%, 850%, 900%, 950%, or 1000% as compared to the number of untreated CNS neurons or surviving neurons in a subject.

[0300] Another strategy for treating a subject with a myelin-related disorder is to administer a therapeutically effective amount of a cell or nucleotide composition described herein and a therapeutically effective amount of one or more oligodendrocyte differentiation and / or proliferation inducers and / or anti-neurodegenerative disease agents. Examples of anti-neurodegenerative disease agents include L-dopa, cholinesterase inhibitors, anticholinergics, dopamine agonists, steroids, and immunomodulators, including interferons, monoclonal antibodies, and glatiramer acetate. Therefore, in another aspect of the present disclosure, the compositions described herein can be administered as part of a combination therapy with an adjuvant therapy for treating neurodegenerative and myelin-related disorders.

[0301] The phrase "combination therapy" encompasses the use of oligodendrocyte precursor differentiation inducing compositions and therapeutic agents described herein as a part of a specific treatment regimen, and the specific treatment regimen is intended to provide a beneficial effect by the joint action of these therapeutic agents. When used as a combination, oligodendrocyte precursor differentiation inducing compounds and therapeutic agents can be formulated into a separate composition. The combination of these therapeutic agents is usually applied in a limited time period (usually a few minutes, hours, days or weeks, depending on the selected combination) and is carried out.

[0302] 7. Pharmaceutical Compositions

[0303] The present disclosure provides a pharmaceutical composition or drug for preventing or treating hereditary or acquired myelin disorders. In some embodiments, the pharmaceutical composition comprises one or more of the RNA molecules, polynucleotides, expression cassettes, expression vectors (e.g., viral vector genomes, expression vectors, rAAV vectors) and host cells described above.

[0304] The pharmaceutical composition further includes a pharmaceutically acceptable carrier, adjuvant, diluent, excipient and / or other medicament. Pharmaceutically acceptable carriers, adjuvants, diluents, excipients or other medicaments are substances without biological or other adverse effects, such as the substance can be applied to a subject without causing adverse biological effects exceeding the favorable biological effects of the substance. Any suitable pharmaceutically acceptable carrier or excipient can be used to prepare a pharmaceutical composition according to the present invention (see, for example, Remington The Science and Practice of Pharmacy, Adeboye Adejare (Academic Press) (ed.), November 2020).

[0305] The pharmaceutical composition is generally sterile, pyrogen-free and stable under the conditions of manufacture and storage. The pharmaceutical composition can be formulated as a solution (e.g., water, saline, glucose solution, buffered solution or other pharmaceutically sterile liquid), microemulsion, liposome or other ordered structure suitable for containing high product (e.g., viral vector particles, microparticles or nanoparticles) concentration.

[0306] In some embodiments, the pharmaceutical composition comprising the RNA molecules, polynucleotides, expression cassettes, expression vectors, vector genomes, host cells or rAAV vectors disclosed herein described above is formulated in water or buffered saline solution. The carrier can be a solvent or a dispersion medium, which contains, for example, water, ethanol, polyols (such as glycerol, propylene glycol and liquid polyethylene glycol, etc.) and a suitable mixture thereof. Appropriate fluidity can be maintained, for example, by using a coating such as lecithin, by maintaining the desired particle size in the case of a dispersion, and by using a surfactant. In some embodiments, isotonic agents such as sugars, polyols (such as mannitol, sorbitol) or sodium chloride may preferably be included in the composition. By including a medicament for delayed absorption in the composition, such as monostearate and gelatin, extended absorption of injectable compositions can be achieved. In some embodiments, nucleic acids, carriers and / or host cells disclosed herein can be administered in a controlled release formulation, for example, in a composition comprising a sustained-release polymer or other carriers for protecting the product from rapid release, including implants and microencapsulation delivery systems.

[0307] In some embodiments, the pharmaceutical composition of the present disclosure is a parenteral pharmaceutical composition, including compositions suitable for intravenous, intraarterial, subcutaneous, intradermal, intraperitoneal, intramuscular, intraarticular, intraparenchymal (IP), intrathecal (IT), intracerebroventricular (ICV) and / or intracisternal (ICM) administration. In some embodiments, the pharmaceutical composition of the present disclosure is formulated for administration by ICV injection. In some embodiments, the rAAV vector (e.g., AAV / Olig001) is formulated in PBS containing 350 mM NaCl and 5% D-sorbitol.

[0308] 8. Application method

[0309] The RNA molecules or polynucleotides or vectors (e.g., vector genomes, rAAV vectors) described above can be administered to a subject (e.g., a patient) to treat the subject. Administration of the vector to a human subject or animal in need can be performed by any means known in the art for administering the vector. The target cells of the vectors disclosed herein include cells of the CNS, preferably oligodendrocytes.

[0310] The vector can be administered in addition to and as an adjunct to standard of care treatment. That is, the vector can be co-administered with another agent, compound, drug, treatment or treatment regimen simultaneously, concurrently, or at a determined dosing interval determined by one skilled in the art using conventional methods. The uses disclosed herein include concurrent administration of the disclosed rAAV vector in addition to and / or according to a dosing regimen that is in parallel with standard of care for a disease known in the art.

[0311] In some embodiments, the combination composition includes one or more immunosuppressants. In some embodiments, the combination composition includes an rAAV vector comprising a transgene (e.g., a polynucleotide encoding an RNA molecule disclosed herein) and one or more immunosuppressants. In some embodiments, the method includes administering or delivering an rAAV vector comprising a transgene to a subject, and administering an immunosuppressant to the subject prophylactically or after administering the vector (i.e., before or after symptoms of a reaction to the vector and / or the protein provided thereby are apparent).

[0312] In one embodiment, the vector (e.g., rAAV vector) of the present disclosure is systemically administered. Exemplary methods of systemic administration include, but are not limited to, intravenous (e.g., portal vein), intraarterial (e.g., femoral artery, hepatic artery), intravascular, subcutaneous, intradermal, intraperitoneal, transmucosal, intrapulmonary, intralymphatic and intramuscular administration, etc., and direct tissue or organ injection. It will be appreciated by those skilled in the art that systemic administration can deliver nucleic acid to all tissues. In certain embodiments, direct tissue or organ administration includes administration to regions (e.g., brain and / or central nervous system) directly affected by oligodendrocyte deficiency. In certain embodiments, the vector of the present disclosure and its pharmaceutical composition are applied to brain parenchyma (i.e., administered in the parenchyma), applied to spinal canal or subarachnoid space to make it reach cerebrospinal fluid (CSF) (i.e., administered intrathecally), applied to ventricles (i.e., administered intraventricularly) and / or applied to the cerebral medullary cistern (i.e., administered intracerebral medullary cistern).

[0313] Therefore, in some embodiments, the vector of the present disclosure is administered by direct injection into the brain (e.g., directly into the parenchyma, cerebral ventricle, cisterna magna, etc.) and / or directly into the CSF (e.g., directly into the spinal canal or subarachnoid space) to treat myelin disorders. The target cells of the vector of the present disclosure include cells located in the cortex, subcortical white matter of the corpus callosum, striatum, and / or cerebellum. In some embodiments, the target cells of the vector of the present disclosure are oligodendrocytes. Additional routes of administration may also include local administration of the vector under direct visualization, such as superficial cortical administration, or other stereotaxic administration.

[0314] In some embodiments, the vectors of the present disclosure are administered by at least two routes. For example, the vector is administered systemically and also directly to the brain. If administered by at least two routes, the administration of the vector can (but need not be) simultaneous or concurrent. Conversely, administration by different routes can be performed separately, with a certain time interval between each administration.

[0315] The RNA molecules described above, or polynucleotides encoding the RNA molecules, or vector genomes or rAAV vectors comprising the polynucleotides can be used for ex vivo transduction of cells or for direct administration to subjects (e.g., directly to the CNS of patients with diseases). In some embodiments, transduced cells (e.g., host cells) are administered to subjects to treat or prevent diseases, disorders, or conditions (e.g., cell therapy for diseases). The rAAV vectors comprising therapeutic nucleic acids (e.g., encoding RNA molecules) are preferably administered to cells in biologically effective amounts. In some embodiments, the biologically effective amount of the vector is an amount sufficient to cause a reduction in the expression of a gene of interest in a target cell.

[0316] In some embodiments, the disclosure includes methods of reducing the level and / or activity of a gene in a cell by administering to the cell (in vivo, in vitro, or ex vivo) a polynucleotide encoding an RNA molecule described herein, either alone or in a vector, including a plasmid, a viral vector, a nanoparticle, a liposome, or any known method for providing nucleic acids to cells.

[0317] The dosage of rAAV vectors depends on, for example, the mode of administration, the disease or condition to be treated, the stage and / or aggressiveness of the disease, the condition of the individual subject (age, sex, weight, etc.), the specific viral vector, the stability of the protein to be expressed, the host's immune response to the vector and / or the gene to be delivered. In general, the dosage range is at least 1×10 40 μg / kg of subject body weight. 8 or more, for example 1×10 9 , 1×10 10 , 1×10 11 , 1×10 12 , 1×10 13 , 1×10 14 , 1×10 15 or more vector genomes (vg) to achieve a therapeutic effect.

[0318] In some embodiments, the polynucleotides encoding the RNA molecules described herein can be administered as a component of a DNA molecule (e.g., a recombinant nucleic acid) having a regulatory element (e.g., a promoter) suitable for expression in a target cell (e.g., an oligodendrocyte). The polynucleotides can be administered as a component of a plasmid or a viral vector (e.g., an rAAV vector). The rAAV vector can be administered in vivo by directly delivering the vector to a patient in need of treatment (e.g., directly to the CNS). The rAAV vector can be administered in vitro to a cell from a donor patient in need of treatment, and then the transduced cells are introduced back into the donor (e.g., cell therapy) and administered to the patient in vitro.

[0319] 9. Test kit

[0320] The present disclosure provides a kit having packaging materials and one or more components thereof. The kit typically includes a label or package insert including a description of the components or instructions for use of the components in vitro, in vivo or ex vivo. The kit may contain a collection of these components, such as the RNA molecules, polynucleotides, nucleic acids, expression cassettes, expression vectors (e.g., viral vector genomes, expression vectors, rAAV vectors) and host cells described above, and an optional second active agent, such as a compound, therapeutic agent, drug or composition.

[0321] Kit refers to a physical structure containing one or more components of the kit. The packaging material can maintain the components in a sterile manner and can be made of materials commonly used for this purpose (e.g., paper, glass, plastic, foil, ampoule, vial, tube, etc.).

[0322] The label or insert may include the identification information, dosage, clinical pharmacology of one or more active ingredients of one or more components, including mechanism of action, pharmacokinetics and pharmacodynamics. The label or insert may include information identifying the manufacturer, batch number, place of manufacture and date, expiration date. The label or insert may include information about a disease (e.g., hereditary or acquired myelin disorders, such as PMD, MSA and H-ABC) that can be treated using kit components. The label or insert may include instructions for clinicians or subjects using one or more of the kit components in methods, purposes or treatment planning (treatment protocol) or therapeutic regimen (therapeutic regimen). Instructions may include dosage, frequency of duration, and instructions for practicing any one of the methods described herein, purposes, treatment planning or prevention or therapeutic regimen.

[0323] The label or insert may include information about potential adverse side effects, complications, or reactions, such as a warning to the subject or clinician regarding circumstances in which a particular composition is not suitable for use.

[0324] 10. Definitions

[0325] As used herein, the terms "nucleic acid sequence", "nucleotide sequence" and "polynucleotide" interchangeably refer to any molecule consisting of or comprising monomeric nucleotides connected by phosphodiester bonds. Nucleic acids can be oligonucleotides or polynucleotides. Nucleic acid sequences are presented herein in a 5' to 3' direction. The nucleic acid sequences (i.e., polynucleotides) disclosed herein can be deoxyribonucleic acid (DNA) molecules or ribonucleic acid (RNA) molecules, and refer to all forms of nucleic acids, such as double-stranded molecules, single-stranded molecules, small or short hairpin RNA (shRNA), micro-interfering RNA or microRNA (miRNA), small or short interfering RNA (siRNA), trans-splicing RNA, antisense RNA, messenger RNA, transfer RNA, ribosomal RNA. When the polynucleotide is a DNA molecule, the molecule can be a gene, cDNA, antisense molecule, or a fragment of any of the aforementioned molecules. Nucleotides are represented herein by single-letter codes: adenine (A), guanine (G), thymine (T), cytosine (C), inosine (I) and uracil (U). Nucleotide sequences can be chemically modified or artificial. Nucleotide sequences include peptide nucleic acids (PNA), morpholinos and locked nucleic acids (LNA), as well as glycol nucleic acids (GNA) and threose nucleic acids (TNA). Each of these sequences differs from naturally occurring DNA or RNA in the change of the molecular backbone. This type of sequence can use phosphorothioate nucleotides. Other deoxynucleotide analogs include methylphosphonates, phosphoramidates, phosphorodithioates, N3'-P5'-phosphoramidates and oligoribonucleotide phosphorothioates and their 2'-O-allyl analogs and 2'-O-methylribonucleotide methylphosphonates, which can be used in the nucleotide sequences of the present disclosure.

[0326] In some embodiments, the protein or nucleic acid is isolated. As used herein, the term "isolated" means artificially produced. As used herein with respect to nucleic acids, the term "isolated" means: (i) amplified in vitro by, for example, polymerase chain reaction (PCR); (ii) produced recombinantly by cloning; (iii) purified by, for example, cleavage and gel separation; or (iv) synthesized by, for example, chemical synthesis. An isolated nucleic acid is a nucleic acid that can be easily manipulated by recombinant DNA techniques well known in the art. Thus, a nucleotide sequence containing known 5' and 3' restriction sites or disclosed polymerase chain reaction (PCR) primer sequences in a vector is considered isolated, but a nucleic acid sequence that exists in its natural state in its natural host is not. An isolated nucleic acid can be substantially purified, but not necessarily. For example, a nucleic acid isolated within a cloning or expression vector is not pure because it may only account for a very small portion of the material in the cell in which it is located. However, such nucleic acids are isolated because the term is used herein because they can be easily manipulated by standard techniques known to those of ordinary skill in the art. As used herein with respect to proteins or peptides, the term "isolated" refers to proteins or peptides that have been separated from their natural environment or artificially produced (e.g., by chemical synthesis, by recombinant DNA technology, etc.). In some embodiments, any one or more thymidine (T) nucleotides or uridine (U) nucleotides in the sequences provided herein can be replaced with any other nucleotides that are suitable for base pairing with adenosine nucleotides (e.g., via Watson-Crick base pairs). For example, T can be replaced with U, and U can be replaced with T.

[0327] "Heterologous" means derived from an entity that is genotypically different from the remainder of the entity to which it is being compared or into which it is introduced or incorporated. For example, a polynucleotide introduced into a different cell type by genetic engineering techniques is a heterologous polynucleotide (and when expressed, may encode a heterologous polypeptide). Similarly, a cellular sequence (e.g., a gene or portion thereof) incorporated into a viral vector is a heterologous nucleotide sequence relative to the vector.

[0328] The term "transgenic" refers to a heterologous polynucleotide that is introduced into a cell and can be transcribed into RNA and optionally translated and / or expressed under appropriate conditions. In various aspects, it imparts the desired properties to the cell into which it is introduced, or otherwise produces a desired therapeutic or diagnostic result. On the other hand, it can be transcribed into a molecule that mediates RNA interference, such as miRNA, siRNA or shRNA.

[0329] As used herein, the term "recombinant" refers to a vector, polynucleotide (e.g., recombinant nucleic acid), polypeptide or cell that is the product of various combinations of cloning, restriction or ligation steps (e.g., associated with the polynucleotides or polypeptides contained therein) and / or other procedures that produce constructs that are different from those found in nature. A recombinant virus or vector (e.g., an rAAV vector) comprises a vector genome that comprises a recombinant nucleic acid (e.g., a nucleic acid comprising a transgene and one or more regulatory elements). The terms include replicas of the original polynucleotide construct and progeny of the original viral construct, respectively.

[0330] As used herein, the term "operably linked" refers to nucleic acid sequence (or polypeptide) elements being linked in a functional relationship. When a nucleic acid is in a functional relationship with another nucleic acid sequence, the nucleic acid is operably linked. For example, if a promoter or other transcriptional regulatory sequence (e.g., enhancer) affects the transcription of a coding sequence, it is operably linked to the coding sequence. In some embodiments, operably linked means that the linked nucleic acid sequences are continuous. In some embodiments, operably linked does not mean that the nucleic acid sequences are continuously linked, but that there are intervening sequences between those linked nucleic acid sequences.

[0331] "Recombinant AAV vector (rAAV vector)" refers to a polynucleotide vector comprising one or more heterologous sequences (i.e., nucleic acid sequences of non-AAV origin) flanked by at least one (and in the embodiment two) AAV reverse terminal repeat sequences. Such rAAV vectors can be replicated and packaged into infectious virions when present in a host cell that has been infected with a suitable helper virus (or expresses a suitable helper function) and expresses AAV rep and cap gene products (i.e., AAV Rep and Cap proteins). When the rAAV vector is incorporated into a larger polynucleotide (e.g., in a chromosome or another vector, such as a plasmid for cloning or transfection), the rAAV vector may be referred to as a "pro-vector", which can be "rescued" by replication and capsid wrapping in the presence of AAV packaging functions and suitable helper functions. The rAAV vector may be any of a variety of forms, including but not limited to plasmids, linear artificial chromosomes, complexed with lipids, encapsulated in liposomes, and encapsulated in viral particles (particularly AAV particles). rAAV vectors can be packaged into AAV viral capsids to produce "recombinant adeno-associated virus particles (rAAV particles)".

[0332] As used herein, the term "vector" refers to a plasmid, a virus (e.g., rAAV), a cosmid, or other vehicles that can be manipulated by inserting or incorporating a nucleic acid (e.g., a recombinant nucleic acid). Vectors can be used for various purposes, including, for example, genetic manipulation (e.g., a cloning vector), introducing / transferring nucleic acids into cells, transcribing or translating nucleic acids inserted into cells. In some embodiments, the vector nucleic acid sequence contains at least a replication origin for propagation in cells. In some embodiments, the vector nucleic acid includes a heterologous nucleic acid sequence, one or more expression control elements (e.g., a promoter, an enhancer), a selectable marker (e.g., antibiotic resistance), a polyadenylic acid (polyA) sequence, and / or an ITR. In some embodiments, when delivered to a host cell, the nucleic acid sequence is propagated. In some embodiments, when delivered to a host cell in vitro or in vivo, the cell expresses a polypeptide encoded by a heterologous nucleic acid sequence. In some embodiments, when delivered to a host cell, a nucleic acid sequence or a portion of a nucleic acid sequence is packaged into a capsid. The host cell can be a separated cell or a cell in a host organism. In addition to the nucleic acid sequence encoding RNA or polypeptide or protein (e.g., transgenic), additional sequences (e.g., regulatory sequences) may be present in the same vector (i.e., connected in cis with the gene) and located on both sides of the gene. In certain embodiments, regulatory sequences may be present on a separate (e.g., second) vector that acts in trans to regulate the expression of the gene. Plasmid vectors may be referred to as "expression vectors" in this article.

[0333] As used herein, the term "vector genome" refers to a recombinant nucleic acid sequence that is packaged or wrapped to form a rAAV vector. Typically, the vector genome includes heterologous polynucleotide sequences that are not initially present in the capsid, such as transgenes, regulatory elements, ITRs. In the case of using a recombinant plasmid to construct or manufacture a recombinant vector (such as an rAAV vector), the vector genome does not include the entire plasmid, but only includes sequences intended to be delivered by a viral vector. This non-vector genome portion of a recombinant plasmid is commonly referred to as a "plasmid backbone", which is very important for cloning, selection and amplification of a plasmid (i.e., the process required for the production of recombinant viral vectors), but it itself is not packaged or wrapped in a rAAV vector.

[0334] As used herein, the term "viral vector" generally refers to a viral particle used as a nucleic acid delivery vehicle and comprising a vector genome (e.g., comprising a transgene other than nucleic acids encoding AAV rep and cap) packaged within a virion (i.e., capsid), and includes, for example, lentiviruses and parvoviruses, including AAV serotypes and variants (e.g., rAAV vectors). Recombinant viral vectors do not comprise a vector genome containing rep and / or cap genes.

[0335] As used herein, "miRNA scaffold" may refer to a polynucleotide containing (i) a double-stranded sequence targeting a gene of interest for gene knockdown by RNAi, and (ii) an additional sequence forming a stem-loop structure similar to the stem-loop structure of an endogenous miRNA. When the polynucleotide is assembled into a miRNA-like secondary structure, the sequence of the gene of interest targeting RNAi (e.g., a short sequence of about 20nt) can be connected to the sequence that produces the miRNA-like stem-loop and the sequence that base pairs with the sequence of interest to form a duplex. As described herein, this duplex may not be completely hybridized, for example, it may contain one or more unpaired or mismatched bases. After this polynucleotide is cut by Dicer, this duplex containing the sequence targeting the gene of interest can be unwound and incorporated into the RISC complex. The miRNA scaffold may refer to the miRNA itself or a DNA polynucleotide encoding the miRNA. An example of a miRNA scaffold is the miR-155 sequence (Lagos-Quintana, M. et al. (2002) Curr. Biol. 12:735-9). Commercially available kits for cloning sequences into miRNA scaffolds are known in the art (e.g., INVITROGEN BLOCK-IT Pol II miR RNAi Expression Vector Kit from Life Technologies, Thermo Fisher Scientific, Waltham, Mass.).

[0336] Functional variants or equivalents of reference peptides, polypeptides or proteins refer to polypeptide derivatives of reference peptides, polypeptides or proteins, such as proteins, fusion proteins or combinations thereof with one or more point mutations, insertions, deletions, truncations. It substantially retains the activity of the reference peptide, polypeptide or protein. Typically, functional equivalents are at least 60% (e.g., any number between 60% and 100%, including, for example, 60%, 70%, 80%, 85%, 90%, 95% and 99%) consistent with the reference peptide, polypeptide or protein. In certain embodiments, point mutations can be conservative modifications.

[0337] As used herein, the term "conservative modification" refers to an amino acid modification that does not significantly affect or change the biological characteristics of a polypeptide or protein. Such conservative modifications include amino acid substitutions, additions, and deletions. Modifications can be introduced into a polypeptide or protein by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are amino acid substitutions in which an amino acid residue is replaced by an amino acid residue with a similar side chain. Families of amino acid residues with similar side chains have been defined in the art. These families include: amino acids with basic side chains (e.g., lysine, arginine, histidine); amino acids with acidic side chains (e.g., aspartic acid, glutamic acid); amino acids with uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan); amino acids with nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine); amino acids with beta-branched side chains (e.g., threonine, valine, isoleucine); and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Non-conservative substitutions will require exchanging a member of one of these classes for a member of another class.

[0338] As used herein, the terms "treat," "treating," or "treatment" refer to the administration of a therapy that partially or completely relieves, ameliorates, alleviates, suppresses, delays the onset of, reduces the severity of, and / or reduces the incidence of one or more symptoms, features, and / or causes of a particular disease, disorder, and / or condition.

[0339] As used herein, the term "improvement" means a detectable or measurable improvement in a disease, disorder or condition, or a symptom or underlying cellular response of a subject. A detectable or measurable improvement includes the occurrence, frequency, severity, progression or duration of a disease, disorder or condition, a subjective or objective reduction, decrease, inhibition, containment, limitation or control of complications caused by or associated with a disease, disorder or condition, an improvement in symptoms of a disease, disorder or condition, or a reversal of a disease, disorder or condition.

[0340] As used herein, the term "associated with" means that one is related to another if the presence, level, and / or form of one is associated with the presence, level, and / or form of another. For example, a particular entity (e.g., a polypeptide, a genetic signature, a metabolite, a microorganism, etc.) is considered to be associated with a disease, disorder, or condition if the presence, level, and / or form of the particular entity (e.g., a polypeptide, a genetic signature, a metabolite, a microorganism, etc.) is associated with the incidence of and / or susceptibility to a particular disease, disorder, or condition (e.g., within a related population).

[0341] As used herein, the term "prevent" or "prevention" refers to delaying the onset of one or more signs or symptoms of a particular disease, disorder or condition (e.g., myelin disease), and / or reducing its frequency and / or severity. In some embodiments, prevention is assessed on a population basis, such that if a statistically significant reduction in the development, frequency and / or intensity of one or more signs or symptoms of the disease, disorder or condition is observed in a population susceptible to a particular disease, disorder or condition, it is considered that the agent can "prevent" the disease, disorder or condition. When the onset of a disease, disorder or condition has been delayed for a predetermined time period, prevention can be considered complete.

[0342] As used herein, the term "subject" refers to an organism, such as a mammal (e.g., a human, a non-human mammal, a non-human primate, a primate, a laboratory animal, a mouse, a rat, a hamster, a gerbil, a cat, a dog). In some embodiments, the subject is a non-human disease model. In some embodiments, the human subject is an adult, a teenager, or a child subject. In some embodiments, the subject suffers from a disease, disorder, or condition, such as a disease, disorder, or condition that can be treated as provided herein. In some embodiments, the subject suffers from a disease, disorder, or condition associated with myelin deficiency or dysfunction. In some embodiments, the subject is susceptible to a disease, disorder, or condition. In some embodiments, susceptible subjects are susceptible to a disease, disorder, or condition, and / or show an increased risk of suffering from a disease, disorder, or condition (compared to the average risk observed in a reference subject or population). In some embodiments, the subject exhibits one or more symptoms of a disease, disorder, or condition. In some embodiments, the subject does not exhibit specific symptoms (e.g., clinical manifestations of the disease) or features of a disease, disorder, or condition. In some embodiments, the subject does not exhibit any symptoms or features of a disease, disorder, or condition. In some embodiments, the subject is a human patient. In some embodiments, the subject is an individual to whom diagnosis and / or therapy is being and / or has been administered.

[0343] As used herein, the term "therapeutically effective amount" refers to an amount administered to produce a desired therapeutic effect. In some embodiments, the term refers to an amount sufficient to treat the disease, disorder or condition when administered to a population suffering from or susceptible to a disease, disorder or condition according to a therapeutic dosing regimen. In some embodiments, a therapeutically effective amount is an amount that reduces the incidence and / or severity of one or more symptoms of a disease, disorder and / or condition, and / or delays its onset. One of ordinary skill in the art will appreciate that the term "therapeutically effective amount" does not actually require successful treatment to be achieved in a particular individual. On the contrary, a therapeutically effective amount can be an amount that provides a specific desired pharmacological response in a large number of subjects when administered to a patient in need of such treatment.

[0344] Examples

[0345] Example 1. Construction of AAV expression cassette

[0346] The 774 base pair coding sequence of human proteolipid protein 1 (PLP1) transcript variant 1 mRNA (NCBI Reference Sequence: NM_000533.5; SEQ ID NO: 1) was obtained from the National Center for Biotechnology Information (NCBI) ncbi.nlm.nih.gov / refseq / .

[0347] Use a non-commercial web-based algorithm-based design tool to identify target regions in the coding sequence of the gene of interest (see Figure 1 , SEQ ID NO:35 sequence in red). These design tools include the Designer of Small Interfering RNA (DSIR) (Vert et al., 2006) and the Genetic Perturbation Platform provided by the Broad Institute. The design tools were used to generate a target sequence of 19 to 21 nucleotides in the human PLP1 coding sequence. A nucleotide sequence that is completely complementary to the target sequence was determined (see Fig. 9 , SEQ ID NO:75 sequence in blue, italic and underlined, 3' to 5' aligned to show complementarity). In this example, human miR-30a was used as the backbone (see Figure 4 ) to design artificial microRNAs that include 3' and 5' flanking regions (50 to 100 nucleotides) and a loop region. A 20 to 22 nucleotide sequence that is fully complementary to the target sequence is immediately followed by the 5' flanking sequence, followed by a loop sequence from a naturally occurring microRNA, a 20 to 22 nucleotide reverse complement of the guide strand (modified by deleting positions 10 to 11 to create a bulge that allows the guide strand to be preferentially loaded into the RISC complex), and then the 3' flanking sequence ( Fig.10 ).

[0348] Web-based software (mfold) for predicting the secondary structure of single-stranded nucleic acids (Zuker, 2003) was used to predict the sequence fold of endogenous miRNA (miR-30a in this experiment) and the designed sequence (miR-30a / PLP1 with SEQ ID NO: 75). In this case, the designed pre-miRNA has the same secondary structure as the natural sequence (with the same framework regions, but with different guide and strand sequences) and has a similar free energy (dG) as the natural sequence ( Fig.11 The resulting hairpin has a conserved loop region and contains the cleavage site required for Dicer to remove the loop and leave behind a dsRNA duplex.

[0349] Synthesize the designed oligonucleotides and clone them into a plasmid containing the chosen promoter. Select a cell line that is able to express the PLP1 gene. A common choice is human embryonic kidney cells HEK293. Perform transfection and assess gene knockdown.

[0350] Example 2. Additional AAV expression cassettes

[0351] Additional AAV expression cassettes were prepared using other suitable endogenous human miRNA structures as backbones or scaffolds in the same manner as described in Example 1. These human miRNA structures include human mir-16-1 NR_029486 (mbase accession number MI0000070) (Han et al., 2006), human miR-21 NC_000017.11 (Yue et al., 2010) (mbase accession number MI0000077), human miR-23a NR_029495 (mbase accession number MI0000079) (Vandenberg et al., 2016), human miRNA-30a NR_029504 (mbase accession number MI0000088) ( Zeng et al., 2002 ), human miR-31 NR_029505.1 (mbase accession number MI0000089) ( Yili et al., 2008 ), human miR-122 NR_029667 (mbase accession number MI0000442) ( Yili et al., 2008 ), human miR-155 NR_030784NC_000021.9 (mbase accession number MI0000681) and human miR-451 NR_029970 (mbase accession number MI0001729) ( Yuda et al., 2013 ).

[0352] Example 3: miRNA downregulation of PLP1 protein expression

[0353] This example demonstrates that miRNA constructs can reduce the expression of target proteins. In the experiment, the ability of three artificial Plp1-miRNAs to inhibit Plp1 protein expression was evaluated in vitro. As described in Example 1 above, a non-commercial design tool based on a web-based algorithm for miRNA formation was used to identify the target region of the PLP1 coding sequence (SEQ ID NO: 1). Three target sequences and guide strands for targeting PLP1 were selected for miRNA targeting (SEQ ID NO: 14 and 54; SEQ ID NO: 34 and 74; and SEQ ID NO: 35 and 75). These three sets of target sequences and guide strands were incorporated into a miR30-based shRNA gene knockdown vector (constructed as a plasmid in Escherichia coli (E. coli)). Each of the three plasmids contained a polycistronic expression cassette consisting of: a CBh promoter, one of the three miR30-based shRNA sequences targeting human Plp1 mRNA, and a codon-optimized enhanced green fluorescent protein (eGFP) cloned between two inverted terminal repeats (ITRs). The fourth plasmid contains scrambled shRNA (not in human or mouse) and is used as a control. Plasmid 1332 contains a miR30-based shRNA knockdown vector with SEQ ID NOs: 35 and 75, plasmid 1333 contains a miR30-based shRNA knockdown vector with SEQ ID NOs: 14 and 54, and plasmid 1336 contains a miR30-based shRNA knockdown vector with SEQ ID NOs: 34 and 74. Plasmid 1307 contains scrambled shRNA (not targeting any mRNA in human or mouse) and is used as a control.

[0354] The gene knockdown efficiency was evaluated in vitro by co-transfection with PLP1 cDNA expression plasmid vector in L cells using Lipofectamine300. After 72 hours, the cell lysate was subjected to Western blot analysis to evaluate the expression of PLP1 protein (using α-tubulin as a control). 10 μg of protein per lane was electrophoresed on SDS-polyacrylamide gel and transferred to nitrocellulose membrane. Western blot analysis was performed using standard techniques known to those skilled in the art. Briefly, the blocked membrane was incubated overnight with rat anti-PLP AA3 (1:250 dilution) and then incubated with anti-rat horseradish peroxidase-coupled secondary antibody (1:1000 dilution). The blot was washed and immunodetected.

[0355] The results of western blot analysis (PLP protein has two bands, one is a monomer, approximately 22 kDa, and the other is a dimer, approximately 44 kDa) showed that at 72 hours, plasmid 1332 (containing a miR30-based shRNA gene knockdown vector with SEQ ID NOs: 35 and 75) and plasmid 1333 (containing a miR30-based shRNA gene knockdown vector with SEQ ID NOs: 14 and 54) achieved a significant reduction in PLP1 expression, while plasmid 1336 (containing a miR30-based shRNA gene knockdown vector with SEQ ID NOs: 34 and 74) did not achieve a significant reduction in PLP1 expression.

[0356] Morphometric analysis was performed using data obtained from 3 replicates to show fold changes, where two PLP1 bands of 22 kD and 44 kD were combined and normalized by eGFP from the blot. The results quantitatively showed that artificial PLP1 miRNA inhibited PLP1 protein expression in vitro. Specifically, plasmids 1332 and 1333 containing miR30-based shRNA knockdown vectors (having SEQ ID NOs: 35 and 75 and SEQ ID NOs: 14 and 54, respectively) achieved a significant reduction in PLP1 expression, while plasmid 1336 containing miR30-based shRNA knockdown vectors (having SEQ ID NOs: 34 and 74) did not achieve a significant reduction in PLP1 expression.

[0357] One-way ANOVA followed by Tukey's multiple comparison test was used to determine statistical significance. Plasmid 1332 and plasmid 1333 both resulted in more than 50% reduction compared to plasmid 1307 (P < 0.01) and plasmid 1336 (P < 0.001). Fig.12 The greatest reduction was achieved with plasmid 1333 (SEQ ID NOs: 14 and 54 shown in FIG. 14 ).

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[0389] The description of the foregoing examples and preferred embodiments should be considered as illustrating rather than limiting the present disclosure as defined by the claims. As will be readily appreciated, many variations and combinations of the above features may be utilized without departing from the present disclosure as set forth in the claims. These variations are not considered to be out of the scope of the present disclosure, and all such variations are intended to be included within the scope of the appended claims. All references cited herein are incorporated by reference in their entirety.

Claims

1. A method for reducing the expression of a target gene in oligodendrocytes or treating a hereditary or acquired myelin disorder, the method comprising: An adeno-associated virus (AAV) particle having a preferential tropism for the surface of oligodendrocytes is provided; wherein the AAV particle encapsidates a nucleic acid, the nucleic acid comprising from 5' to 3': 5′ inverted terminal repeat (ITR), The promoter sequence region, A polynucleotide encoding a pri-miRNA or pre-miRNA targeting the target gene, and a 3′ITR, and The AAV particles are contacted with the oligodendrocytes. 2 . The method of claim 1 , wherein the nucleic acid further comprises one or more of a post-transcriptional regulatory element and a polyA signal sequence region between the polynucleotide and the 3′ ITR.

3. The method according to claim 1, wherein the pri-miRNA or pre-miRNA comprises (a) pri-miRNA or pre-miRNA scaffold, (b) a heterologous guide strand, and (c) Heterologous passenger chain.

4. The method of claim 3, wherein the pri-miRNA or pre-miRNA scaffold is a human pri-miRNA or pre-miRNA scaffold derived from human miRNA.

5. The method according to claim 4, wherein the human microRNA is derived from human mir-16-1, miR-21, miR-23a, miRNA-30a, miR-31, miR-122, miR-155 or miR-451.

6. The method of claim 5, wherein the human pri-miRNA or pre-miRNA scaffold does not include the native sequence of the native guide strand and the native sequence of the native passenger strand of the human miRNA.

7. The method of claim 6, wherein the heterologous guide strand is inserted into the human pri-miRNA or pre-miRNA scaffold as a replacement for the native sequence of the native guide strand.

8. The method of any one of claims 3 to 7, wherein the heterologous guide strand is complementary to the mRNA of the target gene.

9. The method of claim 8, wherein after processing of the pri-miRNA or pre-miRNA by a cytoplasmic nuclease, the heterologous guide strand is incorporated into a RISC complex to allow the RISC complex to target the mRNA of the target gene and downregulate the expression of the target gene.

10. The method of any one of claims 3 to 9, wherein the heterologous guide strand is complementary to an mRNA for a protein, the elimination of which improves the treatment outcome of the inherited or acquired myelin disorder.

11. The method of claim 10, wherein the inherited or acquired myelin disorder is Pelitzaus-Merzbacher disease.

12. The method of claim 11, wherein the protein is PLP1.

13. The method of claim 12, wherein the heterologous guide strand comprises a nucleotide sequence having at least 90% identity to one of SEQ ID NOs: 42 to 80.

14. The method of claim 10, wherein the inherited or acquired myelin disorder is multiple system atrophy.

15. The method of claim 14, wherein the targeted protein is alpha-synuclein.

16. The method of claim 15, wherein the heterologous guide strand comprises a nucleotide sequence having at least 90% identity to one of SEQ ID NOs: 122 to 161.

17. The method of claim 10, wherein the inherited or acquired myelin disorder is hypomyelination with basal ganglia and cerebellar atrophy (H-ABC).

18. The method of claim 17, wherein the targeted protein is the microtubule-associated protein tubulin beta-4a.

19. The method of claim 18, wherein the heterologous guide strand comprises a nucleotide sequence having at least 90% identity to one of SEQ ID NOs: 204 to 244.

20. The method according to any one of claims 13, 16 and 19, wherein the nucleotide sequence is 21 to 30 nucleotides in length.

21. An RNA molecule comprising a first RNA sequence and a second RNA sequence, wherein the first RNA sequence is substantially complementary to the second RNA sequence, wherein the first RNA sequence has a sequence length of at least 19 nucleotides and is at least 90% complementary to a sequence selected from the group consisting of: SEQ ID NOs: 2-40, 82-121 and 163-203.

22. The RNA molecule of claim 21, wherein the RNA molecule is contained in a pre-miRNA scaffold or a pri-miRNA scaffold.

23. The RNA molecule of claim 22, wherein the pri-miRNA or pre-miRNA scaffold is a human pri-miRNA or pre-miRNA scaffold derived from human microRNA.

24. The RNA molecule of claim 23, wherein the human microRNA is human mir-16-1, miR-21, miR-23a, miRNA-30a, miR-31, miR-122, miR-155 or miR-451.

25. The RNA molecule of claim 24, wherein the human pri-miRNA or pre-miRNA scaffold does not include the native sequence of the native guide strand and the native sequence of the native passenger strand of the human microRNA.

26. The RNA molecule of claim 25, wherein the first strand is a heterologous guide strand that is inserted into the human pri-miRNA or pre-miRNA scaffold as a replacement for the native sequence of the native guide strand.

27. A polynucleotide encoding the RNA molecule according to claims 21 to 26.

28. An expression cassette or expression vector comprising the polynucleotide according to claim 27.

29. The expression cassette or expression vector according to claim 28, further comprising from 5' to 3' one or more of the following: 5′ inverted terminal repeat (ITR), The promoter sequence region, Post-transcriptional regulatory elements, The polyA signal sequence region, and 3′ITR.

30. The expression vector of claim 29, wherein the expression vector is a viral vector.

31. The expression vector of claim 30, wherein the viral vector is an AAV vector.

32. The expression vector of claim 31, wherein the AAV vector has a preferential tropism for oligodendrocytes.

33. The expression vector of claim 32, wherein the AAV vector is AAV-Olig001, AAV-Olig002, AAV-Olig003 or AAV9.

34. A host cell comprising the polynucleotide of claim 27 or the expression cassette or expression vector of any one of claims 28 to 33.

35. A pharmaceutical composition comprising The RNA molecule according to claim 21, or the polynucleotide according to claim 27, or the expression cassette or expression vector according to any one of claims 28 to 33, or the host cell according to claim 34, and A pharmaceutically acceptable carrier.

36. A method of treating an inherited or acquired myelin disorder comprising administering to a subject in need thereof an RNA molecule according to claim 21, or a polynucleotide according to claim 27, or an expression cassette or expression vector according to any one of claims 28 to 33, or a host cell according to claim 34, or a pharmaceutical composition according to claim 35.

37. The method of claim 36, wherein the subject is a human.

38. The method of claims 36 to 37, wherein the administration is via injection.

39. The method of any one of claims 36 to 38, wherein the RNA molecule, or the polynucleotide, or the expression cassette, or the expression vector, or the host cell, or the pharmaceutical composition is administered to a region of the central nervous system selected from the group consisting of the brain parenchyma, the spinal canal, the subarachnoid space, the cerebral ventricles, the cisterna magna, and combinations thereof.

40. The method of any one of claims 36 to 38, wherein the RNA molecule, or the polynucleotide, or the expression cassette, or the expression vector, or the host cell, or the pharmaceutical composition is administered by a method selected from the group consisting of intraparenchymal administration, intrathecal administration, intracerebroventricular administration, intracisternal administration, and combinations thereof.

41. The method of claim 10, wherein the disorder is Alexander disease, Mitchell disease, autosomal dominant leukodystrophy with autonomic disease (ADLD), central dysmyelinating leukodystrophy, Waardenburg syndrome, or Hirschsprung disease, adult polyglucan body disease (APBD), hereditary diffuse leukoencephalopathy with spheroids, or Aicardi-Guterres syndrome.

42. The method of claim 10, wherein the disorder is Canavan disease, Krabbe disease, globoid cell leukodystrophy, X-linked adrenoleukodystrophy, metachromatic leukodystrophy, hypomyelinating leukodystrophy-2, Niemann-Pick disease type C, 4H leukodystrophy / Pol III-associated leukodystrophy, Zellweger spectrum disorder, childhood ataxia with central nervous system hypomyelination, encephalotenoid xanthomatosis, SOX10-associated peripheral demyelinating neuropathy, adult Refsum's disease, autism spectrum disorder, Alzheimer's disease, Parkinson's disease, fragile X syndrome, schizophrenia disease, multiple sclerosis, neuromyelitis optica, progressive multifocal leukoencephalopathy, encephalomyelitis, central pontine myelinolysis, adrenoleukodystrophy, Wallerian degeneration, optic neuritis, transverse myelitis, amyotrophic lateral sclerosis, Huntington's disease, spinal cord injury, traumatic brain injury, post-radiation injury, neurologic complications of chemotherapy, stroke, acute ischemic optic neuropathy, vitamin E deficiency, isolated vitamin E deficiency syndrome, Basen-Kortzweig syndrome, Maastricht-Bieder syndrome, metachromatic leukodystrophy, trigeminal neuralgia, acute disseminated encephalitis, Guillain-Barré syndrome, Marie-Charcot-Tooth disease, or Bell's palsy.

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