Compositions and methods useful for treating H-ABC leukodystrophy

Abstract

Compositions and methods for the treatment of H-ABC leukodystrophy are disclosed.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Application No. 63 / 175998, filed April 16, 2021, the entire disclosure of which is specifically incorporated herein by reference.

[0003] (Incorporation by reference of material submitted in electronic form) Incorporated herein by reference in its entirety is a sequence listing submitted via EFS-Web as a text file named SEQLIST.txt, created on April 18, 2022, and having a size of 77,824 bytes.

[0004] (Technical field) The present invention relates to the field of H-ABC leukodystrophy and methods of using small molecule compositions, particularly modified antisense molecules, to ameliorate symptoms thereof. More specifically, the present invention provides modified nucleic acid-based therapeutics that down-modulate Tubulin alpha 4A (TUBB4A), thereby improving dystonia, ataxia, altered gait and motor dysfunction in patients in need thereof. [Background technology]

[0005] Several publications and patent documents are cited throughout this specification to describe the state of the art to which this invention pertains, all of which are incorporated herein by reference in their entirety.

[0006] Hypomyelination with basal ganglia and cerebellar atrophy (also known as H-ABC) is a rare genetic disorder that involves progressive nervous system damage targeting two parts of the brain: the basal ganglia and the cerebellum, which control body movement and movement (Hersheson et al., 2013; Simons et al., 2013). H-ABC is a type of leukodystrophy, a group of disorders that affect the white matter of the brain. As used herein, H-ABC is also intended to refer to a larger group of related disorders, such as TUBB4A-related disorders, with diverse symptoms of dystonia, hypomyelination, and developmental abnormalities (Blumkin et al., 2014; Ferreira et al., 2014; Pizzino et al., 2014). These disorders are associated with impairments in the myelination process, resulting in damage to the myelin sheath that surrounds and protects nerve cells in the brain and spinal cord and speeds up the transmission of messages between cells. Myelination normally occurs during the first few years of life. In H-ABC, the body is unable to produce normal levels of myelin, resulting in hypomyelination, which prevents normal myelination in the brain. In some cases, nerves outside the brain may also be affected. Unfortunately, the size and function of the basal ganglia and cerebellum are also reduced. As a result, H-ABC patients often have movement disorders, including muscle and joint stiffness, reduced muscle tone, difficulty controlling movements, and problems with balance and coordination (Blumkin et al., 2014; Ferreira et al., 2014; Pizzino et al., 2014; Simons et al., 2013).

[0007] H-ABC is caused by a mutation in the TUBB4A gene, which often appears as a random mutation in affected individuals. In these cases, neither parent is a carrier, so the chances of having a child with the condition are extremely low. There is also a small chance that one parent may have the mutation in some cells (mosaicism), which means they will not develop symptoms but can pass the disorder on to other children.

[0008] There is currently no known cure for the disease, and new therapeutic agents are urgently needed to ameliorate the symptoms of H-ABC. Summary of the Invention [Problem to be solved by the invention]

[0009] According to the present invention, a method is provided for reducing TUBB4A levels in target cells, thereby improving symptoms of H-ABC leukodystrophy in a patient in need thereof. An exemplary method includes administering a therapeutically effective amount of a composition comprising a synthetic inhibitory nucleic acid molecule targeting TUBB4A, wherein reducing TUBB4A levels reduces one or more of the following symptoms in said patient: i) motor development delay; ii) cognitive impairment; iii) gait impairment; iv) ataxia; v) intention tremor; vi) dysarthria; viii) dysphonia; and viii) abnormal hypomyelination. In a preferred embodiment, the synthetic nucleic acid molecule is selected from antisense oligonucleotides, shRNAs, siRNAs, and guide strands suitable for CRISPR-edited TUBB4A target nucleic acid. Also provided is a method for treating, delaying onset, improving, and / or alleviating diseases, disorders, and / or conditions associated with H-ABC leukodystrophy, or symptoms thereof, in a patient in need thereof. In one aspect, the method comprises administering to a patient a therapeutically effective amount of a synthetic inhibitory nucleic acid targeting TUBB4A, wherein a disease, disorder and / or condition associated with H-ABC leukodystrophy, or a symptom thereof, is treated, inhibited, delayed onset, ameliorated, and / or alleviated in the patient. As described above, the synthetic nucleic acid molecule is selected from an antisense oligonucleotide, an shRNA, an siRNA, and a guide strand suitable for CRISPR editing. In certain embodiments, the synthetic inhibitory nucleic acid targeting TUBB4A is listed in Table 3 or Table 5 and is modified to increase stability and / or uptake in vivo. In certain embodiments, the synthetic nucleic acid encoding the antisense oligonucleotide is cloned into a vector. In other embodiments, the leukodystrophy is a disease, disorder and / or condition associated with dysmyelination. In other embodiments, the modified oligonucleotide comprises at least one modified internucleoside linkage, at least one nucleoside of the modified oligonucleotide comprises a modified sugar, or at least one nucleoside of the modified oligonucleotide comprises a modified nucleobase.In another embodiment of the invention, the method further comprises administration of an additional active pharmaceutical agent useful in the treatment of leukodystrophy.

[0010] Also provided is a composition for reducing the expression of TUBB4A, comprising a synthesis inhibitory nucleic acid molecule targeted to and specifically hybridized with a nucleic acid encoding TUBB4A, selected from antisense oligonucleotides, shRNA, siRNA, and guide strands suitable for CRISPR editing, in a biologically acceptable carrier. In certain embodiments, the synthesis inhibitory nucleic acid is modified to increase stability in body fluids and / or uptake into cells of interest. In a preferred embodiment, the synthesis inhibitory nucleic acid is listed in Table 3 or Table 5. The nucleic acid is optionally cloned into a vector. Vectors include plasmid vectors, AAV vectors, adenovirus-associated vectors, and the like. In certain embodiments, the oligonucleotide is administered in a lipid nanoparticle complex. The synthesis inhibitory nucleic acid and or vector or carrier comprising it may be present in a pharma- ceutically acceptable carrier, which are formulated via routes of administration, including but not limited to ex vivo cell administration, intracranial administration, parenteral administration, intramuscular administration, and intravenous administration. In a preferred embodiment, the composition is formulated for a single intracerebroventricular (ICV) bolus injection. Kits containing parts suitable for carrying out any of the aforementioned methods are also provided. [Means for solving the problem]

[0011] [1] A method for reducing TUBB4A levels in a target cell, thereby ameliorating a symptom of H-ABC leukodystrophy in a patient in need thereof, comprising administering a therapeutically effective amount of a composition comprising a synthesis-inhibitory nucleic acid molecule targeted to TUBB4A, thereby reducing TUBB4A levels in said cell, and in said patient: i) Motor development delay; ii) cognitive impairment; iii) gait dysfunction; iv) ataxia; v) intention tremor; vi) dysarthria; vii) Difficulty speaking viii) abnormal hypomyelination; Decrease one or more of the following: The method, wherein the synthetic nucleic acid molecule is selected from an antisense oligonucleotide, an shRNA, an siRNA, and a guide strand suitable for the CRISPR-edited TUBB4A target nucleic acid. [2] A method for treating, delaying onset, ameliorating, and / or alleviating a disease, disorder and / or condition associated with H-ABC leukodystrophy, or a symptom thereof, in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of a synthetic nucleic acid targeted to TUBB4A, wherein the disease, disorder and / or condition associated with H-ABC leukodystrophy, or a symptom thereof, is treated, inhibited, delayed onset, ameliorated, and / or alleviated in the patient, and wherein the synthetic nucleic acid molecule is selected from an antisense oligonucleotide, an shRNA, an siRNA, and a guide strand suitable for CRISPR editing. [3] The method of [1] or [2], wherein the synthetic nucleic acid targeting TUBB4A is modified to increase stability and / or uptake in vivo. [4] The method according to [1] or [2], wherein the synthetic nucleic acid targeting TUBB4A is a modified antisense oligonucleotide. [5] The method according to any one of [1] to [4], wherein the synthetic nucleic acid encoding the antisense oligonucleotide is cloned into a vector. [6] The method according to any one of [1] to [5], wherein the vector is selected from a plasmid vector, a lentivirus vector, a retrovirus vector, an AAV vector, and an adenovirus-associated vector. [7] The method according to any of [1] to [6], wherein the disease, disorder and / or condition is associated with dysmyelination. [8] The method according to [4], wherein the synthetic nucleic acid is an antisense oligonucleotide provided in Table 3 or Table 5. [9] The method of any of the preceding claims, wherein the nucleic acid is modified to have a nucleobase sequence that is at least 90%, at least 95%, at least 99%, or 100% complementary to a portion of a human TUBB4A nucleic acid.

[10] The method of [9], wherein the modified oligonucleotide comprises at least one modified internucleoside linkage, at least one nucleoside of the modified oligonucleotide comprises a modified sugar, or at least one nucleoside of the modified oligonucleotide comprises a modified nucleobase.

[11] The method of any of the preceding claims, further comprising an additional active pharmaceutical agent useful for the treatment of leukodystrophy.

[12] A composition for reducing expression of TUBB4A comprising a synthetic nucleic acid molecule selected from antisense oligonucleotides, shRNA, siRNA, and guide strands suitable for CRISPR editing, in a biologically acceptable carrier, that targets and specifically hybridizes to a nucleic acid encoding TUBB4A.

[13] The composition of

[12] , wherein the synthetic nucleic acid is modified to increase stability in body fluids and / or uptake into target cells.

[14] The composition of any one of the preceding claims, wherein the synthetic nucleic acid is listed in Table 3 or Table 5.

[15] The composition according to any one of

[12] to

[14] , wherein the synthetic nucleic acid molecule is present in a vector.

[16] The composition according to any one of

[12] to

[15] , formulated for ex vivo cell administration, intracranial administration, parenteral administration, and intravenous administration.

[17] The composition according to any one of

[12] to

[15] , wherein the composition is formulated for a single intracerebroventricular (ICV) bolus injection.

[18] A kit for carrying out the method according to any one of [1] to

[11] .

[19] The composition of any one of the preceding claims, wherein the synthetic nucleic acid is ASO H14 or ASO H15. [Brief description of the drawings]

[0012] [Figure 1A-1D]The library of ASOs was screened in triplicate using human A549N cells by electroporation. After electroporation of ASOs at different concentrations (0.1 μM, 0.5 μM, 1 μM, 5 μM), RNA was extracted and subjected to qPCR to evaluate the expression level of TUBB4A. TUBB4A expression was normalized to GAPDH (n=3, ***p<0.0001). Figure 1A shows TUBB4A mRNA expression of non-targeting control (NTC) (cells treated with scrambled oligos) and ASOs H1-H5. Figure 1B shows TUBB4A mRNA expression of ASOs H6-H12. Figure 1C shows TUBB4A mRNA expression of ASOs H13-H19. Figure 1D shows TUBB4A mRNA expression in NTC, ASO H2A, ASO H2B, ASO H10A, ASO H10B, ASO H11A, ASO H11B, ASO H15A, and ASO H15B. [Figures 2A-2E] The library of ASOs was screened in triplicate by electroporation using mouse HT-22 cell line. After electroporation of ASOs at different concentrations (0.1 μM, 0.5 μM, 1 μM, 5 μM), RNA was extracted and subjected to qPCR to evaluate TUBB4A expression. TUBB4A expression was normalized to sfrs9 (n=3, ***p<0.0001). Figure 2A shows TUBB4a mRNA expression of ASOs 1-7. Figure 2B shows TUBB4a mRNA expression of ASOs 8-14. Figure 2C shows TUBB4a RNA expression of ASOs H15-H21. Figure 2D shows TUBB4a mRNA expression of ASOs 4A, ASO 4B, ASO 6A, ASO 6B, ASO 7A, and ASO 7B. FIG. 2E shows TUBB4A mRNA expression of ASO 22-24 and NTC ASO1-ASO3. [Figure 3A-3D] Efficient suppression of TUBB4A transcription by ASOs. RT-qPCR data showing the levels of TUBB4A transcripts in the cerebral cortex, cerebellum, and striatum of wild-type mice injected with ASO7A (Figure 3A), ASO7B (Figure 3B), ASO6A (Figure 3C), and ASO6B (Figure 3D) 10 days after injection. [Figure 4]SHYSY5Y cells were plated at 250,000 cells / well and treated with 10 μm Biospring and Microsynth ASOs for 96 h. RNA was extracted and qRT-PCR was performed to examine the levels of TUBB4A. [Diagram 5] SHYSY5Y cells were plated at 250,000 cells / well and treated with 10 μm Biospring and Microsynth ASO for 96 h. To examine the levels of TUBB4A, RNA was extracted and Biospring was performed. [Figure 6A-6C] SHYSY5Y cells were plated at 10,000 cells per well and treated with 10 μM Biospring ASO for 24 h. Digitonin and Staurosporine were used as positive controls to test for viability (Figure 6A), cytotoxicity (Figure 6B), and apoptosis (Figure 6C). [Figure 7A-7C] SHYSY5Y cells were plated at 10,000 cells per well and treated with 10 μM Biospring ASO for 48, 72, and 96 h. Digitonin and Staurosporine were used as positive controls to test for viability (Figure 7A), cytotoxicity (Figure 7B), and apoptosis (Figure 7C). [Figure 8] Selection of potential ASO candidates in control human iPSC-derived neurons using Gymnocys. Human iPSC-derived neurons were treated on day 37 and replenished 4 days later for a total of 1 week of treatment. RNA was extracted and TUBB4A expression was measured using qRT-PCR. [Figure 9A-9C] Toxicity of ASOs. Human iPSC-derived neurons were treated on day 37 and replenished 4 days later for a total of 1 week of treatment. To assess potential toxicity of ASOs, RNA was extracted and the expression of BAX (Figure 9A) and BCL-2 (Figure 9B) was measured using qRT-PCR. A BAX / BCL-2 ratio of 1 or greater indicates high apoptosis, whereas a ratio of less than 1 indicates low apoptosis (Figure 9C). [Figure 10]Dose response of potential ASO candidates against control human iPSC-derived neurons with Gymnocis. Human iPSC-derived neurons were treated on day 1 and replenished after day 4 for a total treatment of 1 week. RNA was extracted and TUBB4A expression was measured by qRT-PCR. [Figures 11A-11C] Toxicity of ASOs. Human iPSC-derived neurons were treated on day 37 and replenished 4 days later for a total of 1 week of treatment. To assess potential toxicity of ASOs, RNA was extracted and the expression of BAX (Figure 11A) and BCL-2 (Figure 11B) was measured using qRT-PCR. A BAX / BCL-2 ratio of 1 or greater indicates high apoptosis, whereas a ratio of less than 1 indicates low apoptosis (Figure 11C). [Figure 12] ASO candidate H15 was dissolved in Tris-EDTA buffer and assessed for downregulation in mutant human iPSC-derived neurons (TUBB4AD249N) using Gymnosys. To confirm previous downregulation results, wild-type medium spiny neurons (MSNs) were re-run: human iPSC-derived medium neurons were treated on d37 and re-fed on d4, for a total of 1 week of treatment. RNA was extracted and qRT-PCR was used to measure the expression of TUBB4A. [Figures 13A-13C] ASO toxicity in iPSC-derived human mutant MSNs. Human iPSC-derived medium spiny neurons were treated on day 37 and re-fed 4 days later. To assess potential toxicity of ASOs, RNA was extracted and the expression of BAX (Figure 13A) and BCL-2 (Figure 13B) was measured using qRT-PCR. A BAX / BCL-2 ratio of 1 or higher indicates high apoptosis, whereas a ratio of <1 indicates low apoptosis (Figure 13C). [Figure 14A-14B] Mouse primary cortical neurons were plated at 200K cells / well and treated with Biospring ASOs at 1 μm and 5 μm for 1 week. RNA was extracted and qRT-PCR was performed to examine the levels of TUBB4A. Figure 14A shows the downregulation of TUBB4A ASOs from ASO6-1 to ASO7-4. Figure 14B shows the downregulation of TUBB4A ASOs from ASO 8-1 to ASO 18-3. [Figure 15] Mouse primary cortical neurons were plated at 150K cells / well and treated with 1 and 5 μm Biospring ASOs for 1 week. RNA was extracted and qRT-PCR was performed to examine TUBB4A levels. [Figure 16A-16B] Mouse cortical neurons were plated at 20,000 cells / well and treated with 5 μM Biospring ASO for 96 h. Viability (Figure 16A) and apoptosis (Figure 16B) were examined using Digitonin and Staurosporine as positive controls. [Figures 17A-17C] To assess the motor outcomes following ASO injection, wild-type mice were subjected to the motor behaviors rotarod (Figure 17A) and grip strength (Figures 17B and 17C) 30 days after ASO ICV injection. [Figure 18A-18B] ASO7-2 (Figure 18A) and 7-3 (Figure 18B) were injected ICV at the age of P60 (adult) and tissues were harvested 23-30 days later. RNA was extracted and TUBB4A downregulation was measured using qRT-PCR. [Figure 19A-19B] ASO8-2 (Figure 19A) and 8-3 (Figure 19B) were injected ICV at the age of P60 (adult), and 23-30 days later tissues were harvested, RNA was extracted, and downregulation of TUBB4A was determined using qRT-PCR. [Figures 20A-20C] ASO18-1 (Figure 20A) and 18-3 (Figure 20B) were injected ICV at the age of P60 (adult), and 23-30 days later tissues were harvested, RNA was extracted, and TUBB4A downregulation was determined using qRT-PCR. The body weights of mice after P90, P97, and P104 injections are shown in Figure 20C. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] Hypomyelination with basal ganglia and cerebellar atrophy (H-ABC) is a rare hypomyelinating leukodystrophy associated with causative mutations in tubulin alpha 4 (TUBB4A). For example, p.Asp249Asn (D249N) is a recurrent mutation found in the majority of affected individuals. Monoallelic mutations in TUBB4A can also result in a broader range of neurological disorders, from early-onset encephalopathy to adult-onset dystonia type 4 (whispered dysphonia) (Blumkin et al., 2014; Ferreira et al., 2014; Pizzino et al., 2014; Simons et al., 2013). H-ABC lies within this spectrum, typically beginning in early childhood and characterized by dystonia, ataxia, gait changes, and progressive motor dysfunction leading to loss of walking by the end of the first decade of life. To date, there is no treatment for this progressive and disabling childhood disease. To understand how TUBB4A mutations cause H-ABC and facilitate the development and preclinical testing of therapeutic strategies, our group used a CRISPR-Cas9 approach to characterize heterozygous (TUBB4A D249N ) , or homozygous (TUBB4A D249N / D249N We developed a knock-in mouse model carrying a TUBB4A mutation in

[0014] In a previous study, we investigated the role of a mouse model of classical H-ABC (TUBB4A D249N / D249N ), which showed reduced survival and progressive motor dysfunction with tremors, abnormal gait, and ataxia, recapitulating the phenotypic characteristics of the disease. D249N / D249N Neuropathological evaluation of mice at postnatal days 14, 21, and 40 using immunolabeling and Western blot showed an initial delay in myelination followed by eventual demyelination. Over time, there was a decrease in myelin protein and a dramatic loss of aspartocyclase activity (ASPA)-positive oligodendrocytes (myelin cells of the central nervous system). Electron microscopy of ultrathin brain sections showed hypomyelination and progressive loss of myelin in the spinal cord and optic nerves of these mice. Furthermore, TUBB4A D249N / D249NIn vitro studies of cultured mouse oligodendrocytes showed a reduction in maturation and myelin markers. Similarly, neuropathology showed severe neuronal loss in striatal and cerebellar granule cells. Furthermore, TUBB4A D249N / D249N In mouse cells, effects on neurons in culture were also observed, including decreased neuronal survival and destabilized microtubule dynamics. D249N / D249N The mouse is a novel mouse model of H-ABC and demonstrates the complexity of cellular physiology in the disease, with possible microtubule instability due to TUBB4A mutations, cell-autonomous effects on oligodendrocytes, striatal neurons, and cerebellar granule cells, and a profound neurodevelopmental phenotype.

[0015] We have also developed novel therapeutic antisense oligonucleotides that effectively reduce the expression of TUBB4A, providing a new approach for the treatment of leukodystrophies.

[0016] definition The subject matter may be more readily understood by reference to the following detailed description, which forms a part of this disclosure: It is to be understood that the invention is not limited to the specific products, methods, conditions or parameters described and / or illustrated herein, and that the terminology used herein is for the purpose of describing particular embodiments by way of example, and is not intended to limit the invention as claimed.

[0017] Unless otherwise defined herein, scientific and technical terms used in connection with this application shall have the meanings commonly understood by those of ordinary skill in the art. Thus, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.

[0018] As used above, and throughout this disclosure, the following terms and abbreviations, unless otherwise indicated, shall be understood to have the following meanings:

[0019] In this disclosure, the singular forms "a," "an," and "the" include plural references, and a reference to a particular numerical value includes at least that particular numerical value unless the context clearly indicates otherwise. Thus, for example, a reference to a "compound" is a reference to one or more of such compounds known to those of skill in the art, and equivalents thereof, and so forth. As used herein, the term "plurality" means one or more. When a range of values ​​is expressed, another embodiment includes from one particular value and / or to the other particular value. Similarly, when values ​​are expressed as approximations, it is understood that the particular value forms another embodiment by use of the antecedent "about." All ranges are inclusive and combinable.

[0020] The words "comprising, having, including" indicate that the claim is open and may be read toward unrecited elements.

[0021] The phrase "consisting essentially of" when referring to a particular nucleotide or amino acid means a sequence having the characteristics of a given SEQ ID NO. For example, when used in reference to an amino acid sequence, the phrase includes the sequence itself and molecular modifications that do not affect the functional and novel characteristics of the sequence.

[0022] The phrase "consisting of" indicates that the claim is directed only to the recited elements.

[0023] As used herein, the terms "component," "composition," "compound composition," "compound," "drug," "pharmaceutical active agent," "active agent," "treatment," "therapy," "treatment," or "pharmaceutical product" are used interchangeably herein and refer to a compound or a composition of matter that, when administered to a subject (human or animal), induces a desired pharmacological and / or physiological effect by local and / or systemic action. The terms "drug" and "test compound" refer to a chemical compound, a mixture of chemical compounds, a biological macromolecule, or an extract made from biological materials such as bacteria, plants, fungi, and animal (especially mammalian) cells and tissues. Biological macromolecules include siRNAs, shRNAs, antisense oligonucleotides, peptides, peptide / DNA complexes, and any nucleic acid-based molecule that exhibits the ability to modulate the activity of a TUBB4A-containing nucleic acid or its encoded protein as described herein.

[0024] As used herein, "TUBB4A" refers to a gene encoding a member of the beta tubulin family. Beta tubulin is one of two core protein families (alpha tubulin and beta tubulin) that heterodimerize and assemble to form microtubules. Mutations in this gene cause leukodystrophy-6, autosomal dominant torsion dystonia-4 and H-ABC, now more commonly referred to as TUBB4A-associated leukoencephalopathy. Reference sequences for TUBB4A include, for example, NM_001289123.1, NM_001289127.1, and NM_001289129.1, and are available in GenBank. Alternative splicing results in multiple transcript variants that code for different isoforms. The sequence of the wild-type TUBB4A protein is available in UniProt P04350-TBB4A_human. Several TUBB4A variants with known associations with human disease have been identified and are listed in Table 1 below. Although the present invention focuses on the D249N mutant, the findings are generalizable to other existing TUBB4A mutants.

[0025] [Table 1]

[0026] Table 2 lists the specific amino acid changes associated with various types of TUBB4A-related leukoencephalopathy. [Table 2]

[0027] As used herein, the terms "treatment" or "therapy" (and their different forms) include preventative (e.g., prophylactic), curative, or palliative treatment. As used herein, the term "treating" includes alleviating or relieving at least one side effect or symptom of a condition, disease, or disorder.

[0028] The terms "subject", "individual" and "patient" are used interchangeably herein and refer to an animal, such as a human, to which treatment, including preventive treatment, is provided by the pharmaceutical composition according to the present invention. The term "subject" as used herein refers to humans and non-human animals. The terms "non-human animals" and "non-human mammals" are used interchangeably herein and include all vertebrates, such as non-human primates, (especially higher primates), sheep, dogs, rodents, (e.g. mice or rats), guinea pigs, goats, pigs, cats, rabbits, cows, horses, and non-mammals such as reptiles, amphibians, chickens, turkeys, etc.

[0029] The terms "polynucleotide", "nucleotide", "nucleotide sequence", "nucleic acid", and "oligonucleotide" are used interchangeably. Refers to a polymer of nucleotides of any length, such as deoxyribonucleotides or ribonucleotides, or their analogs. A polynucleotide may contain one or more modified nucleotides, such as methylated nucleotides or nucleotide analogs. If present, modifications to the nucleotide structure can be made before or after assembly of the polymer. The sequence of nucleotides may be interrupted by non-nucleotide components. A polynucleotide can be further modified after conjugation, such as by conjugation with a labeling component.

[0030] The term "wild type" as used herein is a term understood by those of skill in the art and refers to the typical form of an organism, strain, gene or characteristic occurring in nature, as distinguished from mutant or variant forms. As used herein, the term "mutant" should be interpreted to mean exhibiting a property having a pattern that deviates from the wild type or that includes non-naturally occurring components.

[0031] The terms "non-naturally occurring" and "artificial" refer to the alteration of man and are used interchangeably. When referring to a nucleic acid molecule or polypeptide, the term means that the nucleic acid molecule or polypeptide is at least substantially free of at least one other component with which it is naturally associated, as found in nature.

[0032] The term "effective amount" or "therapeutically effective amount" refers to an amount of an agent sufficient to produce a beneficial or desired result. The therapeutically effective amount may vary depending on one or more of the subject and disease state being treated, the subject's weight and age, the severity of the disease state, the method of administration, etc., which can be easily determined by one of ordinary skill in the art. The term also applies to a dose that provides an image for detection by any one of the imaging methods described herein. The specific dosage will vary depending on one or more of the particular agent selected, the administration regimen to be followed, whether or not it is administered in combination with other compounds, the timing of administration, the tissue to be imaged, and the physical delivery system in which the agent is delivered.

[0033] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics and recombinant DNA, which are within the skill of the art. See: Sambrook, Fritsch and Maniatis, MOLECULAR CLONING:A LABORATORY MANUAL, 2nd edition (1989); CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (FMAusubel, et al. eds., (1987)); the series METHODS IN ENZYMOLOGY (Academic Press, Inc.): PCR 2:A PRACTICAL APPROACH (MJMacPherson, BDHames and GR Taylor eds.(1995)), Harlow and Lane, eds.(1988) ANTIBODIES, A LABORATORY MANUAL, and ANIMAL CELL CULTURE (RIFreshney, ed.(1987)).

[0034] In some embodiments, the vector can drive the expression of one or more sequences in mammalian cells using a mammalian expression vector. Examples of mammalian expression vectors include pCDM8 (Seed, 1987.Nature 329:840) and pMT2PC (Kaufman, et al., 1987.EMBO J.6:187-195). When used in mammalian cells, the control functions of the expression vector are usually provided by one or more control elements. For example, commonly used promoters are derived from polyoma, adenovirus 2, cytomegalovirus, simian virus 40, and others disclosed herein and known in the art. For other expression systems suitable for both prokaryotic and eukaryotic cells, see, for example, chapters 16 and 17 of Sambrook, et al., MOLECULAR CLONING:A LABORATORY MANUAL.2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989.

[0035] The term "vector" refers to a single-stranded or double-stranded circular nucleic acid molecule that can infect, transfect, or transform a cell and replicate within or independently of a host cell genome. A circular double-stranded nucleic acid molecule is cut and thereby linearized when treated with a restriction enzyme. The assortment of vectors, restriction enzymes, and knowledge of the nucleotide sequences targeted by the restriction enzymes are readily available to those skilled in the art, and includes any replicon, such as a plasmid, cosmid, bacmid, phage, or virus, to which another genetic sequence or element (either DNA or RNA) can be attached to effect replication of the attached sequence or element. The nucleic acid molecule of the present invention can be inserted into a vector by cutting the vector with a restriction enzyme and ligating the two fragments.

[0036] In some embodiments, the recombinant mammalian expression vector is capable of directing expression of a nucleic acid (e.g., an antisense oligonucleotide) preferentially in a particular cell type (e.g., tissue-specific regulatory elements are used to express the nucleic acid). Tissue-specific regulatory elements are known in the art. Non-limiting examples of suitable tissue-specific promoters include the albumin promoter (liver-specific; Pinkert et al., 1987. Genes Dev. 1:268-277), lymphocyte-specific promoters (Calame and Eaton, 1988. Adv. Immunol. 43:235-275), in particular promoters of T cell receptors (Winoto and Baltimore, 1989. EMBO J. 8:729-733) and immunoglobulins (Baneiji, et al., 1983. Cell 33:729-740; Queen and Baltimore, 1983. Cell 33:741-748), neuron-specific promoters (e.g., the neurofilament promoter; Byrne and Ruddle, 1989. Proc. Natl. Acad. Sci. USA 86:5473-5477), pancreatic specific promoters (Edlund et al., 1985. Science 230:912-916), and mammary gland specific promoters (e.g., whey promoters, US Pat. No. 4,873,316 and European Patent Publication No. 264,166). For example, mouse hox promoters (Kessel and Gruss, 1990. Science 249:374-379) and the alpha-fetoprotein promoter (Campes and Tilghman, 1989. Genes Dev. 3:537-546). Nucleic acids encoding TUBB4A can be codon optimized to obtain high levels of expression.

[0037] Non-viral methods of delivery of nucleic acids include lipofection, nucleofection, microinjection, biolistics, virosomes, liposomes, immunoliposomes, polycation or lipid:nucleic acid conjugates, naked DNA, artificial virions, drug-enhanced uptake of DNA, etc. Lipofection is described, for example, in U.S. Pat. Nos. 5,049,386, 4,946,787; and 4,897,355, and lipofection reagents are commercially available (e.g., Transfectam). TM and Lipofectin TM ). Cationic and neutral lipids suitable for efficient receptor-recognition lipofection of polynucleotides include those of Feigner, WO 91 / 17424; WO 91 / 16024. Administration can be to cells (e.g., in vitro or ex vivo administration) or to target tissues (e.g., in vivo administration).

[0038] Preparation of lipid:nucleic acid complexes, including targeted liposomes such as immunolipid complexes, is well known to those of skill in the art (see, e.g., Crystal, Science 270:404-410 (1995); Banskota et al., Cell 185:250-265 (2022); Blaese et al., Cancer Gene Ther. 2:291-297 (1995); Behr et al., Bioconjugate Chem. 5:382-389 (1994); Remy et al., Bioconjugate Chem. 5:647-654 (1994); Gao et al., Gene Therapy 2:710-722 (1995); Ahmad et al., Cancer Res. 52:4817-4820 (1992); (U.S. Patent Nos. 4,186,183, 4,217,344, 4,235,871, 4,261,975, 4,485,054, 4,501,728, 4,774,085, 4,837,028, and 4,946,787).

[0039] The use of RNA or DNA virus-based systems to deliver nucleic acids takes advantage of highly evolved processes for targeting viruses to specific cells in the body and transporting the viral payload to the nucleus. Viral vectors can be administered directly to patients (in vivo) or used to treat cells in vitro. The modified cells can then be optionally administered to patients (ex vivo). Traditional virus-based systems include retroviral, lentiviral, adenoviral, adeno-associated viral, and herpes simplex viral vectors for gene transfer. Integration into the host genome is possible with retroviral, lentiviral, and adeno-associated viral gene transfer methods, often resulting in long-term expression of the inserted transgene. Furthermore, high transduction efficiencies have been observed in many different cell types and target tissues.

[0040] The tropism of retroviruses can be altered by incorporating foreign envelope proteins, expanding the potential target population of target cells. Lentiviral vectors are retroviral vectors that can transduce or infect non-dividing cells and usually produce high viral titers. Thus, the choice of retroviral gene transfer system will depend on the target tissue. Retroviral vectors consist of cis-acting long terminal repeats with packaging capacity for up to 6-10 kb of foreign sequences. The minimal cis-acting LTRs are sufficient for vector replication and packaging, and the vector is then used to integrate therapeutic genes into target cells and provide permanent transgene expression. Widely used retroviral vectors include those based on murine leukemia virus (MuLV), gibbon ape leukemia virus (GaLV), simian immunodeficiency virus (SIV), human immunodeficiency virus (HIV), and combinations thereof (see, e.g., Buchscher et al., J. Virol. 66:2731-2739 (1992); Johann et al., J. Virol. 66:1635-1640 (1992); Sommnerfelt et al., Virol. 176:58-59 (1990); Wilson et al., J. Virol. 63:2374-2378 (1989); Miller et al., J. Virol. 65:2220-2224 (1991); see PCT / US94 / 05700).

[0041] For applications where transient expression is desired, adenovirus-based systems can be used. Adenovirus-based vectors are capable of very high transduction efficiency in many cell types and do not require cell division. High titers and expression levels have been obtained with such vectors. The vectors can be produced in large quantities in a relatively simple system. Adeno-associated virus ("AAV") vectors can also be used to introduce target nucleic acids into cells, for example, in in vitro production of nucleic acids and peptides, and in in vivo and ex vivo gene therapy procedures (see, e.g., West et al., Virology 160:38-47 (1987); U.S. Pat. No. 4,797,368; WO 93 / 24641; Kotin, Human Gene Therapy 5:793-893 (1994); Muzyczka, J Clin. No. 4,797,368; WO 93 / 24641; Kotin, Human Gene Therapy 5:793-801 (1994); Muzyczka, J. Clin.Invest. 94:1351 (1994). Construction of recombinant AAV vectors is described in U.S. Pat. No. 5,173,414; Tratschin et al., Mol. 5,173,414; Tratschin et al., Mol. Cell. Biol. 5:3251-3260 (1985); Tratschin et al., Mol. Cell. Biol. 4:2072-2081 (1984); Hermonat & Muzyczka, PNAS 81:6466-6470 (1984); and Samulski et al., J. Virol. 63:03822-3828 (1989).

[0042] Packaging cells are typically used to form viral particles capable of infecting host cells. Such cells include 293 cells for encapsulating adenovirus and ψ2 or PA317 cells for encapsulating retrovirus. Viral vectors used in gene therapy are typically generated by creating cell lines that package nucleic acid vectors into viral particles. The vectors typically contain minimal viral sequences required for packaging and subsequent integration into the host, with other viral sequences being replaced with expression cassettes for the polynucleotides to be expressed. Missing viral functions are typically supplied in trans by the packaging cell line. For example, AAV vectors used in gene therapy typically only have the ITR sequences of the AAV genome required for packaging and integration into the host genome. The viral DNA is packaged in a cell line that contains a helper plasmid that encodes the other AAV genes, namely rep and cap, but lacks the ITR sequences. The cell line can also be infected with adenovirus as a helper. The helper virus facilitates the replication of the AAV vector and the expression of the AAV genes from the helper plasmid. The helper plasmid is not packaged in significant amounts due to the lack of ITR sequences. Adenovirus contamination can be reduced, for example, by heat treatment, to which adenovirus is more sensitive than AAV.

[0043] In some embodiments, host cells are transiently or non-transiently transfected with one or more vectors described herein.In some embodiments, cells are transfected as they naturally occur in a subject.In some embodiments, the transfected cells are taken from a subject.In some embodiments, the cells are derived from cells taken from a subject, such as cell lines.

[0044] In one aspect, the present invention provides a method for modifying a target polynucleotide in a eukaryotic cell, which may be in vivo, ex vivo or in vitro. In some embodiments, the method includes taking a cell or a population of cells from a human or non-human animal and modifying the cell or cells. The culture can be at any stage of ex vivo. The cell can be reintroduced into a human or non-human animal.

[0045] In one aspect, the present invention provides a method for modifying target polynucleotide in eukaryotic cells.In some embodiments, the method comprises: binding CRISPR complex to target polynucleotide, causing the cleavage of said target polynucleotide, thereby modifying said target polynucleotide, wherein CRISPR complex comprises CRISPR enzyme complexed with guide sequence that hybridizes to target sequence in said target polynucleotide, wherein said guide sequence is linked to tracr mate sequence that hybridizes to tracr sequence.

[0046] In one aspect, the present invention provides a kit comprising any one or more of the elements disclosed in the above methods and compositions. In some embodiments, the kit comprises components for a vector system or alternative delivery system as described above. Each element can be provided separately or in combination, and can be provided in any suitable container, such as a vial, bottle, tube, etc. In some embodiments, the kit comprises instructions in one or more languages, for example, in two or more languages.

[0047] In some embodiments, the kit includes one or more reagents for use in a process utilizing one or more elements described herein. The reagents can be provided in any suitable container. For example, the kit can provide one or more reaction or storage buffers. The reagents can be provided in a form that is ready for use in a particular assay or that requires the addition of one or more other components prior to use (e.g., concentrated or lyophilized). The buffer can be any buffer, including, but not limited to, sodium carbonate buffer, sodium bicarbonate buffer, borate buffer, Tris buffer, MOPS buffer, HEPES buffer, and combinations thereof. In some embodiments, the buffer is alkaline. In some embodiments, the buffer has a pH of about 7 to about 10. In some embodiments, the kit includes one or more oligonucleotides corresponding to the guide sequence for insertion into a vector to operably link the guide sequence and the regulatory element. In some embodiments, the kit includes a homologous recombination template polynucleotide.

[0048] Down-modulating or inhibitory nucleic acids include, but are not limited to, antisense molecules, aptamers, ribozymes, triplex-forming molecules, RNA interference (RNAi), CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) RNA (crRNA), external guide sequences, etc. These nucleic acid molecules can act as agonists, inhibitors, regulators, or stimulators of a specific activity of a target molecule, or functional nucleic acid molecules can have de novo activity that is independent of any other molecule. In certain embodiments, inhibitory nucleic acids are employed.

[0049] Antisense molecules are designed to interact with target nucleic acid molecules through canonical or non-canonical base pairing. The interaction between the antisense molecule and the target molecule is designed to promote the destruction of the target molecule, for example, by RNase H-mediated RNA-DNA hybrid degradation. Alternatively, antisense molecules are designed to inhibit the processing functions that would normally occur in the target molecule, such as transcription or replication. Antisense molecules can be designed based on the sequence of the target molecule. There are many ways to optimize antisense efficiency by finding the most accessible regions of the target molecule. For example, in vitro selection experiments and DNA modification studies using DMS or DEPC are included. Antisense molecules can be designed based on the sequence of the target molecule. -6 , 10 -8 , 10 -10 , or 10 -12 The following dissociation constant (K d ) to bind to the target molecule. A representative sample of methods and techniques to aid in the design and use of antisense molecules is set forth in U.S. Patents 5,135,917, 5,294,533, 5,627,158, 5,641,754, 5,691,317, 5,780,607, 5,786,138, 5,849,903, 5,856,103, 5,919,772, 5,955,590, 5,990,088, Nos. 5,994,320, 5,998,602, 6,005,095, 6,007,995, 6,013,522, 6,017,898, 6,018,042, 6,025,198, 6,033,910, 6,040,296, 6,046,004, 6,046,319, and 6,057,437.

[0050] Triplex-forming functional nucleic acid molecules are molecules that can interact with double-stranded or single-stranded nucleic acids. When triplex molecules interact with a target region, a structure called a triplex is formed, in which the three DNA strands form a complex that relies on both Watson-Crick and Hoogsteen base pairing. Triplex molecules are preferred because they can bind to target regions with high affinity and specificity. Triplex-forming molecules can be used in combination with other molecules, such as 10-6 , 10 -8 , 10 -10 , or 10 -12 Less than K dPreferably, triplex-forming molecules bind to target molecules by the triplex-forming molecule. Representative examples of the creation and use of triplex-forming molecules to bind a variety of different target molecules are described in U.S. Patent Nos. 5,176,996, 5,645,985, 5,650,316, 5,683,874, 5,693,773, 5,834,185, 5,869,246, 5,874,566, and 5,962,426. Gene expression can also be effectively silenced in a highly specific manner by RNA interference (RNAi). This silencing was originally observed upon addition of double-stranded RNA (dsRNA) (Fire, A., et al., Nature, 391:806-11 (1998); Napoli, C., et al., Plant Cell, 2:279-89 (1990); Hannon, GJ, Nature, 418:244-51 (2002)). Once inside the cell, dsRNA is cleaved by the RNase III-like enzyme Dicer into double-stranded small interfering RNAs (siRNAs) 21-23 nucleotides in length containing two-nucleotide overhangs at the 3´ end (Elbashir, SM., et al., Genes Dev., 15:188-200 (2001); Bernstein, E., et al., Nature, 409:363-6 (2001); Hammond, SM, et al., Nature, 404:293-6 (2000)). In an ATP-dependent step, siRNAs are incorporated into a multisubunit protein complex commonly known as the RNAi-induced silencing complex (RISC), which guides siRNAs to target RNA sequences (Nykanen, A., et al., Cell, 107:309-21 (2001)). At some point, the siRNA duplex unwinds and the antisense strand remains bound to RISC and appears to direct the degradation of the complementary mRNA sequence by a combination of endonuclease and exonuclease (Martinez, J., et al., Cell, 110:563-74 (2002)). However, the effects of RNAi or siRNA, or their uses, are not limited to any one type of mechanism.

[0051] Small interfering RNA (siRNA) is a double-stranded RNA that can induce sequence-specific post-transcriptional gene silencing, thereby reducing or inhibiting gene expression. In some instances, siRNA induces specific degradation of homologous RNA molecules, such as mRNA, within the region of sequence identity between the siRNA and the target RNA. For example, WO 02 / 44321 discloses siRNAs capable of sequence-specific degradation of target mRNA when base-paired with the 3' overhanging end, and methods for producing these siRNAs are incorporated herein by reference. Sequence-specific gene silencing can be achieved in mammalian cells using synthetic short double-stranded RNAs that mimic the siRNAs produced by the enzyme dicer (Elbashir, SM, et al., Nature, 411:494 498(2001); Ui-Tei, K., et al., FEBS Lett, 479:79-82 (2000)). siRNAs can be chemically or in vitro synthesized or can be the result of short double-stranded hairpin-like RNA (shRNA) processed into siRNAs inside the cell. Synthetic siRNAs are usually designed using algorithms and conventional DNA / RNA synthesizers. Suppliers include Ambion (Austin, TX), ChemGenes (Ashland, MA), Dharmacon (Lafayette, CO), Glen Research (Sterling, VA), MWB Biotech (Esbersberg, Germany), Proligo (Boulder, CO), and Qiagen (Vento, The Netherlands). siRNAs can also be synthesized in vitro using kits such as Ambion's SILENCER.RTM.siRNA. As used herein, when referring to the production of a protein in a host cell, the term "overexpression" means that the protein is produced in greater amounts than it is produced in its naturally occurring environment.

[0052] As used herein, the term "genetic modification" refers to a change from the wild type or reference sequence of one or more nucleic acid molecules. Genetic modification includes, but is not limited to, base pair substitution, addition and deletion of at least one nucleotide from a nucleic acid molecule with a known base sequence.

[0053] As used herein, the term "solid matrix" refers to any format such as beads, microparticles, microarrays, surfaces of microtiter wells or test tubes, dipsticks, filters, etc. Matrix materials include polystyrene, cellulose, latex, nitrocellulose, nylon, polyacrylamide, dextran, agarose, etc.

[0054] "Target nucleic acid" as used herein refers to a predefined region of nucleic acid present in a complex nucleic acid mixture, the defined wild type region containing at least one known nucleotide mutation associated with leukodystrophy.Nucleic acid molecules are isolated from natural sources by cDNA cloning or subtractive hybridization, or are manually synthesized.Nucleic acid molecules may be manually synthesized using triester synthesis methods or may be synthesized using an automated DNA synthesizer.

[0055] The term "complementary" refers to two nucleotides that can form multiple favorable interactions with each other. For example, adenine is complementary because it can form two hydrogen bonds with thymine. Similarly, guanine and cytosine are complementary because they can form three hydrogen bonds. Thus, if a nucleic acid sequence contains the following base sequence, thymine, adenine, guanine, cytosine, then the "complement" of this nucleic acid molecule would be a molecule that contains adenine in place of thymine, thymine in place of adenine, cytosine in place of guanine, and guanine in place of cytosine. A complement can contain a nucleic acid sequence that forms an optimal interaction with the parent nucleic acid molecule, such that the complement can bind to the parent molecule with high affinity.

[0056] The term "promoter element" refers to a nucleotide sequence that, once incorporated into a vector and inside an appropriate cell, can facilitate the binding of transcription factors and / or polymerases and the subsequent transcription of a portion of the vector DNA into mRNA. In one embodiment, a promoter element of the invention precedes the 5' end of a leukodystrophy-specific marker nucleic acid molecule such that the latter is transcribed into mRNA. The host cell machinery then translates the mRNA into a polypeptide.

[0057] Those skilled in the art will recognize that a nucleic acid vector may contain nucleic acid elements other than a promoter element and a TUBB4A down-modulation nucleic acid molecule. These other nucleic acid elements include, but are not limited to, origins of replication, ribosome binding sites, nucleic acid sequences encoding drug resistance enzymes or amino acid metabolic enzymes, and nucleic acid sequences encoding secretion signals, localization signals, or signals useful for polypeptide purification.

[0058] A "replicon" is a gene capable of replication under its own control, e.g., plasmid, cosmid, bacmid, plasmid, phage, virus, etc. A vector can be either RNA or DNA, single-stranded or double-stranded.

[0059] An "expression operon" refers to a nucleic acid segment having transcriptional and translational control sequences, such as promoters, enhancers, translation initiation signals (e.g., ATG or AUG codons), polyadenylation signals, terminators, and the like, that facilitate the expression of a polypeptide coding sequence in a host cell or organism.

[0060] As used herein, the terms "reporter", "reporter system", "reporter gene" or "reporter gene product" refer to an operable genetic system comprising a gene in which the nucleic acid encodes a product that, when expressed, produces a reporter signal that is readily measurable, for example, by biological assay, immunoassay, radioimmunoassay, or colorimetric, fluorescent, chemiluminescent, or other methods. The nucleic acid may be RNA or DNA, linear or circular, single-stranded or double-stranded, and of antisense or sense polarity, and is operably linked to control elements required for expression of the reporter gene product. The necessary control elements will vary depending on the nature of the reporter system and whether the reporter gene is in DNA or RNA form, but may include, but are not limited to, elements such as promoters, enhancers, translation control sequences, poly A addition signals, transcription termination signals, and the like.

[0061] As mentioned above, the introduced nucleic acid may or may not be integrated (covalently linked) into the nucleic acid of the recipient cell or organism. In bacteria, yeast, plants, mammalian cells, etc., the introduced nucleic acid is maintained as an episomal element or an independent replicon, such as a plasmid. Alternatively, the introduced nucleic acid is incorporated into the nucleic acid of the recipient cell or organism, stably maintained in the cell or organism, and further inherited by the progeny cells or organisms of the recipient cell or organism. Finally, the introduced nucleic acid may only be present transiently in the recipient cell or host organism.

[0062] The term "operably linked" means that regulatory sequences essential for expression of the coding sequence are positioned in a DNA molecule in an appropriate position relative to the coding sequence so as to effect expression of the coding sequence. This same definition may also be applied to the arrangement of transcription units and other transcription control elements (e.g. enhancers) in an expression vector.

[0063] As used herein, "gymnosis" refers to the delivery of single-stranded antisense oligodeoxynucleotides to cells in the absence of a carrier (e.g., transfection) or conjugation, resulting in sequence-specific gene silencing.

[0064] The term "modified backbone linkage" includes, but is not limited to, phosphorothioate linkages, methylphosphonate linkages, ethylphosphonate linkages, boron phosphate linkages, sulfonamides, carbonylamides, phosphorodiamidates, phosphorodiamidate linkages containing positively charged side groups, phosphorodithioates, aminoethylglycines, phosphotriesters, aminoalkylphosphotriesters; 3'-alkylenephosphonates; 5'-alkylenephosphonates, chiral phosphonates, phosphinates, 3'-aminophosphoramidates, aminoalkylphosphoramidates, thionophosphoramidates; thionoalkylphosphonates, thionoalkylphosphotriesters, selenophosphates, 2-5' linked boranophosphonate analogs, bonds with reverse polarity, abasic linkages. linkages), short chain alkyl linkages, cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, short chain heteroatom or heterocyclic internucleoside linkages with siloxane backbones, sulfide, sulfoxide, sulfone formacetyl linkages, thioformacetyl linkages, methyleneformacetyl linkages, thioformacetyl linkages, riboacetyl linkages, alkene linkages, sulfamic acid backbones, methyleneimino linkages, methylenehydrazino linkages, sulfonate linkages, amide linkages.

[0065] The phrase "modified sugar" includes, but is not limited to, 2'fluoro, 2'fluoro substituted ribose, 2'-fluoro-D-arabinonucleic acid (FANA), 2'-0-methoxyethyl ribose, 2'-0-methoxyethyl deoxyribose, 2'-O-methyl substituted ribose, morpholino, piperazine, and locked nucleic acid (LNA).

[0066] A "specific binding pair" comprises a specific binding member (sbm) and a binding partner (bp) that have a particular specificity for each other and that bind preferentially to other molecules under normal conditions. Examples of specific binding pairs include antigens and antibodies, ligands and receptors, and complementary nucleotide sequences. Many other examples will be known to those skilled in the art. Furthermore, the term "specific binding pair" also applies when either or both of the specific binding member and binding partner form part of a larger molecule. In embodiments where the specific binding pair comprises nucleic acid sequences, they will be of a length such that they hybridize to each other under the conditions of the assay, preferably 10 nucleotides or more in length, more preferably 15 or 20 nucleotides or more in length.

[0067] "Sample" or "patient sample" or "biological sample" generally refers to a specimen that can be tested for a particular molecule, preferably a leukodystrophy-specific marker molecule, such as those in the table below. Samples include, but are not limited to, cells, body fluids such as blood, serum, plasma, urine, saliva, cerebrospinal fluid, tears, pleural effusions, etc.

[0068] Products and Kits Any of the above products can be incorporated into a kit that includes a TUBB4A-directed down-modulation nucleic acid in a pharma- ceutically acceptable carrier. The nucleic acid may or may not be located in a vector capable of transducing mammalian cells. In other embodiments, the kit includes a vector expressing a nucleic acid encoding human wild-type TUBB4A and / or mutant TUBB4A, which is overexpressed in the target cells of interest. The kit can optionally include a nanoparticle or liposomal formulation that facilitates delivery of the nucleic acid into cells. The kit can also include instructions for use, a container, a container for administration, an assay substrate, or any combination thereof.

[0069] Methods for therapeutic drug development and screening Because the genetic alterations in TUBB4A identified herein are associated with the pathogenesis of H-ABC, methods to identify agents that modulate the activity of the mutated gene and its encoded product should result in the generation of effective therapeutic agents for the treatment of leukodystrophies, particularly H-ABC.

[0070] Molecular modeling should facilitate the identification of specific organic molecules capable of binding to the active site of the altered TUBB4A protein based on key amino acid residues required for conformation and function. A combinatorial chemistry approach will identify highly active molecules for further rounds of screening.

[0071] The polypeptide or fragment used in the drug screening assay may be free in solution, fixed to a solid support, or intracellular. One method of drug screening utilizes eukaryotic or prokaryotic host cells stably transformed with recombinant polynucleotides expressing the polypeptide or fragment, preferably in competitive binding assays. Such cells, either live or fixed, can be used in standard binding assays. For example, complex formation between the polypeptide or fragment and the drug being tested can be determined, or the extent to which the drug being tested disrupts complex formation between the polypeptide or fragment and a known substrate can be examined.

[0072] Another technique for drug screening provides for high throughput screening of compounds with suitable binding affinity for the encoded polypeptide and is described in detail in Geysen, PCT published application WO 84 / 03564, published September 13, 1984. Briefly, large numbers of different small peptide test compounds, as described above, are synthesized on a solid substrate, such as plastic pins. The peptide test compounds are reacted with the target polypeptide and washed. Bound polypeptide is detected by methods well known in the art.

[0073] A further technique for drug screening involves the use of host eukaryotic cell lines or cells (as described above) in which the TUBB4A-related gene is non-functional or modified. These host cell lines or cells are defective at the polypeptide level. The host cell lines or cells are cultured in the presence of a drug compound. The cellular metabolic rate of the host cells is measured to determine whether the compound is capable of modulating the cellular metabolism of the defective cells. Methods for introducing DNA molecules are also well known to those skilled in the art, as described above.

[0074] Host cells expressing the H-ABC-related nucleic acids of the present invention or functional fragments thereof provide a system for screening potential compounds or drugs for their ability to modulate the development of leukodystrophy.Thus, in one embodiment, the nucleic acid molecules of the present invention can be used to generate recombinant cell lines for use in assays to identify drugs that modulate aspects of cellular metabolism related to neuronal signaling and neuronal communication and structure.Also provided herein are methods for screening compounds that can modulate the function of proteins encoded by nucleic acids comprising TUBB4A.

[0075] Another approach is to use phage display libraries engineered to express fragments of the polypeptides encoded by altered TUBB4A nucleic acids on the phage surface. Such libraries are contacted with combinatorial chemical libraries under conditions that allow for detection of binding affinity between the expressed peptides and components of the chemical library. U.S. Patent Nos. 6,057,098 and 5,965,456 provide methods and apparatus for performing such assays. Such compound libraries are commercially available from a number of companies, including, but not limited to: Maybridge Chemical Co. (Trevally, Cornwall, UK), Comgenex (Princeton, NJ), Microsour (New Milford, CT), Aldrich (Milwaukee, WI), Akos Consulting and Solutions GmbH (Basel, Switzerland), Ambinter (Paris, France), Asinex (Moscow, Russia), Aurora (Graz, Austria), BioFocus Chemical Co.), Akos Consulting and Solutions GmbH (Basel, Switzerland), Ambinter (Paris, France), Asinex (Moscow, Russia), Aurora (Graz, Austria), BioFocus DPI (Switzerland), Bionet (Camelford, UK), Chembridge (San Diego, CA), Chem Div (San Diego, CA). Those skilled in the art will be aware of other sources and can readily purchase the same. Therapeutically active compounds identified by the screening assays described herein can be formulated into pharmaceutical compositions and used to treat H-ABC.

[0076] The goal of rational drug design is to generate structural analogs of biologically active polypeptides or small molecule compounds with which they interact (e.g., agonists, antagonists, inhibitors) to generate, for example, more active or stable forms of the polypeptides, or drugs that enhance or inhibit the function of the polypeptides in vivo. See, e.g., Hodgson, (1991) Bio / Technology 9:19-21. In one approach described above, the three-dimensional structure of the protein of interest or, for example, a protein-substrate complex is solved by X-ray crystallography, nuclear magnetic resonance, computer modeling, or most typically, a combination of these approaches. Less often, useful information about the structure of a polypeptide can be obtained by modeling based on the structure of a homologous protein. An example of rational drug design is the development of HIV protease inhibitors (Erickson et al., (1990) Science 249:527-533). Additionally, peptides can be analyzed by alanine scanning (Wells, (1991) Meth.Enzym.202:390-411). In this technique, amino acid residues are replaced with Ala and the effect of this on the activity of the peptide is determined. In this way, each amino acid residue of the peptide is analyzed and the important regions of the peptide are determined.

[0077] It is also possible to isolate target-specific antibodies, selected by functional assays, and solve their crystal structures. In principle, this approach yields a pharmacore on which subsequent drug design can be based.

[0078] It is possible to bypass protein crystallography entirely by generating anti-idiotypic antibodies (anti-ids) to a functional, pharmacologically active antibody. As a mirror image of a mirror image, the binding site of the anti-IDS is predicted to be an analog of the original molecule. This anti-id can then be used to identify and isolate peptides from banks of chemically or biologically generated peptides. Selected peptides then serve as the pharmacore.

[0079] In another embodiment, the availability of modified TUBB4A nucleic acids allows the generation of strains of laboratory mice carrying the leukodystrophy-associated TUBB4A nucleic acids of the invention, as described herein below. Transgenic mice expressing the leukodystrophy-associated nucleic acids of the invention provide a model system to investigate the role of mutant TUBB4A proteins in the development and progression of leukodystrophy. Methods for introducing transgenes into laboratory mice are known to those skilled in the art and are described below. There are three common methods: 1. Incorporation of a retroviral vector encoding a foreign gene of interest into early embryos; 2. Injection of DNA into the pronuclei of newly fertilized eggs; and 3. Incorporation of genetically engineered embryonic stem cells into early embryos. The generation of such transgenic mice facilitates the molecular elucidation of the role that target proteins play in various cellular metabolic and neuronal processes. Such mice are in vivo screening tools for studying potential therapeutics in a whole animal model and are encompassed by the present invention.

[0080] The term "animal" is used herein to include all vertebrate animals, except humans. It also includes individual animals at any stage of development, including embryonic and fetal stages. A "transgenic animal" is an animal that contains one or more cells that have genetic information that has been modified or received, directly or indirectly, by deliberate genetic manipulation at the subcellular level, such as by targeted recombination, microinjection, or infection with a recombinant virus. The term "transgenic animal" does not encompass classical breeding or in vitro fertilization, but rather encompasses animals in which one or more cells have been modified by or received a recombinant DNA molecule. The molecule may be specifically targeted to a defined locus, may be randomly integrated into a chromosome, or may be extrachromosomally replicating DNA. A "germline transgenic animal" refers to a transgenic animal in which a genetic modification or genetic information has been introduced into the germline, thereby conferring the ability to transmit the genetic information to offspring. If such offspring in fact possess some or all of the modification or genetic information, they too are transgenic animals.

[0081] The DNA used to modify the target gene can be obtained by a wide variety of techniques, including, but not limited to, isolation from genomic sources, preparation of cDNA from an isolated mRNA template, direct synthesis, or a combination thereof.

[0082] A preferred target cell for gene transfer is the embryonic stem cell (ES). ES cells can be obtained from preimplantation embryos cultured in vitro (Evans et al., (1981) Nature 292:154-156; Bradley et al., (1984) Nature 309:255-258; Gossler et al., (1986) Proc. Natl. Acad. Sci. 83:9065-9069). Transgenes can be efficiently introduced into ES cells by standard techniques such as DNA transfection or retrovirus-mediated transfer. The resulting transformed ES cells can then be combined with a blastocyst of a non-human animal. The introduced ES cells then colonize the embryo and contribute to the germ lineage of the chimeric animal.

[0083] Techniques are available to inactivate or modify gene regions that harbor desired mutations.

[0084] As used herein, a knock-in animal is, for example, an endogenous mouse gene replaced with the human leukodystrophy-associated TUBB4A gene of the present invention. Such knock-in animals provide an ideal model system for studying the development of leukodystrophy. Knock-out animals can also be generated.

[0085] As used herein, expression of a leukodystrophy-associated nucleic acid, fragment thereof, can be targeted in a "tissue-specific manner" or "cell type-specific manner" using vectors in which a nucleic acid sequence encoding all or a portion of the leukodystrophy-associated nucleic acid is operably linked to regulatory sequences (e.g., promoters and / or enhancers) that direct expression of the encoded protein in a particular tissue or cell type. Such regulatory elements are advantageous for both in vitro and in vivo applications. Promoters for inducing tissue-specific proteins are well known in the art and described herein.

[0086] The present invention also provides a method for using the transgenic mouse of the present invention. The transgenic mouse introduced with the nucleic acid comprising TUBB4A or its encoding protein associated with leukodystrophy is useful for developing screening methods for identifying therapeutic agents that can regulate the onset of leukodystrophy, for example.

[0087] Pharmaceutical Compositions Pharmaceutical compositions containing therapeutic, prophylactic or diagnostic derivatives, such as functional nucleic acid derivatives, can be administered parenterally to subjects in need of such treatment. Parenteral administration can be by subcutaneous, intramuscular or intravenous injection using a syringe, optionally a pen-type syringe. Alternatively, parenteral administration can be performed using an infusion pump. A further option is to administer therapeutic, prophylactic or diagnostic agents intranasally or intrapulmonary, preferably in a composition, powder or liquid specially designed for that purpose.

[0088] Compositions for injection (including intracranial injections) of therapeutic, prophylactic or diagnostic derivatives can be prepared using conventional techniques in the pharmaceutical industry to dissolve and mix the ingredients appropriately to obtain the desired final product. Thus, according to one procedure, the therapeutic, prophylactic or diagnostic derivative can be dissolved in a volume of water that is slightly less than the final volume of the composition to be prepared. If necessary, isotonicity agents, preservatives and buffers can be added, and the pH value of the solution can be adjusted using an acid, e.g., hydrochloric acid, or a base, e.g., aqueous sodium hydroxide, if necessary. Finally, the volume of the solution can be adjusted with water to obtain the desired component concentration.

[0089] In some embodiments, the buffer is selected from the group consisting of sodium acetate, sodium carbonate, citrate, glycylglycine, histidine, glycine, lysine, arginine, sodium dihydrogen phosphate, disodium hydrogen phosphate, sodium phosphate, tris(hydroxymethyl)-aminomethane, bicine, tricine, malic acid, succinic acid, maleic acid, fumaric acid, tartaric acid, disodium hydrogen phosphate, sodium phosphate, and tris(hydroxymethyl)-aminomethane, bicine, tricine, malic acid, succinic acid, maleic acid, fumaric acid, tartaric acid, aspartic acid, or mixtures thereof. Each of these specific buffers and combinations thereof constitutes an alternative embodiment.

[0090] Pharmaceutical preparations can be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include combining active ingredients with pharmaceutical carriers or excipients. In general, the preparation is prepared by uniformly and intimately associating active ingredients with liquid carriers, finely divided solid carriers, or both, and shaping the product as necessary (e.g., to a specific particle size for delivery). In a preferred aspect of the present invention and / or its embodiments, the pharmaceutical preparation is prepared for intramuscular administration with carriers or other agents in a suitable solvent, such as water or normal saline, and optionally in a sterile formulation.

[0091] A "pharmaceutical carrier" or "excipient" can be a pharmaceutically acceptable solvent, suspending agent, or any other pharmaceutically inert vehicle for delivering one or more nucleic acids to an animal, and is known in the art. The excipient can be liquid or solid, and is selected with the intended method of administration in mind, to provide the desired bulk, consistency, etc., when combined with the nucleic acid and other components of a given pharmaceutical composition.

[0092] The compositions provided herein can include two or more antisense compounds. In another related embodiment, the compositions can include one or more antisense compounds, particularly oligonucleotides, that target a first nucleic acid, and one or more additional antisense compounds that target a second nucleic acid target. Alternatively, the compositions provided herein can include two or more antisense compounds that target different regions of the same nucleic acid target. The two or more compounds can be used together or sequentially. The compositions can also be combined with other non-antisense compound therapeutic agents.

[0093] The antisense oligomer compound described herein may be mixed with excipients suitable for the manufacture of aqueous suspensions.Such excipients are suspending agents, such as sodium carboxymethylcellulose, methylcellulose, hydropropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia; dispersing agents or wetting agents can be naturally occurring phosphatides, such as lecithin, or condensation products of alkylene oxides with fatty acids, such as polyoxyethylene stearate, or condensation products of ethylene oxide with long chain aliphatic alcohols, such as heptadecaethyleneoxycetanol, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitols, such as polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides, such as polyethylene sorbitan monooleate.Aqueous suspensions can also contain one or more preservatives, such as ethyl or n-propyl p-hydroxybenzoate. Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water provide the active ingredient in admixture with a dispersing or wetting agent, a suspending agent and one or more preservatives. The antisense oligomer compound composition may be in the form of a sterile injectable aqueous or oily suspension. The suspension may be formulated according to known techniques using suitable dispersing or wetting agents and suspending agents as mentioned above. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, for example, a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that may be used are water, Ringer's solution, and physiological saline. In addition, sterile fixed oils are conventionally used as a solvent or suspending medium. For this purpose, any bland fixed oil may be used, including synthetic mono- or di-glycerides. In addition, fatty acids such as oleic acid are also used in the preparation of injectables.

[0094] The present disclosure also includes antisense oligomeric compound compositions prepared for storage or administration, comprising a pharma- ceutically effective amount of the desired compound in a pharma- ceutically acceptable carrier or diluent. Therapeutic acceptable carriers or diluents are well known in the pharmaceutical arts, and are described, for example, in Remington's Pharmaceutical Sciences (Mack Publishing Co., AR Gennaro edit., 1985). For example, preservatives and stabilizers can be provided, such as sodium benzoate, sorbic acid, esters of p-hydroxybenzoic acid, and the like. In addition, antioxidants and suspending agents can be used.

[0095] The pharmaceutical composition of the present disclosure can also be in the form of oil-in-water emulsion.The oily phase can be vegetable oil, mineral oil, or mixture thereof.Suitable emulsifiers can be naturally occurring gums, such as gum acacia or gum tragacanth, naturally occurring phosphatides, such as soybean, lecithin, and esters or partial esters derived from fatty acids and hexitols, anhydrides, such as sorbitan monooleate, and condensation products of said partial esters with ethylene oxide, such as polyoxyethylene sorbitan monooleate.

[0096] The antisense oligomeric compounds of the present disclosure can be administered to patients by any standard means, with or without stabilizers, buffers, etc., to form a composition suitable for treatment. If liposome delivery mechanism is desired, standard protocols for liposome formation can be followed. Thus, the antisense oligomeric compounds of the present disclosure can be administered in any form, such as intramuscular injection, local injection, systemic injection, or intrathecal injection.

[0097] The disclosure also features the use of antisense oligomeric compound compositions that include surface-modified liposomes (PEG-modified liposomes, or long-circulating liposomes, stealth liposomes) that contain poly(ethylene glycol) lipids. These formulations provide a method for increasing accumulation of antisense oligomeric compounds in target tissues. This class of drug carriers resists opsonization and clearance by the mononuclear phagocyte system (MPS or RES), thereby increasing the blood circulation time and enhancing tissue exposure of the encapsulated antisense oligomeric compounds (Lasic et al, Chem. Rev. 95:2601-2627 (1995) and Ishiwata et al, Chem. Pharm. Bull. 43:1005-1011 (1995). In particular, the long-circulating liposomes improve the pharmacokinetics and pharmacodynamics of antisense oligomeric compounds compared to conventional cationic liposomes, which are known to accumulate in tissues of the MPS (Liu et al., J. Biol. Chem. 42:24864-24870 (1995); Choi et al., PCT Publication No. WO 96 / 10391; Ansell et al., PCT Publication No. WO 96 / 10390; Holland et al., PCT Publication No. WO 96 / 10392). Long-circulating liposomes may also provide greater protection of antisense oligomeric compounds from nuclease degradation compared to cationic liposomes due to their ability to avoid accumulation in metabolically intense MPS tissues such as the liver and spleen.

[0098] After administration of an antisense oligomeric compound composition according to the formulations and methods of the present disclosure, the subject exhibits about a 10% to about 99% reduction in one or more symptoms associated with the disease or disorder being treated, as compared to a placebo-treated subject or other suitable control subject.Several methods for delivery of antisense oligonucleotides for the treatment of neurodegenerative diseases are described in Evers et al. (2015) Advanced Drug Delivery Reviews 87:90-103, which is incorporated herein by reference.

[0099] The following materials and methods are provided to facilitate the practice of the present invention. Preparation of model mice Heterozygous TUBB4A D249N Mice were generated using CRISPR (clustered regularly interspaced short palindromic repeats)-Cas-9 technology by inserting the p.Asp249Asn (c.745G>A) mutation into exon 4 of the TUBB4A gene. The mouse TUBB4A gene is located on chromosome 17 and contains four exons. Cas9 mRNA, gRNA, and oligonucleotides (containing targeting sequences and 120 bp of homologous sequences on both sides) were co-injected into zygotes. The resulting CRISPR knock-in mouse model expresses one allele of the TUBB4A gene (TUBB4A D249N These heterozygous mice were bred to produce heterozygous TUBB4A D249N In addition to animals, homozygous TUBB4A mutations similar to those seen in the taeip rat model ( Li et al., 2003 ). D249N / D249N All analyses included wild-type (WT), TUBB4A D249N and TUBB4A D249N / D249N Mice were included. Animals were genotyped at all experimental steps. Mice were housed in a clean facility with a 12-h light:12-h dark cycle and had free access to food and water. Methods and study protocols conformed to the revised National Institutes of Health Laboratory Animal Welfare Policy.

[0100] Tissue Processing Mice were deeply anesthetized with a mixture of 90-150mg / kg ketamine and 7.5-16mg / kg xylazine based on body weight and perfused transcardially with 4% paraformaldehyde (PFA) in 1X phosphate-buffered saline (PBS) (Thermo Fisher Scientific, USA). Brains were harvested and post-fixed overnight in 4% PFA in 1X PBS, followed by dehydration of the tissue in 30% sucrose in 1X PBS. Brains were embedded in optimal cutting temperature compound (OCTCompound, SAKURA, 4583, USA) and sliced ​​as coronal or sagittal sections (50μm) on a cryostat microtome (CM 3050 S, Leica biosystems, USA).

[0101] Synthesis and screening of antisense oligonucleotides Mouse and human ASOs were designed and synthesized by Microsynth and Biospring. Both types of ASOs were subjected to in vitro screening to identify the optimal ASO design. Mouse HT-22 cells were used for screening mouse ASOs, and human A5499 cells were used for screening human ASOs. One million cells of each cell line were electroporated at 100,000 cells / well in 100 μL of medium at ASO concentrations of 1 μM, 5 μM, and 10 μM at a voltage of 150 V using a NEPA21 electroporation system (NEPA GENE, USA). After electroporation, the cells were transferred to a 96-well plate and placed in an incubator. After 48 hours of treatment, the cells were washed with PBS and then transferred to a PureLink TM RNA extraction was performed using the RNA 96-well Kit (ThermoFisher Scientific, Cat # 12173-011A) according to the manufacturer's instructions. After treatment with DNAase (Invitrogen), 200 ng of RNA was purified using SuperScript TMcDNA was prepared using the IV First-Strand Synthesis System (ThermoFisher Scientific, Cat:18091200). The mRNA expression levels of TUBB4 A, and the expression levels of an endogenous housekeeping gene encoding splicing factor, arginine / serine-rich 9 (sfrs9) as a reference, were quantified using real-time PCR analysis (Taqman chemistry) using an Applied Biosystems Quanta Flex 7 (ThermoFisher Scientific, USA). The results were analyzed by the ΔΔCT method.

[0102] Mouse intracerebroventricular (ICV) injection WT pups (P0-P1) were administered different doses of TUBB4AASO. ASO was administered to cryo-anesthetized mice by ICV injection using a Hamilton 1700 gas-tight syringe (7653-01, Hamilton). The needle was placed between the bregma and the eyeball, 2 / 5 of the distance from the bregma, and inserted to a depth of 2 mm. A total volume of 2 μL was administered into the left ventricle. Mice were allowed to recover on a heating pad and then returned to the dam.

[0103] RNA extraction, cDNA synthesis and dose-response determination using qPCR To investigate the relative expression of TUBB4A in the brain, mice were euthanized 10 days after ICV injection, and different brain regions were harvested, snap frozen, and stored at -80°C. RNA extraction was performed using the RNeasy 96 Universal Tissue kit (Qiagen) according to the manufacturer's instructions. The mRNA expression levels of the endogenous housekeeping gene encoding TUBB4A, and hprt as a reference, were quantified using real-time PCR analysis (Taqman chemistry) using Applied Biosystems Quanta Flex 7 (ThermoFisher Scientific, USA). The results were analyzed by the ΔΔCT method.

[0104] The following examples are provided to illustrate various embodiments of the invention, but are not intended to limit the invention in any way. Example I Antisense oligonucleotide-mediated therapeutic inhibition of TUBB4 A for the treatment of H-ABC leukodystrophy Hypomyelination with basal ganglia and cerebellar atrophy (H-ABC) is a rare leukodystrophy associated with mutations in tubulin alpha 4A (TUBB4A). The p.Asp.249Asn (D249N) mutation is the recurrent mutation found in the majority of H-ABC affected individuals. H-ABC typically presents in infancy and is characterized by dystonia, ataxia, gait changes, and progressive motor dysfunction. We recently published work using a CRISPR knock-in mouse model that harbors this mutation and recapitulates features of H-ABC disease. D249N / D249N Homozygous mutants of TUBB4A exhibit progressive motor dysfunction, ataxia, reduced survival (~P32-P37), severe impaired myelination, and neuronal atrophy in the striatum and cerebellum (Sase et al., 2020). D249N / + ), a reduced phenotype characterized by myelin loss was observed without affecting motor function or survival. D249N / D249N This represents a unique preclinical tool to test new therapeutic strategies for the treatment of H-ABC diseases. Unpublished data show that mice carrying a germline deletion of both copies of TUBB4 A (TUBB4A KO / KO ) develop normally, exhibit normal motor function, and show no myelination or neuronal cell defects. KO / KO TUBB4A D249N / + When crossed with mice, the resulting TUBB4A D249N / KO Mouse, TUBB4A D249N / D249NCompared to control, H-ABC-associated disease showed improved motor deficits, reduced hypomyelination, reduced neuronal cell loss in the neural layer and cerebellum, and increased survival (~P110) (n=8-9, p<0.0001). These results suggest that H-ABC-associated disease correlates with global expression of mutant TUBB4A and relative sparing of wild-type tubulin. Therefore, we propose globally reducing Tubb4a expression as one approach to treat H-ABC. To evaluate the therapeutic potential of TUBB4A inhibition, we designed new antisense oligonucleotides (ASOs) targeting human TUBB4A and mouse TUBB4A. We screened human TUBB4A ASOs in vitro using human cell lines and found promising ASO sequences that reduced human TUBB4A. See Table 3 below.

[0105] [Table 3]

[0106] The library of ASOs was screened in triplicate by electroporation using A549N cells. After ASO electroporation at different concentrations (0.1 μM, 0.5 μM, 1 μM, 5 μM), RNA was extracted and subjected to qPCR to evaluate TUBB4A expression. TUBB4A expression is relative to GAPDH (n=3, ***p<0.0001). The results are shown in Figure 1A-1D. Some of these ASOs were further tested using induced pluripotent stem cells (iPS cells) generated from H-ABC patient cells. These ASOs can be further tested using cells from patients with other TUBB4A mutations. [Table 4]

[0107] Concurrently, mouse ASO sequences were screened in vitro in mouse cell lines, and the ASO sequences are shown in the following table. See Table 4 and Figures 2A-2D.

[0108] The described ASO sequences were tested in vivo in mice using a single intracerebroventricular (ICV) bolus injection of these different ASO sequences into wild-type mice to identify the ASO that maximally reduces TUBB4A levels. This method is expected to be useful in the treatment of H-ABC leukodystrophy symptoms by targeting TUBB4A. D249N / D249N It can also be used on mice.

[0109] Example II Therapeutic inhibition of TUBB4A by antisense oligonucleotides and analysis of toxicity in human cells By screening human TUBB4A ASO in vitro in human cell lines and using the ASO sequences described in Example I, novel antisense oligonucleotides (ASOs) were developed that target human TUBB4A and reduce human TUBB4A. See Table 5.

[0110] These ASO gapmers contain three different modifications. 1. The flanking 5' and 3' terminal sequences are 2'-O-(2-methoxyethyl)-oligoribonucleotides and are indicated by "+". 2. The sequences at the 5' and 3' ends are locked nucleic acids indicated by "@". 3. The sequences at the 5' and 3' ends contain a mixed backbone of locked nucleic acid and 2'-O-(2-methoxyethyl)-oligoribonucleotides. [Table 5] JPEG2024515289000007.jpg23170

[0111] The ASO libraries in Tables 4 and 5 (ASO H2-1 to H15-4) were synthesized by Biospring and screened in SHYSY5Y cells using Gymnosys. Cells were plated at 250,000 cells per well and treated with 10 μm ASO for 96 h. RNA was then extracted and subjected to qPCR to evaluate TUBB4A expression. The results are shown in Figure 4.

[0112] The library of ASOs was further screened in SHYSY5Y cells using Gymnosys. Cells were plated at 250,000 cells per well and treated with 10 μm ASOs for 96 hours. RNA was then extracted and nanostringed to examine the expression levels of TUBB4A. The results are shown in Figure 5.

[0113] The results in Figures 4 and 5 show that ASOs H14 and H15 are particularly suitable for downregulating TUBB4A. The data further indicate that ASOs H14 and H15 are suitable for use as model designs for generating subsequent ASOs. Particularly suitable targets for downregulating TUBB4A and generating optimized ASOs include ASOs H13, H16, and H1.

[0114] After TUBB4A downregulation was confirmed for the ASOs in Tables 3 and 5, these ASOs were subjected to in vitro toxicity testing using the ApoTox-Glo Triplex kit. This kit combines three assay chemistries to assess cell viability, cytotoxicity, and apoptosis in a single assay well. The first part of the assay simultaneously measures two protease activities: one as a marker of cell viability and the other as a marker of cytotoxicity. Cells are then exposed to a second fluorescent cell-impermeable peptide substrate to measure dead cell protease activity, determine lost membrane integrity, and measure the level of ASO-induced apoptosis.

[0115] First, two types of ASOs were selected to detect the in vitro toxicity of ASOs 24 h after administration. SHYSY5Y cells were plated at 10,000 cells per well and treated with 10 μm ASOs for 24 h. As positive controls, two wells were treated with 10 μm Digitonin or Staurosporine. Then, each well was tested for cell viability, cytotoxicity, and apoptosis. The results are shown in Figures 6A-6C. The tested ASOs showed no toxicity in vitro.

[0116] After the preliminary results were confirmed, further in vitro toxicity testing of the ASOs was performed. SHYSY5Y cells were plated at 10,000 cells per well and treated with 10 μm ASOs for 48, 72, and 96 h. As positive controls, wells were treated with 10 μm Digitonin or Staurosporine for 48, 72, and 96 h. Additionally, untreated SHYSY5Y cells and untreated vehicle were tested after 48, 72, and 96 h. The results are shown in Figures 7A-7C. Again, the tested ASOs showed no toxicity in vitro.

[0117] Based on their in vitro TUBB4A downregulation and minimal toxicity, ASOs H10-2, H14, and H15 were tested in human iPSC-derived medium spiny neurons (HiPSC-MSNs). These ASOs were selected because they showed consistent TUBB4A downregulation by both qRT-PCR and Nanostring assays. Results in Figure 4 show that H10-2 knocked down TUBB4A by 51.2% in SH-SY4Y cells, and Figure 5 shows that H10-2 knocked down TUBB4A by 54.4% in SH-SY4Y cells. Results in Figure 4 show that H14 knocked down TUBB4A by 64.0% in SH-SY4Y cells, and Figure 5 shows that H14 knocked down TUBB4A by 60.5% in SH-SY4Y cells. The results in Figure 4 show that H15 knocked down TUBB4A by 53.2% in SH-SY4Y cells, and Figure 5 shows that H15 knocked down TUBB4A by 53.3% in SH-SY4Y cells.

[0118] The expression of the TUBB4A gene in HiPSC-MSNs was examined using Gymnosys. HiPSC-MSNs were treated with ASO candidates on day 37. After 4 days, the cells were re-treated with ASOs. On day 7, RNA was extracted and the expression of TUBB4A was measured using qRT-PCR. The results are shown in Figure 8.

[0119] After the downregulation of TUBB4A by ASO in HiPSC-MSNs was confirmed, a toxicity test of ASO in HiPSC-MSNs was performed. HiPSC-MSNs were treated with ASO candidate substances on day 37. After 4 days, the cells were re-treated with ASO. On day 7, RNA was extracted and the expression of BAX and BCL-2 was measured using qRT-PCR. BCL-2 oncoprotein controls programmed cell death by conferring a survival advantage to rapidly proliferating cells, while BAX protein promotes apoptosis by increasing the sensitivity of cells to apoptotic stimuli. Due to these functions, BAX and BCL-2 proteins are commonly used as indicators of cytotoxicity. A BAX / BCL-2 ratio of 1 or higher indicates more apoptosis, whereas a ratio of less than 1 indicates less apoptosis. The results of this experiment are shown in Figure 9A-C.

[0120] The results of this experiment demonstrated that the tested ASOs were effective in downmodulating TUBB4A and were non-toxic to the treated cells.

[0121] Example III Dose-response analysis of control iPS-MSNs for determining the dosage of antisense oligonucleotides After determining that the ASOs described above were safe and effective in downmodulating TUBB4A, additional experiments were performed to identify the appropriate dosage of ASO. Dose-response of ASO candidates was performed on control HiPS-MSNs using gymnosinase with oligos suspended in biologically compatible buffers such as phosphate-buffered saline or CSF-mimicking buffer. HiPSC-MSNs were treated with 10 μm, 20 μm, and 30 μm ASO H15 on day 37. HiPSC-MSNs were then re-treated 4 days later with the same dosage. Seven days after the first treatment, RNA was extracted and TUBB4A expression was measured using qRT-PCR. The results of this experiment are shown in Figure 10. As the dosage of ASO increased, the relative gene expression decreased.

[0122] After showing increasing downregulation of TUBB4A with increasing dose, the toxicity of ASO was measured in HiPSC-MSNs. HiPSC-MSNs were treated with 10 μm, 20 μm, and 30 μm of ASO H15 on day 37. HiPSC-MSNs were then re-treated 4 days later with the same dose. Seven days after the first treatment, RNA was extracted and the expression of BAX and BCL-2 was measured using qRT-PCR. A BAX / BCL-2 ratio of 1 or higher indicates more apoptosis, whereas a ratio below 1 indicates less apoptosis. The results of this experiment are shown in Figures 11A-11C.

[0123] Furthermore, downregulation of TUBB4A by ASO H15 was confirmed in mutant HiPSC-MSNs (TUBB4A D249N ) were analyzed in Gymnosis. Mutant HiPSC-MSNs were treated with 30 μm ASO H15 on day 37. Wild-type HiPSC-MSNs cells were also treated with 10 μm, 20 μm, and 30 μm ASO H15 on day 37 to confirm the results of the previous downregulation. Mutant and wild-type HiPSC-MSNs were then harvested 4 days later at the same doses. RNA was extracted 7 days after the first treatment and TUBB4A expression was measured using qRT-PCR. ASO H15 showed similar downregulation of TUBB4A in mutant and wild-type cells. The results of this experiment are shown in Figure 12.

[0124] After demonstrating increased downregulation of TUBB4A in mutant HiPSC-MSNs, the toxicity of ASO was confirmed in mutant cells. Mutant HiPSC-MSNs were treated with 30 μm ASO H15 on day 37. Wild-type HiPSC-MSNs cells were also treated with 10 μm, 20 μm, and 30 μm ASO H15 on day 37 to confirm the results of the previous downregulation. Mutant and wild-type HiPSC-MSNs were then harvested 4 days later at the same dosage. RNA was extracted 7 days after the first treatment, and the expression of BAX and BCL-2 was measured using qRT-PCR. The results of this experiment are shown in Figure 13A-13C.

[0125] Example IV Antisense oligonucleotide-mediated therapeutic inhibition of Tubb4a in mouse cells and analysis of toxicity Further antisense oligonucleotides (ASOs) were developed that target human TUBB4A and reduce mouse TUBB4A by screening TUBB4A ASOs in vitro in mouse cell lines and using the ASO sequences described in Example I. See Table 6. [Table 6]

[0126] The mouse ASO lacks homology to human TUBB4A, so we redesigned a combination human ASO sequence that will be screened separately. We will establish proof of concept with the mouse ASO and move the human ASO into clinical trials.

[0127] The library ASOs were screened in mouse cortical primary neuronal cells using Gymnosis. 200,000 or 150,000 cells were plated per well and treated with 1 μm and 5 μm ASOs for 1 week. RNA was then extracted and subjected to qPCR to assess TUBB4A expression. The results of these assays are shown in Figures 14A-14B and 15.

[0128] After TUBB4A downregulation was confirmed for the ASOs in Tables 4 and 6, these ASOs were subjected to in vitro toxicity testing using the ApoTox-Glo Triplex kit. This kit combines three assay chemistries to assess cell viability and apoptosis in a single assay well. The first part of the assay simultaneously measures protease activity as a marker of cell viability. Cells are then exposed to a second fluorescent cell-impermeable peptide substrate to measure dead cell protease activity, determine lost membrane integrity, and measure the level of ASO-induced apoptosis.

[0129] Several ASOs were screened to detect the in vitro toxicity of ASOs 96 hours after administration. Mouse cortical neuron cells were plated at 20,000 cells per well and treated with 5 μm ASOs for 96 hours. As a positive control, two wells were treated with 10 μm Digitonin or Staurosporine. Then, each well was examined for cell viability and apoptosis. The results are shown in Figures 16A-16B. The tested ASOs showed no toxicity in vitro.

[0130] Example V Analysis of antisense oligonucleotide administration to wild-type mice As a result of analyzing the screening assays performed in Examples I and IV, ASOs 7-2, 7-3, 8-2, 8-3, 18-1 and 18-3 were selected for analysis of ASO efficacy and toxicity in wild type mice. A 150 μg / μL solution containing 500 nmole of ASO was prepared.

[0131] Thirty adult P60 mice were injected intracerebroventricularly (ICV) with 15 μg / g or 30 μg / g ASO. Table 7 shows the observations for each WT mouse. [Table 7] JPEG2024515289000010.jpg62170

[0132] The neurological impairment caused by the ASO was assessed in multiple ways. First, mice were weighed (Figure 20C) and observed (listed in Table 7 above) weekly after injection to observe for weight loss or other adverse symptoms. Additionally, general health and physical condition, spontaneous activity, reflexes, and tone were also examined.

[0133] To evaluate motor dysfunction after ASO injection, the rotarod test is performed. In this test, mice are placed on a horizontally rotating cylinder suspended above the cage floor. The mice attempt to stay on the rotating cylinder. The time the mice remain on the cylinder is recorded. This test is performed in three stages. On day 1, the habituation stage, the mice receive one trial for 100 seconds at a constant speed of 5 RPM. On day 2, the practice stage, the mice receive three trials for 300 seconds each at a gradual ramp from 5 to 30 RPM. On day 3, the test stage, the mice receive three trials for 300 seconds each at a gradual ramp from 5 to 30 RPM. The results of the test stage for each mouse 30 days after injection are shown in Figure 17A.

[0134] To further evaluate grip strength after ASO injection, a grip strength test is performed. The grip strength of the forelimbs and hindlimbs is measured in kG / F units. The grip strength of the forelimbs and hindlimbs of ASO-treated mice was tested using a grip strength meter (080312-3 Columbus Instruments, Columbus, OH, USA). In the forelimb test, the mouse was held by the proximal part of the tail and allowed to grasp a horizontal metal bar with both forepaws. The mouse was then steadily pulled away, and the pulling force was recorded when the mouse removed the metal bar. In the hindlimb grip strength measurement, the mouse was allowed to grasp a horizontal bar with its hind forepaws while facing away from the meter, and the tail was pulled toward the meter until the grip strength was broken. Three trials were performed for 2 min each. Figure 17B and 17C show the average grip strength results after three trials.

[0135] The in vivo toxicity of the ASO was analyzed by collecting tissues and blood from each mouse. Tissues removed from the subjects included blood, cerebral cortex, cerebellum, brainstem, midbrain, liver, and residual brain. Once removed, tissues were stored at -80°C until RNA and protein could be extracted. A summary of the toxicology tests and functional observational battery is shown in Table 8. [Table 8]

[0136] TUBB4A downregulation was further assessed using RNA extraction, cDNA generation, and RT-qPCR. Mice were injected with ASO 7-2 ICV at P60 age (adult) as described above. Tissues of interest were harvested 23-30 days after injection. RNA was extracted and TUBB4A downregulation was determined by qRT-PCR. Results are shown in Figure 18A. ASO 7-2 showed toxicity at both doses. At the 30 μg / g dose, WT mice were humanely euthanized at an earlier time point, so the striatal downregulation values ​​are missing at 15 μg / g and 30 μg / g.

[0137] As above, mice were injected ICV with ASO 7-3 at age P60 (adult). Tissues of interest were harvested 23-30 days after injection. RNA was extracted and TUBB4A downregulation was determined by qRT-PCR. Results are shown in Figure 18B. ASO 7-3 demonstrated toxicity at both doses. As above, at the 30 μg / g dose, values ​​for striatal downregulation are missing at 15 μg / g and 30 μg / g because WT mice were humanely euthanized at earlier time points.

[0138] Mice were injected ICV with ASO 8-2 at age P60 (adult). Tissues of interest were harvested 23-30 days after injection. RNA was extracted and TUBB4A downregulation was determined by qRT-PCR. Results are shown in Figure 19A. ASO 8-2 was lethal at a dose of 30 μg / g; therefore, no tissues were harvested at this dose.

[0139] Mice were injected ICV with ASO 8-3 at age P60 (adult) as described above. Tissues of interest were harvested 23-30 days after injection. RNA was extracted and TUBB4A downregulation was determined by qRT-PCR. Results are shown in Figure 19B. ASO 8-3 showed no toxicity at any dose. All tissues were harvested 30 days after injection.

[0140] Mice were injected ICV with ASO 18-1 at age P60 (adult) as described above. Tissues of interest were harvested 23-30 days after injection. RNA was extracted and TUBB4A downregulation was determined by qRT-PCR. Results are shown in Figure 20A. ASO 18-1 showed no toxicity at any dose. All tissues were harvested 30 days after injection.

[0141] Mice were injected ICV with ASO 18-3 at age P60 (adult) as described above. Tissues of interest were harvested 23-30 days after injection. RNA was extracted and TUBB4A downregulation was determined by qRT-PCR. Results are shown in Figure 20B. ASO 18-3 did not show toxicity at any dose. All tissues were harvested 30 days after injection. Notably, ASO 18-3 showed both excellent TUBB4A downregulation and minimal toxicity in vivo.

[0142] Therefore, ASOs 6-5, 7-5, 7-6, 8-5, and 18-5 were synthesized, which, like ASO 18-3, resulted in good TUBB4A downregulation in vivo and minimal toxicity. The results of Tubb4a downregulation are shown in Figure 15. The in vivo toxicity results are shown in Table 9. The toxicity of ASOs was dramatically reduced. [Table 9]

[0143] Example VI Methods of Reducing TUBB4A Activity for the Treatment of H-ABC Leukodystrophy

[0144] H-ABC and related TUBB4A-associated leukodystrophies are currently untreatable. Based on the data provided in the above examples, it is clear that downregulating the expression of TUBB4A reduces the disease symptoms of H-ABC. Furthermore, it is clear that administration of ASOs to patients is non-toxic. Of note, administration of antisense molecules has also been used to treat other neurodegenerative diseases, including spinal muscular atrophy (see clinicaltrials.gov / ct2 / show / NCT02122952). Following this paradigm, patients with symptoms of H-ABC or related TUBB4A-associated leukodystrophies can be treated by injecting an effective amount of an anti-TUBB4A antisense molecule that downregulates TUBB4A expression, using the methods disclosed herein or in Evers et al. (supra), thereby alleviating the symptoms of leukodystrophies.

[0145] References JPEG2024515289000013.jpg126170 While certain features of the invention have been described herein, many modifications, substitutions, changes and equivalents will occur to those skilled in the art. It is therefore to be understood that the appended claims are intended to cover all such modifications and changes as fall within the true spirit of the invention.

Claims

1. Use of a synthesis inhibitory nucleic acid molecule targeted to TUBB4A-expressing cells in the manufacture of a medicament for ameliorating a symptom of H-ABC leukodystrophy in a patient, wherein the synthesis inhibitory nucleic acid molecule reduces TUBB4A levels in the TUBB4A-expressing cells to provide in the patient: i) delayed motor development; ii) cognitive impairment; iii) Gait impairment; iv) ataxia; v) intention tremor; vi) speech disorder; vii) Difficulty speaking viii) Abnormal hypomyelination; Decrease one or more of The use, wherein the synthesis inhibitory nucleic acid molecule is selected from an antisense oligonucleotide, an shRNA, an siRNA, and a guide strand suitable for a CRISPR-edited TUBB4A target nucleic acid.

2. 2. The use of claim 1, wherein symptoms associated with H-ABC leukodystrophy are treated, inhibited, delayed onset, ameliorated and / or alleviated in a patient, and the synthetic inhibitory nucleic acid molecule is selected from an antisense oligonucleotide, an shRNA, an siRNA, and a guide strand suitable for CRISPR editing.

3. The use according to claim 1 or 2, wherein the synthesis-inhibiting nucleic acid molecule targeting TUBB4A is modified to increase stability and / or uptake in vivo.

4. The use according to claim 1 or 2, wherein the synthesis-inhibiting nucleic acid molecule targeting TUBB4A is a modified antisense oligonucleotide.

5. The use according to claim 1 , wherein the synthesis-inhibiting nucleic acid molecule encoding the antisense oligonucleotide is cloned into a vector.

6. The use according to claim 1 , wherein the vector is selected from a plasmid vector, a lentiviral vector, a retroviral vector, an AAV vector, and an adenovirus-associated vector.

7. The use according to claim 1, wherein the disease, disorder and / or condition is associated with dysmyelination.

8. The use of claim 4, wherein the synthesis-inhibiting nucleic acid molecule is an antisense oligonucleotide provided in Table 3 or Table 5.

9. The use according to claim 1 , wherein the nucleic acid is modified and has a nucleobase sequence that is at least 90%, at least 95%, at least 99%, or 100% complementary to a portion of a human TUBB4A nucleic acid.

10. The use of claim 9, wherein the modified oligonucleotide comprises at least one modified internucleoside linkage, at least one nucleoside of the modified oligonucleotide comprises a modified sugar, or at least one nucleoside of the modified oligonucleotide comprises a modified nucleobase.

11. The use of claim 1, wherein the medicament is administered to a patient in combination with an additional active pharmaceutical agent useful for the treatment of leukodystrophy.

12. The use of claim 1, wherein the synthesis-inhibiting nucleic acid molecule is ASO H14 or ASO H15, or an array having at least 90% identity to ASO H14 or ASO H15.

13. A composition for reducing expression of TUBB4A comprising a synthetic nucleic acid molecule selected from an antisense oligonucleotide, an shRNA, an siRNA, and a guide strand suitable for CRISPR editing, in a biologically acceptable carrier, that targets and specifically hybridizes to a nucleic acid encoding TUBB4A.

14. The composition described in claim 13, wherein the synthetic nucleic acid molecule has been modified to increase stability in body fluids and / or uptake into target cells.

15. The composition described in claim 13 or 14, wherein the synthetic nucleic acid molecule is described in Table 3 or Table 5.

16. The composition of claim 15, wherein the synthetic nucleic acid molecule is present in a vector.

17. 14. The composition of claim 13, formulated for ex vivo cell administration, intracranial administration, parenteral administration and intravenous administration.

18. 14. The composition of claim 13, wherein the composition is formulated for a single intracerebroventricular (ICV) bolus injection.

19. The composition of claim 13, wherein the synthetic nucleic acid molecule is ASO H14 or ASO H15, or an array having at least 90% identity to ASO H14 or ASO H15.

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