Spinal cord subpia nebulosa gene delivery system

Abstract

A method and a spinal subpial gene delivery system are provided for delivering nucleic acid molecules into the subpial space of a mammal and effecting spinal cord intraparenchymal infection thereof. [Solution] Provided are a method for delivering a nucleic acid molecule to the subpial space of a subject, the method comprising the steps of exposing a spinal segment of the subject's spine, creating a pial opening in the spinal segment, advancing a catheter into the subpial space through the pial opening created by puncturing the pia mater with an L-shaped stainless steel tube, and delivering the nucleic acid molecule, which is a vector or an antisense oligonucleotide (ASO), to the subject's subpial space, and a system for use in the method.

Description

[Technical Field]

[0001] [Cross-references to related applications] This application claims priority under U.S. Provisional Application No. 62 / 110,340 (filed January 30, 2015) with the U.S. Patent and Trademark Office, which is incorporated herein by reference in its entirety. [Background technology]

[0002] The present invention relates primarily to gene therapy, and more particularly to a method and system for delivering genes and oligonucleotides into the subpia space of mammals to induce spinal transparenchymal infection.

[0003] There are currently two approaches used for delivering vectors or antisense oligonucleotides (ASOs) into the spinal cord parenchyma, but each has substantial drawbacks compared to the present invention.

[0004] Firstly, intrathecal delivery is used when injecting the vector or ASO into the intrathecal space of the spinal cord (i.e., the epipia). With this approach, transgene expression in the deep spinal cord parenchyma is not observed after AAV9 delivery. Because the pia mater does not allow AAV9 to pass through, only subpopulations of A-motor neurons and primary afferent nerves are infected. Although intrathecal delivery of ASO can sometimes result in good penetration of the spinal cord parenchyma, ASO is found throughout the entire spinal cord (i.e., from the cervical to the sacral segments). Therefore, segment-limited distribution of ASO cannot be achieved with intrathecal delivery.

[0005] Secondly, direct injection into the spinal cord parenchyma can be used. This approach allows for segment-specific transgene expression or ASO distribution in the spinal cord parenchyma. However, this technique is invasive because it requires direct needle penetration into the spinal cord parenchyma.

[0006] Therefore, a subpia delivery system is needed that enables nearly complete spinal cord parenchymal AAV9-mediated gene expression or ASO distribution in both white and gray matter. [Overview of the project]

[0007] The effective in vivo use of AAV vectors to achieve gene-specific silencing or upregulation in the central nervous system is limited because it cannot provide anything more than limited deep parenchymal expression in adult animals using the most clinically relevant delivery routes (i.e., intravenous or intrathecal administration). Therefore, according to the present invention, the spinal pia mater is demonstrated to act as a primary barrier that limits effective AAV9 penetration into the spinal parenchyma after intrathecal AAV9 delivery. Accordingly, the present invention provides a method and system for the delivery of genes and oligonucleotides into the spinal parenchyma of macro animals and humans.

[0008] Therefore, in one embodiment, the present invention provides a method for intraspinal infection of a target with a nucleic acid molecule. The method involves administering the nucleic acid molecule into the subpia space of the target. The target may be a mammal such as a human. In various embodiments, the administration step includes exposing a spinal cord segment of the target's vertebra, creating a pia opening within the spinal cord segment, advancing a catheter through the pia opening into the subpia space, and delivering the nucleic acid molecule into the subpia space of the target. The pia opening can be created by puncturing the pia with an L-shaped stainless steel tube, and the catheter is advanced through the tube into the subpia space. In various embodiments, the nucleic acid molecule is administered in a mixture containing about 1-10% dextrose. In various embodiments, the nucleic acid molecule is a vector or an antisense oligonucleotide (ASO). The vector may be a lentiviral vector, an adenovirus vector, or an adeno-associated virus vector (e.g., AAV9 particles). In certain embodiments, the vector comprises a nucleic acid molecule encoding a protein or functional RNA that modulates or treats a neurodegenerative disease (e.g., amyotrophic lateral sclerosis (ALS), Huntington's disease, Alzheimer's disease, Parkinson's disease, etc.).

[0009] In certain embodiments, the nucleic acid molecule is delivered as a single injection. In certain embodiments, the method further includes administering one or more second subpiatric injections of the nucleic acid molecule into another spinal cord segment of the target vertebra. In certain embodiments, the method further includes administering one or more intrathecal injections of the nucleic acid molecule to the target.

[0010] In another embodiment, the present invention provides a gene delivery system. The system comprises an L-shaped guide tube configured to puncture the pia mater of a target; a catheter slidably positioned within the guide tube and configured to advance into the subpia space of a spinal cord segment of the target vertebra; and a reservoir that is in fluid communication with the catheter and contains a composition comprising nucleic acid molecules. In various embodiments, the L-shaped guide tube may be a 16-26G stainless steel tube, and the catheter may be formed from polyethylene tubing (e.g., PE-5 or PE-10).

[0011] In another embodiment, the present invention provides a method for delivering nucleic acid molecules into the subpia space of a target. This method includes exposing a spinal cord segment of the target vertebra, creating a pia opening within the spinal cord segment, positioning the gene delivery system described herein above the spinal cord segment, advancing a catheter through the pia opening into the subpia space, and delivering the nucleic acid molecule into the subpia space of the target. In various embodiments, the nucleic acid molecule is delivered in a mixture containing approximately 1–10% dextrose. The nucleic acid molecule is a vector or an antisense oligonucleotide (ASO). The vector is a lentiviral vector, an adenovirus vector, or an adeno-associated virus vector (e.g., AAV9 particles). The target may be a mammal such as a human. Further aspects of the present invention are described below: [Section 1] A method for intraspinal parenchymal infection of a target with nucleic acid molecules, including the administration of nucleic acid molecules into the subpia space of the target. [Section 2] The administration step is, (a) Expose the spinal cord segments of the target vertebra, (b) To provide a pia mater opening within the spinal cord segment, (c) Advancing the catheter through the pia mater orifice into the subpia space, and (d) Delivering nucleic acid molecules to the target subpia space. The method described in item 1, including the method described in item 1. [Section 3] The method according to item 2, wherein a pia mater opening is created by puncturing the pia mater with an L-shaped stainless steel tube, and a catheter is advanced into the subpia maternal space through the tube. [Section 4] The method according to item 1, wherein nucleic acid molecules are administered in a mixture containing approximately 1-10% dextrose. [Section 5] The method according to item 1, wherein the nucleic acid molecule is a vector or an antisense oligonucleotide (ASO). [Section 6] The method according to item 5, wherein the vector is a lentiviral vector, an adenovirus vector, or an adeno-associated vector. [Section 7] The method described in section 6, wherein the vector is an AAV9 particle. [Section 8] The method according to item 7, wherein the vector comprises a nucleic acid molecule encoding a protein or functional RNA that modulates or treats a neurodegenerative disease. [Section 9] The method described in paragraph 8, wherein the neurodegenerative disease is amyotrophic lateral sclerosis (ALS), Huntington's disease, Alzheimer's disease, or Parkinson's disease. [Section 10] The method described in item 1, wherein nucleic acid molecules are delivered by a single injection. [Section 11] The method according to item 2, further comprising administering one or more second subpiatric doses of nucleic acid molecules into different spinal cord segments of the target vertebra. [Section 12] The method described in item 2, further comprising one or more intrathecal administrations of nucleic acid molecules to a subject. [Section 13] The method described in item 1, wherein the subject is a mammal. [Section 14] The method described in item 13, wherein the subject is a human. [Section 15] A gene delivery system, (a) An L-shaped guide tube configured to puncture the target pia mater, (b) A catheter that is slidably positioned within a guide tube and configured to advance into the subpia space of the spinal cord segment of the target vertebra, (c) A reservoir comprising a composition that is in fluid communication with a catheter and contains nucleic acid molecules, A system that includes this. [Section 16] The method according to item 15, wherein the L-shaped guide tube is a 16-26G stainless steel tube. [Section 17] The method according to item 15, wherein the catheter is formed from polyethylene tubing. [Section 18] A method for delivering nucleic acid molecules to the target subpia space, (a) Expose the spinal cord segments of the target vertebra, (b) To provide a pia mater opening within the spinal cord segment, (c) Positioning the gene delivery system described in item 16 above the spinal cord segment, (d) Advancing the catheter through the pia mater orifice into the subpia space, and (e) Delivering nucleic acid molecules into the target subpia space. Methods that include... [Section 19] A method for treating neurodegenerative disease in a subject requiring treatment, comprising administering a vector or antisense oligonucleotide (ASO) into the subpia space of the subject. [Section 20] The administration step is, (a) Expose the spinal cord segments of the target vertebra, (b) To provide a pia mater opening within the spinal cord segment, (c) Advancing the catheter through the pia mater orifice into the subpia space, and (d) Delivering nucleic acid molecules to the target subpia space. The method described in paragraph 19, including the method described in paragraph 19. [Section 21] The method according to item 20, wherein a pia mater opening is provided by puncturing the pia mater with an L-shaped stainless steel tube, and a catheter is advanced into the subpia maternal space through the tube. [Section 22] The method according to paragraph 19, wherein the neurodegenerative disease is amyotrophic lateral sclerosis (ALS), Huntington's disease, Alzheimer's disease, or Parkinson's disease. [Section 23] The method according to item 19, further comprising one or more second subpial administrations of the vector or ASO into different spinal cord segments of the target vertebra. [Section 24] The method according to item 19, further comprising one or more intrathecal administrations of the vector or ASO to the subject. [Section 25] The method described in paragraph 19, wherein the subject is a mammal. [Section 26] The method described in item 25, wherein the subject is a human. [Brief explanation of the drawing]

[0012] [Figure 1A] This figure shows subpia-mediated AAV9 delivery and macroscopically defined spinal cord surface transgene expression, and is a schematic diagram of a PE-10 catheter placed in the spinal cord, meninges, and subpia in a pig. [Figure 1B] This figure shows subpia AAV9 delivery and macroscopically defined spinal cord surface transgene expression, illustrating a catheter guide tube (18G) with a sharp pia puncture tip (insert). This pia puncture tip (insert) is used for puncturing the pia mater and advancing the PE-10 catheter into the subpia space. [Figure 1C-1E]This figure shows subpia AAV9 delivery and macroscopically defined spinal cord surface transgene expression, illustrating the advancement of the catheter into the subpia space. First, the dura mater is cut and opened (Figure 1C), and the catheter is advanced into the subpia space (Figures 1D and 1E). Air bubbles injected into the subpia space can be seen (Figure 1D - asterisk). [Figure 1F-1J] This figure shows subpia-periarticular AAV9 delivery and macroscopically defined spinal cord surface transgene expression. Figures 1F and 1G are surface GFP fluorescence densitometry images, showing strong signals in both porcine and rat spinal cords with the highest intensity GFP fluorescence observed at the center of lumbar subpia-periarticular injection. The presence of high RFP fluorescence in the spinal cord parenchyma is macroscopically detected in porcine thoracic spinal cord (Figures 1H and 1J). Clearly high levels of RFP expression in the anterior roots are also confirmed (Figure 1H - inset). No fluorescence is observed in control samples that were not injected into the spinal cord (Figure 1I). [Figure 2] This figure shows the insertion of a PE-10 catheter into the subpia space and GFP expression in the entire spinal cord parenchyma and in axons protruding distally from the AAV9 injection segment. [Figure 3] This figure shows effective parenchymal AAV9-mediated transgene expression after a single bolus subpiatric AAV9-UBI-RFP injection in adult pigs. Figures 3A and 3B show horizontal sections of the spinal cord taken from the middle thoracic spinal cord of pigs that had been previously injected with AAV9-UBI-RGF for 6 weeks. High RFP expression can be confirmed throughout the entire region, including white and gray matter. Staining with NeuN antibody (green) revealed that virtually all neurons were also RFP-positive. Figures 3C-3G show cross-sectional images of the spinal cord taken from the subpiatric injection area, showing transversely sectioned RFP+ axons (square inset) in the dorsal (DF), lateral (LF), and ventral (VF) columns. RFP expression is also observed in GFAP-stained astrocytes (inset; RFP / GFAP). High-density RFP+ nerve fiber terminals are observed around RFP-expressing α-motor neurons (Figures 3D and 3E) and interneurons (Figures 3F and 3G) (scale bar: Figures 3A-3C = 500 μm; Figures 3D, 3F = 30 μm). [Figure 4] This study shows strong GFP expression in downward motor axons in the lumbar spinal cord after subpial AAV9 injection into the mid-thoracic spinal cord in pigs. Figures 4A and 4B show spinal cord cross-sections taken from the lumbar spinal cord after 6 weeks of subpial AAV9-UBI-GFP injection into the subpial space of the mid-thoracic spinal cord. High GFP expression is observed in axonal sections in the lateral column (LF) and ventral column (VF) (white asterisks). Relatively low density of GFP+ axons was observed in the dorsal column of the spinal cord (DF). In contrast, high density of GFP+ motor axons can be seen protruding into the gray matter located between the posterior horn (DH) and anterior horn (VH). Figure 4C is a higher-resolution confocal image showing extremely fine branching of GFP+ axons and nerve fiber terminals in the central gray matter. (Scale bar: 4A=1000μm; 4C=30μm), (DH-posterior horn, VH-anterior horn, DF-dorsal chord, LF-lateral chord, VF-ventral chord). [Figure 5] Figures 5A–5L show retrograde transport-mediated GFP expression in the motor center of the brain after AAV9 delivery subpia in the mid-thoracic spinal cord in adult pigs. Figures 5A–5E show retrograde-labeled pyramidal neurons in the motor cortex of pigs after a single AAV9-UBI-GFP injection in the mid-thoracic spinal cord. Figures 5F–5J show equivalent levels of GFP expression in neurons localized in the brainstem. Figure 5K shows the presence of large retrograde-labeled motor GFP+ axons in the medulla oblongata (pyramid). Figure 5L shows high density of anterograde-labeled sensory afferent neurons in the reticular formation (scale bars: Figures 5A–5E and 5G–5J = 50 μm; Figures 5K and 5L = 50 μm). [Figure 6] Figures 6A–6D show retrograde transport-mediated GFP expression in the motor center of the brain after AAV9 delivery to the cervical subpia in adult rats. Figures 6A–6D show bilateral retrograde-GFP-labeled pyramidal neurons in the motor cortex of rats 8 weeks after a single upper cervical AAV9-UBI-GFP injection. Figures 6E–6G show bilateral neuronal GFP expression in the red nucleus (scale bars: Figures 6A, 6B, 6E and 6F = 50 μm; 6C and 6D = 50 μm; 6G = 20 μm). [Figure 7]Figures 7A-7G show segmental differences in spinal cord transgene expression after intrathecal AAV9-UBI-GFP delivery versus subpia-AAV9-UBI-RFP delivery in rats. Figure 7A shows that intrathecal injection of AAV9-UBI-GFP into the lumbar spinal cord results in preferential GFP expression in the spinal cord's dorsal column (DF), dorsal root (DR), and anterior root orifice zone (white square insertion No. 2). Subpia-AAV9-UBI-RFP injection into the upper cervical spinal cord provides clear labeling of the descending motor canal. Figure 7B shows GFP expression in dorsal root ganglion cells (L4) after intrathecal AAV9-UBI-GFP injection into the lumbar spinal cord. Figures 7C and 7D show high GFP expression after intrathecal AAV9-UBI-GFP injection in the dorsal root (DR) and in primary afferent neurites (boutons) in the deeper dorsal horn (white star), but expression was not confirmed in dorsal horn NeuN+ neurons. Figure 7E shows the absence of simultaneous localization of GFP and RFP in the spinal cord dorsal column (DF) (white inset in Figure 7A, No. 1). Figure 7F shows GFP expression in glial cells localized in the anterior root ostial zone due to intrathecal AAV9-UBI-GFP injection (white inset in Figure 7A, No. 2). Figure 7G shows the presence of several retrogradely labeled GFP-expressing α motor neurons surrounded by GFP+ primary Ia afferent neurons in animals injected with AAV9-UBI-GFP (scale bar: Figure 7A = 500 μm; 7B~7G = 30 μm), (DR - dorsal root, DH - dorsal horn, VH - anterior horn, DF - spinal cord dorsal column). [Figure 8]This diagram illustrates subpial AAV9 delivery and is a schematic representation of transgene (GFP) expression throughout the CNS after a single subpial AAV9-UBI-GFP injection. In adult pigs, the AAV9-UBI-GFP virus is delivered into the subpial space using a PE-10 catheter. Upon subpial delivery of AAV9-UBI-GFP, it diffuses and is taken up by segmental neurons (i.e., interneurons and alpha motor neurons) and superior and inferior axons beyond the subpial injection segment. Subsequently, the resulting transgene expression is observed in the following: i) segmental neurons, ii) dorsal root ganglion cells (retrograde infection), iii) motor axons that are neurosensitive to skeletal muscle (anterograde infection), iv) pyramidal neurons in the motor cortex (retrograde infection), and v) brain terminals of spinothalamic neurons (anterograde infection). [Figure 9] This study demonstrates effective parenchymal AAV9-mediated transgene expression after a single bolus injection of lumbar subpia nebula AAV9-UBI-GFP into adult rats. Figures 9A-9D show widespread GFP expression in neurons and white matter tracts in the lower thoracic and upper lumbar spinal cord after L1 subpia nebula AAV9-UBI-GFP injection. Substantially all neurons in the horizontal section (Figure 9A) express GFP. In the transverse section, GFP+ neurons are observed throughout the gray matter between thin membrane layers I-IX (Figures 9B-9D). Numerous NeuN-stained neurons express GFP in the superficial posterior horn (thin membrane layers I-III) and anterior horn. Figures 9E and 9F show high density of GFP+ downward motor fibers in the lumbar spinal cord after upper cervical AAV9-UBI-GFP injection. Figure 9F is a high-density confocal image showing easily recognizable GFP+ nerve fiber terminal boutons in the gray matter. (Scale bars: Figure 9A = 1000 μm; Figures 9B-9D = 30 μm; Figure 9E = 50 μm; Figure 9F = 100 μm), (WM - white matter, GM - gray matter, DH - posterior horn, VH - anterior horn, DF - spinal cord dorsal column). [Modes for carrying out the invention]

[0013] This invention provides a method and system for delivering genes and oligonucleotides into the spinal cord parenchyma of large animals and humans.

[0014] Before describing the compositions and methods of the present invention, it should be understood that the present invention is not limited to the compositions, methods, and experimental conditions described, as the compositions, methods, and experimental conditions themselves can change. Furthermore, since the scope of the present invention is limited only by the appended claims, it should be understood that the terms used herein are for the purpose of describing specific embodiments and are not limiting.

[0015] As used herein and in the appended claims, the singular forms "a," "an," and "the" include the plural form unless the context makes it clear otherwise. Thus, when referring to, for example, "the method," it includes one or more methods and / or steps of the type described herein, for example, as would become apparent to a person skilled in the art reading this disclosure.

[0016] The term “comprising” is used synonymously with “including,” “containing,” or “characterized by,” and is inclusive or open-ended language that does not exclude any further undescribed elements or method steps. The phrase “consisting of” excludes any elements, steps, or components not described in the claims. The phrase “essentially consisting of” limits the claims to the described materials or steps and any that do not essentially affect the basic and novel characteristics of the invention described in the claims. This disclosure intends to describe embodiments of the compositions and methods of the present invention corresponding to the scope of each of these phrases. Thus, a composition or method containing described elements or steps intends to describe a particular embodiment of the composition or method that is essentially consisting of or comprises these elements or steps.

[0017] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention pertains. Any methods and materials similar to or equivalent to those described herein may be used in the practice or testing of the present invention, and preferred methods and materials are described below.

[0018] As used herein, the term “pia mater” refers to the innermost layer of the meninges, the membrane surrounding the brain and spinal cord (Figure 1A). The pia mater is a thin, fibrous tissue that is impermeable to fluids. Therefore, the pia mater can contain cerebrospinal fluid. By containing fluids in this way, the pia mater, along with the other meningeal layers, functions as protection and cushioning for the brain. The spinal pia mater encloses the spinal cord surface or spinal cord and is attached to and anchored to the anterior fissure of the spinal cord. Therefore, the term “subpia mater” means subpia mater or occurring subpia mater.

[0019] As used herein, the term “parenchyma” refers to the functional tissue of an organ, distinct from connective and supporting tissues. Thus, the term “spinal parenchyma” refers to the diverse known anatomical tissues of the spinal cord (non-exclusive examples include gray matter, white matter, dura mater, arachnoid mater, pia mater, posterior and ventral columns, posterior spinocerebellar duct, and anterior spinocerebellar duct).

[0020] As used herein, the term “subject” refers to any individual or patient to whom the Method is applied. Generally, the subject is human, but those skilled in the art will understand that the subject may also be an animal. Thus, other animals (e.g., mammals (e.g., rodents (e.g., mice, rats, hamsters, and guinea pigs), cats, dogs, rabbits, livestock (e.g., cattle, horses, goats, sheep, and pigs), and primates (e.g., monkeys, chimpanzees, orangutans, and gorillas)) are included in the definition of subject.

[0021] As used herein, “treatment” means a clinical intervention performed for a disease, disorder, or physical condition that the patient / subject is characterized by or is susceptible to. Treatment objectives, not limited to these, include the reduction or avoidance of symptoms, the delay or cessation of the progression or worsening of the disease, disorder, or condition, and / or the alleviation of the disease, disorder, or condition. “Treatment” means either or both treatment and preventive or deterrent measures. Subjects requiring treatment include those already suffering from a disease, disorder, or undesirable physical condition, and those who need to avoid a disease, disorder, or undesirable physical condition.

[0022] As used herein, “nucleic acid” and “polynucleotide” are synonymous and refer to any nucleic acid, regardless of whether it is a phosphodiester bond or a modified bond (e.g., phosphotriester bond, phosphoamidite bond, siloxane bond, carbonate bond, carboxymethyl ester bond, acetamidite bond, carbamate bond, thioether bond, cross-linked phosphoamidite bond, cross-linked methylenephosphonate bond, cross-linked phosphoamidite bond, cross-linked phosphoamidite bond, cross-linked methylenephosphonate bond, phosphorothioate bond, methylphosphonate bond, phosphorodioate bond, cross-linked phosphorothioate bond or sultone bond, and combinations of such bonds). “Nucleic acid” and “polynucleotide” specifically also include nucleic acids composed of bases other than the five biologically occurring bases (adenine, guanine, thymine, cytosine, and uracil).

[0023] The terms “polypeptide,” “peptide,” and “protein” are used synonymously herein to refer to polymers of amino acid residues. These terms apply to amino acid polymers, which are artificial chemical mimics in which one or more amino acid residues correspond to naturally occurring amino acids, and also apply to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers.

[0024] The term "amino acid" refers to natural and synthetic amino acids, as well as amino acid analogs and amino acid mimes that function similarly to naturally occurring amino acids. Naturally occurring amino acids refer to amino acids encoded by the genetic code, and those that are later modified (e.g., hydroxyproline, α-carboxyglutamic acid, and O-phosphoserine). Amino acid analogs refer to compounds that have the same basic chemical structure as naturally occurring amino acids (i.e., hydrogen, carboxyl group, amino group, and α-carbon bonded to an R group) (e.g., homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium). Such analogs have a modified R group (e.g., norleucine) or a modified peptide skeleton and retain the same basic chemical structure as naturally occurring amino acids. Amino acid mimes refer to compounds that have a different structure from the general chemical structure of amino acids but function similarly to naturally occurring amino acids.

[0025] In this specification, amino acids may be referred to by their commonly known three-letter symbols or by the single-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides may also be referred to by their commonly accepted single-letter symbols.

[0026] The term "conservatively modified variant" applies to both amino acids and nucleic acid sequences. For a given nucleic acid sequence, a conservatively modified variant refers to a nucleic acid that codes for the same or essentially the same amino acid sequence, or, if the nucleic acid does not code for an amino acid sequence, for an essentially identical sequence. Due to the degeneracy of genetic coding, a number of functionally identical nucleic acids code for any given protein. For example, the codons GCA, GCC, GCG, and GCU all code for the amino acid alanine. Thus, at each position where alanine is specified by a codon, the codon can be changed to one of the corresponding codons described without altering the coded polypeptide. Such nucleic acid mutations are "silent mutations" and constitute a type of conservatively modified mutation. Each nucleic acid sequence in this specification that codes for a polypeptide also represents a possible silent mutation of the nucleic acid. Those skilled in the art will recognize that each codon in a nucleic acid (except AUG, which is generally the sole codon for methionine, and TGG, which is generally the sole codon for tryptophan) can be modified to obtain a functionally identical molecule. Thus, each silent mutation of a polypeptide-coding nucleic acid is implied in each described sequence.

[0027] With regard to amino acid sequences, those skilled in the art will recognize that individual substitutions, deletions, or additions to nucleic acids, peptides, polypeptides, or protein sequences that modify, add, or delete a single amino acid or a few amino acids in the encoded sequence are "conservatively modified variants" and that the resulting change is an amino acid substitution with a chemically similar amino acid. Tables of conservative substitutions that provide functionally similar amino acids are well known in the art. Such conservatively modified variants are added to, and not excluded, the polymorphic variants, interspecific homologs, and alleles of the present invention.

[0028] A "polynucleotide" is a single- or double-stranded polymer of deoxyribonucleotides or ribonucleotide bases read from the 5'-3' ends. Polynucleotides include RNA and DNA and may be isolated from natural sources, synthesized in vitro, or prepared from a combination of natural and synthetic molecules. The size of a polynucleotide is expressed in base pairs (abbreviated as "bp"), nucleotides ("nt"), or kilobases ("kb"). Where the context allows, the latter two terms can describe single- or double-stranded polynucleotides. When applied to a double-stranded molecule, the term refers to the total length and is understood to be equivalent to the term "base pairs." Those skilled in the art will recognize that the two strands of a double-stranded polynucleotide may differ slightly in length, and their ends can be alternated as a result of enzymatic cleavage, so not all nucleotides in a double-stranded polynucleotide molecule can be paired.

[0029] The term “gene” broadly refers to any fragment of nucleic acid associated with a biological function. Therefore, a gene includes the coding and / or regulatory sequences necessary for its expression. For example, a “gene” refers to mRNA, functional RNA, or nucleic acid fragment expressing a specific protein, including its regulatory sequences. A “gene” also includes non-expressed DNA fragments that form recognition sequences for other proteins, for example. A “gene” can be obtained from diverse sources (e.g., cloning from a target source or synthesis from known or predicted sequence information) and may include sequences designed to have desired parameters. An “allele” is one of several alternative forms of a gene that occupies a given locus on a chromosome.

[0030] As used herein, a “vector” is a device capable of introducing a gene sequence into target cells. Typically, “vector construct,” “expression vector,” and “gene transfer vector” refer to any nucleic acid construct capable of directing the expression of a target gene and introducing a gene sequence into target cells. Therefore, this term includes cloning and expression vehicles and embedded vectors.

[0031] Viral vectors can be particularly useful for introducing useful polynucleotides into target cells when performing the method of the present invention. Viral vectors have been developed for use in specific host systems (particularly mammalian systems) and include, for example, retroviral vectors and other lentiviral vectors (e.g., those based on human immunodeficiency virus (HIV), adenovirus vectors (AV), adeno-associated virus vectors (AAV), herpesvirus vectors, and vacciniavirus vectors) (see: Miller and Rosman, BioTechniques 7:980-990, 1992; Anderson et al., Nature 392:25-30 Suppl., 1998; Verma and Somia, Nature 389:239-242, 1997; Wilson, New Engl. J. Med. 334:1185-1187 (1996). These references are incorporated herein by reference). In one embodiment of the present invention, a lentivirus or adenovirus vector is used. Adenoviruses are double-stranded DNA viruses in which both strands of DNA encode genes. The genome encodes approximately 30 proteins. In another embodiment of the present invention, an adeno-associated virus vector is used.

[0032] The term "adenovirus" refers to more than 40 adenovirus subtypes isolated from humans, as well as numerous others from other mammals and birds. See: Strauss, “Adenovirus infections in humans,” in The Adenoviruses, Ginsberg, ed., Plenum Press, New York, NY, pp. 451-596 (1984). Recombinant adenovirus vectors (e.g., those based on human adenovirus 5) (e.g., McGrory WJ, et al., Virology 163: 614-617, 1988) cannot replicate unless grown in a tolerant cell line that provides the deletion gene product in the trans, because they lack essential early genes (usually E1A / E1B) from the adenovirus genome. Instead of the deleted adenovirus genome sequence, the target transgene is cloned and expressed in tissues / cells infected with the replication-deficient adenovirus. Adenovirus-based gene transfer generally cannot stably integrate transgenes into the host genome (less than 0.1% of adenovirus-mediated transfections result in transgene introduction into host DNA). Adenovirus vectors can be transmitted at high titers and can transfect non-replicating cells, but the transgenes are not delivered to daughter cells, making them suitable for gene transfer into non-dividing adult cardiomyocytes. Retroviral vectors offer stable gene transfer, and high titers can now be obtained via retroviral pseudotyping (Burns, et al., Proc. Natl. Acad. Sci. (USA) 90: 8033-8037, 1993). However, current retroviral vectors generally cannot efficiently transduce non-replicating cells.

[0033] Further references describing adenovirus vectors and other viral vectors that cannot be used in the method of the present invention are listed below: Horwitz, MS, Adenoviridae and Their Replication, in Fields, B., et al. (eds.) Virology, Vol. 2, Raven Press New York, pp. 1679-1721, 1990); Graham, F., et al., pp. 109-128 in Methods in Molecular Biology, Vol. 7: Gene Transfer and Expression Protocols, Murray, E. (ed.), Humana Press, Clifton, NJ (1991); Miller, N., et al., FASEB Journal 9:190-199, 1995; Schreier, H, Pharmaceutica Acta Helvetiae 68: 145-159, 1994; Schneider and French, Circulation 88:1937-1942, 1993; Curiel DT, et al., Human Gene Therapy 3: 147-154, 1992; Graham, FL, et al., International Publication No. 95 / 00655 (5 Jan. 1995); Falck-Pedersen, ES, International Publication No. 95 / 16772 (22 Jun. 1995); Denefle, P. et al., International Publication No. 95 / 23867 (8 Sep. 1995); Haddada, H. et al., International Publication No. 94 / 26914 (24 Nov. 1994); Perricaudet, M. et al., International Publication No. 95 / 02697 (26 Jan. 1995); Zhang, W., et al., International Publication No. 95 / 25071 (12 Oct. 1995).A variety of adenovirus plasmids are also available from commercial sources (e.g., Microbix Biosystems, Toronto, Ontario) (see, for example, Microbix Product Information Sheet: Plasmids for Adenovirus Vector Construction, 1996).

[0034] Adeno-associated viruses (AAVs) are small (26 nm) non-enveloped viruses with replication defects that depend on the presence of a second virus (e.g., adenovirus or herpesvirus) for cell proliferation. AAVs are not known to cause disease and induce only very mild immune responses. AAVs can infect both dividing and non-dividing cells and introduce their genome into host cells. An aspect of the present invention provides a method for delivering transgenes to target spinal column tissue using recombinant AAV-based gene transfer.

[0035] Further references describing AAV vectors that may be used in the method of the present invention include: Carter, B., Handbook of Parvoviruses, vol. I, pp. 169-228, 1990; Berns, Virology, pp. 1743-1764 (Raven Press 1990); Carter, B., Curr. Opin. Biotechnol., 3: 533-539, 1992; Muzyczka, N., Current Topics in Microbiology and Immunology, 158: 92-129, 1992; Flotte, TR, et al., Am. J. Respir. Cell Mol. Biol. 7:349-356, 1992; Chatterjee et al., Ann. NY Acad. Sci., 770: 79-90, 1995; Flotte, TR, et al., International Publication No. 95 / 13365 (18 May 1995); Trempe, JP, et al., International Publication No. 95 / 13392 (18 May 1995); Kotin, R., Human Gene Therapy, 5:793-801, 1994; Flotte, TR, et al., Gene Therapy 2:357-362, 1995; Allen, JM, International Publication No. 96 / 17947 (13 Jun. 1996); and Du et al., Gene Therapy 3:254-261, 1996. See also: U.S. Patent No. 8,865,881. This document is incorporated herein by reference.

[0036] The "effective amount" of AAV is an amount sufficient to infect a sufficient number of cells in the target tissue in a subject. The effective amount of AAV can be an amount sufficient to provide a therapeutic benefit in the subject (e.g., extension of the subject's lifespan, improvement of one or more symptoms of a disease in the subject (e.g., symptoms of a neurodegenerative disease)). The effective amount can vary depending on a variety of factors (e.g., species, the subject's age, weight, health status, and target tissue) and thus can vary from subject to subject and from tissue to tissue. The effective amount can also vary depending on the administration form. For example, in targeting CNS tissue by intravascular injection, a different dosage (e.g., a higher dosage) may be required than in the case of targeting CNS tissue by intrathecal injection or intracerebral injection. In some cases, multiple dosages of AAV are administered. The effective amount can also depend on the specific AAV used. For example, the dosage for targeting CNS tissue can depend on the serotype of AAV (e.g., capsid protein). For example, AAV can have an AAV serotype of a capsid protein selected from the group consisting of AAV1, AAV2, AAV5, AAV6, AAV6.2, AAV7, AAV8, AAV9, rh.10, rh.39, rh.43, and CSp3. In certain embodiments, the effective amount of AAV is 10 10 10 11 10 12 10 13 10 14 genome copies per kg. In certain embodiments, the effective amount of AAV is 10 10 10 11 10 12 10 13 10 14 10 15 genome copies per subject.

[0037] Depending on the host cell / vector system used, any of several transcriptional and translational elements (e.g., constitutive and inductive promoters, transcriptional enhancer elements, transcriptional terminators, etc.) can be used in the expression vector (Bitter et al., Meth. Enzymol. 153:516-544, 1987). For example, in cloning in bacterial systems, inductive promoters (e.g., bacteriophage pL, plac, ptrp, ptac (ptrp-lac hybrid promoter)) may be used. In cloning in mammalian cell systems, promoters derived from mammalian cell genomes (e.g., human or mouse metallothionein promoter) or from mammalian viruses (e.g., retroviral long terminal repeat sequences), adenovirus late promoters, or vacciniavirus 7.5K promoters may be used. Recombinant DNA or promoters generated by synthetic techniques may also be used for the transcription of inserted GDF receptor coding sequences.

[0038] As used herein, “reporter gene” or “reporter sequence” refers to any sequence that produces a protein product readily measurable in a standard assay, although this is preferably not strictly necessary. Suitable reporter genes, non-exclusively, include sequences encoding proteins that mediate antibiotic resistance (e.g., ampicillin resistance, neomycin resistance, G418 resistance, puromycin resistance), sequences encoding colored, fluorescent, or luminescent proteins (e.g., green fluorescent protein (GFP), enhanced green fluorescent protein, red fluorescent protein (RFP), luciferase), and proteins that mediate enhanced cell proliferation and / or gene amplification (e.g., dihydrofolate reductase). Examples of epitope tags include, for example, FLAG, His, myc, Tap, HA, or one or more copies of any detectable amino acid sequence. “Expression tags” include sequences encoding reporters that can be operably bound to a desired gene sequence to monitor the expression of a target gene.

[0039] As used herein, the terms “transformation” and “transfect” are used synonymously and refer to the process by which exogenous DNA or RNA is introduced and introduced into a suitable host cell. Furthermore, nucleic acids encoding other heterologous proteins may be introduced into host cells. Such transfected cells include stably transfected cells, where the inserted DNA enables replication within the host cell. Typically, stable transfection requires exogenous DNA introduced with a selectable marker nucleic acid sequence (e.g., a nucleic acid sequence providing antibiotic resistance), thereby enabling the selection of a stable transfectant. This marker nucleic acid sequence may be bound to the exogenous DNA or provided independently by co-transfection with the exogenous DNA. Transfected cells also include transiently expressing cells capable of generating RNA or DNA expression for a limited period. The transfection procedure depends on the host cell being transfected. This may include packaging polynucleotides into the virus and direct incorporation of polynucleotides. As a result of transformation, the inserted DNA may be introduced into the genome of the host cell, or the inserted DNA may be maintained in the host cell in plasmid form. Methods of transformation / transfection are well known in the field and include, but are not limited to, direct injection, e.g., microinjection, viral infection, especially infection with replication-deficient adenoviruses, electroporation, lipofection, and calcium phosphate-mediated direct uptake.

[0040] As used herein, nucleic acid sequences (e.g., coding sequences) and regulatory sequences are said to be operably ligated when they are covalently bonded to each other so that the expression or transcription of the nucleic acid sequence occurs under the influence or control of the regulatory sequence. When it is desired that the nucleic acid sequence be translated into a functional protein, two DNA sequences are said to be operably ligated if: the transcription of the coding sequence occurs as a result of induction of a promoter in the 5' regulatory sequence, and the bonding between the two DNA sequences does not result in (1) the introduction of a frameshift mutation, (2) interference with the ability of the promoter region to direct the transcription of the coding sequence, or (3) interference with the ability of the corresponding RNA transcript to be translated into a protein. Thus, a promoter region is operably ligated to a nucleic acid sequence if the promoter region is capable of causing the transcription of the DNA sequence so that the resulting transcript is translated into a desired protein or polypeptide. Similarly, two or more coding regions are operably ligated if they are bonded so that the expression of two or more proteins translated within a frame is obtained by transcription from a common promoter of two or more coding regions. In some embodiments, operably ligated coding sequences yield a fusion protein. In some embodiments, functional RNA (e.g., shRNA, miRNA) is obtained by operably bound coding sequences.

[0041] In the case of protein-coding nucleic acids, polyadenylated sequences may be inserted after the transgene sequence and before the 3'AAVITR sequence. A useful AAV construction in this invention may also preferably include an intron positioned between the promoter / enhancer sequence and the transgene. One possible intron sequence is derived from SV-40 and is called the SV-40T intron sequence. Another vector element may be used within the internal ribosome entry site (IRES). IRES sequences are used to generate two or more polypeptides from a single-gene transcript. For example, IRES sequences are used to generate proteins containing two or more polypeptide chains. The selection of these and other common vector elements is conventional, and many such sequences are available. In some embodiments, the hand-foot-and-mouth disease virus 2A sequence is included in the polyprotein. This is a small peptide (approximately 18 amino acids in length) that has been shown to be involved in the cleavage of polyproteins (Ryan, MD et al., EMBO, 1994; 4: 928-933; Mattion, NM et al., JVirology, November 1996; p. 8124-8127; Furler, S et al., Gene Therapy, 2001; 8:864-873; and Halpin, C et al., The Plant Journal, 1999; 4: 453-459).The cleavage activity of the 2A sequence has been previously demonstrated in artificial systems including plasmids and gene therapy vectors (AAV and retroviruses) (Ryan, MD et al., EMBO, 1994; 4:928-933; Mattion, NM et al., J Virology, November 1996; p. 8124-8127; Furler, S et al., Gene Therapy, 2001; 8: 864-873; and Halpin, C et al., The Plant Journal, 1999; 4: 453-459; de Felipe, P et al., Gene Therapy, 1999; 6: 198-208; de Felipe, P et al., Human Gene Therapy, 2000; 11: 1921-1931; and Klump, H et al., Gene Therapy, 2001; 8: 811-817).

[0042] The exact nature of regulatory sequences required for gene expression in host cells may vary between species, tissues, or cell types, but generally include 5' untranscribed and 5' untranslated sequences involved in the initiation of transcription and translation, respectively, as needed (e.g., TATA boxes, capping sequences, CAAT sequences, enhancer elements, etc.). In particular, such 5' untranscribed regulatory sequences include promoter regions containing promoter sequences for the transcriptional control of operably linked genes. Regulatory sequences may optionally include enhancer sequences or upstream activator sequences. Vectors of the present invention may selectively include 5' reader or signal sequences. The selection and design of appropriate vectors are within the scope of the skill and discretion of those skilled in the art.

[0043] Inducing promoters enable the regulation of gene expression, which can be modulated by exogenously supplied compounds, environmental factors such as temperature, or the presence of specific physiological conditions (e.g., acute phase, specific cell differentiation state, or only replicating cells). Inducing promoters and induction systems are available from a variety of commercial sources (examples, not limited to, Invitrogen, Clontech, and Ariad). Numerous other systems have been described and can be easily selected by those skilled in the art. Examples of inducing promoters regulated by exogenously supplied promoters are listed below: zinc-induced sheep metallothionein (MT) promoter, dexamethasone (Dex)-induced mouse mammary cancer virus (MMTV) promoter, T7 polymerase promoter system (International Publication No. 98 / 10088); ecdysone insect promoter (No et al, Proc. Natl. Acad. Sci. USA, 93:3346-3351 (1996)); tetracycline suppression system (Gossen et al, Proc. Natl. Acad. Sci. USA, 89:5547-5551 (1992)); tetracycline induction system (Gossen et al, Science, 268:1766-1769 (1995), see also: Harvey et al, Curr. Opin. Chem. Biol., 2:512-518). (1998)), the RU486 induction system (Wang et al, Nat. Biotech., 15:239-243 (1997) and Wang et al, Gene Ther., 4:432-441 (1997)), and the rapamycin induction system (Magari et al, J. Clin. Invest., 100:2865-2872 (1997)). Further types of induction promoters that may be useful in this context include those regulated by specific physiological conditions (e.g., temperature, acute phase, specific differentiation state of cells, or only replicating cells).

[0044] In some embodiments, regulatory sequences confer tissue-specific gene expression capabilities. In some cases, tissue-specific regulatory sequences bind to tissue-specific transcription elements that induce transcription in a tissue-specific manner. Such tissue-specific regulatory sequences (e.g., promoters, enhancers) are well known in the field. Examples of tissue-specific regulatory sequences are listed below without limitation: the following tissue-specific promoters: neuron (e.g., neuron-specific enolase (NSE) promoter) (Andersen et al., Cell. Mol. Neurobiol., 13:503-15 (1993)), neurofilament light chain gene promoter (Piccioli et al., Proc. Natl. Acad. Sci. USA, 88:5611-5 (1991)), and neuron-specific vgf gene promoter (Piccioli et al., Neuron, 15:373-84 (1995)). In some embodiments, the tissue-specific promoter is the promoter of a gene selected from: neuronal nucleus (NeuN), glial cell fibrous acidic protein (GFAP), adenomatous polyposis (APC), and ionized calcium-binding adapter molecule 1 (Iba-1). Other suitable tissue-specific promoters will become apparent to those skilled in the art. In some embodiments, the promoter is the chicken beta-actin promoter.

[0045] In several aspects, the present invention provides AAV vectors to be used in methods for avoiding and treating one or more genetic defects in mammals (e.g., genetic defects, somatic gene mutations) (e.g., genetic defects causing polypeptide deficiency or polypeptide excess in a subject) (in particular, treating or reducing the severity or extent of deficiency in subjects exhibiting CNS-related disorders associated with such polypeptide deficiency in cells and tissues). In some embodiments, these methods involve administering an AAV vector encoding one or more therapeutic peptides, polypeptides, shRNA, microRNA, antisense nucleotides, etc., to a subject in a pharmaceutically acceptable carrier in an amount and duration sufficient to treat CNS-related disorders in a subject having or suspected of having such disorders. "Antisense inhibition" refers to the generation of an antisense RNA transcript capable of suppressing protein expression from endogenous genes or transgenes.

[0046] Therefore, AAV vectors may contain as transgenes nucleic acids encoding proteins or functional RNAs that modulate or treat CNS-related disorders. The following is a non-restrictive list of genes associated with CNS-related disorders: neuronal apoptosis inhibitory protein (NAIP), nerve growth factor (NGF), glial cell-derived neurotrophic factor (GDNF), brain-derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CNTF), tyrosine hydroxylase (TH), GTP cyclohydrolase (GTPCH), aspartoacylase (ASPA), superoxide dismutase (SOD1), and aromatic amino acid decarboxylase (AADC). For example, a useful transgene in the treatment of Parkinson's disease encodes TH, the rate-limiting enzyme in dopamine synthesis. A transgene encoding GTPCH, which produces the TH cofactor tetrahydrobiopterin, may also be used in the treatment of Parkinson's disease. Transgenes encoding GDNF, BDNF, or AADC, which promote the conversion of L-Dopa to DA, may also be used in the treatment of Parkinson's disease. In the treatment of ALS, useful transgenes may encode GDNF, BDNF, or CNTF. Also in the treatment of ALS, useful transgenes may encode functional RNAs (e.g., shRNA, miRNA) that suppress SOD1 expression. In the treatment of ischemia, useful transgenes may encode NAIP or NGF. Transgenes encoding β-glucuronidase (GUS) may be useful in the treatment of certain lysosomal storage disorders (e.g., mucopolysaccharidosis type VII (MPSVII)). Transgenes encoding prodrug activating genes (e.g., HSV-thymidine kinase that inhibits ganciclovir synthesis and converts it to toxic nucleotides that cause cell death) may be useful in the treatment of certain cancers, for example, when administered with a prodrug. Transgenes encoding endogenous opioids such as β-endorphin may be useful in the treatment of pain.Other examples of transgenes that can be used in the AAV vector of the present invention will be apparent to those skilled in the art (see, for example, Costantini LC, et al., Gene Therapy (2000) 7, 93-109).

[0047] Over several decades, several experimental and / or clinical studies have reported the successful use of AAV-based vectors (particularly AAV9) for CNS-targeted gene delivery. These studies have clearly established the value of AAV delivery vectors as a tool for potent gene upregulation or silencing in targeted CNS regions and provide evidence that this treatment approach is effectively usable in the treatment of numerous neurodegenerative diseases (e.g., ALS, SMA, muscle spasms, and chronic pain). Despite these promising data and extensive preclinical animal studies, the detailed mechanisms by which AAV vectors reach the brain or spinal cord parenchyma after use of different AAV delivery pathways (systemic, intrathecal) remain not fully elucidated. These data are crucial in the development of novel and more effective AAV delivery protocols that are equally potent in young and fully developed adult animals and human subjects. Generally, preclinical animal studies can be classified into several groups based on the animals used or the stage of development of the AAV delivery pathway (e.g., systemic or intrathecal). Depending on the parameters used in individual studies, the levels of infected transgene expression and specific cell populations (neurons and / or glial cells) vary considerably.

[0048] Previous studies have shown that systemic intravenous (IV) injection of AAV9-GFP into neonatal mice results in widespread CNSGFP expression, including in dorsal root ganglions, spinal motor neurons (MNs), and neurons in the brain (neocortex, hippocampus, cerebellum). IV-delivered AAV9-GFP in adult mice results in preferential astrocytic cell infection throughout the CNS, but neuronal expression is limited (Foust et al., (2009)). Intravascular AAV9 preferentially targets neonatal neurons and adult astrocytic cells (Nature biotechnology 27: 59-65). Comparable data have been reported showing widespread spinal MNGFP expression after systemic intravenous (iv) delivery of AAV9 in neonatal mice (Duque et al., (2009). Intravenous administration of autocomplementary AAV9 enables transgene delivery to adult motor neurons (Molecular therapy: the journal of the American Society of Gene Therapy 17: 1187-1196). In addition, the same group has shown successful transgene expression in spinal MN after AAV9 delivery to adult mice or cats. Similar to these two studies, Gray et al. reported that while CNS neuronal GFP expression was observed after iv administration of AAV9 in adult mice, the transduction efficiency in young non-human primates was very limited compared to previous studies showing a clear shift from neuronal to glial expression in the brain (Gray, et al. (2011). Preclinical differences in intravascular AAV9 delivery to neurons and glia: a comparative study of adult mice and non-human primates (Molecular therapy: the journal of the American Society of Gene Therapy Gene Therapy 19: 1058-1069)).

[0049] Because neuronal expression after systemic ivAAV9 delivery in adult animals is thus clearly restricted, the use of intrathecal pathways (to bypass the blood-brain barrier) has been investigated in several studies. In 1-year-old, 2kg BW non-human primates (cynomolgus monkeys), a single intrathecal injection of AAV9-CB-GFP into the lumbar spinal cavity resulted in 50-75% MN transduction throughout the spinal cord two weeks after AAV9 injection. Similarly, in young 2-3 year-old non-human primates (cynomolgus monkeys) or young (2-month-old) pigs, potent MN transduction was observed throughout the spinal cord after intracisional or intracisional-lumbar intrathecal AAV9 delivery. In addition, GFP expression was observed in dorsal root ganglion cells, motor cortex, and cerebellar cortex in the same studies. More recent studies have shown comparable lumbar MNGFP expression in non-human primates (cynomolgus monkeys) after intracisional AAV9-GFP injection.

[0050] Interestingly, a review of available historical data across all studies using cisternary or lumbosacral intraspinal delivery pathways revealed a highly peculiar spinal cord transgene expression pattern. This is characterized by high expression in alpha motor neurons, however, anterior horn interneurons remaining near GFP-expressing alpha motor neurons are found to be transgene-negative. Similarly, strong transgene expression is observed in dorsal root ganglion cells and primary afferent neurons. However, transgene expression is absent in small interneurons localized in the superficial dorsal horn or intermediate zone.

[0051] Such high levels of transgene expression selectivity and the lack of infection in deeper gray matter cells suggest the presence of a well-developed regulatory barrier system (in addition to the blood-brain barrier) that prevents viral penetration from the intrathecal space into deeper spinal cord segments. Based on the detailed anatomical composition of meningitis, it is hypothesized that the pia mater is the primary barrier regulating AAV penetration into the spinal cord parenchyma after intrathecal AAV delivery. Therefore, the high levels of transgene expression observed in MN and DRG cells after intrathecal delivery are likely mediated by preferential retrograde and anterograde infection of axons that spread both inside and outside the spinal cord parenchyma and pass through the intrathecal space (i.e., the anterior and posterior roots).

[0052] Therefore, to address this problem, the present invention provides a subpia-vector delivery method in mammals, which, through this delivery pathway, infects an entire population of neurons in the gray matter of the subpia-injected segment by potent intraparenchymal transgene expression. In addition, nearly complete infection of the inferior and superior axons is achieved, coinciding with transgene expression in the brain's central nervous system (i.e., motor cortex, red nucleus).

[0053] Therefore, the methods and delivery systems described herein enable subpial gene therapy (e.g., AAV9 vector or antisense oligonucleotide (ASO) delivery) into the spinal cord parenchyma of large animals or humans. A novel delivery system was designed to include a 90° bent guide tube and catheter (e.g., PE-5 or PE-10) for delivering AAV9 or ASO into the subpial space. This design allows for precise guidance and placement of the subpial catheter into the subpial space of the target spinal cord segment. After catheter placement, AAV9 or ASO was injected for a specified period of time and then removed. In various embodiments, AAV9 or ASO was injected for approximately 2–3 minutes.

[0054] As used herein, “PE-10” refers to polyethylene tubing with an inner diameter of approximately 0.010 inches. In certain embodiments, the inner diameter of PE-10 tubing is approximately 0.011 inches. Similarly, the term “PE-5” refers to polyethylene tubing with an inner diameter of approximately 0.005 inches. In certain embodiments, the inner diameter of PE-5 tubing is approximately 0.008 inches.

[0055] Therefore, the system described in the claims provides subpial delivery (i.e., by passing through the pia mater) that provides nearly complete spinal parenchymal AAV9-mediated gene expression or ASO distribution in both the white and gray matter of the treated area. With currently available non-invasive techniques, a comparable level of spinal parenchymal transgene expression or well-controlled segment-specific gene silencing is not possible.

[0056] To place a subpial catheter in a mammalian subject, several sequential steps may be followed to minimize the possibility of spinal cord injury associated with instrument / catheter manipulation in the vicinity of the exposed “adenata” spinal cord. In various embodiments, the use of caudal and cranial spinal cord clamps (positioned directly above and below the laminectomy) minimizes spinal cord pulsation during catheter placement. Also in various embodiments, an “L”-shaped catheter stainless steel guide tube (e.g., a 16-26G stainless steel tube bent at 90°) mounted on an XYZ manipulator (e.g., as described in U.S. Patent Application Publication No. 2015 / 0224331, which is incorporated herein by reference) is used for subpial catheter placement.

[0057] In certain embodiments, the pia mater is first punctured using a bent 30G needle. When the tip of the penetrating 30G needle is approximately 1–1.5 mm into the subpia space, the pia mater is slightly elevated by 1–2 mm. The subpia catheter is then positioned into the subpia space by advancing the catheter through the guide tube. After advancing the catheter to the target length, the tip of the penetrating needle is removed from the subpia space through the guide tube. After completing the vector injection (typically 2–5 minutes and approximately 3 minutes in some embodiments), the catheter is withdrawn from the subpia space and the dura mater is closed. Using this technical approach, the subpia catheter can be positioned within approximately 3–5 minutes from the moment the dura mater is opened.

[0058] Using this technique, the subpial catheter described herein was successfully implanted in 17 pigs, achieving consistent and damage-free subpial catheter placement. In adult rats, the same technique was used, but with the PE-5 catheter instead. The data obtained using these adult rats and pigs showed: i) potent intraspinal transgene expression in white and gray matter, including neurons and glial cells, after single bolus subpial AAV9 delivery; ii) delivery to nearly all downward motor axons along the entire length of the spinal cord after cervical or thoracic subpial AAV9 injection; iii) potent retrograde transgene expression in the brain's motor centers (motor cortex and brainstem); and iv) safety of this approach, with normal neurological function defined up to 3 months after AAV9 delivery. Thus, subpial delivery of AAV-9 enables gene-based treatment with a wide range of experimental and clinical applications in adult mammals.

[0059] In one embodiment, a 16-26G stainless steel tube bent at 90° was used as a guide cannula for the PE-10 catheter. After positioning this guide tube directly above the spinal pia mater, the PE catheter was advanced into the subpia space through the small pia mater opening (Figures 1A-1E). Subsequently, AAV9 or ASO was injected into the subpia space. In certain embodiments, AAV9 was delivered in a mixture containing 1-10% dextran (10,000-30,000 MW) to allow AAV9 particles to persist in the spinal parenchyma for a longer period. Following AAV9-GFP delivery, consistent GFP expression was observed throughout the spinal parenchyma at the injection level and in axons projecting distally (into the lumbar spinal cord) from the AAV9 injection segment.

[0060] As described herein, a single subpial AAV9 injection resulted in potent intraparenchymal transgene expression rostro-caudally diffusing in multiple segments. Therefore, subpial AAV9 delivery leads to widespread transgene expression in neurons throughout the gray matter and superior and inferior axons within the subpial injection segment. For example, in adult pig spinal cord, consistent transgene diffusion was observed at a distance of approximately 10–15 cm from the injection site. Expression was identified in neurons and glial cells throughout the entire gray matter membrane, as well as in axons in the ventral, lateral, and dorsal columns of the spinal cord, confirming almost complete penetration of the subpial-injected AAV9 vector throughout the spinal cord parenchyma. Analysis of transverse lumbar spinal cord sections from pigs injected with mid-thoracic spinal cord AAV9-UBI-GFP (i.e., approximately 30 cm from the AAV9 delivery site) showed that virtually all inferior motor axons appeared to be labeled 6 weeks after AAV9 injection. Higher-resolution confocal microscopy revealed a high-density network of fine axonal branches, including nerve fiber terminals, throughout the gray matter. Consistent with the level and distribution of infected axons in the white matter, retrograde-infected GFP-expressing neurons were identified in the motor cortex and brainstem. Similarly, centrally projecting sensory axons were identified in the reticular formation and thalamus.

[0061] By comparing transgene expression patterns after subpia and intrathecal AAV9 delivery in rats, the present invention demonstrated substantially different local cellular expression. Therefore, the pia mater represents a primary barrier for effective parenchymal penetration of AAV9 after intrathecal delivery.

[0062] First, after intrathecal delivery, expression was observed only in regions and neuronal glial pools morphologically associated with the dorsal root and anterior root ostial zones. Therefore, potent transgene expression was observed in primary afferent neurons, clearly present in primary afferent neurons in the spinal cord's dorsal column, spinal cord dorsal horn, and Ia afferent neurons projecting into the spinal cord's anterior horn. This transgene expression in primary afferent neurons was consistent with potent expression in dorsal root ganglion cells. Similarly, clear expression was observed around the anterior root ostial zone, including several retrogradely labeled α motor neurons in the anterior horn. In contrast, however, transgene expression was virtually absent in all other neurons between layers I-VII and X of the spinal cord, and also absent in labeled descending motor axons in the lateral or ventral column. Similarly, no transgene expression was observed in axons of the corticospinal tract (localized on the spinal cord dorsal column base) surrounded by GFP-expressing primary afferent neurons in rats. Speaking without being bound by theory, a comprehensive consideration of these data suggests that spinal parenchymal GFP expression (whether in neurons or in protruding primary afferent nerves) may be due to retrograde or anterograde transgene expression, and not to the uptake of AAV9 from the intrathecal space into the spinal parenchyma. This finding is consistent with data from other laboratories showing potent α-motor neuron GFP expression after a single intrathecal or cisternary AAV9-GFP injection in adult non-human primates or young pigs. However, interneuronal GFP expression was not observed in interneurons very close to GFP+α-motor neurons (Meyer et al., (2015). Improved single-dose CSF delivery of AAV9-mediated gene therapy for SMA: Dose-response studies in mice and non-human primates. Molecular therapy: the journal of the American Society of Gene Therapy 23: 477-487; Foust, et al. (2013).Therapeutic AAV9-mediated suppression of mutant SOD1 slows disease progression and extends lifespan in a genetic ALS model (Molecular therapy: the journal of the American Society of Gene Therapy 21: 2148-2159; Passini, et al. (2014)). Translational fidelity of autocomplementary AAV9-surviving motor neuron 1 intrathecal delivery for spinal muscular atrophy (Human gene therapy 25: 619-630; Bell, et al. (2015)). Motor neuron transduction after intracisional delivery of AAV9 in cynomolgus monkeys (Human gene therapy methods 26: 43-44)).

[0063] In contrast, as described above, subpia mater AAV9 delivery was associated with potent transgene expression in gray matter neurons (i.e., alpha motor neurons and interneurons) and in substantially all inferior motor axons and primary afferent neurons of the injection segment. These data clearly demonstrate that the pia mater acts as a primary barrier preventing AAV9 penetration into other spinal cord segments located away from the anterior and posterior root ostial zones. By bypassing the pia mater and depositing AAV9 into the subpia mater, intrawhite matter and gray matter parenchymal infection can be effectively achieved in adult rodents or megazoans.

[0064] Therefore, by using the method and system described in the claims within the subject matter, axonal sprouting after spinal cord injury can be increased by upregulating the expression levels of neurotrophic genes in the downward motor axons. Furthermore, local delivery of ASO in this manner allows for restricted silencing of gene fragments associated with the progression of targeted chronic pain or muscle spasms, while eliminating the upper spinal cord side effects that typically occur after intrathecal ASO delivery.

[0065] The efficacy of subpia-induced infection and neuronal cell populations infecting the spinal cord and brain in adult animals has several high clinical and experimental significances. Firstly, when specific genes should be downregulated through silencing, a single cervical subpia injection of a silencing AAV9 construct provides effective gene silencing in cervical neurons and glial cells across the majority of descending motor axons and ascending sensory fibers throughout the spinal cord. Given the well-characterized neurodegenerative patterns of ALS patients and experimental models of ALS, including the degenerative progression of superior motor neurons and protruding descending motor axons, inferior motor neurons, and spinal interneurons, the ability to achieve broad-spectrum mutant gene silencing is likely to yield substantial benefits in achieving the highest therapeutic efficacy. In addition, a single cervical subpia injection may be combined with one or more additional subpia injections into the lumbar enlargement to target lumbar neuronal / glial populations, and / or intrathecal injections into the lumbar spinal cavity to target the alpha motor neuron pool throughout the thoracic and lumbar spinal cord. Secondly, increased expression of therapeutic genes (e.g., growth factors) associated with axon sprouting can be easily achieved in the downward motor pathway and ascending sensory fibers, allowing for testing of therapeutic efficacy, for example, in spinal cord injury studies. In this case, the AAV9 vector can be administered via a subpia catheter from a single laminectomy site at the center of injury, and the subpia catheter can be advanced systemically rostrally and caudally, targeting the distal end of the severed motor axon and the proximal end of the ascending sensory axon, respectively. Thirdly, near-perfect downward motor pathway labeling, achievable from cervical subpia AAV9-GFP injection, enables axon sprouting and synapse formation between labeled motor axons of the target and spinal cord transplant cells. Such systematically characterizing the level of axon sprouting and / or synaptic progression in cell-transplanted large animal models of spinal cord injury has not been obtained to date.

[0066] As used herein, the advantage of subpiatric AAV9 delivery compared to intrathecal delivery appears to be superior transgene expression within the spinal cord parenchyma. In addition, similarly high levels of transgene expression are fully achieved in adult rats or miniature pigs. Since the spinal cord dimensions in adult pigs weighing 35-40 kg are similar to those of humans, similar parenchymal AAV9 uptake is expected to be achieved in adults as well.

[0067] A relatively limiting aspect of the subpial delivery technique described herein is the need for local laminectomy to access the surface of the posterior horn of the spinal cord where the subpial injection is performed. This requirement for laminectomy may limit its repeated use (in contrast to the repeatability of intrathecal delivery). However, the level of transgene expression achievable after subpial AAV9 delivery appears to balance this limitation (if not outweighed, at least when a clearer and more potent therapeutic effect is observed after using subpial gene delivery in disease modification studies). Furthermore, it has recently been found that high levels of spinal GFP expression persisted in the lumbar spinal cord of untreated control mice 12 months after administration. This strongly suggests that a single subpial delivery of the therapeutic gene yielded a long-lasting effect before considering the need for further gene delivery.

[0068] Using experimental mammalian subjects (e.g., adult pigs and mice), the present invention demonstrates that the subpia-spinal AAV9 delivery technique described herein enables broad transgene expression in the spinal cord parenchyma, descending and ascending axons, without the need for direct spinal cord tissue needle penetration (see Figure 8). In addition to localized spinal cord transgene expression, robust retrograde expression was observed in the brain's motor centers. This technique may be used in preclinical and human clinical trials aimed at upregulation or downregulation of genes in specific spinal cord segments and / or protruding motor and ascending sensory axons. The range of transgene expression in these experimental adult animals suggests that the present invention can be successfully used in adult patient populations targeting a variety of spinal cord neurodegenerative diseases and / or CNS-related disorders. Exemplary neurodegenerative diseases, CNS-related disorders, illnesses, or injuries include, but are not limited to, multiple sclerosis (MS), progressive multifocal leukoencephalopathy (PML), encephalomyelitis (EPL), central pontine myelinolysis (CPM), adrenoleukodystrophy, Alexander disease, Pelizaeus-Merzbacher disease (PMZ), globoid cell leukodystrophy (lysosomal storage disease) and Wallerian degeneration, optic neuritis, transverse myelitis, amyotrophic lateral sclerosis (ALS), Huntington's disease, Alzheimer's disease, Parkinson's disease, Parkinson's-Plas syndrome (e.g., multiple system atrophy, progressive supranuclear palsy, and corticobasal degeneration), surgical resection, spinal cord injury or trauma, CNS injury resulting from tumor resection, transverse myelitis, and optic neuritis. These include Guillain-Barré syndrome (GBS), stroke, traumatic brain injury, post-radiation injury, neurological complications of chemotherapy, anterior ischemic optic neuropathy, vitamin E deficiency, vitamin E deficiency syndrome, Bassen-Kohnzweig syndrome, Marquia-Farber-Bignami disease, metachromatic leukodystrophy, trigeminal neuralgia, glossopharyngeal neuralgia, myasthenia gravis, epilepsy, Bell's palsy, muscular dystrophy, progressive muscular atrophy, primary lateral sclerosis (PLS), pseudomedullary palsy, progressive bulbar palsy, spinal muscular atrophy, progressive bulbar palsy, hereditary muscular atrophy, intervertebral disc disorders (e.g., herniated, burst, and herniated disc syndromes), cervical spondylosis, nerve plexus disorders, thoracic outlet disruption syndrome, peripheral neuropathy, porphyria, mild cognitive impairment, and chronic pain syndromes.

[0069] In some embodiments, particularly high AAV concentrations (e.g., ~10) 13 The AAV composition is formulated to reduce the aggregation of AAV particles in the composition when a GC / ml or higher concentration is present. Methods for reducing AAV aggregation are well known in the art, and examples include the addition of surfactants, pH adjustments, and salt concentration adjusters (see, for example, Wright FR, et al., Molecular Therapy (2005) 12, 171-178. The contents of this document are referenced herein for reference).

[0070] Formulations of pharmaceutically acceptable excipients and carrier solutions are well known to those skilled in the art, as are the development of appropriate dosage and treatment regimens for using the specific compositions described herein in a variety of treatment regimens. Typically, these formulations may contain at least about 0.1% or more of the active ingredient, although of course the percentage of the active ingredient(s) may vary, and for convenience may be about 1 or 2% to about 70% or 80% or more of the total weight or volume of the formulation. Naturally, the amount of the active ingredient in each therapeutically useful composition can be adjusted so that an appropriate dose is obtained at any given unit dose of the compound. As those skilled in the art will know, in the preparation of such pharmaceutical formulations, a variety of dosages and treatment regimens may be desired to take into account factors such as solubility, bioavailability, biological half-life, route of administration, product shelf life, and other pharmacological considerations.

[0071] Examples of pharmaceutical forms suitable for injection include sterile aqueous solutions or dispersions for the immediate preparation of sterile injection solutions or dispersions. Dispersions can be prepared in glycerol, liquid polyethylene glycol, mixtures thereof, and oils. Under normal storage and use conditions, these preparations contain preservatives to prevent microbial growth. In many cases, this form is a sterile fluid to the extent that it passes easily through an injection needle. This form must be stable under manufacturing and storage conditions and must be stored in a manner that prevents contamination by microorganisms such as bacteria and fungi. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, polypropylene glycol, and liquid polyethylene glycol), suitable mixtures thereof, and / or vegetable oils. Appropriate fluidity can be maintained by, for example, the use of coatings such as lecithin, maintaining the required particle size in the case of dispersion, and the use of surfactants.

[0072] For administration of aqueous solutions for injection, for example, the solution should be appropriately buffered as needed, and the liquid diluent should first be isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous, and intraperitoneal administration. In this regard, available sterile aqueous solvents are known to those skilled in the art. For example, one dose is dissolved in 1 ml of isotonic NaCl solution and added to 1000 ml of subcutaneous injection fluid or injected into the proposed injection site (see, e.g., “Remington's Pharmaceutical Sciences” 15th Edition, pages 1035-1038 and 1570-1580). Dosage modifications may be necessary depending on the host's condition. In all cases, the administration officer should determine the appropriate dose for each individual host.

[0073] After preparing the sterile injection solution by taking the required amount of active AAV in a suitable solvent and the required amount of other components listed herein, sterilization is performed by filtration. Generally, dispersions are prepared by introducing various sterile active ingredients into a sterile vehicle containing a basic dispersion medium and other necessary components from those listed above. In the case of sterile powders for the preparation of sterile injection solutions, preferred preparation methods include vacuum drying and freeze-drying techniques, from which the active ingredient powder + any other desired components can be obtained from the solution that has been previously sterile filtered.

[0074] In some embodiments, the drugs, compositions, and / or systems described herein can be assembled as a pharmaceutical kit, diagnostic kit, or research kit to facilitate their use in treatment, diagnostic, or research applications. A kit may include one or more containers containing the components of the present invention and instructions for use. More specifically, such a kit may include a composition containing AAV for administration as described herein, along with instructions describing the intended use and appropriate use of the composition. In certain embodiments, a kit may further include a separate container containing a 90° bent guide tube (e.g., 18 or 23G) and a catheter suitable for a particular use and method of subpial administration of the composition (e.g., PE-5 or PE-10). Kits for research purposes may include components in appropriate concentrations or quantities for performing a variety of experiments. A kit may further include one or more or all of the components necessary for subpial administration of the composition to a control (e.g., syringes, caudal and / or cranial vertebral clamps, XYZ manipulators, etc.).

[0075] As used herein, “Instructions” may define components of instructions and / or promotions and often include written instructions for the packaging of the present invention or related thereto. Instructions may also include any oral or electronic instructions provided in a manner that makes it readily apparent to the user that the instructions are related to the kit (e.g., visual or auditory (e.g., videotape, DVD, etc.), internet and / or web-based communications). Written instructions may take a format prescribed by the government agency that has jurisdiction over the manufacture, use or sale of a pharmaceutical or biological product, and these instructions may also reflect approval by the agency responsible for the manufacture, use or sale of an animal product.

[0076] The following examples are illustrative of the present invention and are not intended to limit it.

[0077] Example 1 material and method Animals and general surgical procedures were used – adult Sprague-Dawley rats (male and female, 250–350 grams, n=16) or adult miniature pigs (both male and female, 30–40 kg; n=6) obtained from crossbreeding Minnesota and Göttingen lines were used. Rats were anesthetized with 5% isoflurane and maintained at 2–3% isoflurane during surgery, depending on respiratory rate and paw pinch response. The rats' backs were then shaved and washed with 2% chlorhexidine. After skin incision, paravertebral muscles around the cervical, thoracic, or lumbar vertebrae were removed, and the animals were fixed to a stoelting frame using Cunningham's vertebral clamp as previously reported (Kakinohana, et al. (2004). Region-specific cell grafts into cervical and lumbar spinal cords in rats: a qualitative and quantitative stereochemical study. Experimental neurology 190: 122-132). To expose the spinal cord, laminectomy of the corresponding vertebrae was performed using a dental drill. Subsequently, the dura mater was incised using a surgical scalpel.

[0078] Miniature pigs were premedicated with intramuscular azaperone (2 mg / kg) and atropine (1 mg / kg; Biotika, SK) before induction with ketamine (20 mg / kg; IV). After induction, the animals were intubated with a 2.5F tracheal tube. Anesthesia was maintained with 1.5% isoflurane in a 50% / 50% air / oxygen mixture at a constant flow rate of 2 L / min. Oxygen saturation was monitored during surgery using a pulse oximeter (Nellcor Puritan Bennett Inc., Ireland). After induction of anesthesia, the animals were placed in a spinal cord fixation device as previously reported (Usvald, et al. (2010). Analysis of drug regimen and reproducibility of intrathecal grafts of human spinal cord stem cells in immunosuppressed miniature pigs. Cell transplantation 19: 1103-1122). Subsequently, laminectomy was performed on the Th5 or L2-L4 vertebrae corresponding to the Th5 and L3-L6 spinal segments, respectively, and the epidural fat was removed by swabbing with cotton. The dura mater was incised and fixed to the surrounding tissue using 6.0 Proline (Figures 1C-1E).

[0079] Subpia Catheter Placement and Subpia AAV9 Injection - An L-shaped catheter guide tube (18 or 23G) was constructed to place the subpia catheter (Figure 1B). This guide tube was attached to an XYZ (Stoelting) manipulator and advanced to the surface of the exposed spinal cord segment. The pia was punctured using a 30G needle that had been pre-bent to 45°. Next, the subpia catheter (PE-10 for pigs and PE-5 for rats) was advanced into the subpia space by manually pushing it from the other end of the guide tube. In rats, the catheter was advanced approximately 1-1.5 cm into the subpia space, and in pigs, approximately 3-6 cm. Subsequently, the virus was injected into the subpia space over 3 minutes using a 50 or 250 μl Hamilton syringe. After injection, the catheter was removed, and the dura mater was closed using 6.0 Proline (dura mater closure was performed only in pigs), after which the animals were allowed to recover.

[0080] Preparation of AAV9 for in vivo injection: A 1.2kb ubiquitin-C (UBC) promoter was synthesized by oligonucleotide synthesis, conjugated with eGFP or DsRed (RFP) and the SV40 polyA signaling molecule, and cloned into a self-complementary double-stranded DNA genomic AAV (scAAV) vector plasmid (Xu, et al. (2012). In vivo gene knockdown mediated by rat dorsal root ganglion via self-complementary adeno-associated virus serotype 5 after intrathecal delivery. PloSone7: e32581). A helper virus-free scAAV9 vector expressing either eGFP or RFP driven by the UBC promoter was generated by transient transfection of HEK293T cells with a vector plasmid, pRep2-Cap9, and a pAd-helper plasmid (Xiao, et al. (1998). Production of high-titer recombinant adeno-associated virus vector in the absence of helper adenovirus. J Virol 72: 2224-2232). The plasmid pRep2-Cap9 was obtained from Vector Core in U. Penn. The AAV vector in cell lysates prepared 72 hours after transfection was purified as previously reported, and its titer was determined by Q-PCR (Xu, et al., previously cited). The final titer was 1.0 x 10⁻¹⁴. 13 The virus was adjusted to genome copies / ml (gc / ml). Immediately before injection, the virus was mixed with dextran (10,000 MW) in a 1:1 ratio to achieve a final dextran concentration of 2.5%. The subpial injection volume was 30 μl for rats and 200 μl for pigs.

[0081] Perfusion fixation of spinal cord and brain sections, postmortem in-situ GFP fluorescence imaging and immunofluorescence – Animals (rats and pigs) were deeply anesthetized with pentobarbital and perfused transcentrally with 200 ml (rats) or 2000 ml (pigs) of heparinized saline, followed by transcentral perfusion with 250 ml (rats) or 4000 ml (pigs) of 4% paraformaldehyde in PBS. The spinal cord and brain were dissected and fixed overnight in 4% formaldehyde in PBS at 4°C, then cryoprotected in 30% sucrose PBS. Transverse or longitudinal sections (30 μm thick) were excised on a cryostat and stored in PBS. Before sectioning, the entire spinal cord was imaged in situ using an IVIS spectral imaging system (Xenogen, Alameda, CA). Sequence acquisition was performed at an excitation wavelength of 465 nm and an emission wavelength of 520 nm. Moderate binning was used, and the exposure time was 3 seconds. Image analysis was performed using Living Image 4.3.1 (Xenogen, Alameda, CA) software. Signal calculations were performed using fixed-volume ROIs. Immunostaining of prepared sections was performed overnight at 4°C in PBS using primary antibodies prepared with 0.2% TritonX-100: rabbit anti-glial cell fibrous acidic protein (GFAP; 1:500, Origene, Rockville, Maryland, USA) and mouse anti-neuronal nuclear antigen (NeuN, 1:1000, Chemicon). After incubation with the primary antibodies, sections were washed three times in PBS and incubated with fluorescently conjugated secondary donkey anti-rabbit and donkey anti-mouse antibodies (AlexaFluor488, 546, or 647, 1:1000, Invitrogen), respectively, as well as DAPI for general nuclear staining. Subsequently, sections were placed on slides, dried at room temperature, and coated with a long-term browning prevention kit (Invitrogen). Fluorescence images were captured using a Zeiss Imager M2 microscope, and confocal images were acquired using an Olympus FV1000 microscope.

[0082] The consistency, high levels of spinal cord parenchymal transgene expression, and feasibility of this approach represent a significant technological advancement compared to current approaches and hold potential for direct clinical applications generally aimed at upregulation or silencing of spinal cord genes. Current approaches utilize intrathecal or invasive direct intraparenchymal AAV injection, which have clear limitations in terms of low levels of parenchymal transgene expression or the invasiveness required to achieve more robust infection effects. Furthermore, this approach allows for unparalleled levels of motor and sensory fiber labeling in segments distal to the AAV9 delivery site, making it an extremely robust tool for research to study the range of synaptic connections and identify regulatory factors (i.e., genes, neurotrophic elements) in target spinal cord segments in both rodents and macro-animal species.

[0083] This specification describes examples illustrating the principle of subpia nephrotic AAV9 delivery and the resulting local parenchymal expression, as well as axonal labeling in segments distal to the AAV9 delivery site in adult pigs two months after AAV9 delivery.

[0084] Example 2 Post-single spinal bolus parenchymal AAV9-mediated transgene expression First, the efficacy of single-dose bolus subpilamentous AAV9-UBI-GFP or AAV9-UBI-RFP delivery was tested in rats and pigs. In animals, AAV9 vectors in 20 μl (rats) or 200 μl (pig) of 2.5% dextran solution were delivered into the subpilamentous space of cervical (C4-6), thoracic (Th6-9), or lumbar (L2-L5) segments. Six to eight weeks after AAV9 delivery, spinal cords were dissected from 4% paraformaldehyde-fixed animals and imaged in situ using the Avis fluorescence system. Next, transverse or horizontal spinal cord sections were excised from the AAV9-injected segments and analyzed for the presence of GFP or RFP, and co-stained with neuronal (NeuN) and glial (GFAP) antibodies. In both rat and pig spinal cords, high GFP or RFP expression was observed on the spinal cord surface and was easily identified by visual inspection as yellow-green or red regions. Figures 1H and 1J show the presence of RFP (red) in transverse pig spinal cord and anterior root (Figure 1H, insert) compared to naive spinal cord (Figure 1I). Correspondingly, spinal cord surface concentration analysis of animals injected with AAV9-UBI-GFP revealed that a broad GFP signal diffused from the subpia maternal AAV9 delivery center to 5–10 cm (Figures 1F and 1G).

[0085] Widespread parenchymal RFP expression was observed in horizontally sectioned thoracic sections (the same spinal cord as shown in Figure 1H) taken from pigs that had been pre-injected subpialally with AAV9-UBI-RFP at the midthoracic level. RFP expression was readily identified in most interneurons and alpha motor neurons and was diffused throughout 4-6 spinal cord segments (Figures 3A and 3B). Similarly, high RFP expression was observed throughout the RFP-expressing gray matter at the interaxonal dendritic branching (Figures 3A and 3B, white asterisks).

[0086] Analysis of transverse spinal cord regions taken from the center point of subpia-AAV9 injection revealed high RFP expression throughout the white and gray matter. In the white matter, punctate RFP expression was observed in most transversely segmented axons (Figure 3C, white asterisks, box insert), and was identified in the vertebral column, lateral column, and ventral column (Figure 3C, DF, LF, VF; box insert). In the gray matter, numerous interneurons were dispersed between the thin membrane layers I-IX and anterior α-motor neurons in the anterior horn, and high RFP expression was observed in the cell body and inter-axonal dendritic branching. High-density RFP expression was also observed at nerve fiber terminals in the gray matter (Figures 3D-3G). Similarly, confocal analysis revealed the presence of RFP signaling in astrocytes (Figure 3C, insert: RFP / GFAP).

[0087] A similar neuronal GFP expression pattern was observed at the L1-L2 level in the rat lumbar spinal cord after subpia-articular AAV9-UBI-GFP injection. High-density GFP+ neurons were identified as being present throughout the L1-L5 lumbar segments and localized throughout the gray matter between the first to IX layers of the thin membrane (Figures 9A-9D).

[0088] Example 3 GFP expression in distal spinal cord segments Next, we characterized the extent of inferior spinal canal GFP expression in the lumbar spinal cord after subpial injection of AAV9-UBI-GFP into the subpial space of the midthoracic (Th6-7) or inferior cervical segments in both rats and pigs. High GFP expression was observed throughout the lumbar spinal cord 3–6 weeks after subpial AAV9 delivery. Using transverse lumbar (L2-L6) spinal cord sections taken from pigs, high-intensity GFP expression in transverse axons in the lateral and ventral columns was readily identified without further GFP immunostaining (Figure 4A, white asterisks). Similar densities of GFP+ axons were observed throughout the white matter in these regions. In contrast to the lateral and ventral columns, relatively few GFP+ axons were observed in the vertebral column (Figure 4A, DF). A high-density network of GFP+ axons terminating in gray matter was observed, consistent with the level of axon labeling seen in the white matter of the lateral and ventral columns (Figures 4A and 4B). These axons were identified between layers III and X of the thin film. A very small number of GFP+ axons were observed in laminas I and III. Using a powerful confocal microscope, a high density of fine GFP+ axons was observed along with numerous nerve fiber terminals in the gray matter (Figure 4C).

[0089] In rats that had been previously injected with AAV9-UBI-GFP subpia in the upper neck, a similar GFP expression pattern was observed in the downward motor axons in the lumbar gray matter (Figures 9E and 9F).

[0090] Example 4 Retrograde transgene expression in brain motor regions after spinal cord subpia delivery Six weeks after subpia-cosal AAV9-UBI-GFP delivery in pigs, analysis of transgene (GFP) expression in the brain's motor centers (motor cortex, red nucleus, and reticular formation) revealed highly GFP-labeled pyramidal neurons in the motor cortex (Figures 5A-5E). Similarly, the localization of numerous GFP+ neurons within the brainstem was identified (Figures 5F-5J). Consistent with the presence of GFP+ neurons in the motor cortex, numerous GFP+ cortical spinal axons were observed in the anterior region of the medulla oblongata (pyramidal formation) (Figure 5K). In addition, high-density anterograde-labeled GFP+ spinal reticular terminals were observed throughout the reticular formation (Figure 5L), and spinothalamic terminals were found in the thalamic nuclei (not shown).

[0091] Similarly, as seen in pigs, high-density GFP+ pyramidal neurons were localized in the bilateral motor cortex in rats (Figures 6A-6D). Similar high levels of GFP expression were also observed in the red nucleus, which were easily identified by the presence of bilaterally localized GFP+ nuclear neuron clusters (Figures 6E-6G).

[0092] Example 5 Localized spinal cord transgene expression after intrathecal versus subpia delivery of AAV9 Next, in the same animal (rats), AAV9 was injected into the intrathecal space of the lumbar region (L1-L2) (AAV9-UBI-GFP) and into the subpia space of the thoracic Th7 segment (AAV9-UBI-RFP), and the distribution of spinal cord transgene expression was compared. Three weeks after intrathecal AAV9 injection in the lumbar region, subpia AAV9 injection was performed, and the expression pattern was analyzed in the transverse lumbar spinal cord region three weeks after subpia AAV9 injection. Intrathecal injection of AAV9-UBI-GFP resulted in high GFP expression in the vertebral chords and primary afferent neurons in the dorsal horn (lamina II-III) and the central part of laminas V-VII. Several GFP+Ia afferent neurons terminating in the anterior horn near CHAT+α motor neurons were also identified. Consistent with the high GFP expression in primary afferent neurons, numerous GFP+ neurons were observed in dorsal root ganglion cells (Figure 7B). A clear increase in GFP expression was consistently observed around the anterior root ostial zone (Figures 7A and 7F). Several GFP-expressing glial cells were found in this region. Similarly, 2-3 GFP+ cells were found in the posterior root ostial zone. In the ventral gray matter, several α-motor neurons showed GFP expression (Figure 7G). Apart from these regions and cell populations showing GFP expression, almost complete absence of neuronal or glial GFP expression was observed in other deeper regions of the gray matter (e.g., the white matter of the posterior horn, intermediate zone, and lateral and ventral columns) (Figure 7A). Interestingly, GFP expression was completely absent in neurons localized in the superficial posterior horn and located very close to the intrathecal space (but separated by the pia mater) (Figures 7C and 7D).

[0093] In contrast to the GFP expression pattern observed after intrathecal AAV9-UBI-GFP delivery, RFP expression resulting from cervical subpia nebulosa AAV9 injection showed a substantially different localized expression pattern when analyzed in the same lumbar spinal cord section. dsRED expression was identified in most axons in the white matter and was present in the lateral and ventral columns. Numerous axons protruding into the gray matter of the posterior horn, intermediate zone (lamina VII), and anterior horn were also observed (Figure 7A). Confocal microscopy revealed that virtually all RFP+ fibers were GFP-negative in either the white or gray matter. Interestingly, numerous RFP+ corticospinal axons were observed remaining on the vertebral column very close to GFP-labeled primary afferent nerves, but RFP+GFP co-localization was not observed in any of these fibers (Figure 7E).

[0094] While the present invention has been described with reference to the above examples, it is understood that modifications and alterations are included within the intent and scope of the present invention. Therefore, the present invention is limited only to the following claims.

Claims

1. A gene delivery system for administering nucleic acid molecules into the target subpia space, (a) An L-shaped guide tube configured to puncture the target pia mater, (b) A catheter configured to be slidably positioned within a guide tube and to be advanced into the subpia space of a spinal cord segment of the target vertebra, and (c) A reservoir that is in fluid communication with a catheter and contains a composition comprising nucleic acid molecules, wherein the composition is delivered to the subpia space of a target via the catheter. A gene delivery system that includes this.

2. The gene delivery system according to claim 1, wherein the L-shaped guide tube is a stainless steel tube of 16 to 26 g.

3. The gene delivery system according to claim 1 or 2, wherein the catheter is formed from polyethylene tubing.

4. A gene delivery system according to any one of claims 1 to 3, configured to advance a catheter into the subpia space through a pia mater opening formed by puncturing the pia mater with an L-shaped stainless steel tube.

5. The gene delivery system according to any one of claims 1 to 4, wherein the nucleic acid molecule is a mixture containing about 1 to 10% dextrose.

6. The gene delivery system according to any one of claims 1 to 5, wherein the nucleic acid molecule is a vector or an antisense oligonucleotide (ASO).

7. The gene delivery system according to claim 6, wherein the vector is a lentiviral vector, an adenovirus vector, or an adeno-associated virus (AAV) vector.

8. The gene delivery system according to claim 7, wherein the vector is an AAV9 vector.

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