Spinal subpial gene delivery systems

Abstract

To provide a method for delivering a nucleic acid molecule to the subpial space of a mammal to achieve spinal parenchymal infection thereof, and a system for spinal subpial gene delivery.SOLUTION: Provided herein is a method for delivering a nucleic acid molecule to the subpial space of a subject comprising the steps of: exposing a spinal segment of the vertebra of the subject; creating a pial opening within the spinal segment by puncturing the pia with an L-shaped stainless steel tube; advancing a catheter through the pial opening and into subpial space; and delivering the nucleic acid molecule to the subpial space of the subject, where the nucleic acid molecule is a vector or antisense oligonucleotide (ASO), and a system used in the method is also provided.SELECTED DRAWING: Figure 1A

Description

[Technical field]

[0001] [CROSS-REFERENCE TO RELATED APPLICATIONS] This application claims the benefit of priority to U.S. Provisional Application No. 62 / 110,340 filed in the United States Patent Office on January 30, 2015, the entire contents of which are incorporated herein by reference. [Background technology]

[0002] The present invention relates generally to gene therapy, and more particularly to methods and systems for delivering genes and oligonucleotides into the subpial space of a mammal to effect spinal trans-parenchymal infection thereof.

[0003] Currently used approaches for delivering vectors or antisense oligonucleotides (ASOs) into the spinal cord parenchyma include two techniques, each of which has substantial disadvantages compared to the present invention.

[0004] First, intrathecal delivery is used to inject vectors or ASOs into the spinal intrathecal space (i.e., extrapial). Using this approach, no transgene expression is observed deep in the spinal cord parenchyma after AAV9 delivery. Because the pia mater is impermeable to AAV9, only a subpopulation of A-motoneurons and primary afferents is infected. Although intrathecal delivery of ASOs may result in good penetration of the spinal cord parenchyma, ASOs are found throughout the spinal cord (i.e., from the cervical to sacral segments). Thus, segmentally restricted distribution of ASOs cannot be achieved with intrathecal delivery.

[0005] Second, direct injection into the spinal cord parenchyma can be used. Using this approach, segment-specific transgene expression or ASO distribution can be achieved in the spinal cord parenchyma. However, this technique is disadvantageous due to its invasiveness, since it requires needle penetration directly into the spinal cord parenchyma.

[0006] Therefore, there is a need for a subpial delivery system that allows for nearly complete spinal cord parenchymal AAV9-mediated gene expression or ASO distribution in both white and gray matter. Summary of the Invention

[0007] Effective in vivo use of AAV-based vectors to achieve gene-specific silencing or upregulation in the central nervous system has been limited by their inability to provide more than limited deep parenchymal expression in adult animals using the most clinically relevant delivery routes (i.e., intravenous or intrathecal administration). Therefore, the present invention demonstrates that the spinal pia mater represents a primary barrier that limits effective AAV9 penetration into the spinal parenchyma after intrathecal AAV9 delivery. Therefore, the present invention provides methods and systems for the delivery of genes and oligonucleotides into the spinal parenchyma of large animals and humans.

[0008] Thus, in one embodiment, the present invention provides a method of intraparenchymal spinal infection of a nucleic acid molecule in a subject. The method includes administering the nucleic acid molecule to the subpial space of the subject. The subject can be a mammal, such as a human. In various embodiments, the administering step includes exposing a spinal segment of the spine of the subject, creating a pial opening in the spinal segment, advancing a catheter through the pial opening into the subpial space, and delivering the nucleic acid molecule to the subpial space of the subject. The pial 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 subpial space. In various embodiments, the nucleic acid molecule is administered in a mixture that includes about 1-10% dextrose. In various embodiments, the nucleic acid molecule is a vector or an antisense oligonucleotide (ASO). The vector can be a lentiviral vector, an adenoviral vector, or an adeno-associated viral 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 comprises administering one or more second subpial injections of the nucleic acid molecule into another spinal segment of the spine of the subject.In certain embodiments, the method further comprises administering one or more intrathecal injections of the nucleic acid molecule to the subject.

[0010] In another embodiment, the present invention provides a gene delivery system. The system includes an L-shaped guide tube configured to puncture the pia mater of a subject, a catheter slidably disposed in the guide tube and configured to be advanced into the subpial space of a spinal segment of a subject's spine, and a reservoir in fluid communication with the catheter and containing a composition comprising a nucleic acid molecule. In various embodiments, the L-shaped guide tube can be a 16-26G stainless steel tube, and the catheter can be formed from polyethylene tubing (e.g., PE-5 or PE-10).

[0011] In another embodiment, the present invention provides a method of delivering a nucleic acid molecule to the subpial space of a subject. The method includes exposing a spinal segment of the spine of a subject, creating a pial opening in the spinal segment, positioning a gene delivery system described herein above the spinal segment, advancing a catheter through the pial opening into the subpial space, and delivering the nucleic acid molecule to the subpial space of the subject. In various embodiments, the nucleic acid molecule is delivered in a mixture that includes about 1-10% dextrose. The nucleic acid molecule is a vector or an antisense oligonucleotide (ASO). The vector is a lentiviral vector, an adenoviral vector, or an adeno-associated viral vector (e.g., AAV9 particles). The subject can be a mammal, such as a human. [Brief description of the drawings]

[0012] [Figure 1A] FIG. 1 shows subpial AAV9 delivery and macroscopically defined spinal cord surface transgene expression. Schematic diagram of spinal cord, meninges and subpial placement of a PE-10 catheter in a pig. [Figure 1B] FIG. 13 shows subpial AAV9 delivery and macroscopically defined spinal cord surface transgene expression, showing a catheter guide tube (18G) with a sharp pial puncture tip (insert) that is used to puncture the pia and advance a PE-10 catheter into the subpial space. [Fig. 1C-1E] 1 shows subpial AAV9 delivery and macroscopically defined spinal cord surface transgene expression. The diagram shows the advancement of catheter placement into the subpial space. First, the dura is cut open (FIG. 1C) and the catheter is advanced into the subpial space (FIGS. 1D and 1E). Air bubbles can be seen injected into the subpial space (FIG. 1D-asterisk). [Fig. 1F-1J] Figure 1F and 1G show surface GFP fluorescence densitometry, which shows strong signals in both porcine and rat spinal cords with the highest intensity of GFP fluorescence seen at the epicenter of the lumbar subpial injection. The presence of high RFP fluorescence in the spinal cord parenchyma is macroscopically detected in the porcine thoracic spinal cord (Figure 1H and 1J). A clear high level of RFP expression in the ventral roots can also be seen (Figure 1H-insert). No fluorescence is seen in the control sample without spinal injection (Figure 1I). [Diagram 2] FIG. 1 shows insertion of a PE-10 catheter into the subpial space and GFP expression throughout the spinal cord parenchyma and in axons projecting distally from the AAV9-injected segment. [Diagram 3] Figure 3 shows effective parenchymal AAV9-mediated transgene expression after a single bolus subpial AAV9-UBI-RFP injection in adult pigs. Figures 3A and 3B show spinal cord horizontal sections taken from the mid-thoracic spinal cord of a pig previously injected 6 weeks with AAV9-UBI-RGF. High RFP expression can be seen throughout the region, including white and grey matter. When stained with NeuN antibody (green), virtually all neurons were also found to be RFP positive. Figures 3C-3G show images of spinal cord transverse sections taken from the subpial injection region, showing transversely sectioned RFP+ axons (square inset) in the dorsal (DF) lateral funiculus (LF) and ventral funiculus (VF). RFP expression is also seen in GFAP-stained astrocytes (inset; RFP / GFAP). High density of RFP+ nerve fiber terminals was observed around RFP-expressing α-motoneurons (Figures 3D and 3E) and interneurons (Figures 3F and 3G) (Scale bars: Figures 3A-3C = 500 μm; Figures 3D, 3F = 30 μm). [Figure 4] Strong GFP expression in the lower motor axons in the lumbar spinal cord after mid-thoracic subpial AAV9 injection in pigs. Figures 4A and 4B show spinal cord cross sections taken from the lumbar spinal cord after 6 weeks of prior subpial AAV9-UBI-GFP injection into the mid-thoracic subpial space. High GFP expression in axonal profiles in the lateral funiculus (LF) and ventral funiculus (VF) is seen (white asterisks). A relatively low density of GFP+ axons was identified in the spinal dorsal funiculus (DF). In contrast, a high density of GFP+ motor axons can be seen projecting into the grey matter located between the dorsal horn (DH) and anterior horn (VH). Figure 4C is a higher resolution confocal image showing extremely fine arborization of GFP+ axons and nerve fiber terminals in the central grey matter. (Scale bars: 4A = 1000 μm; 4C = 30 μm), (DH-dorsal horn, VH-ventral horn, DF-dorsal funiculus, LF-lateral funiculus, VF-ventral funiculus). [Diagram 5] Figures 5A-5L show retrograde transport-mediated GFP expression in the brain motor center after mid-thoracic subpial AAV9 delivery in adult pigs. Figures 5A-5E show retrogradely 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 comparable levels of GFP expression in neurons localized in the brainstem. Figure 5K shows the presence of large retrogradely labeled motor GFP+ axons in the medulla oblongata (pyramids). Figure 5L shows a high density of anterogradely labeled sensory afferents in the reticular formation (scale bars: Figures 5A-5E and 5G-5J = 50 μm; Figures 5K and 5L = 50 μm). [Figure 6] Retrograde transport-mediated GFP expression in the brain motor center after cervical subpial AAV9 delivery in adult rats. Figures 6A-6D show bilateral retrograde-GFP labeled pyramidal neurons in the rat motor cortex 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 differential spinal transgene expression following intrathecal AAV9-UBI-GFP versus subpial AAV9-UBI-RFP delivery in rats. Figure 7A shows that lumbar intrathecal injection of AAV9-UBI-GFP results in preferential GFP expression in the spinal dorsal funiculus (DF), dorsal root (DR) and ventral root entrance zone (white square insert #2). Subpial AAV9-UBI-RFP injection into the upper cervical spinal cord results in distinct labeling of the descending motor tract. Figure 7B shows GFP expression in dorsal root ganglion cells (L4) following lumbar intrathecal AAV9-UBI-GFP injection. Figures 7C and 7D show high GFP expression following intrathecal AAV9-UBI-GFP injection in primary afferent processes (boutons) in the dorsal root (DR) and in the deeper dorsal horn (white star), but no expression in dorsal horn NeuN+ neurons. Figure 7E shows no colocalization of GFP and RFP in the spinal dorsal funiculus (DF) (white inset in Figure 7A, No. 1). Figure 7F shows GFP expression in glial cells localized in the ventral root entrance zone resulting from intrathecal AAV9-UBI-GFP injection (white inset in Figure 7A, No. 2). Figure 7G shows that in animals injected with AAV9-UBI-GFP, there are several retrogradely labeled GFP-expressing α-motor neurons surrounded by GFP+ primary Ia afferents (scale bars: Figure 7A = 500 μm; 7B-7G = 30 μm), (DR-dorsal root, DH-dorsal horn, VH-ventral horn, DF-dorsal funiculus). [Figure 8]FIG. 1 shows subpial AAV9 delivery. Schematic showing transgene (GFP) expression throughout the CNS after a single subpial AAV9-UBI-GFP injection. In adult pigs, AAV9-UBI-GFP virus is delivered into the subpial space using a PE-10 catheter. Subpial delivery of AAV9-UBI-GFP results in diffusion and uptake of the virus beyond the subpial injection segment into segmental neurons (i.e., interneurons and alpha motor neurons) and upward and downward axons. The resulting transgene expression is then seen in: i) segmental neurons, ii) dorsal root ganglion cells (retrograde infection), iii) motor axons innervating skeletal muscles (anterograde infection), iv) pyramidal neurons in the motor cortex (retrograde infection), and v) brain terminals of spinothalamic neurons (anterograde infection). [Figure 9] Figure 9A-9D shows efficient parenchymal AAV9-mediated transgene expression after a single bolus lumbar subpial AAV9-UBI-GFP injection in 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 subpial AAV9-UBI-GFP injection. Virtually all neurons in horizontal sections (Figure 9A) show GFP expression. In transverse sections, GFP+ neurons are found throughout the gray matter between laminae I-IX (Figures 9B-9D). Numerous NeuN-stained neurons are found expressing GFP in the superficial dorsal horn (lamina I-III) and anterior horn. Figures 9E and 9F show a high density of GFP+ descending 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-dorsal horn, VH-ventral horn, DF-dorsal funiculus). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] The present invention provides methods and systems for gene and oligonucleotide delivery 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 these may vary. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention is limited only by the appended claims.

[0015] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include the plural forms unless the context clearly dictates otherwise. Thus, for example, reference to "the method" includes one or more methods and / or steps of the type described herein that would become apparent to one of ordinary skill in the art upon reading this disclosure.

[0016] The term "comprising" is used synonymously with "including," "containing," or "characterized by" and is inclusive or open-ended language and does not exclude additional, unrecited elements or method steps. The phrase "consisting of" excludes any element, step, or ingredient not recited in the claim. The phrase "consisting essentially of" limits the claim to the recited materials or steps and those that do not materially affect the basic and novel characteristics of the claimed invention. The present disclosure contemplates embodiments of the compositions and methods of the invention that fall within the scope of each of these phrases. Thus, a composition or method that includes recited elements or steps contemplates certain embodiments in which the composition or method consists essentially of or consists of those elements or steps.

[0017] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, and suitable methods and materials are described below.

[0018] As used herein, the term "pia mater" refers to the innermost layer of the meninges, the membrane that surrounds the brain and spinal cord (FIG. 1A). The pia mater is a thin fibrous tissue that is impermeable to fluids. As such, it can contain cerebrospinal fluid. By containing fluid, the pia mater, along with the other meningeal layers, acts as a protection and cushion for the brain. The spinal pia mater encloses the spinal cord surface or spinal cord and is attached and anchored to the anterior fissure of the spinal cord. Thus, the term "subpial" refers to occurring under or beneath the pia mater.

[0019] As used herein, the term "parenchyma" refers to the functional tissue of an organ, as distinguished from the connecting and supporting tissues. Thus, the term "spinal cord parenchyma" refers to the various known anatomical tissues of the spinal cord, including, but not limited to, the gray matter, white matter, dura mater, arachnoid mater, pia mater, posterior and ventral columns, posterior spinocerebellar tracts, and anterior spinocerebellar tracts.

[0020] The term "subject" as used herein refers to any individual or patient to whom the method is administered.Generally, the subject is a human, but those skilled in the art 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., cows, horses, goats, sheep, pigs), and primates (e.g., monkeys, chimpanzees, orangutans and gorillas)) are included in the definition of subject.

[0021] As used herein, "treatment" refers to a clinical intervention performed on a disease, disorder or physical condition that a patient / subject manifests or is susceptible to. Non-limiting therapeutic objectives include reducing or avoiding symptoms, slowing or stopping the progression or worsening of a disease, disorder or condition, and / or alleviating a disease, disorder or condition. "Treatment" refers to one or both of treatment and preventive or preventative measures. Subjects in need of treatment include those who already suffer from a disease or disorder or undesirable physical condition, and those who need to avoid a disease or disorder or undesirable physical condition.

[0022] As used herein, "nucleic acid" and "polynucleotide" are synonymous and refer to any nucleic acid, whether phosphodiester or modified (e.g., phosphotriester, phosphoramidite, siloxane, carbonate, carboxymethylester, acetamidite, carbamate, thioether, bridged phosphoramidite, bridged methylene phosphonate, bridged phosphoramidite, bridged phosphoramidite, bridged methylene phosphonate, phosphorothioate, methylphosphonate, phosphorodithioate, bridged phosphorothioate or sultone, and combinations of such bonds). "Nucleic acid" and "polynucleotide" also specifically 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 interchangeably herein to refer to a polymer of amino acid residues. These terms apply to amino acid polymers in which one or more amino acid residues are artificial chemical mimetics of a corresponding naturally occurring amino acid, and to naturally occurring 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 mimetics that function similarly to naturally occurring amino acids. Naturally occurring amino acids refer to amino acids encoded by the genetic code, as well as amino acids 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 (i.e., an α carbon attached to a hydrogen, a carboxyl group, an amino group, and an R group) as naturally occurring amino acids (e.g., homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium). Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as naturally occurring amino acids. Amino acid mimetics refer to compounds that have a structure that differs from the general chemical structure of an amino acid, but that function similarly to a naturally occurring amino acid.

[0025] Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides may also be referred to by their commonly accepted one-letter symbols.

[0026] "Conservatively modified variants" applies to both amino acid and nucleic acid sequences. For a particular nucleic acid sequence, conservatively modified variants refer to nucleic acids that code for identical or essentially identical amino acid sequences, or, if the nucleic acid does not code for an amino acid sequence, to essentially identical sequences. Due to the degeneracy of the genetic code, a large 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 any of the corresponding codons described without changing the encoded polypeptide. Such nucleic acid variations are "silent variations," a type of conservatively modified variation. Each nucleic acid sequence herein that codes for a polypeptide also represents each possible silent variation of the nucleic acid. Those skilled in the art will recognize that each codon in a nucleic acid (except AUG, which is generally the only codon for methionine, and TGG, which is generally the only codon for tryptophan) can be modified to obtain a functionally identical molecule. Thus, each silent variation of a nucleic acid that codes for a polypeptide is implied in each described sequence.

[0027] For amino acid sequences, one of skill in the art will recognize that individual substitutions, deletions, or additions to a nucleic acid, peptide, polypeptide, or protein sequence 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 alteration results in the replacement of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to, and do not exclude, polymorphic variants, interspecies homologs, and alleles of the invention.

[0028] A "polynucleotide" is a single- or double-stranded polymer of deoxyribonucleotide or ribonucleotide bases read out from the 5' to 3' end. 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 as base pairs (abbreviated as "bp"), nucleotides ("nt"), or kilobases ("kb"). When the context permits, the latter two terms may describe a single- or double-stranded polynucleotide. When the term is applied to a double-stranded molecule, it refers to the overall length and is understood as equivalent to the term "base pairs." Those skilled in the art will recognize that not all nucleotides in a double-stranded polynucleotide molecule may be paired, since the two strands of a double-stranded polynucleotide may differ slightly in length and may have their ends interleaved as a result of enzymatic cleavage.

[0029] The term "gene" broadly refers to any fragment of nucleic acid associated with a biological function. Thus, a gene includes coding and / or regulatory sequences necessary for its expression. For example, "gene" refers to a nucleic acid fragment that expresses an mRNA, a functional RNA, or a specific protein, including regulatory sequences. "Gene" also includes non-expressed DNA fragments that, for example, form recognition sequences for other proteins. "Gene" can be obtained from a variety of sources (e.g., cloned from a source of interest or synthesized from known or predicted sequence information) and can include sequences designed to have desired parameters. An "allele" is one of several alternative forms of a gene that occupy a given locus on a chromosome.

[0030] As used herein, a "vector" is capable of introducing a gene sequence into a target cell. Typically, "vector construct", "expression vector" and "gene transfer vector" refer to any nucleic acid construct capable of directing the expression of a gene of interest and introducing a gene sequence into a target cell. Thus, the term includes cloning and expression vehicles, as well as integrating vectors.

[0031] Viral vectors can be particularly useful in introducing polynucleotides useful in carrying out the methods of the present invention into target cells. Viral vectors have been developed for use in specific host systems, particularly mammalian systems, and include, for example, retroviral vectors, other lentiviral vectors, such as those based on human immunodeficiency virus (HIV), adenovirus vectors (AV), adeno-associated virus vectors (AAV), herpes virus vectors, and vaccinia virus 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), each of which is incorporated herein by reference). In one embodiment of the present invention, lentivirus or adenovirus vector is used.Adenovirus is a double-stranded DNA virus, and both strands of DNA code for genes.Genome codes for about 30 proteins.In another embodiment of the present invention, adeno-associated virus vector is used.

[0032] The term "adenovirus" refers to more than 40 adenovirus subtypes isolated from humans, as well as many from other mammalian and avian species. 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., those described in McGrory WJ,et al., Virology 163: 614-617, 1988) lack essential early genes (usually E1A / E1B) from the adenovirus genome and are therefore unable to replicate unless grown in a permissive cell line that provides the deleted gene product in trans. In place of the deleted adenovirus genomic sequences, a transgene of interest is cloned and expressed in tissues / cells infected with a replication-defective adenovirus. Although adenovirus-based gene transfer generally cannot stably integrate the transgene into the host genome (less than 0.1% of adenovirus-mediated transfections result in transgene introduction into the host DNA), adenovirus vectors can propagate at high titers and can transfect non-replicating cells, but the transgene is not passed to daughter cells, making them suitable for gene transfer into non-actively dividing adult cardiomyocytes. Retroviral vectors provide stable gene transfer, and high titers can now be obtained through retroviral pseudotyping (Burns, et al., Proc. Natl. Acad. Sci. (USA) 90: 8033-8037, 1993), but current retroviral vectors generally cannot efficiently transduce non-replicating cells.

[0033] Further references describing adenoviral and other viral vectors that cannot be used in the methods of the present invention include: 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., WO 95 / 00655 (5 Jan. 1995); Falck-Pedersen, ES, WO 95 / 16772(22 Jun. 1995); Denefle, P. et al., WO 95 / 23867(8 Sep. 1995); Haddada, H. et al., WO 94 / 26914(24 Nov. 1994); Perricaudet, M. et al., WO 95 / 02697(26 Jan. 1995); Zhang, W., et al., WO 95 / 25071 (12 Oct. 1995).A variety of adenovirus plasmids are also available from commercial sources (e.g., Microbix Biosystems, Toronto, Ontario) (see, e.g., Microbix Product Information Sheet: Plasmids for Adenovirus Vector Construction, 1996).

[0034] Adeno-associated virus (AAV) is a small (26 nm), replication-defective, non-enveloped virus that depends on the presence of a second virus (e.g., adenovirus or herpesvirus) for cell proliferation. AAV is not known to cause disease and induces a very mild immune response. AAV can infect both dividing and non-dividing cells and transfer its genome into host cells. Aspects of the present invention provide methods for delivering transgenes to spinal tissue in a subject using recombinant AAV-based gene transfer.

[0035] Further references describing AAV vectors that may be used in the methods of the 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., WO 95 / 13365 (18 May 1995); Trempe, JP, et al., WO 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, WO 96 / 17947 (13 Jun. 1996); and Du et al., Gene Therapy 3: 254-261, 1996. See also U.S. Patent No. 8,865,881, which is incorporated herein by reference.

[0036] An "effective amount" of AAV is an amount sufficient to infect a sufficient number of cells of a target tissue in a subject. An effective amount of AAV can be an amount sufficient to provide a therapeutic benefit in a subject (e.g., extending the lifespan of a subject, improving one or more symptoms of a disease in a subject (e.g., symptoms of a neurodegenerative disease)). An effective amount can vary depending on a variety of factors (e.g., species, age, weight, health status and target tissue of a subject, and therefore can vary from subject to subject and tissue. An effective amount can also vary depending on the mode of administration. For example, targeting CNS tissue by intravascular injection may require a different dosage (e.g., a higher dosage) than targeting CNS tissue by intrathecal or intracerebral injection. In some cases, multiple doses of AAV are administered. An effective amount can also depend on the particular AAV used. For example, the dosage to target CNS tissue can depend on the serotype (e.g., capsid protein) of AAV. For example, the AAV can have an AAV serotype with 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, an effective amount of AAV is 10 10 , 10 11 , 10 12 , 10 13 , or 10 14 In certain embodiments, an effective amount of AAV is 10 genome copies per subject. 10 , 10 11 , 10 12 , 10 13 , 10 14 , or 10 15 genome copies.

[0037] Depending on the host cell / vector system used, any of a number of transcription and translation elements (e.g., constitutive and inducible promoters, transcription enhancer elements, transcription terminators, etc.) can be used in the expression vector (Bitter et al., Meth. Enzymol. 153:516-544, 1987). For example, when cloning in bacterial systems, inducible promoters (e.g., bacteriophage pL, plac, ptrp, ptac (ptrp-lac hybrid promoter), etc.) can be used. When cloning in mammalian cell systems, promoters derived from the genome of mammalian cells (e.g., human or mouse metallothionein promoters) or from mammalian viruses (e.g., retroviral long terminal repeats), adenovirus late promoters, or vaccinia virus 7.5K promoters can be used. Promoters produced by recombinant DNA or synthetic techniques can also be used for transcription of the inserted GDF receptor coding sequence.

[0038] As used herein, "reporter gene" or "reporter sequence" refers to any sequence that preferably, but not necessarily, produces a protein product that is easily measured in a routine assay. Non-limiting examples of suitable reporter genes include sequences that code for proteins that mediate antibiotic resistance (e.g., ampicillin resistance, neomycin resistance, G418 resistance, puromycin resistance), sequences that code for colored or 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, one or more copies of FLAG, His, myc, Tap, HA, or any detectable amino acid sequence. Examples of "expression tags" include sequences that code for reporters that can be operably linked to a desired gene sequence to monitor the expression of the gene of interest.

[0039] As used herein, the terms "transformation" or "transfect" are used interchangeably and refer to the process by which exogenous DNA or RNA is introduced and introduced into a suitable host cell. Additionally, nucleic acids encoding other heterologous proteins can be introduced into the host cell. Such transfected cells include stably transfected cells, where the inserted DNA allows for replication in the host cell. Typically, stable transfection requires the exogenous DNA introduced with a selectable marker nucleic acid sequence (e.g., a nucleic acid sequence providing antibiotic resistance), thereby allowing the selection of stable transfectants. This marker nucleic acid sequence may be linked to the exogenous DNA or may be provided independently by simultaneous co-transfection with the exogenous DNA. Transfected cells also include transiently expressing cells that are capable of allowing expression of RNA or DNA to occur for a limited period of time. The transfection procedure depends on the host cell being transfected. It may include packaging of the polynucleotide into a virus or direct uptake of the polynucleotide. Transformation can result in the introduction of the inserted DNA into the genome of the host cell or the maintenance of the inserted DNA in the host cell in the form of a plasmid. Methods of transformation / transfection are well known in the art and include, but are not limited to, direct injection, e.g., microinjection, viral infection, particularly replication-deficient adenovirus infection, electroporation, lipofection, and calcium phosphate-mediated direct uptake.

[0040] As used herein, a nucleic acid sequence (e.g., a coding sequence) and a regulatory sequence are said to be operably linked when they are covalently linked in such a way that expression or transcription of the nucleic acid sequence occurs under the influence or control of the regulatory sequence. When it is desired to translate the nucleic acid sequence into a functional protein, two DNA sequences are said to be operably linked when induction of a promoter in the 5'regulatory sequence results in transcription of the coding sequence and when the binding 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 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 linked to a nucleic acid sequence when it is capable of causing transcription of the DNA sequence such that the resulting transcript is translated into a desired protein or polypeptide. Similarly, two or more coding regions are operably linked when they are joined such that transcription from a common promoter of the two or more coding regions results in expression of two or more proteins translated in frame. In some embodiments, the operably linked coding sequences result in a fusion protein. In some embodiments, the operably linked coding sequence provides a functional RNA (eg, shRNA, miRNA).

[0041] In the case of nucleic acids encoding proteins, a polyadenylation sequence may be inserted after the transgene sequence and before the 3'AAVITR sequence. AAV constructs useful in the present invention may also include an intron, desirably located 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 an 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 that include 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. It is a small peptide (approximately 18 amino acids in length) that has been shown to mediate polyprotein cleavage (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 2A sequences 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] Although the exact nature of the regulatory sequences required for gene expression in a host cell may vary between species, tissues or cell types, they generally include 5' non-transcribed and 5' non-translated sequences involved in the initiation of transcription and translation, respectively, as necessary (e.g., TATA box, capping sequence, CAAT sequence, enhancer element, etc.). In particular, such 5' non-transcribed regulatory sequences include promoter regions that contain promoter sequences for transcriptional control of operably linked genes. Regulatory sequences may optionally include enhancer sequences or upstream activator sequences. Vectors of the present invention may optionally include 5' leader or signal sequences. The selection and design of appropriate vectors is within the ability and discretion of one of ordinary skill in the art.

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

[0044] In some embodiments, the regulatory sequence confers tissue-specific gene expression capability. In some cases, the tissue-specific regulatory sequence binds 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 art. Exemplary tissue-specific regulatory sequences include, but are not limited to, 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 a promoter of a gene selected from the following: neuronal nucleus (NeuN), glial fibrillary acidic protein (GFAP), adenomatous polyposis coli (APC), and ionized calcium-binding adaptor molecule 1 (Iba-1). Other suitable tissue-specific promoters will be apparent to those skilled in the art. In some embodiments, the promoter is a chicken beta-actin promoter.

[0045] In some aspects, the present invention provides AAV vectors for use in methods of avoiding and treating one or more genetic defects (e.g., inherited genetic defects, somatic genetic mutations) in a mammal (e.g., genetic defects causing a polypeptide deficiency or excess in a subject), particularly treating or reducing the severity or extent of the deficiency in a subject exhibiting a CNS-related disorder associated with a deficiency of such polypeptide in cells and tissues. In some embodiments, these methods include administering to a subject an AAV vector encoding one or more therapeutic peptides, polypeptides, shRNAs, microRNAs, antisense nucleotides, etc., in a pharma- tically acceptable carrier in an amount and duration sufficient to treat the CNS-related disorder in a subject having or suspected of having such a disorder. "Antisense inhibition" refers to the production of antisense RNA transcripts capable of suppressing protein expression from an endogenous gene or transgene.

[0046] Thus, AAV vectors can contain as transgenes nucleic acids that code for proteins or functional RNAs that regulate or treat CNS-related disorders. The following is a non-limiting list of genes related to CNS-related disorders: neuronal apoptosis inhibitor protein (NAIP), nerve growth factor (NGF), glial cell line-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 codes for TH, which is the rate-limiting enzyme in dopamine synthesis. A transgene that codes for GTPCH, which produces the TH cofactor tetrahydrobiopterin, can also be used in the treatment of Parkinson's disease. Transgenes encoding GDNF or 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 diseases (e.g., mucopolysaccharidosis type VII (MPSVII)). Transgenes encoding prodrug activating genes (e.g., HSV-thymidine kinase, which converts ganciclovir into toxic nucleotides that disrupt synthesis and 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 vectors 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 the past few decades, several experimental and / or clinical studies have reported on the successful use of AAV-based vectors (especially AAV9) for CNS-targeted gene delivery. These studies clearly establish the value of AAV-delivered vectors as a tool in potent gene upregulation or silencing in targeted CNS regions, providing evidence that this treatment approach can be effectively used in the treatment of a number of neurodegenerative diseases (e.g., ALS, SMA, muscle spasticity, and chronic pain). Despite these encouraging data and extensive preclinical animal studies, the detailed mechanisms of how AAV vectors reach the brain or spinal cord parenchyma after the use of different AAV delivery routes (systemic, intrathecal) have not been fully elucidated. These data are crucial in the development of novel, more effective AAV delivery protocols that are equally potent in young and fully developed adult animals and human subjects. In general, preclinical animal studies can be divided into several groups based on the developmental stage of the animals used or the delivery route of AAV (e.g., systemic or intrathecal). Depending on the parameters used in each individual study, the levels of transgene expression and specific cell populations (neurons and / or glia) infected vary widely.

[0048] Previous studies have shown that systemic intravenous (iv) injection of AAV9-GFP in neonatal mice results in widespread CNS GFP expression, including dorsal root ganglions, spinal motor neurons (MNs), and neurons throughout the brain (neocortex, hippocampus, and cerebellum). In adult mice, iv delivery of AAV9-GFP results in preferential infection of astrocytes throughout the CNS, but with limited neuronal expression (Foust et.al., (2009)). Intravascular AAV9 preferentially targets neonatal neurons and adult astrocytes (Nature biotechnology 27: 59-65). Comparable data have been reported showing widespread spinal MN GFP expression after systemic intravenous (iv) delivery of AAV9 in neonatal mice (Duque et al. (2009)). Intravenous administration of self-complementary AAV9 allows 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 shows successful transgene expression in spinal MNs after AAV9 delivery in adult mice or cats. Similar to these two studies, Gray et al. show CNS neuronal GFP expression after iv administration of AAV9 in adult mice, however, transduction efficiency in juvenile non-human primates is only 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 17: 1187-1196). Gene Therapy 19: 1058-1069)).

[0049] Because of this apparent restriction of neuronal expression after systemic iv AAV9 delivery in adult animals, the use of the intrathecal route (to bypass the blood-brain barrier) has been investigated in several studies. In 1-year-old 2 kg BW non-human primates (cynomolgus monkeys), a single lumbar intrathecal injection of AAV9-CB-GFP resulted in 50-75% MN transduction in the entire spinal cord 2 weeks after AAV9 injection. Similarly, in young 2-3 year-old non-human primates (cynomolgus monkeys) or young (2 month-old) pigs, robust MN transduction in the entire spinal cord was seen after intracisternal or intracisternal-lumbar intrathecal AAV9 delivery. In addition, GFP expression was seen in dorsal root ganglion cells, motor cortex, and cerebellar cortex in the same study. In a more recent study, comparable lumbar MN GFP expression was seen in non-human primates (cynomolgus monkeys) after intracisternal AAV9-GFP injection.

[0050] Interestingly, in all studies using either the cisternal or lumbosacral intrathecal delivery route, a highly idiosyncratic spinal transgene expression pattern was observed, in review of available historical data. This was characterized by high expression in alpha motor neurons, but the remaining anterior horn interneurons in the vicinity of the GFP-expressing alpha motor neurons were found to be transgene negative. Similarly, strong transgene expression was seen in dorsal root ganglion cells and primary afferents, but no transgene expression was seen in small interneurons localized in the superficial dorsal horn or intermediate zone.

[0051] This high level of transgene expression selectivity and lack of infection of deeper gray matter cells indicates 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 segments. Based on the well-described anatomical organization of the spinal cord, it was hypothesized that the pia mater is the primary barrier regulating AAV penetration into the spinal cord parenchyma after intrathecal AAV delivery. Thus, it is speculated that the high level of transgene expression seen in MN and DRG cells after intrathecal delivery is likely mediated by preferential retrograde and anterograde infection of axons that spread within and outside the spinal cord parenchyma and pass through the intrathecal space (i.e., anterior and dorsal roots).

[0052] Therefore, to address this issue, the present invention provides a method for subpial vector delivery in mammals, which route of delivery results in infection of the entire population of neurons in the gray matter of the subpial injection segment with robust intraparenchymal transgene expression in the spinal cord. In addition, nearly complete infection of the descending and ascending axons was achieved, consistent with transgene expression in the central brain (i.e., motor cortex, red nucleus).

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

[0054] As used herein, "PE-10" refers to polyethylene tubing having 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 having an inner diameter of approximately 0.005 inches. In certain embodiments, the inner diameter of PE-5 tubing is approximately 0.008 inches.

[0055] Thus, the claimed system 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 white and gray matter of the treated subject. Currently available non-invasive techniques do not allow for similar levels of spinal parenchymal transgene expression or well-controlled segment-specific gene silencing.

[0056] To place a subpial catheter in a mammalian subject, several sequential procedure steps may be followed to minimize the possibility of spinal cord injury associated with instrument / catheter manipulation in the vicinity of the exposed "dural" spinal cord. In various embodiments, the use of caudal and cranial spinal clamps (placed 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., 16-26G stainless steel tube bent at 90°) mounted on an XYZ manipulator (e.g., as described in U.S. Patent 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 with a bent 30G needle. When the tip of the pierced 30G needle is approximately 1-1.5 mm into the subpial space, the pia mater is slightly elevated by 1-2 mm. The subpial catheter is then positioned in the subpial space by advancing the catheter from the guide tube. After the catheter has been advanced to the target length, the penetrating needle tip of the guide tube is removed from the subpial space. After vector injection is completed (typically for 2-5 minutes and in some embodiments, for about 3 minutes), the catheter is withdrawn from the subpial space and the dura is closed. Using this technical approach, the placement of the subpial catheter can be achieved within about 3-5 minutes from the moment the dura is opened.

[0058] Using this technique, the subpial catheter described herein was successfully placed in 17 pigs, achieving consistent and injury-free subpial catheter placement. In adult rats, the same technique was used, but with a PE-5 catheter instead. The data obtained with these adult rats and pigs showed: i) strong spinal cord parenchymal transgene expression in white and gray matter, including neurons and glial cells, after a single bolus subpial AAV9 delivery, ii) delivery to nearly all of the lower motor axons in the entire length of the spinal cord after cervical or thoracic subpial AAV9 injection, iii) strong retrograde transgene expression in brain motor centers (motor cortex and brainstem), and iv) safety of this approach, with normal neurological function defined for up to 3 months after AAV9 delivery. Thus, subpial delivery of AAV-9 allows gene-based treatments 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 a PE-10 catheter. After placing this guide tube directly above the spinal pia mater, the PE-catheter was advanced into the subpial space through a small pial opening (FIGS. 1A-1E). AAV9 or ASO was then injected into the subpial space. In a specific embodiment, AAV9 was delivered in a mixture containing 1-10% dextran (10,000-30,000 MW) to allow AAV9 particles to persist longer in the spinal cord parenchyma. Following AAV9-GFP delivery, consistent GFP expression was seen throughout the spinal cord parenchyma at the level of injection and in axons projecting distally (into the lumbar spinal cord) from the AAV9-injected segment.

[0060] As shown herein, a single subpial AAV9 injection resulted in robust intraparenchymal transgene expression with rostro-caudally spreading in multiple segments. Thus, subpial AAV9 delivery leads to widespread transgene expression in neurons throughout the grey matter and upper and lower axons in the subpial injected segment. For example, transgene spreading was consistently observed in adult pig spinal cords at a distance of approximately 10-15 cm from the injection site. Expression was identified in neurons and glial cells in the entire grey matter laminae and axons in the spinal cord ventral, lateral and dorsal funiculus, confirming nearly complete penetration of the subpial injected AAV9 vector throughout the spinal cord parenchyma. By analyzing transverse lumbar spinal cord sections in pigs with mid-thoracic AAV9-UBI-GFP injections (i.e., approximately 30 cm from the AAV9 delivery site), virtually all lower motor axons appeared to be labeled 6 weeks after AAV9 injection. Higher resolution confocal microscopy revealed a dense network of fine axonal arborizations containing nerve fiber terminals throughout the gray matter. Consistent with the level and distribution of infected axons in the white matter, retrogradely 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 following subpial versus intrathecal AAV9 delivery in rats, the present invention demonstrated substantially different regional cellular expression, thus indicating that the pia mater represents a primary barrier for effective parenchymal penetration of AAV9 following intrathecal delivery.

[0062] First, after intrathecal delivery, expression was only seen in areas and neuroglial pools morphologically associated with the dorsal and ventral root entry zones. Thus, strong transgene expression was seen in primary afferents, clearly present in the dorsal funiculus, primary afferents in the dorsal horn, and Ia afferents projecting to the ventral horn. This transgene expression in primary afferents coincided with strong expression in dorsal root ganglion cells. Similarly, there was clear expression around the ventral root entry zone, including several retrogradely labeled alpha motor neurons in the ventral horn. In contrast, however, transgene expression was virtually absent in all other neurons between lamina I-VII and X, and no descending motor axons were labeled in the lateral or ventral funiculus. Similarly, no transgene expression was seen in axons of the corticospinal tract (localized at the base of the dorsal funiculus) surrounded by GFP-expressing primary afferents in rats. Without wishing to be bound by theory, these data taken together indicate that spinal cord parenchymal GFP expression may result from retrograde or anterograde transgene expression (whether in neurons or in projecting primary afferents) and not from uptake of AAV9 from the intrathecal space into the spinal cord parenchyma. This finding is consistent with data from other laboratories showing robust α-motor neuron GFP expression after a single intrathecal or cisternal AAV9-GFP injection in adult non-human primates or juvenile pigs. However, no interneuron GFP expression was seen in interneurons in close proximity to GFP+ α-motor neurons (Meyer et al., (2015). Enhancing single-injection CSF delivery of AAV9-mediated gene therapy for SMA: a dose-response study 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 intrathecal delivery of self-complementary AAV9-survival motor neuron 1 for spinal muscular atrophy (Human gene therapy 25: 619-630; Bell, et al. (2015)). Motor neuron transduction following intracisternal delivery of AAV9 in cynomolgus monkeys (Human gene therapy methods 26: 43-44)).

[0063] In contrast, as described above, subpial AAV9 delivery was associated with strong transgene expression in gray matter neurons (i.e., alpha motor neurons and interneurons) and in virtually all of the lower motor axons and primary afferents of the injected segment. These data clearly indicate that the pia mater is a primary barrier that prevents AAV9 penetration into other spinal segments located away from the anterior and dorsal root entrance zones. By bypassing the pia mater and depositing AAV9 into the subpial space, white and gray matter intraparenchymal infection can be effectively achieved in adult rodents or large animals.

[0064] Thus, the claimed method and system can be used in subjects to increase axonal sprouting after spinal cord injury by upregulating the expression level of neurotrophic genes in lower motor axons.Furthermore, this local delivery of ASO allows for the restricted silencing of targeted fragments of genes associated with the progression of chronic pain or muscle spasms, but without the supraspinal side effects that usually occur after intrathecal ASO delivery.

[0065] The efficacy of subpial-induced infection and neuronal cell populations infected in the spinal cord and brain in adult animals has several high clinical and experimental significance. First, when a specific gene is to be silenced or downregulated, a single cervical subpial injection of a silencing AAV9 construct can result in effective gene silencing in cervical neurons and glial cells in 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 upper motor neurons and projecting descending motor axons, lower motor neurons and spinal interneurons, the ability to achieve widespread mutant gene silencing is likely to provide a substantial advantage in achieving the highest therapeutic efficacy. In addition, a single cervical subpial injection may be combined with one or more additional subpial injections into the lumbar enlargement to target lumbar neuronal / glial populations, and / or with lumbar intrathecal injection to target the alpha motor neuron pool throughout the thoracic and lumbar spinal cord. Second, increased expression of therapeutic genes (e.g., growth elements) associated with axonal sprouting can be easily achieved in the lower motor tract and ascending sensory fibers and tested for therapeutic efficacy in, for example, spinal cord injury studies. In this case, AAV9 vectors can be administered using a subpial catheter from a single laminectomy site at the injury epicenter, and this subpial catheter can be systemically extended rostrally and caudally to target the distal ends of severed motor axons and the proximal ends of ascending sensory axons, respectively. Third, the near-complete lower motor tract labeling that can be achieved from cervical subpial AAV9-GFP injections allows axonal sprouting and synapse formation between labeled motor axons of the target and spinal cord transplanted cells. Such data systematically characterizing the progression of axonal sprouting levels and / or synaptic connections in cell-transplanted large animal models of spinal cord injury have not been obtained to date.

[0066] As used herein, the advantage of subpial AAV9 delivery compared to intrathecal delivery appears to be superior spinal cord parenchymal transgene expression. In addition, similar high levels of transgene expression are achieved entirely in adult rats or minipigs. Because the dimensions of the spinal cord in adult 35-40 kg pigs are similar to those in humans, it is expected that similar parenchymal AAV9 uptake will be achieved in adults.

[0067] A relative limitation of the subpial delivery technique described herein is the need to perform a local laminectomy to access the dorsal horn surface of the spinal cord for subpial injection. This need to perform a laminectomy may limit its repeated use (as opposed to the ability to perform repeated intrathecal delivery). However, the level of transgene expression that can be achieved after subpial AAV9 delivery appears to balance, if not surpass, this limitation (although clearer and more robust therapeutic effects have been seen after subpial gene delivery in disease-modifying studies). In addition, it has recently been found that high levels of spinal GFP expression persisted in the lumbar spinal cord in intact control mice 12 months after administration. This strongly suggests that a single subpial delivery of a therapeutic gene results in 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 shows that the subpial spinal AAV9 delivery technique described herein allows widespread transgene expression in spinal parenchyma, descending and ascending axons, without the need for direct spinal tissue needle penetration (see FIG. 8). In addition to spinal cord localized transgene expression, robust retrograde expression was seen in brain motor centers. This technique could potentially be used in preclinical and human clinical trials aimed at upregulating or downregulating genes in specific spinal cord segments and / or in projecting motor and ascending sensory axons. The range of transgene expression in these experimental adult animals suggests that the present invention could be successfully used in adult patient populations to target a variety of spinal cord neurodegenerative diseases and / or CNS-related disorders. Exemplary neurodegenerative diseases, CNS-related disorders, illnesses or trauma 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-plus syndromes (e.g., multiple system atrophy, progressive supranuclear palsy, and corticobasal degeneration), CNS injury resulting from surgical resection, spinal cord injury or trauma, tumor resection, transverse myelitis, optic neuritis , 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-Kornzweig syndrome, Marchiafava-Bignami disease, metachromatic leukodystrophy, trigeminal neuralgia, glossopharyngeal neuralgia, myasthenia gravis, epilepsy, Bell's palsy, muscular dystrophies, progressive muscular atrophy, primary lateral sclerosis (PLS), pseudobulbar palsy, progressive bulbar palsy, spinal muscular atrophy, progressive bulbar palsy, hereditary muscular atrophies, intervertebral disc disorders (e.g., herniated, ruptured and herniated disc syndromes), cervical spondylosis, plexopathies, thoracic outlet destruction 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 aggregation of AAV particles in the composition when a concentration of 1000 ng / ml or more of 1000 ng / ml of AAV is present. Methods of reducing AAV aggregation are well known in the art and include, for example, the addition of detergents, pH adjustments, and salt concentration adjusters (see, e.g., Wright FR, et al., Molecular Therapy (2005) 12, 171-178, the contents of which are incorporated herein by reference).

[0070] The formulation of pharma- ceutically acceptable excipients and carrier solutions is well known to those skilled in the art, as is the development of appropriate dosing and treatment regimens for using the particular compositions described herein in various treatment regimens. Typically, these formulations will contain at least about 0.1% or more of active ingredient, but of course the percentage of active ingredient(s) may vary, conveniently from about 1 or 2% to about 70% or 80% or more of the weight or volume of the total formulation. Of course, the amount of active ingredient in each therapeutically useful composition can be prepared so that an appropriate dosage is obtained in any given unit dose of the compound. In preparing such pharmaceutical formulations, those skilled in the art will understand that various 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] Pharmaceutical forms suitable for injection include sterile aqueous solutions or dispersions for extemporaneous preparation of sterile injectable solutions or dispersions. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof, as well as oils. Under normal storage and use conditions, these preparations contain a preservative to prevent the growth of microorganisms. In many cases, the form is sterile fluid within the range of easy syringability. The form must be stable under the conditions of manufacture and storage, and must be preserved to prevent the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be, for example, a solvent or dispersion medium containing water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, etc.), suitable mixtures thereof, and / or vegetable oils. Proper fluidity can be maintained by the use of a coating, such as lecithin, the maintenance of the required particle size in the case of dispersion, and the use of surfactants.

[0072] For administration of an injectable aqueous solution, for example, the solution is suitably buffered if necessary and the liquid diluent is first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are particularly suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this connection, sterile aqueous media that can be used 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 hypodermic injection fluid or injected at the proposed injection site (see, for example, "Remington's Pharmaceutical Sciences" 15th Edition, pages 1035-1038 and 1570-1580). Depending on the condition of the host, dosage modifications may be required. The person administering the dose will in any event determine the appropriate dosage for the individual host.

[0073] Sterile injection solution is prepared by taking the required amount of active AAV and various other components listed in this specification in a suitable solvent, and then sterilized by filtration.Generally, dispersion is prepared by adding various sterile active components into a sterile vehicle that contains a basic dispersion medium and other components required from the components listed above.In the case of sterile powder for preparing sterile injection solution, the suitable preparation method includes vacuum drying and freeze-drying technology, which obtains powder of active component + any other desired components from the solution that has been previously sterile-filtered.

[0074] In some embodiments, the agents, compositions and / or systems described herein can be assembled into pharmaceutical or diagnostic or research kits to facilitate their use in treatment, diagnosis or research applications. The kits can include one or more containers housing the components of the present invention and instructions for use. In particular, such kits can include a composition comprising AAV for administration as described herein, together with instructions describing the intended use and appropriate use of the composition. In certain embodiments, the kits can further include a separate container containing a 90° bent guide tube (e.g., 18 or 23G) and a catheter (e.g., PE-5 or PE-10) suitable for the particular use and method of subpial administration of the composition. Kits for research purposes can include components in appropriate concentrations or quantities to perform various experiments. The kits can further include one or more or all of the components (e.g., syringes, caudal and / or cranial spinal clamps, XYZ manipulators, etc.) necessary for subpial administration of the composition to subjects.

[0075] As used herein, "instructions" may define an instructional and / or promotional component, and may often include written instructions on or associated with the packaging of the present invention. Instructions may also include any oral or electronic instructions provided to allow a user to easily recognize that the instructions are associated with the kit (e.g., visual, auditory (e.g., videotape, DVD, etc.), internet and / or web-based communication). Written instructions may be in a format prescribed by a government agency having jurisdiction over the manufacture, use, or sale of pharmaceutical or biological products, and these instructions may also reflect approval by the agency responsible for manufacture, use, or sale for animal administration.

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

[0077] Example 1 material and method Animals and general surgical procedures - Adult Sprague-Dawley rats (male and female, 250-350 grams, n=16) or adult minipigs (both males and females, 30-40 kg; n=6) obtained from a crossbreed of Minnesota and Gottingen strains were used. Rats were anesthetized with 5% isoflurane and maintained at 2-3% isoflurane depending on respiratory rate and paw pinch response during surgery. The rats' backs were then shaved and scrubbed with 2% chlorhexidine. After skin incision, the paravertebral muscles around the cervical, thoracic or lumbar vertebrae were removed and the animals were fixed in a spinal immobilization frame (Stoelting) using Cunningham spinal clamps as previously described (Kakinohana, et al. (2004). Region-specific cell grafting into the cervical and lumbar spinal cord in rats: a qualitative and quantitative stereological study. Experimental neurology 190: 122-132). To expose the spinal cord, a spinal laminectomy of the corresponding vertebrae was performed using a dental drill. The dura was then cut open using a scalpel.

[0078] Minipigs 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, 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, animals were placed in a spinal immobilization apparatus as previously described (Usvald, et al. (2010). Analysis of dosing regimens and reproducibility of intrathecal grafts of human spinal cord stem cells in immunosuppressed minipigs. Cell transplantation 19: 1103-1122). Then, a spinal laminectomy was performed at Th5 or L2–L4 vertebrae, corresponding to Th5 and L3–L6 spinal segments, respectively, and epidural fat was removed by wiping with cotton. The dura was cut open and fixed to the surrounding tissues using 6.0 Proline (Figures 1C–1E).

[0079] Subpial Catheter Placement and Subpial AAV9 Injection - To place the subpial catheter, an L-shaped catheter guide tube (18 or 23 G) was constructed (Figure 1B). The guide tube was mounted in an XYZ (Stoelting) manipulator and advanced to the surface of the exposed spinal segment. The pia was punctured with a 30 G needle previously bent at 45°. The subpial catheter (PE-10 for pigs and PE-5 for rats) was then advanced from the guide tube into the subpial space by manually pushing the catheter from the other end of the guide tube. In rats, the catheter was advanced approximately 1-1.5 cm into the subpial space, and in pigs, approximately 3-6 cm. Virus was then injected into the subpial space using a 50 or 250 μl Hamilton syringe over 3 min. After injection, the catheter was removed and the dura was closed with 6.0 Proline (dura was closed in pigs only), after which the animals were allowed to recover.

[0080] Preparation of AAV9 for in vivo injection - A 1.2 kb ubiquitin-C (UBC) promoter was generated by oligonucleotide synthesis, conjugated with eGFP or DsRed (RFP), and SV40 polyA signal, and cloned into a self-complementary double-stranded DNA genomic AAV (scAAV) vector plasmid (Xu, et al. (2012). In vivo gene knockdown in rat dorsal root ganglions mediated by self-complementary adeno-associated virus serotype 5 following intrathecal delivery. PloSone7: e32581). Helper virus-free scAAV9 vectors expressing either eGFP or RFP driven by the UBC promoter were generated by transient transfection of HEK293T cells with the vector plasmid, pRep2-Cap9, and the pAd-helper plasmid (Xiao, et al. (1998). Production of high-titer recombinant adeno-associated virus vectors in the absence of helper adenovirus. J Virol 72: 2224-2232). Plasmid pRep2-Cap9 was obtained from the Vector Core at U. Penn. AAV vectors in cell lysates prepared 72 hours after transfection were purified and titered by Q-PCR as previously described (Xu, et al., supra). Final titers were determined to be 1.0x10 13 The genome copies / ml (gc / ml) was adjusted. Virus was mixed 1:1 with dextran (10,000 MW) immediately before injection to a final dextran concentration of 2.5%. Subpial injection volumes were 30 μl for rats and 200 μl for pigs.

[0081] Perfusion fixation, postmortem in situ GFP fluorescence imaging and immunofluorescence of spinal cord and brain sections - Animals (rats and pigs) were deeply anesthetized with pentobarbital and perfused transcardially with 200 ml (rats) or 2000 ml (pig) of heparinized saline, followed by 250 ml (rats) or 4000 ml (pig) of 4% paraformaldehyde in PBS. Spinal cords and brains were dissected and fixed in 4% formaldehyde in PBS overnight at 4°C, then cryoprotected in 30% sucrose PBS, and transverse or longitudinal sections (30 μm thick) were cut on a cryostat and stored in PBS. Before sectioning, whole spinal cords were imaged in situ using an IVIS Spectral Light Imaging System (Xenogen, Alameda, CA). Sequence acquisition was performed at an excitation wavelength of 465 nm and an emission wavelength of 520 nm. Medium binning was used with an exposure time of 3 seconds. Image analysis was performed using Living Image 4.3.1 (Xenogen, Alameda, CA) software. Signal calculation was performed using fixed volume ROI. Immunostaining of prepared sections was performed overnight at 4 °C with primary antibodies made in PBS with 0.2% TritonX-100: rabbit anti-glial fibrillary 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, the 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, and DAPI for general nuclear staining. The sections were then mounted on slides, dried at room temperature, and covered with long-term browning prevention kit (Invitrogen). Fluorescent image capture was performed using a Zeiss ImagerM2 microscope, and confocal images were taken using an OlympusFV1000 microscope.

[0082] The consistency, high levels of spinal cord parenchymal transgene expression, and feasibility of this approach represent a major technical improvement over current approaches and have potential for direct clinical applications aimed at spinal cord gene upregulation or silencing in general. Current approaches use either intrathecal or invasive direct parenchymal AAV injection, which clearly have limitations in terms of low levels of parenchymal transgene expression or the invasiveness required to achieve a more robust infection effect. This approach also allows for unparalleled levels of motor and sensory fiber labeling in segments distal to the site of AAV9 delivery, making it an extremely robust tool for studies to study the extent of synaptic connectivity and to identify elements (i.e., genes, neurotrophic elements) that regulate axonal sprouting in the targeted spinal cord segment in both rodents and large animal species.

[0083] Described herein are examples demonstrating the principle of spinal subpial AAV9 delivery and the resulting local parenchymal expression and axonal labeling in segments distal to the AAV9 delivery site in adult pigs 2 months after AAV9 delivery.

[0084] Example 2 Parenchymal AAV9-mediated transgene expression after a single spinal cord bolus We first tested the efficacy of single-bolus subpial AAV9-UBI-GFP or AAV9-UBI-RFP delivery in rats and pigs. In animals, AAV9 vectors in 20 μl (rat) or 200 μl (pig) of 2.5% dextran solution were delivered into the subpial space of the 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 cut from 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 seen on the spinal cord surface and was easily identified by visual inspection as yellow-green or red areas. Figures 1H and 1J show the presence of RFP (red) in the transected porcine spinal cord and ventral roots (Figure 1H, insert) compared to naive spinal cord (Figure 1I). Correspondingly, spinal cord surface densitometry analysis of animals injected with AAV9-UBI-GFP revealed extensive GFP signal diffusing up to 5-10 cm from the subpial AAV9 delivery epicenter (Figures 1F and 1G).

[0085] Using horizontally cut thoracic sections (same spinal cord as shown in Figure 1H) taken from pigs previously subpial injected with AAV9-UBI-RFP at the mid-thoracic level, widespread parenchymal RFP expression was seen. RFP expression was easily identified in most interneurons and alpha motor neurons and was diffuse throughout 4-6 spinal segments (Figures 3A and 3B). Similarly, high RFP expression was seen throughout the RFP-expressing gray matter in axon-dendritic arborizations (Figures 3A and 3B, white asterisks).

[0086] Analysis of transected spinal cord sections taken from the epicenter of the subpial AAV9 injection confirmed high RFP expression throughout the white and gray matter. In the white matter, punctate RFP expression was seen in the majority of transversely cut axons (Fig. 3C, white asterisks, box insert) and was identified in the spines, lateral funiculus, and ventral funiculus (Fig. 3C, D-F, D-F, D-F, D-F; box insert). In the gray matter, numerous interneurons were distributed between lamina I-IX and pre-α motor neurons in the ventral horn, with high RFP expression in the cell bodies and axonal dendritic branches. High density RFP expression was also seen in nerve fiber terminals in the gray matter (Fig. 3D-G). Similarly, confocal analysis showed the presence of RFP signal in astrocytes (Fig. 3C, insert: RFP / GFAP).

[0087] A similar pattern of neuronal GFP expression was seen in the rat lumbar spinal cord at the L1-2 level after subpial AAV9-UBI-GFP injection: a high density of GFP+ neurons was identified throughout the L1-L5 lumbar segments and localized throughout the gray matter between laminae I-IX (Figures 9A-9D).

[0088] Example 3 GFP expression in distal spinal cord segments We next characterized the extent of lower spinal canal GFP expression in the lumbar spinal cord after subpial injection of AAV9-UBI-GFP into the subpial space of the mid-thoracic (Th6-7) or lower cervical segments in both rats and pigs. Three to six weeks after subpial AAV9 delivery, high GFP expression was seen throughout the lumbar spinal cord. Using transverse lumbar (L2-L6) spinal cord sections taken from pigs, high intensity GFP expression in transverse axons in the lateral and ventral funiculus was readily identified without additional GFP immunostaining (Figure 4A, white asterisks). In these regions, a similar density of GFP+ axons was seen throughout the white matter. In contrast to the lateral and ventral funiculus, relatively few GFP+ axons were seen in the spinal funiculus (Figure 4A, D-F). Consistent with the levels of axonal labeling seen in the white matter of the lateral and ventral funiculus, a dense network of GFP+ axons terminating in the gray matter was seen (Figures 4A and 4B). These axons were localized in laminae III to X. Very few GFP+ axons were seen in laminae I to III. Using high-power confocal microscopy, a high density of fine GFP+ axons was seen along with numerous nerve fiber endings in the gray matter (Figure 4C).

[0089] A similar pattern of GFP expression in the lower motor axons in the lumbar gray matter was seen in rats that had previously received upper cervical subpial injections of AAV9-UBI-GFP (Figures 9E and 9F).

[0090] Example 4 Retrograde transgene expression in brain motor regions after spinal subpial delivery Analysis of transgene (GFP) expression in brain motor centers (motor cortex, red nucleus, and reticular formation) 6 weeks after subpial AAV9-UBI-GFP delivery in pigs revealed highly GFP-labeled pyramidal neurons in the motor cortex (Figure 5A-5E). Similarly, numerous GFP+ neurons localized within the brainstem were identified (Figure 5F-5J). Consistent with the presence of GFP+ neurons in the motor cortex, numerous GFP+ corticospinal axons were present in the anterior region (pyramids) of the medulla oblongata (Figure 5K). In addition, dense anterogradely labeled GFP+ spinal reticular terminals were found throughout the reticular formation (Figure 5L) and spinothalamic terminals were found in thalamic nuclei (not shown).

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

[0092] Example 5 Regional spinal cord transgene expression following intrathecal versus subpial AAV9 delivery We next compared the distribution of spinal transgene expression after injection of AAV9 into the lumbar (L1-L2) intrathecal space (AAV9-UBI-GFP) and into the subpial space of the thoracic Th7 segment (AAV9-UBI-RFP) in the same animals (rats). Subpial AAV9 injection was performed 3 weeks after lumbar intrathecal AAV9 injection, and expression patterns were analyzed in transverse lumbar spinal cord sites 3 weeks after subpial AAV9 injection. Intrathecal injection of AAV9-UBI-GFP resulted in high GFP expression in the spinal cord, primary afferents in the dorsal horn (lamina II-III) and the central part of laminae V-VII. Several GFP+Ia afferents terminating in the anterior horn in the vicinity of CHAT+α motor neurons were also identified. Consistent with the high GFP expression in primary afferents, numerous GFP+ neurons were seen in dorsal root ganglion cells (Figure 7B). A clear presence of increased GFP expression was consistently seen around the anterior root entry zone (Figures 7A and 7F). In this region, some GFP-expressing glial cells were seen. Similarly, 2-3 GFP+ cells were seen in the dorsal root entry zone. In the ventral gray matter, some α-motor neurons showed GFP expression (Figure 7G). Except for these regions and groups of cells that showed GFP expression, an almost complete absence of neuronal or glial GFP expression was seen in other deeper regions of the gray matter (e.g., dorsal horn, intermediate zone, and white matter of the lateral and ventral funiculus) (Figure 7A). Interestingly, in the case of neurons localized in the superficial dorsal horn and in close proximity to the intrathecal space (but separated by the pia mater), no GFP expression was seen at all (Figures 7C and 7D).

[0093] In contrast to the GFP expression pattern seen after intrathecal AAV9-UBI-GFP delivery, RFP expression resulting from cervical subpial AAV9 injection showed a substantially different regional expression pattern when analyzed in the same lumbar spinal cord section. dsRED expression was identified in the majority of axons in the white matter and was present in the lateral and ventral funiculus. Numerous axons were also found projecting into the gray matter of the dorsal horn, intermediate zone (lamina VII), and anterior horn (Figure 7A). Using confocal microscopy, it was found that virtually all RFP+ fibers were GFP-negative in the white or gray matter. Interestingly, numerous RFP+ corticospinal axons were found remaining on the spinal funiculus in close proximity to GFP-labeled primary afferents, but no RFP+GFP colocalization was seen in any of these fibers (Figure 7E).

[0094] Although the invention has been described with reference to the above examples, it will be understood that variations and modifications are encompassed within the spirit and scope of the invention. Accordingly, the invention is limited only by the scope of the following claims.

Claims

1. A method of intraparenchymal spinal cord infection with a nucleic acid molecule in a subject, comprising administering the nucleic acid molecule to the subpial space of the subject.

2. The administration step comprises: (a) exposing a spinal segment of a subject's spine; (b) creating a pial opening in the spinal segment; (c) advancing the catheter through the pial opening into the subpial space; and (d) delivering the nucleic acid molecule to the subpial space of the subject The method of claim 1 , comprising:

3. 3. The method of claim 2, wherein the pial opening is created by puncturing the pial membrane with an L-shaped stainless steel tube and the catheter is advanced through the tube into the subpial space.

4. The method of claim 1, wherein the nucleic acid molecule is administered in a mixture containing about 1-10% dextrose.

5. The method of claim 1 , wherein the nucleic acid molecule is a vector or an antisense oligonucleotide (ASO).

6. The method of claim 5 , wherein the vector is a lentiviral vector, an adenoviral vector, or an adeno-associated vector.

7. The method of claim 6, wherein the vector is an AAV9 particle.

8. The method of claim 7, wherein the vector comprises a nucleic acid molecule encoding a protein or functional RNA that modulates or treats a neurodegenerative disease.

9. 9. The method of claim 8, wherein the neurodegenerative disease is amyotrophic lateral sclerosis (ALS), Huntington's disease, Alzheimer's disease or Parkinson's disease.

10. The method of claim 1 , wherein the nucleic acid molecule is delivered in a single injection.

11. The method of claim 2, further comprising administering one or more second subpial administrations of the nucleic acid molecule into a different spinal segment of the subject's spine.

12. The method of claim 2, further comprising one or more intrathecal administrations of the nucleic acid molecule to the subject.

13. The method of claim 1 , wherein the subject is a mammal.

14. The method of claim 13 , wherein the subject is a human.

15. 1. A gene delivery system comprising: (a) an L-shaped guide tube configured to puncture the pia mater of a subject; (b) a catheter slidably disposed within the guide tube and configured to be advanced into the subpial space of a spinal segment of the target spine; (c) a reservoir in fluid communication with the catheter and containing a composition comprising a nucleic acid molecule; Including, the system.

16. 16. The method of claim 15, wherein the L-shaped guide tube is a 16-26G stainless steel tube.

17. 16. The method of claim 15, wherein the catheter is formed from polyethylene tubing.

18. 1. A method for delivering a nucleic acid molecule to the subpial space of a subject, comprising: (a) exposing a spinal segment of a subject's spine; (b) creating a pial opening in the spinal segment; (c) positioning the gene delivery system of claim 16 above said spinal segment; (d) advancing the catheter through the pial opening into the subpial space; and (e) delivering the nucleic acid molecule to the subpial space of a subject A method comprising:

19. A method of treating a neurodegenerative disease in a subject in need thereof, comprising administering a vector or an antisense oligonucleotide (ASO) to the subpial space of the subject.

20. The administration step comprises: (a) exposing a spinal segment of a subject's spine; (b) creating a pial opening in the spinal segment; (c) advancing the catheter through the pial opening into the subpial space; and (d) delivering the nucleic acid molecule to the subpial space of the subject 20. The method of claim 19, comprising:

21. 21. The method of claim 20, wherein the pial opening is created by puncturing the pial membrane with an L-shaped stainless steel tube and the catheter is advanced through the tube into the subpial space.

22. 20. The method of claim 19, wherein the neurodegenerative disease is amyotrophic lateral sclerosis (ALS), Huntington's disease, Alzheimer's disease or Parkinson's disease.

23. 20. The method of claim 19, further comprising one or more second subpial administrations of the vector or ASO into a different spinal segment of the subject's spine.

24. 20. The method of claim 19, further comprising one or more intrathecal administrations of the vector or ASO to the subject.

25. 20. The method of claim 19, wherein the subject is a mammal.

26. 26. The method of claim 25, wherein the subject is a human.

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