US20180320176A1 - Intrathecal delivery of nucleic acid sequences encoding abcd1 for treatment of adrenomyeloneuropathy - Google Patents

Intrathecal delivery of nucleic acid sequences encoding abcd1 for treatment of adrenomyeloneuropathy Download PDF

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US20180320176A1
US20180320176A1 US15/773,337 US201615773337A US2018320176A1 US 20180320176 A1 US20180320176 A1 US 20180320176A1 US 201615773337 A US201615773337 A US 201615773337A US 2018320176 A1 US2018320176 A1 US 2018320176A1
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abcd1
vector
aav9
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expression
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Casey A. Maguire
Florian Eichler
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General Hospital Corp
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Definitions

  • X-linked adrenoleukodystrophy a progressive genetic disorder, is caused by mutations in the ABCD1 gene, which encodes a peroxisomal ATP-binding cassette transporter (ABCD1) responsible for transport of CoA-activated very long-chain fatty acids (VLCFA) into the peroxisome for degradation leading to the accumulation of high levels of saturated, very long chain fatty acids (VLCFA) in plasma and tissues of the brain and adrenal cortex.
  • VLCFA very long-chain fatty acids
  • Symptoms can begin in childhood or adulthood.
  • Adult ALD patients typically develop adrenomyeloneuropathy (AMN), a debilitating neurological disorder, in their twenties (Engelen et al., Orphanet J Rare Dis. 2012; 7: 51).
  • AMD adrenomyeloneuropathy
  • the Abcd1 ⁇ / ⁇ mouse develops a phenotype similar to AMN, manifesting spinal cord axon degeneration as well as peripheral neuropathy due to affected dorsal root ganglion neurons (DRGs) (Pujol et al., Hum Mol Genet. 2002; 11:499-505).
  • DRGs dorsal root ganglion neurons
  • rAAV9 recombinant adeno-associated virus serotype 9
  • the invention provides a method of increasing adeno-associated Virus 9 (AAV9) vector titers in transfected producer cells grown in culture, said method comprising the steps of i) incubating a nucleic acid sequence that is complementary to an mRNA encoding ATP binding cassette subfamily D member 1 (ABCD1) with the cells and ii) transfecting an AAV9 vector comprising a nucleotide sequence encoding ABCD1 into the cells (AAV9-ABCD1 vector), wherein the amount of ABCD1 mRNA expressed from the AAV9 vector is decreased, thereby increasing AAV9-ABCD1 vector yield in cell lysate and/or media by about 1 fold to about 50 fold compared to a reference standard.
  • AAV9-ABCD1 vector adeno-associated Virus 9
  • the nucleic acid sequence that is complementary to an mRNA encoding ABCD1 is an interfering RNA.
  • the interfering RNA is an shRNA or siRNA.
  • the siRNA comprises SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7 or a combination thereof.
  • the reference standard comprises AAV9-ABCD1 vector yield in cell lysate and/or media from producer cells that were not incubated with a nucleic acid sequence that is complementary to an mRNA encoding ABCD1.
  • the invention provides a method of treating X-linked adrenoleukodystrophy (X-ALD) in a subject in need thereof comprising administering to the subject a composition comprising purified AAV9-ABCD1 vector obtained from the producer cells having increased AAV9 vector titers compared to a reference standard.
  • X-ALD X-linked adrenoleukodystrophy
  • composition comprising purified AAV9-ABCD1 vector is administered to the subject by intrathecal administration.
  • the invention provides a method of treating X-linked adrenoleukodystrophy (X-ALD) in a subject in need thereof comprising administering to the subject an adeno-associated Virus (AAV) vector encoding an ATP binding cassette subfamily D member 1 (ABCD1), wherein said vector is administered to the subject by intrathecal administration.
  • X-ALD X-linked adrenoleukodystrophy
  • AAV adeno-associated Virus
  • the intrathecal administration is mediated by an osmotic pump.
  • the dose of vector is 0.5 ⁇ 10 11 GC.
  • the AAV is AAV9.
  • the invention provides a method of providing ATP binding cassette subfamily D member 1 (ABCD1) to a subject having X-linked adrenoleukodystrophy (X-ALD) comprising administering to the subject a vector encoding ABCD1, wherein said vector is administered to the subject by intrathecal administration, and wherein ABCD1 expression from said vector in the central nervous system is less than ABCD1 expression from said vector in peripheral organs.
  • ABCD1 ATP binding cassette subfamily D member 1
  • X-ALD X-linked adrenoleukodystrophy
  • the intrathecal administration is mediated by an osmotic pump.
  • the dose of vector is about 1 ⁇ 10 13 GC to about 10 ⁇ 10 13 GC.
  • the vector is an adeno-associated virus (AAV) vector.
  • AAV adeno-associated virus
  • FIG. 1 depicts improved AAV9-ABCD1 vector titers from transfected 293T cells incubated with siRNA specific for ABCD1 mRNA.
  • FIG. 2 depicts reduced ABCD1 protein in AAV-ABCD1 transfected cells incubated with an siRNA pool specific for ABCD1 mRNA.
  • FIG. 3 depicts improved AAV9-ABCD1 vector titers from transfected 293T cells incubated with siRNA specific for ABCD1 mRNA in both cell lysates and conditioned media.
  • FIG. 4 depicts distribution of rAAV9-ABCD1 following intrathecal bolus delivery over 2 minutes.
  • FIG. 5 depicts distribution of rAAV9-ABCD1 following intrathecal pump infusion of rAAV9-ABCD1 over 24 hours.
  • FIG. 6 depicts low dose (0.5 ⁇ 10 11 gc) bolus and pump delivery of AAV9-ABCD1.
  • FIG. 7 depicts higher expression of ABCD1 across peripheral organs (outside the CNS) two weeks after bolus injection of AAV9-ABCD1 compared to pump infusion of AAV9-ABCD1.
  • FIG. 8 depicts a vector map of AAV9-ABCD1.
  • FIG. 9 depicts distribution of endogenous ABCD1 across different organs.
  • FIG. 10 depicts distribution of endogenous ABCD1 across different organs.
  • FIG. 11 depicts expression of ABCD1 after IT pump in Abcd1 ⁇ / ⁇ mouse.
  • FIG. 12 depicts expression of ABCD1 after IT pump in Abcd1 ⁇ / ⁇ mouse.
  • FIG. 13 depicts spinal cord C26:0 level 15 days after IT pump and PT bolus injection.
  • FIG. 14 depicts ABCD1 expression in different cell types after IT pump delivery of AAV9-hABCD1.
  • SC spinal cord;
  • DRG dorsal root ganglion;
  • CD31 endothelial marker;
  • GFAP astrocyte marker;
  • IBA1 microglial marker;
  • TOPRO3 nuclear counterstain;
  • DRG shows expression speckled pattern in neuron and more prominently around neurons (satellite cells).
  • a “subject” is a vertebrate, including any member of the class mammalia, including humans, domestic and farm animals, and zoo, sports or pet animals, such as mouse, rabbit, pig, sheep, goat, cattle and higher primates.
  • the terms “treat,” “treating,” “treatment,” and the like refer to reducing or ameliorating X-ALD, e.g., adrenomyeloneuropathy (AMN), and/or symptoms associated therewith. It will be appreciated that, although not precluded, treating X-ALD or AMN does not require that the disorder, condition or symptoms associated therewith be completely eliminated.
  • X-ALD e.g., adrenomyeloneuropathy
  • a decrease in expression refers to an amount of ABCD1 gene expression or protein expression in peripheral organs of a subject that is at least about 0.05 fold less (for example 0.1, 0.2, 0.3, 0.4, 0.5, 1, 5, 10, 25, 50, 100, 1000, 10,000-fold or more less) than the amount of ABCD1 gene expression or protein expression in the central nervous system of a subject having been administered a vector encoding ABCD1 according to the methods described herein.
  • “Decreased” as it refers to ABCD1 gene expression or protein expression in peripheral organs of a subject also means at least about 5% less (for example 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 99 or 100%) than the amount of ABCD1 gene expression or protein expression in the central nervous system of a subject having been administered a vector encoding ABCD1 according to the methods described herein. Amounts can be measured according to standard methods known in the art for determining amounts of gene expression or protein expression.
  • an increase in vector titers refers to an amount of titer from producer cells transfected with a vector encoding ABCD1 that is at least about 0.05 fold more (for example 0.1, 0.2, 0.3, 0.4, 0.5, 1, 5, 10, 25, 50, 100, 1000, 10,000-fold or more) than the amount of titer from producer cells that were not incubated with a nucleic acid sequence that is complementary to an mRNA encoding ABCD1 according to the methods described herein.
  • “Increased” as it refers to an amount of titer (concentration of AAV vector, often described in genome copies per milliliter) from producer cells transfected with a vector encoding ABCD1 also means at least about 5% more (for example 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 99 or 100%) than the amount of titer from producer cells that were not incubated with a nucleic acid sequence that is complementary to an mRNA encoding ABCD1 according to the methods described herein. Amounts can be measured according to standard methods known in the art for determining amounts of AAV genomes, transgene expression, or protein expression.
  • Ranges provided herein are understood to be shorthand for all of the values within the range.
  • a range of 1 to 50 is understood to include any number, combination of numbers, or sub-range from the group consisting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 (as well as fractions thereof unless the context clearly dictates otherwise).
  • reference level refers to the level of titer in a known sample against which another test sample is compared.
  • a reference level can be obtained, for example, from producer cells that were not incubated with a nucleic acid sequence that is complementary to an mRNA encoding ABCD1 or with a control antisense oligonucleotide or siRNA.
  • a reference level can be obtained, for example, from untreated subjects that do not have X-ALD. “Untreated” refers to the lack of therapy from administration of a vector expressing an ABCD1 transgene.
  • compositions and methods of the invention provide treatments for X-linked adrenoleukodystrophy (X-ALD).
  • X-linked adrenoleukodystrophy is a genetic disorder, caused by mutations in the ABCD1 gene, that occurs primarily in males and mainly affects the nervous system and the adrenal glands. Myelin of the brain and spinal cord deteriorate (demyelination), which reduces the functional ability of the nerves.
  • damage to the outer layer of the adrenal glands (adrenal cortex) causes a shortage of certain hormones (adrenocortical insufficiency).
  • X-linked adrenoleukodystrophy There are several distinct types of X-linked adrenoleukodystrophy, including a childhood cerebral form, an adrenomyeloneuropathy (AMN) type, and a form called Addison disease.
  • X-ALD does not include “neonatal adrenoleukodystrophy,” which belongs to the peroxisomal biogenesis disorders of the Zellweger spectrum and is unrelated to mutations in ABCD1.
  • Methods for diagnosing or identifying subjects with X-ALD or AMN are known in the art and can include measurement of plasma very long chain fatty acid (VLCFA) levels and/or genetic testing; see, e.g., Engelen et al., Orphanet J Rare Dis.
  • VLCFA very long chain fatty acid
  • ABCD1 gene mutations result in a deficiency of ALDP. When this protein is lacking, the transport and subsequent breakdown of VLCFAs is disrupted, causing abnormally high levels of these fats in the body. The accumulation of VLCFAs may be toxic to the adrenal cortex and myelin.
  • ALDP adrenoleukodystrophy protein
  • VLCFAs very long-chain fatty acids
  • ABCD1 adeno-associated virus
  • AAV is adeno-associated virus, and may be used to refer to the recombinant virus vector itself or derivatives thereof. The term covers all subtypes, serotypes and pseudotypes, and both naturally occurring and recombinant forms, except where required otherwise.
  • serotype refers to an AAV which is identified by and distinguished from other AAVs based on its serology, e.g., there are eleven serotypes of AAVs, AAV1-AAV11, and the term encompasses pseudotypes with the same properties. Many of these serotypes have unique biological properties from other AAV serotypes (e.g. cell surface receptor binding, intracellular trafficking).
  • AAV5 serotypes include AAV with the biological properties of AAV5, e.g., a pseudotyped AAV comprising AAV5 capsid and an AAV genome which is not derived or obtained from AAV5 or which genome is chimeric.
  • An “AAV vector” refers to a viral particle composed of at least one AAV capsid protein and an encapsidated polynucleotide. If the particle comprises a heterologous polynucleotide (i.e., a polynucleotide other than a wild-type AAV genome such as a transgene to be delivered to a mammalian cell), it can be referred to as “rAAV (recombinant AAV).”
  • An AAV “capsid protein” includes a capsid protein of a wild-type AAV, as well as modified forms of an AAV capsid protein which are structurally and or functionally capable of packaging an AAV genome and bind to at least one specific cellular receptor which may be different than a receptor employed by wild type AAV.
  • a modified AAV capsid protein includes a chimeric AAV capsid protein such as one having amino acid sequences from two or more serotypes of AAV, e.g., a capsid protein formed from a portion of the capsid protein from AAV5 fused or linked to a portion of the capsid protein from AAV2, and a AAV capsid protein having a tag or other detectable non-AAV capsid peptide or protein fused or linked to the AAV capsid protein, e.g., a portion of an antibody molecule which binds the transferrin receptor may be recombinantly fused to the AAV-2 capsid protein.
  • a chimeric AAV capsid protein such as one having amino acid sequences from two or more serotypes of AAV, e.g., a capsid protein formed from a portion of the capsid protein from AAV5 fused or linked to a portion of the capsid protein from AAV2, and a AAV caps
  • Cells capable of producing AAV include, but are not limited to 293 cells, HeLa cells and insect cells.
  • methods of producing high titers of AAV can be utilized to maximize administration of ABCD1.
  • Transfected producer cells grown in culture can be incubated with a nucleic acid sequence that is complementary to an mRNA encoding ATP binding cassette subfamily D member 1 (ABCD1) and ii) transfected with an AAV vector comprising a nucleotide sequence encoding ABCD1 into the cells (e.g., AAV9-ABCD1 vector).
  • ABCD1 mRNA encoding ATP binding cassette subfamily D member 1
  • AAV9-ABCD1 vector a nucleotide sequence encoding ABCD1 into the cells
  • the amount of ABCD1 mRNA expressed from the AAV vector is decreased, thereby increasing AAV-ABCD1 vector yield in cell lysate and/or media by about 1 fold to about 50 fold compared to a reference standard.
  • the AAV-ABCD1 vector yield in cell lysate and/or media is increased by about 4 fold.
  • Vector titers can be determined according to methods well known in the art. Typically, this is performed using dot blots or quantitative PCR to measure AAV genomes.
  • AAV vector yields can be about 1 ⁇ 10 10 genome copies/ml (gc/ml) to about 1 ⁇ 10 16 gc/ml from cell lysates and from media.
  • the reference standard comprises AAV-ABCD1 vector yield in cell lysate and/or media from producer cells that were not incubated with a nucleic acid sequence that is complementary to an mRNA encoding ABCD1.
  • nucleic acid sequences for use in practicing the methods of the invention and that are complementary to ABCD1 mRNA can be those which inhibit post-transcriptional processing of ABCD1, such as an interfering RNA, including but not limited to an shRNA or siRNA, or an antagomir.
  • Antisense oligonucleotides are typically designed to block expression of a DNA or RNA target by binding to the target and halting expression at the level of transcription, translation, or splicing.
  • Antisense oligonucleotides of the present invention are complementary nucleic acid sequences designed to hybridize under stringent conditions to ABCD1 mRNA. Thus, oligonucleotides are chosen which are sufficiently complementary to the target, i.e., hybridize sufficiently well and with sufficient specificity, to give the desired effect.
  • hybridization means hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleoside or nucleotide bases.
  • adenine and thymine are complementary nucleobases which pair through the formation of hydrogen bonds.
  • Complementary refers to the capacity for precise pairing between two nucleotides. For example, if a nucleotide at a certain position of an oligonucleotide is capable of hydrogen bonding with a nucleotide at the same position of a DNA or RNA molecule, then the oligonucleotide and the DNA or RNA are considered to be complementary to each other at that position.
  • oligonucleotide and the DNA or RNA are complementary to each other when a sufficient number of corresponding positions in each molecule are occupied by nucleotides which can hydrogen bond with each other.
  • “specifically hybridizable” and “complementary” are terms which are used to indicate a sufficient degree of complementarity or precise pairing such that stable and specific binding occurs between the oligonucleotide and the DNA or RNA target.
  • a complementary nucleic acid sequence need not be 100% complementary to that of its target nucleic acid to be specifically hybridizable.
  • a complementary nucleic acid sequence of the invention is specifically hybridizable when binding of the sequence to the target DNA or RNA molecule interferes with the normal function of the target DNA or RNA to cause a loss of activity, and there is a sufficient degree of complementarity to avoid non-specific binding of the sequence to non-target sequences under conditions in which specific binding is desired, i.e., under physiological conditions in the case of in vivo assays or therapeutic treatment, and in the case of in vitro assays, under conditions in which the assays are performed under suitable conditions of stringency.
  • stringent salt concentration will ordinarily be less than about 750 mM NaCl and 75 mM trisodium citrate, preferably less than about 500 mM NaCl and 50 mM trisodium citrate, and more preferably less than about 250 mM NaCl and 25 mM trisodium citrate.
  • Low stringency hybridization can be obtained in the absence of organic solvent, e.g., formamide, while high stringency hybridization can be obtained in the presence of at least about 35% formamide, and more preferably at least about 50% formamide.
  • Stringent temperature conditions will ordinarily include temperatures of at least about 30° C., more preferably of at least about 37° C., and most preferably of at least about 42° C.
  • Varying additional parameters, such as hybridization time, the concentration of detergent, e.g., sodium dodecyl sulfate (SDS), and the inclusion or exclusion of carrier DNA, are well known to those skilled in the art.
  • concentration of detergent e.g., sodium dodecyl sulfate (SDS)
  • SDS sodium dodecyl sulfate
  • Various levels of stringency are accomplished by combining these various conditions as needed.
  • hybridization will occur at 30° C. in 750 mM NaCl, 75 mM trisodium citrate, and 1% SDS.
  • hybridization will occur at 37° C. in 500 mM NaCl, 50 mM trisodium citrate, 1% SDS, 35% formamide, and 100 ⁇ g/ml denatured salmon sperm DNA (ssDNA).
  • hybridization will occur at 42° C. in 250 mM NaCl, 25 mM trisodium citrate, 1% SDS, 50% formamide, and 200 ⁇ g/m1 ssDNA. Useful variations on these conditions will be readily apparent to those skilled in the art.
  • wash stringency conditions can be defined by salt concentration and by temperature. As above, wash stringency can be increased by decreasing salt concentration or by increasing temperature.
  • stringent salt concentration for the wash steps will preferably be less than about 30 mM NaCl and 3 mM trisodium citrate, and most preferably less than about 15 mM NaCl and 1.5 mM trisodium citrate.
  • Stringent temperature conditions for the wash steps will ordinarily include a temperature of at least about 25° C., more preferably of at least about 42° C., and even more preferably of at least about 68° C. In a preferred embodiment, wash steps will occur at 25° C.
  • wash steps will occur at 42° C. in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, wash steps will occur at 68° C. in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. Additional variations on these conditions will be readily apparent to those skilled in the art. Hybridization techniques are well known to those skilled in the art and are described, for example, in Benton and Davis (Science 196:180, 1977); Grunstein and Hogness (Proc. Natl. Acad.
  • the antisense oligonucleotides of the present invention comprise at least 80% sequence complementarity to a target region within the target nucleic acid, moreover that they comprise 90% sequence complementarity and even more preferable to comprise 95% sequence complementarity to the target region within the target nucleic acid sequence to which they are targeted.
  • an antisense compound in which 18 of 20 nucleobases of the antisense oligonucleotide are complementary, and would therefore specifically hybridize, to a target region would represent 90 percent complementarity.
  • Percent complementarity of an antisense compound with a region of a target nucleic acid can be determined routinely using basic local alignment search tools (BLAST programs) (Altschul et al., J. Mol.
  • Antisense and other compounds of the invention which hybridize to ABCD1 mRNA, are identified through experimentation, and representative sequences of these compounds are herein below identified as preferred embodiments of the invention.
  • the nucleic acid sequence that is complementary to ABCD1 mRNA can be an interfering RNA, including but not limited to an shRNA or siRNA.
  • Interfering RNA includes, but is not limited to small interfering RNA (“siRNA”) and small hairpin RNA (“shRNA”). Methods for constructing interfering RNAs are well known in the art.
  • the interfering RNA can be assembled from two separate oligonucleotides, where one strand is the sense strand and the other is the antisense strand, wherein the antisense and sense strands are self-complementary (i.e., each strand comprises nucleotide sequence that is complementary to nucleotide sequence in the other strand; such as where the antisense strand and sense strand form a duplex or double stranded structure); the antisense strand comprises nucleotide sequence that is complementary to a nucleotide sequence in a target nucleic acid molecule or a portion thereof (i.e., an undesired gene) and the sense strand comprises nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof.
  • interfering RNA is assembled from a single oligonucleotide, where the self-complementary sense and antisense regions are linked by means of nucleic acid based or non-nucleic acid-based linker(s).
  • the interfering RNA can be a polynucleotide with a duplex, asymmetric duplex, hairpin or asymmetric hairpin secondary structure, having self-complementary sense and antisense regions, wherein the antisense region comprises a nucleotide sequence that is complementary to nucleotide sequence in a separate target nucleic acid molecule or a portion thereof and the sense region having nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof
  • the interfering can be a circular single-stranded polynucleotide having two or more loop structures and a stem comprising self-complementary sense and antisense regions, wherein the antisense region comprises nucleotide sequence that is complementary to nucleotide sequence in
  • the interfering RNA coding region encodes a self-complementary RNA molecule having a sense region, an antisense region and a loop region.
  • RNA molecule when expressed desirably forms a “hairpin” structure, and is referred to herein as an “shRNA.”
  • the loop region is generally between about 2 and about 10 nucleotides in length. In a preferred embodiment, the loop region is from about 6 to about 9 nucleotides in length.
  • the sense region and the antisense region are between about 15 and about 20 nucleotides in length.
  • the small hairpin RNA is converted into a siRNA by a cleavage event mediated by the enzyme Dicer, which is a member of the RNase III family.
  • Dicer which is a member of the RNase III family.
  • the siRNA is then capable of inhibiting the expression of a gene with which it shares homology.
  • Dicer a member of the RNase III family.
  • the siRNA is then capable of inhibiting the expression of a gene with which it shares homology.
  • siRNAs The target RNA cleavage reaction guided by siRNAs is highly sequence specific.
  • siRNA containing a nucleotide sequences identical to a portion of the target gene are preferred for inhibition.
  • 100% sequence identity between the siRNA and the target gene is not required to practice the present invention.
  • the invention has the advantage of being able to tolerate sequence variations that might be expected due to genetic mutation, strain polymorphism, or evolutionary divergence.
  • siRNA sequences with insertions, deletions, and single point mutations relative to the target sequence have also been found to be effective for inhibition.
  • siRNA sequences with nucleotide analog substitutions or insertions can be effective for inhibition.
  • ABCD1 mRNA is an antagomir.
  • Antagomirs are single stranded, double stranded, partially double stranded and hairpin structured chemically modified oligonucleotides that target a microRNA.
  • an antagomir featured in the invention includes a nucleotide sequence sufficiently complementary to hybridize to a miRNA target sequence of about 10 to 25 nucleotides, preferably about 15 to 20 nucleotides.
  • antagomirs are RNA-like oligonucleotides that harbor various modifications for RNase protection and pharmacologic properties such as enhanced tissue and cellular uptake.
  • An antagomir can differ from normal RNA by having complete 2′-O-methylation of sugar, phosphorothioate backbone and a cholesterol-moiety at 3′-end. Phosphorothioate modifications provide protection against RNase activity and their lipophilicity contributes to enhanced tissue uptake.
  • the antagomir includes six phosphorothioate backbone modifications; two phosphorothioates are located at the 5′-end and four at the 3′-end.
  • Antagomirs of the present invention can also be modified with respect to their length or otherwise the number of nucleotides making up the antagomir.
  • RNA, cDNA, genomic DNA, vectors, viruses or hybrids thereof can be isolated from a variety of sources, genetically engineered, amplified, and/or expressed/generated recombinantly.
  • Recombinant nucleic acid sequences can be individually isolated or cloned and tested for a desired activity. Any recombinant expression system can be used, including e g in vitro, bacterial, fungal, mammalian, yeast, insect or plant cell expression systems.
  • Nucleic acid sequences of the invention can be inserted into delivery vectors and expressed from transcription units within the vectors (e.g., AAV vectors).
  • the recombinant vectors can be DNA plasmids or viral vectors.
  • Generation of the vector construct can be accomplished using any suitable genetic engineering techniques well known in the art, including, without limitation, the standard techniques of PCR, oligonucleotide synthesis, restriction endonuclease digestion, ligation, transformation, plasmid purification, and DNA sequencing, for example as described in Sambrook et al. Molecular Cloning: A Laboratory Manual. (1989)), Coffin et al. (Retroviruses. (1997)) and “RNA Viruses: A Practical Approach” (Alan J.
  • Viral vectors comprise a nucleotide sequence having sequences for the production of recombinant virus in a packaging cell.
  • Viral vectors expressing nucleic acids of the invention can be constructed based on viral backbones including, but not limited to, a retrovirus, lentivirus, adenovirus, adeno-associated virus, pox virus or alphavirus.
  • the recombinant vectors capable of expressing the nucleic acids of the invention can be delivered as described herein, and persist in target cells (e.g., stable transformants).
  • Nucleic acid sequences used to practice this invention can be synthesized in vitro by well-known chemical synthesis techniques, as described in, e.g., Adams (1983) J. Am. Chem. Soc. 105:661; Belousov (1997) Nucleic Acids Res. 25:3440-3444; Frenkel (1995) Free Radic. Biol. Med. 19:373-380; Blommers (1994) Biochemistry 33:7886-7896; Narang (1979) Meth. Enzymol. 68:90; Brown (1979) Meth. Enzymol. 68:109; Beaucage (1981) Tetra. Lett. 22:1859; U.S. Pat. No. 4,458,066.
  • nucleic acid sequences of the invention can be stabilized against nucleolytic degradation such as by the incorporation of a modification, e.g., a nucleotide modification.
  • nucleic acid sequences of the invention include a phosphorothioate at least the first, second, or third internucleotide linkage at the 5′ or 3′ end of the nucleotide sequence.
  • the nucleic acid sequence can include a 2′-modified nucleotide, e.g., a 2′-deoxy, 2′-deoxy-2′-fluoro, 2′-O-methyl, 2′-O-methoxyethyl (2′-O-MOE), 2′-O-aminopropyl (2′-O-AP), 2′-O-dimethylaminoethyl (2′-O-DMAOE), 2′-O-dimethylaminopropyl (2′-O-DMAP), 2′-O-dimethylaminoethyloxyethyl (2′-O-DMAEOE), or 2′-O-N-methylacetamido (2′-O-NMA).
  • the nucleic acid sequence can include at least one 2′-O-methyl-modified nucleotide, and in some embodiments, all of the nucleotides include a 2′-O-methyl modification.
  • nucleic acids used to practice this invention such as, e.g., subcloning, labeling probes (e.g., random-primer labeling using Klenow polymerase, nick translation, amplification), sequencing, hybridization and the like are well described in the scientific and patent literature, see, e.g., Sambrook, ed., MOLECULAR CLONING: A LABORATORY MANUAL (2ND ED.), Vols. 1-3, Cold Spring Harbor Laboratory, (1989); CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Ausubel, ed.
  • ABCD1 vector administration provided by intravenous (IV) or intracerebroventricular (ICV) administration has recently been determined to cause cardiac toxicity.
  • Intrathecal administration is a route of administration comprising injection of desired agents into the subarachnoid space of the spinal canal, thereby providing the agents into the cerebrospinal fluid (CSF).
  • CSF cerebrospinal fluid
  • intrathecal administration ABCD1 expression from a vector within in the central nervous system is less than ABCD1 expression from a vector within peripheral organs, such as the heart. Excess ABCD1 expression in peripheral organs can result in toxicity and therefore, intrathecal administration of ABCD1 vectors comprises an improved method of therapy for X-ALD, e.g., for AMN.
  • the intrathecal administration is via a pump.
  • the pump may be a surgically implanted osmotic pump.
  • the osmotic pump is implanted into the subarachnoid space of the spinal canal to facilitate intrathecal administration.
  • human subjects receive a one-time treatment of intrathecally delivered vector (e.g., AAV9) comprising ABCD1 in an amount of about 1 ⁇ 10 13 GC to about 10 ⁇ 10 13 GC over a period of about 24 hours.
  • intrathecally delivered vector e.g., AAV9
  • the slow continuous intrathecal infusion of the AAV9-hABCD1 can be scaled up to humans by using an osmotically driven pump such as the DUROS® implant, ALZA Corporation (Mountain View, Calif.). See also, J. C. Wright, J. Culwell, Long-term controlled delivery of therapeutic agents by the osmotically driven DUROS® implant, in: M. J. Rathbone, J. Hadgraft, M. S. Roberts (Eds.), Modified-Release Drug Delivery Technology, Informa Healthcare, New York, 2008, pp. 143-149.
  • an osmotically driven pump such as the DUROS® implant, ALZA Corporation (Mountain View, Calif.). See also, J. C. Wright, J. Culwell, Long-term controlled delivery of therapeutic agents by the osmotically driven DUROS® implant, in: M. J. Rathbone, J. Hadgraft, M. S. Roberts (Eds.), Modified-Release Drug Delivery
  • the DUROS® delivery device typically consists of a cylindrical reservoir which contains the osmotic engine, piston, and drug formulation.
  • the reservoir is capped at one end by a controlled-rate water-permeable membrane and capped at the other end by a diffusion moderator through which drug formulation is released from the drug reservoir.
  • the piston separates the drug formulation from the osmotic engine and utilizes a seal to prevent the water in the osmotic engine compartment from entering the drug reservoir.
  • the diffusion moderator is designed, in conjunction with the drug formulation, to prevent body fluid from entering the drug reservoir through the orifice.
  • the DUROS® device releases a therapeutic agent at a predetermined rate based on the principle of osmosis.
  • Extracellular fluid enters the DUROS® device through a semi-permeable membrane directly into a salt engine that expands to drive the piston at a slow and even delivery rate. Movement of the piston forces the drug formulation to be released through the orifice or exit port at a predetermined sheer rate.
  • the reservoir of the DUROS® device is load with a suspension formulation of the present invention, comprising, for example, 1 ⁇ 10 11 gc AAV9-hABCD1, wherein the device is capable of delivering the suspension formulation to a subject over an extended period of time at a pre-determined, therapeutically effective delivery rate.
  • Implantable, drug delivery devices may be used in the practice of the present invention and may include regulator-type implantable pumps that provide constant flow, adjustable flow, or programmable flow of the compound, such as those available from Codman & Shurtleff, Inc. (Raynham, Mass.), Medtronic, Inc. (Minneapolis, Minn.), and Tricumed Medinzintechnik GmbH (Germany).
  • a high amount of cell death/cytopathic effects during production of AAV9-ABCD1 has been previously observed, likely due to overexpression of ABCD1 protein in producer cells. This toxicity reduced AAV vector yields.
  • ABCD1 mRNA was targeted using a pool of siRNAs.
  • AAV packing was carried out as follows. 1 ⁇ 1.5 ⁇ 10 7 293T cells were plated on 15 cm plates in and cultured overnight.
  • Tube B Vector Construct 10 ug 2x Hebs 780 ul Adenovirus helper 26 ug **Serotype plasmid 12 ug 2M CaCl 96.9 2.5 mM Hepes up to 780 ul Total Volume 780 ul Total Volume 780 ul
  • Tube A and Tube B were combined drop-wise while vortexing for 1 minute. The mix was incubated at room temperature for 20 minutes. 1.5 ml of virus mix per 15 cm plate was added, distributing drop-wise over the surface of the plate. Plates were tilted to mix evenly and incubated overnight. At day 3, media was replaced with DMEM 2% FBS 1% p/s. At day 5, half of the plate media volume was removed from each plate. Cells were collected from plates by washing with remaining media or gently using a cell scraper. Cells were spun down at 1300 RPM for 5 minutes. Supernatant was removed.
  • Cells were loosened by flicking the bottom of the tube and re-suspended in 2 ml EDTA PBS per plate of cells and spun at 1300 RPM for 5 minutes. Cells were re-suspended in 1 ml lysis buffer per plate and optionally stored at ⁇ 80 C. Following gradient purification, virus was buffer exchanged into PBS, quantified by qPCR, and used for experimentation.
  • the siRNA protocol has multiple steps:
  • the qPCR protocol has multiple steps:
  • the yield is reported as relative titer in which the AAV9-GFP sample was set arbitrarily at 100% and the other two samples normalized to this value.
  • 293T cells were transfected with control siRNA ( FIG. 2 , lanes 1 , 2 ), siRNA pool against ABCD1 mRNA ( FIG. 2 , lanes 3 - 6 ) or untransfected ( FIG. 2 , lane 7 , “normal” refers to endogenous levels of ABCD1 protein in 293T cells).
  • AAV-ABCD1 plasmid FIG. 2 , lanes 1 - 4
  • AAV-GFP plasmid FIG. 2 , lane 5 , 6
  • Actin blotting was performed for loading control. 8s and is refers to 8 second and 1 second exposure of the radiographic film, respectively.
  • the ABCD1 siRNA reduces the level of overexpressed ABCD1 compared to control siRNA.
  • 293T cells were left untransfected (no siRNA) or transfected with control siRNA or the siRNA pool against ABCD1 mRNA. The following day cells were transfected with AAV plasmids to produce AAV9-ABCD1. On day 3 post transfection of AAV plasmids, qPCR was performed to determine the amount of vector (g.c.) in cell lysate and in the media of the transfected cells. An approximate 3-4 fold increase in AAV9-ABCD1 vector yield in both cell lysate and media was observed ( FIG. 3 ).
  • Self-complementary AAV9 GFP(scAAV9GFP) and rAAV9 encoding ABCD1 were delivered to Abcd1 ⁇ / ⁇ mice intrathecally (IT) either by bolus over a 2 minute duration or by osmotic pump over 24 hour duration with PBS injection as sham control. Two weeks after injection, mice were sacrificed and perfused with 4% PFA. Tissues were then collected, sectioned and stained for immunofluorescence analysis.
  • scAAV9-GFP delivered IT by osmotic pump led to widespread expression across CNS-relevant cell types and DRGs in a dose-dependent manner.
  • ABCD1 expression in the central nervous system was about 3 fold higher than expression of ABCD1 in the central nervous system of an untreated subject that does not have X-ALD (e.g., wild-type).
  • ABCD1 expression in peripheral organs was about 90% less than expression of ABCD1 expression in peripheral organs of an untreated subject that does not have X-ALD (see FIG. 11 , where protein expression among different tissue types in Western blots was normalized to endogenous wild-type levels).
  • rAAV9-mediated ABCD1 gene transfer via intrathecal osmotic pump leads to more uniform and widespread gene delivery to the CNS with reduced leakage into the systemic circulation compared with intrathecal bolus injection.
  • C26:0 is the biochemical hallmark of adrenomyeloneuropathy.
  • VLCFA very long chain fatty acids
  • lipidomic analysis was performed on spinal cord samples. Absolute values of C26:0 and C24:0 as well as ratios of C26:0/C22:0 are reported. It was determined that rAAV9-mediated ABCD1 gene transfer via intrathecal osmotic pump (1 ⁇ 10 11 gc) leads to a 20% reduction in C26:0 levels in the spinal cord ( FIG. 13 ). The levels after intrathecal osmotic pump delivery are comparable to those after intrathecal bolus delivery but avoid systemic leakage.
  • Immunofluorescence staining and confocal microscopy imaging were additionally conducted.
  • tissue section imaging sections of spinal cord (16 ⁇ m) were cut at ⁇ 25° C. using cryostat (Leica) and stored at ⁇ 80° C. Sections were stained with mouse antihuman ABCD1 antibody and then costained with rabbit anti-GFAP (Dako, Carpinteria, Calif.), rabbit anti-IBA1 (Wako, Richmond, Va.) and rabbit anti-CD31 (Abcam) respectively to localize the cell type.
  • TOPRO-3 Thermo Fisher Scientific was used as fluorescent dye for nuclear counterstaining. The slides were imaged by confocal laser microscope and transduced cells counted.
  • ABCD1 transduced cells of each cell type were documented in 20 ⁇ and 40 ⁇ (for microglia) magnification images.
  • rAAV9-mediated ABCD1 gene transfer via intrathecal osmotic pump (1 ⁇ 10 11 gc) targets mainly astrocytes, endothelial cells and a few neurons in the spinal cord ( FIG. 14 ). Within with dorsal root ganglia it targets both satellite cells and neurons ( FIG. 14 ).

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