WO2003053476A1 - Adeno-associated virus-mediated delivery of gdnf to skeletal muscles - Google Patents
Adeno-associated virus-mediated delivery of gdnf to skeletal muscles Download PDFInfo
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Definitions
- the present invention relates generally to compositions and methods for gene delivery.
- the present invention pertains to adeno-associated virus (AAV)-based gene delivery systems for delivering glial cell line-derived neurotrophic factor (GDNF) to skeletal muscle to treat motoneuron diseases such as amyotrophic lateral sclerosis (ALS).
- AAV adeno-associated virus
- Motoneuron neurodegenerative diseases present major public health issues.
- amyotrophic lateral sclerosis is a relentlessly progressive lethal disease that involves selective annihilation of motoneurons.
- ALS amyotrophic lateral sclerosis
- SOD1 Cu/Zn superoxide dismutase
- Transgenic mice overexpressing this mutant gene develop a dominantly inherited adult-onset paralytic disorder that has many of the clinical and pathological features of familial ALS (Gurney et al., Science (1994) 264:1772-1775).
- the molecular mechanisms leading to motoneuron degeneration in ALS and most motor neuron diseases remain poorly understood. Because the mechanism leading to motoneuron degeneration in ALS is not known, there is currently no therapy available to prevent or cure ALS.
- Glial cell line-derived neurotrophic factor has been shown to be the most potent neurotrophic factor for the proliferation, differentiation, and survival of spinal motoneurons.
- GDNF and GDNF mRNA levels have been reported to be up- regulated in denervated muscles as found in ALS, polymyostits (PM) and muscular dystrophy (MD), or after peripheral nerve lesion and the like (Trupp et al., J. Cell Biol. (1995) 130:137-148; Lie and Weis, Neurosci. Lett. (1998) 250: 87-90; Yamamoto et al., Neurochem. Res. (1999) 24:785-790).
- GDNF has been proposed as a therapeutic agent to treat motor neuron disease (Henderson et al., Science (1994) 266:1062-1064; Oppenheim et al., Nature (1995) 373:344-346; Yan et al., Nature (1995) 373:341-344; Sagot et al., J. Neurosci. (1996) 16: 2335-2341 ; Bohn, M.C., Biochem. Pharmacol. (1999) 57:135-142; Mohajeri et al., Hum. Gene Ther. (1999) 10: 1853-1866).
- Neurotrophic factors such as GDNF have been shown to slow motoneuron degeneration and to restore the function of non- functional motoneurons that are still alive (Trupp et al., J. Cell Biol. (1995) 130:137-148; Sagot et al., J. Neurosci. (1998) 18: 1132-1141 ; Lie and Weis, Neurosci. Lett. (1998) 250: 87-90; Baumgartner and Shine, J. Neurosci. Res. (1998) 54: 766-777; Yamamoto et al., Neurochem. Res. (1999) 24:785-790; Biesch and Tuszynski, J. Comp. Neurol. (2001) 436:399-410; Keller-Peck et al., J. Neurosci. (2001) 21:6136-6146.
- GDNF ciliary neurotrophic factor
- BDNF brain-derived neurotrophic factor
- IGF-I insulin-like growth factor-I
- these proteins have a short in vivo plasma half-life, have poor access to spinal cord motoneurons, and cause inflammatory reactions that prevent administration at an adequate dose (Haase et al., Nat. Med.
- Adeno-associated virus has shown promise for delivering genes for gene therapy in clinical trials in humans (see, e.g., Kay et al., Nat. Genet. (2000) 24:257-261).
- AAV Adeno-associated virus
- the AAV genome is a linear, single-stranded DNA molecule containing about 4681 nucleotides.
- the AAV genome generally comprises an internal nonrepeating genome flanked on each end by inverted terminal repeats (ITRs).
- ITRs are approximately 145 base pairs (bp) in length.
- the ITRs have multiple functions, including as origins of DNA replication, and as packaging signals for the viral genome.
- the internal nonrepeated portion of the genome includes two large open reading frames, known as the AAV replication (rep) and capsid (cap) genes.
- the rep and cap genes code for viral proteins that allow the virus to replicate and package into a virion.
- a family of at least four viral proteins are expressed from the AAV rep region, Rep 78, Rep 68, Rep 52, and Rep 40, named according to their apparent molecular weight.
- the AAV cap region encodes at least three proteins, VPI, VP2, and VP3.
- AAV has been engineered to deliver genes of interest by deleting the internal nonrepeating portion of the AAV genome (i.e., the rep and cap genes) and inserting a heterologous gene between the ITRs.
- the heterologous gene is typically functionally linked to a heterologous promoter (constitutive, cell-specific, or inducible) capable of driving gene expression in the patient's target cells under appropriate conditions. Termination signals, such as polyadenylation sites, can also be included.
- AAV is a helper-dependent virus; that is, it requires coinfection with a helper virus (e.g., adenovirus, herpesvirus or vaccinia), in order to form AAV virions.
- a helper virus e.g., adenovirus, herpesvirus or vaccinia
- AAV establishes a latent state in which the viral genome inserts into a host cell chromosome, but infectious virions are not produced.
- Subsequent infection by a helper virus "rescues" the integrated genome, allowing it to replicate and package its genome into an infectious AAV virion.
- the helper virus While AAV can infect cells from different species, the helper virus must be of the same species as the host cell. Thus, for example, human AAV will replicate in canine cells coinfected with a canine adenovirus.
- the present invention provides a potent and effective method for treating neurodegenerative diseases that affect motoneurons.
- skeletal muscle is particularly useful for AAV-mediated GDNF delivery.
- Skeletal muscle is highly transducible, easily accessible and displays a low cell turnover (Wang et al., Proc. Natl. Acad. Sci. USA (2000) 97: 13714-13719; Xiao et al., J. Virol. (1996) 70:8098-8108).
- Nerve terminals are only in contact with myofibers at the neuromuscular junctions (NMJs) at which barriers against various substances is absent, allowing them to reach the central nervous system.
- NMJs neuromuscular junctions
- neurites connect with their targets to gain access to target-derived neurotrophic factors for neuron survival.
- target-derived neurotrophic factor endogenous GDNF produced by skeletal muscle functions via retrograde axonal transport from the target muscle tissue to motoneuronal cell bodies in the spinal cord (Mitsumoto, H., Muscle Nerve (1999) 22: 1000-1021).
- AAV-mediated GDNF gene delivery via intramuscular administration drives substantial and persistent expression of GDNF in large numbers of myofibers.
- expressed GDNF is retrogradely transported to spinal cord motoneurons from nerve terminals in the muscle.
- the invention is directed to a method of delivering a recombinant AAV virion to a muscle cell or muscle tissue of a mammalian subject with a motoneuron disorder.
- the method comprises:
- the muscle cell or tissue is derived from skeletal muscle.
- the recombinant AAV virion is introduced into the muscle cell in vivo, e.g., by intramuscular injection.
- the recombinant AAV virion is introduced into the muscle cell in vitro.
- the motoneuron disorder is amyotrophic lateral sclerosis (ALS).
- the invention is directed to a method of delivering a recombinant AAV virion to a skeletal muscle of a human subject with ALS.
- the method comprises:
- the invention is directed to a method of treating a mammalian subject with a motoneuron disorder comprising administering intramuscularly to the subject recombinant adeno-associated virus (AAV) virions comprising a polynucleotide encoding a GDNF polypeptide operably linked to expression control elements capable of directing the in vivo transcription and translation of the GDNF to provide a therapeutic effect.
- the motoneuron disorder is ALS.
- the subject is human and the polynucleotide encodes a human GDNF, such as human pre-pro-GDNF.
- the control elements can comprise a viral promoter, such as an MLP, CMV, or RSV LTR promoter.
- muscle cells are transduced in vivo, e.g., by administration into skeletal muscle.
- the invention is directed to a method of treating a mammalian subject with ALS.
- the method comprises administering into skeletal muscle of the subject a composition comprising recombinant adeno-associated virus (AAV) virions that comprise a polynucleotide encoding a GDNF polypeptide operably linked to expression control elements capable of directing the in vivo transcription and translation of the GDNF, to provide a therapeutic effect.
- AAV adeno-associated virus
- the subject is a human and the polynucleotide encodes a human GDNF, such as human pre-pro-GDNF.
- the control elements can comprise a viral promoter, such as an MLP, CMV, or RSV LTR promoter.
- Figures 1 A and IB show the GDNF levels in conditioned medium and 293 cell lysate 48 post infection with AAV-GDNF-FLAG or AAV-LacZ as measured by ELISA.
- Figure 2 shows GDNF levels in gastrocnemius muscle of injected mice at various time points post-injection.
- Figure 3 displays percent distribution of muscle fibers of various diameters in wild-type, control ALS and AAV-GDNF-treated ALS mice.
- Figures 4A-4B show the numbers of spinal motoneurons in wild-type, control ALS and AAV-GDNF-treated ALS mice.
- Figure 4A shows the average number of Nissl-stained neurons per anterior horn.
- Figure 4B shows the average number of SMI-32-stained neurons per anterior horn.
- Figures 5 A and 5B display the effect of GDNF on motoneurons that retained axonal projections in wild-type, control ALS and AAV-GDNF-treated ALS mice.
- Figure 5 A shows the survival of CTB-labeled motoneurons per anterior horn. The value represents the CTB/Nissl ratio (average number of neurons per anterior horn).
- Figure 5B shows the percent distribution of muscle fibers of various diameters in wild-type, control ALS and AAV-GDNF-treated ALS mice.
- Figures 6A-6E show the results of experiments demonstrating that GDNF delays the onset of disease, improves motor performance, and prolongs survival in transgenic ALS mice.
- Figure 6 A displays the cumulative probability of onset of rotarod deficits in ALS mice.
- Figure 6B shows performance of ALS mice in the rotarod test at 5 rpm.
- Figure 6C shows performance of ALS mice in the rotarod test at 10 rpm.
- Figure 6D shows performance of ALS mice in the rotarod test at 20 rpm.
- Figure 6E shows the cumulative probability of survival.
- Figure 7 shows the nucleotide sequence and amino acid sequence for a human pre-pro-GDNF.
- the mature GDNF molecule spans amino acid positions 78-211.
- Figure 8 shows the nucleotide sequence and amino acid sequence for a rat pre-pro-GDNF.
- the mature GDNF molecule spans amino acid positions 78-211.
- Figure 9 shows the nucleotide sequence and amino acid sequence for a mouse pre-pro-GDNF.
- the mature GDNF molecule spans amino acid positions 107-240.
- glial cell line-derived neurotrophic factor polypeptide or "GDNF polypeptide” refers to a neurotrophic factor of any origin, which is substantially homologous and functionally equivalent to any of the various known GDNFs.
- Representative GDNFs for three mammalian species are shown in Figures 7, 8 and 9.
- the degree of homology between the rat ( Figure 8, SEQ ID NO:4) and human ( Figure 7, SEQ ID NO:2) protein is about 93% and all mammalian GDNFs have a similarly high degree of homology.
- Such GDNFs may exist as monomers, dimers or other multimers in their biologically active form.
- GDNF polypeptide encompasses active monomeric GDNFs, as well as active multimeric GDNFs, active glycosylated and non-glycosylated forms of GDNF and active truncated forms of the molecule.
- GDNF polypeptide that retains some or all of the biological properties regarding motoneurons, but not necessarily to the same degree, as a native GDNF molecule.
- “Homology” refers to the percent similarity between two polynucleotide or two polypeptide moieties.
- Two polynucleotide, or two polypeptide sequences are "substantially homologous" to each other when the sequences exhibit at least about 50% , preferably at least about 75%, more preferably at least about 80%-85%, preferably at least about 90%, and most preferably at least about 95%-99% or more sequence similarity or sequence identity over a defined length of the molecules.
- substantially homologous also refers to sequences showing complete identity to the specified polynucleotide or polypeptide sequence.
- identity refers to an exact nucleotide-to-nucleotide or amino acid-to-amino acid correspondence of two polynucleotides or polypeptide sequences, respectively. Percent identity can be determined by a direct comparison of the sequence information between two molecules by aligning the sequences, counting the exact number of matches between the two aligned sequences, dividing by the length of the shorter sequence, and multiplying the result by 100. Readily available computer programs can be used to aid in the analysis of similarity and identity, such as ALIGN, Dayhoff, M.O. in Atlas of Protein Sequence and Structure M.O. Dayhoff ed., 5 Suppl.
- nucleotide sequence similarity and identity are available in the Wisconsin Sequence Analysis Package, Version 8 (available from Genetics Computer Group, Madison, WI) for example, the BESTFIT, FASTA and GAP programs, which also rely on the Smith and Waterman algorithm. These programs are readily utilized with the default parameters recommended by the manufacturer and described in the Wisconsin Sequence Analysis Package referred to above. For example, percent similarity of a particular nucleotide sequence to a reference sequence can be determined using the homology algorithm of Smith and Waterman with a default scoring table and a gap penalty of six nucleotide positions.
- Another method of establishing percent similarity in the context of the present invention is to use the MPSRCH package of programs copyrighted by the University of Edinburgh, developed by John F. Collins and Shane S. Sturrok, and distributed by IntelliGenetics, Inc. (Mountain View, CA). From this suite of packages the Smith- Waterman algorithm can be employed where default parameters are used for the scoring table (for example, gap open penalty of 12, gap extension penalty of one, and a gap of six). From the data generated the "Match" value reflects "sequence similarity.”
- Other suitable programs for calculating the percent identity or similarity between sequences are generally known in the art, for example, another alignment program is BLAST, used with default parameters.
- homology can be determined by hybridization of polynucleotides under conditions which form stable duplexes between homologous regions, followed by digestion with single-stranded-specific nuclease(s), and size determination of the digested fragments.
- DNA sequences that are substantially homologous can be identified in a Southern hybridization experiment under, for example, stringent conditions, as defined for that particular system. Defining appropriate hybridization conditions is within the skill of the art. See, e.g., Sambrook et al., supra; DNA Cloning, supra; Nucleic Acid Hybridization, supra.
- GDNF variant is meant a biologically active derivative of the reference GDNF molecule, or a fragment of such a derivative, that retains desired activity, such as neurotrophic activity in the assays described herein.
- the term “variant” refers to compounds having a native polypeptide sequence and structure with one or more amino acid additions, substitutions (generally conservative in nature) and/or deletions, relative to the native molecule, so long as the modifications do not destroy neurotrophic activity.
- the variant has at least the same biological activity as the native molecule. Methods for making polynucleotides encoding for GDNF variants are known in the art and are described further below.
- deletions generally range from about 1 to 30 residues, more usually from about 1 to 10 residues, and typically from about 1 to 5 contiguous residues, or any integer within the stated ranges.
- N-terminal, C-terminal and internal deletions are contemplated.
- Deletions are generally introduced into regions of low homology with other TGF- ⁇ super family members in order to preserve maximum biological activity. Deletions are typically selected so as to preserve the tertiary structure of the GDNF protein product in the affected domain, e.g., cysteine crosslinking.
- Non-limiting examples of deletion variants include truncated GDNF protein products lacking from 1-40 N-terminal amino acids of GDNF, or variants lacking the C-terminal residue of GDNF, or combinations thereof.
- amino acid sequence additions typically include N-and or C-terminal fusions ranging in length from one residue to polypeptides containing a hundred or more residues, as well as internal additions of single or multiple amino acid residues. Internal additions generally range from about 1-10 residues, more typically from about 1-5 residues, and usually from about 1-3 amino acid residues, or any integer within the stated ranges.
- N-terminal addition variants include the fusion of a heterologous N-terminal signal sequence to the N-terminus of GDNF as well as fusions of amino acid sequences derived from the sequence of other neurotrophic factors.
- GDNF substitution variants have at least one amino acid residue of the GDNF amino acid sequence removed and a different residue inserted in its place.
- substitution variants include allelic variants, which are characterized by naturally occurring nucleotide sequence changes in the species population that may or may not result in an amino acid change.
- Particularly preferred substitutions are conservative in nature, i.e., those substitutions that take place within a family of amino acids that are related in their side chains.
- amino acids are generally divided into four families: (1) acidic — aspartate and glutamate; (2) basic ⁇ lysine, arginine, histidine; (3) non-polar — alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; and (4) uncharged polar — glycine, asparagine, glutamine, cysteine, serine threonine, tyrosine. Phenylalanine, tryptophan, and tyrosine are sometimes classified as aromatic amino acids.
- the GDNF molecule may include up to about 5-10 conservative or non-conservative amino acid substitutions, or even up to about 15-25 conservative or non-conservative amino acid substitutions, or any integer between 5- 25, so long as the desired function of the molecule remains intact.
- One of skill in the art may readily determine regions of the molecule of interest that can tolerate change using techniques well known in the art.
- GDNF amino acid sequence may involve modifications to a glycosylation site (e.g., serine, threonine, or asparagine).
- a glycosylation site e.g., serine, threonine, or asparagine.
- the absence of glycosylation or only partial glycosylation results from amino acid substitution or deletion at any asparagine-linked glycosylation recognition site or at any site of the molecule that is modified by addition of an O-linked carbohydrate.
- An asparagine-linked glycosylation recognition site comprises a tripeptide sequence which is specifically recognized by appropriate cellular glycosylation enzymes. These tripeptide sequences are either Asn-Xaa-Thr or Asn-Xaa-Ser, where Xaa can be any amino acid other than Pro.
- a variety of amino acid substitutions or deletions at one or both of the first or third amino acid positions of a glycosylation recognition site (and/or amino acid deletion at the second position) result in non-glycosylation at the modified tripeptide sequence.
- the expression of appropriate altered nucleotide sequences produces variants which are not glycosylated at that site.
- the GDNF amino acid sequence may be modified to add glycosylation sites.
- GDNF amino acid residues or regions for mutagenesis are well known in the art.
- One such method is known as "alanine scanning mutagenesis.” See, e.g., Cunningham and Wells, Science (1989) 244:1081-1085.
- an amino acid residue or group of target residues are identified (e.g., charged residues such as Arg, Asp, His, Lys, and Glu) and replaced by a neutral or negatively charged amino acid (most preferably alanine or polyalanine) to affect the interaction of the amino acids with the surrounding aqueous environment in or outside the cell.
- Those domains demonstrating functional sensitivity to the substitutions are refined by introducing additional or alternate residues at the sites of substitution.
- the target site for introducing an amino acid sequence variation is determined, alanine scanning or random mutagenesis is conducted on the corresponding target codon or region of the DNA sequence, and the expressed GDNF variants are screened for the optimal combination of desired activity and degree of activity.
- the sites of greatest interest for mutagenesis include sites where the amino acids found in GDNF proteins from various species are substantially different in terms of side-chain bulk, charge, and/or hydrophobicity.
- Other sites of interest are those in which particular residues of GDNF-like proteins, obtained from various species, are identical. Such positions are generally important for the biological activity of a protein. Initially, these sites are substituted in a relatively conservative manner. If such substitutions result in a change in biological activity, then more substantial changes (exemplary substitutions) are introduced, and/or other additions or deletions may be made, and the resulting products screened for activity.
- Assays for GDNF activity are known in the art.
- any of the various in vitro model systems, described more fully below, can be used as measures of GDNF activity.
- motoneuron disorder is meant a disease affecting a neuron with motor function, i.e., a neuron that conveys motor impulses.
- Such neurons are also termed “motor neruons.”
- These neurons include, without limitation, alpha neurons of the anterior spinal cord that give rise to the alpha fibers which innervate the skeletal muscle fibers; beta neurons of the anterior spinal cord that give rise to the beta fibers which innervate the extrafusal and intrafusal muscle fibers; gamma neurons of the anterior spinal cord that give rise to the gamma (fusimotor) fibers which innervate the intrafusal fibers of the muscle spindal; heteronymous neurons that supply muscles other than those from which afferent impulses originate; homonymous neurons that supply muscles from which afferent impulses originate; lower peripheral neurons whose cell bodies lie in the ventral gray columns of the spinal cord and whose terminations are in skeletal muscles; peripheral neurons that receive impulses from internuerons; and upper neurons in the
- motoneuron disorders include the various amyotrophies such as hereditary amyotrophies including hereditary spinal muscular atrophy, acute infantile spinal muscular atrophy such as Werdnig-Hoffman disease, progressive muscular atrophy in children such as the proximal, distal type and bulbar types, spinal muscular atrophy of adolescent or adult onset including the proximal, scapuloperoneal, facioscapulohumeral and distal types, amyotrophic lateral sclerosis (ALS) and primary lateral sclerosis (PLS). Also included within the term is motoneuron injury.
- hereditary amyotrophies including hereditary spinal muscular atrophy, acute infantile spinal muscular atrophy such as Werdnig-Hoffman disease, progressive muscular atrophy in children such as the proximal, distal type and bulbar types, spinal muscular atrophy of adolescent or adult onset including the proximal, scapuloperoneal, facioscapulohumeral and dis
- skeletal muscle is meant a striated muscle that is attached to bones and that typically crosses at least one joint. Scientifically, these muscles are often referred to as musculi skeleti.
- vector any genetic element, such as a plasmid, phage, transposon, cosmid, chromosome, virus, virion, etc., which is capable of replication when associated with the proper control elements and which can transfer gene sequences between cells.
- vector includes cloning and expression vehicles, as well as viral vectors.
- an “AAV vector” is meant a vector derived from an adeno-associated virus serotype, including without limitation, AAV-1, AAV-2, AAV-3, AAV-4, AAV- 5, AAV-6, AAV-7 and AAV-8.
- AAV vectors can have one or more of the AAV wild-type genes deleted in whole or part, preferably the rep and/or cap genes, but retain functional flanking ITR sequences. Functional ITR sequences are necessary for the rescue, replication and packaging of the AAV virion.
- an AAV vector is defined herein to include at least those sequences required in cis for replication and packaging (e.g., functional ITRs) of the virus.
- the ITRs need not be the wild-type nucleotide sequences, and may be altered, e.g., by the insertion, deletion or substitution of nucleotides, so long as the sequences provide for functional rescue, replication and packaging.
- AAV helper functions refer to AAV-derived coding sequences which can be expressed to provide AAV gene products that, in turn, function in trans for productive AAV replication.
- AAV helper functions include both of the major AAV open reading frames (ORFs), rep and cap.
- the Rep expression products have been shown to possess many functions, including, among others: recognition, binding and nicking of the AAV origin of DNA replication; DNA helicase activity; and modulation of transcription from AAV (or other heterologous) promoters.
- the Cap expression products supply necessary packaging functions.
- AAV helper functions are used herein to complement AAV functions in trans that are missing from AAV vectors.
- AAV helper construct refers generally to a nucleic acid molecule that includes nucleotide sequences providing AAV functions deleted from an AAV vector which is to be used to produce a transducing vector for delivery of a nucleotide sequence of interest.
- AAV helper constructs are commonly used to provide transient expression of AAV rep and/or cap genes to complement missing AAV functions that are necessary for lytic AAV replication; however, helper constructs lack AAV ITRs and can neither replicate nor package themselves.
- AAV helper constructs can be in the form of a plasmid, phage, transposon, cosmid, virus, or virion. A number of AAV helper constructs and vectors that encode Rep and/or Cap expression products have been described.
- the term "accessory functions" refers to non- AAV derived viral and/or cellular functions upon which AAV is dependent for its replication.
- captures proteins and RNAs that are required in AAV replication including those moieties involved in activation of AAV gene transcription, stage specific AAV mRNA splicing, AAV DNA replication, synthesis of Cap expression products and AAV capsid assembly.
- Viral-based accessory functions can be derived from any of the known helper viruses such as adenovirus, he ⁇ esvirus (other than he ⁇ es simplex virus type-1) and vaccinia virus.
- accessory function vector refers generally to a nucleic acid molecule that includes nucleotide sequences providing accessory functions.
- An accessory function vector can be transfected into a suitable host cell, wherein the vector is then capable of supporting AAV virion production in the host cell.
- accessory function vectors can be in the form of a plasmid, phage, transposon or cosmid.
- adenovirus mutants incapable of DNA replication and late gene synthesis have been shown to be permissive for AAV replication. Ito et al., (1970) J. Gen. Virol. 9:243; Ishibashi et al, (1971) Virology 45:317. Similarly, mutants within the E2B and E3 regions have been shown to support AAV replication, indicating that the E2B and E3 regions are probably not involved in providing accessory functions. Carter et al., (1983) Virology 126:505.
- E2A Haptena et al., (1975) J. Gen. Virol. 29:239; Strauss et al., (1976) J. Virol. 17:140; Myers et al., (1980) J. Virol. 35:665; Jay et al., (1981) Proc. Natl. Acad. Sci. USA 78:2927; Myers et al., (1981) J Biol. Chem. 256:567): E2B (Carter, Adeno-Associated Virus Helper Functions, in I CRC Handbook of Parvoviruses (P.
- accessory function vectors comprise an adenovirus VA RNA coding region, an adenovirus E4 ORF6 coding region, an adenovirus E2A 72 kD coding region, an adenovirus El A coding region, and an adenovirus EIB region lacking an intact ElB55k coding region.
- Such vectors are described in international Publication No. WO 01/83797.
- “capable of supporting efficient rAAV virion production” is meant the ability of an accessory function vector or system to provide accessory functions that are sufficient to complement rAAV virion production in a particular host cell at a level substantially equivalent to or greater than that which could be obtained upon infection of the host cell with an adenovirus helper virus.
- an accessory function vector or system to support efficient rAAV virion production can be determined by comparing rAAV virion titers obtained using the accessory vector or system with titers obtained using infection with an infectious adenovirus. More particularly, an accessory function vector or system supports efficient rAAV virion production substantially equivalent to, or greater than, that obtained using an infectious adenovirus when the amount of virions obtained from an equivalent number of host cells is not more than about 200 fold less than the amount obtained using adenovirus infection, more preferably not more than about 100 fold less, and most preferably equal to, or greater than, the amount obtained using adenovirus infection.
- recombinant virus is meant a virus that has been genetically altered, e.g., by the addition or insertion of a heterologous nucleic acid construct into the particle.
- AAV virion is meant a complete virus particle, such as a wild-type (wt) AAV virus particle (comprising a linear, single-stranded AAV nucleic acid genome associated with an AAV capsid protein coat).
- wt wild-type
- AAV virus particle comprising a linear, single-stranded AAV nucleic acid genome associated with an AAV capsid protein coat.
- single-stranded AAV nucleic acid molecules of either complementary sense, e.g., "sense” or “antisense” strands can be packaged into any one AAV virion and both strands are equally infectious.
- a "recombinant AAV virion,” or “rAAV virion” is defined herein as an infectious, replication-defective virus including an AAV protein shell, encapsidating a heterologous nucleotide sequence of interest which is flanked on both sides by AAV ITRs.
- a rAAV virion is produced in a suitable host cell which has had an AAV vector, AAV helper functions and accessory functions introduced therein. In this manner, the host cell is rendered capable of encoding AAV polypeptides that are required for packaging the AAV vector (containing a recombinant nucleotide sequence of interest) into infectious recombinant virion particles for subsequent gene delivery.
- transfection is used to refer to the uptake of foreign DNA by a cell, and a cell has been "transfected” when exogenous DNA has been introduced inside the cell membrane.
- transfection techniques are generally known in the art. See, e.g., Graham et al. (1973) Virology, 52:456, Sambrook et al. (1989) Molecular Cloning, a laboratory manual, Cold Spring Harbor Laboratories, New York, Davis et al. (1986) Basic Methods in Molecular Biology, Elsevier, and Chu et al. (1981) Gene 13:197.
- exogenous DNA moieties such as a nucleotide integration vector and other nucleic acid molecules
- host cell denotes, for example, microorganisms, yeast cells, insect cells, and mammalian cells, that can be, or have been, used as recipients of an AAV helper construct, an AAV vector plasmid, an accessory function vector, or other transfer DNA.
- the term includes the progeny of the original cell which has been transfected.
- a "host cell” as used herein generally refers to a cell which has been transfected with an exogenous DNA sequence. It is understood that the progeny of a single parental cell may not necessarily be completely identical in mo ⁇ hology or in genomic or total DNA complement as the original parent, due to natural, accidental, or deliberate mutation.
- cell line refers to a population of cells capable of continuous or prolonged growth and division in vitro. Often, cell lines are clonal populations derived from a single progenitor cell. It is further known in the art that spontaneous or induced changes can occur in karyotype during storage or transfer of such clonal populations. Therefore, cells derived from the cell line referred to may not be precisely identical to the ancestral cells or cultures, and the cell line referred to includes such variants.
- heterologous as it relates to nucleic acid sequences such as coding sequences and control sequences, denotes sequences that are not normally joined together, and/or are not normally associated with a particular cell.
- a heterologous region of a nucleic acid construct or a vector is a segment of nucleic acid within or attached to another nucleic acid molecule that is not found in association with the other molecule in nature.
- a heterologous region of a nucleic acid construct could include a coding sequence flanked by sequences not found in association with the coding sequence in nature.
- heterologous coding sequence is a construct where the coding sequence itself is not found in nature (e.g., synthetic sequences having codons different from the native gene).
- a cell transformed with a construct which is not normally present in the cell would be considered heterologous for pu ⁇ oses of this invention. Allelic variation or naturally occurring mutational events do not give rise to heterologous DNA, as used herein.
- a "coding sequence” or a sequence which "encodes” a particular protein is a nucleic acid sequence which is transcribed (in the case of DNA) and translated (in the case of mRNA) into a polypeptide in vitro or in vivo when placed under the control of appropriate regulatory sequences.
- the boundaries of the coding sequence are determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxy) terminus.
- a coding sequence can include, but is not limited to, cDNA from prokaryotic or eukaryotic mRNA, genomic DNA sequences from prokaryotic or eukaryotic DNA, and even synthetic DNA sequences.
- a transcription termination sequence will usually be located 3' to the coding sequence.
- nucleic acid sequence refers to a DNA or RNA sequence.
- the term captures sequences that include any of the known base analogues of DNA and RNA such as, but not limited to 4-acetylcytosine, 8-hydroxy-N6-methyladenosine, aziridinylcytosine, pseudoisocytosine, 5-(carboxyhydroxylmethyl) uracil, 5- fluorouracil, 5-bromouracil, 5-carboxymethylaminomethyl-2-thiouracil, 5-carboxymethylaminomethyluracil, dihydrouracil, inosine, N6-isopentenyladenine, 1-methyladenine, 1-methylpseudouracil, 1-methylguanine, 1-methylinosine, 2,2- dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-methyladenine, 7-methylguanine, 5-methylaminomethyluracil, 5 -
- 2-methylthio-N6-isopentenyladenine 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid, oxybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5- methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, -uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid, pseudouracil, queosine, 2-thiocytosine, and 2,6-diaminopurine.
- control sequences refers collectively to promoter sequences, polyadenylation signals, transcription termination sequences, upstream regulatory domains, origins of replication, internal ribosome entry sites ("IRES"), enhancers, and the like, which collectively provide for the replication, transcription and translation of a coding sequence in a recipient cell. Not all of these control sequences need always be present so long as the selected coding sequence is capable of being replicated, transcribed and translated in an appropriate host cell.
- promoter is used herein in its ordinary sense to refer to a nucleotide region comprising a DNA regulatory sequence, wherein the regulatory sequence is derived from a gene which is capable of binding RNA polymerase and initiating transcription of a downstream (3 '-direction) coding sequence.
- Transcription promoters can include "inducible promoters” (where expression of a polynucleotide sequence operably linked to the promoter is induced by an analyte, cofactor, regulatory protein, etc.), “repressible promoters” (where expression of a polynucleotide sequence operably linked to the promoter is induced by an analyte, cofactor, regulatory protein, etc.), and “constitutive promoters”.
- operably linked refers to an arrangement of elements wherein the components so described are configured so as to perform their usual function.
- control sequences operably linked to a coding sequence are capable of effecting the expression of the coding sequence.
- the control sequences need not be contiguous with the coding sequence, so long as they function to direct the expression thereof.
- intervening untranslated yet transcribed sequences can be present between a promoter sequence and the coding sequence and the promoter sequence can still be considered “operably linked" to the coding sequence.
- isolated when referring to a nucleotide sequence, is meant that the indicated molecule is present in the substantial absence of other biological macromolecules of the same type.
- an "isolated nucleic acid molecule which encodes a particular polypeptide" refers to a nucleic acid molecule which is substantially free of other nucleic acid molecules that do not encode the subject polypeptide; however, the molecule may include some additional bases or moieties which do not deleteriously affect the basic characteristics of the composition.
- a “functional homologue,” or a “functional equivalent” of a given AAV polypeptide includes molecules derived from the native polypeptide sequence, as well as recombinantly produced or chemically synthesized polypeptides which function in a manner similar to the reference AAV molecule to achieve a desired result.
- a functional homologue of AAV Rep68 or Rep78 encompasses derivatives and analogues of those polypeptides—including any single or multiple amino acid additions, substitutions and/or deletions occurring internally or at the amino or carboxy termini thereof—so long as integration activity remains.
- a “functional homologue,” or a “functional equivalent” of a given adenoviral nucleotide region includes similar regions derived from a heterologous adenovirus serotype, nucleotide regions derived from another virus or from a cellular source, as well as recombinantly produced or chemically synthesized polynucleotides which function in a manner similar to the reference nucleotide region to achieve a desired result.
- a functional homologue of an adenoviral VA RNA gene region or an adenoviral E2a gene region encompasses derivatives and analogues of such gene regions—including any single or multiple nucleotide base additions, substitutions and/or deletions occurring within the regions, so long as the homologue retains the ability to provide its inherent accessory function to support AAV virion production at levels detectable above background.
- Convection-enhanced delivery refers to any non-manual delivery of agents.
- examples of convection-enhanced delivery (CED) of AAV can be achieved by infusion pumps or by osmotic pumps. A more detailed description of CED is found below.
- subject refers to a vertebrate, preferably a mammal.
- Mammals include, but are not limited to, murines, simians, humans, farm animals, sport animals and pets.
- an “effective amount” is an amount sufficient to effect beneficial or desired results.
- An effective amount can be administered in one or more administrations, applications or dosages.
- the present invention is based on the su ⁇ rising discovery that AAV vector- mediated GDNF gene delivery results in the durable and substantial expression of GDNF after a single intramuscular injection.
- substantial GDNF expression was achieved in a large number of myofibers and reached as high as nanogram levels in muscle and accumulated at the neuromuscular junctions. Expression persisted for at least 10 months.
- the expressed GDNF was retrogradely transported through axons to corresponding spinal cord motoneurons.
- the transgene GDNF prevented degeneration of motoneurons, preserved the axons innervating the muscle, and inhibited muscle atrophy.
- AAV-GDNF amyotrophic lateral sclerosis
- the method described herein provides for the direct, in vivo injection of recombinant AAV virions into muscle tissue, preferably skeletal muscle, e.g., by intramuscular injection, as well as for the in vitro transduction of muscle cells which can subsequently be introduced into a subject for treatment.
- motoneuron diseases such as any of the various amyotrophies such as hereditary amyotrophies including hereditary spinal muscular atrophy, acute infantile spinal muscular atrophy such as Werdnig-Hoffman disease, progressive muscular atrophy in children such as the proximal, distal type and bulbar types, spinal muscular atrophy of adolescent or adult onset including the proximal, scapuloperoneal, facioscapulohumeral and distal types, amyotrophic lateral sclerosis (ALS) and primary lateral sclerosis (PLS).
- hereditary amyotrophies including hereditary spinal muscular atrophy, acute infantile spinal muscular atrophy such as Werdnig-Hoffman disease, progressive muscular atrophy in children such as the proximal, distal type and bulbar types, spinal muscular atrophy of adolescent or adult onset including the proximal, scapuloperoneal, facioscapulohumeral and distal types, amyotrophic
- GDNF is a protein that may be identified in or obtained from glial cells and that exhibits neurotrophic activity.
- GDNF is an approximately 39 kD glycosylated protein that exists as a homodimer in its native form.
- GDNF is initially translated as a pre-pro-GDNF polypeptide and proteolytic processing of the signal sequence and the "pro" portion of the molecule result in production of a mature form of GDNF.
- a single gene gives rise to alternatively spliced forms. See, e.g., U.S. Patent No. 6,362,319. Both forms contain a consensus signal peptide sequence and a consensus sequence for proteolytic processing.
- GDNF polynucleotides for use in the present AAV vectors may encode either or both of these forms, may encode the entire pre-pro-molecule, the pre-molecule, the pro- molecule, the mature GDNF polypeptide, or biologically active variants of these forms, as defined above.
- GDNF polynucleotide and amino acid sequences are known. Three representative mammalian GDNF sequences are depicted in Figures 7, 8 and 9 herein.
- a human GDNF nucleotide and amino acid sequence is shown in Figure 7 (SEQ ID NOS:l and 2).
- a rat GDNF nucleotide and amino acid sequence is shown in Figure 8 (SEQ ID NOS:3 and 4) and a mouse GDNF nucleotide and amino acid sequence is shown in Figure 9 (SEQ ID NOS:6 and 7).
- the degree of homology between the rat and human proteins is about 93% and all mammalian GDNFs have a similarly high degree of homology.
- GDNF nucleotide and amino acid sequences are known in the art. See, e.g., U.S. Patent Nos. 6,221,376 and 6,363,319, and Lin et al., Science (1993) 260:1130-1132 for rat and human sequences, as well as NCBI accession numbers AY052832, AJ001896, AF053748, AF063586 and L19063 for human sequences; NCBI accession numbers AF184922, AF497634, X92495, NM019139 for rat sequences; NCBI accession number AF516767 for a giant panda sequence; NCBI accession numbers XM122804, NM010275, D88351S1, D49921, U36449, U37459, U66195 for mouse sequences; NCBI accession number AF469665 for a Nipponia nippon sequence; NCBI accession number AF 106678 for a Macaca mulatta sequence; and NC
- AAV-delivered GDNF polynucleotides can be tested in any of a number of animal models of the above diseases, known in the art.
- animal models for the study of motoneuron disorders such as ALS are transgenic mice with an ALS-linked mutant Cu/Zn superoxide dismutase (SOD1) gene (mSODlG93A and or mSODlG37R).
- SOD1 Cu/Zn superoxide dismutase
- mice develop a dominantly inherited adult-onset paralytic disorder with many of the clinical and pathological features of familial ALS. See, e.g., Gurney et al., Science (1994) 264:1772-1775; Nagano et al., Life Sci (2002) 72:541-548.
- mice include two naturally occurring murine models (progressive motor neuronopathy (pmn) and wobbler). See, e.g., Haegggeli and Kato, Neurosci. Lett. (2002) 335:39-43, for descriptions of these mouse models.
- motoneuron diseases such as ALS
- FIG. 10 For a review of various animal models for use in studying motoneuron diseases such as ALS, see, e.g., Jankowsky et al., Curr Neurol Neurosci. Rep. (2002) 2:457-464; Elliott, J.L., Neurobiol. Dis. (1999) 6:310-20; and Borchelt et al., Brain Pathol. (1998) 8:735-757.
- in vitro model systems which use cells, tissue culture and histological methods for studying motoneuron disease.
- a rat spinal cord organotypic slice subjected to glutamate excitotoxicity is useful as a model system to test the effectiveness of neurotrophic factors in preventing motor neuron degeneration.
- in vitro systems for use in studying ALS see, e.g., Bar, P.R., E «r. J. Pharmacol. (2000) 405:285-295; Silani et al., J. Neurol. (2000) 247 Suppl 1:128-36; Martin et al, Int. J. Mol. Med. (2000) 5:3-13.
- Recombinant AAV virions comprising GDNF coding sequences may be produced using a variety of art-recognized techniques described more fully below. Wild-type AAV and helper viruses may be used to provide the necessary replicative functions for producing rAAV virions (see, e.g., U.S. Patent No. 5,139,941). Alternatively, a plasmid, containing helper function genes, in combination with infection by one of the well-known helper viruses can be used as the source of replicative functions (see e.g., U.S. Patent No. 5,622,856 and U.S. Patent No. 5,139,941).
- a plasmid, containing accessory function genes can be used in combination with infection by wild-type AAV, to provide the necessary replicative functions.
- These three approaches when used in combination with a rAAV vector, are each sufficient to produce rAAV virions.
- Other approaches, well known in the art, can also be employed by the skilled artisan to produce rAAV virions.
- a triple transfection method (described in detail in U.S. Patent No. 6,001,650) is used to produce rAAV virions because this method does not require the use of an infectious helper virus, enabling rAAV virions to be produced without any detectable helper virus present.
- the AAV helper function vector encodes the "AAV helper function" sequences (i.e., rep and cap), which function in trans for productive AAV replication and encapsidation.
- the AAV helper function vector supports efficient AAV vector production without generating any detectable wt AAV virions (i.e., AAV virions containing functional rep and cap genes).
- An example of such a vector, pHLP19 is described in U.S. Patent No. 6,001,650.
- the rep and cap genes of the AAV helper function vector can be derived from any of the known AAV serotypes, as explained above.
- the AAV helper function vector may have a rep gene derived from AAV-2 and a cap gene derived from AAV-6; one of skill in the art will recognize that other rep and cap gene combinations are possible, the defining feature being the ability to support rAAV virion production.
- the accessory function vector encodes nucleotide sequences for non- AAV derived viral and/or cellular functions upon which AAV is dependent for replication (i.e., "accessory functions").
- the accessory functions include those functions required for AAV replication, including, without limitation, those moieties involved in activation of AAV gene transcription, stage specific AAV mRNA splicing, AAV DNA replication, synthesis of cap expression products, and AAV capsid assembly.
- Viral-based accessory functions can be derived from any of the well-known helper viruses such as adenovirus, he ⁇ esvirus (other than he ⁇ es simplex virus type-1), and vaccinia virus.
- the accessory function plasmid pLadeno5 is used (details regarding pLadeno5 are described in U.S. Patent No. 6,004,797).
- This plasmid provides a complete set of adenovirus accessory functions for AAV vector production, but lacks the components necessary to form replication-competent adenovirus.
- Recombinant AAV (rAAV) expression vectors are constructed using known techniques to at least provide as operatively linked components in the direction of transcription, control elements including a transcriptional initiation region, the GDNF polynucleotide of interest and a transcriptional termination region.
- the control elements are selected to be functional in a mammalian muscle cell.
- the resulting construct which contains the operatively linked components is bounded (5' and 3') with functional AAV ITR sequences.
- AAV ITR regions The nucleotide sequences of AAV ITR regions are known. See, e.g., Kotin, R.M. (1994) Human Gene Therapy 5 :793-801 ; Berns, K.I. "Parvoviridae and their Replication" in Fundamental Virology, 2nd Edition, (B.N. Fields and D.M. Knipe, eds.) for the AAV-2 sequence.
- AAV ITRs used in the vectors of the invention need not have a wild-type nucleotide sequence, and may be altered, e.g., by the insertion, deletion or substitution of nucleotides.
- AAV ITRs may be derived from any of several AAV serotypes, including without limitation, AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7 and AAV-8, etc.
- 5' and 3' ITRs which flank a selected nucleotide sequence in an AAV expression vector need not necessarily be identical or derived from the same AAV serotype or isolate, so long as they function as intended, i.e., to allow for excision and rescue of the sequence of interest from a host cell genome or vector, and to allow integration of the DNA molecule into the recipient cell genome when AAV Rep gene products are present in the cell.
- Suitable GDNF polynucleotide molecules for use in AAV vectors will be less than about 5 kilobases (kb) in size.
- the selected polynucleotide sequence is operably linked to control elements that direct the transcription or expression thereof in the subject in vivo.
- control elements can comprise control sequences normally associated with the selected gene.
- heterologous control sequences can be employed.
- Useful heterologous control sequences generally include those derived from sequences encoding mammalian or viral genes.
- Examples include, but are not limited to, neuron-specific enolase promoter, a GFAP promoter, the SV40 early promoter, mouse mammary tumor virus LTR promoter; adenovirus major late promoter (Ad MLP); a he ⁇ es simplex virus (HSV) promoter, a cytomegalovirus (CMV) promoter such as the CMV immediate early promoter region (CMVEE), a rous sarcoma virus (RSV) promoter, synthetic promoters, hybrid promoters, and the like.
- HSV he ⁇ es simplex virus
- CMV cytomegalovirus
- CMVEE CMV immediate early promoter region
- RSV rous sarcoma virus
- synthetic promoters hybrid promoters, and the like.
- sequences derived from nonviral genes such as the murine metallothionein gene, will also find use herein.
- Such promoter sequences are commercially available from, e.g., Stratagene (
- control elements include, but are not limited to, those derived from the actin and myosin gene families, such as from the myoD gene family (Weintraub et al. (1991) Science 251:761-766); the myocyte-specific enhancer binding factor MEF-2 (Cserjesi and Olson (1991) Mol. Cell Biol. 11:4854-4862); control elements derived from the human skeletal actin gene (Muscat et al. (1987) Mol. Cell Biol. 7:4089-4099) and the cardiac actin gene; muscle creatine kinase sequence elements (Johnson et al.
- mCK murine creatine kinase enhancer
- control elements derived from the skeletal fast-twitch troponin C gene, the slow-twitch cardiac troponin C gene and the slow-twitch troponin I gene; hypoxia-inducible nuclear factors (Semenza et al. (1991) Proc. Natl. Acad. Sci. USA 88:5680-5684; Semenza et al. J. Biol. Chem. 269:23757-23763): steroid-inducible elements and promoters, such as the glucocorticoid response element (GRE) (Mader and White (1993) Proc.
- GRE glucocorticoid response element
- the AAV expression vector which harbors the GDNF polynucleotide molecule of interest bounded by AAV ITRs can be constructed by directly inserting the selected sequence(s) into an AAV genome which has had the major AAV open reading frames ("ORFs") excised therefrom. Other portions of the AAV genome can also be deleted, so long as a sufficient portion of the ITRs remain to allow for replication and packaging functions.
- ORFs major AAV open reading frames
- Such constructs can be designed using techniques well known in the art. See, e.g., U.S. Patent Nos. 5,173,414 and 5,139,941 ; International Publication Nos. WO 92/01070 (published 23 January 1992) and WO 93/03769 (published 4 March 1993); Lebkowski et al.
- AAV ITRs can be excised from the viral genome or from an AAV vector containing the same and fused 5' and 3' of a selected nucleic acid construct that is present in another vector using standard ligation techniques, such as those described in Sambrook et al., supra.
- ligations can be accomplished in 20 mM Tris-Cl pH 7.5, 10 mM MgCl 2 , 10 mM DTT, 33 ⁇ g/ml BSA, 10 mM-50 mM NaCl, and either 40 ⁇ M ATP, 0.01-0.02 (Weiss) units T4 DNA ligase at 0°C (for "sticky end” ligation) or 1 mM ATP, 0.3-0.6 (Weiss) units T4 DNA ligase at 14°C (for "blunt end” ligation). Intermolecular "sticky end” ligations are usually performed at 30-100 ⁇ g/ml total DNA concentrations (5-100 nM total end concentration).
- AAV vectors which contain ITRs have been described in, e.g., U.S. Patent no. 5,139,941.
- AAV vectors are described therein which are available from the American Type Culture Collection (“ATCC") under Accession Numbers 53222, 53223, 53224, 53225 and 53226.
- chimeric genes can be produced synthetically to include AAV ITR sequences a ⁇ anged 5' and 3' of one or more selected nucleic acid sequences.
- Preferred codons for expression of the chimeric gene sequence in mammalian muscle cells can be used.
- the complete chimeric sequence is assembled from overlapping oligonucleotides prepared by standard methods. See, e.g., Edge (1981) Nature 292:756; Nambair et al. (1984) Science 223:1299; Jay et al. (1984) J. Biol. Chem. (1984) 259:6311.
- suitable host cells for producing rAAV virions from the AAV expression vectors include microorganisms, yeast cells, insect cells, and mammalian cells, that can be, or have been, used as recipients of a heterologous DNA molecule and that are capable of growth in suspension culture.
- the term includes the progeny of the original cell which has been transfected.
- a "host cell” as used herein generally refers to a cell which has been transfected with an exogenous DNA sequence.
- Cells from the stable human cell line, 293 readily available through, e.g., the American Type Culture Collection under Accession Number ATCC CRL1573) are preferred in the practice of the present invention.
- the human cell line 293 is a human embryonic kidney cell line that has been transformed with adenovirus type-5 DNA fragments (Graham et al. (1977) J. Gen. Virol. 36:59), and expresses the adenoviral Ela and Elb genes (Aiello et al. (1979) Virology 94:460).
- the 293 cell line is readily transfected, and provides a particularly convenient platform in which to produce rAAV virions.
- AAV helper functions are generally AAV-derived coding sequences which can be expressed to provide AAV gene products that, in turn, function in trans for productive AAV replication.
- AAV helper functions are used herein to complement necessary AAV functions that are missing from the AAV expression vectors.
- AAV helper functions include one, or both of the major AAV ORFs, namely the rep and cap coding regions, or functional homologues thereof.
- AAV rep coding region is meant the art-recognized region of the AAV genome which encodes the replication proteins Rep 78, Rep 68, Rep 52 and Rep 40. These Rep expression products have been shown to possess many functions, including recognition, binding and nicking of the AAV origin of DNA replication, DNA helicase activity and modulation of transcription from AAV (or other heterologous) promoters. The Rep expression products are collectively required for replicating the AAV genome.
- AAV rep coding region see, e.g., Muzyczka, N. (1992) Current Topics in Microbiol. and Immunol. 158:97-129; and Kotin, R.M. (1994) Human Gene Therapy 5:793-801.
- Suitable homologues of the AAV rep coding region include the human he ⁇ esvirus 6 (HHV-6) rep gene which is also known to mediate AAV-2 DNA replication (Thomson et al. (1994) Virology 204:304- 311).
- AAV cap coding region is meant the art-recognized region of the AAV genome which encodes the capsid proteins VP1, VP2, and VP3, or functional homologues thereof. These Cap expression products supply the packaging functions which are collectively required for packaging the viral genome.
- Muzyczka N.
- Kotin R.M.
- AAV helper functions are introduced into the host cell by transfecting the host cell with an AAV helper construct either prior to, or concurrently with, the transfection of the AAV expression vector.
- AAV helper constructs are thus used to provide at least transient expression of AAV rep and/or cap genes to complement missing AAV functions that are necessary for productive AAV infection.
- AAV helper constructs lack AAV ITRs and can neither replicate nor package themselves.
- constructs can be in the form of a plasmid, phage, transposon, cosmid, virus, or virion.
- a number of AAV helper constructs have been described, such as the commonly used plasmids pAAV/Ad and pBvI29+45 which encode both Rep and Cap expression products. See, e.g., Samulski et al. (1989) J. Virol. 63:3822-3828; and McCarty et al. (1991) J. Virol. 65:2936-2945.
- a number of other vectors have been described which encode Rep and/or Cap expression products. See, e.g., U.S. Patent No. 5,139,941.
- Both AAV expression vectors and AAV helper constructs can be constructed to contain one or more optional selectable markers.
- Suitable markers include genes which confer antibiotic resistance or sensitivity to, impart color to, or change the antigenic characteristics of those cells which have been transfected with a nucleic acid construct containing the selectable marker when the cells are grown in an appropriate selective medium.
- selectable marker genes that are useful in the practice of the invention include the hygromycin B resistance gene (encoding Aminoglycoside phosphotranferase (APH)) that allows selection in mammalian cells by conferring resistance to G418 (available from Sigma, St. Louis, Mo.). Other suitable markers are known to those of skill in the art.
- AAV Accessory Functions The host cell (or packaging cell) must also be rendered capable of providing nonAAV-derived functions, or "accessory functions," in order to produce rAAV virions.
- Accessory functions are nonAAV-derived viral and/or cellular functions upon which AAV is dependent for its replication.
- accessory functions include at least those nonAAV proteins and RNAs that are required in AAV replication, including those involved in activation of AAV gene transcription, stage specific AAV mRNA splicing, AAV DNA replication, synthesis of Cap expression products and AAV capsid assembly.
- Viral-based accessory functions can be derived from any of the known helper viruses.
- accessory functions can be introduced into and then expressed in host cells using methods known to those of skill in the art.
- accessory functions are provided by infection of the host cells with an unrelated helper virus.
- helper viruses include adenoviruses; he ⁇ esviruses such as he ⁇ es simplex virus types 1 and 2; and vaccinia viruses.
- Nonviral accessory functions will also find use herein, such as those provided by cell synchronization using any of various known agents. See, e.g., Buller et al. (1981) J. Virol. 40:241- 247; McPherson et al. (1985) Virology 147:217-222; Schlehofer et al. (1986) Virology 152:110-117.
- accessory functions can be provided using an accessory function vector as defined above. See, e.g., U.S. Patent No. 6,004,797 and International Publication No. WO 01/83797.
- Nucleic acid sequences providing the accessory functions can be obtained from natural sources, such as from the genome of an adenovirus particle, or constructed using recombinant or synthetic methods known in the art. As explained above, it has been demonstrated that the full-complement of adenovirus genes are not required for accessory helper functions. In particular, adenovirus mutants incapable of DNA replication and late gene synthesis have been shown to be permissive for AAV replication. Ito et al., (1970) J. Gen. Virol.
- Ad mutants include: EIB (Laughlin et al. (1982), supra; Janik et al. (1981), supra; Ostrove et al., (1980) Virology 104:502); E2A (Handa et al., (1975) J. Gen. Virol 29:239; Strauss et al., (1976) J. Virol. 17:140; Myers et al., (1980) J. Virol. 35:665; Jay et al., (1981) Proc. Natl. Acad. Sci.
- accessory function vectors encoding various Ad genes.
- Particularly preferred accessory function vectors comprise an adenovirus VA RNA coding region, an adenovirus E4 ORF6 coding region, an adenovirus E2 A 72 kD coding region, an adenovirus El A coding region, and an adenovirus EIB region lacking an intact ElB55k coding region.
- Such vectors are described in international Publication No. WO 01/83797.
- accessory functions are expressed which transactivate the AAV helper construct to produce AAV Rep and/or Cap proteins.
- the Rep expression products excise the recombinant DNA (including the DNA of interest) from the AAV expression vector.
- the Rep proteins also serve to duplicate the AAV genome.
- the expressed Cap proteins assemble into capsids, and the recombinant AAV genome is packaged into the capsids.
- productive AAV replication ensues, and the DNA is packaged into rAAV virions.
- rAAV virions can be purified from the host cell using a variety of conventional purification methods, such as column chromatography, CsCl gradients, and the like. For example, a plurality of column purification steps can be used, such as purification over an anion exchange column, an affinity column and/or a cation exchange column. See, for example, international Publication No. WO 02/12455.
- column purification steps can be used, such as purification over an anion exchange column, an affinity column and/or a cation exchange column. See, for example, international Publication No. WO 02/12455.
- residual helper virus can be inactivated, using known methods. For example, adenovirus can be inactivated by heating to temperatures of approximately 60°C for, e.g., 20 minutes or more.
- This treatment effectively inactivates only the helper virus since AAV is extremely heat stable while the helper adenovirus is heat labile.
- the resulting rAAV virions containing the GDNF nucleotide sequence of interest can then be used for gene delivery using the techniques described below.
- compositions will comprise sufficient genetic material to produce a therapeutically effective amount of the GDNF of interest, i.e., an amount sufficient to reduce or ameliorate symptoms of the disease state in question or an amount sufficient to confer the desired benefit.
- the compositions will also contain a pharmaceutically acceptable excipient.
- excipients include any pharmaceutical agent that does not itself induce the production of antibodies harmful to the individual receiving the composition, and which may be administered without undue toxicity.
- Pharmaceutically acceptable excipients include, but are not limited to, sorbitol, any of the various TWEEN compounds, and liquids such as water, saline, glycerol and ethanol.
- Pharmaceutically acceptable salts can be included therein, for example, mineral acid salts such as hydrochlorides, hydrobromides, phosphates, sulfates, and the like; and the salts of organic acids such as acetates, propionates, malonates, benzoates, and the like.
- auxiliary substances such as wetting or emulsifying agents, pH buffering substances, and the like, may be present in such vehicles.
- One particularly useful formulation comprises recombinant AAV virions in combination with one or more dihydric or polyhydric alcohols, and, optionally, a detergent, such as a sorbitan ester. See, for example, International Publication No. WO 00/32233.
- an effective amount of viral vector which must be added can be empirically determined. Representative doses are detailed below. Administration can be effected in one dose, continuously or intermittently throughout the course of treatment. Methods of determining the most effective means and dosages of administration are well known to those of skill in the art and will vary with the viral vector, the composition of the therapy, the target cells, and the subject being treated. Single and multiple administrations can be carried out with the dose level and pattern being selected by the treating physician.
- transgene can be expressed by the delivered recombinant virion.
- separate vectors each expressing one or more different transgenes, can also be delivered as described herein.
- viral vectors delivered by the methods of the present invention be combined with other suitable compositions and therapies.
- certain systemically delivered compounds such as muristerone, ponasteron, tetracyline or aufin may be administered in order to regulate expression of the transgene.
- Recombinant AAV virions may be introduced into muscle cells using either in vivo or in vitro (also termed ex vivo) transduction techniques. If transduced in vitro, the desired recipient cell, preferably a skeletal muscle cell, will be removed from the subject, transduced with rAAV virions and reintroduced into the subject.
- syngeneic or xenogeneic cells can be used where those cells will not generate an inappropriate immune response in the subject.
- transduced cells can be transduced in vitro by combining recombinant AAV virions with cells to be transduced in appropriate media, and those cells harboring the DNA of interest can be screened using conventional techniques such as Southern blots and or PCR, or by using selectable markers.
- Transduced cells can then be formulated into pharmaceutical compositions, as described above, and the composition introduced into the subject by various techniques as described below, in one or more doses.
- Recombinant AAV virions or cells transduced in vitro may be delivered directly to muscle by injection with a needle, catheter or related device, using techniques known in the art.
- the rAAV virions will be formulated into pharmaceutical compositions and one or more dosages may be administered directly in the indicated manner.
- a therapeutically effective dose will include on the order of from about 10 8 /kg to 10 16 /kg of the rAAV virions, more preferably 10 10 /kg to 10 14 /kg, and even more preferably about 10 u /kg to 10 13 /kg of the rAAV virions (or viral genomes, also termed "vg"), or any value within these ranges.
- CED convection- enhanced delivery
- recombinant virions can be delivered to many cells over large areas of muscle.
- the delivered vectors efficiently express transgenes in muscle cells.
- Any convection-enhanced delivery device may be appropriate for delivery of viral vectors.
- the device is an osmotic pump or an infusion pump. Both osmotic and infusion pumps are commercially available from a variety of suppliers, for example Alzet Co ⁇ oration, Hamilton Co ⁇ oration, Alza, Inc., Palo Alto, California).
- a viral vector is delivered via CED devices as follows.
- a catheter, cannula or other injection device is inserted into appropriate muscle tissue in the chosen subject, such as skeletal muscle.
- appropriate muscle tissue such as skeletal muscle.
- CED delivery see U.S. Patent No. 6,309,634.
- Other modes of administration that will find particular use with muscles use histamine or isolated limb perfusion (a technique where the vascular supply to a limb is isolated from systemic circulation before infusion of the composition in question) for increasing vector spread in the muscle, all well known techniques in the art. See, e.g., Schaadt et al., J. Extra Corpor. Technol. (2002) 34:130-143; Lejeune et al., Surg. Oncol. Clin. N Am. (2001) 10:821-832; Fraser et al., AORNJ. (1999) 70:642-647, 649, 651-653.
- Example 1 Construction of Recombinant AAV Vectors In order to distinguish AAV-delivered GDNF from its endogenous counte ⁇ art, an AAV vector was constructed encoding a recombinant fusion GDNF protein, tagged with the FLAG peptide (AAV-GDNF-FLAG), for recognition by a specific antibody to the FLAG epitope. Briefly, AAV vector plasmid pAAV-GDNF- FLAG was derived from a previously described pAAV-GDNF plasmid (Fan et al., Neurosci. Lett. (1998) 248:61-64).
- This plasmid contains the mouse GDNF cDNA (Matsushita et al., Gene (1997) 203:149-157) tagged by the FLAG sequence (DYKDDDDK (SEQ ID NO: 5) at the carboxyl terminus under the human cytomegalovirus (CMV) immediate-early promoter, with human growth hormone first intron and simian virus 40 (SV40) polyadenylation signal sequence between the inverted terminal repeats (ITR) of the AAV-2 genome.
- CMV cytomegalovirus
- SV40 human growth hormone first intron and simian virus 40 polyadenylation signal sequence between the inverted terminal repeats (ITR) of the AAV-2 genome.
- AAV vector plasmid pAAV-LacZ, auxiliary plasmid pHLP19 and pladenol have previously been described (Fan et al., Neurosci. Lett. (1998) 248:61-64; Shen et al., Hum. Gene Ther. (2000) 11:1509-1519).
- Pladeno5 is described in U.S. Patent No. 6,004,797.
- Subconfluent human 293 cells were transiently transfected with vector plasmid and helper plasmid using the calcium phosphate co-precipitation method. Seventy- two hours after transfection, cells were harvested and lysed by freeze and thaw cycles.
- AAV vectors (AAV-GDNF-FLAG and AAV-LacZ) were purified using two sequential continuous CsCl gradients, as described previously (Matsushita, et al, Gene Therapy (1998) 5:938-945).
- the final particle titer of the AAV-GDNF-FLAG was 1.6 xlO 13 vector genome copies/ml and AAV-LacZ was 2.1 xlO 13 vector genome copies/ml, as estimated by quantitative DNA dot-blot hybridization analysis.
- HEK 293 cells (readily available through, e.g., the American Type Culture Collection under Accession Number ATCC CRL1573) were seeded on 35mm-diameter dishes and allowed to proliferate overnight in complete medium. Cells were transduced with AAV-GDNF-FLAG or AAV-LacZ (0.5-18 x 10 3 vector genome copies/cell) in medium supplied with 2% fetal bovine serum. 48 hours after transduction, conditioned medium (CM) and cell lysates were collected.
- CM conditioned medium
- GDNF levels in CM and 293 cell lysate were measured by ELISA (GDNF E max ImmunoAssay System, Promega).
- lysis buffer 137xl0 3 mol/L NaCl, 20xl0 '3 mol/L Tris [pH 8.0], 1% NP40, 10% glycerol, supplied by Protease Inhibitor Cocktail Tablets Complete Mini [Roche]
- the supernatants were acidified and then neutralized to pH 7.4 before assay. Acidification has been reported to enhance detection of neurotrophic factors (Okragly et al., Exp. Neurol.
- ELISA analysis showed high levels of GDNF expression and secretion in AAV-GDNF-FLAG-transduced cells ( Figures 1A and IB).
- GDNF levels in the CM were much higher than the cell lysate, indicating that the GDNF could be secreted by cells.
- the amount of GDNF in CM and cell lysates showed a vector genome copies per cell/dose-dependent increase.
- GDNF levels were much lower, barely at the detection limit of the ELISA analysis.
- Example 3 In vivo Expression of GDNF in Injected Gastrocnemius Muscles
- the right gastrocnemius muscle was injected with same volume of PBS. No morbidity or morality was observed in nice during the experimental period.
- gastrocnemius muscles were dissected, rapidly frozen in liquid nitrogen-cooled isopentane and stored at -80°C for ELISA analysis or Cryostat sectioning. Mice were then perfused with ice-cold PBS followed by 4% paraphormaldehyde (PFA). Spinal cord was dissected, post-fixed for 4 hours in 4% PFA and cryoprotected by soaking sequentially in 10%, 20% and 30% sucrose at 4°C overnight. Serial transverse Cryostat sections of frozen muscle tissue (10mm) were thawed mounted in slides, coated with gelatin, and completely dried before storing at ⁇ 80°C. Serial transverse sections of lumbar spinal cord were cut on freezing microtome at 30mm thickness, and stored in PBS at 4°C.
- GDNF ELIS As were performed as described above.
- b-galactosidase (b- Gal) histochemistry muscle sections were fixed and stained for 4-6 hours with b-Gal staining solution (500 mg/ml X-Gal, 5mM potassium hexacycanoferrate (HI), 5mM potassium hexacycanoferrate (IT), and 2mM magnesium chloride in PBS) at 37°C.
- Spinal cord samples were stained as free-floating sections and mounted in gelatin- coated slices and dried. Sections were counterstained with eosin for detection.
- ⁇ -Bungarotoxin is a peptide extracted from Bungarus multicinctus, which specifically binds with high affinity to the a-subunit of the nicotinic AchR at the postsynaptic membrane of neuromuscular junctions. Immunofluorescent stained sections were viewed and photographs were captured with a confocal laser scanning microscope (TCS NT; Leica, Heidelberg, Germany).
- Double-immunofluorescent staining with anti-GDNF and anti-FLAG antibodies showed co-localization of GDNF and FLAG immunoreactivity, confirming the presence of a GDNF-FLAG fusion protein. Intense immunoreactivity was concentrated in the vicinity of the sarcolemmas, suggesting its effective secretion by myofibers in vivo. In AAV-LacZ- or PBS-injected muscles, only very weak signals of GDNF could be detected in the vicinity of sarcolemmas, while no immunosignals of FLAG were seen. This is in agreement with ELISA results and indicated that the GDNF detected by ELISA in AAV-LacZ- or PBS-injected muscles was endogenous GDNF.
- neurotrophic factor is synthesized and released by the targets of neurotrophic factor-dependent axons, where it is bound by receptors on axon terminals, taken up, and retrogradely transported to the cell body (DeStefano, P.S., Exp. Neurol. (1993) 124:56-69).
- GDNF-FLAG double-immunofluorescent staining with anti- FLAG antibody and rhodamine-conjugated ⁇ -bungarotoxin was performed on gastrocnemius muscle sections.
- GDNF As a target-derived neurotrophic factor, endogenous GDNF produced by skeletal muscle functions via retrograde axonal transport from the target muscle tissue to motoneuronal cell bodies in the spinal cord (Mitsumoto, H., Muscle Nerve (1999) 22:1000-1021; Leitner et al., J. Neurosci. (1999) 19:9322-9331). Receptor-mediated retrograde transport of GDNF has been described in motoneurons of rats (Leitner et al., J. Neurosci. (1999) 19:9322-9331).
- transgene GDNF-FLAG fusion protein could also be retrogradely transported from muscle to spinal cord motoneurons
- double- immunostaining with anti-FLAG and anti-NeuN (a specific marker of neuron) antibodies was performed on lumber 4 to 6 spinal cord sections co ⁇ esponding with the innervation of gastrocnemius muscles.
- FLAG immunoreactivity was detected in large size NeuN-positive cells of ventral horn ipsilateral to the AAV-GDNF-FLAG injected side. Their large size (with diameters >20mm), ventral horn distribution and NeuN-positive characteristics suggested that these FLAG immunoreactive cells were a-motoneurons.
- transgene GDNF was successfully detected in the ipsilateral ventral horn motoneurons of the spinal cord, following its detection in gastrocnemius muscle after AAV-GDNF-FLAG injection.
- the transgene GDNF might have been delivered to the spinal cord via three different avenues: systemic delivery, retrograde transport of AAV vectors or retrograde transport of the fusion protein itself.
- systemic delivery retrograde transport of AAV vectors
- retrograde transport of the fusion protein itself The restricted distribution and ipsilateral presentation of transgenic GDNF in motoneurons as well as its inability to pass through the blood-brain barrier excludes the possibility of systematic delivery to the spinal cord.
- b-galactosidase signal was not detected in spinal cord motoneurons of AAV-LacZ injected mice, indicating that the AAV vector itself was not delivered by retrograde transport.
- mice with the G93A human SOD1 mutation Male transgenic mice with the G93A human SOD1 mutation (SOD1G93A) were obtained from The Jackson Laboratory (Bar Harbor, ME).
- pAAV-GDNF-FLAG, pAAV-LacZ, auxiliary plasmid pHLP19 and pladenol were as described above.
- AAV vectors were produced in human embryonic kidney (HEK) 293 cells by triple transfection of vector plasmid and helper plasmids listed above as described previously (Wang et al., Gene Ther. (2002) 9:381-389). In brief, subconfluent 293 cells were transiently transfected using the calcium phosphate method.
- AAV vectors were purified by two sequential continuous cesium chloride density gradients and estimated for final particle titer by quantitative DNA dot-blot hybridization.
- mice were first given three days to become acquainted with the rotarod apparatus (Rota-Rod/7650; Ugo Basile, Comerio, Italy) before the test. For detection, mice were placed on the rotating rod at the speeds of 5, 10, and 20 ⁇ m, and the time each mouse remained on the rod was registered automatically. The onset of disease was defined as the time when the mouse could not remain on the rotarod for 7 min at a speed of 20 ⁇ m, as described previously (Li et al., Science (2000) 288:335-339). If the mouse remained on the rod for >7 min, the test was completed and scored as 7 min. Mice were tested every two days until they could no longer perform the task. Mortality was scored as the age of death when the mouse was unable to right itself within 30 sec when placed on its back in a supine position (Li et al., Science (2000) 288:335-339).
- mice were bilaterally injected with neural tracer cholera toxin subunit B (CTB) (0.1% in distilled H 2 O, 3 :1; List Biologic, Campbell, CA) into gastrocnemius muscles to selectively label motoneurons that retained axons innervating the treated muscles.
- CTB neural tracer cholera toxin subunit B
- gastrocnemius muscles were dissected out, weighed, rapidly frozen in liquid nitrogen-cooled isopentane, and then stored at -80°C for immunohistochemistry or GDNF ELISA analysis. After dissecting out the muscles, the mice were perfused with ice-cold PBS, followed by 4% paraformaldehyde (PFA). The spinal cord was dissected out, postfixed for 4 hr in 4% PFA, and then cryoprotected sequentially in sucrose.
- CTB neural tracer cholera toxin subunit B
- GDNF ELISA To determine muscle GDNF levels, tissues were homogenized at a w/v ratio of 100 mg/ml in lysis buffer (137xl0 3 mol /l NaCl, 20x10° mol/ 1 Tris, pH 8.0, 1% NP-40, and 10% glycerol) containing protease and phosphatase inhibitors, ultrasonicated, and then centrifuged at 12,000 x g. The supernatants were acidified and neutralized to pH 7.4 before assaying. The tissue levels of GDNF were measured with an ELISA kit (GDNF Emax ImmunoAssay System; Promega, Madison, WI), according to the protocol of the supplier. The levels of GDNF were expressed as picograms per milligram of protein. The assay sensitivity ranged from 16 to 1000 pg/ml.
- Muscle sections (10 ⁇ m) were fixed in cold acetone, followed by incubation with rabbit anti-FLAG polyclonal antibodies (1:1000; Sigma, St. Louis, MO) as primary antibodies and biotinylated anti-rabbit antibodies as secondary ones (1 :400; Santa Cruz Bio-technology, Santa Cruz, CA). Sections were visualized by the avidin-biotin-peroxidase complex procedure (Vectastain ABC kits; Vector Laboratories, Burlingame, CA) using 3,3-diaminobenzidine as a chromogen.
- Mo ⁇ hometric analysis was performed on images captured with a CCD camera using KS 400 image analysis software (Zeiss, Oberkochen, Germany). The mean area of muscle fibers was calculated from counts of >1000 fibers in randomly selected areas. To compare the number of motoneurons in the spinal cord, neurons were counted in Nissl-stained and SMI-32- and CTB-immunostained sections spanning the cervical and lumbrosacral enlargements in each group, as described previously (Lewis et al., Nat. Genet. (2000) 25:402-405). For each mouse, at least 20 sections in each sixth serial section were subjected to counting. Only large cell profiles meeting the following criteria were included: location in the ventral horn below a lateral line from the central canal, containing a distinct nucleus with a nucleolus, and possession of at least one thick process.
- GDNF transgene expression in muscles of ALS mice The amount of GDNF in gastrocnemius muscles was determined by ELISA using the methods described above.
- the pattern of distribution of transgenic GDNF in muscles was examined by means of immunodetection.
- FLAG was used as a tag to distinguish transgene GDNF from its endogenous counte ⁇ art.
- AAV-GDNF vector-injected mice strong FLAG immunoreactivity was detected in a large number of myofibers, both at 110 days of age and at the end stage of the disease. Punctured and reticular staining was observed in transverse sections of muscles, with intense immunoreactivity mainly localized in the vicinity of the sarcolemma, indicating that transgene-derived GDNF was efficiently secreted into the surrounding regions.
- Substantial FLAG signals could still be detected in atrophied myofibers at the end stage of the disease.
- ⁇ -Bungarotoxin is a molecular probe that specifically binds to the acetylcholine receptor (AChR) with high affinity on the postsynaptic membranes of NMJs.
- AChR acetylcholine receptor
- Retrograde transport of transgenic GDNF into spinal motoneurons Retrograde axonal transport of GDNF into spinal lumbar motoneurons has been demonstrated in adult rats (Leitner et al., J. Neurosci. (1999) 19:9322-9331). Thus, the ability of transgene GDNF to be retrogradely transported to spinal motoneurons in ALS mice was examined. For this pu ⁇ ose, the FLAG tag in transgene GDNF was used to avoid interference of the results by endogenous GDNF. SMI-32 is a well characterized antibody that specifically recognizes nonphosphorylated neurofilaments (NP-NFs) and therefore serves as a reliable marker for motoneurons (Carriedo et al., J.
- transgene GDNF that appeared in the motoneurons could have been derived through three possible ways: systemic delivery, retrograde transport of AAV vectors, or retrograde transport of GDNF fusion protein itself.
- systemic delivery retrograde transport of AAV vectors
- retrograde transport of GDNF fusion protein itself the restricted distribution and ipsilateral presentation of transgenic GDNF in motoneurons, as well as its known inability to pass through the blood-brain barrier, exclude the possibility of its systematic delivery to the spinal cord.
- most reports show that AAV vectors are not retrogradely transported or are transported in only a very limited manner (Chamberlin et al., Brain Res. (1998) 793:169-175; Klein et al., Exp. Neurol.
- transgenic GDNF was abundantly detected in both transduced muscles and the corresponding motoneurons after AAV-GDNF injection. This finding, combined with the previous reports as well as the observation regarding ⁇ - galactosidase activity, indicates that the transgenic GDNF in the motoneurons is mainly derived through retrograde axonal transport of the GDNF protein. This is consistent with previous studies (Kordower et al., Science (2000) 290:767-773; Wang et al., Gene Ther. (2002) 9:381-389), showing that transgenic GDNF is retrogradely transported.
- transgenic GDNF in muscle fibers was predominantly accumulated to the regions of NMJs is also compatible with its retrograde transport hypothesized because it is in the axon terminals in which substances secreted from muscle fibers are taken up to be retrogradely transported.
- Staining for NP-NF is a reliable means of assessing the extent of motoneuron loss in ALS, in which it has been shown that motoneuron degeneration induces dephosphorylation of NP-NF, resulting in SMI-32 staining resistance (Tsang et al., Brain Res. (2000) 861:45-58). Staining was performed on serial sections to evaluate motoneurons with NP-NF. Consistent with the Nissl-staining results, AAV-GDNF vector-treated ALS mice had significantly greater numbers of SMI-32-positive motoneurons compared with in the control ALS group ( Figure 4B).
- CTB can be axonally transported to neuronal cell bodies in a retrograde direction and be detected throughout the neuronal cytoplasm (Llewellyn- Smith et al, J. Neurosci. Meth. (2000) 103:83-90)
- the detection of CTB-positive motoneurons means that they maintain intact axonal connection with the AAV-GDNF vector-injected muscles.
- CTB labeling makes it possible to assess the effect of the transgenic GDNF on the spinal motoneurons more accurately than with Nissl or NP-NF staining alone.
- This method revealed greater numbers of larger spinal motoneurons labeled with CTB in AAV-GDNF vector-treated ALS mice.
- GDNF delays the onset of disease, improves motor performance, and prolongs survival in transgenic ALS mice
- Any group of ALS mice that had AAV-GDNF vector, AAV-LacZ vector, or the vehicle injected in the four limbs at 9 weeks of age showed similar motor performance, as quantified with a rotarod, until 12 weeks of age. Thereafter, it deteriorated quickly in control ALS mice, whereas the performance deterioration was significantly delayed in AAV-GDNF vector-treated mice (p ⁇ 0.05) ( Figures 6A-6D), indicating significantly prolonged maintenance of their motor strength.
- AAV-GDNF vector-treated ALS mice with four-limb injections showed much better behavioral performance, with delayed onset of disease and a prolonged life span, which is in agreement with the attenuation of the motoneuron pathology.
- the therapeutic effects of GDNF on behavioral and pathological features were limited to the same treated side, with obvious deterioration of motor performance on the AAV-LacZ vector-treated side.
- the motor performance on a rotarod and the onset of disease remained similar to those in the control group.
- the therapeutic benefit mostly resulted from direct action of transgenic GDNF on motoneurons after its retrograde transport rather than from the systemic delivery.
- AAV-GDNF vector-treated mice did not prolong the length of time from disease onset to death.
- AAV-GDNF vector-treated mice did not reach the end stage, when mo ⁇ ho logical assessment demonstrated such severe atrophy of myofibers and massive loss of spinal motoneurons as in the control ALS mice. It has been reported that pathological changes occur at asymptomatic stages in ALS mice, and massive motoneuron death occurs at the end stage (Dal Canto and Gurney, Brain Res.
- the transgene GDNF may exhibit its greatest protective function for motoneurons in ALS mice at asymptomatic stages when the ventral horns have a mild pathology. Once the disease develops, however, GDNF gene therapy may not as readily inhibit the massive motoneuron death or interfere with the rapidly inevitable progression of the disease. In the above experiment, treatment was begun at the age of 9 weeks. Administration of GDNF at earlier times and/or together with other neurotrophic factors (Bilak et al., Amyotroph. Lateral Scler. Other Motor Neuron Disord. (2001) 2:83-91) may lead to better results.
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WO2023240236A1 (en) | 2022-06-10 | 2023-12-14 | Voyager Therapeutics, Inc. | Compositions and methods for the treatment of spinal muscular atrophy related disorders |
Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5858775A (en) * | 1994-06-06 | 1999-01-12 | Children's Hospital, Inc. | Adeno-associated virus materials and methods |
US6180613B1 (en) * | 1994-04-13 | 2001-01-30 | The Rockefeller University | AAV-mediated delivery of DNA to cells of the nervous system |
US6325998B1 (en) * | 1996-01-18 | 2001-12-04 | Avigen, Inc. | Methods of treating disease using recombinant adeno-associated virus virions administered to muscle |
Family Cites Families (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5641750A (en) * | 1995-11-29 | 1997-06-24 | Amgen Inc. | Methods for treating photoreceptors using glial cell line-derived neurotrophic factor (GDNF) protein product |
US5858351A (en) * | 1996-01-18 | 1999-01-12 | Avigen, Inc. | Methods for delivering DNA to muscle cells using recombinant adeno-associated virus vectors |
US6222022B1 (en) * | 1996-03-14 | 2001-04-24 | Washington University | Persephin and related growth factors |
CA2277869A1 (en) * | 1997-01-17 | 1998-07-23 | Institut National De La Sante Et De La Recherche Medicale | Adenoviral-vector-mediated gene transfer into medullary motor neurons |
DE19816186A1 (en) * | 1998-04-14 | 1999-10-21 | Univ Muenchen L Maximilians | GDNF-encoding DNA, parts thereof and GDNF variants |
US20020055467A1 (en) * | 1998-07-06 | 2002-05-09 | Johansen Teit E. | Novel neurotrophic factors |
US20030050273A1 (en) * | 2001-08-29 | 2003-03-13 | Keiya Ozawa | Compositions and methods for treating neurodegenerative diseases |
-
2002
- 2002-12-19 WO PCT/US2002/041010 patent/WO2003053476A1/en active Application Filing
- 2002-12-19 CA CA002469658A patent/CA2469658A1/en not_active Abandoned
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- 2002-12-19 EP EP02794348A patent/EP1463530A4/en not_active Withdrawn
- 2002-12-19 US US10/327,620 patent/US7112321B2/en not_active Expired - Fee Related
- 2002-12-19 JP JP2003554232A patent/JP2005516949A/en active Pending
-
2006
- 2006-08-28 US US11/511,102 patent/US20060292123A1/en not_active Abandoned
Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6180613B1 (en) * | 1994-04-13 | 2001-01-30 | The Rockefeller University | AAV-mediated delivery of DNA to cells of the nervous system |
US5858775A (en) * | 1994-06-06 | 1999-01-12 | Children's Hospital, Inc. | Adeno-associated virus materials and methods |
US6325998B1 (en) * | 1996-01-18 | 2001-12-04 | Avigen, Inc. | Methods of treating disease using recombinant adeno-associated virus virions administered to muscle |
Non-Patent Citations (7)
Cited By (11)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2009053536A2 (en) * | 2007-10-25 | 2009-04-30 | Liina Lonka | Splice variants of gdnf and uses thereof |
WO2009053536A3 (en) * | 2007-10-25 | 2009-06-18 | Liina Lonka | Splice variants of gdnf and uses thereof |
EP2551281A1 (en) * | 2007-10-25 | 2013-01-30 | Nevalaita, Lina | Splice variants of GDNF and uses thereof |
US9579362B2 (en) | 2007-10-25 | 2017-02-28 | Liina Nevalaita | Splice variants of GDNF and uses thereof |
US10017553B2 (en) | 2007-10-25 | 2018-07-10 | Liina Nevalaita | Splice variants of GDNF and uses thereof |
WO2012057363A1 (en) | 2010-10-27 | 2012-05-03 | 学校法人自治医科大学 | Adeno-associated virus virions for transferring genes into neural cells |
WO2018131551A1 (en) | 2017-01-13 | 2018-07-19 | 学校法人自治医科大学 | Aav vector for disrupting clotting-related factor gene on liver genome |
WO2019146745A1 (en) | 2018-01-26 | 2019-08-01 | 国立大学法人徳島大学 | Novel adeno-associated virus virion for treating tay-sachs disease and sandhoff disease |
WO2020026968A1 (en) | 2018-07-30 | 2020-02-06 | 株式会社遺伝子治療研究所 | Method for enhancing gene expression by aav vector |
WO2021009805A1 (en) | 2019-07-12 | 2021-01-21 | 株式会社遺伝子治療研究所 | Adeno-associated virus virion for gene transfer to human liver |
WO2022224372A1 (en) | 2021-04-21 | 2022-10-27 | 学校法人自治医科大学 | Adeno-associated virus virion for treating ornithine transcarbamylase deficiency |
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CA2469658A1 (en) | 2003-07-03 |
JP2005516949A (en) | 2005-06-09 |
US7112321B2 (en) | 2006-09-26 |
AU2002359786A1 (en) | 2003-07-09 |
EP1463530A4 (en) | 2006-09-06 |
US20060292123A1 (en) | 2006-12-28 |
EP1463530A1 (en) | 2004-10-06 |
US20030161814A1 (en) | 2003-08-28 |
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