Optimized FIG4 Genes and Expression Cassettes and Their Use

STATEMENT OF PRIORITY

This application claims the benefit, under 35 U.S.C. § 119(e), of U.S. Provisional Application No. 62/755,871, filed on November 5, 2018, the entire contents of which are incorporated by reference herein.

STATEMENT REGARDING ELECTRONIC FILING OF A SEQUENCE LISTING

A Sequence Listing in ASCII text format, submitted under 37 C.F.R. § 1.821, entitled 5470-854WO_ST25.txt, 44,305 bytes in size, generated on November 5, 2019 and filed via EFS-Web, is provided in lieu of a paper copy. This Sequence Listing is hereby incorporated herein by reference into the specification for its disclosures.

FIELD OF THE INVENTION

This invention relates to polynucleotides comprising optimized FIG4 open reading frame (ORF) sequences, vectors comprising the same, and methods of using the same for delivery of the ORF to a cell or a subject and to treat disorders associated with aberrant expression of a FIG4 gene or aberrant activity of a FIG4 gene product in the subject, such as CMT4J.

BACKGROUND OF THE INVENTION

Factor-Induced Gene 4 (FIG4) encodes a polyphosphoinositide phosphatase, also known as phosphatidylinositol 3,5-bisphosphate 5-phosphatase, SAC domain-containing protein 3 (Sac3), or FIG4. Mutations in FIG4 cause the rare autosomal recessive disease Charcot-Marie-Tooth Neuropathy type 4 J (CMT4J), a type of peripheral neuropathy characterized by childhood onset with clumsy gait, limb weakness, and muscle atrophy [Chow et al. 2007 Nature 448:68-72]. Presentation and severity of CMT4J can vary, and patients usually display bi-allelic compound heterozygous mutations in FIG4, where one allele is a null mutant and the other allele is a partial-function missense mutation. Yunis- Varon syndrome is a particularly pathogenic variant of CMT4J, usually caused by bi-allelic pathogenic mutations leading to complete loss of FIG4 function.

FIG4 is highly expressed in myelinating cells, particularly Schwann cells, and dorsal root ganglia sensory neurons [Guo et al. 2012 Neuropathol. Exp. Neurol. 7l(l):28-29]. Schwann cells maintain peripheral nerve myelination. There is consistent demyelination of sensory and motor peripheral nerve axons that are observed in CMT4J patients with depleted FIG4 [Hu et al. 2018 Ann. Neurol. 83:756-770]. Demyelination is non-uniform and is often associated with slowed conduction velocity, temporal dispersion, a sign of dis-synchrony of motor nerve responses, conduction block in nerve conduction studies of patients and macrophage infiltration in the spinal roots. The findings are suggestive of the development of demyelinating polyneuropathy. Both motor and sensory neurons in mice and CMT4J patients are impacted to varying degree. Humans with CMT4J mainly experience weakness and muscle atrophy but no or minimal sensory symptoms [Guo et al. 2012; Zhang et al. 2008 Brain 131(8):1990-2001].

In CMT disorders, phosphoinositide signaling and vesicular trafficking are reported to play a major role [Chow et al 2007; Vaccari et al. 2011 PLoS Genet. 7:el0023l9; McCartney et al. 2014 BioEssays 36:52-64]. Importantly, FIG4 is only functional in complex with two other protein partners Vac 14 and FAB1, and this complex regulates the biosynthesis of phosphatidylinositol 3,5-bisphosphate (PI(3,5)P ₂ ) [Jin et al. 2008 EMBO J. 27:3221-3234; Ikonomov et al. 2009 J. Biol. Chem. 284(36):2396l-2397l], The commonly found I41T missense mutation in CMT4J is located at the protein interface with Vacl4, which leads to its destabilization and proteasome mediated degradation [Lenk et al. 2011 PLoS Genet. 7:31002104] (FIG. 1). Loss of FIG4 results in a drop in the PI(3,5)P ₂ levels [Chow et al 2007; Katona et al. 2011 Eur. J. Neurosci. 33(8): 1401-1410]. PI3,5P ₂ levels are critical for vesicular trafficking and Fig4-/- mice have decreased levels [11] manifesting as excessive lysosomal storage [Katona et al. 2011]. PI(3,5)P ₂ act as gate keepers for Ca ⁺⁺ channels in lysosomal membranes [Dong et al. 2010 Nat. Commun. 1:38]. In Fig4-/- cells the Ca ⁺⁺ channel deactivation impairs lysosomal fission, including Schwann cells that perform myelination of axons [Zou et al. 2015 J. Neurosci. 35:6801-6812].

A recent study in European patients has shown that there were 5 amino acid residues in FIG4 that were highly or very highly conserved between human and species as diverse as yeast, round worm ( Caenorhabditis elegans ), fruit fly, chicken, mouse, rat, dog, macaque, orangutan and chimps [McCartney et al. 2014 BioEssays 36:52-64]. Patients with FIG4 mutations in these amino acid residues had predominantly upper motor neuronal deficits in some cases there was a rapid progression and lower motor neuronal impact.

Current treatment for CMT4J is limited to symptomatic palliative care, and there remains a need in the art for an effective treatment that targets the cause of the disease. The present invention overcomes shortcomings in the art by providing codon-optimized FIG4 genes, expression cassettes, and vectors capable of providing therapeutic levels of FIG4 expression for treating disorders associated with FIG4 expression such as CMT4J.

SUMMARY OF THE INVENTION

The present invention is based, in part, on the development of optimized FIG4 genes, expression cassettes, and vectors capable of providing therapeutic levels of FIG4 expression for treating disorders associated with FIG4 expression such as CMT4J.

Thus, one aspect of the invention relates to a polynucleotide comprising a human FIG4 open reading frame, wherein the human FIG4 open reading frame has been codon- optimized for expression in human cells.

A further aspect of the invention relates to an expression cassette comprising a polynucleotide comprising a human FIG4 open reading frame and vectors, transformed cells, and transgenic animals comprising the polynucleotide of the invention.

Another aspect of the invention relates to a pharmaceutical formulation comprising the polynucleotide, expression cassette, vector, and/or transformed cell of the invention in a pharmaceutically acceptable carrier.

An additional aspect of the invention relates to a method of expressing a FIG4 open reading frame in a cell, comprising contacting the cell with the polynucleotide, expression cassette, and/or vector of the invention, thereby expressing the FIG4 open reading frame in the cell.

A further aspect of the invention relates to a method of expressing a FIG4 open reading frame in a subject, comprising delivering to the subject the polynucleotide, expression cassette, vector, and/or transformed cell of the invention, thereby expressing the FIG4 open reading frame in the subject.

An additional aspect of the invention relates to a method of treating a disorder associated with aberrant expression of an FIG4 gene or aberrant activity of an FIG4 gene product in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the polynucleotide, expression cassette, vector, and/or transformed cell of the invention, such that the FIG4 open reading frame is expressed in the subject.

A further aspect of the invention relates to a method of treating Charcot-Marie-Tooth Neuropathy Type 4 J in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the polynucleotide, expression cassette, vector, and/or transformed cell of the invention, such that the FIG4 open reading frame is expressed in the subject.

Another aspect of the invention relates to a method of treating Yunis-Varon syndrome in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the polynucleotide, expression cassette, vector, and/or transformed cell of the invention, such that the FIG4 open reading frame is expressed in the subject.

An additional aspect of the invention relates to a method of treating amyotrophic lateral sclerosis in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the polynucleotide, expression cassette, vector, and/or transformed cell of the invention, such that the FIG4 open reading frame is expressed in the subject.

A further aspect of the invention relates to a method of treating pediatric-onset multiple sclerosis in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the polynucleotide, expression cassette, vector, and/or transformed cell of the invention, such that the FIG4 open reading frame is expressed in the subject.

Another aspect of the invention relates to a polynucleotide, expression cassette, vector, and/or transformed cell of the invention for use in a method of treating a disorder associated with aberrant expression of a FIG4 gene or aberrant activity of a FIG4 gene product in a subject in need thereof.

These and other aspects of the invention are set forth in more detail in the description of the invention below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows an illustration of the PI(3,5)P ₂ biosynthesis activity of FIG4 in interaction with protein partners FAB1 and VAC 14, and points to the location of the commonly found FIG4 missense mutation 141T.

FIG. 2 shows similarities in FIG4 protein sequence between species. Compared to human (homo) FIG4 protein (SEQ ID NO:7) the mouse (mus; 95.15%; SEQ ID NO:9), rat (rattus; 93.94%; SEQ ID NO: 10) and monkey (Macaca; 99.56%; SEQ ID NO:8) homologues retain high level of amino acid identity. The asterisk (*) annotates a fully conserved amino acid residue, colon (:) annotates strongly similar residues and period (.) annotates weakly similar residues. Amino acids that are not conserved are not annotated. FIG. 3 shows Kaplan-meier survival analysis following AAV9/FIG4 therapy. The mice at Postnatal day 1 (PND1) or 4 (PND4) were dosed intracerebroventricularly (ICV) (n =11 per cohort). Mice at Postnatal days 7 (PND7) or 11 (PND11) were dosed intrathecally (IT) (n > 5 per cohort). The mice were monitored daily for morbidity or mortality. The data are presented as percent survival.

FIG. 4 shows body weight changes. The mice at PND 1 or 4 were dosed ICV (n>l 1 per cohort) and PND 7 or 11 were dosed IT (n = 9 per cohort). The mean body weights (BW) of each cohort are presented.

FIG. 5 shows fore arm and all limb grip strength analysis. AAV9/FIG4 therapy improves muscle strength in Fig4-/- mice. The mice at Postnatal day l(PNDl) or 4 (PND4) were dosed ICV with AAV9-FIG4 (n>9 per cohort). Upper panels show grip strength assessed in PND1 mice at 2 months old. Lower panels show grip strength assessed in PND4 injected mice at 6 months of age. The mean grip strength normalized to body weight are presented. The data are presented as mean±SD. Mann- Whitney t-test, ns p>0.05, * p<0.05.

FIG. 6 shows motor coordination analysis. Motor coordination was assessed by rotarod test in PND1 injected mice at 2 months old (upper panel) and in PND4 injected mice at 6 months old (lower panel) (n>9 per cohort). The graph represents the average latency to fall over 3 trials. The data are presented as mean±SD. Mann- Whitney t-test, ns p>0.05.

FIG. 7 shows overall activity analysis. Overall activity was tested by spontaneous wheel running during 3 nights. PND1 injected mice (first and third panel) were recorded at 2 months old and PND4 injected mice (second and fourth panel) at 6 months old (n>9 per cohort). The total distance travelled is presented as mean±SD.

FIG. 8 shows nerve conduction analysis. Nerve conduction velocity was assessed in PND 1 or PND4 ICV-injected mice at 6 months old (n>8 per cohort). The data are presented as mean±sem. Mann- Whitney t-test, ns p>0.05, * p<0.05, ** pO.Ol.

FIG. 9 shows FIG4 protein analysis. Protein levels of FIG4 (left panel), GFAP (central panel) and p62 (right panel) were analyzed by western blot on whole brain and spinal cord of PND1 ICV-injected mice (n>3 per cohort). The data are presented as mean±sem of the relative fold change (RFC) in each group compared to untreated Fig4 ^+/+ mice. Mann- Whitney t-test, ns p>0.05, ** pO.Ol, **** pO.OOOl.

FIG. 10 shows neuropathology in AAV9-FIG4 treated Fig4 ^/_ mice. Dorsal root ganglion (DRG), thoracic spinal cord and brain sections from PND1 ICV-injected mice were stained by H/E. The arrows indicate the presence of cytoplasmic vacuoles. FIG. 11 shows FIG4 nerve histology analysis. Semi-thin sections of sciatic nerve from PND1 ICV-injected mice showed a significant increase in the frequency of large size axons in Fig4 ^/_ treated vs untreated mice. The graph represent the average frequency of axons vs their area. Images are representative semi-thin sections of sciatic nerves analyzed.

FIG. 12 shows AAV9-FIG4 dose-response analysis. AAV9-FIG4 can rescue

Fig4-/- mice in a dose-dependent fashion. PND7 mice were IT (intrathecal) injected with undiluted AAV9-FIG4 (130.5c10 ¹⁰ vg) or diluted 1 :5, 1 :10 and 1 :100. PND1 were ICV- injected with AAV9-FIG4 at 1 :10 dilution (5.4xl0 ¹⁰ vg). The mice were monitored daily for morbidity or mortality. The data in the top panel represent the survival analysis (n>5 per cohort). In the bottom panel, the mean body weights of each cohort are presented.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is explained in greater detail below. This description is not intended to be a detailed catalog of all the different ways in which the invention may be implemented, or all the features that may be added to the instant invention. For example, features illustrated with respect to one embodiment may be incorporated into other embodiments, and features illustrated with respect to a particular embodiment may be deleted from that embodiment. In addition, numerous variations and additions to the various embodiments suggested herein will be apparent to those skilled in the art in light of the instant disclosure which do not depart from the instant invention. Hence, the following specification is intended to illustrate some particular embodiments of the invention, and not to exhaustively specify all permutations, combinations and variations thereof.

Unless the context indicates otherwise, it is specifically intended that the various features of the invention described herein can be used in any combination. Moreover, the present invention also contemplates that in some embodiments of the invention, any feature or combination of features set forth herein can be excluded or omitted. To illustrate, if the specification states that a complex comprises components A, B and C, it is specifically intended that any of A, B or C, or a combination thereof, can be omitted and disclaimed singularly or in any combination.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. Nucleotide sequences are presented herein by single strand only, in the 5' to 3' direction, from left to right, unless specifically indicated otherwise. Nucleotides and amino acids are represented herein in the manner recommended by the IUPAC-IUB Biochemical Nomenclature Commission, or (for amino acids) by either the one-letter code, or the three letter code, both in accordance with 37 C.F.R. §1.822 and established usage.

Except as otherwise indicated, standard methods known to those skilled in the art may be used for production of recombinant and synthetic polypeptides, antibodies or antigen- binding fragments thereof, manipulation of nucleic acid sequences, production of transformed cells, the construction of rAAV constructs, modified capsid proteins, packaging vectors expressing the AAV rep and/or cap sequences, and transiently and stably transfected packaging cells. Such techniques are known to those skilled in the art. See, e.g., SAMBROOK et al, MOLECULAR CLONING: A LABORATORY MANUAL 2nd Ed. (Cold Spring Harbor, NY, 1989); F. M. AUSUBEL et al CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (Green Publishing Associates, Inc. and John Wiley & Sons, Inc., New York).

All publications, patent applications, patents, nucleotide sequences, amino acid sequences and other references mentioned herein are incorporated by reference in their entirety.

Definitions

As used in the description of the invention and the appended claims, the singular forms "a," "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise.

As used herein, "and/or" refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative ("or").

Moreover, the present invention also contemplates that in some embodiments of the invention, any feature or combination of features set forth herein can be excluded or omitted.

Furthermore, the term "about," as used herein when referring to a measurable value such as an amount of a compound or agent of this invention, dose, time, temperature, and the like, is meant to encompass variations of ± 10%, ± 5%, ± 1%, ± 0.5%, or even ± 0.1% of the specified amount. As used herein, the transitional phrase "consisting essentially of' is to be interpreted as encompassing the recited materials or steps and those that do not materially affect the basic and novel characteristic(s) of the claimed invention. Thus, the term "consisting essentially of' as used herein should not be interpreted as equivalent to "comprising."

The term "consists essentially of' (and grammatical variants), as applied to a polynucleotide or polypeptide sequence of this invention, means a polynucleotide or polypeptide that consists of both the recited sequence (e.g., SEQ ID NO) and a total of ten or less (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) additional nucleotides or amino acids on the 5’ and/or 3’ or N-terminal and/or C-terminal ends of the recited sequence or between the two ends (e.g., between domains) such that the function of the polynucleotide or polypeptide is not materially altered. The total of ten or less additional nucleotides or amino acids includes the total number of additional nucleotides or amino acids added together. The term "materially altered," as applied to polynucleotides of the invention, refers to an increase or decrease in ability to express the encoded polypeptide of at least about 50% or more as compared to the expression level of a polynucleotide consisting of the recited sequence. The term "materially altered," as applied to polypeptides of the invention, refers to an increase or decrease in biological activity of at least about 50% or more as compared to the activity of a polypeptide consisting of the recited sequence.

The term "parvovirus" as used herein encompasses the family Parvoviridae, including autonomously-replicating parvoviruses and dependo viruses. The autonomous parvoviruses include members of the genera Parvovirus, Erythrovirus, Densovirus, Iteravirus, and Contravirus. Exemplary autonomous parvoviruses include, but are not limited to, minute virus of mouse, bovine parvovirus, canine parvovirus, chicken parvovirus, feline panleukopenia virus, feline parvovirus, goose parvovirus, Hl parvovirus, muscovy duck parvovirus, snake parvovirus, and B19 virus. Other autonomous parvoviruses are known to those skilled in the art. See, e.g., FIELDS et al, VIROLOGY, volume 2, chapter 69 (4th ed., Lippincott-Raven Publishers).

The genus Dependovirus contains the adeno-associated viruses (AAV), including but not limited to, AAV type 1, AAV type 2, AAV type 3 (including types 3 A and 3B), AAV type 4, AAV type 5, AAV type 6, AAV type 7, AAV type 8, AAV type 9, AAV type 10, AAV type 11, AAV type 12, AAV type 13, avian AAV, bovine AAV, canine AAV, goat AAV, snake AAV, equine AAV, and ovine AAV. See, e.g., FIELDS et al., VIROLOGY, volume 2, chapter 69 (4th ed., Lippincott-Raven Publishers); and Table 1. The term "adeno-associated virus" (AAV) in the context of the present invention includes without limitation AAV type 1, AAV type 2, AAV type 3 (including types 3 A and 3B), AAV type 4, AAV type 5, AAV type 6, AAV type 7, AAV type 8, AAV type 9, AAV type 10, AAV type 11, avian AAV, bovine AAV, canine AAV, equine AAV, and ovine AAV and any other AAV now known or later discovered. See, e.g., BERNARD N. FIELDS et al, VIROLOGY, volume 2, chapter 69 (4th ed., Lippincott-Raven Publishers). A number of additional AAV serotypes and clades have been identified (see, e.g., Gao et al, (2004) J. Virol. 78:6381-6388 and Table 1), which are also encompassed by the term "AAV."

The parvovirus particles and genomes of the present invention can be from, but are not limited to, AAV. The genomic sequences of various serotypes of AAV and the autonomous parvoviruses, as well as the sequences of the native ITRs, Rep proteins, and capsid subunits are known in the art. Such sequences may be found in the literature or in public databases such as GenBank. See, e.g., GenBank Accession Numbers NC_002077, NC_00l40l, NC_00l729, NC_00l863, NC_00l829, NC_001862, NC_000883, NC 001701, NC_00l5l0, NC_006l52, NC 006261, AF063497, U89790, AF043303, AF028705, AF028704, J02275, J01901, J02275, X01457, AF288061, AH009962, AY028226, AY028223, AY631966, AX753250, EU285562, NC_00l358, NC_00l540, AF513851, AF513852 and AY530579; the disclosures of which are incorporated by reference herein for teaching parvovirus and AAV nucleic acid and amino acid sequences. See also, e.g, Bantel- Schaal et al, (1999) J. Virol. 73: 939; Chiorini et al, (1997) J. Virol. 71 :6823; Chiorini et al, (1999) J. Virol. 73:1309; Gao et al, (2002) Proc. Nat. Acad. Sci. USA 99:11854; Moris et al, (2004) Virol. 33-:375-383; Mori et al, (2004) Virol 330:375; Muramatsu et al, (1996) Virol. 221:208; Ruffing et al, (1994) J. Gen. Virol. 75:3385; Rutledge et al, (1998) J. Virol. 72:309; Schmidt et al, (2008) J Virol. 82:8911; Shade et al, (1986) J. Virol. 58:921; Srivastava et al, (1983) J. Virol. 45:555; Xiao et al, (1999) J. Virol. 73:3994; international patent publications WO 00/28061, WO 99/61601, WO 98/11244; and U.S. Patent No. 6,156,303; the disclosures of which are incorporated by reference herein for teaching parvovirus and AAV nucleic acid and amino acid sequences. See also Table 1. An early description of the AAV1, AAV2 and AAV3 ITR sequences is provided by Xiao, X., (1996), "Characterization of Adeno-associated virus (AAV) DNA replication and integration," Ph.D. Dissertation, University of Pittsburgh, Pittsburgh, PA (incorporated herein it its entirety). A "chimeric" AAV nucleic acid capsid coding sequence or AAV capsid protein is one that combines portions of two or more capsid sequences. A "chimeric" AAV virion or particle comprises a chimeric AAV capsid protein.

The term "tropism" as used herein refers to preferential entry of the virus into certain cell or tissue type(s) and/or preferential interaction with the cell surface that facilitates entry into certain cell or tissue types, optionally and preferably followed by expression ( e.g ., transcription and, optionally, translation) of sequences carried by the viral genome in the cell, e.g., for a recombinant virus, expression of the heterologous nucleotide sequence(s). Those skilled in the art will appreciate that transcription of a heterologous nucleic acid sequence from the viral genome may not be initiated in the absence of trans-acting factors, e.g., for an inducible promoter or otherwise regulated nucleic acid sequence. In the case of a rAAV genome, gene expression from the viral genome may be from a stably integrated provirus and/or from a non-integrated episome, as well as any other form which the virus nucleic acid may take within the cell.

The term "tropism profile" refers to the pattern of transduction of one or more target cells, tissues and/or organs. Representative examples of chimeric AAV capsids have a tropism profile characterized by efficient transduction of cells of the central nervous system (CNS) with only low transduction of peripheral organs ( ee e.g. US Patent No. 9,636,370 McCown et ah, and US patent publication 2017/0360960 Gray et al.).

The term "disorder associated with aberrant expression of a FIG4 gene" as used herein refers to a disease, disorder, syndrome, or condition that is caused by or a symptom of decreased or altered expression of the FIG4 gene in a subject relative to the expression level in a normal subject or in a population.

The term "disorder associated with aberrant activity of a FIG4 gene product" as used herein refers to a disease, disorder, syndrome, or condition that is caused by or a symptom of decreased or altered activity of the FIG4 gene product in a subject relative to the activity in a normal subject or in a population.

As used herein, "transduction" of a cell by a virus vector {e.g, an AAV vector) means entry of the vector into the cell and transfer of genetic material into the cell by the incorporation of nucleic acid into the virus vector and subsequent transfer into the cell via the virus vector.

Unless indicated otherwise, "efficient transduction" or "efficient tropism," or similar terms, can be determined by reference to a suitable positive or negative control {e.g, at least about 50%, 60%, 70%, 80%, 85%, 90%, 95% or more of the transduction or tropism, respectively, of a positive control or at least about 110%, 120%, 150%, 200%, 300%, 500%, 1000% or more of the transduction or tropism, respectively, of a negative control).

Table 1

Similarly, it can be determined if a virus "does not efficiently transduce" or "does not have efficient tropism" for a target tissue, or similar terms, by reference to a suitable control. In particular embodiments, the virus vector does not efficiently transduce (i.e., does not have efficient tropism for) tissues outside the CNS, e.g., liver, kidney, gonads and/or germ cells. In particular embodiments, undesirable transduction of tissue(s) (e.g., liver) is 20% or less, 10% or less, 5% or less, 1% or less, 0.1% or less of the level of transduction of the desired target tissue(s) (e.g., CNS cells).

The terms "5’ portion" and "3’ portion" are relative terms to define a spatial relationship between two or more elements. Thus, for example, a "3’ portion" of a polynucleotide indicates a segment of the polynucleotide that is downstream of another segment. The term "3’ portion" is not intended to indicate that the segment is necessarily at the 3’ end of the polynucleotide, or even that it is necessarily in the 3’ half of the polynucleotide, although it may be. Likewise, a "5’ portion" of a polynucleotide indicates a segment of the polynucleotide that is upstream of another segment. The term "5’ portion" is not intended to indicate that the segment is necessarily at the 5’ end of the polynucleotide, or even that it is necessarily in the 5’ half of the polynucleotide, although it may be.

As used herein, the term "polypeptide" encompasses both peptides and proteins, unless indicated otherwise.

A "polynucleotide," "nucleic acid," or "nucleotide sequence" may be of RNA, DNA or DNA-RNA hybrid sequences (including both naturally occurring and non-naturally occurring nucleotides), but is preferably either a single or double stranded DNA sequence.

The term "open reading frame (ORF)," as used herein, refers to the portion of a polynucleotide, e.g., a gene, that encodes a polypeptide.

The term "codon-optimized," as used herein, refers to a gene coding sequence that has been optimized to increase expression by substituting one or more codons normally present in a coding sequence (for example, in a wild-type sequence, including, e.g., a coding sequence for FIG4) with a codon for the same (synonymous) amino acid. In this manner, the protein encoded by the gene is identical, but the underlying nucleobase sequence of the gene or corresponding mRNA is different. In some embodiments, the optimization substitutes one or more rare codons (that is, codons for tRNA that occur relatively infrequently in cells from a particular species) with synonymous codons that occur more frequently to improve the efficiency of translation. For example, in human codon-optimization one or more codons in a coding sequence are replaced by codons that occur more frequently in human cells for the same amino acid. Codon optimization can also increase gene expression through other mechanisms that can improve efficiency of transcription and/or translation. Strategies include, without limitation, increasing total GC content (that is, the percent of guanines and cytosines in the entire coding sequence), decreasing CpG content (that is, the number of CG or GC dinucleotides in the coding sequence), removing cryptic splice donor or acceptor sites, and/or adding or removing ribosomal entry sites, such as Kozak sequences. Desirably, a codon-optimized gene exhibits improved protein expression, for example, the protein encoded thereby is expressed at a detectably greater level in a cell compared with the level of expression of the protein provided by the wild-type gene in an otherwise similar cell.

The term "sequence identity," as used herein, has the standard meaning in the art. As is known in the art, a number of different programs can be used to identify whether a polynucleotide or polypeptide has sequence identity or similarity to a known sequence. Sequence identity or similarity may be determined using standard techniques known in the art, including, but not limited to, the local sequence identity algorithm of Smith & Waterman, Adv. Appl. Math. 2: 482 (1981), by the sequence identity alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48: 443 (1970), by the search for similarity method of Pearson & Lipman, Proc. Natl. Acad. Sci. USA 55:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, WI), the Best Fit sequence program described by Devereux et al., Nucl. Acid Res. 12:387 (1984), preferably using the default settings, or by inspection.

An example of a useful algorithm is PILEUP. PILEUP creates a multiple sequence alignment from a group of related sequences using progressive, pairwise alignments. It can also plot a tree showing the clustering relationships used to create the alignment. PILEUP uses a simplification of the progressive alignment method of Feng & Doolittle, J. Mol. Evol. 55:351 (1987); the method is similar to that described by Higgins & Sharp, CABIOS 5:151 (1989).

Another example of a useful algorithm is the BLAST algorithm, described in Altschul et al, J. Mol. Biol. 215: 403 (1990) and Karlin et al, Proc. Natl. Acad. Sci. USA 90:5873 (1993). A particularly useful BLAST program is the WU-BLAST-2 program which was obtained from Altschul et al, Meth. Enzymol, 266:460 (1996); blast. wustl/edu/blast/README.html. WU-BLAST-2 uses several search parameters, which are preferably set to the default values. The parameters are dynamic values and are established by the program itself depending upon the composition of the particular sequence and composition of the particular database against which the sequence of interest is being searched; however, the values may be adjusted to increase sensitivity.

An additional useful algorithm is gapped BLAST as reported by Altschul et al, Nucleic Acids Res. 25:3389 (1997).

A percentage amino acid sequence identity value is determined by the number of matching identical residues divided by the total number of residues of the "longer" sequence in the aligned region. The "longer" sequence is the one having the most actual residues in the aligned region (gaps introduced by WU-Blast-2 to maximize the alignment score are ignored).

In a similar manner, percent nucleic acid sequence identity is defined as the percentage of nucleotide residues in the candidate sequence that are identical with the nucleotides in the polynucleotide specifically disclosed herein.

The alignment may include the introduction of gaps in the sequences to be aligned. In addition, for sequences which contain either more or fewer nucleotides than the polynucleotides specifically disclosed herein, it is understood that in one embodiment, the percentage of sequence identity will be determined based on the number of identical nucleotides in relation to the total number of nucleotides. Thus, for example, sequence identity of sequences shorter than a sequence specifically disclosed herein, will be determined using the number of nucleotides in the shorter sequence, in one embodiment. In percent identity calculations relative weight is not assigned to various manifestations of sequence variation, such as insertions, deletions, substitutions, etc.

In one embodiment, only identities are scored positively (+1) and all forms of sequence variation including gaps are assigned a value of "0," which obviates the need for a weighted scale or parameters as described below for sequence similarity calculations. Percent sequence identity can be calculated, for example, by dividing the number of matching identical residues by the total number of residues of the "shorter" sequence in the aligned region and multiplying by 100. The "longer" sequence is the one having the most actual residues in the aligned region.

As used herein, an "isolated" nucleic acid or nucleotide sequence (e.g., an "isolated DNA" or an "isolated RNA") means a nucleic acid or nucleotide sequence separated or substantially free from at least some of the other components of the naturally occurring organism or virus, for example, the cell or viral structural components or other polypeptides or nucleic acids commonly found associated with the nucleic acid or nucleotide sequence.

Likewise, an "isolated" polypeptide means a polypeptide that is separated or substantially free from at least some of the other components of the naturally occurring organism or virus, for example, the cell or viral structural components or other polypeptides or nucleic acids commonly found associated with the polypeptide.

As used herein, the term "modified," as applied to a polynucleotide or polypeptide sequence, refers to a sequence that differs from a wild-type sequence due to one or more deletions, additions, substitutions, or any combination thereof.

As used herein, by "isolate" (or grammatical equivalents) a virus vector, it is meant that the virus vector is at least partially separated from at least some of the other components in the starting material.

By the term "treat," "treating," or "treatment of' (or grammatically equivalent terms) is meant to reduce or to at least partially improve or ameliorate the severity of the subject’s condition and/or to alleviate, mitigate or decrease in at least one clinical symptom and/or to delay the progression of the condition.

As used herein, the term "prevent," "prevents," or "prevention" (and grammatical equivalents thereof) means to delay or inhibit the onset of a disease. The terms are not meant to require complete abolition of disease, and encompasses any type of prophylactic treatment to reduces the incidence of the condition or delays the onset of the condition.

A "treatment effective" amount as used herein is an amount that is sufficient to provide some improvement or benefit to the subject. Alternatively stated, a "treatment effective" amount is an amount that will provide some alleviation, mitigation, decrease or stabilization in at least one clinical symptom in the subject. Those skilled in the art will appreciate that the therapeutic effects need not be complete or curative, as long as some benefit is provided to the subject.

A "prevention effective" amount as used herein is an amount that is sufficient to prevent and/or delay the onset of a disease, disorder and/or clinical symptoms in a subject and/or to reduce and/or delay the severity of the onset of a disease, disorder and/or clinical symptoms in a subject relative to what would occur in the absence of the methods of the invention. Those skilled in the art will appreciate that the level of prevention need not be complete, as long as some benefit is provided to the subject. A "heterologous nucleotide sequence" or "heterologous nucleic acid," with respect to a virus, is a sequence or nucleic acid, respectively, that is not naturally occurring in the virus. Generally, the heterologous nucleic acid or nucleotide sequence comprises an open reading frame that encodes a polypeptide and/or a nontranslated RNA.

As used herein, the term "vector," "virus vector," "delivery vector" (and similar terms) in a specific embodiment generally refers to a virus particle that functions as a nucleic acid delivery vehicle, and which comprises the viral nucleic acid (i.e., the vector genome) packaged within the virion. Virus vectors according to the present invention comprise a chimeric AAV capsid according to the invention and can package an AAV or rAAV genome or any other nucleic acid including viral nucleic acids. Alternatively, in some contexts, the term "vector," "virus vector," "delivery vector" (and similar terms) may be used to refer to the vector genome (e.g., vDNA) in the absence of the virion and/or to a viral capsid that acts as a transporter to deliver molecules tethered to the capsid or packaged within the capsid.

The virus vectors of the invention can further be duplexed parvovirus particles as described in international patent publication WO 01/92551 (the disclosure of which is incorporated herein by reference in its entirety). Thus, in some embodiments, double stranded (duplex) genomes can be packaged.

A "recombinant AAV vector genome" or "rAAV genome" is an AAV genome (i.e., vDNA) that comprises at least one inverted terminal repeat (e.g, one, two or three inverted terminal repeats) and one or more heterologous nucleotide sequences. rAAV vectors generally retain the 145 base terminal repeat(s) (TR(s)) in cis to generate virus; however, modified AAV TRs and non- AAV TRs including partially or completely synthetic sequences can also serve this purpose. All other viral sequences are dispensable and may be supplied in trans (Muzyczka, (1992) Curr. Topics Microbiol. Immunol. 158:97). The rAAV vector optionally comprises two TRs (e.g, AAV TRs), which generally will be at the 5’ and 3’ ends of the heterologous nucleotide sequence(s), but need not be contiguous thereto. The TRs can be the same or different from each other. The vector genome can also contain a single ITR at its 3' or 5' end.

The term "terminal repeat" or "TR" includes any viral terminal repeat or synthetic sequence that forms a hairpin structure and functions as an inverted terminal repeat (i.e., mediates the desired functions such as replication, virus packaging, integration and/or provirus rescue, and the like). The TR can be an AAV TR or a non- AAV TR. For example, a non- AAV TR sequence such as those of other parvoviruses (e.g., canine parvovirus (CPV), mouse parvovirus (MVM), human parvovirus B-19) or the SV40 hairpin that serves as the origin of SV40 replication can be used as a TR, which can further be modified by truncation, substitution, deletion, insertion and/or addition. Further, the TR can be partially or completely synthetic, such as the "double-D sequence" as described in United States Patent No. 5,478,745 to Samulski et al.

Parvovirus genomes have palindromic sequences at both their 5’ and 3’ ends. The palindromic nature of the sequences leads to the formation of a hairpin structure that is stabilized by the formation of hydrogen bonds between the complementary base pairs. This hairpin structure is believed to adopt a "Y" or a "T" shape. See, e.g., FIELDS et al, VIROLOGY, volume 2, chapters 69 & 70 (4th ed., Lippincott-Raven Publishers).

An "AAV terminal repeat" or "AAV TR" may be from any AAV, including but not limited to serotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 or any other AAV now known or later discovered (see, e.g., Table 1). An AAV terminal repeat need not have the native terminal repeat sequence (e.g., a native AAV TR sequence may be altered by insertion, deletion, truncation and/or missense mutations), as long as the terminal repeat mediates the desired functions, e.g., replication, virus packaging, integration, and/or provirus rescue, and the like.

The terms "rAAV particle" and "rAAV virion" are used interchangeably here. A "rAAV particle" or "rAAV virion" comprises a rAAV vector genome packaged within an AAV capsid.

The virus vectors of the invention can further be "targeted" virus vectors (e.g., having a directed tropism) and/or a "hybrid" parvovirus (i.e., in which the viral ITRs and viral capsid are from different parvoviruses) as described in international patent publication WO 00/28004 and Chao et al, (2000) Mol. Therapy 2:619.

Further, the viral capsid or genomic elements can contain other modifications, including insertions, deletions and/or substitutions.

As used herein, the term "amino acid" encompasses any naturally occurring amino acids, modified forms thereof, and synthetic amino acids, including non-naturally occurring amino acids..

Naturally occurring, levorotatory (L-) amino acids are shown in Table 2.

Alternatively, the amino acid can be a modified amino acid residue (nonlimiting examples are shown in Table 3) or can be an amino acid that is modified by post-translation modification (e.g., acetylation, amidation, formylation, hydroxylation, methylation, phosphorylation or sulfatation). Table 2

Table 3: Amino Acid Residue Derivatives

Further, the non-naturally occurring amino acid can be an "unnatural" amino acid as described by Wang et ah, (2006) Annu. Rev. Biophys. Biomol. Struct. 35:225-49. These unnatural amino acids can advantageously be used to chemically link molecules of interest to the AAV capsid protein.

The term "template" or "substrate" is used herein to refer to a polynucleotide sequence that may be replicated to produce the parvovirus viral DNA. For the purpose of vector production, the template will typically be embedded within a larger nucleotide sequence or construct, including but not limited to a plasmid, naked DNA vector, bacterial artificial chromosome (BAC), yeast artificial chromosome (YAC) or a viral vector ( e.g ., adenovirus, herpesvirus, Epstein-Barr Virus, AAV, baculoviral, retroviral vectors, and the like). Alternatively, the template may be stably incorporated into the chromosome of a packaging cell.

As used herein, parvovirus or AAV "Rep coding sequences" indicate the nucleic acid sequences that encode the parvoviral or AAV non-structural proteins that mediate viral replication and the production of new virus particles. The parvovirus and AAV replication genes and proteins have been described in, e.g., FIELDS et al, VIROLOGY, volume 2, chapters 69 & 70 (4th ed., Lippincott-Raven Publishers).

The "Rep coding sequences" need not encode all of the parvoviral or AAV Rep proteins. For example, with respect to AAV, the Rep coding sequences do not need to encode all four AAV Rep proteins (Rep78, Rep 68, Rep52 and Rep40), in fact, it is believed that AAV5 only expresses the spliced Rep68 and Rep40 proteins. In representative embodiments, the Rep coding sequences encode at least those replication proteins that are necessary for viral genome replication and packaging into new virions. The Rep coding sequences will generally encode at least one large Rep protein (i.e., Rep78/68) and one small Rep protein (i. e. , Rep52/40). In particular embodiments, the Rep coding sequences encode the AAV Rep78 protein and the AAV Rep52 and/or Rep40 proteins. In other embodiments, the Rep coding sequences encode the Rep68 and the Rep52 and/or Rep40 proteins. In a still further embodiment, the Rep coding sequences encode the Rep68 and Rep52 proteins, Rep68 and Rep40 proteins, Rep78 and Rep52 proteins, or Rep78 and Rep40 proteins.

As used herein, the term "large Rep protein" refers to Rep68 and/or Rep78. Large Rep proteins of the claimed invention may be either wild-type or synthetic. A wild-type large Rep protein may be from any parvovirus or AAV, including but not limited to serotypes 1, 2, 3a, 3b, 4, 5, 6, 7, 8, 9, 10, 11, or 13, or any other AAV now known or later discovered (see, e.g., Table 1). A synthetic large Rep protein may be altered by insertion, deletion, truncation and/or missense mutations.

Those skilled in the art will further appreciate that it is not necessary that the replication proteins be encoded by the same polynucleotide. For example, for MVM, the NS- 1 and NS -2 proteins (which are splice variants) may be expressed independently of one another. Likewise, for AAV, the pl9 promoter may be inactivated and the large Rep protein(s) expressed from one polynucleotide and the small Rep protein(s) expressed from a different polynucleotide. Typically, however, it will be more convenient to express the replication proteins from a single construct. In some systems, the viral promoters (e.g., AAV pl9 promoter) may not be recognized by the cell, and it is therefore necessary to express the large and small Rep proteins from separate expression cassettes. In other instances, it may be desirable to express the large Rep and small Rep proteins separately, i.e., under the control of separate transcriptional and/or translational control elements. For example, it may be desirable to control expression of the large Rep proteins, so as to decrease the ratio of large to small Rep proteins. In the case of insect cells, it may be advantageous to down-regulate expression of the large Rep proteins (e.g., Rep78/68) to avoid toxicity to the cells (see, e.g., Urabe et al, (2002) Human Gene Therapy 13:1935).

As used herein, the parvovirus or AAV "cap coding sequences" encode the structural proteins that form a functional parvovirus or AAV capsid (i.e., can package DNA and infect target cells). Typically, the cap coding sequences will encode all of the parvovirus or AAV capsid subunits, but less than all of the capsid subunits may be encoded as long as a functional capsid is produced. Typically, but not necessarily, the cap coding sequences will be present on a single nucleic acid molecule.

The capsid structure of autonomous parvoviruses and AAV are described in more detail in BERNARD N. FIELDS et al, VIROLOGY, volume 2, chapters 69 & 70 (4th ed., Lippincott-Raven Publishers).

By "substantially retain" a property, it is meant that at least about 75%, 85%, 90%, 95%, 97%, 98%, 99% or 100% of the property (e.g., activity or other measurable characteristic) is retained.

FIG4 Expression Cassettes and Vectors

The present invention relates to the design of a FIG4 expression cassette to provide therapeutic levels of expression of polyphosphoinositide phosphatase (also known as phosphatidylinositol 3,5-bisphosphate 5 -phosphatase, Sac3, or FIG4), the enzyme encoded by the FIG4 gene, and the use of the expression cassette to achieve therapeutic levels of FIG4 in a subject.

Thus, one aspect of the invention relates to a polynucleotide comprising a human FIG4 open reading frame (ORF), wherein the FIG4 open reading frame has been codon- optimized for expression in human cells. The open reading frame is the portion of the FIG4 gene that encodes FIG4. As used herein, a human FIG4 ORF refers to a nucleotide sequence that encodes human FIG4. Codon optimization is a technique well known in the art and optimal codons for expression in humans are known. The use of a codon-optimized FIG4 sequence allows one to distinguish expression of the transduced sequence from expression of the endogenous FIG4 sequence in a subject.

In some embodiments, the codon-optimized FIG4 open reading frame encodes a FIG4 enzyme that is modified from the wild-type sequence, e.g., comprises, consists essentially of, or consists of an amino acid sequence in which 1, 2, 3, 4, or 5 residues have been substituted, added, and/or deleted compared to the wild-type amino acid sequence.

In some embodiments, the codon-optimized FIG4 open reading frame comprises, consists essentially of, or consists of the nucleotide sequence of SEQ ID NO:l or a sequence at least about 70% identical thereto, e.g., at least about 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical thereto. SEP ID NO:l : Human codon-optimized FIG4 open reading frame

ATGCCAACTGCCGCCGCCCCAATCATTTCATCAGTGCAAAAGCTTGTGCTCTACG

AGACAAGAGCTCGGTATTTCCTGGTCGGCTCCAACAACGCAGAAACCAAGTACA

GAGTGCTTAAGATTGACCGCACCGAACCGAAGGACCTCGTGATTATTGACGATC

GCCACGTGTACACTCAGCAAGAAGTGCGCGAACTCTTGGGACGCCTGGACCTGG

GCAACCGAACCAAAATGGGCCAGAAGGGCTCCTCCGGTCTGTTTCGGGCAGTGT

CGGCTTTCGGCGTCGTGGGATTCGTGCGATTCCTTGAGGGGTACTATATTGTGCT

GATCACGAAGCGGCGCAAGATGGCCGACATCGGAGGACACGCTATATACAAGGT

CGAGGACACCAACATGATCTACATTCCGAACGATTCAGTCAGAGTGACCCACCC

CGATGAAGCCCGCTACCTTAGAATCTTCCAGAACGTGGATCTCTCCTCGAACTTT

TACTTCTCATACTCGTACGACCTGTCCCACTCGCTGCAGTACAACCTGACCGTGC

TGCGGATGCCTCTGGAGATGCTCAAGTCCGAGATGACTCAAAATCGGCAGGAAT

CCTTCGACATCTTCGAGGACGAGGGTCTGATCACTCAAGGAGGGTCGGGAGTGTT

CGGCATTTGCAGCGAACCCTACATGAAATACGTGTGGAACGGAGAGCTCCTGGA

CATTATCAAGAGCACTGTGCACCGGGACTGGCTGCTGTACATCATCCATGGATTC

TGTGGACAGTCAAAGCTCCTGATCTATGGCCGGCCTGTGTACGTGACTCTGATCG

CCCGCCGCTCCTCGAAGTTCGCGGGTACCCGGTTTCTCAAGCGCGGTGCTAACTG

CGAGGGGGATGTGGCCAACGAAGTGGAAACCGAGCAGATCCTCTGTGACGCCTC

CGTGATGAGCTTTACCGCCGGATCCTACTCTTCGTATGTGCAAGTCCGGGGATCC

GTCCCCCTGTACTGGAGCCAGGACATCTCCACTATGATGCCCAAGCCCCCTATCA

CCCTGGACCAGGCCGATCCTTTCGCACACGTGGCTGCCCTGCACTTCGACCAGAT

GTTCCAGCGGTTCGGCTCCCCTATCATCATCCTGAACTTGGTCAAGGAACGGGAG

AAGCGGAAGCATGAGAGGATTCTGTCCGAGGAGCTCGTGGCCGCGGTGACCTAC

CTGAATCAGTTCCTCCCGCCCGAACATACCATCGTGTATATCCCCTGGGATATGG

CCAAGTACACTAAGTCCAAGCTGTGCAATGTGCTGGACCGCCTTAACGTCATCGC

GGAATCCGTGGTCAAGAAAACCGGATTCTTCGTGAATAGACCGGATTCATACTGC

TCCATTCTCCGGCCCGATGAAAAGTGGAACGAACTGGGCGGTTGCGTCATCCCGA

CTGGCCGGCTCCAGACCGGCATCCTTAGGACCAACTGCGTGGACTGCCTGGACA

GAACCAACACGGCCCAATTCATGGTCGGGAAATGTGCCCTGGCCTACCAACTGT

ACTCCCTGGGACTGATCGACAAGCCGAACTTGCAATTCGATACTGACGCCGTGCG

GCTGTTCGAAGAACTGTACGAGGATCACGGCGACACCCTGAGCCTGCAGTACGG

CGGAAGCCAGCTCGTGCATAGAGTGAAAACCTACAGGAAGATTGCTCCGTGGAC

TCAGCACTCGAAGGACATCATGCAGACCCTCAGCCGCTACTACTCCAATGCCTTC TCCGACGCCGACCGCCAGGACTCCATTAACCTCTTCCTGGGAGTGTTCCACCCAA

CCGAGGGAAAGCCCCACCTGTGGGAACTGCCTACTGATTTCTACTTGCATCACAA

GAACACCATGAGACTCCTGCCCACCCGGCGCTCCTACACTTACTGGTGGACCCCT

GAAGTGATCAAGCACCTCCCGCTGCCGTACGACGAGGTCATCTGCGCCGTGAAC

CTGAAAAAGCTGATCGTCAAAAAGTTCCATAAATATGAAGAAGAAATCGACATT

CACAACGAATTCTTCCGGCCATACGAGCTGTCCTCGTTCGACGACACCTTTTGTCT

GGCGATGACCTCATCCGCCCGCGATTTCATGCCTAAGACTGTGGGGATCGACCCC

AGCCCCTTCACCGTCCGCAAGCCTGACGAGACTGGAAAGTCGGTGCTCGGCAAC

AAGAGCAACAGGGAAGAAGCGGTGCTTCAGAGAAAGACTGCTGCCTCGGCGCCA

CCGCCGCCTTCCGAAGAAGCCGTGTCGTCATCCTCGGAAGATGACAGCGGAACC

GACCGCGAGGAGGAGGGGTCCGTGAGCCAGCGGTCCACCCCGGTCAAGATGACT

GACGCCGGGGACTCGGCCAAGGTCACCGAAAACGTGGTGCAACCCATGAAGGA

ACTGTACGGCATCAACCTTAGCGACGGCTTGTCTGAAGAGGACTTCTCCATCTAC

TCTCGGTTTGTGCAGCTGGGGCAGAGCCAGCACAAGCAGGACAAGAATTCCCAA

CAGCCGTGCAGCAGATGCTCCGACGGAGTGATTAAGCTGACTCCGATTAGCGCG

TTCTCGCAAGATAACATCTACGAAGTGCAACCCCCTCGCGTGGACAGGAAGTCC

ACCGAGATTTTCCAGGCCCACATCCAAGCATCCCAGGGAATCATGCAGCCCCTCG

GGAAAGAGGACTCCTCCATGTACCGGGAGTACATCAGAAACCGCTACCTG

Another aspect of the invention relates to an expression cassette comprising a polynucleotide comprising a human FIG4 open reading frame. In certain embodiments, the polynucleotide is a human codon-optimized sequence, e.g., a polynucleotide comprising the nucleotide sequence of SEQ ID NO:l or a sequence at least about 70% identical thereto, e.g., at least about 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical thereto.

The FIG4 open reading frame in the expression cassette may be operably linked to one or more expression elements that may enhance expression of FIG4. In some embodiments, the polynucleotide is operably linked to a promoter, e.g., a chicken beta-actin promoter, e.g., a promoter comprising, consisting essentially of, or consisting of the nucleotide sequence of SEQ ID NO:2 or a sequence at least about 70% identical thereto, e.g., at least about 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical thereto. In some embodiments, the promoter further comprises the chicken beta-actin exon 1 and intron, e.g., comprising, consisting essentially of, or consisting of the nucleotide sequence of SEQ ID NO:3 or a sequence at least about 70% identical thereto, e.g., at least about 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical thereto.

SEQ ID NO:2: Chicken beta-actin promoter

TACGTATTAGTCATCGCTATTACCATGGTCGAGGTGAGCCCCACGTTCTGCTTCA

CTCTCCCCATCTCCCCCCCCTCCCCACCCCCAATTTTGTATTTATTTATTTTTTAAT

TATTTTGTGCAGCGATGGGGGCGGGGGGGGGGGGGGGGCGCGCGCCAGGCGGG

GCGGGGCGGGGCGAGGGGCGGGGCGGGGCGAGGCGGAGAGGTGCGGCGGCAGC

CAATCAGAGCGGCGCGCTCCGAAAGTTTCCTTTTATGGCGAGGCGGCGGCGGCG

GCGGCCCTATAAAAAGCGAAGCGCGCGGCGGGCG

SEP ID NO:3 : Chicken beta-actin exon 1 and intron

GGAGTCGCTGCGACGCTGCCTTCGCCCCGTGCCCCGCTCCGCCGCCGCCTCGCGC

CGCCCGCCCCGGCTCTGACTGACCGCGTTACTCCCACAGGTGAGCGGGCGGGAC

GGCCCTTCTCCTCCGGGCTGTAATTAGCGCTTGGTTTAATGACGGCTTGTTTCTTT

TCTGTGGCTGCGTGAAAGCCTTGAGGGGCTCCGGGAGGGCCCTTTGTGCGGGGG

GGAGCGGCTCGGGGGGTGCGTGCGTGTGTGTGTGCGTGGGGAGCGCCGCGTGCG

GCCCGCGCTGCCCGGCGGCTGTGAGCGCTGCGGGCGCGGCGCGGGGCTTTGTGC

GCTCCGCAGTGTGCGCGAGGGGAGCGCGGCCGGGGGCGGTGCCCCGCGGTGCGG

GGGGGGCTGCGAGGGGAACAAAGGCTGCGTGCGGGGTGTGTGCGTGGGGGGGT

GAGCAGGGGGTATGGGCGCGGCGGTCGGGCTGTAACCCCCCCCTGCACCCCCCT

CCCCGAGTTGCTGAGCACGGCCCGGCTTCGGGTGCGGGGCTCCGTACGGGGCGT

GGCGCGGGGCTCGCCGTGCCGGGCGGGGGGTGGCGGCAGGTGGGGGTGCCGGG

CGGGGCGGGGCCGCCTCGGGCCGGGGAGGGCTCGGGGGAGGGGCGCGGCGGCC

CCCGGAGCGCCGGCGGCTGTCGAGGCGCGGCGAGCCGCAGCCATTGCCTTTTAT

GGTAATCGTGCGAGAGGGCGCAGGGACTTCCTTTGTCCCAAATCTGTGCGGAGCC

GAAATCTGGGAGGCGCCGCCGCACCCCCTCTAGCGGGCGCGGGGCGAAGCGGTG

CGGCGCCGGCAGGAAGGAAATGGGCGGGGAGGGCCTTCGTGCGTCGCCGCGCCG

CCGTCCCCTTCTCCCTCTCCAGCCTCGGGGCTGTCCGCGGGGGGACGGCTGCCTT

CGGGGGGGACGGGGCAGGGCGGGGTTCGGCTTCTGGCGTGTGACCGGCGGCTCT

AGAGCCTCTGCTAACCATGTTCATGCCTTCTTCTTTTTCCTACAGCTCCTGGGCAA

CGTGCTGGTTATTGTGCTGTCTCATCATTTTGGCAAAG In some embodiments, the FIG4 open reading frame is operably linked to an enhancer, e.g., a cytomegalovirus enhancer, e.g., an enhancer comprising, consisting essentially of, or consisting of the nucleotide sequence of SEQ ID NO:4 or a sequence at least about 70% identical thereto, e.g., at least about 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical thereto.

SEP ID NO:4: Cytomegalovirus enhancer

TGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAGTAACG

CCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCC

ACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAA

TGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTT

CCTACTTGGCAGTACATCT

In some embodiments, the FIG4 open reading frame is operably linked to a polyadenylation signal, e.g., a synthetic polyadenylation signal, e.g., a polyadenylation signal comprising, consisting essentially of, or consisting of the nucleotide sequence of SEQ ID NO:5 or a sequence at least about 70% identical thereto, e.g., at least about 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical thereto.

SEP ID NO:5: synthetic polyadenylation signal (SpA)

AATAAAGAGCTCAGATGCATCGATCAGAGTGTGTTGGTTTTTTGTGTG

Those skilled in the art will further appreciate that a variety of promoter/enhancer elements may be used depending on the level and tissue-specific expression desired. The promoter/enhancer may be constitutive or inducible, depending on the pattern of expression desired. The promoter/enhancer may be native or foreign and can be a natural or a synthetic sequence. By foreign, it is intended that the transcriptional initiation region is not found in the wild-type host into which the transcriptional initiation region is introduced.

Promoter/enhancer elements can be native to the target cell or subject to be treated and/or native to the heterologous nucleic acid sequence. The promoter/enhancer element is generally chosen so that it will function in the target cell(s) of interest. In representative embodiments, the promoter/enhancer element is a mammalian promoter/enhancer element. The promoter/enhance element may be constitutive or inducible. Inducible expression control elements are generally used in those applications in which it is desirable to provide regulation over expression of the heterologous nucleic acid sequence(s). Inducible promoters/enhancer elements for gene delivery can be tissue-specific or tissue-preferred promoter/enhancer elements, and include muscle specific or preferred (including cardiac, skeletal and/or smooth muscle), neural tissue specific or preferred (including brain-specific), eye (including retina-specific and cornea-specific), liver specific or preferred, bone marrow specific or preferred, pancreatic specific or preferred, spleen specific or preferred, and lung specific or preferred promoter/enhancer elements. Other inducible promoter/enhancer elements include hormone-inducible and metal-inducible elements. Exemplary inducible promoters/enhancer elements include, but are not limited to, a Tet on/off element, a RU486-inducible promoter, an ecdysone-inducible promoter, a rapamycin- inducible promoter, and a metallothionein promoter.

In embodiments wherein the FIG4 open reading frame is transcribed and then translated in the target cells, specific initiation signals are generally employed for efficient translation of inserted protein coding sequences. These exogenous translational control sequences, which may include the ATG initiation codon and adjacent sequences, can be of a variety of origins, both natural and synthetic.

In certain embodiments, the expression cassette further comprises at least one adeno- associated virus (AAV) inverted terminal repeat (ITR), e.g., two AAV ITRs. The two ITRs may have the same nucleotide sequence or different nucleotide sequences. The AAV ITRs may be from any AAV serotype, e.g., AAV2. Each ITR independently may be the wild-type sequence or a modified sequence. In some embodiments, a modified ITR may have a D- element deletion (WO 01/92551). A D-element deletion is defined as the removal of that portion of the ITR known as the D-element. The D-element can be alternatively referred to or known as a D region, or D sequence, and/or the nucleotides of the ITR that do not form palindromic hairpin structures. In some embodiments, the expression cassette is an AAV genome, e.g., a self-complementary AAV genome.

In certain embodiments, the expression cassette comprises an enhancer, a promoter, a human FIG4 open reading frame, and a polyadenylation site, optionally in the recited order. In certain embodiments, the expression cassette comprises an AAV ITR, an enhancer, a promoter, a human FIG4 open reading frame, a polyadenylation site, and an AAV ITR, optionally in the recited order. In certain embodiments, the expression cassette comprises a CMV enhancer, a chicken beta actin promoter, a human FIG4 open reading frame, and a synthetic polyadenylation site, optionally in the recited order. In certain embodiments, the expression cassette comprises a modified AAV ITR, a CMV enhancer, a chicken beta actin promoter, a human FIG4 open reading frame, a synthetic polyadenylation site, and a wild- type AAV ITR, optionally in the recited order. The aforementioned components are in operable linkage.

In some embodiments, the expression cassette comprise, consists essentially of, or consists of the nucleotide sequence of SEQ ID NO: 6 or a sequence at least about 70% identical thereto, e.g., at least about 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical thereto.

SEP ID NO:6 : FIG4 expression cassette

GGGGGGGGGGGGGGGGGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTG

AGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAG

TGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCT

AGATCTGAATTCGGTACCCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGA

CCGCCCAACGACCCCCGCCCATTGACGTCAATAGTAACGCCAATAGGGACTTTCC

ATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCA

AGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCC

GCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACA

TCTACGTATTAGTCATCGCTATTACCATGGTCGAGGTGAGCCCCACGTTCTGCTTC

ACTCTCCCCATCTCCCCCCCCTCCCCACCCCCAATTTTGTATTTATTTATTTTTTAA

TTATTTTGTGCAGCGATGGGGGCGGGGGGGGGGGGGGGGCGCGCGCCAGGCGGG

GCGGGGCGGGGCGAGGGGCGGGGCGGGGCGAGGCGGAAAGGTGCGGCGGCAGC

CAATCAGAGCGGCGCGCTCCGAAAGTTTCCTTTTATGGCGAGGCGGCGGCGGCG

GCGGCCCTATAAAAAGCGAAGCGCGCGGCGGGCGGGAGTCGCTGCGACGCTGCC

TTCGCCCCGTGCCCCGCTCCGCCGCCGCCTCGCGCCGCCCGCCCCGGCTCTGACT

GACCGCGTTACTCCCACAGGTGAGCGGGCGGGACGGCCCTTCTCCTCCGGGCTGT

A ATT AGCGCTTGGTTT AATGACGGCTT GTTTCTTTT CT GT GGCT GCGT GA AAGCCT

TGAGGGGCTCCGGGAGGGCCCTTTGTGCGGGGGGGAGCGGCTCGGGGGGTGCGT

GCGTGTGTGTGTGCGTGGGGAGCGCCGCGTGCGGCCCGCGCTGCCCGGCGGCTG

TGAGCGCTGCGGGCGCGGCGCGGGGCTTTGTGCGCTCCGCAGTGTGCGCGAGGG

GAGCGCGGCCGGGGGCGGTGCCCCGCGGTGCGGGGGGGGCTGCGAGGGGAACA

AAGGCTGCGTGCGGGGTGTGTGCGTGGGGGGGTGAGCAGGGGGTATGGGCGCGG CGGTCGGGCTGTAACCCCCCCCTGCACCCCCCTCCCCGAGTTGCTGAGCACGGCC

CGGCTTCGGGTGCGGGGCTCCGTACGGGGCGTGGCGCGGGGCTCGCCGTGCCGG

GCGGGGGGTGGCGGCAGGTGGGGGTGCCGGGCGGGGCGGGGCCGCCTCGGGCC

GGGGAGGGCTCGGGGGAGGGGCGCGGCGGCCCCCGGAGCGCCGGCGGCTGTCG

AGGCGCGGCGAGCCGCAGCCATTGCCTTTTATGGTAATCGTGCGAGAGGGCGCA

GGGACTTCCTTTGTCCCAAATCTGTGCGGAGCCGAAATCTGGGAGGCGCCGCCGC

ACCCCCTCTAGCGGGCGCGGGGCGAAGCGGTGCGGCGCCGGCAGGAAGGAAAT

GGGCGGGGAGGGCCTTCGTGCGTCGCCGCGCCGCCGTCCCCTTCTCCCTCTCCAG

CCTCGGGGCTGTCCGCGGGGGGACGGCTGCCTTCGGGGGGGACGGGGCAGGGCG

GGGTTCGGCTTCTGGCGTGTGACCGGCGGCTCTAGAGCCTCTGCTAACCATGTTC

ATGCCTTCTTCTTTTTCCTACAGCTCCTGGGCAACGTGCTGGTTATTGTGCTGTCT

CATCATTTTGGCAAAGACCGGTTCCGGACGCCACCATGCCAACTGCCGCCGCCCC

AATCATTTCATCAGTGCAAAAGCTTGTGCTCTACGAGACAAGAGCTCGGTATTTC

CTGGTCGGCTCCAACAACGCAGAAACCAAGTACAGAGTGCTTAAGATTGACCGC

ACCGAACCGAAGGACCTCGTGATTATTGACGATCGCCACGTGTACACTCAGCAA

GAAGTGCGCGAACTCTTGGGACGCCTGGACCTGGGCAACCGAACCAAAATGGGC

CAGAAGGGCTCCTCCGGTCTGTTTCGGGCAGTGTCGGCTTTCGGCGTCGTGGGAT

TCGTGCGATTCCTTGAGGGGTACTATATTGTGCTGATCACGAAGCGGCGCAAGAT

GGCCGACATCGGAGGACACGCTATATACAAGGTCGAGGACACCAACATGATCTA

CATTCCGAACGATTCAGTCAGAGTGACCCACCCCGATGAAGCCCGCTACCTTAGA

ATCTTCCAGAACGTGGATCTCTCCTCGAACTTTTACTTCTCATACTCGTACGACCT

GTCCCACTCGCTGCAGTACAACCTGACCGTGCTGCGGATGCCTCTGGAGATGCTC

AAGTCCGAGATGACTCAAAATCGGCAGGAATCCTTCGACATCTTCGAGGACGAG

GGTCTGATCACTCAAGGAGGGTCGGGAGTGTTCGGCATTTGCAGCGAACCCTAC

ATGAAATACGTGTGGAACGGAGAGCTCCTGGACATTATCAAGAGCACTGTGCAC

CGGGACTGGCTGCTGTACATCATCCATGGATTCTGTGGACAGTCAAAGCTCCTGA

TCTATGGCCGGCCTGTGTACGTGACTCTGATCGCCCGCCGCTCCTCGAAGTTCGC

GGGTACCCGGTTTCTCAAGCGCGGTGCTAACTGCGAGGGGGATGTGGCCAACGA

AGTGGAAACCGAGCAGATCCTCTGTGACGCCTCCGTGATGAGCTTTACCGCCGGA

TCCTACTCTTCGTATGTGCAAGTCCGGGGATCCGTCCCCCTGTACTGGAGCCAGG

ACATCTCCACTATGATGCCCAAGCCCCCTATCACCCTGGACCAGGCCGATCCTTT

CGCACACGTGGCTGCCCTGCACTTCGACCAGATGTTCCAGCGGTTCGGCTCCCCT

ATCATCATCCTGAACTTGGTCAAGGAACGGGAGAAGCGGAAGCATGAGAGGATT CTGTCCGAGGAGCTCGTGGCCGCGGTGACCTACCTGAATCAGTTCCTCCCGCCCG

AACATACCATCGTGTATATCCCCTGGGATATGGCCAAGTACACTAAGTCCAAGCT

GTGCAATGTGCTGGACCGCCTTAACGTCATCGCGGAATCCGTGGTCAAGAAAAC

CGGATTCTTCGTGAATAGACCGGATTCATACTGCTCCATTCTCCGGCCCGATGAA

AAGTGGAACGAACTGGGCGGTTGCGTCATCCCGACTGGCCGGCTCCAGACCGGC

ATCCTTAGGACCAACTGCGTGGACTGCCTGGACAGAACCAACACGGCCCAATTC

ATGGTCGGGAAATGTGCCCTGGCCTACCAACTGTACTCCCTGGGACTGATCGACA

AGCCGAACTTGCAATTCGATACTGACGCCGTGCGGCTGTTCGAAGAACTGTACGA

GGATCACGGCGACACCCTGAGCCTGCAGTACGGCGGAAGCCAGCTCGTGCATAG

AGTGAAAACCTACAGGAAGATTGCTCCGTGGACTCAGCACTCGAAGGACATCAT

GCAGACCCTCAGCCGCTACTACTCCAATGCCTTCTCCGACGCCGACCGCCAGGAC

TCCATTAACCTCTTCCTGGGAGTGTTCCACCCAACCGAGGGAAAGCCCCACCTGT

GGGAACTGCCTACTGATTTCTACTTGCATCACAAGAACACCATGAGACTCCTGCC

CACCCGGCGCTCCTACACTTACTGGTGGACCCCTGAAGTGATCAAGCACCTCCCG

CTGCCGTACGACGAGGTCATCTGCGCCGTGAACCTGAAAAAGCTGATCGTCAAA

AAGTTCCATAAATATGAAGAAGAAATCGACATTCACAACGAATTCTTCCGGCCAT

ACGAGCTGTCCTCGTTCGACGACACCTTTTGTCTGGCGATGACCTCATCCGCCCG

CGATTTCATGCCTAAGACTGTGGGGATCGACCCCAGCCCCTTCACCGTCCGCAAG

CCTGACGAGACTGGAAAGTCGGTGCTCGGCAACAAGAGCAACAGGGAAGAAGC

GGTGCTTCAGAGAAAGACTGCTGCCTCGGCGCCACCGCCGCCTTCCGAAGAAGC

CGTGTCGTCATCCTCGGAAGATGACAGCGGAACCGACCGCGAGGAGGAGGGGTC

CGTGAGCCAGCGGTCCACCCCGGTCAAGATGACTGACGCCGGGGACTCGGCCAA

GGTCACCGAAAACGTGGTGCAACCCATGAAGGAACTGTACGGCATCAACCTTAG

CGACGGCTTGTCTGAAGAGGACTTCTCCATCTACTCTCGGTTTGTGCAGCTGGGG

CAGAGCCAGCACAAGCAGGACAAGAATTCCCAACAGCCGTGCAGCAGATGCTCC

GACGGAGTGATTAAGCTGACTCCGATTAGCGCGTTCTCGCAAGATAACATCTACG

AAGTGCAACCCCCTCGCGTGGACAGGAAGTCCACCGAGATTTTCCAGGCCCACA

TCCAAGCATCCCAGGGAATCATGCAGCCCCTCGGGAAAGAGGACTCCTCCATGT

ACCGGGAGTACATCAGAAACCGCTACCTGTAGTAACTCGAGAATAAAGAGCTCA

GATGCATCGATCAGAGTGTGTTGGTTTTTTGTGTGACGCGTGCATGCTGGGGAGA

GATCTAGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCG

CTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGACCTTTGGTCGCCCG GCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACCCCCCCCCCCCC

CCCC

A further aspect of the invention relates to a vector comprising the polynucleotide or the expression cassette of the invention. Suitable vectors include, but are not limited to, a plasmid, phage, viral vector ( e.g ., an AAV vector, an adenovirus vector, a herpesvirus vector, an alphavirus vector, or a baculovirus vector), bacterial artificial chromosome (BAC), or yeast artificial chromosome (YAC). For example, the nucleic acid can comprise, consist of, or consist essentially of an AAV vector comprising a 5’ and/or 3’ terminal repeat (e.g., 5’ and/or 3’ AAV terminal repeat). In some embodiments, the vector is a delivery vehicle such as a particle (e.g., a microparticle or nanoparticle) or a liposome to which the expression cassette is attached or in which the expression cassette is embedded. The vector may be any delivery vehicle suitable to carry the expression cassette into a cell.

In some embodiments, the vector is a viral vector, e.g., an AAV vector. The AAV vector may be any AAV serotype, e.g., AAV9. In some embodiments, the AAV vector may comprise wild-type capsid proteins. In other embodiments, the AAV vector may comprise a modified capsid protein with altered tropism compared to a wild-type capsid protein, e.g., a modified capsid protein is liver-detargeted or has enhanced tropism for particular cells.

In some embodiments, the vector is a single-stranded AAV (ssAAV) vector. In some embodiments, the vector is a self-complementary or duplexed AAV (scAAV) vector. scAAV vectors are described in international patent publication WO 01/92551 (the disclosure of which is incorporated herein by reference in its entirety). Use of scAAV to express the FIG4 ORF may provide an increase in the number of cells transduced, the copy number per transduced cell, or both.

An additional aspect of the invention relates to a transformed cell comprising the polynucleotide, expression cassette, and/or vector of the invention. In some embodiments, the polynucleotide, expression cassette, and/or vector is stably incorporated into the cell genome. The cell may be an in vitro, ex vivo, or in vivo cell.

Another aspect of the invention relates to a transgenic animal comprising the polynucleotide, expression cassette, vector, and/or the transformed cell of the invention. In some embodiments, the animal is a laboratory animal, e.g, a mouse, rat, rabbit, dog, monkey, or non-human primate. A further aspect of the invention relates to a pharmaceutical formulation comprising the polynucleotide, expression cassette, vector, and/or transformed cell of the invention in a pharmaceutically acceptable carrier.

In a specific embodiment, the polynucleotide, expression cassette, vector, and/or transformed cell of the invention is isolated.

In another specific embodiment, the polynucleotide, expression cassette, vector, and/or transformed cell of the invention is purified.

Methods of Producing Virus Vectors

The present invention further provides methods of producing virus vectors. In one particular embodiment, the present invention provides a method of producing a recombinant AAV particle, comprising providing to a cell permissive for AAV replication: (a) a recombinant AAV template comprising (i) the polynucleotide or expression cassette of the invention, and (ii) an ITR; (b) a polynucleotide comprising Rep coding sequences and Cap coding sequences; under conditions sufficient for the replication and packaging of the recombinant AAV template; whereby recombinant AAV particles are produced in the cell. Conditions sufficient for the replication and packaging of the recombinant AAV template can be, e.g., the presence of AAV sequences sufficient for replication of the AAV template and encapsidation into AAV capsids (e.g., AAV rep sequences and AAV cap sequences) and helper sequences from adenovirus and/or herpesvirus. In particular embodiments, the AAV template comprises two AAV ITR sequences, which are located 5’ and 3’ to the polynucleotide of the invention, although they need not be directly contiguous thereto.

In some embodiments, the recombinant AAV template comprises an ITR that is not resolved by Rep to make duplexed AAV vectors as described in international patent publication WO 01/92551.

The AAV template and AAV rep and cap sequences are provided under conditions such that virus vector comprising the AAV template packaged within the AAV capsid is produced in the cell. The method can further comprise the step of collecting the virus vector from the cell. The virus vector can be collected from the medium and/or by lysing the cells.

The cell can be a cell that is permissive for AAV viral replication. Any suitable cell known in the art may be employed. In particular embodiments, the cell is a mammalian cell (e.g., a primate or human cell). As another option, the cell can be a trans-complementing packaging cell line that provide functions deleted from a replication-defective helper virus, e.g., 293 cells or other El a trans-complementing cells.

The AAV replication and capsid sequences may be provided by any method known in the art. Current protocols typically express the AAV replcap genes on a single plasmid. The AAV replication and packaging sequences need not be provided together, although it may be convenient to do so. The AAV rep and/or cap sequences may be provided by any viral or non-viral vector. For example, the replcap sequences may be provided by a hybrid adenovirus or herpesvirus vector (e.g., inserted into the El a or E3 regions of a deleted adenovirus vector). EBV vectors may also be employed to express the AAV cap and rep genes. One advantage of this method is that EBV vectors are episomal, yet will maintain a high copy number throughout successive cell divisions (i.e., are stably integrated into the cell as extra-chromosomal elements, designated as an "EBV based nuclear episome," see Margolski, (1992) Curr. Top. Microbiol. Immun. 158:67).

As a further alternative, the replcap sequences may be stably incorporated into a cell.

Typically the AAV replcap sequences will not be flanked by the TRs, to prevent rescue and/or packaging of these sequences.

The AAV template can be provided to the cell using any method known in the art. For example, the template can be supplied by a non-viral (e.g, plasmid) or viral vector. In particular embodiments, the AAV template is supplied by a herpesvirus or adenovirus vector (e.g, inserted into the El a or E3 regions of a deleted adenovirus). As another illustration, Palombo et al, (1998) J. Virology 72:5025, describes a baculovirus vector carrying a reporter gene flanked by the AAV TRs. EBV vectors may also be employed to deliver the template, as described above with respect to the replcap genes.

In another representative embodiment, the AAV template is provided by a replicating rAAV virus. In still other embodiments, an AAV provirus comprising the AAV template is stably integrated into the chromosome of the cell.

To enhance virus titers, helper virus functions (e.g., adenovirus or herpesvirus) that promote a productive AAV infection can be provided to the cell. Helper virus sequences necessary for AAV replication are known in the art. Typically, these sequences will be provided by a helper adenovirus or herpesvirus vector. Alternatively, the adenovirus or herpesvirus sequences can be provided by another non-viral or viral vector, e.g., as a non- infectious adenovirus miniplasmid that carries all of the helper genes that promote efficient AAV production as described by Ferrari et at. , (1997) Nature Med. 3:1295, and U.S. Patent Nos. 6,040,183 and 6,093,570.

Further, the helper virus functions may be provided by a packaging cell with the helper sequences embedded in the chromosome or maintained as a stable extrachromosomal element. Generally, the helper virus sequences cannot be packaged into AAV virions, e.g., are not flanked by ITRs.

Those skilled in the art will appreciate that it may be advantageous to provide the AAV replication and capsid sequences and the helper virus sequences {e.g, adenovirus sequences) on a single helper construct. This helper construct may be a non-viral or viral construct. As one nonlimiting illustration, the helper construct can be a hybrid adenovirus or hybrid herpesvirus comprising the AAV reptcap genes.

In one particular embodiment, the AAV reptcap sequences and the adenovirus helper sequences are supplied by a single adenovirus helper vector. This vector can further comprise the AAV template. The AAV reptcap sequences and/or the AAV template can be inserted into a deleted region {e.g, the Ela or E3 regions) of the adenovirus.

In a further embodiment, the AAV reptcap sequences and the adenovirus helper sequences are supplied by a single adenovirus helper vector. According to this embodiment, the AAV template can be provided as a plasmid template.

In another illustrative embodiment, the AAV reptcap sequences and adenovirus helper sequences are provided by a single adenovirus helper vector, and the AAV template is integrated into the cell as a pro virus. Alternatively, the AAV template is provided by an EBV vector that is maintained within the cell as an extrachromosomal element {e.g., as an EBV based nuclear episome).

In a further exemplary embodiment, the AAV reptcap sequences and adenovirus helper sequences are provided by a single adenovirus helper. The AAV template can be provided as a separate replicating viral vector. For example, the AAV template can be provided by a AAV particle or a second recombinant adenovirus particle.

According to the foregoing methods, the hybrid adenovirus vector typically comprises

/

the adenovirus 5’ and 3’ cis sequences sufficient for adenovirus replication and packaging {i.e., the adenovirus terminal repeats and PAC sequence). The AAV reptcap sequences and, if present, the AAV template are embedded in the adenovirus backbone and are flanked by the 5' and 3' cis sequences, so that these sequences may be packaged into adenovirus capsids. As described above, the adenovirus helper sequences and the AAV rep!cap sequences are generally not flanked by ITRs so that these sequences are not packaged into the AAV virions.

Zhang et ah, ((2001) Gene Ther. 18:704-12) describe a chimeric helper comprising both adenovirus and the AAV rep and cap genes.

Herpesvirus may also be used as a helper virus in AAV packaging methods. Hybrid herpesviruses encoding the AAV Rep protein(s) may advantageously facilitate scalable AAV vector production schemes. A hybrid herpes simplex virus type I (HSV-l) vector expressing the AAV-2 rep and cap genes has been described (Conway et ah, (1999) Gene Ther. 6:986 and WO 00/17377).

As a further alternative, the virus vectors of the invention can be produced in insect cells using baculovirus vectors to deliver the replcap genes and AAV template as described, for example, by Urabe et ah, (2002) Human Gene Ther. 13:1935-43.

AAV vector stocks free of contaminating helper virus may be obtained by any method known in the art. For example, AAV and helper virus may be readily differentiated based on size. AAV may also be separated away from helper virus based on affinity for a heparin substrate (Zolotukhin et ah (1999) Gene Therapy 6:973). Deleted replication-defective helper viruses can be used so that any contaminating helper virus is not replication competent. As a further alternative, an adenovirus helper lacking late gene expression may be employed, as only adenovirus early gene expression is required to mediate packaging of AAV. Adenovirus mutants defective for late gene expression are known in the art (e.g., tslOOK and tsl49 adenovirus mutants).

Methods of Using FIG4 Vectors

The present invention also relates to methods for delivering a FIG4 ORF to a cell or a subject to increase production of FIG4, e.g., for therapeutic or research purposes in vitro, ex vivo, or in vivo. Thus, one aspect of the invention relates to a method of expressing a FIG4 open reading frame in a cell, comprising contacting the cell with the polynucleotide, expression cassette, and/or the vector of the invention, thereby expressing the FIG4 open reading frame in the cell. In some embodiments, the cell is an in vitro cell, an ex vivo cell, or an in vivo cell.

Another aspect of the invention relates to a method of expressing a FIG4 open reading frame in a subject, comprising delivering to the subject the polynucleotide, expression cassette, vector, and/or transformed cell of the invention, thereby expressing the FIG4 open reading frame in the subject. In some embodiments, the subject is an animal model of a disorder associated with aberrant FIG4 gene expression.

A further aspect of the invention relates to a method of treating a disorder associated with aberrant expression of a FIG4 gene or aberrant activity of a FIG4 gene product in a subject in need thereof, comprising delivering to the subject a therapeutically effective amount of the polynucleotide, expression cassette, vector, and/or transformed cell of the invention, thereby treating the disorder associated with aberrant expression of the FIG4 gene or aberrant activity of a FIG4 gene product in the subject. The invention provides a method of treating a disorder associated with aberrant expression of a FIG4 gene or aberrant activity of a FIG4 gene product in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the polynucleotide, the expression cassette, vector, and/or transformed cell of the invention, such that the FIG4 open reading frame is expressed in the subject. In some embodiments, the disorder associated with expression of the FIG4 gene or gene product is CMT4J. In some embodiments, the disorder associated with expression of the FIG4 gene or gene product is Yunis-Varon syndrome. In some embodiments, the disorder associated with expression of the FIG4 gene or gene product is amyotrophic lateral sclerosis. In some embodiments, the disorder associated with expression of the FIG4 gene or gene product is pediatric-onset multiple sclerosis.

The invention further provides a method of treating Charcot-Marie-Tooth Neuropathy Type 4 J in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the polynucleotide, the expression cassette, vector, and/or transformed cell of the invention, such that the FIG4 open reading frame is expressed in the subject.

The invention further provides a method of treating Yunis-Varon syndrome in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the polynucleotide, the expression cassette, vector, and/or transformed cell of the invention, such that the FIG4 open reading frame is expressed in the subject.

The invention further provides a method of treating amyotrophic lateral sclerosis in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the polynucleotide, the expression cassette, vector, and/or transformed cell of the invention, such that the FIG4 open reading frame is expressed in the subject.

The invention further provides a method of treating pediatric-onset multiple sclerosis in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the polynucleotide, the expression cassette, vector, and/or transformed cell of the invention, such that the FIG4 open reading frame is expressed in the subject.

In certain embodiments, the polynucleotide, expression cassette, vector, and/or transformed cell is delivered to the subject, e.g., systemically (e.g, intravenously) or directly to the central nervous system (e.g, to the cerebrospinal fluid by intrathecal or intraventricular injection) of the subject. In some embodiments, the polynucleotide, expression cassette, vector, and/or transformed cell is delivered intravenously. In some embodiments, the polynucleotide, expression cassette, vector, and/or transformed cell is delivered intracerebroventricularly.

Recombinant virus vectors according to the present invention find use in both veterinary and medical applications. Suitable subjects include both avians and mammals. The term "avian" as used herein includes, but is not limited to, chickens, ducks, geese, quail, turkeys, pheasant, parrots, parakeets. The term "mammar as used herein includes, but is not limited to, humans, primates, non-human primates (e.g., monkeys and baboons), cattle, sheep, goats, pigs, horses, cats, dogs, rabbits, rodents (e.g., rats, mice, hamsters, and the like), etc. Human subjects include neonates, infants, juveniles, and adults. Optionally, the subject is "in need of' the methods of the present invention, e.g., because the subject has or is believed at risk for a disorder including those described herein or that would benefit from the delivery of a polynucleotide including those described herein. As a further option, the subject can be a laboratory animal and/or an animal model of disease. Preferably, the subject is a human.

In certain embodiments, the polynucleotide of the invention is administered to a subject in need thereof as early as possible in the life of the subject, e.g., as soon as the subject is diagnosed with aberrant FIG4 expression or activity or any of the above-mentioned diseases or disorders. In some embodiments, the polynucleotide is administered to a newborn subject, e.g., after newborn screening has identified aberrant FIG4 expression or activity. In some embodiments, the polynucleotide is administered to a fetus in utero, e.g., after prenatal screening has identified aberrant FIG4 expression or activity or the presence of one of the above-mentioned diseases or disorders. In some embodiments, the polynucleotide is administered to a subject as soon as the subject develops symptoms associated with aberrant FIG4 expression or activity or is suspected or diagnosed as having aberrant FIG4 expression or activity or one of the above-mentioned diseases or disorders. In some embodiments, the polynucleotide is administered to a subject before the subject develops symptoms associated with aberrant FIG4 expression or activity or disease/disorder, e.g., a subject that is suspected or diagnosed as having aberrant FIG4 expression or activity or one of the above-mentioned diseases or disorders but has not started to exhibit symptoms.

In particular embodiments, the present invention provides a pharmaceutical composition comprising a polynucleotide, expression cassette, vector, and/or transformed cell of the invention in a pharmaceutically acceptable carrier and, optionally, other medicinal agents, pharmaceutical agents, stabilizing agents, buffers, carriers, adjuvants, diluents, etc. For injection, the carrier will typically be a liquid. For other methods of administration, the carrier may be either solid or liquid. For inhalation administration, the carrier will be respirable, and will preferably be in solid or liquid particulate form.

By "pharmaceutically acceptable" it is meant a material that is not toxic or otherwise undesirable, i. e. , the material may be administered to a subject without causing any undesirable biological effects.

One aspect of the present invention is a method of transferring a FIG4 ORF to a cell in vitro. The polynucleotide, expression cassette, and/or vector of the invention may be introduced to the cells in the appropriate amount. The virus vector may be introduced to the cells at the appropriate multiplicity of infection according to standard transduction methods appropriate for the particular target cells. Titers of the virus vector or capsid to administer can vary, depending upon the target cell type and number, and the particular virus vector or capsid, and can be determined by those of skill in the art without undue experimentation. In particular embodiments, at least about 10 infectious units, more preferably at least about 10 infectious units are introduced to the cell.

The cell(s) into which the polynucleotide, expression cassette, and/or vector of the invention, e.g., virus vector, can be introduced may be of any type, including but not limited to neural cells (including cells of the peripheral and central nervous systems, in particular, brain cells such as neurons, oligodendrocytes, glial cells, astrocytes), lung cells, cells of the eye (including retinal cells, retinal pigment epithelium, and corneal cells), epithelial cells (e.g., gut and respiratory epithelial cells), skeletal muscle cells (including myoblasts, myotubes and myofibers), diaphragm muscle cells, dendritic cells, pancreatic cells (including islet cells), hepatic cells, a cell of the gastrointestinal tract (including smooth muscle cells, epithelial cells), heart cells (including cardiomyocytes), bone cells (e.g., bone marrow stem cells), hematopoietic stem cells, spleen cells, keratinocytes, fibroblasts, endothelial cells, prostate cells, joint cells (including, e.g, cartilage, meniscus, synovium and bone marrow), germ cells, and the like. Alternatively, the cell may be any progenitor cell. As a further alternative, the cell can be a stem cell ( e.g ., neural stem cell, liver stem cell). As still a further alternative, the cell may be a cancer or tumor cell. Moreover, the cells can be from any species of origin, as indicated above.

The polynucleotide, expression cassette, and/or vector of the invention, e.g., virus vector, may be introduced to cells in vitro for the purpose of administering the modified cell to a subject. In particular embodiments, the cells have been removed from a subject, the polynucleotide, expression cassette, and/or vector of the invention, e.g., virus vector, is introduced therein, and the cells are then replaced back into the subject. Methods of removing cells from subject for treatment ex vivo, followed by introduction back into the subject are known in the art (see, e.g., U.S. patent No. 5,399,346). Alternatively, the polynucleotide, expression cassette, and/or vector of the invention, e.g., virus vector, is introduced into cells from another subject, into cultured cells, or into cells from any other suitable source, and the cells are administered to a subject in need thereof.

Suitable cells for ex vivo gene therapy are as described above. Dosages of the cells to administer to a subject will vary upon the age, condition and species of the subject, the type of cell, the nucleic acid being expressed by the cell, the mode of administration, and the like. Typically, at least about 10 ² to about 10 ⁸ or about 10 ³ to about 10 ⁶ cells will be administered per dose in a pharmaceutically acceptable carrier. In particular embodiments, the cells transduced with the virus vector ex vivo are administered to the subject in an effective amount in combination with a pharmaceutical carrier.

A further aspect of the invention is a method of administering the polynucleotide, expression cassette, and/or vector of the invention, e.g., virus vector, to a subject. In particular embodiments, the method comprises a method of delivering a FIG4 ORF to an animal subject, the method comprising: administering an effective amount of a virus vector according to the invention to an animal subject. Administration of the virus vectors of the present invention to a human subject or an animal in need thereof can be by any means known in the art. Optionally, the virus vector is delivered in an effective dose in a pharmaceutically acceptable carrier.

Dosages of the virus vectors to be administered to a subject will depend upon the mode of administration, the disease or condition to be treated, the individual subject's condition, the particular virus vector, and the nucleic acid to be delivered, and can be determined in a routine manner. Exemplary doses for achieving therapeutic effects are virus titers of at least about 10 ² , 10 ³ , 10 ⁴ , 10 ⁵ , 10 ⁶ , 10 ⁷ , 10 ⁸ , 10 ⁹ , 10 ¹⁰ , 10 ¹¹ , 10 ¹² , 10 ¹³ , 10 ¹⁴ , 10 ¹⁵ , 10 ¹⁶ transducing units or more, e.g., about 10 ⁷ , 10 ⁸ , 10 ⁹ , 10 ¹⁰ , 10 ¹¹ , 10 ¹² , 10 ¹³ , 10 ¹⁴ , or 10 ¹⁵ transducing units, yet more preferably about 10 ¹⁰ , 10 ¹¹ , 10 ¹² , 10 ¹³ , 10 ¹⁴ , or 10 ¹⁵ transducing units. Doses and virus titer transducing units may be calculated as vector or viral genomes (vg).

In particular embodiments, more than one administration (e.g., two, three, four or more administrations) may be employed to achieve the desired level of gene expression over a period of various intervals, e.g., daily, weekly, monthly, yearly, etc.

Exemplary modes of administration include oral, rectal, transmucosal, topical, intranasal, inhalation (e.g., via an aerosol), buccal (e.g, sublingual), vaginal, intrathecal, intraocular, transdermal, in utero (or in ovo ), parenteral (e.g., intravenous, subcutaneous, intradermal, intramuscular [including administration to skeletal, diaphragm and/or cardiac muscle], intradermal, intrapleural, intracerebral, and intraarticular), topical (e.g., to both skin and mucosal surfaces, including airway surfaces, and transdermal administration), intro- lymphatic, and the like, as well as direct tissue or organ injection (e.g., to liver, skeletal muscle, cardiac muscle, diaphragm muscle or brain). Administration can also be to a tumor (e.g., in or a near a tumor or a lymph node). The most suitable route in any given case will depend on the nature and severity of the condition being treated and on the nature of the particular vector that is being used.

In some embodiments, the viral vector is administered to the CNS, the peripheral nervous system, or both.

In some embodiments, the viral vector is administered directly to the CNS, e.g., the brain or the spinal cord. Direct administration can result in high specificity of transduction of CNS cells, e.g., wherein at least 80%, 85%, 90%, 95% or more of the transduced cells are CNS cells. Any method known in the art to administer vectors directly to the CNS can be used. The vector may be introduced into the spinal cord, brainstem (medulla oblongata, pons), midbrain (hypothalamus, thalamus, epithalamus, pituitary gland, substantia nigra, pineal gland), cerebellum, telencephalon (corpus striatum, cerebrum including the occipital, temporal, parietal and frontal lobes, cortex, basal ganglia, hippocampus and amygdala), limbic system, neocortex, corpus striatum, cerebrum, and inferior colliculus. The vector may also be administered to different regions of the eye such as the retina, cornea or optic nerve. The vector may be delivered into the cerebrospinal fluid (e.g., by lumbar puncture) for more disperse administration of the vector. The delivery vector may be administered to the desired region(s) of the CNS by any route known in the art, including but not limited to, intrathecal, intracerebral, intraventricular, intranasal, intra-aural, intra-ocular (e.g., intra- vitreous, sub-retinal, anterior chamber) and peri-ocular (e.g., sub-Tenon's region) delivery or any combination thereof.

The delivery vector may be administered in a manner that produces a more widespread, diffuse transduction of tissues, including the CNS, the peripheral nervous system, and/or other tissues.

Typically, the viral vector will be administered in a liquid formulation by direct injection (e.g., stereotactic injection) to the desired region or compartment in the CNS and/or other tissues. In some embodiments, the vector can be delivered via a reservoir and/or pump. In other embodiments, the vector may be provided by topical application to the desired region or by intra-nasal administration of an aerosol formulation. Administration to the eye or into the ear, may be by topical application of liquid droplets. As a further alternative, the vector may be administered as a solid, slow-release formulation. Controlled release of parvovirus and AAV vectors is described by international patent publication WO 01/91803.

Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to injection, or as emulsions. Alternatively, one may administer the virus vector in a local rather than systemic manner, for example, in a depot or sustained-release formulation. Further, the virus vector can be delivered dried to a surgically implantable matrix such as a bone graft substitute, a suture, a stent, and the like (e.g., as described in U.S. Patent 7,201,898).

Pharmaceutical compositions suitable for oral administration can be presented in discrete units, such as capsules, cachets, lozenges, or tablets, each containing a predetermined amount of the composition of this invention; as a powder or granules; as a solution or a suspension in an aqueous or non-aqueous liquid; or as an oil-in-water or water-in-oil emulsion. Oral delivery can be performed by complexing a virus vector of the present invention to a carrier capable of withstanding degradation by digestive enzymes in the gut of an animal. Examples of such carriers include plastic capsules or tablets, as known in the art. Such formulations are prepared by any suitable method of pharmacy, which includes the step of bringing into association the composition and a suitable carrier (which may contain one or more accessory ingredients as noted above). In general, the pharmaceutical composition according to embodiments of the present invention are prepared by uniformly and intimately admixing the composition with a liquid or finely divided solid carrier, or both, and then, if necessary, shaping the resulting mixture. For example, a tablet can be prepared by compressing or molding a powder or granules containing the composition, optionally with one or more accessory ingredients. Compressed tablets are prepared by compressing, in a suitable machine, the composition in a free-flowing form, such as a powder or granules optionally mixed with a binder, lubricant, inert diluent, and/or surface active/dispersing agent(s). Molded tablets are made by molding, in a suitable machine, the powdered compound moistened with an inert liquid binder.

Pharmaceutical compositions suitable for buccal (sub-lingual) administration include lozenges comprising the composition of this invention in a flavored base, usually sucrose and acacia or tragacanth; and pastilles comprising the composition in an inert base such as gelatin and glycerin or sucrose and acacia.

Pharmaceutical compositions suitable for parenteral administration can comprise sterile aqueous and non-aqueous injection solutions of the composition of this invention, which preparations are optionally isotonic with the blood of the intended recipient. These preparations can contain anti-oxidants, buffers, bacteriostats and solutes, which render the composition isotonic with the blood of the intended recipient. Aqueous and non-aqueous sterile suspensions, solutions and emulsions can include suspending agents and thickening agents. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like.

The compositions can be presented in unit/dose or multi-dose containers, for example, in sealed ampoules and vials, and can be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example, saline or water-for- injection immediately prior to use.

Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules and tablets of the kind previously described. For example, an injectable, stable, sterile composition of this invention in a unit dosage form in a sealed container can be provided. The composition can be provided in the form of a lyophilizate, which can be reconstituted with a suitable pharmaceutically acceptable carrier to form a liquid composition suitable for injection into a subject. The unit dosage form can be from about 1 ig to about 10 grams of the composition of this invention. When the composition is substantially water- insoluble, a sufficient amount of emulsifying agent, which is physiologically acceptable, can be included in sufficient quantity to emulsify the composition in an aqueous carrier. One such useful emulsifying agent is phosphatidyl choline.

Pharmaceutical compositions suitable for rectal administration can be presented as unit dose suppositories. These can be prepared by admixing the composition with one or more conventional solid carriers, such as for example, cocoa butter and then shaping the resulting mixture.

Pharmaceutical compositions of this invention suitable for topical application to the skin can take the form of an ointment, cream, lotion, paste, gel, spray, aerosol, or oil. Carriers that can be used include, but are not limited to, petroleum jelly, lanoline, polyethylene glycols, alcohols, transdermal enhancers, and combinations of two or more thereof. In some embodiments, for example, topical delivery can be performed by mixing a pharmaceutical composition of the present invention with a lipophilic reagent (e.g, DMSO) that is capable of passing into the skin.

Pharmaceutical compositions suitable for transdermal administration can be in the form of discrete patches adapted to remain in intimate contact with the epidermis of the subject for a prolonged period of time. Compositions suitable for transdermal administration can also be delivered by iontophoresis (see, for example, Pharm. Res. 3:318 (1986)) and typically take the form of an optionally buffered aqueous solution of the composition of this invention. Suitable formulations can comprise citrate or bis\tris buffer (pH 6) or ethanol/water and can contain from 0.1 to 0.2M active ingredient.

The virus vectors disclosed herein may be administered to the lungs of a subject by any suitable means, for example, by administering an aerosol suspension of respirable particles comprised of the virus vectors, which the subject inhales. The respirable particles may be liquid or solid. Aerosols of liquid particles comprising the virus vectors may be produced by any suitable means, such as with a pressure-driven aerosol nebulizer or an ultrasonic nebulizer, as is known to those of skill in the art. See, e.g., U.S. Patent No. 4,501,729. Aerosols of solid particles comprising the virus vectors may likewise be produced with any solid particulate medicament aerosol generator, by techniques known in the pharmaceutical art. Having described the present invention, the same will be explained in greater detail in the following examples, which are included herein for illustration purposes only, and which are not intended to be limiting to the invention.

EXAMPLES

EXAMPLE 1: Identification of hFIG4opt efficacy in in vivo treatment of FIG4 deficiency.

The amino acid sequences of mouse, rat and monkey FIG4 were compared to the optimized human sequence using the Clustal Omega program. The results indicate that FIG4 protein sequence is conserved between species with high-level of amino acid identity (FIG. 2)·

The hFig4 DNA coding sequence of 2721 bp was placed between a 1612 bp chicken beta actin (CBA) promoter with a CMV enhancer and a 48-bp Synthetic polyadenylation (SpA) signal. The CBA promoter and SpA are utilized for their ability to both be small in size as well as drive strong expression allowing for packaging into an AAY vector [Gray et al. 2011 Hum. Gene Ther. 22:1143-1153]. The upstream inverted terminal repeat (ITR; proximal to the promoter) is from AAV2, with the D element deleted to promote packaging of a single- stranded (ss) genome. The downstream ITR (proximal to the poly A) is an intact WT AAV2 ITR.

Preclinical studies were conducted in a mouse model lacking FIG4 expression, which recapitulates the recessive human condition. These mice develop motor neuropathy and other aspects of the human disease, and in fact, the identification of the mouse mutation led to the identification of FIG4 as the causative gene for CMT4J. The human and mouse FIG4 proteins and activities are highly conserved, and preclinical studies conducted in the mouse used a codon optimized human FIG4 gene, delivered by AAV9.

The intra-CSF dose was administered to the animals either intracerebroventricularly (ICV) using a ½ cc insulin syringe, or intrathecally (IT) using a Hamilton® syringe. The age of mice at the initiation of the treatment, dose level and route of administration are presented in Table 4.

Treatment at each age included 5 cohorts: 1) Untreated Fig+/+ mice represent a healthy cohort, 2) AAV9/FIG4-injected Fig+/+ mice represent a non-disease phenotype to monitor safety of the gene therapy, 3) AAV9/FIG4-injected FIG4-/- mice to investigate the efficacy and safety of the gene therapy, 4) vehicle administered Fig4-/~ mice to represent the natural course of the disease, and 5) untreated Fig4-/- controls mice to monitor any effects from injection technique.

Table 4: Preclinical dose administration

Administering this vector directly to the nervous system by either ICV (Pl and P4) or IT (P7 and Pl l) injection produced benefit, as indicated by improved survival (FIG. 3), improved gross motor function (FIGS. 6 and 7) including grip strength (FIG. 5), improved peripheral motor nerve conduction (FIG. 8), and improved histopathological outcomes in spinal cord and dorsal root ganglia, as well as other parts of the central nervous system (FIGS. 9, 10, and 11). These outcomes show that the gene replacement is effective in compensating for the FIG4 loss of function mutation. Importantly, our results do indicate that the benefit is greatest the earlier the FIG4-AAV9 is delivered. Our results also indicate a clear dose-response of the treatment up to the maximum feasible dose in P7 mice, justifying the maximum feasible dose to provide the most effective treatment (FIG. 12). In addition, no adverse effects, either overtly or by histopathological examination, were observed in Fig4 mutant or littermate control mice when treated with the vector (FIGS. 3 and 4).

All references cited herein are incorporated herein by reference in their entireties and for all purposes to the same extent as if each individual publication or patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety for all purposes.

The foregoing examples are illustrative of the present invention, and are not to be construed as limiting thereof. Although the invention has been described in detail with reference to preferred embodiments, variations and modifications exist within the scope and spirit of the invention as described and defined in the following claims.