WO2012109214A1 - Transduction ciblée de vecteurs aav - Google Patents

Transduction ciblée de vecteurs aav Download PDF

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Publication number
WO2012109214A1
WO2012109214A1 PCT/US2012/024108 US2012024108W WO2012109214A1 WO 2012109214 A1 WO2012109214 A1 WO 2012109214A1 US 2012024108 W US2012024108 W US 2012024108W WO 2012109214 A1 WO2012109214 A1 WO 2012109214A1
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cell
aav
aav vector
desialylating
vector
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Aravind Asokan
Shen SHEN
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University of North Carolina at Chapel Hill
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University of North Carolina at Chapel Hill
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/0008—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/0075—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the delivery route, e.g. oral, subcutaneous
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14141—Use of virus, viral particle or viral elements as a vector
    • C12N2750/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • This invention relates to methods of enhancing transduction of cells with adeno-associated virus (AAV) vectors that bind asialoglycans on the cell surface and methods of targeting cells with these vectors by desialyiating cells.
  • the invention further relates to methods, compositions and kits for delivering nucleic acids to cells using the AAV vectors.
  • AAV adeno-associated virus
  • heparan sulfate serves as a primary receptor for Herpesviridae (Akhtar et al., FEBS J. 276:7228 (2009)) as well as certain adenoviruses (Dechecchi et al., J. Virol. 75:8772 (2001)) and parvoviruses
  • AAV are small, single- stranded DNA viruses that belong to the genus Dependovirus of the Parvoviridae family (Bowles et al, (2006) The genus dependovirus. In Parvoviruses pp. 15-24. Edited by J. R. Kerr, S. F. Cotmore, M. E. Bloom et al., New York, Edward Arnold Ltd.). Recombinant AAV vectors, by virtue of their lack of pathogenicity and low immunogenicity, are currently being evaluated as lead candidates in clinical gene therapy trials (Mueller et al, Gene Ther. 75:858 (2008)).
  • AAV4 Kaludov et al, J. Virol 75:6884 (2001)
  • AAV5 Wang et al, J. Biol. Chem. 276:206) 0 (2001)
  • AAV1/6 Wang et al., J. Virol. 50:9093 (2006)
  • Bovine AAV Schot et al, J. Virol. 80:5516 (2006)
  • Sialylated glycans that serve as primary receptors for the latter AAV strains vary at the level of N-acetylneuraminic acid (NeuSAc) linkage to underlying sugars, i.e., a2-3 or 2-6 linked to galactose residues (Wu et al, J. Virol 50:9093 (2006); Kaludov et al, J. Virol 75:6884 (2001)). Further receptor specificity has been demonstrated at the level of N-lmked or CMinked glycans displayed on the cell surface (Wu et al, J. Virol 50:9093 (2006); Kaludov et al, J. Virol 75:6884 (2001)).
  • NeuSAc N-acetylneuraminic acid
  • binding of the AAV vector to the cell is increased relative to binding to a cell that has not been desialylated.
  • Another aspect of the invention relates to a method of targeting an AAV vector that binds asialoglycans to a cell, comprising:
  • a further aspect of the invention relates to a method of restricting AAV vectors that bind asialoglycans to the site of delivery in a subject, comprising:
  • AAV vector is targeted to desialylated cells and systemic dissemination of the AAV vector is restricted.
  • a further aspect of the invention relates to a method of delivering a nucleic acid to a mammalian subject, comprising delivering to the mammalian subject a desialylating agent and an AAV vector that binds asialoglycans and comprises the nucleic acid.
  • An additional aspect of the invention relates to a composition
  • a composition comprising an AAV vector that binds asialoglycans and a desialylating agent.
  • kits comprising an AAV vector that binds asialoglycans and a desialylating agent.
  • Figures 1A-1C show the effect of enzymatic desialylation on AAV9 transduction.
  • Figure 2 shows the effect of enzymatic desialylation on AAV9 binding to different human cell lines.
  • Figure 3 shows the effect of desialylation on the internalization of AAV1 and AAV9 in U87 cells.
  • Figures 4A-4C show the effect of glycan chain composition on cell surface binding and transduction of AAV9.
  • Figures 5A-5C show the effect of glycosylation inhibitors and enzymatic resialylation on AAV9 transduction.
  • Figures 6A-6C show the effect of lectin competition on AAV9 transduction.
  • Figures 7A-7B show the effect of endo- -galactosidase and a-fucosidase on AAV9 transduction efficiency.
  • Figures 10A-10D show the effect of enzymatic desialylation on AAV9 transduction in murine airways in vivo.
  • Figure 11 shows the inhibition of AAV9 infectivity by different glycans.
  • Figures 12A-12B show localized sialidase pretreatment increases AAV9 transgene expression at a low dose of AAV9-CBA-luciferase in joints.
  • Figures 14A-14B show intravitreous sialidase pretreatment increases AAV9 transgene expression in retina.
  • Figure 15 shows galactose levels and viral particle levels in endothelial cells after intravenous administration of sialidase and AAV9.
  • Figures 16A-16E show increased liver transduction efficiency after intravenous injection of sialidase and AAV9.
  • Parvoviridae including autonomously-replicating parvoviruses and dependoviruses.
  • the autonomous parvoviruses include members of the genera Parvovirus, Erythrovirus, Densovirus, Ileravirus, 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, HI parvovirus, muscovy duck parvovirus, snake parvovirus, and B 19 virus (See, e.g., Figs. 20-23).
  • 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., Figs. 8-19; FIELDS et al. VIROLOGY, volume 2, chapter 69 (4th ed., Lippincott-Raven Publishers); and Table 1.
  • AAV adeno-associated viruses
  • AAV adeno-associated virus
  • AAV includes but is 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, snake AAV, avian AAV, bovine AAV, canine AAV, equine AAV, ovine AAV, goat AAV, shrimp AAV, and any other AAV now known or later discovered. See, e.g., FIELDS et al. VIROLOGY, volume 2, chapter 69 (4th ed., Lippincott-Raven Publishers).
  • the term "tropism” as used herein refers to entry of the virus into the cell, optionally 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 sequences(s).
  • 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.
  • gene expression from the viral genome may be from a stably integrated provirus, from a non-integrated episome, as well as any other form in which the virus may take within the cell.
  • transduction or “infection” of a cell by a parvovirus or AAV means that the parvovirus/AAV enters the cell to establish an active (i.e., lytic) infection.
  • transduction of a cell by AAV means that the AAV enters the cell to establish a latent infection. See, e.g., FIELDS et al. VIROLOGY, volume 2, chapter 69 (3d ed., Lippincott-Raven Publishers).
  • a "3 1 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.
  • 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.
  • polypeptide encompasses both peptides and proteins, unless indicated otherwise.
  • a "polynucleotide” is a sequence of nucleotide bases, and may be RNA, DNA or DNA-RNA hybrid sequences (including both naturally occurring and non- naturally occurring nucleotide), and can be either single or double stranded.
  • 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 35:351 (1987); the method is similar to that described by Higgins & Sharp, CABIOS 5 5 ⁇ (1989).
  • BLAST algorithm 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.
  • 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).
  • percent nucleic acid sequence identity with respect to the coding sequence of the polypeptides disclosed herein is defined as the percentage of nucleotide residues in the candidate sequence that are identical with the nucleotides in the polynucleotide specifically disclosed herein.
  • 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.
  • an "isolated" polynucleotide e.g., an “isolated DNA” or an “isolated RNA" means a polynucleotide 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 polynucleotide.
  • 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.
  • an isolated polypeptide is one that is at least about 50, 60, 70, 80, 90, or 95% pure or higher.
  • treat By the terms “treat,” “treating” or “treatment of (and grammatical variations thereof) it is meant that the severity of the subject's condition is reduced, at least partially improved or stabilized and/or that some alleviation, mitigation, decrease or stabilization in at least one clinical symptom is achieved and/or there is a delay in the progression of the disease or disorder.
  • 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.
  • prevention effective amount 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.
  • heterologous nucleotide sequence and “heterologous nucleic acid” are used interchangeably herein and refer to a sequence that is not naturally occurring in the virus.
  • the heterologous nucleic acid comprises an open reading frame that encodes a polypeptide or nontranslated RNA of interest (e.g., for delivery to a cell or subject).
  • virus vector refers to a virus (e.g., AAV) particle that functions as a nucleic acid delivery vehicle, and which comprises the vector genome (e.g., viral DNA [vDNA]) packaged within a virion.
  • vector may be used to refer to the vector genome/vDNA alone.
  • 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).
  • double stranded (duplex) genomes can be packaged into the virus capsids of the invention.
  • a "rAAV vector genome” or "rAAV genome” is an AAV genome (i. e. , vDNA) that comprises one or more heterologous nucleic acid sequences.
  • rAAV vectors generally require only the 145 base ITR in cis to generate virus. All other viral sequences are dispensable and may be supplied in trans (Muzyczka (1 92) Curr. Topics Microbiol.
  • the rAAV vector genome will only retain the one or more ITR sequence so as to maximize the size of the transgene that can be efficiently packaged by the vector.
  • the structural and non-structural protein coding sequences may be provided in trans (e.g., from a vector, such as a plasmid, or by stably integrating the sequences into a packaging cell).
  • the rAAV vector genome comprises at least one ITR sequence (e.g., AAV ITR sequence), optionally two ITRs (e.g., two AAV ITRs), which typically will be at the 5' and 3' ends of the vector genome and flank the heterologous nucleic acid, but need not be contiguous thereto.
  • the ITRs can be the same or different from each other.
  • 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 pro virus rescue, and the like).
  • the ITR can be an AAV ITR or a non- AAV ITR.
  • a non-AAV ITR sequence such as those of other parvoviruses (e.g., canine parvovirus, bovine parvovirus, mouse parvovirus, porcine parvovirus, human parvovirus B-19) or the SV40 hairpin that selves as the origin of SV40 replication can be used as an ITR, which can further be modified by truncation, substitution, deletion, insertion and/or addition.
  • the ITR 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
  • 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 inverted terminal repeat" or "AAV ITR” may be from any AAV, including but not limited to serotypes 1, 2, 3a, 3b, 4, 5, 6, 7, 8, 9, 10, 11, or 13, snake AAV, avian AAV, bovine AAV, canine AAV, equine AAV, ovine AAV, goat AAV, shrimp AAV, or any other AAV now known or later discovered (see, e.g., Table 1).
  • the AAV ITR is from a clade F AAV.
  • An AAV ITR need not have the native terminal repeat sequence (e.g., a native AAV ITR 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 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 ai, (2000) Mot Therapy 2:619.
  • targeted virus vectors e.g. , having a directed tropism
  • a “hybrid” parvovirus i.e., in which the viral ITRs and viral capsid are from different parvoviruses
  • viral capsid or genomic elements can contain other modifications, including insertions, deletions and/or substitutions.
  • amino acid encompasses any naturally occurring amino acids, modified forms thereof, and synthetic amino acids.
  • 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, methyl ation, phosphorylation or sulfatation).
  • post-translation modification e.g., acetylation, amidation, formylation, hydroxylation, methyl ation, phosphorylation or sulfatation.
  • 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).
  • BAC bacterial artificial chromosome
  • YAC yeast artificial chromosome
  • a viral vector e.g., adenovirus, herpesvirus, Epstein-Barr Virus, AAV, baculoviral, retroviral vectors, and the like.
  • the template may be stably incorporated into the chromosome of a packaging cell.
  • 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.
  • 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.
  • 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).
  • 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.
  • 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 f om any 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).
  • the large Rep protein may be from a clade F AAV.
  • a synthetic large Rep protein may be altered by insertion, deletion, truncation and/or missense mutations.
  • small Rep protein refers to Rep52 and/or Rep40.
  • Small Rep proteins of the claimed invention may be either wild-type or synthetic.
  • a wild-type small Rep protein may be from any 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).
  • the small Rep protein may be from a clade F AAV.
  • a synthetic small Rep protein may be altered by insertion, deletion, truncation and/or missense mutations.
  • the replication proteins be encoded by the same polynucleotide.
  • the NS-1 and NS-2 proteins (which are splice variants) may be expressed independently of one another.
  • the p 19 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.
  • the viral promoters may not be recognized by the cell, and it is therefore convenient to express the large and small Rep proteins from separate expression cassettes.
  • AAV vector that binds asialoglycans refers to AAV vectors that bind to asialoglycans on the cell surface as compared to sialoglycans. In certain embodiments, the AAV vector preferentially binds asialoglycans.
  • the term includes naturally occurring AAV particles that bind to asialoglycans as well as AAV vectors that have been engineered to bind to asialoglycans. The ability of an AAV vector to bind to asialoglycans can be quant ated by any method known in the art and as described in the examples below.
  • asialoglycans refers to glycans that do not have a terminal sialic acid group.
  • sialylating agent refers to any compound, molecule, or environmental condition that removes sialyl groups from sialoglycans on the surface of a cell, prevents the synthesis of sialoglycans, and/or prevents the transportation of sialoglycans to the cell surface.
  • the present invention is based on the discovery that Clade F AAV vectors target cells having asialoglycans on the surface and that the presence of sialoglycans on cells inhibits the binding of Clade F AAV vectors to the cells.
  • the present invention provides methods for targeting AAV vectors that bind asialoglycans to cells by desialylating the cells to remove some or substantially all of the sialyl groups on the cell surface.
  • One aspect of the invention relates to a method for increasing transduction of a cell with an adeno-associated virus (AAV) vector that binds asialoglycans, comprising:
  • control cell can be a cell of the same type and/or location as the desialylated cell but has not undergone a desialylation step.
  • the AAV vector is from clade F, e.g., AAV9 (also known as Hu.14), Hu.31 or Hu.32.
  • the AAV vector is a chimeric or hybrid vector comprising a viral genome from any serotype of AAV and a capsid from a clade F AAV.
  • the chimeric or hybrid AAV comprises a capsid made up of at least a portion of a capsid protein from a clade F AAV, e.g., a portion of the capsid protein that provides binding specificity to asialoglycans.
  • the AAV vector is an engineered vector capable of binding asialoglycans.
  • the vector may comprise a modified capsid that has been identified as capable of binding asialoglycans, e.g., by screening capsid protein mutant libraries for binding ability.
  • the engineered vector comprises all or a portion of a naturally-occurring capsid protein that has been identified as capable of binding asialoglycans.
  • the desialylating step comprises removing a portion of the sialyl groups (e.g., one or more sialyl groups but not all of the sialyl groups) from the termini of the cell surface glycans.
  • substantially all of the sialyl groups are removed.
  • the term "substantially all” refers to the removal of at least about 80% of the sialyl groups on the cell surface, e.g., at least about 85, 90, 95, 96, 97, 98, or 99% of the sialyl groups.
  • the cell to be desialylated can be any cell that is to be targeted for AAV transduction.
  • the cell is one that is known in the art to contain or is discovered to contain sialyl groups on the termini of one or more of the cell surface glycans.
  • the cell is an in vitro cell, e.g., a cell in culture.
  • the in vitro cell may be from an established cell line (e.g., CHO, COS, HEK293, U87, Huh-7, or Neuro2a cells) or a primary cell that has been isolated from a subject and cultured.
  • the cell is an ex vivo cell.
  • the ex vivo cell may be one that has been isolated from a subject with the objective of returning the cell back to the subject from which it was isolated or to a different subject, e.g., after the cell has been modified, such as by transduction with an AAV vector.
  • the cell is an in vivo cell, i.e., one that is present in a subject.
  • the subject may be, for example, a patient in need of treatment or prevention of a disorder or an animal model for use in research.
  • the in vitro, ex vivo, or in vivo cell may be any type of cell for which transduction is desired.
  • the cell may be selected from the group consisting of a dendritic cell, T cell, B cell, neural cell, muscle cell, pancreatic cell, hepatic cell, lung cell, retinal cell, epithelial cell, smooth muscle cell, skeletal muscle cell, diaphragm muscle cell, cardiac muscle cell, kidney cell, myocardial cell, bone cell, spleen cell, keratinocyte, fibroblast, endothelial cell, prostate cell, germ cell, progenitor cell, and stem cell.
  • a dendritic cell T cell, B cell, neural cell, muscle cell, pancreatic cell, hepatic cell, lung cell, retinal cell, epithelial cell, smooth muscle cell, skeletal muscle cell, diaphragm muscle cell, cardiac muscle cell, kidney cell, myocardial cell, bone cell, spleen cell, keratinocyte, fibroblast, endothelial cell, prostate cell, germ cell, progenitor cell, and stem cell.
  • the desialylating step can be carried out before, during, and/or after the step of contacting the cell with the AAV vector.
  • the cell can be contacted with a desialylating agent or genetically modified to prevent the production of sialoglycans prior to the cell being contacted with the AAV vector, e.g., about 0.25, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, or 24 hours or more prior to the cell being contacted with the AAV vector.
  • the cell is contacted with the desialylating agent and the AAV vector concurrently.
  • the word "concurrently” means sufficiently close in time to produce a combined effect (that is, concurrently can be simultaneously, or it can be two or more events occurring within a short time period before or after each other).
  • the desialylating agent and the AAV vector are present in the same composition that contacts the cell.
  • the desialylating agent and the AAV vector are in separate compositions that contact the cell concurrently.
  • the cell is contacted with the AAV vector and then contacted with a desialylating agent, e.g., about 0.25, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, or 24 hours or more after the cell is contacted with the AAV vector.
  • the desialylation of a cell comprising sialyl groups can be carried out by any means known in the art. In some embodiments, the desialylation is carried out by removing sialyl groups that are present on the cell surface. In one embodiment, the desialylation is carried out enzymatically using an enzyme known in the art to remove sialyl groups from sialoglycans. In one embodiment, the enzyme is a neuraminidase, e.g., neuraminidase type III from Vibrio cholerae or 2,3,6,8-neuraminidase from Arihrobacter ureafaciens.
  • neuraminidase e.g., neuraminidase type III from Vibrio cholerae or 2,3,6,8-neuraminidase from Arihrobacter ureafaciens.
  • the enzyme is a sialidase, e.g., a bacterial sialidase such as those from Clostridium perfringens, Actinomyces viscosus, Arihrobacter ureafaciens, or Micromonospora viridifaciens, or a mammalian sialidase such as those encoded by the genes NEU2 and NEU4.
  • a sialidase e.g., a bacterial sialidase such as those from Clostridium perfringens, Actinomyces viscosus, Arihrobacter ureafaciens, or Micromonospora viridifaciens
  • a mammalian sialidase such as those encoded by the genes NEU2 and NEU4.
  • the desialylation is carried out chemically.
  • the cell can be contacted with an inhibitor of a sialic acid transporter or sialyltransferase, e.g., swainsonine, soyasaponin, and a-benzyl-O-GalNAc, to inhibit the synthesis of sialoglycans.
  • the sialyltransferase can be, for example, a2,3-(N)- sialyltransferase, 2,6-(N)-sialyltransferase, or a2, 3 -(O)- sialyltransferase.
  • existing sialoglycans can be desialylated under appropriate environmental conditions, such as mildly acidic conditions.
  • the coding and noncoding nucleotide sequences for sialic acid transporters and sialyltransferases are known to those of skill in the art and are readily available in sequence databases such as GenBank.
  • An antisense, siRNA, microRNA, or ribozyme nucleotide sequence or nucleic acid encoding an antisense, siRNA, microRNA, or ribozyme nucleotide sequence can be generated to any portion thereof in accordance with known techniques.
  • the antisense nucleotide sequence can be complementary to the entire nucleotide sequence encoding the polypeptide or a portion thereof of at least 10, 20, 40, 50, 75, 100, 150, 200, 300, or 500 contiguous bases and will reduce the level of polypeptide production.
  • the antisense nucleotide sequence be fully complementary to the target sequence as long as the degree of sequence similarity is sufficient for the antisense nucleotide sequence to hybridize to its target and reduce production of the polypeptide.
  • a higher degree of sequence similarity is generally required for short antisense nucleotide sequences, whereas a greater degree of mismatched bases will be tolerated by longer antisense nucleotide sequences.
  • An antisense nucleotide sequence can be constructed using chemical synthesis and enzymatic ligation reactions by procedures known in the art.
  • an antisense nucleotide sequence can be chemically synthesized using naturally occurring nucleotides or various modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed between the antisense and sense nucleotide sequences, e.g. , phosphorothioate derivatives and acridine substituted nucleotides can be used.
  • the antisense nucleotide sequences further include nucleotide sequences wherein at least one, or all, of the internucleotide bridging phosphate residues are modified phosphates, such as methyl phosphonates, methyl phosphonothioates, phosphoromorpholidates, phosphoropiperazidates and phosphoramidates. For example, every other one of the internucleotide bridging phosphate residues can be modified as described.
  • the antisense nucleotide sequence is a nucleotide sequence in which one, or all, of the nucleotides contain a 2' lower alkyl moiety (e.g., C1 -C4, linear or branched, saturated or unsaturated alkyl, such as methyl, ethyl, ethenyl, propyl, 1-propenyl, 2-propenyl, and isopropyl).
  • a 2' lower alkyl moiety e.g., C1 -C4, linear or branched, saturated or unsaturated alkyl, such as methyl, ethyl, ethenyl, propyl, 1-propenyl, 2-propenyl, and isopropyl.
  • every other one of the nucleotides can be modified as described. See also, Furdon et ah, Nucleic Acids Res. 17:9193 (1989); Ag wal et al. , Proc. Natl. Aca
  • RNA also known as RNA interference (RNAi) . molecules
  • siRNA is a mechanism of post-transcriptional gene silencing in which double-stranded RNA (dsRNA) corresponding to a coding sequence of interest is introduced into a cell or an organism, resulting in degradation of the corresponding mRNA.
  • dsRNA double-stranded RNA
  • the mechanism by which siRNA achieves gene silencing has been reviewed in Sharp et al. , Genes Dev. 75:485 (2001); and Hammond et a , Nature Rev. Gen. 2:110 (2001)).
  • the siRNA effect persists for multiple cell divisions before gene expression is regained.
  • siRNA is therefore a powerful method for making targeted knockouts or "knockdowns" at the RNA level.
  • siRNA has proven successful in human cells, including human embryonic kidney and HeLa cells ⁇ see, e.g., Elbashir et al, Nature 411:494 (2001)).
  • silencing can be induced in mammalian cells by enforcing endogenous expression of RNA hairpins (see Paddison et al, Proc. Natl Acad. Sci. USA i3 ⁇ 4>: 1443 (2002)).
  • transfection of small (21-23 nt) dsRNA specifically inhibits nucleic acid expression (reviewed in Caplen, Trends Biotechnol. 20:49 (2002)).
  • MicroRNA single stranded RNA molecules of about 21-23 nucleotides in length, can be used in a similar fashion to siRNA to modulate gene expression (see U.S. Patent No. 7,217,807).
  • Ribozymes are RNA-protein complexes that cleave nucleic acids in a site-specific fashion. Ribozymes have specific catalytic. domains that possess endonuclease activity (Kim et l, Proc. Natl Acad. Sci. USA 54:8788 (1987); Gerlach etal, Nature 325:802 (1987); Forster and Symons, Cell 49:2 ⁇ (1987)).
  • ribozymes accelerate phosphoester transfer reactions with a high degree of specificity, often cleaving only one of several phosphoesters in an oligonucleotide substrate (Michel and Westhof, J. Mol. Biol. 216:585 (1990); Reinhold-Hurek and Shub, Nature 357:173 (1992)).
  • This specificity has been attributed to the requirement that the substrate bind vi specific base-pairing interactions to the internal guide sequence ("IGS") of the ribozyme prior to chemical reaction.
  • IGS internal guide sequence
  • Ribozyme catalysis has primarily been observed as part of sequence- specific cleavage/ligation reactions involving nucleic acids (Joyce, Nature 338:217 (1989)).
  • U.S. Patent No. 5,354,855 reports that certain ribozymes can act as endonucleases with a sequence specificity greater than that of known ribonucleases and approaching that of the DNA restriction enzymes.
  • sequence- specific ribozyme- mediated inhibition of gene expression may be particularly suited to therapeutic applications (Scanlon et al. , Proc. Natl. Acad. Sci. USA 55:10591 (1991); Sarver ei a/., Science 247:1222 (1990); Sioud et al., J. Mot Biol. 225:831 (1992)).
  • the methods of the present invention can be used to target an AAV vector to a specific cell, tissue, or region in vitro, ex vivo, or in vivo, e.g., by desialylating the specific cell, tissue, or region and then contacting the desialylated cell, tissue, or region with an AAV vector.
  • a method of targeting an AAV vector that binds asialoglycans to a cell comprising:
  • binding of the AAV vector to the cell is increased relative to binding to a suitable control cell, e.g., a cell that has not been desialylated.
  • the cell is a cultured cell.
  • the cell is present in a subject in a specific tissue or region, e.g., selected from the group consisting of eye, ear, nose, joints, thymus, spleen, kidney, lungs, liver, heart, spinal cord, brain, subarachnoid cisternae, ventricles, skeletal muscle, diaphragm, gastrointestinal tract, and pancreas.
  • the method comprises delivering a desialylating agent and the AAV vector to the specific tissue or region.
  • the desialylating agent can be any of the enzymatic, chemical, or genetic agents described above.
  • the desialylating agent is delivered prior to the AAV vector, in another embodiment, the desialylating agent is delivered concurrently with the AAV vector. In a further embodiment, the desialylating agent is delivered after the AAV vector.
  • the desialylating agent and the AAV vector are delivered in one composition. In other embodiments, the desialylating agent and the AAV vector are delivered in separate compositions.
  • the methods of the present invention can be used advantageously in the delivery of AAV vectors to subjects, e.g., for the expression of therapeutic, prophylactic, or diagnostic proteins or polynucleotides.
  • the AAV vectors of the invention may be preferentially targeted to the desialylated cells, tissues, or regions, thereby limiting the spread of the vectors from the site of delivery and minimizing systemic dissemination of the vectors.
  • the site of delivery is selected from the group consisting of spinal column, lung, eye, ear, joints, nose, cranium, subarachnoid cisternae, ventricles, myocardium, pancreatic duct, and intraportal vein.
  • systemic dissemination of the AAV vector is restricted is defined as the presence of less than about 25% (e.g., less that about 20%, 15%, 10%, or 5%) of the delivered AAV vectors (or a nucleic acid delivered by the vector) in the plasma or in a tissue remote from the site of delivery at any time after the AAV vector is delivered to the subject.
  • AAV vector is delivered intrathecally to the central nervous system, less than about 25% of the delivered AAV vectors can be detected circulating in the blood.
  • AAV vectors that binds asialoglycans can be produced by methods well known in the art and described below.
  • the vectors can be produced by a method comprising providing to a cell permissive for AAV replication: (a) a recombinant AAV template comprising (i) a heterologous nucleotide sequence, and (if) AAV ITRs; and (b) a polynucleotide encoding a Rep and Cap proteins; under conditions sufficient for the replication and packaging of the recombinant AAV template; whereby recombinant AAV vectors 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.
  • the AAV template comprises two AAV ITR sequences, which are located 5' and 3' to the heterologous nucleic acid sequence, although they need not be directly contiguous thereto.
  • the recombinant AAV template comprises an ITR that not resolved by Rep to make duplexed AAV vectors as described in international patent publication WO 01/92551.
  • the cell can be a cell that is permissive for AAV viral replication. Any suitable cell known in the art may be employed.
  • the cell is a mammalian cell (e.g., a primate or human ceil).
  • 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.
  • 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.
  • 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, (1 92) Curr. Top. Microbiol. Immun. 158:67).
  • the replcap sequences may be stably incorporated into a cell.
  • 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.
  • the template can be supplied by a non-viral (e.g., plasmid) or viral vector.
  • the AAV template is supplied by a herpesvirus or adenovirus vector (e.g., inserted into the Ela or E3 regions of a deleted adenovirus).
  • Palombo et ai (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.
  • the AAV template is provided by a replicating rAAV virus.
  • an AAV provirus comprising the AAV template is stably integrated into the chromosome of the cell.
  • helper virus functions e.g. , adenovirus or herpesvirus
  • 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.
  • 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 parvovirus production as described by Ferrari et at, (1997) Nature Med. 3:1295, and U.S. Patent Nos. 6,040,183 and 6,093,570.
  • 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.
  • the helper virus sequences cannot be packaged into parvovirus virions, e.g., are not flanked by TRs.
  • helper construct may be a non- viral or viral construct.
  • the helper construct can be a hybrid adenovirus or hybrid herpesvirus comprising the AAV rep/cap genes.
  • the AAV rep/cap sequences and the adenovirus helper sequences are supplied by a single adenovirus helper vector.
  • This vector can further comprise the AAV template.
  • the AAV rep/cap sequences and/or the AAV template can be inserted into a deleted region (e.g., the Ela or E3 regions) of the adenovirus.
  • the AAV rep/cop sequences and the adenovirus helper sequences are supplied by a single adenovirus helper vector.
  • the AAV template can be provided as a plasmid template.
  • the AAV rep/cap 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.
  • the adenovirus helper sequences and the AAV rep/cap sequences are generally not flanked by TRs so that these sequences are not packaged into the AAV virions.
  • 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 al., (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.
  • 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 al. (1999) Gene Therapy 6:973).
  • Deleted replication-defective helper viruses can be used so that any contaminating helper virus is not replication competent.
  • 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. , ts 100 and tsl49 adenovirus mutants).
  • AAV vectors that binds asialoglycans are useful for the delivery of nucleic acids to cells in vitro, ex vivo, and in vivo.
  • the virus vectors can be advantageously employed to deliver or transfer nucleic acids to animal, including mammalian, cells.
  • Any heterologous nucleic acid sequence(s) of interest may be delivered in the virus vectors of the present invention.
  • Nucleic acids of interest include nucleic acids encoding polypeptides, including therapeutic (e.g., for medical or veterinary uses) or immunogenic (e.g., for vaccines) polypeptides.
  • Therapeutic polypeptides include, but are not limited to, cystic fibrosis transmembrane regulator protein (CFTR), dystrophin (including mini- and micro- dystrophins (see, e.g., Vincent et al, (1993) Nature Genetics 5:130; U.S. Patent
  • myostatin propeptide myostatin propeptide, follistatin, activin type II soluble receptor, IGF- 1, anti-inflammatory polypeptides such as the I-kappa B dominant mutant, sarcospan, utrophin (Tinsley et al., (1996) Nature 384:349), mini-utrophin, clotting factors (e.g., Factor VIII, Factor IX, Factor X, etc.), erythropoietin, angiostatin, endostatin, catalase, tyrosine hydroxylase, superoxide dismutase, leptin, the LDL receptor, lipoprotein lipase, ornithine transcarbamylase, ⁇ -globin, a-globin, spectrin, a,i -antitrypsin, adenosine deaminase, hypoxanthine guanine phosphoribosyl transferase, ⁇ -
  • heterologous nucleic acid sequences encode suicide gene products (e.g., thymidine kinase, cytosine deaminase, diphtheria toxin, and tumor necrosis factor), proteins conferring resistance to a drug used in cancer therapy, tumor suppressor gene products (e.g., p53, Rb, Wt-1), TRAIL, FAS-ligand, and any other polypeptide that has a therapeutic effect in a subject in need thereof.
  • AAV vectors can also be used to deliver monoclonal antibodies and antibody fragments, for example, an antibody or antibody fragment directed against myostatin ⁇ see, e.g. , Fang et al, Nature Biotechnol. 23:584-590 (2005)).
  • the heterologous nucleic acid may encode an antisense nucleic acid, a ribozyme ⁇ e.g. , as described in U.S. Patent No. 5,877,022), RNAs that effect spliceosome-mediated trans- splicing ⁇ see, Puttaraju et al., (1999) Nature Biotech. 17:246; U.S. Patent No. 6,013,487; U.S. Patent No.
  • RNAi interfering RNAs
  • siRNA siRNA
  • shRNA miRNA that mediate gene silencing
  • other non-translated RNAs such as "guide” RNAs (Gorman et al, (1998) Proc. Nat. Acad. Sci. USA 95:4929; U.S. Patent No. 5,869,248 to Yuan et al.), and the like.
  • RNAi against a multiple drug resistance (MDR) gene product ⁇ e.g., to treat and/or prevent tumors and/or for administration to the heart to prevent damage by chemotherapy
  • MDR multiple drug resistance
  • myostatin e.g. , for Duchenne muscular dystrophy
  • VEGF vascular endothelial growth factor
  • phospholamban e.g., to treat cardiovascular disease
  • phospholamban inhibitory or dominant-negative molecules such as phospholamban S16E ⁇ e.g., to treat cardiovascular disease, see, e.g., Hoshijima et al. Nat. Med. 8:864-871 (2002)), RNAi to adenosine kinase ⁇ e.g., for epilepsy), RNAi to a sarcoglycan [e.g., ⁇ , ⁇ , ⁇ ], RNAi against myostatin, myostatin propeptide, follistatin, or activin type II soluble receptor, RNAi against anti-inflammatory polypeptides such as the Ikappa B dominant mutant, and RNAi directed against pathogenic organisms and viruses ⁇ e.g. , hepatitis B virus, human immunodeficiency virus, CMV, herpes simplex virus, human papilloma virus, etc.).
  • pathogenic organisms and viruses ⁇ e.g. , hepatitis B
  • the virus vector may also comprise a heterologous nucleic acid that shares homology with and recombines with a locus on a host chromosome. This approach can be utilized, for example, to correct a genetic defect in the host cell.
  • AAV AAV as vaccine vectors
  • the use of AAV as vaccine vectors is known in the art ⁇ see, e.g., Miyamura et o ., (1994) Proc. Nat. Acad. Sci USA 91:8507; U.S. Patent No. 5,916,563 to Young et al, U.S. Patent No. 5,905,040 to Mazzara et al, U.S. Patent No. 5,882,652, U.S. Patent No. 5,863,541 to Samulski et al).
  • the antigen may be presented in the AAV capsid. Alternatively, the antigen may be expressed from a heterologous nucleic acid introduced into a recombinant vector genome. Any immunogen of interest as described herein and/or as is known in the art can be provided by the virus vector of the present invention.
  • An immunogenic polypeptide can be any polypeptide suitable for eliciting an immune response and/or protecting the subject against an infection and/or disease, including, but not limited to, microbial, bacterial, protozoal, parasitic, fungal and/or viral infections and diseases.
  • the immunogenic polypeptide can be an orthomyxovirus immunogen ⁇ e.g., an influenza virus immunogen, such as the influenza virus hemagglutinin (HA) surface protein or the influenza virus nucleoprotein, or an equine influenza virus immunogen) or a lentivirus immunogen ⁇ e.g., an equine infectious anemia virus immunogen, a Simian Immunodeficiency Virus (SIV) immunogen, or a Human Immunodeficiency Virus (HIV) immunogen, such as the HIV or SIV envelope GP160 protein, the HIV or SIV matrix/capsid proteins, and the HIV or SIV gag,pol and env genes products).
  • an influenza virus immunogen such as the influenza virus hemagglutinin (HA) surface protein or the influenza virus nucleoprotein, or an equine influenza virus immunogen
  • a lentivirus immunogen ⁇ e.g., an equine infectious anemia virus immunogen, a Simian Immuno
  • the immunogenic polypeptide can also be an arenavirus immunogen ⁇ e.g., Lassa fever virus immunogen, such as the Lassa fever virus nucleocapsid protein and the Lassa fever envelope glycoprotein), a poxvirus immunogen ⁇ e.g., a vaccinia virus immunogen, such as the vaccinia LI or L8 gene products), a flavivirus immunogen ⁇ e.g., a yellow fever virus immunogen or a Japanese encephalitis virus immunogen), a filovirus immunogen ⁇ e.g., an Ebola virus immunogen, or a Marburg virus immunogen, such as NP and GP gene products), a bunyavirus immunogen (e.g., .
  • Lassa fever virus immunogen such as the Lassa fever virus nucleocapsid protein and the Lassa fever envelope glycoprotein
  • a poxvirus immunogen e.g., a vaccinia virus immunogen, such as the vaccinia LI or L8 gene
  • RVFV right atrial fluorescence virus
  • CCHF CCHF
  • SFS virus immunogens a coronavirus immunogen
  • a coronavirus immunogen e.g., an infectious human coronavirus immunogen, such as the human coronavirus envelope glycoprotein, or a porcine transmissible gastroenteritis virus immunogen, or an avian infectious bronchitis virus immunogen.
  • the immunogenic polypeptide can further be a polio immunogen, a herpes immunogen (e.g., CMV, EBV, HSV immunogens) a mumps immunogen, a measles immunogen, a rubella immunogen, a diphtheria toxin or other diphtheria immunogen, a pertussis antigen, a hepatitis (e.g., hepatitis A, hepatitis B, hepatitis C, etc.) immunogen, and/or any other vaccine immunogen now known in the art or later identified as an immunogen.
  • a herpes immunogen e.g., CMV, EBV, HSV immunogens
  • a mumps immunogen e.g., a mumps immunogen
  • measles immunogen e.g., a measles immunogen
  • a rubella immunogen e.g., a diphtheria toxin or other diphtheria immuno
  • telomerases e.g., telomeres
  • nuclear matrix proteins e.g., telomeres
  • prostatic acid phosphatase e.g., papilloma virus antigens
  • antigens now known or later discovered to be associated with the following cancers: melanoma, adenocarcinoma, thymoma, lymphoma (e.g., non- Hodgkin's lymphoma, Hodgkin's lymphoma), sarcoma, lung cancer, liver cancer, colon cancer, leukemia, uterine cancer, breast cancer, prostate cancer, ovarian cancer, cervical cancer, bladder cancer, kidney cancer, pancreatic cancer, brain cancer and any other cancer or malignant condition now known or later identified (see, e.g., Rosenberg, (1996) Ann. Rev. Med. 47:481 -91).
  • the heterologous nucleic acid can encode any polypeptide that is desirably produced in a cell in vitro, ex vivo, or in vivo.
  • the virus vectors may be introduced into cultured cells and the expressed gene product isolated therefrom.
  • the virus vectors may be introduced into animals to create models of disease.
  • heterologous nucleic acid(s) of interest can be operably associated with appropriate control sequences.
  • the heterologous nucleic acid can be operably associated with expression control elements, such as transcription/translation control signals, origins of replication, polyadenylation signals, internal ribosome entry sites (IRES), promoters, and/or enhancers, and the like.
  • expression control elements such as transcription/translation control signals, origins of replication, polyadenylation signals, internal ribosome entry sites (IRES), promoters, and/or enhancers, and the like.
  • promoter/enhancer elements can be used depending on the level and tissue-specific expression desired.
  • the promoter/enhancer can be constitutive or inducible, depending on the pattern of expression desired.
  • the promoter/enhancer can 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.
  • the promoter/enhancer elements can be native to the target cell or subject to be treated.
  • the promoter/enhancer elements can be native to the target cell or subject to be treated.
  • promoter/enhancer element can be native to the heterologous nucleic acid sequence.
  • the promoter/enhancer element is generally chosen so that it functions in the target cell(s) of interest. Further, in particular embodiments the promoter/enhancer element is a mammalian promoter/enhancer element.
  • the promoter/enhancer element may be constitutive or inducible.
  • 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;
  • heterologous nucleic acid sequence(s) is transcribed and then translated in the target cells
  • specific initiation signals are generally included for efficient translation of inserted protein coding sequences.
  • 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.
  • the virus vectors according to the present invention provide a means for delivering heterologous nucleic acids into a broad range of cells, including dividing and non-dividing cells.
  • the virus vectors can be employed to deliver a nucleic acid of interest to a cell in vitro, e.g., to produce a polypeptide in vitro or for ex vivo gene therapy.
  • the virus vectors are additionally useful in a method of delivering a nucleic acid to a subject in need thereof, e.g., to express an immunogenic or therapeutic polypeptide or a functional RNA. h this manner, the polypeptide or functional RNA can be produced in vivo in the subject.
  • the subject can be in need of the polypeptide because the subject has a deficiency of the polypeptide.
  • the method can be practiced because the production of the polypeptide or functional RNA in the subject may impart some beneficial effect.
  • the virus vectors can also be used to produce a polypeptide of interest or functional RNA in cultured cells or in a subject (e.g., using the subject as a bioreactor to produce the polypeptide or to observe the effects of the functional RNA on the subject, for example, in connection with screening methods).
  • glucose storage diseases e.g., Fabry disease [ -galactosidase] and Pompe disease [lysosomal acid a-glucosidase]
  • Fabry disease [ -galactosidase] and Pompe disease [lysosomal acid a-glucosidase]
  • congenital emphysema a 1 -antitrypsin
  • Lesch-Nyhan Syndrome hyperoxanthine guanine phosphoribosy] transferase
  • Nieman -Pick disease sphingomyelinase
  • Tays Sachs disease lysosomal hexosaminidase A
  • Maple Syrup Urine Disease branched-chain keto acid dehydrogenase
  • retinal degenerative diseases and other diseases of the eye and retina; e.g., PDGF for macular degeneration
  • phenylketonuria phenylalanine hydroxylase
  • the invention can further be used following organ transplantation to increase the success of the transplant and/or to reduce the negative side effects of organ transplantation or adjunct therapies (e.g., by administering immunosuppressant agents or inhibitory nucleic acids to block cytokine production).
  • organ transplantation or adjunct therapies e.g., by administering immunosuppressant agents or inhibitory nucleic acids to block cytokine production.
  • bone morphogenic proteins including BNP 2, 7, etc., RANKL and/or VEGF
  • deficiency states usually of enzymes, which are generally inherited in a recessive manner
  • unbalanced states which may involve regulatory or structural proteins, and which are typically inherited in a dominant manner.
  • gene transfer can be used to bring a normal gene into affected tissues for replacement therapy, as well as to create animal models for the disease using antisense mutations.
  • gene transfer can be used to create a disease state in a model system, which can then be used in efforts to counteract the disease state.
  • virus vectors according to the present invention permit the treatment and/or prevention of genetic diseases.
  • the virus vectors according to the present invention may also be employed to provide a functional NA to a cell in vitro or in vivo.
  • Expression of the functional RNA in the cell can diminish expression of a particular target protein by the cell.
  • functional RNA can be administered to decrease expression of a particular protein in a subject in need thereof.
  • Functional RNA can also be administered to cells in vitro to regulate gene expression and/or cell physiology, e.g., to optimize cell or tissue culture systems or in screening methods.
  • Virus vectors according to the instant invention find use in diagnostic and screening methods, whereby a nucleic acid of interest is transiently or stably expressed in a cell culture system, or alternatively, a transgenic animal model.
  • the virus vectors of the present invention can also be used for various non-therapeutic purposes, including but not limited to use in protocols to assess gene targeting, clearance, transcription, translation, etc., as would be apparent to one skilled in the art.
  • the virus vectors can also be used for the purpose of evaluating safety (spread, toxicity, immunogenicity, etc.). Such data, for example, are considered by the United States Food and Drug Administration as part of the regulatory approval process prior to evaluation of clinical efficacy.
  • virus vectors of the present invention may be used to produce an immune response in a subject.
  • a virus vector comprising a heterologous nucleic acid sequence encoding an immunogenic polypeptide can be administered to a subject, and an active immune response is mounted by the subject against the immunogenic polypeptide.
  • Immunogenic polypeptides are as described hereinabove.
  • a protective immune response is elicited.
  • the virus vector may be administered to a cell ex vivo and the altered cell is administered to the subject.
  • the virus vector comprising the heterologous nucleic acid is introduced into the cell, and the cell is administered to the subject, where the heterologous nucleic acid encoding the immunogen can be expressed and induce an immune response in the subject against the immunogen.
  • the cell is an antigen-presenting cell ⁇ e.g., a dendritic cell).
  • an “active immune response” or “active immunity” is characterized by “participation of host tissues and cells after an encounter with the immunogen. It involves differentiation and proliferation of immunocompetent cells in lymphoreticular tissues, which lead to synthesis of antibody or the development of cell-mediated reactivity, or both.” Herbert B. Herscowitz, Immunophysiology: Cell Function and Cellular Interactions in Antibody Formation, in IMMUNOLOGY: BASIC PROCESSES 117 (Joseph A. Bellanti ed., 1985). Alternatively stated, an active immune response is mounted by the host after exposure to an immunogen by infection or by vaccination.
  • Active immunity can be contrasted with passive immunity, which is acquired through the "transfer of preformed substances (antibody, transfer factor, thymic graft, interIeukin-2) from an actively immunized host to a non-immune host.” Id.
  • virus vector or cell comprising the heterologous nucleic acid can be administered in an immunogenically effective amount, as described below.
  • the virus vectors of the present invention can also be administered for cancer immunotherapy by administration of a virus vector expressing one or more cancer cell antigens (or an immunologically similar molecule) or any other immunogen that produces an immune response against a cancer cell.
  • an immune response can be produced against a cancer cell antigen in a subject by administering a virus vector comprising a heterologous nucleic acid encoding the cancer cell antigen, for example to treat a patient with cancer and/or to prevent cancer from developing in the subject.
  • the virus vector may be administered to a subject in vivo or by using ex vivo methods, as described herein.
  • the cancer antigen can be expressed as part of the virus capsid or be otherwise associated with the virus capsid as described above.
  • any other therapeutic nucleic acid e.g., R Ai
  • polypeptide e.g., cytokine
  • cancer encompasses tumor-forming cancers.
  • cancer tissue encompasses tumors.
  • cancer cell antigen encompasses tumor antigens.
  • Tumor is also understood in the art, for example, as an abnormal mass of undifferentiated cells within a multicellular organism. Tumors can be malignant or benign. In representative embodiments, the methods disclosed herein are used to prevent and treat malignant tumors.
  • treating cancer By the terms “treating cancer,” “treatment of cancer” and equivalent terms it is intended that the severity of the cancer is reduced or at least partially eliminated and/or the progression of the disease is slowed and/or controlled and/or the disease is stabilized. In particular embodiments, these terms indicate that metastasis of the cancer is prevented or reduced or at least partially eliminated and/or that growth of metastatic nodules is prevented or reduced or at least partially eliminated.
  • prevention of cancer or “preventing cancer” and equivalent terms it is intended that the methods at least partially eliminate or reduce and/or delay the incidence and/or severity of the onset of cancer.
  • the onset of cancer in the subject may be reduced in likelihood or probability and/or delayed.
  • cells may be removed from a subject with cancer and contacted with a virus vector according to the instant invention.
  • the modified cell is then administered to the subject, whereby an immune response against the cancer cell antigen is elicited.
  • This method can be advantageously employed with
  • immunomodulatory cytokines e.g., -interferon, ⁇ -interferon, ⁇ -interferon, ⁇ -interferon, ⁇ -interferon, ⁇ -interferon, interleukin-la, interleukin- 1 ⁇ , interIeukin-2, interleukin-3, interleukin-4, interleukin 5, interleukin-6, interleukin-7, interleukin-8, interleukin-9, interleukin-10, interleukin-11, interleukin 12, interleukin-13, interleukin- 14, interleukin- 18, B cell Growth factor, CD40 Ligand, tumor necrosis factor-a, tumor necrosis factor- ⁇ , monocyte chemoattractant protein-1, granulocyte-macrophage colony stimulating factor, and lymphotoxin).
  • immunomodulatory cytokines preferably, CTL inductive cytokines
  • Cytokines may be administered by any method known in the art.
  • compositions comprising an AAV vector that binds asialoglycans and a desialylatirtg agent.
  • the composition comprises a sufficient amount of AAV vector to infect cells.
  • the composition comprises a therapeutically effective amount of the AAV vector.
  • the desialylating agent can be any agent described above.
  • the composition further comprises a physiologically acceptable carrier, in further embodiments, the composition further comprises one or more excipients, such as buffers, stabilizers, etc.
  • the composition can be in the form of a liquid, semi-solid, or solid.
  • kits comprising a desialylating agent and an AAV vector that binds asialoglycans.
  • the desialylating agent and the AAV vector are present in one container.
  • the desialylating agent and the AAV vector are present in separate containers.
  • the kits are useful for carrying out the methods of the invention.
  • the kits can comprise other reagents for delivery of viral vectors and/or detection of expression polypeptides or functional RNAs encoded by the vectors.
  • the reagents may be nucleic acids (e.g., an
  • oligonucleotide that specifically hybridizes to a portion of the vector and can be used as a hybridization probe or an amplification primer), antibodies (e.g., one the specifically binds to a polypeptide encoded by the vector), or other agents that specifically recognize the polynucleotides or polypeptides of the invention.
  • the reagents can be conjugated to a detectable tag or detectable label.
  • a detectable tag can be any suitable tag which allows for detection of the reagents and includes, but is not limited to, any composition or label detectable by spectroscopic,
  • Useful labels in the present invention include biotin for staining with labeled streptavidin conjugate, magnetic beads (e.g., DynabeadsTM), fluorescent dyes (e.g., fluorescein, Texas red, rhodamine, green fluorescent protein, and the like), radiolabels (e.g., 3 H, 12 I, 35 S, 14 C, or 32 P), enzymes (e.g., horse radish peroxidase, alkaline phosphatase and others commonly used in an ELISA), and colorimetric labels such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads.
  • fluorescent dyes e.g., fluorescein, Texas red, rhodamine, green fluorescent protein, and the like
  • radiolabels e.g., 3 H, 12 I, 35 S, 14 C, or 32 P
  • enzymes e.g., horse radish peroxidas
  • reagents can be immobilized on a substrate.
  • a substrate can include any suitable substrate for immobilization of a detection reagent such as would be used in any of the previously described methods of detection.
  • a substrate suitable for immobilization of a detection reagent includes any solid support, such as any solid organic, biopolymer or inorganic support that can form a bond with the detection reagent without significantly effecting the activity and/or ability of the detection reagent to detect the desired target molecule.
  • Exemplary organic solid supports include polymers such as polystyrene, nylon, phenol-formaldehyde resins, acrylic copolymers (e.g., polyacrylamide), stabilized intact whole cells, and stabilized crude whole ceil/membrane homogenates.
  • Exemplary biopolymer supports include cellulose, polydextrans (e.g., Sephadex ® ), agarose, collagen and chitin.
  • Exemplary inorganic supports include glass beads (porous and nonporous), stainless steel, metal oxides (e.g., porous ceramics such as Zr0 2 , Ti0 2 , A1 2 0 3 , and NiO) and sand.
  • kits may further comprise other components useful for delivery of vectors and/or detecting expression or activity, e.g., buffers, cells, culture medium, enzymes, labeling reagents, containers, etc.
  • other components useful for delivery of vectors and/or detecting expression or activity e.g., buffers, cells, culture medium, enzymes, labeling reagents, containers, etc.
  • Virus vectors according to the present invention find use in both veterinary and medical applications. Suitable subjects include both avians and mammals.
  • avian as used herein includes, but is not limited to, chickens, ducks, geese, quail, turkeys, pheasant, parrots, parakeets, and the like.
  • mammal as used herein includes, but is not limited to, humans, non-human primates, bovines, ovines, caprines, equines, felines, canines, lagomorphs, etc. Human subjects include neonates, infants, juveniles and adults.
  • the pharmaceutical composition will further comprise a
  • the carrier will typically be a liquid.
  • the carrier may be either solid or liquid.
  • the carrier will be respirable, and optionally can be in solid or liquid particulate form.
  • 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.
  • the virus vector may be introduced into the cells at the appropriate multiplicity of infection according to standard transduction methods suitable for the particular target cells. Titers of virus vector to administer can vary, depending upon the target cell type and number, and the particular virus vector, and can be determined by those of skill in the art without undue experimentation. In representative embodiments, at least about 10 3 infectious units, more preferably at least about 10 5 infectious units are introduced to the cell.
  • the virus vector may be introduced into the cells before, during and/or after desialylating the cells.
  • the cell(s) into which the virus vector is introduced can 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 and oligodendrocytes), lung cells, ceils of the eye (including retinal cells, retinal pigment epithelium, and corneal cells), blood vessel cells ⁇ e.g., endothelial cells, intimal cells), epithelial cells ⁇ e.g., gut and respiratory epithelial cells), muscle cells ⁇ e.g., skeletal muscle cells, cardiac muscle cells, smooth muscle cells and/or diaphragm muscle cells), dendritic cells, pancreatic cells (including islet cells), hepatic cells, kidney cells, myocardial cells, bone cells (e.g., bone marrow stem cells), hematopoietic stem cells, spleen ceils, keratinocytes, fibroblasts, endothelial ceils, prostate cells, germ cells, and the like.
  • neural cells including
  • the cell can be any progenitor cell.
  • the cell can be a stem cell (e.g., neural stem cell, liver stem cell).
  • the cell can be a cancer or tumor cell.
  • the cell can be from any species of origin, as indicated above.
  • the virus vector can be introduced into cells in vitro for the purpose of administering the modified cell to a subject.
  • the cells have been removed from a subject, the virus vector is introduced therein, and the cells are then administered back into the subject.
  • the desialylating step can occur before and/or after the cells are removed from the subject.
  • the virus vector may be introduced into the cells before, during and/or after desialylating the cells. Methods of removing cells from subject for manipulation ex vivo, followed by introduction back into the subject are known in the art (see, e.g., U.S. Patent No. 5,399,346).
  • the recombinant virus vector can be introduced into cells from a donor subject, into cultured cells, or into cells from any other suitable source, and the cells are administered to a subject in need thereof (i.e., a "recipient" subject).
  • the virus vector is introduced into a cell and the cell can be administered to a subject to elicit an immunogenic response against the delivered polypeptide (e.g. , expressed as a transgene or in the capsid).
  • an immunogenic response against the delivered polypeptide e.g. , expressed as a transgene or in the capsid.
  • a quantity of cells expressing an immunogenically effective amount of the polypeptide in combination with a pharmaceutically acceptable carrier is administered.
  • immunogenic polypeptide is an amount of the expressed polypeptide that is sufficient to evoke an active immune response against the polypeptide in the subject to which the pharmaceutical formulation is administered.
  • the dosage is sufficient to produce a protective immune response (as defined above).
  • the degree of protection conferred need not be complete or permanent, as long as the benefits of administering the immunogenic polypeptide outweigh any disadvantages thereof.
  • a further aspect of the invention is a method of administering the virus vector to subjects.
  • Administration of the virus vectors according to the present invention to a human subject or an animal in need thereof can be by any means known in the art.
  • the virus vector is delivered in a treatment effective or prevention effective dose in a pharmaceutically acceptable carrier.
  • the virus vector may be delivered to the subject before, during and/or after delivering a desialylating agent to the subject.
  • the virus vectors of the invention can further be administered to elicit an immunogenic response (e.g., as a vaccine).
  • immunogenic compositions of the present invention comprise an immunogenically effective amount of virus vector in combination with a pharmaceutically acceptable carrier.
  • the dosage is sufficient to produce a protective immune response (as defined above).
  • the degree of protection conferred need not be complete or permanent, as long as the benefits of administering the immunogenic polypeptide outweigh any disadvantages thereof.
  • Dosages of the virus vector to be administered to a subject depend upon the mode of administration, the disease or condition to be treated and/or prevented, the individual subject' s condition, the particular virus vector, and the nucleic acid to be delivered, and the like, and can be determined in a routine manner.
  • Exemplary doses for achieving therapeutic effects are titers of at least about lO 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 n , 10 12 , 10 13 , 10 14 , 10 15 transducing units, optionally about 10 s - 10 13 transducing units.
  • more than one administration 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, intranasal, inhalation (e.g., via an aerosol), buccal (e.g., sublingual), vaginal, intrathecal, intraocular, transdermal, intraendothelial, in utero (or in ovo), parenteral (e.g., intravenous, intraarterial, intraportal, subcutaneous, intradermal, intracranial,
  • intramuscular including administration to skeletal, diaphragm and/or cardiac muscle], intrapleural, intracerebral, intracisternal, and intraarticular
  • topical e.g., to both skin and mucosal surfaces, including airway surfaces, and transdermal administration
  • intralymphatic e.g., to liver, eye, skeletal muscle, cardiac muscle, diaphragm muscle or brain.
  • Administration can be to any site in a subject, including, without limitation, a site selected from the group consisting of the brain, a joint, a skeletal muscle, a smooth muscle, the heart, the diaphragm, the airway epithelium, the liver, the kidney, the spleen, the pancreas, the skin, and the eye.
  • Administration can also be to a tumor (e.g., in or near a tumor or a lymph node).
  • a tumor e.g., in or 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/or prevented and on the nature of the particular vector that is being used.
  • Administration to cardiac muscle includes administration to the left atrium, right atrium, left ventricle, right ventricle and/or septum.
  • the virus vector can be delivered to cardiac muscle by intravenous administration, intra-arterial administration such as intra-aortic administration, direct cardiac injection (e.g., into left atrium, right atrium, left ventricle, right ventricle), and/or coronary artery perfusion.
  • Delivery to a target tissue can also be achieved by delivering a depot comprising the virus vector.
  • a depot comprising the virus vector is implanted into skeletal, smooth, cardiac and/or diaphragm muscle tissue or the tissue can be contacted with a film or other matrix comprising the virus vector.
  • implantable matrices or substrates are described in U.S. Patent No. 7,201 ,898.
  • a virus vector according to the present invention is administered to skeletal muscle, diaphragm muscle and/or cardiac muscle (e.g., to treat and/or prevent muscular dystrophy or heart disease [for example, PAD or congestive heart failure]).
  • the invention is used to treat and/or prevent disorders of skeletal, cardiac and/or diaphragm muscle.
  • the invention provides a method of treating and/or preventing muscular dystrophy in a subject in need thereof, the method comprising: administering a treatment or prevention effective amount of a virus vector of the invention to a mammalian subject, wherein the virus vector comprises a heterologous nucleic acid encoding dystrophin, a mini-dystrophin, a micro-dystrophin, myostatin propeptide, follistatin, activin type II soluble receptor, IGF-1, anti -inflammatory polypeptides such as the I-kappa B dominant mutant, sarcospan, utrophin, a micro- dystrophin, laminin-a2, a-sarcoglycan, ⁇ -sarcoglycan, ⁇ -sarcoglycan, ⁇ -sarcoglycan, IGF- 1 , an antibody or antibody fragment against myostatin or myostatin propeptide, and/or RNAi against myostat
  • the invention can be practiced to deliver a nucleic acid to skeletal, cardiac or diaphragm muscle, which is used as a platform for production of a polypeptide (e.g., an enzyme) or functional RNA (e.g., RNAi, microRNA, antisense RNA) that normally circulates in the blood or for systemic delivery to other tissues to treat and/or prevent a disorder (e.g., a metabolic disorder, such as diabetes (e.g., insulin), hemophilia (e.g., Factor ⁇ or Factor VIII), a mucopolysaccharide disorder (e.g., Sly syndrome, Hurler Syndrome, Scheie Syndrome, Hurler-Scheie Syndrome, Hunter's Syndrome, Sanfilippo Syndrome A, B, C, D, Morquio Syndrome, Maroteaux-Lamy Syndrome, etc.) or a lysosomal storage disorder (such as Gaucher's disease
  • a metabolic disorder such as diabetes (e.g., insulin), hemophilia (e.g.,
  • the invention further encompasses a method of treating and/or preventing a metabolic disorder in a subject in need thereof, the method comprising: administering a treatment or prevention effective amount of a virus vector of the invention to a subject (e.g. , to skeletal muscle of a subject), wherein the virus vector comprises a heterologous nucleic acid encoding a polypeptide, wherein the metabolic disorder is a result of a deficiency and/or defect in the polypeptide.
  • a subject e.g. , to skeletal muscle of a subject
  • the virus vector comprises a heterologous nucleic acid encoding a polypeptide
  • the metabolic disorder is a result of a deficiency and/or defect in the polypeptide.
  • Illustrative metabolic disorders and heterologous nucleic acids encoding polypeptides are described herein.
  • the virus vectors disclosed herein can be administered to the lungs of a subject by any suitable means, optionally by administering an aerosol suspension of respirable particles comprised of the virus vectors, which the subject inhales.
  • the respirable particles can 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.
  • the virus vectors can be administered to tissues of the CNS (e.g. , brain, eye) and may advantageously result in a more restricted distribution of the virus vector than would be observed in the absence of the present invention.
  • tissues of the CNS e.g. , brain, eye
  • the delivery vectors of the invention may be administered to treat diseases of the CNS, including genetic disorders, neurodegenerative disorders, psychiatric disorders and tumors.
  • diseases of the CNS include, but are not limited to Alzheimer's disease, Parkinson's disease, Huntington's disease, Canavan disease, Leigh's disease, Refsum disease, Tourette syndrome, primary lateral sclerosis, amyotrophic lateral sclerosis, progressive muscular atrophy, Pick's disease, muscular dystrophy, multiple sclerosis, myasthenia gravis, Binswanger's disease, trauma due to spinal cord or head injury, Tay Sachs disease, Lesch-Nyan disease, epilepsy, cerebral infarcts, psychiatric disorders including mood disorders (e.g., depression, bipolar affective disorder, persistent affective disorder, secondary mood disorder), schizophrenia, drug dependency (e.g., alcoholism and other substance dependencies), neuroses (e.g., anxiety, obsessional disorder, somatoform
  • mood disorders e.g.,
  • disorders of the CNS include ophthalmic disorders involving the retina, posterior tract, and optic nerve (e.g., retinitis pigmentosa, diabetic retinopathy and other retinal degenerative diseases, uveitis, age-related macular degeneration, glaucoma).
  • optic nerve e.g., retinitis pigmentosa, diabetic retinopathy and other retinal degenerative diseases, uveitis, age-related macular degeneration, glaucoma.
  • ophthalmic diseases and disorders are associated with one or more of three types of indications: (1) angiogenesis, (2) inflammation, and (3) degeneration.
  • the delivery vectors of the present invention can be employed to deliver anti-angiogenic factors; anti-inflammatory factors; factors that retard cell degeneration, promote cell sparing, or promote cell growth and combinations of the foregoing.
  • Diabetic retinopathy for example, is characterized by angiogenesis.
  • Diabetic retinopathy can be treated by delivering one or more anti-angiogenic factors either intraocularly (e.g., in the vitreous) or periocularly( e.g., in the sub-Tenon's region).
  • One or more neurotrophic factors may also be co-delivered, either intraocularly (e.g. , intravitreally) or periocularly.
  • Uveitis involves inflammation.
  • One or more anti-inflammatory factors can be administered by intraocular (e.g., vitreous or anterior chamber) administration of a delivery vector of the invention.
  • Glaucoma is characterized by increased ocular pressure and loss of retinal ganglion cells.
  • Treatments for glaucoma include administration of one or more neuroprotective agents that protect cells from excitotoxic damage using the inventive delivery vectors.
  • Such agents include N-methyl-D-aspartate (NMD A) antagonists, cytokines, and neurotrophic factors, delivered intraocularly, optionally intravitreally.
  • NMD A N-methyl-D-aspartate
  • cytokines cytokines
  • neurotrophic factors delivered intraocularly, optionally intravitreally.
  • the present invention may be used to treat seizures, e.g., to reduce the onset, incidence or severity of seizures.
  • the efficacy of a therapeutic treatment for seizures can be assessed by behavioral (e.g., shaking, ticks of the eye or mouth) and/or electrographic means (most seizures have signature electrographic abnormalities).
  • the invention can also be used to treat epilepsy, which is marked by multiple seizures over time.
  • somatostatin (or an active fragment thereof) is. administered to the brain using a delivery vector of the invention to treat a pituitary tumor.
  • the delivery vector encoding .
  • somatostatin (or an active fragment thereof) is administered by microinfusion into the pituitary. Likewise, such treatment can be used to treat acromegaly (abnormal growth hormone secretion from the pituitary).
  • the nucleic acid e.g., GenBank Accession No. J00306
  • amino acid e.g., GenBank Accession No. POl 166; contains processed active peptides somatostatin-28 and somatostatin- 14 sequences of somatostatins as are known in the art.
  • the vector can comprise a secretory signal as described in U.S; Patent No. 7,071,172.
  • the virus vector is administered to the CNS (e.g., to the brain or to the eye).
  • the virus vector may be introduced into the spinal cord, brainstem (medulla oblongata, pons), midbrain
  • the virus vector may also be administered to different regions of the eye such as the retina, cornea and/or optic nerve.
  • the virus vector may be delivered into the cerebrospinal fluid (e.g., by lumbar puncture) for more disperse administration of the delivery vector.
  • the virus vector may further be administered intravascularly to the CNS in situations in which the biood-brain barrier has been perturbed (e.g., brain tumor or cerebral infarct).
  • the virus vector can be administered to the desired region(s) of the CNS by any route known in the art, including but not limited to, intrathecal, intra-ocular, intracerebral, intraventricular, intravenous (e.g., in the presence of a sugar such as mannitol), intranasal, intra-aural, intra-ocular (e.g., intra- vitreous, sub-retinal, anterior chamber) and peri-ocular (e.g., sub-Tenon's region) delivery as well as intramuscular delivery with retrograde delivery to motor neurons.
  • intrathecal intra-ocular, intracerebral, intraventricular, intravenous (e.g., in the presence of a sugar such as mannitol), intranasal, intra-aural, intra-ocular (e.g., intra- vitreous, sub-retinal, anterior chamber) and peri-ocular (e.g., sub-Tenon's region) delivery as well as intramuscular delivery
  • the virus vector is administered in a liquid formulation by direct injection (e.g., stereotactic injection) to the desired region or , compartment in the CNS.
  • the virus vector may be provided by topical application to the desired region or by intra-nasal administration of an aerosol formulation. Administration to the eye, may be by topical application of liquid droplets.
  • the virus vector may be administered as a solid, slow-release formulation (see, e.g., U.S. Patent No. 7,201,898).
  • the virus vector can be used for retrograde transport to treat and/or prevent diseases and disorders involving motor neurons (e.g., amyotrophic lateral sclerosis (ALS); spinal muscular atrophy (SMA), etc.).
  • motor neurons e.g., amyotrophic lateral sclerosis (ALS); spinal muscular atrophy (SMA), etc.
  • the virus vector can be delivered to muscle tissue from which it can migrate into neurons.
  • 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.
  • the virus vector can be delivered adhered to or impregnated within a surgically implantable matrix such as a sheet or mesh (e.g., as described in U.S. Patent Publication No. 2004-0013645).
  • Asmids and viruses were obtained from the UNC vector core.
  • the triple plasmid transfection protocol (Gneger et al, Nat. Protoc. 7:1412 (2006)) utilized for production of AAV9 vectors, includes (i) the AAV helper plasmid, pXR9, containing AAV2 Rep and AAV9 Cap genes, (U) the Adenoviral helper plasmid, pXX6- 80 and (iii) the vector genome cassette, pTR-CBA-Luc, containing the firefly luciferase gene driven by the chicken beta-actin (CBA) promoter and flanked by inverted terminal repeats (ITRs) required for packaging.
  • CBA chicken beta-actin
  • ITRs inverted terminal repeats
  • the ITRs are the only elements within the vector genome cassette derived from the wild-type AAV genome, thereby eliminating 96% of viral elements. Recombinant AAV9 vectors generated thus allow quantitation of viral infectivity (transduction efficiency) through luciferase transgene expression assays.
  • HEK293 cells utilized for production of recombinant AAV9 vectors were obtained from the UNC vector core. Sonicated cell lysates and PEG8000 precipitates from supernatant, were pooled and subjected to cesium chloride ultracentrifugation as described earlier (23), Dia!yzed peak fractions were subjected to quantitative PCR using a Roche Light Cycler instrument with luc transgene-specific primers to determine viral vector titers (forward 5'- AAA AGC ACT CTG ATT GAC AAA TAC-3' (SEQ ID NO:l); reverse 5'-CCT TCG CTT CAA AAA ATG GAA C-3' (SEQ ID NO:2)).
  • hepatocarcinoma were obtained from the UNC tissue culture facility and utilized in viral infectivity assays.
  • Chinese hamster ovary (CHO) Pro5 and mutant Lecl, Lec2 cell lines were a gift from Dr. Jude Samulski (UNC-Chapel Hill) and the CHO Lec8 cell line purchased from ATCC. All CHO cells, utilized for viral binding and infectivity assays, were cultured in a-MEM (GIBCO) supplemented with 10% FBS and penicillin, streptomycin, amphotericin B as outlined above.
  • Well-differentiated human airway epithelial (HAE) cultures (4-6 weeks) grown on permeable membrane supports
  • Transduction assays Different cell lines were seeded at 10 5 cells/well in 24-well plates and allowed to adhere overnight at 37°C. Plates were then pre-chilled at 4°C for 30 min and incubated with AAV9 vectors at a multiplicity of infection (MOI) of 1000 vector genomes per cell (vg/cell) to allow binding to the cell surface for 1.5 hrs at 4°C. Unbound virus was then removed by washing three times with ice cold lx phosphate-buffered saline (IxPBS) and 0.5 mL of DMEM added to each well.
  • MOI multiplicity of infection
  • Luciferase transgene expression levels were quantitated after incubation for 24 hrs from cell lysates using a Victor 2 luminometer (Perkin Elmer).
  • a Victor 2 luminometer Perkin Elmer
  • Fluorescence micrographs of green fluorescent protein (GFP) expression in HAE cultures at 2 weeks post-transduction were obtained using an Olympus epifluorescence microscope equipped with a Hamamatsu camera.
  • Cosl cells were seeded at 10 5 cells/well in 24-well plates and pretreated with 50 mU/ml neuraminidase, 3 U/ml heparinase I, 1.5 U/ml heparinase III and 1.5 U/ml chondroitinase ABC in serum-free DMEM at 37°C for 2 hrs.
  • Neuro2a, U87, HEK293 and Huh7 cells were treated with neuraminidase alone.
  • Cells were then washed three times with lxPBS and subjected to AAV9 infection at an MOI of 1000 vg/cell. Luciferase transgene expression assays were carried out as described above at 24 hrs post-infection.
  • CHO Lec2 cells were seeded at 10 s cells/well in 24-well plates and pretreated for 24 hrs with small molecule inhibitors of glycosylation, Swainsonine (10 ⁇ ; Sigma, #S8195) and a-benzyl-GalNAc (1 ⁇ g/mL; Sigma, #B4894) to determine the role of N- and (9-glycans in AAV9 infection.
  • Cells pretreated with chemicals were subjected to AAV9 infection at an MOI of 1000 vg/cell and luciferase transgene expression assays carried out as described above.
  • Olympus 100 microscope Olympus
  • mice Animal studies. All experiments were carried out with 6-8 week old female BALB/c mice (Jackson Labs, Bar Harbor, ME) maintained and treated in accordance with National Institutes of Health guidelines and as approved by IACUC at UNC-Chapel Hill. Mice were administered via intranasal instillation with either 100 ⁇ PBS (50 ⁇ /nostril) or 100 ⁇ Neuraminidase Type III from Vibrio choleras (200 mU, Sigma, St. Louis, MO). At 2 hrs post-treatment, a dose of 5x10 10 AAV9 particles in IxPBS (50 ⁇ /nostril) was administered.
  • Luciferase transgene expression in live animals was obtained using a Xenogen IVIS Lumina® imaging system (Caliper Lifesciences, CA) after intranasal instillation of luciferin substrate (120 mg/kg; Nanolight). Image analysis was carried out using the Living Image software® (Caliper Lifesciences) and luciferase expression reported in relative light units (photons/sec/cm 2 /sr).
  • Neuraminidase treatment selectively increases infectivity of AAV9 in different cell types
  • heparinase III neuraminidase from Vibrio Cholerae and chondroitinase ABC
  • chondroitinase ABC chondroitinase ABC
  • Luciferase transgene expression (Relative Light Units, RLU) was quantified for both studies at 24 hrs post-infection. All experiments were carried out in triplicate. Error bars represent standard error mean.
  • Unbound virions were removed by three washes with ice-cold lx PBS. The amount of cell surface-bound AAV9 virion was quantified using Q-PCR. The amount of bound virions was expressed as vector genome copy numbers (vg) per cell. All experiments were carried out in quadruplicate. Error bars represent standard error mean.
  • sialic acid appears to mask cell surface glycans that selectively facilitate AAV9 infection in vitro. Further, enzymatic desialylation might serve as a facile biochemical strategy to enhance transduction efficiency of AAV9 vectors and might enable detailed analysis of the intracellular trafficking pathways of AAV9 in vitro.
  • FIG. 4A shows a schematic representation of N-glycan compositions of the parental CHO Pro5 cell line and mutants Lec2, Lec8 and Lecl (North et al, J. Biol. Chem. 285:5759 (2010)) using nomenclature proposed by the Consortium for Functional Glycomics nomenclature committee (® Mannose, ⁇ GlcNac, o Galactose, ⁇ Sialic acid).
  • the CHO Lec2 cell line lacks terminal sialic acid due to a defect in CMP- sialic acid transport (Deutscher et al., Cell 39:295 (1984)), while Lec8 and Lecl cell lines are defective in translocation of UDP-galactose and N-acetylglucosaminyltransferase activity (Deutscher et al., J. Biol. Chem. 261:96 (1986); Stanley et al., (1985) Mol. Cell. Biol. 5:1204 (1985)), respectively.
  • cell surface glycans on CHO Lec2 cells contain terminal galactosyl residues, while Lec8 and Lecl cell lines predominantly display terminal N-acetylglucosamine and mannosylated glycans, respectively (FIG. 4A).
  • FIGS. 4B and 4C cell surface binding and infectivity of AAV9 particles on Lec2 cells is significantly increased (> 1 log unit) when compared to the parental Pro5 cell line.
  • no major changes in binding and infectivity are observed in the case of Lec8 and Lecl cells.
  • glycans required for AAV9 infection small molecule inhibitors of glycosylation and sialyltransferases were utilized to modify terminal galactosyl residues on the sialic acid-deficient Lec2 cell surface.
  • CHO Lec2 cells were treated with 50 mU/mL each of 2,3-(N)-sialyltransferase (a2,3NST), a2,6-(N)-sialyltransferase
  • Luciferase transgene expression was quantified at 24 hrs post-infection and expressed as % infectivity with respect to untreated or wild type (CHO Pro5) control (FIG. 5B).
  • CHO Lec2 cells treated with CMP-Sialic acid alone or with CMP-Sialic acid and different sialyltransferases were subjected to lectin staining using FITC-labeled ECL, which exclusively recognizes Gal(pl,4)GlcNAc, or FITC-labeled MAL, which recognizes ⁇ x2,3-Sialylated GaI(pl ,4)GlcNAc (FIG. 5C).
  • Untreated wild type CHO Pro5 cells, which show high levels of FITC-MAL I staining and untreated Lec2 cells, which show high levels of FITC-ECL staining were included as controls. All experiments were carried out in triplicate. Error bars indicate standard error mean.
  • Swainsonine (Elbein et al., Proc. Natl. Acad. Sci. U. S. A. 75:7393 (1981)) and a-benzyl-O-GalNAc (Kuan et al., J. Biol. Chem. 264:19271 (1989)) are chemical inhibitors of N-linked and O-linked glycosylation, respectively. Treatment with these reagents results in a corresponding decrease in cell surface expression of N-linked glycans and O-linked glycans. As seen in FIG.
  • AAV9 infection is significantly blocked by swainsonine ( ⁇ 75%), while ct-benzyl-0-GalNAc has a modest inhibitory effect ( ⁇ 25%).
  • Terminal galactosyl residues are critical for AAV9 infection
  • WGA Wheat germ agglutinin
  • N-GlcNAc N-acetylglucosamine
  • Concanavalin A Con A
  • the SNA lectin had no effect on AAV9 infection and can be explained by low levels of a2,6-sialylated glycans in both cell lines of rodent (hamster) origin (Stults et al. , J. Biol. Chem. 264:19956 (1989)).
  • the MAL I lectin demonstrated 5 to 10-fold inhibition of AAV9 infection in both the Pro5 and Lec2 cell lines. More importantly, a striking difference in AAV9 infectivity was observed in the case of ECL- treated cells with 5-fold inhibition in parental Pro5 cells and nearly 200-fold inhibitory activity in the Lec2 cell line demonstrating the importance of core Gal ⁇ -linked residues.
  • the SNA lectin which recognizes a2,6-sialic acid does not stain either Pro5 or Lec2 cells confirming the lack (or modest expression) of a2,6-sialylated glycans on these hamster-derived cell lines.
  • Cos- 1 cells were untreated (-) or pretreated (+) with Neuraminidase Type III from Vibrio Choler e followed by no treatment (-) or treatment (+) with (A) endo-P-galactosidase (80 mU/ml, from Pseudomonas sp., Sigma #G6920) and (B) a-fucosidase (50 mU/ml, from bovine kidney, Prozyme #GKX-5006).
  • Sialic acid-deficient Lec2 cells B; untreated wild type Pro5 cells ( ⁇ ) and sialidase-treated Pro5 cells (o) were prechilled and incubated with AAV9 particles at different MOI ranging from 100 to 500,000 (across 4.5 orders of magnitude) at 4°C to allow binding, without cellular uptake (FIG. 8).
  • Quantitative analysis of dose-dependent AAV9 binding to cell surface asialo N-glycans was carried out by generating binding curves using a single-site binding model. The inset shows linear range of the binding curve from X-axis values ranging from 100 to 10,000 vg/cell. Calculated binding parameters are listed in Table 4. All experiments were carried out in triplicate. Error bars represent standard error mean.
  • Sialidase pretreatment increases AAV9 gene transfer efficiency in HAE and murine airways
  • C Representative live animal bioluminescent images of luciferase expression in Balb/c mice pretreated with intranasally administered PBS or neuraminidase (200 ⁇ / ⁇ / ⁇ ). Intranasal instillation of AAV9-CBA-Luciferase vectors (5xl0 10 vg/50 ⁇ l/nostr ⁇ l) was carried out 2 hrs post-sialidase treatment and bioluminescent images obtained at 4 weeks post-administration.
  • D Bioluminescence intensity was quantified using Living Image ® software and expressed as relative light units (RLU). Error bars represent standard error mean.
  • luciferase transgene expression in the nasal cavity of Balb/C mice pretreated with PBS (control) or Neuraminidase from Arthrobacter Ureafaciens (200 mU/50uL/nostril) was monitored at 4weeks post-administration with AAV9-CBA-Luciferase vectors (5xl0 10 vg/mouse) via intranasal instillation.
  • Xenogen 1VIS Lumina® system Caliper Lifesciences
  • luciferin substrate 120 mg/kg; Nanolight
  • Image analysis was carried out using Living Image software (FIGS. 10A-10D).
  • LN LacNAc
  • MOI 10 4 vg/cell
  • Glycomic profiles of major N-and O-glycans expressed in the parental CHO Pro5 and the CHO Lec2 mutant were instrumental towards delineating the nature of glycans that play a role in cell surface binding and infection by AAV9 vectors.
  • the N-glycan profile of CHO Pro5 cells has been shown to possess complex bi-, tri-, and tetra-antennary structures bearing multiple N-acetyllactosamine (LacNAc) extensions, capped with sialic acid (NeuAc) residues.
  • the 0-glycan profile contains Gal- ( i,3)-GalNAc core structures that are mono- or di-sialylated.
  • the most abundant glycans produced by the sialic acid-deficient Lec2 cell line are asialo N-glycans, possessing between 2 and 7 LacNAc units.
  • the Oglycan profile is known to be similarly affected by altered sialylation (North et al., J. Biol. Chem. 285:5759 (2010)). These observations suggest that LacNAc units might serve as cell surface attachment factors for AAV9.
  • LacNAc (LN), but not a2,3- sialylated LN (3'-SLN) or a2,3-sialylated di-LN (3'-S-Di-LN) glycans appear to selectively block AAV9 infection (FIG. 11).
  • Carbohydrate receptors utilized by members of different AAV clades appear to fall under two classes, namely, heparan sulfate proteoglycans and sialylated glycans (Wu et ah, Moh Ther. 14:316 (2006)).
  • AAV2 a Clade B member utilizes heparan sulfate proteoglycan as a primary receptor (Summerford et ah, J. Virol. 72:1438 (1998)).
  • the closely related strains, AAVl and AAV6 of Clade A appear to equally prefer a2,3- and 0,2,6-N-linked sialic acid for infection (Wu et ah, J. Virol.
  • AAV6 has been shown to bind heparin implying a potential dual mechanism of interaction with cell surface glycans (Wu et ah, J. Virol. 50:11393 (2006)).
  • the current study identifies a third class of glycan receptors lacking terminal sialic acid utilized by the AAV strain Hu.l4/AAV9 for infection.
  • the latter serotype has been classified under Clade F within the AAV phylogenetic tree (Gao et ah, J. Virol. 75:6381 (2004)).
  • the major capsid protein (VP3, viral protein subunit 3) of other AAV isolates within Clade F is largely similar to Hu. l4/AAV9 (GenBank# AY530579.1).
  • isolate Hu.31 (GenBank# AY530596.1) differs from Hu.l4/AAV9 by 2 amino acid residues (S386G, N716S), while the VP3 subunit of isolate Hu.32 (GenBank# AY530597.1) is identical to
  • co-administration or pretreatment of different tissue types in animal models such as the lung, CNS or eye with recombinant sialidase might serve as (i) a strategy to expose high avidity glycan binding sites and consequently restrict AAV9 transduction to these specific tissue types; (ii) a facile biochemical strategy to increase gene transfer efficiency of AAV9 vectors and (iii) evaluate AAV9 vectors in desialylated preclinical animal models eliminating cross-species variation in sialic acid linkage patterns.
  • Luciferase expression levels are represented as RLU. All experiments were carried out in triplicate. Error bars represent standard error.
  • FIG. 13A shows representative live animal bioluminescent images of luciferase expression.
  • FIG. 13B shows quantification of luciferase transgene expression levels at 7 days post injection in liver, leg muscle (gastrocnemius), and joint. Luciferase expression levels are represented as RLU.
  • FIG. 13C shows vector genome copy numbers of AAV9-CBA-luciferase at 7 days post injection in liver, leg muscle, and joint. Vector genome copy numbers are normalized per ⁇ g of genomic DNA. All experiments were carried out in triplicate. Error bars represent standard error.
  • mice were co-administered AAV9 vectors with PBS or sialidase from Vibrio cholera.
  • AAV9-CBA-luciferase vectors (1 x 10 9 vg) were premixed with PBS or 4 mU of sialidase to reach a total volume of 1 ⁇ for each injection.
  • the left eye of each mouse was injected with AAV9/sialidase while the right eye was injected with AAV9/PBS as a control. Experiments were carried out in duplicate as two separate groups.
  • Bioluminescent images were obtained at 16 days and 157 days post injection.
  • FIG. 14A shows representative live animal bioluminescent images of luciferase expression.
  • FIG. I4B shows quantification of luciferase transgene expression levels at 4 weeks post injection in retina and sclera tissues. Four weeks post intravitreous injection of AAV9/PBS and AAV9/sialidase mixture into mice eyeballs, the animals were sacrificed for tissue harvesting. Retina and sclera tissues were separated, minced and ground before measuring their luciferase activity. Luciferase expression levels are represented as RLU. Error bars represent standard error.
  • FIG. 16A live animal bioluminescence imaging
  • FIGS. 16C and 16E quantitation of luciferase activity in the liver and the heart

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Abstract

L'invention concerne des procédés permettant de renforcer la tranduction de cellules avec des vecteurs de virus adéno-associés (AAV) qui lient des asialoglycans sur la surface desdites cellules ainsi que des procédés permettant de cibler des cellules avec ces vecteurs par désialylation des cellules. L'invention concerne en outre des procédés, des compositions et des trousses permettant d'administrer des acides nucléiques à des cellules à l'aide des vecteurs AAV.
PCT/US2012/024108 2011-02-07 2012-02-07 Transduction ciblée de vecteurs aav Ceased WO2012109214A1 (fr)

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JP2014507154A (ja) * 2011-02-17 2014-03-27 ザ・トラステイーズ・オブ・ザ・ユニバーシテイ・オブ・ペンシルベニア 組織特異性を改変し、ａａｖ９媒介遺伝子導入を改善するための組成物および方法
CN110885854A (zh) * 2013-03-15 2020-03-17 北卡罗来纳-查佩尔山大学 双重聚糖结合aav载体的方法和组合物
CN114126667A (zh) * 2019-05-17 2022-03-01 全国儿童医院研究所 使用糖苷水解酶的基因疗法载体向视网膜细胞的改进递送
WO2022125601A1 (fr) * 2020-12-09 2022-06-16 The University Of North Carolina At Chapel Hill Lignées cellulaires pour la production de virus adéno-associé
WO2023018674A1 (fr) * 2021-08-09 2023-02-16 Amicus Therapeutics, Inc. Détermination de la puissance de transduction des gènes dans les cellules de type neuronal
WO2025098308A1 (fr) * 2023-11-07 2025-05-15 康霖生物科技（杭州）有限公司 Activateur de transduction in vitro et procédé de détection d'efficacité d'une infection

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WO2005005610A2 (fr) * 2003-06-30 2005-01-20 The Regents Of The University Of California Virions de virus adeno-associes mutants et procedes d'utilisation
US20050287122A1 (en) * 2001-01-05 2005-12-29 Children's Hospital Inc. AAV vectors and methods

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US20050287122A1 (en) * 2001-01-05 2005-12-29 Children's Hospital Inc. AAV vectors and methods
WO2005005610A2 (fr) * 2003-06-30 2005-01-20 The Regents Of The University Of California Virions de virus adeno-associes mutants et procedes d'utilisation

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2014507154A (ja) * 2011-02-17 2014-03-27 ザ・トラステイーズ・オブ・ザ・ユニバーシテイ・オブ・ペンシルベニア 組織特異性を改変し、ａａｖ９媒介遺伝子導入を改善するための組成物および方法
US9884071B2 (en) 2011-02-17 2018-02-06 The Trustees Of The University Of Pennsylvania Compositions and methods for altering tissue specificity and improving AAV9-mediated gene transfer
US10406173B2 (en) 2011-02-17 2019-09-10 Trustees Of The University Of Pennsylvania Compositions and methods for altering tissue specificity and improving AAV9-mediated gene transfer
US10918658B2 (en) 2011-02-17 2021-02-16 The Trustees Of The University Of Pennsylvania Compositions and methods for altering tissue specificity and improving AAV9-mediated gene transfer
US11766448B2 (en) 2011-02-17 2023-09-26 The Trustees Of The University Of Pennsylvania Compositions and methods for altering tissue specificity and improving AAV9-mediated gene transfer
CN110885854A (zh) * 2013-03-15 2020-03-17 北卡罗来纳-查佩尔山大学 双重聚糖结合aav载体的方法和组合物
CN114126667A (zh) * 2019-05-17 2022-03-01 全国儿童医院研究所 使用糖苷水解酶的基因疗法载体向视网膜细胞的改进递送
WO2022125601A1 (fr) * 2020-12-09 2022-06-16 The University Of North Carolina At Chapel Hill Lignées cellulaires pour la production de virus adéno-associé
WO2023018674A1 (fr) * 2021-08-09 2023-02-16 Amicus Therapeutics, Inc. Détermination de la puissance de transduction des gènes dans les cellules de type neuronal
WO2025098308A1 (fr) * 2023-11-07 2025-05-15 康霖生物科技（杭州）有限公司 Activateur de transduction in vitro et procédé de détection d'efficacité d'une infection

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