WO2004099423A1 - Vp2-modified raav vectors and uses thereof - Google Patents
Vp2-modified raav vectors and uses thereof Download PDFInfo
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- WO2004099423A1 WO2004099423A1 PCT/US2004/005205 US2004005205W WO2004099423A1 WO 2004099423 A1 WO2004099423 A1 WO 2004099423A1 US 2004005205 W US2004005205 W US 2004005205W WO 2004099423 A1 WO2004099423 A1 WO 2004099423A1
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- expression system
- recombinant adeno
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Classifications
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- 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
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
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- 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
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
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- 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
- C12N2750/14122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
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- 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
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
- C12N2750/00011—Details
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- C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
- C12N2750/14141—Use of virus, viral particle or viral elements as a vector
- C12N2750/14145—Special targeting system for viral vectors
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- C12N2810/00—Vectors comprising a targeting moiety
- C12N2810/50—Vectors comprising as targeting moiety peptide derived from defined protein
Definitions
- the present invention relates generally to the fields of molecular biology and virology, and in particular, to the development of gene delivery vehicles.
- the invention provides VP2-modified recombinant adeno-associated virus (rAAV) vectors that, while deleted for VP2, are still fully virulent. Methods are provided for preparing and using these modified rAAV-based vector constructs in a variety of viral-based gene therapies, and in particular, in the treatment, amelioration, and/or prevention of human diseases.
- rAAV recombinant adeno-associated virus
- AAV adeno-associated virus
- HSPG heparin sulfate proteoglycan
- the present invention overcomes these and other limitations inherent in the prior art by providing new rAAV-based genetic constructs that encode one or more mammalian therapeutic polypeptides for the prevention, treatment, and/or amelioration of various disorders resulting from a deficiency in one or more of such polypeptides.
- the invention provides AAV-based genetic constructs encoding one or more mammalian therapeutic proteins, polypeptides, peptides, antisense oligonucleotides, and ribozymes, as well as variants, and/or active fragments thereof, for use in the treatment and prophylaxis of a variety of conditions and mammalian diseases and disorders.
- AAV2 targeting strategies involve inserting DNA sequences that code for specific receptor ligands within the capsid open reading frame of the pIM45 plasmid. While this approach has identified surface positions capable of tolerating peptide insertions, there are certain limitations. Because the three capsid proteins share the same open reading frame and stop codon, the amino acid sequence of the major capsid protein, VP3, and any peptide ligands inserted in this region of the open reading frame, are contained within the 2 larger and significantly less abundant capsid proteins, VP1 and VP2.
- the inventors In order to target peptide ligands to a specific capsid protein, the inventors have investigated an alternative method for the production of recombinant AAV2 vectors. By mutating the capsid proteins' start codons the inventors have generated pIM45 plasmids that only express one capsid protein: pIM45-VPl, pIM45-VP2 (acg/atg), and pIM45-VP3. Such plasmids can be complemented with plasmids that express the remaining 2 capsid proteins (pIM45-VP2,3, ⁇ IM45-VPl,3, and ⁇ IM45-VPl,2, respectively) in order to produce viable recombinant AAV2 vectors.
- the plasmid, pIM45-VPl,3 is also capable of producing infectious virions in the absence of VP2 expression.
- Expression of the capsid proteins in this manner allows for the genetic modification of a specific capsid protein across its entire sequence. As a result, more control of the position and number of expressed peptide insertions is obtained in producing recombinant AAV2 vectors.
- This system allows for the production of novel targeted recombinant AAV2 vectors containing significantly larger peptide insertions in an individual capsid protein without disruption of the remaining capsid structure.
- the invention concerns rAAV vectors that comprise a nucleic acid segment modified to express functional VP1 and VP3 capsid proteins substantially in the absence of functional VP2 protein.
- rAAV vectors that comprise a nucleic acid segment modified to express functional VP1 and VP3 capsid proteins substantially in the absence of functional VP2 protein.
- the inventors have shown that such a vector can produce an infectious virion in the absence of exogenous VP2 protein.
- the lack or substantial absence of functional VP2 protein may be the result of at least a first mutation in the capsid gene sequence region that comprises the VP2 start codon, or alternatively in the VP2 start codon itself.
- An exemplary vector described herein is pIM45-VPl,3.
- the invention concerns rAAV vectors that comprise a nucleic acid segment modified to express functional VPl and VP2 capsid proteins substantially in the absence of functional VP3 protein. Although such vector cannot produce an infectious virion in the absence of exogenous VP3 protein, if a second helper vector that encodes a functional VP3 protein is employed to coinfect cells with this vector, infectious virions can be obtained.
- the lack or substantial absence of functional VP3 protein may be the result of at least a first mutation in the capsid gene sequence region that comprises the VP3 start codon, or alternatively in the VP3 start codon itself.
- An exemplary vector described herein is pIM45-VPl,2.
- the invention concerns rAAV vectors that comprise a nucleic acid segment modified to express functional VP2 and VP3 capsid proteins substantially in the absence of functional VPl protein.
- rAAV vectors that comprise a nucleic acid segment modified to express functional VP2 and VP3 capsid proteins substantially in the absence of functional VPl protein.
- a second helper vector that encodes a functional VPl protein is employed to coinfect cells with this vector, infectious virions can be obtained.
- the lack or substantial absence of functional VPl protein may be the result of at least a first mutation in the capsid gene sequence region that comprises the VPl start codon, or alternatively in the VPl start codon itself.
- An exemplary vector described herein is pIM45-VP2,3.
- a yet further embodiment of the invention is an expression vector that expresses an rAAV capsid protein selected from the group consisting of VPl, VP2, and VP3, each in the absence of substantially any other rAAV protein, such as the other capsid proteins or helper functions.
- This expression vector may comprise, for example, a mutation at position 1 of the cap gene, a mutation at position 138 of the cap gene, or a mutation at position 203 of the cap gene.
- Exemplary such vectors provided herein are pIM45-VPl, pIM45-VP2, or pIM45-VP3, which produce substantially a single VPl, VP2, or VP3 protein, respectively.
- Another embodiment of the invention is an expression vector that expresses: (a) rAAV capsid proteins VPl and VP2 in the absence of substantial amounts of VP3 protein; (b) rAAV capsid proteins VPl and VP3 in the absence of substantial amounts of VP2 protein; or (c) rAAV capsid proteins VP2 and VP3 in the absence of substantial amounts of VPl protein.
- Such vectors typically comprise: (a) at least one mutation in the start codon of the VPl protein and at least one mutation in the start codon of the VP2 protein; (b) at least one mutation in the start codon of the VPl protein and at least one mutation in the start codon of the VP3 protein; or (c) at least one mutation in the start codon of the VP2 protein and at least one mutation in the start codon of the VP3 capsid protein.
- the vector may comprise: (a) at least one mutation at position 1 and at least one mutation at position 138 of the cap gene, (b) at least one mutation at position 1 and at least one mutation at position 203 of the cap gene; or (c) at least one mutation at position 138 and at least one mutation at position 203 of the cap gene.
- Vectors pIM45-VPl,2; pIM45-VPl,3; and pIM45-VP2,3 described herein, are representative examples of each of such vectors, respectively.
- the invention also provides in an important embodiment, an rAAV expression system substantially lacking in expression of VP2 protein.
- This VP2-free system comprises:(a) at least a first rAAV vector comprising at least a first heterologous nucleic acid segment inserted into the capsid sequence region, with the segment encoding at least a first heterologous peptide; and (b) at least a second expression vector that expresses functional VPl and VP3 capsid proteins in the absence of substantial quantities of VP2 protein, or at least a second and a third expression vector that separately express functional VPl and VP3 capsid proteins, each of these second and third plasmids expressing a single VPl or VP3 protein, both in the absence of substantial amounts of VP2 protein.
- the system will preferably comprise, consist essentially of, or consist of, at least a first rAAV vector that substantially lacks VP2 expression.
- Such expression systems will give rise to infectious virions, so long as the helper plasmids provide sufficient exogenous VPl and VP3 protein to permit the rAAV vector to form the capsid.
- the sequence of the capsid gene when it is desirable to "target" particular cells, cell surfaces, or cell surface ligands or receptors, it may be desirable to alter the sequence of the capsid gene through the addition of one or more relatively short nucleic acid segments that encode at least 1 or more targeting peptides that, when these heterologous peptides are expressed on the- surface of an rAAV virion comprising the vector, the peptide sequence contained within the altered capsid protein will permit the selective targeting of the rAAV virions comprising them to one or more specific types of cells, cell surfaces, or cell surface receptors when the particles are used to transfect a plurality, population, or subpopulation of selected host cells.
- Such methods could be particularly desirable in altering the native affinity of one or more of the various known serotypes of AAV to one or more host cells not previously capable of efficient transfection by one or more particular serotypes.
- an rAAV serotype 1 vector may be able to efficiently transfect a cell line not readily transfected by wild-type rAAVl vectors.
- an rAAV serotype 2 vector may be sufficiently modified by addition of appropriate targeting ligands to effectively transfect one or more cell lines, cells types, tissues, or organs, not previously capable of efficient transfection using the unmodified wild-type rAAV2 vector.
- preferred embodiments include those VP2-free rAAV expression systems, wherein at least a first peptide inserted into one or more of the capsid protein sequences, permits the rAAV virion to transfect a specific organ tissue, or host cell, with a higher efficiency than an unmodified rAAV vector.
- the VP2-free rAAV expression systems of the invention may utilize any rAAV vector, including those of serotypes 1, 2, 3, 4, 5, or 6, and may employ at least two helper plasmids such as pIM45-VPl, pIM45-VP2, or pIM45-VP3, as the second and third expression vectors required in the system to provide exogenous VPl, VP2, and/or VP3 as may be required for efficient virion formation by the rAAV vectors.
- helper plasmids such as pIM45-VPl, pIM45-VP2, or pIM45-VP3, as the second and third expression vectors required in the system to provide exogenous VPl, VP2, and/or VP3 as may be required for efficient virion formation by the rAAV vectors.
- a single vector may be employed such as, for example, pIM45-VPl,3.
- a fully functional, fully virulent rAAV virion may be reconstituted from the disclosed expression system, either in the presence of functional VP2 protein, or alternatively, substantially in the absence of any endogenously- or exogenously-provided VP2 protein.
- vectors When the use of such vectors is contemplated for introduction of one or more exogenous proteins, polypeptides, peptides, ribozymes, and/or antisense oligonucleotides, to a particular cell transfected with the vector, one may employ the rAAV vectors or the VP2- free rAAV expression systems disclosed herein by genetically modifying the vectors to further comprise at least a first exogenous polynucleotide operably positioned downstream and under the control of at least a first heterologous promoter that expresses the polynucleotide in a cell comprising the vector to produce the encoded peptide, protein, polypeptide, ribozyme, or antisense oligonucleotide.
- Such constructs may employ heterologous promoters that are constitutive, inducible, or even cell-specific promoters.
- exemplary such promoters include, but are not limited to, a CMV promoter, a ⁇ -actin promoter, a hybrid CMV promoter, a hybrid ⁇ -actin promoter, an EF1 promoter, a Ula promoter, a Ulb promoter, a Tet-inducible promoter and a VP16-LexA promoter.
- the vectors or expression systems may also further comprise a second nucleic acid segment that comprises, consists essentially of, or consists of, one or more enhancers, regulatory elements, transcriptional elements, to alter or effect transcription of the heterologous gene cloned in the rAAV vectors.
- the rAAV vectors of the present invention may further comprise a second nucleic acid segment that comprises, consists essentially of, or consists of, at least a first CMV enhancer, a synthetic enhancer, or a cell- or tissue-specific enhancer.
- the second nucleic acid segment may also further comprise, consist essentially of, or consist of one or more intron sequences, post- transcriptional regulatory elements, or such like.
- the vectors and expression systems of the invention may also optionally further comprise a third nucleic acid segment that comprises, consists essentially of, or consists of, one or more polylinker or multiple cloning regions to facilitate insertion of selected genetic elements, polynucleotides, and the like into the vectors and expression constructs at convenient restriction sites.
- a third nucleic acid segment that comprises, consists essentially of, or consists of, one or more polylinker or multiple cloning regions to facilitate insertion of selected genetic elements, polynucleotides, and the like into the vectors and expression constructs at convenient restriction sites.
- the invention concerns methods for altering, reducing, or eliminating, the binding of particular rAAV vectors for particular ligands.
- the invention provides rAAV vectors that have altered affinity for heparin, heparin sulfate, and heparin sulfate proteoglycan.
- This vector comprises at least a first mutation in the capsid gene, wherein the mutation substantially reduces or eliminates the affinity of a viral particle comprising the vector for binding to heparin, heparin sulfate, or heparin sulfate proteoglycan.
- these rAAV vectors comprise one or more Arginine to Alanine mutations, and particularly one or more Arginine to Alanine mutations at position 585 or position 588 of the capsid polypeptide sequence.
- rAAV vectors comprising either a single R585A or R588A mutation, or a double mutant comprising both the R585A and the R588A mutations, affinity for heparin sulfate binding by the vector was eliminated.
- Such vectors are therefore important when one wishes to design improved rAAV vectors that comprise particular capsid protein mutations that either have increased or reduced affinity for one or more particular ligands.
- the exogenous polynucleotides that are comprised within one or more of the improved rAAV vectors disclosed herein will be of mammalian origin, with polynucleotides of human, primate, murine, porcine, bovine, ovine, feline, canine, equine, epine, caprine, or lupine origin being particularly preferred.
- the exogenous polynucleotide will preferably encode one or more proteins, polypeptides, peptides, ribozymes, or antisense polynucleotides, oligonucleotides, PNA molecules, or a combination of two or more of these therapeutic agents.
- the exogenous polynucleotide may encode two or more such molecules, or a plurality of such molecules as may be desired.
- two or more different molecules may be produced from a single rAAV expression system, or alternatively, a selected host cell may be transfected with two or more unique rAAV expression systems, each of which may comprise a distinct polynucleotide.
- the invention also concerns the disclosed rAAV vectors comprised within an infectious adeno-associated viral particle or virion, or pluralities thereof, which may also be further comprised within one or more pharmaceutical vehicles, formulated for administration to a mammal such as a human for therapeutic, and/or prophylactic gene therapy regimens.
- Such vectors, virus particles, virions, and pluralities thereof may also be provided in excipient formulations that are acceptable for veterinary administration to selected livestock, exotic or domesticated animals, pets, and the like.
- the invention also concerns host cells that comprise at least one of the disclosed rAAV vectors or expression systems.
- host cells are particularly mammalian host cells, with human host cells being particularly highly preferred, and may be either isolated, in cell or tissue culture, or even within the body of the animal itself.
- non-human host cells, or isolated human host cells that comprise one or more of the disclosed AAV vectors is also contemplated to be useful for a variety of diagnostic, and laboratory protocols, including, for example, means for the production of large-scale quantities of the rAAV vectors described herein.
- virus production methods are particularly desirable to obtain the often high-titer viral stocks required by many gene therapy protocols.
- compositions comprising one or more of the disclosed rAAV vectors, expression systems, infectious AAV particles, or host cells also form part of the present invention, and particularly those compositions that further comprise at least a first pharmaceutically- acceptable excipient for use in the manufacture of medicaments and methods involving therapeutic administration of such rAAV vectors.
- Such pharmaceutical compositions may optionally further comprise liposomes, a lipid, a lipid complex; or the rAAV vectors may be comprised within a microsphere or a nanoparticle.
- Pharmaceutical formulations suitable for intramuscular, intravenous, or direct injection into an organ or tissue or a plurality of cells or tissues of a human or other mammal are particularly preferred.
- compositions that comprise one or more of the AAV vectors disclosed herein, such as for example pharmaceutical formulations of the vectors intended for administration to a mammal through suitable means, such as, by intramuscular, intravenous, or direct injection to cells, tissues, or organs of a selected mammal.
- suitable means such as, by intramuscular, intravenous, or direct injection to cells, tissues, or organs of a selected mammal.
- compositions may be formulated with pharmaceutically-acceptable excipients as described hereinbelow, and may comprise one or more liposomes, lipids, lipid complexes, microspheres or nanoparticle formulations to facilitate administration to the selected organs, tissues, and cells for which therapy is desired.
- Kits comprising one or more of the disclosed vectors, virions, viral particles, transformed host cells or pharmaceutical compositions comprising such; and (ii) instructions for using the kit in a therapeutic, diagnostic, or clinical embodiment also represent preferred aspects of the present disclosure.
- kits may further comprise one or more reagents, restriction enzymes, peptides, therapeutics, pharmaceutical compounds, or means for delivery of the compositions to host cells, or to an animal, such as syringes, injectables, and the like.
- kits may be therapeutic kits for treating, preventing, or ameliorating the symptoms of particular diseases, and will typically comprise one or more of the modified AAV vector constructs, expression systems, virion particles, or therapeutic compositions described herein, and instructions for using the kit.
- kits may also be used in large-scale production methodologies to produce large quantities of the viral vectors.
- Another important aspect of the present invention concerns methods of use of the disclosed vectors, virions, expression systems, compositions, and host cells described herein in the preparation of medicaments for preventing, treating or ameliorating the symptoms of various diseases, dysfunctions, or deficiencies in an animal, such as a vertebrate mammal.
- Such methods generally involve administration to a mammal, or human in need thereof, one or more of the disclosed vectors, virions, viral particles, host cells, compositions, or pluralities thereof, in an amount and for a time sufficient to prevent, treat, or lessen the symptoms of such a disease, dysfunction, or deficiency in the affected animal.
- the methods may also encompass prophylactic treatment of animals suspected of having such conditions, or administration of such compositions to those animals at risk for developing such conditions either following diagnosis, or prior to the onset of symptoms.
- FIG. 1A, FIG. IB and FIG. IC show generation of plasmids that express two capsid proteins through missense mutation of individual capsid protein start codons.
- FIG. 1A shows mutations required to eliminate VPl and VP2 expression. Immunoblot of whole cell lysates using Bl antibody that recognizes all three capsids following transfection of plasmids, pIM45(lanel); pIM45-VP2,3 (lane2); pIM45-VPl,3 (lane3); and pIM45-M203L (lane 4). Note, lane 4 is the initial attempt to produce plasmid that expresses only VPl and VP2. Further mutations are required.
- FIG. IB shows mutations required to eliminate VP3 expression.
- FIG. IC shows alternative mutation used to eliminate VP3 expression while maximizing expression of VP2 protein.
- FIG. 2 shows generation of plasmids that express a single capsid protein. Immunoblot of whole cell lysates using Bl antibody that recognizes all three capsid proteins following transfection of ⁇ IM45 (lanel); ⁇ IM45-VPl (lane2); pIM45-VP2 (lane3) ⁇ IM45- VP2A (lane4); pIM45-VP3 (lane5).
- FIG. 3A and FIG. 3B show production and purification of rAAV2-like particles that lack expression of specific capsid proteins.
- FIG. 3A shows analysis of effects of missense mutations required to eliminate VP3 expression.
- Left panel shows immunoblot using Bl antibody that recognizes all three capsid proteins of purified particle stocks from pIM45 (lanel); ⁇ IM45-M203L (lane2); pIM45-M211L (lane3); pIM45-M235L (lane4), ⁇ IM45- M203,211,235 (lane 5).
- Right panel shows dot blot autoradiograph of DNA extracted from same particle stocks.
- FIG. 3B shows analysis of effects of eliminating a single capsid on the production and purification of virus particles.
- Left panel shows immunoblot using Bl antibody that recognizes all three capsid proteins of purified particle stocks from pIM45 (lanel); ⁇ IM45-VPl,2 (lane2); ⁇ IM45-VPl,3 (lane3); and ⁇ IM45-VP2,3 (lane4).
- Right panel shows dot blot autoradiograph of DNA extracted from same particle stocks. Aliquots from an iodixinal step gradient were with incubated with DNAsel, inactivated with EDTA, digested with proteinase K, phenol hloroform extracted, and precipitated with ethanol. DNA was transferred to nitrocellulose and probed with radiolabelled GFP probe.
- FIG. 4 shows complementation capsid plasmid groups employed to produce viable rAAV2 particle preparations.
- Group VPO is a control group consisting of pIM45 and pIM45- VPO (all capsid expression eliminated).
- Group VPl is group consisting of pIM45-VPl and pIM45-VP2,3 in which expression of VPl is isolated.
- Group VP2/VP2A is group consisting of ⁇ IM45-VP2 or ⁇ IM45-VP2A and ⁇ IM45-VPl,3 in which expression of VP2 is isolated, and in case of pIM45-VP2A, VP2 expression is maximized.
- Group VP3 is group consisting of pIM45-VP3 and pIM45-VPl,2 in which expression of VP3 is isolated. Isolation of specific capsid proteins allows genetic modification of the isolated capsid without further modifying remaining capsids. Alternatively, genetic modification of two capsids can be accomplish without further modification of remaining capsid. These groups are cotransfected with pXX6 (Ad helper functions) and pTR-UF5 (terminal repeats flanking expression cassette with CMV promoter driving expression of GFP) to produce rAAV vectors.
- pXX6 Ad helper functions
- pTR-UF5 terminal repeats flanking expression cassette with CMV promoter driving expression of GFP
- FIG. 5A and FIG. 5B show production and purification of rAAV2-like particles from complementation groups described in FIG. 4.
- FIG. 5A, right panel, shows immunoblot using
- FIG. 5A Right panel shows dot blot autoradiograph of DNA extracted from same particle stocks. Aliquots from an iodixinal step gradient were with incubated with DNAsel, inactivated with EDTA, digested with proteinase K, phenolxhloroform extracted, and precipitated with ethanol. DNA was transferred to nitrocellulose and probed with radiolabelled GFP probe.
- FIG. 5A Right panel shows dot blot autoradiograph of DNA extracted from same particle stocks. Aliquots from an iodixinal step gradient were with incubated with DNAsel, inactivated with EDTA, digested with proteinase K, phenolxhloroform extracted, and precipitated with ethanol. DNA was transferred to nitrocellulose and probed with radiolabelled GFP probe.
- FIG. 5B left panel, shows immunoblot using Bl antibody that recognizes all three capsid proteins of purified particle stocks from transfection of pIM45-VP2A and pIM45-VP3 showing production of rAAV2-like particles composed of VP2 and VP3 with increased VP2 levels relative to VP3.
- FIG. 6A, FIG. 6B and FIG. 6C depict production of rAAV2-like particles with large peptide insertions in VPl and VP2 capsid proteins.
- FIG. 6A shows production scheme for insertion of large peptides in VPl and VP2 (top) involves insertion of peptide immediately after amino acid 138 in a plasmid that expresses only VPl and VP2 (pIM45-VPl,2A) and complementing this plasmid with plasmid, pIM45-VP3, to produce particles.
- FIG. 6B shows immunoblot of purified rAAV2-like particles produced by above production schemes with protein, leptin, inserted in VPl and VP2 or only in VP2.
- FIG. 6C shows immunoblot of purified rAAV2-like particles produced by above production schemes with protein, GFP, inserted in VPl and VP2 or only in VP2.
- Lane 1 pIM45; Lane 2: pIM45-VPl,2A-GFP/pIM45-VP3; Lane 3: pIM45-VP2A- GFP/pIM45-VPl,3; Lane 4: pIM45-VP3 only; pIM45-VPl,3 only.
- FIG. 7 shows Western blot of iodixanol virus stocks. Equal volumes of virus stock were separated by 10% SDS-PAGE and analyzed by Western blot using the Bl antibody.
- FIG. 8 shows heparin-agarose binding profiles of mutant capsids.
- FIG. 9A and FIG. 9B show production and purification of AAV serotypes.
- FIG. 9A shows equivalent amounts of iodixanol purified AAV1, AAV2 and AAV5 were separated by 10% PAGE and analyzed by Western blot using the Bl antibody.
- FIG. 9B shows heparin- agarose binding properties of AAV2, AAVl and AAV5. Abbreviations are the same as FIG. 8.
- FIG. 10 shows particle-to-infectivity ratios of mutants relative to wild type.
- the particle-to-infectivity ratio for each mutant was calculated by dividing the average genomic titer by the average green cell assay titer (Table 2).
- the P/I ratio of each mutant was then normalized to wild type by dividing the P I of each mutant by the P/I of wild type rAAV2, and the logio value of the ratio was plotted. Wild type, therefore, equals one and is indicated by the dashed line.
- Grey bars mutant viruses with infectivity comparable to wild type; Black bars, mutant viruses that are heparin binding deficient; White bars, mutant viruses with an undetermined block to infectivity; Asterisks indicate those mutants for which no green cells were scored.
- the green cell assay titer used was the limit of detection in the assay.
- the log difference is a minimum estimate.
- FIG. 11 shows GFP transduction ability of mutants in HeLa C12 cells.
- FIG. 12A and FIG. 12B show binding and uptake of rAAV2 and R585A/R588A genomes in Hela C12 cells.
- FIG. 12b shows the percent bound/internalized DNA was calculated by dividing the total DNA present in both the media and the cell pellet by the amount bound/internalized for each time point. The average of three determinations is shown. Error bars indicate a standard deviation.
- FIG. 13A, FIG. 13B, FIG. 13C and FIG. 13D show modifying the heparin binding properties of AAV5.
- FIG. 13A shows alignment of AAV2 amino acid residues 585 through 590 to residues predicted by amino acid alignment to be structurally equivalent in AAV5.
- FIG. 13B shows Western blot of iodixanol virus stocks. Equal volumes of virus were separated by 10% SDS-PAGE and analyzed by Western blot using the Bl antibody.
- FIG. 13C shows novel heparin binding properties of AAV5-HS. Heparin-agarose binding was performed as described in FIG. 8. See FIG. 8 for abbreviations.
- FIG. 17D shows, the log of the particle-to-infectivity ratio of the rAAV5 variants normalized to wild type rAAV2 as described in FIG. 10.
- FIG. 14 shows an immunoslotblot of total capsid protein from novel production system following standard purification procedures. Immunoslotblot was probed with anti- VP1,2,3 monoclonal antibody. 1. pIM45/ ⁇ IM45-VP0; 2. pIM45-VPl/ ⁇ IM45-VP2,3; 3. pIM45-VP2acg/pIM45-VPl,3; 4. pIM45-VP2atg/pIM45-VPl,3; 5. pIM45-VP3/pIM45- VP1,2.
- FIG. 15 shows a dot blot autoradiograph of DNA extracted from pTR-UF5 and system plasmid combinations. Numbering scheme is the same as described in FIG. 14. Equal volume aliquots from an iodixinol step gradient were with incubated with DNAsel, inactivated with EDTA, digested with proteinase K, phenol hloroform extracted, and precipitated with ethanol. DNA was transferred to nitrocellulose and probed with radiolabeled GFP probe.
- FIG. 16 shows the in vivo transduction ability of recombinant AAV vectors produced using various system components. GFP fluorescence microscopy was performed on Hela C12 infected at an MOI of 1000 genomes/cell 24 hours post infection.
- FIG. 17 shows the Immunoblot and dot blot autoradiograph of virions produced from ⁇ TR-UF5; pIM45-VPl,2; ⁇ IM45-VPl,3; and ⁇ IM45-VP2,3 plasmids following standard purification protocols.
- the capsid proteins VPl, VP2, and VP3 are indicated. No virions were obtained in 40% iodixanol fraction from plasmid pIM45-VPl,2.
- FIG. 18 shows the in vivo transduction ability of recombinant AAV vectors containing only two capsid proteins. GFP fluorescence microscopy was performed on Hela C12/24 hours post infection.
- FIG. 19 depicts an immunoblot of protein fractions collected from iodixinol purified passed over a heparin-agarose column. Immunoblot was probed with anti-VPl,2,3 monoclonal antibody. C, 5E+10 virus particles loaded directly onto blot, FT, flowthrough fraction, W, wash fraction, E, 2M NaCI fraction
- FIG. 20 shows a dot blot autoradiograph of DNA extracted from pTR-UF5 and rAAV R585A, R588A. Equal volume aliquots from an iodixinal step gradient were with incubated with DNAsel, inactivated with EDTA, digested with proteinase K, pheno chloroform extracted, and precipitated with ethanol. DNA was transferred to nitrocellulose and probed with radiolabeled GFP probe.
- FIG.21 shows the in vivo transduction ability of ⁇ TR-UF5 and R585A, R588A.
- GFP fluorescence microscopy was performed on Hela C12 and HEK 293 cells infected at an MOI of 1000 genomes/cell 24 hours post infection.
- FIG. 22 shows a slot blot autoradiograph of an in vivo DNA tracking time course experiment of pTR-UF5, rAAV R585A, R588A.
- Media and cells infected with ⁇ TR-UF5 and rAAV R585A, R588A were collected at 1,4, and 20 hours post infection.
- Hirt DNA was extracted, transferred to nitrocellulose and probed with a radiolabeled GFP probe.
- FIG. 23 shows a schematic diagram of the pIM45 vector showing the rep and cap sequences.
- FIG. 24A and FIG. 24B show Western blot analysis of AAV capsid proteins in 293 cell lysates (FIG. 24A) and iodixanol purified virus stocks (FIG. 24B) following insertion of FKN or LEP peptides after residue 138 in the Eagl/Mlul cloning site engineered in the VP1/2 overlap region. Equal volumes of lysates or virus stocks were separated by SDS- 10% polyacrylamide gel electrophoresis and analyzed by Western blot using the Bl antibody. The diagram illustrates the position of the insertion of the E/M cloning site and the FKN and LEP ligands.
- FIG. 25A, FIG. 25B and FIG. 25C show mutants that express only two capsid proteins.
- FIG. 25 A shows the missense mutations within the start codons of the three capsid proteins (MIL, T138L, and M203L) are illustrated along with the capsid proteins expressed from each mutant on an SDS acrylamide gel blotted with Bl antibody.
- FIG. 25B shows the VP3-like proteins that result from read-through translation.
- FIG. 25C shows an alternative approach to eliminating VP3 expression while maximizing VP2 expression.
- ⁇ VPl,2A contains a standard ATG start codon for VP2 instead of ACG, a T138M mutation, thereby increasing VP2 expression and eliminating VP3 expression (compare pVPl,2A in FIG. 25C to pVPl,2 in FIG. 25B).
- FIG. 26 shows mutants that express only a single capsid protein. Equal volumes of 293 cell extracts transfected with capsid mutants that express a single capsid protein were separated by SDS-10% polyacrylamide gel electrophoresis and analyzed by Western blot using Bl antibody. The diagram illustrates the missense mutation(s) in each construct.
- FIG. 27A, FIG. 27B and FIG. 27C show which capsid mutants can make a virus particle.
- FIG. 27A shows the effect of the M203L, M211L, and M235L mutations on particle formation.
- FIG. 27B shows particle formation from mutants that lack a specific capsid protein.
- FIG. 27C shows particle formation from mutants that express a single capsid protein.
- FIG. 28A and FIG. 28B show complementation of mutants that make a single capsid protein.
- FIG. 28A shows Western blot analysis of AAV particles purified by iodixanol step gradients and heparin column chromatography following transfection of 293 cells with complementation groups described in Table 8.
- FIG. 28B shows Western blot analysis of iodixanol fractions of particles obtained from transfection with pVP2A, pVP3 or both plasmids. Equal volumes of purified virus stocks were separated by SDS-10% acrylamide gel electrophoresis and analyzed by Western blot using the Bl antibody.
- FIG. 29A, FIG. 29B and FIG. 29C show capsid complementation strategy for creating particles with large peptide insertions in the VP1/VP2 overlap region.
- FIG. 29A shows a diagram of constructs used to complement insertions at amino acid 138 in both VPl and VP2A or just VP2A.
- FIG. 29B shows particles with the FKN insertion were purified by iodixanol gradients and probed on SDS- 10% polyacrylamide gels with anti-capsid (Bl) antibody or anti-FKN antibody.
- FIG. 29C shows particles with the LEP insertion were purified by iodixanol gradients and probed on SDS-10% polyacrylamide gels with anti-capsid (Bl) antibody or anti-LEP antibody.
- FIG. 30A, FIG. 30B and FIG. SOC show capsid protein stoichiometry and infectivity of AAV virus stocks missing a capsid protein or containing a ligand insertion.
- FIG. 30A shows Western blot of virus stocks purified by iodixanol gradients and heparin sulfate column chromatography. Approximately 1 X 10 11 AAV-like particles were separated by SDS-10% polyacrylamide gel electrophoresis and analyzed by Western blot using the Bl antibody.
- FIG. 30B shows particle to infectivity ratios of AAV-like particles relative to that of pIM45.
- the particle to infectivity (P/I) ratio for each particle was calculated by dividing the average genomic titer by the average FCA titer (see Table 7). The P/I ratio for each type of virus was then normalized to that of wild type virus (pIM45) by dividing the P/I of each AAV-like particle by the P/I of pIM45, and the loglO value of the ratio was plotted. The wild type pIM45 ratio equals zero and is indicated by the dashed line. Grey bars, particles with infectivity comparable to pIM45 (within 1 log); white bars, particles with significantly reduced infectivity (1-4 logs lower infectivity), black bars, particles that were essentially non-infectious (> 4 logs). FIG.
- FIG. 30C shows Western blot of approximately 1 X 10 11 AAV-like particles with GFP inserted in the capsid.
- Virus samples were purified as in FIG. 30A above, fractionated by SDS-10% polyacrylamide gel electrophoresis and analyzed by Western blot using the Bl antibody.
- FIG. 31 shows time course of VP1,2A-GFP + VP3 particle trafficking following infection in the absence (top panel) and presence (bottom panel) of Ad 5.
- HeLa cells were infected with AAV containing a GFP insertion at an MOI of 10,000 ⁇ Ad 5 at an MOI of 20.
- Vectors remained on the cells for the duration of the time course.
- the input capsids appear green from the native GFP fluorescence of the capsid, the nuclei are stained red with propidium iodide and the early endosomal antigen, EER1, is stained blue.
- Adeno-associated virus 2 (Muzyczka and Berns, 2001) requires the assembly of 60 individual structural proteins into a non-enveloped, T-l icosahedral lattice capable of protecting a 4.7 kb single-stranded DNA genome (Kronenberg et ah, 2001; Xie et al., 2002).
- Purified infectious AAV particles contain three major structural proteins designated VPl, VP2 and VP3 (87, 73 and 62 kDa, respectively) in an approximate ratio of 1:1:18 (Buller and Rose, 1978).
- the three AAV capsid proteins are produced in an overlapping fashion from the cap ORF using alternative mRNA splicing of the transcript and alternative translational start codon usage (Becerra et al., 1988 Becerra et al., 1985; Cassinotti et al., 1988; Janik et al., 1984; McPherson and Rose, 1983; Rose et al., 1971; Trempe and Carter, 1988; Weger et al., 1997).
- a common stop codon is employed for all three proteins (Srivastava et al., 1983).
- AAV capsid protein expression in SF9 cells also suggests an essential role for VP2 in particle formation.
- the requirement for either VPl or VP2 for capsid assembly seems to correlate with a lower nuclear localization of VP3, the most abundant capsid protein (Hoque et al., 1999; Ruffing et al., 1992; Steinbach et al., 1997).
- AAV2 helper-dependent human parvovirus
- the mature 20 nm diameter AAV2 particle is composed of three structural proteins designated VPl, VP2, and VP3 (molecular masses of 87, 73, and 62 kDa respectively) in a ratio of 1:1:18.
- VPl proteins three are VP2 proteins
- VP3 proteins fifty-four are VP3 proteins.
- AAV serotypes unique among parvoviruses, as all others known package their genomes within icosahedral particles composed of only two capsid proteins.
- the anti- parallel ⁇ -strand barreloid arrangement of these 60 capsid proteins results in a particle with a defined tropism that is highly resistant to degradation.
- the AAV2 genome contains two large open reading frames (ORF), rep and cap, flanked by inverted terminal repeats.
- the AAV2 capsid proteins are produced in an overlapping fashion from the cap ORF; arising through alternative mRNA splicing of the transcript (initiated at the p40 promoter), with subsequent alternative translational start codon usage.
- a common stop codon is employed for all three capsid proteins.
- Correct capsid protein stoichiometry is maintained by translating VPl from the 2.4 KB mRNA, while VP2 and VP3 arise from the 2.3-kB mRNA using a weaker ACG start codon for VP2 protein production with resultant read-through translation for the production of the VP3 protein.
- VP3 is contained within the significantly less abundant and longer VPl and VP2 proteins.
- VPl's unique 137 amino acid N-terminal extension of VP2 contains a phospholipase enzymatic activity important for viral infectivity.
- VP2 extends the N-terminus of VP3 by 64 amino acids with this VP1/VP2 overlap region possessing a putative nuclear localization signal (NLS) involved in the nuclear translocation of VP2.
- NLS nuclear localization signal
- the VP3 region common to all three capsid proteins contains the critical ⁇ -barrel structural motifs characteristic of all parvoviruses and particle surface loops involved in determining viral tropism.
- AAV2 particle Great interest in the assembly, structure, and mutability of the AAV2 particle results from its promise as a recombinant gene delivery vehicle (rAAV2) in vivo.
- Manipulation of the rAAV2 particle in order to control its cellular receptor interactions is essential for vector targeting.
- the feasibility of various targeting strategies based on AAV cap ORF mutagenesis is currently an area of active investigation.
- a better understanding of the AAV2 particle surface architecture through systematic scanning-alanine and insertional mutagenesis of the AAV cap ORF and recent publication of the AAV2 crystal structure has identified several amino acid regions on the surface of the particle that tolerate sequence alteration without loss of capsid stability or integrity.
- sequence mutation of the overlapping cap ORF is that mutation of only one capsid protein across its entire sequence is currently not possible. The full potential in manipulation of the particle is not reached with direct alteration of regions of capsid overlap. Predicted surface regions of capsid overlap leading to defective phenotypes upon mutagenesis may allow production of viable particles if such mutations were only in one or two of the capsid proteins.
- An additional degree of flexibility in modifying the rAAV2 particle would result from the ability to mutate the entire coding region of a specific capsid protein without altering the remaining two capsid proteins.
- the N-terminal 137 amino acids of VPl are the only region of the cap ORF where mutations are restricted to a single capsid protein. Successful insertions within this region have included HA and serpin.
- the VP1/VP2 overlap region (amino acid 138-202) also has been receptive to sequence modification. Insertions in this region have included HA, serpin and luetinizing hormone receptor ligand sequences immediately following amino acid 138 in the cap ORF.
- the success of inserting sequences to the VPl and VP1/VP2 regions may be due in part to less disruption of the integrity of the particle compared to insertion in the VP3 region of capsid overlap (amino acid 203-735). It is important to note that these mutant particles would require further mutation of the putative heparin-binding motif to restrict infection to the target cell. Not surprisingly, since it is the longest region of capsid protein overlap, contains many critical structural motifs, and targeting sequences in this region have 60 representatives in the rAAV particle, mutations in the VP3 region of the AAV2 cap ORF have resulted in the highest number of defective phenotypes. Yet, one location within the
- VP3 region has received much attention for the successful insertion of small targeting sequences in all three capsid proteins (amino acid 587).
- amino acid 587 One major advantage of targeting insertions to this position is that the resultant mutant particle also has lost the ability to bind its native receptor.
- Viable mutations in the VP3 region of the cap ORF have been restricted in size ( ⁇ 30 amino acids).
- rAAV2 particles One caveat of creating genetically-targeted rAAV2 particles, is the consideration that many cell surface receptors have ligands whose coding sequence are much larger than those successfully inserted directly into the overlapping cap ORF. Due to the modest size of this ORF ( ⁇ 2kB), the insertion of larger peptide sequences into the capsid coding sequences may result in serious disruption of splicing, read-through translation, capsid structure and/or stability. The insertion of large sequences into the rAAV2 particle have been limited to a study involving the fusion of the CD34 single chain antibody coding sequence with the N- termini of the individual capsid proteins following isolation of their expression to separate CMV promoters.
- Viable CD34-retargeted rAAV particles of extremely low titer were produced only when this fusion was to VP2 protein, and co-expression of wild-type VP2 protein was required. Nonetheless, the fusion of large peptide sequences to the N-terminus of VP2 does not interfere with the incorporation of this capsid protein into the rAAV2 particle.
- AAV adeno-associated virus type 2
- cap ORF capsid open reading frame
- the invention has produced a method that can be used to insert large (up to 30 kDa) peptide ligands into the AAV particle. This system allows greater flexibility than current approaches in genetically manipulating the composition of the AAV particle, and, in particular, may allow vector retargeting to alternative receptors requiring interaction with full length conformation dependent peptide ligands.
- the adeno-associated virus type-2 uses heparan sulfate proteoglycan (HSPG) as its primary cellular receptor.
- HSPG heparan sulfate proteoglycan
- biochemical information about heparin/heparin sulfate (HS), AAV serotype protein sequence alignments, and data from previous capsid studies was used to select residues for mutagenesis.
- charged-to- alanine substitution mutagenesis was performed on individual and combinations of basic residues for the production and purification of recombinant viruses that contained a GFP reporter gene cassette.
- Intact capsids were assayed for their ability to bind to heparin-agarose in vitro and virions that packaged DNA were assayed for their ability to transduce normally permissive cell lines. It was found that mutation of arginine residues at position 585 or 588 eliminated binding to heparin-agarose. Mutation of residues R484, R487, and K532 showed partial binding to heparin-agarose.
- the genetic constructs of the present invention may be prepared in a variety of compositions, and may also be formulated in appropriate pharmaceutical vehicles for administration to human or animal subjects.
- the AAV molecules of the present invention and compositions comprising them provide new and useful therapeutics for the treatment, control, and amelioration of symptoms of a variety of disorders.
- pharmaceutical compositions comprising one or more of the nucleic acid compounds disclosed herein provide significant advantages over existing conventional therapies - namely, (1) their reduced side effects, (2) their increased efficacy for prolonged periods of time, (3) their ability to increase patient compliance due to their ability to provide therapeutic effects following as little as a single administration of the selected therapeutic AAV composition to affected individuals. Exemplary pharmaceutical compositions and methods for their administration are discussed in significant detail hereinbelow.
- compositions comprising one or more of the disclosed vectors, expression systems, virions, viral particles; or mammalian cells.
- compositions may further comprise a pharmaceutical excipient, buffer, or diluent, and may be formulated for administration to an animal, and particularly a human being.
- Such compositions may further optionally comprise a liposome, a lipid, a lipid complex, a microsphere, a microparticle, a nanosphere, or a nanoparticle, or may be otherwise formulated for administration to the cells, tissues, organs, or body of a mammal in need thereof.
- compositions may be formulated for use in therapy, such as for example, in the amelioration, prevention, or treatment of conditions such as peptide deficiency, polypeptide deficiency, tumor, cancer or other malignant growth, neurological dysfunction, autoimmune diseases, lupus, cardiovascular disease, pulmonary disease, ischemia, stroke, cerebrovascular accidents, diabetes and diseases of the pancreas, neural diseases, including Alzheimer's, Huntington's, Tay-Sach's, and Parkinson's diseases, memory loss, trauma, motor impairment, and the like, as well as biliary, renal or hepatic disease or dysfunction, as well as musculoskeletal diseases including, for example, arthritis, cystic fibrosis (CF), amyotrophic lateral sclerosis (ALS), multiple sclerosis (MS), muscular dystrophy (MD), and such like, to name only a few.
- conditions such as peptide deficiency, polypeptide deficiency, tumor, cancer or other malignant growth, neurological dysfunction, autoimmune diseases, lup
- the present invention concerns formulation of one or more of the rAAV compositions disclosed herein in pharmaceutically acceptable solutions for administration to a cell or an animal, either alone or in combination with one or more other modalities of therapy, and in particular, for therapy of human cells, tissues, and diseases affecting man.
- nucleic acid segments, RNA, DNA or PNA compositions that express one or more of therapeutic gene products may be administered in combination with other agents as well, such as, e.g., proteins or polypeptides or various pharmaceutically-active agents, including one or more systemic or topical administrations of therapeutic polypeptides, biologically active fragments, or variants thereof.
- agents such as, e.g., proteins or polypeptides or various pharmaceutically-active agents, including one or more systemic or topical administrations of therapeutic polypeptides, biologically active fragments, or variants thereof.
- the rAAV compositions may thus be delivered along with various other agents as required in the particular instance.
- Such compositions may be purified from host cells or other biological sources, or alternatively may be chemically synthesized as described herein.
- compositions may further comprise substituted or derivatized RNA, DNA, or PNA compositions.
- Formulation of pharmaceutically-acceptable excipients and carrier solutions is well- known to those of skill in the art, as is the development of suitable dosing and treatment regimens for using the particular compositions described herein in a variety of treatment regimens, including e.g., oral, parenteral, intravenous, intranasal, and intramuscular administration and formulation.
- these formulations may contain at least about 0.1% of the active compound or more, although the percentage of the active ingredient(s) may, of course, be varied and may conveniently be between about 1 or 2% and about 70% or 80% or more of the weight or volume of the total formulation.
- the amount of active compound(s) in each therapeuticaUy- useful composition may be prepared is such a way that a suitable dosage will be obtained in any given unit dose of the compound. Factors such as solubility, bioavailability, biological half-life, route of administration, product shelf life, as well as other pharmacological considerations will be contemplated by one skilled in the art of preparing such pharmaceutical formulations, and as such, a variety of dosages and treatment regimens may be desirable.
- compositions disclosed herein either subcutaneously, intraocularly, intravitreally, parenterally, subcutaneously, intravenously, intracerebro-ventricularly, intramuscularly, intrathecally, orally, intraperitoneally, by oral or nasal inhalation, or by direct injection to one or more cells, tissues, or organs by direct injection.
- the methods of administration may also include those modalities as described in U. S. Patent 5,543,158; U. S. Patent 5,641,515 and U. S. Patent 5,399,363 (each specifically incorporated herein by reference in its entirety).
- Solutions of the active compounds as freebase or pharmacologically acceptable salts may be prepared in sterile water and may also suitably mixed with one or more surfactants, such as hydroxypropylcellulose. Dispersions may also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
- the pharmaceutical forms of the AAV-based viral compositions suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions (U. S. Patent 5,466,468, specifically incorporated herein by reference in its entirety).
- the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi.
- the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and/or vegetable oils.
- polyol e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like
- suitable mixtures thereof e.g., vegetable oils
- vegetable oils e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like
- suitable mixtures thereof e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like
- vegetable oils e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like
- Proper fluidity may be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion
- isotonic agents for example, sugars or sodium chloride.
- Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.
- the solution may be suitably buffered, if necessary, and the liquid diluent first rendered isotonic with sufficient saline or glucose.
- aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration.
- a sterile aqueous medium that can be employed will be known to those of skill in the art in light of the present disclosure.
- one dosage may be dissolved in 1 ml of isotonic NaCI solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion, (see for example, "Remington's Pharmaceutical Sciences” 15th Edition, pages 1035- 1038 and 1570-1580).
- Some variation in dosage will necessarily occur depending on the condition of the subject being treated.
- the person responsible for administration will, in any event, determine the appropriate dose for the individual subject.
- preparations should meet sterility, pyrogenicity, and the general safety and purity standards as required by FDA Office of Biologies standards.
- Sterile injectable solutions are prepared by incorporating the active AAV vector- delivered therapeutic polypeptide-encoding DNA fragments in the required amount in the appropriate solvent with several of the other ingredients enumerated above, as required, followed by filtered sterilization.
- dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above.
- the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
- compositions disclosed herein may also be formulated in a neutral or salt form.
- Pharmaceutically-acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimemylamine, histidine, procaine and the like.
- solutions Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective.
- the formulations are easily administered in a variety of dosage forms such as injectable solutions, drug-release capsules, and the like.
- carrier includes any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, and the like.
- carrier includes any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, and the like.
- the use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated. Supplementary active ingredients can also be inco ⁇ orated into the compositions.
- compositions that do not produce an allergic or similar untoward reaction when administered to a human, and in particular, when administered to the human eye.
- aqueous composition that contains a protein as an active ingredient is well understood in the art.
- injectables either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid prior to injection can also be prepared.
- the preparation can also be emulsified.
- AAV compositions and time of administration of such compositions will be within the purview of the skilled artisan having benefit of the present teachings. It is likely, however, that the administration of therapeutically-effective amounts of the disclosed compositions may be achieved by a single administration, such as for example, a single injection of sufficient numbers of infectious particles to provide therapeutic benefit to the patient undergoing such treatment. Alternatively, in some circumstances, it may be desirable to provide multiple, or successive administrations of the AAV vector compositions, either over a relatively short, or a relatively prolonged period of time, as may be determined by the medical practitioner overseeing the administration of such compositions. For example, the number of infectious particles administered to a mammal may be on the order of about 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , 10 12 ,
- infectious particles/ml given either as a single dose, or divided into two or more administrations as may be required to achieve therapy of the particular disease or disorder being treated.
- the inventors contemplate the use of liposomes, nanocapsules, microparticles, microspheres, lipid particles, vesicles, and the like, for the introduction of the compositions of the present invention into suitable host cells.
- the rAAV vector delivered gene therapy compositions of the present invention may be formulated for delivery either encapsulated in a lipid particle, a liposome, a vesicle, a nanosphere, or a nanoparticle or the like.
- Such formulations may be preferred for the introduction of pharmaceutically acceptable formulations of the nucleic acids or the rAAV constructs disclosed herein.
- the formation and use of liposomes is generally known to those of skill in the art (see for example, Couvreur et al.,
- liposomes were developed with improved serum stability and circulation half-times (Gabizon and Papahadjopoulos, 1988; Allen and Choun, 1987; U. S. Patent 5,741,516, specifically incorporated herein by reference in its entirety). Further, various methods of liposome and liposome like preparations as potential drug carriers have been reviewed (Takakura, 1998;
- Liposomes have been used successfully with a number of cell types that are normally resistant to transfection by other procedures including T cell suspensions, primary hepatocyte cultures and PC 12 cells (Renneisen et al, 1990; Muller et al, 1990). In addition, liposomes are free of the DNA length constraints that are typical of viral-based delivery systems. Liposomes have been used effectively to introduce genes, drugs (Heath and Martin, 1986; Heath etal,
- Liposomes are formed from phospholipids that are dispersed in an aqueous medium and spontaneously form multilamellar concentric bilayer vesicles (also termed multilamellar vesicles (MLVs).
- MLVs generally have diameters of from 25 nm to 4 ⁇ m. Sonication of MLVs results in the formation of small unilamellar vesicles (SUVs) with diameters in the range of 200 to 500 A, containing an aqueous solution in the core.
- SUVs small unilamellar vesicles
- Liposomes bear resemblance to cellular membranes and are contemplated for use in connection with the present invention as carriers for the peptide compositions. They are widely suitable as both water- and lipid-soluble substances can be entrapped, i.e. in the aqueous spaces and within the bilayer itself, respectively. It is possible that the drug-bearing liposomes may even be employed for site-specific delivery of active agents by selectively modifying the liposomal formulation.
- Phospholipids can form a variety of structures other than liposomes when dispersed in water, depending on the molar ratio of lipid to water. At low ratios the liposome is the preferred structure.
- the physical characteristics of liposomes depend on pH, ionic strength and the presence of divalent cations. Liposomes can show low permeability to ionic and polar substances, but at elevated temperatures undergo a phase transition which markedly alters their permeabihty.
- phase transition involves a change from a closely packed, ordered structure, known as the gel state, to a loosely packed, less-ordered structure, known as the fluid state. This occurs at a characteristic phase-transition temperature and results in an increase in permeability to ions, sugars and drugs.
- soluble proteins such as cytochrome c
- Cholesterol inhibits this penetration of proteins, apparently by packing the phospholipids more tightly. It is contemplated that the most useful liposome formations for antibiotic and inhibitor delivery will contain cholesterol.
- SUVs unilamellar vesicles
- LUVs large unilamellar vesicles
- Polar compounds are trapped in the aqueous spaces and nonpolar compounds bind to the lipid bilayer of the vesicle. Polar compounds are released through permeation or when the bilayer is broken, but nonpolar compounds remain affiliated with the bilayer unless it is disrupted by temperature or exposure to lipoproteins. Both types show maximum efflux rates at the phase transition temperature.
- Liposomes interact with cells via four different mechanisms: Endocytosis by phagocytic cells of the reticuloendothelial system such as macrophages and neutrophils; adsorption to the cell surface, either by nonspecific weak hydrophobic or electrostatic forces, or by specific interactions with cell-surface components; fusion with the plasma cell membrane by insertion of the lipid bilayer of the liposome into the plasma membrane, with simultaneous release of liposomal contents into the cytoplasm; and by transfer of liposomal lipids to cellular or subcellular membranes, or vice versa, without any association of the liposome contents. It often is difficult to determine which mechanism is operative and more than one may operate at the same time.
- liposomes The fate and disposition of intravenously injected liposomes depend on their physical properties, such as size, fluidity, and surface charge. They may persist in tissues for h or days, depending on their composition, and half lives in the blood range from min to several h. Larger liposomes, such as MLVs and LUVs, are taken up rapidly by phagocytic cells of the reticuloendothelial system, but physiology of the circulatory system restrains the exit of such large species at most sites. They can exit only in places where large openings or pores exist in the capillary endothelium, such as the sinusoids of the liver or spleen. Thus, these organs are the predominate site of uptake.
- MLVs and LUVs are taken up rapidly by phagocytic cells of the reticuloendothelial system, but physiology of the circulatory system restrains the exit of such large species at most sites. They can exit only in places where large openings or pores exist in the ca
- SUVs show a broader tissue distribution but still are sequestered highly in the liver and spleen. In general, this in vivo behavior limits the potential targeting of liposomes to only those organs and tissues accessible to their large size. These include the blood, liver, spleen, bone marrow, and lymphoid organs.
- Targeting is generally not a limitation in terms of the present invention. However, should specific targeting be desired, methods are available for this to be accomplished.
- Antibodies may be used to bind to the liposome surface and to direct the antibody and its drug contents to specific antigenic receptors located on a particular cell-type surface.
- Carbohydrate determinants may also be used as recognition sites as they have potential in directing liposomes to particular cell types.
- intravenous injection of liposomal preparations would be used, but other routes of administration are also conceivable.
- the invention provides for pharmaceutically acceptable nanocapsule formulations of the AAV vector-based polynucleotide compositions of the present invention.
- Nanocapsules can generally entrap compounds in a stable and reproducible way (Henry- Michelland etal, 1987; Quintanar-Guerrero et al., 1998; Douglas et al., 1987).
- ultrafine particles sized around 0.1 ⁇ m
- Biodegradable polyalkyl- cyanoacrylate nanoparticles that meet these requirements are contemplated for use in the present invention.
- Such particles may be are easily made, as described (Couvreur etal, 1980; Couvreur, 1988; zur Muhlen et al, 1998; Zambaux et al. 1998; Pinto-Alphandry et al., 1995 and U. S. Patent 5,145,684, specifically inco ⁇ orated herein by reference in its entirety).
- the following techniques are also contemplated as alternative methods of delivering the disclosed rAAV vector based polynucleotide compositions to target cells or selected tissues and organs of an animal, and in particular, to cells, organs, or tissues of a vertebrate mammal, and more particularly, to a primate, such as a human being.
- Sonophoresis i.e., ultrasound
- U. S. Patent 5,656,016 specifically inco ⁇ orated herein by reference in its entirety
- Other drug delivery alternatives contemplated are intraosseous injection (U. S.
- expression vector or construct means any type of genetic construct containing a nucleic acid in which part or all of the nucleic acid encoding sequence is capable of being transcribed.
- expression includes transcription of the nucleic acid, for example, to generate a biologically-active therapeutic agent(s), such as, for example, one or more peptides, polypeptides, proteins, enzymes, or an antisense polynucleotide or oligonucleotide, or catalytic RNA molecules such as ribozymes, from a selected nucleic acid segment that encodes the therapeutic agent or agents.
- a biologically-active therapeutic agent(s) such as, for example, one or more peptides, polypeptides, proteins, enzymes, or an antisense polynucleotide or oligonucleotide, or catalytic RNA molecules such as ribozymes
- Particularly useful vectors are contemplated to be those vectors in which the nucleic acid segment to be transcribed is positioned under the transcriptional control of one or more promoter and/or enhancer elements that are capable of directing synthesis of the encoded therapeutic in a selected cell into which the vectors have been introduced.
- a “promoter” refers to a DNA sequence recognized by the synthetic machinery of the cell, or introduced synthetic machinery, required to initiate the specific transcription of a gene.
- the phrases “operatively positioned,” “operably positioned” “operably linked” "under control” or “under transcriptional control” means that the promoter is in the correct location and orientation in relation to the nucleic acid to control RNA polymerase initiation and expression of the selected nucleic acid segment encoding the therapeutic agent.
- a recombinant or heterologous promoter is intended to refer to a promoter that is not normally associated with the gene in its natural environment.
- Such promoters may include promoters normally associated with other genes, and/or promoters isolated from bacterial, viral, eukaryotic, or mammalian cells.
- promoter that effectively directs the expression of the encoded therapeutic agent in the cell type, organism, or even animal, chosen for expression.
- the use of promoter and cell type combinations for protein expression is generally known to those of skill in the art of molecular biology, for example, see Sambrook et al (1989), inco ⁇ orated herein by reference.
- the promoters employed may be constitutive, or inducible, and can be used under the appropriate conditions to direct high-level expression of the introduced polynucleotide segment, or the promoters may direct tissue- or cell-specific expression of the therapeutic constructs, such as, for example, an islet cell- or pancreas-specific promoter such as the insulin promoter.
- At least one module in a promoter functions to position the start site for RNA synthesis.
- the best-known example of this is the TATA box, but in some promoters lacking a TATA box, such as the promoter for the mammalian terminal deoxynucleotidyl transferase gene and the promoter for the SV40 late genes, a discrete element overlying the start site itself helps to fix the place of initiation. Additional promoter elements regulate the frequency of transcriptional initiation. Typically, these are located in the region 30-110 bp upstream of the start site, although a number of promoters have been shown to contain functional elements downstream of the start site as well.
- the spacing between promoter elements frequently is flexible, so that promoter function is preserved when elements are inverted or moved relative to one another.
- the spacing between promoter elements can be increased to 50 bp apart before activity begins to decline.
- individual elements can function either co-operatively or independently to activate transcription.
- the particular promoter that is employed to control the expression of a nucleic acid is not believed to be critical, so long as it is capable of expressing the se ⁇ in or cytokine- polypeptide encoding nucleic acid segment in the targeted cell.
- a human cell is targeted, it is preferable to position the nucleic acid coding region adjacent to and under the control of a promoter that is capable of being expressed in a human cell.
- a promoter might include either a human or viral promoter, such as a CMV or an HSV promoter.
- ⁇ -actin, and in particular, chicken ⁇ -actin promoters have been shown to be particularly preferred for certain embodiments of the invention.
- the human cytomegalovirus (CMV) immediate early gene promoter, the SV40 early promoter and the Rous sarcoma virus long terminal repeat can be used to obtain high-level expression of transgenes.
- CMV cytomegalovirus
- the use of other viral or mammalian cellular or bacterial phage promoters that are well known in the art to achieve expression of a transgene is contemplated as well, provided that the levels of expression are sufficient for a given pu ⁇ ose.
- a variety of promoter elements have been described in Tables 1 and 2 that may be employed, in the context of the present invention, to regulate the expression of the present se ⁇ in or cytokine-encoding nucleic acid segments comprised within the recombinant AAV vectors of the present invention.
- Enhancers were originally detected as genetic elements that increased transcription from a promoter located at a distant position on the same molecule of DNA. This ability to act over a large distance had little precedent in classic studies of prokaryotic transcriptional regulation. Subsequent work showed that regions of DNA with enhancer activity are organized much like promoters. That is, they are composed of many individual elements, each of which binds to one or more transcriptional proteins.
- enhancers The basic distinction between enhancers and promoters is operational. An enhancer region as a whole must be able to stimulate transcription at a distance; this need not be true of a promoter region or its component elements. On the other hand, a promoter must have one or more elements that direct initiation of RNA synthesis at a particular site and in a particular orientation, whereas enhancers lack these specificities. Promoters and enhancers are often overlapping and contiguous, often seeming to have a very similar modular organization.
- Eukaryotic Promoter Data Base EPDB any promoter/enhancer combination (as per the Eukaryotic Promoter Data Base EPDB) could also be used to drive expression.
- Use of a T3, T7 or SP6 cytoplasmic expression system is another possible embodiment.
- Eukaryotic cells can support cytoplasmic transcription from certain bacterial promoters if the appropriate bacterial polymerase is provided, either as part of the delivery complex or as an additional genetic expression construct.
- Troponin I (TN I) Yx ⁇ zey etal, 1989
- MMTV mimmary Glucocorticoids Huang et al, 1981; Lee et al, tumor virus
- Majors and Varmus 1983; Chandler et al, 1983; Lee et al, 1984; Ponta etal, 1985; Sakai et al, 1988 ⁇ -Interferon poly(rI)x Tavernier et al, 1983 ⁇ oly(rc)
- engineered and recombinant cells are intended to refer to a cell into which an exogenous DNA segment, such as DNA segment that leads to the transcription of a biologically-active se ⁇ in or cytokine polypeptide or a ribozyme specific for such a biologically-active se ⁇ in or cytokine polypeptide product, has been introduced. Therefore, engineered cells are distinguishable from naturally occurring cells, which do not contain a recombinantly introduced exogenous DNA segment. Engineered cells are thus cells having DNA segment introduced through the hand of man.
- an rAAV expression vector that comprises a biologically- active se ⁇ in or cytokine polypeptide-encoding nucleic acid segment under the control of one or more promoters.
- a sequence "under the control of a promoter one positions the 5' end of the transcription initiation site of the transcriptional reading frame generally between about 1 and about 50 nucleotides "downstream" of (i.e., 3' of) the chosen promoter.
- the "upstream" promoter stimulates transcription of the DNA and promotes expression of the encoded polypeptide. This is the meaning of "recombinant expression" in this context.
- Particularly preferred recombinant vector constructs are those that comprise an rAAV vector. Such vectors are described in detail herein.
- modified nucleotide compositions such as, for example, in the generation of the nucleic acid segments that encode either parts of the AAV vector itself, or the promoter, or even the therapeutic gene delivered by such rAAV vectors.
- Site-specific mutagenesis is a technique useful in the preparation and testing of sequence variants by introducing one or more nucleotide sequence changes into the DNA.
- Site-specific mutagenesis allows the production of mutants through the use of specific oligonucleotide sequences which encode the DNA sequence of the desired mutation, as well as a sufficient number of adjacent nucleotides, to provide a primer sequence of sufficient size and sequence complexity to form a stable duplex on both sides of the deletion junction being traversed.
- a primer of about 17 to 25 nucleotides in length is preferred, with about 5 to 10 residues on both sides of the junction of the sequence being altered.
- the technique of site-specific mutagenesis is well known in the art.
- the technique typically employs a bacteriophage vector that exists in both a single stranded and double stranded form.
- Typical vectors useful in site-directed mutagenesis include vectors such as the M13 phage. These phage vectors are commercially available and their use is generally well known to those skilled in the art.
- Double stranded plasmids are also routinely employed in site directed mutagenesis, which eliminates the step of transferring the gene of interest from a phage to a plasmid.
- site-directed mutagenesis is performed by first obtaining a single-stranded vector, or melting of two strands of a double stranded vector that includes within its sequence a DNA sequence encoding the desired ribozyme or other nucleic acid construct.
- An oligonucleotide primer bearing the desired mutated sequence is synthetically prepared.
- This primer is then annealed with the single-stranded DNA preparation, and subjected to DNA polymerizing enzymes such as E. coli polymerase I Klenow fragment, in order to complete the synthesis of the mutation-bearing strand.
- E. coli polymerase I Klenow fragment DNA polymerizing enzymes
- a heteroduplex is formed wherein one strand encodes the original non-mutated sequence and the second strand bears the desired mutation.
- This heteroduplex vector is then used to transform appropriate cells, such as E. coli cells, and clones are selected that include recombinant vectors bearing the mutated sequence arrangement.
- sequence variants of the selected nucleic acid sequences using site- directed mutagenesis is provided as a means of producing potentially useful species and is not meant to be limiting, as there are other ways in which sequence variants may be obtained.
- recombinant vectors encoding the desired gene may be treated with mutagenic agents, such as hydroxylamine, to obtain sequence variants.
- nucleic acid amplification techniques it may be necessary to employ one or more nucleic acid amplification techniques to produce the nucleic acid segments of the present invention.
- Various methods are well-known to artisans in the field, including for example, those techniques described herein:
- Nucleic acid used as a template for amplification, may be isolated from cells contained in the biological sample according to standard methodologies (Sambrook etal, 1989).
- the nucleic acid may be genomic DNA or fractionated or whole cell RNA. Where RNA is used, it may be desired to convert the RNA to a complementary DNA.
- the RNA is whole cell RNA and is used directly as the template for amplification.
- primers that selectively hybridize to nucleic acids corresponding to the ribozymes or conserved flanking regions are contacted with the isolated nucleic acid under conditions that permit selective hybridization.
- the term "primer”, as defined herein, is meant to encompass any nucleic acid that is capable of priming the synthesis of a nascent nucleic acid in a template-dependent process.
- primers are oligonucleotides from ten to twenty base pairs in length, but longer sequences can be employed.
- Primers may be provided in double- stranded or single-stranded form, although the single-stranded form is preferred.
- the nucleic acid:primer complex is contacted with one or more enzymes that facilitate template-dependent nucleic acid synthesis. Multiple rounds of amplification, also referred to as "cycles,” are conducted until a sufficient amount of amplification product is produced.
- the amplification product is detected.
- the detection may be performed by visual means.
- the detection may involve indirect identification of the product via chemiluminescence, radioactive scintigraphy of inco ⁇ orated radiolabel or fluorescent label or even via a system using electrical or thermal impulse signals (e.g., Affymax technology).
- PCR polymerase cham reaction
- two primer sequences are prepared that are complementary to regions on opposite complementary strands of the marker sequence.
- An excess of deoxynucleoside triphosphates is added to a reaction mixture along with a DNA polymerase, e.g., Taq polymerase. If the marker sequence is present in a sample, the primers will bind to the marker and the polymerase will cause the primers to be extended along the marker sequence by adding on nucleotides.
- the extended primers will dissociate from the marker to form reaction products, excess primers will bind to the marker and to the reaction products and the process is repeated.
- a reverse transcriptase PCRTM amplification procedure may be performed in order to quantify the amount of mRNA amplified.
- Methods of reverse transcribing RNA into cDNA are well known and described in Sambrook etal. (1989).
- Alternative methods for reverse transcription utilize thermostable, RNA-dependent DNA polymerases. These methods are described in Int. Pat. Appl. Publ. No. WO 90/07641 (specifically inco ⁇ orated herein by reference). Polymerase chain reaction methodologies are well known in the art.
- LCR ligase chain reaction
- Q ⁇ Replicase (Q ⁇ R), described in Int. Pat. Appl. No. PCT/US87/00880, inco ⁇ orated herein by reference, may also be used as still another amplification method in the present invention.
- a replicative sequence of RNA that has a region complementary to that of a target is added to a sample in the presence of an RNA polymerase.
- the polymerase will copy the replicative sequence that can then be detected.
- An isothermal amplification method in which restriction endonucleases and ligases are used to achieve the amplification of target molecules that contain nucleotide 5'-[ ⁇ -thio]- triphosphates in one strand of a restriction site may also be useful in the amplification of nucleic acids in the present invention.
- Strand Displacement Amplification (SDA), described in U. S. Patent Nos. 5,455,166, 5,648,211, 5,712,124 and 5,744,311, each inco ⁇ orated herein by reference, is another method of carrying out isothermal amplification of nucleic acids which involves multiple rounds of strand displacement and synthesis, i.e., nick translation.
- a similar method, called Repair Chain Reaction (RCR) involves annealing several probes throughout a region targeted for amplification, followed by a repair reaction in which only two of the four bases are present. The other two bases can be added as biotinylated derivatives for easy detection.
- RCR Repair Chain Reaction
- Target specific sequences can also be detected using a cyclic probe reaction (CPR).
- a probe having 3' and 5' sequences of non-specific DNA and a middle sequence of specific RNA is hybridized to DNA that is present in a sample.
- the reaction is treated with RNase H, and the products of the probe identified as distinctive products that are released after digestion.
- the original template is annealed to another cycling probe and the reaction is repeated.
- nucleic acid amplification procedures include transcription-based amplification systems (TAS), including nucleic acid sequence based amplification (NASBA) and 3SR Gingeras etal, Int. Pat. Appl. Publ. No. WO 88/10315, inco ⁇ orated herein by reference.
- TAS transcription-based amplification systems
- NASBA nucleic acid sequence based amplification
- 3SR Gingeras etal Int. Pat. Appl. Publ. No. WO 88/10315, inco ⁇ orated herein by reference.
- the nucleic acids can be prepared for amplification by standard phenol/chloroform extraction, heat denaturation of a clinical sample, treatment with lysis buffer and minispin columns for isolation of DNA and RNA or guanidinium chloride extraction of RNA.
- amplification techniques involve annealing a primer that has target specific sequences.
- DNA/RNA hybrids are digested with RNase H while double stranded DNA molecules are heat denatured again.
- the single stranded DNA is made fully double stranded by addition of second target specific primer, followed by polymerization.
- the double-stranded DNA molecules are then multiply transcribed by an RNA polymerase such as T7 or SP6.
- the RNAs are reverse transcribed into single stranded DNA, which is then converted to double stranded DNA, and then transcribed once again with an RNA polymerase such as T7 or SP6.
- T7 or SP6 an isothermal cyclic reaction
- the resulting products whether truncated or complete, indicate target specific sequences.
- ssRNA single-stranded RNA
- dsDNA double-stranded DNA
- the ssRNA is a template for a first primer oligonucleotide, which is elongated by reverse transcriptase (RNA-dependent DNA polymerase).
- RNA-dependent DNA polymerase reverse transcriptase
- the RNA is then removed from the resulting DNA:RNA duplex by the action of ribonuclease H (RNase H, an RNase specific for RNA in duplex with either DNA or RNA).
- RNase H ribonuclease H
- the resultant ssDNA is a template for a second primer, which also includes the sequences of an RNA polymerase promoter (exemplified by T7 RNA polymerase) 5' to its homology to the template.
- This primer is then extended by DNA polymerase (exemplified by the large "Klenow" fragment of E. coli DNA polymerase I), resulting in a double-stranded DNA (“dsDNA”) molecule, having a sequence identical to that of the original RNA between the primers and having additionally, at one end, a promoter sequence.
- This promoter sequence can be used by the appropriate RNA polymerase to make many RNA copies of the DNA. These copies can then re-enter the cycle leading to very swift amplification. With proper choice of enzymes, this amplification can be done isothermally without addition of enzymes at each cycle. Because of the cyclical nature of this process, the starting sequence can be chosen to be in the form of either DNA or RNA.
- WO 89/06700 disclose a nucleic acid sequence amplification scheme based on the hybridization of a promoter/primer sequence to a target single-stranded DNA ("ssDNA”) followed by transcription of many RNA copies of the sequence. This scheme is not cyclic, i.e., new templates are not produced from the resultant RNA transcripts.
- Other amplification methods include "RACE” and "one-sided PCRTM” (Frohman, 1990, specifically inco ⁇ orated herein by reference).
- Methods based on ligation of two (or more) oligonucleotides in the presence of nucleic acid having the sequence of the resulting "di-oligonucleotide,” thereby amplifying the di- oligonucleotide, may also be used in the amplification step of the present invention.
- amplification products are separated by agarose, agarose-acrylamide or polyacrylamide gel electrophoresis using standard methods (see e.g., Sambrook etal, 1989).
- chromatographic techniques may be employed to effect separation.
- Amplification products must be visualized in order to confirm amplification of the marker sequences.
- One typical visualization method involves staining of a gel with ethidium bromide and visualization under UV light.
- the amplification products are integrally labeled with radio- or fluorometrically-labeled nucleotides, the amplification products can then be exposed to x-ray film or visualized under the appropriate stimulating spectra, following separation.
- visualization is achieved indirectly.
- a labeled, nucleic acid probe is brought into contact with the amplified marker sequence.
- the probe preferably is conjugated to a chromophore but may be radiolabeled.
- the probe is conjugated to a binding partner, such as an antibody or biotin, and the other member of the binding pair carries a detectable moiety.
- detection is by Southern blotting and hybridization with a labeled probe.
- the techniques involved in Southern blotting are well known to those of skill in the art and can be found in many standard books on molecular protocols. See Sambrook et al, 1989. Briefly, amplification products are separated by gel electrophoresis. The gel is then contacted with a membrane, such as nitrocellulose, permitting transfer of the nucleic acid and non- covalent binding. Subsequently, the membrane is incubated with a chromophore-conjugated probe that is capable of hybridizing with a target amplification product. Detection is by exposure of the membrane to x-ray film or ion-emitting detection devices.
- RNA, DNA, PNAs and/or substituted polynucleotide compositions disclosed herein will be used to transfect an appropriate host cell.
- Technology for introduction of PNAs, RNAs, and DNAs into cells is well known to those of skill in the art.
- the present invention contemplates a variety of AAV-based expression systems, and vectors.
- the preferred AAV expression vectors comprise at least a first nucleic acid segment that encodes a therapeutic peptide, protein, or polypeptide.
- the preferred AAV expression vectors disclosed herein comprise at least a first nucleic acid segment that encodes an antisense molecule.
- a promoter is operatively linked to a sequence region that encodes a functional mRNA, a tRNA, a ribozyme or an antisense RNA.
- operatively linked means that a promoter is connected to a functional RNA in such a way that the transcription of that functional RNA is controlled and regulated by that promoter.
- Means for operatively linking a promoter to a functional RNA are well known in the art.
- a vector useful in practicing the present invention is capable of directing the expression of the functional RNA to which it is operatively linked.
- RNA polymerase transcribes a coding DNA sequence through a site where polyadenylation occurs. Typically, DNA sequences located a few hundred base pairs downstream of the polyadenylation site serve to terminate transcription. Those DNA sequences are referred to herein as transcription-termination regions. Those regions are required for efficient polyadenylation of transcribed messenger RNA (mRNA).
- mRNA messenger RNA
- a variety of methods have been developed to operatively link DNA to vectors via complementary cohesive termini or blunt ends. For instance, complementary homopolymer tracts can be added to the DNA segment to be inserted and to the vector DNA. The vector and DNA segment are then joined by hydrogen bonding between the complementary homopolymeric tails to form recombinant DNA molecules.
- one of the key aspects of the present invention is the creation of one or more mutations into specific polynucleotide sequences that encode one or more of the therapeutic agents encoded by the disclosed rAAV constructs.
- the resulting polypeptide sequence is altered by these mutations, or in other cases, the sequence of the polypeptide is unchanged by one or more mutations in the encoding polynucleotide to produce modified vectors with improved properties for effecting gene therapy in mammalian systems.
- the amino acid changes may be achieved by changing one or more of the codons of the encoding DNA sequence, according to Table 3.
- amino acids may be substituted for other amino acids in a protein structure without appreciable loss of interactive binding capacity with structures such as, for example, antigen-binding regions of antibodies or binding sites on substrate molecules. Since it is the interactive capacity and nature of a protein that defines that protein's biological functional activity, certain amino acid sequence substitutions can be made in a protein sequence, and, of course, its underlying DNA coding sequence, and nevertheless obtain a protein with like properties. It is thus contemplated by the inventors that various changes may be made in the polynucleotide sequences disclosed herein, without appreciable loss of their biological utility or activity. TABLE 3
- the hydropathic index of amino acids may be considered.
- the importance of the hydropathic amino acid index in conferring interactive biologic function on a protein is generally understood in the art (Kyte and Doolittle, 1982, inco ⁇ orate herein by reference). It is accepted that the relative hydropathic character of the amino acid contributes to the secondary structure of the resultant protein, which in turn defines the interaction of the protein with other molecules, for example, enzymes, substrates, receptors, DNA, antibodies, antigens, and the like.
- Each amino acid has been assigned a hydropathic index on the basis of their hydrophobicity and charge characteristics (Kyte and Doolittle, 1982), these are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and argierine (-4.5).
- hydrophilicity values have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0 ⁇ 1); glutamate (+3.0 ⁇ 1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); Tooonine (-0.4); proline (-0.5 ⁇ 1); alanine (-0.5); histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine (- 1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4).
- an amino acid can be substituted for another having a similar hydrophilicity value and still obtain a biologically equivalent, and in particular, an immunologically equivalent protein.
- substitution of amino acids whose hydrophilicity values are witiiin ⁇ 2 is preferred, those that are within ⁇ 1 are particularly preferred, and those within ⁇ 0.5 are even more particularly preferred.
- amino acid substitutions are generally therefore based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like.
- Exemplary substitutions which take several of the foregoing characteristics into consideration are well known to those of skill in the art and include: arginine and lysine; glutamate and aspartate; serine and threonine; glutamine and asparagine; and valine, leucine and isoleucine.
- the invention also encompasses one or more of the modified rAAV vector compositions described herein together with one or more pharmaceutically-acceptable excipients, carriers, diluents, adjuvants, and/or other components, as may be employed in the formulation of particular rAAV-polynucleotide delivery formulations, and in the preparation of therapeutic agents for administration to a mammal, and in particularly, to a human.
- kits may comprise one or more of the disclosed rAAV compositions in combination with instructions for using the viral vector in the treatment of such disorders in a mammal, and may typically further include containers prepared for convenient commercial packaging.
- preferred animals for administration of the pharmaceutical compositions disclosed herein include mammals, and particularly humans.
- Other preferred animals include murines, bovines, equines, porcines, canines, and felines.
- the composition may include partially or significantly purified rAAV compositions, either alone, or in combination with one or more additional active ingredients, which may be obtained from natural or recombinant sources, or which may be obtainable naturally or either chemically synthesized, or alternatively produced in vitro from recombinant host cells expressing DNA segments encoding such additional active ingredients.
- kits may also be prepared that comprise at least one of the compositions disclosed herein and instructions for using the composition as a therapeutic agent.
- the container means for such kits may typically comprise at least one vial, test tube, flask, bottle, syringe or other container means, into which the disclosed rAAV composition(s) may be placed, and preferably suitably aliquoted.
- the kit may also contain a second distinct container means into which this second composition may be placed.
- the plurality of therapeutic biologically active compositions may be prepared in a single pharmaceutical composition, and may be packaged in a single container means, such as a vial, flask, syringe, bottle, or other suitable single container means.
- the kits of the present invention will also typically include a means for containing the vial(s) in close confinement for commercial sale, such as, e.g., injection or blow-molded plastic containers into which the desired vial(s) are retained.
- one aspect of the invention concerns the use of the modified capsid vectors to deliver catalytic RNA molecules (ribozymes) to selected mammalian cells and tissues to effect a reduction or elimination of expression of one or more native DNA or mRNA molecules, so as to prevent or reduce the amount of the translation product of such mRNAs.
- catalytic RNA molecules ribozymes
- Ribozymes are biological catalysts consisting of only RNA. They promote a variety of reactions involving RNA and DNA molecules including site-specific cleavage, ligation, polymerization, and phosphoryl exchange (Cech, 1989; Cech, 1990). Ribozymes fall into three broad classes: (1) RNAse P, (2) self-splicing introns, and (3) self-cleaving viral agents. Self- cleaving agents include hepatitis delta virus and components of plant virus satellite RNAs that sever the RNA genome as part of a rolling-circle mode of replication. Because of their small size and great specificity, ribozymes have the greatest potential for biotechnical applications.
- ribozymes have already been used to cleave viral targets and oncogene products in living cells (Koizumi et al, 1992; Kashani- Sabet et al, 1992; Taylor and Rossi, 1991; von-Weizsacker et al, 1992; Ojwang et al, 1992; Stephenson and Gibson, 1991; Yu et al, 1993; Xing and Whitton, 1993; Yu et al, 1995; Little and Lee, 1995).
- ribozymes Two kinds have been employed widely, hai ⁇ ins and hammerheads. Both catalyze sequence-specific cleavage resulting in products with a 5' hydroxyl and a 2',3'-cyclic phosphate.
- Hammerhead ribozymes have been used more commonly, because they impose few restrictions on the target site. Hai ⁇ in ribozymes are more stable and, consequently, function better than hammerheads at physiologic temperature and magnesium concentrations.
- Ribozymes are RNA- protein complexes that cleave nucleic acids in a site-specific fashion. Ribozymes have specific catalytic domains that possess endonuclease activity (Kim and Cech, 1987; Gerlach et al, 1987; Forster and Symons, 1987).
- ribozymes accelerate phosphoester transfer reactions with a high degree of specificity, often cleaving only one of several phosphoesters in an oligonucleotide substrate (Cech et al, 1981; Michel and Westhof, 1990; Reinhold-Hurek and Shub, 1992).
- This specificity has been attributed to the requirement that the substrate bind via 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, 1989; Cech et al, 1981).
- 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, 1991; Sarver et al, 1990).
- ribozymes elicited genetic changes in some cells lines to which they were applied; the altered genes included the oncogenes H-ras, c-fos and genes of HTV. Most of this work involved the modification of a target mRNA, based on a specific mutant codon that is cleaved by a specific ribozyme.
- enzymatic nucleic acids act by first binding to a target RNA. Such binding occurs through the target binding portion of a enzymatic nucleic acid which is held in close proximity to an enzymatic portion of the molecule that acts to cleave the target RNA. Thus, the enzymatic nucleic acid first recognizes and then binds a target RNA through complementary base pairing, and once bound to the correct site, acts enzymatically to cut the target RNA.
- RNA Strategic cleavage of such a target RNA will destroy its ability to direct synthesis of an encoded protein. After an enzymatic nucleic acid has bound and cleaved its RNA target, it is released from that RNA to search for another target and can repeatedly bind and cleave new targets.
- ribozyme The enzymatic nature of a ribozyme is advantageous over many technologies, such as antisense technology (where a nucleic acid molecule simply binds to a nucleic acid target to block its translation) since the concentration of ribozyme necessary to affect a therapeutic treatment is lower than that of an antisense oligonucleotide.
- This advantage reflects the ability of the ribozyme to act enzymatically.
- a single ribozyme molecule is able to cleave many molecules of target RNA.
- the ribozyme is a highly specific inhibitor, with the specificity of inhibition depending not only on the base pairing mechanism of binding to the target RNA, but also on the mechanism of target RNA cleavage.
- ribozyme Single mismatches, or base- substitutions, near the site of cleavage can completely eliminate catalytic activity of a ribozyme. Similar mismatches in antisense molecules do not prevent their action (Woolf etal, 1992). Thus, the specificity of action of a ribozyme is greater than that of an antisense oligonucleotide binding the same RNA site.
- the enzymatic nucleic acid molecule may be formed in a hammerhead, hai ⁇ in, a hepatitis ⁇ virus, group I intron or RNaseP RNA (in association with an RNA guide sequence) or Neurospora VS RNA motif.
- hammerhead motifs are described by Rossi et al
- hai ⁇ in motifs are described by Hampel etal. (Eur. Pat. Appl. Publ. No. EP 0360257), Hampel and Trite (1989), Hampel etal (1990) and U. S. Patent 5,631,359 (specifically inco ⁇ orated herein by reference).
- An example of the hepatitis ⁇ virus motif is described by Perrotta and Been (1992); an example of the RNaseP motif is described by Guerrier-Takada et l (1983); Neurospora VS RNA ribozyme motif is described by Collins (Saville and Collins, 1990; Saville and Collins, 1991; Collins and Olive, 1993); and an example of the Group I intron is described in U. S.
- Patent 4,987,071 (specifically inco ⁇ orated herein by reference). All that is important in an enzymatic nucleic acid molecule of this invention is that it has a specific substrate binding site which is complementary to one or more of the target gene RNA regions, and that it have nucleotide sequences within or surrounding that substrate binding site which impart an RNA cleaving activity to the molecule. Thus the ribozyme constructs need not be limited to specific motifs mentioned herein.
- enzymatic cleaving agents that exhibit a high degree of specificity for the RNA of a desired target, such as one of the sequences disclosed herein.
- the enzymatic nucleic acid molecule is preferably targeted to a highly conserved sequence region of a target mRNA.
- Such enzymatic nucleic acid molecules can be delivered exogenously to specific cells as required, although in preferred embodiments the ribozymes are expressed from DNA or RNA vectors that are delivered to specific cells.
- Small enzymatic nucleic acid motifs may also be used for exogenous delivery.
- the simple structure of these molecules increases the ability of the enzymatic nucleic acid to invade targeted regions of the mRNA structure.
- catalytic RNA molecules can be expressed within cells from eukaryotic promoters (e.g., Scanlon etal, 1991; Kashani-Sabet etal, 1992; Dropulic etal, 1992; Weerasinghe etal, 1991; Ojwang etal, 1992; Chen etal, 1992; Sarver etal, 1990).
- any ribozyme can be expressed in eukaryotic cells from the appropriate DNA vector.
- the activity of such ribozymes can be augmented by their release from the primary transcript by a second ribozyme (Int. Pat. Appl. Publ. No. WO 93/23569, and Int. Pat. Appl. Publ. No. WO 94/02595, both hereby inco ⁇ orated by reference; Ohkawa etal, 1992; Taira et al, 1991; and Ventura et al, 1993).
- Ribozymes may be added directly, or can be complexed with cationic lipids, lipid complexes, packaged within liposomes, or otherwise delivered to target cells.
- the RNA or RNA complexes can be locally administered to relevant tissues ex vivo, or in vivo through injection, aerosol inhalation, infusion pump or stent, with or without their inco ⁇ oration in biopolymers.
- Ribozymes may be designed as described in Int. Pat. Appl. Publ. No. WO 93/23569 and Int. Pat. Appl. Publ. No. WO 94/02595 (each specifically inco ⁇ orated herein by reference) and synthesized to be tested in vitro and in vivo, as described. Such ribozymes can also be optimized for delivery. While specific examples are provided, those in the art will recognize that equivalent RNA targets in other species can be util ed when necessary.
- Hammerhead or hai ⁇ in ribozymes may be individually analyzed by computer folding (Jaeger etal, 1989) to assess whether the ribozyme sequences fold into the appropriate secondary structure, as described herein. Those ribozymes with unfavorable intramolecular interactions between the binding arms and the catalytic core are eliminated from consideration. Varying binding arm lengths can be chosen to optimize activity. Generally, at least 5 or so bases on each arm are able to bind to, or otherwise interact with, the target RNA.
- Ribozymes of the hammerhead or hai ⁇ in motif may be designed to anneal to various sites in the mRNA message, and can be chemically synthesized.
- the method of synthesis used follows the procedure for normal RNA synthesis as described in Usman et al. (1987) and in Scaringe et al (1990) and makes use of common nucleic acid protecting and coupling groups, such as dimethoxytrityl at the 5'-end, and phosphoramidites at the 3'-end. Average stepwise coupling yields are typically >98 .
- Hai ⁇ in ribozymes may be synthesized in two parts and annealed to reconstruct an active ribozyme (Chowrira and Burke, 1992).
- Ribozymes may be modified extensively to enhance stability by modification with nuclease resistant groups, for example, 2'-amino, 2'-C-allyl, 2'-flouro, 2'-o-methyl, 2'-H (for a review see e.g., Usman and Cedergren, 1992). Ribozymes may be purified by gel electrophoresis using general methods or by high-pressure liquid chromatography and resuspended in water.
- nuclease resistant groups for example, 2'-amino, 2'-C-allyl, 2'-flouro, 2'-o-methyl, 2'-H (for a review see e.g., Usman and Cedergren, 1992).
- Ribozymes may be purified by gel electrophoresis using general methods or by high-pressure liquid chromatography and resuspended in water.
- Ribozyme activity can be optimized by altering the length of the ribozyme binding arms, or chemically synthesizing ribozymes with modifications that prevent their degradation by serum ribonucleases (see e.g., Int. Pat. Appl. Publ. No. WO 92/07065; Perrault et al, 1990; Pieken etal, 1991; Usman and Cedergren, 1992; Int. Pat. Appl. Publ. No. WO 93/15187; Int. Pat. Appl. Publ. No. WO 91/03162; Eur. Pat. Appl. Publ. No. 92110298.4; U.S. Patent 5,334,711; and Int. Pat. Appl.
- a preferred means of accumulating high concentrations of a ribozyme(s) within cells is to inco ⁇ orate the ribozyme-encoding sequences into a DNA expression vector. Transcription of the ribozyme sequences are driven from a promoter for eukaryotic RNA polymerase I (pol I), RNA polymerase ⁇ (pol H), or RNA polymerase HI (pol UT). Transcripts from pol ⁇ or pol IJJ promoters will be expressed at high levels in all cells; the levels of a given pol II promoter in a given cell type will depend on the nature of the gene regulatory sequences (enhancers, silencers, etc.) present nearby.
- Prokaryotic RNA polymerase promoters may also be used, providing that the prokaryotic RNA polymerase enzyme is expressed in the appropriate cells (Elroy-Stein and Moss, 1990; Gao and Huang, 1993; Lieber etal, 1993; Zhou etal, 1990). Ribozymes expressed from such promoters can function in mammalian cells (Kashani-Sabet etal, 1992; Ojwang etal, 1992; Chen etal, 1992; Yu etal, 1993; L'Huillier etal, 1992; Lisziewicz etal, 1993).
- transcription units can be inco ⁇ orated into a variety of vectors for introduction into mammalian cells, including but not restricted to, plasmid DNA vectors, other viral DNA vectors (such as adenovirus vectors), or viral RNA vectors (such as retroviral, semliki forest virus, Sindbis virus vectors).
- Ribozymes may be admimstered to cells by a variety of methods known to those familiar to the art, including, but not restricted to, encapsulation in liposomes, by iontophoresis, or by inco ⁇ oration into other vehicles, such as hydrogels, cyclodextrins, biodegradable nanocapsules, and bioadhesive microspheres.
- ribozymes may be directly delivered ex vivo to cells or tissues with or without the aforementioned vehicles.
- the RNA/vehicle combination may be locally delivered by direct inhalation, by direct injection or by use of a catheter, infusion pump or stent.
- routes of delivery include, but are not limited to, intravascular, intramuscular, subcutaneous or joint injection, aerosol inhalation, oral (tablet or pill form), topical, systemic, ocular, intraocular, retinal, subretinal, intraperitoneal, intracerebroventricular, intrathecal delivery, and/or direct injection to one or more tissues of the brain. More detailed descriptions of ribozyme and rAAV vector delivery and administration are provided in Int. Pat. Appl. Publ. No. WO 94/02595 and Int. Pat. Appl. Publ. No. WO 93/23569, each specifically inco ⁇ orated herein by reference.
- Ribozymes and the AAV vectored-constructs of the present invention may be used to inhibit gene expression and define the role (essentially) of specified gene products in the progression of one or more neural diseases, dysfunctions, cancers,, and/or disorders. In this manner, other genetic targets may be defined as important mediators of the disease.
- combination therapies e.g., multiple ribozymes targeted to different genes, ribozymes coupled with known small molecule inhibitors, or intermittent treatment with combinations of ribozymes and/or other chemical or biological molecules).
- the AAV constructs of the invention will find utility in the delivery of antisense oligonucleotides and polynucleotides for inhibiting the expression of a selected mammalian mRNA in neural cells.
- the letters, A, G, C, T, and U respectively indicate nucleotides in which the nucleoside is Adenosine (Ade), Guanosine (Gua), Cytidine (Cyt), Thymidine (Thy), and Uridine (Um).
- compounds that are "antisense” to a particular PNA, DNA or mRNA "sense" strand are nucleotide compounds that have a nucleoside sequence that is complementary to the sense strand. It will be understood by those skilled in the art that the present invention broadly includes polynucleotides and smaller oligonucleotide compounds that are capable of binding to the selected DNA or mRNA sense strand.
- mRNA includes not only the ribonucleotide sequences encoding a protein, but also regions including the 5 '-untranslated region, the 3 '-untranslated region, the 5 -cap region and the intron/exon junction regions.
- the invention includes compounds which are not strictly antisense; the compounds of the invention also include those polynucleotides and oligonucleotides that may have some bases that are not complementary to bases in the sense strand provided such compounds have sufficient binding affinity for the particular DNA or mRNA for which an inhibition of expression is desired.
- base modifications or the use of universal bases such as inosine in the oligonucleotides of the invention are contemplated within the scope of the subject invention.
- C 1-4 alkyl means a branched or unbranched hydrocarbon having 1 to 4 carbon-atoms.
- substituted acridine means any acridine derivative capable of intercalating nucleotide strands such as DNA.
- Preferred substituted acridines are 2-memoxy-6-cWoro-9-pentylarmnoacridine, N-(6-cMoro-2-methoxyacridinyl)- O-methoxydusopropylammophosphmyl-3-aminopropanol, and N-(6-chloro-
- nucleotides includes nucleotides in which the phosphate moiety is replaced by phosphorothioate or alkylphosphonate and the nucleotides may be substituted by substituted acridines.
- the antisense compounds of the invention differ from native DNA by the modification of the phosphodiester backbone to extend the life of the antisense molecule.
- the phosphates can be replaced by phosphorothioates.
- the ends of the molecule may also be optimally substituted by an acridine derivative that intercalates nucleotide strands of DNA. Intl. Pat. Appl. Publ. No. WO 98/13526 and U. S.
- Patent 5,849,902 (each specifically inco ⁇ orated herein by reference in its entirety) describe a method of preparing three component chimeric antisense compositions, and discuss many of the currently available methodologies for synthesis of substituted oligonucleotides having improved antisense characteristics and/or half- life.
- the reaction scheme involves 1 H-tetrazole-catalyzed coupling of phosphoramidites to give phosphate intermediates that are subsequently reacted with sulfur in 2,6-lutidine to generate phosphate compounds.
- Oligonucleotide compounds are prepared by treating the phosphate compounds with thiophenoxide (1:2:2 thiophenol/triethylarr ⁇ ne/tetrahydrofuran, room temperature, 1 hr). The reaction sequence is repeated until an oligonucleotide compound of the desired length has been prepared. The compounds are cleaved from the support by treating with ammonium hydroxide at room temperature for 1 hr and then are further deprotected by heating at about 50°C overnight to yield preferred antisense compounds.
- antisense compositions specific for a given gene sequence is based upon analysis of the chosen target sequence and determination of secondary structure, T m , binding energy, relative stability, and antisense compositions were selected based upon their relative inability to form dimers, hai ⁇ ins, or other secondary structures that would reduce or prohibit specific binding to the target mRNA in a host cell.
- Highly preferred target regions of the mRNA are those that are at or near the AUG translation initiation codon, and those sequences that were substantially complementary to 5' regions of the mRNA.
- polynucleotides, nucleic acid segments, nucleic acid sequences, and the like include, but are not limited to, DNAs (including and not limited to genomic or extragenomic DNAs), genes, peptide nucleic acids (PNAs) RNAs (including, but not limited to, rRNAs, mRNAs and tRNAs), nucleosides, and suitable nucleic acid segments either obtained from natural sources, chemically synthesized, modified, or otherwise prepared or synthesized in whole or in part by the hand of man.
- DNAs including and not limited to genomic or extragenomic DNAs
- genes include peptide nucleic acids (PNAs) RNAs (including, but not limited to, rRNAs, mRNAs and tRNAs), nucleosides, and suitable nucleic acid segments either obtained from natural sources, chemically synthesized, modified, or otherwise prepared or synthesized in whole or in part by the hand of man.
- PNAs peptide nucleic acids
- Expression The combination of intracellular processes, including transcription and translation undergone by a polynucleotide such as a structural gene to synthesize the encoded peptide or polypeptide.
- Promoter a term used to generally describe the region or regions of a nucleic acid sequence that regulates transcription.
- Regulatory Element a term used to generally describe the region or regions of a nucleic acid sequence that regulates transcription.
- exemplary regulatory elements include, but are not limited to, enhancers, post-transcriptional elements, transcriptional control sequences, and such like.
- Structural gene A polynucleotide, such as a gene, that is expressed to produce an encoded peptide, polypeptide, protein, ribozyme, catalytic RNA molecule, or antisense molecule.
- Transformation A process of introducing an exogenous polynucleotide sequence (e.g., a viral vector, a plasmid, or a recombinant DNA or RNA molecule) into a host cell or protoplast in which the exogenous polynucleotide is inco ⁇ orated into at least a first chromosome or is capable of autonomous replication within the transformed host cell.
- an exogenous polynucleotide sequence e.g., a viral vector, a plasmid, or a recombinant DNA or RNA molecule
- Transfection, electroporation, and "naked" nucleic acid uptake all represent examples of techniques used to transform a host cell with one or more polynucleotides.
- Transformed cell A host cell whose nucleic acid complement has been altered by the introduction of one or more exogenous polynucleotides into that cell.
- Transgenic eel Any cell derived or regenerated from a transformed cell or derived from a transgenic cell, or from the progeny or offspring of any generation of such a transformed host cell.
- a nucleic acid molecule (typically comprised of DNA) capable of replication in a host cell and/or to which another nucleic acid segment can be operatively linked so as to bring about replication of the attached segment.
- a plasmid, cosmid, or a virus is an exemplary vector.
- substantially corresponds to denotes a characteristic of a nucleic acid or an amino acid sequence, wherein a selected nucleic acid or amino acid sequence has at least about 70 or about 75 percent sequence identity as compared to a selected reference nucleic acid or amino acid sequence. More typically, the selected sequence and the reference sequence will have at least about 76, 77, 78, 79, 80, 81, 82, 83, 84 or even 85 percent sequence identity, and more preferably at least about 86, 87, 88, 89, 90, 91, 92, 93, 94, or 95 percent sequence identity.
- highly homologous sequences often share greater than at least about 96, 97, 98, or 99 percent sequence identity between the selected sequence and the reference sequence to wliich it was compared.
- the percentage of sequence identity may be calculated over the entire length of the sequences to be compared, or may be calculated by excluding small deletions or additions which total less than about 25 percent or so of the chosen reference sequence.
- the reference sequence may be a subset of a larger sequence, such as a portion of a gene or flanking sequence, or a repetitive portion of a chromosome.
- the reference sequence will typically comprise at least about 18-25 nucleotides, more typically at least about 26 to 35 nucleotides, and even more typically at least about 40, 50, 60, 70, 80, 90, or even 100 or so nucleotides.
- the extent of percent identity between the two sequences will be at least about 80%, preferably at least about 85%, and more preferably about 90% or 95% or higher, as readily determined by one or more of the sequence comparison algorithms well-known to those of skill in the art, such as e.g., the FASTA program analysis described by Pearson and Lipman (1988).
- naturally occurring refers to the fact that an object can be found in nature.
- a polypeptide or polynucleotide sequence that is present in an organism (including viruses) that can be isolated from a source in nature and which has not been intentionally modified by the hand of man in a laboratory is naturally-occurring.
- laboratory strains of rodents that may have been selectively bred according to classical genetics are considered naturally occurring animals.
- heterologous is defined in relation to a predetermined referenced gene sequence.
- a heterologous promoter is defined as a promoter which does not naturally occur adjacent to the referenced structural gene, but which is positioned by laboratory manipulation.
- a heterologous gene or nucleic acid segment is defined as a gene or segment that does not naturally occur adjacent to the referenced promoter and/or enhancer elements.
- Transcriptional regulatory element refers to a polynucleotide sequence that activates transcription alone or in combination with one or more other nucleic acid sequences.
- a transcriptional regulatory element can, for example, comprise one or more promoters, one or more response elements, one or more negative regulatory elements, and/or one or more enhancers.
- transcription factor recognition site and a “transcription factor binding site” refer to a polynucleotide sequence(s) or sequence motif(s) which are identified as being sites for the sequence-specific interaction of one or more transcription factors, frequently taking the form of direct protein-DNA binding.
- transcription factor binding sites can be identified by DNA footprinting, gel mobility shift assays, and the like, and/or can be predicted on the basis of known consensus sequence motifs, or by other methods known to those of skill in the art.
- operably linked refers to a linkage of two or more polynucleotides or two or more nucleic acid sequences in a functional relationship.
- a nucleic acid is “operably linked” when it is placed into a functional relationship with another nucleic acid sequence.
- a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the coding sequence.
- “Operably linked” means that the nucleic acid sequences being linked are typically contiguous, or substantially contiguous, and, where necessary to join two protein coding regions, contiguous and in reading frame.
- Transcriptional unit refers to a polynucleotide sequence that comprises at least a first structural gene operably linked to at least a first czs-acting promoter sequence and optionally linked operably to one or more other cts-acting nucleic acid sequences necessary for efficient transcription of the structural gene sequences, and at least a first distal regulatory element as may be required for the appropriate tissue-specific and developmental transcription of the structural gene sequence operably positioned under the control of the promoter and/or enhancer elements, as well as any additional cis sequences that are necessary for efficient transcription and translation (e.g., polyadenylation site(s), mRNA stability controlling sequence(s), etc.
- Transcriptional unit refers to a polynucleotide sequence that comprises at least a first structural gene operably linked to at least a first czs-acting promoter sequence and optionally linked operably to one or more other cts-acting nucleic acid sequences necessary for efficient transcription of the structural gene sequences,
- substantially complementary when used to define either amino acid or nucleic acid sequences, means that a particular subject sequence, for example, an oligonucleotide sequence, is substantially complementary to all or a portion of the selected sequence, and thus will specifically bind to a portion of an mRNA encoding the selected sequence.
- sequences will be highly complementary to the mRNA "target" sequence, and will have no more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 base mismatches throughout the complementary portion of the sequence.
- highly complementary sequences will typically bind quite specifically to the target sequence region of the mRNA and will therefore be highly efficient in reducing, and/or even inhibiting the translation of the target mRNA sequence into polypeptide product.
- Substantially complementary oligonucleotide sequences will be. greater than about 80 percent complementary (or '% exact-match') to the corresponding mRNA target sequence to which the oligonucleotide specifically binds, and will, more preferably be greater than about 85 percent complementary to the corresponding mRNA target sequence to which the oligonucleotide specifically binds.
- the oligonucleotide sequences will be greater than about 90 percent complementary to the corresponding mRNA target sequence to which the oligonucleotide specifically binds, and may in certain embodiments be greater than about 95 percent complementary to the corresponding mRNA target sequence to which the oligonucleotide specifically binds, and even up to and mcluding 96%, 97%, 98%, 99%, and even 100% exact match complementary to all or a portion of the target mRNA to which the designed oligonucleotide specifically binds.
- Percent similarity or percent complementary of any of the disclosed sequences may be determined, for example, by comparing sequence information using the GAP computer program, version 6.0, available from the University of Wisconsin Genetics Computer Group (UWGCG).
- the GAP program utilizes the alignment method of Needleman and Wunsch (1970). Briefly, the GAP program defines similarity as the number of aligned symbols (i.e., nucleotides or amino acids) that are similar, divided by the total number of symbols in the shorter of the two sequences.
- the preferred default parameters for the GAP program include: (1) a unary comparison matrix (containing a value of 1 for identities and 0 for non-identities) for nucleotides, and the weighted comparison matrix of Gribskov and Burgess (1986), (2) a penalty of 3.0 for each gap and an additional 0.10 penalty for each symbol in each gap; and (3) no penalty for end gaps.
- constructs are transfected into an appropriate cell type along with a construct containing a transgene expression cassette flanked by the AAV terminal repeats (e.g., pTR-UF5).
- AAV terminal repeats e.g., pTR-UF5
- This example describes an rAAV2 production system based on modifications of the triple plasmid transfection method. In this system, the expression of a specific capsid protein is restricted to one pIM45 plasmid and complemented in trans with the remaining two capsid proteins expressed from a second pIM45 plasmid. This approach maintains expression of the capsid proteins in their genomic context while providing a platform for the genetic modification of a specific capsid protein or two of the capsid proteins across their entire coding sequence.
- Missense mutation of the capsid proteins' start codons generated pIM45 plasmids that express a single capsid protein: pIM45-VPl, pIM45-VP2 (ACG or ATG start codon), and pIM45-VP3.
- Such plasmids can be complemented with plasmids expressing the remaining 2 capsid proteins ( ⁇ IM45-VP2,3, pIM45-VPl,3, and ⁇ IM45-VPl,2 (ACG or ATG start codon), respectively) in order to produce viable rAAV2 vectors.
- rAAV2-like particles are produced as long as the VP3 protein is present (VP1+2+3, VPl+3, VP2+3, and VP3 only). Focusing on large peptide insertions in the VPl and VP2 proteins without altering the critical VP3 protein, the utility of this system is demonstrated through the production of viable rAAV2 particles containing 8-, 15-, and 29-kDa proteins inserted immediately following amino acid 138 in both VPl and VP2 proteins or in VP2 protein alone. Finally, rAAV2-like particles can be produced with altered capsid protein stoichiometry if VP2 is significantly over expressed.
- missense mutation of the AAV2 cap ORF start codons was employed as previously described.
- the VPl start codon was mutated to leucine to generate the construct, pIM45-VP2,3, the VP2 start codon to alanine to generate the construct, pIM45-
- the expression of VPl and VP2 could be eliminated by missense mutation of their start codons (FIG. 1A, lanes 2 and 3), and, in contrast, mutation of the VP3 start codon resulted in expression of a smaller VP3-like fragment (VP3a) (FIG. 1A, lane 4).
- VP3 start codon allows translational initiation to occur downstream at the next available
- 523, 558, and 604 are in a context that is predicted favorable by Kozak. Since the VP3a fragment is slightly smaller than wildtype VP3, the contribution of continued read through translational initiation to the appearance of the VP3a fragment was examined by mutating the next two available ATG codons (M211 and M235) on a pIM45-M203L template yielding the plasmids, pIM45-M203L, pIM45-M203,211L and pIM45-M203,211,235L (FIG. 2A).
- FIG. IB again demonstrates the formation of VP3a following the mutation M203L.
- Combined mutation of M203 and M211 allowed less robust expression of a second still shorter VP3-like fragment (VP3b, FIG. IB, lane 3).
- Subsequent mutation of M235 in the pIM45-M203,211L background led to disappearance of this VP3b fragment generating pIM45-VPl,2 (FIG. IB, lane 4).
- VP2 proteins (pIM45-VPl,2, M203,211,235L).
- VP1,2A with one point mutation in both the VPl and VP2 proteins (T138M). Presumably, this stronger VP2 start codon prevents efficient translational initiation at the downstream
- VP3 start codon The VP2 start codon was mutated to ATG on a pIM45 template (pIM45-
- VP1,2A (FIG. IC)) as an alternative means of eliminating VP3 protein (while maximizing
- pIM45 plasmids that express a single capsid protein were generated next.
- the plasmids pIM45-VPl, ⁇ IM45-VP2, pIM45-VP2A, and pIM45-VP3 (FIG. 2) were also generated.
- pIM45-VPl has the VP2 start codon mutated to alanine and M203, M211, and M235 mutated to L in the expressed VPl protein .
- pIM45-VP2 has the VPl start codon mutated to leucine and M203, M211, and M235 mutated to L.
- the expressed VP2 protein contains only M203, M211, and M235 mutations.
- ⁇ IM45-VP3 has the VPl start codon mutated to leucine and the VP2 start codon mutated to alanine. Like all VP3 protein in these complementary groups, the VP3 coding sequence is not mutated.
- pIM45-VP2A has the VPl start codon mutated to leucine and the VP2 start codon mutated to methionine resulting in the single T138M modification of the VP2 protein being expressed.
- the plasmids pIM45-M203L, pIM45-M211L, pIM45-M235L, and pIM45M-203,211,235L were cotransfected separately with ⁇ TR-UF5 and pXX6 in a 1:1:8 molar ratio in 293 cells and 72 hrs later the cells were harvested and particles were purified as previously reported.
- Western blotting of capsid protein expression and dot blot analysis of genome containing particles was carried out on the mutant virus preparations (FIG. 3A). No particles were recovered from pIM45-M203L (lane 2) indicating that the combination of VPl, VP2, and VP3a does not able form a stable AAV particle.
- M211 Equally important in the formation of the particle is M211 (lane 3), as this mutation also prevented particle recovery. Whether it is the M211L in VPl, VP2, or VP3 that leads to this defective phenotype is unclear. This issue is addressed infra when pIM45- VP1,2 is complemented with pIM45-VP3 to produce AAV particles (Fig 4 #5). Finally, particles were obtained from pIM45-M235L (Fig 3A, lane 4) that package DNA efficiently.
- ⁇ IM45-VPl, pIM45-VP2, pIM45- VP2A, and pIM45-VP3 were transfected separately into 293 cells with pTR-UF5 and pXX6 in a 1:1:8 molar ratio and harvested cells 72 hrs later and purified particles as previously described.
- Western blotting of capsid proteins, A20 ELISA, and dot blot analysis of virus preparations were carried out with no detectable AAV-like particles obtained from pIM45- VP1, ⁇ IM45-VP2, or pIM45-VP2A (FIG.
- FIG. 4 To control for twice the Rep expression resulting from two pIM45 plasmids, an additional plasmid was constructed, pIM45-VP0, that expresses no capsid proteins as a result of 5 point mutations (MIL, T138A, M203,211,235L).
- Complementary group VPO (FIG. 4, #1) includes pIM45 and pIM45-VP0, group VPl includes ⁇ IM45-VPl and pIM45-VP2,3 (FIG.
- group VP2 includes ⁇ IM45-VP2 and pIM45-VPl,3 (FIG. 4, #3), group VP2A includes pIM45-VP2A and pIM45-VPl,3 (FIG. 4, #4), and group VP3 includes ⁇ IM45-VP3 and ⁇ IM45-VPl,2 (FIG. 4, #5).
- Western blotting of capsid proteins, A20 ELISA, and dot blot analysis of virus preparations were carried out following transfection of the individual groups into 293 cells with pTR-UF5 and pXX6 in a 1:1:8 molar ratio. 72 hrs post transfection the cells were harvested and particles were purified as previously described.
- Cotransfection of the pIM45-VP2A and pIM45-VP3 plasmid should yield a particle with an altered VP2:VP3 ratio if such a defective interfering particle contributes to the low titer of this group.
- an AAV-like particle with an overrepresentation of VP2 protein was purified that resembled the VP2,3 and VP3 only particles with respect to infectivity (FIG. 5B, lane 4).
- VP1/VP2 overlap region has been shown to be on the surface of the particle and flexible in the acceptance of targeting epitopes, the ability of this region to accept larger insertions was examined. Presumably, large insertions in the VP3 protein would decrease ones success in obtaining a particle due to steric hindrances in assembling the 60 modified capsid subunits. Sixty ligands were considered excessive when inserting large molecules into the AAV particle, so the strategy employed was to focus larger insertions to VPl and/or
- 138 may have less steric constraints but may produce particles with defective trafficking due to the juxtaposition of a large insertion to the putative phospholipase motif in VPl protein.
- VPl essentially an N-terminal fusion of 137 amino acids to VP2, and a CD 34 sc antibody VP2 protein fusion are readily inco ⁇ orated into an AAV particle
- insertion of large epitopes only at the N-terminus of VP2 may have advantages.
- genetic modification of the VP2 protein exclusively has not been accomplished from within a pIM45 based AAV production scheme.
- directional cloning sites were generated immediately after amino acid 138 in plasmids that express VPl and VP2 or VP2 only (FIG. 6A). The choice of
- Eagl/Mlul restriction sites resulted in two amino acids insertions on either side of further inserted sequence (RP/RT).
- pIM45-VPl,2A and pIM45-VP2A were chosen as templates for engineering Eagl/Mlul restriction sites immediately after amino acid 138 in VPl and VP2 or in VP2 only (pIM45VPl,2AEM138, pIM45-VP2AEM138, FIG. 6A). Templates with VP2 protein over expression were used to help ensure that genetically-modified VP2 would be present at sufficient levels for assembly.
- the pIM45-VPl,2AEM138 plasmid was complemented with pIM45-VP3, while the pIM45-VP2AEM138 plasmid was complemented with pIM45-VPl,3 for the production of rAAV particles carrying insertion sequences.
- VPl,2Alep pIM45-VP2Alep
- pIM45-VPl,2AGFP pIM45-VP2AGFP
- pIM45-VP2AGFP pIM45-VP2AGFP
- This example increases the flexibility in probing the surface of the particle by isolating the expression of a given capsid protein to a separate plasmid.
- Such an approach allows for manipulation of this capsid protein only within the produced particle and allows for retesting regions of capsid overlap for the acceptance of sequence modification.
- the system also allows for the modification of only two of the capsid proteins while leaving the third protein unmodified.
- characterization of the catalogue of plasmids required for this system yielded interesting results concerning the role of each capsid protein in the assembly of AAV-like particles.
- Elimination of VP3-like fragments illustrates importance of VP3 N terminus, as particles with these mutations in the VPl and VP2 proteins were recovered following complementation of pIM45-VPl,2 with pIM45-VP3.
- Evidence that ability to modify individual capsid proteins in regions of overlap may allow production of particles that were defective for production when mutations are in all three proteins. Increases the flexibility in manipulation of the particle for targeting pu ⁇ oses.
- isolation of the expression of a C-terminal modified VP3 separately allowed for modification of the c-terminus of VP3 with his tag and production of viable recombinant virus follow nickel chromatography.
- the present system allows for complementation and recovery of rAAV2 particles with all capsid proteins present. Since it allows for the genetic modification of only one or two of the capsid proteins, it can also be used for studies of previously reported lethal mutations in overlapping capsid sequences to see if mutations at the same positions in fewer capsid proteins rescue the position for particle manipulation. Important genetic modification would include insertion of genetic sequence for retargeting the virus, purification of the virus, monitoring of the virus particle following infection, or presentation of immunogenic epitopes on the surface of the virus particle.
- This example is the first report of the purification of an AAV-like particle containing a mutation in the VP2 protein exclusively.
- VP2 overexpression may have ensured the inclusion of modified VP2 protein large insertions with VP2 acg start codon produced significantly less modified VP2 proteins.
- charged-to-alanine substitution mutants were made to analyze the effects of single and combinatorial mutations in the capsid gene. New point mutants that result in assembly, packaging, and receptor binding deficiencies have been discovered. Importantly, five amino acids, arginines 484, 487, 585, and 588, and one lysine at position 532 have been identified that appear to mediate the natural affinity of AAV for HSPG. Those observations contribute to the current map of the AAV capsid and provide a reagent for the discovery of novel, heparin independent targeting ligands.
- Plasmid pIM45 (previously called pIM29-45) contains the Rep and Cap coding sequences from AAV with expression controlled by their natural promoters (McCarty et al, 1991). It was used as the parent template for construction of all the AAV2 mutant vectors.
- Plasmid pXX6 supplies the adenovirus helper gene products in trans to allow rAAV production in an adenovirus free environment (Xiao et al, 1998).
- Plasmid pTR2-UF5 supplies the recombinant AAV DNA to be packaged. It contains a cytomegalovirus promoter driving expression of a green fluorescent protein (GFP) reporter gene flanked by AAV2 terminal repeats (Klein et al, 1998). Plasmid pTR5-UFll was constructed using an expression cassette consisting of a strong constitutive CBA promoter (Xu et al, 2001), GFP reporter gene (Zolotukhin et al, 1996), woodchuck hepatitis virus posttranscriptional regulatory element WPRE (Donello et al, 1998) and bovine growth hormone gene polyadenylation signal. The cassette was assembled using standard molecular biology techniques and substituted for the la L cassette in the plasmid backbone pAAV5RntocZ containing AAV5 terminal repeats (Chiorini et al, 1999).
- GFP green fluorescent protein
- Plasmids pXYZl, ⁇ XYZ5 contain the AAV1 and AAV5 Cap coding sequences, respectively, in addition to AAV2 Rep coding sequence with an ACG start codon under control of the AAV2 p5 promoter (Zolotukhin et al, 2002). Plasmid pAAV5-2 contains the AAV5 nucleotides 260 to 4448 without terminal repeats (Chiorini et al, 1999).
- Transfection quality plasmid DNA was produced by standard alkaline lysis method of a 1 -liter TB culture followed by PEG precipitation and cesium chloride gradient purification.
- AAV2 virions To produce AAV2 virions, low passage 293 's were seeded so that they were approximately 75% confluent at transfection time.
- a triple plasmid transfection protocol (Xiao et al, 1998) was followed that included pIM45 to supply Rep and mutated capsid genes, ⁇ TR2-UF5 (Klein et al, 1998) to supply recombinant DNA with AAV2 terminal repeats and a CMV driven GFP reporter gene, and pXX6 (Xiao et al, 1998) to supply the adenovirus helper functions in trans.
- a total of 60 ⁇ g of plasmid DNA in a 1:1:1 molar ratio was transfected by lipofectamine (Invitrogen).
- rAAVl and rAAV5 particles To produce pseudotyped rAAVl and rAAV5 particles, a total of 60 ⁇ g of pXYZl or pXYZ5 (Zolotukhin et al, 2002) was co-transfected with ⁇ TR2-UF5 plasmid DNA in a 1:1 molar ratio as above. To produce rAAV5 and rAAV5-HS virions a total of 60 ⁇ g of pAAV5 or ⁇ AAV5-HS was co-transfected with pTR5-UFll.
- the A20 ELIZA American Research Bioproducts
- the A20 antibody detects intact, fully assembled particles, both full and empty (Wistuba et al, 1995). Iodixinal purified stocks were serially diluted and processed by the manufacturer's recommended protocol. Only readings within the linear range of the kit standard were used.
- RT-PCRTM Reactions were extracted with phenol/chloroform/isoamyl-alcohol (25:24:1) and undigested DNA was precipitated overnight with ethanol and glycogen carrier. Precipitated DNA pellets were resuspended in 100 ⁇ l of water. Five ⁇ l was used for RT-PCRTM analysis in a reaction mixture that included 900 nM each of GFP forward and reverse primers, 250 nM Taqman probe, IX Taqman universal PCR master mix in a total volume of 50 ⁇ l. Cycling parameters were 1 cycle each of 50°C, 5 mins, and 95°C, 10 mins, followed by 40 cycles of 95°C, 15 sec and 60°C, 1 min. Only values within the linear portion of a standard curve having a coefficient of linearity greater than .98 were accepted. The average RT-PCRTM titer was calculated from virus preparations assayed three times.
- GCA green cell assay
- the average GCA titer was calculated by averaging the number of green cells counted in individual wells from two or three virus preparations assayed three times. Particle to infectivity ratios were calculated by dividing the average RT-PCRTM titer by the average GCA titer. In some figures, this number was expressed as a logio value with rAAV2 arbitrarily set to one.
- Bio-Rad microspin columns were treated with silicon dioxide to minimize nonspecific binding of the virus to the column wall.
- Bound virus was eluted with IX TD + 2M NaCI. The entire volume of the flow through, wash, and eluate fractions were pooled separately, denatured by boiling in SDS, and slot blotted onto nitrocellulose for immunoblot analysis.
- the membrane (Osmonics) was blocked in PBS/0.05% Tween-20 + 5% dry milk, and incubated with Bl antibody (Wistuba et al, 1997) at a 1:3000 dilution for 18 hrs at 4°C.
- Anti-mouse IgG-horse radish peroxidase was used to detect bands by enhanced chemiluminesence (Amersham- Pharmacia).
- HeLa C12 cells were seeded in 6 well plates so that they were approximately 75% confluent at infection time.
- FACS analysis for GFP expression was done in the ICBR Flow Cytometry lab of the University of Florida on a Becton-Dickinson FACScan.
- Cells were incubated at 37°C in a 5% CO 2 atmosphere until harvesting. At indicated time points, the infection media was removed and saved and the cells were washed four times with PBS before being scraped. Low molecular weight DNA from the infection media and the cell pellet was extracted by the Hirt procedure (Hirt, 1967). DNA pellets were resuspended in 0.2M NaOH, incubated at 37°C for 20 mins, and slot blotted onto nitrocellulose.
- DNA was UV cross- linked to the nitrocellulose and probed at 65°C for 18 hrs with [ - 32 P]-dATP labeled GFP probe in hybridization buffer (7% SDS, 10 mM EDTA and .5M Na 2 HPO 4 ).
- Membranes were washed twice in 2X SSC/0.1%SDS, 0.2XSSC/0.1%SDS, 0.1X SSC/0.1%SDS, and rinsed with water. The membranes were then exposed to film and quantitated using a BAS- 1000 phosphor imager (Fuji).
- rAAV4 and rAAV5 do not bind heparin and instead recognize 2,3 O-linked and 2,6 N-linked sialic acid moieties (Kaludov et al, 2001). Indeed, this may account for their different cellular tropisms. It was reasoned that residues conserved among all five serotypes were probably not participating directly in receptor discrimination and binding and were excluded from further consideration. Additionally, a number of charge to alanine substitution mutants in the AAV capsid had been identified, and these had been characterized for their ability to bind heparin sulfate columns (Wu et al, 2000) and amino acid positions that did not affect heparin binding or had been shown to be assembly mutants were excluded from further study.
- Residues selected for mutagenesis were generated by a sequence alignment of the VPl capsid protein from each serotype using the Clustal W algorithm (Vector NTi 5.2, Informax).
- b Amino acids are represented by their one letter abbreviation. Blue letters represent positively charged, basic amino acids. Red letters represent any other amino acid.
- a series of single and combinatorial capsid mutants were generated from the pool of candidate residues in the AAV2 capsid gene (Table 4). To designate the mutant viruses, the number of the mutated amino acid based on its position in VPl was used. Iodixanol purified virus stocks were checked by western blot using the monoclonal antibody Bl. The Bl antibody recognizes a linear epitope in the extreme carboxyl terminus of all three VP proteins from AAV serotypes 1, 2, 3 and 5 (Rabinowitz et al, 2002; Wobus et al, 2000). With the exception of H358A, capsid proteins were detected in all virus stocks (FIG. 7).
- the particle concentration was measured with an A20 antibody ELISA (Table 5).
- the A20 antibody recognizes a structural epitope that is found only on assembled capsids with or without packaged DNA (Grimm et al, 1998). Although there was some variability between stocks due to different transfection efficiencies and purification recoveries, only the H358A mutant was negative by A20 ELISA assay. Excluding H358A, a particle concentration range was determined that spanned 1.5 logs and correlated reasonably well with the Bl antibody results (FIG. 7; Table 5).
- capsid subunits containing these mutations (i) form intact particles inefficiently, (ii) are unstable during purification or (iii) formed a particle with a partially disrupted A20 epitope. Since none of these mutations fell within the antigenic regions that have been mapped for A20 (Wobus et al, 2000), these results suggested that the A20 epitope had probably not been modified but rather the stability or assembly of some of the mutants was altered so that fewer particles were recovered after iodixanol centrifugation (FIG. 7; Table 5).
- Mutant virus A20/ml Genome/ml (IU/ml) infectivity binding 6 Full g d rAAV2 (WT) 1.5 x 10 12 4.6 x 10 11 1.8 x 10 10 25 + 3.4
- A20 particle titers were determined as described using the A20 ELISA assay. Genomic titers were determined by RT-PCRTM .
- Infectious titers were determined by green cell assay as described by counting GFP fluorescent cells.
- d Particle-to-infectivity ratio was calculated by dividing the average genomic titer as determined by RT-PCRTM by the average green cell assay titer.
- e Determined by heparin-agarose binding assay. +, >95% virus recovered in the eluate; +/-, >50 recovered in the eluate; -, ⁇ 5% of virus recovered in the eluate.
- f N/D not determined.
- Empty-to-full ratio was determined by dividing the A20 particle titer by the average genomic titer.
- titer of DNA containing virions was determined by real-time (RT) PCRTM (Clark et al, 1999; Veldwijk et al, 2002) (Table 5) and confirmed by DNA dot blot hybridization. Although there was variation between preparations, the majority of the capsid mutants were able to package detectable DNA (Table 5). As expected, H358A was negative for DNA packaging, as it did not produce virus particles. It was concluded that none of the capsids in the mutant panel that made A20 positive particles were completely defective for DNA packaging.
- mutants of mutant R459A contained 40-fold more empty particles than wild type rAAV2. Thus, R459 could have a role in DNA packaging. Although less dramatic, mutants R447A, R566A, R587A, R585K, and R585K/R588K had approximately 10-fold more empty particles than rAAV2. The remainder of the virus preparations packaged DNA at levels comparable to wild type AAV2 (Table 5).
- mutant capsids to bind heparin sulfate
- a modification of an assay previously described by Wu et al. (2002) was used.
- Virus preparations that had been purified by iodixanol step gradients were loaded on heparin agarose columns and the entire volume of the flow through, wash, and eluate fractions were pooled separately, denatured, and slot blotted onto nitrocellulose for immunoblot analysis with Bl antibody.
- a representative Western analysis for each mutant is shown in FIG. 8. As expected, wild type AAV2 was not observed in the flow through or wash fractions and most of the virus bound to the column was recovered at the elution step.
- R447A, R459A, H509A, R513A, K544A, K566A, N587A, and H526A/K527A had a heparin-agarose binding phenotype indistinguishable from wild type.
- the results with R513A confirmed a previous report (Wu et al, 2000) in which a double mutant at positions 513 and 514 was positive for heparin binding. In marked contrast, it was observed that any capsid harboring a non- conservative mutation at position 585 or 588 was detected only in the flow through and wash.
- iodixanol stocks were tested for their ability to transduce HeLa C12 cells by performing a green cell assay (GCA).
- GCA green cell assay
- Twenty-four hours post-infection (hpi) the number of GFP expressing cells in individual wells were counted and a GCA titer was calculated (Table 5). The detection limit of this assay was approximately 10 4 transducing units/ml.
- the GCA titers were then normalized to genome containing physical particles by calculating a particle to infectivity (P/I) ratio. This ratio is equivalent to the number of genomes required to transduce one cell (Table 5). To get a measure of the relative impact of a particular mutation on viral infectivity, the P/l ratio of each mutant was divided by the wild type capsid P/l ratio and the logio of this value was plotted in FIG. 10. This provided a simple comparison of how many genome-containing particles of each mutant were required to achieve the same number of transduced cells as the wild type virus.
- Mutants R477A, K544A, and K566A were virtually identical to wild type, and mutants R513A, N587A, R585K, and R585K/R588K were only slightly defective (approximately 1 log). These seven mutants were found previously to bind heparin sulfate to the same extent as wild type rAAV2 (FIG.
- R459A was the most severe example of three mutants (R459A, H509A, and H526A/K527A) that were essentially wild type for heparin binding but defective for transduction (FIG. 10). These mutants were presumably defective in some late stage of viral infection.
- Hela C12 cells were transfected and the location of viral DNA tracked.
- the infection media was removed and saved, and the cells were washed extensively to remove any residual unbound virus.
- the cells were then harvested and low molecular weight DNA was extracted from both the infection media (unbound) and the cell pellet (bound or internalized) by the Hirt procedure and transferred to nitrocellulose for Southern hybridization with an [ - 32 P]-dATP labeled GFP probe (FIG. 12A and FIG. 12B).
- rAAV5 and rAAV5-HS packaged viruses were generated that contained a recombinant AAV5 genome in which the GFP reporter gene was flanked by AAV5 terminal repeats.
- the infectivity of these viruses was compared to rAAV2 in a GCA assay and particle-to-infectivity ratios were calculated as before (FIG. 13D).
- rAAV5 was able to transduce Hela C12 cells at a low efficiency, approximately 2.5 logs lower than AAV2. However, no transduction was seen with AAV5-HS ( ⁇ 1 x 10 4 IU/ml) (Table 5; FIG. 13D).
- This example describes the identification of amino acids in the capsid of AAV2 that mediate binding to heparin sulfate proteoglycans.
- HSPG serves as the primary receptor for AAV2. Inhibition of AAV2 infection can be demonstrated by competition with soluble analogs, GAG desulfation by sodium chlorate treatment, antibody competition, enzymatic removal of heparin, and use of mutant cell lines that express varying levels of HSPG (Handa et al, 2000; Qiu et al., 2000; Summerford and Samulski, 1998; Wu et al, 2000).
- Binding to heparin sulfate is usually the result of electrostatic charge interactions between basic amino acids (R, K, or H) and negatively charged sulfate residues (Hileman et al, 1998; Mulloy and Linhardt, 2001).
- basic amino acids R, K, or H
- sulfate residues Hileman et al, 1998; Mulloy and Linhardt, 2001.
- many of the basic amino acids in the AAV2 capsid that could potentially contribute to heparin sulfate binding were eliminated (Wu et al, 2000).
- the remaining basic residues were examined by looking at their conservation in AAV serotypes 1-5. Those that were present in all five serotypes were not likely to contribute significantly to heparin binding.
- capsids with mutations at residue 484, 487, 532, 585 or 588 were partially or completely defective for heparin-agarose binding.
- the most severe defect was found with mutations in R585 and R588.
- No binding to heparin sulfate columns could be detected with either mutant (FIG. 8), and both mutations reduced the particle-to- infectivity ratio by two to three logs (Table 5). Mutants that contained substitutions at both positions had even lower infectivity.
- R487A, R585A, and R588A were probably due largely to defective heparin binding.
- the double mutant R585A/R588A was approximately 500 fold more defective in cell binding and internalization than wild type (FIG. 12B) when corrected for the MOI, and approximately 2000 fold less infectious (Table 5), as judged by the change in particle-to-infectivity ratio.
- Another indication that heparin binding was primarily responsible for the defects in R585 and R588 was the fact that conservative mutations at these two positions (R585K and R585K/R588K) produced virus particles with properties similar to wild type (FIG. 8; FIG. 10; FIG. 11; Table 5).
- R484A and K532A two other mutants were found that were defective for heparin binding, R484A and K532A.
- R484A and K532A like R487A, had a more modest effect on binding to heparin sulfate, but unlike the other heparin binding mutants, these two mutations had a dramatic effect on transduction efficiency.
- Both R484A and R532A were more than 5 logs less infectious than wild type capsids (Table 5; FIG. 10). This severe defect is presumably due to a different block in the infection process that is unrelated to heparin binding, but as yet it has not been identified.
- Mut 37 had a phenotype identical to K532A in that it produced full virus particles that were non-infectious and more recently has been shown to have a modest defect (approximately 5 fold) in heparin binding and internalization. This potentially maps this defect to a single amino acid.
- the apparent dissociation constant (Kd) of AAV2 and heparin sulfate was determined by competition analysis to be 2 x 10 9 M (Qiu et al, 2000). Although this is higher than some heparin-protein interactions, it is sufficiently strong to suggest cooperative binding by one HS glycosaminoglycan chain to multiple attachment points. This example does not address whether heparin sulfate could form a bridge between basic residues in one of the threefold spikes to those in another.
- H538A was defective for particle assembly.
- mutations that disrupt AAV2 particle formation several of which are located in the conserved ⁇ -strand regions (Rabinowitz et al, 1999; Shi et al, 2001; Wu et al, 2000). Since H358 is neither surface accessible nor in a conserved ⁇ -strand, it is possible that it acts internally to stabilize the monomer subunit structure.
- mutant R459A produced a 40-fold excess of empty capsid particles compared to rAAV2.
- Other studies have reported that short insertions at positions 323, 339, 466, 520, 540, 595, 597 that did not interfere with capsid formation still reduced DNA packaging to levels detectable only by PCRTM amplification (Shi et al, 2001).
- a point mutant R432A prevents DNA packaging (Wu et al., 2000). Although the relationship between these mutations and their mechanism of action is unclear, it is possible that they disrupt protein-capsid or DNA-capsid interactions.
- FIG. 14 shows the results of an immunoslotblot of total capsid protein following standard purification procedures of a representative expression system of the invention.
- FIG. 15 shows a dot blot autoradiograph of DNA extracted from pTR-UF5 and the system plasmid combinations.
- FIG. 16 shows the in vivo transduction ability of recombinant AAV vectors produced using various system components.
- FIG. 17 shows an Immunoblot and dot blot autoradiograph of virions produced from ⁇ TR-UF5; pIM45-VPl,2; pIM45-VPl,3; and ⁇ IM45-VP2,3 plasmids following standard purification protocols.
- FIG. 18 shows the in vivo transduction ability of recombinant AAV vectors containing only two capsid proteins, while FIG. 19 depicts an immunoblot of protein fractions collected from iodixinol purified passed over a heparin- agarose column. Using an anti-VPl,2,3 monoclonal antibody.
- FIG. 18 shows the in vivo transduction ability of recombinant AAV vectors containing only two capsid proteins
- FIG. 19 depicts an immunoblot of protein fractions collected from iodixinol purified passed over a heparin- agarose column. Using an anti-VPl,2,3 monoclonal antibody.
- FIG. 20 shows a dot blot autoradiograph of DNA extracted from ⁇ TR-UF5 and rAAV R585A, R588A
- FIG. 21 summarizes an exemplary system that demonstrates the in vivo transduction ability of pTR- UF5 and R585A, R588A
- FIG. 22 shows a slot blot autoradiograph of an in vivo DNA tracking time course experiment of pTR-UF5, rAAV R585A, R588A
- FIG. 23 shows a schematic diagram of the pIM45 vector showing the rep and cap sequences.
- AAV particle Interest in the composition, assembly, and atomic structure of the AAV particle stems in part from its promise as a recombinant gene delivery vehicle in vivo. However, further clinical development of AAV for gene therapy will require the ability to target specific tissue types. A better understanding of the particle's surface architecture has been obtained through systematic alanine-scanning (Wu et al., 2000) and insertional mutagenesis
- the insertion of targeting sequences in the VPl/2 and VP3 capsid overlap regions of the cap ORF have produced AAV with alternative cellular receptor usage. Insertions after residue 138 (N-terminus of VP2) expand the tropism of AAV (Loiler et al., 2003; Shi et al, 2001; Wu et al., 2000), as they do not disturb the capsid residues involved in binding cellular heparan sulfate proteoglycan (Kern et al, 2003; Opie et al, 2003).
- Ligands inserted after residue 587 reside at the particle's threefold axis between critical residues involved in cell binding via heparan sulfate proteoglycan (Kern et al, 2003; Opie et al, 2003; Xie et al, 2002), the primary viral receptor.
- these insertions can simultaneously restrict viral entry and redirect it to an alternative receptor. Still, these inserted sequences have been restricted in size ( ⁇ 30 amino acids) consisting of linear receptor binding epitopes.
- One limitation to manipulating the cap ORF in the direct insertion approach is that modification of only one capsid across its entire sequence, leaving the remaining two capsids unaltered, is not possible. Only one region of the cap ORF allows for modification of a single capsid (VPl, residues 1-137) and this region contains a phospholipase A motif that is critical for efficient viral infection (Girod et al, 1999).
- a single report (Yang et al, 1998) has shown that a significantly larger single chain antibody coding sequence can be incorporated into recombinant particles if it is fused to the N-terminus of VP2 and co-expressed with wild type VPl, VP2, and VP3 capsids. These particles were capable of retargeting the vector to the CD34 molecule but recombinant titers were extremely low.
- plasmids were generated that expressed only one or two of the capsid proteins, and their ability to produce AAV particles was tested.
- AAV-like particles are produced as long as VP3 is present. Characterization of the physical titers of these AAV-like particles that lacked specific capsid proteins demonstrated that the VP2 protein is apparently redundant and is not essential for viral infectivity.
- a method of producing AAV-like particles with large peptide insertions in VPl and VP2 or VP2 exclusively was described, by expressing the modified protein separately, and providing the remaining wild type capsids in trans.
- AAV-like particles could be produced with altered capsid composition if VP2 is significantly over-expressed.
- Plasmid, pIM45 contains the rep and cap coding sequences of AAV with their expression controlled by their native promoters (McCarty et al., 1991). It was used as a parent template for construction of all mutant plasmids. Plasmid pXX6 (Xiao t al., 1998) supplies the adenovirus helper gene products in trans to allow rAAV production in an adenovirus free environment and was supplied by Jude Samulski. Plasmid pTR-UF5 (Zolotukhin et al, 1996) supplies the rAAV DNA to be packaged. It contains a cytomegalovirus promoter driving expression of a GFP reporter gene flanked by the AAV terminal repeats. Plasmid pTRdsRed is identical to pTR-UF5 except that the GFP coding sequence is substituted with the red fluorescent protein (RFP) coding sequence.
- RFP red fluorescent protein
- Site directed mutagenesis was performed on plasmid pIM45 as per the manufacturer's instructions. For each mutant plasmid, two complementary PCRTM primers containing a missense mutation in the individual capsid protein start codons were used to introduce changes in the cap ORF of pIM45. The oligonucleotides used for mutagenesis are listed in Table 6. These plasmids were screened for restriction sites inserted by silent mutations, and the mutations were confirmed by DNA sequencing.
- VP2A-E/M138 aggaacctgttaagATGCGGCCGACGCGTgctccgggaaaaaagag (SEQ ID NO:8)
- FKN insert b cgCGGCCGtctggttcaggtagcggttctggtcagcacctcggcatgacgaaatgc (+) (SEQ ID NO:9) cgACGCGTaccgctgccagaacctgagccgctaccatttctagtcagggcagcggt (-) (SEQ ID NO: 10) LEP insert cgCGGCCGgtgcccatccaaaaagtccaagat (+) (SEQ ID NO: 11) cgACGCGTgcacccagggctgaggtccagctg (-) (SEQ ID NO:12) GFP insert cgCGGCCGatgagcaagggcgagggaactg (+) (SEQ ID NO:13) cgACGCGTcttgtacagctcgtccatgcc (-) (SEQ ID NO:14) a top group: + complementary oligon
- the same site-directed mutagenesis strategy was used to insert an Eagl/Mlul cloning site immediately after amino acid position 138 in pIM45.
- the same oligonucleotide pair with an additional T138M mutation was used to introduce these sites into pVPl,2A and ⁇ VP2A.
- the resulting plasmids were called ⁇ IM45-E/M138, ⁇ VPl,2A-E/M138, and pVP2A-E/M138.
- the cDNA for the rat fractalkine chemokine domain (FKN, CX3CL1, accession: NM134455), the human hormone leptin (LEP, accession: BC060830), and the green fluorescent protein (GFP, accession: U50963) flanked by Eagl and Mlul restriction sites were generated using PCRTM (Table 6).
- the PCRTM products were cloned into pIM45-E/M138, pVPl,2A-E/M138, and pVP2A-E/M138.
- low passage 293 cells were transfected at ⁇ 80% confluence using a modification of the triple transfection protocol (Li et al., 1997; Xiao et al., 1998; Zolotukhin et al., 1996). All plasmids were transfected in equivalent molar ratios using Lipofectamine Plus reagent (Invitrogen) according to the manufacturer's suggestions.
- A20 ELISA American Research Bioproducts
- the A20 antibody detects intact, fully assembled particles, both full and empty (Grimm et al, 1999; Grimm et al., 1998).
- Iodixanol purified stocks were serially diluted and processed by the manufacturer's recommended protocol. Only readings within the linear range of the assay were averaged.
- the reactions were then extracted with phenol/chloroform/ isoamyl-alcohol (25:24:1) and the packaged DNA was precipitated overnight with ethanol and glycogen carrier.
- the precipitated DNA pellets were dissolved in 100 ⁇ l of water and 5 ⁇ l was used for real-time PCRTM analysis in a reaction mixture that included 900 nM each of GFP forward and reverse primers, 250 nM Taqman probe, and IX Taqman universal PCRTM master mix in a total volume of 50 ⁇ l.
- the cycling parameters were 1 cycle each of 50°C, 5 min, and 95°C, 10 min, followed by 40 cycles of 95°C, 15 sec and 60°C, 1 min. Only values within the linear portion of a standard curve having a coefficient of linearity greater than 0.98 were accepted.
- the average real-time PCRTM tiler was calculated from virus preparations assayed three times.
- the RFP gene from pTR-dsRed was packaged and particle titers were determined by dot blot as described previously (Zolotukhin et al, 1999). Equal volume aliquots of the vector preparations were incubated with DNasel, inactivated with EDTA, digested with proteinase K, phenol: chloroform extracted, and precipitated with ethanol. The DNA was then transferred to nitrocellulose and probed with radiolabelled RFP probe.
- FCA fluorescent cell assay
- the average FCA titer was calculated by averaging the number of green fluorescent cells (or red fluorescent cells in the case of virus that contained a GFP insert in the particle) from preparations assayed three times. Particle to infectivity ratios were calculated by dividing the average DNA titer by the average FCA titer.
- HeLa cells were seeded in 8 chamber tissue culture slides (Falcon) 24 hours prior to infection with VP1,2A-GFP particles at an MOI of 10,000 in the absence and presence of
- BSA bovine serum albumin
- PBS Phosphate buffered saline
- RT room temperature
- EER1 The primary rabbit anti-Early Endosomal Antigen 1
- Sections were examined with a confocal laser scanning microscope (Bio-Rad Olympus) illuminated by three lasers (argon, "green” helium-neon, and “red” helium-neon), which supply excitation lines at 458, 488, 514, 543, and 633 nm.
- Cells on each slide were examined first for GFP staining. The focal plane was adjusted so that the number of detectable cell bodies was maximized and the green GFP image was then stored in memory. The procedure was repeated for the red PI image and the blue Cy5 image. Finally, a superimposition of the three colored images was made and stored. All manipulations of contrast and illumination on color images were made using Adobe PhotoShop ® 6.0 software on a PC.
- Residue 138 was chosen because ligands inserted at this position are present on the surface of the particle and result in alternative receptor recognition by AAV vectors (Loiler et al., 2003; Shi et al, 2001; Wu et al, 2000). Furthermore, this position does not directly interrupt the phospholipase A2 motif of VPl (Girod et al, 1999) or interfere with the structurally critical VP3 ⁇ -barrel arrangement (Xie et al, 2002). To test the direct insertion of larger peptides into cap, the directional cloning sites Eagl and Mlul were inserted immediately after residue 138 of the cap ORF in the plasmid pIM45, which contains the wild type rep and cap sequences.
- restriction enzymes meant that ligands inserted into the resulting plasmid (pIM45-E/M138) were flanked by arg and pro on the N-terminal side and arg and thr on the C-terminal side. These additional four amino acids had little effect on capsid expression, particle formation or titers
- the parental plasmid pIM45-E/M138 which had a 4 amino acid insertion in VPl and VP2 produced virus with approximately the same yield of particles and particle to infectivity ratio as pIM45, which contained wild type capsid proteins.
- VPl, 3 a A20 particle titers were determined as described in Materials and Methods using the A20 ELISA assay. Genomic titers were determined by RT-PCRTM . b Infectious titers were determined by fluorescent cell assay as described. c Particle to infectivity ratio was calculated by dividing the average genomic titer as determined by RT-PCR ⁇ by the average green cell assay titer. d Empty to full ratio was determined by dividing the A20 particle titer by the average genomic titer.
- VP3 would have to be provided in trans to complement the ligand-extended VPl and VP2.
- a complementary capsid protein expression system was generated that would allow for a single capsid protein to be modified in a region of sequence overlap (e.g. , genetic modifications of VP2 exclusively at residue 138).
- the VPl start codon, MIL was mutated generating the construct pVP2,3, which should only make VP2 and VP3 (FIG. 25A, Table 8).
- the VP2 start codon, T138A was mutated, generating the construct pVPl,3 that would make only VPl and VP3.
- the VP3 start codon, M203L was mutated in an initial attempt to generate the construct, pVPl,2 (FIG. 25A).
- Western blotting analysis of capsid protein expression in 293 cell lysates demonstrated that, while the expression of VPl and VP2 were eliminated by single point mutations (Fig.
- pM203L expressed a VP3-like species that migrated slightly faster than VP3 (VP3a) (see FIG. 25A, lane pM203L). This had been seen previously by Ruffing et al. (1992), who had used a similar strategy to eliminate VP3 expression.
- VP3a VP3-like species that migrated slightly faster than VP3
- transfection of pM203,211L resulted in weaker expression of a second yet smaller VP3-like species, VP3b (FIG. 25B, lane pM203,211L), while expression of all VP3-like species was eliminated in the triple mutant M203,211,235L, finally generating the plasmid, pVPl,2 (pM203,211,235L), which makes only VPl and VP2 (FIG. 25B, lane ⁇ VPl,2).
- Weak doublets present at the VP3 position in the ⁇ VPl,2 lane are due to cellular proteins that cross react with the Bl antibody (data not shown).
- capsid mutants that express a single capsid protein was accomplished by sequential mutation of start codons in the mutants that express two capsid proteins (FIG. 26).
- the construct that expressed only VPl had the VP2 start codon mutated, T138A, and the M203, 211,235L mutations that were required to eliminate VP3-like species (Table 8).
- the construct pVP2 had the VPl start codon mutation, MIL, and the M203,211,235L mutations, while construct pVP2A (to over-express VP2 alone) had the VPl start codon mutation, MIL, and the VP2 start codon mutation, T 8M.
- the construct pVP3 had the VPl start codon mutation, MIL, and the VP2 start codon mutation, T138A.
- Western blot analysis of capsid protein expression in 293 cells transfected with these plasmids showed that indeed these constructs expressed only a single capsid protein as expected (FIG. 26).
- the construct pVP2A significantly increased expression of VP2 in the absence of VPl or VP3 (FIG. 26).
- Modified Capsid Protein Complementing Plasmid pVPO (MIL; T138A; M203,211,235L) pIM45 (WT) pVPl (T138A; M203,211,235L) pVP2,3 (MIL) pVP2 (MIL; M203,211,235L) pVPl,3 (T138A) pVP2A (T138M) pVPl,3 (T138A) pVP3 (MIL; T138A) pVPl,2 (M203,211,235L)
- Capsid mutant complementation groups are co-transfected with pXX6 and pTRUF5 in 293 cells to produce particles.
- Plasmids that made only one or two of the capsid proteins allowed reexaminatuib of the ability of various combinations of VPl, 2, and 3 to make viable AAV particles.
- AAV-like particles composed of VP2 and VP3 were obtained following transfection of pVP2,3. Due to the lack of the capsid sequences unique to VPl, these particles displayed the lip phenotype with a particle to infectivity ratio approximately 3 logs lower than wild type (Table 7). This has been shown previously (Girodet al., 1999; Hermonat et al., 1984; Tratschin et al., 1984; Wu et al., 2000) and is presumably due to the absence of the VPl phospholipase A activity.
- an AAV-like particle formed in the absence of the previously reported critical VP2 capsid protein (Hoque et al., 1999; Muralidhar et al., 1994; Ruffing et al, 1992) when VPl and VP3 were present (FIG. 27B, pVPl,3 lane). Furthermore, these VP2 negative particles had virtually the same properties and yield as wild type particles (Table 7). Finally, the constructs that made only VPl and VP2 (pVPl,2 and pVPl,2A) were unable to assemble a particle in the absence of VP3, irrespective of the level of VP2 expression (FIG. 27B, Table 7).
- the capsid protein complementation groups include: pIM45 + pVPO, which makes wild type capsid proteins; constructs pVPl + pVP2,3, which allows for exclusive modification of VPl; constructs pVP2 + pVPl,3, which allows for exclusive modification of the VP2; constructs pVP2A + pVPl,3, which allows for exclusive modification of and significant over- expression of VP2; and constructs pVP3 + pVPl,2 which allows for exclusive modification of VP3.
- Eagl/Mlul cloning sites were again inserted after amino acid position 138 in pVPl,2A and pVP2A as described earlier for pIM45 to create the plasmids pVPl,2AE/M138 and pVP2A-E/M.
- the VP2 over-expressing background was chosen to increase the incorporation of VP2-ligand fusion proteins into viral particles.
- Both the FKN and LEP coding sequences were inserted into pVPl,2A E/M138 and pVP2A-E/M138 to make pVPl,2A-FKN, pVP2A-FKN, pVPl,2A-LEP, and pVP2A-LEP (FIG. 29A, FIG. 29B, FIG. 29C, Table 7).
- These plasmids were transfected into 293 cells in combination with pVP3 or pVPl,3, and pXX6 and pTRUF5 at equivalent molar ratios, and the resulting virus particles were purified with iodixanol step gradients.
- the particles were titered by the A20 ELISA assay (to estimate the total particles, empty and full), the real-time PCRTM assay (to determine the titer of genome containing DNase resistant full particles), and the fluorescent cell assay (to determine the infectious particle titer). These assays were all performed on the iodixanol purified stocks (Table 7) and then the log relative infectivity was calculated (FIG. 30B).
- the AAV particle is capable of transducing a wide range of dividing and non- dividing cell types.
- the promiscuity of this gene therapy vector is due in part to the widespread distribution of its primary receptors (Kern et al., 2003; Opie et al., 2003; Qing et al, 1999; Summerford et al., 1999; Summerford and Samulski, 1998) and the strong electrostatic interaction between cell surface heparan sulfate and the spike protrusion at the particle's three-fold axes (Kern et al, 2003; Opie et al., 2003; Summerford and Samulski, 1998).
- retargeting sequences can result in the incorporation of differing numbers of ligands per particle. For instance, insertions immediately after residue 138 in the VP1/VP2 region have been shown to expand the tropism of the virus (Shi et al, 2001; Wu et al., 2000) following the incorporation of approximately 6 modified capsid proteins (3 VPl and 3 VP2).
- VPS-LH E PROTEINS CAN BE TRANSLATED FROM 3 DIFFERENT METHIONINE CODONS AND THE FIRST 8 AMMO ACIDS OF 3 APPEAR TO BE ESSENTIAL FOR 3 CAPSID ASSEMBLY
- M203L results in an N-terminal truncation of VP3 (VP3a), while M211L is a point mutation in full length VP3.
- VP3a N-terminal truncation of VP3
- M211L is a point mutation in full length VP3.
- These mutations are present in all three capsid proteins, but appear to be critical to VP3 as the combination of pVPl,2 + pVP3 produced essentially wild type recombinant particles.
- the formation of particles from the complementation groups are examples of positional rescue of mutations at the VP3 N-terminus, as the M203L and M211L mutations that are required to eliminate VP3 expression (FIG. 25B) abolish particle formation (FIG.
- the AAV-like particle composed of only VPl and VP3 had infectious titers within a factor of 4 of wild type (FIG. 27B and FIG. 30B, Table 6), and particle to infectivity ratios which were identical to wild type.
- VP2 appeared to be a redundant capsid that is not essential for infectivity. This made it an ideal candidate for the insertion of large peptides for the purpose of retargeting the particle.
- VP2 negative particles (VPl, 3) appear to be functionally wild type
- VP2 may perform some function in AAV infection.
- a comparison of the characteristics of the VP3 particle with the VP2,3 particle suggests that the additional VP2 residues may facilitate transduction in the absence of VPl.
- the basic residues that cluster in the VP2 N-terminal extension of VP3 which are capable of being nuclear localization signals (Hoque et al., 1999) play a role.
- VP2A,3 particle is less infectious than VP2,3 showing that the inclusion of more VP2 unique sequence into the particle is detrimental (FIG. 30B, Table 7).
- 5.3.33 VP3 is THE ONLY CAPSID PROTEIN REQUIRED TO FORM GENOME CONTAINING PARTICLES
- AAV-like particles were obtained from any combination of capsid proteins or capsid mutants as long as VP3 was present (FIG. 30A and FIG. SOC, Table 8). Furthermore, VP3 alone was sufficient to make viral particles. Viral particles composed of VP2,3, VPl, 3, and VP3 were obtained only at wild type particle titers (both full and empty) (FIG. 27B, FIG. 27C, FIG. 28, FIG. 30A and Table 7). As expected, particles that were missing VPl (VP2,3, VP2A,3 and VP3) were severely defective for infectivity (FIG. 30B, Table 7).
- VP2 is non-essential and tolerates large peptide insertions at its N-terminus.
- the stoichiometry of the particle can be altered if VP2 is significantly over-expressed in the presence of native levels of VPl and VP3.
- the inserted sequences studied here are themselves potential targeting ligands, this system could also be applied to the insertion of large conjugate-based linker sequences (Ponnazhagan et al., 2002; Ried et al, 2002) or for the presentation of large immunogenic peptides for vaccine development using empty particles formed with this system as the platform for epitope presentation.
- FKN and LEP particles will involve testing their ability to bind their respective receptors.
- the GFP containing particles may have potential use in real time in vivo fluorescent monitoring of events that occur during infection. It is evident that optimal retargeting of these particles with insertions at the N-terminus of VP2 may require manipulation of linker sequences between the inserted ligand and VP2 to optimize presentation of the ligand binding domain. Furthermore, mutation of the recently identified residues involved in binding heparan sulfate proteoglycan (Kern et al, 2003; Opie et al, 2003) will also be required to restrict these vectors to cellular entry via the targeting ligand/receptor interaction.
- the system described here for modifying capsid proteins with larger peptide insertions in specific capsid proteins should facilitate development of retargeted AAV vectors for clinically relevant cell types and be applicable to all AAV serotypes and chimeric type particles (Bowles et al, 2003; Gao et al, 2003; Hauck et al, 2003; Hildinger et al, 2001; Rabinowitz et al, 2002).
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WO2007046703A3 (en) * | 2005-10-20 | 2007-06-07 | Amsterdam Molecular Therapeutics Bv | Improved aav vectors produced in insect cells |
US8802080B2 (en) | 2002-05-01 | 2014-08-12 | University Of Florida Research Foundation, Inc. | Raav expression systems for genetic modification of specific capsid proteins |
WO2019183579A1 (en) * | 2018-03-23 | 2019-09-26 | Life Technologies Corporation | Methods and kits to detect viral particle heterogeneity |
CN110691846A (en) * | 2018-05-07 | 2020-01-14 | 北卡罗来纳-查佩尔山大学 | Rational polyploid adeno-associated virus vectors and methods of making and using same |
CN111727243A (en) * | 2017-12-19 | 2020-09-29 | 北卡罗来纳-查佩尔山大学 | Methods and compositions for delivery of viral vectors across the blood-brain barrier |
US11702676B2 (en) | 2017-03-15 | 2023-07-18 | The University Of North Carolina At Chapel Hill | Rational polyploid adeno-associated virus vectors for the treatment of disease |
US11819539B2 (en) | 2017-09-22 | 2023-11-21 | The Trustees Of The University Of Pennsylvania | Gene therapy for treating Mucopolysaccharidosis type II |
US11821009B2 (en) | 2018-05-15 | 2023-11-21 | Cornell University | Genetic modification of the AAV capsid resulting in altered tropism and enhanced vector delivery |
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AU2004226961A1 (en) | 2004-11-18 |
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AU2004226961B2 (en) | 2009-06-11 |
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