Abstract: In vitro methods for making a recombinant adenoviral genome, as well as kits for practicing the same and the recombinant adenovirus vectors produced thereby, are provided. In the subject methods, the subject genomes are prepared from first and second vectors. The first vector includes an adenoviral genome having an E region deletion and three different, non-adenoviral restriction endonuclease sites located in the E region. The second vector is a shuttle vector and includes an insertion nucleic acid flanked by two of the three different non-adenoviral endonuclease sites present in the first vector. Cleavage products are prepared from the first and second vectors using the appropriate restriction endonucleases. The resultant cleavage products are then ligated to produce the subject recombinant adenovirus genome. The subject adenoviral genomes find use in a variety of applications, including as vectors for use in a variety of applications, including gene therapy.

Abstract: A helper dependent adenoviral vector system is provided. The subject helper dependent adenoviral vector system is made up of: (1) a “gutless” adenoviral vector that include cis-acting human stuffer DNA that provides for in vivo long term, high level expression of a coding sequence present on the vector; (2) an adenoviral helper vector that is characterized by having an adenoviral genome region flanked by recombinase recognition sites, where the helper vectors further include a non-mammalian endonuclease recognition site positioned outside of the adenoviral genome region; and (3) a mammalian cell that expresses the corresponding recombinase and endonuclease, as well as the adenoviral preterminal and polymerase proteins. Also provided are methods of using the subject systems to produce virions having the subject helper dependent adenoviral vectors encapsulated in an adenoviral capsid. In addition, kits for use in practicing the subject methods are provided.

Abstract: The instant invention provides methods of expressing polynucleotides in the cells of the liver comprising administering viral particles comprising a recombinant AAV vector into a mammal, preferably a human.

Abstract: Circular nucleic acid vectors that provide for persistently high levels of protein expression are provided. The circular vectors of the subject invention are characterized by being devoid of expression-silencing bacterial sequences, where in many embodiments the subject vectors include a unidirectional site-specific recombination product hybrid sequence in addition to an expression cassette. Also provided are methods of using the subject vectors for introduction of a nucleic acid, e.g., an expression cassette, into a target cell, as well as preparations for use in practicing such methods. The subject methods and compositions find use in a variety of different applications, including both research and therapeutic applications. Also provided is a highly efficient and readily scalable method for producing the vectors employed in the subject methods, as well as reagents and kits/systems for practicing the same.

Abstract: A helper dependent adenoviral vector system is provided. The subject helper dependent adenoviral vector system is made up of: (1) a “gutless” adenoviral vector, which in certain embodiments includes cis-acting human stuffer DNA that provides for in vivo long term, high level expression of a coding sequence present on the vector, where in certain embodiments the vector includes an integrating domain; (2) an adenoviral helper vector that is characterized by having an adenoviral genome region flanked by recombinase recognition sites, where the helper vectors further include a non-mammalian endonuclease recognition site positioned outside of the adenoviral genome region and in certain embodiments a third adenoviral inverted terminal repeat (ITR) sequence positioned between first and second terminal ITRs; and (3) a mammalian cell that expresses the corresponding recombinase and endonuclease, as well as the adenoviral preterminal and polymerase proteins.

Abstract: Non-human mammalian hosts are provided, comprising functional human hepatocytes. Isolated human hepatocytes or fragments of human hepatic tissue are introduced into the xenogeneic host in conjunction with one or more agent that stimulates human hepatocyte growth factor receptor. The human hepatocytes are maintained in the host by administration of one or more agent that stimulates human hepatocyte growth factor receptor, either continuously (e.g., via an implanted catheter or intravenous apparatus) or in discrete, regular dosages of the agent (e.g., via intravenous injections or oral dosages). The human hepatocytes are able to survive and function in the host animal for a period of at least 5 months.

Abstract: In vitro methods for making a recombinant adenoviral genome, as well as kits for practicing the same and the recombinant adenovirus vectors produced thereby, are provided. In the subject methods, the subject genomes are prepared from first and second vectors. The first vector includes an adenoviral genome having an E region deletion and three different, non-adenoviral restriction endonuclease sites located in the E region. The second vector is a shuttle vector and includes an insertion nucleic acid flanked by two of the three different non-adenoviral vectors present in the first vector. Cleavage products are prepared from the first and second vectors using the appropriate restriction endonucleases. The resultant cleavage products are then ligated to produce the subject recombinant adenovirus genome. The subject adenoviral genomes find use in a variety of application, including as vectors for use in a variety of applications, including gene therapy.

Abstract: Methods and compositions for introducing a nucleic acid into the genome of at least one cell of a multicellular organism are provided. In the subject methods, a Sleeping Beauty transposon that includes the nucleic acid is administered to the multicellular organism along with a source of a Sleeping Beauty transposase activity. Administration of the transposon and transposase results in integration of the transposon, as well as the nucleic acid present therein, into the genome of at least one cell of the multicellular organism The subject methods find use in a variety of different applications, including the in vivo transfer of genes for use in, among other applications, gene therapy applications.

Abstract: Methods are provided for the in vivo introduction of an expression cassette into a target cell of a vascularized organism, e.g., a mammal, in manner such that the encoded protein of the introduced expression cassette is persistently expressed at a high level in the target cell. In the subject methods, an aqueous formulation of a minimal plasmid vector that includes the expression cassette is administered into the vascular system of the organism. The minimal plasmid vector employed in the subject methods is one that provides for persistent and high level expression of an expression cassette that is present on the vector in a manner that is substantially expression cassette sequence and direction independent Also provided are the minimal plasmid vectors employed in the subject methods.

Abstract: Methods for introducing recombinant adeno-associated virus (rAAV) virions into the liver of a mammal are provided. In these methods, the liver is partially or completely isolated from its blood supply, a catheter is introduced into the liver via a peripheral blood vessel, and rAAV virions are then infused through the catheter to the liver. The methods described herein may be used, for example, to deliver heterologous genes encoding therapeutic proteins to the hepatocytes of humans. This can be accomplished, for example, by introducing the catheter into a femoral artery, threading the catheter into the hepatic artery, and infusing rAAV virions through the catheter and into the liver. Exemplary examples of heterologous genes include those coding for blood coagulation factors.

Abstract: Non-human mammalian hosts are provided, comprising functional human hepatocytes. Isolated human hepatocytes or fragments of human hepatic tissue are introduced into the xenogeneic host in conjunction with one or more agent that stimulates human hepatocyte growth factor receptor. The human hepatocytes are maintained in the host by administration of one or more agent that stimulates human hepatocyte growth factor receptor, either continuously (e.g., via an implanted catheter or intravenous apparatus) or in discrete, regular dosages of the agent (e.g., via intravenous injections or oral dosages). The human hepatocytes are able to survive and function in the host animal for a period of at least 5 months.

Abstract: Methods and compositions for introducing a nucleic acid into the genome of at least one cell of a multicellular organism are provided. In the subject methods, a Sleeping Beauty transposon that includes the nucleic acid is administered to the multicellular organism along with a source of a Sleeping Beauty transposase activity. Administration of the transposon and transposase results in integration of the transposon, as well as the nucleic acid present therein, into the genome of at least one cell of the multicellular organism The subject methods find use in a variety of different applications, including the in vivo transfer of genes for use in, among other applications, gene therapy applications.

Abstract: Methods and compositions are provided for modulating, e.g., reducing, coding sequence expression in mammals. In the subject methods, an effective amount of an RNAi agent, e.g., an interfering ribonucleic acid (such as an siRNA or shRNA) or a transcription template thereof, e.g., a DNA encoding an shRNA, is administered to a non-embryonic mammal, e.g., via a hydrodynamic administration protocol. Also provided are RNAi agent pharmaceutical preparations for use in the subject methods. The subject methods and compositions find use in a variety of different applications, including academic and therapeutic applications.

Abstract: Methods and compositions are provided for modulating, e.g., reducing, viral coding sequence expression in mammals and mammalian cells. In the subject methods, an effective amount of an RNAi agent, e.g., an interfering ribonucleic acid (such as an siRNA or shRNA) or a transcription template thereof, e.g., a DNA encoding an shRNA, is introduced into a target cell, e.g., by being administered to a mammal that includes the target cell, e.g., via a hydrodynamic administration protocol. Also provided are RNAi agent pharmaceutical preparations for use in the subject methods. The subject methods and compositions find use in a variety of different applications, including academic and therapeutic applications.

Abstract: A helper dependent adenoviral vector system is provided. The subject helper dependent adenoviral vector system is made up of: (1) a “gutless” adenoviral vector that include cis-acting human stuffer DNA that provides for in vivo long term, high level expression of a coding sequence present on the vector; (2) an adenoviral helper vector that is characterized by having an adenoviral genome region flanked by recombinase recognition sites, where the helper vectors further include a non-mammalian endonuclease recognition site positioned outside of the adenoviral genome region; and (3) a mammalian cell that expresses the corresponding recombinase and endonuclease, as well as the adenoviral preterminal and polymerase proteins. Also provided are methods of using the subject systems to produce virions having the subject helper dependent adenoviral vectors encapsulated in an adenoviral capsid. In addition, kits for use in practicing the subject methods are provided.

Abstract: The instant invention provides methods of expressing polynucleotides in the cells of the liver comprising administering viral particles comprising a recombinant AAV vector into a mammal, preferably a human.

Abstract: Methods and compositions for introducing a nucleic acid into the genome of at least one cell of a multicellular organism are provided. In the subject methods, a Sleeping Beauty transposon that includes the nucleic acid is administered to the multicellular organism along with a source of a Sleeping Beauty transposase activity. Administration of the transposon and transposase results in integration of the transposon, as well as the nucleic acid present therein, into the genome of at least one cell of the multicellular organism The subject methods find use in a variety of different applications, including the in vivo transfer of genes for use in, among other applications, gene therapy applications.

Abstract: In one aspect, the present invention provides nucleic acid expression cassettes that are predominantly expressed in the mammalian liver. The present invention also provides vectors comprising a nucleic acid expression cassette that is predominantly expressed in the mammalian liver. The present invention also provides methods of ameliorating the symptoms of a disease, the methods including the steps of introducing into the liver of a mammalian subject a vector comprising a nucleic acid expression cassette that encodes a polypeptide, and expressing a therapeutic amount of the polypeptide in the liver.

Abstract: Methods and compositions are provided for introducing an expression cassette into a cell. In the subject methods, a population of at least two distinct adeno-associated viral particles is provided, where each distinct type of viral particle in the population comprises a different portion of the expression cassette to be introduced into the cell. The target cell is contacted with population of adeno-associated viral vectors under conditions sufficient to produce a hetero-concatemer in the cell, where the hetero-concatemer includes a functional expression cassette having an intron that includes an ITR sequence. Also provided by the subject invention are vector preparations for practicing the subject methods as well as kits for use in producing the vectors employed in the subject methods. The subject methods find use in a variety of different gene transfer applications, including both in vivo and in vitro gene transfer applications, and are particularly suited for use in the transfer of long genes.

Abstract: In vitro methods for making a recombinant adenoviral genome, as well as kits for practicing the same and the recombinant adenovirus vectors produced thereby, are provided. In the subject methods, the subject genomes are prepared from first and second vectors. The first vector includes an adenoviral genome having an E region deletion and three different, non-adenoviral restriction endonuclease sites located in the E region. The second vector is a shuttle vector and includes an insertion nucleic acid flanked by two of the three different non-adenoviral restriction endonucleases sites present in the first vector. Cleavage products are prepared from the first and second vectors using the appropriate restriction endonucleases. The resultant cleavage products are then ligated to produce the subject recombinant adenovirus genome. The subject adenoviral genomes find use in a variety of application, including as vectors for use in a variety of applications, including gene therapy.