University of Connecticut

Targetech, Inc. a Wholly Owned Subsidiary of the Immune Response

George Y Wu

Catherine H Wu

Nidhi Gupta

Ellen Carmichael

George L Spitalny

Ying Zhang

Juan Ruiz

The present disclosure relates to, inter alia, compositions and kits comprising an asialoglycoprotein covalently attached to a polycation, and functional mammalian mitochondria that are at least partially purified and are electrostatically bound to the AsG-polycation molecule; as well as methods of their preparation and use.

Targeted transplantation of mitochondria to hepatocytes

The present invention relates to the preparation of non-human animals having chimeric livers, whereby some or substantially all of the hepatocytes present are human hepatocytes. It is based, at least in part, on the discovery that rats, tolerized in utero against human hepatocytes, were found to serve as long-term hosts for human hepatocytes introduced post-natally, and the introduced hepatocytes maintained their differentiated phenotype, as evidenced by continued production of human albumin. The present invention further relates to the use of such animals as models of various liver diseases, including viral invention. Such embodiments are based on the discovery that transplanted human hepatocytes in chimeric livers were found to be susceptible to Hepatitis B virus and Hepatitis C virus infection.

Propagation of human hepatocytes in non-human animals

The present invention relates to the preparation of non-human animals having chimeric livers, whereby some or substantially all of the hepatocytes present are human hepatocytes. It is based, at least in part, on the discovery that rats, tolerized in utero against human hepatocytes, were found to serve as long-term hosts for human hepatocytes introduced post-natally, and the introduced hepatocytes maintained their differentiated phenotype, as evidenced by continued production of human albumin.

Propagation of human hepatocytes in non-human mammals

Molecular complexes for targeting oligonucleotides, such as antisense oligonucleotides or ribozymes, to a specific cell to block expression of a gene or genes in the cell are described. The single-stranded poly- or oligonucleotide is complexed to a conjugate of a cell-specific binding agent and a poly- or oligonucleotide-binding agent. The cell-specific binding agent is specific for a cellular surface structure which mediates internalization of the complex. An example is the asialoglycoprotein receptor of hepatocytes. The poly- or oligodeoxy-nucleotide-binding agent is a compound such as a polycationic protein which stably complexes the oligonucleotide under extracellular conditions and releases it under intracellular conditions so that it can hybridize with the target RNA. The molecular complex is stable and soluble in physiological fluids and can be used to selectively introduce antisense oligonucleotides, ribozymes or other single-stranded oligonucleotides into a cell to inhibit expression of a gene within the cell. The oligonucleotide can be directed against cellular genes (e.g., cellular oncogenes) or genes of noncellular origin (e.g., viral oncogenes, genes of an infecting pathogen).

Targeted delivery of poly- or oligonucleotides to cells

The invention pertains to a multimeric self-cleaving ribozyme comprising multiple repeating units, wherein each unit comprises a ribozyme containing a catalytic domain and an antisense domain, and a target recognition sequence or portion thereof recognized by the antisense domain of the ribozyme and comprising a ribozyme cleavage site to facilitate self-cleavage. The invention also pertains to constructs comprising DNA encoding the multimeric self-cleaving ribozyme, cells stably transformed with these constructs, and methods of cleaving RNA, regulating gene expression and generating molecular weight ladders utilizing the multimeric self-cleaving ribozyme.

Multimeric self-cleaving ribozyme

A targetable gene delivery system is provided for introducing foreign genes into mamallian cells. The system employs a soluble targetable DNA complex and utilizes receptor-mediated endocytosis to endow cell specificity. The soluble DNA-carrying complex is formed by non-covalently binding a ligand conjugate with the foreign gene. The conjugate, in turn, is formed by bonding receptor-specific ligands such as asialoglycoproteins to polycations such as polylysine through covalent bonds such as disulfide bonds. The system exhibits a high degree of cell specificity and offers potential for the treatment of inherited genetic disorders.

Soluble molecular complex for the introduction of genes into mammalian

An improved soluble molecular complex for targeting a polynucleotide to a specific cell is disclosed. The molecular complex comprises (a) a polynucleotide (b) a carrier made up of a polynucleotide binding agent and a cell-specific binding agent which binds to a surface molecule of the cell and is internalized into an endosome, and (c) a bacterial component or fragment thereof which lyses the endosome and causes the polynucleotide to be released into the cytoplasm of the cell. In a preferred embodiment of the invention the polynucleotide binding agent is polylysine, the cell-specific binding agent is an asialoglycoprotein, and the bacterial component is listeriolysin O. The disclosed soluble molecular complex and methods of use can be used therapeutically to deliver genes and antisense polynucleotides to specific cells in vivo.

Use of a bacterial component to enhance targeted delivery of

The expression of polynucleotide introduced into a cell by means of a targeted complex of the polynucleotide linked to a cell-specific binding agent can be enhanced and prolonged by inhibiting translocation or fusion of endosomes to lysosomes using colchicine.

Method of enhancing and/or prolonging expression of gene introduced into

A targetable gene delivery system is provided for introducing foreign genes into mammalian cells. The system employs a soluble targetable DNA complex and utilizes receptor-mediated endocytosis to endow cell specificity. The soluble DNA-carrying complex is formed by non-covalently binding a ligand conjugate with the foreign gene. The conjugate, in turn, is formed by bonding receptor-specific ligands such as asialoglycoproteins to polycations such as polylysine through covalent bonds such as disulfide bonds. The system exhibits a high degree of cell specificity and offers potential for the treatment of inherited genetic disorders.

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Avon, CT, United States of America

George Y Wu