University of Pittsburgh

University of Michigan

Other

The United States Government As Represented by the Department of Veterans Affairs

Joseph C Glorioso

Paul David Robbins

Christopher H Evans

David Krisky

Darren Wolfe

David J Fink

Andrea Gambotto

William F Goins

Zhibao Mi

Raymond Frizzell

Richard Kane

Geethani Bandara

Steven C Ghivizzani

Richard Kang

The invention provides a cell line complementing HSV ICP4, ICP27, and UL55 genes.

Cell line

The invention provides an expression cassette comprising a DNA sequence encoding amino acids 1-99 of human preproenkephalin, a DNA sequence encoding a precursor of a carboxy-amidated peptide flanked by dibasic cleavage sites and optionally a DNA sequence encoding a marker protein (such as Enhanced Green Fluorescent Protein (GFP)) all in operable linkage and under control of a promoter. Where the encoded precursor of a carboxy-amidated peptide is an agonist for an opioid receptor, the invention further provides a method of treating neuropathic pain by administering the gene transfer vector comprising such an expression cassette to a patient. The invention also provides a method for detecting a peptide having a desired effect comprising introducing a library of DNA sequences encoding one or more precursors of carboxy-amidated peptides into host cells; expressing the carboxy-amidated peptides encoded in the library to provide expression products; and screening from the polypeptide expression products for the desired effect.

Method of amidated peptide biosynthesis and delivery in vivo: endomorphin-2 for pain therapy

The invention provides a herpes simplex virus vector comprising deletions in ICP4, ICP27, and UL55.

Herpes simplex virus vector

Methods for introducing at least one gene encoding a product into at least one target cell of a mammalian host for use in treating the mammalian host are disclosed. These methods include employing recombinant techniques to produce a vector molecule that contains the gene encoding for the product, and infecting the target cells of the mammalian host using the DNA vector molecule. A method to produce an animal model for the study of connective tissue pathology is also disclosed.

Gene transfer for studying and treating a connective tissue of a mammalian host

The present invention relates to internalizing peptides which facilitate the uptake and transport of cargo into the cytoplasm and nuclei of cells as well as methods for the identification of the peptides, and methods of use for the peptides. The internalizing peptides of the present invention are selected for their ability to efficiently internalize cargo into a wide variety of cell types both in vivo and in vitro.

Identication of peptides that facilitate uptake and cytoplasmic and/or nuclear transport of proteins, DNA and virues

The subject invention concerns a method of introducing at least one DNA sequence expressing a protein or protein fragment which substantially alleviates articular cartilage defects. This method involves in vitro culture of chondrocytes, transfection of the chondrocytes with a recombinant vector housing the DNA sequence to be expressed, and delivery of the transfected chondrocytes to the damaged cartilage region. This method can also be used in tandem with synovial cell delivery techniques of the present invention. This method is also useful as a model in animal studies regarding joint pathologies.

Cartilage alterations by administering to joints chondrocytes comprising a heterologous polynucleotide

The subject invention concerns a method of introducing at least one gene encoding a product into at least one cell of a connective tissue of a mammalian host for use in treating the mammalian host including employing recombinant techniques to produce a DNA vector molecule which contains the gene encoding for the product and infecting the connective cell of the mammalian host using the DNA vector molecule using the gene coding for the product. A method is provided for introducing at least one gene encoding a product into at least one cell of a connective tissue of a mammalian host employing non-viral methods. A method to produce an animal model for the study of connective tissue pathology is also disclosed. Additionally, this invention provides a method of using in vivo a gene encoding and extracellular interleukin-1 binding domain of an interleukin-1 receptor.

Viral vectors to inhibit leukocyte infiltration or cartilage degradation of joints

Methods for treating a connective tissue disorder by introducing at least one gene encoding a product into at least one target cell of a mammalian host for use in treating the mammalian host are disclosed. These methods include employing recombinant techniques to produce a vector molecule containing the DNA sequence encoding for the product and infecting the target cell of the mammalian host using the vector. The injection can be done in vivo, by directly injecting the vector into the host, or can be done in vitro by transfecting a population of cultured target cells with the vector and transplanting them each into the host. Nonviral means can also be used to introduce the DNA sequence to the host. Administration of more than one gene of interest results in an enhanced therapeutic benefit. Also disclosed is a method for treating a connective tissue disorder by introducing at least one gene encoding a product into at least one target cell of a joint of a host for use in treating multiple joints of the host. Injection of a vector molecule containing the DNA sequence encoding for a product of interest, or non-viral introduction of such a DNA sequence, to one join of a mammalian host results in a therapeutic benefit in that joint as well as other joints in the host

Viral vectors to inhibit leukocyte infiltration or cartilage degradation

Gene transfer for studying and treating a connective tissue of a

The present invention provides a method for preparing HSV vectors. The method comprises co-transfecting a source vector and a mutating cassette together into a population of appropriate host cells, such that homologous recombination occurs between the mutating cassette and the source vector whereby the mutating cassette replaces a region of the HSV genome. The mutating cassette has a unique restriction site not present in the sequence of the vector. The method further comprises plaquing the co-transfected host cells, selecting plaques in which recombination has occurred between the source vector and the mutating cassette, and isolating the viral DNA from the plaques. The isolated viral DNA is digested with a restriction endonuclease appropriate for cleaving the viral DNA at the unique restriction site within the mutating cassette to produce two viral polynucleotides. Following purification, the two viral polynucleotides can be ligated to form an HSV vector comprising the two viral polynucleotides. Alternatively, the two isolated viral polynucleotides can be recombined with an insertion cassette to form an HSV vector comprising the insertion cassette at the former locus of the unique restriction site. The present invention further provides a mutant vector, particularly an HSV vector constructed in accordance with the method for the present invention. The present invention further provides a multigene HSV vector, particularly a multigene HSV vector for cancer therapy.

Multigene vectors

The subject invention concerns a method of introducing at least one gene encoding a product into at least one cell of a connective tissue of a mammalian host for use in treating the mammalian host including employing recombinant techniques to produce a DNA vector molecule which contains the gene encoding for the product and infecting the connective cell of the mammalian host using the DNA vector molecule using the gene coding for the product. A method is provided for introducing at least one gene encoding a product into at least one cell of a connective tissue of a mammalian host employing non-viral means. In a specific example, isolated synovial cells were infected with a retrovirus comprising a DNA sequence encoding IRAP and the transfected cells administered to an arthritic joint. A method to produce an animal model for the study of connective tissue pathology is also disclosed. Additionally, this invention provides a method of using in vivo a gene encoding and extracellular interleukin-1 binding domain of an interleukin-1 receptor.

IRAP gene as treatment for arthritis

A gene transfer vector for transferring genetic material to a host tissue and expressing a gene product therefrom includes a virus capable of establishing extra chromosomal viral latency in the host tissue. The strain includes at least one foreign gene recombined into the virus which is foreign to the viral genome and a promoter for initiating and driving transcription of the foreign gene during latency of the virus in the host tissue.

HSV-1 vector containing a lat promoter

The application discloses the structure of a herpes virus promoter that by means of which one can obtain the transcription of a non-herpes gene in a cell latently infected with a herpes virus. An HSV-1 vector comprising a LAP2 promoter encoded by SEQ ID NO:1, or fragments of SEQ ID NO:1 which have LAP 2 promoter activity regulating the expression of a heterologous gene is particularly disclosed. The herpes virus is preferably used to obtain such expression in the neurons of the peripheral nerves, and the ganglion cells of the cranial nerves. The invention teaches that specific diseases and pathological conditions and the corresponding non-herpes genes wherein such expression has practical value.

Growing community of inventors

Cheswick, PA, United States of America

Joseph C Glorioso