Abstract: A composition for treatment of HSV-related pathologies including an expression vector for altering expression of a target sequence in an HSV-infected cell by production of single-stranded cDNA (ssDNA) in the cell in vivo suspended for topical application to an affected site in a suitable delivery vehicle. The expression vector is comprised of a cassette comprising a sequence of interest, an inverted tandem repeat, and a primer binding site 3? to the inverted tandem repeat, and a reverse transcriptase/RNAse H coding gene, and is transfected into the infected cells for inhibition of HSV replication. The resulting ssDNA binds to the target sequence to alter expression of the target sequence for such purposes as gene activation or inactivation using duplex or triplex binding of nucleic acids, site-directed mutagenesis, interruption of cellular function by binding to specific cellular proteins, or interfering with RNA splicing functions.

Abstract: The invention provides an improved cell free amplification method capable of producing large quantities of therapeutic-quality nucleic acids and methods of using the synthesized nucleic acid in research, therapeutic and other applications—The methods combine several different state-of-the-art procedures and coordinate their applications to affordably synthesize nucleic acids for therapeutic purposes. It combines in vitro rolling circle amplification, high fidelity polymerases, high affinity primers, and streamlined template specifically designed for particular applications. For expression purposes, the templates contain an expression cassette including a eukaryotic promoter, the coding sequence for the gene of interest, and a eukaryotic termination sequence.

Abstract: A composition for treatment of HSV-related pathologies including an expression vector for altering expression of a target sequence in an HSV-infected cell by production of single-stranded cDNA (ssDNA) in the cell in vivo suspended for topical application to an affected site in a suitable delivery vehicle. The expression vector is comprised of a cassette comprising a sequence of interest, an inverted tandem repeat, and a primer binding site 3? to the inverted tandem repeat, and a reverse transcriptase/RNAse H coding gene, and is transfected into the infected cells for inhibition of HSV replication. The resulting ssDNA binds to the target sequence to alter expression of the target sequence for such purposes as gene activation or inactivation using duplex or triplex binding of nucleic acids, site-directed mutagenesis, interruption of cellular function by binding to specific cellular proteins, or interfering with RNA splicing functions.

Abstract: The current invention is directed to oligonucleotide sequences isolated from a sequence designated rbl-1 [SEQ ID NO. 19] that either kill or inhibit growth, or prevent the production of endogenously expressed toxin, of microorganisms. These ssDNA sequences, identified through use of a screening method, appear to act as modulators of essential growth functions which may act at the level of triplex formation, antisense inhibition, or as aptamers that alter gene function. The sequences, referred to as minimum functional regions, or MFRs, are useful inter alia as therapeutic agents for treatment of sepsis and other pathologies caused by microorganisms such as sepsis and/or in which microorganisms are contributory agents.

Abstract: Methods and compositions comprising a DNA expression cassette for producing ss-cDNA inside a host cell (in vivo). The expression system optionally contains a reverse transcriptase/RNAse H coding gene, and a restriction endonuclease gene. The cassette carries cloning sites in two distinct locations for cloning and expressing a sequence of interest. The mRNA then serves as a template for reverse transcriptase and synthesis of the ss-cDNA. In one embodiment, the ss-cDNA folds and forms a dsDNA “stem-loop” structure which can be designed to contain restriction endonuclease recognition site the stem portion for cutting off the ssDNA loop containing the SOI having minimal flanking sequence attached. In another embodiment, the mRNA template folds prior to reverse transcription and serves as a termination signal for the RT. Again, a ss-cDNA is formed having only minimal flanking sequence. The data shows the usefulness of this system for producing ss-DNA inside a host/target cell.

Abstract: A selectively inducible, single-stranded DNA (ssDNA) expression library, a method for constructing a ssDNA expression library, a method for screening ssDNA using the expression library, and a method for identifying ssDNA molecules that alter expression of bacterial and fungal gene(s) related to cell growth and toxin production and secretion. The screening library is used to, among other things, identify ODNs effective in stopping cell growth, killing bacteria or fungi, or preventing bacteria and/or fungi from synthesizing and secreting their toxins, and/or to discover ODNs effective in eukaryotic (e.g., mammalian) cells for targeted alteration of gene function. The library is also useful for identifying ssDNAs or ODNs that are used as therapeutic agents for, for instance, providing a method for treatment of bacterial infections such as sepsis.

Abstract: A selectively inducible, single-stranded DNA (ssDNA) expression library, a method for constructing a ssDNA expression library, a method for screening ssDNA using the expression library, and a method for identifying ssDNA molecules that alter expression of bacterial gene(s) related to cell growth and toxin production and secretion. The screening library is used to, among other things, identify ODNs effective in stopping bacterial growth, killing bacteria or preventing bacteria from synthesizing and secreting their toxins, and/or to discover ODNs effective in eukaryotic (e.g., mammalian) cells for targeted alteration of gene function. The library is also useful for identifying ssDNAs or ODNs that are used as therapeutic antibacterial reagents, for identifying essential bacterial genes that can serve as targets for antibiotic discovery, and for providing a method for treatment of bacterial infections.

Abstract: A method for producing ODNs in bacterial or fungal cells in vivo for treatment of sepsis so that, when the ODNs reach and knock down their target genes, and thereby kill bacterial or fungal cells or inhibit their growth, the bacterial or fungal accumulation in the bloodstream is held constant or diminished and the sepsis syndrome is reduced or eliminated. The invention also contemplates of certain ODNs for use in treatment of sepsis.

Abstract: A selectively inducible, single-stranded DNA (ssDNA) expression library, a method for constructing a ssDNA expression library, a method for screening ssDNA using the expression library, and a method for identifying ssDNA molecules that switch on or off bacterial gene(s) related to cell growth and toxin production and secretion. The screening library is used to, among other things, identify ODNs effective in stopping bacterial growth, killing bacteria or preventing bacteria from synthesizing and secreting their toxins is the focus of the present invention and/or to discover ODNs effective in eukaryotic (e.g., mammalian) cells for targeted gene down regulation. The library is also useful for identifying ssDNAs or ODNs that are used as therapeutic antibacterial reagents, for identifying essential bacterial genes that can serve as targets for antibiotic discovery, and for providing a method for treatment of bacterial infections.

Abstract: A composition for treatment of HSV-related pathologies including an expression vector for altering expression of a target sequence in an HSV-infected cell by production of single-stranded cDNA (ssDNA) in the cell in vivo suspended for topical application to an affected site in a suitable delivery vehicle. The expression vector is comprised of a cassette comprising a sequence of interest, an inverted tandem repeat, and a primer binding site 3′ to the inverted tandem repeat, and a reverse transcriptase/RNAse H coding gene, and is transfected into the infected cells for inhibition of HSV replication. The resulting ssDNA binds to the target sequence to alter expression of the target sequence for such purposes as gene activation or inactivation using duplex or triplex binding of nucleic acids, site-directed mutagenesis, interruption of cellular function by binding to specific cellular proteins, or interfering with RNA splicing functions.

Abstract: An expression vector for altering expression of a target nucleic acid sequence in a host cell by production of single-stranded cDNA (ssDNA) in the host cell in vivo. The expression vector is comprised of a cassette comprising a sequence of interest, an inverted tandem repeat, and a primer binding site 3′ to the inverted tandem repeat, and a reverse transcriptase/RNAse H coding gene, and may be transfected into the host cell. Transcription of the cassette by the host cell produces an RNA template which is reverse transcribed with the product of the RT coding gene to produce ssDNA of a specified sequence. The ssDNA is modified to remove flanking vector sequences by taking advantage of the “stem-loop” structure of the ssDNA, which forms as a result of the inverted tandem repeat that allows the ssDNA to fold back on itself, forming a double stranded DNA stem.