University of Utah Research Foundation

Genta Incorporated

Salus Therapeutics, Inc.

Zhidong Chen

Duane E Ruffner

Michael Pierce

Ramesh Prakash

Richard Koehn

Inhibitory oligonucleotides are disclosed which are targeted to three specific target regions and subsequences of the target regions found on nucleic acids encoding Bcl-2. These inhibitory oligonucleotides are generally of from about 8 to about 50 nucleotides in length. Specific preferred oligonucleotides are disclosed. The oligonucleotides of the invention may be incorporated into compositions such as pharmaceutical compositions, and may be used in methods for inhibiting the expression of Bcl-2 in a cell or tissue, methods for treating conditions susceptible to modulation of Bcl-2 expression in an organism, and methods for detecting nucleic acid encoding BCL-2.

Inhibitory oligonucleotides targeted to Bcl-2

A method for generating and amplifying closed circular DNA having a specific sequence in vitro in a cell-free system is disclosed. Prior to the invention of this method, closed circular DNA could only be amplified in vivo in appropriate host cells. The essence of the method is the inclusion of a thermostable DNA ligase in a PCR reaction. This procedure is referred to as ligation-during-amplification (LDA), in which the fully extended DNA strands are ligated by the DNA ligase and used as templates for subsequent amplification. Closed circular DNA having a specific sequence can be selectively amplified exponentially by the use of two sequence-specific primers in the LDA reaction. In addition, one or more site-specific mutations can be introduced into a closed circular DNA by the use of one or more mutagenic primers in the LDA reaction. Various thermostable DNA polymerases and thermostable ligases can be used for LDA amplification. Any primer position and orientation, either inward or outward, can be used in LDA amplification, as long as there is at least one primer complementary to each strand of the circular DNA. This method has applications in the areas of mutagenesis, cloning, DNA detection, DNA modification, gene hunting, gene therapy, and cell-free DNA production.

Method for in vitro amplification of circular DNA

A method for making a directed antisense library against a target transcript is described. A cDNA of the target transcript is cloned in an appropriate cloning vector. Next, a plurality of deletion derivatives of the cloned cDNA is prepared such that the deletions serially extend into the cDNA from one end thereof. The resulting deletion library is then treated such that cDNA is removed from the other end of each cDNA insert, thus obtaining a fragment library having fragments of a selected size. A catalytic core is then inserted into each fragment of the fragment library, resulting in the directed antisense library. An illustrative antisense gene in the hammerhead ribozyme catalytic core. Plasmids for making the antisense library, plasmids and methods for making the fragment library, and a method for identifying target sites for antisense-mediated gene inhibition are also described.

Directed antisense libraries

A method for the automated molecular cloning and amplification of closed circular clones in vitro in a cell-free system using a pair of particularly designed cloning primers in a ligation-during-amplification (LDA) process, in which the fully extended DNA strands containing the donor sequence are ligated by DNA ligase and then used as templates for subsequent amplification. By designing particular cloning primers, the present invention can be used to obtain amplified closed circular nucleic acid clones with inverted or non-inverted insertions or substitutions with a donor nucleic acid fragment, or deletions of a fragment of the closed circular nucleic acid.

Growing community of inventors

Salt Lake City, UT, United States of America

Zhidong Chen