Chemistry: molecular biology and microbiology – Process of mutation – cell fusion – or genetic modification – Introduction of a polynucleotide molecule into or...
Reexamination Certificate
2001-04-18
2002-06-25
McKelvey, Terry (Department: 1636)
Chemistry: molecular biology and microbiology
Process of mutation, cell fusion, or genetic modification
Introduction of a polynucleotide molecule into or...
C435S456000, C435S458000, C435S468000, C435S471000, C514S04400A
Reexamination Certificate
active
06410327
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to selective expression of genes useful in gene therapy protocols via photoactivation. In particular, the present invention provides a means of selectively expressing genes in specific cells, comprising delivering “caged” (inactivated) nucleic acid to cells and “uncaging” (activating) the nucleic acid by exposure of the targeted cells to light, thereby allowing temporally controlled expression of exogenous nucleic acid only in targeted cells or selectively regulating endogenous gene expression.
2. Background Art
The as yet unrealized goal of in vivo gene therapy is the expression of exogenous genetic material within only a target cell population. Successful in vivo gene therapy must overcome two challenges: 1) delivery of genes to the specific target cell population and 2) subsequent expression only within these cells. Viral and non-viral technologies for targeted delivery of genes have been evaluated. These include localized injection in skeletal muscle (Manthrope et al., 1993; reviewed in Brown et al., 1996), targeting of liposomes by incorporating antibodies to unique cell surface markers in the liposome outer surface (reviewed in Torchilin, 1996) and use of viruses with naturally selected sub-population targets, such as adenovirus for the bronchial epithelium (Rosenfeld et al., 1993). Although all of these strategies require that each target cell population be uniquely defined, the potential utility has kept interest high. Because of immune responses with adenovirus, safety issues with retroviruses and the poor targeting ability of liposomes, none of these strategies has proven suitable in its current form for targeted delivery and expression of genes.
In addition, targeted post-delivery expression strategies have been attempted (reviewed by Yarranton, 1992). These strategies involve delivery of nucleic acids comprising elements which can be broadly classified into 1) inducers triggered by changes in the cellular environment (cell milieu inducers) and 2) promoters which induce expression only within specific tissues. Cell milieu inducers can include promoters sensitive to metal concentration (Searle et al., 1985; Mayo et al., 1982), tetracycline (Furth et al., 1994; Gossen et al., 1995), hormones (Hynes et al., 1981; Andres et al., 1987) and the insect molting hormone ecdysone (No et al., 1996). However, the cell milieu inducers cannot be used to target sub-populations of cells, since all transfected cells respond to such changes in the cellular environment. Furthermore, unique tissue-specific promoters (reviewed by Hart, 1996; Stein et al., 1996) must be developed for each individual target cell population.
Photosensitive precursors or “caging” groups are molecules which bind an “effector” molecule through a covalent bond to the photosensitive precursor group, thereby reversibly rendering the effector molecule inert (McCray et al., 1989). The term “caged” is merely descriptive of the photo release property of these groups and does not refer to physical trapping of the inactivated substance within a crystal lattice. Caging groups have been used in a number of biological studies to study cell motility, muscle fibers, active transport proteins, biological membranes and other intracellular responses (e.g. Ishihara et al., 1997; Lee et al., 1997; Patton et al., 1991; see review by McCray et al., 1989). Caging groups have also been used in the caging of nucleotide analogues (Walker et al., 1988) and the synthesis of bio-chip arrays (McGall et al., 1996). Classically, caging groups have been used to study the time course of cellular responses induced by a step change in a local concentration of caged and subsequently, inactivated bio-chemical species, e.g. caged ATP. A rapid localized increase in concentration or activity of the caged substance is achieved by application of a directed pulse of light, which releases the bio-chemical inactivating group and returns the caged species to its biologically active state. In the case of caged ATP, this results in a localized high concentration of ATP. However, neither the caging of nucleic acids for selective regulation of gene expression nor the use of caged nucleic acids in therapeutic applications such as gene therapy have been described.
The present invention overcomes previous shortcomings in gene therapy technology by providing methods whereby caged genes or caged proteins can be delivered nonspecifically to cells and the genes or proteins in selected cells can be activated by exposure to light, thereby limiting the expression of genes or activity of exogenous proteins to selected cells. The methods of this invention can be employed to treat a variety of disease states and genetic disorders.
SUMMARY OF THE INVENTION
The present invention provides an isolated nucleic acid covalently linked to a photolabile caging group which reversibly prevents expression of the nucleic acid.
The present invention further provides a method of selectively expressing a nucleic acid in a cell, comprising: a) covalently linking the nucleic acid to a photolabile caging group which reversibly prevents expression of the nucleic acid; b) introducing the nucleic acid of step (a) into the cell; and c) exposing the cell of step (b) to light, whereby exposure to the light unlinks the nucleic acid and the caging group and the nucleic acid is selectively expressed in the cell.
The present invention additionally provides a method of selectively regulating the expression of an endogenous nucleic acid comprising: a) covalently linking a nucleic acid encoding an antisense nucleic acid to a photolabile caging group which reversibly prevents expression of the nucleic acid; b) introducing the nucleic acid of step (a) into the cell; and c) exposing the cell of step (b) to light, whereby exposure to the light unlinks the nucleic acid and the caging group and the nucleic acid is selectively expressed in the cell as an antisense nucleic acid which can bind to and inactivate a complementary nucleic acid within the cell.
Various other objectives and advantages of the present invention will become apparent from the following detailed description.
DETAILED DESCRIPTION OF THE INVENTION
As used herein, “a” or “an” can mean multiples.
The present invention provides a novel strategy for localized targeting of gene expression based on delivering reversibly inactivated genes to cells which can be selectively expressed in targeted cells upon activation of the genes by exposure to light. Transcription of the genes in cells is blocked by biochemical modification of the plasmid nucleic acid with a “caging” group. Activation of transcription is achieved by “uncaging” the plasmid by exposure to light.
Thus, the present invention provides an isolated nucleic acid covalently linked to a photolabile caging group which reversibly prevents expression of the nucleic acid. As used herein, “nucleic acid” refers to single- or double-stranded molecules which may be DNA, comprising two or more nucleotides comprised of the nucleotide bases A, T, C and G, or RNA, comprised of the bases A, U (substitute for T), C and G. The nucleic acid may represent a coding strand or its complement. Nucleic acids may be identical in sequence to a sequence which is naturally occurring or may include alternative codons which encode the same amino acid as that which is found in a naturally occurring sequence (Lewin, 1994). Furthermore, nucleic acids may include codons which represent conservative substitutions of amino acids as are well known in the art. With regard to gene therapy applications, the nucleic acid can comprise a nucleotide sequence which encodes a gene product which is meant to function in the place of a defective gene product and restore normal function to a cell which functioned abnormally due to the defective gene product. Alternatively, the nucleic acid may encode a gene product which was not previously present in a cell or was not previously present in the cell at a therapeutic concentration, whereby the presence of t
Alexander J. Steven
Haselton, III Frederick R.
McKelvey Terry
Needle & Rosenberg P.C.
Vanderbilt University Office of Technology Transfer
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