Chemistry: molecular biology and microbiology – Process of mutation – cell fusion – or genetic modification – Introduction of a polynucleotide molecule into or...
Reexamination Certificate
2001-08-22
2003-09-23
Horlick, Kenneth R. (Department: 1637)
Chemistry: molecular biology and microbiology
Process of mutation, cell fusion, or genetic modification
Introduction of a polynucleotide molecule into or...
C514S04400A, C536S023100, C536S024500
Reexamination Certificate
active
06623966
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates generally to the transfection of eukaryotic cells, the regulation of gene expression, and gene therapy. In particular, the present invention relates to novel compositions and uses thereof including, but not limited to: the regulation of nucleic acid expression, the transfection of eukaryotic cells, gene therapy, the creation of transgenic animals, the biological production of pharmaceuticals, and the treatment of a variety of human diseases and disorders.
2. Description of Related Art
The State of the Technology: Gene Transfer
One of the most utilized and important techniques employed in the biological sciences today is the transfer of foreign nucleic acids into cells in vitro and in vivo. This technology is the foundation for gene therapy. A primary obstacle to the successful implementation of gene transfer technology is that cellular membranes provide a significant barrier to the translocation of nucleic acid. Currently, techniques exist for the transfer of nucleic acids across the cytoplasmic membrane of prokaryotic (transformation) and eukaryotic cells (transfection) including chemical, physical, and biological methods such as: calcium phosphate co-precipitation, DEAE dextran treatment, electroporation, microinjection, biolistic bombardment, viral infection, and liposomal based methods. The nuclear membrane of eukaryotic cells, however, proves to be a more formidable challenge. This membrane is a barrier to the passive movement of macromolecules larger than 15,000 kDa. Empirically, transfection protocols are limited to rapidly dividing cells. The hypothesis for these observations is that transfected nucleic acids have access to the nuclear compartment only when the nuclear membrane is dissolved during mitosis. Without a nuclear membrane, the transfected nucleic acids are thought to distribute throughout the volume of the cell, and a portion of these nucleic acids might remain in the nucleus after the nuclear membrane reforms.
Various strategies have been attempted to circumvent an intact nuclear membrane during transfection. Several viral vectors are under study as gene transfer agents, but all of them have major disadvantages. Viral vectors are a biohazard, it is usually necessary to employ procedures to limit viral replication by eliminating certain viral genes and using helper virus strains, the gene of interest must be cloned into the viral vector, and adeno-associated vectors (AAV) are difficult to produce in large quantities and have a limited capacity for accepting large transgenes. The transfection efficiency of most viral vectors is dependent upon proliferation of the host cell which, again, limits the utility of such vectors. Replication deficient adenoviral vectors have been used extensively in the lungs, but trigger an acute inflammatory response and a chronic immune response. Retroviral vectors, which insert randomly into the host genome, have the potential to disrupt normal genes and carry a risk of inducing malignancies. Thus, viral vectors show limited utility for in vivo therapy.
Several non-viral strategies to circumvent an intact nuclear membrane during transfection have been investigated, but none has proven to be efficient. One attempt is described in U.S. Pat. No. 5,827,705 to Dean. The Dean U.S. Pat. No. 5,827,705 patent utilizes a nucleic acid segment, referred to as a nuclear transport sequence (NTS), found to promote the transport of plasmid DNA into the nucleus of several cell types following microinjection of the plasmid into the cell. This approach has several major drawbacks including the need to clone the gene of interest into this specialized plasmid and the need for microinjection into individual cells as part of the transfection procedure.
Another attempt was described by Rossi et al. (1993). A plasmid DNA was covalently linked to a nuclear localization sequence (NLS) for nuclear import; however, this system damages the DNA making it non-viable for expression or replication.
Still another transfection method was described in U.S. Pat. No. 5,807,746 to Lin et al. In the Lin U.S. Pat. No. 5,807,746 patent, an importation competent signal peptide (ICSP) was demonstrated to promote the passage of a biologically active peptide through the cytoplasmic membrane. The ICSP was also made in conjunction with an NLS (ICSP-NLS). The ICSP-NLS promoted the passage of the biologically active peptide into the nuclear compartment. The Lin U.S. Pat. No. 5,807,746 patent suggested methods for linking nucleic acids to the ICSP-NLS using charge association or covalent linkage by thioester bonds. However, charge-association is too weak of an interaction for efficient transfection (Fritz et al. (1996) Human Gene Therapy 7:1395-1404) and covalent linkage eliminates the functionality of the nucleic acid (Rossi et al. (1993) Molecular & General Genetics 239:345-353).
A second major challenge to the successful implementation of gene transfer technologies involves the phenomenon of gene silencing or transient expression of transferred nucleic acids. This effect is characterized by the termination of transgene expression within three or four days of transfection. One explanation for this phenomenon is that the foreign nucleic acids are transported out of the nucleus by a cellular process (Alwine (1985) Mol Cell Biol 5:1034-1042). The common redress in vitro for transient expression is to employ selection methods to kill cells that do not express a co-transfected detoxifying agent. What is needed is a system that retains viable nucleic acids and plasmids in the nucleus which are capable of long-term expression without the need for toxic selective agents.
The State of the Technology: Peptide Nucleic Acid
Peptide nucleic acids (PNAs) are analogs of nucleic acids in which the ribose-phosphate backbone has been replaced with a backbone that is held together by amide bonds as described in U.S. Pat. No. 5,539,082 to Nielsen et al. PNAs have several interesting features, including the ability to hybridize to complementary DNA, RNA, or peptide nucleic acid (PNA) sequences. The hybridization of a PNA to a complementary sequence is generally greater than that of nucleic acid to nucleic acid hybrids (Good and Nielsen (1997) Antisense and Nuc Acid Drug Dev (7)431-7). Furthermore, PNAs are resistant to proteases and nucleases, and exhibit an ability to modulate transcription in a positive direction when hybridized to the non-coding strand of a promoter region and in a negative direction when hybridized to the coding strand of a promoter region (N. E. Mollegaard (1994) Proc. Natl. Acad. Sci. 91(9)3892-3895; Good and Nielsen, 1997). Specific PNA molecules have been used as antisense oligonucleotides (Good and Nielsen (1998) Nature Biotechnology (16)355-358), transcription enhancers and transcription repressors (N. E. Mollegaard, 1994; Good and Nielsen, 1997; Wang et al. (1999) Nuc Acids Res 27:2806-2813), and PCR™ clamping reagents (U.S. Pat. No. 5,656,461 to Demers).
Deficiencies Inherent in the State of the Technology
Current transfection methods are inefficient. Particularly lacking are methods for transfecting cells with an intact nuclear membrane. Methods for transfecting mitotic cells (in which the nuclear membrane has dissolved) are also limited because only a few percent of cells in a given population are mitotic and because transgene silencing eliminates expression within a few days without selective agents and the additional transfection of selection resistance genes. Improved transfection methods would benefit both basic scientific research and clinical medicine. The limited success of gene therapy thus far can be attributed to procedures that target actively dividing cell populations and likely achieve expression in less than one percent of targeted cells. Improved transfection methods that permit the efficient transfection of nucleated cells would immediately yield dramatic improvements in basic science, clinical medicine, and especially gene therapy. Benefits would include: the ability t
Schelling Douglas W.
Vanderbilt University
Waddey & Patterson , P.C.
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