Hepatocellular chimeraplasty

Details Hepatocellular chimeraplasty Hepatocellular chimeraplasty

1998-06-30

2003-02-25

Guzo, David (Department: 1636)

Drug, bio-affecting and body treating compositions

Preparations characterized by special physical form

Liposomes

C514S04400A, C536S023100, C536S024100, C536S024300

Reexamination Certificate

active

06524613

ABSTRACT:

1. FIELD OF THE INVENTION
The invention concerns methods and compositions for the use of recombinagenic oligonucleobases in vivo for the correction of disease causing genetic defects and the prevention of disease by introducing genetic modifications into the genes that encode Apolipoprotein B (Apo B) and Apolipoprotein E (Apo E)
2. BACKGROUND TO THE INVENTION
2.1 The Use of Chimeric Mutational Vectors to Effect Genetic Changes in Cultured Cells
The inclusion of a publication or patent application in this specification is not an admission that the publication or the invention, if any, of the application occurred prior to the present invention or resulted from the conception of a person other than the present inventors.
The published examples of recombinagenic oligonucleobases are termed Chimeric Mutational Vectors (CMV) or chimeraplasts because they contain both 2′-O-modified ribonucleotides and deoxyribonucleotides.
An oligonucleotide having complementary deoxyribonucleotides and ribonucleotides and containing a sequence homologous to a fragment of the bacteriophage M13mp19, was described in Kmiec, E. B., et al., November 1994, Mol. and Cell. Biol. 14, 7163-7172. The oligonucleotide had a single contiguous segment of ribonucleotides. Kmiec et al. showed that the oligonucleotide was a substrate for the REC2 homologous pairing enzyme from
Ustilago maydis.
Patent publication WO 95/15972, published Jun. 15, 1995, and counterpart U.S. Pat. No. 5,565,350 (the '350 patent) described duplex CMV for the introduction of genetic changes in eukaryotic cells. Examples in a
Ustilago maydis
gene and in the murine ras gene were reported. The latter example was designed to introduce a transforming mutation into the ras gene so that the successful mutation of the ras gene in NIH 3T3 cells would cause the growth in soft agar of a colony of cells (“transformation”). The '350 patent reported that the maximum rate of transformation of NIH 3T3 was less than 0.1%, i.e., about 100 transformants per 10
6
cells exposed to the ras duplex CMV. In the
Ustilago maydis
system the rate of transformants was about 600 per 10
6
. A chimeric vector designed to introduce a mutation into a human bcl-2 gene was described in Kmiec, E. B., February 1996, Seminars in Oncology 23, 188.
A duplex CMV designed to repair the mutation in codon 12 of K-ras was described in Kmiec, E. B., December 1995, Advanced Drug Delivery Reviews 17, 333-40. The duplex CMV was tested in Capan 2, a cell line derived from a human pancreatic adenocarcinoma, using LIPOFECTIN™ to introduce the duplex CMV into the Capan 2 cells. Twenty four hours after the duplex CMV was introduced, the cells were harvested and genomic DNA was extracted; a fragment containing codon 12 of K-ras was amplified by PCR and the rate of conversion estimated by hybridization with allele specific probes. The rate of repair was reported to be approximately 18%.
A duplex CMV designed to repair a mutation in the gene encoding liver/bone/kidney type alkaline phosphatase was reported in Yoon, K., et al., March 1996, Proc. Natl. Acad. Sci. 93, 2071. The alkaline phosphatase gene was transiently introduced into CHO cells by a plasmid. Six hours later the duplex CMV was introduced. The plasmid was recovered at 24 hours after introduction of the duplex CMV and analyzed. The results showed that approximately 30 to 38% of the alkaline phosphatase genes were repaired by the duplex CMV.
WO 97/41411 and counterpart U.S. Pat. No. 5,760,012 to E. B. Kmiec, A. Cole-Strauss and K. Yoon, and the publication Cole-Strauss, A., et al., September 1996, SCIENCE 273, 1386 disclose duplex CMV that are used in the treatment of genetic diseases of hematopoietic cells, e.g., Sickle Cell Disease, Thalassemia and Gaucher Disease. U.S. Pat. No. 5,731,181 to E. B. Kmiec describes duplex CMV having non-natural nucleotides for use in specific, site-directed mutagenesis. The duplex CMV described in the applications and certain of the publications of Kmiec and his colleagues contain a central segment of DNA:DNA homoduplex and flanking segments of RNA:DNA hybrid-duplex or 2′-OMe-RNA:DNA hybrid-duplex.
The work of Kmiec and his colleagues concerned cells that are mitotically active, i.e., proliferating cells, at the time they are exposed to CMV. Kmiec and colleagues used a CMV/liposomal macromolecular carrier complex in which the CMV were mixed with a pre-formed liposome or lipid vesicle. In such a complex the CMV are believed to adhere to the surface of the liposome.
Kren et al., June 1997, Hepatology 25, 1462-1468, reported the successful use of a CMV in non-replicating, primary tissue-cultured rat hepatocytes to mutate the coagulation factor IX gene. Kren et al., March 1998, Nature Medicine 4, 285 reported the use of a CMV in vivo to introduce a genetic defect in the same gene.
2.2 The Use of a Polyethylenimine Macromolecular Carrier for In Vivo and In Vitro Transfection
Branched chain polyethylenimine has been used as a carrier to introduce nucleic acids into eukaryotic cells both in vivo and in vitro. Boussif, O., et al., 1995, Proc. Natl. Acad. Sci. 92, 7297; Abdallah, B. et al., 1996, Human Gene Therapy 7, 1947. Boletta, A., et al., 1997, 8, 1243-1251. The in vitro use of galactosylated polyethylenimine to introduce DNA into cultured HepG2 hepatocarcinoma cell lines is reported by Zanta, et al., Oct. 1, 1997, Bioconjugate Chemistry 8, 839-844. The coupling of a protein ligand, transferrin, to polyethylenimine and its use to introduce a test gene into cultured cells by use of the transferrin receptor is described in Kircheis, R., et al., 1997, Gene Therapy 4, 409-4-18. Branched chain polyethylenimines contain secondary and tertiary amino groups having a broad range of pK's and, consequently these polyethylenimines have a substantial buffering capacity at a pH where polylysine has little or no capacity, i.e., less than about 8. Tang, M. K., & Szoka, F. C., 1997, Gene Therapy 4, 823-832. The use of branched chain polyalkanylimines, including polyethylenimine as carriers for the introduction of nucleic acids into cells is described in WO 96/02655 to J-P. Behr et al.
The successful in vivo and in vitro use of linear polyethylenimine to transfect a gene is reported by Ferrari, S., et al., 1997, Gene Therapy 4, 1100-1106. Compositions comprising a linear polyalkanylimine and a nucleic acid as disclosed in patent publication WO 93/20090 to S. Stein et al.
2.3 The Use of a Liposomal Carrier for In Vivo Transfection
The use of liposomes or lipid vesicles to introduce DNA encoding a foreign protein into cells has been described. The most frequently used techniques adhere the DNA to the surface of a positively charged liposome, rather than encapsulating the DNA, although encapsulated DNA techniques were known. U.S. Pat. Nos. 4,235,871 and 4,394,448 are relevant. The field is reviewed by Smith, J. G., et al., 1993, Biochim. Biophy. Acta 1154, 327-340 and Staubinger, R. M., et al., 1987, Methods in Enzymology 185, 512. The use of DOTAP, a cationic lipid in a liposome to transfect hepatic cells in vivo is described in Fabrega, A. J., et al., 1996, Transplantation 62, 1866-1871. The use of cationic lipid-containing liposomes to transfect a variety of cells of adult mice is described in Zhu, N., et al., 1993, Science 261, 209. The use of phosphatidylserine containing lipids to form DNA encapsulating liposomes for transfection is described in Fraley, R., et al., 1981, Biochemistry 20, 6978-87.
2.4 The Use of the Asialoglycoprotein Receptor for Hepatoceelular Specific Transfection
U.S. Pat. Nos. 5,166,320 and 5,635,383 disclose the transfection of hepatocytes by forming a complex of a DNA, a polycationic macromolecular carrier and a ligand for the asialoglycoprotein receptor. In one embodiment, the macromolecular carrier was polylysine. The use of a lactosylcerebroside containing liposome to transfect a hepatocyte in vivo is described by Nandi, P. K., et al., 1986, J. Biol. Chem. 261, 16722-16722. The use of asialofetuin-labeled liposomes to transfect liver cells with a reporter plasmid

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