Patent Application
20100297219
The present invention related generally to the chimeric proteins, compositions and products containing one or more chimeric proteins, as well as the use thereof to modify apolipoprotein B processing, to treat or prevent atherogenic diseases or disorders, and to modify the intravascular lipoprotein population.
Cholesterol is carried in blood by specific carrier proteins called apolipoproteins and from one tissue to another as lipoprotein particles. Apolipoprotein B is an integral and non-exchangeable structural component of lipoprotein particles referred to as chylomicrons, very low density lipoprotein (“VLDL”), and low density lipoprotein (“LDL”). Apolipoprotein B circulates in human plasma as two isoforms, apolipoprotein B100 and apolipoprotein B48. Apolipoprotein B48 is generated by an RNA editing mechanism which changes codon 2153 (CAA) to a translation stop codon (UAA) (Chen et al., “Apolipoprotein B-48 is the product of a messenger RNA with an organ-specific in-frame stop codon,” Science 238:363-366 (1987); Powell et al., “A novel form of tissue-specific RNA processing produces apolipoprotein-B48 in intestine,” Cell 50:831-840 (1987)). Editing is a site-specific deamination event catalyzed by apolipoprotein B mRNA editing catalytic subunit 1 (known as APOBEC-1) (Teng et al., “Molecular cloning of an apo B messenger RNA editing protein,” Science 260:18116-1819 (1993)) with the help of auxiliary factors (Teng et al., “Molecular cloning of an apo B messenger RNA editing protein,” Science 260:18116-1819 (1993); Yang et al., “Partial characterization of the auxiliary factors involved in apo B mRNA editing through APOBEC-1 affinity chromatography,” J. Biol. Chem. 272:27700-27706 (1997); Yang et al., “Multiple protein domains determine the cell type-specific nuclear distribution of the catalytic subunit required for apo B mRNA editing,” Proc. Natl. Acad. Sci. USA 94:13075-13080 (1997); Lellek et al., “Purification and Molecular cloning of a novel essential component of the apo B mRNA editing enzyme complex,” J. Biol. Chem. 275:19848-19856 (2000); Mehta et al., “Molecular cloning of apobec-1 complementation factor, a novel RNA-binding protein involved in the editing of apolipoprotein B mRNA,” Mol. Cell. Biol. 20:1846-1854 (2000); Yang et al., “Induction of cytidine to uridine editing on cytoplasmic apolipoprotein B mRNA by overexpressing APOBEC-1,” J. Biol. Chem. 275:22663-22669 (2000); Blanc et al., “Identification of GRY-RBP as an apoB mRNA binding protein that interacts with both apobec-1 and with apobec-1 complementation factor (ACF) to modulate C to U editing,” J. Biol. Chem. 276:10272-10283 (2001)) as a holoenzyme or editosome (Smith et al. “In vitro apolipoprotein B mRNA editing: Identification of a 27S editing complex,” Proc. Natl. Acad. Sci. USA 88:1489-1493 (1991); Harris et al., “Extract-specific heterogeneity in high-order complexes containing apo B mRNA editing activity and RNA-binding proteins,” J. Biol. Chem. 268:7382-7392 (1993)). Apolipoprotein B100 and apolipoprotein B48 play different roles in lipid metabolism, most importantly, apolipoprotein B100-associated lipoproteins (VLDL and LDL) are much more atherogenic than apolipoprotein B48-associated lipoproteins (chylomicrons and their remnants and VLDL).
Specifically, the apolipoprotein B48-associated lipoproteins are cleared from serum more rapidly than the apolipoprotein B100-associated lipoproteins. As a result, apolipoprotein B48-VLDL usually are not present in serum for an amount of time sufficient for serum lipases to convert the VLDL to LDL. In contrast, the apolipoprotein B100-VLDL are present in the serum for sufficient amounts of time, allowing serum lipases to convert the VLDL to LDL. Elevated serum levels of LDL are of particular biomedical significance as they are associated with an increased risk of atherogenic diseases or disorders. Lipoprotein analyses have shown that the ability of mammalian liver to edit results in a lowering of the VLDL+LDL:HDL ratio. Therefore, it would be desirable to identify an approach for modifying apolipoprotein B editing which would favor an increase in the relative concentration of apolipoprotein B48 in proportion to apolipoprotein B100 (or total apolipoprotein concentration), thereby clearing a greater concentration of lipoproteins from serum and minimizing the atherogenic risks associated with high serum levels of VLDL and LDL.
Current lipid-lowering therapies include statins and bile-acid-binding resins. Statins are competitive inhibitors of hydroxymethylglutaryl-coenzyme A (HMG-CoA) reductase, which catalyzes the committed step in the synthesis of cholesterol (Davignon et al., “HMG-CoA reductase inhibitors: a look back and a look ahead,” Can. J. Cardiol. 8:843-64 (1992)). Bile-acid-binding resins sequester bile acids in the intestine, thereby interrupting the enterohepatic circulation of bile acids and increasing the elimination of cholesterol from the body. These are effective therapies for some patients with hyperlipidermia; however, adverse effects have been observed in up to 30% of the patients, suggesting the need for alternative therapies. Mutations in the gene encoding the LDL-receptor or apolipoprotein B can cause a human genetic disease known as familial hypercholesterolemia, characterized by an elevated level of cholesterol and early atherosclerosis due to the defect in LDL-receptor mediated cholesterol uptake by cells (Goldstein et al., Familial hypercholesterolemia,” In The Metabolic and Molecular Bases of Inherited Disease, Vol. 2., p1981-2030, Scriver et al. (eds.), McGraw-Hill, New York (1995)). Therapy for children with this disorder is needed in order to prevent morbidity or mortality, however the National Cholesterol Education Program (NCEP) recommends consideration of drug treatment only for children 10 years of age or older due to the risk that prolonged drug therapy may impair growth and pubertal development. Developing alternative approaches for lowering serum LDL levels is therefore essential for the sectors of the population still at risk.
Stimulating hepatic apolipoprotein B mRNA editing is a means of reducing serum LDL through the reduction in synthesis and secretion of apolipoprotein B100 containing VLDL. In most mammals (including humans), apolipoprotein B mRNA editing is carried out only in the small intestine. The presence of substantial editing in liver (found in 4 species) is associated with a less atherogenic lipoprotein profile compared with animals that do not have liver editing activity (Greeve et al., “Apolipoprotein B mRNA editing in 12 different mammalian species: hepatic expression is reflected in low concentrations of apoB-containing plasma lipoproteins,” J. Lipid Res. 34:1367-1383 (1993)). APOBEC-1 is expressed in all tissues that carry out apolipoprotein B mRNA editing (Teng et al., “Molecular cloning of an apo B messenger RNA editing protein,” Science 260:18116-1819 (1993)). Human liver does not express APOBEC-1 but it does express sufficient auxiliary proteins to complement exogenous APOBEC-1 in apolipoprotein B mRNA editing in transfected cells (Teng et al., “Molecular cloning of an apo B messenger RNA editing protein,” Science 260:18116-1819 (1993); Sowden et al., “Apolipoprotein B RNA Sequence 3′ of the mooring sequence and cellular sources of auxiliary factors determine the location and extent of promiscuous editing,” Nucleic Acids Res. 26:1644-1652 (1998)).
Transgenic experiments aiming to enhance hepatic editing through apobec-1 gene transfer have shown a marked lowering of plasma apolipoprotein B100 and significant reduction of serum LDL (Teng et al., “Adenovirus-mediated gene transfer of rat apolipoprotein B mRNA editing protein in mice virtually eliminates apolipoprotein B100 and normal low density lipoprotein production,” J. Biol. Chem. 269:29395-29404 (1994); Hughs et al., “Gene transfer of cytidine deaminase APOBEC-1 lowers lipoprotein(a) in transgenic mice and induces apolipoprotein B mRNA editing in rabbits,” Hum. Gene Ther. 7:39-49 (1996); Nakamuta et al., “Complete phenotypic characterization of the apobec-1 knockout mice with a wild-type genetic background and a human apolipoprotein B transgenic background, and restoration of apolipoprotein B mRNA editing by somatic gene transfer of Apobec-1,” J. Biol. Chem. 271:25981-25988 (1996); Kozarsky et al., “Hepatic expression of the catalytic subunit of the apolipoprotein B mRNA editing enzyme ameliorates hypercholesterolemia in LDL receptor-deficient rabbits,” Hum. Gene Ther. 7:943-957 (1996); Farese et al., “Phenotypic analysis of mice expressing exclusively apolipoprotein B48 or apolipoprotein B100,” Proc. Natl. Acad. Sci. USA 93:6393-6398 (1996); Qian et al., “Low expression of the apolipoprotein B mRNA editing transgene in mice reduces LDL but does not cause liver dysplasia or tumors,” Arteriosc. Thromb. Vasc. Biol. 18:1013-1020 (1998); Wu et al., “Normal perinatal rise in serum cholesterol is inhibited by hepatic delivery of adenoviral vector expressing apolipoprotein B mRNA editing enzyme in rabbits,” J. Surg. Res. 85:148-157 (1999)). Apolipoprotein B 100 is not essential for life as mice that synthesize exclusively apolipoprotein B48 (apolipoprotein B48-only mice) generated through targeted mutagenesis developed normally, were healthy and fertile. Compared with wild-type mice fed on a chow diet, the level of LDL-cholesterol was lower in apolipoprotein B48-only mice (Farese et al., “Phenotypic analysis of mice expressing exclusively apolipoprotein B48 or apolipoprotein B100,” Proc. Natl. Acad. Sci. USA 93:6393-6398 (1996)). However, the induction of apolipoprotein B mRNA editing activity through apobec-1 gene transfer and tissue-specific overexpression poses a significant challenge in that it has induced hepatocellular dysplasia and carcinoma in transgenic mice and rabbits (Yamanaka et al., “Apolipoprotein B mRNA editing protein induces hepatocellular carcinoma and dysplasia in transgenic animals.,” Proc. Natl. Acad. Sci. USA 92: 8483-8487 (1995); Yamanaka et al., “Hyperediting of multiple cytidines of apolipoprotein B mRNA by APOBEC-1 requires auxiliary protein(s) but not a mooring sequence motif;” J. Biol. Chem. 271:11506-11510 (1996); Yamanaka et al., “A novel translational repressor mRNA is edited extensively in livers containing tumors caused by the transgene expression of the apoB mRNA editing enzyme,” Genes & Dev. 11:321-333 (1997)). This was proposed to be due to persistent high levels of APOBEC-1 expression resulting in unregulated and nonspecific mRNA editing (Sowden et al., “Overexpression of APOBEC-1 results in mooring-sequence-dependent promiscuous RNA editing,” J. Biol. Chem. 271:3011-3017 (1996); Yamanaka et al., “A novel translational repressor mRNA is edited extensively in livers containing tumors caused by the transgene expression of the apoB mRNA editing enzyme,” Genes & Dev. 11:321-333 (1997); Sowden et al., “Apolipoprotein B RNA Sequence 3′ of the mooring sequence and cellular sources of auxiliary factors determine the location and extent of promiscuous editing,” Nucleic Acids Res.
26:1644-1652 (1998)). Adverse effects were not observed in transgenic animals with low to moderate levels of APOBEC-1 expression (Teng et al., “Adenovirus-mediated gene transfer of rat apolipoprotein B mRNA editing protein in mice virtually eliminates apolipoprotein B 100 and normal low density lipoprotein production,” J. Biol. Chem. 269:29395-29404 (1994); Qian et al., “Low expression of the apolipoprotein B mRNA editing transgene in mice reduces LDL but does not cause liver dysplasia or tumors,” Arteriosc. Thromb. Vasc. Biol. 18:1013-1020 (1998); Wu et al., “Normal perinatal rise in serum cholesterol is inhibited by hepatic delivery of adenoviral vector expressing apolipoprotein B mRNA editing enzyme in rabbits,” J. Surg. Res. 85:148-157 (1999)). Despite the limited success of apobec-1 gene therapy in modifying apolipoprotein B mRNA editing, such gene therapy poses too great a risk of adverse effects stemming from either persistent elevated levels of APOBEC-1 expression or problems associated with the use of infective transformation vectors (e.g., adenoviral vectors).
For these reasons, it would be desirable to identify an approach to achieve apolipoprotein B mRNA editing, where its induction can be maintained at low levels and importantly, achieved in a transient manner. Moreover, it would be desirable to identify an approach to achieve apolipoprotein B mRNA editing which is substantially free of the side-effects observed with reported gene therapy approaches. The present invention is directed to overcoming these and other deficiencies in the art.
A first aspect of the present invention relates to a chimeric protein including: a first polypeptide that includes a protein transduction domain and a second polypeptide that includes APOBEC-1 or a fragment thereof which can edit mRNA encoding apolipoprotein B.
A second aspect of the present invention relates to a chimeric protein including: a first polypeptide that includes a protein transduction domain; and a second polypeptide that includes APOBEC-1 Complementation Factor (“ACF”) or a fragment thereof which can bind to apolipoprotein B mRNA to facilitate editing of the mRNA by APOBEC-1.
Third and fourth aspects of the present invention relate to DNA molecules which encode one of the chimeric proteins of the present invention. DNA constructs, expression vectors, and recombinant host cells including such DNA molecules are also disclosed.
A fifth aspect of the present invention relates to a composition which includes: a pharmaceutically acceptable carrier and a chimeric protein of the present invention.
A sixth aspect of the present invention relates to a composition which includes: a first chimeric protein including a first polypeptide that includes a protein transduction domain and a second polypeptide that includes APOBEC-1 or a fragment thereof which can edit mRNA encoding apolipoprotein B; and a second chimeric protein including a first polypeptide that includes a protein transduction domain and a second polypeptide that includes ACF or a fragment thereof which can bind to apolipoprotein B mRNA to facilitate editing of the mRNA by APOBEC-1 or the fragment thereof.
A seventh aspect of the present invention relates to a delivery device which includes either a chimeric protein of the present invention or a composition of the present invention.
An eighth aspect of the present invention relates to a method of modifying apolipoprotein B mRNA editing in vivo which includes: contacting apolipoprotein B mRNA in a cell with a chimeric protein including a first polypeptide that includes a protein transduction domain and a second polypeptide that includes APOBEC-1 or a fragment thereof which can edit mRNA encoding apolipoprotein B, under conditions effective to increase the concentration of apolipoprotein B48 which is secreted by the cell as compared to the concentration of apolipoprotein B100 which is secreted by the cell, relative to an untreated cell.
A ninth aspect of the present invention relates to a method of reducing serum LDL levels which includes: delivering into one or more cells of a patient, without genetically modifying the cells, an amount of a protein comprising APOBEC-1 or a fragment thereof which can edit mRNA encoding apolipoprotein B, which amount is effective to increase the concentration of VLDL-apolipoprotein B48 that is secreted by the one or more cells into serum and, consequently, reduce the serum concentration of LDL.
A tenth aspect of the present invention relates to a method of treating or preventing an atherogenic disease or disorder which includes: administering to a patient an effective amount of a protein including APOBEC-1 or a fragment thereof which can edit mRNA encoding apolipoprotein B, wherein upon said administering the protein is taken up by one or more cells of the patient that can synthesize and secrete VLDL-apolipoprotein B under conditions which are effective to increase the concentration of VLDL-apolipoprotein B48 that is secreted by the one or more cells into serum, whereby rapid clearing of VLDL-apolipoprotein B48 from serum decreases the serum concentration of LDL to treat or prevent the atherogenic disease or disorder.
An eleventh aspect of the present invention relates to a liposome or niosome which is targeted for uptake by a liver cell, the liposome or niosome containing (i) APOBEC-1 or a fragment thereof which is effective to edit apolipoprotein B mRNA, (ii) ACF or a fragment thereof which is effective to bind apolipoprotein B mRNA, or (iii) a combination thereof. Compositions which include the liposome or niosome are also disclosed.
The present invention demonstrates the efficacy of protein-mediated delivery to increase intracellular APOBEC-1 in cells which produce and secrete VLDL-apolipoprotein B. By increasing the extent of apolipoprotein B mRNA editing in vivo, it is possible to modify the ratio of VLDL-apolipoprotein B48 to VLDL-apolipoprotein B100 which is secreted by such cells, specifically increasing the relative serum concentration of VLDL-apolipoprotein B48 and decreasing the relative serum concentration of VLDL-apolipoprotein B100. Due to the nature of these complexes, the B48 complex is cleared much more rapidly from serum, minimizing the conversion of VLDL into LDL, a major atherogenic disease factor. By minimizing the amount of VLDL-apolipoprotein B100 and increasing the amount of VLDL-apolipoprotein B48, it is possible to both treat and prevent atherogenic diseases or disorders. Moreover, by using protein delivery, it is possible to avoid the apparently unavoidable side effects of gene therapy. These results presented here open new possibilities for the treatment of hyperlipidemia through the induction of precisely controlled hepatic editing activity.
The present invention relates to protein-mediated approaches for regulating apolipoprotein B mRNA editing and, therefore, regulating the relative concentration of secreted apolipoprotein B derivatives, which offers an approach for controlling the serum levels of atherogenic disease factors such as low density lipoproteins (“LDL”) which associates with apolipoprotein B and its derivatives.
According to one aspect of the present invention, a first chimeric protein is provided for such uses. The first chimeric protein includes a first polypeptide that includes a protein transduction domain and a second polypeptide that includes APOBEC-1 or a fragment thereof which can edit mRNA encoding apolipoprotein B.
The first polypeptide can be any protein, or polypeptide fragment thereof, which is suitable for inducing cellular uptake of the chimeric protein.
By way of example, protein transduction domains from several known proteins can be employed, including without limitation, HIV-1 Tat protein, Drosophila homeotic transcription factor (ANTP), and HSV-1 VP22 transcription factor (Schwarze et al., “In vivo protein transduction: Intracellular delivery of biologically active proteins, compounds, and DNA,” TiPS 21:45-48 (2000), which is hereby incorporated by reference in its entirety).
A preferred protein transduction domain is the protein transduction domain of the human immunodeficiency virus (“HIV”) tat protein. An exemplary HIV tat protein transduction domain has an amino acid sequence of SEQ ID No: 9 as follows:
This protein transduction domain has also been noted to be a nuclear translocation domain (HIV Sequence Compendium 2000, Kuiken et al. (eds.), Theoretical Biology and Biophysics Group, Los Alamos National Laboratory, which is hereby incorporated by reference in its entirety). One DNA molecule which encodes the tat protein transduction domain has a nucleotide sequence of SEQ ID No: 10 as follows:
Variations of these tat sequences can also be employed. Such sequence variants have been reported in HIV Sequence Compendium 2000, Kuiken et al. (eds.), Theoretical Biology and Biophysics Group, Los Alamos National Laboratory, which is hereby incorporated by reference in its entirety.
Other cellular uptake polypeptides and their use have been described in the literature, including membrane-permeable sequences of the SN50 peptide, the Grb2 SH2 domain, and integrin β3, β1, and αIIb cytoplasmic domains (Hawiger, “Noninvasive intracellular delivery of functional peptides and proteins,” Curr. Opin. Chem. Biol. 3:89-94 (1999), which is hereby incorporated by reference in its entirety).
The second polypeptide can be either a full length APOBEC-1 or a fragment thereof which includes the catalytic domain thereof. The APOBEC-1 protein or fragment thereof is a mammalian APOBEC-1 protein or fragment thereof, including without limitation, human, rat, mouse, etc.
The full length human APOBEC-1 has an amino acid sequence according to SEQ ID No: 11 as follows:
This human APOBEC-1 sequence is reported at Genbank Accession No. NP—001635, which is hereby incorporated by reference in its entirety. The full length human APOBEC-1 is believed to include a putative bipartite nuclear localization signal between amino acid residues 15-34, a catalytic center between amino acid residues 61-98, and a putative cytoplasmic retention signal between amino acid residues 173-229. A cDNA sequence which encodes the full length human APOBEC-1 is set forth as SEQ ID No: 12 as follows:
The full length rat APOBEC-1 has an amino acid sequence according to SEQ ID No: 13 as follows:
This rat APOBEC-1 sequence is reported at Genbank Accession No. P38483, which is hereby incorporated by reference in its entirety. Recombinant studies using rat APOBEC-1 have demonstrated that an N-terminal region, containing the putative nuclear localization signal, is required for nuclear distribution of APOBEC-1 while a C-terminal region, containing a putative cytoplasmic retention signal (Yang et al., “Multiple protein domains determine the cell type-specific nuclear distribution of the catalytic subunit required for apolipoprotein B mRNA editing,” Proc. Natl. Acad. Sci. USA 94:13075-13080 (1997), which is hereby incorporated by reference in its entirety. A cDNA sequence which encodes the full length rat APOBEC-1 is set forth as SEQ ID No: 14 as follows:
The cDNA molecule is reported at Genbank Accession No. L07114, which is hereby incorporated by reference in its entirety.
The full length mouse APOBEC-1 has an amino acid sequence according to SEQ ID No: 15 as follows:
This mouse APOBEC-1 sequence is reported at Genbank Accession No. NP—112436, which is hereby incorporated by reference in its entirety. A cDNA sequence which encodes the full length mouse APOBEC-1 is set forth as SEQ ID No: 16 as follows:
The cDNA molecule is reported at Genbank Accession No. NM—031159, which is hereby incorporated by reference in its entirety.
The first chimeric protein of the present invention can also include one or more other polypeptide sequences, including without limitation: (i) a polypeptide that includes a cytoplasmic localization protein or a fragment thereof which, upon cellular uptake of the first chimeric protein, localizes the first chimeric protein to the cytoplasm; (ii) a polypeptide that includes a plurality of adjacent histidine residues; and (iii) a polypeptide that includes an epitope tag.
The polypeptide that includes a cytoplasmic localization protein or a fragment thereof can be any protein, or fragment thereof, which can effectively retain the first chimeric protein within the cytoplasm of a cell into which the first chimeric protein has been translocated. One such protein is chicken muscle pyruvate kinase (“CMPK”), which has an amino acid sequence of SEQ ID No: 17 as follows:
A DNA molecule encoding the full length CMPK has a nucleotide sequence according to SEQ ID No: 18 as follows:
The amino acid sequence and nucleotide sequence for the full length CMPK is reported at Genbank Accession Nos. AAA49021 and J00903, respectively, each of which is hereby incorporated by reference in its entirety.
Fragments of CMPK which afford cytoplasmic retention of the first chimeric protein include, without limitation, polypeptides containing at a minimum residues 1-479 of SEQ ID No: 18.
The polypeptide that includes a plurality of histidine residues preferably contains a sufficient number of histidine residues so as to allow the first chimeric protein containing such histidine residues to be bound by an antibody which recognizes the plurality of histidine residues. One type of DNA molecule encoding Hn is (cac)n, where n is greater than 1, but preferably greater than about 5. This His region can be used during immuno-purification, which is described in greater detail below.
The polypeptide that includes an epitope tag can be any epitope tag that is recognized with antibodies raised against the epitope tag. An exemplary epitope tag is a hemagglutinin (“HA”) domain. The HA domain is present only when it is desirable to examine, i.e., in vitro, localization of the first chimeric protein within cells that have translocated it. One suitable HA domain has an amino acid sequence according to SEQ ID No: 19 as follows:
This HA sequence is encoded by a DNA molecule having a nucleotide sequence according to SEQ ID No: 20 as follows:
An exemplary first chimeric protein of the present invention which is suitable for use in humans, designated TAT-hAPOBEC-CMPK, is set forth in
An exemplary first chimeric protein of the present invention which is suitable for use in rats, designated TAT-rAPOBEC-CMPK, is set forth in
According to a second aspect of the present invention, a second chimeric protein is provided for use in combination with the first chimeric protein described above. The second chimeric protein includes a first polypeptide that includes a protein transduction domain and a second polypeptide the includes ACF or a fragment thereof which can bind to apolipoprotein B mRNA.
The first polypeptide of the second chimeric protein can be a protein transduction domain of the type described above. The protein transduction domain of the second chimeric protein can be the same or different from the protein transduction domain of the first chimeric protein.
The second polypeptide of the second chimeric protein, as noted above, includes ACF or a fragment thereof which can bind to apolipoprotein B mRNA. Although it has been proposed that a number of different proteins assist APOBEC-1 in editing apolipoprotein B mRNA, ACF has been identified as the minimal protein complement for editing in vitro in the human system (Mehta et al., Molecular cloning of apobec-1 complementation factor, a novel RNA binding protein involved in the editing of apo B mRNA,” Mol. Cell. Biol. 20:1846-1854 (2000), which is hereby incorporated by reference in its entirety). In accordance with the present invention, therefore, the second chimeric protein binds apolipoprotein B mRNA at the mooring sequence and through its interactions with the first chimeric protein, sequesters the first chimeric protein to the cytidine of the apolipoprotein B mRNA to be edited (i.e., at position 6666), thereby resulting in its conversion to a uridine. As noted above, this conversion results in a stop codon that contributes to expression of the apolipoprotein B48 derivative.
Recent studies have suggested that APOBEC-1 requires a chaperone for its nuclear localization (Yang et al., “Intracellular trafficking determinants in APOBEC-1, the catalytic subunit for cytidine to uridine editing of apolipoprotein B mRNA,” Exp. Cell Res. 267:153-164 (2001), which is hereby incorporated by reference in its entirety). More recently, however, it has been learned that APOBEC-1 is most likely associated with ACF throughout the cell and, therefore, it may import to the nucleus as an APOBEC-1/ACF complex. A bipartite nuclear localization signal is predicted in ACF (see below).
ACF is expressed at sufficient levels within the hepatic cells of rat (Dance et al., “Two proteins essential for apolipoprotein B mRNA editing are expressed from a single gene through alternative splicing,” J. Biol. Chem., electronically published as manuscript M111337200 (2002), which is hereby incorporated by reference in its entirety), such that augmenting of the intracellular ACF concentration is not needed. However, to optimize apolipoprotein B mRNA editing, in some instances it may be desirable to increase the intracellular concentration of ACF.
The full length rat ACF has an amino acid sequence according to SEQ ID No: 21 as follows:
A DNA molecule encoding the full length rat ACF has a nucleotide sequence according to SEQ ID No: 22 as follows:
The amino acid sequence and nucleotide sequence for the full length rat ACF65 is reported at Genbank Accession Nos. AAK50145 and AY028945, respectively, each of which is hereby incorporated by reference in its entirety. In addition, it should be noted that a short isoform of rat ACF64 exists, as identified at Genbank Accession No. AF290984, which is hereby incorporated by reference in its entirety.
The full length human ACF has an amino acid sequence according to SEQ ID No: 23 as follows:
A DNA molecule encoding the full length human ACF has a nucleotide sequence according to SEQ ID No: 24 as follows:
The amino acid sequence and nucleotide sequence for the full length human ACF is reported at Genbank Accession Nos. AAF76221 and AF271789, respectively, each of which is hereby incorporated by reference in its entirety.
In comparing the human and rat ACF homologs, it is apparent that these proteins share 93.5 percent identity at the amino acid level and, moreover, antibodies raised against the human ACF also recognize rat ACF. Is has been reported that functional complementation of apolipoprotein B mRNA editing by APOBEC-1 involves the N-terminal 380 residues of ACF (Blanc et al., “Mutagenesis of Apobec-1 complementation factor reveals distinct domains that modulate RNA binding, protein-protein interaction with Apobec-1, and complementation of C to U RNA-editing activity,” J. Biol. Chem. 276(49): 46386-46393 (2001), which is hereby incorporated by reference in its entirety).
The second chimeric protein of the present invention can also include one or more other polypeptide sequences, including without limitation: (i) a polypeptide that includes a cytoplasmic localization protein or a fragment thereof which, upon cellular uptake of the second chimeric protein, localizes the second chimeric protein to the cytoplasm; (ii) a polypeptide that includes a plurality of adjacent histidine residues; and (iii) a polypeptide that includes a hemagglutinin domain. Each of these has been described above with respect to the first chimeric protein.
An exemplary second chimeric protein of the present invention which is suitable for use in humans, designated TAT-hACF, is set forth in
An exemplary second chimeric protein of the present invention which is suitable for use in rats, designated TAT-rACF, is set forth in
DNA molecules encoding the above-identified first and second chimeric proteins can be assembled using conventional molecular genetic manipulation for subcloning gene fragments, such as described by Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Springs Laboratory, Cold Springs Harbor, N.Y. (1989), and Ausubel et al. (ed.), Current Protocols in Molecular Biology, John Wiley & Sons (New York, N.Y.) (1999 and preceding editions), each of which is hereby incorporated by reference in its entirety. In conjunction therewith, desired fragments of the APOBEC-1, ACF, or CMPK encoding DNA molecules can be obtained using the PCR technique together with specific sets of primers chosen to represent particular portions of the protein. Erlich et al., Science 252:1643-51 (1991), which is hereby incorporated by reference in its entirety.
Once the desired DNA molecules have been assembled, DNA constructs can be assembled by ligating together the DNA molecule encoding the first or second chimeric protein with appropriate regulatory sequences including, without limitation, a promoter sequence operably connected 5′ to the DNA molecule, a 3′ regulatory sequence operably connected 3′ of the DNA molecule, as well as any enhancer elements, suppressor elements, etc. The DNA construct can then be inserted into an appropriate expression vector. Thereafter, the vector can be used to transform a host cell, typically although not exclusively a prokaryote, and the recombinant host cell can express the first or second chimeric protein of the present invention.
When a prokaryotic host cell is selected for subsequent transformation, the promoter region used to construct the DNA construct (i.e., transgene) should be appropriate for the particular host. The DNA sequences of eukaryotic promoters, as described infra for expression in eukaryotic host cells, differ from those of prokaryotic promoters. Eukaryotic promoters and accompanying genetic signals may not be recognized in or may not function in a prokaryotic system and, further, prokaryotic promoters are not recognized and do not function in eukaryotic cells.
Similarly, translation of mRNA in prokaryotes depends upon the presence of the proper prokaryotic signals which differ from those of eukaryotes. Efficient translation of mRNA in prokaryotes requires a ribosome binding site called the Shine-Dalgarno (“SD”) sequence on the mRNA. This sequence is a short nucleotide sequence of mRNA that is located before the start codon, usually AUG, which encodes the amino-terminal methionine of the protein. The SD sequences are complementary to the 3′-end of the 16S rRNA (ribosomal RNA) and probably promote binding of mRNA to ribosomes by duplexing with the rRNA to allow correct positioning of the ribosome. For a review on maximizing gene expression, see Roberts and Lauer, Methods in Enzymology, 68:473 (1979), which is hereby incorporated by reference in its entirety.
Promoters vary in their “strength” (i.e., their ability to promote transcription). For the purposes of expressing a cloned gene, it is desirable to use strong promoters in order to obtain a high level of transcription and, hence, expression of the gene. Depending upon the host cell system utilized, any one of a number of suitable promoters may be used. For instance, when cloning in E. coli, its bacteriophages, or plasmids, promoters such as the T7 phage promoter, lac promoter, trp promoter, recA promoter, ribosomal RNA promoter, the PR and PL, promoters of coliphage lambda and others, including but not limited, to lacUV5, ompF, bla, lpp, and the like, may be used to direct high levels of transcription of adjacent DNA segments. Additionally, a hybrid trp-lacUV5 (tac) promoter or other E. coli promoters produced by recombinant DNA or other synthetic DNA techniques may be used to provide for transcription of the inserted gene.
Bacterial host cell strains and expression vectors may be chosen which inhibit the action of the promoter unless specifically induced. In certain operons, the addition of specific inducers is necessary for efficient transcription of the inserted DNA. For example, the lac operon is induced by the addition of lactose or IPTG (isopropylthio-beta-D-galactoside). A variety of other operons, such as trp, pro, etc., are under different controls.
Specific initiation signals are also required for efficient gene transcription and translation in prokaryotic cells. These transcription and translation initiation signals may vary in “strength” as measured by the quantity of gene specific messenger RNA and protein synthesized, respectively. The DNA expression vector, which contains a promoter, may also contain any combination of various “strong” transcription and/or translation initiation signals. For instance, efficient translation in E. coli requires a Shine-Dalgarno (“SD”) sequence about 7-9 bases 5′ to the initiation codon (“ATG”) to provide a ribosome binding site. Thus, any SD-ATG combination that can be utilized by host cell ribosomes may be employed. Such combinations include, but are not limited to, the SD-ATG combination from the cro gene or the N gene of coliphage lambda, or from the E. coli tryptophan E, D, C, B or A genes. Additionally, any SD-ATG combination produced by recombinant DNA or other techniques involving incorporation of synthetic nucleotides may be used.
Mammalian cells can also be used to recombinantly produce the first or second chimeric proteins of the present invention. Suitable mammalian host cells include, without limitation: COS (e.g., ATCC No. CRL 1650 or 1651), BHK (e.g., ATCC No. CRL 6281), CHO (ATCC No. CCL 61), HeLa (e.g., ATCC No. CCL 2), 293 (ATCC No. 1573), CHOP, and NS-1 cells. Suitable expression vectors for directing expression in mammalian cells generally include a promoter, as well as other transcription and translation control sequences known in the art. Common promoters include, without limitation, SV40, MMTV, metallothionein-1, adenovirus Ela, CMV, immediate early, immunoglobulin heavy chain promoter and enhancer, and RSV-LTR.
Regardless of the selection of host cell, once the DNA molecule coding for a first or second chimeric protein has been ligated to its appropriate regulatory regions using well known molecular cloning techniques, it can then be introduced into a suitable vector or otherwise introduced directly into a host cell using transformation protocols well known in the art (Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Press, NY (1989), which is hereby incorporated by reference in its entirety).
The recombinant DNA molecule can be introduced into host cells via transformation, particularly transduction, conjugation, mobilization, or electroporation. Suitable host cells include, but are not limited to, bacteria, virus, yeast, mammalian cells, insect, plant, and the like. The host cells, when grown in an appropriate medium, are capable of expressing the chimeric protein, which can then be isolated therefrom and, if necessary, purified. The first or second chimeric protein is preferably produced in purified form (preferably at least about 80%, more preferably 90%, pure) by conventional techniques, including immuno-purification techniques. Immuno-isolation followed by metal-chelating affinity chromatography and cationic exchange chromatography is described in Example 1 infra.
A further aspect of the present invention relates to a number of compositions, preferably pharmaceutical compositions, which include the first and/or second chimeric protein of the present invention.
According to one embodiment, a composition includes a pharmaceutically acceptable carrier and the first chimeric protein of the present invention. The first chimeric protein is preferably present in an amount which is effective to modify apolipoprotein B mRNA editing in cells which uptake the first chimeric protein.
According to a second embodiment, a composition includes the first and second chimeric proteins of the present invention. This composition can also include a pharmaceutically acceptable carrier in which the first and second chimeric proteins are dispersed. Preferably, the first chimeric protein is present in an amount which is effective to modify apolipoprotein B mRNA editing in cells which uptake the first chimeric protein and the second chimeric protein is present in an amount which is effective to bind apolipoprotein B mRNA and assist the first chimeric protein in modifying apolipoprotein B mRNA in cells which uptake the first and second chimeric proteins.
The compositions of the present invention can also include suitable excipients, or stabilizers, and can be in solid or liquid form such as, tablets, capsules, powders, solutions, suspensions, or emulsions. Typically, the compositions will contain from about 0.01 to 99 percent, preferably from about 20 to 75 percent of the chimeric protein(s), together with the carrier, excipient, stabilizer, etc.
The solid unit dosage forms can be of the conventional type. The solid form can be a capsule, such as an ordinary gelatin type containing the first and/or second chimeric protein(s) of the present invention and a carrier, for example, lubricants and inert fillers such as, lactose, sucrose, or cornstarch. In another embodiment, these first and/or second chimeric protein(s) are tableted with conventional tablet bases such as lactose, sucrose, or cornstarch in combination with binders like acacia, cornstarch, or gelatin, disintegrating agents, such as cornstarch, potato starch, or alginic acid, and a lubricant, like stearic acid or magnesium stearate.
The first and/or second chimeric protein(s) of the present invention may also be administered in injectable or topically-applied dosages by solution or suspension of these materials in a physiologically acceptable diluent with a pharmaceutical carrier. Such carriers include sterile liquids, such as water and oils, with or without the addition of a surfactant and other pharmaceutically and physiologically acceptable carrier, including adjuvants, excipients or stabilizers. Illustrative oils are those of petroleum, animal, vegetable, or synthetic origin, for example, peanut oil, soybean oil, or mineral oil. In general, water, saline, aqueous dextrose and related sugar solution, and glycols, such as propylene glycol or polyethylene glycol, are preferred liquid carriers, particularly for injectable solutions.
For use as aerosols, the first and/or second chimeric protein(s) of the present invention in solution or suspension may be packaged in a pressurized aerosol container together with suitable propellants, for example, hydrocarbon propellants like propane, butane, or isobutane with conventional adjuvants. The compositions of the present invention also may be administered in a non-pressurized form such as in a nebulizer or atomizer.
Depending upon the treatment being effected, the compounds of the present invention can be administered orally, topically, transdermally, parenterally, subcutaneously, intravenously, intramuscularly, intraperitoneally, by intracavitary or intravesical instillation, intraocularly, intraarterially, intralesionally, or by application to mucous membranes, such as, that of the nose, throat, and bronchial tubes. In most instances, subcutaneous, intravenous, intramuscular, intraperitoneal, and intraarterial routes are preferred.
Compositions within the scope of this invention include all compositions wherein the first and/or second chimeric proteins of the present invention is contained in an amount effective to achieve its intended purpose, noted above. While individual needs vary, determination of optimal ranges of effective amounts of each of the first and second chimeric proteins is within the skill of the art. Typical dosages comprise about 0.01 to about 100 mg/kg·body wt. The preferred dosages comprise about 0.1 to about 100 mg/kg·body wt. The most preferred dosages comprise about 1 to about 100 mg/kg·body wt.
The amounts of the first and second chimeric proteins can be determined by one of ordinary skill in the art using routine testing to optimize the dosage levels of the first and second chimeric proteins in accordance with the desired degree of apolipoprotein B mRNA editing. Based on May 2001 guidelines by the National Institutes of Health's National Cholesterol Education Program (NCEP), individuals at low risk for a heart attack should have LDL levels under 160 mg/dL, while those at highest risk should aim for LDLs under 100 mg/dL. Treatment regimen for the administration of the first and/or second chimeric proteins of the present invention can also be determined readily by those with ordinary skill in art.
Typically, the first and/or second chimeric proteins (or compositions which contain one or both of the chimeric proteins of the present invention) can be administered via a drug delivery device which includes a chimeric protein or a composition of the present invention. Exemplary delivery devices include, without limitation, liposomes, niosomes, transdermal patches, implants, and syringes.
Liposomes are vesicles comprised of one or more concentrically ordered lipid bilayers which encapsulate an aqueous phase. They are normally not leaky, but can become leaky if a hole or pore occurs in the membrane, if the membrane is dissolved or degrades, or if the membrane temperature is increased to the phase transition temperature. Current methods of drug delivery via liposomes require that the liposome carrier ultimately become permeable and release the encapsulated drug at the target site. This can be accomplished, for example, in a passive manner wherein the liposome bilayer degrades over time through the action of various agents in the body. Every liposome composition will have a characteristic half-life in the circulation or at other sites in the body and, thus, by controlling the half-life of the liposome composition, the rate at which the bilayer degrades can be somewhat regulated.
In contrast to passive drug release, active drug release involves using an agent to induce a permeability change in the liposome vesicle. Liposome membranes can be constructed so that they become destabilized when the environment becomes acidic near the liposome membrane (see, e.g., Proc. Natl. Acad. Sci. USA 84:7851 (1987); Biochemistry 28:908 (1989), which is hereby incorporated by reference in its entirety). When liposomes are endocytosed by a target cell, for example, they can be routed to acidic endosomes which will destabilize the liposome and result in drug release.
Alternatively, the liposome membrane can be chemically modified such that an enzyme is placed as a coating on the membrane which slowly destabilizes the liposome. Since control of drug release depends on the concentration of enzyme initially placed in the membrane, there is no real effective way to modulate or alter drug release to achieve'“on demand” drug delivery. The same problem exists for pH-sensitive liposomes in that as soon as the liposome vesicle comes into contact with a target cell, it will be engulfed and a drop in pH will lead to drug release.
This liposome delivery system can also be made to accumulate at a target organ, tissue, or cell via active targeting. In accordance with the present invention, liposomes can be targeted to liver cells by incorporating into the liposome bilayer a molecule which target hepatocyte receptors. One such molecule is the asialoglycoprotein asialofetuin, which targets the asialoglycoprotein receptor of hepatocytes. The incorporation of asialofetuin into the liposome bilayer can be performed according to the procedures set forth in Wu et al., “Increased liver uptake of liposomes and improved targeting efficacy by labeling with asialofetuin in rodents,” Hepatology 27(3):772-778 (1998), which is hereby incorporated by reference in its entirety.
Niosomes are vesicles formed by amphiphilic materials. Non-ionic surfactants were the first materials studied (Iga et al., “Membrane modification by negatively charged stearylpolyoxyethylene derivatives for thermosensitive liposomes: Reduced liposomal aggregation and avoidance of reticuloendothelial system uptake,” J. Drug Target 2:259-67 (1994), which is hereby incorporated by reference in its entirety) and a large number of surfactants have since been found to self assemble into closed bilayer vesicles (Ahl et al., “Enhancement of the in vivo circulation lifetime of L-alpha-distearoylphosphatidylcholine liposomes: Importance of liposomal aggregation versus complement opsonization,” Biochim Biophys Acta 1329:370-82 (1997), which is hereby incorporated by reference in its entirety). These niosomal materials may be used for delivery of the first or second chimeric protein or for delivery of APOBEC-1 or fragments thereof alone or in combination with ACF or fragments thereof.
For example, 200 nm doxorubicin niosomes with a polyoxyethylene (molecular weight 1,000) surface have been shown to be rapidly taken up by the liver (Uchegbu et al., “Distribution, metabolism and tumoricidal activity of doxorubicin administered in sorbitan monostearate (Span 60) niosomes in the mouse,” Pharm. Res. 12:1019-24 (1995), which is hereby incorporated by reference in its entirety), allowing polymeric drug conjugates to be formed for delivery of the drug (see Duncan, “Drug polymer conjugates—potential for improved chemotherapy,” Anti-Cancer Drugs 3:175-210 (1992), which is hereby incorporated by reference in its entirety). These techniques can be readily adapted for delivery of the first and second chimeric proteins or, alternatively, APOBEC-1 or a fragment thereof alone or in combination with ACF or a fragment thereof.
Compositions including the liposomes or niosomes in a pharmaceutically acceptable carrier are also contemplated.
Transdermal delivery devices have been employed for delivery of low molecular weight proteins by using lipid-based compositions (i.e., in the form of a patch) in combination with sonophoresis. However, as reported in U.S. Pat. No. 6,041,253 to Ellinwood, Jr. et al., which is hereby incorporated by reference in its entirety, transdermal delivery can be further enhanced by the application of an electric field, for example, by iontophoresis or electroporation. Using low frequency ultrasound which induces cavitation of the lipid layers of the stratum corneum, higher transdermal fluxes, rapid control of transdermal fluxes, and drug delivery at lower ultrasound intensities can be achieved. Still further enhancement can be obtained using a combination of chemical enhancers and/or magnetic field along with the electric field and ultrasound.
Implantable or injectable protein depot compositions can also be employed, providing long-term delivery of, e.g., the first and second chimeric proteins. For example, U.S. Pat. No. 6,331,311 to Brodbeck et al., which is hereby incorporated by reference in its entirety, reports an injectable depot gel composition which includes a biocompatible polymer, a solvent that dissolves the polymer and forms a viscous gel, and an emulsifying agent in the form of a dispersed droplet phase in the viscous gel. Upon injection, such a gel composition can provide a relatively continuous rate of dispersion of the agent to be delivered, thereby avoiding an initial burst of the agent to be delivered.
Other suitable protein delivery system which are known to those of skill in the art can also be employed to achieve the desired delivery and, thus, modification in the editing of apolipoprotein B mRNA and its concomitant effects.
By virtue of the first chimeric protein being able to edit apolipoprotein B mRNA, the present invention affords a method of modifying apolipoprotein B mRNA editing in vivo. This aspect of the present invention can be carried out by contacting apolipoprotein B mRNA in a cell with the first chimeric protein of the present invention under conditions effective to increase the concentration of apolipoprotein B48 which is secreted by the cell as compared to the concentration of apolipoprotein B100 which is secreted by the cell, relative to an untreated cell (i.e., which has not taken up the first chimeric protein). Basically, the contacting is carried out by exposing the cell to the first chimeric protein under conditions effective to induce cellular uptake of the first chimeric protein. Because the first chimeric protein includes the first polypeptide (i.e., which includes a protein transduction domain), the first chimeric protein is taken up by the cell. In addition, the same cell can also be contacted with the second chimeric protein of the present invention, causing the second chimeric protein also to be taken up by the cell. As a result, the apolipoprotein B mRNA in the cell is contacted by the second chimeric protein, binding the apolipoprotein mRNA (as described above) so as to facilitate editing thereof by the first chimeric protein. The cell in which the apolipoprotein B mRNA editing is modified can be any cell which can synthesize and secrete VLDL with apolipoprotein B or its derivatives. Exemplary cells of this type include liver cells and intestinal cells, although preferably liver cells. The cell can also be in a mammal, preferably a human.
Likewise, the present invention also affords a method of reducing serum LDL levels. This aspect of the present invention can be carried out by delivering into one or more cells of a patient, without genetically modifying the cells, an amount of a protein comprising APOBEC-1 or a fragment thereof which can edit mRNA encoding apolipoprotein B, which amount is effective to increase the concentration of VLDL-apolipoprotein B48 that is secreted by the one or more cells into serum and, consequently, reduce the serum concentration of LDL. In accordance with this aspect of the present invention, the patient is a mammal, preferably a human, and the one or more cells are preferably liver cells, intestinal cells, or a combination thereof.
To sustain the reduced serum LDL levels, delivery of the protein into the one or more cells is preferably repeated periodically (i.e., following a delay of from about 1 to about 7 days).
Delivery of the protein into the one or more cells can be carried out by exposing the one or more cells to the protein under conditions effective to cause cellular uptake of the protein. Preferably, the protein which includes APOBEC-1 or a fragment thereof is actually the first chimeric protein of the present invention and the protein transduction domain induces cellular uptake by the one or more cells. In addition to delivering the protein, a second protein can also be delivered simultaneously into the one or more cells of the patient, without genetically modifying the cells, where the second protein includes ACF or a fragment thereof which can bind to apolipoprotein B mRNA. Preferably, the second protein is the second chimeric protein of the present invention and the protein transduction domain induces cellular uptake by the one or more cells.
Alternatively, APOBEC-1 can be delivered directly into one or more liver cells by contacting each of them with liposomes including a molecule which binds to a hepatocyte receptor (e.g., asialofetuin), thereby inducing uptake of the liposomes and degradation thereof intracellularly to empty their contents into the one or more liver cells. In addition, ACF or a fragment thereof which can bind to apolipoprotein B mRNA can also be delivered via the liposomes.
By increasing the ratio of apolipoprotein B48 to apolipoprotein B100 which is secreted by the one or more cells, the present invention also relates to a method of treating or preventing an atherogenic disease or disorder. This aspect of the present invention can be carried out by administering to a patient an effective amount of a protein comprising APOBEC-1 or a fragment thereof which can edit mRNA encoding apolipoprotein B, wherein upon said administering the protein is taken up by one or more cells of the patient that can synthesize and secrete VLDL-apolipoprotein under conditions which are effective to increase the concentration of VLDL-apolipoprotein B48 that is secreted by the one or more cells into serum, whereby rapid clearing of VLDL-apolipoprotein B48 from serum decreases the serum concentration of LDL to treat or prevent the atherogenic disease or disorder. In accordance with this aspect of the present invention, the patient is a mammal, preferably a human, and the one or more cells are preferably liver cells.
Administration of the protein can be carried out according to any of the above-identified approaches. Continued preventative or therapeutic treatment can be effected by repeatedly administering the APOBEC-1 protein periodically (i.e., following a delay of from about 1 to about 7 days).
Preferably, the protein which includes APOBEC-1 or a fragment thereof is actually the first chimeric protein of the present invention and the protein transduction domain induces cellular uptake by the one or more cells. As with the above-described methods, a second protein that includes ACF or a fragment thereof which can bind to apolipoprotein B mRNA can also be delivered simultaneously. Preferably, the second protein is the second chimeric protein of the present invention and the protein transduction domain induces cellular uptake by the one or more cells.
Alternatively, using a liposome delivery vehicle, APOBEC-1 and optionally ACF can be delivered directly into one or more liver cells by contacting each of them with a liposome including a molecule which binds to a hepatocyte receptor, thereby inducing uptake of the liposomes and degradation thereof intracellularly to empty their contents into the one or more liver cells.
The following examples are intended to illustrate, but by no means are intended to limit, the scope of the present invention as set forth in the appended claims.
The induction of hepatic apolipoprotein B mRNA editing was sought through TAT mediated APOBEC-1 protein transduction into liver cells. It has been shown that linking an 11-amino-acid protein transduction domain (PTD) of HIV-1 TAT protein to heterologous protein conferred the ability to transduce into cells (Nagahara et al., “Transduction of full-length TAT fusion proteins into mammalian cells: TAT-p27Kip1 induces cell migration,” Nature Med. 4:1449-1452 (1998); Schwarze et al., “In vivo protein transduction: delivery of a biologically active protein into the mouse,” Science 285:1569-1572 (1999); Vocero-Akbani et al., “Killing HIV-infected cells by transduction with an HIV protease-activated caspase-3 protein,” Nature Med. 5:29-33 (1999), each of which is hereby incorporated by reference in its entirety). PTD-linked protein transduced into ˜100% of cells and the transduction process occurred in a rapid and concentration-dependent but receptor- and transporter-independent manner (Schwarze et al., “Protein transduction: unrestricted delivery into all cells,” Trends Cell Biol. 10:290-295 (2000), which is hereby incorporated by reference in its entirety). Liver cells have been shown to be susceptible to transduction (Nagahara et al., “Transduction of full-length TAT fusion proteins into mammalian cells: TAT-p27Kip1 induces cell migration,” Nature Med. 4:1449-1452 (1998), which is hereby incorporated by reference in its entirety). In order to produce in-frame TAT fusion protein from E. coli, a prokaryotic expression vector was constructed that has an N-terminal PTD flanked by glycine residues for free bond rotation of the domain (Schwarze et al., “In vivo protein transduction: delivery of a biologically active protein into the mouse,” Science 285:1569-1572 (1999), which is hereby incorporated by reference in its entirety), an hemagglutinin (HA) tag and a C-terminal 6-histidine tag. Using this vector as a backbone, a plasmid was constructed to encode full-length TAT-rAPOBEC-CMPK protein, SEQ ID No: 4 (
A double-stranded oligomeric nucleotide encoding the 9-amino acid TAT domain flanked by glycine residues (sense strand shown below, SEQ ID No: 25)
and a PCR product encoding HA-rAPOBEC-CMPK (SEQ ID No: 26 as set forth below)
or HA-CMPK (SEQ ID No: 27 as set forth below)
(Yang et al., “Induction of cytidine to uridine editing on cytoplasmic apolipoprotein B mRNA by overexpressing APOBEC-1,” J. Biol. Chem. 275:22663-22669 (2000), which is hereby incorporated by reference in its entirety) were inserted into NdeI/XhoI digested pPROEX vector (Life, Gaithersburg, Md.). The entire constructs (TAT-rAPOBEC-CMPK (SEQ ID No: 3) or TAT-CMPK (SEQ ID No: 28 as set forth below)
were inserted into pET-24b (Novagen, Madison, Wis.) vector to take advantage of the C-terminal His6 tag. TAT fusion proteins (referred to as TAT-CMPK, the expression product of SEQ ID No: 28, and TAT-rAPOBEC-CMPK, SEQ ID No: 4) were purified from BL-21(DE3) codon plus cells (Stratagene, La Jolla, Calif.). Two to four 1-liter cultures were inoculated with a 10 ml overnight culture each and induced by 0.1 mM IPTG at 30° C. for 1 hour. Soluble proteins were obtained by French press in 25 ml of buffer A (8M urea, 10 mM Tris pH 8, 100 mM NaH2PO4). Cellular lysates were cleared by centrifugation, loaded onto a 5-ml Ni-NTA column (Qiagen, Valencia, Calif.) in buffer A with 10-20 mM imidazole, washed and eluted with imidazole in buffer A ‘stepwise’ (100, 175 and 250 mM) and loaded onto a HiTrap SP column (Amersham Pharmacia, Piscataway, N.J.). The column was washed and eluted with 1 M NaCl in buffer A. The urea and high salt were removed from the relevant fractions by rapid dialysis against buffer B (30 mM Tris pH=8.5, 50 mM NaCl, 10 μM zinc acetate, 5% glycerol). The elution profile was analyzed by SDS-PAGE. Gels were stained with silver according to manufacture's recommendations (Bio-Rad, Hercules, Calif.).
Recombinant proteins were solubilized in 8M urea buffer from bacterial cells so as to maximize their yield from inclusion bodies. Previous studies have shown that denatured proteins could transduce as well as native proteins (Schwarze et al., “In vivo protein transduction: delivery of a biologically active protein into the mouse,” Science 285:1569-1572 (1999), which is hereby incorporated by reference in its entirety). The proteins were purified through metal-chelating affinity chromatography followed by cationic exchange chromatography. The urea was removed by rapid dialysis and the purity of full-length 86 kDa TAT-rAPOBEC-CMPK, SEQ ID No: 4, was apparent as shown by silver staining (
The uptake of TAT-rAPOBEC-CMPK, SEQ ID No: 4, into McArdle cells was evaluated using an antibody reactive with the HA epitope and fluorescence microscopy.
McArdle RH7777 cells were obtained from ATCC (Manassas, Va.) and cultured as described previously (Yang et al., “Partial characterization of the auxiliary factors involved in apo B mRNA editing through APOBEC-1 affinity chromatography,” J. Biol. Chem. 272:27700-27706 (1997), which is hereby incorporated by reference in its entirety). McArdle cells, grown on six well cluster plates were treated with either TAT-rAPOBEC-CMPK or TAT-CMPK for the indicated times. Cells were then washed extensively with PBS and subsequently fixed with 2% paraformaldehyde, permeabilized with 0.4% Triton X100, blocked with 1% BSA and reacted with affinity purified anti-HA (Babco, Berkeley, Calif.) and affinity purified FITC conjugated goat anti-mouse secondary antibody (Organon Teknika, West Chester, Pa.), each at 1:1000 dilution. Fluorescence was observed and electronic images captured on an inverted, fluorescence Olympus microscope.
Recombinant APOBEC-1 has a tendency to aggregate, a property which persists in TAT-rAPOBEC-CMPK, apparent as aggregates of HA antibody-reactive material attached to the surface of cells 1 h following the addition of the protein to the media (
Within 6 h following treatment, both TAT-rAPOBEC-CMPK (
Given that TAT-CMPK entered McArdle cells, as demonstrated in Example 2, an evaluation was made as to whether this would affect apolipoprotein B mRNA editing activity (
Total cellular RNA was isolated from cells with Tri-Reagent (Molecular Research Center, Cincinnati, Ohio) according to manufacture's recommendations. Purified RNAs were digested with RQ-DNase I (Promega, Madison, Wis.) and with RsaI (Promega) restriction enzyme that has a recognition site between the PCR annealing sites of target substrates to ensure the removal of the contaminating genomic DNA.
Editing activity was determined by the reverse transcriptase-polymerase chain reaction (RT-PCR) methodology described previously (Smith et al. “In vitro apolipoprotein B mRNA editing: Identification of a 27S editing complex,” Proc. Natl. Acad. Sci. USA 88:1489-1493 (1991), which is hereby incorporated by reference in its entirety). First strand cDNA was generated using oligo dT-primed total cellular RNA. Specific PCR amplification of rat apolipoprotein B sequence surrounding the editing site was accomplished using ND1/ND2 primer pairs set forth below:
PCR products were gel isolated and the editing efficiency was determined by poisoned primer extension assay using 32P ATP (NEN, Boston, Mass.) end-labeled DD3 primer (SEQ ID No: 31) as follows:
under high concentration of dideoxy GTP as described previously (Smith et al. “In vitro apolipoprotein B mRNA editing: Identification of a 27S editing complex,” Proc. Natl. Acad. Sci. USA 88:1489-1493 (1991); Sowden et al., “Overexpression of APOBEC-1 results in mooring-sequence-dependent promiscuous RNA editing,” J. Biol. Chem. 271:3011-3017 (1996), each of which is hereby incorporated by reference in its entirety). Primer extension products were resolved on a 10% denaturing polyacrylamide gel, autoradiographed, and then quantified by a laser densitometric scanning (Molecular Dynamics, Sunnyvale, Calif.). Percent editing was calculated as the counts in the UAA (edited) band divided by the sum of the counts in UAA and those in the CAA (unedited) bands and multiplied by 100.
No change in the percent editing of apolipoprotein B mRNA relative to untreated cells (see
In contrast, editing activity increased in McArdle cells with 360 nM (62 μg protein/ml media) TAT-rAPOBEC-CMPK following 6 h and continued to a peak by 24 h, a more than 3-fold increase over the level of editing observed in control cells (
To determine if the results obtained using McArdle cells would be applicable in primary liver cells, cultured rat primary hepatocytes were prepared and then treated with TAT-rAPOBEC-CMPK. The rat primary hepatocytes were prepared from unfasted, male Sprague-Dawley rats (250-275 g body weight, Taconic Farm) fed ad libitum normal rat chow as described previously (Van Mater et al., “Ethanol increases apolipoprotein B mRNA editing in rat primary hepatocytes and McArdle cells,” Biochem. Biophys. Res. Comm. 252:334-339 (1998), which is hereby incorporated by reference in its entirety). Recombinant TAT fusion protein was added directly to the cell culture media after dialysis.
It has been shown that the editing efficiency in primary rat hepatocytes decreased as a result of proliferation after 72 hours in culture (Van Mater et al., “Ethanol increases apolipoprotein B mRNA editing in rat primary hepatocytes and McArdle cells,” Biochem. Biophys. Res. Comm. 252:334-339 (1998), which is hereby incorporated by reference in its entirety). Together with the fact that TAT-rAPOBEC-CMPK maximally increased editing 24 hours after treatment in McArdle cells, a decision was made to evaluate dose response for a fixed time rather than study kinetics. Primary hepatocytes were treated with the indicated amounts of TAT-rAPOBEC-CMPK and analyzed for edited apolipoprotein B mRNA 24 hours afterwards. Analysis of apolipoprotein B mRNA was carried out a described in Example 3 above.
The editing activity of hepatocytes increased in proportion to the amount of TAT-rAPOBEC-CMPK added to the cell culture media relative to cells treated with buffer alone (
Promiscuous editing of additional cytidines in rat apolipoprotein B mRNA of transfected cells (Sowden et al., “Overexpression of APOBEC-1 results in mooring-sequence-dependent promiscuous RNA editing,” J. Biol. Chem. 271:3011-3017 (1996); Yamanaka et al., “Hyperediting of multiple cytidines of apolipoprotein B mRNA by APOBEC-1 requires auxiliary protein(s) but not a mooring sequence motif,” J. Biol. Chem. 271:11506-11510 (1996); Sowden et al., “Apolipoprotein B RNA Sequence 3′ of the mooring sequence and cellular sources of auxiliary factors determine the location and extent of promiscuous editing,” Nucleic Acids Res. 26:1644-1652 (1998), each of which is hereby incorporated by reference in its entirety) or hyper-editing of other mRNAs in transgenic mice and rabbits (Yamanaka et al., “Hyperediting of multiple cytidines of apolipoprotein B mRNA by APOBEC-1 requires auxiliary protein(s) but not a mooring sequence motif,” J. Biol. Chem. 271:11506-11510 (1996); Yamanaka et al., “A novel translational repressor mRNA is edited extensively in livers containing tumors caused by the transgene expression of the apoB mRNA editing enzyme,” Genes & Dev. 11:321-333 (1997), each of which is hereby incorporated by reference in its entirety) has been observed in response to very high levels of APOBEC-1 expression. Editing of cytidines 5′ of the wild type editing site (C6666) was a bellwether for the loss of editing site fidelity in rat cells and could be used to monitor the induction of promiscuous editing in relation to changes in APOBEC-1 expression (Sowden et al., “Apolipoprotein B RNA Sequence 3′ of the mooring sequence and cellular sources of auxiliary factors determine the location and extent of promiscuous editing,” Nucleic Acids Res. 26:1644-1652 (1998); Siddiqui et al., “Disproportionate relationship between APOBEC-1 expression and apoB mRNA editing activity,” Exp. Cell Res. 252:154-164 (1999), each of which is hereby incorporated by reference in its entirety). Promiscuous editing of cytidine 3′ C6666 in apolipoprotein B mRNA did not occur to a significant extent in rat cells and hyperediting of mRNAs other than apolipoprotein B was not a characteristic of APOBEC-1 overexpression in rat cells (Sowden et al., “Apolipoprotein B RNA Sequence 3′ of the mooring sequence and cellular sources of auxiliary factors determine the location and extent of promiscuous editing,” Nucleic Acids Res. 26:1644-1652 (1998), which is hereby incorporated by reference in its entirety).
Despite the high level of editing activity in treated primary hepatocytes, promiscuous editing (evident as additional primer extension products above UAA (Sowden et al., “Determinants involved in regulating the proportion of edited apolipoprotein B RNAs,” RNA 2:274-288 (1996); Sowden et al., “Apolipoprotein B RNA Sequence 3′ of the mooring sequence and cellular sources of auxiliary factors determine the location and extent of promiscuous editing,” Nucleic Acids Res. 26:1644-1652 (1998), each of which is hereby incorporated by reference in its entirety) was not observed (
To further confirm the efficacy of this method, secreted apolipoprotein B protein was evaluated in primary rat hepatocytes that were long-term metabolically labeled with [35S]-methionine and [35S]-cysteine after TAT-rAPOBEC-CMPK treatment.
Twelve to eighteen hour rat primary hepatocytes grown in Waymouth's 752/1 media (Sigma, St. Louis, Mo.) were treated for 11 hours with TAT-rAPOBEC-CMPK and then incubated for 1 hour in DMEM deficient medium (without methionine, cysteine and L-glutamine) (Sigma, St. Louis, Mo.) containing 0.2% (w/v) BSA, 0.1 nM insulin, 100 μg/ml streptomycin and 50 μg/mlgentamicin. The medium was replaced with fresh labeling medium containing 0.7 μCi/ml L-[35S]-Methionine and L-[35S]-Cysteine using EXPRE35S35S protein labeling mix (NEN, Boston, Mass.). Cells were incubated in the labeling medium for 30 minutes. One volume of Waymouth's medium with cold cysteine and methionine was added to cells and the labeling continued for an additional 12 hours, after which cell culture medium was collected for the isolation and analysis of secreted apolipoprotein B protein and RNAs. (RNA analysis was conducted as in Example 3 above.)
Immunoprecipitation of apolipoprotein B from cell culture medium was performed as described previously (Sparks et al., “Insulin-mediated inhibition of apolipoprotein B secretion requires an intracellular trafficking event and phosphatidylinositol 3-kinase activation: studies with brefeldin A and wortmannin in primary cultures of rat hepatocytes,” Biochem. J. 313:567-574 (1996), which is hereby incorporated by reference in its entirety). A rabbit polyclonal antibody raised against rat apolipoprotein B and reactive with the N-terminus of apolipoprotein B100 and apolipoprotein 1348 (obtained from Drs. J. D. Sparks and C. E. Sparks, University of Rochester) was used to precipitate apolipoprotein B. The immunoprecipitants were separated by SDS-PAGE on 5% gel. The gel was dried and exposed to film to reveal the secreted apolipoprotein B containing lipoprotein profile which represents the secreted apolipoprotein B48 and apolipoprotein B100 during the 12 hour labeling period.
The secreted [35S]-labeled apolipoprotein B lipoproteins were isolated from the cell culture media exposed to cells for 12 hours followed by immunoprecipitation, and analyzed by autoradiography after SDS-PAGE separation. The signal on the gel was in direct proportion to the number of cysteine and methionine residues in apolipoprotein B100 and apolipoprotein B48. Since apolipoprotein B48 was the N-terminal 48% of apolipoprotein B100, stronger signal was expected from apolipoprotein B100 in control cells. However, as the editing efficiency approached 90% due to TAT-rAPOBEC-CMPK treatment, an increasing amount of apolipoprotein B48 was secreted, and apolipoprotein B100 became almost undetectable (
It is believed that the present invention offers a novel approach to curtail hepatic output of apolipoprotein B100 associated atherogenic factors through up-regulating apolipoprotein B mRNA editing by using protein transduction into target (e.g., liver) cells. The PTD, amino acid residues 49-57, of HIV-1 TAT protein has been used in other systems to deliver functional full-length protein molecules into cells (Nagahara et al., “Transduction of full-length TAT fusion proteins into mammalian cells: TAT-p27Kip1 induces cell migration,” Nature Med. 4:1449-1452 (1998); Schwarze et al., “In vivo protein transduction: delivery of a biologically active protein into the mouse,” Science 285:1569-1572 (1999); Vocero-Akbani et al., “Killing HIV-infected cells by transduction with an HIV protease-activated caspase-3 protein,” Nature Med. 5:29-33 (1999), each of which is hereby incorporated by reference in its entirety). Some of these fusion molecules, when introduced into mice, entered all tissue cells, even crossing the blood brain barrier (Schwarze et al., “In vivo protein transduction: delivery of a biologically active protein into the mouse,” Science 285:1569-1572 (1999), which is hereby incorporated by reference in its entirety). Although the detailed mechanism for the cellular uptake of the fusions remains unknown, denaturing of the protein during membrane transduction is thought to be a rapid process and the rate limiting event is the renaturing of the transduced protein once inside of cells (Schwarze et al., “Protein transduction: unrestricted delivery into all cells,” Trends Cell Biol. 10:290-295 (2000), which is hereby incorporated by reference in its entirety).
In this regard, the protein transduction method may have limitations in that some proteins may not be able successfully to adopt an active conformation after they have been unfolded. It is significant, therefore, that the above Examples demonstrate that both TAT-CMPK (expression product of SEQ ID No: 28) and TAT-rAPOBEC-CMPK (SEQ ID No: 4) had the capacity to enter hepatocytes and that TAT-rAPOBEC-CMPK activated editing within 6 hours of its addition to the media.
Similar kinetics have been observed with TAT-rAPOBEC-CMPK prepared under native conditions.
Importantly, TAT-CMPK could not stimulate editing activity, demonstrating that the observed changes in editing were specific to APOBEC-1 containing recombinant proteins. Considering the tendency for APOBEC-1 containing proteins to aggregate, part of the lag in entering cells could have been due to the inability of these multimeric complexes to cross the plasma membrane and the time it took for TAT-rAPOBEC-CMPK monomers to dissociate from the aggregates and cross the membrane. This is supported by the finding that TAT-CMPK, which did not appear to form large aggregates, appeared to accumulate within the cells with more rapid kinetics than that observed for TAT-rAPOBEC-CMPK. The six hour lag before an increase in editing activity could be measured may have also been due to the time required for the transduced protein to refold and assemble editosomes.
Apolipoprotein B mRNA editing occurs in the cell nucleus despite the fact that editing factors can also be demonstrated in the cytoplasm (Yang et al., “Induction of cytidine to uridine editing on cytoplasmic apolipoprotein B mRNA by overexpressing APOBEC-1,” J. Biol. Chem. 275:22663-22669 (2000), which is hereby incorporated by reference in its entirety). The mechanism responsible for APOBEC-1's distribution in the nucleus is not understood (Yang et al., “Intracellular Trafficking Determinants in APOBEC-1, the Catalytic Subunit for Cytidine to Uridine Editing of ApoB mRNA,” Exp. Cell Res. 267:163-184 (2001), which is hereby incorporated by reference in its entirety), however its mass appeared to be important as the chimeric protein APOBEC-CMPK was excluded from the nucleus (Yang et al., “Multiple protein domains determine the cell type-specific nuclear distribution of the catalytic subunit required for apo B mRNA editing,” Proc. Natl. Acad. Sci. USA 94:13075-13080 (1997); Yang et al., “Induction of cytidine to uridine editing on cytoplasmic apolipoprotein B mRNA by overexpressing APOBEC-1,” J. Biol. Chem. 275:22663-22669 (2000), each of which is hereby incorporated by reference in its entirety). TAT-rAPOBEC-CMPK's ability to distribute in both the cytoplasm and the nucleus was consistent with the proposed ability of the TAT PTD to act also as a nuclear localization signal (Schwarze et al., “In vivo protein transduction: delivery of a biologically active protein into the mouse,” Science 285:1569-1572 (1999), which is hereby incorporated by reference in its entirety). Although TAT-rAPOBEC-CMPK's distribution mimicked that of the wild type enzyme's distribution (Yang et al., “Multiple protein domains determine the cell type-specific nuclear distribution of the catalytic subunit required for apo B mRNA editing,” Proc. Natl. Acad. Sci. USA 94:13075-13080 (1997), which is hereby incorporated by reference in its entirety), uncertainty remains as to whether all of the transduced TAT-rAPOBEC-CMPK molecules were active in editing, as well as whether cytoplasmic or nuclear transcripts were edited. Nonetheless, regardless of the degree of activity or its localization within the cell, a positive reduction in apolipoprotein B100 lipoprotein was demonstrated.
Enhancement of editing activity by overexpression of APOBEC-1 through gene transfer has been shown to be associated with promiscuous editing on both nuclear and cytoplasmic transcripts (Sowden et al., “Overexpression of APOBEC-1 results in mooring-sequence-dependent promiscuous RNA editing,” J. Biol. Chem. 271:3011-3017 (1996); Yang et al., “Induction of cytidine to uridine editing on cytoplasmic apolipoprotein B mRNA by overexpressing APOBEC-1,” J. Biol. Chem. 275:22663-22669 (2000), each of which is hereby incorporated by reference in its entirety). Metabolic stimulation of apolipoprotein B mRNA editing always retained fidelity (Wu et al., “ApoB mRNA editing: validation of a sensitive assay and developmental biology of RNA editing in the rat,” J. Biol. Chem. 265:12312-12316 (1990); Greeve et al., “Apolipoprotein B mRNA editing in 12 different mammalian species: hepatic expression is reflected in low concentrations of apoB-containing plasma lipoproteins,” J. Lipid Res. 34:1367-1383 (1993); Phung et al., “Regulation of hepatic apoB RNA editing in the genetically obese Zucker rat,” Metabolism 45:1056-1058 (1996); von Wronski et al., “Insulin increases expression of apobec-1, the catalytic subunit of the apoB B mRNA editing complex in rat hepatocytes,” Metabolism Clinical & Exp. 7:869-873 (1998), each of which is hereby incorporated by reference in its entirety). It is highly significant, therefore, that the fidelity of the editing activity was retained with TAT-rAPOBEC-CMPK even when editing was enhanced to >90%. This level of high fidelity editing could not be achieved without hyper-editing in apobec-1 transgenic animals (Yamanaka et al., “Hyperediting of multiple cytidines of apolipoprotein B mRNA by APOBEC-1 requires auxiliary protein(s) but not a mooring sequence motif,” J. Biol. Chem. 271:11506-11510 (1996); Yamanaka et al., “A novel translational repressor mRNA is edited extensively in livers containing tumors caused by the transgene expression of the apoB mRNA editing enzyme,” Genes & Dev. 11:321-333 (1997); Sowden et al., “Overexpression of APOBEC-1 results in mooring-sequence-dependent promiscuous RNA editing,” J. Biol. Chem. 271:3011-3017 (1996); Sowden et al., “Apolipoprotein B RNA Sequence 3′ of the mooring sequence and cellular sources of auxiliary factors determine the location and extent of promiscuous editing,” Nucleic Acids Res. 26:1644-1652 (1998); each of which is hereby incorporated by reference in its entirety). There was no pathology in transgenic animals in which induction of hepatic apolipoprotein B mRNA editing was achieved at a low level of apobec-1 expression and these animals had a markedly lower serum apolipoprotein B100 and significantly reduced serum LDL compared to controls (Teng et al., “Adenovirus-mediated gene transfer of rat apolipoprotein B mRNA editing protein in mice virtually eliminates apolipoprotein B-100 and normal low density lipoprotein production,” J. Biol. Chem. 269:29395-29404 (1994); Hughs et al., “Gene transfer of cytidine deaminase APOBEC-1 lowers lipoprotein(a) in transgenic mice and induces apolipoprotein B mRNA editing in rabbits,” Hum. Gene Ther. 7:39-49 (1996); Kozarsky et al., “Hepatic expression of the catalytic subunit of the apolipoprotein B mRNA editing enzyme ameliorates hypercholesterolemia in LDL receptor-deficient rabbits,” Hum. Gene Ther. 7:943-957 (1996); Farese et al., “Phenotypic analysis of mice expressing exclusively apolipoprotein B48 or apolipoprotein B100,” Proc. Natl. Acad. Sci. USA 93:6393-6398 (1996); Qian et al., “Low expression of the apolipoprotein B mRNA editing transgene in mice reduces LDL but does not cause liver dysplasia or tumors,” Arteriosc. Thromb. Vasc. Biol. 18:1013-1020 (1998); Wu et al., “Normal perinatal rise in serum cholesterol is inhibited by hepatic delivery of adenoviral vector expressing apolipoprotein B mRNA editing enzyme in rabbits,” J. Surg. Res. 85:148-157 (1999), each of which is hereby incorporated by reference in its entirety). Interestingly, apobec-1 gene transfer into apobec-1 gene knockout mice restored editing and reduced serum LDL levels (Nakamuta et al., “Complete phenotypic characterization of the apobec-1 knockout mice with a wild-type genetic background and a human apolipoprotein B transgenic background, and restoration of apolipoprotein B mRNA editing by somatic gene transfer of Apobec-1,” J. Biol. Chem. 271:25981-25988 (1996), which is hereby incorporated by reference in its entirety), demonstrating that APOBEC-1 has therapeutic potential in livers with no prior editing activity. The induction of hepatic editing of apolipoprotein B mRNA in apobec-1 transgenic rabbits with an LDL receptor deficiency also ameliorated hypercholesterolemia (Kozarsky et al., “Hepatic expression of the catalytic subunit of the apolipoprotein B mRNA editing enzyme ameliorates hypercholesterolemia in LDL receptor-deficient rabbits,” Hum. Gene Ther. 7:943-957 (1996), which is hereby incorporated by reference in its entirety). Taken together, these studies suggested that apolipoprotein B mRNA editing could be safely targeted as a mechanism for reducing serum LDL and the risk of atherogenic diseases.
However, controlling a low level of apobec-1 expression using gene therapy is difficult and, quite often, unpredictable. For all of these reasons, despite the limited success of gene therapy approaches, gene therapy using apobec-1 does not appear to be a promising avenue which can be pursued for preventative or therapeutic control over atherogenic disease factors. The advantage of protein transduction therapy is that the dose can be modulated relative to the desired response and that the effect on editing can be terminated by withdrawing therapy.
The PTD should allow protein to enter all cells of the body, even if the protein is delivery intravenously (Schwarze et al., “In vivo protein transduction: delivery of a biologically active protein into the mouse,” Science 285:1569-1572 (1999), which is hereby incorporated by reference in its entirety). Ideally the liver should be specifically targeted with TAT-rAPOBEC-CMPK and an intraperitoneal injection can be utilized to accomplish a first pass clearance, transducing most of the protein into hepatocytes. Even though APOBEC-1 is not widely expressed in tissues (Teng et al., “Molecular cloning of an apo B messenger RNA editing protein,” Science 260:18116-1819 (1993), which is hereby incorporated by reference in its entirety), its generalized expression in transgenic animals did not induce pathology (Teng et al., “Adenovirus-mediated gene transfer of rat apolipoprotein B mRNA editing protein in mice virtually eliminates apolipoprotein B-100 and normal low density lipoprotein production,” J. Biol. Chem. 269:29395-29404 (1994); Hughs et al., “Gene transfer of cytidine deaminase APOBEC-1 lowers lipoprotein(a) in transgenic mice and induces apolipoprotein B mRNA editing in rabbits,” Hum. Gene Ther. 7:39-49 (1996); Kozarsky et al., “Hepatic expression of the catalytic subunit of the apolipoprotein B mRNA editing enzyme ameliorates hypercholesterolemia in LDL receptor-deficient rabbits,” Hum. Gene Ther. 7:943-957 (1996); Farese et al., “Phenotypic analysis of mice expressing exclusively apolipoprotein B48 or apolipoprotein B100,” Proc. Natl. Acad. Sci. USA 93:6393-6398 (1996); Qian et al., “Low expression of the apolipoprotein B mRNA editing transgene in mice reduces LDL but does not cause liver dysplasia or tumors,” Arteriosc. Thromb. Vasc. Biol. 18:1013-1020 (1998); Wu et al., “Normal perinatal rise in serum cholesterol is inhibited by hepatic delivery of adenoviral vector expressing apolipoprotein B mRNA editing enzyme in rabbits,” J. Surg. Res. 85:148-157 (1999), each of which is hereby incorporated by reference in its entirety).
Uptake of TAT-rAPOBEC-CMYK or TAT-hAPOBEC-CMPK is unlikely to induce any side effects. Aside from one study suggesting that overexpression of APOBEC-1 in liver can lead to editing of mRNAs other than apolipoprotein B (Yamanaka et al., “A novel translational repressor mRNA is edited extensively in livers containing tumors caused by the transgene expression of the apoB mRNA editing enzyme,” Genes & Dev. 11:321-333 (1997), which is hereby incorporated by reference in its entirety) no other mRNA substrates for APOBEC-1 have been found (Skuse et al., “Neurofibromatosis type I mRNA undergoes base-modification RNA editing,” Nucleic Acids Res. 24:478-486 (1996); Sowden et al., “Apolipoprotein B RNA Sequence 3′ of the mooring sequence and cellular sources of auxiliary factors determine the location and extent of promiscuous editing,” Nucleic Acids Res. 26:1644-1652 (1998), each of which is hereby incorporated by reference in its entirety). Furthermore, apobec-1 gene knock out studies have shown that there were no other editing enzymes capable of editing apolipoprotein B mRNA and that APOBEC-1 was not required for life (Hirano et al., “Targeted disruption of the mouse apobec-1 gene abolishes apolipoprotein B mRNA editing and eliminates apolipoprotein B48,” J. Biol. Chem. 271:9887-9890 (1996); Nakamuta et al., “Complete phenotypic characterization of the apobec-1 knockout mice with a wild-type genetic background and a human apolipoprotein B transgenic background, and restoration of apolipoprotein B mRNA editing by somatic gene transfer of Apobec-1,” J. Biol. Chem. 271:25981-25988 (1996), each of which is hereby incorporated by reference in its entirety). Taken together the data suggest that mRNA editing by APOBEC is self-limited due to its specificity for apolipoprotein B mRNA and, therefore, neither TAT-rAPOBEC-CMPK nor TAT-hAPOBEC-CMPK is likely to have effects in tissues other than those which express apolipoprotein B mRNA and auxiliary proteins.
Current cholesterol-lowering therapies target circulating cholesterol at the level of enhanced elimination or reduced production. A sector of the population remains at risk for atherosclerosis due to side effects from current therapies in some of these patients and the inability of others with defects in apolipoprotein B and/or the LDL receptor mediated uptake pathway to completely benefit from conventional cholesterol lowering therapies. Hypercholesterolemia is an early onset disease yet the restricted usage of conventional therapies among children due to the potential of interfering with pubertal development has not been resolved. Protein based therapies such as insulin or growth hormone have been extensively used among children to treat Type I diabetes or pituitary dwarfism, respectively. To the patient or the parent of the patient, the reversible nature of protein based therapy may be more appealing than gene therapy. To this end, the above results illustrate an alternative to conventional or gene therapy approaches for reducing the risk of atherosclerosis in the sectors of population at risk.
Although preferred embodiments have been depicted and described in detail herein, it will be apparent to those skilled in the relevant art that various modifications, additions, substitutions, and the like can be made without departing from the spirit of the invention and these are therefore considered to be within the scope of the invention as defined in the claims which follow.
This application claims the benefit of U.S. Provisional Patent Application Ser. No. 60/271,856, filed Feb. 27, 2001, which is hereby incorporated by reference in its entirety.
This invention was made, at least in part, using funding received from the U.S. Public Health Service, grant DK43739. The U.S. government may have certain rights in this invention.
| Number | Date | Country | |
|---|---|---|---|
| 60271856 | Feb 2001 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 10468987 | Jan 2004 | US |
| Child | 12784310 | US |
