The present application provides GB virus C sequences (GBV-C or hepatitis G virus) and methods of using the sequences.

SUMMARY OF THE INVENTION

The compositions and methods of the present invention take advantage of the discovery of an isolated and purified nucleic acid molecule encoding an infectious GBV-C.

"Isolated and purified" indicates the nucleic acid molecule is not part of an intact GBV-C virus. These nucleic acid molecules have been produced in the form of a DNA construct or expression construct, as well as an infectious full-length GBV-C RNA transcript expressed from the DNA construct (collectively referred to as "recombinant GBV-C"). A cDNA clone made from the full-length or a less-than full-length transcript is also contemplated within the scope of the invention.

A nucleic acid sequence of the entire GBV-C genome is represented by SEQ ID NO:19, which corresponds to Genbank accession number AY196904. The sequence is provided as a DNA sequence, however, it may also be an RNA sequence with the corresponding thymidines (T) being substituted with uracils (U). The protein sequence encoded by this nucleic acid sequence is represented in SEQ ID NO:20. While nucleic acid molecules comprising all of SEQ ID NO: 19 are contemplated, smaller transcripts and clones containing less than the full-length of GBV-C sequences are considered within the present invention; particularly useful are transcripts or clones containing less than the entire-GBV-C sequence but also capable of producing an infectious GBV-C virus particle. Such viral particles made by recombinant techniques would be considered "recombinant." Viruses isolated from serum, for example, whose genome has not been altered recombinantly are considered non-recombinant. While the present invention is directed at recombinant forms of GVB-C, in some methods of the invention, non-recombinant viruses may be used as recombinant viruses.

In some embodiments of the invention, infectious GBV-C nucleic acid molecules and GBV-C viral particles produced from these molecules contain heterologous nucleic acid sequences. These heterologous sequences encode non-GBV-C sequences. For example, the heterologous sequence could encode HCV sequences, such that a chimeric virus is produced. GBV-C can be used a viral vector to provide a cell with an exogenous nucleic acid sequence. Alternatively, the compositions of the invention may be used as a vaccine to evoke an immune response against either GBV-C or a polypeptide or polypeptides encoded by heterologous sequences in the GBV-C nucleic acid molecules. These heterologous sequences could encode any sequence with therapeutic, preventative, or diagnostic functions. They could encode for antisense sequences, ribozymes, peptides, or polypeptides. Furthermore, they could be derived from non-GBV-C viruses, prokaryotes, or eukaryotes, such as mammals, or even humans. Transcription of a heterologous sequence may be controlled by a regulatory region, such as a promoter and/or enhancer, that is from GBV-C or a heterologous region. In some cases, the control region may be endogenous to the host cell, or it may be the control region that is normally associated with the heterologous sequence. The promoter and enhancers for use with the present invention may be eukaryotic, such as from a mammal, or it may be prokaryotic, such as T3, T7, and Sp6, or viral. In a further embodiment, the infectious GBV-C nucleic acid molecule exhibits resistance to interferon.

The present invention is also directed to methods of preparing or producing an infectious GBV-C. In some embodiments, an infectious GBV-C is prepared by incubating a nucleic acid molecule containing GBV-C sequence under conditions effective to allow transcription of at least a portion of the GBV-C sequence, collecting the RNA transcript, and providing the RNA transcript to a cell. A cell, which can be a prokaryotic or eukaryotic cell, can be provided with the transcript by a number of ways, including transfection methods, which are well known to those of skill in the art. In other methods, the transfected cell is incubated in appropriate media with or without serum to allow the cell to live. In any of the methods of the present invention, the cell may be prokaryotic or eukaryotic; the cell may be a mammalian cell. In other examples, the cell is a lymphocyte, while in still further examples, the cell is a CD4+ lymphocyte cell. A lymphocyte cell may be a T cell or a B cell. In the methods of the present invention, after sufficient time to allow the virus to propagate has passed, the supernatant can be collected from the cell. Any of the compositions described above and herein may be used to prepare infectious GBV-C. Any and all progeny GBV-C particles produced using the method and compositions of the present invention are encompassed by the invention.

In further aspects of the present invention, methods of producing infectious GBV-C are provided. Such methods may be accomplished by providing to a cell any composition of the present invention such as an isolated and purified nucleic acid molecule encoding an infectious GBV-C, or a GBV-C produced from such a molecule. In some cases the molecule will further comprise a heterologous sequence, with or without a heterologous, exogenous, or endogenous promoter. This cell may then be incubated under conditions that will permit replication and/or integration of viral nucleic acid molecules encoding an infectious GBV-C. The transfected or infected cell will eventually produce viral particles that can be collected from the supernatant.

The methods of the present invention may also include the steps of taking the supernatant from an infected or transfected cell and contacting a second cell with the supernatant of the first infected or transfected cell; incubating the second cell under conditions to permit replication of a GBV-C viral genome; and collecting the supernatant from the second cell.

The invention includes methods of expressing a heterologous nucleic acid sequence by providing to a cell an isolated and purified nucleic acid molecule encoding an infectious GBV-C sequence and the heterologous nucleic acid sequence. These methods can be utilized in vitro or in vivo.

Because the compositions of the invention can comprise a heterologous sequence encoding a polypeptide, they can be used to produce an immune response as well as antibodies in a subject given these compositions. For example, methods of producing an immune response in a subject can be accomplished by administering to the subject an effective amount of an expression construct comprising GBV-C sequences and a heterologous nucleic acid sequence operably linked to a promoter, such that the heterologous nucleic acid sequence encodes a polypeptide that elicits an immune response against the polypeptide.

In other methods of the invention, HIV disease progression (AIDS) is inhibited or reduced in a subject infected with HIV. This can be accomplished by administering to the subject an effective amount of an isolated and purified nucleic acid molecule encoding an infectious GBV-C sequence. The nucleic acid molecule may be RNA or DNA, or it may be a virus produced by such an isolated and purified nucleic acid molecule. The molecule may also contain the sequence of SEQ ID NO:1, be 9.3-9.7 kb in length, or comprise a portion of SEQ ID NO:1. These methods may be implemented in conjunction with other AIDS treatments such as AZT, HAART, or at least one protease inhibitor. Alternatively, these methods can be used to prevent HIV infection in an uninfected subject as well. Such methods could be employed by administering an effective amount of an isolated and purified nucleic acid molecule encoding an infectious GBV-C to a subject. This could be used to prevent HIV infection of a person's CD4+ cell.

Other embodiments of the invention include methods of treating a subject infected with HIV comprising administering to a cell of the subject an effective amount of an infectious GBV-C comprising a heterologous nucleic acid sequence. The method may be practiced in vitro or in vivo. If cells are treated in vitro, they may then be placed in a subject. In some embodiments, a recombinant infectious GVB-C is employed, while in others non-recombinant GVB-C is employed.

Methods of treating a subject infected with HIV may be implemented according to the present invention by administering to the subject an effective amount of an expression construct comprising a GBV-C sequence, such that the subject is provided a therapeutic benefit. Other ways of practicing the treatment methods of the invention include administering to the subject other AIDS treatments before, after, or concurrently with the expression construct. In some embodiments, a cell infected with HIV is contacted with an isolated and purified nucleic acid molecule comprising a nucleic acid sequence of SEQ ID NO:1, such that all or part of a GBV-C polypeptide is expressed in the cell. The polypeptide may cause HIV replication to be inhibited. It is contemplated that the isolated and purified nucleic acid molecule may encode an infectious GBV-C and inhibit HIV replication in the HIV-infected cell. The cell may be in an animal, such as a human. It is further contemplated that the cell may be one typically infected by HIV such as a CD4+ cell. Traditional AIDS therapy such as AZT or a protease inhibitor or HAART may be implemented in combination with any of the treatment methods described herein.

In other embodiments, a subject may be evaluated for cytokine inductions. Cytokine levels in a subject may be assayed before and/or after exposure to an infectious GVB-C sequence. In some embodiments, IL-2, IL-1B, IL-8, or IL-15 may be assayed by techniques well known to those of skill in the art.

The cells of the various methods may be eukaryotic or prokaryotic. In some cases, the cells are mammalian. In other cases, the cells are lymphocytes or are PBMCs. Alternatively, the cells may be CD4+. In still further embodiments, the cell may be any cell that supports the infection and/or propagation of a GBV-C, such as HepG2, Daudi, MT-2, and PH5CH cells. Cells may also be sustained in culture or in an organism.

In any of the compositions or methods of the present invention, a heterologous sequence may be comprised within a nucleic acid molecule encoding GBV-C sequences. The heterologous sequence is any nucleic acid sequence that does not encode GBV-C sequences. It may encode more than one gene or regulatory region. A heterologous sequence may encode an untranslated RNA such as an antisense construct or ribozyme, or a polypeptide that has therapeutic, preventative, or diagnostic uses. It may also encode a selectable or screenable marker by itself or in conjunction with another heterologous coding region. The untranslated RNA or polypeptide may be derived from an eukaryote, prokaryote, or virus. Examples of RNA and polypeptides encoded by heterologous sequences are provided below, but the invention should not be limited to those examples.

It is also contemplated that the invention covers all subsequent generations of GBV-C produced using the compositions and methods of the present invention. For example, if an isolated and purified nucleic acid molecule encoding a GBV-C virus is introduced into a cell such that the cell produces infectious GBV-C particles (first generation), the invention covers not only the particles, but also the viruses produced from the first generation particles, which would include viruses from generations 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, and later.

In other embodiments of the invention, an effective amount of an infectious GVB-C may be administered to cells or a subject to induce expression of IL-2, IL-1B, IL-8, or IL-15. Alternatively, an effective amount of an infectious GBV-C may be administered to reduce or inhibit the expression of IL-13. It is contemplated that GBV-C may be administered to cells or a subject to alter the cytokines listing in FIG. 14 (see Original Patent).

In some embodiments of the invention, an infectious GBV-C can be used to inhibit or prevent apoptosis in a cell. An effective amount of GBV-C may be administered to a cell in vitro or in vivo to prevent or delay apoptosis.

The use of the word "a" or "an" when used in conjunction with the term "comprising" in the claims and/or the specification may mean "one," but it is also consistent with the meaning of "one or more," "at least one," and "one or more than one." Furthermore, where multiple steps of a method of process are cited, it is understood that the steps are not required to be performed in the particular order recited unless one of skill in the art is not be able to practice the method in a different order.

The DNA of the invention may encode GBV-C 765. The vaccine may also contain an immunological adjuvant. The terms "protein," "peptide" and "polypeptide" are used interchangeably herein.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

While several full-length GBV-C sequences have been submitted to Genbank, the present invention provides a cDNA clone encoding full-length GBV-C RNA transcripts that are infectious in peripheral blood mononuclear cell (PBMC) cultures. Unlike most previous full-length HCV constructions, the present invention is not a product of a consensus sequence, but instead, an authentic GBV-C sequence obtained from direct amplification of viral RNA. Replication was demonstrated by serial passage of culture supernatants, expression of the envelope glycoprotein E2, RNA replication (positive and negative strand RNA synthesis), and the detection of viral particles by sucrose gradient centrifugation and immune electron microscopy.

Thus, the present invention concerns the discovery of an infectious GBV-C clone. GBV-C is the most closely related virus to HCV, the cause of hepatitis C.

It was recently reported that GBV-C infection in HIV-infected patients correlates with a delayed onset of HIV (LeFrere et al., 1999). This report, however, is in conflict with a previous study by Sabin et al. (Sabin et al., 1998). Thus, there existed confusion in the art at this time about whether or not there is a correlation between the onset of AIDS and a GBV-C infection. Furthermore, any evidence that did exist was merely correlative, as opposed to involving an evaluation of whether GBV-C was involved in the observed delay of HIV.

The experiments described herein demonstrate a correlation. Unlike the previously reported studies, only subjects with viremia, as demonstrated by the detection of GBV-C RNA by RT-PCR, were evaluated; people with GBV-C anti-E2 antibody were not included. The studies disclosed herein also show that GBV-C inhibits HIV replication, but they significantly extend the observation of a correlation and provide a mechanism by which GBV-C delays the onset of HIV. Since this study employed an infectious GBV-C clone, they further indicate the advantage of the GBV-C clones of the present invention. Therefore, an infectious GBV-C of the present invention can be implemented in preventative or therapeutic treatments for HIV infection and the development of AIDS.

I. GBV-C

Like other members of the Flaviviridae, GBV-C is a positive-strand RNA virus that encodes a single long open reading frame (Leary et al., 1996). As discussed above, it does not cause acute or chronic hepatitis, yet it is the family member most closely related to HCV, the cause of hepatitis C. While sequences of GBV-C have been previously reported, for example in U.S. Pat. No. 5,874,563, an infectious GBV-C clone has not been previously described.

The GBV-C polyprotein is predicted to be cleaved into two envelope proteins (E1 and E2), an RNA helicase, a trypsin-like serine protease, and an RNA-dependent RNA polymerase. A major difference between GBV-C and HCV is in the amino terminus of the polyprotein. In many isolates, this region is truncated, and no core (or nucleocapsid) protein is present (Simons et al., 1995; Xiang et al., 1999). In vitro translation experiments suggest that the AUG immediately upstream of the putative E1 protein is preferentially used to initiate translation, although there may be as many as four AUG's in frame with the polyprotein upstream of this AUG (Simons et al., 1996). In addition, the mutation frequency in codon position 1 and 2 of the region upstream of this AUG suggest that it is a non-coding region (Okamoto et al., 1997). These observations have led to speculation that GBV-C may not have a core protein or nucleocapsid (Dickens and Lenon, 1997; Simons et al., 1996). However, the inventors and others have shown that the sedimentation profiles of GBV-C particles are consistent with the presence of a nucleocapsid (Melvin et al., 2000; Xiang et al., 1998), and electron microscopy of plasma-derived GBV-C demonstrated enveloped particles with a nucleocapsid structure (Xiang et al., 1999). Although the amino acid composition of the nucleocapsid remains undefined, some infected individuals have antibody to a peptide representing amino acids upstream of the predicted E1 protein in frame with the polyprotein (Xiang et al., 1998). Thus, this region may encode the nucleocapsid.

Simons et al., 1996 demonstrated that the AUG codon at the amino terminus of putative E1 protein (AUG-554 in the isolate) was capable of initiating translation, whereas the upstream AUG's were not (Simons et al., 1996). In many isolates, the amino terminus of the predicted HGV polyprotein is truncated or absent (Leary et al., 1996; Linnen et al., 1996; Okamoto et al., 1997), and the frequency of polymorphisms in codon position 1 and 2 in the upstream ORF suggests that the region is not a coding region (Okamoto et al., 1997). Thus, it has been suggested that GBV-C may not have a core protein (Dickens and Lemon, 1997). It was previously shown that GBV-C particles have similar densities and sedimentation characteristics in sucrose and cesium chloride gradients as HCV (Xiang et al., 1998); subsequently particles of approximately 65 nm particle were shown with a 50 nm nucleocapsid structure (Xiang et al., 1999). In this study, two GBV-C particles types were identified with densities of 1.07 and 1.18 g/ml, consistent with virions and nucleocapsids respectively (Xiang et al., 1998). Furthermore, electron dense structures approximately 50-55 nm in size were visualized within the enveloped particle. Thus, the data support previous work identifying a nucleocapsid for GBV-C. The truncation of the polyprotein upstream of AUG-554 would be abolished if most isolates did not contain a single nucleotide deletion at position 381. Given the fact that all sequences produced thus far utilized nested RT-PCR, this may represent a polymerase artifact. Nevertheless, propagation of GBV-C in culture should allow the production of sufficient virus for ultimate characterization of the protein content of the GBV-C nucleocapsid. With the exception of the 5' ntr region, the remaining GBV-C sequences are highly conserved among geographically diverse isolates. Although there is less than 50% sequence homology in the 3' ntr region between GBV-C, GBV-B and HCV, the predicted secondary structures of these viruses bear striking similarities. GBV-C does not include a polypyrimidine tract, but does have three stem-loop structures at the extreme 3' end. This indicates that the polypyrimidine regions of HCV and GBV-B are not requirements of hepacivirus replication.

The site of GBV-C replication has not been clearly identified, but it appears that replication in the hepatocyte, if it occurs, is not the primary source of virus in infected individuals (Laskus et al., 1998; Pessoa et al., 1998; Seipp et al., 1999). Recently, there were reports that human peripheral blood mononuclear cells (PBMC's) and interferon-resistant Daudi cells are permissive for GBV-C replication (Fogeda et al., 1999; Shimizu, 1999). In addition, transient replication of GBV-C was described in MT-2 cells (a human T-cell line), and PH5CH (a human hepatocyte line immortalized with simian virus 40 large T antigen) (Seipp et al., 1999).

A. Polynucleotides

1. Infectious GBV-C

The present invention concerns infectious GBV-C polynucleotides or nucleic acid molecules, isolatable and purifiable from GBV-C or mammalian cells infected with GBV-C, indicating they are free from total viral genomic RNA and proteins and are capable of infecting cells and propagating infectious GBV-C particles. It is contemplated that an isolated and purified infectious GBV-C nucleic acid molecule may take the form of RNA or DNA. An infectious GBV-C nucleic acid molecule refers to an RNA or DNA molecule that is capable of yielding an infectious GBV-C particle from a transfected cell.

As used herein, the term "RNA transcript" refers to an RNA molecule that has been isolated free of total genomic viral RNA and virus proteins and that is the product of transcription from a nucleic acid molecule for which at least one strand is DNA. A "full-length RNA transcript" refers to an RNA transcript that is full-length when compared to the genomic coding region, for example of GBV-C. Therefore, a full-length GBV-C RNA transcript encoding the GBV-C genome refers to an RNA segment that contains GBV-C sequences capable of producing an infectious GBV-C, yet is isolated away from, or purified free from, total GBV-C viral genomic RNA and GBV-C proteins. Such a full-length transcript may encode for one or more polypeptides, as well as contain regions controlling the regulation, e.g., transcription, translation, and RNA stability, of these polypeptides.

As used in this application, the term "polynucleotide" refers to a nucleic acid molecule, RNA or DNA, that has been isolated free of total genomic nucleic acid. Therefore, a "polynucleotide encoding an infectious GBV-C" refers to a nucleic acid segment that contains GBV-C coding sequences, yet is isolated away from, or purified and free of, total viral genomic RNA and proteins; similarly, a "polynucleotide encoding full-length GBV-C" refers to a nucleic acid segment that contains full-length GBV-C coding sequences yet is isolated away from, or purified and free of, total viral genomic RNA and protein. Therefore, when the present application refers to the function or activity of an infectious GBV-C that is encoded by a GBV-C polynucleotide, it is meant that the polynucleotide encodes a molecule that has the ability to propagate an infectious GBV-C virus particle from a cell. It is contemplated that an infectious GBV-C polynucleotide may refer to a GBV-C RNA transcript that is able to propagate an infectious GBV-C virus particle after introduction to a cell or to a GBV-C expression construct, clone, or vector composed of double-stranded DNA or DNA/RNA hybrid that is similarly capable.

The term "cDNA" is intended to refer to DNA prepared using RNA as a template. The advantage of using a cDNA, as opposed to genomic RNA or an RNA transcript is stability and the ability to manipulate the sequence using recombinant DNA technology (See Maniatis, 1989; Ausubel, 1994). There may be times when the full or partial genomic sequence is preferred. Alternatively, cDNAs may be advantageous because it represents coding regions of a polypeptide and eliminates introns and other regulatory regions.

It also is contemplated that a given GBV-C from a given cell may be represented by natural variants or strains that have slightly different nucleic acid sequences but, nonetheless, encode the same viral polypeptides. Consequently, the present invention also encompasses derivatives of GBV-C with minimal amino acid changes in its viral proteins, but that possess the same activities.

The term "gene" is used for simplicity to refer to a functional protein, polypeptide, or peptide-encoding unit. As will be understood by those in the art, this functional term includes genomic sequences, cDNA sequences, and smaller engineered gene segments that express, or may be adapted to express, proteins, polypeptides, domains, peptides, fusion proteins, and mutants. The nucleic acid molecule encoding GBV-C may contain a contiguous nucleic acid sequence encoding one or more GBV-C genes and regulatory regions and be of the following lengths: about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 441, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, 1000, 1010, 1020, 1030, 1040, 1050, 1060, 1070, 1080, 1090, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900, 4000, 4100, 4200, 4300, 4400, 4500, 4600, 4700, 4800, 4900, 5000, 5100, 5200, 5300, 5400, 5500, 5600, 5700, 5800, 5900, 6000, 6100, 6200, 6300, 6400, 6500, 6600, 6700, 6800, 6900, 7000, 7100, 7200, 7300, 7400, 7500, 7600, 7700, 7800, 7900, 8000, 8100, 8200, 8300, 8400, 8500, 8600, 8700, 8800, 8900, 9000, 9100, 9200, 9300, 9400, 9500, 9600, 9700, 9800, 9900, 10000, 10100, 10200, 10300, 10400, 10500, 10600, 10700, 10800, 10900, 11000, 11100, 11200, 11300, 11400, 11500, 11600, 11700, 11800, 11900, 12000 or more nucleotides, nucleosides, or base pairs. Such sequences may be identical or complementary to SEQ ID NO:1 or Genbank Accession number AF070476.

"Isolated substantially away from other coding sequences" means that the gene of interest forms part of the coding region of the nucleic acid segment, and that the segment does not contain large portions of naturally-occurring coding nucleic acid, such as large chromosomal fragments or other functional genes or cDNA coding regions. Of course, this refers to the nucleic acid segment as originally isolated, and does not exclude genes or coding regions later added to the segment by human manipulation.

In particular embodiments, the invention concerns isolated nucleic acid segments and recombinant vectors incorporating DNA sequences that encode GBV-C polypeptides or peptides that include within its amino acid sequence a contiguous amino acid sequence in accordance with, or essentially corresponding to GBV-C polypeptides.

In other embodiments, the invention concerns isolated nucleic acid segments and DNA recombinant vectors incorporating nucleic acid sequences that encode GBV-C polypeptides or peptides, particularly those necessary for infection, that include within its amino acid sequence a contiguous amino acid sequence in accordance with, or essentially corresponding to all strains of GBV-C polypeptides.

The nucleic acid segments used in the present invention, regardless of the length of the coding sequence itself, may be combined with other DNA or RNA sequences, such as promoters, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, other coding segments, and the like, such that their overall length may vary considerably. It is therefore contemplated that a nucleic acid fragment of almost any length may be employed, with the total length preferably being limited by the ease of preparation and use in the intended recombinant DNA protocol.

It is contemplated that the nucleic acid constructs of the present invention may encode full-length GBV-C or an infectious GBV-C, with or without heterologous sequences. A "heterologous" sequence refers to a sequence that is foreign or exogenous to the remaining sequence. A heterologous gene refers to a gene that is not found in nature adjacent to the sequences with which it is now placed.

In a non-limiting example, one or more nucleic acid constructs may be prepared that include a contiguous stretch of nucleotides identical to or complementary to GBV-C. A nucleic acid construct may be about 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1,000, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, 11,000, 12,000, 13,000, 14,000, 15,000, 20,000, 30,000, 50,000, 100,000, 250,000, about 500,000, 750,000, to about 1,000,000 nucleotides in length, as well as constructs of greater size, up to and including chromosomal sizes (including all intermediate lengths and intermediate ranges), given the advent of nucleic acids constructs such as a yeast artificial chromosome are known to those of ordinary skill in the art. It will be readily understood that "intermediate lengths" and "intermediate ranges," as used herein, means any length or range including or between the quoted values (i.e., all integers including and between such values). Non-limiting examples of intermediate lengths include about 11, about 12, about 13, about 16, about 17, about 18, about 19, etc.; about 21, about 22, about 23, etc.; about 31, about 32, etc.; about 51, about 52, about 53, etc.; about 101, about 102, about 103, etc.; about 151, about 152, about 153, about 97001, about 1,001, about 1002, about 50,001, about 50,002, about 750,001, about 750,002, about 1,000,001, about 1,000,002, etc. Non-limiting examples of intermediate ranges include about 3 to about 32, about 150 to about 500,001, about 3,032 to about 7,145, about 5,000 to about 15,000, about 20,007 to about 1,000,003, etc.

The nucleic acid segments used in the present invention encompass biologically functional equivalent GBV-C proteins and peptides. Such sequences may arise as a consequence of codon redundancy and functional equivalency that are known to occur naturally within nucleic acid sequences and the proteins thus encoded. Alternatively, functionally equivalent proteins or peptides may be created via the application of recombinant DNA technology, in which changes in the protein structure may be engineered, based on considerations of the properties of the amino acids being exchanged. Changes designed by human may be introduced through the application of site-directed mutagenesis techniques, e.g., to introduce improvements to the antigenicity of the protein or to test mutants in order to examine DNA binding activity at the molecular level.

2. Vectors Encoding Infectious GBV-C

The present invention encompasses the use of vectors to encode for an infectious GBV-C. The term "vector" is used to refer to a carrier nucleic acid molecule into which a nucleic acid sequence can be inserted for introduction into a cell where it can be replicated. A nucleic acid sequence can be "exogenous," which means that it is foreign to the cell into which the vector is being introduced or that the sequence is homologous to a sequence in the cell but in a position within the host cell nucleic acid in which the sequence is ordinarily not found. Vectors include plasmids, cosmids, viruses (bacteriophage, animal viruses, and plant viruses), and artificial chromosomes (e.g., YACs). One of skill in the art would be well equipped to construct a vector through standard recombinant techniques, which are described in Maniatis et al., 1988 and Ausubel et al., 1994.

The term "expression vector" or "expression construct" refers to a vector containing a nucleic acid sequence coding for at least part of a gene product capable of being transcribed. In some cases, RNA molecules are then translated into a protein, polypeptide, or peptide. In other cases, these sequences are not translated, for example, in the production of antisense molecules or ribozymes. Expression vectors can contain a variety of "control sequences," which refer to nucleic acid sequences necessary for the transcription and possibly translation of an operably linked coding sequence in a particular host organism. In addition to control sequences that govern transcription and translation, vectors and expression vectors may contain nucleic acid sequences that serve other functions as well and are described infra. It is contemplated that the infectious GBV-C particles of the present invention may arise from a vector containing GBV-C sequence or RNA encoding GBV-C sequence into a cell. Either of these, or any other nucleic acid molecules of the present invention may be constructed with any of the following nucleic acid control sequences. Thus, the full-length RNA transcript may contain the benefit of recombinant DNA technology such that it contains exogenous control sequences or genes.

a. Promoters and Enhancers

A "promoter" is a control sequence that is a region of a nucleic acid sequence at which initiation and rate of transcription are controlled. It may contain genetic elements at which regulatory proteins and molecules may bind such as RNA polymerase and other transcription factors. The phrases "operatively positioned," "operatively linked," "under control," and "under transcriptional control" mean that a promoter is in a correct functional location and/or orientation in relation to a nucleic acid sequence to control transcriptional initiation and/or expression of that sequence. A promoter may or may not be used in conjunction with an "enhancer," which refers to a cis-acting regulatory sequence involved in the transcriptional activation of a nucleic acid sequence.

A promoter may be one naturally associated with a gene or sequence, as may be obtained by isolating the 5' non-coding sequences located upstream of the coding segment and/or exon. Such a promoter can be referred to as "endogenous." Similarly, an enhancer may be one naturally associated with a nucleic acid sequence, located either downstream or upstream of that sequence. Alternatively, certain advantages will be gained by positioning the coding nucleic acid segment under the control of a recombinant or heterologous promoter, which refers to a promoter that is not normally associated with a nucleic acid sequence in its natural environment. A recombinant or heterologous enhancer refers also to an enhancer not normally associated with a nucleic acid sequence in its natural environment. Such promoters or enhancers may include promoters or enhancers of other genes, and promoters or enhancers isolated from any other prokaryotic, viral, or eukaryotic cell, and promoters or enhancers not "naturally occurring," i.e., containing different elements of different transcriptional regulatory regions, and/or mutations that alter expression. In addition to producing nucleic acid sequences of promoters and enhancers synthetically, sequences may be produced using recombinant cloning and/or nucleic acid amplification technology, including PCR.TM., in connection with the compositions disclosed herein (see U.S. Pat. No. 4,683,202, U.S. Pat. No. 5,928,906). Furthermore, it is contemplated the control sequences that direct transcription and/or expression of sequences within non-nuclear organelles such as mitochondria, chloroplasts, and the like, can be employed as well.

Naturally, it will be important to employ a promoter and/or enhancer that effectively directs the expression of the nucleic acid segment in the cell type, organelle, and organism chosen for expression. Those of skill in the art of molecular biology generally know the use of promoters, enhancers, and cell type combinations for protein expression, for example, see Sambrook et al. (1989). The promoters employed may be constitutive, tissue-specific, inducible, and/or useful under the appropriate conditions to direct high level expression of the introduced DNA segment, such as is advantageous in the large-scale production of recombinant proteins and/or peptides. The promoter may be heterologous or exogenous, i.e., from a different source than GBV-C sequence. In some examples, a prokaryotic promoter is employed for use with in vitro transcription of a desired sequence. Prokaryotic promoters for use with many commercially available systems include T7, T3, and Sp6.

Table 1 (see Original Patent) lists several elements/promoters that may be employed, in the context of the present invention, to regulate the expression of a gene. This list is not intended to be exhaustive of all the possible elements involved in the promotion of expression but, merely, to be exemplary thereof. Table 2 (see Original Patent) provides examples of inducible elements, which are regions of a nucleic acid sequence that can be activated in response to a specific stimulus.

The identity of tissue-specific promoters or elements, as well as assays to characterize their activity, is well known to those of skill in the art. Examples of such regions include the human LIMK2 gene (Nomoto et al. 1999), the somatostatin receptor 2 gene (Kraus et al., 1998), murine epididymal retinoic acid-binding gene (Lareyre et al., 1999), human CD4 (Zhao-Emonet et al., 1998), mouse alpha2 (XI) collagen (Tsumaki, et al., 1998), D1A dopamine receptor gene (Lee, et al., 1997), insulin-like growth factor II (Wu et al., 1997), human platelet endothelial cell adhesion molecule-1 (Almendro et al., 1996).

b. Initiation Signals and Internal Ribosome Binding Sites

A specific initiation signal also may be required for efficient translation of coding sequences. These signals include the ATG initiation codon or adjacent sequences. Exogenous translational control signals, including the ATG initiation codon, may need to be provided. One of ordinary skill in the art would readily be capable of determining this and providing the necessary signals. It is well known that the initiation codon must be "in-frame" with the reading frame of the desired coding sequence to ensure translation of the entire insert. The exogenous translational control signals and initiation codons can be either natural or synthetic. The efficiency of expression may be enhanced by the inclusion of appropriate transcription enhancer elements.

In certain embodiments of the invention, the use of internal ribosome entry sites (IRES) elements are used to create multigene, or polycistronic, messages. IRES elements are able to bypass the ribosome scanning model of 5' methylated Cap dependent translation and begin translation at internal sites (Pelletier and Sonenberg, 1988). IRES elements from two members of the picornavirus family (polio and encephalomyocarditis) have been described (Pelletier and Sonenberg, 1988), as well an IRES from a mammalian message (Macejak and Sarnow, 1991). IRES elements can be linked to heterologous open reading frames. Multiple open reading frames can be transcribed together, each separated by an IRES, creating polycistronic messages. By virtue of the IRES element, each open reading frame is accessible to ribosomes for efficient translation. Multiple genes can be efficiently expressed using a single promoter/enhancer to transcribe a single message (see U.S. Pat. Nos. 5,925,565 and 5,935,819).

c. Multiple Cloning Sites

Vectors can include a multiple cloning site (MCS), which is a nucleic acid region that contains multiple restriction enzyme sites, any of which can be used in conjunction with standard recombinant technology to digest the vector. (See Carbonelli et al., 1999, Levenson et al., 1998, and Cocea, 1997) "Restriction enzyme digestion" refers to catalytic cleavage of a nucleic acid molecule with an enzyme that functions only at specific locations in a nucleic acid molecule. Many of these restriction enzymes are commercially available. Use of such enzymes is widely understood by those of skill in the art. Frequently, a vector is linearized or fragmented using a restriction enzyme that cuts within the MCS to enable exogenous sequences to be ligated to the vector. "Ligation" refers to the process of forming phosphodiester bonds between two nucleic acid fragments, which may or may not be contiguous with each other. Techniques involving restriction enzymes and ligation reactions are well known to those of skill in the art of recombinant technology.

d. Splicing Sites

Most transcribed eukaryotic RNA molecules will undergo RNA splicing to remove introns from the primary transcripts. Vectors containing genomic eukaryotic sequences may require donor and/or acceptor splicing sites to ensure proper processing of the transcript for protein expression. (See Chandler et al., 1997).

e. Termination Signals

The vectors or constructs of the present invention will generally comprise at least one termination signal. A "termination signal" or "terminator" is comprised of the DNA sequences involved in specific termination of an RNA transcript by an RNA polymerase. Thus, in certain embodiments a termination signal that ends the production of an RNA transcript is contemplated. A terminator may be necessary in vivo to achieve desirable message levels.

In eukaryotic systems, the terminator region may also comprise specific DNA sequences that permit site-specific cleavage of the new transcript so as to expose a polyadenylation site. This signals a specialized endogenous polymerase to add a stretch of about 200 A residues (polyA) to the 3' end of the transcript. RNA molecules modified with this polyA tail appear to more stable and are translated more efficiently. Thus, in other embodiments involving eukaryotes, it is preferred that that terminator comprises a signal for the cleavage of the RNA, and it is more preferred that the terminator signal promotes polyadenylation of the message. The terminator and/or polyadenylation site elements can serve to enhance message levels and/or to minimize read through from the cassette into other sequences.

Terminators contemplated for use in the invention include any known terminator of transcription described herein or known to one of ordinary skill in the art, including but not limited to, for example, the termination sequences of genes, such as for example the bovine growth hormone terminator or viral termination sequences, such as for example the SV40 terminator. In certain embodiments, the termination signal may be a lack of transcribable or translatable sequence, such as due to a sequence truncation.

f. Polyadenylation Signals

For expression, particularly eukaryotic expression, one will typically include a polyadenylation signal to effect proper polyadenylation of the transcript. The nature of the polyadenylation signal is not believed to be crucial to the successful practice of the invention, and/or any such sequence may be employed. Preferred embodiments include the SV40 polyadenylation signal and/or the bovine growth hormone polyadenylation signal, convenient and/or known to function well in various target cells. Polyadenylation may increase the stability of the transcript or may facilitate cytoplasmic transport.

g. Origins of Replication

In order to propagate a vector in a host cell, it may contain one or more origins of replication sites (often termed "ori"), which is a specific nucleic acid sequence at which replication is initiated. Alternatively an autonomously replicating sequence (ARS) can be employed if the host cell is yeast.

h. Selectable and Screenable Markers

In certain embodiments of the invention, the cells containing a nucleic acid construct of the present invention may be identified in vitro or in vivo by including a marker in the expression vector. Such markers would confer an identifiable change to the cell permitting easy identification of cells containing the expression vector. Generally, a selectable marker is one that confers a property that allows for selection. A positive selectable marker is one in which the presence of the marker allows for its selection, while a negative selectable marker is one in which its presence prevents its selection. An example of a positive selectable marker is a drug resistance marker.

Usually the inclusion of a drug selection marker aids in the cloning and identification of transformants, for example, genes that confer resistance to neomycin, puromycin, hygromycin, DHFR, GPT, zeocin and histidinol are useful selectable markers. In addition to markers conferring a phenotype that allows for the discrimination of transformants based on the implementation of conditions, other types of markers including screenable markers such as GFP, whose basis is colorimetric analysis, are also contemplated. Alternatively, screenable enzymes such as herpes simplex virus thymidine kinase (tk) or chloramphenicol acetyltransferase (CAT) may be utilized. One of skill in the art would also know how to employ immunologic markers, possibly in conjunction with FACS analysis. The marker used is not believed to be important, so long as it is capable of being expressed simultaneously with the nucleic acid encoding a gene product. Further examples of selectable and screenable markers are well known to one of skill in the art.

3. Host Cells

As used herein, the terms "cell," "cell line," and "cell culture" may be used interchangeably. All of these terms also include their progeny, which refers to any and all subsequent generations. It is understood that all progeny may not be identical due to deliberate or inadvertent mutations. In the context of expressing a heterologous nucleic acid sequence, "host cell" refers to a prokaryotic or eukaryotic cell, and it includes any transformable organisms that is capable of replicating a vector and/or expressing a heterologous gene encoded by a vector. A host cell can, and has been, used as a recipient for vectors. A host cell may be "transfected" or "transformed," which refers to a process by which exogenous nucleic acid is transferred or introduced into the host cell. A transformed cell includes the primary subject cell and its progeny.

Characterization of PBMC subsets identified the CD4+ T cells as the cells supporting GBV-C replication. Although early studies suggested that GBV-C replicates in the liver, most reported studies indicate that GBV-C is not hepatotropic (Kiyosawa and Tanaka, 1999; Laskus et al., 1998). The inability to demonstrate infection of HepG2 cells is consistent with this, although the inventors were also unable to demonstrate persistent replication in the CD4+ T cell line (MOLT-4). Thus, host cell factors in primary cells may be necessary for replication. Studies are underway with primary hepatocyte cultures to test this hypothesis. Nevertheless, several studies have found GBV-C replication in PBMC's, and the concentration of virus in plasma relative to liver tissues suggests that the hepatocyte is not a prominent source of virus (Kobayashi et al., 1999). Taken together, these data suggest that GBV-C may be lymphotropic. As such, any lymphocyte-derived cell or cell line, particularly a CD4+ cell, is preferred for use with the present invention, however, any other cell line that permits transfection and/or propagation of an infectious GBV-C nucleic acid molecule is contemplated for use with the present invention. In other embodiments, the CD4+ cell may be infected with HIV, and such cells are contemplated to be targets for treatment to prevent or inhibit the progression of AIDS.

Nonetheless, host cells may be derived from prokaryotes or eukaryotes, depending upon whether the desired result is replication of the vector, expression of part or all of the vector-encoded nucleic acid sequences, or production of infectious viral particles. Numerous cell lines and cultures are available for use as a host cell, and they can be obtained through the American Type Culture Collection (ATCC), which is an organization that serves as an archive for living cultures and genetic materials (see the world wide web at atcc.org). An appropriate host can be determined by one of skill in the art based on the vector backbone and the desired result. A plasmid or cosmid, for example, can be introduced into a prokaryote host cell for replication of many vectors. Bacterial cells used as host cells for vector replication and/or expression include DH5.alpha., JM109, and KC8, as well as a number of commercially available bacterial hosts such as SURE.RTM. Competent Cells and SOLOPACK.TM. Gold Cells (STRATAGENE.RTM., La Jolla). Alternatively, bacterial cells such as E. coli LE392 could be used as host cells for phage viruses.

Examples of eukaryotic host cells for replication and/or expression of a vector include HeLa, NIH3T3, Jurkat, 293, Cos, CHO, Saos, and PC12. Many host cells from various cell types and organisms are available and would be known to one of skill in the art. Similarly, a viral vector may be used in conjunction with either an eukaryotic or prokaryotic host cell, particularly one that is permissive for replication or expression of the vector.

Some vectors may employ control sequences that allow it to be replicated and/or expressed in both prokaryotic and eukaryotic cells. One of skill in the art would further understand the conditions under which to incubate all of the above described host cells to maintain them and to permit replication of a vector. Also understood and known are techniques and conditions that would allow large-scale production of vectors, as well as production of the nucleic acids encoded by vectors and their cognate polypeptides, proteins, or peptides.

4. Expression Systems

Numerous expression systems exist that comprise at least a part or all of the compositions discussed above. Prokaryote- and/or eukaryote-based systems can be employed for use with the present invention to produce nucleic acid sequences, or their cognate polypeptides, proteins and peptides. Many such systems are commercially and widely available.

The insect cell/baculovirus system can produce a high level of protein expression of a heterologous nucleic acid segment, such as described in U.S. Pat. Nos. 5,871,986 and 4,879,236, which can be bought, for example, under the name MAXBAC.RTM. 2.0 from INVITROGEN.RTM. and BACPACK.TM. BACULOVIRUS EXPRESSION SYSTEM from CLONTECH.RTM..

Other examples of expression systems include STRATAGENE.RTM.'S COMPLETE CONTROL.TM. Inducible Mammalian Expression System, which involves a synthetic ecdysone-inducible receptor, or its pET Expression System, an E. coli expression system. Another example of an inducible expression system is available from INVITROGEN.RTM., which carries the T-REX.TM. (tetracycline-regulated expression) System, an inducible mammalian expression system that uses the full-length CMV promoter. The Tet-On.TM. and Tet-Off.TM. systems from CLONTECH.RTM. can be used to regulate expression in a mammalian host using tetracycline or its derivatives. The implementation of these systems is described in Gossen et al., 1992 and Gossen et al., 1995, and U.S. Pat. No. 5,650,298.

INVITROGEN.RTM. also provides a yeast expression system called the Pichia methanolica Expression System, which is designed for high-level production of recombinant proteins in the methylotrophic yeast Pichia methanolica. One of skill in the art would know how to express a vector, such as an expression construct, to produce a nucleic acid sequence or its cognate polypeptide, protein, or peptide.

5. Non-Translated Nucleic Acid Sequences

In some embodiments of the present invention, a GBV-C clone or infectious GBV-C nucleic acid molecule encodes a heterologous nucleic acid sequence that is transcribed into RNA but that is not translated. Examples of this type of heterologous nucleic acid sequence include antisense molecules or sequences and ribozymes.

a. Antisense Constructs

Antisense methodology takes advantage of the fact that nucleic acids tend to pair with "complementary" sequences. By complementary, it is meant that polynucleotides are those which are capable of base-pairing according to the standard Watson-Crick complementarity rules. That is, the larger purines will base pair with the smaller pyrimidines to form combinations of guanine paired with cytosine (G:C) and adenine paired with either thymine (A:T) in the case of DNA, or adenine paired with uracil (A:U) in the case of RNA. Inclusion of less common bases such as inosine, 5-methylcytosine, 6-methyladenine, hypoxanthine and others in hybridizing sequences does not interfere with pairing.

Targeting double-stranded (ds) DNA with polynucleotides leads to triple-helix formation; targeting RNA will lead to double-helix formation. Antisense polynucleotides, when introduced into a target cell, specifically bind to their target polynucleotide and interfere with transcription, RNA processing, transport, translation and/or stability. Antisense RNA constructs, or DNA encoding such antisense RNA's, may be employed to inhibit gene transcription or translation or both within a host cell, either in vitro or in vivo, such as within a host animal, including a human subject.

Antisense constructs may be designed to bind to the promoter and other control regions, exons, introns or even exon-intron boundaries of a gene. It is contemplated that the most effective antisense constructs will include regions complementary to intron/exon splice junctions. Thus, it is proposed that a preferred embodiment includes an antisense construct with complementarity to regions within 50-200 bases of an intron-exon splice junction. It has been observed that some exon sequences can be included in the construct without seriously affecting the target selectivity thereof. The amount of exonic material included will vary depending on the particular exon and intron sequences used. One can readily test whether too much exon DNA is included simply by testing the constructs in vitro to determine whether normal cellular function is affected or whether the expression of related genes having complementary sequences is altered.

As stated above, "complementary" or "antisense" means polynucleotide sequences that are substantially complementary over their entire length and have very few base mismatches. For example, sequences of fifteen bases in length may be termed complementary when they have complementary nucleotides at thirteen or fourteen positions. Naturally, sequences that are completely complementary will be sequences which are entirely complementary throughout their entire length and have no base mismatches. Other sequences with lower degrees of homology also are contemplated. For example, an antisense construct that has limited regions of high homology, but also contains a non-homologous region (e.g., ribozyme; see below) could be designed. These molecules, though having less than 50% homology, would bind to target sequences under appropriate conditions.

It may be advantageous to combine portions of genomic DNA with cDNA or synthetic sequences to generate specific constructs. For example, where an intron is desired in the ultimate construct, a genomic clone will need to be used. The cDNA or a synthesized polynucleotide may provide more convenient restriction sites for the remaining portion of the construct and, therefore, would be used for the rest of the sequence.

Particular oncogenes that are targets for antisense constructs are ras, myc, neu, raf erb, src, fms, jun, trk, ret, hst, gsp, bcl-2, and abl. Also contemplated to be useful are anti-apoptotic genes and angiogenesis promoters. Other antisense constructs can be directed at genes encoding viral or microbial genes to reduce or eliminate pathogenicity, such as HCV or HIV genes. Specific constructs target genes such as viral env, pol, gag, rev, tat, taf or coat or capsid genes, or microbial endotoxin, recombination, replication, or transcription genes.

b. Ribozymes

Although proteins traditionally have been used for catalysis of nucleic acids, another class of macromolecules has emerged as useful in this endeavor. Ribozymes are RNA-protein complexes that cleave nucleic acids in a site-specific fashion. Ribozymes have specific catalytic domains that possess endonuclease activity (Kim and Cook, 1987; Gerlach et al., 1987; Forster and Symons, 1987). For example, a large number of ribozymes accelerate phosphoester transfer reactions with a high degree of specificity, often cleaving only one of several phosphoesters in an oligonucleotide substrate (Michel and Westhof, 1990; Reinhold-Hurek and Shub, 1992). This specificity has been attributed to the requirement that the substrate bind via specific base-pairing interactions to the internal guide sequence ("IGS") of the ribozyme prior to chemical reaction.

Ribozyme catalysis has primarily been observed as part of sequence-specific cleavage/ligation reactions involving nucleic acids (Joyce, 1989; Cook et al., 1981). For example, U.S. Pat. No. 5,354,855 reports that certain ribozymes can act as endonucleases with a sequence specificity greater than that of known ribonucleases and approaching that of the DNA restriction enzymes. Thus, sequence-specific ribozyme-mediated inhibition of gene expression may be particularly suited to therapeutic applications (Scanlon et al., 1991; Sarver et al., 1990). Recently, it was reported that ribozymes elicited genetic changes in some cells lines to which they were applied; the altered genes included the oncogenes H-ras, c-fos and genes of HIV. Most of this work involved the modification of a target mRNA, based on a specific mutant codon that is cleaved by a specific ribozyme. Targets for this embodiment will include angiogenic genes such as VEGFs and angiopoietins as well as the oncogenes (e.g., ras, myc, neu, raf, erb, src, fms, jun, trk, ret, hst, gsp, bcl-2, EGFR, grb2 and abl). Other constructs will include overexpression of anti-apoptotic genes such as bcl-2, as well as microbial genes directed to viral or bacterial genes.

c. Other Heterologous Sequences

As the present invention is directed in some embodiments to the delivery of a sequence that is heterologous either to the virus or to a transduced cell, a variety of heterologous sequences are envisioned as part of the invention. Nucleic acid molecules that inhibit infection by non-GBV-C viruses are contemplated to be such sequences, for example, nucleotide inhibitors of HIV such as inhibitors of reverse transcriptase or integrase. In addition to encoding nucleic acid molecules, some embodiments of the invention concern the expression of a heterologous sequence as a polypeptide, and this would include proteinaceous inhibitors such as peptidic protease inhibitors, such as inhibitors of proteases associated with viral infection.

6. Introduction of Nucleic Acids into Cells

There are a number of ways in which nucleic acid molecules such as expression vectors may be introduced into cells. In certain embodiments of the invention, the expression vector comprises a GBV-C infectious particle or engineered vector derived from a GBV-C genome. The ability of certain viruses to enter cells via receptor-mediated endocytosis, to integrate into host cell genome and express viral genes stably and efficiently have made them attractive candidates for the transfer of foreign genes into mammalian cells (Ridgeway, 1988; Nicolas and Rubenstein, 1988; Baichwal and Sugden, 1986; Temin, 1986). The first viruses used as gene vectors were DNA viruses including the papovaviruses (simian virus 40, bovine papilloma virus, and polyoma) (Ridgeway, 1988; Baichwal and Sugden, 1986) and adenoviruses (Ridgeway, 1988; Baichwal and Sugden, 1986). These have a relatively low capacity for foreign DNA sequences and have a restricted host spectrum. Furthermore, their oncogenic potential and cytopathic effects in permissive cells raise safety concerns. They can accommodate only up to 8 kb of foreign genetic material but can be readily introduced in a variety of cell lines and laboratory animals (Nicolas and Rubenstein, 1988; Temin, 1986).

"Viral expression vector" is meant to include those vectors containing sequences of that virus sufficient to (a) support packaging of the vector and (b) to express a polynucleotide that has been cloned therein. In this context, expression may require that the gene product be synthesized. The present invention encompasses the use of an infectious GBV-C clone as a viral vector to transport and express a heterologous sequence in a host cell. Alternatively, a viral expression vector could be used to generate RNA transcripts encoding viral packing sequences and a heterologous gene, such that transfection of the transcripts into a host cell yield infectious viral particles containing the heterologous sequence.

A number of such viral vectors have already been thoroughly researched, including adenovirus, adeno-associated viruses, retroviruses, herpesviruses, and vaccinia viruses.

Adenovirus vectors have been used in eukaryotic gene expression (Levrero et al., 1991; Gomez-Foix et al., 1992) and vaccine development (Grunhaus and Horwitz, 1992; Graham and Prevec, 1992). Recently, animal studies suggested that recombinant adenovirus could be used for gene therapy (Stratford-Perricaudet and Perricaudet, 1991; Stratford-Perricaudet et al., 1990; Rich et al., 1993). Studies in administering recombinant adenovirus to different tissues include trachea instillation (Rosenfeld et al., 1991; Rosenfeld et al., 1992), muscle injection (Ragot et al., 1993), peripheral intravenous injections (Herz and Gerard, 1993) and stereotactic inoculation into the brain (Le Gal La Salle et al., 1993).

The retroviruses are a group of single-stranded RNA viruses characterized by an ability to convert their RNA to double-stranded DNA in infected cells by a process of reverse-transcription (Coffin, 1990). The resulting DNA then stably integrates into cellular chromosomes as a provirus and directs synthesis of viral proteins. The integration results in the retention of the viral gene sequences in the recipient cell and its descendants. The retroviral genome contains three genes, gag, pol, and env that code for capsid proteins, polymerase enzyme, and envelope components, respectively. A sequence found upstream from the gag gene contains a signal for packaging of the genome into virions. Two long terminal repeat (LTR) sequences are present at the 5' and 3' ends of the viral genome. These contain strong promoter and enhancer sequences and also are required for integration in the host cell genome (Coffin, 1990).

In order to construct a retroviral vector, a nucleic acid encoding a gene of interest is inserted into the viral genome in the place of certain viral sequences to produce a virus that is replication-defective. In order to produce virions, a packaging cell line containing the gag, pol, and env genes but without the LTR and packaging components is constructed (Mann et al., 1983). When a recombinant plasmid containing a cDNA, together with the retroviral LTR and packaging sequences is introduced into this cell line (by calcium phosphate precipitation for example), the packaging sequence allows the RNA transcript of the recombinant plasmid to be packaged into viral particles, which are then secreted into the culture media (Nicolas and Rubenstein, 1988; Temin, 1986; Mann et al., 1983). The media containing the recombinant retroviruses is then collected, optionally concentrated, and used for gene transfer. Retroviral vectors are able to infect a broad variety of cell types. However, integration and stable expression require the division of host cells (Paskind et al., 1975).

A novel approach designed to allow specific targeting of retrovirus vectors was recently developed based on the chemical modification of a retrovirus by the chemical addition of lactose residues to the viral envelope. This modification could permit the specific infection of hepatocytes via sialoglycoprotein receptors.

A different approach to targeting of recombinant retroviruses was designed in which biotinylated antibodies against a retroviral envelope protein and against a specific cell receptor were used. The antibodies were coupled via the biotin components by using streptavidin (Roux et al., 1989). Using antibodies against major histocompatibility complex class I and class II antigens, they demonstrated the infection of a variety of human cells that bore those surface antigens with an ecotropic virus in vitro (Roux et al., 1989).

Other viral vectors may be employed as expression constructs in the present invention. Vectors derived from viruses such as vaccinia virus (Ridgeway, 1988; Baichwal and Sugden, 1986; Coupar et al., 1988) adeno-associated virus (AAV) (Ridgeway, 1988; Baichwal and Sugden, 1986; Hermonat and Muzycska, 1984) and herpesviruses may be employed. They offer several attractive features for various mammalian cells (Friedmann, 1989; Ridgeway, 1988; Baichwal and Sugden, 1986; Coupar et al., 1988; Horwich et al., 1990).

With the recent recognition of defective hepatitis B viruses (HBV), new insight was gained into the structure-function relationship of different viral sequences. In vitro studies showed that the virus could retain the ability for helper-dependent packaging and reverse transcription despite the deletion of up to 80% of its genome (Horwich et al., 1990). This suggested that large portions of the genome could be replaced with foreign genetic material. The hepatotropism and persistence (integration) were particularly attractive properties for liver-directed gene transfer. Chang et al., recently introduced the chloramphenicol acetyltransferase (CAT) gene into duck hepatitis B virus genome in the place of the polymerase, surface, and pre-surface coding sequences. It was co-transfected with wild-type virus into an avian hepatoma cell line. Culture media containing high titers of the recombinant virus were used to infect primary duckling hepatocytes. Stable CAT gene expression was detected for at least 24 days after transfection (Chang et al., 1991). A GBV-C viral vector may be constructed and propagated in a manner similar to HBV.

7. Methods of Gene Transfer

In order to effect expression of gene constructs, the expression vector or RNA transcripts must be delivered into a cell. This delivery may be accomplished in vitro, as in laboratory procedures for transforming cells lines, or in vivo or ex vivo, as in the treatment of certain disease states. One mechanism for delivery is via viral infection where the expression vector is encapsidated in an infectious viral particle. These methods are described above.

Several non-viral methods for the transfer of expression vectors into cultured mammalian cells also are contemplated by the present invention. These include calcium phosphate precipitation (Graham and Van Der Eb, 1973; Chen and Okayama, 1987; Rippe et al., 1990) DEAE-dextran (Gopal, 1985), electroporation (Tur-Kaspa et al., 1986; Potter et al., 1984), direct microinjection (Harland and Weintraub, 1985), DNA-loaded liposomes (Nicolau and Sene, 1982; Fraley et al., 1979) and lipofectamine-DNA complexes, cell sonication (Fechheimer et al., 1987), gene bombardment using high velocity microprojectiles (Yang et al., 1990), and receptor-mediated transfection (Wu and Wu, 1987; Wu and Wu, 1988). Some of these techniques may be successfully adapted for in vivo or ex vivo use.

Once the expression vector or RNA transcript has been delivered into the cell the nucleic acid encoding the gene of interest may be positioned and expressed at different sites. In certain embodiments, the nucleic acid encoding a gene or genes may be stably integrated into the genome of the cell. This integration may be in the cognate location and orientation via homologous recombination (gene replacement) or it may be integrated in a random, non-specific location (gene augmentation). In yet further embodiments, the nucleic acid may be stably maintained in the cell as a separate, episomal segment of DNA. Such nucleic acid segments or "episomes" encode sequences sufficient to permit maintenance and replication independent of or in synchronization with the host cell cycle. How the expression vector is delivered to a cell and where in the cell the nucleic acid remains is dependent on the type of expression vector employed.

Transfer of a nucleic acid molecule may be performed by any of the methods mentioned above which physically or chemically permeabilize the cell membrane. This is particularly applicable for transfer in vitro but it may be applied to in vivo use as well. Dubensky et al. (1984) successfully injected polyomavirus DNA in the form of calcium phosphate precipitates into liver and spleen of adult and newborn mice demonstrating active viral replication and acute infection. Benvenisty and Neshif (1986) also demonstrated that direct intraperitoneal injection of calcium phosphate-precipitated plasmids results in expression of the transfected genes. It is envisioned that DNA encoding a gene of interest also may be transferred in a similar manner in vivo and express the gene product.

An embodiment of the claimed invention transfers RNA transcripts or a combination of transcripts into cells via perfusion. Continuous perfusion of an expression construct or a viral construct also is contemplated. The amount of construct or peptide delivered in continuous perfusion can be determined by the amount of uptake that is desirable. The present invention discloses an example of perfusion whereby a cell culture with an initial concentration of 10.sup.6 cells/ml can first be labeled, washed, and then incubated with 100 .mu.g of isolated RNA for two hours.

In still another embodiment of the invention for transferring a nucleic acid molecule into cells may involve particle bombardment. This method depends on the ability to accelerate DNA-coated microprojectiles to a high velocity allowing them to pierce cell membranes and enter cells without killing them (Klein et al., 1987). Several devices for accelerating small particles have been developed. One such device relies on a high voltage discharge to generate an electrical current, which in turn provides the motive force (Yang et al., 1990). The microprojectiles used have consisted of biologically inert substances such as tungsten or gold beads.

Selected organs including the liver, skin, and muscle tissue of rats and mice have been bombarded in vivo (Yang et al., 1990; Zelenin et al., 1991). This may require surgical exposure of the tissue or cells, to eliminate any intervening tissue between the gun and the target organ, i.e., ex vivo treatment. Again, nucleic acid encoding a particular gene such as GBV-C packing polypeptides may be delivered via this method and still be incorporated by the present invention.

In a further embodiment of the invention, the nucleic acid molecule may be entrapped in a liposome. Liposomes are vesicular structures characterized by a phospholipid bilayer membrane and an inner aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self-rearrangement before the formation of closed structures and entrap water and dissolved solutes between the lipid bilayers (Ghosh and Bachhawat, 1991). Also contemplated are lipofectamine-nucleic acid complexes.

Liposome-mediated nucleic acid delivery and expression of foreign nucleic acid in vitro has been very successful. Wong et al., (1980) demonstrated the feasibility of liposome-mediated delivery and expression of foreign DNA in cultured chick embryo, HeLa and hepatoma cells. Nicolau et al., (1987) accomplished successful liposome-mediated gene transfer in rats after intravenous injection.

In certain embodiments of the invention, the liposome may be complexed with a hemagglutinating virus (HVJ). This has been shown to facilitate fusion with the cell membrane and promote cell entry of liposome-encapsulated DNA (Kaneda et al., 1989). In other embodiments, the liposome may be complexed or employed in conjunction with nuclear non-histone chromosomal proteins (HMG-1) (Kato et al., 1991). In yet further embodiments, the liposome may be complexed or employed in conjunction with both HVJ and HMG-1. In that such expression vectors have been successfully employed in transfer and expression of nucleic acid in vitro and in vivo, then they are applicable for the present invention. Where a bacterial promoter is employed in the DNA vector, it also will be desirable to include within the liposome an appropriate bacterial polymerase.

Lipid based non-viral formulations provide an alternative to adenoviral gene therapies. Although many cell culture studies have documented lipid based non-viral gene transfer, systemic gene delivery via lipid based formulations has been limited. A major limitation of non-viral lipid based gene delivery is the toxicity of the cationic lipids that comprise the non-viral delivery vehicle. The in vivo toxicity of liposomes partially explains the discrepancy between in vitro and in vivo gene transfer results. Another factor contributing to this contradictory data is the difference in lipid vehicle stability in the presence and absence of serum proteins. The interaction between lipid vehicles and serum proteins has a dramatic impact on the stability characteristics of lipid vehicles (Yang and Huang, 1997). Cationic lipids attract and bind negatively charged serum proteins. Lipid vehicles associated with serum proteins are either dissolved or taken up by macrophages leading to their removal from circulation. Current in vivo lipid delivery methods use subcutaneous, intradermal, intratumoral, or intracranial injection to avoid the toxicity and stability problems associated with cationic lipids in the circulation. The interaction of lipid vehicles and plasma proteins is responsible for the disparity between the efficiency of in vitro (Felgner et al., 1987) and in vivo gene transfer (Zhu et al., 1993; Philip et al., 1993; Solodin et al., 1995; Liu et al., 1995; Thierry et al., 1995; Tsukamoto et al., 1995; Aksentijevich et al., 1996).

Recent advances in lipid formulations have improved the efficiency of gene transfer in vivo (Smyth-Templeton et al. 1997; WO 98/07408). A novel lipid formulation composed of an equimolar ratio of 1,2-bis(oleoyloxy)-3-(trimethyl ammonio)propane (DOTAP) and cholesterol significantly enhances systemic in vivo gene transfer, approximately 150 fold. The DOTAP:cholesterol lipid formulation is said to form a unique structure termed a "sandwich liposome." This formulation is reported to "sandwich" DNA between an invaginated bi-layer or `vase` structure. Beneficial characteristics of these lipid structures include a positive .rho., colloidal stabilization by cholesterol, two dimensional DNA packing and increased serum stability.

The production of lipid formulations often is accomplished by sonication or serial extrusion of liposomal mixtures after (I) reverse phase evaporation (II) dehydration-rehydration (III) detergent dialysis and (IV) thin film hydration. Once manufactured, lipid structures can be used to encapsulate compounds that are toxic (chemotherapeutics) or labile (nucleic acids) when in circulation. Lipid encapsulation has resulted in a lower toxicity and a longer serum half-life for such compounds (Gabizon et al., 1990). Numerous disease treatments are using lipid based gene transfer strategies to enhance conventional or establish novel therapies.

In certain embodiments of the invention, the lipid vehicle may be complexed with a hemagglutinating virus (HVJ). This has been shown to facilitate fusion with the cell membrane and promote cell entry of lipid-encapsulated DNA (Kaneda et al., 1989). In other embodiments, the lipid vehicle may be complexed or employed in conjunction with nuclear non-histone chromosomal proteins (HMG-1) (Kato et al., 1991). In yet further embodiments, the lipid vehicle may be complexed or employed in conjunction with both HVJ and HMG-1.

Protamine may also be used to form a complex with an expression construct. Such complexes may then be formulated with the lipid compositions described above for adminstration to a cell. Protamines are small highly basic nucleoproteins associated with DNA. Their use in the delivery of nucleic acids is described in U.S. Pat. No. 5,187,260.

Other expression vectors that can be employed to deliver a nucleic acid encoding a particular gene into cells are receptor-mediated delivery vehicles. These take advantage of the selective uptake of macromolecules by receptor-mediated endocytosis in almost all eukaryotic cells. Because of the cell type-specific distribution of various receptors, the delivery can be highly specific (Wu and Wu, 1993).

Receptor-mediated gene targeting vehicles generally consist of two components: a cell receptor-specific ligand and a DNA-binding agent. Several ligands have been used for receptor-mediated gene transfer. The most extensively characterized ligands are asialoorosomucoid (ASOR) (Wu and Wu, 1987) and transferrin (Wagner et al., 1990). A synthetic neoglycoprotein, which recognizes the same receptor as ASOR, has been used as a gene delivery vehicle (Ferkol et al., 1993; Perales et al., 1994) and epidermal growth factor (EGF) also has been used to deliver genes to squamous carcinoma cells (Myers, EPO 0273085).

In other embodiments, the delivery vehicle may comprise a ligand and a liposome. For example, Nicolau et al., (1987) employed lactosyl-ceramide, a galactose-terminal asialganglioside, incorporated into liposomes and observed an increase in the uptake of the insulin gene by hepatocytes. Thus, it is feasible that a nucleic acid encoding a particular gene also may be specifically delivered into a cell type such as lung, epithelial or tumor cells, by any number of receptor-ligand systems with or without liposomes. For example, epidermal growth factor (EGF) may be used as the receptor for mediated delivery of a nucleic acid encoding a gene in many tumor cells that exhibit upregulation of EGF receptor. Mannose can be used to target the mannose receptor on liver cells. Also, antibodies to CD5 (CLL), CD22 (lymphoma), CD25 (T-cell leukemia) and MAA (melanoma) can similarly be used as targeting moieties.

In certain embodiments, gene transfer may more easily be performed under ex vivo conditions. Ex vivo gene therapy refers to the isolation of cells from an animal, the delivery of a nucleic acid into the cells in vitro, and then the return of the modified cells back into an animal. This may involve the surgical removal of tissue/organs from an animal or the primary culture of cells and tissues.

Continuous perfusion of an expression vector or a viral vector also is contemplated. The amount of vector or peptide delivered in continuous perfusion can be determined by the amount of uptake that is desirable.

Primary mammalian cell cultures may be prepared in various ways. In order for the cells to be kept viable while in vitro and in contact with the nucleic acid molecule, it is necessary to ensure that the cells maintain contact with the correct ratio of oxygen and carbon dioxide and nutrients but are protected from microbial contamination. Cell culture techniques are well documented and are disclosed herein by reference (Freshner, 1992).

During in vitro culture conditions the expression construct may then deliver and express a nucleic acid encoding GBV-C proteins and/or a heterologous gene(s) into the cells. Finally, the cells may be reintroduced into the original animal, or administered into a distinct animal, in a pharmaceutically acceptable form by any of the means described below. Thus, providing an ex vivo method of treating a mammal with a pathologic condition is within the scope of the invention.

8. Nucleic Acid Detection

In addition to their use in yielding GBV-C proteins, polypeptides and/or peptides, the nucleic acid sequences disclosed herein have a variety of other uses. For example, they have utility as probes or primers for embodiments involving nucleic acid hybridization, such as sequence comparison and detection of infection.

a. Hybridization

The use of a probe or primer of between 13 and 100 nucleotides, preferably between 17 and 100 nucleotides in length, or in some aspects of the invention up to 1-2 kilobases or more in length, allows the formation of a duplex molecule that is both stable and selective. Molecules having complementary sequences over contiguous stretches greater than 20 bases in length are generally preferred, to increase stability and/or selectivity of the hybrid molecules obtained. One will generally prefer to design nucleic acid molecules for hybridization having one or more complementary sequences of 20 to 30 nucleotides, or even longer where desired. Such fragments may be readily prepared, for example, by directly synthesizing the fragment by chemical means or by introducing selected sequences into recombinant vectors for recombinant production.

Accordingly, the nucleotide sequences of the invention may be used for their ability to selectively form duplex molecules with complementary stretches of DNAs and/or RNAs or to provide primers for amplification of DNA or RNA from samples. Depending on the application envisioned, one would desire to employ varying conditions of hybridization to achieve varying degrees of selectivity of the probe or primers for the target sequence.

For applications requiring high selectivity, one will typically desire to employ relatively high stringency conditions to form the hybrids. For example, relatively low salt and/or high temperature conditions, such as provided by about 0.02 M to about 0.10 M NaCl at temperatures of about 50.degree. C. to about 70.degree. C. Such high stringency conditions tolerate little, if any, mismatch between the probe or primers and the template or target strand and would be particularly suitable for isolating specific genes or for detecting specific mRNA transcripts. It is generally appreciated that conditions can be rendered more stringent by the addition of increasing amounts of formamide.

For certain applications, for example, site-directed mutagenesis, it is appreciated that lower stringency conditions are preferred. Under these conditions, hybridization may occur even though the sequences of the hybridizing strands are not perfectly complementary, but are mismatched at one or more positions. Conditions may be rendered less stringent by increasing salt concentration and/or decreasing temperature. For example, a medium stringency condition could be provided by about 0.1 to 0.25 M NaCl at temperatures of about 37.degree. C. to about 55.degree. C., while a low stringency condition could be provided by about 0.15 M to about 0.9 M salt, at temperatures ranging from about 20.degree. C. to about 55.degree. C. Hybridization conditions can be readily manipulated depending on the desired results.

In other embodiments, hybridization may be achieved under conditions of, for example, 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl.sub.2, 1.0 mM dithiothreitol, at temperatures between approximately 20.degree. C. to about 37.degree. C. Other hybridization conditions utilized could include approximately 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl.sub.2, at temperatures ranging from approximately 40.degree. C. to about 72.degree. C.

In certain embodiments, it will be advantageous to employ nucleic acids of defined sequences of the present invention in combination with an appropriate means, such as a label, for determining hybridization. A wide variety of appropriate indicator means are known in the art, including fluorescent, radioactive, enzymatic or other ligands, such as avidin/biotin, which are capable of being detected. In preferred embodiments, one may desire to employ a fluorescent label or an enzyme tag such as urease, alkaline phosphatase or peroxidase, instead of radioactive or other environmentally undesirable reagents. In the case of enzyme tags, colorimetric indicator substrates are known that can be employed to provide a detection means that is visibly or spectrophotometrically detectable, to identify specific hybridization with complementary nucleic acid containing samples.

In general, it is envisioned that the probes or primers described herein will be useful as reagents in solution hybridization, as in PCR, for detection of expression of corresponding genes, as well as in embodiments employing a solid phase. In embodiments involving a solid phase, the test DNA (or RNA) is adsorbed or otherwise affixed to a selected matrix or surface. This fixed, single-stranded nucleic acid is then subjected to hybridization with selected probes under desired conditions. The conditions selected will depend on the particular circumstances (depending, for example, on the G+C content, type of target nucleic acid, source of nucleic acid, size of hybridization probe, etc.). Optimization of hybridization conditions for the particular application of interest is well known to those of skill in the art. After washing of the hybridized molecules to remove non-specifically bound probe molecules, hybridization is detected, and/or quantified, by determining the amount of bound label. Representative solid phase hybridization methods are disclosed in U.S. Pat. Nos. 5,843,663, 5,900,481 and 5,919,626. Other methods of hybridization that may be used in the practice of the present invention are disclosed in U.S. Pat. Nos. 5,849,481, 5,849,486 and 5,851,772.

b. Amplification of Nucleic Acids

Nucleic acids used as a template for amplification may be isolated from cells, tissues or other samples according to standard methodologies (Sambrook et al., 1989). In certain embodiments, analysis is performed on whole cell or tissue homogenates or biological fluid samples without substantial purification of the template nucleic acid. The nucleic acid may be genomic DNA or fractionated or whole cell RNA. Where RNA is used, it may be desired to first convert the RNA to a complementary DNA.

The term "primer," as used herein, is meant to encompass any nucleic acid that is capable of priming the synthesis of a nascent nucleic acid in a template-dependent process. Typically, primers are oligonucleotides from ten to twenty and/or thirty base pairs in length, but longer sequences can be employed. Primers may be provided in double-stranded and/or single-stranded form, although the single-stranded form is preferred.

Pairs of primers designed to selectively hybridize to nucleic acids corresponding to GBV-C sequences are contacted with the template nucleic acid under conditions that permit selective hybridization. Depending upon the desired application, high stringency hybridization conditions may be selected that will only allow hybridization to sequences that are completely complementary to the primers. In other embodiments, hybridization may occur under reduced stringency to allow for amplification of nucleic acids contain one or more mismatches with the primer sequences. Once hybridized, the template-primer complex is contacted with one or more enzymes that facilitate template-dependent nucleic acid synthesis. Multiple rounds of amplification, also referred to as "cycles," are conducted until a sufficient amount of amplification product is produced.

The amplification product may be detected or quantified. In certain applications, the detection may be performed by visual means. Alternatively, the detection may involve indirect identification of the product via chemiluminescence, radioactive scintigraphy of incorporated radiolabel or fluorescent label or even via a system using electrical and/or thermal impulse signals (Affymax technology; Bellus, 1994).

A number of template dependent processes are available to amplify the oligonucleotide sequences present in a given template sample. One of the best known amplification methods is the polymerase chain reaction (referred to as PCR) which is described in detail in U.S. Pat. Nos. 4,683,195, 4,683,202 and 4,800,159, and in Innis et al., 1990.

A reverse transcriptase PCR amplification procedure may be performed to quantify the amount of mRNA amplified. Methods of reverse transcribing RNA into cDNA are well known and described in Sambrook et al., 1989. Alternative methods for reverse transcription utilize thermostable DNA polymerases. These methods are described in WO 90/07641. Polymerase chain reaction methodologies are well known in the art. Representative methods of RT-PCR are described in U.S. Pat. No. 5,882,864.

Another method for amplification is ligase chain reaction ("LCR"), disclosed in European Application No. 320 308. U.S. Pat. No. 4,883,750 describes a method similar to LCR for binding probe pairs to a target sequence. A method based on PCR and oligonucleotide ligase assay (OLA), disclosed in U.S. Pat. No. 5,912,148, may also be used.

Alternative methods for amplification of target nucleic acid sequences that may be used in the practice of the present invention are disclosed in U.S. Pat. Nos. 5,843,650, 5,846,709, 5,846,783, 5,849,546, 5,849,497, 5,849,547, 5,858,652, 5,866,366, 5,916,776, 5,922,574, 5,928,905, 5,928,906, 5,932,451, 5,935,825, 5,939,291 and 5,942,391, GB Application No. 2 202 328, and in PCT Application No. PCT/US89/01025.

Qbeta Replicase, described in PCT Application No. PCT/US87/00880, may also be used as an amplification method in the present invention. In this method, a replicative sequence of RNA that has a region complementary to that of a target is added to a sample in the presence of an RNA polymerase. The polymerase will copy the replicative sequence which may then be detected.

An isothermal amplification method, in which restriction endonucleases and ligases are used to achieve the amplification of target molecules that contain nucleotide 5'-[alpha-thio]-triphosphates in one strand of a restriction site may also be useful in the amplification of nucleic acids in the present invention (Walker et al., 1992). Strand Displacement Amplification (SDA), disclosed in U.S. Pat. No. 5,916,779, is another method of carrying out isothermal amplification of nucleic acids which involves multiple rounds of strand displacement and synthesis, i.e., nick translation.

Other nucleic acid amplification procedures include transcription-based amplification systems (TAS), including nucleic acid sequence based amplification (NASBA) and 3SR (Kwoh et al., 1989; Gingeras et al., PCT Application WO 88/10315). Davey et al., European Application No. 329 822 disclose a nucleic acid amplification process involving cyclically synthesizing single-stranded RNA ("ssRNA"), ssDNA, and double-stranded DNA (dsDNA), which may be used in accordance with the present invention.

Miller et al., PCT Application WO 89/06700 disclose a nucleic acid sequence amplification scheme based on the hybridization of a promoter region/primer sequence to a target single-stranded DNA ("ssDNA") followed by transcription of many RNA copies of the sequence. This scheme is not cyclic, i.e., new templates are not produced from the resultant RNA transcripts. Other amplification methods include "race" and "one-sided PCR" (Frohman, 1990; Ohara et al., 1989a).
 

Claim 1 of 14 Claims

1. An isolated and purified nucleic acid molecule encoding an infectious GBV-C, wherein the nucleic acid molecule encodes SEQ ID NO:20, or a variant thereof, wherein said variant is 99% identical to SEQ ID NO:20.

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