# Patent

## Title
Identification of a human gyrovirus and applications

## Abstract

The present invention relates to HGyV, a human gyrovirus related to the chicken anemia virus (CAV). The present invention also relates to a new proteins encoded by HGyV, which proteins display some homology to CAV proteins. Among these new proteins, H-apoptin is of particular interest as it is herein found for the first time in a human virus and can be used for treating cancer. Also provided are methods for detecting the HGyV virus in a subject.

## Background
This application is the U.S. National Stage of International Application No. PCT/EP2011/071086, filed Nov. 25, 2011, which claims the benefit of European Application No. 10306307.9, filed Nov. 26, 2010, and of U.S. Provisional Application No. 61/449,930, filed Mar. 7, 2011.


The present invention first relates to the identification of a human gyrovirus HGyV, related to the chicken anemia virus (CAV). The present invention also relates to new proteins encoded by HGyV, which proteins display some homology to CAV proteins. Among these new proteins, H-apoptin is of particular interest as it is found for the first time in a human virus and can be used for treating cancer. Also provided are methods for detecting the HGyV virus in biological samples.
INTRODUCTION
During the course of a research program looking for pathogens present in biological samples, we have identified and isolated for the first time a new human virus that we have designated as Human Gyrovirus (HGyV) because of its homology with the chicken anemia virus, the only previously known virus in the Gyrovirus genus.
The chicken anemia virus (CAV) is highly contagious and causes severe anemia, hemorrhaging, and depletion of lymphoid tissue through the destruction of bone marrow erythroblastoid cells in young chicken. CAV is the only species of the genus Gyrovirus, which are a part of the family of Circoviridae, and consists of a non-enveloped, round, icosahedral capsid, 19-27 nm in diameter. The CAV genome consists of a single molecule of circular, single-stranded negative-sense DNA that forms a closed circle. The complete genome is 2290-2320 nucleotides long and contains three partially overlapping open reading frames which produce a single polycistronic unspliced mRNA encoding three proteins VP1, VP2, and VP3. The 5′-nontranscribed sequences of the CAV genome are the sole promoter enhancer for CAV; they comprise 6 direct repeats of sequence AGCTCA similar to the estrogen response element (ERE) consensus half-sites (A)GGTCA (Noteborn et al., Gene, 150: 313-318, 1994; Miller et al., J. Virol., 79(5): 2859-2868, 2005).
The VP1 protein is a 51-kDa capsid protein (WO 96/01116). In addition to its structural function, it also contains motifs for rolling circle replication in the C-terminal region. VP2 is a 24-kDa non-structural protein with dual-specificity phosphatase activity. Virions with mutations in SP2 are still replication competent; however, their cytopathic effects are highly attenuated. VP3, also designated apoptin, is a 13-kDa protein that has been shown to independently induce apoptosis in chicken cells both in vitro and in vivo (Koch et al., Vaccine, 13: 763-770, 1995; Noteborn et al., J. Virol., 65: 3131-3139, 1991; Noteborn et al., J. Virol., 68: 346-351, 1994; Todd et al., J. Gen. Virol., 71(Pt 4): 819-823, 1990).
Apoptin is composed of 121 amino acids, and does not show significant homology with known cellular proteins. Several proteins motifs, however, can be readily identified in the protein. For example, the C-terminus of apoptin contains a bipartite nuclear-localization sequence (NS) and a putative nuclear export sequence (NES) (Tavassoli et al., Apoptosis, 10: 717-724, 2005; Los et al., Biochim Biophys Acta, 1793(8): 1335-42, 2009). A phosphorylation site (Thr-108) is located close to the NES; phosphorylation at this site may regulate tumor-specific nuclear accumulation of the protein via inactivation of the NES (WO 02/32954; Rohn et al., J. Biol. Chem., 277: 50820-50827, 2002; Tavassoli et al., Apoptosis, 10: 717-724, 2005; Los et al., Biochim Biophys Acta, 1793(8): 1335-42, 2009). Apoptin also harbors in its N-terminus a short hydrophobic leucine-rich stretch which is required for self-association as well as binding to other partners (Leliveld et al., J. Biol. Chem., 278: 9042-9051, 2003; Teodoro et al., Genes Dev., 18: 1952-1957, 2004; Maddika et al., J Cell Sci, 118(Pt 19): 4485-4493, 2005; Maddika et al., Cell Prolif, 40(6): 835-848, 2008; Maddika et al., Oncogene, 27: 3060-3065, 2008; Los et al., Biochim Biophys Acta, 1793(8): 1335-42, 2009).
Apoptin is capable of inducing apoptosis in human malignant and transformed cell lines, but not in untransformed cell cultures (Danen-van Oorschot et al., Proc. Natl. Acad. Sci. U.S.A., 94: 5843-5847, 1997). In vitro and in vivo, apoptin fails to induce apoptosis in a variety of normal cells, including human endothelial cells, hepatocytes and hematopoietic stem cells. Moreover, the safety of apoptin is confirmed by the fact that continuous expression of apoptin in transgenic mice does not interfere with development and proliferation of both lymphocytes and melanocytes (Pietersen et al., J. Med. Mol. Biol., 2: 321-330, 2005; Los et al., Biochim Biophys Acta, 1793(8): 1335-1342, 2009; Xiao et al., Mol. Cancer, 9: 10, 2010).
In normal cells, apoptin was found predominantly in the cytoplasm, whereas in transformed or malignant cells, it is located in the nucleus (Tavassoli et al., Apoptosis, 10: 717-724, 2005). Nuclear localization of apoptin appears to be important for its cell killing activity (Danen-van Oorschot et al., J. Biol. Chem., 278(30): 27729-27736, 2003; Tavassoli et al., Apoptosis, 10: 717-724, 2005; Heilman et al., J. Virol., 80(15): 7535-7545, 2006; Los et al., Biochim Biophys Acta, 1793(8): 1335-1342, 2009).
Apoptin-induced apoptosis is largely independent of p53 function. Indeed, apoptin expression is capable of inducing cell-cycle arrest and apoptosis in p53 null cells (WO 96/41191; Teodoro et al., Genes Dev., 18: 1952-1957, 2004; Zuang et al., Cancer Res., 55: 486-489, 1995). The p53 pathway is the major mechanism by which cancer cells are destroyed by chemotherapy and radiotherapy (Soussi and Lozano, Biochem. Biophys. Res. Commun., 331: 834-842, 2005). However, because the p53 gene is mutated in approximately half of all human tumors, cancer cells are often refractory to these forms of therapy (Scherr, Cell, 116: 235-246, 2004). The p53-independent and tumor-specific properties associated with apoptin thus make it a very important candidate for cancer therapy.
However, apoptin is derived from a bird-specific virus and therefore, may not be as efficient in inducing apoptosis in human tumor cells as a genuine human protein. It can be anticipated that a human apoptin should be more potent in mammals than its avian counterpart.
Considering the above, we now provide a new human virus which presents oncolytic activities specific to proliferative cells and thus which is directly useful in the treatment of cancer, especially in human. In addition, we also provide the human apoptin protein which can be directly used as a biotherapeutic to treat cancer in human and animals.
DESCRIPTION
In a first aspect, the present invention provides a new human virus homologous to the CAV. This new virus, designated HGyV (for Human Gyrovirus), was identified at the surface of the skin from healthy people. Its genome shows homology with the CAV genome, both at the nucleotide and organizational levels. By “CAV genome”, it is herein referred to a genome having a sequence such as in e.g. Genbank accession No M55918 and represented in SEQ ID NO: 8, and comprising a 5′ region with promoter features and three overlapping open reading frames. The said open reading frames have nucleotide sequences represented by SEQ ID: 9, SEQ ID: 10, and SEQ ID: 11. The said open reading frames code for the VP1, VP2 and apoptin polypeptides respectively; as an example, such polypeptides may have sequences represented by e.g. SEQ ID: 12, SEQ ID: 13, and SEQ ID: 14. Whereas CAV is specific for avian cells, HGyV is capable of replicating in human cells.
The present invention thus provides an isolated polynucleotide sequence, wherein said polynucleotide is a human virus genome and said sequence comprises a 5′ promoter region and/or three partially open reading frames.
In a preferred embodiment, the genome of the HGyV virus contains a 5′ region with promoter features. A “promoter” as used herein is a sequence which is capable of directing expression of an open reading frame operationally linked to said sequence. Specific regulatory DNA sequences (cis-elements) in the promoter serve as binding sites for transcription factors (trans-acting factors). The cis-elements found in gene promoters can be divided in two categories. The first category comprises those cis-elements which are involved in initiation of transcription, such as the TATA box and the CAAT box. The second category is composed of cis-elements which are involved in temporal and spatial regulation of gene expression; examples of such cis-elements include the direct repeats of sequence represented by SEQ ID NO: 29: AGCTCA found in the CAV promoter.
Preferably, the said 5′ HGyV promoter region includes repeated cis-elements having sequences homologous to the estrogen response element (ERE) consensus half-sites represented by SEQ ID NO 30: (A)GGTCA. More preferably, the said 5′ region comprises repeated cis-elements which sequences show at least 85%, preferably at least 90%, more preferably at least 95%, yet still more preferably at least 98% identity with the sequence represented by SEQ ID NO: 31: ACGTCA. In the most preferred embodiment, the said 5′ region comprises repeated cis-elements having the sequence represented by SEQ ID NO: 31. Preferably, the said 5′ region contains at least 2 repeated cis-elements as defined above, more preferably at least 3, even more preferably at least 4. The said 5′ HGyV promoter region may further include a cis-element consisting of a palindromic sequence; preferably, the said palindromic sequence is the sequence represented by SEQ ID NO: 17: CAATCAGAATTG.
According to the present invention, the genome of HGyV virus of the invention may also comprise three partially overlapping open reading frames. The present inventors found in particular that the said open-reading frames show homology at the nucleotide level with the CAV VP1, VP2, and VP3 (apoptin) genes, respectively.
Thus, the invention provides an H-VP1 open reading frame which has a sequence sharing at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 87.5%, 90%, 92.5%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% identity with the sequence represented by SEQ ID NO: 2 or with the sequence represented by SEQ ID NO: 34 or with the sequence represented by SEQ ID NO: 38. In a preferred embodiment, the said H-VP1 open reading frame has the sequence represented by SEQ ID NO: 2 or the sequence represented by SEQ ID NO: 34 or with the sequence represented by SEQ ID NO: 38. More preferably, the said H-VP1 open reading frame has the sequence represented by SEQ ID NO: 34. The invention also relates to an H-VP2 gene which sequence has at least at least 65%, 70%, 75%, 80%, 85%, 87.5%, 90%, 92.5%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% identity with the sequence represented by SEQ ID NO: 4. In another preferred embodiment, the said H-VP2 open reading frame has the sequence represented by SEQ ID NO: 4. The invention also refers to an H-VP3 open reading frame which has a sequence with at least 65%, 70%, 75%, 80%, 85%, 87.5%, 90%, 92.5%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% identity with the sequence represented by SEQ ID NO: 6. In still another preferred embodiment, the said H-VP3 gene has a sequence represented by SEQ ID NO: 6. The invention thus relates to an isolated polynucleotide, wherein the said polynucleotide is selected from the group consisting of:

    
    
        a H-VP1 gene having a sequence with at least 55% identity with the sequence represented by SEQ ID NO: 2 or with the sequence represented by SEQ ID NO: 34 or with the sequence represented by SEQ ID NO: 37;
        a H-VP2 gene having a sequence with at least 65% identity with the sequence represented by SEQ ID NO: 4; and
        a H-VP3 gene having a sequence with at least 65% identity with the sequence represented by SEQ ID NO: 6.
    
    


The HGyV virus of the invention may contain all three of the H-VP1, the H-VP2, and the H-VP3 genes. In a preferred embodiment, the HGyV virus of the invention has a sequence displaying at least 60%, 65%, 70%, 75%, 80%, 85%, 87.5%, 90%, 92.5%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% identity with the sequence represented by SEQ ID NO: 1. In another preferred embodiment, the HGyV virus of the invention has a sequence displaying at least 60%, 65%, 70%, 75%, 80%, 85%, 87.5%, 90%, 92.5%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% identity with the sequence represented by SEQ ID NO: 37. This sequence corresponds to another HGyV molecular clone, wherein a 60-nucleotide insertion is present between positions 2263 and 2264 of the sequence represented by SEQ ID NO: 1 In the most preferred embodiment of the invention, the HGyV virus of the invention has a sequence selected from the group consisting of the sequences represented by SEQ ID NO: 1 and SEQ ID NO: 37.
It also relates to the HGyV virus, in a recombinant form (meaning that the virus displays at least one modification from naturally occurring virus), to the HGyV virus in the form of complementary sequence of SEQ ID NO: 1 or SEQ ID NO: 37, or even to the HGyV virus in the form of complete cDNA of SEQ ID NO: 1 or SEQ ID NO: 37. The invention also encompasses parts of said HGyV virus as defined above comprising at least 100, 200 or 500 consecutive nucleotides of the sequence as defined above (SEQ ID NO: 1 or SEQ ID NO: 37, complementary sequence thereof or cDNA thereof). In another aspect, the invention also relates to an HGyV viral particle containing the said HGyV virus.
The term “sequence identity” refers to the identity between two peptides or between two nucleic acids. Identity between sequences can be determined by comparing a position in each of the sequences which may be aligned for the purposes of comparison. When a position in the compared sequences is occupied by the same base or amino acid, then the sequences are identical at that position. A degree of sequence identity between nucleic acid sequences is a function of the number of identical nucleotides at positions shared by these sequences. A degree of identity between amino acid sequences is a function of the number of identical amino acid sequences that are shared between these sequences. Since two polypeptides may each (i) comprise a sequence (i.e. a portion of a complete polynucleotide sequence) that is similar between two polynucleotides, and (ii) may further comprise a sequence that is divergent between two polynucleotides, sequence identity comparisons between two or more polynucleotides over a “comparison window” refers to the conceptual segment of at least 20 contiguous nucleotide positions wherein a polynucleotide sequence may be compared to a reference nucleotide sequence of at least 20 contiguous nucleotides and wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e. gaps) of 20 percent or less compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences.
To determine the percent identity of two amino acids sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison. For example, gaps can be introduced in the sequence of a first amino acid sequence or a first nucleic acid sequence for optimal alignment with the second amino acid sequence or second nucleic acid sequence. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences. Hence % identity=number of identical positions/total number of overlapping positions ×100.
In this comparison the sequences can be the same length or can be different in length. Optimal alignment of sequences for determining a comparison window may be conducted by the local homology algorithm of Smith and Waterman (J. Theor. Biol., 91(2): 370-380, 1981), by the homology alignment algorithm of Needleman and Wunsch (J. Mol. Biol, 48(3): 443-453, 1972), by the search for similarity via the method of Pearson and Lipman (Proc. Natl. Acad. Sci. U.S.A., 85(5): 2444-2448, 1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA and TFASTA in the Wisconsin Genetics Software Package Release 7.0, Genetic Computer Group, 575, Science Drive, Madison, Wis.) or by inspection. The best alignment (i.e. resulting in the highest percentage of identity over the comparison window) generated by the various methods is selected.
The term “sequence identity” means that two polynucleotide or polypeptide sequences are identical (i.e. on a nucleotide by nucleotide or an amino acid by amino acid basis) over the window of comparison. The term “percentage of sequence identity” is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base (e.g. A, T, C, G, U, or 1) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e. the window size) and multiplying the result by 100 to yield the percentage of sequence identity. The same process can be applied to polypeptide sequences. The percentage of sequence identity of a nucleic acid sequence or an amino acid sequence can also be calculated using BLAST software (Version 2.06 of September 1998) with the default or user defined parameter.
The term “sequence similarity” means that amino acids can be modified while retaining the same function. It is known that amino acids are classified according to the nature of their side groups and some amino acids such as the basic amino acids can be interchanged for one another while their basic function is maintained.
The CAV virus mostly infects young chicks of around 10-14 days of age. Outbreaks of the disease are characterized by anemia, thymus atrophy, bone marrow aplasia and immunosuppression. The HGyV virus of the invention should function in a similar way, i.e. young children are expected to be at risk of being affected by an HGyV infection. It is therefore important to be capable of detecting the presence or not of the HGyV virus of the invention, in particular in young children. Detection of the HGyV virus of the infection in pregnant women is also crucial in order to monitor the risk of vertical transmission from the mother to the child.
The invention thus also relates to an in vitro method of detection of an HGyV virus in a a subject, comprising the steps of:
a) determining the presence of the HGyV virus in a biological sample of the said subject.
A “biological sample” may be any sample that may be taken from a subject, and thus includes, but is not limited to, for example, blood, serum, plasma, sputum, urine, stool, skin, cerebrospinal fluid, saliva, gastric secretions, semen, seminal fluid, breast milk, and tears. A sample can be obtained by an oropharyngeal swab, nasopharyngeal swab, throat swab, nasal aspirate, nasal wash, fluid collected from the ear, eye, mouth, or respiratory airway, spinal tissue or fluid, cerebral fluid, trigeminal ganglion sample, a sacral ganglion sample, adipose tissue, lymphoid tissue, placental tissue, upper reproductive tract tissue, gastrointestinal tract tissue, male genital tissue and fetal central nervous system tissue. A sample can also be a pool of individual samples, especially those made during the process of manufacturing of biological samples obtained from humans (blood or urine derived products, for example), or any intermediate product sampled during the manufacturing of such products. Such sample must allow the determination of the presence of HGyV through the methods of the invention.
The presence of the HGyV virus may be determined by any technology known to a man skilled in the art. In particular, the HGyV virus may be detected at the genomic and/or nucleic and/or protein level. The method according to the invention may thus comprise another preliminary step, between the taking of the sample from the patient and step a) as defined above, corresponding to the transformation of the biological sample into a genomic DNA sample, or into an mRNA (or corresponding cDNA) sample, or into a protein sample, which is then ready to use for in vitro detection of HGyV in step a). Once a ready-to-use genomic DNA, mRNA (or corresponding cDNA) or protein sample is available, the detection of the HGyV virus may be performed, depending on the type of transformation and the available ready-to-use sample, either at the genomic DNA (i.e. based on the presence of at least one sequence consisting of at least a part of the HGyV genome as defined above), mRNA (i.e. based on the mRNA content of the sample) or at the protein level (i.e. based on the protein content of the sample).
Methods for detecting a genomic nucleic acid in a biological sample include inter alia hybridization with a labeled probe, genomic PCR, nucleic microarrays, high-throughput sequencing, and all other methods known to the person of skills in the art. The amount of nucleic acid transcripts can be measured by any technology known by the skilled person. In particular, the measure may be carried out directly on an extracted messenger RNA (mRNA) sample, or on retrotranscribed complementary DNA (cDNA) prepared from extracted mRNA by technologies well-known in the art. From the mRNA or cDNA sample, the amount of nucleic acid transcripts may be measured using any technology known by a person skilled in the art, including nucleic microarrays, quantitative PCR, and hybridization with a labeled probe.
In a preferred embodiment, the presence of the said virus is determined by hybridization of probes specific for the said virus or parts thereof with the biological sample. In another embodiment, amplification and/or sequencing of the HGyV sequences is performed in order to assess the presence of the said virus. In yet another embodiment, the presence of the HGyV virus is determined by detecting a protein produced by at least one of the three open reading frames, H-VP1, H-VP2, and H-VP3.
Another object of the invention therefore relates to a probe capable of hybridizing to the genomic DNA of HGyV. By “probe capable of hybridizing”, one should understand that the said probe is substantially complementary to at least part of the HGyV virus genome. For example, the said probe comprises a nucleotide sequence displaying at least 50%, 55%, 65%, 70%, 75%, 80%, 85%, 87.5%, 90%, 92.5%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% identity with at least a part of a sequence of the genomic DNA of HGyV. The probe of the invention comprises at least 12 nucleotides, more preferably at least 15 nucleotides, even more preferably at least 20 nucleotides. According to a specific embodiment, the method of the invention is performed by hybridization with the probes of the invention. Detection of a hybridization signal is thus indicative of the presence of a HGyV virus in the biological sample. It is advantageous to use labelled probes in this embodiment.
The present invention also includes primers specific for the HGyV virus. Preferably, the said primers have the sequences as laid out in SEQ ID NOS: 21-28. The said primers can be used for amplification of specific regions of the HGyV virus of the invention. The amplification may be carried out directly on genomic DNA, on an extracted messenger RNA (mRNA) sample, or on retrotranscribed complementary DNA (cDNA) prepared from extracted mRNA by technologies well-know in the art. The said primers of the invention can also be used for sequencing the HGyV virus. Alternatively, the said HGyV virus is detected by high-throughput sequencing. Many such methods are already known to the man of skills in the art; according to some of the methods, amplification of the template prior to sequencing may be required (see, for a few examples, Mitreva & Mardis, Methods Mol Biol., 533:153-87, 2009; Mardis, Genome Med., 1(4): 40, 2009; Cloonan et al., Nat Methods, 5(7): 613-619, 2008; Valouev et al., Genome Res., 18(7):1051-63, 2008, Valouev et al., Nat Methods., 5(9):829-34, 2008; Orscheln et al., Clin Infect Dis., 49(4):536-42, 2009; Walter et al., Proc Natl Acad Sci USA., 106(31):12950-5, 2009; Mardis et al., N Engl J Med., 361(11):1058-66, 2009, Hutchinson, Nucl. Acids Res., 35(18): 6227-6237, 2007; Shendure & Ji, Nat Biotechnol., 26(10):1135-45. 2008; Pihlak et al., Nat Biotechnol., 26(6): 676-684, 2008; Fuller et al., Nature Biotechnol., 27(11): 1013-1023, 2009; Mardis, Genome Med., 1(4): 40, 2009; Metzker, Nature Rev. Genet., 11(1): 31-46, 2010).
An embodiment of the present invention thus provides a method of detection of an HGyV virus comprising a step of amplification and/or sequencing of the said virus using the primers of the invention. In this particular embodiment, amplification or sequencing of nucleic acid using the primers of the invention is indicative of the presence of the HGyV virus in the said sample. When referring to sequencing, it is within the scope of the invention to detect HGyV in samples, or to screen for HGyV in biological materials, with deep sequencing techniques, such as pyro-sequencing.
When the HGyV virus is detected at the protein level, it may be notably performed using specific antibodies, in particular using well known technologies such as cell membrane staining using biotinylation or other equivalent techniques followed by immunoprecipitation with specific antibodies, western blot, ELISA or ELISPOT, antibodies microarrays, or tissue microarrays coupled to immunohistochemistry. Other suitable techniques include FRET or BRET, single cell microscopic or histochemistery methods using single or multiple excitation wavelength and applying any of the adapted optical methods, such as electrochemical methods (voltametry and amperometry techniques), atomic force microscopy, and radio frequency methods, e.g. multipolar resonance spectroscopy, confocal and non-confocal, detection of fluorescence, luminescence, chemiluminescence, absorbance, reflectance, transmittance, and birefringence or refractive index (e.g., surface plasmon resonance, ellipsometry, a resonant mirror method, a grating coupler waveguide method or interferometry), cell ELISA, flow cytometry, radioisotopic, magnetic resonance imaging, analysis by polyacrylamide gel electrophoresis (SDS-PAGE); HPLC-Mass Spectroscopy; Liquid Chromatography/Mass Spectrometry/Mass Spectrometry (LC-MS/MS)). All these techniques are well known in the art and need not be further detailed here.
In another aspect, the invention provides a HGyV protein produced by the HGyV virus of the invention. By “HGyV protein”, it is herein referred to a protein encoded by one of the open reading frames of the human virus of the invention. The HGyV protein of the invention is thus a protein encoded by anyone of the polynucleotide described above. In particular, the HGyV protein of the invention is encoded by anyone of the three open reading frames of the HGyV virus of the invention, H-VP1, H-VP2, and H-VP3, as defined above.
In a preferred embodiment, the HGyV protein is H-VP1p and has a sequence showing at least 45%, 50%, 55%, 65%, 70%, 75%, 80%, 85%, 87.5%, 90%, 92.5%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% identity with a sequence represented by SEQ ID NO: 3 or with the sequence represented by SEQ ID NO: 35 or with the sequence represented by SEQ ID NO: 39. More preferably, the H-VP1p protein of the invention has the sequence represented by SEQ ID NO: 3 or the sequence represented by SEQ ID NO: 35 or the sequence represented by SEQ ID NO: 39. Even more preferably, the H-VP1p protein of the invention has the sequence represented by SEQ ID NO: 35. In another preferred embodiment, the HGyV protein is a H-VP2p and has a sequence showing at least 50%, 55%, 65%, 70%, 75%, 80%, 85%, 87.5%, 90%, 92.5%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% identity with a sequence represented by SEQ ID NO: 5. Still more preferably, the H-VP2p protein of the invention has the sequence represented by SEQ ID NO: 5. In yet another preferred embodiment, the HGyV protein is H-apoptin and has a sequence showing at least HGyV protein has a sequence showing at least 35%, 40%, 45%, 50%, 55%, 65%, 70%, 75%, 80%, 85%, 87.5%, 90%, 92.5%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% identity with a sequence represented by SEQ ID NO: 7. In a further preferred embodiment, the H-apoptin protein of the invention may comprise one or several protein motifs similar to the ones found in the avian apoptin protein. In particular, the H-apoptin of the invention may contain a leucine-rich stretch, a nuclear exportation signal (NES) and/or at least one nuclear localization signal. Advantageously, the said H-apoptin protein contains in addition a phosphorylation site close to the NES. Preferably, the said phosphorylation site corresponds to the threonine residue at position 110 in SEQ ID NO: 7. In a more preferred embodiment, the H-apoptin protein of the invention has the sequence represented by SEQ ID NO: 7.
The invention thus relates to an isolated polypeptide, wherein the said polypeptide is selected from the group consisting of:

    
    
        a H-VP1p polypeptide having a sequence with at least 45% identity with the sequence represented by SEQ ID NO: 3 or with the sequence represented by SEQ ID NO: 35 or with the sequence represented by SEQ ID NO: 39;
        a H-VP2 polypeptide having a sequence with at least 50% identity with the sequence represented by SEQ ID NO: 5; and
        a H-apoptin polypeptide having a sequence with at least 35% identity with the sequence represented by SEQ ID NO: 7.
    
    


In a particular embodiment, the polypeptide of the invention is associated to a cell-penetrating peptide (CPP). As used herein, “CPP” refers to a small (10-30 residues in length), often positively charged sequence of amino acids which has the ability to enter cells independent of a membrane receptor, and show no cell-type specificity (see Richard et al., J. Biol. Chem, 278(1): 585-590, 2003; Patel et al., Pharm Res, 24(11): 1977-1992, 2007). The CPPs of the invention facilitate cellular uptake of the said polypeptide, preferentially H-apoptin, through endocytosis with the said polypeptide delivered to the endosomes of living mammalian cells. In a preferred embodiment, the CPP of the invention include such sequences as the nuclear transcription activator Tat protein, Tat-(47-57) (SEQ ID NO: 40; YGRKKRRQRRR), the regulator of expression of virion Rev protein, HIV-1 Rev-(34-50) (SEQ ID NO: 41; TRQARRNRRRR WRERQR), both encoded by HIV-1, the Drosophila Antennapedia protein, Antp-(43-58) (SEQ ID NO: 42; RQIKIYFQNRRMKWKK), flock house virus (FHV) coat-(35-49) (SEQ ID NO: 43; RRRRNRTRRNRRRVR), small oligoarginine, (R)n, small oligolysine, (K)n, the model amphipathic peptide MAP, (SEQ ID NO: 44; KLALKLALKALKAALKLA), transportan (SEQ ID NO: 45; GWTLNSAGYLLGKINLKALAALAKKIL), pisl (SEQ ID NO: 46; RVIRVWFQNKRCKDKK), Pep-1 (SEQ ID NO: 47; KETWWVETWWVTEWSQPKKKRRV), the protein transduction domain 4, PTD4 (SEQ ID NO: 48: YARAAARQARA), and the protein transduction domain PTD (SEQ ID NO: 49; ARAAAAQARA). In a further preferred embodiment, the CPP of the invention is PTD4 or PTD. In the most preferred embodiment, the CPP of the invention is PTD.
The CPP of the invention can be associated to the said polypeptide either through non-covalent interactions or through a covalent bond. Preferably, the CPP is associated with the polypeptide of the invention through a covalent bond.
Advantageously, the association of the CPP with the polypeptide of the invention results from a fusion at the genetic level. In this embodiment, the genes of the CPP and of the polypeptide of the invention form a single transcriptional and translational unit leading to the synthesis of a CPP fusion protein. Thus the present invention is also directed to an isolated polynucleotide comprising an open reading frame encoding a CPP fused in frame to an H-apoptin (H-VP3) gene. The invention also provides a fusion protein comprising a CPP domain fused to an H-apoptin polypeptide.
Advantageously, the CPP fusion protein of the invention comprises a linker domain between the CPP and the polypeptide of the invention. Such a linker domain is an amino acid stretch that adopts an extended conformation to allow for maximal flexibility. The linker thus prevents one moiety of the said fusion protein from negatively affecting the function of the other. Linkers have often been used in the art. The skilled person may, for example, refer to the LINKER web server (Xue et al., Nucleic Acids Res, 32 (Web Server issue): W562-W565, 2004). A preferred embodiment of the invention is thus directed to an isolated polynucleotide comprising an open reading frame encoding a CPP, an open reading frame encoding a linker, and an H-apoptin (H-VP3) gene, the said polynucleotide encoding a CPP-linker-H-apoptin fusion protein. The invention thus also provides a CPP-linker-H-apoptin fusion protein.
Preferably, the linker has the sequence GGSGGS (SEQ ID NO: 50).
Preferably, the polypeptide of the invention which is associated with CPP is H-apoptin. Apoptin induces apoptosis in tumor cells. The association between H-apoptin and CPP thus improves delivery of apoptin across the cell membrane, into the tumor cell where it exerts its activity. In a further preferred embodiment, H-apoptin is fused to a CPP. Such a CPP-H-apoptin fusion polypeptide is especially useful for cancer therapy. In an even further preferred embodiment, H-apoptin is fused to a CPP which is not PTD4. More preferably, H-apoptin is fused to PTD. Even more preferably, the PTD-H-apoptin fusion comprises a linker. In the most preferred embodiment, the PTD-linker-H-apoptin has the sequence of SEQ ID NO: 58, and is encoded by a polynucleotide having the sequence of SEQ ID NO: 57.
The invention provides recombinant vectors comprising at least one, two or three of the polynucleotides of the invention as defined above, especially at least SEQ ID NO: 7. The polynucleotide of the invention may be inserted into a replicable vector for cloning (amplification of the DNA) or for expression. Various vectors are publicly available. The vector may, for example, be in the form of a plasmid, cosmid, viral particle, or phage. The appropriate nucleic acid sequence may be inserted into the vector by a variety of procedures. In general, DNA is inserted into an appropriate restriction endonuclease site(s) using techniques known in the art (see, for example, the techniques described in Sambrook et al., 1990, Molecular Cloning, A Laboratory Manual, 2d Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. and Ausubel et al., eds., 1998, Current Protocols in Molecular Biology, John Wiley & Sons, NY). Vector components generally include, but are not limited to, one or more of a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence. Construction of suitable vectors containing one or more of these components employs standard ligation techniques which are known to the skilled artisan. Thus, it is within the scope of the invention to provide a vector for expressing any of the above defined a H-VP1p polypeptide, a H-VP2p polypeptide, a H-apoptin polypeptide, or a CPP-H-apoptin fusion polypeptide, in particular a H-apoptin polypeptide or a CPP-H-apoptin fusion polypeptide.
In order to express the HGyV proteins or CPP fusion proteins of the invention, the polynucleotides encoding said proteins are inserted into expression vectors such that the genes are operatively linked to transcriptional and translational sequences. Expression vectors include plasmids, YACs, cosmids, retrovirus, adenovirus, EBV-derived episomes, and all the other vectors that the skilled man will know to be convenient for ensuring the expression of the protein of interest. Both expression and cloning vectors contain a nucleic acid sequence that enables the vector to replicate in one or more selected host cells. Such sequences are well known for a variety of bacteria, yeast, and viruses. The origin of replication from the plasmid pBR322 is suitable for most Gram-negative bacteria, the 2p plasmid origin is suitable for yeast, and various viral origins (SV40, polyoma, adenovirus, VSV or BPV) are useful for cloning vectors in mammalian cells. Expression and cloning vectors will typically contain a selection gene, also termed a selectable marker. Typical selection genes encode proteins that (a) confer resistance to antibiotics or other toxins, e.g., ampicillin, neomycin, methotrexate, or tetracycline, (b) complement auxotrophic deficiencies, or (c) supply critical nutrients not available from complex media, e.g., the gene encoding D-alanine racemase for Bacilli.
Polynucleotides of the invention and vectors comprising these molecules can be used for the transformation of a suitable host cell. Transformation can be performed by any known method for introducing polynucleotides into a cell host. Such methods are well known of the man skilled in the art and include dextran-mediated transformation, calcium phosphate precipitation, polybrene-mediated transfection, protoplast fusion, electroporation, encapsulation of the polynucleotide into liposomes, biolistic injection and direct microinjection of DNA into nuclei. Therefore, the invention also encompasses a host cell comprising a vector of the invention.
Preferably, the said host cell is a bacterial cell; more preferably, it is a eukaryotic cell; even more preferably it is a mammalian cell. The nature of the host cell will be dictated by the intended use of the vector of the invention. For example, a cloning vector will usually be maintained and propagated in bacterial cells. On the other hand, it will be advantageous to transform an expression vector in a mammalian cell in order to express the HGyV proteins or CPP fusion proteins of the invention. For long-term, high-yield production of recombinant proteins, stable expression is preferred. For example, cell lines which stably express the protein of the invention may be engineered. Rather than using expression vectors which contain viral origins of replication, host cells can be transformed with DNA controlled by appropriate expression control elements (e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.), and a selectable marker. Following the introduction of the foreign DNA, engineered cells may be allowed to grow for 1-2 days in an enriched media, and then are switched to a selective media. The selectable marker in the recombinant plasmid confers resistance to the selection and allows cells to stably integrate the plasmid into their chromosomes and grow to form foci which in turn can be cloned and expanded into cell lines. This method may advantageously be used to engineer cell lines which express the HGyV protein or CPP fusion protein of the invention. Such engineered cell lines may be particularly useful in screening and evaluation of compounds that interact directly or indirectly with the said HGyV protein.
The protein of the invention may be prepared by growing a culture transformed host cells under culture conditions necessary to express the desired protein. The resulting expressed protein may then be purified from the culture medium or cell extracts. Soluble forms of the protein of the invention can be purified from conditioned media. Membrane-bound forms of protein of the invention can be purified by preparing a total membrane fraction from the expressing cell and extracting the membranes with a non-ionic detergent such as Triton X-100.
The protein can be purified using methods known to those skilled in the art. For example, the protein of the invention can be concentrated using a commercially available protein concentration filter, for example, an Amicon or Millipore Pellicon ultrafiltration unit. Following the concentration step, the concentrate can be applied to a purification matrix such as a gel filtration medium. Alternatively, an anion exchange resin can be employed, for example, a matrix or substrate having pendant diethylaminoethyl (DEAE) or polyetheyleneimine (PEI) groups. The matrices can be acrylamide, agarose, dextran, cellulose or other types commonly employed in protein purification.
Alternatively, a cation exchange step can be employed. Suitable cation exchangers include various insoluble matrices comprising sulfopropyl or carboxymethyl groups. Sulfopropyl groups are preferred (e.g., S-Sepharose B columns). The purification of the MU-1 protein from culture supernatant may also include one or more column steps over such affinity resins as concanavalin A-agarose, heparin-Toyopearl or Cibacrom blue 3GA Sepharose B; or by hydrophobic interaction chromatography using such resins as phenyl ether, butyl ether, or propyl ether; or by immunoaffinity chromatography. Finally, one or more reverse-phase high performance liquid chromatography (RP-HPLC) steps employing hydrophobic RP-HPLC media, e.g., silica gel having pendant methyl or other aliphatic groups, can be employed to further purify the protein of the invention.
Affinity columns including antibodies to the protein of the invention can also be used in purification in accordance with known methods. Some or all of the foregoing purification steps, in various combinations or with other known methods, can also be employed to provide a substantially purified isolated recombinant protein. Preferably, the isolated protein of the invention is purified so that it is substantially free of other mammalian proteins.
It is thus also an aspect of the invention to provide a method for producing a recombinant HGyV protein or a CPP fusion protein of the invention. According to a particular embodiment, the method of the invention comprises the steps of:
(a) introducing a nucleic acid encoding the recombinant HGyV protein or a CPP fusion protein into one of the host cell described above;
(b) growing the transfected host cell to produce the said HGyV protein or CPP fusion protein; and
(c) isolating the recombinant HGyV protein or CPP fusion protein from the host cell.
Proteins of the invention may be used to screen for agents which are capable of binding to the said protein. Binding assays using a desired binding protein, immobilized or not, are well known in the art and may be used for this purpose using the protein of the invention. Purified cell based or protein based (cell free) screening assays may be used to identify such agents. For example, H-apoptin may be immobilized in purified form on a carrier and binding or potential ligands to purified H-apoptin may be measured.
The CAV-encoded A-apoptin has been shown to induce apoptosis in a tumor cells, but not in wild-type cells (Danen-van Oorschot et al., Proc. Natl. Acad. Sci. U.S.A., 94: 5843-5847, 1997). Preclinical studies with A-apoptin showed that gene transfer into tumor cells, using virus methods including viral transfer, either in cell culture or in vivo, led to the rapid death of cells, and that this effect was specific for the tumor cells. For example, in vivo adenovirus-mediated delivery of A-apoptin was efficient against a primary squamous cell carcinoma as a model of head and neck cancers (Schloop et al., Cancer Biol Ther, 7(9): 1368-1373, 2008; Schloop et al., J Mol Histol, 40(3): 177-81, 2009). A-apoptin sensitizes the activation of apoptosis in radioresistant tumor SQD9 cells, resulting in an additive cytotoxic effect of irradiation and apoptin (Olijslagers et al., Basic Clin Pharmacol Toxicol, 100(2): 127-131, 2007). Also, liver, cervix and gastric and hepatocarcinoma carcinoma xenografts treated with A-apoptin exhibited a significant tumor growth inhibition in vivo (Sun et al., Int. J. Cancer, 124: 2973-2981, 2009).
The apoptosis-inducing activity of A-apoptin is associated with its nuclear localization, which has been shown to be dependent upon specific motifs found in the protein, e.g. the NLS and NES sequences, the LRS domain, and the phosphorylation site. Since these elements are also found in the sequence of H-apoptin, the said H-apoptin behaves like the CAV protein and thus has pro-apoptotic activity in tumor cells only.
In another aspect, the invention thus relates to a pharmaceutical composition comprising a protein or fusion protein of the invention, purified from cells or recombinantly produced, combined with a pharmaceutically acceptable carrier. Advantageously, H-apoptin is used as a pharmaceutical composition, because of its pro-apoptotic activity in tumor cells. Even more advantageously, CPP-H-apoptin is used as a pharmaceutical composition, because of its facilitated delivery to the tumor cells. In this case, the H-apoptin protein or CPP-H-apoptin fusion protein is administered directly to a subject in need thereof.
As an alternative, it is possible to administer the H-apoptin gene of the invention to the said subject using gene therapy techniques. In this case, the pharmaceutical composition of the invention may contain the HGyV virus. Alternatively, the pharmaceutical composition of the invention may comprise the H-apoptin gene carried by a vector suitable for administration to a patient. Such vectors may be either derived from a virus or from a non-viral origin.
Non-viral vectors include plasmids. Such a plasmid may be a conditionally replicating plasmid that is incapable of replicating in the patients for safety reasons. These plasmids may be based on the plasmids described in the patent PCT applications WO 97/10343 and WO 2009/027351. Naked plasmid DNA can be directly injected into muscle cells (Wolff et al, Science, 247: 1465-1468, 1990) or attached to gold particles that are bombarded into the tissue (Cheng et al, Proc. Natl. Acad. Sci. U.S.A., 90: 4455-4459, 1993). Though not very efficient, this can result in prolonged low level expression in vivo. The plasmid DNA can also be transfected into the cell with the use of non-viral gene delivery vectors, termed “self-assembled” systems, based on cationic molecules, which form spontaneous complexes with negatively charged nucleic acids (Eliyahu et al., Molecules, 10: 34-64, 2005).
In another aspect of the invention, the vector is a viral vector. By replacing genes that are needed for the replication phase of the virus life cycle (the non-essential genes) with foreign genes of interest, the recombinant viral vectors can transduce the cell type it would normally infect. To produce such recombinant viral vectors the non-essential genes are provided in trans, either integrated into the genome of the packaging cell line or on a plasmid. Several vectors based on viruses such as adenovirus, adeno-associated virus (AAV), lentivirus, or herpes simplex virus 1 (HSV1), are available for gene therapy. All of them are encompassed within this invention.
Adenoviral vectors are currently the most frequently used viral vectors in gene therapy in humans. So-called third-generation (or “gutless”) adenoviral vectors (Lindermann and Schnittler, Thromb. Haemost., 102: 1135-1143, 2009) are preferred for use in the present invention. Said vectors need not be detailed here, since the skilled person is fully aware of the characteristics and uses of said adenoviral vectors.
Alternatively the skilled person may use a lentiviral vector to deliver the H-apoptin of the invention. Preferentially, the said lentiviral is a self-inactivating (SIN) lentivirus. In a further preferred embodiment, the lentiviral vector genome comprises, as an inserted cis-acting fragment, at least one polynucleotide consisting in the DNA flap (Zennou et al., Cell, 101: 173-185, 2000; WO 99/55892; WO 01/27304; WO 2009/019612) or containing such DNA flap. In a particular embodiment, the DNA flap is inserted upstream of the polynucleotide of interest, advantageously but not necessarily to be located in an approximate central position in the vector genome. Nevertheless, any lentiviral vector can be used in the context of the present invention. The construction and the manipulation of lentiviral vectors are well known to the skilled person.
The preferred viral vectors according to the invention are based on adenoviral-associated virus or AAV. Amongst the 8 serotypes, the AAV used for treating a neuromuscular disease according to the invention is preferentially an AAV1, i.e. its capsid is of the serotype 1. AAV1 has been shown to be the most efficient for muscle cells transduction. On the other hand, the sequences of a viral origin, and in particular the ITRs, associated to the transgene are preferably of AAV2 origin. The resulting AAV-based vector of the invention has, preferentially, a 2/1 pseudotype. The skilled person will easily realize, however, that the invention is not restricted to this particular vector; in fact, all AAV serotypes are equally suited for use in this invention. For example, AAV6, AAV8 or AAV9 also effectively transduce striated muscle cells, while AAV5 is highly efficient in transducing neural cells in the brain (Markakis et al., Molecular Therapy, 18: 588-593, 2010); all of them can therefore be used successfully in the context of the invention. Like adenoviral and lentiviral vectors, the AAV-based vectors have already been used extensively by the skilled person for gene therapy purposes (see e.g. Michelfelder and Trepel, Adv Genet., 67: 29-60, 2009); there is thus no need for detailing methods for constructing and using the said AAV vectors.
The pharmaceutical composition of the invention may contain, in addition to the carrier and H-apoptin or CPP-H-apoptin protein or gene or HGyV virus, various diluents, fillers, salts, buffers, stabilizers, solubilizers, and other materials well known in the art.
As used herein, “pharmaceutically acceptable carrier” includes any and all solvents, buffers, salt solutions, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. The type of carrier can be selected based upon the intended route of administration. In various embodiments, the carrier is suitable for intravenous, intraperitoneal, subcutaneous, intramuscular, topical, transdermal or oral administration. Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. The use of media and agents for pharmaceutically active substances is well known in the art. As detailed herebelow, additional active compounds can also be incorporated into the compositions, such as anti-cancer and/or anti-angiogenesis agents; in particular, the additional active compound can be an anti-angiogenic agent, a chemotherapeutic agent, or a low-molecular weight agent. A typical pharmaceutical composition for intravenous infusion could be made up to contain 250 ml of sterile Ringer's solution, and 100 mg of the combination. Actual methods for preparing parenterally administrable compounds will be known or apparent to those skilled in the art and are described in more detail in for example, Remington's Pharmaceutical Science, 17th ed., Mack Publishing Company, Easton, Pa. (1985), and the 18th and 19th editions thereof, which are incorporated herein by reference.
H-apoptin or CPP-H-apoptin protein or gene or HGyV virus in the composition preferably is formulated in an effective amount. An “effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired result, such as induction of apoptosis in tumor cells. A “therapeutically effective amount” means an amount sufficient to influence the therapeutic course of a particular disease state. A therapeutically effective amount is also one in which any toxic or detrimental effects of the agent are outweighed by the therapeutically beneficial effects.
For therapeutic applications, the H-apoptin or CPP-H-apoptin protein or gene or HGyV virus of the invention is administered to a mammal, preferably a human, in a pharmaceutically acceptable dosage form such as those discussed above, including those that may be administered to a human intravenously as a bolus or by continuous infusion over a period of time, by intramuscular, intraperitoneal, intracerebrospinal, subcutaneous, intraarticular, intrasynovial, intrathecal, oral, topical, or inhalation routes. The H-apoptin or CPP-H-apoptin protein is also suitably administered by intratumoral, peritumoral, intralesional, or perilesional routes, to exert local as well as systemic therapeutic effects. The intraperitoneal route is expected to be particularly useful, for example, in the treatment of ovarian tumors.
Dosage regimens may be adjusted to provide the optimum response. For example, a single bolus may be administered, several divided doses may be administered over time, or the dose may be proportionally reduced or increased. The compositions of the invention can be administered to a subject to effect cell growth activity in a subject. As used herein, the term “subject” is intended to include living organisms in which apoptosis can be induced, and specifically includes mammals, such as rabbits, dogs, cats, mice, rats, monkey transgenic species thereof, and preferably humans.
The H-apoptin protein encoded by the H-VP3 gene of the invention has therapeutic properties, because of its apoptotic activity in tumor cells. The invention thus also relates to the H-apoptin or CPP-H-apoptin protein or gene or HGyV virus as a medicament. More specifically, the H-apoptin or CPP-H-apoptin protein or gene or HGyV virus and the pharmaceutical compositions of the invention are useful in the treatment or prevention of a variety of cancers, including (but not limited to) the following: carcinoma, including that of the bladder, breast, colon, head and neck, kidney, including renal cell carcinoma, liver, lung, ovary, pancreas, stomach, cervix, thyroid and skin; including squamous cell carcinoma; hematopoietic tumors of lymphoid lineage, including leukemia, acute lymphocytic leukemia, acute lymphoblastic leukemia, B-cell lymphoma, T-cell lymphoma, Burkitt's lymphoma; hematopoietic tumors of myeloid lineage, including acute and chronic myelogenous leukemias and promyelocytic leukemia; tumors of mesenchymal origin, including fibrosarcoma and rhabdomyoscarcoma; other tumors, including melanoma, seminoma, tetratocarcinoma, neuroblastoma and glioma; tumors of the central and peripheral nervous system, including astrocytoma, neuroblastoma, glioma, and schwannomas; tumors of mesenchymal origin, including fibrosarcoma, rhabdomyosarcoma, and osteosarcoma; and other tumors, including melanoma, xeroderma pigmentosum, keratoactanthoma, seminoma, thyroid follicular cancer and teratocarcinoma, and other cancers yet to be determined. In a preferred embodiment, the H-apoptin protein of the invention is used to treat melanoma, leukemia, renal cancer, colon cancer, ovarian cancer, prostate cancer, lung cancer, bladder cancer, breast cancer, or head and neck cancer.
The present invention thus relates to the H-apoptin or CPP-H-apoptin protein or gene or HGyV virus described above for use in treating or inhibiting cancer-related diseases in a subject. It is an aspect or object of the present invention to provide a method of treating diseases and processes that result from cancer cell proliferation, and a composition for treating or repressing the growth of a cancer by induction of apoptosis. Yet another aspect of the invention is to provide compositions and methods useful for gene therapy for the modulation of cancer. The method of the present invention may be used in particular for the treatment of melanoma, leukemia, renal cancer, colon cancer, ovarian cancer, prostate cancer, lung cancer, bladder cancer, breast cancer, or head and neck cancer.
The present invention also includes kits, e.g., comprising one or more described H-apoptin or CPP-H-apoptin protein and/or gene and/or HGyV virus and instructions for the use of the said H-apoptin or CPP-H-apoptin protein and/or gene and/or HGyV virus for treating cancer. The instructions may include directions for using the H-apoptin or CPP-H-apoptin protein and/or gene and/or HGyV virus in vitro, in vivo or ex vivo. Typically, the kit will have a compartment containing the H-apoptin or CPP-H-apoptin protein and/or gene and/or HGyV virus. The H-apoptin or CPP-H-apoptin protein and/or gene and/or HGyV virus may be in a lyophilized form, liquid form, or other form amendable to being included in a kit. The kit may also contain additional elements needed to practice the method described on the instructions in the kit, such a sterilized solution for reconstituting a lyophilized powder, additional agents for combining with the H-apoptin or CPP-H-apoptin protein and/or gene and/or HGyV virus prior to administering to a patient, and tools that aid in administering the H-apoptin or CPP-H-apoptin protein and/or vector to a patient.
The effectiveness of the H-apoptin or CPP-H-apoptin protein and/or gene and/or HGyV virus in preventing or treating disease may be improved by administering said protein or gene serially or in combination with another agent that is effective for those purposes, such as one or more conventional therapeutic agents such as, for example, alkylating agents, folic acid antagonists, anti-metabolites of nucleic acid metabolism, antibiotics, pyrimidine analogs, 5-fluorouracil, cisplatin, purine nucleosides, amines, amino acids, triazol nucleosides, or corticosteroids. In another aspect of the invention, the administration is combined with an administration of therapeutically effective amount of chemotherapeutic agent, such as for example, taxol (paclitaxel) or taxotere (docetaxel).
Chemotherapeutic agents include without any limitations, anti-microtubule agents such as diterpenoids and vinca alkaloids; platinum coordination complexes; alkylating agents such as nitrogen mustards, oxazaphosphorines, alkylsulfonates, nitrosoureas, and triazenes; antibiotic agents such as anthracyclins, actinomycins and bleomycins; topoisomerase II inhibitors such as epipodophyllotoxins; antimetabolites such as purine and pyrimidine analogues and antifolate compounds; topoisomerase I inhibitors such as camptothecins; hormones and hormonal analogues; signal transduction pathway inhibitors; non-receptor tyrosine kinase angiogenesis inhibitors; immunotherapeutic agents; proapoptotic agents; and cell cycle signaling inhibitors. In addition, the methods of the invention can be combined with another anti-cancer treatment, anti-angiogenic agent, or chemotherapeutic agent or radiation therapy. A preferred example is docetaxel or taxotere. Other examples include, gemcitabine, cisplatin diterpenoids and vinca alkaloids, paclitaxel, vinblastine, vincristine, and vinorelbine, carboplatin, cyclophosphamide, melphalan, and chlorambucil, busulfan, carmustine, dacarbazine, cyclophosphamide, melphalan, chlorambucil, busulfan, carmustine, dacarbazine, anti-neoplastic agents including, but not limited to, actinomycins such as dactinomycin, anthrocyclins such as daunorubicin and doxorubicin, bleomycins, epipodophyllotoxins, etoposide and teniposide; antimetabolite neoplastic agents, 5-fluorouracil, methotrexate, cytarabine, mecaptopurine, thioguanine, camptothecins, irinotecan HCl, and topotecan HCl.
A variety of different chemotherapeutic agents or anti-cancer polypeptides can also be selected. Information sources such as www.clinicaltrials.gov, vww.ncbi.nlm.nih, and www.druqs.com, include references to polypeptides and agents that can be selected.
Such other agents, e.g. anti-angiogenic agents or chemotherapeutic agents may be present in the composition being administered or may be administered separately. In one aspect of the invention, the administration is performed with the other active principle, either simultaneously, separately or sequentially over time. When the administration is performed simultaneously, the two active principles may be combined in a single pharmaceutical composition, comprising the two compositions, such as a tablet or a gel capsule. On the other hand, the two active principles may, whether or not they are administered simultaneously, be present in separate pharmaceutical compositions. To this end, the combination may be in the form of a kit comprising, on the one hand, the H-apoptin or CPP-H-apoptin protein and/or a H-apoptin-encoding vector, including HGyV, as described above and, on the other hand, the second active principle, the H-apoptin or CPP-H-apoptin protein and/or a H-apoptin-encoding vector as described above and the second active principle being in separate compartments and being intended to be administered simultaneously, separately, or sequentially over time.
The combination according to the present invention can be administered especially for tumor therapy in combination with chemotherapy, protein therapy (i.e. using a therapeutic agent such as an antibody or recombinant protein), gene therapy, radiotherapy, immunotherapy, surgical intervention, or a combination of these. Long-term therapy is equally possible as is adjuvant therapy in the context of other treatment strategies, as described above.
The examples that follow are merely exemplary of the scope of this invention and content of this disclosure. One skilled in the art can devise and construct numerous modifications to the examples listed below without departing from the scope of this invention.



FIGURE LEGENDS
FIG. 1: Nucleotide sequence of HGyV and principal features. Principal features are described by homology with the annotated sequence of CAV (accession number M55918)
FIG. 2: Nucleotide alignment between CAV and HGyV. HGyV was aligned with the CAV sequence (accession number M55918) using the CLC program. The consensus sequence (SEQ ID NO: 18).
FIG. 3: Nucleotide sequence of the promoter region of HGyV (SEQ ID NO: 15 and principal features (see text). Direct repeats (DRa to DRe) of 22 nt are shown with putative estrogen responsive elements ACGTCA (SEQ ID NO.: 31) that are underlined. Putative SP1 sites are depicted. The palindromic CAAT CAGA ATTG (SEQ ID NO: 17) possible estrogen responsive element is depicted.
FIG. 4: Comparison between CAV and HGyV VP1 at the amino acids level. HGyV VP1 was aligned with its CAV homolog (accession number P54088) using the CLC program. The consensus sequence (SEQ ID NO: 36).
FIG. 5: Comparison between CAV and HGyV VP2 at the amino acids level. HGyV VP1 was aligned with its CAV homolog (accession number P69485) using the CLC program. The consensus sequence (SEQ ID NO: 20).
FIG. 6: Comparison between CAV and HGyV apoptin at the amino acids level. HGyV apoptin was aligned with its CAV homolog (accession number P54094) using the CLC program. Principal domains of the A-apoptin protein are shown (LRS: leucine rich domain, NLS 1 and 2: Nuclear localization signal, NES: putative nuclear exportation signal). The amino acid Thr in position 108 of A-apoptin is depicted by a red arrow. The phosphorylation site at amino acid Thr-111 of H-apoptin predicted by the Netphos program (www.cbs.dtu.dk/services/NetPhos/) is also shown.



EXPERIMENTAL EXAMPLES
Isolation and Sequencing of HGyV
DNA samples extracted with an automatic EasyMag apparatus (BioMérieux, Marcy I'Etoile, France) from cutaneous swabs taken from healthy people were screened and amplified by the bacteriophage phi29 polymerase based multiple displacement amplification (MDA) assay using random primers. The reaction was performed essentially with the REPLI-g Midi kit (Qiagen) according to the manufacturer's instructions. This provides concateners of high molecular weight DNA.
Sequencing was conducted by an Illumina High Seq sequencer: 5 μg of high molecular weight DNA resulting from isothermal amplification were fragmented into 200 to 350 nt fragments, to which were ligated adapters; 7588712 reads of 100 nt were derived from the sample.
Sorting out the flow of Illumina sequences was first done by a subtractive database comparison procedure. To this end, the whole host genome sequence (NCBI build 37.1/assembly hg19) was scanned with the reads using SOAPaligner (remaining: 4664094 reads). A quick and very restrictive BLASTN study was also performed to eliminate additional host reads (remaining: 4469243 reads). The best parameters to be used have been determined previously. A number of assembly programs dedicated to short or medium-sized reads (Velvet, SOAPdenovo, CLC) have been tested for their efficiency in our pipeline. Optimal parameters have been set. The comparison of the single reads and contigs with already known genomic and taxonomic data was done on dedicated specialized viral, bacterial and generalist databases maintained locally (GenBank viral and bacterial databases, nr). The aforementioned databases were scanned using BLASTN and BLASTX. Binning (or taxonomic assignment) was based on the lowest common ancestor from the best hits among reads with a significant e-value.
Among different contigs found harboring a size compatible with a viral genome, a contig of 2315 nt was more deeply analyzed because it showed some homology with the members of the Circoviridae family and more specifically with the only known member of the genus Gyrovirus, i.e. the Chicken Anemia Virus (CAV). Although CAV was described and isolated more than 30 years ago in chicken showing severe anemia (Yuasa et al., Avian Dis, 23: 366-385, 1979), no homolog virus is currently known in humans and thus this result was unexpected. We thus conducted detailed analysis, which showed that this contig corresponded to the full or quasi full length-genome of a new virus species we have named HGyV (Human Gyrovirus), on the basis of the characteristics shown below.
Based on the sequence of this contig, we have defined a set of primers (Table 1) and confirmed the sequence of the whole genome by the Sanger method. The resulting sequence is presented in FIG. 1, which also presents its principal features. Alignment of the whole genome with that of CAV is depicted in FIG. 2. The overall nucleotide identity between the two genomes is low: in the zone of maximal homology, between nt 100 and 700 (CAV accession number M55918), the overall homology is around 70%.
This virus shares the general organization of the CAV genome. It contains:

    
    
        a 5′ region with the general features of the CAV promoter/enhancer (Miller et al., J. Virol., 79(5): 2859-2868, 2005) (see FIG. 3): like the CAV promoter, it includes repeated regions of 22 nt which were designated DRa to DRe. Like in CAV, the DR regions contain the sequence ACGTCA (AGCTCA in CAV), which both vary by one nucleotide from the estrogen response element (ERE) half site, (A)GGTCA. In fact, there is also one less conserved repetition just upstream of DRa (SEQ ID NO: 16: C/A G/T GTACAGGGGGGTACGTCA T/C C/A, position nt 153-174, that also contains the ACGTCA sequence, not shown in FIG. 3). In HGyV, these DRs are repeated without any space between them. In CAV, these repeats are in the number of 4 to 5 in function of the strains and are separated by insert sequences of 15 bp between the first two or three and the last two. This is not the case for HGyV. We did not evidence the Estrogen Responsive Elements (ERE) found around nt 50 in CAV; nevertheless, we found downstream of DRd a palindromic sequence (CAATCAGAATTG) (SEQ ID NO: 17) which could reveal similar characteristics: in fact ERE generally consists of pentamer or hexamers (A)GGTCA separated by a 2-3 by insert, but this consensus could vary widely (Aumais et al., J Biol Chem, 271: 12568-12577, 1996). We have identified Transcription factor binding sites with the help of the programs TFSEARCH and TFBIND. As in CAV several SP1 sites were found (see FIG. 3).
        three partially overlapping open reading frames corresponding to different proteins. We have used the names of the CAV counterparts (see details in Table 2). For reason of clarity we have named the CAV protein “avian proteins” (a-proteins) and the HGyV counterpart “human proteins” (h-proteins):
        
            VP1, the protein of nucleocapsid: the alignment of h-VP1 and a-VP1 is shown in FIG. 4.
            VP2, the phosphatase: the alignment of h-VP2 with a-VP2 is shown in FIG. 5.
            VP3, the non-structural protein, referred in CAV as apoptin. The alignment of H-apoptin with A-apoptin is shown in FIG. 6, together with the domains of A-apoptin that have been shown to be important in functions (review in Los et al., Biochim Biophys Acta, 1793(8): 1335-1342, 2009). The overall similarity with A-apoptin is low (less than 40%).
        
        
    
    


The A-apoptin alone can induce apoptosis in a broad range of transformed and cancer cells but not in non-transformed or primary cells (for a review see Los et al., Biochim Biophys Acta, 1793(8): 1335-1342, 2009). It induces the apoptosis by a mechanism implicating the mitochondrial (intrinsic) pathway and is thus independent of the death receptor (extrinsic) pathway. As shown in FIG. 5, the so-called Leucine Rich Stretch seems conserved with a high contents of hydrophobic aliphatic leucine or isoleucine aa. This region seems to interact with different cellular proteins that seem important for its functions (summarized in Los et al., Biochim Biophys Acta, 1793(8): 1335-1342, 2009). One of the NLS signal (NLS2) seems also conserved, together with the putative nuclear exportation signal (NES). Importantly, the Netphos 2.0 server predicts a phosphorylation site at position 110, which is located between NLS2 and NES and seems homolog to position Thr-108 of A-apoptin. In fact nuclear accumulation of A-apoptin, which is important for its pro-apoptotic activity, is dependent on the phosphorylation of Thr-108. This phosphorylation is mediated by a kinase active in tumors and transformed cells but not in normal cells.







TABLE 1







primers used for 


resequencing the HGyV genome with the method of Sanger








Primer sequence (5′→3′)
Expected size









Forward
Reverse
(bp)





SEQ ID NO: 21:
SEQ ID NO: 22:
642


5′-tccgttgcagtgtgctcgttg-3′
5′-gcacttagagggcttcccag-3′



 


SEQ ID NO: 23:
SEQ ID NO: 24:
654


5′-ctcatctgccggtgcattgg-3′
5′-ccgggctcctatgtggtaag-3′



 


SEQ ID NO: 25:
SEQ ID NO: 26:
698


5′-aaagcttgagcctccggaatg-3′
5′-taggcaagaggggcaacttgg-3′



 


SEQ ID NO: 27:
SEQ ID NO: 28:
711


5′-tagttgttgctcgtcggcgag-3′
5′-ctttcaccacgcaccaaggg-3′
















TABLE 2







Nucleotide positions in the HGyV genome


of the three main open reading frames.


HGyV Open Reading Frames











ORF
Position Start
Position End















H-apoptin
508
882



H-VP1
884
2281



H-VP2
381
1076










Replication of HGyV in Human Cells
Because of the high homology of HGyV with CAV, it was investigated whether HGyV can replicate in human cells. CAV is known not to be capable of such replication.
Cloning of the HGyV Virus
From the high molecular weight DNA resulting from isothermal amplification from the same swab sample, a full length genome of HGyV was amplified, using the following primers:








HGyV.12F (SEQ ID NO: 51):
GAAAGCGCTGCAAAGCTGGGG


 


HGyV.12R (SEQ ID NO: 52):
TGCAGCGCTTTCTGCTATGGG






The genome was cloned in the plasmid PCR2.1 (Invitrogen, TA cloning kit) so as Eco47III sites were located at both sides of the genome. We have named this molecular clone HGyV-PTQ1 and generated its sequence by Sanger sequencing. This sequence, represented by SEQ ID NO.: 37, is identical to the prototypal HGyV sequence, excepted for an insertion of 60 nt between positions 2263 et 2264 of the sequence represented by SEQ ID NO.: 1. This insertion introduces a stop codon, leading to the deletion of 5 amino acids at the C-term part of VP1 (SEQ ID NO.: 39).
Preparation of Circularized HGyV DNA Genome.
Plasmid DNA from HGyV-PTQ1 containing the complete HGyV DNA genome (30 μg) was digested with Eco47III and self-ligated by T4 DNA ligase treatment to circularize the genome (DNA was diluted at 10 ng/μL in order to favor intramolecular ligation). Ligation products were analyzed on a 0.8% agarose gel.
Electroporation Transfection.
Two clones of the lymphoblastoid cell line JURKAT (E6.1 and J77.20) were cotransfected by the Neon Transfection System (Invitrogen) using 3 μg of circularized HGyV DNA in addition to 7 μg of pEGFP-N1 (a plasmid expressing the Green Fluorescent Protein, Clontech Laboratories) allowing for estimating the transfection efficiency. At 48 h after transfection, medium was removed and replaced by new medium in order to reduce the amount of free-non-transfected DNA. Serials passages were made every three days and 2×106 cells were collected every time for detecting the presence of intracellular HGyV mRNAs. Cells from passage 4 to 7 were analyzed by extraction of total RNA and RT-PCR using the following primers, targeting respectively the VP1 and the VP2 protein:









HGyV-VP1-F2q (SEQ ID NO: 53): 



TGCTTGCAACAATGCCTTTAGA



 



HGyV-VP1-R2q (SEQ ID NO: 54): 



CGCTGGGTCTTTTTGTTTCTG



 



HGyV-VP2-F3q (SEQ ID NO: 55): 



GGCATACGAGCATCCAACAA



 



HGyV-VP2-R3q (SEQ ID NO: 56): 



TCGTCCGCAGGTGCAGAT






VP1 and the VP2 transcripts were detected in the cells. No PCR amplification was obtained in controls in which the reverse transcription step (RT) was omitted, which demonstrates that RNAs were evidenced and not residual amounts of genomic DNA. Because HGyV is a DNA virus, the sustained presence of HGyV RNAs during seven passages is a demonstration of viral replication. The maximum amount of these transcripts was obtained at the 6th passage. No transcript of VP1 or VP2 was detected in non-transfected cells.
In Vivo Inhibition of Tumor Growth by PTD4-H-Apoptin Protein Fusion
Expression of Avian and Human Apoptins:
Fusion proteins were constructed by inserting a protein transduction domain (PTD) based on the PTD4 domain described by Ho et al (Cancer Res, 61(2): 474-477, 2001), at the N-terminus of the apoptin protein, in order to facilitate apoptin entry into cells. A GGSGGS linker was inserted between the PTD domain and the apoptin moiety. The same constructions were made for H-apoptin and A-apoptin, with the goal of comparing the activity of H-apoptin with A-apoptin (Sun et al., Int J Cancer, 124(12): 2973-2981, 2009).
The following fusion proteins were designed:







PTD GGSGGS-H-apoptin


(SEQ ID NO: 58)


ARAAAAQARAGGSGGSTPRSRRRATTTQSELLTAYEHPTSSSPPAET


TSIEIQIGIGSTIITLSLPGYASVRVLTTRSAPADDGGVTGSRRLVD


LSHRRPRRTSSPEIYVGFAAKEKQQKENLITLRENGPPIKKLRL*


 


PTD-GGSGGS-A-apoptin


(SEQ ID NO: 60)


ARAAAAQARAGGSGGSNALQEDTPPGPSTVFRPPTSSRPLETPHCRE


IRIGIAGITITLSLCGCANARAPTLRSATADNSESTGFKNVPDLRTD


QPKPPSKKRSCDPSEYRVSELKESLITTTPSRPRTARRCIRL*






The genes were synthesized following codon optimisation (GeneArt AG, Regensburg, Germany) for production in E. coli. Negative cis-acting sites (such as splice sites, TATA-boxes, etc.) which may negatively influence expression were eliminated wherever possible. GC content was adjusted to prolong mRNA half-life. Codon usage was adapted to the bias of Escherichia coli. In addition, a 5′ NheI and a 3′ BamHI restriction sites were introduced. The resulting genes have the sequences represented by SEQ ID NOs: 57 and 58, respectively.
Each gene was cloned in the NheI and BamHI sites of the plasmid pET-28, downstream of the His-tag sequence. Both proteins were expressed and purified from E. coli using standard procedures either from the soluble or non-soluble fraction.
PTD-Apoptin Protein Induces Cell Death in Tumor Cells.
In order to test the pro-apoptotic activity of each fusion protein, the tumor cytotoxicity of PTD GGSGGS-H-apoptin and PTD GGSGGS-A-apoptin is determined in vitro on tumor primary cells isolated from a melanoma cancer xenograft model. The said tumor cells are responsive to adriamycin but not to 5-FU.
A primary cell culture is obtained from a frozen stock of explanted and dissociated xenografts. After thawing, cells are grown in complete growth medium (F12/DMEM supplemented with 8% fetal bovine serum, 100 μg/ml penicillin G sodium, 100 μg/ml streptomycin sulphate). After centrifugation and resuspension in complete growth medium, the cells are plated at a density of ca. 140 000 cells/cm2 in 75 cm2 cell culture flasks and maintained at 37° C. in a humidified atmosphere with 5% CO2 for at least one week. Then, the cells are harvested and seeded in 96-wells plates at a density of 2.5×103 cells/well for cytotoxicity assays. Cells are incubated 48 h at 37° C. prior to addition of the test molecules and selected comparison compounds and vehicle.
Test drugs are added at desired final concentrations and further incubated for 72 hr.
Cell viability is assessed before drugs' addition (TO) and 72 hrs. after drugs' addition by measuring ATP cell content using CellTiter-Glo® Luminescent Cell Viability Assay (Promega) according to the manufacturer's instructions. Luciferase activity is measured on a luminometer (PerkinElmer® EnVisionTM)
Each of the fusion protein is diluted in DMEM/F12 medium at the following concentrations: 1000 nM; 333.33 nM; 111.11 nM; 37.04 nM; 12.35 nM; 4.12 nM; 1.37 nM; 0.46 nM; 0.15 nM; and 0.05 nM. Each concentration of the 2 compounds is tested in triplicate and three different experiments are performed. In addition, a positive control (adriamycin) as well as a negative control (vehicle alone) is included in the experiment.
The IC50 is the concentration of drug that inhibits cell viability by 50%. Cell viability is calculated as a percentage of ATP value compared to vehicle-treated controls. IC50 is determined for each measurement using GraphPad Prism software. IC50 determination demonstrates conclusively that the PTD GGSGGS-H-apoptin fusion protein shows at least equivalent or superior activity than the corresponding avian fusion protein.

## Claims

1. A recombinant fusion protein comprising a cell penetrating peptide (CPP), a linker, and a polypeptide comprising a sequence at least 80% identical to SEQ ID NO: 7.

2. The recombinant fusion protein of claim 1, wherein the CPP comprises the amino acid sequence of SEQ ID NO: 40.

3. The recombinant fusion protein of claim 1, wherein the CPP comprises the amino acid sequence of SEQ ID NO: 41.

4. The recombinant fusion protein of claim 1, wherein the CPP comprises the amino acid sequence of SEQ ID NO: 42.

5. The recombinant fusion protein of claim 1, wherein the CPP comprises the amino acid sequence of SEQ ID NO: 43.

6. The recombinant fusion protein of claim 1, wherein the CPP comprises the amino acid sequence of SEQ ID NO: 44.

7. The recombinant fusion protein of claim 1, wherein the CPP comprises the amino acid sequence of SEQ ID NO: 45.

8. The recombinant fusion protein of claim 1, wherein the CPP comprises the amino acid sequence of SEQ ID NO: 46.

9. The recombinant fusion protein of claim 1, wherein the CPP comprises the amino acid sequence of SEQ ID NO: 47.

10. The recombinant fusion protein of claim 1, wherein the CPP comprises the amino acid sequence of SEQ ID NO: 48.

11. The recombinant fusion protein of claim 1, wherein the CPP comprises the amino acid sequence of SEQ ID NO: 49.

12. The recombinant fusion protein of claim 1, wherein the linker comprises the amino acid sequence represented by SEQ ID NO: 50.

13. The recombinant fusion protein of claim 1, wherein the recombinant fusion protein comprises the amino acid sequence represented by SEQ ID NO: 58.

14. The recombinant fusion protein of claim 1, wherein the recombinant fusion protein is encoded by the sequence represented by SEQ ID NO. 57.