Abstract:
The present disclosure relates to identification of genes upregulated by interferon-α administration, in particular the human genes corresponding to the cDNA sequences in GenBank designated g4586459, g2342476, g3327161 and g4529886. Determination of expression products of these genes is proposed as having utility in predicting responsiveness to treatment with interferon-α and other interferons which act at the Type 1 interferon receptor. Therapeutic use of the proteins encoded by the same genes is also envisaged.

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS  
       [0001]     This application is a continuation of U.S. application Ser. No. 10/203,311 filed Aug. 6, 2002, which was a national stage filing under 35 U.S.C. 371 of PCT/GB01/000541 filed Feb. 9, 2001, which International Application was published by the International Bureau in English on Aug. 16, 2001 and claims foreign priority from Great Britain applications 0002979.3 filed Feb. 9, 2000, 0002980.1 filed Feb. 9, 2000, 0002982.7 filed Feb. 9, 2000, and 0002981.9 filed Feb. 9, 2000; each of which is hereby incorporated herein in its entirety by reference. 
     
    
     BACKGROUND OF THE INVENTION  
       [0002]     1. Field of the Invention  
         [0003]     The present invention relates to identification of genes upregulated by interferon-α (IFN-α) administration. Detection of expression products of these genes may thus find use in predicting responsiveness to IFN-α and other interferons which act at the Type 1 interferon receptor. Therapeutic use of the proteins encoded by the same genes is also envisaged.  
         [0004]     2. Background of the Invention  
         [0005]     IFN-α is widely used for the treatment of a number of disorders. Disorders which may be treated using IFN-α include neoplastic diseases such as leukemia, lymphomas, and solid tumours, AIDS-related Kaposi&#39;s sarcoma and viral infections such as chronic hepatitis. IFN-α has also been proposed for administration via the oromucosal route for the treatment of autoimmune, mycobacterial, neurodegenerative, parasitic and viral disease. In particular, IFN-α has been proposed, for example, for the treatment of multiple sclerosis, leprosy, tuberculosis, encephalitis, malaria, cervical cancer, genital herpes, hepatitis B and C, HIV, HPV and HSV-1 and 2. It has also been suggested for the treatment of arthritis, lupus and diabetes. Neoplastic diseases such as multiple myeloma, hairy cell leukemia, chronic myelogenous leukemia, low grade lymphoma, cutaneous T-cell lymphoma, carcinoid tumours, cervical cancer, sarcomas including Kaposi&#39;s sarcoma, kidney tumours, carcinomas including renal cell carcinoma, hepatic cellular carcinoma, nasqpharyngeal carcinoma, haematological malignancies, colorectal cancer, glioblastoma, laryngeal papillomas, lung cancer, colon cancer, malignant melanoma and brain tumours are also suggested as being treatable by administration of IFN-via the oromucosal route, i.e. the oral route or the nasal route.  
         [0006]     IFN-α is a member of the Type 1 interferon family, which exert their characteristic biological activities through interaction with the Type 1 interferon receptor. Other Type 1 interferons include IFN-β, IFN-ω and IFN-τ.  
         [0007]     Unfortunately, not all potential patients for treatment with a Type 1 interferon such as interferon-α, particularly, for example, patients suffering from chronic viral hepatitis, neoplastic disease and relapsing remitting multiple sclerosis, respond favourably to Type 1 interferon therapy and only a fraction of those who do respond exhibit long-term benefit. The inability of the physician to confidently predict the therapeutic outcome of Type 1 interferon treatment raises serious concerns as to the cost-benefit ratio of such treatment, not only in terms of wastage of an expensive biopharmaceutical and lost time in therapy, but also in terms of the serious side effects to which the patient is exposed. Furthermore, abnormal production of IFN-α has been shown to be associated with a number of autoimmune diseases. For these reasons, there is much interest in identifying Type 1 interferon responsive genes since Type 1 interferons exert their therapeutic action by modulating the expression of a number of genes. Indeed, it is the specific pattern of gene expression induced by Type 1 interferon treatment that determines whether a patient will respond favourably or not to the treatment.  
       SUMMARY OF THE INVENTION  
       [0008]     It has now been found that the human genes corresponding to the cDNA sequences in GenBank assigned accession nos. g4586459, g2342476, g3327161 and g4529886, correspond to a mouse gene upregulated by administration of IFN-α by an oromucosal route or intravenously. These human genes are thus now also designated an IFN-α upregulated gene.  
         [0009]     The proteins corresponding to GenBank cDNAs g4586459, g2342476, g3327161 and g4529886 have previously had no assigned function. These proteins (referred to below as HuIFRG-1, HuIFRG-2, HuIFRG-3 and HuIFRG-4 proteins respectively), and functional variants thereof, are now envisaged as therapeutic agents, in particular for use as an anti-viral, anti-tumour or immunomodulatory agent. For example, they may be used in the treatment of autoimmune, mycobacterial, neurodegenerative, parasitic or viral disease, arthritis, diabetes, lupus, multiple sclerosis, leprosy, tuberculosis, encephalitis, malaria, cervical cancer, genital herpes, hepatitis B or C, HIV, HPV, HSV-1 or 2, or neoplastic disease such as multiple myeloma, hairy cell leukemia, chronic myelogenous leukemia, low grade lymphoma, cutaneous T-cell lymphoma, carcinoid tumours, cervical cancer, sarcomas including Kaposi&#39;s sarcoma, kidney tumours, carcinomas including renal cell carcinoma, hepatic cellular carcinoma, nasopharyngeal carcinoma, haematological malignancies, colorectal cancer, glioblastoma, laryngeal papillomas, lung cancer, colon cancer, malignant melanoma or brain tumours. In other words, such proteins may find use in treating any Type 1 interferon treatable disease.  
         [0010]     Determination of the level of HuIFRG-1, HuIFRG-2, HuIFRG-3 or HuIFRG-4 proteins or a naturally-occurring variant thereof, or the corresponding mRNA, in cell samples of Type 1 interferon-treated patients, e.g. patients treated with IFN-α, e.g. such as by the oromucosal route or intravenously, may also be used to predict responsiveness to such treatment. It has additionally been found that alternatively and more preferably, such responsiveness may be judged, for example, by treating a sample of human peripheral blood mononuclear cells in vitro with a Type 1 interferon and looking for upregulation or downregulation of an expression product, preferably mRNA, corresponding to the same gene.  
         [0011]     According to a first aspect of the invention, there is thus provided an isolated polypeptide comprising; 
        (i) the amino acid sequence of SEQ ID NO: 2, SEQ ID NO:4, SEQ ID NO:6 or SEQ ID NO: 8;     (ii) a variant thereof having substantially similar function, e.g. an immunomodulatory activity and/or an anti-viral activity and/or an anti-tumour activity; or     (iii) a fragment of (i) or (ii) which retains substantially similar function, e.g. an immunomodulatory activity and/or an anti-viral activity and/or an anti-tumour activity 
 
 for use in therapeutic treatment of a human or non-human animal, more particularly for use as an anti-viral, anti-tumour or immunomodulatory agent. As indicated above, such use may extend to any Type 1 interferon treatable disease. 
       
 
         [0015]     According to another aspect of the invention, there is provided an isolated polynucleotide, e.g. in the form of an expression vector, which directs expression in vivo of a polypeptide as defined above for use in therapeutic treatment of a human or non-human animal, more particularly for use as an anti-viral, anti-tumour or immunomodulatory agent. Such a polynucleotide will typically include a sequence comprising: 
        (a) the nucleic acid of SEQ. ID. NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO: 7 or the coding sequence thereof;     (b) a sequence which hybridises, e.g. under stringent conditions, to a sequence complementary to a sequence as defined in (a);     (c) a sequence that is degenerate as a result of the genetic code to a sequence as defined in (a) or (b); or     (d) a sequence having at least 60% identity to a sequence as defined in (a), (b) or (c); 
 
 such that the polypeptide encoded by said sequence is capable of expression in vivo. 
       
 
         [0020]     In a further aspect, the invention provides a method of predicting responsiveness of a patient to treatment with a Type 1 interferon, e.g. IFN-α treatment (such as IFN-α treatment by the oromucosal route or a parenteral route, for example, intravenously, subcutaneously or intramuscularly), which comprises determining the level of one or more proteins selected from the proteins defined by the sequences set forth in SEQ ID NO: 2, SEQ ID NO:4, SEQ ID NO:6 or SEQ ID NO: 8, and naturally-occurring variants thereof, e.g. allelic variants, or one or more of the corresponding mRNAs, in a cell sample from said patient, e.g. a blood sample, wherein said sample is obtained from said patient following administration of a Type 1 interferon, e.g. IFN-α by an oromucosal route or intravenously, or is treated prior to said determining with a Type 1 interferon such as IFN-α in vitro. Such determining may be combined with determination of any other protein or mRNA whose expression is known to be affected in human cells by Type 1 interferon administration e.g. IFN-α administration.  
         [0021]     The invention also provides: 
        a pharmaceutical composition comprising the protein defined by the amino acid sequence set forth in SEQ ID NO: 2, SEQ ID NO:4, SEQ ID NO:6 or SEQ ID NO: 8, or a functional variant thereof as defined above, and a pharmaceutically acceptable carrier or diluent;     a method of treating a subject having a Type 1 interferon treatable disease, which method comprises administering to the said patient an effective amount of such a protein; 
            use of such a protein in the manufacture of a medicament for use in therapy as an anti-viral or anti-tumour or immunomodulatory agent, more particularly for use in treatment of a Type 1 interferon treatable disease;     a pharmaceutical composition comprising a polynucleotide as defined above and a pharmaceutically acceptable carrier or diluent;     a method of treating a subject having a Type 1 interferon treatable disease, which method comprises administering to said patient an effective amount of such a polynucleotide;     use of such a polynucleotide in the manufacture of a medicament, e.g. a vector preparation, for use in therapy as an anti-viral, anti-tumour or immunomodulatory agent, more particularly for use in treating a Type 1 interferon treatable disease;     a polynucleotide capable of expressing in vivo an antisense sequence to a coding sequence for the amino acid sequence defined by SEQ ID NO: 2, SEQ ID NO:4, SEQ ID NO:6 or SEQ ID NO: 8, or a naturally-occurring variant of said coding sequence, for use in therapeutic treatment of a human or non-human animal and pharmaceutical compositions comprising such a polynucleotide in combination with a pharmaceutically acceptable carrier or diluent;     an antibody to the protein defined by the amino acid sequence set forth the in SEQ ID NO: 2, SEQ ID NO:4, SEQ ID NO:6 or SEQ ID NO: 8 for use in therapeutic treatment of a human or animal body and corresponding pharmaceutical compositions.    
               
 
       BRIEF DESCRIPTION OF THE SEQUENCES  
       [0030]     SEQ. ID. No. 1 is the amino acid sequence of human protein HuIFRG-1 and its encoding cDNA.  
         [0031]     SEQ. ID. No.2 is the amino acid sequence alone of HuIFRG-1 protein.  
         [0032]     SEQ. ID. No.3 is the amino acid sequence of human protein HuIFRG-2 and its encoding cDNA.  
         [0033]     SEQ. ID. No.4 is the amino acid sequence alone of HuIFRG-2 protein.  
         [0034]     SEQ. ID. No.5 is the amino acid sequence of human protein HuIFRG-3 and its encoding cDNA.  
         [0035]     SEQ. ID. No.6 is the amino acid sequence alone of HuIFRG-3 protein.  
         [0036]     SEQ. ID. No.7 is the amino acid sequence of human protein HuIFRG-4 and its encoding cDNA.  
         [0037]     SEQ. ID. No.8 is the amino acid sequence alone of HuIFRG-4 protein. 
     
    
     DETAILED DESCRIPTION OF THE INVENTION  
       [0038]     As indicated above, human proteins HuIFRG-1, HuIFRG-2, HuIFRG-3 or HuIFRG-4 and functional variants thereof are now envisaged as therapeutically useful agents, more particularly for use as an anti-viral, anti-tumour or immunomodulatory agent.  
         [0039]     A variant of HuIFRG-1, HuIFRG-2, HuIFRG-3 or HuIFRG-4 protein for this purpose may be a naturally-occurring variant, either an allelic variant or a species variant, which has substantially the same functional activity as HuIFRG-1, HuIFRG-2, HuIFRG-3 or HuIFRG-4 protein and is also upregulated in response to administration of IFN-α, e.g oromucosal or intravenous administration of IFN-α. Alternatively, a variant of HuIFRG-1, HuIFRG-2, HuIFRG-3 or HuIFRG-4 protein for therapeutic use may comprise a sequence which varies from SEQ. ID. No. 2 but which is a non-natural mutant.  
         [0040]     The term “functional variant” refers to a polypeptide which has the same essential character or basic function of HuIFRG-1, HuIFRG-2, HuIFRG-3 or HuIFRG-4 protein. The essential character of HuIFRG-1, HuIFRG-2, HuIFRG-3 or HuIFRG-4 protein may be deemed to be as an immunomodulatory polypeptide. A functional variant polypeptide may show additionally or alternatively anti-viral activity and/or anti-tumour activity.  
         [0041]     Desired anti-viral activity may, for example, be tested for as follows. A sequence encoding a variant to be tested is cloned into a retroviral vector such as a retroviral vector derived from the Moloney murine leukemia virus (MoMuLV) containing the viral packaging signal , and a drug-resistance marker. A pantropic packaging cell line containing the viral gag, and pol, genes is then co-transfected with the recombinant retroviral vector and a plasmid, pVSV-G, containing the vesicular stomatitis virus envelope glycoprotein in order to produce high-titre infectious replication-incompetent virus (Burns et al., Proc. Natl., Acad. Sci. USA 84, 5232-5236). The infectious recombinant virus is then used to transfect interferon sensitive fibroblasts or lymphoblastoid cells and cell lines that stably express the variant protein are then selected and tested for resistance to virus infection in a standard interferon bio-assay (Tovey et al., Nature, 271, 622-625, 1978). Growth inhibition using a standard proliferation assay (Mosmann, T., J. Immunol. Methods, 65, 55-63, 1983) and expression of MHC class I and class II antigens using standard techniques may also be determined.  
         [0042]     A desired functional variant of HuIFRG-1, HuIFRG-2, HuIFRG-3 or HuIFRG-4 protein may consist essentially of the sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 or SEQ ID NO: 8. A functional variant of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 or SEQ ID NO: 8 may be a polypeptide which has a least 60% to 70% identity, preferably at least 80% or at least 90% and particularly preferably at least 95%, at least 97% or at least 99% identity with the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 or SEQ ID NO: 8 over a region of at least 20, preferably at least 30, for instance at least 100 contiguous amino acids or over the full length of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 or SEQ ID NO: 8. Methods of measuring protein identity are well known in the art.  
         [0043]     Amino acid substitutions may be made, for example from 1, 2 or 3 to 10, 20 or 30 substitutions. Conservative substitutions may be made, for example according to the following Table. Amino acids in the same block in the second column and preferably in the same line in the third column may be substituted for each other.  
                                                           ALIPHATIC   Non-polar   G A P                   I L V               Polar-uncharged   C S T M                   N Q               Polar-charged   D E                   K R           AROMATIC       H F W Y                      
 
         [0044]     Variant polypeptide sequences for therapeutic use in accordance with the invention may be shorter polypeptide sequences, for example, a peptide of at least 20 amino acids or up to 50, 60, 70, 80, 100, 150 or 200 amino acids in length is considered to fall within the scope of the invention provided it retains appropriate biological activity of HuIFRG-1, HuIFRG-2, HuIFRG-3 or HuIFRG-4 protein. In particular, but not exclusively, this aspect of the invention encompasses the situation when the variant is a fragment of a complete naturally-occurring protein sequence.  
         [0045]     Variant polypeptides for therapeutic use in accordance with the invention may be chemically modified, e.g. post-translationally modified. For example, they may be glycosylated and/or comprise modified amino acid residues. They may also be modified by the addition of a sequence either at the N-terminus and/or C-terminus. Polypeptides for therapeutic use in accordance with the invention may be made synthetically or by recombinant means. Such polypeptides may be modified to include non-naturally occurring amino acids, e.g. D amino acids. Variant polypeptides for use in accordance with the invention may have modifications to increase stability in vitro and/or in vivo. When the polypeptides are produced by synthetic means, such modifications may be introduced during production. The polypeptides may also be modified following either synthetic or recombinant production.  
         [0046]     A number of side chain modifications are known in the protein modification art and may be present in variants for therapeutic use according to the invention. Such modifications include, for example, modifications of amino acids by reductive alkylation by reaction with an aldehyde followed by reduction with NaBH 4 , amidination with methylacetimidate or acylation with acetic anhydride.  
         [0047]     Polypeptides for use in accordance with the invention will be in substantially isolated form. It will be understood that the polypeptides may be mixed with carriers or diluents which will not interfere with the intended purpose of the polypeptide and still be regarded as substantially isolated.  
         [0000]     Polynucleotide Therapy  
         [0048]     As an alternative to administration of HuIFRG-1, HuIFRG-2, HuIFRG-3 or HuIFRG-4 protein, or a functional variant thereof as described above, an isolated polynucleotide may be administered, e.g. in the form of an expression vector such as a viral vector, which directs expression of the desired polypeptide in vivo. Hence, as indicated above, in a further embodiment the invention provides an isolated polynucleotide, which directs expression in vivo of a polypeptide as defined above, which polynucleotide includes a sequence comprising: 
        (a) the nucleic acid of SEQ. ID. NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO: 7 or the coding sequence thereof;     (b) a sequence which hybridises, e.g under stringent conditions, to a sequence complementary to a sequence as defined in (a);     (c) a sequence that is degenerate as a result of the genetic code to a sequence as defined in (a) or (b); or     (e) a sequence having at least 60% identity to a sequence as defined in (a), (b) or (c) 
 
 for use in therapeutic treatment of a human or non-human animal, more particularly for use as an anti-viral, anti-tumour or immunomodulatory agent. 
       
 
         [0053]     Preferably, such a polynucleotide will be a DNA. The coding sequence for HuIFRG-1, HuIFRG-2, HuIFRG-3 or HuIFRG-4 protein or a variant thereof may be provided by a cDNA sequence or a genomic DNA sequence. Polynucleotides comprising an appropriate coding sequence can be isolated from human cells or synthesised according to methods well known in the art, as described by way of example in Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, 2 nd  edition, Cold Spring Harbor Laboratory Press.  
         [0054]     Polynucleotides for use in accordance with the invention may include within them synthetic or modified nucleotides. A number of different types of modification to polynucleotides are known in the art. These include methylphosphonate and phosphothioate backbones, addition of acridine or polylysine chains at the 3′ and/or 5′ ends of the molecule. Such modifications may be incorporated to enhance the in vivo activity or life span of the polynucleotide as a therapeutic agent.  
         [0055]     Typically, a polynucleotide for use in accordance with the invention will include a sequence of nucleotides, which may preferably be a contiguous sequence of nucleotides, which is capable of hybridising under selective conditions to the complement of the coding sequence of SEQ. ID. NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO: 7. Such hybridisation will occur at a level significantly above background. Background hybridisation may occur, for example, because of other cDNAs present in a cDNA library. The signal level generated by the interaction between a desired coding sequence and the complement of the coding sequence of SEQ. ID. NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO: 7 will typically be at least 10 fold, preferably at least 100 fold, as intense as interactions between other polynucleotides and the target sequence. The intensity of interaction may be measured, for example, by radiolabelling the nucleic acid selected for probing, e.g. with  32 P. Selective hybridisation may typically be achieved using conditions of low stringency (0.3M sodium chloride and 0.03M sodium citrate at about 40° C.), medium stringency (for example, 0.3M sodium chloride and 0.03M sodium citrate at about 50° C.) or high stringency (for example, 0.03M sodium chloride and 0.003M sodium citrate at about 60° C.).  
         [0056]     The coding sequence of SEQ. ID. NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO: 7 may be modified for incorporation into a polynucleotide as defined above by nucleotide substitutions, for example from 1, 2 or 3 to 10, 25, 50 or 100 substitutions. Degenerate substitutions may, for example, be made and/or substitutions may be made which would result in a conservative amino acid substitution when the modified sequence is translated, for example as shown in the table above. The coding sequence of SEQ. ID. NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO: 7 may alternatively or additionally be modified by one or more insertions and/or deletions and/or by an extension at either or both ends provided it encodes a polypeptide with the appropriate functional activity compared to HuIFRG-1, HuIFRG-2, HuIFRG-3 or HuIFRG-4 protein.  
         [0057]     A nucleotide sequence capable of selectively hybridising to the complement of SEQ. ID. NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO: 7, or at least the coding sequence thereof, will be generally at least 70%, preferably at least 80 or 90% and more preferably at least 95% or 97%, homologous to such a DNA sequence. This homology may typically be over a region of at least 20, preferably at least 30, for instance at least 40, 60 or 100 or more contiguous nucleotides of the said DNA sequence.  
         [0058]     Any combination of the above mentioned degrees of homology and minimum size may be used to define nucleic acids comprising desired coding sequences, with the more stringent combinations (i.e. higher homology over longer lengths) being preferred. Thus for example a polynucleotide which is at least 80% homologous over 25, preferably over 30 nucleotides may be found suitable, as may be a polynucleotide which is at least 90% homologous over 40 nucleotides.  
         [0059]     Homologues of polynucleotide or protein sequences as referred to herein may be determined in accordance with well-known means of homology calculation, e.g. protein homology may be calculated on the basis of amino acid identity (sometimes referred to as “hard homology”). For example the UWGCG Package provides the BESTFIT program which can be used to calculate homology, for example used on its default settings, (Devereux et al. (1984)  Nucleic Acids Research  12, p387-395). The PILEUP and BLAST algorithms can be used to calculate homology or line up sequences or to identify equivalent or corresponding sequences, typically used on their default settings, for example as described in Altschul S. F. (1993) J Mol Evol 36:290-300; Altschul, S, F et al. (1990) J Mol Biol 215:403-10.  
         [0060]     Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/). This algorithm involves first identifying high scoring sequence pair (HSPs) by identifying short words of length W in the query sequence that either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighbourhood word score threshold (Altschul et al, supra). These initial neighbourhood word hits act as seeds for initiating searches to find HSP=s containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Extensions for the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T and X determine the sensitivity and speed of the alignment. The BLAST program uses as defaults a word length (W) of 11, the BLOSUM62 scoring matrix (see Henikoff and Henikoff(1992)  Proc. Natl. Acad. Sci.  USA 89: 10915-10919) alignments (B) of 50, expectation (E) of 10, M=5, N=4, and a comparison of both strands.  
         [0061]     The BLAST algorithm performs a statistical analysis of the similarity between two sequences; see e.g., Karlin and Altschul (1993)  Proc. Natl. Acad. Sci. USA  90: 5873-5787. One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a sequence is considered similar to another sequence if the smallest sum probability in comparison of the first sequence to the second sequence is less than about 1, preferably less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001.  
         [0062]     As indicated above, a polynucleotide for use in accordance with the invention in substitution for direct administration of HuIFRG- 1, HuIFRG-2, HuIFRG-3 or HuIFRG-4 protein or a functional variant thereof may preferably be in the form of an expression vector. Expression vectors are routinely constructed in the art of molecular biology and may, for example, involve the use of plasmid DNA and appropriate initiators, promoters, enhancers and other elements, such as for example polyadenylation signals which may be necessary, and which are positioned in the correct orientation, in order to allow for protein expression. Such vectors may be viral vectors. Examples of suitable viral vectors include herpes simplex viral vectors, replication-defective retroviruses, including lentiviruses, adenoviruses, adeno-associated virus, HPV viruses (such as HPV-16 and HPV-18) and attenuated influenza virus vectors. Other suitable vectors would be apparent to persons skilled in the art. By way of further example in this regard reference is made again to Sambrook et al., 1989 (supra).  
         [0063]     A polynucleotide capable of expressing in vivo an antisense sequence to a coding sequence for the amino acid sequence defined by SEQ. ID. No. 2, or a naturally-occurring variant thereof, for use in therapeutic treatment of a human or non-human animal is also envisaged as constituting an additional aspect of the invention. Again, such a polynucleotide may preferably be in the form of an expression vector. Such a polynucleotide will find use in treatment of diseases associated with upregulation of HuIFRG-1, HuIFRG-2, HuIFRG-3 or HuIFRG-4 protein.  
         [0064]     It will be appreciated that antibodies to HuIFRG-1, HuIFRG-2, HuIFRG-3 or HuIFRG-4 protein and antigen-binding fragments thereof may find similar use.  
         [0000]     Pharmaceutical Compositions  
         [0065]     A polypeptide for use in accordance with the invention is typically formulated for administration with a pharnaceutically acceptable carrier or diluent. The pharmaceutical carrier or diluent may be, for example, an isotonic solution. For example, solid oral forms may contain, together with the active compound, diluents, e.g. lactose, dextrose, saccharose, cellulose, corn starch or potato starch; lubricants, e.g. silica, talc, stearic acid, magnesium or calcium stearate and or polyethelene glycols; binding agents, e.g. starches, arabic gums, gelatin, methyl cellulose, carboxymethylcellulose or polyvinyl pyrrolidone; desegregating agents, e.g. starch, alginic acid, alginates or sodium starch glycolate; effervescing mixtures; dyestuffs; sweeteners; wetting agents, such as lecithin, polysorbates, laurylsulphates; and, in general, non-toxic and pharmacologically inactive substances used in pharmaceutical formulations. Such pharmaceutical preparations may be manufactured in known manner, for example, by means of mixing, granulating, tableting, sugar-coating, or film coating processes.  
         [0066]     Liquid dispersions for oral administration may be syrups, emulsions and suspensions. The syrups may contain as carriers, for example, saccharose or saccharose with glycerine and/or mannitol and/or sorbitol.  
         [0067]     Suspensions and emulsions may contain as carrier, for example a natural gum, agar, sodium alginate, pectin, methyl cellulose, carboxymethylcellulose, or polyvinyl alcohol. The suspensions or solutions for intramuscular injections may contain, together with the active compound, a pharmaceutically acceptable carrier, e.g. sterile water, olive oil, ethyl oleate, glycols, e.g. propylene glycol, and if desired, a suitable amount of lidocaine hydrochloride.  
         [0068]     Solutions for intravenous injection or infusions may contain as carrier, for example, sterile water or preferably they may be in the form of sterile, aqueous, isotonic saline solutions.  
         [0069]     The dose of polypeptide for use in accordance with the invention may be determined according to various parameters, especially according to the substance used; the age, weight and condition of the patient to be treated; the route of administration; and the required regimen. A physician will be able to determine the required route of administration and dosage for any particular patient. A typical daily dose is from about 0.1 to 50 mg per kg, preferably from about 0.1 mg/kg to 10 mg/kg of body weight, according to the activity of the specific active compound, the age, weight and condition of the subject to be treated, and the frequency and route of administration. Preferably, daily dosage levels are from 5 mg to 2 g.  
         [0070]     A polynucleotide for use in accordance with the invention will also typically be formulated for administration with a pharmaceutically acceptable carrier or diluent. Such a polynucleotide may be administered by any known technique whereby expression of the desired polypeptide can be attained in vivo. For example, the polynucleotide may be delivered intradermally, subcutaneously, or intramuscularly. Alternatively, the polynucleotide may be delivered across the skin using a particle-mediated delivery device. A polynucleotide for use in accordance with the invention may be administered by intranasal or oral administration.  
         [0071]     A non-viral vector for use in accordance with the invention may be packaged into liposomes or into surfactant. Uptake of nucleic acid constructs for use in accordance with the invention may be enhanced by several known transfection techniques, for example those including the use of transfection agents. Examples of these agents include cationic agents, for example calcium phosphate and DEAE dextran and lipofectants, for example lipophectam and transfectam. The dosage of the nucleic acid to be administered can be varied. Typically, the nucleic acid is administered in the range of from 1 pg to 1 mg, preferably from 1 pg to 10 □g nucleic acid for particle-mediated gene delivery and from 10 □g to 1 mg for other routes.  
         [0000]     Prediction of Type 1 Interferon Responsiveness  
         [0072]     As also indicated above, in a still further aspect the present invention provides a method of predicting responsiveness of a patient to treatment with a Type 1 interferon, e.g. IFN-α treatment such as IFN-α treatment by an oromucosal route or intravenously, which comprises determining the level of one or more of HuIFRG-1, HuIFRG-2, HuIFRG-3, HuIFRG-4 protein and naturally-occurring variants thereof, or one or more corresponding mRNAs, in a cell sample from said patient, wherein said sample is taken from said patient following administration of a Type 1 interferon or is treated prior to said determining with a Type 1 interferon in vitro.  
         [0073]     Preferably, the Type 1 interferon for testing responsiveness will be the Type 1 interferon selected for treatment. It may be administered by the proposed treatment route and at the proposed treatment dose. Preferably, the subsequent sample analysed may be, for example, a blood sample or a sample of peripheral blood mononuclear cells (PBMCs) isolated from a blood sample.  
         [0074]     More conveniently and preferably, a sample obtained from the patient comprising PBMCs isolated from blood may be treated in vitro with a Type 1 interferon, e.g. at a dosage range of about 1 to 10,000 IU/ml. Such treatment may be for a period of hours, e.g. about 7 to 8 hours. Preferred treatment conditions for such in vitro testing may be determined by testing PBMCs taken from normal donors with the same interferon and looking for upregulation of an appropriate expression product. Again, the Type 1 interferon employed will preferably be the Type 1 interferon proposed for treatment of the patient, e.g. recombinant IFN-α. PBMCs for such testing may be isolated in conventional manner from a blood sample using Ficoll-Hypaque density gradients. An example of a suitable protocol for such in vitro testing of Type 1 interferon responsiveness is provided in Example 6 below.  
         [0075]     The sample, if appropriate after in vitro treatment with a Type 1 interferon, may be analysed for the level of HuIFRG-1, HuIFRG-2, HuIFRG-3 or HuIFRG-4 protein or a naturally-occurring variant thereof. This may be done using an antibody or antibodies capable of specifically binding one or more of HuIFRG-1, HuIFRG-2, HuIFRG-3 or HuIFRG-4 protein and naturally-occurring variants thereof, eg. allelic variants thereof. Preferably, however, the sample will be analysed for mRNA encoding HuIFRG-1, HuIFRG-2, HuIFRG-3 or HuIFRG-4 protein or a naturally-occurring variant thereof. Such mRNA analysis may employ any of the techniques known for detection of mRNAs, e.g. Northern blot detection or mRNA differential display. A variety of known nucleic acid amplification protocols may be employed to amplify any mRNA of interest present in the sample, or a portion thereof, prior to detection. The mRNA of interest, or a corresponding amplified nucleic acid, may be probed for using a nucleic acid probe attached to a solid support. Such a solid support may be a micro-array carrying probes to determine the level of further mRNAs or amplification products thereof corresponding to Type 1 interferon upregulated genes, e.g. such genes identified as upregulated in response to oromucosal or intravenous administration of IFN-α. Methods for constructing such micro-arrays (also referred to commonly as nucleic acid, probe or DNA chips) are well-known (see, for example, EP-B 0476014 and 0619321 of Affymax Technologies N.V. and Nature Genetics Supplement January 1999 entitled “The Chipping Forecast”).  
         [0000]     The Following Examples Illustrate the Invention:  
       EXAMPLES  
     Example 1  
       [0076]     Previous experiments had shown that the application of 5 μl of crystal violet to each nostril of a normal adult mouse using a P20 Eppendorf micropipette resulted in an almost immediate distribution of the dye over the whole surface of the oropharyngeal cavity. Staining of the oropharyngeal cavity was still apparent some 30 minutes after application of the dye. These results were confirmed by using  125 I-labelled recombinant human IFN-□1-8 applied in the same manner. The same method of administration was employed to effect oromucosal administration in the studies which are described below.  
         [0077]     Six week old, male DBA/2 mice were treated with either 100,000 IU of recombinant murine interferon α (IFN α) purchased from Life Technologies Inc, in phosphate buffered saline (PBS), 10 μg of recombinant human interleukin 15 (IL-15) purchased from Protein Institute Inc, PBS containing 100 μg/ml of bovine serum albumin (BSA), or left untreated. Eight hours later, the mice were sacrificed by cervical dislocation and the lymphoid tissue was removed surgically from the oropharyngeal cavity and snap frozen in liquid nitrogen and stored at −80° C. RNA was extracted from the lymphoid tissue by the method of Chomczynski and Sacchi (Anal. Biochem. (1987) 162, 156-159) and subjected to mRNA Differential Display Analysis (Lang, P. and Pardee, A. B., Science, 257, 967-971).  
         [0000]     Differential Display Analysis  
         [0078]     Differential display analysis was carried out using the “Message Clean” and “RNA image” kits of the GenHunter Corporation essentially as described by the manufacturer. Briefly, RNA was treated with RNase-free DNase, and 1 μg was reverse-transcribed in 100 μl of reaction buffer using either one or the other of the three one-base anchored oligo-(dT) primers A, C, or G. RNA was also reverse-transcribed using one or the other of the 9 two-base anchored oligo-(dT) primers AA, CC, GG, AC, CA, GA, AG, CG, GC. All the samples to be compared were reverse transcribed in the same experiment, separated into aliquots and frozen. The amplification was performed with only 1 μl of the reverse transcription sample in 10 μl of amplification mixture containing Taq DNA polymerase and α- 33 P dATP (3,000 Ci/mmole). Eighty 5′ end (HAP) random sequence primers were used in combination with each of the (HT11) A, C, G, AA, CC, GG, AC, CA, GA, AG, CG or GC primers. Samples were then run on 7% denaturing polyacrylamide gels and exposed to authoradiography. Putative differentially expressed bands were cut out, reamplified according to the instructions of the supplier, and further used as probes to hybridise Northern blots of RNA extracted from the oropharyngeal cavity of IFN treated, IL-15 treated, and excipient treated animals.  
         [0000]     Cloning and Sequencing  
         [0079]     Re-amplified bands from the differential display screen were cloned in the Sfr 1 site of the pPCR-Script SK(+) plasmid (Stratagene), and cDNA amplified from the rapid amplification of cDNA ends were isolated by TA cloning in the pCR3 plasmid (Invitrogen). DNA was sequenced using an automatic di-deoxy sequencer (Perkin Elmer ABI PRISM 377).  
         [0000]     Identification of Human cDNA  
         [0080]     Differentially expressed murine 3′ sequences identified from the differential display screen were compared with random human expressed sequence tags (EST) present in the dbEST database of GenBank™ of the United States National Center for Biotechnology Information (NCBI). The sequences potentially related to the murine EST isolated from the differential display screen were combined in a contig and used to construct a human consensus sequence corresponding to a putative cDNA.  
         [0081]     One such cDNA was found to correspond to GenBank cDNA sequence g4586459. The corresponding polypeptide sequence is GenBank sequence g4586460, not assigned in GenBank any function.  
         [0082]     Other mouse genes upregulated in lymphoid tissue in response to oromucosal administration of IFN-α as described above have also been found to be upregulated in the spleen of mice in response to intravenous administration of IFN-α. A similar result is anticipated in respect of the mouse gene corresponding to the human gene identified by Genbank cDNA accesssion no. g4586459 when intravenous administration of IFN-α is carried out as described in Example 5 below.  
         [0083]     Furthermore, mRNAs corresponding to human gene analogues of mouse genes found to be upregulated in response to oromucosal and intravenous administration of IFN-α have been found to be enhanced in human peripheral blood mononuclear cells following treatment with IFN-α in vitro. The same result is anticipated for mRNA corresponding to the cDNA as set forth in SEQ ID NO: 1 when human peripheral blood mononuclear cells are treated with IFN-α as described in Example 6 below.  
       Example 2  
       [0084]     Previous experiments had shown that the application of 5 μl of crystal violet to each nostril of a normal adult mouse using a P20 Eppendorf micropipette resulted in an almost immediate distribution of the dye over the whole surface of the oropharyngeal cavity. Staining of the oropharyngeal cavity was still apparent some 30 minutes after application of the dye. These results were confirmed by using  125 I-labelled recombinant human IFN-□1-8 applied in the same manner. The same method of administration was employed to effect oromucosal administration in the studies which are described below.  
         [0085]     Six week old, male DBA/2 mice were treated with either 100,000 IU of recombinant murine interferon α (IFN α) purchased from Life Technologies Inc, in phosphate buffered saline (PBS), 10 μg of recombinant human interleukin 15 (IL-15) purchased from Protein Institute Inc, PBS containing 100 μg/ml of bovine serum albumin (BSA), or left untreated. Eight hours later, the mice were sacrificed by cervical dislocation and the lymphoid tissue was removed surgically from the oropharyngeal cavity and snap frozen in liquid nitrogen and stored at −80° C. RNA was extracted from the lymphoid tissue by the method of Chomczynski and Sacchi (Anal. Biochem. (1987) 162, 156-159) and subjected to mRNA Differential Display Analysis (Lang, P. and Pardee, A. B., Science, 257, 967-971).  
         [0000]     Differential Display Analysis  
         [0086]     Differential display analysis was carried out using the “Message Clean” and “RNA image” kits of the GenHunter Corporation essentially as described by the manufacturer. Briefly, RNA was treated with RNase-free DNase, and 1 μg was reverse-transcribed in 100 μl of reaction buffer using either one or the other of the three one-base anchored oligo-(dT) primers A, C, or G. RNA was also reverse-transcribed using one or the other of the 9 two-base anchored oligo-(dT) primers AA, CC, GG, AC, CA, GA, AG, CG, GC. All the samples to be compared were reverse transcribed in the same experiment, separated into aliquots and frozen. The amplification was performed with only 1 μl of the reverse transcription sample in 10 μl of amplification mixture containing Taq DNA polymerase and α- 33 P dATP (3,000 Ci/mmole). Eighty 5′ end (HAP) random sequence primers were used in combination with each of the (HT11) A, C, G, AA, CC, GG, AC, CA, GA, AG, CG or GC primers. Samples were then run on 7% denaturing polyacrylamide gels and exposed to authoradiography. Putative differentially expressed bands were cut out, reamplified according to the instructions of the supplier, and further used as probes to hybridise Northern blots of RNA extracted from the oropharyngeal cavity of IFN treated, IL-15 treated, and excipient treated animals.  
         [0000]     Cloning and Sequencing  
         [0087]     Re-amplified bands from the differential display screen were cloned in the Sfr 1 site of the pPCR-Script SK(+) plasmid (Stratagene), and cDNA amplified from the rapid amplification of cDNA ends were isolated by TA cloning in the pCR3 plasmid (Invitrogen). DNA was sequenced using an automatic di-deoxy sequencer (Perkin Elmer ABI PRISM 377).  
         [0000]     Identification of Human cDNA  
         [0088]     Differentially expressed murine 3′ sequences identified from the differential display screen were compared with random human expressed sequence tags (EST) present in the dbEST database of GenBank™ of the United States National Center for Biotechnology Information (NCBI). The sequences potentially related to the murine EST isolated from the differential display screen were combined in a contig and used to construct a human consensus sequence corresponding to a putative cDNA.  
         [0089]     One such cDNA was found to correspond to GenBank cDNA sequence g2342476. The corresponding polypeptide sequence is GenBank sequence g2342477, not assigned in GenBank any function.  
         [0090]     Other mouse genes upregulated in lymphoid tissue in response to oromucosal administration of IFN-α as described above have also been found to be upregulated in the spleen of mice in response to intravenous administration of IFN-α. A similar result is anticipated in respect of the mouse gene corresponding to the human gene identified by Genbank cDNA accesssion no. g2342476 when intravenous administration of IFN-α is carried out as described in Example 5 below.  
         [0091]     Furthermore, mRNAs corresponding to human gene analogues of mouse genes found to be upregulated in response to oromucosal and intravenous administration of IFN-α have been found to be enhanced in human peripheral blood mononuclear cells following treatment with IFN-α in vitro. The same result is anticipated for mRNA corresponding to the cDNA as set forth in SEQ. ID. No. 3 when human peripheral blood mononuclear cells are treated with IFN-α as described in Example 6 below.  
       Example 3  
       [0092]     Previous experiments had shown that the application of 5 μl of crystal violet to each nostril of a normal adult mouse using a P20 Eppendorf micropipette resulted in an almost immediate distribution of the dye over the whole surface of the oropharyngeal cavity. Staining of the oropharyngeal cavity was still apparent some 30 minutes after application of the dye. These results were confirmed by using  125 I-labelled recombinant human IFN-□1-8 applied in the same manner. The same method of administration was employed to effect oromucosal administration in the studies which are described below.  
         [0093]     Six week old, male DBA/2 mice were treated with either 100,000 IU of recombinant murine interferon α (IFN α) purchased from Life Technologies Inc, in phosphate buffered saline (PBS), 10 μg of recombinant human interleukin 15 (IL-15) purchased from Protein Institute Inc, PBS containing 100 μg/ml of bovine serum albumin (BSA), or left untreated. Eight hours later, the mice were sacrificed by cervical dislocation and the lymphoid tissue was removed surgically from the oropharyngeal cavity and snap frozen in liquid nitrogen and stored at −80° C. RNA was extracted from the lymphoid tissue by the method of Chomczynski and Sacchi (Anal. Biochem. (1987) 162, 156-159) and subjected to mRNA Differential Display Analysis (Lang, P. and Pardee, A. B., Science, 257, 967-971).  
         [0000]     Differential Display Analysis  
         [0094]     Differential display analysis was carried out using the “Message Clean” and “RNA image” kits of the GenHunter Corporation essentially as described by the manufacturer. Briefly, RNA was treated with RNase-free DNase, and 1 μg was reverse-transcribed in 100 μl of reaction buffer using either one or the other of the three one-base anchored oligo-(dT) primers A, C, or G. RNA was also reverse-transcribed using one or the other of the 9 two-base anchored oligo-(dT) primers AA, CC, GG, AC, CA, GA, AG, CG, GC. All the samples to be compared were reverse transcribed in the same experiment, separated into aliquots and frozen. The amplification was performed with only 1 μl of the reverse transcription sample in 10 μl of amplification mixture containing Taq DNA polymerase and α- 33 P dATP (3,000 Ci/mmole). Eighty 5′ end (HAP) random sequence primers were used in combination with each of the (HT11) A, C, G, AA, CC, GG, AC, CA, GA, AG, CG or GC primers. Samples were then run on 7% denaturing polyacrylamide gels and exposed to authoradiography. Putative differentially expressed bands were cut out, reamplified according to the instructions of the supplier, and further used as probes to hybridise Northern blots of RNA extracted from the oropharyngeal cavity of IFN treated, IL-15 treated, and excipient treated animals.  
         [0000]     Cloning and Sequencing  
         [0095]     Re-amplified bands from the differential display screen were cloned in the Sfr 1 site of the pPCR-Script SK(+) plasmid (Stratagene), and cDNA amplified from the rapid amplification of cDNA ends were isolated by TA cloning in the pCR3 plasmid (Invitrogen). DNA was sequenced using an automatic di-deoxy sequencer (Perkin Elmer ABI PRISM 377).  
         [0000]     Identification of Human cDNA  
         [0096]     Differentially expressed murine 3′ sequences identified from the differential display screen were compared with random human expressed sequence tags (EST) present in the dbEST database of GenBank™ of the United States National Center for Biotechnology Information (NCBI). The sequences potentially related to the murine EST isolated from the differential display screen were combined in a contig and used to construct a human consensus sequence corresponding to a putative cDNA.  
         [0097]     One such cDNA was found to correspond to GenBank cDNA sequence g3327161. The corresponding polypeptide sequence is GenBank sequence g3327162, not assigned in GenBank any function.  
         [0098]     Other mouse genes upregulated in lymphoid tissue in response to oromucosal administration of IFN-α as described above have also been found to be upregulated in the spleen of mice in response to intravenous administration of IFN-α. A similar result is anticipated in respect of the mouse gene corresponding to the human gene identified by Genbank cDNA accesssion no. g3327161 when intravenous administration of IFN-α is carried out as described in Example 5 below.  
         [0099]     Furthermore, mRNAs corresponding to human gene analogues of mouse genes found to be upregulated in response to oromucosal and intravenous administration of IFN-α have been found to be enhanced in human peripheral blood mononuclear cells following treatment with IFN-α in vitro. The same result is anticipated for mRNA corresponding to the cDNA as set forth in SEQ. ID. No. 5 when human peripheral blood mononuclear cells are treated with IFN-α as described in Example 6 below.  
       Example 4  
       [0100]     Previous experiments had shown that the application of 5 μl of crystal violet to each nostril of a normal adult mouse using a P20 Eppendorf micropipette resulted in an almost immediate distribution of the dye over the whole surface of the oropharyngeal cavity. Staining of the oropharyngeal cavity was still apparent some 30 minutes after application of the dye. These results were confirmed by using  125 I-labelled recombinant human IFN-□1-8 applied in the same manner. The same method of administration was employed to effect oromucosal administration in the studies which are described below.  
         [0101]     Six week old, male DBA/2 mice were treated with either 100,000 IU of recombinant murine interferon α (IFN α) purchased from Life Technologies Inc, in phosphate buffered saline (PBS), 10 μg of recombinant human interleukin 15 (IL-15) purchased from Protein Institute Inc, PBS containing 100 μg/ml of bovine serum albumin (BSA), or left untreated. Eight hours later, the mice were sacrificed by cervical dislocation and the lymphoid tissue was removed surgically from the oropharyngeal cavity and snap frozen in liquid nitrogen and stored at −80° C. RNA was extracted from the lymphoid tissue by the method of Chomczynski and Sacchi (Anal. Biochem. (1987) 162, 156-159) and subjected to mRNA Differential Display Analysis (Lang, P. and Pardee, A. B., Science, 257, 967-971).  
         [0000]     Differential Display Analysis  
         [0102]     Differential display analysis was carried out using the “Message Clean” and “RNA image” kits of the GenHunter Corporation essentially as described by the manufacturer. Briefly, RNA was treated with RNase-free DNase, and 1 μg was reverse-transcribed in 100 μl of reaction buffer using either one or the other of the three one-base anchored oligo-(dT) primers A, C, or G. RNA was also reverse-transcribed using one or the other of the 9 two-base anchored oligo-(dT) primers AA, CC, GG, AC, CA, GA, AG, CG, GC. All the samples to be compared were reverse transcribed in the same experiment, separated into aliquots and frozen. The amplification was performed with only 1 μl of the reverse transcription sample in 10 μl of amplification mixture containing Taq DNA polymerase and α- 33 P dATP (3,000 Ci/mmole). Eighty 5′ end (HAP) random sequence primers were used in combination with each of the (HT11) A, C, G, AA, CC, GG, AC, CA, GA, AG, CG or GC primers. Samples were then run on 7% denaturing polyacrylamide gels and exposed to authoradiography. Putative differentially expressed bands were cut out, reamplified according to the instructions of the supplier, and further used as probes to hybridise Northern blots of RNA extracted from the oropharyngeal cavity of IFN treated, IL-15 treated, and excipient treated animals.  
         [0000]     Cloning and Sequencing  
         [0103]     Re-amplified bands from the differential display screen were cloned in the Sfr 1 site of the pPCR-Script SK(+) plasmid (Stratagene), and cDNA amplified from the rapid amplification of cDNA ends were isolated by TA cloning in the pCR3 plasmid (Invitrogen). DNA was sequenced using an automatic di-deoxy sequencer (Perkin Elmer ABI PRISM 377).  
         [0000]     Identification of Human cDNA  
         [0104]     Differentially expressed murine 3′ sequences identified from the differential display screen were compared with random human expressed sequence tags (EST) present in the dbEST database of GenBank™ of the United States National Center for Biotechnology Information (NCBI). The sequences potentially related to the murine EST isolated from the differential display screen were combined in a contig and used to construct a human consensus sequence corresponding to a putative cDNA.  
         [0105]     One such cDNA was found to correspond to GenBank cDNA sequence g4529886. The corresponding polypeptide sequence is GenBank sequence g4529888, not assigned in GenBank any function.  
         [0106]     Other mouse genes upregulated in lymphoid tissue in response to oromucosal administration of IFN-α as described above have also been found to be upregulated in the spleen of mice in response to intravenous administration of IFN-α. A similar result is anticipated in respect of the mouse gene corresponding to the human gene identified by Genbank cDNA accesssion no. g4529886 when intravenous administration of IFN-α is carried out as described in Example 5 below.  
         [0107]     Furthermore, mRNAs corresponding to human gene analogues of mouse genes found to be upregulated in response to oromucosal and intravenous administration of IFN-α have been found to be enhanced in human peripheral blood mononuclear cells following treatment with IFN-α in vitro. The same result is anticipated for mRNA corresponding to the cDNA as set forth in SEQ. ID. No. 7 when human peripheral blood mononuclear cells are treated with IFN-α as described in Example 6 below.  
       Example 5  
       [0000]     Intravenous Administration of IFN-α 
         [0108]     Male DBA/2 mice are injected intravenously with 100,000 IU of recombinant murine IFN-α purchased from Life Technologies Inc. in 200:1 of PBS or treated with an equal volume of PBS alone. Eight hours later the animals are sacrificed by cervical dislocation and the spleen was removed using conventional procedures. Total RNA was extracted by the method of Chomczynski and Sacchi (Anal. Biochem. (1987) 162, 156-159) and 10.0 :g of total RNA per sample is subjected to Northern blotting in the presence of glyoxal and hybridised with a cDNA probe for the mRNA of interest as described by Dandoy-Dron et al. (J. Biol. Chem. (1998) 273, 7691-7697). The blots are first exposed to autoradiography and then quantified using a Phospholmager according to the manufacturer&#39;s instructions.  
       Example 6  
       [0000]     Testing Type 1 Interferon Responsiveness In Vitro  
         [0109]     Human peripheral blood mononuclear cells (PBMC) from normal donors are isolated on Ficoll-Hypaque density gradients and treated in vitro with 10,000 IU of recombinant human IFN-α2 (intron A from Schering-Plough) in PBS or with an equal volume of PBS alone. Eight hours later the cells are centrifuged (800×g for 10 minutes) and the cell pellet recovered. Total RNA is extracted from the cell pellet by the method of Chomczynski and Sacchi and 10.0 :g of total RNA per sample is subjected to Northern blotting as described in Example 5 above.  
         [0110]     The same procedure can be used to predict Type 1 interferon responsiveness using PBMC taken from a patient proposed to be treated with a Type 1 interferon.