Abstract:
The present invention relates to improved methods for the treatment or prevention of disease with gene therapy or vaccines comprising host optimized GM-CSF nucleic acid sequences. More particularly, the invention provides methods for codon optimized GM-CSF with enhanced expression and adjuvant activity ex vivo and in vivo. The present invention also provides for methods of optimizing homologous gene expression ex vivo and in vitro or in transgenic animals.

Description:
BACKGROUND OF THE INVENTION 
     1. Field of the Invention 
     The present invention relates to improved recombinant mammalian, particularly human and murine, nucleic acid sequences for granulocyte macrophage-colony stimulating factor (GM-CSF) and their use in improving response to vaccines or gene therapy used for treating or preventing disease. 
     2. Background of the Invention 
     Granulocyte macrophage-colony stimulating factor, a soluble secreted glycoprotein, is a potent immunomodulatory cytokine known to facilitate development and prolongation of both humoral and cellular mediated immunity. Additional activities include, for example, activation and enhanced maturation of antigen presenting cells, increasing the expression of MHC class II antigens, activation of mature granulocytes, macrophages and monocytes, and proliferation and differentiation of hematopoietic progenitor cells. These and other diverse immunomodulatory activities have made GM-CSF an attractive investigational cytokine for use as a vaccine adjuvant for improving the immune response to vaccines, including those used for the treatment of cancer and HIV. The cDNA for GM-CSF is known and its expression in cultured cells described previously. 
     Several strategies have been investigated using GM-CSF as an adjuvant. GM-CSF is a soluble, secreted protein. Vaccine strategies have been described wherein soluble GM-CSF is expressed in vitro (e.g., in yeast, bacteria, or cultured mammalian cells; U.S. Pat. Nos. 5,298,603 and 5,679,356), purified, and administered concomitantly with an immunogen, which may be derived from pathogens (e.g., viruses or bacteria), or molecules associated with cancer, such as melanoma antigen (MAGE). This approach has several problems, including the cost associated with heterologous expression of GM-CSF, non-native glycosylation patterns, and the short half-life observed with systemically administered proteins, which are degraded rapidly or diffuse away from the site of the immunogen. 
     Fusion proteins which comprise GM-CSF in tandem with a second protein, such as an antigen as described in U.S. Pat. No. 5,108,910, have also been described for use as an adjuvant, however, these compositions suffer the same problems described above with respect to the systemic administration of a purified protein, such as limited half-life, in addition to difficulties arising from unpredictable changes in tertiary structure that may affect the biologic activity of GM-CSF. An alternative approach to administering GM-CSF protein is gene therapy wherein DNA encoding GM-CSF protein is administered and subsequently taken up by the receipient&#39;s cells and GM-CSF protein produced in vivo. This approach offers the advantages of locally sustained release of the cytokine and has proven successful in murine models (Warren T L et al., “Uses of granulocyte-macrophage colonly-stimulating factor in vaccine development,”  Curr. Opin. in Hematology  2000;7:168-173. In addition, recipient cells can be removed from the organism, transfected with GM-CSF DNA, and then reintroduced into the organism at the desired location. The expression levels observed from these approaches may be insufficient for providing the intended immunomodulatory adjuvant activity. Recombinant forms of human GM-CSF have been created with the goal of improving the activity or permitting large scale protein production, however, these sequences alter the amino acid sequence and have been associated with adverse immunologic responses, presumably due to formation of novel epitopes that result from recombinant genes or exposed native protein backbone which is normally blocked by glycosylation (Gribben et al., “Development of antibodies to unprotected glycosylation sites on recombinant human GM-CSF,”  Lancet  1990 Feb. 24; 335(8687):434-7). For example, sargramostim differs from the native human GM-CSF by one amino acid at position 23 and a different carbohydrate moiety. In contrast, the present invention provides for improved human and murine GM-CSF encoding nucleic acid sequences that encode the identical amino acid sequence as the native gene but with improved in vivo adjuvant activity. The GM-CSF nucleic acid sequences of the present invention alter select nucleotides within the native sequence without altering the amino acid sequence of the native protein. 
     It is well know that most amino acids are specified by more than one trinucleotide codon and that codon usage varies across species with G+C bias in mammals compared with A+T usage in microorganisms, yeast, and viruses. Selective codon usage has been further characterized by the differences in gene expression observed across different genes from the same species. Genes that are expressed with high frequency include a bias toward particular codons. The codons associated with highly expressed genes also vary with species. The different frequency of codons observed across and within species has been described and categorized by frequency of appearance in the genome (e.g., Nakamura Y., et al., “Codon usage tabulated from the international DNA sequences databases: status for the year 2000 , ”Nucl. Acids Res . 28, 292 (2000)). These differences, such as A and T preferences in the third position of a codon observed in bacteria, have been incorporated into human genes, for example, for enhancing expression of heterologous genes in microorganisms (N. D. Connell, “Expression systems for use in actinomycetes and related organisms,”  Curr. Opin. Biotechnol ., 2001, October:12(5):446-9). U.S. Pat. No. 6,288,303 discusses optimizing the codon usage of mammalian genes for optimal expression in plant cells. 
     As described in detail herein below, the present invention provides for human and murine GM-CSF nucleic acid sequences that comprise codons associated with high expression levels in humans and mice, respectively, and surprisingly result in improved adjuvant activity compared with the native sequence. 
     SUMMARY OF THE INVENTION 
     The codon optimized mammalian, particularly human and murine GM-CSF nucleic acid sequences of the present invention, although encoding the same amino acid sequence as the native human or murine GM-CSF, provide enhanced adjuvant activity when expressed in vivo compared to the native GM-CSF gene sequence. The present invention provides modified GM-CSF DNA sequences wherein codons in the native gene have been replaced with trinucleotide codons identified in highly expressed genes of the same host (i.e., humans or mice). 
     The codon optimized GM-CSF sequences of the present invention preserve the native amino acid sequence encoded by the host, and therefore, all native glycosylation sites and processing signals, but result in improved biologic responses when used as a vaccine adjuvant compared to the native gene or recombinant GM-CSF genes of the prior art. The codon optimized GM-CSF nucleic acid sequences are utilized, according to the present invention, for improved biologic responses in support of prevention and treatment of disease by vaccines and gene therapy, and for research purposes. 
    
    
     BRIEF DESCRIPTION OF THE FIGURES 
     FIG.  1 —Alignment of the native murine GM-CSF cDNA sequence with the codon optimized murine sequences of the present invention. The more preferred embodiment is shown immediately above the native sequence. 
     FIG.  2 —Wild type (“wt”) and codon optimized (“c”) murine GM-CSF expression in transfected COS-7 cells. The COS-7 cells were transfected with wild type or codon optimized murine GM-CSF DNA constructs. Cell lysates from control vector pcDNA3.1-, pwt-GMCSF- and pc-GMCSF-transfected cells were separated by SDS-PAGE, transferred to nitrocellulose membranes, and analyzed by immunoblotting with a rabbit anti-murine GM-CSF antibody (top panels). Soluble GM-CSF released from transfected cells was monitored by ELISA (bottom panel). 
     FIG.  3 —Murine GM-CSF expression in transfected 293 and HeLa cells. The 293 and HeLa cells were transfected with wild type or codon optimized murine GM-CSF DNA constructs. Cell lysates from control vector pcDNA3.1-, pwt-GMCSF- and pc-GMCSF-transfected cells were separated by SDS-PAGE, transferred to nitrocellulose membranes, and analyzed by immunoblotting with a rabbit anti-murine GM-CSF antibody (top panels). Soluble GM-CSF released from transfected cells was monitored by ELISA (bottom panel). 
     FIG.  4 —Immune responses to HIV-1 GAG in BALB/c mice coinjected with pGMCSF/pcGMCSF and pGag DNA plasmids. Mice were coinjected i.m. with 50 μg pGag and a total of 50 μg pwt-GMCSF or pc-GMCSF and or with corresponding control vector plasmids respectively, as indicated. Plasmid doses were divided in half between the two gastrocnemius muscles. Anti-Gag antibodies were evaluated after vaccinia virus challenge. Serum IgG reactivity to pGag was measured by ELISA using recombinant HIV-1 Gag as coating antigen. Sera were tested as a pool of 3 sera diluted 1:300. Vaccinia/Gag, sera from mice infected with recombinant vaccinia virus/Gag as a positive control. Background OD values of preimmune serum at 1:300+2 SD (0.1) were subtracted. 
     FIG.  5 —ELISPOT assay. Mice were coinjected i.m. twice two weeks apart with 50 μg pGag and a total of 50 μg pwt-GMCSF or pc-GMCSF and or with corresponding control vector plasmids respectively, as indicated. Plasmid doses were divided in half between the two gastrocnemius muscles. Immunized mice were i.p. challenged with a recombinant vaccinia virus vector encoding HIV-1 Gag (10 7  PFU per mouse) at 35 days after the final DNA vaccination. Splenocytes were harvested 5 days after the recombinant vaccinia virus challenge. The spot numbers are the means of the triplicates. Error bars indicate the standard deviations from triplicated cultures. 
     FIG.  6 —Intracellular cytokine staining and flow cytometry analysis. Mice were coinjected i.m. twice two weeks apart with 50 μg pGag and a total of 50 μg pwt-GMCSF or pc-GMCSF and or with corresponding control vector plasmids respectively, as indicated. Plasmid doses were divided in half between the two gastrocnemius muscles. 
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     The wild type human and murine GM-CSF cDNA sequences were optimized by first identifying codons within the cDNA which are not associated with the codon usage in highly expressed genes in humans and mice, respectively. Each sub-optimal codon is replaced with those identified from highly expressed genes of the same species, which are known in the art and available online, for example, at http://www.kazusa.or.jp/codon/. This usually requires only by a single base change in the third position but may also require changes in the second position. The codon optimized sequences can be prepared entirely by cDNA synthesis or created from wild type cDNA using molecular cloning methods well known in the art (e.g., Maniatis, T. 1989. “Molecular Cloning: A Laboratory Manual,” Cold Spring Harbor Laboratory Press, NY, vol 1, 2, 3). 
     This invention embraces the optimized gene sequences encoding mammalian GM-CSF together with their use as an adjuvant in gene therapy or in connection with vaccines to obtain a stronger immunoresponse to expressed proteins. The methods, per se, do not constitute part of the invention, save for the use of the novel optimized sequences addressed herein. Representative formulations, dosages and administration, such as those established for human patients, can be found with respect to GM-CSF in Hamm et al,  J. Clin. Oncol . December 1994; 12(12):2667-76 and Bergmann et al.,  Eur. J. Cancer  December 1995; 31A(13-14):2164. The utilization of the inventive subject matter is in manner not distinguishable per se from conventional DNA delivery techniques in connection with gene therapy and vaccines. Both ex vivo and in vivo utilities are embraced by the invention. 
     In particular, the sequences of this invention can be used in conjunction with a wide variety of DNA vaccines, where the effective composition includes as effective elements DNA, either naked or in the form of modified vectors, together with an appropriate carrier and the codon optimized GM-CSF DNA of the invention. Appropriate carriers include everything from saline solutions for conventional IM preparations to gene guns and preparations therefor to liposome complexes. 
     The inventive sequences of this invention are also suitable for use in ex vivo and in vivo cancer therapies, including radiation and chemotherapeutic treatment of cancer cells, particularly tumor cells, which have been transduced/transfected with GM-CSF vectors, commonly in the form of adenoviral vectors carrying GM-CSF as a heterologous transgene. Other transfer means are set forth, e.g., in U.S. Pat. No. 5,910,488. 
     Those of skill in the art will appreciate that there is a large, but finite number of combinations of codon bases that can be utilized to optimize expression. The invention embraces any optimization of mammalian, particularly human and murine, GM-CSF sequences. Among prefexred embodiments is that set forth in FIG.  1 . 
     EXAMPLE 
     This invention is further illustrated by reference to the example set forth below. In the example, the murine GM-CSF sequence has been codon optimized. The same methods can be used to optimize other mammalian sequences encoding GM-CSF, including, preferably, human with predictably similar results. The degree of enhanced activity will depend, in part, on the completeness of optimization. Optimization of a single codon may have limited impact, in contrast with the optimization of a plurality of codons of a given sequence. All the alternatives are embraced within the invention exemplified herein. 
     Experimental Protocol and Results 
     Construction of murine GM-CSF expression vectors. The native sequences coding for the 153 amino acids (aa) of murine GM-CSF (GenBank accession no. X02333; FIG. 1 upper sequence) was amplified by PCR and cloned into pcDNA3.1(−) downstream from the human CMV immediate-early promoter (Pcmv) sequence, using BamH I sites to generate pm-wtGMCSF. To generate pm-cGMCSF, the codon modified form of murine GMCSF, we changed the codon usage to that utilized by highly expressed human genes as described recently for HIV-1 gp120 and gag-pol (Haas, J., E. Park, and B. Seed. 1996. Codon usage limitation in the expression of HIV-1 envelope glycoprotein. Curr. Biol. 6:315-32; Kotsopoulou E, Kim V N, Kingsman A J, ingsman S M, and Mitrophanous K A. 2000. A Rev-independent human immunodeficiency virus type 1 (HIV-1)-based vector that exploits a codon-optimized HIV-1 gag-pol gene. J Virol 74(10):4839-52). 
     The resulting modified murine GM-CSF encoding a protein without any amino acid change (FIG. 1 lower sequence). The resulting 480-bp gene cassette included the XbaI and Xho I cloning sites and was constructed synthetically by the Aptagen Company (Herndon, Va.). This modified murine GM-CSF sequence was cloned into the XbaI and Xho I restriction sites of the eukaryotic expression vector pcDNA3.1(−) that employs the cytomegalovirus (CMV) immediate-early enhancer/promoter and bGH terminator (Invitrogen, San Diego, Calif.), resulting in the plasmid pm-cGMCSF. The CMV promoter provides a high level of constitutive expression in a range of mammalian cells. The BGHpA signal provides efficient transcription termination and polyadenylation of mRNA. 
     Comparison of murine GM-CSF expression by the various constructs in primate cells. We initially tested the protein expression by various murine GM-CSF expression vectors in transfected COS-7 cells, an African green monkey kidney-derived cell line. When cell lysates from transfected COS-7 cells were analyzed by immunoblotting with a rabbit anti-murine GM-CSF antibody, GM-CSF precursor protein was not detected in pcDNA3.1-transfected COS-7 cells, as expected (FIG. 2, lane 1, top panel). Also, GM-CSF protein was not detected in pm-wtGMCSF-transfected COS-7 cells (FIG. 2, lane 2, top panel). GM-CSF protein was detected in transfected COS-7 cells with the pm-cGMCSF constructs (FIG. 2, top panel). 
     The secreted GM-CSF protein was also detected in culture supernatants of pm-cGMCSF-transfected COS-7 cells by the rabbit anti-murine GM-CSF antibody (FIG. 2, bottom panel). As expected, no GM-CSF protein was detected in culture supernatants of pcDNA3.1 (lane 1)-transfected COS-7 cells (FIG. 2, bottom panel). Again, the pm-cGMCSF-transfected COS-7 cells produced much more GM-CSF proteins than the pm-wtGMCSF-transfected COS-7 cells (FIG. 2, bottom panel). These results suggest that the mutations modifying the codon usages in the murine GM-CSF coding sequence increase GM-CSF expression in COS-7 cells. 
     Comparison of HIV-1 Gag expression by the various constructs in human cells. GM-CSF expression was also evaluated with the various constructs in transfected 293 cells, a human embryonal kidney epithelial cell line and HeLa cells. Immunoblot analysis of cell lysates prepared from transfected 293 cells is shown in FIG. 3 (top panel). Transfection of the 293 cells with pcDNA3.1 or pm-wtGMCSF did not produce detectable levels of GM-CSF protein by immunoblot analysis (FIG. 3, top panel, lanes 1 and 2). On the other hand, pm-cGMCSF-transfected 293 cells produced detectable GM-CSF (FIG.  3 ). GM-CSF protein was also detected in the supematants of pm-cGMCSF and pm-wtGMCSF-transfected 293 cells (FIG. 3, bottom panel) but no GM-CSF was detected in the supematants of pcDNA3.1-transfected 293 cells (FIG. 3, bottom panel). Again, the pm-cGMCSF-transfected 293 cells produced much more GM-CSF proteins than the pm-wtGMCSF-transfected 293 cells. 
     To compare levels of GM-CSF protein expression in human cancer cells by the various murine GM-CSF constructs, we performed transient transfection experiments using HeLa cells. Three days after transfection, cell lysates from transfected HeLa cells were analyzed by immunoblotting for the presence of cell-associated GM-CSF molecules (FIG. 3, top panel). GM-CSF was only detected in the cell lysates of HeLa cells transfected by pm-cGMCSF (lane 6, FIG. 3, top panel). No GM-CSF was detected in the cell lysates of pcDNA3.1- and pm-wtGMCSF-transfected HeLa cells by rabbit anti-murine GM-CSF antibody (FIG. 3, top panel, lanes 4 and 5). GM-CSF was also detected in the supernatants of pm-cGMCSF-transfected HeLa cells (FIG. 3, bottom panel). No GM-CSF was detected in the supernatants of pcDNA3.1-transfected HeLa cells (FIG. 3, bottom panel). 
     Anti-Gag antibody responses in mice immunized with naked DNA vaccine. BALB/c mice were intramuscularly injected with total 100 μg of plasmid DNA two times at 0 and 2 weeks. Anti-Gag antibodies were measured after virus challenge. Sera were collected from the mice injected with the different DNA constructs and analyzed by immunoblot using purified Gag p55 recombinant protein from  Escherichia coli . As shown in FIG. 4, anti-Gag antibodies were detected in mice vaccinated with either pGAG/wt-GM (lane 3) or pGAG/cGM DNA mixture (lane 4) and in mice infected with recombinant vaccinia virus/Gag (lane 5). In contrast, no anti-Gag antibodies were detected in mice immunized with pcDNA3.1 and pGAG/vector DNA mixture (FIG. 4, lanes 1 and 2). Administration of pGAG plasmid alone did not induced detectable IgG antibody responses. The co administration of the wild type GM-CSF plasmid induced only a modest increase in total IgG anti-Gag serum titers. By comparison, co administration of the codon modified GM-CSF plasmid significantly increased total IgG serum titers. 
     ELISPOT assay in DNA-vaccinated mice. Antigen-specific CD8 +  T cell responses were measured by two methods: IFN-γ ELISPOT assay and Intracellular cytokine staining and flow cytometry analysis. Mice were immunized with the various DNA constructs as described above. At Day 5 after recombinant vaccinia virus challenge, splenocytes from the mice in each group were harvested and pooled, and the IFN-γ ELISPOT assay specific to HIV-1 Gag peptide were measured following antigen stimulation. As expected, those plasmids that did not express Gag p55 in vitro, such as pcDNA3.1 did not elicit an antigen-specific CD8 +  T cell response against HIV-1 Gag (FIG.  5 ). A 3-fold increase in numbers of cells secreting Ag-specific IFN-γ to either peptide was detected in mice immunized with the addition of wild type GM-CSF plasmid (500 SFU vs. 170 SFU, FIG. 5, lane 3 vs. 2). The group of vaccinated mice that received pm-cGMCSF developed a slightly higher level of HIV-1 Gag-specific CD8 +  activity than those that received pm-wtGMCSF. These results showed that intramuscular injection of the mixture of pGAG and codon modified GM-CSF plasmids had larger numbers of IFN-γ secreting cells that remained elevated longer after the second immunization and can induce both humoral and cellular immune responses against HIV-1 Gag in a murine model. Intracellular cytokine staining and flow cytometry analysis. CD8 +  T lymphocytes are one of the most crucial components of antiviral effector cells. We therefore assessed the number of HIV-1 Gag-specific CD8 +  T cells expressing IFN-γ in the spleens of mice immunized with DNA vector and challenged with vP1287. These cells were measured by brief stimulation in vitro for 24 h with MHC class I-restricted HIV p24 peptide (amino acid sequence: AMQMLKETI), followed by staining for CD8 and intracellular IFN-γ. 
     Intracellular cytokine staining and ELISPOT are sensitive functional assays used to measure the IFN-γ production at the single-cell level. We observed a high level of Gag-specific CD8 +  effector cells in the spleens of mice co immunized with the mixture pGAG plasmid and pm-cGMCSF plasmid (1.14% of the total splenocytes and 10.2% of the total CD8 +  T cells) within 5 days of vP1287 challenge (FIG.  6 ). In contrast, less than 0.02% of the total splenocytes were CD8 +  T cells expressing IFN-γ in control vector-immunized mice. The level of Gag-specific CD8 +  effector cells in the spleens of mice immunized with pm-wtGMCSF plasmid (0.79% of the total splenocytes and 8% of the total CD8 +  T cells) was lower than that obtained with pm-c-GMCSF. The background level in mice immunized with pcDNA3.1 and challenged with recombinant vaccinia virus expressing HIV-1 Gag was low, suggesting that Gag-specific CD8 +  IFN-γ-producing cells were generated from memory T cells. 
     For application of the above example to other codons and other optimization solutions, reference to the following sequences may be of value. 
     Sequences 
     Bolded trinucleotides, shown below in the native and codon optimzied sequences, are representative of those codons which may be modified according to the present invention. 
     
       
         
               
               
             
               
               
               
             
               
               
             
               
               
               
             
               
               
             
               
               
               
             
           
               
                 Native Human Exon 1: 
                   
               
               
                 Seq. ID NO: 1: 
               
               
                 ATGTGGCTGCAGAGCCTGCTGCTC TTG GGC ACT GTGGCCTGCAGCATC 
               
               
                   
               
               
                   TCTGCA CCCGCCCGCTCGCCCAGCCCCAGCACGCAGCCCTGGGAG CAT   
               
               
                   
               
               
                 GTG AAT GCCATCCAGGAGGCC CGGCGTCTC CTGAACCTG AGTAGA GAC 
               
               
                   
               
               
                 
                   ACT 
                 
               
               
                   
               
               
                 Codon Optimized Human Exon 1: 
               
               
                 Seq. ID NO: 2: 
               
               
                 atgtggctgcagagcctgctcctg ctg ggc acc gtggcctgcagcatc 
               
               
                   
               
               
                   agcgct cccgccagaagccccagcccctccacccagccctgggag cac   
               
               
                   
               
               
                 gtg aac gccatccaggaggcc agacggctg ctgaacctg tccaga gac 
               
               
                   
               
               
                 
                   acc 
                 
               
               
                   
               
               
                 Seq. ID NO:3: 
               
               
                 atgtggctgcagagcctgctgctc ctg ggc acc gtggcctgcagcatc 
               
               
                   
               
               
                   agcgcc cccgcccgctcgcccagccccagcacgcagccctgggag cac   
               
               
                   
               
               
                 gtg aac gccatccaggaggcccgg gcg ctcctgaacctg agccgc gac 
               
               
                   
               
               
                 
                   acc 
                 
               
               
                   
               
               
                 Seq. ID NO:4: 
               
               
                 atgtggctgcagagcctgctgctc ctc ggc act gtggcctgcagcatc 
               
               
                   
               
               
                   tccgca cccgcccgctcgcccagccccagcacgcagccctgggagcat 
               
               
                   
               
               
                 gtgaatgccatccaggaggcccggcgtctcctgaacctg tcc agagac 
               
               
                   
               
               
                 
                   act 
                 
               
               
                   
               
               
                 Seq. ID NO:5: 
               
               
                 atgtggctgcagagcctgctgctc ctc ggc acc gtggcctgcagcatc 
               
               
                   
               
               
                   tccgcc cccgcccgctcgcccagccccagcacgcagccctgggag cac   
               
               
                   
               
               
                 gtg aac gccatccaggaggcccgg gcg ctcctgaacctg tcccgc gac 
               
               
                   
               
               
                 
                   acc 
                 
               
               
                   
               
               
                 Native Human Exon 2: 
               
               
                 Seq. ID NO:6: 
               
               
                   GCTGCTGAGATGAATGAAACAGTAGAA GTCATC TCAGAA ATG TTT GACCTCCAG 
               
               
                   
               
               
                 Codon Optimized Human Exon 2: 
               
               
                 Seq. ID NO: 7: 
               
               
                   gccgcc gagatg aacgagaccgtggaggtg atc agcgag atg ttc gacctgcag 
               
               
                   
               
               
                 Seq. ID NO: 8: 
               
               
                   gccgcc gagatg aacgaaaccgtggag gtcatc agcgaa atg ttt gacctccag 
               
               
                   
               
               
                 Seq. ID NO: 9: 
               
               
                   gccgcc gagatg aatgagaccgtggaa gtcatc agcgag atg ttc gacctccag 
               
               
                   
               
               
                 Native Human Exon 3: 
               
               
                 Seq. ID NO: 10: 
               
               
                 GAG CCG ACC TGCCTA CAGACC CGC CTGGAGCTGTACAAGCAG GGC CTG 
               
               
                   
               
               
                   CGG GGCAGC CTC ACCAAGCTCAAG GGC CCC TTG ACCATGATGGCCAGC 
               
               
                   
               
               
                 CACTACAAGCAGCACTGC CCTCCAACCCCG   
               
               
                   
               
               
                 Codon Optimized Human Exon 3: 
               
               
                 Seq. ID NO: 11: 
               
               
                 gag ccc acc tgtctg cagacc cgg ctggagctgtacaagcag gga ctg 
               
               
                   
               
               
                 cggggcagc ctg accaag ctg aag gga ccc ctg accatgatggccagc 
               
               
                   
               
               
                 cactacaagcagcactgccct cccacaccc   
               
               
                   
               
               
                 Seq. ID NO: 12: 
               
               
                 gagccgacctgc ctg cagacccgcctggagctgtacaagcagggcctg 
               
               
                   
               
               
                   cgc ggcagcctcaccaagctcaagggcccc ctg accatgatggccagc 
               
               
                   
               
               
                 cactacaagcagcactgc cccccc accccg 
               
               
                   
               
               
                 Seq. ID NO: 13: 
               
               
                 gagccgacctgc ctc cagacccgcctggagctgtacaagcagggcctg 
               
               
                   
               
               
                   cgc ggcagcctcaccaagctcaagggcccc ctc accatgatggccagc 
               
               
                   
               
               
                 cactacaagcagcactgc cccccc accccg 
               
               
                   
               
               
                 Seq. ID NO: 14: 
               
               
                 gagccgacctgc ctg cagacccgcctggagctgtacaagcagggcctg 
               
               
                   
               
               
                   cgc ggcagcctcaccaagctcaagggcccc ctc accatgatggccagc 
               
               
                   
               
               
                 cactacaagcagcactgc cccccc accccg 
               
               
                   
               
               
                 Native Human Exon 4 encoding Ile at position 117: 
               
               
                 Seq. ID NO: 15: 
               
               
                   GAAACTTCCTGTGCA ACCCAG ATT ATCACC TTTGAAAGT TTC AAA GAG 
               
               
                   
               
               
                 AACCTGAAGGAC TTT CTG CTTGTCAT CCCC TTT GACTGCTGGGAG CCA   
               
               
                   
               
               
                   GTC CAGGAGTGA 
               
               
                   
               
               
                 Codon Optimized Human Exon 4: 
               
               
                 Seq. ID NO: 16: 
               
               
                   gagaccagctgcgcc acccag atc atcacc ttcgagagc ttc aag gag 
               
               
                   
               
               
                 aacctgaaggac ttc ctg ctggtg atcccc ttc gactgctgggag ccc   
               
               
                   
               
               
                   gtg caggagtga 
               
               
                   
               
               
                 Seq. ID NO: 17: 
               
               
                   gagacc tcc tgcgcc acccag atc atcacc ttcgagagc ttc aag gag 
               
               
                   
               
               
                 aacctgaaggac ttc ctg ctg gtcatcccc ttc gactgctgggag ccc   
               
               
                   
               
               
                 gtccaggagtga 
               
               
                   
               
               
                 Seq. ID NO: 18: 
               
               
                   gagacc tcc tgcgcc acccag atc atcacc ttcgagagc ttc aag gag 
               
               
                   
               
               
                 aacctgaaggac ttc ctg ctc gtcatcccc ttc gactgctgggag ccc   
               
               
                   
               
               
                 gtccaggagtga 
               
               
                   
               
               
                 Seq. ID NO: 19: 
               
               
                   gagacc tcc tgcgcc acccag atc atcacc ttcgagtcc ttc aag gag 
               
               
                   
               
               
                 aacctgaaggac ttc ctg ctg gtcatcccc ttc gactgctgggag ccc   
               
               
                   
               
               
                 gtccaggagtga 
               
               
                   
               
               
                 Seq. ID NO: 20: 
               
               
                 gaa acc tcc tgtgcc acccagatc atc acc ttc gaa agc ttc aag gag 
               
               
                   
               
               
                 aacctgaaggac ttc ctg ctg gtcatcccc ttc gactgctgggag ccc   
               
               
                   
               
               
                 gtccaggagtga 
               
               
                   
               
               
                 Seq. ID NO: 21: 
               
               
                   gagacc tcc tgcgcc acccag atc atcacc ttcgagtcc ttc aag gag 
               
               
                   
               
               
                 aacctgaaggac ttc ctg ctc gtcatcccc ttc gactgctgggagccc 
               
               
                   
               
               
                 gtccaggagtga 
               
               
                   
               
               
                 Native Human Exon 4 encoding Thr at position 117: 
               
               
                 SEQ. ID NO. 28: 
               
               
                   GAAACTTCCTGTGCA ACCCAGACTATCACC TTTGAAAGT TTC AAA GAG 
               
               
                   
               
               
                 AACCTGAAGGAC TTT CTG CTT GTCATCCCC TTT GACTGCTGGGAG CCA   
               
               
                   
               
               
                 GTCCAGGAGTGA 
               
               
                   
               
               
                 Codon Optimized Human Exon 4: 
               
               
                 Seq. ID NO: 22: 
               
               
                   gagaccagctgcgcc acccag acc atcacc ttcgagagc ttc aag gag 
               
               
                   
               
               
                 aacctgaaggac ttc ctg ctggtg atcccc ttc gactgctgggag ccc   
               
               
                   
               
               
                   gtg caggagtga 
               
               
                   
               
               
                 Seq. ID NO: 23: 
               
               
                   gagacc tcc tgcgcc acccag acc atcacc ttcgagagc ttc aag gag 
               
               
                   
               
               
                 aacctgaaggac ttc ctg ctg gtcatcccc ttc gactgctgggag ccc   
               
               
                   
               
               
                 gtccaggagtga 
               
               
                   
               
               
                 Seq. ID NO: 24: 
               
               
                   gagacc tcc tgcgcc acccag acc atcacc ttcgagagc ttc aag gag 
               
               
                   
               
               
                 aacctgaaggac ttc ctg ctc gtcatcccc ttc gactgctgggag ccc   
               
               
                   
               
               
                 gtccaggagtga 
               
               
                   
               
               
                 Seq. ID NO: 25: 
               
               
                   gagacc tcc tgcgcc acccag acc atcacc ttcgagtcc ttc aag gag 
               
               
                   
               
               
                 aacctgaaggac ttc ctg ctg gtcatcccc ttc gactgctgggag ccc   
               
               
                   
               
               
                 gtccaggagtga 
               
               
                   
               
               
                 Seq. ID NO: 26: 
               
               
                 gaa acc tcc tgtgcc acccag atc atcacc ttc gaa agc ttc aag gag 
               
               
                   
               
               
                 aacctgaaggac ttc ctg ctg gtcatcccc ttc gactgctgggag ccc   
               
               
                   
               
               
                 gtccaggagtga 
               
               
                   
               
               
                 Seq. ID NO: 27: 
               
               
                   gagacc tcc tgcgcc acccag acc atcacc ttcgagtcc ttc aag gag 
               
               
                   
               
               
                 aacctgaaggac ttc ctg ctc gtcatcccc ttc gactgctgggag ccc   
               
               
                   
               
               
                 gtccaggagtga 
               
               
                   
               
               
                 Codon optimized (co) (SEQ ID NO.: 29) murine GM-CSF compared 
               
               
                 to wild type (wt) (SEQ ID NO.: 30) murine 
               
               
                 GM-CSF (Genbank accession no. X02333) 
               
             
          
           
               
                 co 
                 atggctcacgagcggaaggctaaggtgctgcgcagaatgtggctgcagaacctgctgttc 
                   
               
               
                   
               
               
                 wt 
                 atggcccacgagagaaaggctaaggtcctgaggaggatgtggctgcagaatttacttttc 
               
               
                   
               
               
                 co 
                 ctgggcatcgtggtgtacagcctgagcgcccccaccagaagccccatcaccgtgaccaga 
               
               
                   
               
               
                 wt 
                 ctgggcattgtggtctacagcctctcagcacccacccgctcacccatcactgtcacccgg 
               
               
                   
               
               
                 co 
                 ccctggaagcacgtggaggccatcaaggaagctctgaacctgctggacgacatgcccgtg 
               
               
                   
               
               
                 wt 
                 ccttggaagcatgtagaggccatcaaagaagccctgaacctcctggatgacatgcctgtc 
               
               
                   
               
               
                 co 
                 accctgaacgaggaggtggaggtggtgagcaacgagtttagctttaagaagctgacctgc 
               
               
                   
               
               
                 wt 
                 acattgaatgaagaggtagaagtcgtctctaacgagttctccttcaagaagctaacatgt 
               
               
                   
               
               
                 co 
                 gtgcagacccggctgaagatcttcgagcagggactgcggggcaactttaccaagctgaag 
               
               
                   
               
               
                 wt 
                 gtgcagacccgcctgaagatattcgagcagggtctacggggcaatttcaccaaactcaag 
               
               
                   
               
               
                 co 
                 ggagccctgaacatgaccgccagctactaccagacctactgccctcccacacccgagacc 
               
               
                   
               
               
                 wt 
                 ggcgccttgaacatgacagccagctactaccagacatactgccccccaactccggaaacg 
               
               
                   
               
               
                 co 
                 gactgtgaaacccaggtgaccacctacgccgactttatcgacagcctgaagaccttcctg 
               
               
                   
               
               
                 wt 
                 gactgtgaaacacaagttaccacctatgcggatttcatagacagccttaaaacctttctg 
               
               
                   
               
               
                 co 
                 accgacatccccttcgagtgtaagaagcccgtgcagaag tga ctcgagcgg 
               
               
                   
               
               
                 wt 
                 actgatatcccctttgaatgcaaaaaaccagtccaaaaatga 
               
               
                   
               
             
          
           
               
                 Codon optimized (co) (SEQ ID NO.: 31) human GM-CSF compared 
                   
               
               
                 to wild type (wt) (SEQ ID NO.: 30) human GM-CSF 
               
               
                 encoding Ile at position 117 (Genbank accession no. X03021) 
               
             
          
           
               
                 co 
                 atgtggctgcagagcctgctcctgctgggcaccgtggcctgcagcatcagcgctcccgcc 
                   
               
               
                   
               
               
                 wt 
                 atgtggctgcagagcctgctgctcttgggcactgtggcctgcagcatctctgcacccgcc 
               
               
                   
               
               
                 co 
                 agaagccccagcccctccacccagccctgggagcacgtgaacgccatccaggaggccaga 
               
               
                   
               
               
                 wt 
                 cgctcgcccagccccagcacacagccctgggagcatgtgaatgccatccaggaggcccgg 
               
               
                   
               
               
                 co 
                 cggctgctgaacctgtccagagacaccgccgccgagatgaacgagaccgtggaggtgate 
               
               
                   
               
               
                 wt 
                 cgtctcctgaacctgagtagagacactgctgctgagatgaatgaaacagtagaagtcatc 
               
               
                   
               
               
                 co 
                 agcgagatgttcgacctgcaggagcccacctgtctgcagacccggctggagctgtacaag 
               
               
                   
               
               
                 wt 
                 tcagaaatgtttgacctccaggagccgacctgcctacagacccgcctggagctgtacaag 
               
               
                   
               
               
                 co 
                 cagggactgcggggcagcctgaccaagctgaagggacccctgaccatgatggccagccac 
               
               
                   
               
               
                 wt 
                 cagggcctgcggggcagcctcaccaagctcaagggccccttgaccatgatggccagccac 
               
               
                   
               
               
                 co 
                 tacaagcagcactgccctcccacacccgagaccagctgcgccacccagatcatcaccttc 
               
               
                   
               
               
                 wt 
                 tacaaacagcactgccctccaaccccggaaacttcctgtgcaacccagattatcaccttt 
               
               
                   
               
               
                 co 
                 gagagcttcaaggagaacctgaaggacttcctgctggtgatccccttcgactgctgggag 
               
               
                   
               
               
                 wt 
                 gaaagtttcaaagagaacctgaaggactttctgcttgtcatcccctttgactgctgggag 
               
               
                   
               
               
                 co 
                 cccgtgcaggagtga 
               
               
                   
               
               
                 wt 
                 ccagtccaggagtga 
               
               
                   
               
             
          
           
               
                 Codon optimized (co) (SEQ ID NO.: 33) human GM-CSF compared 
                   
               
               
                 to wild type (wt) (SEQ ID NO.: 34) human GM-CSF 
               
               
                 encoding Thr at position 117 (Genbank accession no. M10663) 
               
             
          
           
               
                 co 
                 atgtggctgcagagcctgctcctgctgggcaccgtggcctgcagcatcagcgctcccgcc 
                   
               
               
                   
               
               
                 wt 
                 atgtggctgcagagcctgctgctcttgggcactgtggcctgcagcatctctgcacccgcc 
               
               
                   
               
               
                 co 
                 agaagccccagcccctccacccagccctgggagcacgtgaacgccatccaggaggccaga 
               
               
                   
               
               
                 wt 
                 cgctcgcccagccccagcacacagccctgggagcatgtgaatgccatccaggaggcccgg 
               
               
                   
               
               
                 co 
                 cggctgctgaacctgtccagagacaccgccgccgagatgaacgagaccgtggaggtgatc 
               
               
                   
               
               
                 wt 
                 cgtctcctgaacctgagtagagacactgctgctgagatgaatgaaacagtagaagtcatc 
               
               
                   
               
               
                 co 
                 agcgagatgttcgacctgcaggagcccacctgtctgcagacccggctggagctgtacaag 
               
               
                   
               
               
                 wt 
                 tcagaaatgtttgacctccaggagccgacctgcctacagacccgcctggagctgtacaag 
               
               
                   
               
               
                 co 
                 cagggactgcggggcagcctgaccaagctgaagggacccctgaccatgatggccagccac 
               
               
                   
               
               
                 wt 
                 cagggcctgcggggcagcctcaccaagctcaagggccccttgaccatgatggccagccac 
               
               
                   
               
               
                 co 
                 tacaagcagcactgccctcccacacccgagaccagctgcgccacccagaccatcaccttc 
               
               
                   
               
               
                 wt 
                 tacaaacagcactgccctccaaccccggaaacttcctgtgcaacccagactatcaccttt 
               
               
                   
               
               
                 co 
                 gagagcttcaaggagaacctgaaggacttcctgctggtgatccccttcgactgctgggag 
               
               
                   
               
               
                 wt 
                 gaaagtttcaaagagaacctgaaggactttctgcttgtcatcccctttgactgotgggag 
               
               
                   
               
               
                 co 
                 cccgtgcaggagtga 
               
               
                   
               
               
                 wt 
                 ccagtccaggagtga 
               
             
          
         
       
     
     The invention claimed herein has been described generically, and by reference to specific embodiments. Examples and specific features are not intended to be limiting unless so indicated above. Modifications will occur to those of skill in the art, particularly in the selection of the source of mammalian GM-CSF, the specific codons optimized, the specific bases within the codons replaced to effect optimization, and the number and placement of the codons optimized, without departing from the invention, except as excluded by the claims set forth below. 
     
       
         
           
             34 
           
           
             1 
             147 
             DNA 
             Homo sapiens 
           
            1
atgtggctgc agagcctgct gctcttgggc actgtggcct gcagcatctc tgcacccgcc     60
cgctcgccca gccccagcac gcagccctgg gagcatgtga atgccatcca ggaggcccgg    120
cgtctcctga acctgagtag agacact                                        147
 
           
             2 
             147 
             DNA 
             Homo sapiens 
           
            2
atgtggctgc agagcctgct cctgctgggc accgtggcct gcagcatcag cgctcccgcc     60
agaagcccca gcccctccac ccagccctgg gagcacgtga acgccatcca ggaggccaga    120
cggctgctga acctgtccag agacacc                                        147
 
           
             3 
             147 
             DNA 
             Homo sapiens 
           
            3
atgtggctgc agagcctgct gctcctgggc accgtggcct gcagcatcag cgcccccgcc     60
cgctcgccca gccccagcac gcagccctgg gagcacgtga acgccatcca ggaggcccgg    120
gcgctcctga acctgagccg cgacacc                                        147
 
           
             4 
             147 
             DNA 
             Homo sapiens 
           
            4
atgtggctgc agagcctgct gctcctcggc actgtggcct gcagcatctc cgcacccgcc     60
cgctcgccca gccccagcac gcagccctgg gagcatgtga atgccatcca ggaggcccgg    120
cgtctcctga acctgtccag agacact                                        147
 
           
             5 
             147 
             DNA 
             Homo sapiens 
           
            5
atgtggctgc agagcctgct gctcctcggc accgtggcct gcagcatctc cgcccccgcc     60
cgctcgccca gccccagcac gcagccctgg gagcacgtga acgccatcca ggaggcccgg    120
gcgctcctga acctgtcccg cgacacc                                        147
 
           
             6 
             54 
             DNA 
             Homo sapiens 
           
            6
gctgctgaga tgaatgaaac agtagaagtc atctcagaaa tgtttgacct ccag           54
 
           
             7 
             54 
             DNA 
             Homo sapiens 
           
            7
gccgccgaga tgaacgagac cgtggaggtg atcagcgaga tgttcgacct gcag           54
 
           
             8 
             54 
             DNA 
             Homo sapiens 
           
            8
gccgccgaga tgaacgaaac cgtggaggtc atcagcgaaa tgtttgacct ccag           54
 
           
             9 
             54 
             DNA 
             Homo sapiens 
           
            9
gccgccgaga tgaatgagac cgtggaagtc atcagcgaga tgttcgacct ccag           54
 
           
             10 
             126 
             DNA 
             Homo sapiens 
           
            10
gagccgacct gcctacagac ccgcctggag ctgtacaagc agggcctgcg gggcagcctc     60
accaagctca agggcccctt gaccatgatg gccagccact acaagcagca ctgccctcca    120
accccg                                                               126
 
           
             11 
             126 
             DNA 
             Homo sapiens 
           
            11
gagcccacct gtctgcagac ccggctggag ctgtacaagc agggactgcg gggcagcctg     60
accaagctga agggacccct gaccatgatg gccagccact acaagcagca ctgccctccc    120
acaccc                                                               126
 
           
             12 
             126 
             DNA 
             Homo sapiens 
           
            12
gagccgacct gcctgcagac ccgcctggag ctgtacaagc agggcctgcg cggcagcctc     60
accaagctca agggccccct gaccatgatg gccagccact acaagcagca ctgccccccc    120
accccg                                                               126
 
           
             13 
             126 
             DNA 
             Homo sapiens 
           
            13
gagccgacct gcctccagac ccgcctggag ctgtacaagc agggcctgcg cggcagcctc     60
accaagctca agggccccct caccatgatg gccagccact acaagcagca ctgccccccc    120
accccg                                                               126
 
           
             14 
             126 
             DNA 
             Homo sapiens 
           
            14
gagccgacct gcctgcagac ccgcctggag ctgtacaagc agggcctgcg cggcagcctc     60
accaagctca agggccccct caccatgatg gccagccact acaagcagca ctgccccccc    120
accccg                                                               126
 
           
             15 
             108 
             DNA 
             Homo sapiens 
           
            15
gaaacttcct gtgcaaccca gattatcacc tttgaaagtt tcaaagagaa cctgaaggac     60
tttctgcttg tcatcccctt tgactgctgg gagccagtcc aggagtga                 108
 
           
             16 
             108 
             DNA 
             Homo sapiens 
           
            16
gagaccagct gcgccaccca gatcatcacc ttcgagagct tcaaggagaa cctgaaggac     60
ttcctgctgg tgatcccctt cgactgctgg gagcccgtgc aggagtga                 108
 
           
             17 
             108 
             DNA 
             Homo sapiens 
           
            17
gagacctcct gcgccaccca gatcatcacc ttcgagagct tcaaggagaa cctgaaggac     60
ttcctgctgg tcatcccctt cgactgctgg gagcccgtcc aggagtga                 108
 
           
             18 
             108 
             DNA 
             Homo sapiens 
           
            18
gagacctcct gcgccaccca gatcatcacc ttcgagagct tcaaggagaa cctgaaggac     60
ttcctgctcg tcatcccctt cgactgctgg gagcccgtcc aggagtga                 108
 
           
             19 
             108 
             DNA 
             Homo sapiens 
           
            19
gagacctcct gcgccaccca gatcatcacc ttcgagtcct tcaaggagaa cctgaaggac     60
ttcctgctgg tcatcccctt cgactgctgg gagcccgtcc aggagtga                 108
 
           
             20 
             108 
             DNA 
             Homo sapiens 
           
            20
gaaacctcct gtgccaccca gatcatcacc ttcgaaagct tcaaggagaa cctgaaggac     60
ttcctgctgg tcatcccctt cgactgctgg gagcccgtcc aggagtga                 108
 
           
             21 
             108 
             DNA 
             Homo sapiens 
           
            21
gagacctcct gcgccaccca gatcatcacc ttcgagtcct tcaaggagaa cctgaaggac     60
ttcctgctcg tcatcccctt cgactgctgg gagcccgtcc aggagtga                 108
 
           
             22 
             108 
             DNA 
             Homo sapiens 
           
            22
gagaccagct gcgccaccca gaccatcacc ttcgagagct tcaaggagaa cctgaaggac     60
ttcctgctgg tgatcccctt cgactgctgg gagcccgtgc aggagtga                 108
 
           
             23 
             108 
             DNA 
             Homo sapiens 
           
            23
gagacctcct gcgccaccca gaccatcacc ttcgagagct tcaaggagaa cctgaaggac     60
ttcctgctgg tcatcccctt cgactgctgg gagcccgtcc aggagtga                 108
 
           
             24 
             108 
             DNA 
             Homo sapiens 
           
            24
gagacctcct gcgccaccca gaccatcacc ttcgagagct tcaaggagaa cctgaaggac     60
ttcctgctcg tcatcccctt cgactgctgg gagcccgtcc aggagtga                 108
 
           
             25 
             108 
             DNA 
             Homo sapiens 
           
            25
gagacctcct gcgccaccca gaccatcacc ttcgagtcct tcaaggagaa cctgaaggac     60
ttcctgctgg tcatcccctt cgactgctgg gagcccgtcc aggagtga                 108
 
           
             26 
             108 
             DNA 
             Homo sapiens 
           
            26
gaaacctcct gtgccaccca gatcatcacc ttcgaaagct tcaaggagaa cctgaaggac     60
ttcctgctgg tcatcccctt cgactgctgg gagcccgtcc aggagtga                 108
 
           
             27 
             108 
             DNA 
             Homo sapiens 
           
            27
gagacctcct gcgccaccca gaccatcacc ttcgagtcct tcaaggagaa cctgaaggac     60
ttcctgctcg tcatcccctt cgactgctgg gagcccgtcc aggagtga                 108
 
           
             28 
             108 
             DNA 
             Homo sapiens 
           
            28
gaaacttcct gtgcaaccca gactatcacc tttgaaagtt tcaaagagaa cctgaaggac     60
tttctgcttg tcatcccctt tgactgctgg gagccagtcc aggagtga                 108
 
           
             29 
             471 
             DNA 
             Mus musculus 
           
            29
atggctcacg agcggaaggc taaggtgctg cgcagaatgt ggctgcagaa cctgctgttc     60
ctgggcatcg tggtgtacag cctgagcgcc cccaccagaa gccccatcac cgtgaccaga    120
ccctggaagc acgtggaggc catcaaggaa gctctgaacc tgctggacga catgcccgtg    180
accctgaacg aggaggtgga ggtggtgagc aacgagttta gctttaagaa gctgacctgc    240
gtgcagaccc ggctgaagat cttcgagcag ggactgcggg gcaactttac caagctgaag    300
ggagccctga acatgaccgc cagctactac cagacctact gccctcccac acccgagacc    360
gactgtgaaa cccaggtgac cacctacgcc gactttatcg acagcctgaa gaccttcctg    420
accgacatcc ccttcgagtg taagaagccc gtgcagaagt gactcgagcg g             471
 
           
             30 
             462 
             DNA 
             Mus musculus 
           
            30
atggcccacg agagaaaggc taaggtcctg aggaggatgt ggctgcagaa tttacttttc     60
ctgggcattg tggtctacag cctctcagca cccacccgct cacccatcac tgtcacccgg    120
ccttggaagc atgtagaggc catcaaagaa gccctgaacc tcctggatga catgcctgtc    180
acattgaatg aagaggtaga agtcgtctct aacgagttct ccttcaagaa gctaacatgt    240
gtgcagaccc gcctgaagat attcgagcag ggtctacggg gcaatttcac caaactcaag    300
ggcgccttga acatgacagc cagctactac cagacatact gccccccaac tccggaaacg    360
gactgtgaaa cacaagttac cacctatgcg gatttcatag acagccttaa aacctttctg    420
actgatatcc cctttgaatg caaaaaacca gtccaaaaat ga                       462
 
           
             31 
             435 
             DNA 
             Homo sapiens 
           
            31
atgtggctgc agagcctgct cctgctgggc accgtggcct gcagcatcag cgctcccgcc     60
agaagcccca gcccctccac ccagccctgg gagcacgtga acgccatcca ggaggccaga    120
cggctgctga acctgtccag agacaccgcc gccgagatga acgagaccgt ggaggtgatc    180
agcgagatgt tcgacctgca ggagcccacc tgtctgcaga cccggctgga gctgtacaag    240
cagggactgc ggggcagcct gaccaagctg aagggacccc tgaccatgat ggccagccac    300
tacaagcagc actgccctcc cacacccgag accagctgcg ccacccagat catcaccttc    360
gagagcttca aggagaacct gaaggacttc ctgctggtga tccccttcga ctgctgggag    420
cccgtgcagg agtga                                                     435
 
           
             32 
             435 
             DNA 
             Homo sapiens 
           
            32
atgtggctgc agagcctgct gctcttgggc actgtggcct gcagcatctc tgcacccgcc     60
cgctcgccca gccccagcac acagccctgg gagcatgtga atgccatcca ggaggcccgg    120
cgtctcctga acctgagtag agacactgct gctgagatga atgaaacagt agaagtcatc    180
tcagaaatgt ttgacctcca ggagccgacc tgcctacaga cccgcctgga gctgtacaag    240
cagggcctgc ggggcagcct caccaagctc aagggcccct tgaccatgat ggccagccac    300
tacaaacagc actgccctcc aaccccggaa acttcctgtg caacccagat tatcaccttt    360
gaaagtttca aagagaacct gaaggacttt ctgcttgtca tcccctttga ctgctgggag    420
ccagtccagg agtga                                                     435
 
           
             33 
             435 
             DNA 
             Homo sapiens 
           
            33
atgtggctgc agagcctgct cctgctgggc accgtggcct gcagcatcag cgctcccgcc     60
agaagcccca gcccctccac ccagccctgg gagcacgtga acgccatcca ggaggccaga    120
cggctgctga acctgtccag agacaccgcc gccgagatga acgagaccgt ggaggtgatc    180
agcgagatgt tcgacctgca ggagcccacc tgtctgcaga cccggctgga gctgtacaag    240
cagggactgc ggggcagcct gaccaagctg aagggacccc tgaccatgat ggccagccac    300
tacaagcagc actgccctcc cacacccgag accagctgcg ccacccagac catcaccttc    360
gagagcttca aggagaacct gaaggacttc ctgctggtga tccccttcga ctgctgggag    420
cccgtgcagg agtga                                                     435
 
           
             34 
             435 
             DNA 
             Homo sapiens 
           
            34
atgtggctgc agagcctgct gctcttgggc actgtggcct gcagcatctc tgcacccgcc     60
cgctcgccca gccccagcac acagccctgg gagcatgtga atgccatcca ggaggcccgg    120
cgtctcctga acctgagtag agacactgct gctgagatga atgaaacagt agaagtcatc    180
tcagaaatgt ttgacctcca ggagccgacc tgcctacaga cccgcctgga gctgtacaag    240
cagggcctgc ggggcagcct caccaagctc aagggcccct tgaccatgat ggccagccac    300
tacaaacagc actgccctcc aaccccggaa acttcctgtg caacccagac tatcaccttt    360
gaaagtttca aagagaacct gaaggacttt ctgcttgtca tcccctttga ctgctgggag    420
ccagtccagg agtga                                                     435