Patent Application: US-19494900-A

Abstract:
the present invention relates to polynucleotides comprising a dna sequence encoding an hcv protein and fragments thereof that contain codons optimized for expression in a vertebrate host . uses of the polynucleotides include eliciting an immune response specifically recognizing hcv .

Description:
this invention relates to novel fonnulations of nucleic acid pharmaceutical products , specifically nucleic acid vaccine products . the nucleic acid vaccine products , when introduced directly into muscle cells , induce the production of immune responses which specifically recognize hepatitis c virus ( hcv ). non - a , non - b hepatitis ( nanbh ) is a transmissible disease ( or family of diseases ) that is believed to be virally induced , and is distinguishable from other forms of virus - associated liver disease , such as those caused by hepatitis a virus ( hav ), hepatitis b virus ( hbv ), delta hepatitis virus ( hdv ), cytomegalovirus ( cmv ) or epstein - barr virus ( ebv ). epidemiologic evidence suggests that there may be three types of nanbh : the water - bome epidemic type ; the blood or needle associated type ; and the sporadically occurring ( community acquired ) type . however , the number of causative agents is unknown . recently , a new viral species , hepatitis c virus ( hcv ) has been identified as the primary ( if not only ) cause of blood - associated nanbh ( bb - nanbh ). hepatitis c appears to be the major form of transfusion - associated hepatitis in a number of countries , including the united states and japan . there is also evidence implicating hcv in induction of hepatocellular carcinoma . thus , a need exists for an effective method for preventing or treating hcv infection : currently , there is none . the hcv may be distantly related to the flaviviridae . the flavivirus family contains a large number of viruses which are small , enveloped pathogens of man . the morphology and composition of flavivirus particles are known , and are discussed in m . a . brinton , in “ the viruses : the togaviridae and flaviviridae ” ( series eds . fraenkel - conrat and wagner , vol . eds . schlesinger and schlesinger , plenum press , 1996 ), pp . 327 - 374 . generally , with respect to morphology , flaviviruses contain a central nucleocapsid surrounded by a lipid bilayer . virions are spherical and have a diameter of about 40 - 50 nm . their cores are about 25 - 30 nm in diameter . along the outer surface of the virion envelope are projections measuring about 5 - 10 nm in length with terminal knobs about 2 nm in diameter . typical examples of the family include yellow fever virus , west nile virus , and dengue fever virus . they possess positive - stranded rna genomes ( about 11 , 000 nucleotides ) that are slightly larger than that of hcv and encode a polyprotein precursor of about 3500 amino acids . individual viral proteins are cleaved from this precursor polypeptide . the genome of hcv appears to be single - stranded rna containing about 10 , 000 nucleotides . the genome is positive - stranded , and possesses a continuous translational open reading frame ( orf ) that encodes a polyprotein of about 3 , 000 amino acids . in the orf , the structural proteins appear to be encoded in approximately the first quarter of the n - terminal region , with the majority of the polyprotein attributed to non - structural proteins . when compared with all known viral sequences , small but significant co - linear homologies are observed with the nonstructural proteins of the flavivirus family , and with the pestiviruses ( which are now also considered to be part of the flavivirus family ). intramuscular inoculation of polynucleotide constructs , i . e ., dna plasmids encoding proteins halve been shown to result in the generation of the encoded protein in situ in muscle cells . by using cdna plasmids encoding viral proteins , both antibody and ctl responses were generated , providing homologous and heterologous protection against subsequent challenge with either the homologous or cross - strain protection , respectively . each of these types of immune responses offers a potential advantage over existing vaccination strategies . the use of pnvs ( polynucleotide vaccines ) to generate antibodies may result in an increased duration of the antibody responses as well as the provision of an antigen that can have both the exact sequence of the clinically circulating strain of virus as well as the proper post - translational modifications and conformation of the native protein ( vs . a recombinant protein ). the generation of ctl responses by this means offers the benefits of cross - strain protection without the use of a live potentially pathogenic vector or attenuated virus . the standard techniques of molecular biology for preparing and purifying dna constructs enable the preparation of the dna therapeutics of this invention . while standard techniques of molecular biology are therefore sufficient for the production of the products of this invention , the specific constructs disclosed herein provide novel therapeutics which surprisingly produce cross - strain protection , a result heretofore unattainable with standard inactivated whole virus or subunit protein vaccines . the amount of expressible dna to be introduced to a vaccine recipient will depend on the strength of the transcriptional and translational promoters used in the dna construct , and on the immunogenicity of the expressed gene product . in general , an immunologically or prophylactically effective dose of about 1 μg to 1 mg , and preferably about 10 μg to 300 μg is administered directly into muscle tissue . subcutaneous injection , intradermal introduction , impression through the skin , and other modes of administration such as intraperitoneal , intravenous , or inhalation delivery are also contemplated . it is also contemplated that booster vaccinations are to be provided . the dna may be naked , that is , unassociated with any proteins , adjuvants or other agents which impact on the recipients immune system . in this case , it is desirable for the dna to be in a physiologically acceptable solution , such as , but not limited to , sterile saline or sterile buffered saline . alternatively , the dna may be associated with surfactants , liposomes , such as lecithin liposomes or other liposomes known in the art , as a dna - liposome mixture , ( see for example wo93 / 24640 ) or the dna may be associated with an adjuvant known in the art to boost immune responses , such as a protein or other carrier . agents which assist in the cellular uptake of dna , such as , but not limited to , calcium ions , detergents , viral proteins and other transfection facilitating agents may also be used to advantage . these agents are generally referred to as transfection facilitating agents and as pharmaceutically acceptable carriers . as used herein , the term gene refers to a segment of nucleic acid which encodes a discrete polypeptide . the term pharmaceutical , and vaccine are used interchangeably to indicate compositions useful for inducing immune responses . the terms construct , and plasmid are used interchangeably . the term vector is used to indicate a dna into which genes may be cloned for use according to the method of this invention . the following examples are provided to further define the invention , without limiting the invention to the specifics of the examples . v1j is derived from vectors v1 and puc18 , a commercially available plasmid . v1 was digested with sspi and ecori restriction enzymes producing two fragments of dna . the smaller of these fragments , containing the cmvinta promoter and bovine growth hormone ( bgh ) transcription termination elements which control the expression of heterologous genes , was purified from an agarose electrophoresis gel . the ends of this dna fragment were then “ blunted ” using the t4 dna polymerase enzyme in order to facilitate its ligation to another “ blunt - ended ” dna fragment . puc18 was chosen to provide the “ backbone ” of the expression vector . it is known to produce high yields of plasmid , is well - characterized by sequence and function , and is of minimum size . we removed the entire lac operon from this vector , which was unnecessary for our purposes and may be detrimental to plasmid yields and heterologous gene expression , by partial digestion with the haeii restriction enzyme . the remaining plasmid was purified from an agarose electrophoresis gel , blunt - ended with the t4 dna polymerase , treated with calf intestinal alkaline phosphatase , and ligated to the cmvinta / bgh element described above . plasmids exhibiting either of two possible orientations of the promoter elements within the puc backbone were obtained . one of these plasmids gave much higher yields of dna in e . coli and was designated v1j . this vector &# 39 ; s structure was verified by sequence analysis of the junction regions and was subsequently demonstrated to give comparable or higher expression of heterologous genes compared with v1 . the ampicillin resistance marker was replaced with the neomycin resistance marker to yield vector v1jneo . an sfi i site was added to v1jneo to facilitate integration studies . a commercially available 13 base pair sfi i linker ( new england biolabs ) was added at the kpn i site within the bgh sequence of the vector . v1jneo was linearized with kpn i , gel purified , blunted by t4 dna polymerase , and ligated to the blunt sfi i linker . clonal isolates were chosen by restriction mapping and verified by sequencing through the linker . the new vector was designated v1jns . expression of heterologous genes in v1jns ( with sfi i ) was comparable to expression of the same genes in v1jneo ( with kpn i ). vector v1ra ( sequence is shown in fig1 ; map is shown in fig2 ) was derived from vector v1r , a derivative of the v1jns vector . multiple cloning sites ( bglii , kpni , ecorv , ecori , sali , and noti ) were introduced into v1r to create the v1ra vector to improve the convenience of subcloning . v1ra vector derivatives containing the tpa leader sequence and ubiquitin sequence were generated ( vtpa ( fig3 ) and vub ( fig4 ), respectively ). expression of viral antigen from vtpa vector will target the antigen protein into the exocytic pathway , thus producing a secretable form of the antigen proteins . these secreted proteins are likely to be captured by professional antigen presenting cells , such as macrophages and dendritic cells , and processed and presented by class ii molecules to activate cd4 + th cells . they also are more likely to efficiently simulate antibody responses . expression of viral antigen through vub vector will produce a ubiquitin and antigen fusion protein . the uncleavable ubiquitin segment ( glycine to alanine change at the cleavage site , butt et al ., jbc 263 : 16364 , 1988 ) will target the viral antigen to ubiquitin - associated proteasomes for rapid degradation . the resulting peptide fragments will be transported into the er for antigen presentation by class i molecules . this modification is attempted to enhance the class i molecule - restricted ctl responses against the viral antigen ( townsend et al , jem 168 : 1211 , 1988 ). gene segments were converted to sequences having identical translated sequences ( except where noted ) but with alternative codon usage as defined by r . lathe in a research article from j . molec . biol . vol . 183 , pp . 1 - 12 ( 1985 ) entitled “ synthetic oligonucleotide probes deduced from amino acid sequence data : theoretical and practical considerations ”. the methodology described below was based on our hypothesis that the known inability to express a gene efficiently in mammalian cells is a consequence of the overall transcript composition . thus , using alternative codons encoding the same protein sequence may remove the constraints on hcv gene expression . inspection of the codon usage within hcv genome revealed that a high percentage of codons were among those infrequently used by highly expressed human genes . the specific codon replacement method employed may be described as follows employing data from lathe et al . : 2 . compare wild type codon for observed frequency of use by human genes ( refer to table 3 in lathe et al .). 3 . if codon is not the most commonly employed , replace it with an optimal codon for high expression based on data in table 5 . 4 . inspect the third nucleotide of the new codon and the first nucleotide of the adjacent codon immediately 3 ′- of the first . if a 5 ′- cg - 3 ′ pairing has been created by the new codon selection , replace it with the choice indicated in table 5 . 5 . repeat this procedure until the entire gene segment has been replaced . 6 . inspect new gene sequence for undesired sequences generated by these codon replacements ( e . g ., “ attta ” sequences , inadvertent creation of intron splice recognition sites , unwanted restriction enzyme sites , etc .) and substitute codons that eliminate these sequences . the consensus core sequence of hcv was adopted from a generalized core sequence reported by bukh et al . ( pnas , 91 : 8239 , 1994 ). this core sequence contains all the identified ctl epitopes in both human and mouse . the gene is composed of 573 nucleotides and encodes 191 amino acids . the predicted molecular weight is about 23 kda . the codon replacement was conducted to eliminate codons which may hinder the expression of the hcv core protein in transfected mammalian cells in order to maximize the translational efficiency of dna vaccine . twenty three point two percent ( 23 . 2 %) of nucleotide sequence ( 133 out of 573 nucleotides ) were altered , resulting in changes of 61 . 3 % of the codons ( 117 out 191 codons ) in the core antigen sequence . the optimized nucleotide sequence of hcv core is shown in fig5 . the optimized hcv core gene ( fig5 ) was constructed as a synthetic gene annealed from multiple synthetic oligonucleotides . to facilitate the identification and evaluation of the synthetic gene expression in cell culture and its immunogenicity in mice , a ctl epitope derived from influenza virus nucleoprotein residues 366 - 374 and an antibody epitope sequence derived from sv40 t antigen residues 684 - 699 were tagged to the carboxyl terminal of the core sequence ( fig6 ). for clinical use it may be desired to express the core sequence without the nucleoprotein 366 - 374 and sv40 t 684 - 698 sequences . for this reason , the sequence of the two epitopes is flanked by two ecori sites which will be used to excise this fragment of sequence at a later time . thus an embodiment of the invention for clinical use could consist of the v1ra . hcv1corepab , vtpa . hcv1corepab , or vub . hcv1corepab plasmids that had been cut with ecori , annealed , and ligated to yield plasmids v1ra . hcv1core , vtpa . hcv1core , and vub . hcv1core . the synthetic gene was built as three separate segments in three vectors , nucleotides 1 to 80 in v1ra , nucleotides 80 to 347 ( bstx1 site ) in puc18 , and nucleotides 347 to 573 plus the two epitope sequence in puc18 . all the segments were verified by dna sequencing , and joined together in v1ra vector . in each case , the junction sequences from the 5 ′ promoter region ( cmvinta ) into the cloned gene is shown . the position at which the junction occurs is demarcated by a “/”, which does not represent any discontinuity in the sequence . the nomenclature for these constructs follows the convention : “ vector name - 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