Patent Application: US-83729701-A

Abstract:
the present invention relates to novel hepatitis b virus vectors for use in gene therapy which can deliver therapeutic genes to liver cells . the invention also provides methods for the production of novel recombinant hepatitis b viruses . the recombinant viruses produced by this invention can deliver therapeutic genes specifically to liver cells either through in vivo or ex vivo therapy protocols . this vector can be used not only to treat liver diseases but also genetic diseases .

Description:
to facilitate understanding of the invention , a number of terms are defined below . as used herein , the term “ cis ” is used in reference to the presence of genes on the same chromosome . the term “ trans - acting ” is used in reference to the controlling effect of a regulatory gene on a gene present on a different chromosome . as used herein , the term “ in trans ” is used in reference to indicate the complementation effect of a gene product on a gene present on a different chromosome . the term “ cis - acting ” is used in reference to the controlling effect of a regulatory gene on a gene present on the same chromosome . nucleotide sequences of hbv genome were numbered according to galibert et al . ( galibert et al ., 1979 ), unless otherwise indicated . in this numbering system , the 5 ′- end of the pregenomic rna is at nt . 1820 ( nassal et al ., 1990 ). on the other hand , nucleotide sequences of the plasmids included in seq were numbered from the 5 ′- end of the pregenomic rna . hepatitis b virus ( hbv ), the causative agent of chronic hepatitis in man , is the prototype member of the hepadnaviridae ( ganem , 1996 ). related members of the hepadnavirus family include woodchuck hepatitis virus ( whv ), ground squirrel hepatitis virus ( gshv ), and duck hepatitis b virus ( dhbv ). hbv genome is a circular dna of only 3 . 2 k bp in length . the viral genome is a partially duplex circular dna , possessing a single - stranded gap region in plus - strand dna . although hbv has a dna genome , it replicates through reverse transcription of an rna intermediate , the pregenomic rna ( pgrna ), within the subviral core particle . there are four major open reading frames ( orfs ), all encoded in same strand ( fig1 ). inspection of the sequence led to the recognition of conserved repeat elements that play important roles in the genome replication . these direct repeats ( denoted dr1 and dr2 ) are located near the 5 ′ end of the minus and plus dna strands ( fig1 ). the hepadnaviral life cycle is outlined in fig2 ( ganem et al ., 1987 ). hepadnaviruses are thought to enter the hepatocytes through receptor - mediated endocytosis . upon entry , a partial duplex genome is repaired to a covalently closed circular dna ( ccc ), which is the template for transcription . four viral transcripts are synthesized and transported to cytoplasm . the 3 . 5 k bp rna , also called pregenomic rna , serves as a template for reverse transcription as well as for translation of the core ( c ) and polymerase ( p ). despite of the general similarities to retroviruses , many steps in its replication are distinct ( nassal et al ., 1996 ). the first step of hbv genome replication is the encapsidation of the pregenomic rna into core particles . core particle assembly involves the interactions of the structural proteins , core ( c ) and polymerase ( p ) with the pregenomic rna . incorporation of p protein as well as the pregenomic rna into assembling core particles is essential for viral dna synthesis . the cis - acting element for encapsidation , termed ε , has been defined within 85 nucleotides ( nt ) near the 5 ′ end of pgrna , which is necessary and sufficient to direct encapsidation of heterologous rna sequences into viral core particle ( junker - niepmann et al ., 1990 ; hirsch et al ., 1991 ). the epsilon element can fold into a stem - loop structure , which is highly conserved among hepadnaviruses . reverse transcription mechanism of hbv polymerase is quite complicated , as expected from its peculiar genome structure . a process described as template switching is required for the successful synthesis of a double - stranded dna product ( fig3 ). first , minus - strand dna synthesis is initiated near the 5 ′- end of its template , the pregenomic rna ( loeb et al ., 1995 ). the viral polymerase is both the primer and polymerase for minus - strand dna synthesis ( wang et al ., 1992 ). following template switching to an acceptor site near the 3 ′ end of the pregenomic rna , minus - strand dna synthesis resumes at this position , resulting in a genome - length , minus - strand dna . upon completion of the synthesis of minus - strand dna , the final rnase h cleavage product , the 18 - nt rna fragment , serves as a primer for the initiation of plus - strand dna synthesis ( loeb et al ., 1991 ). upon translocation to dr2 , the rna primer is used for the initiation of plus - strand dna synthesis . for the plus - strand dna initiated at dr2 , a third template switch , termed circularization , is required to generate a mature relaxed circular dna . molecular analysis revealed that several elements play a role in the viral dna synthesis ( nassal et al ., 1996 ). the list of cis - acting elements includes : 5 ′ epsilon , encapsidation signal ( junker - niepmann et al ., 1990 ; hirsch et al ., 1991 ); dr1 and dr2 , primer acceptor sites for primer translocation step during viral dna synthesis ( nassal et al ., 1996 ); r ( repeat ) for circularization ( loeb et al ., 1997 ); and pre , posttranscriptional rna processing element ( huang et al ., 1995 ). all of these known elements are located to either side of the hbv pregenome ( fig1 ). in the case of dhbv , three additional elements , termed 3e , m and 5e , have been reported to be essential for template switching during plus - strand dna synthesis ( havert et al ., 1997 ). however , it is not known whether any other elements in the middle of the viral genome are essential for hbv genome replication . hbv genome has not been explored as a gene therapy vector , primarily due to the lack of information on its cis - acting elements . having identified all cis - acting elements required for hbv genome replication , it is possible to design a gene therapy vector that can accomodate a heterologous gene sequence without compromising its ability to replicate , if trans - acting factors are provided in trans . briefly , the hbv vector encodes all cis - acting elements that are essential for the viral genome replication , but lacks expression of the viral proteins . nonetheless , the recombinant virus can be produced if the viral proteins ( i . e ., core , polymerase , surface antigens ) are provided in trans via a helper plasmid or packaging cell lines ( fig4 ). several issues need to be considered for the design of a gene therapy vector including insertion site , size of insert ( s ), promoter to drive transgene transcription . first of all , two insertion sites were selected ; one between 5 ′ epsilon and the α element , the other site located between the α element and dr2 ( fig8 ). these two insertion sites were selected since these sequences are dispensable for viral genome replication . regarding insert size , fragments of up to 0 . 90 k bp and 1 . 7 k bp , respectively , can be inserted into these two insertion sites without significantly exceeding the wild - type genome size . two endogenous viral promoters ( i . e ., core promoter and pre - s2 / s promoter ), conveniently located just upstream of these two insertion sites , are employed to drive transcription . further , this hbv vector can be used as a bicistronic expression vector , if two insertion sites are used simultaneously ( fig8 ). most of the techniques used for vector construction and cell transfection are widely practiced in the art , and most practitioners are familiar with standard resource materials describing specific conditions and procedures . construction of the vectors of the invention employs standard ligation and restriction techniques which are well understood in the art ( see sambrook et al ., 2001 ). in this experimental disclosure , the following abbreviations are applied : m ( molar ), mm ( millimolar ), ml ( milliliters ), μg ( micrograms ), mg ( milligrams ), peg ( polyethylene glycol ), orf ( open reading frame ), the following examples are presented to illustrate the present invention and to assist one of ordinary skill in making and using the same . the examples are not intended in any way to otherwise limit the scope of the disclosure . to design a hbv gene therapy vector , it is prerequisite to map all the cis - acting elements that are essential for the viral genome replication . to achieve this , a replication - competent plasmid that can lead to the production of infectious hbv particles upon transfection was constructed . the fact that a heterologous promoter driven rna transcript analogous to the pregenomic rna can lead to the production of infectious viral particles are well understood in the art ( nassal et al ., 1990 ). thus , a pregenomic rna expression plasmid was designed such that the 5 ′- end of the transcripts would be identical to that of wild - type hbv . specifically , the position of 5 ′- epsilon element is 30 nucleotides away from the 5 ′- end ( jeong et al ., 2000 ). the nucleotide sequence of the hbv genome was numbered starting at the unique eco ri site of hbv ayw subtype , according to the method of galibert et al . ( galibert et al ., 1979 ). nucleotide numbers ( nt .) indicate the hbv sequence number , unless otherwise indicated . in this number system , the 5 ′ end of the pregenomic rna is at nt . 1820 ( nassal et al ., 1990 ). to generate a replication - competent hbv construct , the greater - than - genome - length viral genome should be inserted downstream of the promoter element to maintain terminal redundancy of the pregenomic rna ( see fig1 ; ganem et al ., 1987 ). the genome of hepatitis b virus was derived from psv2a - neo ( hbv ) 2 plasmid that contains a dimer of hbv ayw subtype ( shih et al ., 1989 ). the greater - than - genome - length fsp i ( nt . 1804 )- to - xba i ( nt . 1992 ) fragment ( 3354 nt ) of hbv ayw subtype ( galibert et al ., 1979 ) was inserted into eco rv and xba i sites in the multiple cloning site of pcdna1 / amp plasmid ( invitrogen , u . s . a . ): r402 plasmid ( pcmv - hbv / 164 ). the hbv transcript made from this plasmid ( pcmv - hbv / 164 ) has a vector - derived 134 nt at the 5 ′ end relative to that of wild - type pregenomic rna . to make a rna expression plasmid that can transcribe the hbv pregenomic rna that is almost identical to the wild - type pregenomic rna with respect to the position of the epsilon element , a small deletion was introduced into r402 plasmid ( pcmv - hbv / 164 ). thus , r015 plasmid ( pcmv - hbv / 30 ) was made by removing this pcdna1 / amp plasmid - derived 134 nucleotides by a pcr - mediated method ( jeong et al ., 2000 ). briefly , a fragment was made by polymerase chain reaction using a forward primer of the sequence 5 - ccc gagctc tctggctaactaactttttcacctctgcc - 3 ( saci site underlined ) and a reverse primer of the sequence 5 - cccaagcttctattgttcccaagaatatgg - 3 ( nt 2839 to 2822 ) with r402 ( pcmv - hbv / 164 ) as a template . the resulting pcr fragment was digested by saci and bspei and then inserted between the saci ( nt . 2894 of pcdna1 / amp ) and bspei ( nt . 2331 ) site of r402 ( pcmv - hbv / 164 ). briefly , pcr was carried out using a forward primer with ecor i site and a reverse primer with xho i site to generate the ecor i - to - xho i fragment ( nt . 1903 - to - 2454 ) then , the 0 . 5 k bp pcr product was inserted into pcdna3 ( invitrogen , u . s . a ) via ecor i , xho i restriction sites to make r062 plasmid . next , the bspe i ( nt . 233 1 )- to - apa i of r062 plasmid was substituted by 2 . 6 k bp bspe i ( nt . 2331 )- to - apa i of r015 plasmid . this r063 plasmid lacking encapsidation signal , epsilon , was employed as helper plasmid to provide the viral proteins ( i . e ., core , polymerase , surface antigen ) essential for the viral replication and assembly . forward primer : 5 ′- catg gaattc atggacatcgaccct - 3 ( ecor i site underlined ) reverse primer : 5 ′- ccg ctcgag ctaacattgagattcccgaga - 3 ′ ( xho i site underlined ) forward primer : 5 ′- catg gaattc atggacatcgaccct - 3 ( ecor i site underlined ) reverse primer : 5 ′- ccg ctcgag ctaacattgagattcccgaga - 3 ′ ( xho i site underlined ) demonstration of replication - competency of wild - type pregenomic rna expression plasmid , r015 plasmid ( pcmv - hbv / 30 ). human hepatoma cells , designated huh7 cells were grown in dmem ( gibco - brl ) supplemented with 10 % fetal bovine serum ( gibco - brl ) and 10 □ of gentamicin per ml and were split every third day . the day before transfection , cells were plated at a confluency of 75 %. on the following day , cells were washed twice with phosphate - buffered saline ( pbs ) and given fresh media . after 2 hours , cells were transfected with 10 □ of supercoiled plasmid dna per 60 mm plate by the capo4 coprecipitation technique . 2 - 2 . southern blot analysis of the viral replication - intermediate from cytoplasmic core particles . three days after transfection , viral dnas were extracted from intracellular core particles by peg precipitation as described previously ( staprans et al ., 1991 ). briefly , transfected cells from a 100 - mm plate were lysed in lysis buffer [ 10 mm tris ( ph 7 . 5 ), 1 mm edta , 50 mm nacl , 8 % sucrose , 0 . 25 % nonidet p - 40 ]. nuclei were removed by centrifugation for 3 min in a microcentrifuge , and the cytoplasmic extract was adjusted to 6 mm mgcl 2 and digested with dnase i ( 50 □/□) for 30 min at 37 ° c . cores were precipitated by centrifugation for 4 min after adding 4 × pne buffer [ 26 % peg , 1 . 4 m nacl , 25 mm edta ], and incubating at 4 ° c . for 30 min . core particles resuspended in buffer [ 10 mm tris ( ph 7 . 5 ), 6 mm mgcl 2 ] were then digested with dnase i for an additional 15 min at 37 ° c ., followed by the addition of 5 mm edta , 1 % sds , and 500 □ of proteinase k per □ and were incubated for 1 h at 37 ° c . core nucleic acid was extracted twice with phenol / chcl 3 ( 1 : 1 ) and precipitated with ethanol , then resuspended in 50 □ of te [ 10 mm tris ( ph 7 . 5 ), 1 mm edta ]. extracted viral dna were subjected to agarose gel electrophoresis , followed by southern blot analysis , which are well known to those skilled in the art ( current protocols in molecular biology , ausubel , f . et al ., eds ., wiley and sons , new york , 1995 ). a series of small deletion mutants were generated by standard recombinant dna technology ( sambrook et al ., 2001 ). plasmid r059 ( pbs + δ1910 - 1992 ) was made in which the sac i - to - ecor i ( nt . 3182 ) fragment of hbv ayw subtype , but lacking the nt . 1910 - 1992 fragment ( galibert et al ., 1979 ) was subcloned into pbluescript sk (+) plasmid ( stratagene , usa ). subsequently , the sac i - to - ecor i fragment of r015 was replaced by the sac i - to - ecor i fragment of r059 to generate r060 plasmid . first , plasmid r046 was made in which the sac i - to - ecor i ( nt . 3182 ) fragment of hbv ayw subtype ( galibert et al ., 1979 ) was subcloned into pch110 ( pharmacia ). then , r047 plasmid was generated by deleting the 151 bp xba i fragment ( nt . 1992 - 2143 ). subsequently , the sac i - to - ecor i fragment of r015 plasmid was replaced by the sac i - to - ecor i fragment of r047 to generate r048 plasmid . first , plasmid r049 was made in which the sac i - to - ecor i ( nt . 3182 ) fragment of hbv ayw subtype ( galibert et al ., 1979 ) was subcloned into pbluescript ii ks (+) ( stratagene , u . s . a .). the sty i ( nt . 1884 )- to - sty i ( nt . 2459 ) fragment of r049 was replaced by a pcr product of sty i ( nt . 1884 )- to - xba i ( nt . 2143 ) fragment encoding sty i restriction site at the 5 ′- end of the reverse primer to make r051 plasmid . subsequently , the sac i - to - ecor i fragment of r015 was replaced by the sac i - to - ecor i fragment of r051 plasmid to generate the r056 deletion mutant . first , plasmid r407 was made in which the sac i - to - xho i ( nt . 129 ) fragment of r015 plasmid was subcloned into pbluebachis2 plasmid ( invitrogen , u . s . a .). then , the sty i ( nt . 2459 )- to - bste ii ( nt . 2817 ) fragment of r407 was deleted and filled in by klenow fragment to make r018 plasmid . subsequently , the bspe i ( nt . 2331 )- to - ecor i ( nt . 3182 ) fragment of r015 was replaced by the bspe i - to - ecor i fragment of r018 to generate r021 deletion mutant . to make r022 , the bste ii ( nt . 2662 )- to - ecor i ( nt . 3182 ) fragment of r015 plasmid was deleted and filled in by klenow fragment to make r022 plasmid . first , plasmid r701 was made in which the bste ii ( nt . 2817 )- to - sph i ( nt . 1239 ) fragment of r015 plasmid was subcloned into pgem - 4z plasmid ( promega , u . s . a ). then , the bgl ii ( nt . 2839 )- to - ecor i ( nt . 3182 ) fragment of r701 was deleted and filled in by klenow fragment to make r043 plasmid . subsequently , the bstx i ( nt . 2817 )- to - bstx i ( nt . 620 ) fragment of r015 was replaced by the corresponding 642 bp bstx i fragment of r043 to generate r045 deletion mutant . the bsu36 i ( nt . 3052 )- to - ecor i ( nt . 3182 ) fragment of r701 was deleted and filled in by klenow fragment to make r042 plasmid . subsequently , the bstx i ( nt . 2817 )- to - bstx i ( nt . 620 ) fragment of r015 was replaced by the corresponding 855 bp bstx i fragment of r042 plasmid to generate r044 deletion mutant . to make r023 , the ecor i ( nt . 3182 )- to - xho i ( nt . 129 ) fragment of r015 was deleted and filled in by klenow fragment to make r023 plasmid . first , plasmid r037 was made in which the ecor i ( nt . 3182 )- to - sph i ( nt . 1239 ) fragment of r015 plasmid was subcloned into pgem - 4z plasmid ( promega , u . s . a ). the xho i ( nt . 129 )- to - bamh i ( nt . 490 ) fragment of r037 plasmid was deleted and filled in by klenow fragment to make r038 plasmid . subsequently , the ecor i ( nt . 3182 )- to - sph i ( nt . 1238 ) fragment of r015 plasmid was replaced by the corresponding 877 bp ecor i - to - sph i fragment of r038 plasmid to generate r040 deletion mutant . the bamh i ( nt . 490 )- to - acc i ( nt . 827 ) fragment of r037 was deleted and filled in by klenow fragment to make r039 plasmid . subsequently , the ecor i ( nt . 3182 )- to - sph i ( nt . 1238 ) fragment of r015 plasmid was replaced by the corresponding 897 bp ecor i - to - sph i fragment of r039 to generate r041 deletion mutant . first , a plasmid r050 was made in which the ecor i ( nt . 3182 )- to - apa i fragment of r015 plasmid was subcloned into pbluescript ii ks (+) ( stratagene , u . s . a .). the acc i ( nt . 827 )- to - sph i ( nt . 1238 ) fragment of r050 plasmid was deleted and filled in by t4 dna polymerase to make r008 plasmid . subsequently , the ecor i ( nt . 3182 )- to - apa i fragment of r015 was replaced by the corresponding 1591 bp ecor i - to - apa i fragment of r008 to generate r025 deletion mutant . the sph i ( nt . 1238 )- to - nco i ( nt . 1374 ) fragment of r050 was deleted and filled in by t4 dna polymerase to make r009 plasmid . subsequently , the ecor i ( nt . 3182 )- to - apa i fragment of r015 plasmid was replaced by the corresponding 1866 bp ecor i - to - apa i fragment of r009 plasmid to generate r026 deletion mutant . the nco i ( nt . 1374 )- to - aat ii ( nt . 1419 ) fragment of r050 plasmid was deleted and filled in by t4 dna polymerase to make r012 plasmid . subsequently , the ecor i ( nt . 3182 )- to - apa i fragment of r015 was replaced by the corresponding 1957 bp ecor i - to - apa i fragment of r012 to generate r027 deletion mutant . the aat ii ( nt . 1419 )- to - fsp i ( nt . 1804 ) fragment of r050 was deleted and filled in by t4 dna polymerase to make r013 plasmid . subsequently , the ecor i ( nt . 3182 )- to - apa i fragment of r015 was replaced by the corresponding 1617 bp ecor i - to - apa i fragment of r013 to generate r028 deletion mutant . the aat ii ( nt . 1419 )- to - apa i fragment of r050 was replaced by the pcr product of aat ii ( nt . 1592 )- to - apa i fragment encoding aat ii restriction site at the 5 ′- end of the forward primer to make r052 plasmid . subsequently , the ecor i - to - apa i fragment of r015 was replaced by the ecor i - to - apa i fragment of r052 to generate r053 deletion mutant . the ecor i ( nt . 3182 )- to - bsa i ( nt . 1607 ) blunted fragment of r050 plasmid was ligated with the ecor i ( nt . 3182 )- to - fsp i ( nt . 1804 ) of r015 plasmid to make r035 plasmid . the fsp i ( nt . 1804 )- to - sty i ( nt . 1884 ) fragment of r050 was deleted and filled in by klenow polymerase to make r010 plasmid . subsequently , the ecor i ( nt . 3182 )- to - apa i fragment of r015 was replaced by the corresponding 1922 bp ecor i - to - apa i fragment of r010 to generate r029 deletion mutant . transfection , dna extraction and southern blots were performed as described in example 2 - 2 . to complement trans - acting viral proteins , a helper plasmid ( pcmv - cps ) that provides core protein and polymerase was cotransfected along with each deletion mutant during transfection . fig1 showed a typical southern blot result . as described above , three species of the hbv replication - intermediates can be seen in this southern blot of core - associated viral dna : ss ( single - stranded dna ), dl ( double - stranded linear dna ), and rc ( relaxed circular dna ). rc form is the mature product of viral genome replication found in virions . thus , lack of the rc form dna in southern blots would indicate that a cis - acting element essential for the viral genome synthesis is deleted in the mutants . a series of deletion mutants was generated that encompass the entire hbv genome . each deletion lacks a fragment of between 0 . 05 - 0 . 52 k bp . southern analysis indicated that only ss dna was detected from cells transfected by the r022 mutant ( pcmv - ayw δ2662 - 3182 / 0 ). to delineate the region deleted in mutant r022 , two addition mutants were made : r045 ( pcmv - ayw δ2839 - 3182 / 0 ) and r044 ( pcmv - ayw δ3052 - 3182 / 0 ). as a result , rc dna as well as ss dna and dl dna were detected in cells transfected by r044 mutants . thus , a sequence deleted in the r044 mutant is not essential for viral genome synthesis . on the other hand , only trace amount of ss dna was detected in southern blots of cells transfected by mutant r045 . thus , a sequence deleted in r045 is essential for the viral genome replication . taken together , a novel cis - element essential for hbv genome replication , termed α element ( nt 2662 - 3052 ), was identified . in addition , mutant r028 ( pcmv - ayw δ1419 - 1804 ) lacking the dr2 element ( nt . 1592 - 1602 ) was made . southern blot analysis of cells transfected by r028 ( pcmv - ayw δ1419 - 1804 ) showed the detection of only ss dna , but not rc dna . this result is consistent with published reports on the role of dr2 on minus - strand dna synthesis in dhbv ( loeb et al ., 1996 ; condreay et al ., 1992 ). to delineate this region further , mutant r053 ( pcmv - ayw δ1419 - 1592 ) lacking sequence upstream of dr2 element was made . southern blot analysis indicated detection of rc dna from cells transfected by mutant r053 . this result indicated that the sequence lacking in the r053 mutant is dispensable for hbv genome replication . to further delineate this region , r035 mutant ( pcmv - ayw δ1607 - 1804 ) lacking a sequence between dr2 and dr1 elements was made . even ss dna was not detected from cells transfected by the r035 mutant . thus , the sequence between dr2 and dr1 element , termed β element , is essential for the minus - strand dna synthesis . in addition , r029 mutant ( pcmv - ayw δ1804 - 1884 ) lacking dr1 element was made . consistent with published data , no ss dna was detected ( condreay et al ., 1992 ). in contrast , sequences deleted in some of the deletion mutants turned out to be dispensable . these mutants included r060 mutant ( pcmv - ayw δ1910 - 1992 ), r048 mutant ( pcmv - ayw δ1992 - 2143 ), r056 mutant ( pcmv - ayw δ2143 - 2459 ), r021 mutant ( pcmv - ayw δ2459 - 2817 ), r044 mutant ( pcmv - ayw δ3052 - 3182 ), r023 mutant ( pcmv - ayw δ3182 - 129 ), r040 mutant ( pcmv - ayw δ120 - 490 ), r041 ( pcmv - ayw δ490 - 827 ), r025 mutant ( pcmv - ayw δ827 - 1238 ), r026 mutant ( pcmv - ayw δ1238 - 1374 ), r027 mutant ( pcmv - ayw δ1374 - 1419 ), and r053 ( pcmv - ayw δ1419 - 1592 ). in summary , the present invention reveals two novel cis - acting elements that are essential for the hbv genome replication . the complete mapping of cis - acting elements that are essential for hbv genome replication allowed us to design a prototype hbv vector . the present invention reveals two novel cis - acting elements that are essential for the hbv genome replication . in literature , a half dozen elements have been reported to be essential in various stages of hbv genome synthesis . among these are : 5 ′- epsilon for encapsidation ( hirsh et al ., 1991 ; junker - niepmann et al ., 1990 ), dr2 element ( condreay et al ., 1992 ; loeb et al ., 1996 ), dr1 element ( seeger et al ., 1991 ), r ( repeat ) element for circularization ( loeb et al ., 1997 ), and pre element for post - transcriptional rna processing ( yen , 1998 ). in addition to these , the two elements identified in the invention , termed α and β , complete the mapping of cis - acting elements essential for hbv viral genome replication ( fig6 ). based on the information on these cis - acting elements , a prototype hbv gene therapy vector was designed ( fig8 ). the critical parameters of this gene therapy vector are the size and position of the inserts . in this prototype vector , two insertion sites were identified . first , the sequence ( nt 1909 - 2816 ) between 5 ′ epsilon and the α element could be substituted by a heterologous gene of interest . at the very least , the 0 . 9 k bp fragment can be substituted in this site . since this insertion site overlaps the core open reading frame , the core promoter can then be used to drive the heterologous gene inserted . secondly , the sequence ( nt . 3052 - 1592 ) between a element and dr2 element can be substituted by a heterologous gene . a fragment up to 1 . 7 k bp can be substituted in this site . similarly , this insertion site overlaps the pre - s2 / s gene . thus , the pre - s2 / s promoter can be used to derive the heterologous gene inserted . to test feasibility of the prototype vector described in example 5 , a hbv vector was made by the insertion of gfp ( green fluorescent protein ) gene at the site proposed : r711 ( pcmv - hbv / gfp ) ( fig9 ). the insertion site was determined by considering following points : ( i ) all cis - acting elements essential for hbv genome replication should be kept intact , ( ii ) the endogenous viral promoter needs to be employed to maximize the coding capacity of the vector without exceeding the maximal packaging limit . 6 - 2 . insertion of gfp ( green fluorescent protein ) gene into the hbv vector insertion of the 0 . 7 k bp gfp fragment was facilitated by polymerase chain reaction ( pcr ). restriction sites were created at the end of the pcr fragment by an appropriately designed pcr primer . first of all , r709 ( pcmv - hbv / δps2gfp ) construct was made by substitution of bsu36 i ( nt . 3052 )- to - ecor i ( nt . 3182 ) fragment of r015 with the 0 . 7 k bp bsu36 i - to - ecor i fragment of pcr product encoding the gfp ( green fluorescent protein ) gene . the primers used for pcr were : in this subcloning process , a subset of the pre - s2 / s promoter was deleted . to make up this deletion , the bsu36 i ( nt . 3052 )- to - bsu36 i ( nt . 3166 ) fragment was inserted into r709 plasmid to create r710 ( pcmv - prehbv / gfp ). subsequently , to construct r711 ( pcmv - hbv / gpf ), the ecor i ( nt . 3182 )- to - sph i ( nt . 1238 ) fragment was deleted by restriction digestion . taken together , the 1 . 3 k bp fragment ( ecor i - to - sph i ) of hbv genome was substituted by the 0 . 7 k bp gfp fragment . thus , the size of the genome is approximately 0 . 6 k bp smaller than the wild - type . feasibility of the recombinant hbv vector was examined by testing replication competency of the hbv vector . huh7 cells were transfected by r711 plasmid , along with a helper , pcmv - cps . dna extraction and southern blots were performed as described in example 2 - 2 . dna extracted from hepg2 2 . 2 . 15 cells was included as a control ( sells et al ., 1988 ). fig1 a indicated that rc dna was detected from cells transfected by r711 . further , the amount of rc dna and the relative amount of three species of replication - intermediate dna was comparable to that of r015 , the wild - type hbv clone . in addition , the replication of the hbv - gfp vector was further confirmed by using gfp probe ( fig1 b ). all publications and patent applications cited in the above specification are herein incorporated by reference . various modifications and variations of the described method and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention . although the invention has been described in connection with specific preferred embodiments , it should be understood that the invention as claimed should not to be unduly limited to such specific embodiments . indeed , various modifications of the described modes for carrying out the invention which are obvious to those skilled in molecular biology or related fields are intended to be within the scope of the following claims . ausubel , f . et al . eds ., current protocols in molecular biology , wiley and sons , new york , 1995 . chaisomchit , s ., d . l . j . tyrrell , and l . j . chang . gene therapy 4 : 1330 - 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