Patent Application: US-201415029898-A

Abstract:
there is disclosed a retroviral vector comprising a primer binding site , a long terminal repeat and an rna packaging sequence , wherein the rna packaging sequence is located 3 ′ of the long terminal repeat and no long terminal repeat is located 3 ′ of the rna packaging sequence such that reverse transcription initiated at the primer binding site does not lead to reverse transcription of the rna packaging sequence into vector dna in a target cell . also described is a host cell , a virion , a pharmaceutical composition , a method and uses including or involving the vector described above . further , a cell or transgenic animal produced by using the vector is also described .

Description:
lentiviral transfer vector cis elements such as the hiv - 1 rna packaging signal ( ψ ) and rev response element ( rre ) are essential for viral rna genome packaging into virions in producer cells . in standard lentiviral vectors , these cis elements are reverse transcribed into dna along with the transgene expression cassette and persist in target cells after transduction . this persistence creates several known and potential problems for lentiviral vector gene therapy applications . splice sites within cis elements have been shown to splice with nearby host genes , creating aberrant fusion transcripts . the cpg island within the rna packaging sequence undergoes dna methylation in some target cells , potentially contributing to transgene silencing . the rna packaging sequence also enables remobilisation of lentiviral vector genomes in cells expressing lentiviral proteins , which could be problematic in hiv - positive patients . large packaging sequences within the reverse transcript may reduce the size of the transgene cassette which can be accommodated . in standard lentiviral vectors , essential cis elements are located between the two viral long terminal repeats ( ltrs ) and so are within the region of the vector that is reverse transcribed . the inventors have developed a novel transfer vector in which the rna packaging signal and the rre are located downstream of the 3 ′ ltr so are present in the rna genome during virion packaging but are outside of the region of the genome that is reverse transcribed into dna in the target cell . these vectors can be produced to high titre ( 2 . 6 × 10 8 tu / ml by egfp flow cytometry of 293ts , pccl parallel preparation 1 . 3 × 10 9 tu / ml ) and egfp expression is maintained to 14 days post - transduction . it is suggested that the use of this configuration to eliminate most of the remaining viral dna from target cell proviruses could be a feature of the next generation of gene therapy vectors based on hiv - 1 and other retroviruses . in existing lentiviral vectors , cis elements such as the rna packaging signal and the rre are located between the two viral ltrs and so are within the region of the genome that is reverse transcribed . in the ltr1 vector described in this invention , the cis elements are located downstream of the 3 ′ ltr ( fig2 ). these elements are therefore present in the viral rna genome so that it can be packaged into virions , but are outside of the region of the genome that is reverse transcribed and so are not present in the vector dna in the target cell . the name “ ltr1 ” was chosen because these vectors contain only one ltr in the transfer vector plasmid rather the two ltrs present in a conventional lentiviral vector . to understand the mechanism behind this invention it is useful to outline with the classical model of retroviral reverse transcription employed by third generation lentiviral vectors such as rrl or ccl ( fig3 ). in these vectors ( as in those from previous generations ), the substrate for reverse transcription is a viral rna genome located in the target cell . a host - derived trna molecule bound to the pbs acts as a primer for the initiation of minus strand dna synthesis by the viral reverse transcriptase enzyme . once initiated , minus strand synthesis proceeds to the end of the 5 ′ r region . as the minus strand is synthesised , the rnaseh activity of reverse transcriptase degrades the rna strand of the resulting rna - dna double stranded hybrid . upon reaching the end of the 5 ′ r region , minus strand transfer occurs via homology between the r regions on the minus strand dna and the 3 ′ end of the rna genome . minus strand synthesis continues from this region . rnaseh degradation continues , but a short ppt within the rna genome immediately upstream of the u3 region is not degraded . the ppt acts as a primer for the initiation of plus strand dna synthesis . plus strand synthesis continues into the trna primer and stops after the first 18 bp due to the presence of a modified rna nucleotide at the next position in the trna which reverse transcriptase cannot use as a template . plus strand transfer then occurs via homology between the complementary pbs sequences on each dna strand , and synthesis of both dna strands continues until a full length double stranded dna provirus is created . the ltr1 vector described in this invention uses a different mechanism for reverse transcription ( fig4 ). in this vector , the trna primer is predicted to be bound to a pbs located closer to the 3 ′ end of the rna genome . a trna molecule bound to the 5 ′ pbs cannot initiate minus strand synthesis because there is no template upstream from this position . after minus strand synthesis initiates at the 3 ′ pbs , it proceeds to the end of the 5 ′ pbs without a minus strand transfer step taking place . plus strand synthesis initiates at the ppt as before and plus strand transfer occurs via homology between the complementary pbs sequences before synthesis of the double stranded dna provirus . the 3 ′ end of the rna genome including the rna packaging signal and rre is predicted not to be reverse transcribed as it lacks homology with both the minus and plus strands , so cannot be primed for reverse transcription . instead it is predicted to be separated from the reverse transcription complex and eventually degraded by target cell rnases . a number of reports confirm the novel configuration of the ltr1 genome is competent for packaging into virions and reverse transcription in target cells . firstly , it has been shown that the hiv - 1 rna packaging signal and rre remain capable of efficient packaging into virions when relocated close to the centre of heterologous rna molecules ( 8 ). secondly , it has been shown that a pbs flanked by the appropriate local hiv secondary structure remains capable of efficient initiation of minus strand synthesis when relocated to the centre of a lentiviral vector genome ( 29 ). the practical development of a lentiviral vector which employs this novel reverse transcription mechanism to eliminate cis elements such as the rna packaging signal and the rre from target cell proviruses is described herein . the parental plasmids for the constructs described in this invention were the prrl . sin and pccl . sin lentiviral transfer vector plasmids for hiv - 1 components , the pgk promoter , the egfp cdna and the wpre ( 3 ), the pci plasmid for the sv40 late polyadenylation signal ( promega ), and gene synthesis of the gapdh promoter ( life technologies ) based on the university of california , santa cruz ( ucsc ) human genome build hg19 between the primer sequences described in ( 30 ). the sequences for all plasmids are provided in the appendix . plasmids pcmv - dr8 . 74 and pmdg2 were generated and distributed by didier trono at the ecole polytechnique federale de lausanne . 1 . 2 × 10 7 hek293t ( 293t ) cells were seeded in t175 flasks in 20 ml dulbecco &# 39 ; s modified eagle &# 39 ; s medium supplemented with 10 % fetal calf serum and 1 % pencillin - streptomycin ( complete dmem ) one day before transfection to reach & gt ; 90 % confluence . for each flask , 50 μg vector plasmid , 32 . 5 μg packaging plasmid pcmv - dr8 . 74 , and 17 . 5 μg vesicular stomatitis virus envelope plasmid pmdg2 was added to 5 ml optimem ( gibco ) and 0 . 22 μm filtered . 1 μl 10 mm pei ( sigma - aldrich 40872 - 7 ) was added to 5 ml optimem and 0 . 22 μm filtered . the two mixtures were combined and allowed to complex for 20 min . the complex was added to the t175 flask containing 293t cells and incubated at 37 ° c . with 5 % co 2 for 4 hours . the complex was removed and replaced with 20 ml complete dmem . 24 hours later , the medium was replaced . 48 hours after transfection the medium was removed , 0 . 22 μm filtered , and centrifuged at 50 , 000g for 2 hours . virus pellets were resuspended in 1500 optimem and aliquots were stored at − 80 ° c . 10 5 293t cells were seeded in 250 μl of complete dmem into each well of a 24 - well plate one day before transduction . for transduction , a 5 - fold dilution series of virus was performed in 50 μl aliquots of optimem . each aliquot of diluted virus was added to the medium in one well . transgene expression was measured by flow cytometry 14 days post - transduction . expression titre was calculated by selecting the well in which 5 - 15 % of transduced cells expressed the transgene of interest and dividing the number of transduced cells in this well by the volume of virus used to transduce them . briefly , hek 293t cells were transduced at high moi with ltr1 . 20 / ampr - ori , containing an ampicillin resistance gene and bacterial origin of plasmid replication , and left to culture for 1 week before recovery of cell pellets . genomic dna was extracted using a commercial kit ( qiagen ) and quantified by nanodrop . 10 μg of gdna was digested with xbai restriction endonuclease , which does not target any sequence within the provirus , but exists within the human genome at an incidence of approximately 279 . 3 sites per megabase pair . the digested gdna was then column purified before ligation to circularise any released lentiviral backbone fragments , followed by transformation of electro - competent escherichia . coli . individual bacterial colonies were selected and grown in preparation for sequence analysis ; these would only grow if they received a re - circularised copy of the vector backbone containing ampr and ori . recovered dna was sequenced using the primers targetting the rre region ( which should be omitted from the final provirus ), the bacterial origin of replication and the ampicillin resistance gene . the production of infectious lentiviral virions requires co - assembly of the viral genomic rna with viral gag , gag - pol and envelope proteins . previous reports of codon - optimised gag - pol expression plasmids improved the intracellular expression of these proteins but did not result in higher titres ( 21 ; 31 ). it is likely that expression of the viral rna genome is generally the limiting factor for viral titre in many vector preparation protocols . therefore , in order to maximise the titre obtainable with the ltr1 vector experiments were undertaken to optimise the elements used to drive viral rna genome transcription . efficient polyadenylation is associated with higher steady - state levels of rna polymerase ii ( pol ii ) transcripts , and it has been shown that the simian virus 40 ( sv40 ) late polyadenylation ( polya ) signal acts as a stronger polyadenylation signal than the sv40 early polya signal ( 32 ). the inventors therefore generated two ltr1 configurations : ltr1 . 0 / pgk - egfp - wpre ( ltr1 . 0 / pew ) uses the sv40 early polya signal present in the parental rrl backbone while ltr1 . 5 / pew uses an sv40 late polya signal derived from pci ( fig5 ). lentiviral vector was prepared in parallel using these two constructs and a conventional rrl / pew construct and titred on 293t cells by flow cytometry . the vector titres were calculated as ltr1 . 0 pew 2 . 3 × 10 4 transducing units / ml ( tu / ml ), ltr1 . 5 pew 4 . 5 × 10 4 tu / ml , rrl pew 6 . 9 × 10 7 tu / ml . as the sv40 late polya signal yielded a higher titre , this element was used in all subsequent ltr1 constructs . continued egfp expression 14 days after transduction and following at least four passages of dividing cells suggests that the ltr1 vectors are competent for integrase - mediated chromosomal integration . high steady - state levels of pol ii transcripts can also be achieved through the use of a strong promoter , so constructs were generated in which the rous sarcoma virus ( rsv ) promoter derived from the parental rrl backbone was replaced by a human cytomegalovirus immediate early ( cmv ) promoter derived from a ccl plasmid . in order to optimise the alignment of the cmv promoter and the ltr1 5 ′ pbs , three constructs were generated in which the pbs was positioned precisely on the reported transcription start site for the cmv promoter ( 33 ) ( ltr1 . 7 . 672 / pew ), 1 bp upstream ( ltr1 . 7 . 671 / pew ) or 1 bp downstream ( ltr1 . 7 . 673 / pew ). lentiviral vector was prepared in parallel from these constructs , ltr1 . 5 / pew and rrl / pew and titred on 293t cells by flow cytometry . the replacement of the rsv promoter with the cmv promoter resulted in a 25 - fold increase in ltr1 vector titre ( ltr1 . 5 pew 2 . 3 × 10 4 tu / ml , ltr1 . 7 . 671 pew 5 . 6 × 10 5 tu / ml , ltr1 . 7 . 672 pew 5 . 4 × 10 5 tu / ml , ltr1 . 7 . 673 pew 4 . 7 × 10 5 tu / ml , rrl pew 5 . 8 × 10 7 tu / ml ). as the ltr1 . 7 . 671 cmv configuration yielded the highest titre , this promoter and alignment were used in subsequent ltr1 constructs . lentiviral rna genomes acquire a 5 ′ 7 - methylguanylate cap ( m7g ) cap during transcription by polii and hence are competent for translation by producer cell ribosomes . in the scanning model of translation initiation in mammalian cells , the ribosome loads onto the 5 ′ cap and scans in a 5 ′- 3 ′ direction until it reaches the 5 ′- most atg codon , at which point translation can initiate . in rrl and ccl rna genomes the 5 ′- most atg occurs at the start of the truncated gag element and is followed by a 21 codon open reading frame . in the ltr1 vector configuration , the 5 ′- most atg will be found within the transgene expression cassette , probably at a cryptic atg within the internal promoter . for example , in the ltr1 . 7 . 671 pew vector the 5 ′- most atg occurs close to the middle of the pgk promoter and is in frame with the downstream egfp coding sequence with no intervening stop codons . since ltr1 . 7 . 671 pew was found to yield vector titres around 10 2 - fold lower than an rrl pew vector it was hypothesised that this cryptic translation product might be interfering with virion production in producer cells . to test this hypothesis the pgk promoter was replaced with the human gapdh promoter which lacks atg codons entirely . in ltr1 . 7 . 671 gapdh - egfp - wpre ( ltr1 . 7 . 671 gew ) the 5 ′- most atg is at the start of the egfp open reading frame , so translation of full length vector rna should produce egfp only . lentiviral vector was prepared from rrl pew , rrl gew , ltr1 . 7 . 671 pew and ltr1 . 7 . 671 gew and titred on 293t cells by flow cytometry . the resulting titres were rrl pew 6 . 4 × 10 7 tu / ml , rrl gew 1 . 6 × 10 8 tu / ml , ltr1 . 7 . 671 pew 7 . 1 × 10 5 tu / ml and ltr1 . 7 . 671 gew 7 . 6 × 10 5 tu / ml . cryptic translation initiation does not appear to have been the limiting factor in this ltr1 vector preparation , but the gapdh promoter was used in subsequent constructs as the mean fluorescence intensity indicates that it is a stronger promoter than the pgk promoter so is a better reporter of successful transduction . the sin ltr located near the midpoint in the ltr1 vector configuration contains the hiv - 1 polyadenylation signal . although a previous report in which the hiv - 1 ltr was relocated to the midpoint of a vector reported efficient full length transcription ( 8 ), it was decided to investigate the possibility that polyadenylation within the sin ltr of an ltr1 vector might be preventing production of full length rna genomes and hence causing the observed reduction in titre relative to third generation lentiviral configurations . in order to prevent cleavage and polyadenylation at the ltr polya signal , the aataaa motif which binds cleavage and polyadenylation specificity factor ( cpsf ) was mutated to aacaaa in construct ltr1 . 11 . 0 gew . this mutation was previously reported to abolish cleavage and polyadenylation at the hiv - 1 polya signal ( 34 ). as a functional polya signal is required for reporter gene expression in target cells , a fragment of the hiv - 1 genome covering the region from the 5 ′ r to the major splice donor ( msd ) stem loop was introduced at the 5 ′ end of this construct and the 3 ′ pbs was deleted . this vector is predicted to initiate minus strand synthesis at the 5 ′ pbs and undergo minus strand transfer and subsequent stages of reverse transcription in the same way as a third generation lentiviral vector so that the 3 ′ mutated polya signal is replaced by the functional 5 ′ polya signal . construct ltr1 . 11 . 1 gew is the same as ltr1 . 11 . 0 except that it retains a functional aataaa motif . lentiviral vector prepared from ltr1 . 11 . 0 gew and ltr1 . 11 . 1 gew yielded titres of 2 . 5 × 10 8 tu / ml and 1 . 7 × 10 8 tu / ml respectively compared to a ccl gew parallel control titre of 1 . 3 × 10 9 tu / ml . this suggests that the midpoint polya signal is not efficiently used in an ltr1 configuration . it is possible that the msd located downstream of the ltr is able to block polyadenylation at this site as occurs at the 5 ′ polya signal in a conventional lentiviral vector ( 35 ). 2 . 4 . effects of minus strand transfer and a 5 ′ major splice donor the greatly improved titres observed with the ltr1 . 11 configuration suggested two possible explanations . firstly , initiation of minus strand synthesis at the 5 ′ pbs and / or minus strand transfer might improve the efficiency of reverse transcription . secondly , the presence of the msd at the 5 ′ end of the vector might improve the efficiency of rna genome transcription . in order to test these hypotheses construct ltr1 . 13 . 0 gew was generated which resembles ltr1 . 7 . 671 except that the 5 ′ end of the rna includes the full hiv - 1 genome between the pbs and the msd stem loop instead of only the 18 bp pbs . in this construct initiation of minus strand dna synthesis is predicted to take place at the midpoint of the vector . construct ltr1 . 13 . 1 gew is identical to ltr1 . 13 . 0 gew except that the sequence between the 5 ′ pbs and the msd stem loop has been deleted . lentiviral vector was prepared from ltr1 . 13 . 0 gew , ltr1 . 13 . 1 gew and ltr1 . 7 . 671 gew and titred by flow cytometry . these constructs yielded titres of 2 . 6 × 10 8 tu / ml , 9 . 2 × 10 7 tu / ml and 3 . 2 × 10 7 tu / ml respectively compared to a ccl gew parallel control titre of 1 . 3 × 10 9 tu / ml . these results suggest that the elimination of minus strand transfer from reverse transcription has no negative impact on viral titre and that the large increase in titre observed with the ltr1 . 11 configuration was due to the presence of the full sequence between the 5 ′ pbs and the msd stem loop . it has been previously reported that splice sites close to the promoter activate transcription of mammalian genes ( 36 ) and previous attempts to mutate the major splice donor in lentiviral vectors resulted in reduced vector titres ( 20 ; 21 ). therefore it appears that the presence of splicing factors close to the 5 ′ end of the vector rna genome is required for the production of higher titre lentiviral vectors in both a conventional and ltr1 configuration . in order to mimic the transcriptional activating effect of the msd in ltr - 1 vectors while reducing hiv sequence , it was replaced by a chimeric intron from the pci expression plasmid , inserted between the pbs and cppt , to produce pltr1 . 20 / gapdh - egfp - wpre . as the wild - type 5 ′ ltr has been removed from ltr - 1 the hiv msd no longer functions to regulate the production of subgenomic rnas in this vector and it can be exchanged . the use of the strong heterologous intron from pci offers advantages over the inclusion of the msd given that no flanking splice enhancers are required and it also includes a splice acceptor which facilitates its removal from viral rna in producer cells . addition of the pci chimeric intron in the pltr1 . 20 / gapdh - egfp - wpre vector led to a 3 - fold improvement in titre , as calculated by facs . to demonstrate the structure of reverse transcribed proviral dna from the pltr1 . 7 . 671 / gapdh - egfp - wpre vector , hek293t cells were transduced at a multiplicity of infection of 10 . genomic dna was extracted 1 week after transduction . the full - length provirus was amplified by pcr using the oligos designated below to give a 2 . 1 kb amplicon , which was extracted following separation on a 1 % agarose gel and ta - cloned ( lifetechnologies ) prior to sequencing . sequence analysis showed that the provirus contained the correct 5 ′ and 3 ′ ltrs following reverse transcription , displaying the expected sequence with the gag - rre region removed , when the provirus was examined . in order to stringently investigate the sequences of ltr - 1 proviruses and confirm the absence of the deleted hiv - 1 packaging sequences , a technique was employed in which the ampicillin resistance marker and bacterial origin of replication were removed from the pltr1 . 20 plasmid backbone and inserted within the transgenic region , between the ltrs . this enables pltr1 . 20 / amp1r - ori proviral dna to be excised from transduced hek 293t cells , recircularised and transformed into escherichia . coli bacteria which would replicate and form colonies upon uptake of a vector genome containing an ampicillin resistance gene ( ampr ) and bacterial origin of replication ( ori ) between the ltrs . propagation of proviral sequences in bacteria allows the reverse transcribed sequence to be studied in detail . sequencing of the proviral dna rescued in bacteria reads through the ltr and into a region within , host cell chromosomal dna which was identified by blat search . the internal sequence of the provirus confirmed the mechanism of reverse transcription did result in the expected structure as shown in fig4 . it was not possible to sequence the rre element , suggesting that it was absent from the integrated provirus as predicted . to demonstrate ltr - 1 vector function in vivo , 1 day old neonatal cd - 1 mice were injected intravenously with 4 . 5x10 5 vector particles of ltr1 . 20 / sffv - egfp - wpre . mice were sacrificed and livers were dissected 1 week after vector administration and imaged revealing gfp - positive cells visible in the livers of injected animals demonstrating that ltr - 1 is capable of gene deliver to cells in vivo . a number of different retroviruses have been developed as retroviral vectors for mammalian gene transfer ( 1 - 5 ). the largest change which takes place during vectorisation of a retrovirus is the separation of viral components into cis elements which must remain present on the nucleic acid being transferred into the target cell and trans elements which need only be provided in producer cells to enable production of infectious particles . examples of retroviral cis elements include the hiv - 1 rna packaging signal ( ψ ) and rev response element ( rre ) which must remain covalently attached to the transgene expression cassette in order for it to be packaged into virions , while trans elements include coding sequences for the gag and gag - pol polyproteins and the viral envelope glycoprotein . as with other retroviral vectors , the cis elements within hiv - 1 - based lentiviral vectors are located between the viral long terminal repeats ( ltrs ) so are reverse transcribed and remain associated with the transgene expression cassette within the target cell . the presence of these cis elements within target cell proviruses creates a number of empirically observed and theoretically possible problems for the practical application of lentiviral vectors , particularly for gene therapy . the cis elements contain splice sites able to splice with and dysregulate host genes involved in the control of cell proliferation ( 14 ), cpg islands able to undergo dna methylation ( 16 ), large untranscribed regions associated with transcriptional silencing of episomal dna molecules ( 17 ), and rna packaging signals able to mediate vector remobilisation ( 15 ). lentiviral cis elements also occupy up to 2 kb of the reverse transcript which may reduce the size of transgene expression cassettes which can be delivered with these vectors . in this report , an approach to eliminating hiv - 1 - derived cis elements from the dna delivered to target cells is described . in the novel ltr1 configuration the cis elements are located downstream of the 3 ′ ltr . these elements are therefore present in the viral rna genome so that it can be packaged into virions , but are outside of the region of the genome that is reverse transcribed and so are not present in the vector dna in the target cell . in the first configuration tested , transcription of the ltr1 rna genome was driven by an rsv promoter and sv40 early polya signal cassette . this configuration produced functional vector titres 3000 - fold lower than a conventional rrl vector . a modified cassette using the cmv promoter and an sv40 late polyadenylation signal improved titres significantly to approximately 100 - fold lower than a conventional rrl vector . by relocating the hiv - 1 - derived 5 ′ leader sequences the new configuration raised the possibility of aberrant translation products originating from within the transgene cassette . it was suggested that aberrant translation could be reducing the vector titres obtained with these constructs . the inventors addressed this possibility by replacing the pgk promoter which contains a cryptic start codon with a gapdh promoter which lacks atg codons . no difference in titre was observed . another potential problem for vector titre is the presence of a functional polyadenylation signal within the ltr located near the midpoint of the vector . termination of transcription at this site would prevent production of full length transfer vector rna competent for packaging into virions . a point mutation was introduced to the aataaa hexamer which was previously shown to abolish cleavage and polyadenylation at the hiv - 1 polya signal but observed no increase in titre . the inventors hypothesise that the hiv - 1 major splice donor ( msd ) located downstream of the ltr in the ltr1 configuration is able to block cleavage and polyadenylation at this site just as it does in wildtype hiv - 1 and conventional lentiviral vectors ( 35 ). the inventors then reintroduced sequences between the pbs and the msd stem loop to the 5 ′ end of the ltr1 genomic rna and observed a significant increase in titre to within 5 - fold of a ccl - based construct . the presence of splicing factors close to the 5 ′ end of the vector genomic rna appears to aid the production of high titre lentiviral vectors in both the conventional and ltr1 configurations but is not essential ( 20 ; 21 ). the ltr1 vector configuration described in this report could potentially replace all third generation hiv - 1 - based lentiviral vectors in the applications in which they are currently used . furthermore , the mechanism of reverse transcription is highly conserved among retroviruses so an ltr1 configuration could be applied to vectors based on retroviruses other than hiv - 1 . 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