the examples below describe two embodiments of stable delivery and expression of - hiv cell entry inhibitor proteins via viral gene transfer . the embodiments exploit the ability of raav to effect long - term delivery to , expression of genes in , and secretion of proteins from mature skeletal muscle . the goal of secretion of hiv - 1 cell entry inhibitors into circulation is to inhibit hiv - 1 replication . the examples illustrating embodiments of the invention include example 1 describing use of rantes chemokine derivatives to inhibit hiv - 1 infection via ccr5 co - receptor blockade and example 2 describing the delivery and expression of genes encoding hiv - 1 fusion inhibitor peptides to inhibit hiv - 1 replication and growth . use of rantes chemokine derivatives to inhibit hiv - 1 infection via ccr5 co - receptor blockade since primary hiv - 1 isolates almost exclusively utilize ccr5 as the co - receptor for initial infection of cells , the chemokine rantes ( a natural ccr5 ligand ) represents an ideal candidate for competitive blockade of the ccr5 co - receptor . the present inventors contemplate that elevated plasma levels of the rantes chemokine will significantly attenuate or prevent hiv - 1 infection of cd4 + cells and that the approach will be well - tolerated in vivo , since individuals who are deficient in ccr5 signaling are healthy and lack obvious immunological defects . as described below , rhesus rantes genes ( wild - type and a non - signaling mutant ) have been cloned into raav - 1 vectors and are delivered into mouse muscle tissue . in order to maximize circulating rhrantes expression levels as well as decease its proinflammatory activities , optimized molecular constructs were generated . first , an optimized leader sequence was added onto the n - terminus of rhrantes to increase the efficiency of protein secretion from muscle cells into the systemic circulation . second , a mutant rhrantes ( c1c5 ) was constructed that retains the ability to associate with the ccr5 co - receptor but lacks chemotactic properties to minimize the potential for undesirable inflammatory responses imparted by rantes overexpression on the cell - signaling cascade in vivo . c1c5 has two ser → cys substitutions at positions 1 and 5 . polo et al ., eur . j . immunol ., 30 : 3190 - 3198 ( 2000 ) demonstrated that this mutated form of rantes ( c1c5 ) has a reduced ability to induce chemotaxis , but increased hiv - 1 blocking activity when compared to wild - type rantes . third , to maximize gene transfer levels in muscle raav - 1 serotype rather than aav - 2 serotype vectors were constructed . fourth , to enhance the specific activity ( potency ) of the raav / chemokine vectors , self - complementary raav / chemokine vectors were constructed . mccarty et al ., gene ther ., 10 : 2112 - 2118 ( 2003 ) showed that hairpin vectors rapidly form transcriptionally active double - stranded templates within a transduced cell , resulting in increased expression levels ( typically 10 - fold ) and expression kinetics compared to standard single - strand raav vectors . macaca mulatta ( rhesus ) specific rantes pcr primers were designed and used to pcr amplify both wild - type and mutant forms of the rhesus rantes from a plasmid encoding the human rantes cdna ( porf - hrantes ; invivogen inc .). forward primers for both wild - type and mutant forms were designed to contain an optimized synthetic leader sequence . furthermore , all primers were engineered with not i restriction enzyme sites to facilitate molecular cloning . fig1 illustrates the amino acid differences between the mature human and rhesus ( wild - type and c1c5 mutant ) rantes . the pcr products were subsequently cloned into pcr2 . 1 topo ta cloning vector ( invitrogen ) and constructs containing the 300 bp rhrantes amplification product were confirmed by dna sequencing . upon dna sequence confirmation , one pcr2 . 1 clone each for the wild - type and c1c5 mutant were digested with not i restriction enzyme to release the rantes coding region which was then ligated into a not i digested ptp - 1 / β - gal cloning vector . the approach was similar in concept to that previously published in clark et al ., human gene therapy , 6 : 1329 - 1341 ( 1995 ). specific modifications to construct the ptp - 1 / β - gal plasmid construct were as follows : first , a raav vector carrying the e . coli lac z transgene was generated and was termed paav / cmv / β - gal . the base raav vector was derived from psub201 [ samulski et al ., j . virol ., 61 : 3096 - 3101 ( 1987 )], which contains a wild - type aav genome that has been altered to contain convenient restriction enzyme sites ( xba i ) that facilitate removal of the rep and cap genes and insertion of a 4 . 5 kb cmv promoter — e . coli lac z — sv40 poly a transgene expression cassette ( released by pst i restriction enzyme digestion ) from plasmid pcmvβ ( clontech ). the resulting raav vector was 4 . 9 kb in length ( including aav2 inverted terminal repeats ), which was 104 . 7 % of wild - type genome length . secondly , a plasmid dna construct designated pbs / rep2 - cap1 / neotk was generated . in brief , a 4 . 4 kb restriction fragment containing the aav2 rep and aav1 cap sequences was obtained by not i and ngom iv restriction enzyme digestion of plasmid pxr1 ( rabinowitz et al ., j . virol ., 76 ( 2 ): 791 - 801 ( 2001 ). the rep2 - cap1 dna fragment was blunt end ligated into xba i restricted plasmid pbs / neotk vector ( contains the sv40 early promoter — neomycin phosphotransferase gene — thymidine kinase polyadenylation site cassette cloned into pbluescript ks −) lastly , the construction of a tripartite plasmid was accomplished by removing the rep2 - cap1 / neo r cassette from pbs / rep2 - cap1 / neotk ( via not i and cla i restriction enzyme digestion ) and inserting it into the unique ngom i site in paav / cmv / β - gal . thus , the ptp - 1 / β - gal tripartite plasmid contained : ( i ) a raav vector genome ( raav / β - gal ), ( ii ) rep2 - cap1 , and ( iii ) the neo r gene . recombinant clones containing the rantes coding regions were identified by not i restriction and confirmed by dna sequencing . two correct and two reverse orientation rhrantes ( wild - type and c1c5 mutant ) clones ( 4 total ) were chosen for further analysis . to construct the sc raav - 1 producer plasmids , the rhrantes transgenes ( the wild - type or c1c5 mutant ) were cloned into a self - complementary raav - 1 producer plasmid ( ptp - 1 / sc - x5 ) using not i restriction sites . the ptp - 1 / sc - x5 plasmid was made in several steps . first , the x5 scfv coding sequence ( 800 bp ) was pcr amplified with not i restriction site ends from plasmid pcombx / x5 scfv ( gift from dr . dennis burton , the scripps research institute , la jolla calif .) and cloned into paav / cmv / β - gal following removal of the 3 . 4 kb lac z gene by not i restriction enzyme digestion to yield plasmid paav / cmv / x5 . next , the 1 . 8 kb cmv - x5 - sv40 poly a cassette was pcr amplified with hpa i and xba i restriction site ends and sequence identity confirmed by sequencing . this dna fragment was cloned into plasmid phpa7 ( gift of r . jude samulski , university of north carolina ) that was restricted with hpa i and xba i . phpa7 plasmid contains a deletion in the 5 ′ aav2 inverted terminal repeat that results in the packaging of double - stranded self - complementary vectors ( mccarty et al ., gene ther ., 10 ( 26 ): 2112 - 2118 ( 2002 ). the resulting plasmid was termed paav / cmv / sc - x5 . lastly , the construction of a tripartite plasmid was accomplished by removing the rep2 - cap1 / neo r cassette from pbs / rep2 - cap1 / neotk ( via not i and cla i restriction enzyme digestion ) and inserting it into the unique swa i site in paav / cmv / sc - x5 . thus , the ptp - 1 / sc - x5 tripartite plasmid contained : ( i ) a raav vector genome with a mutated 5 ′ inverted terminal repeat ( raav / cmv / sc - x5 ), ( ii ) rep2 - cap1 , and ( iii ) the neo r gene . correct constructs containing the rhrantes transgenes were confirmed by restriction enzyme digestion and dna sequencing . additionally , dna sequence analysis confirmed that the sc vectors contained the expected hpa i - xba i deletion in the 5 ′ viral inverted terminal repeat ( itr ). to demonstrate the ptp - 1 / rhrantes vectors were functional , bhk - 21 ( baby hamster kidney ) cells were transfected with the rhrantes plasmid clones using superfect transfection reagent ( qiagen inc .). forty - eight hours post - transfection , cell culture supernatant was analyzed for the presence of rantes ( ng / ml ) using a commercial human rantes elisa ( r & amp ; d systems , inc .). as seen in fig2 wherein “*” denotes reverse orientation and “#” denotes correct orientation , bhk - 21 cells produced significant amounts of secreted rhrantes compared to negative control plasmid transfections ( cmv - egfp and ptp - 1 / β - gal ). furthermore , raav - 1 / rantes plasmids containing the transgene in the reverse orientation produced background levels of rantes . the amount of c1c5 rhrantes found in the cell supernatant was consistently lower than the wild - type rhrantes , but this is likely due to reduced antibody affinity in the commercial elisa for the mutated c1c5 form . the recombinant sc plasmids were also competent for rantes production . rhrantes production from single - stranded ( ptp - 1 ) and double - stranded sc raav1 / rhrantes production plasmids were compared in bhk - 21 or hela cells . forty - eight hours after plasmid transfection , the supernatant was assayed by elisa for rantes protein expression . as shown in fig3 , the sc vectors produced significant amounts of secreted rantes compared to the negative control dna plasmid transfections ( raav - 1 - β - gal and raav1 - x5 ). again , the level of wild - type rantes production was greater than that observed for the c1c5 mutant constructs . data are the average of 3 separate transfections . to demonstrate the raav producer plasmids were able to replicate and generate infectious raav particles efficiently , a passage assay was performed . briefly , hela cell were transfected with the raav1 / rhrantes plasmids and subsequently infected with adenovirus type 5 ( ad5 ) at an moi = 20 . forty - eight hours later , cells were harvested and crude cell lysates were prepared . following heat inactivation ( 56 ° c ., 30 min ) of the ad5 , a 1 : 10 dilution of the clarified cell lysate was added to c12 cells ( aav2 rep expressing cell line ) in the presence of a ad5 . forty - eight hours later , low molecular weight dna was extracted from the cells . following gel electrophoresis , southern blot dna hybridization was performed to visualize aav replication intermediates . as shown in fig4 , detectable monomeric ( 1 . 7 kb ) and dimeric ( 3 . 4 kb ) replication forms were observed in the c12 dna indicative of infectious raav1 formation in the hela cell clarified lysate . in fig4 , lane 1 is ptp - 1 / wt rhrantes + ad5 and lane 2 is ptp - 1 / c1c5 rhrantes + ad5 . hybridizing dna fragments were detected that corresponded to the expected sizes of replication competent virus : momomeric form = 1 . 7 kb , dimeric form = 3 . 4 kb . replication forms were present in neither hela cells infected with ad5 nor hela cells transduced with cell lysates prepared from ptp - 1 / rhrantes transfected hela cells minus ad5 infection . similar results were obtained for the sc raav1 / rhrantes recombinant vectors , confirming the ability of the plasmid constructs to generate infectious raav1 / rhrantes particles ( data not shown ). g . raav1 / rhrantes particles are infectious and produce rantes following transduction of cells in culture . to document the ability of the raav1 / rhrantes viral vectors to mediate secreted rantes expression following infection of cells in culture , a small - scale viral preparation ( via transient plasmid transfection ) was generated and used to infect c12 cells ( moi = 1 , 000 dnase resistant particles per cell ) in absence or presence of ad5 ( moi = 20 ). rantes elisa was performed on the cell culture supernatants from c12 cells transduced with raav1 / rhrantes vectors ( wild - type and c1c5 mutant ) or self - complementary derivatives ( wild - type and c1c5 mutant ). as shown in fig5 , all 4 raav1 / rhrantes vectors ( ss wild - type , ss c1c5 , sc wild - type , and sc c1c5 ) produced significantly greater amounts of rantes compared to raav1 / vector negative controls ( raav1 / β - gal , raav1 / gfp , and sc raav1 / x5 ). consistent with the plasmid transfection data , the wild - type vectors appear to produce greater levels of rantes compared to the c1c5 mutant vectors . similar levels and patterns of expression were observed in hela and bhk - 21 cells after of raav1 / rhrantes vector infection ( data not shown ). to facilitate large - scale viral production , hela - based cell lines were constructed by plasmid dna transfection and drug resistance selection using the four ptp - 1 / rhrantes plasmids ( ss wild - type , ss c1c5 mutant , sc wild - type , and sc c1c5 mutant ). optimal producer cell lines were selected essentially as described by clark et al ., hum . gene ther ., 6 : 1329 - 1341 ( 1995 ). positive cell lines containing a replicating raav genome were expanded and the dnase resistant particles ( drp ) per cell productivity determined by quantitative taqman pcr analysis . table 1 shows the drp per cell values for the clones that were subsequently sent to the viral vector core for cell - cube production . a second attractive target for hiv - 1 entry inhibition is the final step of the hiv - 1 infection process , fusion of the viral envelope with the cell membrane . fusion is mediated by the gp41 envelope glycoprotein and a model of gp41 - mediated membrane fusion analogous to the “ spring - loaded ” mechanism of influenza virus has been proposed . the sequence of gp41 contains two heptad - repeat regions termed hr1 and hr2 that denote the presence of hydrophobic regions found in 2 alpha - helical “ coiled - coil ” structures . significantly , mutations in these hr regions interfere with the fusion property of gp41 . the model predicts that the gp120 - gp41 trimer holds each gp41 moiety in a high - energy configuration , with the fusion peptide pointed inward , toward the viral surface . the binding of gp 120 to cd4 and chemokine co - receptors appears to release gp41 from this configuration , causing the fusion peptide to “ spring ” outward toward the cell membrane . the hr1 regions then fold over into the hydrophobic groove formed by the three corresponding hr2 regions , forming a stable six - helix bundle , resulting in the juxtaposition of viral and cellular membranes and ultimately fusion . two gp41 hr2 peptides t - 20 and t - 1249 are currently being studied as small molecule inhibitors of hiv - 1 fusion . t - 20 and t - 1249 partially overlap , but t - 1249 extends into a “ deep - pocket ” region of hr1 that is important for the formation of the six - helix structure required for fusion ( fig6 ). these competitive inhibitors are thought to bind to the hr1 region and “ lock ” it into a non - fusogenic conformation . both peptides appear to possess broad activity against x4 , r5 , and dual tropic variants of hiv - 1 . importantly , oral treatment with this large peptide is not feasible and daily intravenous doses of peptide are required for therapeutic effect . the present inventors contemplate that therapeutic levels of circulating t - 20 / t - 1249 can be achieved via raav mediated muscle - targeted gene transfer . towards this objective , raav - 1 based vectors expressing the t - 20 or t - 1249 peptides have been constructed as described below . in order to maximize circulating t - 20 and t - 1249 expression levels , constructs were first engineered to include an optimized synthetic leader sequence to increase the efficiency of protein secretion from muscle cells into the systemic circulation . second , the t - 20 dna was synthesized by retrogen inc . using optimal human codon usage to enhance gene expression . third , to maximize gene transfer levels into myocytes , raav - 1 serotype vectors were constructed . fourth , self - complementary raav / chemokine vectors were generated . a synthetic , codon - optimized t - 20 oligonucleotide ( seq id no : 7 ) was generated by retrogen inc . the sequence generated was as follows : the gene possesses flanking not i restriction sites ( lower case letters ) to facilitate cloning and a 5 ′ kozak consensus sequence ( ccacc ) for efficient translation initiation . additionally , the construct encodes a 21 amino acid synthetic secretory leader sequence that provides increased secretion . the t - 20 gene was cloned into ( via not i restriction sites ) the raav - 1 producer plasmid ptp - 1 / β - gal to yield ptp - 1 / sl - t20 and recombinant clones confirmed by dna sequencing . similarly , the t - 20 gene was cloned into the sc raav - 1 producer plasmid ( ptp - 1 / sc - x5 ) using not i restriction sites to generate the hairpin vector ( ptp - 1 / sc / sl - t20 ). taking advantage of significant sequence overlap between the t - 20 and t - 1249 sequences , pcr amplification was used to generate the t - 1249 gene using the t - 20 gene as the pcr template . producer plasmids were generated that included the optimized synthetic leader sequence or the native leader sequences one of two highly secreted proteins , cystatin and alpha - 1 anti - trypsin . three forward pcr primers were synthesized to incorporate the appropriate leader sequence and add the 9 additional n - terminal amino acids to the t - 20 template sequence . the primer sequences were : the three pcr products were cloned into ptp - 1 / β - gal as described above using not i restriction sites . to compare the efficiency of t - 1249 secretion into cell culture media , hela cells were transfected ( superfect ; qiagen inc .) with the ptp - 1 / t - 1249 plasmids and t - 1249 levels quantified in cell culture supernatants ( 1 : 5 dilution ) 48 hr post - transfection . levels were determined by extrapolation from a standard curve generated using a t - 20 peptide standard and an hiv - 1 neutralizing monoclonal antibody ( 2f5 ) that recognizes a linear epitope ( eldkwa ) present within both peptides . western dot blot assay sensitivity was determined to be 20 ng t - 20 / dot . to normalize for transfection efficiency , a second plasmid encoding the e . coli lacz gene was included in the transfection ( pcmv / β - gal ) and β - galactosidase levels quantified using a commercial kit ( all - in - one β - gal kit , pierce chemical ). normalization for the transfection efficiency allowed for comparison of relative t - 1249 levels . the experiment was performed in duplicate and data are the average of the 2 experiments . as seen in fig7 , the synthetic leader was approximately 2 - 4 fold better at mediating peptide secretion compared to the native cellular leader peptides . d . production of infectious raav - 1 particles and transduction of cells in culture a viral passage assay was then performed to confirm the ability of plasmids ptp - 1 / sl - t20 and ptp - 1 / sl - t1249 to generate infectious raav1 particles , similar to that described for the rantes vectors . optimal raav - 1 hela producer cell lines were then isolated and productivity assessed using quantitative taqman pcr ( 3 . 6 × 10 4 drp / ml , t - 20 and 1 . 7 × 10 4 drp / ml , t - 1249 ). a small - scale raav1 / sc / sl - t20 vector stock was generated by wild - type ad5 infection ( moi = 20 ) and virus purified by iodixanol gradient fractionation and anion - exchange chromatography . a purified raav1 / sl - t20 vector was used to infected hela cells ( 2 × 10 6 cells ) at the moi indicated in fig8 . forty - eight hr post - transduction cell culture supernatant was collected and a cell lysate was generated by detergent lysis . both the supernatant ( 1 : 5 dilution of 0 . 2 ml ) and cell lysate ( 1 × 10 5 cell equivalents ) were blotted onto a nitrocellulose membrane and t - 20 levels visualized using the human 2f5 antibody ( 1 : 1 , 000 dilution ) as the primary antibody . as seen in fig8 , a dose response relationship was observed at various raav - 1 / t20 inputs ( moi = 1 , 000 drp or 10 , 000 drp ), with robust t - 20 secretion into the cell culture supernatant . while the present invention has been described in terms of specific embodiments , it is understood that variations and modifications will occur to those skilled in the art . accordingly , only such limitations as appear in the claims should be placed on the invention .
the present invention relates generally to the use of recombinant adeno - associated viruses for gene delivery and more specifically to the use of raav to deliver genes encoding human immunodeficiency virus entry inhibitors to target cells in mammals .