Patent Abstract:
genetic modification of pluripotent hemopoietic stem cells of primates by transduction of p - phsc with a recombinant adeno - associated virus . tile genomc of the recombinant aav comprises a dna sequence flanked by the inverted terminal repeats of aav . the dna sequence will normally comprise regulatory sequences which are functional in hemopoictic cells and , controlled by these regulatory sequences , a sequence coding for a protein or rna with a therapeutic property when introduced into hemopoietic cells . preferred examples of dna sequences are the human lysosomal glococerebrosidase gene , a globin gene from the human β - globin gene cluster , a dna sequence encoding an rna or protein with anti - viral activity , the α1 - antitrypsin gene and the human multidrug resistance gene i . the invention provides for effective gene therapy with phsc of primates , in particular humans .

Detailed Description:
the present invention is based on the discovery that adeno - associated virus - derived vectors efficiently transduce primate pluripotent hemopoietic stem cells . adeno - associated virus has not been reported to transduce pluripotent hemopoietic stem cells of primates and aav - derived vectors have not been shown to transduce hemopoietic cells with in vivo repopulating ability , in addition , it is surprising that the vector integrates with high efficiency into p - phsc , even though most of the p - phsc are not actively dividing at the time of infection . this is surprising , since it has been established that raav integration in dividing cells occurs 200 times more efficiently in dividing , as opposed to nondividing cells [ 38 ]. also , it was reported that primary cells are much less efficiently transduced by raav than immortalized cell lines [ 47 ]. in addition , it was reported that orf 6 from adenovirus e4 - region stimulates transduction by recombinant aav [ 48 ]. in a gene therapy setting , it is undesirable to have functionally active adenovirus present due to toxicity problems caused by the virus directly or the immune system of the patient . at the keystone symposium on molecular and cellular siology , taos , n . mex . feb . 4 - 10 , 1996 , prof . a . nienhuis presented a paper stating that they transduced rhesus monkey cd34 + cells and , subsequently , autologously transplanted the infected cells [ 49 ]. analysis of the peripheral blood cells circulating in blood with a polymerase chain reaction specific for the raav revealed that cells carrying the raav were only detected up until 7 days post transplantation [ 49 ], i . e . p - phsc were not transduced by raav in their experiment . nonetheless , the present invention demonstrates that an adeno - associated virus - derived vector may be used to deliver exogenous dna efficiently to cells of the hemopoietic system with long term repopulating ability . the current perception of aav - integration into the cellular host chromosome is that the pre - integration complex is stable in cells . although integration occurs more efficiently in dividing cells , the pre - integration complex is stable in non - dividing cells and integrates when the cell is triggered to undergo cell cycling [ 38 , 60 ]. the primate - derived hemopoietic stem cells and committed progenitor cells upon autologous transplantation into an irradiated recipient are triggered into cycle to repopulate the destroyed hemopoietic system . for this reason , it is generally believed that the hemopoietic cells need not be triggered in vitro . for this reason , protocols to transduce hemopoietic progenitor cells with raav do not involve culturing the cells in the presence of hemopoietic growth factors . although this reasoning is very plausible with the current information , we devised experiments to investigate the effect of in vitro culture of hemopoietic stem cells and the in vitro stimulation with hemopoietic growth factors . as used herein , the term “ recombinant aav vector ” means a dna sequence flanked at each end by an aav - itr or functional equivalent or part thereof . the recombinant aav vector can be used directly or be packaged into a complex before use , as used herein , the term “ complex ” is defined as a combination of two or more components physically linked to each other through hydrophobic , hydrophilic or electrostatic interactions or covalent bonds , whereby one component of the complex at least is a recombinant aav molecule . other components of the complex can comprise , but are not limited to , one or a combination of liposomes , calcium phosphate precipitate , polylysine , adenovirus , adenovirus proteins , rep78 , rep68 , aav capsids or the aav capsid proteins vp1 , vp2 or vp3 . in a preferred embodiment the complex consists of the recombinant aav vector and the aav capsid proteins . this complex can be , but is not limited to , the form of an intact virion or particle where the recombinant aav vector is packaged inside an aav capsid or functional analogs thereof . as used herein , the term “ functional analogs ” refers to the same activity in kind , but not in amount or degree , i . e . not quantitatively . when the recombinant aav is packaged into aav particles , the size of the dna sequence will be limited by the size constraints for packaging into aav particles which , with the current state of the technology , is about 5 kb . the dna fragment preferably does not contain sequences functionally analogous to the terminal resolution site in the aav - itr as this might interfere with the stability of the recombinant vector . the dna sequence can be any sequence with therapeutic properties when introduced into hemopoietic stem cells , but the dna sequence preferably encodes one or more proteins or rna with therapeutic properties when expressed in hemopoietic cells . non - limiting examples of such sequences are the human β - globin gene operably linked to cis - acting sequences for erythroid specific physiological expression , the human lysosomal glucocerebrosidase gene ( e . c3 . 2 . 1 . 45 ), the α1 - antitrypsin gene , a dna sequence encoding an rna or protein with anti - viral activity or the multidrug resistance gene i ( mdri ). aav - itr sequences may be obtained from aav serotypes 1 , 2 , 3 , 4 or 5 . alternatively , mutant or recombinant itr sequences can be used , which retain the essential properties of the aav - itr prototype , examples of which are described in lefebvre et al , [ 50 ]. packaging of raav into aav - virions can be achieved using a variety of different methods . all methods are based on bringing the necessary proteins and raav - containing dna in an environment that supports the replication and packaging of raav , one method relies on the transfection of adenovirus 5 infected human cells with a plasmid carrying the raav - dna together with a plasmid containing expression cassettes for the aav - genes rep and cap . upon continued culture of the manipulated cells , raav is replicated and packaged . after three days , the cells are harvested and the accumulated recombinant virions are released from the cells [ 15 - 19 ]. a variation on the packaging system described above is the use of packaging cells that carry all or part of the relevant sequences stably integrated in their genome ( i . e . the recombinant aav vector , the rep - gene , the cap - gene , and the relevant protein coding domains of the helper virus ). when only partial packaging cells are used , the missing packaging functions have to be supplied externally via transtections of plasmids carrying the functions or virus infection . the helper virus functions are required for efficient packaging of recombinant aav . for most applications , the helper virus is inactivated or separated physically from the recombinant aav virions before using the recombinant aav virions for the transduction of cells [ 15 - 19 ]. recombinant aav vectors can be packaged by adding the recombinant aav - dna to protein extracts or mixtures of protein extracts of cells that expressed all or part of the relevant proteins for the replication and packaging of recombinant aav . when protein extracts are used from cells expressing only some of the relevant proteins for packaging of recombinant aav , the missing proteins can be supplied externally in purified form . the rep - gene can be derived from aav serotypes 1 - 5 or functional analogues thereof either obtained through non - essential mutations in the rep - genes or through the isolation of genes with similar capabilities such as the human herpesvirus 6 aav - 2 rep gene homologue [ 58 ]. the cap - gene can be derived from aav serotypes 1 - 5 or functional analogues thereof obtained through non - essential mutations in the cap - genes . alternatively , the cap - gene sequences can be altered through the replacement or addition of sequences rendering the produced virion new or altered target cell specificities . recombinant aav virions produced by the methods described above can be purified and concentrated using biological , physical or chemical separation techniques such as , but not limited to , antibody affinity purification , density gradient centrifugation or ion exchange chromatography . alternatively , the virions produced can be used in an unpurified form . as used herein , pluripotent hemopoietic stem cells from primates ( p - phsc ) are functionally defined as cells from primates with the capability to form and maintain an entire hemopoietic system , ranging from mature t - cells , b - cells , macrophages or erythrocytes to new p - phsc . p - phsc display this capability in unmanipulated primates or upon their autologous transplantation . sources of p - phsc are the bone marrow , the peripheral blood or cord blood . p - phsc can be collected from unmanipulated primates or from primates treated with compounds such as , but not limited to , cytostatic drugs or hemopoiatic growth factors to activate , recruit or otherwise potentiate the p - phsc . transduction of p - phsc is preferably performed ex vivo , following harvesting of the p - phsc from a suitable source , and after the transduction the transduced cells are autologously transplanted . in a preferred embodiment of the invention , the p - phsc are cultured during their ex vivo transduction , where it is most preferred that during this culture the p - phsc are stimulated with at least one hemopoietic growth factor , such as , e . g ., interleukin - 3 . alternatively , p - phsc transduction is performed in vivo when suitable methods have been developed to target the recombinant aav vector in vivo to p - phsc . table 1 key properties of adeno - associated virus vectors and amphotropic retrovirus vectors . table 2 characterization of recombinant aav preparations useful for the transduction of primate phsc . fig1 a recombinant aav - vectors useful for the transduction of primate phsc . lcr = core sequences from hypersensitive sites 4 , 3 and 2 from the β - globin locus control region . − 103 = human β - globin gene promoter fragment extending − 103 upstream of the transcription start site . − 265 = human β - globin gene promoter fragment extending − 265 upstream of the transcription start site . β - globin = human β - globin gene with modified intron 2 ( see text and 21 ). tkprom = herpes simplex virus thymidine kinase gene promoter ( approx . 500 bp nari - bgiii fragment ) pa = polyadenylation signal from herpes simplex virus thymidine kinase gene λapprox . 500 bp smai - nari fragment ). β *- globin = human β - globin gene with in the 5 ′ untranslated region three point mutations that generate two restriction enzyme sites ( see fig1 b ). δmo + pyf101 a moloney murine leukemia virus long terminal repeat fragment in which the moloney enhancer is replaced by an enhancer from a mutant polyoma virus that was selected to grow on embryonal carcinoma cells [ 2 , 51 , 52 , 53 ]. fig1 b nucleotide sequence of the 5 ′ untranslated region ( utr ) of the normal ( β ) and the marked ( β *) human β - globin gene . fig2 detection of recombinant aav in rhesus monkey peripheral blood cells . blood cells were collected as described in the text . peripheral blood mononuclear cells ( wbc ) were separated from the granulocytes ( gran ) and a neospecific nested pcr was performed on the dna of both cell types . dna from the nested pcr was analyzed on agarose gels and compared to positive and negative control samples . the sensitivity of the nested pcr was such that approximately one raav - vector could be detected in a background of 10 5 negative cells . (+) indicates the presence of a neo - specific band and (−) the absence of a neo - specific band in the agarose gel . fig3 a - 3b graphic representation of direct and nested neo - specitic pcr data from monkeys bb94 and a94 ( fig3 a ) and monkeys 9128 en 9170 ( fig3 b ). the data on the latter two monkeys shown in fig2 are included in fig3 as well . for clarity , negative pcr - results were not included in the graphs . closed circles ( pbmc ) and closed squares ( granulocytes ) indicate the time - points after transplantation at which the vector was detected . arrows in fig3 b indicate the time - points at which docetaxel ( taxotere ) was administered . fig4 detection of neo - specific sequences in hemopoietic cells from rh bb94 at 16 months post transplantation . bm ( bone marrow ) , pbmc ( peripheral blood mononuclear cells ), gran ( granulocytes ). fig5 detection of vector specific globin sequences in rhesus monkey peripheral blood cells ( samples from 2 months ( a94 ) and 6 months ( bb94 ) post - transplantation ) with this pcr , the two vectors ig - cft and ig - cft * are discriminated since the size of the ig - cft * fragment is approximately 150 pb . longer than the fragment specific for ig - cft . ligation of recombinant aav vectors containing the human β - globin gene and / or the neo r gene in order to determine whether recombinant aav could transduce p - phsc , it was necessary to generate appropriate vectors . we generated three different recombinant aav - vectors , which are schematically represented in fig1 a . the ligation of the vector ig - cft containing a human β - globin gene together with sequences from the β - globin locus control region and the neo r - gene is described in [ 21 ], ig - cft * differs from ig - cft in the size of the human β - globin promoter and in the presence of three point mutations in the 5 ′ untranslated region ( utr ) of the human β - globin gene , in ig - cft *, the promoter driving β - globin expression extends 265 bp upstream of the transcription start site instead of the 103 bp in ig - cft . in ig - cft *, three point mutations in the 5 ′ utr of the human β - globin gene created two new restriction sites , one for xbai and one for hindiii , see also fig1 b . ig - δmoneo ( depicted in fig1 a ) contains the raav - backbone ( xbai - fragment ) from psub201 [ 51 ], the nhei - smai promoter - fragment from the δmo + pyf101 ltr [ 53 ], the bglii - smai fragment from the tn5 - derived neo r - gene followed by the smai - nari poly - adenylation signal from herpes simplex virus ( hsv ) thymidine kinase ( tk ) gene [ 54 ]. the elements were linked together using the polylinker from pbluescript sk + ( stratagene ). the 293 cell line [ 55 ], which is a human embryonic kidney cell line transformed with ads dna , the a549 cell line , which is a human bronchial carcinoma cell line , and the c88 cell line [ 56 ], which is a murine erythroleukemia ( mel ) cell line , were maintained in dmem ( gibco - brl ) containing 10 % fetal calf serum ( fcs ), 100 μg / ml streptomycin and 100 u / ml penicillin . recombinant aav was produced by transfecting a raav packaging plasmid and a vector plasmid into approx . 90 % confluent permissive 293 cells . the cells were made permissive for aav - replication by transfecting them with a plasmid capable of expressing all the relevant early genes from adenovirus but not the late genes or by infecting them with adenovirus ts149 with a multiplicity of infection of 20 . the packaging plasmid was either paav / ad [ 15 ] or pim45 , which contains sequences 146 to 4493 from wtaav2 in the polylinker of pbluescript , the ratio of vector plasmid dna to packaging plasmid dna was 1 : 10 to accommodate the fact that the recombinant aav vector upon expression from the packaging plasmid replicates , whereas the packaging plasmid does not replicate . for crude virus stocks , the cells were harvested in their own culture medium after two to three days and subjected to three freeze / thaw cycles . the latter was performed by intermittent freezing and thawing in liquid nitrogen and a 37 ° c . water bath . cell debri was subsequently pelleted . ( 10 min , 200 g ) and the supernatant was incubated at 56 ° c . for 1 hour to inactivate residual adenovirus . concentrated high titer recombinant aav stocks were prepared by harvesting the cells in there own culture medium , and washing in pbs ( max . 10 7 cells / ml ). the virus was released from the cells by 3 freeze / thaw cycles and / or 30 sonication pulses of 1 second on ice to prevent warming . cell debri was spun down and the supernatant was made a density of 1 . 4 by adding solid cscl . after o / n centrifugation ( 50 . 000 r . p . m ., 20 ° c ., using a vti ti65 . 1 rotor in a beckman ultracentrifuge ), fractions were collected and raav was determined . fractions containing raav were pooled and further concentrated in a centricon concentrator ( amicon ) according to manufacturer &# 39 ; s specifications . after concentration , the medium containing the virus was changed by two successive washes in the centricon concentrator , using optimem culture medium ( gibco - brl ). to determine the effect of the different methods of virus preparation and the different processing steps on the quality of the various raav - batches , we characterized them for 5 parameters : 1 ) the capacity to deliver the desired dna to the nucleus of the target cell by means of a replication center assay ( rca ) described below , 2 ) the capacity to stably transduce cells and express the neo r - gene by means of a limiting dilution on mel cells followed by g418 selection , 3 ) the wild - type aav titer in the batches by a rca without added wtaav , 4 ) the amount of replication proficient adenovirus in each preparation , and 5 ) the concentration of cscl in the raav preparations that were purified using cscl radients ( see table 2 ). the replication center assay ( rca ) takes advantage of the fact that in a lytic infection of aav up to 10 6 aav , genomes are produced inside a cell . this amount of dna is sufficient for the radioactive detection of infected cells . to accomplish this , 293 cells were seeded in a flat bottom 96 wells plate such that they reached near confluence the following day . for a titration of recombinant aav , the cells were infected with dilutions of recombinant virus stock , adenovirus ts149 ( m . o . i . 20 ) and wtaav - 2 ( m . o . i . 2 ). for a titration of the wild type aav , the cells were infected with dilutions of recombinant virus stock and adenovirus ts149 ( m . o . i . 20 ). the cells were subsequently incubated at 39 ° c . the next day , after 24 hours , the medium was replaced by ice - cold pbs containing 5 mm edta . after 5 to 20 min . on ice , a single cell suspension was made by rigorous pipetting . the cells were diluted in 5 ml pbs and sucked onto hybond n + filter circles ( pore size 0 . 22 μm ) of 3 . 6 cm diameter . filters were incubated for 5 min in denaturation solution ( 0 . 4 m naoh ; 0 . 6 m nacl ) and 5 min in renaturation buffer ( 1 , 5 m nacl ; 1 m tris - hcl , ph 7 ). filters were washed and stored in 5xsspe until hybridization . filters were hybridized with a recombinant aav specific probe for the determination of the recombinant aav titer and hybridized with a wild type aav specific probe for the determination of the wild - type aav titer . 1 . 5 × 10 5 mel cells were seeded in 2 ml culture medium per well ( 24 wells plate , falcon ) and the appropriate dilution of raav virus was added . the cells were collected the next day and reseeded in 30 ml culture medium in a 75 cm 2 flask ( falcon ) . after three days , the medium was replaced by selection medium by spinning down the cells ( 200 g , rt ) and resuspending the cells in fresh medium containing 1 mg / ml ( dry weight ) g418 ( gibco ). medium was replaced every three to four days . after fourteen days , the cultures were scored . when the cells had grown to confluency , the cultures were scored positive since the specific virus dilution contained raav capable of stably transducing mel cells . specific virus dilutions were scored negative when , after fourteen days , confluency had not been reached . adenovirus was determined by serial dilutions of the aav virus stock on a549 ( human bronchial carcinoma ) cells . dilutions were scored positive when cytopathic effect was visible after 6 days . wild - type adenovirus 5 stocks with a known titer were used as positive controls . cscl concentrations in the aav preparations were determined by flame photometry . a summary of the characterization is given in table 2 . the infectious particle ( ip ) concentration , i , e . the capacity - to deliver raav - dna to the nucleus of target cells determined in the pca varied considerably among the different batches . also the transducing particle ( tp ) concentration and the amount of wild - type aav contamination varied considerably . three batches had a ip to tp ratio of 10 4 , the 248 crude batch had a much lower ratio of 200 . the animals used for transplantation were 3 - 5 kg rhesus monkeys ( macaca mulatta ), bred at the biomedical primate research centre ( bprc ), rijswijk , the netherlands . three weeks before transplantation , the animals were transferred to a laminar flow unit and selectively decontaminated in the digestive tract by treatment with metronidazole ( 40 mg / kg / day ), during 5 days , followed by daily oral administration of ciprofloxacin ( 6 . 5 mg / kg / day ), polymixin b ( 10 mg / kg / day ) and nystatin ( 40 ku / monkey / day ). a94 and bb94 received one administration of ivermectine 200 μg / kg anti - worm treatment approximately two weeks prior to transplantation . the monkeys were kept under barrier nursing and antimicrobial treatment until leukocyte counts exceeded a value of 1 × 10 9 / liter . the day before transplantation , the monkeys received 5 gy total - body x - ray irradiation . for this purpose , the animals were placed in a cylindrical polycarbonate cage which rotated 6 rpm around its vertical axis during irradiation from two opposing beams ( physical parameters : 300 kv , 7 ma , 0 . 26 gy / min dose rate , 0 . 80 m average focus - to - skin distance ). bone - marrow grafts were infused into a peripheral vein in a volume of 7 . 5 ml 0 . 9 % nacl . supportive care after transplantation included blood transfusions of 15 gray - irradiated thrombocytes when thrombocyte counts were below 40 × 10 9 / liter , subcutaneous fluid upon indicationl , and red blood cell transfusions when hematocrit levels dropped below 0 . 2 l / l . monkey 9128 was administered daily baytrill s . c . for 2 weeks , 9 months after transplantation , as treatment of a salmionella infection . monkeys bb94 and a94 were treated for streptococci septis and received cefamandolnafaat 50 mg / kg / day and tobramycine 3 mg / kg / day . a94 was additionally treated for streptococci sepsis with amoxiline 9 mg / kg / day , clavulanic acid 2 . 5 mg / kg / day and ceftriaxone 50 mg / kg / day and with amphotericin b 8 mg / kg / day for a yeast infection . selective decontamination was stopped a few days after hemopoietic repopulation of the monkeys . sepsis treatment was stopped 4 days after the body temperature had returned to normal and serum cultures were found to be sterile . docetaxel ( taxotere ®) treatment was given to monkeys rh9128 and rh9170 at indicated times ( fig3 ) at a dose of 50 mg / m 2 . in monkey rh9128 , around 14 months post transplantation 4 docetaxel doses were given of 10 mg / m 2 . the appropriate amount of docetaxel was diluted in 50 ml pbs - glucose ( nppi , the netherlands ) and was administered by iv injection at a rate of 1 ml / min . 40 ml of bone marrow aspirate was obtained by puncturing both femoral shafts under total anesthesia . bone marrow cells were collected in hanks &# 39 ; basic salt solution containing heparin at 100 units per ml and deoxyribonuclease - i and subjected to ficoll - hypaque ( sigma ) cenitrifugation . cd34 + selection was performed using a smallscale ceprate lc column ( cellpro , bothell , wash .). from 5 × 10 4 to 50 × 10 4 cells were incubated at 4 ° c . for 30 min in 0 . 1 ml pbs and 1 % bovine serum albumin ( bsa ) with 5 ml of a phycoerythrin - conjugated anti - cd34 antibody ( 563 . f ) or unconjugated anti - cd34 antibody ( 566 ). cells incubated with the antibody 566 were washed ( pbs , 0 . 1 * bsa ) and further incubated with percp conjugated rabbit anti - mouse igg1 ( becton - dickinson , cat no . 340272 ). after washing , cells were acquired on a facsort ( becton - dickinson ) flow cytometer . cells were analyzed with the lysis ii software program . the percentage of cd34 + cells was calculated as the ratio of cd34 + cells to total number of cells and multiplied by 100 . for rhesus monkeys 9128 and 9170 , the enriched cd34 + cells were immediately processed for transduction . for rhesus monkeys a94 and bb94 the enriched cd34 + cells were split into two equal fractions and stored in liquid nitrogen . transduction of cd34 + cells was done as described below . a summary of the experimental conditions is given in table 3 . rhesus monkey 9129 and 9170 : four days prior to transplantation the cds34 + enriched cells were split in two equal fractions and cultured at a density of 10 6 cells per ml in low density bmc culture medium supplemented with recombinant rhesus monkey interleukin - 3 ( rhil - 3 ; burger et al ., 1990 ) as described in [ 57 ], on day 2 and day 3 , one fraction of cultured cd34 + cells was exposed to the crude raav preparation of ig - cft and the other fraction was exposed to a crude raav - preparation of ig - δmoneo by adding an equal volume of virus preparation to the medium of the cultured cd34 + cells . after three to five hours , the cells were collected by centrifugation ( 7 min , 200 g ) and resuspended into fresh rhil - 3 supplemented low density bmc culture medium in the same volume as the culture was started in . on day four , the cells were collected by centrifugation ( 7 min , 200 g ) and resuspended in an equal volume of 0 . 9 % nacl and separately transplanted into autologous rhesus monkeys by iv injection . rhesus monkey a94 and bb94 : four days prior to transplantation , one fraction of the frozen cd34 + enriched cells was thawed and subsequently washed with hanks balanced salt solution . live cells were collected by ficoll - hypaque ( sigma ) centrifugation and cultured at a density of 10 6 cells per ml in iscove &# 39 ; s modified eagles medium ( imdm , gibco - brl ) supplemented with fetal calf &# 39 ; s serum ( fcs , 10 %) and recombinant rhesus monkey interleukin - 3 ( rhil - 3 ; burger et al ., 1990 ). on day 2 and day 3 , cells were collected by centrifugation ( 7 min , 200 g ) and resuspended in 10 to 200 μl of imdm + 10 % fcs and rhil - 3 and subsequently exposed to a purified raav preparation of ig - cft ( monkey a94 ) or ig - cft * ( monkey b994 ). after two hours , the cells were washed with imdm + 10 % fcs and reseeded in imdm + 10 % fcs and rh - il - 3 . at day four , the cells were collected by centrifugation and suspended in 0 . 9 % nacl . also , on day four , the other fraction of cd34 + cells was thawed and washed with hanks balanced salt solution . live cells were collected by ficoll - hypaque ( sigma ) centrifugation , resuspended in 10 to 200 μl of imdm + 10 % fcs and rhil - 3 and subsequently exposed to a purified raav - preparation of ig - cft ( monkey bb94 ) or ig - cft * ( monkey a94 ). after two hours , the cells were collected by centrifugation and suspended in 0 . 9 % nacl . after collection in nacl ( 0 . 9 %), the cells were separately transplanted into autologous irradiated rhesus monkeys by iv injection . daily observation of clinical signs . weekly complete physical examination and determination of body weight . blood chemistry analysis was performed before and after x - ray irradiation . hematology was performed weekly . bone marrow samples were punctured from the femoral shafts under total anesthesia . heparine blood samples were taken weekly for pcr analysis . pbmc and granulocytes were isolated from peripheral blood samples , as described previously by ficoll hypaque centrifugation ( van seusechem et al ., 1992 ). circulating t - and b - cells were purified from pbmc by sorting cd2 and cd20 positive cells , respectively . fitc labeled cd2 ( clone s 5 . 2 ; becton - dickinson , california ) or cd20 ( clone l27 ; becton - dickinson , california ) antibodies were incubated with pbmc according to the manufacturers protocols . labeled cells were separated using the macs ® column and anti - fitc beads ( miltenyi , germany ) according to the manufacturers protocol . re - analyses of the sorted cells on facs ® ( becton - dickinson , usa ) showed that the sorted cells were more then 95 % pure populations . rh912b and rh9170 hemopoietic cells were plated in duplicate at 5 × 10 3 / ml ( cd34 + selected ) or 1 × 10 5 / ml ( post - ficoll ) in 1 ml methylcellulose medium , as described in [ 57 ], supplemented with 30 ng / ml rhil - 3 and 25 ng / ml gm - csf . rh a94 and bb94 hemopoietic cells were seeded for colony formation in methylcellulose medium containing 50 ng / ml scf , 10 ng / ml gm - csf , 10 ng / ml il - 3 and 3 u / ml epo ( methocult gf h4434 , stemcell technologies inc , vancouver , canada ). for cell lysis , pellets were incubated ( 10 7 cells / ml ) in nonionic detergent lysis buffer ( 0 . 5 % np40 , 0 . 5 % tween 20 , 10 mm tris ph 8 . 3 , 50 mm kcl , 0 . 01 % gelatin , 2 . 5 mm mgcl 2 ) containing proteinase k ( 60 mg / ml ) at 56 ° c . for 1 hour , lysates were then heated at 95 ° c . for 10 min to inactivate the proteinase k . two different pcr detections were performed . one was a nested neo r - specific pcr and one was a β - globin specific pcr . the protocol for the neo r - specific pcr will be described first . the first amplification was performed on 10 μl lysates in a total volume of 50 μl with 2 u of supertaq polymerase ( ht biotechnology , cambridge , england ) in a reaction mix ( final concentration : 200 mm each of 2 ′- deoxyadenosine - 5 ′- triphosphate , 2 ′- deoxycytidine - 5 ′- triphosphate , 2 ′- deoxyguanosine - 5 ′- triphosphate , 2 ′- deoxythymidine - 5 ′- triphosphate ( pharmacia , roosendaal , the netherlands ), 0 . 2 μm each of 5 ′ neo - 1 and the antisense primer 3 ′ neo - 2 and the reaction buffer supplied by the manufacturer ( ht biotechnology , cambridge , england ). the nested amplification was performed on 5 μl of the first reaction in a total volume of 50 μl with 2 u of supertaq polymerase ( ht biotechnology , cambridge , england ) in a reaction mix ( final concentration ; 200 mm each of 2 ′- deoxyadenosine - 5 ′- triphosphate , 2 ′- deoxycytidine - 5 ′- triphosphate , 2 ′- deoxyguanosine - 5 ′- triphosphate , 2 ′- deoxythymidine - 5 ′- triphosphate ( pharmacia , roosendaal , the netherlands ), 0 . 2 μm each of 5 ′ neo - 2 and the antisense primer 3 ′ neo - 1 and the reaction buffer supplied by the manufacturer ( ht biotechnology , cambridge , england ). primers were chosen to selectively amplify the neo r gene . amplification conditions were the same for the first and the nested amplification and were performed in a trio thermocycler ( biometra , göttingen , germany ) temperature cycling apparatus , the conditions chosen were : 95 ° c . for 5 minutes , then 30 cycles of 94 ° c . for 30 seconds , 55 ° c . for 30 seconds , 72 ° c . for 1 minute , followed by extension at 72 ° c . for 10 minutes . five to ten microliters of the nested reaction were separated on 2 % agarose gel ( pronarose , hispanagar , burgos , spain ). each assay included titrations of a murine erythroid leukemia cell line c88 - c1 , containing a single provirus integration of ig - cft [ 21 ] and / or a titration of a pool of g418 selected mel cells infected with ig - cft *. for practical reasons , we developed an alternative pcr method to detect the neo - cassette in the raav - vectors ig - cft , ig - cft * and ig - δmo + neo . the sequences of the primers were as follows ; neo - 1s : 5 ′- tagcgttggctacccgtgat - 3 ′ ( seq id no5 ), and neo - 4as : 5 ′- tgccgtcatagcgcgggtt - 3 ′ ( seq id no . 6 ). reaction mixtures were prepared as described above and the reaction temperature was 95 ° c . for 3 minutes followed by 30 cycles of 95 ° c . for 30 seconds , 65 ° c . for 30 seconds and 72 ° c . for 1 minute . the completion of the 30 cycles was followed by an extension of 5 minutes at 72 ° c . five to ten microliter of the pcr - reaction was run on a 2 % agarose gel , blotted and hybridized to a 157 bp . specific probe isolated from a bstbi - smai digest of ig - cft . the β - globin specific pcr was carried out in essentially the same way as the first reaction of the neo r - specific pcr . but instead of the neo r - primers , the primers listed below , specific . for the 3 ′ part of the hs - 2 fragment and β - globin intron i , were added . the sequences of the primers are : the temperatures for the cycling were : 95 ° c . for 3 minutes and then 30 cycles of 95 ° c . for 30 seconds , 55 ° c . for 30 seconds , 72 ° c . for 30 seconds . following the 30 cycles , an extension at 72 ° c . for 5 minutes was performed . samples were run on 2 % agarose gels , which were blotted and hybridized to a ncoi - clai β - globin promoter specific probe using standard techniques . the survival and the selection of the purification and transduction procedure of cd34 + rhesus monkey bone marrow cells was controlled by determining the number of cfu - c present at different stages in the procedure . the cd34 selection for rh9128 and rh9170resulted in a 13 - 19 fold enrichment of cfu - c resp . for a94 and bb94 , the enrichment for cfu - c was 37 - 92 fold resp . ( table 4 ). the number of cfu - c did not vary by more then a factor of 2 during culture or upon transduction , with the exception of monkey bb94 where the decrease in the number of cfu - c was considerable upon culture and infection with ig - cft . this was due to a direct toxicity of the cscl purified ig - cft batch , as determined by a titration of the batch on human cord blood post ficoll bone marrow which resulted in a dilution factor dependent toxicity on cfu - c ( not shown ). since it is known that cscl is a very toxic substance , we determined the cscl concentration in the two cscl purified raav preparations . both contained considerable amounts of cscl , enough to account for the observed toxicity ( table 2 ). due to the observed toxicity on cfu - c in this experiment the two grafts that rh - bb94 received were very different in size . whereas the cultured graft wasostill considerable , the graft - size for the short transduction protocol was very small ( table 4 ). however , since stem cells are not measured in a cfu - c assay and are indeed more resistant to a large variety of drugs and agents , it is possible that many of them survived the high concentration of cscl . to determine whether the engrafted cells had been transduced by the recombinant aav vectors , approx . 3 ml of blood was collected each week from every monkey . granulocytes and mononuclear cells were purified , as described in ( 57 ), and the dna was released and analyzed by pcr for the presence of raav - sequences . two different pcr reactions - were performed . on the samples from all four monkeys , pcr reactions specific for the neo r - gene were performed . the neo r - gene is present in all the vectors , so this pcr detects all recombinant aav - genomes present in the cells . on the samples from monkeys rh - a94 and rh - bb94 , also a β - globin specific pcr was performed . this pcr utilizes the size difference in the β - globin promoter in vectors ig - cft and ig - cft *. these vectors were used to transduce the p - phsc via two different protocols , the effect of the two different protocols can thus be read out by the prevalence of one of the two vectors in the peripheral blood cells of the monkeys . the results of the neo - pcr are depicted in fig2 and 3 . all monkeys were negative for raav before transplantation and became positive for raav after transplantation . the presence of the vector varied from week to week . some samples were positive for the vector , others were negative , indicating that the frequency of transduced cells averaged around the detection limit of the pcr - reaction which was determined to be at 1 copy in 10 5 nucleated cells for the neo - specific pcr . monkey bb94 was positive in all samples immediately after transplantation and regeneration of the hemopoietic system , indicating a more efficient transduction of early progenitors during the ex vivo handling of the cells . in monkeys bb94 and 9128 , vector containing cells could be detected for at least more then one year after transplantation . bone marrow samples taken from these animals at 2 and 6 months ( 9128 ) or 14 months ( bb94 ) post transplantation also contained vector transduced cells . in bb94 , the vector was detected in pbmc , granulocytes , bone marrow and purified populations of b - and t - cells ( fig4 ). this result demonstrated the transduction of stem cells which had repopulated both the myeloid lineage ( granulocytes ) and the lymphoid lineage ( t - and b - cells ). the granulocytes , t cells , and b cells were still pcr positive more than 15 months post - transplantation , indicating the transduction of cells with extensive self - renewal capacity , the transduction of primate cells with ( 1 ) an extremely long - term in vivo stability after transplantation , and ( 2 ) the capability of multiple - lineage repopulation long after transplantation , provides strong evidence for transduction of p - phsc . rhesus monkey 9128 received treatments with taxotere , a cytostatic drug , to ablate the mature cells in the circulation , inducing periodic regrowth from immature hemopoietic cells residing in the bone marrow . recombinant aav transduced cells were detected in circulating cells fter a series of treatments with taxotere , over a period of 14 months post transplantation . the persistence of transduced cells in peripheral blood cells and the resistance to taxotere treatment provides convincing evidence of the transduction of p - phsc . the experiment with monkeys bb94 and a94 was designed to quantify the success of two different transduction protocols . for each monkey , the transplant was split in two equal fractions and each fraction was transduced in a different way . to be able to discriminate which protocol resulted in a better transduction , we used a different vector for each transduction . we compared the efficiency of transduction of cultured p - phsc versus that of non - cultured p - phsc . for the transduction of p - phsc from monkey bb94 , we used the purified virus ig - gft for the non - cultured p - phsc and the purified virus ig - cft * for the cultured p - phsc . to exclude a possible role of quality differences between the virus batches , we switched the two virus batches for the transduction protocols for monkey a94 ; we used ig - gft for its cultured p - phsc and ig - gft * for its non - cultured p - phsc . following transplantation and repopulation of the . hemopoietic system of the monkeys , we performed the β - globin specific pcr to determine which transduction procedure resulted in the highest frequency of gene modified circulating cells . for both monkeys , we were able to detect only the virus used to transduce the cultured p - phsc , i . e ., ig - gft * for monkey bb94 and ig - gft for monkey a94 ( fig5 ). thus , in vitro stimulation of p - phsc results in a more efficient transduction with recombinant aav vectors . this result was not expected . it is generally accepted that culture of p - phsc promotes progressive loss of the grafting potential of the p - phsc , presumably due to differentiation . hence , if both procedures resulted in similar p - phsc transduction efficiencies , we would expect the progeny of the non - cultured p - phsc co prevail among the circulating blood cells due to grafting advantages . since we observed the opposite , the stable transduction efficiency of the cultured p - phsc must be significantly higher than that of the noncultured p - phsc . it is known that aav - vectors integrate with higher efficiency in cycling cells then in non - cycling cells ( 38 ) however , in non - cycling cells the vector remains in the nucleus and retains its ability to integrate when the cell is triggered into cycle ( 60 ). once transplanted , the p - phsc start to divide and repopulate the ablated hemopoietic system . considering the enormous amount of cells that need to be produced in a short time , it is presumed that the p - phsc start to divide within a couple of days once inside the body . therefore , a difference in transducibility of cultured versus non - cultured cells is not expected when only replication of the target cells is the enhancing factor . we infer that culture and exposure to hemopoietic growth factors such as il - 3 could in other ways potentiate the transduction with recombinant aav . one possible explanation is the up - regulation or activation of receptors for the virus on the surface of the p - phsc . another is the induction of proteins inside the p - 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