lentiviral vectors are promising gene transfer agents for ex vivo correction of inherited or acquired genetic defects . hematopoietic disorders are well suited as targets for this approach , due to the relative ease with which these cells are harvested , transduced ex vivo with lentivirus , and reinfused into patients . however , even with robust gene transfer efficiencies , the modified cells and their therapeutic potential are diluted upon reinfusion into the large population of endogenous , unmodified cells . however , few inherited hematopoietic disorders , when genetically corrected , provide an inherent survival advantage at the hematopoietic stem cell level . to overcome this limitation , genes that permit selective expansion of transduced cells in vivo several drug resistance genes have been assessed for in vivo stem cell selection [ 14 ] . however , the most stringent in vivo stem cell selection results have been achieved with point mutants of o - methylguanine - dna methyltransferase ( mgmt ) [ 5 , 6 ] . agt repairs o - alkylguanine dna lesions induced by methylating and chloroethylating agents , such as , temozolomide and 1,3-bis(2-chloroethyl)-nitrosourea ( bcnu ) , respectively ( figure 1 ) . specific agt point mutations , including p140k and g156a , are resistant to the wild - type agt inhibitor , o - benzylguanine ( bg ) [ 710 ] . inactivation of wild - type agt with bg sensitizes untransduced bone marrow cells to o - alkylating agents , allowing the resistant mgmt mutant - transduced cells to expand and repopulate . sustained high - level expression of mgmt has been demonstrated with both single - gene retroviral and lentiviral vectors [ 5 , 6 , 12 , 13 ] . dual - gene vectors containing mutant mgmt have also been shown to efficiently select stem cells in vivo [ 1416 ] ; however , higher mois are often required to compensate for the reduced expression efficiency of one or both genes in dual - gene lentivectors . this strategy allows two genes to be rapidly evaluated without a need for dual gene vector construction . cotransduction with separate vectors has been used extensively for in vitro models [ 17 , 18 ] , or for derivation of induced pluripotent stem cells [ 19 , 20 ] . frimpong and spectro demonstrated that separate vsv - g - pseudotyped lentiviruses could be used to cotransduce cell lines or primary human neurons . they also demonstrated efficient cotransduction of cells with two bicistronic vectors , each with a unique drug resistance gene , for in vitro selection of only dual - positive cells . the efficiency of hematopoietic cell cotransduction , and whether the cotransduced cells can be enriched in vivo , has not been evaluated . therefore , we evaluated whether separate single - gene lentivectors , one expressing mgmt - p140k , and the other expressing gfp , could efficiently cotransduce hematopoietic cells at low mois to produce dual - positive populations that could be enriched with bg and bcnu selection . cotransduction efficiencies were first assessed in the human erythroleukemia k562 cell line , using various virus ratios , selection stringencies , and total mois . this information was then applied to murine bone marrow cell cotransductions to selectively amplify dual - positive cells in vivo . since insertional mutagenesis has been demonstrated as a potential risk associated with gene therapy , these studies involved significantly reduced mois compared to mois reported for in vivo selection with dual - gene lentiviral vectors [ 2224 ] . our data demonstrate that cotransduction , coupled with mgmt - mediated selection , allows enrichment of dual - positive cells in vivo . further , we show that mgmt - mediated hsc enrichment can be coupled to lineage - specific transgene expression from a separate cotransduced lentivector . selective in vivo expansion of hematopoietic cells cotransduced with two separate single - gene lentivectors , one of which provides drug resistance , has not been evaluated . as a first approach to evaluating the efficiency of this strategy , cotransduction and selection were carried out in vitro using the human k562 erythroleukemia cell line . a self - inactivating lentiviral vector containing the internal mnd promoter   was obtained from donald kohn ( ucla , santa monica , ca ) . the woodchuck hepatitis virus posttranscriptional regulatory element ( pre ) and a multiple cloning site were introduced for subsequent generation of vectors expressing agt - p140k or the green fluorescence protein ( gfp ) ; pmnd - mgmt and pmnd - gfp , respectively . both vectors express at high levels in k562 cells , and distinct agt , gfp , and dual - positive populations can be detected in cotransduced k562 cells . the cotransduction efficiency of k562 cells was first evaluated with the total moi held at 0.5 , varying the individual mgmt and gfp virus proportions ( figure 2(a ) ) . the degree of cotransduction was proportional to the total agt and total gfp expression percentages (  total  refers to both the single - positive and the dual - positive cells ) . k562 cells cotransduced with an equivalent mgmt and gfp moi mix ( 0.25  :  0.25 ) resulted in the highest agt and gfp total expression percentages . the degree of cotransduction was lowest ( 4% ) when an mgmt  :  gfp moi mix of 0.05  :  0.45 was used . however , cells transduced with this moi mixture had the highest proportion of dual - positive cells ( 49% ) after treatment with 10  m bg and 15  m bcnu ( figure 2(b ) ) . drug treatment enriched agt cells to 95% in each culture , with the level of enrichment inversely proportional to the moi of mgmt used . both the agt only and agt - gfp dual - positive populations were expanded to the same extent , indicating that although both vectors utilize the same promoter , the level of agt expressed in both populations was equally protective . the percentage of cells expressing gfp did not change after treatment , since the total proportion of gfp cells in the culture is equivalent to the proportion of gfp cells expressing agt . although gfp expression was maintained in the gfp - only - transduced cultures after drug treatment , survival was markedly reduced and limiting cell numbers were available for analysis . the initially high cotransduction rates achieved using an moi of 0.25 of each virus had comparatively lower percentages of dual - positive populations after selection ( 29% ) , due to the large proportion of mgmt singly transduced cells present in the culture prior to drug treatment . as expected in the setting of drug selection , the highest percentages of dual - positive cells were obtained with virus mixtures composed of low mgmt and high gfp moi proportions . slightly higher than expected dual - positive percentages were obtained in the k562 cells , compared to calculated values based on the total agt and gfp expression percentages . to evaluate whether this was the result of transcomplementation of virus proteins during transduction , or the inability to completely resolve the transduced populations , k562 cells were cotransduced with gfp virions at an moi of 0.25 and mgmt virus mois of 0 to 10 . the proportion of gfp cells was then evaluated by flow cytometry in the absence of agt staining . as shown in figure 2(c ) , the transduction efficiency of gfp is independent from the mgmt transduction levels ( determined separately by agt staining ) , indicating that the slightly elevated cotransduction levels are due to the inability to completely resolve the single- and dual - positive populations by flow cytometry . cotransduction of k562 cells with mois greater than 1 resulted in a high proportion of dual - positive cells in the absence of selection . however , since high mois are associated with an increased risk of insertional mutagenesis , we evaluated whether cotransduction with an optimal mgmt  :  gfp ( 0.05  :  0.6 ) moi ratio at a low total moi would result in higher percentages of dual - positive cells , compared to that obtained with a 1  :  1 mixture of the two viruses at a higher total moi ( = 1 ; 0.5 of each virus ) . before selection , the level of cotransduction obtained with the highest total moi was over 4 times that obtained with the 0.05 mgmt and 0.6 gfp virus moi mix , 21% versus 4.6% , respectively ( figure 3(a ) ) . however , after selection , cultures transduced with the 0.05 mgmt and 0.6 gfp virus moi mixture had the highest percentage of dual - positive cells ( 66% ) , due to the increased rate of dual - positive cell enrichment ( figure 3(b ) ) . these data indicate that selective expansion of cells cotransduced with optimal virus proportions can overcome the reduced transduction rates obtained with low mois .   drug selection of cells transduced with dual gene vectors can lead to preferential expression of only the drug resistance marker . to evaluate whether mgmt singly transduced cells out compete dual transduced populations under more stringent expansion conditions , the untreated cotransduction cultures from figure 3(a ) were diluted into a 50-fold excess of untransduced k562 cells and selected with two sequential rounds of 10  m bg and 15  m bcnu treatment . as shown in figure 3(c ) , the fraction of dual - positive cells in the total agt population is equivalent before and after two rounds of drug selection , demonstrating that preferential expansion of mgmt singly expressing cells did not occur . we next evaluated the cotransduction efficiency of murine bone marrow ( bm ) cells using the single - gene mnd - mgmt and mnd - gfp vectors . progenitor populations derived from 5-fluorouracil - treated donors , or enriched by sca / kit / lineage ( skl ) selection , were cotransduced with equal or low mgmt  :  gfp virus proportions at constant total mois . higher mois were used on primary cell cultures to overcome the reduced transduction efficiencies , compared to human cell lines , but well within the range of mois used in typical hsc transduction protocols [ 2224 ] . the transduced cells were drug treated and expanded in culture for 10 days to determine agt and gfp expression levels by flow cytometry ( figures 4(a ) and 4(b ) ) or plated in methylcellulose to calculate the survival percentages of hematopoietic progenitors ( figures 4(c ) and 4(d ) ) . although the mgmt mois used to transduce skl cells were 210-fold higher than those used for the bm cultures , the agt expression percentages obtained in the unselected skl populations were half of those observed in the bm cells . nevertheless , increased progenitor survival percentages were obtained with skl cells , compared to that of the bm cells , at higher bcnu doses ( figures 4(c ) and 4(d ) ) . the dual - positive percentages in both the skl and bm cultures were much higher than expected values , based on total agt and gfp cell percentages . in addition , the progenitor survival rates obtained with equivalent or staggered mgmt and gfp moi ratios were not dramatically different in either the skl or the bm cell assays . these data suggest that progenitor populations that are permissive to transduction at the time of virus addition are efficiently transduced with separate vectors at relatively low mois . insertion of two large genes into the same vector can reduce titers and leave little space for other beneficial sequences , such as , posttranscriptional regulatory elements , insulators , or matrix attachment regions . cotransduction may be particularly applicable to situations in which the transcription of two genes is targeted to different cell types . to evaluate whether codelivery of a ubiquitous mgmt expression vector and a lineage - restricted gfp virus would allow both stem cell selection and expression of gfp in the correct hematopoietic compartment , we used a modified lentiviral gfp reporter vector ( prrl - gata - gfp ) that restricts gfp expression to ter119 murine erythroblasts . our in vitro cotransduction results demonstrated that the reduced transduction efficiency achieved with lower mois of the mgmt virus can be circumvented by increased selection stringencies . thus , as a first approach , skl cells were cotransduced with an mnd - mgmt : gata - gfp moi mixture of 5  :  15 and transplanted into 6 lethally irradiated recipients .   at five weeks posttransplant agt pbmc percentages in the cohort of 6 untreated animals averaged 19% ( 651% ) , while gfp detection in ter119 erythroblasts averaged 18% ( 929% ) ( figure 5(a ) ) . agt pbmc averages increased to 41% ( 1465% ) in the 4 animals selected with three rounds of bg and bcnu treatment , compared to 27% in 2 untreated controls . detection of gfp cells in ter119 bm mononucleocytes averaged 57% ( 196% ) in the 4 drug selected animals , compared to 344% in the 2 untreated controls . as expected , gfp expression was restricted to ter119 erythroblasts ( figure 5(b ) ) , and the animals with the highest agt cell enrichment also had the highest percentage of gfp cells in the ter119 bm fraction ( figure 5(c ) ) . copy number analysis revealed an average of 14 copies of each vector per cell ( figure 5(d ) ) . cotransduction with total mois equivalent to that used for dual - gene vectors , results in approximately equivalent vector insertion numbers ( data not shown ) . these data indicate that cotransduction allows efficient lineage - specific gene expression to be coupled to progenitor selection in vivo . a second transplant experiment was carried out to determine whether this strategy is efficient with limiting mnd - mgmt and gata - gfp mois . whole bm harvested from 5-fu - treated donor mice was transduced with each virus at an moi of 3 . following a 12-hour transduction , 2 posttransplant , agt pbmc levels in the cohort of 6 untreated animals ranged from 627% , while little to no gfp expression was detected ( figure 6 ) . of the four animals selected with three rounds of 30  mg / kg bg and 10  mg / kg bcnu selection , only two showed evidence of transduced cell engraftment . the agt pbmc expression in these two animals in addition , the gfp percentage detected in ter119 pb erythroblasts was 2642% in the drug - treated animals , compared to 35% in the untreated animals . the two - drug - treated animals had high percentages of both agt mononuclear cells ( 69% and 55% ) and gfp erythroblasts ( 74% and 56% , resp . ) . the number of mgmt and gfp vector insertions in these two animals ranged from 13 and 14 copies per cell , respectively . these data demonstrate that mgmt - mediated selection permits expansion of cells cotransduced with limiting mois and suggests this strategy may serve as first approach to evaluating lineage - specific therapeutic vectors in vivo . we evaluated whether cotransduction of hematopoietic cells with two separate vectors , one of which allows selection , would provide an efficient alternative to dual - gene vectors . these studies involved significantly reduced mois than previously reported for in vivo selection with bicistronic lentiviral vectors . we found that 1 ) the level of cotransduction is proportional to the transduction efficiencies of each vector ; 2 ) at limiting total mois , low mgmt  :  gfp virus proportions resulted in the highest level of dual - positive cells after drug selection ; 3 ) cotransduction allows mgmt - mediated progenitor cell expansion to be coupled to lineage - specific transgene expression . the use of cotransduction for dual - gene delivery eases vector insertion size limits and cis - acting complications that limit the expression efficiency of one or both genes in dual - gene constructs . given that cotransduction involves two genes delivered with separate vectors , we evaluated in vitro the stoichiometry of the two viruses used , the total moi , and the effect of drug selection . when the total moi was low and held constant , the level of cotransduction in k562 cells was found to be proportional to the transduction efficiency of each vector . therefore , equivalent levels of each vector resulted in the highest percentage of dual - positive cells in the absence of selection . with drug treatment the proportion of gfp cells in the total population is equivalent to the proportion of gfp cells in the drug - resistant population . therefore , at a constant moi , low mgmt  :  gfp virus ratios resulted in the highest level of dual - positive cells after selection . as previously reported with other vectors , the level of cotransduction was slightly higher than the expected values based on the individual transduction efficiencies . however , in the absence of mgmt staining , the transduction efficiency of the gfp lentivirus was unaffected by increasing mois of the mgmt virus . thus the slightly higher dual - positive values seen when agt and gfp were measured simultaneously are likely due to the inability to completely resolve the single- and dual - expressing populations by flow cytometry . the selection stringency must be taken into account when enriching cells that are cotransduced at low mois . the degree of expansion obtained with stringent selection conditions can overcome reduced cotransduction rates obtained with limiting mois . low mgmt to gfp virus ratios at a low total moi resulted in higher levels of dual - positive cells after selection than that obtained with equal mixtures of the two viruses at a higher total moi . for dual - gene vectors , highly stringent drug treatments have been shown to preferentially select for drug resistance gene expression at the expense of the other gene [ 26 , 29 ] . however , using a two vector system , mgmt - gfp - transduced cells were enriched to the same extent as mgmt singly transduced cells , even after stringent selection conditions . the cotransduction efficiency of primary bm cells is more difficult to predict since it consists of a mixed population of cell types that also differ in their cycling status . higher mois were used in these studies than with human cell lines to overcome the reduced transduction rates obtained with murine bm cells . although the initial expression percentages were low , the percentage of dual - positive cells obtained after drug treatment was over two times higher than the expected levels calculated from the individual expression percentages . these data indicate that specific cells may be more or less susceptible to transduction and thus , the high mois used to increase transduction efficiency of primary cells will likely result in higher integration rates in the susceptible cell populations . cotransduction may have the most potential for situations in which the two genes are transcriptionally targeted to different cell types . this is especially true for severe hemoglobinopathies , including beta - thalassemia major and sickle cell anemia , in which bulky regulatory elements are required for lineage - restricted therapeutic gene expression [ 30 , 31 ] . we report here the ability to couple mgmt - mediated selection to erythroblast - specific gfp expression using separate single - gene vectors . selection for engrafted mgmt- and gata - gfp - positive cells was efficient in both skl and whole bm transplant recipients . this strategy should be useful for evaluating the therapeutic potential of low - copy gene replacement used to correct hematopoietic disease models . since the gene expression levels are independent with this strategy , a wide range of therapeutic vectors could be rapidly assessed by cotransduction with mgmt . although cotransduction requires at least two insertions for dual - gene expression , the enhanced expression efficiency achieved with single - gene vectors suggests that fewer total copies may be required to achieve expression levels equivalent to that of dual - gene vectors . the self - inactivating lentiviral vector ( pcso - rre - cppt - mcu3-luc ) containing an internal mnd promoter , and the central polypurine tract / central termination sequence ( cppt / cts ) was kindly provided by d. kohn ( ucla , santa monica , ca).the luciferase gene was removed from this vector by ncoi / ecori digestion and replaced with a multiple cloning site cassette . the woodchuck hepatitis virus posttranscriptional regulatory element ( obtained from t. hope ) was inserted into the unique ecori site . the resulting vector was restricted with ncoi / bamhi for insertion of mgmt - p140k or gfp . briefly , 293 t cells were triple transfected with the packaging vector ( pcmvdeltar8.91 ) , the vsv - g pseudotyping vector ( pmd.g ) , and either the pmnd - mgmt - p140kpre , pmnd - gfppre , or prrl - gata - gfp transfer vectors at a mass ratio of 3  :  1  :  3 , using lipofectamine 2000 ( invitrogen ) according to the manufacturer 's instructions . virus produced 2448 hours after transfection was harvested in dulbecco 's modified eagle medium ( dmem ; cellgro ) containing 10% heat - inactivated fbs ( cellgro ) and 2  mm glutamax ( invitrogen ) . virus - enriched media was filtered through 0.45  m syringe filter units ( millipore ) and stored at 80c . expression titers were determined on k562 cells using virus dilutions that resulted in less than 10% transduction . titers ranged from 6  10 to 3  10 expression units / ml . virus preps were premixed at the specified moi ratios prior to the addition of cells . k562 cells were cotransduced in iscove 's medium ( cellgro ) containing 10% heat - inactivated fbs , 2  mm glutamax , and 8  g / ml polybrene ( sigma , st . whole bm and sorted stem cell populations were transduced for 12  hrs in alpha - mem containing 20% heat - inactivated fbs , 2  mm glutamax , 6  g / ml polybrene , in the presence of 20  ng / ml murine il-3 and 50  ng / ml of murine il-6 , and scf ( r&d systems inc . , minneapolis , mn , usa ) . the apparent mois used for mouse cell transductions were based on expression titers established in k562 cells . the transduced cells were transplanted immediately after transduction . for whole bm transplants , donor marrow , was obtained from 6- to 8-week - old c57bl/6j mice ( jackson laboratories , bar harbor , me , usa ) 2 days after treatment with 150  mg / kg 5-fu . six - week - old recipient mice were lethally irradiated with 850  cgy using a cs source and transplanted with 2  10 5-fu enriched cells per mouse or 1,000 skl cells supported with 2  10 lineage cells per mouse . bg was synthesized by dr robert moschel at the frederick cancer research institute ( frederick , md , usa ) , and bcnu was obtained from the drug synthesis and chemistry branch of the national cancer institute ( nci ; bethesda , md ) . all in vitro drug treatment incubations were carried out in serum - free media at 37c . cells were pretreated with bg for 1 hour , followed by a 2-hour treatment in bcnu . drug treatments for in vitro murine bm selections were carried out with 25  m bg and 0 , 5 , 15 , or 30  m bcnu in alpha - mem containing 1.2% spleen - cell - conditioned media ( stem cell technologies ) . drug - treated murine bm samples were expanded 10 days in liquid culture using the same media formulation as described for transduction , without polybrene . progenitor survival assays were performed as previously described . for in vivo selection , bg was dissolved to 3  mg / ml in 40% polyethylene glycol ( union carbide corp . , danbury , ct , usa ) and 60% pbs ( ph 8.0 ) , and bcnu was dissolved in ethanol and diluted to 1  mg / ml in pbs . mice were injected intraperitoneally with 30  mg / kg bg , followed by 10  mg / kg bcnu an hour later . three rounds of selection were carried out at 3-week intervals , beginning at 3 weeks for animals transplanted with whole bm and 5 weeks for animals transplanted with skl cells . all flow cytometry results were obtained with an lsr flow cytometer ( becton dickinson ) . gfp expression was measured in k562 cells after a single wash in pbs . for agt detection , k562 cells were fixed in 2% paraformaldehyde for 30 minutes at 4c , permeabilized in 1% tween-20 for 30 minutes at 37c , and blocked in 10% normal goat serum for 15 minutes at room temperature . the cells were then immunolabeled with the mouse anti - human mgmt monoclonal antibody mt3.1 ( kamiya biomedical , seattle , wa , usa ) and an ( apc- ) conjugated goat anti - mouse secondary antibody ( caltag laboratories , burlingame , ca , usa ) . dual detection of agt and gfp expression was carried out with the same staining protocol as that used for agt alone . red blood cells were lysed in murine pb and bm samples prior to preparation for flow cytometry . vector insertions per cell were evaluated using a lightcycler instrument and the lightcycler faststart dna master sybr green i kit from roche diagnostics ( basel , switzerland ) . vector copy numbers were determined using proviral - specific mgmt primers ; sense 5-acgtctatatcatggccg-3 , antisense 5-tgaggatcttgacaggatt-3 and gfp primers ; sense 5-acgtctatatcatggccg-3 and antisense 5-tgtgatcgcgcttctc-3. genomic copy numbers were determined using gapdl4-specific primers , as previously described . mgmt , gfp , and gapdl4 copy numbers were assessed using a standard curve consisting of murine genomic pbmnc dna spiked with known vector copy numbers , and verified using k562 cell clones containing a single mgmt - gfp vector insertion . one diploid copy of the mouse genome was assumed to contain two copies of gapdl4 and 5.9  pg of dna . ten cfu were analyzed per animal and the average copy number per cfu was compared to that of total bm from each animal .