sickle cell disease ( scd ) is caused by an at mutation that changes the negatively charged glutamic acid to hydrophobic valine in position 6 ( e6v ) of the -globin subunit of adult hemoglobin ( hba ) . when deoxygenated , hydrophobic patches of sickle hemoglobin ( hbs ) molecules interact with each other , forming tubular hbs fibers that increase the rigidity of the red blood cells ( rbcs ) . sickle rbcs ( ss - rbcs ) have a shortened lifespan in the circulation ; they adhere to and activate endothelium , promote inflammation , and interact with leukocytes , leading to episodes of painful sickle cell crisis and progressive organ damage.1 , 2 scd treatment is mainly supportive and focuses on alleviating disease complications . hydroxyurea , which induces fetal -globin and inhibits hbs polymerization,4 , 5 , 6 is the only approved drug for managing scd with demonstrated reductions in morbidity and mortality.7 , 8 however , concerns about its potential carcinogenic properties have contributed to its underuse . current life expectancy of patients with sickle cell anemia in the united states is roughly three decades shorter than that of the general population.10 , 11 allogeneic hematopoietic stem cell ( hsc ) transplant ( hsct ) can be curative for scd , but only 18% of scd patients have matched sibling donors . receiving a graft from an unmatched donor is associated with a higher risk for mortality and morbidity . clinical trials utilizing autologous bone marrow ( bm ) cd34 cells transduced with lentiviral vectors carrying anti - sickling globin genes are currently under way , but so far have been met with mixed outcomes.14 , 15 , 16 another lenti - based short hairpin rna ( shrna ) gene therapy approach that selectively knock downs b cell lymphoma / leukemia 11a ( bcl11a ) , one of the key suppressors of anti - sickling fetal -globin,17 , 18 , 19 , 20 in erythroid cells is actively under investigation.21 , 22 other novel gene therapy strategies such as correcting the sickle mutation in induced pluripotent stem cells ( ipscs)23 , 24 or in primary human cd34 hematopoietic stem and progenitor cells ( hspcs)25 , 26 have been explored pre - clinically . however , fully functional hscs have yet to be derived from ipscs , and achieving efficient levels of gene correction in long - term repopulating hscs via homology - directed repair ( hdr ) pathways without utilizing a selectable marker has remained challenging , limiting the clinical relevance of such approaches . in contrast to hdr , gene disruption via non - homologous end joining ( nhej ) repair pathways occurs at a high frequency in hscs . therefore , reactivating fetal -globin while reducing sickle adult -globin expression via the disruption of bcl11a , whose only known function in the erythroid lineage appears to be mediating fetal to adult globin switching,17 , 18 , 29 , 30 offers a potential strategy for developing an effective treatment for scd . here , we employed zinc finger nucleases ( zfns ) to disrupt expression of the bcl11a gene in bm - derived cd34 cells from healthy volunteers . through a combination of in  vitro and in  vivo studies , we show that targeted disruption of the gataa motif in the erythroid - specific enhancer of bcl11a can both reactivate fetal -globin to levels expected to prevent hbs polymerization and produce edited hspcs capable of long - term multilineage engraftment in immunodeficient mice . together , these data provide a compelling rationale to pursue genome editing of bcl11a erythroid - specific enhancer for autologous cell therapy for scd patients . zfns targeting exon 2 ( coding zfns ) or the gataa motif31 , 32 within an intronic erythroid - specific enhancer ( enhancer zfns ) of the bcl11a gene were engineered ( figure  1a ) . introduction of zfn mrna via electroporation into bm - cd34 cells induced double - stranded dna breaks that were repaired by the nhej dna repair pathway . this produced a spectrum of small insertions or deletions ( indels ) centered at the targeted cleavage site , which was quantitated by targeted amplicon sequencing ( figures 1b and 1c ) . when primary bm - cd34 cells were transfected with escalating amounts of mrnas encoding the zfns , increased levels of indels were detected until a plateau ( 60% of total alleles ) was reached ( figure  2a , left panel ) . when these transfected cd34 cells were further cultured under erythroid conditions , they gave rise to erythroid cells with corresponding increases in indels ( figure  2a , middle panel ) and in their fetal globin expression , which reached as high as 35% of total -like globin chains ( g  + a  +   +  ) in both groups , as gauged by reverse phase high - performance liquid chromatography ( hplc ) ( figure  2a , right panel ) . the bcl11a knockout ( ko ) alleles , defined as having indels that caused frameshift mutations in exon 2 ( figure  1b ) or disruption of the gataa motif in the erythroid - specific enhancer ( figure  1c ) , were quantitated . ko alleles were found to be less frequent at the population level after erythroid differentiation than in the starting pool of the treated bm - cd34 cells . this difference was more pronounced in samples treated with the coding zfns than those treated with the enhancer zfns ( figure  2b ; figure  s1 ) . these results suggest that frameshift mutations in the coding sequence of bcl11a may be counter - selected during erythroid differentiation . because erythroid cells derived from bulk cd34 cell cultures originated from a pool of heterogeneous edited cells ( figure  1 ) , they did not provide sufficient resolution to assess whether erythroid differentiation was affected by the editing and whether fetal globin was upregulated to levels predicted to inhibit hbs polymerization . to address this issue , we sorted zfn mrna - treated bm - cd34 cells onto 96-well plates to allow for clonal derivation of mature erythroid cells from individual burst - forming unit - erythroid ( bfu - e ) progenitors . when binned based on the genotype of each erythroid colony , it was revealed that by 1718  days post erythroid culture , erythroid colonies with bi - allelic knockout ( ko / ko ) of exon 2 expressed predominantly fetal globin ( g  + a ) , accounting for 80% of total -like globin chains as measured by ultra - performance liquid chromatography ( uplc ) , with reciprocal reduction in adult -globin ( figure  3a , left panel ; figures s2 and s3 ) . the fetal globin levels expressed in ko / ko colonies were significantly higher than that in either wt / ko or wt / wt colonies ( p  < 0.0001 ) . however , the coding ko / ko erythroid progenitors gave rise to fewer erythroid cells than the wt / wt or wt / ko erythroid progenitors ( two out of three donors ) ( figure  3a , middle panel ; figure  s2b , middle panel ) , which may have partially contributed to the aforementioned decrease in ko indel frequency after erythroid differentiation ( figure  2b ; figure  s1 ) . in addition , coding ko / ko colonies had significantly lower mean enucleation rates ( three out of three donors ) than colonies identified as wt / ko or wt / wt ( p  < 0.0001 ) ( figure  3a , right panel ; figure  s2b , right panel ) . this reduced enucleation efficiency was specific to coding ko / ko genotype , because cells with bi - allelic in - frame mutations enucleated as efficiently as unedited colonies ( figure  s2c ) . consistent with the flow - based enucleation assessment , cytospins of day-18 erythroid cells showed mostly enucleated rbcs in coding wt / wt and wt / ko colonies , but mostly nucleated erythroblasts in ko / ko colonies ( figure  4a , top row ) . the majority of erythroblasts , regardless of the genotype , resembled orthochromatic erythroblasts morphologically ( figure  4a ) and expressed low levels of alpha 4 integrin and high levels of rhd blood group antigen , a hallmark of late erythroblasts ( figure  4b ) . these data suggest that coding ko / ko erythroid progenitors were capable of progressing to the stage of orthochromatic erythroblasts but were less efficient in nuclear extrusion than their wt / wt or wt / ko counterparts . when the impact of zfn - mediated bcl11a erythroid enhancer gataa motif disruption was assessed , increased levels of fetal globin expression , averaging 37% of total -like globin chains based on data from five individual donors , were observed in gataa motif ko / ko erythroid cells compared with gataa motif wt / wt or wt / ko cells ( figure  3b , left panel ; figures s2 and s3 ) . however , in contrast to the adverse effects of bcl11a exon 2 editing on enucleation , the gataa motif ko / ko mutations did not impact enucleation efficiency ( figure  3b , right panel ; figure  s2b ) . cytospins confirmed the flow - based findings and showed that similar to gataa motif wt / wt and wt / ko erythroid cells , gataa motif ko / ko day-18 erythroid cells were mostly rbcs ( figure  4a , bottom row ) . the majority of the remaining erythroblasts had low alpha 4 integrin and high rhd blood group antigen expression and resembled orthochromatic erythroblasts regardless of their genotype ( figure  4b ) . these data indicate that the disruption of bcl11a erythroid - enhancer gataa motif significantly increases fetal globin expression in terminally matured erythroid cells without interfering with the differentiation or maturation of erythroid progenitors . to determine the engraftment potential of genome - edited hspcs , we transplanted zfn - treated bm - cd34 cells into nod / scid / il2rg null ( nsg ) mice ( figure  5 ) . repopulated human cells ( hcd45 ) were readily detectable in the peripheral blood ( pb ) of all transplanted animals at week 8 post - transplant ( figure  5a ) . however , a sharp decline in indel frequency post - transplant was observed in the coding zfn - treated group , but much less so in the enhancer zfn - treated group ( figure  5b ) , despite that the cd34 cell viabilities were comparable after electroporation at the time of harvesting ( table s1 ) . this is consistent with the recently described role of bcl11a in maintaining hsc functions.22 , 35 , 36 taken together , the negative impact of exon 2 disruption of the bcl11a gene on both terminal erythroid enucleation as well as on hsc function strongly argues against disruption of the coding sequence of bcl11a as a therapeutic strategy . in order to more thoroughly examine the impact of bcl11a enhancer zfn treatment , we performed additional long - term engraftment studies of enhancer zfn - edited bm - cd34 cells ( figure  6 ) . human chimerism in circulation was similar between control and enhancer zfn - treated groups for at least 16  weeks ( figure  6a ) . cd19 b cell lineage was the primary human cell type in bm , followed by cd33 myeloid lineage . low levels of cd3 t  cells , cd56 nk cells , and cd34 hspcs were also detected . human chimerism levels in bm were largely indistinguishable between control and enhancer zfn - treated groups except for minor but statistically significant differences in cd19 and cd33 populations . importantly , indels within the bcl11a enhancer were maintained at approximately 45%50% in pb ( figure  6b ) , reproducing the data reported in figure  5 . in addition , similar indel retention was observed in total bm , individually sorted cd3 , cd19 , and cd33 cell populations , as well as in human granulocytes or macrophages ( gm ) , and erythroid cells differentiated ex  vivo from the chimeric bm of primary recipients at 16  weeks post - transplant ( figure  6b ) . the high levels of indels in ex - vivo - derived erythroid cells from the enhancer zfn - treated group coincided with an increased percentage of erythroid cells expressing fetal globin ( f cells ) and elevated overall fetal globin levels compared with cells from the control group , whereas comparable levels of enucleation were attained ( figure  6c ) . because any human bfu - e erythroid progenitors present in chimeric bm at 16  weeks post - transplant were likely derived from engrafted long - term hscs , it was estimated that roughly 26% of the long - term engrafting hscs from the input bm - cd34 cells were gataa motif ko / ko and 14% were gataa motif wt / ko ( figure  6d ) . finally , the bm cells of primary recipient mice at 16  weeks post - transplant were injected into secondary recipients . low , yet comparable levels of human chimerism was achieved in control versus enhancer zfn - treated groups at 8  weeks post - secondary transplant ( figure  6e ) . these data provide strong evidence that the bcl11a erythroid enhancer in human long - term engrafting hscs can be edited by zfns . fetal hemoglobin ( hbf , 22 ) is recognized as one of the most important disease modifiers for scd because it not only inhibits hbs polymerization , but also reduces mean corpuscular hbs concentration.5 , 37 , 38 , 39 , 40 hbf is the predominant hemoglobin at birth , but it typically falls below 1% of total hemoglobin within a year and is replaced by adult hemoglobin ( 22 ) as the switch from - to -globin in the -globin locus occurs . pharmacologic reactivation of hbf presents one strategy to treat scd and has received tremendous attention in the field . conceptually it is also possible to permanently reactivate fetal globin expression by interfering with the fetal globin silencing machinery via genomic manipulation of a patient s autologous hscs . the -globin locus itself , bcl11a , and the hbs1l - myb intergenic region have been associated with hereditary persistence of fetal hemoglobin ( hpfh ) in genome - wide association studies ( gwas)19 , 41 , 42 , 43 and provide tangible molecular targets for  such an approach . indeed , two recent studies have utilized crispr - cas9-mediated genome editing to recapitulate known hpfh mutations in the -globin locus in the primary human cd34 cells.44 , 45 although upregulated fetal globin expression in the erythroid progeny of cd34 cells is observed , it remains to be seen at what frequency long - term repopulating hscs have been targeted by these genome - editing strategies . in this study , we used the zfn genome - editing technology to target bcl11a in primary human bm - derived cd34 cells instead of mobilized pb - derived cd34 cells because of the potentially life - threatening complications when mobilizing scd patients with granulocyte colony - stimulating factor ( g - csf ) . bcl11a is a key suppressor of fetal globin expression,17 , 18 , 20 , 48 and the conditional knockout of bcl11a in the erythroid lineage significantly increases fetal globin expression and cured a mouse model of scd . here because exon 2 is shared by all bcl11a isoforms , frameshift mutations in this region should completely inactivate bcl11a expression from the edited alleles . although bjurstrom and colleagues show that bcl11a exon 2-edited mobilized pb - cd34 can successfully engraft nsg mice , we found the engraftment by edited bm - cd34 cells to be severely impaired ( figure  6 ) . because it has recently been reported that bcl11a not only is important for lymphoid development , but also plays a critical role in maintaining vital hsc functions,22 , 35 , 36 a defect in the engraftment of exon 2 ko / ko cells was therefore not unexpected . in contrast to being essential for preserving hsc functions , it has been reported that bcl11a is dispensable for rbc production based on data obtained from erythroid - specific knockout of bcl11a in mice and shrna knockdown of bcl11a in primary human erythroblasts.22 , 30 although we found that bcl11a coding ko / ko erythroid progenitors were capable of differentiating and maturing into orthochromatic erythroblasts , the enucleation efficiency of coding ko / ko erythroid cells was significantly lower than their wt / wt or wt / ko counterparts ( figures 3 and 4 ; figure  s2 ) . the reason behind the suboptimal enucleation in coding ko / ko cells is not clear . however , this is unlikely an outcome of potential off - target editing because impaired enucleation was observed only in cells with bi - allelic out - of - frame mutations , and not in those with bi - allelic in - frame mutations at bcl11a exon 2 . in any case , suboptimal engraftment of exon 2-edited cd34 cells and reduced erythroid enucleation suggest that genome editing of the coding region of bcl11a is not a sustainable strategy for scd . as an alternative , we targeted an erythroid - specific enhancer within the second intron of the bcl11a gene that controls bcl11a expression exclusively within that cell lineage . more specifically , zfns were engineered to target the gataa motif , a key regulatory element situated in the  + 58 dnasei hypersensitive site of the enhancer.31 , 32 , 33 unlike bcl11a exon 2 editing , bi - allelic knockout of the gataa motif did not affect enucleation efficiency ( figures 3 and 4 ; figure  s2 ) , presumably because of residual bcl11a expression.22 , 31 although the induction of fetal globin expression in enhancer ko / ko erythroid cells was less robust than that in coding ko / ko erythroid cells , the levels of fetal globin expression , averaging 37% of total -like globins with a reciprocal reduction in adult -globin , should nevertheless be sufficient to inhibit hbs polymerization if maintained in  vivo.4 , 50 it should be noted that the quantification of fetal globin protein expression was done within clonally derived erythroid cells . this is particularly important because fetal globin expression at the individual cell level , and not at the population level , determines whether the erythrocytes can be protected from hbs polymerization . furthermore , the enhancer wt / ko erythroid cells had a moderate increase of fetal globin expression , which can also be disease - modifying given the inverse relationship between fetal globin level and scd mortality . it is also noteworthy that mono - allelic gataa motif ko erythroid cells , as well as mono - allelic exon 2 ko erythroid cells , expressed fetal globin at levels similar to those detected in patients with bcl11a haploinsufficiency,51 , 52 which validates the relevancy of our in  vitro system . finally , in contrast to bcl11a exon 2 editing , the enhancer - edited bm - cd34 cells achieved robust , long - term engraftment in nsg mice . genotyping of bfu - e colonies derived from the repopulated human cells in chimeric bm provided much - needed quantification of the editing efficiency in long - term repopulating hscs , which could only be assayed in nsg mice or similar in  vivo model systems . based on allogenic hsct data,53 , 54 mouse studies,55 , 56 and mathematical modeling,57 , 58 non - s hsc chimerism as low as 10%20% may lead to clinical improvement . if gene - edited , high fetal globin - expressing scd rbcs have a similar survival advantage as normal rbcs , a frequency of 26% gataa motif ko / ko hscs and 14% wt / ko hscs should offer long - term , substantial alleviation of symptoms in scd patients . altogether , our data provide in  vitro and in  vivo evidence that the gataa motif in the erythroid - specific enhancer of bcl11a gene is an effective and safe target compared with the bcl11a coding region for gene disruption for the treatment of scd . zfn - mediated editing of this gataa motif occurs efficiently in functional long - term repopulating hscs and does not interfere with terminal erythroid maturation . furthermore , gataa - motif disruption produces erythroid cells that express fetal globin at levels predicted to be effective in inhibiting hbs polymerization . therefore , our results provide a strong preclinical proof - of - concept that supports the clinical development of genome editing to disrupt the bcl11a erythroid - specific enhancer in autologous hscs as a potentially curative therapy for scd patients . zfns that target either the exon 2 ( coding zfns ) or the gataa motif in an erythroid - specific enhancer ( enhancer zfns ) of the bcl11a gene31 , 32 , 33 ( figure  1 ) were engineered by sangamo biosciences as previously described . bm - cd34 cells were isolated from de - identified bm aspirates of healthy volunteers ( hemacare ) . briefly , de - identified bm aspirates from multiple abo - matched donors were pooled , rbc - depleted with hetastarch , and then enriched with the clinimacs prodigy using clinimacs cd34 microbeads per the manufacturer s instructions ( miltenyi biotec ) . enriched bm - cd34 cells were cryopreserved in cryostor cs10 ( sigma - aldrich ) using a controlled rate freezer and stored in the vapor phase of a liquid nitrogen dewar . alternatively , cryopreserved bm - cd34 cells were purchased from lonza ( hopkinton , ma ) , stemcell technologies , or hemacare . to edit bm - cd34 cells , we thawed and cultured cryopreserved cells in cd34 maintenance media , comprised of x - vivo-10 ( lonza ) , 1x glutamax , 100  u / ml penicillin , 100  g / ml streptomycin ( all three from thermo fisher scientific ) , and recombinant human thrombopoietin ( tpo ) , fms - like tyrosine kinase 3 ligand ( flt3l ) , and stem cell factor ( scf ) ( 100  ng / ml each ; peprotech ) for 1 or 2  days at 37c , 5% co2 in a humidified incubator . electroporation of zfn mrna was performed either with a btx ecm 830 square wave electroporator ( harvard apparatus ) or a maxcyte device per the manufacturer s instructions . after transfection , cells were incubated in the cd34 maintenance media at 30c for 16  hr and then at 37c for 24  hr . the treated bm - cd34 cells were either analyzed directly or cryopreserved . cd34 cells were differentiated into erythroid cells using a three - step differentiation protocol developed by luc douay s group . in brief , cd34 cells were cultured for 7  days in step-1 media consisting of iscove s modified dulbecco s medium ( imdm ) ( thermo fisher scientific ) supplemented with 1x glutamax , 100  u / ml penicillin , 100  g / ml streptomycin , 5% human ab plasma , 330  g human holo - transferrin , 20  g / ml human insulin , 2  u / ml heparin , 1  m hydrocortisone ( all four from sigma - aldrich ) , 3  u / ml recombinant human erythropoietin ( epo ) ( thermo fisher scientific ) , 100  ng / ml scf , and 5  ng / ml interleukin-3 ( il-3 ) ( all three from peprotech ) . on day 7 , cells were transferred to step-2 media , which was identical to step 1 media except the absence of hydrocortisone and il-3 , and cultured for 34  days . next , cells were cultured for 79  days in step 3 media , which was similar to step 2 media but without the addition of scf . to determine the enucleation rate , we stained cells with nucred ( thermo fisher scientific ) per the manufacturer s instruction . cells were acquired using a facscanto , and analysis was done using flowjo software v10 ( tree star ) . to determine the percentage of hbf - positive cells , cells were fixed with 4% formaldehyde ( sigma - aldrich ) , permeabilized with ice - cold acetone ( thermo fisher scientific ) , washed with pbs ( thermo fisher scientific ) supplemented with 0.5% bsa ( sigma - aldrich ) , and stained with fluorescein isothiocyanate ( fitc)-conjugated anti--globin antibody ( santa cruz ) according to the method by thorpe et  al . globin chain expression was measured by uplc or hplc , modified from a published protocol . for uplc , 5  l of hemolysate was injected onto a waters acquity uplc protein beh c4 column ( 300a , 1.7 micron , 2.1   100  mm ) . elution was obtained at room temperature with a flow rate of 0.2  ml / min using an 18-min linear gradient of 38%42.5% acetonitrile in water ( both from thermo fisher scientific ) . trifluoroacetic acid was added to both water and acetonitrile to maintain a constant concentration of 0.1% . elution was followed at 220  nm with no reference wavelength and 280  nm with reference at 360  nm . area percentage for specific globin chains : a , g ,  ,  , or  , representing the relative amount of each specific globin chain , was quantitated using agilent openlab software . for hplc , 20  l of hemolysate was injected onto a grace vydac c4 column ( 5 micron , 4.6   250  mm ) . elution was obtained at room temperature with a flow rate of 1  ml / min using a 30-min linear gradient of 38.4%43.8% acetonitrile in water with trifluoroacetic acid constant at 0.1% . area percentage for specific globin chains was determined at 220  nm using agilent chemstration software . for single - cell studies , transfected cd34 cells were sorted into step 1 erythroid culture media ( 200  l / well ) in 96-well u - bottom non - tc - treated plates ( corning ) at two cells / well using facsaria iii because a pilot experiment showed that less than 20% of the cd34 cells gave rise to erythroid colonies . after 7  days of culture , 150  l of media per well was removed and replaced with 100  l step 2 erythroid culture media . after 4 additional days of culture , 100  l media per well was removed and replaced with 100  l step 3 media . on day 14 post - differentiation , furthermore , 100  l of media per well was removed and replaced with 100  l of fresh step 3 media . remaining cells were cultured for 34 more days when 50  l of cell suspension per well was collected , stained with an equal volume of nucred ( 2 drops / ml in pbs with 0.5% bsa ) , and analyzed with guava easycyte for cell count and enucleation rate . remaining cells were spun down , washed once with pbs , and lysed in 20  l hplc - grade water . cell debris was removed by centrifugation ( 10,000   g , 15  min , 4c ) . hemolysate was stored at 80c until ready for globin chain analysis by uplc as described above . for cell morphology examination , day-18 cultures were spun onto glass slides ( thermo fisher scientific ) using a shandon 4 cytospin ( thermo fisher scientific ) and stained with protocol hema 3 fixative and solution ( thermo fisher scientific ) per the manufacturer s instructions . to characterize surface marker expression by erythroid colonies , we harvested day-18 erythroid cells and stained them with antibodies against glycophorin a63 , 64 ( catalog no . f0870 ; dako , agilent pathology solutions ) , alpha 4 integrin ( category no . 563458 ; bd biosciences ) and rhd blood group antigen ( catalog no . 130 - 104 - 819 ; miltenyi biotec ) . genomic dna was subjected to targeted pcr amplification using human bcl11a - specific primers that also bear sample - specific barcodes and the illumina flow cell - specific sequences ( p5 and p7 ) ( table 1 ) . libraries were sequenced for 150x2 paired - end reads on the miseq sequencing system ( illumina ) following the manufacturer s instructions . after the miseq run , the forward and reverse paired - end reads were merged using paired - end read merger ( pear ) . the primers and several following bases were trimmed off to eliminate sequencing and pcr errors in the beginning of a read . next , fragments with the same length and high similarity were merged to further eliminate sequencing or pcr errors . to identify the mutations in the fragments , we compared reads that passed the above filters with the reference amplicon sequence via single - gap alignment . female nod.cg-prkdcscid il2rgtm1wjl / szj ( nsg ) mice ( jackson laboratories ) at the age of 67  weeks were sub - lethally irradiated at 300  rad 1  day before transplant . for primary recipients , human bm - cd34 cells were injected into the mice via the tail vein at approximately 1 million viable cells per mouse . human chimerism in pb was evaluated by flow cytometry at weeks 8 , 12 , and 16 post - transplant . mouse pb was sampled from the submandibular vein with edta as the anti - coagulant agent . rbcs were lysed using ack lysing buffer ( thermo fisher scientific ) , and cells were washed with pbs supplemented with 0.5% bsa . a fraction of washed cells was spun down and stored at 20c for dna extraction and deep sequencing . for flow cytometry 422302 and 101320 ; biolegend ) for 15  min , followed by the incubation with hcd45-allophycocyanin ( apc)-cy7 antibody ( catalog no . bm cells were collected from femurs , tibias , and pelvic bones for cell sorting , flow cytometry , ex  vivo cultures , burst - forming unit - erythroid ( bfu - e ) cultures , and secondary transplant . human cd45 cells were identified as positive by staining with the hcd45-bv510 antibody ( catalog no . specific lineages such as b cell ( cd19 ) , t  cell ( cd3 ) , and myeloid precursors ( cd33 ) within the hcd45 population were identified by the corresponding fluorochrome - conjugated human specific antibodies ( cd19-r - phycoerythrin [ pe ] , catalog no . granulocyte - monocyte / macrophage cell differentiation was performed by culturing bm cells in imdm supplemented with 1x glutamax , 100  u / ml penicillin , 100  g / ml streptomycin , 5% human ab plasma , 2  u / ml heparin , 50  ng / ml scf , 5  ng / ml il-3 , 10  ng / ml granulocyte macrophage colony - stimulating factor ( gm - csf ) , and 20  ng / ml g - csf ( all recombinant human cytokines were purchased from peprotech ) for 14  days . for bfu - e cultures , bm cells were plated in semi - solid methylcellulose culture media supplemented with human growth factors ( methocult h4434 ; stemcell technologies ) . individual hemoglobinized erythroid colonies were genotyped 14  days after culture . for secondary transplant , the remaining bm cells from five primary recipients were pooled , enriched for the repopulated human cells by depleting b220 cells , ter119 cells , and mouse cd117 cells , and then injected via tail vein into three irradiated recipients . pb and bm were harvested for human chimerism determination and deep sequencing as described above . all procedures were performed in accordance with protocols approved by the biogen institutional animal care and use committee ( iacuc ) . multiple samples were statistically compared by the kruskal - wallis test followed by the dunn s multiple comparisons test . are or were full - time employees and equity owners of biogen , and a.r . are or were full - time employees and equity owners of sangamo biosciences when these studies were conducted .
to develop an effective and sustainable cell therapy for sickle cell disease ( scd ) , we investigated the feasibility of targeted disruption of the bcl11a gene , either within exon 2 or at the gataa motif in the intronic erythroid - specific enhancer , using zinc finger nucleases in human bone marrow ( bm ) cd34 + hematopoietic stem and progenitor cells ( hspcs ) . 
 both targeting strategies upregulated fetal globin expression in erythroid cells to levels predicted to inhibit hemoglobin s polymerization . 
 however , complete inactivation of bcl11a resulting from bi - allelic frameshift mutations in bcl11a exon 2 adversely affected erythroid enucleation . 
 in contrast , bi - allelic disruption of the gataa motif in the erythroid enhancer of bcl11a did not negatively impact enucleation . 
 furthermore , bcl11a exon 2-edited bm - cd34 + cells demonstrated a significantly reduced engraftment potential in immunodeficient mice . 
 such an adverse effect on hspc function was not observed upon bcl11a erythroid - enhancer gataa motif editing , because enhancer - edited cd34 + cells achieved robust long - term engraftment and gave rise to erythroid cells with elevated levels of fetal globin expression when chimeric bm was cultured ex  vivo . 
 altogether , our results support further clinical development of the bcl11a erythroid - specific enhancer editing in bm - cd34 + hspcs as an autologous stem cell therapy in scd patients .