HUMAN GENE THERAPY 9:1157-1164 (May 20, 1998) Mary Ann Liebert, Inc.

M u r i n e B o n e M a r r o w E x p r e s s i n g the N e o m y c i n R e s i s t a n c e

Gene Has No Competitive Disadvantage Assessed In Vivo

TONG WU, MICHAEL L. BLOOM, JIAN-MEI YU, JOHN F. TISDALE, and CYNTHIA E. DUNBAR

ABSTRACT

The neomycin phosphotransferase (neo) gene is one of the most common mariner genes used in gene transfer

experimentation, but potential effects of neo gene expression in vivo have not been systematically investigated.

Several early clinical retroviral gene transfer studies have suggested that neo gene expression could have dele­

terious effects on hematopoiesis, owing to a discrepancy between the level of «eo-marked transduced marrow

progenitor cells compared with mature circulating progeny cells posttransplantation (Brenner et al., 1993;

K o h n et al., 1995; Brenner, 1996b). W e examined the long-term in vivo repopulating ability of bone marrow

from transgenic mice expressing neo from a strong constitutive promoter using a competitive repopulation

assay. Different ratios of neo transgenic and wild-type congenic marrow cells were cotransplanted into W / W ^

recipient mice. The percentages of blood cells containing the neo transgene in each group of recipient mice

monitored for 4 months posttransplantation closely matched the input ratios of neo transgenic to congenic

control m a r r o w cells. Similar concordances of engraftment with input ratios of neo transgenic cells were also

found in spleen, thymus, and whole marrow of recipient mice at 4 months posttransplantation. Analysis of the /3-hemoglobin phenotype (/3*'"8'* for the neo transgenic and C 5 7 control cells and /S iffuse Jq^ jj,g congenic

competitor H W 8 0 cells) in recipients confirmed erythroid repopulation from neo transgenic marrow cells at

levels matching the input ratios. W e conclude that hematopoietic cells expressing neo had no engraftment or

maturation defects detectable in vivo. These results suggest that the low-level contribution of vector-marked

cells to circulating populations in clinical trials is not due to direct deleterious effects of neo gene expression

on hematopoiesis.

O V E R V I E W S U M M A R Y cages is being pursued as a promising method for treatment of many congenital and acquired diseases (Dunbar and Young,

There has been concern based on indirect evidence that ex- 1996; Brenner, 1996a). Most preclinical in vitro and animal pression of the neomycin resistance phosphotransferase models, as well as all completed or ongoing clinical trials, have gene, included as a selectable marker in many gene trans- utilized replication-incompetent retroviral vectors to transfer fer vectors, could be toxic to or affect the differentiation of exogenous genes into hematopoietic cells (Miller, 1992). The hematopoietic cells. Using a murine competitive repopula- bacterial neomycin phosphotransferase (neo) gene, which con-tion model, we have shown that marrow repopulating cells fers resistance to the antibiotic G418, has been included in the from neo transgenic mice compete normally in terms of en- vast majority of these retroviral vectors for a number of rea-graftment and production of progeny cells of multiple lin- sons (Southem and Berg, 1982). The neo gene product allows cages. for rapid isolation of retroviral producer cell clones. In addi­

tion, straightforward measurement of vector production by pro­ducer cells can be carried out, and gene transfer efficiency in

I N T R O D U C T I O N eukaryotic target cells or their progeny can be measured easily by detection of unique neo sequences or by growth of trans-

E N G R A F T M E N T OF hematopoietic long-term repopulating duced cells in G418. cells modified with corrective or therapeutic genes and ex- Despite widespread use of the neo gene as a selectable

pression of the gene products in progeny cells from various lin- marker in a myriad of in vitro stadies without obvious effects

Hematology Branch, National Heart, Lung, and Blood Institute, National Instimtes of Health, Bethesda, M D 20892.

1157

1158 W U ET AL.

on cellular behavior, there are a small number of reports of per­turbations of proliferation or differentiation of cell lines, neo expression was shown to delay differentiation of human HL60 promyelocytic leukemia cells and modify the glycolytic path­way in 3T3 fibroblasts and FT0-2B hepatoma cells (von Melch-ner and Housman, 1988; Valera et al, 1994). Of more concrete concem are findings from two clinical gene transfer studies uti­lizing retroviral vectors containing the neo gene. In one study, three newborns with severe combined immunodeficiency were infused with autologous cord blood cells transduced with a vec­tor containing neo and the corrective adenosine deaminase gene (Kohn et al, 1995). Posttransplantation, significantly higher percentages of marrow colony-forming units (CFU-C) or puri­fied CD34''' cells contained the vector as compared with cir­culating mature progeny cells. A discrepancy between gene transfer levels in marrow C F U versus differentiated circulating cells was also noted in a gene-marking study involving chil­dren undergoing autologous transplantation for neuroblastoma or acute leukemia (Brenner etal, 1993; Brenner, 1996a). These observations suggested that neo gene expression could have a deleterious effect on differentiation of hematopoietic progeni­tors or that neo-expressing cells have a competitive disadvan­tage, either due to immune destmction of maturing cells or di­rect toxicity. Further examination of this issue is of great importance to the interpretation of clinical and preclinical stud­ies using vectors containing the neo gene, and the design of fu-tare protocols and strategies.

W e used the murine marrow transplantation model lo study systematically the effect of neo gene expression on hemato­poietic stem and progenitor cells and their progeny in vivo. To assess quantitatively the contribution of neo-expressing hema­topoietic cells to engraftment, we analyzed the long-term com­petitive repopulating ability of bone marrow cells from neo transgenic mice. By using marrow cells from neo transgenic mice instead of retroviral vector-transduced martow cells, we could precisely quantify the percentage of input «eo-express-ing marrow repopulating cells transplanted, and thus analyze the relationship of this variable to the percentage of neo cells in various hematopoietic tissues posttransplantation. W e found that neo gene expression had no direct deleterious effects on hematopoietic engraftment, differentiation, or overall contribu­tion to hematopoiesis.

MATERIALS AND METHODS

Mice

Adult female C57BL/6J (abbreviated C57), B6.C-H-lb/ByJ (abbreviated H W 8 0 ) , and WBB6F,-W/W^ (abbreviated W/W') mice were purchased from the Jackson Laboratory (Bar Har­bor, M E ) . C57BL/6J-TgN(pPGKneobpA)3Ems tieo transgenic mice expressing the neomycin resistance phosphotransferase gene from a constitutive phosphoglycerate kinase (pgk) pro­moter were also purchased from the Jackson Laboratory.

Harvest of bone marrow

Bone marrow (BM) cells were collected by flushing the con­tents of femurs and tibias into Dulbecco's minimal essential medium (GIBCO-BRL, Gaithersburg, M D ) with 1 0 % fetal

bovine semm (Atianta Biologicals, Norcross, G A ) , 4 m M of L-glutamine, penicUlin (50 mg/ml), and streptomycin (50 mg/ml) (all from GIBCO-BRL). Single-cell suspensions were obtained by passing martow cells through a 21-gauge needle.

CFU-C assay

Unfractionated marrow cells from donor neo and C57 mice or W / W recipient mice transplanted with either neo or C57 marrow cells were plated at concentrations of 1.5 X 10"* cells/dish and 5 X lO'* cells/dish in MethoCuU M3434 (Stem-Cell Technologies, Vancouver, BC, Canada) methylcellolose medium in the presence of a 0- to 2.5-mg/ml concentration of (active) G418 (GIBCO-BRL) at 36.5°C in 5 % CO2. Nine days later colonies of more than 30 cells were scored, using an in­verted microscope.

Competitive repopulation assay

The competitive repopulation assay was performed as pre­viously described (Harrison, 1980; Harrison etal, 1993). Fresh B M cells from donor neo, C57, and H W 8 0 mice were mixed at various experimental ratios, and 1-5 X 10^ total nucleated cells were transplanted into each nonirradiated W / W recipient mouse via tail vein injection. Peripheral blood (PB) was col­lected from the retroorbital sinus in heparinized capillary tubes (Fisher Scientific, Vienna, V A ) every 2 to 4 weeks. Hemoglo­bin (Hb) phenotype was determined by electrophoresis using reagents and the CliniScanll instrument obtained from Helena Laboratories (Beaumont, T X ) as previously described (Whit­ney, 1978). The relative repopulating ability of neo transgenic or C57 marrow cells compared with H W 8 0 was determined by the relative proportion of the /3*>"8ie m , ^^j^^ transgenic or C57) versus 3*'"''= Hb (HW80) bands.

PCR analysis

DNA was extracted from peripheral blood samples every 4 weeks and from thymus, spleen, and bone marrow 16 weeks posttransplantation using the QIAamp blood or tissue kit (Qia­gen, Chatsworth, C A ) , respectively. Polymerase chain reaction (PCR) was carried out as duected using the buffers, nucleotides, and Taq polymerase from the GeneAmp kit (Perkin-Elmer/Ce-tus, Norwalk, CT), widi the addition of 0.125 /nl/reaction of [3-P]deoxycytidine triphosphate (25 ^iCi/ml; Amersham, Ar­lington Heights, IL). One hundred nanograms of each genomic D N A sample mixed widi the P C R reaction reagents listed above was divided equally into P C R tubes containing either the neo or /3-actin oligonucleotide primers (final primer concentration 10 p M ) . neo primers were 5'-TCC A T C A T G G C T G A T G C A ATG CGG C and 5'GAT AGA AGG CGA TGC GCT GCG AAT CG. /3-Actin primers were 5'-CAT TGT GAT GGA CTC CGG AGA CGG and 5'-CAT CTC CTG CTC GAA CTC TAG AGC. Amplification conditions were 95°C for 3 min, followed by 23 cycles (neo) or 26 cycles (actio) of 95°C for 1 min, 55°C for 1 min, and 72°C for 2 min, followed by an 8-min 72°C ex­tension. P C R products were separated on 8 % nondenaturing polyacrylamide gels. The expected band size was 433 bp for the neo product and 232 bp for the /3-actin product. Negative controls included no D N A , and D N A extracted from nontrans-genic C57 blood or tissues concurrently with the test samples.

neo E X P R E S S I O N H A S N O E F F E C T O N H E M A T O P O I E S I S 1159

Serial dilutions of neo transgenic mouse D N A into C57 D N A were used as positive controls. Band intensity was quantitated using a Phosphorlmager (Molecular Dynamics, Synnyvale, CA). All neo reactions were run under conditions optimized to give linear results between 1 and 9 0 % neo cells, and actin re­actions were optimized to be linear for the range of total D N A concentrations used. The ratios of neo to actin band intensities were used to calculate estimated percent contribution of neo transgenic cells compared with the neo transgenic control dilu­tions.

Expression of neo m R N A was determined by reverse tran­scriptase-polymerase chain reaction (RT-PCR). Total R N A was isolated from donor or recipient P B or other tissues using the R N A STAT-60 reagent (Tel-Test B, Friend wood, T X ) as di­rected, followed by treatment with DNase I (GIBCO-BRL). c D N A was synthesized from 700 ng of R N A using the R N A P C R core kit and random hexamer priming (Perkin-Elmer, Branchburg, NJ). Each sample was run with a concurrent con­trol without reverse transcriptase to document lack of D N A con­tamination. Synthetic conditions were 42°C for 15 min, 99°C for 5 min, and 4°C for 5 min, then each c D N A sample was di­vided equally into P C R tubes containing buffer, magnesium chloride, Taq, and either the neo or /3-actin primer listed above. P C R was performed by denaturation for 2 min at 95°C, fol­lowed by 38 cycles (neo) or 30 cycles (actin) of 1 min at 95 °C, 1.5 min at 60°C, 2 min at 72°C, and a final extension for 8 min at 72°C. All amplified products were separated by 1.5% agarose gel. R N A from W f W " recipients transplanted with C57 marrow cells was used as a negative control, and R N A from neo trans­genic mouse and G l N a producer cells as the positive controls.

Statistical analysis

Analysis of significance using the two-tailed Student t test and regression analysis were performed using the SigmaPlot for Windows software (SPSS A S G , Erkrath, Gemiany).

RESULTS

Hematopoietic cells from neo transgenic mice and W I W recipients of neo transgenic m a r r o w cells express the neomycin phosphotransferase gene

The neo transgenic mouse strain C57BL/6J-TgN (pPGK-neobpA)3Ems expresses the neomycin resistance gene from a constitutive phosphoglycerate kinase (pgk) promoter. Expres­sion of the neo gene and conferral of a G418-resistant pheno­type has been documented in a wide variety of tissues from these mice (E. Simpson, the Jackson Laboratory, unpublished data, 1997). W e verified expression of the neo gene in hematopoietic cells from neo donor transgenic mice and W / W " recipients of neo donor transgenic marrow by assaying the growth of hematopoietic progenitor cells and their progeny in the C F U - C assay with and without G418 (Fig. 1). B M cells from neo and C57 mice or W / W recipients injected with neo or C57 marrow cells were plated in methylcellulose medium at a range of G418 concentrations. Figure 1A shows that there was a significant shift to the right in the killing curve of the neo ti-ansgenic C F U - C compared with control C 5 7 CFU-C. This was also tine for the W / W " recipients of neo marrow versus C57

1 2 G418(mg/ml)

0

bp

603-310-194-

603-310-194-

o ^ ra S

1 Q. 'o o X 1- 1-

•

•

•

1 2 G418(mg/ml)

Recipient 2

(-) Control

Donor

(+) Control

No RNA

H H I-I-KI-P-I-I-K CC CCOCCECC (TCC CCCCQ;

• • • • i l l

• D

• p-actin

5 6 7 8 9101112

FIG. 1. Documentation of neo gene expression by G418-re-sistant C F U - C assay and RT-PCR. The martow cells from donor neo transgenic (O) and C57BL/6J (•) mice (A) or W / W " re­cipients of neo or C57 B M harvested 8 weeks after transplan­tation (B) were cultured in methylcellulose medium at the con­centrations of G418 shown. Each point represents the mean colony (CFU) number for at least three separate experiments. The number of G418-resistant C F U - C was significantiy higher in the tieo donor marrow or the neo recipient marrow compared with control C57 donor marrow or recipients of C57 marrow at a 1-mg/ml concentration of G418 [p < 0.05 for both (A) and (B)]. (C) Eight weeks posttransplantation, P B R N A from W / W " recipients transplanted with neo or C57 marrow cells was re­verse transcribed, and analyzed by P C R for neo sequences, with ;S-actin as a control for R N A quality. Each sample was ana­lyzed with reverse transcriptase: -l-RT (lanes 1, 3, 5, 7, 9, and 11), and without reverse transcriptase: - R T (lanes 2, 4, 6, 8, 10, and 12). Recipients 1 and 2: two recipient mice transplanted with neo transgenic B M . (—) control: recipient mouse receiv­ing C57 B M . Donor: neo transgenic mouse. (-I-) control: G l N a producer cells.

1160 W U ET AL.

Hemoglobin Pattern

Harvest BM

Transplant

Hemoglobin Pattern

Donors

i C57BU6J HW80

- ^ / 3

-single

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1 \

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1

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Recipient

neo transgenic mice W / W ^

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C57/HW80 neo/HW80 neo/HW80

1:99

neo/HWBO

^ X ^ ^ X ^ _ - 0 . - = £ 3 1x107 1x10^ 1x10'' 5x10^

-single

S diffuse

FIG. 2. Experimental design of competitive repopulation assay. Marrow cells were collected from donor C57 (Hb '"*'* ), neo transgenic (Hb ™8''=), and H W 8 0 (Hb'''ff"'«=) mice, and mixtures of varying ratios of the cells were made: C57/HW80, 1:1; neofWNSO, 1:1; neo/HW80, 1:9, neofHWSO, 1:99. A total of 1-5 X 10' nucleated manrow cells of the various mixtares was in­jected into each recipient W / W " mouse.

marrow (Fig. IB), indicating that there was no extinction in ex­pression owing simply to the transplantation procedure. In these mice, the plating efficiency at I m g of active G418 per milli­liter was 28 for neo recipients versus 1 for C57 recipients per 10' B M cells plated (p < 0.05). There was no difference in the characteristics of the neo versus C57 marrow cells plated in the absence of G418: plating efficiency, myeloid:erythroid distrib­ution, appearance, and size of the colonies were indistinguish­able.

W e also documented neo transgene expression in the PB of the neo transgenic mice and in W / W " recipinets of neo trans­genic marrow by RT-PCR. After reconstitation, neo m R N A ex­pression was detected in PB of neo recipients 8 weeks after transplantation, as well as in neo transgenic donors (Fig. IC).

Design of competitive repopulation assay

To determine whether neo-expressing marrow has a com­petitive disadvantage in vivo, the murine competitive repopu­lation assay was used (Harrison, 1980). As shown in Fig. 2 and described above, B M cells were collected from donor mice, and neo cells were mixed at three ratios (1:1, 1:9, and 1:99) with congenic H W 8 0 competitor cells disparate only at the jS-he-moglobin locus. B M cells from C57 mice were mixed with H W 8 0 cells at a ratio of 1:1 as a control. Each cell mixture was transplanted into five recipient W / W " mice, with each individ­ual mouse receiving 1-5 X 10' nucleated cells. C57 mice or neo transgenic mice (C57 background) and H W 8 0 mice are ge­netically distinguishable by their ;8-hemoglobin alleles: C57 mice are homozygous for the /3'*i"gi'= allele, and H W 8 0 mice are homozygous for the jgd'ff se allele. After hematopoietic recon­

stitation of the W / W " recipients documented by normalization of hematocrit and mean corpuscular volume, the relative re­populating abilities of the donor congenic marrows can be quan­titatively assessed by measuring the percentages of single and diffuse ^-hemoglobin in the PB. W h e n more than 10^ cells/mouse are transplanted, this assay is quantitative and ac­curate with an experimental error of 3 to 5 % (Harrison, 1980, 1993). Erythroid reconstitution as measured by H b elec­trophoresis is accompanied by similar ratios of reconstitution in other hematopoietic lineages (Barker et al, 1991; Dunbar et al, 1991). P C R analysis for neo gene sequences was used to assess the relative contribution of the neo transgenic marrow to hematopoiesis, specifically measuring the contribution to cir­culating leukocytes, diymic cells, splenocytes, and marrow.

Repopulating ability of neo gene-expressing hematopoietic cells

After mixtures of /teo:HW80 or C57:HW80 marrow cells were injected into W / W " recipient mice, replacement of en­dogenous erythropoiesis was completed by 4 weeks post&ans-plantation as assessed by normalization of hematocrit values and mean corpuscular volume (data not shown). P C R analysis of recipient PB leukocyte D N A demonstrated that the percent­age of circulating cells derived from the neo as compared widi the H W 8 0 donor marrow matched the input ratios ofneo.HWSO cells closely: at 16 weeks posttransplantation 46.3, 14.7, and 1.1% neo cells in the groups receiving the 1:1, 1:9, and 1:99 competitive mixtures, respectively (Figs. 3 and 4). Linear re­gression analysis revealed a high correlation between input per­centage of neo transgenic marrow cells and the resulting per-

neo EXPRESSION HAS NO EFFECT ON HEMATOPOIESIS 1161

0) o + o c

70-

60-

50-

40-

30-

20-

10-

• 1:1 neo/HW80 O1:9neo/HW80 • 1:99 neo/HW80

6 a 10 12 A Weeks Post-transplantation

16

FIG. 3. P C R analysis of neo gene sequences in peripheral blood posttransplantation. Peripheral blood was obtained monthly and analyzed by P C R for neo sequences, as well as -actin sequences as a control for amplifiable D N A . Each point represents the mean ± standard deviation for each group of four or five mice. Percent neo contribution was obtained by plotting the ratio of neo to actin signal for each point on a regression curve of simultaneously amplified D N A extracted from mix-tares of neo transgenic and control murine cells.

centage of circulating P B cells containing the neo gene post­transplantation (Fig. 5).

As shown in Fig. 6, the contribution of neo marrow to ery­thropoiesis was assessed by H b phenotyping. A s compared with C57 wild-type marrow, the neo marrow competed somewhat better against the H W 8 0 cells in the 1:1 mixtures (at 16 weeks the percentage H b ;8= ™«' was 68.9 ± 6.0% in the neo:HW80 group and 38.4 ± 6.6% in the C 5 7 ; H W 8 0 group, p < 0.001). The repoulating ability of the neo marrow assessed by hemo­globin phenotype matched the input ratios of B M cells in the 1:9 and 1:99 groups, with 14.5 ± 2.5% and 1.4 ± 1.0% H b singie respectively.

W e also examined long-term reconstitation in other hematopoietic lineages 4 months after transplantation. After sacrifice, P C R analysis was used to quantify the percentage of neo transgenic donor-derived cells in thymus, spleen, and bone marrow (Fig. 7). A s with the PB, there was a close correlation with the input percentage of neo marrow cells (r values of 0.90, 0.79, and 0.94, respectively), although the presence of recipi­ent-derived thymic and splenic nonhematopoietic stromal ele­ments, as well as long-lived lymphoid cells, m a y have decreased the relative contribution of the neo marrow-derived cells to these tissues, especially in the 1:1 ratio group of recipient mice.

DISCUSSION

In this study, we demonstrated that murine «eo-expressing hematopoietic cells did not have detectable intrinsic engraft­ment or differentiation defects in vivo. The percentage of cir­culating hematopoietic cells of erythroid and nonerythroid lin­eages, thymic cells, splenic B and T cells, and whole bone marrow was strongly correlated with the percentage of neo transgenic bone marrow cells transplanted. This suggests that low gene transfer levels reported in circulating or mature hematopoietic cells in vivo after transplantation with trans­duced progenitor/stem cells result from another mechanism be­sides direct deleterious effects of neo gene expression on hematopoiesis. The close correlation between input percentages of neo transgenic marrow cells and posttransplantation circu­lating levels of neo transgenic progeny cells also does not ex­plain the discrepancy between levels of neo gene-marked C F U or CD34^ cells postengraftment compared with differentiated circulating gene-marked cells reported in some (Brenner et al, 1993; Kohn et al, 1995; Dunbar et al, 1996) but not all hu­man and large animal studies (Dunbar et al, 1995; Cometta et al, 1996; Emmons et al, 1997).

There are a number of possible explanations for these dif­ferences, neo gene expression may have different effects on cel­lular viability or differentiation of murine as compared with hu­man cells. However, neo has been used as a selectable marker

1:1 neo/HW80

neo-

46.3%t 6.8%

1:9 neo/HWBO

14.7%±6.3%

1:99 neo/Hwso

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FIG. 4. P C R analysis of neo and actin sequences in peripheral blood of recipient mice 16 weeks posttransplantation. Each lane represents D N A from one recipient mouse. Groups of recipient mice receiving the same ratio of donor cells are labeled above the lanes with the ratio of neo transgenic to H W 8 0 donor cells used. (-) control: concurtendy extracted C57 P B D N A . (-I-) con­trols: D N A extracted from mixtures of weo-ttansgenic and C57 cells at the indicated percentage ratios. The mean percentage of neo''' cells shown below each group was calculated from individual values of Neo/actin band intensities plotted on the regres­sion curve generated from (-I-) controls.

1162 W U ET AL.

c 0) u o a. m Q.

O

120

100

0 20 40 60 80 100 120 %Neo-Transgenic Marrow Cells Transplanted

FIG. 5. Relationship between percentage neo transgenic mar­row cells transplanted and percentage neo P B cells at 16 weeks posttransplantation: The regression line with 9 5 % confidence intervals is shown, plotting percentage neo transgenic marrow cells transplanted versus percentage neo transgenic P B cells (from P C R analysis) in individual mice at 16 weeks posttrans­plantation. The correlation coefficient (r) is 0.96, and the re­gression coefficient (slope) is 0.82.

in vast numbers of experiments on both human and murine cell lines and primary cells in vitro, and with the exception of two reports involving relatively subtle changes in cellular physiol­ogy of murine (3T3) or human (HL60) cells, no other reports of direct toxicity or effects on differentiation have been pub­lished (von Melchner and Housman, 1988; Valera et al, 1994). The effects of neo expression in both of those studies may have resulted from clonal variations or effects of prolonged in vitro

100

o c (?) n X

W e e k s Post-transplantation

FIG. 6. Contribution of neo-transgenic or C57 (Hb'""^''^) mar­row cells to erythropoiesis posttransplantation. Peripheral blood was collected every 2 to 4 weeks and analyzed by H b elec­trophoresis. Each point shows the mean and standard deviation of the percentage of Hb'""s''= (derived from either neo transgenic or control C57 marrow cells) in each group of mice transplanted with the designated mixture of donor marrow cells.

d m g selection compared with the parental cell lines: in one study no controls using an altemative selectable marker were

included (Valera et al, 1994). L o w levels of circulating genetically altered cells in vivo m a y

result instead from immune responses agamst the nonself gene product (McCarthy, 1996). Differentiated cu-culating hemato­poietic cells may be more prone to immune destruction than marrow C F U or C D 3 4 + cells, owing to differences in the level of neo or other transgene expression or inefficient immuno­genicity of primitive cells owing to lack of required co-stimu­latory signals (Egner and Hart, 1995). There are lunited pub­lished data on immune responses specifically against the neo gene product. One patient receiving multiple infusions of gene-modified T lymphocytes was shown to develop anti-neo and anti-herpes thymidine kinase cell-mediated immune responses that coincided with rapid disappearance of transduced cells in vivo (Bonini et al. 1997). In several murine gene ttansfer stud­ies, retrovirally transduced marrow cells selected ex vivo for neo expression by cultare in G418 showed poor and only tran­sient engraftment in vivo, potentially implicating an immune re­sponse against infused cells selected for high-level neo expres­sion (Dick et al, 1985; W o n g et al, 1989). More likely is that prolonged ex vivo culture greatiy decreased repopulating po­tential, or that neo was not expressed highly enough in the most primitive repopulating cells to allow ex vivo selection. The en­graftment of transduced primitive murine progenitor/stem cells as opposed to lymphocytes or other cell types has been demon­strated to induce tolerance to foreign transgenes, at least in ir­radiated recipients (Sykes et al, 1993). In irradiated primates, we have to date found no significant difference in the engraft­ment or persistence of circulating hematopoietic cells derived from primitive hematopoietic cells transduced with neo-ex-pressing compared with nonexpressing vectors (Y. Hanazono,

• Thymus D Spleen D B M 0PB

L i A ^ 1:1 neo/HW80 1:9neo/HW80 1:99 neo/HW80

FIG. 7. P C R analysis of neo-transgenic contribution to non­erythroid hematopoietic lineages. D N A was exti-acted from diy-mus, spleen, B M , and P B after sacrifice of recipient mice 16 weeks posttransplantation and analyzed by P C R for neo se­quences, as well as -actin sequences as a control for amplifi­able D N A . Each bar represents the mean ± standard deviation for each group of four or five mice. Percent neo contribution was obtained by comparing the ratio of neo to actin signal for each point to a standard curve of simultaneously amplified D N A extracted from mixtures of neo ttansgenic and conttol murine cells.

neo E X P R E S S I O N H A S N O E F F E C T O N H E M A T O P O I E S I S 1163

unpublished data, 1998). The results in this article do not sug­gest an anti-«eo immune response in the model system used. However, diese mice may more easOy develop tolerance to for­eign gene products expressed in hematopoietic cells owing to eventual complete thymic engraftment with the progeny of transplanted cells in die absence of irradiation (Barker et al, 1991).

Another potential explanation for lower levels of circulating genetically modified cells than marrow C F U or CD34+ cells may be that efficientiy ttansduced committed progenitors home to the marrow microenvironment and persist for long periods, but do not contribute to ongoing production of mature hematopoietic cells in vivo. Instead, less efficientiy transduced stem cells may be completely responsible for replenishment of the circulating populations. One murine study found that trans­plantation of sex-mismatched syngeneic cells into nonablated recipients resulted in much higher levels of engraftment of com­mitted CFU-S compared with stem cells responsible for long-term hematopoiesis (Wu and Keating, 1993). W e have contin­ued to stady this issue in the nonhuman primate model. Vectors with and without neo have been used to transduce rhesus mon­key repopulating cells, and animals have been followed sys­tematically posttransplantation to assess levels of genetically modified CD34''" cells, marrow CFU, and mature circulating cells. No difference in levels of vector-containing cells have been seen in any animals long term (S. Sellers, unpublished data, 1998).

Even if the neo gene product is not toxic or responsible for immune rejection of progeny of transduced hematopoietic stem cells, it seems pmdent to remove any unnecessary genes from vectors designed for clinical or in vivo gene transfer trials. Im­provements in rettoviral vector production technology have al­lowed rapid isolation of high-titer producer clones even with­out inclusion of selectable markers within the vector backbone (Pear et al, 1993; Finer et al, 1994). However, disappointing levels of gene ttansfer reported in early human clinical trials directed at repopulating stem cells more likely resulted from poor ttansduction of primitive cells, and were not simply a con­sequence of having included the neo gene in gene transfer vectors.

ACKNOWLEDGMENTS

We thank Daniel Dunn for helpful discussions and Karen Stoos and Stephanie Sellers for technical assistance.

REFERENCES

BARKER, J.E., GREER, J., BACON, S., and COMPTON, S.T. (1991). Temporal replacement of donor erythrocytes and leukocytes in nonanemic W44j/W44j and severely anemic W/Wv mice. Blood 78, 1432-1437.

BONINI, C, FERRARI, G., VERZLETTI, S., SERVIDA, P., ZAP-PONE, E., RUGGIERI, L., PONZONI, M., ROSSINI, S., MAVILIO, F., TRAVERSARI, C, and BORDIGNON, C. (1997). HSV-TK gene transfer into donor lymphocytes for control of allogeneic graft-ver-sus-leukemia. Science 276, 1719-1724.

BRENNER, M. (1996a). Gene marking. Hum. Gene Ther. 7, 1927-1936.

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Address reprint requests to: Dr. Cynthia E. Dunbar

Hematology Branch, NHLBI, N I H Building 10, R o o m 7C103

9000 Rockville Pike Bethesda, M D 20892

Received for publication November 20, 1997; accepted after re­vision March 6, 1998.