Proc. Nall. Acad. Sci. USAVol. 86, pp. 8912-8916, November 1989Genetics

Production of human glucocerebrosidase in mice after retroviralgene transfer into multipotential hematopoietic progenitor cells

(gene therapy/retroviral vectors/Gaucher disease/spleen colony-forming units/stem cells)

PAMELA H. CORRELLt, JOHN K. FINKt, ROSCOE 0. BRADY, LELAND K. PERRYt, AND STEFAN KARLSSONtttMolecular and Medical Genetics Section, Developmental and Metabolic Neurology Branch, National Institute of Neurological Disorders and Stroke, NationalInstitutes of Health, Bethesda, MD 20892

Contributed by Roscoe 0. Brady, August 18, 1989

ABSTRACT The human glucocerebrosidase (GC) genehas been transferred efficiently into spleen colony-forming unit(CFU-S) multipotential hematopoietic progenitor cells, andproduction of human GC RNA and protein has been achievedin transduced CFU-S colonies. High-titer retroviral vectorscontaining the human GC cDNA were constructed. Mouse bonemarrow cells were stimulated with hematopoietic growth fac-tors, infected by coculture with producer cells, and injectedinto lethally irradiated animals. Four vectors were comparedwith respect to gene-transfer efficiency into CFU-S progeni-tors. One vector (G vector) required high concentrations ofinterleukins 3 and 6 during stimulation and coculture forefficient transduction of CFU-S progenitors. The remainingthree vectors (NTG, GTN, and GI vectors) transduced theseprogenitors at infection frequencies approaching 100% usinglow concentrations of hematopoietic growth factors to stimulatecell division prior to and during the infection. Vectors using theviral long terminal repeat enhancer/promoter to drive thehuman GC cDNA produced high levels of human GC RNA inthe progeny of CFU-S progenitors after gene transfer. When aninternal herpes simplex thymidine kinase promoter assisted bya mutant polyoma enhancer was used to drive the human GCcDNA (NTG vector), little or no human GC RNA was detectedin transduced CFU-S colonies. All three vectors producinghuman GC RNA in CFU-S colonies can generate human GC asdetected by immunochemical analysis of CFU-S colonies. NTGvector-infected bone marrow cells were transplanted into W/W' recipients to generate long-term reconstituted mice. Thecapacity of the viral long terminal repeat and the internalthymidine kinase promoter to direct synthesis of RNA intransduced bone marrow and spleen cells 5 months after bonemarrow transplantation reflected the performance of thesepromoters in NTG-transduced CFU-S colonies.

Gaucher disease, the most frequent sphingolipid storagedisorder in humans, is an autosomal recessive disordercaused by deficient activity of lysosomal f3-glucocerebrosi-dase [GC, EC 3.2.1.45] (1). Glucocerebroside accumulationin cells of the monocyte-macrophage lineage results in he-pato- and splenomegaly and bone marrow infiltration withlipid-laden macrophages. Neuronopathic forms ofthe disease(Gaucher disease types 2 and 3) are characterized by centralnervous system involvement in addition to the organomegalyand bone marrow infiltration seen in the most common form,type 1 Gaucher disease (2, 3).Macrophages are hematopoietic cells that originate from

bone marrow stem cells. Heterologous bone marrow trans-plantation (BMT) as a treatment for Gaucher disease hasproduced complete resolution of the hepatosplenomegaly (4)and regression of the radiological changes in the bones (5).The first successfully transplanted patient (type 3) was

healthy with complete correction of the organomegaly butlittle if any improvement of the central nervous systemsymptoms 5 years after BMT (6). This remarkable successindicates that BMT can reverse most or all symptoms of type1 Gaucher disease. Currently, however, BMT carries a highrisk of morbidity and mortality (5, 7).The cDNA encoding human GC has been cloned and

sequenced (8-10). When two of these cDNAs were trans-ferred into fibroblasts of Gaucher patients using retroviralvectors, they corrected the enzyme deficiency (11, 12). SinceBMT can reverse the symptoms of Gaucher disease, it islikely that transfer of the cloned normal GC gene into thepatient's own hematopoietic stem cells will also reverse thesymptoms of the disease after autologous transplantation oftransduced bone marrow cells. Experiments using retroviralgene transfer have successfully accomplished transfer offunctionally intact genes into multipotential hematopoieticprogenitors and repopulating bone marrow stem cells ofmice. These include the genes for neomycin resistance(NeoR) (13, 14), human adenosine deaminase (15), and human,B-globin (16-18). Tissue-specific expression of the human,B-globin gene was seen in long-term reconstituted animals(16, 18).The present study reports the generation of high-titer stable

retroviral vectors that contain the human GC cDNA. Effi-cient transfer of the human GC gene into murine spleencolony-forming unit (CFU-S) multipotential hematopoieticprogenitor cells was accomplished. Three of four vectorstested resulted in human GC mRNA production in theprogeny of CFU-S progenitors, and human GC (cross-reacting material) was detected in CFU-S-derived coloniesby human-specific anti-GC antibodies. Successful transfer ofthe human GC gene into repopulating stem cells of the mouseis also presented.

MATERIALS AND METHODSCells and Viruses. PA317 and P-2 packaging cells and

thymidine kinase-negative 3T3 cells were grown as described(19). Viruses containing the NeoR gene were titered bystandard techniques (19), and helper virus assays were per-formed on D-56 cells (17). The titers of the GI virus producers(Fig. 1) were indirectly estimated by determining the con-centration of interleukin (IL) 3 in their supernatants in asimple proliferation assay using the IL-3-dependent cell line,32-D. 32-D cells were grown in RPMI 1640, 10% (vol/vol)fetal bovine serum, and WEHI-3D cell-conditioned medium,as a source of IL-3. COS cells, normal human fibroblasts, and

Abbreviations: CFU-S, spleen colony-forming units; NeoR, neomy-cin-resistance gene; IL, interleukin; BMT, bone marrow transplan-tation; GC, glucocerebrosidase; Htk, herpes simplex thymidinekinase; Py*, mutant polyoma virus enhancer; LTR, long terminalrepeat.tTo whom reprint requests should be addressed at: Building 10,Room 3D04, National Institutes of Health, Bethesda, MD 20892.

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The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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SD SA

SD SA

SD SA SA

SD SA

NTG

GTN

GI

G

FIG. 1. Vectors used. Boxes at the left and right represent the 5'and 3' LTRs. Arrows indicate promoters and SD and SA indicatedonor and acceptor splice sites, respectively. NEO, NeoR gene; TK,the Py* enhancer plus the herpes simplex thymidine kinase (Htk)promoter (20); GC, GC cDNA (8); IL-3, IL-3 cDNA (21).

fibroblasts from type 2 Gaucher patients (GM-877) and GM-1260) were grown in Dulbecco's modified Eagle's medium(DMEM) and 10% fetal bovine serum. COS cells were

transfected by the calcium phosphate technique (22).Vectors. All retroviral vectors used were derived from N2

(23). To simplify constructions the 3.2-kilobase (kb) Sac Ifragment from the 9.6-kb N2 plasmid was cloned into the SacI site ofpLSDL (24) to generate a 6.5-kb vector plasmid, pN7,containing the N2 viral sequences. The 5' long terminalrepeat (LTR) in pN7 contains the Moloney murine sarcoma

virus sequences upstream from the Sac I site. The NTGvector (Fig. 1) was generated by inserting the 273-base-pairXho I-Pst I fragment containing a polyoma virus mutantenhancer (Py*) and the Htk promoter (20) in front of the2.3-kb EcoRI GC cDNA (8) in an intermediary cloningvector. The 2.6-kb transcription unit, Py* Htk GC cDNA,was then cloned into the Xho I site of pN7. To generate theG virus (Fig. 1), the 1.5-kb EcoRI fragment containing theNeoR gene was removed from pN7 and replaced by the 2.3-kbGC cDNA. To construct the GTN and GI vectors, the 1.3-kbEcoRI-Sal I fragment containing the NeoR gene was removedfrom pN7 and replaced by the GC cDNA. These constructspreserve the Xho I cloning site from N2. A 1.2-kb Xho Ifragment from pCD-IL3 (21) containing two splice acceptorsites and the murine IL-3 cDNA was inserted into the Xho Icloning site to create the GI virus (Fig. 1). The GTN virus(Fig. 1) was made by inserting the Py* Htk NeoR transcriptionunit (Xho I-Sal I fragment) from MCI Neo (20) into the sameXho I cloning site. The G and GI virus plasmids do notcontain a NeoR gene. They were cotransfected into 4I-2 cellswith pSV2neo and the primary transfectants were used as

producer lines. These viruses contain a 5' LTR from Moloneymurine sarcoma virus. The other viruses (NTG and GTN)were produced as described (25) and have Moloney murineleukemia virus promoters and enhancers after viral replica-tion.Bone Marrow Infection and Transplantation. Bone marrow

infection was performed as described (17) with the followingmodifications. Bone marrow was harvested from donor mice2-5 days after i.v. administration of 5-fluorouracil (150 mg/kg). Bone marrow was stimulated [15% (vol/vol) WEHI 3Dcell conditioned medium and IL-3 and IL-6, each at 20-200units/ml] for 2 days and cocultivated with viral producer cellsfor 48 hr. Recombinant murine IL-3 and IL-6 were generatedin COS cells by transfection of plasmids pCDIL-3 andpCDIL-6 (kindly provided by Frank Lee of DNAX). Purifiedmurine IL-3 (courtesy of J. Ihle, National Cancer Institute)and human IL-6 (S. Clark, Genetics Institute) were also used.

Coculture was done in the presence of Polybrene and stim-ulation medium. Lethally irradiated WBB6F1 mice wereinjected with 1-10 x 105 bone marrow cells. Spleens conflu-ent with CFU-S colonies and individual colonies were har-vested 14 days after BMT. W/WV mice were injected with 1-2x 106 viable bone marrow cells for long-term reconstitution.The nature and source of animals has been described (17).DNA and RNA Analysis. Southern blot analysis was per-

formed using standard techniques. Total cellular RNA wasextracted by guanidine thiocyanate (26, 27) and separated ona formaldehyde/agarose gel. The RNA was transferred tonitrocellulose filters, prehybridized, hybridized, and washedas described (10).GC Assay. Cell pellets were extracted in 50 mM potassium

citrate/potassium phosphate (pH 5.9) containing Triton X-100 (2 mg/ml) and sonicated on ice. Cleared cellular lysateswere assayed in a buffer containing 4.8 mM 4-methylumbel-liferyl glucopyranoside, Triton X-100 (1.5 mg/ml), and so-dium taurocholate (1.25 mg/ml) using a fluorimeter.Immunocytochemical Analysis. Hematopoietic cells were

transferred to microscope slides in a cytocentrifuge. Immu-nochemical detection was performed as described by vanDongen et al. (28). The mouse monoclonal anti-human GCantibody 8E4 was used to detect human GC (29). Fluores-cein-conjugated goat anti-mouse immunoglobins (VectorLaboratories) were used as secondary antibodies.Polymerase Chain Reaction. Peripheral blood cells were

lysed (30), the DNA was denatured (940C, 5 min), andamplified (940C, 1.5 min; 560C, 2 min; 720C, 3 min; 30 cycles).Oligonucleotide primers that amplified a 790-base-pair seg-ment homologous to the NeoR gene were as follows: 5'-CAA-GAT-GGA-TTG-CAC-GCA-GG-3' (sense) and 5'-CCC-GCT-CAG-AAG-AAC-TCG-TC-3' (antisense).

RESULTSGC Vectors. To determine whether the human GC cDNA

encoded a functionally active enzyme, the GC cDNA wascloned into pMT2, which produces high levels of protein(from the transferred gene) after transfection into COS cells(31). COS cells transfected with 10, 30, and 50 ,ug of pMT2-GC showed 3-, 6-, and 7-fold increases, respectively, in GCactivity. Moreover, retroviral infection of Gaucher type 2fibroblasts resulted in normalization of GC activity (32).These results agree with the data of Sorge et al. (12) and provethat the cDNA we obtained was intact.Two categories of vectors were constructed and used (Fig.

1). The NTG vector contains the NeoR gene driven by theMoloney murine leukemia virus LTR promoter and the GCgene is driven by the Htk promoter assisted by the polyomamutant enhancer. This enhancer/promoter has been reportedto function well in embryonic stem cells (20). The secondcategory of vectors uses the viral LTR as an enhancer/promoter to drive the GC gene. Three LTR vectors wereconstructed (GTN, GI, and G). Splice acceptor sites in frontof the IL-3 coding sequence in the GI vector allow thegeneration of a monocistronic IL-3 mRNA. Cells that gaingrowth advantage through high-level IL-3 production aftertransplantation of GI-transduced cells would be expected toproduce high levels ofGC, since both genes, GC and IL-3, aredriven by the LTR promoter.Ten to 20 clones of viral producer cells were generated for

each vector and evaluated with respect to rearrangements,titer, and RNA production. Supernatants from freshlythawed producer cells did not contain helper virus (S+L-assay). The NTG and GTN vectors were titered on 3T3 cellsand colonies resistant to G418 were counted. The titer of GIand G virus colonies was estimated indirectly by IL-3 pro-duction in the supernatants (GI virus) and RNA analysis.

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GTN GI G1 2 34 5 1 2 3 1 2

9.5-6.64.3-

2.3 -- -

1.35-

D-G2.3 kb i

Eco RI Eco RI

FIG. 2 Southern blot analysis of DNA from individual CFU-Scolonies. (A) DNA from NTG vector-infected CFU-S colonies cutwith Xho I, which releases a 2.6-kb fragment that hybridizes with theGC probe. 4r, negative-control DNA from T-2 cells. (B) DNA fromCFU-S colonies whose CFU-S progenitors had been exposed to theGTN, GI, and G vectors. All three vector DNAs release a 2.3-kbfragment that hybridizes with the GC probe. The probe was afull-length GC cDNA. Molecular sizes are given as kb x 10-3. N,NeoR; T, Htk and Py*; G, GC.

Finally, a proper titer was performed on CFU-S progenitorcells.Gene Transfer into CFU-S Progenitors. On day 14 after

BMT, individual CFU-S colonies were dissected from recip-ient spleens. DNA from the colonies was examined bySouthern blot analysis to determine whether proviral DNAcould be detected (Fig. 2). Table 1 summarizes the genetransfer efficiency data from eight experiments performedwith the four vectors described in Fig. 1. Efficient gene

transfer with the NTG, GTN, and GI vectors (100%, 60-75%,and 52-100%, respectively, ofCFU-S colonies) was achieveddespite a low concentration of growth factors during infec-tion. Infection efficiency can vary up to 2-fold in differentexperiments using the same vector under similar infectionconditions.One of the vectors, the G virus, has by far the lowest

infection efficiency (12%) when low concentrations of IL-3and IL-6 are used. When recombinant murine IL-3 and IL-6(each at 200 units/ml) are added during prestimulation andcoculture, the infection efficiency is increased 6-fold (72%).It is, therefore, clear that one can increase infection effi-ciency of primitive hematopoietic progenitors significantly

NTGV SI S2

G

V SI S2

GTNV S

Table 1. Gene transfer efficiency into CFU-S multipotentialprogenitor cells

Growth No. positive/Vector Exp. factors total no. %

NTG 1 Low 31/31 100GTN 1 Low 9/12 75

2 Low 15/25 60GI 1 Low 8/9 89

2 Low 13/25 523 Low 13/13 100

G 1 Low 1/8 122 High 8/11 72

Donor bone marrow cells from 5-fluorouracil-treated C57BL/6Jmice were prestimulated and infected by coculture using the fourvectors shown. Fourteen days after transplantation into irradiatedWBB6F1 ±/± recipients, the spleen was harvested and individualCFU-S colonies were dissected. Their DNA was extracted andexamined by Southern blot analysis (Fig. 2). Low growth factorsindicate 15% WEHI conditioned medium and 20 units of recombinanthuman IL-6. Twenty units of purified mouse IL-3 per ml of mediumwas added when available. High growth factors indicate 15% WEHIconditioned medium and recombinant murine IL-3 and IL-6 fromCOS cell supernatants each at 200 units/ml.

when high concentrations of IL-3 and IL-6 are used tostimulate cell division prior to and during infection.Production of Human GC RNA in CFU-S Colonies. We

asked whether the retrovirally transferred gene was ex-pressed in CFU-S colonies. Spleens confluent with coloniesfrom infected bone marrow were harvested on day 14. TheRNA was extracted and examined by Northern blot analysis(Fig. 3). RNA from vector producer cells served as a positivecontrol. In general, the main RNA species seen from all fourvectors is the viral genomic RNA driven by the LTR pro-moter. The NTG vector produces two main species in theproducer cells detected by the human GC probe, the viralgenomic RNA (5.8 kb) and the human GC mRNA initiated bythe Py*/Htk enhancer/promoter. The viral genomic mRNAis also seen in the CFU-S colonies but the internal Htkpromoter is either not active or has very low activity inCFU-S colonies compared to the Moloney murine leukemiavirus LTR. Very weak signals or low-intensity smearing at 3kb, seen in some of the NTG transduced spleen samples, mayindicate low activity of the Htk promoter (Fig. 3, sample S2,NTG vector).The other three vectors (G, GTN, and GI) generate mainly

the unspliced viral genomic RNA as detected by the humanGC probe (Fig. 3). Splicing of the genomic viral RNA is notprominent in any of the samples. The GI vector generates a

GI HSI S2 S3 V F

795-9.:5 9,-9.5 9.5LTR- 9.5 7.5 LTR lss* 7.5

- .~~ ~~~~7I5LTAW-4.4v745 LTR- -44 44K LTR -4E4

24.140 2.4

2.4 201.4 14

1.4-- 1.4 .

D- }-E5.8

3.1-

PROBE

4.0 O-- 5.6 - 5.6

El

FIG. 3. Northern blot analysis of RNA isolatedfrom spleens confluent with CFU-S colonies (lanesS, S1, S2, and S3) and virus producer cells (lanesV). The transplanted bone marrow had been ex-posed to the NTG, G, GTN, and GI vectors, asshown above the autoradiographs. Diagrams of thevectors used are shown below. Wavy lines repre-sent RNAs generated by vectors that will hybridizewith a GC probe. The length of each RNA moleculein kb is indicated. Vector-generated RNAs that donot hybridize with the full-length GC cDNA probeare not shown. LTR, RNAs initiated by the viralLTR promoter; TK, RNA initiated by the Htkpromoter. Molecular sizes are given as kb x 10-3.l, IL-3. Other abbreviations are as in Fig. 2.

A

9.5 -6.6 --4.3 -

V1 2 3 4 5 6 7 8

B

2.3-2.0 -1.35-

tL- N T1Gi 2.6 kb t

Xhol Xhol

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EA D C

FIG. 4. Production of human GC in individual- 7.5 CFU-S colonies as detected by a monoclonal anti-

^ body. (A-D) Immunofluorescent staining of cells- 4.4 from individual CFU-S colonies with antibody 8E4.

(A) Uninfected. (B) G virus infected. (C) G0 virus- 2.4 infected. (D) GTN virus infected. (E) Northern blot

analysis of RNA isolated from the cells shown in A,1 4 C, and D. Lanes: A, RNA from uninfected CFU-S

colony (A): C, RNA from a Gl virus infected CFU-Scolony (C); D, RNA from a GTN-infected colony(D). RNA was not isolated from the G virus-infected cells in B. For probes and viral diagramssee Fig. 3. Molecular sizes are in kb x 10-i.

smaller monocistronic IL-3 mRNA by splicing. This splicedRNA is not detected by the GC probe as expected but is seenwith an IL-3 probe (data not shown). Collectively the datapresented in Fig. 3 demonstrate that the viral LTR promoter(Moloney murine leukemia virus in NTG and GTN; Moloneymurine sarcoma virus in G and GI) generates high steady-state levels of human GC mRNA in CFU-S colonies. Incontrast, the internal Py*/Htk enhancer/promoter does notwork effectively in the NTG vector after transduction intoCFU-S progenitors despite its good function in mouse fibro-blast producer cells (Fig. 3). A weakly hybridizing 1.8-kbRNA species is seen in the producer cells of NTG, G, andGTN. This is the mouse GC RNA that cross-hybridizes to thehuman GC probe.

Production of Human GC in CFU-S Colonies. Cells fromindividual CFU-S colonies were stained with a mouse mono-clonal antibody, 8E4, which reacts with human but not mouseGC (29). Fig. 4 shows examples of CFU-S colonies infectedwith G, GTN, and GI vectors. Each vector produced humanGC (cross-reacting material) in the differentiated progeny ofCFU-S progenitor cells, as detected by the 8E4 antibody.Uninfected colonies did not show any staining. Vector gen-erated human GC RNA was found in the antibody-positivecolonies that were tested by Northern blot analysis (Fig. 4).

Transfer of the Human GC Gene into Long-Term Recon-stituted Mice. The helper virus-free vector, NTG, was used toinfect donor bone marrow cells that were subsequentlytransplanted into W/W8' recipients. Analysis ofDNA from 12

V B S

9.5-7.5 U- LTR

2.4

FIG. 5. Northern blot analysis of RNA isolated from a mouse 5months after BMT with NTG vector-infected bone marrow cells.Lanes: V, NTG virus producer cells; B, bone marrow; S, spleen.LTR, RNA initiated by the viral TR promoter; TK, RNA initiated bythe Htk promoter (a very weak signal is seen in the B and S samples).Molecular sizes are given as kb x 10-3. For the probe and the NTGvector diagram, see Fig. 3.

mice using the polymerase chain reaction demonstratedvector DNA in 9 mice more than 5 months after BMT (datanot shown). RNA from nonadherent cells ofthe bone marrowand spleen of a vector-positive mouse was examined byNorthern blot analysis (Fig. 5). Both tissues contain onemajor RNA species corresponding to the expected size of theviral genomic RNA. This species was also seen in CFU-Scolonies (Fig. 3). A faint band migrating as. a 3-kb RNAspecies detected in both bone marrow and spleen mayrepresent 3.1-kb human GC RNA initiated by the Py*/Htkenhancer/promoter. The nature of the intermediate bands(4.4 kb and 3.5 kb) seen in Fig. 5, lane B, is unknown. Theycould represent unspecific mRNA bands around the 28SrRNA species but the 4.4-kb band could also be due toabnormal viral RNA splicing.

DISCUSSIONWe have demonstrated efficient transfer of the human GCcDNA into multipotential hematopoietic progenitor cells ofmice, as detected by DNA analysis of CFU-S colonies.Proviral genomes in transduced CFU-S colonies are func-tional, and three of four vectors tested produced high levelsof human GC RNA in the CFU-S-derived cells. The viralLTR directs production of the human GC RNA efficiently,but the internal Py*/Htk enhancer/promoter does not. Sim-ilarly, the LTR promoter is much more active in the hema-topoietic tissues of long-term reconstituted mice than theinternal Py*/Htk enhancer/promoter ofthe NTG vector. Theresults from this study and previous data on f-globin vectorexpression (17, 18) suggest that retrovirally transduced genesexpressed in CFU-S colonies will also be functionally activein the differentiated progeny of repopulating hematopoieticstem cells transduced with the same vector. All three vectorsusing the LTR promoter to drive the GC cDNA can causeproduction of human GC protein (cross-reacting material) inCFU-S colonies.The efficiency of CFU-S progenitor cell transduction de-

pends on vector titer, the density of the target cells, and theircell cycle phase at the time of infection. Three of the vectorsused were able to transduce a great majority of the CFU-Sprogenitors when a low concentration of growth factors wasused prior to and during infection. These three vectors (NTG,GTN, and GI), therefore, have a high titer on CFU-S pro-genitors, and one (NTG) has been demonstrated to infectrepopulating stem cells efficiently (9 of 12 mice are vector-positive). The transduction efficiency of one low-titer vector(G virus) was increased 6-fold by high concentrations of IL-3and IL-6 before and during the infection. Synergistic prolif-eration effects of IL-3 and IL-6 on murine multipotentialprogenitors were reported and postulated to be due, in partat least, to a decrease in the Go period of individual stem cells

A

C

B

D

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(33). Similarly, Bodine et al. (18) demonstrated an increase inthe number ofCFU-S progenitors and repopulating stem cellsand increased retroviral transduction efficiencies of thesecells under the proliferative stimulation effects ofmurine IL-3and IL-6.To obtain optimal expression of the transferred gene in the

progeny of the initially transduced progenitor or stem cell, itis important to select carefully the transcriptional regulatoryelements (enhancers and promoters) to drive the transferredgene within the vector. The viral LTR can direct the pro-duction of human GC RNA in mature hematopoietic cells invivo after gene transfer into CFU-S progenitors and hema-topoietic stem cells. These results agree with earlier studiesemploying the viral LTR to drive the NeoR gene (13, 14) andthe IL-3 gene (34). The Py* enhancer linked to the Htkpromoter was reported to function well after gene transferinto embryonic cells (20). Our results demonstrate that thistranscription regulatory unit functions poorly in the progenyof NTG vector transduced hematopoietic stem cells. Thismay be due to poor function of these regulatory elements inthe biological system used here or it may reflect the positionof the Py*/Htk enhancer/promoter within the retroviraltranscription unit in the NTG vector. Hantzopoulos et al. (35)have demonstrated that an adenosine deaminase promoterfunctions poorly within, but well outside, the retroviraltranscription unit, and other observations using plasmids totransfer genes have shown that active upstream promoterscan exert an inhibitory effect on a promoter placed down-stream (36). However, there are examples of retroviral vec-tors with internal promoters that direct RNA synthesis effi-ciently after transfer into primitive hematopoietic cells (15).The experiments described here represent initial steps to

develop gene replacement therapy for Gaucher disease. Type1 Gaucher disease is particularly well suited for gene therapysince heterologous BMT has proven to offer causal therapyof the disease, but potentially fatal complications have dis-couraged widespread use of this treatment (4-7). Gene trans-fer and subsequent autologous BMT is expected to causefewer and less severe therapeutic complications. It is notclear at present whether gene therapy can reverse neurologicsymptoms in type 2 and type 3 Gaucher disease since thepathogenesis of the neurological manifestations is poorlyunderstood and BMT has so far failed to improve neurolog-ical symptoms in a patient with type 3 disease (6). Recentadvances in molecular diagnosis of the disease suggest thatprognosis may be predicted according to the patient's exactgenotype (37-39). If proven reliable, these techniques wouldhelp to identify suitable candidates for gene therapy beforethe development of serious symptoms that may hampertherapeutic success.

We thank Dr. C. T. Noguchi and Dr. N. S. Young for commentson the manuscript, Drs. E. Beutler and J. Sorge for the GC cDNA,Dr. A. W. Nienhuis for use of his mouse facility, Dr. D. Bodine foradvice on mice and helpful discussions, Dr. J. Leonardo for valuablehelp with plasmid constructions, Dr. F. Lee for a gift of plasmidspCDIL-3 and pCDIL-6, and Dr. R. Kaufman for a gift of plasmidpMT2. This research was supported in part by the National GaucherFoundation by a grant (NGF 26) to S.K. This work was performedin partial fulfillment of the requirements for the Ph.D. degree ingenetics at The George Washington University, Washington, DC, byP.H.C.

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