Proc. Natl. Acad. Sci. USAVol. 92, pp. 7272-7276, August 1995Medical Sciences

Resistance to human immunodeficiency virus type 1 infectionconferred by transduction of human peripheral bloodlymphocytes with ribozyme, antisense, or polymerictrans-activation response element constructsLUN QUAN SUN, JAGDEESH PYATI, JASON SMYTHE, LI WANG, JANET MACPHERSON, WAYNE GERLACH,AND GEOFF SYMONDS*The R. W. Johnson Pharmaceutical Research Institute-Sydney, 74 McLachlan Avenue, Rushcutters Bay, New South Wales 2011, Australia

Communicated by Neal L. First, University of Wisconsin, Madison, WI, April 10, 1995 (received for review September 30, 1994)

ABSTRACT Human peripheral blood lymphocytes(PBLs) were transduced with a number of recombinant ret-roviruses including RRz2, an LNL6-based virus with a ri-bozyme targeted to the human immunodeficiency virus (HIV)tat gene transcript inserted within the 3' region of theneomycin-resistance gene; RASH5, an LNHL-based viruscontaining an antisense sequence to the 5' leader region ofHIV-1 downstream of the human cytomegalovirus promoter;and R2OTAR, an LXSN-based virus with 20 tandem copies ofthe HIV-1 trans-activation response element sequence drivenby the Moloney murine leukemia virus long terminal repeat.After G418 selection, transduced PBLs were challenged withthe HIV-1 laboratory strain IIIB and a primary clinical isolateof HIV-1, 82H. Results showed that PBLs from differentdonors could be transduced and that this conferred resistanceto HIV-1 infection. For each of the constructs, a reduction of'70% in p24 antigen level relative to the correspondingcontrol-vector-transduced PBLs was observed. Molecularanalyses showed constitutive expression of all the transducedgenes from the retroviral long terminal repeat, but no detect-able transcript was seen from the internal human cytomeg-alovirus promoter for the antisense construct. Transductionof, and consequent transgene expression in, PBLs did notimpact on the surface expression of either CD4+/CD8+(measured by flow cytometry) or on cell doubling time (ex-amined by [3H]thymidine uptake). These results indicate thepotential utility of these anti-HIV-1 gene therapeutic agentsand show the preclinical value of this PBL assay system.

The human immunodeficiency virus (HIV) has been identifiedas the etiological agent ofAIDS and its associated disorders (1,2). At present, there is no adequate treatment for this disease;however, the use of genetic manipulation to inhibit HIVreplication appears to be a promising approach to AIDStherapy. Possible gene therapeutic approaches to intervene inaspects of HIV-1 replication include the use of ribozymeexpression to catalytically cleave and thus inactivate HIV-1RNA; antisense RNA expression to inhibit reverse transcrip-tion, processing, and translation of HIV RNA; expression ofmutant HIV structural or regulatory genes with dominantrepression activity; and expression of RNA decoys to inhibitHIV-1 transcription, processing, and packaging.To date, retroviral vectors have been the chosen delivery

method for the introduction of transgenes and gene therapeu-tic anti-HIV-1 agents. These vectors have been tested inhuman hematopoietic T-lymphocytic cell lines, such as CEM,SupTl, and MOLT-4 (3-12), that have several desirable char-acteristics, including unlimited growth potential for in vitroassays, but have the disadvantage of being transformed cells.

Therefore, it is necessary to test efficacy of anti-HIV genetherapeutic agents in primary human cells, such as peripheralblood lymphocytes (PBLs). By using PBLs, one can predict notonly the efficacy of antiviral constructs but also the transduc-tion efficiency and the level and duration of expression oftransgenes in the relevant cells. However, an in vitro assay ofPBLs has yet to be developed, although recent studies (13, 14)have shown the potential of PBLs in testing anti-HIV-1constructs.We have conducted a comprehensive study on human PBLs

to (i) test anti-HIV agents, including ribozyme, antisense, andRNA trans-activation response element (TAR) decoys, and(ii) establish the conditions for PBL transduction, G418 se-lection, and HIV-1 challenge with laboratory and clinicalHIV-1 isolates. The present report demonstrates that trans-duction of primary PBLs with retroviral constructs expressinga ribozyme targeted to the HIV-1 tat gene, an antisensesequence complementary to the 5' leader region of HIV-1, ora 20-copy TAR RNA decoy conferred substantial resistance toHIV-1 infection. This assay system is an improvement uponprevious assays of anti-HIV retroviral constructs and serves tocomplement present T-cell line assays. By using this system, wehave generated significant data of clinical relevance to HIVgene therapy.

MATERIALS AND METHODSCell Lines. Packaging cell lines q2 (15) and PA317 (ATCC

CRL 9078) were cultured in Dulbecco's modified Eagle'smedium (DMEM) containing 10% (vol/vol) fetal bovineserum. PA317 cells were subjected to selection (5-7 days)every 6 weeks in hypoxanthine/aminopterine/thymidine(HAT) medium. CRE and qiCRIP (16) were grown inDMEM plus 10% bovine calf serum.

Retroviral Vector Constructions. A chemically synthesizedhammerhead ribozyme targeted to the HIV-1 tat gene tran-script (nt 5865-5882 of HIV-1 IIIB, GGAGCCAGLTAGATC-CTA) was cloned into a Sal I site of the LNL6 vector (17)within the 3' untranslated region of the neomycin-resistance(neor) gene (Fig. 1A). This construct was named RRz2. Forthe antisense construct, a 550-bp BamHI fragment of theHXB2 clone-containing part of R, U5, and the 5' portion of thegag gene (nt 78-628) was cloned in an antisense orientationinto a BamHI site of the LNHL vector (Fig. 1B), which wasderived from the pNHP-1 vector by removing the humanHPRT cDNA at the BamHI site (18). The resultant antisense

Abbreviations: LTR, long terminal repeat; PBL, peripheral bloodlymphocyte; TAR, trans-activation response (element); HIV, humanimmunodeficiency virus; neor, neomycin resistance; CMV, cytomeg-alovirus; FACS, fluorescence-activated cell sorter; RCR, replication-competent retrovirus.*To whom reprint requests should be addressed.

7272

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

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Rz 2

Sal I

B Antisense

_\r -- ~~~-a-E20 TAR

Xhio I B._

HI

RRz2(LNL6)

RASH-5(LNHL)

R20TAR(LXSN)

FIG. 1. Schematic representation of the retroviral vector con-

structs and their consequent expression (not drawn to scale). (A)Ribozyme construct RRz2. (B) Antisense construct RASH5. (C)PolyTAR construct R20TAR. Parental retroviral vectors are denotedin parentheses. HCMV-P, human CMV promoter; SV40-P, simianvirus 40 promoter.

construct was called RASH5. The polymeric-TAR (polyTar)construct was made by inserting a 20-copy TAR fragment (20tandem copies) into Xho I and BamHI sites within the LXSNvector (19) and was termed R20TAR (Fig. 1C). The sequence

integrity and orientation of the constructs were confirmed byDNA sequencing or restriction enzyme mapping.

Production of Amphotropic Retroviruses. RetrovirusesLNL6 and RRz2 were produced by transinfection involving thepackaging cell lines q2 and PA317 cells. Approximately 80%confluent 4i2 cells were transfected with 10,ug of the constructDNA by using calcium precipitation and incubating in DMEMcontaining 10% fetal bovine serum (DMEM growth medium)for 14 hr. This medium was then removed, replaced with freshDMEM growth medium, and incubated overnight at 37°C in5% C02/95% air. Ecotropic viral supernatant was then col-lected from the transfected q2 cells and used to infect sub-confluent (60-80%) PA317 cells in DMEM growth medium inthe presence of Polybrene (4,ug/ml). After a 24-hr incubationat 37°C in 5% C02/95% air, the infected PA317 cells were

trypsinized and split 1:20 into DMEM growth medium con-

taining G418 (750 ,tg/ml). The medium was changed every

3-4 days until colonies formed. Ten to 20 clones from each ofthe constructs were picked and expanded for viral titer (neo-mycin-resistant colony assay) and replication-competent ret-rovirus (RCR) assays (19). NIH 3T3 cells were used fortitration of retroviruses and RCR assays. Retroviruses LNHL,RASH5, LXSN, and R20TAR were produced by transfecting4,CRE and infecting 4vCRIP cells. Twenty to 96 clones of eachconstruct were isolated for titer and RCR assays.

Transduction of Human PBLs. PBLs were prepared fromleukopacks of healthy donors by Ficoll/Hypaque gradientcentrifugation. CD4+ cells were enriched by depletion ofCD8+ cells with a MicroCELLector flask (Applied ImmuneSciences, Menlo, CA) by the manufacturer's instructions. Totalor CD4+-enriched PBLs (5 x 105 cells per ml) were stimulatedwith phytohemagglutinin (Sigma; 5 ,kg/ml) or the OKT3monoclonal antibody (Janssen-Cilag; 10 ng/ml) in RPMI 1640medium supplemented with 10% fetal bovine serum andhuman recombinant interleukin 2 at 20 units/ml (RPMIgrowth medium) for 48-72 hr. The simulated PBLs were

transduced by exposure of the cells to a producer cell-freeretroviral stock for 18 hr in the presence of Polybrene at 4,.g/ml (at a multiplicity of infection of 0.5). Forty-eight hoursafter transduction, PBLs were selected in RPMI growth me-

dium containing G418 at 300-500 p,g/ml for 10-14 days. Toachieve complete selection, a two-step procedure was em-

ployed. For each batch of PBLs, a G418 toxic dose assay wasset up and, simultaneously, a base-line G418 concentration of300,ug/ml was applied to the transduced PBLs in the initial 7-9days. After this initial period, the G418 concentration wasadjusted to that determined within the toxic dose assay. Aftertransduction and selection, the PBLs were then cultured infresh medium without G418 for a week. This recovery step isimportant to enhance cell viability and increase cell numbers(a 3-5 times increase was found relative to that seen withG418) for the subsequent HIV-1 challenge assays.

HIV-1 Infection. The infectious titers [tissue culture 50%infective dose (TCID5o)] of the HIV-1 laboratory strain IIIBand clinical isolate 82H were determined on human PBLs asdescribed (20). The primary clinical isolate 82H was directlyobtained from a male patient through cocultivation withnormal PBLs and only passaged once in HUT78 cells. It hasbeen characterized as a T-cell tropic and syncytia-inducingisolate. Approximately 5 x 105 transduced PBLs were infectedwith 100 TCID50 of HIV for 2 hr at 37°C followed by washingcells twice with RPMI 1640 and resuspending cells in 5 ml ofRPMI growth medium. Every 3-4 days, aliquots of the super-natant were sampled for p24 antigen ELISA (Coulter).

Southern Blot Analysis. Cellular DNA was extracted fromnormal PBLs, transduced selected PBLs, and transduced non-selected PBLs by using a DNA extraction kit (Promega). DNA(10,ug) was digested with HindIII and EcoRI and separated ona 0.7% agarose gel. A neor_specific probe was used to detectintegrated retroviral vectors.RNA Analysis. Total cellular RNA was extracted by the

guanidium isothiocyanate method (21) from transduced PBLs.RNA (15 jig) was fractionated on a 1% agarose/formaldehydegel, transferred to a nylon membrane (Hybond-N), and hy-bridized with 32P-labeled neor_specific probe, the 550-bpBamHI fragment of HIV-1 HXB2, or the 20-TAR fragmentfor detection of neor-ribozyme, antisense, and TAR expres-sion, respectively.

Fluorescence-Activated Cell Sorter (FACS) Analysis ofTransduced PBLs. Approximately 1 x 105 transduced PBLswere incubated for 20 min at 4°C with CD4- or CD8-specificfluorescein isothiocyanate (FITC)-conjugated monoclonal an-tibodies (Becton Dickinson) or with a control antibody (FITC-mouse IgGl; Becton Dickinson). After two washes in PBS, thecells were analyzed on a Becton Dickinson FACScan.

[lH]Thymidine Uptake Assay. PBLs were transduced asdescribed above. After selection in G418 and recovery in freshRPMI growth medium, viability was assessed by trypan blueexclusion, and cell numbers were adjusted to 1 X 106 viablecells per ml. Triplicate wells (Corning 24-well plates) wereseeded with 1 x 106 cells per well and 1 ,uCi of [6-3H]thymidine(5 Ci/mmol; Amersham; 1 Ci = 37 GBq) was added to eachwell. After 48 hr in culture, cells were transferred to glass fiberfilters under vacuum, washed three times with ice-cold PBS,and precipitated three times with S ml of ice-cold 10% (wt/vol)trichloroacetic acid. Filters were rinsed with ethanol andsubjected to (3-scintillation counting. Statistical analysis wasperformed by using Student's t test.

RESULTSGeneration of High-Titer Amphotropic Retroviruses Con-

taining Various Transgenes. Three retroviruses expressing thetransgenes (ribozyme, antisense, or polyTAR) were con-structed based on the different vector backbones. RRz2 wasconstructed by inserting an anti-HIV tat ribozyme gene intothe 3' untranslated region of the neor gene driven by theMoloney murine leukemia virus long terminal repeat (LTR) inthe LNL6 vector (Fig. 1A). A chimeric neors-ribozyme RNAtranscript is expected from this retrovirus. In the RASH5retrovirus (Fig. 1B), the antisense sequence could be tran-scribed from either the viral LTR or the internal human

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cytomegalovirus (CMV) promoter. In the R20TAR construct,polyTAR is expressed from the viral LTR (Fig. 1C). The threeretroviral constructs and the corresponding control vectorswere used to generate amphotropic producer cell lines. Viraltiters were within the range of 1 x 105 to 5 x 106 colony-forming units (cfu)/ml, as measured (19). In general, retroviraltiters >106 cfu/ml were used in transduction experiments. Allthe viral stocks were tested and confirmed to be free of RCRand stored at -80°C.

Retroviral Transduction of PBLs. To optimize the stimula-tion of PBLs for retroviral transduction, the responses ofCD4+-enriched PBLs to phytohemagglutinin or the OKT3antibody were compared. No difference was observed with thecultures by using phytohemagglutinin or OKT3 in terms of celldoubling time, viability, and transduction capacity (data notshown). In the present experiments, OKT3 was used becauseit has been approved for use in humans. Stimulated PBLs werethen transduced with the amphotropic retroviruses at a mul-tiplicity of infection of 0.5. Determination of the relativetransduction efficiency was based on the number of cells thatsurvived G418 selection. The overall transduction efficiencyvaried from 2 to 7%, depending on the donor blood packs.

Integration and Expression of the Transgenes in the Trans-duced PBLs. To confirm and correlate the presence of retro-viral constructs in G418-resistant cells, RRz2- and LNL6-transduced cells were chosen for Southern blot analysis. Asshown in Fig. 2, the integration of the constructs was demon-strated in RRz2- and LNL6-transduced PBLs. Stronger signalswere seen in G418-selected PB3Ls, as expected, indicating aselection for cells containing retrovirus. The expression ofribozyme, antisense, or TAR sequence in the transduced PBLswas also evaluated. Fig. 3 shows the representative pattern ofNorthern blot analysis. In RRz2- and LNL6-transduced cells(Fig. 3A), both spliced and unspliced transcripts containingneor-ribozyme or neor mRNAs were detected with a neor-specific probe (3.2 kb and 2.4 kb). The predominant RNAspecies was the unspliced transcript. When the blot washybridized with a ribozyme-specific probe, the same patternwas observed for RRz2 RNA only (data not shown). InRASH5-transduced PBLs, two transcripts from the 5' LTR(spliced and unspliced) were detected with the 550-bp probeand confirmed to be the expected sizes (4.8 kb and 4.0 kb) (Fig.3B). However, the shorter transcript expected from the inter-nal CMV promoter was not expressed, indicating that theCMV promoter had been shut off in this construct. TheR20TAR vector generated an unspliced 4.6-kb transcript fromthe 5' LTR hybridizing to the TAR probe as expected (Fig. 3C)due to inactivation of the splice donor in LXSN (19).

-G418 +G418

(D c4 (CD C4_. N _J N .Jz cc z c m

-J:> a.

Pi: 's ''

*.:.'.m~~~~~~~~~~~~~~~~~~~~~~~~~~.... ... ......

*. ... ,...,.....<...

~~~~~~~~~~~~... .... ..... ....... .... ....... ..;

FIG. 2. Southern blot analysis of G418-selected PBLs transducedwith RRz2 and LNL6. Genomic DNA samples were digested withEcoRI and HindTIT to detect a 1.43-kb neor fragment from LNL6vector or a 1.48-kb fragment containing both neor and the ribozymegene from RRz2. Non-G418-selected (-G418) or G418-selected(+G418) PBLs or PBLs alone, as indicated, were probed.

A

-i , Nm z X0. -' &

B C

-i -l ) a0 Z < X XC. d A. -J m

FIG. 3. Expression of the retroviral constructs in PBLs transducedwith LXSN or R20TAR compared to control PBLs. The expression ofribozyme (A), antisense (B), and 20TAR RNA (C) was examined byNorthern blot analysis by using the corresponding 32P-labeled probes:a neor_specific fragment for RRz2 and LNL6 to detect neor-ribozymechimeric RNA; a 550-bp fragment from the HIV-1 5' LTR for RASH5and LNHL; a fragment of five tandem TAR copies for 20TAR andLXSN.

Inhibition ofHIV-1 Replication in Human PBLs. To analyzethe relevance of the PBL assay system to the study of HIV-1gene therapy, HIV challenge experiments were conducted ontransduced PBLs by using both the laboratory strain (IIIB) anda primary clinical isolate (82H). The infections were done induplicate and repeated with three to five batches of PBLs.HIV-1 replication was monitored at various times by measur-ing p24 antigen levels in the culture supernatant. In thechallenge experiments using HIV-1 IIIB, p24 production wasmarkedly reduced (70%) in the PBLs expressing ribozyme(Fig. 4A), antisense (Fig. 4B), and TAR decoy (Fig. 4C)relative to PBLs transduced with corresponding control vec-tors. Transduced and selected PBLs were also assessed fortheir resistance to the infection with a primary HIV-1 isolate.The replication of 82H (assayed by p24 antigen ELISA) wasinhibited to a level similar to that seen in HIV-1 IIIB-infectedPBLs (Fig. 4 D-F). These results indicate that these transgenesdelivered into human PBLs through retroviral vectors caninhibit HIV-1 replication in primary hematopoietic cells.

Analysis of Transduced PBLs. To investigate potentialeffects of transduction and construct expression on PBLdoubling time, [3H]thymidine-uptake assays were performedon all the transformed and selected PBLs. When comparedwith nontransduced normal PBLs, there were no obviousdeleterious effects in transgene construct- or vector-trans-duced PBLs (P < 0.02) (Fig. 5). In addition, FACS analysisrevealed that there was no difference of CD4 surface markerbetween RRz2- and LNL6-vector-transduced cells (Table 1).Thus, the inhibitory effect observed in HIV-1 challenge assayswas not due to a reduction in the number of CD4 receptors onthe transduced PBLs.

- DISCUSSIONFor gene therapy to be used to inhibit HIV-1 replication invivo, such gene therapeutic approaches must first be examinedin experimental systems. To date, these experimental systemshave mainly involved the use of human T-lymphocytic celllines, which are biologically different from primary PBLs.Despite the success of RNA-based or protein-based anti-HIVapproaches in T-cell lines, the clinical relevance of the dataremains to be clarified. To generate preclinical data, it isimportant to test anti-HIV-1 gene therapeutic agents in pri-mary hematopoietic cells. These cells are the major targets for

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B4000

3000 -

2000

1000

2

4 6 8 10 12 14 2

2.I----- .

4 6 8 10 12Time, days

E3000-

2000-

1o000

C

4 6 8 10 12 14 2 4 6 8 10 12 14

F

1

14 2 4 6 8 10 12

Time, days

1500-

1o000

500-

14 4 6 8 10Time, days

12 14

FIG. 4. Inhibition of HIV-1 replication in transduced PBLs. The transduced and selected PBLs were challenged with HIV-1 IIIB (A-C) or a

clinical isolate 82H (D-F) and assayed. Data plotted are the means from five donors (A and B) or two donors (C-F). (A) Curves: 1, LNL6; 2, RRz2.(B) Curves: 1, LNHL; 2, RASH5. (C) Curves: 1, LXSN; 2, R20TAR. (D) Curves: 1, LNL6; 2, RRz2. (E) Curves: 1, LNHL; 2, RASH5. (F) Curves:1, LXSN; 2, R20TAR.

HIV-1 infection and replication and there are significantdifferences in growth characteristics, response to in vitromanipulation, and reactivity to HIV-1 infection between celllines and PBLs. The CD4+ PBL population is depleted inAIDS and, thus, represents a major target-cell population forHIV-1 gene therapy. We note that all recently proposedclinical trials for HIV-1 gene therapy have involved use ofCD4+ PBLs as target cells (for review, see ref. 22). For thesereasons, we have conducted the present study to establish aPBL assay system.

In this study, three retroviral vectors expressing ribozyme,antisense, or TAR decoy genes have been tested for theirantiviral efficacy in human PBLs. Although they were con-

structed in different retroviral vectors, all were shown to besimilarly effective in HIV-1 protection assays with no apparentcytotoxicity, as measured by [3H]thymidine incorporation as-

say and FACS analysis. The anti-tat ribozyme construct hasbeen designed to target the AUG initiator codon region of thetat gene, which has generally been shown more accessible toantisense oligomers (23, 24). Studies in in vitro cleavage and in

120-

0 U)~ 100-

800

L 60-Cco

40

HE]0PBL only LXSN R-20TAR LNHL RASH5 LNL6 RRz2

FIG. 5. [3H]Thymidine uptake assays of the transduced PBLs. Dataare the mean ± SD (n = 3). Bars: PBLs only, untransduced PBLs;LXSN, R20TAR, LNHL, RASH5, LNL6, and RRz2, transduced PBLswith the corresponding retroviruses.

T-cell line assays have demonstrated this ribozyme to be veryeffective in cleaving the tat transcript and inhibiting HIV-1replication (L.Q.S., L.W., W.G., and G.S., unpublished re-

sults). This efficacy is further demonstrated in a PBL system,which indicates the potential utility of this anti-tat ribozyme inclinical studies of HIV-1 gene therapy.

Considering the points in the HIV-1 replication cycle atwhich the inhibition of HIV-1 replication by ribozyme, anti-sense RNA, or polyTAR occurs, they may act by the differentmechanisms. Ribozymes have been shown to mainly cleaveand, perhaps, eliminate viral RNA. Previous reports havesuggested that expression of ribozyme genes interfered withboth early and late events in HIV-1 replication (6, 25). Sinceantisense RNA can bind to both DNA and RNA molecules,multilevel action for antisense RNA would also be likely,potentially affecting RNA entry, reverse transcription, tran-scription, and RNA processing or translation (7). PolyTARsequences can act as a decoy to bind Tat and/or cellularfactors, thereby inhibiting HIV replication (26). However, toour knowledge, none of these inhibitory mechanisms has beenexploited in the PBL system and it would be valuable if suchinformation could be provided from studies in primary PBLs,such as the points where inhibition occurs, cellular localizationof the transgenes, and potential involvement of cellular factors.Furthermore, combinational use of these transgenes for HIV-1gene therapy may be advantageous over their single use based

Table 1. CD4/CD8 surface markers on total PBLs as measuredby FACS analysis

% cells

PBLs CD4+ CD8+

Total 43.35 23.41LNL6 34.41 27.00RRz2 33.06 30.37

Total PBLs, untransduced total PBLs; LNL6 PBLs, total PBLstransduced with LNL6 virus; RRz2 PBLs, total PBLs transduced withRRz2 virus.

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on their ability to target different sites and points in thereplicative cycle.To utilize PBLs as either an assay system or as therapeutic

target cells, several points should be noted. A high viral titeris crucial to achieve efficient transduction of PBLs. This isespecially true for clinical purposes where it may not bedesirable to select transduced PBLs with G418. For in vitroassay, we have developed a two-step G418 selection procedureby which complete selection can be readily achieved. Theseprocedures minimize the time of in vitro culture, therebyreducing modification to the T-cell population. In our hands,continuous culture of PBLs in vitro for 2 weeks did notsignificantly impact the surface markers (CD4+ and CD8+).This period (2 weeks) may be sufficient for any ex vivomanipulation of PBLs for therapeutic purposes.

Retroviral vector design is another important aspect forefficient gene transfer and expression. Although no directcomparison can be made among the three vector designs usedin this study, two observations should be noted. (i) All thetransgenes controlled by the viral LTR (but not from the CMVinternal promoter for one construct) were efficiently expressedin a constitutive manner in primary human hematopoieticcells. (ii) The strategy whereby a ribozyme gene is inserted intothe 3' untranslated region of a gene such as neor in theretroviral vector appears to be as efficient in PBLs as it is inT-cell lines (25). These observations may be useful for futureimprovements in gene therapeutic design.

In conclusion, transduction of primary human PBLs andtheir protection from HIV-1 infection ex vivo can be accom-plished by using the protocols presented here. This not onlywill provide a useful system for assessment of gene therapeuticagents in vitro but also forms the basis for HIV-1 gene therapytargeted to CD4+ lymphocytes.

We thank A. Minter, S. Houghton, C. Witherington, and A. King fortechnical assistance; M. Miller for FACS analysis; and R. Penny andD. Cooper for advice and facilities. We are also grateful to Dr. B. Wylieof Sydney Red Cross Blood Transfusion Service for the donation of theleukopacks. This work was supported by a Research and DevelopmentGrant from Gene Shears Proprietary Ltd.

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